Mesenchymal stem cells (MSCs) derived from human pluripotent stem cells (hPSC-derived MSCs) will be one promising alternative cell source for MSC-based therapies. Here, an efficient protocol is demonstrated for generating hPSC-derived MSCs under a feeder-free culture system by regulating signaling pathways. Simultaneous treatments with Activin A, BIO (6-bromoindirubin-3¢-oxime), and bone morphogenetic protein 4 (ABB) activated the transcription of mesoderm-lineage genes such as T, MIXL1, and WNT3 in hPSCs. The ABBtreated hPSCs could develop into CD105+ cells with a high efficiency of 20% in the MSC-induction medium. The properties of the hPSC-derived CD105 + cells were similar to those of adult MSCs in terms of surface antigens. Also, hPSC-derived MSCs had the potential to differentiate into adipocytes, osteoblasts, and chondrocytes in vitro. The results demonstrated that functional MSCs could be generated efficiently from hPSCs by the combined modulation of signaling pathways.
Constitutively active Ras (CA-Ras) is known to enhance cell growth through the induction of various signaling cascades including the phosphoinositide 3-kinase (PI3K)/Akt and mitogen-activated protein kinase (MAPK)/ERK signaling pathways, although the cellular response is highly dependent on the cell type. To evaluate the effect of CA-Ras overexpression on cell growth in recombinant Chinese hamster ovary (rCHO) cells, an erythropoietin (EPO)-producing rCHO cell line with regulated CA-Ras overexpression (EPO-off-CA-Ras) was established using the Tet-off system. The CA-Ras expression level in EPO-off-CA-Ras cells was tightly regulated by doxycycline addition. Although CA-Ras overexpression slightly increased the viable cell concentration during the late exponential phase, it did not increase the maximum viable cell concentration or specific growth rate to a significant degree. Unexpectedly, CA-Ras overexpression in rCHO cells led only to the enhancement in the activation of the MAPK/ERK signaling pathway and not the PI3K/Akt signaling pathway. Taken together, CA-Ras overexpression in rCHO cells did not significantly affect cell growth; it also had no critical impact on viable cell concentration or EPO production, possibly due to a failure to activate the PI3K/Akt signaling pathway.
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