Fig. 4. APs in human organoids ancestralized for KIF18a and KNL1 have a shorter metaphase than modern human controls.

(A) Human H9 ESCs were gene edited using CRISPR technology to convert the indicated three amino acids in KIF18a and KNL1 from the modern human (h) to the ancestral (a) Neanderthal-like states, and used to grow cerebral organoids (see Materials and Methods). (B and C) Live-tissue imaging of the indicated mitotic phases of APs in organotypic slice cultures of days 27 to 30 cerebral organoids grown from the indicated control (B) and ancestralized (C) line. Zero (0) minute is metaphase plate onset. Time-lapse intervals are 2.08 min. Red lines indicate the duration of metaphase, white dashed lines the ventricular surface. Scale bar, 5 μm. (D) Times between the start of chromosome congression and metaphase plate onset (referred to as Congression), between metaphase plate onset and chromatid segregation onset (referred to as “Metaphase plate”), and between chromatid segregation onset and general chromosome decondensation onset (referred to as “Anaphase”), for APs in the two types of organoids as described for line 1 in (B) and (C). Data are the means ± SEM for organoids grown from the two control and the two ancestralized lines (L1, L2) and comprise ≥119 APs from four independent experiments, with ≥7 organoids, for each of the lines. Bracket with ****P < 0.0001 (Mann-Whitney U test).