Cell replacement therapy for type 1 diabetes (T1D), affecting millions of people worldwide, requires the immunoisolation of insulin-producing islets by encapsulation with a semi-impermeable material. Due to the shortage of donor islets, human pluripotent stem cell (hPSC) derived islets are an attractive alternative. However, properties of the encapsulating substrate are known to influence hPSC cell fate. In this work, we determine the effect of substrate stiffness on growth and pancreatic fate of encapsulated hPSCs. We precisely identify the range of substrate properties conducive for pancreatic cell fate, and also the mechanism by which substrate properties modify the cell signaling pathways and hence cell fate. Such information will be critical in driving regenerative cell therapy for long term treatment of T1D.

This study provides a detailed experimental and mathematical analysis of the impact of the initial pathway of definitive endoderm (DE) induction on later stages of pancreatic maturation. Human embryonic stem cells (hESCs) were induced to insulin-producing cells following a directed-differentiation approach. DE was induced following four alternative pathway modulations. DE derivatives obtained from these alternate pathways were subjected to pancreatic progenitor (PP) induction and maturation and analyzed at each stage. Results indicate that late stage maturation is influenced by the initial pathway of DE commitment. Detailed quantitative analysis revealed WNT3A and FGF2 induced DE cells showed highest expression of insulin, are closely aligned in gene expression patterning and have a closer resemblance to pancreatic organogenesis. Conversely, BMP4 at DE induction gave most divergent differentiation dynamics with lowest insulin upregulation, but highest glucagon upregulation. Additionally, we have concluded that early analysis of PP markers is indicative of its potential for pancreatic maturation.

Whole organ engineering has emerged as a promising alternative avenue to fill the gap of donor organ shortage in organ transplantation. Recent breakthroughs in the decellularization of solid organs and repopulation with desired cell populations have generated neo-organ constructs with promising functional outcomes. The realization of this goal requires engineering advancement in the perfusion-based bioreactors to (i) efficiently deliver decellularization agents, followed by (ii) its reconstruction with relevant cell types and (iii) maintenance of viability and function of the repopulated organ. In this study, we report the development and assembly of a perfusion bioreactor with the potential to enable regenerative reconstruction of pancreas. The assembled bioreactor is versatile to efficiently decellularize multiple organs, as demonstrated by complete decellularization of pancreas, liver and heart in the same set-up. Further, the same system is amenable to support organ repopulation with diverse cell types. Using our in-house bioreactor system, we demonstrate pancreas repopulation with both immortalized MIN-6 beta cells and differentiating human pluripotent stem cells. Importantly, we show the significant advantage of perfusion culture over static culture in enhancing cell engraftment, viability and phenotypic maintenance of the repopulated pancreas. In addition, this study is a significant step forward for whole organ engineering as it will facilitate cost-effective and easy assembly of perfusion bioreactors to enable rapid advancement in regenerative organ reconstruction.