Stem Cell Technology Provides Novel Tools to Understand the Impact of Human Variation on Malaria

Stem Cell Technology Provides Novel Tools to Understand the Impact of Human Variation on Malaria | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Stem Cell Technology Provides Novel Tools to Understand the Impact of Human Variation on Malaria Alena Pance, Bee Ling Ng, Kioko Mwikali, Manousos Koutsourakis, and 6 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-1808642/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Plasmodium falciparum parasites have a complex life cycle, but the most clinically relevant stage of the disease is the invasion of erythrocytes and the proliferation of the parasite in the blood. The influence of human genetic traits on malaria has been known for a long time, however understanding the role of the proteins involved is hampered by the anuclear nature of erythrocytes that makes them inaccessible to genetic tools. Here we overcome this limitation with a differentiation protocol to derive erythroid cells in- vitro from a diversity of human stem cells and an adaptation of flow cytometry to detect hemozoin. We combine this strategy with genome editing to show that deletion of basigin ablates invasion while deletion of ATP2B4 has a minor effect and that erythroid cells from reprogrammed patient-derived HbBart α-thalassemia samples poorly support infection. This approach offers vast potential for understanding the impact human polymorphisms on malaria. Biological sciences/Stem cells/Stem-cell differentiation Biological sciences/Microbiology/Parasitology/Parasite biology Biological sciences/Stem cells Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Introduction Malaria is an infectious disease caused by several species of Plasmodium parasites that are transferred between humans by female Anopheline mosquitoes. The parasite life cycle is complex and involves multiple organs in each host, with a wide range of interactions between parasite and host at each step. Nevertheless, the pathology and severe complications of malaria infection in humans result from the blood stages, 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 formation of the parasitophorous vacuole, 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 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 frequently either undeciphered or disputed. This is mainly due to the limitations 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, which makes their long-term culture impossible. Thus, research has almost exclusively relied on clinical samples, with inherent difficulties of donor availability and variability as well as the impact of storage and transport on sample quality which impose limitations on the ability to perform detailed cellular studies. Secondly and perhaps most significantly for mechanistic studies, mature erythrocytes are anucleated and therefore gene editing technologies cannot be applied. Attempts to overcome these limitations have been developed in recent years using a variety of stem cell technologies. siRNA knock-down techniques in Haematopoietic Stem Cells (HSCs) have been used to study the specific role of Glycophorin A (GYPA) ( 12 ) and Basigin (BSG) ( 13 ) as well as to screen more broadly for erythrocyte proteins involved in P. falciparum growth and development ( 14 ). While a significant step forward, the applicability of this approach is curtailed by the restricted availability of HSCs, their limited proliferation capacity and the variable levels of knock-down 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 explore 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 ). One potential 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. Critically, studies using stem cell lines engineered to facilitate differentiation towards erythropoiesis do not offer the possibility of exploring specific genetic characteristics or complex genetic traits in their original genomic context, such as those found in patients or particular human populations. 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 ), could provide a versatile alternative with additional advantages. Stem cells have the potential to differentiate into any cell type, which would allow examination of the same genomic background on all parasite stages. Furthermore, the development of reprogramming techniques that revert terminally differentiated cells to pluripotency ( 21 ) makes it possible to generate iPSCs from any specific individual. In this way, complex genotypes and rare variants, including non-viable mutations, can be brought into the lab and stored for unlimited studies. Genome editing to change or correct the mutations also becomes possible, offering a direct confirmation of their physiological role. In this work we developed a differentiation protocol to drive both ESCs and iPSCs towards erythropoiesis and produce cells that are competent for P. falciparum infection. This approach makes it possible to study a wide range of patient-derived cells simultaneously as well as make use of existing iPS lines, while also allowing the potential to incorporate genome editing of specific host genes and compare their effect on multiple different genomic backgrounds. Our protocol mimics more closely natural development by driving the pluripotent cells to mesoderm first, thus avoiding the commonly used embryoid body formation with the associated cell loss, and leading to a better yield of erythroid cells. To assess invasion of the in-vitro -derived cells, we established an assay to accurately quantify parasitaemia using an adaptation of flow cytometry based on the refractive properties of haemozoin, a pigment produced by malaria parasites after digestion of haemoglobin (( 22 ). These protocols represent versatile tools to explore the impact of host genetic variation on Plasmodium parasites. Understanding the mechanisms of host-parasite interactions at a molecular level may identify new targets for therapeutic intervention. Results Differentiation protocol that allows the in-vitro generation of erythroid cells from a wide variety of human stem cells. The main objective was to establish a stem cell differentiation protocol to generate erythroid cells able to support parasite invasion and growth, in sufficient numbers to allow parasite invasion assays to be performed. With this in mind, we aimed to avoid the widely used embryoid body stage to minimise manipulation and cell loss. Our protocol (Fig. S1), directs adherent pluripotent cells towards the mesoderm path, based on that described by Vallier et al ( 23 ) by exposing them to two steps of specific cytokines. First the cells are exposed to low levels of Activin A while inhibiting Fibroblast Growth Factor 2 (FGF2) to suppress neuroectoderm and then to a combination of Bone Morphogenetic Protein 4 (BMP4), FGF2 and inhibition of Activin A signalling with SB431542 to favour mesoderm differentiation over endoderm fate. Interleukin 3 (IL-3) is added at this stage in order to direct the forming mesoderm towards haematopoiesis. As the cells start differentiating, qRT-PCR analysis shows decline of pluripotency gene expression while mesoderm markers increase (Fig 1A), before induction of the crucial transcription factors involved in driving the myeloid line of haematopoiesis towards megakaryocyte-erythroid progenitors (MEP). The modulation of gene expression is concomitant with a visible morphological change of the cells. Microarray analysis confirmed the transition of the transcriptome from early mesoderm towards haematopoiesis through this stage of the differentiation process (Fig. 1B). Expression of mesoderm specification genes ( eomes, BMP2/4, BMP receptor 1B (BMPR1B), Cripto (CFC1) ) became evident early during the mesoderm phase, peaking through the Meso-Ery transition, while the major drivers of haematopoiesis ( Brachyury (T), Tal1, GATA1and MIXL1, ) peaked later. Based on the increased expression of haematopoietic genes towards the end of this stage of the protocol, the length of this step was set at 8 – 12 days. During this stage the cells spontaneously detach from the plates and can be harvested from the supernatant avoiding potentially damaging trypsinisation. The suspension of detached cells is then guided towards erythropoiesis by exposure to Erythropoietin (EPO) and IL-3 to drive differentiation into erythroblasts. Dexamethasone is added to halt the process at the erythroblast stage and improve the homogeneity of the culture. Cell numbers are increased by expanding the erythroblastic cells with Stem Cell Factor (SCF) and differentiation is completed by removing dexamethasone (Fig. S1). At the end of the differentiation process, analysis of the cells shows expression of the major erythrocytic marker Glycophorin A (GYPA) as well as adult haemoglobins A and B (Fig. 2A). Most cells also express the transferrin receptor (CD71), foetal haemoglobins Gamma and Epsilon are also detected, and approximately 70% stain positively for nucleic acids (Hoechst33342), indicating that the cells still have some form of nucleus or nucleic acid content (nucleus or fragments thereof) (Fig 2A). Comparison with primay erythocytes (Fig. S3A) indicate that the cells generated correspond to immature erythroid cells earlier in the erythropoietic differentiation pathway. Microarray analysis of the transcriptome changes shows induction of major erythrocytic genes including structural proteins (EPB41, SCL4A1), functional proteins such as components of the haeme cycle (PO, CPOS, PPOX, UROS, UROD) and membrane transporters (ATP2B4, ATP2B1) as well as surface markers (GYPC, CD34, CD44, CD99, CD47). It is worth noting that induction of many erythrocytic genes occurs in the last stages of the meso/ery transition and the very early erythroid differentiation (Ery I). As erythropoiesis proceeds, the cells become less metabolically active, start extruding organelles and their RNA rapidly degrades. As a consequence, transcripts for many proteins, such as GYPA and TRFC (CD71) that we identify on the cell surface by flow cytometry (Fig. 2A) are no longer detectable as we see in the microarrays. The full transcriptome changes through the differentiation process are shown in Fig. S2 as well as similar analyses on blood samples for comparison. The versatility of the differentiation protocol was tested using a variety of cell lines of different origin, including human embryonic stem cell (hESC) lines (Shef3 and Shef6), as well as Induced Pluripotent Stem Cell (hIPSC) lines derived from both fibroblasts (RH1, SF2 and K4) and blood (CD3, CD5 and GB1, GB4). The efficacy of the process was assessed by expression of surface markers GYPA and CD71 by flow cytometry (Fig. 3A) and the haemoglobin genes by qRT-PCR (Fig. 3B). Though variations in the differentiation efficiency were observed particularly in the levels of GYPA, all the lines generated erythroid cells expressing these genes (Fig. 3). The overall expression of erythroid genes as revealed by microarray analysis was broadly similar across all the lines tested (Fig. 3C). Malaria parasites use a range of erythrocyte surface proteins as invasion receptors. Plasmodium falciparum in particular is well-known for its ability to use multiple invasion pathways and even switch between them to adapt to host polymorphisms or evade the immune response (6). In this context, we assessed expression of genes reported to be important for invasion of erythrocytes (24) using microarray analysis (Fig. 3D). While some genes (e.g. ATP2B4, CR1 and GYPC) were expressed in all the cell lines used, others (e.g. CD55) showed more variable expression at the transcript level. This highlights the need to assess the cell lines chosen for these types of studies in detail to ensure their suitability. Haemozoin depolarisation can quantify parasitaemia in invasion assays The in-vitro -derived erythroid cells maintain some nucleic acid content, revealed by Hoechst33342 staining (Fig. 2A). So to assess parasite invasion into these cells, our assay resorts to an alternative method to quantify parasitaemia. This approach is based on the detection of haemozoin (Hz) by flow cytometry as it accumulates in the parasite when it metabolises haemoglobin in the invaded cells, using its biophysical property to depolarise light. To assess whether depolarisation caused by Hz reflects parasitaemia in our system, a culture of parasites was labelled with Sybr Green (SG) to verify detection of Hz with parasite quantification (Fig. 4A). The gates were set up with uninfected blood (Fig. 4A top row) and applied to the parasite culture. SG fluorescence (Fig. 4A middle plot) shows several subpopulations of increasing SG intensity, deemed to reflect rings and brighter later stages. The depolarisation capacity (Fig. 4A third plot) showed 8.15% positive events, which corresponded well with the 8.14% of SG-positive cells and the 7.3% manual count of the corresponding Giemsa-stained slide. Examining the depolarising population for SG staining confirmed that 93.8% of this population corresponds to SG-labelled parasites. The non-depolarising population contains mostly uninfected erythrocytes and 1.98% of low intensity SG cells, corresponding to small rings that have not yet accumulated enough Hz to be identified by depolarisation. Sequential dilution of the culture and comparison between depolarisation and SG staining (Fig. 4B) showed good correspondence between depolarisation and SG staining as well as Giemsa slide counting and calculation of the original culture. The detection limit was determined as a 0.05 dilution that corresponds to 0.4% parasitaemia beyond which the quantification is unreliable for both, the depolarisation and SG staining. The invasion assays consist of purified late-stage parasites co-cultured with target cells in a 96 well plate. The target cells are labelled with a membrane dye (DDAO) to distinguish the freshly invaded cells from the purified free parasites and any carry-over erythrocytes. In order to determine the best timing for the invasion assays, a time course of infection was followed (Fig. 5). After eliminating debris and doublets the population of target cells is identified by the membrane staining (Fig. 5A DDAO+) and this population is examined for depolarisation. To confirm that the depolarising events are indeed cells infected with the parasites, the assays were stained with Sybr Green (SG) and the DDAO+ population was also examined for SG signal. A sample of the invasion assay was smeared on a slide and stained with Giemsa to check the stage of the parasites. Parasitaemia can be detected as soon as 2 hours post-invasion, though the increase observed at 6 hours indicates that invasion is still occurring. The difference between Hz and SG quantification 6 hours post-infection shows that accumulation of Hz is still below detection in about half of the parasites. At 24 hours, the levels of parasitaemia are maintained, with detection by Hz reaching similar levels as SG, confirming parasite growth and metabolic activity. Parasitaemia increases at 48 hours and an underestimation of Hz quantification compared to SG indicates that reinvasion has occurred with small parasites appearing in the assays as shown in the Giemsa slides. Our protocol can generate substantial numbers of cells (10-20 million) from a 10 cm plate of pluripotent stem cell cultures. However, this not sufficient to emulate regular erythrocyte cultures at 2.5% haematocrit (approximately 35 million erythrocytes per assay), as each invasion assay includes duplicates or triplicates and a sample of cells alone per time point. In order to do this as well as increase the invasion rates for accurate quantification, we defined one million cells per assay (estimated at approximately 0.13% haematocrit) and compared invasion at these two levels of haematocrit (Fig. 5B). As expected, higher invasion levels were observed at the lower haematocrit and this is more noticeable at the second time point. The similar parasitaemia quantified by Hz and SG shows that the events detected are indeed infected erythrocytes because haemozoin alone does not contain nucleic acids. The high parasitaemias observed at 48 hours post infection suggest some level of re-infection in the cultures, confirmed by the higher SG signal compared to Hz and the presence of small rings in the Giemsa slides. We conclude this level of haematocrit is suitable for assessing differences in invasion capacity between cells of diverse origin. Based on this data, the time points chosen for the assays were 18 hours aiming to detect all invasion events and 42 hours aiming to evaluate development before egress and reinvasion. Stem cell-derived erythroid cells support invasion by Plasmodium falciparum. The accurate quantification of the capacity of different cell lines to support P. falciparum infection (Fig. S4), depends on the effective exclusion of non-invading parasites. For this, lines of 3D7 P. falciparum expressing fluorochromes of different wavelengths were generated ( 25 ) to allow visualisation of the free parasites on the flow cytometry plots (Fig. S5A) and separate them from the labelled cells so that Hz is quantified only in the target cells reflecting the % parasitaemia (Fig. S5). The wide range of fluorescence observed in the parasite population comes from non-fluorescent carry over cells and also from varying levels of fluorochrome expression by the parasites. . All samples are analysed first on side and forward scatter to eliminate debris, then on pulse width to eliminate doublets and select the single cell population (Fig S5B). Single cells are then analysed for fluorescence using the cell label (DFFA in this case) and parasite fluorescence (mCherry in this case) to select the labelled population of cells (Fig S5C). The labelled cell population is then analysed for depolarisation capacity. The analysis is first performed with the uninfected labelled cells to establish the depolarisation gate (Fig. S5C). The invasion assays are analysed in the same way (Fig. S5D), applying the gates established with the controls that allow quantification of the number of labelled erythroid cells containing metabolically active parasites. Overlay of the depolarising population on a plot of non-infected cells and free parasites confirms that the haemozoin-containing events correspond to stem cell-derived erythroid cells infected with the parasite (Fig. S5E). Because the quantification of depolarising events is applied to the whole population of labelled cells, this proportion reflects the parasitaemia of the culture. Since haemozoin is the metabolic product of the parasite’s digestion of haemoglobin ( 26 ), it only accumulates in the food vacuole as a result of parasite metabolism, and therefore its detection reflects live, active parasites in the differentiated erythroid cells. Several fluorochromes and membrane labels were tested to find the combinations leading to the clearest parasitaemia determination and show the versatility of this system. Labelling the cells with the far-red dye DDAO and infecting them with Midori-ishi cyan- or tagBFP-expressing parasites (Fig. S6 and S7) was equally effective at determining parasitaemia in the invasion assays. 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 assays with the in-vitro -generated erythroid cells showed successful invasion upon examination of Giemsa-stained slides (Fig. 6A), and quantification of 50.000 events using our flow cytometry strategy. Erythroid cells differentiated from all nine stem cells lines were effectively invaded by P. falciparum. The parasitaemia ranged between 5 and 8% (Fig. 6B), similar to the levels observed in control assays with labelled blood in regular culture conditions (2.5 %) (Fig. S7A). At 42 hours parasitaemia rose in all cell lines to 7–10%, likely reflecting the growth of small parasites that went undetected at the 18 hour timepoint (Fig. 4B). The blood controls showed a significantly higher increase in parasitaemia at the 42-hour time point ( P <.001) with an average of 20% (Fig. S8A), likely due to the higher cell-to-parasite ratio in these controls. The concomitant increase in Hz intensity with life cycle progression is a useful tool to measure parasite growth ( 26, 27 ). Indeed, synchronised parasite cultures show a clear shift in Hz signal intensity as they progress from rings to schizonts (Fig. S8B), and this is replicated in our bespoke invasion assays (Fig. S8C), albeit a wider 42-hour peak resulting from the characteristics of the assays as explained above. The gate established by the shift in Hz intensity between the ring and schizont stages (M1 in Fig. S8B), was used to quantify the percentage of mature parasites in the cultures (Fig. S8C), which showed an average of 64% (Fig. S8D). Applying the same gate to the assays with the in-vitro -generated erythroid cells, we observed similar levels of mature parasites between 60 and 80% (Fig. 6C). Genome editing of human stem cells reveals a role for specific genes in malaria invasion. The potential to derive edited erythrocytes by introducing targeted modifications in the stem cell lines was tested using CRISPR/Cas9 technology in the control line RH1 (Fig. S9). 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 ( 28 ). The edited clones were genotyped (Fig. S10A) and verified by sequencing (Fig. S10B) 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 by flow cytometry and microarray analysis of the pluripotent and differentiated cells (Fig 7A). When challenged with the parasites, erythroid cells differentiated from two independent Basigin-null cell lines (B5 and C5) showed strongly decreased invasion compared to the parental non-edited RH1 cells (Fig 7B), consistent with the role of Basigin as an essential receptor for P. falciparum ( 13 ). The proportion of mature parasites after 42 hours of culture was similar to the control RH1 cells (Fig. 7B), indicating that the few parasites that invaded the modified cells could achieve some growth. Deletion of ATP2B4 (Fig. 7A) showed a tendency to lower invasion levels compared to RH1 cells, but this decrease was significant for only one clone (Fig 7C). The proportion of mature parasites in the ATP2B4 KO cultures at the 42-hour time point was similar to that observed in the control RH1 line, 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 Figure 8,both thalassaemiccell 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. 8). Discussion This work presents a protocol that effectively differentiates a variety of human stem cell lines towards erythropoiesis generating cells able to support Plasmodium falciparum infection. A total of 9 stem cell lines of diverse origin were studied, showing that while differentiation efficiency does vary 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 ( 29, 30 ) we observed levels of 20-30%, Bearing in mind that cells with positive nucleic acid staining include cells with nuclear fractionation, incomplete nuclear extrusion (Fig. 1B) and remnant nucleic acid content. 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 (Hz) and stored in the food vacuole ( 22 ), becoming a distinct feature of intra-erythrocytic Plasmodium parasites ( 27 ). Comparing parasitaemia quantification by Hz with Sybr Green (SG) DNA staining of the parasites confirms the depolarising events as parasites and the accuracy of the method. Intracellular Hz accumulates at 12 to 18 hours post-infection and both crystal size and number increase with progression of the blood cycle ( 31, 32 ), though the sensitivity of flow cytometry can detect Hz earlier, which can help deciphering effects on early development as shown in Figure 5A. Though, smaller rings can be missed as indicated by the underestimation of Hz+ events compared to SG particularly at 6 hours, depolarisation detection is high and accurate enough to potentially distinguish between invasion and early inhibition in these types of assays. . 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 when for simplicity, routine cultures are used. Therefore, the increase in the number of Hz-positive events at 42 hours is likely due to the maturation of parasites that invaded slightly later and escaped detection at the 18 hour time point, also confirming the viability of the parasites. The use of fluorescent parasites constitutes an additional control for the accurate quantification of parasitaemia, however detection of haemozoin makes it possible to perform this type of studies with any chosen parasite line as demonstrated in Figures 4 and 5. It is useful to use fluorescent control parasites in parallel to non-fluorescent ones to exclude the population of free parasites from quantification.. 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 ). The low levels of invasion detected are likely due to the high sensitivity and additional controls of the quantification strategy used here and consistent with observations in the laboratory and in the field. Natural variants in ATP2B4 have been associated with resistance to malaria in various studies ( 10, 33 ), 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 ( 34 ). As a consequence, expression levels of the protein are 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 ( 35 ). A role of calcium in the invasion process of P. falciparum has been suggested ( 36, 37 ) and it is also possible that impairment of calcium homeostasis affects survival and development of the parasite in the erythrocyte ( 34, 38, 39 ) . However, a knock-out of ATP2B4 in our system did not show a major effect on P. falciparum invasion or growth, though a tendency towards a reduction in both parameters was observed. A compensatory effect of ATP2B1 ( PMCA1 ), which represents 20% of erythrocytic Calcium ATPases, could explain the minimal effect of deleting ATP2B4 . The ubiquitous expression of ATP2B4 throughout the body, could also imply other effects on the disease, such as the interaction of infected erythrocytes with endothelial cells or with the brain, as has been suggested ( 28 ) . We further demonstrate the adaptability of this strategy by reprogramming iPS cells from haemoglobinopathy patients, a trait known to confer protection against malaria. 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 because primary erythrocytes with this genotype are unavailable to perform laboratory assays, thus highlighting the advantages of stem cell technology. Both reprogrammed cell lines are null for alpha globin, presenting –SEA/–SEA (GN03433) ( 40 ) and –SEA/–Fil (GM10796) ( 41 ) genotypes. When differentiated, both cell lines showed a significantly reduced ability to support P. falciparum infection, consistent with reported effects of haemoglobinopathies on malaria ( 42, 43 ). 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 ( 44 ). 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 ( 45 ). 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, correlating with the MCHC and mean corpuscular volume (MCV) of these cell types ( 46 ). This novel application of stem cell technology for the study of malaria represents a new and exciting option to study complex genetic traits as well as multiple mutations in their full genomic context. Genome editing can 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. Though in this work we aimed at developing a strategy with minimal handling that can be potentially scaled up for screening purposes, it can also be easily adapted to more detailed studies by the possibility of labelling receptors of interest or sorting the erythroid cells from the in-vitro -differentiated population and assessing any parasite strain. This versatility allows to examine rare and non-viable genotypes and identify impact on cell differentiation as well as parasite invasion and development. The potential to use existing resources of banked available cell lines as well as reprogramming iPS lines from easily obtainable blood samples from patients or individuals with specific genotypes offers access to the study and preservation of a wide range of genetic characteristics. 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 cell-based system for a wide range of applications and potential identification of therapeutic targets. Methods 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/EE/0253 and the Wellcome Sanger Institute Human Materials and Data Management Committee HMDMC 15/076. The use of human embryonic stem cell lines was approved by the Steering Committee for the UK Stem Cell Bank and for the use of Stem Cell Lines (ref. SCSC11-23) and the Wellcome Sanger Institute Human Materials and Data Management Committee. The Human Embryonic Stem cell lines were obtained from the Centre for Stem Cell Biology, University of Sheffield, Sheffield, UK. The fibroblast lines from haemoglobinopathy patients were obtained from the NIGMS human genetic cell repository of the Coriell Institute for Medical Research, USA. Reprogramming of Induced Pluripotent Stem cell lines Human IPS lines were derived and verified at the Wellcome Sanger Institute as described ( 47-50 ). 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% CO 2 . 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% CO 2 and medium was changed regularly. Pluripotent cells were passaged using 0.5 mM EDTA (Gibco, UK) and 10µM Rock inhibitor. Erythropoietic differentiation Stem cells were taken off feeder cells with 0.5 mM EDTA (GIBCO) and seeded on gelatin-coated 10 cm plates pre-conditioned with MEF medium over-night and cultured in CDM-PVA supplemented with 12 nM hbFGF (Cell guidance systems, UK) and 10 nM hActivin-A (Source Bioscience, UK). CDM-PVA: 50% IMDM (Invitrogen), 50% advanced DMD-F12 (GIBCO) with 1g/l Poly(vinyl alcohol) PVA (SIGMA), Penicillin/Streptomycin 1x (GIBCO), 1-thioglycerol MTG (SIGMA), Insulin-Transferrin-Selenium 1x (ITS, Life Technologies), Cholesterol 1x (SyntheChol, SIGMA). As a first step of differentiation, the cells were taken towards the mesoderm germline: Mesoderm 2 Days : CDM-PVA medium supplemented with 5 nM hActivin-A and 2μM SU5402 (SIGMA) Meso/Ery transition 8-12 Days : CDM-PVA medium supplemented with 20ng/ml bFGF, 10nM IL-3 (SIGMA), 10nM BMP4 (R&D Systems), 5μM SB431542 (SIGMA), 5μM CHIR99021 (Axon, The Netherlands), 5μM LY294002 (SIGMA). During this stage, the detached cells are recovered, washed with PBS and transferred to the erythrocytic differentiation stage, performed in a basic erythrocytic medium (BEM): CellGRO SCGM (CellGenix, Germany) supplemented with ITS, cholesterol, 40ng/ml IGF-1 (Abcam), Penicillin/Streptomycin, 1µM 4-hydroxy 5-methytetrahydrofolate (SIGMA). Ery I 4-5 Days : BEM supplemented with 10ng/ml IL-3, 50ng/ml SCF (Life Technologies), 1µM dexamethasone (SIGMA), 2U/ml EPO (SIGMA), 10ng/ml FLT3 (R&D Systems). Ery II 4-5 Days : BEM supplemented with 50ng/ml SCF, 1µM dexamethasone, 2U/ml EPO Diff Ery minimum 4 Days : BEM supplemented with 2U/ml EPO, 1µM Triiodo-L-Thyronine (T3, SIGMA) 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) HbB 5’-gtctgccgttactgccctgtgg 5’-agcatcaggagtggacagatcc 136 HbA 5’-ggtgctgtctcctgccgac 5’-cctgggcagagccgtggctc 164 HbG 5’-cctgtcctctgcctctgcc 5’-cacagtgcagttcactcagc 140 HbE 5’-gctgccgtcactagcctgtg 5’-gcccaggatggcagagg 144 TAL1 5’-atgccttccctatgttcaccacca 5’-tgaagatacgccgcacaactttgg 108 Brach 5’-acaaagagatgatggaggaacccg 5’-aggatgaggatttgcaggtggaca 110 GATA1 5’-cctctcccaagcttcgtggaac 5’-caggcgttgcataggtagtggc 127 KLF1 5’-ccggacacacaggatgacttcc 5’-ctggtcctcagacttcacgtggag 114 GAPDH 5’-gcctcctgcaccaccaactgc 5’-ggcagtgatggcatggactg 102 OCT4 5’-ctgccgctttgaggctctgcagc 5’-cctgcacgagggtttctgc 134 NANOG 5’-ccagctgtgtgtactcaatgatag 5’-ctctggttctggaaccaggtcttc 123 GYPA 5’-ccactgaggtggcaatgcac 5’-cttcatgagctctaggagtggctgc 120 GYPC 5’-ggacattgtcgtcattgcaggtg 5’-gcctcattggtgtggtacgtgc 117 BSG 5’-ccatgctggtctgcaagtcagag 5’-cacgaagaacctgctctcggag 116 TfR 5’-gggctggcagaaaccttg 5’-cagttggagtgctggagact 145 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. Parasite culture Fluorescent P. falciparum parasites were cultured in complete RPMI medium (GIBCO) at 2.5% haematocrit with O- RBCs sourced from NHSBT, Cambridge. Cultures were maintained at 37°C in malaria gas (1% O 2 , 3% CO 2 and 96% N 2 ). Fluorescent parasites Parasites were engineered to express a variety of fluorochromes for detection at different wavelengths ( 25 ). 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)( 51 ). Staining Procedures DDAO labelling of cells In-vitro -differentiated erythroid cells were centrifuged (1100xg for 4’) and resuspended in 1 ml of Diff Ery medium containing 1 µM DDAO-HS dye for 1 h at 37 0 C. Cells were spun again and resuspended in 1ml DDAO-free medium and incubated for 30 minutes at 37 0 C. After a final spin, cells were resuspended in parasite culture medium at a concentration of 10 6 cells per 75 µl. Giemsa staining of slides Cells, parasite cultures and invasion assays were stained with Giemsa for microscopic examination. Five µl of culture were dropped on a glass slide and spread with a pipette tip or smeared with a glass slide and dried. The slides were fixed with methanol for a few seconds, dried and incubated with 1x Giemsa stain solution for 5-10 minutes. The stain solution as drained away, the slides were washed with tap water and dried before microscopic examination. Sybr Green staining of parasite DNA Fixed culture samples were incubated in 100 µl of a 1X solution (1/10000 dilution) of Sybr Green nucleic acid gel stain 10000X (Invitrogen) for 30 minutes at 37 0 C. 200 µl of PBS were added to each sample before flow cytometry analysis using a 488 nm laser with a 530/40 nm bandpass filter. Invasion Assays In-vitro -differentiated labelled erythroid cells were counted and 75 μl containing 1 million cells were dispensed into a 96 well plate, including a well of cells alone for controls. Further controls included, primary erythrocytes obtained from the blood bank that 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 for 20’, 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: CD71-APC Biolegend #334108 GYPA-PE Southern Biotech #9861-09 BSG-FITC MACS Miltenyi #130-104-489 ATP2B4-FITC LSBio #LS-C446496 A MoFlo flow cytometer (Beckman Coulter, USA) was adapted for detection of laser light depolarisation produced by parasite hemozoin ( 26 ). 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-in-air 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. Invitrogen Bigfoot cell sorter (Thermo Fisher Scientific, Inc.) was also used for the detection of laser light depolarisation produced by parasite hemozoin. The cell sorter is equipped with six spatially separated solid state lasers but only two of the lasers would be turn ON and used in the assay. The laser power for 488 nm was set at 125 mW and 640 nm was set at 100 mW. The laser 488 nm, 640 nm was used to excite Sybr Green and DDAO respectively. Fluorescence emitted from SYBR Green and DDAO was collected using a 507/19 nm and 670/30 nm band pass filter respectively. The instrument is also equipped with default polarisers at the 488nm laser light path which can be switch ‘ON’ during the analysis. This optic set up allows the measurement of normal SSC (488 SSC, Area Linear) and depolarised SSC (488 SSC Polar, Area Linear) (See figure below). A total of 20,000 events was acquired and analysed using FCSExpressv7 (De Novo Software, Inc.). Statistical Analysis Results are presented as means and standard deviation. The reported significance was calculated using a two-tailed unpaired Student’s T test analysis. Genome Editing Genomic modification to ablate the genes chosen for this study was performed in the RH1 cell line, as described previously ( 52 ) and shown schematically in supplementary Fig. S9. Briefly, a CRISPR/Cas9 strategy was used targeting a critical exon in each gene (exon 5 in BSG and exon 11 in ATP2B4) for substitution with a selection cassette as depicted in Fig. S9. The puromycin resistance in the cassette was used to select correctly targeted clones which were examined for damage to the second allele as shown in Fig. S10. Microarray data analysis All microarray datasets were put through “neqc” background correction followed by quantile normalization using the limma R package ( 53 ). 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 GSE63703 gene expression matrix from GREIN (https://shiny.ilincs.org/grein) was used to identify erythrocyte and erythroid progenitor specific genes. Declarations DATA AVAILABILITY All data needed to evaluate the conclusions in the paper are present in the paper and supplementary materials and on request from the corresponding author. AKNOWLEDGEMENTS This work was funded by the Wellcome Trust. The authors wish to thank Aleš Kilpatrick for helping with imaging. AUTHOR CONTRIBUTIONS AP conception and design of the project, performing of experiments and writing of the manuscript; JCR conception of the project and writing of the manuscript; BL setting up and running of flow cytometry approach. KM bioinformatics analysis; HP handling of the data and analysis; MK genome editing of BSG and ATP2B4; CA and FR repogramming of iPSC lines; RM help and guidance with flow cytometric analysis; FL help and advice for stem cell culture and maintenance. COMPETING INTERESTS The authors declare that they have no competing interests. MTA The data can be provided, pending scientific review and completed transfer agreement. Requests should be submitted to Alena Pance. References C. Aurrecoechea et al. , PlasmoDB: a functional genomic database for malaria parasites. Nucleic Acids Res 37 , D539-543 (2009). T. W. Lo et al. , Precise and heritable genome editing in evolutionarily diverse nematodes using TALENs and CRISPR/Cas9 to engineer insertions and deletions. 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Supplementary Files PANCEetalDATA.xlsx data set PANCESupplementaryFigures.pdf Suplementary figures PANCESupplementaryFigures.pdf Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-1808642","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":558721887,"identity":"cd549e2c-d639-4347-b61d-5ffc0d2dc77b","order_by":0,"name":"Alena Pance","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA3ElEQVRIiWNgGAWjYBADOQMEm404LcZALYwNJGlJ3EC0Fv4G3oOfC2ps0rez9z5/+IPBTp5BIi0BrxaJA3zJ0jOOpeXu7Dlu2MzDkGzYIJF2AL81B3gMpHkbDuduuJHG2MzAwJzAIJHegFeH/AEe49+8Df/TDe4/Y2z8wVBPWIvBAR4zoC0HEgxusDE28DAcBmoh4DDDw3xp1jzHkg03nEljnM1jcNywjedZAl4tcsd7D9/mqbGTNzh+jOHjj4pqeX72NAO8WhiYeVDcSVRE8hBWMgpGwSgYBSMcAACKRz3jgok0uQAAAABJRU5ErkJggg==","orcid":"https://orcid.org/0000-0002-9017-2644","institution":"University of Hertfordshire","correspondingAuthor":true,"prefix":"","firstName":"Alena","middleName":"","lastName":"Pance","suffix":""},{"id":558721888,"identity":"0f0048ac-8e6a-4b3c-8313-ea3d18947f3e","order_by":1,"name":"Bee Ling Ng","email":"","orcid":"https://orcid.org/0000-0002-8338-4390","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Bee","middleName":"Ling","lastName":"Ng","suffix":""},{"id":558721889,"identity":"df5f18ce-5887-4ffe-9bac-23989df6e0db","order_by":2,"name":"Kioko Mwikali","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Kioko","middleName":"","lastName":"Mwikali","suffix":""},{"id":558721890,"identity":"f2e421ae-bc18-4aa2-9338-fb79324223b1","order_by":3,"name":"Manousos Koutsourakis","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Manousos","middleName":"","lastName":"Koutsourakis","suffix":""},{"id":558721891,"identity":"039a4598-ae29-4704-9e4d-996c15866119","order_by":4,"name":"Chukwuma Agu","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Chukwuma","middleName":"","lastName":"Agu","suffix":""},{"id":558721892,"identity":"edd88626-3bb4-4920-b16c-8cc5246daad5","order_by":5,"name":"Foad Rouhani","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Foad","middleName":"","lastName":"Rouhani","suffix":""},{"id":558721893,"identity":"36e37a97-c48b-4dff-b6b5-3495b01e17db","order_by":6,"name":"Ruddy Montandon","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Ruddy","middleName":"","lastName":"Montandon","suffix":""},{"id":558721894,"identity":"ac438013-4369-4357-af59-3c6d4cd42050","order_by":7,"name":"Frances Law","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Frances","middleName":"","lastName":"Law","suffix":""},{"id":558721895,"identity":"01008538-4bbc-437a-b2c9-1e2b7e455362","order_by":8,"name":"Hannes Ponstingl","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Hannes","middleName":"","lastName":"Ponstingl","suffix":""},{"id":558721896,"identity":"d7f2a7c0-b83c-444a-b1a0-a71fc03b0e68","order_by":9,"name":"Julian Rayner","email":"","orcid":"https://orcid.org/0000-0002-9835-1014","institution":"University of Cambridge","correspondingAuthor":false,"prefix":"","firstName":"Julian","middleName":"","lastName":"Rayner","suffix":""}],"badges":[],"createdAt":"2022-06-29 16:20:33","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-1808642/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-1808642/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":98432197,"identity":"ab8326bb-ed78-4511-9fd6-479188965e2d","added_by":"auto","created_at":"2025-12-17 16:49:13","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":43917,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eDifferentiation of RH1 stem cells towards mesoderm.\u003c/strong\u003e\u003c/em\u003e\u003cem\u003e \u003c/em\u003e\u003cem\u003e\u003cstrong\u003ea:\u003c/strong\u003e\u003c/em\u003e\u003cem\u003eExpression of pluripotency (POU5f2 (Oct 4), Nanog and c-myc), mesoderm (Sox17, MESP1 and Brach) and haematopoiesis (Mixl1, GATA1, Tal1, KLF1) markersis measured by qRT-PCR (at least 3 biological replicates are represented as a mean and standard deviation). Morphological changes of the differentiating cells are documented by light microscopy (200x: 20x objective, 10x ocular) during regular culture of pluripotent cells (undifferentiated), after 2 days of mesoderm differentiation (mesoderm) and after 8 days of meso/ery differentiation (meso/ery transition) (Cell differentiation is routinely followed by microscopic observation, of which the pictures presented are representative examples). \u003c/em\u003e\u003cem\u003e\u003cstrong\u003eb: \u003c/strong\u003e\u003c/em\u003e\u003cem\u003eMicroarray transcriptomic analysis of Mesoderm and haemato/erythropoiesis driver and signalling genes through the mesoderm steps of the differentiation protocol. Meso: 2 days mesoderm differentiation, Meso/Ery: mesoderm/erythropoiesis transition after 4, 8 and 12 days of differentation (the heatmap is the average of 3 data sets).\u003c/em\u003e\u003c/p\u003e","description":"","filename":"OnlinePANCEFigure1.png","url":"https://assets-eu.researchsquare.com/files/rs-1808642/v1/774afce46c9663fa7a742375.png"},{"id":98181398,"identity":"d1977834-b0bc-4638-8812-7cd648ee2377","added_by":"auto","created_at":"2025-12-15 01:10:37","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":27886,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eErythropoietic stage of RH1 differentiation.\u003c/strong\u003e\u003c/em\u003e\u003cem\u003e \u003c/em\u003e\u003cem\u003e\u003cstrong\u003ea:\u003c/strong\u003e\u003c/em\u003e\u003cem\u003e Expression of the main erythrocyte markers at the end of the differentiation process: Glicophorin A (GYPA) and transferring receptor (CD71), and DNA labelling with Hoechst33342 were assessed by flow cytometry; expression of the haemoglobins was quantified by qRT-PCR (average of 3 samples and SD). Morphology of the cells is shown by Giemsa staining and light microscopy (1000x). \u003c/em\u003e\u003cem\u003e\u003cstrong\u003eb:\u003c/strong\u003e\u003c/em\u003e \u003cem\u003eMicroarray analysis of genes characteristic of haemato/erythropoiesis over time through the mesoderm/erythropoiesis transition (8 and 12 days of differentiation) step, the onset of erythropoiesis (Ery I: 4 days of the first step of erythropoiesis) and final erythrocytic differentiation (Diff Ery: 5 days of final erythropoietic differentiation) steps of the protocol (average of 3 data sets).\u003c/em\u003e\u003c/p\u003e","description":"","filename":"OnlinePANCEFigure2.png","url":"https://assets-eu.researchsquare.com/files/rs-1808642/v1/1d49178698e5c1649cb377da.png"},{"id":98181404,"identity":"6917f7a3-de44-4877-a494-356a5e43ff33","added_by":"auto","created_at":"2025-12-15 01:10:37","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":72356,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eCharacterisation of a variety of stem cell lines differentiated to erythrocytes in-vitro at the end of the differentiation protocol. a:\u003c/strong\u003e\u003c/em\u003e\u003cem\u003eerythrocytic surface markers Glycophorin A (GYPA) and the transferrin receptor (CD71) by flow cytometry (average of 3 replicates, SD). \u003c/em\u003e\u003cem\u003e\u003cstrong\u003eb:\u003c/strong\u003e\u003c/em\u003e\u003cem\u003e expression of the haemoglobins (adult A and B; foetal G and E) assessed by qRT-PCR (average of three replicates, SD). \u003c/em\u003e\u003cem\u003e\u003cstrong\u003ec:\u003c/strong\u003e\u003c/em\u003e\u003cem\u003e comparison of gene expression by microarray analysis at Undifferentiated and final erythropoietic differentiation stage. \u003c/em\u003e\u003cem\u003e\u003cstrong\u003ed:\u003c/strong\u003e\u003c/em\u003e\u003cem\u003eexpression of the main erythrocyte surface receptors important for malaria parasites invasion in differentiated cells detected by microarray (average of 3 data sets).\u003c/em\u003e\u003c/p\u003e","description":"","filename":"OnlinePANCEFigure3.png","url":"https://assets-eu.researchsquare.com/files/rs-1808642/v1/2b3cf9483f23af8601ecebaf.png"},{"id":98430694,"identity":"266b7e79-b172-4f03-bf89-9978dc22122a","added_by":"auto","created_at":"2025-12-17 16:46:03","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":48276,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eHaemozoin-based parasitaemia quantitation. a: \u003c/strong\u003e\u003c/em\u003e\u003cem\u003eFlow cytometry analysis parasite cultures. Uninfected erythrocytes are used to set the gates for defining the depolarisation and Sybr Green boundaries (top row plots). The parasite culture (right plot) is analysed with the set gates for Sybr Green staining (middle plot) and depolarisation (left-hand plot). Sybr Green staining of the depolarising (far right-hand top plot) and non-depolarising events (far right-hand bottom plot). \u003c/em\u003e\u003cem\u003e\u003cstrong\u003eb:\u003c/strong\u003e\u003c/em\u003e\u003cem\u003e sequential dilution of the parasite culture (Giemsa image) comparing parasitaemia quantitation by depolarisation, Sybr Green staining and calculated from manual counting.\u003c/em\u003e\u003c/p\u003e","description":"","filename":"OnlinePANCEFigure4.png","url":"https://assets-eu.researchsquare.com/files/rs-1808642/v1/a6f852d690e07d7965de811c.png"},{"id":98181402,"identity":"88907c82-0ddf-4413-b208-1c1a3d95ba2e","added_by":"auto","created_at":"2025-12-15 01:10:37","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":54673,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eInvasion assay. a: \u003c/strong\u003e\u003c/em\u003e\u003cem\u003etime course of parasitaemia. The purified parasites (Percoll preparation) are mixed with DDAO-stained erythrocytes and incubated for the reported times. Parasitaemia is quantified by Haemozoin depolarisation as well as Sybr Green labelling and Giemsa-stained images of each time point are presented. The overall results are shown as mean and SEM of 3 biological replicates. \u003c/em\u003e\u003cem\u003e\u003cstrong\u003eb: \u003c/strong\u003e\u003c/em\u003e\u003cem\u003eHaematocrit levels: the Haematocrit levels of regular cultures (2.5%) are compared with low levels (0.13%) more amenable to in-vitro-derived cells. Results are presented as mean and SEM of 3 replicates and Giemsa images are shown for the 0.13% haematocrit assays for the time points specified.\u003c/em\u003e\u003c/p\u003e","description":"","filename":"OnlinePANCEFigure5.png","url":"https://assets-eu.researchsquare.com/files/rs-1808642/v1/fe2eb55a9cbbd33b267eb19f.png"},{"id":98181406,"identity":"1b9b70a8-0261-47fd-ae63-27b130101ffb","added_by":"auto","created_at":"2025-12-15 01:10:38","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":35406,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eInvasion of in-vitro-generated erythrocytes with P. falciparum. a:\u003c/strong\u003e\u003c/em\u003e\u003cem\u003e examples of Giemsa staining of invasion assays on slide smears. \u003c/em\u003e\u003cem\u003e\u003cstrong\u003eb:\u003c/strong\u003e\u003c/em\u003e\u003cem\u003equantification of invasion by flow cytometry of a variety of differentiated cell lines at two time points of culture, 18h (invasion) and 42 hours (development) (results are shown as mean of at least 3 replicates and SD). \u003c/em\u003e\u003cem\u003e\u003cstrong\u003ec:\u003c/strong\u003e\u003c/em\u003e\u003cem\u003equantification of mature parasites percentage at 42 hours (mean of at least 3 replicates, SD).\u003c/em\u003e\u003c/p\u003e","description":"","filename":"OnlinePANCEFigure6.png","url":"https://assets-eu.researchsquare.com/files/rs-1808642/v1/5522d926809ce140d57411b7.png"},{"id":98181407,"identity":"976583ab-a7db-4056-9c67-6a66392c2b95","added_by":"auto","created_at":"2025-12-15 01:10:38","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":32323,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eGene editing in stem cells to study specific genes: Basigin (right) and ATP2B4 (left).\u003c/strong\u003e\u003c/em\u003e\u003cem\u003e Each gene was deleted in the RH1 cell line using CRISPR/Cas9 and clones were isolated. \u003c/em\u003e\u003cem\u003e\u003cstrong\u003ea:\u003c/strong\u003e\u003c/em\u003e\u003cem\u003e expression of the protein corresponding to the deleted gene was measured on the cell surface with specific antibodies by flow cytometry (representative example of 3 replicates) and confirmed by microarrays of the cell lines transcriptomes in undifferentiated and differentiated states (average and SD from 3 data sets). \u003c/em\u003e\u003cem\u003e\u003cstrong\u003eb:\u003c/strong\u003e\u003c/em\u003e\u003cem\u003einvasion of the parental line RH1 (CT) and the Basigin KO clones B5 and C5 ** P\u0026lt;.001 and percentage of mature parasites at 42 hours (right two panels) and invasion of the parental RH1 line (CT) and ATP2B4 KO clones E6 and F5 * P\u0026lt;.05 and percentage of mature parasites at 42 hours (left two panels). (Dot plots represent individual replicates with average and SD)\u003c/em\u003e\u003c/p\u003e","description":"","filename":"OnlinePANCEFigure7.png","url":"https://assets-eu.researchsquare.com/files/rs-1808642/v1/ecff2ba12bd2d6fa411c1fab.png"},{"id":98181405,"identity":"7d9f7e83-78d2-455a-9443-eaed6597f3f1","added_by":"auto","created_at":"2025-12-15 01:10:37","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":22496,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eReprogramming of IPS cells from patients with α-thalassemia haemoglobinopathy. \u003c/strong\u003e\u003c/em\u003e\u003cem\u003eFibroblasts from α-thalassemia major samples were reprogrammed to IPS cell lines (EM and FJ) that were differentiated to erythroid cells and exposed to P. falciparum to assess invasion in parallel to control cell lines (CT) and percentage of mature parasites at 42 hours (results are presented as mean and SD of at least 3 independent experiments) * P\u0026lt;.05; ** P\u0026lt;.005.\u003c/em\u003e\u003c/p\u003e","description":"","filename":"OnlinePANCEFigure8.png","url":"https://assets-eu.researchsquare.com/files/rs-1808642/v1/830867b26e2017771e8bdca0.png"},{"id":98623011,"identity":"647ecf5e-7f9d-43fe-b42e-275e4da58eb1","added_by":"auto","created_at":"2025-12-19 17:04:02","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1969859,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-1808642/v1/8036fddf-0d44-4a7f-a4d6-637f52d48e9c.pdf"},{"id":98432238,"identity":"2d9be3dd-2c58-436f-8388-feb951993cb9","added_by":"auto","created_at":"2025-12-17 16:49:16","extension":"xlsx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":35903,"visible":true,"origin":"","legend":"data set","description":"","filename":"PANCEetalDATA.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-1808642/v1/72d3192284337c199d55a416.xlsx"},{"id":98431429,"identity":"dd6db99f-6fb9-4d7c-8715-2653c291fbd5","added_by":"auto","created_at":"2025-12-17 16:47:40","extension":"pdf","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":1286667,"visible":true,"origin":"","legend":"Suplementary figures","description":"","filename":"PANCESupplementaryFigures.pdf","url":"https://assets-eu.researchsquare.com/files/rs-1808642/v1/08c735841418f7cd4446bfc7.pdf"},{"id":98181408,"identity":"9ca0d98b-ac75-4c33-b6fc-56a62d11a3c3","added_by":"auto","created_at":"2025-12-15 01:10:38","extension":"pdf","order_by":10,"title":"","display":"","copyAsset":false,"role":"supplement","size":1286667,"visible":true,"origin":"","legend":"","description":"","filename":"PANCESupplementaryFigures.pdf","url":"https://assets-eu.researchsquare.com/files/rs-1808642/v1/7827457003f99e24ab0f1710.pdf"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e Competing Interest.","formattedTitle":"\u003cp\u003eStem Cell Technology Provides Novel Tools to Understand the Impact of Human Variation on Malaria\u003c/p\u003e","fulltext":[{"header":"Introduction","content":"\u003cp\u003eMalaria is an infectious disease caused by several species of \u003cem\u003ePlasmodium\u003c/em\u003e parasites that are transferred between humans by female Anopheline mosquitoes. The parasite life cycle is complex and involves multiple organs in each host, with a wide range of interactions between parasite and host at each step. Nevertheless, the pathology and severe complications of malaria infection in humans result from the blood stages, during which parasites invade and develop inside erythrocytes. The completion of reference genomes (\u003cem\u003e1\u003c/em\u003e), the development of genome editing technologies (\u003cem\u003e2, 3\u003c/em\u003e) and their adaptation to the parasite (\u003cem\u003e4, 5\u003c/em\u003e) 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 (\u003cem\u003e6\u003c/em\u003e), as well as the formation of the parasitophorous vacuole, remodelling of the erythrocyte (\u003cem\u003e7, 8\u003c/em\u003e), 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 \u003cem\u003ePlasmodium\u003c/em\u003e \u003cem\u003efalciparum\u003c/em\u003e, the most virulent species affecting humans (\u003cem\u003e9, 10\u003c/em\u003e). 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 (\u003cem\u003e11\u003c/em\u003e). However, despite population studies providing compelling evidence for protective effects of multiple human protein variants, the molecular mechanisms of these effects are frequently either undeciphered or disputed. This is mainly due to the limitations in accessing primary cell samples, and the technical challenges of reproducing variants \u003cem\u003ein-vitro\u003c/em\u003e in order to perform tightly controlled cellular studies.\u003c/p\u003e\n\u003cp\u003eThere 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, which makes their long-term culture impossible. Thus, research has almost exclusively relied on clinical samples, with inherent difficulties of donor availability and variability as well as the impact of storage and transport on sample quality which impose limitations on the ability to perform detailed cellular studies. Secondly and perhaps most significantly for mechanistic studies, mature erythrocytes are anucleated and therefore gene editing technologies cannot be applied. Attempts to overcome these limitations have been developed in recent years using a variety of stem cell technologies. siRNA knock-down techniques in Haematopoietic Stem Cells (HSCs) have been used to study the specific role of Glycophorin A (GYPA) (\u003cem\u003e12\u003c/em\u003e) and Basigin (BSG) (\u003cem\u003e13\u003c/em\u003e) as well as to screen more broadly for erythrocyte proteins involved in \u003cem\u003eP. falciparum\u0026nbsp;\u003c/em\u003egrowth and development (\u003cem\u003e14\u003c/em\u003e). While a significant step forward, the applicability of this approach is curtailed by the restricted availability of HSCs, their limited proliferation capacity and the variable levels of knock-down that can be achieved. More recently, an immortalised adult erythroblast line able to proliferate and differentiate \u003cem\u003ein-vitro\u003c/em\u003e, was established by transformation of erythroid progenitor cells with the human papilloma virus HPV16-dervied proteins HPV16-E6/E7 (\u003cem\u003e15\u003c/em\u003e). One such line (BEL-A) was combined with genome editing technologies to explore the mechanisms of Basigin involvement in \u003cem\u003eP. \u0026nbsp;falciparum\u003c/em\u003e invasion (\u003cem\u003e16\u003c/em\u003e). This approach was also applied to peripheral blood samples and shown to generate cells permissive to \u003cem\u003eP. falciparum\u003c/em\u003e and \u003cem\u003eP. vivax\u003c/em\u003e invasion and amenable to genome editing studies (\u003cem\u003e17\u003c/em\u003e). One potential 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. Critically, studies using stem cell lines engineered to facilitate differentiation towards erythropoiesis do not offer the possibility of exploring specific genetic characteristics or complex genetic traits in their original genomic context, such as those found in patients or particular human populations.\u003c/p\u003e\n\u003cp\u003eOther approaches, such as the use of established Embryonic and induced Pluripotent Stem Cell lines (ESCs and iPSCs) that can be cultured (\u003cem\u003e18\u003c/em\u003e) and genetically manipulated (\u003cem\u003e19\u003c/em\u003e) while maintaining their pluripotency (\u003cem\u003e20\u003c/em\u003e), could provide a versatile alternative with additional advantages. Stem cells have the potential to differentiate into any cell type, which would allow examination of the same genomic background on all parasite stages. Furthermore, the development of reprogramming techniques that revert terminally differentiated cells to pluripotency (\u003cem\u003e21\u003c/em\u003e) makes it possible to generate iPSCs from any specific individual. In this way, complex genotypes and rare variants, including non-viable mutations, can be brought into the lab and stored for unlimited studies. Genome editing to change or correct the mutations also becomes possible, offering a direct confirmation of their physiological role.\u003c/p\u003e\n\u003cp\u003eIn this work we developed a differentiation protocol to drive both ESCs and iPSCs towards erythropoiesis and produce cells that are competent for \u003cem\u003eP. falciparum\u003c/em\u003e infection. This approach makes it possible to study a wide range of patient-derived cells simultaneously as well as make use of existing iPS lines, while also allowing the potential to incorporate genome editing of specific host genes and compare their effect on multiple different genomic backgrounds. Our protocol mimics more closely natural development by driving the pluripotent cells to mesoderm first, thus avoiding the commonly used embryoid body formation with the associated cell loss, and leading to a better yield of erythroid cells. To assess invasion of the \u003cem\u003ein-vitro\u003c/em\u003e-derived cells, we established an assay to accurately quantify parasitaemia using an adaptation of flow cytometry based on the refractive properties of haemozoin, a pigment produced by malaria parasites after digestion of haemoglobin ((\u003cem\u003e22\u003c/em\u003e). These protocols represent versatile tools to explore the impact of host genetic variation on \u003cem\u003ePlasmodium\u0026nbsp;\u003c/em\u003eparasites. Understanding the mechanisms of host-parasite interactions at a molecular level may identify new targets for therapeutic intervention.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003eDifferentiation protocol that allows the \u003cem\u003ein-vitro\u003c/em\u003e generation of erythroid cells from a wide variety of human stem cells.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe main objective was to establish a stem cell differentiation protocol to generate erythroid cells able to support parasite invasion and growth, in sufficient numbers to allow parasite invasion assays to be performed. With this in mind, we aimed to avoid the widely used embryoid body stage to minimise manipulation and cell loss.\u0026nbsp;Our protocol (Fig. S1), directs adherent pluripotent cells towards the mesoderm path, based on that described by Vallier et al (\u003cem\u003e23\u003c/em\u003e) by exposing them to two steps of specific cytokines. First the cells are exposed to low levels of Activin A while inhibiting Fibroblast Growth Factor 2 (FGF2) to suppress neuroectoderm and then to a combination of Bone Morphogenetic Protein 4 (BMP4), FGF2 and inhibition of Activin A signalling with SB431542 to favour mesoderm differentiation over endoderm fate. Interleukin 3 (IL-3) is added at this stage in order to direct the forming mesoderm towards haematopoiesis. As the cells start differentiating, qRT-PCR analysis shows decline of pluripotency gene expression while mesoderm markers increase (Fig 1A), before induction of the crucial transcription factors involved in driving the myeloid line of haematopoiesis towards megakaryocyte-erythroid progenitors (MEP). The modulation of gene expression is concomitant with a visible morphological change of the cells.\u003c/p\u003e\n\u003cp\u003eMicroarray analysis confirmed the transition of the transcriptome from early mesoderm towards haematopoiesis through this stage of the differentiation process (Fig. 1B). Expression of mesoderm specification genes (\u003cem\u003eeomes, BMP2/4, BMP receptor 1B (BMPR1B), Cripto (CFC1)\u003c/em\u003e) became evident early during the mesoderm phase, peaking through the Meso-Ery transition, while the major drivers of haematopoiesis (\u003cem\u003eBrachyury (T), Tal1, GATA1and MIXL1,\u003c/em\u003e) peaked later. Based on the increased expression of haematopoietic genes towards the end of this stage of the protocol, the length of this step was set at 8 \u0026ndash; 12 days. During this stage the cells spontaneously detach from the plates and can be harvested from the supernatant avoiding potentially damaging trypsinisation.\u003c/p\u003e\n\u003cp\u003eThe suspension of detached cells is then guided towards erythropoiesis by exposure to Erythropoietin (EPO) and IL-3 to drive differentiation into erythroblasts. Dexamethasone is added to halt the process at the erythroblast stage and improve the homogeneity of the culture. Cell numbers are increased by expanding the erythroblastic cells with Stem Cell Factor (SCF) and differentiation is completed by removing dexamethasone (Fig. S1). At the end of the differentiation process, analysis of the cells shows expression of the major erythrocytic marker Glycophorin A (GYPA) as well as adult haemoglobins A and B (Fig. 2A). Most cells also express the transferrin receptor (CD71), foetal haemoglobins Gamma and Epsilon are also detected, and approximately 70% stain positively for nucleic acids (Hoechst33342), indicating that the cells still have some form of nucleus or nucleic acid content (nucleus or fragments thereof) (Fig 2A). Comparison with primay erythocytes (Fig. S3A) indicate that the cells generated correspond to immature erythroid cells earlier in the erythropoietic differentiation pathway.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eMicroarray analysis of the transcriptome changes shows induction of major erythrocytic genes including structural proteins (EPB41, SCL4A1), functional proteins such as components of the haeme cycle (PO, CPOS, PPOX, UROS, UROD) and membrane transporters (ATP2B4, ATP2B1) as well as surface markers (GYPC, CD34, CD44, CD99, CD47). It is worth noting that induction of many erythrocytic genes occurs in the last stages of the meso/ery transition and the very early erythroid differentiation (Ery I). As erythropoiesis proceeds, the cells become less metabolically active, start extruding organelles and their RNA rapidly degrades. As a consequence, transcripts for many proteins, such as GYPA and TRFC (CD71) that we identify on the cell surface by flow cytometry (Fig. 2A) are no longer detectable as we see in the microarrays. The full transcriptome changes through the differentiation process are shown in Fig. S2 as well as similar analyses on blood samples for comparison.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe versatility of the differentiation protocol was tested using a variety of cell lines of different origin, including human embryonic stem cell (hESC) lines (Shef3 and Shef6), as well as Induced Pluripotent Stem Cell (hIPSC) lines derived from both fibroblasts (RH1, SF2 and K4) and blood (CD3, CD5 and GB1, GB4). The efficacy of the process was assessed by expression of surface markers GYPA and CD71 by flow cytometry (Fig. 3A) and the haemoglobin genes by qRT-PCR (Fig. 3B). Though variations in the differentiation efficiency were observed particularly in the levels of GYPA, all the lines generated erythroid cells expressing these genes (Fig. 3). The overall expression of erythroid genes as revealed by microarray analysis was broadly similar across all the lines tested (Fig. 3C).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eMalaria parasites use a range of erythrocyte surface proteins as invasion receptors. Plasmodium falciparum in particular is well-known for its ability to use multiple invasion pathways and even switch between them to adapt to host polymorphisms or evade the immune response (6). In this context, we assessed expression of genes reported to be important for invasion of erythrocytes (24) using microarray analysis (Fig. 3D). While some genes (e.g. ATP2B4, CR1 and GYPC) were expressed in all the cell lines used, others (e.g. CD55) showed more variable expression at the transcript level. This highlights the need to assess the cell lines chosen for these types of studies in detail to ensure their suitability.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eHaemozoin depolarisation can quantify parasitaemia in invasion assays\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe \u003cem\u003ein-vitro\u003c/em\u003e-derived erythroid cells maintain some nucleic acid content, revealed by Hoechst33342 staining (Fig. 2A). So to assess parasite invasion into these cells, our assay resorts to an alternative method to quantify parasitaemia. This approach is based on the detection of haemozoin (Hz) by flow cytometry as it accumulates in the parasite when it metabolises haemoglobin in the invaded cells, using its biophysical property to depolarise light. To assess whether depolarisation caused by Hz reflects parasitaemia in our system, a culture of parasites was labelled with Sybr Green (SG) to verify detection of Hz with parasite quantification (Fig. 4A). The gates were set up with uninfected blood (Fig. 4A top row) and applied to the parasite culture. SG fluorescence (Fig. 4A middle plot) shows several subpopulations of increasing SG intensity, deemed to reflect rings and brighter later stages. The depolarisation capacity (Fig. 4A third plot) showed 8.15% positive events, which corresponded well with the 8.14% of SG-positive cells and the 7.3% manual count of the corresponding Giemsa-stained slide. Examining the depolarising population for SG staining confirmed that 93.8% of this population corresponds to SG-labelled parasites. The non-depolarising population contains mostly uninfected erythrocytes and 1.98% of low intensity SG cells, corresponding to small rings that have not yet accumulated enough Hz to be identified by depolarisation.\u003c/p\u003e\n\u003cp\u003eSequential dilution of the culture and comparison between depolarisation and SG staining (Fig. 4B) showed good correspondence between depolarisation and SG staining as well as Giemsa slide counting and calculation of the original culture. The detection limit was determined as a 0.05 dilution that corresponds to 0.4% parasitaemia beyond which the quantification is unreliable for both, the depolarisation and SG staining.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe invasion assays consist of purified late-stage parasites co-cultured with target cells in a 96 well plate. The target cells are labelled with a membrane dye (DDAO) to distinguish the freshly invaded cells from the purified free parasites and any carry-over erythrocytes. In order to determine the best timing for the invasion assays, a time course of infection was followed (Fig. 5). After eliminating debris and doublets the population of target cells is identified by the membrane staining (Fig. 5A DDAO+) and this population is examined for depolarisation. To confirm that the depolarising events are indeed cells infected with the parasites, the assays were stained with Sybr Green (SG) and the DDAO+ population was also examined for SG signal. A sample of the invasion assay was smeared on a slide and stained with Giemsa to check the stage of the parasites. Parasitaemia can be detected as soon as 2 hours post-invasion, though the increase observed at 6 hours indicates that invasion is still occurring. The difference between Hz and SG quantification 6 hours post-infection shows that accumulation of Hz is still below detection in about half of the parasites. At 24 hours, the levels of parasitaemia are maintained, with detection by Hz reaching similar levels as SG, confirming parasite growth and metabolic activity. Parasitaemia increases at 48 hours and an underestimation of Hz quantification compared to SG indicates that reinvasion has occurred with small parasites appearing in the assays as shown in the Giemsa slides.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eOur protocol can generate substantial numbers of cells (10-20 million) from a 10 cm plate of pluripotent stem cell cultures. However, this not sufficient to emulate regular erythrocyte cultures at 2.5% haematocrit (approximately 35 million erythrocytes per assay), as each invasion assay includes duplicates or triplicates and a sample of cells alone per time point. In order to do this as well as increase the invasion rates for accurate quantification, we defined one million cells per assay (estimated at approximately 0.13% haematocrit) and compared invasion at these two levels of haematocrit (Fig. 5B). As expected, higher invasion levels were observed at the lower haematocrit and this is more noticeable at the second time point. The similar parasitaemia quantified by Hz and SG shows that the events detected are indeed infected erythrocytes because haemozoin alone does not contain nucleic acids. The high parasitaemias observed at 48 hours post infection suggest some level of re-infection in the cultures, confirmed by the higher SG signal compared to Hz and the presence of small rings in the Giemsa slides. We conclude this level of haematocrit is suitable for assessing differences in invasion capacity between cells of diverse origin.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eBased on this data, the time points chosen for the assays were 18 hours aiming to detect all invasion events and 42 hours aiming to evaluate development before egress and reinvasion.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStem cell-derived erythroid cells support invasion by \u003cem\u003ePlasmodium falciparum.\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe accurate quantification of the capacity of different cell lines to support \u003cem\u003eP. falciparum\u003c/em\u003e infection (Fig. S4), depends on the effective exclusion of non-invading parasites. For this, lines of 3D7 \u003cem\u003eP. falciparum\u003c/em\u003e expressing fluorochromes of different wavelengths were generated (\u003cem\u003e25\u003c/em\u003e) to allow visualisation of the free parasites on the flow cytometry plots (Fig. S5A) and separate them from the labelled cells so that Hz is quantified only in the target cells reflecting the % parasitaemia (Fig. S5). The wide range of fluorescence observed in the parasite population comes from non-fluorescent carry over cells and also from varying levels of fluorochrome expression by the parasites.\u003c/p\u003e\n\u003cp\u003e.\u003c/p\u003e\n\u003cp\u003eAll samples are analysed first on side and forward scatter to eliminate debris, then on pulse width to eliminate doublets and select the single cell population (Fig S5B). Single cells are then analysed for fluorescence using the cell label (DFFA in this case) and parasite fluorescence (mCherry in this case) to select the labelled population of cells (Fig S5C). The labelled cell population is then analysed for depolarisation capacity. The analysis is first performed with the uninfected labelled cells to establish the depolarisation gate (Fig. S5C). The invasion assays are analysed in the same way (Fig. S5D), applying the gates established with the controls that allow quantification of the number of labelled erythroid cells containing metabolically active parasites. Overlay of the depolarising population on a plot of non-infected cells and free parasites confirms that the haemozoin-containing events correspond to stem cell-derived erythroid cells infected with the parasite (Fig. S5E). Because the quantification of depolarising events is applied to the whole population of labelled cells, this proportion reflects the parasitaemia of the culture. Since haemozoin is the metabolic product of the parasite\u0026rsquo;s digestion of haemoglobin (\u003cem\u003e26\u003c/em\u003e), it only accumulates in the food vacuole as a result of parasite metabolism, and therefore its detection reflects live, active parasites in the differentiated erythroid cells.\u003c/p\u003e\n\u003cp\u003eSeveral fluorochromes and membrane labels were tested to find the combinations leading to the clearest parasitaemia determination and show the versatility of this system. Labelling the cells with the far-red dye DDAO and infecting them with Midori-ishi cyan- or tagBFP-expressing parasites (Fig. S6 and S7) was equally effective at determining parasitaemia in the invasion assays. 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.\u003c/p\u003e\n\u003cp\u003eInvasion assays with the \u003cem\u003ein-vitro\u003c/em\u003e-generated erythroid cells showed successful invasion upon examination of Giemsa-stained slides (Fig. 6A), and quantification of 50.000 events using our flow cytometry strategy. Erythroid cells differentiated from all nine stem cells lines were effectively invaded by \u003cem\u003eP. falciparum.\u0026nbsp;\u003c/em\u003eThe parasitaemia ranged between 5 and 8% (Fig. 6B), similar to the levels observed in control assays with labelled blood in regular culture conditions (2.5 \u0026nbsp;%) (Fig. S7A). At 42 hours parasitaemia rose in all cell lines to 7\u0026ndash;10%, likely reflecting the growth of small parasites that went undetected at the 18 hour timepoint (Fig. 4B). The blood controls showed a significantly higher increase in parasitaemia at the 42-hour time point (\u003cem\u003eP\u003c/em\u003e\u0026lt;.001) with an average of 20% (Fig. S8A), likely due to the higher cell-to-parasite ratio in these controls.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe concomitant increase in Hz intensity with life cycle progression is a useful tool to measure parasite growth (\u003cem\u003e26, 27\u003c/em\u003e). Indeed, synchronised parasite cultures show a clear shift in Hz signal intensity as they progress from rings to schizonts (Fig. S8B), and this is replicated in our bespoke invasion assays (Fig. S8C), albeit a wider 42-hour peak resulting from the characteristics of the assays as explained above. The gate established by the shift in Hz intensity between the ring and schizont stages (M1 in Fig. S8B), was used to quantify the percentage of mature parasites in the cultures (Fig. S8C), which showed an average of 64% (Fig. S8D). Applying the same gate to the assays with the \u003cem\u003ein-vitro\u003c/em\u003e-generated erythroid cells, we observed similar levels of mature parasites between 60 and 80% (Fig. 6C).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eGenome editing of human stem cells reveals a role for specific genes in malaria invasion.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe potential to derive edited erythrocytes by introducing targeted modifications in the stem cell lines was tested using CRISPR/Cas9 technology in the control line RH1 (Fig. S9). Two target genes were chosen , Basigin (\u003cem\u003eBSG\u003c/em\u003e, CD147) which is known to be a universal receptor for \u003cem\u003eP. falciparum\u003c/em\u003e invasion (\u003cem\u003e13\u003c/em\u003e) as a proof of principle, and \u003cem\u003eATP2B4\u003c/em\u003e (\u003cem\u003ePMCA4\u003c/em\u003e) since natural variation in this gene has been correlated to resistance to severe malaria (\u003cem\u003e28\u003c/em\u003e). The edited clones were genotyped (Fig. S10A) and verified by sequencing (Fig. S10B) 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 by flow cytometry and microarray analysis of the pluripotent and differentiated cells (Fig 7A).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eWhen challenged with the parasites, erythroid cells differentiated from two independent Basigin-null cell lines (B5 and C5) showed strongly decreased invasion compared to the parental non-edited RH1 cells (Fig 7B), consistent with the role of Basigin as an essential receptor for \u003cem\u003eP. falciparum\u003c/em\u003e (\u003cem\u003e13\u003c/em\u003e). The proportion of mature parasites after 42 hours of culture was similar to the control RH1 cells (Fig. 7B), indicating that the few parasites that invaded the modified cells could achieve some growth. Deletion of ATP2B4 (Fig. 7A) showed a tendency to lower invasion levels compared to RH1 cells, but this decrease was significant for only one clone (Fig 7C). The proportion of mature parasites in the ATP2B4 KO cultures at the 42-hour time point was similar to that observed in the control RH1 line, indicating that disruption of this gene does not have a major effect on the development of the parasite.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eReprogramming IPS lines from haemoglobinopathy patients shows the versatility of this system.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFibroblast lines isolated from patients with \u0026alpha;-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 Figure 8,both thalassaemiccell 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. 8).\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThis work presents a protocol that effectively differentiates a variety of human stem cell lines towards erythropoiesis generating cells \u0026nbsp;able to support \u003cem\u003ePlasmodium falciparum\u0026nbsp;\u003c/em\u003einfection. A total of 9 stem cell lines of diverse origin were studied, showing that while differentiation efficiency does vary 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 \u003cem\u003ein-vitro\u003c/em\u003e (\u003cem\u003e29, 30\u003c/em\u003e) we observed levels of 20-30%, Bearing in mind that cells with positive nucleic acid staining include cells with nuclear fractionation, incomplete nuclear extrusion (Fig. 1B) and remnant nucleic acid content. Importantly, stem cells differentiated with this protocol are capable of supporting invasion by \u003cem\u003eP. falciparum\u003c/em\u003e without the need to sort or purify the differentiated cells.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eDuring the blood cycle, haemoglobin is the main source of amino acids for the parasite\u0026rsquo;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: \u0026beta;-hematin or haemozoin (Hz) and stored in the food vacuole (\u003cem\u003e22\u003c/em\u003e), becoming a distinct feature of intra-erythrocytic Plasmodium parasites (\u003cem\u003e27\u003c/em\u003e). Comparing parasitaemia quantification by Hz with Sybr Green (SG) DNA staining of the parasites confirms the depolarising events as parasites and the accuracy of the method. Intracellular Hz accumulates at 12 to 18 hours post-infection and both crystal size and number increase with progression of the blood cycle (\u003cem\u003e31, 32\u003c/em\u003e), though the sensitivity of flow cytometry can detect Hz earlier, which can help deciphering effects on early development as shown in Figure 5A. Though, smaller rings can be missed as indicated by the underestimation of Hz+ events compared to SG particularly at 6 hours, depolarisation detection is high and accurate enough to potentially distinguish between invasion and early inhibition in these types of assays. . 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 when for simplicity, routine cultures are used. Therefore, the increase in the number of Hz-positive events at 42 hours is likely due to the maturation of parasites that invaded slightly later and escaped detection at the 18 hour time point, also confirming the viability of the parasites. The use of fluorescent parasites constitutes an additional control for the accurate quantification of parasitaemia, however detection of haemozoin makes it possible to perform this type of studies with any chosen parasite line as demonstrated in Figures 4 and 5. It is useful to use fluorescent control parasites in parallel to non-fluorescent ones to exclude the population of free parasites from quantification..\u003c/p\u003e\n\u003cp\u003eThe successful manipulation of genes implicated in malaria infection was demonstrated by deletion of \u003cem\u003eBasigin\u003c/em\u003e, which resulted in a dramatic decrease of infection as expected given the known role of this protein in invasion (\u003cem\u003e13\u003c/em\u003e). The low levels of invasion detected are likely due to the high sensitivity and additional controls of the quantification strategy used here and consistent with observations in the laboratory and in the field. Natural variants in ATP2B4 have been associated with resistance to malaria in various studies (\u003cem\u003e10, 33\u003c/em\u003e), 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 (\u003cem\u003e34\u003c/em\u003e). As a consequence, expression levels of the protein are 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 (\u003cem\u003e35\u003c/em\u003e). A role of calcium in the invasion process of \u003cem\u003eP. falciparum\u003c/em\u003e has been suggested (\u003cem\u003e36, 37\u003c/em\u003e) and it is also possible that impairment of calcium homeostasis affects survival and development of the parasite in the erythrocyte (\u003cem\u003e34, 38, 39\u003c/em\u003e)\u003cstrong\u003e. However, a knock-out of \u003cem\u003eATP2B4\u003c/em\u003e in our system did not show a major effect on \u003cem\u003eP. falciparum\u0026nbsp;\u003c/em\u003einvasion or growth, though a tendency towards a reduction in both parameters was observed. A compensatory effect of ATP2B1 (\u003cem\u003ePMCA1\u003c/em\u003e), which represents 20% of erythrocytic Calcium ATPases, could explain the minimal effect of deleting \u003cem\u003eATP2B4\u003c/em\u003e. The ubiquitous expression of \u003cem\u003eATP2B4\u003c/em\u003e throughout the body, could also imply other effects on the disease, such as the interaction of infected erythrocytes with endothelial cells or with the brain, as has been suggested\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003e(\u003cem\u003e28\u003c/em\u003e)\u003c/strong\u003e\u003cstrong\u003e.\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eWe further demonstrate the adaptability of this strategy by reprogramming iPS cells from haemoglobinopathy patients, a trait known to confer protection against malaria.\u0026nbsp;\u003c/strong\u003eAlpha-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 \u0026alpha;-globin genes, known as \u0026alpha;-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 because primary erythrocytes with this genotype are unavailable to perform laboratory assays, thus highlighting the advantages of stem cell technology. Both reprogrammed cell lines are null for alpha globin, presenting \u0026ndash;SEA/\u0026ndash;SEA (GN03433) (\u003cem\u003e40\u003c/em\u003e)\u003cstrong\u003e\u0026nbsp;and \u0026ndash;SEA/\u0026ndash;Fil (GM10796)\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003e(\u003cem\u003e41\u003c/em\u003e)\u003c/strong\u003e genotypes. When differentiated, both cell lines showed a significantly reduced ability to support \u003cem\u003eP. falciparum\u003c/em\u003e infection, consistent with reported effects of haemoglobinopathies on malaria (\u003cem\u003e42, 43\u003c/em\u003e). 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 \u0026alpha;-thalassemias impair erythrocyte deformability (\u003cem\u003e44\u003c/em\u003e). It was shown that erythrocyte deformability is lower in samples of \u0026alpha;-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 \u003cem\u003eP. falciparum\u003c/em\u003e invasion (\u003cem\u003e45\u003c/em\u003e). 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 \u003cem\u003eP. falciparum\u0026nbsp;\u003c/em\u003eparasites produce significantly lower numbers of merozoites in alpha and beta thalassemia trait cells, correlating with the MCHC and mean corpuscular volume (MCV) of these cell types (\u003cem\u003e46\u003c/em\u003e).\u003c/p\u003e\n\u003cp\u003eThis novel application of stem cell technology for the study of malaria represents a new and exciting option to study complex genetic traits as well as multiple mutations in their full genomic context. Genome editing can 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. Though in this work we aimed at developing a strategy with minimal handling that can be potentially scaled up for screening purposes, it can also be easily adapted to more detailed studies by the possibility of labelling receptors of interest or sorting the erythroid cells from the \u003cem\u003ein-vitro\u003c/em\u003e-differentiated population and assessing any parasite strain. This versatility allows to examine rare and non-viable genotypes and identify impact on cell differentiation as well as parasite invasion and development. The potential to use existing resources of banked available cell lines as well as reprogramming iPS lines from easily obtainable blood samples from patients or individuals with specific genotypes offers access to the study and preservation of a wide range of genetic characteristics. 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 cell-based system for a wide range of applications and potential identification of therapeutic targets.\u0026nbsp;\u003c/p\u003e"},{"header":"Methods","content":"\u003cp\u003e\u003cstrong\u003eEthics Statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe use of primary erythrocytes for the culture of Plasmodium falciparum was approved by the NHS Cambridgeshire 4 Research Ethics Committee REC ref. 15/EE/0253 and the Wellcome Sanger Institute Human Materials and Data Management Committee HMDMC 15/076.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe use of human embryonic stem cell lines was approved by the Steering Committee for the UK Stem Cell Bank and for the use of Stem Cell Lines (ref. SCSC11-23) and the Wellcome Sanger Institute Human Materials and Data Management Committee. The Human Embryonic Stem cell lines were obtained from the Centre for Stem Cell Biology, University of Sheffield, Sheffield, UK.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe fibroblast lines from haemoglobinopathy patients were obtained from the NIGMS human genetic cell repository of the Coriell Institute for Medical Research, USA.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eReprogramming of Induced Pluripotent Stem cell lines\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eHuman IPS lines were derived and verified at the Wellcome Sanger Institute as described (\u003cem\u003e47-50\u003c/em\u003e). Briefly, 5x10\u003csup\u003e5\u003c/sup\u003e cells were transduced with Sendai virus carriers of the Yamanaka factors: hOCT4, hSOX2, hKLF4 and hc-MYC overnight at 37\u003csup\u003e0\u003c/sup\u003eC in 5% CO\u003csub\u003e2\u003c/sub\u003e. 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% \u0026beta; 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.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCell Culture\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll 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 \u0026deg;C, 5% CO\u003csub\u003e2\u003c/sub\u003e and medium was changed regularly. Pluripotent cells were passaged using 0.5 mM EDTA (Gibco, UK) and 10\u0026micro;M Rock inhibitor.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eErythropoietic differentiation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eStem cells were taken off feeder cells with 0.5 mM EDTA (GIBCO) and seeded on gelatin-coated 10 cm plates pre-conditioned with MEF medium over-night and cultured in CDM-PVA supplemented with 12 nM hbFGF (Cell guidance systems, UK) and 10 nM hActivin-A (Source Bioscience, UK). CDM-PVA: 50% IMDM (Invitrogen), 50% advanced DMD-F12 (GIBCO) with 1g/l Poly(vinyl alcohol) PVA (SIGMA), Penicillin/Streptomycin 1x (GIBCO), 1-thioglycerol MTG (SIGMA), Insulin-Transferrin-Selenium 1x (ITS, Life Technologies), Cholesterol 1x (SyntheChol, SIGMA).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAs a first step of differentiation, the cells were taken towards the mesoderm germline:\u003c/p\u003e\n\u003cp\u003e\u003cu\u003eMesoderm 2 Days\u003c/u\u003e: CDM-PVA medium supplemented with 5 nM hActivin-A and 2\u0026mu;M SU5402 (SIGMA)\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cu\u003eMeso/Ery transition 8-12 Days\u003c/u\u003e: CDM-PVA medium supplemented with 20ng/ml bFGF, 10nM IL-3 (SIGMA), 10nM BMP4 (R\u0026amp;D Systems), 5\u0026mu;M SB431542 (SIGMA), 5\u0026mu;M CHIR99021 (Axon, The Netherlands), 5\u0026mu;M LY294002 (SIGMA).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eDuring this stage, the detached cells are recovered, washed with PBS and transferred to the erythrocytic differentiation stage, performed in a basic erythrocytic medium (BEM): CellGRO SCGM (CellGenix, Germany) supplemented with ITS, cholesterol, 40ng/ml IGF-1 (Abcam), Penicillin/Streptomycin, 1\u0026micro;M\u0026nbsp;4-hydroxy 5-methytetrahydrofolate (SIGMA).\u003c/p\u003e\n\u003cp\u003e\u003cu\u003eEry I 4-5 Days\u003c/u\u003e: BEM supplemented with 10ng/ml IL-3, 50ng/ml SCF (Life Technologies), 1\u0026micro;M dexamethasone (SIGMA), 2U/ml EPO (SIGMA), 10ng/ml FLT3 (R\u0026amp;D Systems).\u003c/p\u003e\n\u003cp\u003e\u003cu\u003eEry II 4-5 Days\u003c/u\u003e: BEM supplemented with 50ng/ml SCF, 1\u0026micro;M dexamethasone, 2U/ml EPO\u003c/p\u003e\n\u003cp\u003e\u003cu\u003eDiff Ery minimum 4 Days\u003c/u\u003e: BEM supplemented with 2U/ml EPO, 1\u0026micro;M Triiodo-L-Thyronine (T3, SIGMA)\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eRNA extraction, qRT-PCR and Microarrays\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eRNA was extracted using the Isolate II RNA Mini kit (Bioline, UK). 1-3 \u0026micro;g were reverse transcribed with a MuLV reverse transcriptase (Applied Biosystems, UK) using random primers (Bioline, UK). One \u0026micro;l 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:\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003egene\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;forward primer \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;reverse primer\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;length (bp)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eHbB\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;5\u0026rsquo;-gtctgccgttactgccctgtgg\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;5\u0026rsquo;-agcatcaggagtggacagatcc\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;136\u003c/p\u003e\n\u003cp\u003eHbA\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;5\u0026rsquo;-ggtgctgtctcctgccgac\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;5\u0026rsquo;-cctgggcagagccgtggctc\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;164\u003c/p\u003e\n\u003cp\u003eHbG\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;5\u0026rsquo;-cctgtcctctgcctctgcc \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;5\u0026rsquo;-cacagtgcagttcactcagc \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;140\u003c/p\u003e\n\u003cp\u003eHbE\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;5\u0026rsquo;-gctgccgtcactagcctgtg\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;5\u0026rsquo;-gcccaggatggcagagg\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;144\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTAL1\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;5\u0026rsquo;-atgccttccctatgttcaccacca\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;5\u0026rsquo;-tgaagatacgccgcacaactttgg\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;108\u003c/p\u003e\n\u003cp\u003eBrach\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;5\u0026rsquo;-acaaagagatgatggaggaacccg\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;5\u0026rsquo;-aggatgaggatttgcaggtggaca\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;110\u003c/p\u003e\n\u003cp\u003eGATA1\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;5\u0026rsquo;-cctctcccaagcttcgtggaac 5\u0026rsquo;-caggcgttgcataggtagtggc 127\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eKLF1\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;5\u0026rsquo;-ccggacacacaggatgacttcc\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;5\u0026rsquo;-ctggtcctcagacttcacgtggag\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;114\u003c/p\u003e\n\u003cp\u003eGAPDH\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;5\u0026rsquo;-gcctcctgcaccaccaactgc 5\u0026rsquo;-ggcagtgatggcatggactg 102\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eOCT4\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;5\u0026rsquo;-ctgccgctttgaggctctgcagc\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;5\u0026rsquo;-cctgcacgagggtttctgc 134\u003c/p\u003e\n\u003cp\u003eNANOG\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;5\u0026rsquo;-ccagctgtgtgtactcaatgatag\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;5\u0026rsquo;-ctctggttctggaaccaggtcttc \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;123\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eGYPA\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;5\u0026rsquo;-ccactgaggtggcaatgcac\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;5\u0026rsquo;-cttcatgagctctaggagtggctgc \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;120\u003c/p\u003e\n\u003cp\u003eGYPC\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;5\u0026rsquo;-ggacattgtcgtcattgcaggtg\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;5\u0026rsquo;-gcctcattggtgtggtacgtgc 117\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eBSG\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;5\u0026rsquo;-ccatgctggtctgcaagtcagag\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;5\u0026rsquo;-cacgaagaacctgctctcggag \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;116\u003c/p\u003e\n\u003cp\u003eTfR\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;5\u0026rsquo;-gggctggcagaaaccttg\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;5\u0026rsquo;-cagttggagtgctggagact \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;145\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eFor 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.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eParasite culture\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFluorescent \u003cem\u003eP. falciparum\u0026nbsp;\u003c/em\u003eparasites were cultured in complete RPMI medium (GIBCO) at 2.5% haematocrit with O- RBCs sourced from NHSBT, Cambridge. Cultures were maintained at 37\u0026deg;C in malaria gas (1% O\u003csub\u003e2\u003c/sub\u003e, 3% CO\u003csub\u003e2\u003c/sub\u003e and 96% N\u003csub\u003e2\u003c/sub\u003e).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFluorescent parasites\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eParasites were engineered to express a variety of fluorochromes for detection at different wavelengths (\u003cem\u003e25\u003c/em\u003e). The chosen fluorochromes: tagBFP, Midori-ishi cyan, Kusabira Orange and mCherry were individually inserted into the XhoI / AvrII site of an \u003cem\u003eattP\u003c/em\u003e-containing vector under regulation by the calmodulin promoter and bearing blasticidin resistance as a selection marker. The NF54 \u003cem\u003eattB\u003c/em\u003e strain of \u003cem\u003ePlasmodium falciparum\u003c/em\u003e was transfected with each fluorochrome vector together with an expression vector for Bxb1 integrase and transfectants were selected with blasticidin (2 ugr/ml)(\u003cem\u003e51\u003c/em\u003e).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStaining Procedures\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDDAO labelling of cells\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eIn-vitro\u003c/em\u003e-differentiated erythroid cells were centrifuged (1100xg for 4\u0026rsquo;) and resuspended in 1 ml of Diff Ery medium containing 1\u0026nbsp;\u0026micro;M DDAO-HS dye for 1 h at 37\u003csup\u003e0\u003c/sup\u003eC. Cells were spun again and resuspended in 1ml DDAO-free medium and incubated for 30 minutes at 37\u003csup\u003e0\u003c/sup\u003eC. After a final spin, cells were resuspended in parasite culture medium at a concentration of 10\u003csup\u003e6\u003c/sup\u003e cells per 75\u0026nbsp;\u0026micro;l.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eGiemsa staining of slides\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCells, parasite cultures and invasion assays were stained with Giemsa for microscopic examination. Five\u0026nbsp;\u0026micro;l of culture were dropped on a glass slide and spread with a pipette tip or smeared with a glass slide and dried. The slides were fixed with methanol for a few seconds, dried and incubated with 1x Giemsa stain solution for 5-10 minutes. The stain solution as drained away, the slides were washed with tap water and dried before microscopic examination.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSybr Green staining of parasite DNA\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFixed culture samples were incubated in 100\u0026nbsp;\u0026micro;l of a 1X solution (1/10000 dilution) of Sybr Green nucleic acid gel stain 10000X (Invitrogen) for 30 minutes at 37\u003csup\u003e0\u003c/sup\u003eC. 200\u0026nbsp;\u0026micro;l of PBS were added to each sample before flow cytometry analysis using a 488 nm laser with a 530/40 nm bandpass filter.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eInvasion Assays\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eIn-vitro\u003c/em\u003e-differentiated labelled erythroid cells were counted and 75 \u0026mu;l containing 1 million cells were dispensed into a 96 well plate, including a well of cells alone for controls. Further controls included, primary erythrocytes obtained from the blood bank that were labelled in the same way, adding 75\u0026nbsp;\u0026micro;l at 5% haematocrit per assay.\u003c/p\u003e\n\u003cp\u003eAsynchronous cultures of fluorescent parasites at a parasitaemia of 1.5 \u0026ndash; 2% mature parasites were used to purify schizonts: 10 ml of culture were centrifuged (1100xg for 5\u0026rsquo; brake 3), resuspended in 1 ml of medium and loaded onto a 63% Percoll cushion. Centrifugation at 1300xg for 11\u0026rsquo; 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 \u0026mu;l/well.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eOne plate per time point was prepared and plates were placed in a gas chamber filled with malaria gas and left in a 37\u0026deg;C incubator for the appropriate length of time.\u003c/p\u003e\n\u003cp\u003eAfter the incubation time, the plate was removed, adding 200 \u0026mu;l of PBS/well and spinning at 1100xg 1\u0026rsquo;. Three \u0026mu;l 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 \u0026mu;l 4% paraformaldehyde for 20\u0026rsquo;, washed and resuspended in PBS to be analysed by flow cytometry.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFlow Cytometry\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eExpression 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 \u0026amp; Co, NJ) and SUMMIT V3.1. Antibodies:\u003c/p\u003e\n\u003cp\u003eCD71-APC\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Biolegend #334108\u003c/p\u003e\n\u003cp\u003eGYPA-PE\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Southern Biotech #9861-09\u003c/p\u003e\n\u003cp\u003eBSG-FITC\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;MACS Miltenyi #130-104-489\u003c/p\u003e\n\u003cp\u003eATP2B4-FITC\u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;LSBio #LS-C446496\u003c/p\u003e\n\u003cp\u003eA MoFlo flow cytometer (Beckman Coulter, USA) was adapted for detection of laser light depolarisation produced by parasite hemozoin (\u003cem\u003e26\u003c/em\u003e).\u0026nbsp;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-in-air 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 \u0026nbsp;520/36nm, 580/30nm, 615/20nm, 447/60nm and 671/28nm band pass filter respectively.\u0026nbsp;An optical modification\u0026nbsp;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).\u0026nbsp;A total of 50000 events was acquired and analysed using Flojo V10.3.\u003c/p\u003e\n\u003cp\u003eInvitrogen Bigfoot cell sorter (Thermo Fisher Scientific, Inc.) was also used for the detection of laser light depolarisation produced by parasite hemozoin. The cell sorter is equipped with six spatially separated solid state lasers but only two of the lasers would be turn ON and used in the assay. The laser power for 488 nm was set at 125 \u0026nbsp;mW and 640 nm was set at 100 mW. The laser 488 nm, 640 nm was used to excite Sybr Green and DDAO respectively. Fluorescence emitted from SYBR Green and DDAO was collected using a 507/19 nm and 670/30 nm band pass filter respectively. The instrument is also equipped with default polarisers at the 488nm laser light path which can be switch \u0026lsquo;ON\u0026rsquo; \u0026nbsp;during the analysis. This optic set up allows the measurement of normal SSC (488 SSC, Area Linear) and depolarised \u0026nbsp;SSC (488 SSC Polar, Area Linear) (See figure below). A total of 20,000 events was acquired and analysed using FCSExpressv7 (De Novo Software, Inc.).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStatistical Analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eResults are presented as means and standard deviation. The reported significance was calculated using a two-tailed unpaired Student\u0026rsquo;s T test analysis.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eGenome Editing\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eGenomic modification to ablate the genes chosen for this study was performed in the RH1 cell line, as described previously (\u003cem\u003e52\u003c/em\u003e) and shown schematically in supplementary Fig. S9. Briefly, a CRISPR/Cas9 strategy was used targeting a critical exon in each gene (exon 5 in BSG and exon 11 in ATP2B4) for substitution with a selection cassette as depicted in Fig. S9. The puromycin resistance in the cassette was used to select correctly targeted clones which were examined for damage to the second allele as shown in Fig. S10.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMicroarray data analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll microarray datasets were put through \u0026ldquo;neqc\u0026rdquo; background correction followed by quantile normalization using the limma R package (\u003cem\u003e53\u003c/em\u003e). \u0026nbsp;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 GSE63703 gene expression matrix from GREIN (https://shiny.ilincs.org/grein) was used to identify erythrocyte and erythroid progenitor specific genes.\u0026nbsp;\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eDATA AVAILABILITY\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data needed to evaluate the conclusions in the paper are present in the paper and supplementary materials and on request from the corresponding author.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAKNOWLEDGEMENTS\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was funded by the Wellcome Trust. The authors wish to thank Ale\u0026scaron;\u0026nbsp;Kilpatrick for helping with imaging.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAUTHOR CONTRIBUTIONS\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAP conception and design of the project, performing of experiments and writing of the manuscript; JCR conception of the project and writing of the manuscript; BL setting up and running of flow cytometry approach. KM bioinformatics analysis; HP handling of the data and analysis; MK genome editing of BSG and ATP2B4; CA and FR repogramming of iPSC lines; RM help and guidance with flow cytometric analysis; FL help and advice for stem cell culture and maintenance.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCOMPETING INTERESTS\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMTA\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe data can be provided, pending scientific review and completed transfer agreement. Requests should be submitted to Alena Pance.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eC. Aurrecoechea\u003cem\u003e\u0026nbsp;et al.\u003c/em\u003e, PlasmoDB: a functional genomic database for malaria parasites. \u003cem\u003eNucleic Acids Res\u003c/em\u003e \u003cstrong\u003e37\u003c/strong\u003e, D539-543 (2009).\u003c/li\u003e\n \u003cli\u003eT. W. 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Ritchie\u003cem\u003e\u0026nbsp;et al.\u003c/em\u003e, limma powers differential expression analyses for RNA-sequencing and microarray studies. \u003cem\u003eNucleic Acids Res\u003c/em\u003e \u003cstrong\u003e43\u003c/strong\u003e, e47 (2015).\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-1808642/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-1808642/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"Plasmodium falciparum parasites have a complex life cycle, but the most clinically relevant stage of the disease is the invasion of erythrocytes and the proliferation of the parasite in the blood. The influence of human genetic traits on malaria has been known for a long time, however understanding the role of the proteins involved is hampered by the anuclear nature of erythrocytes that makes them inaccessible to genetic tools. Here we overcome this limitation with a differentiation protocol to derive erythroid cells in- vitro from a diversity of human stem cells and an adaptation of flow cytometry to detect hemozoin. We combine this strategy with genome editing to show that deletion of basigin ablates invasion while deletion of ATP2B4 has a minor effect and that erythroid cells from reprogrammed patient-derived HbBart α-thalassemia samples poorly support infection. This approach offers vast potential for understanding the impact human polymorphisms on malaria.","manuscriptTitle":"Stem Cell Technology Provides Novel Tools to Understand the Impact of Human Variation on Malaria","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-12-15 01:10:33","doi":"10.21203/rs.3.rs-1808642/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"3a524f25-35b7-4806-acd8-1d3673f73c72","owner":[],"postedDate":"December 15th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":59579359,"name":"Biological sciences/Stem cells/Stem-cell differentiation"},{"id":59579360,"name":"Biological sciences/Microbiology/Parasitology/Parasite biology"},{"id":59579361,"name":"Biological sciences/Stem cells"}],"tags":[],"updatedAt":"2025-12-15T01:10:33+00:00","versionOfRecord":[],"versionCreatedAt":"2025-12-15 01:10:33","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-1808642","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-1808642","identity":"rs-1808642","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}