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

Abstract

BackgroundInduced 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.MethodsWe 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.ResultsGenerated 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.ConclusionsEfficiency 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.Electronic supplementary materialThe online version of this article (doi:10.1186/s12967-016-1097-0) contains supplementary material, which is available to authorized users.

Establishment of Insulin-Producing Cells From Human Embryonic Stem Cells Underhypoxic Condition for Cell Based Therapy

“…This finding suggests the presence of a common progenitor of neurons and insulin-positive cells during in vitro differentiation (Soria, 2001 ). Moreover, the IPC derived from induced pluripotent stem cells could be enhanced by overexpression of Pdx1 and Nkx6.1 factors (Walczak et al, 2016 ). In the present study, the expression of Nkx6.1 was detected starting at DE formation stage and then increased until the later stages of differentiation.…”

Episomal Induced Pluripotent Stem Cells: Functional and Potential Therapeutic Applications

“…Two components of the oriP and Epstein-Barr nuclear antigen-1 (EBNA-1) are also used widely in episomal plasmids [32][33][34][35][36][37][38] . Yu et al reported that human iPSCs that are completely free of plasmids and transgene sequences could be derived from fibroblasts by a single transfection with oriP/ EBNA-1-based episomal plasmids [39][40][41][42] .…”

“…The human cells reported to have been successfully reprogrammed into EiPSCs include fibroblasts, epithelial cells, keratinocytes, mononuclear cells from adult peripheral blood, cord blood cells, amniotic fluid stem cells, mesenchymal stromal cells, lymphoblasts, lamina propria progenitor cells from oral mucosa, and urothelial cells obtained from urine. A summary of these reported human EiPSC sources is shown in Table 1 8,20,21,[32][33][34][35][36][37][38][39][40][41][42][43][47][48][49][50][51][52]55,56,58, . The variety of cell lineages that can be reprogrammed by episomal techniques demonstrates the versatility of this technique, which has been used in many laboratories around the world.…”

Continuous inhibition of Sonic hedgehog signaling effectively leads to differentiation of human-induced pluripotent stem cells into functional insulin-producing β cells

“…This protocol involves the addition of various differentiation-inducing factors to the culture media at each differentiation stage, because of which the expression of transcription factors that are speci c to that particular pancreatic developmental stage is induced. Small molecules are also being widely used as differentiation-inducing factors, and IPCs generated using small molecules have shown robust function in vivo and in vitro [9][10][11][12].…”