Two waves of CD45⁺ HSPCs in IAC cells. (A) PCA plot of a subset of data containing IACs, illustrating the trajectory of IAC differentiation from HE along the PC1 axis. (B) Expression of Gja5, Hey1, and Rac2 illustrating the maturation of IAC cells along the trajectory. (C) PCA plot showing the separation of 10.5 and 11.5 dpc IAC cells along the PC3 axis. (D) 10.5 and 11.5 dpc IAC cells, 10.5 dpc CD45⁺ IAC cells, and 11.5 dpc pre-HSCs plotted separately to visualize their relative distribution along the PC3 axis. A k-nearest-neighbor classifier (k = 3 with PC1-10 as feature input) was trained using 10.5 dpc CD45⁺ IAC cells and 11.5 dpc pre-HSCs to determine the fraction of pre-HSCs (red) in 10.5 and 11.5 dpc IAC cells. (E) Heatmap showing top differentially expressed genes in 10.5 dpc CD45⁺ IAC cells vs 11.5 dpc pre-HSCs. (F) Preferential expression of Mecom in 11.5 dpc pre-HSCs and IAC cells vs Myc, Il7r, and Gata1 in 10.5 dpc CD45⁺ IAC enriched for lymphomyeloid-biased progenitors and in 10.5 dpc IAC cells. (G) Reactome pathway analysis comparing 11.5 dpc pre-HSC and 10.5 dpc CD45⁺ IAC cells. Color indicates pathway activity score computed using the AUCell package.⁶³  (H) Methylcellulose (colony forming unit-culture, CFU-C) assay performed in the presence of stem cell factor (SCF), interleukin 3 (IL-3), IL-6, and erythropoietin (EPO) to measure the frequency of committed erythroid and myeloid progenitors in 10.5 dpc CD45⁺ IAC cells, CD45⁻ IAC cells, and 9.5 dpc yolk sac EMPs. BFU-E, burst forming unit-erythroid; GEMM, granulocyte/erythroid/monocyte/megakaryocyte; GM, granulocyte/macrophage; Mac, macrophage; MK, megakaryocyte. Values and error bars are mean ± SD; n= 3 experiments. Frequencies of total progenitors are indicated above the bars. (I) Limiting dilution assays on OP9 stromal cells to determine the frequencies of progenitors in purified 10.5 dpc CD45⁺ IAC and 10.5 dpc CD45⁻ IAC cells yielding B (CD45⁺CD19⁺B220^mid/lo), myeloid (M) (Gr1⁺Mac1⁺ or Gr1⁺Mac1⁻), and B+myeloid (B/M) cells in culture. Also shown are frequencies of progenitors in purified 10.5 dpc CD45⁺ IAC and 10.5 dpc CD45⁻ IAC cells that produced T cells (CD90⁺ CD25⁺) when cultured on OP9 cells expressing the Notch ligand delta-like 1. Error bars; mean ± SD. Values and error bars are mean ± SD; n = 7. Frequencies of all progenitors are indicated above the bars. (J) Percentage of wells at the limiting cell dose containing B, M, or B/M cells from experiments in panel I. Values and error bars are mean ± SD; n = 8 experiments.