Culture Media For The Differentiation of Mesenchymal Stromal Cells

Acta Biomaterialia 7 (2011) 463477
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Acta Biomaterialia
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Review
Culture media for the differentiation of mesenchymal stromal cells

Corina Vater, Philip Kasten, Maik Stiehler
Department of Orthopedic Surgery, University Hospital Carl Gustav Carus, Dresden, Germany
a r t i c l e
i n f o
a b s t r a c t
Mesenchymal stromal cells (MSCs) can be isolated from various tissues such as bone marrow aspirates, fat or umbilical cord blood. These cells have the ability to proliferate in vitro and differentiate into a series of mesoderm-type lineages, including osteoblasts, chondrocytes, adipocytes, myocytes and vascular cells. Due to this ability, MSCs provide an appealing source of progenitor cells which may be used in the eld of tissue regeneration for both research and clinical purposes. The key factors for successful MSC proliferation and differentiation in vitro are the culture conditions. Hence, we here summarize the culture media and their compositions currently available for the differentiation of MSCs towards osteogenic, chondrogenic, adipogenic, endothelial and vascular smooth muscle phenotypes. However, optimal combination of growth factors, cytokines and serum supplements and their concentration within the media is essential for the in vitro culture and differentiation of MSCs and thereby for their application in advanced tissue engineering. 2010 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.
Article history: Received 7 April 2010 Received in revised form 20 July 2010 Accepted 27 July 2010 Available online 3 August 2010 Keywords: Mesenchymal stromal cell Cell culture medium Differentiation Proliferation Osteoblast
1. Introduction Since Owen and Friedensteins work [1] it has been assumed that bone marrow contains special cells that can be expanded in vitro and differentiated into various mesoderm-type lineages, including bone, fat, cartilage, muscle, tendon, haematopoiesis-sup-
Abbreviations: 1,25-D3, 1,a25-dihydroxyvitamin D3; a2Col6, a chain 2 of type 6 collagen; ALP, alkaline phosphatase; asc, ascorbic acid; Asc-2-P, ascorbic acid 2-phosphate; AT, adipose tissue; BM, bone marrow; BM-MSCs, bone marrowderived mesenchymal stromal cells; BMP, bone morphogenetic protein; BSP, bone sialoprotein; C/EBP, CCAAT-enhancer-binding proteins; cAMP, cyclic adenosine monophosphate; Col10, collagen type 10; Col11, collagen type 11; Col1a1, collagen type 1 alpha-1; Col2, collagen type 2; Col9, collagen type 9; CREB/p300, cAMP response element-binding protein/E1A binding protein p300; dex, dexamethasone; EC, endothelial cell; ECM, extracellular matrix; FABP4/aP2, fatty acid-binding protein-4; FCS, fetal calf serum; FGF, broblast growth factor; Gata-6, GATA-binding protein 6; GPDH, glycerol-3-phosphate dehydrogenase; IBMX, 3-isobutyl-1-methylxanthine; IGF, insulin-like growth factor; LDL, low-density lipoprotein; LPL, lipoprotein lipase; mRNA, messenger ribonucleic acid; MSC, mesenchymal stromal cell; OC, osteocalcin; ON, osteonectin; OPN, osteopontin; Osx, osterix; PDGF, platelet-derived growth factor; PPARc2, peroxisome proliferation-activated receptor c2; PRP, platelet-rich plasma; Runx-2, runt-related transcription factor-2; SCID, severe combined immunodeciency; SM22-a, transgelin; SMMHC, smooth muscle myosin heavy chain; Sox9, SRY-related high-mobility group box 9; SRF, serum response factor; T3, triiodothyonine; TGF-b, transforming growth factor-beta; UCB, umbilical cord blood; UCWJ, umbilical cord Whartons jelly; VEGF, vascular endothelial growth factor; vitD3, vitamin D3; VSMC, vascular smooth muscle cell; a-SMA, a-smooth muscle actin; b-GP, beta-glycerophosphate. Corresponding author. Address: Department of Orthopedic Surgery, University Hospital Carl Gustav Carus, Fetscherstr. 74, D-01307 Dresden, Germany. Tel.: +49 (0)351 458 3137; fax: +49 (0)351 449 210 419. E-mail address: maik.stiehler@uniklinikum-dresden.de (M. Stiehler).
porting stroma and vasculature [26]. We presently refer to these nonhematopoietic cells as mesenchymal stromal cell (MSC; Fig. 1) [7]. In addition, researchers have recently transdifferentiated MSCs into non-mesodermal cell types such as neuronal-like cells [811] and pancreatic cell progenitors [1215]. To date, MSCs have been isolated not only from bone marrow but also from many other tissues and organs, including adipose tissue, umbilical cord blood, placental tissue, liver, spleen, testes, menstrual blood, amniotic uid, pancreas and periosteum [16 20]. When cultured in vitro on polystyrene surfaces, MSCs reveal morphological heterogeneity. These cells can be narrow spindleshaped, large polygonal or even cuboidal-shaped when growing into a conuent monolayer [21]. In adult human, MSCs lack the hematopoietic surface antigens, e.g. CD11, CD14, CD34 and CD45 [22]. Meanwhile numerous molecular markers have been found on MSC surface, but none of them is specic to MSCs. Despite this, molecules such as CD44, CD73, CD90, STRO-1 and CD105/SH2 [3,2325] are still currently used to identify MSCs. MSCs are considered to be nonimmunogenic since these cells have been transplanted into allogeneic hosts even without using any immunosuppressive drugs [22,26]. Furthermore, it has been reported that MSCs actually possess immunosuppressive properties by modulating the function of T-cells [27,28], dendritic cells and B-cells [2932]. For the characterization of MSC plasticity, their ability to differentiate in vitro into osteoblasts, chondrocytes and adipocytes is currently treated as the gold standard. This, in combination with the advantages that MSCs have no immunogenicity and can be easily isolated from different tissues and expanded in vitro, enables MSCs to be a promising source of stem cells. Hence MSCs have
1742-7061/$ - see front matter 2010 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.actbio.2010.07.037
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C. Vater et al. / Acta Biomaterialia 7 (2011) 463477
Fig. 1. Morphology of human immortalized single cell-derived BM-MSCs at different stages of conuency (A: 10%; B: 90%). Cells were cultured in a-MEM containing 1% penicillin/streptomycin and 10% FCS (BM-MSCs, bone marrow-derived MSCs).
been used in the therapy of diseases such as extended osseous defects [33], acute myocardial infarction [34], leukemia [35] and diabetes [36]. In addition, the homing capability endows MSCs with further potential applications. For example, MSCs may be used for supporting tissue regeneration [37], correcting congenital disorders (e.g. osteogenesis imperfecta [38]) and controlling chronic inammatory diseases [39,40], and have even employed as vehicles for the delivery of biological agents [22] and as probes in the biocompatibility test of new implant materials. A prerequisite for the therapeutic application of MSCs is to develop efcient and standardized protocols so that MSCs can be induced to differentiate along the way as required. Therefore, we here present an overview of the optimized protocols for MSC differentiation towards the osteogenic, chondrogenic, adipogenic, endothelial and vascular smooth muscle phenotypes. 2. Osteogenic differentiation 2.1. Background
trix (ECM), mainly composed of collagen type I, and in a later stage form aggregates or nodules that can be stained positively by alizarin red and von Kossa techniques. Increased expression of alkaline phosphatase (ALP; Fig. 2A) and calcium accumulation are observed in MSCs during osteogenic differentiation [3,22]. The enzymatic activity of ALP as well as the calcium content can be quantied by colorimetric assays [42]. At the molecular level, osteogenic differentiation of MSCs is controlled by interactions between distinct hormones and transcription factors. Runt-related transcription factor-2 (Runx-2) effectuates the expression of bone-specic genes, e.g. osterix (Osx), collagen type 1 alpha-1 (Col1a1), osteocalcin (OC) and bone sialoprotein (BSP), by binding to the promoters of these genes [4348]. Generally, Runx-2, ALP, Col1a1, transforming growth factor-beta 1 (TGF-b1), osteonectin (ON) and bone morphogenetic protein-2 (BMP-2) are known to be early markers of osteoblastic differentiation, whereas OC and osteopontin (OPN) are expressed later in the differentiation process [4952]. 2.3. Differentiation protocols
Bone diseases are major socioeconomic issues. The World Health Organization has acknowledged this fact by declaring the years 20002010 The Bone and Joint Decade. The development of innovative bone-healing strategies is a prerequisite for the successful treatment of a variety of patients suffering from local bone defects caused by trauma, tumour, infection, degenerative joint disease, congenital crippling disorders or periprosthetic bone loss. Furthermore, bone graft material is frequently needed for spinal fusion, joint revision surgery, corrective osteotomy procedures and bone reconstruction in the eld of oral and maxillofacial surgery. Bone grafting is one of the most common orthopaedic procedures with autologous bone graft providing osteoinductive growth factors, bone-forming cells and structural support for new bone ingrowth. However, the use of autologous bone graft is associated with the disadvantages of limited graft availability and donor site morbidity, e.g. pain, infection, pelvic fractures or neurovascular injury. The implantation of sterilized bone allograft material usually derived from femoral heads during joint replacement procedures as a widely used alternative bone-lling material may result in failure rates of up to 30% due to insufcient osseointegration of the graft, requiring further surgical intervention [41]. Insufcient bone-healing therefore remains a challenging issue. In this context, innovative cell-based strategies using MSCs are promising for both site-specic and systemic bone regeneration. 2.2. Morphology and differentiation markers When being differentiated into osteoblasts, MSCs transform from a broblastic to a cuboidal shape, produce extracellular ma-
The classical method for osteogenic differentiation of MSCs in vitro involves incubating a conuent monolayer of MSCs with combinations of dexamethasone (dex), beta-glycerophosphate (bGP) and ascorbic acid (asc) for several weeks. In addition, combinations of vitamin D3 (vitD3), transforming growth factor-beta (TGFb) and bone morphogenetic proteins (BMPs) are used for osteogenic differentiation. In the following these supplements will be described in detail. Dex is a synthetic glucocorticoid and has been reported to be an essential requirement for osteoprogenitor cell differentiation in MSCs [53,54]. While MSCs that were cultured in basal medium without osteogenic supplements express increased levels of ALP, they fail to express mineralized ECM as well as other osteogenic markers such as Col1 [55]. Although the precise mechanisms of action of dex on stem cell differentiation and skeletal function are not known, it is supposed that dex induces transcriptional effects. In rat osteoblast-like cells, for instance, dex induces transcription of BSP by binding on a glucocorticoid response element in the promoter region of the BSP gene [56]. On the other hand, dex improves the expression of the b-catenin-like molecule TAZ (transcriptional coactivator with PDZ-binding motif) as well as integrin a5, which both promote osteoblastic differentiation of MSCs by activating Runx-2-dependent gene transcription [57,58]. However, glucocorticoids in supraphysiological amounts have deleterious effects on bone in vivo, resulting in inhibition of osteoblast function [59]. In a study by Walsh et al. MSCs were cultured in the presence and absence of dex at concentrations between 10 pM and 1 lM for up to 28 days [60]. The authors suggest that the critical effective
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Fig. 2. Differentiation potential of MSCs. For osteogenic differentiation human BM-MSCs were seeded on glass slides and cultured in a-MEM containing 1% penicillin/ streptomycin, 10% FCS, 10 nM dex, 3.5 mM b-GP and 10 nM 1,25-D3. After 21 days of cultivation immunouorescent staining for alkaline phosphatase (shown in green) and uorescence-labeled phalloidin staining for actin (shown in red) was performed (A). To induce chondrogenic differentiation porcine BM-MSCs were cultured in pellet in DMEM containing 1% ITS, 1% penicillin/streptomycin, 10 ng ml1 TGF-b3, 10 nM dex and 210 lM ascorbic acid. After 21 days, differentiation into the chondrogenic lineage was visualized by immunohistochemical staining for ColX (shown in brown) (B). Human immortalized single-cell-derived BM-MSCs were seeded on tissue culture polystyrene and induced to differentiate into adipocytes by culturing the cells in DMEM containing 10% FCS, 1 mM dex, 500 lM IBMX, 1 lg ml1 insulin, and 100 lM indomethacin for 21 days. The appearance of intracellular lipid-rich vacuoles was conrmed by oil red O staining (shown in red) (C). Tubular-like structures formed by human immortalized single-cell-derived BM-MSCs in brin-based gels after 7 days of endothelial differentiation in a-MEM containing 1% penicillin/streptomycin, 10% FCS and 50 ng ml1 VEGF was visualized by bright-eld microscopy (D). Immunouorescent staining for calponin (shown in green) and uorescent staining for actin (shown in red) and nuclei (shown in blue) in porcine BM-MSCs differentiated towards the vascular smooth muscle phenotype (E). Therefore cells were cultured in a-MEM containing 1% penicillin/streptomycin, 10% FCS and 10 ng ml1 TGF-b1.
concentration of dex is 10 nM, corresponding to its physiological concentration. At higher concentrations (of the order of 100 nM) dex inhibits osteoblast differentiation in MSCs and leads to glucocorticoid-induced osteoporosis [57]. In addition, proliferation is negatively affected, mainly due to the inhibitory effect of glucocorticoids on collagen synthesis [61]. However, when MSCs are cultured in the presence of asc, the effects of glucocorticoids on collagen production are markedly masked [62]. Song et al. even reported on a reductive effect of dex on density-related apoptosis in MSC cultures [63]. When implanted subcutaneously into SCID mice, pretreatment of MSCs with 100 nM dex for up to 5 weeks resulted in enhanced bone tissue formation compared with untreated controls [42]. For complete induction of osteogenic differentiation of in vitro cultivated MSCs at least 3 weeks of continuous treatment with dex is required [42]. For matrix mineralization the presence of both calcium and phosphate ions is essential. b-GP, which is enzymatically hydro-
lyzed by alkaline phosphatase, serves as a crucial source of inorganic phosphate [64]. Chung et al. showed that cultivation of osteoblast-like cells in culture medium containing b-GP leads to mineral formation, lactate generation, increased ALP activity, as well as protein and phospholipid synthesis, indicating enhanced osteogenic differentiation [65]. Usually 510 mM b-GP is used for osteogenic differentiation of MSCs [55,65,66]. Asc plays an important role as a cofactor for the hydroxylation of proline and lysine residues in collagens, which are the most abundant group of ECM proteins in the body [67,68]. One difculty concerning the handling of asc is its instability in solution, especially under standard culture conditions (pH 7.5, 37 C, humidied atmosphere, 5% CO2) [69,70]. Thus it is recommended to use the long-acting vitamin C derivative ascorbic acid 2-phosphate (Asc2-P) which was found to be stable under conventional culture conditions [71]. Recent studies showed that in the presence of Asc-2-P MSCs demonstrate upregulation of genes related to cell cycle and
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mitosis, whereas absence of Asc-2-P leads to reduced ALP expression and inhibition of calcium accumulation [72]. The mitogenic effect of Asc-2-P can be ascribed either to the role of the Asc-2-Pinduced ECM as a growth factor reservoir or to a direct activation of mitogenic pathways. In the absence of collagens, integrin signalling is impaired and downstream signals that are needed for mitogen-activated protein kinase activation are absent. No inhibitory effect on cell growth could be found when supplementing the culture media with up to 10 mM ascorbic acid. Usually, concentrations ranging from 50 up to 500 lM are used to induce osteogenic phenotype of MSCs [42,69,73]. vitD3 is a secosteroid hormone which is essential for the function of osteoblasts. Its active form, 1a,25-dihydroxyvitamin D3 (1,25-D3), demonstrates potent antiproliferative effects by blocking the transition from the G1- to the S-phase during the cell cycle. Thereby osteogenic differentiation of MSCs is supported [74,75]. Non-genomic effects of 1,25-D3, which occur through the interaction of the hormone with a membrane-bound receptor, include an increase in cyclic adenosine monophosphate (cAMP) and the opening of calcium channels [76]. Through interaction with a nuclear vitamin D receptor, 1,25-D3 has been shown to stimulate the expression of bone-related transcription factors, i.e. Runx-2 and Osx, in addition to osteoblast differentiation markers, e.g. ALP, Col1a1, OC and OPN [70]. Although 1,25-D3 synergized with both dex and bone morphogenetic protein-2 promoted expression of osteoblastic markers, this agent alone was unable to induce matrix mineralization [59,77]. In contrast to this, Jaiswal et al. reported that dex may reduce vitamin D receptor expression in osteoblastic cells [78], leading to a reduced uptake ability for 1,25-D3 and therefore decreased expression of differentiation markers such as OC. As well as using dex, b-GP, Asc-2-P and 1,25-D3, the addition of distinct growth factors can enhance osteogenic differentiation of MSCs in vitro. TGF-b exists in three isoforms, whereas TGF-b1 is the major form found in bone [79,80]. TGF-b1 inuences cell growth and plays an essential role in the control of bone formation by modulating the synthesis and degradation of several bone matrix components, e.g. collagen type 1 and non-collagenous proteins [81,82]. Notably, although TGF-b1 stimulates the expression of Runx-2, it inhibits osteoblast differentiation in the late stages [77,83]. BMPs are also members of the TGF-superfamily and can, in contrast to TGF-b1, induce ectopic bone formation in developed tissues [84,85]. Recent reports investigating the role of BMPs in osteogenesis [8688] showed that there may be species-specic differences in the effect of BMPs in vitro. In both mice and rats, BMPs promote osteoblast differentiation [8991]. Interestingly, there is body of evidence that BMP-2, BMP-4 and BMP-7 fail to induce osteogenic differentiation of human MSCs [87,92]. In contrast,
several studies demonstrated that in cells of the osteoblast lineage, BMP-2, BMP-4 and BMP-7 are capable of inducing expression of ALP, Col1a1, OPN, BSP and other non-collagenous bone proteins found in osteoid [55,91,9396]. Representative protocols used for in vitro osteogenic differentiation of MSCs including dex, b-GP, Asc-2-P, vitD3, TGF-b and BMPs as well as commercially available culture media are summarized in Tables 1 and 2.
3. Chondrogenic differentiation 3.1. Background Cartilage defects have only a limited intrinsic healing capacity. For instance, partial thickness defects that do not penetrate the subchondral bone usually do not repair spontaneously [97]. Small full-thickness defects can repair spontaneously, resulting in hyaline-like cartilage. However, larger defects only regenerate by production of brous tissue or brocartilage, which as biomechanically inferior compared with physiological hyaline cartilage. The cell-based regeneration of (osteo)chondral defects presents a major challenge. Although, in principle, autologous chondrocytes can be used for cartilage tissue-engineering applications, their limited availability, quantity and viability are major drawbacks [98]. In this context, MSCs are promising candidates for cartilage regeneration [99]. 3.2. Morphology and differentiation markers During chondrogenic differentiation MSCs change from a characteristic broblast-like morphology to a large round shape. The cells are surrounded by abundant ECM, consisting of a highly organized network of collagen (predominantly type 2 collagen) and aggregating proteoglycans and glycosaminoglycans of high molecular weight [100102] that can be detected by, for example, Alcian blue, Toluidine blue and Safranin O stainings and quantied by specic assays [72,103]. The protein SRY-related high-mobility group box 9 (Sox9) is an early transcription factor of chondrogenesis and controls the expression of the genes collagen type 2 (Col2), collagen type 9 (Col9), collagen type 10 (Col10; Fig. 2B), collagen type 11 (Col11), aggrecan and cartilage link protein [104108]. By binding to the promoter of these genes, Sox9 forms transactivating complexes with other proteins, e.g. Sox5/Sox6, cAMP response element-binding protein/E1A binding protein p300 (CREB/p300) and c-musculoaponeurotic brosarcoma [104,109,110]. Col10 is known to be synthesized during chondrocyte hypertrophy in the growth plate
Table 1 Representative protocols used for in vitro osteogenic differentiation of MSCs (asc, ascorbic acid; bFGF, basic broblast growth factor; BMP, bone morphogenetic protein; dex, dexamethasone; FCS, fetal calf serum; ITS, insulin-transferrin-selenious acid; TGF-b, transforming growth factor-beta; vitD3, vitamin D3; b-GP, beta-glycerophosphate; BM, bone marrow; UCB, umbilical cord blood; UCWJ, umbilical cord Whartons jelly). Reference Oswald et al. [5] Jrgensen et al. [59] Friedman et al. [262] FCS 10% 10% dex 100 nM 100 nM 100 nM asc 200 lM 25 lg ml1 b-GP 10 mM 5 mM Other vitD3 (1 nM) BMP-2 (100 ng ml1) ITS (1%) bFGF (25 ng ml1) FGF-8/FGF-10 (50 ng ml1) TGF-b (200 pM) BMP-2/4/6/7/14 (20 nM) vitD3 (10 nM) BMP-2 (100 nM) Cell source Human BM Human BM Human BM
Stiehler et al. [263] Hildebrandt et al. [55] Pytlk et al. [73] Song et al. [42] Chen et al. [66]
10% 10% 10% 10%
100 nM 10 nM 100 nM 100 nM 100 nM 100 nM
290 nM 300 lM 500 lM 50 lM 200 lM
5 mM 5 mM 10 mM 10 mM 10 mM
Human Human Human Human Human
BM UCB BM BM BM/UCWJ
C. Vater et al. / Acta Biomaterialia 7 (2011) 463477 Table 2 Media commercially available for osteogenic differentiation of MSCs. Company Invitrogen (Carlsbad, USA) Trevigen (Gaithersburg, USA) Thermo Scientic (Waltham, USA) R&D Systems (Minneapolis, USA) Miltenyi Biotec (Bergisch Gladbach, Germany) Cellular Engineering Technologies Inc. (Coralville, USA) Provitro GmbH (Berlin, Germany) PromoCell GmbH (Heidelberg, Germany) Product STEMPRO Osteogenesis Differentiation Kit Mesenchymal Stem Cell Osteogenic Differentiation Kit HyClone AdvanceSTEM Osteogenic Differentiation Kit Human/Mouse StemXVivo Osteogenic/Adipogenic Base Media Human/Mouse StemXVivo Osteogenic Supplement NH OsteoDiff Medium Osteogenic Differentiation Media hMSC osteogenesis induction medium Mesenchymal Stem Cell Osteogenic Differentiation Medium
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of long bones and is used to identify terminally differentiated hypertrophic chondrocytes [111114]. 3.3. Differentiation protocols In 1998 Johnstone et al. rst described a dened cell culture medium for in vitro chondrogenesis of MSCs [115]. The two main principles involved in enhancing chondrogenic differentiation in vitro are close cell-to-cell contact, usually achieved by cell pellet or micromass culture [115,116], and the addition of chondrogenic bioactive factors, e.g. dex, asc, TGF-b, BMPs, broblast growth factor (FGF) and insulin-like growth factor (IGF). Among these dex, asc and TGF-b have been shown to be most effective [102,117120]. The effects of these factors on MSCs will be discussed in more detail below. Glucocorticoids are used for the in vitro differentiation of MSCs into multiple lineages, including stroma, fat, bone and cartilage [121]. Glucocorticoid function is mediated by the cytoplasmic glucocorticoid receptor, which inuences various differentiation processes by inducing transcriptional actions. In a study by Derfoul et al., dex upregulated gene expression and protein levels of several cartilage matrix markers, in particular Col11 [121]. However, in contrast to combination with TGF-b, dex alone has little effects on the expression of chondrogenic markers such as aggrecan, cartilage oligomeric matrix protein and Col2. For human primary bone marrow-derived MSCs, successful chondrogenic differentiation was shown in medium containing 100 nM dex [122]. Asc and its derivative Asc-2-P causes collagen hydroxylation via modication of proline and lysine residues. Its addition to the culture medium leads to increased MSC proliferation and enhances production of collagen type 2, which is one of the most important ECM proteins in cartilage [120]. TGF-b interacts with a heteromeric receptor complex comprising two structurally related serinethreonine kinases, types I and II receptors, and transforms its signals intracellularly via Smad proteins [123,124]. Together with dex, TGF-b represents an indispensable factor for chondrogenic differentiation of human MSCs [118]. Although Barry et al. state that TGF-b2 and TGF-b3 are more effective than TGF-b1 in promoting glycosaminoglycan and type 2 collagen accumulation [122], others have concluded that any of the TGF-b subtypes are equally active chondrogenic factors and that lot- rather than subtype-specic differences seem to exist [125]. Whilst chondrogenic differentiation of MSCs in monolayer culture appears to be dependent on TGF-b concentration and on cultivation time, a concentration of 10 ng ml1 TGF-b is sufcient for successful MSC differentiation in pellet culture [122,126]. Although TGF-b supports early- and intermediate-stage chondrogenesis, it is known to retain chondrocytes in the prehypertrophic state. Therefore TGF-b most likely represses terminal in vitro differentiation of MSCs [127]. BMPs play an important role during bone morphogenesis by initiating chondro-progenitor cell determination and differentiation [128]. Although BMP-2, BMP-4, BMP-6 and BMP-7 act synergisti-
cally with TGF-b by enhancing ECM deposition, they are not sufcient to stimulate in vitro chondrogenesis of human MSCs in conventional pellet culture with dex-supplemented medium [129,130]. In a study by Richter et al. MSCs from bone marrow required only dex and TGF-b for successful chondrogenic differentiation, whereas the differentiation of MSCs derived from adipose tissue or synovial tissue was dependent on the additional supplementation with BMP-6 [122]. Sekiya et al. demonstrated that the chondrogenic effect of BMP-2 on bone-marrow-derived MSCs is greater than that of BMP-4 and BMP-6 as measured by increased pellet weight, superior production of proteoglycans and Col2 [131]. In vitro treatment of MSCs with FGF, dex, or combinations of both, increased pellet content of collagen type 2 and glycosaminoglycans as well as mRNA expression of aggrecan [132,133]. Jingushi et al. found that repeated injections of FGF into the fracture site in rats induced cartilaginous callus formation enlargement [117]. The chondrogenic differentiation potential of MSCs treated with IGF has been investigated extensively. IGF stimulates MSC proliferation, regulates cell apoptosis, induces the expression of chondrocyte markers, increases the synthesis of matrix proteins including collagen type 2 and proteoglycans, and promotes the survival, development and maturation of chondrocytes in vitro [102,134 136]. IGF-preconditioned autologous bone-marrow-derived MSCs enhanced chondrogenesis in full-thickness cartilage lesions in the femoropatellar articulations of young mature horses [137]. Although some studies did not show any effect of isolated IGF application on in vivo MSC differentiation, Indrawattana et al. showed that IGF is capable of inducing chondrogenic differentiation on a level comparable to TGF-b [138140]. However, Matsuda et al. reported that the combination of TGF-b, dex and IGF was the most effective cocktail to stimulate differentiation of MSCs to chondrocytes [141]. Usually chondrogenic differentiation of MSCs is performed in serum-free media in order to reduce serum-induced cellular apoptosis [142]. In this context, platelet-rich plasma (PRP) seems to be a promising supplementary agent. PRP is dened as plasma enriched for platelets and contains effective growth factors such as TGF-b1 and vascular endothelial growth factor (VEGF) [143]. Recent in vitro investigations have conrmed that both cellular proliferation as well as mRNA levels of Runx-2, Sox9 and aggrecan were signicantly higher in PRP-treated cells compared to fetal calf serum (FCS)-treated MSC cultures [144]. Furthermore, the level of chondrogenic potential of MSCs is dependent on the tissue source the cells are isolated from. Studies showed that bone-marrow-derived MSCs demonstrated higher levels of in vitro chondrogenic potential compared to adipose-derived MSCs [145148]. Sakaguchi et al. even demonstrated that MSCs isolated from synovium showed superior chondrogenic potential than MSCs isolated from other mesenchymal tissues [149]. Tables 3 and 4 give an overview about representative protocols used for in vitro chondrogenic differentiation of MSCs including dex, asc, TGF-b, BMPs and IGF as well as the respective commercially available media.
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Table 3 Representative protocols used for in vitro chondrogenic differentiation of MSCs (asc, ascorbic acid, BSA, bovine serum albumin; BMP, bone morphogenetic protein; dex, dexamethasone; FCS, fetal calf serum; IGF, insulin-like growth factor; ITS, insulin-transferrin-selenious acid; TGF-b, transforming growth factor-beta; BM, bone marrow; UCWJ, umbilical cord Whartons jelly; AT, adipose tissue). In all protocols cells were cultured under three-dimensional conditions (pellet, scaffold). Reference Matsuda et al. [141] Sekiya et al. [131] FCS 10% dex 39 ng ml1 100 nM asc 50 lg ml1 50 lg ml1 TGF-b 100 ng ml1 100 ng ml1 Other IGF (100 ng ml1) BSA (1.25 mg ml1) ITS (50 mg ml1) linoleic acid (5.35 mg ml1) BMP-2/4/6 (500 ng ml1) BSA (1.25 mg ml1) insulin (5 lg ml1) transferring (5 lg ml1) selenous acid (5 lg ml1) BSA (1.25 mg ml1) ITS (1%) linoleic acid (5.35 lg ml1) BMP-7 (100 ng ml1) BSA (1.25 mg ml1) ITS (1%) ITS (50 lg ml1) BMP-6 (500 ng ml1) ITS (1%) BMP-6 (10/500 ng ml1) Cell source Human BM Human BM
Vogel et al. [264]
100 nM
170 lM
10 ng ml1
Human BM
Shen et al. [265]
100 nM
50 lg ml1
10 ng ml1
Human BM
Chen et al. [66] Fernandes et al. [72] Diekman et al. [266]
100 nM 100 nM 100 nM
50 lg ml1 200 lM 37.5 lg ml

1
10 ng ml1 10 ng ml1 10 ng ml
1
Human BM/UCWJ Human BM Human BM/AT
Table 4 Media commercially available for chondrogenic differentiation of MSCs. Company Invitrogen (Carlsbad, USA) R&D Systems (Minneapolis, USA) Miltenyi Biotec (Bergisch Gladbach, Germany) Cellular Engineering Technologies Inc. (Coralville, USA) Provitro GmbH (Berlin, Germany) PromoCell GmbH (Heidelberg, Germany) Product StemPro Chondrogenesis Differentiation Kit Human/Mouse StemXVivo Chondrogenic Base Media Human/Mouse StemXVivo Chondrogenic Supplement NH ChondroDiff Medium Chondrogenic Differentiation Media hMSC chondrogenesis induction medium Mesenchymal Stem Cell Chondrogenic Differentiation Medium
4. Adipogenic differentiation 4.1. Background The formation of adipocytes and their aggregation in order to form adipose tissue denotes an important step in the evolution of vertebrates, as it guarantees independence from food intake over longer periods of time [150]. In plastic and reconstructive surgical procedures, adipose tissue is frequently needed to repair soft tissue defects that result from traumatic injury (i.e. signicant burns), tumour resections (i.e. mastectomy and carcinoma removal), and congenital defects [151]. The American Society of Plastic Surgeons reported that over 5 million reconstructive procedures were performed in 2004, approximately 4 million of which were due to tumour removal [152]. Strategies to repair soft tissue defects, e.g. breast reconstruction procedures, include the use of implants and llers [153,154]. However, there exists no void-lling material that will satisfy all clinical needs. The use of autologous fat tissue has not been consistently successful in patients [155,156] due to the fact that fat transplants lack sufcient vascularization, i.e. central graft blood ow is not adequate for long-term survival of the tissue, and often leads to tissue resorption [157]. In this context, tissue engineering using MSCs is an appealing strategy to provide adipose tissue grafts. 4.2. Morphology and differentiation markers Adipose cell differentiation is a multistep process characterized by a sequence of events during which preadipocytes divide until conuence [158]. When being differentiated into adipocytes, broblastic MSCs are converted to a spherical shape expressing several types of ECM proteins, including bronectin, laminin, and types 1,
3, 4, 5 and 6 collagen. Initially a bronectin network develops, followed by the formation of a type I collagen network [159 161]. Finally, differentiation of MSCs into adipocytes leads to accumulation of intracellular lipid-rich vacuoles that can be stained positively by oil red O (Fig. 2C). Quantitative analyses can be performed either by staining the cells with oil red O and extracting the dye from the cells with isopropanol [103] or by determining the activity of glycerol-3-phosphate dehydrogenase (GPDH) [162164]. The adipocyte-specic peroxisome proliferation-activated receptor c2 (PPARc2) belongs to the PPARs family, and is, together with CCAAT-enhancer-binding proteins (C/EBP), one of the transcription factors that regulate expression of genes responsible for induction and progression of adipogenesis [165,166]. Lipoprotein lipase (LPL) and the a chain 2 of type 6 collagen (a2Col6) are known to be early markers of adipogenic differentiation, whereas leptin, fatty acid-binding protein-4 (FABP4/aP2) and adiponectin are expressed during later stages of differentiation [158,167170]. 4.3. Differentiation protocols A routinely used method to stimulate adipogenic differentiation of preadipocytes and MSCs is the cultivation of these cells until conuency, followed by incubation with dex, 3-isobutyl-1-methylxanthine (IBMX), insulin and indomethacin, or combinations of these. In addition to these agents, triiodothyonine (T3), Asc-2-P and basic FGF (bFGF-2) are also used for adipogenic differentiation as will be described in detail below. The glucocorticoid dex induces the accumulation of transcription factors including C/EBP and PPARc which are crucial for adipose conversion of precursor cells [171]. In a study conducted by Cui and coworkers, adipogenesis (as quantied by the presence
469
of triglyceride-containing vesicles and by expression of FABP4) was induced in a murine MSC cell line by treating the cells with dex [172]. The positive effect of glucocorticoids on adipogenic differentiation can be amplied by cAMP-elevating agents such as IBMX [173]. While inhibiting the activity of phosphodiesterase and tumour necrosis factor-a (TNF-a), IBMX enhances the expression of PPARc2 and LPL and down-regulates the expression of osteogenic marker genes such as Runx-2 and OPN by activation of protein kinase A [170,174176]. Among the commonly used adipogenesis-inducing drugs, insulin is known to be the most potent [177]. It stimulates adipogenesis through the Akt-TSC2-mTORC1 pathway by increasing and accelerating triglyceride accumulation [178,179]. To maintain in vitro adipogenic differentiation of MSCs, the effect of insulin can be enhanced by insulin sensitizers, e.g. rosiglitazone or troglitazone, which act by stimulating PPARc [180182]. The nonsteroidal anti-inammatory drug indomethacin is a cyclooxygenase inhibitor and blocks osteoclast and osteoblast differentiation, while promoting hMSC commitment to the adipogenic lineage [183,184]. Furthermore, indomethacin was found to bind directly to the PPARc receptors and to act as a PPARc agonist, leading in conuent MSC monolayers to the expression of adipogenic markers such as adipose differentiation-related protein [185,186]. Several other growth factors, including thyroid hormones, e.g. T3, Asc-2-P and FGF-2, have been shown to positively inuence adipogenesis [187189]. While T3 multiplies the effects of insulin, media supplementation with 500 lM Asc-2-P results in the accumulation of the highest number of oil droplets compared to control medium without Asc-2-P [103]. FGF-2, which belongs to the family of heparin-binding growth factors, increases cellular proliferation
and exerts a direct effect on adipogenic differentiation by inducing the expression of PPARc [190193]. Matrigel functionalized with FGF-2 has been found to be proadipogenic when injected subcutaneously alone [188] or in combination with gelatin microspheres into mice [194]. Within the implantation period, a visible fat pad was formed at the injection site, which is probably due to preadipocyte and endothelial cell migration to the injection site [188,195]. The process of adipogenic differentiation of MSCs starts upon cellular monolayer conuence and is induced by the factors mentioned above. To reach conuence MSCs are often cultured in medium containing FCS. However, it has been shown that extended maintenance of MSCs in FCS-supplemented medium markedly and irreversibly reduces the capability for an adipogenic conversion to mature adipocytes, most likely due to the effect of serum factors present in the FCS, e.g. TNF-a or TGF-b [196]. Therefore Hemmrich et al. recommended the use of human serum which, in contrast to FCS, allows substantial proliferation and differentiation even after long cell expansion periods [163]. Representative protocols used for in vitro adipogenic differentiation of MSCs, including dex, IBMX, insulin, indomethacin, T3 and asc, as well as commercially available culture media, are summarized in Tables 5 and 6. 5. Endothelial differentiation 5.1. Background A major aim in tissue engineering is to ensure adequate vascularization of articial tissue-like constructs. Previous studies have showed that vascularization within in vitro engineered tissues
Table 5 Representative protocols used for in vitro adipogenic differentiation of MSCs (asc, ascorbic acid; FCS, fetal calf serum; dex, dexamethasone; IBMX, 3-isobutyl-1-methylxanthine; T3, triiodothyonine; AT, adipose tissue; BM, bone marrow; UCWJ, umbilical cord Whartons jelly). Reference van Harmelen et al. [267] FCS dex Insulin 66 nM IBMX 500 lM Indo-methacin Other Transferrin (10 lg ml ) T3 (200 pM) cortisol (100 nM) Transferrin (10 lg ml1) pioglitazone (100 lg ml1) T3 (1 nM) asc (50 lM) T3 (200 pM) transferring (100 nM) calcium pantotenate (17 mM) biotin (33 mM) asc (250 lM) T3 (200 pM) rosiglitazone (1 lM)
1
Cell source Human AT
Hemmrich et al. [163]
100 nM
66 nM
500 lM
Human AT
Bunnell et al. [268] Choi et al. [103] Tsuji et al. [164]
20% 10%
500 nM 1 mM 100 nM
10 mg ml 50 lM
500 lM 500 lM
50 lM 10 mM
Rat AT Human BM Human AT
Vashi et al. [269] Chen et al. [66] Pytlik et al. [73] Vallee et al. [161]
10% 10% 3%
1 mM 1 mM 100 nM 1 lM
10 mM 5 mg ml 10 lg ml 100 nM
500 lM 500 lM 500 lM 250 lM
200 mM 60 mM 200 lM
Rat BM Human BM/UCWJ Human BM Human AT
Table 6 Media commercially available for adipogenic differentiation of MSCs. Company Invitrogen (Carlsbad, USA) Trevigen (Gaithersburg, USA) Thermo Scientic (Waltham, USA) R&D Systems (Minneapolis, USA) Miltenyi Biotec (Bergisch Gladbach, Germany) Cellular Engineering Technologies Inc. (Coralville, USA) Provitro GmbH (Berlin, Germany) PromoCell GmbH (Heidelberg, Germany) Product STEMPRO Adipogenesis Differentiation Kit Mesenchymal Stem Cell Adipogenic Differentiation Kit, HyClone AdvanceSTEM Adipogenic Differentiation Kit Human/Mouse StemXVivo Osteogenic/Adipogenic Base Media Human/Mouse StemXVivo Adipogenic Supplement NH AdipoDiff Medium Adipogenic Differentiation Media hMSC adipogenesis induction medium Mesenchymal Stem Cell Adipogenic Differentiation Medium
470
using mature endothelial cells (ECs) improved blood perfusion and cell viability during and after transplantation [197,198]. Blood vessels primarily consist of three cell types. While ECs can be found in the innermost layer of a vessel, vascular smooth muscle cells (VSMCs) are typically located in the intermediate layer and broblast-like cells in the adventitial outermost layer. However, mature ECs and SMCs have limited proliferation potential and their use for generating vascular conduits would require laborious autologous cell isolation. In contrast, the fact that MSCs can be easily isolated and differentiated into ECs makes them a favourable alternative cell source. In addition, the use of allogeneic or even xenogenic MSCs may be feasible without the aid of extended medical immunosuppression, thereby further alleviating the availability of MSCs as cellular component for tissue-engineering applications [199,200]. 5.2. Morphology and differentiation markers When being differentiated in semi-solid medium (see below) into endothelial cells, MSCs form three-dimensional capillary-like tubular structures (Fig. 2D) and have the ability to take up acetylated low-density lipoproteins (LDLs) via special receptors. The incorporation of dye-conjugated LDLs, e.g. 1,10-dioctadecyl-13,3,30,30-tetramethyl-indo-carbocyanine perchlorate conjugated to acetylated-LDL (e.g. DiI-Ac-LDL, Invitrogen, Carlsbad, USA), can be determined by uorescence microscopy and uorescence-activated cell sorting [66,200]. Vascular endothelial growth factor (VEGF)-receptor 2 is one of the two major VEGF receptors and is expressed during the early stage of endothelial differentiation of MSCs [201,202]. Markers that characterize later stages of endothelial differentiation include tissue-type plasminogen activator, von Willebrand factor, platelet-endothelial cell adhesion molecule 1 (CD31) and vascular endothelial-cadherin [200,202]. 5.3. Differentiation protocols A commonly used method to stimulate the differentiation of MSCs into endothelial cells is the cultivation of these cells in socalled semi-solid medium (e.g. Matrigel, BD Biosiences, Franklin Lakes, USA). The presence of VEGF the most common supplementary agent to date markedly enhances endothelial differentiation of MSCs [5]. The VEGF family comprises seven members (i.e. VEGFA/B/C/D/E/F and placental growth factor) that all have a common VEGF homology domain. Alternative splicing of the VEGF-A gene generates six isoforms of VEGF-A composed of 121, 145, 165, 183, 189 and 206 amino acids, respectively. Of these, VEGF165 is the most commonly expressed isoform [203,204]. VEGF-A is typically synthesized by vascular and tumour cells, and has been shown to be expressed by primary human MSCs as well [200]. To test whether the MSC-secreted VEGF is able to contribute to angiogenesis, Beckermann et al. added supernatant from cultured MSCs to human umbilical vein endothelial cells [205]. This resulted in strong induction of sprouting in similar intensity as observed with the addition of recombinant VEGF alone. In addition, VEGF induces vascular hyperpermeability needed, for example, during wound healing [206] and is known to be a chemoattractant for MSCs [207], for ECs [208,209] and for osteoclasts [210,211]. In the cornea and in bone grafts, VEGF induces growth of capillary sprouts from preexisting blood vessels [212], whereas osteogenic commitment is directly prevented by VEGF by inhibition of BMP-2 expression [213]. Other factors that can be used for endothelial differentiation of MSCs include FGF and platelet-derived growth factor (PDGF). FGF stimulates EC proliferation [190] and migration [214] as well as production of plasminogen activator and collagenase, which play a role in the clotting system [215]. After implantation on the mes-
enteric membrane in rat peritoneum, poly-(lactic-co-glycolic acid)based microspheres releasing FGF induced the formation of large and mature blood vessels [216]. However, these angiogenic effects of FGF contrast with the fact that vascular development is not hampered in FGF-decient mice, suggesting highly restricted roles for FGF under normal developmental and physiological conditions [217]. Within the PDGF family four members have been identied: the classical PDGFs, PDGF-A and PDGF-B, which have been studied intensively for more than 20 years; and the novel PDGFs, PDGF-C and PDGF-D, which were only recently discovered [218]. PDGF-A and PDGF-B bind as disulphide-linked homo- or heterodimers to their tyrosine kinase receptors (PDGF receptor a and PDGF receptor b) and are physiologically synthesized by platelets, monocytes, macrophages and endothelial cells [219,220]. Capillary endothelial cells stimulated by PDGF-BB not only increase DNA synthesis [221], but also form angiogenic sprouts in vitro [222,223]. Representative protocols used for in vitro endothelial differentiation of MSCs including VEGF as well as commercially available media are shown in Tables 7 and 8. 6. Vascular smooth muscle differentiation 6.1. Background VSMCs play an important role in angiogenesis, mechanical support of vessels and blood pressure control [224]. Thus, generating a functional VSMC layer is a prerequisite for successful construction of tissue-engineered blood vessels. The replicative ability of autologous VSMCs derived from older donors, representing the majority of potential recipients of vascular grafts for treatment of cardiovascular diseases, is limited [225,226]. Thus, MSCs represent an appealing source of smooth muscle progenitor cells for vascular engineering approaches. 6.2. Morphology and differentiation markers VSMCs demonstrate both a synthetic and a contractile phenotype. The synthetic phenotype demonstrates broblast-like morphology, high proliferation rate and production of ECM comprised of collagen, elastin and proteoglycans, which allows for elasticity and improved compliance [227]. In contrast, contractile VSMCs are spindle-shaped with a prominent, centrally located nucleus, proliferate at an extremely low rate but are capable of contraction [228]. Contraction is predominantly mediated by
Table 7 Representative protocols used for in vitro endothelial differentiation of MSCs (EGF, epidermal growth factor; FCS, fetal calf serum; VEGF, vascular endothelial growth factor; BM, bone marrow; UCWJ, umbilical cord Whartons jelly). Reference Jiang et al. [270] Oswald et al. [5] Chen et al. [66] FCS VEGF 2% 5%
1
Other
Cell Source Human BM Human BM Human BM/UCWJ
10 ng ml 50 ng ml1 1 100 ng ml EGF (50 ng ml1) hydrocortisone (1 lg ml1)
Table 8 Media commercially available for endothelial differentiation of MSCs. Company PromoCell GmbH (Heidelberg, Germany) Cellular Engineering Technologies Inc. (Coralville, USA) Product Endothelial Progenitor Medium Endothelial Progenitor Cell Differentiation Media
471
Ca2+-sensitive ion channels [229,230]. Microscopically, contractility can be measured by a collagen gel lattice contraction assay in combination with stimulative agents, e.g. potassium chloride or carbachol [227,231]. On the molecular level there are several markers commonly used to assess VSMC phenotype, including a-smooth muscle actin (a-SMA), transgelin (SM22-a), calponin (Fig. 2E), caldesmon, smooth muscle myosin heavy chain (SMMHC) and smoothelin [232234]. a-SMA and SM22-a are early markers of smooth muscle differentiation, whereas calponin, caldesmon and SMMHC are markers of late-stage VSMC differentiation. Smoothelin, a cytoskeletal protein that is only found in contractile VSMCs, is known to be a marker of mature VSMCs [233]. Transcriptional activation of the VSMC marker genes described above is mediated by myocardin, GATA-binding protein 6 (Gata-6) and serum response factor (SRF) [230]. 6.3. Differentiation protocols The most potent inducer of VSMC differentiation of MSCs is TGF-b1. During the differentiation process it plays a role in both initial commitment and further differentiation of VSMCs [235]. Through activation of the transcription factors Gata-6 and SRF, TGF-b1 mediates the upregulation of the VSMC marker genes a-SMA, SM22-a, calponin and SMMHC in MSCs [236,237]. SMC-specic transcription is regulated by SRF, which binds to CArG (CC(A=T)6GG) cis elements that are found in the promoters of almost all SMC marker genes [238241]. Gong et al. showed that a concentration of 0.110 ng ml1 TGF-b1 inhibited human MSC proliferation, but increased calponin expression in a dose-dependent manner [234], correlating well with expression levels found in human coronary arterial smooth muscle cells. TGF-b1 deciency is correlated with atherosclerosis and stenosis of the major coronary vessels [242]. In contrast to TGF-b1, disruption of the TGF-b2 gene results in 100% mortality just prior to or just after birth, and affects developmental processes that are involved in epithelialmesenchymal transitions, cell growth, ECM production and tissue remodeling [243]. These studies indicate that the presence of all TGF-b isoforms is important for proper maturation of smooth muscle cells [244]. PDGF is known to be a growth factor inuencing VSMC differentiation of MSCs. However, the role of PDGF-BB in SMC differentia-
tion is controversial. During embryonic development, PDGF-BB is involved in the recruitment of pericytes and SMC precursors towards endothelial cells [245]. Postnatally, PDGF-BB is associated with SMC proliferation, e.g. in response to vascular injury and atherosclerosis [224]. Thus, PDGF-BB appears to be responsible for phenotypic modulation and dedifferentiation of SMCs in vivo. There is also evidence that PDGF-BB suppresses expression of markers consistent with terminal differentiation in vitro, including SM22-a and SMMHC [246], and induces a proliferative and synthetic VSMC phenotype [224]. However, studies by Dandre et al. show that PDGF-BB-induced inhibition of SMC marker expression is only observed in low-density cultures but not when cells are growth-arrested by culture at subconuent density in serum-free conditions or when maintained at high cell density in serum-rich conditions [246]. In addition to TGF-b1 and PDGF-BB, factors such as angiotensin II [247], sphingosylphosphorylcholine [248] and ascorbic acid [249] have also been applied for VSMC differentiation. Furthermore, close cell-to-cell contact is required for VSMC differentiation [250]. Several protocols are available for the differentiation of MSCs into VSMCs. Representative protocols for this, including TGF-b1 and PDGF-BB, are shown in Table 9. Selected commercially available media for VSMC differentiation of MSCs are listed in Table 10. 7. General aspects 7.1. Cell source MSCs can be isolated from various tissues, including bone marrow, adipose tissue, umbilical cord blood, placental tissue [251], liver, spleen [20], testes [19], menstrual blood, amniotic uid [17], pancreas [16], dermis [252], dental pulp [253], periosteum [254] and even lung [255]. Within these, bone marrow is believed to be the enriched reservoir of MSCs and the major source for these precursor cells, which populate other adult tissues and organs [256]. Because of their abundant availability and easy accessibility, MSCs derived from adipose tissue, in particular, are considered to be an attractive alternative to MSCs from bone marrow. However, in terms of their osteogenic potential, adipose tissue-derived MSCs seem to be inferior to bone-marrow-derived MSCs [199]. For MSCs from umbilical cord blood (UCB) conicting results in terms of
Table 9 Representative protocols used for in vitro vascular smooth muscle differentiation of MSCs (asc, ascorbic acid; bFGF, basic broblast growth factor; BMP-4, bone morphogenetic protein-4; FCS, fetal calf serum; HGF, hepatocyte growth factor; PDGF, platelet-derived growth factor; TGF-b1, transforming growth factor-beta 1; BM, bone marrow; AT, adipose tissue). Vascular smooth muscle cells demonstrate a synthetic and a contractile phenotype that is characterized by morphology, proliferation rate, and the expression of special marker proteins. However, most of the protocols listed below mainly focus on the expression of smooth muscle-specic genes and proteins, respectively. Only a few researchers have also addressed the contraction ability of differentiated MSCs (e.g. Wang et al. [231]). Reference Kanematsu et al. [271] Ross et al. [230] Shukla et al. [272] Narita et al. [249] Gong et al. [234] FCS 0.5% 0.5/5% 20% 10% TGF-b1 5 ng ml1 2.5 ng ml1 5 ng ml1 0.1/1/10 ng ml1 0.01/0.1/1/10 ng ml1 Other HGF (40 ng ml1) PDGF-BB (0100 ng ml1) PDGF-BB (25 ng ml1) asc (30/300 lM) PDGF-BB (10 ng ml1) PDGF-CC (10 ng ml1) bFGF (10 ng ml1) asc (50 lg ml1) Copper sulfate (3 ng ml1) proline (50 mg ml1) glycine (50 mg ml1) ascorbic acid (50 mg ml1) alanine (20 mg ml1) Heparin (7.5 U ml1) angiotensin II (2 lM) sphingosylphosphorylcholine (2 lM) BMP-4 (2.5 ng ml1) Cell source Rat BM Murine/rat/human/porcine BM Porcine BM Human BM Human BM
Kurpinski et al. [273] Gong et al. [6]
10% 10%
5 ng ml1 1 ng ml1
Human MSCs (Cambrex) Human BM
Harris et al. [227]
13%
2 ng ml1
Human AT
Wang et al. [231]
1%
5 ng ml1
Human AT
472
Table 10 Media commercially available for vascular smooth muscle differentiation of MSCs. Company PromoCell GmbH (Heidelberg, Germany) Invitrogen (Carlsbad, USA) BD Biosciences (Bedford, USA) Product Smooth Muscle Cell Growth Medium 2 Medium 231 Smooth Muscle Differentiation Supplement (SMDS) SMC Differentiation Medium
Disclosure of interests The authors indicate no potential conict of interests. Acknowledgements We thank Peter Bernstein, MD, Juliane Rauh, PhD, Angela Jacobi, PhD, and Mike Tipsword for proof-reading of this manuscript. Thanks a lot to Cornelia Liebers and Suzanne Manthey for assisting with cell culture and histology. M.S. and C.V. are nancially supported by the German Academic Exchange Service/German Federal Ministry of Education and Research (Grant No. D/09/04774) and by the Center for Regenerative Therapies Dresden, Germany (Seed Grant No. 09-08).
success rate of MSC isolation have been initially reported, probably because of their low frequency in UCB [257,258]. In general, MSCs are a minor fraction in bone marrow and other tissues; the exact frequency is difcult to calculate because of the different methods of harvest and separation. However, the frequency in human bone marrow has been estimated to be of the order of 0.0010.01% of the total nucleated cells, and therefore about 10-fold less abundant than haematopoietic stem cells. Furthermore, the frequency of MSCs declines with age, from 1/10,000 nucleated marrow cells in a newborn to about 1/10,00,000 nucleated marrow cells in a 80 year old person [259]. 7.2. Use of FCS
Appendix A. Figures with essential colour discrimination Certain gures in this article, particularly Figures 1 and 2, are difcult to interpret in black and white. The full colour images can be found in the on-line version, at doi:10.1016/j.actbio.2010.07.037. References
Most of the protocols used today recommend the use of FCSsupplemented medium for the differentiation of MSCs because of the nutrients and growth factors present in the FCS. While MSCs serum concentrations in the range of 1020% are used for expansion, differentiation of MSCs occurs mainly in medium supplemented with 210% FCS. However, there is a considerable batch-to-batch variation within the FCS, which may result in a signicant variation in MSC properties and differentiation behavior, respectively. Therefore it is important to perform a FCS batch test prior to a study to nd the batch that is most suitable for the respective experiment. In addition, it must be kept in mind that FCS is an undesirable additive to cells that are expanded and differentiated for therapeutic purposes in humans because the use of FCS carries the risk of transmitting viral and prion diseases and proteins that may initiate xenogeneic immune responses. Because of this, various alternatives have been considered, including, for example, (autologous) human platelet lysates or serum [260,261] as well as formulated dened media. 8. Summary MSCs are a promising source of precursor cells which may be applied in various tissue-engineering strategies. By using differentiation-specic protocols, MSCs can be induced to differentiate towards a variety of mature target cells. In this context, the composition of the cell culture media used is crucial. Currently, different commercial culture media with or without FCS are available for MSC differentiation. More importantly, however, particular supplementary agents need to be added to the media for lineage-specic differentiation of MSCs. In general, culture supplements required for osteogenic differentiation include dex, b-GP, asc, vitD3, TGF-b and BMPs. Chondrogenic differentiation of MSCs is achieved by growing cells in pellet with media containing dex, asc, TGF-b, BMPs and IGF. Supplements including dex, IBMX, insulin, indomethacin and T3 have been shown to be effective in adipogenic differentiation. In order to induce MSC differentiation towards endothelial cell lineage VEGF is routinely used as supplementary agent. MSC differentiation towards the VSMC phenotype is achieved by stimulating the cells with TGF-b1 and PDGF-BB. In practice, the combination of FCS and these supplementary agents such as growth factors and cytokines and their concentration should be optimized to meet each individual need.
[1] Owen M, Friedenstein AJ. Stromal stem cells: marrow-derived osteogenic precursors. Ciba Found Symp 1988;136:4260. [2] Mackay AM, Beck SC, Murphy JM, Barry FP, Chichester CO, Pittenger MF. Chondrogenic differentiation of cultured human mesenchymal stem cells from marrow. Tissue Eng 1998;4:41528. [3] Pittenger MF, Mackay AM, Beck SC, Jaiswal RK, Douglas R, Mosca JD, et al. Multilineage potential of adult human mesenchymal stem cells. Science 1999;284:1437. [4] Drost AC, Weng S, Feil G, Schfer J, Baumann S, Kanz L, et al. In vitro myogenic differentiation of human bone marrow-derived mesenchymal stem cells as a potential treatment for urethral sphincter muscle repair. Ann NY Acad Sci 2009;1176:13543. [5] Oswald J, Boxberger S, Jrgensen B, Feldmann S, Ehninger G, Bornhuser M, et al. Mesenchymal stem cells can be differentiated into endothelial cells in vitro. Stem Cells 2004;22:37784. [6] Gong Z, Calkins G, Cheng E, Krause D, Niklason LE. Inuence of culture medium on smooth muscle cell differentiation from human bone marrowderived mesenchymal stem cells. Tissue Eng Part A 2009;15:31930. [7] Dominici M, Le Blanc K, Mueller I, Slaper-Cortenbach I, Marini F, Krause D, et al. Minimal criteria for dening multipotent mesenchymal stromal cells. The International Society for Cellular Therapy position statement. Cytotherapy 2006;8:3157. [8] Kokai LE, Rubin JP, Marra KG. The potential of adipose-derived adult stem cells as a source of neuronal progenitor cells. Plast Reconstr Surg 2005;116:145360. [9] Safford KM, Rice HE. Stem cell therapy for neurologic disorders: therapeutic potential of adipose-derived stem cells. Curr Drug Targets 2005;6:5762. [10] Phinney DG, Isakova I. Plasticity and therapeutic potential of mesenchymal stem cells in the nervous system. Curr Pharm Des 2005;11:125565. [11] Kassem M. Mesenchymal stem cells: biological characteristics and potential clinical applications. Cloning Stem Cells 2004;6:36974. [12] Di Gioacchino G, Di Campli C, Zocco MA, Piscaglia AC, Novi M, Santoro M, et al. Transdifferentiation of stem cells in pancreatic cells: state of the art. Transplant Proc 2005;37:26623. [13] Timper K, Seboek D, Eberhardt M, Linscheid P, Christ-Crain M, Keller U, et al. Human adipose tissue-derived mesenchymal stem cells differentiate into insulin, somatostatin, and glucagon expressing cells. Biochem Biophys Res Commun 2006;341:113540. [14] Moriscot C, de Fraipont F, Richard M, Marchand M, Savatier P, Bosco D, et al. Human bone marrow mesenchymal stem cells can express insulin and key transcription factors of the endocrine pancreas developmental pathway upon genetic and/or microenvironmental manipulation in vitro. Stem Cells 2005;23:594603. [15] Chen L, Jiang X, Yang L. Differentiation of rat marrow mesenchymal stem cells into pancreatic islet beta-cells. World J Gastroenterol 2004;10:301620. [16] Kruse C, Kajahn J, Petschnik AE, Maass A, Klink E, Rapoport DH, et al. Adult pancreatic stem/progenitor cells spontaneously differentiate in vitro into multiple cell lineages and form teratoma-like structures. Ann Anat 2006;188:50317. [17] De Coppi P, Bartsch GJ, Siddiqui MM, Xu T, Santos CC, Perin L, et al. Isolation of amniotic stem cell lines with potential for therapy. Nat Biotechnol 2007;25:1006. [18] Meng X, Ichim TE, Zhong J, Rogers A, Yin Z, Jackson J, et al. Endometrial regenerative cells: a novel stem cell population. J Transl Med 2007;5:57.
C. Vater et al. / Acta Biomaterialia 7 (2011) 463477 [19] Guan K, Nayernia K, Maier LS, Wagner S, Dressel R, Lee JH, et al. Pluripotency of spermatogonial stem cells from adult mouse testis. Nature 2006;440:1199203. [20] int Anker P, Noort W, Scherjon S, Kleijburg-van der Keur C, Kruisselbrink A, van Bezooijen R, et al. Mesenchymal stem cells in human second-trimester bone marrow, liver, lung, and spleen exhibit a similar immunophenotype but a heterogeneous multilineage differentiation potential. Haematologica 2003;88:84552. [21] Javazon EH, Beggs KJ, Flake AW. Mesenchymal stem cells: paradoxes of passaging. Exp Hematol 2004;32:41425. [22] Chamberlain G, Fox J, Ashton B, Middleton J. Concise review: mesenchymal stem cells: their phenotype, differentiation capacity, immunological features, and potential for homing. Stem Cells 2007;25:273949. [23] Haynesworth S, Baber M, Caplan A. Cell surface antigens on human marrowderived mensenchymal cells are detected by monoclonal antibodies. Bone 1992;13(1):6980. [24] Lodie TA, Blickarz CE, Devarakonda TJ, He C, Dash AB, Clarke J, et al. Systematic analysis of reportedly distinct populations of multipotent bone marrow-derived stem cells reveals a lack of distinction. Tissue Eng 2002;8:73951. [25] Suva D, Garavaglia G, Menetrey J, Chapuis B, Hoffmeyer P, Bernheim L, et al. Non-hematopoietic human bone marrow contains long-lasting, pluripotential mesenchymal stem cells. J Cell Physiol 2004;198:1108. [26] Niemeyer P, Seckinger A, Simank HG, Kasten P, Sdkamp N, Krause U. Allogenic transplantation of human mesenchymal stem cells for tissue engineering purposes: an in vitro study. Orthopade 2004;33:134653. [27] Di Nicola M, Carlo-Stella C, Magni M, Milanesi M, Longoni PD, Matteucci P, et al. Human bone marrow stromal cells suppress T-lymphocyte proliferation induced by cellular or nonspecic mitogenic stimuli. Blood 2002;99:383843. [28] Bartholomew A, Sturgeon C, Siatskas M, Ferrer K, McIntosh K, Patil S, et al. Mesenchymal stem cells suppress lymphocyte proliferation in vitro and prolong skin graft survival in vivo. Exp Hematol 2002;30:428. [29] Aggarwal S, Pittenger MF. Human mesenchymal stem cells modulate allogeneic immune cell responses. Blood 2005;105:181522. [30] Jiang X, Zhang Y, Liu B, Zhang S, Wu Y, Yu X, et al. Human mesenchymal stem cells inhibit differentiation and function of monocyte-derived dendritic cells. Blood 2005;105:41206. [31] Beyth S, Borovsky Z, Mevorach D, Liebergall M, Gazit Z, Aslan H, et al. Human mesenchymal stem cells alter antigen-presenting cell maturation and induce T-cell unresponsiveness. Blood 2005;105:22149. [32] Corcione A, Benvenuto F, Ferretti E, Giunti D, Cappiello V, Cazzanti F, et al. Human mesenchymal stem cells modulate B-cell functions. Blood 2006;107:36772. [33] Bernstein P, Bornhuser M, Gnther K, Stiehler M. Bone tissue engineering in clinical application: assessment of the current situation. Orthopde 2009;38(11):102937. [34] Orlic D, Kajstura J, Chimenti S, Limana F, Jakoniuk I, Quaini F, et al. Mobilized bone marrow cells repair the infarcted heart, improving function and survival. Proc Natl Acad Sci USA 2001;98:103449. [35] Lee ST, Jang JH, Cheong J, Kim JS, Maemg H, Hahn JS, et al. Treatment of highrisk acute myelogenous leukaemia by myeloablative chemoradiotherapy followed by co-infusion of T cell-depleted haematopoietic stem cells and culture-expanded marrow mesenchymal stem cells from a related donor with one fully mismatched human leucocyte antigen haplotype. Br J Haematol 2002;118:112831. [36] Urbn VS, Kiss J, Kovcs J, Gcza E, Vas V, Monostori E, et al. Mesenchymal stem cells cooperate with bone marrow cells in therapy of diabetes. Stem Cells 2008;26:24453. [37] Guhathakurta S, Subramanyan UR, Balasundari R, Das CK, Madhusankar N, Cherian KM. Stem cell experiments and initial clinical trial of cellular cardiomyoplasty. Asian Cardiovasc Thorac Ann 2009;17:5816. [38] Horwitz EM, Gordon PL, Koo WKK, Marx JC, Neel MD, McNall RY, et al. Isolated allogeneic bone marrow-derived mesenchymal cells engraft and stimulate growth in children with osteogenesis imperfecta: implications for cell therapy of bone. Proc Natl Acad Sci USA 2002;99:89327. [39] Constantin G, Marconi S, Rossi B, Angiari S, Calderan L, Anghileri E, et al. Adipose-derived mesenchymal stem cells ameliorate chronic experimental autoimmune encephalomyelitis. Stem Cells 2009;27:262435. [40] Newman RE, Yoo D, LeRoux MA, Danilkovitch-Miagkova A. Treatment of inammatory diseases with mesenchymal stem cells. Inamm Allergy Drug Targets 2009;8:11023. [41] Sorger J, Hornicek F, Zavatta M, Menzner J, Gebhardt M, Tomford W, et al. Allograft fractures revisited. Clin Orthop Relat Res 2001;Jan(382):6674. [42] Song I, Caplan AI, Dennis JE. In vitro dexamethasone pretreatment enhances bone formation of human mesenchymal stem cells in vivo. J Orthop Res 2009;27:91621. [43] Ducy P, Zhang R, Geoffroy V, Ridall AL, Karsenty G. Osf2/Cbfa1: a transcriptional activator of osteoblast differentiation. Cell 1997;89:74754. [44] Ducy P, Starbuck M, Priemel M, Shen J, Pinero G, Geoffroy V, et al. A Cbfa1dependent genetic pathway controls bone formation beyond embryonic development. Genes Dev 1999;13:102536. [45] Kern B, Shen J, Starbuck M, Karsenty G. Cbfa1 contributes to the osteoblast-specic expression of type I collagen genes. J Biol Chem 2001;276:71017. [46] Nakashima K, Zhou X, Kunkel G, Zhang Z, Deng JM, Behringer RR, et al. The novel zinc nger-containing transcription factor osterix is required for osteoblast differentiation and bone formation. Cell 2002;108:1729.
473
[47] Lian JB, Stein GS, Javed A, van Wijnen AJ, Stein JL, Montecino M, et al. Networks and hubs for the transcriptional control of osteoblastogenesis. Rev Endocr Metab Disord 2006;7:116. [48] Romero-Prado M, Blzquez C, Rodrguez-Navas C, Muoz J, Guerrero I, Delgado-Baeza E, et al. Functional characterization of human mesenchymal stem cells that maintain osteochondral fates. J Cell Biochem 2006;98:145770. [49] Spector JA, Luchs JS, Mehrara BJ, Greenwald JA, Smith LP, Longaker MT. Expression of bone morphogenetic proteins during membranous bone healing. Plast Reconstr Surg 2001;107:12434. [50] Zhu JX, Sasano Y, Takahashi I, Mizoguchi I, Kagayama M. Temporal and spatial gene expression of major bone extracellular matrix molecules during embryonic mandibular osteogenesis in rats. Histochem J 2001;33:2535. [51] Long MW. Osteogenesis and bone-marrow-derived cells. Blood Cells Molecules Dis 2001;27:67790. [52] Young MF. Bone matrix proteins: their function, regulation, and relationship to osteoporosis. Osteoporos Int 2003;14(Suppl. 3):S3542. [53] Leboy PS, Beresford JN, Devlin C, Owen ME. Dexamethasone induction of osteoblast mRNAs in rat marrow stromal cell cultures. J Cell Physiol 1991;146:3708. [54] Herbertson A, Aubin JE. Dexamethasone alters the subpopulation make-up of rat bone marrow stromal cell cultures. J Bone Miner Res 1995;10:28594. [55] Hildebrandt C, Bth H, Thielecke H. Inuence of cell culture media conditions on the osteogenic differentiation of cord blood-derived mesenchymal stem cells. Ann Anat 2009;191:2332. [56] Ogata Y, Yamauchi M, Kim R, Li J, Freedman L, Sodek J. Glucocorticoid regulation of bone sialoprotein (BSP) gene expression. Identication of a glucocorticoid response element in the bone sialoprotein gene promoter. Eur J Biochem 1995;230(1):18392. [57] Hong D, Chen H, Xue Y, Li D, Wan X, Ge R, et al. Osteoblastogenic effects of dexamethasone through upregulation of TAZ expression in rat mesenchymal stem cells. J Steroid Biochem Mol Biol 2009;116:8692. [58] Hamidouche Z, Fromigu O, Ringe J, Hupl T, Vaudin P, Pags J, et al. Priming integrin alpha5 promotes human mesenchymal stromal cell osteoblast differentiation and osteogenesis. Proc Natl Acad Sci USA 2009;106(44): 1858791. [59] Jrgensen NR, Henriksen Z, Srensen OH, Civitelli R. Dexamethasone, BMP-2, and 1, 25-dihydroxyvitamin D enhance a more differentiated osteoblast phenotype: validation of an in vitro model for human bone marrow-derived primary osteoblasts. Steroids 2004;69:21926. [60] Walsh S, Jordan GR, Jefferiss C, Stewart K, Beresford JN. High concentrations of dexamethasone suppress the proliferation but not the differentiation or further maturation of human osteoblast precursors in vitro: relevance to glucocorticoid-induced osteoporosis. Rheumatology (Oxford) 2001;40:7483. [61] Lukert BP. Clinical and basic aspects of glucocorticoids action in bone. In: Bilezikian JP, Raisz LG, Rodan GA, editors. Principles of bone biology. San Diego: Academic Press; 1996. p. 53348. [62] Gundle R. Microscopical and biochemical studies of mineralized matrix production by human bone derived cells. Ph.D. Thesis, University of Oxford; 1995. [63] Song I, Caplan AI, Dennis JE. Dexamethasone inhibition of conuence-induced apoptosis in human mesenchymal stem cells. J Orthop Res 2009;27:21621. [64] Chang YL, Stanford CM, Keller JC. Calcium and phosphate supplementation promotes bone cell mineralization: implications for hydroxyapatite (HA)enhanced bone formation. J Biomed Mater Res 2000;52:2708. [65] Chung C, Golub E, Forbes E, Tokuoka T, Shapiro I. Mechanism of action of beta-glycerophosphate on bone cell mineralization. Calcif Tissue Int 1992;51(4):30511. [66] Chen M, Lie P, Li Z, Wei X. Endothelial differentiation of Whartons jellyderived mesenchymal stem cells in comparison with bone marrow-derived mesenchymal stem cells. Exp Hematol 2009;37:62940. [67] Kielty C, Hopkinson I, Grant M. Collagen: the collagen family: structure, assembly, and organization in the extracellular matrix. In: Royce SB, Steinmann B, editors. Connective tissue and its heritable disorders. Molecular, genetic, and medical aspects. New York: Wiley-Liss; 1993. p. 10347. [68] Prockop DJ, Kivirikko KI. Collagens: molecular biology, diseases, and potentials for therapy. Annu Rev Biochem 1995;64:40334. [69] Takamizawa S, Maehata Y, Imai K, Senoo H, Sato S, Hata R. Effects of ascorbic acid and ascorbic acid 2-phosphate, a long-acting vitamin C derivative, on the proliferation and differentiation of human osteoblast-like cells. Cell Biol Int 2004;28:25565. [70] Maehata Y, Takamizawa S, Ozawa S, Kato Y, Sato S, Kubota E, et al. Both direct and collagen-mediated signals are required for active vitamin D3-elicited differentiation of human osteoblastic cells: roles of osterix, an osteoblastrelated transcription factor. Matrix Biol 2006;25:4758. [71] Hata R, Senoo H. L-Ascorbic acid 2-phosphate stimulates collagen accumulation, cell proliferation, and formation of a three-dimensional tissue-like substance by skin broblasts. J Cell Physiol 1989;138:816. [72] Fernandes H, Mentink A, Bank RA, Stoop R, van Blitterswijk C, De Boer J. Endogenous collagen inuences differentiation of human multipotent mesenchymal stromal cells. Tissue Eng Part A 2009;16(5):1693702. [73] Pytlk R, Stehlk D, Soukup T, Kalbcov M, Rypcek F, Trc T, et al. The cultivation of human multipotent mesenchymal stromal cells in clinical grade medium for bone tissue engineering. Biomaterials 2009;30: 341527.
474
C. Vater et al. / Acta Biomaterialia 7 (2011) 463477 [101] Chang Y, Ueng SWN, Lin-Chao S, Chao CC. Involvement of Gas7 along the ERK1/2 MAP kinase and SOX9 pathway in chondrogenesis of human marrowderived mesenchymal stem cells. Osteoarthritis Cartilage 2008;16:140312. [102] An C, Cheng Y, Yuan Q, Li J. IGF-1 and BMP-2 induces differentiation of adipose-derived mesenchymal stem cells into chondrocytes-like cells. Ann Biomed Eng 2010;38(4):164754. [103] Choi K, Seo Y, Yoon H, Song K, Kwon S, Lee H, et al. Effect of ascorbic acid on bone marrow-derived mesenchymal stem cell proliferation and differentiation. J Biosci Bioeng 2008;105:58694. [104] Lefebvre V, Huang W, Harley VR, Goodfellow PN, de Crombrugghe B. SOX9 is a potent activator of the chondrocyte-specic enhancer of the pro alpha1(II) collagen gene. Mol Cell Biol 1997;17:233646. [105] Bridgewater LC, Lefebvre V, de Crombrugghe B. Chondrocyte-specic enhancer elements in the Col11a2 gene resemble the Col2a1 tissue-specic enhancer. J Biol Chem 1998;273:149985006. [106] Sekiya I, Tsuji K, Koopman P, Watanabe H, Yamada Y, Shinomiya K, et al. SOX9 enhances aggrecan gene promoter/enhancer activity and is upregulated by retinoic acid in a cartilage-derived cell line, TC6. J Biol Chem 2000;275:1073844. [107] Zhang P, Jimenez SA, Stokes DG. Regulation of human COL9A1 gene expression. Activation of the proximal promoter region by SOX9. J Biol Chem 2003;278:11723. [108] Kou I, Ikegawa S. SOX9-dependent and -independent transcriptional regulation of human cartilage link protein. J Biol Chem 2004;279:509428. [109] Huang W, Lu N, Eberspaecher H, De Crombrugghe B. A new long form of cMaf cooperates with Sox9 to activate the type II collagen gene. J Biol Chem 2002;277:5066875. [110] Tsuda M, Takahashi S, Takahashi Y, Asahara H. Transcriptional co-activators CREB-binding protein and p300 regulate chondrocyte-specic gene expression via association with Sox9. J Biol Chem 2003;278:272249. [111] Schmid TM, Linsenmayer TF. Immunohistochemical localization of short chain cartilage collagen (type X) in avian tissues. J Cell Biol 1985;100:598605. [112] Ornitz DM, Marie PJ. FGF signaling pathways in endochondral and intramembranous bone development and human genetic disease. Genes Dev 2002;16:144665. [113] Shen G. The role of type X collagen in facilitating and regulating endochondral ossication of articular cartilage. Orthod Craniofac Res 2005;8:117. [114] Mwale F, Stachura D, Roughley P, Antoniou J. Limitations of using aggrecan and type X collagen as markers of chondrogenesis in mesenchymal stem cell differentiation. J Orthop Res 2006;24:17918. [115] Johnstone B, Hering TM, Caplan AI, Goldberg VM, Yoo JU. In vitro chondrogenesis of bone marrow-derived mesenchymal progenitor cells. Exp Cell Res 1998;238:26572. [116] 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;46:334960. [117] Jingushi S, Heydemann A, Kana SK, Macey LR, Bolander ME. Acidic broblast growth factor (aFGF) injection stimulates cartilage enlargement and inhibits cartilage gene expression in rat fracture healing. J Orthop Res 1990;8:36471. [118] Yoo JU, Barthel TS, Nishimura K, Solchaga L, Caplan AI, Goldberg VM, et al. The chondrogenic potential of human bone-marrow-derived mesenchymal progenitor cells. J Bone Joint Surg Am 1998;80:174557. [119] Mastrogiacomo M, Cancedda R, Quarto R. Effect of different growth factors on the chondrogenic potential of human bone marrow stromal cells. Osteoarthritis Cartilage 2001;9(Suppl. A):S3640. [120] Na K, Choi S, Kim S, Sun BK, Woo DG, Chung H, et al. Enhancement of cell proliferation and differentiation by combination of ascorbate and dexamethasone in thermo-reversible hydrogel constructs embedded with rabbit chondrocytes. Biotechnol Lett 2007;29:14537. [121] Derfoul A, Perkins GL, Hall DJ, Tuan RS. Glucocorticoids promote chondrogenic differentiation of adult human mesenchymal stem cells by enhancing expression of cartilage extracellular matrix genes. Stem Cells 2006;24:148795. [122] Richter W. Mesenchymal stem cells and cartilage in situ regeneration. J Intern Med 2009;266:390405. [123] Miyazono K. Positive and negative regulation of TGF-beta signaling. J Cell Sci 2000;113(Pt. 7):11019. [124] Lee JW, Kim YH, Kim S, Han SH, Hahn SB. Chondrogenic differentiation of mesenchymal stem cells and its clinical applications. Yonsei Med J 2004;45(Suppl.):417. [125] Barry F, Boynton RE, Liu B, Murphy JM. Chondrogenic differentiation of mesenchymal stem cells from bone marrow: differentiation-dependent gene expression of matrix components. Exp Cell Res 2001;268:189200. [126] Bosnakovski D, Mizuno M, Kim G, Ishiguro T, Okumura M, Iwanaga T, et al. Chondrogenic differentiation of bovine bone marrow mesenchymal stem cells in pellet cultural system. Exp Hematol 2004;32:5029. [127] Zhang X, Ziran N, Goater JJ, Schwarz EM, Puzas JE, Rosier RN, et al. Primary murine limb bud mesenchymal cells in long-term culture complete chondrocyte differentiation: TGF-beta delays hypertrophy and PGE2 inhibits terminal differentiation. Bone 2004;34:80917. [128] Pogue R, Lyons K. BMP signaling in the cartilage growth plate. Curr Top Dev Biol 2006;76:148. [129] Xu D, Gechtman Z, Hughes A, Collins A, Dodds R, Cui X, et al. Potential involvement of BMP receptor type IB activation in a synergistic effect of chondrogenic promotion between rhTGFbeta3 and rhGDF5 or rhBMP7 in human mesenchymal stem cells. Growth Factors 2006;24:26878.
[74] Wang QM, Jones JB, Studzinski GP. Cyclin-dependent kinase inhibitor p27 as a mediator of the G1-S phase block induced by 1,25-dihydroxyvitamin D3 in HL60 cells. Cancer Res 1996;56:2647. [75] Verlinden L, Verstuyf A, Convents R, Marcelis S, Van Camp M, Bouillon R. Action of 1,25(OH)2D3 on the cell cycle genes, cyclin D1, p21 and p27 in MCF7 cells. Mol Cell Endocrinol 1998;142:5765. [76] Nakagawa K, Tsugawa N, Okamoto T, Kishi T, Ono T, Kubodera N, et al. Rapid control of transmembrane calcium inux by 1alpha, 25-dihydroxyvitamin D3 and its analogues in rat osteoblast-like cells. Biol Pharm Bull 1999;22:105863. [77] Fromigu O, Marie PJ, Lomri A. Differential effects of transforming growth factor beta2, dexamethasone and 1, 25-dihydroxyvitamin D on human bone marrow stromal cells. Cytokine 1997;9:61323. [78] Jaiswal N, Haynesworth SE, Caplan AI, Bruder SP. Osteogenic differentiation of puried, culture-expanded human mesenchymal stem cells in vitro. J Cell Biochem 1997;64:295312. [79] Bonewald LF, Wakeeld L, Oreffo RO, Escobedo A, Twardzik DR, Mundy GR. Latent forms of transforming growth factor-beta (TGF beta) derived from bone cultures: identication of a naturally occurring 100-kDa complex with similarity to recombinant latent TGF beta. Mol Endocrinol 1991;5:74151. [80] Dallas SL, Park-Snyder S, Miyazono K, Twardzik D, Mundy GR, Bonewald LF. Characterization and autoregulation of latent transforming growth factor beta (TGF beta) complexes in osteoblast-like cell lines. Production of a latent complex lacking the latent TGF beta-binding protein. J Biol Chem 1994;269:681521. [81] Centrella M, McCarthy TL, Canalis E. Transforming growth factor beta is a bifunctional regulator of replication and collagen synthesis in osteoblastenriched cell cultures from fetal rat bone. J Biol Chem 1987;262:286974. [82] Centrella M, McCarthy TL, Canalis E. Transforming growth factor-beta and remodeling of bone. J Bone Joint Surg Am 1991;73:141828. [83] Jger M, Sager M, Knipper A, Degistirici O, Fischer J, Kgler G, et al. In vivo and in vitro bone regeneration from cord blood derived mesenchymal stem cells. Orthopde 2004;33:136172. [84] Hogan BL. Bone morphogenetic proteins in development. Curr Opin Genet Dev 1996;6:4328. [85] Holleville N, Quilhac A, Bontoux M, Monsoro-Burq AH. BMP signals regulate Dlx5 during early avian skull development. Dev Biol 2003;257:17789. [86] Hanada K, Dennis JE, Caplan AI. Stimulatory effects of basic broblast growth factor and bone morphogenetic protein-2 on osteogenic differentiation of rat bone marrow-derived mesenchymal stem cells. J Bone Miner Res 1997;12:160614. [87] Diefenderfer DL, Osyczka AM, Reilly GC, Leboy PS. BMP responsiveness in human mesenchymal stem cells. Connect Tissue Res 2003;44(Suppl. 1):30511. [88] Knippenberg M, Helder MN, Zandieh Doulabi B, Wuisman PIJM, Klein-Nulend J. Osteogenesis versus chondrogenesis by BMP-2 and BMP-7 in adipose stem cells. Biochem Biophys Res Commun 2006;342:9028. [89] Abe E, Yamamoto M, Taguchi Y, Lecka-Czernik B, OBrien CA, Economides AN, et al. Essential requirement of BMPs-2/4 for both osteoblast and osteoclast formation in murine bone marrow cultures from adult mice: antagonism by noggin. J Bone Miner Res 2000;15:66373. [90] Lou J, Tu Y, Li S, Manske PR. Involvement of ERK in BMP-2 induced osteoblastic differentiation of mesenchymal progenitor cell line C3H10T1/2. Biochem Biophys Res Commun 2000;268:75762. [91] Cheng H, Jiang W, Phillips FM, Haydon RC, Peng Y, Zhou L, et al. Osteogenic activity of the fourteen types of human bone morphogenetic proteins (BMPs). J Bone Joint Surg Am 2003;85-A:154452. [92] Osyczka AM, Diefenderfer DL, Bhargave G, Leboy PS. Different effects of BMP2 on marrow stromal cells from human and rat bone. Cells Tissues Organs 2004;176:10919. [93] Cheifetz S, Li IW, McCulloch CA, Sampath K, Sodek J. Inuence of osteogenic protein-1 (OP-1;BMP-7) and transforming growth factor-beta 1 on bone formation in vitro. Connect Tissue Res 1996;35:718. [94] Li IW, Cheifetz S, McCulloch CA, Sampath KT, Sodek J. Effects of osteogenic protein-1 (OP-1, BMP-7) on bone matrix protein expression by fetal rat calvarial cells are differentiation stage specic. J Cell Physiol 1996;169:11525. [95] Lecanda F, Avioli LV, Cheng SL. Regulation of bone matrix protein expression and induction of differentiation of human osteoblasts and human bone marrow stromal cells by bone morphogenetic protein-2. J Cell Biochem 1997;67:38696. [96] Locklin RM, Riggs BL, Hicok KC, Horton HF, Byrne MC, Khosla S. Assessment of gene regulation by bone morphogenetic protein 2 in human marrow stromal cells using gene array technology. J Bone Miner Res 2001;16:2192204. [97] Fuller JA, Ghadially FN. Ultrastructural observations on surgically produced partial-thickness defects in articular cartilage. Clin Orthop Relat Res 1972;86:193205. [98] Nesic D, Whiteside R, Brittberg M, Wendt D, Martin I, Mainil-Varlet P. Cartilage tissue engineering for degenerative joint disease. Adv Drug Deliv Rev 2006;58:30022. [99] Koga H, Engebretsen L, Brinchmann JE, Muneta T, Sekiya I. Mesenchymal stem cell-based therapy for cartilage repair: a review. Knee Surg Sports Traumatol Arthrosc 2009;17:128997. [100] Hardingham TE, Oldershaw RA, Tew SR. Cartilage, SOX9 and Notch signals in chondrogenesis. J Anat 2006;209:46980.
C. Vater et al. / Acta Biomaterialia 7 (2011) 463477 [130] Hennig T, Lorenz H, Thiel A, Goetzke K, Dickhut A, Geiger F, et al. Reduced chondrogenic potential of adipose tissue derived stromal cells correlates with an altered TGFbeta receptor and BMP prole and is overcome by BMP-6. J Cell Physiol 2007;211:68291. [131] Sekiya I, Larson BL, Vuoristo JT, Reger RL, Prockop DJ. Comparison of effect of BMP-2, -4, and -6 on in vitro cartilage formation of human adult stem cells from bone marrow stroma. Cell Tissue Res 2005;320:26976. [132] Solchaga LA, Penick K, Porter JD, Goldberg VM, Caplan AI, Welter JF. FGF-2 enhances the mitotic and chondrogenic potentials of human adult bone marrow-derived mesenchymal stem cells. J Cell Physiol 2005;203:398409. [133] Stewart AA, Byron CR, Pondenis HC, Stewart MC. Effect of dexamethasone supplementation on chondrogenesis of equine mesenchymal stem cells. Am J Vet Res 2008;69:101321. [134] Luyten FP, Hascall VC, Nissley SP, Morales TI, Reddi AH. Insulin-like growth factors maintain steady-state metabolism of proteoglycans in bovine articular cartilage explants. Arch Biochem Biophys 1988;267:41625. [135] Longobardi L, ORear L, Aakula S, Johnstone B, Shimer K, Chytil A, et al. Effect of IGF-I in the chondrogenesis of bone marrow mesenchymal stem cells in the presence or absence of TGF-beta signaling. J Bone Miner Res 2006;21:62636. [136] Nixon AJ, Goodrich LR, Scimeca MS, Witte TH, Schnabel LV, Watts AE, et al. Gene therapy in musculoskeletal repair. Ann NY Acad Sci 2007;1117:31027. [137] Nixon AJ, Fortier LA, Williams J, Mohammed H. Enhanced repair of extensive articular defects by insulin-like growth factor-I-laden brin composites. J Orthop Res 1999;17:47587. [138] Weiss S, Hennig T, Bock R, Steck E, Richter W. Impact of growth factors and PTHrP on early and late chondrogenic differentiation of human mesenchymal stem cells. J Cell Physiol 2010;223:8493. [139] Milne M, Quail JM, Rosen CJ, Baran DT. Insulin-like growth factor binding proteins in femoral and vertebral bone marrow stromal cells: expression and regulation by thyroid hormone and dexamethasone. J Cell Biochem 2001;81:22940. [140] Indrawattana N, Chen G, Tadokoro M, Shann LH, Ohgushi H, Tateishi T, et al. Growth factor combination for chondrogenic induction from human mesenchymal stem cell. Biochem Biophys Res Commun 2004;320:9149. [141] Matsuda C, Takagi M, Hattori T, Wakitani S, Yoshida T. Differentiation of human bone marrow mesenchymal stem cells to chondrocytes for construction of three-dimensional cartilage tissue. Cytotechnology 2005;47:117. [142] Wang C, Chen L, Kuo P, Chang J, Wang Y, Hung S. Apoptosis in chondrogenesis of human mesenchymal stem cells: effect of serum and medium supplements. Apoptosis 2010;15(4):43949. [143] Eppley BL, Woodell JE, Higgins J. Platelet quantication and growth factor analysis from platelet-rich plasma: implications for wound healing. Plast Reconstr Surg 2004;114:15028. [144] Mishra A, Tummala P, King A, Lee B, Kraus M, Tse V, et al. Buffered plateletrich plasma enhances mesenchymal stem cell proliferation and chondrogenic differentiation. Tissue Eng Part C Methods 2009;15:4315. [145] Colter DC, Sekiya I, Prockop DJ. Identication of a subpopulation of rapidly self-renewing and multipotential adult stem cells in colonies of human marrow stromal cells. Proc Natl Acad Sci USA 2001;98:78415. [146] Huang JI, Kazmi N, Durbhakula MM, Hering TM, Yoo JU, Johnstone B. Chondrogenic potential of progenitor cells derived from human bone marrow and adipose tissue: a patient-matched comparison. J Orthop Res 2005;23:13839. [147] Liu TM, Martina M, Hutmacher DW, Hui JHP, Lee EH, Lim B. Identication of common pathways mediating differentiation of bone marrow- and adipose tissue-derived human mesenchymal stem cells into three mesenchymal lineages. Stem Cells 2007;25:75060. [148] Prockop DJ, Gregory CA, Spees JL. One strategy for cell and gene therapy: harnessing the power of adult stem cells to repair tissues. Proc Natl Acad Sci USA 2003;100(Suppl. 1):1191723. [149] Sakaguchi Y, Sekiya I, Yagishita K, Muneta T. Comparison of human stem cells derived from various mesenchymal tissues: superiority of synovium as a cell source. Arthritis Rheum 2005;52:25219. [150] Hauner H, Lfer G. Adipose tissue development: the role of precursor cells and adipogenic factors. Part I: Adipose tissue development and the role of precursor cells. Klin Wochenschr 1987;65:80311. [151] Langstein HN, Robb GL. Reconstructive approaches in soft tissue sarcoma. Semin Surg Oncol 1999;17:5265. [152] Gomillion CT, Burg KJ. Stem cells and adipose tissue engineering. Biomaterials 2006;27:605263. [153] Ashinoff R. Overview: soft tissue augmentation. Clin Plast Surg 2000;27:47987. [154] Klein AW, Elson ML. The history of substances for soft tissue augmentation. Dermatol Surg 2000;26:1096105. [155] Patrick CWJ. Adipose tissue engineering: the future of breast and soft tissue reconstruction following tumor resection. Semin Surg Oncol 2000;19:30211. [156] Patrick CWJ. Tissue engineering strategies for adipose tissue repair. Anat Rec 2001;263:3616. [157] Patrick CW, Mikos AG, McIntire LV. Frontiers in tissue engineering. 1st ed. Oxford and New York: Pergamon Press; 1998. [158] Dani C. Embryonic stem cell-derived adipogenesis. Cells Tissues Organs 1999;165:17380.
475
[159] Kubo Y, Kaidzu S, Nakajima I, Takenouchi K, Nakamura F. Organization of extracellular matrix components during differentiation of adipocytes in longterm culture. In Vitro Cell Dev Biol Anim 2000;36:3844. [160] Rosen ED, MacDougald OA. Adipocyte differentiation from the inside out. Nat Rev Mol Cell Biol 2006;7:88596. [161] Valle M, Ct J, Fradette J. Adipose-tissue engineering: taking advantage of the properties of human adipose-derived stem/stromal cells. Pathol Biol 2009;57:30917. [162] Pairault J, Green H. A study of the adipose conversion of suspended 3T3 cells by using glycerophosphate dehydrogenase as differentiation marker. Proc Natl Acad Sci USA 1979;76:513842. [163] Hemmrich K, von Heimburg D, Cierpka K, Haydarlioglu S, Pallua N. Optimization of the differentiation of human preadipocytes in vitro. Differentiation 2005;73:2835. [164] Tsuji W, Inamoto T, Yamashiro H, Ueno T, Kato H, Kimura Y, et al. Adipogenesis induced by human adipose tissue derived stem cells. Tissue Eng Part A 2008;15:8393. [165] Tontonoz P, Hu E, Spiegelman BM. Stimulation of adipogenesis in broblasts by PPAR gamma 2, a lipid-activated transcription factor. Cell 1994;79:114756. [166] Rosen ED, Sarraf P, Troy AE, Bradwin G, Moore K, Milstone DS, et al. PPAR gamma is required for the differentiation of adipose tissue in vivo and in vitro. Mol Cell 1999;4:6117. [167] Zhang Y, Proenca R, Maffei M, Barone M, Leopold L, Friedman JM. Positional cloning of the mouse obese gene and its human homologue. Nature 1994;372:42532. [168] Gregoire FM, Smas CM, Sul HS. Understanding adipocyte differentiation. Physiol Rev 1998;78:783809. [169] Zechner R, Strauss J, Frank S, Wagner E, Hofmann W, Kratky D, et al. The role of lipoprotein lipase in adipose tissue development and metabolism. Int J Obes Relat Metab Disord 2000;24(Suppl. 4):S536. [170] Obregon M. Thyroid hormone and adipocyte differentiation. Thyroid 2008;18:18595. [171] Shugart EC, Umek RM. Dexamethasone signaling is required to establish the postmitotic state of adipocyte development. Cell Growth Differ 1997;8:10918. [172] Cui Q, Wang GJ, Balian G. Steroid-induced adipogenesis in a pluripotential cell line from bone marrow. J Bone Joint Surg Am 1997;79:105463. [173] Hauner H, Schmid P, Pfeiffer EF. Glucocorticoids and insulin promote the differentiation of human adipocyte precursor cells into fat cells. J Clin Endocrinol Metab 1987;64:8325. [174] Prawitt J, Niemeier A, Kassem M, Beisiegel U, Heeren J. Characterization of lipid metabolism in insulin-sensitive adipocytes differentiated from immortalized human mesenchymal stem cells. Exp Cell Res 2008;314:81424. [175] Deree J, Martins JO, Melbostad H, Loomis WH, Coimbra R. Insights into the regulation of TNF-alpha production in human mononuclear cells: the effects of non-specic phosphodiesterase inhibition. Clinics (Sao Paulo) 2008;63:3218. [176] Yang D, Tsay H, Lin S, Chiou S, Li M, Chang T, et al. CAMP/PKA regulates osteogenesis, adipogenesis and ratio of RANKL/OPG mRNA expression in mesenchymal stem cells by suppressing leptin. PLoS One 2008;3:e1540. [177] Klemm DJ, Leitner JW, Watson P, Nesterova A, Reusch JE, Goalstone ML, et al. Insulin-induced adipocyte differentiation. Activation of CREB rescues adipogenesis from the arrest caused by inhibition of prenylation. J Biol Chem 2001;276:284305. [178] Lfer G, Hauner H. Adipose tissue development: the role of precursor cells and adipogenic factors. Part II: The regulation of the adipogenic conversion by hormones and serum factors. Klin Wochenschr 1987;65:8127. [179] Zhang HH, Huang J, Dvel K, Boback B, Wu S, Squillace RM, et al. Insulin stimulates adipogenesis through the Akt-TSC2-mTORC1 pathway. PLoS One 2009;4:e6189. [180] Rydn M, Dicker A, Gtherstrm C, Astrm G, Tammik C, Arner P, et al. Functional characterization of human mesenchymal stem cell-derived adipocytes. Biochem Biophys Res Commun 2003;311:3917. [181] Skurk T, Birgel M, Lee Y, Hauner H. Effect of troglitazone on tumor necrosis factor alpha and transforming growth factor beta expression and action in human adipocyte precursor cells in primary culture. Metabolism 2006;55:30916. [182] Leyvraz C, Suter M, Verdumo C, Calmes J, Paroz A, Darimont C, et al. Selective effects of PPARgamma agonists and antagonists on human pre-adipocyte differentiation. Diabetes Obes Metab 2010;12(3):195203. [183] Ringold GM, Chapman AB, Knight DM, Torti FM. Hormonal control of adipogenesis. Ann NY Acad Sci 1986;478:10919. [184] Kellinsalmi M, Parikka V, Risteli J, Hentunen T, Leskel H, Lehtonen S, et al. Inhibition of cyclooxygenase-2 down-regulates osteoclast and osteoblast differentiation and favours adipocyte formation in vitro. Eur J Pharmacol 2007;572:10210. [185] Lehmann JM, Lenhard JM, Oliver BB, Ringold GM, Kliewer SA. Peroxisome proliferator-activated receptors alpha and gamma are activated by indomethacin and other non-steroidal anti-inammatory drugs. J Biol Chem 1997;272:340610. [186] Ye H, Serrero G. Stimulation of adipose differentiation related protein (ADRP) expression by ibuprofen and indomethacin in adipocyte precursors and in adipocytes. Biochem J 1998;330(Pt. 2):8039.
476
C. Vater et al. / Acta Biomaterialia 7 (2011) 463477 incorporating controlled release basic broblast growth factor microspheres. J Biomed Mater Res A 2003;65:48997. Miller DL, Ortega S, Bashayan O, Basch R, Basilico C. Compensation by broblast growth factor 1 (FGF1) does not account for the mild phenotypic defects observed in FGF2 null mice. Mol Cell Biol 2000;20:22608. Li X, Eriksson U. Novel PDGF family members: PDGF-C and PDGF-D. Cytokine Growth Factor Rev 2003;14:918. Solheim E. Growth factors in bone. Int Orthop 1998;22:4106. Papetti M, Herman IM. Mechanisms of normal and tumor-derived angiogenesis. Am J Physiol Cell Physiol 2002;282:C94770. Bar RS, Boes M, Booth BA, Dake BL, Henley S, Hart MN. The effects of plateletderived growth factor in cultured microvessel endothelial cells. Endocrinology 1989;124:18418. Battegay EJ, Rupp J, Iruela-Arispe L, Sage EH, Pech M. PDGF-BB modulates endothelial proliferation and angiogenesis in vitro via PDGF beta-receptors. J Cell Biol 1994;125:91728. Nicosia RF, Nicosia SV, Smith M. Vascular endothelial growth factor, plateletderived growth factor, and insulin-like growth factor-1 promote rat aortic angiogenesis in vitro. Am J Pathol 1994;145:10239. Owens GK, Kumar MS, Wamhoff BR. Molecular regulation of vascular smooth muscle cell differentiation in development and disease. Physiol Rev 2004;84:767801. McKee JA, Banik SSR, Boyer MJ, Hamad NM, Lawson JH, Niklason LE, et al. Human arteries engineered in vitro. EMBO Rep 2003;4:6338. Poh M, Boyer M, Solan A, Dahl SLM, Pedrotty D, Banik SSR, et al. Blood vessels engineered from human cells. Lancet 2005;365:21224. Harris LJ, Abdollahi H, Zhang P, McIlhenny S, Tulenko TN, DiMuzio PJ. Differentiation of adult stem cells into smooth muscle for vascular tissue engineering. J Surg Res 2009 [Epub ahead of print]. Chan-Park MB, Shen JY, Cao Y, Xiong Y, Liu Y, Rayatpisheh S, et al. Biomimetic control of vascular smooth muscle cell morphology and phenotype for functional tissue-engineered small-diameter blood vessels. J Biomed Mater Res A 2009;88:110421. Moosmang S, Lenhardt P, Haider N, Hofmann F, Wegener JW. Mouse models to study L-type calcium channel function. Pharmacol Ther 2005;106:34755. Ross JJ, Hong Z, Willenbring B, Zeng L, Isenberg B, Lee EH, et al. Cytokineinduced differentiation of multipotent adult progenitor cells into functional smooth muscle cells. J Clin Invest 2006;116:313949. Wang C, Cen L, Yin S, Liu Q, Liu W, Cao Y, et al. A small diameter elastic blood vessel wall prepared under pulsatile conditions from polyglycolic acid mesh and smooth muscle cells differentiated from adipose-derived stem cells. Biomaterials 2010;31:62130. Solway J, Seltzer J, Samaha FF, Kim S, Alger LE, Niu Q, et al. Structure and expression of a smooth muscle cell-specic gene, SM22 alpha. J Biol Chem 1995;270:134609. van der Loop FT, Schaart G, Timmer ED, Ramaekers FC, van Eys GJ. Smoothelin, a novel cytoskeletal protein specic for smooth muscle cells. J Cell Biol 1996;134:40111. Gong Z, Niklason LE. Small-diameter human vessel wall engineered from bone marrow-derived mesenchymal stem cells (hMSCs). FASEB J 2008;22:163548. Sinha S, Hoofnagle MH, Kingston PA, McCanna ME, Owens GK. Transforming growth factor-beta1 signaling contributes to development of smooth muscle cells from embryonic stem cells. Am J Physiol Cell Physiol 2004;287:C15608. Hirschi KK, Rohovsky SA, DAmore PA. PDGF, TGF-beta, and heterotypic cell cell interactions mediate endothelial cell-induced recruitment of 10T1/2 cells and their differentiation to a smooth muscle fate. J Cell Biol 1998;141:80514. Deaton RA, Su C, Valencia TG, Grant SR. Transforming growth factor-beta1induced expression of smooth muscle marker genes involves activation of PKN and p38 MAPK. J Biol Chem 2005;280:3117281. Madsen CS, Hershey JC, Hautmann MB, White SL, Owens GK. Expression of the smooth muscle myosin heavy chain gene is regulated by a negativeacting GC-rich element located between two positive-acting serum response factor-binding elements. J Biol Chem 1997;272:633240. Li L, Liu Z, Mercer B, Overbeek P, Olson EN. Evidence for serum response factor-mediated regulatory networks governing SM22alpha transcription in smooth, skeletal, and cardiac muscle cells. Dev Biol 1997;187:31121. Herring BP, Smith AF. Telokin expression in A10 smooth muscle cells requires serum response factor. Am J Physiol 1997;272:C1394404. Miano JM, Carlson MJ, Spencer JA, Misra RP. Serum response factordependent regulation of the smooth muscle calponin gene. J Biol Chem 2000;275:981422. Bttinger EP, Letterio JJ, Roberts AB. Biology of TGF-beta in knockout and transgenic mouse models. Kidney Int 1997;51:135560. Sanford LP, Ormsby I, Gittenberger-de Groot AC, Sariola H, Friedman R, Boivin GP, et al. TGFbeta2 knockout mice have multiple developmental defects that are non-overlapping with other TGFbeta knockout phenotypes. Development 1997;124:265970. Hungerford JE, Little CD. Developmental biology of the vascular smooth muscle cell: building a multilayered vessel wall. J Vasc Res 1999;36:227. Hellstrm M, Kaln M, Lindahl P, Abramsson A, Betsholtz C. Role of PDGF-B and PDGFR-beta in recruitment of vascular smooth muscle cells and pericytes during embryonic blood vessel formation in the mouse. Development 1999;126:304755.
[187] Mandrup S, Lane MD. Regulating adipogenesis. J Biol Chem 1997;272:536770. [188] Kawaguchi N, Toriyama K, Nicodemou-Lena E, Inou K, Torii S, Kitagawa Y. De novo adipogenesis in mice at the site of injection of basement membrane and basic broblast growth factor. Proc Natl Acad Sci USA 1998;95:10626. [189] Katz AJ, Llull R, Hedrick MH, Futrell JW. Emerging approaches to the tissue engineering of fat. Clin Plast Surg 1999;26:587603 [viii]. [190] Gospodarowicz D, Ferrara N, Schweigerer L, Neufeld G. Structural characterization and biological functions of broblast growth factor. Endocr Rev 1987;8:95114. [191] Neubauer M, Fischbach C, Bauer-Kreisel P, Lieb E, Hacker M, Tessmar J, et al. Basic broblast growth factor enhances PPARgamma ligand-induced adipogenesis of mesenchymal stem cells. FEBS Lett 2004;577:27783. [192] Inoue S, Hori Y, Hirano Y, Inamoto T, Tabata Y. Effect of culture substrate and broblast growth factor addition on the proliferation and differentiation of human adipo-stromal cells. J Biomater Sci Polym Ed 2005;16:5777. [193] Kakudo N, Shimotsuma A, Kusumoto K. Fibroblast growth factor-2 stimulates adipogenic differentiation of human adipose-derived stem cells. Biochem Biophys Res Commun 2007;359:23944. [194] Kimura Y, Ozeki M, Inamoto T, Tabata Y. Time course of de novo adipogenesis in matrigel by gelatin microspheres incorporating basic broblast growth factor. Tissue Eng 2002;8:60313. [195] Tabata Y, Miyao M, Inamoto T, Ishii T, Hirano Y, Yamaoki Y, et al. De novo formation of adipose tissue by controlled release of basic broblast growth factor. Tissue Eng 2000;6:27989. [196] Hauner H, Entenmann G, Wabitsch M, Gaillard D, Ailhaud G, Negrel R, et al. Promoting effect of glucocorticoids on the differentiation of human adipocyte precursor cells cultured in a chemically dened medium. J Clin Invest 1989;84:166370. [197] Levenberg S, Rouwkema J, Macdonald M, Garfein ES, Kohane DS, Darland DC, et al. Engineering vascularized skeletal muscle tissue. Nat Biotechnol 2005;23:87984. [198] Tsigkou O, Pomerantseva I, Spencer JA, Redondo PA, Hart AR, ODoherty E, et al. Regenerative medicine special feature: engineered vascularized bone grafts. Proc Natl Acad Sci USA 2010;107(8):33116. [199] Niemeyer P, Fechner K, Milz S, Richter W, Suedkamp NP, Mehlhorn AT, et al. Comparison of mesenchymal stem cells from bone marrow and adipose tissue for bone regeneration in a critical size defect of the sheep tibia and the inuence of platelet-rich plasma. Biomaterials 2010;31(13):35729. [200] Huang NF, Li S. Mesenchymal stem cells for vascular regeneration. Regen Med 2008;3:87792. [201] Ferrara N. Vascular endothelial growth factor: basic science and clinical progress. Endocr Rev 2004;25:581611. [202] Bai K, Huang Y, Jia X, Fan Y, Wang W. Endothelium oriented differentiation of bone marrow mesenchymal stem cells under chemical and mechanical stimulations. J Biomech 2010;43(6):117681. [203] Dvorak HF, Brown LF, Detmar M, Dvorak AM. Vascular permeability factor/ vascular endothelial growth factor, microvascular hyperpermeability, and angiogenesis. Am J Pathol 1995;146:102939. [204] Robinson CJ, Stringer SE. The splice variants of vascular endothelial growth factor (VEGF) and their receptors. J Cell Sci 2001;114:85365. [205] Beckermann BM, Kallifatidis G, Groth A, Frommhold D, Apel A, Mattern J, et al. VEGF expression by mesenchymal stem cells contributes to angiogenesis in pancreatic carcinoma. Br J Cancer 2008;99:62231. [206] Nagy JA, Benjamin L, Zeng H, Dvorak AM, Dvorak HF. Vascular permeability, vascular hyperpermeability and angiogenesis. Angiogenesis 2008;11:10919. [207] Ball SG, Shuttleworth CA, Kielty CM. Mesenchymal stem cells and neovascularization: role of platelet-derived growth factor receptors. J Cell Mol Med 2007;11:101230. [208] Streeten EA, Brandi ML. Biology of bone endothelial cells. Bone Miner 1990;10:8594. [209] Gerber HP, Vu TH, Ryan AM, Kowalski J, Werb Z, Ferrara N. VEGF couples hypertrophic cartilage remodeling, ossication and angiogenesis during endochondral bone formation. Nat Med 1999;5:6238. [210] Engsig MT, Chen QJ, Vu TH, Pedersen AC, Therkidsen B, Lund LR, et al. Matrix metalloproteinase 9 and vascular endothelial growth factor are essential for osteoclast recruitment into developing long bones. J Cell Biol 2000;151:87989. [211] Nakagawa M, Kaneda T, Arakawa T, Morita S, Sato T, Yomada T, et al. Vascular endothelial growth factor (VEGF) directly enhances osteoclastic bone resorption and survival of mature osteoclasts. FEBS Lett 2000;473:1614. [212] Connolly DT, Heuvelman DM, Nelson R, Olander JV, Eppley BL, Delno JJ, et al. Tumor vascular permeability factor stimulates endothelial cell growth and angiogenesis. J Clin Invest 1989;84:14708. [213] Schnmeyr BH, Soares M, Avraham T, Clavin NW, Gewalli F, Mehrara BJ. VEGF inhibits BMP2 expression in rat mesenchymal stem cells. Tissue Eng Part A 2010;16(2):65362. [214] Terranova VP, DiFlorio R, Lyall RM, Hic S, Friesel R, Maciag T. Human endothelial cells are chemotactic to endothelial cell growth factor and heparin. J Cell Biol 1985;101:23304. [215] Millauer B, Wizigmann-Voos S, Schnrch H, Martinez R, Mller NP, Risau W, et al. High afnity VEGF binding and developmental expression suggest Flk-1 as a major regulator of vasculogenesis and angiogenesis. Cell 1993;72:83546. [216] Perets A, Baruch Y, Weisbuch F, Shoshany G, Neufeld G, Cohen S. Enhancing the vascularization of three-dimensional porous alginate scaffolds by
[217]
[218] [219] [220] [221]
[222]
[223]
[224]
[225] [226] [227]
[228]
[229] [230]
[231]
[232]
[233]
[234]
[235]
[236]
[237]
[238]
[239]
[240] [241]
[242] [243]
[244] [245]
C. Vater et al. / Acta Biomaterialia 7 (2011) 463477 [246] Dandr F, Owens GK. Platelet-derived growth factor-BB and Ets-1 transcription factor negatively regulate transcription of multiple smooth muscle cell differentiation marker genes. Am J Physiol Heart Circ Physiol 2004;286:H204251. [247] Kim YM, Jeon ES, Kim MR, Jho SK, Ryu SW, Kim JH. Angiotensin II-induced differentiation of adipose tissue-derived mesenchymal stem cells to smooth muscle-like cells. Int J Biochem Cell Biol 2008;40:248291. [248] Jeon ES, Moon HJ, Lee MJ, Song HY, Kim YM, Bae YC, et al. Sphingosylphosphorylcholine induces differentiation of human mesenchymal stem cells into smooth-muscle-like cells through a TGF-betadependent mechanism. J Cell Sci 2006;119:49945005. [249] Narita Y, Yamawaki A, Kagami H, Ueda M, Ueda Y. Effects of transforming growth factor-beta 1 and ascorbic acid on differentiation of human bonemarrow-derived mesenchymal stem cells into smooth muscle cell lineage. Cell Tissue Res 2008;333:44959. [250] Galmiche MC, Koteliansky VE, Brire J, Herv P, Charbord P. Stromal cells from human long-term marrow cultures are mesenchymal cells that differentiate following a vascular smooth muscle differentiation pathway. Blood 1993;82:6676. [251] Chang Y, Hwang S, Tseng C, Cheng F, Huang S, Hsu L, et al. Isolation of mesenchymal stem cells with neurogenic potential from the mesoderm of the amniotic membrane. Cells Tissues Organs 2010;192:93105. [252] Sellheyer K, Krahl D. Cutaneous mesenchymal stem cells: status of current knowledge, implications for dermatopathology. J Cutan Pathol 2010;37:62434. [253] Pierdomenico L, Bonsi L, Calvitti M, Rondelli D, Arpinati M, Chirumbolo G, et al. Multipotent mesenchymal stem cells with immunosuppressive activity can be easily isolated from dental pulp. Transplantation 2005;80:83642. [254] Ringe J, Leinhase I, Stich S, Loch A, Neumann K, Haisch A, et al. Human mastoid periosteum-derived stem cells: promising candidates for skeletal tissue engineering. J Tissue Eng Regen Med 2008;2:13646. [255] Sabatini F, Petecchia L, Tavian M, Jodon de Villeroch V, Rossi GA, BroutyBoy D. Human bronchial broblasts exhibit a mesenchymal stem cell phenotype and multilineage differentiating potentialities. Lab Invest 2005;85:96271. [256] Prockop DJ. Marrow stromal cells as stem cells for nonhematopoietic tissues. Science 1997;276:714. [257] Erices A, Conget P, Minguell JJ. Mesenchymal progenitor cells in human umbilical cord blood. Br J Haematol 2000;109:23542. [258] Bieback K, Kern S, Klter H, Eichler H. Critical parameters for the isolation of mesenchymal stem cells from umbilical cord blood. Stem Cells 2004;22:62534. [259] Caplan AI. The mesengenic process. Clin Plast Surg 1994;21:42935. [260] Prins H, Rozemuller H, Vonk-Grifoen S, Verweij VGM, Dhert WJA, SlaperCortenbach ICM, et al. Bone-forming capacity of mesenchymal stromal cells
477
[261]
[262]
[263]
[264]
[265]
[266]
[267]
[268] [269]
[270]
[271]
[272] [273]
when cultured in the presence of human platelet lysate as substitute for fetal bovine serum. Tissue Eng Part A 2009;15:374151. Shahdadfar A, Frnsdal K, Haug T, Reinholt FP, Brinchmann JE. In vitro expansion of human mesenchymal stem cells: choice of serum is a determinant of cell proliferation, differentiation, gene expression, and transcriptome stability. Stem Cells 2005;23:135766. Friedman MS, Long MW, Hankenson KD. Osteogenic differentiation of human mesenchymal stem cells is regulated by bone morphogenetic protein-6. J Cell Biochem 2006;98:53854. Stiehler M, Bnger C, Baatrup A, Lind M, Kassem M, Mygind T. Effect of dynamic 3-D culture on proliferation, distribution, and osteogenic differentiation of human mesenchymal stem cells. J Biomed Mater Res A 2009;89:96107. Vogel JP, Szalay K, Geiger F, Kramer M, Richter W, Kasten P. Platelet-rich plasma improves expansion of human mesenchymal stem cells and retains differentiation capacity and in vivo bone formation in calcium phosphate ceramics. Platelets 2006;17:4629. Shen B, Wei A, Whittaker S, Williams LA, Tao H, Ma DDF, et al. The role of BMP-7 in chondrogenic and osteogenic differentiation of human bone marrow multipotent mesenchymal stromal cells in vitro. J Cell Biochem 2010;109(2):40616. Diekman BO, Rowland CR, Caplan AI, Lennon D, Guilak F. Chondrogenesis of adult stem cells from adipose tissue and bone marrow: induction by growth factors and cartilage derived matrix. Tissue Eng Part A 2010;16(2):52333. van Harmelen V, Rhrig K, Hauner H. Comparison of proliferation and differentiation capacity of human adipocyte precursor cells from the omental and subcutaneous adipose tissue depot of obese subjects. Metabolism 2004;53:6327. Bunnell BA, Flaat M, Gagliardi C, Patel B, Ripoll C. Adipose-derived stem cells: isolation, expansion and differentiation. Methods 2008;45:11520. Vashi AV, Keramidaris E, Abberton KM, Morrison WA, Wilson JL, OConnor AJ, et al. Adipose differentiation of bone marrow-derived mesenchymal stem cells using Pluronic F-127 hydrogel in vitro. Biomaterials 2008;29:5739. Jiang Y, Jahagirdar BN, Reinhardt RL, Schwartz RE, Keene CD, Ortiz-Gonzalez XR, et al. Pluripotency of mesenchymal stem cells derived from adult marrow. Nature 2002;418:419. Kanematsu A, Yamamoto S, Iwai-Kanai E, Kanatani I, Imamura M, Adam RM, et al. Induction of smooth muscle cell-like phenotype in marrow-derived cells among regenerating urinary bladder smooth muscle cells. Am J Pathol 2005;166:56573. Shukla D, Box GN, Edwards RA, Tyson DR. Bone marrow stem cells for urologic tissue engineering. World J Urol 2008;26:3419. Kurpinski K, Chu J, Wang D, Li S. Proteomic proling of mesenchymal stem cell responses to mechanical strain and TGF-beta1. Cell Mol Bioeng 2009;2:60614.

Culture Media For The Differentiation of Mesenchymal Stromal Cells

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Acta Biomaterialia 7 (2011) 463477

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Culture media for the differentiation of mesenchymal stromal cells

C. Vater et al. / Acta Biomaterialia 7 (2011) 463477

C. Vater et al. / Acta Biomaterialia 7 (2011) 463477

C. Vater et al. / Acta Biomaterialia 7 (2011) 463477

10% 10% 10% 10%

100 nM 10 nM 100 nM 100 nM 100 nM 100 nM

290 nM 300 lM 500 lM 50 lM 200 lM

Human Human Human Human Human

C. Vater et al. / Acta Biomaterialia 7 (2011) 463477

Vogel et al. [264]

Shen et al. [265]

Chen et al. [66] Fernandes et al. [72] Diekman et al. [266]

100 nM 100 nM 100 nM

50 lg ml1 200 lM 37.5 lg ml

Human BM/UCWJ Human BM Human BM/AT

C. Vater et al. / Acta Biomaterialia 7 (2011) 463477

Cell source Human AT

Hemmrich et al. [163]

Bunnell et al. [268] Choi et al. [103] Tsuji et al. [164]

Rat AT Human BM Human AT

500 lM 500 lM 500 lM 250 lM

Rat BM Human BM/UCWJ Human BM Human AT

C. Vater et al. / Acta Biomaterialia 7 (2011) 463477

Cell Source Human BM Human BM Human BM/UCWJ

10 ng ml 50 ng ml1 1 100 ng ml EGF (50 ng ml1) hydrocortisone (1 lg ml1)

C. Vater et al. / Acta Biomaterialia 7 (2011) 463477

Kurpinski et al. [273] Gong et al. [6]

Human MSCs (Cambrex) Human BM

Harris et al. [227]

Wang et al. [231]

C. Vater et al. / Acta Biomaterialia 7 (2011) 463477

[218] [219] [220] [221]

[225] [226] [227]

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