Photo-click hydrogels for 3D in situ differentiation of pancreatic progenitors from induced pluripotent stem cells

Abstract

Background Induced pluripotent stem cells (iPSC) can be differentiated to cells in all three germ layers, as well as cells in the extraembryonic tissues. Efforts in iPSC differentiation into pancreatic progenitors in vitro have largely been focused on optimizing soluble growth cues in conventional two-dimensional (2D) culture, whereas the impact of three-dimensional (3D) matrix properties on the morphogenesis of iPSC remains elusive. Methods In this work, we employ gelatin-based thiol-norbornene photo-click hydrogels for in situ 3D differentiation of human iPSCs into pancreatic progenitors (PP). Molecular analysis and single-cell RNA-sequencing were utilized to elucidate on the distinct identities of subpopulations within the 2D and 3D differentiated cells. Results We found that, while established soluble cues led to predominately PP cells in 2D culture, differentiation of iPSCs using the same soluble factors led to prominent branching morphogenesis, ductal network formation, and generation of diverse endoderm populations. Through single-cell RNA-sequencing, we found that 3D differentiation resulted in enrichments of pan-endodermal cells and ductal cells. We further noted the emergence of a group of extraembryonic cells in 3D, which was absent in 2D differentiation. The unexpected emergence of extraembryonic cells in 3D was found to be associated with enrichment of Wnt and BMP signaling pathways, which may have contributed to the emergence of diverse cell populations. The expressions of PP signature genes PDX1 and NKX6.1 were restored through inhibition of Wnt signaling at the beginning of the posterior foregut stage. Conclusions To our knowledge, this work established the first 3D hydrogel system for in situ differentiation of human iPSCs into PPs.

Soft Polyethylene Glycol Hydrogels Support Human PSC Pluripotency and Morphogenesis

“…Moreover, we observed that with decreasing hydrogel stiffness, more than 90% of aggregates in soft and stiff hydrogels showed the presence of a single lumen (Figure A,B). Similar results were reported by Arkenberg et al., where iPSCs were cultured in gelatin-PEG norbornene hydrogels with a G ′ of 500 and 1000 Pa . Cells cultured in the softer matrices had greater viability and an increased prevalence of morphogenic features like lumens.…”

“…Studies in 2D have shown that culturing cells on low adherence substrates upregulates pluripotency . Indeed, several studies using nonadhesive 3D culture matrices have reported an impressive ability to support hPSC pluripotency. ,, While other studies using PEG-based hydrogels have reported similar directed differentiations results, they were performed in the presence of matrix adhesion. ,, While outside the scope of this work, the effects of the hydrogel elastic modulus on pluripotency and differentiation may also be investigated using this system, holding promise as a future area of work.…”

“… 34 , 43 , 72 While other studies using PEG-based hydrogels have reported similar directed differentiations results, they were performed in the presence of matrix adhesion. 32 , 47 , 49 While outside the scope of this work, the effects of the hydrogel elastic modulus on pluripotency and differentiation may also be investigated using this system, holding promise as a future area of work.…”