Single-nucleus multi-omics of human stem cell-derived islets identifies deficiencies in lineage specification

Abstract

Insulin-producing β cells created from human pluripotent stem cells have potential as a therapy for insulin-dependent diabetes, but human pluripotent stem cell-derived islets (SC-islets) still differ from their in vivo counterparts. To better understand the state of cell types within SC-islets and identify lineage specification deficiencies, we used single-nucleus multi-omic sequencing to analyse chromatin accessibility and transcriptional profiles of SC-islets and primary human islets. Here we provide an analysis that enabled the derivation of gene lists and activity for identifying each SC-islet cell type compared with primary islets. Within SC-islets, we found that the difference between β cells and awry enterochromaffin-like cells is a gradient of cell states rather than a stark difference in identity. Furthermore, transplantation of SC-islets in vivo improved cellular identities overtime, while long-term in vitro culture did not. Collectively, our results highlight the importance of chromatin and transcriptional landscapes during islet cell specification and maturation.

Novel MAFA+, ISL1+, NKX6-1-stem cell derived pancreatic cell clusters secrete insulin and control blood glucose in rodent models of diabetes

“…Grafts of NKX6-1-high clusters (first protocol) revealed a loss of C-peptide expression in the NKX6-1 positive population over time (figure 5A-D). This contrasts with previous reports that PDX-1/NKX6-1 co-positive cells mature in a hyperglycemic background in vivo to generate insulin-secreting cells homologous to native beta cells in this time frame [3, 16] Clusters from both protocols were found on explant to have been remodeled with host tissue to form structures with abundant, human endocrine tissue comingled with remodeled host tissue (figure 5 A-H). Although the clusters were delivered as a bolus, explants revealed islet like structures that were separated from each other by tissue that resembled ducts and parenchyma.…”

“…Protocols for guiding pancreatic maturation of pluripotent stem cells aim to trigger pancreatic fate choice and restrict non-pancreatic lineages. These protocols create populations comprised of cells with functional pancreatic features and off-target cells [3-5]. We hypothesize that the presence of cells without appropriate endocrine specification can diminish the clinical potency and safety of the population.…”

“…NKX6-1 is activated in early pancreatic endocrine cell commitment and is again expressed in insulin-secreting beta cells [11]. Among populations of differentiating pluripotent stem cells, it has been suggested that PDX-1+/NKX6-1+ are fated to form functional insulin-secreting beta cells, while PDX-1+/NKX6-1-cells form polyhormonal cells that either resolve in vivo to glucagon-secreting alpha cells or non-functional off-target cells [3, 11-14]. NKX6-1 also has an important role in the specification of the enterochromaffin population of serotonin-producing enteroendocrine cells of the gut [15].…”

“…NKX6-1 also has an important role in the specification of the enterochromaffin population of serotonin-producing enteroendocrine cells of the gut [15]. Pancreatic and enterochromaffin populations expressing NKX6-1 emerge from a common NGN3-expressing progenitor population and share similar functional features [3, 5, 15, 16]. Because serotonin-expressing cells are common among populations of stem cell-derived pancreatic cells, it is speculated that stem cell-derived pancreatic populations contain off target cells with enterochromaffin identity [16-19].…”

Scaling Insulin-Producing Cells by Multiple Strategies

“…In contrast, single cell transcriptomic analysis complemented by single cell transposase-accessible chromatin sequencing on β-like cells, revealed that the presence of enterochromaffin cells may represent an intermediary transitory state of pancreatic endocrine cells and intestinal cells [48]. This finding suggests the possibility of transdifferentiating enterochromaffin cells into a β cell identity by modulating the chromatin states, timing, and expression of key transcription factors and signals, similar to the process of transdifferentiating stomach cells into β-like cells [49,50].…”