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. 2020 May 7;11:2241. doi: 10.1038/s41467-020-16049-3

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© The Author(s) 2020

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 1 — a Embryonic stem cell differentiation to SC-beta cells results in a heterogeneous population. In this differentiation, 8.9% of cells coexpress markers for insulin and glucagon. b Schematic of directed differentiation protocol from hPSC into pre-alpha cells. The factors added at each step are noted below, catalog numbers are provided in Supplementary Table 1. c In the resulting pre-alpha cell differentiation, 63% of the cells stain for glucagon and insulin proteins. d Immunofluorescent cross-sectional staining of a cluster for insulin and glucagon. Scale bar = 50 μm. e Single-cell RNAseq analysis of pre-alpha cells from the SC-alpha protocol. Expression data are visualized with a tSNE plot where cells are clustered based on the similarity of transcript expression patterns. The expression profile for cells in the pre-alpha cell cluster is similar to the expression profile of human islet alpha cells (Supplementary Fig. 17b). Population ratios by this analysis are consistent with the flow cytometry plot in c. f Histograms of expression levels (as violin plots) for pancreatic hormones of SC-alpha cells and cadaveric human islets²⁷. g Pre-alpha cells secrete glucagon, but do not secret insulin. Representative ELISA measurements of secreted glucagon (left panel) from HUES8 differentiated pre-alpha cells or human islets challenged sequentially with 2.8 or 20 mM glucose with a 60-min incubation for each concentration. Representative ELISA measurement of secreted full processed human insulin (right panel) from HUES8 differentiated pre-alpha cells or human islets challenged sequentially with 2.8 or 20 mM glucose with a 60-min incubation for each concentration. Data presented as mean glucagon or insulin secreted per 1000 total cells ± SEM (n = 3 biologically independent samples) Significance calculated using a two-tailed paired Student’s t test. ESC: embryonic stem cell, DE: definitive endoderm, GTE: gut tube endoderm, PP: pancreatic progenitor, EP: endocrine progenitor, PA: pre-alpha cell, KGF: keratinocyte growth factor, LDN: LDN193189, Alk5i: Alk5 inhibitor II, Repl.: replicating cells.