Efficient Generation of Glucose-Responsive Beta Cells from Isolated GP2+ Human Pancreatic Progenitors

Abstract

Stem cell-based therapy for type 1 diabetes would benefit from implementation of a cell purification step at the pancreatic endoderm stage. This would increase the safety of the final cell product, allow the establishment of an intermediate-stage stem cell bank, and provide a means for upscaling β cell manufacturing. Comparative gene expression analysis revealed glycoprotein 2 (GP2) as a specific cell surface marker for isolating pancreatic endoderm cells (PECs) from differentiated hESCs and human fetal pancreas. Isolated GP2⁺ PECs efficiently differentiated into glucose responsive insulin-producing cells in vitro. We found that in vitro PEC proliferation declines due to enhanced expression of the cyclin-dependent kinase (CDK) inhibitors CDKN1A and CDKN2A. However, we identified a time window when reducing CDKN1A or CDKN2A expression increased proliferation and yield of GP2⁺ PECs. Altogether, our results contribute tools and concepts toward the isolation and use of PECs as a source for the safe production of hPSC-derived β cells.

Keywords: CDKN1A; CDKN2A; GP2; cell surface marker; cell-cycle regulators; differentiation; human embryonic stem cells; insulin-producing beta cells; pancreatic progenitors; type 1 diabetes.