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However, when confluent cells were maintained in culture in the presence or absence of 5-AzaC without passage for more than 21 d, expression of specific skeletal muscle markers such as MyoD and fast-switch myosin was observed (Figure 3A).
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15971941
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More rapid myogenic differentiation was obtained in the presence of 24-h-conditioned medium from the murine myoblastic cell line C2C12 previously induced to form myotubes [14].
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Direct coculture of hESMPCs with C2C12 cells led to the formation of hESMPC-derived myotubes, as visualized by expression of human nuclear antigen (Figure 3B), similar to those formed by host C2C12 cells.
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After 1 wk of coculture, myotubes composed of human nuclei accounted for more than 10% of the total number of human cells present, and each human myotube was composed of up to ten human nuclei.
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Human cell contribution to myotubes in coculture was confirmed by expression of human muscle-specific transcripts such as MyoD, myosin heavy chain IIa, and myogenin (data not shown).
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These data demonstrate that hESMPCs can give rise to mesenchymal derivatives typically obtained from primary adult MSCs, as well as to cells expressing markers of skeletal muscle.
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One concern for the clinical application of hESC-derived progeny in regenerative medicine is the risk of teratoma formation due to the presence of residual undifferentiated ES cells among the differentiated progeny.
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We did not detect markers of undifferentiated hESCs, such as Nanog [15] or Oct-4 [16], in any of the hESMPCs by RT-PCR (see Figure 2D) and immunocytochemistry (data not shown), suggesting the lack of any undifferentiated ES cells in hESMPC cultures.
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However, future in vivo studies are required to rule out the potential of these cells for teratoma formation.
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Previous studies have demonstrated the derivation of neural cells [1–3], hematopoietic [17] and endothelial lineages [18], and cardiomyocytes [19] from hESCs.
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This study presents the induction of paraxial mesoderm with the generation of multipotent mesenchymal precursors.
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We calculate that under these conditions a single undifferentiated hESC yields an average of one CD73+ cell at day 40 of differentiation, suggesting a balance between cell proliferation and cell selection.
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There were no obvious differences in marker and gene-expression profile or in differentiation behavior among the five hESMPC lines generated.
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However, some of the lines (e.g., hESMPC9.1) exhibited a tendency of spontaneous osteogenic differentiation after long-term propagation.
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Directed differentiation of hESCs into somatic stem-cell-like precursors represents a substantial advancement in harnessing the developmental potential of hESCs.
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The high purity, unlimited availability, and multipotentiality of hESMPCs will provide the basis for future therapeutic efforts using these cells in preclinical animal models of disease.
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Such in vivo studies will also be required to properly assess the safety profile of these cells.
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Furthermore, our system also offers a novel platform to study basic mechanisms of mesodermal induction and differentiation during early human development.
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