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Telomere dynamics in hematopoietic stem cells Gylfadottir, Valgerdur 2007

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T E L O M E R E D Y N A M I C S IN HEMATOPOIETIC STEM CELLS by V A L G E R D U R GYLFADOTTIR B.Sc , University of Iceland, 2001 A THESIS SUBMITTED IN PARTIAL F U L F I L L M E N T OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE In THE F A C U L T Y OF G R A D U A T E STUDIES (Experimental Medicine) THE UNIVERSITY OF BRITISH C O L U M B I A August 2007 © Valgerdur Gylfadottir, 2007 ABSTRACT Hematopoietic stem cells (HSCs) are known to possess an exceptionally high replicative potential (Szilvassy SJ 2003). However, although they express detectable levels of telomerase, previous murine studies have suggested that HSCs still experience telomere shortening and are not immortal (Allsopp RC 2003). To further clarify the role of aging and clonal exhaustion in HSCs we used murine models to investigate the relationship between the number of cells transplanted, telomerase status, the length of their telomeres and their long term exhaustion. We initiated our studies by investigating the replicative potential of titrated numbers of highly purified HSCs. Results showed that bone marrow stemming from a single HSC could only be serially transplanted twice whereas bone marrow stemming from 10 HSCs could be serially transplanted at least three times. In order to better understand HSC replicative potential, we modified Flow-FISH for murine telomere length measurements. Our Flow-FISH protocol enables us to measure telomere lengths at different timepoints within individual mice, providing us with a unique insight into the telomere length status. With the primary goal of clarifying the role of aging and clonal exhaustion in HSC we aimed to investigate the relationship between telomerase status and HSC exhaustion. We set out to characterize the HSCs of TERT-KO mice that lack the mTERT gene encoding the telomerase enzyme (Erdmann N 2004). A comparison of the frequency of Kit+Sca+Lineage" cells between the TERT-KO and its -wild type counterpart revealed no difference. To our knowledge no studies have explored the rate of telomere shortening of HSC within individual mice over time. Using our optimised Flow-FISH based method we studied the rate of telomere shortening within primary B M transplant recipients, in relation to telomerase status. We find that telomere n length is maintained over a nine month period post transplantation, regardless of telomerase status. Furthermore, serially transplanting titrated numbers of wild type and telomerase deficient W B M does not result in telomere shortening. However, despite the apparent maintenance of telomere length, reconstitution levels decrease with each transplantation regardless of transplant dose and telomerase status. These findings suggest the presence of telomere independent barriers, HSC dilution (Iscove N N 1999), alternative lengthening of telomeres or low HSC turnover following W B M transplantation. 111 TABLE OF CONTENTS ABSTRACT ii TABLE OF CONTENTS iv LIST OF TABLES vii LIST OF FIGURES viii ABBREVIATIONS ....... x ACKOWLEDGEMENTS .xiii CHAPTER 1 INTRODUCTION 1.1 Hematopoietic stem cells : 1 1.2 Telomeres 3 1.2.1 Telomere structure and function 3 1.2.2 Loss of telomeric D N A 4 1.2.3 Telomerase 5 1.2.4 Telomeres, telomerase and human diseases 8 1.2.5 Telomere length dynamics 10 1.2.6 Telomere length measurements 11 1.2.7 Bone marrow transplantations 14 1.3 Murine telomeres 17 1.3.1 Mouse telomere biology 17 1.3.2 Telomerase deficiency in mice.... 18 1.4 Murine serial transplantation studies 20 1.4.1 Telomere shortening accompanies increased cell cycle activity during serial transplantation of hematopoietic stem cells 20 iv 1.4.2 Telomerase is required to slow telomere shortening and extend replicative lifespan of HSCs during serial transplantation. 22 1.4.3 Effect of TERT over-expression on the long-term transplantation capacity of hematopoietic stem cells 24 1.4.4 Contradicting results 26 1.5 Thesis objectives 27 C H A P T E R 2 M A T E R I A L S A N D M E T H O D S 2.1 Mice 28 2.2 Single cell transplantation 28 2.3 TERT-KO K S L characterization 30 2.4 Competitive bone marrow repopulation assay 30 ' 2.5 Longitudinal studies 30 2.6 Serial transplantation -.31 2.7 Flow Cytometry..... 32 2.8 M A C S 32 2.9 Bone marrow aspiration 33 2.10 Flow FISH 33 2.11 D N A isolation 34 2.12 Real Time Quantitative PCR .> .34 C H A P T E R 3 R E S U L T S 3.1 Bone marrow stemming from a single HSC can only be serially transplanted twice whereas bone marrow stemming from 10 HSCs can be serially transplanted at a minimum three times 36 v 3.2 Mouse telomere lengths can be accurately measured using a Flow-FISH based method 41 3.3 Mouse telomere lengths can not be accurately measured using a PCR based method : 49 3.4 Characterization of telomerase deficient HSC 54 3.5 Telomere lengths are maintained over a 9 month period post transplantation regardless of telomerase status , 57 3.6 Telomere lengths are maintained throughout serial transplantations regardless of telomerase status and original transplant dose 60 C H A P T E R 4 DISCUSSIONS 66 C H A P T E R 5 R E F E R E N C E S 78 vi L I S T O F T A B L E S Table 3.1 Primary, secondary and tertiary transplant 39 Table 3.2 Reconstitution of primary, secondary and tertiary recipients 40 vii LIST OF FIGURES Figure 1.1 Schematic representation of telomere structure 4 Figure 1.2 Telomere replication 6 Figure 1.3 Measurement of mean terminal restriction fragment (TRF) length during HSC serial transplantation : 21 Figure 1.4 Analysis of telomere length and replicative capacity during serial transplantation of HSCs from telomerase-deficient and wild type mice 23 Figure 1.5 Analysis of telomere length and replicative capacity during serial transplantation of HSCs that overexpress the telomerase enzyme 25 Figure 3.1 Single HSC transplantation 37 Figure 3.2 Isolation of cKit+Scal"""Lineage" Hoechst side population cells by Flow Cytometry 37 Figure 3.3 HSC serial transplantation 38 Figure 3.4 Reconstitution of primary, secondary and tertiary recipients 40 Figure 3.5 Blood sampling results in sufficient white blood cell numbers for Flow-FISH based telomere length measurements 43 Figure 3.6 Blood sampling results in insufficient granulocyte numbers for Flow-FISH based telomere length measurements 45 Figure 3.7 Bone marrow aspiration results in sufficient granulocyte numbers for Flow-FISH based telomere length measurements 46 Figure 3.8 Effect of bone marrow aspiration on telomere length 47 Figure 3.9 Optimized Flow-FISH protocol 48 Figure 3.10 Agarose gel electrophoresis following PCR with telomere primers 51 vni Figure 3.11 Agarose gel electrophoresis following PCR with 36B4 primers 52 Figure 3.12 Acrylamide gel electrophoresis following PCR with 36B4 primers 52 Figure 3.13 Comparison of telomere length measurements with PCR vs Flow-FISH 53 Figure 3.14 Characterization of first generation TERT-KO Kit+Sca+Lineage" cells 55 Figure 3.15 Competitive bone marrow transplantation 56 Figure 3.16 Longitudinal studies 58 Figure 3.17 Purification of donor derived granulocytes..... 58 Figure 3.18 Telomere length donor derived granulocytes 0 days, 6 weeks, 3 months and 9 months after transplantation :. 59 Figure 3.19 Whole bone marrow serial transplantation 61 Figure 3.20 Reconstitution of W B M cells at 3 months post primary, secondary and tertiary transpl antation .62 Figure 3.21 Purification of donor derived granulocytes 63 Figure 3.22 Telomere lengths of donor derived granulocytes 3 months after primary, secondary and tertiary transplantation 63 Figure 3.23 Flow-FISH detects contaminant endogenous Gr l cells in low purity samples ...64 Figure 3.24 Flow FISH effectively distinguishes between donor and recipient cells in low purity samples : 65 ix ABBREVIATIONS HSC hematopoietic stem cell HSCs hematopoietic stem cells W B M whole bone marrow D N A deoxyribonucleic acid RNA ribonucleic acid Kb kilobases K O knock out A L T Alternative Lengthening of Telomeres TERT telomerase reverse transcriptase TERC telomerase R N A component TR telomerase RNA component D K C Dyskeratosis Congenita Flow-FISH Flow-Fluorescence In Situ Hybridization Q-FISH Quantitative-Fluorescence In Situ Hybridization PCR Polymerase Chain Reaction PNA peptide nucleic acid FACS fluorescence-activated cell sorting M A C S magnetic-activated cell sorting TRF terminal restriction fragment B6 mice Black 6 mice CD45.1 cluster of differentiation 45.1 CD45.2 cluster of differentiation 45.2 X c-Kit CD117 antibody Seal stem cell antigen Lin lineage markers K S L Kit+Sca+Lineage" SP side population SP-KSL Kit+Sca+Lineage" Hoechst side population PE PhycoErythrin A P C AlloPhycoCyanin FITC Fluorescein Isothiocyanate GFP Green Fluorescein Protein Gr l Granulocyte antigen Macl Macrophage antigen B220 B-lymphocyte antigen CD3 T-lymphocyte antigen PBS Phosphate Buffer Saline EDTA Ethylene Diamine Tetra Acetate FCS Fetal Calf Serum Tris trishydroxymethylaminomethane s.c. sub cutenous i.v. intra venous LDS751 Laser Dye Styryl-751 CP crossing point MgCh magnesium chloride xi Fw forward Rv reverse ACKNOWLE DGEMENTS I am deeply grateful to my supervisor, Dr. Fabio Rossi for the opportunity to do graduate training at the Biomedical Research Centre and for his continuous support and guidance throughout this project. I would like to thank Dr. Peter Lansdorp, a collaborator and a graduate committee member, for his Flow-FISH expertise and assistance in project discussions and Dr. Kelly McNagny who also served on my graduate committee. Special gratitude goes to members of the Rossi lab (Aaron, Lin, Leslie, Yasmine, Bahareh, Stephane, Bernie, Mike and Hans Peter) for their vibrant discussions, constant encouragement and technical support. I would like to specifically thank Dr. Stephane Corbel and Lin Y u for their assistance during bone marrow transplantations and lima Vulto for telomere length measurements. Lastly, I would like to thank the CIHR/HSF Strategic Training Program in Transfusion Science and the Stem Cell Network Training Program for their financial support. Xlll CHAPTER 1 INTRODUCTION 1.1 Hematopoietic stem cells Hematopoietic stem cells (HSCs) are adult stem cells that reside in the bone marrow and are responsible for giving rise to and maintaining the entire repertoire of mature blood cells. HSCs are multipotent cells with a frequency of less than 1 in 10,000 nucleated cells in the bone marrow, and like other stem cells, HSCs are defined by their capacity to undergo both differentiation and self-renewal. This is a key property of HSCs as the limited lifespan of mature blood cells requires their continuous production and only HSC self-renewal can ensure that sufficient number of stem cells is available to meet the demands of hematopoiesis over a normal lifespan (Lansdorp 1997). To compensate for the continual loss of differentiated blood cells, it is estimated that an adult human needs to produce between 101 1 and 10 1 2 mature blood cells per day (Elwood NJ 2004), this includes B and T lymphocytes, erythrocytes, megakaryocytes, platelets, basophils, mast cells, eosinophils, neutrophils, granulocytes, monocytes and macrophages (Abramason S 1977; Dick JE 1985; Keller G 1985; Jordan CT 1990; Szilvassy SJ 1994). Furthermore, in addition to regular cell turnover, the hematopoietic system must be able to respond rapidly to illness and trauma. As a result, HSCs possess an exceptionally high replicative potential. It is estimated that in normal humans there are approximately 50 million HSCs, some of which can generate up to 10 1 3 mature blood cells over a normal lifespan (Szilvassy SJ 2003). Moreover, murine studies have shown that a single stem cell can regenerate and maintain the entire hematopoietic system following 1 transplantation into an irradiated host (Morrison SJ 1995; Osawa M 1996; Krausse DS 2001). It is clear that under normal circumstances, HSCs are highly capable of maintaining blood cell production throughout a normal lifespan. However, under certain conditions, including bone marrow transplantations, HSCs may possibly reach their replicative potential (Elwood NJ 2004). The transplanted HSCs are limited in number and thus need to proliferate extensively in order to meet the requirements of the host. If further demands are placed on the hematopoietic system, such as following a chronic infection or i f too few donor stem cells are transplanted, long-term survivors of stem cell grafts may be at risk of developing anemia due to HSC exhaustion (Lansdorp P M 2005). Hematopoietic cell transplantations have become a routine procedure for treating patients with hematological malignancies, such as myeloma and leukaemia. Furthermore, there is a growing interest in using hematopoietic stem cell transplantation to treat solid tumours and non-malignant diseases, including P-thalassaemia and sickle-cell anaemia (Hoppe CC 2001). It is therefore exceedingly important to investigate the effect transplantation procedures have on the rate of aging of the transplanted cells. 2 1.2 Telomeres 1.2.1 Telomere structure and function Telomeres are a complex of guanine-rich repeat sequences (TTAGGG) and associated proteins, located at the end of every eukaryotic chromosome. Its primary function is to enable the D N A repair machinery to distinguish between chromosome ends and double strand brakes and thus protect the chromosome ends against chromosomal fusion, recombination and terminal D N A degradation (Blackburn EH 2001). This chromosomal capping mechanism is accomplished through a unique structure known as the T-loop. Although much of the telomere is a duplex D N A structure, the telomere is characterized by its 3 'strand overhang, a single-strand overhang that folds back into the duplex telomeric D N A to from a protective loop structure, the T-loop, which hides the end of the telomere from normal D N A repair mechanisms (Griffith JD 1999). Telomeric D N A also serves as a binding site for a number of DNA-binding proteins, including the telomere repeat factors (TRF) 1 and 2, hPOTl and various other proteins that indirectly associate with the telomere through the telomere-binding proteins (Figure 1.1). These proteins all serve critical functions in the maintenance of telomere function and structural integrity (De Lange T 2002). 3 Figure 1.1 Schematic representation of telomere structure. The T-loop of telomeric DNA is formed by invasion of its duplex region by the 3 'overhang. Illustrated are some of the reported telomere associated proteins (Shin JS 2006). 1.2.2 Loss of telomeric DNA Over 40 years ago, Hayflick and Moorhead suggested that most normal cells are programmed for a given number of cell divisions and cannot divide indefinitely (Hayflick L 1961). 10 years later Watson (Watson JD 1972) and Olovnikov (Olovnikov A M 1973) identified the telomere end replication problem which is believed to be the primary cause of the limited replicative potential of most normal somatic cells. Every time a cell divides, both the leading strand and the lagging strand need to be synthesized. However due to limitations of the D N A polymerase, it can not initiate lagging strand synthesis from the very end of linear D N A and no known mechanism exists for replicating terminal R N A primers, resulting in an incompletely replicated 5'strand (Greenwood M J 2003). A s a result, the telomeres of most cells continuously shorten with each cell division. When a cell reaches its replicative potential, also known as the Hayflick limit, at least some o f its telomeres have become critically short and thus can no longer effectively cap 4 their chromosome. This leads to chromosomal instability and the cell normally stops dividing and becomes senescent or dies by apoptosis. Furthermore, this continuous telomere shortening as cells proliferate, suggests that telomere length may serve as a kind of a mitotic clock, ticking off the passage of time with each cell division and thus providing a measure of the replicative history of a cell (Rando TA 2006). This makes telomere length measurements an attractive tool when studying aging of cells and their replicative potential. In fact, previous studies have shown that humans, who normally have 5 - 20 kb of telomeric DNA, lose 50 to 200 base pairs with each successive round of cell division (Greenwood MJ 2003). However, nature is seldom this simple and indeed several other mechanisms have emerged that also cause telomere shortening including oxidative damage, the failure to unwind or correctly process higher-order structures of G-rich telomeric D N A and the deletion of T loops by homologous recombination (Crabbe L 2004; Ding H 2004; Wang RC 2004). The relative importance of these different telomere erosion pathways and their effect on the telomere shortening of diverse cell types has not yet been established. 1.2.3 Telomerase Although the replicative potential of most cells is believed to be limited due to telomere erosion, there are several cell types that surpass this limitation by expressing an enzyme known as telomerase (Figure 1.2). These cells include germ-line cells, early embryonic stem cells and most cancer cells, all highly proliferating cell types that need to conserve their telomere length in order to maintain proliferative capacity and avoid replicative senescence. In addition to telomerase expression some cells may generate 5 telomeric D N A through recombination events, a mechanism known as alternative lengthening of telomeres ( A L T ) . However the ALT-related mechanisms have only been clearly identified in abnormal situations and most normal and malignant cells seem to utilize the telomerase-dependent pathway (Henson JD 2002). -rift;<.y.v - A S y . ^ . T - A i . : y — A l : . 3 - M D X O V 7 ^.m>CCCAAC \ l n t e m a t 1 ,r ~j— RNA \ template "fetomerase DNA synthesis 3 ' Enzyme trans to cation r v DNA synthesis S i / 3 ' A A U C C C 5 ' Multiple synthesis steps by telomerase 5 ' TTAGGQTTA- i 3 ' AAUCCCS' )NA synthesis Elongation of opposite strand by DNA polymerase a 5 ' T T A G < 3 G ~ ~ A • • l l H I I H v ; - ^ m M < - mmm •? 3 / A A l . C C C V v 5 i Key: { • 5 ' G G G T T A 3 ' \m3 C C C A A U 5 Figure 1.2 Telomere replication. Shown here are the reactions involved in synthesizing the repeating G-rich sequences that form the telomeres. Telomerase extends the TG-rich strand of telomeres by DNA synthesis using an internal RNA template (Strachan 1999). 6 The telomerase enzyme is a large ribonucleoprotein consisting of both a catalytically active reverse transcriptase protein (TERT) and a RNA template (TERC or TR) (Yu G L 1990). Both components are essential for normal telomerase activity. In humans the R N A template is 445 nucleotides long with an 11 nucleotide template sequence complementary to the human telomere sequence (Feng J 1995). Cells that express sufficient levels of the telomerase enzyme avoid the end replication problem by extending 3 'chromosomal ends through the addition of single stranded T T A G G G repeats. As a result, telomerase activity and telomere maintenance are associated with the immortality of cancer cells, germ-line cells and embryonic stem cells. Nevertheless, it should be noted that several in vitro studies have shown that cultured rodent cells eventually stop dividing and acquire a senescence-like phenotype, even though they successfully maintain both high telomerase activity and long telomeres (Hanahan D 2000; Sherr SJ 2000; Wright WE 2000). However, it has been suggested that this ' telomere-independent arrested state may simply reflect a cell cycle checkpoint response to inappropriate culture rather than an intrinsic limitation imposed by a cell-division counting mechanism (Tang DG 2001). Although most healthy somatic cells do not express telomerase and thus experience telomere shortening with each cell division, there are a few exceptions. In the hematopoietic department this includes some types of lymphocytes and also hematopoietic stem cells, all of which are characterized by a need for a high proliferative capacity. However, although HSCs do express detectable levels of the telomerase enzyme, previous murine studies have shown that these cells still experience telomere shortening and are not immortal (Allsopp RC 2003a). This suggests that the level of 7 telomerase expression in HSC is not sufficient to completely prevent telomere erosion. This notion has been further supported by telomere length measurements of human peripheral blood lymphocytes and granulocytes which show an age dependent telomere loss. Furthermore, the rate of telomere loss of hematopoietic cells is also age dependent. Studies investigating the rate of telomere shortening of. human peripheral blood cells suggest that telomere erosion is at its highest level within the first year of life but thereafter continues at a slow decline until 50 to 60 years of age, after which the decline again accelerates (Rufer N 1999; Verfaillie C M 2002). 1.2.4 Telomeres, telomerase and human diseases The importance of telomerase expression is highlighted by the severe consequences of telomerase deficiency. Dyskeratosis Congenita (DKC) is a rare human genetic disorder characterized by defects in highly proliferative tissues including hematopoietic tissue, gut epithelium and chromosomal instability (Greenwood MJ 2003). In this disease, a defect of the telomerase RNA template gene results in the absence of telomerase activity and premature telomere shortening. Positional cloning identified the gene affected in many patients with X-linked D K C as the human homologue of yeast CBF5, termed D K C 1 , encoding a protein, dyskerin. Furthermore, the association of the telomerase complex with dyskerin suggested that the D K C phenotype may be the result of altered telomerase activity (Vulliamy T 2001). Later, the discovery of a 3'deletion in the gene encoding TERC in a single large family with autosomal dominant D K C confirmed the importance of telomerase deficiency as the aetiology for D K C and highlighted the importance of telomerase in maintaining cellular lifespan and replicative 8 potential in human organs (Hiyama E 2007). Patients with DKS frequently develop bone marrow failure or malignancy before they reach the age of 50. Furthermore, chromosomal rearrangements are common and peripheral blood cells from these patients demonstrate markedly shortened telomeres over age-matched controls (Marrone A 2003). Aplastic anaemia is yet another disease that further confirms the importance of maintaining HSC replicative potential. This disease is thought to stem from severe and prolonged damage to the HSC department, forcing the remaining HSCs to undergo additional cell divisions. As a result, patients suffering from aplastic anaemia have unusually short telomeres in their peripheral blood granulocytes and monocytes and eventually their HSCs fail to produce sufficient quantities of all the hematopoietic lineages (Greenwood MJ 2003). Furthermore, telomeres and telomerase expression are believed to be major players in cancer biology. Current data support the idea that telomere shortening with age evolved as a tumor suppressor mechanism in long-lived species as it limits the lifespan of cells and thus strives to prevent uncontrollable proliferation (Baerlocher G M 2004). By suppressing immortal growth, younger and healthier cells are recruited into action to replace older cells that may have accumulated damaging mutations. However, this tumor suppressor mechanism has proven to be a double edge sword. Under normal circumstances when a telomere becomes critically short, the chromosome becomes unstable, it triggers a D N A damage response and the cell stops dividing and becomes senescent or dies by apoptosis. On the other hand, i f a cell already has accumulated damaging mutations such as a mutation in the tumor suppressive gene p53 or in other key players of the DNA damage response, the cells fate may be a very different one. Due to 9 the block in the D N A damage response the cell fails to exit the cell cycle and the chromosomal instability may well cause the accumulation of new mutations through chromosomal fusion or recombination (Shay JW 2002). Telomere shortening can therefore end up facilitating cancer formation instead of preventing it. Nevertheless, human cancer cells do eventually have to overcome the restrictions telomeres impose on their proliferative potential. This is typically achieved by.up-regulation of telomerase activity. Telomerase is reactivated in more than 90% of all types of human tumors, thereby rescuing short telomeres and perpetuating cells with short telomeres and high chromosomal instability (Chin L 1999; Lin SY 2003). Furthermore, most metastases also contain telomerase-positive cells, which indicates that telomerase is required to sustain their growth (Blasco M A 2005). 1.2.5 Telomere length dynamics Human telomeres are generally between 5 and 20 kb long. However, there is a great variation between different individuals, between different cell types, between different chromosomes within the same cell and even between different chromosome ends on the same chromosome. Studies show that chromosome 17p typically contains the smallest number of telomere repeats, an interesting finding since that happens to be the location of the tumor suppressor gene p53 (Martens U M 1998). It is important to note that it seems to be the shortest telomere rather than the average telomere length within a cell that dictates cell viability and chromosome stability (Hemann M T 2001). Therefore, the proliferative potential of cells may be limited by the chromosome with the shortest length of telomere repeats (Moyzis R K 1988; Lansdorp P M 1995). However, 10 experimental evidence does suggest that short telomeres accumulate on multiple chromosomes prior to senescence and that replicative senescence is not triggered by the first telomere to reach a critical minimal threshold length (Lansdorp P M 2000). In addition, studies have shown that telomere shortening is not uniform but instead it is proportional to the size of the G-rich telomeric 3 'overhang such that cells with long overhangs lose more telomeric repeats with each cell division than those with shorter overhangs (Huffman K E 2000). As a result, telomere length measurements should be focused on recognizing the shortest telomeres rather than the average telomere length as it is the accumulation of short telomeres that dictates cell viability and chromosomal stability (Elwood NJ 2004). 1.2.6 Telomere length measurements Telomere length dynamics have been shown to be important for the regulation of the replicative life span of cells and consequently for aging and tumorigenesis. As a result, several methods have been developed to measure telomere lengths including Southern blot, Quantitative-Fluorescence In Situ Hybridization (Q-FISH), Flow-FISH and a Polymerase Chain Reaction (PCR)-based method. Telomere length measurements by Southern blot is a fairly well established method but has its limitations. First D N A is isolated and digested by a restriction enzyme, known to be located close, but not within telomeric DNA. Next the resulting D N A fragments are separated based on their size using gel electrophoresis. Finally the telomere fragments are visualized by hybridization with telomere probes and the average telomere length estimated (Baerlocher G M 2004). However, there are several limitations 11 to using Southern blot as a way of evaluating telomere lengths. First, there are no restriction enzyme sites located exactly at the end of the telomeric repeats. As a result, variable amounts of subtelomeric D N A are bound to be included in the telomeric fractions and telomeric length will therefore be overestimated. Second, large amount of cells are needed (105-106 cells) and last but not least,, southern blot measures average telomere lengths (Baerlocher G M 2004). This is a major limitation as studies have shown that it is the shortest telomere length but not the average telomere length that limits the lifespan of a cell. Q-FISH uses digital images of metaphase chromosomes, after in situ hybridization with a fluorescently labelled telomere probe, to evaluate telomere lengths. First, cells are stimulated in culture, arrested in metaphase and mounted onto slides. Then in situ hybridization is performed, using fluorescently labelled telomere peptide nucleic acid (PNA) probe along with a DNA counter-stain. Finally, telomere lengths are analysed using digital images. Q-FISH is a very sensitive method that allows the length of individual chromosomal ends to be evaluated. However, Q-FISH is very expensive and time-consuming and since the method requires cells to be in metaphase, Q-FISH can not be used to measure telomere lengths in non-dividing cells (Baerlocher G M 2004). Basic Flow-FISH requires isolation of nucleated cells from whole blood or tissue. Cells are fixed and D N A denatured and hybridized with fluorescently labelled telomere-PNA probe along with a D N A counter-stain. Lastly, the resulting cells are acquired and their telomere fluorescence analysed on the flow cytometer (Baerlocher G M 2004). This method has several advantages, cells can be quiescent, many cells can be analyzed within a short period and subpopulations of cells can be analyzed separately based on 12 differences in light scatter properties. Problems include interference of hemoglobin with telomere fluorescence and the loss of cells and also the loss of most cell-surface antigens through the fixation of cells with heat and formamide (Baerlocher G M 2004). Telomere length measurements using flow-FISH have been further improved in Dr. Peter Lansdorp's lab resulting in a method known as the Automated Multicolor Flow-FISH. The improvements include the automation of most cell processing steps by a robotic washing station, the utilization of bovine thymocytes as an internal control and a limited immunophenotyping step. By combining flow-FISH with immunophenotyping it is possible to measure telomere lengths of different subpopulations within a sample. However, due to the apparent loss of surface epitopes through the fixation step, only a limited number of cell types can effectively be immunophenotyped (Baerlocher G M 2004). Recently a PCR-based method was developed for measuring telomere lengths (Cawthon R M 2002). It had been presumed impossible to measure telomere lengths by PCR amplification since the primers designed to hybridize to the T T A G G G and C C C T A A repeats would only result in primer-dimer derived products; However, in 2002 Dr. Richard Cawthon introduced a primer pair that eliminated this problem. Each primer was designed to allow the D N A polymerase to extend from its 3'end when it is hybridized to telomere hexamer repeats but not when it is hybridized to the other primer. The method includes the following steps: Quantitative PCR is performed separately using the telomere primers and primers that recognize a single copy gene for each sample. Standard dilution curves are prepared using reference D N A along with either the telomere primers or the single copy gene primers. Finally, the telomere lengths are 13 evaluated by calculating the factor by which the test sample differed from the reference D N A sample in its ratio of telomere repeat copy number to single copy gene copy number (Cawthon R M 2002). This method has some obvious advantages; it is simple, rapid and cheap and can be performed on a high-throughput scale. Furthermore, it does not require high cell numbers and can therefore easily be used for rare cell types such as HSCs. 1.2.7 Bone marrow transplantations Since HSC presumably loose telomere repeats upon proliferation and may therefore have a limited replicative potential, concerns have been raised regarding its effect on the long term success of bone marrow transplantations. Studies have shown that the telomere length of donor hematopoietic cells preceding transplant is greater than that of the donor cells found in the recipient following transplant (Notaro R 1997). Furthermore, Wynn et al reported an average shortening of around 0.4 kb and suggested that this telomere deficit translates into an additional stem cell aging effect caused by the transplantation procedure in the range of 15 years (Wynn RF 1998). This dramatic telomere shortening is established within the first year post transplant but thereafter the telomeres seem to follow the normal rate of erosion (Lee J 1999; Briimmendorf TH 2001; Rufer N 2001; De Pauw Es 2002; Thornley I 2002). In addition, by measuring the telomere lengths of chromosome 7q in granulocytes, Notaro et al detected a significant inverse correlation between telomere shortening and the number of mono-nucleated cells infused, supporting the notion that telomere shortening indicates expansion of the stem 14 cell compartment associated with repopulation of the host bone marrow by donor cells post-transplant (Notaro R 1997; Briimmendorf TH 2006). Although the degree of telomere shortening following bone marrow transplantation does not appear to reach a level that compromises bone marrow function and there is little evidence that suggest telomere shortening will result in an epidemic of marrow failure in HSC transplant recipients, caution still remains warranted when the number of cells transplanted is limited or the donor telomere lengths are short (Lansdorp P M 2005). Long-term survivors of stem cell grafts may be at risk of stem cell exhaustion i f the transplant dose is too low, the cells come from an old donor or patients with telomerase deficiencies and therefore have unusually short telomeres or i f increased demands are placed on the hematopoietic system, such as following a.chronic infection. At least two cases have been published in which telomere shortening following bone marrow transplantation appears to have played a role in poor graft function (Awaya N 2002). In the first case, bone marrow from a 61 year old donor was transplanted into a 7 year old boy with acute lymphocytic leukaemia. Although the engraftment of the donor cells was successful, the patient experienced poor marrow function 25 months post-transplant. The telomere length of donor cells harvested from the patient was shown to be 2 kb shorter than that of the original donor cells and the young recipient had a hematopoietic system with telomere lengths significantly shorter than those of other children his age. In the second case, a 13 year old boy with a severe aplastic anaemia underwent transplantation with bone marrow from his 14 year old sister. Although the transplantation appeared successful, 25 years later abnormal blood counts revealed that 15 the patient's bone marrow was hypo-cellular. Furthermore, the telomere length of the patients HSCs proved to be approximately 2.2 kb shorter than that of the donor. As a result of these and other examples, studying the effect bone marrow transplantations and consequent telomere shortening have on the replicative potential of the transplanted HSCs remains important. 16 1.3 Murine telomeres 1.3.1 Mouse telomere biology A l l research presented is this thesis was performed using mouse models. There are several facts that need to be taken into consideration when using mouse telomere biology as a model for human telomere biology. One is that mice contain telomerase activity in most tissues but humans retain telomerase activity in undifferentiated germ-line or highly proliferating cells (Prowse K R 1995; Greider CW 1998; Martin-Rivera L 1998; Venkatesan R N 1998; Kishi S 2003). More importantly, telomere shortening with age is believed to have evolved as a tumor suppressor mechanism in humans but not in mice. As a result, mice possess much longer telomeres than humans do. Human telomeres are usually 5-20 kb long whereas laboratory mouse telomeres are ranging from 20 to lOOkb, although it does depend heavily on the mouse strain (Kipling D 1990; Starling JA 1990; Prowse K R 1995; Hemann M 2000). Since mice have such long telomeres, HSC exhaustion through a single bone marrow transplantation is not expected. As a result, when studying HSC replicative potential in mice it is necessary to perform serial bone marrow transplantations. Previous studies suggest HSCs can be serially transplanted 4-6 times before the HSCs exhaust and can no longer accomplish reconstitution (Harrison DE 1982). 17 1.3.2 Telomerase deficiency in mice The core components of the telomerase enzyme are the reverse transcriptase (TERT) and the RNA template (TR or TERC). Knockout mice have been developed and analysed for both TERT and TR resulting in a functionally equivalent phenotype (Erdmann N 2004). Whereas a modest twofold reduction in telomerase activity levels in humans results in premature death from complications of aplastic anaemia or immune deficiency, generations of mice completely lacking either TERT or TR appear normal and healthy (Dokal I 2001; Collins K 2002; Fogarty PF 2003). This lag of several generations before the appearance of abnormalities in mice is thought to be due to the fact that laboratory mice have extremely long telomeres and several generations are thus needed to reach the degree of telomere shortening sufficient to cause complications. However, after three to four generations the ongoing telomere shortening leads to infertility, reduced viability and defects in highly proliferative tissues such as the hematopoietic system, skin and digestive system (Lee HW 1998; Herrera E 1999). Late generation telomerase deficient mice generally suffer from genomic instability, poor wound healing, an increase in end-to-end chromosomal fusions and an increased incidence of tumors in older mice (Blasco M A 1997; Liu Y 2000). Furthermore, the mice may experience heart failure, immuno-senescence-related diseases and various tissue atrophies (Blasco M A 2005). Telomerase deficient mice are usually bred on either a mixed C57BL6/129Sv background or on a pure C57BL6 background. The pure C57BL6 background mice have shorter telomeres and can only be bred for four generations while mixed background mice can be bred for six. Furthermore, fifty percent of pure background fourth generation mice die at only five months of age, whereas the 18 late generation mixed background mice are viable to adulthood despite defects in their hematopoietic system (Herrera E 1999). Previous studies have shown that mice heterozygous for TERT do experience increased telomere loss compared to C57BL6 wild type mice. However, despite short average telomere lengths, TERT +/- mice remain fertile and do not exhibit telomere signal free ends. As a result Erdmann et al concluded that one functional allele of TERT is sufficient to maintain minimal telomere D N A at all chromosome ends even though it is not sufficient to maintain overall average telomere length (Erdmann N 2004). In addition, TERT +/+ littermates also possess shorter telomeres than wild type C57BL6 mice suggesting haplo-insufficiency of TERT cannot be completely corrected within one wild type generation (Erdmann N 2004). 19 1.4 Murine serial transplantation studies Several studies have been published that are highly relevant to the studies presented in this thesis. This chapter serves to outline these experiments and their results. 1.4.1 Telomere shortening accompanies increased cell cycle activity during serial transplantation of hematopoietic stem cells In 2001, Allsopp et al set out to investigate whether telomere shortening might play a role in the limitation of HSC division capacity in vivo (Allsopp RC 2001). With that aim, they serially transplanted HSCs into lethally irradiated primary and secondary recipients and analyzed their telomere lengths. 2-3 month old C57B1/Ka-Thyl.l (CD45.1) were used as HSC donors and the congenic C57B1/Ka-Thyl.2 (CD45.2) were used as recipients. Furthermore, the donor strain bone marrow was found to have an initial telomere length of 10-40kb. Donor HSCs were sorted by an initial enrichment of c-kit positive cells by M A C S , after which the HSCs were further purified on a dual-laser FACS Vantage as c-kit +Sca-l +Thyl .1 l o L i n \ Serial transplantations were performed using either 100-200 sorted HSCs as described above or whole bone marrow supposedly containing 100-200 HSCs. Secondary transplantation was performed 4 months later. A l l transplant recipient mice used for telomere length analysis were at least 80% reconstituted. Bone marrow was collected at the time of the initial transplantation, 4 months post primary transplantation and 4 months post secondary transplantation. Whole bone marrow (WBM) D N A was isolated and telomere lengths were measured by Southern blot. Results showed that after one round of transplantation the average drop in telomere length of W B M was a moderate 1.5 kb (Figure 1.3). However between the primary and secondary transplantation the telomeres dropped by and additional 5.5kb. 20 Allsopp et al suggested this increase in telomere erosion was due to increased HSC turnover in the secondary recipient as the HSCs were already cycling at time of isolation and may thus experience reduced ability to home to and settle in the bone marrow. P=0.02 P=0.004 Figure 1.3 Measurement of mean terminal restriction fragment (TRF) length during HSC serial transplantation. The mean TRF length was calculated for a total of 9 sibling adult B A mice (C57B1/Ka-Thyl.l), 11 primary recipients and 10 secondary recipients and averaged for all experiments. Error bars (standard deviation) and P values (Student's t test) are shown (Allsopp RC 2001). In order to measure the telomere length of HSCs directly, they sorted 1000 donor derived c-kit +Sca-l +Thyl .l'°Lin" cells, cytospun them onto glass slides and measured their telomere lengths with Q-FISH. Results showed a 30% reduction in signal intensity after two rounds of transplantation. To assess whether the extent of reduction in telomere length was dependent on the initial dose of transplanted HSCs, they performed a second transplantation experiment were they compared the mean terminal restriction fragment length (Southern blot) for primary recipients reconstituted with either 30 or 3000 HSCs (c-kit +Sca-l +Thyl.l'°Lin~). At four months after transplant, mice reconstituted with 30 HSCs had lkb shorter telomeres than mice reconstituted with 3000 HSCs (21.7kb vs 22.7kb). Allsopp et, al concluded that the difference was significant and that these results further supported that the apparent telomere erosion was due to increased HSC division. 21 1.4.2 Telomerase is required to slow telomere shortening and extend replicative lifespan of HSCs during serial transplantation The same research group published a follow up study in 2003 where they aimed to assess the relevance of telomerase to the long-term replicative potential of HSCs in vivo (Allsopp RC 2003a). In this study, they serially transplanted HSCs from telomerase deficient mice and wild type siblings until exhaustion and monitored their telomere lengths. They used two different strains of telomerase deficient donor mice, TR 7 " mice that do not express the telomerase R N A component or TERT 7 " mice that lack expression of the telomerase reverse transcriptase. The TR 7 " mice had been backcrossed 6 times and the TERT"" mice 5 times to the C57B1/Ka-Thyl.l (Ly5.1) strain. HSCs were isolated as previously described (c-kit +Sca-l +Thyl.l , 0Lin") and 150 double sorted HSCs were used in each round of transplantation. Lethally irradiated C57B1/Ka-Thyl.2 (Ly5.2) recipient mice were serially transplanted at an interval of at least four months. Furthermore, donor derived HSCs were sorted at the point of each transplantation and their telomeres measured with Q-FISH. According to their results, wild type HSCs could be serially transplanted five times whereas TR 7 " HSCs could only be serially transplanted twice (Figure 1.4). Furthermore, the rate of telomere loss was approximately two-fold higher during transplantation of TR 7 " HSCs compared with T R + / + HSCs. Deficiency of the telomerase reverse transcriptase showed a similar pattern. A Q-FISH analysis revealed a 1.7 fold higher rate of telomere loss in TERT 7 " HSCs compared to T E R T + / + HSCs in addition to a reduced transplantation capacity of TERT 7 " HSCs. 22 ^ 2 . 0 1 2 3 4 Transplantation no. 1 2 Transplantation no. 1100 B 1000 c 900 a. BOO >. 700 "S 600' § 500 £ 400 til SP 10041* m i t « » < --2.0 I1 1 2 3 . Transplantation no. . 1 2 3 < s Transplantation no. Figure 1.4 Analysis of telomere length and replicative capacity during serial transplantation of HSCs from telomerase-deficient and wild type mice. A) Q-FISH analysis of telomere length during serial transplantation of HSCs from TR + / + and TR"'" mice. B) The average frequency of donor-derived TR + / + and TR"'" HSCs from long-term reconstituted mice, transplantation of HSCs from TERT C) + ' + and TERT"'" Q-FISH analysis of telomere length during serial ice. D) The average frequency of donor-derived TERT + / + and TERT"'" HSCs from long-term reconstituted mice. C-D) Dotted line: 1 of 8 mTERT"'" 2°recipients showed an increase in HSC telomere length and hematopoietic reconstitution, despite no measurable telomerase activity. Authors suggest that a telomerase-independent alternative mechanism for telomere replication has been activated in these HSCs. (Allsopp RC 2003a). This study also revealed an elevated frequency of signal-free chromosome ends and end-to-end dicentric chromosomes in donor derived secondary recipient TR"7" HSCs. From these studies, Allsopp et al concluded that one function of telomerase in HSCs is most likely to increase the replicative potential of HSCs. This is accomplished by reducing the rate of telomere shortening during cell division and thereby preventing premature critical shortening of telomeres and loss of telomere function. 23 1.4.3 Effect of TERT over-expression on the long-term transplantation capacity of hematopoietic stem cells In a third publication, Allsopp et al published a very similar experiment except this time they studied the effect of TERT over-expression instead of deficiency, on the long-term transplantation capacity of HSCs (Allsopp RC 2003b). They developed a transgenic mouse strain in which TERT is over-expressed in HSCs. The TERT transgene expression was driven by the major histocompatibility class I gene H-2K b promoter and had detectable transgene expression and an approximately' four-fold increase in telomerase activity. However the promoter was not specific to HSCs and allowed transgene expression throughout the hematopoietic system causing a mild splenomegaly of all splenic lymphocytes in young adult transgenic mice. HSCs were purified by FACS from transgenic and non-transgenic siblings as c-kit +Sca-l +Thyl.l'°Lin". 150-200 HSCs or an equivalent W B M was serially transplanted into lethally irradiated recipients at a 4 month interval. Terminal restriction fragment (TRF) length was analyzed by Southern blot 4 months after each round of W B M transplantation. After 4 rounds of serial transplantation the overall decrease in mean TRF length of non-transgenic mice was 10.6 kb, corresponding to at least 40% of the total telomeric DNA (Figure 1.5). Furthermore, in an independent experiment where HSCs were purified and serially transplanted, telomeres were analysed in donor derived HSCs with Q-FISH. With this method they observed a 50% decrease in telomere signal intensity of non-transgenic HSCs between first and fourth serial transplantation. On the contrary, telomere length remained stable during serial transplantation of both W B M and HSCs from transgenic mice. However, even though over-expressing TERT led to stable telomere lengths, it did not lead to an 24 extension of HSC transplantation capacity. Interestingly, for both transgenic and non-transgenic mice, HSCs could be serially transplanted no more than four times. A B C Transplantation no. Transplantation no. Transplantation no. Figure 1.5 Analysis of telomere length and replicative capacity during serial transplantation of HSCs that overexpress the telomerase enzyme. A) Mean TRF length was calculated and averaged for long-term reconstituted mice at each stage of serial transplantation. B) Q-FISH analysis of telomere length. C) Average frequency of donor-derived HSCs was measured for all mice used in the mean TRF length analysis. HSC frequency is shown relative to the fraction of donor-derived cells. Error bars represent standard deviation. Blue square = transgenic; green circle = non-transgenic (Allsopp RC 2003b). Furthermore, since it is the shortest telomere length rather than the average telomere length that limits replicative potential, they also used Q-FISH to assess the distribution of telomere lengths. Results showed no detectable shift in telomere length to shorter sizes nor was there an increase in chromosomal ends lacking detectable telomere signal during serial transplantation of transgenic HSCs. Allsopp et al concluded that stabilization of telomere length is not sufficient to sustain indefinite transplantation capacity and telomere-independent barriers may also limit the transplantation capacity of HSCs. 25 1.4.4 Contradicting results Several research groups have challenged the above findings. In 2002, Samper et al performed a serial transplantation with 5x10 s W B M from either wild type mice or generation three TR"A B6 mice (Samper E 2002). Telomere lengths were evaluated using Southern blot, Q-FISH and Flow-FISH. Surprisingly the wild type bone marrow showed a significant reduction in telomere fluorescence (38%) after the first transplantation whereas the transplanted TR"~ B6 bone marrow cells showed only a slight non-significant reduction in the telomeric signal (9%). The authors suggest this may reflect the existence of alternative mechanisms of telomere maintenance or the clonal selection of bone marrow repopulation cells with longer telomeres. Furthermore, Iscove et al argued that the failure of bone marrow to reconstitute recipient mice beyond the fourth serial transplantation is due to HSC dilution rather than HSC exhaustion (Iscove N N 1999). When HSCs are transplanted they proliferate and self-renew greatly in the recipient. However, they never reach normal levels of HSC but plateau after reaching only 4% of normal HSC numbers. As a result, when a second transplantation is performed, Iscove claims the number of long-term reconstituting cells and thus stem cells is far lower than in the first transplant. In 1999, Iscove et al published a study where a serial transplantation was performed with increasing numbers of bone marrow cells in order to keep the number of long-term reconstituting cells constant. Their results showed an increase in long-term reconstituting cells without evidence of exhaustion supporting their hypothesis that the apparent HSC exhaustion is simply due to HSC transplant dilution. 26 1.5 Thesis objectives The experiments presented in this thesis were driven by the primary goal to clarify the role of aging and clonal exhaustion in hematopoietic stem cells. Previous studies suggest the telomeres of HSCs shorten as a result of proliferation, despite detectable telomerase acitivity. Furthermore, HSCs can only be serially transplanted 4-6 times in mice before the HSCs exhaust and can no longer accomplish reconstitution (Harrison DE 1982). HSCs are therefore believed to have a limited replicative potential, raising concerns regarding its effect on the long term success of bone marrow transplantations. In this study we set out to investigate the relationship between the number of cells transplanted, telomerase status and their long term exhaustion. Our hypothesis was that murine HSCs loose telomeric D N A upon serial transplantation and the rate of telomere loss is dependent on original transplant dose and telomerase status. The main goals were the following: 1) To investigate the replicative potential of titrated numbers of highly purified HSCs using murine serial transplantations 2) To investigate telomere length in relation to telomerase status using longitudinal transplantation studies 3) To investigate telomere length in relation to the number of cells transplanted and telomerase status using murine serial transplantations 27 CHAPTER 2 MATERIALS AND METHODS 2.1 Mice Mice were bred and maintained at the Biomedical Research Centre animal facility according to the guidelines of the Canadian Council on Animal Care. Transplant donor and recipients pairs were either chosen based on their GFP genotype or their CD45 cell surface marker genotype. Transplant donor and recipient pairs were the following: C57B16 (3-actin GFP that express GFP and C57B16 that do not express GFP; C57B16 that express CD45.2 and C57B16/J S J L that express CD45.1; or TERT-KOxC57B16 that express CD45.2 and C57B16/J S J L that express CD45.1. The TERT-KO strain which lacks telomerase expression was received as a gift from Lee Harrington where it was backcrossed over 10 times on a C57B16 background (Erdmann N 2004). TERT-KO mice were maintained by heterozygous breeding at the Biomedical Research Centre. 2.2 Single cell transplantation HSCs where isolated as cKit+Scal+Lineage" Hoechst side population cells (SP-KSL). Briefly, bone marrow was harvested from a C57B16 P-actin GFP mouse, red blood cells lysed with ammonium chloride and the remaining white blood cells stained with Hoechst 33342 at 37°C for 90 minutes. Subsequent samples were stained with lineage-PE-Texas red, cKit-PE and Scal-APC antibodies at 4°C for 20 minutes. Using FACS Vantage, single SP-KSL cells were double sorted into a 96 well plate and individual wells checked by fluorescence microscope to confirm the presence of a single cell. In addition, a second set of wells received 10 SP-KSL cells. Support cells were harvested from a C57B16/J S J L mouse and depleted of Sca l + cells using AutoMACS. 35 28 C57B16/J primary recipient mice were lethally irradiated (1100 rads) and transplanted with 1 or 10 SP-KSL cells, along with 1 million GFP negative supporter cells by tail vein injections. Thirty mice received 1 SP-KSL cell whereas five mice received 10 SP-KSL cells. One month later peripheral blood was analysed for donor derived cells. One year after primary transplantation, bone marrow was harvested from the primary recipients. Red blood cells were lysed and one million donor derived GFP positive cells FACS purified from each mouse, and subsequently transplanted into lethally irradiated (1100 rads) secondary recipients. Three mice received bone marrow stemming from one SP-K S L cell and three mice received bone marrow stemming from 10 SP-KSL cells. One month later peripheral blood was analysed for donor derived cells. One year after secondary transplantation, peripheral blood was again analysed for donor derived cells. Only four mice had enough reconstitution to qualify for a tertiary transplantation. These mice included two that had received one SP-KSL and two that had received 10 SP-KSL cells in the primary transplant. The bone marrow was harvested from the secondary recipients. The red blood cells were lysed and all donor derived GFP positive cells sorted from each mouse using FACS Vantage and transplanted into lethally irradiated (1100 rads) tertiary recipients, along with 500,000 Seal depleted helper cells. One month after the tertiary transplantation peripheral blood was examined for donor derived cells. Reconstitution was calculated as a percentage of donor derived white blood cells. 29 2.3 TERT-KO KSL characterization Fresh bone marrow was harvested by flushing the femora of a 8-11 week old female C57B16 and a TERT-KOxC57B16 mouse. Red blood cells were lysed with ammonium chloride and the remaining white blood cells stained with Lineage-PE-Cy7, cKit-FITC and Scal-PE. Cells were washed once with PBS + 2mM EDTA + 2% FCS and analysed on FACS Area. 2.4 Competitive bone marrow repopulation assay Fresh bone marrow was harvested by flushing the femora of a 21-22 week old male C57B16 and TERT-KOxC57B16 mouse. A second, 16 week old male C57B16/J S J L mouse was also harvested. Red blood cells were lysed and the remaining whole bone marrow (WBM) cells counted. Groups of five lethally irradiated C57B16/J S J L mice (1100 rads) received transplants of 100,000 C57B16 or TERT-KOxC57B16 W B M cells, which were complimented with 100,000 C57B16/J S J L competitor W B M cells. Sixteen weeks post transplant, peripheral blood was lysed and stained for the presence of viable CD45.2 B-lymphoid (B220+), myeloid (Macl /Grl + ) and T-cells (CD3 +). 2.5 Longitudinal studies Bone marrow was harvested by flushing the femora of a 21-22 week old C57B16 and TERT-KOxC57B16 mouse. Red blood cells were lysed and the remaining white ' II blood cells counted. Two groups of five C57B16/J recipient mice were lethally irradiated (1100 rads) and transplanted with either 1,000,000 TERT-KO W B M cells or wild type W B M cells. One month later, peripheral blood was analysed for donor derived 30 granulocytes and the reconstitution evaluated. At six and 12 weeks post transplant, bone marrow was aspirated from recipient mice and red blood cells lysed. Donor derived granulocytes were FACS purified and stored in liquid nitrogen for telomere length measurements. Nine months post transplant, recipients were sacrificed and their bone marrow harvested. Once more, donor derived granulocytes were FACS purified and the telomere lengths of all granulocytes samples measured by Flow-FISH. 2.6 Serial transplantation Bone marrow was harvested by flushing the femora of a 21-22 week old C57B16 and TERT-KOxC57B16 mouse. Red blood cells were lysed and the remaining white blood cells counted. Forty C57B16/J S J L primary recipients were lethally irradiated (1100 rads) and transplanted with titrated wild type or TERT-KO W B M numbering one hundred thousand, one million or ten million cells. Ten mice received the smallest transplant dose but five mice received each of the larger doses. With the aid of FACS Vantage, donor derived W B M was sorted from the three primary recipients showing the highest level of reconstitution within each group after three months. Secondary recipients were lethally irradiated (1100 rads) and transplanted with five million W B M cells each. Tertiary transplantation was performed after an additional three months by, once more, FACS purifying donor derived W B M and transplanting each tertiary recipient with five million W B M cells. Furthermore, donor derived granulocytes were FACS purified at the time of each transplantation and three months after the tertiary transplantation and their telomere lengths measured by Flow-FISH. Peripheral blood was 31 analysed for donor derived granulocytes to evaluate reconstitution level one week prior to each transplantation. 2.7 Flow Cytometry Flow Cytometry was used to analyse the reconstitution of transplanted mice. lOOpl of peripheral blood was harvested from the tail vein of each mouse and the red blood cells lysed with ammonium chloride. Thereafter, the remaining leukocytes where incubated on ice for 20 minutes with the following antibodies: CD45.1-FITC, CD45.2-APC and Grl -PE; or in the case of a competitive assay: either Grl -PE and CD3-FITC or Macl-PE and B220-FITC. The cells were washed once with PBS + 2mM EDTA + 2% FCS and analysed on FACS Calibur. 2.8 MACS Magnetic beads were used in an attempt to isolate granulocytes for telomere measurements and to deplete the support cells of Sca l + cells. Bone marrow was harvested by flushing the femur or aspirating bone marrow. The red blood cells were then lysed with ammonium chloride and the remaining leukocytes incubated on ice for 20 minutes with either Scal-PE or Grl -PE. Thereafter, cells were washed once with PBS + 2mM EDTA + 2% FCS and subsequently incubated on ice for 20 min with lOpl of M A C S anti-PE microbeads per 10 total cells. The cells then received a second wash. Finally, the cells were either depleted of Sca l + cells or enriched for G r l + cells using Auto-MACS before being analysed on FACS Calibur for purity. 32 2.9 Bone marrow aspiration Mice were kept asleep with 3.5% isoflorine and 96.5% oxygen. A syringe needle was placed into the femur and the bone marrow aspirated. Following the procedure, mice were injected s.c. with 1.2pg buprenorphine for pain relief. 2.10 Flow FISH Bone marrow was harvested by either bone marrow aspiration or by sacrificing the mouse and flushing its femur and red blood cells lysed with ammonium chloride. Donor derived granulocytes were sorted on FACS Vantage and stored in liquid nitrogen. To measure telomere lengths with Flow-FISH, each sample (of 2 x l 0 5 - l x l 0 6 cells) was split in half and mixed with 2x10 s fixed cow thymocytes of known telomere length. One sample was to be stained, leaving the second unmarked to account for auto-fluorescence. D N A was then denatured for 15 minutes in 75% formamide at 87°C, followed by a 90 minute, room temperature, hybridization with 0.3pg/ml FITC-labelled protein nucleic acid which recognizes the telomere sequence. Excess probe was removed by several washes in a Hydra robotic washing station. The first four washes were performed with 75% formamide while the fifth contained only PBS. Next, the D N A was counterstained with LDS 751 at O.Olpg/ml for at least 20 minutes to visually separate the granulocytes from the cow thymocytes. Finally, the cells were analysed on FACS Calibur and telomere lengths calculated in Microsoft Excel. 33 2.11 DNA isolation D N A was isolated from FACS purified granulocytes using phenol extraction and ethanol precipitation. Briefly, cells were lysed for one hour at 55°C in ImM Tris, pH7.6, lOmM EDTA, 0.5% Sarkosyl, lOmM NaCI and lOOpg/pl Protease K. This was followed by a protease inactivation at 95°C for 20 minutes. 1 x volume of phenol:chloroform:isolamyl alcohol (25:24:1) was mixed thoroughly with the cell lysate and the D N A separated and extracted by centrifugation. One tenth volume of 3M sodium acetate, pH5.2 and 2.5 volume of 100% Ethanol was added and the D N A precipitated overnight at -20°C. This was followed by a 70% ethanol step and centrifugation. Lastly the D N A pellet was air-dried, resuspended in dF^O overnight and the D N A quality and quantity measured on a Nanodrop spectrophotometer. 2.12 Real Time Quantitative PCR Real time kinetic quantitative PCR measures the fractional number of cycles (CP) needed for a sample's fluorescence to cross a set threshold (Higuchi R 1993). A plot of CP versus the logarithm of the amount of input target D N A is seen to be linear. This allows for a simple relative quantification of unknowns by comparing their CP value to a standard curve derived from amplification of serial dilutions for reference D N A in the same plate (Cawthon R M 2002). Telomere PCRs and single copy gene PCRs were performed in separate 96 well plates. Two master mixes of PCR reagents were prepared, one with the telomere primer pair, the other with the single copy gene (36B4) primer pair. Each telomere reaction contained the following: 20ng DNA, 3mM MgCl2, 0.2uM telomere forward primer, 0.2pM telomere reverse primer, one fifth Light Cycler Fast 34 Start Master SYBR Green Kit and ddH 20 up to 20pl. Each single copy gene reaction contained the following: 20ng D N A , 3mM M g C l 2 , 0.3pM 36B4 forward primer, 0.3pM 36B4 reverse primer, one fifth Light Cycler Fast Start Master SYBR Green Kit arid ddF^O up to 20pl. Each sample was completed in triplicate. A telomere and 36B4 standard curve was performed for each of their respective plates at a 15-fold range from 3.75 to 56.25 ng/well. The telomere primers were as follows: telomere forward: 5' CGG TTT GTT TGG GTT TGG GTT TGG GTT TGG GTT TGG GTT 3', telomere reverse: 5' GGC TTG CCT T A C CCT T A C CCT T A C CCT T A C CCT T A C CCT 3'. The single copy gene primers were: 36B4 forward: 5' A C T GGT CTA G G A CCC G A G A A G 3', 36B4 reverse: 5' TCA A T G GTG CCT CTG G A G ATT 3'. A l l PCRs were performed on the Roche LightCycler LC480. The thermal cycling profile was performed as follows: Telomere: 95°C for 10 min, 35 cycles of 95°C for 5 seconds, 54°C for 10 seconds and 72°C for 60 seconds followed by a cooling to 4°C. For the 36B4 it was: 95°C for 10 min, 35 cycles of 95°C for 15 seconds, 56°C for 20 seconds and 72°C for 30 seconds followed by a cooling to 4°C. For each sample the LightCycler 480 Software was then used to generate the standard curve for each plate and to determine the dilution factors corresponding to the telomere and single copy gene amounts. Microsoft Excel was used to calculate the T/S ratio - the relative telomere to single copy gene ratio. 35 CHAPTER 3 RESULTS 3.1 Bone marrow stemming from a single HSC can only be serially transplanted twice whereas bone marrow stemming from 10 HSCs can be serially transplanted a minimum of three times The primary goal of this research was to clarify the role of aging and clonal exhaustion in hematopoietic stem cells. As such we began our studies by investigating the replicative potential of titrated highly purified HSCs using murine serial transplantation. Previous studies suggest that HSCs can only be serially transplanted 4-6 times in mice before they exhaust and can no longer accomplish reconstitution (Harrison DE 1982). As a result, HSCs are believed to have a limited replicative potential, raising concerns over its effect on the long term success of bone marrow transplantation. By serially transplanting titrated HSCs we aimed to investigate the relationship between the number of HSCs transplanted and their long term exhaustion. HSCs were isolated as cKit+Scal+Lineage" Hoechst side population cells (SP-KSL) from a C57B16 P-actin GFP donor mouse (Figure 3.1 and 3.2). Using FACS Vantage, single SP-KSL cells were, sorted into a 96 well plate and individual wells checked by fluorescent microscopy to confirm the presence of single cells. A second set of wells received 10 SP-KSL cells. c I I Furthermore, support cells were harvested from a C57B16/J mouse and depleted of Sca l + cells by Auto-MACS. Support cells were co-transplanted with one or 10 HSCs to provide the recipients with enough mature blood cells and progenitors to see them through the first two weeks following transplantation. This is necessary since the lethal irradiation destroys the recipient's hematopoiesis and the transplanted HSCs require 36 approximately 10 days to produce mature blood cells and establish a healthy hematopoietic system. Moreover, it is necessary to deplete the support cells of Scal + cells to remove all indigenous HSCs and prevent competition with the test HSCs. Figure 3.1 Single HSC transplantation. HSCs where isolated as cKit +Scal +Lineage" Hoechst side population cells (SP-KSL) from a GFP/CD45.1 donor mouse. Using FACS Vantage, single SP-KSL cells were sorted into a 96 well plate and individual wells checked by fluorescent microscope to confirm the presence of single cells. Supporter cells were harvested from a CD45.1 mouse and depleted of Seal 4 cells using Auto-MACS. Finally, lethally irradiated CD45.2 primary recipients were injected with 1 or 10 SP-KSL cells, along with 1 million GFP" support cells. m •» 6 7 5 Hoechst Figure 3.2 Isolation of cKit +Scal +Lineage" Hoechst side population cells by Flow Cytometry. After harvesting the bone marrow, red blood cells where lysed and the remaining bone marrow cells stained with Hoechst 33342 at 37°C for 90 minutes. Subsequent samples were stained with lineage-PE-Texas red, Kit-PE and Sca-APC antibodies at 4°C for 20 minutes. Using FACS Vantage, HSCs were purified cKit +Scal +Lineage" Hoechst side population cells. 37 1 or 10 HSC + •1-million, gfp-supporter cells 1 million GFP+ BM Total GFP+ BM Donor mouse GFP+ Primary recipient wt Secondary recipient wt Tertiary recipient wt Day 0 Month 11 Month 24 Figure 3.3 H S C serial transplantation. To examine the replicative potential of HSCs, SP-KSL cells were FACS purified and murine serial bone marrow transplantations performed. The primary recipients received either 1 or 10 highly purified HSCs (SP-KSL) while the secondary recipients received 1 million bone marrow cells originating from the initial donor. Finally, the tertiary recipients received the total GFP + cells present in each secondary recipient. After sorting single SP-KSL cells, lethally irradiated primary recipient mice were transplanted with either one or ten HSCs along with one million support cells (Figure 3.3 and table 3.1). One month after the transplantation, reconstitution was evaluated by investigating peripheral blood for donor derived white blood cells (Table 3.2). Mice were considered reconstituted if over 5% of the total white blood cells were donor derived. By this criterion seven out of the 30 recipients that originally received a single HSC transplant were reconstituted, with an average reconstitution of 19.3%. In the same manner four of the five mice that originally received 10 HSCs were reconstituted, with an average reconstitution of 43%. This level of reconstitution suggests only a fraction of the 10 KSL-SP cells successfully homed into and repopulated the recipient bone marrow. One year after the primary transplantation, a secondary transplantation was performed. The three most highly reconstituted mice were chosen from each group and one million 38 GFP whole bone marrow cells sorted from each mouse and transplanted into a single recipient each. One month later, peripheral blood was investigated for reconstitution. Although all secondary recipients received equal cell numbers there was a dramatic difference in reconstitution depending on the initial transplant. The repopulation was an average 12% when the cells originally stemmed from a single HSC whereas the repopulation was a strong 73% when the transplant originally stemmed from 10 HSCs, suggesting HSC proliferative history affects their proliferative capacity. At the time of tertiary transplantation both the single HSC group and the 10 HSC group had lost one member. As a result, the tertiary transplant was performed by sorting the total GFP + cells from the remaining two single HSC and two 10 HSC secondary recipients and transplanting them into one lethally irradiated recipient each (Table 3.1). The GFP + cells were transplanted along with 500,000 Seal" support cells. One month later reconstitution was evaluated by investigating peripheral blood for donor derived white blood cells. The mice that received bone marrow originating from a single HSC were not successfully reconstituted, whereas, the mice that received the bone marrow originating from the 10 HSCs showed 44% reconstitution (Table 3.2 and Figure 3.4). We concluded that this preliminary data suggest a strong connection between transplant dose and long-term repopulation capacity. Table 3.1 Primary, secondary and tertiary transplant Primary transplant Secondary transplant Tertiary transplant* 1 GFP + KSL-SP + 500,000 support cells 1,000,000 GFP + W B M 370,000 GFP + W B M + 500,000 support cells 180,000 GFP + W B M + 500,000 support cells 10 GFP + KSL-SP + 500,000 support cells 1,000,000 GFP + W B M 800,000 GFP + W B M + 500,000 support cells 420,000 GFP + W B M + 500,000 support cells *Total GFP cells were sorted from each secondary recipient and transplanted into one tertiary recipient each. 39 Table 3.2 Reconstitution of primary, secondary and tertiary recipients Primary recipients (only>5% reconstituted recipients are shown) Reconstitution (%) 1 KSL-SP (n=30) 32 2.X 12 11 10 KSL-SP (n=5) 60 39 17 Secondary recipients 1 KSL-SP* (n=3) 10 KSL-SP* (n=3) .X5 •X (>4 Tertiary recipients 1 KSL-SP* (n=2) 0 10KSL-SP* (n=2) 58 0 30 Peripheral blood was analysed 1 month post transplant and reconstitution calculated as a percentage of GFP + white blood cells. Shaded boxes represent mice used as donors for subsequent transplantation. *Original transplant Reconstitution of primary, secondary and tertiary recipients 100 1 2 3 T r an sp l an ta t i on # Figure 3.4 Reconstitution of primary, secondary and tertiary recipients. Peripheral blood was examined for donor derived cells. The bone marrow that stemmed from the single HSC could only be serially transplanted twice while the 10 HSC bone marrow could be serially transplanted at least three times. 40 3.2 Mouse telomere lengths can be accurately measured using a Flow-FISH based method In view of the fact that most somatic cells loose telomeric D N A upon proliferation, telomere length measurements have emerged as a valuable tool to evaluate replicative history. Consequently, in order to better understand HSC replicative potential, a reliable method to effectively measure the telomere lengths of murine HSCs was needed. In collaboration with Dr. Lansdorp at the Terry Fox Laboratory, U B C Vancouver, the Automated Multicolor Flow-FISH, a method used regularly in his laboratory was optimized and applied to murine HSCs. Automated Multicolor Flow-FISH is used routinely to measure the telomere lengths of human peripheral blood cells. The technique is used as follows. Nucleated cells are isolated from peripheral blood and, as an internal control, mixed with fixed cow thymocytes of a known telomere length. Next, the cells are fixed, D N A denatured and hybridized with a fluorescently labelled telomere-PNA probe that specifically recognizes the telomere D N A sequence. This is followed by an antibody staining step that allows individual cell groups to be analyzed seperately. However, due to the apparent loss of surface epitopes through the fixation step, only a limited number of cell types can effectively be immunophenotyped. Lastly, D N A is counterstained in order to visually separate the test cells from the cow thymocytes within each sample. The cells are then analysed by Flow Cytometry and telomere lengths calculated. This method has several important advantages, cells can be quiescent, many cells can be analyzed within a short period of time and subpopulations of cells can be analyzed separately based on differences in their light scatter properties and immunophenotype. Furthermore, most cell processing steps have been automated using a 41 robotic washing station which, in addition to the utilization of bovine thymocytes as an internal control, results in exceptionally accurate measurements (Baerlocher G M 2004). The Automated Multicolor Flow-FISH was originally set up and optimized for measuring the telomere lengths of human peripheral blood cells. However, the goal for this study was to use the Flow-FISH based method to measure telomere lengths in mice. Furthermore, it was desirous to perform the measurements at different time points within each mouse. As a result, there were two major challenges to be faced: First, laboratory mice have extremely long telomeres and second, the number of cells available for analysis is very limited. Our initial hope was that sampling blood from the tail-vein of a mouse would provide adequate cell numbers for telomere length measurements. Small amounts, of peripheral blood could be harvested at different time points providing valuable information on both HSC replicative history and potential within each mouse. With this in mind it was set out to explore whether bleeding a mouse provides sufficient cell quantities for telomere length measurements. Three hundred microliters of peripheral blood were collected from the tail vein of a single mouse. The red blood cells were lysed and the telomere lengths of the remaining peripheral blood cells measured. Results showed that the 300pl blood sample resulted in 3.75x105 cells which proved sufficient for telomere length measurements (Figure 3.5). 42 Total white blood cells 37 Kb 0 200 400 <S0O 800 .FS&Ht&t Figure 3.5 Blood sampling results in sufficient white blood cells for Flow-FISH based telomere length measurements. Peripheral blood was sampled from a mouse, red blood cells lysed and the telomere lengths of the remaining total white blood cells measured by Flow-FISH. Each sample is mixed with fixed cow thymocytes and stained with LDS 751 to visually separate the mouse cells from the bovine cells. Telomeres are hybridized with a FITC labelled telomere-PNA probe. Telomere lengths are calculated by comparing the FITC signal intensity of total white blood cells with that of the cow thymocytes of a known telomere length. An unstained sample is also used in the comparison to account for autofluorescence. 300ul of blood resulted in 3;75xl05 cells which proved to be sufficient for telomere length measurement. Up to this point the telomere measurements were performed on total white blood cells, however of more interest was the specific measurement of HSC telomere lengths. Unfortunately, due to exceedingly low HSC numbers it was not possible to directly measure the telomere lengths by Flow-FISH and it was decided to use granulocytes as substitutes for the HSCs. Granulocytes represent approximately 20% of the peripheral white blood cells and 80% of whole bone marrow. As opposed to T and B cells, all granulocytes are short-lived cells and do not express the telomerase enzyme. They can therefore be used as a surrogate marker for the telomere length of HSCs, assuming the number of cell divisions it takes for a HSC to give rise to a granulocyte is relatively constant and that telomere shortening in stem cells is primarily from replication and relatively constant with each cell division (Verfaillie C M 2002). The next objective was 43 to specifically measure the telomere lengths of granulocytes. After several attempts to stain the granulocytes at the antibody staining step of the Automated Multicolor Flow-FISH protocol it was evident that like most epitopes, the Gr l epitope is destroyed during the protocol's denaturation step. This results from the harsh denaturation and fixation condition which involves incubating cells in 75% formamide at 87°C. It became apparent that a different approach was needed and the decision was made to purify the granulocytes prior to measuring their telomere lengths. With that aim, granulocytes were labelled with magnetic beads and sorted on a magnetic column. Unfortunately, the majority of granulocytes were lost in the process, resulting in very low cell numbers that could not be used for Flow-FISH. The next attempt was to sort the granulocytes by FACS. The granulocytes were labelled with a fluorescent antibody and sequentially sorted on FACS Vantage. From 300pl of blood we retrieved approximately 30,000 granulocytes and measured their telomere lengths with Flow-FISH (Figure 3.6). The results showed that although there was not much variation between the two samples, 44 kb vs 44.7 kb, the cell numbers were still critically low. Consequently, it was not possible to accurately distinguish the small granulocyte population from debris and fixed bovine thymocytes. This meant that the resulting analysis was unreliable. This was a concern as we planned to use this method to measure the telomere lengths of donor derived granulocytes within bone marrow recipients. Since most bone marrow recipients are generally not 100% reconstituted the actual granulocyte numbers are expected to be even lower. 44 Granulocytes 44 Kb FSC.H*i£tt. Figure 3.6 Blood sampling resulted in insufficient granulocyte numbers for Flow-FISH based telomere length measurement. 300ul of peripheral blood was sampled from a mouse, red blood cells lysed and 30,000 granulocytes sorted on FACS Vantage. The granulocyte telomere lengths were measured by Flow-FISH. Due to low cell numbers, it was not possible to accurately distinguish the small granulocyte population from debris and fixed bovine thymocytes within each sample, resulting in unreliable analysis. Since blood sampling resulted in critically low cell numbers, the next approach was to retrieve granulocytes through bone marrow aspiration. There are several important advantages and disadvantages to this approach. Most importantly, bone marrow aspiration results in greatly elevated cell numbers, furthermore 80% of bone marrow cells are granulocytes. On the other hand, this procedure is more invasive and time consuming than blood sampling and can only be performed a limited number of times for each mouse. Bone marrow aspirations generally resulted in a yield of 1-2 million whole bone marrow cells. After labelling the granulocytes and sorting them on FACS we retrieved, on average, 1 million granulocytes from a non-transplanted mouse. This is considered enough to successfully measure telomere lengths when using Flow-FISH. The next step was to verify that the optimized Flow-FISH method could accurately measure the long telomeres laboratory mice possess. With that aim, bone 45 marrow was aspirated from a two month old wild type mouse and an eight month old TERT-KO mouse that was lacking telomerase expression and was therefore expected to have shorter telomere lengths. The red blood cells were lysed and granulocytes sorted on FACS Vantage. We retrieved 1.7 million granulocytes from the TERT-KO mouse and 1.2 million granulocytes from the wild type mouse. Their telomere lengths were measured by Flow-FISH. The results showed that the method is sensitive enough to detect the differences in telomere length and that the long mouse telomeres do not cause a problem (Figure 3.7). Wild type bone marow Granulocytes aoo eoo FSC-HeigM Tert-KO bone marow Granulocytes Figure 3.7 Bone marrow aspiration results in sufficient granulocyte numbers for Flow-FISH based telomere length measurements. To verify the optimized protocol, bone marrow was aspirated from a wild type mouse and a TERT-KO mouse which lacks the telomerase enzyme. As expected, the results show that the granulocytes from TERT-KO mice have shorter telomeres than those of wild type mice. 46 One of the concerns was that aspirating bone marrow may, by itself, cause increased telomere shortening. Removing large numbers of bone marrow cells may cause the remaining cells to proliferate in order to compensate for the cell loss, possibly resulting in greater telomere erosion. In order to test the validity of this concern, bone marrow was aspirated from the left femur of three mice. Three months later the mice were sacrificed and their bone marrow harvested by flushing the left and right femur separately. Granulocytes were sorted from each femur and their telomere lengths measured by Flow-FISH as previously described. Effect of bone marrow aspiration on telomere length 50 45 -\ 40 2 35 -] £ 30 cn •2 25 -! 0) a> 20 o £ 15 10 5 0 Right Left/aspirated Leg F igure 3.8 Effect of bone m a r r o w aspiration on telomere length. Bone marrow was aspirated from the left femur of 3 mice. 3 months later bone marrow was flushed from both left and right femurs and the telomere lengths of their granulocytes measured by Flow-FISH. The results show no relevant difference of telomere length between aspirated and non-aspirated femurs. 47 The results showed no relevant difference between the telomere lengths of the granulocytes from aspirated and non-aspirated femurs, suggesting the number of bone marrow cells aspirated is small enough and does therefore not affect telomere erosion to a measurable degree (Figure 3.8). It was concluded that the Flow-FISH method was fully optimized and can be used reliably in murine HSC aging research. Furthermore, the optimized protocol enables the measurement of telomere lengths of granulocytes at different timepoints within individual mice, providing a unique insight into the telomere length status (Figure 3.9). Bone marrow aspiration granulocytes Stained granulocyte* + cow thymocytes Unstained granulocytes + cow thymocytes Rbc lysis Sort granulocytes DNA denaturation -135°C Hybridization with telomere PNA Wash excess probe DNA counterstaining LDS 751 FACS Figure 3.9 Optimized Flow-FISH protocol. In collaboration with Dr. Peter Lansdorp at the Terry Fox Laboratory the Automated Multicolor F l o w - F I S H method was optimized for use in murine H S C aging research. Bone marrow is harvested by bone marrow aspiration and the red blood cells lysed. Granulocytes are sorted on F A C S Vantage and stored in liquid nitrogen. Each sample is split in two and mixed with fixed bovine thymocytes o f a known telomere length. D N A is then denatured and hybridized with a FITC-label led protein nucleic acid which binds specifically to the telomere sequence. Following washing the D N A is counterstained to separate the granulocytes from the bovine thymocytes. Finally, the cells are analysed on F A C S Calibur and telomere lengths calculated. 48 3.3 Mouse telomere lengths can not be accurately measured using a PCR based method In addition to measuring telomere lengths with Flow-FISH it was planned to use a PCR based method to further support our results. This method has some important advantages since it is simple, rapid and cheap yet does not require high cell numbers. The latter quality was especially important since murine bone marrow aspirations generally result in relatively low cell numbers. However, it had been presumed impossible to measure telomere lengths by PCR amplification since primers designed to hybridize to the T T A G G G and C C C T A A repeats would mostly result in primer-dimer derived products. Nevertheless, in 2002 Dr. Cawthon introduced a primer pair that resolved this problem and developed a PCR-based assay that successfully measured telomere lengths of human peripheral blood cells (Cawthon R M 2002). Furthermore, in 2006 Callicott et al attempted to adapt the human assay for use in mice (Callicott RJ 2006). To confirm the reliability of the optimized assay Callicott et al measured the telomere lengths of two mouse strains known to have very different telomere lengths: M. musculus that has greater than 20 kb of telomeric D N A and M. spretus that has only 5-10 kb of telomeric D N A (Coviello-McLaughlin G M 1997; Hemann M 2000; Kim S 2003). Moreover, Callicott et al compared the results with terminal restriction fragment analysis for the same samples.' The results revealed that the PCR-based method was sensitive enough to detect the difference in telomere length and correlated well with the Southern blot. However, the PCR measurements of the longer telomeres varied to a greater degree than the shorter telomere samples. This was of concern since most laboratory mice, including the strains used in the experiments detailed herein, have over 20 kb of 49 telomeric repeats. Throughout the optimization the work of Cawthon and Callicott was used as the basis for our PCR settings and reactions. In addition, since both Cawthon and Callicott used the Applied Biosystems Prism 7700 sequence detection system to perform their Real time PCR, the results of Gil et al (Gil M E 2004), detailing how Cawthon's method was modified for the Roche LightCycler were also implemented. D N A was isolated from test cells and the control cells using ethanol precipitation and phenol extraction. For each sample quantitative PCR was performed separately, using the telomere primers and primers that recognize a single copy gene, 36B4. Each telomere reaction contained 20ng of DNA, 3mM MgCh, 0.2pM telomere forward and reverse primers and one fifth by volume of Light Cycler Fast Start Master SYBR green Kit. The single copy gene reactions were identical except for the use of 0.3pM of 36B4 forward and reverse primers instead of the telomere primers Standard dilution curves were prepared at a 15-fold dilution range from 3.75 to 56.25 ng/well, using reference D N A along with either the telomere primers or the single copy gene primers. In the end, the telomere lengths were evaluated by calculating how much the test sample differed from the reference D N A sample in its ratio of telomere repeat copy number to single copy gene number. In order to optimize the PCR-based method, the optimal annealing temperature and cycle number for both primer pairs was sought. PCR was performed on a gradient PCR machine using temperatures of 52°C, 54°C, 56°C, 58°C, 60°C & 62°C for telomere primers and 46°C, 48°C, 50°C, 52°C, 54°C & 56°C for single copy gene primers. Furthermore, 5pl aliquots were taken at cycles 20, 25, 30 & 35 and then run on a 0.8% 50 agarose gel. The results of the telomere reaction showed the optimal telomere annealing temperature to be 54°C (Figure 3.10). , k h 20 cycles 25 cycles ladder 52 54 56 58 60 62 52 54 56 58 60 62 ladder 52 54 56 58 60 62 52 54 56 58 60 62 Figure 3.10 Agarose gel electrophoresis following PCR with telomere primers. Annealing temperature ranged from 52°C - 62°C and cycle number ranged from 20-35. 1 kb ladder from Invitrogen. The single copy gene reaction revealed two bands at the lower temperatures including a very low molecular weight band that was consistent at all annealing temperatures (Figure 3.11). In order to determine the actual size of the smaller band the 35 cycle product was run on an 8M Urea, 15% polyacrylamide gel, confirming that the smaller band was around 75bp (Figure 3.12). This confirmed that this was the correct band but not a result of primer-dimer pairs. It also served to confirm that the larger band appearing at the lower annealing temperatures was unspecific and as such it could be concluded that the optimal 36B4 annealing temperature was 56°C. Furthermore, it was decided to use 35 cycles for both the telomere and single copy gene reaction in order to 51 guarantee that all samples would reach their crossing point, this being the fractional number of cycles needed for a sample's fluorescence to cross a set threshold. 1 kb 20 cycles 25 cycles 1 kb 30 cycles 35 cycles Figure 3.11 Agarose gel electrophoresis following PCR with 36B4 primers. Annealing temperature ranged from 46°C - 56°C and cycle number ranged from 20-35. 1 kb ladder from Fermentas. 50 bp l a d d e r 35 cycles Figure 3.12 Acrylamide gel electrophoresis following PCR with 36B4 primers. Annealing temperature ranged from 48°C - 56°C. The 36B4 primers amplify a 75 bp band. 50 bp ladder from Fermentas. 52 To evaluate whether PCR could be used to measure the telomere lengths of laboratory mice which have greater than 20kb long telomeres, a set of samples was measured with both our optimized PCR-based method and Flow-FISH. Six. granulocyte samples were measured from telomerase deficient mice and eight samples from C57B16 mice. A l l samples were taken from secondary bone marrow recipients. The,results clearly showed that whereas the Flow-FISH method was sensitive enough to detect the difference in telomere length, 24 kb in TERT-KO granulocytes and 34 kb in C57B16 mice, the PCR-based measurements did not detect any difference (Figure 3.13). It was therefore concluded that PCR can not be used to measure the long telomeres most laboratory mice possess. 35 -30 -CO 25 -8 20 -o 15 -10 -5.-0 -PCR versusFlov/FISH o 12 • F lo* Fish Wild ' type ——v Telomerase deficient 16 Figure 3.13 Comparison of telomere length measurements with PCR vs Flow-FISH. Bone marrow was harvested from secondary recipients and the donor derived granulocytes sorted. The original donor was either a wild type (C57B16) mouse or a TERT-KO mouse. Flow-FISH measures telomere lengths in kb whereas PCR measures it as a T/S ratio, the factor by which the test sample differed from the reference DNA sample in its ratio of telomere repeat copy number to single copy gene copy number.. The T/S ratio was multiplied by a factor of ten in order to more efficiently compare it to the Flow-FISH results. The results suggest that PCR-based methods are not sensitive enough to measure the length of the long telomeres found in laboratory mice. 53 3.4 Characterization of telomerase deficient HSC With the primary goal of clarifying the role of aging and clonal exhaustion in HSC, this study planned to investigate the relationship between telomerase status and HSC exhaustion. TERT-KO mice were received as a kind gift from Lee Harrington of The Ontario Cancer Institute, Toronto (Erdmann N 2004). The TERT-KO strain had been backcrossed over 10 times on a C57B16 background after which it was maintained by heterozygous breeding. TERT-KO mice lack the mTERT gene encoding the reverse transcriptase, a core component of the telomerase enzyme (Erdmann N 2004). Whereas a modest twofold reduction in telomerase levels in humans results in premature death from complications of aplastic anaemia or immune deficiency, first generation TERT-KO mice appear normal and healthy, thanks to the long telomeres mice possess (Dokal I 2001; Collins K 2002; Fogarty PF 2003). However, after three to four generations of homozygous TERT-KO breeding, the ongoing telomere shortening leads to infertility, reduced viability and defects in highly proliferative tissues such as the hematopoietic system, skin and digestive system (Lee HW 1998; Herrera E 1999). With the aim of characterizing first generation telomerase deficient HSC, the TERT-KO cKit+Scal+Lineage" population was compared with its wild type counterpart. Results revealed that TERT-KO K S L cells represent 0.121% of W B M and wild type K S L cells represent 0.126% of W B M cells (Figure 3.14). This suggests similar frequency of HSCs in the two mouse strains. 54 Wild type 101 HP ioJ ioq Lineage T E R T - K O 101 10' 10J Lineage 2.55% "'•'Iff ' ' ' • 1 ' ' ' i "VI ' ' ' 1 ' 1 ' 0° 101 102 103 1Q Seal • 2.12% 1 ' ' '""1 1 1 ' 1' 1 r . f . i ' i . Seal Figure 3.14 Characterization of first generation TERT -KO cKit +Scal +Lineage" cells. First generation KSL cells were compared with wild type KSL cells. TERT-KO KSL cells represent 0.121% of W B M and wild type KSL cells represent 0.126% of W B M cells. Furthermore, in order to determine whether wild type bone marrow has a competitive advantage over telomerase deficient bone marrow, under circumstances where telomere lengths should be sufficient, we set up a competitive primary transplantation. Groups of five lethally irradiated (1100 rads) C57B16/JSJL recipients received chimeric transplants that contained 100,000 wild type C57B16 WBM cells or TERT-KO telomerase deficient WBM; along with 100,000 C57B16/JSJL competitor WBM cells. Sixteen weeks post transplant, peripheral blood was stained for the presence of viable CD45.2 granulocytes, macrophages, T-cells and B-cells (Figure 3.15). 55 Competitive bone marrow transplantation 70 -, Wild Wild Wild Wild Wild TERT- TERT- TERT- TERT- TERT-type type type type type KO KO KO KO KO Cells competing with 100,000 Ly5.1 whole bone marrow Figure 3.15 Competitive bone marrow transplantation. 100,000 wild type or telomerase deficient CD45.2 W B M was transplanted along with 100,000 CD45.1 W B M cells into five lethally irradiated CD45.1 mice each. Results showed that the wild type bone marrow had no obvious competitive advantage over the telomerase deficient bone marrow. The results showed that neither the C57B16 W B M nor the TERT-KO W B M cells were out competed by the C57B16/J S J L W B M cells and that the wild type bone marrow had no obvious competitive advantage over the telomerase deficient bone marrow. However, it was noted that the telomerase deficient bone marrow had unusually high levels of granulocytes and macrophages but low levels of lymphocytes. Nevertheless, even though there are slight differences in repopulation patterns the results did not show an obvious competitive disadvantage of telomerase deficient bone marrOw in a primary transplantation setting. 56 3.5 Telomere lengths are maintained over a 9 month period post transplantation regardless of telomerase status Previous studies suggest the telomeres of HSCs shorten as a result of proliferation, despite detectable telomerase activity (Allsopp RC 2001). Furthermore, the telomerase enzyme is believed to be required to slow down telomere shortening and to extend the replicative lifespan of HSCs (Allsopp RC 2003a). As a result, HSCs are assumed to have a limited replicative potential, raising concerns over its effect on the long term success of bone marrow transplantation. Earlier murine studies focusing on HSC telomere shortening and exhaustion have all utilized serial transplantation models (Iscove N N 1999; Allsopp RC 2001; Samper E 2002; Allsopp RC 2003a; Allsopp RC 2003b). To the author's knowledge no studies have explored the rate of telomere shortening of HSC within individual mice. Using the optimized Flow-FISH method detailed previsously, bone marrow can be sampled for telomere length measurement at different time points within individual mice. This provides a unique insight into the rate of telomere shortening under more ordinary circumstances. As a result, the optimized Flow-FISH method was utilized to address questions regarding the rate of telomere shortening in relation to telomerase status. With that aim, lethally irradiated mice (1100 rads) were transplanted with l x l O 6 W B M cells from either wild type or TERT-KO mice, resulting in an 83±5% reconstitution, regardless of bone marrow origin (Figure 3.16). The bone marrow was sampled by bone marrow aspiration at weeks six and 12 and donor derived granulocytes FACS purified for telomere length measurement (Figure 3.17). Furthermore, nine months post transplant, recipient mice were finally sacrificed, bone 57 marrow harvested and donor derived granulocytes FACS purified for telomere length measurement. "P106wbm Donor mouse Primary recipient Bone marrow CD45.2or CD45.1 aspiration TERT-KO/CD45.2 Day 0 Week 6 Bone marrow aspiration Month 3 Bone marrow aspiration Month 9 Figure 3.16 Longitudinal studies. Longitudinal studies were performed on mice receiving l x lO 6 whole bone marrow cells from either wild type or TERTKO mice. Granulocytes were FACS sorted from bone marrow biopsies at different time points within individual mice, for Flow FISH telomere length measurement. CD45.2 1tr 1cr CD45.2 1 0 H Figure 3.17 Purification of donor derived granulocytes. A. Bone marrow was aspirated from recipient mice and donor derived (CD45.2) Gr l positive cells were sorted on FACS Vantage. B. The purity of the sorted CD45.2 + Grl + cells was generally over 95%. 58 Average telomere length 40 -i 35 H E f 30 4 c 1,000,000 wild type — 1,000,000 TERT-KO .2 20 J 10 -0 Donor 6 weeks 3 months 9 months Tii me posttransplantation Figure 3.18 Telomere length of donor derived granulocytes 0 days, 6 weeks, 3 months and 9 months after transplantation. Bone marrow was sampled by bone marrow aspiration and donor derived granulocytes were sorted on FACS Vantage and their telomere lengths measured by Flow-FISH. Results showed no relevant telomere shortening throughout the 9 month period, regardless of telomerase status. Error bars represent standard deviation. The results show that the two mice strains have very different telomere lengths, C57B16 has 34 kb of telomere repeats whereas TERT-KO mice have only 24 kb (Figure 3.18). However, contradictory to published studies, our data showed no shortening of telomeres within the 9 month period post transplantation, regardless of telomerase status (Allsopp RC 2001; Allsopp RC 2003a; Allsopp RC 2003b). 59 3.6 Telomere lengths are maintained throughout serial transplantations regardless of telomerase status and original transplant dose In the spirit of the primary goal to clarify the role of aging and clonal exhaustion in HSCs, it was decided that using serial transplantations to further investigate the relationship between the number of cells transplanted, telomerase status and their long term exhaustion would be beneficial. The initial assumption was that the low transplant doses would force the HSCs to proliferate extensively in order to meet the demands of the recipient's hematopoietic system. This would cause accelerated shortening of telomeres and possibly premature clonal exhaustion. Furthermore, the lack of telomerase expression may further increase the rate of telomere erosion and consequently, clonal exhaustion. In order to address these questions regarding telomere length and clonal exhaustion relating to the number of cells transplanted and telomerase status, bone marrow cells were titrated and murine serial bone marrow transplantations performed. Primary recipients were transplanted with l x l O 5 , l x l O 6 or l x l O 7 W B M cells from either wild type or TERT knock out mice which lack the mTERT gene encoding the telomerase enzyme (Figure 3.19). It is estimated that HSCs have a frequency of one in ten thousand to one in a hundred thousand total W B M cells (Bonnet 2002). As a result, the transplant doses used for the primary transplantation were equivalent to approximately 1-10, 10-100 and 100-1000 HSCs, respectively. A secondary transplant was performed three months later with 5xl0 6 donor-derived W B M cells and a tertiary transplant was performed at the six month time-point, also with 5x106 donor-derived W B M cells. 60 r i 0 5 w b m n o 6 wbm n o 7 wbm 5 million Ly5.2 bm 5 million Ly5.2 bm Donor mouse CD45.2 or TERT-KO/CD45.2 Primary recipient CD45.1 Secondary recipient Tertiary recipient CD45.1 CD45.1 Day 0 Month 3 Month 6 Figure 3.19 Whole bone marrow serial transplantation. To address questions regarding telomere length in relation to the number of cells transplanted and telomerase status, the number of W B M cells was titrated and murine serial bone marrow transplantations performed, transplanting the primary recipients with lx lO 5 , l x lO 6 or l x l 0 7 W B M cells from either wild type or TERT knock out mice. A secondary transplant was performed three months later with 5x l0 6 donor-derived W B M cells from the 3 primary recipients showing the highest level of reconstitution within each group. A tertiary transplantation was performed three months later with 5x l0 6 donor-derived W B M cells. A l l recipient mice were lethally irradiated (1100 rads). Donor derived granulocytes were sorted for telomere length measurements, at the time of each transplantation. Peripheral blood was examined for donor derived cells three months after each transplantation and reconstitution calculated as a percentage of donor derived granulocytes (Figure 3.20). The results showed no relevant relationship between the number of cells transplanted, telomerase status and their long term exhaustion. However, it may be argued that the level of reconstitution in the primary recipients was slightly affected by the transplant dose. Furthermore, at the lowest transplant dose lack of telomere expression appeared to negatively affect the reconstitution levels. 61 B Reconstitution of primary recipients at Z months post transplant 100.330 1,000 000 10.000.000 Transplantalion dose Reconstitution of secondary recipients at 3 months post transplant Transplantation dose D Reconstitution of tertiary recipients at 3 months post transplant LL 1.000.000 Orignial transplant Average reconstitution - • - 100 000 wild type —• -1.000.000 v»ld type —*— 10.000 000 vald type - - • 100,000 TERT-KO —1 -1.OO0.000 TERT-KO — « — 10.000.000 TERT-KO Primary Secondary Recipient Tertiary Figure 3.20 Reconstitution of W B M cells at 3 months post primary, secondary and tertiary transplantation. Peripheral blood was examined for donor derived cells and reconstitution calculated as a percentage of donor derived granulocytes. A. Reconstitution of primary recipients. The three most highly repopulated primary recipients from each group were used as donors for the secondary transplantation. B. Reconstitution of secondary recipients. C. Reconstitution of tertiary recipients. A-C. Each mouse is represented by a single column. Grey columns represent mice whose original transplant was wild type W B M cells, while the black columns represent mice whose original transplant were TERT-KO W B M cells. D . Average reconstitution of primary, secondary and tertiary recipients. Error bars represent standard deviation. In addition to measuring reconstitution, telomere length measurements were used to evaluate HSC replicative history and potential clonal exhaustion. Bone marrow was harvested at the time of transplantation and donor derived granulocytes sorted from the recipients and their telomere lengths measured by Flow-FISH (Figure 3.21 and 3.22). 62 A B CD45.2 CD45.2 Figure 3.21 Purification of donor derived granulocytes. Bone marrow was harvested at the time of transplantation and cells sorted for telomere length measurements and subsequent transplantations. A . Donor derived (CD45.2) Gr l positive cells were sorted on FACS Vantage for telomere length measurements. B. The purity of the sorted CD45.2 +Grl +cells was generally over 95% C. Donor derived (CD45.2) W B M cells were sorted for subsequent transplantation. D. The purity of the sorted CD45.2+ cells was generally over 95%. A v e r a g e T e l o m e r e l e n g t h 40 35 30 I 2 5 1 20 0 I 1 5 10 5 0 -•—100,000 wild type 1,000,000 wild type 10,000,000 wild type + 100,000 TERT-KO • 1,000,000 TERT-KO * 10,000,000 TERT-KO Donor 1 "recipient 2°recipient 3°recipient Mice Figure 3.22 Telomere lengths of donor derived granulocytes 3 months after primary, secondary and tertiary transplantation. Bone marrow was harvested and donor derived granulocytes were sorted on FACS Vantage and their telomere lengths measured by Flow-FISH. The results showed no relevant telomere shortening throughout the serial transplantation, regardless of telomerase status and original transplant dose. Error bars represent standard deviation. 63 Interestingly, although reconstitution levels decreased with each transplantation, the telomere data shows no relevant telomere shortening . throughout the serial transplantation, regardless of telomerase status and original transplant dose. It is worth noting an interesting phenomenon that further supports the reliability of Flow-FISH based telomere length measurement. In samples with low purity, the contaminant endogenous G r l + cells were easily detected as a distinct subset with longer telomeres (Figure 3.23). 1,000,000 wild type 10,000,000 wild type i d - F t T C s t ^ l T C 100,000 TERT-KO 1,000,000 TERT-KO :tW=!TC t tS-FlTC Figure 3.23 Flow-FISH detects contaminant endogenous G r l + cells in low purity samples. Donor derived granulocytes were sorted and telomere lengths measured by Flow-FISH. Results showed two distinct peaks representing recipient and donor cells in low purity samples. To confirm that the second Grl population was due to low purity and did not reflect the existence of alternative mechanisms of telomere maintenance or the clonal selection of bone marrow repopulation cells with longer telomeres, the recipient (C57B16/JSJL) telomere length was independently established and. estimated to be approximately 45 kb (Figure 3.7). Furthermore, only weakly repopulated tertiary recipients resulted in impure FACS sorts and sample purity consistently corresponded with the size of the second Gr l population (Figure 3.24). 64 Figure 3.24 Flow FISH effectively distinguishes between donor and recipient cells in low purity samples. After sorting for donor derived granulocytes, the purity of the sorted CD45.2 + Grl + cells was evaluated. The size of the contaminant endogenous cells corresponded consistently with the size of the second G r l + population, confirming the recipient origin. In conclusion, although our preliminary SP-KSL transplantation data suggested a connection between transplant dose and long-term repopulation capacity, both our longitudinal studies and serial transplantation studies showed no relevant relationship between the number of cells transplanted, telomerase status and their long term exhaustion. Average telomere length remained constant throughout the longitudinal studies and serial transplantations, regardless of original transplant dose and telomerase status. However, even though telomere lengths were maintained throughout the serial transplantations, the reconstitution efficiency decreased with each transplantation. 65 CHAPTER 4 DISCUSSION Hematopoietic stem cells (HSCs) are adult stem cells that reside in the bone marrow and are responsible for giving rise to and maintaining the entire repertoire of mature blood cells. HSCs are defined by their capacity to undergo both differentiation and self-renewal. This is a key property of HSCs as the limited lifespan of mature blood cells requires their continuous production and only HSC self-renewal can ensure that sufficient number of stem cells is available to meet the demands of hematopoiesis over a normal lifespan (Lansdorp 1997). In addition to regular cell turnover, the hematopoietic system must be able to respond rapidly to illness and trauma. As a result, HSCs possess an exceptionally high replicative potential (Szilvassy SJ 2003). It is clear that under normal circumstances, HSCs are highly capable of maintaining blood cell production throughout a normal lifespan. However, under certain conditions, including bone marrow transplantations, HSCs may possibly reach the limit of their replicative potential (Elwood NJ 2004). Transplanted HSCs are limited in number and thus need to proliferate extensively in order to meet the requirements of the host. If further demands are placed on the hematopoietic system, such as following a chronic infection or i f too few donor stem cells are transplanted, long-term survivors of stem cell grafts may be at risk of developing anemia due to HSC exhaustion (Lansdorp P M 2005). HSC do express detectable levels of the telomerase enzyme. Nevertheless, previous murine studies have shown that these cells still experience telomere shortening and are not immortal (Allsopp RC 2003a). This suggests that the level of telomerase expression in HSC is not sufficient to completely prevent telomere erosion and concerns have been raised regarding its effect on the long term success of bone marrow transplantations. The primary goal of the 66 experiments presented in this thesis was to clarify the role of aging and clonal exhaustion in hematopoietic stem cells using murine transplantations. As mice possess significantly longer telomeres than humans do (5-20 kb in humans vs 20-100 kb in mice), HSC exhaustion is not expected through a single bone marrow transplantation (Kipling D 1990; Starling JA 1990; Prowse K R 1995; Hemann M 2000). Previous studies suggest HSCs can be serially transplanted 4-6 times before the HSCs exhaust and can no longer accomplish reconstitution (Harrison DE 1982). Furthermore, telomerase is believed to be required to slow telomere shortening and extend the replicative lifespan of HSCs, although telomerase over-expression is not sufficient to extend their transplantation capacity (Allsopp RC 2003a; Allsopp RC 2003b). In our study we set out to investigate the relationship between the number of cells transplanted, the length of their telomeres and their long term exhaustion. We initiated our studies by investigating the replicative potential of titrated numbers of HSCs. HSCs were isolated as Kit+Sca+Lineage" Hoechst side population cells and lethally irradiated recipients were transplanted with either 1 or 10 HSCs along with supporter cells (Figure 3.1-3.3). The secondary transplantation was performed with 1 million donor derived cells stemming from either 1 or 10 HSCs and finally the tertiary recipients received the total donor derived cells present in each secondary recipient. The results showed that bone marrow stemming from a single HSC could only be serially transplanted twice whereas bone marrow stemming from 10 HSCs could be serially transplanted at least three times (Figure 3.4). There are two possible explanations for these results, one supporting HSC limited replicative potential and the other HSC dilution. If HSCs have a limited replicative potential, a smaller transplant dose would 67 force each HSC to proliferate to a greater extent in order to meet the needs of the recipient. The small transplant dose would therefore reach its replicative limit sooner, explaining the apparent premature exhaustion of bone marrow stemming from a single HSC. However, since HSCs fail to regenerate to normal levels after even a single transplantation (Harrison DE 1982; Harrison DE 1990), the evident connection between transplant dose and long-term,repopulation capacity may be due to HSC dilution (Iscove N N 1999). Even though all secondary transplantations were performed with 1 million donor derived W B M cells, it is possible that the same number of W B M cells from different recipients does not contain the same number of HSCs. Bone marrow stemming from 1 HSC could contain fewer long-term repopulating cells then bone marrow stemming from 10 HSCs. As a result, the tertiary transplant originating from 1 HSCs may not have contained enough HSC numbers to successfully repopulate its recipient. The apparent HSC exhaustion may therefore simply be due to HSC dilution. In view of the fact that most somatic cells loose telomeric D N A upon proliferation, telomere length measurements have emerged as a valuable tool to evaluate replicative history. Consequently, in order to better understand HSC replicative potential, a reliable method was needed to effectively measure the telomere lengths of murine HSCs. In collaboration with Dr. Lansdorp at the Terry Fox Laboratory we optimized the Automated Multicolor Flow-FISH used in his laboratory. The-Flow-FISH based method was originally set up and optimized for measuring the telomere lengths of human peripheral blood cells. However, our goal was to use Flow-FISH to measure telomere lengths in mice. As a result, we were faced with two major challenges: First, laboratory mice have extremely long telomeres and second, cell numbers are limited. We 68 successfully overcame these problems and established an optimized protocol that can be used effectively in murine HSC aging research (Figure 3.9). Bone marrow is sampled by bone marrow aspiration and granulocytes sorted and their telomere lengths measured by Flow-FISH. Granulocyte telomeres can be used as a surrogate for HSC telomeres, assuming the number of cell divisions it takes for a HSC to give rise to a granulocyte is relatively constant and that telomere shortening in stem cells is primarily resulting from replication and relatively constant with each cell division (Verfaillie C M 2002). To prevent infection and limit growth factor mediated granulocyte expansion, mice were kept in pathogen free environment. The optimized protocol enables us to measure telomere lengths at different timepoints within individual mice, providing us with a unique insight into the telomere length status. The minimum detectable difference in telomere length and the reproducibility of the method are in the range of 0.2-0.5 kb. Furthermore, since mice are thought to lose 50 to '100 base pairs of telomeric D N A with each successive round of cell division (Prowse K R 1995; Allsopp RC 2001), we expected to reliably detect shortening of telomeres after approximately 10 cell division rounds. In addition to measuring telomere lengths using Flow-FISH the plan was to use a PCR based method to further support our results. In 2002, Dr. Cawthon developed a PCR-based assay that effectively measured telomere lengths of human peripheral blood cells (Cawthon R M 2002). We attempted to optimize his method for use in murine HSC aging research, however the long telomeres most laboratory mice possess, proved to be an obstacle that we could not overcome. In our hands, mouse telomere lengths can not be accurately measured using a PCR based method (Figure 3.13). 69 With the primary goal of clarifying the role of aging and clonal exhaustion in HSC we planned to investigate the relationship between telomerase status and HSC exhaustion. We were fortunate to receive TERT-KO mice as a gift from Lee Harrington and we set out to characterize their HSCs. TERT-KO mice lack the mTERT gene encoding the telomerase enzyme (Erdmann N 2004). The TERT-KO Kit+Sca+Lineage" population was compared with its wild type counterpart. Results revealed that TERT-KO K S L cells represent 0.121% of W B M and wild type K S L cells represent 0.126% of W B M cells (Figure 3.14). This suggests similar frequency of HSCs in the two mouse strains. Furthermore, in order to determine whether wild type bone marrow has a competitive advantage over telomere deficient bone marrow, under circumstances where telomere lengths should be sufficient, a competitive primary transplantation was performed. Groups of five lethally irradiated (1100 rads) C57B16/J S J L recipients received chimeric transplants that contained 100,000 wild type C57B16 W B M cells or TERT-KO telomerase deficient W B M ; along with 100,000 C57B16/J S J L competitor W B M cells (Figure 3.15). Results showed that the wild type bone marrow had no obvious competitive advantage over the telomerase deficient bone marrow. However, it was noted that the telomerase deficient bone marrow had unusually high levels of granulocytes and macrophages but low levels of lymphocytes. Furthermore, the impaired B lymphopoiesis and increased myelopoiesis was present 16 weeks after being transplantated into young wild type recipients. This contradicts published studies that claim that the impaired B lymphocyte development and increased myelopoiesis seen in TERT-KO mice is an environmental effect and that both the B lymphopoiesis and 70 myelopoiesis become normalized when transplanted into young wild type mice (Ju Z 2007). To our knowledge no studies have explored the rate of telomere shortening of HSC within individual mice over time. Using our optimised Flow-FISH based method we studied the rate of telomere shortening within primary B M transplant recipients, in relation to telomerase status. Lethally irradiated mice were transplanted with l x l 0 6 W B M from either a wild type or a TERT-KO donor (Figure 3.16). Telomere lengths of donor derived granulocytes were measured at the time of transplantation (day 0) and 6 weeks , 3 months and 9 months after transplant. Surprisingly, our data showed no shortening of telomeres within the 9 month period post transplantation, both in wild type and telomerase K O B M (Figure 3.18). This surprising finding is in contradiction with previous studies (Allsopp RC 2001; Allsopp RC 2003a; Allsopp RC 2003b), and potential causes of this apparent discrepancy will be discussed below. With the aim of further investigating the relationship between the number of cells transplanted, telomerase status and their long term exhaustion, we measured telomere lengths in donor granulocytes in mice subjected to serial transplantation. Lethally irradiated primary recipients were transplanted with 1x10s, l x l O 6 or 1,107 W B M cells from either a wild type or a TERT-KO donor (Figure 3.19). The secondary and the tertiary transplantations were performed at a three month interval with 5xl0 6 donor derived W B M cells. Interestingly, results showed no relevant relationship between the number of cells transplanted, telomerase status and their long term exhaustion. However, reconstitution efficiency defined as the percentage of donor derived granulocytes did decrease with each transplantation, regardless of original transplant dose and telomerase 71 status (Figure 3.20). Furthermore, average telomere length remained constant throughout the serial transplantations, regardless of original transplant dose and telomerase status (Figure 3.22). In conclusion, even though telomere lengths were maintained throughout the serial transplantations, the reconstitution levels decreased. There are several possible explanations for these surprising results, the first being HSC dilution. When HSCs are transplanted into a lethally irradiated recipient, they proliferate and self-renew extensively to meet the needs of the recipient. However, they never reach normal levels of HSCs but are believed to platform after reaching only 4% of normal HSC numbers (Iscove N N 1999). As a result, when a secondary transplantation is performed, the number of long-term reconstituting cells may be far lower than in the first transplant and the number of HSCs in the tertiary transplant is even lower. This would explain why reconstitution levels decrease with each transplantation even though HSC telomere lengths are maintained. Nevertheless, i f HSC dilution plays a major role, we would have expected the reconstitution levels to be more dependent on the original transplant dose. The second explanation involves telomere-independent barriers. It can not be ruled out that HSC replicative potential may be limited by more then just the length of their telomeres. In 2003, Allsopp et al studied the effect of TERT over-expression on the long-term transplantation capacity of HSC and noted that although the transgenic HSCs maintained their telomere lengths they could still only be serially transplanted 4 times (Allsopp RC 2003b). Furthermore, since it is the shortest telomere length rather than the average telomere length that limits replicative potential, they used Q-FISH to assess the distribution of telomere lengths and noted no detectable shift in telomere length to shorter 72 sizes. They concluded that stabilization of telomere length is not sufficient to sustain indefinite transplantation capacity and telomere-independent barriers may also limit the transplantation capacity of HSCs. Telomere independent intracellular timers are well known in other cell types, including oligodendrocyte precursor cells, where the accumulation of some intracellular proteins such as p27 and p i 8 and progressive decrease of Id4 allows them to control the number of times they divide before they stop and terminally differentiate into postmitotic oligodendrocytes (Durand B 1997; Kondo T 2000; Tokumoto Y M 2002; Raff 2006). The third explanation focuses on the level of HSC turnover after bone marrow transplantation. In adults, the more primitive the population, the greater the fraction that are quiescent. Whereas most terminally differentiating erythroid and myeloid cells are actively cycling, HSC are naturally quiescent cells (Eaves CJ 2006) Furthermore, the quiescent state is believed to be a key property for the maintenance of HSCs (Arai F 2004). It is possible that HSC proliferate only moderately following bone marrow transplantation, and that it is the more differentiated progenitors they give rise to that proliferate extensively in order to meet the recipient's needs. Under these circumstances, HSC turnover would be slow and their telomere lengths would not be compromised. Nevertheless, decreased reconstitution levels would still have to be explained by HSC dilution or telomere-independent barriers. There is no doubt that our telomere data strongly contradicts published studies. Previous studies by Allsopp et al suggested both wild type and telomerase deficient HSC telomeres shorten as a result of serial transplantation, furthermore they concluded the rate of telomere shortening was dependent on telomerase status and 73 transplant dose (Allsopp RC 2001; Allsopp RC 2003a; Allsopp RC 2003b). This disagreement may partly be explained by differences in study designs and methods used for telomere length measurements. Whereas Allsopp et al measured the telomere lengths of Sca+Kit+Lineage" cells with Q-FISH and W B M cells with Southern blot, we used granulocytes as surrogates for HSCs and measured their telomere lengths with Flow-FISH. A l l measurements were performed blindly and all samples were processed in parallel. Q-FISH, however, uses digital images of metaphase chromosomes, after in situ hybridization with a fluorescently labelled telomere probe, to evaluate telomere lengths. It requires cells to be stimulated in culture, arrested in metaphase and mounted onto slides (Baerlocher G M 2004). Q-FISH measures telomere lengths of individual chromosomes within a limited number of cells and is therefore less quantitative than Flow-FISH. Also, errors are common due to intersession variability of signal intensity and lack of homogeneity of the light field over time (Canela A 2007). Southern blot was used to measure W B M telomere lengths as the method requires high cell numbers. In addition, southern blot is known to generally overestimate telomere lengths as there are no restriction enzyme sites directly at the end of the telomere repeats (Baerlocher G M 2004). The fact that the telomere data from both our longitudinal studies and our serial transplantations complement each other remarkably well and show consistent telomere maintenance, verifies the reliability of the Flow-FISH method and strongly suggests that the results presented here are real. Furthermore, based on published literature, we trusted Flow-FISH to reliably detect the expected shortening of telomeres. According to the literature, mice are believed to lose 50 to 100 base pairs of telomeric D N A with each successive round of cell 74 division (Prowse K R 1995; Allsopp RC 2001). Furthermore, transplanted HSCs have been shown to increase 10-20-fold over the number initially transplanted, after which they are maintained at a plateau level (Sauvageau G 1995; Pawliuk R 1996; Iscove N N 1997; Sauvageau G 2004). Serial transplantation studies have shown that when HSCs are taken from reconstituted mice in the steady state and again injected into secondary recipients, they once more increase 10-fold over the number injected. Four successive serial transplantations were shown to achieve a cumulative 8400-fold increase over the number of HSCs initially transplanted (Iscove N N 1997). Our primary recipients were transplanted with l x l O 5 , l x l 0 6 and l x l O 7 W B M cells followed by a secondary and tertiary transplant of 5x106 donor-derived W B M cells. It is estimated that HSCs have a frequency of one in ten thousand to one in a hundred thousand total W B M cells (Bonnet 2002). As a result, the transplant doses used for the primary transplantation were equivalent to approximately 1-10, 10-100 and 100-1000 HSCs, respectively. Since four population doublings are needed for the transplanted HSCs to increase 10-20-fold over the number initially transplanted, we expected a telomere shortening within the primary recipient of approximately 0.2-0.4 kb from the self-renewal divisions alone. By the end of the tertiary transplantation, a total telomere shortening of at least 0.55-1.1 kb was expected to result from wild type HSC self-renewal divisions. However, these calculations do not take into account asymmetric divisions, and lack of telomerase expression was expected to result in increased telomere shortening. Since the Flow-FISH minimum detectable difference in telomere length is in the range of 0.2-0.5 kb, based on current literature we expected to reliably detect the expected shortening of telomeres^ Nevertheless, expected telomere shortening does does not exceed the minimum 75 detectable difference in telomere length by much. As a result, current literature need only moderately overestimate HSC cycling following transplantation in order for the expected results to fall outside the sensitivity of the Flow-FISH method Finally, it can not be excluded that HSC telomeres may stay long because of a telomerase independent mechanism active in stem cells. According to the literature, some cells are believed to generate telomeric D N A through recombination events, a mechanism known as alternative lengthening of telomeres (ALT). A L T is believed to involve inter-telomeric copying, where single-stranded D N A at one telomere terminus invades another telomere and uses it as a copy template resulting in net increase in telomeric sequence (Henson JD 2002). Conversely, the ALT-related mechanisms have only been clearly identified in abnormal situations, including human tumors, immortalized human cell lines but also telomerase-null mouse cell lines and late generation telomerase-null mice (Bryan T M 1995; Hande MP 1999; Herrera E 2000; Niida H 2000). There is some evidence that recombinational telomere lengthening may occur in some mouse cells in vivo under exceptional conditions. Herrera et al studied mice that had lost telomerase activity due to a knockout mutation. They found that the germinal centre lymphocytes lost 7 kb of telomere repeats post immunization, consistent with the proliferation in the absence of telomerase. However, in later generations of telomerase-null mice there were only a few germinal centers, but the lymphocytes had elongated their telomeres by an average of 12 kb, suggesting the utilization of an A L T -like mechanism (Herrera E 2000). However, A L T cells are generally characterized by a great heterogeneity of telomere size (Henson JD 2002) which we did not observe in our experiments. 76 In conclusion, although our preliminary SP-KSL transplantation data suggested a connection between transplant dose and long-term repopulation capacity, both our longitudinal studies and serial transplantation studies showed no relevant relationship between the number of cells transplanted, telomerase status and their long term exhaustion. Using our optimized Flow-FISH protocol we find that telomere length is maintained over a nine month period post transplantation. 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