Browsing by Subject "Photo-click chemistry"

Now showing 1 - 1 of 1
  • Thumbnail Image
    Item
    Photo-click hydrogels for 3D in situ differentiation of pancreatic progenitors from induced pluripotent stem cells
    (BMC, 2023-08-30) Arkenberg, Matthew R.; Ueda, Yoshitomo; Hashino, Eri; Lin, Chien‑Chi; Otolaryngology -- Head and Neck Surgery, School of Medicine
    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.