Capsule stiffness regulates the efficiency of pancreatic differentiation of human embryonic stem cells.

Encapsulation of donor islets using a hydrogel material is a well-studied strategy for islet transplantation, which protects donor islets from the host immune response. Replacement of donor islets by human embryonic stem cell (hESC) derived islets will also require a means of immune-isolating hESCs by encapsulation. However, a critical consideration of hESC differentiation is the effect of surrounding biophysical environment, in this case capsule biophysical properties, on differentiation. The objective of this study, thus, was to evaluate the effect of capsule properties on growth, viability, and differentiation of encapsulated hESCs throughout pancreatic induction. It was observed that even in the presence of soluble chemical cues for pancreatic induction, substrate properties can significantly modulate pancreatic differentiation, hence necessitating careful tuning of capsule properties. Capsules in the range of 4-7kPa supported cell growth and viability, whereas capsules of higher stiffness suppressed cell growth. While an increase in capsule stiffness enhanced differentiation at the intermediate definitive endoderm (DE) stage, increased stiffness strongly suppressed pancreatic progenitor (PP) induction. Signaling pathway analysis indicated an increase in pSMAD/pAKT levels with substrate stiffness likely the cause of enhancement of DE differentiation. In contrast, sonic hedgehog inhibition was more efficient under softer gel conditions, which is necessary for successful PP differentiation. STATEMENT OF SIGNIFICANCE: 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.

Potential for pancreatic maturation of differentiating human embryonic stem cells is sensitive to the specific pathway of definitive endoderm commitment.

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.