Impact of source tissue and ex vivo expansion on the characterization of goat mesenchymal stem cells

Journal of Animal Science and Biotechnology

James D Murray³  Elizabeth A Maga¹  Pablo J Ross¹  Nuradilla Mohamad-Fauzi² 

[1] Department of Animal Science, University of California, Davis, California 95616, USA;Institute of Ocean and Earth Sciences, University of Malaya, 50603 Kuala Lumpur, Malaysia;Department of Population Health and Reproduction, University of California, Davis, California 95616, USA

关键词: Mesenchymal stem cells;    Goat;    Differentiation;    Characterization;    Bone marrow;    Adipose;   

Others  :  1135812
DOI  :  10.1186/2049-1891-6-1

 received in 2014-08-28, accepted in 2014-12-18,  发布年份 2015

【 摘 要 】

Background

There is considerable interest in using goats as models for genetically engineering dairy animals and also for using stem cells as therapeutics for bone and cartilage repair. Mesenchymal stem cells (MSCs) have been isolated and characterized from various species, but are poorly characterized in goats.

Results

Goat MSCs isolated from bone marrow (BM-MSCs) and adipose tissue (ASCs) have the ability to undergo osteogenic, adipogenic and chondrogenic differentiation. Cytochemical staining and gene expression analysis show that ASCs have a greater capacity for adipogenic differentiation compared to BM-MSCs and fibroblasts. Different methods of inducing adipogenesis also affect the extent and profile of adipogenic differentiation in MSCs. Goat fibroblasts were not capable of osteogenesis, hence distinguishing them from the MSCs. Goat MSCs and fibroblasts express CD90, CD105, CD73 but not CD45, and exhibit cytoplasmic localization of OCT4 protein. Goat MSCs can be stably transfected by Nucleofection, but, as evidenced by colony-forming efficiency (CFE), yield significantly different levels of progenitor cells that are robust enough to proliferate into colonies of integrants following G418 selection. BM-MSCs expanded over increasing passages in vitro maintained karyotypic stability up to 20 passages in culture, exhibited an increase in adipogenic differentiation and CFE, but showed altered morphology and amenability to genetic modification by selection.

Conclusions

Our findings provide characterization information on goat MSCs, and show that there can be significant differences between MSCs isolated from different tissues and from within the same tissue. Fibroblasts do not exhibit trilineage differentiation potential at the same capacity as MSCs, making it a more reliable method for distinguishing MSCs from fibroblasts, compared to cell surface marker expression.

【 授权许可】

   
2015 Mohamad-Fauzi et al.; licensee BioMed Central.

【 图 表 】

Figure 1.

Figure 2.

Figure 3.

Figure 4.

Figure 5.

Figure 6.

Figure 7.

Figure 8.

Figure 9.

Figure 10.

【 参考文献 】

• [1]da Silva Meirelles L, Chagastelles PC, Nardi NB: Mesenchymal stem cells reside in virtually all post-natal organs and tissues. J Cell Sci 2006, 119:2204-13.
• [2]Caplan AI: Mesenchymal stem cells. J Orthop Res 1991, 9:641-50.
• [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, 284:143-7.
• [4]Zuk PA, Zhu M, Mizuno H, Huang J, Futrell JW, Katz AJ, et al.: Multilineage cells from human adipose tissue: implications for cell-based therapies. Tissue Eng 2001, 7:211-28.
• [5]Dominici M, Le Blanc K, Mueller I, Slaper-Cortenbach I, Marini F, Krause D, et al.: Minimal criteria for defining multipotent mesenchymal stromal cells. The International Society for Cellular Therapy position statement. Cytotherapy 2006, 8:315-7.
• [6]Koga H, Muneta T, Nagase T, Nimura A, Ju YJ, Mochizuki T, et al.: Comparison of mesenchymal tissues-derived stem cells for in vivo chondrogenesis: suitable conditions for cell therapy of cartilage defects in rabbit. Cell Tissue Res 2008, 333:207-15.
• [7]Im GI, Shin YW, Lee KB: Do adipose tissue-derived mesenchymal stem cells have the same osteogenic and chondrogenic potential as bone marrow-derived cells? Osteoarthritis Cartilage 2005, 13:845-53.
• [8]Monaco E, Sobreira de Lima A, Bionaz M, Maki A, Wilson SM, Hurley WL, et al.: Morphological and transcriptomic comparison of adipose and bone marrow derived porcine stem cells. Open Tissue Eng Regen Med J 2009, 2:20-33.
• [9]Spencer ND, Chun R, Vidal MA, Gimble JM, Lopez MJ: In vitro expansion and differentiation of fresh and revitalized adult canine bone marrow-derived and adipose tissue-derived stromal cells. Vet J 2012, 191:231-9.
• [10]Bourin P, Bunnell BA, Casteilla L, Dominici M, Katz AJ, March KL, et al.: Stromal cells from the adipose tissue-derived stromal vascular fraction and culture expanded adipose tissue-derived stromal/stem cells: a joint statement of the International Federation for Adipose Therapeutics and Science (IFATS) and the International Society for Cellular Therapy (ISCT). Cytotherapy 2013, 15:641-8.
• [11]De Ugarte DA, Alfonso Z, Zuk PA, Elbarbary A, Zhu M, Ashjian P, et al.: Differential expression of stem cell mobilization-associated molecules on multi-lineage cells from adipose tissue and bone marrow. Immunol Lett 2003, 89:267-70.
• [12]Proffen BL, McElfresh M, Fleming BC, Murray MM: A comparative anatomical study of the human knee and six animal species. Knee 2012, 19:493-9.
• [13]Liu X, Li X, Fan Y, Zhang G, Li D, Dong W, et al.: Repairing goat tibia segmental bone defect using scaffold cultured with mesenchymal stem cells. J Biomed Mater Res B Appl Biomater 2010, 94:44-52.
• [14]Vertenten G, Lippens E, Girones J, Gorski T, Declercq H, Saunders J, et al.: Evaluation of an injectable, photopolymerizable, and three-dimensional scaffold based on methacrylate-endcapped poly(D, L-lactide-co-epsilon-caprolactone) combined with autologous mesenchymal stem cells in a goat tibial unicortical defect model. Tissue Eng A 2009, 15:1501-11.
• [15]Coburn JM, Gibson M, Monagle S, Patterson Z, Elisseeff JH: Bioinspired nanofibers support chondrogenesis for articular cartilage repair. Proc Natl Acad Sci U S A 2012, 109:10012-7.
• [16]Toh WS, Lim TC, Kurisawa M, Spector M: Modulation of mesenchymal stem cell chondrogenesis in a tunable hyaluronic acid hydrogel microenvironment. Biomaterials 2012, 33:3835-45.
• [17]Murphy JM, Fink DJ, Hunziker EB, Barry FP: Stem cell therapy in a caprine model of osteoarthritis. Arthritis Rheum 2003, 48:3464-74.
• [18]Zhu S, Zhang B, Man C, Ma Y, Hu J: NEL-like molecule-1-modified bone marrow mesenchymal stem cells/poly lactic-co-glycolic acid composite improves repair of large osteochondral defects in mandibular condyle. Osteoarthritis Cartilage 2011, 19:743-50.
• [19]Sun XD, Jeng L, Bolliet C, Olsen BR, Spector M: Non-viral endostatin plasmid transfection of mesenchymal stem cells via collagen scaffolds. Biomaterials 2009, 30:1222-31.
• [20]Ren Y, Wu H, Zhou X, Wen J, Jin M, Cang M, et al.: Isolation, expansion, and differentiation of goat adipose-derived stem cells. Res Vet Sci 2012, 93:404-11.
• [21]Knippenberg M, Helder MN, Doulabi BZ, Semeins CM, Wuisman PI, Klein-Nulend J: Adipose tissue-derived mesenchymal stem cells acquire bone cell-like responsiveness to fluid shear stress on osteogenic stimulation. Tissue Eng 2005, 11:1780-8.
• [22]Qiu P, Bai Y, Liu C, He X, Cao H, Li M, et al.: A dose-dependent function of follicular fluid on the proliferation and differentiation of umbilical cord mesenchymal stem cells (MSCs) of goat. Histochem Cell Biol 2012, 138:593-603.
• [23]Azari O, Babaei H, Derakhshanfar A, Nematollahi-Mahani SN, Poursahebi R, Moshrefi M: Effects of transplanted mesenchymal stem cells isolated from Wharton’s jelly of caprine umbilical cord on cutaneous wound healing; histopathological evaluation. Vet Res Commun 2011, 35:211-22.
• [24]Hematti P: Mesenchymal stromal cells and fibroblasts: a case of mistaken identity? Cytotherapy 2012, 14:516-21.
• [25]Alt E, Yan Y, Gehmert S, Song YH, Altman A, Gehmert S, et al.: Fibroblasts share mesenchymal phenotypes with stem cells, but lack their differentiation and colony-forming potential. Biol Cell 2011, 103:197-208.
• [26]Potchoiba MJ, Lu CD, Pinkerton F, Sahlu T: Effects of all-milk diet on weight gain, organ development, carcass characteristics and tissue composition, including fatty acids and cholestrol contents, of growing male goats. Small Rumin Res 1990, 3:583-92.
• [27]Hausman GJ, Dodson MV, Ajuwon K, Azain M, Barnes KM, Guan LL, et al.: Board-invited review: the biology and regulation of preadipocytes and adipocytes in meat animals. J Anim Sci 2009, 87:1218-46.
• [28]Du M, Yin J, Zhu MJ: Cellular signaling pathways regulating the initial stage of adipogenesis and marbling of skeletal muscle. Meat Sci 2010, 86:103-9.
• [29]Izadpanah R, Trygg C, Patel B, Kriedt C, Dufour J, Gimble JM, et al.: Biologic properties of mesenchymal stem cells derived from bone marrow and adipose tissue. J Cell Biochem 2006, 99:1285-97.
• [30]Wagner W, Horn P, Castoldi M, Diehlmann A, Bork S, Saffrich R, et al.: Replicative senescence of mesenchymal stem cells: a continuous and organized process. PLoS One 2008, 3:e2213.
• [31]Halfon S, Abramov N, Grinblat B, Ginis I: Markers distinguishing mesenchymal stem cells from fibroblasts are downregulated with passaging. Stem Cells Dev 2011, 20:53-66.
• [32]Tsai CC, Su PF, Huang YF, Yew TL, Hung SC: Oct4 and Nanog directly regulate Dnmt1 to maintain self-renewal and undifferentiated state in mesenchymal stem cells. Mol Cell 2012, 47:169-82.
• [33]Kretlow JD, Jin YQ, Liu W, Zhang WJ, Hong TH, Zhou G, et al.: Donor age and cell passage affects differentiation potential of murine bone marrow-derived stem cells. BMC Cell Biol 2008, 9:60. BioMed Central Full Text
• [34]Pittenger MF: Mesenchymal stem cells from adult bone marrow. Methods Mol Biol 2008, 449:27-44.
• [35]Behboodi E, Bondareva A, Begin I, Rao K, Neveu N, Pierson JT, et al.: Establishment of goat embryonic stem cells from in vivo produced blastocyst-stage embryos. Mol Reprod Dev 2011, 78:202-11.
• [36]He S, Pant D, Schiffmacher A, Bischoff S, Melican D, Gavin W, et al.: Developmental expression of pluripotency determining factors in caprine embryos: novel pattern of NANOG protein localization in the nucleolus. Mol Reprod Dev 2006, 73:1512-22.
• [37]Li JW, Guo XL, He CL, Tuo YH, Wang Z, Wen J, et al.: In vitro chondrogenesis of the goat bone marrow mesenchymal stem cells directed by chondrocytes in monolayer and 3-dimetional indirect co-culture system. Chin Med J 2011, 124:3080-6.
• [38]Wang YH, Bower NI, Reverter A, Tan SH, De Jager N, Wang R, et al.: Gene expression patterns during intramuscular fat development in cattle. J Anim Sci 2009, 87:119-30.
• [39]Wei S, Zan LS, Wang HB, Cheng G, Du M, Jiang Z, et al.: Adenovirus-mediated interference of FABP4 regulates mRNA expression of ADIPOQ, LEP and LEPR in bovine adipocytes. Genet Mol Res 2013, 12:494-505.
• [40]Livak KJ, Schmittgen TD: Analysis of relative gene expression data using real-time quantitative PCR and the 2(−Delta Delta C(T)) Method. Methods 2001, 25:402-8.
• [41]Schmittgen TD, Livak KJ: Analyzing real-time PCR data by the comparative C(T) method. Nat Protoc 2008, 3:1101-8.
• [42]Khan DR, Dube D, Gall L, Peynot N, Ruffini S, Laffont L, et al.: Expression of pluripotency master regulators during two key developmental transitions: EGA and early lineage specification in the bovine embryo. PLoS One 2012, 7:e34110.
• [43]Cauffman G, Liebaers I, Van Steirteghem A, Van de Velde H: POU5F1 isoforms show different expression patterns in human embryonic stem cells and preimplantation embryos. Stem Cells 2006, 24:2685-91.
• [44]Kern S, Eichler H, Stoeve J, Kluter H, Bieback K: Comparative analysis of mesenchymal stem cells from bone marrow, umbilical cord blood, or adipose tissue. Stem Cells 2006, 24:1294-301.
• [45]Lorenz K, Sicker M, Schmelzer E, Rupf T, Salvetter J, Schulz-Siegmund M, et al.: Multilineage differentiation potential of human dermal skin-derived fibroblasts. Exp Dermatol 2008, 17:925-32.
• [46]Sorensen AL, Jacobsen BM, Reiner AH, Andersen IS, Collas P: Promoter DNA methylation patterns of differentiated cells are largely programmed at the progenitor stage. Mol Biol Cell 2010, 21:2066-77.
• [47]Boquest AC, Noer A, Collas P: Epigenetic programming of mesenchymal stem cells from human adipose tissue. Stem Cell Rev 2006, 2:319-29.
• [48]Aldridge A, Kouroupis D, Churchman S, English A, Ingham E, Jones E: Assay validation for the assessment of adipogenesis of multipotential stromal cells–a direct comparison of four different methods. Cytotherapy 2013, 15:89-101.
• [49]Chen FG, Zhang WJ, Bi D, Liu W, Wei X, Chen FF, et al.: Clonal analysis of nestin(−) vimentin(+) multipotent fibroblasts isolated from human dermis. J Cell Sci 2007, 120:2875-83.
• [50]Delikatny EJ, Chawla S, Leung DJ, Poptani H: MR-visible lipids and the tumor microenvironment. NMR Biomed 2011, 24:592-611.
• [51]Bosch P, Pratt SL, Stice SL: Isolation, characterization, gene modification, and nuclear reprogramming of porcine mesenchymal stem cells. Biol Reprod 2006, 74:46-57.
• [52]Vacanti V, Kong E, Suzuki G, Sato K, Canty JM, Lee T: Phenotypic changes of adult porcine mesenchymal stem cells induced by prolonged passaging in culture. J Cell Physiol 2005, 205:194-201.
• [53]Izadpanah R, Joswig T, Tsien F, Dufour J, Kirijan JC, Bunnell BA: Characterization of multipotent mesenchymal stem cells from the bone marrow of rhesus macaques. Stem Cells Dev 2005, 14:440-51.
• [54]Jaiswal RK, Jaiswal N, Bruder SP, Mbalaviele G, Marshak DR, Pittenger MF: Adult human mesenchymal stem cell differentiation to the osteogenic or adipogenic lineage is regulated by mitogen-activated protein kinase. J Biol Chem 2000, 275:9645-52.
• [55]Beresford JN, Bennett JH, Devlin C, Leboy PS, Owen ME: Evidence for an inverse relationship between the differentiation of adipocytic and osteogenic cells in rat marrow stromal cell cultures. J Cell Sci 1992, 102(Pt 2):341-51.
• [56]Zhao LJ, Jiang H, Papasian CJ, Maulik D, Drees B, Hamilton J, et al.: Correlation of obesity and osteoporosis: effect of fat mass on the determination of osteoporosis. J Bone Miner Res 2008, 23:17-29.
• [57]Lecka-Czernik B, Moerman EJ, Grant DF, Lehmann JM, Manolagas SC, Jilka RL: Divergent effects of selective peroxisome proliferator-activated receptor-gamma 2 ligands on adipocyte versus osteoblast differentiation. Endocrinology 2002, 143:2376-84.
• [58]Schellenberg A, Stiehl T, Horn P, Joussen S, Pallua N, Ho AD, et al.: Population dynamics of mesenchymal stromal cells during culture expansion. Cytotherapy 2012, 14:401-11.
• [59]Pochampally R: Colony forming unit assays for MSCs. Methods Mol Biol 2008, 449:83-91.
• [60]Russell KC, Phinney DG, Lacey MR, Barrilleaux BL, Meyertholen KE, O’Connor KC: In vitro high-capacity assay to quantify the clonal heterogeneity in trilineage potential of mesenchymal stem cells reveals a complex hierarchy of lineage commitment. Stem Cells 2010, 28:788-98.
• [61]Digirolamo CM, Stokes D, Colter D, Phinney DG, Class R, Prockop DJ: Propagation and senescence of human marrow stromal cells in culture: a simple colony-forming assay identifies samples with the greatest potential to propagate and differentiate. Br J Haematol 1999, 107:275-81.
• [62]Eslaminejad MB, Nadri S: Murine mesenchymal stem cell isolated and expanded in low and high density culture system: surface antigen expression and osteogenic culture mineralization. In Vitro Cell Dev Biol Anim 2009, 45:451-9.
• [63]Meirelles Lda S, Nardi NB: Murine marrow-derived mesenchymal stem cell: isolation, in vitro expansion, and characterization. Br J Haematol 2003, 123:702-11.
• [64]Lapi S, Nocchi F, Lamanna R, Passeri S, Iorio M, Paolicchi A, et al.: Different media and supplements modulate the clonogenic and expansion properties of rabbit bone marrow mesenchymal stem cells. BMC Res Notes 2008, 1:53. BioMed Central Full Text
• [65]Kisselbach L, Merges M, Bossie A, Boyd A: CD90 Expression on human primary cells and elimination of contaminating fibroblasts from cell cultures. Cytotechnology 2009, 59:31-44.
• [66]Ishii M, Koike C, Igarashi A, Yamanaka K, Pan H, Higashi Y, et al.: Molecular markers distinguish bone marrow mesenchymal stem cells from fibroblasts. Biochem Biophys Res Commun 2005, 332:297-303.
• [67]Ramkisoensing AA, Pijnappels DA, Askar SF, Passier R, Swildens J, Goumans MJ, et al.: Human embryonic and fetal mesenchymal stem cells differentiate toward three different cardiac lineages in contrast to their adult counterparts. PLoS One 2011, 6:e24164.
• [68]Roura S, Farre J, Soler-Botija C, Llach A, Hove-Madsen L, Cairo JJ, et al.: Effect of aging on the pluripotential capacity of human CD105+ mesenchymal stem cells. Eur J Heart Fail 2006, 8:555-63.
• [69]Lysy PA, Smets F, Sibille C, Najimi M, Sokal EM: Human skin fibroblasts: From mesodermal to hepatocyte-like differentiation. Hepatology 2007, 46:1574-85.
• [70]Duff SE, Li C, Garland JM, Kumar S: CD105 is important for angiogenesis: evidence and potential applications. FASEB J 2003, 17:984-92.
• [71]Li C, Hampson IN, Hampson L, Kumar P, Bernabeu C, Kumar S: CD105 antagonizes the inhibitory signaling of transforming growth factor beta1 on human vascular endothelial cells. FASEB J 2000, 14:55-64.
• [72]Vincent EB, Runyan RB, Weeks DL: Production of the transforming growth factor-beta binding protein endoglin is regulated during chick heart development. Dev Dyn 1998, 213:237-47.
• [73]Li L, Chopp M, Ding GL, Qu CS, Li QJ, Lu M, et al.: MRI measurement of angiogenesis and the therapeutic effect of acute marrow stromal cell administration on traumatic brain injury. J Cereb Blood Flow Metab 2012, 32:2023-32.
• [74]Quertainmont R, Cantinieaux D, Botman O, Sid S, Schoenen J, Franzen R: Mesenchymal stem cell graft improves recovery after spinal cord injury in adult rats through neurotrophic and pro-angiogenic actions. PLoS One 2012, 7:e39500.
• [75]Mitchell JB, McIntosh K, Zvonic S, Garrett S, Floyd ZE, Kloster A, et al.: Immunophenotype of human adipose-derived cells: temporal changes in stromal-associated and stem cell-associated markers. Stem Cells 2006, 24:376-85.
• [76]Varma MJ, Breuls RG, Schouten TE, Jurgens WJ, Bontkes HJ, Schuurhuis GJ, et al.: Phenotypical and functional characterization of freshly isolated adipose tissue-derived stem cells. Stem Cells Dev 2007, 16:91-104.
• [77]Guo CL, Liu L, Jia YD, Zhao XY, Zhou Q, Wang L: A novel variant of Oct3/4 gene in mouse embryonic stem cells. Stem Cell Res 2012, 9:69-76.
• [78]Wang X, Zhao Y, Xiao Z, Chen B, Wei Z, Wang B, et al.: Alternative translation of OCT4 by an internal ribosome entry site and its novel function in stress response. Stem Cells 2009, 27:1265-75.
• [79]Lee J, Kim HK, Rho JY, Han YM, Kim J: The human OCT-4 isoforms differ in their ability to confer self-renewal. J Biol Chem 2006, 281:33554-65.
• [80]Violini S, Ramelli P, Pisani LF, Gorni C, Mariani P: Horse bone marrow mesenchymal stem cells express embryo stem cell markers and show the ability for tenogenic differentiation by in vitro exposure to BMP-12. BMC Cell Biol 2009, 10:29. BioMed Central Full Text
• [81]Ock SA, Jeon BG, Rho GJ: Comparative characterization of porcine mesenchymal stem cells derived from bone marrow extract and skin tissues. Tissue Eng Part C Methods 2010, 16:1481-91.
• [82]Zuk PA: The intracellular distribution of the ES cell totipotent markers OCT4 and Sox2 in adult stem cells differs dramatically according to commercial antibody used. J Cell Biochem 2009, 106:867-77.
• [83]Lengner CJ, Camargo FD, Hochedlinger K, Welstead GG, Zaidi S, Gokhale S, et al.: Oct4 expression is not required for mouse somatic stem cell self-renewal. Cell Stem Cell 2007, 1:403-15.
• [84]Vasquez KM, Marburger K, Intody Z, Wilson JH: Manipulating the mammalian genome by homologous recombination. Proc Natl Acad Sci U S A 2001, 98:8403-10.
• [85]Haleem-Smith H, Derfoul A, Okafor C, Tuli R, Olsen D, Hall DJ, et al.: Optimization of high-efficiency transfection of adult human mesenchymal stem cells in vitro. Mol Biotechnol 2005, 30:9-20.
• [86]Mets T, Verdonk G: In vitro aging of human bone marrow derived stromal cells. Mech Ageing Dev 1981, 16:81-9.
• [87]Colter DC, Sekiya I, Prockop DJ: Identification of a subpopulation of rapidly self-renewing and multipotential adult stem cells in colonies of human marrow stromal cells. Proc Natl Acad Sci U S A 2001, 98:7841-5.
• [88]Neuhuber B, Swanger SA, Howard L, Mackay A, Fischer I: Effects of plating density and culture time on bone marrow stromal cell characteristics. Exp Hematol 2008, 36:1176-85.
• [89]Mizrahi O, Sheyn D, Tawackoli W, Kallai I, Oh A, Su S, et al.: BMP-6 is more efficient in bone formation than BMP-2 when overexpressed in mesenchymal stem cells. Gene Ther 2013, 20:370-7.
• [90]Sheyn D, Kallai I, Tawackoli W, Cohn Yakubovich D, Oh A, Su S, et al.: Gene-modified adult stem cells regenerate vertebral bone defect in a rat model. Mol Pharm 2011, 8:1592-601.
• [91]Dembinski JL, Wilson SM, Spaeth EL, Studeny M, Zompetta C, Samudio I, et al.: Tumor stroma engraftment of gene-modified mesenchymal stem cells as anti-tumor therapy against ovarian cancer. Cytotherapy 2013, 15:20-32.
• [92]Benabdallah BF, Allard E, Yao S, Friedman G, Gregory PD, Eliopoulos N, et al.: Targeted gene addition to human mesenchymal stromal cells as a cell-based plasma-soluble protein delivery platform. Cytotherapy 2010, 12:394-9.
• [93]Li H, Zhang B, Lu Y, Jorgensen M, Petersen B, Song S: Adipose tissue-derived mesenchymal stem cell-based liver gene delivery. J Hepatol 2011, 54:930-8.
• [94]Janderova L, McNeil M, Murrell AN, Mynatt RL, Smith SR: Human mesenchymal stem cells as an in vitro model for human adipogenesis. Obes Res 2003, 11:65-74.
• [95]Shimba S, Wada T, Hara S, Tezuka M: EPAS1 promotes adipose differentiation in 3 T3-L1 cells. J Biol Chem 2004, 279:40946-53.
• [96]Poulos SP, Dodson MV, Hausman GJ: Cell line models for differentiation: preadipocytes and adipocytes. Exp Biol Med 2010, 235:1185-93.
• [97]Boucher S, Lakshmipathy U, Vemuri M: A simplified culture and polymerase chain reaction identification assay for quality control performance testing of stem cell media products. Cytotherapy 2009, 11:761-7. 767 e761-762