**Skeletogenesis and the Hematopoietic Niche**

Elizabeth Sweeney and Olena Jacenko *University of Pennsylvania USA* 

#### **1. Introduction**

146 Advances in Hematopoietic Stem Cell Research

Zhou S, Schuetz JD, Bunting KD, Colapietro AM, Sampath J, Morris JJ, Lagutina I, Grosveld

determinant of the side-population phenotype. *Nat Med.* Sep;7(9):1028-34.

GC, Osawa M, Nakauchi H, Sorrentino BP. (2001) The ABC transporter Bcrp1/ABCG2 is expressed in a wide variety of stem cells and is a molecular

> The reciprocal regulation of the skeletal and the immune systems has been clinically appreciated for years. In particular, factors produced by immune cells during homeostasis and activation markedly affect the skeleton, which in turn affects the marrow niche environments (as reviewed in (Compston 2002). This relationship also extends to an interdependence between bone and hematopoiesis during immune cell development, however the critical cell types and extracellular matrix components involved in establishing and maintaining hematopoietic niches within the bone marrow are only recently beginning to be defined. Indeed, some immuno-osseous disorders with hematopoietic defects such as bone marrow failure and immune dysfunction, as well as certain cancers, may result from a defective hematopoietic niche (Spranger et al. 1991; Kuijpers et al. 2004; Hermanns et al. 2005; Walkley et al. 2007; Walkley et al. 2007; Raaijmakers et al. 2010). Likewise during aging, a progressive decline in cell replacement and repair manifests in both the skeletal and hematopoietic systems with reduced bone mass and diminished blood cell formation respectively (as reviewed in (Rossi et al. 2008) and (Gruver et al. 2007). Further, this altered hematopoiesis due to aging leads to deficient immune function and increased incidence of malignancies (Rossi et al. 2005; Janzen et al. 2006; Mayack et al. 2010). Thus, the dynamic relationship between skeletal and hematopoietic maintenance throughout life suggests that these clinical outcomes may ensue from cell signaling deficiencies or from defects in the structural environment supporting hematopoiesis. This chapter provides an overview of our current understanding of how hematopoietic niches may be established, how they promote hematopoiesis, and how the skeletal status may modulate niche function.

#### **2. Coordinate skeletal and hematopoietic development**

The vertebrate skeleton develops by one of two essential processes, endochondral (EO) and intramembranous (IO) ossification mechanisms (as reviewed in (Chan, D. and Jacenko 1998). The direct differentiation of ectomesenchymal cells to osteoblasts in IO represents the rudimentary mechanism through which many skull bones and all periosteal bones form. The IO-derived bone is referred to as "dense", "compact" or "cortical", and as the names imply, is a solid bone with primary functions relating to weight bearing and protection (**Fig. 1C**). In contrast, EO relies on the generation of a cartilaginous skeletal blueprint that is gradually replaced by a "trabecular", "spongy", "cancellous" bone and a marrow capable of sustaining hematopoiesis (Chan, D. and Jacenko 1998; Mackie et al. 2008) (**Fig. 1C**). This replacement mechanism of EO is responsible for the formation of the vertebrate axial and appendicular skeleton, as well as certain cranial bones (Jacenko et al. 1991; Chan, D. and Jacenko 1998).

Skeletogenesis and the Hematopoietic Niche 149

Fig. 1. Architecture of the endochondral bone. A) A schematic of a developing long bone illustrating its architecture. The epiphysis, or the bulbous end, lined by articular cartilage and containing the secondary ossification center with marrow, is supported by the flared metaphysis, which in turn rests upon the slender cylindrical shaft of the diaphysis. The growth plate separates the primary and secondary ossification centers, and consists of a gradient of differentiating chondrocyte zones; the proliferative cartilage (PC) and

hypertrophic cartilage (HC) zones are marked, as well as the hybrid trabecular bone (TB) protruding into the marrow. The locations of the two-layered periosteal membrane surrounding the diaphysis and the inner endosteal network are marked. B) A longitudinal tibial section from a week-3 wild type mouse stained with safranin-orange, hematoxylin & eosin (H&E) and counterstained with fast green. Using these stains, the negatively charged

cartilaginous matrix appears orange while the bone stains light blue-green; mature erythrocytes stain green, while other marrow elements stain pink-purple with H&E. The boxed inset is a high magnification of the chondro-osseous junction containing hypertrophic chondrocytes, bone and marrow with vascular. The hybrid nature of the trabecular bone can

As EO initiates during embryogenesis, its distinctive feature is the emergence of hypertrophic cartilage, which is present in all skeletal elements that will develop a marrow cavity, e.g. long bones, hips, vertebrae, ribs, certain skull bones. The eventual replacement of cartilage by bone and marrow via EO relies on the sequential maturation of chondrocytes from resting, to proliferating, to hypertrophic (**Fig. 1A**). Chondrocyte hypertrophy manifests with a dramatic increase in cell size, cessation of proliferation, and synthesis of a new repertoire of differentiation-specific gene products (Godman and Porter 1960; Chan, D. and Jacenko 1998; Alvarez et al. 2001; James et al. 2010). Among these is the matrix protein collagen X, which represents the predominant biosynthetic product of hypertrophic cartilage (Gibson and Flint 1985; Schmid and Linsenmayer 1985). Concomitant with hypertrophy is a transformation from a non-calcified avascular cartilage matrix, to a calcifiable one that is permissive to vascular invasion. Morphometric analysis suggests that before vascular invasion, the terminal hypertrophic chondrocytes undergo either autophagy (Srinivas and Shapiro 2006; Bohensky et al. 2007) or apoptosis (Farnum and Wilsman 1989), the rate of which controls longitudinal growth of the skeletal element, as well as the transition from cartilage to trabecular bone and marrow (Farnum and Wilsman 1989).

Subsequent vascular entry into hypertrophic cartilage is critical to skeleto-hematopoietic development, since it leads to an influx of mesenchymal cells, hematopoietic precursors, and chondro/osteoclasts. This influx of cells, together with growth factors, cytokines and hormones, establishes the primary center of ossification and the marrow environment where hematopoiesis ensues (**Fig. 1**). Specifically, while chondro/osteoclasts degrade hypertrophic cartilage, multipotent stromal cells, including mesenchymal and perivascular reticular cells, form the marrow stroma, a meshwork of non-hematopoietic cells supporting hematopoiesis by providing structural scaffolding and producing hematopoietic factors (Taichman et al. 1996; Bianco et al. 1999). As hypertrophic cartilage continues to be degraded, matrix remnants serve as scaffolds upon which differentiating osteoblasts deposit bone matrix, thus forming trabecular bony spicules with hypertrophic cartilage cores (**Fig. 1B**) (Chan, D. and Jacenko 1998). Of note, the origin of the trabecular bone osteoblasts at the junction between marrow and the hypertrophic cartilage, termed the chondro-osseous junction, is still debated (Roach 1992; Roach et al. 1995; Roach and Erenpreisa 1996; Nakamura et al. 2006; Hilton et al. 2007; Maes et al. 2010). Following the formation of the primary ossification zones in the central or diaphyseal regions of skeletal elements, the establishment of secondary ossification centers at outer epiphyseal ends of bones defines the growth plate regions at the metaphysis (**Fig. 1A**). The growth plates occupy the narrow space that separates the marrow of the primary and secondary ossification centers, and are composed of a gradient of differentiating chondrocytes culminating in a zone of hypertrophic chondrocytes **(Fig. 1A & B)** (as reviewed in (Lefebvre and Smits 2005). The continual replacement of the hypertrophic chondrocytes by trabecular bone and marrow allows for longitudinal skeletal growth, robust hematopoiesis, and the progression of EO without consumption of the skeletal model until maturity, when in most non-rodent vertebrates EO ceases and growth plates close (**Fig. 1C**) (Kilborn et al. 2002). Thus, the end result of EO is a porous network of primary trabecular bone, consisting of a hybrid hyptertrophic cartilagebone matrix, and engulfed by a hematopoietic marrow (**Fig. 1B & C**). Subsequent bone remodeling gradually leads to a complete replacement of the hybrid primary bone by mature secondary bone, and is coincident with a gradual decline in lymphopoiesis and the onset of immunosenescence (**Fig. 1C**) (as reviewed in (Compston 2002; Gruver et al. 2007).

As EO initiates during embryogenesis, its distinctive feature is the emergence of hypertrophic cartilage, which is present in all skeletal elements that will develop a marrow cavity, e.g. long bones, hips, vertebrae, ribs, certain skull bones. The eventual replacement of cartilage by bone and marrow via EO relies on the sequential maturation of chondrocytes from resting, to proliferating, to hypertrophic (**Fig. 1A**). Chondrocyte hypertrophy manifests with a dramatic increase in cell size, cessation of proliferation, and synthesis of a new repertoire of differentiation-specific gene products (Godman and Porter 1960; Chan, D. and Jacenko 1998; Alvarez et al. 2001; James et al. 2010). Among these is the matrix protein collagen X, which represents the predominant biosynthetic product of hypertrophic cartilage (Gibson and Flint 1985; Schmid and Linsenmayer 1985). Concomitant with hypertrophy is a transformation from a non-calcified avascular cartilage matrix, to a calcifiable one that is permissive to vascular invasion. Morphometric analysis suggests that before vascular invasion, the terminal hypertrophic chondrocytes undergo either autophagy (Srinivas and Shapiro 2006; Bohensky et al. 2007) or apoptosis (Farnum and Wilsman 1989), the rate of which controls longitudinal growth of the skeletal element, as well as the transition from cartilage to trabecular bone and marrow (Farnum and Wilsman 1989).

Subsequent vascular entry into hypertrophic cartilage is critical to skeleto-hematopoietic development, since it leads to an influx of mesenchymal cells, hematopoietic precursors, and chondro/osteoclasts. This influx of cells, together with growth factors, cytokines and hormones, establishes the primary center of ossification and the marrow environment where hematopoiesis ensues (**Fig. 1**). Specifically, while chondro/osteoclasts degrade hypertrophic cartilage, multipotent stromal cells, including mesenchymal and perivascular reticular cells, form the marrow stroma, a meshwork of non-hematopoietic cells supporting hematopoiesis by providing structural scaffolding and producing hematopoietic factors (Taichman et al. 1996; Bianco et al. 1999). As hypertrophic cartilage continues to be degraded, matrix remnants serve as scaffolds upon which differentiating osteoblasts deposit bone matrix, thus forming trabecular bony spicules with hypertrophic cartilage cores (**Fig. 1B**) (Chan, D. and Jacenko 1998). Of note, the origin of the trabecular bone osteoblasts at the junction between marrow and the hypertrophic cartilage, termed the chondro-osseous junction, is still debated (Roach 1992; Roach et al. 1995; Roach and Erenpreisa 1996; Nakamura et al. 2006; Hilton et al. 2007; Maes et al. 2010). Following the formation of the primary ossification zones in the central or diaphyseal regions of skeletal elements, the establishment of secondary ossification centers at outer epiphyseal ends of bones defines the growth plate regions at the metaphysis (**Fig. 1A**). The growth plates occupy the narrow space that separates the marrow of the primary and secondary ossification centers, and are composed of a gradient of differentiating chondrocytes culminating in a zone of hypertrophic chondrocytes **(Fig. 1A & B)** (as reviewed in (Lefebvre and Smits 2005). The continual replacement of the hypertrophic chondrocytes by trabecular bone and marrow allows for longitudinal skeletal growth, robust hematopoiesis, and the progression of EO without consumption of the skeletal model until maturity, when in most non-rodent vertebrates EO ceases and growth plates close (**Fig. 1C**) (Kilborn et al. 2002). Thus, the end result of EO is a porous network of primary trabecular bone, consisting of a hybrid hyptertrophic cartilagebone matrix, and engulfed by a hematopoietic marrow (**Fig. 1B & C**). Subsequent bone remodeling gradually leads to a complete replacement of the hybrid primary bone by mature secondary bone, and is coincident with a gradual decline in lymphopoiesis and the onset of immunosenescence (**Fig. 1C**) (as reviewed in (Compston 2002; Gruver et al. 2007).

Fig. 1. Architecture of the endochondral bone. A) A schematic of a developing long bone illustrating its architecture. The epiphysis, or the bulbous end, lined by articular cartilage and containing the secondary ossification center with marrow, is supported by the flared metaphysis, which in turn rests upon the slender cylindrical shaft of the diaphysis. The growth plate separates the primary and secondary ossification centers, and consists of a gradient of differentiating chondrocyte zones; the proliferative cartilage (PC) and hypertrophic cartilage (HC) zones are marked, as well as the hybrid trabecular bone (TB) protruding into the marrow. The locations of the two-layered periosteal membrane surrounding the diaphysis and the inner endosteal network are marked. B) A longitudinal tibial section from a week-3 wild type mouse stained with safranin-orange, hematoxylin & eosin (H&E) and counterstained with fast green. Using these stains, the negatively charged cartilaginous matrix appears orange while the bone stains light blue-green; mature erythrocytes stain green, while other marrow elements stain pink-purple with H&E. The boxed inset is a high magnification of the chondro-osseous junction containing hypertrophic chondrocytes, bone and marrow with vascular. The hybrid nature of the trabecular bone can

Skeletogenesis and the Hematopoietic Niche 151

Table 1. Mouse models with altered hematopoetic niche enviroments.

be appreciated by the orange staining of the cartilaginous core, with green-blue bone matrix deposited on the surface (magnification, 10x). C) The inorganic mineralized matrix of a mature zebra bone illustrates the structural differences between the EO-derived trabecular /spongy/cancellous bone and the IO-derived compact/dense/cortical bone. Boxed is a high magnification of the EO- and IO-derived bone tissues. Note the mesh-like structure of trabecular bone for hematopoietic cell support.

Taken together, the proper differentiation of chondrocytes, vascular invasion and the gradual replacement of the cartilaginous anlagen by trabecular bone and marrow through EO, underscore the intricate orchestration of skeleto-hematopoiteic development. Moreover, the coincident establishment and localization of trabecular bone within the site of active hematopoiesis likely reflects a critical hematopoietic niche in the chondro-osseous region (**Fig. 1B boxed**) (Jacenko et al. 1993; Nilsson et al. 1997; Gress and Jacenko 2000; Nilsson et al. 2001; Jacenko et al. 2002; Yoshimoto et al. 2003; Arai, F. et al. 2004; Balduino et al. 2005; Sweeney et al. 2008; Kohler et al. 2009; Lo Celso et al. 2009; Xie et al. 2009; Sweeney et al. 2010). This skeleto-hematopoietic link is strongly supported by several animal models where alterations in process of EO leads to hematopoietic defects (**Table 1**), including mouse models with altered: collagen X (Jacenko et al. 1993; Gress and Jacenko 2000; Jacenko et al. 2002; Sweeney et al. 2008; Sweeney et al. 2010), parathyroid hormone related protein (PTHrP) receptor in osteoblasts (Calvi et al. 2001; Calvi et al. 2003; Kuznetsov et al. 2004; Wu et al. 2008), osteoblast numbers (Visnjic et al. 2001; Visnjic et al. 2004; Zhu et al. 2007), bone morphogenic protein (BMP) receptor type 1A in marrow cells (Zhang, J. et al. 2003), osteoclast function (Blin-Wakkach et al. 2004; Mansour et al. 2011), retinoic acid receptor gamma (Purton et al. 2006; Walkley et al. 2007), Gs in ostoblasts (Wu et al. 2008), Dicer in ostoblasts (Raaijmakers et al. 2010), glypican-3 (Viviano et al. 2005), and perlecan (Rodgers et al. 2008). **Table 1** presents a list of mouse models with defects in hematopoiesis due to alterations in a component within the niche environment. Only those mouse models are summarized that were proven, by and large, via bone marrow transplantation experiments to have an aberrant niche environment, since wild type marrow cells could not rescue the disease phenotype of the host.
