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The role of Wnt10b in post natal bone homeostasis and maintenance of mesenchymal progenitor cells

Dissertation
Author: Jennifer Renee Stevens
Abstract:
Mesenchymal Progenitor Cells (MPCs) contribute to the development and maintenance of bone, fat, muscle and cartilage in mammals. Several lines of evidence implicate canonical Wnt signaling in maintenance of these stem cell populations. Wnt10b is a canonical Wnt ligand expressed in developing bone, calvarial osteoblasts, and multipotential mesenchymal progenitors isolated from adult bone marrow. Here we demonstrate that Wnt10b null mice exhibit defects in both intramembranous and endochondral ossification that present as cranial suture agenesis at post natal day four and an age progressive loss of trabecular bone starting between one and two months of age. Maintenance of normal adult bone requires both copies of the Wnt10b gene as heterozygous animals express similar reductions in trabecular bone density in aged mice. No difference was observed in osteoclast number or activity as assessed by analysis of serum CTx levels indicating that the age progressive osteopenia in Wnt10b null mice is not due to increased bone resorption but rather is the result of decreased bone deposition. Using in vitro colony forming unit assays we show that the loss in trabecular bone and suture agenesis is associated with a reduction in the number of bone marrow derived mesenchymal progenitors and MPC lineage derived osteoblasts and adipoblasts and calvaria derived osteoprogenitors. Analysis of osteogenic gene expression in primary bone marrow stromal cells and calvarial osteoblasts demonstrates reductions in expression of several osteoblast differentiation markers but no change in Runx2, an osteoblast associated transcription factor expressed in multi-potent MPC. Additionally, Wnt10b null bone marrow derived progenitors and calvarial derived osteoblasts express reduced levels of bone morphogenic proteins (Bmp's) and Bmp response genes. Stimulation of the Wnt pathway in primary calvarial osteoblasts results in up regulation of Bmp4 and associated Bmp responsive genes showing that Wnt10b is sufficient to induce expression of Bmp4 and Bmp target genes in calvarial osteoblasts. Taken together, this work indicates that Wnt10b is a critical Wnt signaling ligand, required for mesenchymal progenitor activity in both calvarial morphogenesis and maintenance of adult bone and suggest a role for Wnt10b in maintenance of immature osteo and mesenchymal progenitors capable of depositing bone.

TABLE OF CONTENTS Page List of Figures vii List of Tables ix List of Abbreviations x Acknowledgments xi Vita xiii Abstract of the Dissertation xv Chapter 1 Introduction to Bone Development, Post Natal Homeostasis, and Mesenchymal Progenitor Cells 1.1 Introduction to Bone Development 2 1.2 Intramembranous bone development and cranial vault morphogenesis 2 1.3 Enodchondral Bone development 3 1.4 Endochondral Bone Remodeling and Post Natal Homeostasis 4 1.5 Mesenchymal Stem Cells 6 1.5.1 Introduction to Mesenchymal Stem Cells 6 1.5.2 Osteoblastogenesis 8 1.6 References 9 Chapter 2 Wnt Signaling in Bone Development, Post Natal Homeostasis, and Mesenchymal Progenitor Cells 2.1 Introduction to Wnt Signal Transduction 14 2.2 Wnts in Development and Postnatal Homeostasis of Bone 16 2.2.1 Introduction 16 2.2.2 Wnt co-receptors Lrp5/6 18 2.2.3 Wnt signaling transcription factor P-catenin 19 2.2.4 Negative regulators of Wnt Signaling in Skeletogenesis 21 2.2.5 Wnt Ligands 23 2.3 Wnt Signaling in Mesenchymal Stem and Progenitor Cells 24 2.3.1 Introduction 24 2.3.2 Canonical and non-canonical Wnt signaling in maintenance and differentiation of mesenchymal stem and progenitor cells. 24 in

2.4 WntlOb in Maintenance of Mesenchymal Stem and Progenitor Cells 27 2.5 Summary of Results Presented In Subsequent Chapters 29 2.6 References 31 Chapter 3 Wntl Ob Deficiency Results in Age Dependent Loss of Bone Mass and Progressive Reduction of Mesenchymal Progenitor Cells 38 3.1 Introduction 39 3.2 Results 41 3.2.1 WntlOb null mice appear morphologically normal and have normal growth rates. 41 3.2.2 WntlOb null mice exhibit enhanced early maturation followed by age progressive loss of trabecular bone (osteopenia) 43 3.2.3 Age progressive osteopenia in WntlOb null mice is not due to abnormal bone resorption. 46 3.2.4 Primary bone marrow stromal cells from WntlOb null mice have fewer osteoprogenitors by 6 months of age. 47 3.2.5 WntlOb null animals have fewer mesenchymal progenitors at 6 months. 50 3.3 Discussion 52 3.4 Materials and Methods 57 3.4.1 Generation and genotyping of WntlOb null mice 57 3.4.2 Quantitative rt-PCR 58 3.4.3 Histomorphometry and Microcomputerized Tomography (uCT) 58 3.4.4 Primary bone marrow stromal cell isolation, osteo/adipogenic differentiation and histological staining 59 3.4.5 Enumeration and differentiation of mesenchymal stem cells harvested in mesencultâ„¢ 60 3.4.6 Serology 60 3.5 References 61 IV

Chapter 4 Cranial Vault Morphogenesis Defects in WntlOb Null Mice are Linked to Misregulation of Bone Morphogenic Proteins (Bmp's) and Bmp Target Genes In Calvarial Osteoblasts. 65 4.1 Introduction 66 4.2 Results 68 4.2.1 WntlOb null mice show delayed suture closure and are defective for osteogenesis in vitro. 68 4.2.2 Reduced in vitro differentiation of WntlOb null osteoblasts is associated with decreased numbers of Bmp4 responsive osteoblasts in primary calvarial cell isolates. 70 4.2.3 Wnt signaling induces expression of mRNA for Bmp4 and Bmp4 target genes. 73 4.2.4 WntlOb null primary bone marrow stromal cells are deficient for expression of Bmp4 mRNA and this decrease is associated with decreased numbers of osteogenic progenitor cells. 75 4.3 Discussion 77 4.4 Materials and Methods 81 4.4.1 Generation and genotyping of WntlOb null mice 81 4.4.2 Alcian Blue/Alizarin Red Skeletal Staining and Microcomputerized Tomography (uCT) 81 4.4.3 Calvarial osteoblast isolation and differentiation 81 4.4.4 Primary bone marrow stromal cell isolation and osteogenic differentiation 83 4.4.5 Quantitative rt-PCR 84 4.5 References 85 Appendices Work in Progress Introduction to Appendices 90 Appendix A Wnt3a inhibits Ossificatoin in WT and WntlOb null Bone marrow stromal Cell Culture 92 Appendix B WntlOb null mice have fewer linVScal +/CD347CD44" and lin"/Scal+/CD347CD44+ putative mesenchymal progenitors at 2, 3, and 6 six months of age. 96 Appendix C Generation of Vectors for to create WntlOb reporter mice and Wntl Ob reporter lentivirus 102 v

Appendix D Primers For Quantitative Real Time PCR 109 References for Appendices 111

LIST OF FIGURES Page 2.1 Wnt Signaling Cascades. 15 2.2 Canonical and Non canonical Wnt Signaling Pathways in Maintenance and Differentiation of Mesenhymal Stem and Progenitor Cells. 30 3.1 Wntl0b-mx\\ mice appear morphologically normal and have normal growth rates. 42 3.2 WntlOb-rmW mice show an age dependent loss of trabecular bone and loss of one allele is sufficient to generate severe osteopenia. 44 3.3 Dynamic properties of bone formation in WT and WntlOb null mice. 45 3.4 The age progressive osteopenia in WntlOb null mice is not due to abnormal bone resorption, but is associated with decreased numbers of osteogenic and adipogenic progenitor cells. 48 3.5 WntlOb-null animals have reduced numbers bone-derived mesenchymal progenitors. 51 4.1 WntlOb null mice show delayed suture closure and are defective for osteogenesis in vitro. 69 4.2 Reduced in vitro differentiation of WntlOb null osteoblasts is associated with decreased numbers of Bmp4 responsive osteoblasts in primary calvarial cell isolates. 71 4.3 Wnt signaling induces expression of mRNA for Bmp4 and Bmp4 target genes. 74 4.4 WntlOb null primary bone marrow stromal cells are deficient for expression of Bmp4 mRNA and this decrease is associated with decreased numbers of osteogenic progenitor cells. 76 A.l Wnt3a inhibits ossification of WT and WntlOb null bone marrow stromal cells. Stimulation with Bmp4 overcomes inhibition by Wnt3a but does not restore WntlOb null CFU-Ob to WT levels. 94 vn

B. 1 WntlOb null mice have fewer bone-derived mesenchymal progenitors in vivo 99 C. 1 Wntl Ob reporter lenti-virus and Wntl Ob Knock-in Constructs 103 C.2 Wntl Ob-2300 NLSemGFP lentivirus directs expression of nuclear localized GFP in HeLa cells. 104 vni

LIST OF TABLES 2.1 Development and Post Natal Bone Acquisitions Phenotypes in Wnt Mutant Mice 17 D.l Quantitiave Real Time PCR Primers Designed 109 IX

LIST OF ABBREVIATIONS ALP BV/TV CFU-A CFU-F CFU-Ob CTx DNA FACS FBS LRP MAR MPC MS/BS MSC OSCN PBMSC RNA Tb.N Tb.Sp. uCT Alkaline Phosphatase Bone volume/total volume Colony Forming Unit Adipocyte Colony Forming Unit Fibroblast Colony Forming Unit Osteoblast C-terminal cross-linking telopeptide of type I collagen Deoxyribonucleic Acid Flouresence Activated Cell Sorting Fetal Bovine Serum LDL-receptor related protein Matrix Aposition rate Mesenchymal Progenitor Cell Mineralizing surface/bone surface Mesenchymal Stem Cell Osteocalcin Primary Bone Marrow Stromal Cells Ribonucleic Acid Trabecular Number Trabecular Spacing microcomputerized tomography X

ACKNOWLEDGMENTS Chapter 3 is a version of a "Stevens J.R, Miranda-Carboni G.A., Singer M.A., Brugger S.M, Lyons K.M., Lane T.F. WntlOb deficiency results in age dependent loss of bone mass and progressive reduction of mesenchymal progenitor cells." A manuscript in press with the Journal of Bone and Mineral Research. The authors would like to thank Kathleen Yee, and Cate Sullivan for technical contributions, and to the members of the Lane Lab for their support and great effort. We would like to thank Drs. Susan Krum and John Adams for discussions. JRS is supported by NRSA fellowships HL69766. The work has been supported by grants from the National Cancer Institute (R01-CA107002-01A1), the American Cancer Society (RSG-05-034-01-CSM), and through fellowship support from the Margaret E Early Foundation and Department of Defense, C.D.M.R.P DAMD17-02-1-0327 (TFL). Chapter 4 is a version of a "Stevens J.R, Singer M.A, and Lane T.F. "Cranial Vault Morphogenesis Defects in WntlOb Null Mice are Linked to Misregulation of Bone Morphogenic Proteins (Bmp's) and Bmp Target Genes In Calvarial Osteoblasts." A manuscript in preparation for submission. The authors would like to thank Kathleen Yee, for technical contributions, and to the members of the Lane Lab for their support and great effort. We would like to thank xi

Dr. Aykut Uren for his kind gift of Wnt3a and WntlOb conditioned media. We would like to thank Drs. Susan Krum and Karen Lyons for comments on the manuscript. The work has been supported by grants from the National Cancer Institute (R01- CA107002-01A1), the American Cancer Society (RSG-05-034-01-CSM), and through fellowship support from the Margaret E Early Foundation and Department of Defense, C.D.M.R.P DAMD17-02-1-0327 (TFL). JRS is supported by NRSA fellowships HL69766. xii

VITA March 27, 1973 Born, Los Angeles, California 2002 B.S., Microbiology, Magna Cum Laude California Polytechnic State University San Luis Obispo, California 2002 2nd place, Cal-State University Undergraduate Research Competition Biological and agricultural sciences category Cal-State University, Long Beach 2003,2004 Honorable Mention National Science Foundation Fellowship Competition 2003,2004 Teaching Assistant Department of Microbiology, Immunology, and Molecular Genetics University of California, Los Angeles 2002-2005 Research Associate Department of Microbiology, Immunology, and Molecular Genetics University of California, Los Angeles 2005 M.S. Microbiology, Immunology, and Molecular Genetics University of California, Los Angeles 2005-2010 Research Associate Department of Biological Chemistry University of California, Los Angeles 2008 Teaching Assistant Department of Molecular, Cellular, and Developmental Biology University of California, Los Angeles xiii

PUBLICATIONS AND PRESENTATIONS Stevens J.R, Miranda-Carboni, G.A., Singer, M.A., Brugger, S.M., Lyons, K.M., & Lane, T.F. WntlOb deficiency results in age dependent loss of bone mass and progressive reduction of mesenchymal progenitor cells. (In Press) Journal of Bone and Mineral Research. Vredevoe, L.K., Stevens, J.R., Schneider B.S., 2004. Detection and Characterization of Borrelia bissettii in Rodents from the Central California Coast. J. Med. Entomol. 41(4): 736-745 Stevens, J.R., and Vredevoe, L. (2002) Detection and Identification of Borrelia burgdorferi sensu lato in San Luis Obispo County Rodents. 2002 West Coast Biological Sciences Undergraduate Research Conference, Loyola Marymount University, Los Angeles. Vredevoe L., Stevens J.R., Schneider B. (2002). Detection and Cultivation of Borrelia bissettii in Rodents from the Central California Coast. 2002 General Meeting. American Society for Microbiology. Salt Lake City, UT. Bortz, E., Jia, Q., Whitelegge J.P., Stevens J.R., Atanasov, I., Hong Zhou Z., and Sun, R. (2003). Identification of Virion Proteins Associated With Murine Gammaherpesvirus-68. 28th International Herpesvirus Workshop, Madision, WI. Stevens, J.R. and Lane T.F. (2009) Multiple Defects of Skeletogenesis in WntlOb null Mice suggest a roll for WntlOb in maintenance and self-renewal of mesenchymal progenitor cells. American Society of Cell Biology General Meeting, San Diego, California, USA. Poster Presentation. Stevens, J.R., Lane T.F. (2010) Role of WntlOb in Post Natal Bone Homeostasis and maintenance of osteoprogenitors. Keystone Symposia on Stem Cell Differentiation and Dedifferentiation, Keystone, Colorado, USA. Poster Presentation. xiv

ABSTRACT OF THE DISSERTATION The Role of WntlOb in Post Natal Bone Homeostasis and Maintenance of Mesenchymal Progenitor Cells By Jennifer Renee Stevens Doctor of Philosophy in Biological Chemistry University of California, Los Angeles, 2010 Professor Timothy F. Lane, Chair Mesenchymal Progenitor Cells (MPCs) contribute to the development and maintenance of bone, fat, muscle and cartilage in mammals. Several lines of evidence implicate canonical Wnt signaling in maintenance of these stem cell populations. WntlOb is a canonical Wnt ligand expressed in developing bone, calvarial osteoblasts, and multipotential mesenchymal progenitors isolated from adult bone marrow. Here we demonstrate that WntlOb null mice exhibit defects in both intramembranous and endochondral ossification that present as cranial suture agenesis at post natal day four and an age progressive loss of trabecular bone starting between one and two months of age. Maintenance of normal adult bone requires both copies of the WntlOb gene as heterozygous animals express similar reductions in trabecular bone density in aged mice.

No difference was observed in osteoclast number or activity as assessed by analysis of serum CTx levels indicating that the age progressive osteopenia in WntlOb null mice is not due to increased bone resorption but rather is the result of decreased bone deposition. Using in vitro colony forming unit assays we show that the loss in trabecular bone and suture agenesis is associated with a reduction in the number of bone marrow derived mesenchymal progenitors and MPC lineage derived osteoblasts and adipoblasts and calvaria derived osteoprogenitors. Analysis of osteogenic gene expression in primary bone marrow stromal cells and calvarial osteoblasts demonstrates reductions in expression of several osteoblast differentiation markers but no change in Runx2, an osteoblast associated transcription factor expressed in multi-potent MPC. Additionally, WntlOb null bone marrow derived progenitors and calvarial derived osteoblasts express reduced levels of bone morphogenic proteins (Bmp's) and Bmp response genes. Stimulation of the Wnt pathway in primary calvarial osteoblasts results in up regulation of Bmp4 and associated Bmp responsive genes showing that WntlOb is sufficient to induce expression of Bmp4 and Bmp target genes in calvarial osteoblasts. Taken together, this work indicates that WntlOb is a critical Wnt signaling ligand, required for mesenchymal progenitor activity in both calvarial morphogenesis and maintenance of adult bone and suggest a role for WntlOb in maintenance of immature osteo and mesenchymal progenitors capable of depositing bone. xvi

Chapter 1 Introduction to Bone Development, Post Natal Homeostasis, and Mesenchymal Progenitor Cells Summary The mammalian skeleton develops via two distinct mechanisms called intramembranous and endochondral ossification. The process by which skeletal elements develop and are maintained throughout the life of an organism involves complex interactions between several key developmental pathways. While the primary focus of this dissertation is on the role of Wnt signaling in development and postnatal homeostasis of bone a discussion of how bone develops and is maintained is provided to assist readers unfamiliar with bone biology. To that end the sections in Chapter 1 will provide a brief overview of both intramembranous and endochondral ossification followed by a discussion of bone remodeling and postnatal homeostasis of endochondral bone. Additionally, an introduction to the discovery and biology of mesenchymal stem cells which give rise to bone forming osteoprogenitors will be discussed. 1

1.1 Introduction to Bone development Mammalian skeletal elements are derived from the paraxial mesoderm, lateral plate mesoderm, and the neural crest (reviewed in (Chung, et al., 2004)). Bones derived from the neural crest form the facial skeleton and frontal skull plates through a process called intramembranous bone formation. The vast majorities of bones, including those of the axial and appendicular skeleton are derived from paraxial and lateral mesoderm and form through a process called endochondral bone formation. Both intramembranous and endochondral bone formation begin with mesenchymal condensations (reviewed in (Kronenberg, 2003)). In the former case, multipotent mesenchymal cells (MSCs) in these condensations differentiate directly into osteoblasts which secrete and subsequently ossify matrix. However, the process of endochondral bone formation is not as direct. In this case, MSCs first differentiate into chondrocytes and lay down cartilaginous molds that are subsequently invaded by mesenchymal stem cell derived osteoblasts which replace the cartilage mold with mineralized matrix. 1.2 Intramembranous bone development and cranial vault morphogenesis Development of the cranial vault begins during embryogenesis and continues into adulthood. It is composed of two groups of bones, the viscerocranium and neurocranium. The viscerocranium, comprised of the bones surrounding the oral cavity and pharanx, derive from neural crest while bones of the neurocranium are derived from the paraxial mesoderm (reviewed in (Lynne, 2000)). At birth the bones of the cranial vault are separated by fibrous suture structures that remain pliable (patent) to allow for continued 2

growth of the brain and are the primary sites of bone formation in the cranial vault (Cohen, 2000). Although the bones of the neurocranium and axial skeleton are both derived from paraxial mesoderm the bones of the cranial vault undergo a process of intramembranous ossification while those of the axial skeleton undergo a process called endochondral ossification (discussed below). Similar to that seen for endochondral ossification the process of intramembranous ossification begins with mesenchymal condensations. In contrast to that seen for endochondral bone formation multipotent mesenchymal stem cells (MSC) within these condensations differentiate directly into osteoblasts which secrete and subsequently ossify matrix. As the neurocranium enlarges cues from the underlying dura matter result in growth of cranial bones along the osteogenic fronts of the skull plates. Growth of bones in the cranial vault continues until neural growth subsides. Upon cessation of neural growth sutures lose patency and become fusing sutures. Wnts, Bone morphogenic proteins (Bmps) and Fibroblast growth factors (FGFs) have been shown to play important roles in maintenance of suture patency and initiation of suture fusion. Although Indian hedgehog (Ihh) is indispensable for development of the long bones of the axial skeleton it is not required for formation of the cranial vault (St- Jacques, et al., 1999). 1.3 Enodchondral Bone development Endochondral bone formation begins with the formation of mesenchymal condensations composed of multipotent mesenchymal stem cells (MSC's). In contrast to 3

that seen for intramembranous ossification (discussed above) these cells first differentiate into chondrocytes which are proliferative, secrete typell collagen and express a variety of genes driven by the transcription factor Sox9 (Ikeda, et al, 2005). Indian hedgehog (Ihh), considered a master regulator of endochondral bone formation, drives chondrocyte proliferation and enlargement of the cartilage mold by stimulating the production of parathyroid hormone related protein (PTHrP). PTHrP functions to maintain chondrocytes in a proliferative state. The importance of Ihh in bone development was determined by examination of the skeletal defects in the long bones of Ihh null mice. These defects were shown to be the result of reduced chondrocyte proliferation and differentiation and a failure to synthesize PTHrP (St-Jacques, et al, 1999). As the cartilage mold develops, chondrocytes in the center of the mold stop responding to PTHrP signaling and differentiate into the more mature non-proliferative hypertrophic chondrocytes. These cells recruit blood vessels to the developing bone through secretion of vascular endothelial growth factor (VEGF) (Gerber, et al., 1999) and direct cells in the surrounding perichondrium to differentiate into osteoblasts which form the bone collar, a precursor of cortical bone. Upon apoptotic death of hypertrophic chondrocytes, osteoblasts invade and ossify the cartilaginous matrix forming the primary spongiosa, a precursor to the formation of trabecular bone. 1.4 Endochondral Bone Remodeling and Post Natal Homeostasis Throughout adulthood the bones of mammals undergoe a continuous process of remodeling. The process of bone remodeling begins with resorption of bone by 4

osteoclasts derived from the macrophage monocyte lineage followed by new bone synthesis carried out by mesenchymal progenitor cell derived osteoblasts. This cycle of degradation followed by deposition of bone is tightly coupled by receptor activator of NF-kappa-B/receptor activator of NF-kappa-B ligand (RANK/RANKL) interactions between RANK expressing osteoclast precursors and RANKL secreting osteoblasts. This coupling ensures that the amount of new bone generated is equal to the amount degraded. Imbalances in the amount of bone resorbed to the amount of bone replaced can cause either osteoporosis (low bone mass) or osteopetrosis (high bone mass). The bone remodeling cycle begins with recruitment of osteoclast progenitors from peripheral blood borne mononuclear cells to sites of bone remodeling. Production of two haematopoietic factors necessary for osteoclastogenesis, the tumor necrosis factor-related cytokine RANKL and colony stimulating factor-1 (CSF-1) (Lacey, et al., 1998, Yasuda, et al., 1998) by bone marrow stromal cells activates RANK which is expressed on the surface of haematopoietic and osteoclast precursor cells (Hsu, et al., 1999, Nakagawa, et al., 1998). Activation results in fusion of 10-20 mononuclear cells to form a fused multinucleate polykaryon known as the osteoclast. RANKL exists as both a type II transmembrane protein found on the surface of expressing cells and as a proteolytically released soluble form (Anderson, et al., 1997, Lacey, et al., 1998, Wong, et al., 1997). Expression of RANKL by osteoblasts (Hofbauer, et al., 2000, Theill, et al., 2002) activates osteoclasts at the sites of bone remodeling to adhere to the bone surface and begin releasing bone resorptive lytic enzymes tartrate- resistant acid phosphatase (TRAP), and cathepsin K (CATK) (Li, et al, 1999). 5

As bone is degraded by osteoclasts growth factors bound to extracellular matrix (ECM) are released and it has long been thought that this may play an important role in coupling of bone resporption to bone deposition during the process of bone remodeling. TGF-B1 is stored in bone ECM and release and activation of TGF-fl during bone resporption by osteoclasts (Dallas, et al., 2002, Hauschka, et al., 1986, S M Seyedin, 1985) can stimulate recruitment and proliferation of osteoblast precursors (Bonewald, 1999). Osteoblasts express osteoprotegrin (OPG), a decoy receptor for RANKL, in response to oestrogen and bone morphogenetic proteins (BMP) (Schoppet, et al., 2002, Udagawa, et al., 2000). Secretion of OPG binds and sequesters RANKL from interacting with RANK on osteoclasts. The result of this is apoptosis of osteoclasts and a retreat from the lytic phase of bone remodeling (Simonet, et al, 1997). This allows the the anabolic phase of bone remodeling in which osteoblasts undergo differentiation to become mineralizing osteoblasts to begin. 1.5 Mesenchymal Stem Cells 1.5.1 Introduction to Mesenchymal Stem Cells The concept of mesenchmyal stem cells (MSC) first arose with the observation that transplantation of bone marrow to extramedullary sites resulted in reconstitution of the hemopoietic and trabecular structures that was the result of proliferation and differentiation of stromal cells into osteoblasts (Tavassoli and Crosby, 1968). These stromal cells were later identified as a small subset of bone marrow cells that were distinct from haematopoietic cells and could be identified as plastic adherent fibroblast- 6

like cells that formed discreet colonies when seeded at clonal density. These colonies were observed to give rise to bone, cartilage, and adipose tissues (Friedenstein, et al., 1970, Friedenstein, et al, 1974, Friedenstein, 1980). While STRO-1 has been identified as a marker for human mesenchymal stem cells (Gronthos, et al., 1994, Simmons and Torok-Storb, 1991) the surface marker phenotype of mouse MSC remains controversial. Several groups have attempted to characterize the surface phenotype of culture expanded plastic adherent stromal cells in mice with varying results. One group found that cultured mouse mulitpotent adult progenitor cells (mMAPCs) are CD34-/44-/45-/c-kit-/Scal+ (Jiang, et al., 2002). Chimeric mice generated by injection of isolated mMAPCs from ROSA26 mice into 3.5 day old C57BL/6 blastocysts showed that cells bearing this cell surface phenotype contribute to many tissues including skeletal muscle, bone marrow, and blood (Jiang, et al., 2002). Another group showed that cultured primary plastic adherent bone marrow cells immunodepleted for lymphocyte markers CD34/45/1 lb are c-kit negative, and variably express CD44 and Seal (Baddoo, et al., 2003). This group went on to show that immunodepleted mMSC differentiate into adipocytes, chondrocytes, and osteoblasts in- vitro, and osteocytes in-vivo (Baddoo, et al., 2003). Still another group showed that cultured primary plastic adherent bone marrow cells that are Seal positive, CD34/45/44 negative, can differentiate into adipocytes, chondrocytes, and osteoblasts in vitro while those that are CD34/45 negative, CD44/Scal positive can differentiate into adipocytes and osteoblasts in vitro. While these studies are of value for determining the phenotype of mesenchymal progenitors in ex vivo culture the identification of the cell surface 7

phenotype of MSC from fresh bone marrow along with the elucidation of their niche is critical for studying the molecular mechanisms responsible for self renewal and differentiation of these cell populations. 1.5.2 Osteoblastogenesis Mesenchymal stem cell (MSC) derived osteoprogenitors develop into osteoblasts, responsible for deposition and ossification of bone matrix (reviewed in (Aubin, 1998) and (Westendorf, et al., 2004)). Osteoprogenitors are proliferative and express low levels of osteoblast markers, type 1 collagen (Coll) and bone alkaline phosphatase (bALP). Differentiation of osteoprogenitor cells gives rise to pre-osteoblasts which have limited proliferative capacity, and express increasing levels of bALP and parathyroid hormone related protein (PTHrP). Upon leaving the cell cycle, these cells become osteoblasts, which express type 1 collagen and function to lay down and ossify matrix. Osteoblast specific genes include osteocalcin (OCN), a variety of non-collagenous proteins, and high levels of bALP. Osteoprogenitors can be isolated from bone marrow stroma and calvaria and stimulated to complete osteogenesis in vitro. 8

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Chapter 2 Wnt Signaling in Bone Development, Post Natal Homeostasis, and Mesenchymal Progenitor Cells Summary Wnts represent a superfamily of extracellular growth factors that have been implicated in the development of all organ systems. In addition to bone morphogenic proteins (BMPs), there is increasing information of the role of a variety of signaling pathways on the development of mesenchymal derivatives and bone. Wnts have been shown to play important roles in development and maintenance of bone. Increasingly, wnts are understood to play crucial roles in the maintenance of adult stem cells and have been implicated in both repair and aging. The sections in chapter 2 will provide a brief summary of the wnt signaling pathway, with a specific focus on components shown to be important for development and homeostasis of bone followed by an in depth examination of Wnt signaling in these processes. Additionally, a discussion of Cannonical and Non canonical Wnt signaling in maintenance of mesenchymal stem and progenitor Cells will be presented. 13

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Abstract: Mesenchymal Progenitor Cells (MPCs) contribute to the development and maintenance of bone, fat, muscle and cartilage in mammals. Several lines of evidence implicate canonical Wnt signaling in maintenance of these stem cell populations. Wnt10b is a canonical Wnt ligand expressed in developing bone, calvarial osteoblasts, and multipotential mesenchymal progenitors isolated from adult bone marrow. Here we demonstrate that Wnt10b null mice exhibit defects in both intramembranous and endochondral ossification that present as cranial suture agenesis at post natal day four and an age progressive loss of trabecular bone starting between one and two months of age. Maintenance of normal adult bone requires both copies of the Wnt10b gene as heterozygous animals express similar reductions in trabecular bone density in aged mice. No difference was observed in osteoclast number or activity as assessed by analysis of serum CTx levels indicating that the age progressive osteopenia in Wnt10b null mice is not due to increased bone resorption but rather is the result of decreased bone deposition. Using in vitro colony forming unit assays we show that the loss in trabecular bone and suture agenesis is associated with a reduction in the number of bone marrow derived mesenchymal progenitors and MPC lineage derived osteoblasts and adipoblasts and calvaria derived osteoprogenitors. Analysis of osteogenic gene expression in primary bone marrow stromal cells and calvarial osteoblasts demonstrates reductions in expression of several osteoblast differentiation markers but no change in Runx2, an osteoblast associated transcription factor expressed in multi-potent MPC. Additionally, Wnt10b null bone marrow derived progenitors and calvarial derived osteoblasts express reduced levels of bone morphogenic proteins (Bmp's) and Bmp response genes. Stimulation of the Wnt pathway in primary calvarial osteoblasts results in up regulation of Bmp4 and associated Bmp responsive genes showing that Wnt10b is sufficient to induce expression of Bmp4 and Bmp target genes in calvarial osteoblasts. Taken together, this work indicates that Wnt10b is a critical Wnt signaling ligand, required for mesenchymal progenitor activity in both calvarial morphogenesis and maintenance of adult bone and suggest a role for Wnt10b in maintenance of immature osteo and mesenchymal progenitors capable of depositing bone.