RBCs in the placental villous stroma are of the primitive erythroid lineage. (A) Intracellular flow cytometry confirmed that the CD235⁺CD71⁺ erythroid cells in the early first trimester placenta were ζ-globin expressing primitive RBCs (colored cells are the same in both FACS plots). IHC of adjacent tissue sections demonstrated that the RBCs observed in the placental villous stroma were of the primitive lineage. In precirculation placentas (i-i′), no ζ-globin⁺CD235⁺ cells were found in placental blood vessels (arrowheads). The first cells in the vasculature (arrowheads) were CD235⁺ (II) and ζ-globin⁺ (ii′). At 5 weeks of age, all RBCs in the placenta expressed ζ-globin (iii vs iii′) and some were localized to the placental stroma (arrows). By 6 weeks many more primitive RBCs were observed in the villous stroma (arrows in iv-iv′). At 11 weeks, the placental RBC pool comprised mostly ζ-globin⁻ RBCs that were contained in blood vessels (arrowheads in v-v′). Images were acquired at 400× original magnification. (B) Immunofluorescence using 3 different endothelial markers, CD31, Flk1, and CD34, confirmed the extravascular localization of placental RBCs (white boxes). V indicates blood vessels; scale bars, 20 μm. (C) Staining of yolk sac and fetal liver at the developmental stages when RBC extravasation was prominent in the placenta did not reveal major populations of ζ-globin⁺ extravascular primitive RBCs (i-iii). By 11 weeks, the liver had generated ζ-globin⁻ definitive RBCs (iv-iv′). Arrowheads mark circulating RBCs; arrows mark RBCs in the fetal liver parenchyma. Images were acquired at 200× original magnification.