Fig. 3: Validation and lineage potential assessment of human CD34⁺CD127⁺IL-3RA⁺ and CD34⁺CD127⁺IL-3RA^– lymphoid progenitors in fetal liver.

a Visualization of HSPC, LP1, LP2, ILCP, ETP and pre pro-B cell clusters by trimap assay. b Partition-based graph abstraction (PAGA) topology tree of cell clusters. The width of line in PAGA indicates the strength of connectivity between cell clusters. The colors represent cell identities in a and b. c Heatmap shows the highly expressed TF-encoding genes in progenitor clusters. d Dot plots show the expression level of top 10 DEGs in HSPC, LP1, LP2, ILCP, ETP and pre pro-B cell cluster in liver. Colors represent the gene expression level and size encodes the proportion of gene-expressing cells. IL3RA highlighted in red is highly expressed in LP1 and LP2 cells. e Flow cytometry plots show stronger CD34 expression in IL-3RA⁺ than that in IL-3RA^–Lin^–CD45⁺CD34⁺CD127⁺ lymphoid progenitors of human fetal liver. Gating strategy for IL-3RA was based on Fluorescence Minus One (FMO) control (right panel). f, h IL-3RA⁺ and IL-3RA^–Lin^–CD45⁺CD34⁺CD127⁺ lymphoid progenitors (150 cells for each subset) were sorted and cocultured with SCF, FL, IL-7 and IL-15 on OP9 stromal cells (f) and OP9-DL4 stromal cells (h). The IL-3RA⁺ lymphoid progenitors generate more CD45⁺ hematopoietic cells and have more NK, myeloid (upper panel in f) and T lineage (upper panel in h) potential while IL-3RA^– lymphoid progenitors show B lineage commitment (below panel in f) suggested by coculture experiments. B, NK, myeloid and T cells were identified as positive for its respective lineage markers and negative for other lineage markers. g, i Box plots show the numbers of indicated lineage cells, and the percentage of B cells in total CD45⁺ hematopoietic cells after cocultured for 8 days on OP9 stromal cells (g) or 12 days on OP9-DL4 stromal cells (i). j Fetal liver ILCP (Lin^–CD45⁺CD7⁺CD127⁺CD117⁺, upper panel), IL-3RA⁺ (middle panel) and IL-3RA^–Lin^–CD45⁺CD34⁺CD127⁺ lymphoid progenitors (below panel) were cultured on OP9-DL4 stromal cells for 10–14 days under ILC-induction condition (IL-2, IL-7, IL-1β, IL-23, IL-25 and IL-33). After stimulation with PMA/ionomycin 4 h, cytokine production was analyzed by flow cytometry analysis. IL-17α and IL-22 producing cells were analyzed after gating on cells negative for IFN-γ or IL-13. k Box plots show numbers of ILC1, ILC2 and ILC3 production of the indicated populations. ILC1, ILC2 and ILC3 were identified as shown in j.