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Phenotypic characterization of mouse mammosphere-initiating cells (Ma-ICs) indicates they represent a… Tegzeš, Andrea 2006

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P H E N O T Y P I C C H A R A C T E R I Z A T I O N O F M O U S E M A M M O S P H E R E -I N I T I A T I N G C E L L S (MA-ICS) I N D I C A T E S T H E Y R E P R E S E N T A N O V E L P R O G E N I T O R P O P U L A T I O N by: A N D R E A T E G Z E S B.Sc. Okanagan University College, 2003 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE In THE FACULTY OF GRADUATE STUDIES (Experimental Medicine) UNIVERSITY OF BRITISH COLUMBIA April 2006 ©Andrea Tegzes, 2006 A B S T R A C T Elucidation of the mechanisms regulating the development of the mammary gland would be facilitated by quantitative in vitro assays for mammary epithelial stem cells. Recent studies have shown that tight clusters of cells containing primitive clonogenic mammary cells are produced when dissociated human breast cells are cultured in serum-free liquid suspension cultures containing epidermal growth factor (EGF) and fibroblast growth factor (FGF). Single cell suspensions prepared from mouse breast tissue formed similar "mammospheres" under these conditions but cell aggregation was a significant factor in determining, their size and numbers. Addition of 1% methylcellulose to the culture medium prevented cell aggregation and resulted in the generation of smaller mammospheres over a 7-14-day period at a constant frequency of ~1 per 100 cells plated over a 100-fold range of cell concentrations. Interestingly, when subsets of antibody-stained cells in suspensions of freshly dissociated mouse mammary glands were first isolated by fluorescence activated cell sorting, the mammospheres they generated in semi-solid cultures were not from fractions containing highly purified cells with either mammary epithelial stem cell or progenitor activity (as identified by an ability to repopulate a cleared mammary fat pad, or generate colonies in standard 2D cultures containing feeder layers). These findings provide a quantitative assay for a previously uncharacterized clonogenic cell that is present in mouse breast tissue that may facilitate the survival or propagation of primititive mammary cells in vitro but whose developmental relationship to the mammary epithelial hierarchy has yet to be established. ii T A B L E O F C O N T E N T Abstract ii Table of Content iii List of Figures '. , v Abbreviation . vii Acknowledgements viii Dedication ix Chapter 1. Introduction 1 1.1. Overall objective and rationale 1 1.2. Background 2 1.2.1. Histological description of the mammary gland 2 The major types of epithelial cells that constitute the mammary gland 2 Basement membrane and stroma 4 1.2.2. Mammary gland development 7 Ontogeny , '. 7 Extrinsic regulation 10 Intrinsic regulation 13 1.2.3. Concept of a differentiation hierarchy of mammary epithelial cells 15 Mammary stem cells 15 Mammary progenitors 19 Mammospheres 21 1.3. Thesis objectives 25 Chapter 2. Materials and Methods 26 2.1. Mice 26 iii 2.2. Preparation of mammary cell suspensions...., .26 2.3. Isolation of subsets of mammary cells 27 2.4. Mammosphere culture 28 2.5. Ma-CFC Assay . '. 29 Chapter 3. Results. , : .' 30 3.1. AIM 1 - To investigate the relationship between mammosphere formation and cell aggregation 30 3.2. AIM 2 - To investigate whether semi-solid mammosphere cultureS can support the survival of Ma-CFCs 34 3.3. AIM 3 - To analyze the distribution of Ma-ICs in different phenotypically defined subsets of mammary cells 36 3.4. AIM 4 - To investigate the capacity of generate Ma-CFCs 41 Chapter 4. Discussion and conclusion .- ...42 Bibliography 47 iv L I S T O F F I G U R E S Figure 1-1. Whole mount preparation of a developing murine mammary gland 3 Figure 1-2. Schematic representation of mammary acinus 3 Figure 1-3. Schematic diagram depicting the generation of mammary ducts 5 Figure 1 -4. Diagram illustrating the pre-pubertal (linear) and adult (cyclical) phases of mammary gland development [20] 8 Figure 1-5. Composite of whole-mount stained mammary glands found at different stages development 10 Figure 1-6. A model describing some of the key endocrine and paracrine signals that regulate mammary branching morphogenesis 11 Figure 1-7. Experimental design of the retroviral marking study that demonstrated the presence of stem cells within the mouse mammary gland 16 Figure 1-8. FACS profiles of mammary epithelial cells distributed according to their expression of a (A) or P (B) integrin and CD24 18 Figure 1-9. Mammary epithelial colonies 19 Figure 1-10. A putative hierarchy of mammary epithelial differentiation 21 Figure 1-11. 2° and 3° neural colony forming ability of cells different sized primary colonies. '. 22 Figure 1-12. High proliferative potential of cells derived from colonies of >2 mm diameter suggests self-renewal ability of the initial CFC [38] 23 Figure 3-1. Differing yields of mammospheres generated in liquid and semi-solid media.... 31 Figure 3-2. Contrasting size of 7 day-old mammospheres generated in semi-solid and liquid cultures 32 Figure 3-3. Mammosphere yields in methylcellulose cultures 33 Figure 3-4. Maintenance of Ma-CFCs in mammosphere culture 35 Figure 3-5. FACS plot showing the 6 populations assayed for Ma-ICs and Ma-CFCs 37 Figure 3-6. Distribution of Ma-ICs among different subpopulations of mammary cells 38 Figure 3-7. Distribution of Ma-CFCs among different subpopulations of mammary cells.. 39 Figure 3-8. Ma-ICs of the same phenotype give rise to mammospheres seen after either 7 or 21 days of growth 40 v i A B B R E V I A T I O N r ADAM a disintegrin and metalloproteinase AREG Amphiregulin BM Basement membrane CFC Colony-forming cell ECM Extracellular Matrix EGF Epidermal Growth Factor EGFR Epidermal growth factor receptor EpCAM Epithelial Cell Adhesion Molecule ER Estrogen receptor ESA Epithelial Specific Antigen FGF Fibroblast growth factor FGFR FGF receptor GH Growth hormone GHR GH receptor Hh Hedghehog IGF Insulin-like growth factor IGF1R IGF-1 receptor IGFBPs • IGF-binding proteins Ma-CFC Mammary colony-forming cell Ma-IC Mammosphere-initiating cell Ma-SC Mammary stem cell MMP Matrix metalloproteinase MRU Mammary repopulating unit MUC1 Mucin 1 PR Progesterone receptor SMA Smooth muscle actin SP Serine protease TEB Terminal end bud TGF-p Transforming growth factor (3 TIMP Tissue inhibitor of metalloproteinases v i i A C K N O W L E D G E M E N T S I would like to thank the following people for their support on this project: Connie Eaves Allen Haves Afshin Raouf John Stingl Sharon Louis PeterEirew Special thanks to: Kaveh Eskandar Afshari Sanja Sekulovic Darcy Wilkinson Pavle Vrljicak . I would also like to thank the National Science and Engineering Research Council for awarding me an Industrial M.Sc. Studentship and Stem Cell Technologies, Inc. for additional support. Operating funds for this project from the Stem Cell Network are also acknowledged. V l l l D E D I C A T I O N Mojim najbli^ima %apunupodrsku i neiscrpni i^yor snage i samopou^danja. C H A P T E R 1. I N T R O D U C T I O N 1.1. O V E R A L L O B J E C T I V E A N D R A T I O N A L E Adult tissue stem cells are characterized by their ability to undergo self-renewal and multilineage differentiation [1]. Differentiation results in the generation of progeny that perform the specialized functions of the tissue, whereas stem cell self-renewal allows a lifelong supply of the cells required to produce and maintain the functional integrity of the tissue [1]. -The investigation of mammary stem cells (Ma-SCs) and their regulation has been gready limited by a lack of methods to prospectively isolate and propagate these cells "in vitro. Over the years, many studies have been devoted to addressing these issues. As described in detail below, there has been major recent progress in identifying mammary cells with stem cell properties as displayed in jn vivo transplantation assays [2, 3]. Evidence that primitive human mammary cells may be maintained in suspension cultures that promote the formation of "mammospheres" has also been reported [4]. These findings raised the possibility that such mammospheres might represent colonies derived from mammary stem cells and their detection might establish the basis of a quantitative in vitro assay for murine mammary stem cells. The overall goal of this thesis was to explore these possibilities. 1 1.2. B A C K G R O U N D 1.2.1. H I S T O L O G I C A L D E S C R I P T I O N O F T H E M A M M A R Y G L A N D T H E MAJOR T Y P E S O F E P I T H E L I A L C E L L S T H A T C O N S T I T U T E T H E M A M M A R Y G L A N D The major function of the mammary gland is to produce and secrete milk — a proteinaceous fluid composed of a and (3 caseins, lactoferrin, growth factors, long chain of polyunsaturated fatty acids, bile salt-stimulated lipase, and anti-infectious oligosaccharides and glycoconjugates [5]. Structurally, the mammary gland is a tubuloalveolar epithelial organ (Figure 1-1) that consists of a double layered network of 2 major differentiated cell types: a centrally located layer of tightly packed cuboidal luminal cells and a surrounding sparser outer layer of myoepithelial cells with contractile properties [6] (Figure 1-2). 2 Figure 1-1. Whole mount preparation of a developing murine mammary gland. The gland consists of a ductal network that protrudes throughout the fat pad. The large round black structure on the left side of the gland is a lymph node. (Taken from[2]). Current Opinion in Cell Biology Figure 1-2. Schematic representation of mammary acinus. The structure is comprised of a layer of cuboidal epithehal cells and a surrounding layer of contractile myoepithelial cells. The acinus is enveloped by a protein-rich basement membrane (BM). (Taken from [7]). 3 In the alveoli, luminal cells differentiate further into milk-secreting cells.. Immunostaining has shown that smooth muscle actin (SMA) and cytokeratin 14 are found exclusively in the basally located myoepithelial cells whose function is to force the milk through the ducts and out of the mammary gland [8, 9]. In contrast, luminal cells express cytokeratins 8, 18 and 19, and epithelial cell adhesion molecule (EpCAM). ErbB2 (the EGF receptor, EGFR) is expressed at higher levels on luminal epithelial cells, but weak expression of ErbB2 can also be seen on myoepithelial cells [10, 11]. CD49 (oc6 integrin) is expressed on the cells found adjacent to the basement membrane (BM) [11, 12]. Cytokeratin 6 has been found to be expressed by a small percentage of luminal epithelial cells in the subtending ducts [6]. B A S E M E N T M E M B R A N E A N D S T R O M A The mammary gland itself is embedded in a complex environment that actually makes up approximately 80% of the breast tissue [9]. Separating the mammary epithelium from the rest of the breast tissue is a BM consisting primarily of collagen IV, laminin, fibronectin and heparan sulfate [9]. The BM is encircled by an outer ring of extracellular matrix (ECM) which, in turn, is surrounded by loose connective tissue. It is thought that the BM is synthesized in early development by cells within the terminal end buds [13] and in the adult by the myoepithelial cells [9, 14] (Figure 1-3). 4 luminal MM fibroblasts body coils Figure 1-3. Schematic diagram depicting the generation of mammary ducts. The ducts develop as a result of the proliferation of terminal end bud (TEB) cells. Ducts are surrounded by a layer of BM and a collar of fibroblasts. Thinning of the BM (dotted lines) is observed at the tips of TEBs. (Taken from [13]). Both the BM and ECM elements have been implicated in the regulation of mammary gland branching morphogenesis [15]. The rest of the breast stroma consists of vascularized adipose tissue, fibroblasts and immune cells. The spatial relationship of mammary epithelial cells to their surroundings affects many aspects of mammary epithelial cell development and function [13]. Extracellular matrix, receptors for components of the matrix as well as ECM-degrading enzymes [13] influence the location and functional integration of mammary epithelial cells. During ductal 5 development luminal epithelial cells establish basal, lateral, and free apical surfaces via cell-cell, cell-matrix and associated cytoskeletal interactions [16]. In the absence of polarity, luminal epithelial cells are unable to form ducts and secrete milk proteins [15]. Evidence for this comes from the studies that demonstrated the failure of isolated luminal cells to form properly polarized hollow spheres when cultured in type I collagen gels unless myoepithelial cells were added. Myoepithelial cells are known to secrete laminin-1, one of the key components of BM and so it is interesting that small amounts of reconstituted BM containing laminin-1 appeared able to rescue the polarity of luminal cells in the absence of myoepithelial cells. In addition to cell-matrix adhesion, cell-cell interactions through desmosomal adhesions are thought to play an important role in establishing ductal polarity [17]. For example, alveolar morphogenesis was shown to be abrogated by peptides that directly interfered with desmosomal cell-cell interactions [13, 17]. ECM also regulates the architecture of the mammary ductal network by providing physical barriers whose remodelling by matrix degrading enzymes (such as matrix metalloproteases (MMPs) and serine proteases (SPs)) allows the protrusion of ducts through the mammary stroma [13, 18]. The deposition of matrix proteins, such as fibronectin, is believed to create ductal branch-point selection. During pregnancy, the degradation of the ECM is thought to be important for the development of the acinar structures that will later produce milk [18]. ECM can also affect the development of the mammary gland by binding and sequestering signaling molecules such as amphkegulin (AREG), fibroblast growth factors 6 (FGFs), transforming growth factor-(3 (TGF-p), and insulin-Hke growth factor (IGF)-binding proteins 1 -6. The remodeling of the ECM can thus be envisaged to be involved in controlling the interactions of these cytokines with their cognate receptors on mammary epithelial cells. 1.2.2. M A M M A R Y G L A N D D E V E L O P M E N T O N T O G E N Y The mammary gland can be viewed as a modified apocrine sweat gland [19] that develops in 2 distinct phases as illustrated in Figure 1-4: a linear phase (ontogeny) and repeated cyclical phases related to pregnancy and lactation, and to a lesser degree, the menstrual cycles. 7 Embryonic (E10-Birth) • Prepubescent Immature (e.g. 3 week, growth queiscent) T Postpubescent Immature (e.g. 5 week, growth active) T Mature (10-12 week) (growth quiescent) Involution ( \ Pregnancy (Apoptosis, I The Mammary Cycle I (Alveolar regresion and \ / growth and remodelina) V J secretory ^ *y Lactation (Milk secretion) Figure 1-4. Diagram illustrating the pre-pubertal (linear) and adult (cyclical) phases of mammary gland development [20]. The formation of the mammary gland in the mouse begins on embryonic day 10 (E10) and is characterized by the initial development of the mammary streaks — two lines of epidermally-derived thickened epithelium that run anterior to posterior, symmetrically displaced off the ventral midline [20]. At E l l , the presumptive epithelium forms the precursor of the nipple region and becomes associated with the underlying condensed mammary mesenchyme. Mammary mesenchyme is comprised of concentrically oriented fibroblasts that are sensitive to testosterone and estrogen [21]. The bud stage, that appears on E12.5, is marked by the generation of ductal progenitors [20] that are responsible for the invasion of another type of mesenchymal tissue — the forerunner of the fat pad, which is 8 comprised of pre-adipocytes. Both the mammary mesenchyme and the fat pad are integral to the development of a normal mammary gland [21]. Three distinct phases of mammary gland development are recognized in female mice after their birth: a pre-pubescent growth phase (0-5 weeks), a post-pubescent growth phase (5-10 weeks) and an adult, stable phase (10-12 weeks) [20]. Before puberty, the elongation of mammary ducts through the mammary fat pad occurs at a pace that is analogous to the animal's overall growth rate [8]. At puberty, the gonadal hormones accelerate the growth of the mammary gland and lead to the development of large club-shaped TEBs at the tips of the ducts [8]. These TEBs are comprised of 4 to 6 layers of undifferentiated "body cells" and a surrounding layer of "cap cells" (Figure 1-3) [8, 22]. These 2 populations have been postulated to contain mammary stem cells as well as precursors of luminal and myoepithelial. cells. TEBs then appear to undergo invagination to form two TEBs, both of which give rise to subtending ducts [21]. This continues until the ductal system reaches the limits of the fat pad space. The TEBs then regress leaving a complete ductal system inplace [20, 22] (Figure!-5). 9 Figure 1-5. Composite of whole-mount stained mammary glands found at different stages development. From left to right and top to bottom: Newborn, 4 week virgin, 6 week virgin, 10 week virgin, 9th day of pregnancy, and 16th day of pregnancy. (Reproduced from [23]). The characteristic pattern of cytokeratin expression by luminal and myoepithelial cells is maintained throughout the pubescent period and also in nulliparous adult females. In early pregnancy, many of the proliferating luminal cells in the alveolae express cytokeratins 6 and 14 but the expression of these then disappears at the end of lactation [6]. E X T R I N S I C R E G U L A T I O N Post-pubertal development of the mammary gland is highly influenced by ovarian and pituitary hormones. It is believed that pituitary gonadotropic hormone (GH) acts on mammary stromal cells to elicit the expression of insulin-like growth factor-1 (IGF-1) which 10 then stimulates TEB formation and epithelial branching. During puberty, estrogens and progesterone further enhance the activity of IGF-1 to stimulate side branching (Figure 1-6) [24]. Figure 1-6. A model describing some of the key endocrine and paracrine signals that regulate mammary branching morphogenesis. Estrogen also affects expression of the epidermal growth factor receptor (EGFR) - a receptor tyrosine kinase that binds several ligands that promote mammary development. One of them is AREG, a previously mentioned transmembrane precursor whose expression is upregulated during puberty [25]. AREG is proteolytically cleaved by transmembrane 11 metalloproteinase ADAM (a disintegrin and metallo-proteinase)-17 (TNF-a-converting enzyme; TACE) to activate EGFR on nearby stromal cells [26]. AD AM 17 and AREG are expressed on mammary epithelial cells, while EGFR is present in the stroma [26] thus illustrating the type of epithelial-stromal cross-talk that is thought to regulate mammary gland development. However, FGFs 2 and 7 support the growth and branching of cultured EGFR-null mammary organoids, whereas EGFR agonists and FGFs fail to support the growth of organoids lacking FGF receptor 2 (FGFR2)[13]. FGFR2b is expressed on mammary epithelial cells and is required for forming embryonic mammary structures, as is stromal FGF 10 [13]. The roles of stromal FGFs and their epithelial receptors have been described in the branching of mammalian lung, salivary gland and kidney epithelia [13] suggesting that similarly mechanisms may be involved in mammary gland development. When pregnancy and lactation occur, mammary epithelial cells undergo a peak in rate magnitude of proliferative activity [20]. These changes are directed first by estrogen and progesterone produced by the ovary, then by estrogen, progesterone and somatotropin produced by the placenta, prolactin from the pituitary gland and adrenocorticoids from the adrenal gland. In combination with local growth factors, cellular/extracellular matrix interactions and cell-cell interactions [21], an extensive network of milk-secreting alveolae develop at the ends of the ducts [23]. Then when lactation ceases, a massive wave of apoptosis occurs and the mammary gland reverts to its pre-pregnant tubular structure. 12 I N T R I N S I C R E G U L A T I O N Pathways that govern development of normal mammary gland are speculated to be the same ones that are deregulated during mammary carcinogenesis. Based on many studies involving malignant transformation, several pathways were found to play key roles in mammary gland development. The most prominent of these are the Hedgehog (Hh), Notch and Wnt signaling pathways. Hh signaling was initially discovered as responsible for Drosophila embryo patterning [27] and subsequendy was recognized to be a significant regulator of development in mammals. In mammary development, disruption of either the receptor protein Patched-1 (Ptchl) or the transcription factor Gli-2, a downstream target of the Hh signaling cascade, led to defects in ductal morphogenesis and pre-cancerous growth patterns [13] consistent with major roles in epithelial-stromal interactions during ductal development [9]. The Notch group of receptors represents another set of molecules found to play a crucial role in cell fate determination of many tissues [13, 28], Overexpression of constimtively expressed Notch4 under the control of the mouse mammary tumor virus (MMTV) promoter was found to inhibit the differentiation of normal breast epithelial cells, and result in the development of poorly differentiated adeno-carcinomas [13, 28], The Wnt family of secreted proteins acts, in part, through a complex of receptors that cause stabilization of intracellular P-catenin [13, 29, 30]. P-catenin is then translocated to the nucleus where it binds and activates a variety of transcription factors. Inhibition of p-catenin 13 signaling blocks mammary development and pregnancy-induced proliferation [13, 16, 28]. Retroviral transduction of activated P-catenin in transgenic mice produced epithelial cancers, similarly to MMTV activated expression of Wnt [13]. In addition to signaling pathways, transcription factors (TFs) are also major intrinsic regulators of many aspects of mammary gland development. Among the families of TFs thus far implicated, Hox proteins have attracted particular interest. Homeobox genes represent one of the largest families of nuclear proteins that regulate multiple aspects of morphogenesis and cell differentiation to establish spatial patterns [20]. Many of these Tfs are organized in four gene clusters (HoxA-HoxD). The homeobox domain that they all share includes a 183bp sequence that contains a helix-loop-helix (HLH) motif. The HLH domain binds DNA by inserting the recognition helix into the major groove of the DNA double helix and its ammo-terminal arm into the minor one. The activity of homeobox proteins. appears to be regulated by FGF activation and sonic hedgehog (SHH) signaling [20]. In the mammary gland, expression of several homeobox genes, including Hoxc-6,, Hoxc-8, Hoxd-8, Hoxd-9 and.Hoxd-10 appears to be regulated developmentally and in response to the level of estrogen [20, 31]. Disruption of normal development and function of the mammary gland was demonstrated in mutant mice carrying various deletions of the paralogous genes Hoxa-9, Hoxb-9 and Hoxd-9 [32]. While single mutant lines lacking Hoxa-9 or Hoxb-9 showed only a small decrease in newborn survival, Hoxd-9 disruption alone reduced survival to below 50%. Double mutant combinations showed synergistic 14 effects and further reduced survival, suggesting functional cooperativity [32, 33]. When examined throughout postnatal development Hoxd-9 was expressed highly in periductal fibroblasts and in the ductal epithelium in the vkgin female, but was only weakly expressed in these cell types during pregnancy. Hoxd-9 was also upregulated in the developing alveolar epithelium relative to subtending ducts, further supporting the notion that it plays a role in regulating alveolar cell differentiation [31]. 1.2.3. C O N C E P T O F A D I F F E R E N T I A T I O N H I E R A R C H Y O F M A M M A R Y E P I T H E L I A L C E L L S M A M M A R Y S T E M C E L L S The large output of mammary cells required to allow the mammary gland to repeatedly expand during successive pregnancies is thought to reflect the activity of subpopulations of progenitor cells that ultimately derive from a persisting population of undifferentiated multipotent Ma-SCs [28]. The earliest indication of such a multi-step process was obtained from electron microscopic observations [34]. These studies identified a population of "small light cells" that were postulated to include both stem cells and derivative progenitors with more restricted proliferative and differentiative abilities (sometimes referred to as transit amplifying cells) [34]. Subsequently, in 1998, formal evidence of murine Ma-SCs was obtained from experiments in which retrovirally marked mammary tissue fragments were transplanted into the pre-cleared mammary fat pads of syngeneic recipients [35] (Figure 1-7). 15 Figure 1-7. Experimental design of the retroviral marking study that demonstrated the presence of stem cells within the mouse mammary gland. Small fragments of mammary epithelium from Czechll MMTV-infected mice were transplanted into cleared mammary fat pads of syngeneic hosts. About 80% of each of the reconstituted glands was analyzed using Southern blots to examine the pattern of viral integrations, and the rest was used for further transplants into secondary mice. If the glands had been derived from several different progenitors, no clear pattern of MMTV-insertions would have been detected. In 20 out of 30 different outgrowths, a distinct and easily detected pattern of MMTV was seen, suggesting a clonal origin of the outgrowths. Fragments of the regenerated glands transplanted into new hosts were shown to produce new outgrowths that 16 displayed the same pattern of MMTV insertion sites as the original outgrowth, providing evidence of self-renewal of the original stem cell. Drawing from [35]. In these experiments, it was found that complete mammary structures displaying a common integration pattern were obtained suggesting their origin from a single cell [35]. Similarly, the demonstration that the same patterns were obtained upon transfer of cells from the primary regenerated glands to the cleared fat pads of secondary mice showed that the original cell could also execute self-renewal divisions. More recently, this transplantation assay has been successfully adapted for single cell suspensions of mouse mammary epithelial cells and limiting dilution analysis has been used to allow quantification of the cells that regenerate the -.complete mammary structures obtained. The cells thus detected are thus detected in this in vivo transplant assay are referred to operationally as mammary repopulating units (MRUs). MRUs are assumed to represent the Ma-SCs of the mammary gland as they have also been shown to have extensive self-renewal activity in serial transplantation experiments [2, 3]. MRUs (Ma-SCs) constitute ~0.1% of the cells in the breast of an adult virgin female mouse (i.e. ~1,400 MRUs in a breast containing 2 million cells [2]). Mouse MRUs have been characterized phenotypically by assaying subpopulations of dissociated mammary cells stained with antibodies to various cell surface markers and then isolated by fluorescent-activated cell sorting. (FACS) [12]. Stingl et al found that MRUs express intermediate levels of CD24, high levels of CD49f and low levels of stem cell antigen 17 (Sca-1). On the basis of these markers, MRUs could be obtained at a purity of approximately 5% (Figure 1-8) [2]. Similarly, Shackelton et al [3] found that MRUs were CD29++CD24+ and could be isolated in that population at a similar purity (Figure 1-8). CD29 is a pi integrin that forms a heterodimer with CD49f (a6 integrin) that is thought to required for expression of the integrin on the cell surface [36], and these concordant results are consistent with this concept. Figure 1-8. FACS profiles of mammary epithelial cells distributed according to their expression of a (A) or p (B) integrin and CD24. The functionally defined mammary repopulating unit (MRU)-rich fraction is found to be in the subset of cells expressing high levels of the a6 or its pi integrin partner and intermediate levels of CD24 [2, 3]. The left panel is reproduced from Reference 2 and the right panel from Reference 3. 18 Future experiments will likely identify additional markers that allow this rare fraction of cells to be obtained at even higher purities that will then enable more in-depth studies of the mechanisms that determine their self-renewal and differentiation properties. M A M M A R Y P R O G E N I T O R S The growth and differentiation of mammary cells in vitro has also been studied. Colonies of adherent epithelial cells can be obtained when mammary cells are seeded at low densities onto irradiated fibroblasts and cultured in a supportive medium (Figure 1-9) [11]. In assays of human mammary cells, the differentiation of luminal and myoepithelial lineages is preserved and hence colonies can be typed according to the mature cells they contain. However, when colonies are generated from murine cells, the cells produced within the colonies display evidence of epithelial to mesenchymal transition and thus cannot be reliably used to infer the lineage commitment of the cells from which the colonies arose. Figure 1-9. Mammary epithelial colonies. A . A colony generated from a mammary colony-forming cell (Ma-CFC) in the 2D assay. B . A colony generated by a multipotent progenitor in the 3D matrigel assay. The bar in (B) is 10 \im [2]. 19 Ma-CFCs have been recently shown to be present in the mouse mammary gland at a 20-fold higher frequency than MRUs and hence comprise a larger population in each gland (—3.0 x 104 in a gland containing 2 million cells) [2, 3]. Murine Ma-CFCs have also been characterized in cell sorting experiments. Like MRUs, Ma-CFCs in the virgin adult female mammary gland express both CD24 and CD49f, but the levels of CD24 are higher and the levels of CD49f are lower levels on the Ma-CFCs, which allows their almost complete separation from MRUs (Figure 1-8) [2]. When single cell suspensions of mammary cells are cultured in semi-solid "matrigel" cultures (so-called 3D cultures) containing prolactin, acinar-like structures are produced (Figure 1-9 B). , These structures have been reported to contain both luminal and myoepithelial cells organized into lobuloalveolar and ductal structures containing milk proteins [4]. ("Matrigel" is a reconstituted extract of the mouse Englebreth-Holm-Swarm tumor and consists mosdy of laminin [15]). Taken together, these findings suggest a hierarchical model of murine mammary cell differentiation as outlined in Figure 1-10. However, many features of this model have yet to be established. In the human system, cell separation and serial culture experiments have clearly established the existence of distinct populations of luminal-restrictedmyoepithelial restricted and bipotent progenitor populations. However, as noted above, this has not yet been possible with murine cells. 20 1 I , Mammary stem cell Detected by in vivo fat pad assay ( # Multipotent progenitor r Detected by in vitro 2D and 3D assays Lineage-restricted | | progenitors Luminal Mfmpithelmt Differentiated progeny Figure 1-10. A putative hierarchy of mammary epithelial differentiation. Mammary stem cells give rise to multipotent progenitors. These then produce lineage-restricted progeny that ultimately differentiate into either luminal or myoepithelial cells. M A M M O S P H E R E S In 1992, Reynolds et al inoculated cells of the subventricular zone of mouse forebrain into a liquid media and cultured them under low adhesion conditions [37]. Some of the cells in the heterogeneous cell clusters obtained (which they called neurospheres) appeared to be multipotent neural precursor cells since they could generate similar structures that also 21 contained multipotent neural precursors for several passages [19]. Subsequent experiments in collagen showed that neural colonies could be derived from single cells (Neural Colony-Forming Cells) [38] and that the size of these colonies varied according the proliferative potential of the cell of origin. Specifically, neural stem-like cells were found to form larger spheres than cells with more restricted neural progenitor activity (Figures 1-11 and 1-12) [39]. >2 mm 1-2 mm 0.5-1 mm < 0.5 mm colony size Figure 1-11. 2° and 3° neural colony forming ability of cells different sized primary colonies. Only cells isolated from neurospheres larger than 2 mm in diameter always formed tertiary neurospheres (from [38]). 22 16,00 -r 14.00 -12.00 -"g 10.00 -I 8.00-3 6.00 • 4.00-2.00 • o.oo 4 — i — i — i — i — i — i — i — i — i — i — P3 P4 P5 P6 P7 P8 P9 P10 P11 P12 Passage # Figure 1-12. High proliferative potential of cells derived from colonies of >2 mm diameter suggests self-renewal ability of the initial CFC [38]. More recendy, several groups have reported the generation of similar cell clusters when dissociated cells from breast tissue are cultured under similar low adhesion conditions and they have called the structures obtained "mammospheres" [4]. The protocol described by Dontu et al resulted in the generation of multicellular structures containing human mammary epithelial cells, including cells expressing CD49f and CD 10, both of which have been recendy implicated as potential markers of primitive human mammary epithelial cells [40]. Furthermore, when cells from these mammospheres were transferred to a collagen substratum, some then differentiated into colonies of cells that expressed human luminal 23 (ESA, MUC1, and cytokeratin 18) and/or myoepithelial cell-specific markers (CD 10, SMA, and cytokeratin 14) (as referenced [14]). Dontu et al also showed that single mammosphere-derived cells would generate structures with a lobuloalveolar-like morphology when plated in 3D assays and the structures contained cells that could be induced to secrete milk proteins under proper stimulation. In addition, they demonstrated that subsequent generations of mammospheres contained bipotent Ma-CFCs [4]. These findings suggested the human mammospheres obtained were derived from mammary epithelial cells with multi-lineage differentiation potential and possibly some self-renewal ability. However, the likelihood that aggregation was contributing to the formation of mammospheres in liquid cultures was not addressed. In addition, no study has provided any evidence that mammospheres are clonally derived which makes the properties of the putative mammosphere initiating cells (Ma-ICs) or their relationship to Ma-SCs unclear. However, the results reported by Dontu et al, strongly suggested that Ma-ICs represent a population of primitive human mammary epithelial progenitors whose ability to survive and. proliferate in vitro may be dependent on their suspension in an anchorage-independent system. Alternatively, it could be envisaged that mammospheres contain a mixture of developmentally unrelated cells, that when physically associated allow primitive mammary cells to survive and proliferate. 24 1.3. THESIS O B J E C T I V E S The goals of this thesis were: (1) to determine whether clusters of cells with the appearance of "mammospheres" could be derived from cells present in single cell suspensions of mouse breast tissue by culturing these in a semi-solid medium containing the same growth factors and nutrients used to generate mammospheres in liquid culture; and if so, (2) to characterize the cell of origin and its relationship to known types of mammary progenitors. To pursue this goal, I designed experiments to address the following questions: . Can mammospheres be generated in methylcellulose media containing single cell suspensions consistent with their origin from single cells; i.e., mammosphere initiating cells (Ma-ICs)? Can a quantitative assay for Ma-ICs be devised and what is their frequency? How do Ma-ICs compare phenotypically and functionally to MRUs and Ma-CFCs? 25 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. M I C E FVB mice were generated from breeders obtained from Taconic (Germantown, NY, USA). They were bred and maintained in the BC Cancer Research Centre Animal Facility according to institutional guidelines. All experiments were approved by the Animal Care Committee Office of Research Services, University of British Columbia (Vancouver, BC, Canada). 2.2. P R E P A R A T I O N O F M A M M A R Y C E L L S U S P E N S I O N S Number 3 and 4 mammary glands from 8 to 20 week-old female FVB mice were removed aseptically, digested for 8-10 hours at 37°C with 300 U/mL collagenase and 100 U/mL hyaluronidase in EpiCult-B™ medium supplemented with 5% fetal bovine serum (FBS) (all from StemCell Technologies, Vancouver, BC). The cell aggregates thus obtained' were then further dissociated by vortexing. The resulting cell suspension was centrifuged at 450 g for 5 minutes, the supernatant was discarded and contaminating red blood cells were lysed in fetal bovine serum (FBS):NH4C1 (StemCell Technologies). Following another centrifugation, the cell pellets were suspended in 2-5 mL of 0.25% trypsin (StemCell Technologies) pre-warmed to 37°C and the cells further dissociated by gentle pipetting of the cell pellets for 1-2 minutes. The trypsin was then inactivated by the addition of 10 mL of Hank's Balanced Salt Solution (StemCell Technologies) supplemented with 2% FBS (HF), and the cells were then washed, resuspended in HF with 5 mg/mL dispase II (StemCell; 26 Technologies) and 0.1 mg/mL deoxyribonuclease I (DNAse; Sigma Chemicals, St. Louis, MO)and incubated for 2 minutes at 37°C. The resultant cell suspension was then further dissociated by repeated pipetting, diluted in HF and filtered first through a 100 um mesh (StemCell Technologies) and then through a 40 urn mesh (StemCell Technologies) to obtain a single-cell suspension free of most cell clumps. 2.3. I S O L A T I O N O F S U B S E T S O F M A M M A R Y C E L L S Hematopoietic (CD45+/Terll9+) and endothelial (CD31+) cells were removed by pre-incubating freshly dissociated cells in 2 ug/mL Fc receptor antibody 2.4G2 (American Type Culture Collection, Rockville, MD) followed by a 10-minute incubation at room temperature with a 1:500 dilution of the biotin-conjugated StemSep™ Murine/Human Chimera Cocktail (StemCell Technologies), 1 ug/mL biotinylated CD31 (clone MEC13.3, Pharmingen, San Diego, CA) and then removal of the labeled cells using an EasySep™ Biotin Selection Kit (StemCell Technologies) as recommended by the supplier. For further subdivision of these CD45/Terll9/CD31-depleted cells into additional subpopulations, they were incubated in 1 |ag/mL of R-phycoeryfhrin (PE)-conjugated anti-CD24 antibody (clone Ml/69, Pharmingen) and a 1:40 dilution PE or fluorescein-isothiocyanate (FITC)-conjugated anti-CD49f antibody (clone GoH3, Pharmingen) for 30-45 minutes at room temperature and then washed twice with HF including l|u.g/mL propidium iodide (PI, Sigma) in the second wash. Cells were then kept on ice in the dark until being sorted in a FACS Vantage (Becton Dickinson, San Jose, CA) using gates set to exclude dead (PI+) cells and >99.9% of cells labeled with isotype-matched control antibodies conjugated with the corresponding 27 fluorochromes. The cells were collected in HF and then centfifuged before transfer into SF7-supplemented 5% FBS or semi-solid mammosphere medium (described below). In some cases, isolated CD24"CD49f cells were resorted prior to use. , 2 .4. M A M M O S P H E R E C U L T U R E Cell suspensions were suspended by vortexing in a semi-solid serum-free medium containing 1% methylcellulose and various supplements optimized for the generation of mammospheres (StemCell Technologies). Aliquots were then plated into ultra-low attachment plates or 35 mm petri dishes (StemCell Technologies). After incubation of the cultures for 7 days (unless otherwise indicated) at 37°C in a humidified atmosphere of air containing 5% C0 2 , clusters of >20 cells (mammospheres) were scored using an inverted microscope. Preliminary experiments showed that these were not seen within the first 2 days after plating and failed to form if the cells had been irradiated with 50 Gy prior to plating (data not shown). For the replacing experiments, the contents of the entire, culture were collected in a 10-fold excess volume of the same medium without methylcellulose and the cells centrifuged at 450 g for 5-7 minutes. The spheres were dissociated by adding 100-200 uL of 0.25% trypsin (StemCell Technologies) and triturating the suspension vigorously for 1-2 minutes with 200 uL pipette. 800 uL of HF was then added to inactivate the tryspin and the cells were washed and plated in new methyl ceUulose-containing medium. 28 2.5. M A - C F C A S S A Y Ma-CFCs were assayed as previously described for human Ma-CFCs [41], with the modification that the colonies were scored after only 5-6 days of incubation at 37°C. Briefly, this involved plating the cells on subconfluent irradiated (50 Gy) NIH3T3 fibroblasts in SF7 medium plus 5% FBS for the first 2 days and then in serum-free SF7 medium for another 3-4 days. The medium was then removed and the colonies were fixed with a 1:1 mixture of acetone and methanol for 5 seconds at room temperature. The plates were then air-dried, rinsed with tap water, stained with Wright's Giemsa (Fisher Scientific, Vancouver, BC, Canada) for 30-60 seconds, rinsed again with water and left to air-dry. Colonies containing >30 cells were scored using an inverted microscope. 29 C H A P T E R 3. R E S U L T S 3.1. A I M 1 - T O I N V E S T I G A T E T H E R E L A T I O N S H I P B E T W E E N M A M M O S P H E R E F O R M A T I O N A N D C E L L A G G R E G A T I O N A first series of experiments were performed to determine the number of mammospheres that would be obtained in liquid cultures of bulk mammary cells plated at densities of 105 cells per mL. Within 4 days numerous dense clusters of cells (mammospheres) were already evident. By 7 days, many such clusters, notably localized toward the centre of the culture plate (Figure 3-1) contained more than 100 cells. In contrast, when the cells were inoculated at the same seeding density into the same growth media but made highly viscous with 1% methylcellulose, the generation of cell clusters with the same appearance progressed more slowly over time. On day 7, 4 times as many containing >20 cells, were detected in the memylceUulose-containing medium as compared to the liquid cultures (Figure 3-1). The mammospheres produced in the semi-solid cultures were also evenly distributed throughout the plate and were on average much smaller (<50 cells each) (Figure 3-2). The rapidity with which mammospheres appeared in liquid culture strongly suggested that cell aggregation was playing a large role in their formation whereas the slower formation of mammospheres in the semi-solid medium was consistent with their development due to the mitotic activity of single isolated cells. This was later confirmed by time-lapse photography (in collaboration with Dr. E. Jervis, University of Waterloo, ON, data not shown). 30 2000 1% Methyl cellulose 1600 . CL Q> *"* S Q1200 o "<5 8 0 0 "-o 400 Figure 3-1. Differing yields of mammospheres generated in liquid and semi-solid media. Values shown are the mean ± standard deviation of results from 6 experiments. The difference between the 2 conditions is significant (p<0.001, Students, t-test). 31 Figure 3-2. Contrasting size of 7 day-old mammospheres generated in semi-solid and liquid cultures. A. Random field showing small mammospheres obtained in semi-solid medium. B. Large mammospheres obtained in liquid suspension cultures, concentrated in the centre of the dish. Both photographs were taken at the same magnification. To assess whether aggregation might also occur in semi-solid media, I examined the relationship between mammosphere yield and input cell concentration over a 100-fold range. Accordingly 103, 3 x 104, 6 x 104 and 105 freshly dissociated breast tissue cells were plated per mL and the number of mammospheres present was again scored 7 days later. The data show that the efficiency of mammosphere formation was not affected by the input seeding density over the range studied (Figure 3-3). The linearity of the relationship between mammosphere yield and number of cells plated also suggests that neither aggregation nor paracrine factors were significant contributing factors to mammosphere formation under the 32 conditions tested. The results in Figure 3-3 indicate a frequency of Ma-ICs of 1.6 per 100 freshly dissociated breast tissue cells cultured. If mammospheres are derived from mammary 5.0 CO O * , 4.0 \ Figure 3-3. Mammosphere yields in methylcellulose cultures. Mammosphere output is linearly related to the number of cells assayed independent of their input density. The grey line indicates the mammosphere-forming efficiency of bulk mammary cells, and the black line depicts the same for the same population after removal of cells that were CD45+, Terll9 + or CD31+. Error bars indicate standard deviations from 3 replicate dishes from 1 experiment. 33 epithelial cells, then it would be predicted that removal of contaminating hematopoietic (CD45+ and Terll9+) and endothelial (CD31+) cells would give a similar linear relationship between the number of cells plated and the yield of mammospheres obtained, but the values would be relatively higher. To test this prediction, freshly dissociated cells were first depleted of CD45+, Terll9+ and CD31+ ceUs and were then plated at 103, 3 x 104, 6 x 104 and 105 cells per mL of semi-solid medium. The results - also shown in Figure 3-3 (black line) - are consistent with this prediction and indicate that 2.4-fold enrichment in Ma-ICs had been achieved by the removal of hematopoietic and endothelial cells. 3.2. A I M 2 - T O I N V E S T I G A T E W H E T H E R S E M I - S O L I D M A M M O S P H E R E C U L T U R E S C A N S U P P O R T T H E S U R V I V A L O F M A - C F C S One of the defining functional characteristics of mammary stem cells is their ability to generate more differentiated progeny that can be detected using the previously described 2D and 3D assays. As a first test of whether Ma-ICs have the ability to generate Ma-CFCs, an experiment was designed to determine whether Ma-CFCs could be recovered from serially passed cultures containing mammospheres. Accordingly, variable volumes of 105 bulk starting cells per mL were cultured for 7 days under standard conditions for generating mammospheres in methylcellulose-supplemented medium. At the end of this time, the contents of the entire culture were harvested and a single cell suspension prepared. From this suspension, aliquots of 2 x 104 cells were removed and assayed for Ma-CFCs in standard 2D assays (at 2 x 104 cells per 2 mL culture). The remaining cells (-98% of the primary mammosphere culture) were then Used to initiate secondary mammosphere culture and the 34 protocol then repeated. The yields of Ma-CFCs in the harvested aliquots were used to calculate the total Ma-CFCs present in the primary and secondary mammosphere cultures and the values were then compared with the number of Ma-CFCs present in the initial starting population assayed separately. The results for 2 independent experiments are shown in Figure 3-4. It can be seen that Ma-CFCs continued to be detected through 2 serial mammosphere cultures, albeit in decreasing numbers. However, this experimental design does not establish their origin. While it is possible that Ma-CFCs were contained within the mammospheres, it is also possible that they were maintained independendy. Figure 3-4. Maintenance of Ma-CFCs in mammosphere culture. The number of Ma-CFCs gradually declines with prolonged culture through sequential passages; however, Ma-CFCs could still be detected after 14 days in culture. 35 3.3. A I M 3 - T O A N A L Y Z E T H E D I S T R I B U T I O N O F M A - I C S I N D I F F E R E N T P H E N O T Y P I C A L L Y D E F I N E D S U B S E T S O F M A M M A R Y C E L L S Thanks to the recently published works of Shackelton and Stingl [2], the phenotypes of MRUs and Ma-CFCs have been identified. It was therefore of interest to determine the phenotype of Ma-ICs with respect to the markers used to purify and distinguish MRU and Ma-CFCs according to Stingl's published method for isolating these cells. Using the combined technology of immunomagnetic cell separation and flow cytometry, 6 populations of (CD45/Terll9/CD31)" cells were isolated based on their co-expression of CD49 and CD24 (Figure 3-5). Each of these populations were assayed for their Ma-IC content and, as a control for the separation method, the Ma-CFC content of each fraction was also measured. The results of these experiments showed that 96+1 % of all the Ma-ICs recovered were in the CD24CD49 fraction; 3 ± 1 % were found in the CD24lowCD49" fraction, 1 ± 1 % were in the CD24lowCD49+ fraction, 0.5 ± 0.3 % were in the CD24CD49+ fraction, 0.06 ± 0.06 % were found to be CD24+CD49+ fraction and none were detected in the CD24+CD49 fraction (Figure 3-6). Thus almost no Ma-ICs. were present in any fraction associated with MRU or Ma-CFC activity. 36 Figure 3-5. FACS plot showing the 6 populations assayed for Ma-ICs and Ma-CFCs. The vertical gate and the lower horizontal gate were set using isotype controls. 37 100 Figure 3-6. Distribution of Ma-ICs among different subpopulations of mammary cells. Values per fraction are expressed as a % of total number of Ma-ICs in 105 (CD45/Terll9/CD31)" initially sorted cells. The numbers on the horizontal axis refer to the gated populations indicated in Figure 3-5. The error bars represent the SEM of the means of data from 3 independent experiments. In contrast, the results of the 2D colony assays showed that only 3.0 ± 0.7 % of the Ma-CFCs were recovered from the CD24CD49" fraction. 0.03% ± 0.03 % of the Ma-CFCs were found in the CD24"CD49+ fraction, 19 ± 1 % were in the CD24lowCD49+ fraction, 25 ± 1 % were in the CD24lowCD49- fraction, 17. ± 1 % were in the CD24+CD49+ fraction, and 38 36 ± 1 % were CD24+CD49" (Figure 3-7). Thus the phenotypes of the Ma-CFCs were as expected and different from Ma-ICs. Figure 3-7. Distribution of Ma-CFCs among different subpopulations of mammary cells. Values per fraction are expressed as a % of total number of Ma-CFCs in 105 (CD45/Terll9/CD31)" initially sorted cells. The numbers on the horizontal axis refer to the gated populations indicated in Figure 3-5. The error bars represent the SEM of the means of data from 3 independent experiments. 39 To determine whether prolongation of the culture' time might detect a different distribution of Ma-ICs (perhaps more similar to Ma-CFCs), the Ma-ICs cultures were returned to the incubator for another 2 weeks and a second sphere count was then performed. Although many of the spheres had increased in size during the extra week of culture, the same phenotype distribution of Ma-ICs was seen (Figure 3-8), suggesting that the same spheres were being scored at both time points. Figure 3-8. Ma-ICs of the same phenotype give rise to mammospheres seen after either 7 or 21 days of growth. Values shown are the Ma-IC frequencies on each fraction expressed as a % of the cells plated. Results are from 2 independent experiments. The numbers on the horizontal axis refer to the gated populations indicated in Figure 3-5. Error bars represent standard deviations. 40 3.4. A I M 4 - T O I N V E S T I G A T E T H E C A P A C I T Y O F M A - I C S T O G E N E R A T E M A - C F C S To further test the possibility that Ma-ICs are prirnitive precursors of Ma-CFCs, a series of experiments were initiated in which double-sorted CD45~Terll9~CD3rCD24~CD49~ cells were assayed in equal cell numbers for Ma-CFCs and Ma-ICs. After 7 days in mammosphere media, the cells in these cultures were harvested, dissociated and assayed for Ma-CFCs. The results of these experiments yielded no detectable Ma-CFCs either before (n=3) or after the generation of the mammospheres (n=2). 41 C H A P T E R 4. D I S C U S S I O N A N D C O N C L U S I O N The study of somatic stem cells in solid tissues has been largely modelled after the decades of research undertaken to characterize hematopoietic stem cells (HSCs). These investigations have shown that adult bone marrow maintains a population of cells that can each individually differentiate into all of the various lineages of blood cells and sustain their output for long periods of time. In addition, these HSCs can generate daughter cells with the same pluripotent properties, as demonstrated in sequential transplantation experiments. The study of HSCs has also indicated the existence of an extensive hierarchy of stem cell and progenitor compartments encompassing the early stages of lineage restriction and loss of self-renewal potential [42]. The emerging model of mammary gland development similarly reflects a postulated hierarchical structure similar to that established for the hematopoietic system (see Figure 1-10). In this model, mammary epithelial stem cells give rise to transient amplifying cells that may still be unrestricted in their mammary differentiation potential but lack the ability to extensively self-renew. These latter cells, in turn, would then give rise to lineage-restricted progenitors that finally generate the majority populations of terminally differentiated luminal and myoepithelial cells that have a finite lifespan and are incapable of further proliferation [28]. This model is almost certainly an oversimplified description of how mammary cell differentiation occurs, but further progress in its elucidation has been hindered by a lack of robust methods for quantifying and distinguishing different types of mammary cell progenitors in vitro. 42 For example, in many systems data has been accumulated to indicate that the stem cells in the adult are normally characterized by low mitotic activity. However, recent studies of the cycling status of the Ma-SC population in the adult virgin female mouse mammary gland indicates that most of these cells, like the majority of the Ma-CFCs in the same tissue are rapidly cycling [2]. This observation raises 2 very interesting possibilities. One would be that only cycling Ma-SCs have the ability to successfully engraft a cleared mammary fat pad assay. If this were true, then the entire subset of non-cycling Ma-SCs, despite thek putative potential, would not be detected whenever this assay was used. On the other hand, Ma-SCs may not normally include a significant quiescent fraction, in which case other mechanisms of cell population control would be particularly important in regulating breast growth and regression throughout life. Similarly, the 2D Ma-CFC assay demands that the progenitors attach to the tissue culture dish and generate colonies over a course of 6 days when cultured in the presence of a defined set of supplements and growth factors [12]. Pre-culturing of dissociated human mammary epithelial cells is a method currendy used in the enrichment of such progenitor populations. The detected ~5-fold increased frequency of progenitors can be attributed to the elimination of differentiated cells that lack the ability to adhere, or perhaps the attrition of cells damaged during the sample preparation [43]. Or perhaps, the pre-culturing period allows another set of progenitors that are initially unable to generate colonies to complete the necessary steps and enter the mitotic cycle upon the introduction of FBS that acts as an inducer of differentiation. 43 All of these considerations underscore the need for new methods for detecting and propagating different types of mammary progenitors in vitro. For this reason, designing a culture system that would closely mimic the in vivo environment of the mammary, gland has been an attractive goal of many studies. Thus, it was recognized that reconstituted basement membrane containing multiple stromal matrix proteins as well as growth factors has the ability to support the growth of acinar-like structures that upon stimulation with lactogenic hormones synthesize milk proteins [15], Dontu et al [4] also found that culturing human mammary epithelial cells in liquid medium in the absence of FBS supported the development of mammospheres that contained matrix proteins suggesting that this might create unique conditions for stimulating Ma-SC self-renewal divisions. The hypothesis Dontu et al put forward is that mammospheres represent clonal expansions of mammary progenitors that appear to contain stem-cell like properties. My work demonstrates that under liquid conditions, this is highly unlikely. Notably, smaller and more numerous spheres were obtained in semi-solid cultures by comparison to liquid cultures. Moreover, these results provide strong evidence that significant aggregation of plated single cell suspensions does occur even in semi-solid media. Culturing mammary cells in liquid suspension thus allows cells at various states of differentiation to aggregate and generate a common microenvironment. It is possible, therefore, that the nature of the created niche varies depending on the progenitor content of the aggregate. Even if Ma-ICs do not belong to the mammary' epithelial hierarchy, as suggested here at least for murine cells, their ability to generate mammospheres and associate 44 with progenitors may provide the primitive mammary cells with a suitable environment for their maintenance in vitro. Phenotypic studies performed on murine Ma-ICs revealed that these did not co-purify with either of the described primitive populations of the murine mammary epitheUum. Functional studies also showed that Ma-ICs do not possess the ability to generate epithelial colonies or Ma-CFCs, as detected by the in vitro 2D assay. Thus based on these 2 criteria, Ma-ICs appear to be progenitors that may not belong to the mammary epithelial differentiation hierarchy. . Nevertheless, my experiments also showed that primitive mammary epithelial cells remain detectable in semi-solid cultures for at least 14 days. This finding supports the alternative possibility that Ma-ICs may help to create an environment that supports mammary progenitor maintenance, even though they are not, themselves, developmentally related to the mammary epitheUum. An experiment that could distinguish these alternative explanations, could involve the injection of highly purified (single) MRUs into the cleared fat pads of congenic recipients. The cells from subsequendy harvested grafts could then be assayed for the presence of Ma-ICs and the host or donor origin of the mammospheres produced, determined. If the resultant mammospheres were of donor origin, this would indicate that Ma-ICs can be produced by MRU and are thus part of the mammary epithelial hierarchy. The opposite result would suggest that they are more likely to be stromal in origin. If no mammospheres were obtained, this might suggest that Ma-ICs are more primitive than Ma-SCs and cannot read out in the currentiy used in vivo MRU assay. 45 In order to investigate a putative supporting role of Ma-ICs in the maintenance of mammary progenitors, a mixing experiment could be set up. By combining various identified fractions of mammary progenitors with the Ma-IC enriched fraction and allowing it to aggregate in liquid mammosphere media, it might be possible to maintain the functional integrity of progenitors and/or stem cells in vitro for an extended period of time and longer than under any other known mammary culture conditions. It should also be noted that there may be significant differences between the human and murine systems, as noted for other tissues. Even though the mouse is widely used as a model for mammalian development and function, species-specific differences are well recognized and as such present challenges in research and especially for clinical investigations. Nevertheless, the mouse model offers many advantages in terms of tissue availability, homogeneous genetic and environmental backgrounds and ease of manipulation. 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