Extended Data Fig. 11: HARE5 knock-in hiPSC/hESC lines and cortical NPCs/organoid differentiation.
From: A human-specific enhancer fine-tunes radial glia potency and corticogenesis
a, Experimental paradigm for genotyping after CRISPR editing. b, EcoRI enzyme digestion shows the successful mutation between Hs-HARE5 and Pt-HARE5 (gained EcoRI site within the 3rd substitution) in the control and mutant cell lines. Blue arrow, Pt, Red arrow, Hs. c, Sanger sequencing showing the successful knock-in with all 4 mutations in Hs-HARE5Pt/Pt lines and Hs-HARE5Hs/Pt lines. This is representative of genotyping of 200 colonies. d,e, Colonies stained with OCT4 (red) and DAPI (blue). This is representative of multiple stainings. f, Experimental paradigm for human NPC differentiation. g-i, Human NPC identity characterization by RT-qPCR on D12-14 of control (n = 4), HARE5Pt/Pt (n = 7) NPCs from 2 and 3 cell lines, respectively, across 3 differentiations, showing FOXG1 (g) PAX6 (h) and OCT4 (i) *P = 0.0239 (i). RNA levels normalized with D0 controls. j-k, RT-qPCR of FZD8 (j) and CCNY (k) of D12-14 human NPC of control (n = 4), HARE5Pt/Pt (n = 7) NPCs from 2 and 3 cell lines, respectively, across 3 differentiations. Each mutant line normalized with controls. **P = 0.0098. l, Experimental paradigm for chimpanzee NPC differentiation. m-o, Chimpanzee NPC identity characterization by RT-qPCR on D15 of controls and Pt-HARE5Hs/Hs lines of control (n = 9) and Pt-HARE5Hs/Hs (n = 5) NPCs from 2 and 1 cell lines, respectively, across 5 differentiations, showing FOXG1 (m) PAX6 (n) and OCT4 (o). RNA levels normalized with D0 controls. p-q, RT-qPCR of FZD8 (p) and CCNY (q) mRNA of D15 NPCs of control (n = 9) and Pt-HARE5Hs/Hs (n = 5) chimpanzee NPCs from 2 and 1 cell lines, respectively, across 5 individual differentiations. Each mutant line normalized with controls. *P = 0.0491 (p); *P = 0.0306 (q). r, Experimental paradigm for human 3D cortical organoids differentiation. s, D30 cortical organoids in bright field. t, RT-qPCR of FOXG1 mRNA on D30 human cortical organoids of controls (n = 14) and Hs-HARE5Pt/Pt (n = 12) from 2 and 2 cell lines, respectively, across 2 differentiations. Normalized with D0 H9. u, Images showing different neural rosette fields for control and Hs-HARE5Pt/Pt cortical organoids (n = 2 lines each shown), stained for SOX2 (green), PH3 (magenta) and DAPI (blue). This is representative of 16 independent stainings. Scale bars: 100 µm (d,e,s), 50 µm (u). All values represent the mean ± s.d. Statistics on independent NPC experiments (g-q), organoids (t); colors, cell lines; shapes, differentiations. P values were calculated based on the student’s unpaired, two-tailed t-test (g-k, m-q, t).
