Journal: Disease Models & Mechanisms

Article Title: Truncated CD19 as a selection marker for the isolation of stem cell-derived β-cells

doi: 10.1242/dmm.052376

Figure Lengend Snippet: Generation and characterization of hESCs with INS-2A-CD19-mScarlet. (A) Schematic overview of the targeting strategy using CRISPR/Cas9 to knock-in add-on 2A-CD19-mScarlet downstream of INS exon 3. The DNA donor vector features a proconvertase (PC) 1/3 recognition site, a 2A fragment to facilitate cleavage, and a fused sequence truncated, signaling-deficient form of CD19 (CD19Δ) and mScarlet red fluorescent protein. (B) Bar graphs showing the results of relative qPCR analysis of cell-specific pancreatic endocrine marker genes in cells of INS-2A-CD19Δ-mScarlet (orange) and H1 (gray) cell lines on day 29 (d29) of differentiation. D0, undifferentiated control. Data represent n ≥4 independent biological replicates. * P <0.05, unpaired t -test. (C) Flow cytometry analysis of C-peptide expression in cells of INS-2A-CD19-mScarlet and H1 cell lines on d29 of differentiation. Bar plots show quantitative representation of the data for INS-2A-CD19Δ-mScarlet (orange) and H1 (gray) cell lines. Data represent n =3 independent biological replicates. Statistical significance was assessed using unpaired t -test. (D) Representative flow cytometry analysis of mScarlet and C-peptide expression on INS-2A-CD19Δ-mScarlet SCβ-cells. The proportion of C-peptide+ cells (red) within the total mScarlet+ population (blue) is shown in INS-2A-CD19Δ-mScarlet cells. n =3 independent biological replicates. (E) Western blot analysis of pro-insulin, insulin, mScarlet and CD19 protein levels on d29-d32 of differentiation for the INS-2A-CD19-mScarlet and H1 cell lines; human islets (H-is) included as a control. D0, INS-2A-CD19Δ-mScarlet hESC line at d0 of differentiation (before starting induction); CD19, INS-2A-CD19Δ-mScarlet line. ns, not significant. Error bars indicate the mean±s.e.m.

Article Snippet: Cells were stained with mouse anti-human CD19 antibody (Miltenyi Biotec, cat# 130-113-730; 1:50) or mouse anti-human connecting (C)-peptide antibody (BD Pharmingen, #565831; 1:100) in the dark for 25 min.

Techniques: CRISPR, Knock-In, Plasmid Preparation, Sequencing, Marker, Control, Flow Cytometry, Expressing, Western Blot

Journal: Disease Models & Mechanisms

Article Title: Truncated CD19 as a selection marker for the isolation of stem cell-derived β-cells

doi: 10.1242/dmm.052376

Figure Lengend Snippet: CD19Δ-mScarlet localizes to the cell surface of SCβ-cells. (A) HEK293 cells express CD19Δ-mScarlet 72 h post transfection. HEK293 cells were transfected with the CD19Δ-mScarlet construct with or without 2A at the N-terminus. Cells were dissociated and assayed for CD19 and mScarlet expression with flow cytometry after 72 h. n =3 independent biological replicates. (B) Representative flow cytometry analysis of CD19 expression on unstained, surface stained, and surface and intracellularly stained d25 SCβ-cells. n =4 independent biological replicates. *** P <0.001, **** P <0.0001 (one-way ANOVA followed by Tukey's multiple comparisons test). Error bars indicate the mean±s.e.m. (C) Immunofluorescence images of unsorted stage 6, d25 immature INS-2A-CD19Δ-mScarlet SCβ-cell clusters expressing INS-2A-CD19Δ-mScarlet (red). Third image: Boxed areas on the left and right indicate magnified representative views of bottom left and top right, respectively. Staining was against insulin (green); nuclei of clustered cells were stained with DAPI (blue). Scale bars: 50 µm.

Article Snippet: Cells were stained with mouse anti-human CD19 antibody (Miltenyi Biotec, cat# 130-113-730; 1:50) or mouse anti-human connecting (C)-peptide antibody (BD Pharmingen, #565831; 1:100) in the dark for 25 min.

Techniques: Transfection, Construct, Expressing, Flow Cytometry, Staining, Immunofluorescence

Journal: Disease Models & Mechanisms

Article Title: Truncated CD19 as a selection marker for the isolation of stem cell-derived β-cells

doi: 10.1242/dmm.052376

Figure Lengend Snippet: MACS enriches CD49+ cells in immature INS-GFP SCβ-cells. (A) Representative flow cytometry analysis of CD49 expression on day 25 in SCβ-cells that were unsorted (Unsorted CTRL), sorted once (CD49 once), or sorted twice (CD49 sorted twice). (B) Bar plot quantification of flow cytometry data showing the proportions of CD49 + cells and CD49 + INS-GFP + cells in the Unsorted, CD45 sorted, and CD45 sorted twice groups. n =3 independent biological replicates. * P <0.05, **** P <0.0001 (one-way ANOVA followed by Tukey's multiple comparisons test). (C) Recovery rate was calculated by dividing yield by expected yield. Statistical significance was determined with the unpaired t -test. n =3 independent biological replicates. (D) Live confocal imaging of unsorted and sorted stage 6, d35 immature INS-2A-CD19Δ-mScarlet SCβ-cell clusters expressing INS-GFP (green). Scale bar: 50 µm. Quantitative presentation of the flow cytometry data from panel A, highlighting the percentage of GFP-expressing cells in unsorted and CD49-sorted populations. (E) Static secretion of C-peptide in response to stimulation with 2.8 mM glucose, 16.7 mM glucose and 40 mM KCl in an in vitro assay for sorted stage 6, d35 immature INS-2A-CD19Δ-mScarlet SCβ-cell clusters. Total C-peptide was collected by ethanol acid extraction. n =4 independent biological replicates. **** P <0.0001 (one-way ANOVA followed by Tukey's multiple comparisons test). Error bars indicate the mean±s.e.m.

Article Snippet: Cells were stained with mouse anti-human CD19 antibody (Miltenyi Biotec, cat# 130-113-730; 1:50) or mouse anti-human connecting (C)-peptide antibody (BD Pharmingen, #565831; 1:100) in the dark for 25 min.

Techniques: Flow Cytometry, Expressing, Imaging, In Vitro, Extraction

Journal: Disease Models & Mechanisms

Article Title: Truncated CD19 as a selection marker for the isolation of stem cell-derived β-cells

doi: 10.1242/dmm.052376

Figure Lengend Snippet: MACS enriches CD19+ mScarlet+ and C-peptide+ cells from immature INS-2A-CD19Δ-mScarlet SCβ-cells. (A) Representative flow cytometry analysis of CD19 expression on d35 INS-2A-CD19Δ-mScarlet SCβ-cells that were unsorted and CD19 sorted. (B) Bar plot quantification of flow cytometry data showing the proportions of CD19+ cells and CD19+ mScarlet+ cells in the unsorted and MACS CD19 sorted groups. n =3; black circles indicate individual biological replicates. ** P <0.01 (one-way ANOVA followed by Tukey's multiple comparisons test.). (C) Representative flow cytometry analysis of C-peptide expression on d25 SCβ-cells that were unsorted and CD19 sorted. n =2 independent biological replicates. (D) Recovery rate was calculated by dividing yield by expected yield. Statistical significance was determined with the unpaired t -test. n =3; black circles indicate individual biological replicates. * P <0.05 (one-way ANOVA with multiple comparisons). (E) Live confocal imaging of unsorted and CD19 sorted stage 6, d35 immature INS-2A-CD19Δ-mScarlet SCβ-cell clusters expressing INS-2A-CD19-ΔmScarlet (red). Scale bars: 50 µm. (F) Static secretion of C-peptide in response to stimulation with 2.8 mM glucose, 16.7 mM glucose and 40 mM KCl in an in vitro assay. Total C-peptide was collected by ethanol acid extraction. Fold-stimulation was calculated as C-peptide secretion at high glucose divided by C-peptide secretion at low glucose. n =3; each colored circle denotes a biological replicate, with identical colors indicating the same sample. * P <0.05, ** P <0.01 (one-way ANOVA followed by Tukey's multiple comparisons test). Error bars indicate the mean±s.e.m.

Article Snippet: Cells were stained with mouse anti-human CD19 antibody (Miltenyi Biotec, cat# 130-113-730; 1:50) or mouse anti-human connecting (C)-peptide antibody (BD Pharmingen, #565831; 1:100) in the dark for 25 min.

Techniques: Flow Cytometry, Expressing, Imaging, In Vitro, Extraction

Journal: Disease Models & Mechanisms

Article Title: Truncated CD19 as a selection marker for the isolation of stem cell-derived β-cells

doi: 10.1242/dmm.052376

Figure Lengend Snippet: Gene expression analysis of unsorted, CD49 sorted and CD19 sorted SCβ-cells using NanoString analysis. Plotted are the expression counts of selected genes as indicated, with key marker genes from islets (top), endocrine (middle), mesodermal lineage and pancreatic immune cells (bottom). CD49+, CD49 sorted SCβ-cells; CD19+, CD19 sorted SCβ-cells. n =3 independent biological replicates. * P <0.05, ** P <0.01, *** P <0.001, **** P <0.0001 (one-way ANOVA followed by Tukey's multiple comparisons test.); ns, not significant. Error bars indicate the mean±s.e.m.

Article Snippet: Cells were stained with mouse anti-human CD19 antibody (Miltenyi Biotec, cat# 130-113-730; 1:50) or mouse anti-human connecting (C)-peptide antibody (BD Pharmingen, #565831; 1:100) in the dark for 25 min.

Techniques: Gene Expression, Expressing, Marker