The main advantage of the induced pluripotent stem cell (iPSC) approach to medicine is the ability to provide customized, patient-derived stem cells that can serve as a renewable source of replacement cells for autologous transplantation. We report the generation of iPSCs from adult tail-tip fibroblasts derived from wild-type and transgenic Pdx1-GFP knock-in mice by the retroviral transduction of three pluripotency factors Oct4, Sox2, and Klf4. iPSC clones highly resemble mouse embryonic stem cells (ESCs) in their defining characteristics: expression of pluripotency markers both at mRNA and protein levels, retroviral silencing capacity, and differentiation potential both in vitro and in vivo through the formation of embryoid bodies and teratomas, respectively. Despite its potential benefits, iPSCs have yet to be robust differentiated in vitro towards relevant cell types whose dysfunction drives the pathogenesis of diseases, like diabetes.
Diabetes mellitus, notably type 1 diabetes, can be potentially targeted by such iPSC-based therapies as it results from the destruction of one particular cell type in the adult, the insulin-secreting ß-cell. Islet transplantation has rendered some T1D patients insulin-independent, but requires toxic immunosuppressive regimens and large amounts of donor islets. Hence, the transplantation of iPSC-derived pancreatic islet or ß-cells appears to be the most favorable strategy. We differentiated Pdx1-GFP knock-in iPSCs towards the pancreas in vitro based on the external cues that drive pancreas specification in embryonic development. iPSCs. With high Activin-A treatment, iPSCs generate flat, epithelial cells that resemble the definitive endoderm, positive for FoxA2, Sox17, and Cxcr4. They further undergo pancreas specification upon the induction of markers Pdx1 and Hnf6. During the last stages, cells express endocrine hormone markers Insulin, Glucagon, and Somatostatin. Sorting for Pdx1-GFP+ cells purifies pancreatic progeny expressing endocrine hormone markers including Insulin2, confirming their pancreatic identity, albeit immature.
Finally, microRNAs (miRNAs) are a novel class of small, non-coding RNAs that fine-tune gene expression by inhibiting mRNA translation. The elucidation of their role during cell fate specification may improve reprogramming and differentiation. We describe that iPSCs assume an ESC-like miRNA signature upon reprogramming, marked by the upregulation of ESC-enriched clusters miR-290-295 and miR-302/367. On the other hand, upon treatment with 100 ng/ml Activin-A, we observe upregulation in 13 miRNAs that may play a role in endoderm induction, notably miR-141 and miR-218.
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