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

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.

The following data sets were generated
The following previously published data sets were used

Article and author information

Author details

  1. Sevim Kahraman

    Islet Cell and Regenerative Biology, Joslin Diabetes Center, Boston, United States
    Competing interests
    Sevim Kahraman, S.K. is an employee of Boehringer Ingelheim Pharmaceuticals, Inc..
  2. Kimitaka Shibue

    Islet Cell and Regenerative Biology, Joslin Diabetes Center, Boston, United States
    Competing interests
    No competing interests declared.
  3. Dario F De Jesus

    Islet Cell and Regenerative Biology, Joslin Diabetes Center, Boston, United States
    Competing interests
    No competing interests declared.
  4. Hyunki Kim

    Islet Cell and Regenerative Biology, Joslin Diabetes Center, Boston, United States
    Competing interests
    No competing interests declared.
  5. Jiang Hu

    Islet Cell and Regenerative Biology, Joslin Diabetes Center, Boston, United States
    Competing interests
    No competing interests declared.
  6. Debasish Manna

    Chemical Biology and Therapeutics Science Program, Broad Institute, Cambridge, United States
    Competing interests
    No competing interests declared.
  7. Bridget K Wagner

    Chemical Biology and Therapeutics Science Program, Broad Institute, Cambridge, United States
    Competing interests
    No competing interests declared.
  8. Amit Choudhary

    Chemical Biology and Therapeutics Science Program, Broad Institute, Cambridge, United States
    Competing interests
    No competing interests declared.
  9. Rohit N Kulkarni

    Islet Cell and Regenerative Biology, Joslin Diabetes Center, Boston, United States
    For correspondence
    rohit.kulkarni@joslin.harvard.edu
    Competing interests
    Rohit N Kulkarni, is on the Scientific Advisory Board of Novo Nordisk, Biomea and Inversago Therapeutics..
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-5029-6119

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.

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  1. Sevim Kahraman
  2. Kimitaka Shibue
  3. Dario F De Jesus
  4. Hyunki Kim
  5. Jiang Hu
  6. Debasish Manna
  7. Bridget K Wagner
  8. Amit Choudhary
  9. Rohit N Kulkarni
(2023)
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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https://doi.org/10.7554/eLife.85056

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