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: Embryonic stem cells (ESCs) and mesenchymal stem cells (MSCs) have been studied for years as primary cell sources for regenerative biology and medicine. MSCs have been derived from cell and tissue sources, such as bone marrow (BM), and more recently from ESCs. This study investigated MSCs derived from BM, H1- and H9-ESC lines in terms of morphology, surface marker and growth factor receptor expression, proliferative capability, modulation of immune cell growth and multipotency, in order to evaluate ESC-MSCs as a cell source for potential regenerative applications. The results showed that ESC-MSCs exhibited spindle-shaped morphology similar to BM-MSCs but of various sizes, and flow cytometric immunophenotyping revealed expression of characteristic MSC surface markers on all tested cell lines except H9-derived MSCs. Differences in growth factor receptor expression were also shown between cell lines. In addition, ESC-MSCs showed greater capabilities for cell proliferation, and suppression of leukocyte growth compared to BM-MSCs. Using standard protocols, induction of ESC-MSC differentiation along the adipogenic, osteogenic, or chondrogenic lineages was less effective compared to that of BM-MSCs. By adding bone morphogenetic protein 7 (BMP7) into transforming growth factor beta 1 (TGFβ1)-supplemented induction medium, chondrogenesis of ESC-MSCs was significantly enhanced. Our findings suggest that ESC-MSCs and BM-MSCs show differences in their surface marker profiles and the capacities of proliferation, immunomodulation, and most importantly multi-lineage differentiation. Using modified chondrogenic medium with BMP7 and TGFβ1, H1-MSCs can be effectively induced as BM-MSCs for chondrogenesis.Cell and Tissue Research 06/2014; 358(1). · 3.33 Impact Factor
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ABSTRACT: Mesenchymal stem cells (MSCs) have a high potential for therapeutic efficacy in treating diverse musculoskeletal injuries and cardiovascular diseases, and ameliorating the severity of graft-versus-host and autoimmune diseases. While most of these clinical applications require substantial cell quantities, the number of MSCs that can be obtained initially from a single donor is limited. Reports on the derivation of MSC-like cells from pluripotent stem cells (PSCs) are thus of interest, since the infinite proliferative capacity of PSCs opens the possibility to generate large amounts of uniform batches of MSCs. However, characterization of such MSC-like cells is currently inadequate, especially concerning the question of whether these cells are equivalent or identical to MSCs. In this study, we have derived MSC-like cells (iMPCs) using four different methodologies from a newly established induced PSC line reprogrammed from human bone marrow stromal cells (BMSCs), and compared the iMPCs directly to the originating parental BMSCs. The iMPCs exhibited typical MSC/fibroblastic morphology and MSC-typical surface marker profile, and were capable of differentiation in vitro along the osteogenic, chondrogenic, and adipogenic lineages. However, compared to the parental BMSCs, iMPCs displayed a unique expression pattern of mesenchymal and pluripotency genes and were less responsive to traditional BMSC differentiation protocols. We therefore conclude that iMPCs generated from PSCs via spontaneous differentiation represent a distinct population of cells that comprise MSC-like characteristics.Stem cells and development 03/2014; · 4.15 Impact Factor
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ABSTRACT: The chemical, mechanical, and topographical features of the extracellular matrix (ECM) have all been documented to influence cell adhesion, gene expression, migration, proliferation, and differentiation. Topography plays a key role in the architecture and functionality of various tissues in vivo, thus raising the possibility that topographic cues can be instructive when incorporated into biomaterials for regenerative applications. In the literature, there are discrepancies regarding the potential roles of nanotopography to enhance the osteogenic phenotype of mesenchymal stem cells (MSC). In this study, we used thin film substrates of poly(methyl methacrylate) (PMMA) with nanoscale gratings to investigate the influence of nanotopography on the osteogenic phenotype of MSCs, focusing in particular on their ability to produce mineral similar to native bone. Topography influenced focal adhesion size and MSC alignment, and enhanced MSC proliferation after 14 days of culture. However, the osteogenic phenotype was minimally influenced by surface topography. Specifically, alkaline phosphatase (ALP) expression was not increased on nanotopographic films, nor was calcium deposition improved after 21 days in culture. Ca: P ratios were similar to native mouse bone on films with gratings of 415 nm width and 200 nm depth (G415) and 303 nm width and 190 nm depth (G303). Notably, all surfaces had Ca∶P ratios significantly lower than G415 films. Collectively, these data suggest that, PMMA films with nanogratings are poor drivers of an osteogenic phenotype.PLoS ONE 03/2014; 9(3):e90719. · 3.53 Impact Factor