Directed differentiation of human iPSC into insulin producing cells is improved by induced expression of PDX1 and NKX6.1 factors in IPC progenitors.

BACKGROUND: Induced pluripotent stem cells (iPSC) possess an enormous potential as both, scientific and therapeutic tools. Their application in the regenerative medicine provides new treatment opportunities for numerous diseases, including type 1 diabetes. In this work we aimed to derive insulin producing cells (IPC) from iPS cells established in defined conditions. METHODS: We optimized iPSC generation protocol and created pluripotent cell lines with stably integrated PDX1 and NKX6.1 transgenes under the transcriptional control of doxycycline-inducible promoter. These cells were differentiated using small chemical molecules and recombinant Activin A in the sequential process through the definitive endoderm, pancreatic progenitor cells and insulin producing cells. Efficiency of the procedure was assessed by quantitative gene expression measurements, immunocytochemical stainings and functional assays for insulin secretion. RESULTS: Generated cells displayed molecular markers characteristic for respective steps of the differentiation. The obtained IPC secreted insulin and produced C-peptide with significantly higher hormone release level in case of the combined expression of PDX1 and NKX6.1 induced at the last stage of the differentiation. CONCLUSIONS: Efficiency of differentiation of iPSC to IPC can be increased by concurrent expression of PDX1 and NKX6.1 during progenitor cells maturation. Protocols established in our study allow for iPSC generation and derivation of IPC in chemically defined conditions free from animal-derived components, which is of the utmost importance in the light of their prospective applications in the field of regenerative medicine.

Generation of human iPSCs from cells of fibroblastic and epithelial origin by means of the oriP/EBNA-1 episomal reprogramming system.

INTRODUCTION: The prospect of therapeutic applications of the induced pluripotent stem cells (iPSCs) is based on their ability to generate virtually any cell type present in human body. Generation of iPSCs from somatic cells has opened up new possibilities to investigate stem cell biology, to better understand pathophysiology of human diseases, and to design new therapy approaches in the field of regenerative medicine. In this study, we focus on the ability of the episomal system, a non-viral and integration-free reprogramming method to derive iPSCs from somatic cells of various origin. METHODS: Cells originating from neonatal and adult tissue, renal epithelium, and amniotic fluid were reprogrammed by using origin of replication/Epstein-Barr virus nuclear antigen-1 (oriP/EBNA-1)-based episomal vectors carrying defined factors. The iPSC colony formation was evaluated by using immunocytochemistry and alkaline phosphatase assay and by investigating gene expression profiles. The trilineage formation potential of generated pluripotent cells was assessed by embryoid body-mediated differentiation. The impact of additionally introduced factors on episome-based reprogramming was also investigated. RESULTS: Reprogramming efficiencies were significantly higher for the epithelial cells compared with fibroblasts. The presence of additional factor miR 302/367 in episomal system enhanced reprogramming efficiencies in fibroblasts and epithelial cells, whereas the downregulation of Mbd3 expression increased iPSC colony-forming efficiency in fibroblasts solely. CONCLUSIONS: In this study, we performed a side-by-side comparison of iPSC colony-forming efficiencies in fibroblasts and epithelial cells transiently transfected with episomal plasmids and demonstrated that iPSC generation efficiency was highest when donor samples were derived from epithelial cells. We determined that reprogramming efficiency of episomal system could be further improved. Considering results obtained in the course of this study, we believe that episomal reprogramming provides a simple, reproducible, and efficient tool for generating clinically relevant pluripotent cells.