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

Author:

Kahraman Sevim123ORCID,Shibue Kimitaka123,De Jesus Dario F123,Kim Hyunki123,Hu Jiang1,Manna Debasish45,Wagner Bridget4,Choudhary Amit45,Kulkarni Rohit N123ORCID

Affiliation:

1. Islet Cell and Regenerative Biology, Joslin Diabetes Center

2. Department of Medicine, Beth Israel Deaconess Medical Center, Harvard Medical School

3. Harvard Stem Cell Institute, Harvard Medical School

4. Chemical Biology and Therapeutics Science Program, Broad Institute of MIT and Harvard

5. Divisions of Renal Medicine and Engineering, Brigham and Women’s Hospital

Abstract

Pancreatic α-cells secrete glucagon, an insulin counter-regulatory peptide hormone critical for the maintenance of glucose homeostasis. Investigation of the function of human α-cells remains a challenge due to the lack of cost-effective purification methods to isolate high-quality α-cells from islets. Here, we use the reaction-based probe diacetylated Zinpyr1 (DA-ZP1) to introduce a novel and simple method for enriching live α-cells from dissociated human islet cells with ~95% purity. The α-cells, confirmed by sorting and immunostaining for glucagon, were cultured up to 10 days to form α-pseudoislets. The α-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 α-cell identity genes were sustained in culture while some of the genes such as DLK1, GSN, SMIM24 were altered in α-pseudoislets in a time-dependent manner. In conclusion, we report a method to sort human primary α-cells with high purity that can be used for downstream analyses such as functional and transcriptional studies.

Funder

National Institutes of Health

Publisher

eLife Sciences Publications, Ltd

Subject

General Immunology and Microbiology,General Biochemistry, Genetics and Molecular Biology,General Medicine,General Neuroscience

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