Pezzolla, D. | Search bioRxiv

bioRxiv2023

Regulation of multiple signaling pathways promotes the consistent expansion of human pancreatic progenitors in defined conditions

The unlimited expansion of human progenitor cells in vitro could unlock many prospects for regenerative medicine. However, it remains an important challenge as it requires the decoupling of the mechanisms supporting progenitor self-renewal and expansion from those mechanisms promoting their differentiation. This study focuses on the expansion of human pluripotent stem (hPS) cell derived pancreatic progenitors (PP) to advance novel therapies for diabetes. We obtained mechanistic insights into PP expansion requirements and, through a hypothesis-driven iterative approach, identified conditions for the robust and unlimited expansion of hPS cell derived PP cells under GMP-compliant conditions. We show that the combined stimulation of specific mitogenic pathways, suppression of retinoic acid signaling and inhibition of selected branches of the TGF{beta} and Wnt signaling pathways are necessary for the effective decoupling of PP proliferation from differentiation. This enabled the reproducible, 2000-fold, over ten passages and 40-45 days, expansion of PDX1+/SOX9+/NKX6-1+ PP cells. Transcriptome analyses confirmed the stabilisation of PP identity and the effective suppression of differentiation. Using these conditions, PDX1+/SOX9+/NKX6-1+ PP cells, derived from different, both XY and XX, hPS cells lines, were enriched to nearly 90% homogeneity and expanded with very similar kinetics and efficiency. Furthermore, non-expanded and expanded PP cells, from different hPS cell lines, were differentiated in microwells into homogeneous islet-like clusters (SC-islets) with very similar efficiency. These clusters contained abundant {beta}-cells of comparable functionality as assessed by glucose-stimulated insulin secretion assays. These findings established the signaling requirements to decouple PP proliferation from differentiation and allowed the consistent expansion of hPS cell derived PP cells. They will enable the establishment of large banks of GMP-derived PP cells derived from diverse hPS cell lines. This approach will streamline SC-islet production for further development of the differentiation process, diabetes research, personalized medicine and cell therapies.

developmental biology↗

bioRxiv2023

Multi-organ single-cell RNA-sequencing reveals early hyperglycaemia responses that converge on fibroblast dysregulation

Diabetes causes a range of complications that can affect multiple organs. Hyperglycaemia is an important driver of diabetes-associated complications, mediated by biological processes such as dysfunction of endothelial cells, fibrosis and alterations in leukocyte number and function. Here, we dissected the transcriptional response of key cell types to hyperglycaemia across multiple tissues using single-cell RNA-seq (scRNA-seq) and identified conserved, as well as organ-specific, changes associated with diabetes complications. By studying an early timepoint of diabetes, we focus on biological processes involved in the initiation of the disease, before the later organ-specific manifestations had supervened. We used a mouse model of type 1 diabetes and performed scRNA-seq on cells isolated from the heart, kidney, liver and spleen of streptozotocin-treated and control mice after 8 weeks and assessed differences in cell abundance, gene expression, pathway activation and cell signalling across organs and within organs. In response to hyperglycaemia, endothelial cells, macrophages and monocytes displayed organ-specific transcriptional responses, whereas fibroblasts showed similar responses across organs, exhibiting a myofibroblast-like phenotype with altered metabolic gene expression and increased differentiation of myeloid-derived fibroblasts. Further, we found evidence of endothelial dysfunction in the kidney, and of endothelial to mesenchymal transition in streptozotocin-treated mouse organs. In summary, our study represents the first single-cell and multi-organ analysis of early dysfunction in type 1 diabetes-associated hyperglycaemia, and our large-scale dataset (comprising 67,611 cells) will serve as a starting point, reference atlas, and resource for further investigating the events leading to early diabetic disease.

cell biology↗