Human mesenchymal stromal cells (hMSCs) have generated significant interest due to their potential use in clinical applications. hMSCs are present at low frequency in vivo, but after isolation can be expanded considerably, generating clinically useful numbers of cells. In this study, we demonstrate the use of a defined embryonic stem cell expansion medium, mTeSR (Stem Cell Technologies), for the expansion of bone-marrow-derived hMSCs. The hMSCs grow at comparable rates, demonstrate tri-lineage differentiation potential, and show similar surface marker profiles (CD29+, CD44+, CD49a+, CD73+, CD90+, CD105+, CD146+, CD166+, CD34−, and CD45−) in both the fetal bovine serum (FBS)-supplemented medium and mTeSR. However, expression of early differentiation transcription factors runt-related transcription factor 2, sex-determining region Y box 9, and peroxisome proliferator-activated receptor gamma changed significantly. Both runt-related transcription factor 2 and sex-determining region Y box 9 were upregulated, whereas peroxisome proliferator-activated receptor gamma was downregulated in mTeSR compared with FBS. Although osteogenic and chondrogenic differentiation was comparable in cells grown in mTeSR compared to FBS, adipogenic differentiation was significantly decreased in mTeSR-expanded cells, both in terms of gene expression and absolute numbers of adipocytes. The removal of the FBS from the medium and the provision of a defined medium with disclosed composition make mTeSR a superior study platform for hMSC biology in a controlled environment. Further, this provides a key step toward generating a clinical-grade medium for expansion of hMSCs for clinical applications that rely on osteo- and chondroinduction of MSCs, such as bone repair and cartilage generation.
RenG, ZhangL, ZhaoX, XuG, ZhangY, RobertsAI, ZhaoRC, ShiY. 2008. Mesenchymal stem cell-mediated immunosuppression occurs via concerted action of chemokines and nitric oxide cell. Stem Cell, 2:141–150.
4.
MaitraB, SzekelyE, GjiniK, LaughlinMJ, DennisJ, HaynesworthSE, KocON. 2004. Human mesenchymal stem cells support unrelated donor hematopoietic stem cells and suppress T-cell activation. Bone Marrow Transplant, 33:597–604.
5.
SegersVFM, LeeRT. 2008. Stem-cell therapy for cardiac disease. Nature, 451:937–942.
6.
MurryCE, FieldLJ, MenascheP. 2005. Cell-based cardiac repair: reflections at the 10-year point. Circulation, 112:3174–3183.
7.
Le BlancK, SamuelssonH, GustafssonB, RembergerM, SundbergB, ArvidsonJ, LjungmanP, LonniesH, NavaS, RingdenO. 2007. Transplantation of mesenchymal stem cells to enhance engraftment of hematopoietic stem cells. Leukemia, 21:1733–1738.
8.
KocON, GersonSL, CooperBW, DyhouseSM, HaynesworthSE, CaplanAI, LazarusHM. 2000. Rapid hematopoietic recovery after coinfusion of autologous-blood stem cells and culture-expanded marrow mesenchymal stem cells in advanced breast cancer patients receiving high-dose chemotherapy. J Clin Oncol, 18:307–316.
9.
CovasDT, PanepucciRA, FontesAM, SilvaWAJr., OrellanaMD, FreitasMCC, NederL, SantosARD, PeresLC, JamurMC, ZagoMA. 2008. Multipotent mesenchymal stromal cells obtained from diverse human tissues share functional properties and gene-expression profile with CD146+ perivascular cells and fibroblasts. Exp Hematol, 36:642–654.
10.
SchallmoserK, RohdeE, ReinischA, BartmannC, ThalerD, DrexlerC, ObenaufAC, LanzerG, LinkeschW, StrunkD. 2008. Rapid large-scale expansion of functional mesenchymal stem cells from unmanipulated bone marrow without animal serum. Tissue Eng Part C Methods, 14:185–196.
11.
SakamotoN, TsujiK, MuulLM, LawlerAM, PetricoinEF, CandottiF, MetcalfJA, TavelJA, LaneHC, UrbaWJ, FoxBA, VarkiA, LunneyJK, RosenbergAS. 2007. Bovine apolipoprotein B-100 is a dominant immunogen in therapeutic cell populations cultured in fetal calf serum in mice and humans. Blood, 110:501–508.
12.
SundinM, RingdenO, SundbergB, NavaS, GotherstromC, Le BlancK. 2007. No alloantibodies against mesenchymal stromal cells, but presence of anti-fetal calf serum antibodies, after transplantation in allogeneic hematopoietic stem cell recipients. Haematologica, 92:1208–1215.
13.
SelvaggiTA, WalkerRE, FleisherTA. 1997. Development of antibodies to fetal calf serum with arthus-like reactions in human immunodeficiency virus-infected patients given syngeneic lymphocyte infusions. Blood, 89:776–779.
14.
CapelliC, DomenghiniM, BorleriG, BellavitaP, PomaR, CarobbioA, MicoC, RambaldiA, GolayJ, IntronaM. 2007. Human platelet lysate allows expansion and clinical grade production of mesenchymal stromal cells from small samples of bone marrow aspirates or marrow filter washouts. Bone Marrow Transplant, 40:785–791.
15.
DoucetC, ErnouI, ZhangY, LlenseJ-R, BegotL, HolyX, LatailladeJ-J. 2005. Platelet lysates promote mesenchymal stem cell expansion: a safety substitute for animal serum in cell-based therapy applications. J Cell Physiol, 205:228–236.
16.
LangeC, CakirogluF, SpiessA-N, Cappallo-ObermannH, DierlammJ, ZanderAR. 2007. Accelerated and safe expansion of human mesenchymal stromal cells in animal serum-free medium for transplantation and regenerative medicine. J Cell Physiol, 213:18–26.
17.
SchallmoserK, BartmannC, RohdeE, ReinischA, KashoferK, StadelmeyerE, DrexlerC, LanzerG, LinkeschW, StrunkD. 2007. Human platelet lysate can replace fetal bovine serum for clinical-scale expansion of functional mesenchymal stromal cells. Transfusion, 47:1436–1446.
18.
KocaoemerA, KernS, KlüterH, BiebackK. 2007. Human AB serum and thrombin-activated platelet-rich plasma are suitable alternatives to fetal calf serum for the expansion of mesenchymal stem cells from adipose tissue. Stem Cells, 25:1270–1278.
19.
TurnovcovaK, RuzickovaK, VanecekV, SykovaE, JendelovaP. 2009. Properties and growth of human bone marrow mesenchymal stromal cells cultivated in different media. Cytotherapy, 11:874–885.
20.
GronthosS, SimmonsPJ. 1995. The growth factor requirements of STRO-1-positive human bone marrow stromal precursors under serum-deprived conditions in vitro. Blood, 85:929–940.
21.
ApelA, GrothA, SchlesingerS, BrunsH, SchemmerP, BüchlerMW, HerrI. 2009. Suitability of human mesenchymal stem cells for gene therapy depends on the expansion medium. Exp Cell Res, 315:498–507.
22.
NgF, BoucherS, KohS, SastryKSR, ChaseL, LakshmipathyU, ChoongC, YangZ, VemuriMC, RaoMS, TanavdeV. 2008. PDGF, TGF-{beta}, and FGF signaling is important for differentiation and growth of mesenchymal stem cells (MSCs): transcriptional profiling can identify markers and signaling pathways important in differentiation of MSCs into adipogenic, chondrogenic, and osteogenic lineages. Blood, 112:295–307.
23.
BattulaVL, BareissPM, TremlS, ConradS, AlbertI, HojakS, AbeleH, ScheweB, JustL, SkutellaT, BühringH-J. 2007. Human placenta and bone marrow derived MSC cultured in serum-free, b-FGF-containing medium express cell surface frizzled-9 and SSEA-4 and give rise to multilineage differentiation. Differentiation, 75:279–291.
24.
BianchiG, BanfiA, MastrogiacomoM, NotaroR, LuzzattoL, CanceddaR, QuartoR. 2003. Ex vivo enrichment of mesenchymal cell progenitors by fibroblast growth factor 2. Exp Cell Res, 287:98–105.
25.
MartinI, MuragliaA, CampanileG, CanceddaR, QuartoR. 1997. Fibroblast growth factor-2 supports ex vivo expansion and maintenance of osteogenic precursors from human bone marrow. Endocrinology, 138:4456–4462.
26.
MuragliaA, CanceddaR, QuartoR. 2000. Clonal mesenchymal progenitors from human bone marrow differentiate in vitro according to a hierarchical model. J Cell Sci, 113:1161–1166.
27.
ZaragosiL-E, AilhaudG, DaniC. 2006. Autocrine fibroblast growth factor 2 signaling is critical for self-renewal of human multipotent adipose-derived stem cells. Stem Cells, 24:2412–2419.
28.
SolchagaLA, PenickK, PorterJD, GoldbergVM, CaplanAI, WelterJF. 2005. FGF-2 enhances the mitotic and chondrogenic potentials of human adult bone marrow-derived mesenchymal stem cells. J Cell Physiol, 203:398–409.
29.
LudwigTE, LevensteinME, JonesJM, BerggrenWT, MitchenER, FraneJL, CrandallLJ, DaighCA, ConardKR, PiekarczykMS, LlanasRA, ThomsonJA. 2006. Derivation of human embryonic stem cells in defined conditions. Nat Biotechnol, 24:185–187.
30.
LudwigTE, BergendahlV, LevensteinME, YuJ, ProbascoMD, ThomsonJA. 2006. Feeder-independent culture of human embryonic stem cells. Nat Methods, 3:637–646.
31.
SongL, WebbNE, SongY, TuanRS. 2006. Identification and functional analysis of candidate genes regulating mesenchymal stem cell self-renewal and multipotency. Stem Cells, 24:1707–1718.
32.
NethP, CiccarellaM, EgeaV, HoeltersJ, JochumM, RiesC. 2006. Wnt signaling regulates the invasion capacity of human mesenchymal stem cells. Stem Cells, 24:1892–1903.
33.
EtheridgeSL, SpencerGJ, HeathDJ, GeneverPG. 2004. Expression profiling and functional analysis of wnt signaling mechanisms in mesenchymal stem cells. Stem Cells, 22:849–860.
SchubertD, KimuraH. 1991. Substratum-growth factor collaborations are required for the mitogenic activities of activin and FGF on embryonal carcinoma cells. J Cell Biol, 114:841–846.
36.
GronthosS, SimmonsPJ, GravesSE, RobeyPG. 2001. Integrin-mediated interactions between human bone marrow stromal precursor cells and the extracellular matrix. Bone, 28:174–181.
37.
GronthosS, ZannettinoACW, HaySJ, ShiS, GravesSE, KortesidisA, SimmonsPJ. 2003. Molecular and cellular characterisation of highly purified stromal stem cells derived from human bone marrow. J Cell Sci, 116,Pt 9:1827–1835.
38.
SimmonsPJ, Torok-StorbB. 1991. Identification of stromal cell precursors in human bone marrow by a novel monoclonal antibody, STRO-1. Blood, 78:55–62.
39.
RiderD, NalathambyT, NurcombeV, CoolS. 2007. Selection using the alpha-1 integrin (CD49a) enhances the multipotentiality of the mesenchymal stem cell population from heterogeneous bone marrow stromal cells. J Mol Histol, 38:449–458.
40.
GangEJ, BosnakovskiD, FigueiredoCA, VisserJW, PerlingeiroRCR. 2007. SSEA-4 identifies mesenchymal stem cells from bone marrow. Blood, 109:1743–1751.
41.
TaipaleJ, Keski-OjaJ. 1997. Growth factors in the extracellular matrix. FASEB J, 11:51–59.
42.
ZhouG, ZhengQ, EnginF, MunivezE, ChenY, SebaldE, KrakowD, LeeB. 2006. Dominance of SOX9 function over RUNX2 during skeletogenesis. Proc Natl Acad Sci USA, 103:19004–19009.
43.
JaquiéryC, SchaerenS, FarhadiJ, Mainil-VarletP, KunzC, ZeilhoferH-F, HebererM, MartinI. 2005. In vitro osteogenic differentiation and in vivo bone-forming capacity of human isogenic jaw periosteal cells and bone marrow stromal cells. Ann Surg, 242:859–868.
44.
BiancoP, KuznetsovSA, RiminucciM, Gehron RobeyP. 2006. Postnatal skeletal stem cells. Methods in Enzymology. KlimanskayaI, LanzaR. Academic Press: San Diego, CA, 117–148.
45.
MartinI. et al.2001. Quantitative analysis of gene expression in human articular cartilage from normal and osteoarthritic joints. Osteoarthritis Cartilage, 9:112–118.
46.
SelvamuruganN, KwokS, PartridgeNC. 2004. Smad3 Interacts with JunB and Cbfa1/Runx2 for transforming growth factor-Î1-stimulated collagenase-3 expression in human breast cancer cells. J Biol Chem, 279:27764–27773.
47.
FrithJE, ThomsonB, GeneverP. 2009. Dynamic three-dimensional culture methods enhance mesenchymal stem cell properties and increase therapeutic potential. Tissue Eng Part C Methods[Epub ahead of print].
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