An insulin hypersecretion phenotype precedes pancreatic ß cell failure in MODY3 patient-specific cells.

MODY3 is a monogenic hereditary form of diabetes caused by mutations in the transcription factor HNF1A. The patients progressively develop hyperglycemia due to perturbed insulin secretion, but the pathogenesis is unknown. Using patient-specific hiPSCs, we recapitulate the insulin secretion sensitivity to the membrane depolarizing agent sulfonylurea commonly observed in MODY3 patients. Unexpectedly, MODY3 patient-specific HNF1A+/R272C ß cells hypersecrete insulin both in vitro and in vivo after transplantation into mice. Consistently, we identified a trend of increased birth weight in human HNF1A mutation carriers compared with healthy siblings. Reduced expression of potassium channels, specifically the KATP channel, in MODY3 ß cells, increased calcium signaling, and rescue of the insulin hypersecretion phenotype by pharmacological targeting ATP-sensitive potassium channels or low-voltage-activated calcium channels suggest that more efficient membrane depolarization underlies the hypersecretion of insulin in MODY3 ß cells. Our findings identify a pathogenic mechanism leading to ß cell failure in MODY3.

Enhanced OCT4 transcriptional activity substitutes for exogenous SOX2 in cellular reprogramming.

Adenoviral early region 1A (E1A) is a viral gene that can promote cellular proliferation and de-differentiation in mammalian cells, features required for the reprogramming of somatic cells to a pluripotent state. E1A has been shown to interact with OCT4, and as a consequence, to increase OCT4 transcriptional activity. Indeed, E1A and OCT4 are sufficient to revert neuroepithelial hybrids to pluripotency, as demonstrated in previous cell fusion experiments. However, the role that E1A might play in the generation of induced pluripotent stem cells (iPSCs) has not been investigated yet. In this report, we show that E1A can generate iPSCs in combination with OCT4 and KLF4, thus replacing exogenous SOX2. The generated iPSCs are bona fide pluripotent cells as shown by in vitro and in vivo tests. Overall, our study suggests that E1A might replace SOX2 through enhancing OCT4 transcriptional activity at the early stages of reprogramming.

Epigenetic Aberrations Are Not Specific to Transcription Factor-Mediated Reprogramming.

Somatic cells can be reprogrammed to pluripotency using different methods. In comparison with pluripotent cells obtained through somatic nuclear transfer, induced pluripotent stem cells (iPSCs) exhibit a higher number of epigenetic errors. Furthermore, most of these abnormalities have been described to be intrinsic to the iPSC technology. Here, we investigate whether the aberrant epigenetic patterns detected in iPSCs are specific to transcription factor-mediated reprogramming. We used germline stem cells (GSCs), which are the only adult cell type that can be converted into pluripotent cells (gPSCs) under defined culture conditions, and compared GSC-derived iPSCs and gPSCs at the transcriptional and epigenetic level. Our results show that both reprogramming methods generate indistinguishable states of pluripotency. GSC-derived iPSCs and gPSCs retained similar levels of donor cell-type memory and exhibited comparable numbers of reprogramming errors. Therefore, our study demonstrates that the epigenetic abnormalities detected in iPSCs are not specific to transcription factor-mediated reprogramming.

Dissecting the role of distinct OCT4-SOX2 heterodimer configurations in pluripotency.

The transcription factors OCT4 and SOX2 are required for generating induced pluripotent stem cells (iPSCs) and for maintaining embryonic stem cells (ESCs). OCT4 and SOX2 associate and bind to DNA in different configurations depending on the arrangement of their individual DNA binding elements. Here we have investigated the role of the different OCT4-SOX2-DNA assemblies in regulating and inducing pluripotency. To this end, we have generated SOX2 mutants that interfere with specific OCT4-SOX2 heterodimer configurations and assessed their ability to generate iPSCs and to rescue ESC self-renewal. Our results demonstrate that the OCT4-SOX2 configuration that dimerizes on a Hoxb1-like composite, a canonical element with juxtaposed individual binding sites, plays a more critical role in the induction and maintenance of pluripotency than any other OCT4-SOX2 configuration. Overall, the results of this study provide new insight into the protein interactions required to establish a de novo pluripotent network and to maintain a true pluripotent cell fate.

Counteracting activities of OCT4 and KLF4 during reprogramming to pluripotency.

Differentiated cells can be reprogrammed into induced pluripotent stem cells (iPSCs) after overexpressing four transcription factors, of which Oct4 is essential. To elucidate the role of Oct4 during reprogramming, we investigated the immediate transcriptional response to inducible Oct4 overexpression in various somatic murine cell types using microarray analysis. By downregulating somatic-specific genes, Oct4 induction influenced each transcriptional program in a unique manner. A significant upregulation of pluripotent markers could not be detected. Therefore, OCT4 facilitates reprogramming by interfering with the somatic transcriptional network rather than by directly initiating a pluripotent gene-expression program. Finally, Oct4 overexpression upregulated the gene Mgarp in all the analyzed cell types. Strikingly, Mgarp expression decreases during the first steps of reprogramming due to a KLF4-dependent inhibition. At later stages, OCT4 counteracts the repressive activity of KLF4, thereby enhancing Mgarp expression. We show that this temporal expression pattern is crucial for the efficient generation of iPSCs.

Reprogramming to pluripotency through a somatic stem cell intermediate.

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.

Optimal reprogramming factor stoichiometry increases colony numbers and affects molecular characteristics of murine induced pluripotent stem cells.

Somatic cells can be reprogrammed toward pluripotency by overexpression of a set of transcription factors, yielding induced pluripotent stem cells (iPSCs) with features similar to embryonic stem cells. Little is known to date about stoichiometric requirements of the individual reprogramming factors (RFs) for efficient reprogramming and especially about whether stoichiometry also influences the quality of derived iPSCs. To address this important issue, we chose bicistronic lentiviral vectors coexpressing fluorescent reporters (eGFP, dTomato, Cerulean, or Venus) along with the canonical RFs to transduce a bulk of murine embryonic fibroblasts (MEFs). Using a flow cytometric approach, we were able to independently and proportionally quantify all fluorophores in multiple-infected MEFs and more importantly could sort these cells into all 16 stoichiometric combinations of high or moderate expression of the four factors. On average, we obtained about 600 alkaline phosphatase-expressing colonies from 20,000 seeded cells. Interestingly, only seven different stoichiometric ratios gave rise to any colonies at all. The by far most colonies were obtained from those fractions, where Oct4 was in excess over the other three factors (2,386 colonies/20,000 cells), or where both Oct4 and c-Myc were in excess over Sox2 and Klf4 (1,593 colonies/20,000 cells). Our findings suggest that increased Oct4 levels opposite to modest ones for Sox2 and Klf4 are required for satisfying reprogramming efficiencies and that these stoichiometries are also highly beneficial for achieving a stable pluripotent state independent of ectopic RF expression. Finally, the eligible Oct4(high) , Sox2(low) , and Klf4(low) subpopulation only resembles a small fraction of cells targeted by equal vector amounts, suggesting the necessity to address stoichiometry also in alternative approaches for iPSC generation or between different experimental systems.