1. Ethical approval/declaration
To generate liver organoids in this study, we used human iPSC lines established in previous studies (26, 27). Human erythrocytes were obtained from the peripheral venous blood of the subjects after obtaining informed consent. Protocols related to iPSCs, blood collection and cell preparation were approved by the Human Research Protection Unit, Faculty of Medicine Siriraj Hospital, Mahidol University (COA no. Si 953/2023). P. vivax sporozoites were used in accordance with biosafety guidelines, and the corresponding protocols were approved by the Faculty of Medicine Siriraj Hospital, Mahidol University (approval no. SI 2024-003).
2. Culture of human iPSCs
Two human iPSC lines were used: MUi019 (26) and MUSIi001-A (27). Human iPSCs were cultured in a cell culture plate coated with 2 µg/mL Matrigel (Growth Factor Reduced; Corning, BD Bioscience). The culture medium used was Essential E8 medium (Gibco, Waltham, Massachusetts) supplemented with 10,000 units/mL penicillin and 10,000 µg/mL streptomycin (Invitrogen, Waltham, Massachusetts). The cells were passaged every 5–6 days using 0.5 mM EDTA (Invitrogen, Waltham, Massachusetts) to detach the cells from the plate and dissociate the cells into clumps. The culture medium was supplemented with 10 μM Y-27632 (StemCell Technologies, Vancouver, Canada) to prevent cell death after cell passage or thawing of liquid nitrogen-frozen cells.
3. Generation of hepatic endoderm cells
To obtain hepatic endoderm cells, two sequential steps were performed. For endoderm specification, human iPSCs were cultured in RPMI medium supplemented with 100 ng/mL activin A (Peprotech, Cranbury, New Jersey) and 50 ng/mL Wnt family member 3a (Peprotech, Cranbury, New Jersey) and 1x B27 (Gibco, Waltham, Massachusetts), hereafter called RPMI/B27 medium. The cells were incubated under ambient oxygen and 5% CO2 for 6 days. The culture medium was replaced every other day. In the second phase, the culture medium was changed to RPMI/B27 medium containing 20 ng/mL bone morphogenetic protein 4 (Peprotech, Cranbury, New Jersey) and 10 ng/mL basic fibroblast growth factor (Gibco, Waltham, Massachusetts). The cells were cultured under ambient O2 and 5% CO2 for another 2 days.
4. Generation of endothelial progenitors
To generate endothelial progenitor cells, human iPSCs were first differentiated into mesoderm cells via cultivation in DMEM/F12 supplemented with 1X B27, 1X GlutaMAX (Gibco, Waltham, Massachusetts), 8 μM CHIR99021 (Sigma‒Aldrich, St. Louis, Missouri), and 25 ng/mL BMP4 (PeproTech, Cranbury, New Jersey) for 4 days under ambient O2 and 5% CO2. Then, the culture medium was changed to endothelial induction medium, which consists of StemPro34-SFM (Gibco, Waltham, Massachusetts) supplemented with 200 ng/mL vascular endothelial growth factor (PeproTech, Cranbury, New Jersey) and 2 μM forskolin (StemCell Technologies, Vancouver, Canada), for another 4 days under the same conditions.
5. Generation of septum transversum mesenchyme cells
Regarding mesenchymal cells, septum transversum mesenchyme (28) cells are derived from the mesoderm and were shown to be responsible for liver budding in a mouse model (23). STM generation was divided into three sequential steps. In the first step, human iPSCs were cultured with DMEM/F12 culture medium containing 1× B27 (Gibco, Waltham, Massachusetts), 1× GlutaMAX, 8 μM CHIR99021 (Sigma‒Aldrich, St. Louis, Missouri), and 25 ng/mL bone morphogenetic protein 4 for 4 days. Next, the culture medium was changed to DMEM/F12 supplemented with 1× B27, 1× GlutaMAX, 2 ng/mL activin A, and 10 ng/mL platelet-derived growth factor-BB (Miltenyi Biotec, Bergisch Gladbach, Germany) at a ratio of 1:1, and the cells were cultured for an additional 2 days. In the last step, the cells were cultured in STM induction medium consisting of StemPro34-SFM, 10 ng/mL basic fibroblast growth factor, and 10 ng/mL platelet-derived growth factor-BB for 2 days. All three steps involved incubation under ambient oxygen and 5% CO2.
6. Liver organoid formation
Liver organoids were generated following a previous study, with modifications, to obtain three different cell types on the same day. Hepatic endoderm cells, endothelial progenitors, and STM cells were detached from a well of a 24-well plate using 0.25% trypsin/EDTA (Gibco, Waltham, Massachusetts). Each type of cell was resuspended in liver organoid medium containing hepatocyte culture medium (Lonza, Basel, Switzerland) and endothelial growth medium (StemPro-34 SFM containing 50 ng/mL vascular endothelial growth factor) at a 1:1 ratio. Then, the liver organoid medium was supplemented with 20 ng/mL hepatocyte growth factor (Peprotech, Cranbury, New Jersey), 10 ng/mL oncostatin-M (Peprotech, Cranbury, New Jersey), 0.1 μM dexamethasone (Peprotech, Cranbury, New Jersey), and 2% fetal bovine serum (Gibco, Waltham, Massachusetts). Then, the hepatic endoderm cells, endothelial progenitor cells, and STM cells were mixed at a ratio of 100:70:20. Next, 190 μL of the mixed cells were seeded on top of presolidified Matrigel, which was prepared by mixing with liver organoid medium at a ratio of 1:1, in a well of a 96-well cell culture plate. The cells were cultured under ambient O2 and 5% CO2 in a 37 °C incubator for 13 days. The culture medium was renewed every other day. On day 21, the culture medium was changed to hepatocyte culture medium (Lonza, Basel, Switzerland), and the cells were further cultured until day 33.
4. Sporozoite invasion and development
Liver organoids fully develop at 33 days post iPSC differentiation, based on the release of albumin and the expression of hepatocyte-specific transcripts. Thus, 33-day liver buds were subjected to coculture with sporozoites. Cryopreserved P. vivax sporozoites obtained from the Shoklo Malaria Research Unit were thawed by dripping them in a water bath at 37 °C for 30 sec and resuspending them in hepatocyte culture medium. One hundred microliters of sporozoite suspension at a density of 5x104 cells/mL was added to each well of a 96-well plate and incubated at 37 °C for 6 h. After 3 h of incubation, the culture medium was removed to remove the noninvaded sporozoites, and liver organoid medium was added. The infected hepatocyte culture was maintained at 37 °C, and the medium was changed daily. On days 3 and 9 post sporozoite invasion, liver-stage parasites and exoerythrocytic forms were monitored using an indirect immunofluorescence assay and quantitative reverse transcription PCR. To evaluate sporozoite infectivity among batches, the percentage of infected hepatocytes among the total number of inoculated sporozoites was examined, since complete development of the exoerythrocytic forms indicates the release of hepatic or exoerythrocytic merozoites from a hepatocyte. Thus, human reticulocytes were added to each well on days 9 to 12. After 48 hours of incubation, the reticulocytes were collected and attached to glass slides via cytocentrifugation. Giemsa staining was performed daily to observe the ring-shaped trophozoites.
5. Sporozoite gliding motility assay
To verify the survival rate of the sporozoites after thawing, a gliding motility assay was performed following a previous report (29). After the sporozoites were thawed and mixed with hepatocyte culture medium, 50 μL of each sporozoite was transferred to a well of a 96-well plate. The sporozoites were activated by adding 50 μL of 6% bovine serum albumin (BSA) prepared in hepatocyte culture medium. The plates were centrifuged at 200 × g for 3 min at room temperature. The movement of the sporozoites was observed under a light microscope and recorded via video for 3 min. The percentage of motile sporozoites was calculated for each batch. When more than 80% motility was observed, the liver organoids were considered to be infected.
6. Immunofluorescence staining
In the wells, liver buds were soaked in cold HPBS to solubilize the Matrigel. Then, the liver organoids were washed 2-3 times to remove the solubilized Matrigel. The liver buds were fixed with 4% paraformaldehyde in PBS at room temperature for 30 min, followed by paraffin embedding and sectioning. Each liver bud section was attached to a glass slide and air-dried. For analysis of single cells, cells were attached to a glass slide via cytocentrifugation. Before immunostaining, liver bud sections or cells were fixed with 4% paraformaldehyde in PBS at room temperature for 30 min and then permeabilized with 0.25% Triton X-100 in PBS for 15 min. After washing with PBS, the samples were incubated with 1% BSA in PBS for 15 min at room temperature, followed by incubation with the following antibodies at the optimal concentrations: rabbit anti-human polyclonal HNF4 antibody (1:200, Sigma‒Aldrich), mouse anti-human monoclonal α-fetoprotein antibody (1:200, Sigma‒Aldrich), rabbit anti-human polyclonal albumin antibody (1:1000, Sigma‒Aldrich), rabbit anti-human polyclonal CYP3A43 antibody (1:100, Abcam), rabbit anti-human polyclonal SR-BI antibody (1:200, Abcam) and mouse anti-human monoclonal CD81 antibody (1:100, Abcam). All antibodies were diluted in 1% BSA, applied to glass slides and incubated overnight at 4 °C. After washing with PBS, the cells were incubated with Alexa Fluor 488-conjugated goat anti-mouse IgG (Invitrogen) and Alexa Fluor 488-conjugated goat anti-rabbit IgG (Invitrogen). The glass slides were mounted with DAPI-containing antifading medium and covered with a glass coverslip. Images of the cells were acquired using a confocal microscope (ECLIPSE Ti-Cl 4 Laser Unit, Nikon).
7. Quantitative real-time PCR
RNA was extracted using an RNA extraction kit (Invitrogen), and cDNA was prepared using a cDNA synthesis kit (Biotech Rabbit, Berlin, Germany). The primer sets used were obtained from previous reports (30-36) and are shown in Table 1. For PCR, Luna® Universal qPCR Master Mix (New England BioLabs, Ipswich, Massachusetts) was used at a concentration of 1 mM for forward and reverse primers. The thermal cycling parameters were as follows: initial denaturation at 95 °C for 3 min; 30 cycles of denaturation at 95 °C for 10 sec, annealing at 60 °C for 10 sec, and extension at 72 °C for 10 sec; and a final extension at 72 °C for 1 min. Transcripts of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) served as internal controls for normalization of gene expression levels. The threshold cycle (CT) of each sample was used to calculate the relative expression level via the 2-ΔΔCT method (37). Gene expression data were obtained from three independent experiments, and each real-time PCR analysis was carried out in duplicate.
8. Lipid storage
Lipids stored in cells were visualized using fat-soluble Oil Red O following the manufacturer’s instructions (Abcam). The cells were attached to a glass slide via cytocentrifugation. After air drying, the glass slide was placed in propylene glycol for 2 min and then incubated with Oil Red O solution for 6 min. The glass slide was immersed in 85% propylene glycol in distilled water for 1 min and rinsed twice with distilled water. The cells were then stained with hematoxylin for 1-2 min and rinsed thoroughly in tap water. Then, the glass slide was rinsed twice with distilled water and air-dried.
9. Glycogen storage
The cells were treated with 4% paraformaldehyde in PBS and permeabilized with 0.5% Triton X-100 in PBS. Cells treated with 1 mg/mL diastase in PBS (Sigma) were used as the negative control. The cells were then incubated with periodic acid for 5 min, washed with distilled water, and incubated with freshly prepared Schiff’s solution for 15 min. Finally, the cells were rinsed, and the nuclei were stained with hematoxylin. After rinsing with water, the cells were incubated with a bluing reagent for 30 sec and then incubated with Light Green Solution for 2 min. The glass slide was immersed in absolute alcohol for dehydration and air drying.
10. Enzyme-linked immunosorbent assay (ELISA) for human albumin
The amount of human albumin secreted into the culture medium was assessed using the Human Albumin/Serum albumin ELISA Kit (Millipore, Burlington, Massachusetts) following the manufacturer's instructions. Hepatocyte culture medium was used as a background control. Each independent experiment was performed in triplicate.
11. Statistical analysis
GraphPad Prism 10.2.2 (GraphPad Software, Boston, Massachusetts) was used for statistical analysis. The data are presented as the means ± standard deviations (SD). Differences were statistically evaluated using Student’s t test. P values less than 0.05 were considered to indicate statistical significance.