title: Native extracellular matrix preserves mesenchymal stem cell "stemness" and differentiation potential under serum-free culture conditions.
abstract: BACKGROUND
  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.
authors.affiliation: Department of Comprehensive Dentistry, University of Texas Health Science Center at San Antonio, 7703 Floyd Curl Drive, San Antonio, TX, 78229-3900, USA. rakianr@livemail.uthscsa.edu.
authors.forename: Rubie
authors.initials: R
authors.lastname: Rakian
authors.affiliation: Department of Comprehensive Dentistry, University of Texas Health Science Center at San Antonio, 7703 Floyd Curl Drive, San Antonio, TX, 78229-3900, USA. blockt@livemail.uthscsa.edu.
authors.forename: Travis J
authors.initials: TJ
authors.lastname: Block
authors.affiliation: Department of Comprehensive Dentistry, University of Texas Health Science Center at San Antonio, 7703 Floyd Curl Drive, San Antonio, TX, 78229-3900, USA. shannanmjohnson@gmail.com.|Periodontics Graduate Program, Wilford Hall 59th Medical Wing, 2133 Pepperrell Street, Building 3352, Joint Base San Antonio, Lackland, TX, 78236, USA. shannanmjohnson@gmail.com.
authors.forename: Shannan M
authors.initials: SM
authors.lastname: Johnson
authors.affiliation: Department of Comprehensive Dentistry, University of Texas Health Science Center at San Antonio, 7703 Floyd Curl Drive, San Antonio, TX, 78229-3900, USA. marinkovic@livemail.uthscsa.edu.
authors.forename: Milos
authors.initials: M
authors.lastname: Marinkovic
authors.affiliation: Department of Orthodontics, Fourth Military Medical University, School of Stomatology, 145 West Chang-le Road, Xi'an, Shaanxi Province, 710032, P.R. China. wujunjiedds@163.com.
authors.forename: Junjie
authors.initials: J
authors.lastname: Wu
authors.affiliation: Department of Comprehensive Dentistry, University of Texas Health Science Center at San Antonio, 7703 Floyd Curl Drive, San Antonio, TX, 78229-3900, USA. daiq@uthscsa.edu.
authors.forename: Qiuxia
authors.initials: Q
authors.lastname: Dai
authors.affiliation: Department of Comprehensive Dentistry, University of Texas Health Science Center at San Antonio, 7703 Floyd Curl Drive, San Antonio, TX, 78229-3900, USA. deand@uthscsa.edu.
authors.forename: David D
authors.initials: DD
authors.lastname: Dean
authors.affiliation: Department of Comprehensive Dentistry, University of Texas Health Science Center at San Antonio, 7703 Floyd Curl Drive, San Antonio, TX, 78229-3900, USA. chenx4@uthscsa.edu.|Research Service, Audie Murphy VA Medical Center, South Texas Veterans Health Care System, 7400 Merton Minter Boulevard, San Antonio, TX, 78229-4404, USA. chenx4@uthscsa.edu.
authors.forename: Xiao-Dong
authors.initials: XD
authors.lastname: Chen
mesh_terms: D000328:Adult; D002454:Cell Differentiation; D049109:Cell Proliferation; D016895:Culture Media, Serum-Free; D005109:Extracellular Matrix; D006801:Humans; D059630:Mesenchymal Stem Cells / Q000166:cytology*; D055815:Young Adult
chemical_list: D016895:Culture Media, Serum-Free