An Isogenic Human ESC Platform for Functional Evaluation of Genome-wide-Association-Study-Identified Diabetes Genes and Drug Discovery

Abstract

Genome-wide association studies (GWASs) have increased our knowledge of loci associated with a range of human diseases. However, applying such findings to elucidate pathophysiology and promote drug discovery remains challenging. Here, we created isogenic human ESCs (hESCs) with mutations in GWAS-identified susceptibility genes for type 2 diabetes. In pancreatic beta-like cells differentiated from these lines, we found that mutations in CDKAL1, KCNQ1, and KCNJ11 led to impaired glucose secretion in vitro and in vivo, coinciding with defective glucose homeostasis. CDKAL1 mutant insulin+ cells were also hypersensitive to glucolipotoxicity. A high-content chemical screen identified a candidate drug that rescued CDKAL1-specific defects in vitro and in vivo by inhibiting the FOS/JUN pathway. Our approach of a proof-of-principle platform, which uses isogenic hESCs for functional evaluation of GWAS-identified loci and identification of a drug candidate that rescues gene-specific defects, paves the way for precision therapy of metabolic diseases.

Figure 1. Biallelic mutation of CDKAL1, KCNQ1 or KCNJ11 does not affect differentiation or the expression of mature pancreatic beta cell markers

(A) qRT-PCR experiments confirmed the expression of CDKAL1, KCNQ1, and KCNJ11 in insulin-GFP⁺ (INS-GFP⁺) cells derived from INS^GFP/W HES3 cells (n=4 independent experiments, error bars indicate S.D.) The expression level of CDKAL1, KCNQ1, and KCNJ11 transcripts in primary human beta cells was calculated by dividing the expression level in primary human islets by the percentage of insulin⁺ cells. (B) Western blotting analysis of wildtype (wt) and isogenic mutant hESC-derived D30 cells. (C) Representative flow cytometry analysis and quantification of wt and isogenic mutant hESC-derived cells at day 30, n=3. (D) Immunocytochemistry analysis of wt and isogenic mutant hESC-derived D30 cells. The insulin⁺ cells express mature beta cell markers, including PDX1, NKX6.1 and NKX2.2. Scale bar = 100 µm. (E) Intracellular FACS analysis of D30 cells. (F) Total c-peptide content per 1 k insulin-GFP⁺ cells as measured by ELISA, n=3. Total c-peptide content in primary human beta cells was calculated by dividing the total c-peptide in primary human islets by the percentage of insulin⁺ cells. Clone #1 and #2 are two independent isogenic hESC clones carrying different frameshift mutations. hESCs were differentiated using protocol 2. The data is presented as mean±S.D. p values calculated by unpaired two-tailed Student’s t-test were *p<0.05, **p<0.01, ***p<0.001. See also Figure S1.

Figure 2. Biallelic mutation of CDKAL1, KCNQ1 or KCNJ11 impairs insulin secretion upon various stimulations

(A and B) Human c-peptide (% of content) (A) and fold change (B) of wildtype (wt) and isogenic mutant cells at day 30 with or without 30 mM KCl stimulation in the presence of 2 mM D-glucose, n=3. (C and D) Human c-peptide (% of content) (C) and fold change (D) of wildtype and isogenic mutant cells at day 30 with and without 10 mM arginine stimulation in the presence of 2 mM D-glucose, n=3. (E and F) Human c-peptide (% of content) (E) and fold change (F) of wildtype and isogenic mutant cells at day 30 with or without 20 µM forskolin and 50 µM IBMX stimulation in the presence of 2 mM D-glucose, n=3. (G and H) Human c-peptide (% of content) (G) and fold change (H) of wildtype and isogenic mutant cells at day 30 with 2 mM or 20 mM D-glucose, n=3. Arg: arginine; forsk: forskolin; LG: 2 mM D-glucose; HG: 20 mM D-glucose. Human c-peptide secretion was calculated by dividing the secreted c-peptide by the total c-peptide of insulin-GFP⁺ cells or primary human beta cells. Clones #1 and #2 are two independent isogenic hESC clones carrying different frameshift mutations. hESCs were differentiated using protocol 2. The data is presented as mean±S.D. n.s. indicates a non-significant difference. p values calculated by unpaired two-tailed Student’s t-test were *p<0.05, **p<0.01, ***p <0.001, ****p <0.0001. See also Figure S2.

Figure 3. CDKAL1^−/− insulin-GFP⁺ cells are hypersensitive to glucotoxicity and lipotoxicity

(A and B) Immunocytochemistry analysis (A) and quantification of the percentage (B) of PI⁺/insulin⁺ cells in wildtype (wt) or CDKAL1^−/− insulin⁺ cells cultured in the presence of 2 mM D-Glucose (ctrl-g), 35 mM D-Glucose (glu), no palmitate (ctrl-p) or 1 mM palmitate (palm). PI⁺/insulin⁺ cells are highlighted by arrows. (C and D) Flow cytometry analysis (C) and quantification of the percentage (D) of Annexin V⁺ cells in wildtype and CDKAL1^−/− insulin-GFP⁺ cells cultured as in (A). (E) Heat map representing the expression profiles of ER-stress related genes comparing wildtype and CDKAL1^−/− insulin⁺ cells cultured in the absence or presence of 1 mM palmitate. (F) Ingenuity Pathway Analysis of genes that are >2 fold upregulated in CDKAL1^−/− insulin⁺ cells cultured in the presence of 1 mM palmitate. INS: insulin; PI: propidium iodide. n=3 independent biological replicates. n.s. indicates a non-significant difference. Clone #1 and #2 are two independent isogenic hESC clones carrying different frameshift mutations. hESCs were differentiated using protocol 2. p values calculated by unpaired two-tailed Student’s t-test were *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. Scale bar = 100 µm. See also Figure S3.

Figure 4. CDKAL1^−/−, KCNQ1^−/− and KCNJ11^−/− cells show defective glucose stimulated insulin secretion and impaired ability to maintain glucose homeostasis after transplantation into streptozotocin-treated immunodeficient mice

(A) human insulin GSIS at 2 weeks after transplantation of the mutant cells compared to wildtype cells (wt). (B) GSIS secretion of SCID-beige mice carrying human cells at 6 weeks after transplantation. p values calculated by one-way repeated measures ANOVA. (C and D) Intraperitoneal glucose tolerance test (IPGTT) (C) and area under the curve (AUC) (D) of STZ treated mice 6 weeks after transplantation. p values calculated by two-way repeated measures ANOVA with a Bonferroni test for multiple comparisons between wt and mutant cells. n=8 mice for each condition. hESCs were differentiated using protocol 2. n.s. indicates a non-significant difference. p values were *p<0.05 and ** p<0.01, *** p<0.001, **** p<0.0001. See also Figure S4.

Figure 5. A high content chemical screen identifies a drug candidate that rescues glucolipotoxicity caused specifically by mutations in CDKAL1

(A) Chemical structure of T5224. (B and C) Efficacy curve of T5224 on the number of insulin⁺ cells (B) and the percentage of PI⁺INS⁺ cells (C). PI: propidium iodide. (D and E) Immunocytochemistry analysis (D) and quantification of the percentage (E) of PI⁺/insulin⁺ cells in wildtype (wt) and CDKAL1^−/−, insulin⁺ cells treated with 30 µM T5224 when cultured in the presence of 2 mM D-Glucose (ctrl-g), 35 mM D-Glucose (glu), no palmitate (ctrl-p) or 1 mM palmitate (palm). PI⁺/insulin⁺ cells are highlighted by arrows. (F and G) Flow cytometry analysis (F) and quantification (G) of apoptotic rate for wt or CDKAL1^−/− insulin-GFP⁺ cells treated with DMSO or T5224. (H and I) T5224 also rescues the impaired forskolin-induced (H) and glucose-induced insulin secretion (I). Experiments in Figure 5A–C were performed using cells derived from protocol 1. Experiments in Figure 5D–I were performed using cells derived from protocol 2. n=3 independent biological replicates for each condition. n.s. indicates a non-significant difference. p values calculated by unpaired two-tailed Student’s t-test were *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. Scale bar = 50 µm. See also Figure S5.

Figure 6. T5224 rescues beta cell defects caused by CDKAL1 mutation through inhibiting the FOS/JUN pathway

(A) Pathway enrichment analysis on up/down-regulated genes in CDKAL1^−/− insulin-GFP⁺ cells using the DAVID function annotation tool. (B) Heat map of Focal Adhesion pathway associated genes comparing wildtype (wt) and CDKAL1^−/− insulin-GFP⁺ cells. (C) Heat map of FOS/JUN pathway associated genes comparing wt and CDKAL1^−/− insulin-GFP⁺ cells. (D) Top 20 upregulated genes in CDKAL1^−/− insulin-GFP⁺ cells as compared to wildtype cells. (E) qRT-PCR analysis of JUNB, FOS, FOSB expression in wildtype and CDKAL1^−/− insulin-GFP⁺ cells. (F) Western blotting analysis of FOS protein in wildtype and CDKAL1^−/− cells at D30 of differentiation. (G) Targeted mutation of FOS rescues the high death rate in CDKAL1^−/− insulin-GFP⁺ cells in the presence of 35 mM D-glucose or 1 mM palmitate. (H and I) Flow cytometry analysis (H) and quantification of apoptotic rate (I) of CDKAL1^−/− insulin-GFP⁺ cells expressing Cas9 and either scrambled sgRNA or sgFOS. (J and K) Mutation of FOS rescues the impaired forskolin-induced (J) and glucose-induced (K) insulin secretion that is caused by mutation of CDKAL1. SgFOS 1# and 2# represent two independent sgRNAs targeting different locations of exon1 of c-FOS. Scramble sgRNA #1 and Scramble #2 “target” controls were designed to have low homology to the human genome and are used as non-targeting controls. hESCs were differentiated using protocol 2. The data is presented as mean±S.D. n.s. indicates a non-significant difference. p values calculated by unpaired two-tailed Student’s t-test were *p<0.05, **p<0.01, ***p<0.001. See also Figure S6.

Figure 7. T5224 or loss of FOS rescues the function of CDKAL1^−/− cells in SCID-beige mice carrying human cells

(A) Human insulin GSIS at 10 weeks after transplantation of mutant cells compared to wildtype cells (wt). (B) GSIS secretion of SCID-beige mice carrying human cells after glucose stimulation 48 hours after treatment with 300 mg/kg T5224 or vehicle. (C and D) IPGTT (C) and AUC (D) of mice transplanted with CDKAL1^−/− cells treated with 300 mg/kg T5224 or vehicle. (E) GSIS secretion of SCID-beige mice carrying human cells after glucose stimulation after treatment with T5224 or vehicle twice a week for four weeks. (F and G) IPGTT (F) and AUC (G) of mice transplanted with CDKAL1^−/− cells treated with 300 mg/kg T5224 or vehicle twice a week for 4 weeks. (H) GSIS secretion of SCID-beige mice transplanted with CDKAL1^−/− cells carrying scramble sgRNA or CDKAL1^−/− cells carrying sgFOS. (I and J) IPGTT (I) and AUC (J) of mice transplanted with CDKAL1^−/− cells carrying scramble sgRNA or CDKAL1^−/− cells carrying sgFOS at 6 weeks after transplantation. n=8 mice for each condition. hESCs were differentiated using protocol 2. In GSIS assay, p values were calculated by one-way repeated measures ANOVA. In IPGTT assay, p values were calculated by two-way repeated measures ANOVA with a Bonferroni test for multiple comparisons between DMSO and T5224 treated conditions. p values were *p<0.05, ** p<0.01, ***p<0.001. See also Figure S7.