Mehmeti, I. | Search bioRxiv

bioRxiv2021

Pluripotent stem cell SOX9 and INS reporters facilitate differentiation into insulin-producing cells

Differentiation of human pluripotent stem cells into insulin-producing stem cell-derived beta cells harbors great potential for research and therapy of diabetes. The SOX9 gene 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 genes into the SOX9 locus and a P2A-mCherry reporter gene into the INS locus mediated by CRISPR/CAS9-technology. The knock-ins enable co-expression of the endogenous genes and reporter genes, report the endogenous gene expression and enable the purification of pancreatic progenitors and insulin-producing cells using FACS or MACS. 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.

developmental biology↗

bioRxiv2016

TriPer, an optical probe tuned to the endoplasmic reticulum, tracks H2O2 consumption by glutathione

The fate of H2O2 in the endoplasmic reticulum (ER) has been inferred indirectly from the activity of ER localized thiol oxidases and peroxiredoxins, in vitro, and the consequences of their genetic manipulation, in vivo. Here we report on the development of TriPer, a vital optical probe sensitive to changes in the concentration of H2O2 in the thiol-oxidizing environment of the ER. Consistent with the hypothesized contribution of oxidative protein folding to H2O2 production, ER-localized TriPer detected an increase in the luminal H2O2 signal upon induction of pro-insulin (a disulfide bonded protein of pancreatic {beta}-cells), which was attenuated by the ectopic expression of catalase in the ER lumen. Interfering with glutathione production in the cytosol by buthionine sulfoximine (BSO) or enhancing its localized destruction by expression of the glutathione-degrading enzyme ChaC1 in lumen of the ER, enhanced further the luminal H2O2 signal and eroded {beta}-cell viability. Tracking ER H2O2 in live cells points to an unanticipated role for glutathione in H2O2 turnover.\n\nSignificance statementThe presence of millimolar glutathione in the lumen of the endoplasmic reticulum has been difficult to understand purely in terms of modulation of protein-based disulphide bond formation in secreted proteins. Over the years hints have suggested that glutathione might have a role in reducing the heavy burden of hydrogen peroxide (H2O2) produced by the luminal enzymatic machinery for disulphide bond formation. However, limitations in existing in vivo H2O2 probes have rendered them all but useless in the thiol-oxidizing ER, precluding experimental follow-up of glutathiones role ER H2O2 metabolism.\n\nHere we report on the development and mechanistic characterization of an optical probe, TriPer that circumvents the limitations of previous sensors by retaining specific responsiveness to H2O2 in thiol-oxidizing environments. Application of this tool to the ER of an insulin-producing pancreatic b-cells model system revealed that ER glutathione antagonizes locally-produced H2O2 resulting from the oxidative folding of pro-insulin.\n\nThis study presents an interdisciplinary effort intersecting cell biology and chemistry: An original redox chemistry concept leading to development of a biological tool, broadly applicable for in vivo studies of H2O2 metabolism in the ER. More broadly, the concept developed here sets a precedent for applying a tri-cysteine relay system to discrimination between various oxidative reactants, in complex redox milieux.

cell biology↗