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.

Highlights d scRNA-seq of hESC differentiation into b cells in vitro d Progenitor and endocrine compartments express different WNT pathway components d APC deletion in mouse endocrine precursors prevents endocrine differentiation d Small molecule inhibition of the WNT pathway in vitro increases endocrine yield

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