Fluorescein-based sensors to purify human a-cells for functional and transcriptomic analyses
- Joslin Diabetes Center, United States
- Broad Institute, United States
Abstract
Pancreatic a-cells secrete glucagon, an insulin counter-regulatory peptide hormone critical for the maintenance of glucose homeostasis. Investigation of the function of human a-cells remains a challenge due to the lack of cost-effective purification methods to isolate high-quality a-cells from islets. Here, we use the reaction-based probe diacetylated Zinpyr1 (DA-ZP1) to introduce a novel and simple method for enriching live a-cells from dissociated human islet cells with ~ 95% purity. The a-cells, confirmed by sorting and immunostaining for glucagon, were cultured up to 10 days to form a-pseudoislets. The a-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 a-cell identity genes were sustained in culture while some of the genes such as DLK1, GSN, SMIM24 were altered in a-pseudoislets in a time-dependent manner. In conclusion, we report a method to sort human primary a-cells with high purity that can be used for downstream analyses such as functional and transcriptional studies.
Data availability
RNA-seq data have been deposited under accession code GSE199412. Further information and requests for resources and reagents should be directed to the corresponding author.
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Using Zinc-based probes to generate pseudoislets from purified human alpha-cells for functional analysesNCBI Gene Expression Omnibus, GSE199412.
Article and author information
Author details
Funding
National Institutes of Health (U01 DK123717)
- Bridget K Wagner
- Rohit N Kulkarni
National Institutes of Health (UC4 DK116255)
- Bridget K Wagner
- Amit Choudhary
- Rohit N Kulkarni
National Institutes of Health (R01 067536)
- Rohit N Kulkarni
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
Reviewing Editor
- Lori Sussel, University of Colorado Anschutz Medical Campus, United States
Version history
- Received: November 21, 2022
- Preprint posted: November 28, 2022 (view preprint)
- Accepted: September 11, 2023
- Accepted Manuscript published: September 21, 2023 (version 1)
- Version of Record published: October 11, 2023 (version 2)
- Version of Record updated: January 12, 2024 (version 3)
Copyright
© 2023, Kahraman et al.
This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.
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Fluorescein-based sensors to purify human a-cells for functional and transcriptomic analyses
eLife 12:e85056.
https://doi.org/10.7554/eLife.85056
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