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

Abstract

Differentiation of insulin-producing pancreatic β cells from induced pluripotent stem cells (iPSCs) derived from patients with diabetes promises to provide autologous cells for diabetes cell replacement therapy. However, current approaches produce patient iPSC-derived β (SC-β) cells with poor function in vitro and in vivo. Here, we used CRISPR-Cas9 to correct a diabetes-causing pathogenic variant in Wolfram syndrome 1 (WFS1) in iPSCs derived from a patient with Wolfram syndrome (WS). After differentiation to β cells with our recent six-stage differentiation strategy, corrected WS SC-β cells performed robust dynamic insulin secretion in vitro in response to glucose and reversed preexisting streptozocin-induced diabetes after transplantation into mice. Single-cell transcriptomics showed that corrected SC-β cells displayed increased insulin and decreased expression of genes associated with endoplasmic reticulum stress. CRISPR-Cas9 correction of a diabetes-inducing gene variant thus allows for robust differentiation of autologous SC-β cells that can reverse severe diabetes in an animal model.

Fig. 1.. CRISPR/Cas9 correction of WFS1 generates functional WS SC-β cells in vitro.

(A) Schematic summary of iPSC generation from patients with WS. (B) Information on the 3 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 WS4^unedit and WS13^unedit iPSCs targeted for CRISPR/Cas9 correction. (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 WS4^unedit (n=7), WS9^unedit (n=6), and WS13^unedit (n=5), WS4^corr (n=15) and WS4^corr-B (n=4), and WS13^corr (n=4) stage 6 cells in an in vitro static GSIS assay. Data with WS4^unedit and WS13^unedit 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. Act A, 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 co-expressing pancreatic β cell or islet markers for WS4^unedit (n=4-7) and WS4^corr (n=4-8) stage 6 cells. **p<0.01, ****p<0.0001 by two-way unpaired t-test. (C) Immunostaining of sectioned WS4^corr and WS4^unedit stage 6 clusters stained for β cell or islet markers. Scale bar, 100 μm. (D) Western blot (left) and quantified intensity (right) of WFS1 protein in stage 0 and stage 6 WS4^corr (n=3) and WS4^unedit (n=3) cells. **p<0.01, ***p<0.001 by two-way unpaired t-test. (E) Dynamic human insulin secretion of WS4^corr (n=5) and WS4^unedit (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 gene 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 WS4^unedit stage 6 cells (STZ, WS4^unedit Txp; 5x10⁶ cells; n=6; blue), diabetic mice transplanted with human islets (STZ, HI Txp; 4000 IEQ; n=4; dark grey), diabetic mice transplanted with WS4^corr stage 6 cells (STZ, WS4^corr Txp; 5x10⁶ cells; n=10; red), and non-diabetic mice with a sham transplant (No STZ, No Txp; n=5; light grey). (C) Immunostaining of sectioned kidney explanted from a mouse that had received WS4^corr stage 6 cells 2 wk prior. Scale bar, 100 μm. (D) Glucose tolerance test (GTT) 9 d and 10 wk after Txp. Five groups were studied: STZ, No Txp (n=7 at 9 d and 10 wk; black); STZ, WS4^unedit Txp (n=6 at 9 d; n=5 at 10 wk; blue); STZ, HI Txp (n=4 at 9 d; dark grey); STZ, WS4^corr Txp (n=10 at 9 d; n=9 at 10 wk; red); and No STZ, No Txp (n=5 at 9 d and 10 wk; light grey). (E) Area under the curve (AUC) quantification of GTT data. **p<0.01, ***p<0.001 by Mann-Whitney two-tailed nonparametric test. (F) In vivo GSIS secretion 2 and 10 wk after transplantation for STZ, WS4^unedit Txp (n=6 and 5 at 2 and 10 wk; blue); STZ, WS4^corr Txp (n=10 and 9 at 2 and 10 wk; red); and STZ, HI Txp (n=4; dark grey) mice at 0 and 60 min after 2 g/kg glucose injection. ns=not significant, **p<0.01 by one-way paired t-test. †p<0.05, †††p<0.001 by two-way unpaired t-test compared to WS4^unedit 60 min measurements. (G) Molar ratio of serum human proinsulin to insulin for STZ, WS4^unedit Txp (n=5; blue) and STZ, WS4^corr Txp (n=9; red) 60 min after 2 g/kg glucose injection 10 wk after transplantation. ***p<0.001 by two-way unpaired t-test. (H) Blood glucose measurements before and after nephrectomy of two STZ, WS4^corr Txp (orange) mice compared to remaining non-nephrectomized STZ, WS4^corr Txp (n=6; red). CP, C-peptide; GCG, glucagon; SST, somatostatin.

Fig. 4.. Single cell transcriptional analysis reveals WS4^corr and WS4^unedit SC-β cell populations and off-targets.

(A) tSNE projection from unsupervised clustering of transcriptional data from scRNA-seq of WS4^unedit and WS4^corr stage 6 cells. (B) Calculated percentages of defined cluster populations for WS4^unedit and WS4^corr stage 6 cells. (C) Heat map of key β cell population gene markers (insulin [INS], chromogranin A [CHGA], SPINK1, ID3) with low/none (grey), medium (yellow), and high (red) expression. NP1, neural progenitor 1; NP2, neural progenitor 2; NP3, 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 WS4^unedit (blue) and WS4^corr (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 WS4^unedit (n=6-13; blue) and WS4^corr (n=7-14; red). **p<0.01, ***p<0.001, ****p<0.0001 by Man-Whitney two-tailed nonparametric test. (C), Immunostaining of single-cell dispersed WS4^corr and WS4^unedit 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 WS4^unedit (blue) and WS4^corr (red) SC-β cell populations defined in Fig. 4. Log fold-change and adjusted p-values for violin plots available in table S3B. (B) Representative transmission electron microscopy images of ER (top) and mitochondria (bottom) for WS4^unedit, WS4^corr SC-β cells, and human islets. White dotted 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 WS4^unedit (n=7; blue) and WS4^corr (n=9; red) stage 6 cells. **p<0.01, ****p<0.0001 by two-way unpaired t-test. (D) Mitochondrial respiration of WS4^unedit stage 6 (n=11; blue), WS4^corr stage 6 (n=9; red), and human islets (n=6; dark grey) 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. ns, not significant. (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<0.01, ***p<0.001 by two-way unpaired t-test compared to ctrl. ctrl=control.