Live Cell Monitoring and Enrichment of Stem Cell‐Derived β Cells Using Intracellular Zinc Content as a Population Marker

Abstract

Our laboratory and others have developed protocols to generate glucose‐responsive stem cell–derived β cells in vitro. The cells resulting from these protocols could supplement or replace the use of human cadaveric islets for cell‐based therapy for diabetes. The combination of an unlimited supply of pluripotent stem cell–derived β cells and gene‐editing approaches will facilitate numerous in vitro studies not possible with cadaveric islets. Here, we describe a protocol for fluorescent labeling and isolation of stem cell–derived β cells. This purification of SC‐β cells is based on intracellular zinc content and is a simple method to complement other approaches for generating and assaying these cells. © 2019 The Authors. Basic Protocol: Fluorescent labeling and isolation of stem cell‐derived β cells

Glucose Response by Stem Cell-Derived β Cells In Vitro Is Inhibited by a Bottleneck in Glycolysis

“…TSQ High cells are observable by flow cytometry, and the size of this population reflects the total size of the insulin + population in SC-islets ( Figure S1A ). Using this approach, it is possible to enrich the endocrine SC-β population from differentiations in pluripotent cell lines as well as cadaveric islets ( Figure S1B ) ( Davis et al, 2019 ).…”

“…This analysis allows for measuring both mass and position of labeled carbons to infer contribution from distinct metabolic pools derived from labeled glucose. Utilizing TSQ enrichment of SC-β cells as described earlier ( Davis et al, 2019 ), reaggregates of 5,000 cells were cultured after sorting for 72 h. The resulting enriched clusters contained a majority of SC-β cells in addition to a smaller population of poly-hormonal cells. Clusters composed of more than half SC-β cells and of this total cell number/cluster are similar to the composition of unsorted cadaveric islets that were used as controls ( Hart and Powers, 2019 ; Brissova et al, 2005 ) ( Figure S1B ).…”

Identification of a LIF-Responsive, Replication-Competent Subpopulation of Human β Cells

“…All the experiments were performed using the human embryonic stem cell line HUES8 (NIH hESC registry #09-0021; male) and the iPSC 1016 Insulin reporter line developed by Davis et al (2019). All cell lines were tested regularly for mycoplasma contamination and normal karyotype.…”

Integrin αvβ5 heterodimer is a specific marker of human pancreatic beta cells

“…There is a dearth of cell surface markers available that specifically identify pancreatic beta cells—instead, enrichment strategies based on intracellular zinc granules (for example TSQ 1 or Newport Green 2 or negative selection are currently used. Given that integrins are often used to distinguish and isolate specific cell types from a variety of sources 3 , 4 , we sought to determine whether this group of proteins could identify specific sub-populations within human islets.…”