Genome Editing of Lineage Determinants in Human Pluripotent Stem Cells Reveals Mechanisms of Pancreatic Development and Diabetes

Abstract

Directed differentiation of human pluripotent stem cells (hPSCs) into somatic counterparts is a valuable tool for studying disease. However, examination of developmental mechanisms in hPSCs remains challenging given complex multi-factorial actions at different stages. Here, we used TALEN and CRISPR/Cas-mediated gene editing and hPSC-directed differentiation for a systematic analysis of the roles of eight pancreatic transcription factors (PDX1, RFX6, PTF1A, GLIS3, MNX1, NGN3, HES1, and ARX). Our analysis not only verified conserved gene requirements between mice and humans but also revealed a number of previously unsuspected developmental mechanisms with implications for type 2 diabetes. These include a role of RFX6 in regulating the number of pancreatic progenitors, a haploinsufficient requirement for PDX1 in pancreatic β cell differentiation, and a potentially divergent role of NGN3 in humans and mice. Our findings support use of systematic genome editing in hPSCs as a strategy for understanding mechanisms underlying congenital disorders.

Figure 1. Gain-of-function studies in hESCs

A) Schematics of the doxycycline-inducible gene expression platform in hESCs. Homology directed repair of the DNA DSB induced by a pair of TALENs led to targeted integration of the transgene and M2rtTA into the AAVS1 locus. After the establishment of a clonal line, transgene expression can be induced upon doxycycline treatment. SA: Splice acceptor; 2A: Self-cleavage 2A peptide; Puro: Puromycin resistant gene; TRE: tetracycline response element; Neo: Neomycin resistant gene; CAG, constitutive synthetic promoter; M2rtTA, reverse tetracycline transactivator; DOX: doxycycline (also indicated by the red dot). B, C) Southern blotting (B) and qRT-PCR (C) analysis of the iNotchIC and iNGN3 lines. Correctly targeted lines without random integrations are indicated in red. WT: Wild-type control; 3’ EXT: 3’ external probe; 5’ INT: 5’ external probe; hESC: undifferentiated hESC; PP: pancreatic progenitor. D) Representative immunofluorescence staining of iNotchIC and iNGN3 cells at PH-β cell stage with or without doxycycline treatment at pancreatic progenitor stage. DE: Definitive endoderm; PP: pancreatic progenitors; PH-β: Polyhormonal β cells; CPEP; C-peptide; GCG: glucagon; SST: somatostatin. E, F) Representative FACS plots (E) and quantification of iNGN3 hESC-derived INS+ cells (F) with or without doxycycline treatment. Number in the FACS plots indicates the percentage of target cells. n = 4: two independent experiments were performed on 2 iNGN3 lines. G) qRT-PCR analysis of endocrine markers and endocrine specific transcription factors in iNGN3 hESCs without and with doxycycline treatment. n = 4. Unless otherwise indicated, scale bar = 100 µm in all figures; error bars indicate standard error of the mean (SEM); and P values by unpaired two-tailed student t-test <0.05, 0.01, and 0.0001 are indicated by one, two, and four asterisks, respectively. For qRT-PCR results, P values are not indicated in graphs due to the large number of bars, but are mentioned in text when relevant. (See also Figure S1)

Figure 2. iCRISPR-mediated creation of knockout hESC lines for loss-of-function studies

A) Schematics of the iCRISPR system. In established iCas9 hESCs, Cas9 expression is induced by doxycycline treatment. After transfection of gRNA, Cas9 is guided to the target locus via Watson-Crick base pairing and induces DNA DSBs. In the absence of repair templates, error-prone non-homologous end joining often results in Indel (insertion/deletion) mutations. In the presence of repair templates, HDR can generate patient-specific mutations or correct a mutation in mutant lines. B) Schematics of the experiment design. 18 CRISPR/gRNAs were used to target 8 genes to generate 62 hESC mutants using iCRISPR. By stepwise differentiation of the hESC mutants into pancreatic lineage, the effects of these mutations were extensively examined by immunofluorescence staining, flow cytometry, qRT-PCR and Western blotting at each developmental step. C) Percentage of hESC clonal lines carrying different types of mutations generated with CRISPR gRNA targeting. +/+: no mutation in either allele; +/Indel: with Indels in one allele; Indel/Indel: with Indels in both alleles or Indel/Y in the case of ARX. *: ARX is on the X chromosome and the parental HUES8 line is from a male donor. D) Western blot analysis for validation of the loss of wild-type proteins in hESC knockout mutants in PDX1, MNX1 and HES1. Clonal names and mutant genotypes are labeled on the top, and the CRISPR/gRNAs used to generate the mutants are labeled in the bottom. The PDX1 antibody detected two protein bands likely associated with post-translational modification of PDX1 (Frogne et al., 2012). (See also in Figure S2)

Figure 3. Phenotypic analysis of hESCs mutants to determine the developmental basis for PNDM

A) FACS quantification of CXCR4+ cells, PDX1+ cells and INS+ cells in hESC mutant and wild-type (WT) cells at the DE, PP and PH-β cell stage respectively. FACS quantification results were normalized to the wild-type controls from the same experiment and combined together for statistic analysis. n = 8 for all genes except for HES1 (n = 4). Results from 4 (2 for HES1) clonal lines each with two differentiation experiments were combined. B) Representative immunofluorescence staining of hESC mutants and wild-type cells at the PH-β cell stage. C) Representative FACS plots of INS+ cells in hESC mutants and wild-type cells at the endocrine cell stage. D) qRT-PCR analysis of endocrine markers in hESC mutants and wild-type cells at the endocrine cell stage. n = 8 for all genes except for HES1 (n = 4). (See also in Figure S3)

Figure 4. RFX6 and PDX1 in pancreatic differentiation

A) Representative immunofluorescence staining of PDX1, Phospho-Histone H3 (PHH3) and cleaved Caspase 3 (Casp3) expression in wild-type and RFX6^−/− mutants at the pancreatic progenitor stage. B, C) FACS quantification of the percentage of PHH3 (B) and Casp3-expressing cells (C) in PDX1+ cells formed from wild-type and RFX6^−/− mutants at the PP stage. n = 4 for wild-type lines and n = 8 for RFX6^−/− lines. D) FACS histogram for PDX1 expression in wild-type and RFX6^−/− mutants at the pancreatic progenitor stage. E) Schematics illustrating the protein structure of the wild-type PDX1 protein and the predicted truncated proteins expressed from PDX1^L36fs and PDX1^A34fs alleles. Transactivation and DNA-binding homeobox domains are indicated in blue and red respectively. New sequences resulting from frameshift translation are indicated in hatched boxes. F, G) Representative immunofluorescence staining (F) and FACS plots (G) of the wild-type, PDX1^−/− (PDX1^L36fs/L36fs and PDX1^L36fs/A34fs) and PDX1^+/− (PDX1^L36fs/+ and PDX1^A34fs/+) mutant cells at the PH-β cell stage. H) FACS quantification of INS+ cells in wild-type, PDX1^−/− and PDX1^+/− mutants at the PH-β cell stage. n = 4: two independent experiments were performed on 2 lines for each genotype. I) qRT-PCR analysis of endocrine markers in wild-type control, PDX1^−/− and PDX1^+/− mutants at the PH-β cell stage. n = 4. J-L) Representative immunofluorescence (J) Western blot analysis (K) and FACS analysis (L) is shown for the wild-type PDX1^+/+ control, PDX1^−/− (PDX1^L36fs/L36fs and PDX1^L36fs/A34fs) and PDX1^+/− (PDX1^L36fs/+ and PDX1^A34fs/+) mutant cells The red bar indicates differential PDX1 expression levels in the FACS histogram. (See also in Figure S4)

Figure 5. An important but not essential requirement for NGN3 in human endocrine differentiation

A) Schematics showing the genomic sequences of the wild-type NGN3 allele (in NGN3^+/+), the NGN3 mutant allele with a 526 bp deletion (in NGN3^Δ/Δ), the NGN3 Indel mutant alleles generated using NGN3-cr3, cr4, cr5 and cr6 (in NGN^−/−) and the NGN3 allele carrying a patient-specific mutation (in NGN3^R107S/R107S). Coding sequences are indicated in grey, with the sequence corresponding to the bHLH DNA-binding domain highlighted in red. C>A substitution (red, also indicated by an asterisk) was introduced using the ssDNA HDR template, resulting in an R107S amino acid substitution. B) Sanger sequencing results of NGN3 wild-type cells and NGN3 mutants homozygous for NGN3^R107S. C>A substitution is labeled in red box. C) Representative FACS plots of INS+ cells showing the appearance of INS+ cells only in NGN3^Δ/Δ but not PDX1^−/− mutants. D) Representative immunofluorescence staining showing the residue GCG+ and/or INS+ cells in NGN3^−/− (generated using NGN3-cr3 and cr4), NGN3^Δ/Δ and NGN3^R107S/R107S cells. Arrows point to the same cells in corresponding images in the upper and lower panel. Intact nuclei DAPI staining (without fragmentation) argues against non-specific staining from dead cells. (See also in Figure S4 and S5)

Figure 6. NGN3 is important for the generation of glucose-responsive β-like cells

A) Schematics and Sanger sequencing results demonstrating the generation of knockout NGN3^−/− hESC lines, and the subsequent creation of the corrected NGN3^Cr/Cr hESC lines, in which both NGN3 mutant alleles were reversed to wild-type. B) FACS quantification of CXCR4+, PDX1+NKX6.1+ and CPEP+ (total and monohormonal) cells formed from wild-type, NGN3^Cr/Cr and NGN3^−/− lines at the DE, PP2 and β-like stage, respectively. n = 4. Results from 2 clonal lines each with 2 differentiation experiments were combined. C, D) Representative immunofluorescence staining (C) and FACS plots (D) for pancreatic endocrine hormone expression in wild-type, NGN3^Cr/Cr lines and NGN3^−/− mutants at the β-like stage. E, F) Representative immunofluorescence staining (E) and FACS plots (F) for C-peptide, PDX1 and NKX6.1 expression in wild-type, NGN3^Cr/Cr lines and NGN3^−/− mutants at the β-like stage. G) Glucose-stimulated insulin secretion assay for wild-type, NGN3^Cr/Cr and NGN3^−/− hESCs at the β-like stage. The fold change of insulin secretion with high glucose (16.7 mM) relative to low glucose (2.8 mM) treatment is shown. H, I) FACS quantification of the percentage of monohormonal and polyhormonal cells (H) and NKX6.1+ cells (I) in CPEP+ cells in wild-type, NGN3^Cr/Cr and NGN3^−/− hESCs. n = 4. (See also in Figure S5 and S6)

Figure 7. Temporal control of NGN3 activity in NGN3^−/− mutants

A) Schematics illustrating gene correction and temporal control of NGN3 expression in NGN3^−/− hESCs. B) Schematics of temporal regulation of NGN3 expression at different stages of pancreatic differentiation in the NGN3^−/− mutant background. C, D) Representative immunofluorescence staining (C) and FACS plots (D) for the induction of pancreatic endocrine cells corresponding to NGN3 expression at different differentiation stages. E) Quantification of the FACS results in panel D. n = 6: results from 3 clonal lines each with two differentiation experiments were combined. Statistic analysis was indicated for both monohormonal and total CPEP+ cells. (See also in Figure S7)