Differentiated somatic cells can be reprogrammed into pluripotent stem cells by transduction of exogenous reprogramming factors. After induced pluripotent stem (iPS) cells are established, exogenous genes are silenced. In the pluripotent state, retroviral genes integrated in the host genome are kept inactive through epigenetic transcriptional regulation. In this study, we tried to determine whether exogenous genes remain silenced or are reactivated upon loss of pluripotency or on differentiation using an in vitro system. We induced differentiation of iPS cells into neural stem cells (NSCs) in vitro; the NSCs appeared morphologically indistinguishable from brain-derived NSCs and stained positive for the NSC markers Nestin and Sox2. These iPS cellderived NSCs (iPS-NSCs) were also capable of differentiating into all three neural subtypes. Interestingly, iPS-NSCs spontaneously formed aggregates on long-term culture and showed reactivation of the Oct4-GFP marker, which was followed by the formation of embryonic stem cell-like colonies. The spontaneously reverted green fluorescent protein (GFP)-positive (iPS-NSC-GFP 1 ) cells expressed high levels of pluripotency markers (Oct4 and Nanog) and formed germline chimeras, indicating that iPS-NSC-GFP 1 cells had the same pluripotency as the original iPS cells. The reactivation of silenced exogenous genes was tightly correlated with the downregulation of DNA methyltransferases (Dnmts) during differentiation of iPS cells. This phenomenon was not observed in doxycycline-inducible iPS cells, where the reactivation of exogenous genes could be induced only by doxycycline treatment. These results indicate that pluripotency can be regained through reactivation of exogenous genes, which is associated with dynamic change of Dnmt levels during differentiation of iPS cells.
Pluripotent stem cells, such as embryonic stem (ES) cells, can differentiate into all cell types. So, these cells can be a biological resource for regenerative medicine. However, ES cells known as standard pluripotent cells have problem to be used for cell therapy because of ethical issue of the origin and immune response on the graft. Hence, recently reprogrammed pluripotent cells have been suggested as an alternative source for regenerative medicine. Somatic cells can acquire the ES cell-like pluripotency by transferring somatic cell nuclei into oocytes, by cell fusion with pluripotent cells. Retroviral-mediated introduction of four factors, Oct4, Sox2, Klf4 and c-Myc can successfully reprogram somatic cells into ES cell-like pluripotent stem cells, known as induced pluripotent stem (iPS) cells. These cells closely resemble ES cells in gene expression pattern, cell biologic and phenotypic characteristics. However, to reach the eventual goal of clinical application, it is necessary to overcome the major drawbacks such as low reprogramming efficiency and genomic alterations due to viral integration. In this review, we discuss the current reprogramming techniques and mechanisms of nuclear reprogramming induced by transcription factor transduction.
Somatic cells are reprogrammed to induced pluripotent stem cells (iPSCs) by overexpression of a combination of defined transcription factors. We generated iPSCs from mouse embryonic fibroblasts (with Oct4-GFP reporter) by transfection of pCX-OSK-2A (Oct4, Sox2, and Klf4) and pCX-cMyc vectors. We could generate partially reprogrammed cells (XiPS-7), which maintained more than 20 passages in a partially reprogrammed state; the cells expressed Nanog but were Oct4-GFP negative. When the cells were transferred to serum-free medium (with serum replacement and basic fibroblast growth factor), the XiPS-7 cells converted to Oct4-GFP-positive iPSCs (XiPS-7c, fully reprogrammed cells) with ESC-like properties. During the conversion of XiPS-7 to XiPS-7c, we found several clusters of slowly reprogrammed genes, which were activated at later stages of reprogramming. Our results suggest that partial reprogrammed cells can be induced to full reprogramming status by serum-free medium, in which stem cell maintenance- and gamete generation-related genes were upregulated. These long-term expandable partially reprogrammed cells can be used to verify the mechanism of reprogramming.
Pluripotent stem cells can be derived from preimplantation and postimplantation mouse embryos. Embryonic stem cells (ESCs) derived from blastocysts are in a "naive" pluripotent state and meet all of the criteria for pluripotency, including the ability to generate live pups through tetraploid complementation. Epiblast stem cells (EpiSCs) derived from postimplantation epiblasts are in a "primed" pluripotent state. ESCs and EpiSCs show different phenotypes and gene expression patterns, and EpiSCs are thought to be less pluripotent than ESCs. In this study, we addressed whether EpiSCs can be differentiated into specialized cell types in vitro. To do this, we first derived EpiSCs from E5.5-6.5 mouse embryos containing the Oct4-GFP transgene. We found that EpiSCs expressed pluripotency markers and differentiated into all three germ layers in intro and in vivo. Interestingly, EpiSCs also efficiently differentiated into a homogenous population of neural stem cells (NSCs) in vitro. The EpiSC-derived NSCs (EpiSC-NSCs) expressed NSC markers (Nestin, Sox2, and Musashi), self-renewed for more than 20 passages, and differentiated into neuronal and glial neural cell subtypes in vitro. We then transplanted the EpiSC-NSCs into the neonatal mouse brains, and found that they were able to survive and differentiate into robust neurons and glial cells in the mouse brains, demonstrating that primed pluripotent EpiSCs efficiently form functional NSCs. We compared the global gene expression patterns of NSCs differentiated from EpiSC-NSCs, ESCs, and brain tissue and found that the expression patterns of most genes, including pluripotency and NSC specificity, were similarly clustered, but that the developmental process-related genes were distantly clustered. Moreover, the global gene expression pattern of brain-derived NSCs was more similar to that of ESC-derived NSCs than that of EpiSC-derived NSCs. Taken together, these results indicate that although NSCs, regardless of their origins, display very similar in vitro and in vivo differentiation properties, their global gene expression profiles may differ, depending on the pluripotency state, i.e., naive or primed.
BackgroundDifferentiated cells can be reprogrammed into pluripotency by transduction of four defined transcription factors. Induced pluripotent stem cells (iPS cells) are expected to be useful for regenerative medicine as well as basic research. Recently, the report showed that mouse embryonic fibroblasts (MEF) cells are not essential for reprogramming. However, in using fibroblasts as donor cells for reprogramming, individual fibroblasts that had failed to reprogram could function as feeder cells.Methodology/Principal FindingHere, we show that adult mouse neural stem cells (NSCs), which are not functional feeder cells, can be reprogrammed into iPS cells using defined four factors (Oct4, Sox2, Klf4, and c-Myc) under feeder-free conditions. The iPS cells, generated from NSCs expressing the Oct4-GFP reporter gene, could proliferate for more than two months (passage 20). Generated and maintained without feeder cells, these iPS cells expressed pluripotency markers (Oct4 and Nanog), the promoter regions of Oct4 and Nanog were hypomethylated, could differentiated into to all three germ layers in vitro, and formed a germline chimera. These data indicate that NSCs can achieve and maintain pluripotency under feeder-free conditions.Conclusion/SignificanceThis study suggested that factors secreted by feeder cells are not essential in the initial/early stages of reprogramming and for pluripotency maintenance. This technology might be useful for a human system, as a feeder-free reprogramming system may help generate iPS cells of a clinical grade for tissue or organ regeneration.
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