Chapter Seven – TGF-β1 Regulates Differentiation of Bone Marrow Mesenchymal Stem Cells

7/27/2019 Chapter Seven TGF-1 Regulates Differentiation of Bone Marrow Mesenchymal Stem Cells

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C H A P T E R S E V E N

TGF-b1 Regulates Differentiation of

Bone Marrow Mesenchymal

Stem Cells

Longmei Zhao and Basil M. Hantash

Contents

I. Bone Marrow Mesenchymal Stem Cells 128A. Overview 128

B. Heterogeneous nature 128

C. Differentiation potential 129

II. The Role of TGF-b1 in Differentiation of Bone Marrow MSCs 131

A. TGF-b1 and TGF-b signaling 131

B. TGF-b1 induces chondrogenic differentiation of

bone marrow MSCs 133

C. TGF-b1 regulates osteogenic differentiation of


D. TGF-b1 inhibits adipogenic differentiation of


E. TGF-b1 mediates bone marrow MSC differentiation into

other lineages 135

F. The molecular mechanism underlying TGF-b1-mediated

MSC differentiation 136

III. Summary 137

Acknowledgments 137

References 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 cardiomyo-

cytes. Multiple signaling networks orchestrate MSCs differentiating into functional

Vitamins and Hormones, Volume 87 # 2011 Elsevier Inc.

ISSN 0083-6729, DOI: 10.1016/B978-0-12-386015-6.00042-1 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 differenti-

ation 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-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. 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 twocharacteristic properties: unlimited or prolonged self-renewal and potentialfor differentiation. Multipotent stem cell populations found in adult tissueshave been of great interest because they serve as reservoirs for tissue repairand regeneration after trauma, disease, and aging. One important type ofadult stem cell is mesenchymal stromal/stem cells (MSCs), a small popula-tion of stromal cells present in most adult connective tissues. MSCs werefirst indentified by Friedenstein et al. who demonstrated that a rare popula-tion of plastic-adherent cells (1 in 10,000 nucleated cells) in bone marrowwere able to form single cell-derived colonies at low cell density(Friedenstein et al., 1970). The colonies consist of spindle-shaped cellsknown as colony-forming unit-fibroblasts (Fig. 7.1).

Although MSCs were originally isolated from bone marrow, similarpopulations 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-characterizedMSCs remain adult bone marrow MSCs.

B. Heterogeneous nature

Bone marrow is a complex tissue comprising hematopoietic precursors andstromal cells, the latter of which are a heterogeneous mixture of cells includingadipocytes, reticulocytes, endothelial cells, and fibroblastic cells (Bruderet al.,1997). Direct plating from bone marrow aspirates is the generally acceptedmethod of bone marrow MSC isolation and expansion. The direct plating

128 Longmei Zhao and Basil M. Hantash


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method and heterogeneous nature of bone marrow lead to heterogeneity ofbone marrow MSCs with respect to cell phenotype, colony size, and differen-tiation potential. Four types of cells are observed in primary culture of bonemarrow MSCs: spindle-shaped cells, star-shaped cells, large flat cells (Xiaoet al., 2010), and small round cells (Colter et al., 2001). Even single-cell-derived clonal MSC populations are also highly heterogeneous in their pro-

liferative and differentiation potentials (De Bari et al., 2008; Phinney andProckop, 2007). In addition, the lack of unified practice for the culture andpropagation of bone marrow MSCs results in a wide diversity in MSC isolatesacross the many research laboratories and clinics that handle them. At present,there remains no single unique specific cell surface marker to identify these cellpopulations. Thus, The International Society for Cellular Therapy providedthe following minimum criteria for defining multipotent human MSCs: (1)plastic-adherent under standard culture conditions; (2) positive for expressionof CD105, CD73, and CD90, and negative for expression of hematopoietic

cell surface markers CD34, CD45, CD11a, CD19, and HLA-DR; (3) underspecific stimuli, cells should differentiate into adipocytes, osteoblasts, andchondrocytes in vitro (Horwitz et al., 2005).

C. Differentiation potential

Bone marrow MSCs are maintained in a relative state of quiescence in vivobut, in response to a variety of physiological and pathological stimuli, arecapable of proliferation then differentiation. It is well established that MSCsare able to differentiate into the chondrogenic, osteogenic, and adipogeniclineages (Fig. 7.2). During chondrogenic differentiation, bone marrowMSCs change from a characteristic fibroblast-like morphology to a largeround shape and produce extracellular matrix (ECM), containing a highly

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.

TGF-b1 and Mesenchymal Stem Cell Differentiation 129


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organized type II collagen and proteoglycans and glycosaminoglycans(Vater et al., 2011). When being differentiated into osteoblasts, bone mar-row MSCs transform to a cubic shape and produce ECM, mainly composed

of type I collagen (Vater et al., 2011). Increased expression of alkalinephosphatase (ALP) and calcium accumulation is observed in MSCs duringosteogenic differentiation. When being differentiated into adipocytes,fibroblastic MSCs are converted to a spherical shape expressing severaltypes of ECM proteins, including fibronectin, laminin, and types I, III,IV, V, and VI collagen (Vater et al., 2011). Accumulation of intracellularlipid-rich vacuoles inside the cells can be stained positively by oil red O.

Under appropriate conditions, MSCs can also differentiate into othermesenchymal 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 (Gwaket al., 2009), and tenocytes (De Bari et al., 2003; Hoffmann et al., 2006).In addition, researchers have recently transdifferentiated MSCs into non-mesodermal 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 potencyof MSCs is, however, questioned because, for example, MSC-derivedneuron-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 differen-tiation of MSCs into functional mesenchymal lineages. Among these, trans-forming growth factor-b (TGF-b) proteins have emerged as key players inthe 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 ofBone Marrow MSCs

A. TGF-b1 and TGF-b signaling

TGF-b1 is a 25-kDa disulfide-linked homodimeric peptide, belonging tothe TGF-b family. TGF-b1 has two closely related mammalian isoforms(TGF-b2 and -b3) and shares a 6485% amino acid sequence homologywith them. Despite this high-sequence homology, they are functionallynonredundant (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 islocated in 19q13. Unlike the other two isoforms, TGF-b1 is extensivelyexpressed in almost all tissues (Massague, 1990; Moses and Serra, 1996).



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TGF-b1 is a multifunctional growth factor, which regulates a broad

range of biological processes, including cell proliferation, cell survival, celldifferentiation, cell migration, and production of ECM (Massague et al.,2000; Siegel and Massague, 2003). These combined actions mediate theglobal effect of TGF-b1 on many developmental processes and maintenanceof normal tissue homeostasis (Massague, 1998). Combined with its stimula-tory effect on MSC proliferation, TGF-b1 signaling thus allows for expan-sion 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 complexinduces a conformational change that induces phosphorylation and activa-tion of type I receptors by type II receptors. The activated receptorssubsequently phosphorylate the effectors Smad2/Smad3. PhosphorylatedSmad2/Smad3 then form complexes with the common Smad (Smad4)and translocate into the nucleus, where they interact at the promoter withother transcription factors at DNA sequence-specific binding sites ATF2(activating transcription factor-2) and SBE (Smad binding element) toregulate gene expression. The heteromeric Smad complex in the nucleusalso interacts with various transcriptional coactivators or corepressors result-ing in the activation or the repression of downstream target genes (Derynck,1998; Massague, 1998). TGF-b1 also induces non-SMAD signalingpathways by activation of the mitogen-activated protein kinase (MAPK)pathway (extracellular signal-regulated kinase-1 (ERK-1), c-Jun N-terminal

Mesenchymal stem cell

TGF-b1

Osteoblast

Adipocyte

Chondrocyte

Smooth muscle cell/cardiomyocyte

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



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

B. TGF-b1 induces chondrogenic differentiation ofbone marrow MSCs

The natural mesenchymal propensity of MSCs has prompted researchersto devote attention to their chondrogenic and osteogenic differentiationpotential. TGF-b1 plays an important role in cartilage development and is awell-documented potent chondrogenic factor. One of the earliest identifiedactivities of TGF-b1 was the induction of chondrogenesis in primitive ratmesenchymal cells in vitro (Rosen et al., 1986; Seyedin et al., 1986). This

chondrogenic effect induced by TGF-b1 has since been observed in rabbitchondrocyte 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-b1induces chondrogenic differentiation of bone marrow MSCs in vitro(Johnstone et al., 1998). They developed a pellet culture system thatallows cellcell interactions analogous to those that occur in precartilagecondensation during embryonic development. Rabbit bone marrow cell

pellet preparations was cultured in the defined medium supplied with TGF-b1 and 10% fetal bovine serum. The induction of chondrogenesis wasaccompanied by enhanced mRNA levels of both type IIA and IIB collagen,two of the most important ECM components in cartilage, and increasedALP activity of the aggregated cells (Johnstone et al., 1998). Since then,the role of TGF-b1 in cartilage tissue engineering has been investigatedextensively by cultivating bone marrow MSCs with various biomaterials inthree-dimensional (3D) systems. Bosnakovski et al. demonstrated that chon-drogenic capacity of bovine bone marrow MSCs was greatly enhancedwhen cultured in type II collagen hydrogel with media containing TGF-b1,even though the hydrogel alone had the potential to induce and maintainMSC chondrogenesis (Bosnakovski et al., 2006). The study of Parks groupshowed that chondrogenesis was only evident in rabbit bone marrow MSCsencapsulated in the hydrogel containing TGF-b1-loaded gelatin micropar-ticles, and chondrocyte-specific gene expression was varied with TGF-b1concentration in a dose-dependent manner (Park et al., 2007). The hydrogelmay function as both the scaffold of MSCs and the matrix of TGF-b1release, resulting in enhanced MSC aggregation and the consequent pro-motion of cell proliferation and chondrogenic differentiation (Ogawa et al.,2010). In the study by Xia et al., bone marrow MSCs adenovirally trans-duced with the human TGF-b1 gene were encapsulated into a biodegradedscaffold 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 collagenand sulfated proteoglycans (a late chondrogenic marker) in neocartilagetissue. In vivo, sustained production of TGF-b1, albeit at lower levels, was

sufficient for the induction of chondrogenic differentiation of bone marrowMSCs.

C. TGF-b1 regulates osteogenic differentiation ofbone marrow MSCs

TGF-b1 plays a pivotal role in bone regeneration because it was proven toaffect both bone formation and bone resorption (Janssens et al., 2005).TGF-b1 is secreted by osteoblasts as well as by bone marrow MSCs and isstored in bone matrix (Liu et al., 1999; Robey et al., 1987). TGF-b1effectively 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. Bonemarrow MSCs are a major source of osteoprogenitor cells (Owen andFriedenstein, 1988). Thus, TGF-b1 was believed to promote bone forma-tion through stimulation of proliferation and differentiation of bone mar-row MSCs, the osteoblast precursors. Although this seems straightforward,the story is much more complicated because many seemingly contradictoryreports have been published.

The direct positive effect of TGF-b1 on osteogenic differentiation ofbone marrow MSCs in vitro was reported by Zhao et al. (2010). After 14 daysof treatment with TGF-b1 (10 ng/mL), murine bone marrow MSCsunderwent osteogenic differentiation by increasing Runx-2 (a global regu-lator of osteogenesis), type I collagen and osteopontin (two osteoblastdifferentiation markers) as well as ALP activity in monolayer culture (Zhaoet al., 2010). Several investigators reported a biphasic effect of TGF-b1 onosteogenic capacity of bone marrow MSCs synergistically with other oste-ogenic inducers. Low concentrations of TGF-b1 (0.11 ng/mL) stimulatedosteogenic differentiation, while high concentrations of TGF-b1 (10 ng/mL)were inhibitory (Lieb et al., 2004; Liu et al., 1999). Liu et al. reported thatALP activity was enhanced in human bone marrow MSCs by cotreatmentwith 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 byhigh doses of TGF-b1 (single treatment at 10 ng/mL) in rat bone marrowMSCs cultivated with dexamethasone, ascorbic acid, b-glycerol phosphate,common chemical osteogenic inducers (Lieb et al., 2004). From the above, itis clear that the effect of TGF-b1 on in vitro osteogenic differentiation ofbone marrow MSCs is highly dependent on a broad range of experimentalconditions 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 ofbone 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 tomediate the suppressive effects of genistein (and 17b-estradiol) on adipogenicdifferentiation of human bone marrow MSCs. Blocking the TGF-b1 pathwayabolished the genistein-induced decrease in proteinlevels of adipocyte-specificperoxisome proliferation-activated receptorg2 (PPARg2), one of the tran-scription factors that regulates expression of genes responsible for inductionand progression of adipogenesis (Devine et al., 1999; Rosen et al., 1999). Thisled 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 inmurine bone marrow MSC monolayer cultures (Zhao et al., 2010).

E. TGF-b1 mediates bone marrow MSC differentiation intoother lineages

TGF-b1 modulation of MSC differentiation involves not only the three

lineages mentioned above but also other lineages such as SMCs and cardio-myocytes. TGF-b1 signaling contributes to development of SMCs fromembryonic stem cells (Beckeret al., 2008) and upregulates a variety of SMCdifferentiation markers in cultured SMCs derived from mature blood vessels(Kennard et al., 2008). The first evidence of TGF-b1s role in SMC differenti-ation of bone marrow MSCs was from Kinners study. They found that TGF-b1 significantly increased alpha-smooth muscle actin (a-SMA, an early markerof SMC differentiation) expression and the contractility of human bonemarrow MSCs (Kinneret al., 2002). Gong et al. showed that a concentrationof 0.110 ng/mL TGF-b1 inhibited human MSC proliferation but increasedcalponin (a late-stage SMC differentiation marker) expression in a dose-dependent manner (Gong and Niklason, 2008), indicating that TGF-b1 notonly initiates SMC differentiation but also promotes further differentiation.Similar to chondrogenesis, cellcell contact plays an important role in TGF-b1-induced SMC differentiation. Rat bone marrow MSCs plated on type IVcollagen-coated surfaces and exposed to TGF-b1 differentiated into a homo-geneous 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 mouseembryonic stem cells (Behfar et al., 2002). When treated with TGF-b1,both murine bone marrow MSCs and rat adipose-derived MSCs increasedexpression 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-mediatedMSC differentiation

TGF-b1-mediated MSC chondrogenic differentiation was revealed throughnon-SMAD signaling pathways, MAPK signaling and Wnt signaling cascades(Tuli et al., 2003). During chondrogenic differentiation, TGF-b1 treatmentactivates p38 and ERK-1 to promote sox9 expression, which in turn formtransactivating complexes with other proteins, for example, Sox5/Sox6 (Vateretal., 2011), to control expression of the chondrocyte-specific genes (collagen,

aggrecan, and cartilage link proteins). TGF-b1-mediated MAPK activationalso controls WNT-7A gene expression and Wnt-mediated signaling throughthe intracellularb-cateninTCF pathway, which regulates N-cadherin expres-sion and subsequent N-cadherin-mediated celladhesion complexes duringthe early steps of MSC chondrogenesis (Tuli et al., 2003).

At the molecular level, the central regulation of bone differentiation andformation is controlled by the transcriptional activity of Runx2 and TAZ(transcriptional coactivator with PDZ-binding motif ). Runx2 is a transcrip-tion factor required for bone formation (Komori et al., 1997; Otto et al.,

1997) and is a common target of bone morphogenetic protein- and TGF-b

-induced osteoblast-specific genes expression in pluripotent mesenchymalprecursor cells (Lee et al., 2000; Zhao et al., 2010). TAZ functions as atranscriptional modulator to control R-SmadSmad4 complex nucleocy-toplasmic shuttling as well as stem cell self-renewal and differentiation(Hong et al., 2005; Varelas et al., 2008). The net effect of TGF-b1 stimulatingosteogenic differentiation while simultaneously blocking the differentiationof MSCs into fat occurs through activating TAZ by the Smad-dependentpathway. The direct interaction between TAZ and the transcription factorsRunx2 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, GATA-binding protein 6 (GATA-6), and serum response factor (SRF), the latterbinds to CArG (CC(AT)6GG) cis elements that are found in the promo-ters of almost all SMC marker genes. Deaton et al. reported that TGF-b1induced SMC differentiation of MSCs through the activation of a smallGTPase RhoA-driven signaling cascade (Deaton et al., 2005). The signalingpathway involves RhoA/threonine protein kinase N-mediated activationof p38 MAPK, which in turn activates GATA and SRF leading to upregu-lation of SMC marker gene expression. Other downstream targets of TGF-b1 and RhoA, such as Rho kinase and Smads, may also play a role inmediating 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 appliedin various tissue engineering strategies. By using differentiation-specificprotocols, MSCs can be induced to differentiate towards a variety of maturetarget cells. TGF-b1 signaling plays an important role in the regulation ofMSCs at both transcriptional and posttranscriptional levels (Kurpinski et al.,2009), and a precise combination of microenvironmental cues may promoteor inhibit MSC differentiation. In general, TGF-b1 can induce eitherchondrogenic or SMC differentiation of MSCs in vitro. However, it requirescellcell and cellmatrix interactions, similar to development of these tissues

in vivo. The effect of TGF-b1-regulated osteogenic differentiation of MSCsin vitro depends on the specific culture conditions involved. TGF-b1 inhi-bits adipogenic differentiation of MSCs in monolayer culture. The presenceof other growth factors in the environment influences the exact outcome ofTGF-b1 functioning because TGF-b1 signaling through Smads cross talkwith many other signaling pathways such as Wnt, and Smads interact with amultitude of DNA-binding transcription factors, which themselves aretargeted by signaling pathways. In order to develop more effective regener-ative therapies with MSCs the combination of serum and supplementary

agents such as TGF-b1 or other growth factors and cytokines and theirconcentration should be optimized to meet each individual need.

ACKNOWLEDGMENTS

We apologize to those researchers whose work was not included in the review becauseof 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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Chapter Seven – TGF-β1 Regulates Differentiation of Bone Marrow Mesenchymal Stem Cells