All experiments using patient cells were carried out as approved by Institutional Ethics Committee of ICMR-National Institute for Malaria Research (NIMR) and Health Ministry Screening Committee of Indian Council of Medical Research (ICMR). All stem cell experiments were carried out under Institutional Committee of Stem Cell Research, Institute for Stem Cell Biology and Regenerative medicine (inStem), and the National Apex Committee for Stem Cell Research (NAC-SCR). All experiments of transfection and infection were carried out after approval of Institutional Biosafety and Bio-Ethics Committee of NCBS and InStem.
Plasmodium vivax patient screening and blood collection
Patient screening was conducted at Wenlock District Government Hospital, Mangalore by NIMR. A written informed consent was obtained from each patient participating in this study. Plasmodium vivax mono-infected patients were screened from patients diagnosed with malaria by Giemsa-staining of blood smears and Falcivax® rapid diagnostic test with P. vivax specific lactate dehydrogenase. Additionally, the patients were confirmed to be negative for HIV and HCV. For mosquito blood feeding, the P. vivax infected blood samples were directly transferred into standard membrane feeding cup maintained at 37 °C.
Production and isolation of Plasmodium vivax sporozoites
Anopheles stephensi mosquitoes were reared as described previously . Briefly, the mosquitoes were maintained at 27 °C and 75–80% humidity with a 12 h light–dark cycle. Larvae were reared on yeast and dog biscuit in water (70:30 Brewer’s yeast : dog food (Pedigree brand; chicken and vegetable mixed). Pupae were segregated for adult emergence, and freshly emerged adult mosquitoes were fed on 10% d-glucose solution (Sigma-Aldrich) containing 0.05% para-aminobenzoic acid (PABA)(Sigma Aldrich)  and 10 µg/ml penicillin–streptomycin antibiotic cocktail (Invitrogen). For egg production, adult female were allowed to take non infected human blood through membrane feeding. To infect the aseptic mosquitoes with P. vivax, freshly collected P. vivax mono-infected venous blood in anticoagulant heparin sulfate-coated vacutainers (BD Bioscience) were either directly fed or washed the packed P. vivax infected red blood cells with AB + ve human serum (haematocrit adjusted to 50%), prior to feeding 3–4 days old female mosquitoes using membrane feeding apparatus maintained at 37 °C using a circulating water bath. Subsequently, the fully engorged mosquitos were transferred to arthropod containment facility and maintained at temperate conditions as described above. Subsequently, the mosquitoes were fed with water containing 10 µg/mL penicillin–streptomycin in 10% d-glucose to prevent the risk of contamination in the later hepatocyte infection assays [28, 29]. The infection and development of P. vivax in the mosquitoes was confirmed by detection of oocysts in the mid gut of the mosquitoes using 1% mercurochrome staining at day 8–10 post infection as described previously . 14 days post blood feeding, salivary glands of the infected mosquitoes were dissected and mature P. vivax sporozoites were released by mechanical rupture. Insectary operations were carried out in NIMR, Bangalore.
Preparation of primary human hepatocyte (PHH) monolayers
Cryopreserved Primary Human Hepatocyte (PHH) from Bio IVT, formerly Bioreclamation IVT, USA was used in all the studies . Characterized PHH cells (UG4) that are known to be infected by P. vivax  were used. The cells were thawed according to manufacturer’s instructions and cell viability was assessed by trypan blue exclusion. The PHH cells were diluted using in vitro growth CP media (Bio IVT) containing the following antibiotics: Penicillin-50U/ml, Streptomycin-50 µg/ml and Neomycin-100 µg/ml. Cells were seeded onto Pre-collagen coated 384 well plates  at a density of 20,000 cells/well. In some experiments, hepatocytes were incubated with additional broad-spectrum antibiotic Moxifloxacin at 0.5-2 µg/ml and Amphotericin B at 2 µg/ml concentration for various periods to prevent early contamination and increase longevity of plated hepatocytes. The plates are incubated at 37 °C, 5% CO2 to develop confluent PHH cell monolayers prior to infection.
HC04 cell line seeding and culture
HC04 (14,16) and other hepatoma-like cell lines tested (HepG2, Chang Liver, WRL-68, PLC/PRF) were maintained in MEM/F12 medium with 1 × anti-anti (Thermo Fisher) at 37 °C, 5% CO2. Viable cells quantified by trypan blue exclusion. HCO4 cells were seeded at a density of 25,000 cells per well in 384 well plate while other cells mentioned were seeded at 20,000 cells per well in 384 well plate or 80,000 cells per well in 96 well plate before infection.
iPSC derivation from vivax patient PBMC
Three vivax patients’ PBMCs were processed for iPSC generation using CytoTune®-iPS 2.0 Sendai Reprogramming Kit (Thermo Fisher Scientific, Waltham, MA, USA) according to manufacturer’s Instruction. Briefly, the PBMCs were thawed, cultured for 5 days in a selection media to enrich CD34+ hematopoietic progenitor cells, and transfected with reprogramming factors, namely Oct4, Sox2, Klf4 and cMyc as well as the reporter of transfection (GFP) gene in Sendai virus non-genome integrated vectors, to generate iPSCs. After 16 days of culture in iPSC culture medium, iPSC colonies emerged. The colonies were then cloned and expanded to establish the iPSC lines. At least three morphologically relevant and stable iPSC lines per patient were established from each PBMCs. Non-patient iPSC lines were derived from commercially available PBMCs from a healthy donor (AccuCell, Frederick, MD, USA). The iPSCs were expanded and stocked after confirmation of pluripotency marker expression and normal karyotype.
Nine patient iPSCs (BG1, BG2 and BG3 iPSC line from patient 1; NG1, NG3 and NG4 iPSC lines from patient 2; and S2, S4 and S5 iPSC lines from patient 3), non-patient iPSCs (K iPSC line from commercially available PBMCs) and H9 ESC (WA09, WiCell, Madison, WI, USA) were routinely cultured using standard feeder-dependent or feeder-free conditions as described previously . Briefly, in feeder-dependent condition, cells were cultured in ESC media (DMEM/F12 medium) supplemented with 20% Knockout Serum Replacement (Thermo Fisher Scientific), MEM nonessential amino acids (Thermo Fisher Scientific), l-glutamine (Thermo Fisher Scientific), penicillin/streptomycin (Thermo Fisher Scientific) and bFGF (4 ng/ml; Thermo Fisher Scientific) on mitotically inactivated mouse embryonic fibroblasts (MEFs). In feeder-free condition, the cells were cultured on hESC-verified Matrigel (Becton, Dickinson and Company) coated plates in mTeSR1 media (Stem Cell Technologies, Vancouver, Canada). The feeder free condition was used for at least two to three passages prior to hepatic differentiation. Absence of mycoplasma contamination in all cells was confirmed at the time of freezing, after thawing and after every 2 months of culture by MycoAlert Mycoplasma Detection Kit (Lonza, Basel, Switzerland).
Characterization of iPSCs
Pluripotency marker expression and normal karyotype was confirmed as described previously . Briefly, the cells were fixed with 4% paraformaldehyde (PFA), washed with PBS, permeabilized with 0.1% Triton-X 100/PBS and blocked with 2% BSA/PBS. The cells were then incubated with primary antibodies: NANOG (4903S, Cell Signaling, Danver, MA,USA, 1:100), SOX2 (Y17, sc- 17320, Santa Cruz, Dallas, TX, USA, 1:100), OCT4 (C10, sc-5279, Santa Cruz, 1:100), SSEA3 (631, sc-21703, Santa Cruz, 1:100), SSEA4 (MC813, sc-59368, Santa Cruz, 1:100), TRA-1-60 (sc-21705, Santa Cruz), TRA-1-81 (sc-21706, Santa Cruz), followed by incubation with Alexa Fluor 594- or Alexa Fluor 488- conjugated secondary antibody (Thermo Fisher Scientific), and mounted with Vectashield with DAPI (Vector Laboratory, Burlingame, CA, USA). Alkaline phosphatase activity was detected by Vector Blue Alkaline Phosphatase Substrate Kit (Vector Laboratory) according to the manufacturer’s protocol. For karyotype analysis, the cells were arrested in metaphase with colcemide (KaryoMAX, Sigma-Aldrich, St. Louis, MO, USA), dissociated with trypsin/EDTA, treated with a hypotonic solution, and fixed with Carnoy’s fixative solution. Chromosomal G-bands were stained with Leishman’s staining solution (L6254, Sigma Aldrich).
Hepatocyte differentiation from iPSCs and characterization
iPSC’s were differentiated to hepatocytes by step-by-step sequential differentiation protocol with oxygen control. Directed differentiation was achieved by sequential exposure to Activin A (120-14E, Peprotech, Rehovot, Israel) at 20% O2 to differentiate to definitive endoderm (DE stage), BMP4 (bone morphogenic protein 4, PHC 9534, Invitrogen), FGF2 (100-18B, Peprotech) HGF (100-39, Peprotech) at 4% O2 for hepatoblast (HB) and hepatocyte (HC) differentiation, and finally with Dexamethasone (D-2925, Sigma) and OncostatinM (OSM, 295-OM, R & D Systems, Minneapolis, MN, USA) at 20% O2 for hepatocyte maturation. Maturation of hepatocytes was further achieved via treatment with free fatty acids, high density lipoprotein and small molecules FH1 and FPH133, under ambient oxygen. At each stage mentioned, differentiation was confirmed by morphology, immunostaining and RT-qPCR with stage specific markers as described previously . All specific probe/primes for RT-qPCR are TaqMan gene expression and assays performed following the manufacturer’s instructions (Applied Biosystems, Foster City CA, USA); OCT4 (POU5F1) (Hs01895061_u1), NANOG (Hs02387400_g1), SOX17 (Hs00751752_s1), GATA4 (Hs00171403_m1), FOXA2 (Hs00232764_m1), HHEX (Hs00242160_m1), HNF4A (Hs00230853_m1), PROX1 (Hs00896294_m1), AFP (Hs00173490_m1), ALB (Hs00910225_m1) and A1AT (SERPINA)(Hs01097800_m1). The primary antibodies used for Immunostaining were; FOXA2 (AB4125, Millipore, Billerica MA, USA), SOX17 (AF1924, R&D Systems), CXCR4 (MAB173, R&D Systems), HNF1b (C-20, Santa Cruz), HNF4a (H1415, R&D Systems), AFP (ab75705, Abcam, Cambridge, UK), A1AT (ab17438, Abcam, Cambridge, UK), ALB (MAB1455, R&D Systems), CK18 (SAB3300015, Sigma-Aldrich), CD81 (ab59477, Abcam), BSEP (PA5-27742, Thermo Fisher Scientific), OATP2/8 (ab15441, Abcam), MRP2 (ab3373, Abcam) and SRB1 (ab52629, Abcam).
Further characterization of iPSC-derived hepatocytes was achieved using functional assays. Stored lipids were detected by Oil Red O staining. The hepatocytes were fixed with 4% PFA and incubated in 0.5% Oil red O solution (O1391, Sigma-Aldrich). The cells were rinsed and nuclei were counter-stained with haematoxylin.
Stored glycogen was detected by Periodic Acid Schiff (PAS) staining. The cells were fume-fixed with 4% PFA and permeabilized with 0.1% Triton X-100, following which they were incubated with Periodic acid, and then Schiff’s solution. The cells were counter-stained with haematoxylin. Low-density lipoprotein uptake and regulation was assessed by LDL Uptake Assay Kit (ab133127, Abcam) according to the manufacturer’s protocol. Albumin and Alpha-1 Anti trypsin secretion was measured by human albumin ELISAKit (ab108788, Abcam) and Alpha 1 Antitrypsin ELISA Kit (ab108799 Abcam), respectively, using culture supernatants of the cells, according to the manufacturer’s protocol. Bile transporter activity was assessed by indocyanine green (ICG) (I2633, Sigma-Aldrich) uptake and release through the Liver-Specific organic anion Transporter-1 (LST1). Cellular uptake and release of ICG was examined in 1 h and overnight incubation, respectively. Cytochrome P450 (CYP) activity was measured by P450-Glo CYP3A4 Assay (V9001, Promega, Madison WI, USA) with non-lytic cell-based assay according to the manufacture’s protocol. The hepatocytes were transferred into 96-well plate at 30,000 cells/well for infection assays.
Infection of the hepatocytes (PHH, HC04, iPSCs derived from vivax patient and non-patient) with the P. vivax sporozoites
Plasmodium vivax sporozoites were prepared by dissecting the infective salivary gland of Anopheles stephensi on day 14 post membrane feeding. The P. vivax sporozoites were diluted in dissection medium (without serum) and counted using haemocytometer. The ratio of either 2 sporozoite for 1 hepatocyte (2:1) or 1 sporozoite for 1 hepatocyte (1:1) was typically used for infection assays, unless mentioned otherwise. Accordingly, the desired number of sporozoites were adjusted in appropriate volume of dissection medium and added per well in either 384 well or 96 well plate formats having the respective hepatocyte cells (PHH, HC04 or iPSCs derived hepatocytes). Additionally, different hepatoma cell lines, namely HepG2, Chang liver, WRL-68 and PLC/PRF were also cultured and assessed for their susceptibility to P. vivax sporozoite infection. All cell types were seeded 24 h prior to infection with sporozoites and checked for cell confluency prior to infection. After infecting the respective wells with P. vivax sporozoites, the culture plates were centrifuged at 200 g for 3 min and incubated at 37 °C, 5% CO2 for 4 h followed by washing infected hepatocytes with complete medium to remove uninvaded sporozoites and salivary gland debris. Next, the P. vivax sporozoite infected cultures were maintained at 37 °C, 5% CO2 with everyday media change until fixed by 4% PFA for 15 min at room temperature at defined time points for respective set of experiments.
The P. vivax infection in the fixed hepatocyte cells were analysed by immunostaining to detect the presence of small and large forms of the parasite, using P. vivax specific antibodies, namely Plasmodium HSP70 and PvUIS4. DAPI was used as nuclear stain. All the images were acquired in Nikon Ti2 series microscope at 40X magnification and the image analysis was performed using FIJI (ImageJ). Hepatocytes were considered infected if the following criteria were met, a) positive signal for UIS4 antibodies showing the characteristic ring-like shape of P. vivax liver stage forms, b) presence of the signal inside the hepatocyte cell, as assessed by bright field images, c) DAPI staining for the parasite nuclei, which gives a distinctive staining pattern compared to host nucleus.
Compounds were added once per day along with the media (MEM/F12 +10% FBS + 1X Anti Anti + Amikacin 200 μg/ml) until four days after infection starting from 4 h after infection. MMV390048  was used in the concentration of 10 µM whereas Torin was used in the concentration of 250 nM .
Plasmodium vivax infection experiments were performed in three or more independent batches consisting of differentiated hepatocytes from a minimum of three iPSC clones/patient along with ES- and non-patient iPSC-derived hepatocytes and HCO4 hepatoma cell line in triplicate or more for each condition. Data from representative batches are presented. For all comparisons between two conditions, statistical significance was assessed by Student’s t test were performed.