differentiation-human-pluripotent-stem-cells-into-insulin-producing

Differentiation of Human Pluripotent Stem Cells into Insulin-Producing Islet Clusters

Differentiation of human pluripotent stem cells (hPSCs) into insulin-secreting beta cells provides material for investigating beta cell function and ...

University of British Columbia

In early stage of diabetes, insulin secretion from pancreatic β-cells is increased to deal with the elevated blood glucose. Previous studies have reported that islet-produced carbon monoxide (CO) is associated with increased glucose-stimulated insulin secretion from β-cells. However, this compensatory mechanism by which CO may act to enhance β-cell function remain unclear. In this study, we revealed that CO promoted intracellular calcium ([Ca]) elevation and glucose-stimulated insulin secretion (GSIS) from pancreatic β-cells in leptin receptor deficient db/db mice but not in C57 mice. The stimulatory effects of CO on β-cell function in db/db mice was blocked by inhibition of Phospholipase C (PLC) signaling pathway. We further demonstrated that CO triggered [Ca] transients and enhanced GSIS in C57 islets when β-cells overexpressed with PLCγ1 and PLCδ1, but not PLCβ1. On the other hand, reducing PLCγ1 and PLCδ1 expressions in db/db islets dramatically attenuated the stimulatory effects of CO on β-cell function, whereas interfering PLCβ1 expression had no effects on CO-induced β-cell function enhancement. Our findings showing that CO elevated [Ca] and enhanced GSIS by activating PLC signaling through PLCγ1 and PLCδ1 isoforms in db/db pancreatic β-cells may suggest an important mechanism by which CO promotes β-cell function to prevent hyperglycemia. Our study may also provide new insights into the therapy for type II diabetes and offer a potential target for therapeutic applications of CO.

Carbon monoxide enhances calcium transients and glucose-stimulated insulin secretion from pancreatic β-cells by activating Phospholipase C signal pathway in diabetic mice.

Visualizing the function of pancreatic β-cells  has been a long-sought goal for β-cell researchers. Unlike imaging of β-cells in mammalian species with conventional positron emission tomography and single-photon emission computed tomography, which only provides limited spatial-temporal resolution, transparent zebrafish embryos are a unique model that allows high-resolution fluorescent imaging of β-cells in their native physiological microenvironment . Here, we detail a protocol for real-time visualization of individual β-cell function  in a non-invasive manner, through combination of a novel transgenic zebrafish reporter line Tg () with both a commercial spinning-disc confocal microscope and an in-house developed super-resolution microscope (2P3A-DSLM). The protocol described here allows for the longitudinal monitoring of dynamic calcium activities from heterogeneous β-cells in early developing zebrafish embryos and is readily adaptable for use in imaging other important processes in islet biology, as well as screening new compounds that can promote β-cell function or maturation using a living whole organism system.

 Imaging of Calcium Activities from Pancreatic β-cells in Zebrafish Embryos Using Spinning-disc Confocal and Two-photon Light-sheet Microscopy.

The generation of functional β-cells from human pluripotent stem cells (hPSCs) for cell replacement therapy and disease modeling of diabetes is being investigated by many groups. We have developed a protocol to harvest and aggregate hPSC-derived pancreatic progenitors generated using a commercially available kit into near uniform spheroids and to further differentiate the cells toward an endocrine cell fate in suspension culture. Using a static suspension culture platform, we could generate a high percentage of insulin-expressing, glucose-responsive cells. We identified FGF7 as a soluble factor promoting aggregate survival with no inhibitory effect on endocrine gene expression. Notch inhibition of pancreatic progenitor cells during aggregation improved endocrine cell induction in vitro and improved graft function following implantation and further differentiation in mice. Thus we provide an approach to promote endocrine formation from kit-derived pancreatic progenitors, either through extended culture or post implant.

Protocol development to further differentiate and transition stem cell-derived pancreatic progenitors from a monolayer into endocrine cells in suspension culture.

Orbital shaker-based suspension culture systems have been in widespread use for differentiating human pluripotent stem cell (hPSC)-derived pancreatic progenitors toward islet-like clusters during endocrine induction stages. However, reproducibility between experiments is hampered by variable degrees of cell loss in shaking cultures, which contributes to variable differentiation efficiencies. Here, we describe a 96-well-based static suspension culture method for differentiation of pancreatic progenitors into hPSC-islets. Compared with shaking culture, this static 3D culture system induces similar islet gene expression profiles during differentiation processes but significantly reduces cell loss and improves cell viability of endocrine clusters. This static culture method results in more reproducible and efficient generation of glucose-responsive, insulin-secreting hPSC-islets. The successful differentiation and well-to-well consistency in 96-well plates also provides a proof of principle that the static 3D culture system can serve as a platform for small-scale compound screening experiments as well as facilitating further protocol development.

Article	Year
Carbon monoxide enhances calcium transients and glucose-stimulated insulin secretion from pancreatic β-cells by activating Phospholipase C signal pathway in diabetic mice. Biochemical and biophysical research communications\| PubMed ID: 34678590	2021
Imaging of Calcium Activities from Pancreatic β-cells in Zebrafish Embryos Using Spinning-disc Confocal and Two-photon Light-sheet Microscopy. Bio-protocol\| PubMed ID: 35005090	2021
Protocol development to further differentiate and transition stem cell-derived pancreatic progenitors from a monolayer into endocrine cells in suspension culture. Scientific reports\| PubMed ID: 37264038	2023
Differentiation of stem cell-derived pancreatic progenitors into insulin-secreting islet clusters in a multiwell-based static 3D culture system. Cell reports methods\| PubMed ID: 37323565	2023

Shenghui Liang

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