Persistence of Plasmodium cynomolgi hypnozoites in cynomolgus monkey iPS-derived hepatocytes

Plasmodium cynomolgi (Pc) is one of the few parasite species that forms quiescent liver stage parasites known as hypnozoites and is therefore a suitable model for Plasmodium vivax. Very little is known about liver stage dormancy, which hampers the search for compounds with anti-hypnozoite activity. Here, we present the development of a Pc in vitro infection model using stem cell-derived hepatocytes from Macaca fascicularis. IPS cells were established on feeder free condition and differentiated into hepatocytes via inducible overexpression of key transcription factors. The generated hepatocytes were infected with Pc sporozoites and hypnozoite formation as well as schizont development were confirmed by immunofluorescence. This system is a promising tool to study the mechanisms underlying liver stage dormancy and facilitate drug discovery against hypnozoites.


Main Text:
In 2018, malaria had an estimated global incidence of 228,000,000 and a death toll of 405,000 (WHO, 2019). Thus, in spite of the recent successes in the fight against malaria (Rabinovich et al., 2017), the disease remains to be a main killer in the tropics. Malaria being anthroponotic, elimination and even eradication are theoretically possible. However, in addition 5 to drug-resistant Plasmodium parasites and insecticide-resistant Anopheles mosquitoes (Ashley, Pyae Phyo, & Woodrow, 2018), there is an ultimate hindrance to malaria eradication: the hypnozoites (Campo, Vandal, Wesche, & Burrows, 2015). Hypnozoites are small, uninucleated, intracellular stages that stay dormant for months or even years in the liver of infected carriers (Dembele et al., 2014;Krotoski et al., 1982), building a hidden reservoir of parasites that, upon 10 reactivation, causes relapsing malaria. Primaquine and tafenoquine are the only drugs that eliminate hypnozoites (Llanos-Cuentas et al., 2019), but their use is limited due to severe side effects in glucose-6-phosphate dehydrogenase-deficient patients. Therefore, new molecules with anti-hypnozoite activity are needed.
Hypnozoite biology is elusive and the mechanisms underlying dormancy and reactivation 15 remain to be elucidated. Most studies on Plasmodium vivax (Pv) hypnozoites have been conducted with human primary hepatocytes as host cells. These, however, present problems regarding quality, donor-to-donor variability, and ethical considerations. Moreover, working with Pv is challenging as there is no long term in vitro culture system and sporozoite sourcing is extremely limited (Tachibana et al., 2012). The best surrogate organism for Pv is Plasmodium cynomolgi 20 (Pc), a closely related and more accessible malaria parasite of non-human primates that also produces hypnozoites (Krotoski et al., 1980). Here we present for the first time a solution that overcomes the limitations of both Pv in vitro work and primary host cell usage by establishing an in vitro system for Pc hypnozoites that uses cynomolgus monkey hepatocyte-like cells generated by iPS (induced pluripotent stem cells) technology.
Cynomolgus iPS (cynIPS) cells were generated from dermal fibroblasts from two different monkeys by reprogramming, using Sendai viruses bearing the transcription factors Oct3/4, Sox2, Klf4, and c-Myc (Takahashi & Yamanaka, 2006). Pluripotent colonies appeared two to three 5 weeks after infection and were kept on irradiated mouse embryonic fibroblasts. After several passages, the iPS cells were transferred on Matrigel, but spontaneous differentiation occurred on this feeder-less system (  15 The generation of human hepatocytes from stem cells is a well-known process and diverse protocols have been developed (Chen et al., 2012;Gao et al., 2017;Si-Tayeb et al., 2010), but the translation of this process to cynomolgus cells required extensive tailoring. The stepwise approach, which consists of adding growth factors mimicking liver embryogenesis, was not robust and reproducible enough (data not shown). Therefore, we used a direct differentiation approach to 20 generate cynomolgus hepatocyte-like cells (cynHLCs) by overexpressing essential transcription factors for hepatic lineage formation (Huang et al., 2014). Using an inducible construct based on a tetracycline ON/OFF system (Figure 1-figure supplement 6), HNF4A (hepatocyte nuclear factor 4 4 homeobox A) and FOXA3 (forkhead box protein A3) were overexpressed in cynIPS cells to drive endoderm formation and hepatocyte maturation Yoon et al., 2018).
Briefly, confluent cynIPS cells were induced for 4 days with doxycycline (Dox), and hepatic progenitors were further matured for 2 days to generate cynHLCs ( Figure 1A). CynHLCs produced from clone HNF4A/FOXA3-#13.5 displayed the best hepatic characteristics, such as ALB 5 (albumin), HNF1A (hepatocyte nuclear factor 1 homeobox A), HNF4A, A1AT (α1-antitrypsin), KRT18 (cytokeratin 18), AFP (α-fetoprotein), and FOXA2 (Forkhead Box A2) expression ( Figure   1B and Figure 1-figure supplement 7) and high levels of albumin secretion ( Figure 1C). Additional characterization methods revealed accumulation of lipid droplets (Oil Red O staining) and glycogen storage (Periodic Acid Schiff staining), which are attributes of hepatocytes ( Figure 1D). showed a high similarity on the mRNA expression profile. While differentiating, the obtained cynHLCs lost expression of pluripotent markers and expressed typical hepatic progenitor genes and mature hepatic genes including efflux transporters at day 7. We also assessed the expression 10 of known Plasmodium host entry receptors such as CD81 (Cluster of Differentiation 81) and SRB1 (Scavenger Receptor class B type 1) in the generated cynHLCs. Expression of SRB1 and CD81 was confirmed in cynHLCs at the mRNA level during the differentiation process ( Figure 2B). CD81 was gradually expressed in iPS (day 0) and differentiating cells. In contrast, SRB1 was weakly expressed in iPS cells but highly and constantly after day 3, suggesting that cynHLCs 15 become permissive to Plasmodium only when they acquire a hepatic signature (Ng et al., 2015). Therefore, cynHLCs were infected when they expressed hallmark hepatic markers like ALB, HNF4A, and AFP in addition to the host entry receptors SRB1 and CD81 ( Figure 2C). Although SRB1 was already highly expressed at day 3, sporozoite inoculation was performed at day 4 to avoid potential parasite inhibition due to doxycycline (Gaillard, Madamet, & Pradines, 2015;Pang, 20 Limsomwong, & Singharaj, 1988). CYP metabolism). Expression values in transcripts per million (tpm) are colored relative to the maximum (red) and minimum (white) expression value for each gene. B, Line chart representing mRNA levels in tpm of SRB1 and CD81 at day 0, 3, 4, 5, and 6. Error bars represent standard deviation. n=3. C, Immunofluorescence of cynHLCs at day 4 with antibodies to the hepatic markers ALB (green), HNF4A (red), and AFP (red) and to the host entry receptors SRB1 (red) and 5 CD81 (green). Nuclei were visualized with Hoechst (blue). Scale bars, 50 μm.
While the differentiation process was extremely fast with the cynomolgus cells, the maintenance of the hepatic stage was challenging. After 8 days of differentiation, cynHLCs started to detach and died. In order to improve cell survival, we conducted a screen against an in-house chemical library of well-characterized compounds. Three hit compounds that improved cell 10 viability were identified: a p38 MAPK (mitogen-activated protein kinase) inhibitor named Doramapimod, a DHODH (dihydroorotate dehydrogenase) inhibitor, and a Raf inhibitor (  Infection of 4-days differentiated cynHLCs was performed with freshly isolated Pc sporozoites from Anopheles stephensi mosquitoes. We detected the first LS parasites at 2 days 10 post-infection (dpi) after fixation and staining with Pc anti-Hsp70 (70 kilodalton heat shock proteins) antibodies. An additional staining with antibodies against UIS4 (up regulated in infective sporozoites gene 4) confirmed that the parasitophorous vacuole membrane (PVM) of LS parasites was formed (Bertschi et al., 2018). At 2 dpi, we observed a uniform population of uninucleated parasites with a size of 3 µm ( Figure 4A). Despite the low infection rate of less than 10 parasites per well in a 96 well plate, two populations of LS parasites were clearly distinguishable by 4 dpi.
The first ( Figure 4B) was composed of stationary parasites that stayed uninucleated over time (Dembele et al., 2014). These parasites were defined as hypnozoites based on their small size 5 (diameter < 8 µm) and their unique nucleus. The second population instead ( Figure 4C) was composed of growing and multinucleated forms with a larger diameter, which would lead to the formation of liver schizonts. Developing schizonts and hypnozoites were observed until 12 dpi.
As previously described (Mikolajczak et al., 2015), the hypnozoites were slightly increasing in size (from 3 µm at 4 dpi to 5 µm at 12 dpi), but the single nucleus observed by Hoechst staining 10 confirmed the quiescent state of these LS parasites. The current conditions did not allow us to visualize hypnozoite reactivation and subsequent development into mature liver schizonts as described for Pc in primary cynomolgus hepatocytes (Dembele et al., 2014). parasites with antibodies specific for Pc-Hsp70 (green) and Pc-UIS4 (red) at 2 dpi (A), 4-12 dpi small forms (B) and 4-12 dpi large forms (C). Cell nuclei and parasite DNA were visualized with Hoechst (blue). Scale bars, 20 µm. 5 In summary, we report for the first time the generation of cynHLCs from the Pc natural host Macaca fascicularis. Infection of these host cells with Pc sporozoites resulted in successful formation of hypnozoites and persistence until 12 dpi. Moreover, combining the iPS technology with in vitro cultured Pc erythrocytic stages would make the Pc system more accessible and more importantly, largely independent of primates. In conclusion, this iPS-based in vitro system 5 provides a promising alternative to investigate the dormant stage of malaria in the liver and may facilitate drug screening for compounds with activity against hypnozoites.

Primary cells
Cynomolgus monkey fibroblasts used for iPS cell generation were obtained from ZenBio (monkey PDF091812) and Novartis (monkey 5501). Cynomolgus monkey primary hepatocytes were purchased from Biopredic. Human dermal fibroblasts used for iPS and hepatocyte generation were

Ectoderm differentiation
Neuronal precursors were differentiated from cynIPS cells using a modified dual Smad inhibition protocol. 6E04 undifferentiated iPS cells were seeded onto a 96-well ULA (ultra low attachment) plate (Costar, 7007) in 0.1 mL StemFlex medium with 10 ng/mL penicillin/streptomycin and 10 15 μM rock inhibitor to prevent apoptosis. 24 hours after seeding, Embryoid Bodies (EBs) were formed and 0.1 mL fresh StemFlex medium were added to the wells. The next day, 30 EBs were transferred to a 4-well Matrigel coated plate with StemFlex medium (7 or 8 EBs seeded per well).  20 The next day, medium was changed with CDM supplemented with 1 µg/mL Dox. On day 2, medium was switched to William's E medium (no phenol red, Gibco, A1217601) with Primary Hepatocyte Maintenance Supplements (Gibco, CM4000) and 1 µg/mL Dox. Medium was refreshed the next day. On day 4, Dox induction was stopped and William's E medium containing Primary Hepatocyte Maintenance Supplements was supplemented with 5 µM of a combination of compounds (Doramapimod, and inhibitors of DHODH and Raf) to maintain cynHLCs until 17 days of differentiation. Medium was refreshed daily. 5

Generation of human hepatocyte-like cells (hHLCs)
Human iPS cells were harvested and seeded onto a 96-well Laminin 521 coated plate (8E04 cells/well) in mTeSR1 medium supplemented with 100 µg/mL G418 and 10 µM rock inhibitor.
The next day (day 0), cells were washed with PBS and differentiated for 3 days in CDM supplemented with 1 µg/mL Dox (medium was refreshed daily). On day 3, medium was switched 10 to William's E medium with Primary Hepatocyte Maintenance Supplements and 1 µg/mL Dox.
Medium was refreshed on day 4 and 5. On day 6, Dox induction was stopped and cells were further differentiated in William's E medium containing Primary Hepatocyte Maintenance Supplements until day 25. Medium was refreshed every other day. 15 Immunofluorescence analysis Cells were fixed with 4% paraformaldehyde in PBS, permeabilized with Triton X-100 (Sigma, T8787) in PBS and stained with primary antibodies visualized with appropriate fluorescently labeled secondary antibodies (Alexa Fluor). Cultures were counterstained with the nuclear marker Hoechst (Invitrogen, 33342) and immunostained samples were imaged using a Zeiss LSM 700 20 microscope. Primary antibodies references can be found in Table S1.
Periodic Acid-Schiff (PAS) staining (Sigma, Fixed samples were rinsed with deionized water and then placed in 0.5% periodic acid aqueous solution for 5 min at RT. Samples were then rinsed with running tap water for 3 min and quickly rinsed with deionized water before being placed in Schiff reagent solution for 15 min. The same rinsing steps as described above were used and samples were then counterstained with 5 Hematoxylin for 2 min. Samples were rinsed with deionized water for 3 min. Samples were rinsed twice with 70% ethanol, twice with 96% ethanol and twice with 100% ethanol for 1 min each.
Samples were covered with PBS before imaging.
Oil Red O staining 10 Fixed samples were washed twice with deionized water and placed in 60% isopropanol for 5 min.

mL Oil Red O Stock Solution (60 mg Oil Red O (BioVision, K580-24-3) in 20 mL 100%
isopropanol) was mixed with 20 mL deionized water and filtered using a 0.2 µm syringe filter to make Oil Red O Working Solution. Samples were incubated with working solution for 15 min and then washed two to five times with deionized water until excess stain was no longer apparent. 15 Samples were counterstained with Hematoxylin (BioVision, K580-24-2) for 1 min. Samples were washed two to five times with deionized water before imaging.

ELISA assays
To assess albumin secretion capacity of hepatocytes, cynIPS cells were harvested and seeded onto 20 a 24-well Laminin 521 coated plate (7.5E05 cells/well) and differentiated as previously described.
Culture medium was collected every 24 hours before the next medium change. ELISA assay was performed with the collected supernatants according to the provider standard protocol (Immunology Consultants Laboratory, E-80AL).

Hepatocyte survival screen
CynIPS cells were harvested and 2.5E04 cells/well were seeded using a multidrop dispenser onto 5 384-well Laminin 521 coated plates (Corning,3712) in StemFlex medium supplemented with 100 µg/mL G418, and 10 µM rock inhibitor. Cells were differentiated as described above until day 4.
At day 4, Dox induction was stopped, medium was replaced by William's E medium with Primary Hepatocyte Maintenance Supplements and cells were treated with the Novartis compound library in the Echo ® Liquid Handling platform (Labcyte Inc.). Cells were returned to overnight incubation 10 at 37 °C, 5% CO2. Cells were treated with the Novartis compound library every other day after medium change. At day 13, bright field images of all plates were acquired with a high-throughput high-content imaging system (Operetta, Perkin-Elmer) and CellTiter-Glo luminescent cell viability assay (Promega, G7570) was performed. Luminescent readings were obtained using an EnVision multilabel plate reader (Perkin-Elmer) after 10 min incubation at RT. Generated data 15 were analyzed in TIBCO Spotfire ® (TIBCO Software Inc.  RS-122-2001) and sequenced using the Illumina HiSeq2500 platform. Samples were sequenced to a length of 2 x 76 base pairs. Read pairs from cynomolgus iPS, HLCs, and primary hepatocytes were mapped to the Macaca fascicularis genome and the cynomolgus gene transcripts from Refseq by using an in-house gene quantification pipeline (Schuierer & Roma, 2016). The human genome (hg38) was used for the mapping of human HLCs, primary hepatocytes, and liver related cancer cell lines. Genome and transcript alignments were used to calculate gene counts based on Ensembl gene IDs. Radboud, Department of Medical Microbiology) were fed with this blood using an ex vivo glass feeder system.

Sporozoite infection of cynHLCs
Sporozoite inoculation of cynHLCs was performed at Novartis Institutes for Biomedical Research (NIBR, CH) according to the methods of Dembélé et al. (5). Pc sporozoites were kindly provided by Anne-Marie Zeeman (BPRC, NL) and shipped in Leibovitz L15 medium (Invitrogen, 11415-056) with 3% FCS and 2% penicillin/streptomycin at 4 °C to ensure good sporozoite 5 infectiousness. Upon arrival, 4-days differentiated cynHLCs were infected with different sporozoite densities in 96-well plates. Cultures were kept at 37 °C in 5% CO2 with daily medium changes. To evaluate the development of Pc liver stages, cultures were fixed with 4% paraformaldehyde at the indicated time points. 10 Immunofluorescence staining of LS parasites Infected hepatocytes were fixed with 4% paraformaldehyde for 30 min, followed by overnight incubation at 4 °C with rabbit primary anti-PcHsp70 antibodies and rat primary anti-PcUIS4 antibodies both diluted in 1% BSA and 0.3% Triton X-100 in PBS solution. Primary antibodies were kindly provided by Anne-Marie Zeeman (BPRC, NL). Subsequently, donkey secondary IgG 15 Alexa Fluor ® 555-conjugated anti-rabbit antibodies (1:1,000 dilution, Invitrogen, A31572), chicken secondary IgG Alexa Fluor ® 594-conjugated anti-rat antibodies (1:1,000 dilution, Invitrogen, A21471) were added for three hours at RT. Nuclei were counterstained with Hoechst for 10 min at RT, samples were finally covered with PBS and viewed under an inverted microscope (Leica DMI6000 or Zeiss LSM 700). Image acquisition and post processing were performed with 20 proprietary Leica or Zeiss software, LAS X or Zen 2012 respectively. Primary and secondary antibodies references can be found in Table 1.
In Figure 3C, one-way ANOVA was used to compare the groups followed by Dunnett post hoc 5 test.

Acknowledgments: We thank Isabelle Fruh, Bettina Leonhard, Carole Manneville, and Annick
Werner for continuous support with iPS technology; Nicole van der Werff, Ivonne Nieuwenhuis, and Lars Vermaat for mosquito dissection and sporozoite preparation; Annemarie Voorberg-van der Wel for helpful discussions regarding infection assay and critical reading of the manuscript; Isabelle Claerr for technical assistance with high content imaging; Olaf Galuba for support in