Mitochondrial development and remodeling occur at pancreatic progenitor stage during induction of stem cell-derived...

Despite decades of research, there is still no effective cure for type 1 diabetes, except islet transplantation, which is limited by healthy islet donors' shortage. Transplantation of surrogate insulin-producing β-cells from human pluripotent stem cells (hPSC) may provide an alternative solution when endogenous β-cells are nearly depleted. However, most differentiation protocols generate immature β-cells with impaired glucose-stimulated insulin secretion (GSIS) compared to primary β-cells. During β-cell development, there is a metabolic shift from glycolysis to OXPHOS metabolism to meet the increasing energy demand, suggesting that mitochondrial function is essential for hPSC-derived β-cell differentiation. This study investigated mitochondrial development and function during the differentiation process of hPSC into islet organoids. Two hPSC cell lines (HUES8 and iPSC824) were differentiated into islet organoids using a 6-stage differentiation protocol. Relevant stage-specific markers and GSIS were measured. Mitochondrial biogenesis, dynamics, morphological remodeling, and function were assessed to investigate mitochondria role in β-cell development. Mitochondrial contribution to total ATP production and lactate production were measured to assess the metabolic shift. The results showed, in both cell lines, >96% OCT4⁺ cells (pluripotency marker) at S0, >70% PDX1⁺ cells (pancreatic progenitor marker) induction at S3 and β-cells at S6 evidenced by NKX6.1⁺/insulin⁺ cells. The stage-specific markers measured by flow were confirmed by immunofluorescence, and functionality was assessed by responding to glucose stimulation. Moreover, our results demonstrate that the metabolic shift from glycolysis to OXPHOS occurred at pancreatic progenitor stage as evidenced by an increase in mitochondrial intensity. This shift in mitochondrial intensity coincided with (i-) enhance in mitochondrial biogenesis and dynamics (ii-) mitochondrial morphology remodeling from round shaped to elongated and well-defined cristae (iii-) increase in mitochondrial function related genes, and proteins as well as increased OXPHOS contribution to total ATP production (iv-) downregulation of "disallowed" glycolytic genes and proteins that ultimately promote functional islets.