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.

Stem cell-derived tissues offer platforms to study organ development, model physiology during health and disease, and test novel therapies. We describe methods to isolate cells at successive stages during in vitro differentiation of human stem cells into the pancreatic endocrine lineage. Using flow cytometry, we purify live lineage intermediates in numbers not available by fetal biopsy. These include pancreatic and endocrine progenitors, isolated based on known surface markers, and we report a strategy that leverages intracellular zinc content and DPP4/CD26 expression to separate monohormonal insulin + β cells from polyhormonal counterparts. These methods enable comprehensive molecular profiling during human islet lineage progression.BASIC PROTOCOL 1: In vitro isolation of human islet developmental intermediates

Stem cell-derived tissues that recap endogenous physiology are key for regenerative medicine. Yet, most methods yield products that function like fetal, not adult tissues. Organoids are typically grown in constant environments, while our tissues mature along with behavioral cycles. Here, we show that inducing circadian rhythms in pancreatic islet organoids, by entraining them to daily feeding-fasting cycles, elicits their metabolic maturation. Our results show that rhythms can be harnessed to further functional maturation of organoids destined for human therapeutics.