Osteogenesis and Bone-Marrow-Derived Cells

M. W. Long

Blood Cells, Molecules, and Diseases (2001) 27(3) May/June: 677– 690 doi:10.1006/bcmd.2001.0431, available online at http://www.idealibrary.com on

Osteogenesis and Bone-Marrow-Derived Cells Submitted 05/11/01 (Communicated by M. Lichtman, M.D., 05/11/01)

Michael W. Long1 ABSTRACT: This paper addresses some of the important aspects of stem cell commitment to the bone cell lineage examining the various types of precursor cells, their responses to cytokines and other extracellular influences, and recent observations on the biochemical and molecular control of lineage-specific gene expression. The process of osteopoiesis involves the proliferation and maturation of primitive precursor cells into functional osteoblasts. The bone cells purportedly originate from mesenchymal stem cells that commit to the osteogenic cell lineage becoming osteoprogenitor cells, preosteoblasts, osteoblasts, and osteocytes. Further understanding of this developmental process requires that lineage-specific markers be identified for the various populations of bone cells and their precursors, that cell separation techniques be established so that cells of the osteogenic lineage can be purified at different stages of differentiation, and that these isolated cells are studied under serum-free, chemically defined conditions. © 2001 Academic Press

INTRODUCTION

CFU-f from both mice and human (6 –12). The relationship of these cells to both MSC and of CD34⫺ cell populations is unknown. To date, reports show that osteopoietic cells are found in bone marrow MSC populations, as well as CD34negative and positive populations. This paper focuses on cell populations with know osteogenic potential, and their extrinsic and intrinsic regulation.

Recently, a great deal of recent attention has focused on the ability of bone marrow derived cells to contribute to multiple tissues. A number of these studies indicate that bone marrow derived cells have osteogenic potential. It is not clear whether multiple lineages have osteogenic potential, or whether a single primordial stem cell gives raise to these and other cell types. The majority of these investigations point to mesenchymal stems cells as undergoing osteopoiesis2 when cultured in the presence of bone-active cytokines (1– 4). MSC, as currently used, are a heterogeneous population of cells isolated by plastic adherence, and propagated by low-density passage. Nonetheless, a recent publication indicates the clonal nature of cell fate outcomes in MSC indicating that a single MSC cell can give rise two or three mesenchymal lineages one of which is usually bone cells (5). These studies are consistent with earlier reports that demonstrated the osteogenic potential of bone marrow stromal cells, in particular the so-called

BONE CELL POPULATION HIERARCHY (OSTEOPOIESIS) The cellular hierarchy of bone precursor cells is best understood if bone precursor cell development is artificially divided into a number of developmental stages: mesenchymal stem cells (MSC), osteoprogenitor cells, preosteoblasts, and osteoblasts (Fig. 1). Such an artificial “compartmentalization” serves to categorize and describe the characteristics of subpopulations of all developing cell lineages, but belies the fact that these cells exist as a developmental continuum having no distinct boundaries and, hence, no distinct

Correspondence and reprint requests to author: Room 3570B, MSRB-II, 1150 West Medical Center, Ann Arbor, MI 48109-0688. E-mail: [email protected]. 1 Department of Pediatrics and The Comprehensive Cancer Center, University of Michigan, Ann Arbor, Michigan 48109. 2 Osteopoiesis ( . . . etic) is used to refer to the bone cell developmental process leading from the MSC 3 osteoprogenitor cell 3 preosteoblast 3 osteoblast. The term osteogenesis ( . . . genic) is reserved for the generation of mineralized matrix/bone by maturing osteoblasts. 1079-9796/01 $35.00 Copyright © 2001 by Academic Press All rights of reproduction in any form reserved

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FIG. 1. Bone cell populations. The derivation of these cells from bone marrow cell populations and the proliferation/ differentiation of these cells are referred to as osteopoiesis. This is distinctly different from the deposition of new bone, which is known as osteogenesis.

compartments. Mesenchymal stem cells are a pluripotent population capable of generating multiple stromal cell lineages. It is unclear whether these are derived from more primitive progenitors. Osteoprogenitor cells are committed to the bone cell lineage, being responsible for the expansion of osteoblast numbers, and proliferate in response to a number of mitotic growth factors. The preosteoblasts are transitional in nature, bridging the progenitor cells with the mature and osteoblasts. Osteoblasts are the cells of this lineage responsible for bone formation [for reviews, see (4, 13)]. Osteocytes are embedded within the bone lacunae/canniculi, and are felt to be the mature progeny of osteoblasts that play a role in calcium/ phosphate homeostasis and mechanosensation/ functional adaptation. These latter two cell types are not discussed in this review.

eages of cells, and as having the capacity for self-renewal. This is best demonstrated by the hematopoietic stem cell that gives rise to eight lineages of blood cells, and is capable of selfrenewal (as demonstrated in serial transplantation experiments). The adult bone marrow also contains a putatively separate population of cells (i.e., MSCs) that generate cells of bone, cartilage, muscle, adipose, and tendon phenotype. While many investigators have adopted the MSC nomenclature to refer to the mesenchymal nature of marrow stromal cell populations, only recently has the clonal nature of MSC been proven. Thus, singlecell isolation of human MSC generated clones that express the same surface phenotype as unfractionated MSC (5). Interestingly, of the six MSC clones evaluated, two retained osteogenic, chrondrogenic and adipogenic potential; others were bipotent (either osteo- plus chondrogenic potential, or osteo-adipocytic potential) or were unilineage (chondrocyte). This suggests that MSC themselves are heterogeneous in nature (although

Mesenchymal Stem Cells Stem cells are classically defined as cells having the potential to clonally produce multiple lin678

M. W. Long

Blood Cells, Molecules, and Diseases (2001) 27(3) May/June: 677– 690 doi:10.1006/bcmd.2001.0431, available online at http://www.idealibrary.com on

culture conditions also may have led to loss of lineage potential). To date, the self-renewal capacity of MSC remains in question. Nonetheless, these in vitro studies and other in vivo studies (14 –16) show that MSC can commit to the bone cell lineage and develop to the state of matrix mineralization in vitro, or bone formation in vivo.

potential (18, 27). However, only portions of these are alkaline phosphatase-positive CFU-f, and only a portion of the alkaline-positive CFU-f are truly osteogenic CFC (17). When cultured under mineralizing conditions, these colonies allow the evaluation of osteoprogenitor cells as demonstrated by the formation of mineralized foci of bone cell development (i.e., “bone-nodules”). Such progenitor cells also are referred to as bone nodule-forming cells, or, simply, osteoprogenitor cells. These cultures have the advantage of containing the complete repertoire of cell types (both osteogenic and non-osteogenic) necessary for bone-nodule formation (28, 29). They were first used to delineate the process of osteogenesis, and to define the developmental hierarchy of murine bone lineage cells (30). These assays also demonstrate the presence of differing classes of osteoprogenitor cells as indicated by their colony characteristics and differential sensitivity to dexamethasone (31, 32). Interestingly, neither CFU-f nor CFU-osteoblast demonstrate linearity in limiting dilution analysis until very high plating densities are reached, thus showing a requirement for accessory cells in osteoprogenitor cell development. Thus, the addition of nonadherent bone marrow cells (or their conditioned media) to developing osteoprogenitor cells, restores colony clonality (31). As well, mineralizing colonies of osteoprogenitor cells (in this case using cells from fetal rat calvaria) are heterogeneous, being classified as fibroblastic or osteoblastic (33). Singlecell and single-colony studies demonstrated that different repertoires of osteoblast markers are expressed in different cells, again suggesting marked heterogeneity in the osteoprogenitor cell and/or osteoblast phenotype. Human studies have confirmed and extended the observations made in rodent systems. Importantly, human allogeneic bone marrow transplants (BMT) have been used to treat osteogenesis imperfecta (34), thus demonstrating the presence of osteopoietic stem cells (of unknown origin) in the transplanted marrow, and the clinical utility of BMT in treating bone disorders. Recent in vitro investigations address changes occurring during aging and in disease. Osteoprogenitor cell num-

Osteoprogenitor Cells A progenitor cell is functionally defined as being able to clonally generate cells of one or more lineages, but as lacking in self-renewal capacity. The in vitro correlate of this function is that progenitor cells give rise to colonies of cells that show a zero-intercept, linear relationship with the number of cells cultured (i.e., one cell gives raise to one colony at limiting cell dilutions). It remains unclear whether bi- and tripotential restricted stromal progenitor cells exist for the mesenchymal cell lineages, as they do for hematopoietic progenitor cells. While such cells have been reported, problems with plating density or lack of clonality studies make interpretation of the data difficult [for a review, see (17)]. Clearly, the MSC data discussed above suggest that such cells exist. However, it is difficult to determine the extent to which progenitor cell plasticity plays a role in these observations. For example, hypertrophic chondrocytes express a number of bone cell proteins such as osteopontin, bone sialoprotein, osteonectin, PTH receptor, and alkaline phosphatase (17) Finally, it needs emphasis that many osteoprogenitor cell assays differ in source of cells (e.g., bone outgrowth, fetal calvaria, bone marrow), type of growth factors added, and species used, all of which makes comparisons between laboratories difficult. Led by the studies of Friedenstein and Owen, a number of reports documented the in vitro and in vivo osteogenic capacity of rodent bone marrow-derived stromal cells (6, 18 –26). These cells are characterized as a subpopulation of adherent bone marrow cells that form colonies of fibroblast-like cells, hence the term the fibroblast colony-forming cell (i.e., the CFU-f). In culture, the CFU-f generate a heterogeneous population of progeny, some of which have clear osteogenic 679

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bers appear to decrease sharply with age and in osteoporosis (35–37). Human osteoprogenitor cells have been cultured from both adherent (35, 36, 38) and nonadherent (39 – 42) populations of human bone marrow, although the relationship between the two phenotypes is unknown. Human osteoprogenitor cells show considerable developmental heterogeneity. They are antigenically distinct subpopulations (see below) (12, 40, 43– 46), and show differential responsiveness to growth factors (40, 47, 48). Importantly, this population of human bone marrow-derived cells is negative for the hematopoietic marker CD34, as shown by flow cytometry and RT-PCR (40, 45). These properties therefore demonstrate the existence of functionally distinct subpopulations of cells. Using limiting dilution, clonal colony assays, our laboratory has identified two maturational classes of human osteoprogenitor cells, distinguished by their proliferative potential, and responsiveness to stimuli that are analogous to cluster-forming and colonyforming hematopoietic progenitor cells (40). Other studies from our laboratories, using highly purified populations (⬵5000-fold purification) of human bone precursor cells (a population of immunologically isolated osteoprogenitor cells and preosteoblasts) (45, 46), demonstrate that there is an obligate requirement for bone marrow accessory cells in the early phases of human osteopoiesis (46). These cells, termed osteopoietic accessory cells (OAC), are purified from human nonadherent bone marrow cells and lack antigenic markers of bone cells, or those typically found on bone marrow accessory cells such as macrophages, T-cells and NK-cells. In the absence of OAC, human bone precursor cells die, despite the presence of multiple bone-active cytokines. The OAC produce a soluble regulator that reconstitutes both the proliferation and differentiation of purified human bone precursor cells. Lastly, it should be emphasized that, while animal models are an important part of the discovery process, they do not faithfully mimic human biology. Indeed, a recent careful study by Robey and co-workers demonstrated distinct differences in the in vivo capacities of murine and human bone progenitor cells, with human bone

M. W. Long

formation being more dependent on prior ex vivo expansion, and implantation vehicle (49). Preosteoblasts Preosteoblasts represent a transitional state between the highly proliferative osteoprogenitor cell and the mature osteoblast. As such, they express relatively low levels of bone cell proteins such as alkaline phosphatase (12, 40), and/or other bone proteins such as osteonectin, osteopontin, etc. (50, 51). Preosteoblasts are predominately defined morphologically, based on their localization adjacent to active osteoblasts (either in tissue sections or in vitro) (52, 53). A number of studies have used rodent cell lines to characterize preosteoblast development (54 –58). These have provided important information as regarding growth factor responsiveness (43, 59, 60), signal transduction (54, 61), and mineralization (55). As with osteoprogenitor cells, preosteoblasts also show distinct age-related changes in the expression of bone proteins such as osteonectin, osteopontin, and osteocalcin (45, 62). To date, studies of isolated preosteoblast populations are limited. As discussed in the next section, our laboratory and others have used or defined preosteoblast antigenic determinants for purification studies. Phenotypic Markers of Bone Progenitor Cells Unlike the blood or immune systems, a large number of developmental stage-specific antigenic markers are not available for bone cells. However, some do exist, and have been used to isolate purified populations of cells. Turksen and Aubin first demonstrated that immune adhesion, using monoclonal antibodies to alkaline phosphatase, resulted in the segregation of rodent bone noduleforming cells in the alkaline phosphatase-positive fraction, with few spontaneously forming bone nodule-forming cells observed in the alkaline phosphatase-negative fraction (63). Interestingly, the alkaline phosphatase-negative fraction of cells contained greater numbers of dexamethasone-sensitive bone nodule-forming cells (6-fold increase over the alkaline phosphatase-positive fraction), demonstrating the presence of a more primitive 680

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Blood Cells, Molecules, and Diseases (2001) 27(3) May/June: 677– 690 doi:10.1006/bcmd.2001.0431, available online at http://www.idealibrary.com on

population of osteoprogenitor cells which requires dexamethasone stimulation to proliferate and/or differentiate. Murine cells that express the murine hematopoietic antigen Sca-1, and avidly bind the lectin Wheat Germ Agglutinin, show an osteogenic nature (64). These cells were observed to be large (high forward-angle light scatter during flow cytometry) and have an increased intracellular complexity (as indicated by light side-scatter characteristics). Unfortunately, the reported isolation procedure caused cell-damage, limiting the functional assessment of the role of SCA-1⫹ cells in osteogenesis (based on flow cytometry data from our laboratory, these cells would be defined as osteoblasts— given their high forward-angle scatter and side-scatter characteristics). Two recent studies have identified (uncharacterized) antigenic determinants that seemingly distinguish human osteoprogenitor cells from osteoblasts. The SH2, SH3, and SH4 antibodies, derived using MSC as an immunogen, react with bone progenitor cells, but not with osteoblast or osteocytes (65). Likewise, hOP-26, derived using bone marrow fibroblast cultures, identifies marrow osteoprogenitor cells and not blood cells (66). An antigenic determinant known as STRO-1 marks human bone marrow stromal cells (67). STRO-1 identifies clonogenic bone marrow stromal progenitor cells (CFU-f) in adult bone marrow (11). STRO-1-positive CFU-f are heterogeneous, giving rise to fibroblasts, adipocytes, and smooth muscle cells (67). When placed in mineralizing conditions (inorganic phosphorous, dexamethasone, ascorbic acid) a portion of STRO-1positive CFU-f become alkaline phosphatase positive, respond to 1,25-dihydroxy vitamin D3 (1,25-OH D3) with increased osteocalcin production, and within 4 weeks undergo mineralization (11). Thus, some STRO-1-positive bone marrow CFU-f are clearly osteogenic in nature. Combined Stro-1 and alkaline phosphatase (AP) based cell sorting allows further characterization of these cells (12). These data show that preosteoblasts (characterized by decreased proliferative potential and matrix protein expression) were Stro-1pos and APneg, whereas osteoblasts were either APpos, or negative for both markers. Our laboratory has demonstrated the isolation

and purification of human bone precursor cells utilizing immunology-based technology (39, 45, 46, 68). These cells are purified from nonadherent, low-density (NALD) population of bone marrow cells, lack the hematopoietic marker CD34, or the megakaryocyte/platelet marker, glycoprotein IIb/IIIa (40), and cannot be isolated by alkaline phosphatase-based immune adhesion. In contrast to the murine Sca-1-positive cells, human bone precursor cells are characterized as smallsized cells that express low amounts of bone proteins (osteocalcin, osteonectin, and alkaline phosphatase) and have a low degree of internal complexity (i.e., laser light side scatter characteristic). When stimulated to differentiate with TGF␤1, these preosteoblast-like cells become osteoblast-like in their appearance, size, antigenic expression, and internal structure (39, 40), Immune isolation of human bone precursor cells also cosegregates a osteoprogenitor cell population. Interestingly, when cultured under conditions leading to three-dimensional tissue-like cell growth, these human preosteoblast-like cells (referred to herein as human bone precursor cells) are capable of generating microcrystalline human bone ex vivo (68). OSTEOGENIC GROWTH FACTORS Like other developing tissues, bone responds to bone-specific, and other soluble growth factors. It is therefore important to understand that the appropriate growth factors are required to discover the osteogenic potential of isolated cell populations. TGF-␤ is a member of a family of polypeptide growth regulators which affects cell growth and differentiation during developmental processes such as embryogenesis and tissue repair (69). TGF-␤ strongly inhibits proliferation of normal and tumor-derived epithelial cells, blocks adipogenesis, myogenesis, and hematopoiesis (69). However, in bone, TGF-␤ is a positive regulator (69). In addition to TGF-␤, other growth factors or cytokines are implicated in bone development. A number of bone morphogenic proteins (BMP), originally identified in an extract of demineralized human bone matrix (70), have now been cloned (71, 72). Additional growth factors also regulate 681

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bone development, but seem to function at a different level from the BMPs that may be lateacting regulators (73). For example, bone-derived growth factors (BDGF) stimulate bone cells to proliferate in serum-free media (74, 75). Basic fibroblast growth factor (bFGF) plays a role in both in vivo and in vitro bone cell development (76 –78). Recombinant human bFGF stimulates osteoprogenitor cell proliferation (79), and also stimulates fracture-repair (in rats), increasing the volume and mineral content of the bone in a dose-dependent fashion (78). Of the multiple cytokines implicated in osteogenesis, two factors play a central role in bone cell development: TGF-␤ and insulin-like growth factor (IGFs). IGF-II and TGF-␤ thus represent the first and second most abundant mitogens in human bone extracellular matrix (ECM). TGF-␤ is localized in active centers of bone differentiation (cartilage canals and osteocytes) (80), and is found in high quantity in bone, suggesting that bone contains the greatest total amount of TGF-␤ (80). During bone formation, TGF-␤ promotes chondrogenesis (80), an effect presumably related to its ability to stimulate the deposition of matrix components (81). Besides stimulating cartilage formation, TGF-␤ is synthesized and secreted in bone cell cultures, and stimulates the growth of sub-confluent layers of fetal bovine bone cells, thus showing it to be an autocrine (or paracrine) regulator of bone cell development (69). Like TGF-␤, the IGFs are found in high concentrations in bone. In fact, IGFs are the most abundant growth factors in bone [(82) and references therein]. Both TGF-␤ and the IGF proteins are involved in the coupling of bone reabsorption to bone formation. TGF-␤ is present in bone as a latent complex in bone ECM, and is believed to be activated by the osteoclast’s acidic environment (83, 84). Increased bone reabsorption thus results in an increased release and activation of TGF-␤ which subsequently stimulates osteogenic cells. On a cellular basis, IGFs function to stimulate osteoblast proliferation (85– 87), activating MAP kinases such as ERK-1 and ERK-2 (88) and targeting early response genes such as c-myc (89). Importantly, IGFs are dysregulated in postmenopausal osteoporosis (90), and are decreased dur-

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ing caloric restriction (91). IGFs are synthesized by osteoblasts and as such may serve autocrine or paracrine functions (87). Moreover, osteoblasts respond to hormonal signals such as increased growth hormone or calcitonin by increasing IGF synthesis (87, 92). The roles of IGFs in bone formation are complex, and reports are often contradictory. It is well understood that IGFs function in relation to their binding proteins (IGFBPs) IGFs are found in bone, but are complexed with IGF-binding proteins (82, 93). These IGF-binding proteins (IGFBPs) inhibit the biological actions (proliferation and matrix synthesis) of IGF in a dose-dependent manner (93). All six of the IGFBPs bind IGF with high affinity and, therefore, inhibit IGF function. Interestingly, the IGFBPs show skeletal or sitedependent differences in distribution (94), with human trabecular osteoblasts producing IGFBP3, IGFBP4, and IGFBP5 (92). TGF-␤ may be part of this regulatory loop as it is known to both stimulate osteoblast development (69) and to inhibit the production of IGFBPs 4 and 5 (92). IGFBPs also are reported to augment IGF actions on osteoblast development. Thus, IGFBP3 internalization and processing is reported to markedly enhance IGF receptor signaling, a process that requires phosphatidylinositol-3 kinase activation but not the activation of MAP kinase (95). These affects require pretreatment with IGFBP3 that, alone, had no effect. Likewise, IGFBP5 stimulates osteoblast proliferation in vitro or in vivo, either alone or in combination with IGF (96). Therefore, bone reabsorption (as with TGF-␤) releases active IGFs that subsequently stimulate osteogenic cells in a paracrine manner (82). IGFs are synthesized by osteoblasts and as such may serve autocrine or paracrine functions (87). Moreover, osteoblasts respond to hormonal signals such as increased growth hormone or calcitonin by increasing IGF synthesis (87, 92). Bone extracellular matrix also contains both collagenous and non-collagenous proteins that are involved in bone formation. Osteonectin is a 32kDa protein which, binding to calcium, hydroxyapatite, and collagen, is believed to form a link between the mineral and organic phases of bone tissue (97). In vivo analysis of osteonectin mes682

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sage reveals its presence in a variety of developing tissues (98, 99). However, it is present in its highest levels in bones of the axial skeleton, skull, and the blood platelet (megakaryocyte) (98). Osteocalcin, also known as bone gla protein (BGP), is a vitamin K-dependent, 5700-Da calcium binding bone protein, that normally functions to limit bone formation without impairing resorption or mineralization (100 –102). AS mentioned, bone ECM also is the site of localization for a number of the growth factors mentioned above such as TGF-␤, IGF-I, or IGF-II. These activities are capable of stimulating the proliferation of mesenchymal target cells (BALB/c 3T3 fibroblasts, capillary endothelial cells, and rat fetal osteoblasts) (73, 75, 82, 93, 103, 104).

teins, and is expressed in mesenchymal cells that develop into chondrocytes or osteoblasts (107) [for reviews, see (14, 108), and references therein]. Cbfa1 haploinsufficiency in mutant mice causes skeletal abnormalities consistent with cleidocraniodysplasia and, indeed, humans with this disorder have Cbfa1 mutants (109). Homozygous Cbfa1-null mice show normal skeletal patterning, but the skeleton is cartilaginous as osteoblast differentiation fails to occur in these animals (90, 110). TWIST is a basic helix–loop– helix (HLH) transcription factor that also seems to play a role in osteoprogenitor cells. In human SaOS2 cells, its overexpression maintains a more osteoprogenitor cell-like phenotype, whereas inhibition by antisense oligonucleotides increases alkaline phosphatase and Type I collagen expression (111). TWIST is also expressed in preosteoblasts and is upregulated by FGF (60). Interestingly, another transcription factor, Id, a dominant negative HLH believed to suppress bHLH transcription factors, is regulated by BMP-2, suggesting that these two transcription factors and cytokines are part of a single regulatory circuit (112). The homeobox gene Msx2 is implicated in osteoprogenitor cell function. Msx2a a homologue of the Drosophila muscle segment Msh gene, is expressed primarily in proliferating cells of the bone lineage (i.e., osteoprogenitor cells and preosteoblasts) (113) and is seen embryologically in early developing bone (105). Mice deficient in Msx2 show a marked delay in ossification of the bones of the skull (114). Msx2 also appears to be an upstream regulator of Cbfa1, as it is downregulated in Msx2-mutant mice. Likewise, the Bapx1 transcription factor seems to play a role in chondrocyte condensations, the skeletal patterning of the vertebral bodies, and bones at the base of the skull (115, 116). Finally, some mention must be made of Dlx5 a murine member of the Distal-less gene family. Although restricted to osteoblast in its expression, it is the only known transcription factor that distinguishes mineralizing osteoblasts and thus serves an inverse role to that of Msx2. While transcription factor regulation of commitment and differentiation is important, downstream regulators such as signal transduction molecules undoubtedly play a role in maintaining the

Molecular and Biochemical Control Ultimately, full understanding of bone cell development and bone formation rests on understanding the molecular events occurring during these processes. Recent studies demonstrate that transcription factors regulate the expression or maintenance of the osteo-phenotype, and that homeobox genes are important in the control of skeletal pattern formation. Together with biochemical studies of the control of osteoprogenitor cell proliferation and differentiation, these investigations begin to build a picture of osteopoiesis and osteogenesis at the molecular level. Transcriptional factors are nuclear proteins that regulate the activation state of multiple other genes, typically as part of a larger regulatory protein complex. The regulated genes usually correspond to those required for lineage-specific differentiation. Certain transcription factors, such as MyoD1, function as master regulatory switches capable of conferring lineage phenotype even when expressed in heterogeneous cell types. Within the bone cell lineage, the transcription factor Cbfa1 plays an important role in maintaining the osteo-phenotype (105), although a distinct “master” gene remains to be identified. Cbfa1 (core-binding factor-1 also know as AML-3 or PEBP2␣A) is the first osteoblast-specific transcription factor to be identified (106). It is a homologue of the Drosophila runt and lozenge pro683

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osteoblast phenotype. For example, Fgd1 is the gene responsible for Aarskog syndrome (faciogenital dysplasia; FGDY), an X-linked disorder that adversely affects multiple skeletal structures. It was identified as a Rho/Rac guanine nucleotide exchange factor that activates Cdc42 (117). The murine ortholog of Fgd1 is initially expressed during the onset of embryonic ossification in regions of active bone formation: in the trabeculae and diaphyseal cortices of developing long bones (118).

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bone. Studies of embryonic chick (calvarial or limb-bud) cells also confirm the cell density-mediated induction of chondrogenesis, and demonstrate an obligate requirement for cell:cell interaction in this process (121, 124). During embryonic/fetal osteogenesis primordial cells differentiate into preosteoblasts and then undergo cellular condensation prior to ossification (125). Likewise, in vitro calvarial or bone marrow-derived osteogenic cells grown on two-dimensional (i.e., planar) surfaces eventually lead to a localized piling of confluent cells into “bone nodules” (i.e., three-dimensional adherent cell arrays) that mineralize the surrounding ECM (28). Such observations strongly suggest that cell-density plays a role in the process of bone formation. Confirming this, we recently reported that serumfree TGF-␤1 treatment of human bone cells derived from multiple sources results in the formation of spherical three-dimensional cellular condensations (herein termed bone cell spheroids), and the upregulation of bone proteins such as osteonectin and alkaline phosphatase (68). At appropriate cell densities, these bone cell spheroids produce crystalline structures (termed microspicules) consisting of organized human bone. This study clearly demonstrates that the three-dimensional growth of human osteoblasts in tissue-like aggregates is a prerequisite for bone formation, and provides a model system for evaluating cell populations capable of bone formation.

THREE-DIMENSIONAL BONE TISSUE DEVELOPMENT The development of bone requires the concerted action of a number of microenvironmental signals: cytokines/growth factors, extracellular matrix (ECM) molecules, and cell:cell interactions (119). Such regulatory signals, expressed in a temporal and spatial order, result in a developmental microenvironment that facilitates threedimensional cell growth. Cellular condensation, a process of cell aggregation mediated by mesenchymal:epithelial cell interactions, plays a crucial role during skeletogenesis affecting both chondrogenesis and osteogenesis (53). During osteogenesis, preosteoblast condensation precedes osteoblast differentiation and matrix mineralization (53). Studies of perichondrocytes also demonstrate that cell condensation is cytokine-mediated, and that cytokines induce changes in the expression of a number of developmentally important genes. Either TGF-␤1 or BMP2 stimulate chondrocytic condensation and upregulate ECM proteins (120). This requisite step of cellular condensation during mesenchymal chondrogenesis is mimicked in vitro in chondrocyte cultures where high cell density results in the formation of three dimensional spheroid structures that are cartilaginous in nature (121–123). These chondrocytic cellular-condensations are associated with the upregulation of extracellular matrix components such as Type II collagen and cartilage link protein (121). Notably, these manipulations do not result in the chondrocytes differentiating into osteoblasts, the mineralization of matrix, nor the formation of either bone nodules or microcrystalline

CONCLUSIONS AND PERSPECTIVES An increased understanding of the cellular and molecular basis of bone precursor cells and the lineages they are derived from will have an immediate impact on clinical medicine. While the molecular and/or biochemical basis of many bone disorders remains unknown, a number of studies (addressed above) demonstrate that a molecular or cellular basis or component is involved. To effectively control bone progenitor cells production and differentiation, both normal and abnormal development must be examined at the cellular, biochemical and molecular level. This requires that the various lineages of cells that contribute to osteopoiesis/osteogenesis be defined. Similarly, 684

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the signal transduction pathways activated by these osteopoietic/osteogenic signals must be understood. The understanding of bone formation thus requires the identification of those genes that regulate mesenchymal cell commitment, osteoprogenitor cell proliferation, differentiation, and mineralization. The ability to purify various bone cell phenotypes, when combined with the power of molecular analyses such as transcriptisome profiling, will greatly advance our understanding of these processes. Likewise, the use of tissue-like systems to study bone cell development, or bone tissue engineering, also is an emerging area that requires an understanding of bone progenitor cells and their microenvironment [see articles and reviews in Science (289(No. 5484), Sept. 1, 2000) and Nature Biotechnology (18(No. 9), Sept. 2000)]. Finally, studies of aging populations or bone disorders will provide important information regarding the physical, biological, and molecular mechanisms of bone formation.

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ACKNOWLEDGMENT This paper is based on a presentation made at the Focused Workshop on Stem Cell Plasticity sponsored by The Leukemia & Lymphoma Society and the Great Basin Foundation for Biological Research in Santa Barbara, California, on May 4 and 5, 2001.

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