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.
-
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.
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.
Metrics
-
- 1,453
- views
-
- 274
- downloads
-
- 0
- citations
Views, downloads and citations are aggregated across all versions of this paper published by eLife.
Download links
A two-part list of links to download the article, or parts of the article, in various formats.
Downloads (link to download the article as PDF)
Open citations (links to open the citations from this article in various online reference manager services)
Cite this article (links to download the citations from this article in formats compatible with various reference manager tools)
Fluorescein-based sensors to purify human a-cells for functional and transcriptomic analyses
eLife 12:e85056.
https://doi.org/10.7554/eLife.85056
