Extended Data Fig. 3: CTCF induction during differentiation using doxycycline inducible CRISPRa stem cells. Control represents cells without doxycycline treatment.
a, qPCR, plotting mean ± s.e.m. (n = 4 biologically independent samples), showing upregulation of dCas9 (gRNA sequence 1, P = 4.2 × 10−8; gRNA sequence 2, P = 7.8 × 10−4), CTCF overexpression (gRNA sequence 1, P = 1.4 × 10−6; gRNA sequence 2, P = 0.014), and changes in expression (gRNA sequence 1, INS (P = 5.6 × 10−4) ISL1 (P = 1.1 × 10−6), SLC18A1 (P = 0.034), FEV (P = 0.012), CHGA (ns, P = 0.12); gRNA sequence 2, INS (P = 4.4 × 10−6), ISL1 (P = 7.8 × 10−8), SLC18A1 (P = 0.042), FEV (P = 0.0069), CHGA (P = 0.0050), upon doxycycline. Statistical significance assessed by unpaired two-sided t-test. b, qPCR analysis, plotting mean ± s.e.m. (n = 4 biologically independent samples), of CTCF overexpressed (INS, P = 0.0010; IAPP, P = 1.5 × 10−7; ISL1, P = 3.0 × 10−5; DLK1, P = 3.7 × 10−6), EC cells (SLC18A1, P = 1.6 × 10−4; FEV, P = 0.0019; DDC, P = 1.2 × 10−4; TPH1, P = 0.0047; LMX1A, P = 4.6 × 10−4; PDX1, P = 0.0031; NEUROD1, P = 6.2 × 10−5; NKX2-2, P = 0.0047; NKX6-1, P = 7.5 × 10−5; CHGA, P = 6.8 × 10−5; GCG, P = 5.9 × 10−6; SST, P = 0.0046). Statistical significance assessed by unpaired two-sided t-test. c, Immunocytochemistry of SC-islets with CTCF overexpression showing C-peptide (green), NKX6-1 (red), and SLC18A1 (red). Individual image representative of 6 biologically independent samples. d, Protein quantification plot showing mean ± s.e.m. (by ELISA, n = 4 biologically independent samples) after CTCF overexpression (P = 2.2 × 10−4). Statistical significances assessed by unpaired two-sided t-test. e, Insulin secretion, plotting mean ± s.e.m. (by ELISA, n = 3 biologically independent samples), of control (P = 0.0095) and CTCF overexpression (P = 0.0032). Statistical significance assessed using paired two-sided t-test and unpaired two-sided t-test respectively. f, qPCR analysis, plotting mean ± s.e.m. (n = 4 biologically independent samples), comparing CTCF overexpression during Stage 5 of protocol or at Stage 6 of protocol (dCAS9, P = 6.13 × 10−4; CTCF, P = 2.0 × 10−5; INS, P = 0.10; DLK1, P = 0.0023; ISL1, P = 0.18; IAPP, P = 3.3 × 10−4; LMX1A, P = 0.97; SLC18A1, P = 0.12; NEUROG3, P = 9.3 × 10−4; FEV, P = 0.30; DDC, P = 0.27; TPH1, P = 0.21) Statistical significance assessed by unpaired two-sided t-test. g, UMAPs (12467 cells from 2 independent biological samples; integration of all samples) showing cell types in SC-islets induced with CTCF overexpression. h, Heatmap showing normalized gene expression associated with CTCF experiment. i, Dot plots highlighting expression of CTCF and endocrine-associated genes. j, Violin plot of β-cell associated gene expressions of CTCF overexpression in the SC-β cell population. k, Chromatin accessibility around INS genomic region of SC-β cells. l, Feature plots showing decreased accessibility of β-cell motifs and increased accessibility to CTCF motifs in endocrine populations with CTCF overexpression. EC, enterochromaffin cells; ns, not significant.
