culture media for the differentiation of mesenchymal stromal cells

Acta Biomaterialia 7 (2011) 463–477

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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

Article history:Received 7 April 2010Received in revised form 20 July 2010Accepted 27 July 2010Available online 3 August 2010

Keywords:Mesenchymal stromal cellCell culture mediumDifferentiationProliferationOsteoblast

1742-7061/$ - see front matter � 2010 Acta Materialdoi:10.1016/j.actbio.2010.07.037

Abbreviations: 1,25-D3, 1,a25-dihydroxyvitamin Dcollagen; ALP, alkaline phosphatase; asc, ascorbic2-phosphate; AT, adipose tissue; BM, bone marrowderived mesenchymal stromal cells; BMP, bone morpsialoprotein; C/EBP, CCAAT-enhancer-binding protemonophosphate; Col10, collagen type 10; Col11, collatype 1 alpha-1; Col2, collagen type 2; Col9, collageresponse element-binding protein/E1A binding proteiEC, endothelial cell; ECM, extracellular matrix; FABP4tein-4; FCS, fetal calf serum; FGF, fibroblast growth fprotein 6; GPDH, glycerol-3-phosphate dehydrogenasylxanthine; IGF, insulin-like growth factor; LDL, lolipoprotein lipase; mRNA, messenger ribonucleic acidcell; OC, osteocalcin; ON, osteonectin; OPN, osteoplatelet-derived growth factor; PPARc2, peroxisome ptor c2; PRP, platelet-rich plasma; Runx-2, runt-relatedsevere combined immunodeficiency; SM22-a, transgemyosin heavy chain; Sox9, SRY-related high-mobiliresponse factor; T3, triiodothyonine; TGF-b, transformumbilical cord blood; UCWJ, umbilical cord Whaendothelial growth factor; vitD3, vitamin D3; VSMC,a-SMA, a-smooth muscle actin; b-GP, beta-glyceroph⇑ Corresponding author. Address: Department of O

Hospital Carl Gustav Carus, Fetscherstr. 74, D-01307(0)351 458 3137; fax: +49 (0)351 449 210 419.

E-mail address: maik.stiehler@uniklinikum-dresde

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 seriesof 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 field oftissue regeneration for both research and clinical purposes. The key factors for successful MSC prolifer-ation and differentiation in vitro are the culture conditions. Hence, we here summarize the culture mediaand their compositions currently available for the differentiation of MSCs towards osteogenic, chondro-genic, adipogenic, endothelial and vascular smooth muscle phenotypes. However, optimal combination ofgrowth factors, cytokines and serum supplements and their concentration within the media is essentialfor the in vitro culture and differentiation of MSCs and thereby for their application in advanced tissueengineering.

� 2010 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

1. Introduction porting stroma and vasculature [2–6]. We presently refer to these

Since Owen and Friedenstein’s work [1] it has been assumedthat bone marrow contains special cells that can be expandedin vitro and differentiated into various mesoderm-type lineages,including bone, fat, cartilage, muscle, tendon, haematopoiesis-sup-

ia Inc. Published by Elsevier Ltd. A

3; a2Col6, a chain 2 of type 6acid; Asc-2-P, ascorbic acid; BM-MSCs, bone marrow-

hogenetic protein; BSP, boneins; cAMP, cyclic adenosinegen type 11; Col1a1, collagenn type 9; CREB/p300, cAMPn p300; dex, dexamethasone;/aP2, fatty acid-binding pro-actor; Gata-6, GATA-bindinge; IBMX, 3-isobutyl-1-meth-w-density lipoprotein; LPL,

; MSC, mesenchymal stromalpontin; Osx, osterix; PDGF,roliferation-activated recep-transcription factor-2; SCID,

lin; SMMHC, smooth musclety group box 9; SRF, seruming growth factor-beta; UCB,rton’s jelly; VEGF, vascularvascular smooth muscle cell;osphate.rthopedic Surgery, UniversityDresden, Germany. Tel.: +49

n.de (M. Stiehler).

nonhematopoietic cells as mesenchymal stromal cell (MSC; Fig. 1)[7]. In addition, researchers have recently transdifferentiated MSCsinto non-mesodermal cell types such as neuronal-like cells [8–11]and pancreatic cell progenitors [12–15].

To date, MSCs have been isolated not only from bone marrowbut also from many other tissues and organs, including adipose tis-sue, umbilical cord blood, placental tissue, liver, spleen, testes,menstrual blood, amniotic fluid, pancreas and periosteum [16–20]. When cultured in vitro on polystyrene surfaces, MSCs revealmorphological heterogeneity. These cells can be narrow spindle-shaped, large polygonal or even cuboidal-shaped when growinginto a confluent monolayer [21]. In adult human, MSCs lack thehematopoietic surface antigens, e.g. CD11, CD14, CD34 and CD45[22]. Meanwhile numerous molecular markers have been foundon MSC surface, but none of them is specific to MSCs. Despite this,molecules such as CD44, CD73, CD90, STRO-1 and CD105/SH2[3,23–25] are still currently used to identify MSCs.

MSCs are considered to be nonimmunogenic since these cellshave been transplanted into allogeneic hosts even without usingany immunosuppressive drugs [22,26]. Furthermore, it has beenreported that MSCs actually possess immunosuppressive proper-ties by modulating the function of T-cells [27,28], dendritic cellsand B-cells [29–32].

For the characterization of MSC plasticity, their ability to differ-entiate in vitro into osteoblasts, chondrocytes and adipocytes iscurrently treated as the gold standard. This, in combination withthe advantages that MSCs have no immunogenicity and can be eas-ily isolated from different tissues and expanded in vitro, enablesMSCs to be a promising source of stem cells. Hence MSCs have

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Fig. 1. Morphology of human immortalized single cell-derived BM-MSCs at different stages of confluency (A: 10%; B: 90%). Cells were cultured in a-MEM containing 1%penicillin/streptomycin and 10% FCS (BM-MSCs, bone marrow-derived MSCs).

464 C. Vater et al. / Acta Biomaterialia 7 (2011) 463–477

been used in the therapy of diseases such as extended osseous de-fects [33], acute myocardial infarction [34], leukemia [35] and dia-betes [36]. In addition, the homing capability endows MSCs withfurther potential applications. For example, MSCs may be usedfor supporting tissue regeneration [37], correcting congenital dis-orders (e.g. osteogenesis imperfecta [38]) and controlling chronicinflammatory diseases [39,40], and have even employed as vehi-cles for the delivery of biological agents [22] and as probes in thebiocompatibility test of new implant materials. A prerequisite forthe therapeutic application of MSCs is to develop efficient andstandardized protocols so that MSCs can be induced to differenti-ate along the way as required. Therefore, we here present an over-view of the optimized protocols for MSC differentiation towardsthe osteogenic, chondrogenic, adipogenic, endothelial and vascularsmooth muscle phenotypes.

2. Osteogenic differentiation

2.1. Background

Bone diseases are major socioeconomic issues. The WorldHealth Organization has acknowledged this fact by declaring theyears 2000–2010 ‘‘The Bone and Joint Decade”. The developmentof innovative bone-healing strategies is a prerequisite for the suc-cessful treatment of a variety of patients suffering from local bonedefects caused by trauma, tumour, infection, degenerative jointdisease, congenital crippling disorders or periprosthetic bone loss.Furthermore, bone graft material is frequently needed for spinalfusion, joint revision surgery, corrective osteotomy proceduresand bone reconstruction in the field of oral and maxillofacial sur-gery. Bone grafting is one of the most common orthopaedic proce-dures with autologous bone graft providing osteoinductive growthfactors, bone-forming cells and structural support for new bone in-growth. However, the use of autologous bone graft is associatedwith the disadvantages of limited graft availability and donor sitemorbidity, e.g. pain, infection, pelvic fractures or neurovascular in-jury. The implantation of sterilized bone allograft material – usu-ally derived from femoral heads during joint replacementprocedures – as a widely used alternative bone-filling materialmay result in failure rates of up to 30% due to insufficient osseoin-tegration of the graft, requiring further surgical intervention [41].Insufficient bone-healing therefore remains a challenging issue.In this context, innovative cell-based strategies using MSCs arepromising for both site-specific and systemic bone regeneration.

2.2. Morphology and differentiation markers

When being differentiated into osteoblasts, MSCs transformfrom a fibroblastic to a cuboidal shape, produce extracellular ma-

trix (ECM), mainly composed of collagen type I, and in a later stageform aggregates or nodules that can be stained positively by aliza-rin red and von Kossa techniques. Increased expression of alkalinephosphatase (ALP; Fig. 2A) and calcium accumulation are observedin MSCs during osteogenic differentiation [3,22]. The enzymaticactivity of ALP as well as the calcium content can be quantifiedby colorimetric assays [42]. At the molecular level, osteogenic dif-ferentiation of MSCs is controlled by interactions between distincthormones and transcription factors.

Runt-related transcription factor-2 (Runx-2) effectuates theexpression of bone-specific genes, e.g. osterix (Osx), collagen type1 alpha-1 (Col1a1), osteocalcin (OC) and bone sialoprotein (BSP),by binding to the promoters of these genes [43–48]. Generally,Runx-2, ALP, Col1a1, transforming growth factor-beta 1 (TGF-b1),osteonectin (ON) and bone morphogenetic protein-2 (BMP-2) areknown to be early markers of osteoblastic differentiation, whereasOC and osteopontin (OPN) are expressed later in the differentiationprocess [49–52].

2.3. Differentiation protocols

The classical method for osteogenic differentiation of MSCsin vitro involves incubating a confluent monolayer of MSCs withcombinations of dexamethasone (dex), beta-glycerophosphate (b-GP) and ascorbic acid (asc) for several weeks. In addition, combina-tions of vitamin D3 (vitD3), transforming growth factor-beta (TGF-b) and bone morphogenetic proteins (BMPs) are used for osteo-genic differentiation. In the following these supplements will bedescribed in detail.

Dex is a synthetic glucocorticoid and has been reported to be anessential requirement for osteoprogenitor cell differentiation inMSCs [53,54]. While MSCs that were cultured in basal mediumwithout osteogenic supplements express increased levels of ALP,they fail to express mineralized ECM as well as other osteogenicmarkers such as Col1 [55]. Although the precise mechanisms of ac-tion of dex on stem cell differentiation and skeletal function are notknown, it is supposed that dex induces transcriptional effects. Inrat osteoblast-like cells, for instance, dex induces transcription ofBSP by binding on a glucocorticoid response element in the pro-moter region of the BSP gene [56]. On the other hand, dex improvesthe expression of the b-catenin-like molecule TAZ (transcriptionalcoactivator with PDZ-binding motif) as well as integrin a5, whichboth promote osteoblastic differentiation of MSCs by activatingRunx-2-dependent gene transcription [57,58]. However, glucocor-ticoids in supraphysiological amounts have deleterious effects onbone in vivo, resulting in inhibition of osteoblast function [59]. Ina study by Walsh et al. MSCs were cultured in the presence andabsence of dex at concentrations between 10 pM and 1 lM forup to 28 days [60]. The authors suggest that the critical effective

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 immunofluorescent staining for alkaline phosphatase (shown in green) andfluorescence-labeled phalloidin staining for actin (shown in red) was performed (A). To induce chondrogenic differentiation porcine BM-MSCs were cultured in pellet inDMEM containing 1% ITS, 1% penicillin/streptomycin, 10 ng ml�1 TGF-b3, 10 nM dex and 210 lM ascorbic acid. After 21 days, differentiation into the chondrogenic lineagewas visualized by immunohistochemical staining for ColX (shown in brown) (B). Human immortalized single-cell-derived BM-MSCs were seeded on tissue culturepolystyrene and induced to differentiate into adipocytes by culturing the cells in DMEM containing 10% FCS, 1 mM dex, 500 lM IBMX, 1 lg ml�1 insulin, and 100 lMindomethacin for 21 days. The appearance of intracellular lipid-rich vacuoles was confirmed by oil red O staining (shown in red) (C). Tubular-like structures formed by humanimmortalized single-cell-derived BM-MSCs in fibrin-based gels after 7 days of endothelial differentiation in a-MEM containing 1% penicillin/streptomycin, 10% FCS and50 ng ml�1 VEGF was visualized by bright-field microscopy (D). Immunofluorescent staining for calponin (shown in green) and fluorescent 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 ml�1 TGF-b1.

C. Vater et al. / Acta Biomaterialia 7 (2011) 463–477 465

concentration of dex is 10 nM, corresponding to its physiologicalconcentration. At higher concentrations (of the order of 100 nM)dex inhibits osteoblast differentiation in MSCs and leads to gluco-corticoid-induced osteoporosis [57]. In addition, proliferation isnegatively affected, mainly due to the inhibitory effect of glucocor-ticoids on collagen synthesis [61]. However, when MSCs are cul-tured in the presence of asc, the effects of glucocorticoids oncollagen production are markedly masked [62]. Song et al. even re-ported on a reductive effect of dex on density-related apoptosis inMSC cultures [63]. When implanted subcutaneously into SCIDmice, pretreatment of MSCs with 100 nM dex for up to 5 weeks re-sulted in enhanced bone tissue formation compared with un-treated controls [42]. For complete induction of osteogenicdifferentiation of in vitro cultivated MSCs at least 3 weeks of con-tinuous treatment with dex is required [42].

For matrix mineralization the presence of both calcium andphosphate ions is essential. b-GP, which is enzymatically hydro-

lyzed by alkaline phosphatase, serves as a crucial source of inor-ganic phosphate [64]. Chung et al. showed that cultivation ofosteoblast-like cells in culture medium containing b-GP leads tomineral formation, lactate generation, increased ALP activity, aswell as protein and phospholipid synthesis, indicating enhancedosteogenic differentiation [65]. Usually 5–10 mM b-GP is used forosteogenic differentiation of MSCs [55,65,66].

Asc plays an important role as a cofactor for the hydroxylationof proline and lysine residues in collagens, which are the mostabundant group of ECM proteins in the body [67,68]. One difficultyconcerning the handling of asc is its instability in solution, espe-cially under standard culture conditions (pH 7.5, 37 �C, humidifiedatmosphere, 5% CO2) [69,70]. Thus it is recommended to use thelong-acting vitamin C derivative ascorbic acid 2-phosphate (Asc-2-P) which was found to be stable under conventional culture con-ditions [71]. Recent studies showed that in the presence of Asc-2-PMSCs demonstrate upregulation of genes related to cell cycle and


mitosis, whereas absence of Asc-2-P leads to reduced ALP expres-sion and inhibition of calcium accumulation [72]. The mitogenic ef-fect of Asc-2-P can be ascribed either to the role of the Asc-2-P-induced ECM as a growth factor reservoir or to a direct activationof mitogenic pathways. In the absence of collagens, integrin signal-ling is impaired and downstream signals that are needed for mito-gen-activated protein kinase activation are absent. No inhibitoryeffect on cell growth could be found when supplementing the cul-ture media with up to 10 mM ascorbic acid. Usually, concentra-tions ranging from 50 up to 500 lM are used to induceosteogenic phenotype of MSCs [42,69,73].

vitD3 is a secosteroid hormone which is essential for the func-tion of osteoblasts. Its active form, 1a,25-dihydroxyvitamin D3(1,25-D3), demonstrates potent antiproliferative effects by block-ing 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 interac-tion of the hormone with a membrane-bound receptor, include anincrease in cyclic adenosine monophosphate (cAMP) and the open-ing of calcium channels [76]. Through interaction with a nuclearvitamin D receptor, 1,25-D3 has been shown to stimulate theexpression of bone-related transcription factors, i.e. Runx-2 andOsx, in addition to osteoblast differentiation markers, e.g. ALP,Col1a1, OC and OPN [70]. Although 1,25-D3 synergized with bothdex and bone morphogenetic protein-2 promoted expression ofosteoblastic markers, this agent alone was unable to induce matrixmineralization [59,77]. In contrast to this, Jaiswal et al. reportedthat dex may reduce vitamin D receptor expression in osteoblasticcells [78], leading to a reduced uptake ability for 1,25-D3 andtherefore decreased expression of differentiation markers such asOC.

As well as using dex, b-GP, Asc-2-P and 1,25-D3, the addition ofdistinct growth factors can enhance osteogenic differentiation ofMSCs in vitro. TGF-b exists in three isoforms, whereas TGF-b1 isthe major form found in bone [79,80]. TGF-b1 influences cellgrowth and plays an essential role in the control of bone formationby modulating the synthesis and degradation of several bone ma-trix components, e.g. collagen type 1 and non-collagenous proteins[81,82]. Notably, although TGF-b1 stimulates the expression ofRunx-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 developedtissues [84,85]. Recent reports investigating the role of BMPs inosteogenesis [86–88] showed that there may be species-specificdifferences in the effect of BMPs in vitro. In both mice and rats,BMPs promote osteoblast differentiation [89–91]. Interestingly,there is body of evidence that BMP-2, BMP-4 and BMP-7 fail to in-duce osteogenic differentiation of human MSCs [87,92]. In contrast,

Table 1Representative protocols used for in vitro osteogenic differentiation of MSCs (asc, ascorbidexamethasone; FCS, fetal calf serum; ITS, insulin-transferrin-selenious acid; TGF-b, transfomarrow; UCB, umbilical cord blood; UCWJ, umbilical cord Wharton’s jelly).

Reference FCS dex asc

Oswald et al. [5] 10% 100 nM 200 lMJørgensen et al. [59] 10% 100 nM –

Friedman et al. [262] – 100 nM 25 lg ml–1

Stiehler et al. [263] 10% 100 nM 290 nMHildebrandt et al. [55] – 10 nM 100 nM 300 lMPytlík et al. [73] 10% 100 nM 500 lMSong et al. [42] 10% 100 nM 50 lMChen et al. [66] 10% 100 nM 200 lM

several studies demonstrated that in cells of the osteoblast lineage,BMP-2, BMP-4 and BMP-7 are capable of inducing expression ofALP, Col1a1, OPN, BSP and other non-collagenous bone proteinsfound in osteoid [55,91,93–96].

Representative protocols used for in vitro osteogenic differenti-ation of MSCs including dex, b-GP, Asc-2-P, vitD3, TGF-b and BMPsas well as commercially available culture media are summarized inTables 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 thesubchondral bone usually do not repair spontaneously [97]. Smallfull-thickness defects can repair spontaneously, resulting inhyaline-like cartilage. However, larger defects only regenerate byproduction of fibrous tissue or fibrocartilage, which as biomechan-ically inferior compared with physiological hyaline cartilage. Thecell-based regeneration of (osteo)chondral defects presents a ma-jor challenge. Although, in principle, autologous chondrocytescan be used for cartilage tissue-engineering applications, their lim-ited availability, quantity and viability are major drawbacks [98].In this context, MSCs are promising candidates for cartilage regen-eration [99].


During chondrogenic differentiation MSCs change from a char-acteristic fibroblast-like morphology to a large round shape. Thecells are surrounded by abundant ECM, consisting of a highly orga-nized network of collagen (predominantly type 2 collagen) andaggregating proteoglycans and glycosaminoglycans of high molec-ular weight [100–102] that can be detected by, for example, Alcianblue, Toluidine blue and Safranin O stainings and quantified byspecific assays [72,103].

The protein SRY-related high-mobility group box 9 (Sox9) is anearly transcription factor of chondrogenesis and controls theexpression 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 [104–108]. By binding to thepromoter of these genes, Sox9 forms transactivating complexeswith other proteins, e.g. Sox5/Sox6, cAMP response element-bind-ing protein/E1A binding protein p300 (CREB/p300) and c-musculo-aponeurotic fibrosarcoma [104,109,110]. Col10 is known to besynthesized during chondrocyte hypertrophy in the growth plate

c acid; bFGF, basic fibroblast growth factor; BMP, bone morphogenetic protein; dex,rming growth factor-beta; vitD3, vitamin D3; b-GP, beta-glycerophosphate; BM, bone

b-GP Other Cell source

10 mM – Human BM– vitD3 (1 nM)

BMP-2 (100 ng ml�1)Human BM

5 mM ITS (1%)bFGF (25 ng ml�1)

FGF-8/FGF-10 (50 ng ml�1)TGF-b (200 pM)BMP-2/4/6/7/14 (20 nM)

Human BM

5 mM vitD3 (10 nM) Human BM5 mM BMP-2 (100 nM) Human UCB10 mM – Human BM10 mM – Human BM10 mM – Human BM/UCWJ

Table 2Media commercially available for osteogenic differentiation of MSCs.

Company Product

Invitrogen (Carlsbad, USA) STEMPRO� Osteogenesis Differentiation KitTrevigen (Gaithersburg, USA) Mesenchymal Stem Cell Osteogenic Differentiation KitThermo Scientific (Waltham, USA) HyClone™ AdvanceSTEM™ Osteogenic Differentiation KitR&D Systems (Minneapolis, USA) Human/Mouse StemXVivo Osteogenic/Adipogenic

Base Media Human/Mouse StemXVivo Osteogenic SupplementMiltenyi Biotec (Bergisch Gladbach, Germany) NH OsteoDiff MediumCellular Engineering Technologies Inc. (Coralville, USA) Osteogenic Differentiation MediaProvitro GmbH (Berlin, Germany) hMSC osteogenesis induction mediumPromoCell GmbH (Heidelberg, Germany) Mesenchymal Stem Cell Osteogenic Differentiation Medium


of long bones and is used to identify terminally differentiatedhypertrophic chondrocytes [111–114].


In 1998 Johnstone et al. first described a defined cell culturemedium for in vitro chondrogenesis of MSCs [115]. The two mainprinciples involved in enhancing chondrogenic differentiationin vitro are close cell-to-cell contact, usually achieved by cell pelletor micromass culture [115,116], and the addition of chondrogenicbioactive factors, e.g. dex, asc, TGF-b, BMPs, fibroblast growth fac-tor (FGF) and insulin-like growth factor (IGF). Among these dex, ascand TGF-b have been shown to be most effective [102,117–120].The effects of these factors on MSCs will be discussed in more de-tail below.

Glucocorticoids are used for the in vitro differentiation of MSCsinto multiple lineages, including stroma, fat, bone and cartilage[121]. Glucocorticoid function is mediated by the cytoplasmic glu-cocorticoid receptor, which influences various differentiation pro-cesses by inducing transcriptional actions. In a study by Derfoulet al., dex upregulated gene expression and protein levels of severalcartilage matrix markers, in particular Col11 [121]. However, incontrast to combination with TGF-b, dex alone has little effectson the expression of chondrogenic markers such as aggrecan, car-tilage oligomeric matrix protein and Col2. For human primary bonemarrow-derived MSCs, successful chondrogenic differentiationwas shown in medium containing 100 nM dex [122].

Asc and its derivative Asc-2-P causes collagen hydroxylation viamodification of proline and lysine residues. Its addition to the cul-ture medium leads to increased MSC proliferation and enhancesproduction of collagen type 2, which is one of the most importantECM proteins in cartilage [120].

TGF-b interacts with a heteromeric receptor complex compris-ing two structurally related serine–threonine kinases, types I andII receptors, and transforms its signals intracellularly via Smad pro-teins [123,124]. Together with dex, TGF-b represents an indispens-able factor for chondrogenic differentiation of human MSCs [118].Although Barry et al. state that TGF-b2 and TGF-b3 are more effec-tive than TGF-b1 in promoting glycosaminoglycan and type 2 col-lagen accumulation [122], others have concluded that any of theTGF-b subtypes are equally active chondrogenic factors and thatlot- rather than subtype-specific differences seem to exist [125].Whilst chondrogenic differentiation of MSCs in monolayer cultureappears to be dependent on TGF-b concentration and on cultiva-tion time, a concentration of 10 ng ml�1 TGF-b is sufficient for suc-cessful MSC differentiation in pellet culture [122,126]. AlthoughTGF-b supports early- and intermediate-stage chondrogenesis, itis known to retain chondrocytes in the prehypertrophic state.Therefore TGF-b most likely represses terminal in vitro differentia-tion of MSCs [127].

BMPs play an important role during bone morphogenesis by ini-tiating 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 suffi-cient to stimulate in vitro chondrogenesis of human MSCs inconventional pellet culture with dex-supplemented medium[129,130]. In a study by Richter et al. MSCs from bone marrow re-quired only dex and TGF-b for successful chondrogenic differentia-tion, whereas the differentiation of MSCs derived from adiposetissue or synovial tissue was dependent on the additional supple-mentation with BMP-6 [122]. Sekiya et al. demonstrated that thechondrogenic effect of BMP-2 on bone-marrow-derived MSCs isgreater than that of BMP-4 and BMP-6 as measured by increasedpellet weight, superior production of proteoglycans and Col2 [131].

In vitro treatment of MSCs with FGF, dex, or combinations ofboth, increased pellet content of collagen type 2 and glycosamino-glycans as well as mRNA expression of aggrecan [132,133]. Jingushiet al. found that repeated injections of FGF into the fracture site inrats induced cartilaginous callus formation enlargement [117].

The chondrogenic differentiation potential of MSCs treated withIGF has been investigated extensively. IGF stimulates MSC prolifer-ation, regulates cell apoptosis, induces the expression of chondro-cyte markers, increases the synthesis of matrix proteins includingcollagen type 2 and proteoglycans, and promotes the survival,development and maturation of chondrocytes in vitro [102,134–136]. IGF-preconditioned autologous bone-marrow-derived MSCsenhanced chondrogenesis in full-thickness cartilage lesions in thefemoropatellar articulations of young mature horses [137].Although some studies did not show any effect of isolated IGFapplication on in vivo MSC differentiation, Indrawattana et al.showed that IGF is capable of inducing chondrogenic differentia-tion on a level comparable to TGF-b [138–140]. However, Matsudaet al. reported that the combination of TGF-b, dex and IGF was themost effective cocktail to stimulate differentiation of MSCs tochondrocytes [141].

Usually chondrogenic differentiation of MSCs is performed inserum-free media in order to reduce serum-induced cellular apop-tosis [142]. In this context, platelet-rich plasma (PRP) seems to be apromising supplementary agent. PRP is defined as plasma enrichedfor platelets and contains effective growth factors such as TGF-b1and vascular endothelial growth factor (VEGF) [143]. Recentin vitro investigations have confirmed that both cellular prolifera-tion as well as mRNA levels of Runx-2, Sox9 and aggrecan were sig-nificantly higher in PRP-treated cells compared to fetal calf serum(FCS)-treated MSC cultures [144]. Furthermore, the level of chon-drogenic potential of MSCs is dependent on the tissue source thecells are isolated from. Studies showed that bone-marrow-derivedMSCs demonstrated higher levels of in vitro chondrogenic poten-tial compared to adipose-derived MSCs [145–148]. Sakaguchiet al. even demonstrated that MSCs isolated from synoviumshowed superior chondrogenic potential than MSCs isolated fromother mesenchymal tissues [149].

Tables 3 and 4 give an overview about representative protocolsused for in vitro chondrogenic differentiation of MSCs includingdex, asc, TGF-b, BMPs and IGF as well as the respective commer-cially available media.

Table 3Representative 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 Wharton’s jelly; AT, adipose tissue). In all protocols cells were cultured under three-dimensional conditions (pellet, scaffold).

Reference FCS dex asc TGF-b Other Cell source

Matsuda et al. [141] 10% 39 ng ml�1 50 lg ml�1 100 ng ml�1 IGF (100 ng ml�1) Human BMSekiya et al. [131] – 100 nM 50 lg ml�1 100 ng ml�1 BSA (1.25 mg ml�1)

ITS (50 mg ml�1)linoleic acid (5.35 mg ml�1)BMP-2/4/6 (500 ng ml�1)

Human BM

Vogel et al. [264] – 100 nM 170 lM 10 ng ml�1 BSA (1.25 mg ml�1)insulin (5 lg ml�1)transferring (5 lg ml�1)selenous acid (5 lg ml�1)

Human BM

Shen et al. [265] – 100 nM 50 lg ml�1 10 ng ml�1 BSA (1.25 mg ml�1)ITS (1%) linoleic acid (5.35 lg ml�1)BMP-7 (100 ng ml�1)

Human BM

Chen et al. [66] – 100 nM 50 lg ml�1 10 ng ml�1 BSA (1.25 mg ml�1)ITS (1%)

Human BM/UCWJ

Fernandes et al. [72] – 100 nM 200 lM 10 ng ml�1 ITS (50 lg ml�1)BMP-6 (500 ng ml�1)

Human BM

Diekman et al. [266] – 100 nM 37.5 lg ml�1 10 ng ml�1 ITS (1%)BMP-6 (10/500 ng ml�1)

Human BM/AT

Table 4Media commercially available for chondrogenic differentiation of MSCs.

Company Product

Invitrogen (Carlsbad, USA) StemPro� Chondrogenesis Differentiation KitR&D Systems (Minneapolis, USA) Human/Mouse StemXVivo Chondrogenic Base Media

Human/Mouse StemXVivo Chondrogenic SupplementMiltenyi Biotec (Bergisch Gladbach, Germany) NH ChondroDiff MediumCellular Engineering Technologies Inc. (Coralville, USA) Chondrogenic Differentiation MediaProvitro GmbH (Berlin, Germany) hMSC chondrogenesis induction mediumPromoCell GmbH (Heidelberg, Germany) Mesenchymal Stem Cell Chondrogenic Differentiation Medium


4. Adipogenic differentiation

4.1. Background

The formation of adipocytes and their aggregation in order toform adipose tissue denotes an important step in the evolutionof vertebrates, as it guarantees independence from food intakeover longer periods of time [150]. In plastic and reconstructive sur-gical procedures, adipose tissue is frequently needed to repair softtissue defects that result from traumatic injury (i.e. significantburns), tumour resections (i.e. mastectomy and carcinoma re-moval), and congenital defects [151]. The American Society of Plas-tic Surgeons reported that over 5 million reconstructive procedureswere performed in 2004, approximately 4 million of which weredue to tumour removal [152]. Strategies to repair soft tissue de-fects, e.g. breast reconstruction procedures, include the use of im-plants and fillers [153,154]. However, there exists no void-fillingmaterial that will satisfy all clinical needs. The use of autologousfat tissue has not been consistently successful in patients[155,156] due to the fact that fat transplants lack sufficient vascu-larization, i.e. central graft blood flow is not adequate for long-termsurvival of the tissue, and often leads to tissue resorption [157]. Inthis context, tissue engineering using MSCs is an appealing strat-egy to provide adipose tissue grafts.


Adipose cell differentiation is a multistep process characterizedby a sequence of events during which preadipocytes divide untilconfluence [158]. When being differentiated into adipocytes, fibro-blastic MSCs are converted to a spherical shape expressing severaltypes of ECM proteins, including fibronectin, laminin, and types 1,

3, 4, 5 and 6 collagen. Initially a fibronectin network develops,followed by the formation of a type I collagen network [159–161]. Finally, differentiation of MSCs into adipocytes leads to accu-mulation of intracellular lipid-rich vacuoles that can be stainedpositively by oil red O (Fig. 2C). Quantitative analyses can be per-formed either by staining the cells with oil red O and extractingthe dye from the cells with isopropanol [103] or by determiningthe activity of glycerol-3-phosphate dehydrogenase (GPDH)[162–164].

The adipocyte-specific peroxisome proliferation-activatedreceptor c2 (PPARc2) belongs to the PPARs family, and is, togetherwith CCAAT-enhancer-binding proteins (C/EBP), one of the tran-scription factors that regulate expression of genes responsible forinduction and progression of adipogenesis [165,166]. Lipoproteinlipase (LPL) and the a chain 2 of type 6 collagen (a2Col6) areknown to be early markers of adipogenic differentiation, whereasleptin, fatty acid-binding protein-4 (FABP4/aP2) and adiponectinare expressed during later stages of differentiation [158,167–170].


A routinely used method to stimulate adipogenic differentiationof preadipocytes and MSCs is the cultivation of these cells untilconfluency, followed by incubation with dex, 3-isobutyl-1-methyl-xanthine (IBMX), insulin and indomethacin, or combinations ofthese. In addition to these agents, triiodothyonine (T3), Asc-2-Pand basic FGF (bFGF-2) are also used for adipogenic differentiationas will be described in detail below.

The glucocorticoid dex induces the accumulation of transcrip-tion factors including C/EBP and PPARc which are crucial for adi-pose conversion of precursor cells [171]. In a study conducted byCui and coworkers, adipogenesis (as quantified by the presence


of triglyceride-containing vesicles and by expression of FABP4) wasinduced in a murine MSC cell line by treating the cells with dex[172].

The positive effect of glucocorticoids on adipogenic differentia-tion can be amplified by cAMP-elevating agents such as IBMX[173]. While inhibiting the activity of phosphodiesterase and tu-mour necrosis factor-a (TNF-a), IBMX enhances the expression ofPPARc2 and LPL and down-regulates the expression of osteogenicmarker genes such as Runx-2 and OPN by activation of protein ki-nase A [170,174–176].

Among the commonly used adipogenesis-inducing drugs, insu-lin is known to be the most potent [177]. It stimulates adipogenesisthrough the Akt-TSC2-mTORC1 pathway by increasing and acceler-ating triglyceride accumulation [178,179]. To maintain in vitro adi-pogenic differentiation of MSCs, the effect of insulin can beenhanced by insulin sensitizers, e.g. rosiglitazone or troglitazone,which act by stimulating PPARc [180–182].

The nonsteroidal anti-inflammatory drug indomethacin is acyclooxygenase inhibitor and blocks osteoclast and osteoblast dif-ferentiation, while promoting hMSC commitment to the adipogeniclineage [183,184]. Furthermore, indomethacin was found to bind di-rectly to the PPARc receptors and to act as a PPARc agonist, leadingin confluent MSC monolayers to the expression of adipogenic mark-ers 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 influenceadipogenesis [187–189]. While T3 multiplies the effects of insulin,media supplementation with 500 lM Asc-2-P results in the accu-mulation of the highest number of oil droplets compared to controlmedium without Asc-2-P [103]. FGF-2, which belongs to the familyof heparin-binding growth factors, increases cellular proliferation

Table 5Representative protocols used for in vitro adipogenic differentiation of MSCs (asc, ascorbicT3, triiodothyonine; AT, adipose tissue; BM, bone marrow; UCWJ, umbilical cord Wharton

Reference FCS dex Insulin IBMX

van Harmelen et al. [267] – – 66 nM 500 lM

Hemmrich et al. [163] – 100 nM 66 nM 500 lM

Bunnell et al. [268] 20% 500 nM – 500 lMChoi et al. [103] 10% 1 mM 10 mg ml 500 lMTsuji et al. [164] – 100 nM 50 lM –

Vashi et al. [269] – 1 mM 10 mM 500 lMChen et al. [66] 10% 1 mM 5 mg ml 500 lMPytlik et al. [73] 10% 100 nM 10 lg ml 500 lMVallee et al. [161] 3% 1 lM 100 nM 250 lM

Table 6Media commercially available for adipogenic differentiation of MSCs.

Company

Invitrogen (Carlsbad, USA)Trevigen (Gaithersburg, USA)Thermo Scientific (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)

and exerts a direct effect on adipogenic differentiation by inducingthe expression of PPARc [190–193]. Matrigel functionalized withFGF-2 has been found to be proadipogenic when injected subcuta-neously alone [188] or in combination with gelatin microspheresinto mice [194]. Within the implantation period, a visible fat padwas formed at the injection site, which is probably due to prea-dipocyte and endothelial cell migration to the injection site[188,195].

The process of adipogenic differentiation of MSCs starts uponcellular monolayer confluence and is induced by the factors men-tioned above. To reach confluence MSCs are often cultured in med-ium containing FCS. However, it has been shown that extendedmaintenance of MSCs in FCS-supplemented medium markedlyand irreversibly reduces the capability for an adipogenic conver-sion to mature adipocytes, most likely due to the effect of serumfactors present in the FCS, e.g. TNF-a or TGF-b [196]. ThereforeHemmrich et al. recommended the use of human serum which,in contrast to FCS, allows substantial proliferation and differentia-tion even after long cell expansion periods [163].

Representative protocols used for in vitro adipogenic differenti-ation of MSCs, including dex, IBMX, insulin, indomethacin, T3 andasc, as well as commercially available culture media, are summa-rized in Tables 5 and 6.

5. Endothelial differentiation

5.1. Background

A major aim in tissue engineering is to ensure adequate vascu-larization of artificial tissue-like constructs. Previous studies haveshowed that vascularization within in vitro engineered tissues

acid; FCS, fetal calf serum; dex, dexamethasone; IBMX, 3-isobutyl-1-methylxanthine;’s jelly).

Indo-methacin Other Cell source

– Transferrin (10 lg ml�1)T3 (200 pM)

cortisol (100 nM)

Human AT

– Transferrin (10 lg ml�1)pioglitazone (100 lg ml�1)T3 (1 nM)

Human AT

50 lM – Rat AT10 mM asc (50 lM) Human BM– T3 (200 pM)

transferring (100 nM)calcium pantotenate (17 mM)biotin (33 mM)

Human AT

200 mM – Rat BM60 mM – Human BM/UCWJ200 lM – Human BM– asc (250 lM)

T3 (200 pM)rosiglitazone (1 lM)

Human AT

Product

STEMPRO� Adipogenesis Differentiation KitMesenchymal Stem Cell Adipogenic Differentiation Kit,HyClone AdvanceSTEM Adipogenic Differentiation KitHuman/Mouse StemXVivo Osteogenic/Adipogenic Base MediaHuman/Mouse StemXVivo Adipogenic SupplementNH AdipoDiff MediumAdipogenic Differentiation MediahMSC adipogenesis induction mediumMesenchymal Stem Cell Adipogenic Differentiation Medium

Table 7Representative protocols used for in vitro endothelial differentiation of MSCs (EGF,epidermal growth factor; FCS, fetal calf serum; VEGF, vascular endothelial growthfactor; BM, bone marrow; UCWJ, umbilical cord Wharton’s jelly).

Reference FCS VEGF Other Cell Source

Jiang et al. [270] – 10 ng ml�1 – Human BMOswald et al. [5] 2% 50 ng ml�1 – Human BMChen et al. [66] 5% 100 ng ml�1 EGF (50 ng ml�1)

hydrocortisone(1 lg ml�1)

Human BM/UCWJ

Table 8Media commercially available for endothelial differentiation of MSCs.

Company Product

PromoCell GmbH(Heidelberg, Germany)

Endothelial Progenitor Medium

Cellular Engineering Technologies Inc.(Coralville, USA)

Endothelial Progenitor CellDifferentiation Media


using mature endothelial cells (ECs) improved blood perfusion andcell viability during and after transplantation [197,198]. Blood ves-sels primarily consist of three cell types. While ECs can be found inthe innermost layer of a vessel, vascular smooth muscle cells(VSMCs) are typically located in the intermediate layer and fibro-blast-like cells in the adventitial outermost layer. However, matureECs and SMCs have limited proliferation potential and their use forgenerating vascular conduits would require laborious autologouscell isolation. In contrast, the fact that MSCs can be easily isolatedand differentiated into ECs makes them a favourable alternativecell source. In addition, the use of allogeneic or even xenogenicMSCs may be feasible without the aid of extended medical immu-nosuppression, thereby further alleviating the availability of MSCsas cellular component for tissue-engineering applications[199,200].


When being differentiated in semi-solid medium (see below)into endothelial cells, MSCs form three-dimensional capillary-liketubular structures (Fig. 2D) and have the ability to take up acety-lated low-density lipoproteins (LDLs) via special receptors. Theincorporation of dye-conjugated LDLs, e.g. 1,10-dioctadecyl-1-3,3,30,30-tetramethyl-indo-carbocyanine perchlorate conjugatedto acetylated-LDL (e.g. DiI-Ac-LDL, Invitrogen, Carlsbad, USA), canbe determined by fluorescence microscopy and fluorescence-acti-vated cell sorting [66,200]. Vascular endothelial growth factor(VEGF)-receptor 2 is one of the two major VEGF receptors and isexpressed during the early stage of endothelial differentiation ofMSCs [201,202]. Markers that characterize later stages of endothe-lial differentiation include tissue-type plasminogen activator, vonWillebrand factor, platelet-endothelial cell adhesion molecule 1(CD31) and vascular endothelial-cadherin [200,202].


A commonly used method to stimulate the differentiation ofMSCs into endothelial cells is the cultivation of these cells in so-called semi-solid medium (e.g. Matrigel™, BD Biosiences, FranklinLakes, USA). The presence of VEGF – the most common supplemen-tary agent to date – markedly enhances endothelial differentiationof MSCs [5]. The VEGF family comprises seven members (i.e. VEGF-A/B/C/D/E/F and placental growth factor) that all have a commonVEGF homology domain. Alternative splicing of the VEGF-A genegenerates six isoforms of VEGF-A composed of 121, 145, 165,183, 189 and 206 amino acids, respectively. Of these, VEGF165 isthe most commonly expressed isoform [203,204]. VEGF-A is typi-cally synthesized by vascular and tumour cells, and has beenshown to be expressed by primary human MSCs as well [200]. Totest whether the MSC-secreted VEGF is able to contribute to angi-ogenesis, Beckermann et al. added supernatant from cultured MSCsto human umbilical vein endothelial cells [205]. This resulted instrong induction of sprouting in similar intensity as observed withthe addition of recombinant VEGF alone. In addition, VEGF inducesvascular hyperpermeability needed, for example, during woundhealing [206] and is known to be a chemoattractant for MSCs[207], for ECs [208,209] and for osteoclasts [210,211]. In the corneaand in bone grafts, VEGF induces growth of capillary sprouts frompreexisting blood vessels [212], whereas osteogenic commitmentis directly prevented by VEGF by inhibition of BMP-2 expression[213].

Other factors that can be used for endothelial differentiation ofMSCs include FGF and platelet-derived growth factor (PDGF). FGFstimulates EC proliferation [190] and migration [214] as well asproduction of plasminogen activator and collagenase, which playa 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 largeand mature blood vessels [216]. However, these angiogenic effectsof FGF contrast with the fact that vascular development is nothampered in FGF-deficient mice, suggesting highly restricted rolesfor FGF under normal developmental and physiological conditions[217].

Within the PDGF family four members have been identified: theclassical PDGFs, PDGF-A and PDGF-B, which have been studiedintensively for more than 20 years; and the novel PDGFs, PDGF-Cand PDGF-D, which were only recently discovered [218]. PDGF-Aand PDGF-B bind as disulphide-linked homo- or heterodimers totheir tyrosine kinase receptors (PDGF receptor a and PDGF recep-tor b) and are physiologically synthesized by platelets, monocytes,macrophages and endothelial cells [219,220]. Capillary endothelialcells 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 differenti-ation of MSCs including VEGF as well as commercially availablemedia are shown in Tables 7 and 8.

6. Vascular smooth muscle differentiation

6.1. Background

VSMCs play an important role in angiogenesis, mechanical sup-port of vessels and blood pressure control [224]. Thus, generating afunctional VSMC layer is a prerequisite for successful constructionof tissue-engineered blood vessels. The replicative ability of autol-ogous VSMCs derived from older donors, representing the majorityof potential recipients of vascular grafts for treatment of cardiovas-cular diseases, is limited [225,226]. Thus, MSCs represent anappealing source of smooth muscle progenitor cells for vascularengineering approaches.


VSMCs demonstrate both a synthetic and a contractile pheno-type. The synthetic phenotype demonstrates fibroblast-likemorphology, high proliferation rate and production of ECMcomprised of collagen, elastin and proteoglycans, which allowsfor elasticity and improved compliance [227]. In contrast, contrac-tile VSMCs are spindle-shaped with a prominent, centrally locatednucleus, proliferate at an extremely low rate but are capable ofcontraction [228]. Contraction is predominantly mediated by


Ca2+-sensitive ion channels [229,230]. Microscopically, contractil-ity can be measured by a collagen gel lattice contraction assay incombination with stimulative agents, e.g. potassium chloride orcarbachol [227,231].

On the molecular level there are several markers commonlyused 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[232–234]. a-SMA and SM22-a are early markers of smooth mus-cle differentiation, whereas calponin, caldesmon and SMMHC aremarkers of late-stage VSMC differentiation. Smoothelin, a cytoskel-etal protein that is only found in contractile VSMCs, is known to bea marker of mature VSMCs [233]. Transcriptional activation of theVSMC marker genes described above is mediated by myocardin,GATA-binding protein 6 (Gata-6) and serum response factor (SRF)[230].


The most potent inducer of VSMC differentiation of MSCs isTGF-b1. During the differentiation process it plays a role in bothinitial 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 genesa-SMA, SM22-a, calponin and SMMHC in MSCs [236,237].SMC-specific transcription is regulated by SRF, which binds toCArG (CC(A=T)6GG) cis elements that are found in the promotersof almost all SMC marker genes [238–241]. Gong et al. showed thata concentration of 0.1–10 ng ml�1 TGF-b1 inhibited human MSCproliferation, but increased calponin expression in a dose-depen-dent manner [234], correlating well with expression levels foundin human coronary arterial smooth muscle cells. TGF-b1 deficiencyis correlated with atherosclerosis and stenosis of the majorcoronary vessels [242]. In contrast to TGF-b1, disruption of theTGF-b2 gene results in 100% mortality just prior to or just afterbirth, and affects developmental processes that are involved in epi-thelial–mesenchymal transitions, cell growth, ECM production andtissue remodeling [243]. These studies indicate that the presenceof all TGF-b isoforms is important for proper maturation of smoothmuscle cells [244].

PDGF is known to be a growth factor influencing VSMC differen-tiation of MSCs. However, the role of PDGF-BB in SMC differentia-

Table 9Representative protocols used for in vitro vascular smooth muscle differentiation of MSCsprotein-4; FCS, fetal calf serum; HGF, hepatocyte growth factor; PDGF, platelet-derived growtissue). Vascular smooth muscle cells demonstrate a synthetic and a contractile phenotypemarker proteins. However, most of the protocols listed below mainly focus on the expressihave also addressed the contraction ability of differentiated MSCs (e.g. Wang et al. [231])

Reference FCS TGF-b1

Kanematsu et al. [271] 0.5% 5 ng ml�1

Ross et al. [230] - 2.5 ng ml�1

Shukla et al. [272] 0.5/5% 5 ng ml�1

Narita et al. [249] 20% 0.1/1/10 ng ml�1

Gong et al. [234] 10% 0.01/0.1/1/10 ng ml�1

Kurpinski et al. [273] 10% 5 ng ml�1

Gong et al. [6] 10% 1 ng ml�1

Harris et al. [227] 13% 2 ng ml�1

Wang et al. [231] 1% 5 ng ml�1

tion is controversial. During embryonic development, PDGF-BB isinvolved in the recruitment of pericytes and SMC precursors to-wards endothelial cells [245]. Postnatally, PDGF-BB is associatedwith SMC proliferation, e.g. in response to vascular injury and ath-erosclerosis [224]. Thus, PDGF-BB appears to be responsible forphenotypic modulation and dedifferentiation of SMCs in vivo.There is also evidence that PDGF-BB suppresses expression ofmarkers consistent with terminal differentiation in vitro, includingSM22-a and SMMHC [246], and induces a proliferative and syn-thetic VSMC phenotype [224]. However, studies by Dandre et al.show that PDGF-BB-induced inhibition of SMC marker expressionis only observed in low-density cultures but not when cells aregrowth-arrested by culture at subconfluent density in serum-freeconditions or when maintained at high cell density in serum-richconditions [246].

In addition to TGF-b1 and PDGF-BB, factors such as angiotensinII [247], sphingosylphosphorylcholine [248] and ascorbic acid[249] have also been applied for VSMC differentiation. Further-more, close cell-to-cell contact is required for VSMC differentiation[250].

Several protocols are available for the differentiation of MSCsinto VSMCs. Representative protocols for this, including TGF-b1and PDGF-BB, are shown in Table 9. Selected commercially avail-able 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 bonemarrow, adipose tissue, umbilical cord blood, placental tissue[251], liver, spleen [20], testes [19], menstrual blood, amniotic fluid[17], pancreas [16], dermis [252], dental pulp [253], periosteum[254] and even lung [255]. Within these, bone marrow is believedto be the enriched reservoir of MSCs and the major source for theseprecursor 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 tobe an attractive alternative to MSCs from bone marrow. However,in terms of their osteogenic potential, adipose tissue-derived MSCsseem to be inferior to bone-marrow-derived MSCs [199]. For MSCsfrom umbilical cord blood (UCB) conflicting results in terms of

(asc, ascorbic acid; bFGF, basic fibroblast growth factor; BMP-4, bone morphogeneticth factor; TGF-b1, transforming growth factor-beta 1; BM, bone marrow; AT, adiposethat is characterized by morphology, proliferation rate, and the expression of special

on of smooth muscle-specific genes and proteins, respectively. Only a few researchers.

Other Cell source

HGF (40 ng ml�1) Rat BMPDGF-BB (0–100 ng ml�1) Murine/rat/human/porcine BMPDGF-BB (25 ng ml�1) Porcine BMasc (30/300 lM) Human BMPDGF-BB (10 ng ml�1)PDGF-CC (10 ng ml�1)bFGF (10 ng ml�1)asc (50 lg ml�1)

Human BM

– Human MSCs (Cambrex)Copper sulfate (3 ng ml�1)proline (50 mg ml�1)glycine (50 mg ml�1)ascorbic acid (50 mg ml�1)alanine (20 mg ml�1)

Human BM

Heparin (7.5 U ml�1)angiotensin II (2 lM)sphingosylphosphorylcholine (2 lM)

Human AT

BMP-4 (2.5 ng ml�1) Human AT

Table 10Media commercially available for vascular smooth muscle differentiation of MSCs.

Company Product

PromoCell GmbH(Heidelberg, Germany)

Smooth Muscle Cell Growth Medium 2

Invitrogen (Carlsbad, USA) Medium 231 Smooth MuscleDifferentiation Supplement (SMDS)

BD Biosciences (Bedford, USA) SMC Differentiation Medium


success rate of MSC isolation have been initially reported, probablybecause of their low frequency in UCB [257,258].

In general, MSCs are a minor fraction in bone marrow and othertissues; the exact frequency is difficult to calculate because of thedifferent methods of harvest and separation. However, the fre-quency in human bone marrow has been estimated to be of the or-der of 0.001–0.01% of the total nucleated cells, and therefore about10-fold less abundant than haematopoietic stem cells. Further-more, the frequency of MSCs declines with age, from 1/10,000nucleated marrow cells in a newborn to about 1/10,00,000 nucle-ated marrow cells in a 80 year old person [259].

7.2. Use of FCS

Most of the protocols used today recommend the use of FCS-supplemented medium for the differentiation of MSCs because ofthe nutrients and growth factors present in the FCS. While MSCsserum concentrations in the range of 10–20% are used forexpansion, differentiation of MSCs occurs mainly in medium sup-plemented with 2–10% FCS. However, there is a considerablebatch-to-batch variation within the FCS, which may result in a sig-nificant variation in MSC properties and differentiation behavior,respectively. Therefore it is important to perform a FCS batch testprior to a study to find the batch that is most suitable for therespective experiment. In addition, it must be kept in mind thatFCS is an undesirable additive to cells that are expanded and differ-entiated for therapeutic purposes in humans because the use ofFCS carries the risk of transmitting viral and prion diseases andproteins that may initiate xenogeneic immune responses. Becauseof this, various alternatives have been considered, including, forexample, (autologous) human platelet lysates or serum [260,261]as well as formulated defined media.

8. Summary

MSCs are a promising source of precursor cells which may be ap-plied in various tissue-engineering strategies. By using differentia-tion-specific protocols, MSCs can be induced to differentiatetowards a variety of mature target cells. In this context, the compo-sition of the cell culture media used is crucial. Currently, differentcommercial culture media with or without FCS are available forMSC differentiation. More importantly, however, particular supple-mentary agents need to be added to the media for lineage-specificdifferentiation of MSCs. In general, culture supplements requiredfor osteogenic differentiation include dex, b-GP, asc, vitD3, TGF-band BMPs. Chondrogenic differentiation of MSCs is achieved bygrowing cells in pellet with media containing dex, asc, TGF-b, BMPsand IGF. Supplements including dex, IBMX, insulin, indomethacinand T3 have been shown to be effective in adipogenic differentiation.In order to induce MSC differentiation towards endothelial cell line-age VEGF is routinely used as supplementary agent. MSC differenti-ation towards the VSMC phenotype is achieved by stimulating thecells with TGF-b1 and PDGF-BB.

In practice, the combination of FCS and these supplementaryagents such as growth factors and cytokines and their concentra-tion should be optimized to meet each individual need.

Disclosure of interests

The authors indicate no potential conflict 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 assistingwith cell culture and histology. M.S. and C.V. are financially sup-ported by the German Academic Exchange Service/German FederalMinistry of Education and Research (Grant No. D/09/04774) and bythe Center for Regenerative Therapies Dresden, Germany (SeedGrant No. 09-08).

Appendix A. Figures with essential colour discrimination

Certain figures in this article, particularly Figures 1 and 2, are dif-ficult to interpret in black and white. The full colour images can befound in the on-line version, at doi:10.1016/j.actbio.2010.07.037.

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culture media for the differentiation of mesenchymal stromal cells

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