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Merck

Fluorescein-based sensors to purify human ฮฑ-cells for functional and transcriptomic analyses.

eLife (2023-09-21)
Sevim Kahraman, Kimitaka Shibue, Dario F De Jesus, Hyunki Kim, Jiang Hu, Debasish Manna, Bridget Wagner, Amit Choudhary, Rohit N Kulkarni
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Pancreatic ฮฑ-cells secrete glucagon, an insulin counter-regulatory peptide hormone critical for the maintenance of glucose homeostasis. Investigation of the function of human ฮฑ-cells remains a challenge due to the lack of cost-effective purification methods to isolate high-quality ฮฑ-cells from islets. Here, we use the reaction-based probe diacetylated Zinpyr1 (DA-ZP1) to introduce a novel and simple method for enriching live ฮฑ-cells from dissociated human islet cells with ~95% purity. The ฮฑ-cells, confirmed by sorting and immunostaining for glucagon, were cultured up to 10 days to form ฮฑ-pseudoislets. The ฮฑ-pseudoislets could be maintained in culture without significant loss of viability, and responded to glucose challenge by secreting appropriate levels of glucagon. RNA-sequencing analyses (RNA-seq) revealed that expression levels of key ฮฑ-cell identity genes were sustained in culture while some of the genes such as DLK1, GSN, SMIM24 were altered in ฮฑ-pseudoislets in a time-dependent manner. In conclusion, we report a method to sort human primary ฮฑ-cells with high purity that can be used for downstream analyses such as functional and transcriptional studies.

MATERIALS
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Sigma-Aldrich
Monoclonal Anti-Glucagon antibody produced in mouse, clone K79bB10, ascites fluid
Sigma-Aldrich
Anti-SMIM24 antibody produced in rabbit, Prestige Antibodiesยฎ Powered by Atlas Antibodies, affinity isolated antibody, buffered aqueous glycerol solution
Sigma-Aldrich
Anti-GSN antibody produced in rabbit, Prestige Antibodiesยฎ Powered by Atlas Antibodies, affinity isolated antibody