Forward programming of hiPSCs towards beta-like cells using Ngn3, Pdx1, and MafA

Abstract

Abstract Transplantation of stem cell-derived β-cells is a promising therapeutic advancement in the treatment of type 1 diabetes mellitus. A current limitation of this approach is the long differentiation timeline of up to two months that generates a heterogeneous population of pancreatic endocrine cells. To address this limitation, an inducible lentiviral overexpression system of mature β-cell markers was introduced into human induced-pluripotent stem cells (hiPSCs). Following selection of the successfully transduced hiPSCs, the cells were treated with doxycycline in pancreatic progenitor induction medium to support their transition towards the pancreatic lineage. Cells cultured with doxycycline presented the markers of interest, NGN3, PDX1, and MAFA, after five days of culture, and glucose-stimulated insulin secretion assays demonstrated that the cells were glucose-responsive in a monolayer culture. When cultured as a spheroid, the markers of interest and insulin secretion in a parallel glucose-stimulated insulin secretion assay were maintained; however, insulin secretion upon consecutive glucose challenges was limited. Comparison to human fetal and adult donor tissues identified that although the hiPSC-derived spheroids present similar markers to adult insulin-producing cells, they are functionally representative of fetal development. Together, these results suggest that with optimization of the temporal expression of these markers, forward programming of hiPSCs towards insulin-producing cells could be a possible alternative for islet transplantation.