Introduction: While excellent glycemic control without insulin injections can be achieved by transplantation of cadaveric islets, this strategy is severely limited by donor availability thus hampering its broader application to diabetes treatment. Differentiation of human pluripotent stem cells (hPSCs) to insulin-secreting beta cells offers an alternative, unlimited source of transplantable material. However, stem cell-derived beta cells made using current approaches typically remain functionally immature and do not fully resemble bona fide natural beta cells. 

Method: Building upon our previous studies in developing methods of generating islet cells from hPSCs, in this work we employed a hybrid strategy – combination of the STEMdiff^TM Pancreatic Progenitor Kit (from stage 1-4) and a modified Rezania’s 2014 Nature Biotechnology method (termed as “R-protocol” hereafter from stage 5-7) – in order to further improve the differentiation consistency and outcomes. 

Results: With the STEMdiff^TM Pancreatic Progenitor Kit, we consistently differentiated Mel1 INS^GFP/W hESCs (an insulin reporter line) into pancreatic progenitors in which over 80% of cells co-express key markers PDX1 and NKX6.1. Following the planar culture from stage 1-4, we used AggreWell^TM plates to generate pancreatic progenitor clusters with uniform size and morphology. With modifications, we used R-protocol to further differentiate pancreatic progenitors into islet clusters and switched the cultures to a static suspension format for endocrine differentiation. At the end of differentiation, flow cytometry and immunostaining analysis showed that these islet clusters were comprised of three major islet cell types including insulin-expressing beta cells, glucagon-expressing alpha cells and somatostatin-expressing delta cells, as well as expressed transcription factors key in beta cell maturation. In vitro functional assessment further showed that the islet clusters displayed dynamic insulin secretion in response to glucose and an incretin hormone as well as depolarization challenge, yet did not fully mimic primary human islets. 

Conclusion: Future efforts will focus on continued optimization of the differentiation protocols and increasing the scale to manufacture more mature islet cells, demonstrating applications of these stem cell-derived islet clusters in disease modeling, drug screening, and ultimately diabetes reversal.