Fluorescein-based sensors to purify human α-cells for functional and transcriptomic analyses
- Islet Cell and Regenerative Biology, Joslin Diabetes Center, United States
- Department of Medicine, Beth Israel Deaconess Medical Center, Harvard Medical School, United States
- Harvard Stem Cell Institute, Harvard Medical School, United States
- Chemical Biology and Therapeutics Science Program, Broad Institute of MIT and Harvard, United States
- Divisions of Renal Medicine and Engineering, Brigham and Women’s Hospital, United States
Figures
Figure 1 with 1 supplement
Isolation of live human pancreatic α-cells after staining with diacetylated Zinpyr1 (DA-ZP1) by fluorescence activated cell sorting (FACS).
(a) Experimental outline. (b) Representative FACS plot showing three cell populations with low, intermediate, or bright fluorescence. The plot represents the data collected from Donor-1 islets. …
Figure 1—figure supplement 1
Gating strategy for isolation of live human pancreatic α-cells.
(a) Starting cell population was determined by SSC-A/FSC-A gating. Single human islet cells were gated according to FSC-A/FSC-W gating. Treatment of the single cells with diacetylated Zinpyr1 …
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Figure 1—figure supplement 1—source data 1
Number of live cells collected by FACS using 15,000 islet equivalents (IEQs) determined by trypan blue staining.
- https://cdn.elifesciences.org/articles/85056/elife-85056-fig1-figsupp1-data1-v3.xlsx
Figure 2 with 1 supplement
α-Pseudoislets are viable and able to proliferate in vitro post-sorting.
(a) The single islet cells were seeded in round-bottom wells (1k cells per well) after sorting to allow re-aggregation. (b, c) Bright-field images of intermediate (sorted α-cells) (b) and unsorted …
Figure 2—figure supplement 1
α-Pseudoislets tended to form tighter clusters.
Bright-field images of α-pseudoislets (a) and unsorted pseudoislets (b) 5 days post-sorting. 1k (top panel) or 2k (bottom panel) single cells were seeded per well. Scale bar, 100 μm. n=3 biological …
Figure 3
Glucagon secretion in response to glucose challenge.
(a) α-Pseudoislets, unsorted pseudoislets, or native islets were preincubated in Krebs-Ringer bicarbonate (KRB) buffer with 16.7 mM glucose followed by the incubation in KRB buffer with 3.3 mM …
Figure 4 with 1 supplement
Changes in gene expression levels driven by dissociation and re-aggregation of human islet cells.
(a) Heatmap showing expression levels of α-cell enriched and β-cell enriched genes in α-pseudoislets, unsorted pseudoislets, and native islets on days 0, 5, and 10. Asterisks show genes associated …
Figure 4—figure supplement 1
Changes in expression levels of α-cell enriched and β-cell enriched genes in α-pseudoislets, unsorted pseudoislets, and native islets on days 0, 5, 10.
Pie charts show percentage of genes altered on day 5 or 10 compared to day 0 (FDR < 0.1; FC > 2 upregulated or FC < –2 downregulated). n=4; α-pseudoislets d0, d5, d10, unsorted pseudoislets d5, …
Figure 5
Time-dependent changes in transcriptome of α-pseudoislets.
(a) Venn diagram shows number of genes that progressively up- or downregulated in α-pseudoislets, unsorted pseudoislets, and native islets in culture over the period from day 0 to day 5 to day 10. (b…
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Antibody | Anti-C-peptide (Rat monoclonal) | Developmental Studies Hybridoma Bank | Cat# GN-ID4, RRID: AB_2255626 | IF (1:200) |
| Antibody | Anti-Ki67 (Mouse monoclonal) | BD Biosciences | Cat# BD550609, RRID: AB_393778 | IF (1:100) |
| Antibody | Anti-Insulin (Guinea Pig polyclonal) | Abcam | Cat# ab7842, RRID: AB_306130 | IF (1:400) |
| Antibody | Anti-Glucagon (Mouse monoclonal) | MilliporeSigma | Cat# G2654, RRID: AB_259852 | IF (1:10,000) |
| Antibody | Anti-Glucagon (Rabbit monoclonal) | Abcam | Cat# ab92517, RRID: AB_10561971 | IF (1:5000) |
| Antibody | Anti-DLK1 (Rabbit polyclonal) | Abcam | Cat# ab21682, RRID: AB_731965 | IF (1:1000) |
| Antibody | Anti-GSN (Rabbit polyclonal) | MilliporeSigma | Cat# HPA054026, RRID: AB_2682347 | IF (1:50) |
| Antibody | Anti-SMIM24 (Rabbit polyclonal) | MilliporeSigma | Cat# HPA045046, RRID: AB_10964444 | IF (1:50) |
| Antibody | Anti-Guinea Pig AF594 (Donkey polyclonal) | Jackson ImmunoResearch | Cat# 706-586-148, RRID: AB_2340475 | IF (1:400) |
| Antibody | Anti-Rat AF488 (Donkey polyclonal) | Jackson ImmunoResearch | Cat# 712-546-153, RRID: AB_2340686 | IF (1:400) |
| Antibody | Anti-Mouse AF594 (Donkey polyclonal) | Jackson ImmunoResearch | Cat# 715-586-150, RRID: AB_2340857 | IF (1:400) |
| Antibody | Anti-Mouse AF488 (Donkey polyclonal) | Jackson ImmunoResearch | Cat# 715-545-150, RRID: AB_2340846 | IF (1:400) |
| Antibody | Anti-Rabbit AF594 (Donkey polyclonal) | Jackson ImmunoResearch | Cat# 711-586-152, RRID: AB_2340622 | IF (1:400) |
| Antibody | Anti-Rabbit AF488 (Donkey polyclonal) | Jackson ImmunoResearch | Cat# 711-545-152, RRID: AB_2313584 | IF (1:400) |
| Biological sample (Homo sapiens) | Primary human pancreatic islets | Integrated Islet Distribution Program (IIDP), Prodo Laboratories Inc, ADI Islet Core | http://iidp.coh.org; RRID: SCR_014387, https://prodolabs.com/ | Freshly isolated |
| Chemical compound | DA-ZP1 | Laboratory of Amit Choudhary | Broad Institute of MIT and Harvard | |
| Chemical compound | ZIGIR | Laboratory of Wen-hong Li | University of Texas, Dallas | |
| Chemical compound | NPG | Thermo | N7991 | |
| Other | DAPI, dilactate | Sigma | D9564 | IF (1:6600) |
| Other | Miami Media #1A | Cellgro | 98-021-CV | Islet cell culture |
| Other | TrypLE | Thermo Fisher Scientific | 12604-013 | Islet cell dissociation |
| Other | FBS | Thermo Fisher Scientific | 10437028 | Islet cell dissociation |
| Other | DPBS | Thermo Fisher Scientific | 14190250 | Islet cell dissociation |
| Other | D-Glucose | Sigma-Aldrich | G8769 | Glucagon secretion assay |
| Other | FFA-BSA | Sigma-Aldrich | 3117057001 | Glucagon secretion assay |
| Other | DMSO | Sigma-Aldrich | D2650-100 | FACS |
| Other | Penicillin-streptomycin | Corning | 30-002-Cl | Islet cell culture |
| Other | 4% PFA | Wako | 163-20145 | Embedding islets in agar |
| Other | Antibody diluent | Abcam | Ab64211 | IF |
| Other | Low melting agarose | Scientific Laboratory Supplies | NAT1030 | Embedding islets in agar |
| Other | TRIzol reagent | Invitrogen | 15596026 | RNA isolation |
| Commercial assay or kit | Glucagon ELISA kit | Mercodia | 10-1271-01 | |
| Commercial assay or kit | MycoAlert Mycoplasma Test Kit | Lonza | LT07-318 | |
| Commercial assay or kit | CellTiter-Glo Luminescent Cell Viability Assay Kit | Promega | G7570 | |
| Commercial assay or kit | ApopTag Peroxidase In Situ Apoptosis Detection Kit | MilliporeSigma | S7100: RRID:AB_2661855 | |
| Commercial assay or kit | QIAGEN RNeasy micro kit | QIAGEN | 74004 | |
| Software, algorithm | Prism v.7.0 | GraphPad Software | http://www.graphpad.com; RRID: SCR_002798 | |
| Software, algorithm | Flowjo-v10 | FlowJo Software | http://www.flowjo.com; RRID: SCR_008520 | |
| Software, algorithm | ImageJ | ImageJ Software | https://imagej.net: RRID:SCR_003070 | |
| Software, algorithm | R version 4.1.0 | R Software | https://www.r-project.org/ |
Additional files
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Supplementary file 1
Single-cell RNA-sequencing (scRNA-seq) analysis of GSE84133 dataset revealed genes that are differentially expressed between islet α-cells and β-cells.
- https://cdn.elifesciences.org/articles/85056/elife-85056-supp1-v3.xlsx
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Supplementary file 2
Changes in expression levels of α-cell enriched and β-cell enriched genes in sorted α-cells on day 0.
- https://cdn.elifesciences.org/articles/85056/elife-85056-supp2-v3.xlsx
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Supplementary file 3
Changes in expression levels of α-cell enriched and β-cell enriched genes in α-pseudoislets, unsorted pseudoislets, and native islets on day 5 and day 10 vs day 0.
- https://cdn.elifesciences.org/articles/85056/elife-85056-supp3-v3.xlsx
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Supplementary file 4
Differentially expressed genes between unsorted pseudoislets versus native islets with fold change >2 and <–2 (log2 FC >1 and <–1) and false discovery rate (FDR)<0.1.
- https://cdn.elifesciences.org/articles/85056/elife-85056-supp4-v3.xlsx
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Supplementary file 5
Pathway analysis of commonly downregulated genes on days 5 and 10 in the unsorted pseudoislets compared to native islets.
- https://cdn.elifesciences.org/articles/85056/elife-85056-supp5-v3.xlsx
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Supplementary file 6
Progressively up- or downregulated genes over the period from day 0 to day 10.
- https://cdn.elifesciences.org/articles/85056/elife-85056-supp6-v3.xlsx
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Supplementary file 7
Altered pathways in α-pseudoislets and unsorted pseudoislets except native islets with time.
- https://cdn.elifesciences.org/articles/85056/elife-85056-supp7-v3.xlsx
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Supplementary file 8
Altered pathways exclusively in α-pseudoislets with time.
- https://cdn.elifesciences.org/articles/85056/elife-85056-supp8-v3.xlsx
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Supplementary file 9
Donor information.
- https://cdn.elifesciences.org/articles/85056/elife-85056-supp9-v3.xlsx
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MDAR checklist
- https://cdn.elifesciences.org/articles/85056/elife-85056-mdarchecklist1-v3.docx
