Supplementary Figure 2: Defining HSCs and LMPPs based on Gata1.
From: Distinct myeloid progenitor–differentiation pathways identified through single-cell RNA sequencing
(a-d) 22 weeks in vivo peripheral blood reconstitution after transplantation of 100 FACS purified GE− or GE+ LSKCD150+CD48− cells together with 250,000 congenic whole BM cells. Values show reconstitution of the compartment analyzed (mean ±SD); n=3 recipients/population in one experiment. WBC; white blood cells. (e) For evaluation of megakaryocyte (Mk) potential, LSKFlt3hi GE− and GE+ cells were manually plated at 1 cell per well into 60 well Terasaki plates and cultured for 8 or 12 days. Megakaryocytes were evaluated using an inverted microscope. Results are from 2 experiments, shown as percentage of colonies containing Mk cells of the total number of colonies formed. n=240 cells/population in 2 experiments. (f) Histogram plot showing expression of Flt3 and Gata1 in single sorted GE− (left) and GE+ (right) pre-GMs. Expression is shown as -(Ct(gene)-Ct(Kit)). (g) Combinations of one GE− pre-GM and three GE+ pre-GMs were cultured for 8 days. Single CD48−HSCs were cultured for 25 days. Myeloid lineage potential readout of the cultures was based on morphology analysis after cytospins and MGG stain of the cultures. Number of cultures analyzed in indicated. Mo: monocytes, PMN: polymorphonuclear cells, Ma: mast cell. (h) Representative morphology of cells from cultures described in (g). Monocytes (Mo), polymorphonuclear granulocytes (PMN), and mast cells (Ma) are indicated. Scale bars: 25μm.
