Research ArticleDiabetes

Gene-edited human stem cell–derived β cells from a patient with monogenic diabetes reverse preexisting diabetes in mice

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Science Translational Medicine  22 Apr 2020:
Vol. 12, Issue 540, eaax9106
DOI: 10.1126/scitranslmed.aax9106
  • Fig. 1 CRISPR-Cas9 correction of WFS1 facilitates generation of functional in vitro WS SC-β cells.

    (A) Schematic of iPSC generation from patients with WS. (B) Information on the three patients, including the genetic location of autosomal recessive pathogenic variants in WFS1 and age of symptom onset. N, none; M, male; F, female; DM, diabetes mellitus; OA, optic atrophy; DI, diabetes insipidus. (C) Gene variants in WFS1 in WS4unedit and WS13unedit iPSCs targeted for CRISPR-Cas9 correction. WT, wild type. (D) Schematic of differentiation protocol mimicking embryonic development of pancreatic β cells. (E) Bright-field images of stage 6 clusters produced from all cell lines in this study. Scale bar, 500 μm. (F) Static GSIS functional assessment of WS4unedit (n = 7), WS9unedit (n = 6), and WS13unedit (n = 5), WS4corr (n = 15) and WS4corr-B (n = 4), and WS13corr (n = 4) stage 6 cells in an in vitro static GSIS assay. Data with WS4unedit and WS13unedit cells are regraphed to help with comparisons. Y axes differ between panels. *P < 0.05, ****P < 0.0001 by one-way paired t test. †††P < 0.001, ††††P < 0.0001 by two-way unpaired t test comparing to high glucose in unedited cells. iPSC, induced pluripotent stem cell. DE, definitive endoderm; PGT, primitive gut tube; PP, pancreatic progenitor; EP, endocrine progenitor; AA, activin A; CHIR, CHIR99021; KGF, keratinocyte growth factor; RA, retinoic acid; LDN, LDN193189; T3, triiodothyronine; Alk5i, Alk5 inhibitor type II; ESFM, enriched serum-free medium.

  • Fig. 2 In vitro characterization of unedited and corrected β cells from iPSCs derived from an individual with WS.

    (A) Representative flow cytometry dot plots and (B) quantified fraction of cells expressing or coexpressing pancreatic β cell or islet markers for WS4unedit (n = 4 to 7) and WS4corr (n = 4 to 8) stage 6 cells. **P < 0.01, ****P < 0.0001 by two-way unpaired t test. (C) Immunostaining of sectioned WS4corr and WS4unedit stage 6 clusters stained for β cell or islet markers. Scale bar, 100 μm. DAPI, 4′,6-diamidino-2-phenylindole. (D) Western blot (left) and quantified intensity (right) of WFS1 protein in stage 0 and stage 6 WS4corr (n = 3) and WS4unedit (n = 3) cells. **P < 0.01, ***P < 0.001 by two-way unpaired t test. ns, not significant. (E) Dynamic human insulin secretion of WS4corr (n = 5) and WS4unedit (n = 4) stage 6 cells and primary human islets (17). Clusters were perfused with 2 mM glucose except where indicated by high glucose (20 mM). CP, C-peptide; GCG, glucagon; SST, somatostatin.

  • Fig. 3 Transplantation of WFS1-edited patient-derived β cells into mice reverses preexisting diabetes.

    (A) Schematic of diabetes induction with streptozotocin (STZ), transplantation of stage 6 cells containing WS SC-β cells, and nephrectomy of the transplanted mice. (B) Blood glucose measurements before and after STZ treatment and after transplantation with SC-β cells or human islets. Five groups were studied: diabetic mice without a transplant (STZ, No Txp; n = 7; black), diabetic mice transplanted with WS4unedit stage 6 cells (STZ, WS4unedit Txp; 5 × 106 cells; n = 6; blue), diabetic mice transplanted with human islets (STZ, HI Txp; 4000 IEQ; n = 4; dark gray), diabetic mice transplanted with WS4corr stage 6 cells (STZ, WS4corr Txp; 5 × 106 cells; n = 10; red), and nondiabetic mice with sham transplant (No STZ, No Txp; n = 5; light gray). (C) Immunostaining of sectioned kidney explanted from a mouse that had received WS4corr stage 6 cells 2 weeks prior. Scale bar, 100 μm. (D) Glucose tolerance test (GTT) 9 days and 10 weeks after Txp. Five groups were studied: STZ, No Txp (n = 7 at 9 days and 10 weeks; black); STZ, WS4unedit Txp (n = 6 at 9 days; n = 5 at 10 weeks; blue); STZ, HI Txp (n = 4 at 9 days; dark gray); STZ, WS4corr Txp (n = 10 at 9 days; n = 9 at 10 weeks; red); and No STZ, No Txp (n = 5 at 9 days and 10 weeks; light gray). (E) Area under the curve (AUC) quantification of GTT data. **P < 0.01, ***P < 0.001 by two-tailed Mann-Whitney test. (F) In vivo GSIS secretion 2 and 10 weeks after transplantation for STZ, WS4unedit Txp (n = 6 and 5 at 2 and 10 weeks; blue); STZ, WS4corr Txp (n = 10 and 9 at 2 and 10 weeks; red); and STZ, HI Txp (n = 4; dark gray) mice at 0 and 60 min after glucose injection (2 g/kg). **P < 0.01 by one-way paired t test. †P < 0.05, †††P < 0.001 by two-way unpaired t test compared to WS4unedit 60 min measurements. (G) Molar ratio of serum human proinsulin to insulin for STZ, WS4unedit Txp (n = 5; blue) and STZ, WS4corr Txp (n = 9; red) 60 min after glucose injection (2 g/kg) 10 weeks after transplantation. ***P < 0.001 by two-way unpaired t test. (H) Blood glucose measurements before and after nephrectomy of two STZ, WS4corr Txp (orange) mice compared to remaining non-nephrectomized STZ, WS4corr Txp (n = 6; red).

  • Fig. 4 Single-cell transcriptional analysis reveals WS4corr and WS4unedit SC-β cell populations and off-targets.

    (A) t-Distributed Stochastic Neighbor Embedding (tSNE) projection from unsupervised clustering of transcriptional data from scRNA-seq of WS4unedit and WS4corr stage 6 cells. (B) Calculated percentages of defined cluster populations for WS4unedit and WS4corr stage 6 cells. (C) Heat map of key β cell population gene markers [insulin (INS), chromogranin A (CHGA), SPINK1, and ID3] with low/none (gray), medium (yellow), and high (red) expression. NP 1, neural progenitor 1; NP 2, neural progenitor 2; NP 3, neural progenitor 3; PH, polyhormonal; EC, enterochromaffin.

  • Fig. 5 CRISPR-Cas9 correction of WFS1 improves β cell gene expression in differentiated cells.

    (A) Violin plots detailing log-normalized gene expression of β cell and islet markers in the WS4unedit (blue) and WS4corr (red) SC-β cell populations defined in Fig. 4. Log fold change and P values for violin plots are available in table S3A. (B) Real-time PCR analysis of the total stage 6 population measuring expression of β cell and islet genes for WS4unedit (n = 6 to 13; blue) and WS4corr (n = 7 to 14; red). **P < 0.01, ***P < 0.001, ****P < 0.0001 by Mann-Whitney two-tailed nonparametric test. (C) Immunostaining of single-cell dispersed WS4corr and WS4unedit stage 6 cells stained for indicated pancreatic and β cell markers. Scale bar, 50 μm.

  • Fig. 6 CRISPR-Cas9 correction of WFS1 reduces WS SC-β cell stress.

    (A) Violin plots detailing log-normalized expression of stress genes in the WS4unedit (blue) and WS4corr (red) SC-β cell populations defined in Fig. 4. Log fold change and adjusted P values for violin plots are available in table S3B. (B) Representative transmission electron microscopy images of ER (top) and mitochondria (bottom) for WS4unedit, WS4corr SC-β cells, and human islets. White dashed lines outline the ER and mitochondria in the cell cytoplasm. Scale bar, 500 nm. (C) Human insulin content (left) and proinsulin/insulin content ratio (right) of WS4unedit (n = 7; blue) and WS4corr (n = 9; red) stage 6 cells. **P < 0.01, ****P < 0.0001 by two-way unpaired t test. (D) Mitochondrial respiration of WS4unedit stage 6 (n = 11; blue), WS4corr stage 6 (n = 9; red), and human islets (n = 6; dark gray) represented as percentage of baseline oxygen consumption rate measurements. Respiration was interrogated by measuring changes in relative OCR after injection with oligomycin (OM), FCCP, and antimycin A (AA)/rotenone (R). (E) Static GSIS functional assessment of stage 6 cells treated with DMSO or thapsigargin (Tg). n = 3. ***P < 0.001 by two-way unpaired t test. (F) Real-time PCR analysis of bulk stage 6 population measuring expression of stress genes after treatment with cytokine mixture (CM), high glucose (Glu), or Tg. n = 4. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, by two-way unpaired t test compared to control (Ctrl).

Supplementary Materials

  • stm.sciencemag.org/cgi/content/full/12/540/eaax9106/DC1

    Materials and Methods

    Fig. S1. Additional patient and cell line information.

    Fig. S2. Additional WS4 stage 6 and human islet analysis.

    Fig. S3. Differentiation progression and efficiency for WS4corr and WS4unedit lines.

    Fig. S4. WFS1 expression during SC-β cell differentiation in WS4corr and WS4unedit lines.

    Fig. S5. GSIS normalized to β cell population.

    Fig. S6. Additional analysis of WS4corr and WS4unedit SC-β cell transplantations in diabetic mice.

    Fig. S7. Additional analysis of WS4corr and WS4unedit SC-β cell scRNA-seq.

    Fig. S8. Additional analysis of WS4corr and WS4unedit SC-β cell scRNA-seq for off-targets.

    Fig. S9. Additional analysis of differences in SC-β cell β and islet markers for WS4corr clones and WS4unedit lines.

    Fig. S10. Additional analysis of ER stress gene expression.

    Fig. S11. Additional analysis of TEM and mitochondrial respiration.

    Fig. S12. Treatment of SC-β cells with chemical stressors.

    Fig. S13. Stress marker measurements of WS4corr-B and human islets.

    Table S1. CRISPR sequences.

    Table S2. Additional analysis of WS4corr and WS4unedit SC-β cell scRNA-seq and population up-regulated genes.

    Table S3. Log fold change values between WS4corr and WS4unedit SC-β cells for markers in Figs. 5A and 6A.

    Table S4. Differentiation protocol.

    Table S5. Differentiation factors.

    Table S6. Media and buffer formulations.

    Table S7. Antibody list.

    Table S8. Primers used for real-time PCR.

    Data file S1. Individual-level data for all figures.

    References (72, 73)

  • The PDF file includes:

    • Materials and Methods
    • Fig. S1. Additional patient and cell line information.
    • Fig. S2. Additional WS4 stage 6 and human islet analysis.
    • Fig. S3. Differentiation progression and efficiency for WS4corr and WS4unedit lines.
    • Fig. S4. WFS1 expression during SC-β cell differentiation in WS4corr and WS4unedit lines.
    • Fig. S5. GSIS normalized to β cell population.
    • Fig. S6. Additional analysis of WS4corr and WS4unedit SC-β cell transplantations in diabetic mice.
    • Fig. S7. Additional analysis of WS4corr and WS4unedit SC-β cell scRNA-seq.
    • Fig. S8. Additional analysis of WS4corr and WS4unedit SC-β cell scRNA-seq for off-targets.
    • Fig. S9. Additional analysis of differences in SC-β cell β and islet markers for WS4corr clones and WS4unedit lines.
    • Fig. S10. Additional analysis of ER stress gene expression.
    • Fig. S11. Additional analysis of TEM and mitochondrial respiration.
    • Fig. S12. Treatment of SC-β cells with chemical stressors.
    • Fig. S13. Stress marker measurements of WS4corr-B and human islets.
    • Table S1. CRISPR sequences.
    • Table S2. Additional analysis of WS4corr and WS4unedit SC-β cell scRNA-seq and population up-regulated genes.
    • Table S3. Log fold change values between WS4corr and WS4unedit SC-β cells for markers in Figs. 5A and 6A.
    • Table S4. Differentiation protocol.
    • Table S5. Differentiation factors.
    • Table S6. Media and buffer formulations.
    • Table S7. Antibody list.
    • Table S8. Primers used for real-time PCR.
    • References (72, 73)

    [Download PDF]

    Other Supplementary Material for this manuscript includes the following:

    • Data file S1 (Microsoft Excel format). Individual-level data for all figures.

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