Characterization of human erythroblast maturation by susceptibility to Plasmodium falciparum malaria infection and by transcriptional response. (Ai) Percentage of day 5, 10, and 16 erythroblasts infected by Plasmodium falciparum. No infection is detected in pro-/basophilic erythroblasts (day 5). At day 10, when the population is largely at the polychromatic stage a few early orthochromatic cells are detected that can also be infected. (ii) Infection is seen in early/mid (day 14, ■) and late (day 16, ■) orthochromatic erythroblasts (N indicates nucleated; and E, enucleated), whereas only minor infection can be detected in polychromatic erythroblasts (day 10). represent the percentage of nucleated/enucleated cells in the population, and ■ represent the proportion infected (marked with numbers) in each subset. Under these conditions, in cultures containing only mature erythrocytes we detect 60% infection. Error bars indicate SEM; n = 3 for day 10; n = 3 for day 14; n = 2 for day 16. (B) Brightfield images of Giemsa-stained infected erythroblast cells at 22 hours after invasion (hpi indicates hours of intracellular parasite growth; day 10, i; day 14, iii; day 16, v) and at 40 hpi (day 10, ii; day 14, iv; day 16, vi). On day 10 at 22 hpi, a few cells contain ring-stage parasites (i, ). Judging from staining of condensed nuclei, these cells are at the early orthochromatic stage. At 40 hpi, no mature schizonts are seen, but ring vacuoles (ii, ) may remain. On day 14, at 22 hpi, more than half the cells show rings (iii, ), which fail to mature to schizonts, but ring vacuoles (iv, ) are prominently detected. Half of these cells become vacuolated by 40 hpi (iv, []). On day 16, at 22 hpi and 40 hpi, respectively, ring-stage parasites (v, ) and schizont-stage parasites with hemozoin crystals (vi, ) are evident in nucleated, late orthochromatic erythroblasts. Photomicrographs were taken by light microscopy with a Zeiss Axioskop upright microscope and Nuance spectral camera/unmixing system (Cambridge Research and Instrumentation) using a 100× objective. Scale represents 5 μm. (C) Transcriptional profile of day 14 versus day 10 erythroblasts. Up-regulated transcripts are denoted with red squares; down-regulated, with blue diamonds; unchanged, with gray crosses. As expected, hemoglobins a and b were highly transcribed (spot 1, purple asterisks), erythrocyte protein band 4.1 (epb41) was up-regulated, and adducin 2 (add2) was down-regulated (inset, black squares, spots 12 and 13, respectively), suggesting assembly of the cytoskeleton was still in progress. Beta-actin (actb, spot 2), glycophorins (gypc spot 3, gypa spot 4, gypb spot 6), spectrin (spta1 spot 5, sptb spot 9), ankyrin (ank1 spot 7), band 3 (slc4a1 spot 8), Duffy (darc spot 10), and adducin 3 (add3 spot 11) are all expressed but show no significant change (fold change < 2, black squares). In addition erythropoietin receptor (epor spot 14, inset) transcript is reduced relative to transferrin receptor (cd71 spot 15, green circles) but nonetheless detectable. Bcl2-like 1 (bcl2l1 or bcl-x_L; spot 18), a marker for terminal stages of erythropoiesis,¹⁰  is up-regulated 2.6-fold (P = .006, q = 0.005). In addition, it appears early orthochromatic cells are exiting the cell cycle because both cyclins D3 and E2 (ccnd3, ccne2; spots 16 and 17, respectively) are down-regulated ∼ 3-fold compared with polychromatic cells (P = .002, q = 0.003 and P = .02, q > 0.007, respectively). (D) Diagram displaying differential susceptibility of erythroblasts to P falciparum infection. Whereas orthochromatic cells can support parasite entry, only enucleating erythroblasts/nascent reticulocytes support intracellular parasite growth.