Primitive RBCs eject their nuclei in the placental villous stroma. (A) H&E staining of 7-week placenta (i) suggested the presence of free RBC nuclei (pyrenocytes) in the extravascular stroma of placental villi (top inset arrow) as well as less frequent pyrenocytes and primitive RBCs with pyknotic nuclei in circulation (bottom inset, arrow and arrowhead, respectively). Staining for ζ-globin and the nuclear counterstain Fast red (ii) demonstrated that pyrenocytes in the villous stroma were associated with ζ-globin⁺ primitive RBCs (solid boxed inset arrowhead) or contained a small amount of ζ-globin (dotted boxed inset). Images were acquired at 400× original magnification. (B) The presence of primitive pyrenocytes in the placenta was further verified by cell sorting using the nucleic acid dye DRAQ5 in conjunction with CD235. Subgating for small cells in the CD235^medDRAQ5⁺ (dotted gate 4) fraction permitted purification of primitive pyrenocytes from the placenta. Arrow marks pyrenocytes; arrowhead points to an erythroblast that cosorted with pyrenocytes. Images were acquired at 400× original magnification. (C) The same sorting strategy allowed the isolation of definitive pyrenocytes from the fetal liver. Solid arrow marks pyrenocytes, and dashed arrow indicates an enucleated erythrocyte that cosorted with the pyrenocytes. Images were acquired at 400× original magnification.