"Medicine, Faculty of"@en . "DSpace"@en . "UBCV"@en . "Carter, Lindsay A."@en . "2009-08-20T18:39:21Z"@en . "2002"@en . "Master of Science - MSc"@en . "University of British Columbia"@en . "In the olfactory epithelium (OE), new olfactory receptor neurons (ORNS) are\r\ncontinually generated throughout mammalian adulthood. Given this substantial neuronal\r\nturnover, a stem cell is proposed to reside within the basal compartment of the OE, which\r\ngenerates ORNs on demand when stimulated by changes in its microenvironment.\r\nAlthough previous studies have identified possible candidates for the olfactory stem cell,\r\nits exact identity is as yet unknown. We hypothesize that a population o f horizontal basal\r\ncells (HBCs), situated upon the basement membrane of the OE, contains stem cells that\r\ncontribute to olfactory neurogenesis.\r\nA major impediment to the study of these cells is the lack of reliable cell surface\r\nmolecular markers to distinguish them from other OE cell types. By screening a panel of\r\nselected clusters of differentiation (CD) antigens, we have identified three new cell\r\nsurface markers for the HBC population, namely intercellular adhesion molecule -1\r\n(ICAM-1), \u00CE\u00B2\u00E2\u0082\u0081 integrin and \u00CE\u00B2\u00E2\u0082\u0084 integrin. Using these markers to characterize the HBC\r\nlayer following bulbectomy-induced ORN loss, we have provided evidence of stem cell\r\ntraits in vivo, including proliferative quiescence relative to OE progenitors, response to\r\nlesion, and possible molecular heterogeneity within the HBC compartment. In addition,\r\nthese studies indicate changes in the populational and subcellular distribution of HBC\r\nmarkers upon loss of ORNs, suggesting a role for these adhesion receptors in the\r\nregulation of HBC function in addition to highlighting possible molecular similarities to\r\nstem cells of other self-renewing tissues. We have developed a method to select for\r\nHBCs in vitro using magnetic activated cell sorting (MACS) and by exploiting their\r\nexpression of ICAM-1. Using in vitro colony-forming analyses, we obtained evidence\r\n\r\nthat the ICAM-1+ population is enriched for progenitor activity. Further, the efficiency\r\nof colony formation can be modulated in vitro by growth factors and adhesive substrates.\r\nLastly, immunohistochemical analysis demonstrated that globose basal cell (GBC)\r\nprogenitors, ORNs and olfactory ensheathing glia (OEGs) are generated by the ICAM-1+\r\nfraction in clonal culture. Based on these results, we conclude that ICAM-1+ HBCs\r\ncontribute to the progenitor cell compartment, possibly as stem cells, during olfactory\r\nneurogenesis and that the function of these cells may be modulated via adhesion and\r\ngrowth factor signaling by components resident within their in vivo microenvironment."@en . "https://circle.library.ubc.ca/rest/handle/2429/12405?expand=metadata"@en . "13134576 bytes"@en . "application/pdf"@en . "Olfactory Epithelial Horizontal Basal Cells: An Assessment of Stem Cell Candidacy and Behavioural Regulation in vivo and in vitro by Lindsay A . Carter B.Sc. (Hons.), University o f British Columbia, 1999 A THESIS S U B M I T T E D I N P A R T I A L F U L F I L M E N T OF T H E R E Q U I R E M E N T F O R T H E D E G R E E O F M A S T E R O F S C I E N C E in T H E F A C U L T Y O F G R A D U A T E S T U D I E S (The Graduate Program in Neuroscience) We accept this thesis as conforming to the required standard T H E U N I V E R S I T Y O F B R I T I S H C O L U M B I A July 2002 \u00C2\u00A9 Lindsay A . Carter, 2002 Friday, July 26, 2002 UBC Rare Books and Special Collections - Thesis Authorisation Form Page: 1 i n presenting this thesis i n p a r t i a l fulfilment of the requirements for an advanced degree at the University of B r i t i s h Columbia, I agree that the Library sh a l l make i t freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It i s understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department of Vancouver, Canada http://www.library.ubc.ca/spcoll/thesauth.html A B S T R A C T In the olfactory epithelium (OE), new olfactory receptor neurons (ORNS) are continually generated throughout mammalian adulthood. Given this substantial neuronal turnover, a stem cell is proposed to reside within the basal compartment of the O E , which generates O R N s on demand when stimulated by changes in its microenvironment. Although previous studies have identified possible candidates for the olfactory stem cell, its exact identity is as yet unknown. We hypothesize that a population o f horizontal basal cells (HBCs) , situated upon the basement membrane of the O E , contains stem cells that contribute to olfactory neurogenesis. A major impediment to the study of these cells is the lack of reliable cell surface molecular markers to distinguish them from other O E cell types. B y screening a panel of selected clusters o f differentiation (CD) antigens, we have identified three new cell surface markers for the H B C population, namely intercellular adhesion molecule -1 ( ICAM-1) , PJ integrin and P4 integrin. Using these markers to characterize the H B C layer following bulbectomy-induced O R N loss, we have provided evidence of stem cell traits in vivo, including proliferative quiescence relative to O E progenitors, response to lesion, and possible molecular heterogeneity within the H B C compartment. In addition, these studies indicate changes in the populational and subcellular distribution of H B C markers upon loss of ORNs, suggesting a role for these adhesion receptors in the regulation of H B C function in addition to highlighting possible molecular similarities to stem cells of other self-renewing tissues. We have developed a method to select for H B C s in vitro using magnetic activated cell sorting ( M A C S ) and by exploiting their expression of I C A M - 1 . Using in vitro colony-forming analyses, we obtained evidence i i that the I C A M - 1 + population is enriched for progenitor activity. Further, the efficiency of colony formation can be modulated in vitro by growth factors and adhesive substrates. Lastly, immunohistochemical analysis demonstrated that globose basal cell ( G B C ) progenitors, O R N s and olfactory ensheathing glia (OEGs) are generated by the I C A M - 1 + fraction in clonal culture. Based on these results, we conclude that I C A M - 1 + H B C s contribute to the progenitor cell compartment, possibly as stem cells, during olfactory neurogenesis and that the function of these cells may be modulated via adhesion and growth factor signaling by components resident within their in vivo microenvironment. i i i TABLE OF CONTENTS ABSTRACT i i TABLE OF CONTENTS iv LIST OF FIGURES viii LIST OF TABLES x LIST OF ABBREVIATIONS x i ACKNOWLEDGEMENTS x i i i CHAPTER I Introduction and Research Objectives 1 1.1 The Olfactory System 1 1.2 Ce l l Replacement via Stem Cells 9 1.3 Manifestations of Stem Cells in Three Physiologically Different Tissues. . .16 1.4 The Cellular Constituents of Olfactory Neurogenesis 19 1.5 The Molecular Regulation of Olfactory Neurogenesis 25 1.6 The H B C vs. G B C as O E Stem Cel l Controversy. 29 1.7 Working Hypothesis and Research Objectives 30 CHAPTER II Materials and Methods 32 2.1 Olfactory Bulbectomies and Tissue Preparation 32 2.2 B r d U Incorporation and Detection 33 2.3 Immunohistochemistry 33 2.4 Antibodies 34 2.5 Primary Culture of Basal Cells 35 iv 2.6 I C A M - 1 Immunornagnetic Selection of Basal Cells 36 2.7 Test Conditions for Optimizing Colony-Forming Efficiency 38 2.8 Assessment of Adhesion Kinetics 39 2.9 Immunocytochemistry 39 C H A P T E R III An initial screen of CD antibodies reveals three cell surface markers for horizontal basal cells within the mouse olfactory epithelium 41 3.1 Introduction 41 3.2 I C A M - 1 , P i integrin and P4 integrin are expressed in basal cells apposed to the basement membrane within the adult olfactory epithelium 42 3.3 I C A M - 1 expression directly overlaps with that of the horizontal basal cell marker, Keratin 903 45 3.4 a integrins are expressed in a complimentary fashion to the P i and P4 integrins ... 45 3.5 C D antigens are expressed within other cell types within the olfactory mucosa 47 3.6 Summary 47 C H A P T E R IV An in vivo characterization of HBCs: response to bulbectomy and examination of potential stem cell traits 51 4.1 Introduction 51 4.2 The removal of the olfactory bulb induces O R N loss and basal cell proliferation in the epithelium 51 4.3 G B C s , negative for both I C A M - 1 and N C A M expression, are depleted regionally following lesion 52 4.4 Adhesion receptor positive H B C s divide post-bulbectomy, but remain relatively quiescent compared to G B C s 54 4.5 The expression and/or distribution of I C A M - 1 , P i and P4 integrins is altered post-bulbectomy 57 v 4.6 H B C s display some heterogeneity in the complement of adhesion receptor they express, an observation which is exaggerated post-bulbectomy 57 4.7 Summary 60 C H A P T E R V Immunomagnetic selection in conjunction with in vitro progenitor assays and lineage-specific marker expression support an H B C contribution to the olfactory progenitor cell compartment 63 5.1 Introduction 63 5.2 Preliminary in vitro findings 64 5.3 Ce l l surface antigen selection and sorting of H B C s 64 5.4 The I C A M - 1 + fraction possesses a superior colony-forming ability at clonal density 67 5.5 Determination of optimal media conditions with respect to I C A M - 1 + colony forming efficiency 69 5.6 Effect of E C M substrate on the overall colony-forming ability of I C A M - 1 selected cells 71 5.7 Effect of E C M components on the incidence of small, medium and large colonies within clonal cultures of I C A M - 1 + cells 73 5.8 Adhesion kinetics assay on different E C M components demonstrates an overall preference for collagen 76 5.9 Effects of growth factor addition on overall colony forming efficiency of I C A M - 1 + cells 78 5.10 Effect of growth factors on the incidence of small, medium and large colonies within clonal cultures of I C A M - 1 + cells 79 5.11 Cultured I C A M - 1 + cells produce a mosaic of differentiated olfactory cell phenotypes 82 5.12 I C A M - 1 positive colonies contain cells possessing a mature olfactory neuron phenotype 84 5.13 Summary 87 v i C H A P T E R VI Discussion 88 C H A P T E R VII Concluding Remarks 113 B I B L I O G R A P H Y 116 v i i LIST O F FIGURES Figure 1.1 The primary olfactory neuraxis 2 Figure 1.2 The three major cell compartments and their relative positions within the olfactory epithelium 4 Figure 1.3 Antigenic markers and relative height of cells within the O E 7 Figure 1.4 The classical stem cell->transit amplifier->differentiated progeny hierarchy 13 Figure 3.1 I C A M - 1 , Pi integrin and P4 integrin are detected within H B C s of adult olfactory epithelium 44 Figure 3.2 H B C expression of I C A M - 1 is confirmed via Keratin 903 co-localization 46 Figure 3.3 Potential a integrin pairing partners are identified for the Pi and P4 integrin subunits 48 Figure 3.4 Other screened C D antigens are detected in non-HBC cells in the olfactory mucosa 49 Figure 4.1 The loss of ORNs following bulbectomy induces proliferation within the basal cell compartment of the O E 53 Figure 4.2 G B C progenitors are depleted locally within discrete regions of O E following bulbectomy 55 Figure 4.3 H B C s proliferate in response to bulbectomy, but remain quiescent relative to robustly proliferating G B C s 56 Figure 4.4 Changes are detected in the populational uniformity of I C A M - 1 , Pi and P4 integrin expression within the H B C layer post-bulbectomy 58 Figure 4.5 The subcellular distributions of ICAM -1 and P4 integrin are altered in some cells post-bulbectomy, while that of Pi integrin remains constant...59 Figure 4.6 The observed heterogeneity of H B C adhesion receptor expression in normal O E is exaggerated post-bulbectomy 61 Figure 5.1 Preliminary evidence of progenitor activity in heterogenous cultures o f OE-derived cells 65 v i i i Figure 5.2 In vitro immunomagnetic sorting of H B C s on the basis of I C A M - 1 antigenicity 66 Figure 5.3 The M A C S selected I C A M - 1 + fraction displays a superior colony forming efficiency in vitro 68 + Figure 5.4 Effect of media condition on the colony forming efficiency of I C A M - 1 cells at clonal density 70 Figure 5.5 Effect of substrate on overall colony forming efficiency of I C A M - 1 + cells at clonal density 72 Figure 5.6 Representative small, medium and large colonies at 14 D I V 74 Figure 5.7 Effect of substrate on the incidence of small, medium and large colonies seeded by I C A M - 1 + , M A C S selected cells at clonal density 75 Figure 5.8 M A C S selected I C A M - 1 + cells display different kinetics of adhesion when plated on different substrates 77 Figure 5.9 Effect of growth factor on the overall colony forming efficiency of I C A M -1 + cells at clonal density 80 Figure 5.10 Effect of growth factor on the incidence of small, medium and large colonies seeded by I C A M - 1 + cells at clonal density 81 Figure 5.11 Large colonies contain cells expressing markers of olfactory differentiation, while retaining I C A M - l + / p 4 integrin + H B C s as well 83 Figure 5.12 Some neurons present at the perimeter of these colonies possess a mature olfactory neuron phenotype 85 Figure 5.13 Some neurons displayed a distinctly non-olfactory phenotype 86 ix LIST OF TABLES Table 3.1 Expression and functional properties of selected C D antigens within stem/progenitor cell hierarchies of other self-renewing tissues 43 x LIST O F ABBREVIATIONS ACIII adenylate cyclase III B M P bone morphogenic protein B D N F brain derived neurotrophic factor B r d U bromodeoxyuridine C D clusters of differentiation antigens Cdc2 kinase cell-division-cycle2 kinase C F E colony forming efficiency C N S central nervous system D A P I 4',6-Diamidine-2'-phenylindole dihydrochloride D I V day in vitro D M E M Dulbecco's modified Eagle's medium E embryonic day E C M extracellular matrix E G F epidermal growth factor ES embryonic stem cell F B S fetal bovine serum F G F fibroblast growth factor F G F R fibroblast growth factor receptor G A P -4 3 growth associated protein-43 G B C globose basal cell G - O l f olfactory G-protein G F A P glial acidic fibrillary protein 3 H tritiated thymidine H B C horizontal basal cell I C A M - 1 intercellular adhesion molecule-1 I L - l p interleukin-lp INP immediate neuronal precursor x i I R N immature receptor neuron K - S F M keratinocyte serum-free media LFA-1 leukocyte function associated-1 M A C S magnetic activated cell sorting Mash-1 mammalian achaete scute homologue LIF leukemia inhibitory factor L I F R leukemia inhibitory factor receptor L P lamina propria N C A M neural cell adhesion molecule NT-3 neurotrophin-3 N G F nerve growth factor N S T Neuron specific tubulin O E olfactory epithelium O E G olfactory ensheathing glia O M P olfactory marker protein O R N olfactory receptor neuron P postnatal day P B S phosphate buffered saline P F A paraformaldehyde R T - P C R reverse transcriptase polymerase chain reaction SE standard error T A transit amplifying cell T G F - a transforming growth factor-a TGF-( i transforming growth factor-P V L A - 4 very late activating antigen-4 V L A - 5 very late activating antigen-5 ZnSCv zinc sulphate x i i Acknowledgements Firstly, I would like to thank my husband, Derrick, who specifically asked that I refer to him as \"wonderful\" and \"nice\" in my acknowledgements. He is indeed. I would also like to thank family and friends for their support and encouragement, especially my brother, M i k e , sister-in-law, Johanna, and long-time friend, Suzanne. I wish to acknowledge my lab mates, past and present, for their support and friendship and thank my supervisor, Dr. Jane Roskams, for providing me with the opportunity to conduct this research in her lab. Finally, I would like to thank the Rick Hansen Institute for awarding me a neurotrauma studentship. Lastly, I would like to dedicate this work to the memory of my mother. A s threshing separates the wheat from the chaff, so does affliction purify virtue. Sir Richard Francis Burton x i i i C H A P T E R I: Introduction and Research Objectives 1.1 The Olfactory System The olfactory epithelium (or OE), a columnar, pseudo-stratified epithelium located peripherally within the nasal cavity, is both the birthplace and residence of mature olfactory receptor neurons (ORNs) (Figure 1.1; Graziadei and Graziadei, 1979a; Graziadei and Graziadei, 1979b; Moulton, 1974; Suzuki and Takeda, 1993). ORNs , the singular neuronal cell type of the O E , generate nerve impulses upon interaction with chemical odorants detected in the environment. A s such, ORNs are crucial for our sense of smell. The dendrites of ORNs exit the O E apically to access these odorant molecules in the airborne environment, while their axons are herded into discrete axon bundles upon their basal passage out of the O E (Farbman, 1992). O R N axons are then guided (in part by glia and stromal cells) through to their synaptic target- the olfactory bulb, which forms the most rostral portion of the C N S (Doucette, 1990; Doucette, 1991; Farbman, 1992). A s a consequence of their direct interaction with the outside environment, O R N s are prone to physical and environmental damage (Farbman, 1992). Within the adult rat O E , O R N s typically have a lifespan of about 4-6 weeks (Farbman, 1992; Murray and Calof, 1999). Environmental, as opposed to genetic, determination of O R N lifespan is supported by the finding that animals housed in a \"dirty\" environments exhibit higher rates of O R N turnover than do those residing in sterile cage environments (Hinds et al, 1984). In order to cope with this innate vulnerability of ORNs, a mechanism, unique in the mammalian nervous system, exists to actively regenerate ORNs lost due to environmental insult in order to sustain the critical sensory function of olfaction. 1 Figure 1.1: The pr imary olfactory neuraxis. Olfactory receptor neurons (ORNs) within the peripherally located olfactory epithelium (OE) send their axons through the cribiform plate to synapse with target cells within the glomeruli of the olfactory bulb. (Margolis et al., 1991). 2 The O E is akin to other epithelia in that it displays a topographical gradient of maturity, with the most primitive, developmentally immature cells situated at the base o f the O E , while functionally mature cell types are located in the upper reaches of the epithelium with intermediates existing in between. The cell types of the O E can be distinguished from one another by either cell morphology, relative position in the O E , or by the differential expression of protein markers (Schwob, 2002). The O E can be subdivided into three compartments: the apical, middle and basal compartments as diagrammed in Figure 1.2 (Calof et al., 1998). The apical layer is composed of nuclei belonging to a single cell type, the sustentacular, or supporting, cells. In the rat O E , sustentacular cells comprise roughly 15-20% of the total epithelium (Farbman et al., 1988). These elongated macroglial cells contact the basement membrane at the base of the O E and extend microvil l i out into the nasal cavity, thereby spanning the entire O E proper in length (Farbman, 1992). Although relatively few studies have examined their function, it is known that sustentacular cells phagocytose dead neurons following injury (Suzuki et al., 1995; Suzuki et al., 1996). Further proposed roles include a detoxification function (Ding and Coon, 1988; Chen et al., 1992) and the regulation of the passage of compounds between the O E proper and its underlying lamina propria (Rafols and Getchell, 1983). These cells are identified by their expression of cytokeratins 8 and 18 and several monoclonal antibodies of unidentified antigenicity (Goldstein and Schwob, 1996; Goldstein et al., 1997; Jang and Schwob, 2001). The next section of O E , the middle compartment, contains the cell bodies of olfactory receptor neurons, both immature and fully functional, which can be further segregated according to maturity such that immature ORNs are situated basal to mature 3 HB CFU Ml INP ORN MORN Su Figure 1.2: The three major cell compartments and their relative positions within the olfactory epithelium. The O E can be subdivided into three topographical compartments, namely: apical, middle, and basal compartments. The apical compartment comprises sustentacula cells (Su), while the middle compartment contains the cell bodies of mature O R N s ( M O R N ) and immature ORNs (ORN). The basal compartment contains horizontal (HB) and globose (GB) basal cells. In this diagram, globose basal cells are further sub-divided into neuronal colony forming units (CFU) , Mash-1 expressing cells ( M l ) , and immediate neuronal precursors (INP). (Calof et al., 1998) 4 ORNs. Both stages of O R N constitute 75-80% of the rat O E (Farbman et al., 1988). The period of time necessary for a newborn neuron to achieve functional maturity and attain its mature height in the O E is approximately 1 week (Miragall and Mont i Graziadei, 1982; Schwob et al., 1992). ORNs are bipolar cells and can be identified, in general, by the expression of neural cell adhesion molecule ( N C A M ) (Miragall et al., 1988; Caggiano et al. , 1994; Calof and Chikaraishi, 1989). To identify immature neurons, those whose axons have not yet made contact with the olfactory bulb, laboratories typically use antibodies generated against neuron specific tubulin (NST), growth associated protein-43 (GAP-43), and the tyrosine receptor kinase Trk B (Verhaagen et al., 1989; Roskams et al., 1996; Roskams et al., 1998). Another panel of antibodies is utilized to identify mature olfactory neurons, including olfactory marker protein (OMP) , and components of the olfactory signal transduction cascade, most commonly adenylate cyclase III and G-o l f (Margolis, 1972; Keller and Margolis, 1975). Lastly, the base of the O E consists of the basal compartment and houses the proliferative region of the O E and is the region from which the O E derives its regenerative potential. This compartment contains two cell types: globose basal cells (GBCs) and horizontal basal cells (HBCs) , which together account for no greater than 10% of the total O E proper (Farbman et al., 1988). These two cell types can be distinguished morphologically as the H B C s are typically flattened and tightly apposed to the basement membrane, while G B C s display a polyhedral phenotype and are situated one cell layer removed from the basement membrane, sitting atop the H B C s . H B C s can also be identified antigenically via their expression of cytokeratin 5/6 (Calof and Chikaraishi, 1989; Suzuki and Takeda, 1993). In earlier studies of the O E , G B C s were 5 defined on the basis that they were negative for antigenic markers of neighbouring cell types. For example, an O E cell situated within the basal portion of the epithelium that was cytokeratin* and N C A M \" negative was designated a G B C (Calof et al., 1998). More recently, however, laboratories have employed the so-called G B C antibodies generated by Goldstein and Schwob (1996). Although the identities of the antigens that these \" G B C \" antibodies recognize are as yet unknown, they serve as the only global markers of the G B C population. The G B C layer can be further subdivided according to function and antigenicity. The immediate neuronal precursor (FNP) of the O E is a G B C that expresses the neuronal differentiation transcription factor neurogeninl (Cau et al., 1997). The so-called Mash-1-expressing cell is believed to function as a transit amplifying cell situated upstream from INPs and downstream of a hypothesized O E stem cell (Gordon et al., 1995; Cau et al., 1997). To date, there is considerable debate concerning whether the G B C or H B C layer contains the olfactory stem cell. Some assert that H B C s play a purely non-regenerative supporting role within the O E proper, while others purport that they are the ultimate source of the OE ' s regenerative capacity. Suggested H B C supporting roles include a function in the maintenance of O E tissue integrity and a role as signaling cells to report the status of ORNs, via their proximity to O R N axon bundles, to the overlying O E (Farbman, 1992; Holbrook et al., 1995). The cell types of the O E , together with their phenotypical markers are diagrammed in Figure 1.3. The lamina propria (LP) of the olfactory mucosa, situated beneath the O E proper, houses a diversity of cell types which function in the support of O R N related activities, including a unique class of glia, the olfactory ensheathing glia (OEGs), and most connective tissue cell types, such as mast cells, leukocytes, macrophages and fibroblasts. 6 H B C G B C Immature Mature Sustenta-cular cell Sus-4 Keratin G B C - 1 , O R N 5/6 -2, and N C A M ORNs O M P ACIII G-o l f N C A M -3 N S T GAP-43 TrkB Keratin 8 and 18 Figure 1.3 Antigenic markers and relative height of cells within the O E . The most commonly used marker for H B C s is cytokeratin 5/6, while G B C s are recognized by several monoclonal antibodies of unknown antigenicity. Immature O R N markers include neuron specific tubulin (NST), growth associated protein-43 (GAP-43), and the tyrosine receptor kinase Trk B . Mature ORNs are recognized by olfactory marker protein (OMP) , adenylate cyclase III (ACIII), and the olfactory g-protein, G-olf. Neural cell adhesion molecule ( N C A M ) is expressed in both immature and mature ORNs. Finally, sustentacular cells are identified by their expression of keratins 8 and 18, in addition to Sus-4, a monoclonal antibody of unknown antigenicity. (Farbman, 1992) O E G s are of particular interest on account of their growth-promoting properties (Ramon-Cueto and A v i l a , 1998). Through their entire length from O E to olfactory bulb, O R N axons are in continual contact with OEGs . OEGs ensheathe ORNs to provide a permissive substrate for growth, pathfinding guidance to the olfactory bulb and trophic support (Ramon-Cueto and Av i l a , 1998). In stark contrast to the primary olfactory neuraxis, the mature C N S is not permissive of axonal regeneration and elongation. A s such, it appears as though O E G s are the olfactory system's answer for a continual need for axonal regeneration throughout adult life. OEGs can be discriminated both in vivo and in vitro by the expression of glial fibrillary acidic protein (GFAP) , the calcium-binding protein SlOOp or P75 (Ramon-Cueto and Av i l a , 1998). Other structural elements of the L P include the Bowman's glands, which secrete mucus exterior to the O E via ducts in order to protect the tissue from desiccation and other environmental insults (Farbman, 1992). Also inhabiting the L P are relatively large caliber blood vessels with thin walls, which permit considerable blood flow to the surrounding tissue (Farbman, 1992). The olfactory mucosa, as a whole, is comprised o f cellular and structural elements supportive of O R N s ' innate situational vulnerability, providing both protective and renewal mechanisms to ensure that the sense of olfaction remains intact. One question that has received much attention in the field of olfaction is the ultimate source of new ORNs . Like other self-renewing tissues, the O E is proposed to contain a stem cell that serves as the basis of its unique neuronal regenerative capacity. 8 1.2 C e l l Replacement v i a Stem Cells A s cells terminally differentiate and acquire more specialized functions, they often lose the ability to self-replicate. This inability can be attributed to a variety of causes, ranging from a simple unsuitability of the cell's morphological form, as is the case with neurons, to an incompatibility of the molecular pathways regulating mitosis and differentiation (Alberts et al., 1994). When such cells reside in tissues that exhibit continual turnover, such as the epidermis, the lining of the gut, and blood-forming tissues, they must rely on stem cells for their replenishment. Unique within the adult organism, stem cells possess a distinctive ensemble of cellular behaviours reminiscent of those expressed by embryonic progenitors, those cells which initially produced differentiated cell types within the pre-natal animal (Hall and Watt, 1989; Potten and Loeffler, 1990). For the most part, however, these embryonic progenitors are transient and progressively lose their multipotency as development proceeds until they effectively disappear via terminal differentiation. In contrast, stem cells are everlasting with respect to the lifespan of an organism (Hall and Watt, 1989; Potten and Loeffler, 1990). This is an essential stem cell characteristic as the requirement for the generation of new, functionally mature cells proceeds throughout adult life. The cell biological basis of this classical stem parameter is a proliferative process termed self-renewal, whereby stem cells divide in order to maintain their persistence in the organism. Although there is often much dispute surrounding the exact definition of stem cells and which traits should be included in such a definition, self-renewal is unambiguous in this regard as this feature is solidly present in any discussion of stem cell parameters. Counterintuitive to their high proliferative potential, at any given singular moment of 9 time, stem cells are typically slow-cycling, such that these cells are conserved and their function spread out over time to preserve tissue homeostasis throughout the lifetime of an organism. In addition, it is suggested that an infrequently cycling, non-transient cell would be desirable as it would accumulate less D N A replication-related errors, and, hence, would present less risk to the animal than a permanent, fast-cycling cell (Lavker and Sun, 2000). A second classical function of stem cells is the ability to give rise to daughter cells that are more differentiated than themselves and which typically embark upon a course that ultimately leads to terminal differentiation. B y performing this function, stem cells are able to attend to the cellular replacement needs of their resident tissue. To summarize, the ability to proliferate, self-renew, and to generate differentiated daughter cells are the hallmarks of stem cell behaviour. Cells which exhibit reduced capacities to carry out these features (i.e. limited self-renewal and proliferative capacities and restricted differentiation potential) are more appropriately termed progenitor cells. In addition, the term progenitor may be broadly applied to include \"potential\" stem cells in addition to true committed progenitors (Hall and Watt, 1989; Potten and Loeffler, 1990). Philosophical definitions of what exactly constitutes a stem cell are often subject to much debate. Oftentimes, traits are included in stem cell definitions that are perhaps secondary to their function as potent self-renewers and differentiated cell generators. One such trait is that stem cells are, by nature, undifferentiated cells with respect to both morphology and in molecular makeup (Potten and Loeffler, 1990). However this is often considered a weak parameter of stem cells, as it is only relative, in a qualitative manner, to their mature descendants. Another trait is that of stem pluripotency, or the ability to produce a range of differentiated cell phenotypes (Hall and Watt, 1989; Potten and 10 Loeffler, 1990). Most tissues contain a variety of specialized cells, which in their maturity serve as effectors of the function of a particular tissue. Many stem cells possess the ability to regenerate all of the component cell types in their resident tissue. A corollary to this characteristic is a stem parameter that holds that stem cells should be able to completely reconstitute their resident tissue when challenged by injury. Although not a prerequisite according to most definitions, some stem cells certainly do possess this capability. A final secondary parameter of stem cells is the ability to regulate their planes of mitosis, such that both symmetric and asymmetric divisions are available when required by the tissue. Decisions regarding the plane of mitosis affect the outcome of a particular cell division, such that a symmetric division results in either two stem cells or two more mature cells, while an asymmetric mitotic event yields one of each (Potten and Loeffler, 1990; Gage, 2000). In very early studies of self-renewing tissues, it was believed that every proliferating cell detected in vivo was a stem cell (reviewed in Jones, 1997). However, it is now understood that stem cells can indeed be very slowly cycling cells, and the cells exhibiting rapid rates of proliferation in vivo are transitory, committed intermediates, termed transit amplifying cells (Potten and Loeffler, 1990). A s such, regenerative pathways in self-renewing tissues are initiated by stem cells and proceed through committed transit amplifying progenitor stages to culminate with the production of differentiated progeny. Stem cell divisions result, on average, one transit amplifying cell and one stem cell, either by populational or individual asymmetry (Hall and Watt, 1989; Watt and Hogan, 2000). In mammals, this decision is highly regulated by environmental cues, and does not appear to be strictly predetermined (Watt and Hogan, 2000). The 11 primary function of transit amplifying cells is to decrease the proliferative burden from stem cells such that a single stem cell division can ultimately yield a large number of differentiated progeny (see Figure 1.4). In addition, the presence of transit amplifying cells within the stem cell hierarchy serves to segregate different routes of commitment, such that one population of transit amplifying cells is lineally committed to one specialized cell phenotype, while its sister population is dedicated to generating another. In general, one can envision these hierarchies as gradients of stem cell to mature progeny function (Potten and Loeffler, 1990). The first such gradient involves proliferative capacity: stem cells display the highest degree of this property, while terminally differentiated cells at the bottom of the hierarchy are incapable of cell division. Transit amplifying cells, while capable of proliferation, have a finite number of possible mitotic rounds. Further, the differentiative capacity decreases in a stepwise fashion down the hierarchy. Stem cells generally possess the potential to produce a variety of functionally mature descendants, while transit amplifying cells display progressively less potential in this regard as the hierarchy nears its mature, differentiated endpoint (Potten and Loeffler, 1990). In order to ensure the continual fulfillment of stem cell responsibilities, the choice between stem cell maintenance and differentiation must, intuitively, be a highly regulated process (Jones, 2001). The niche model predicts that growth factors, extracellular matrix components and intercellular contact with neighbouring cell types form a specific microenvironment for stem cells that provide the necessary cues for the management of stem cell function in the adult (Hall and Watt, 1989). A s such, a change in the local niche environment w i l l elicit a functional change in the stem population resident within 12 Current Opinion in Genelica 4 09velopment| Figure 1.4 The classical stem cell->transit amplifier-> differentiated progeny hierarchy. A stem cell (S) divides asymmetrically to produce one stem daughter and one transit amplifying progenitor (T). Amplification divisions within the transit progenitor population produces a total number of 8 terminally differentiated cells (TD) for each single stem cell mitosis. (Watt, 2001) 13 that region. In contrast to functional modulation, apoptosis can be employed to cull the stem cell pool, such that cells excluded from the optimal stem cell niche are eliminated (Domen, 2001). Hence, apoptosis serves as a mechanism to regulate the number of stem cells, which are often produced in excess of what is required by the surrounding tissue. A pervasive challenge in stem cell biology is in detecting the components of the niche responsible for directing stem cell function in vivo (Fuchs and Segre, 2000). Though stem cells are regulated by a combination of intrinsic and extrinsic factors, this control is considerably influenced by the extracellular environment. In the past, studies concerning environmental signaling were primarily focused on secreted growth factors and cytokines, which can be released to instruct the needs of a tissue to its stem cell population. For example, TGF-P inhibits proliferation of epidermal keratinocytes by causing growth arrest in G l . TGF-P is produced by stem cell keratinocytes, thereby providing an autocrine mechanism by which these cells may control their own growth (Akhurst et al., 1988). In contrast, the growth factors EGF and TGF-a stimulate proliferation of epidermal stem cells (Barrandon and Green, 1987; Coffey et al., 1987). Hence, via the secretion of growth factors, proliferation within the stem cell compartment can be controlled to cater to the needs of the tissue. However, in recent years, it has become apparent that growth factor signaling accounts for only a portion of the picture (Sastry and Horwitz, 1996). Evidence arising from a variety of stem cell systems conclusively demonstrates that cell adhesion molecules, most notably the integrins, are also key players in the management of stem cell activity. For example, expression of the integrins o^Pi (or very late activating antigen-4 [VLA-4]) and CC5P1 (VLA-5) mediate adhesion of CD34 + hematopoietic 14 progenitor cells to fibronectin and vascular adhesion molecule ( V C A M ) , both found in the bone marrow microenvironment (Brakebusch et a l , 1997). Co-culturing C D 3 4 + cells with fibronectin inhibits entry into S-phase (Hurley et al., 1995). Furthermore, the addition of an anti-o^Pi antibody to long-term cultures of bone marrow cells causes an inhibition of the myeloid and lymphoid lineages (Miyake et al., 1991). These antibodies also result in the appearance of hematopoietic progenitors within the peripheral blood, likely as a consequence of lost adhesion to the bone marrow stroma (Papayannopoulou and Nakamoto, 1993). The final major component of the stem cell niche is intercellular contact, whereby adjacent cells influence one another via cell surface protein interaction. A n example of this class of stem cell control is provided by the Notch family of receptors. Notch receptors, present on the surfaces of subsets of cells, bind their ligands, present on neighbouring cells (Weinmaster, 2000). Both receptor and ligand are transmembrane, cell surface proteins. Upon ligation, Notch elicits signaling pathways within the cell to regulate gene expression. In parallel to other studies of Notch action within stem cells, Hitoshi and colleagues (2002) recently demonstrated that Notch activation functions in the maintenance of the stem cell phenotype in ES-derived neurospheres. A s Notch signaling is initiated via intercellular contact, the preceding is a further example o f mechanisms by which stem cells can self-regulate their in vivo population. Nonetheless, neither of the three modes of signaling functions in isolation in the in vivo animal and together they possess the means to faithfully regulate stem cell behaviour. Further dissecting the stem cell niche for factors responsible for the management of stem cell activity in their in vivo environment w i l l permit the development of precise culture 15 conditions used to control stem cell maintenance and to specifically direct their differentiation in vitro for potential therapeutic purposes (Jones, 2001). 1.3 Manifestations of Stem Cells in Three Physiologically Different Tissues In order to illustrate the function and characteristics of stem cells noted above, three different stem cell systems, at various degrees in their characterization, are discussed below. Hematopoiesis, the process of blood cell formation, is the most characterized of all adult stem cell systems. Early in the study of this process, researchers revealed that healthy bone marrow cells could reconstitute the entire blood system when injected into lethally irradiated mice (Ford et al., 1956; T i l l and McCul loch , 1961). Later, this astonishing regenerative ability was attributed to single pluripotent, clonogenic stem cells with high capacities for self-renewal, resident within the bone marrow (Weissman et al., 2001). Primitive hematopoietic stem cells can commit to become either the common myeloid or lymphoid progenitors which, in turn, eventually produce a total of 9 separate lineages (Chan and Watt, 2001). The magnitude of this process is highlighted by the estimate that homeostasis requires the production of 10 1 1 blood cells per day in humans (Domen, 2001). The primitive adult stem cells of this system reside within the bone marrow, which serves as their regulatory niche. Herein, bone marrow stromal cells secrete regulatory factors and communicate intercellularly with stem cells to influence decisions concerning proliferation, survival and commitment to differentiation (Chan and Watt, 2001). 16 The complexity of the hematopoietic lineages clearly illustrates the importance of possessing reliable cell surface markers that recognize distinct cellular steps within a stem cell hierarchy. Stem cells and committed progenitor populations can be fractionated in vitro by exploiting their known antigenic characteristics (Weissman et al., 2001). Fractionated cells can then be studied using in vitro clonogenic assays and in vivo repopulation assays to assess stem cell parameters. When compared to the multifaceted lineage o f the hematopoietic stem cell system, that of the epidermis appears relatively simple. However, it possesses a comparable regenerative capacity as the human epidermis turns over every two weeks (Fuchs and Segre, 2000). The epidermis is a multi-layered epithelium consisting of 4 principle cellular layers which differ in their degree of differentiation, with the least mature cells located in apposition to the basement membrane and the most mature located in the upper layer of the epidermis (Jones, 1997). The stem cells within this system are believed to reside within the population of basal cells in close apposition to the basement membrane at the base of the epidermis (Turksen and Troy, 1998). The most commonly utilized marker of these cells is pi integrin, which is expressed at high levels on the surfaces of keratinocyte stem cells (Jones et al., 1995). In the interfollicular epidermis, only one fate is offered for keratinocyte stem cells (Jones et al., 1997). Transit amplifying cells are also located within the basal cell population, albeit typically at slightly higher levels within the epidermal epithelium (Turksen and Troy, 1998). The regulation of epidermal stem cell activity is known to rely heavily on the interaction with the basement membrane, upon which the epidermal stem cells reside. Basement membranes are specialized sheets of extracellular protein matrices that are 17 found surrounding or adjacent to a wide variety of cells (Timpl, 1996). A key function of basement membranes is to compartmentalize different types of tissue like, for example, the epidermis and the mesenchyme beneath it, in order to prevent cell mixing. More importantly in the context of this discussion, the basement membrane functions as a platform upon which the regulation of stem cell activity depends. Epidermal stem cells adhere to a basement membrane, which includes in its composition the E C M proteins fibronectin, laminin, type IV collagen, and heparan sulphate proteoglycan (Watt, 1987). It is these E C M proteins, concentrated within the basement membrane, that provide the majority of the known cues employed by cell adhesion molecules to carry out their task. Lastly, neural stem cells can be roughly defined as any cell that can generate neurons and glia (via asymmetric division) and has some capacity for self-renewal (Gage, 2000). In the rodent hippocampus, it is estimated that one neuron is produced every day for every 2000 pre-existing granule cells (Gage, 2000). Perhaps the most characterized adult neural stem cells are those derived from the subependymal layer of the germinal zone which lines the lateral ventricles (Morshead and van der Kooy, 2001). Previous to their discovery, it was generally thought that the mammalian brain was entirely post-mitotic. These cells, when cultured in the absence of any coating substrate together with E G F , form clonal cell aggregates termed neurospheres (Reynolds and Weiss, 1996). Spontaneous differentiation of these spheres yields a number of mature phenotypes, including neurons and glia (Reynolds and Weiss, 1992). In addition, neurospheres can be serially passaged, indicating their robust potential for self-renewal (Chiasson et al., 1999). A major obstacle in the study of these neural stem cells is the lack of established stem cell markers, making their exact identification in vivo impossible (Gage, 2000; 18 Morshead and van der Kooy, 2001). In culture, these cells are identified according to the aforementioned stem traits. However, further insight into the subependymal neural stem cell hierarchy would be greatly accelerated i f reliable cell surface antigens were revealed for stem/progenitor cell candidates that could be utilized both in vivo and in vitro. In addition, as there is still some question as to the exact location of these cells in vivo, their niche is as yet poorly defined. Although insight has been gained towards the complement of growth factors beneficial to their expansion and differentiation in vitro, these potential niche components have not been confirmed in their natural residence. Finally, in contrast to blood-forming and epidermal tissues, tissues that house neural stem cells are not believed to be regenerative in the sense of replacing tissue lost to cell death or injury. Rather, the consensus concerning the in vivo function of neural stem cells within the mammalian C N S is that they produce neurons to add onto the existing cytoarchitecture of areas of the brain involved in learning and memory, such as the hippocampus, olfactory bulb and neocortex (Gage, 2000; Magavi et al., 2000). 1.4 The Cellular Constituents of Olfactory Neurogenesis In parallel to the epidermis and blood-forming tissues, the olfactory epithelium is likewise a self-renewing tissue. Stem cells are, as above, proposed to reside in the O E in order to replace neurons that are continually lost due to environmental influence (Calof et al., 1998; Schwob, 2002). Initial studies directed towards the identification of possible O R N stem/progenitors utilized 3H-thymidine incorporation coupled with autoradiography (Moulton et al., 1970: Graziadei and Metcalf, 1971; Graziadei, 1973; Moulton, 1974; Graziadei and Mont i Graziadei, 1978). A t early time-points after incorporation, labeled 19 nuclei were present within the basal cell compartment of the O E . Later, however, the 3 H -thymidine labeled cells were discovered in a more apical location in the O E within the neuronal layer. It was therefore concluded that basal cells divide and migrate apically to produce O R N s Studies conducted at a number of laboratories have contributed to the notion that neurogenesis in the O E , as in other stem cell systems, is an exquisitely regulated process (Schwob, 2002). In the normal, unperturbed animal, olfactory neurons undergo a continual cycle of death and rebirth from basally located precursors, as noted above. Although the rate at which this cycle occurs is slow in the normal animal, several techniques have been developed to dramatically enhance the pace of degeneration/regeneration in the O E , thereby easing the dissection of events that affect the regulation of olfactory neurogenesis. A t present, there exist three general methods of inducing the degeneration of olfactory neurons: 1) chemical destruction of the O E by various agents (e.g. methyl bromide inhalation, Triton X-100 or ZnS04 infusion; 2) transection of the olfactory nerve; and 3) removal of the olfactory bulb (also termed olfactory bulbectomy) (Costanzo, 1984, 1985; Costanzo and Graziadei, 1983; Mont i Graziadei, 1983; Schwob et al., 1992; Schwob et al., 1995). Following each type o f experimental injury, animals are allowed to recover and are typically injected with thymidine analog prior to sacrifice at set time-points in order to establish a timeline of degeneration/regeneration. A l l three result in the immediate or eventual destruction of ORNs. The bulbectomy and nerve transection procedures eliminate only ORNs, while chemical lesions typically eliminate the entire O E , sparing, for the most part, only the most basally situated cell types, namely 20 G B C s and H B C s . Hence, the regenerative response elicited by chemical lesion is often referred to as \"epitheliopoiesis\", while that caused by axotomy or bulbectomy is described by the familiar term \"neurogenesis\" (Goldstein et al., 1998). In all three paradigms, there is a marked increase in both the number of dying cells and the number of proliferating basal cells post-lesion, although the kinetics of these degenerative and regenerative waves differ (Schwartz Levey et al., 1991; Carr and Farbman, 1992, 1993; Schwob et al., 1992; Schwob et al., 1995). In addition, G B C s contribute more substantially to the proliferative compartment than do H B C s in each lesion paradigm. It was also discovered that the lesion model that produced the most H B C proliferation was that which eliminated the highest variety of cell types (fully 90% of the epithelium), namely chemical traumatization by methyl bromide inhalation (Schwob et al., 1995). The methyl bromide inhalation paradigm was also instructive as to the possible identity of the hypothesized olfactory stem cell as only basal cells were spared post-lesion. Since O R N s eventually repopulate the treated O E , it was inferred that they must arise from one or the other population of basal cells. Lineage analysis using replication incompetent retroviruses has also contributed greatly to the current model o f olfactory neurogenesis. Confirming the results o f the thymidine labeling experiments discussed above, lineage analysis in bulbectomized animals demonstrated a clear relationship between ORNs and G B C s (Caggiano et al., 1994; Schwob et al., 1994). Lineage relationships were also surveyed during methyl bromide induced \"epitheliopoiesis\" in which Huard et al. (1998) arrived at a number of important conclusions regarding tissue reconstitution in the O E . In addition to confirming that G B C s and neurons are lineally related, the data revealed that H B C s and 21 sustentacular cells should also be included in this lineage (Huard et al., 1998). Further, they demonstrated that the complement of O E cell types generated depends on which cell phenotypes are lost due to lesion (Huard et al., 1998). However, these studies did not examine O E G marker co-incidence with retroviral labeling, leaving this portion of the lineage unsolved. The first distinct stage of the olfactory hierarchy was also examined using an embryonic O E explant culture system coupled with [ H]-thymidine uptake analysis and immunohistochemical identification of cell types (Calof and Chikaraishi, 1989). The immediate neuronal precursors (INPs) express the neuronal differentiation factor neurogeninl, divide symmetrically to produce neurons and have a limited capacity for self-renewal (DeHamer et al., 1994; Cau et al., 1997). A s such, FNPs exhibit characteristics of transit amplifying cells. A further contribution of the G B C population to olfactory neurogenesis was illuminated by a study of Mash-1 knockout mice (Guillemot et al., 1993). Mash-1 is a transcription factor that is expressed within a subpopulation of G B C progenitors, located upstream of the neurogeneninl-expressing INPs (Gordon et al., 1995; Cau et al., 1997 ). A targeted mutation in the Mash-1 gene leads to a dramatic reduction in the number of olfactory neurons (Guillemot et al., 1993). It is hypothesized that the Mash-1 expressing cells occupy a compartment once removed from the INPs described previously by this laboratory using the same culture system (Gordon et al., 1995; DeHamer et al., 1994). Studies involving the transplantation of a stem c e l l f r o m its home niche to a foreign environment are utilized to assess whether a particular candidate cell type exhibits the prerequisite functional characteristics of stem cells and are often instructive 22 in determining the extent of intrinsic versus extrinsic regulation of differentiative and proliferative potentials. Furthermore, such studies can yield information as to whether a stem cell has a pre-determined commitment to its native lineage, or whether a stem cell can adapt to its new environment and generate new lineages accordingly. Several such transplantation studies have been performed with respect to the stem/progenitor cell of the O E . Magrassi and Graziadei (1996) showed that both the adult and developing E l 5 rat olfactory epithelia contain cells that possess the potential to generate neurons and glia with a distinct C N S phenotype after transplantation to the embryonic C N S . Constitutively LacZ-expressing O E donor cells were transplanted as single cell suspensions into several C N S sites within the developing rat E l 5 embryo (Magrassi and Graziadei, 1996). Within the first day post-transplant, it was observed that some transplanted cells aggregated into groups, while others integrated into the host tissue as individual cells. Interestingly, only the aggregated donor cells produced neurons possessing an olfactory phenotype, as determined by olfactory marker protein (OMP) immunopositivity. This class of donor cell did not produce cells with a C N S phenotype, while the scattered, individual cells were capable of differentiating into neurons and glia with a central phenotype, as determined via immunohistochemistry and electron microscopic examination of morphology. A s such, it was concluded that the lineage specification o f the O E donor cells is dependent upon cellular interaction between host-donor and donor-donor pairings (Magrassi and Graziadei, 1996). One criticism of the above study is that the donor cells were not purified. Rather, a heterogenous \"slurry\" of cells was transplanted into the donor embryos (Magrassi and Graziadei, 1996). Goldstein et al. (1998) circumvented this problem by selecting for 23 G B C s as their donor material. This was achieved by retroviral infection of dividing cells with a LacZ vector at a time post-bulbectomy when the majority of proliferating (> 90%) cells are G B C s (Goldstein et al., 1998). When bulbectomized OE-derived donor cells were transplanted homotypically into the olfactory epithelia of bulbectomized hosts, only neurons were generated. This study, and that noted in the preceding paragraph, indicate differences in the differentiative potential of G B C s and the unidentified O E stem cell. In vitro studies focused at dissecting olfactory neurogenesis can be divided into two general categories: 1) assays directed at characterizing stem or progenitor cells in the O E ; and 2) assays used to test the overall effects of growth factors and cytokines on olfactory neurogenesis as a whole. There are presently very few published reports concerning the first group of examination. Most notably, M u m m and colleagues (1996) developed a neuronal colony-forming assay to test for stem and progenitor cells in dissociated primary cultures of embryonic O E . To enrich for progenitors, N C A M + neurons were removed from O E cell suspensions via immunological panning. When plated in adherent culture, >95% of the cells differentiated into neurons within the first 2 days in vitro. Most cells die by 7 days in vitro. However, surviving cells at this time-point typically appear in tightly associated colonies of 5 or more cells, which contain subsets of cells that express N C A M . Using thymidine incorporation in tandem with N C A M immunocytochemistry, it was demonstrated that these colonies contain the immediate neuronal precursors (INPs) of ORNs. In addition to INPs, the authors proposed the existence of rare cells in these cultures, which are capable of prolonged neurogenesis up to 7 days in vitro (Mumm et al., 1996). 24 The second category of in vitro examination involves the analysis of secreted factors on the regulation of olfactory neurogenesis. A t their endpoints, these studies typically quantified the total numbers of neurons or the number of thymidine analog-incorporating cells in order to determine the effects of the substances supplied to O E cell cultures. The nature of the starting cultures is varied, ranging from primary explant, semi- and fully-dissociated cells to immortalized cell lines (Mahanthappa and Schwarting, 1993; Shou et al., 2000; Goldstein et al., 1997). Although instructive in the regulation o f neurogenesis as a whole, these studies provide us with little information concerning the identities of stem or progenitor cells within the O E . The results of these assays are discussed further below. 1.5 The Molecu la r Regulation of Olfactory Neurogenesis Olfactory neurogenesis appears highly regulated, such that the production of new O E cells is likely subject to strict control. This is corroborated by evidence arising from several lesion paradigms (as discussed above) that indicate that the rate and extent of neurogenesis is influenced by the density of neurons in proximity to olfactory stem /progenitor cells (Schwob, 2002). In parallel, it was also shown that neurogenesis is inhibited in vitro when O E progenitors are co-cultured with exogenous olfactory neurons, indicating the existence of some feedback mechanism that halts the generation of neurons when no neuronal replenishment is required (Mumm etal . , 1996). These initial, broad observations incited a more detailed examination of the control of olfactory neurogenesis. M u c h information has been revealed by in vivo and in vitro experimentation regarding the role of growth factors and cytokines in the control of this regenerative process. 25 Several studies have implicated fibroblast growth factor-2 (FGF-2) in the control of G B G progenitor cell divisions. Although the exact cell types that express F G F receptors are, as yet, still unknown, R T - P C R indicates that the transcripts for FGFr-1 and FGFr-2 are present within the O E (DeHamer et al., 1994). In the olfactory system, F G F -2 stimulates G B C proliferation when added to dissociated primary cultures of OE-derived cells and to O E explant cultures (Dehamer et al., 1994; Newman et al., 2000). In addition, F G F - 2 inhibits neuronal differentiation when supplemented to OE-derived cell lines (Goldstein et al., 1997). Together, these two effects of FGF-2 may permit the fine-tuned control of the number of ORNs produced by regulating the rounds of progenitor cell division prior to terminal differentiation. In vivo, F G F 2 is detected within olfactory neurons and sustentacular cells within the rat O E (Goldstein et al., 1997). Epidermal growth factor (EGF) and transforming growth factor-oc (TGF-a) are two additional examples of growth factors that exert mitogenic effects upon O E cells. In the O E , T G F - a is present in basal cells, sustentacular cells, and in Bowman's glands beneath the O E by immunohistochemistry, while no immunoreactivity to E G F is detected (Farbman and Bucholz, 1996). The epidermal growth factor receptor (EGFR) , which binds both E G F and T G F - a , is located within the H B C population in rats (Farbman and Bucholz, 1996). The mitogenic effects of T G F - a upon H B C s has been determined both in vivo and in vitro. One in vivo study utilized transgenic mice that overexpressed the T G F - a gene driven by the keratin-14 promoter (Getchell et al., 2000). The olfactory epithelia of these transgenic mice contained more T G F - a protein and evidenced a considerable increase in H B C proliferation when compared with nontransgenic controls, as determined by B r d U incorporation (Getchell et al., 2000). This finding is 26 complimented by in vitro studies, which have shown that proliferation is stimulated upon the addition of T G F - a to explant, semi- and fully-dissociated cultures of O E cells (Mahanthappa and Schwarting, 1993; Farbman and Bucholz, 1996). A further example of growth factor regulatory influences is provided by leukemia inhibitory factor (LIF), which exerts an apparent stimulatory effect on olfactory neurogenesis. The LIF receptor expression is upregulated on G B C s following the neuronal loss induced by bulbectomy (Nan et al., 2001). In vitro, L IF enhances G B C proliferation in primary cultures of O E cells (Satoh and Yoshida, 1997). Hence, several factors are known to positively influence the generation of neurons within the O E . In contrast to the stimulatory influences of the above growth factors, bone morphogenic proteins (BMPs) are believed to negatively regulate olfactory neurogenesis (Shou et al., 1999). Using a neuronal colony-forming assay, it was determined that when B M P s 2, 4, or 7 are added to dissociated cultures of embryonic O E , neurogenesis is inhibited (Shou et al., 1999). To determine which stage of the O R N lineage B M P s target and how their action is manifested, B M P 4 was supplied to the media at different times following plating. The early addition of B M P 4 inhibited neuronal colony formation, while its late addition had no effect. Furthermore, it was demonstrated that B M P 4 causes a rapid decrease in M A S H - 1 immunoreactivity in O E explants, a result that prompted an examination of the cause of M A S H - 1 disappearance (Shou.et al., 1999). It was revealed that exposure to B M P s caused a flurry of new gene expression which resulted in the proteolysis of the M A S H - 1 transcription factor, thereby terminating the progression of the neuronal lineage in those cells (Shou et al., 1999). Although B M P s are expressed in O E tissue, their exact cellular localization is unknown (Shou et al., 2000). 27 In addition to molecules that serve in policing proliferation of O E progenitors, several growth factors function in promoting the neural phenotype. One such growth factor is transforming growth factor-13 (TGF-(3). TGF-P promotes neuronal differentiation when added to semi- and fully-dissociated primary cultures and O E cell lines (Mahanthappa and Schwarting, 1993; Newman et al., 2000). Hence, mechanisms that both positively and negatively regulate O R N formation have been revealed. A complement of brain-specific growth factors, the neurotrophins, also influence the progression o f neuron production in the O E . The neurotrophins are a family o f peptides which exert their effects by interacting with high and low affinity receptors on the surfaces of responsive cells, and have been implicated in several stages of neuronal development throughout the central and peripheral nervous systems. N G F , B D N F , and N T - 3 likely regulate aspects of survival and differentiation within the O E (Roskams et al., 1996). The studies detailed above suggest that O E progenitors are keenly tuned to the requirements of their resident tissue, which secrete signals to instruct the appropriate mode of action according to current needs. However, in contrast to growth factor and cytokine signaling in the O E , the role of adhesive and intercellular regulation of olfactory neurogenesis has remained largely unstudied. One study, however, noted that when cultures of O E cells are biochemically or mechanically stressed, the disruption of cell surface contacts results in the promotion of differentiation in primary O E cultures (Feron et al., 1999). Hence, given the roles of these means of cellular communication in the regulation of stem cells in other self-renewing tissues, it is likely that intercellular and adhesive modes of signaling are active in regulating olfactory neurogenesis. 28 1.6 The H B C vs. G B C as O E Stem C e l l Controversy In vivo anatomical studies indicate that the hypothesized O E stem cell likely resides within the basal compartment of the O E . Retroviral lineage and in vitro studies have confirmed that a subset of G B C s function as immediate neuronal precursors (INPs) and suggest the presence of a slightly more primitive transit amplifying subset of G B C s , located immediately upstream of the INPs. A persistent controversy within the field o f olfactory neurogenesis, however, concerns whether H B C s or G B C s are the stem cells of the O E . Although H B C s and G B C s are lineally related (Caggiano et a l , 1994; Huard et al., 1998), the direction of this relationship is unknown. Many who argue that G B C s are the most likely stem cell candidates appeal to the fact that G B C s are detected earlier than H B C s both developmentally in the early embryo and during methyl bromide induced regeneration of ventral O E (Holbrook et al., 1995; Schwob et al., 1995). However, one could argue that the poor antigenic characterization of these two cell types has influenced the above conclusion. Alternatively, with respect to the embryonic situation, proponents of the H B C hypothesis argue that recent developments in the non-olfactory stem cell field indicate that stem cells may arise in late development, just in time to initiate their role as regulators of post-developmental tissue homeostasis (van der Kooy and Weiss, 2000). This new model hypothesizes that the cells present to create an embryonic tissue and those present to generate an adult tissue are not the same cells (van der Kooy and Weiss, 2000). In contrast, those who favour the H B C argument draw support for their hypothesis from the discovery of cells intermediate in morphology to H B C s and G B C s (Graziadei and Mont i Graziadei, 1979; Holbrook et al., 1995). 29 Nonetheless, it is apparent upon surveying the extant olfactory literature that relatively little assessment of stem cell traits has been performed with respect to either population of cells, although a considerable amount has been revealed regarding the progenitor role of G B C s . For instance, no study has isolated a cell with extensive proliferative properties or potent clonogenic capacity. Indeed, the most commonly utilized culture systems contain cells that are capable of only a few rounds of cell division prior to terminal differentiation (DeHamer et al., 1994). In addition, although multipotency of the proposed stem cell is apparent from transplantation and lineage analyses (Caggiano et al., 1994; Magrassi and Graziadei, 1996; Goldstein et al., 1998; Huard etal . , 1998), this has not been shown in vitro from an isolated candidate O E cell. Furthermore, there has been a lack of reliable cell surface markers to fractionate candidate stem and/or progenitor cells in order to assay these cells individually. A s such, culture methods, at their most selective, are limited to negative selection via immunopanning utilizing N C A M to remove ORNs (DeHamer et al., 1994). Otherwise, heterogenous cell suspensions or explants derived from the O E and consisting of an array of O E cell types have typically been used to test for neurogenic ability in the O E . A s such, the field of olfactory neurogenesis has much to gain by mirroring the methods utilized in the study of other, better characterized, self-renewing tissues. 1.7 W o r k i n g hypothesis and Research Objectives Working hypothesis: The horizontal basal cell (HBC) layer of the olfactory epithelium (OE) contains stem cells that contribute to olfactory neurogenesis. 30 The aims of the current study were 4-fold: 1) To identify new antigenic markers of the H B C population via a screen of antibodies generated against clusters of differentiation (CD) antigens of interest; 2) To develop methods for the enrichment of H B C s in vitro; 3) To assess H C B stem/progenitor cell candidacy, both in vivo and in vitro, via the examination of several stem and progenitor parameters; 4) To examine and identify growth factors and extracellular matrix components ( E C M ) components that influence H B C function. 31 C H A P T E R II. Materials and Methods 2.1 Olfactory Bulbectomies and Tissue Preparation For unilateral bulbectomies, adult C D - I mice (aged 6 weeks) were anaesthetized with Xylaket [25% ketamine HC1, 100 mg/mL; 2.5% Xylazine (Bayer Inc., Etobicoke, Ontario), 100 mg/mL; 14.2% ethanol in 0.9% saline)]. The right olfactory bulb was exposed via partial dorsal craniotomy and was ablated by suction. Care was taken to avoid damage to the contralateral olfactory bulb. The ablation cavity was filled with gelfoam (Pharmacia & Upjohn Inc., Don M i l l s Ontario) to prevent invasion of frontal cortex into this region that could provide an alternative target for regenerating olfactory axons. The skin above the lesion was sutured and the animals were allowed to recover under a heat lamp. The presence of complete lesion was verified both visually and microscopically/After recovery from anaesthesia, animals were maintained according to standard Animal Care and Use protocols until sacrificed at 6 days post-bulbectomy. Prior to sacrifice, mice were anaesthetized with Xylaket and perfused transcardially with phosphate buffered saline (PBS) followed with 4% paraformaldehyde. The brain and olfactory epithelium were dissected out, immersion fixed in 4% paraformaldehyde for two hours, and then sequentially bathed in 10% and 30% sucrose to cryoprotect. Tissue was embedded in plastic moulds with O C T compound (Tissue-Tek, Baxter, Columbia, M D ) over l iquid nitrogen. Cryostat sections (10-14 mm) were then cut and frozen until needed. , 32 2.2 BrdU Incorporation and Detection For in vivo studies, mice were injected with 30mg/kg Bromodeoxyuridine (BrdU) (Sigma) at 3 hrs and 1 hour prior to sacrifice. In vitro, 7 day-old cultures were incubated with 10 m M B r d U for 2 days, at which point they were fixed with 4% paraformaldehyde. Tissue sections and cells were processed according to standard immunohistochemistry and immunocytochemistry protocols (see below) with the exception that a 20 minute treatment in 4 M HC1 was required before the incubation with primary antibody. 2.3 Immunohistochemistry Frozen sections were rehydrated in P B S for 5 minutes, permeabilized in 0.1% Triton-X 100 (Sigma) in P B S for 20 minutes, blocked with 4% normal serum, and incubated at 4\u00C2\u00B0C for 12-20 hours in primary antibody. Tissue sections were washed and incubated with either biotinylated (30 minutes) or fluorophore-conjugated secondary antibodies (60 minutes) at room temperature. Sections processed using the horseradish peroxidase method were treated with 0.5% H2O2 in P B S for 10 minutes to quench endogenous peroxidase activity, incubated with avidin-biotin-peroxidase kit (Vectastain A B C kit, Vector laboratories, Burlingame, C A ) to enzyme label the biotinylated secondary antibody, and then developed with either V I P , diaminobenzidine (both from Vector labs), or i f a fluorescent label was desired, Amplex red (Molecular Probes Inc, Eugene, OR) . In some cases, a 10 minute incubation with 4',6-Diamidine-2'-phenylindole dihydrochloride (DAPI , 1:15 000; Roche) was performed to visualize nuclei. Slides were then coverslipped with aquapolymount mounting media (Polysciences) or, i f fluorescently labeled, with Vectashield mounting media (Vector Laboratories). In all 33 instances, negative controls, consisting of an incubation in P B S rather than primary antibody, were included to assess non-specific staining. In addition, each primary antibody utilized either possessed distinct staining patterns when compared to other antibodies generated against antigens of the same donor species, or evidenced no staining when utilized on irrelevant tissue or cells. A n additional 10 minute incubation in 0.1 M N a O H step was added prior to the Triton X permeabilization step for the a integrin antibodies, in order to increase signal. 2.4 Antibodies The following primary antibodies were utilized for immunohistochemistry: monoclonal unconjugated mouse anti-BrdU G3G4 (1:500; Developmental Studies Hybridoma Bank, Iowa city, IA), monoclonal hamster anti-mouse I C A M - 1 (CD54; 1:100; Pharmingen, San Diego C A ) rat anti-mouse P i integrin subunit (CD29; l:100;Pharmingen), monoclonal rat anti-mouse P4 integrin subunit (CD 104; 1:100; Pharmingen), monoclonal rat anti-mouse CD34 (1:100; Pharmingen), monoclonal rat anti-mouse CD43 (1:100, Pharmingen), monoclonal rat anti-mouse CD44 (1:100, Pharmingen), monoclonal mouse anti-rat Beta-Ill neuron-specific tubulin (NST; 1:500; T u J l ; BabCo, Richmond, C A ) , polyclonal rabbit anti-bovine glial fibrillary acidic protein, G F A P (1:5; Incstar, Stillwater, Minnesota), monoclonal mouse anti-bovine S100p (1:1000; Sigma), polyclonal rabbit anti-mouse P75 (1:1000; Chemicon international Inc., Temecula, C A ) polyclonal goat anti-rat O M P (1:5000; gift from F. Margolis), polyclonal rabbit anti-chicken N C A M (1:500; Chemicon international, inc.), monoclonal mouse anti-rat G B C - 2 (undiluted; gift from J. Schwob), polyclonal goat 34 aintegrins 1 (rat origin), 3 (human origin) and 6 (human origin) (1:100; Santa Cruz biotech, Santa Cruz, C A ) , polyclonal rabbit anti-rat adenylate cyclase III (1:200; Santa Cruz Biotech), polyclonal rabbit anti-rat Gas /o l f (1:1000; Santa Cruz Biotech), and monoclonal mouse anti-human keratin 903 (undiluted; Enzo diagnostics Inc, Farmingdale, N Y ) . Biotinylated secondary antibodies used for peroxidase immunohistochemistry were horse anti-hamster (Pharmingen), goat anti-rat (Vector labs), goat anti-rabbit (Vector labs), rabbit anti-goat (Vector Labs) and horse-anti mouse (Vector labs). Secondary antibodies used for immunofluorescence were goat anti-hamster Cy-3 (Jackson ImmunoResearch Laboratories Inc., West Grove, P A ) , donkey anti-goat, goat anti-rabbit, goat anti-mouse, goat anti-rat Alexa 488 and Alexa 596 (Molecular probes Inc). 2.5 Primary Culture of Basal Cells For each basal cell preparation, the olfactory epithelium was carefully dissected from a litter of postnatal day (P) 5-9 C D - I mice into lOmLs of D M E M / F 1 2 (1:1; Gibco) pre-warmed to 37 \u00C2\u00B0C. During the dissection procedure, care was taken to ensure that the forceps tips were not pushed past the cribiform plate (to avoid removing olfactory bulb tissue), nor past the olfactory epithelium (to avoid removing adjacent optic tissue). Olfactory tissue was minced with a sterile razor blade and scissors to 1 mm in size, and spun at 1 l g for 10 minutes to reduce the number of fibroblasts in the desired cell fraction. The supernatant was aspirated and lOmls of D M E M / F 1 2 was added to resuspend the pellet. The suspension was triturated with a PI000 plastic tip pipettor and spun at 43 g for 35 5 minutes, after which the supernatant was aspirated and the pellet was resuspended in 10 mis o f fresh D M E M / F 1 2 . The tissue suspension was then dissociated enzymatically by incubation with Liberase blendzyme I (0.45 mg/mL; Roche), hyaluronidase (1 mg/mL; Sigma) and D N A s e I (1 mg/mL; Roche) for 1 hour in a 37\u00C2\u00B0C water bath with occasional swirling to resuspend tissue. Initial experiments determined that these digestion conditions produced the best yield of basal cells in culture. Following enzymatic dissociation, the suspension was triturated and filtered through a sterile 80 urn wire mesh to remove larger pieces of undissociated tissue. The flow-through was centrifuged at 250g for 5 minutes, after which the supernatant was aspirated and the pellet resuspended D M E M / F 1 2 . After additional trituration, the suspension was filtered through a 40 um cell strainer to remove non-dissociated cell aggregates. Cel l fractions from each of the filtrates and flow-through steps were cultured in D M E M / F 1 2 + 10% F B S supplemented with fungizone and penstrep (2.5 mg/mL and 100 mg/mL, respectively, Gibco), and it was determined that the highest number of basal cells was present among cells remaining on top of the 40 um cell strainer. These cells were used for initial plating experiments, prior to the selection of I C A M - 1 + cells using M A C S . 2.6 I C A M - 1 Immunomagnetic Selection of Basal Cells: The culture method utilized for immunomagnetic selection of basal cells was the same as that detailed above for initial experiments, except that the post- 80 um suspension was treated in 2 m M E D T A in P B S for 10 minutes at 37\u00C2\u00B0C to further dissociate cells, such that more basal cells could pass through the 40 um cell strainer. 36 The resulting single cell suspension obtained from the 40 urn flow-through was spun at 250 g for 5 minutes and then blocked in 2% F B S in P B S for 10 minutes at room temperature to prevent non-specific binding of primary antibody. The suspension was spun at 250 g for 5 minutes once again, the supernatant aspirated and the cells treated with a 1:1000 dilution of biotinylated anti-ICAM-1 (CD54) antibody (Pharmingen) in P B S for 30 minutes on ice. The cells were washed twice in 2 m M E D T A / P B S and then incubated with magnetic activated cell sorting ( M A C S ) streptavidin microbeads (Miltenyi biotec, Auburn, C A ) for 20 minutes at 4\u00C2\u00B0 C. Labelled cells were separated using a M A C S M S separation column (Miltenyi biotec) placed in the magnetic field o f the M A C S stand (Miltenyi biotec). The yield of I C A M - 1 + , I C A M - 1 \" and unselected fractions (taken prior to sorting) was then determined using a haemocytometer. For clonal density plating, cells were plated at 6000 viable cells per 10 cm petri dish. Cel l viability was assessed using trypan blue exclusion during haemocytometer counts. During the counting procedure, the presence o f a single cell suspension was also visually inspected prior to plating the cells. Standard plating medium was D M E M / F 1 2 + 10% F B S supplemented with fungizone and penstrep (2.5 mg/mL and 100 mg/mL, respectively, Gibco). The standard coating substrate was rat tail collagen, a source which contains primarily which type I collagen (5 ug/cm 2; Roche). Cells were incubated at 37\u00C2\u00B0 C with 5% CO2 for 2 weeks, at which point small (containing <30 cells), medium (>30 and <150 cells) and large (>150 cells) colonies were counted by scanning dishes upon a grid using an inverted microscope at 50X magnification. Colony-forming efficiencies were determined by dividing the number of total colonies scored by the number of cells 37 initially plated. 2.7 Test Conditions for Optimizing Colony Forming Efficiency Media: Alternative media tested for effect on colony-forming efficiency included O p t i - M E M supplemented with 4% F B S , Keratinocyte-Serum free media, D M E M / F 1 2 supplemented with 10% F B S , R P M I supplemented with 10% F B S (all media and supplements are from Gibco). A l l media conditions were supplemented with fungizone and penstrep and were performed in triplicate for each independent experiment. I C A M -1 + cells were plated on collagen-coated 10 cm petri dishes at 6000 cells/dish. A t 14 D I V , colonies were scored and colony-forming efficiency was determined for each condition. Growth factors tested were used to supplement O p t i - M E M / 4 % F B S : E G F (lOng/mL; Roche), T G F - a (20ng/mL; Sigma), and LIF (20 ng/mL; Sigma), individually and in combination. A s cell growth at clonal density does not require frequent replenishment o f the media (Freshney, 2000), the media and growth factors were refreshed weekly until scoring at 2 weeks in vitro. A s above, cells were plated in triplicate for each independent experiment at a density of 6000 cells per collagen-coated 10 cm dish. Extracellular matrix components tested for colony-forming efficiencies of I C A M - 1 + cells: 10 cm dishes were coated with collagen (5 ug/cm 2; Roche), laminin (3 ug/cm 2; Roche), fibronectin (5 ug/cm ;Roche) and laminin/collagen and fibronectin/collagen combinations. The concentrations of collagen, fibronectin, and laminin used for the substrate mixture experiments were as follows: 3.4 ug/cm collagen and 1 ug/cm laminin (2collagen.T laminin), 1.7ug/cm2 collagen and 2 ug/cm 2 laminin 38 (lcollagen:21aminin), 3.4 u.g/cm2 collagen and 1.7 ug/cm 2 fibronectin, and 1.7 ug/cm2 collagen with 3.3 ug/cm fibronectin. I C A M - 1 cells were plated in triplicate at 6000 cells per 10 cm dish and cultured in D M E M / F 1 2 + 10% F B S for 14 D I V , at which time small, medium and large colonies were scored and colony-forming efficiencies determined for each condition. A l l graphs were plotted using Cricketgraph and student's t tests were performed using Microsoft Excel . 2.8 Assessment of Adhesion Kinetics I C A M - 1 + cells were plated at high density (between 7.5 X 10 4 and 1.1 X 10 s cells per well) in D M E M / F 1 2 + 10% F B S onto duplicate 6-well tissue culture plates coated with collagen (5 ug/cm ), laminin (3 pg/cm ), and fibronectin (5 ug/cm). Media from these wells was removed after 4 or 24 hours (one time-point per well), wells were washed 3 times in P B S and non-adherent cells were counted with a haemocytometer. The data were analyzed statistically as above. 2.9 Immunocytochemistry Cells were fixed for 10 minutes in 4% paraformaldehyde, permeabilized in 0.1% Triton-X 100 for 15 minutes, and blocked in 4% normal serum for 30 minutes before an incubation in primary antibody (or P B S for negative controls) for 12-18 hours at 4\u00C2\u00B0C. After washes in P B S , the cells were then incubated in the appropriate fluorescently conjugated secondary antibody for 1 hour at room temperature. Prior to coverslipping in vectashield (Vector labs), the cells were treated with the nuclear counterstain D A P I (Roche). A l l images were visualized with an Axioskop 2 M O T microscope (Zeiss, Jena 39 G E R ) and a SPOT camera (Diagnostic Instruments Inc., Sterling Heights MI) with Northern Eclipse software (Empix Imaging Inc., Mississauga, ON) and were compiled using Adobe Photoshop 6.0. 40 C H A P T E R III. An initial screen of CD antibodies reveals three cell surface markers for horizontal basal cells within the mouse olfactory epithelium 3.1 Introduction The main focus of this study was to characterize horizontal basal cells (HBCs) in order to determine whether they may function as stem cells within the mouse olfactory epithelium (OE). A common impediment to the study of adult neural stem cells is a lack of readily identifiable undifferentiated markers for positively identifying and selecting quiescent neural stem cells from within an adult tissue (Gage, 2000; Morshead and van der Kooy, 2001). The same quandary holds true for H B C s , which have previously been characterized by the lack of antigenic markers of differentiation in conjunction with the presence of cytokeratin 5/6, an often unreliable intermediate filament marker (Calof and Chikaraishi, 1989; Suzuki and Takeda, 1993). The dearth of dependable molecular markers for neural stem cells differs from the situation within the epidermal and hematopoietic systems, where populations enriched in stem cells or committed transit amplifying cells can be distinguished on the basis of marker expression both in vivo and in vitro (Fuchs and Segre, 2000; Weissman, 2000). Furthermore, given that many of these antigens are present at the cell surface, in vitro sorting techniques can be exploited to fractionate populations within the stem/progenitor lineage, thereby by enabling their individual in vitro study. Hence, initial efforts of this study were focused at revealing potential cell surface antigens of the olfactory H B C population via an immunohistochemical screen of selected clusters of differentiation (CD) antigens. These antigens were chosen according to their reported expression and function on stem and progenitor cells of different origins. 41 3.2 ICAM-1, P i integrin and P4 integrin are expressed in basal cells apposed to the basement membrane within the adult olfactory epithelium To identify new cell surface markers for the H B C layer, an immunohistochemical screen of select C D antigens was performed. Antibodies included in the screen were selected from a list of mouse C D antigens and were chosen on the basis of reported function in regulating proliferation and/or differentiation and expression in stem/progenitor cells of other systems (Table 3.1). Immunohistochemistry was performed on P F A fixed coronal sections of adult O E . Intercellular adhesion moleule-1 ( I C A M - 1 ; CD54) is uniformly expressed within the bottom-most basal cell layer of the O E (Figure 3.1, A ) . Immunoreactivity is confined to those cells situated directly on top of the basement membrane separating the O E proper from the lamina propria. These cells resemble H B C s in their morphology and position within the epithelium (Holbrook et al., 1995). The pattern of I C A M - 1 immunoreactivity, with respect to individual basal cells, appears to represent a homogenous, pericellular distribution encompassing the entire surface of the cell (Figure 3.1, A ) . Likewise, the P i and P4 integrin subunits (CD29 and C D 104, respectively) are expressed in a uniform fashion upon the basal cells directly apposed to the basement membrane (Figure 3.1, B and C) . However, the intensity of the P i integrin signal was markedly weaker than those of I C A M - 1 and P4 integrin. Furthermore, in contrast to the apparent \"full ce l l \" distribution of the I C A M - 1 signal, P i and P4 integrin immunoreactivity appeared to be concentrated to the basal surfaces of these cells. Negative, secondary antibody only controls for I C A M - 1 and the integrins are represented in Figure 3.1, D and E , respectively. 42 C D antigen Recognizes subsets of stem and/or progenitor cells in: Reported function of interest CD29 (Pi integrin subunit) hematopoiesis; epidermis; prostate epithelium regulation of proliferation, differentiation, survival; most commonly used marker for epidermal stem cells CD34 hematopoiesis Adhesion; a common marker for hematopoietic progenitors CD43 hematopoiesis adhesion CD44 hematopoiesis; epidermis; prostate epithelium Extracellular adhesion; binds hyaluronic acid; tumour metastasis CD54 ( ICAM-1) hematopoiesis; expressed in response to injury in epidermal keratinocytes Adhesion; regulation of proliferation and survival; proposed role in differentiation CD104 (P4 integrin subunit) hematopoiesis; epidermis; prostate epithelium adhesion to basement membrane; associates with integrin c i 6 subunit to from a laminin-binding receptor; regulation of proliferation, survival, differentiation and survival Table 3.1: Expression and functional properties of selected CD antigens within stem/progenitor cell hierarchies of other self-renewing tissues. (Nievers et al., 1999; Collins etal. , 2001; Mason et al., 2001) 43 Figure 3.1: I C A M - 1 , Pi integrin and p% integrin are detected within H B C s of adult olfactory epithelium. A screen of selected C D antibodies detected three cell surface proteins upon horizontal basal cells (HBCs) , situated on top of the basement membrane (dotted line) within the O E . (A) I C A M - 1 immunoreactivity is detected uniform fashion on H B C s (arrow) . (B, C) Likewise, the integrin subunits pi and p 4 are expressed in a continuous manner upon H B C s (arrows). Immunoreactivity for Pi and P 4 integrin subunits was also detected in the lamina propria (LP) in a pattern indicative of olfactory ensheathing glia (arrowheads, B and C) . (D and E) Secondary only negative controls for I C A M - 1 and the integrins, respectively. Magnification 400X. 44 The P i and P4 integrins are also detected within other non-HBC cells situated within the lamina propria (LP), beneath the olfactory epithelium proper (Figure 3.1, B and C , arrowheads). These integrin positive cells possess a staining pattern indicative of olfactory ensheathing glia (OEGs) (Ramon-Cueto and Av i l a , 1998). 3.3 ICAM-1 expression directly overlaps with that of the horizontal basal cell marker, Keratin 903 A s the bulk of all ensuing experiments directed at testing the H B C population for stem characteristics hinges on the correct identity of the adhesion receptor-expressing cells, we next wished to confirm that these proteins are localized to the H B C population. Towards this end, double immunofluorescence was performed using I C A M - 1 , as a case in point, in combination with the common H B C marker from the literature (Calof and Chikaraishi, 1989; Suzuki and Takeda, 1993). A s demonstrated in Figure 3.2, the I C A M -1 immunoreactivity (A) directly overlaps with that of keratin (B; overlap in C with D A P I nuclear stain in blue) verifying that I C A M - 1 does in fact label H B C s . 3.4 a integrins are expressed in a complimentary fashion to the P i and P4 integrins Integrins are heterodimeric cell adhesion molecules formed by the non-covalent binding of a and p subunits (Hynes, 1992). Given that we have demonstrated that two p integrin subunits are expressed upon H B C s , we next wished to determine the identities o f the a integrin subunits with which they pair to form functional extracellular matrix (ECM)-binding receptor pairs in vivo. To this end, immunohistochemistry was performed on P F A fixed adult O E tissue with antibodies directed against 6 of the a integrin subunits. 45 Figure 3.2: H B C expression of I C A M - 1 is conf i rmed v i a K e r a t i n 903 co-localization. The I C A M - 1 signal (A) directly overlaps with the H B C marker keratin 903 (B); merged in (C),with D A P I nuclear stain, confirming the identity o f the I C A M - 1 + population. Magnification 400X. 46 We detected expression of oci, 0 C 3 , and ct6 integrin subunits upon the surfaces of H B C s (Figure 3.3, A , B , and C) . The 0 : 3 subunit expression was also observed on subsets of olfactory receptor neuron (ORN) axons within the axon bundles of the L P . In addition, 0C6 integrin subunit was expressed within cells around the perimeter of axon bundles, likely olfactory ensheathing glia. Negative, secondary antibody only control is shown in Figure 3.3 D . 3.5 C D antigens are expressed within other cell types within the olfactory mucosa In addition to yielding new markers against the H B C population, the C D antibody screen revealed the presence of C D antigens amongst other cell types within the olfactory mucosa. CD34 is detected within endothelial cells of L P blood vessels (Figure 3.4, A ) . CD43 expression is confined to the L P in single punctate cells scattered throughout. The expression pattern of CD43 is akin to that of macrophages resident beneath the O E proper (Suzuki et al., 1995). However, due to the frequency of positive cells observed, it is likely that other L P cells express CD43 as well . Finally, CD44 is present in a pattern similar to that of CD43 , in that immunoreactive cells were detected scattered throughout the L P . In addition some discrete immunoreactive cells were occasionally observed within the basal cell compartment. The identities of these C D 4 3 + cells are unknown, though they likely represent macrophages. 3.6 Summary In order to identify new cell surface markers of horizontal basal cells (HBCs) , an immunohistochemical screen of select C D antigens was performed on adult mouse O E tissue. Three markers for H B C s were revealed, namely: I C A M - 1 (CD54), Pi integrin 47 Figure 3.3: Potential a integrin pairing partners are identified for the p\ and pV, integrin subunits. a and (3 subunits associate non-covalently to form functional integrin receptors. We wished to identify potential a subunit binding partners for the |3 submits found upon H B C s . a , , a 3 , and a 6 integrin subunit expression is likewise detected on H B C s (A, B , C ; arrows). In addition, a 3 subunit expression is present within O R N axons (B; arrowhead), while a6 subunit immunoreactivity is detected in O E G s surrounding axon bundles within the lamina propria (C; arrowhead). Secondary only, negative control is shown in D . Magnification 400X. 48 A \"JS !;\u00C2\u00BB y Figure 3.4: Other screened C D antigens are detected in non-HBC cells in the olfactory mucosa. CD34 expression is localized to endothelial cells l ining the blood vessels of the lamina propria (A; arrow), while CD43 and CD44 expression is restricted to single, discrete cells within the L P (B and C; arrows). CD44 expression is also present within unidentified cells within the O E proper (C). Magnification 400X. 49 (CD29), and 04 integrin (CD104). The discovery of these new H B C markers w i l l aid in the characterization of this candidate stem cell both in vivo and in vitro, and suggests that olfactory H B C s share common signaling pathways with stem/progenitor cells of other systems. Most significantly, this discovery w i l l enable us to select for H B C s on the basis of their antigenicity using sorting procedures in vitro. Also , the detection of these new markers yields information regarding how H B C behaviour might be regulated via I C A M -1, Pi integrin, and P4 integrin. 50 C H A P T E R IV. An in vivo characterization of HBCs: response to bulbectomy and examination of potential stem cell traits. 4.1 Introduction Our next aim involved the use of these new markers to further characterize horizontal basal cells (HBCs) in their in vivo residence. The olfactory epithelium (OE) can be coaxed into an enhanced regenerative state via removal of the olfactory bulb, a surgical procedure termed the bulbectomy. Upon the removal of their synaptic target, upon which they depend for trophic support, olfactory receptor neurons (ORNs) undergo apoptosis and are subsequently replaced from progenitors within the O E proper (Costanzo and Graziadei, 1983; Schwartz-Levey et al., 1991; Cowan et al., 2001). Given the proposed role of H B C s as olfactory stem cells, we hypothesized that changes in this cell population, as detected by expression of I C A M - 1 , (31 and (34 integrins, would be evident following O R N cell death. In addition, in our search for corroborating evidence to support a stem cell role for olfactory H B C s , we examined several characteristics of stem cells in other self-renewing tissues. 4.2 The removal of the olfactory bulb induces O R N loss and basal cell proliferation in the epithelium Within the field of olfactory biology, the unilateral olfactory bulbectomy is commonly used to facilitate the study of cells that contribute to neurogenesis (Costanzo and Graziadei, 1983; Mont i Graziadei, 1983; Schwob et al., 1992). We first wished to examine the relationship between O R N abundance and proliferation of basal cells within the lesioned O E in order to standardize other published bulbectomy studies with our proceeding results. 51 Bulbectomized mice were injected with B r d U at 3 and 1 hours pre-sacrifice and then processed for immunohistochemistry as discussed in Materials and Methods. A s the 6-day post-bulbectomy time-point coincides with the peak of basal cell proliferation, this time-point was utilized for all subsequent examinations (A. So and J. Roskams, unpublished data). Double immunofluorescence was performed on normal, unlesioned and bulbectomized tissue with antibodies directed against the mature O R N marker O M P or the marker for both ORNs and IRNs, N C A M , in conjunction with B r d U . In the normal, quiescent O E , B r d U + basal cells (likely G B C s , according to their rounded rather than flattened nuclear morphology) were occasionally detected in some regions of the O E that appeared to have a full complement of O M P + ORNs and N C A M + IRNs/ORNs (Figure 4.1, C and E). The loss of >90% of the mature O M P + O R N population by 6d following bulbectomy, leads to the induction of mitosis in the lower third of the O E (Figure 4.1, D and F). Also of note, is that the general thickness of the O E is greater in the normal tissue than in lesioned, due to the loss of neuronal cell bodies (Figure 4.1, A and B) . These results identify an inverse relationship between the abundance o f olfactory neurons and the number of dividing cells, in keeping with earlier reports (Monti Graziadei and Graziadei, 1979; Costanzo and Graziadei, 1983; Schwartz-Levey et al., 1991; Holcomb et al., 1995). N o fluorescent signal was detected in secondary antibody only negative controls. 4.3 GBCs, negative for both ICAM-1 and N C A M expression, are depleted regionally following lesion Given the observed relationship between the frequency of dividing cells in the O E and the abundance of O M P + mature olfactory neurons, we next compared the 52 Figure 4.1. The loss of ORNs following bulbectomy induces proliferation within the basal cell compartment of the O E . The thickness o f the O E is substantially reduced at 6 days post-bulbectomy (B) relative to normal, unlesioned O E (A). This reduction of epithelial thickness is attributed to the loss o f O R N s elicited by bulbectomy. The unlesioned (UL) O E contains a full complement o f O M P + mature O R N s (C; light gray) and N C A M + neurons (E; light gray), yet rarely contains B r d U + , dividing basal cells (C and E ; dark gray). A t 6 days post-bulbectomy (L), the O M P + and N C A M + populations are greatly reduced in number (D and F ; light gray), while basal cells exhibit robust B r d U uptake (D and F; dark gray). Magnification 400X. 53 immunoreactivity of N C A M with that of I C A M - 1 . In unlesioned tissue, I C A M - 1 and N C A M label distinct populations o f cells, the H B C s adjacent to the basement membrane and neurons, respectively (Figure 4.2, A and B ; overlap in C). These two populations are separated by one full cell layer of non-immunoreactive cells, presumable globose basal cell ( G B C ) progenitor cells. However, at 6 days post-bulbectomy, within discrete sections of O E , this unlabelled population of separating cells apparently disappears, leaving the I C A M - 1 and N C A M labelled populations in close apposition to one another (Figure 4.2, E) . This phenomenon occurs within short segments of O E and is not observed within normal, unlesioned tissue. Negative, secondary antibody only controls were void of fluorescent signal. 4 . 4 Adhesion receptor positive HBCs divide post-bulbectomy, but remain relatively quiescent compared to GBCs Stem cells, by nature, must undergo mitosis in order to achieve self-maintenance or generate differentiated cells to ensure tissue homeostasis (Hall and Watt, 1989; Potten and Loeffler, 1990). To determine whether the I C A M - 1 expressing H B C population contains dividing cells, we used B r d U incorporation and immunofluorescence to identify cells in S-phase within the regenerating epithelium. A s above, many cells in the basal portion of the O E incorporate B r d U following bulbectomy. However, I C A M - 1 + H B C s were only rarely B r d U + (Figure 4.3, A ) . In contrast, >90% of proliferating cells were G B C s , defined according to nuclear morphology and position in the O E (B). Negative, secondary antibody only controls contained no observable signal. 54 Figure 4 .2. G B C progenitors are depleted locally within discrete regions of O E following bulbectomy. In normal, unlesioned O E , I C A M - 1 (A) and N C A M (B) label distinct populations of cells, namely H B C s and neurons respectively. (D) Phase contrast o f ( A - C ) . Double immunofluorescence indicates a single, un-labelled layer o f cells located between the I C A M - T and N C A M + populations. These cells are l ikely G B C s according to their position and antigenicity. Fo l lowing bulbectomy, this layer o f unreactive cells disappears in discrete sections o f O E , while they persist in others (E). Also o f note is the upregulation o f I C A M - 1 in endothelial cells o f the blood vessels post-bulbectomy (E). (F) phase contast o f (E). ( A - D ) Magnif icat ion 20x. (E-F) Magnification 400X. 55 Figure 4.3. HBCs proliferate in response to bulbectomy, but remain quiescent relative to robustly proliferating GBCs. Robust proliferation is evident in the basal cel l compartment fol lowing bulbectomy. I C A M - 1 + H B C s ( A , dark gray) rarely incorporate B r d U ( A , light gray; overlap, arrow). In contrast, G B C s , residing immediately above the I C A M - 1 + H B C layer (B, dark gray), proliferate extensively, as demonstrated by B r d U uptake (B, light gray). Magnification 200X. 56 4.5 The expression and/or distribution of ICAM-1, P i and P4 integrins is altered post-bulbectomy To determine whether the pattern of I C A M - 1 expression is altered during induced synchronous regeneration of olfactory neurons, we next performed immunohisto-chemistry with antibodies directed against I C A M - 1 , P i and P4 integrins on normal and lesioned O E tissue. A s demonstrated in the previous chapter, I C A M - 1 , P i and P4 integrin expression is uniform within the H B C layer in normal, quiescent O E (Figure 3.1). For the most part, the continuity of adhesion receptor staining is unchanged within bulbectomized tissue. However, small breaks in the uniformity o f staining are detected at intervals along the OE ' s length for each of these H B C markers (Figure 4.4). In addition, changes were detected with respect to the localization of these markers within individual H B C s . In normal O E , I C A M - 1 and P4 integrin are localized in a pericellular fashion upon H B C s , with a slight concentration of P4 integrin to the basal surface (Figure 4.5, A - B ) . In bulbectomized O E , these proteins are strongly concentrated to the basal surfaces of many H B C s (D-E). In contrast, there was no observable difference in the subcellular distribution of P i integrin in lesioned tissue (C and F). Secondary antibody only, negative controls produced no signal. 4.6 HBCs display some heterogeneity in the complement of adhesion receptor they express, an observation which is exaggerated post-bulbectomy A sub-feature of stem cell populations is that they are often comprised of cells with different probabilities of self-maintenance and differentiation (Hall and Watt, 1989). In this respect, they are said to be a heterogenous population of cells. To determine whether H B C s may be subdivided on the basis of their adhesion molecule expression, we 57 ICAM-1 A A * B pi integrin 4 A ^0, p mp \u00E2\u0080\u00A2 \u00C2\u00AB A A A C f f \u00E2\u0080\u00A2 M M (Hi j A f c f y \u00E2\u0080\u00A2V-WW)HAM< * J (34 integrin Figure 4.4. Changes are detected in the populational uniformity of ICAM-1, P i and P 4 integrin expression within the HBC layer post-bulbectomy. In contrast to their expression in the normal OE, discrete breaks in the uniformity of HBC ICAM-1, (3i and p4 integrin expression are detected in lesioned OE (A-C, arrowheads). Magnification 200X. 58 ICAM-1 (34 integrin (31 integrin Figure 4.5. The subcellular distributions of ICAM-1 and p 4 integrin are altered in some cells post-bulbectomy, while that of p\ integrin remains constant. In normal, unlesioned (UL) tissue, ICAM-1 displays a pericellular distribution upon HBCs (A), while Pi integrin is localized to the basal surfaces of HBCs (C). P4 integrin expression, though pericellular in general, is also concentrated to the basal surfaces of HBCs (B). Following bulbectomy (L), expression of ICAM-1 and P4 integrin are further concentrated to the basal surfaces within many cells, leaving no immunoreactivity upon the apical and lateral sides (D and E). No change is noted in the expression of Pi integrin post-bulbectomy (F). Magnification 400X. 59 performed double immunofluorescence with different combinations of these antibodies on fixed sections of olfactory tissue. In normal, quiescent tissue, co-incident expression of either I C A M - l / P i integrin or I C A M - I / P 4 integrin appeared to be complete. However, in each case, rare cells were observed that did not co-express both proteins, but expressed adhesion receptor markers singly (Figure 4.6, A and C) . These findings suggest that there exists a heterogeneity within the H B C compartment, in that not all of this population expresses every adhesion receptor equivalently. Following lesion, there are more cells that express single markers, indicating an increase in the heterogeneity of marker expression (Figure 4.6, B and D). This experiment also further demonstrated the pattern of adhesion receptor expression with respect to the individual cell level, in that it is evident that the integrins are concentrated basally while I C A M - 1 signal appears to be present throughout in normal tissue. 4.7 Summary Upon the transition from a quiescent to regenerative olfactory epithelium (OE) following bulbectomy and neuronal loss, several trends are detected within the horizontal basal cell ( H B C ) population. There is a pronounced proliferative heterogeneity with respect to the two basal cell layers: I C A M - 1 + H B C s divide infrequently, while I C A M - 1 \" globose basal cells contribute robustly to neurogenesis. Proliferative heterogeneity is frequently observed during cell kinetic analyses of stem cells in other regenerative tissues (Potten and Loeffler, 1990). Changes are also detected at both the populational and subcellular levels with respect to H B C marker expression following bulbectomy. Firstly, the uniformity of marker expression is disrupted, as distinct regions of the H B C layer are devoid of marker expression. Secondly, I C A M - 1 and P4 integrin proteins appear to shift 60 UL ICAM/ p4 c w D - * Figure 4.6. The observed heterogeneity of H B C adhesion receptor expression in normal O E is exaggerated post-bulbectomy. Double immunofluoresence with I C A M -1/pi integrin (green and red, respectively, A and B ) and I C A M - I / P 4 integrin (light and dark gray, respectively, C and D)was performed in order to detect differences in marker overlap in both normal ( U L ) and lesioned (L) tissue. In normal tissue, the overlap of expression for both combinations o f proteins was complete in most cells (A and C) . However, rare cells which expressed only single markers were also detected (A and C, arrowheads). In bulbectomized tissue, there is an increase in the number on cells which express only one marker within both combinations surveyed (B and D , arrowheads). Magnification 200X. 61 from a pericellular distribution in the quiescent O E , to a concentration to the basal surfaces of H B C s . Lastly, H B C s display some heterogeneity in the complement of adhesion markers present on their cell surfaces in the normal O E . This apparent heterogeneity is more pronounced post-bulbectomy. Together, these results may provide insight into the in vivo function of I C A M - 1 , P i and P4 integrins upon H B C s . 62 C H A P T E R V . Immunomagnetic selection in conjunction with in vitro progenitor assays and lineage-specific marker expression support an H B C contribution to the olfactory progenitor cell compartment 5.1 Introduction The overall objective of this study was to assess the horizontal basal cell ( H B C ) contribution to the olfactory stem cell compartment and, in turn, to determine how these cells might be regulated. Stem cells classically possess high proliferative potentials, which are exploited to achieve populational self-maintenance and the production of multiple differentiated daughter cell types (Hall and Watt, 1989; Potten and Loeffler, 1990). We define progenitors as cells with reduced capacities for the above traits (i.e. limited proliferative and self-renewal capacity and restricted differentiation potential) and include in this definition \"potential\" stem cells, that is, cells which exhibit certain stem parameters while others are either presently undetected or unknown. To test for these traits, in vitro colony-forming analyses and an immunohistochemical survey of markers of olfactory differentiation were performed on fractionated H B C s in clonal culture. The in vivo microenvironment, or niche, provides instructive and selective cues to faithfully regulate stem cell function (Adams and Watt, 1993; Fuchs and Segre, 2000). Published reports in the olfactory literature in conjunction with results from this study, indicate the role of growth factors and extracellular matrix ( E C M ) components as regulators of H B C function. We tested the ability of these substances to influence the colony-forming efficiency of clonal cultures of I C A M - 1 + , magnetic activated cell sorting ( M A C S ) selected cells in order to illuminate the effects of resident in vivo niche components on H B C behaviour in vitro. 63 5.2 Preliminary in vitro findings Initial primary cell culture experiments separated fractions of olfactory epithelial (OE) cells v ia differential adhesive properties and cell size. Briefly, O E tissue dissected from neonatal mice was dissociated enzymatically and passed through a series of filters of decreasing pore size. The cellular fraction retained on the 40pm cell strainer contained clumps of undissociated cells which acquired a basal cell-like morphology upon expansion in adherent culture (Figure 5.1). These clusters contained H B C s (as identified by I C A M - 1 , P i , and P4 integrin expression), incorporated B r d U , and were co-incident with the appearance of neurons and glia at later time-points in culture. These results provided preliminary evidence that H B C s may contribute neuro- and gliogenesis in the olfactory system. 5.3 Cell surface antigen selection and sorting of HBCs We next wished to increase the stringency of our experiments via the development of an immunomagnetic selection and culture protocol for H B C s , exploiting the discovery of cell surface H B C expression of I C A M - 1 . A neonatal, OE-derived single cell suspension was labeled with a primary biotinylated antibody directed against the I C A M - 1 protein, followed by treatment with magnetic activated cell sorting ( M A C S ) streptavidin magnetic microbeads. Within a strong magnetic field, the labeled suspension was poured through a M A C S column. The I C A M - 1 \" cells were washed through the column while magnetically labeled I C A M - 1 + cells were retained. After washing, the column was removed from the magnetic field and the positive fraction was eluted (see 64 Figure 5.1. PreUminary evidence of progenitor activity in heterogenous cultures of OE-derived cells. Cohesive clusters o f cells possessing a basal cell-like morphology expand in culture (A->C) and appear to generate process-bearing cells later in vitro (C). These clusters express I C A M - 1 (D-F, red), p\ integrin (E, green), and (34 integrin (F, green) and incorporate B r d U (D, red). Early in culture, (34 integrin + clusters are devoid of process-bearing cells (G). Later, larger p*4 integrin* clusters (H) and co-incident with neurons and glia (I: N S T in green, G F A P in red). Magnification ( A - B ) 200X, (C) 100X, (D-F) 400X, (G-I) 200X. 65 Step 1: Label cells with biotinylated anti-ICAM-1 antibody (\u00E2\u0080\u00A2) Step 2: Step 3: Incubate with avidin conjugated MACS magnetic microbeads ( \u00E2\u0080\u00A2 \u00C2\u00B0 ) When the cells pass through a magnetic column, the ICAM-1 positive microbead labelled fraction is retained within the column, while the ICAM-1 negative fraction is eluted. ICAM- negative fraction Step 4: ICAM-positive(TT) fraction o o ol The ICAM-1 positive fraction is then eluted via removal from the magnetic field. Figure 5.2 In vitro immunomagnetic sorting of HBCs on the basis of ICAM-1 antigenicity. 66 Figure 5.2). A n average of 25,000 I C A M - 1 + cells was isolated by M A C S from each dissected mouse epithelium. This represents 2.31 \u00C2\u00B1 0.49% (mean \u00C2\u00B1 SE, n=5) of the total unfractionated cell suspension prior to M A C S selection. Prior to plating, the presence of a single cell suspension was visually inspected during the haemocytometer cell counting procedure. When plated at very low, or clonal density (6000 viable cells per 10 cm petri dish), the single cell suspension produced symmetrical, expanding clusters of cohesive cells. Hence, we assume that the probability that these clusters represent colonies (i.e. derived from single cells) is high. However, genuine clonality can only be proven via micromanipulation of single cells during the plating procedure (Freshney, 2000). 5.4 The I C A M - 1 + fraction possesses a superior colony-forming ability at clonal density A s we hypothesize that I C A M - 1 expressing olfactory H B C s contribute to the genesis of O E cell types, we predicted that the I C A M - 1 + fraction would express culture characteristics consistent with a progenitor phenotype. To test this hypothesis, we utilized a common progenitor assay which exploits the fact that only stem and progenitor cells produce colonies under low density, or clonal, plating conditions (Jones et al., 1995). Colony-forming efficiency refers to the number of colonies formed after 14 days in vitro (DIV) divided by the number of I C A M - 1 + cells initially plated. Towards this purpose, we define colony as a cohesive cluster of more than 2 cells. However, in practice, colonies typically exceeded this lower 2-cell limit, and contained >10 cells per colony. 67 0.4 g 0.3 OE Cell Fraction Figure 5.3 The M A C S selected ICAM-1 + fraction displays a superior colony-forming efficiency in vitro. To assess the ability to seed colonies, I C A M - 1 + and I C A M -1\" fractions resulting from immunomagentic sorting were plated at clonal density on collagen coated 10 cm petri dishes. Likewise, the unselected cell suspension, prior to M A C S enrichment, was also assayed. A l l cells were cultured in D M E M / F 1 2 + 10% F B S for 14 D I V , at which point colonies (cohesive clusters containing >2 cells) were scored. Colony-forming efficiency was determined by dividing the number of colonies by the number of cells initially plated (mean\u00C2\u00B1SE). Student's t-test relative to unselected fraction: I C A M - 1 + (p=0.0089), I C A M - 1 \" (p=0.0060). 68 The I C A M - 1 + fraction exhibited a significant increase in colony-forming efficiency when compared to the unselected cells (0.25 \u00C2\u00B1 0.057 and 0.033 \u00C2\u00B1 0.0055, respectively; p=0.0089, n=4) indicating an enrichment of progenitor activity within the I C A M - 1 + fraction (Figure 5.3). In contrast, the I C A M - T fraction possessed a lower cloning efficiency than that of the unselected fraction (0.0083 \u00C2\u00B1 0.0036, p=0.0060, n=4), which suggests that colony-initiating cells have been depleted from this fraction. When positive and negative fractions are examined as a combined total, 96.88 \u00C2\u00B1 1.11 % o f the colony-forming cells were found in the I C A M - 1 + fraction. 5.5 Determination of optimal media conditions with respect to I C A M - 1 + colony-forming efficiency A higher colony-forming efficiency, for the purpose of future study of these cells and statistical power, was desirable. Hence, media, substrate and growth factor conditions were examined for increases in overall colony-forming ability in tandem with any possible functional consequences which might be informative as to the regulation of I C A M - 1 + H B C s in culture. The first such experiment was directed at determining the optimal media conditions of I C A M - 1 + H B C s . These were tested by plating I C A M - 1 + cells on collagen at clonal density (6000 cells per 10 cm dish) in a variety of different media formulations. A t 2 weeks in vitro, colony-forming efficiency was then assayed. Preliminary experiments (Figure 5.4) demonstrated that D M E M / F 1 2 + 10% F B S produced the greatest colony formation, followed by O p t i - M E M supplemented with 4% F B S . The calcium-free media conditions R P M I + 10% F B S and keratinocyte-serum free media (K-69 0.6 DMF12 O-Mem RPMI K-SFM Serum% 10 4 10 0 Growth Media Figure 5.4 Effect of media condition on the colony forming-efficiency of I C A M - 1 + cells at clonal density. Cells were plated at clonal density on collagen coated 10 cm petri dishes and cultured in different media formulations. A t 14 D I V , colonies (cohesive clusters o f >2 cells) were counted and the colony forming efficiency (CFE) for each condition was calculated. Data are represented as mean\u00C2\u00B1SE. D M E M / F 1 2 (DMF12) and O p t i - M E M (O-Mem) produced the highest colony-forming efficiencies, while R P M I and Keratinoctye serum-free media ( K - S F M ) yielded no colonies. 70 S F M ) produced no colonies. Consequently, D M E M / F 1 2 + 10% F B S was used as the preferred culture media in subsequent experiments, except where noted otherwise. 5.6 Effect of E C M substrate on the overall colony-forming ability of ICAM -1 selected cells A s demonstrated in the preceding results sections, horizontal basal cells express the integrin subunits Pi and p4. Depending on the a/p subunit pair formed, these integrins can bind collagen, laminin, or fibronectin in order to manipulate a variety of stem cell behaviours, including proliferation, differentiation, and cell survival (Fuchs et al., 1997; Brakebusch et al., 1997). These E C M components are present in the basement membrane underlying the O E , directly beneath the H B C layer (Julliard and Hartmann, 1998). To determine whether these cell matrix components have an effect on H B C colony-forming ability in vitro, we cultured I C A M - 1 + cells at clonal density on 10 cm petri dishes coated with fibronectin, collagen, laminin singly and with combinations o f collagen/laminin and collagen/fibronectin. Among the single substrate preparations, I C A M - 1 + cells plated on collagen exhibited the highest relative colony-forming efficiency (100%), while laminin and fibronectin were roughly half as efficient at promoting colony production (54.93 \u00C2\u00B1 8.67%., p-0.0067 and 42.50 \u00C2\u00B1 1.84%, p=0.00050, respectively; Figure 5.5). When collagen and laminin are mixed, either in a 2:1 or 1:2 ratio, a significant increase in colony-forming efficiency is observed relative to collagen alone (143.33 + 11.45%, p=0.016 and 162.59 \u00C2\u00B1 22.06%, p=0.033). In contrast, no significant change in overall colony-forming efficiency is detected when collagen is mixed with fibronectin (2:1, 120.53 \u00C2\u00B1 22.84 and 1:2, 91.07 \u00C2\u00B1 3.34). 71 Figure 5.5 Effect of substrate on overall colony-forming efficiency of I C A M - 1 + cells at clonal density. Cells were plated on 10 cm petri dishes coated with collagen (C), laminin (L), fibronectin (F), or mixed collagen/laminin (C+L) and collagen/fibronectin (C+F) substrates. A l l cells were cultured in D M E M / F 1 2 + 10% F B S for 14 D I V . Colonies, defined as cohesive clusters containing >2 cells, were counted and colony forming efficiency was determined for each condition. Results are represented as a percent of the collagen control (mean\u00C2\u00B1SE). Student's t-test relative to collagen control: L , p=0.0067; C+L (2:1), p=0.016; C+L (1:2), p=0.033; F, p-0.00050. 72 5 . 7 Effect of E C M components on the incidence of small, medium and large colonies within clonal cultures of I C A M - 1 + cells After 14 D I V , it became clear that the I C A M - 1 + cell fraction produced colonies that could be sub-categorized according to size. This observation parallels what is seen in other colony-forming efficiency assays (Barrandon and Green, 1988; Collins et al., 2001). For all subsequent analyses, size categories were assigned as small (<30 and >2 cohesive cells per colony), medium (>30 and <150 cells) and large (>150 cells per colony). Representative small, medium, and large colonies are shown in Figure 5.6. In order to determine the effects of substrate condition on colony size, I C A M - 1 + cells were plated at clonal density on dishes coated with fibronectin, collagen, laminin singly and with combinations of collagen/laminin and collagen/fibronectin. A t 14 D I V , small, medium and large colonies were scored and colony-forming efficiencies were determined for each size category. On collagen, the different sizes of colony were generally equally represented (Figure 5.7, A ) . On laminin, however, the majority of colonies at 14 D I V were small colonies (<30 cells). The increase in colony-forming efficiency on a mixed collagen:laminin substrate appears to largely reflect an increase in the proportion and number of large colonies (Figure 5.7B). When compared with collagen as a singular plating substrate, large colony formation was increased 4-fold on both mixed collagemlaminin matrices, while laminin alone yielded roughly half the number of large colonies compared to collagen singly. 73 Figure 5.6 Representative small , medium and large colonies at 14 D I V . At 14 DIV, colonies produced by the I C A M - 1 + cell fraction displayed substantial differences in size. These colonies were scored as small (A; containing <30 cells), medium (B; between 30 and 150 cells) and large (C; >150 cells) for all subsequent experiments evaluating colony size. Magnification 100X (A and B ) ; 50X (C). 74 Substrate Composi t ion Figure 5.7. Effect of substrate on the incidence of small, medium and large colonies seeded by ICAM-1 + , M A C S selected cells at clonal density. Cells were plated on 10cm petri dishes coated with collagen (C), laminin (L), or collagen/laminin mixtures (C+L). A l l cells were cultured in D M E M / F 1 2 + 10% F B S for 14 D I V , at which point small (<30 cells), medium (>30 and <150 cells) and large (>150 cells) colonies were scored. Data in (A) are represented as small, medium or large colonies as percent of the total number of colonies within each condition (mean\u00C2\u00B1SE) . Data for each condition in (B) are represented as a - fo ld difference in large colonies over a collagen control (mean \u00C2\u00B1SE). 75 5.8 Adhesion kinetics assay on different E C M components demonstrates an overall preference for collagen Several studies demonstrate that stem cells, a population characteristically rich in adhesion receptor expression, exhibit rapid adherence to collagen, while transit amplifying progenitors, typically characterized by low adhesion receptor expression, possess slower kinetics of adhesion to this E C M component. Indeed, some studies have exploited this rapid adherence to substrate to fractionate stem and transit amplifying compartments in culture, as part of standard protocol (Jones et al., 1995; Collins et al., 2000). Given that we have shown that H B C s exhibit high adhesion receptor expression both in vivo and in vitro and respond differentially to E C M components in culture, we wished to test whether our I C A M - 1 + cells display similar adhesion kinetic properties to those discussed above. Furthermore, we wished to determine whether the observed differences in colony-forming efficiency on collagen, laminin and fibronectin could reflect differences in the number of cells that initially adhere to these substrates. To this end, I C A M - 1 + cells were plated at high density in wells coated with collagen, laminin, or fibronectin. A t 4 and 24 hours post-plating, non-adherent cells were removed and quantified. In each condition, the majority of I C A M - 1 + cell adherence occurred within the first 4 hours in culture, as indicated by the steep slope of the graph (Figure 5.8). The rate of adherence then decreased markedly between the 4-hour and 24 hour time-points. This experiment also demonstrated that the I C A M - 1 + population as a whole adheres most completely to collagen (8.85 \u00C2\u00B1 4.05 % non-adherent cells), in effect demonstrating a preference for this substrate. In contrast, adhesion to laminin is less complete (59.8 \u00C2\u00B1 15.56 % non-adherent cells), while fibronectin elicits an intermediate response (37.23 \u00C2\u00B1 5.84%) with respect to adhesion within the first 24 hours of culture. 76 - Collagen -A-Laminin 0 4 8 12 16 20 24 Time (hrs) Figure 5.8 M A C S selected ICAM-1 + cells display different kinetics of adhesion when plated on different E C M components. To reveal possible differences in initial adhesion to matrix, I C A M - 1 + cells were plated at high density on duplicate wells coated with collagen, laminin, or fibronectin. A t 4 and 24 hours post-plating, non-adherent cells were removed and counted. Data are represented as non-adherent cells as a percent of the total cells plated per well . I C A M - 1 + cell adherence to collagen occurred most rapidly and was optimal for matrix adhesion. Student's t-test relative to collagen at 24 hour time-point: laminin, p=0.036 and fibronectin, p=0.0T2. 77 Both laminin and fibronectin were significantly different from collagen at the 24 hour time-point (collagen vs. laminin, p=0.036; collagen vs. fibronectin: p=0.012; n=3) 5.9 Effects of growth factor addition on overall colony-forming efficiency of I C A M -1+ cells In the rodent olfactory epithelium, the receptor for both E G F and T G F - a , the E G F receptor, is expressed within the H B C layer, while both these growth factors function in vitro to promote olfactory neurogenesis (Rama Krishna et al., 1996; Ezeh and Farbman, 1998; Calof et al., 1991; Mahanthappa and Schwarting, 1993; Farbman and Bucholz, 1996). We thus hypothesized that E G F and T G F - a , either singly or in combination, would stimulate the ability of I C A M - 1 + cells to proliferate and form colonies. In addition, previous work with olfactory epithelial derived primary cultures and cell lines indicates that leukemia inhibitory factor (LIF) influences neurogenesis by promotion of progenitor proliferation (Satoh and Yoshida, 1997; Nan et al., 2001). LIF was also of interest because of its ability to maintain the stem phenotype in long-term culture when supplemented with E G F to neurospheres in vitro (Shimazaki et al., 2001). To test the effects of these growth factors on the colony-forming ability of selected cells, I C A M - 1 + cells were plated immediately after M A C S enrichment onto collagen coated 10 cm dishes at clonal density (6000 cells/10 cm dish). The base medium was O p t i - M E M supplemented with 4% F B S , which also served as the control. O p t i - M E M with 4% F B S was the preferred medium in this series of experiments as the addition of growth factor to D M E M / F 1 2 + 10% F B S showed no increase in colony-forming efficiency, likely due to sufficient levels of other growth factors within the serum. Addit ion of E G F , T G F - a , or both growth factors resulted in a significant increase 78 in the colony-forming efficiency relative to the control (EGF: 224.93 + 49.88 %, p=0.020, n=8; T G F : 188.71 \u00C2\u00B1 30.65%, p=0.031, n=4; EGF+TGF: 289.29 \u00C2\u00B1 66.67%, p=0.033, n=4; Figure 5.9). The addition of LIF to I C A M - 1 + cells had no significant effect on the overall ability of these cells to form colonies (117.30 \u00C2\u00B1 16.29%, n=6; Figure 5.9). When subjected to a combined E G F / L I F treatment, I C A M - 1 + cells yielded a comparable colony-forming efficiency to control values (144.66 \u00C2\u00B1 35.45%, n=6), but appeared to decrease relative to that of E G F singly. 5.10 Effect of growth factors on the incidence of small, medium, and large colonies within clonal cultures of ICAM-1 + cells In order to identify the effects of growth factor addition on the incidence of small, medium and large colonies, colonies of different size were scored at 14 D I V as per the E C M experiments. The observed increase in overall colony-forming efficiency with respect to E G F and T G F - a appears to largely reflect an increase in the number and proportion of large colonies, with little change in the incidence of small or medium colonies (Figure 5.10, A and B) . When compared with the no growth factor control, large colony formation increased roughly 9-fold with E G F , 5-fold with T G F - a , and 4-fold with LIF (Figure 5.10, B) . With respect to the growth factor mixtures, E G F / T G F - a and E G F / L I F treated cultures yielded approximately a 5-fold and 2-fold increase in the formation of large colonies, respectively (Figure 5.10, B) . 79 400 1 _3\u00C2\u00B0\u00C2\u00B0 \u00C2\u00A3P 8 200 I o (f) co 100 o o o C E G F L I F E G F T G F a E G F + L I F + T G F a Growth Factor Figure 5.9 Effect of growth factor on the overall colony-forming efficiency of I C A M - 1 + cells at clonal density. Cells were plated onto collagen coated 10 cm petri dishes and cultured in O p t i - M E M + 4% F B S with supplemented E G F , LIF , EGF+LIF , TGF-a, or EGF+TGF-a or without growth factor (control; C). A t 14 D I V , colonies (> 2 cells) were counted and colony-forming efficiency was calculated for each condition. Data are represented as a percentage o f the \"no growth factor\" control (mean\u00C2\u00B1SE). Student's t-tests relative to control: E G F , p=0.020; TGF-a, p=0.031, EGF+TGF-a, p=0.033. 80 A (0 0> \"E o o o - i 1 0 0 T 8 0 6 0 B ~ \u00E2\u0080\u00A2 S m a l l a Medium B Large O 4 0 J >\u00C2\u00BB o c O) i_ CO - J 2*. 6 CD CO 4 0) 1 .2 O 0 C EGF LIF EGF TGFa EGF +LIF +TGFa Growth Factor Figure 5.10 Effect of growth factor on the incidence of small, medium and large colonies seeded by I C A M - 1 + cells at clonal density. I C A M - 1 + cells were plated onto collagen coated 1 0 cm petri dishes and cultured in O p t i - M E M + 4 % F B S with E G F , L IF , EGF+LIF, T G F - a , EGF+ T G F - a or without growth factor as a control (C). A t 1 4 D I V , \u00E2\u0080\u00A2small ( < 3 0 cells), medium ( > 3 0 and <150 cells) and large ( > 1 5 0 cells) colonies were counted. Data in (A) are represented as small, medium, or large colonies as percent of total colonies formed within each condition (mean\u00C2\u00B1SE). Data in (B) are displayed as a -fold increase in large colony formation relative to the \"no growth factor\" control (mean\u00C2\u00B1SE). 81 5.11 Cultured I C A M - 1 + cells produce a mosaic of differentiated olfactory cell phenotypes Our previous assessment of heterogenous, unselected olfactory epithelial derived cultures indicated that neurons and glia are present at later culture time-points and are co-incident with basal cell clusters. To strengthen the argument that cultured H B C s possess the ability to generate differentiated cells of the olfactory lineage, we tested for the expression of stage-specific markers of the olfactory lineage in colonies produced by the I C A M - 1 + cell fraction. Immunohistochemistry was carried out on fixed large colonies derived from I C A M - 1 + clonal cultures, between 14 and 28 DIV. Expression of G B C - 2 , a marker for a presumptive transit amplifying subpopulation of globose basal cells, is detected in clustered subsets of cells within colonies generated by the I C A M - 1 + cell fraction (Figure 5.11, A ) . To assess the presence of neurons, we utilized the immature neuronal marker, neuron specific tubulin (NST). Figure 5.11 (B) demonstrates the presence of N S T + cells within our colonies. Olfactory ensheathing glia, as determined the co-expression of S100P and P75 (Ramon-Cueto and A v i l a , 1998), are found in abundance within these colonies (C). N S T + neurons possessed a migratory, non-cohesive appearance and were localized either to the colony periphery or on top of a bed of cells within the colony proper. A similar localization was observed for glia, although they sometimes formed semi-cohesive clusters forming a core within the colony. In addition to assaying for differentiated cell types, it was of interest to determine whether H B C s are represented within colonies after 14 D I V , since classical stem cells must display self-maintenance in order to protect themselves from exhaustion (Hall and 82 A ^ GBC-2IB 4\" K ^ ^ ^ ^ ^ ^ D CD54 E CD104 i \u00E2\u0080\u00A2 . \u00E2\u0080\u00A2\u00E2\u0080\u00A2\u00E2\u0080\u00A2\u00E2\u0080\u00A2 \u00E2\u0080\u00A2 # : , \" * ii \u00C2\u00AB\u00C2\u00BB\" Figure 5.11 Large colonies contain cells expressing markers of olfactory differentiation, while retaining I C A M - l + / p 4 integrin+ HBCs as well. To determine whether differentiated cells are represent within large colonies at 14-28 days in clonal culture, immunohistochemistry was performed with markers of O E differentiation. G B C -2, a marker for G B C s , is present within subsets of cells (A) . Neuron specific tubulin (NST) expression, indicative o f neurons, is detected within cells at the colony perimeter, or on top of the colonies (B). Cells resembling olfactory ensheathing glia were are detected (C), either in slightly cohesive groupings or individually. Large colonies retain cells possessing an H B C phenotype (CD54: I C A M - 1 and CD29 : p\ integrin; D and E , respectively). (A, C,D-E) D A P I nuclear stain. Magnification 200X (A-C) and 1 0 0 X ( D -E). 83 Watt, 1989; Potten and Loeffler, 1990). Following 14 D I V on a collagen substrate, I C A M - 1 immunoreactive cells were detected in approximately 100% of large colonies, 83%o of medium colonies, and in 75% of small colonies. Large colonies were heterogenous with respect to the proportion of I C A M - 1 + cells in a given colony, ranging from 5-100% with an estimated mean of between 25-50%. Within large colonies, expression of I C A M - 1 and P4 integrin was detected within cohesive subsets within the colony (Figure 5.11, D and E) 5.12 ICAM-1 positive colonies contain cells possessing a mature olfactory neuron phenotype Given that N S T + neurons are found in later cultures of I C A M - 1 + cells, we next wished to determine whether these neurons are olfactory receptor neurons (ORNs). Immunohistochemistry was again performed on I C A M - 1 + fraction-derived large colonies (14-28 DIV) with antibodies directed against olfactory marker protein (OMP) , adenylate cylcase III (ACIII) and olfactory G protein (G-olf), the latter two being components o f the olfactory signal transduction cascade and the former a classical marker for mature ORNs . Cells expressing each of these proteins were found in I C A M - 1 + cell cultures (Figure 5.12, A - D ) . Interestingly, during the course of examining neuronal morphology and antigenicity, we observed NST-expressing neurons possessing a distinctly non-olfactory phenotype (Figure 5.13). In contrast to the streamlined bipolar morphology o f the typical O R N (as seen in Figure 5.12, D), these neurons possessed multiple, elaborate processes. 84 A 4 vACIII % ' i B Golf ,4 \ \u00E2\u0080\u00A2 L \u00E2\u0080\u00A2 1 G i \u00E2\u0080\u00A2 QMP J m. % mi, %ll * % Hhi s ' \u00C2\u00AB \u00E2\u0080\u00A2 '*ti _ IMP ^ Oft D OMP Figure 5.12 Some neurons present at the perimeter of large colonies possess a mature olfactory neuron phenotype. The mature olfactory neuronal phenotype was assessed using adenylate cyclase III (ACIII; A ) , the olfactory G-protein (Golf; B) and olfactory marker protein ( O M P ; C and D) . Cells expressing these markers were typically streamlined and bipolar. (C and D) also show D A P I nuclear stain. Magnification (A , C -D) 400X and (B) 200X. 85 Figure 5.13. Some neurons displayed a distinctly non-olfactory phenotype. Several NST-expressing neurons found in proximity to large colonies possessed a distinctly non-olfactory morphology, as they exhibited multiple elaborate processes as opposed to being streamlined and bipolar (A and B). Negative \"secondary only\" control in C. Magnification 400X. 86 5.13 Summary B y integrating magnetic associated cell sorting ( M A C S ) with in vitro colony-forming assays, we obtained evidence that the horizontal basal cell (HBC) layer is enriched for progenitor activity, as evidenced by the I C A M - 1 + fraction's superior colony-forming ability. Colonies resulting from these cultures contained up to 40, 000 cells, an observation suggestive of a substantial H B C proliferative potential. We have identified factors that influence the colony-forming ability of M A C S -selected, I C A M - 1 + cells. A l l of the substances tested are detected in close proximity to H B C s within their in vivo microenvironment. I C A M - 1 + cells adhere preferentially to collagen and yield the most colonies when plated on collagen or collagen/laminin mixtures. These substrate treatments also appear to produce the most \"large\" colonies. With respect to growth factors, E G F and T G F - a , both singly and in combination, produced the most significant increase in overall colony-forming efficiency, while LIF produced no observable effect. However, all three growth factors appeared to increase the incidence of large colonies, likely a reflection of their reported roles in promoting olfactory neurogenesis. In addition, we provide immunohistochemical evidence of olfactory-specific differentiation, in that globose basal cells, olfactory receptor neurons and olfactory ensheathing glia are produced from clonal cultures of I C A M - 1 + H B C s and that H B C s themselves are maintained within these colonies. 87 C H A P T E R V I . Discussion A pervasive challenge within the field of olfactory neurogenesis is to reveal the ultimate source of newborn olfactory receptor neurons (ORNs), which are generated on demand within the epithelium both in response to injury and throughout the adult lifespans o f mammals. Several studies indicate two possible stem cell candidates, globose basal cells (GBCs) and horizontal basal cells (HBCs) , within the basal compartment of the O E . To date, there is still much debate over which of these cell populations contains the olfactory neural stem cells. Our overall hypothesis is that these stem cells are resident within the H B C layer and that the functions of these cells can be controlled by microenvironmental cues. Using a combination of in vivo and in vitro approaches, this study sought to assess the H B C population for stem cell traits and to test its behavioural regulation. This study commenced with an immunohistochemical screen of clusters of differentiation (CD) antigens commonly expressed by various stem/progenitor cells, with an aim to identify new cell surface markers of the H B C population of the olfactory epithelium (OE). Three adhesion receptors were detected in a uniform manner upon H B C s : intercellular adhesion molecule-1 ( ICAM-1) , P i integrin and P4 integrin (Figure 3.1). The yield of data resulting from this antigen screen serves a utility, as the presence of these cell surface markers enables us to selectively enrich these cells in an in vitro milieu, and is also insightful, as their presence upon H B C s w i l l l ikely yield information into the regulation of the behaviour of these cells. Before speculating on their possible 88 regulatory roles in the O E , their known function in various cell types must first be surveyed. These traits are noted in the ensuing discussion. Intercellular adhesion molecule-1, or I C A M - 1 , is a cell surface glycoprotein belonging to the immunoglobulin superfamily and is expressed on a wide variety of cell types (Hubbard and Rothlein, 2000). A s its name suggests, I C A M - 1 is involved in intercellular adhesion, a property carried out via binding ligand present on the surfaces of neighbouring cells. I C A M - 1 can also ligate extracellular substances, such as hyaluronic acid, a component of the extracellular matrix ( E C M ) that is typically enriched in extracellular matrices (van der Stolpe and van der Saag, 1996; Tammi et al., 2002). Intracellular^, the cytoplasmic domain of I C A M - 1 interacts with actin-binding proteins in association with the cytoskeleton, by which I C A M - 1 activity within the cell is thought to be mediated. Since its discovery, I C A M - 1 has been thought to function via a purely adhesive mechanisms, such that a cell expressing I C A M - 1 ligand simply adheres to, and is immobilized by, an I C A M - 1 expressing cell (Hubbard and Rothlein, 2000). Such is the case for I C A M - 1 's most prolific role, that of leukocyte trafficking, where I C A M - 1 expression is induced in endothelial cells by pro-inflammatory cytokines to signal the location of a site of injury. Leukocytes, which combat infection and may digest cellular debris, adhere to these I C A M - 1 expressing cells, and subsequent immobilization and transendothelial migration into the inflamed tissue ensue (Springer, 1994; Hayflick et al. , 1998; Hubbard and Rothlein, 2000). This process is mediated by a purely adhesive role attributed to I C A M - 1 . 89 More recently, however, it has become apparent that I C A M - 1 also has signaling functions within the cell. For instance, studies directed at revealing I C A M - 1 intracellular signaling pathways have demonstrated that I C A M - 1 antibody cross-linking leads to a rapid increase in tyrosine phosphorylation. N o intrinsic kinase ability has yet been ascribed to I C A M - 1 (Hayflick et al., 1998). However, an alternative to intrinsic kinase ability is to mediate signaling events via association with cytoplasmic tyrosine kinases. Several such proteins have been found to be activated following I C A M - 1 ligation, including the Src family kinase p53/p56 l y n, mitogen-activated protein kinases, Raf-1, E R K - 1 and cdc2 kinase (Holland and Owens, 1997; Chirathaworn et al., 1995). This ensemble of activated kinases has reported activity in mediating a large variety of cellular events, which typically culminate in changes in gene transcription. With respect to I C A M - 1 , recruitment of these cytoplasmic kinases might explain the observed activation of the transcription factor of AP-1 (the Jun/Fos heteromer), the activation of Rho and transcription of I L - i p following I C A M - 1 crosslinking (Koyama et al., 1996; Etienne et al., 1998). The reported possible endpoints of these I C A M - 1 activated intracellular signaling pathways include the control of proliferation, protection against apoptosis and regulation of differentiation (Shimamoto et al., 2000;Gao et a l , 2000). It is currently not known how I C A M - 1 might instigate the signaling events described above. Wi th respect to stem cell systems, I C A M - 1 is present at very low levels in keratinocytes within the normal epidermis (De Panfilis et al., 1992). However, in certain disease and inflammatory conditions, such as psoriasis, I C A M - 1 is markedly upregulated in these cells (Griffiths and Nickoloff, 1989; van Pelt et al., 1998). Furthermore, keratinocytes exhibiting increased I C A M - 1 expression in psoriasis are hyperproliferative. 90 Although the reported function of I C A M - 1 is an immunological one, some authors speculate that I C A M - 1 performs a secondary function in skin remodeling (Muller-Rover etal., 2000). Within our tissue of interest, the olfactory epithelium, I C A M - 1 is constitutively expressed at high levels upon H B C s (Figure 3.1). Two possible scenarios exist regarding the function of I C A M - 1 within olfactory H B C s . Firstly, a classical immunological role for I C A M - 1 in H B C s is possible. Indeed, the I C A M - 1 protein expressed upon endothelial cells, which is upregulated after olfactory bulbectomy, undoubtably plays a role in recruiting macrophages and other leukocytes to the site of injury. Several studies, including our own, have reported that macrophages expressing the I C A M - 1 ligand, Mac-1, are recruited into the olfactory mucosa following lesion (Carter and Roskams, unpublished observations; Suzuki et al., 1995; Nan et al., 2001). A s in other inflammatory responses, these macrophages are proposed to function in phagocytosing cellular debris and in secreting growth factors and cytokines to promote regeneration of the tissue. A s such, H B C I C A M - 1 expression might function in attracting and immobilizing macrophages to the stem/progenitor layer of the O E in order to ensure a local enrichment of macrophage-secreted growth factors. Additionally, H B C I C A M - 1 may serve a function in the migration of these cells into the O E proper by facilitating the crossing of the basement membrane barrier in an analogous manner to transendothelial migration of leukocytes (Springer, 1990). In other tissues, I C A M - 1 expression is typically induced by inflammatory cytokines in cell types which previously displayed low or undetectable expression of this protein (Springer, 1990; van der Stolpe and van der Saag, 1996). A s such, I C A M - 1 91 expression is induced only when immunologically required. However, in the O E , I C A M -1 is constitutively expressed upon H B C s , suggesting a non-immune role for this cell adhesion molecule. Hence, we speculate that I C A M - 1 may mediate intracellular signaling cascades to influence cellular decisions including proliferation, survival and differentiation, as has been reported in other cell types (Hubbard and Rothlein, 2000). To date there are five known ligands for I C A M - 1 . These include the P2 integrins Mac-1 and L F A - 1 , the sialomucin CD43, the soluble factor fibrinogen, and the E C M component hyaluronan (van de Stope and van der Saag, 1996). Previous and current experimentation have indicated that L F A - 1 , Mac-1, (L. Carter and J. Roskams, unpublished observations) and CD43 are unlikely candidate ligands for I C A M - 1 within the olfactory system, leaving fibrinogen and hyaluronan as the remaining known potential ligands. It appears that hyaluronan is the most probable candidate ligand for I C A M - 1 within the olfactory system, a conjecture that is supported by the fact that in preliminary culture experiments directed at determining the optimal combination of cell dissociation enzymes, the mixture containing hyaluronidase produced the most basal cell clusters. Although the expression of this ligand has not been examined within the epithelium, we expect to observe its localization within the basement membrane, placing it in an optimal setting for interaction with I C A M - 1 + H B C s . The true function of I C A M - 1 in the O E , however, requires further exploration. Nonetheless, the expression of this protein upon H B C s provides an additional milieu for the study of this adhesion molecule in general and may provide insight into its role in olfactory neurogenesis. Two of the remaining cell surface H B C markers identified in this study belong to the integrin family of cell adhesion receptors. These proteins are ubiquitously expressed 92 throughout the developing and adult organism and serve as a bridge between the extracellular environment and the cytoskeleton within the cell, in addition to activating intracellular signaling cascades (Howe et al., 1998). Integrins are heterodimeric proteins consisting of an a subunit non-covalently linked to a (3 subunit. A diversity of these subunits abound, giving rise to a multitude of different possible a and P combinations. However, not all a subunits can associate with all p subunits and vice versa. In addition, ligand binding specificities and response to ligation for each particular intact integrin is determined by the combination of a and P subunits from which it is fashioned in conjunction with cellular context. The current model of integrin-ligand interaction is that a subunits inhibit P subunits from interacting with the cytoskeleton. Upon ligand binding this inhibition is relieved, thereby permitting intracellular signaling events (van der Flier and Sonnenberg, 2001). Two varieties of cellular communication are transduced by integrin signaling: \"outside-in\" and \"inside-out signaling\". \"Inside-out\" signaling refers to the ability of the cellular machinery to regulate an integrin's affinity for ligand, thereby changing the response of the cell to its extracellular environment. On the other hand, \"outside-in\" signaling is initiated by ligand binding outside the cell, and elicits a series of intracellular events, beginning with the clustering of integrin receptors within specialized complexes of proteins called focal adhesions. The typically short cytoplasmic domains of integrins are void of any enzymatic ability, but activate intracellular signaling and cytoskeletal remodelling via the association with adaptor proteins, which function in connecting integrins to the cytoskeleton, cytoplasmic kinases and growth factor receptors (van der Flier and Sonnenberg, 2001). Several classical second messengers are activated, including G-proteins and tyrosine kinases, which in turn 93 trigger a variety of possible regulatory pathways, including those that mediate cell growth and migration, survival, and differentiation. Integrins, together with their E C M ligands, transmit strong regulatory influences to stem cells of different origins, upon which integrins are commonly expressed. A variety of different integrin heterodimers are expressed in primitive hematopoietic cells and in epidermal stem cells, wherein they participate in the control of proliferation, differentiation and survival (Watt, 2000; Chan and Watt, 2001). Indeed, in the epidermis, the current model of keratinocyte differentiation highlights the pivotal role of integrins in the control of this developmental process. Epidermal stem cells express Pi integrin with which they utilize to interact with the underlying E C M - r i c h basement membrane. Pi integrin signaling maintains stem characteristics in these cells by actively inhibiting differentiation and by influencing decisions regarding proliferative status (Adams and Watt, 1993; Levy et al., 2000; Zhu et al., 1999). Loss o f p i integrin expression elicits the exit from the stem cell compartment by disrupting the integrin-mediated protective effects and via reduced adhesion. Apica l migration and terminal differentiation of keratinocytes subsequently follows (Watt, 2001). In the O E , we have demonstrated that a i , 0:3, ae integrin subunits are expressed in H B C s (Figure 3.3). A l l of these a subunits are potential pairing partners for the Pi integrin detected within this population of cells, while only ct6 is capable of associating with p 4 integrin subunits. The discovery of these candidate pairings reveals potential components of the H B C niche, or microenvironment, within which the regulation of these cells is expected to occur. A pairing of Pi with C M forms a receptor for collagens I-IV and laminin, while 013 associates with Pi to make bind a range of substrates, including 94 laminin, collagen and fibronectin (van der Flier and Sonnenberg, 2001). Lastly, o^Pi forms a receptor for laminin (van der Flier and Sonnenberg, 2001). Given that collagen, fibronectin and laminin are components of the O E basement membrane situated immediately beneath the H B C layer (Julliard and Hartman, 1998), it is likely that these factors activate integrin signaling within H B C s . However, the determination of which E C M components activate which functional integrin receptor pairs w i l l require further experimentation. Nonetheless, with respect to the potential function of these integrins within the O E , perhaps the most obvious role for Pi integrin is adhesion to the basement membrane on account of its restriction to the basal surfaces of H B C s (Figure 3.1 and 4.5). This adhesive role would not only permit the interaction of other integrins with E C M components, but would also promote H B C binding of growth factors typically sequestered in basement membrane sheets. Further, strong integrin mediated adhesion would immobilize H B C s within a particular microenvironment to potentially act as a barrier to differentiation and, as such, might serve in the maintenance of the stem cell phenotype via both immobilization and intrinsic signalling. The presence of P4 integrin subunits upon H B C s likely indicates similar roles to those of P i integrins, as discussed above. However, P4 integrin subunits possess some structural and functional peculiarities that predict alternative functions within H B C s as well . In contrast to the actin cytoskeletal linkage of other integrins, the P4 integrin is unique in that it exerts its intracellular effects via an association with the intermediate filament cytoskeleton (Mercurio and Rabinovitz, 2001). In addition, the P4 subunit possesses a much larger cytoplasmic domain than other p integrins and bears no 95 significant homology to these shorter cytoplasmic domains (Hogervorst et al., 1990). We have detected as integrin subunit expression in H B C s within the normal O E (Figure 3.3). Since this is the only known a subunit to which 04 integrin associates, it is likely that the 0:604 pairing is a functional integrin receptor in H B C s . CC604, a laminin-binding receptor, is one of the principle transmembrane components o f hemidesmosomes. Hemidesmosomes are specialized c e l l - E C M adhesion sites on the basal surfaces of epithelial cells that anchor keratin intermediate filaments within the cell to the underlying basement membrane (Nievers et al., 1999). In other epithelial tissues, hemidesmosomes function in the maintenance of tissue integrity and in defining epithelial boundaries (Nievers et al., 1999). However, it is not yet known whether hemidesmosomes function as signaling units in the absence of 01604. Interestingly, the presence of hemidesmosomes has been previously described in olfactory H B C s (Holbrook et al., 1995). Hence, it is anticipated that 066 04 may be found to contribute to the structural framework of these adhesions in H B C s . However, ct604 is not likely restricted to hemidesmosomes since expression of this receptor's component parts is detected on the apical and lateral surfaces of H B C s in the normal O E , in addition to strong basal staining. A similar phenomenon is reported in a study concerning epidermal keratinocyte integrin expression, wherein the authors propose two sites of oi604 localization: one within hemidesmosomes and one without (Hertle et al., 1991). Thus, in parallel to the epidermis, H B C ct604 expression may serve different functions intracellularly, namely: a role in cementing H B C s to the basement membrane via hemidesmosome adhesions and a role in intracellular signaling whereby proliferation, differentiation and survival are regulated. 96 A l l three of the adhesion receptor H B C markers identified in this study are expressed upon stem cells and/or progenitors in other self-renewing tissues. In particular, as noted by Mahanthappa and Schwarting (1993), the O E and epidermis share many similarities with respect to tissue organization, basal location of stem/progenitor cells and molecular regulation of the stem cell hierarchy, likely owing to the fact that they share a common ectodermal origin. Our findings that basal cells of these two tissues express both Pi and P4 integrins further demonstrates this point. Given these facts, it is interesting that I C A M - 1 is constitutively expressed at high levels in H B C s of the normal, unlesioned O E , while in its epidermal counterpart I C A M - 1 is only expressed in non-normal states. These observations highlight some notable similarities and differences between olfactory H B C s and stem cells in other self-renewing tissues. The discovery of these adhesion receptor molecules upon H B C s prompted us to query the expression of these proteins after surgical removal of the olfactory bulb in order to yield further insight into their in vivo function. Several trends were observed with regard to changes in the cellular constituents of the O E in addition to changes in the expression and localization of the identified H B C markers post-bulbectomy. In accordance with other published studies (Costanzo and Graziadei, 1983; Mont i Graziadei, 1983; Schwob et al., 1992), our data show an inverse relationship between the number of dividing basal cells and the abundance of neurons within the lesioned O E (Figure 4.1). This result was of significance as it highlights the dynamic, highly regulated pattern of neurogenesis elicited by the removal of the olfactory bulb. In addition, this result served as a baseline experiment indicating that O R N loss and 97 replenishment is occurring following our in-house lesion paradigm, in keeping with published bulbectomy studies from other laboratories. Using the bulbectomy paradigm, we wished to examine the adhesion receptor expression of H B C s with an aim to illuminate any functional contribution of these receptors during neurogenesis. In the normal quiescent O E , I C A M - 1 expression in H B C s is pericellular. A t 6d post-bulbectomy, corresponding to the peak of basal cell proliferation, the I C A M - 1 signal is concentrated at the basal surfaces of many H B C s (Figure 4.5). In other regions of the H B C layer, I C A M - 1 expression is lost altogether (Figure 4.4). A similar phenomenon was observed for the p4 integrin subunit, where the protein, though concentrated basally, has a general pericellular distribution in the quiescent O E , which is further concentrated to the basal surface following bulbectomy. In contrast, no observable change in Pi integrin distribution is observed following lesion, though the same immuno-negative patches of O E are detected. We hypothesize that depriving cells of basement membrane contact, via dowregulation of adhesion receptor expression, provides a stimulus to migrate apically and further drive the differentiation program, in a series of events parallel to those which occur during keratinocyte differentiation (Watt, 2000). A s such, disruptions in the continuity of I C A M - 1 , Pi and P4 integrins within the lesioned O E might represent cells which have committed to differentiation. On the other hand, the downward concentration of I C A M - 1 and P4 integrin to the basal surfaces of H B C s following bulbectomy suggests that these proteins function in strengthening static adhesion to the basement membrane. A redistribution of adhesion receptors to the basal surface would also increase the likelihood of receptor ligation with their ligands within the basement membrane. More 98 ligated receptors would potentiate intracellular adhesion signaling and could possibly ensure that an inappropriate or undesirable response to secreted neurogenic factors does not occur. A s such, a reinforcement of HBC-basement membrane adhesion would serve to maintain the stem cell compartment. The decision between downregulation and redistribution in this model is likely made according to cues in the cell's immediate environment. For example, when local O R N numbers are low, adhesion receptors are downregulated to permit H B C differentiation. In contrast, a differentiative response from all H B C s would not be desirable, as it could deplete the local stock of stem cells. Hence, some H B C s redistribute adhesion receptors to strengthen adhesion to the basement membrane and adhesion signaling within the cell in order to maintain the stem cell phenotype for future regenerative activity within the O E . To initiate testing of this model, function-blocking antibodies directed against the integrins could be supplied to cultures of isolated H B C s and then assayed for differences in proliferation, differentiation and survival. Further, to test for an effect of local differentiated cells on H B C expression of Pi and p4 integrins, neurons could be added in excess to purified H B C cultures. According to the above model, we would expect to observe an upregulation of integrin expression in H B C s in the neuron-treated cultures. When assayed for changes in H B C proliferation, differentiation, and survival, we would predict that the neuron-treated H B C s would evidence effects opposite to those of the function-blocking antibody experiments. Another trend observed in H B C s following lesion is the apparent increased heterogeneity of I C A M - l / P i integrin and I C A M - I / P 4 integrin expression as detected via double immunofluorescence (Figure 4.6). These differences highlight the dynamic nature 99 of H B C s and may represent a continuum of stem potentials such that those H B C s which express a full complement of these markers, for example, may possess a more primitive stem phenotype than those expressing a sole marker. Such is the case of both hematopoietic and epidermal lineages and is a trait that is exploited by researchers in these fields to fractionate cells at various points down the stem/progenitor hierarchy In addition to potential adhesion receptor mediated regulatory events, our bulbectomy studies revealed traits of the H B C population suggestive of a stem cell phenotype. Firstly, a pronounced proliferative heterogeneity was observed in the basal cell layer, with G B C s showing robust cell division while H B C s divided rarely (Figure 4.3). We can thus conclude that, although I C A M - 1 + H B C s are capable of proliferation, they are relatively quiescent with respect to the more apically situated G B C layer. This is in keeping with the proposed role of G B C s as transit amplifying cells and lends supporting evidence towards the argument of H B C s as stem cells (Schwob et al., 2002). Proliferative heterogeneity is a common trait of self-renewing tissues, within which it is thought to have evolved as a mechanism to preserve stem cells from proliferative exhaustion (Lavker and Sun, 2000). A further piece of evidence which serves in arguing against the hypothesis of a stem cell resident in the G B C population was again derived from immunohistochemical analysis. In discrete regions of the lesioned O E , the single layer of immuno-negative G B C s , separating H B C s and immature neurons in normal quiescent tissue, disappears (Figure 4.2). Two possible explanations exist for this observation. Firstly, transit amplifying G B C s may be locally depleted owing to a strong neurogenic force imposed by the complete loss of neurons by 3 days post-bulbectomy (Cowan et al., 2001). That 100 G B C s might be exhausted, albeit in confined regions of O E , does not bode well for its candidacy for O E stem cell. On the other hand, or perhaps simultaneously, G B C s might be induced to express markers of neuronal differentiation prematurely which, again, would discount their candidacy as stem cell owing to the fact that stem cells are defined as having an undifferentiated phenotype. At no point did the H B C layer express markers of differentiation. In summary, the in vivo portion of this study provided support for the H B C as stem cell argument as well as providing information regarding the potential regulation of these cells within their in vivo microenvironment. The focus of the in vitro portion of this study was to initiate the development of a method for isolating H B C s in vitro and to determine whether H B C s display traits o f stem/progenitor cells in culture. B y fractionating neo-natal mouse O E suspensions according to cell size and differential adhesive properties, we have isolated a semi-dissociated fraction that produces tightly adhesive clusters of H B C s in vitro, as confirmed by immunohistochemistry with antibodies directed against I C A M - 1 , Pi integrin and p 4 integrin (Figure 5.1). These clusters also incorporate B r d U and are co-incident with neurons and glia at late time-points in culture. These preliminary results prompted us to examine stem/progenitor cell traits using a culture system that more stringently fractionates the H B C and non-HBC population on the basis of I C A M - 1 antigenicity. The magnetic activated cell sorting ( M A C S ) H B C culture method developed for this study represents a departure from in vitro progenitor isolation techniques commonly used in the olfactory literature in that it is the first to employ positive selection (via 101 immunomagnetic labeling) to specifically enrich for candidate stem cells. In addition, ours is one the few studies which uses a single cell suspension, rather than semi-dissociated or explant culture to test for stem characteristics in candidate olfactory cell types. A s such, we have developed a culture method that can be utilized to test a single candidate cell population for stem/progenitor cell characteristics. Finally, ours is the first reported study to exploit the cell surface antigen expression of candidate cells to sort out in vitro in any post-natal neural stem cell population utilizing a reliable marker for the cell type to be assayed. Stem cells are defined according to their functional attributes (Potten and Loeffler, 1990). A s such, assays have been developed in order to test for stem cell functional traits. One such assay, which examines colony-forming efficiency, tracks the ability of a given cell population to seed colonies that expand under low density culture conditions. Within the extant literature, there is an agreement that stem cells should exhibit clonogenicity (Potten and Loeffler, 1990; Zheng et al., 2000;Seery and Watt, 2000). However, colony-forming assays do not screen for stem cells only, as committed progenitors may also form colonies under these conditions. Hence, in the ensuing discussion, the term \"progenitor\" is used, in the broad sense, to include \"potential\" stem cells in addition to \"true\" committed progenitors. Our results indicate that M A C S selected I C A M - 1 + cells display a far superior colony-forming efficiency than do their I C A M - T counterparts (Figure 5.3). This result supports the hypothesis that H B C s possess progenitor activity. Furthermore, the observation that the I C A M - 1 - fraction is depleted in apparent colony-forming units with respect to the total unselected population of O E cells, circumvents the argument that 102 other stem cell candidates (for example, GBCs) are unfairly represented due to the abundance of other cell types within negative fractions. One caveat, however, could be that the I C A M - 1 \" contains cells that are inhibitory to colony-formation by I C A M - 1 \" progenitors. Indeed, a previous study of olfactory progenitors in culture demonstrated that neurogenesis is inhibited when an excess of neurons is added to these cultures (Mumm et al., 1996). However, it seems unlikely that this would be the case in our cultures, as they are plated at very low density (6000 cells per 10 cm dish). Thus, intercellular contact between an \"inhibitory\" cell and a progenitor would be virtually non-existent, and secreted factors would be immediately diluted upon exit from the cell , making it unlikely that these would influence progenitor activity. Nonetheless, to test for an inhibitory effect in the I C A M - 1 \" fraction, N C A M + neurons could be depleted from this fraction using cell sorting techniques, and the resulting I C A M - 1 \" / N C A M \" fraction could then be tested for colony-forming efficiency and compared to the original I C A M - 1 \" fraction. Dissociation and cell counting of colonies containing >150 cells yielded an average of 5363 cells per colony, with an upper range of 40, 000 cells per colony. Assuming that the observed colonies were seeded by single cells, this data would indicate the considerable proliferative potential of the I C A M - 1 founding cell, a finding that highlights its potential as olfactory stem cell. Previous culture studies report that the alternative stem cell candidate, the G B C , evidences a much lower proliferative capacity, forming colonies with a size that would lead us to characterize them as \"small\" colonies (<30 cells per colony), according to our size definitions. Also , one colony was formed 103 for every 1000 G B C s plated at high density (Mumm et al., 1996), indicating a lower colony-forming ability relative to our study of H B C s at clonal density. Typically, the culture of stem/progenitor cells at clonal density yields very low colony-forming efficiencies as a result of reduced intercellular interaction, especially during the early development of culture conditions for a particular cell type. For example, Kaur and L i (2000) reported that human epidermal keratinocytes have a colony forming efficiency of 0.45%, while embryonic striatal neurospheres exhibit a colony forming efficiency of 1% when viable cells are plated at low density (Reynolds and Weiss, 1996). Defining the in vivo stem/progenitor niche is often instructive in determining the optimal factors that might be supplemented to the culture of these cells to promote optimal colony formation. In addition, further insight can be gained concerning the regulation of stem/progenitor cells by factors within their resident in vivo niche. In order to increase the colony-forming efficiency of our I C A M - 1 + M A C S selected cells, cells were plated in different media formulations commonly used in the culture of stem cells (Figure 5.4). D M E M / F 1 2 + 10% F B S proved the most effective at supporting colony growth, with O p t i - M E M + 4% F B S approximately 70% of the D M E M / F 1 2 value. The fact that no colonies formed when I C A M - 1 + positive cells are cultured in either R P M I or K - S F M , both of which contain low concentrations of calcium, suggests that higher levels of calcium are required for H B C colony formation. This concurs with what is known to date regarding the function of cell adhesion molecules, most of which, including the integrins and, depend on calcium ions for optimal signaling and adhesion to the substratum (Alberts et al., 1994). Hence, a deficiency o f calcium ions in the medium might interfere with adhesion receptor function upon H B C s and force 104 them to differentiate. Based on these results, we preferentially used D M E M / F 1 2 + 10% F B S for all ensuing culture experiments except in cases where growth factor was added, in which case the reduced serum O p t i - M E M + 4% F B S was utilized. Given that H B C s express ECM-bind ing integrin receptors in vivo and in vitro, and owing to a previous report of several common E C M components within the O E ' s underlying basement membrane, we tested the effect of collagen, laminin and fibronectin on the colony-forming efficiency of I C A M - 1 + cells. Plating on collagen produced the highest colony-forming efficiency, while both fibronectin and laminin yielded efficiencies roughly half of that of collagen (Figure 5.5). When cells are plated on mixtures o f collagen and laminin, overall colony-forming efficiency surpasses that o f collagen alone. However, the collagen/fibronectin mixtures, though higher than fibronectin individually, are more comparable to collagen applied singly. Wi th respect to the incidence of large colonies, both mixtures of collagen and laminin yielded a 4-fold increase with respect to the cells plated on collagen (Figure 5.7). The collagen- and laminin-dependent increases in colony-forming efficiency might be mediated by any of the integrin receptor pairings discussed above, including ociPi, OC3P1, 0^4, and OC6P4. Although alternative, unidentified fibronectin receptors might exist on H B C s , the only potential H B C integrin that is reported to bind fibronectin is 0C3PI. The results o f this series of colony-forming efficiency assays indicates a role in the promotion of colony formation for collagen and laminin. With regard to fibronectin, these assays did not provide any particularly insightful information. It is difficult to ascertain whether significant fibronectin signaling is occurring within these cells, or whether the effects o f fibronectin are simply not detectable using our assay system (one that recognizes 105 differences in colony initiation and proliferation). We can, however, exclude an inhibitory effect of fibronectin on colony expansion, as is the case in epidermal keratinocytes (Adams and Watt, 1989), as a collagen/fibronectin mixed substrate does not decrease overall colony-forming efficiency relative to collagen alone. To further illuminate the influence of collagen, laminin and fibronectin on the colony-forming ability of I C A M - 1 + cells, an analysis of the kinetics of adhesion to these substrates was performed (Figure 5.8). In all three cases, the majority of adhesion to substrate occurs within the first 4 hours in culture. Also , overall, I C A M - 1 + selected cells display a clear preference for collagen, and roughly equal, but lesser, adherence to laminin and fibronectin. One might argue that the results of the adhesion kinetics experiment indicate that the large difference in overall colony formation between substrate conditions is a consequence of the capability of the cells to adhere to the substratum. Hence, a cell population that does not adhere to fibronectin, for example, w i l l not form many adherent colonies. In addition, the increase in overall colony-forming efficiency with the collagen/laminin mixed substrate likely reflects an additive adhesive effect of the two substrates individually. However, an additive effect is not observed for collagen/fibronectin mixtures. It is likely that the differences in adhesion account, at least in part, for the observed differences in overall colony formation in vitro. However, with respect to the formation of large colonies specifically, the effects of a mixed collagen/laminin substrate does not appear to reflect a simple additive effect o f the two substrates individually. A s such, it is likely that a post-adhesive mechanism, such as the promotion of survival or proliferation, is promoting the formation of more large colonies on the mixed matrices. To summarize, we conclude that these E C M components 106 differentially affect I C A M - 1 + cell adhesion in vitro and likely contribute to intracellular adhesion signaling to influence proliferation and/or survival of integrin-bearing cells. Further experimentation, using assays specific for the above cellular functions, w i l l be required to clarify the endpoints of integrin signaling in H B C s . In addition to E C M proteins, we also tested the effects of several resident O E growth factors on the efficiency of I C A M - 1 + cell colony formation. Both E G F and T G F -a increase thymidine labeling indices by stimulating mitosis in O E cultures derived from fetal rat (Calof et al., 1991; Mahanfhappa and Schwarting, 1993; Farbman and Bucholz, 1996). E G F R , the receptor for both these growth factors is localized to the H B C layer in the rat in vivo (Rama Krishna et al., 1996; Ezeh and Farbman, 1998). A s such, we assayed the effects of these growth factors on I C A M - 1 + selected cells at clonal density and demonstrated that E G F and T G F a , both singly and i n combination, significantly increased the number of total colonies formed (Figure 5.9). Furthermore, these growth factors appeared to stimulate the formation of large colonies at the expense of small ones (Figure 5.10). Given their reported function both within the olfactory system and elsewhere, this increase in overall colony formation in tandem with enhanced formation of large colonies can likely be attributed to the reported mitogenic effects of these growth factors. A s such, single undividing cells which would not have been included in the colony count in control cultures at 2 weeks in vitro, would be stimulated to divide by E G F and/or T G F - a , thereby increasing overall colony-forming efficiency. Likewise, small and medium sized colonies are coaxed to increase in cell number, resulting in the observed frequency of large colonies. We therefore conclude that we have detected a means to enhance the expansion of I C A M - 1 + H B C s in vitro. Furthermore, the observed 107 responsiveness to E G F and T G F - a of M A C S selected I C A M - 1 + cells in vitro further supports the conclusion that these cells are the in vitro equivalents of olfactory H B C s . Leukemia inhibitory factor (LIF) was a factor of interest owing to the fact that the olfactory literature indicates its role in olfactory neurogenesis. Within the O E proper, the LIF receptor (LIFR) is detected infrequently in the odd G B C (Nan et al., 2001). The incidence of L I F R + G B C s is elevated following olfactory bulbectomy, implicating LIF signaling in the generation of new ORNs. A s well , in vitro studies show that L IF increases the B r d U labeling index of immediate neuronal precursors (INPs) (Satoh and Yoshida, 1997). Our interest was further piqued by the report that subependymal-derived neurospheres can be maintained in a \"primitive\", proliferative stem state via the addition of LIF and E G F to the culture medium (Shimazaki et al., 2001). Our results, however, indicate that the addition of LIF to clonal cultures of I C A M - 1 + selected cells has no effect on the overall ability of these cells to initiate colonies (Figure 5.9). Nonetheless, LIF appears to influence the incidence of large colonies at 14 D I V , in that there is roughly a 4-fold increase i n this category o f colonies when LIF is added to the medium (Figure 5.10). Given these results, two models of LIF action are possible. Firstly, in accordance with its proposed role as G B C mitogen, LIF may be increasing the size of existing colonies via G B C expansion. A potential explanation for the stagnant overall colony-forming efficiency of LIF-treated cultures is that the I C A M - 1 + cells initially seeded are unresponsive to LIF. Hence, the only LIF-responsive cells possible in our culture system are daughter cells of H B C s , formed by the division of H B C s irrespective of LIF supplementation. Alternatively, the addition of LIF might achieve large colonies by promoting the survival of G B C s formed after plating, such that the higher incidence of 108 large colonies reflects decreased cell death. When LIF and E G F were added to cultures in combination, far fewer large colonies were formed relative to the E G F only treatment, although there does not appear to be a significant difference between overall colony formation. One possible explanation for this observation would be the existence of a paracrine mechanism to control H B C proliferation, induced by ligation of L I F R upon G B C s . If this was indeed the case, it would provide a mechanism whereby a single factor might simultaneously control both the stem and transit amplifying compartments, thereby ensuring the appropriate response to neuronal loss as dictated by the O E microenvironment. Stem cells, by definition, generate daughter cells which terminally differentiate in order to maintain functional tissue homeostasis (Hall and Watt, 1989; Potten and Loeffler, 1990). Homeostasis also implies that a population of stem cells is maintained in a given tissue. To address whether H B C are capable of these traits, we examined the expression of markers of olfactory differentiation in combination with the new H B C markers identified in this study within older cultures of I C A M - 1 + , M A C S selected cells. Single I C A M - 1 + cells can generate colonies that contain up to 40, 000 cells over 14 D I V , an observation that exemplifies H B C s ' proliferative potential in vitro. B y surveying the phenotypic makeup of these colonies, we found that cores of I C A M - 1 7 p 4 integrin cells were maintained in all large colonies (Figure 5.11). The combined expression of I C A M -1 with P 4 integrin, indicative only of H B C s within the O E , suggests that cores within the colonies contain a significant representation from an expanded population of H B C s . Given that cores of adhesion receptor positive H B C s are maintained within large colonies 109 at later culture time-points, it is possible that H B C s are demonstrating the stem trait o f self-maintenance to ensure that stem cells are not depleted in vitro. Globose basal cells (GBCs) , the proposed transit amplifying cells and proven immediate precursors of olfactory receptor neurons, are also represented in large colonies in vitro, as evidenced by the expression of G B C - 2 within subsets of cells (Figure 5.11). Given their presence, it is likely that they are daughter cells of horizontal basal cells, and serve in recapitulating the proposed in vivo stem/progenitor cell hierarchy in vitro. Our results also show that olfactory neurons are generated and mature in cultures of I C A M - 1 + immunomagnetically selected cultures of O E cells (Figure 5.11 and 5.12). The latter characteristic is of interest due to previous reports that olfactory neurons achieve maturity with difficulty in vitro ( A J Roskams, pers. comm.). In addition, we can infer that neurons are being produced even at later time-points in culture given that previous in vitro studies report that O R N s do not survive past 7 D I V (Calof and Chikaraishi, 1989; Calof and Lander, 1991; Ronnett et al., 1991; Mahanthappa and Schwarting, 1993; Pixley et al., 1994; Farbman and Bucholz, 1996; Roskams et al., 1996). Our results also indicate the presence of cells indicative of olfactory ensheathing glia (OEGs) within older cultures of M A C S selected cells (Figure 5.11). The presence of dual neuronal and glial lineages from progenitors obtained from the O E has not been previously reported for expanded G B C s in vitro, and indicates that H B C s may have a greater degree of pluripotency than G B C s . Furthermore, we propose the existence of an INP equivalent for the glial lineage, such that olfactory stem cells produce daughters committed to a particular lineage in order to segregate the production of differentiated cells, as is the case in stem/progenitor hierarchies in other multi-lineage tissues. The 110 potential pluripotency of H B C s is further demonstrated by the detection of neurons with distinctly non-olfactory phenotypes (Figure 5.13). Future experiments directed at increasing the stringency of H B C culture experiments include the addition of a micromanipulation step prior to plating H B C s . For example, H B C s could be sorted according to I C A M - 1 antigenicity by fluorescence associated cell sorting ( F A C S ) with a machine equipped with a single cell depositor. B y seeding I C A M - 1 + cells singly in individual wells, we could prove that the \"assumed\" colonies detailed in this study are true colonies (i.e. seeded by single cells). In addition, as F A C S is a more powerful sorting strategy than M A C S , the purification of H B C s would be maximized and we could further fractionate H B C s according to I C A M - 1 , Pi and P4 integrin expression using tri-colour F A C S . In this manner, subsets of H B C s could be compared with respect to progenitor activity. Finally, in order to elevate H B C status from progenitor to stem cell, the requisite stem cell trait of self-renewal must be satisfied. Secondary cloning assays, where single colonies generated by the I C A M - 1 + fraction are dissociated, replated and assayed for colony-formation, are currently being performed in the laboratory. Preliminary results indicate that secondary colonies are produced only rarely and are generated from large primary colonies (>150 cells) only (L. Carter, A . Griffiths and J. Roskams, unpublished observations). In addition, there is preliminary evidence that tertiary cloning is also possible by plating the colonies generated by secondary cloning (A. Griffiths and J. Roskams, unpublished observations). We hypothesize that the low efficiencies of preliminary secondary cloning experiments are a consequence of a rapid loss of stem cell traits in vitro, as is observed in the culture of other stem cells (Domen, 2001). The observation that our cultures of I C A M - 1 + , M A C S 111 selected cells produce colonies with an abundance of differentiated cells supports this hypothesis. Future experiments w i l l be directed at optimizing the I C A M - 1 + H B C culture conditions to ensure that the stem cell phenotype is maintained in culture. 112 C H A P T E R V I I . Conc lud ing Remarks B y immunohistochemically screening a panel of selected clusters of differentiation (CD) antigens, we have revealed three new cell surface markers of olfactory horizontal basal cells (HBCs) , namely: intercellular adhesion molecule-1 ( ICAM-1) , Pi and P4 integrin. Potential pairing partners for the P integrin subunits include cci, 0 C 3 , and 0C6 subunits, as all are detected in H B C s . The discovery of these markers was of significance as it has enabled us to selectively enrich for H B C s in vitro by exploiting their molecular phenotype. Further, since these proteins are also present on stem and progenitor cells in other self-renewing tissues, their presence on H B C s suggests that these stem cells share common signaling pathways. After olfactory bulbectomy, a procedure which results in the loss of olfactory receptor neurons (ORNs) from the olfactory epithelium (OE), changes are detected with respect to populational and subcellular expression of I C A M - 1 , Pi and P4 integrin. Breaks in the continuity of marker expression are observed in addition to an apparent redistribution of I C A M - 1 and P4 integrin to the basal surfaces of H B C s . These changes in marker expression suggest a role for these adhesion receptors in the regulation of H B C function during neurogenesis. We also detected evidence of stem cell traits within the H B C population in vivo: 1) proliferative heterogeneity with respect to robustly proliferating G B C s ; 2) molecular heterogeneity within the H B C compartment (which becomes more pronounced post-bulbectomy); and 3) response to lesion. 113 For in vitro studies, a method was developed to immunomagnetically sort O E -derived cells according to I C A M - 1 antigenicity using magnetic activated cell sorting ( M A C S ) . A n experiment to compare the ability to form colonies at very low, or clonal, plating density demonstrated that the I C A M - 1 + fraction has superior colony-forming ability relative to unselected and I C A M - 1 \" fractions. These results suggest that the I C A M - 1 + fraction is enriched for progenitor activity, while the I C A M - 1 \" fraction is depleted of these colony-forming cells. In order to optimize the colony-forming efficiency of M A C S selected, I C A M - 1 + cells, several growth factors and extracellular matrix ( E C M ) components, all resident in proximity to H B C in vivo, were tested via clonal analysis. Collagen and collagen/laminin mixtures produced the highest overall colony-forming efficiencies and also yielded the highest formation of large colonies, while fibronectin and laminin singly resulted in the lowest overall colony-forming efficiencies and the lowest large colony formation. With regard to the growth factors tested, E G F and T G F - a , both individually and in combination, produced the highest overall colony-forming ability, while LIF produced no observable effect. However, all growth factors served in promoting large colonies in culture. B y examining the expression of markers of olfactory differentiation, our results show that cultures of I C A M - 1 + cells produce large colonies containing globose basal cell progenitors, olfactory receptor neurons and olfactory ensheathing glia under clonal conditions. In addition, the H B C phenotype is maintained within cores of these large colonies as evidenced by I C A M - 1 and p% integrin immnohistochemistry. We conclude that H B C s possess traits suggestive of a stem cell phenotype in vivo and exhibit 114 progenitor activity in vitro. A s such, we conclude that H B C s likely contribute to the genesis of olfactory cell types, potentially as the O E stem cell. Further, the function of H B C s is likely modulated via adhesive and growth factor signaling in a manner parallel to stem cells i n other, better characterized tissues. Further experiments are currently being performed in order to test for H B C self-renewal, the final requisite stem cell trait. 115 B I B L I O G R A P H Y Adams, J. C. and F. M . Watt (1989). \"Fibronectin inhibits the terminal differentiation of human keratinocytes.\" Nature 340(6231): 307-9. Adams, J. C. and F. M . Watt (1993). \"Regulation o f development and differentiation by the extracellular matrix.\" Development 117(4): 1183-98. Akhurst, R. J., F. Fee, et al. (1988). \"Localized production of TGF-beta m R N A in tumour promoter-stimulated mouse epidermis.\" Nature 331(6154): 363-5. Alberts B , Bray D , Lewis J et al (eds). (1994). Molecular biology o f the cell. 3 r d edition. Garland Publishing, Inc.: New York. Anderson, D . J., F . H . Gage, et al. (2001). \"Can stem cells cross lineage boundaries?\" Nat M e d 7(4): 393-5. Ark in , S., B . Naprstek, et al. (1991). \"Expression of intercellular adhesion molecule-1 (CD54) on hematopoietic progenitors.\" Blood 77(5): 948-53. Barrandon, Y . and H . Green (1987). \"Three clonal types of keratinocyte with different capacities for multiplication.\" Proc Natl Acad Sci U S A 84(8): 2302-6. Brakebusch, C , E . Hirsch, et al. (1997). \"Genetic analysis of betal integrin function: confirmed, new and revised roles for a crucial family o f cell adhesion molecules.\" J Cel l Sci 110(Pt 23): 2895-904. Caggiano, M . , J. S. Kauer, et al. (1994). \"Globose basal cells are neuronal progenitors in the olfactory epithelium: a lineage analysis using a replication-incompetent retrovirus.\" Neuron 13(2): 339-52. Calof, A . L . and D . M . Chikaraishi (1989). \"Analysis of neurogenesis in a mammalian neuroepithelium: proliferation and differentiation of an olfactory neuron precursor in vitro.\" Neuron 3(1): 115-27. Calof, A . L . , A . D . Lander, et al. (1991). \"Regulation of neurogenesis and neuronal differentiation in primary and immortalized cells from mouse olfactory epithelium.\" Ciba Found Svmp 160: 249-65; discussion 265-76. Calof, A . L . , J. S. Mumm, et al. (1998). \"The neuronal stem cell o f the olfactory epithelium.\" J Neurobiol 36(21: 190-205. Carr, V . M . and A . I. Farbman (1992). \"Ablation o f the olfactory bulb up-regulates the rate o f neurogenesis and induces precocious cell death in olfactory epithelium.\" Exp Neurol 115m: 55-9. 116 Cau, E . , G . Gradwohl, et al. (1997). \" M a s h l activates a cascade o f b H L H regulators in olfactory neuron progenitors.\" Development 124(8): 1611-21. Chan, J. Y . and S. M . Watt (2001). \"Adhesion receptors on haematopoietic progenitor cells.\" B r J Haematol 112(3): 541-57. Chen, Y . , M . L . Getchell, et al. (1992). \"Immunolocalization o f two cytochrome P450 isozymes in rat nasal chemosensory tissue.\" Neuroreport 3(9): 749-52. Chiasson, B . J., V . Tropepe, et al. (1999). \"Adult mammalian forebrain ependymal and subependymal cells demonstrate proliferative potential, but only subependymal cells have neural stem cell characteristics.\" J Neurosci 19(11): 4462-71. Chirathaworn, C , S. A . Tibbetts, et al. (1995). \"Cross-linking o f I C A M - 1 on T cells induces transient tyrosine phosphorylation and inactivation o f cdc2 kinase.\" J Immunol 155(12): 5479-82. Coffey, R. J., Jr., R. Derynck, et al. (1987). \"Production and auto-induction of transforming growth factor-alpha in human keratinocytes.\" Nature 328(6133): 817-20. Collins, A . T., F . K . Habib, et al. (2001). \"Identification and isolation of human prostate epithelial stem cells based on alpha(2)beta(l)-integrin expression.\" J Cel l Sci 114(Pt21): 3865-72. Costanzo, R. M . (1984). \"Comparison of neurogenesis and cell replacement in the hamster olfactory system with and without a target (olfactory bulb).\" Brain Res 307(1-2): 295-301. Costanzo, R. M . (1985). \"Neural regeneration and functional reconnection following olfactory nerve transection in hamster.\" Brain Res 361(1-2): 258-66. Costanzo, R. M . and P. P. Graziadei (1983). \" A quantitative analysis of changes in the olfactory epithelium following bulbectomy in hamster.\" J Comp Neurol 215(4): 370-81. Cowan, C. M . , J. Thai, et al. (2001). \"Caspases 3 and 9 send a pro-apoptotic signal from synapse to cell body in olfactory receptor neurons.\" J Neurosci 21(18): 7099-109. De Panfilis, G . , G . C. Manara, et al. (1992). \"Adhesion molecules on the plasma membrane of epidermal cells. IV. Immunolocalization of the intercellular adhesion molecule-1 ( I C A M - 1 , CD54) on the cell surface of a small subpopulation of keratinocytes freshly isolated from normal human epidermis.\" Reg Immunol 4(3): 119-29. 117 DeHamer, M . K . , J. L . Guevara, et al. (1994). \"Genesis of olfactory receptor neurons in vitro: regulation of progenitor cell divisions by fibroblast growth factors.\" Neuron 13(5): 1083-97. Ding, X . X . and M . J. Coon (1988). \"Purification and characterization o f two unique forms o f cytochrome P- 450 from rabbit nasal microsomes.\" Biochemistry 27(22): 8330-7. Domen J. The role of apoptosis in regulating hematopoietic stem cell numbers. (2001) Apoptosis. 6(4):239-52. Doucette, R. (1990). \" G l i a l influences on axonal growth in the primary olfactory system.\" Gl i a 3(6): 433-49. Doucette, R. (1991). \" P N S - C N S transitional zone o f the first cranial nerve.\" J Comp Neurol 312(3): 451-66. Etienne, S., P. Adamson, et al. (1998). \" I C A M - 1 signaling pathways associated with Rho activation in microvascular brain endothelial cells.\" J Immunol 161(10): 5755-61. Ezeh, P. I. and A . I. Farbman (1998). \"Differential activation of ErbB receptors in the rat olfactory mucosa by transforming growth factor-alpha and epidermal growth factor in vivo.\" J Neurobiol 37(2): 199-210. Farbman, A . I. (1990). \"Olfactory neurogenesis: genetic or environmental controls?\" Trends Neurosci 13(9): 362-5. Farbman A l . (1992). Ce l l biology of olfaction. Cambridge University Press: Cambridge. Farbman, A . I., P. C. Brunjes, et al. (1988). \"The effect of unilateral naris occlusion on cell dynamics in the developing rat olfactory epithelium.\" J Neurosci 8(9): 3290-5. Farbman, A . I. and J. A . Buchholz (1996). \"Transforming growth factor-alpha and other growth factors stimulate cell division in olfactory epithelium in vitro.\" J Neurobiol 30(2): 267-80. Feron, F., A . Mackay-Sim, et al. (1999). \"Stress induces neurogenesis in non-neuronal cell cultures o f adult olfactory epithelium.\" Neuroscience 88(2): 571-83. Ford C E , Hamerton JL , Barnes D W H , Loutit JF. (1956). \"Cytological identification of radiation chimeras\". Nature 177: 452-454. 118 Freshney, RI. (2000). Culture of Animal Cells: A Manual of Basic Technique. 4th edition. Wiley-Liss, Inc: New York. Fuchs, E. , J. Dowling, et al. (1997). \"Integrators of epidermal growth and differentiation: distinct functions for beta 1 and beta 4 integrins.\" Curr Opin Genet Dev 7(5): 672-82. Fuchs, E. and J. A. Segre (2000). \"Stem cells: a new lease on life.\" CeU 100(1): 143-55. Gage, F. H. (2000). \"Mammalian Neural Stem Cells.\" Science 287(5457): 1433-1439. Gao, Y. , I. Morita, et al. (2000). \"Expression of adhesion molecules LFA-I and ICAM-I on osteoclast precursors during osteoclast differentiation and involvement of estrogen deficiency.\" Climacteric 3(4): 278-87. Getchell, T. V., R. K. Narla, et al. (2000). \"Horizontal basal cell proliferation in the olfactory epithelium of transforming growth factor-alpha transgenic mice.\" Cell Tissue Res 299f21: 185-92. Gilbert, SF. (1997). Developmental biology. 5th edition. Sinauer Associates, Inc: Massachusetts. Goldstein, B. J., H. Fang, et al. (1998). \"Transplantation of multipotent progenitors from the adult olfactory epithelium.\" Neuroreport 9(7): 1611-7. Goldstein, B. J. and J. E. Schwob (1996). \"Analysis of the globose basal cell compartment in rat olfactory epithelium using GBC-1, a new monoclonal antibody against globose basal cells.\" J Neurosci 16(12): 4005-16. Goldstein, B. J., B. L. Wolozin, et al. (1997). \"FGF2 suppresses neuronogenesis of a cell line derived from rat olfactory epithelium.\" J Neurobiol 33(4): 411-28. Gordon, M . K., J. S. Mumm, et al. (1995). \"Dynamics of M A S H 1 expression in vitro and in vivo suggest a non-stem cell site of MASH 1 action in the olfactory receptor neuron lineage.\" Mol Cell Neurosci 6(4): 363-79. Graziadei, G. A. and P. P. Graziadei (1979). \"Neurogenesis and neuron regeneration in the olfactory system of mammals. II. Degeneration and reconstitution of the olfactory sensory neurons after axotomy.\" J Neurocytol 8(2): 197-213. Graziadei, P. P. and G. A. Graziadei (1979). \"Neurogenesis and neuron regeneration in the olfactory system of mammals. I. Morphological aspects of differentiation and structural organization of the olfactory sensory neurons.\" J Neurocytol 8(1): 1-18. Graziadei, P. P. and A. G. Monti Graziadei (1983). \"Regeneration in the olfactory system of vertebrates.\" Am J Otolaryngol 4(4): 228-33. 119 Griffiths, C. E . and B . J. Nickoloff (1989). \"Keratinocyte intercellular adhesion molecule-1 ( ICAM-1) expression precedes dermal T lymphocytic infiltration in allergic contact dermatitis (Rhus dermatitis).\" A m J Pathol 135(6): 1045-53. Guillemot, F., L . C . L o , et al. (1993). \"Mammalian achaete-scute homolog 1 is required for the early development o f olfactory and autonomic neurons.\" Cel l 75(3): 463-76. Hal l , P. A . and F. M . Watt (1989). \"Stem cells: the generation and maintenance of cellular diversity.\" Development 106(4): 619-33. Hayflick, J. S., P. Kilgannon, et al. (1998). \"The intercellular adhesion molecule ( I C A M ) family o f proteins. New members and novel functions.\" Immunol Res 17(3): 313-27. Hinds, J. W. , P. L . Hinds, et al. (1984). \" A n autoradiographic study of the mouse olfactory epithelium: evidence for long-lived receptors.\" Anat Rec 210(2): 375-83. Hitoshi, S., T. Alexson, et al. (2002). \"Notch pathway molecules are essential for the maintenance, but not the generation, of mammalian neural stem cells.\" Genes Dev 16(7): 846-58. Hogervorst, F. , I. Kuikman, et al. (1990). \"Cloning and sequence analysis of beta-4 c D N A : an integrin subunit that contains a unique 118 kd cytoplasmic domain.\" Embo J 9(3): 765-70. Holbrook, E . H . , K . E . Szumowski, et al. (1995). \" A n immunochemical, ultrastructural, and developmental characterization of the horizontal basal cells o f rat olfactory epithelium.\" J Comp Neurol 363(1): 129-46. Holland, J. and T. Owens (1997). \"Signaling through intercellular adhesion molecule 1 ( ICAM-1) in a B cell lymphoma line. The activation o f L y n tyrosine kinase and the mitogen-activated protein kinase pathway.\" J B i o l Chem 272(14): 9108-12. Howe, A . , A . E . Ap l in , et al. (1998). \"Integrin signaling and cell growth control.\" Curr Opin Cel l B i o l 10(2): 220-31. Huard, J. M . , S. L . Youngentob, et al. (1998). \"Adult olfactory epithelium contains multipotent progenitors that give rise to neurons and non-neural cells.\" J Comp Neurol 400(4): 469-86. Hubbard, A . K . and R. Rothlein (2000). \"Intercellular adhesion molecule-1 ( ICAM-1) expression and cell signaling cascades.\" Free Radic B i o l M e d 28(9): 1379-86. 120 Hurley, R. W. , J. B . McCarthy, et al. (1995). \"Direct adhesion to bone marrow stroma via fibronectin receptors inhibits hematopoietic progenitor proliferation.\" J C l i n Invest 96(1): 511-9. Hynes, R. O. (1992). \"Integrins: versatility, modulation, and signaling in cell adhesion.\" Ce l l 69(1): 11-25. Jones, L . (2001). \"Stem cells: so what's in a niche?\" Curr B i o l 11(12): R484-6. Jones, P. H . (1997). \"Epithelial stem cells.\" Bioessays 19(8): 683-90. Jones, P. H . , S. Harper, et al. (1995). \"Stem cell patterning and fate in human epidermis.\" C e l l 80(1): 83-93. Julliard, A . K . and D . J. Hartmann (1998). \"Spatiotemporal patterns of expression o f extracellular matrix molecules in the developing and adult rat olfactory system.\" Neurosrience 84(4): 1135-50. Kaur, P. and A . L i (2000). \"Adhesive properties of human basal epidermal cells: an analysis of keratinocyte stem cells, transit amplifying cells, and postmitotic differentiating cells.\" J Invest Dermatol 114(3): 413-20. Keller, A . and F. L . Margolis (1975). \"Immunological studies of the rat olfactory marker protein.\" J Neurochem 24(6): 1101-6. Koyama, Y . , Y . Tanaka, et al. (1996). \"Cross-linking of intercellular adhesion molecule 1 (CD54) induces AP-1 activation and IL-lbeta transcription.\" J Immunol 157(11): 5097-103. Krishna, N . S., S. S. Little, et al. (1996). \"Epidermal growth factor receptor m R N A and protein are expressed in progenitor cells o f the olfactory epithelium.\" J Comp Neurol 373(2): 297-307. Lavker, R. M . and T. T. Sun (2000). \"Epidermal stem cells: properties, markers, and location.\" Proc Natl Acad Sci U S A 97(25): 13473-5. Levy, L . , S. Broad, et al. (2000). \"betal integrins regulate keratinocyte adhesion and differentiation by distinct mechanisms.\" M o l B i o l Ce l l 11(2): 453-66. Magavi SS, Leavitt B R , Macklis JD. (2000). Induction o f neurogenesis in the neocortex o f adult mice. Nature. 405(6789):951-5. Magrassi, L . and P. P. Graziadei (1996). \"Lineage specification o f olfactory neural precursor cells depends on continuous cell interactions.\" Brain Res Dev Brain Res 96(1-2): 11-27. 121 Mahanthappa, N . K . and G . A . Schwarting (1993). \"Peptide growth factor control of olfactory neurogenesis and neuron survival in vitro: roles of E G F and TGF-beta s.\" Neuron 10(2): 293-305. Margolis, F . L . (1982). \"Olfactory marker protein ( O M P ) . \" Scand J Immunol Suppl 9: 181-99. Margolis, F. L . , J. Verhaagen, et al. (1991). \"Regulation of gene expression in the olfactory neuroepithelium: a neurogenetic matrix.\" Prog Brain Res 89: 97-122. Mason D , Simmons, D , Buckley C et al., (eds). (2001). Leucocyte Typing VII . Oxford University Press: Oxford. Mercurio, A . M . , I. Rabinovitz, et al. (2001). \"The alpha 6 beta 4 integrin and epithelial cell migration.\" Curr Opin Cel l B i o l 13(5): 541-5. Miragall , F. , G . Kadmon, et al. (1988). \"Expression of cell adhesion molecules in the olfactory system o f the adult mouse: presence of the embryonic form of N - C A M . \" D e v B i o l 129(2): 516-31. Miragall , F . and G . A . Mont i Graziadei (1982). \"Experimental studies on the olfactory marker protein. II. Appearance of the olfactory marker protein during differentiation of the olfactory sensory neurons o f mouse: an immunohistochemical and autoradiographic study.\" Brain Res 239(1): 245-50. Miyake, K . , I. L . Weissman, et al. (1991). \"Evidence for a role of the integrin V L A - 4 in lympho-hemopoiesis.\" J Exp M e d 173(3): 599-607. Morshead, C . M . and D . van der K o o y (2001). \" A new 'spin' on neural stem cells?\" Curr Opin Neurobiol 11(1): 59-65. Moulton, D . G . (1974). \"Dynamics o f cell populations in the olfactory epithelium.\" A n n N Y Acad Sci 237(0): 52-61. Moulton D G , Celebi G , Fink RP . (1970). \"Olfaction in mammals-two aspects: proliferation of cells in the olfactory epithelium and sensitivity to odours\". In: Ciba foundation symposium on taste and smell in vertebrates. Eds., G E W Wolstenholme and J Knight. Churchill , London. Muller-Rover, S., S. Bulfone-Paus, et al. (2000). \"Intercellular adhesion molecule-1 and hair follicle regression.\" J Histochem Cytochem 48(4): 557-68. Mumm, J. S., J. Shou, et al. (1996). \"Colony-forming progenitors from mouse olfactory epithelium: evidence for feedback regulation of neuron production.\" Proc Natl Acad Sci U S A 93(20): 11167-72. 122 Murray, R. C. and A . L . Calof (1999). \"Neuronal regeneration: lessons from the olfactory system.\" Semin Ce l l Dev B i o l 10(4): 421-31. Nan, B . , M . L . Getchell, et al. (2001). \"Leukemia inhibitory factor, interleukin-6, and their receptors are expressed transiently in the olfactory mucosa after target ablation.\" Journal o f Comparative Neurology 435(1): 60-77. Newman, M . P., F . Feron, et al. (2000). \"Growth factor regulation o f neurogenesis in adult olfactory epithelium.\" Neuroscience 99(2): 343-50. Nievers, M . G . , R. Q. Schaapveld, et al. (1999). \"Biology and function o f hemidesmosomes.\" Matrix B i o l 18(1): 5-17. Papayannopoulou, T. and B . Nakamoto (1993). \"Peripheralization of hemopoietic progenitors in primates treated with an t i -VLA4 integrin.\" Proc Natl Acad Sci U S A 90(20): 9374-8. Pixley, S. K . , M . Bage, et al. (1994). \"Olfactory neurons in vitro show phenotypic orientation in epithelial spheres.\" Neuroreport 5(5): 543-8. Potten, C . S. and M . Loeffler (1990). \"Stem cells: attributes, cycles, spirals, pitfalls and uncertainties. Lessons for and from the crypt.\" Development 110(4): 1001-20. Rafols, J. A . and T. V . Getchell (1983). \"Morphological relations between the receptor neurons, sustentacular cells and Schwann cells in the olfactory mucosa o f the salamander.\" Anat Rec 206(1): 87-101. Ramon-Cueto, A . and J. A v i l a (1998). \"Olfactory ensheathing glia: properties and function.\" Brain Res B u l l 46(3): 175-87. Reynolds, B . A . and S. Weiss (1992). \"Generation o f neurons and astrocytes from isolated cells of the adult mammalian central nervous system.\" Science 255(5052): 1707-10. Reynolds, B . A . and S. Weiss (1996). \"Clonal and population analyses demonstrate that an EGF-responsive mammalian embryonic C N S precursor is a stem cel l .\" Dev B i o l 175(1): 1-13. Ronnett, G . V . , L . D . Hester, et al. (1991). \"Primary culture of neonatal rat olfactory neurons.\" J Neurosci 11(5): 1243-55. Roskams, A . J., M . A . Bethel, et al. (1996). \"Sequential expression of Trks A , B , and C in the regenerating olfactory neuroepithelium.\" J Neurosci 16(4): 1294-307. 123 Roskams, A . J., X . Cai , et al. (1998). \"Expression of neuron-specific beta-Ill tubulin during olfactory neurogenesis in the embryonic and adult rat.\" Neuroscience 83(1): 191-200. Sastry, S. K . and A . F. Horwitz (1996). \"Adhesion-growth factor interactions during differentiation: an integrated biological response.\" Dev B i o l 180(2): 455-67. Satoh, M . and T. Yoshida (1997). \"Promotion of neurogenesis in mouse olfactory neuronal progenitor cells by leukemia inhibitory factor in vitro.\" Neurosci Lett 225(3): 165-8. Schwartz Levey, M . , D . M . Chikaraishi, et al. (1991). \"Characterization of potential precursor populations in the mouse olfactory epithelium using immunocytochemistry and autoradiography.\" J Neurosci 11(11): 3556-64. Schwob, J. E . (2002). \"Neural regeneration and the peripheral olfactory system.\" Anat Rec 269(1): 33-49. Schwob, J. E . , J. M . Huard, et al. (1994). \"Retroviral lineage studies of the rat olfactory epithelium.\" Chem Senses 19(6): 671-82. Schwob, J. E . , K . E . Szumowski, et al. (1992). \"Olfactory sensory neurons are trophically dependent on the olfactory bulb for their prolonged survival.\" J Neurosci 12(10): 3896-919. Schwob, J. E . , S. L . Youngentob, et al. (1995). \"Reconstitution of the rat olfactory epithelium after methyl bromide- induced lesion.\" J Comp Neurol 359(1): 15-37. Seery, J. P. and F. M . Watt (2000). \"Asymmetric stem-cell divisions define the architecture of human oesophageal epithelium.\" Curr B i o l 10(22): 1447-50. Shimamoto, T., K . Ohyashiki, et al. (2000). \"Overexpression of the homeobox gene D L X - 7 inhibits apoptosis by induced expression o f intercellular adhesion molecule-1.\" Exp Hematol 28(4): 433-41. Shimazaki, T., T. Shingo, et al. (2001). \"The ciliary neurotrophic factor/leukemia inhibitory factor/gpl30 receptor complex operates in the maintenance of mammalian forebrain neural stem cells.\" J Neurosci 21(19): 7642-53. Shou, J., R. C. Murray, et al. (2000). \"Opposing effects o f bone morphogenetic proteins on neuron production and survival in the olfactory receptor neuron lineage [In Process Citation].\" Development 127(24): 5403-13. Shou, J., P. C . R i m , et al. (1999). \" B M P s inhibit neurogenesis by a mechanism involving degradation of a transcription factor [see comments].\" Nat Neurosci 2(4): 339-45. 124 Springer, T. A . (1990). \"Adhesion receptors of the immune system.\" Nature 346(6283): 425-34. Springer, T. A . (1994). \"Traffic signals for lymphocyte recirculation and leukocyte emigration: the multistep paradigm.\" Ce l l 76(2): 301-14. Suzuki, Y . , J. Schafer, et al. (1995). \"Phagocytic cells in the rat olfactory epithelium after bulbectomy.\" Exp Neurol 136(2): 225-33. Suzuki, Y . and M . Takeda (1993). \"Basal cells in the mouse olfactory epithelium during development: immunohistochemical and electron-microscopic studies.\" Brain Res Dev Brain Res 73m: 107-13. Suzuki, Y . , M . Takeda, et al. (1996). \"Supporting cells as phagocytes in the olfactory epithelium after bulbectomy.\" J Comp Neurol 376(4): 509-17. Tammi, M . I., A . J. Day, et al. (2002). \"Hyaluronan and homeostasis: a balancing act.\" J B i o l Chem 277(7): 4581-4. T i l l JE and McCul loch E A . (1961). \" A direct measurement of the radiation sensitivity of normal mouse bone marrow cells\". Radiat. Res. 14: 213-222. Timpl , R. (1996). \"Macromolecular organization o f basement membranes.\" Curr Opin C e U B i o l 8(5): 618-24. Turksen, K . and T. C . Troy (1998). \"Epidermal cell lineage.\" Biochem Cel l B i o l 76(6): 889-98. van de Stolpe, A . and P. T. van der Saag (1996). \"Intercellular adhesion molecule-1.\" J M o l M e d 74(1): 13-33. van der Flier, A . and A . Sonnenberg (2001). \"Function and interactions of integrins.\" Cel l Tissue Res 305(3): 285-98. van der Kooy, D . and S. Weiss (2000). \"Why stem cells?\" Science 287(5457): 1439-41. van Pelt, J. P., S. H . Kuijpers, et al. (1998). \"The CD1 l b / C D 18-integrin in the pathogenesis o f psoriasis.\" J Dermatol Sci 16(2): 135-43. Verhaagen, J., A . B . Oestreicher, et al. (1989). \"The expression o f the growth associated protein B50 /GAP43 in the olfactory system of neonatal and adult rats.\" J Neurosci 9(2): 683-91. Watt, F . M . (2001). \"Stem cell fate and patterning in mammalian epidermis.\" Curr Opin Genet Dev 11(4): 410-7. 125 Watt, F. M . , P. Boukamp, et al. (1987). \"Effect of growth environment on spatial expression of involucrin by human epidermal keratinocytes.\" A r c h Dermatol Res 279(5): 335-40. Watt, F . M . and B . L . Hogan (2000). \"Out of Eden: Stem Cells and Their Niches.\" Science 287(5457): 1427-1430. Weinmaster, G . (2000). \"Notch signal transduction: a real rip and more.\" Curr Opin Genet Dev 10(4): 363-9. Weissman, I. L . (2000). \"Stem cells: units o f development, units of regeneration, and units in evolution.\" C d l 100(1): 157-68. Zheng Y W , Taniguchi A , Suzuki K et al. (2000). \"Effects of combined growth factors on clonal growth and albumin secretion o f murine fetal hepatocytes in low density culture\". Transplant Proc 32: 2372. Zhu, A . J., I. Haase, et al. (1999). \"Signaling via betal integrins and mitogen-activated protein kinase determines human epidermal stem cell fate in vitro.\" Proc Natl Acad Sci U S A 96(12): 6728-33. Zuckerman K S , Prince C W , Gray S. (1989). \"The hematopoetic extracellular matrix\". In: Handbook of the hematopoetic microenvironement. Tavassoli M (ed). Humana Press: New Jersey. 126 "@en . "Thesis/Dissertation"@en . "2002-11"@en . "10.14288/1.0090380"@en . "eng"@en . "Neuroscience"@en . "Vancouver : University of British Columbia Library"@en . "University of British Columbia"@en . "For non-commercial purposes only, such as research, private study and education. Additional conditions apply, see Terms of Use https://open.library.ubc.ca/terms_of_use."@en . "Graduate"@en . "Olfactory epithelial horizonal basal cells : an assessment of stem cell candidacy and behavioural regulation in vivo and in vitro"@en . "Text"@en . "http://hdl.handle.net/2429/12405"@en .