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STEM CELL TECHNOLOGY PROVIDES NOVEL TOOLS TO UNDERSTAND HUMAN VARIATION IN Plasmodium falciparum MALARIA

Plasmodium falciparum interacts with several human cell types during their complex life cycle, including erythrocytes and hepatocytes. The enuclated nature of erythrocytes makes them inaccessible to genetic tools, which in turn makes studying erythrocyte proteins involved in malaria invasion and development particularly difficult. Here we overcome this limitation using stem cell technology to develop a universal differentiation protocol for in vitro derivation of erythrocytes from a variety of stem cell lines of diverse origin. This allows manipulation of erythrocytic genes and examination of their impact on the parasite by flow cytometric detection of parasite haemozoin. Deletion of Basigin, the essential receptor for P. falciparum, abrogates invasion, while other less studied proteins such as ATP2B4 have a minor effect. Reprogramming of induced pluripotent stem cells from α-thalassemia primary samples shows reduced infection levels, demonstrating this approach is useful for understanding the effect of natural human polymorphisms on the disease.


INTRODUCTION
Malaria is an infectious disease caused by several species of Plasmodium parasites that are transferred between humans by female Anopheline mosquitoes. The life cycle of the parasite is complex and involves various organs in each host, with a wide range of interactions between parasite and host cell at each step. Nevertheless, the pathology and severe complications of malaria infection in humans result from the blood stages of the infection, during which parasites invade and develop inside erythrocytes. The completion of reference genomes (1), the development of genome editing technologies (2,3) and their adaptation to the parasite (4,5) have revolutionised our understanding of the parasite side of the blood cycle. These advances have enabled the identification of many parasite proteins involved in invasion (6), as well as the subsequent formation of the parasitophorous vacuole and remodelling of the erythrocyte (7,8), and a much broader understanding of parasite blood-stage biology. On the host side, genome-wide association studies (GWAS) have identified multiple human genetic variants associated with differences in the severity of disease caused particularly by Plasmodium falciparum, the most virulent species of parasite affecting humans (9,10). These include many genes implicated in erythrocyte structure and function, such as the membrane protein Band 3, the red blood cell enzyme Glucose-6-Phosphate Dehydrogenase and the Haemoglobins, amongst others (11). However, despite population studies providing compelling evidence for protective effects of multiple human protein variants, the molecular mechanisms of these effects are still largely undeciphered. This is mainly due to the difficulties in accessing primary cell samples and the technical challenges of reproducing variants in vitro in order to perform tightly controlled cellular studies.
There are two major hurdles for identifying host proteins that interact with malaria parasites and understanding their function. Firstly, erythrocytes are non-proliferative, terminally differentiated cells with a limited life span, making long-term culture of the same cells impossible. Thus, research has relied on clinical samples, with the inherent difficulties of donor availability and variability as well as the impact of storage and transport on sample quality. Perhaps most significantly for mechanistic studies, mature erythrocytes are enucleated and therefore gene editing technologies cannot be applied. These limitations have been partly overcome using siRNA knock-down techniques in Haematopoietic Stem Cells (HSCs) to study the role of Glycophorin A (GYPA) (12), Basigin (BSG) (13) and to perform a screen for erythrocyte-specific proteins (14). However, this approach is curtailed by the restricted availability of HSCs, their limited proliferation capacity and the variable efficiency of the knock-down levels that can be achieved. More recently, an immortalised adult erythroblast line able to proliferate and differentiate in vitro, was established by transformation of erythroid progenitor cells with the human papilloma virus HPV16-dervied proteins HPV16-E6/E7 (15). One such line (BEL-A) was combined with genome editing technologies to successfully unpick the mechanisms of Basigin involvement in P. falciparum invasion (16). This approach was also applied to peripheral blood samples and shown to generate cells permissive to P. falciparum and P. vivax invasion and amenable to genome editing studies (17). A disadvantage of this strategy is the viral transformation of the cells with its inherent genetic consequences that might be limiting on the long term, particularly when addressing natural genomic variation.
While these are clearly robust and useful tools, other approaches, such as the use of established Embryonic and induced Pluripotent Stem Cell lines (ESCs and iPSCs) that can be cultured (18) and genetically manipulated (19) while maintaining their pluripotency (20), can provide a versatile alternative with additional advantages. Stem cells have the potential to differentiate into any cell type, which allows examination of the same genomic background as well as any modification or editing on different cell types in all parasite stages. Furthermore, the development of reprogramming techniques that revert terminally differentiated cells to pluripotency (21) makes it possible to generate iPSCs from specific individuals. In this way, complex genotypes and rare variants, including non-viable mutations, can be brought into the lab and stored for future studies. Genome editing to change or correct the mutations also become possible, offering a direct confirmation of their physiological role.
In this work we developed a differentiation protocol to drive any stem cell line, ESCs and iPSCs, towards erythropoiesis and produce cells that are competent for P. falciparum infection. This approach makes it possible for the first time to study patient-derived cells, while also allowing the potential to incorporate genome editing of specific host genes on multiple different genomic backgrounds. To complete the pipeline we established an in vitro assay to accurately quantify invasion of the stem cell-derived erythrocytes by the parasite using an adaptation of flow cytometry based on the refractive properties of haemozoin, a pigment produced by malaria parasites after digestion of haemoglobin. This approach represents a versatile tool to explore a wide variety of host aspects influencing malaria infection and potentially open new avenues for therapeutic intervention.

Ethics Statement
The use of primary erythrocytes for the culture of Plasmodium falciparum was approved by the NHS Cambridgeshire 4 Research Ethics Committee REC ref. 15

Reprogramming of Induced Pluripotent Stem cell lines
Human IPS lines were derived and verified at the Wellcome Sanger Institute as described (22)(23)(24)(25). Briefly, 5x10 5 cells were transduced with Sendai virus carriers of the Yamanaka factors: hOCT4, hSOX2, hKLF4 and hc-MYC overnight at 37 0 C in 5% CO2. After a medium change the next day, the cells were cultured for 4 days and from then on maintained in Stem Cell medium: advanced DMEM/F-12 (Gibco, UK) supplemented with 2 mM Glutamax (Gibco), 0.01% β mercapto ethanol (sigma), 4 nM human FGF-basic-147 (Cambridge Bioscience, UK) and 20% KnockOut serum replacement (Gibco, UK), changing medium daily. Ten to 21 days post-transduction, formation of pluripotent colonies was evident, the visible colonies were handpicked and transferred to 12 well plates with MEF feeders. Colonies were expanded into 6 well feeder plates and passaged every 5 to 7 days depending on confluence.

Cell Culture
All stem cell lines used in this study were cultured on feeder cells (irradiated mouse embryonic fibroblasts MEFS (Global Stem) in the Stem Cell medium described above. The cultures were kept at 37 °C, 5% CO2 and medium was changed regularly. Pluripotent cells were passaged using 0.5 mM EDTA (Gibco, UK) and 10µM Rock inhibitor.
As a first step of differentiation, the cells were taken towards the mesoderm germline:

RNA extraction, qRT-PCR and Microarrays
RNA was extracted using the Isolate II RNA Mini kit (Bioline, UK). 1-3 µg were reverse transcribed with a MuLV reverse transcriptase (Applied Biosystems, UK) using random primers (Bioline, UK). One µl of cDNA was specifically and quantitatively amplified using Biotool 2x SybrGreen qPCR master mix (Stratech, UK) following the cycling parameters established by the manufacturer on a light cycler 480 II (Roche) and using GAPDH as a control for normalisation. The primers used (IDT, Belgium) were: gene forward primer reverse primer length (bp) For microarray analyses, RNA was extracted as above, the Illumina TotalPrep RNA amplification kit (Ambion Life technologies) was used to process the samples, and gene expression analysis was assessed on Illumina HumanHT-12v4 chips following the instructions of the manufacturer.

Fluorescent parasites
Parasites were engineered to express a variety of fluorochromes for detection at different wavelengths (26). The chosen fluorochromes: tagBFP, Midori-ishi cyan, Kusabira Orange and mCherry were individually inserted into the XhoI / AvrII site of an attP-containing vector under regulation by the calmodulin promoter and bearing blasticidin resistance as a selection marker. The NF54 attB strain of Plasmodium falciparum was transfected with each fluorochrome vector together with an expression vector for Bxb1 integrase and transfectants were selected with blasticidin (2 ugr/ml) (27).

Invasion Assays
Differentiated human stem cells were counted and 1 million cells per assay to be performed were labelled with 1 μM DDAO-HS membrane dye for 2h. After a 30 min wash with medium, the cells were centrifuged (1100 x g for 4'), resuspended in complete parasite culture medium at 75 μl/well and dispensed into a 96 well plate, including a well of cells alone for controls. For controls, primary erythrocytes obtained from the blood bank were labelled in the same way, adding 75 µl at 5% haematocrit per assay.
Asynchronous cultures of fluorescent parasites at a parasitaemia of 1.5 -2% mature parasites were used to purify schizonts: 10 ml of culture were centrifuged (1100xg for 5' brake 3), resuspended in 1 ml of medium and loaded onto a 63% Percoll cushion. Centrifugation at 1300xg for 11' no brake separated the mature parasites at the Percoll interface. These were recovered, washed with parasite medium and resuspended in 3 ml of parasite medium. The parasite suspension, which due to the asynchronous nature of the starting culture contained a relatively broad window of late-stage parasites, from late trophozoites to mature schizonts, was added to the cells in the 96 well plate at 75 μl/well.
One plate per time point was prepared and plates were placed in a gas chamber filled with malaria gas and left in a 37°C incubator for the appropriate length of time.
After the incubation time, the plate was removed, adding 200 μl of PBS/well and spinning at 1100xg 1'. Three μl were taken from the bottom of the wells and smeared on slides to be stained with Giemsa and the supernatant was removed. The pellets were fixed with 100 μl 4% paraformaldehyde for20', washed and resuspended in PBS to be analysed by flow cytometry.

Flow Cytometry
Expression of proteins on the membrane of stem cell-derived erythrocytes was measured using specific fluorochrome-tagged antibodies and quantitation by flow cytometry on a LSFORTESSA BD analyser using Flowjo V10.3 (Bcton Dickinson & Co, NJ) and SUMMIT V3.1. Antibodies: A MoFlo flow cytometer (Beckman Coulter, USA) was adapted for detection of laser light depolarisation produced by parasite hemozoin (28). The Mo-Flo flow cytometer has a Z-configuration optics platform and is equipped with four solid state lasers (488nm, 561nm, 405nm, 640nm) spatially separated at the stream-inair flow chamber with 488nm primarily assigned as the first laser. The laser power for 561nm, 405nm, 640nm were all set at 100mW and 488nm was set at 50mW. The laser 488nm, 561nm, 405nm, 640nm was used to excite the Cyan and PE, mcherry, BFP and DDAO respectively. Fluorescence emitted from Cyan, PE, mcherry, BFP and DDAO was collected using a 520/36nm, 580/30nm, 615/20nm, 447/60nm and 671/28nm band pass filter respectively. An optical modification was made on the primary laser detection pod so that the scattered light from 488nm laser light was split into two using a 50/50 beam splitter to measure the normal SSC (vertical) and depolarised SSC (Horizontal) by placing a polarizer (Chroma Technology Corp) with its polarisation axis horizontal to the polarisation plane of the laser light. Both SSC detectors have a 488/10nm band pass filter (Fig. S2). A total of 50000 events was acquired and analysed using Flojo V10.3.

Genome Editing
Genomic modification to ablate the genes chosen for this study was performed in the RH1 cell line, as described previously (29) and shown schematically in supplementary Fig. S8.

Microarray data analysis
All microarray datasets were put through "neqc" background correction followed by quantile normalization using the limma R package (30). Inter-plate variation (batch effects) were adjusted using combat algorithm https://pubmed.ncbi.nlm.nih.gov/16632515/ [pubmed.ncbi.nlm.nih.gov]. Differential expression analysis was performed to obtain a subset of significant probes (those that change between two or more conditions), FDR adjusted P value of 0.05 was chosen as the cut-off using limma R package. Heatmaps were plotted using Complexheatmap R package https://pubmed.ncbi.nlm.nih.gov/27207943/ [pubmed.ncbi.nlm.nih.gov]. The

A variety of human stem cells differentiate into erythroid cells in vitro.
A protocol to differentiate human stem cells into erythrocytes was developed (Fig. S1), in which the adherent pluripotent cells are first taken towards the mesoderm path, as described by Vallier et al (31) by exposing the cells to two steps of specific cytokines. As the cells start differentiating, the pluripotency markers decrease while expression of mesoderm markers increases (Fig 1A) followed by the induction of the crucial transcription factors involved in driving the myeloid line of haematopoiesis towards megakaryocyteerythroid progenitors (MEP). The modulation of gene expression is concomitant with a morphological change of the cells. For our goals, the final stage of this phase was extended to 8 -12 days during which the cells detach spontaneously from the plate. The suspension of cells detaching from the plate is then taken through the erythropoietic phase of the protocol during which the cells are exposed to Erythropoietin (EPO) and IL-3 to drive differentiation into erythroblasts. The cells are kept in dexamethasone in order to halt the process at the erythroblast stage and homogenise the culture. Next, the erythroblasts are expanded and differentiation is completed by removing dexamethasone (Fig. 1B). At the end of the differentiation process, analysis of the characteristics of the cells show surface expression of Glycophorin A (GYPA), a major marker of erythrocytes and expression of adult haemoglobins A and B (Fig. 1B). The cells also express the transferrin receptor (CD71) and foetal haemoglobins Gamma and Epsilon and the great majority of them (70 to 80%) stain positively with a DNA dye (Hoechst33342), indicating that they still have some form of nucleus or nucleic acid content (Fig 1B). These features indicate that the cells generated correspond to immature erythroid cells earlier in the differentiation pathway to mature erythrocytes. The full transcriptome changes as the cells differentiate were analysed using microarrays, showing increasing expression of erythrocytic genes including membrane proteins, components of the haeme cycle and the haemoglobins (Fig. 1B). The microarrays also confirmed that extension of the mesoderm/erythroid transition step of the mesoderm stage improves erythrocytic differentiation and that a duration of 8 or 12 days leads to similar gene expression patterns. To establish the versatility of the differentiation protocol, a variety of cell lines of different origins were tested. This included human embryonic stem cell (hESC) lines (Shef3 and Shef6), as well as Induced Pluripotent Stem Cell (hIPSC) lines derived from fibroblasts (RH1, SF2 and K4) and from blood (CD3, CD5 and GB1, GB4). The efficacy of the process was assessed according to the expression of surface markers GYPA and CD71 by flow cytometry (Fig. 2A) and the haemoglobin genes by qRT-PCR (Fig. 2B). Figure 2 shows that though there are variations between the lines tested, they all differentiate in a similar fashion. The overall expression of erythroid genes revealed by microarray analysis confirmed a similar response by all cell lines tested to the differentiation protocol (Fig. 2C).

Stem cell-derived erythroid cells support invasion by Plasmodium falciparum.
The capacity of the different cell lines to support P. falciparum infection was assessed with a purposely developed assay. The in vitro differentiated cells are fluorescently labelled to distinguish them from any primary erythrocytes co-purified with the parasites, and cultured with percolled parasites from fluorochrome-expressing P. falciparum lines (Fig. S1). The use of purified fluorescent late-stage parasites ensures prompt release of merozoites for invasion and avoids the traditional labelling with DNA dyes that is incompatible with the presence of nucleic acids in the in vitro-derived erythrocytes. To detect invasion, we used a flow cytometry approach specifically adapted to detect haemozoin (Fig S3), the metabolic product of the parasite's digestion of haemoglobin (28). The strategy for the quantification is shown in Figure S4: briefly, after eliminating debris and doublets, the infected differentiated erythrocyte population is selected based on the fluorescence of both cells and parasites, and then examined for depolarisation using uninfected cells to establish the gates. Since light depolarisation is an exclusive property of haemozoin produced by the parasite, the events detected correspond to parasites that successfully invaded labelled in vitrodifferentiated cells. Because the quantification of depolarising events is applied to the selected population of labelled cells, the proportion reflects the parasitaemia of the culture. To determine the optimal combination of fluorescent labels for the assay, parasites expressing a range of fluorochromes were generated (26) and different cell dyes were tested. Invasion of cells labelled with the green cell dye DFFA with mCherry-parasites (Fig. S4) was equally effective for quantification of parasitaemia as labelling cells with the far-red dye DDAO and infecting them with Midori-ishi cyan-or tagBFP-parasites ( Fig. S5 and S6). The combination of DDAO-stained cells and tagBFP parasites was chosen for all following experiments because these two labels are very strong with the best separation of their excitation/emission spectra.
Invasion is first quantified after 18 hours to allow enough time for haemozoin accumulation and fluorescent protein expression. As haemozoin synthesis increases throughout the blood cycle, a second time point towards the end of the erythrocytic cycle at 42 hours is also measured to assess parasite growth. Examination of Giemsa-stained slides under the microscope confirmed successful invasion (Fig 3A). All cell lines differentiated were effectively invaded by P. falciparum reaching a parasitaemia between 5 and 8% (Fig. 3B), similar to the levels observed in control assays with labelled blood (4.55%) (Fig. S7A). At 42 hours parasitaemia rose in all cell lines to 7-10%, reflecting the growth of small parasites that went undetected at the 18 hour timepoint (Fig. 3B). The blood controls showed a significantly higher increase in parasitaemia at the 42-hour time point (P<.001) with an average of 20% (Fig. S7A), likely due to the much higher cell-toparasite ratio in these control wells. The purified parasites contain a range of late stages from late trophozoites to mature schizonts, meaning that some parasites will mature and rupture within a 10-12 hour window after the set-up of the assay (see Methods section). The 2.5% haematocrit of the blood assays represents an approximate ratio of 30:1 erythrocytes to parasites offering ample availability for continuous invasion throughout the assay. By contrast, because the number of in vitro-derived erythrocytes is limited, these assays have a ratio close to 1:1, restricting invasion to the initial merozoites released in the assays. The concomitant increase in haemozoin intensity with life cycle progression is a useful tool to measure parasite growth (28,32). Indeed, synchronised parasite cultures show a clear shift in haemozoin signal intensity as they progress from rings to schizonts (Fig. S7B), and this is replicated in our bespoke invasion assays (Fig. S7C), albeit a wider 42-hour peak resulting from the characteristics of the assays as explained above. The gate established by the shift in haemozoin intensity between the ring and schizont stages (M1 in Fig. S7B), was used to quantify the percentage of mature parasites in the cultures (Fig. S7C), which showed an average of 64% (Fig. S7D). Applying the same gate to the assays with the in vitro-generated erythrocytes, we observed similar levels of mature parasites between 60 and 80% (Fig. 3C).

Genome editing of human stem cells reveals a role for specific genes in malaria invasion.
The potential to derive edited erythrocytes with this approach was tested with the control line RH1 using CRISPR/Cas9 technology (Fig. S8). Two target genes were chosen, Basigin (BSG, CD147) which is known to be a universal receptor for P. falciparum invasion (13) as a proof of principle, and ATP2B4 (PMCA4) since natural variation in this gene has been correlated to resistance to severe malaria (33). The edited clones were genotyped (Fig. S9A) and verified by sequencing (Fig. S9B) confirming small deletions in the critical exon that generate a stop codon downstream. The lack of protein expression was confirmed with specific FITC-labelled antibodies for flow cytometry (Fig 4A). When challenged with the parasites, neither of the independent Basigin-null cell lines (B5 and C5) supported invasion (Fig 4B), consistent with its role as an essential receptor for P. falciparum (13). The proportion of mature parasites after 42 hours of culture was similar to the control cells (Fig. 4B), indicating that the few parasites that invaded the modified cells could achieve some growth. Deletion of ATP2B4 (Fig. 4A) showed a tendency to lower invasion levels, marginally significant for only one clone (Fig 4C). The proportion of mature parasites in the ATP2B4 KO cultures at the 42-hour time point was similar to that observed in the control lines, indicating that disruption of this gene does not have a major effect on the development of the parasite.

Reprogramming IPS lines from haemoglobinopathy patients shows the versatility of this system.
Fibroblast lines isolated from patients with α-thalassemia (HbBart) haemoglobinopathies were sourced from the Coriell repository and reprogrammed to generate IPS lines (Coriell Cat# GM10796, RRID:CVCL_N352 called Euml (EM); Coriell Cat# GM03433, RRID:CVCL_N008, called Fijo (FJ)). The IPS lines obtained were differentiated in parallel with our reference cell lines and exposed to fluorescent parasites in our invasion assays. As shown in Fig 5, both thalassaemic cell lines showed significantly decreased efficiency of invasion. Furthermore, the proportion of later stages of parasites in these cultures at the 42-hour time point was also reduced compared to the control cell lines (Fig. 5).

Figure 5: Reprogramming of IPS cells from patients with αthalassemia haemoglobinopathy.
Fibroblasts from αthalassemia major samples were reprogrammed to IPS cell lines that were differentiated and exposed to P. falciparum to assess invasion and percentage of mature parasites at 42 hours * P<.05; ** P<.005.

DISCUSSION
In this work we present a protocol that effectively differentiates a variety of human stem cell lines towards erythropoiesis and generates cells that are susceptible to infection with Plasmodium falciparum parasites. A total of 9 stem cell lines of diverse origin were studied, showing that though the differentiation efficiency does vary slightly between lines, they all generate erythroid cells as demonstrated by upregulation of erythrocytic genes and expression of erythrocytic proteins. Despite enucleation being notoriously difficult to achieve in vitro (34,35) we observed enucleation levels of 20-30%. In fact, the cells with positive nucleic acid staining also include cells with nuclear fractionation and some in which extrusion of the nucleus is incomplete, as can be seen in Figure 1B. Importantly, stem cells differentiated with this protocol are capable of supporting invasion by P. falciparum without the need to sort or purify the differentiated cells.
During the blood cycle, haemoglobin is the main source of amino acids for the parasite's metabolic needs. Degradation of haemoglobin releases free haem, which represents a major toxic insult to the parasite. In a detoxification mechanism the oxidised iron group is compacted into an insoluble crystalline form: β-hematin or haemozoin and stored in the food vacuole (36), becoming a distinct feature of intra-erythrocytic Plasmodium parasites (32). Intracellular haemozoin formation is reliably detected 12 to 18 hours postinfection and both crystal size and number increase with progression of the blood cycle (37,38). The flow cytometry strategy to detect depolarisation of light caused by hemozoin therefore ensures that the events detected correspond to parasites that have successfully invaded differentiated cells, survived and started growing. However, because haemozoin only becomes detectable at the late ring stage, smaller rings can be missed at the first time point of 18 hours. The use of schizonts for the assays ensures quick invasion after the co-culture is set up, but complete synchrony is difficult to achieve as there will always be a contribution of less mature parasites in the schizont preparations, particularly from routine asynchronous cultures. Therefore, the increase in the number of haemozoin-positive events at 42 hours is likely due to the maturation of late-invading parasites that become detectable at the later time.
The successful manipulation of genes implicated in malaria infection was demonstrated by deletion of Basigin, which resulted in a dramatic decrease of infection as expected given the known role of this protein in invasion (13). Natural variants in ATP2B4 have been associated with resistance to malaria in various studies (10,39), but the mechanism of protection is not known. A number of variant SNPs have been identified in this gene, mostly in Linkage Disequilibrium (LD), and though it is not clear whether all these SNPs play a role in protection against severe malaria, one of them was shown to disrupt a GATA-1 site in the promoter of the gene (40). As a consequence, the expression levels of the protein are markedly reduced giving rise to changes in erythrocyte parameters such as mean corpuscular haemoglobin concentration (MCHC) and size. ATP2B4 is the main membrane Calcium ATPase of erythrocytes that removes calcium from the cytosol to maintain the low levels necessary for calcium-dependent signalling to occur (41). A role of calcium in the invasion process of P. falciparum has been suggested (42,43) and it is also possible that impairment of calcium homeostasis will affect survival and development of the parasite in the erythrocyte (44). Furthermore, reduced levels of ATP2B4 lead to a reduced volume and MCHC (40,45), and these parameters are also predicted to impact the development of the parasite. However, a knock-out of ATP2B4 in our system did not show a major effect on P. falciparum invasion or on growth, though a tendency towards a reduction in both parameters was observed. A compensatory effect of PMCA1, which represents 20% of erythrocytic Calcium ATPases, could explain the minimal effect of deleting ATP2B4. It is also possible that the natural impact of ATP2B4 variation on P. falciparum growth is relatively subtle and cannot be detected in only one erythrocytic cycle as measured in our assays, but could make the tendency observed important over the time of an infection. Alternatively, as ATP2B4 is widely expressed throughout the body, variations in the gene might affect other aspects of the disease, such as the interaction of infected erythrocytes with endothelial cells or with the brain, as has also been suggested (33).
We further demonstrate the adaptability of this strategy by reprogramming iPS cells from haemoglobinopathy patients. For this we chose one of the traits that is known to confer protection against malaria: thalassemia. Alpha-thalassemia results from a variety of large deletions affecting one or more of the duplicated alpha globin genes and the severity of the disease depends on how many of the four genes are affected. Loss of all 4 α-globin genes, known as α-thalassemia major, can occur in the common South East Asian deletion, leading to the lethal HbBarts hydrops foetalis. Alpha-thalassemia major was chosen for these studies because of the extreme phenotype and also because primary erythrocytes with this genotype are unavailable to perform laboratory assays, thus highlighting the advantages of stem cell technology. Both of our derived reprogrammed cell lines are null for alpha globin, presenting -SEA/-SEA (GN03433) (46) and -SEA/-Fil (GM10796) (47) genotypes. When differentiated, both cell lines showed a significantly reduced ability to support P. falciparum infection, consistent with reported effects of haemoglobinopathies on malaria (48,49). Though several mechanisms have been proposed, it is still unclear how haemoglobin deficiencies impact the parasite and their study is complicated by the variety of genetic changes underlying these traits as well as the difficulty in obtaining samples of primary erythrocytes. It is known that the imbalance in the synthesis of globin chains in alpha and beta thalassemias result in impairment of the assembly of haemoglobin tetramers. This leads to the formation of haemoglobin precipitates (Heinz bodies), which together with the increased hydration occurring in α-thalassemias impair erythrocyte deformability (50). It was shown that erythrocyte deformability is lower in samples of α-thalassemia traits in which 2 alpha globin genes are inactivated and the decrease is much stronger in Haemoglobin H disease in which 3 alpha globin genes are missing. Furthermore, this decrease in deformability was directly corelated to decreasing P. falciparum invasion (51). It is reasonable to predict an even greater deformability defect in the total absence of haemoglobin alpha of the cell lines used here, which is consistent with the dramatic decrease in invasion we observed. Additionally, it was shown that P. falciparum parasites produce significantly lower numbers of merozoites in alpha and beta thalassemia trait cells, which correlates with the MCHC and mean corpuscular volume (MCV) of these cell types (52).
The strategy presented here is based on a universal differentiation protocol that allows the in vitro derivation of erythrocytes from a variety of stem cells lines amenable to the study of host factors involved in malaria infection. Stem cells of any origin can be used, including existing resources of ESC and iPSC lines and also offers the possibility to generate and study novel patient-derived iPS lines. The latter is a novel application of stem cell technology for the study of malaria that presents the only option to study complex genetic traits as well as multiple mutations in their full genomic context. Genome editing can also be applied to introduce or correct specific changes to identify human factors involved in the disease and understand the mechanisms of the impact of genomic variation. The cell lines generated have unlimited proliferation potential and can be stored, thus preserving the genetic characteristics. They also have the potential to be differentiated into a variety of cell types allowing examination of the same genotypes in different stages of the disease. This approach is a powerful tool for the understanding of this disease, circumventing limitations such as availability and access to primary cells with certain traits and complex polymorphisms. A universal differentiation protocol such as we present here greatly increases the versatility and power of this stem cellbased system for a wide range of applications for future malaria research. Figure S1. Protocol for Stem cell differentiation. Stem cells can be reprogrammed or edited and differentiated into erythrocytes and then challenged with fluorescent parasites to assess invasion and growth.

Figure S7. Invasion of blood controls.
Every assay had control wells with labelled blood that were analysed in the same way as in vitro derived erythrocytes (A). Synchronized parasites were used to assess the shift in haemozoin intensity (B), which was also measured in the regular assays (C and D).