In vitro differentiation of human stem cells can produce pancreatic β cells; the loss of this insulin-secreting cell type underlies type 1 diabetes. Here, as a step towards understanding this differentiation process, we report the transcriptional profiling of over 100,000 human cells undergoing in vitro β-cell differentiation, and describe the cells that emerged. We resolve populations that correspond to β cells, α-like poly-hormonal cells, non-endocrine cells that resemble pancreatic exocrine cells and a previously unreported population that resembles enterochromaffin cells. We show that endocrine cells maintain their identity in culture in the absence of exogenous growth factors, and that gene-expression changes associated with in vivo β-cell maturation are recapitulated in vitro. We implement a scalable re-aggregation technique to deplete non-endocrine cells and identify CD49a (also known as ITGA1) as a surface marker for the β-cell population, which allows magnetic sorting to a purity of 80%. Finally, we use a high-resolution sequencing time course to characterize gene-expression dynamics during human pancreatic endocrine induction, from which we develop a lineage model of in vitro β-cell differentiation. This study provides a deep perspective on human stem cell differentiation, and will guide future endeavours that focus on the differentiation of pancreatic islet cells, and applications in regenerative medicine.

Polymorphic HLAs form the primary immune barrier to cell therapy. In addition, innate immune surveillance impacts cell engraftment, yet a strategy to control both, adaptive and innate immunity, is lacking. Here we employed multiplex genome editing to specifically ablate the expression of the highly polymorphic HLA-A/-B/-C and HLA class II in human pluripotent stem cells. Furthermore, to prevent innate immune rejection and further suppress adaptive immune responses, we expressed the immunomodulatory factors PD-L1, HLA-G, and the macrophage “don’t-eat me” signal CD47 from the AAVS1 safe harbor locus. Utilizing in vitro and in vivo immunoassays, we found that T cell responses were blunted. Moreover, NK cell killing and macrophage engulfment of our engineered cells were minimal. Our results describe an approach that effectively targets adaptive as well as innate immune responses and may therefore enable cell therapy on a broader scale.

Highlights d Epigenome dynamics of human-stem-cell-derived islet differentiation and maturation d Pioneer factors coordinate pervasive enhancer priming to steer endocrine cell fates d Core regulatory circuits identify LMX1B as critical for endocrine cell generation d Circadian rhythms trigger islet maturation via clockcontrolled metabolic cycles SUMMARY Stem-cell-derived tissues could transform disease research and therapy, yet most methods generate functionally immature products. We investigate how human pluripotent stem cells (hPSCs) differentiate into pancreatic islets in vitro by profiling DNA methylation, chromatin accessibility, and histone modification changes. We find that enhancer potential is reset upon lineage commitment and show how pervasive epigenetic priming steers endocrine cell fates.Modeling islet differentiation and maturation regulatory circuits reveals genes critical for generating endocrine cells and identifies circadian control as limiting for in vitro islet function. Entrainment to circadian feeding/fasting cycles triggers islet metabolic maturation by inducing cyclic synthesis of energy metabolism and insulin secretion effectors, including antiphasic insulin and glucagon pulses. Following entrainment, hPSC-derived islets gain persistent chromatin changes and rhythmic insulin responses with a raised glucose threshold, a hallmark of functional maturity, and function within days of transplantation. Thus, hPSC-derived tissues are amenable to functional improvement by circadian modulation.