Extended Data Fig. 6: Downregulation of CD32 signalling impairs hematopoietic development.

a) Quiver plot depicting summarized cell-state transition vectors, as simulated by CellOracle from clusters 0, 1, 2, 11, 16, and 17, with arrows indicating the predicted direction of developmental flow; b) Quiver plot illustrating the simulated cell-state shift vectors resulting from an in-silico CD32 KO, as modeled by CellOracle, with arrows highlighting the predicted alterations in developmental flow due to the FCGR2B knockout; c) Digitized grid representation of the CD32 KO simulation by CellOracle, with each section colored relying on Perturbation Scores (PS). Green indicates a negative PS, suggesting that the transcription factor perturbation inhibits differentiation, while purple signifies a positive PS, indicating promotion of differentiation. Single-cell transition vectors are aggregated at specific grid points; d) Representative map of the donor plasmid used to engineer H1 hPSCs to express 3x shRNAs to silence FCGR2B expression. Donor plasmid was inserted in AAVS1 locus by nucleofection with eCas9, the T2 gRNA plasmid, and p53 dominant negative (DD); e) Representative flow cytometry analysis showing CD32 and CD34 expression in day 8 wild-type (left panel) or CD32 KD (right panel) WNTd hPSC-derived CD34⁺CD43^neg cells. Gated on SSC/FSC/Live/CD34⁺CD43^neg. n = 4, independent; f) Bar plot showing the frequency of CD32⁺ within CD34⁺CD43^neg cells at day 8 of WNTd hPSC-derived hematopoietic culture as in e). Mean ± SEM. One-tail paired Student’s t-test, nonparametric, for all biological replicates (n = 4,), *p = 0.02203. g) Bar plot showing the frequency of CD45⁺ derived from CD34⁺ cells isolated at day 8 of WNTd hPSC-derived hematopoietic culture as in Fig. 4g. Mean ± SEM. One-tail paired Student’s t-test, nonparametric, for all biological replicates (n = 5), *p = 0.0312.

Source data