Recapitulating endocrine cell clustering in culture promotes maturation of human stem-cell-derived β cells

Abstract

Despite advances in the differentiation of insulin-producing cells from human embryonic stem cells, the generation of mature functional β cells in vitro has remained elusive. To accomplish this goal, we have developed cell culture conditions to closely mimic events occurring during pancreatic islet organogenesis and β cell maturation. In particular, we have focused on recapitulating endocrine cell clustering by isolating and reaggregating immature β-like cells to form islet-sized enriched β-clusters (eBCs). eBCs display physiological properties analogous to primary human β cells, including robust dynamic insulin secretion, increased calcium signalling in response to secretagogues, and improved mitochondrial energization. Notably, endocrine cell *

Live Cell Monitoring and Enrichment of Stem Cell‐Derived β Cells Using Intracellular Zinc Content as a Population Marker

“…Purification of stem cell-derived β cells for culture or experimental analysis has been reported recently by several groups, including our own (Nair et al, 2019;Veres et al, 2019). In both of these reports, enrichment of the differentiated endocrine population improves glucose-stimulated insulin secretion, making this a desirable process for experiments utilizing SC-β cells from a diverse collection of pluripotent stem cell sources.…”

“…In both of these reports, enrichment of the differentiated endocrine population improves glucose-stimulated insulin secretion, making this a desirable process for experiments utilizing SC-β cells from a diverse collection of pluripotent stem cell sources. For this reason, zinc-based enrichment offers a more rapid and broadly applicable method for enriching SC-β cells than genetically encoded reporters (Nair et al, 2019). Alternatively, CD49a enrichment is also useful in a number of different backgrounds, although this requires use of antibodies and needs more time and preparation for enrichment.…”

“…Here, we describe the use of live-cell zinc dyes for isolation and monitoring of SC-β cells. Recent reports have also suggested that enrichment of the SC-β population for extended culture may improve insulin secretory profiles of SC-β cells using genetic reporters (Nair et al, 2019;Veres et al, 2019). Here, we describe a method to enrich SC-β cells without the need for gene editing, allowing studies of SC-β biology across multiple genetic backgrounds and human islets.…”

Establishing cell-intrinsic limitations in cell cycle progression controls graft growth and promotes differentiation of pancreatic endocrine cells

“…Substantial progress has been made to differentiate pluripotent stem cells to insulin-producing cells [46][47][48][49] . In common to these studies is the activation or inhibition of developmental signaling pathways to promote differentiation to specific cell types.…”

“…Several strategies have been developed to reduce the risk of such outcomes. A flow-cytometry based method can be used to purify stem cell-derived insulin-producing cells labeled with a fluorescent reporter to prevent teratomas 48 . Furthermore, genetically engineering a suicide gene in a stem cell line can efficiently kill non-insulin-producing cells upon drug administration at the end of differentiation 55 .…”