Abstract
Differentiation of human pluripotent stem cells into insulin-producing stem cell-derived beta cells harbors great potential for research and therapy of diabetes. SOX9 plays a crucial role during development of the pancreas and particularly in the development of insulin-producing cells as SOX9+ cells form the source for NEUROG3+ endocrine progenitor cells. For the purpose of easy monitoring of differentiation efficiencies into pancreatic progenitors and insulin-producing cells, we generated new reporter lines by knocking in a P2A-H-2Kk-F2A-GFP2 reporter gene into the SOX9-locus and a P2A-mCherry reporter gene into the INS-locus mediated by CRISPR/CAS9-technology. The knock-ins enabled co-expression of the endogenous and reporter genes and report on the endogenous gene expression. Furthermore, FACS and MACS enabled the purification of pancreatic progenitors and insulin-producing cells. Using these cell lines, we established a new differentiation protocol geared towards SOX9+ cells to efficiently drive human pluripotent stem cells into glucose-responsive beta cells. Our new protocol offers an alternative route towards stem cell-derived beta cells, pointing out the importance of Wnt/beta-catenin inhibition and the efficacy of EGF for the development of pancreatic progenitors, as well as the significance of 3D culture for the functionality of the generated beta cells.Graphic
Highlights
The SOX9 protein belongs to a family of high-mobility domain transcription factors with pleiotropic functions during development, cellular maintenance and disease development [1]
The aims of this study were to generate two types of reporter cell lines, namely a SOX9 and a SOX9/INS knock-in cell line and a differentiation protocol optimized towards the generation of SOX9+ multipotent pancreatic progenitor cells (MPCs)
The human pluripotent stem cell (hPSC) lines Hes-3 (‘ESC’) from ES Cell International and Phoenix (‘iPSC’, MHHi001-A) [12] were cultured on cell culture plastic coated with Matrigel (Corning, Amsterdam, Netherlands) and mTeSR1 (Stem Cell Technologies, Cologne, Germany) or StemMACSTM iPS-Brew XF medium (Miltenyi Biotec, Bergisch Gladbach, Germany) in an incubator with 37 °C and 5% C O2
Summary
The SOX9 protein belongs to a family of high-mobility domain transcription factors with pleiotropic functions during development, cellular maintenance and disease development [1]. SOX9 haploinsufficiency leads to campomelic dysplasia with pancreatic dysmorphogenesis [2]. These and other results have shown that SOX9 maintains the multipotent pancreatic progenitor pool and belongs to the group of master regulators of pancreatic development [3,4,5,6]. SOX9 expression during the initial forming of the dorsal and ventral pancreatic buds co-localizes with PDX1 in mouse and man [6, 7]. In mice, when the early unpolarized epithelium branches into a plexus with proximal trunk and distal tip domains, Sox is co-expressed with Cpa and Pdx in the tip domain or with Nkx6.1 and Pdx in the trunk domain [6].
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Disclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.