New hPSC SOX9 and INS Reporter Cell Lines Facilitate the Observation and Optimization of Differentiation into Insulin-Producing Cells

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 Abstract

Islets in the body are never flat: transitioning from two-dimensional (2D) monolayer culture to three-dimensional (3D) spheroid for better efficiency in the generation of functional hPSC-derived pancreatic β cells in vitro

“…Furthermore, culturing cells as 3D aggregates increased the expression of pancreatic progenitor marker gene (PDX1) by threefold as compared to 2D [ 45 ]. Consistently, Dettmer et al [ 52 ] found a highest expression of Sox9 (a known transcription factor for pancreatic progenitors) in 3D compared to 2D using a reporter cell line, indicating that 3D protocol enhances the efficiency of pancreatic progenitors’ induction.…”

Proinflammatory cytokines induce rapid, NO-independent apoptosis, expression of chemotactic mediators and interleukin-32 secretion in human pluripotent stem cell-derived beta cells

“…Human cell culture Routine culturing of the mycoplasmanegative human reporter ESC lines HES-3 SC30 ICNC4 and HES-3 SC30 [22] and the parental cell line HES-3 ('ESC', ES Cell International [ESIBI], Singapore) was performed on Matrigel-coated (Corning, Amsterdam, the Netherlands) sixwell plates using mTeSR1 medium (Stem Cell Technologies, Cologne, Germany). The culture of EndoC-βH1 cells was performed according to the standard protocol [23].…”

“…The culture of EndoC-βH1 cells was performed according to the standard protocol [ 23 ]. Differentiation was performed as described before [ 22 ] (Fig. 1a ) and differentiation efficiency was monitored with the help of the reporter cell lines via flow cytometry (Fig.…”

“…However, directed differentiation does not exclusively generate SC-beta cells but also other pancreatic and non-pancreatic cell populations [ 21 ]. In order to address the problem of heterogeneity, we have recently developed dual reporter hESC lines (SOX9 [SRY-box transcription factor 9]-GFP2/H-2K K [H-2 class I histocompatibility antigen, K-K alpha chain], insulin [INS]-mCherry) [ 22 ]. This allowed us to assign the effects of our experiments to SC-beta cells in a highly specific manner and to differentiate them from other cell types.…”

Cross-regulation between SOX9 and the canonical Wnt signalling pathway in stem cells

“…Another study indicated that miR-690 induces Sox9 silencing and inactivates Wnt signalling, which leads to the arrested differentiation of β-cells from iPSC-derived insulin-producing cells ( Xu et al, 2019 ). Nevertheless, the activation of Wnt signalling arrests the generation of β-cells by attenuating SOX9-dependent multipotent pancreatic progenitor cells ( Dettmer et al, 2021 ). These findings collectively suggest that during SOX9-dependent pancreatic development and β-cell differentiation, the cross-regulation between SOX9 and Wnt signalling prevents the over-activation of SOX9.…”