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Purification of Specific Cell Population by Fluorescence Activated Cell Sorting FACS

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Published on: July 10, 2010

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

Sevim Kahraman^1,2,3, Kimitaka Shibue^1,2,3, Dario F De Jesus^1,2,3

¹Islet Cell and Regenerative Biology, Joslin Diabetes Center, Boston, United States.

|September 21, 2023

Summary

Researchers developed a simple method using DA-ZP1 to purify human pancreatic alpha-cells (~95% purity). These purified cells form viable pseudoislets for studying glucagon secretion and gene expression in glucose homeostasis.

Keywords:

biochemistry cell biology chemical biology diabetes human human pancreatic islets pseudoislets α-cells

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Published on: June 4, 2019

Area of Science:

Endocrinology
Cell Biology
Biochemistry

Background:

Pancreatic alpha-cells produce glucagon, essential for glucose homeostasis.
Isolating pure human alpha-cells for research is difficult due to purification challenges.

Purpose of the Study:

To develop a cost-effective and efficient method for purifying live human pancreatic alpha-cells.
To establish a method for culturing purified alpha-cells for functional and transcriptional studies.

Main Methods:

Utilized the reaction-based probe diacetylated Zinpyr1 (DA-ZP1) for cell enrichment.
Employed cell sorting and immunostaining to confirm alpha-cell purity and identity.
Cultured sorted alpha-cells to form pseudoislets for up to 10 days.

Main Results:

Achieved ~95% purity of live human alpha-cells using the DA-ZP1 method.
Established viable alpha-pseudoislets that maintained glucagon secretion in response to glucose.
RNA-sequencing showed sustained expression of key alpha-cell genes in culture, with time-dependent alterations in specific genes like DLK1.

Conclusions:

A novel, simple, and high-purity method for isolating human primary alpha-cells was established.
The method enables downstream functional and transcriptional analyses of human alpha-cells.
Cultured alpha-pseudoislets provide a valuable model for studying alpha-cell biology.