Fig 2: Dynamics of CD127- and CD127+ ELPs in the BM of HSC-engrafted mice(A) Experimental design: neonatal CD34hiCD45RA−CD38lo/−Lin- progenitors (Lin: CD3, CD4, CD10, CD19, CD24, CD56, CD115, CD116, CD123, CD127, ITGB7; thereafter referred to as HSCs) pooled from different donors were transduced with egfp-reporter lentiviruses (1.5 × 105 cells/mouse) before mixing with non-transduced bulk CD34+ HSPCs (1.5 × 105 cells/mouse) and intravenous injection to irradiated NSG mice. Note that non-transduced bulk CD34+ HSPCs were used as carriers to ensure full comparability with mice reconstituted with LMDPs (see also Figures S1E and S1F). To follow multi-lineage reconstitution, hu-CD45+egfp+ cells harvested from the BM of mice sacrificed at weekly endpoints were analyzed by multiparameter flow cytometry; percentages of corresponding cell subsets are indicated; results from individual mice are representative of 3 independent experiments.(B) Density plots showing the kinetics of monocytes (Mo; CD116+CD123-) (upper panels; blue rectangles), dendritic cells (DC; CD116+CD123+ (medium panels; blue rectangles) and B lymphocytes (BL CD19+) (lower panels; blue rectangles) in the BM of xenografted mice.(C) Asynchronous in vivo emergence of CD127- and CD127+ ELPs. Upper density plots show the kinetics of CD34hiCD45RAhiLin− HSPCs (red circles) in xenografted mice; gates are set on Lin− negative cells (Lin: CD115, CD116, CD123, CD10, CD19). Lower contour plots show the dynamics of CD127- (green) and CD127+ (red) ELPs; blue lines correspond to LMDPs; red arrows show the gating strategy.(D) Asynchronous in vitro emergence of CD127- and CD127+ ELPs. LMDPs cultured under standard scf condition were analyzed by flow cytometry at the indicated time points; gates are set on CD45RA+My−CD19− cells (My−: CD15−, CD115-, CD116-, CD123-; see also Figure S1C). Bidimensional density plots show the kinetics of differentiation of CD127- and CD127+ ELPs. Data are from ≥ 10 concatenated wells; percentages of the corresponding populations are indicated. Results are from 1 representative experiment.(E–G) Time-dependent changes in the lymphoid potential of cultured LMDPs. (E) Experimental design: LMDPs sorted from NSG mice at week-3 post-grafting were cultured by 100 cell-pools for 7 days onto OP9 stromal cells before analysis by flow cytometry (see also Figure S1C for the gating procedure). Condition-1 (d0-LMDPs; left panel): LMDPs are cultured for 7-day under the standard scf condition. Condition-2 (d2-LMDPs; medium panel): LMDPs sorted from cultures at day-2 are seeded for 5 more days in secondary cultures under the standard scf condition; Condition-3 (d5-LMDPs; right panel): LMDPs sorted from cultures at day-5 are seeded for 2 more days in secondary cultures under the standard scf condition. (F) Shown are absolute cell yields (upper panel) and median percentages of ELPs (lower panel); circles correspond to individual wells. (G) Stacked bar plot shows normalized ratios of CD127- (green) vs. CD127+ (red) ELPs; median numbers of ELPs per well are indicated (upper row); positivity threshold for ELP detection is set arbitrarily at ≥ 10 ELPs/well. Results are expressed as medians of 4–9 replicates from a representative experiment.(H and I) CD127- and CD127+ ELPs emerge independently from LMDPs. (H) Density plot shows the gating strategy used for fractionation of CD34hiCD45RAhiCD127−Lin- HPCs (Lin: CD10, CD19, CD115, CD116, CD123) from HSC-xenografted mice (week-3). (I) The indicated ITGB7neg; int; hi cellular fractions were sorted from the BM of mice at week-3 after grafting and then cultured for 3 or 7 days under the under standard scf condition before analysis by flow cytometry. Density plots show the percentages of in vitro-generated CD127+ ELPs (blue rectangles); gates are set as aforementioned; results from one representative experiment are pooled from 10 concatenated wells. ITGB7neg cells correspond to LMDPs.

Fig 3: Differentiation of erythroid-myeloid progenitors (EMPs) from human iPSCs(A) Schematic of EMP differentiation.(B) Representative cellular morphologies of EMP differentiation. On day 0, iPSC colonies formed with a sparse density. Arrowhead: cell clusters protruding with differentiating hematopoietic progenitors.(C) Representative flow cytometry plots of the generation of KDR+CD34+ mesodermal progenitors on differentiation days 3 and 5. Reprinted and adapted with permission from Li et al., 2024.(D) Representative flow cytometry plots of the expression of CD43, CD34, and CD115 in iPSC-derived EMPs harvested on days 8 to 10.