C/EBPα poises B cells for rapid reprogramming into induced pluripotent stem cells
One of the major goals of current stem cell research is understanding the mechanism of somatic cell reprogramming by Oct4, Sox2, Klf4 and Myc (OSKM) into induced pluripotent stem cells (iPSCs). However, the finding that only a small proportion of the cells become reprogrammed, typically requiring &g...
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| Formato: | tesis doctoral |
| Estado: | Versión publicada |
| Fecha de publicación: | 2014 |
| País: | España |
| Recursos: | CBUC, CESCA |
| Repositorio: | TDR. Tesis Doctorales en Red |
| OAI Identifier: | oai:www.tdx.cat:10803/283484 |
| Acesso em linha: | http://hdl.handle.net/10803/283484 |
| Access Level: | acceso abierto |
| Palavra-chave: | Stem cells Cell reprogramming iPS cells Pluripotent stem cells Embryonic stem cells Transdifferentiation Hematopoiesi Células madre Reprogramación celular Células iPS Células madre pluripotentes Células madre embrionaria Transdiferenciación Hematopoyeisis 576 |
| Resumo: | One of the major goals of current stem cell research is understanding the mechanism of somatic cell reprogramming by Oct4, Sox2, Klf4 and Myc (OSKM) into induced pluripotent stem cells (iPSCs). However, the finding that only a small proportion of the cells become reprogrammed, typically requiring >12 days, has hampered progress towards this goal. C/EBPα is a transcription factor specifically expressed in myelomonocytic cells within the hematopoietic system whose forced expression in B cells efficiently induces transdifferentiation into macrophages. We have now found that an 18-hour pulse of C/EBPα expression followed by OSKM activation induces an approximately 100-fold increase in the iPSC reprogramming efficiency, involving up to 95% of the cells within a week. Concomitantly, the cells undergo an epithelial-mesenchymal transition and pluripotency genes become upregulated to levels comparable to embryonic stem and iPS cells. In serum-free conditions the process is further accelerated, with 60% of the poised and OSKM induced B cells becoming Oct4-GFP positive within 2 days. These results are consistent with the idea that the C/EBPα pulse helps to overcome the stochastic phase of iPSC reprogramming. In addition, our work shed new light on the role of C/EBPα in induced pluripotency. Our data indicate that C/EBPα acts as a pathbreaker, at least in part mediated by the dioxygenase Tet2. C/EBPα binds to the Tet2 gene, induces its expression and translocates the protein to the nucleus. Here Tet2 binds to regulatory regions of pluripotency genes and converts methylated cytosine residues into hydroxymethylated cytosines. The pulse also renders the chromatin at regulatory sites of pluripotency genes accessible to DNase I digestion and, following OSKM induction, leads to local demethylation and to the binding of Oct4, correlating with the observed rapid upregulation of pluripotency genes. In line with an important role of Tet2 as a mediator of reprogramming, coexpression of the gene with OSKM enhanced B cell reprogramming substantially. The rapid and highly efficient iPSC reprogramming approach described herein should help to fully elucidate the early events of reprogramming to pluripotency and, if applicable to human cells, could have potential clinical applications. |
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