CDX4 is expressed at the onset of definitive hematopoietic progenitor specification within mesoderm. (A) Differentiation schematic and hematopoietic progenitor identification. hPSCs are differentiated using a serum-free, stroma-free approach, with stage-specific application of WNT signal manipulation. Inhibition of WNT signaling within mesendoderm with 3 μM IWP2 leads to the generation of KDR⁺CD235a⁺ mesodermal population, which gives rise to CD43⁺ primitive hematopoietic progenitors, whereas WNT activation with 3 μM CHIR99021 generates a KDR⁺CD235a⁻ mesodermal population that gives rise to CD34⁺CD43⁻CD73⁻CD184⁻ HE. No manipulation of WNT signaling leads to a heterogeneous population of primitive and definitive hematopoietic progenitors. (B) Representative cell-sorting strategy employed for RNA-seq analyses. Mesoderm harboring definitive (blue) or primitive (red) progenitors were isolated by FACS. (C) Heatmap of CDX gene expression within different mesodermal populations, as determined by RNA-seq. n = 4. (D) qRT-PCR analyses of CDX1 (top), CDX2 (middle), and CDX4 (bottom) expression during the first 6 days of differentiation as in panel A. Period of WNT manipulation is shaded in light blue. n ≥ 3 mean ± standard error of the mean (SEM). Student t test compared with DMSO control: *P < .05. (E) Representative flow cytometric analysis of CD73 and CD184 expression, gated on CD34⁺CD43⁻ cells following either CHIR99021 (CHIR) treatment or CHIR + 1 μM PD173074 (FGFRi) treatment as in panel A. (F) qRT-PCR analyses of CDX1 (left), CDX2 (middle), and CDX4 (right) expression on day 3 of differentiation, following treatment with either vehicle (DMSO), CHIR99021 (CHIR), IWP2, or PD173074 (FGFRi) as in panel A. Normalized to CHIR treatment. n = 3 mean ± SEM. Student t test compared with CHIR treatment: *P < .05; **P < .01. BMP4, bone morphogenetic protein 4; DMSO, dimethylsulfoxide; EPO, erythropoietin; IGF-1, insulin-like growth factor 1; IL-6, interleukin-6; qRT-PCR, quantitative reverse transcription polymerase chain reaction; RNA-seq, RNA sequencing; SCF, stem cell factor; TPM, transcripts per million; VEGF, vascular endothelial growth factor.