Derivation Conditions Impact X-Inactivation Status in Female Human Induced Pluripotent Stem Cells

Gladstone Institute of Cardiovascular Disease, University of California, San Francisco, San Francisco, CA 94158, USA.
Cell stem cell (Impact Factor: 22.27). 07/2012; 11(1):91-9. DOI: 10.1016/j.stem.2012.05.019
Source: PubMed


Female human induced pluripotent stem cell (hiPSC) lines exhibit variability in X-inactivation status. The majority of hiPSC lines maintain one transcriptionally active X (Xa) and one inactive X (Xi) chromosome from donor cells. However, at low frequency, hiPSC lines with two Xas are produced, suggesting that epigenetic alterations of the Xi occur sporadically during reprogramming. We show here that X-inactivation status in female hiPSC lines depends on derivation conditions. hiPSC lines generated by the Kyoto method (retroviral or episomal reprogramming), which uses leukemia inhibitory factor (LIF)-expressing SNL feeders, frequently had two Xas. Early passage Xa/Xi hiPSC lines generated on non-SNL feeders were converted into Xa/Xa hiPSC lines after several passages on SNL feeders, and supplementation with recombinant LIF caused reactivation of some of X-linked genes. Thus, feeders are a significant factor affecting X-inactivation status. The efficient production of Xa/Xa hiPSC lines provides unprecedented opportunities to understand human X-reactivation and -inactivation.

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    • "Taken together, these reports lead to the conclusion that hPSCs and EpiSCs share a similar pluripotency state characterised by their dependency on Activin/Nodal signalling. Nevertheless, hESCs and EpiSCs are not strictly identical: contrary to EpiSCs, hESCs express pre-implantation markers such as REX1 (Chan et al., 2009) and not post-implantation markers such as FGF5 (Vallier et al., 2004), and also can exhibit X activation, indicative of pre-implantation stages (Lengner et al., 2010; Tomoda et al., 2012). These observations could underline species divergence. "
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    Full-text · Article · Feb 2015 · Development
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    • "Somatic stem cells reside in niches, and environmental changes, such as temperature, extracellular matrix proteins, stromal cell contacts, and oxygen tension, have a great influence on stem cell function and differentiation. For example, Tomoda et al. have recently reported that culturing conditions can influence the X chromosome inactivation status in hiPSC1213. X-chromosome reactivation (XaXa), a characteristic of naïve pluripotent state cells, rarely occurs during the reprogramming of human female somatic cells to induced pluripotent stem cells. Furthermore, mammalian embryonic epiblasts reside in a physiologically hypoxic environment, and culturing ESCs in a hypoxic environment is known to prevent differentiation of human ESCs and enhance generation of human and mouse iPS colonies141516. "
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    ABSTRACT: Standard culture of human induced pluripotent stem cells (hiPSCs) requires basic Fibroblast Growth Factor (bFGF) to maintain the pluripotent state, whereas hiPSC more closely resemble epiblast stem cells than true naïve state ES which requires LIF to maintain pluripotency. Here we show that chemokine (C-C motif) ligand 2 (CCL2) enhances the expression of pluripotent marker genes through the phosphorylation of the signal transducer and activator of transcription 3 (STAT3) protein. Moreover, comparison of transcriptomes between hiPSCs cultured with CCL2 versus with bFGF, we found that CCL2 activates hypoxia related genes, suggesting that CCL2 enhanced pluripotency by inducing a hypoxic-like response. Further, we show that hiPSCs cultured with CCL2 can differentiate at a higher efficiency than culturing with just bFGF and we show CCL2 can be used in feeder-free conditions in the absence of LIF. Taken together, our finding indicates the novel functions of CCL2 in enhancing its pluripotency in hiPSCs.
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    • "Two clones of dfD551K1-iPSC showed expression of the TT SNP GRPR of the parental fibroblasts (Tchieu et al., 2010). We found that six dfD551K1-iPSC clones expressed both AA and TT alleles of GRPR that may have acquired two active X chromosomes via X chromosome reactivation (XCR) during reprogramming (Tomoda et al., 2012). However , unexpectedly, two clones of dfD551K1-iPSC expressed GRPR with the AA allele. "
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    ABSTRACT: Induced pluripotent stem cells (iPSCs) acquire embryonic stem cell (ESC)-like epigenetic states, including the X chromosome. Previous studies reported that human iPSCs retain the inactive X chromosome of parental cells, or acquire two active X chromosomes through reprogramming. Most studies investigated the X chromosome states in established human iPSC clones after completion of reprogramming. Thus, it is still not fully understood when and how the X chromosome reactivation occurs during reprogramming. Here, we report a dynamic change in the X chromosome state throughout reprogramming, with an initial robust reactivation of the inactive X chromosome followed by an inactivation upon generation of nascent iPSC clones. iPSCs with two active X chromosomes or an eroded X chromosome arise in passaging iPSCs. These data provide important insights into the plasticity of the X chromosome of human female iPSCs and will be crucial for the future application of such cells in cell therapy and X-linked disease modeling.
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