Native extracellular matrix preserves mesenchymal stem cell “stemness” and differentiation potential under serum-free culture conditions

Stem Cell Research & Therapy

Xiao-Dong Chen⁴  David D. Dean³  Qiuxia Dai³  Junjie Wu²  Milos Marinkovic³  Shannan M. Johnson¹  Travis J. Block³  Rubie Rakian³ 

[1]Periodontics Graduate Program, Wilford Hall 59th Medical Wing, 2133 Pepperrell Street, Building 3352, Joint Base San Antonio, Lackland 78236, TX, USA

[2]Department of Orthodontics, Fourth Military Medical University, School of Stomatology, 145 West Chang-le Road, Xi’an 710032, Shaanxi Province, P.R. China

[3]Department of Comprehensive Dentistry, University of Texas Health Science Center at San Antonio, 7703 Floyd Curl Drive, San Antonio 78229-3900, TX, USA

[4]Research Service, Audie Murphy VA Medical Center, South Texas Veterans Health Care System, 7400 Merton Minter Boulevard, San Antonio 78229-4404, TX, USA

关键词: Stem cell expansion;    Serum-free media;    Extracellular matrix;    Mesenchymal stem cells;   

Others  :  1235094
DOI  :  10.1186/s13287-015-0235-6

 received in 2015-07-24, accepted in 2015-11-10,  发布年份 2015

【 摘 要 】

Introduction

Bone marrow-derived mesenchymal stem cells (BM-MSCs) for clinical use should not be grown in media containing fetal bovine serum (FBS), because of serum-related concerns over biosafety and batch-to-batch variability. Previously, we described the preparation and use of a cell-free native extracellular matrix (ECM) made by bone marrow cells (BM-ECM) which preserves stem cell properties and enhances proliferation. Here, we compare colony-forming ability and differentiation of MSCs cultured on BM-ECM with a commercially available matrix (CELLstart™) and tissue culture plastic (TCP) under serum-free conditions.

Methods

Primary MSCs from freshly isolated bone marrow-derived mononuclear cells or passaged MSCs (P1) were grown in serum-containing (SCM) or serum-free (SFM) media on BM-ECM, CELLstart™, or TCP substrates. Proliferation, cell composition (phenotype), colony-forming unit replication, and bone morphogenetic protein-2 (BMP-2) responsiveness were compared among cells maintained on the three substrates.

Results

Proliferation of primary BM-MSCs was significantly higher in SCM than SFM, irrespectively of culture substrate, suggesting that the expansion of these cells requires SCM. In contrast, passaged cells cultured on BM-ECM or CELLstart™ in SFM proliferated to nearly the same extent as cells in SCM. However, morphologically, those on BM-ECM were smaller and more aligned, slender, and long.

Cells grown for 7 days on BM-ECM in SFM were 20–40 % more positive for MSC surface markers than cells cultured on CELLstart™. Cells cultured on TCP contained the smallest number of cells positive for MSC markers. MSC colony-forming ability in SFM, as measured by CFU-fibroblasts, was increased 10-, 9-, and 2-fold when P1 cells were cultured on BM-ECM, CELLstart™, and TCP, respectively. Significantly, CFU-adipocyte and -osteoblast replication of cells grown on BM-ECM was dramatically increased over those on CELLstart™ (2X) and TCP (4-7X). BM-MSCs, cultured in SFM and treated with BMP-2, retained their differentiation capacity better on BM-ECM than on either of the other two substrates.

Conclusions

Our findings indicate that BM-ECM provides a unique microenvironment that supports the colony-forming ability of MSCs in SFM and preserves their stem cell properties. The establishment of a robust culture system, combining native tissue-specific ECM and SFM, provides an avenue for preparing significant numbers of potent MSCs for cell-based therapies in patients.

【 授权许可】

   
2015 Rakian et al.

【 图 表 】

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【 参考文献 】

• [1]Pittenger MF, Mosca JD, McIntosh KR: Human mesenchymal stem cells: progenitor cells for cartilage, bone, fat and stroma. Curr Top Microbiol Immunol. 2000, 251:3-11.
• [2]Deng J, Petersen BE, Steindler DA, Jorgensen ML, Laywell ED: Mesenchymal stem cells spontaneously express neural proteins in culture and are neurogenic after transplantation. Stem Cells. 2006, 24:1054-64.
• [3]Engler AJ, Sen S, Sweeney HL, Discher DE: Matrix elasticity directs stem cell lineage specification. Cell. 2006, 126:677-89.
• [4]Petersen BE, Bowen WC, Patrene KD, Mars WM, Sullivan AK, Murase N, et al.: Bone marrow as a potential source of hepatic oval cells. Science. 1999, 284:1168-70.
• [5]Friedenstein AJ, Latzinik NW, Grosheva AG, Gorskaya UF: Marrow microenvironment transfer by heterotopic transplantation of freshly isolated and cultured cells in porous sponges. Exp Hematol. 1982, 10:217-27.
• [6]Wexler SA, Donaldson C, Denning-Kendall P, Rice C, Bradley B, Hows JM: Adult bone marrow is a rich source of human mesenchymal ‘stem’ cells but umbilical cord and mobilized adult blood are not. Br J Haematol. 2003, 121:368-74.
• [7]Shahdadfar A, Fronsdal K, Haug T, Reinholt FP, Brinchmann JE: In vitro expansion of human mesenchymal stem cells: choice of serum is a determinant of cell proliferation, differentiation, gene expression, and transcriptome stability. Stem Cells. 2005, 23:1357-66.
• [8]Schallmoser K, Rohde E, Reinisch A, Bartmann C, Thaler D, Drexler C, et al.: Rapid large-scale expansion of functional mesenchymal stem cells from unmanipulated bone marrow without animal serum. Tissue Eng Part C Methods. 2008, 14:185-96.
• [9]Tonti GA, Mannello F: From bone marrow to therapeutic applications: different behaviour and genetic/epigenetic stability during mesenchymal stem cell expansion in autologous and foetal bovine sera? Int J Dev Biol. 2008, 52:1023-32.
• [10]Agata H, Watanabe N, Ishii Y, Kubo N, Ohshima S, Yamazaki M, et al.: Feasibility and efficacy of bone tissue engineering using human bone marrow stromal cells cultivated in serum-free conditions. Biochem Biophys Res Commun. 2009, 382:353-8.
• [11]Chase LG, Lakshmipathy U, Solchaga LA, Rao MS, Vemuri MC: A novel serum-free medium for the expansion of human mesenchymal stem cells. Stem Cell Res Ther. 2010, 1:8. BioMed Central Full Text
• [12]Swamynathan P, Venugopal P, Kannan S, Thej C, Kolkundar U, Bhagwat S, et al.: Are serum-free and xeno-free culture conditions ideal for large scale clinical grade expansion of Wharton’s jelly derived mesenchymal stem cells? A comparative study. Stem Cell Res Ther. 2014, 5:88. BioMed Central Full Text
• [13]Swistowski A, Peng J, Han Y, Swistowska AM, Rao MS, Zeng X: Xeno-free defined conditions for culture of human embryonic stem cells, neural stem cells and dopaminergic neurons derived from them. PLoS One. 2009, 4:e6233.
• [14]Fuchs E, Tumbar T, Guasch G: Socializing with the neighbors: stem cells and their niche. Cell. 2004, 116:769-78.
• [15]Moore KA, Lemischka IR: Stem cells and their niches. Science. 2006, 311:1880-5.
• [16]Chen XD, Dusevich V, Feng JQ, Manolagas SC, Jilka RL: Extracellular matrix made by bone marrow cells facilitates expansion of marrow-derived mesenchymal progenitor cells and prevents their differentiation into osteoblasts. J Bone Miner Res. 2007, 22:1943-56.
• [17]Lai Y, Sun Y, Skinner CM, Son EL, Lu Z, Tuan RS, 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, 19:1095-107.
• [18]Gospodarowicz DJ: Extracellular matrices and the control of cell proliferation and differentiation in vitro. Prog Clin Biol Res. 1984, 145:103-28.
• [19]Lukashev ME, Werb Z: ECM signalling: orchestrating cell behaviour and misbehaviour. Trends Cell Biol. 1998, 8:437-41.
• [20]Cukierman E, Pankov R, Stevens DR, Yamada KM: Taking cell-matrix adhesions to the third dimension. Science. 2001, 294:1708-12.
• [21]Abbott A: Cell culture: biology’s new dimension. Nature. 2003, 424:870-2.
• [22]Lin H, Yang G, Tan J, Tuan RS: Influence of decellularized matrix derived from human mesenchymal stem cells on their proliferation, migration and multi-lineage differentiation potential. Biomaterials. 2012, 33:4480-9.
• [23]Bae SE, Bhang SH, Kim BS, Park K: Self-assembled extracellular macromolecular matrices and their different osteogenic potential with preosteoblasts and rat bone marrow mesenchymal stromal cells. Biomacromolecules. 2012, 13:2811-20.
• [24]Prewitz MC, Seib FP, von Bonin M, Friedrichs J, Stiβel A, Niehage C, et al.: Tightly anchored tissue-mimetic matrices as instructive stem cell microenvironments. Nat Methods. 2013, 10:788-94.
• [25]Papadimitropoulos A, Scotti C, Bourgine P, Scherberich A, Martin I: Engineered decellularized matrices to instruct bone regeneration processes. Bone. 2015, 70:66-72.
• [26]Hughes CS, Radan L, Betts D, Postovit LM, Lajoie GA: Proteomic analysis of extracellular matrices used in stem cell culture. Proteomics. 2011, 11:3983-91.
• [27]Bara JJ, Richards RG, Alini M, Stoddart MJ: Concise review: Bone marrow-derived mesenchymal stem cells change phenotype following in vitro culture: implications for basic research and the clinic. Stem Cells. 2014, 32:1713-23.
• [28]Baker N, Boyette LB, Tuan RS: Characterization of bone marrow-derived mesenchymal stem cells in aging. Bone. 2015, 70:37-47.
• [29]Sanchez-Hidalgo M, Lu Z, Tan DX, Maldonado MD, Reiter RJ, Gregerman RI: Melatonin inhibits fatty acid-induced triglyceride accumulation in ROS17/2.8 cells: implications for osteoblast differentiation and osteoporosis. Am J Physiol Regul Integr Comp Physiol 2007, 292:R2208-15.
• [30]Menon NV, Chuah YJ, Phey S, Zhang Y, Wu Y, Chan V, et al.: A microfluidic assay to study the combinatorial impact of substrate properties on mesenchymal stem cell migration. ACS Appl Mater Interfaces. 2015, 7:17095-103.
• [31]Brunner D, Frank J, Appl H, Schoffl H, Pfaller W, Gstraunthaler G: Serum-free cell culture: the serum-free media interactive online database. ALTEX. 2010, 27:53-62.
• [32]Chen XD, Fisher LW, Robey PG, Young MF: The small leucine-rich proteoglycan biglycan modulates BMP-4-induced osteoblast differentiation. FASEB J. 2004, 18:948-58.
• [33]Ducy P: Cbfa1: a molecular switch in osteoblast biology. Dev Dyn. 2000, 219:461-71.
• [34]Ruoslahti E, Yamaguchi Y: Proteoglycans as modulators of growth factor activities. Cell. 1991, 64:867-9.
• [35]Delany AM, Canalis E: The metastasis-associated metalloproteinase stromelysin-3 is induced by transforming growth factor-beta in osteoblasts and fibroblasts. Endocrinology. 2001, 142:1561-6.
• [36]Bi Y, Stuelten CH, Kilts T, Wadhwa S, Iozzo RV, Robey PG, et al.: Extracellular matrix proteoglycans control the fate of bone marrow stromal cells. J Biol Chem. 2005, 280:30481-9.