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: 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: Skeletal muscle (SkM) comprise approximately 40% of human body weight. Injury or damage to this important tissue can result in physical disability, and in severe cases is difficult for its endogenous stem cell-the satellite cell-to reverse effectively. Mesenchymal stem cells (MSC) are post-natal progenitor/stem cells that possess multilineage mesodermal differentiation capacity, including towards SkM. Adult bone marrow (BM) is the best-studied source of MSCs; however, aging also decreases BMMSC numbers and can adversely affect differentiation capacity. Therefore, we asked whether human sources of developmentally early-stage mesenchymal stem cells (hDE-MSCs) isolated from embryonic stem cells, fetal bone, and term placenta could be cellular sources for SkM repair. Under standard muscle-inducing conditions, hDE-MPCs differentiate towards a SkM lineage rather than cardiomyocytic or smooth muscle lineages, as evidenced by increased expression of SkM-associated markers and in vitro myotube formation. In vivo transplantation revealed that SkM-differentiated hDE-MSCs can incorporate into host SkM tissue efficiently in a mouse model of SkM injury. In contrast, adult BMMSCs do not express SkM-associated genes after in vitro SkM differentiation nor engraft in vivo. Further investigation of possible factors responsible for this difference in SkM differentiation potential revealed that, compared to adult BMMSCs, hDE-MSCs expressed higher levels of serum response factor (SRF), a transcription factor critical for SkM lineage commitment. Moreover, knockdown of SRF in hDE-MSCs resulted in decreased expression of SkM-related genes after in vitro differentiation and decreased in vivo engraftment. Our results implicate SRF as a key factor in age-related SkM differentiation capacity of MSCs, and demonstrate that hDE-MSCs are possible candidates for SkM repair.Stem cells and development 02/2014; · 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 01/2014; 9(3):e90719. · 3.53 Impact Factor