Extended Data Fig. 1. Characterization of primitive hematopoiesis up to Day 10 of differentiation.

A) FACS analysis shows that addition of erythropoietin (EPO) from Day 6 to Day 10 of differentiation causes the emergence of CD235A+ erythrocytes at Day 10 of differentiation, as well as a reduction in the percentage of Macro (macrophage precursor) fated cells. B) Brightfield images over days 4–10 of the differentiation show that round hematopoietic cells progressively proliferate in semi-suspension through day 10, black arrows point to hematopoietic cells. Scale bar = 100 μM (Day 4, 10) and 50uM (Day 7). C) IF shows VE-cadherin+ hemogenic endothelium at Day 10 of differentiation. Scale bar = 100 μM. D) Imputed gene expression trends over pseudotime as calculated by Palantir of HOXA genes in the 3 differentiation trajectory arms show nearly absent HOXA1–7 expression, and minimal HOXA9 and 10 expression at low pseudotimes (õne log fold lower than genes in Fig. 2C). PMAC (PMAC arm, green), MK (megakaryocytic arm, pink), and ERY (erythrocyte arm, orange). E) Unimputed gene expression trends of HOXA1–10 genes show similar trends to imputed trends shown in A, nearly absent HOXA1–7 expression and minimal HOXA9–10 expression only at low pseudotimes in all differentiation trajectory arms. PMAC (PMAC arm, green), MK (megakaryocytic arm, pink), and ERY (erythrocyte arm, orange). F) Unimputed gene expression trends of key signature genes over pseudotime as calculated by Palantir show similar trends to imputed gene trends shown in Fig. 2C. Separate differentiation arms increasingly express their corresponding identity markers over pseudotime: ERY (GYPA, HBE1), MK (ITGA2B, ITGB3, GP1BA), and PMAC (CX3CR1, CSF1R, SPI1, PTPRC). MY (PMAC arm, green), MK (megakaryocytic arm, pink), and ERY (erythrocyte arm, orange). n = 6743 and error bars = SD for (A)-(C).