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.
Highlights
Despite ongoing progress, it is at present still not possible to generate mature insulin-producing cells from human induced pluripotent stem cells that capture all aspects of endogenous β-cell in vitro
For modulating Wnt-signaling in human induced pluripotent stem cells (hiPSCs)-derived stage 7 (S7) cells, we chose to use similar concentrations as previously used for the WNT-ligands to stimulate Wnt-signaling in murine micro islets, human islets, as well as human β-cell line EndoC-BH1 [7]
By comparing S6 cells corresponding to immature β-like cells [1] to S7 cells, we found as expected that the level of bi-hormonal cells decreased during in vitro maturation of hiPSC-derived cells (Figure 3C)
Summary
It is at present still not possible to generate mature insulin-producing cells from human induced pluripotent stem cells (hiPSCs) that capture all aspects of endogenous β-cell in vitro. It remains unclear to date, which signaling pathways will promote the last steps of β-cell differentiation and functional maturation, as well as whether these mechanisms can be activated in vitro. The Wnt signaling pathways are a group of highly conserved pathways that regulate key aspects of cell fate decisions, migration, polarity, patterning and organogenesis during embryonic development [8,9,10,11,12]. Stage-specific signaling through Wnt regulates patterning and pancreas specification of human pluripotent stem cells, and canonical Wnt signaling has been found to induce a posterior endoderm fate and to enhance the development of pancreatic linage cells [17]
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