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Olfactory epithelial horizonal basal cells : an assessment of stem cell candidacy and behavioural regulation… Carter, Lindsay A. 2002

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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 IN P A R T I A L F U L F I L M E N T OF THE REQUIREMENT FOR THE DEGREE OF  M A S T E R OF SCIENCE  in  T H E F A C U L T Y OF G R A D U A T E STUDIES (The Graduate Program i n Neuroscience)  W e accept this thesis as conforming to the required standard  THE UNIVERSITY OF BRITISH C O L U M B I A July 2002  © Lindsay A . Carter, 2002  Friday, July 26, 2002  UBC Rare Books and Special Collections - Thesis Authorisation Form  i n presenting t h i s t h e s i s i n p a r t i a l f u l f i l m e n t of the requirements f o r an advanced degree at the U n i v e r s i t y of B r i t i s h Columbia, I agree that the L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r reference and study. I further agree that permission f o r extensive copying of t h i s t h e s i s f o r s c h o l a r l y purposes may be granted by the head of my department or by h i s or her representatives. I t i s understood that copying or p u b l i c a t i o n of t h i s t h e s i s f o r f i n a n c i a l gain s h a l l not be allowed without my w r i t t e n permission.  Department of Vancouver, Canada  http://www.library.ubc.ca/spcoll/thesauth.html  Page: 1  ABSTRACT In the olfactory epithelium (OE), new olfactory receptor neurons ( O R N S ) are continually generated throughout mammalian adulthood. Given this substantial neuronal turnover, a stem cell is proposed to reside within the basal compartment o f 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. W e hypothesize that a population o f horizontal basal cells ( H B C s ) , situated upon the basement membrane o f the O E , contains stem cells that contribute to olfactory neurogenesis. A major impediment to the study o f these cells is the lack o f reliable cell surface molecular markers to distinguish them from other O E cell types. B y screening a panel o f selected clusters o f differentiation ( C D ) antigens, we have identified three new cell surface markers for the H B C population, namely intercellular adhesion molecule -1 ( I C A M - 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 o f 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 i n the populational and subcellular distribution o f H B C markers upon loss o f O R N s , suggesting a role for these adhesion receptors i n the regulation o f H B C function in addition to highlighting possible molecular similarities to stem cells o f 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 o f I C A M - 1 .  Using in vitro colony-forming analyses, we obtained evidence  ii  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  +  +  HBCs  contribute to the progenitor cell compartment, possibly as stem cells, during olfactory neurogenesis and that the function o f these cells may be modulated v i a adhesion and growth factor signaling by components resident within their in vivo microenvironment.  iii  T A B L E OF CONTENTS  ABSTRACT  ii  T A B L E OF CONTENTS  iv  viii  LIST OF FIGURES LIST OF TABLES  x  LIST OF ABBREVIATIONS  xi  ACKNOWLEDGEMENTS  xiii  CHAPTER I  Introduction and Research Objectives  1  1.1  The Olfactory System  1  1.2  C e l l Replacement v i a Stem Cells  9  1.3  Manifestations o f Stem Cells i n Three Physiologically Different Tissues. ..16  1.4  The Cellular Constituents o f Olfactory Neurogenesis  19  1.5  The Molecular Regulation o f Olfactory Neurogenesis  25  1.6  The H B C vs. G B C as O E Stem C e l 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 o f Basal Cells  35  iv  2.6  I C A M - 1 Immunornagnetic Selection o f Basal Cells  36  2.7  Test Conditions for Optimizing Colony-Forming Efficiency  38  2.8  Assessment o f Adhesion Kinetics  39  2.9  Immunocytochemistry  39  C H A P T E R III  A n initial screen of C D antibodies reveals three cell surface markers for horizontal basal cells within the mouse olfactory epithelium 41  3.1  Introduction  3.2  I C A M - 1 , P i integrin and P4 integrin are expressed i n 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 o f the horizontal basal cell marker, Keratin 903 45  3.4  a integrins are expressed i n a complimentary fashion to the P i and P 4 integrins ... 45  3.5  C D antigens are expressed within other cell types within the olfactory mucosa 47  3.6  Summary  C H A P T E R IV  41  47  A n in vivo characterization of HBCs: response to bulbectomy and examination of potential stem cell traits 51  4.1  Introduction  4.2  The removal o f the olfactory bulb induces O R N loss and basal cell proliferation i n 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 o f I C A M - 1 , P i and P4 integrins is altered post-bulbectomy 57  51  v  4.6  H B C s display some heterogeneity in the complement o f adhesion receptor they express, an observation which is exaggerated post-bulbectomy 57  4.7  Summary  CHAPTER V  60  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  5.2  Preliminary in vitro findings  5.3  C e l l surface antigen selection and sorting o f H B C s  5.4  The I C A M - 1 density  5.5  Determination o f optimal media conditions with respect to I C A M - 1 forming efficiency  5.6  Effect o f E C M substrate on the overall colony-forming ability o f I C A M - 1 selected cells 71  5.7  Effect o f E C M components on the incidence o f small, medium and large colonies within clonal cultures o f I C A M - 1 cells 73  63  +  64 64  fraction possesses a superior colony-forming ability at clonal 67 +  colony 69  +  5.8  Adhesion kinetics assay on different E C M components demonstrates an overall preference for collagen 76  5.9  Effects o f growth factor addition on overall colony forming efficiency o f I C A M - 1 cells 78 +  5.10 Effect o f growth factors on the incidence o f small, medium and large colonies within clonal cultures o f I C A M - 1 cells +  5.11 Cultured I C A M - 1 phenotypes  +  79  cells produce a mosaic o f differentiated olfactory cell 82  5.12 I C A M - 1 positive colonies contain cells possessing a mature olfactory neuron phenotype 84 5.13 Summary  87  vi  C H A P T E R VI  Discussion  C H A P T E R VII  Concluding Remarks  88  113  BIBLIOGRAPHY  116  vii  LIST O F F I G U R E S  Figure 1.1  The primary olfactory neuraxis  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 o f cells within the O E  Figure 1.4  The classical hierarchy  Figure 3.1  I C A M - 1 , Pi integrin and P4 integrin are detected within H B C s o f adult olfactory epithelium 44  Figure 3.2  H B C expression o f I C A M - 1 is confirmed v i a 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 n o n - H B C cells i n the olfactory mucosa 49  Figure 4.1  The loss o f O R N s following bulbectomy induces proliferation within the basal cell compartment o f the O E 53  Figure 4.2  G B C progenitors are depleted locally within discrete regions o f O E following bulbectomy 55  Figure 4.3  H B C s proliferate i n response to bulbectomy, but remain quiescent relative to robustly proliferating G B C s 56  Figure 4.4  Changes are detected i n the populational uniformity o f 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 o f I C A M - 1 and P4 integrin are altered i n some cells post-bulbectomy, while that o f Pi integrin remains constant...59  Figure 4.6  The observed heterogeneity o f H B C adhesion receptor expression i n normal O E is exaggerated post-bulbectomy 61  Figure 5.1  Preliminary evidence o f progenitor activity i n heterogenous cultures o f OE-derived cells 65  stem  cell->transit  viii  2  amplifier->differentiated  7 progeny 13  Figure 5.2  In vitro immunomagnetic sorting o f H B C s on the basis o f antigenicity  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 o f media condition on the colony forming efficiency o f I C A M - 1 + cells at clonal density 70  Figure 5.5  Effect o f substrate on overall colony forming efficiency o f I C A M - 1 at clonal density  Figure 5.6  Representative small, medium and large colonies at 14 D I V  Figure 5.7  Effect o f substrate on the incidence o f small, medium and large colonies seeded by I C A M - 1 , M A C S selected cells at clonal density 75  ICAM-1 66  +  +  cells 72 74  +  Figure 5.8  M A C S selected I C A M - 1 cells display different kinetics o f adhesion when plated on different substrates 77  Figure 5.9  Effect o f growth factor on the overall colony forming efficiency o f I C A M 1 cells at clonal density 80  +  +  Figure 5.10  Effect o f growth factor on the incidence o f 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 o f 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 o f these colonies possess a mature olfactory neuron phenotype 85  Figure 5.13  Some neurons displayed a distinctly non-olfactory phenotype  ix  86  LIST OF TABLES Table 3.1  Expression and functional properties o f selected C D antigens within stem/progenitor cell hierarchies o f other self-renewing tissues 43  x  LIST O F A B B R E V I A T I O N S  ACIII  adenylate cyclase III  BMP  bone morphogenic protein  BDNF  brain derived neurotrophic factor  BrdU  bromodeoxyuridine  CD  clusters o f differentiation antigens  Cdc2 kinase  cell-division-cycle2 kinase  CFE  colony forming efficiency  CNS  central nervous system  DAPI  4',6-Diamidine-2'-phenylindole dihydrochloride  DIV  day i n vitro  DMEM  Dulbecco's modified Eagle's medium  E  embryonic day  ECM  extracellular matrix  EGF  epidermal growth factor  ES  embryonic stem cell  FBS  fetal bovine serum  FGF  fibroblast growth factor  FGFR  fibroblast growth factor receptor  GAP-43  growth associated protein-43  GBC  globose basal cell  G-Olf  olfactory G-protein  GFAP  glial acidic fibrillary protein  3  H  tritiated thymidine  HBC  horizontal basal cell  ICAM-1  intercellular adhesion molecule-1  IL-lp  interleukin-lp  INP  immediate neuronal precursor  xi  IRN  immature receptor neuron  K-SFM  keratinocyte serum-free media  LFA-1  leukocyte function associated-1  MACS  magnetic activated cell sorting  Mash-1  mammalian achaete scute homologue  LIF  leukemia inhibitory factor  LIFR  leukemia inhibitory factor receptor  LP  lamina propria  NCAM  neural cell adhesion molecule  NT-3  neurotrophin-3  NGF  nerve growth factor  NST  Neuron specific tubulin  OE  olfactory epithelium  OEG  olfactory ensheathing glia  OMP  olfactory marker protein  ORN  olfactory receptor neuron  P  postnatal day  PBS  phosphate buffered saline  PFA  paraformaldehyde  RT-PCR  reverse transcriptase polymerase chain reaction  SE  standard error  TA  transit amplifying cell  TGF-a  transforming growth factor-a  TGF-(i  transforming growth factor-P  VLA-4  very late activating antigen-4  VLA-5  very late activating antigen-5  ZnSCv  zinc sulphate  xii  Acknowledgements Firstly, I would like to thank my husband, Derrick, who specifically asked that I refer to h i m 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 i n her lab. Finally, I would like to thank the R i c k Hansen Institute for awarding me a neurotrauma studentship. Lastly, I would like to dedicate this work to the memory o f my mother.  A s threshing separates the wheat from the chaff, so does affliction purify virtue. Sir Richard Francis Burton  xiii  C H A P T E R I: Introduction and Research Objectives  1.1  T h e Olfactory System The olfactory epithelium (or O E ) , a columnar, pseudo-stratified epithelium  located peripherally within the nasal cavity, is both the birthplace and residence o f mature olfactory receptor  neurons  (ORNs) (Figure 1.1; Graziadei and Graziadei, 1979a;  Graziadei and Graziadei, 1979b; Moulton, 1974; Suzuki and Takeda, 1993). O R N s , the singular neuronal cell type o f the O E , generate nerve impulses upon interaction with chemical odorants detected in the environment. A s such, O R N s are crucial for our sense o f smell. The dendrites o f O R N s 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 o f 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 o f the C N S (Doucette, 1990; Doucette, 1991; Farbman, 1992). A s a consequence o f 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 o f about 4-6 weeks (Farbman, 1992; Murray and Calof, 1999). Environmental, as opposed to genetic, determination o f O R N lifespan is supported by the finding that animals housed i n a "dirty" environments exhibit higher rates o f O R N turnover than do those residing in sterile cage environments (Hinds et al, 1984). In order to cope with this innate vulnerability o f O R N s , a mechanism, unique i n the mammalian nervous system, exists to actively regenerate O R N s lost due to environmental insult in order to sustain the critical sensory function o f olfaction.  1  F i g u r e 1.1: T h e p r i m a r y 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 o f the olfactory bulb. (Margolis et al., 1991).  2  The O E is akin to other epithelia in that it displays a topographical gradient o f maturity, with the most primitive, developmentally immature cells situated at the base o f the O E , while functionally mature cell types are located i n the upper reaches o f the epithelium with intermediates existing in between.  The cell types o f the O E can be  distinguished from one another by either cell morphology, relative position i n the O E , or by the differential expression o f protein markers (Schwob, 2002). subdivided into three compartments:  The O E can be  the apical, middle and basal compartments as  diagrammed i n Figure 1.2 (Calof et al., 1998). The apical layer is composed o f nuclei belonging to a single cell type, the sustentacular, or supporting, cells.  In the rat O E ,  sustentacular cells comprise roughly 15-20% o f the total epithelium (Farbman et al., 1988). These elongated macroglial cells contact the basement membrane at the base o f the O E and extend microvilli out into the nasal cavity, thereby spanning the entire O E proper i n 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 o f the passage o f compounds between the O E proper and its underlying lamina propria (Rafols and Getchell, 1983). These cells are identified by their expression o f cytokeratins 8 and 18 and several monoclonal antibodies o f unidentified antigenicity (Goldstein and Schwob, 1996; Goldstein et al., 1997; Jang and Schwob, 2001). The next section o f O E , the middle compartment, contains the cell bodies o f olfactory receptor neurons, both immature and fully functional, which can be further segregated according to maturity such that immature O R N s 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 O R N s (ORN). The basal compartment contains horizontal ( H B ) and globose ( G B ) basal cells. In this diagram, globose basal cells are further sub-divided into neuronal colony forming units ( C F U ) , Mash-1 expressing cells ( M l ) , and immediate neuronal precursors (INP). (Calof et al., 1998)  4  O R N s . Both stages o f O R N constitute 75-80% o f the rat O E (Farbman et al., 1988). The period o f 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 M o n t i Graziadei, 1982; Schwob et al., 1992). O R N s are bipolar cells and can be identified, i n general, by the expression o f neural cell adhesion molecule ( N C A M ) (Miragall et al., 1988; Caggiano et al., 1994; C a l o f and Chikaraishi, 1989). T o 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 o f antibodies is utilized to identify  mature olfactory neurons, including olfactory marker protein ( O M P ) , and components o f the olfactory signal transduction cascade, most commonly adenylate cyclase III and G - o l f (Margolis, 1972; Keller and Margolis, 1975). Lastly, the base o f the O E consists o f the basal compartment and houses the proliferative region o f 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  ( G B C s ) and horizontal basal cells (HBCs), which together account for no greater than 10% o f 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 v i a their expression o f cytokeratin 5/6 (Calof and Chikaraishi, 1989; Suzuki and Takeda, 1993). In earlier studies o f the O E , G B C s were  5  defined on the basis that they were negative for antigenic markers o f neighbouring cell types.  For example, an O E cell situated within the basal portion o f 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 o f the antigens that these  " G B C " antibodies recognize are as yet unknown, they serve as the only global markers o f the G B C population. The G B C layer can be further subdivided according to function and antigenicity. The immediate neuronal precursor (FNP) o f the O E is a G B C that expresses the neuronal differentiation transcription factor neurogeninl (Cau et al., 1997). The socalled Mash-1-expressing cell is believed to function as a transit amplifying cell situated upstream from INPs and downstream o f a hypothesized O E stem cell (Gordon et al., 1995; C a u et al., 1997). T o 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 o f the O E ' s regenerative capacity. Suggested H B C supporting roles include a function i n the maintenance o f O E tissue integrity and a role as signaling cells to report the status o f O R N s , via their proximity to O R N axon bundles, to the overlying O E (Farbman, 1992; Holbrook et al., 1995). The cell types o f the O E , together with their phenotypical markers are diagrammed in Figure 1.3. The lamina propria (LP) o f the olfactory mucosa, situated beneath the O E proper, houses a diversity o f cell types which function i n the support o f O R N related activities, including a unique class o f glia, the olfactory ensheathing glia (OEGs), and most connective tissue cell types, such as mast cells, leukocytes, macrophages and fibroblasts.  6  HBC Keratin 5/6  GBC Immature GBC-1, ORN -2, and N C A M -3 NST GAP-43 TrkB  Mature ORNs OMP ACIII G-olf NCAM  Sustentacular cell Sus-4 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 o f 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 O R N s are recognized by olfactory marker protein ( O M P ) , adenylate cyclase III (ACIII), and the olfactory g-protein, G-olf. Neural cell adhesion molecule ( N C A M ) is expressed i n both immature and mature O R N s . Finally, sustentacular cells are identified by their expression o f keratins 8 and 18, i n addition to Sus-4, a monoclonal antibody of unknown antigenicity. (Farbman, 1992)  O E G s are o f particular interest on account o f their growth-promoting properties (RamonCueto and A v i l a , 1998). Through their entire length from O E to olfactory bulb, O R N axons are i n continual contact with O E G s .  O E G s ensheathe O R N s to provide a  permissive substrate for growth, pathfinding guidance to the olfactory bulb and trophic support (Ramon-Cueto and A v i l a , 1998).  In stark contrast to the primary olfactory  neuraxis, the mature C N S is not permissive o f 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.  O E G s can be discriminated both in vivo  and in vitro by the expression o f glial fibrillary acidic protein ( G F A P ) , the calciumbinding protein SlOOp or P75 (Ramon-Cueto and A v i l a , 1998). Other structural elements o f the L P include the Bowman's glands, which secrete mucus exterior to the O E v i a 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 o f O R N s ' innate situational vulnerability, providing both protective and renewal mechanisms to ensure that the sense o f olfaction remains intact. One question that has received much attention in the field o f olfaction is the ultimate source o f new ORNs.  L i k e other self-renewing tissues, the O E is proposed to contain a stem cell that  serves as the basis o f 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 o f  causes, ranging from a simple unsuitability o f the cell's morphological form, as is the case with neurons, to an incompatibility o f 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 o f 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 o f cellular behaviours reminiscent o f 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 proceeds until they effectively disappear via terminal differentiation.  development  In contrast, stem  cells are everlasting with respect to the lifespan o f an organism (Hall and Watt, 1989; Potten and Loeffler, 1990). This is an essential stem cell characteristic as the requirement for the generation o f new, functionally mature cells proceeds throughout adult life.  The  cell biological basis o f this classical stem parameter is a proliferative process termed selfrenewal, whereby stem cells divide in order to maintain their persistence in the organism. Although there is often much dispute surrounding the exact definition o f 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 o f stem cell parameters. Counterintuitive to their high proliferative potential, at any given singular moment o f  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 o f 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 o f 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 o f their resident tissue. To summarize, the ability to proliferate, self-renew, and to generate differentiated daughter cells are the hallmarks o f 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 i n addition to true committed progenitors (Hall and Watt, 1989; Potten and Loeffler, 1990). Philosophical definitions o f 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 i n molecular makeup (Potten and Loeffler, 1990). However this is often considered a weak parameter o f stem cells, as it is only relative, in a qualitative manner, to their mature descendants.  Another trait is that o f stem pluripotency, or the ability to  produce a range o f differentiated cell phenotypes (Hall and Watt, 1989; Potten and  10  Loeffler, 1990).  Most tissues contain a variety o f specialized cells, which i n their  maturity serve as effectors o f the function o f a particular tissue. M a n y stem cells possess the ability to regenerate all o f 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 o f 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 o f mitosis affect the outcome o f a particular cell division, such that a symmetric division results i n either two stem cells or two more mature cells, while an asymmetric mitotic event yields one o f each (Potten and Loeffler, 1990; Gage, 2000). In very early studies o f 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 o f proliferation in vivo are transitory, committed intermediates, termed transit amplifying cells (Potten and Loeffler, 1990).  A s such, regenerative  pathways i n self-renewing tissues are initiated by stem cells and proceed through committed transit amplifying progenitor stages to culminate with the production o f 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).  11  The  primary function o f 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 o f differentiated progeny (see Figure 1.4).  In addition, the presence o f transit amplifying  cells within the stem cell hierarchy serves to segregate different routes o f commitment, such that one population o f 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 o f stem cell to mature progeny function (Potten and Loeffler, 1990). capacity:  The first such gradient involves proliferative  stem cells display the highest degree o f this property, while terminally  differentiated cells at the bottom o f the hierarchy are incapable o f cell division. Transit amplifying cells, while capable o f proliferation, have a finite number o f possible mitotic rounds.  Further, the differentiative capacity decreases i n a stepwise fashion down the  hierarchy. Stem cells generally possess the potential to produce a variety o f functionally mature descendants, while transit amplifying cells display progressively less potential i n this regard as the hierarchy nears its mature, differentiated endpoint (Potten and Loeffler, 1990). In order to ensure the continual fulfillment o f 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 o f 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 o f 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 E G F and T G F - 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  14  +  hematopoietic  progenitor cells to fibronectin and vascular adhesion molecule ( V C A M ) , both found i n 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 o f an anti-o^Pi antibody to long-term cultures o f bone marrow cells causes an inhibition o f the myeloid and lymphoid lineages (Miyake et al., 1991). These antibodies also result in the appearance o f hematopoietic progenitors within the peripheral blood, likely as a consequence o f lost adhesion to the bone marrow stroma (Papayannopoulou and Nakamoto, 1993). The final major component o f the stem cell niche is intercellular contact, whereby adjacent cells influence one another via cell surface protein interaction. A n example o f this class o f stem cell control is provided by the Notch family o f receptors.  Notch  receptors, present on the surfaces o f subsets o f cells, bind their ligands, present on neighbouring cells (Weinmaster, 2000).  Both receptor and ligand are transmembrane,  cell surface proteins. U p o n ligation, Notch elicits signaling pathways within the cell to regulate gene expression. In parallel to other studies o f Notch action within stem cells, Hitoshi and colleagues (2002) recently demonstrated that Notch activation functions in the maintenance o f 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 o f the three modes o f 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 o f stem cell activity i n their in vivo environment w i l l permit the development o f 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 o f stem cells noted above, three different  stem cell systems, at various degrees in their characterization,  discussed below.  are  Hematopoiesis, the process o f blood cell formation, is the most  characterized o f all adult stem cell systems.  Early in the study o f 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 M c C u l l o c h , 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 o f 9 separate lineages  (Chan and Watt, 2001).  The magnitude o f this process is highlighted by the  estimate that homeostasis requires the production o f 1 0 (Domen, 2001).  11  blood cells per day i n humans  The primitive adult stem cells o f 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 o f the hematopoietic lineages clearly illustrates the importance o f 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 o f 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 o f 4  principle cellular layers which differ in their degree o f differentiation, with the least mature cells located i n apposition to the basement membrane and the most mature located in the upper layer o f the epidermis (Jones, 1997). The stem cells within this system are believed to reside within the population o f basal cells in close apposition to the basement membrane at the base o f the epidermis (Turksen and Troy, 1998). The most commonly utilized marker o f these cells is p i integrin, which is expressed at high levels on the surfaces o f 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 o f 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 o f extracellular protein matrices that are  17  found surrounding or adjacent to a wide variety o f cells (Timpl, 1996). A key function o f basement membranes is to compartmentalize different types o f tissue like, for example, the epidermis and the mesenchyme beneath it, i n order to prevent cell mixing.  More  importantly i n the context of this discussion, the basement membrane functions as a platform upon which the regulation o f 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 I V collagen, and heparan sulphate proteoglycan (Watt, 1987). It is these E C M proteins, concentrated within the basement membrane, that provide the majority o f 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 o f 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 postmitotic.  These cells, when cultured in the absence o f any coating substrate together with  E G F , form clonal cell aggregates termed neurospheres (Reynolds and Weiss, 1996). Spontaneous differentiation o f these spheres yields a number o f 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 o f these neural stem cells is the lack o f 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 o f 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 o f replacing tissue lost to cell death or injury. Rather, the consensus concerning the in vivo function o f neural stem cells within the mammalian C N S is that they produce neurons to add onto the existing cytoarchitecture o f areas o f 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 i n the O E i n order to replace neurons that are continually lost due to environmental influence (Calof et al., 1998; Schwob, 2002). Initial studies directed towards the identification o f possible O R N stem/progenitors utilized H-thymidine incorporation coupled with autoradiography 3  (Moulton et al., 1970: Graziadei and Metcalf, 1971; Graziadei, 1973; Moulton, 1974; Graziadei and M o n t i Graziadei, 1978). A t early time-points after incorporation, labeled  19  nuclei were present within the basal cell compartment o f the O E . Later, however, the H 3  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 o f laboratories have contributed to the notion that neurogenesis i n the O E , as i n other stem cell systems, is an exquisitely regulated process (Schwob, 2002).  In the normal, unperturbed animal, olfactory neurons undergo a  continual cycle o f death and rebirth from basally located precursors, as noted above. Although the rate at which this cycle occurs is slow i n the normal animal, several techniques  have  been  developed  to  dramatically  enhance  the  pace  of  degeneration/regeneration in the O E , thereby easing the dissection o f events that affect the regulation o f olfactory neurogenesis. A t present, there exist three general methods o f inducing the degeneration o f olfactory neurons:  1) chemical destruction o f the O E by various agents (e.g. methyl  bromide inhalation, Triton X - 1 0 0 or ZnS04 infusion; 2) transection o f the olfactory nerve; and 3) removal o f the olfactory bulb (also termed olfactory bulbectomy) (Costanzo, 1984, 1985; Costanzo and Graziadei, 1983; M o n t 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 o f degeneration/regeneration.  A l l three  result i n the immediate or eventual destruction o f O R N s . The bulbectomy and nerve transection procedures eliminate only O R N s , 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 i n both the number o f dying cells and the number o f proliferating basal cells post-lesion, although the kinetics o f 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 i n 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 o f cell types (fully 90% o f 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 o f 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 i n bulbectomized animals demonstrated a clear relationship between O R N s 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 o f important conclusions regarding tissue reconstitution i n 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 o f 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 o f the lineage unsolved. The first distinct stage o f the olfactory hierarchy was also examined using an embryonic O E explant culture system coupled with [ H]-thymidine uptake analysis and immunohistochemical identification o f cell types (Calof and Chikaraishi, 1989). immediate  neuronal precursors  (INPs) express  the neuronal differentiation  The 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 o f transit amplifying cells. A further contribution o f the G B C population to olfactory neurogenesis  was illuminated by a study o f Mash-1 knockout mice  (Guillemot et al., 1993).  Mash-1 is a transcription factor that is expressed within a  subpopulation o f G B C progenitors, located upstream o f 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 i n the number o f 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 o f 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 o f stem cells and are often instructive  22  in determining the extent o f intrinsic versus extrinsic regulation o f 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 o f 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 ( O M P ) immunopositivity. This class o f donor cell did not produce cells with a C N S phenotype, while the scattered, individual cells were capable o f differentiating into neurons and glia with a central phenotype, as determined via immunohistochemistry and electron microscopic examination o f morphology.  A s such, it was concluded that the lineage  specification o f the O E donor cells is dependent upon cellular interaction between hostdonor and donor-donor pairings (Magrassi and Graziadei, 1996). One criticism o f the above study is that the donor cells were not purified. Rather, a heterogenous "slurry" o f 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 o f dividing cells with a L a c Z vector at a time post-bulbectomy when the majority o f 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 o f bulbectomized hosts, only neurons were generated. This study, and that noted in the preceding paragraph, indicate differences i n the differentiative potential o f 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 o f growth factors and cytokines on olfactory neurogenesis as a whole.  There are presently very few published reports  concerning the first group o f examination. Most notably, M u m m and colleagues (1996) developed a neuronal colony-forming assay to test for stem and progenitor cells i n dissociated primary cultures o f embryonic O E .  To enrich for progenitors,  NCAM  +  neurons were removed from O E cell suspensions via immunological panning. When plated i n adherent culture, >95% o f 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 o f 5 or more cells, which contain subsets o f cells that express N C A M .  Using thymidine incorporation i n tandem with  N C A M immunocytochemistry, it was demonstrated immediate neuronal precursors (INPs) o f O R N s .  that these colonies contain the In addition to INPs, the authors  proposed the existence o f rare cells in these cultures, which are capable o f prolonged neurogenesis up to 7 days in vitro ( M u m m et al., 1996).  24  The second category o f in vitro examination involves the analysis o f secreted factors on the regulation o f olfactory neurogenesis.  A t their endpoints, these studies  typically quantified the total numbers o f neurons or the number o f thymidine analogincorporating cells i n order to determine the effects o f the substances supplied to O E cell cultures. semi-  The nature o f the starting cultures is varied, ranging from primary explant,  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 o f stem or progenitor cells within the O E . The results o f these assays are discussed further below.  1.5  T h e M o l e c u l a r Regulation of Olfactory Neurogenesis Olfactory neurogenesis appears highly regulated, such that the production o f 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 o f neurogenesis is influenced by the density o f neurons i n 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 o f some feedback mechanism that halts the generation o f neurons when no neuronal replenishment is required ( M u m m e t a l . , 1996). These initial, broad observations incited a more detailed examination o f the control o f olfactory neurogenesis. M u c h information has been revealed by in vivo and in vitro experimentation regarding the role o f growth factors and cytokines i n the control o f this regenerative process.  25  Several studies have implicated fibroblast growth factor-2 (FGF-2) i n the control o f 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 F G F r - 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 o f 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 o f F G F - 2 may permit the finetuned control o f the number o f O R N s produced by regulating the rounds o f 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 ( E G F ) and transforming growth factor-oc ( T G F - a ) are two additional examples o f growth factors that exert mitogenic effects upon O E cells. In the O E , T G F - a is present i n 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 ( E G F R ) , which  binds both E G F and T G F - a , is located within the H B C population i n rats (Farbman and Bucholz, 1996). The mitogenic effects o f 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 o f 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  26  al., 2000).  This finding is  complimented by in vitro studies, which have shown that proliferation is stimulated upon the addition o f T G F - a to explant, semi- and fully-dissociated cultures o f O E cells (Mahanthappa and Schwarting, 1993; Farbman and Bucholz, 1996). A further example o f growth factor regulatory influences is provided by leukemia inhibitory factor (LIF), neurogenesis.  which exerts an apparent stimulatory effect on olfactory  The L I F receptor expression is upregulated on G B C s following the  neuronal loss induced by bulbectomy (Nan et al., 2001). In vitro, L I F enhances G B C proliferation i n primary cultures o f O E cells (Satoh and Yoshida, 1997). Hence, several factors are known to positively influence the generation o f neurons within the O E . In contrast to the stimulatory influences o f the above growth factors, bone morphogenic proteins ( B M P s ) are believed to negatively regulate olfactory neurogenesis (Shou et al., 1999). U s i n g a neuronal colony-forming assay, it was determined that when B M P s 2, 4, or 7 are added to dissociated cultures o f embryonic O E , neurogenesis is inhibited (Shou et al., 1999). To determine which stage o f 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 o f 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 i n M A S H - 1 immunoreactivity in O E explants, a result that prompted an examination o f the cause o f M A S H - 1 disappearance (Shou.et al., 1999). It was revealed that exposure to B M P s caused a flurry o f new gene expression which resulted in the proteolysis o f the M A S H - 1 transcription factor, thereby terminating the progression o f the neuronal lineage in those cells (Shou et al., 1999). Although B M P s are expressed i n O E tissue, their exact cellular localization is unknown (Shou et al., 2000).  27  In addition to molecules that serve i n policing proliferation o f O E progenitors, several growth factors function in promoting the neural phenotype. factor  is  transforming  growth  factor-13  (TGF-(3).  TGF-P  One such growth  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 o f brain-specific growth factors, the neurotrophins, also influence the progression o f neuron production i n the O E . The neurotrophins are a family o f peptides which exert their effects by interacting with high and l o w affinity receptors on the surfaces o f responsive cells, and have been implicated in several stages o f neuronal development throughout the central and peripheral nervous systems. N G F , B D N F , and N T - 3 likely regulate aspects o f 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 o f their resident tissue, which secrete signals to instruct the appropriate mode o f action according to current needs. However, i n contrast to growth factor and cytokine signaling i n the O E , the role o f adhesive and intercellular regulation o f olfactory neurogenesis has remained largely unstudied.  One study, however, noted that when  cultures o f O E cells are biochemically or mechanically stressed, the disruption o f cell surface contacts results in the promotion o f differentiation in primary O E cultures (Feron et al., 1999). Hence, given the roles o f these means o f cellular communication i n the regulation o f stem cells in other self-renewing tissues, it is likely that intercellular and adhesive modes o f signaling are active in regulating olfactory neurogenesis.  28  1.6 T h e 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 o f the O E . Retroviral lineage and in vitro studies have confirmed that a subset o f G B C s function as immediate neuronal precursors (INPs) and suggest the presence o f a slightly more primitive transit amplifying subset o f G B C s , located immediately upstream o f 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 o f 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 o f 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 o f ventral O E (Holbrook et al., 1995; Schwob et al., 1995). However, one could argue that the poor antigenic characterization o f these two cell types has influenced the above conclusion. Alternatively, with respect to the embryonic situation, proponents o f the H B C hypothesis argue that recent developments i n the non-olfactory stem cell field indicate that stem cells may arise in late development, just i n time to initiate their role as regulators o f post-developmental tissue homeostasis (van der K o o y 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 K o o y and Weiss, 2000).  In contrast, those who favour the H B C argument draw support for their  hypothesis from the discovery o f cells intermediate in morphology to H B C s and G B C s (Graziadei and M o n t i Graziadei, 1979; Holbrook et al., 1995).  29  Nonetheless, it is apparent upon surveying the extant olfactory literature that relatively little assessment o f stem cell traits has been performed with respect to either population o f cells, although a considerable amount has been revealed regarding the progenitor role o f 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 o f only a few rounds o f cell division prior to terminal differentiation (DeHamer et al., 1994). In addition, although multipotency o f the proposed stem cell is apparent from transplantation and lineage analyses (Caggiano et al., 1994; Magrassi and Graziadei, 1996; Goldstein et al., 1998; Huard e t a l . , 1998), this has not been shown in vitro from an isolated candidate O E cell. Furthermore, there has been a lack o f reliable cell surface markers to fractionate candidate stem and/or progenitor cells i n order to assay these cells individually. A s such, culture methods,  at their most  selective, are  limited  to negative  selection v i a  immunopanning utilizing N C A M to remove O R N s (DeHamer et al., 1994). Otherwise, heterogenous cell suspensions or explants derived from the O E and consisting o f an array of O E cell types have typically been used to test for neurogenic ability i n the O E . A s such, the field o f olfactory neurogenesis has much to gain by mirroring the methods utilized in the study o f 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 ( H B C ) layer o f the olfactory epithelium (OE) contains stem cells that contribute to olfactory neurogenesis.  30  The aims o f the current study were 4-fold: 1) To identify new antigenic markers o f the H B C population v i a a screen o f antibodies generated against clusters o f differentiation (CD) antigens o f interest; 2) To develop methods for the enrichment o f H B C s in vitro; 3) To assess H C B stem/progenitor cell candidacy, both in vivo and in vitro, v i a the examination o f 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.  2.1  Materials and Methods  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 v i a 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., D o n M i l l s Ontario) to prevent invasion o f 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 o f complete lesion was verified both visually and microscopically/After recovery from anaesthesia, animals were maintained according to standard A n i m a l 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 i n 4% paraformaldehyde for  two hours, and then sequentially bathed i n 10% and 30% sucrose to cryoprotect. Tissue was embedded i n plastic moulds with O C T compound (Tissue-Tek, Baxter, Columbia, M D ) over liquid nitrogen. Cryostat sections (10-14 mm) were then cut and frozen until needed.  ,  32  2.2 B r d U 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 i n 4 M HC1 was required before the incubation with primary antibody.  2.3 Immunohistochemistry Frozen sections were rehydrated i n P B S for 5 minutes, permeabilized i n 0.1% Triton-X 100 (Sigma) i n P B S for 20 minutes, blocked with 4% normal serum, and incubated at 4°C for 12-20 hours i n 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 ABC  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, O R ) . In some cases, a 10 minute incubation with 4',6-Diamidine-2'-phenylindole dihydrochloride ( D A P I , 1:15 000; Roche) was performed to visualize nuclei. Slides were then  coverslipped  with  aquapolymount  mounting  media  (Polysciences)  fluorescently labeled, with Vectashield mounting media (Vector Laboratories).  33  or,  if  In all  instances, negative controls, consisting o f an incubation i n 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 o f the same donor species, or evidenced no staining when utilized on irrelevant tissue or cells. A n additional 10 minute incubation i n 0.1 M N a O H step was added prior to the Triton X permeabilization step for the a integrin antibodies, i n order to increase signal.  2.4 Antibodies The following primary antibodies were utilized for monoclonal unconjugated  mouse  immunohistochemistry:  anti-BrdU G 3 G 4 (1:500; Developmental  Studies  Hybridoma Bank, Iowa city, I A ) , monoclonal hamster anti-mouse I C A M - 1 ( C D 5 4 ; 1:100; Pharmingen,  San Diego C A ) rat  anti-mouse  l:100;Pharmingen), monoclonal rat anti-mouse  P i integrin subunit  (CD29;  P4 integrin subunit ( C D 104; 1:100;  Pharmingen), monoclonal rat anti-mouse C D 3 4 (1:100; Pharmingen), monoclonal rat anti-mouse  C D 4 3 (1:100, Pharmingen), monoclonal rat anti-mouse  CD44  (1:100,  Pharmingen), monoclonal mouse anti-rat Beta-Ill neuron-specific tubulin ( N S T ; 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 S100p  (1:1000;  Sigma), polyclonal rabbit  anti-mouse  P75  (1:1000;  anti-bovine 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 G a s / 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 A l e x a 488 and A l e x a 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 o f postnatal day (P) 5-9 C D - I mice into lOmLs o f D M E M / F 1 2 (1:1; Gibco) pre-warmed to 37 °C. 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 m m i n size, and spun at 1 l g for 10 minutes to reduce the number o f fibroblasts in the desired cell fraction. The supernatant was aspirated and lOmls o f 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 i n 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 i n a 37°C water bath with occasional swirling to resuspend  tissue. Initial experiments  determined that these digestion  conditions produced the best yield o f basal cells i n culture.  Following enzymatic  dissociation, the suspension was triturated and filtered through a sterile 80 urn wire mesh to remove larger pieces o f 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.  C e l l fractions from each o f 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 o f basal cells was present among cells remaining on top o f the 40 um cell strainer. These cells were used for initial plating experiments, prior to the selection o f I C A M - 1 cells using M A C S . +  2.6 I C A M - 1 Immunomagnetic Selection of B a s a l Cells: The culture method utilized for immunomagnetic selection o f basal cells was the same as that detailed above for initial experiments, except that the post- 80 u m suspension was treated i n 2 m M E D T A i n P B S for 10 minutes at 37°C 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 i n 2 % F B S i n P B S for 10 minutes at room temperature to prevent non-specific binding o f 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 o f biotinylated anti-ICAM-1 (CD54) antibody (Pharmingen) i n P B S for 30 minutes on ice. The cells were washed twice i n 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° 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 o f 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. C e l 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 ; Roche). Cells were incubated at 37° C with 2  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  5 0 X magnification. Colony-forming efficiencies  determined by dividing the number o f total colonies scored by the number o f cells  37  were  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 i n triplicate for each independent experiment.  ICAM-  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 L I F (20 ng/mL; Sigma), individually and i n 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 i n  triplicate for each independent experiment at a density o f 6000 cells per collagen-coated 10 c m dish. Extracellular matrix components tested for colony-forming efficiencies o f I C A M - 1  +  cells: 10 c m dishes were coated with collagen (5 ug/cm ; Roche), laminin (3 ug/cm ; 2  Roche), fibronectin (5 ug/cm combinations.  ;Roche) and laminin/collagen and fibronectin/collagen  The concentrations o f collagen, fibronectin, and laminin used for the  substrate mixture experiments were as follows: laminin  2  (2collagen.T laminin),  1.7ug/cm  2  38  3.4 ug/cm  collagen  and  collagen and 1 ug/cm 2  ug/cm  2  laminin  (lcollagen:21aminin), 3.4 u.g/cm collagen and 1.7 ug/cm fibronectin, and 1.7 ug/cm 2  2  collagen with 3.3 ug/cm fibronectin. I C A M - 1  2  cells were plated i n 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 and 1.1 X 10 cells +  4  s  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). M e d i a 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 ° C . 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 i n 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 S P O T camera (Diagnostic Instruments Inc., Sterling Heights M I ) with Northern Eclipse software (Empix Imaging Inc., Mississauga, O N ) and were compiled using Adobe Photoshop 6.0.  40  C H A P T E R III. A n initial screen of C D antibodies reveals three cell surface markers for horizontal basal cells within the mouse olfactory epithelium  3.1  Introduction The main focus o f this study was to characterize horizontal basal cells ( H B C s ) i n  order to determine whether they may function as stem cells within the mouse olfactory epithelium (OE). A common impediment to the study o f adult neural stem cells is a lack o f 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 o f antigenic markers o f differentiation in conjunction with the presence o f cytokeratin 5/6, an often unreliable intermediate filament marker (Calof and Chikaraishi, 1989; Suzuki and Takeda, 1993). The dearth o f dependable molecular markers for neural stem cells differs from the situation within the epidermal and hematopoietic systems, where populations enriched i n stem cells or committed transit amplifying cells can be distinguished on the basis o f marker expression both in vivo and in vitro (Fuchs and Segre, 2000; Weissman, 2000). Furthermore, given that many o f 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 o f this study were focused at revealing potential cell surface antigens o f the olfactory H B C population v i a an immunohistochemical screen o f selected clusters o f differentiation ( C D ) antigens.  These antigens were chosen according to their reported expression and  function on stem and progenitor cells o f different origins.  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 To identify new cell surface markers for the H B C layer, an immunohistochemical  screen o f select C D antigens was performed.  Antibodies included i n the screen were  selected from a list o f mouse C D antigens and were chosen on the basis o f reported function  in  regulating  proliferation  and/or  differentiation  and  expression  in  stem/progenitor cells o f other systems (Table 3.1). Immunohistochemistry was performed on P F A fixed coronal sections o f adult O E . Intercellular adhesion moleule-1 ( I C A M - 1 ; C D 5 4 ) is uniformly expressed within the bottom-most basal cell layer o f the O E (Figure 3.1, A ) . Immunoreactivity is confined to those cells situated directly on top o f 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 o f I C A M - 1 immunoreactivity, with respect to individual basal cells, appears to represent a homogenous, pericellular distribution encompassing the entire surface o f 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 o f the P i integrin signal was markedly weaker than those o f I C A M - 1 and P 4 integrin.  Furthermore,  i n contrast to the apparent "full  cell"  distribution o f the I C A M - 1 signal, P i and P4 integrin immunoreactivity appeared to be concentrated to the basal surfaces o f these cells.  Negative, secondary antibody only  controls for I C A M - 1 and the integrins are represented i n Figure 3.1, D and E , respectively.  42  C D antigen  CD29 (Pi integrin subunit)  Recognizes subsets of stem and/or progenitor cells in: hematopoiesis; epidermis; prostate epithelium  CD34  hematopoiesis  CD43 CD44  hematopoiesis hematopoiesis; epidermis; prostate epithelium  CD54 (ICAM-1)  hematopoiesis; expressed in response to injury i n epidermal keratinocytes  Adhesion; regulation o f 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 o f proliferation, survival, differentiation and survival  Reported function of interest  regulation o f proliferation, differentiation, survival; most commonly used marker for epidermal stem cells Adhesion; a common marker for hematopoietic progenitors adhesion Extracellular adhesion; binds hyaluronic acid; tumour metastasis  Table 3.1: Expression and functional properties of selected C D 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 o f selected C D antibodies detected three cell surface proteins upon horizontal basal cells ( H B C s ) , situated on top o f 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 i n a continuous manner upon H B C s (arrows). Immunoreactivity for Pi and P 4 integrin subunits was also detected in the lamina propria ( L P ) i n a pattern indicative o f 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 n o n - H B C 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 o f olfactory ensheathing glia (OEGs) (Ramon-Cueto and A v i l a , 1998).  3.3  I C A M - 1 expression directly overlaps with that of the horizontal basal cell marker, Keratin 903 A s the bulk o f all ensuing experiments directed at testing the H B C population for  stem characteristics hinges on the correct identity o f 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 o f keratin (B; overlap in C with D A P I nuclear stain i n 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 o f 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 o f the a integrin subunits.  45  F i g u r e 3.2: H B C expression o f I C A M - 1 is c o n f i r m e d v i a K e r a t i n 903 colocalization. The I C A M - 1 signal ( A ) directly overlaps with the H B C marker keratin 903 (B); merged i n (C),with D A P I nuclear stain, confirming the identity o f the I C A M - 1 population. Magnification 4 0 0 X . +  46  W e detected expression o f oci, 0 C 3 , and ct6 integrin subunits upon the surfaces o f 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 ( O R N ) axons within the axon bundles o f the L P . In addition, 0C6 integrin subunit was expressed within cells around the perimeter o f axon bundles, likely olfactory ensheathing glia. Negative, secondary antibody only control is shown i n 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 o f C D antigens amongst other cell types within the olfactory mucosa. C D 3 4 is detected within endothelial cells o f L P blood vessels (Figure 3.4, A ) . C D 4 3 expression is confined to the L P in single punctate cells scattered throughout. The expression pattern o f C D 4 3 is akin to that o f macrophages resident beneath the O E proper (Suzuki et al., 1995). However, due to the frequency o f positive cells observed, it is likely that other L P cells express CD43 as well. Finally, C D 4 4 is present in a pattern similar to that o f C D 4 3 , 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 o f these C D 4 3 cells are unknown, though they likely +  represent macrophages.  3.6 Summary In order to identify new cell surface markers o f horizontal basal cells ( H B C s ) , an immunohistochemical screen o f 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), P i 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. W e wished to identify potential a subunit binding partners for the |3 submits found upon H B C s . a , , a , and a integrin subunit expression is likewise detected on H B C s ( A , B , C ; arrows). In addition, a subunit expression is present within O R N axons ( B ; arrowhead), while a subunit immunoreactivity is detected i n O E G s surrounding axon bundles within the lamina propria (C; arrowhead). Secondary only, negative control is shown i n D . Magnification 400X. 3  6  3  6  48  A  "JS  !;»  y  Figure 3.4: Other screened C D antigens are detected in non-HBC cells in the olfactory mucosa. C D 3 4 expression is localized to endothelial cells lining the blood vessels o f the lamina propria ( A ; arrow), while C D 4 3 and C D 4 4 expression is restricted to single, discrete cells within the L P (B and C ; arrows). C D 4 4 expression is also present within unidentified cells within the O E proper (C). Magnification 400X.  49  (CD29), and 04 integrin (CD104). The discovery o f these new H B C markers w i l l aid i n the characterization o f 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 o f 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.  A l s o , the detection o f these new  markers yields information regarding how H B C behaviour might be regulated v i a I C A M 1, Pi integrin, and P4 integrin.  50  C H A P T E R IV. A n in vivo characterization of HBCs: response to bulbectomy and examination of potential stem cell traits.  4.1  Introduction Our next aim involved the use o f these new markers to further characterize  horizontal basal cells ( H B C s ) i n their in vivo residence. The olfactory epithelium (OE) can be coaxed into an enhanced regenerative state via removal o f the olfactory bulb, a surgical procedure termed the bulbectomy. Upon the removal o f 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 o f H B C s as olfactory stem cells, we hypothesized that changes i n this cell population, as detected by expression o f I C A M - 1 , (31 and (34 integrins, would be evident following O R N cell death. In addition, i n our search for corroborating evidence to support a stem cell role for olfactory H B C s , we examined several characteristics o f stem cells i n 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 o f olfactory biology, the unilateral olfactory bulbectomy is  commonly used to facilitate the study o f cells that contribute to neurogenesis (Costanzo and Graziadei, 1983; M o n t i Graziadei, 1983; Schwob et al., 1992). We first wished to examine the relationship between O R N abundance and proliferation o f 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 i n Materials and Methods. A s the 6-day post-bulbectomy time-point coincides with the peak o f 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 O R N s and IRNs, N C A M , i n 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 i n some regions o f the O E that appeared to have a full complement o f O M P (Figure 4.1, C and E).  +  O R N s and N C A M  The loss o f >90% o f the mature O M P  +  +  IRNs/ORNs  O R N population by 6d  following bulbectomy, leads to the induction o f mitosis in the lower third o f the O E (Figure 4.1, D and F). A l s o o f note, is that the general thickness o f the O E is greater i n the normal tissue than i n lesioned, due to the loss o f 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 o f 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 i n secondary antibody only negative controls.  4.3  GBCs, negative for both ICAM-1 and N C A M expression, are depleted regionally following lesion G i v e n the observed relationship between the frequency o f dividing cells i n the O E  and the abundance o f 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 o f epithelial thickness is attributed to the loss o f O R N s elicited b y bulbectomy. The unlesioned ( U L ) 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 i n 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 o f N C A M with that o f 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 i n C). These two populations are separated by one full cell layer o f non-immunoreactive cells, presumable globose basal cell ( G B C ) progenitor cells. However, at 6 days post-bulbectomy, within discrete sections o f O E , this unlabelled population o f separating cells apparently disappears, leaving the I C A M - 1 and N C A M labelled populations i n close apposition to one another (Figure 4.2, E ) . This phenomenon occurs within short segments o f O E and is not observed within normal, unlesioned tissue. Negative, secondary antibody only controls were void o f 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 i n S-phase within the regenerating epithelium. A s above, many cells i n the basal portion o f the O E incorporate B r d U following bulbectomy. However, I C A M - 1 were only rarely B r d U (Figure 4.3, A ) . In contrast, +  +  HBCs  >90% o f 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 o f 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 likely G B C s according to their position and antigenicity. F o l l o w i n g bulbectomy, this layer o f unreactive cells disappears i n discrete sections o f O E , while they persist i n others (E). A l s o o f note is the upregulation o f I C A M - 1 i n endothelial cells o f the blood vessels postbulbectomy (E). (F) phase contast o f (E). ( A - D ) M a g n i f i c a t i o n 20x. ( E - F ) Magnification 400X. +  55  Figure 4.3. H B C s proliferate in response to bulbectomy, but remain quiescent relative to robustly proliferating G B C s . Robust proliferation is evident i n the basal cell compartment following 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 o f I C A M - 1 expression is altered during induced  synchronous regeneration  o f 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 i n 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 i n the uniformity o f staining are detected at intervals along the O E ' s length for each o f these H B C markers (Figure 4.4). In addition, changes were detected with respect to the localization o f these markers within individual H B C s . In normal O E , I C A M - 1 and P4 integrin are localized i n a pericellular fashion upon H B C s , with a slight concentration o f P4 integrin to the basal surface (Figure 4.5, A - B ) . In bulbectomized O E , these proteins are strongly concentrated to the basal surfaces o f many H B C s (D-E).  In contrast, there was no observable  difference in the subcellular distribution o f P i integrin in lesioned tissue (C and F ) . Secondary antibody only, negative controls produced no signal.  4.6  H B C s display some heterogeneity in the complement of adhesion receptor they express, an observation which is exaggerated post-bulbectomy A sub-feature o f stem cell populations is that they are often comprised o f cells  with different probabilities o f self-maintenance and differentiation (Hall and Watt, 1989). In this respect, they are said to be a heterogenous population o f cells.  To determine  whether H B C s may be subdivided on the basis o f their adhesion molecule expression, we  57  A ICAM-1 A  *  B pi integrin  p mp  ^0,  A  A  4  • « A  A  C (34  integrin f  f  f •MM  (Hi jAfc  y •V-WW)HAM<  *  J  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 p integrin expression are detected in lesioned OE (A-C, arrowheads). Magnification 200X. 4  58  ICAM-1  (34 integrin  (31 integrin  Figure 4.5. The subcellular distributions of ICAM-1 and p integrin are altered in some cells post-bulbectomy, while that of p\ integrin remains constant. In normal, 4  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 o f these antibodies on fixed sections o f olfactory tissue. In normal, quiescent tissue, co-incident expression o f 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 o f this population expresses every adhesion receptor equivalently. Following lesion, there are more cells that express single markers, indicating an increase i n the heterogeneity o f marker expression (Figure 4.6, B and D).  This experiment also further demonstrated the  pattern o f 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 i n normal tissue.  4.7  Summary U p o n 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 o f 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 o f marker expression is disrupted, as distinct regions o f the H B C layer are devoid o f marker expression. Secondly, I C A M - 1 and P4 integrin proteins appear to shift  60  UL  c ICAM/  p4  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 i n order to detect differences i n marker overlap in both normal ( U L ) and lesioned (L) tissue. In normal tissue, the overlap o f expression for both combinations o f proteins was complete i n 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 i n the quiescent O E , to a concentration to the basal surfaces o f H B C s .  Lastly, H B C s display some heterogeneity i n the complement o f  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 o f I C A M - 1 , P i and  62  P4  integrins upon H B C s .  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 o f 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 o f multiple differentiated daughter cell types (Hall and Watt, 1989; Potten and Loeffler, 1990). W e 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 i n 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 o f markers  o f 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 o f growth factors and extracellular matrix ( E C M ) components regulators o f H B C function.  as  We tested the ability o f these substances to influence the  colony-forming efficiency o f clonal cultures o f I C A M - 1 , magnetic activated cell sorting +  ( M A C S ) selected cells in order to illuminate the effects o f resident components on H B C behaviour  in  vitro.  63  in  vivo  niche  5.2 Preliminary in vitro findings Initial primary cell culture experiments separated fractions o f olfactory epithelial (OE) cells v i a differential adhesive properties and cell size. Briefly, O E tissue dissected from neonatal mice was dissociated enzymatically and passed through a series o f filters of decreasing pore size. The cellular fraction retained on the 40pm cell strainer contained clumps o f 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 o f neurons and glia at later time-points i n 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 o f our experiments  v i a the  development o f an immunomagnetic selection and culture protocol for H B C s , exploiting the discovery o f cell surface H B C expression o f 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  64  (see  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 i n 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 (3 integrin (F, green) and incorporate B r d U (D, red). Early in culture, (3 integrin clusters are devoid o f process-bearing cells ( G ) . Later, larger p* integrin* clusters (H) and co-incident with neurons and glia (I: N S T i n green, G F A P i n red). Magnification ( A - B ) 200X, (C) 100X, (D-F) 400X, (G-I) 200X. 4  +  4  4  65  Step 1: Label cells with biotinylated anti-ICAM-1 antibody (•) Step 2: Incubate with avidin conjugated M A C S magnetic microbeads ( • ° ) Step 3:  ICAM- negative fraction  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.  o o ICAM-positive(TT) ol fraction Step 4:  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 o f 25,000 I C A M - 1  +  cells was isolated by M A C S from each  dissected mouse epithelium. This represents 2.31 ± 0.49% (mean ± S E , n=5) o f the total unfractionated cell suspension prior to M A C S selection. Prior to plating, the presence o f 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 o f 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 o f 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 o f O E cell types, we predicted that the I C A M - 1  +  characteristics consistent with a progenitor phenotype.  fraction would express culture 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 o f colonies formed after 14 days in  vitro  (DIV) divided by the number o f I C A M - 1  +  cells initially plated.  Towards this  purpose, we define colony as a cohesive cluster o f more than 2 cells. However, i n 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 I C A M - 1 fraction displays a superior colonyforming 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 i n 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 o f colonies by the number o f cells initially plated (mean±SE). 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  ICAM-1  fraction  +  exhibited a significant increase  in colony-forming  efficiency when compared to the unselected cells (0.25 ± 0.057 and 0.033 ± 0.0055, respectively; p=0.0089, n=4) indicating an enrichment o f progenitor activity within the ICAM-1  +  fraction (Figure 5.3).  In contrast, the I C A M - T fraction possessed a lower  cloning efficiency than that o f the unselected fraction (0.0083 ± 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 ± 1.11 % o f the colony-forming cells were found i n the I C A M - 1  +  fraction.  5.5 Determination of optimal media conditions with respect to I C A M - 1 colonyforming efficiency +  A higher colony-forming efficiency, for the purpose o f future study o f 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 o f I C A M - 1 H B C s i n culture. +  The first such experiment conditions o f I C A M - 1  +  HBCs.  was directed at determining the optimal media  These were tested by plating I C A M - 1  +  cells on collagen  at clonal density (6000 cells per 10 cm dish) i n a variety o f different media formulations. At  2 weeks  experiments  in  vitro,  colony-forming efficiency was  (Figure 5.4) demonstrated that D M E M / F 1 2  then  assayed.  Preliminary  + 10% F B S produced  greatest colony formation, followed by O p t i - M E M supplemented with 4% F B S .  the The  calcium-free media conditions R P M I + 10% F B S and keratinocyte-serum free media ( K -  69  0.6  Serum%  DMF12 10  O-Mem RPMI 4 10  K-SFM 0  Growth Media Figure 5.4 Effect of media condition on the colony formingefficiency 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 i n different media formulations. A t 14 D I V , colonies (cohesive clusters o f >2 cells) were counted and the colony forming efficiency ( C F E ) for each condition was calculated. Data are represented as m e a n ± S E . D M E M / F 1 2 ( D M F 1 2 ) 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 I C A M - 1 selected cells A s demonstrated i n 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 o f 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. A m o n g 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 ± 8.67%., p-0.0067 and 42.50 ± 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 i n colony-forming efficiency is observed relative to collagen alone (143.33 + 11.45%, p=0.016 and 162.59 ± 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 ± 22.84 and 1:2, 91.07 ± 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 i n 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 o f the collagen control (mean±SE). 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 could be sub-categorized according to size.  +  cell fraction produced colonies that  This observation parallels what is seen i n  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. O n collagen, the different sizes o f colony were generally equally represented (Figure 5.7, A ) . O n 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 colonies (Figure 5.7B).  an increase i n the proportion and number of large  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 o f large colonies compared to collagen singly.  73  F i g u r e 5.6 Representative small, medium and large colonies at 14 D I V . A t 14 D I V , colonies produced by the I C A M - 1 cell fraction displayed substantial differences i n 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 ) ; 5 0 X (C). +  74  Substrate C o m p o s i t i o n  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 i n 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 o f the +  total number o f colonies within each condition ( m e a n ± S E ) . Data for each condition i n (B) are represented as a - f o l d difference i n large colonies over a collagen control (mean ±SE).  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 i n adhesion receptor  expression, exhibit rapid adherence  to collagen, while  transit  amplifying progenitors, typically characterized by low adhesion receptor expression, possess slower kinetics o f 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 o f 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 i n 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 i n colony-forming efficiency on collagen, laminin and fibronectin could reflect differences in the number o f 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 o f I C A M - 1  +  cell adherence  occurred within the first 4 hours in culture, as indicated by the steep slope o f the graph (Figure 5.8). The rate o f 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 ± 4.05 % non-adherent cells), i n effect demonstrating a preference for this substrate.  In contrast, adhesion to laminin is less  complete (59.8 ± 15.56 % non-adherent cells), while fibronectin elicits an intermediate response (37.23 ± 5.84%) with respect to adhesion within the first 24 hours o f culture.  76  -  Collagen  -A-Laminin  0  4  8  12  16  20  24  Time (hrs) Figure 5.8 M A C S selected I C A M - 1 cells display different kinetics of adhesion when plated on different E C M components. T o reveal possible differences i n 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 o f 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; C a l o f et al., 1991; Mahanthappa and Schwarting, 1993; Farbman and Bucholz, 1996). W e thus hypothesized that E G F and T G F - a , either singly or i n combination, would stimulate the ability o f 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 o f progenitor proliferation (Satoh and Yoshida, 1997; N a n et al., 2001).  L I F was also o f interest  because o f its ability to maintain the stem phenotype i n long-term culture when supplemented with E G F to neurospheres in vitro  (Shimazaki et al., 2001).  To test the effects o f these growth factors on the colony-forming ability o f selected cells, I C A M - 1  +  cells were plated immediately after M A C S enrichment onto  collagen coated 10 c m 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 o f experiments as the addition o f growth factor to D M E M / F 1 2 + 10% F B S showed no increase i n colonyforming efficiency, likely due to sufficient levels o f other growth factors within the serum. Addition o f E G F , T G F - a , or both growth factors resulted i n a significant increase  78  in the colony-forming efficiency relative to the control (EGF: p=0.020, n=8; T G F : 188.71 ± 30.65%, p=0.031, n=4; E G F + T G F : p=0.033, n=4; Figure 5.9).  The addition o f L I F to I C A M - 1  +  224.93 + 49.88 % , 289.29 ± 66.67%,  cells had no significant  effect on the overall ability o f these cells to form colonies (117.30 ± 16.29%, n=6; Figure 5.9).  W h e n 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 ± 35.45%, n=6), but appeared to decrease relative to that o f E G F singly.  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 +  In order to identify the effects o f growth factor addition on the incidence o f small, medium and large colonies, colonies o f 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 i n the number and proportion o f large colonies, with little change in the incidence o f 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 4fold with L I F (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 i n the formation o f large colonies, respectively (Figure 5.10, B ) .  79  400  1 _°° 3  £P I  200  8  o  (f)  co 100 o o  o C  E G F  L I F  E G F  T G F a  + L I F  E G 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 c m petri dishes and cultured in O p t i - M E M + 4% F B S with supplemented E G F , L I F , E G F + L I F , 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 ( m e a n ± S E ) . 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  100  T  •Small  60  O >» o c  40J  3  20  <D  a Medium B Large  80  CT CD  C  EGF  LIF  EGF TGFa EGF +LIF +TGFa  Growth Factor  B  ~  (0  "E o 0  10  0> O) i_ CO  -J  2*.  6  CD CO  4  1  .2  0)  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 c m petri dishes and cultured i n O p t i - M E M + 4 % F B S with E G F , L I F , E G F + L I F , T G F - a , E G F + T G F - a or without growth factor as a control (C). A t 1 4 D I V , •small ( < 3 0 cells), medium ( > 3 0 and <150 cells) and large ( > 1 5 0 cells) colonies were counted. Data i n ( A ) are represented as small, medium, or large colonies as percent o f total colonies formed within each condition (mean±SE). Data in (B) are displayed as a fold increase i n large colony formation relative to the "no growth factor" control (mean±SE). +  +  81  5.11 Cultured I C A M - 1 cells produce a mosaic of differentiated olfactory cell phenotypes +  Our previous assessment o f heterogenous, unselected olfactory epithelial derived cultures indicated that neurons and glia are present at later culture time-points and are coincident with basal cell clusters. To strengthen the argument that cultured H B C s possess the ability to generate differentiated cells o f the olfactory lineage, we tested for the expression o f stage-specific markers o f the olfactory lineage i n colonies produced by the ICAM-1  +  cell fraction. Immunohistochemistry was carried out on fixed large colonies  derived from I C A M - 1 clonal cultures, between 14 and 28 D I V . +  Expression  of  GBC-2,  a  marker  for  a  presumptive  transit  amplifying  subpopulation o f globose basal cells, is detected in clustered subsets o f cells within colonies generated by the I C A M - 1 cell fraction (Figure 5.11, A ) . To assess the presence +  o f neurons, we utilized the immature neuronal marker, neuron specific tubulin (NST). Figure 5.11 (B) demonstrates the presence o f N S T cells within our colonies. Olfactory +  ensheathing glia, as determined the co-expression o f S100P and P75 (Ramon-Cueto and A v i l a , 1998), are found i n 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 o f a bed o f 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 o f 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  CD54  D  CD104  E  i • .  ii #  :  ,  •••• •  «»"  " *  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. To determine whether differentiated cells are represent within large colonies at 14-28 days in clonal culture, immunohistochemistry was performed with markers o f O E differentiation. G B C 2, a marker for G B C s , is present within subsets o f cells ( A ) . Neuron specific tubulin (NST) expression, indicative o f neurons, is detected within cells at the colony perimeter, or on top o f the colonies (B). Cells resembling olfactory ensheathing glia were are detected (C), either i n slightly cohesive groupings or individually. Large colonies retain cells possessing an H B C phenotype ( C D 5 4 : I C A M - 1 and C D 2 9 : p \ integrin; D and E , respectively). ( A , C , D - E ) D A P I nuclear stain. Magnification 2 0 0 X ( 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 i n approximately 100% o f large colonies, 83%o o f medium colonies, and i n 75% o f small colonies.  Large colonies were  heterogenous with respect to the proportion o f I C A M - 1 cells i n a given colony, ranging +  from 5-100% with an estimated mean o f between 25-50%.  Within large colonies,  expression o f 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 G i v e n that N S T  +  neurons are found i n later cultures o f 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 D I V ) with antibodies directed against olfactory marker protein ( O M P ) , 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 o f these proteins were found in I C A M - 1  +  cell cultures  (Figure 5.12, A - D ) . Interestingly, during the course o f examining neuronal morphology and antigenicity, we observed NST-expressing neurons possessing a distinctly nonolfactory 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  G  Golf  4 vACIII B % '  J  •  i m.  mi,  s  Hhi _  ^  QMP  D  ,4  \  •  L•  1  OMP  %  * '  i  «•  %ll  % '*ti  IMP Oft  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) 4 0 0 X and (B) 200X.  85  Figure 5.13. Some neurons displayed a distinctly non-olfactory phenotype. Several NST-expressing neurons found i n proximity to large colonies possessed a distinctly nonolfactory morphology, as they exhibited multiple elaborate processes as opposed to being streamlined and bipolar ( A and B). Negative "secondary only" control i n 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 ( H B C ) 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 o f a substantial H B C proliferative potential. We have identified factors that influence the colony-forming ability o f M A C S selected, I C A M - 1  +  cells. A l l o f the substances tested are detected in close proximity to  H B C s within their in vivo  microenvironment.  ICAM-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.  W i t h respect to growth factors, E G F and T G F - a , both singly and in combination, produced the most significant increase i n overall colony-forming efficiency, while L I F produced no observable effect.  However, all three growth factors appeared to increase  the incidence o f large colonies, likely a reflection o f their reported roles i n promoting olfactory neurogenesis. In addition, we provide immunohistochemical evidence o f olfactory-specific differentiation, i n that globose basal cells, olfactory receptor neurons and olfactory ensheathing glia are produced from clonal cultures o f I C A M - 1 themselves are maintained within these colonies.  87  +  H B C s and that H B C s  C H A P T E R V I . Discussion  A pervasive challenge within the field o f olfactory neurogenesis is to reveal the ultimate source o f 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 ( G B C s ) and horizontal basal cells (HBCs), within the basal compartment o f the O E . To date, there is still much debate over which o f 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 o f these cells can be controlled by microenvironmental cues.  Using a combination o f 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 o f clusters o f differentiation (CD) antigens commonly expressed by various stem/progenitor cells, with an aim to identify new cell surface markers o f the H B C population o f the olfactory epithelium (OE). HBCs:  Three adhesion receptors were detected i n a uniform manner upon  intercellular adhesion molecule-1 ( I C A M - 1 ) , P i integrin and P4 integrin (Figure  3.1). The yield o f data resulting from this antigen screen serves a utility, as the presence o f these cell surface markers enables us to selectively enrich these cells i n an in  vitro  milieu, and is also insightful, as their presence upon H B C s w i l l likely yield information into the regulation o f the behaviour o f these cells. Before speculating on their possible  88  regulatory roles i n 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 o f cell types (Hubbard and Rothlein, 2000).  A s its name suggests, I C A M - 1 is involved i n  intercellular adhesion, a property carried out via binding ligand present on the surfaces o f neighbouring cells. I C A M - 1 can also ligate extracellular substances, such as hyaluronic acid, a component o f the extracellular matrix ( E C M ) that is typically enriched i n extracellular matrices (van der Stolpe and van der Saag, 1996; Tammi et al., 2002). Intracellular^, the cytoplasmic domain o f 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 o f leukocyte trafficking, where I C A M - 1 expression is induced in endothelial cells by pro-inflammatory cytokines to signal the location o f a site o f 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 , mitogen-activated protein kinases, Raf-1, lyn  E R K - 1 and cdc2 kinase (Holland and Owens, 1997; Chirathaworn et al., 1995). This ensemble o f activated kinases has reported activity in mediating a large variety o f cellular events, which typically culminate in changes in gene transcription.  With respect to  I C A M - 1 , recruitment o f these cytoplasmic kinases might explain the observed activation of the transcription factor o f A P - 1 (the Jun/Fos heteromer), the activation o f Rho and transcription o f I L - i p following I C A M - 1 crosslinking (Koyama et al., 1996; Etienne et al., 1998).  The reported possible endpoints o f these I C A M - 1 activated intracellular  signaling pathways include the control o f proliferation, protection against apoptosis and regulation o f 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. W i t h 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 o f 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 o f 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 o f I C A M - 1 within olfactory H B C s . Firstly, a classical immunological role for  ICAM-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 o f injury. Several studies, including our own, have reported that macrophages expressing the I C A M - 1 ligand, M a c 1, are recruited into the olfactory mucosa following lesion (Carter and Roskams, unpublished observations;  Suzuki et al., 1995; N a n et al., 2001).  A s in other  inflammatory responses, these macrophages are proposed to function i n phagocytosing cellular debris and in secreting growth factors and cytokines to promote regeneration o f 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 o f the O E i n order to ensure a local enrichment o f macrophage-secreted growth factors.  Additionally, H B C I C A M - 1  may serve a function in the migration o f these cells into the O E proper by facilitating the crossing o f the basement membrane barrier in an analogous manner to transendothelial migration o f leukocytes (Springer, 1990). In other tissues,  I C A M - 1 expression is typically induced by  inflammatory  cytokines i n cell types which previously displayed low or undetectable expression o f this protein (Springer, 1990; van der Stolpe and van der Saag, 1996).  91  A s such, I C A M - 1  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 i n 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 C D 4 3 , the soluble factor fibrinogen, and the E C M component hyaluronan (van de Stope and van der Saag, 1996). experimentation have indicated that L F A - 1 ,  Previous and current  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 o f cell dissociation enzymes, the mixture containing hyaluronidase produced the most basal cell clusters. Although the expression o f this ligand has not been examined within the epithelium, we expect to observe its localization within the basement membrane, placing it i n an optimal setting for interaction with I C A M - 1  +  H B C s . The true function o f I C A M - 1 i n the O E ,  however, requires further exploration. Nonetheless, the expression o f this protein upon H B C s provides an additional milieu for the study o f this adhesion molecule i n general and may provide insight into its role in olfactory neurogenesis. Two o f the remaining cell surface H B C markers identified i n this study belong to the integrin family o f 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 o f an a subunit non-covalently linked to a (3 subunit.  A diversity o f these  subunits abound, giving rise to a multitude o f 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 o f a and P subunits from which it is fashioned in conjunction with cellular context. The current model o f integrin-ligand interaction is that a subunits inhibit P subunits from interacting with the cytoskeleton.  U p o n ligand  binding this inhibition is relieved, thereby permitting intracellular signaling events (van der Flier and Sonnenberg, 2001).  Two varieties o f cellular communication are  transduced by integrin signaling: "outside-in" and "inside-out signaling".  "Inside-out"  signaling refers to the ability o f the cellular machinery to regulate an integrin's affinity for ligand, thereby changing the response o f the cell to its extracellular environment. O n the other hand, "outside-in" signaling is initiated by ligand binding outside the cell, and elicits a series o f intracellular events, beginning with the clustering o f integrin receptors within specialized complexes o f proteins called focal adhesions.  The typically short  cytoplasmic domains o f integrins are void o f any enzymatic ability, but activate intracellular signaling and cytoskeletal remodelling v i a 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 i n turn  93  trigger a variety o f 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 o f different origins, upon which integrins are commonly expressed.  A  variety o f different integrin heterodimers are expressed in primitive hematopoietic cells and in epidermal stem cells, wherein they participate in the control o f proliferation, differentiation and survival (Watt, 2000; Chan and Watt, 2001). Indeed, in the epidermis, the current model o f keratinocyte differentiation highlights the pivotal role o f integrins i n the control o f 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; L e v y et al., 2000; Z h u 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 v i a reduced adhesion.  A p i c a l migration and terminal differentiation o f  keratinocytes subsequently follows (Watt, 2001). In the O E , we have demonstrated that a i , 0:3, ae integrin subunits are expressed i n H B C s (Figure 3.3).  A l l o f these a subunits are potential pairing partners for the Pi  integrin detected within this population o f cells, while only ct6 is capable o f associating with p  4  integrin subunits.  The discovery o f these candidate pairings reveals potential  components o f the H B C niche, or microenvironment, within which the regulation o f these cells is expected to occur. A pairing o f Pi with C M forms a receptor for collagens I-IV and laminin, while 013 associates with Pi to make bind a range o f 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  o f 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 o f 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 o f these integrins within the O E , perhaps the most obvious role for P i integrin is adhesion to the basement membrane on account o f its restriction to the basal surfaces o f H B C s (Figure 3.1 and 4.5). This adhesive role would not only permit the interaction o f other integrins with E C M components,  but would also promote  H B C binding o f growth factors  typically  sequestered i n 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 o f the stem cell phenotype v i a both immobilization and intrinsic signalling. The presence o f P4 integrin subunits upon H B C s likely indicates similar roles to those o f 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 o f other integrins, the P4 integrin is  unique i n that it exerts its intracellular effects v i a 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 i n H B C s . CC604, a laminin-binding receptor, is  one  of  the  principle  transmembrane  components  of  hemidesmosomes.  Hemidesmosomes are specialized c e l l - E C M adhesion sites on the basal surfaces o f 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 i n the maintenance o f tissue integrity and in defining epithelial boundaries (Nievers et al., 1999). However, it is not yet known whether hemidesmosomes function as signaling units i n the absence o f 01604. Interestingly, the presence o f 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 o f these adhesions i n H B C s . However, ct604 is not likely restricted to hemidesmosomes since expression o f this receptor's component parts is detected on the apical and lateral surfaces o f H B C s i n the normal O E , i n addition to strong basal staining.  A similar phenomenon is reported i n a  study concerning epidermal keratinocyte integrin expression, wherein the authors propose two sites o f 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 i n 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 o f 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 o f stem/progenitor cells and molecular regulation o f the stem cell hierarchy, likely owing to the fact that they share a common ectodermal origin. Our findings that basal cells o f 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 o f the normal, unlesioned O E , while i n 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 i n other self-renewing tissues.  The discovery o f these adhesion receptor molecules upon H B C s prompted us to query the expression o f these proteins after surgical removal o f the olfactory bulb i n order to yield further insight into their in vivo  function. Several trends were observed with  regard to changes in the cellular constituents o f the O E i n addition to changes i n the expression and localization o f the identified H B C markers post-bulbectomy. In accordance with other published studies (Costanzo and Graziadei, 1983; M o n t i Graziadei, 1983; Schwob et al., 1992), our data show an inverse relationship between the number o f dividing basal cells and the abundance o f neurons within the lesioned O E (Figure 4.1).  This result was o f significance as it highlights the dynamic, highly  regulated pattern o f neurogenesis elicited by the removal o f 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 o f H B C s with an aim to illuminate any functional contribution o f these receptors during neurogenesis. In the normal quiescent O E , I C A M - 1 expression i n H B C s is pericellular.  A t 6d post-bulbectomy, corresponding to the peak o f basal  cell  proliferation, the I C A M - 1 signal is concentrated at the basal surfaces o f many H B C s (Figure 4.5). In other regions o f 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 i n 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 o f O E are detected. We  hypothesize that depriving cells o f basement membrane  contact, v i a  dowregulation o f adhesion receptor expression, provides a stimulus to migrate apically and further drive the differentiation program, in a series o f events parallel to those which occur during keratinocyte differentiation (Watt, 2000).  A s such, disruptions in the  continuity o f I C A M - 1 , Pi and P4 integrins within the lesioned O E might represent cells which have committed to differentiation. O n the other hand, the downward concentration of I C A M - 1 and P4 integrin to the basal surfaces o f H B C s following bulbectomy suggests that these proteins function in strengthening static adhesion to the basement membrane. A redistribution o f adhesion receptors to the basal surface would also increase the likelihood o f receptor ligation with their ligands within the basement membrane.  98  More  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 o f HBC-basement membrane adhesion would serve to maintain the stem cell compartment.  The decision between downregulation and  redistribution i n 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 o f 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 . T o initiate testing o f this model, function-blocking antibodies directed against the integrins could be supplied to cultures o f isolated H B C s and then assayed for differences in proliferation, differentiation and survival.  Further, to test for an effect o f local  differentiated cells on H B C expression o f Pi and p4 integrins, neurons could be added i n excess to purified H B C cultures.  According to the above model, we would expect to  observe an upregulation o f integrin expression i n H B C s i n 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 o f the function-blocking antibody experiments. Another trend observed i n H B C s following lesion is the apparent increased heterogeneity o f I C A M - l / P i integrin and I C A M - I / P 4 integrin expression as detected v i a double immunofluorescence (Figure 4.6).  These differences highlight the dynamic nature  99  of H B C s and may represent a continuum o f stem potentials such that those H B C s which express a full complement o f these markers, for example, may possess a more primitive stem phenotype than those expressing a sole marker.  Such is the case o f both  hematopoietic and epidermal lineages and is a trait that is exploited by researchers i n 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 o f the H B C population suggestive o f 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 o f proliferation, +  they are relatively quiescent with respect to the more apically situated G B C layer. This is in keeping with the proposed role o f G B C s as transit amplifying cells and lends supporting evidence towards the argument o f H B C s as stem cells (Schwob et al., 2002). Proliferative heterogeneity is a common trait o f 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 o f evidence which serves in arguing against the hypothesis o f a stem cell resident i n the G B C population was again derived from immunohistochemical analysis.  In discrete regions o f the lesioned O E , the single layer o f immuno-negative  G B C s , separating H B C s and immature neurons in normal quiescent tissue, disappears (Figure 4.2).  T w o 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 o f neurons by 3 days post-bulbectomy (Cowan et al., 2001).  100  That  G B C s might be exhausted, albeit i n confined regions o f O E , does not bode well for its candidacy for O E stem cell. O n the other hand, or perhaps simultaneously, G B C s might be induced to express markers o f 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. A t no point did the H B C layer express markers of differentiation. In summary, the in vivo portion o f this study provided support for the H B C as stem cell argument as well as providing information regarding the potential regulation o f these cells within their in vivo microenvironment.  The focus o f the in vitro  portion o f this study was to initiate the development o f a  method for isolating H B C s in vitro stem/progenitor cells in culture.  and to determine whether H B C s display traits o f B y fractionating neo-natal mouse O E suspensions  according to cell size and differential adhesive properties, we have isolated a semidissociated fraction that produces tightly adhesive clusters o f H B C s in vitro,  as confirmed  by immunohistochemistry with antibodies directed against I C A M - 1 , Pi integrin and p integrin (Figure 5.1).  4  These clusters also incorporate B r d U and are co-incident with  neurons and glia at late time-points i n 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 n o n - H B C population on the basis o f 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 i n 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 semidissociated or explant culture to test for stem characteristics i n 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 o f candidate cells to sort out in vitro  i n 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 i n order to test for stem cell  functional traits. One such assay, which examines colony-forming efficiency, tracks the ability o f a given cell population to seed colonies that expand under l o w 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, i n the broad sense, to include "potential" stem cells i n 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 o f O E cells, circumvents the argument that  102  other stem cell candidates (for example, G B C s ) are unfairly represented due to the abundance o f 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 o f olfactory progenitors i n culture demonstrated  that neurogenesis is inhibited when an excess o f neurons is added to these cultures ( M u m m et al., 1996).  However, it seems unlikely that this would be the case i n 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 nonexistent, 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 o f colonies containing >150 cells yielded an average o f 5363 cells per colony, with an upper range o f 40, 000 cells per colony. Assuming that the observed colonies were seeded by single cells, this data would indicate the considerable proliferative potential o f 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. A l s o , one colony was formed  103  for every 1000 G B C s plated at high density ( M u m m et al., 1996), indicating a lower colony-forming ability relative to our study o f H B C s at clonal density. Typically, the culture o f stem/progenitor cells at clonal density yields very l o w colony-forming efficiencies as a result o f reduced intercellular interaction, especially during the early development o f culture conditions for a particular cell type.  For  example, Kaur and L i (2000) reported that human epidermal keratinocytes have a colony forming efficiency o f 0.45%, while embryonic striatal neurospheres exhibit a colony forming efficiency o f 1% when viable cells are plated at l o w density (Reynolds and Weiss, 1996).  Defining the in vivo  stem/progenitor  niche is often instructive i n  determining the optimal factors that might be supplemented to the culture o f these cells to promote optimal colony formation. In addition, further insight can be gained concerning the regulation o f stem/progenitor cells by factors within their resident in vivo niche. In order to increase the colony-forming efficiency o f our I C A M - 1  +  MACS  selected cells, cells were plated i n different media formulations commonly used i n the culture o f 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% o f 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 i n either R P M I or K - S F M , both o f which contain low concentrations o f calcium, suggests that higher levels o f calcium are required for H B C colony formation.  This  concurs with what is known to date regarding the function o f cell adhesion molecules, most o f 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 i n 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 i n 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. G i v e n that H B C s express E C M - b i n d i n g integrin receptors in vivo and in vitro,  and  owing to a previous report o f several common E C M components within the O E ' s underlying basement membrane, we tested the effect o f collagen, laminin and fibronectin on the colony-forming efficiency o f I C A M - 1 highest  colony-forming  efficiency,  while  +  cells. Plating on collagen produced the both  fibronectin  efficiencies roughly half o f that of collagen (Figure 5.5).  and  laminin  yielded  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. W i t h respect to the incidence o f large colonies, both mixtures o f 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 i n colony-forming efficiency might be mediated by any o f 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 o f colony-forming efficiency assays indicates a role i n the promotion o f colony formation for collagen and laminin.  W i t h 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  105  recognizes  differences  i n colony initiation and proliferation). We can, however, exclude  inhibitory effect  an  o f 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 o f collagen, laminin and fibronectin on the colony-forming ability o f I C A M - 1 cells, an analysis o f the kinetics o f adhesion to these +  substrates was performed (Figure 5.8).  In all three cases, the majority o f adhesion to  substrate occurs within the first 4 hours in culture. A l s o , overall, I C A M - 1 selected cells +  display a clear preference for collagen, and roughly equal, but lesser, adherence to laminin and fibronectin. experiment  One might argue that the results o f the adhesion kinetics  indicate that the large difference  i n overall colony formation  between  substrate conditions is a consequence o f the capability o f 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 o f the two substrates individually. However, an additive effect is not observed for collagen/fibronectin mixtures. It is likely that the differences i n adhesion account, at least in part, for the observed differences i n overall colony formation in vitro. respect to the  formation  o f large colonies specifically, the  effects  However, with o f 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 o f survival or proliferation, is promoting the formation o f 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 o f integrin-bearing cells. Further experimentation, using assays specific for the above cellular functions, w i l l be required to clarify the endpoints o f integrin signaling in H B C s . In addition to E C M proteins, we also tested the effects o f several resident O E growth factors on the efficiency o f 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 i n the rat in vivo  (Rama Krishna et al., 1996; Ezeh and Farbman, 1998).  assayed the effects o f these growth factors on I C A M - 1  +  A s such, we  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 o f total colonies formed (Figure 5.9).  Furthermore, these growth  factors appeared to stimulate the formation o f large colonies at the expense o f 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 o f large colonies can likely be attributed to the reported mitogenic effects o f these growth factors.  A s such, single undividing cells which would not have been included i n the  colony count i n 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 i n cell number, resulting in the observed frequency o f large colonies.  W e therefore conclude that we have detected a  means to enhance the expansion o f I C A M - 1 H B C s in vitro. +  107  Furthermore, the observed  responsiveness to E G F and T G F - a o f M A C S selected I C A M - 1 supports the conclusion that these cells are the in vitro  +  cells in vitro  further  equivalents o f olfactory H B C s .  Leukemia inhibitory factor (LIF) was a factor o f interest owing to the fact that the olfactory literature indicates its role in olfactory neurogenesis. Within the O E proper, the L I F receptor (LIFR) is detected infrequently i n the odd G B C (Nan et al., 2001). incidence o f L I F R  +  The  G B C s is elevated following olfactory bulbectomy, implicating L I F  signaling i n the generation o f new O R N s .  A s well, in vitro  studies show that L I F  increases the B r d U labeling index o f immediate neuronal precursors (INPs) (Satoh and Yoshida, 1997). Our interest was further piqued by the report that subependymal-derived neurospheres can be maintained i n a "primitive", proliferative stem state v i a the addition o f L I F and E G F to the culture medium (Shimazaki et al., 2001). Our results, however, indicate that the addition o f L I F to clonal cultures o f I C A M - 1 selected cells has no effect +  on the overall ability o f these cells to initiate colonies (Figure 5.9). Nonetheless, L I F appears to influence the incidence o f large colonies at 14 D I V , in that there is roughly a 4-fold increase i n this category o f colonies when L I F is added to the medium (Figure 5.10). Given these results, two models o f L I F action are possible. Firstly, in accordance with its proposed role as G B C mitogen, L I F may be increasing the size o f existing colonies v i a G B C expansion.  A potential explanation for the stagnant overall colony-  forming efficiency o f LIF-treated cultures is that the I C A M - 1 cells initially seeded are +  unresponsive to L I F . Hence, the only LIF-responsive cells possible in our culture system are daughter cells o f H B C s , formed by the division o f H B C s irrespective o f L I F supplementation.  Alternatively, the addition o f L I F might achieve large colonies by  promoting the survival o f G B C s formed after plating, such that the higher incidence o f  108  large colonies reflects decreased cell death. When L I F and E G F were added to cultures i n 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 o f a  paracrine mechanism to control H B C proliferation, induced by ligation o f 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  OE  microenvironment. Stem cells, by definition, generate daughter cells which terminally differentiate i n order to maintain functional tissue homeostasis (Hall and Watt, 1989; Potten and Loeffler, 1990). Homeostasis also implies that a population o f stem cells is maintained in a given tissue.  T o address whether H B C are capable o f these traits, we examined the  expression o f markers o f olfactory differentiation i n combination with the new H B C markers identified in this study within older cultures o f 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 o f these colonies, we found that cores o f I C A M - 1 7 p integrin cells 4  were maintained i n all large colonies (Figure 5.11). The combined expression o f I C A M 1 with P 4 integrin, indicative only o f H B C s within the O E , suggests that cores within the colonies contain a significant representation from an expanded population o f H B C s . Given that cores o f 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 ( G B C s ) , the proposed transit amplifying cells and proven immediate precursors o f olfactory receptor neurons, are also represented i n large colonies in vitro,  as evidenced by the expression o f G B C - 2 within subsets o f cells (Figure 5.11).  G i v e n their presence, it is likely that they are daughter cells o f 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 i n cultures of I C A M - 1  +  immunomagnetically selected cultures o f O E cells (Figure 5.11 and 5.12).  The latter characteristic is o f 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; C a l o f 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 o f cells indicative o f olfactory ensheathing glia (OEGs) within older cultures o f M A C S selected cells (Figure 5.11). The presence o f 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 o f pluripotency than G B C s . Furthermore, we propose the existence o f an I N P equivalent for the glial lineage, such that olfactory stem cells produce daughters committed to a particular lineage in order to segregate the production o f differentiated cells, as is the case in stem/progenitor hierarchies i n other multi-lineage tissues.  110  The  potential pluripotency o f H B C s is further demonstrated by the detection o f neurons with distinctly non-olfactory phenotypes (Figure 5.13). Future  experiments  directed at increasing the  stringency o f H B C culture  experiments include the addition o f 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 i n 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 o f 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 o f H B C s could be compared with respect to progenitor activity. Finally, i n order to elevate H B C status from progenitor to stem cell, the requisite stem cell trait o f 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 i n 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 o f preliminary secondary cloning experiments are a consequence o f a rapid loss o f stem cell traits in vitro,  as is observed in the culture o f  other stem cells (Domen, 2001). The observation that our cultures o f I C A M - 1 , M A C S +  111  selected cells produce colonies with an abundance o f 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 i n culture.  112  C H A P T E R V I I . Concluding Remarks  By  immunohistochemically  screening  a  panel  of  selected  clusters  of  differentiation ( C D ) antigens, we have revealed three new cell surface markers o f olfactory horizontal basal cells (HBCs), namely: intercellular adhesion molecule-1 ( I C A M - 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 o f these  markers was o f significance as it has enabled us to selectively enrich for H B C s in vitro exploiting their molecular phenotype.  by  Further, since these proteins are also present on  stem and progenitor cells i n 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 i n the loss o f olfactory receptor neurons (ORNs) from the olfactory epithelium (OE), changes are detected with respect to populational and subcellular expression o f I C A M - 1 , Pi and P4 integrin. Breaks in the continuity o f marker expression are observed in addition to an apparent redistribution o f I C A M - 1 and P4 integrin to the basal surfaces o f H B C s . These changes in marker expression suggest a role for these adhesion receptors i n the regulation o f H B C function during neurogenesis. We also detected evidence o f 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 postbulbectomy); 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 (MACS).  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. ICAM-1  +  These results suggest that the  fraction is enriched for progenitor activity, while the I C A M - 1 " fraction is  depleted o f these colony-forming cells. In order to optimize the colony-forming efficiency o f M A C S selected, I C A M - 1  +  cells, several growth factors and extracellular matrix ( E C M ) components, all resident i n 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 o f large colonies, while fibronectin and laminin singly resulted i n 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 i n combination, produced the highest overall colony-forming ability, while L I F produced no observable effect.  However, all growth factors served in promoting large colonies i n  culture. B y examining the expression o f markers o f olfactory differentiation, our results show that cultures o f 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 o f these large  colonies as evidenced by I C A M - 1 and p% integrin immnohistochemistry. W e conclude that H B C s possess traits suggestive o f a stem cell phenotype in vivo  114  and exhibit  progenitor activity in vitro.  A s such, we conclude that H B C s likely contribute to the  genesis o f olfactory cell types, potentially as the O E stem cell.  Further, the function o f  H B C s is likely modulated v i a adhesive and growth factor signaling i n a manner parallel to stem cells i n other, better characterized tissues.  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