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Analysis of stem and progenitor cell responses to variations in bioprocess culture variables 2006

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Analysis of Stem and Progenitor Cell Responses to Variations in Bioprocess Culture Variables by Muhammad Arshad S. Chaudhry B.Sc . (Engineering), University o f Engineering & Technology, Lahore, Pakistan, 1992 M . S c , K i n g Fahd University o f Petroleum & Minerals, Dhahran, K S A , 1995 A THESIS S U B M I T T E D I N P A R T I A L F U L F I L L M E N T O F T H E R E Q U I R E M E N T S F O R T H E D E G R E E O F D O C T O R O F P H I L O S O P H Y In T H E F A C U L T Y O F G R A D U A T E S T U D I E S (Department of Chemical & Biological Engineering) T H E U N I V E R S I T Y O F B R I T I S H C O L U M B I A August 2006. © Muhammad Arshad S. Chaudhry, 2006 Abstract The realization o f many potential cell therapies w i l l depend on the development o f culture protocols that consistently expand stem and progenitor cell numbers while retaining their developmental properties. Hematopoietic cell lines were used to develop predictive mathematical models o f cytokine-dependent proliferation. First, Hill-type empirical models provided a quantitative understanding of how cytokines (IL-3 and G M - CSF) individually influenced the growth rate. Then a competitive model o f cytokine interactions was developed that effectively predicted the growth rates for TF-1 and M 0 7 e cells exposed to combinations o f IL-3 and G M - C S F , over wide concentration ranges. Mouse embryonic stem cells (mESC) were used to study the influence o f various culture variables on their proliferation and embryoid body (EB) formation potential. The medium p H , osmolality, basal medium composition, as wel l as serum and serum replacement (SR) concentrations were found to modulate m E S C responses. For most o f these variables, conventional cultivation methods did not expose the cells to levels that significantly decreased culture output. However, for feeder-containing cultures, the osmolality increased to over 400 mOsm/kg and, when E S C were cultured in such a medium, the E B yield was decreased by more than 50% compared to media at 300 and 335 mOsm/kg. Likewise, p H dose response experiments revealed that, within 48 h, compared to a medium initially at p H 7.3, the E B yield decreased by approximately 50% when R l cells were cultured at either p H 7.15 or 7.45, with even lower yields at more extreme p H conditions. The m E S C responses were also found to be serum concentration- dependent with maximal results at 5% serum in D M E M compared to the commonly used 15%. In D M E M : F 1 2 , a reduced dependency on serum was observed. When S R was used, both the proliferation and E B formation potential were maximal with 5% S R in D M E M . In D M E M : F 1 2 , 1.7% S R was sufficient to maintain the E B yield o f m E S C lines with a similar growth rate. Taken together, these results demonstrate that multiple critical culture variables can profoundly influence the responses o f m E S C and that these variables need to be kept within defined ranges to maintain the m E S C in culture without compromising their developmental potential. i i Table of Contents Abstract i i Table o f Contents i i i List o f Tables v i i List o f Figures , v i i i List o f Abbreviations and Symbols x i i i Acknowledgements x v i Dedication x v i i i 1 Introduction and Literature Review 1 1.1 Hematopoiesis 3 1.1.1 Hematopoietic Cytokines 4 1.2 Origin and Derivation of Embryonic Stem Cells 6 1.3 Maintenance o f Undifferentiated State of m E S C 8 1.4 Characterization of m E S C Responses 10 1.5 Molecular Mechanisms Underlying Self-Renewal of m E S C 11 1.6 Human E S C Lines 13 1.7 Embryoid Body Formation Based Differentiation o f m E S C 16 1.8 Culture Environment and Maintenance o f Stem Ce l l Potential 18 1.8.1 Serum 19 1.8.2 Culture Osmolality 20 1.8.2.1 Regulation o f Volume Control 21 1.8.2.2 Organic Osmolytes 22 1.8.2.3 Molecular Mechanism of Cellular Protection from Hyperosmolality.. 24 1.8.3 Culture p H 26 1.8.3.1 Effect o f Culture Medium on Intracellular p H (pHj) 28 1.8.3.2 Regulation o f p H ; 29 1.8.3.3 Influence of p H on Cellular Functions and Development 31 1.9 Mouse E S C as a Mode l System 32 1.10 Thesis Obj ectives 35 i i i 1.11 References 45 2 Empirical Models of the Proliferative Response of Cytokine Dependent Hematopoietic Ce l l Lines 76 2.1 Introduction 76 2.2 Materials and Methods 80 2.2.1 Ce l l Lines 80 2.2.2 Ce l l Proliferation Assay 80 2.2.3 Cytokine Depletion Analysis by E L I S A 81 2.2.4 Calculation of Ce l l Specific Cytokine Uptake Rate 81 2.2.5 Mathematical Model ing of Cytokine Dependent Proliferation 82 2.2.5.1 Initial Single Cytokine Mode l 82 2.2.5.2 Improved Single Cytokine Mode l 82 2.2.5.3 Cytokine Interaction Models 83 2.3 Results 85 2.3.1 Dose-Response Analysis of M 0 7 e and TF-1 cells 85 2.3.2 Determination of Cytokine Interactions 86 2.4 Discussion 88 2.5 Conclusions 92 2.6 References 104 3 Culture p H and Osmolality Modulate Proliferation and Embryoid Body Formation Potential o f Murine Embryonic Stem Cells 109 3.1 Introduction 109 3.2 Materials and Methods 115 3.2.1 Growth of Mouse Embryonic Fibroblasts ( M E F ) 115 3.2.2 Growth of Mouse Embryonic Stem Cells (mESC) 115 3.2.3 Determination of Growth Rate 116 3.2.4 Preparation of Medium at Desired p H or Osmolality 116 3.2.5 Measurement o f p H or Osmolality 117 3.2.6 Measurement o f Glucose, Lactate and Glutamine Concentrations 118 3.2.7 Preparation o f m E S C for p H or Osmolality Experiments 119 iv 3.2.8 Embryoid Body Formation Assay 120 3.2.9 Flow Cytometry 121 3.2.10 Statistics 122 3.3 Results 123 3.3.1 Culture Variables during Routine Passaging o f m E S C 123 3.3.2 Osmolality Dose-Response Experiments 124 3.3.3 p H Dose-Response Experiments 125 3.3.4 Influence o f p H on Metabolism o f m E S C Line, R l 127 3.3.5 Variation of Ce l l Size as a Function o f p H 127 3.3.6 Variation of Oct-4 Positive and Annexin-V Positive Cells with p H 128 3.3.7 Reversibility of p H Effects 128 3.4 Discussion 130 3.5 Conclusions 134 3.6 References 154 4 Basal Medium Composition and Serum or Serum Replacement Concentration Modulate Responses o f Murine Embryonic Stem Cells 160 4.1 Introduction 160 4.2 Materials and Methods 165 4.2.1 Growth o f Mouse Embryonic Fibroblasts ( M E F ) 165 4.2.2 Growth of Mouse Embryonic Stem Cells (mESC) 165 4.2.3 Determination of Growth Rate 166 4.2.4 Preparation of Media for Serum or Serum Replacement Dose-Response Experiments 166 4.2.5 Measurement o f Glucose, Lactate and Glutamine 167 4.2.6 Preparation o f m E S C for Dose-Response Experiments 167 4.2.7 Embryoid Body Formation Assay 167 4.2.8 Statistics ..167 4.3 Results 168 4.4 Discussion 172 4.5 Conclusions 175 v 4.6 References 182 5 Conclusions and Future Directions 187 5.1 Conclusions 187 5.2 Future Directions 191 5.3 References 194 vi List of Tables Table 1.1 Characteristic Features of Cytokines 38 Table 1.2: Examples of hematopoietic cytokines and their actions 39 Table 2.1 %2/DOF statistics for goodness-of-fit test comparing Monod-type model Eq. (2.3), Hill-type function Eq. (2.4) with exponent 'n ' fixed (n = 0.5) as well as Hi l l function with variable exponents against experimental growth rates of TF-1 cells grown in the presence of IL-3, IL-6 or GM-CSF alone 93 Table 2.2 Hi l l model kinetic parameters for cell lines, TF-1 and M07e cells growing in IL-3, IL-6 and G M CSF a 94 Table 2.3 %2fDOF statistics for goodness-of-fit test comparing the two cytokine interaction models against experimental growth rates of TF-1 cells grown in the presence of combination of IL-3, IL-6 and GM-CSF 95 Table 2.4 The values of interaction parameter y computed to compare the additive interaction model (Equation 2.5) against experimental growth rates of TF-1 cells grown in the presence of combination of IL-3 and GM-CSF 96 Table 3.1 Average initial, final and logarithmic averaged pH values that R l cells were exposed to during the final 24 h (24 - 48 h) period in culture during pH dose-response experiments reported in Figure (3.9). Values shown are drawn from a subset of five experiments. 135 Table 3.2 Summary Table of 2-way A N O V A to determine the influence of pH and inoculum growth history on EB yield and growth rate of R l cells 136 vn List of Figures Figure 1.1 Hematopoietic hierarchy 40 Figure 1.2 Stem cell hierarchy. Adopted from Wobus et al. (2005) Physiol Rev 85, 635- 678. 41 Figure 1.3 Regulation o f self-renewal of m E S C by Oct-3/4, Nanog, BMP-dependent S M A D and L I F dependent J A K / S T A T 3 signaling pathways. Adopted from Wobus et al. (2005) Physiol Rev 85, 635-678 42 Figure 1.4 Schematic representing the potential interactions between the environment o f the embryo, either in vitro or in vivo, the embryo's short-term responses, and their long- term consequences. Adopted from Fleming et al.(2004) B i o l Reprod 71, 1046-1054 43 Figure 1.5 Summary of the main acid/base transporters of mammalian cells. Receptor activation by mitogens induces activation of the N a + / H + exchanger (NHE) leading to alkalanization o f the cytosol. The transporter H + -ATPase , N H E , lactate~-H+ symporters, N a + / H C C >3 - cotransporter ( N B C ) , C 1 7 H C 0 3 - exchanger (AE) and N a + dependent C 1 7 H C 0 3 - exchanger ( N D C B E ) all are involved in regulation o f pHj. Adopted from Schreiber (2005) J Membr B i o l 205, 129-137 44 Figure 2.1 Comparison o f the growth rate o f TF-1 cells cultured for either 72 or 120 h in the presence o f (a) IL-3 , (b) G M - C S F and (c) IL-6 with predictions of Monod- and Hill-type models. Data are mean ± standard deviation of three independent experiments. Legend applies to all three panels 97 Figure 2.2 Comparison of the growth rate of TF-1 cells cultured for either 72 or 120 h in the presence o f (a) IL-3, (b) G M - C S F and (c) IL-6 with prediction o f a Hill-type model with n fixed at 0.5 or varied according to the value obtained from non-linear regression o f the data. Data are mean ± standard deviation of three independent experiments. The same legend applies to all three panels 98 Figure 2.3 Ce l l concentration and G M - C S F uptake rate as a function of final G M - C S F concentration for 72 h cultures of TF-1 cells.. The rate of G M - C S F uptake was calculated from the rate o f G M - C S F depletion from the medium as determined by E L I S A . Data are the mean ± standard deviation of two independent experiments 99 Figure 2.4 Growth rates o f M 0 7 e cells cultured for 72 h in the presence o f IL-3 alone, G M - C S F alone or IL-3 and G M - C S F together at a ratio of [ IL-3] / [GM-CSF] = 2. Abscissa scale is initial IL-3 concentration when IL-3 and G M - C S F were used in combination. Data are mean ± standard deviation of three independent experiments. . .100 v i i i Figure 2.5 Comparison of the growth rate of TF-1 cell cultured in the presence o f both IL-3 and G M - C S F for (a) 72 and (b) 120 h with the predictions o f an additive Hill-type model (Eq. 2.5). In each set of experiments, the [IL-3] / [GM-CSF] ratio remained constant at fixed values of 2, 3.5 and 10.5. Data are mean ± standard deviation o f three independent experiments. The legend applies to both panels 101 Figure 2.6 Comparison of the growth rate o f TF-1 cells cultured in the presence o f both IL-3 and G M - C S F for (a) 72 and (b) 120 h with the predictions of a competitive Hill-type model (Eq. 2.6). In each set o f experiments, the [ IL-3] / [GM-CSF] ratio remained constant at fixed values of 2, 3.5 or 10. Data are mean ± standard deviation o f three independent experiments. Figure legend applies to both panels 102 Figure 2.7 Comparison of the growth rate o f TF-1 cells cultured in the presence o f (a) IL-3 + IL-6 or (b) IL-6 + G M - C S F for 72 and 120 hours, with the predictions o f a competitive Hill-type model (Eq. 2.6). In each set o f experiments, the [IL-6]/[IL-3] and [ IL-6] / [GM-CSF] ratios remained constant at fixed values o f 8 and 16 respectively. Data are mean ± standard deviation of three independent experiments. Figure legend applies to both panels 103 Figure 3.1 Pictures of 3-dimensional cellular aggregates that were considered and counted as E B s 137 Figure 3.2 Pictures of cellular aggregates that were not considered as E B s 138 Figure 3.3 Metabolic, p H and osmolality profiles o f R l mouse E S C grown on irradiated feeders following the conventional protocol o f exchanging medium at 24 h and harvesting the cells at 48 h after inoculation in maintenance medium of p H 7.6. Panel (a) shows glutamine and ammonium profiles, panel (b) depicts p H and osmolality profiles, and, panel (c) shows the glucose and lactate profiles. Values shown are mean ± S E M o f three independent experiments 139 Figure 3.4 Metabolic, p H and osmolality profiles o f R l mouse E S C grown on 0.1 % gelatin coated dishes following the conventional protocol o f exchanging medium at 24 h and harvesting the cells at 48 h after inoculation in maintenance medium o f p H 7.6. Panel (a) shows glutamine, panel (b) depicts p H and osmolality profiles, and, panel (c) shows the glucose and lactate profiles. Values shown are mean ± S E M of three independent experiments 140 Figure 3.5 Growth rate and mean EBs/init ial cell inoculated o f mouse E S C line R l as a function o f (a) initial glucose concentration (b) initial glutamine concentration and (c) initial ammonium concentration following conventional protocol o f exchanging medium 24 h following inoculation and harvesting the cells 48 h after inoculation.. The standard maintenance medium contains 20-22 m M glucose, 4 m M glutamine and generally around 1.5 m M ammonium. Values shown are mean ± S E M of seven experiments (* p < 0.05 compared to (a) 20 m M glucose (b) 4 m M glutamine and (c) 1.5 m M ammonium by paired t-test). Figure legend applies to all panels 141 ix Figure 3 .6 Growth rate and mean EBs/ini t ial cell inoculated of m E S C line R l as functions of medium osmolality following conventional protocol o f exchanging medium 2 4 h following inoculation and harvesting the cells 4 8 h after inoculation. Values shown are mean ± S E M of fifteen experiments (* p < 0 . 0 5 compared to osmolality of 3 0 0 mOsm/kg by paired t-test) 1 4 2 Figure 3 .7 Growth rate and mean EBs/ini t ial cell inoculated o f R l mouse E S C as functions of initial p H following conventional protocol of exchanging medium, pre- equilibrated at the same p H , 2 4 h following inoculation and harvesting the cells 4 8 h after inoculation. Values shown are mean ± S E M of thirteen experiments (* p < 0 . 0 5 compared to p H 7.3 by paired t-test) 1 4 3 Figure 3 .8 Growth rate and mean % E B s o f R l mouse E S C as functions o f initial p H following conventional protocol o f exchanging medium, pre-equilibrated at the same p H , 2 4 h following inoculation and harvesting the cells 4 8 h after inoculation. Values shown are mean ± S E M of thirteen experiments (* p < 0 . 0 5 compared to p H 7.3 by paired t-test). 1 4 4 Figure 3 .9 Dose response analysis o f growth rate and mean EBs/ini t ial cell inoculated o f m E S C line R l as functions of initial p H for two passages ( 9 6 h total exposure to various pH) following conventional protocol of exchanging medium, pre-equilibrated at the same p H , at 2 4 and 7 2 h following inoculation and harvesting the cells 4 8 and 9 6 h after inoculation. Values shown are mean ± S E M of seven experiments (* p < 0 . 0 5 compared to p H 7.3 by paired t-test) 1 4 5 Figure 3 . 1 0 Growth rate and mean EBs/init ial cell inoculated o f m E S C line E F C as functions o f initial p H following conventional protocol of exchanging medium, pre- equilibrated at the same p H , 2 4 h following inoculation and harvesting the cells 4 8 h after inoculation. Values shown are mean ± S E M of five experiments (* p < 0 . 0 5 compared to p H 7.3 by paired t-test) 1 4 6 Figure 3 . 1 1 (a) Mean number of E B s obtained per initial cell inoculated as a function o f initial p H when the inoculum R l mouse E S C were previously grown on 0 . 1 % gelatin- coated dishes or on irradiated feeders. Two-way A N O V A analyses found significant differences both in growth rate and in mean EB/ini t ia l cell as a function o f p H as wel l as previous growth history, (b) Growth rate as a function o f initial p H when the inoculum R l mouse E S C were previously grown on 0 . 1 % gelatin-coated dishes or on irradiated feeders. A l l values shown are mean ± S E M of at least five independent experiments 1 4 7 Figure 3 . 1 2 (a) Ce l l specific average glucose uptake rate (sGUR) and lactate production rate (sLPR) as functions o f initial p H of m E S C line R l used in the p H dose-response analyses depicted in Figure ( 3 . 5 ) . (b) Lactate to glucose yield coefficient ( Y ^c /G I U ) and average glutamine uptake rate (sGlnUR) as functions of initial p H o f R l mouse E S C used in the p H dose-response analyses depicted in Figure ( 3 . 5 ) Values shown are mean ± S E M of a subset o f seven experiments 1 4 8 x Figure 3.13 Variation o f average diameters of R l mouse E S C as a function o f initial p H (at initial osmolality o f 300 mOsm/kg for cultures with initial p H o f 6.7 - 7.3 and initial osmolality o f 320 - 345 mOsm/kg for cultures with initial p H 7.45 - 7.75) used in the dose-response analyses depicted in Figures 3.5 and 3.6. Values shown are mean ± S E M of a subset o f ten independent experiments. (* p < 0.05 compared to p H 7.3 by paired t- test). 149 Figure 3.14 (a) Variation o f %Oct-4 positive cells from m E S C line R l as a function o f initial p H during the dose-response experiments carried out for 48 and 96 h. (b) Variation o f % Annexin-V positive cells from m E S C line R l as a function o f initial p H during the dose-response experiments carried out for 48 and 96 h. Values shown are mean ± S E M of five experiments. (* p < 0.05 compared to p H 7.3 by paired t-test) 150 Figure 3.15 Reversibility of p H effect on responses of R l cells. Variation o f initial p H (6.7 and 7.75) as a function of time 151 Figure 3.16 Reversibility of p H effect on responses of R l cells. Average normalized growth rates o f R l cells as functions of p H and duration of exposure. Cells were harvested after 48 h o f setting up the experiment and cells were further grown for 48 or 96 h under p H 7.3 conditions. Values shown are mean ± S E M of an experiment carried out in triplicate. (* p < 0.05 compared to normali=zed p H 7.3 by paired t-test) 152 Figure 3.17 Reversibility of p H effect on responses of R l cells. Average normalized % E B s o f R l cells as functions of p H and duration o f exposure. Cells were harvested after 48 h of setting up the experiment and cells were further grown for 48 or 96 h under p H 7.3 conditions. Values shown are mean ± S E M of an experiment carried out in triplicate. (* p < 0.05 compared to normalized p H 7.3 by paired t-test) 153 Figure 4.1 Average growth rate observed when R l (open symbols) and E F C (closed symbols) cells were inoculated in the presence o f (a) serum with D M E M as the basal medium, (b) serum with D M E M : F 1 2 as the basal medium, (c) serum replacement with D M E M as the basal medium, and (d) serum replacement with D M E M : F 1 2 as the basal medium. Values shown are mean ± S E M of six independent experiments. (* p < 0.05 compared to 15% serum or S R by paired t-test) 176 Figure 4.2 Mean number of E B s obtained per initial cell inoculated when R l (open symbols) and E F C (closed symbols) cells were inoculated in the presence of (a) serum with D M E M as the basal medium, (b) serum with D M E M : F 1 2 as basal the medium, (c) serum replacement with D M E M as the basal medium, and (d) serum replacement with D M E M : F 1 2 as the basal medium. Values shown are mean ± S E M of six independent experiments. (* p < 0.05 compared to 15% serum or S R by paired t-test) 177 Figure 4.3 Ce l l specific glucose uptake rate (sGUR) obtained when R l (open symbols) and E F C (closed symbols) cells were inoculated in the presence of (a) serum with D M E M as the basal medium, (b) serum with D M E M : F 1 2 as the basal medium, (c) serum replacement with D M E M as the basal medium, and (d) serum replacement with x i D M E M : F 1 2 as the basal medium. Values shown are mean ± S E M of six independent experiments. (* p < 0.05 compared to 15% serum or S R by paired t-test) 178 Figure 4.4 C e l l specific lactate production rate (sLPR) obtained when R l (open symbols) and E F C (closed symbols) cells were inoculated in the presence of (a) serum with D M E M as the basal medium, (b) serum with D M E M : F 1 2 as the basal medium, (c) serum replacement with D M E M as the basal medium, and (d) serum replacement with D M E M : F 1 2 as the basal medium. Values shown are mean ± S E M of six independent experiments. (* p < 0.05 compared to 15% serum or S R by paired t-test) 179 Figure 4.5 Ce l l specific glutamine uptake rate (sGlnUR) obtained when R l (open symbols) and E F C (closed symbols) cells were inoculated in the presence o f (a) serum with D M E M as the basal medium, (b) serum with D M E M : F 1 2 as the basal medium, (c) serum replacement with D M E M as the basal medium, and (d) serum replacement with D M E M : F 1 2 as the basal medium. Values shown are mean ± S E M of six independent experiments. (* p < 0.05 compared to 15% serum or S R by paired t-test) 180 Figure 4.6 Mean number of E B s obtained per initial cell inoculated and growth rate o f R l cells when inoculated with three different serum replacement lots not used in previous experiments reported i n Figures (4.1-4.5) (a) 5% S R with D M E M (b) 15% S R with D M E M (c) 5% S R with D M E M : F 1 2 (d) 15% S R with D M E M : F 1 2 . Legend applies to all panels. Values shown are mean ± S E M of two independent duplicated experiments. (* p < 0.05 compared to 15% serum or SR by paired t-test) 181 x i i List of Abbreviations and Symbols b F G F Basic fibroblast growth factor (also known as FGF-2) B M P Bone morphogenetic protein B S A Bovine serum albumin C F C Colony forming assay C R U Competitive repopulation unit D M E M Dulbecco's modified Eagle's medium E B Embryoid Bodies E C Embryonal carcinoma E C M Extracellular matrix E G F Epidermal growth factor E G F R Epidermal growth factor receptor E L I S A Enzyme linked immunosorbent assay Epo Erythropoietin ( a cytokine) E S C Embryonic stem cells F B S Fetal bovine serum F C S Fetal calf serum Flt-3 Flt-3 ligand ( a cytokine) G - C S F Granulocyte-colony stimulating factor ( a cytokine) G M - C S F Granulocyte Macrophage-colony stimulating factor ( a cytokine) G M - C S F R Granulocyte Macrophage-colony stimulating factor receptor G P C Glycerophosphorylcholine h E S C Human embryonic stem cells HIL-6 Hyper-Interleukin-6 ( a cytokine) H S C Hematopoietic stem cells I C M Inner cell mass IL-11 Interleukin-11 ( a cytokine) IL-2 Interleukin-2 ( a cytokine) x i i i IL-3 Interleukin-3 ( a cytokine) IL-5 Interleukin-5 ( a cytokine) IL-6 Interleukin-6 ( a cytokine) I M D M Iscove's modified Dulbecco medium J A K Janus Kinase L I F Leukemia inhibitory factor ( a cytokine) L I F R Leukemia inhibitory factor receptor L T C - I C Long term culture-initiating cell M - C S F Macrophage-colony stimulating factor ( a cytokine) M D C K Madin-Darby canine kidney M E F Mouse embryonic fibroblasts m E S C Mouse embryonic stem cells M T G Monothioglycerol P B Peripheral blood p H e Extracellular p H pH; Intracellular p H R V D Regulatory volume decrease R V I Regulatory volume increase SF Stem Cel l or Steel Factor (a cytokine) S S E A Stage specific embryonic antigen S T A T Signal transducer and activation of transcription T R A Tumor recognition antigen V E G F Vascular endothelial growth factor Wnt Wingless Average specific growth rate (h"1) Average saturation specific growth rate (h"1) IL-3 average saturation specific growth rate (h"1) G M - C S F average saturation specific growth rate (h"1) Symbols M Msa, Msat IL-3 MsatGM xiv «1 Ratio of IL-3 half-saturation constant to G M - C S F half-saturation constant Ratio o f G M - C S F half-saturation constant to IL-3 half-saturation constant HCO; Sodium bicarbonate concentration (mM) Gin Glutamine concentration (pM) Glu Glucose concentration (pM) KGM G M - C S F half saturation constant (ng/mL) KIU IL-3 half saturation constant (ng/mL) KL Cytokine half saturation constant (ng/mL) L Cytokine concentration (ng/mL or pg/mL) Lac Lactate concentration (pM) n H i l l ' s exponent (-) pC02 Partial pressure o f carbon dioxide (mm Hg) Ce l l specific cytokine uptake rate (pg/h-10 6 cells) sGlnUR Cel l specific glutamine uptake rate (pmol/h-10 6 cells) sGUR Cel l specific glucose uptake rate (pmol/h-10 6 cells) sLPR Cel l specific lactate production rate (pmol/h-10 6 cells) t Time (h) X0 Initial cell concentration (cells/mL) xv Viable cell concentration (cells/mL) X V Acknowledgements I express my deepest gratitude and appreciation to Jamie and Bruce for providing me with the opportunity to work on this exciting project as well as for their vision, support and encouragement. Their guidance, generosity and optimism contributed greately to the completion o f this thesis. They provided a supportive environment that allowed me to develop skills both personal and professional that w i l l continue to provide me with strong guidance and knowledge in my future career. I have felt previliged to be supervised by Connie Eaves. She has inspired me with her intelligence and dedication to science. She provided stimulating, productive and often in-depth discussions during our group meetings and her constructive criticism helped a great deal to improve my work. M y other committee member, Sue Baldwin, is gratefully acknowledged for the valuable feedback she provided. Cl ive Glover helped me in many different ways during the course o f this research. Our discussions have always been very productive and his intelligence and advice has been invaluable on multiple occasions. His help with the statistical software package, R , is gratefully acknowledged. I have benefited immensely from interactions with my colleagues in the lab. Julie Audet, Jason D o w d and Y i t a Lee have provided valuable feedback and support early on during the course o f mathematical modeling and culture of hematopoietic cell lines. Faiz Gurgis introduced me to mammalian cell culture and I 'm thankful to him for that. Chris Sherwood has been very invaluable during my stay in the lab and his friendship is greatly appreciated. Nicolas Caron, Conine Hoesli , Marta Szabat, Shariar Imami and others have provided very valuable feedback during my group meeting presentations. I thank everyone who was in the lab during my tenure as a grad student. Their friendship is appreciated and w i l l never be forgotten. I would also like to thank Michael O'Conner, Melanie Kardel, Diane Reid and David Youssef in Connie Eave's lab for their wonderful support, help and guidance with human embryonic stem cell cultures that I performed in their lab. x v i I sincerely thank Jamie for providing me a chance to supervise co-op and undergraduate thesis students during my tenure in the lab. It is a valuable experience that w i l l benefit me immensely during my professional career. I am greatly indebted to Karyn Ho , Kenneth Park and Anna Maslennikove who helped me a great deal as co-op students. What I am today is in large part due to my parents, especially my mother's, efforts and sacrifices. I am sure my mother would have been very proud o f me today had she survived her Hepatitis B infection. I am greately indebted to Mona for her unconditional love and unwavering support. Even though by the grace o f Almighty A l l a h (God), I was able to ensure that free energy change for my thesis completion remains negative, I had much reduced impact on the rate of progress. It was Mona 's patience, commitment, endurance and constant encouragement that in large part made it possible to finish this thesis. I would also like to thank my family, relatives and friends for their prayers and support. Finally, I would like to gratefully acknowledge the support and facilities provided by Michael Smith Laboratories where I conducted my experiments, stayed t i l l late at night and made new friends. Financial support provided by International Council for Canadian Studies in the form of a Commonwealth Scholarship, U B C ' s University Graduate Fellowship as well as grants from N S E R C , Stemcell Technologies Inc. and National Centers o f Excellence of Canada (Stemcell Network and M I T A C S ) are thankfully acknowledged. x v i i Dedication H a v e they never considered the funct ioning of the heavens a n d the earth, a n d have they never observed closely any th ing that A l l a h (God) has created? A n d has i t never occurred to them that their life t e rm m i g h t a l ready have d r a w n near to its end? T h e n after this w a r n i n g of the Messenger wha t else can there be i n w h i c h they w i l l believe? W h o m e v e r A l l a h depr ives of guidance, there is no guidance for h i m , for A l l a h leaves such people w a n d e r i n g about b l i n d l y i n their contumacy. The Q u r a n (Chapter 7 (A l -Ara f ) , verses 185-186) To My Teachers, Parents, Mona, Hassan, Safivan, Suha and Rayyan xv i i i 1 Introduction and Literature Review Stem cell biology has come of age. Unequivocal proof that stem cells exist in the hematopoietic system has given way to the prospective isolation of many tissue-specific stem and progenitor cells, the initial delineation of their properties and expressed genetic programs, and much work developing their utility in regenerative medicine. B y definition, stem cells have the ability to both perpetuate themselves through self-renewal divisions and generate mature cells through differentiation. Although it seems reasonable to propose that each tissue arises from a tissue-specific stem cell, the rigorous identification and isolation o f the somatic stem cells has been accomplished only in a few instances. For example, hematopoietic stem cells (HSC) have been isolated from mice and humans, and have been shown to be responsible for generation and regeneration o f the blood forming and immune systems. H S C have already been used extensively in therapeutic settings1. The recent isolation and growth of human embryonic stem cells (hESC) in culture has caused much excitement and has raised hopes that in future it may be possible to generate, in vitro, new tissues and organs to replace those damaged by age and disease. The ability o f stem cells to undergo stable genetic transformations and produce altered gene products for the lifetime o f the host also makes them especially useful targets for gene therapy. Considerable progress has already been made in bioengineering relatively simple tissues such as skin, cartilage, and bone. To grow complex organs composed of many different cell types w i l l l ikely require manipulating stem cells, which have a greater developmental potential. A prerequisite for the clinical use of stem cells is a thorough understanding o f in vitro conditions that help determine stem cell self-renewal (i.e., the ability o f stem cells to renew themselves by dividing into the same non-specialized cell type), differentiation (i.e., the process whereby the stem cells acquire the features of specialized cells such as liver, heart or muscle cell), apoptosis (i.e., programmed cell death) or lineage commitment decisions that must be identified, defined and optimized in order to realize the potentials o f regenerative medicine. Strategies and techniques that favor the selection, isolation and enrichment o f specific cell populations must also be developed. 1 In the absence o f such detailed understanding, the clinical potential o f stem cells w i l l l ikely remain unexploited. A body o f evidence suggests the possibility to expand in vitro the desired functional cells for implantation into humans by manipulating specific environmental signals that control the process o f tissue formation. Soluble or membrane-bound cytokines control cell proliferation, differentiation and survival by interacting with cells v ia specific trans- membrane receptors and trigger signal cascades within the cells that alter gene expression and determine or modify cell function. Also , the 3-dimensional extracellular matrix conveys information that can control cell growth and development. The orchestration of stem cell self-renewal, proliferation, differentiation and apoptosis in vitro w i l l require not only the identification o f key molecular interactions but also the spatial and temporal aspects of these interactions. This represents one challenge faced by the emerging field o f stem cell bioengineering. Another equally important challenge is to be able to expand stem cells to large numbers in culture while maintaining their developmental potential. For example, as many as 3 x 1 0 n tumor infiltrating lymphocytes were needed to mediate beneficial effects in clinical trials 2, while 2 * 1 0 H myeloid cells would be necessary for a treatment to prevent neutropenia following chemotherapy3. It is therefore reasonable to assume that for clinical realization o f stem cell based therapies, it w i l l be necessary to grow and maintain relatively large numbers o f undifferentiated stem cells. Human embryonic stem cells (hESC) have a doubling time of ~ 40 h 4 and extensive cell expansion (in large-scale bioreactor cultures) would be needed to produce clinically relevant numbers o f cells. Expanding the h E S C numbers with maintenance o f developmental potential in a large-scale bioreactor would be challenging as the p H , osmolality and composition of the culture medium (serum containing vs. serum-free, for example), shear stress, dissolved oxygen tension, nutrient consumption and inhibitory metabolite accumulation, mode of reactor operation (for example batch or fed-batch vs. perfusion), etc., all become important variables to be optimized for a given system to realize the success o f the culture. This requires that stem cell responses to variations in these culture variables be first thoroughly understood in 2 small scale cultures with the hope that results from these studies would then guide large scale culture development. 1.1 Hematopoiesis Hematopoiesis is a complex, tightly-regulated process by which a variety of specialized blood cell types are generated from a relatively small population o f pluripotent hematopoietic stem cells (HSC). Mature blood cells have diverse functions ranging from oxygen transport and blood clotting to infection fighting. Because the terminal blood cells have limited life spans and are unable to replicate, hematopoiesis is necessary to sustain adult life, even in the absence of disease. The production o f mature blood cells from their precursors involves a series of differentiation events that take place over a large number o f cell generations. This results in a recognizable hierarchy o f hematopoietic progenitor cells of decreasing proliferative and differentiative potentials (Figure 1.1). The H S C are defined functionally by their ability to generate and sustain multi-lineage hematopoiesis after transplantation into a hematologically-compromised host. To maintain the stem cell population and generate more mature cells at the same time, a stem cell has to preserve a balance between self-renewal on the one hand, and lineage commitment followed by terminal differentiation into erythroid, myeloid or lymphoid cells on the other. During the latter process, the progenitors maintain the capacity to undergo cell division, but their differentiation potential becomes more and more restricted. A n intricate network o f cytokines and cell-cell interactions constantly ensures that the appropriate quantities of the different cell types are produced. Failure of these control mechanisms can result in various malignancies. Although phenotypic and functional properties have been extensively characterized (reviewed in references5"7), the fundamental question o f how H S C self-renewal is regulated remains unanswered. In most cases, combinations o f growth factors that can induce potent proliferation cannot prevent the differentiation of H S C in long-term cultures. Although some progress has been made in identifying culture conditions that maintain H S C activity in culture (for 3 example, see references8"1 0), it has proved exceedingly difficult to identify combinations o f defined growth factors that cause a significant expansion in culture in the number o f progenitors with transplantable H S C activity. 1.1.1 Hematopoietic Cytokines Cytokines are secreted or membrane-bound protein molecules that promote intercellular communication and mediate a number of biological functions such as cell growth and differentiation as well as immunity and inflammatory responses (Table 1.1). The most studied cytokines can be categorized into three general families based on their receptors: the tyrosine kinase receptor family, hematopoietic growth factor receptor family and the gpl30 receptor family. Flt-3 Ligand (Flt-3) and Steel Factor (SF) are examples of cytokines binding to receptors with tyrosine kinase activity. The receptors for cytokines belonging to the hematopoietic growth factor family do not possess an intrinsic tyrosine kinase domain but bind to cytoplasmic kinases. Interleukin-3 (IL-3), granulocyte-colony stimulating factor (G-CSF) and granulocyte-macrophage colony- stimulating factor ( G M - C S F ) are examples o f this family. The gpl30 family includes interleukin-6 (IL-6), interleukin-11 (IL-11) and leukemia inhibitory factor (LIF) among others. Receptors for these cytokines share the common receptor chain, gpl30. One characteristic that many cytokines share is an ability to maintain the viability o f their respective target cells by suppressing apoptosis in vitrou. A second function shared by cytokines is an ability to simulate the proliferation o f the appropriate progenitor cell populations, i.e., to act as mitogens. The effect o f the cytokines on the survival o f hematopoietic cells can be uncoupled from proliferation. For example, exposure to low concentrations of macrophage-colony stimulating factor ( M - C S F ) promotes survival (but not proliferation) of macrophages while higher concentrations promote both survival and proliferation 1 2. 4 Another characteristic o f cytokines is their ability to provide the conditions whereby multipotent and lineage-restricted progenitors undergo differentiation and development to produce mature cells . Some cytokines play a crucial role in the production o f specific lineages such that, for example, antibodies against erythropoietin (Epo) cause anemia when injected into animals. Other factors such as IL-3 and G M - C S F exert actions on a variety o f lineages. Table (1.2) lists some o f the important cytokines and their actions. A number o f these cytokines are known to be produced by fibroblasts and endothelial cells as well as macrophages and lymphocytes, all o f which help form the unique and heterogeneous microenvironment o f the bone marrow. It is clear from Table (1.2) that more than one cytokine can act on a particular lineage. There are several reasons for this redundancy: 1) Sequential Action: Cytokines may act at specific phases in the differentiation sequence o f specific lineages. For example, Epo acts on more mature erythroid precursors whereas IL-3 and SF act on early progenitors. 2) Recruitment: Adult stem cells appear to require a combination of two or more growth factors for proliferation, e.g., SF + Flt-3 + IL-11. SF alone maintains the survival of stem cells but induces little proliferation unless an additional cytokine is present. 3) Synergy: Enhancement o f proliferation by combinations o f growth factors such as SF + G - C S F or G M - C S F results in a significant increase in the number o f cells per colony compared to either growth factor alone. The overall picture o f cytokine regulation of hematopoiesis that has emerged from extensive research in this field is that most of the primitive hematopoietic cells in the adult are in a dormant non-cycling state. Their recruitment into a cycling state is influenced by early acting cytokines (e.g., IL-6, G - C S F , and SF). The proliferation o f these cycling multipotent cells is controlled by intermediate growth factors (e.g., IL-3 , G M - C S F ) . The late-acting lineage specific cytokines (e.g., Epo, IL-5, M - C S F ) control the survival and maturation of committed lineage specific progenitors 1 4. 5 Receptors for some o f the cytokines, such as those of IL-3, IL-6, L I F and G M - C S F , are comprised o f two membrane-spanning subunits; a specific a chain that binds the ligand with low affinity and a common P (or gpl30) chain that is shared by these cytokines and is required for high-affinity binding and signal transduction. Ligand binding results in dimerization of the two subunits, followed by rapid and reversible tyrosine phosphorylation o f a number o f cellular proteins including the P subunit of the receptor. Janus kinases ( JAKs) that are constitutively associated with the a chain mediate this phosphorylation. Upon activation, J A K s phosphorylate several substrates including the signal transducer and activator of transcription (STAT) proteins and tyrosine residues o f the p c chain that serve as docking sites for various signaling proteins. Further details about the receptor structure and activation mechanisms can be found in reviews 1 5 " 1 9 . 1.2 Origin and Derivation of Embryonic Stem Cells Several discoveries in the areas o f cell and developmental biology during the past 30 years proved to be pivotal events that have profoundly influenced our view o f life: i) the establishment of embryonic stem cells (ESC) derived from mouse 2 0 ' 2 1 and human 22 embryos , ii) the creation o f genetic mouse models of disease through homologous recombination in E S C , iii) the reprogramming o f somatic cells after nuclear transfer into enucleated eggs 2 3, and iv) the demonstration o f germ-line development of E S C in vitro24' 2 6 . Because o f these breakthroughs, cell therapies based on an unlimited, renewable source o f cells have become an attractive concept in regenerative medicine. Many of these advances are based on developmental studies of mouse embryogenesis. The fertilized egg has the ability to generate an entire organism. This capacity, defined as totipotency, is retained by the early progeny of the zygote up to the eight-cell stage o f the morula (Figure 1.2). Subsequently, cell differentiation results in the formation of a blastocyst, composed o f outer trophoblast cells and the inner cell mass ( ICM). Cells of the I C M are pluripotent, i.e., they retain the ability to develop into all the cell types that comprise the embryo 6 proper. In vivo, the cells of the I C M give rise to multipotent progenitors o f the three germ layers, i.e., the ectoderm, mesoderm and endoderm. Embryonic stem cell lines are - derived from the blastocyst state embryo. These cells can be maintained undifferentiated in vitro for prolonged periods of time and, upon exposure to appropriate culture conditions, can be induced to differentiate to produce the cells o f the three germ layers. Embryonic stem cell research dates back to the early 1970s, when a striking observation was reported: early mouse embryos grafted into adult mice produced teratocarcinomas - malignant multi-differentiated tumors containing a significant population o f undifferentiated ce l l s 2 7 ' 2 8 . Previously, studies of spontaneously occurring terato- carcinomas had established that the undifferentiated component, embryonal carcinoma (EC), could be propagated in culture. This also proved to be true o f embryo-derived teratocarcinomas, and E C cells were established as cell l i ne s 2 9 ' 3 0 . After transplantation to extrauterine sites of appropriate mouse strains, E C cells produced either benign teratomas or malignant teratocarcinomas 2 8 ' 3 1 . Clonally isolated E C cells retained their capacity for differentiation and could produce derivatives of all three primary germ layers. E C cells, however, showed chromosomal aberrations 3 2, lost their ability to differentiate 3 3 or differentiated in vitro only under the influence o f chemical inducers 3 4. Maintenance o f the undifferentiated state relied on cultivation with feeder ce l l s 3 5 and, after transfer into early blastocysts, E C cells only sporadically colonized the germ l ine 3 6 . These data suggested that the E C cells did not retain the pluripotent capacities o f early embryonic cells and had undergone cellular changes during the transient tumorigenic state in vivo (see review by Andrews 3 7 ) . The direct in vitro propagation of mouse embryonic cells was the logical next step and, in 1981, two groups succeeded in cultivating pluripotent cell lines from mouse blastocysts using either a feeder layer o f mouse embryonic fibroblasts ( M E F ) 2 0 or an embryonic carcinoma cell-conditioned medium 2 1 . These cell lines, later termed mouse embryonic stem cells (mESC), originate from the I C M of the blastocyst (Figure 1.2). 7 The protocols for m E S C derivation are relatively simple and remain unchanged to the present day. Embryos at the expanded blastocyst stage are plated, either intact or following immunosurgical isolation o f the I C M , onto a feeder layer. After several days o f culture, the cell mass is disaggregated and replated onto fresh feeders. Various types o f differentiated colonies arise along with colonies o f a characteristic undifferentiated morphology. The latter are individually dissociated and replated. I f secondary colonies o f undifferentiated cells arise, these can generally be expanded further and continuous E S cell lines established. These cells proliferate rapidly in culture and clonal populations can be readily initiated from single cells. The pluripotency o f m E S C was demonstrated in vivo by their introduction into blastocysts. The resulting mouse chimeras demonstrated that m E S C could contribute to all cell lineages including the germ l i n e 3 8 ' 3 9 . In vitro, m E S C showed the capacity to reproduce the various somatic cell t y p e s 2 0 ' 4 0 ' 4 1 and only recently were found to develop into cells o f the germ l ine 2 4 " 2 6 . The establishment o f human ES cell lines (hESC) from in vitro fertilized embryos 2 2 and the demonstration of their in vitro developmental potential 4 2" 4 6 have evoked widespread discussions concerning not only future applications o f h E S C in regenerative medicine but also about their ethical and legal implications. 1.3 Maintenance of Undifferentiated State of mESC The efficiency o f m E S C derivation proved strain dependent, and inbred mice, like the 129 strain, demonstrated the highest rates of success for the generation of ES cel ls 4 7 . Once established as permanent lines, m E S C displayed an almost unlimited proliferation capacity in vitro48 and retained the ability to contribute to all cell lineages. In vitro, the m E S C maintained a relatively normal and stable karyotype and were characterized by a relatively short generation time of ~ 12 - 15 h with a short G l cell-cycle phase 4 9" 5 1. However, maintenance of the undifferentiated stem cell phenotype is not cell- autonomous. Media containing all necessary metabolites and nutrients are not sufficient to support either derivation or maintenance o f m E S C . Co-culture with a feeder layer was originally considered essential. Subsequently, it was discovered that the feeders could be 8 substituted by conditioned media preparations . This suggested that the critical requirement is to provide trophic stimulation, without which the E S C differentiate. This interpretation was confirmed by the subsequent finding that a single cytokine, leukemia inhibitory factor (LEF), could sustain m E S C self-renewal in the absence o f feeders 5 3 ' 5 4 . L I F is produced by feeder cells, and its expression is stimulated by the presence o f E S C 5 5 . Furthermore, feeders lacking a functional L IF gene do not support E S C propagation effectively 5 6. In m E S C , L IF predominantly activates S T A T 3 . On withdrawal of L I F or feeders, or the inactivation o f S T A T 3 , proliferation continues but differentiation is induced, and stem cells do not persist beyond a few days 5 7 . Studies on hematopoietic stem cell expansion had suggested that the number o f ligand- receptor complexes on the cell surface could influence the stem cell fate decision 5 8 . According to this model, when a relevant ligand-receptor interaction falls below a certain threshold, the probability o f differentiation is increased; otherwise, self-renewal is favored. Examination o f E S C over a range o f L I F concentrations demonstrated that L I F supplementation had little effect on growth rates, but it significantly altered the probability o f cells undergoing self-renewal versus differentiation 5 9. To further address this question, a designer cytokine (a fusion protein of sIL-6/sIL-6R linked to a flexible peptide chain) called Hyper-IL-6 ( H I L - 6 ) 6 0 together with LEF were employed to experimentally and computationally test their capacity to sustain E S C self-renewal. Quantitative measurements o f ES cell phenotypic markers, functional assays (embryoid body (EB) formation), and transcription factor (Oct-3/4) expression over a range o f L I F and HIL-6 concentrations demonstrated a superior ability of L I F to maintain E S cell pluripotentiality at higher concentrations (500 p M ) 6 1 . These results supported a ligand/receptor signaling threshold model o f E S C fate modulation that requires appropriate types and levels of cytokine stimulation to maintain self-renewal 5 8. 9 1.4 Characterization of mESC Responses m E S C are identified by a number of in vitro phenotypic, functional and gene expression assays. m E S C exhibit a high nuclear to cytoplasm ratio, grow in compact colonies and have a relatively short G i cell cycle phase 5 0. It is now wel l established that undifferentiated m E S C express a specific cell surface antigen, stage specific embryonic antigen-1 (SSEA-1) , which is first expressed on cells o f the preimplantation embryo at around the 8-cell stage and is present on most o f the I C M cel ls 6 2 ' 6 3 . Expression o f S S E A - 1 increases as the E S C form primitive ectoderm cells, but diminishes upon further differentiation 6 2 , 6 3 . The precise function of SSEA-1 is not known; a suggested role includes the mediation o f mouse embryo compaction 6 4 . Although SSEA-1 has not been identified as a marker for any particular cell lineage, it is also found in the adult mouse brain, kidney and sperm 6 3 . Another phenotypic marker that is differentially expressed as m E S C undergo differentiation is E-cadherin which is a Ca 2 +-dependent cell adhesion molecule that mediates cell-cell interactions in the formation o f the trophectoderm and compaction o f preimplantation mouse embryos 6 5" 6 8 . Pluripotent cells o f the I C M and the primitive ectoderm express E-cadherin, but expression is downregulated during mesoderm formation at gastrulat ion 6 9 ' 1 0 . In addition to L I F R and gpl30, m E S C also express the receptor for stem cell factor, c - k i t 7 1 ' 7 2 , and possess high activities for the enzymes alkaline phosphatase (AP) and telomerase 7 3. Differentiation is accompanied by a fall in alkaline phosphatase activity 7 4 . In vivo functional assays for m E S C include their abilities, on ectopic transplantation, to give rise to teratocarcinomas containing a wide range of differentiated cells and to contribute to normal embryonic development, resulting in germline competent chimeric mice, when injected into mouse embryos 3 8 . One in vitro functional assay used to measure m E S C pluripotentiality is the assessment of their ability to form multiple lineages in E B formation cultures under appropriate conditions. In addition, undifferentiated m E S C have higher E B formation efficiencies than differentiated cells. A s the ability to form E B s correlates with other E S C markers such as SSEA-1 expression and A P activity 5 9 , this assay can be used to assess m E S C developmental potential. m E S C 10 differentiation may also be analyzed by assaying the germline specific Oct-3/4 protein, an octamer-binding transcription factor 7 5" 7 7 that acts on multiple target genes and regulates early developmental events 7 8. Oct-3/4 is expressed at high levels in undifferentiated cells and is downregulated upon differentiation both in vivo and in vitro19. Another homeobox-containing gene, ehox, has recently been identified as having differential expression during m E S C differentiation. In contrast to Oct-3/4, ehox expression is upregulated upon E S C differentiation 8 0. 1.5 Molecular Mechanisms Underlying Self-Renewal of mESC In vivo, zygotic expression o f the transcription factor, Oct-3/4, is essential for the initial development o f pluripotentiality in the I C M as mutant null embryos develop only to the blastocyst stage and do not form I C M 7 7 ' 8 1 . In m E S C , continuous function of Oct-3/4 is necessary to maintain pluripotency 8 2. A less than two-fold increase in expression causes differentiation into primitive endoderm and mesoderm, whereas downregulation o f Oct- 3/4 induces the formation o f trophectoderm concomitant with a loss of pluripotency (see Figure 1.3(B)) 8 2. Maintenance of Oct-3/4 expression is not in itself sufficient to sustain the pluripotent phenotype. Propagation o f the pluripotent m E S C phenotype requires both the intrinsic activity of Oct-3/4 and the cytokine-induced action o f S T A T 3 . Recently, two groups identified the homeodomain protein Nanog as another key regulator o f pluripotential i ty 8 3 ' 8 4 . In preimplantation embryos, its expression is restricted to and required in I C M cells from which ES cells can be derived. The dosage of Nanog is a critical determinant o f cytokine-independent colony formation, and forced expression of this protein confers constitutive self-renewal in ES cells without gpl30 stimulation. Nanog may therefore act to restrict the differentiation-inducing potential o f Oct-3/4. L I F , when applied to serum-free ES cell cultures, is insufficient to maintain pluripotency or block (neural) differentiation. In combination with bone morphogenetic protein ( B M P ) , L I F sustains self-renewal, multilineage differentiation, chimera colonization, and germ- line transmission properties. The critical contribution of B M P is to induce expression o f Id (inhibitor o f differentiation) genes via the S M A D pathway (Figure 1.3 (A)). Forced 11 expression of Id genes liberates ES cells from B M P or serum dependence and allows self- renewal in LEF alone. Blockade o f lineage-specific transcription factors by Id proteins enables the self-renewal response to L I F / S T A T 3 signaling 8 5 . Somewhat surprisingly, activation o f the E R K mitogen-activated protein kinase ( M A P K ) pathway, seems not to be required for ES cell self-renewal 8 6 ' 8 7 . E R K activation is normally a key signal for cell-cycle progression through the cyclin D / C D K checkpoint in G i 8 8 . In fact, inhibition of the ERK-activating enzyme M E K actually enhances self-renewal, implying that there is a pro-differentiative effect o f E R K activation (Figure 1.3(A)). This may relate to growth factors and other inductive stimuli that signal through the R a s - R a f - M E K - E R K cascade and also to integrin-mediated E R K activation 7 1. Overall, the self-renewal of E S cells appears to depend on a balance between conflicting intracellular signals. The dual requirements o f achieving and maintaining a high level o f S T A T 3 activation and a low level o f E R K activity may underlie some of the difficulties experienced in ES cell derivation, where the alternative outcome o f differentiation is generally favored. In this context, application o f the M E K inhibitor PD05809 appears to increase the efficiency o f ES cell establishment by promoting expansion o f primary stem OA cell colonies . However, it remains unclear how this pathway interacts with Nanog, Oct- 3/4, and L I F signaling to regulate pluripotentiality (see Figure 1.3). Finally, a recent study has implicated Wnt-signaling pathways in the maintenance o f E S C pluripotency. Wnt pathway activation by a specific pharmacological inhibitor (BIO; 6- bromoindirubin-3'-oxime) of glycogen synthase kinase-3p (GSK-3 P) maintains the undifferentiated phenotype in both mouse and human ES cells and sustains expression o f the pluripotent stage-specific transcription factors Oct-3/4 and Nanog 9 0 . Whether or not Wnt signaling has an effect on h E S C self-renewal over longer periods through multiple passages remains to be determined. The reversibility of the BlO-mediated Wnt- activation in h E S C also suggests a practical application o f GSK-3P-specific inhibitors to regulate early steps of differentiation, which may prove valuable for the derivation o f cells suitable for regenerative medicine. 12 The m E S C property o f self-renewal therefore depends on a stoichiometric balance among various signaling molecules, and an imbalance in any one can cause the identity o f the m E S C to be lost. Other molecular markers potentially defining pluripotentiality include R e x l 9 1 , Sox2 9 2 , Genesis 9 3 , G B X 2 9 4 , U T F 1 9 5 ' 9 6 , and Pern 9 7 ' 9 8 . A l l o f these have been shown to be expressed in the I C M of blastocysts and are downregulated upon differentiation; however, they are not exclusively expressed by pluripotent embryonic stem cells and can be found in other cell types in the soma. Their potential role in maintaining pluripotentiality or self-renewal remains to be determined 1.6 Human ESC Lines The techniques used to isolate and culture m E S C proved critical to the generation o f h E S C lines from preimplantation "spare" embryos produced by in vitro fer t i l iza t ion 2 2 , 4 6 ' 9 9 and after in vitro culture o f blastocysts 1 0 0. The resulting h E S C shared some fundamental characteristics o f murine lines, such as Oct-3/4 expression, telomerase activity, and the formation o f teratomas containing derivatives of all three primary germ layers in immunodeficient mice 2 2 ' 4 6 . Similar to m E S C , h E S C maintained proliferative potential for prolonged periods o f culture and retained a normal karyotype 1 0 1 . In contrast to m E S C , h E S C formed mainly cystic E B s 1 0 2 and displayed proteoglycans ( T R A - 1 - 6 0 , T R A - 1 - 8 1 , G C T M - 2 ) and different subtypes o f stage-specific antigens (SSEA-3 , S S E A - 4), which were absent from mouse E S cell lines. Several potentially important differences exist between mouse and human E S C . h E S C have a longer average population doubling time than m E S C (30-35 h vs. 12-15 h 1 0 1 ) . Wi th murine cells, it is possible to substitute the feeder layer of embryonic fibroblasts with recombinant L I F . In contrast, L IF is insufficient to inhibit the differentiation o f h E S C 2 2 ' 4 6 which continue to be routinely cultured on feeder layers o f M E F or feeder cells from human tissues. The identity o f the essential self-renewal signals provided to h E S C by M E F feeder cells remain i l l defined. The cultivation o f h E S C on extracellular matrix proteins, such as Matrigel (a complex mixture o f E C M proteins isolated from Engelbreth- Holm-Swarm tumor) or laminin with MEF-conditioned media 1 0 3 causes h E S C to express 13 high levels o f S S E A - 3 along with oi6- and Pi-integrins, which are involved in cell interactions with l amin in 1 0 3 . These results show that application o f extracellular matrix- associated factors can be employed to improve the culture and maintenance o f pluripotent h E S C . h E S C lines have now been cultivated both on human feeders to avoid xenogenic contamination 1 0 4 ' 1 0 5 and in the absence o f feeder cells under serum-free conditions 1 0 5 . These technological advances suggest that new h E S C lines free from potential retroviral infections w i l l be prepared and that these cells, unlike most o f those currently available, might be suitable for eventual therapeutic applications. Although the principal techniques necessary to culture (up to 80 and more passages) and manipulate h E S C have been established (cell c lon ing 1 0 1 , cryo-preservation 1 0 6, transfection 1 0 7, and gene targeting by homologous recombination 1 0 8), other methods (single cell dissociation and proliferation) are still not yet optimal. Because o f the variabilities among h E S C lines (growth characteristics, differentiation potential, and culturing techniques), it w i l l be important to define a reliable set o f molecular and cellular markers that characterize the undifferentiated pluripotent (sternness) or differentiated state o f h E S C . Recent attempts to define molecular markers o f undifferentiated cells, however, indicate similar pattern o f marker expression among four h E S C lines maintained in a feeder-free culture system 1 0 9 and examined after long-term culture 4. It is evident that the present data, although limited, does allow an unambiguous molecular definition of pluripotent stem cell properties. The application o f transcriptome profiling with proteomic analyses to ES cell lines may prove useful to define which lines and growth conditions are optimal for human ES cells in vitro. Formulation of optimal culture conditions for h E S C w i l l depend on whether the cell line w i l l be used as a cell source for therapeutics or as a model to study early human development. For the former application, the cell line w i l l need to have enough proliferative capacity to provide a sufficient quantity o f cells, reliably differentiate into an appropriate cell population and remain karyotypically stable during expansion, 14 differentiation as wel l as after transplantation. Xenogenic-free culture conditions would need to be developed for these cells. In addition, therapeutic applications w i l l require that the cell line be compatible with FDA-type regulations and hence it is l ikely that there w i l l be a change to the production o f h E S C under current Good Manufacturing Practices ( cGMP) regulations. A l l methods w i l l need to be wel l defined and subjected to quality control procedures. Bulk culture systems for h E S C are still in their infancy and much bioprocess R & D is needed to improve these systems for optimal production o f h E S C and their derivatives. If h E S C are to be used as a model to study early human development, the most desirable characteristics w i l l be appropriate proliferation and cell cycle regulation, gene expression and ability to appropriately respond to developmental cues. Pluripotency is a key feature as these cells w i l l be used to study cell fate choices. For both o f the above goals, generating and maintaining the cells in defined culture conditions w i l l allow the establishment o f more reproducible cultures that can be consistently maintained in multiple laboratories. These conditions w i l l include a defined matrix and medium supplemented with recombinant proteins, and passaging which allows cell seeding at a consistent cell density. Moreover, the culture conditions should maintain cells phenotypically and karyotypically stable for the required time period and allow them to retain the capacity for appropriate and reproducible differentiation. The molecular regulation o f h E S C self-renewal is less well understood than for m E S C . Wi th current protocols, h E S C can be maintained either on feeder cells in a serum-free medium supplemented with basic fibroblast growth factor ( b F G F ) 1 1 0 or on matrigel or laminin-coated plates in the presence of b F G F and M E F conditioned medium 1 0 3 . Cells grown under these conditions for > 100 population doublings retained normal karyotypes and stem cell characteristics, including their in vitro and in vivo differentiation potential. h E S C express both Oct-4 and N a n o g 9 0 ' 1 1 1 suggesting that this aspect o f their regulation may be similar to that observed in m E S C . A requirement for Nanog in the direction o f self-renewal o f h E S C seems probable but remains to be established 1 1 2. Oct-3/4 appears 15 to be required to suppress extra-embryonic differentiation in h E S C 1 1 3 ' 1 1 4 . Extrinsic signals that support h E S C self-renewal have yet to be definitively identified but may be different from those effective on m E S C . Although h E S C can self-renew in the absence o f exogenously added L IF , this does not necessarily mean that S T A T 3 activation is not important for the self renewal o f hESC. It is possible that autocrine signaling through gpl30 occurs in these cultures and that the extent of this is sufficient for human but not for mouse E S C . Alternatively, h E S C express a higher effective level o f Nanog than m E S C and hence may not require a cooperative interaction between S T A T 3 and endogenous Nanog in order to self-renew effectively 1 1 2 . A trivial explanation for the reported lack o f effect of LEF on h E S C is that most studies used recombinant mouse L I F which does not bind to the human LIF receptor 1 1 5. Further studies are therefore needed to define the molecules needed for the maintenance of h E S C and uncover the molecular mechanisms that regulate their self-renewal. 1.7 Embryoid Body Formation Based Differentiation of mESC In 1985, an in vitro model of mouse embryogenesis based on differentiating m E S C was presented for the first t ime 4 0 . When grown in the absence o f feeder cells and L I F , in suspension, in bacterial dishes, in spinner flasks, in a semisolid methylcellulose medium or as hanging drops, m E S C are able to differentiate spontaneously 4 0 ' 1 1 6 ' U 7 . Because o f the ability o f the differentiated cells to form spheroid aggregates mimicking postimplantation embryonic tissues, they were termed embryoid bodies (EBs). Fol lowing aggregation, the outer layer o f the E B s is specified as extra-embryonic primitive endoderm and it further differentiates into visceral endoderm 4 0. Extracellular matrix, which is secreted from the primitive endoderm layer, then forms a thick basement membrane, separating the primitive endoderm from the inner mass o f the E B s . A long the inside o f the basement membrane, a primitive ectoderm layer is formed and cavitation occurs in the core o f the EBs . This series of processes is considered to be an in vitro model recapitulating early embryonic development from the blastocyst stage to the egg cylinder stage (E3.5-E6.5) 1 1 8 . In contrast to tumor cell derived spheroids, oxygenation in E B s was shown to be very efficient 1 1 9 . Cells within developing E B s differentiate to more 16 advanced stages o f embryogenesis, resulting in various committed cell types including hematopoietic precursors 1 2 0 ' 1 2 1 , card iomyocytes 1 1 7 , 1 2 2 , skeletal muscle ce l l s 1 2 3 and many others. Establishment o f E B s has facilitated the investigation of several aspects o f mouse development in vitro. First, E B s are a powerful tool for characterizing the function o f precursor cells, which is very difficult to perform in vivo as it is nearly impossible to isolate early stage cells from developing embryos. Second, E B s derived from m E S C in which both alleles of a specific gene have been disrupted by targeted mutagenesis often represents a fast alternative for investigating the impact o f a given null mutation. The same holds true i f the deletion of a given gene induces early embryo death. It is wel l known that m E S C derivation is strain dependent with 38% o f the explanted blastocysts from the 129Sv strain giving rise to m E S C while only 1% o f the C57B1/6J I C M strain generally develops into a m E S C line in vitro124. It has proved impossible so far to isolate a single m E S C line from F V B blastocysts. These results suggest that the genetic background and perhaps even minor epigenetic differences between strains and perhaps profound differences among species might predetermine whether the I C M cells can survive on feeder cells in the presence o f L I F or not. It is also wel l known that self-renewal capacity and differentiation control are highly variable among even isogenic m E S C lines generated in different laboratories. E B development is therefore influenced not only by the genetic background o f the m E S C line used but also by the complex conditions under which that m E S C line had been generated. In addition, both the pattern and the efficiency o f differentiation are affected by parameters like m E S C density, medium components (high glucose concentration, i.e., at least 4.5 g glucose/L is required) and amino acids, growth factors and extracellular matrix ( E C M ) proteins, p H and osmolality, and the quality o f the fetal calf serum ( F C S ) 1 2 5 . Because the differentiation efficiency depends on the presence o f F C S , and even the "batch" o f serum used, many efforts have been taken to avoid the resulting uncertainties, e.g., by using chemically defined m e d i a 1 2 6 ' 1 2 7 or, more recently, by substitution o f F C S 17 with B S A fraction V 1 2 8 . Furthermore, different ES cell lines display unique developmental properties in vitro125,129. Additional parameters that influence m E S C potential and E B differentiation include the developmental stage o f the embryo at the time of m E S C generation, the time required to reach confluency in vitro, the type o f feeder cells, the source and batch o f serum and, last but not least, the handling of the cells by the investigator. Furthermore, the self-renewal capacity strongly influences E B development 1 3 0. The initial proliferation of m E S C , for example, determines the number of E S C which aggregate to form E B s , and the final number o f cells in the compact E B aggregate strongly influences cardiomyogenesis and hematopoiesis 1 3 1 , 1 3 2 . Thus, m E S C proliferation and maintenance o f pluripotency significantly contribute to the subsequent differentiation potential in E B s . It is generally accepted that m E S C are reprogrammed to delete all epigenetic modi f i ca t ions 1 3 3 ' 1 3 4 , but examples of epigenetic inheritance exist 1 3 5 ' 1 3 6 and may not only influence the differentiation potential but additionally contribute to the divergence o f the differentiation potential observed in E B development. 1.8 Culture Environment and Maintenance of Stem Cell Potential Cellular homeostasis (maintenance o f a stable internal environment) is an important regulator of many cell functions such as cell division, protein synthesis, differentiation, cell-to-cell communication and cytoskeleton dynamics, as wel l as metabolism 1 3 7 . Therefore, any disruptions that may occur in cellular homeostasis as a result o f trauma or stress can have significant implications for normal growth and development. Stress can be induced by using inappropriate media formulations, oxygen or carbon dioxide levels, p H , osmolality or even the presence o f visible light. Several studies using animal models have shown that preimplantation embryos are sensitive to environmental conditions that can affect future growth and developmental potential, both pre- and post-natally. These studies were initiated with the observation that cultured mouse embryos, after transfer to surrogate mothers, resulted in reduced fetal growth compared with that of their in vivo 18 counterparts 1 3 8 ' 1 3 9 . Further studies on the mouse have confirmed this effect and identified possible culture conditions which may act deleteriously 1 4 0" 1 4 4 . These and other similar studies have given rise to the notion that immediate manifestations o f culture- induced stress include altered homeostasis and perturbed metabolism, which lead to altered cell function and impaired energy production, respectively 1 4 5 " 1 4 7 . A downstream effect o f culture-induced stress is altered gene function 1 4 8 . The potential influences o f culture environment on the embryo's responses are summarized in Figure 1.4. 1.8.1 Serum Most ex vivo expansions o f hematopoietic cultures have been carried out in media containing 10-20% fetal calf serum or horse serum. A number o f studies have also reported expansion in serum-free media (see Sandstrom et a l . 1 4 9 for a review). Serum functions as a source of hormones and essential nutrients. It also can protect cells from environmental damage by providing enzymes, enzyme inhibitors and other molecules that react with or bind to toxins. Serum alters the physiological/physicochemical properties o f the culture environment by coating surfaces with macromolecules and buffering or altering the p H , osmolarity, surface tension and viscosity. Addit ion of serum to culture medium, however, makes its composition undefined, adds uncontrolled variability and complicates the clinical use of cells. Serum-containing media are superior for expansion of the granulocyte and monocyte l ineages 1 5 0 ' 1 5 1 . Serum-free media, on the other hand, promote greater expansion o f erythroid and megakaryocyte lineages primarily because serum contains transforming growth factor (3 (TGF0), a potent inhibitor to the expansion of these lineages . Autologous serum or plasma, however, can partly alleviate this shortcoming o f serum-free media 1 5 0 . Serum has also been reported to either increase or decrease the expression o f various genes in several cell types. For instance, addition o f serum to the culture medium of beta T C I cells (a pancreatic cell line) increased the m R N A levels of c-fos and c-jun153'154. Increasing the serum concentration from 2.5% to 10% in cultures of H L 6 0 cells increased the CD13 receptor surface expression by 100% 1 5 5 . This increase in CD13 surface expression was correlated with a 30% increase in CD13 m R N A levels. Serum also protected H L 6 0 cells 19 from hydrodynamic damage under conditions of high agitation 1 5 5 . In contrast, reducing the serum concentration from 5% to 0.5% increased the m R N A and protein levels o f progesterone receptor in M C F - 7 human breast cancer ce l l s 1 5 6 . Similarly, decreasing the serum concentration from 10% to 0.5% increased the collagen m R N A levels in smooth muscle ce l l s 1 5 7 . Mammalian embryos are not exposed to serum in vivo148. Studies using more defined embryo culture systems have shown that serum induces premature blastulation in domestic animal embryos, affects embryo morphology and leads to perturbations in the ultra-structure, energy metabolism and cryotolerance of blastocysts 1 5 8" 1 6 1 . Extensive work in recent years has helped to elucidate possible mechanisms behind these observations. Specifically, fetal overgrowth has been associated with reduced fetal methylation and expression of IGF2R, indicating that there were epigenetic alterations in imprinted genes during the preimplantation period in vitro when embryos were exposed to serum 1 6 2 . 1.8.2 Culture Osmolality Most measured values reported in the literature are osmolality (osmoles per kilogram, Osm/kg) rather than osmolarity (osmoles per liter, OsM) . However, in the relatively dilute solutions that are physiologically relevant, these two measures differ negligibly. Both osmolality and osmolarity are intrinsic properties o f a solution and are distinguishable from tonicity which describes the osmotic effect o f a solution on a particular entity such as a cell and is thus context specific. Animal cells regulate their size precisely. This is true not only for somatic cells but also for the cells of eggs and early embryos. Oocytes grow to a precise diameter, characteristic o f each species, and the egg maintains that diameter for an extended period o f time, implying that mechanisms exist by which an egg can determine and control its size. Subsequently, as the embryo cleaves into successively smaller cells, each embryonic stage possesses blastomeres that are maintained at characteristic dimensions. 20 The membranes of animal cells are generally highly permeable to water and cannot tolerate substantial hydrostatic pressure gradients. Water movement across the membranes is in large part dictated by osmotic pressure gradients. Thus any imbalance o f intracellular and extracellular osmolality is paralleled by respective water movement across the cell membrane and subsequent alteration of the cell volume. Cells accumulate a number o f substances such as proteins, amino acids and carbohydrate metabolites. The concentration of these substances is higher within the cells than in the extracellular fluid. The excess cellular concentrations o f these organic substances are counterbalanced by lower intracellular ion concentrations. Most cells extrude N a + in exchange for K + by the N a + - K + - ATPase pump. The cell membrane is only slightly permeable to N a + , and the exclusion o f impermeable N a + outweighs the cellular osmolality created by the impermeant organic solutes [double Donnan hypothesis 1 6 3]. O n the other hand, the cell membrane is highly permeably to K + . The exit o f K + creates an outside-positive cell membrane potential that drives C l ~ out of the cell. The low intracellular C l ~ concentration compensates for the excess intracellular concentration o f organic substances. 1.8.2.1 Regulation of Volume Control Delicately balanced mechanisms are brought into play to maintain a normal volume after the cells are exposed either to hypotonic or hypertonic solutions. Three sequential responses occur when cells are exposed to a hypertonic extracellular fluid. A n initial rapid response involves the movement o f water out of the cell, causing it to shrink. A second slower response follows in which ions and/or organic osmolytes move from the environment into the cell such that cells approach the original volume (regulatory cell volume increase, R V I ) . If the volume is still not restored, a very slow third response occurs in which genes are activated to stimulate the synthesis of organic osmolytes intracellularly. Exposure to a hypotonic solution results in the same sequence o f responses, each in reverse. It should be kept in mind, however, that exposure o f cells to 21 anisotonic extracellular fluid does not only modify the cell volume but also the volume o f intracellular organelles such as the mitochondria 1 6 4 . Furthermore, in parallel to cellular osmolality, the intracellular ionic strength is altered even i f the extracellular ionic strength is kept constant. Thus the sequelae o f osmotic alterations of cell volume are not necessarily identical to the consequences of isotonic alterations of cell vo lume 1 6 5 . During cell swelling, cells extrude ions thus accomplishing regulatory volume decrease ( R V D ) , whereas during cell shrinkage, cells accumulate ions to achieve R V I . The activation o f ion release during R V D is paralleled by inhibition of ion uptake mechanisms, and the ion uptake during R V I is accompanied by inhibition of ion release mechanisms. Thus, the simultaneous stimulation o f ionic mechanisms for R V D and R V I is largely avoided 1 6 6 . 1.8.2.2 Organic Osmolytes The cellular accumulation o f electrolytes after cell shrinkage is limited, as high ion concentrations interfere with the structure and function o f macromolecules including proteins 1 6 7 " 1 7 0 . Furthermore, alterations of ion gradients across the cell membrane would affect the respective transporters. A n increase o f intracellular N a + activity, for instance, would reverse N a + / C a + exchange and thus increase intracellular C a + activity, which would in turn affect a multitude o f cellular funct ions 1 7 1 ' 1 7 2 . To circumvent the untoward effects o f disturbed ion composition, cells rely on so-called osmolytes - molecules that correct osmolality without compromising other cellular funct ions 1 7 3 ' 1 7 4 . A large body o f work convincingly demonstrates that these osmolytes are compatible with biochemical function even at high (molar) concentrations. Three groups of osmolytes are used in mammalian cells: (i) polyalcohols such as sorbitol, glycerophosphorylcholine (GPC) and inositol; (ii) methylamines such as betaine; and (iii) amino acids and their derivatives such as glycine, glutamine, glutamate aspartate and taurine. Beyond their function in cell volume regulation, osmolytes can be protective against the destructive effects of excessive temperature 1 7 5 ' 1 7 6 and des icca t ion 1 7 7 ' 1 7 8 . Furthermore, they have been found to ease cell membrane assembly 1 7 9 . 22 Sorbitol and G P C are synthesized within the cell in which they are accumulated as organic osmolytes. However, most organic osmolytes are instead transported into the cell from the environment via specific transporters. Four such osmolyte transport systems have been identified in mammals: the betaine transporter, (3-amino acid or the taurine transporter (system P), the Na + /myo-inositol transporter and system A , which transports neutral amino acids. A l l o f these transport systems respond to increased tonicity by increasing their rate o f substrate transport 1 7 4 ' 1 8 ° . In each case, the increased rate o f transport is due to an increase in the maximal transport velocity ( V m a x ) 1 8 1 " 1 8 3 . The rate o f osmolyte transport increases slowly, reaching a maximal level only hours to days after the external osmolality is increased 1 8 4 ' 1 8 5 . V a n Winkle et al. first proposed that mammalian embryos utilize organic osmolytes and their presence protects embryos against increased osmolality. Evidence has since accumulated that early mammalian embryos use a range of organic compounds for osmoprotection. McKiernan et a l . 1 8 7 examined the effect o f each o f the common amino acids and taurine, separately and in combination, on the development o f hamster one-cell embryos to blastocysts. Three amino acids - glutamine, glycine and taurine - were found to greatly stimulate development and several others were found to stimulate development in the presence of glutamine. Glutamine has been found to be especially beneficial for the culture o f preimplantation stage embryos o f mouse, hamster, cow and human " . A n extensive body of work has shown that, when added as a group, Eagle's nonessential amino acids ( N E A A ) plus glutamine and taurine are stimulatory to cleavage stage mouse, bovine and human embryo development 1 4 6 ' 1 9 3 " 1 9 6 . This group overlaps considerably with the stimulatory amino acids identified in the hamster 1 8 7. Many o f the amino acids that have been found to be protective o f osmotic stress and beneficial to embryo development, especially glutamine, taurine, glycine and alanine, are established organic osmolytes in somatic cells as w e l l 1 6 9 ' 1 9 7 . In Madin-Darby canine kidney ( M D C K ) cells, in which the effect of osmolality on intracellular amino acid content has been studied most systematically, taurine, alanine, glutamine, glycine, proline and serine were found to accumulate in response to hypertonicity and to act as organic 23 183 198 osmolytes ' . Comparing the amino acids accumulated in M D C K cells with those found beneficial to preimplantation embryo development reveals that the two groups are almost identical. This raises the possibility that one o f the major functions o f beneficial amino acids is to act as organic osmolytes. 1.8.2.3 Molecular Mechanism of Cellular Protection from Hyperosmolality Hypertonicity stimulates a diverse array o f signal transduction pathways. C e l l swelling has been shown to stimulate protein kinase C 1 9 9 , focal adhesion and phosphatidylinositol 3-kinases ( P I 3 K ) 2 0 0 , as wel l as to trigger the mitogen activated protein kinase ( M A P K ) cascade leading to the activation o f cJun-N-terminal kinase ( I N K ) or extracellular signal regulated kinases ( E R K 1 and E R K 2 ) 2 0 1 " 2 0 4 . Similarly, osmotic cell shrinkage has been shown to activate protein kinase C 1 9 9 , the M A P K cascade leading to ribosomal S6 protein kinase 2 0 5 , 2 0 6 , and either p38 M A P K or I N K 2 0 7 ' 2 0 8 . These signal transduction pathways eventually result in increase or decrease in expression o f a wide variety o f genes. In somatic cells, both cell swelling and cell shrinkage markedly influence the expression o f a wide variety o f genes. Hypertonicity increases sorbitol concentration by raising the amount and activity o f aldose reductase - the enzyme that catalyzes the synthesis o f 9ftQ 911 sorbitol from glucose " . The enzyme is upregulated by hypertonic extracellular addition o f N a C l or raffinose but not o f membrane permeable solutes such as urea or glycerol. It is presumably an increase in intracellular ionic strength that stimulates the aldose reductase transcription ra te 2 1 0 ' 2 1 2 . The aldose reductase transcription rate peaks at a level approximately 18-fold higher than control after 24 h o f hypertonicity; then, as sorbitol and other organic osmolytes accumulate, the transcription rate falls to a steady- state level approximately 5 times that of the control 2 1 3 . Ce l l shrinkage has been shown to stimulate the expression of heat shock proteins that serve to stabilize the proteins and thus to counteract the detrimental effects o f increased salt concentrat ions 2 1 4 ' 2 1 5 . Increased intracellular ionic strength also stimulates transcription of genes for betaine and inositol 24 transporters 2 1 6. Ce l l shrinkage stimulates Na +-coupled transport of neutral amino a c i d s 2 1 7 ' 2 1 8 and p ro teo lys i s 2 1 9 ' 2 2 0 and inhibits protein synthesis 2 2 1. Ce l l shrinkage also stimulates the expression o f genes with diverse functions not obviously related to R V I such as P-glycoprotein , cyclooxygenase-2 , c-fos ' , phosphoenolpyruvate carboxykinase 2 2 5 and tyrosine hydroxylase 2 2 6 , to name a few. Ce l l shrinkage is frequently observed to alkalinize cells, at least partially due to activation o f the volume I | 227 228 regulatory N a / H exchanger ' . Alkalanization after cell shrinkage should stimulate g l y c o l y s i s 2 2 9 ' 2 3 0 . Conversely, cell swelling stimulates the expression of immediate early genes, c-fos and c- jun23X'232, a-actin233 and tublin234, M A P K cascades in cardiac myocytes 2 3 1 , and T N F - a in macrophages 2 3 5. Ce l l swelling inhibits proteolysis and stimulates protein synthes i s 2 2 1 ' 2 3 6 and leads to cytosolic acidification 2 3 7 ' 2 3 8 , which has been explained by the exit of bicarbonate ion through anion channels 2 3 9 . Swelling also increases glycogen synthesis and inhibits g l y c o l y s i s 2 4 0 ' 2 4 1 . Information on the mechanisms triggering altered gene expression remains scanty. Some evidence points to the involvement of the cytoskeleton 2 4 2 . The expression o f aldose- reductase is regulated by a distinct osmolality-responsive e lement 2 0 9 ' 2 4 3 . The stimulation o f c-fos expression by swelling of cardiomyocytes depends on tyrosine phosphorylation 2 3 2 . The c-jun transcription after swelling of hepatocytes is at least partially the result o f M A P K activation followed by phosphorylation of c- jun 2 4 4 ' 2 4 5 . Hypertonicity has also been shown to alter karyotype 2 4 6 . Ce l l proliferation has been shown to correlate with increases of cell volume in f ib rob las t s 2 4 7 ' 2 4 8 , lymphocytes 2 4 9 , 2 5 0 , H L - 6 0 ce l l s 2 5 1 ' 2 5 2 , hybridoma ce l l s 2 5 3 and H e L a ce l l s 2 5 4 . In contrast to cell proliferation, cell differentiation is accompanied by cell shrinkage in erythroleukemia ce l l s 2 5 5 " 2 5 7 , H L - 6 0 leukemia ce l l s 2 5 8 ' 2 5 9 and E M T 6 / r o mouse mammary sarcoma ce l l s 2 6 0 . 25 In summary, cells are able to control their size precisely in the face o f an osmolality challenge by taking up organic osmolytes from the medium as well as by inducing expression of a wide variety of genes. Increases or decreases in osmolality o f medium also profoundly influence the proliferation of cells. 1.8.3 Culture pH The ability to regulate ionic homeostasis is essential for normal cell function. Intracellular levels o f ions such as calcium (Ca; + 2 ) , hydrogen (Hj + or pHj), magnesium (Mgj + 2 ) and phosphate ( P 0 4 r 3 ) regulate a multitude of cellular functions such as cell division, protein synthesis, cytoskeletal dynamics, metabolism and energy production. It is also known that the difference in ionic composition between the cell and its microenvironment is a universal source o f cellular potential energy 2 6 1 . Transmembrane ionic gradients - particularly that o f sodium - are the driving force used to accomplish both organ-specific cellular functions (e.g., action potential in nerves, reabsorption and secretion by kidney and cerebrospinal fluid formation), and general cellular functions essential for cell growth and division (e.g., entry o f nutrients and amino acids, as wel l as regulation of pHj and free calcium). Therefore, it is essential for normal cell development that ionic homeostasis be tightly regulated. Aberrations in cellular homeostasis result in perturbed cell function and loss of developmental competence. This has significant implications for E S C cultures where the target is to expand E S C numbers without loss o f developmental potential. To ensure this, E S C culture must be performed under conditions where cellular homeostatic stress is not too great. Developmental biologists generally divide the genome into two parts: one consisting o f genes directly involved in determination, differentiation and morphogenesis and the other part consisting of genes that regulate metabolic functions. Genes involved in metabolic control are generally not considered important from a developmental point-of-view 2 6 2 . However, it is increasingly being recognized that ions and small molecules operating in complex networks are likely involved in the sequential control of developmentally important genes 2 6 3 , 2 6 4 . Ionic signaling occurs when an ovum is activated at fertilization, 26 and ion transport systems become active at specific times to produce fluid-filled cavities in the embryo such as the blastocoel and the amnion 2 6 5 . Similarly, two specific physiological systems in which ions are involved in developmentally important phenomena in the early embryo are: i) regulation of pHj which emphasizes the fact that some transport systems change with development according to the embryo's needs at each stage o f the life cycle, and ii) regionalization of the N a + / K + - ATPase in the embryo which results in the formation o f the blastocyst through cavity formation, thereby establishing the earliest patterned structure in mammalian development 2 6 3. In general, the intracellular concentrations o f N a + and K + are held constant. However, they are maintained at concentrations very different from those found in the extracellular microenvironment. The intracellular concentration of N a + is low while its extracellular concentration is high. The reverse is true for K + . Consequently, large electrochemical gradients due to these two ions exist across the cell membrane. Both o f these ions are however, in a constant state o f flux. A t any given time, their flux is the net result o f the rate at which the ion is lost by leaking down its electrochemical gradient and the rate at which it is retrieved by N a + / K + pumps which expend chemical energy derived from the hydrolysis o f A T P . The energy, in the form o f these ion gradients (mainly o f N a + ) , is then used for other energy-requiring transports, such as co- and counter-transport o f protons, chloride, phosphate, glucose and amino acids. Further discussion of these dynamic homeostatic systems can be found in standard references 2 6 1 ' 2 6 6 . The normal functions of cellular proteins depend on the degree of ionization of their component amino acids. The ionization in turn depends on the presence o f a stable acid- base status in the cells. The distribution o f H + ions across the plasma membrane o f most cells is such that the internal p H (pHj) is much higher than that predicted i f H + were passively distributed. For an average membrane potential of -60 m V and for an external p H o f 7.4, a pHj value of 6.4 would be expected i f the hydrogen ions are in electrochemical equilibrium across the membrane 2 6 7 ' 2 6 8 . Therefore, all cells have mechanisms for H + extrusion that maintain pH; at a value which is wel l above equilibrium and is compatible with the necessities of cytoplasmic reactions. This fact has 27 also been demonstrated in mammalian embryos including the human, where it is observed that the p H of the external medium (pH e) does not equate to pHj. Numerous studies performed during the last two decades have demonstrated that changes in pHj • • "5/̂ 0 9*71 occur during metabolic and developmental transitions in a large variety of cells " . Ac id ic cytoplasmic conditions are usually associated with a quiescent or dormant cellular state while an increase in pHj is often a signal for cellular ac t iva t ion 2 7 1 ' 2 7 2 . 1.8.3.1 Effect of Culture Medium on Intracellular pH (pHj) Some components o f the culture medium have been shown to have significant effects on pHj regulation by somatic cells as wel l as embryos. For example, levels o f both lactate and amino acids in the medium have been shown to alter pHj. L o w levels o f lactate in the medium (5 m M D/L-lactate) significantly reduced the pHj of mouse embryos by around 0.2 u n i t s 2 7 3 ' 2 7 4 . Although only L-lactate can be metabolized by cells, both D - and L - lactate affect p H 2 7 3 ' 2 7 4 . The other important components of the medium for pH; regulation are the amino acids. Proportions of some o f the N E A A such as taurine and glycine exist as zwitterions at physiological p H . Zwitterions can move readily across the membrane and bind protons and therefore buffer pHj. Taurine, glycine and glutamine are present at high concentrations in the female reproductive tract 2 7 5 and quite l ikely these amino acids are able to regulate pHj in vivo. The capacity o f amino acids in the culture medium to buffer pHj has been demonstrated in mouse pronuclear stage embryos. Incubation o f embryos in the presence o f the weak acid, 5,5-dimethyl-2,4- oxazolidinedione ( D M O ) , results in a significant decrease in pHj. Addit ion o f N E A A to a culture medium containing D M O reduced the resultant acidification and therefore increased the ability of embryos to buffer protons and maintain p H ; 2 7 6 . Furthermore, addition of amino acids to the culture medium prevents the efflux o f endogenous amino acids and would therefore assist in maintaining the intrinsic buffering capacity o f the cytoplasm 2 7 7 . A s discussed in Section 1.7.3, N E A A can also protect cells against stress caused by hyperosmolality. 28 1.8.3.2 Regulation of pHj In order to maintain pH; at a constant and steady level, the cell must be able to regulate and neutralize intracellular acids such as lactic acid - a by-product o f metabolism. Cells have two mechanisms to regulate p H f : i) short-term regulation and (ii) long-term regulation. Short-term regulation o f changes in pHj is achieved by physiochemical buffering o f the cytoplasm as wel l as buffering by the organelles themselves. The intrinsic buffering capacity can be measured and expressed as the millimoles of protons that can buffered by the cytoplasm o f the cell. A higher value indicates an increased ability o f the cytoplasm to buffer against a p H challenge. Typically, the intrinsic buffering capacity o f cells is reported to range from 12 to 30 m M / A p H . Hamster oocytes, however, have been reported to have a much higher buffering capacity (51 m M / A p H ) 2 7 8 " 2 8 0 . This increased buffering capacity o f hamster oocytes may be a mechanism to compensate for the reduced capacity to restore p H due to lack o f functional membrane transporters. The intrinsic buffering capacity o f the cytoplasm can buffer pHj in a matter o f seconds. For long-term stability and regulation o f pHj, it is necessary to either extrude the protons or buffer them by the import of bicarbonate ions. This is achieved by specific transporters in the cell membrane including proton pumps ( H + ATPases ) 2 8 1 , proton channels 2 8 2 and ion transporters that drive H + or equivalent H + and HC03~ ions into and out o f the cell (Figure 1.5). The latter include an N a + / H + exchanger encoded by members o f the sodium hydrogen exchanger (NHE) gene family ( N H E l - 9 ) 2 8 3 ' 2 8 4 , N a + - dependent H C C V co-transporters ( N B C ) 2 8 5 , an Na+-independent C 1 7 H C 0 3 " transporter ( A E ) 2 8 6 ' 2 8 7 , Na+-dependent C I T H C C V transporter ( N D C B E ) and the recently described C 1 7 0 H 288 exchanger . Other membrane transporters such as the lactate-proton co-transporter may also participate in intracellular p H regula t ion 2 8 9 ' 2 9 0 (Figure 1.5). N H E is an electroneutral ion exchanger that pumps H + out o f the cell when it is turned on by intracellular acidosis. The H + efflux is driven by the inwardly directed N a + 29 electrochemical gradient. First discovered in kidney and intestine , the antiporter has now been found in most tissues of eukaryotic cells. So far, nine members o f the N H E family sharing 30 - 60% homology have been cloned. The activity o f the exchanger increases exponentially to restore pHj as acidification increases 2 7 8. The exchanger extrudes protons in exchange for N a + ions and regulates pH; in the acid to neutral range. The amino-terminal domain is sufficient to transport the ions 2 9 1 ' 2 9 2 . The C-terminal region features an A T P and other protein binding sites in addition to many phosphorylation sites and is thus involved in neurohormonal regulation o f the ion- exchange activity^ " . The N H E 1 isoform is ubiquitously expressed in somatic cells and its activity has also been demonstrated in embryos from several strains o f m i c e 2 7 4 as wel l as from h a m s t e r 2 7 8 , 2 8 ° , c o w 2 9 6 , and human 2 9 7 . The kinetic features o f the N H E isoforms have been studied after stable transfection in antiporter-deficient fibroblast cell lines. N H E 1-3 typically show similar Michaelis constants for external N a + ( K m N a = 4 - 1 8 m M ) . The kinetics for intracellular H + are also similar and fit a sigmoidal curve which suggests the presence o f an intracellular H + modifier site in addition to the H + transport s i te 2 9 8 . The N H E 1-3 exhibit half-maximal activity at intracellular p H values between 6.2 and 6.8. The p H sensitivity o f N H E 1 is mechanistically complex. It is regulated by an autoinhibitory domain (residues 636-656) that prevents protonation o f the p H sensor 2 9 9" 3 0 1 and another domain that is more directly responsible for the maintenance o f p H sensitivity (residues 567 - 635) 2 9 1 ' 2 9 5 . Amino acids 515-595 in the C-terminal domain are particularly critical for the p H sensitivity o f the antiporter 3 0 1. A l l N H E isoforms show dependence on A T P , as a drop in intracellular A T P dramatically decreases their activity in several cell t ypes 3 0 1 ' 3 0 2 . The A T P dependence o f N H E 1 cannot be explained entirely by the requirement o f phosphorylation, as mutation o f all known phosphorylation sites does not abolish the metabolic regulation o f the antiporter 2 9 4. The cytoskeleton is l ikely to participate in this process and a non-diffusible effector that binds A T P is probably required to activate the antiporter in the presence o f A T P 3 0 3 , 3 0 4 . 30 The N H E is also regulated by cell volume ' . N H E 1 is stimulated by hypertonicity and, under similar conditions, the activity o f N H E 2 is also increased. Under hypotonic conditions, the activity o f both isoforms is inhibited. In contrast, N H E 3 is markedly inhibited by hypertonic cell shrinkage but is unaffected by h y p o t o n i c i t y 2 2 7 ' 3 0 2 ' 3 0 5 . A phosphorylation-independent mechanism likely underlies the volume regulation o f the antiporter and hence the "volume sensitive site" is different from the site(s) postulated to mediate the stimulatory effects of calcium and growth factors 3 0 6. The HC03~/Cr exchanger transports bicarbonate ions out of cytoplasm in exchange for CP and regulates pH; in the neutral to alkaline range. When the pHj o f the cell rises above the physiological set point, the H C C V / C r exchanger is activated and acidifies the cytoplasm until the pHj is restored to its physiological l e v e l 3 0 7 . The H C O 3 7CF exchanger has been extensively studied in erythrocytes. HC03~/Cr exchangers are members o f the anion exchanger (AE) gene family ubiquitously expressed in vertebrate tissues and are expressed very early during embryogenesis. A s early as the two-cell stage, embryos are able to recover from an alkaline load by switching on this exchanger 3 0 8 ' 3 0 9 . The H C 0 3 7 C r exchanger is utilized by mouse 3 1 0 , hamster 2 7 9, c o w 2 9 6 and human 2 9 7 embryos to regulate pHj in the alkaline range. Both the N H E and H C O 3 7 C T exchangers can take several minutes to restore pH; to its physiological level. 1.8.3.3 Influence of pH on Cellular Functions and Development It is wel l recognized that pHj is an important epigenetic regulator o f somatic cell functions ' . In mammalian cells, p H e is easily disturbed as weak acids and bases from cell metabolism can accumulate significantly in the culture media o f long-term and high cell density cultures. In somatic cells, p H e has been shown to have many diverse effects, including those on proliferation r a t e 3 1 1 ' 3 1 2 , differentiation 3 1 3" 3 1 5 , metabolism and C a homeostasis ' " , protein synthesis, degradation and glycosylation " , cell motility, contractility and cell-cell coupl ing 3 2 3 , cell adhesion 3 2 4 , expression o f various cell surface receptors 3 1 6, glucose as wel l as amino acid transport 3 2 5, binding o f growth factors 31 to extracellular matrix proteins ' , expression of matrix metalloproteinase-9 and cell 329 330 death ' . Clinically, the acidic p H e has been shown to modulate the sensitivity o f solid tumors to radiation and chemotherapeutic agents 3 3 1 ' 3 3 2 . In addition, acidic p H e increases the expression o f i) platelet-derived endothelial cell growth factor/thymidine phosphorylase in human breast tumor ce l l s 3 3 3 , ii) vascular endothelial growth factor ( V E G F ) in glioma and gliobastoma 3 3 4 , 3 3 5 , and i i i) IL-8 in human pancreatic adenocarcinoma 3 3 6" 3 3 9 and ovarian carcinoma cells 3 4 0 through activation o f transcription factors N F - k B and/or A P - 1 . Given the extensive work that has been done on the influence o f p H in somatic cells, we might expect that alterations in pHj w i l l have important consequences for mammalian embryos and that different medium formulations that influence pHj might enhance or reduce embryo development. Exposure o f preimplantation embryos to culture conditions that result in the cytoplasm becoming either weakly acidic or weakly basic causes significant reductions in the ability of the embryos to develop in c u l t u r e 2 7 3 ' 2 7 8 ' 2 7 9 ' 3 4 1 . Furthermore, exposure o f two-cell embryos to either acidic or basic conditions results in disruptions in mitochondrial distribution and microfilament organization which in turn are correlated with perturbed development in culture 3 4 1 " 3 4 3 . Embryonic stem cells could also be profoundly influenced by changes in external p H . 1.9 Mouse ESC as a Model System Although yeasts, worms and flies are excellent models for studying the cell cycle and many developmental processes, mice are far better tools for probing the immune, endocrine, nervous, cardiovascular, skeletal and other complex physiological systems that mammals share. L ike humans and many other mammals, mice naturally develop diseases that affect these systems, including cancer, atherosclerosis, hypertension, diabetes, osteoporosis and glaucoma. In addition, certain diseases that afflict humans but normally do not strike mice, such as cystic fibrosis and Alzheimer's, can be induced by manipulating the mouse genome and environment. Adding to the mouse's appeal as a model for biomedical research is the animal's relatively low cost o f maintenance and its 32 ability to quickly multiply, reproducing as often as every nine weeks. A substantial proportion o f scientific breakthroughs has been as a result o f experiments conducted on l iving mice, their organs or cells. Both the human 3 4 4 and the m o u s e 3 4 5 ' 3 4 6 genomes have been sequenced and comparative maps illustrate the high degree of homology between the two genomes. It is reported that, although the mouse genome is 14% smaller than the human genome, over 90% of the two genomes can be partitioned into regions o f conserved synteny and both contain approximately thirty thousand genes 3 4 6 . The mouse is an ideal model organism for human disease. Not only are mice physiologically similar to humans, but a large genetic reservoir of potential models o f human disease has been generated through the identification o f >1000 spontaneous, radiation- or chemically induced mutant loci . In addition, a number o f recent technological advances have dramatically increased our ability to create mouse models o f human disease. These technological advances include the development o f high resolution genetic and physical linkage maps of the mouse genome, which in turn are facilitating the identification and cloning o f mouse disease loci . Furthermore, transgenic technologies that allow one to ectopically express or make germ-line mutations in virtually any gene in the mouse genome have been developed, as wel l as methods for analyzing complex genetic diseases. Further, to date, >100 mouse models of human disease where the homologous gene has been shown to be mutated in both human and mouse have been developed. In the vast majority of these models, the mouse mutant phenotype very closely resembles the human disease phenotype and these models therefore provide valuable resources to understand how the diseases develop and to test ways to prevent or treat these diseases. However, in certain cases, the knockout mice may reproduce only some o f the human disease phenotype, may be more severely affected than human cases, or may have no clinical phenotype at all . Under these circumstances, the disease pathology can become more complex, causing the researcher to evaluate basic differences in mouse and human biology as well as questions of genetic background, alternate pathways, and possible gene interactions. Similarly, for many human infectious diseases (e.g., H I V ) despite the use o f immunodeficient severe combined immunodeficiency (SCID) mice, the mouse remains, at best, an imperfect model to study the disease 33 pathogenesis. Some would, therefore, argue that human variation and complex diseases can be studied and modeled only in our own species. Certainly there are common human diseases that do not occur naturally in the mouse, such as mental illness, autism, and Alzheimer's. However, using technologies to genetically alter their genomes, researchers have created mice that recapitulate many, but not al l , o f the features o f human Alzheimer 's 3 4 7 . Using detailed behavioral phenotyping and standardized tests, investigators are defining traits in the mouse that may be equivalent to human mental illness and behavioral disorders 3 4 8 . A n advantage o f many o f these mouse models is the ability to dissect out parts of a complex human disorder and study the contributions of individual components to the complete phenotype. The influence of environmental factors on a genetic trait can be studied under controlled conditions in the mouse. This characteristic is extremely important in cancer, where effects o f diet and chemical exposures on an underlying genetic predisposition may be difficult to discern. Investigators have discussed how the genetic resources o f inbred mouse strains with varying cancer susceptibilities, recombinant inbreds, consomics, and congenics can be applied to study human cancer or other complex traits 3 4 9 . Thus, it may be concluded that, mouse models are useful in many ways and similar to human diseases and this similarity allows the mouse to be a good model system for human biomedical research. The field o f stem cell research has benefited greatly by having access to al l stages o f the developing mouse embryo. A considerable amount of knowledge regarding human hematopoietic stem cells, for example, has been garnered from studying the fetal murine bone marrow and liver. M i c e have also been used in gene therapy applications to prove that inserted genes are functioning properly, that they do not have a deleterious effect on the organism and that they correct the phenotypic defect 3 5 0. Mouse models o f other human diseases such as Parkinson's and Alzheimer's have also been developed. m E S C are readily available and their responses can be characterized by both phenotypic and functional assays. Since mice have been extensively used in biomedical research as an acceptable model for humans and murine embryonic stem cells have been extensively 34 studied and characterized, they w i l l be used in this study to develop an understanding o f how culture variables influence the maintenance o f stem cell potential. 1.10 Thesis Objectives Extensive research is rapidly developing our understanding of the molecular control o f proliferation and differentiation o f stem and progenitor cells. These advances have given hope for the cellular therapy o f various disorders for which no efficacious treatments currently exist. In sharp contrast, related bioengineering developments are still in their infancy. The available tissue culture technology for primary cells is limited and scale-up capability is little explored. Yet many o f the promises o f new treatments cannot be realized unless a transplantable number of cells can be generated and manipulated ex vivo. Recently bioengineers have started developing protocols for large-scale expansion and maintenance o f neural 3 5 1 " 3 5 4 and mouse and human embryonic stem and derived ce l l s 3 5 5 " 3 5 9 . However, growing cells in a large bioreactor is a complex task. Protocols would have to be developed to maintain the expansion o f cells for an extended period o f time. Ranges of different culture variables have to be determined within which such cultures could be performed. The dynamics of large-scale cultures are entirely different from those of the well plates or T-flasks that are commonly used to grow these cells in the laboratory. The ability to adequately provide oxygen to cells often becomes a key issue. Furthermore, the selection of an appropriate growth medium, concentration gradients o f p H and metabolites, shear stress, growth factor requirements, etc., become important factors in the design and successful operation o f these large-scale bioreactors. The bioreactor microenvironment may therefore be completely different than that found in vivo. The challenge is to increase the stem cell content without compromising their developmental potential. From the information presented thus far, it is evident that both mouse and human E S C can be cultured in the lab while maintaining their developmental properties. It is also becoming clear that these cells may play a crucial therapeutic role in the treatment o f various currently non-curable disorders in the near future. However in order to treat 3 5 potentially millions of patients, it w i l l be necessary to develop methods to generate h E S C in large quantities. To date, relatively few studies have been published highlighting the large-scale production of terminally differentiated cells from mouse E S C 3 5 5 ' 3 5 7 ' 3 6 0 . Only one study has so far reported on the maintenance of mouse E S C in a small-scale perfusion bioreactor 3 5 9 . The research presented in this thesis w i l l examine the influence o f environmental variables on the maintenance o f stem cell potential and the ranges o f these variables within which these cultures can be conducted in small- and potentially large-scale cultures. Specifically, this project is concerned with developing a quantitative understanding of cytokine dependent proliferation of cells using hematopoietic cell lines as a model system as wel l as developing an understanding of the impact such culture variables as basal medium composition, p H , osmolality, serum and serum replacement concentration can have on the maintenance of stem cell potential using mouse E S C as a model. In order to be able to grow primary hematopoietic cells in large-scale bioreactors for transplantation and other applications, it is instructive to study how single and multiple cytokines and their concentrations w i l l influence their proliferation. Primary hematopoietic cultures, in general, have various limitations: they are inherently heterogeneous in nature with a variety o f different cell types differing in their differentiation and proliferative status present at any given time in the culture. These cultures are labor intensive and subject to donor-to-donor variability. Besides, they require three to four months to assess changes in their most primitive progenitor numbers (using the long term culture-initiating cell (LTC-IC) or competitive repopulation unit ( C R U ) assays). These issues posed major obstacles to the use of primary cells for initial experimental studies as well as mathematical model development. We therefore chose to use cytokine-dependent cell lines for initial studies since they are homogenous in nature, yield reproducible results, and their responses can be analyzed within days. The first specific objective o f this project was to investigate the effect of two cytokines (IL-3 and G M - C S F ) and their interactions on proliferation of hematopoietic cell lines TF-1 and M 0 7 e as a function of cytokine concentration by means of dose-response experiments as wel l as to develop empirical mathematical models of cytokine-dependent growth o f both 36 cell lines when cytokines are present alone and in combination. This work is described in Chapter 2. The second specific objective of this project was to study the influence o f various environmental variables on the proliferation (measured by the mean o f growth rate) and maintenance o f stem cell potential (using the E B formation assay) o f the m E S C lines, R l and E F C . To this end, dose-response experiments were carried out to study the influence o f the p H and osmolality o f the medium (Chapter 3), as wel l as the basal medium composition and serum as well as serum replacement concentrations (Chapter 4) on the proliferation and maintenance of stem cell potential of m E S C . 37 Table 1.1 Characteristic Features of Cytokines > Cytokines are (glyco) proteins that function as signaling molecules with molecular weights in the range o f 6 - 30 kDa . Many cytokines form higher molecular weight oligomers. > Cytokines are potent and are active at nanomolar or even picomolar concentrations. > Cytokines are not constitutively produced; various inducing stimuli regulate their production at the transcription or translational level. > Cytokines exert their action via autocrine or paracrine mechanisms. > Binding o f cytokine to their cognate receptors is a high affinity process with a dissociation constant, Kd, in the range o f 10"9 - 10"12 M . > Cytokine actions may be classified as: Pleiotropic: The cytokine tends to have multiple target cells and multiple actions Redundant: The same effect may be produced by different cytokines Synergistic: Cellular response to two cytokines is greater than the sum o f responses observed in the presence o f each cytokine alone. 38 Table 1.2: Examples of hematopoietic cytokines and their actions Cytokine Molecular Wt. (kDa) Epo 18-32 G - C S F 19-25 G M - C S F 22 IL-3 IL-6 SF 28 26 20-35 Origin Kidney, fetal liver Monocyte, macrophage Macrophage or endothelial cells T cells, mast cells T cells, B cells, monocytes, endothelial cells Marrow stromal, fibroblasts Receptor EpoR; 507 amino acid polypeptide G - C S F R , 130 k D a high affinity oligomer L o w affinity a subunit (85 kDa); high affinity P subunit common to IL-3 and IL-5 L o w affinity a subunit (70 kDa); high affinity P subunit common to G M - C S F and IL-5 IL-6R; low affinity a chain (80 kDa); gpl30 (P sub unit) common to other family members c-Kit , 145 kDa, intrinsic tyrosine kinase Action Stimulation o f C F U - E , proliferation and differentiation o f B F U - E Proliferation and differentiation o f neutrophils; used for mobilization o f stem cells in peripheral blood (PB) Growth and differentiation o f C F U - G M , C F U - M k ; activation o f neutrophils, eosinophils and endothelial cells Stimulation o f C F U - G M , B F U - E , C F U - M k Proliferation and differentiation o f C F U - G M , B F U - M , C F U - M k Stimulation o f B F U - E , C F U - G M , pluripotent stem cells as wel l as leukemic cells 39 Differentiation, Amplification CRU , L-fC-IC j CFC Long- term HSC Short- term HSC Mature cells Myeloid lineage progenitor / \ Lymphoid lineage progenitor — *" Erythrocytes s * - * Megakaryocytes/Platelets l ^ X | Neutrophils ^ ^ „ £ — • f l f Monocytes/Macrophages * T-Cell *"B-Cell Figure 1.1 Hematopoietic hierarchy. 40 Zygote M o r u l a I C M E S C Blastocyst In vitro Ectoderm -> Mesoderm Endoderm In vivo Ectoderm — • Mesoderm Multipotent ~* Progenitors * ° r ^ s Endoderm Figure 1.2 Stem cell hierarchy. Adopted from Wobus et al. (2005) Physiol Rev 85, 635-678. 41 Figure 1.3 Regulation of self-renewal of mESC by Oct-3/4, Nanog, BMP-dependent SMAD and LIF dependent JAK/STAT3 signaling pathways. Adopted from Wobus et al. (2005) Physiol Rev 85, 635-678. 42 In vitro culture Protein suppl. Media composition Embryo Environment Glucose, energy substrates, amino acids, growth factors, steroid hormones, cytokines, metabolic regulators In vivo environment Diet Body composition Potential short term responses 'developmental plasticity' Epigentic modifications Altered intracellular signaling Metabolic stress Gene expression changes Disturbed proliferation & Apoptois Potential long term responses Reduced implantation capacity Unbalanced fetal/placental allocations Altered maternal nutrient provisions Abnormal fetal growth rate Abnormal birth weight and postnatal growth Cardiovascular and metabolic syndromes Figure 1.4 Schematic representing the potential interactions between the environment of the embryo, either in vitro or in vivo, the embryo's short-term responses, and their long-term consequences. Adopted from Fleming et al.(2004) Biol Reprod 71,1046-1054.. 43 Figure 1.5 Summary of the main acid/base transporters of mammalian cells. 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J . ; Lehmann, J.; Zweigerdt, R., Differentiation and lineage selection of mouse 74 embryonic stem cells in a stirred bench scale bioreactor with automated process control. Biotechnol Bioeng 2005, 92, (7), 920-33. 75 2 Empirical Models of the Proliferative Response of Cytokine Dependent Hematopoietic Cell Lines3 2.1 Introduction Identifying conditions that support the ex vivo expansion of human hematopoietic stem cells without compromising their developmental potential remains a challenging task. Recently, the use of engineering principles to analyze and optimize various culture parameters has made significant contributions towards achieving this goal 1" 5 . However, many aspects o f these processes are still poorly understood. A central role in this regulatory process is played by cytokines that are critical mediators o f cellular proliferation, differentiation and survival. Many o f the cytokines required by stem cells have now been cloned and purified to homogeneity. For therapeutic culture processing involving stem and progenitor cells, optimization of likely complex cytokine cocktails w i l l be needed to derive the most consistent and effective use of these valuable materials. In order to optimize ex vivo expansion protocols, there is a need for more quantitative information about how changing cytokine concentrations alter primitive hematopoietic cell proliferation and differentiation responses. Although substantial progress has been made in elucidating the molecular basis o f cell growth regulation, there has been limited development of reliable mathematical models that predict the growth response of cells to mitogenic cytokines. Probably the simplest and most widely used approach in this regard has been to apply the 2-parameter empirical Monod model to describe the growth rate dependence of cells on the concentration o f serum 6 or insulin 7 added to the medium. a A version o f this chapter has been published. Chaudhry, M . A . S . , Bowen, B . D . , Eaves, C.J . and Piret, J . M . (2004) Empirical models o f the proliferative response o f cytokine- dependent hematopoietic cell lines. Biotechnology & Bioengineering 88(3) 348-358. 76 Other specific cytokine effects that have been modeled include the interleukin-2 (IL-2)- stimulated proliferation of T cells 8 and the epidermal growth factor (EGF)-stimulated proliferation o f fibroblasts9. Cantrell and Smith 8 proposed a conceptual model for the cell cycle progression o f T cells that accounted for the observed effects of changes in I L - 2 concentration on the IL-2 receptor density and the duration o f receptor activation. Their study suggested that a threshold number of ligand-receptor interactions must occur before quiescent T cells w i l l initiate D N A synthesis. Knauer et a l . 9 developed a mechanistic model that relates fibroblast proliferation in response to E G F R binding by E G F . Assuming a simple reversible bimolecular receptor-ligand interaction, a first-order internalization rate and negligible recycling, they derived an expression relating the total number o f occupied receptors to E G F uptake. Again it was proposed that the mitogenic response depended on a minimum threshold o f receptor occupancy followed by a simple linear increase in the probability o f activating D N A synthesis according to the number o f signaling complexes formed. When the maximum number o f cells that could be stimulated to enter the S-phase was achieved, a ceiling was reached. Lauffenburger and coworkers 1 0" 1 2 further extended this model by including the hypothesis that the mitogenic activity o f E G F - E G F R binding on the cell surface w i l l be attenuated by receptor down- regulation or depletion o f the ligand from the medium by cellular internalization and degradation. There are many complex mathematical models in the theoretical biology and biochemical engineering literature 1 3. However, most practicing biologists and biochemical engineers have not found these to be very useful. A major practical obstacle has been to obtain the large number of parameters required for each new cell population and each new cell environment. Most often these parameters are determined by using pooled data from different cell types, and there have been only a few efforts to critically test the predictive capability o f such complex mode ls 1 2 ' 1 4 . Microbiologists as well as environmental engineers have extensively used unstructured models to describe the growth of microorganisms on a limiting substrate. Given the simplicity o f these empirical models, it is a concern that they may not adequately capture 77 the complexity o f biological responses. Recently, however, we have shown how systematic experimentation and empirical modeling can greatly simplify the analysis o f multiple interacting cytokine effects on a mixed cell culture system 1 5 . A central composite experimental design was used to measure the concentration effects o f three cytokines on murine hematopoietic stem, progenitor and total cell numbers generated by 14-day serum-free cultures. These data were used to derive both polynomial and modified Monod models that fit the results for all three populations. The common models for the different cell types greatly simplified the perceived complexity o f the multiple interacting cytokines in mixed culture systems by revealing characteristic qualitative trends. The model parameters varied considerably between the cell types providing a coherent view o f the quantitative differences between their responses. IL-3 and G M - C S F are members of the type I cytokine superfamily and their receptors consist o f a ligand-specific, but low-affinity a-chain, and a common p c-chain to which IL-3 and G M - C S F bind competitively when complexed with their a-chains 1 6 , 1 7 . IL-6 is a member of a different cytokine family that utilizes gp 130 as a common signaling element in the receptor complex. Thus, at the cell surface, interactions between IL-3 and G M - C S F receptors would be expected whereas interactions o f either o f these cytokines with IL-6 receptors would not be predicted. Given recent progress in the field of ex vivo expansion o f hematopoietic stem and progenitor cells, it has become increasingly important to develop predictive semi- empirical models that can assist with the analysis o f such complex systems for clinical cell cultures. To investigate the interactions of different cytokines and their influence on hematopoietic cells, we have selected a relatively simple biological system provided by two immortal, but cytokine-dependent hematopoietic cell lines: M 0 7 e and TF-1 cells. Because o f their stability as relatively homogeneous populations, these cell lines are widely utilized for bioassays of various cytokines and as model systems to investigate mitogenic signal transduction pathways. The central objective of this work was to develop practical predictive models for the growth rates o f these cells as a function o f 78 cytokine doses and interactions. B y not taking into account the complex details o f ligand binding and intracellular signaling, much simpler models having only a limited number o f parameters could be formulated. These relatively simple models, developed in terms o f readily measurable variables, should provide a foundation for future empirical or mechanistic additions that may include cytokine dependent self-renewal, differentiation and apoptosis. 79 2.2 Materials and Methods 2.2.1 Cell Lines M 0 7 e cells were originally derived from a patient with acute megakaryocyte leukemia while TF-1 cells were derived from a patient with severe pancytopenia 1 9. Both lines were maintained in Iscove's medium ( I M D M , Invitrogen Life Technologies, Burlington, O N ) with 5% fetal calf serum (FCS) (Invitrogen) and 5 ng/mL o f either human IL-3 or human G M - C S F (gifts from Novartis, Basel, Switzerland). Both cell lines were grown in the presence of various concentrations o f IL-3 and G M - C S F alone as wel l as in combination such that the ratio [ IL-3] / [GM-CSF] was kept constant at 2, 3.5 and 10.5 respectively to cover a relatively wide range of these cytokines. TF-1 cells were also grown in the presence of various concentrations o f human IL-6 alone (gift from Novartis, Basel, Switzerland) as well as in combination with IL-3 or G M - C S F . 2.2.2 Cell Proliferation Assay Exponentially growing M 0 7 e or TF-1 cells were harvested from T-flasks and washed twice in I M D M + 5% F C S by centrifuging and resuspending i n fresh medium to remove residual cytokines from the inoculum culture, and incubated overnight without any cytokine additions. Fifty u L o f cells suspended in I M D M + 5% F C S (at a final concentration of 5 x l 0 4 cells mL" 1 ) were then added to an equal volume o f the same medium containing the desired concentrations o f IL-3, G M - C S F and IL-6 (alone or in combination as described above) in triplicate individual wells of a 96-well microtitre plate. The cultures were incubated for 72 or 120 h at 37°C in a humidified atmosphere o f 5% C O 2 . A t the end o f the incubation period, 20 u L o f M T S / P M S solution (Promega, Madison, WI) was added to all the wells and the plate was incubated for another hour prior to measuring the absorbance at 490 nm using a micro-plate reader (Molecular Devices, Sunnyvale, C A ) . The absorbance values were converted to viable cell concentrations using appropriate standard curves prepared for either the TF-1 or M 0 7 e 80 exponentially growing cells. The average specific growth rate (ju ) was calculated using the equation: X.. 'X.\ (2.1) In At where Xv is the viable cell concentration (cells/mL) at the end of the growth period (72 or 120 h), X0 is the inoculum viable concentration (cells/mL) and At is the time interval (h). 2.2.3 Cytokine Depletion Analysis by ELISA Human IL-3 and G M - C S F concentrations were determined using sandwich enzyme- linked immunosorbent assays ( E L I S A ) 2 0 with capture and detection antibodies against human IL-3 and G M - C S F from B D Biosciences (Mississauga, ON) . 2.2.4 Calculation of Cell Specific Cytokine Uptake Rate The average cell specific cytokine uptake rate, ^ (pg/h-10 6 cells), was calculated from: AL At f \ X-X„ x l 0 ° In X., 'X. (2.2) where AL is the change in cytokine concentration (pg/mL), At is the time interval (h) and Xv and X0 are the final and initial cell concentrations (No. o f cells/mL) respectively. 81 2.2.5 Mathematical Modeling of Cytokine Dependent Proliferation 2.2.5.1 Initial Single Cytokine Model Both M 0 7 e and TF-1 cells exhibit a saturation-type o f population growth kinetics as a function of cytokine concentration. Hence, the following Monod-type equation was initially employed to model the average specific growth rate o f these cell lines as a function o f the cytokine concentration. where jusat (h"1) is the average specific growth rate when the cytokine concentration approaches saturation; L is the initial concentration of cytokine (ng/mL) and KL is the average apparent half-saturation constant (ng/mL). The statistical software package, R, (http://www.r-proiect.org/) was employed to determine the kinetic parameters, fisat and K L , using a nonlinear least-squares method. 2.2.5.2 Improved Single Cytokine Model The Monod-type model, Eq . (2.3), showed a consistent and systematic lack o f fit to the growth rates measured at intermediate and high cytokine concentrations. The following Hill-type function was therefore tested next: 77= M s a t L " (2.4) KNL +Ln where n is H i l l ' s exponent. When initially developed, the H i l l equation sought to describe the binding o f oxygen to hemoglobin . However, it is now widely regarded as an empirical description o f complex binding phenomena. The H i l l coefficient, n, describes the shape o f the curve. When n > 1, the curve shows a sigmoidal behavior with n > 5 representing a step change in response. When n < 1, the curve exhibits a more gradual rise to the maximum value. H i l l ' s function reduces to the Monod model Eq . (2.3) when n = 1. A l l o f the parameters of Eq . (2.4) were obtained via nonlinear regression analysis as described above. 82 2.2.5.3 Cytokine Interaction Models Few attempts to mathematically model cytokine interactions have been reported, although there is considerable experience with modeling drug interactions in the pharmaceutical f ie ld 2 2 . However, the methods used are diverse and can predict very different results when applied to the same set o f data, such that a given drug combination may be synergistic according to one method and antagonistic according to another 2 2. In the work reported here, we define a synergistic interaction as one where "the effect o f the combination is greater than the sum of the effects of its individual constituents". It is important to note that, although numerous articles have reported synergistic effects o f cytokines on hematopoietic cells in culture 2 3" 2 5, many of the results obtained do not in fact satisfy this definition. To model the growth rates observed in the presence of two cytokines, two independent approaches were adopted. First, the following mathematical relationship, developed from an adaptation o f a pharmacodynamic model 2 6 , was used to accommodate the possibility of synergistic cytokine interactions: _ Msat IL3 Ln/L2 Msat GM ^GM u = Lt^- + + y AIL3 + ^IL3 GM ^GM Msat IL3 L"L3 VsatGM LGM . IL3+^IL3 GM GM J (2.5) where y is an interaction parameter. y> 0 indicates a synergistic interaction as the overall growth rate becomes greater than the sum of the growth rates obtained with single cytokines. y< 0 implies an overlapping of the effects of the two cytokines. The value o f y was determined by minimizing the sum o f squared differences between the experimental and predicted growth rates using the "Solver" function in Excel 2000 (Microsoft, Redmond, W A ) . The second modeling approach was based on an analogy to the situation where the presence o f one substrate inhibits the utilization of another 2 7. Given that IL-3 and G M - C S F bind to a 83 common p c chain and a competition between IL-3 and G M - C S F for p c has been observed experimental ly 2 8 , 2 9 , the following competitive model was developed: _ VsatIL3LIL3 MsatGM LGM + — " — (2-6) KIL3+LIL3+a2LGM KGM +LGM +al LIL3 where a2 = ™/ a n d a^ = h KGM 7 2 Here, the third term in each denominator predicts the competitive influence o f another cytokine based on the ratio o f KL values obtained from the single cytokine responses. This model is fully predictive in the sense that all o f the parameters can be determined from independent measurements in single-cytokine experiments. 84 2.3 Results 2.3.1 Dose-Response Analysis of M07e and TF-1 cells Dose-response experiments were performed with M 0 7 e and TF-1 cell lines in the presence o f IL-3 , G M - C S F and IL-6. Since the two cell lines exhibited similar responses, primarily the results for the TF-1 cells are shown. Figure 2.1 compares the experimentally determined average growth rates o f TF-1 cells with the results predicted by Eqs. (2.3) and (2.4) for various doses of IL-3, G M - C S F or IL-6. It can be seen that the Hill-type model, Eq . (2.4), provided a better fit to the measured growth rates for both the 72 h and 120 h experiments than the Monod-type model, Eq . (2.3). The Monod-type model overestimated the growth rates at intermediate cytokine concentrations, underestimated the growth rates at low and high cytokine concentrations and gave a 5 to 200 times higher %2/DOF goodness-of-fit statistics (Table 2.1). The improved fit o f the H i l l equation was due to the additional parameter, n. However, for TF-1 cells, the fit was not significantly altered when the parameter n was kept constant at a value o f 0.5 (i.e., approximately the average n determined for TF-1 cells in response to IL-3, G M - C S F and IL-6 for both the 72 h and 120 h time points) (Figure 2.2 and Table 2.1). The constant value of n, however, varied depending on the cell line and for M 0 7 e cells at 72 h, n ~ 1.57 for G M - C S F and n ~ 1.35 for IL-3 (Table 2.2). Both o f these values o f n decreased by approximately 60% for the 120 h data. Dose-response analyses also revealed that for the 72 h TF-1 cultures, the apparent half- saturation constant o f G M - C S F was about two times lower than that o f IL-3 (Table 2.2). For M 0 7 e cells, the apparent half-saturation constant of G M - C S F was also consistently lower than that o f IL-3. This suggests that G M - C S F is a more potent stimulator than IL-3 for both o f these cell lines as has also been reported 3 0 where it was found that the doubling time of TF-1 cells grown in G M - C S F was significantly lower than that o f cells grown in IL-3 . 85 The M 0 7 e apparent half-saturation constant for both IL-3 and G M - C S F also decreased with duration o f the culture by 73% and 16%, respectively for IL-3 and G M - C S F between 72 h and 120 h and for TF-1 cells, by 84% and 83% respectively during the same interval. Figure 2.3 shows the cell concentration and the cell-specific G M - C S F uptake rate for T F - 1 cells cultured at various cytokine concentrations for 72 h. It can be seen that the uptake rate increased with increasing G M - C S F concentration in contrast to the growth rate that saturated quickly as a function of the cytokine concentration. These results show that cytokine uptake rate and cell proliferation are uncoupled processes in these cells. To check that G M - C S F was not degraded when incubated at 37°C or lost by adhesion to the tissue culture plastic, G M - C S F was in incubated in culture media for 120 hours at 37°C and the level o f G M - C S F was found not to decrease (data not shown). 2.3.2 Determination of Cytokine Interactions Figure 2.4 shows the growth rates o f M 0 7 e cells over a 72 h period when IL-3 and G M - C S F were present alone or together. The overall growth rate of cells exposed to IL-3 and G M - C S F in combination was less than the sum of the growth rates achievable with IL-3 or G M - C S F separately, i.e., the mitogenic activity o f IL-3 and G M - C S F on this cell line was not synergistic. Two models were developed to describe the interactions o f the cytokines on the growth rates o f M 0 7 e and TF-1 cells, as described in the mathematical modeling section. A s shown in Figure 2.5, Eq . (2.5) provided only a rough fit o f the experimental data for TF-1 cells when IL-3 and G M - C S F were added in ratios of 2, 3.5 and 10.5 at 72 h (see Table (2.3) for % / D O F values). However, the agreement of Eq . (2.5) with experimental data at 120 hour is reasonable. The value of the interaction parameter, y, estimated to be between -23.7 to -37.3 for both IL-3 and G M - C S F for the 72 and 120 h data (Table 2.4), indicates a non- synergistic but overlapping effect of these two cytokines. 86 The competitive model (Eq. 2.6) provided a good fit (Table 2.3) o f the experimentally observed growth rates of both cell lines as illustrated in Figure 2.6 for TF-1 cells cultured for 72 and 120 h with IL-3 and G M - C S F concentrations at ratios of 2, 3.5 and 10.5. To test the wider applicability o f this model for a case where receptor subunits are not shared, the interactive effects on TF-1 cells of a third cytokine, IL-6, with either IL-3 or G M - C S F were investigated. A s is evident from the results shown in Figure (2.7a) for IL- 3+IL-6 and in Figure (2.7b) for IL -6+GM-CSF, the cell growth rates observed did not fit the competitive model, Eq . (2.6), consistent with the different receptor specificities of IL- 6. 87 2.4 Discussion Analyses o f the cytokine dose-responses o f both M 0 7 e and TF-1 cells to IL-3 and G M - C S F revealed that the Monod-type model did not fit the results obtained at intermediate and high cytokine concentrations but that this could be corrected by using the more general Hill-type model. In order to correct for the more gradual approach to saturation exhibited by the TF-1 cells, the values o f n required were consistently ~ 0.5. However, values o f n > 1 were required to fit the model to the 72 h M 0 7 e dose-response data and these decreased to n<\ for the 120 h data where cells reached the declining growth phase o f culture. The value o f n is therefore cell line and cytokine specific and may vary with the culture conditions. It is remarkable that the Hill-type model continued to fit better than the Monod-type model even as growth rates were reduced by non-cytokine factors. The dose response data and relative KL values in Table 2.2 show that G M - C S F stimulated a higher growth rate of TF-1 cells than did the same concentration o f IL-3 . This may be due to a greater number o f G M - C S F receptors on the surface o f these cells. Radiolabeled ligand-receptor binding assays have indicated that, for TF-1 cells, the number o f low affinity G M - C S F receptors (a-chain receptors) to be ~9000 per c e l l 3 1 ' 3 2 , while the number of low affinity IL-3 receptors has been reported to be -340 per c e l l 3 2 . The equilibrium dissociation constant for G M - C S F is 189 p M while that o f IL-3 is 244 p M 3 3 . G M - C S F also appears to have a greater ability to recruit the p c chain than I L - 3 2 8 ' 3 4 . G M - C S F therefore appears to be a more potent growth stimulator for TF-1 cells than IL-3 . A s the cytokine concentration was lowered, there was a concomitant decrease in the rate at which the population expanded. This may be due to insufficient stimulation to initiate entry into the S phase o f cell cycle and/or initiation o f apoptotic mechanisms. Comparison o f the 72 and 120 h dose-response data also revealed that the growth rate o f TF-1 and M 0 7 e cells decreased with culture duration as the cells passed from the exponential to the declining growth phase. This may also be partially due to cytokine 88 depletion as was shown to occur when TF-1 cells were cultured in a GM-CSF-conta ining medium. These decreases were most l ikely caused by the accumulation o f inhibitory metabolites like lactate and ammonium and a decrease in p H . A n important conclusion indicated by the present studies is that proliferation and cytokine uptake appeared to be uncoupled processes. For instance, while cellular proliferation was observed to saturate quickly as the cytokine concentration was increased, cytokine uptake rate continued to increase dramatically. Evidence in the literature suggests the existence o f a soluble form of the G M - C S F receptor a-chain ( s o l G M R c c ) 3 5 ' 3 6 . The solGMRct is secreted by a variety of cell types including myeloid 37 38 9̂ cell lines ' , bone marrow progenitors, monocytes, and macrophages and its secretion is upregulated by G M - C S F in normal human monocytes in a dose-dependent manner 3 9. The s o l G M R a binds to G M - C S F and is able to interact with the common p c-chain such that high affinity binding of s o l G M R a with p c is indistinguishable from that o f transmembrane G M - C S F receptor a-chain (trmGMRoc) with p c 4 0 . The addition of G M - C S F to TF-1 cells starved o f cytokines for 24 h elicited either a marked increase 4 1 or virtually no change 4 2 in the expression o f p c , whereas there was a marked down- modulation o f t r m G M R a . Therefore, s o l G M R a may also be involved in the uptake o f G M - C S F from the extracellular medium in these studies and thereby contributes to the uptake o f G M - C S F at concentrations that have already reached the levels required to maximize the number o f cells stimulated to proliferate. These studies also revealed that the apparent half-saturation constant constants (KL) o f both cytokines varied with duration of culture and could depend on culture conditions. A decrease in KL values would mean that lower cytokine concentrations are required to maximize growth over longer batch culture times - an observation that might have bioprocessing implications for the expansion and maintenance of progenitor and differentiating cells. However, in stem cell cultures, it has been shown that higher cytokine concentrations can be required for the maintenance o f self-renewal and suppression o f differentiation 4 3 ' 4 4 . The variation o f KL values could be explained on the basis o f the increasing cell density per well such that the metabolic activity o f reducing 89 M T S per individual cell decreased as has been previously observed . These findings also signify a need for further investigations to determine the impact that bioprocess variables have on effective cytokine utilization. For example, additional experiments could be carried out to study how changes in the extra-cellular p H as wel l as a build-up o f inhibitory metabolites like lactate and ammonia alter cytokine activity or cytokine- receptor binding affinity, and how conditioned medium affects cytokine stability. The results reported here indicate that there is an overlapping interaction between IL-3 and G M - C S F as they affected the growth rates o f the cell lines employed in this study. The expression pattern o f IL-3 and G M - C S F receptors on TF-1 may therefore explain these observations. Since the number of G M - C S F R a-chains is far greater than that o f I L - 3R, as discussed above, at high initial concentrations of G M - C S F it is l ikely that most o f the p c chains are complexed with G M - C S F R a molecules allowing them to out-compete I L - 3 R a molecules for the p c chains. However, when the initial G M - C S F concentration is low, it is rapidly depleted from the culture medium thereby making the IL-3 molecules more competitive. A report by Gesner et a l . 4 6 that G M - C S F competition with IL-3 on K G - 1 cells was concentration-dependent supports this view. Mult iple cytokines are often simultaneously administered to hematopoietic as wel l as tissue engineering cultures to stimulate cell survival, proliferation and differentiation responses. These cytokines can interact in synergistic, overlapping or antagonistic ways. The empirical modeling approach using H i l l ' s function was extended to address the cytokine interaction and its influence on the growth rates o f the cell lines used in the study. The additive interaction model developed agreed well with the experimental data obtained at three different [ IL-3] / [GM-CSF] ratios for 120 h cultures o f TF-1 cells. The competitive model developed has the advantage that it is based on parameters obtained from single cytokine measurements and hence can 'predict' the outcome when both cytokines are simultaneously present. The model was tested at various IL-3 to G M - C S F concentration ratios and found to predict the experimentally observed results. Also when parameters ( « / and ai) were obtained from a particular IL-3 to G M - C S F concentration ratio in the single cytokine experiments, they could be used to predict the results obtained at a different IL-3 to G M - 90 C S F concentration ratio (data not shown). A s expected, the competitive model did not fit the growth response data obtained in the presence o f IL-3 and IL-6 or G M - C S F and IL-6, where these cytokine pairs do not share receptor subunits. These cytokine interaction models are not mechanistic and do not provide detailed information about ligand-receptor interactions or intracellular signaling processes. However, this approach does has the advantage that it depends only on extracellular parameters that can be relatively easily measured and does not necessitate the determination o f an unpractically large number of parameters, as is required by many mechanistic cellular models. 91 2.5 Conclusions Models o f hematopoietic cell growth rate dependence on medium cytokine additions have been developed. A n empirical approach was adopted because it depended only on extracellular parameters that can be relatively easily measured, compared to much more complex mechanistic cellular models. Interestingly, a Monod-type model was poorly fit to IL-3 , IL-6 or G M - C S F dose-response data. Instead, it was proposed that a Hill-type relationship provides a more appropriate model, although it requires one additional parameter. When IL-3 and G M - C S F were added together, it was shown that their effects on growth did not interact synergistically or even additively. A competitive model was developed by modifying the Hill-function to include the competition between IL-3 and G M - C S F for their common receptor (p c) subunit. This model had no new parameters beyond those obtained from the single cytokine cultures and provided good prediction o f the growth rates for both cell lines when they are exposed to combinations of IL-3 and G M - C S F , over a wide range o f concentration. The competitive receptor system provided a clear rationale for the predictive model developed, suggesting that this modeling approach may be effective for a wide range of other shared receptor subunit systems. Although empirical, these H i l l ' s and competitive mathematical models both could be used to quantitatively predict the proliferative responses o f hematopoietic cells to a wide range o f cytokine stimulation. 92 Table 2.1 x 2/DOF statistics for goodness-of-fit test comparing Monod-type model Eq. (2.3), Hill-type function Eq. (2.4) with exponent '«' fixed (« = 0.5) as well as Hill function with variable exponents against experimental growth rates of TF-1 cells grown in the presence of IL-3, IL-6 or GM-CSF alone. X 2 / D O F Cytokine Monod Model Hill function (n = variable) Hill function (n = fixed at 0.5) 72 h IL-3 1.3 x 10"4 2.0 x 10"6 9.7 x 10"6 IL-6 4.4 x 10"5 4.1 x 10"6 4.1 x 10"6 GM-CSF 2.8 x 10"4 2.6 x 10"6 2.9 x 10"5 120 h IL-3 4.8 x 10"5 9.7 x 10~7 9.6 x 10"7 IL-6 5.4 x 10"5 2.9 x 10"6 5.2 x 10"6 GM-CSF 1.7 x 10"4 7.9 x 10"7 8.2 x 10"7 93 Table 2.2 Hill model kinetic parameters for cell lines, TF-1 and M07e cells growing in IL-3, IL-6 and GM-CSF' IL-3 G M - C S F 72 h 120 h 72 h 120 h Cell Msat KL n Msat KL n Msat KL n Msat KL n Line h"' ng/mL (-) ng/mL (-) h ' ng/mL (-) ng/mL (-) TF-1 0.044 ± 0.003 0.040 ± 0.002 0.40 + 0.06 0.028 + 0.004 0.0062 ± 0.0007 0.48 ± 0.02 0.045 ± 0.003 0.017 ± 0.004 0.68 + 0.05 0.030 ± 0.003 0.0031+ 0.0004 0.49 + 0.03 M07e 0.039 ± 0.004 0.84 + 0.02 1.35 ± 0.05 0.024 + 0.003 0.23 + 0.02 0.50 + 0.04 0.042 + 0.005 0.092 + 0.003 1.57 ± 0.02 0.036 ± 0.003 0.077 + 0.002 0.62 ± 0.07 IL-6 72 h 120 h Cell Line Msat KL n Msat KL n ng/mL (-) ng/mL (-)' TF-1 0.026 ± 0.004 0.09 ± 0.005 0.49 ± 0.03 0.018 + 0.004 0.364 + 0.02 0.57 + 0.06 a the values are mean ± std. dev o f three independent experiments 94 Table 2.3 x2fDOF statistics for goodness-of-fit test comparing the two cytokine interaction models against experimental growth rates of TF-1 cells grown in the presence of combination of IL- 3, IL-6 and GM-CSF. Cytokine Interaction Model Cytokine Cone. Ratio X 2 / D O F 72 h Equation (2.5) [IL-3] / [GM-CSF] = --2 4.98 x 10"4 Equation (2.5) [IL-3] / [GM-CSF] = 3.5 2.69 x lO"4 Equation (2.5) [IL-3] / [GM-CSF] = 10 3.03 x 10"4 Equation (2.6) [IL-3] / [GM-CSF] = = 2 1.77 x 10"4 Equation (2.6) [IL-3] / [GM-CSF] = 3.5 5.4 x 10"5 Equation (2.6) [IL-3] / [GM-CSF] = 10 7.60 x 10"5 Equation (2.6) [IL-6]/[IL-3] = 8 5.30 x 10"3 Equation (2.6) [IL-6] / [GM-CSF] = 16 8.97 x 10 - 4 120 h Equation (2.5) [IL-3] / [GM-CSF] = --2 2.37 x 10"4 Equation (2.5) [IL-3] / [GM-CSF] = 3.5 8.80 x 10"5 Equation (2.5) [IL-3] / [GM-CSF] = 10 1.03 x 10"4 Equation (2.6) [IL-3] / [GM-CSF] = = 2 1.13 x 10"4 Equation (2.6) [IL-3] / [GM-CSF] = 3.5 2.83 x 10"5 Equation (2.6) [IL-3] / [GM-CSF] = 10 4.03 x 10"5 Equation (2.6) [IL-6]/[IL-3] = 8 6.63 x 10"4 Equation (2.6) [IL-6] / [GM-CSF] = 16 5.20 x 10"4 95 Table 2.4 The values of interaction parameter y computed to compare the additive interaction model (Equation 2.5) against experimental growth rates of TF-1 cells grown in the presence of combination of IL-3 and GM-CSF. Cytokine Cone. Ratio 72 h y(h) [ IL-3] / [GM-CSF] = 2 -23.70 [IL-3] / [GM-CSF] = 3.5 -25.65 [IL-3] / [GM-CSF] = 10 120 h -24.79 [IL-3] / [GM-CSF] = 2 -36.46 [IL-3] / [GM-CSF] = 3.5 -37.33 [IL-3] / [GM-CSF] = 10 -28.0 96 Figure 2.1 Comparison of the growth rate of TF-1 cells cultured for either 72 or 120 h in the presence of (a) IL-3, (b) GM-CSF and (c) IL-6 with predictions of Monod- and Hill-type models. Data are mean ± standard deviation of three independent experiments. Legend applies to all three panels. 97 0.050 -| 0 .045- 0.040 - s 15 0 .035- or. 0 .030- > O 0 .025- <u O ) 0 .020- £5 a) > 0 .015- < 0 .010- 0.050 -• 0.045 - .—. 0.040 - £ 0 .035- CO a: . c 0.030 - ro wt  0 .025- o 0.020 - (a) 43 0.005 0.1 1 10 Initial IL-3 Concentration (ng/mL) (b) 1 10 100 Initial IL-6 Concentration (ng/mL) 0.050 -, 0 .045- 0 .040- aj to 0 .035- CC w th  0.030- p i _ O 0 .025- CD O) 0 .020- 0) > 0 .015-< 0.010 0.01 0.1 1 10 Initial GM-CSF Concentration (ng/mL) Experimental Growth Rate (72 h) Experimental Growth Rate (120 h) • Hill Model (n = variable) Hill model (n = 0.5) Figure 2.2 Comparison of the growth rate of TF-1 cells cultured for either 72 or 120 h in the presence of (a) IL-3, (b) GM-CSF and (c) IL-6 with prediction of a Hill-type model with n fixed at 0.5 or varied according to the value obtained from non-linear regression of the data. Data are mean ± standard deviation of three independent experiments. The same legend applies to all three panels. 98 10S 0) o O •2 10s o o i o 4 i^ — A — Cell Concentration - • - GM-CSF Uptake Rate I I I I l l l l i i i n i l ] i i i 111i i | i — i | i i i 111i i | 1E-4 1E-3 0.01 0.1 1 10 7500^ 1 5000 8 2500 o O) 140 120 100 w 80 60 40 20 0 O J* Q. o 100 1000 o Final GM-CSF Concentration (ng/mL) Figure 2 J Cell concentration and GM-CSF uptake rate as a function of final GM-CSF concentration for 72 h cultures of TF-1 cells.. The rate of GM-CSF uptake was calculated from the rate of GM-CSF depletion from the medium as determined by ELISA. Data are the mean ± standard deviation of two independent experiments. 99 0.05-1 S 0.04 <D * 0.03 o O 0.02 <D O) ro |S 0.01 0.00 • Growth Rate (GM-CSF) • Growth Rate (IL-3) A Growth Rate (IL-3+GM-CSF) 0.1 1 10 100 Initial Cytokine Concentration (ng/mL) Figure 2.4 Growth rates of M07e cells cultured for 72 h in the presence of IL-3 alone, G M - CSF alone or IL-3 and GM-CSF together at a ratio of [IL-3]/[GM-CSF] = 2. Abscissa scale is initial IL-3 concentration when IL-3 and GM-CSF were used in combination. Data are mean ± standard deviation of three independent experiments. 100 0 . 0 5 ^ 0 . 0 4 CO 0£ 2 o 0 . 0 3 0 . 0 2 0 . 0 1 (a) Growth Rate (Ratio=2) — Model Eq. 2.5 (Ratio=2) Growth Rate (Ratio=3.5) Model Eq. 2.5 (Ratio=3.5) * Growth Rate (Ratio=10.5) Model Eq. 2.5 (Ratio=10.5) 0 . 0 1 0.1 1 0 0 . 0 5 0 . 0 4 0 . 0 3 0 . 0 2 0 . 0 1 (b) 0 . 0 1 0.1 1 0 Initial IL-3 Concentration (ng/mL) Figure 2.S Comparison of the growth rate of TF-1 cell cultured in the presence of both IL-3 and GM-CSF for (a) 72 and (b) 120 h with the predictions of an additive Hill-type model (Eq. 2.5). In each set of experiments, the [IL-3]/[GM-CSF] ratio remained constant at fixed values of 2,3.5 and 10.5. Data are mean ± standard deviation of three independent experiments. The legend applies to both panels. 101 Initial IL-3 Concentration (ng/mL) Figure 2.6 Comparison of the growth rate of TF-1 cells cultured in the presence of both IL-3 and GM-CSF for (a) 72 and (b) 120 h with the predictions of a competitive Hill-type model (Eq. 2.6). In each set of experiments, the [IL-3]/[GM-CSF] ratio remained constant at fixed values of 2,3.5 or 10. Data are mean ± standard deviation of three independent experiments. Figure legend applies to both panels. 102 0.05 -, 0.04 ^ 0.03 (0 1 2 o 0.02 0.01 A 0.00 (a) 0.05 0.04 0.03 0.02 0.01 ™r 0.1 1 10 100 0.1 1 10 100 Initial IL-6 Concentration (ng/mL) M M 0.00 10 100 0.1 (b) Growth Rate(72h) Hill Model ( 72h) Growth Rate (120h) Hill Model (120h) Figure 2.7 Comparison of the growth rate of TF-1 cells cultured in the presence of (a) IL-3 + IL-6 or (b) IL-6 + GM-CSF for 72 and 120 hours, with the predictions of a competitive Hill-type model (Eq. 2.6). In each set of experiments, the [IL-6]/[IL-3] and [IL-6]/[GM-CSF] ratios remained constant at fixed values of 8 and 16 respectively. Data are mean ± standard deviation of three independent experiments. Figure legend applies to both panels. 103 2.6 References 1. da Silva, C. L . ; Goncalves, R.; Lemos, F. ; Lemos, M . A . ; Zanjani, E . D . ; Almeida-Porada, G . ; Cabral, J. M . , Model l ing of ex vivo expansion/maintenance o f hematopoietic stem cells. Bioprocess Biosyst Eng 2003, 25, (6), 365-9. 2. Koller , M . R.; Bradley, M . S.; Palsson, B . O., Growth-Factor Consumption and Production in Perfusion Cultures o f Human Bone-Marrow Correlate with Specific Ce l l Production. Experimental Hematology 1995, 23, (12), 1275-1283. 3. Sandstrom, C . E . ; Collins, P. C ; McAdams, T. A . ; Bender, J. G . ; Papoutsakis, E . T.; Mi l l e r , W . 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J Cell Physiol 1988, 136, (3), 493-499. 108 3 Culture pH and Osmolality Modulate Proliferation and Embryoid Body Formation Potential of Murine Embryonic Stem Cells b 3.1 Introduction Pluripotent embryonic stem cells (ESC) have been derived from the inner cell mass o f blastocyst stage embryos o f murine, primate as well as human sources1"4. These cells exhibit indefinite replicative potential and the ability to differentiate into derivatives o f all three germ layers. The manipulation o f stem cells is expected to provide new means to treat conditions that are currently incurable. Stem cells are subject to a wide variety o f control mechanisms and hence progress in harnessing these cells to repair damaged tissues w i l l rely upon identifying the critical genetic and culture factors that influence these regulatory mechanisms. Whereas human embryonic stem cell-derived cells have tremendous potential in many experimental and therapeutic applications, they have a doubling time of the order o f 35- 40 h 5 . The realization o f stem cell clinical therapies depends on the development of consistent, scalable protocols to expand their numbers without compromising their developmental potential. Maintaining a large pool o f high purity stem cells in culture represents a great challenge as, although mouse or human E S C can be readily expanded in culture, many types o f differentiated cells are also produced in these cultures. To maximize E S C numbers and minimize the fraction of diverse differentiated cells, culture conditions that favor E S C self-renewal need to be defined. b A version of this chapter will be submitted for publication. Chaudhry, M.A.S . , Bowen, B.D. , Eaves, C.J. and Piret, J .M. (2006) Culture pH and Osmolality Modulate Proliferation and Embryoid Body Formation Potential of Murine Embryonic Stem Cells. 109 Murine E S C are pluripotent cells derived from the early mouse embryo. When cultured in the presence of mouse embryonic fibroblasts ( M E F ) and/or leukemia inhibitory factor (LIF), these cells can proliferate while maintaining their capacity to differentiate into a wide variety o f other cell types 1. Removal of M E F or L IF causes E S C to spontaneously differentiate, following a reproducible temporal pattern of development that in many ways recapitulates early embryogenesis6. A s E S C differentiate in culture, they typically form three-dimensional aggregates of cells called embryoid bodies (EBs). Over time, E B s increase in cell number and complexity as cells from the three germ layers are formed. E B formation has been shown to recapitulate the formation o f a three- dimensional architecture wherein cell-cell and cell-matrix interactions are thought to support the development of the three embryonic germ layers and their derivatives 6 ' 7. Generation o f a wide variety of cell types, e.g., cardiac myocytes, hematopoietic cells, neurons and pancreatic cells, from E S C have been reported 8 - 1 3 . Mouse and human E S C can be cultured in the lab while maintaining their developmental properties. It is also becoming clear that human E S C may play a crucial therapeutic role in the treatment of various currently non-curable disorders in the near future. However in order to treat potentially millions of patients, it w i l l be necessary to develop methods to generate these cells in large quantities. To date, relatively few studies have been published highlighting the large-scale production o f undifferentiated as wel l as terminally differentiated cells from mouse E S C 1 4 " 1 7 . To scale up these bioreactors, it w i l l be necessary to determine the ranges o f culture variables within which these cells can be maintained in an undifferentiated state. Murine embryonic stem cells (mESC) provide a practical model for stem cell culture process research as they can be readily obtained at relatively high purities and can be propagated indefinitely. However, stem cell bioengineering is still in its infancy and a thorough understanding of environmental tolerance ranges o f either mouse or human E S C is still lacking. Because the mouse is widely used as a model for conducting in vitro and in vivo studies to gain insight into the developmental biology and regenerative medicine that w i l l eventually benefit humans, it is important to develop an understanding of the influence o f the culture environment on 110 the maintenance o f the stem cell potential o f m E S C . The insight gained through m E S C should also be beneficial towards developing methods for the expansion o f human embryonic stem cells (hESC) i n culture without loss o f their developmental potential. E S C are normally cultured in well-plates, dishes or T-flasks with daily replacement o f medium such that they are subject to variations in the cellular microenvironment. The rapid exhaustion o f cultivation area in these systems necessitates frequent passaging o f cells to prevent the spontaneous differentiation that otherwise ensues. Most importantly, the batch-type approach does not provide steady-state operating conditions, resulting in temporal variation of the culture microenvironment that can potentially influence the cellular output o f E S C cultures. In the case of mammalian cells, weak acids from cell metabolism normally accumulate in longer-term and high density cultures. Culture extracellular p H has been shown to have diverse influences on cells, including effects on proliferation rate ' , differentiation " , » 71 7S 1f\ 78 metabolism " , protein synthesis, degradation and glycosylation " , glucose transport and glycolysis, branched chain amino acid oxidation 2 9 as wel l as expression o f cell 71 « i n surface receptors and amino acid transport . A t low extracellular p H , there may be nutrient uptake limitations and cells may not be able to maintain sufficient intracellular levels of nutrients for growth 3 1 . A stimulation o f glucose transport in alkaline p H conditions has been reported for many cell systems, including rat adipocytes and liver cel ls 3 2 . Under alkaline p H , nutrient molecules may have the wrong ionic charges for uptake and cell surface proteins may assume the wrong configuration for their proper function 3 1 . Raised osmolality has also been shown to affect both cell growth and recombinant protein production by C H O 3 3 ' 3 4 as well as hybridoma c e l l s 3 5 , 3 6 . Osmotic stress affects cells by the mechanical consequences o f changes in volume or turgor pressure and by the 111 effects on macromolecules of changes in ionic strength and molecular crowding. Adaptive responses to osmotic stress include the acute influx of salts and water (called regulatory volume increase, R V I ) , the chronic accumulation of organic osmolytes and the activation of the genes for heat shock proteins, aldose reductase and the transporters o f betaine, inositol and taurine. The increased expression of aldose reductase occurs when the cell volume falls and the intracellular concentrations of N a + and K + salts rise. Apparently, it is the increased ion concentration, not the decreased volume that is the 37 38 stimulus ' . B y the same reasoning, the osmotic induction o f heat shock genes may also depend on the intracellular inorganic ion concentration. Increased expression of HSP-70 m R N A in response to a high N a C l concentration is reduced when betaine and inositol are present in the medium (and the cells), compared to the much greater and more prolonged elevation when there is no betaine or inositol in the medium 3 9 . The development o f a mouse zygote to a blastocyst is severely impaired when the concentration of N a C l exceeds 115 m M (-290 mOsm/kg). The development o f the embryo is not affected i f the N a C l concentration is as low as 80 m M (230 mOsm/kg) 4 0 . Two key enzymes o f glycolysis, L D H and pyruvate kinase, become essentially inoperable in the presence o f high salt concentrations 4 1. Moreover, a decreased rate o f protein synthesis and the disruption of gene expression with increasing salt concentration has also been reported in mammalian embryos 4 2 ' 4 3 . Hypo- or hyper-osmotic conditions activate a variety o f kinases, including stress- and mitogen-activated kinases 4 4" 4 6 and Rho kinase 4 7 among others. Hyperosmotic stress has been shown to induce proteosome-mediated degradation o f cyclin D l in a lymphoma cell l ine 4 8 . Janus kinase 2 and calmodulin have been shown to activate the N a + / H + exchanger in C H O - K 1 cel ls 4 9 . Unl ike hematopoietic stem cells, for which quantitative functional assays o f stem cell potential have been defined and validated, such assays for mouse or human E S C are less wel l developed. Self-renewal is generally measured by the ability o f mouse E S C to continuously proliferate in culture while maintaining undifferentiated colony morphology. The most rigorous in vivo assay to establish the functionality o f cultured mouse E S C is blastocyst injection and measurement of their ability to give rise to 112 chimeric mice, since an E S C contribution is required for all adult tissue, including germ cel ls 5 0 . Two in vitro assays have been used extensively as surrogates for chimera formation when testing culture reagents or examining the consequences o f genetic manipulation. The colony-forming cell (CFC) assay is used to determine the plating efficiency o f E S C populations under various conditions and thus may be considered indicative o f their self-renewal potential. Formation of E B s can be performed at a clonal level in vitro and reflects multilineage differentiation potential 5 1. Assessment o f pluripotency o f mouse E S C has also relied on the expression o f selected molecular markers. These have included alkaline phosphatase, the P O U transcription factor Oct-4, and stage-specific embryonic antigen 1 (SSEA-1) . We have recently demonstrated the superiority o f the embryoid body formation assay over phenotypic characterization by Oct-4 and SSEA-1 staining to monitor the loss o f mouse E S C potential 5 2. To successfully grow the number o f cells required for clinical applications, it is essential to know how the pluripotentiality o f h E S C is affected by changes in various bioprocessing variables such as oxygen tension, p H , osmolality, shear, glucose and glutamine concentrations, etc. In addition, the ranges of these bioprocess variables within which such cultures could be performed without compromising the h E S C developmental potential need to be determined. Once the acceptable ranges o f these various culture variables have been found, optimization of culture conditions to maximize yields can be carried out. To this end, we have used m E S C as a model system as they are readily available in high numbers and purities and are more readily maintained and manipulated in culture compared to h E S C . For example, m E S C are passaged as single cells while h E S C , at present, require passaging as small aggregates such that even cell numbers are difficult to enumerate and control. Physiochemical and nutritional culture parameters are increasingly being recognized as important determinants of culture performance for recombinant protein production as well as for hematopoietic cultures in cellular therapy applications. Culture p H is an important parameter whose effect on embryonic stem cell culture performance remains 113 largely unexplored. In the present study, the m E S C lines, R l and E F C , have been used as model systems to study the effect o f two important culture variables, medium p H and osmolality, on proliferation and E B formation potential. Most m E S C differentiation protocols consider E B formation as the first step. Hence, it is important to define those culture conditions that would maximize the E B yield (total number o f E B s obtained per initial m E S C inoculated) from differentiating m E S C . We have examined the changes in p H and other culture variables in m E S C cultures following the conventional passaging protocol and show that proliferation and E B formation potential are greatly influenced both by p H as well as the previous growth history of inoculum cells, i.e., whether they were grown on irradiated feeders or in gelatinized dishes. Dose-response experiments then show that both p H and osmolality influence the growth rate and E B forming potential o f both R l and E F C cells. In addition, both metabolism and cell size exhibit a strong correlation with p H . Flow cytometric analysis is then used to enumerate the Oct-4 and Annexin-V positive cells under various p H conditions. 114 3.2 Materials and Methods A l l reagents were obtained from StemCell Technologies Inc. [STI], Vancouver, B C , unless otherwise indicated. 3.2.1 Growth of Mouse Embryonic Fibroblasts (MEF) Day 12.5 M E F from a C D - I mouse strain were thawed onto a gelatinized dish and maintained at 37°C in humidified air with 5% CO2 in Dulbecco's modified Eagle's medium ( D M E M ) supplemented with 10% F B S , 100 U / m L penicillin, 100 ug/mL streptomycin, and 100 u M monothioglycerol ( M T G ) (Sigma-Aldrich, Oakville, O N ) . Cells to be irradiated were trypsinized, resuspended in 4 m L of medium, and exposed to 60 G y from an x-ray source before replating for use as feeder cells. 3.2.2 Growth of Mouse Embryonic Stem Cells (mESC) The R l 5 3 , and E F C 5 4 murine E S C were routinely maintained in 37°C humidified air with 5% CO2 on a layer of irradiated M E F and fed daily with a complete change o f E S C maintenance medium. The maintenance medium consisted of high-glucose D M E M supplemented with 15% ESC-qualified fetal bovine serum (FBS), 0.1 m M nonessential amino acids, 2 m M glutamine, 1,000 U / m L leukemia inhibitory factor (LIF), 100 U / m L penicillin, 100 ug/mL streptomycin, and 100 u M M T G . R l cells were from passage 17 while E F C cells were from passage 11 and had been frozen at 10 6 cells per vial . Both cell lines were used within 5-8 passages of initial thawing. Cells were passaged every second day in maintenance cultures. To passage the cells, a single-cell suspension was generated by treatment with 0.25% trypsin and 1 m M E D T A (T/E) (Invitrogen Life Technologies, Burlington, ON) for 5 min until the cells detached from the culture vessel surface. T / E activity was then quenched with D M E M supplemented with 10% F B S . The cells were centrifuged at 1,200 rpm for 7 min and resuspended in E S C maintenance 115 medium. Viable cells were plated at ~2 x 10 4 cells/cm 2 in either 10 cm irradiated M E F dish (10 m L o f maintenance medium) or a 6 cm gelatinized dish ( 4 m L maintenance medium) and cultured for a further 48 h at 37°C, 5% CO2 before harvesting for the p H or osmolality experiments. 3.2.3 Determination of Growth Rate The viable cell concentration was determined by either counting cells on a hemocytometer or by using an automated cell counter, Cedex (Innovative Directions, Pinole, C A ) which, in addition to providing viable cell numbers and viability values, also computed the distribution of cell diameters. The average specific growth rate (ju ) o f m E S C under various p H and osmolality conditions was calculated using the equation: where Xv (cells/mL) is the viable cell concentration at the end o f the culture period (48 h), X0 (cells/mL) is the inoculum viable cell concentration and At (h) is the time interval. 3.2.4 Preparation of Medium at Desired pH or Osmolality The p H o f the E S C maintenance medium was adjusted to different values ranging from 6.7 to 7.9 by adding 5 N HC1 or 5 N N a O H and then incubating the medium overnight to equilibrate in a 5% CO2 atmosphere. In some experiments, a powdered D M E M formulation without glucose, glutamine, sodium bicarbonate, sodium pyruvate and phenol red (Sigma-Aldrich, Oakville, ON) was used to prepare the maintenance medium and sodium bicarbonate was added to obtain the desired p H since the equilibrium bicarbonate concentration ( [ H C O 3 ] , m M ) at 37°C is related to the medium p H and the partial pressure of CO2 (pC02, mmHg) in the gas phase via the following equation 5 5: (3.1) l o g [ H C O ; ] = P H + log[pC02] - 7.543. (3.2) 116 A custom-made D M E M formulation ( JRH Biosciences, Lenexa, K S ) with a low N a C l content (500 mg/L vs. standard 6400 mg/L) was used to prepare the E S C maintenance medium and then appropriate volumes o f 1 M N a C l (prepared in D M E M ) were added to adjust the osmolality o f the medium to between 250 and 500 mOsm/kg. Both the p H and osmolality experiments were carried out on gelatinized 60 mm tissue culture dishes (Sarstedt, Montreal, PQ). 3.2.5 Measurement of pH or Osmolality A l l medium samples were measured after equilibration for several hours in an incubator containing 5% CO2. Samples o f equilibrated media were aseptically removed as quickly as feasible from the culture dishes into 1.7 m L Eppendorf tubes, which were filled completely and capped. The p H measurements were conducted using a Bayer Rapidlab 348 gas analyzer. Prior to each use, the electrodes were calibrated using p H 6.8 and 7.3 buffer solutions. Visual inspection of the culture plates was also done at the start o f the experiment, before medium exchange at 24 or 72 h as well as before culture harvesting at 48 or 96 h. The medium contained phenol red which changes color from red (neutral pH) to yellow (acidic pH) or magenta (basic pH), allowing an approximate confirmation that the p H was in the correct range. Osmolality is a measure of the total solute concentration in a solution. The medium osmolality was measured by freezing point depression using an Advanced Osmometer 3D3 (Advanced Instruments, Norwood, M A ) . The osmometer was cooled using a mixture o f ethylene glycol and several other unspecified chemicals. It was calibrated using 100, 290 and 1500 mOsm/kg standard solutions (Advanced Instruments). The instrument was operated using a sample volume of 250 p L . A l l osmolality values were calculated as the average measurement from duplicate samples. 117 3.2.6 Measurement of Glucose, Lactate, Glutamine and Ammonium Concentrations Glucose and glutamine are the primary carbon and energy sources in mammalian cell cultures. Glucose, lactate, glutamine and ammonium concentrations were determined using an Y S I 7100MBS analyzer (Yel low Springs Instruments, Ye l l ow Springs, OH) . The instrument was operated according to the instructions provided by the manufacturer. The system solutions included a reagent pack with all the necessary standards and buffers. The average cell specific glucose uptake rate was calculated from: *GUR = - ^ x l O 6 (3.3) log mean Xv At where sGUR is the average cell specific glucose uptake rate (pmol/10 6 cell-h), AGlu is the change in glucose concentration (pM), log mean Xv is the logarithmic mean average viable cell concentration (cells/mL) and At is time interval (h). Similarly, the average cell specific lactate production rate, sLPR (pmol/10 6 cell-h) was determined from: sLPR= l- ^ x l O 6 (3.4) log mean Xv At where ALac is the change in lactate concentration. The average cell specific glutamine uptake rate, sGlnUR (pmol/10 6 cell-h) was determined using: sGlnUR= — xlO 6 (3.5) log mean Xv At 118 where AGln is the change in glutamine concentration. Since measurements were made on samples collected every 24 h, spontaneous glutamine degradation in culture was assumed to be negligible (since the first-order degradation constant, k = 0.004675/h 5 6). The yield o f lactate from glucose (YLac/Giu) was calculated from the equation: _ _ Mac AGlu 3.2.7 Preparation of mESC for pH or Osmolality Experiments The mouse E S C were thawed and cultured for 2 passages on irradiated feeders as described above. To prepare the cells for exposure to various p H or osmolality solutions, they were harvested by trypsinization as described above, resuspended in E S C maintenance medium, and preplated on tissue culture plates for 15-20 minutes at 37°C, 5% C O 2 , in order to deplete contaminating M E F . A t the end of this pre-plating step, the non-adherent and loosely adherent E S C were collected by gently washing the surface o f the tissue culture plate. The cells were then centrifuged, resuspended in E S C maintenance medium and viable cell numbers were counted on a hemocytometer with trypan blue dye or on an automated cell counter (Cedex, Innovative Directions, Pinole, C A ) to distinguish live from dead cells. The frequency o f contaminating M E F in the undifferentiated (day 0) E S C samples was estimated to be less than 0.2% based on cell size during counting ( M E F cells are easily distinguishable from m E S C as they are bigger in size (generally >16 um in diameter) and appear to have irregular morphology). A l l cultures for the various p H and osmolality experiments initially contained ~2 x 10 4 cells/cm in a 6-cm dish (4 m L medium). The conditioned medium from each of these cultures was collected after 24 hours and replaced with fresh medium at the same p H or osmolality. The following day, the cultures were harvested by trypsinization and the cell concentrations were determined as described above. 119 3.2.8 Embryoid Body Formation Assay To determine the effect o f maintenance medium p H or osmolality on the ability of mouse E S C to form EBs , the latter were differentiated in a semi-solid medium consisting o f 0.9% methylcellulose, 15% F B S , 2 m M glutamine, 150 u M M T G and the remainder I M D M . Single-cell suspensions were collected on day 0, or prepared after harvesting the cells from the p H and osmolality experiments. Defined numbers o f cells (1000 for E F C cells, 2500 for R l cells) were plated in 35 mm low adherence Greiner dishes in the E S C differentiation methyl-cellulose medium described above to determine the efficiency o f E B formation. E B numbers were determined microscopically after 5-6 days o f culture. It is important to note that the p H of the methylcellulose medium was not adjusted. Figure (3.1) shows a representative picture o f 3-dimensional cell aggregates that were considered as E B s and counted as such, while Figure (3.2) shows the cell aggregates that were not considered to be E B s . Morphologically, only those aggregates were considered as E B s that had a 3-dimensional (more or less spherical) shape, fairly smooth margins and a minimum diameter of around 130-150 um. Considerable variation in the sizes o f counted E B s was observed and the mean diameter was 230 ± 73 um. The largest E B s counted were -350 um in diameter. The E B output as a function of initial input number o f cells, in the range of 500 - 5000 cells/mL, remained more or less linear between. A t higher initial cell densities, both the E B output and the morphology o f the resulting E B s varied considerably (data not shown). The percent E B formation efficiency was calculated by dividing the total number of E B s observed by the number of cells plated, and multiplying by 100. Typically % E B formation efficiencies varied between 0.5 - 10%. The E B yield per initial cell for each p H or osmolality culture was determined by calculating the number o f E B s that would have formed i f all the cells harvested had been inoculated in E B cultures and dividing that number by the total viable cell density at the start o f the culture. 120 3.2.9 Flow Cytometry The antibody used for apoptosis analysis was purified fluorescein isothiocyanate- conjugated Annexin-V (BD Pharmingen). Single-cell suspensions collected on day 0 or prepared from R l cells exposed to various p H and osmolality conditions were washed twice in cold P B S and then resuspended in I X binding buffer at a concentration o f 10 6 cells/mL. Five u L Annexin-V were added to a 100 u L aliquot of cells. The cells were gently mixed and incubated for 15 minutes at room temperature in the dark. Four hundred u L of binding buffer were added to each tube and the samples were analyzed by flow cytometry using a FACSCa l ibu r flow cytometer and C E L L Quest software ( B D Pharmingen, Mississauga, ON) . The forward- versus side-scatter profile was used to gate on viable cells, and an unstained sample (treated with the test cells under otherwise identical conditions) was employed to determine the appropriate gating for quantification o f expression. Cells to be stained for Oct-4 were resuspended in 100 u L of Hanks' buffered saline solution plus 2% F B S (HF) and fixed with 100 p L of Intra Prep Permeabilization Reagent 1 (Immunotech, Westbrook, M E ) for 15 minutes at room temperature. The cells were then washed with H F and permeabilized with IntraPrep Permeabilization Reagent 2 for 5 minutes before incubation with a 1:100 dilution of mouse anti-mouse Oct3/4 monoclonal antibody (Transduction Laboratories, Lexington, K Y ) for 15 minutes at room temperature. The cells were washed with H F before staining with allophycocyanin- labeled anti-mouse immunoglobulin G l (BD Pharmingen). A sample stained with secondary antibody only was used to determine the appropriate gating for quantification o f expression. The samples were analyzed by flow cytometry as described below. F low cytometry analyses were conducted using a Becton Dickinson F A C S C a l i b u r instrument ( B D Biosciences, San Jose, C A ) . This device is a non-sorting analytical flow cytometer that uses an air-cooled argon ion laser to deliver 488 nm light at approximately 15 m W . The flow cytometer was calibrated using Calibrite beads ( B D Pharmingen). A 121 2 m L single-cell sample was placed in a 5 m L plastic tube and air pressure was used to transport the cells into the flow chamber. From the chamber, the cells were injected into a flow cell while suspended in a sheath fluid. The device was set to analyze 10 4 viable cells gated appropriately. 3.2.10 Statistics Data are reported as mean ± standard error o f the mean (SEM) , unless otherwise noted. Statistical comparisons were performed using a two-tailed paired Student's t-test. A n asterisk (*) was used to denote statistical significance (p < 0.05). 122 3.3 Results 3.3.1 Culture Variables during Routine Passaging of mESC This study examined the influence of culture variables on the growth rate and E B formation potential o f m E S C . First R l mESC-conditioned media were analyzed to determine the temporal variations over the course o f conventional cultures, inoculated at 2.5 x 10 4 cells/cm 2 on irradiated feeder cells with daily feeding o f maintenance medium. During the first 24 h, glucose, glutamine, ammonium, p H and osmolality (Figure 3.3) showed relatively modest changes that were increased 2- to 3-fold in the next 24 h, consistent with the increased cell concentration before passaging at 48 h. Gelatinized surfaces can replace feeder cells though the E S C colonies appear increasingly differentiated after two successive passages on gelatin. Nonetheless, this is a useful experimental system for assessing culture variable impacts on E S C for 1 or 2 passages in the absence o f any confounding effects of the variables on the feeder cells. To compare these cultures, R l m E S C were grown on gelatinized dishes and it can be seen (Figure 3.4) that the measured culture variables consistently changed less than in the feeder- containing cultures. Irradiated feeders alone decrease the p H only by 0.05 units and consume less than 3 m M glucose over a 48 h period (data not shown). In most cases, these reductions were smaller than those seen in Figure (3.3), consistent with the absence o f the feeder metabolism and their stimulatory effects on E S C . The one exception was that the osmolality increased from 330 to over 400 mOsm/kg in the feeder cultures compared with only up to 365 mOsm/kg in the gelatinized cultures. This is probably due to cell lysis and hydrolysis of the irradiated feeder cell components. The greatest changes (Figure 3.4) in culture variables occurred when the cell concentrations were highest during the final 24 h (24 - 48 h) o f culture prior to passaging. 123 Dose-response experiments were carried out with glucose, glutamine and ammonium as variables. The growth rate o f the R l m E S C was independent o f the glucose concentration except at its lowest initial value o f 1.25 m M where the glucose decreased to ~ 0 m M while the growth rate decreased by only--12% (Figure 3.5a). Compared to control cultures containing 20 m M glucose, the E B formation potential was also significantly decreased (p < 0.05) when the initial glucose concentrations was 2.5 m M or lower. There was a more gradual increase in E B yield between 2.5 and 20 m M such that 5 m M or more glucose would appear to maximize the E B yield. A similar dependence on glutamine was observed (Figure 3.5b) with E B yields significantly decreased (p < 0.05) in cultures initially at 1 m M glutamine and the maximum yields were obtained from 2 m M glutamine or more present in the medium. Both the growth rate and the E B forming ability were significantly lower (p < 0.01) when glutamine was not added to the initial medium compared to the control culture with 4 m M glutamine. Ammonium dose-response experiments, depicted in Figure (3.5c), show that the R l m E S C growth rates were significantly decreased (p < 0.05) when ammonium was present at a concentration o f 4.5 m M or higher compared to the control culture containing 1.5 m M ammonium. The E B yield was significantly decreased (p < 0.05) only when ammonium was present at a concentration o f 6 m M or higher. Due to the presence o f ammonium in the serum (15% of medium), it was not possible to test ammonium at 0 m M . The data shown in Figure (3.4) clearly illustrated the fact that, following the conventional passaging protocol, the glucose and glutamine concentrations never fell to the threshold levels described above while the ammonium build-up was never more than 3 m M (data not shown). Based on these observations, these variables were considered not to influence the proliferation and E B formation potential of m E S C lines and hence were not further explored. 3.3.2 Osmolality Dose-Response Experiments The influence of the osmolality of the medium on the growth rate and E B yield o f R l cells was determined by means o f dose-response experiments carried out in the range o f 124 200 - 500 mOsm/kg at an initial p H of 7.3. The R l cells grew relatively slowly at the low and high ends of this range compared with the conventional osmolality o f 300 mOsm/kg (Figure 3.6). The E B yield per initial cell increased linearly when the osmolality was raised from 200 to 300 mOsm/kg and then declined sharply between 335 and 500 mOsm/kg. This was due to a decline in the number o f cells capable o f forming E B s at low and high osmolalities with the result that there was almost a 5-fold decline in E B yield at 200 and 500 mOsm/kg compared to the culture with an osmolality o f 300 mOsm/kg. The E B yield of R l cells showed a plateau in the range o f 300 - 335 mOsm/kg. Since the feeder containing cultures had over 400 mOsm/kg levels, these results favored the used of feeder-free gelatinized dish cultures to study p H effects without influences from high osmolality. 3.3.3 pH Dose-Response Experiments The gelatinized dish based p H dose-response experiments determined the effect o f initial culture p H on the growth rate and E B yield o f R l cells exposed to a range o f p H values for 48 h (Figure 3.7). The initial and final average p H values as wel l as the corresponding logarithmic average p H values to which cells were exposed during the final 24 h in culture are shown in Table (3.1). The R l m E S C growth rate as wel l as the E B yield was strongly influenced at both the low and high initial p H values compared to an initial p H 7.3. There was almost a 3-fold decline in the E B yield at p H 6.7 and 7.7 compared to that at p H 7.3. A n initial p H o f 7.3 maximized the growth rate and E B yield o f R l cells, except that the growth rate o f these cells did not significantly decrease at p H 7.6. The growth rate declined rapidly in cultures exposed to p H 7.75 and higher with virtually no viable cells remaining in cultures with a p H o f 7.9. The reduced E B yield from low and high p H cultures appeared to be due to both the reduced growth rate as wel l as the reduced fraction o f cells capable o f forming EBs , i.e., lower frequency (% EBs) o f cells capable of forming E B s (Figure 3.8). The p H dose-response was refined to include more p H values as wel l as the culture time extended to 96 h, to assess the influence o f prolonged exposure o f R l cells to different 125 p H conditions. A s depicted in Figure (3.9), data with trends similar to the 48 h cultures were obtained. Again, a p H 7.3 environment maximized the E B yield. Even though the growth rate o f R l cells was not influenced by p H in the range of 7.15 to 7.6, the E B yield was significantly higher at p H 7.3 compared to 7.15 or 7.45. These results demonstrate that p H is an important culture parameter that strongly influences the performance o f m E S C cultures within the ranges of standard maintenance cultures conditions (Figure 3.4). A n additional p H dose-response analysis was performed with the E F C cells, a different m E S C line. The growth rate and mean E B yield o f E F C cells was significantly higher at all p H values compared to R l cells. The E B yield o f E F C cells increased almost linearly with increasing p H up to 7.3 and then declined at p H 7.6 (Figure 3.10). A s was the case with the R l cells, p H 7.3 produced the maximum E B yield and the E B output was significantly less for cultures exposed to either p H 7.0 or 7.6. L ike R l cells, the growth rate of E F C cells was not significantly different between p H 7.3 and 7.6. A systematic comparison of the p H dose-response o f R l cells in which the inoculum cells were taken from cultures that were maintained either on feeders or on gelatinized dishes was also performed. A s shown in Figure (3.11), over the range of p H values tested (6.7 - 7.7), the growth rate as well as the E B formation potential o f R l cultures inoculated with cells grown on feeders is significantly higher than those cultures that were initiated with cells taken from gelatinized dishes. Table (3.2) summarizes the results o f 2-way A N O V A analysis showing that both p H and surface used for inoculum growth significantly influenced the growth rate as wel l as E B formation potential (EBs/init ial cell) o f R l cells. N o interaction was, however, detected between the p H and the growth surface (gelatin or feeders) on either the E B formation potential or the growth rate o f R l cells. These results further strengthen the observations stated above regarding the metabolic data (Figures 3.3 and 3.4) that m E S C are better maintained on feeders than gelatin. 126 3.3.4 Influence of pH on Metabolism of mESC Line, R1 Glucose, lactate and glutamine concentrations were measured and the corresponding metabolic rates were determined during a 48 h culture of R l cells. L ike growth rate and E B yield, the cellular metabolism o f m E S C was strongly influenced by variations in p H . A s shown in Figure (3.12a), the average sGUR and sLPR both increased monotonically with increasing p H . When the initial culture p H was raised from 6.7 to 7.6, sGUR increased more than two-fold from 53 pmol/10 6 cell-h to 124 pmol/10 6 cell-h. The corresponding increase in sLPR was amost five-fold from 41 pmol/10 6 cell-h to 193 pmol/10 6 cell-h. Thus, Figure (3.12b) shows that the lactate to glucose yield coefficient, YLac/ciu, increased from 0.84 ± 0.2 at p H 6.7 to 1.62 ± 0.05 at p H 7.3. The glutamine metabolism was also influenced by p H . The cell specific glutamine uptake rate, sGlnUR, was highest at p H 6.7 (38 ± 5 pmol/10 6 cell-h) and lowest at p H 7.3 (27 ± 2 pmol/10 6 cell-h). 3.3.5 Variation of Cell Size as a Function of pH Changes in cell size provide another indication of how the cells respond to a particular culture environment. Figure (3.13) shows the variation of average cell diameter as a function of p H for R l cells exposed to different p H environments for 48 or 96 h. A dose- dependent increase in average viable cell diameter with p H was observed. Cells exposed to p H 6.7 for 48 and 96 h had mean diameters of 13.27 ± 0.17 and 13.28 ± 0.15 um, respectively, while at the other extreme, p H 7.75, the mean diameters were 15.03 ± 0.16 and 15.15 ± 0.21 um, respectively. For both the 48 and 96 h cultures, R l cells exposed to the p H range 6.7 - 7.15 had similar average diameters that were significantly lower than those o f cells at p H 7.3. Similarly, the cells exposed to media at p H 7.45 - 7.75 had average diameters that were significantly larger than at p H 7.3. 127 3.3.6 Variation of Oct-4 Positive and Annexin-V Positive Cells with pH Flow cytometric analysis was carried out to determine whether exposure to extremes o f p H causes a down-regulation of Oct-4 - a transcription factor that is highly expressed by E S C but not by their differentiated progeny. In addition, cells were stained with Annexin- V - a marker of early apoptotic cells - to determine whether exposure to extreme p H values induced cell death. Figure (3.14a) shows that there were no statistically significant differences in the percentage o f Oct-4+ cells after 48 h o f exposure to acidic (6.7) p H compared to the control at p H 7.3. Exposure to alkaline p H (7.75), however, decreased the percentage o f Oct-4 positive cells after 48 h o f exposure. After 96 h o f exposure to both p H 6.7 and 7.75, however, there were significant declines in Oct-4+ cells compared to cells exposed to p H 7.3. Figure (3.14b) demonstrates that exposure o f R l cells to either acidic (6.7) or alkaline (7.75) media during the first 48 h induced cell death, as the percentage o f Annexin-V positive cells at these conditions was significantly higher than at p H 7.3. However, there is an apparent adaptation to both acidic and alkaline p H conditions, as the number of Annexin-V positive cells at 96 h was not significantly different across the range o f p H values tested. 3.3.7 Reversibility of pH Effects The other aspect o f p H influence on the proliferation and E B forming potential o f m E S C addressed in these studies was to determine whether the observed p H effects are permanent or i f cellular responses would be restored back to their original values once the medium p H was returned to 7.3. R l cells were seeded in 60 mm gelatinized dishes in triplicate and exposed to p H values of 6.7, 7.3 and 7.75. The initial p H variation during this time course experiment is shown in Figure (3.15). For the R l cells with 24 h exposure to p H 6.7 and 7.75, the medium was removed at 24 h and replaced with a medium at p H 7.3. For cells exposed for 48 h, their medium was exchanged at 24 h at their respective p H values. A t the end o f 48 h, all cells were harvested, counted and E B assays were initiated. A l l the cultures were then passaged twice in p H 7.3 medium, i.e., cultured for 96 h using the conventional maintenance protocol. The normalized growth 128 rate and % E B s formed, with respect to values obtained at p H 7.3, are shown in Figures (3.16) and (3.17), respectively. A t 48 h, as expected, cells exposed to p H 7.3 had the highest growth rate as wel l as % EBs . The 24 h or 48 h exposures to p H 6.7 significantly (p < 0.05) decreased the growth rate as wel l as the frequency o f stem cells in those cultures while the 24 h exposure to p H 7.75 had a relatively less detrimental effect on either stem cell frequency or growth rate compared to the 48 h exposure where both the growth rate and % E B formed were significantly (p < 0.05) decreased. The influence o f p H on m E S C responses is not permanent as cells begin to recover their original state soon after their return to p H 7.3 conditions. A l l the cultures (previously exposed to different p H conditions) that had been allowed to grow in p H 7.3 medium for another 96 h had either restored or started to recover their growth rate and stem cell frequency (% EBs) compared to the p H 7.3 control condition, thereby showing that the influence o f p H on m E S C is not permanent. 129 3.4 Discussion R l cells growing on feeders typically show a 10-fold expansion in cell concentration over a 48 h culture period. Consequently, due to this large increase in cell numbers, metabolic and physiochemical variables also indicate large variations. It is important to note that the larger variations in culture variables seen when R l cells were grown on feeders is due to the stimulation provided by feeders to the R l cells and is not due to the metabolism o f the feeders. In addition to feeders, m E S C can also be maintained on gelatinized surfaces where the p H , osmolality and metabolic profiles are relatively less changed compared to when these cells are grown on feeders. R l cells can be maintained on gelatin, but at a lower growth rate and with decreasing E B formation potential. This is l ikely due to the lack o f feeder secreted soluble fac tors 5 7 ' 5 8 in addition to extracellular matrix proteins deposited by feeders that can also play an important role in cell-cell signaling and maintenance o f pluripotency o f mouse E S C 5 9 . Based on the variation with time o f p H and osmolality seen in these experiments, it is suggested that, although not very convenient, a medium exchange at around 36 h after culture initiation would help minimize the variation in culture variables and subsequent detrimental effects on the proliferation o f mouse E S C and their ability to form EBs . This study has shown that E B yields from m E S C cultures were very sensitive to the medium p H . The 96 h experiments showed that the E B yield was significantly lower even at 0.15 p H units higher or lower than 7.3. The m E S C growth rates were maintained over a relatively broader range o f p H but more extreme p H values reduced growth rates. The trends were confirmed with another m E S C line, E F C , derived from a 129J strain o f mice on gelatinized surfaces 5 4 ( R l cells were derived on feeders from a 129/Sv strain). Due to their derivation on gelatin in the presence o f LEF, E F C cells can be propagated on gelatin for multiple passages without an appreciable decline in either their growth rate or E B forming potential. R l cells, on the other hand, retain a higher E B formation potential when grown on feeders and show a decline both in growth rate and E B forming potential when transferred from feeders to gelatinized surface. Since most experiments in this 130 study were conducted on gelatinized dishes, E F C cells therefore had much higher growth rates and E B yields compared to R l cells. From a bioprocessing point o f view, a feedback control of p H would probably improve the culture's E B yield and maintain the growth rates. L ike p H , the results obtained from osmolality experiments (Figure 3.6) also show that the growth rate and E B yield o f m E S C cultured on gelatinized surfaces is influenced by extremes o f osmolality. For R l m E S C , initial medium osmolality values o f 300 and 335 mOsm/kg resulted in both growth rate and E B yields being maintained. Although experiments were not conducted to determine the intracellular p H (pHj) o f cells when exposed to various osmolality or extracellular p H (pH e) conditions, both o f these variables can theoretically influence pHj. When p H e is decreased, the elimination o f protons by the N a + / H + exchanger can become less efficient (as indicated by an increased counter-gradient o f protons across the cell membrane 6 0), thereby leading to a lower pHj. A reverse situation might be expected in the case o f an increased medium osmolality. The increased extracellular concentration of N a + ions can facilitate the exchange o f protons by the exchanger, as a consequence o f the increased N a + gradient across the cell membrane. This might induce a cytoplasmic alkalization leading to an increase in p H , 6 0 . The increase in glucose and lactate metabolism at high p H values, observed for m E S C lines (Figure 3.12), has also been reported for transformed ce l l s 6 1 , 6 2 . One possible explanation lies in the increase o f the activity o f glycolytic enzymes under alkaline conditions. It is known, for example, that hexokinase has an optimal p H o f 8 6 3 . A n increase in pH; stimulates the activity o f phosphofructokinase, P F K 6 4 . The activity o f P F K isolated from mouse embryos can be significantly altered by changes in p H o f around 0.2 units 6 5 . Another explanation is that increasing the p H alters the membrane potential, changing the glucose transport rate through the membrane 6 6. The lactate production rate increased because of a higher glycolytic activity under alkaline p H conditions. A third explanation is that lactate transport out of the cell is mediated via a lactate/H + co-transporter which is only slightly sensitive to pHj but is strongly dependent on p H e . It has been shown that alkaline p H enhances 6 7 and acidic p H i n h i b i t s 6 7 , 6 8 the rate 131 of lactate exit from the cell. The depression of lactate release results in an inhibition o f the glycolytic enzymes through a feedback mechanism which increases the concentration o f glycolytic intermediates and hence lowers the overall lactate production rate. The yield coefficient o f lactate from glucose increased at elevated p H values. This may suggest a self-regulation to keep the internal p H constant. The greater amount of lactate formed allows the cell to survive under alkaline conditions. Similarly, less lactate is produced at low p H as has been observed for other cell l ines 2 5 . The glutamine consumption rate had a minimum at p H 7.3. The glutamine uptake rate was higher at low p H where glucose utilization was minimal. Glutamine could be used preferentially as an energy substrate at low p H . The glutamine uptake rate increased at high p H values possibly due to the increased glutaminase activity which is known to have a p H optimum of around 8. The mean viable cell diameter (and hence cell volume) was found to change as a function o f extracellular p H (Figure 3.13). The volume of cells exposed to p H 7.75 was 32% greater than those o f cells exposed to p H 7.3 which in turn were 13% greater in volume compared to cells exposed to p H 6.7. It is therefore possible that variation in glycolysis rates reported in Figure (3.12) is partly due to this change in cell volume. A n increase in cell diameter with p H has also been reported for hematopoietic cel ls 2 3 . Analysis o f cells by flow cytometry for detection o f early apoptotic cells (Annexin-V+) revealed that, during the first 48 h, exposure to acidic or alkaline p H induced apoptosis in cells (Figure 3.14). There was no apparent increase in the number o f floating (dead) cells or cellular debris found in the supernatant collected before cells were harvested for flow cytometry. However, there was an apparent adaptation with time as there was no significant difference in Annexin-V positive cells exposed to these different p H conditions for another 48 h. It is also plausible that under extreme p H conditions, cells that acquire tolerance to low or high p H were being selected. A phenotypic analysis o f mouse E S C was also done to determine the expression o f Oct-4, a key transcription factor highly expressed by these cells. There was a decline in number of Oct-4 positive cells at p H 6.7 but only after 96 h of exposure to this condition. This result confirmed our 132 previously reported observation that Oct-4 is a lagging indicator o f loss o f pluripotency o f mouse E S C 5 2 . The influence o f p H on m E S C responses is not permanent and once the cells exposed to extreme p H conditions are returned to normal conditions, they gradually recover their growth rate as wel l as E B formation potential. This demonstration of reversibility o f p H effects on m E S C responses implies that perhaps cells capable o f forming E B s are not permanently lost under extreme p H conditions. However, even the short term influence o f p H on m E S C responses is still significant from the subsequent differentiation point o f view as it is quite possible that cells exposed to adverse p H conditions may not generate fully functional differentiated progeny. The short term p H effects are also important from the point o f view o f expansion of m E S C in culture, as adverse p H conditions both reduce the growth rate as well as final cell number generated in those cultures. 133 3.5 Conclusions The conventional m E S C maintenance protocol of daily medium exchange and passaging cells every other day resulted in glucose and glutamine concentrations that remained within ranges that should have little influence on the growth rate or E B yields. Similarly, ammonium was not found in m E S C cultures at the elevated concentrations that would influence the m E S C responses. However, the p H and osmolality did vary considerably during the conventional passaging protocol. Based on the dose-response experiments, it was found that m E S C growth rates did not significantly decrease for relatively broad p H and osmolality ranges while the E B yield was more sensitive to p H and osmolality variations. Hence to maximize E B formation from m E S C lines, the initial medium p H should be maintained close to 7.3 and the osmolality within the range o f 300 - 335 mOsm/kg. Maximiz ing E B yield is important as it is generally the first step in differentiation protocols. The influence o f p H on m E S C responses does not appear to be permanent and, once cells exposed to extreme p H conditions are returned to p H 7.3, they gradually restore their growth rate as wel l as E B forming ability. These studies indicate that, for culturing h E S C , particular attention also be paid to determining the optimal p H and osmolality conditions. 134 Table 3.1 Average initial, final and logarithmic averaged pH values that R l cells were exposed to during the final 24 h (24 - 48 h) period in culture during pH dose-response experiments reported in Figure (3.9). Values shown are drawn from a subset of five experiments. Average Initial p H ± SD Average Final p H ± SD (at 48 h) Logarithmic Average p H during 24-48 h period 6.72 ± 0.02 6.80 ± 0 . 1 6.72 6.86 ± 0 . 0 3 6.80 ± 0 . 0 7 6.83 7.01 ± 0.02 6.84 ± 0.04 6.92 7.15 ± 0 . 0 5 6.97 ± 0.06 7.06 7.30 ± 0.04 7.05 ± 0.03 7.17 7.45 ± 0.03 7.24 ± 0.04 7.34 7.62 ± 0 . 0 4 7.39 ± 0 . 0 7 7.5 7.75 ± 0.02 7.75 ± 0.06 7.75 135 Table 3.2 Summary Table of 2-way ANOVA to determine the influence of pH and inoculum growth history on EB yield and growth rate of R l cells. Number of EBs/Initial Cell inoculated Degrees of freedom Sum of Squares Mean Sq F value Pr (>F) p H 4 0.394 0.0986 3.590 0.01* Surface 1 0.168 0.168 6.125 0.0158* (PEF or Gelatin) p H : Surface 4 0.078 0.019 0.712 0.587 Residuals 69 1.895 0.0275 Growth Rate P H 4 0.0058 0.00145 8.574 1.122e-05 * Surface 1 0.0016 0.0016 9.45 0.0030 * (PEF or Gelatin) p H : Surface 4 0.00015 0.000037 0.2161 0.93 Residuals 69 0.012 0.00017 136 Figure 3.1 Pictures of 3-dimensional cellular aggregates that were considered and counted as EBs. 137 9 Figure 3.2 Pictures of cellular aggregates that were not considered as EBs. 138 T 1 1 1 1 ' 1 ' 1 - 0 10 20 30 40 Time (h) Figure 3.3 Metabolic, pH and osmolality profiles of R l mouse ESC grown on irradiated feeders following the conventional protocol of exchanging medium at 24 h and harvesting the cells at 48 h after inoculation in a maintenance medium of pH 7.6. Panel (a) shows glutamine and ammonium profiles, panel (b) depicts pH and osmolality profiles, and, panel (c) shows the glucose and lactate profiles. Values shown are mean ± SEM of three independent experiments. 139 Figure 3.4 Metabolic, pH and osmolality profiles of R l mouse ESC grown on 0.1% gelatin coated dishes following the conventional protocol of exchanging medium at 24 h and harvesting the cells at 48 h after inoculation in maintenance medium of pH 7.6. Panel (a) shows glutamine, panel (b) depicts pH and osmolality profiles and panel (c) shows the glucose and lactate profiles. Values shown are mean ± SEM of three independent experiments. 140 0.9 0.8- 0.7 3 0.6 I 0.5- g 0.4. 1 0 3 5 0.2- 0.1 o.o. 1 1 T" 1 1 I . 1 1 (a) — ~9 i A i 1 10 15 Initial Glucose Cone. (mM) 20 0.00 25 2 4 6 Initial Ammonium Cone. (mM) 2 4 6 8 Initial Glutamine Cone. (mM) - EBs/lnitial Cell - Growth Rate Figure 3.5 Growth rate and mean EBs/initial cell inoculated of mouse ESC line R l as a function of (a) initial glucose concentration (b) initial glutamine concentration and (c) initial ammonium concentration following conventional protocol of exchanging medium 24 h following inoculation and harvesting the cells 48 h after inoculation. The standard maintenance medium contains 20-22 mM glucose, 4 mM glutamine and generally around 1.5 mM ammonium. Values shown are mean ± SEM of seven experiments (* p < 0.05 compared to (a) 20 mM glucose (b) 4 mM glutamine and (c) 1.5 mM ammonium by paired t-test). Figure legend applies to all panels. 141 Figure 3.6 Growth rate and mean EBs/initial cell inoculated of mESC line R l as functions of medium osmolality following conventional protocol of exchanging medium 24 h following inoculation and harvesting the cells 48 h after inoculation. Values shown are mean ± SEM of fifteen experiments (* p < 0.05 compared to osmolality of 300 mOsm/kg by paired t-test). 142 0.08 ~ 0.06 -J —•— EB/initial cell — a — Growth Rate 0.30 h0.25 ho.20 o ra 1-0-15 g LU ho.10 | h0.05 0.00 Initial pH Figure 3.7 Growth rate and mean EBs/initial cell inoculated of R l mouse ESC as functions of initial pH following conventional protocol of exchanging medium, pre-equilibrated at the same pH, 24 h following inoculation and harvesting the cells 48 h after inoculation. Values shown are mean ± SEM of thirteen experiments (* p < 0.05 compared to pH 7.3 by paired t-test). 143 7.8 8.0 Initial pH Figure 3.8 Growth rate and mean % EBs of R l mouse ESC as functions of initial pH following conventional protocol of exchanging medium, pre-equilibrated at the same pH, 24 h following inoculation and harvesting the cells 48 h after inoculation. Values shown are mean ± SEM of thirteen experiments (* p < 0.05 compared to pH 7.3 by paired t-test). 144 CD •4—» CO o 0.05 0.04-J 0.03-^ 0.02 H 0.01 4 0.00 CD o 15 0Q LU c CO 0 Initial pH Figure 3.9 Dose response analysis of growth rate and mean EBs/initial cell inoculated of mESC line R l as functions of initial pH for two passages (96 h total exposure to various pH) following conventional protocol of exchanging medium, pre-equilibrated at the same pH, at 24 and 72 h following inoculation and harvesting the cells 48 and 96 h after inoculation. Values shown are mean ± SEM of seven experiments (* p < 0.05 compared to pH 7.3 by paired t-test). 145 Figure 3.10 Growth rate and mean EBs/initial cell inoculated of mESC line EFC as functions of initial pH following conventional protocol of exchanging medium, pre-equilibrated at the same pH, 24 h following inoculation and harvesting the cells 48 h after inoculation. Values shown are mean ± SEM of five experiments (* p < 0.05 compared to pH 7.3 by paired t-test). 146 0.08 Initial pH (-) Initial p H (.) Figure 3.11 (a) Mean number of EBs obtained per initial cell inoculated as a function of initial pH when the inoculum R l mouse ESC were previously grown on 0.1% gelatin-coated dishes or on irradiated feeders. Two-way ANOVA analyses found significant differences both in growth rate and in mean EB/initial cell as a function of pH as well as previous growth history, (b) Growth rate as a function of initial pH when the inoculum R l mouse ESC were previously grown on 0.1% gelatin- coated dishes or on irradiated feeders. All values shown are mean ± SEM of at least five independent experiments. 147 250 CD O 2200-j _ - _ o E a. 1*150 0 i—1 , i , (a) Glu uptake rate -•— Lac prod rate .6 6.8 7.0 7.2 7.4 7.6 Initial pH (-) 2.0 1.8- 1.6- ri.4H c CD ? 1 . 2 -3= cu o o CD >- 1.0- 0.8- 0.6- 0.4 ~i—•—r t—•—r —•—Yield Coefficient —±— Gin Uptake rate T 1 • 1 1 1 1 J 1 1 6.6 6.8 7.0 7.2 7.4 7.6 Initial pH (-) Figure 3.12 (a) Cell specific average glucose uptake rate (sGUR) and lactate production rate (sLPR) as functions of initial pH of mESC line R l used in the pH dose-response analyses depicted in Figure (3.5). (b) Lactate to glucose yield coefficient ( Y ^ g i , , ) and average glutamine uptake rate (sGlnUR) as functions of initial pH of R l mouse ESC used in the pH dose-response analyses depicted in Figure (3.5) Values shown are mean ± SEM of a subset of seven experiments. 148 16 * -1 1 1 < 1 1 1 1 1 < 1 • r~ 6.6 6.8 7.0 7.2 7.4 7.6 7.8 Initial pH (-) Figure 3.13 Variation of average diameters of R l mouse ESC as a function of initial pH (at initial osmolality of 300 mOsm/kg for cultures with initial pH of 6.7 - 7.3 and initial osmolality of 320 - 345 mOsm/kg for cultures with initial pH 7.45 - 7.75) used in the dose-response analyses depicted in Figures 3.5 and 3.6. Values shown are mean ± SEM of a subset often independent experiments. (* p < 0.05 compared to pH 7.3 by paired t-test). 149 Initial pH (-) Initial pH (-) Figure 3.14 (a) Variation of %Oct-4 positive cells from mESC line R l as a function of initial pH during the dose-response experiments carried out for 48 and 96 h. (b) Variation of % Annexin-V positive cells from mESC line R l as a function of initial pH during the dose-response experiments carried out for 48 and 96 h. Values shown are mean ± SEM of five experiments. (* p < 0.05 compared to pH 73 by paired t-test). 150 7.8-, 7.7 A 7.6-1 x Q. : E 7.5-1 7.4 A 7.3 A 7.75_48h 7.75 24h 7.3-1 7.2 7.1-1 x Q. 1 7.0 6.9 6.8 • 6.7-1 i 1 i 1 i 1 i 1 i 1 i • i • i 0 24 48 72 96 120144168 6.7_48h 6.7 24h i—1—i—1—i 1 i • i •—i—'—i—>—r 0 24 48 72 96 120144168 Time (h) Time (h) Figure 3.15 Reversibility of pH effect on responses of R l cells. Variation of initial pH (6.7 and 7.75) as a function of time. 151 Figure 3.16 Reversibility of pH effect on responses of R l cells. Average normalized growth rates of R l cells as functions of pH and duration of exposure. Cells were harvested after 48 h of setting up the experiment and cells were further grown for 48 or 96 h under pH 7.3 conditions. Values shown are mean ± SEM of an experiment carried out in triplicate. 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Physiol. 1985, 248, (3), C337-C347. 159 4 Basal Medium Composition and Serum or Serum Replacement Concentration Modulate Responses of Murine Embryonic Stem Cells 0 4.1 Introduction Stem cells are defined as cells that, at the single-cell level, are capable o f both self- renewal and differentiation to specialized cell types'. Pluripotent embryonic stem cells (ESC) have been derived from the inner cell mass o f blastocyst stage embryos o f murine, porcine, primate as well as human sources. Human embryonic stem cells (hESC) exhibit indefinite replicative and developmental potential to differentiate into derivatives o f al l three germ layers even after prolonged culture. Whereas hESC-derived cells have tremendous potential in many experimental and therapeutic applications, they have a doubling time of the order o f 35-40 h 2 , 3 and extensive cell expansion w i l l be required to meet the requirements for human therapies. Maintaining a large pool o f uncommitted stem cells without compromising their developmental potential in culture represents a great bioprocessing challenge. Murine E S C (mESC) can self-renew in vitro with leukemia inhibitory factor (LIF) in the presence of serum or mouse feeder layer cells, to obtain daughter cells that maintain their multilineage potential 4 ' 5. Similarly, derivation and propagation o f h E S C and establishment of permanent lines were first carried out in the presence o f serum and mouse embryonic fibroblasts as feeder cells 6 ' 7 . When m E S C are maintained i n feeder- free conditions, the fraction of undifferentiated cells begins to decline rapidly with consecutive passaging ' , and cells with a non-ES cell morphology quickly arise in culture, despite the presence o f LEF. Thus, additional factors and/or the cell-cell contact provided by the feeders appear to be required to fully support the self-renewal o f m E S C . c A version of this chapter will be submitted for publication. Chaudhry, M.A.S . , Bowen, B.D. , Eaves, C.J. and Piret, J .M. (2006) Basal Medium Composition and Serum or Serum Replacement Concentration Modulate Responses of Murine Embryonic Stem Cells 160 Removal o f both L IF and feeders cause m E S C to spontaneously differentiate, following a reproducible pattern o f development that in many ways recapitulates early embryogenesis, including the formation o f a complex three-dimensional architecture wherein cell-cell and cell-matrix interactions are thought to support the development o f the three embryonic germ layers 1 0 ' n . A s m E S C differentiate in suspension or semi-solid culture, they typically form three-dimensional aggregates called embryoid bodies (EBs). Over time, these E B s increase in cell number and complexity as cells from the three germ layers are formed. Generation o f a wide variety o f apparent cell types - cardiac myocytes, hematopoietic cells, neurons, pancreatic islet cells, etc. - from m E S C has been 12 15 reported . In an ideal scenario, differentiation of E S C could be directed to a pure population o f the desired cell type. Culture conditions have been described that exclusively permit the formation of neural progenitor cells from m E S C , albeit at very low cell frequencies16. In most cases, however, the knowledge to precisely control the mouse or human E S C fate decisions is still far from complete. Consequently, the most robust method for generating the most differentiated cell types is through the embryoid body formation step. Standard methods of E B formation include hanging drop, l iquid suspension and methylcellulose based semi-solid cultures. These culture systems are thought to maintain a balance between the E S C aggregation necessary for E B formation and preventing E B agglomeration for efficient cell growth and differentiation 1 7. The media commonly used in mammalian cell culture provide essential nutrients that are incorporated into dividing cells, such as amino acids, fatty acids, sugars, ions, trace elements, vitamins and co-factors, as well as molecules that are necessary for maintaining a proper chemical environment for the cells. Most media (e.g., minimal essential medium, Dulbecco's modified Eagle's medium ( D M E M ) ) were developed specifically for use with serum supplementation and high-density growth of c e l l s 1 8 ' 1 9 . In contrast, Ham's nutrient mixtures F12 and F10 were tailored towards growing specific cells (e.g., C H O , fibroblasts) at low density with a minimal amount o f undefined protein added so that the effects of different nutrient components could be s tud ied 2 0 , 2 1 . The D M E M : F 1 2 (1:1 vol/vol) medium was originally developed for growing cells in defined serum-free • • 22 conditions and can support high cell densities with serum or defined growth factor 161 supplements in serum-free media. These supplements, depending on the cell type and expected outcome of the culture, include cytokines, buffers, antioxidants, surfactants and shear protectants. The theoretical basis for medium development 2 3 as wel l as the protocols for developing serum-free m e d i a 2 1 ' 2 4 - 2 6 have been reviewed. In addition to serum or serum substitutes, the basal medium formulation can also profoundly influence the expression of differentiated functions as has been demonstrated for the case o f primary hepatocytes 2 7" 3 2 and the human intestinal cell line C a c o - 2 3 3 ' 3 4 . It is wel l known that the conditions to which pre-implantation stage mammalian embryos are exposed in culture can have a profound effect on their subsequent development including physiology 3 5 , metabolism 3 6 , 3 7 and v iab i l i ty 3 8 , 3 9 . Inappropriate media formulations, such as those lacking amino acids, have been shown to have profound negative effects on many aspects o f embryo physiology. This was highlighted in a series of studies examining the rate of m R N A synthesis in pre-implantation embryos cultured in a medium at low (85 m M ) or at high (125 m M ) sodium chloride concentrations. It was determined that m R N A synthesis was reduced by 20% at the higher salt concentration and, furthermore, the resultant m R N A was less stable 4 0. It was subsequently shown that the expression o f nine genes in a medium containing amino acids was closer to that o f in Wvo-developed embryos than blastocysts developed in a simple medium lacking amino acids 4 1 . Micro-array technology has demonstrated that, out of a total of 22690 transcripts and variants assessed, on average only 39% were expressed by in vz'vo-developed mouse blastocysts. Whereas blastocysts cultured in a medium lacking amino acids had aberrant expression o f 114 genes compared with in v/'vo-developed embryos, those blastocysts cultured in an amino acid-containing medium had only 29 genes misexpressed 4 2. Similarly, the imprinting status of the H19 gene was shown to be aberrant in a medium lacking amino acids compared to an optimized medium containing amino acids 4 3 . A s has been extensively reviewed 4 4 , 4 5 , serum functions as a source o f hormones and essential nutrients, and alters the physiological/physiochemical properties o f the medium. The use o f serum to culture human cells for clinical applications is highly undesirable due to the risk o f exposure to various pathogens. The general culture conditions for m E S C 162 maintenance are well established. The most critical component o f an E S C maintenance medium is serum. Serum can be highly variable from lot to lot, and can contain a variety o f growth factors and other undefined components that can affect in vitro E S C plating efficiency, growth and can also promote differentiation o f m E S C . Hence only pre- screened batches of serum are used to maintain m E S C in an undifferentiated state. Pre- screening serum, however, is both cost and labor intensive and considerably increases the overall cost o f m E S C culture. A commercial serum-free formulation, Knockout™ serum replacement (SR), has found widespread use both in mouse and human E S C culture as it directly replaces serum and maintains E S C in an undifferentiated state. The product is a better-defined (although the complete formulation is not disclosed) growth supplement and results in less E S C spontaneous differentiation. The manufacturer (Invitrogen, Carlsbad, C A ) recommends it to be used for growth and maintenance o f undifferentiated E S C at the same concentration as serum is used, generally 15%. Compared to hematopoietic stem cells, for which quantitative functional assays o f potential have been defined and validated, relatively less wel l developed assays exist for mouse or human E S C . Self-renewal can be measured by the ability o f E S C to proliferate in culture while maintaining an undifferentiated colony morphology and teratoma formation capacity. The most rigorous in vivo assay to establish the functionality o f cultured m E S C is blastocyst injection followed by the measurement of their ability to give rise to chimeric mice. This assay provides proof that there has been E S C contributions to all adult tissue, including germ cel ls 4 6 . Two in vitro assays have been used extensively as surrogates for chimera formation when testing culture reagents or examining the consequences o f genetic manipulation. The colony-forming cell (CFC) assay is used to determine the plating efficiency of E S C populations under various conditions and thus may be considered indicative o f self-renewal potential. Formation o f embryoid bodies (EBs) can be performed at a clonal level in vitro and reflects multilineage differentiation potential 4 7. Assessment o f the pluripotency of mouse E S C has also relied on the expression of selected molecular markers. These have included alkaline phosphatase, the P O U transcription factor Oct-4, and the stage-specific embryonic antigen 1 (SSEA-1) . We have recently demonstrated that the embryoid body 163 formation assay more rapidly detects decreasing m E S C numbers than phenotypic characterization by Oct-4 and SSEA-1 under differentiating culture conditions 4 8 . In the present study, the murine E S cell lines, R l and E F C , have been used as model systems to study the influence o f basal medium composition and concentration o f serum or S R on proliferation and maintenance o f E B forming ability. Since E B formation is generally considered as the first step in most m E S C differentiation protocols, it is important to study those culture variables that would influence the E B yield from differentiating m E S C . In addition, defining the ranges of different variables that maximize the E B yield would accelerate bioprocess R & D in this field. We have examined the effect o f basal medium composition along with serum or S R concentration in m E S C cultures following the conventional passaging protocol and show that growth rate, E B formation potential and metabolism are greatly influenced by these variables. In addition, dose-response experiments were carried out to determine the optimum serum or S R concentration when two different basal media are present. These studies led to a series o f time course experiments to show that the widely-used S R concentration o f 15% is not optimal especially when an enriched basal medium such as D M E M : F 1 2 is employed. 164 4.2 Materials and Methods A l l reagents were obtained from StemCell Technologies Inc. [STI], Vancouver, B C , unless otherwise indicated. 4.2.1 Growth of Mouse Embryonic Fibroblasts (MEF) M E F were cultured as described in Chapter 3 section 3.2.1. 4.2.2 Growth of Mouse Embryonic Stem Cells (mESC) The m E S C were thawed and cultured for 2 passages on irradiated feeders in the maintenance medium ( D M E M + 15% serum). To prepare the cells for exposure to serum or S R concentrations, they were harvested by trypsinization, resuspended in E S C maintenance medium, and preplated on tissue culture plates for 15-20 minutes at 37°C, 5% C O 2 , in order to deplete contaminating M E F . A t the end o f this pre-plating step, the non-adherent and loosely adherent E S C were collected by gently washing the surface o f the tissue culture plate. The cells were then centrifuged, resuspended in E S C maintenance medium and viable cell numbers were counted on a hemocytometer with trypan blue dye or on an automated cell counter (Cedex, Innovative Directions, Pinole, C A ) to distinguish live from dead cells. The frequency o f contaminating M E F in the undifferentiated (day 0) E S C samples was estimated to be less than 0.2% based on cell size during counting ( M E F cells are easily distinguishable from m E S C as they are bigger in size (generally >16 urn in diameter) and appear to have irregular morphology). A l l cultures for the serum or S R experiments initially contained 2 x 10 4 cells/cm 2 in 4 m L o f medium in a 60-mm gelatinized dish. The conditioned medium from each o f these cultures was collected after 24 hours and replaced with fresh medium at the same serum or S R concentration. The following day, the cultures were harvested by trypsinization and the cell concentrations were determined as described above. 165 4.2.3 Determination of Growth Rate The growth rate o f both m E S C lines was determined as described in Chapter 3 section 3.2.3. (Eq. 3.1) 4.2.4 Preparation of Media for Serum or Serum Replacement Dose- Response Experiments The E S maintenance medium was prepared as described above using either D M E M or D M E M : F 1 2 (Invitrogen Life Technologies, Burlington, ON) as the basal medium. Serum or S R was added at three different concentration levels: 1.67, 5 or 15% (v/v). To ensure that the p H values of both the D M E M - and DMEM:F12-based media are the same, a powdered D M E M formulation without glucose, glutamine, sodium bicarbonate, sodium pyruvate and phenol red (Sigma-Aldrich, Oakville, O N , Cat #D5030) was used. The sodium bicarbonate concentration was then adjusted to obtain the desired p H since the equilibrium bicarbonate concentration ([HC03~]; m M ) at 37°C can be related to the partial pressure o f CO2 (pC02 , mmHg) in the gas phase and the medium p H via the following equation 4 9: log[HC03-] = p H + log[pC02] - 7.54. (4.1) Both the D M E M - and DMEM:F12-based media were adjusted to equilibrate at p H 7.3 i n a 5% CO2 atmosphere. When D M E M was used as the basal medium, the average osmolality of the ES maintenance medium containing 15% ES qualified serum was 334±5 mOsm/kg and, with 15% SR, it was 328±7 mOsm/kg. Wi th D M E M : F 1 2 as the basal medium, the 15% serum-containing medium had an average osmolality o f 327±4 mOsm/kg while the medium containing 15% S R had an average osmolality of 322±5 mOsm/kg. On average, the changes in osmolality due to the lower concentrations o f serum or S R used were within 10% of the average osmolality o f the E S maintenance media with 15% serum or SR. Based on the osmolality dose-response experiments carried out separately (data shown in Chapter 3), such small changes in medium osmolality should not affect the proliferation and E B forming ability o f either the R l or 166 E F C cells. Both the serum and SR experiments were carried out on gelatinized 60-mm tissue culture dishes (Sarstedt, Montreal, PQ). 4.2.5 Measurement of Glucose, Lactate and Glutamine These measurements were taken and metablic rates were determined as described in Chapter 3, section 3.2.5. 4.2.6 Preparation of mESC for Dose-Response Experiments m E S C lines were maintained and used for these experiments as described in Chapter 3, section 3.2.6. 4.2.7 Embryoid Body Formation Assay The E B assays were initiated exactly as described in Chapter 3, section 3.2.7. 4.2.8 Statistics Data are reported as mean ± standard error of the mean (SEM) , unless otherwise noted. Statistical comparisons were performed using a two-tailed paired Student's t-test. A n asterisk (*) was used to denote statistical significance (p < 0.05). 167 4.3 Results The influence o f serum, S R and basal medium ( D M E M or D M E M : F 1 2 ) on the 48 h proliferation response o f both R l and E F C m E S C is shown in Figure (4.1). The E F C cells displayed a higher growth rate compared to the R l cells under all conditions tested. In D M E M , both cell lines exhibited an increase in growth rate as a function o f serum concentration that saturated at approximately the 5% level Figure (4.1a). The growth rate at the 1.67% serum level was significantly (p < 0.05) lower than at the recommended 15% level. A reduced dependence on serum concentration was observed when D M E M : F 1 2 was used as the basal medium (Figure 4.1b). Even 1.67% serum was sufficient to maintain the growth rate of both cell lines within - 8 5 % of the maximum observed with 15% serum. D M E M : F 1 2 supported higher growth rates of both cell lines compared to D M E M at all concentrations o f serum tested. In the dose-response experiments using D M E M with S R (Figure 4.1c), 5% S R again was sufficient for maximal growth, with even a significantly decreased (p < 0.05) growth rate at 15% S R for the E F C cells. The growth rate of both cell lines also decreased slightly at 15% S R with D M E M : F 1 2 as the basal medium (Figure 4.Id). The ability o f both R l and E F C m E S C to form E B s (represented by the E B yield which is defined as the mean number of EBs/init ial cell inoculated) was also tested for the two basal media ( D M E M and D M E M : F 1 2 ) and as a function o f the serum or S R level. The E F C cells had much higher E B forming capacities than R l cells under all conditions tested (Figure 4.2). In D M E M , the R l cells exhibited a serum dose-dependent increase in E B yield while, for the E F C cells, the yield saturated at approximately a 5% serum concentration (Figure 4.2a). The response o f both cell lines to S R dosages with D M E M as the basal medium is, however, quite different and exhibited an apparent high dose inhibition at a 15% S R concentration, e.g., the E B yield o f the E F C cell line showed a decline at this level that was significantly lower (p < 0.05) than at either the 1.67 or 5% S R levels (Figure 4.2c). For both cell lines, 5% S R maximized the E B yield. The results o f the serum dose-response experiments for both the R l and E F C cells in D M E M : F 12 are shown in Figure (4.2b). The E F C cells exhibited a dose-dependent increase in E B 168 formation potential while the response of the R l cells was essentially independent o f the serum concentration used. The E B yields of both cell lines were higher at 1.67% S R i n D M E M : F 12 compared to D M E M (Figures 4.2d vs. 4.2c). The E F C cells had a decreased ability to form E B s with increasing S R concentration with D M E M : F 1 2 , with a significantly (p < 0.05) lower E B yield at 15% S R compared to either 1.67 or 5% S R (Figure 4.2d). The 1.67% SR maintained the maximal E B forming capacity o f E F C cells with higher concentrations of S R reducing the E B yield. The response of the R l cells under these conditions, on the other hand, was essentially similar to that observed when these cells were exposed to serum with D M E M : F 1 2 . Again as was the case with growth rate, D M E M : F 1 2 , compared to D M E M , supported higher E B yields for both cell lines at all concentrations o f serum tested. The metabolism of both cell lines is influenced depending on whether serum or S R is used in conjunction with D M E M or D M E M : F 1 2 as the basal medium. The changes in the cell specific glucose uptake rate (sGUR) o f both cell lines when exposed to serum or S R with the two different basal media are shown in Figure (4.3). Contrary to the growth rate and E B formation data shown in Figures (4.1) and (4.2), R l cells generally display a higher s G U R compared to E F C cells. Figure (4.3a) shows a dose-dependent increase in the s G U R o f R l cells with serum concentration in D M E M that saturates at the 5% serum level. The s G U R of E F C cells, on the other hand, is virtually independent o f the serum concentration used. Both cell lines exhibit almost identical s G U R profiles when serum is used with D M E M : F 1 2 (Figure 4.3b). Under these conditions, the glucose uptake rates are essentially independent of the serum concentration. When S R is present with D M E M , both cell lines show a dose-dependent increase in s G U R that appears to saturate at the 5% level as shown in Figure (4.3c). The response of both cell lines to S R with D M E M : F 1 2 is similar to serum with D M E M : F 1 2 - s G U R is independent o f the S R concentration used (Figure 4.3d). The cell-specific lactate production rates (sLPR) o f both cell lines when exposed to various concentrations o f serum or S R with D M E M or D M E M : F12 as the basal medium are shown in Figure (4.4). Since the R l cells displayed higher s G U R under all conditions 169 tested as shown in Figure (4.3), they also exhibited higher s L P R values compared to the E F C cells. The R l cells show an almost linear increase in s L P R as a function o f serum concentration when D M E M is used as the basal medium, whereas the response o f the E F C cells under these conditions is a decline in s L P R with increasing serum concentration (Figure 4.4a). Compared to Figure (4.3b) where both cell lines show almost identical s G U R responses, when serum is used in the presence of D M E M : F 1 2 , the s L P R response is quite different (Figure 4.4b). The R l cells show a decline in s L P R as a function of serum concentration that appears to stabilize around 170 pmol/10 6 cell-h. The s L P R o f the E F C cells, on the other hand, is unaffected by the serum concentration used and remains constant around 75 pmol/10 6 cell-h. In the presence o f S R with D M E M as the basal medium, both cell lines display s L P R profiles that are essentially independent o f the S R concentration (Figure 4.4c). When S R is used in conjunction with D M E M : F 1 2 , the s L P R of the R l cells exhibited an apparent maximum at 5% SR, with lower s L P R values at both lower and higher SR concentrations. For the E F C cells, s L P R appeared to be almost independent o f the S R concentration (Figure 4.4d). Glutamine is used by most cell lines as a source o f nitrogen and energy. The cell specific glutamine uptake rate (sGlnUR) of both the R l and E F C cells is depicted in Figure (4.5). In the presence o f serum with D M E M , the R l cells showed an apparent linear increase in s G l n U R with serum concentration while the sGlnUR of the E F C cells was almost constant and independent of the serum concentration (Figure 4.5a). When D M E M : F 1 2 is employed as the basal medium with serum, both cell lines exhibit s G l n U R values that are virtually independent o f the serum concentration (Figure 4.5b). When S R is used, the s G l n U R o f both cell lines is essentially independent o f the SR concentration regardless o f whether D M E M or D M E M : F 1 2 is present as the basal medium. However, the magnitude o f s G l n U R is quite different for the two basal media. For the R l cells in D M E M , s G l n U R is around 12 pmol/10 6 cell-h while, for the E F C cells, it is around 6 pmol/10 6 cell-h (Figure 4.5c). However, in D M E M : F 1 2 , the sGlnUR of both cell lines is around 5 pmol/10 6 cell-h (Figure 4.5d). 170 The finding that a higher concentration (15%) of S R can be inhibitory for both the cell proliferation and E B forming ability o f both the R l and E F C cells was then further investigated. Three different SR lots (not previously used in our experiments) were acquired and the R l cells were grown with 5% or 15% S R in either D M E M or D M E M :F 12 for three consecutive passages to determine to what extent the observed responses might be sustained up to 144 h and in different S R lots. In D M E M , there was a gradual loss o f E B forming ability as wel l as a decline in growth rate o f R l cells with consecutive passaging on gelatin, with all three S R lots behaving similarly (Figure 4.6a). Compared to the results obtained with 5% SR, there was a decline in both the growth rate and the E B yield o f R l cells when 15% S R was used (Figure 4.6b). On average, there was almost a 2-fold or more decline in the E B forming capacity o f R l cells exposed to 15% S R compared to 5% SR. The growth rate of R l cells exposed to 15% S R was also lower by 22 - 25% (on average) compared to cells exposed to 5% SR. However, as was the case with 5% SR, the three different lots of S R behaved similarly at the 15% level. It was therefore clear that high concentrations of S R are inhibitory to both proliferation and E B formation in m E S C lines. There was relatively little lot-to-lot variability. In D M E M : F 1 2 with 5% S R (Figure 4.6c), all three SR lots behaved similarly in terms o f both the E B yield and growth rate o f the R l cells throughout the time course and there was a slow decline in both the E B yield and the growth rate of the R l cells as a function o f time due to consecutive passaging o f these cells on gelatin. A s shown in Figure (4.6d), when the R l cells were exposed to 15% SR in D M E M : F 1 2 , there was on average a 2.5- fold or more decline in their E B forming capacity (compared to 5% SR) while the growth rate o f these cells also declined by ~25%. However, the results obtained with all three S R lots were statistically indistinguishable at both the 5% and 15% S R levels. In DMEM- .F12 with both 5% and 15% SR, the growth rate o f the R l cells declined significantly (p < 0.05) at 144 h compared to 48 h. 171 4.4 Discussion Derivation and propagation of m E S C have generally been carried out in the presence o f fetal bovine serum (FBS) with mouse embryonic fibroblasts as feeder cells. Considerable attention is devoted to screening serum lots or purchasing pre-screened ESC-qualified serum prior to use because of lot-to-lot serum variability as wel l as its ability to maintain the m E S C in an undifferentiated state. Serum is a very expensive component added to E S C maintenance media and, hence, a reduction in serum concentration while maintaining the pluripotent potential of m E S C would be highly desirable. Recently, S R has increasingly been used in both mouse and human E S C cultures to maintain these stem cells in an undifferentiated state. It is recommended that the SR be added to the medium at the same concentration as serum is generally used, i.e., 15% for m E S C . The results o f this study indicate that SR actually provides a stronger growth stimulatory effect at lower concentrations (1.67% and 5%) than serum at similar levels when D M E M is used as the basal medium. Similarly, E B yields are higher at lower (1.67% and 5%) S R concentrations compared to similar serum levels in D M E M . S R therefore appears to provide higher levels of factors (than serum) that stimulate higher growth rates and increase the E B yields o f both the R l and E F C cells. It would be interesting to perform cell cycle analysis o f m E S C grown in 5% S R and compare them with cells grown in either 5% or 15% serum to see i f the stimulatory effect o f S R actually reduces the overall cell cycle time by reducing the duration o f the gap ( G l or G2) phases or i f it affects the survival of the cells. The results o f this study also demonstrate the importance o f basal medium selection for the maintenance o f m E S C in culture. While high glucose D M E M is the conventional basal medium used for m E S C , the results presented here make a strong case for D M E M :F 12 being a superior basal medium, in most cases increasing growth rate and E B yields. D M E M and Ham's F12 are often mixed to combine benefits from each formulation. For example, F12 provides small amounts o f trace metals (copper and zinc) and some non-essential amino acids not present in D M E M . F12 also contains 172 hypoxanthine, linoleic acid, lipoic acid and thymidine that are absent from D M E M . D M E M has generally higher levels of vitamins. Hence, being an enriched medium, D M E M : F 1 2 likely provides more of the components that cells require to grow and, hence, is able to reduce serum or S R dependence. Serum replacement, on the other hand, is a propritory supplement which consists of lipid-rich bovine serum albumin or albumin substitute (Albumax-I) and one or more ingredients selected from the following groups: amino acids, vitamins, antioxidants, insulin or insulin substitute, collagen precursors and trace elements. Since both D M E M : F 1 2 and S R have many components in common, adding 15% S R to D M E M : F 1 2 results in high-dose inhibition effect on both the proliferation and E B forming ability o f m E S C . L ike growth rate and E B forming capacity, the metabolism of both cell lines was influenced by the different basal media used with serum or SR. The cell specific glucose uptake (Figure 4.3), lactate production (Figure 4.4) and glutamine uptake (Figure 4.5) rates o f both cell lines varied considerably depending on the basal medium present with serum or SR. Unlike the growth rate and EBs/ini t ial cell , the R l cells, under all conditions tested, had higher rates of all three of these metabolic rates compared to the E F C cells. Variations in s G U R as a function of serum concentration were similar to H e L a cells that have been reported to exhibit a decrease in s G U R as a function o f serum concentration 5 0 while increasing concentrations of serum had no effect on glucose consumption for hybridoma cel ls 5 1 . Neither cell line exhibited a dependence o f s G U R on S R concentration regardless of whether D M E M or D M E M : F 1 2 was present as the basal medium. It is also interesting to note that in D M E M , the s G U R values o f R l cells were higher than those o f E F C cells both in the presence of serum and SR. But, with D M E M : F 1 2 , both cell lines approach similar values of s G U R with either serum or SR. s L P R (Figure 4.4) and sGlnUR (Figure 4.5) show trends similar to s G U R for both cell lines. L ike serum, S R can have lot-to-lot variability problems. For instance, the osmolality o f S R has been reported to influence the successful derivation o f h E S C lines (e.g. Mel ton lab h E S C culture protocol, see footnote on final page in the H U E S manual accessed at: 173 http:/ /www.mcb.harw In most cases, the recommended 15% S R levels resulted in sub-maximal growth rates and E B yields (especially for E F C in D M E M : F 1 2 ) . It may be that by optimizing the level o f S R added this apparent high dose inhibition effect could be avoided and the actual variability o f optimized S R additions reduced. Wi th S R at levels such as 1.67 or 5%, instead o f 15%, the expenses o f these cultures would also be greatly decreased. h E S C are now routinely cultured in D M E M : F 1 2 with 20% SR. It would be interesting to determine i f lower S R concentrations could also sustain h E S C in an undifferentiated state over multiple passages. However, the high dose inhibition effect of S R observed with m E S C might not be observed with h E S C as they are normally cultured on M E F feeders and the feeder "conditioning" of the medium could lower the concentration o f components that caused inhibition to m E S C . In an ideal scenario, both mouse and human E S C should be cultured in completely defined serum-free conditions. In order to develop a serum-free medium, it would be advisable to perform experiments comparing new serum-free media to those supplemented with serum or S R at optimized levels (e.g. 5%) so as to adjust the concentrations o f the serum-free components with a more appropriate positive control. 174 4.5 Conclusions The results presented in this chapter highlight the fact that selection o f the basal medium and the supplements can have a profound impact on the maintenance o f the proliferation and E B formation potentials o f m E S C . The growth rate o f both R l and E F C cells show dependence on serum concentration when D M E M is used as a basal medium. However, this serum dependence can partially be removed by using an enriched basal medium such as D M E M : F 1 2 . S R at 5% levels seems to optimally maintain both proliferation and E B forming capacity o f both cell lines and higher (15%) concentration appears to be inhibitory to both growth and E B yields of both cell lines. S R at 5% levels also maintains higher growth rate and E B yields than serum with D M E M thereby showing that it probably provides components o f similar nature to cells that are also present i n D M E M : F 1 2 . Hence a combination o f high S R concentration and D M E M : F 1 2 appear to cause a high dose inhibition to both growth rate and E B yields of both R l and E F C cells. Metabolism o f cells is also strongly influenced by the basal medium present along with serum or SR. There is apparently no lot-to-lot variability associated with S R and it is the high concentration being recommended to be used in m E S C culture that causes problems. 175 0.06 5 o O <D I 0.00 0.06 0.05 0.04 0.03 0.02 0.01 0.00 (a) 0 5 10 15 Serum Concentration (%) (b) * I I — ' — 1 i 1 I i 1 0 1 5 10 15 0.06-r 0.05- 0.04- a: 0.03- G ro w  0.02- 'ag e 0.01- Av ei  0.00- 0 5 10 15 Serum Replacement Concentration (%) Serum Concentration (%) 0 5 10 15 Serum Replacement Concentration (%) Figure 4.1 Average growth rate observed when R l (open symbols) and E F C (closed symbols) cells were inoculated in the presence of (a) serum with D M E M as the basal medium, (b) serum with DMEM:F12 as the basal medium, (c) serum replacement with D M E M as the basal medium, and (d) serum replacement with DMEM:F12 as the basal medium. Values shown are mean ±SEM of six independent experiments. (* p < 0.05 compared to 15% serum or SR by paired t-test). 176 0 5 10 15 Serum Concentration (%) =£ 0.5 tn 00 Wfj.08 ro0.06 20.04 0.02 0.00 0 5 10 15 Serum Replacement Concentration (%) o 5 10 15 Serum Concentration (%) 0 5 10 15 Serum Replacement Concentration (%) Figure 4.2 Mean number of EBs obtained per initial cell inoculated when R l (open symbols) and E F C (closed symbols) cells were inoculated in the presence of (a) serum with D M E M as the basal medium, (b) serum with DMEM:F12 as basal the medium, (c) serum replacement with D M E M as the basal medium, and (d) serum replacement with DMEM:F12 as the basal medium. Values shown are mean ±SEM of six independent experiments. (* p < 0.05 compared to 15% serum or SR by paired t- test) 177 —150 S125-I CO O ^1 o E oT Z> O </) c CD CD (a) [ * / r 1 [ i T ] i 150 -I ce ll 125 CO o 100 o E 75- cc 50 CD co c 25- CD CD 0 5 10 15 Serum Concentration (%) 5 10 15 Serum Concentration (%) 5 10 15 Serum Replacement Concentration (%) (d) —— I 5 10 15 Serum Replacement Concentration (%) Figure 4.3 Cell specific glucose uptake rate (sGUR) obtained when R l (open symbols) and EFC (closed symbols) cells were inoculated in the presence of (a) serum with D M E M as the basal medium, (b) serum with DMEM:F12 as the basal medium, (c) serum replacement with D M E M as the basal medium, and (d) serum replacement with DMEM:F12 as the basal medium. Values shown are mean ±SEM of six independent experiments. (* p < 0.05 compared to 15% serum or SR by paired t- test). 178 350 0 5 10 15 Serum Replacement Concentration (%) 10 15 i Concentration (%) 0 5 10 15 Serum Replacement Concentration (%) Figure 4.4 Cell specific lactate production rate (sLPR) obtained when R l (open symbols) and E F C (closed symbols) cells were inoculated in the presence of (a) serum with D M E M as the basal medium, (b) serum with DMEM:F12 as the basal medium, (c) serum replacement with D M E M as the basal medium, and (d) serum replacement with DMEM:F12 as the basal medium. Values shown are mean ±SEM of six independent experiments. (* p < 0.05 compared to 15% serum or SR by paired t-test). 179 0 5 10 15 Serum Replacement Concentration (%) 0 5 10 15 Serum Concentration (%) 0 5 10 15 Serum Replacement Concentration (%) Figure 4.5 Cell specific glutamine uptake rate (sGlnUR) obtained when R l (open symbols) and EFC (closed symbols) cells were inoculated in the presence of (a) serum with D M E M as the basal medium, (b) serum with DMEM:F12 as the basal medium, (c) serum replacement with D M E M as the basal medium, and (d) serum replacement with DMEM:F12 as the basal medium. Values shown are mean ±SEM of six independent experiments. (* p < 0.05 compared to 15% serum or SR by paired t-test). 180 100 Time (h) Figure 4.6 Mean number of EBs obtained per initial cell inoculated and growth rate of R l cells when inoculated with three different serum replacement lots not used in previous experiments reported in Figures (4.1-4.5) (a) 5% SR with D M E M (b) 15% SR with D M E M (c) 5% SR with DMEM:F12 (d) 15% SR with DMEM:F12. Legend applies to all panels. Values shown are mean ±SEM of two independent duplicated experiments. (* p < 0.05 compared to 15% serum or SR by paired t-test). 181 4.6 References 1. Weissman, I. L . , Stem cells: units o f development, units o f regeneration, and units in evolution. Cell 2000, 100, (1), 157-68. 2. Denning, C ; Allegrucci, C ; Priddle, H . ; Barbadillo-Munoz, M . D . ; Anderson, D . ; Self, T.; Smith, N . M . ; Parkin, T.; Young, L . E . , Common culture conditions for maintenance and cardiomyocyte differentiation of the human embryonic stem cell lines, BG01 and H U E S - 7 . 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This in turn requires that the proliferation and the maintenance of the stem cell potential of these cells be carefully evaluated as a function o f the culture environment to determine the range of variables within which such cultures should be carried out without compromising stem cell properties. For cellular therapy applications, both adult (such as hematopoietic) and embryonic stem cells could be used. A s discussed in Chapter 1, the proliferation and stem cell potential o f hematopoietic stem cells is strongly dependent on the kind and level of cytokines present in the medium. To expedite the bioprocessing research and development in this exciting area, there is a strong need to develop and validate mathematical models that can predict the influence o f the culture environment and especially cytokine utilization and cytokine- dependent cell proliferation. The research presented in this thesis has therefore examined the influence o f environmental variables on proliferation and maintenance o f stem and progenitor cell potentials. Specifically, this project was concerned with developing a quantitative understanding o f the cytokine-dependent proliferation o f cells using hematopoietic cell lines as model systems. Due to various reasons, studies o f the cytokine-dependent differentiation o f hematopoietic cell lines are relatively difficult to perofrom and get any meaningful results. The m E S C , on the other hand, were shown to differentiate readily under the influence of variety o f different stimuli. The proliferation o f m E S C is however, cytokine independent and differentiation is generally carried out using non-cytokine agents such as retinoic acid (a vitamin A derivate) ascorbic acid (vitamin-C), and chemicals such as dimethylsulfoxide and 3-methoxybezene. Hence two m E S C lines were chosen as model systems to develop an understanding o f the impact o f such environmental variables as basal medium composition, p H , osmolality, as wel l as serum and serum substitute concentration on the proliferation and maintenance o f stem cell potential. 187 Mathematical models o f the proliferation and differentiation responses o f stem cells as a function of culture variables can greatly accelerate the bioprocess research and development needed before stem cells can safely be used in clinical settings. In the studies presented in this thesis, empirical mathematical models were developed to predict the proliferation response o f two hematopoietic cell lines as a function o f cytokine concentration when one or more cytokines are simultaneously present (Chapter 2). This modeling approach resulted in the development of a 'competitive model ' to represent the competition reported in the literature between the two cytokines, IL-3 and G M - C S F , for the common receptor (p c) subunit. This model had no new parameters beyond those obtained when the cells were cultured with single cytokines and provided good prediction of the growth rates for both cell lines exposed to combinations of IL-3 and G M - C S F , over a wide range of concentrations. The competitive receptor hypothesis provided a clear rationale for the predictive model developed, suggesting that this modeling approach may be effective for a wide range o f other shared receptor subunit systems. The experiments reported here did not reveal a synergistic interaction between IL-3 and G M - C S F probably for two reasons: (i) at the signal transduction level, since IL-3 and G M - C S F share a common receptor subunit (p c), common signal transduction molecules downstream o f the receptors are likely employed by both cytokines, and (ii) growth rate is being used as the response variable. A synergistic increase in cell proliferation w i l l probably occur only when two cytokines that employ completely different signal transduction mechanisms are employed. Also , since the calculation of growth rate involves a logarithmic transformation, it dampens the effect of any increase in cell concentration and hence a synergistic growth rate response may not be observed. Hematopoietic stem cells are difficult to maintain and expand in culture and little expansion of H S C with a long term in vivo repopulating ability has so far been obtained. Consequently, mouse embryonic stem cells were employed as a model to further study the influence of culture environment on the proliferation and maintenance o f stem cell potential. Biologists working with m E S C agree to the notion that these cells require very careful handling and exposing them to inappropriate culture conditions results in loss o f 188 the pluripotentiality o f these cells. However there are no systematic reports in the literature about the responses of m E S C to various culture conditions when varied over a wide range. In addition, the ranges of culture variables within which m E S C cultures can be performed without adversely affecting the cells has not been established. The studies reported here demonstrate that both the p H and the osmolality o f the medium are important modulators o f m E S C proliferation and E B formation potential. Results o f dose-response experiments revealed that, within 48 h, the growth rate and yield o f E B s decreased substantially when R l or E F C cells were cultured under suboptimal p H or osmolality conditions. A s has been reported for cell lines used in recombinant protein production processes, changes in the metabolism o f both m E S C lines also showed a strong correlation with p H . The average cell diameter was also found to be a strong function of p H . Extreme p H conditions induced apoptosis following 48 h o f exposure, however, an apparent adaptation to these extreme p H values seemed to take place as, following 96 h of exposure to various p H conditions, there was no significant difference in the proportion of cells that were positive for Annexin-V. The p H o f the m E S C maintenance medium is conventionally around 7.3 at the beginning of each culture and the medium is exchanged the next day (i.e., after 24 h). Although not very convenient, another medium exchange could be performed after 36 h o f culture since m E S C start to grow exponentially after 24 h and their metabolism is most active during the last 24 h o f the typically culture cycle where the cells are harvested and passaged after 48 h to avoid overgrowth. Such a procedure would, reduce the p H decrease due to accumulation o f lactic acid. However, the influence o f p H on the proliferation and E B forming capacity o f m E S C is not permanent and these cellular responses were restored to their original values within 96 h o f culture under optimal conditions. In contrast to p H , the osmolality o f the medium could be varied over a relatively wide range (between 300 - 335 mOsm/kg) without significantly influencing the responses of m E S C . In addition to p H and osmolality, serum (or serum replacement) concentration and the basal medium used to prepare the m E S C maintenance medium were found to profoundly influence the m E S C responses. For both cell lines employed in these studies, the basal medium used turned out to be an important factor in determining the responses o f cell 189 lines to different serum or serum replacement concentrations. For instance, a 5% serum concentration appears to be optimal in the presence of D M E M , while even 1.67% serum is sufficient to maintain both proliferation and E B formation potential with D M E M : F 1 2 as the basal medium. Similar results were obtained when serum was replaced with serum replacement. Hence, using an enriched basal medium such as D M E M : F 1 2 is beneficial to the maintenance o f proliferation and stem cell potential o f m E S C . A striking observation o f these studies was that the currently recommended level of S R to be used in preparing m E S C maintenance media (15%) is actually inhibitory to both the proliferation and E B yield of m E S C , and the effect becomes more pronounced when S R is added to D M E M : F 1 2 , where optimal responses were obtained at 1.67% SR. This was further confirmed by conducting independent experiments with R l cells and three new lots o f SR. The results o f these experiments strongly argue that it is the high S R concentration used to culture m E S C that lowers the growth rate and E B formation potential and that lot- to-lot variability is actually minimal. A similar high dose inhibition effect o f S R has not been reported in h E S C cultures as the 'conditioning' of S R containing h E S C medium by M E F s eithers removes or lowers the concentration o f those components that cause this effect. It is therefore quite possible that when m E S C are cultured on M E F s , the high dose inhibition effect o f S R would also be minimal or not detected. It is therefore recommended that to culture m E S C on gelatinized surfaces, the maintenance medium be routinely prepared using D M E M : F 1 2 as the basal medium supplemented either with serum or S R at the 5% level. The medium, after the addition of all the supplements, should be equilibrated to a p H o f around 7.3 in order to optimize both the proliferation and E B forming potential o f m E S C . If S R is used instead o f serum, this w i l l help significantly in reducing the cost of E S C culture media and bioprocess development. Developing conditions that would minimize the spontaneous differentiation o f m E S C that ensue when they are plated on gelatin would also be very valuable. Different extracellular matrix ( E C M ) proteins such as laminin, fibronectin, collagen, etc., or their combinations, could replace gelatin and should be experimentally tested. A s described i n the literature review (Chapter 1), addition of M A P Kinase inhibitor PD05809 has been shown to enhance the derivation o f m E S C lines. It would be valuable to test this 190 compound's influence on the routine maintenance o f different m E S C lines and test whether m E S C grown in its presence have a similar or altered differentiation potential. 5.2 Future Directions The studies reported in this thesis have addressed the issue of the impact o f culture variables on the m E S C responses of proliferation and E B formation potential, but didn't investigate the mechanisms responsible for the observed responses. For example, whether p H influences only the metabolic enzymes or also the other signaling pathways that interact with these metabolic enzymes is currently unknown. Chronic exposure o f cells to suboptimal culture conditions (such as low pH) would invariably invoke the cellular stress response. H o w this stress response interacts with the signaling pathways and the genes influencing the cellular fate decisions o f self-renewal vs. differentiation is also currently unknown and experiments conducted to answer these questions could provide important insights about how the culture environment shapes these cellular responses. Most studies reported in the literature on the influence o f extracellular p H on intracellular p H and the resulting cellular responses were short term and did not consider the issue o f chronic exposure o f cells to suboptimal extracellular p H and its effect on intracellular p H as wel l as other cellular functions. Further, the p H experiments reported in this thesis did not address the mechanistic details of how various cell surface transporters or intracellular signaling molecules are being affected by different p H conditions. Most of the experiments reported in the literature on pHj regulation in somatic cells as wel l as in embryos did not look at the chronic exposure o f cells to low or high p H e values and the resulting influence, for example, on the expression as well as function o f various cell surface exchangers (like N H E 1 or C17HC03 - ) . Furthermore, studies performed with cell lines used in recombinant protein production processes also reported an influence o f p H on metabolism as both glucose uptake and lactate production rates changed as a function 191 of medium p H . None of these studies have reported any mechanistic details about how metabolism is affected by the changing p H conditions. It w i l l be important to perform such mechanistic experiments, especially with h E S C , as a detailed characterization o f how these cells respond intracellularly to imposed chronic p H challenges w i l l shed light on whether it is just the metabolism or also the signaling pathways that regulate cellular fate decisions that are being affected by variations in p H . Even i f p H just influences the metabolism o f E S C , it is quite possible that the metabolic enzymes interact with other signaling pathways operating in the cells and, hence, indirectly influence the proliferation and/or differentiation response. A recent study2 that demonstrated the involvement o f G S K 3 p \ an enzyme with metabolic as well as other diverse functions, in the maintenance of the pluripotentiality of both human and mouse E S C supports this notion. A related aspect is the formal demonstration o f the expression and functional activation of one or more sodium-hydrogen exchanger (NHE) isoforms as well as other exchangers involved in pHj regulation in h E S C under both short- and long-term exposures to suboptimal p H conditions. Our emphasis in the p H experiments reported in this thesis was to assess the m E S C responses when they were exposed to various p H values under the standard conditions used to maintain these cells, i.e., medium exchange after 24 h of culture inoculation and cell harvesting after 48 h. We only investigated the influence o f the variation of different culture variables on the E B yield of these cells as an indirect measure of their self- renewal ability, since a culture that contains a higher proportion of stem cells should give rise to more EBs . We didn't address the question of what might be the influence of the variation of such culture variables as p H , osmolality, as wel l as serum or serum replacement concentration on the cellular functions (e.g. differentiation, cell-cell adhesion or migration) o f these cells. It would therefore be very informative, for example, to subject the mouse or human E S C (previously exposed to various p H or osmolality conditions for the duration o f one passage - 48 h for m E S C and 7 days for hESC) to differentiation by L IF (or bFGF) removal or addition of retinoic acid or D M S O 192 to the culture medium and determine whether the differentiation of these cells is affected compared to a control culture. It is conceivable that lineage commitment decision are influenced by prior exposure to different p H or osmolality conditions, resulting in enhanced or reduced production of one type of cells at the expense o f others. The adhesion molecules, such as E-cadherin or gap junctions, expressed on the cell surface could be influenced by high or low p H conditions to which cells are exposed and hence could influence the plating efficiency o f these cells in maintenance cultures as wel l as in E B and C F C assays. Similarly, the p H or osmolality of the methylcellulose-based differentiation medium was not altered in our experiments. Preparation o f methylcellulose-based media adjusted to various p H or osmolality values could also influence the differentiation of cells and result in enhanced or reduced production o f a particular type o f cell. If this happens, it could be a relatively easy way to produce a desired cell type without going through the trouble of genetic alterations to enrich for that cell type. During the course of this project, dose-response and factorial design experiments o f glucose and glutamine concentrations were also used to study the influence of these variables on the proliferation and E B formation potential of m E S C (Chapter 3). These experiments demonstrated that glutamine is far more important than glucose for the maintenance o f the stem cell potential of m E S C . The m E S C proliferation as wel l as E B formation potential were significantly reduced when glutamine was absent from the medium compared to the control conditions o f 4 m M glutamine even though the medium contained 20 m M of glucose under both conditions. On the other hand, the cellular responses, were not as badly affected when glucose was present at low levels (1.25 m M ) with 4 m M of glutamine in the medium. Hence, glutamine can apparently substitute for glucose deprivation, but the reverse is not true. Two-factor interactions between p H , glucose and glutamine were also significant when a 3x3 factorial experiment was performed (data not shown). It would therefore be important to thoroughly investigate these variables using both mouse and human E S C as models since a better understanding o f their responses to systematic variations o f p H , glucose and glutamine in a factorial 193 type experiment would help in developing an optimized medium for both o f these cell types. The production and maintenance o f m E S C is not o f clinical significance. It w i l l eventually be necessary to apply all that has been learned from m E S C to the human system. The influence o f p H , osmolality and other culture variables on the proliferation and differentiation potential of h E S C has to be studied before these cells can be produced on a large scale under c G M P guidelines. Although human E S C w i l l be used in clinical settings, the use o f m E S C for further experimentation should not be abandoned. Rather, they should be used to investigate issues such as the development of large-scale bioreactor systems. They could also be used to study the influence o f other culture variables, such as temperature, dissolved oxygen and shear rate that have not been addressed in this thesis. Not only are these culture variables important when studied alone, their potential interactions could also impact the responses of E S C . Hence, factorial design and response surface methodologies could be used to understand the impact o f these interactions and to further optimize the levels of these variables. 5.3 References 1. Palmqvist, L . ; Glover, C. H . ; Hsu, L . ; L u , M . ; Bossen, B . ; Piret, J. M . ; Humphries, R. K . ; Helgason, C. D . , Correlation of murine embryonic stem cell gene expression profiles with functional measures o f pluripotency. Stem Cells 2005, 23, (5), 663-680. 2. Sato, N . ; Meijer, L . ; Skaltsounis, L . ; Greengard, P.; Brivanlou, A . H . , Maintenance o f pluripotency in human and mouse embryonic stem cells through activation of Wnt signaling by a pharmacological GSK-3-specific inhibitor. Nat Med 2004, 10, (1), 55-63. 194

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