Harnessing reaction-based probes to preferentially target pancreatic β-cells and β-like cells.

Sevim Kahraman,Basudeb Maji,Ercument Dirice,Rohit N Kulkarni,Bridget K Wagner,Jonnell Small,Debasish Manna,Amit Choudhary

doi:10.26508/lsa.202000840

Sevim Kahraman, Basudeb Maji + Show 6 more

Open Access

https://doi.org/10.26508/lsa.202000840

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Abstract

Highly sensitive approaches to target insulin-expressing cells would allow more effective imaging, sorting, and analysis of pancreatic β-cells. Here, we introduce the use of a reaction-based probe, diacetylated Zinpyr1 (DA-ZP1), to image pancreatic β-cells and β-like cells derived from human pluripotent stem cells. We harness the high intracellular zinc concentration of β-cells to induce a fluorescence signal in cells after administration of DA-ZP1. Given its specificity and rapid uptake by cells, we used DA-ZP1 to purify live stem cell-derived β-like cells as confirmed by immunostaining analysis. We tested the ability of DA-ZP1 to image transplanted human islet grafts and endogenous mouse pancreatic islets in vivo after its systemic administration into mice. Thus, DA-ZP1 enables purification of insulin-secreting β-like cells for downstream applications, such as functional studies, gene-expression, and cell-cell interaction analyses and can be used to label engrafted human islets and endogenous mouse islets in vivo.

Highlights

Restoring normoglycemia independent of exogenous insulin injections can be achieved by islet cell replacement therapy
Current protocols for making Human pluripotent stem cells (hPSCs)-derived β-like cells result in cell cultures that consist of a mixture of cell types, including non-β endocrine cells and cells with tumorigenic potential, which could develop into tumors after transplantation (Nair et al, 2019; Veres et al, 2019)
The use of fluorescent zinc probes for sorting insulin-positive cells is promising because it satisfies several important criteria: (1) it offers high selectivity for insulin-positive β-cells compared with insulin-negative non-β cells; (2) it is nontoxic to the cells; and (3) it elicits a significant and rapid fluorescent response upon zinc binding, which can be captured by flow cytometry, minimizing autofluorescence and avoiding UVinduced tissue damage, as might be caused by zinc probes described in earlier reports such as TSQ (Huang & Lippard, 2012)

Summary

Introduction

Restoring normoglycemia independent of exogenous insulin injections can be achieved by islet cell replacement therapy. The second concern is the known variability across hPSC lines, which results in generation of variable numbers of insulin-expressing cells at the final stage of the differentiation protocol (Thatava et al, 2013). To circumvent these issues, several groups have developed methods to isolate and purify insulin-secreting β-like cells for transcriptional and functional analyses. Several groups have developed methods to isolate and purify insulin-secreting β-like cells for transcriptional and functional analyses One such approach uses INSGFP/w human embryonic stem cells (hESCs) to facilitate isolation of insulin-expressing cells (Micallef et al, 2012). Magnetic cell sorting of β-like cells using the cell surface marker, CD49a, has been reported to enrich β-like cells derived from hPSCs; this method requires antibody staining (Veres et al, 2019)

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