Persistent Donor Cell Gene Expression among Human Induced Pluripotent Stem Cells Contributes to Differences with Human Embryonic Stem Cells

University of Calgary, Canada
PLoS ONE (Impact Factor: 3.23). 02/2010; 5(2):e8975. DOI: 10.1371/journal.pone.0008975
Source: PubMed


Human induced pluripotent stem cells (hiPSCs) generated by de-differentiation of adult somatic cells offer potential solutions for the ethical issues surrounding human embryonic stem cells (hESCs), as well as their immunologic rejection after cellular transplantation. However, although hiPSCs have been described as "embryonic stem cell-like", these cells have a distinct gene expression pattern compared to hESCs, making incomplete reprogramming a potential pitfall. It is unclear to what degree the difference in tissue of origin may contribute to these gene expression differences. To answer these important questions, a careful transcriptional profiling analysis is necessary to investigate the exact reprogramming state of hiPSCs, as well as analysis of the impression, if any, of the tissue of origin on the resulting hiPSCs. In this study, we compare the gene profiles of hiPSCs derived from fetal fibroblasts, neonatal fibroblasts, adipose stem cells, and keratinocytes to their corresponding donor cells and hESCs. Our analysis elucidates the overall degree of reprogramming within each hiPSC line, as well as the "distance" between each hiPSC line and its donor cell. We further identify genes that have a similar mode of regulation in hiPSCs and their corresponding donor cells compared to hESCs, allowing us to specify core sets of donor genes that continue to be expressed in each hiPSC line. We report that residual gene expression of the donor cell type contributes significantly to the differences among hiPSCs and hESCs, and adds to the incompleteness in reprogramming. Specifically, our analysis reveals that fetal fibroblast-derived hiPSCs are closer to hESCs, followed by adipose, neonatal fibroblast, and keratinocyte-derived hiPSCs.

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    • "Several studies have suggested that iPS cells might retain a certain degree of epigenetic memory similar to somatic cells from which they are derived from [34] [35] [36] [37]. This retention of epigenetic memory might involve histone modification [36] and DNA methylation [37]. "
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    ABSTRACT: Embryonal carcinoma (EC) cells, which are considered to be malignant counterparts of embryonic stem cells, comprise the pluripotent stem cell component of teratocarcinomas, a form of testicular germ cell tumors (GCT). Nevertheless, many established human EC cell lines are nullipotent with limited or no capacity to differentiate under normal circumstances. In this study, we tested whether an over-expression of Yamanaka's reprogramming factors OCT4, SOX2, c-MYC and KLF4 might enable differentiation of the human nullipotent EC cells N2102Ep. Using OCT4 knockdown differentiated N2102Ep cells, we are able to derive reprogrammed N2102Ep cell lines. The induced pluripotency of N2102Ep allows the cells to differentiate toward neural lineage by retinoic acid; expression of SSEA3 and SSEA4 is down-regulated, whereas that of neural surface markers is up-regulated. Consistent with the up-regulation of neural surface markers, expression of the master neuroectodermal transcription factor PAX6 is also induced in reprogrammed N2102Ep. We next investigated whether PAX6 might induce spontaneous differentiation of nullipotent stem cells N2102Ep. However, while an ectopic expression of PAX6 promotes differentiation of NTERA2, it induces cell death in N2102Ep. We nevertheless find that upon induction of retinoic acid, the reprogrammed N2102Ep cells form mature neuronal morphology similar to differentiated pluripotent stem cells NTERA2 as determined by TUJ1 expression, which is absence in N2102Ep parental cells. Altogether, we conclude that the nullipotent state of human EC cells can be reprogrammed to acquire a more relaxed state of differentiation potential by Yamanaka's factors.
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    • "Recent research has shown than the continuous passage of iPSCs can attenuate transcriptional, epigenetic, and functional differences (Hanna et al., 2010; Sullivan et al., 2010). Another study has identified that donor cell–specific gene expression patterns of human iPSCs in early passages are different from those in latepassage cells (Ghosh et al., 2010), suggesting an influence of continuous passage on the molecular properties of the resultant iPSCs. However, both studies only focused on the expression of differentiated genes and did not examine the effect on the expression of pluripotent genes in iPSCs by continuous passage. "
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    ABSTRACT: Abstract Induced pluripotent stem cells (iPSCs) are usually generated by reprogramming somatic cells through transduction with a transcription factor cocktail. However, the low efficiency of this procedure has kept iPSCs away from the study of the clinical application of stem cell biology. Our research shows that continuous passage increases the efficiency of reprogramming. Compared with conventional method of establishment of iPSCs, more embryonic stem cell (ESC)-like clones are generated by continuous passage during early reprogramming. These inchoate clones, indistinguishable from genuine ESC clones, are closer to fully reprogrammed cells compared with those derived from classical iPSC induction, which increased the expression of pluripotent gene markers and the levels of demethylation of Oct4 and Nanog. These results suggested that full reprogramming is a gradual process that does not merely end at the point of the activation of endogenous pluripotency-associated genes. Continuous passage could increase the pluripotency of induced cells and accelerate the process of reprogramming by epigenetic modification. In brief, we have provided an advanced strategy to accelerate the reprogramming and generate more nearly fully reprogrammed iPSCs efficiently and rapidly.
    01/2014; 16(1). DOI:10.1089/cell.2013.0067
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    • "However, these observations remain controversial, as another report detected residual donor-cell type memory in human iPSCs even at late passages [10]. It is intriguing that all the studies reporting a permanent memory of the cell of origin have been conducted in human iPSCs [8,10,14,15]. Thus, the discrepancy between these studies could be due to the different naïve and primed pluripotent cell stages exhibited by mouse and human iPSCs, respectively [16]. "
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    ABSTRACT: Transcription factor-based reprogramming can lead to the successful switching of cell fates. We have recently reported that mouse embryonic fibroblasts (MEFs) can be directly reprogrammed into induced neural stem cells (iNSCs) after the forced expression of Brn4, Sox2, Klf4, and Myc. Here, we tested whether iNSCs could be further reprogrammed into induced pluripotent stem cells (iPSCs). The two factors Oct4 and Klf4 were sufficient to induce pluripotency in iNSCs. Immunocytochemistry and gene expression analysis showed that iNSC-derived iPSCs (iNdiPSCs) are similar to embryonic stem cells at the molecular level. In addition, iNdiPSCs could differentiate into cells of all three germ layers, both in vitro and in vivo, proving that iNdiPSCs are bona fide pluripotent cells. Furthermore, analysis of the global gene expression profile showed that iNdiPSCs, in contrast to iNSCs, do not retain any MEF transcriptional memory even at early passages after reprogramming. Overall, our results demonstrate that iNSCs can be reprogrammed to pluripotency and suggest that cell fate can be redirected numerous times. Importantly, our findings indicate that the induced pluripotent cell state may erase the donor-cell type epigenetic memory more efficiently than other induced somatic cell fates.
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