Fluorescein-based sensors to purify human α-cells for functional and transcriptomic analyses
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 islet...
| Main Authors: | , , , , , , , , |
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| Format: | Article |
| Language: | English |
| Published: |
eLife Sciences Publications Ltd
2023-09-01
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| Series: | eLife |
| Subjects: | |
| Online Access: | https://elifesciences.org/articles/85056 |
| _version_ | 1797356556058099712 |
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| author | Sevim Kahraman Kimitaka Shibue Dario F De Jesus Hyunki Kim Jiang Hu Debasish Manna Bridget Wagner Amit Choudhary Rohit N Kulkarni |
| author_facet | Sevim Kahraman Kimitaka Shibue Dario F De Jesus Hyunki Kim Jiang Hu Debasish Manna Bridget Wagner Amit Choudhary Rohit N Kulkarni |
| author_sort | Sevim Kahraman |
| collection | DOAJ |
| description | 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. |
| first_indexed | 2024-03-08T14:29:21Z |
| format | Article |
| id | doaj.art-b75a7103ed324acca5108365e616c62c |
| institution | Directory Open Access Journal |
| issn | 2050-084X |
| language | English |
| last_indexed | 2024-03-08T14:29:21Z |
| publishDate | 2023-09-01 |
| publisher | eLife Sciences Publications Ltd |
| record_format | Article |
| series | eLife |
| spelling | doaj.art-b75a7103ed324acca5108365e616c62c2024-01-12T15:54:44ZengeLife Sciences Publications LtdeLife2050-084X2023-09-011210.7554/eLife.85056Fluorescein-based sensors to purify human α-cells for functional and transcriptomic analysesSevim Kahraman0https://orcid.org/0000-0002-2880-6589Kimitaka Shibue1Dario F De Jesus2Hyunki Kim3Jiang Hu4Debasish Manna5Bridget Wagner6Amit Choudhary7Rohit N Kulkarni8https://orcid.org/0000-0001-5029-6119Islet Cell and Regenerative Biology, Joslin Diabetes Center, Boston, United States; Department of Medicine, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, United States; Harvard Stem Cell Institute, Harvard Medical School, Boston, United StatesIslet Cell and Regenerative Biology, Joslin Diabetes Center, Boston, United States; Department of Medicine, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, United States; Harvard Stem Cell Institute, Harvard Medical School, Boston, United StatesIslet Cell and Regenerative Biology, Joslin Diabetes Center, Boston, United States; Department of Medicine, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, United States; Harvard Stem Cell Institute, Harvard Medical School, Boston, United StatesIslet Cell and Regenerative Biology, Joslin Diabetes Center, Boston, United States; Department of Medicine, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, United States; Harvard Stem Cell Institute, Harvard Medical School, Boston, United StatesIslet Cell and Regenerative Biology, Joslin Diabetes Center, Boston, United StatesChemical Biology and Therapeutics Science Program, Broad Institute of MIT and Harvard, Cambridge, United States; Divisions of Renal Medicine and Engineering, Brigham and Women’s Hospital, Boston, United StatesChemical Biology and Therapeutics Science Program, Broad Institute of MIT and Harvard, Cambridge, United StatesChemical Biology and Therapeutics Science Program, Broad Institute of MIT and Harvard, Cambridge, United States; Divisions of Renal Medicine and Engineering, Brigham and Women’s Hospital, Boston, United StatesIslet Cell and Regenerative Biology, Joslin Diabetes Center, Boston, United States; Department of Medicine, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, United States; Harvard Stem Cell Institute, Harvard Medical School, Boston, United StatesPancreatic 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.https://elifesciences.org/articles/85056α-cellspseudoisletshuman pancreatic isletsdiabetes |
| spellingShingle | Sevim Kahraman Kimitaka Shibue Dario F De Jesus Hyunki Kim Jiang Hu Debasish Manna Bridget Wagner Amit Choudhary Rohit N Kulkarni Fluorescein-based sensors to purify human α-cells for functional and transcriptomic analyses eLife α-cells pseudoislets human pancreatic islets diabetes |
| title | Fluorescein-based sensors to purify human α-cells for functional and transcriptomic analyses |
| title_full | Fluorescein-based sensors to purify human α-cells for functional and transcriptomic analyses |
| title_fullStr | Fluorescein-based sensors to purify human α-cells for functional and transcriptomic analyses |
| title_full_unstemmed | Fluorescein-based sensors to purify human α-cells for functional and transcriptomic analyses |
| title_short | Fluorescein-based sensors to purify human α-cells for functional and transcriptomic analyses |
| title_sort | fluorescein based sensors to purify human α cells for functional and transcriptomic analyses |
| topic | α-cells pseudoislets human pancreatic islets diabetes |
| url | https://elifesciences.org/articles/85056 |
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