Differentiation of human pluripotent stem cells to islet organoids (SC-islets) containing functional β cells is a promising source of replacement cells for diabetes cellular therapy. While there has been significant successes in recent years generating these cells in vitro, stem cell-derived β (SC-β) cells are still functionally and transcriptionally immature when compared to donor primary β cells. Here, we identify and characterize the transcriptional changes that occur after transplantation of SC-islets derived from human embryonic stem (hESC) and induced pluripotent stem cell (hiPSC) lines. This was accomplished by developing methodologies to generate SC-β cells capable of undergoing dynamic glucose-stimulated insulin secretion in vitro along with the successful retrieval of high purity and viable transplanted cells. Using single-cell RNA sequencing of SC-islets before and after transplantation into diabetic mice, we identified 5,867 genes that were differentially expressed in SC-β cells. Genes and gene sets associated with β cell maturation were much more highly expressed after SC-β cell transplantation, including INS, MAFA, G6PC2, and UCN3 now matching primary β cells. SC-β cells had substantially reduced expression of the non-β cell hormone genes GCG and SST along with other off-target or immaturity genes, including ALDH1A1 and CRYBA2. Overall, the transcriptome of transplanted SC-β cells matured to closely resemble that of primary β cells from 3 donors. Single-cell analysis revealed that not only SC-β cells but also SC-α cells underwent drastic transcriptional changes to more closely resemble primary α cells. Similar results were observed with hESC- and hiPSC-derived islet organoids. This study provides an extensive indexing of transcriptional maturation of SC-islets through transplantation and serves as an important resource to understanding maturation of differentiated cell types to enable improved protocols for generating replacement cells.

Disclosure

P. Augsornworawat: None. K.G. Maxwell: None. J.R. Millman: Employee; Spouse/Partner; Acera Surgical, Inc. Other Relationship; Self; Harvard University, Washington University.

Funding

National Institutes of Health (R01DK114233, T32DK108742); JDRF (5-CDA-2017-391-A-N); Washington University in St. Louis

© 2020 by the American Diabetes Association
2020
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