The Skeletal Stem Cell

C H A P T E R

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The Skeletal Stem Cell Dongsu Park*, Jonathan Hoggatt*, Francesca Ferraro*, David T. Scadden Center for Regenerative Medicine, Massachusetts General Hospital, Boston, MA; Department of Stem Cells and ­ egenerative Biology, Harvard University, Cambridge, MA; Harvard Stem Cell Institute, Cambridge, MA, USA R *Equal contribution

INTRODUCTION The lineage relationships and kinetics of the cellular components of bone are the focus of this chapter. Tissues in mammals are generally maintained by one of two schemas, mature cell replacement by mature cell division or mature cell replenishment from a more immature stem or progenitor population. It is the latter model that applies to bone and we will review here the definition of the skeletal stem cell (SSC), its relationship to descendent semi-committed osteoprogenitors, and the terminally differentiated mesenchymal cells (MC) that make up the mature bone. In addition, we will discuss the migration of mesenchymal osteoprogenitors, their kinetics in regulation of life-long bone production, and the interaction of bone cells with hematopoietic cells in the bone marrow.

DEFINING SKELETAL STEM CELLS The human skeleton is formed during embryogenesis and is estimated to undergo complete turnover four to five times in an average human lifetime [1]. Maintenance of bone tissue is thought to be accomplished by a pool of stem cells. Stem cells are defined by two cardinal features: their ability to self-renew, thereby providing durable replenishment of themselves and their descendants, and their ability to differentiate to achieve specific mature cell fates. Stem cells are therefore functionally defined. Their most rigorous experimental definition requires that they achieve their two cardinal functions in vivo and on a clonal (single cell) level. This has been achieved with relatively few stem cell types to date and is best exemplified by the hematopoietic system. In that

Osteoporosis. http://dx.doi.org/10.1016/B978-0-12-415853-5.00007-8

context, single cells have been shown on transplantation to repopulate all blood lineages in a recipient including the production of new stem cells that can repopulate a second recipient. It is serial transplantation that is used as an indicator of in vivo self-renewal. The accomplishment of the experimental definition of a stem cell is not possible in humans and therefore much of what we understand about stem cells in general and SSCs in particular is derived from animal models. The mouse has been the most common animal model used either by examining mouse cell functions in vivo or studying human cells transplanted into immunodeficient mice. The information discussed in this chapter should, therefore, be viewed with these caveats in mind and recognition that not all conclusions that will be presented from experimental work can be rigidly assumed to hold true in humans. It is also important to realize that the term “stem cells” is often applied to cells that have not met the rigorous definitions indicated above. This is particularly problematic with bone-derived MCs where the term “mesenchymal stem cells” (MSC) has been applied to a wide range of human and mouse cells. Many cells called MSCs have been so labeled because of their functions in vitro not in vivo. Such assessment may or may not reflect the cell's function in the body as MCs are notoriously plastic when cultured. A note of caution to the reader then on equating an MSC with true, functionally defined SSCs: these cells cannot, and should not, be assumed to be the same. Because the MCs that are grown ex vivo have extensive medical testing and interest, but lack rigorous confirmation that they reflect an in vivo stem cell population, they will be termed here as mesenchymal cells (MC).

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Copyright © 2013 Elsevier Inc. All rights reserved.

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When considering SSCs, it should also be recognized that there may be different types of SSCs. Multiple cell types comprise the bone and they may be derived from shared or distinct stem cell types. For example, stem cells may produce all the mesenchymal lineages of the bone, as is proposed for putative “MC” or they may produce only a single cell type, such as osteolineage cells, a heterogeneous cell population comprised of bone-lining cells across a spectrum of differentiation and cells incorporated within calcified bone (osteocytes). In this chapter, we will define a “skeletal stem cell” (SSC) as a cell that is harvested from bone and meets the experimental criteria for a stem cell in vivo. Where rigorous experimental definition is lacking, we will point out the limitations. The cells will often also be further delineated as “multipotent-SSCs”, those capable of giving rise to skeletal tissues including osteogenic, adipogenic, and chondrogenic cells, or “osteogenic-SSCs”, those restricted to osteogenic differentiation, but maintaining stem cell features. The distinction between stem cells and progenitor cells also merits comment. Progenitor cells are often highly proliferative components of a tissue that are intermediate in their differentiation state. They are destined to differentiate and do not generally have self-renewal capability. Progenitor cells are, therefore, distinct from stem cells in their self-renewing ability. Because they are intermediate in differentiation state and are often capable of rapid proliferation, progenitors are referred to as a transit, amplifying population. Clear boundaries between the stem and progenitor pools of cells exist conceptually, but are often difficult to define experimentally or by cell markers; therefore, it is common for primitive cells to be clumped together in one heterogeneous description as stem/progenitor cells.

Developmental Origin of Bone and Skeletal Stem Cells The skeletal system derives from the mesodermal layer of the embryonic disc. Intra-embryonic bone is formed either by direct ossification of embryonic connective tissue (intramembranous ossification) or by replacement of hyaline cartilage (endochondral or intracartilaginous ossification) [2]. Intramembranous ossification forms the skeletal infrastructure of the head (roof of the skull and most bone of the face) and begins in the primordial mesenchyme. As the primordial mesenchyme becomes increasingly vascularized, proliferating MCs give rise to osteoprogenitors, which subsequently develop into mature osteoblasts. As osteoblasts collect, they begin to lay down the osteoid, an organic unmineralized portion of the bone matrix. The osteoid is then mineralized with hydroxyapatite crystals in a centrifugal manner beginning at the center of ossification. Concurrently, the

spicules of spongy bone develop and group together to form the trabeculae. As the bone compacts, osteoblasts become entrapped in the newly formed bone and are then referred to as osteocytes. Endochondral ossification takes place at the base of the skull, vertebrae, hips, and limbs through the replacement of a cartilaginous rudiment with bone. The process begins with the formation of the primary ossification center, localized in the diaphysis. Here, the cartilage cells become hypertrophic and the cartilage matrix calcifies. In parallel, the outside portion of the chondrogenic blastema evolves into a perichondrium that subsequently develops into the primitive periosteum. Osteoprogenitors in the periosteum contribute to the formation of a bony collar, and then subsequently certain areas of the calcified matrix disintegrate, opening cavities that then communicate with connective tissue and vessels at the surface that invade the developing marrow cavity. The bone collar holds together the shaft of the bone, which has been weakened by the disintegration of the cartilage. Once bone growth is completed, SSCs capable of forming bone, cartilage, and fat remain in the bone marrow and contribute to renewal of the skeleton throughout life [3].

Isolation and Identification of Skeletal Stem Cells The first experiments demonstrating that postnatal SSCs might exist were performed in the late 1960s, showing that transplantation of bone marrow-derived cells in heterotopic sites was capable of generating a bone template that was then repopulated by marrow cells, thus recapitulating embryonic development [4,5]. In 1970, Friedenstein identified that osteogenic potential was specifically attributable to a small subset of bone marrow cells that were adherent, spindle-shaped, and capable of forming fibroblast colonies (colony-forming unit-fibroblast (CFU-F)) [6]. Much like studies exploring hematopoietic stem and progenitor cells (HSPC) at the time, the identification of a CFU-F allowed for the establishment of clonogenicity, demonstrating that resultant experimental effects were due to a single cell. These cells, after in vitro culture forming CFU-F, were implanted subcutaneously in mice and demonstrated the ability to produce bone (ossicles) [4,6–8]. These cells were also capable of serial transplantation into mice with formation of new ossicles, presumably demonstrating their self-renewal ability [9]. More recently, it has been demonstrated that the cell type in humans that forms colonies, can be transplanted and generates bone and bone marrow and is capable of secondary colony formation, is marked by the expression of the antigen, CD146 [10]. These cells were capable of serial transplantation and ossicle formation at heterotypic sites at a clonal level in immunodeficient mice [10]. This is the most compelling evidence for an

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Migration of Skeletal Stem Cells

SSC in humans. The only caveat in interpreting these studies is that the cells were assessed after culture rather than as freshly isolated cells. It is, therefore, possible that characteristics of the cells changed during the time of in vitro culture. Also of note, the cells that were generated both in vivo and in vitro were of the osteolineage. Whether the cells also made other MC types in vivo was not assessed and, therefore, it would be proper to consider the CD146+ cell as an osteogenic-SSC. The use of antigenic markers to subselect for cells is exceptionally important for studies of stem cell function and number. The study of CD146+ cells therefore represents an important advance in that it validated the functions of the antigen bearing cells. Other markers have also been studied though with perhaps less rigor and are summarized in Table 7.1. The reader is reminded that the antigens studied have enabled fractionation of bone marrow cells to enrich for cells with SSC features by flow cytometric cell sorting. However, there is not a clear method by which to purify SSC to unity (each cell having SSC features). Some of these markers have also been used to evaluate the location of SSCs in the bone by immunohistochemistry. For example, human cells expressing STRO-1 [11,12], CD105 [13], and CD146 surround blood vessels in vivo (Fig. 7.1). A perivascular location for the SSC is now an accepted concept in the field. It has raised the issue of whether SSCs are equivalent to, or are a subset of, so-called pericytes. It has also raised the issue of whether the cells are capable of trafficking into and out of the vasculature. As might be expected from the interest in SSC and the relative ease of using the model, much work has been done on SSC in the mouse. In one such example, green fluorescent protein (GFP) was driven by the type I collagen (ColI) promoter in a genetically engineered mouse. Whole bone marrow taken from the animal was transplanted into lethally irradiated recipient mice to test whether transplanted SSC could engraft in a manner similar to blood stem cells [93]. GFP-positive cells were tracked within the bone of the transplanted animals and could be seen lining endosteal and trabecular bone, suggesting that SSC transfer had occurred. There was no evidence for the cells progressing to osteocytes, and when the cells were cultured, they did not form mineralized bone nodules, but these studies were indicative of a transplantable osteolineage cell [93]. Others have used flow cytometry to define subpopulations of bone marrow cells and evaluated their ability to form osteoblasts and adipocytes as evidence for SSC. Using the markers, PDGFRα+, Sca-1+, CD45–, and TER119–, it was possible to demonstrate that this population could engraft and form osteogenic and adipogenic tissue, again supporting the notion of a transplantable SSC [94]. Others have tried to use in vivo labeling of defined subsets of MCs to track whether they are capable of

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functioning as stem cells in the mouse. These so-called lineage tracing studies have indicated that Nestin [95,96] and Mx1 [96] labeled populations of bone marrow perivascular cells with characteristics of SSCs. When the progeny of adult Nestin-expressing bone marrow cells were tracked by using a genetically engineered mouse, the cells were found to contribute to skeletal remodeling through differentiation into cartilage- and bone-forming cells [95]. Interestingly, their contribution to osteoblasts and chondrocytes required 8 months to be visualized, suggesting that these cells may be quiescent and that there may be distinct stem/progenitors that more readily and rapidly contribute to osteogenesis. Perivascular Nestin-positive cells have also been described in histological sections of human bone marrow biopsies [97] (Fig. 7.2). Mx1 is an interferon-inducible promoter that has been used to label cells that contribute to durable osteogenesis over time and can be serially transplantable. This may, therefore, also be a marker for endogenous SSCs [96]. A difference between these two models is that, unlike the delayed appearance of osteoblastic progeny from Nestin-labeled bone marrow cells, Mx1labeled SSCs can replenish the vast majority of osteogenic cells within 20 days and can do so durably over time [96]. Therefore, Mx1-labeled cells may be more rapidly dividing cells. However, Mx1-labeled cells do not appear to contribute to chondrogenesis, suggesting that Mx1 can label active osteogenic-SSCs that supply the majority of new osteoblasts over time, whereas Nestin may mark more quiescent, multipotent-SSCs. Heterogeneity among stem cells within other tissues has also been defined and may reflect a general principle for how the stem cell compartment has distinctive subsets of cells performing related, but different functions [277].

MIGRATION OF SKELETAL STEM CELLS If SSCs are capable of migration then they may participate in bone repair at a distance, opening the possibility of a systemically administered SSC therapy. As early as the beginning of the 20th century, the presence of circulating mesenchymal progenitors was suggested by the presence of CFU-F in the peripheral blood (as reviewed in [98]). Numerous reports confirmed the presence of circulating fibroblastic progenitors in mouse [98–101], adult humans [27,98,99,102–107], and human umbilical cord blood [108–113]. One study reported that some of these circulating cells express osteocalcin (Ocn) and form bone nodules in vitro, although their functional contribution to bone remodeling in vivo is not clear [114]. In a study where skin flaps between animals are joined, so-called parabiotic animals, it is possible to test if cells from one genetically tagged animal can traverse the ­circulatory system and populate the parabiotic partner.

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TABLE 7.1  Phenotypic Markers for Human Skeletal Stem Cell Identification Markers

Molecules

References (+) or (–)

CD4

T4

(–) [14]

CD5

LEU1

(–) [15]

CD9

Non-descript tetraspanin

(+) [14]

CD10

Membrane metallo-endopeptidase (Neprilysin)

(+) [16–18]

CD11a

Integrin α L chain

(–) [14,19]

CD11ba

Integrin α M chain

(–) [20]

CD13

Aminopeptidase N

(+) [15–18,21–23]

CD14a

Monocyte differentiation antigen

(–) [14–16,21–27]

CD15

Fucosyltransferase 4

(–) [14]

CD18

Integrin β 2 chain

(–) [14,19]

CD25

Interleukin-2 receptor α

(–) [14]

CD29

Integrin β 1 chain

(+) [14,15,19,21–25,28–31]

CD31

Platelet/endothelial cell adhesion molecule (PECAM)

(–) [10,14,21,22,32,33]

CD34a

Nondescript sialomucin

(–) [14,26,34–36] (–/+) [37,38]

CD38

Cyclic ADP ribose hydrolase

(–)[21]

CD44

Hyaluronic acid receptor

(+) [14,21,23,25,28–32,39–42] (–) [43]b

CD45

Protein tyrosine phosphatase, receptor type, C (PTPRC)

(–) [14,35,37,44–49] (dim) [50,51]

CD49a

Integrin α 1 chain

(+) [14,48,50,52–54]

CD49b

Integrin α 2 chain

(+) [14] (low) [55]

CD49c

Integrin α 3 chain

(+) [14]

CD49d

Integrin α 4 chain

(–) [14]

CD49e

Integrin α 5 chain

(+) [14] (low) [47]

CD50

Intercellular adhesion molecule 3 (ICAM3)

(–) [14]

CD51

Integrin α V chain

(+) [14,55]

CD54

Intercellular adhesion molecule 1 (ICAM1)

(+) [14,23,24] (–) [19]

CD56

Neural cell adhesion molecule 1 (NCAM1)

(+) [56–58] (–) [15,24,33,40]

CD58

Lymphocyte-function associated antigen 3 (LFA-3)

(+) [14,19]

CD59

Membrane inhibitor of reactive lysis (MIRL); protectin

(+) [59]

CD61

Integrin β 3 chain

(+) [14] (low) [19,24]

CD62E

E-selectin

(–) [14,19]

CD62L

L-selectin

(+) [14]

CD62P

P-selectin

(–) [14,19]

CD63

TSPAN30

(+) [52]

CD71

Transferrin receptor

(+) [14]

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Defining Skeletal Stem Cells

TABLE 7.1  Phenotypic Markers for Human Skeletal Stem Cell Identification—Cont’d Markers

Molecules

References (+) or (–)

CD73a

5'-nucleotidase, ecto

(+) [14,55,60–62]

CD90a

Thy-1 cell surface antigen

(+) [14,50,63,64] (low) [55]

CD102

Intercellular adhesion molecule 2 (ICAM2)

(+) [14] (–) [19]

CD104

Integrin β 4 chain

(+) [14] (low) [19]

CD105a

Endoglin

(+) [14–17,21–25,27,30–32,39–42, 44,49,59,61,62,65–73] (low) [55]

CD106

Vascular cell adhesion molecule 1 (VCAM1)

(+) [14,19,21,24,29,39,70,74–76]

CD109

Non-descript GPI-linked glycoprotein

(+) [18,77]

CD117

C-kit; Stem cell factor receptor

(–) [18] (–/+) [38]

CD119

Interferon gamma receptor 1 (IFNGR1)

(+) [14]

CD120a

Tumor necrosis factor receptor superfamily, member 1A

(+) [14]

CD120b

Tumor necrosis factor receptor superfamily, member 1B

(+) [14]

CD121a

Interleukin-1 receptor, type I (IL-1R)

(+) [14]

CD123

Interleukin-3 receptor, α (IL-3Rα)

(+) [14]

CD124

Interleukin-4 receptor (IL-4R)

(+) [14]

CD126

Interleukin-6 receptor (IL-6R)

(+) [14]

CD127

Interleukin-7 receptor (IL-7R)

(+) [14]

CD130

Interleukin-6 signal transducer (gp130); oncostatin M receptor

(+) [55]

CD133

Prominin 1

(–) [16,18,22,23,32] (+) [54]c

CD140a

Platelet-derived growth factor receptor, α polypeptide (PDGFRα)

(+) [18]

CD140b

Platelet-derived growth factor receptor, β polypeptide (PDGFRβ)

(+) [17,18]

CD144

Cadherin 5, type 2 (vascular endothelium) (VE-cadherin)

(–) [14,66,70]

CD146

Melanoma cell adhesion molecule (MCAM)

(+) [55,74,78]

CD164

Endolyn

(+) [18]

CD166

Activated leukocyte cell adhesion molecule (ALCAM)

(+) [14,21,23,28,31,41,52,79]

CD172a

Signal-regulatory protein α (SIRPA)

(+) [18]

CD178

Fas ligand

(–) [14]

CD200

Orexin (OX-2)

(+) [55]

CD235a

GlycophorinA

(–) [12,35,44,45,48]

CD271

Nerve growth factor receptor (NGFR)

(+) [17,46,56–58,71,80–83]

CD309

Vascular endothelial growth factor-receptor 2 (VEGF-R2)

(+) [26]

CD340

Erythroblastic leukemia viral oncogene homolog 2 (ERBB2)

(+) [17]

α-SMA

α-Smooth muscle actin

(+) [38,74,84]

ALDH

Aldehyde dehydrogenase

(bright) [85]d

CNN1

Calponin

(+) [84] (Continued)

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TABLE 7.1  Phenotypic Markers for Human Skeletal Stem Cell Identification—Cont’d Markers

Molecules

References (+) or (–)

CDCP1

CUB domain-containing protein 1

(+) [86]

D7-FIB

Orphan antigen (fibroblast)

(+) [16,46]

DES

Desmin

(+) [84]

EGFR-3

Epidermal growth factor receptor 3

(–) [14]

FGFR

Fibroblast growth factor receptor

(+) [14]

GD2

Ganglioside

(+) [87]

HSP90β

Heat-shock protein 90 β

(+) [88]

NES

Nestin

(+) [18,89]

SSEA4

Stage-specific embryonic antigen

(+) [90]

Stro-1

Orphan antigen

(+) [12]

TGF-β1R

Transforming growth factor receptor β1 receptor

(+) [14]

TGF-β2R

Transforming growth factor receptor β2 receptor

(+) [14]

TNAP

Tissue nonspecific alkaline phosphatase

(+) [36,56–58] (–) [91]e

VIM

Vimentin

(+) [18,89]

vWF

Von Willebrand factor

(–) [14]

W5C5

Orphan antigen

(+) [17]

a 

Markers as part of the International Society for Cellular Therapy recommendations [92]. CD44 – Qian et al. argue that primary SSCs do not express CD44 and that the CD44-negative fraction contains the clonogenic activity and that in vitro culture results in expression of CD44. c  CD133 – Gindraux et al. suggest that native SSCs express CD133 while cultured SSCs lose expression. d  ALDH – assessed using aldefluor staining for enzyme activity. e  TNAP – Kim et al. suggest that true multipotent-SSC are TNAP negative, and as SSCs progress down osteogenesis they express TNAP. b 

This experimental system was used to test whether osteogenic stem/progenitor cells can traffic from one animal to a bone morphogenic protein-2 (BMP-2)-treated collagen scaffold implanted in the partner [115]. This experiment quite convincingly confirms that osteogenic cells indeed move via the circulation. To determine whether the intravascular migration of osteogenic stem/progenitor cells can be induced, studies have tested the impact of injury or growth factors. A dramatic increase in the number of circulating osteogenic stem/progenitor cells was noted when an animal was primed with vascular injury or high BMP stimulation [116]. These data suggest that inflammatory and chemoattractant signals may enhance the mobility of endogenous SSCs. In addition, the release of transforming growth factor (TGF)-β1 during bone resorption has been shown to induce the migration of skeletal stem/ progenitor cells to bone resorptive sites [117], thereby demonstrating that circulating skeletal stem/progenitor cells even participate in steady-state bone maintenance. Therefore, skeletal stem/progenitor cells do appear to move through the blood and may do so in an inducible manner in response to physiologic challenges.

KINETICS OF SKELETAL CELL TURNOVER Osteoblast Kinetics Early studies of osteoblastic cell kinetics were estimated by the labeling of proliferating cells and the persistence of labeled cells on growing bone surfaces before cells became embedded in matrix. This was done by simultaneous single dose injection of tritiated glycine (3H-glycine) and tritiated thymidine (3H-thymidine) into growing rabbits [118]. Glycine marked the newly produced extracellular matrix protein and therefore, the position of the bone surface and thymidine-labeled cells that made new deoxyribonucleic acid (DNA) as part of the cell replicative process. Autoradiographic analysis of femur bone sections revealed that labeled cells spend about 10 days on the bone surface before becoming embedded in the bone as osteocytes [118]. Later, a study defining the lifetime of osteoblasts in the mouse periodontium demonstrated that periodontal proliferating progenitors go through a single S-phase and become osteoblasts that last for about 20 days [119]. Although these studies did not

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Kinetics of Skeletal Cell Turnover

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FIGURE 7.1  Double immunostaining of CD34 (brown, peroxidase) and CD146 (red, alkaline phosphatase) in human bone marrow. While CD34 consistently labels the endothelial lining (A and B), CD146 is present in the subendothelial aspect of sinusoids (A) and arteries (B, C), and in loose microvascular-like structures (A). The presence of CD146 in mural cells composing the arteriolar wall is best appreciated in obliquely oriented vessels (B). C: red and brown rectangles at the extremities of a longitudinally oriented artery inscribe a rare individual CD146-positive (D) and a CD34-positive cell. Scale bars: A–C = 20 μm; D and E = 10 μm. (See color plate.) Source: Ferraro F.

FIGURE 7.2  Detection of nestin in human bone marrow. A: immunoperoxidase staining showing two adjacent cells labeled by nestin (brown) laying within the hematopoietic tissue (scale bar 50 μm). B: the elongated shape of nestin-positive cells is better appreciated at higher magnification of the same microscopic field as in A (scale bar 20 μm). C: red arrow points to a pericyte-like appearance of a nestin-positive cell located between two bone trabeculae (BT) and asterisk indicates a small nestin-positive cell. (See color plate.) Source: Ferraro F.

define with precision the cell types involved in proliferation and had limited ability to measure the turnover rate of nonproliferating cells, they strongly suggested that a fraction of cells in growing bones have a relatively short duration on the bone surface and rapidly become boneembedding osteocytes (10–20 days). Human osteoblast function and average lifespan have been determined independently. After double tetracycline labeling, dividing the total wall width (the amount of bone made by osteoblasts) by the mineral apposition rate was used to examine the osteoblast lifespan in humans. These very indirect studies concluded that the average lifespan of normal human osteoblasts is about 150 days [120,121], though a proportion of osteoblasts become

osteocytes during this process considerably shortening their life span as an osteoblast. The estimates of cell durability in these studies were based on a calculation derived from the measurements of new bone synthesis, which is highly variable in different types of bone and different species, possibly accounting for the wide difference seen in mouse and human studies. In addition, these studies did not precisely define cell types, broadly classifying all cuboidal cells localized along the bone surface as osteoblasts. However, despite the limitations of these studies and the discrepancies between species, the data indicated that osteoblasts have a limited lifespan and implied that they are postmitotic, therefore replaced by unspecified immature cells [65,66,69].

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More modern techniques have taken advantage of the ability to label specific subsets of cells genetically at a particular time and follow them by sequential in vivo imaging [122,123]. This technology is obviously limited to mice at this time, but indicates that mature osteoblast populations have a high rate of basal turnover with a durability of about 60 days. These studies also evaluated the cell populations that replenish the osteoblasts and will be discussed below. Overall, it appears that mature osteoblasts in adult bones turn over rapidly in homeostasis

Osteoblasts are Postmitotic Osteoblasts are a short-lived cell and the basis for their replenishment is a topic of some interest in understanding how the tissue is organized as a system. Multiple studies have suggested the possibility that mature osteoblasts are capable of proliferation in bones exposed to stress conditions or under early developmental processes. For example, when mice were irradiated and proliferation of the cells along the endosteal surface assessed by 5-bromo-2-deoxyuridine (Brd-U) administration (a pyrimidine analog incorporated into newly synthesized DNA), over 10% of the ColI-expressing cells were Brd-U positive compared with about 2% of controls, suggesting a rapid expansion of the resident cell population [124]. Interestingly, most of the increases in ColI and Ocn-expressing cells were observed in the metaphyses of bones. Similar results have been reported in developing bones [125]. Therefore, under certain stress conditions and at particular ages, osteoblasts along endosteal surfaces appear to gain proliferative potential. However, the studies must be interpreted with caution as Brd-U labeling may occur in proliferating osteoprogenitors upstream of the mature osteoblasts, and the observed increase in the Brd-U-positive ColI-expressing osteoblasts could be the outcome of rapid differentiation from a progenitor pool, rather than from mature osteoblast proliferation. This point is raised in particular because in other studies using genetic marking where only mature, Ocn-expressing osteoblasts were labeled, no observable proliferation of mature osteoblasts was noted [96]. This was confirmed in long-term homeostasis in adult animals and in models of injury providing fairly definitive evidence for the osteoblast as a postmitotic cell [96]. However, it cannot be excluded that under particular kinds of stress not tested in the model or at particular ages, a subset of mature osteolineage cells can divide.

Turnover of Other Osteolineage Cell Types Evaluation of what cells do divide to replace osteoblasts in part depends upon the ability to define osteolineage

cells. In bone development, osteoblast differentiation from osteogenic progenitors can be distinguished by changes in stage-specific genes. Runx2 is one of the earliest genetic markers of osteo/chondrogenic progenitors, followed by osterix (Osx) in osteoprogenitors and ColI and Ocn in maturing osteoblastic cells [126]. Unfortunately, in adult animals, these genetic markers do not correlate with reliable surface-expressed phenotypic markers to label skeletal stem and progenitor cells clearly. Furthermore, while the temporal expression of these genetic markers makes it tempting to place these populations of cells in a linear relationship to each other, with Runx2 expression representing an early stem cell to Ocn expression representing a mature cell, with various restricted progenitors inbetween, the relationship amongst these populations may be more complex. Therefore, it is difficult to measure definitively the subsets of cells involved in the replenishment of osteoblasts in adults. However, a number of studies have provided useful information. The anatomic sites where bone-forming cells are thought to reside include the inner layer of the periosteum (inner cambial layer) [127], the endosteum [1], osteonal (Harvarsian) canals, and perivascular locations within bone marrow [95]. Engineered mice where cells can be fluorescently tagged at a particular time and followed kinetically (so called cellular pulse-chase experiments) have provided information as to which cells divide and replace mature cells in adult animals. In the example provided above, the promoter that is only activated at the level of the mature osteoblast (Ocn) was used to drive activation of a cell label and indicated that Ocn-expressing osteoblasts do not divide and do not replace themselves [96]. Rather, they appeared to be replaced by cells expressing Osx as evident by another similarly engineered mouse using the Osx promoter to drive a fluorescent tag. The Osx-expressing cells were also minimally proliferative however, as indicated by Ki-67 staining. Therefore, a more primitive population of cells appeared to be required. These could be marked by induction of Mx1 in mice engineered such that the Mx1 promoter would activate a fluorescent tag [96]. These Mx1-labeled cells generated new osteoblasts under settings of both homeostasis and after microfracture. Mx1-labeled stem/progenitor cells appeared at the site of fracture within 2–5 days and dramatically increased in number at 12 days. They did differentiate into fully mature osteoblasts as evident by Ocn expression and the ability to lay down new bone in vivo. Similar dynamics and contributions of osteogenic progenitors have been observed in injury models using a smooth muscle α-actin promoter (αSMA) transgenic mouse model [128], another promoter also hypothesized to denote SSCs. Therefore, osteoblast replenishment in adults appears to be mediated by the stem/progenitor cell pool without evidence for mature cells replacing other mature cells.

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Potential Use of Skeletal Stem Cells as Therapy

POTENTIAL USE OF SKELETAL STEM CELLS AS THERAPY If bone cells depend upon a dynamic population of SSCs that can migrate interstitially and by the circulation [96], then it is biologically feasible to consider cellbased bone therapies. This issue has prompted much of the attention on the putative SSC that has been called MSC by in vitro features as discussed above. While the in vitro cultured population that is termed MSC or here, MC, may not have close correspondence with bona fide endogenous SSCs, it may be valuable as a source of cells with osteolineage potential. The latter has been extensively shown through the use of the cells in vitro in bone constructs, in vivo by homotopic transplant into bone, and even by systemic administration of an allogeneic bone marrow graft in children with osteogenesis imperfecta (OI) [129]. Several aspects of the biology of these cells are important considerations for cell therapy. Practical clinical utility of cell therapy will likely require intravenous infusion and SSC homing to the bone marrow microenvironment although several animal models of osteoporosis have shown some success with intrabone injections of MC [130,131]. The homing ability of primary or ex vivo expanded MC has been rigorously studied [94,132,133]. These studies used varying approaches to isolate and expand MCs in vitro and found that the engraftment efficiency toward bone marrow or target tissues of ex vivo expanded MC was variable and dependent on the source of cells, prior culture conditions, passage number, and route of delivery. In general, studies utilizing cultured MC showed poor engraftment in target tissues after systemic administration, confirmed by a comparative study demonstrating that primary murine SSCs have significantly higher efficiency of homing to the bone marrow compared to cultured MC [133]. The downregulation of cell adhesion molecules and/or chemokine receptors (CXCR4, receptor for stromal cell-derived factor (SDF)-1α) in cultureexpanded MC is a possible cause of poor migration and homing. To overcome these limitations of SSCs to home and migrate appropriately in vivo, and thus improve the engraftment of SSCs in a potential clinical setting, multiple approaches have been employed to enhance the homing response of MC. Since the SDF-1α and CXCR4 axis is a key mechanism of SSC homing, retroviral overexpression of CXCR4 in MC resulted in increased MC homing to inflammatory sites or ischemic myocardium [134,135]. Similarly, studies employing either an adenoassociated viral vector (AAV) to transiently cause ectopic expression of the α4 integrin on murine MC [136] or by chemical modification of the cell surface with a peptidomimetic ligand against α4 integrin on the MC cell surface coupled to bisphosphonate [137], leads to substantially increased homing of MC into the bone marrow

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of recipient mice and a greater than 10-fold increase in new bone formation compared to control. Sackstein et al., using human MC preparations, chemically modified the cell surface of MC via an α-1,3-fucosyltransferase preparation to convert the native CD44 glycoform into hematopoietic cell E-selectin/L-selectin ligand (HCELL) [138]. This allowed for the chemically modified MC to have potent E-selectin binding without effects on cell viability or multipotency. When transplanted into immunocompromised mice, the chemically modified MC were capable of infiltrating the marrow within hours of infusion, with foci of endosteally localized cells and human osteoid generation [138]. These novel approaches may eventually lead to therapeutic strategies allowing for the prospective harvesting of SSCs and directed targeting of those cells to sites of injury or disease to coordinate bone formation and repair. As continued advancements are made in directing SSCs to the bone marrow after systemic delivery, the next phase of clinical therapy will be how best to apply SSCs to treat disease. SSCs from aged patients or donors are undoubtedly reduced in number and have a reduced capacity for osteogenic potential [139–142], and hence therapeutic efficacy, compared to younger SSCs. Therefore, autologous transplantation of SSCs, without modification, may have little long-term value. Peripheral, circulating osteogenic-SSCs are of five times greater abundance in adolescents than the aged, and are also present in higher numbers after injury [114]. In a mouse study, aged mice transplanted with MC from young donors had increased bone density and a longer period of survival [142]. Therefore, allogeneic transplantation from younger, healthier donors might be preferred, but graft tolerance and immunosuppression issues create additional complications. Therefore, if SSCs from patients can be modified or corrected, then autologous transplantation of modified cells, absent of graft rejection issues, may represent the best option. One example where this approach is moving toward clinical testing is in the treatment of OI. Clinical trials using allogeneic bone marrow transplants or SSC-enriched transplants have demonstrated improvement in children with OI [129,143,144]. Given that OI is normally associated with a mutation in a single gene, ColI, it is a highly attractive target for gene therapy. Using an AAV approach, Chamberlain and colleagues have demonstrated the ability to correct gene defects in OI patient SSCs and restore osteogenic potential in vitro [145,146]. In a similar approach, the laboratory-generated induced pluripotent stem cells (iPSCs) from AAVcorrected SSCs and demonstrated that they were able to form bone in immunocompromised mice [147]. The transformation to an iPSC state theoretically allows for unlimited expansion of the cells before differentiation to acquire sufficient numbers for a robust transplant, and

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the iPSC reprogramming transgenes could be removed by activating Cre recombinase [147]. As gene-targeting therapies evolve, and our understanding of the genetic and molecular pathways governing bone degenerative diseases increases, a combinatorial gene therapy/SSC transplant strategy may offer new opportunities for bone therapeutics.

INTERACTIONS WITH THE HEMATOPOIETIC SYSTEM The Endosteal Hematopoietic Niche The bone marrow has long been recognized as the primary source of hematopoiesis in adult mammals. The notion of a bone marrow transplant relies on the fact that bone is a source of HSPCs capable of repopulating myeloablated hosts. Intriguingly, one of the studies describing cells that behaved like osteogenic-SSCs was actually seeking to determine why hematopoiesis was located in the bone [5]. Tavassoli and Crosby placed bone marrow fragments into nonbony tissues to see if the addition on nonhematopoietic bone cells could enable engraftment of hematopoietic cells in those tissues. Hematopoiesis occurred only if a bony ossicle with microcirculation formed. They observed that a “reticular” cell that could differentiate into osteoblasts was required for the bone and bone marrow formation, thus presaging the recognition of a bone-forming SSC and an important relationship between bone and hematopoiesis. Within bone marrow, the endosteal surfaces of bone were recognized as a rich source of primitive hematopoietic cells by studies where tissue at progressive distances from the endosteum was preserved and tested [148,149]. These studies suggested that endosteum was a site of enrichment for hematopoietic stem/progenitor cells and therefore, the location of the HSC niche. This issue is controversial, however. A number of histologic studies have indicated that HSC are perivascular in the unmanipulated mouse bone marrow (see below). This may be different from the bone marrow in the setting of stem cell transplantation. Several different imaging approaches have demonstrated that transplanted HSCs localize to endosteal surfaces, while more mature hematopoietic progenitor cells (HPCs) localize further away [122,150–152]. Hematopoietic stem cells that were closely associated with the endosteum were also shown to have greater transplantation activity than those HSC contained in the central marrow cavity [153]. Given that osteolineage cells line the endosteum, they were theorized as a potential supportive mediator of the HSC niche. When HSCs were acquired from aged donors, it was found that they localize to sites further away from the endosteum than HSCs from younger donors [150], suggesting

that HSC localization within the bone may reflect the difference in HSC function observed with the aging process. Studies by Taichman and colleagues [154,155] demonstrated that osteolineage cells produce many supportive growth factors including granulocyte colony stimulating factor (G-CSF), granulocyte macrophage colony stimulating factor (GM-CSF), interleukin (IL)1, IL-6, and TGF-β. Later, in vivo studies using various genetic approaches demonstrated that osteolineage cells are a significant regulatory component of hematopoiesis [156–159]. In one of these studies where a constitutively active parathyroid hormone (PTH)/parathyroid hormone-related protein (PTHrP) receptor was driven by an osteolineage promoter, the effect of osteolineage cells on HSC expansion was mediated, at least in part, by the Notch ligand Jagged1. Notch signaling regulates cell fate decisions including HSC self-renewal [160–162], thus by changing self-renewal versus differentiation decisions, Notch can increase HSC number without differentiation or increase in HPC or mature cells [160,162]. This regulatory relationship of Notch ligand expression by osteolineage cells may only be important with PTH/PTHrP receptor activation, however, as studies on Notch in homeostasis in adult animals do not support an important role in HSC function [163]. Within the endosteal niche, HSCs are thought to be retained through a variety of adhesion molecule interactions, many of which are likely redundant systems. Early studies exploring the role of osteolineage cells in maintaining HSCs suggested that N-cadherin interactions mediated the positive effects on HSCs [157]; however, more recent studies have contradicted these findings [164,165]. Numerous other adhesion molecules have been implicated as contributing to HSC and HPC tethering, including, but not limited to, the integrins α4β1 – very late antigen (VLA)-4 [166–171], α5β1 –VLA-5 [167,168,170,172], α4β7 – lymphocyte Peyer's patch adhesion molecule-1 (LPAM-1) [173], the alfa 6 integrins (laminins) [174,175], CD44 [167,176], E-selectins [177–179], the angiopoietin receptor tyrosine kinase with immunoglobulin-like and EGF-like domains-2 (Tie-2) [159], endolyn (CD164) [180], the calcium-sensing receptor (CaR) [181], stromal-derived factor-1α (SDF-1α) [182], and osteopontin (OPN) [183,184]. Intriguingly, OPN is also a negative regulator of HSC pool size within the bone marrow niche [184,185], and knockout of OPN in mice results in endogenous hematopoietic mobilization to the periphery and increases the mobilization response to G-CSF [183]. In 2011, osteolineage cells were also found to express agrin, which mediates cell–cell contact with short-term HSCs and initiates proliferation, perhaps functioning as an opposing signal to OPN [186]. These results suggest that OPN (a target for modulation of the endosteal HSC niche) and agrin receptors (targets on HSCs) may serve as future therapeutic strategies

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to modify and enhance hematopoietic transplant and recovery, in line with previously described strategies targeting other adhesion interactions in the niche [187–192].

Hematopoietic Regulation of the Endosteal Niche Up to this point, the focus has been on the role of osteolineage cells and their associated regulation of hematopoietic cells. However, cells of hematopoietic origin also support and regulate osteolineage cells. Cell–cell contact between osteolineage cells and HPC has been shown to be important for HPC survival [193]. Using an in vitro system, Gillette et al. demonstrated that human CD34+ cells (an HPC/HSC population of cells) co-cultured with osteolineage cells made cell–cell contact and exchanged a portion of their membrane with the osteolineage cells, creating a signaling endosome [194]. This signaling endosome caused osteolineage cells to downregulate Smad signaling and increase production of SDF-1α, suggesting that hematopoietic cells may instruct osteolineage cells to create a more habitable environment, allowing them to affect their own microenvironment directly. These results may suggest that HSCs help to create their own niches, perhaps by instructing neighboring stromal cells to produce supportive factors. Mature hematopoietically derived cells also have the ability to alter the endosteal lining osteolineage cells. One therapeutic manipulation of the hematopoietic niche involves treatment with G-CSF to “mobilize” HSCs and HPCs out of the bone marrow and into the peripheral blood, where they can subsequently be collected by apheresis and used in hematopoietic transplantation. Treatment with G-CSF causes significant suppression of osteolineage cells [195–197], causing apoptosis [196] and a characteristic “flattening” of osteolineage cells [197]. In 2010, Winkler et al. [195] reported that a population of macrophage cells, characterized as F4/80+ Ly-6 G+ CD11b+ and termed “osteomacs” [198], line the endosteal surfaces of bone and that G-CSF treatment resulted in a trafficking, and reduction in number, of osteomacs [195]. Presumably, this reduction in osteomacs was responsible for the attenuation of function of osteolineage cells. To confirm this hypothesis, macrophages were depleted using Mafia transgenic mice or with clodronate-loaded liposomes (Clo-lip) treatment, causing significant mobilization of HSC and HPC. A similar report from Chow et al. also demonstrated that depletion of macrophages with Clo-lip treatment results in hematopoietic mobilization [199], describing a Gr-1negative F4/80+ CD115mid CD169+ macrophage population as a regulator of osteolineage cells. Similar to the osteomac population, the authors suggest that the CD169+ macrophages express a soluble, yet to be identified, factor(s) that positively supports niche cells. In comparable studies by Christopher et al., a soluble factor produced by

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monocyte/macrophage populations supported osteolineage function [200], suggesting that manipulation of monocyte/macrophage populations or the factor(s) produced by them is a future therapeutic strategy to manipulate the endosteal niche, and possibly suggest a novel strategy to affect bone cells therapeutically. Osteoclasts are also monocyte-derived cells that are present along endosteal surfaces of bone. Osteoblasts and osteoclasts regulate bone formation and bone resorption, respectively, within the bone marrow niche, and pre-osteoclasts cause retraction of osteoblasts [201], theoretically creating transient “holes” in the HSC supporting endosteal niche. Given that treatment with G-CSF causes increases in osteoclast number and activity [202,203], it was hypothesized that osteoclasts may regulate the endosteal niche and alter HSC retention and localization. Kollet et al. treated mice with RANK ligand, which increased osteoclast activity and correlated with a moderate increase in HPC mobilization [204], consistent with an independent report [205]. Correspondingly, stress models such as bleeding or LPS treatment also increased the number of osteoclasts in the endosteal niche and HPC in the peripheral blood, supporting a role for osteoclasts in niche regulation. Kollet et al. suggested that osteoclast-derived proteolytic enzymes, such as cathepsin K, degraded important niche retention molecules, thereby facilitating egress of HSC and HPC [204]. A more recent study by the same laboratory showed decreased osteoclast maturation and activity in CD45 knockout mice, with reduced mobilization to RANK ligand and G-CSF [206], further suggesting osteoclasts are important in regulation of the endosteal niche. In contrast to these studies, Takamatsu et al. earlier reported that while G-CSF treatment increased osteoclast number and bone resorption in both BALB/c mice and humans, the increase in osteoclasts did not occur until 10–15 days or 6–8 days, respectively, after treatment with G-CSF [203], which has also been observed by other groups using similar systems [207,208]. Since niche clearance by G-CSF is typically observed 4 to 5 days after treatment, the importance of osteoclasts remains unclear. Similarly, treatment of mice with osteoclast-inhibiting bisphosphonates does not result in an impaired mobilization response to G-CSF [195,203,209], and in fact increases HSC egress [195,209]. Intriguingly, the endosteal surface of bone, particularly underneath resorbing osteoclasts, is a significant source of soluble extracellular calcium and previous studies by our laboratory demonstrated that HSC expression of calcium-sensing receptors mediates a chemoattraction to soluble Ca2+ [181]. When the calciumsensing receptor was knocked out, mice had reduced HSC content within the bone marrow niche and increased HSC in peripheral blood. These results might suggest that the bone resorbing, and subsequent Ca2+ releasing activity of osteoclasts, may be involved in niche retention.

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Hematopoietic transplantation normally requires conditioning of the bone marrow niche, or a clearance of resident HSC and HPC, to allow for successful engraftment. Conditioning is normally accomplished by treatment with chemotherapeutic agents and/or irradiation to remove HSC and HPC; however, these agents can also affect niche cells themselves. A study from Dominici et al. demonstrated that following a lethal dose of irradiation, megakaryocytes migrated toward the endosteal surfaces of bones and induced a two-fold expansion in osteolineage cells, presumably increasing the available niches for subsequent HSC engraftment [124]. These results are in close alignment with other studies in which mice deficient in GATA-1 and NF-E2 transcription factors had significantly increased megakaryocyte production, leading to a six-fold expansion of osteolineage cells [210], a process requiring direct cell– cell contact between megakaryocytes and osteolineage cells [211,212]. Further support for blood cells affecting bone biology are the observations that excessive blood loss, causing stress on the hematopoietic system, leads to osteoporosis in mice [213] and that splenectomy in mice results in an expansion of megakaryocytes with reduced GATA-1 expression and increases in bone formation [214]. These intriguing findings suggest that there is much yet to be learned about the bidirectional regulatory relationships of blood and bone. Further exploration of these is clearly warranted particularly since there is a substantial prevalence of bone disorders following hematopoietic transplantation and its associated conditioning regimens [215–222].

Endosteal Age and Turnover The endosteal surface of bone, as described earlier, is a dynamic tissue with a high rate of turnover of osteoblasts, which are replenished by earlier osteogenic precursors and SSCs. Several reports, using in vitro co-culture studies have shown that HSCs cultured on more immature osteolineage cells have a higher activity and repopulating ability than HSCs cultured on more mature osteoblasts [223,224], suggesting that the maturational status of osteolineage cells may be a key determinate of HSC niche support. Indeed, several in vivo reports described niche cells responsible for HSC maintenance that resembled SSCs and their immediate downstream precursors [10,225], and in studies in which terminally differentiated osteoblasts were ablated, hematopoiesis was unaffected [226,227]. These findings suggest that the HSC niche is composed of an immature osteolineage cell subset. The aforementioned Nestin+ multipotent-SSCs express high levels of hematopoietic supportive molecules, suggesting that these cells may form a unique bone marrow niche [95] compromised of heterologous stem cell pairs. Intriguingly, these Nestin+

cells were found to wrap around endothelial cells within the bone marrow near the endosteum, but also toward the central marrow, suggesting an alternate HSC niche localization further from the endosteum.

Vascular and Perivascular Cells – the Vascular Niche While it is clear that HSCs reside and thrive within the endosteal bone marrow niche, evidence, including the localization of CXCL12-abundant reticular cells [225,228] and Nestin+ SSCs [95], suggests that the endosteum is not the exclusive and may not even be the predominant niche for hematopoiesis. Stem cell localization studies utilizing in vivo imaging out of our laboratory [122] and others [152] found that while HSCs were near the endosteum, they were not exclusively adjacent to osteolineage cells. Studies assessing the localization of signaling lymphocyte activation molecule (SLAM; CD150+, CD48–, and CD244–) cells, which are highly enriched for HSC, have shown a greater proportion of SLAM cells adjacent to bone marrow sinusoidal blood vessels, suggesting a “vascular niche” for HSC [165,229]. This is supported by the fact that endothelial cells, very similarly to osteolineage cells, can support hematopoiesis both in vitro and in vivo [230–232]. We have also described small endothelial microdomains that express E-selectin and SDF-1α, and act as a point of entry for HSC after transplantation [123]. Regeneration of sinusoidal endothelial cells has been reported to be essential for hematopoietic reconstitution following myelosuppression [233], and bone marrow endothelial cells have been reported to support the growth and expansion of HSC through angiocrine factors [234]. Interestingly, similar to the HSC cell-to-cell contact with osteolineage cells altering the endosteal niche function [194], Slayton et al. reported that donor hematopoietic cells migrated to sites of vascular sinusoidal injury within bones and contributed to repair following transplantation [235], further highlighting the complexity and interconnectedness of the bone marrow microenvironment. While it is not completely clear what factors influence the particular niche location of HSCs, the fact that reticular cells with high production of SDF-1α are present in both the endosteal and vascular niches, may provide a common mechanism of HSC support in both niches [225,228,236]. One report has demonstrated that HSC, shortly after transplantation into nonmyeloablated recipients, preferentially localize to the endosteum in metaphyseal regions of bone in close association with blood vessels, and that this requires hyaluronic acid [237]. Given that the endosteal region is itself highly vascularized may suggest that the HSC niche actually encompasses overlapping regions within the bone.

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The Bone Marrow Environment is Hypoxic (see Chapter 8 for effects of hypoxia on osteoclasts) Several reports have demonstrated that HSC are present in regions of hypoxia within the bone marrow [238– 240], and those HSC contained in hypoxic areas have greater hematopoietic repopulating ability than those in more perfused areas [241], suggesting that perhaps the overlapping regions of the endosteal and vascular biomes contains the right mix of atmospheric (oxygen and other chemical mediators) conditions to support hematopoiesis. Cellular exposure to hypoxia results in stabilization of hypoxia inducible factor 1-α (HIF-1α), which is a transcriptional regulator of erythropoietin (EPO) production [242]; numerous cell proliferation and survival genes [243–245]; the angiogenic growth factor, vascular endothelial growth factor (VEGF) [246]; and other genes. In mice where the HIF-1α responsive element is mutated on the Vegfa promoter, thereby preventing upregulated expression in hypoxic conditions, HSCs were found to have defects in their function [247], supporting a role of hypoxia-induced gene expression in the niche for maintenance of HSCs. HIF-1α also increases production of SDF-1α [248] and CXCR4 receptor expression [249], and prevents hematopoietic cell damage caused by overproduction of reactive oxygen species (ROS) [250]. Therefore, HSCs residing in areas of low oxygen will have increased activity of HIF-1α, thereby maintaining their fitness and stemness [251], a hypothesis that is supported by the fact that in vitro hypoxic conditions expand human HSC [252] and HPC populations [253–255]. In 2010, using HIF-1α deficient mice, Takubo and colleagues demonstrated that HSC cell cycle quiescence was lost, and HSC numbers diminished after stressful insults [256]. Additional studies exploring the protein stabilization of HIF-1α demonstrated that a precise regulation of HIF-1α activity was necessary for optimal HSC function. Miharada et al. also reported that HIF-1α-deficient mice have reduced expression of Cripto, also known as teratocarcinoma-derived growth factor-1 (TDGF-1), on endosteal osteolineage cells and a reduction of one of Cripto's cognate receptors, GRP78, on HSCs [257]. The authors demonstrated that the Cripto/ GRP78 signaling axis is an important regulator of HSC quiescence downstream of HIF-1α signaling, and postulated that GRP78 can be used as a marker to distinguish between quiescent and active HSCs. Advanced technologies that are able to measure oxygen tension accurately within the bone marrow microenvironment at high resolution overlaid with imaging assessing HSC localization are likely to be highly informative, and perhaps will resolve some discrepancies and unanswered questions as to the specifications of the HSC niche. Oxygen tension is also important in regulating osteolineage cell function. Bone marrow MSCs cultured in hypoxic conditions demonstrated increased self-renewal division

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and increased integrin expression allowing for more robust migration compared to their normoxic-cultured controls [258], consistent with reports that hypoxic conditioning can increase osteogenic, adipogenic, and chondrogenic differentiation potential [259–263], though other reports propose opposite effects [264] suggesting that regulation of SSCs by hypoxia may be highly dynamic. One report has also demonstrated that VEGF production by osteolineage cells is cooperatively regulated by Osx and Hif-1α [265], suggesting hematopoietic and angiogenic support by osteolineage cells is also regulated by oxygen tension. Further exploration into these mechanisms is likely to lead to advancements in therapies, particularly in terms of bioengineering scaffolds and ex vivo culture conditions for cellular therapeutics of bone degeneration [262,266–268].

Nervous System Mediators While it is clear that cellular and molecular constituents within the microenvironment interact and coregulate each other, the question arises as to whether there is a global regulator of the entire bone system, able to alter numerous components of the niche to respond as needed rapidly. Nestin+ MSCs are highly intriguing, as nestin, an intermediate filament protein, is normally restricted to nerve cells. Intriguingly, work by the same laboratory demonstrated that HSC and HPC mobilization by G-CSF required peripheral β2-adrenergic signals [197] in studies utilizing chemically sympathectomized mice treated with 6-hydroxydopamine; mice treated with the β-blocker propranolol; or mice genetically deficient in the gene for dopamine β-hydroxylase (Dbh), an enzyme that converts dopamine into norepinephrine. They also showed that treatment with the β2-adrenergic agonist clenbuterol reversed the phenotype of Dbh knockout mice [197]. The Frenette laboratory also demonstrated that the nervous system regulates HSC niche retention via circadian rhythms [269,270]. Signaling via β3-adrenergic stimulations causes rhythmic oscillations controlling norepinephrine release, CXCR4 expression, and SDF-1α production, causing regular transient trafficking from the bone marrow niche. Nervous system signaling has also been demonstrated to have direct effects on HSC, as human CD34+ hematopoietic cells express β2-adrenergic and dopamine receptors and neurotransmitters, such as norepinephrine, serve as direct chemoattractants for HSC [271], demonstrating nervous system global control over multiple cellular components of the hematopoietic ecologic system. One report showed that β2-adrenergic signaling causes upregulation of the vitamin D receptor (VDR) on osteolineage cells and that VDR signaling was necessary for osteolineage responses to G-CSF treatment [272]. VDR expression is also regulated by circadian rhythms [273] demonstrating further

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interconnectivity of the nervous system control over the bone microenvironmental system. Yamazaki et al. described a population of nonmyelinating Schwann cells that ensheath sympathetic nerves in the bone marrow [274]. These cells were a primary producer of TGF-β, which they and others have demonstrated is a quiescent signal for HSC [275,276], and when the bone was denervated, TGF-β signaling was reduced leading to a reduction in the HSC pool [274]. Given that TGF-β is also capable of attracting SSCs [117], this may suggest that the nervous system can coordinate SSC trafficking and migration to sites of need, though further exploration is required.

SUMMARY The relationship of bone and bone marrow remains a highly intriguing biologic paradigm for how heterologous cells interrelate to accomplish complex and highly divergent physiologic processes. While the hematopoietic system has been more accessible and more extensively studied in terms of cellular hierarchies and stem cell function, applying approaches and concepts learned from it have again revealed marked connectedness between bone and bone marrow. The bone, like the blood, appears to be organized in a hierarchical manner where stem and progenitor cells are required for replenishment of short-lived mature cells. Indeed, the turnover times for mature bone cells, osteoblasts, and mature red cells are similar in both mouse and man. In addition, both skeletal and hematopoietic stem cell compartments appear to be heterogeneous and both stem cell populations appear capable of migration, particularly through the vascular tree. Further exploration of the similarities and differences between these systems promises to both teach important cell and organismal biology and to offer the potential of defining novel therapeutic strategies for diseases in each system.

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[7]  Friedenstein AJ, Latzinik NW, Grosheva AG, Gorskaya UF. Marrow microenvironment transfer by heterotopic transplantation of freshly isolated and cultured cells in porous sponges. Exp Hematol 1982;10:217–27. [8]  Friedenstein AJ, Chailakhyan RK, Gerasimov UV. Bone marrow osteogenic stem cells: in vitro cultivation and transplantation in diffusion chambers. Cell Tissue Kinet 1987;20:263–72. [9]  Friedenstein AJ, Chailakhyan RK, Latsinik NV, Panasyuk AF, Keiliss-Borok IV. Stromal cells responsible for transferring the microenvironment of the hemopoietic tissues. Cloning in vitro and retransplantation in vivo. Transplantation 1974;17:331–40. [10] Sacchetti B, Funari A, Michienzi S, Di CS, Piersanti S, Saggio I, et al. Self-renewing osteoprogenitors in bone marrow sinusoids can organize a hematopoietic microenvironment. Cell 2007;131:324–36. [11]  Gronthos S, Graves SE, Ohta S, Simmons PJ. The STRO-1+ fraction of adult human bone marrow contains the osteogenic precursors. Blood 1994;84:4164–73. [12] Simmons PJ, Torok-Storb B. Identification of stromal cell precursors in human bone marrow by a novel monoclonal antibody, STRO-1. Blood 1991;78:55–62. [13] Robledo MM, Hidalgo A, Lastres P, Arroyo AG, Bernabeu C, Sanchez-Madrid F, et al. Characterization of TGF-beta 1-binding proteins in human bone marrow stromal cells. Br J Haematol 1996;93:507–14. [14] Pittenger MF, Mackay AM, Beck SC, Jaiswal RK, Douglas R, ­Mosca JD, et al. Multilineage potential of adult human mesenchymal stem cells. Science 1999;284:143–7. [15] Schallmoser K, Bartmann C, Rohde E, Reinisch A, Kashofer K, ­Stadelmeyer E, et al. Human platelet lysate can replace fetal ­bovine serum for clinical-scale expansion of functional mesenchymal stromal cells. Transfusion 2007;47:1436–46. [16] Jones EA, Kinsey SE, English A, Jones RA, Straszynski L, ­Meredith DM, et al. Isolation and characterization of bone marrow multipotential mesenchymal progenitor cells. Arthritis Rheum 2002;46:3349–60. [17] Buhring HJ, Battula VL, Treml S, Schewe B, Kanz L, Vogel W. Novel markers for the prospective isolation of human MSC. Ann N Y Acad Sci 2007;1106:262–71. [18] Vogel W, Grunebach F, Messam CA, Kanz L, Brugger W, Buhring HJ. Heterogeneity among human bone marrow-derived mesenchymal stem cells and neural progenitor cells. Haematologica 2003;88:126–33. [19] Majumdar MK, Keane-Moore M, Buyaner D, Hardy WB, ­Moorman MA, McIntosh KR, et al. Characterization and functionality of cell surface molecules on human mesenchymal stem cells. J Biomed Sci 2003;10:228–41. [20] Meirelles LS, Nardi NB. Murine marrow-derived mesenchymal stem cell: isolation, in vitro expansion, and characterization. Br J Haematol 2003;123:702–11. [21] Lu LL, Liu YJ, Yang SG, Zhao QJ, Wang X, Gong W, et al. Isolation and characterization of human umbilical cord mesenchymal stem cells with hematopoiesis-supportive function and other potentials. Haematologica 2006;91:1017–26. [22] Bartmann C, Rohde E, Schallmoser K, Purstner P, Lanzer G, Linkesch W, et al. Two steps to functional mesenchymal stromal cells for clinical application. Transfusion 2007;47:1426–35. [23] Soncini M, Vertua E, Gibelli L, Zorzi F, Denegri M, Albertini A, et al. Isolation and characterization of mesenchymal cells from human fetal membranes. J Tissue Eng Regen Med 2007;1:296–305. [24] Conget PA, Minguell JJ. Phenotypical and functional properties of human bone marrow mesenchymal progenitor cells. J Cell Physiol 1999;181:67–73. [25] Mareschi K, Ferrero I, Rustichelli D, Aschero S, Gammaitoni L, Aglietta M, et al. Expansion of mesenchymal stem cells isolated from pediatric and adult donor bone marrow. J Cell Biochem 2006;97:744–54.

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Summary

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II.  CELLULAR, MOLECULAR, AND DEVELOPMENTAL BIOLOGY OF BONE