Dissecting mechanisms of human islet differentiation and maturation through epigenome profiling

Abstract

Investigating pancreatic islet differentiation from human stem cells in vitro provides a unique opportunity to dissect mechanisms that operate during human development in utero. We developed methods to profile DNA methylation, chromatin accessibility, and histone modifications from pluripotent stem cells to mature pancreatic islet cells, uncovering widespread epigenome remodeling upon endocrine commitment. Key lineage-defining loci are epigenetically primed before activation, foreshadowing cell fate commitment, and we show that priming of αcell-specific enhancers steers polyhormonal cells toward an α-cell fate. We further dissect pioneer factors and core regulatory circuits across islet cell differentiation and maturation stages, which identify LMX1B as a key regulator of in vitro-derived endocrine progenitors. Finally, by contrasting maturing stem cell-derived to natural β-cells, we discover that circadian metabolic cycles trigger rhythmic control of insulin synthesis and release and promote mature insulin responsiveness via an increased glucose threshold. These findings form a basis for understanding mechanisms orchestrating human islet cell specification and maturation. 4 domains, putative pioneer factors that establish them, and their state dynamics throughout human islet development. We find that endocrine specification involves widespread enhancer resetting and is foreshadowed by the priming of lineage-specifying loci. Accordingly, we show that priming of α-cell-specific enhancers in PH cells precedes resolution toward an α-cell fate. We further infer core regulatory circuits for each islet differentiation stage, capturing both known and unexpected factors including LMX1B, which we validate as key for in vitro-derived endocrine progenitors. Finally, we contrast regulatory landscapes in SC-β with their natural counterparts to uncover a role for circadian rhythms in fostering mature insulin responsiveness.Metabolically synchronized cadaveric/SC-islets gain cyclic transcription of genes controlling insulin synthesis/release and rhythmic insulin responses with an increased glucose threshold, a hallmark of functional maturity. These data form a basis for understanding genetic and epigenetic mechanisms controlling human islet cell fate and function. RESULTS In vitro isolation of human islet developmental intermediatesWe used the directed differentiation of human pluripotent stem cells (hPSC) into functional islet cells as a model to study human islet development (Figures 1A and S1A). Based on marker genes, we used flow cytometry to isolate cells at defined differentiation stages representing key intermediates during islet lineage progression (Figures S1B and S1C): undifferentiated hPSC, SOX17 + definitive endoderm (DE) cells, PDX1 + early pancreatic progenitors (PP1), PDX1 + NKX6.1 + late progenitors (PP2), NGN3 + endocrine 5 progenitors (EN), and monohormonal (INS + GCG -) SC-β as well as polyhormonal (INS + GCG + )PH cells. To isolate live SC-β and PH, we developed a strategy that combines endocrine cell enric...

β-Cell Maturation and Identity in Health and Disease

“…They unveiled genomic loci fundamental for the α- and β-cell lineage specification and regulatory circuits necessary for the differentiation towards β-cells. Finally, they stretched the importance of the circadian metabolic cycles as critical factors for the achievement of functional mature β-cells [164].…”