Supplementary Figure 4: In vitro differentiation of hematopoietic progenitors.
From: Distinct myeloid progenitor–differentiation pathways identified through single-cell RNA sequencing
(a) Schematic overview of culture conditions used. Indicated progenitor populations were cultured for 3 days with mSCF and mIL-3, allowing the cells to reach a GMP stage, as defined by FcγRII/III expression. (b) Morphological analysis of EGFP-immunostained cytospins from cultures of the indicated in vitro generated EGFP− and EGFP+ FcγRII/III+ cells after manually plating at a density of 5 cells/well and cultured for 8 days. Cell types are shown as a percentage of total number of cultures analyzed (numbers above bars). (c) Flow cytometry analysis demonstrate expression of CD55, Ly6C, integrin beta 7 (Itgb7) and interleukin 1 receptor-like 1 (Il1rl1) with Gata1-EGFP in pre-GMs and GMPs. Numbers indicate the percentage of cells within each quadrant. Representative plots of 2 (CD55, Ly6C and Il1rl1) or 1 experiment (Itgb7) are shown. (d) Quantification of identified cell population after 8-day cultures of CD55+ or Ly6C+ pre-GM and GMP by FACS as in Figure 4a. n=6-8 biological replicates. Error bars show SD. Mo: monocyte, Ne: neutrophil, Eo: eosinophil, Ma: mast cell. (e) Flow cytometry analysis of T-cell cultures (OP9-DL1 stroma) of LMPP and pre-GM GE− cells cultured for 21 days (10 cells/culture). Plots show live cells. Stromal cells were gated out on FSC-SSC plot. Percentages are averages of cells in the gates or quadrants (+/− SD). (n=5 for LMPP, n=19 for pre-GM GE−). Panels on the right show Thy1.2 vs CD25 expression profile on cells first gated as CD4−CD8−.
