Figure 1.

Immature gametocytes develop properly within human primary erythroblasts. (A) Diagram illustrating the erythroblast infection protocol. Burst forming unit-erythroid cells (BFU-E) expressing CD34 were cultivated for 7 days to generate colony forming unit-erythroid cells (CFU-E) expressing CD36 and then CFU-E were allowed to differentiate into late erythroblasts for 5 to 7 days. Synchronized GFP-expressing schizonts at 40 hpi were added to erythroblasts at an MOI of 2. After several days of culture, infected erythroblasts were analyzed by fluorescent microscopy and flow cytometry. (B) Distribution of the different erythroblast stages at the time of infection. Morphological analysis of erythroblasts was performed by May-Grünwald Giemsa staining of cytospin from 3 independent experiments. (C) Erythroblast infection was observed by May-Grünwald Giemsa staining at 1 dpi (ring stage, upper left panel), 2 dpi (trophozoite stage, upper right panel and schizont stage, lower left panel), and 6 dpi (stage III gametocyte, lower right panel). Scale bars, 5 μm. (D) Gametocytes within erythroblasts at 6 dpi were observed by differential interference contrast microscopy: stage III gametocyte (left panel) and stage IV gametocyte (right panel). Scale bars, 5 μm. (E) Transmission electron microscopy shows that immature gametocytes within infected erythroblasts present typical sexual structures as the microtubular network (MT) and the inner membrane complex (IMC). Scale bars, 1 µm (left panels); 200 nm (right panels). (F) Immunofluorescence analysis of paraformaldehyde-fixed gametocyte-infected erythroblast at 6 dpi stained with anti–Pf11-1 antibodies (green) and with anti-GPA labeling the erythroblast membrane (red). DNA is stained with Hoechst 33342 (blue). Scale bars, 5 μm. (G) Infection with the Hsp70-GFP line (upper panels) and with the Pfs16-GFP line (lower panels) was observed by fluorescent microscopy at 2 dpi (upper panels and lower left panel) and at 6 dpi (lower right panel). Erythroblast membrane is stained with PKH-26 (red). DNA is stained with Hoechst 33342 (blue). Scale bars, 5 μm. (H) Infection of erythrocytes or erythroblasts with the Hsp70-GFP line was evaluated by flow cytometry at 2 dpi. (I) Infection of erythroblasts with the Hsp70-GFP and Pfs16-GFP lines was evaluated by flow cytometry at 2 dpi. (H-I) Circles indicate the number of independent experiments (n = 6) that were performed on erythroblasts derived from cytaphereses (n = 5) or bone marrow aspirates (n = 1) from 4 independent donors. Error bars show the standard error of the mean. **P < .01. BASO, basophilic stage; DIC, differential interference contrast; EC, erythroblast cytosol; EN, erythroblast nucleus; ES, exomembrane system; GC, gametocyte cytosol; GN, gametocyte nucleus; ORTHO, orthochromatic stage; POLY, polychromatic stage; PPM, parasite plasma membrane; PRO-E, proerythroblast stage; PVM: parasitophorous vacuole membrane; RETIC, reticulocyte stage.