Improve cellular fitness of human stem cell-derived islets under hypoxia

Stem cell-derived islet (SC-islet) cell therapy holds immense potential for the treatment of Type 1 Diabetes. However, low oxygen supply leads to cell dysfunction post-transplantation, especially in subcutaneous spaces and encapsulation devices. The response of SC-islets to hypoxia and effective strategies to alleviate its detrimental effects remain poorly understood. This study investigates the impact of hypoxia on SC-islets and elucidate the dynamics of hypoxia-induced stress. We performed transcriptional profiling of human SC-islets exposed to hypoxic conditions, and drew the transcriptomic and epigenetic profiles of single cells. Our findings demonstrate that ß cells within SC-islets gradually undergo a decline in cell identity and metabolic function in response to hypoxia. The loss of expression from immediate early genes, specifically EGR1, FOS, and JUN, results in the downregulation of key transcription factors (TFs) that are essential for maintaining SC-islet cell identity under hypoxic conditions. By comparing SC-islets under low and high oxygen conditions, we identified genes that play a role in maintaining the fitness of SC-islets in a low-oxygen environment. Notably, the expression of EDN3, a potent vasoconstrictor gene that is enriched in native pancreatic ß cells, significantly aids in preserving ß cell identity under hypoxic conditions. Elevated expression of EDN3 in SC-islets effectively mitigated the deleterious effects of hypoxia by modulating genes involved in SC-islet maturation including genes associated with glucose sensing and regulation in ß cells. These insights improve the understanding of SC-islets under hypoxic conditions, offering a potential point of intervention for future clinical applications in the treatment of Type 1 Diabetes Overall design: Human embryonic stem cell maintenance and differentiation was carried out with Human Embryonic Stem Cells 8 (HUES8) as previously described 33. SC-islet cell differentiations were initiated 72 h after initial passage by aspirating mTeSR1 (STEMCELL Technologies, 85850) and replenished with stage- and day-specific media supplemented with the appropriate small molecules or growth factors as previously described. SC-islets after enrichment and reaggregation were maintained in 30 mL dispensable bioreactor to ensure gas equilibration and cultured in incubators (Eppendorf, C170i) with three different oxygen tensions-21%, 5% and 2% O2. All cell culture was maintained in a humidified incubator at 5% CO2 and 37 °C. Tissue culture incubators were adapted to maintain different oxygen tensions. A nitrogen compressor was used to pulse N2 into incubators with a feedback loop to maintain the specified oxygen tension. All tissue culture handling was done outside of hypoxia incubators (at environmental oxygen tensions of 21% O2). Enriched SC-islets were fed with fresh S6 medium before hypoxia challenge and every other day during hypoxic incubation.