Large-scale expansion of clinical-grade human adipose-derived stem cells using the extracellular matrix

doi:10.3969/j.issn.2095-4344.2014.10.008

Abstract

Abstract:

BACKGROUND: Large-scale expansion of undifferentiated and multipotential adipose-derived stem cells using serum-free culture system is a difficult issue to be resolved.
OBJECTIVE: To establish an in vitro culture system combined with the extracellular matrix in order to investigate the efficiency, effectiveness and security of extracellular matrix on expanding adipose-derived stem cells.
METHODS: In vitro isolated adipose-derived stem cells were seeded in traditional two-dimensional plastic plates and extracellular matrix-coated plates supplemented with serum-free medium respectively. After in vitro expansion, total cell number, expression of cell surface markers, cell senescence degree and multipotent differentiation ability (adipogenic, osteoblastic and chondrogenic differentiation) of adipose-derived stem cells cultured under both conditions were detected and compared. Moreover, the clinical safety of adipose-derived stem cells expanded in extracellular matrix-coated plates was investigated.
RESULTS AND CONCLUSION: Total cell number of passage 5 adipose-derived stem cells cultured in extracellular matrix-coated plates was 10 times more than that in traditional two-dimensional plastic plates. Flow-cytometric analysis showed that adipose-derived stem cells cultured with extracellular matrix expressed stem cell surface markers. Cellular senescence examination showed that almost all of passage 15 adipose-derived stem cells cultured with extracellular matrix showed no aging, while most passage 5 adipose-derived stem cells cultured by the two-dimensional system aged and lost their proliferation ability. Multidirectional induction of adipose-derived stem cells showed that passage 15 adipose-derived stem cells cultured with extracellular matrix could still differentiate into adipocytes, osteoblasts and chondrocytes as passage 5 adipose-derived stem cells did, which performed much better than the induced differentiations of passage 5 adipose-derived stem cells cultured by the two-dimensional system. Karyotype analysis and in vivo invasion experiment insured the clinical safety of adipose-derived stem cells expanded with extracellular matrix. All above results suggest a safe and more efficient expansion system of extracellular matrix for clinical application using the serum-free culture system combined with extracellular matrix.

中国组织工程研究杂志出版内容重点：干细胞；骨髓干细胞；造血干细胞；脂肪干细胞；肿瘤干细胞；胚胎干细胞；脐带脐血干细胞；干细胞诱导；干细胞分化；组织工程

全文链接：

Key words: stem cells, mesenchymal stem cells, extracellular matrix, serum, cell culture techniques

CLC Number:

R394.2

Su Yue-han, Wei Chao, Lv Pin-lei, Cao Yun, Qiu Yun, Zheng Qing, Xiao Shu-dong, Wang Zheng . Large-scale expansion of clinical-grade human adipose-derived stem cells using the extracellular matrix[J]. Chinese Journal of Tissue Engineering Research, 2014, 18(10): 1521-1531.

Figures/Tables 9

References

[1]Guilak F, Lott KE, Awad HA, et al. Clonal analysis of the differentiation potential of human adipose-derived adult stem cells. J Cell. Physiol. 2006;206(1): 229-237.

[2]Zuk PA, Zhu M, Ashjian P, et al. Human adipose tissue is a source of multipotent stem cells. Mol Biol Cell. 2002;13(12): 4279-4295.

[3]Rodriguez AM, Elabd C, Amri EZ, et al. The human adipose tissue is a source of multipotent stem cells. Biochimie. 2005; 87(1):125-128.

[4]Zuk PA, Zhu M, Mizuno H, et al. Multilineage cells from human adipose tissue: implications for cell-based therapies. Tissue Eng. 2001;7(2): 211-228.

[5]Dragoo JL, Carlson G, McCornick F, et al. Healing full-thickness cartilage defects using adipose-derived stem cells. Tissue Eng. 2007;13(7): 1615-1621.

[6]Safford KM, Rice HE. Stem cell therapy for neurologic disorders: therapeutic potential of adipose-derived stem cells. Current Drug Targets. 2005;6(1): 57-62.

[7]Banas A, Teratani T, Yamamoto Y, et al. Rapid hepatic fate specification of adipose-derived stem cells and their therapeutic potential for liver failure. J Gastroenterol and Hepatol. 2009;24(1): 70-77.

[8]Mizuno H. Adipose-derived stem cells for tissue repair and regeneration: ten years of research and a literature review. J Nippon Med Sch. 2009;76(2): 56-66.

[9]Park IS, Rhie JW, Kim SH. A novel three-dimensional adipose-derived stem cell cluster for vascular regeneration in ischemic tissue. Cytotherapy. 2013;S1465-3249(13): 00681-00686.

[10]Wang YL, Li G, Zou XF, et al. Effect of autologous adipose-derived stem cells in renal cold ischemia and reperfusion injury. Transplant Proc. 2013;45(9): 3198-3202.

[11]Lee HJ, Jung J, Cho KJ, et al. Comparison of in vitro hepatogenic differentiation potential between various placenta-derived stem cells and other adult stem cells as an alternative source of functional hepatocytes. Differentiation. 2012;84(3): 223-231.

[12]Bunnell BA, Flaat M, Gagliardi C, et al. Adipose-derived stem cells - isolation, expansion and differentiation. Methods. 2008; 45(2): 115-120.

[13]Cheng NC, Chen SY, Li JR, et al. Short-term spheroid formation enhances the regenerative capacity of adipose- derived stem cells by promoting stemness, angiogenesis, and chemotaxis. Stem Cells Transl Med. 2013;2(8): 584-594.

[14]Murphy MB, Moncivais K, Caplan AI. Mesenchymal stem cells: environmentally responsive therapeutics for regenerative medicine. Exp Mol Med. 2013;45: e54.

[15]Fernández Vallone VB, Romaniuk MA, et al. Mesenchymal stem cells and their use in therapy: what has been achieved? Differentiation. 2013;85(1-2): 1-10.

[16]Doorn J, Moll G, Le Blanc K, et al. Therapeutic applications of mesenchymal stromal cells: paracrine effects and potential improvements. Tissue Eng Part B Rev. 2012;18(2): 101-115.

[17]Mark P, Kleinsorge M, Gaebel R, et al. Human mesenchymal stem cells display reduced expression of CD105 after culture in serum-free medium. Stem Cells Int. 2013;2013: 698076.

[18]Tsang WP, Shu Y, Kwok PL, et al. CD146(+) human umbilical cord perivascular cells maintain stemness under hypoxia and as a cell source for skeletal regeneration. PLoS One. 2013; 8(10): e76153.

[19]Zhang D, Kilian KA. The effect of mesenchymal stem cell shape on the maintenance of multipotency. Biomaterials. 2013;34(16): 3962-3969.

[20]Scadden DT. The stem-cell niche as an entity of action. Nature. 2006;441(7097): 1075-1079.

[21]Sharma MB, Limaye LS, Kale VP. Mimicking the functional hematopoietic stem cell niche in vitro: recapitulation of marrow physiology by hydrogel-based three-dimensional cultures of mesenchymal stromal cells. Haematologica. 2012; 97(5): 651-660.

[22]Frantz C, Stewart KM, Weaver VM. The extracellular matrix at a glance. J Cell Sci. 2010;123: 4195-4200.

[23]Brizzi MF, Tarone G, Defilippi P. Extracellular matrix, integrins, and growth factors as tailors of the stem cell niche. Curr Opin Cell Biol. 2012;24(5): 645-651.

[24]Kazanis I, ffrench-Constant C. Extracellular matrix and the neural stem cell niche. Dev Neurobiol. 2011;71(11): 1006- 1017.

[25]Watt FM, Huck WT. Role of the extracellular matrix in regulating stem cell fate. Nat Rev Mol Cell Biol. 2013;14(8): 467-473.

[26]Taylor-Weiner H, Schwarzbauer JE, Engler AJ. Defined extracellular matrix components are necessary for definitive endoderm induction. Stem Cells. 2013;31(10): 2084-2094.

[27]Kurtz A, Oh SJ. Age related changes of the extracellular matrix and stem cell maintenance. Prev Med. 2012;54(Suppl): S50-56.

[28]Chen XD. Extracellular matrix provides an optimal niche for the maintenance and propagation of mesenchymal stem cells. Birth Defects Res C Embryo Today. 2010;90(1): 45-54.

[29]Young DA, Choi YS, Engler AJ, et al. Stimulation of adipogenesis of adult adipose-derived stem cells using substrates that mimic the stiffness of adipose tissue. Biomaterials. 2013;34(34): 8581-8588.

[30]Peterbauer-Scherb A, Danzer M, Gabriel C, et al. In vitro adipogenesis of adipose-derived stem cells in 3D fibrin matrix of low component concentration. J Tissue Eng Regen Med. 2012;6(6): 434-442.

[31]Schubert T, Xhema D, Vériter S, et al. The enhanced performance of bone allografts using osteogenic- differentiated adipose-derived mesenchymalstem cells. Biomaterials. 2011;32(34): 8880-8891.

[32]Hicok KC, Du Laney TV, Zhou YS, et al. Human adipose-derived adult stem cells produce osteoid in vivo. Tissue Eng. 2004;10(3-4):371-380.

[33]Huang JI, Zuk PA, Jones NF, et al. Chondrogenic potential of multipotential cells from human adipose tissue. Plast Reconstr Surg. 2004;113(2): 585-594.

[34]Hamid AA, Idrus RB, Saim AB, et al. Characterization of human adipose-derived stem cells and expression of chondrogenic genes during induction ofcartilage differentiation. Clinics (Sao Paulo). 2012;67(2): 99-106.

[35]Mizuno H, Hyakusoku H. Mesengenic potential and future clinical perspective of human processed lipoaspirate cells. J Nippon Med Sch. 2003;70(4): 300-306.

[36]Gronthos S, Franklin DM, Leddy HA, et al. Surface protein characterization of human adipose tissue-derived stromal cells. J Cell Physiol. 2001;189(1): 54-63.

[37]Mitchell JB, McIntosh K, Zvonic S, et al. Immunophenotype of human adipose-derived cells: temporal changes in stromal-associated and stem cell-associated markers. Stem Cells. 2006;24(2): 376-385.

[38]Jiang S, Zu Y, Fu Y, et al. Activation of the mitochondria-driven pathway of apoptosis in human PC-3 prostate cancer cells by a novel hydrophilic paclitaxel derivative, 7-xylosyl-10-deacetylpaclitaxel. Int J Oncol. 2008; 33(1): 103-111.

[39]Tao Q, Lv B, Qiao B, et al. Immortalization of ameloblastoma cells via reactivation of telomerase function: Phenotypic and molecular characteristics. Oral Oncol. 2009;45(12): 239-244.

[40]Lai Y, Sun Y, Skinner CM, et al. Reconstitution of marrow-derived extracellular matrix ex vivo: a robust culture system for expanding large-scale highly functional human mesenchymal stem cells. Stem Cells Dev. 2010;19(7): 1095-1107.

[41]Bendall SC, Stewart MH, Menendez P, et al. IGF and FGF cooperatively establish the regulatory stem cell niche of pluripotent human cells in vitro. Nature. 2007;448(7157): 1015-1021.

[42]Stewart MH, Bendall SC, Bhatia M. Deconstructing human embryonic stem cell cultures: niche regulation of self-renewal and pluripotency. J Mol Med (Berl). 2008;86(8): 875-886.

[43]Bendall SC, Stewart MH, Bhatia M. Human embryonic stem cells: lessons from stem cell niches in vivo. Regen Med. 2008; 3(3): 365-376.