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

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    The manuscript by Kahraman et al. describes the use of a fluorescent dye for purifying and analyzing human islet alpha cells. The study provides solid evidence that the alpha cells can be purified using this method and the cells remained viable and functional after culturing for several days. The significance of the study is access to a new tool that will be useful for islet biologists and researchers studying diabetes mechanisms.

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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.

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  1. eLife assessment

    The manuscript by Kahraman et al. describes the use of a fluorescent dye for purifying and analyzing human islet alpha cells. The study provides solid evidence that the alpha cells can be purified using this method and the cells remained viable and functional after culturing for several days. The significance of the study is access to a new tool that will be useful for islet biologists and researchers studying diabetes mechanisms.

  2. Reviewer #1 (Public Review):

    The study by Kahraman et al describes the application of a reaction-based probe "diacetylated Zinpyr1" (DA-ZP1) that was developed for the enrichment of human islet beta cells (Lee et al. 2020 to purify human cadaveric alpha cells. The probe binds zinc with high enough affinity to allow the authors to separate beta cells from alpha cells based on the fluorescence intensity; beta cells had high intensity and alpha cells had medium intensity. FACs sorting of cells with intermediate fluorescent intensity were enriched for glucagon expression indicating they were alpha cells. They went on to reaggregate the purified alpha cells into pseudo-islets to test for viability, proliferation, ability to secrete glucagon and transcriptome analysis. These studies demonstrated that the pseudo-alpha cell islets were able to be maintained in culture for up to 10 days without losing their function and with only minor changes in gene expression.

    The strengths of the manuscript include:
    1. The description and characterization of a novel tool with which to purify human islet alpha cells
    2. The ability to use the same DA-ZP1 probe to purify both human alpha and beta cells
    3. The functional analysis to show that purified alpha cells retain their identity and maintain function even after in vitro culturing.
    4. Providing a comparison of the transcriptome between whole islets, unsorted islets and sorted alpha cell pseudo-islets. The data is strengthened by the use of four donor islets and several timepoints for the transcriptomic analysis.
    5. The quality of the data and data presentation

    Weaknesses include:
    1. Lack of a comparison with other published methods to purify human alpha cells
    2. Unbiased transcriptome analysis of the sorted "high" vs. "medium" fluorescent populations to assess the degree of cross contamination between the 2 populations
    3. Use of only one donor islet for functional analyses

    Overall, this study represents a solid characterization of a new tool for purifying cadaveric human alpha cells that will be useful to researchers in the islet biology and diabetes fields.

  3. Reviewer #2 (Public Review):

    In the manuscript by Kahraman et al. the authors tested a recently developed Zn2+ indicator fluorogenic sensor as a tool to sort and purify human alpha cells from cadaveric donor islets, for downstream transcriptional and functional analysis. They demonstrate that their previously published sensor DA-ZP1, which was used to sort adult human islet beta cells in their previous work (Lee et al. 2020) they have now adapted for sorting alpha cells based on the 'intermediate' fluorescence intensity of these cells during staining. FACS purification of DA-ZP1-intermediate cells reveals they are strongly enriched for GCG+ cells (alpha cells). The sorted alpha cells can be reaggregated into alpha-pseudoislets for further studies. They carry out a variety of assays to characterize the viability, proliferation, apoptosis, glucagon secretion and transcriptomic changes in their sort purified alpha cells as compared with unsorted islet cells and intact islets. They conclude that sorting alpha cells with DA-ZP1 staining does not alter their function or transcriptome and allows stable maintenance of alpha-pseudoislets in culture for up to 10 days with no deleterious effects.

    1. The study is a nice resource for the field, particularly with the ongoing interest in studying alpha cell biology and function relevant to health and diabetes. The probe that they have previously published can now be used to simultaneously sort alpha and beta cells, which would be a great approach for the field. The results are generally supportive of the conclusions.

    2. The study used several human cadaveric donor islet preparations (four in total) representing different ancestries, limiting bias and inter-donor variation. A variety of cellular/molecular assays are employed to provide detailed phenotypic information.

    3. The transcriptomic profiling are very strong and provide solid evidence that the reaggregated alpha-pseudoislets are not dedifferentiating or losing function during prolonged (10 day) culture times.

    4. Visual presentation is clear and easy to follow for non-specialists.


    1. The authors are presenting a previously developed probe/tool and also mention that other probes have been developed that can perform a very similar function, so the overall novelty is limited. They did not provide experimental evidence of how their probe is comparable or superior to other probes (e.g. ZIGIR, Newport Green).

    2. The authors performed glucagon secretion assays to monitor the function of the sort purified and reaggregated alpha-pseudoislets, but this was only done on 1 of the 4 human islet donors, limiting the generalizability of the conclusions. Also very few experiments were performed to examine alpha cell function in the sort purified cells.

  4. Reviewer #3 (Public Review):

    This study presents a new method to highly purify live human pancreatic α cells using the zinc-based reaction probe DA-ZP1. After demonstrating this probe is capable of separating β and α cells from other islet and non-islet cells based on florescence intensity, the authors employ a variety of experimental approaches to demonstrate that these isolated α cells are functional and capable of maintaining their viability and identity in culture over time. The authors also investigate the impact of islet dissociation and cell reaggregation on the islet cell transcriptome, where they primarily identified downregulation of pathways associated with extracellular matrix organization, cell surface interactions, and focal adhesion. Overall, this study adds an additional tool to isolate human α cells to the islet biology community, which may aid in further understanding of human α cell biology under both normal and diabetic conditions. However, some caveats of this study include:

    1. While the authors claim that this method improves human α cell yield over antibody-based approaches, they provide no direct comparison between the two methods.
    2. The strength of studies determining cell fraction purity and α cell characteristics (function, viability, proliferation, and apoptosis rates) would be strengthened by performing these studies across multiple donors rather than multiple replicates from the same donor.
    3. Given the heterogeneous nature of the human islet, the use of bulk RNA-sequencing makes the interpretation of genes obtained via the comparison of α-pseudoislets and unsorted pseudoislets difficult. Some cell-specific signals will be missed or masked by differences in cell mixture between groups. It is unclear whether these expression changes are due to α-intrinsic changes or simply the loss of other cell types.
    4. Supplementary files concerning bulk sequencing data is not transparent, with only the direction of the gene expression noted.