Efficient Derivation and Concise Gene Expression Profiling of Human Embryonic Stem Cell-Derived Mesenchymal Progenitors (EMPs)
ABSTRACT New potential sources of stem cells for clinical application include bone marrow mesenchymal stem cells (BMMSCs), human embryonic stem cells (hESCs), and induced pluripotent stem cells (iPS). However, each source is not without its own concerns. While research continues in an effort to overcome these problems, the generation of mesenchymal progenitors from existing hESC lines may circumvent many of these issues. We report here a simple and efficient method of generating hESC-derived mesenchymal progenitors (EMPs) and transcriptome profiling using a concise, custom-designed, oligomnucleotide gene expression microarray. Characterization of EMPs shows that these cells are similar to BMMSCs in terms of differentiation capacity as well as cell surface marker expression. In addition, EMPs express several ESC markers and HLA-G, a nonclassical MHC class I molecule with immunomodulatory properties. Morevoer, EMPs possess significantly enhanced proliferative ability over BMMSCs during which karyotypic stability was maintained. Although derived from hESCs, EMPs do not form any tumors in immunocompromised mice. To efficiently profile gene expression in multiple samples, we designed an oligoarray to probe just over 11,000 genes highly expressed in stem cells. We found that the transcriptome of EMPs is more similar to BMMSCs than hESCs. Both cell types highly express genes involved in processes related to the cytoskeleton, extracellular matrix, and cell adhesion, but EMPs show higher expression of genes involved in cell proliferation whereas BMMSCs showed higher expression of immune-related genes. Based on our data, EMPs may be an accessible source of mesenchymal progenitor for therapeutic use.
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ABSTRACT: 3-dimensional microcarrier (3D-MC) cell culture systems are often used for expansion of stem cells including mesenchymal stem cells (MSCs) for high cell volumes required in clinical applications. However, compared to 2-dimensional (2D) cell culture, effects of 3D-MC systems on MSC differentiation have not been well studied. In this study, the behavior of various sources of MSCs from two species was observed and compared on 3D collagen I-coated-MCs (COL-MC) versus 2D culture. Proliferation of all MSCs cultured on 3D COL-MC was much decreased compared to 2D culture. Unexpectedly, COL-MC-cultured MSCs underwent spontaneous osteogenesis without exogenous addition of biochemical factors, as evidenced by increased osteogenic genes expression, ALP activity, calcium deposition, and collagen I secretion. Furthermore, MSCs cultured on 3D-MC alone without collagen I coating is sufficient to induce osteogenesis. The spontaneous lineage commitment induced by 3D-MC culture was mediated by increased cytoskeletal tension and actomyosin contraction of MSCs, which could be prevented by latrunculin B and blebbistatin, inhibitors of cytoskeletal tension and actomyosin contraction respectively. Our findings show that the combination of bioengineered MC and MSCs alone can induce specific lineage commitment very efficiently. These data have strong implications in simplifying tissue engineering strategies for therapeutic applications.Biomaterials 01/2012; 33(2):556-64. DOI:10.1016/j.biomaterials.2011.09.090 · 8.31 Impact Factor
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ABSTRACT: Large-scale production and noninvasive methods for harvesting mesenchymal stem cells (MSCs), particularly in elderly individuals, has prompted researchers to find new patientspecific sources for MSCs in regenerative medicine. This study aims to produce mesenchymal stem cells (MSCs) from human induced pluripotent stem cells (hiPSCs) and to evaluate their therapeutic effects in a CCl₄-induced mouse model of fulminant hepatic failure (FHF). hiPSCMSCs have shown MSCs morphology, antigen profile and differentiation capabilities, and improved hepatic function in our model. hiPSC-MSCs transplanted animals provide significant benefit in terms of survival, serum LDH, total bilirubin, and lipid peroxidation. hiPSC-MSCs therapy resulted in a one-third reduction of histologic activity index and a threefold increase in the number of proliferating hepatocytes. This was accompanied by a significant decrease in the expression levels of collagen type I, Mmp13, Mmp2, and Mmp9 genes and increase in Timp1 and Timp2 genes in transplanted groups. hiPSC-MSCs secreted hepatocyte growth factor (HGF) in vitro and also expressed HGF in evaluated liver sections. Similar results were observed with human bone marrow (hBM)-derived MSCs. In conclusion, our results have demonstrated that hiPSC-MSCs might be valuable appropriate alternatives for hBM-MSCs in FHF liver repair and support liver function by cell therapy with a large-scale production capacity, patient-specific nature and no invasive MSC harvesting.Cell Transplantation 02/2013; 22(10). DOI:10.3727/096368912X662462 · 3.57 Impact Factor
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ABSTRACT: Mesenchymal stem cells (MSCs) display multipotent characteristics that make them ideal for potential therapeutic applications. MSCs are typically cultured as monolayers on tissue culture plastic, but there is increasing evidence suggesting that they may lose their multipotency over time in vitro, and eventually cease to retain any resemblance to in vivo resident MSCs. Three dimensional (3D) culture systems that more closely recapitulate the physiological environment of MSCs and other cell types are increasingly explored for their capacity to support and maintain cell phenotypes. In much of our own work, we have utilized fibrin, a natural protein-based material that serves as the provisional extracellular matrix (ECM) during wound healing. Fibrin has proven to be useful in numerous tissue engineering applications, and has been used clinically as a hemostatic material. Its rapid self-assembly driven by thrombin-mediated alteration of fibrinogen makes fibrin an attractive 3D substrate in which cells can adhere, spread, proliferate, and undergo complex morphogenetic programs. However, there is a significant need for simple, cost-effective methods to safely retrieve cells encapsulated within fibrin hydrogels in order to perform additional analyses or use the cells for therapy. Here, we present a safe and efficient protocol for the isolation of MSCs from 3D fibrin gels. The key ingredient of our successful extraction method is nattokinase, a serine protease of the subtilisin family that has strong fibrinolytic activity. Our data show that MSCs recovered from 3D fibrin gels using nattokinase are not only viable, but also retain their proliferative and multilineage potential. Demonstrated for MSCs, this method can be readily adapted to retrieve any other cell type from 3D fibrin gel constructs for various applications, including expansion, bioassays, and in vivo implantation.Tissue Engineering Part C Methods 06/2013; 20(3). DOI:10.1089/ten.TEC.2013.0051 · 4.64 Impact Factor