Reprogramming Pluripotency Research Articles

LncRNA Osilr9 coordinates promoter DNA demethylation and the intrachromosomal loop structure required for maintaining stem cell pluripotency

Nuclear reprogramming of somatic cells into a pluripotent status has the potential to create patient-specific induced pluripotent stem cells for regenerative medicine. Currently, however, the epigenetic mechanisms underlying this pluripotent reprogramming are poorly understood. To delineate this epigenetic regulatory network, we utilized a chromatin RNA in situ reverse transcription sequencing (CRIST-seq) approach to identify long noncoding RNAs (lncRNAs) embedded in the 3-dimensional intrachromosomal architecture of stem cell core factor genes. By combining CRIST-seq and RNA sequencing, we identified Oct4-Sox2 interacting lncRNA 9 (Osilr9) as a pluripotency-associated lncRNA. Osilr9 expression was associated with the status of stem cell pluripotency in reprogramming. Using short hairpin RNA (shRNA) knockdown, we showed that this lncRNA was required for the optimal maintenance of stem cell pluripotency. Overexpression of Osilr9 induced robust activation of endogenous stem cell core factor genes in fibroblasts. Osilr9 participated in the formation of the intrachromosomal looping required for the maintenance of pluripotency. After binding to the Oct4 promoter, Osilr9 recruited the DNA demethylase ten-eleven translocation 1, leading to promoter demethylation. These data demonstrate that Osilr9 is a critical chromatin epigenetic modulator that coordinates the promoter activity of core stem cell factor genes, highlighting the critical role of pluripotency-associated lncRNAs in stem cell pluripotency and reprogramming.

CDK1-PDK1-PI3K/Akt signaling pathway regulates embryonic and induced pluripotency

The mechanisms of how signaling pathways are coordinated and integrated for the maintenance of the self-renewal of human embryonic stem cells (hESCs) and the acquisition of pluripotency in reprogramming are still only partly understood. CDK1 is a key regulator of mitosis. Recently, CDK1 has been shown to be involved in regulating self-renewal of stem cells, even though the mechanistic role of how CDK1 regulates pluripotency is unknown. In this report, we aim to understand how CDK1 can control pluripotency by reducing CDK1 activity to a level that has no effect on cell cycle progression. We demonstrated that high levels of CDK1 is associated with the pluripotency stage of hESCs; and decreased CDK1 activity to a level without perturbing the cell cycle is sufficient to induce differentiation. CDK1 specifically targets the phosphorylation of PDK1 and consequently the activity of PI3K/Akt and its effectors ERK and GSK3β. Evidence of the reversion of inactive CDK1-mediated differentiation by the inhibition of Akt signaling effectors suggests that the CDK1-PDK1-PI3K/Akt kinase cascade is a functional signaling pathway for the pluripotency of hESCs. Moreover, cyclin B1-CDK1 complexes promote somatic reprogramming efficiency, probably by regulating the maturation of induced pluripotent stem cells (iPSCs), as cyclin B1 stimulates a higher cellular level of LIN28A, suggesting that monitoring iPSC factors could be a new path for the enhancement of reprogramming efficiency. Together, we demonstrate an essential role for the CDK1-PDK1-PI3K/Akt kinase signaling pathway in the regulation of self-renewal, differentiation, and somatic reprogramming, which provides a novel kinase cascade mechanism for pluripotency control and acquisition.

Epigenetic marks in somatic chromatin are remodelled to resemble pluripotent nuclei by amphibian oocyte extracts

Reprogramming pluripotency after nuclear transplantation shows that molecules in oocytes can remodel somatic chromatin to a stem cell state. Here we report on an ex-ovo system using axolotl oocyte extracts to remodel epigenetic marks of somatic chromatin. Molecules present in axolotl oocyte extracts induce the reduction of the overall levels of H3K9me3, HP1α, and DNA methylation of somatic cells, and they increase the levels of H3K9ac. The levels of signal intensity detected in treated differentiated cells resemble those detected in embryonic stem cells, which are, in contrast, unaffected by these extracts. Analysis of specific genome sequences shows that somatic cells exposed to oocyte extracts undergo demethylation of LINE-1 repeats but Major Satellite repeats and the imprinted gene H19 remain unchanged. In addition, they induce demethylation of the Oct-4 promoter. Finally, the kinetics of activation of Oct-4 and Nanog expression from MEF nuclei treated in extracts suggests that these genes are subject to different levels of epigenetic control. The results demonstrate that axolotl oocyte extracts are a useful tool for studying epigenetic remodelling of somatic cells to a stem cell configuration, and for elucidating oocyte specific mechanisms of nuclear reprogramming.