Fig 2: Identification of iPSC-derived HSPCs and erythroid cellsA. Schematic diagram of the protocol used to generate HSPCs and erythroid cells from iPSCs in vitro. B. Histogram showing the counts of iPSCs on day 0 and SCs collected on day 14 per round-bottom 96-well plate. Error bar represents SD. C. Percentage of CD34+ and/or CD45+ cells in the total SCs (mean ± SD). D. Count of colonies growing from SCs collected on day 14. E. The morphology of hematopoietic CFUs growing from SCs. Scale bar = 1000 μm. F. Flow cytometry results showing cells expressing CD71 and CD235a on day 14, 17, 23, and 28 during the erythroid differentiation of SCs. G. qPCR of β-chain and γ-chain globin expression on day 4, day 14 + 3, and day 14 + 9 during the entire system differentiation. H. The morphology of erythroid cells on day 28. Scale bar = 20 μm. iPSC, induced pluripotent stem cell; HSPC, hematopoietic stem progenitor cell; EB, embryoid body; SC, suspension cell; SFM, serum-free medium; BMP4, bone morphogenetic protein 4; FGF-2, fibroblast growth factor-2; SCF, stem cell factor; VEGF, vascular endothelial growth factor; EPO, erythropoietin; CFU, colony-forming unit; IL3, interleukin 3; CFU-E, colony-forming unit erythroid; CFU-G, colony-forming unit granulocyte; CFU-GM, colony-forming unit granulocyte macrophage; CFU-M, colony-forming unit macrophage.

Fig 3: CD55 is required for P. falciparum invasion.(A) Schematic of CD55 gene structure showing targeting sites of two single guide RNAs (sgRNAs). Vertical red lines indicate the positions of CD55 exons. (B) Expression of CD55 on mutant (CD55-CRISPR) cRBCs generated with one sgRNA (left) or two sgRNAs (right), as compared to control (Cas9) cRBCs. (C) Invasion efficiency of P. falciparum 3D7 in Day 18 CD55-CRISPR cRBCs compared to isogenic controls (Cas9), relative to the invasion efficiency in peripheral blood erythrocytes (N = 4 biological replicates; n = 3 technical replicates; error bars indicate SEM; **p<0.01). CD55-null cRBCs were generated using dual sgRNAs: sgRNA1, CD55-Cr1 and sgRNA2, CD55-Cr8. (D) Time course of expression of CD71 on cRBCs harvested on different days of differentiation. Enucleated versus nucleated cells were gated using a nuclear dye. (E) Invasion efficiency of P. falciparum strain 3D7 in CD71-negative, CD55-CRISPR cRBCs compared to CD71-negative isogenic controls (Cas9). Invasion efficiency is presented relative to the mean of Cas9 control. Three independent biological replicates are shown; error bars represent SEM (n = 2 or three technical replicates; **p<0.01, ***p<0.005). CD55-null cRBCs were generated using dual sgRNAs. (F) Parasitemia in population of CD55-CRISPR cRBCs, in which ~ 90% of the cells lack CD55 (CD55-negative), and the remaining are CD55-positive, as quantified by immunofluorescence assays. (G) Representative images of CD55-CRISPR cRBCs, including one P. falciparum-infected cell (magnified). Images are brightfield with fluorescence overlaid. Blue, dapi. Red, anti-CD55-PE. Scale bars indicate 10 µm.Figure 1—source data 1.Source data for invasion assays.

Fig 4: Functional characterization of sexually committed schizonts and early sexual stages.(A) Expression of RhopH complex genes and rap1 (control) in committed schizonts of the NF54 line. Values are the FC between inducing (−choline) and noninducing (+choline) conditions. Transcript levels were normalized against rama. In all panels, data are presented as the average and SEM of independent biological replicates. N = 4. (B) Western blot analysis of E5ind culture supernatant or mature schizont pellets with antibodies against CLAG3 and the loading controls AMA1 or HSP70. The bar chart shows the fold decrease in normalized band intensity between induced and noninduced cultures. N = 3 (supernatant) or 4 (pellet). (C) Sorbitol resistance assay performed ~30 to 40 hpi at the cycle after induction. Values are the percentage of pigmented parasites surviving sorbitol treatment. Shield 1 (Shld) stabilizes PfAP2-G in E5-PfAP2-G-DD cultures. N = 3. (D) Uptake of 5-ALA in induced and noninduced E5ind cultures. Representative fluorescence microscopy images and quantification of the proportion of positive parasites are shown. N = 3. Scale bars, 10 μm. (E) Flow cytometry analysis of invasion preference by committed and noncommitted merozoites. Reticulocytes were identified by CD71 signal and parasites by Hoechst signal. Representative dot plots are shown. PE, phycoerythrin. (F) Distribution of parasites between erythrocytes and reticulocytes after invasion by committed and noncommitted merozoites. N = 2.

Fig 5: Characterization of plasma-derived extracellular vesicles from P. vivax patients.a BCA (protein concentration) of circulating EVs from healthy donors (hEVs) and P. vivax patients (PvEVs) (n = 10, individual samples). Data show mean ± SD. Two-sided, Mann–Whitney test, *p = 0.0232 (GraphPad). b P. vivax proteins identified by different unique peptides (sequences below the description of corresponding proteins). UniProtKB accession numbers and gene name corresponding to the P. vivax PvP01 strain are shown. c Western blot analysis of PvEVs obtained from different individual patients (Pv1-7 and Pv10) and human donors (H1 and H3) using anti P. vivax MSP3.1 (upper membrane) and PHISTc (bottom membrane) antibodies. MSP3.1 and PHISTc recombinant truncated-proteins fused to GST, were used as positive controls. Molecular weight in kDaltons (kDa) is shown to the left. M: molecular size marker. Image representative of three independent experiments. d Nanoparticle tracking analysis (NTA) profile (size [nm] versus concentration [particles/mL]) of pooled hEVs and PvEVs was analysed using NanoSight LM10-12. Table shows mean of three measurement of pooled hEVs and PvEVs quantified by NTA (particles concentration) and BCA (protein concentration). e Beads-based flow cytometry analysis of pooled hEVs and PvEVs using six EV markers (CD9, CD63, CD81, GAL3, CD5L and CD71). Data show median fluorescence intensity (MFI) of each antibody and control antibodies (rabbit and mouse-isotype) ± SD (technical replicates, n = 3). Unpaired and two-sided, t-test **p = 0.0032 (CD63), ***p = 0.0006 (CD81, CD71), ****p < 0.0001 (CD9) (GraphPad). Source data are provided as a Source data file and Supplementary Data 2, 3 and 4.