222-OR: Overcoming Islet Scarcity through In Vitro Differentiation of Human Pluripotent Stem Cells

The potential of cell therapy for Type 1 Diabetes (T1D) has been constrained by the scarcity of islets and the dependence on obtaining them from deceased donors. The in vitro differentiation of islets from human pluripotent stem cells (hPSCs) could overcome this limitation to provide a sustainable cell source, facilitating the broad availability of tissues for therapeutic purposes. Challenges face this approach, including large-scale biomanufacturing of high-quality stem cell-derived islets (SC-islets) and limited understanding of transitions of key cellular identities. Here we have developed a scaled differentiation process that produces over one billion cells by first generating β cells that co-express C-peptide and NKX6-1 along with other endocrine cell types on large-surface area culture flasks using combinations of growth factors and small molecules. These cells can be single-cell dispersed and cryopreserved using a controlled-rate freezer and subsequently thawed and aggregated into islet-like clusters. These clusters are glucose-responsive and able to reverse diabetes when transplanted into a streptozotocin-induced diabetes mouse model. Using single-nuclei multiomic sequencing, we have mapped the entire differentiation process epigenetically and transcriptionally. Using this data, we defined the cellular transition states and timings for the generation of β, α, and δ cells. In addition, we have identified the transcription factor LMX1A as a key determinant in the generation of a serotonin-producing enterochromaffin-like cell state. These results demonstrate larger-scale biomanufacturing of SC-islets and provide a novel epigenomic roadmap of their differentiation.