The Effect of Wnt Pathway Modulators on Human iPSC-Derived Pancreatic Beta Cell Maturation

Abstract

Current published protocols for targeted differentiation of human stem cells toward pancreatic β-cells fail to deliver sufficiently mature cells with functional properties comparable to human islet β-cells. We aimed to assess whether Wnt-modulation could promote the final protocol stages of β-cell maturation, building our hypothesis on our previous findings of Wnt activation in immature hiPSC-derived stage 7 (S7) cells compared to adult human islets and with recent data reporting a link between Wnt/PCP and in vitro β-cell maturation. In this study, we stimulated canonical and non-canonical Wnt signaling in hiPSC-derived S7 cells using syntetic proteins including WNT3A, WNT4, WNT5A and WNT5B, and we inhibited endogenous Wnt signaling with the Tankyrase inhibitor G007-LK (TKi). Whereas neither canonical nor non-canonical Wnt stimulation alone was able to mature hiPSC-derived S7 cells, WNT-inhibition with TKi increased the fraction of monohormonal cells and global proteomics of TKi-treated S7 cells showed a proteomic signature more similar to adult human islets, suggesting that inhibition of endogenous Wnt contributes toward final β-cell maturation.

In vivo hyperglycaemia exposure elicits distinct period‐dependent effects on human pancreatic progenitor differentiation, conveyed by oxidative stress

“…To investigate the in vivo impact of hyperglycaemia on islet cells differentiation, we xenotransplanted (TX) alginate‐encapsulated hiPSC‐derived pancreatic progenitor cells (here forth S5‐cells) in either normal or diabetic humanized NSG rat insulin promoter (RIP)‐diphtheria toxin receptor (DTR) mice (Figure A). For β‐cell differentiation we used the protocol designed by Rezania et al with slight modifications as previously described . The encapsulated cells were exposed to either a brief (1 week, here on 1w‐postTX) or an extended period of time (4 weeks, here on 4w‐postTX) in the in vivo hyperglycaemic environment.…”

“…Encapsulated cells were lysed directly in the alginate beads, in a buffer containing 8 mol/L Urea, 200 mmol/L EPPS pH8.5 and protease inhibitors (Roche complete with EDTA) and sonicated (30 seconds × 3 times at 30% power). Human islets were processed as previously described, is short by boiling in 4% SDS buffer before sonication. Chloroform‐Methanol precipitation was performed on alginate beads as previously described, involving adding methanol, chloroform and water before centrifugation thereafter the alginate were removed as best as possible.…”

“…Clusters 0 and for 1 W and 0, 5 and 8 for 4 W were manually curated. The pathway analyses were generated by QIAGEN's IPA program (IPA®, QIAGEN, http://www.qiagen.com/ingenuity) as previously described, here using 35 molecules/network; 25 networks/analysis for generating the interaction networks.…”

“…For β-cell differentiation we used the protocol designed by Rezania et al 9 with slight modifications as previously described. 5,14 The encapsulated cells were exposed to either a brief (1 week, here on 1w-postTX) or an extended period of time (4 weeks, here on 4w-postTX) in the in vivo hyperglycaemic environment. These time points were selected to allow both the examination of the initial changes in the cells' regulatory landscape, known to occur rapidly in response to altered cell signalling or environmental cues, 15,16 and the assessment of the long-term effect of hyperglycaemia on the cells.…”

“…Subsequent separation and acquisition were performed as previously described. 5,14 Samples were analysed in duplicate, one with advanced peak determination (ADP) activated and a second run with this option off. Both analyses used the real-time search algorithm.…”