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TGF-b1 Regulates Differentiation of Bone Marrow Mesenchymal Stem Cells

Longmei Zhao and Basil M. Hantash Contents
I. Bone Marrow Mesenchymal Stem Cells A. Overview B. Heterogeneous nature C. Differentiation potential II. The Role of TGF-b1 in Differentiation of Bone Marrow MSCs A. TGF-b1 and TGF-b signaling B. TGF-b1 induces chondrogenic differentiation of bone marrow MSCs C. TGF-b1 regulates osteogenic differentiation of bone marrow MSCs D. TGF-b1 inhibits adipogenic differentiation of bone marrow MSCs E. TGF-b1 mediates bone marrow MSC differentiation into other lineages F. The molecular mechanism underlying TGF-b1-mediated MSC differentiation III. Summary Acknowledgments References 128 128 128 129 131 131 133 134 135 135 136 137 137 137

Abstract
Mesenchymal stromal/stem cells (MSCs) are a small population of stromal cells present in most adult connective tissues, such as bone marrow, fat tissue, and umbilical cord blood. MSCs are maintained in a relative state of quiescence in vivo but, in response to a variety of physiological and pathological stimuli, are capable of proliferating then differentiating into osteoblasts, chondrocytes, adipocytes, or other mesoderm-type lineages like smooth muscle cells (SMCs) and cardiomyocytes. Multiple signaling networks orchestrate MSCs differentiating into functional
Escape Therapeutics, Inc., San Jose, California, USA Vitamins and Hormones, Volume 87 ISSN 0083-6729, DOI: 10.1016/B978-0-12-386015-6.00042-1
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2011 Elsevier Inc. All rights reserved.

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mesenchymal lineages. Among these, transforming growth factor-b1 (TGF-b1) has emerged as a key player. Hence, we summarize the effects of TGF-b1 on differentiation of MSCs toward different lineages. TGF-b1 can induce either chondrogenic or SMC differentiation of MSCs in vitro. However, it requires cellcell and cellmatrix interactions, similar to development of these tissues in vivo. The effect of TGF-b1regulated osteogenic differentiation of MSCs in vitro depends on the specific culture conditions involved. TGF-b1 inhibits adipogenic differentiation of MSCs in monolayer culture. Using this information, we may optimize the culture conditions to differentiate MSCs into desired lineages. 2011 Elsevier Inc.

I. Bone Marrow Mesenchymal Stem Cells

A. Overview
Stem cells are undifferentiated multipotent precursor cells that share two characteristic properties: unlimited or prolonged self-renewal and potential for differentiation. Multipotent stem cell populations found in adult tissues have been of great interest because they serve as reservoirs for tissue repair and regeneration after trauma, disease, and aging. One important type of adult stem cell is mesenchymal stromal/stem cells (MSCs), a small population of stromal cells present in most adult connective tissues. MSCs were first indentified by Friedenstein et al. who demonstrated that a rare population of plastic-adherent cells ( 1 in 10,000 nucleated cells) in bone marrow were able to form single cell-derived colonies at low cell density (Friedenstein et al., 1970). The colonies consist of spindle-shaped cells known as colony-forming unit-fibroblasts (Fig. 7.1). Although MSCs were originally isolated from bone marrow, similar populations have been reported in other tissues. Besides bone marrow, human MSCs have been isolated from adipose tissue (Fig. 7.1) (Zuk et al., 2001), umbilical cord blood (Erices et al., 2000), peripheral blood (Marinova-Mutafchieva et al., 2000), amniotic fluid (Int Anker et al., 2003b), placenta (Int Anker et al., 2004), liver (Campagnoli et al., 2001), lung (Int Anker et al., 2003a), dermis (Toma et al., 2001), skeletal muscle ( Jiang et al., 2002), and others. However, the most well-characterized MSCs remain adult bone marrow MSCs.

B. Heterogeneous nature
Bone marrow is a complex tissue comprising hematopoietic precursors and stromal cells, the latter of which are a heterogeneous mixture of cells including adipocytes, reticulocytes, endothelial cells, and fibroblastic cells (Bruder et al., 1997). Direct plating from bone marrow aspirates is the generally accepted method of bone marrow MSC isolation and expansion. The direct plating

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Figure 7.1 Morphology of human bone marrow MSCs (A) and adipose-derived MSCs (B). Cells were cultured in a-MEM (A) or in DMEM (B) containing 1% penicillin/ streptomycin and 10% FBS.

method and heterogeneous nature of bone marrow lead to heterogeneity of bone marrow MSCs with respect to cell phenotype, colony size, and differentiation potential. Four types of cells are observed in primary culture of bone marrow MSCs: spindle-shaped cells, star-shaped cells, large flat cells (Xiao et al., 2010), and small round cells (Colter et al., 2001). Even single-cellderived clonal MSC populations are also highly heterogeneous in their proliferative and differentiation potentials (De Bari et al., 2008; Phinney and Prockop, 2007). In addition, the lack of unified practice for the culture and propagation of bone marrow MSCs results in a wide diversity in MSC isolates across the many research laboratories and clinics that handle them. At present, there remains no single unique specific cell surface marker to identify these cell populations. Thus, The International Society for Cellular Therapy provided the following minimum criteria for defining multipotent human MSCs: (1) plastic-adherent under standard culture conditions; (2) positive for expression of CD105, CD73, and CD90, and negative for expression of hematopoietic cell surface markers CD34, CD45, CD11a, CD19, and HLA-DR; (3) under specific stimuli, cells should differentiate into adipocytes, osteoblasts, and chondrocytes in vitro (Horwitz et al., 2005).

C. Differentiation potential
Bone marrow MSCs are maintained in a relative state of quiescence in vivo but, in response to a variety of physiological and pathological stimuli, are capable of proliferation then differentiation. It is well established that MSCs are able to differentiate into the chondrogenic, osteogenic, and adipogenic lineages (Fig. 7.2). During chondrogenic differentiation, bone marrow MSCs change from a characteristic fibroblast-like morphology to a large round shape and produce extracellular matrix (ECM), containing a highly

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Figure 7.2 Differentiation potential of MSCs. Human bone marrow MSCs were seeded on tissue culture plate and induced to differentiate into adipocytes by culturing the cells in adipogenic media (Cell Applications, San Diego, CA) (B), or regular a-MEM media (A, control) for 14 days and then stained with Oil red O. For osteogenic differentiation, human adipose-derived MSCs were seeded on tissue culture plates and cultured in osteogenic media (Cell Applications) (D), or regular DMEM media (C, control) for 21 days and then stained with alizarin red. Cellular nuclei were countstained with hematoxylin (C and D). To induce chondrogenic differentiation, human adipose-derived MSCs were cultured as pellets in chondrogenic media (Cell Applications) (E). After 21 days, differentiation into the chondrogenic lineage was visualized.

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organized type II collagen and proteoglycans and glycosaminoglycans (Vater et al., 2011). When being differentiated into osteoblasts, bone marrow MSCs transform to a cubic shape and produce ECM, mainly composed of type I collagen (Vater et al., 2011). Increased expression of alkaline phosphatase (ALP) and calcium accumulation is observed in MSCs during osteogenic differentiation. When being differentiated into adipocytes, fibroblastic MSCs are converted to a spherical shape expressing several types of ECM proteins, including fibronectin, laminin, and types I, III, IV, V, and VI collagen (Vater et al., 2011). Accumulation of intracellular lipid-rich vacuoles inside the cells can be stained positively by oil red O. Under appropriate conditions, MSCs can also differentiate into other mesenchymal lineages such as smooth muscle cells (SMCs) (Gong and Niklason, 2008; Kanematsu et al., 2005; Kinner et al., 2002; Seruya et al., 2004), skeletal myocytes (Wakitani et al., 1995), cardiomyocytes (Gwak et al., 2009), and tenocytes (De Bari et al., 2003; Hoffmann et al., 2006). In addition, researchers have recently transdifferentiated MSCs into nonmesodermal cell types such as neuronal-like cells (Black and Woodbury, 2001) and pancreatic cell progenitors (Moriscot et al., 2005; Timper et al., 2006). The clinical relevance of the presumptive nonmesenchymal potency of MSCs is, however, questioned because, for example, MSC-derived neuron-like cells were unable to generate action potentials and, therefore, to function as neurons (Hofstetter et al., 2002). Multiple signaling networks orchestrate the development and differentiation of MSCs into functional mesenchymal lineages. Among these, transforming growth factor-b (TGF-b) proteins have emerged as key players in the self-renewal, maintenance of stem cells in their undifferentiated state, and the progression of differentiation along an individual lineage. Here, we illustrate the role of TGF-b1 in differentiation of bone marrow MSCs (see Fig. 7.3 for overview).

II. The Role of TGF-b1 in Differentiation of Bone Marrow MSCs

A. TGF-b1 and TGF-b signaling
TGF-b1 is a 25-kDa disulfide-linked homodimeric peptide, belonging to the TGF-b family. TGF-b1 has two closely related mammalian isoforms (TGF-b2 and -b3) and shares a 6485% amino acid sequence homology with them. Despite this high-sequence homology, they are functionally nonredundant (Dickson et al., 1995; Geiser et al., 1998; Kulkarni et al., 1993; Proetzel et al., 1995; Shull et al., 1992). The gene encoding TGF-b1 is located in 19q13. Unlike the other two isoforms, TGF-b1 is extensively expressed in almost all tissues (Massague, 1990; Moses and Serra, 1996).

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TGF-b1 Mesenchymal stem cell

Smooth muscle cell/ cardiomyocyte Chondrocyte

Adipocyte

Osteoblast

Figure 7.3 Schematic overview of the effects (stimulation or inhibition) of TGF-b1 on differentiation of MSCs toward different lineage. # indicates stimulation, ? indicates inhibition.

TGF-b1 is a multifunctional growth factor, which regulates a broad range of biological processes, including cell proliferation, cell survival, cell differentiation, cell migration, and production of ECM (Massague et al., 2000; Siegel and Massague, 2003). These combined actions mediate the global effect of TGF-b1 on many developmental processes and maintenance of normal tissue homeostasis (Massague, 1998). Combined with its stimulatory effect on MSC proliferation, TGF-b1 signaling thus allows for expansion of MSCs and their progenitors (Chen et al., 2004). TGF-b1 initiates signaling by its extracellular domain binding two types (type I and type II) of transmembrane receptor serinethreonine kinases, which form a complex at the cell surface. Ligand binding to this complex induces a conformational change that induces phosphorylation and activation of type I receptors by type II receptors. The activated receptors subsequently phosphorylate the effectors Smad2/Smad3. Phosphorylated Smad2/Smad3 then form complexes with the common Smad (Smad4) and translocate into the nucleus, where they interact at the promoter with other transcription factors at DNA sequence-specific binding sites ATF2 (activating transcription factor-2) and SBE (Smad binding element) to regulate gene expression. The heteromeric Smad complex in the nucleus also interacts with various transcriptional coactivators or corepressors resulting in the activation or the repression of downstream target genes (Derynck, 1998; Massague, 1998). TGF-b1 also induces non-SMAD signaling pathways by activation of the mitogen-activated protein kinase (MAPK) pathway (extracellular signal-regulated kinase-1 (ERK-1), c-Jun N-terminal

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kinase, and p38) through upstream mediators such as TGF-b-activated kinase (TAK1) (Hocevar et al., 1999; Yu et al., 2002).

B. TGF-b1 induces chondrogenic differentiation of bone marrow MSCs

The natural mesenchymal propensity of MSCs has prompted researchers to devote attention to their chondrogenic and osteogenic differentiation potential. TGF-b1 plays an important role in cartilage development and is a well-documented potent chondrogenic factor. One of the earliest identified activities of TGF-b1 was the induction of chondrogenesis in primitive rat mesenchymal cells in vitro (Rosen et al., 1986; Seyedin et al., 1986). This chondrogenic effect induced by TGF-b1 has since been observed in rabbit chondrocyte cultures (Kato et al., 1988), chicken mesenchymal cells (Leonard et al., 1991), and bovine nasal and articular chondrocytes culture (Xu et al., 1996). In 1998, Johnstone and colleagues first demonstrated that TGF-b1 induces chondrogenic differentiation of bone marrow MSCs in vitro ( Johnstone et al., 1998). They developed a pellet culture system that allows cellcell interactions analogous to those that occur in precartilage condensation during embryonic development. Rabbit bone marrow cell pellet preparations was cultured in the defined medium supplied with TGFb1 and 10% fetal bovine serum. The induction of chondrogenesis was accompanied by enhanced mRNA levels of both type IIA and IIB collagen, two of the most important ECM components in cartilage, and increased ALP activity of the aggregated cells ( Johnstone et al., 1998). Since then, the role of TGF-b1 in cartilage tissue engineering has been investigated extensively by cultivating bone marrow MSCs with various biomaterials in three-dimensional (3D) systems. Bosnakovski et al. demonstrated that chondrogenic capacity of bovine bone marrow MSCs was greatly enhanced when cultured in type II collagen hydrogel with media containing TGF-b1, even though the hydrogel alone had the potential to induce and maintain MSC chondrogenesis (Bosnakovski et al., 2006). The study of Parks group showed that chondrogenesis was only evident in rabbit bone marrow MSCs encapsulated in the hydrogel containing TGF-b1-loaded gelatin microparticles, and chondrocyte-specific gene expression was varied with TGF-b1 concentration in a dose-dependent manner (Park et al., 2007). The hydrogel may function as both the scaffold of MSCs and the matrix of TGF-b1 release, resulting in enhanced MSC aggregation and the consequent promotion of cell proliferation and chondrogenic differentiation (Ogawa et al., 2010). In the study by Xia et al., bone marrow MSCs adenovirally transduced with the human TGF-b1 gene were encapsulated into a biodegraded scaffold and implanted into the mouse dorsa subcutaneous tissue (Xia et al., 2009). At 3-weeks post-implantation, TGF-b1 significantly increased the

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volume of neocartilage tissue and the amount of type II collagen and sulfated proteoglycans (a late chondrogenic marker) in neocartilage tissue. In vivo, sustained production of TGF-b1, albeit at lower levels, was sufficient for the induction of chondrogenic differentiation of bone marrow MSCs.

C. TGF-b1 regulates osteogenic differentiation of bone marrow MSCs

TGF-b1 plays a pivotal role in bone regeneration because it was proven to affect both bone formation and bone resorption ( Janssens et al., 2005). TGF-b1 is secreted by osteoblasts as well as by bone marrow MSCs and is stored in bone matrix (Liu et al., 1999; Robey et al., 1987). TGF-b1 effectively stimulates the formation of collagen I (Arnold et al., 2002; Hock et al., 1990; Liu et al., 1999), the main matrix protein of bone (Anselme, 2000), which already was proved by various studies. Bone marrow MSCs are a major source of osteoprogenitor cells (Owen and Friedenstein, 1988). Thus, TGF-b1 was believed to promote bone formation through stimulation of proliferation and differentiation of bone marrow MSCs, the osteoblast precursors. Although this seems straightforward, the story is much more complicated because many seemingly contradictory reports have been published. The direct positive effect of TGF-b1 on osteogenic differentiation of bone marrow MSCs in vitro was reported by Zhao et al. (2010). After 14 days of treatment with TGF-b1 (10 ng/mL), murine bone marrow MSCs underwent osteogenic differentiation by increasing Runx-2 (a global regulator of osteogenesis), type I collagen and osteopontin (two osteoblast differentiation markers) as well as ALP activity in monolayer culture (Zhao et al., 2010). Several investigators reported a biphasic effect of TGF-b1 on osteogenic capacity of bone marrow MSCs synergistically with other osteogenic inducers. Low concentrations of TGF-b1 (0.11 ng/mL) stimulated osteogenic differentiation, while high concentrations of TGF-b1 (10 ng/mL) were inhibitory (Lieb et al., 2004; Liu et al., 1999). Liu et al. reported that ALP activity was enhanced in human bone marrow MSCs by cotreatment with TGF-b1 (0.110 ng/mL) plus Vitamin D3 [1,25(OH)2D3] (Liu et al., 1999). Both ALP activity and osteocalcin expression were suppressed by high doses of TGF-b1 (single treatment at 10 ng/mL) in rat bone marrow MSCs cultivated with dexamethasone, ascorbic acid, b-glycerol phosphate, common chemical osteogenic inducers (Lieb et al., 2004). From the above, it is clear that the effect of TGF-b1 on in vitro osteogenic differentiation of bone marrow MSCs is highly dependent on a broad range of experimental conditions such as cell density, the dosage of TGF-b1, the presence of serum, amongst others.

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D. TGF-b1 inhibits adipogenic differentiation of bone marrow MSCs

TGF-b1 has been shown to be a strong inhibitor of adipogenesis in 3T3 fibroblasts (Ignotz and Massague, 1985). TGF-b1 pathway was reported to mediate the suppressive effects of genistein (and 17b-estradiol) on adipogenic differentiation of human bone marrow MSCs. Blocking the TGF-b1 pathway abolished the genistein-induced decrease in protein levels of adipocyte-specific peroxisome proliferation-activated receptor g2 (PPARg2), one of the transcription factors that regulates expression of genes responsible for induction and progression of adipogenesis (Devine et al., 1999; Rosen et al., 1999). This led to a reduction in the proliferation rate of precursor cells (Heim et al., 2004). Recently, Zhao et al. demonstrated a direct inhibition effect of TGF-b1 on adipogenic differentiation of murine bone marrow MSCs (Zhao et al., 2010). PPARg2 and adipsin (a late adipogenic marker) were decreased by TGF-b1 in murine bone marrow MSC monolayer cultures (Zhao et al., 2010).

E. TGF-b1 mediates bone marrow MSC differentiation into other lineages

TGF-b1 modulation of MSC differentiation involves not only the three lineages mentioned above but also other lineages such as SMCs and cardiomyocytes. TGF-b1 signaling contributes to development of SMCs from embryonic stem cells (Becker et al., 2008) and upregulates a variety of SMC differentiation markers in cultured SMCs derived from mature blood vessels (Kennard et al., 2008). The first evidence of TGF-b1s role in SMC differentiation of bone marrow MSCs was from Kinners study. They found that TGFb1 significantly increased alpha-smooth muscle actin (a-SMA, an early marker of SMC differentiation) expression and the contractility of human bone marrow MSCs (Kinner et al., 2002). Gong et al. showed that a concentration of 0.110 ng/mL TGF-b1 inhibited human MSC proliferation but increased calponin (a late-stage SMC differentiation marker) expression in a dosedependent manner (Gong and Niklason, 2008), indicating that TGF-b1 not only initiates SMC differentiation but also promotes further differentiation. Similar to chondrogenesis, cellcell contact plays an important role in TGF-b 1-induced SMC differentiation. Rat bone marrow MSCs plated on type IV collagen-coated surfaces and exposed to TGF-b1 differentiated into a homogeneous population expressing a-SMA and calponin (Seruya et al., 2004). TGF-b1 is also known to play a key role in embryonic heart development (Akhurst et al., 1990) and to induce cardiomyocyte differentiation of mouse embryonic stem cells (Behfar et al., 2002). When treated with TGF-b1, both murine bone marrow MSCs and rat adipose-derived MSCs increased expression of cardiac-specific markers, such as troponin I, troponin T, cardiac myosin heavy chain, and a-sarcomeric actin, suggesting that TGF-b1

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may also promote MSC cardiomyogenic differentiation in vitro (Gwak et al., 2009; Li et al., 2005).

F. The molecular mechanism underlying TGF-b1-mediated MSC differentiation

TGF-b1-mediated MSC chondrogenic differentiation was revealed through non-SMAD signaling pathways, MAPK signaling and Wnt signaling cascades (Tuli et al., 2003). During chondrogenic differentiation, TGF-b1 treatment activates p38 and ERK-1 to promote sox9 expression, which in turn form transactivating complexes with other proteins, for example, Sox5/Sox6 (Vater et al., 2011), to control expression of the chondrocyte-specific genes (collagen, aggrecan, and cartilage link proteins). TGF-b1-mediated MAPK activation also controls WNT-7A gene expression and Wnt-mediated signaling through the intracellular b-cateninTCF pathway, which regulates N-cadherin expression and subsequent N-cadherin-mediated celladhesion complexes during the early steps of MSC chondrogenesis (Tuli et al., 2003). At the molecular level, the central regulation of bone differentiation and formation is controlled by the transcriptional activity of Runx2 and TAZ (transcriptional coactivator with PDZ-binding motif ). Runx2 is a transcription factor required for bone formation (Komori et al., 1997; Otto et al., 1997) and is a common target of bone morphogenetic protein- and TGF-binduced osteoblast-specific genes expression in pluripotent mesenchymal precursor cells (Lee et al., 2000; Zhao et al., 2010). TAZ functions as a transcriptional modulator to control R-SmadSmad4 complex nucleocytoplasmic shuttling as well as stem cell self-renewal and differentiation (Hong et al., 2005; Varelas et al., 2008). The net effect of TGF-b1 stimulating osteogenic differentiation while simultaneously blocking the differentiation of MSCs into fat occurs through activating TAZ by the Smad-dependent pathway. The direct interaction between TAZ and the transcription factors Runx2 and PPARg results in transcriptional enhancement and repression, respectively, of selective programs of gene expression (Hong and Yaffe, 2006; Hong et al., 2005; Zhao et al., 2010). SMC-specific transcription is regulated by transcription factors, GATAbinding protein 6 (GATA-6), and serum response factor (SRF), the latter binds to CArG (CC(AT)6GG) cis elements that are found in the promoters of almost all SMC marker genes. Deaton et al. reported that TGF-b1 induced SMC differentiation of MSCs through the activation of a small GTPase RhoA-driven signaling cascade (Deaton et al., 2005). The signaling pathway involves RhoA/threonine protein kinase N-mediated activation of p38 MAPK, which in turn activates GATA and SRF leading to upregulation of SMC marker gene expression. Other downstream targets of TGFb1 and RhoA, such as Rho kinase and Smads, may also play a role in mediating these effects on MSCs (Deaton et al., 2005).

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III. Summary
MSCs are a promising source of precursor cells which may be applied in various tissue engineering strategies. By using differentiation-specific protocols, MSCs can be induced to differentiate towards a variety of mature target cells. TGF-b1 signaling plays an important role in the regulation of MSCs at both transcriptional and posttranscriptional levels (Kurpinski et al., 2009), and a precise combination of microenvironmental cues may promote or inhibit MSC differentiation. In general, TGF-b1 can induce either chondrogenic or SMC differentiation of MSCs in vitro. However, it requires cellcell and cellmatrix interactions, similar to development of these tissues in vivo. The effect of TGF-b1-regulated osteogenic differentiation of MSCs in vitro depends on the specific culture conditions involved. TGF-b1 inhibits adipogenic differentiation of MSCs in monolayer culture. The presence of other growth factors in the environment influences the exact outcome of TGF-b1 functioning because TGF-b1 signaling through Smads cross talk with many other signaling pathways such as Wnt, and Smads interact with a multitude of DNA-binding transcription factors, which themselves are targeted by signaling pathways. In order to develop more effective regenerative therapies with MSCs the combination of serum and supplementary agents such as TGF-b1 or other growth factors and cytokines and their concentration should be optimized to meet each individual need.

ACKNOWLEDGMENTS
We apologize to those researchers whose work was not included in the review because of space constraints. We thank Chris Nye for providing the pictures of MSC differentiation. We thank Dr. Yiou Li for assistance with preparing Fig. 7.3.

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