�����ƽ��

Neuronal conversion from human fibroblasts can be induced by lineage-specific transcription factors; however, the introduction of ectopic genes limits the therapeutic applications of such induced neurons (iNs). Here, we report that human fibroblasts can be directly converted into neuronal cells by a chemical cocktail of seven small molecules, bypassing a neural progenitor stage. These human chemical-induced neuronal cells (hciNs) resembled hiPSC-derived neurons and human iNs (hiNs) with respect to morphology, gene expression profiles, and electrophysiological properties. This approach was further applied to generate hciNs from familial Alzheimer's disease patients. Taken together, our transgene-free and chemical-only approach for direct reprogramming of human fibroblasts into neurons provides an alternative strategy for modeling neurological diseases and for regenerative medicine.

Human induced pluripotent stem (hiPS) cells have potential uses for drug discovery and cell therapy, including generation of pancreatic β-cells for diabetes research and treatment. In this study, we developed a simple protocol for generating insulin-producing cells from hiPS cells. Treatment with activin A and a GSK3β inhibitor enhanced efficient endodermal differentiation, and then combined treatment with retinoic acid, a bone morphogenic protein inhibitor, and a transforming growth factor-β (TGF-β) inhibitor induced efficient differentiation of pancreatic progenitor cells from definitive endoderm. Expression of the pancreatic progenitor markers PDX1 and NGN3 was significantly increased at this step and most cells were positive for anti-PDX1 antibody. Moreover, several compounds, including forskolin, dexamethasone, and a TGF-β inhibitor, were found to induce the differentiation of insulin-producing cells from pancreatic progenitor cells. By combined treatment with these compounds, more than 10% of the cells became insulin positive. The differentiated cells secreted human c-peptide in response to various insulin secretagogues. In addition, all five hiPS cell lines that we examined showed efficient differentiation into insulin-producing cells with this protocol.

The embryonic stem cell-specific cell cycle-regulating (ESCC) family of microRNAs (miRNAs) enhances reprogramming of mouse embryonic fibroblasts to induced pluripotent stem cells. Here we show that the human ESCC miRNA orthologs hsa-miR-302b and hsa-miR-372 promote human somatic cell reprogramming. Furthermore, these miRNAs repress multiple target genes, with downregulation of individual targets only partially recapitulating the total miRNA effects. These targets regulate various cellular processes, including cell cycle, epithelial-mesenchymal transition (EMT), epigenetic regulation and vesicular transport. ESCC miRNAs have a known role in regulating the unique embryonic stem cell cycle. We show that they also increase the kinetics of mesenchymal-epithelial transition during reprogramming and block TGFβ-induced EMT of human epithelial cells. These results demonstrate that the ESCC miRNAs promote dedifferentiation by acting on multiple downstream pathways. We propose that individual miRNAs generally act through numerous pathways that synergize to regulate and enforce cell fate decisions.