Improving hematopoietic differentiation from human induced pluripotent stem cells by the modulation of Hippo signaling with a diarylheptanoid derivative.

BACKGROUND: The diarylheptanoid ASPP 049 has improved the quality of adult hematopoietic stem cell (HSC) expansion ex vivo through long-term reconstitution in animal models. However, its effect on hematopoietic regeneration from human induced pluripotent stem cells (hiPSCs) is unknown. METHOD: We utilized a defined cocktail of cytokines without serum or feeder followed by the supplementation of ASPP 049 to produce hematopoietic stem/progenitor cells (HSPCs). Flow cytometry and trypan blue exclusion analysis were used to identify nonadherent and adherent cells. Nonadherent cells were harvested to investigate the effect of ASPP 049 on multipotency using LTC-IC and CFU assays. Subsequently, the mechanism of action was explored through transcriptomic profiles, which were validated by qRT-PCR, immunoblotting, and immunofluorescence analysis. RESULT: The supplementation of ASPP 049 increased the number of phenotypically defined primitive HSPCs (CD34+CD45+CD90+) two-fold relative to seeded hiPSC colonies, indicating enhanced HSC derivation from hiPSCs. Under ASPP 049-supplemented conditions, we observed elevated HSPC niches, including CD144+CD73- hemogenic- and CD144+CD73+ vascular-endothelial progenitors, during HSC differentiation. Moreover, harvested ASPP 049-treated cells exhibited improved self-renewal and a significantly larger proportion of different blood cell colonies with unbiased lineages, indicating enhanced HSC stemness properties. Transcriptomics and KEGG analysis of sorted CD34+CD45+ cells-related mRNA profiles revealed that the Hippo signaling pathway is the most significant in responding to WWTR1/TAZ, which correlates with the validation of the protein expression. Interestingly, ASPP 049-supplemented HSPCs upregulated 11 genes similarly to umbilical cord blood-derived HSPCs. CONCLUSION: These findings suggest that ASPP 049 can improve HSC-generating protocols with proliferative potentials, self-renewal ability, unbiased differentiation, and a definable mechanism of action for the clinical perspective of hematopoietic regenerative medicine.

Trio fluorophore-based phenotypic assay for the detection of artemisinin-induced growth-arrested Plasmodium falciparum in human erythrocytes.

Artemisinin combination therapy remains effective for the treatment of falciparum malaria. However, Plasmodium falciparum can escape the effects of artemisinin by arresting their growth. The growth-arrested parasites cannot be distinguished from nonviable parasites with standard microscopy techniques due to their morphological similarities. Here, we demonstrated the efficacy of a new laboratory assay that is compatible with the artemisinin susceptibility test. As a result of the differential cell permeabilities of two DNA-binding fluorophores, growth-arrested P. falciparum can be distinguished from parasites killed by artemisinin, since the latter lose cell membrane permeability. This fluorescence-based assay increased the sensitivity and specificity of the ring survival assay in the assessment of artemisinin susceptibility. When combined with a third fluorophore-conjugated anti-human leukocyte antibody, this trio fluorophore assay became more useful in identifying growth-arrested parasites in mock human blood samples. This novel assay is a simple and rapid technique for monitoring artemisinin resistance with greater sensitivity and accuracy compared with morphology-based observations under a light microscope.

Thrombopoietin-independent generation of platelet-like particles from megakaryoblastic cells.

The use of megakaryoblastic leukemia MEG-01 cells can help reveal the mechanisms of thrombopoiesis. However, conventional in vitro activation of platelet release from MEG-01 cells requires thrombopoietin, which is costly. Here, we aim to develop a more straightforward and affordable method. Synchronization of the MEG-01 cells was initially performed using serum-free culture, followed by spontaneous cell differentiation in the presence of serum. Different stages of megakaryoblast differentiation were classified based on cell morphology, DNA content, and cell cycle. The MEG-01 cells released platelet-like particles at a level comparable to that of the thrombopoietin-activated MEG-01 cells. The platelet-like particles were distinguishable from PLP-derived extracellular vesicles and could express P-selectin following ADP activation. Importantly, the platelet-like particles induced fibrin clotting in vitro using platelet-poor plasma. Therefore, this thrombopoietin-independent cell synchronization method is an effective and straightforward method for studying megakaryopoiesis and thrombopoiesis.

Phenotypic assay for cytotoxicity assessment of Balamuthia mandrillaris against human neurospheroids.

Introduction: The phenotypic screening of drugs against Balamuthia mandrillaris, a neuropathogenic amoeba, involves two simultaneous phases: an initial step to test amoebicidal activity followed by an assay for cytotoxicity to host cells. The emergence of three-dimensional (3D) cell cultures has provided a more physiologically relevant model than traditional 2D cell culture for studying the pathogenicity of B. mandrillaris. However, the measurement of ATP, a critical indicator of cell viability, is complicated by the overgrowth of B. mandrillaris in coculture with host cells during drug screening, making it challenging to differentiate between amoebicidal activity and drug toxicity to human cells. Methods: To address this limitation, we introduce a novel assay that utilizes three-dimensional hanging spheroid plates (3DHSPs) to evaluate both activities simultaneously on a single platform. Results and discussion: Our study showed that the incubation of neurospheroids with clinically isolated B. mandrillaris trophozoites resulted in a loss of neurospheroid integrity, while the ATP levels in the neurospheroids decreased over time, indicating decreased host cell viability. Conversely, ATP levels in isolated trophozoites increased, indicating active parasite metabolism. Our findings suggest that the 3DHSP-based assay can serve as an endpoint for the phenotypic screening of drugs against B. mandrillaris, providing a more efficient and accurate approach for evaluating both parasite cytotoxicity and viability.

Mitochondrial genome diversity of Balamuthia mandrillaris revealed by a fatal case of granulomatous amoebic encephalitis.

Introduction: Balamuthia (B.) mandrillaris is a free-living amoeba that can cause rare yet fatal granulomatous amoebic encephalitis (GAE). However, efficacious treatment for GAE is currently unavailable, especially when genomic studies on B. mandrillaris are limited. Methods: In this study, B. mandrillaris strain KM-20 was isolated from the brain tissue of a GAE patient, and its mitochondrial genome was de novo assembled using high-coverage Nanopore long reads and Illumina short reads. Results and Discussion: Phylogenetic and comparative analyses revealed a range of diversification in the mitochondrial genome of KM-20 and nine other B. mandrillaris strains. According to the mitochondrial genome alignment, one of the most variable regions was observed in the ribosomal protein S3 (rps3), which was caused by an array of novel protein tandem repeats. The repeating units in the rps3 protein tandem region present significant copy number variations (CNVs) among B. mandrillaris strains and suggest KM-20 as the most divergent strain for its highly variable sequence and highest copy number in rps3. Moreover, mitochondrial heteroplasmy was observed in strain V039, and two genotypes of rps3 are caused by the CNVs in the tandem repeats. Taken together, the copy number and sequence variations of the protein tandem repeats enable rps3 to be a perfect target for clinical genotyping assay for B. mandrillaris. The mitochondrial genome diversity of B. mandrillaris paves the way to investigate the phylogeny and diversification of pathogenic amoebae.

Glucose-6-phosphate dehydrogenase is dispensable for human erythroid cell differentiation in vitro.

Glucose-6-phosphate dehydrogenase (G6PD) deficiency impairs cellular processes under oxidative stress. Individuals with severe G6PD deficiency still produce sufficient numbers of erythrocytes. Nevertheless, the G6PD independence of erythropoiesis remains questionable. This study elucidates the effects of G6PD deficiency on the generation of human erythrocytes. Peripheral blood-derived CD34-positive hematopoietic stem and progenitor cells (HSPCs) of human subjects with normal, moderate, and severe G6PD activities were cultured in two distinct phases: erythroid commitment and terminal differentiation. Regardless of G6PD deficiency, HSPCs were able to proliferate and differentiate into mature erythrocytes. There was no impairment in erythroid enucleation among the subjects with G6PD deficiency. To our knowledge, this study is the first report of effective erythropoiesis independent of G6PD deficiency. The evidence firmly indicates that the population with the G6PD variant could produce erythrocytes to an extent similar to that in healthy individuals.

An Optical and Chemiluminescence Assay for Assessing the Cytotoxicity of Balamuthia mandrillaris against Human Neurospheroids.

A spheroid is a cell aggregate in a three-dimensional context; thereby, it recapitulates the cellular architecture in human tissue. However, the utility of spheroids as an assay for host-parasite interactions remains unexplored. This study demonstrates the potential use of neurospheroids for assessing the cytotoxicity of the life-threatening pathogenic amoeba Balamuthia mandrillaris. The neuroblastoma SH-SY5Y cells formed a spheroid in a hanging drop of culture medium. Cellular damage caused by B. mandrillaris trophozoites on human neuronal spheroids was observed using microscopic imaging and ATP detection. B. mandrillaris trophozoites rapidly caused a decrease in ATP production in the spheroid, leading to loss of neurospheroid integrity. Moreover, 3D confocal microscopy imaging revealed interactions between the trophozoites and SH-SY5Y neuronal cells in the outer layer of the neurospheroid. In conclusion, the neurospheroid allows the assessment of host cell damage in a simple and quantitative manner.

Balamuthia mandrillaris trophozoites ingest human neuronal cells via a trogocytosis-independent mechanism.

BACKGROUND: Environmental protozoa need an adaptation mechanism to survive drastic changes in niches in the human body. In the brain parenchyma, Balamuthia mandrillaris trophozoites, which are causative agents of fatal brain damage, must acquire nutrients through the ingestion of surrounding cells. However, the mechanism deployed by the trophozoites for cellular uptake remains unknown. METHODS: Amoebic ingestion of human neural cell components was investigated using a coculture system of clinically isolated B. mandrillaris trophozoites and human neuroblastoma SH-SY5Y cells. Cell-to-cell interactions were visualized in a three-dimensional manner using confocal and holotomographic microscopes. RESULTS: The B. mandrillaris trophozoites first attached themselves to human neuroblastoma SH-SY5Y cells and then twisted themselves around the cytoplasmic bridge. Based on fluorescence-based cell tracking, the B. mandrillaris trophozoites then inserted invadopodia into the cytoplasm of the human cells. Subsequently, the human protein-enriched components were internalized into the trophozoites in the form of nonmembranous granules, whereas the human lipids were dispersed in the cytoplasm. Intervention of trogocytosis, a process involving nibbling on parts of the target cells, failed to inhibit this cellular uptake. CONCLUSIONS: Human cell ingestion by B. mandrillaris trophozoites likely differs from trogocytosis, suggesting that a pathogen-specific strategy can be used to ameliorate brain damage.

Peptide of Trichinella spiralis Infective Larval Extract That Harnesses Growth of Human Hepatoma Cells.

Trichinella spiralis, a tissue-dwelling helminth, causes human trichinellosis through ingestion of undercooked meat containing the parasite's infective larvae. However, benefits from T. spiralis infection have been documented: reduction of allergic diseases, inhibition of collagen-induced arthritis, delay of type 1 diabetes progression, and suppression of cancer cell proliferation. Since conventional cancer treatments have limited and unreliable efficacies with adverse side effects, novel adjunctive therapeutic agents and strategies are needed to enhance the overall treatment outcomes. This study aimed to validate the antitumor activity of T. spiralis infective larval extract (LE) and extricate the parasite-derived antitumor peptide. Extracts of T. spiralis infective larvae harvested from striated muscles of infected mice were prepared and tested for antitumor activity against three types of carcinoma cells: hepatocellular carcinoma HepG2, ovarian cancer SK-OV-3, and lung adenocarcinoma A549. The results showed that LE exerted the greatest antitumor effect on HepG2 cells. Proteomic analysis of the LE revealed 270 proteins. They were classified as cellular components, proteins involved in metabolic processes, and proteins with diverse biological functions. STRING analysis showed that most LE proteins were interconnected and played pivotal roles in various metabolic processes. In silico analysis of anticancer peptides identified three candidates. Antitumor peptide 2 matched the hypothetical protein T01_4238 of T. spiralis and showed a dose-dependent anti-HepG2 effect, not by causing apoptosis or necrosis but by inducing ROS accumulation, leading to inhibition of cell proliferation. The data indicate the potential application of LE-derived antitumor peptide as a complementary agent for human hepatoma treatment.

Impairment of Invasion and Maturation and Decreased Selectivity of Plasmodium falciparum in G6PD Viangchan and Mahidol Variants.

BACKGROUND: Protection against Plasmodium falciparum is observed in a population deficient in glucose-6-phosphate dehydrogenase (G6PD), particularly in African and Mediterranean regions. However, such protection remains unknown among G6PD-deficient individuals in Southeast Asia. METHODS: In this study, we assessed the invasion and maturation of P falciparum K1 in a culture of erythrocytes isolated from Thai subjects carrying Viangchan (871G > A) and Mahidol (487G > A). RESULTS: We found that the parasites lost their ability to invade hemizygous and homozygous G6PD-deficient erythrocytes of Viangchan and Mahidol variants in the second and third cycles of intraerythrocytic development. It is interesting to note that P falciparum parasites selectively grew in erythrocytes from hemi- and homozygous genotypes with normal G6PD activity. Moreover, externalization of phosphatidylserine upon P falciparum infection was significantly increased only in Viangchan hemizygous variant cells. CONCLUSIONS: This study is the first to show that blockage of invasion in long-term culture and potentially enhanced removal of parasitized erythrocytes were observed for the first time in erythrocytes from Viangchan and Mahidol G6PD-deficient individuals.

Progress and Challenges in the Use of a Liver-on-a-Chip for Hepatotropic Infectious Diseases.

The liver is a target organ of life-threatening pathogens and prominently contributes to the variation in drug responses and drug-induced liver injury among patients. Currently available drugs significantly decrease the morbidity and mortality of liver-dwelling pathogens worldwide; however, emerging clinical evidence reveals the importance of host factors in the design of safe and effective therapies for individuals, known as personalized medicine. Given the primary adherence of cells in conventional two-dimensional culture, the use of these one-size-fit-to-all models in preclinical drug development can lead to substantial failures in assessing therapeutic safety and efficacy. Advances in stem cell biology, bioengineering and material sciences allow us to develop a more physiologically relevant model that is capable of recapitulating the human liver. This report reviews the current use of liver-on-a-chip models of hepatotropic infectious diseases in the context of precision medicine including hepatitis virus and malaria parasites, assesses patient-specific responses to antiviral drugs, and designs personalized therapeutic treatments to address the need for a personalized liver-like model. Second, most organs-on-chips lack a monitoring system for cell functions in real time; thus, the review discusses recent advances and challenges in combining liver-on-a-chip technology with biosensors for assessing hepatocyte viability and functions. Prospectively, the biosensor-integrated liver-on-a-chip device would provide novel biological insights that could accelerate the development of novel therapeutic compounds.

Next-Generation Human Liver Models for Antimalarial Drug Assays.

Advances in malaria prevention and treatment have significantly reduced the related morbidity and mortality worldwide, however, malaria continues to be a major threat to global public health. Because Plasmodium parasites reside in the liver prior to the appearance of clinical manifestations caused by intraerythrocytic development, the Plasmodium liver stage represents a vulnerable therapeutic target to prevent progression. Currently, a small number of drugs targeting liver-stage parasites are available, but all cause lethal side effects in glucose-6-phosphate dehydrogenase-deficient individuals, emphasizing the necessity for new drug development. Nevertheless, a longstanding hurdle to developing new drugs is the availability of appropriate in vitro cultures, the crucial conventional platform for evaluating the efficacy and toxicity of drugs in the preclinical phase. Most current cell culture systems rely primarily on growing immortalized or cancerous cells in the form of a two-dimensional monolayer, which is not very physiologically relevant to the complex cellular architecture of the human body. Although primary human cells are more relevant to human physiology, they are mainly hindered by batch-to-batch variation, limited supplies, and ethical issues. Advances in stem cell technologies and multidimensional culture have allowed the modelling of human infectious diseases. Here, current in vitro hepatic models and toolboxes for assaying the antimalarial drug activity are summarized. Given the physiological potential of pluripotent and adult stem cells to model liver-stage malaria, the opportunities and challenges in drug development against liver-stage malaria is highlighted, paving the way to assess the efficacy of hepatic plasmodicidal activity.

Progress and challenges in the use of fluorescence-based flow cytometric assays for anti-malarial drug susceptibility tests.

Drug-resistant Plasmodium is a frequent global threat in malaria eradication programmes, highlighting the need for new anti-malarial drugs and efficient detection of treatment failure. Plasmodium falciparum culture is essential in drug discovery and resistance surveillance. Microscopy of Giemsa-stained erythrocytes is common for determining anti-malarial effects on the intraerythrocytic development of cultured Plasmodium parasites. Giemsa-based microscopy use is conventional but laborious, and its accuracy depends largely on examiner skill. Given the availability of nucleic acid-binding fluorescent dyes and advances in flow cytometry, the use of various fluorochromes has been frequently attempted for the enumeration of parasitaemia and discrimination of P. falciparum growth in drug susceptibility assays. However, fluorochromes do not meet the requirements of being fast, simple, reliable and sensitive. Thus, this review revisits the utility of fluorochromes, notes previously reported hindrances, and highlights the challenges and opportunities for using fluorochromes in flow cytometer-based drug susceptibility tests. It aims to improve drug discovery and support a resistance surveillance system, an essential feature in combatting malaria.

Molecular identification of native Wolbachia pipientis in Anopheles minimus in a low-malaria transmission area of Umphang Valley along the Thailand-Myanmar border.

BACKGROUND: Wolbachia, obligate intracellular bacteria, infect the majority of arthropods, including many mosquito species of medical importance. Some Wolbachia strains interfere with the development of Plasmodium parasites in female Anopheles, a major vector of malaria. The use of Wolbachia as a means to block malaria transmission is an emerging vector control strategy in highly endemic areas. Hence, identification of native Wolbachia strains in areas where malaria transmission is low may uncover a particular Wolbachia strain capable of Plasmodium interference. This study aims to identify native Wolbachia strains in female Anopheles spp. that are predominant in a low-malaria transmission area in mainland Southeast Asia. METHODS: Following a 2-year survey of malaria vectors in Umphang Valley of Tak Province, Thailand, DNA extracts of female An. minimus, An. peditaeniatus, and An. maculatus were subjected to amplification of the conserved region of the 16S rRNA-encoding gene. The DNA sequences of the amplicons were phylogenetically compared with those of known Wolbachia strains. RESULTS: Among three Anopheles spp., amplification was detected in only the DNA samples from An. minimus. The DNA sequencing of amplicons revealed 100% similarity to Wolbachia pipientis, confirming the specificity of amplification. The Wolbachia-positive An. minimus samples were devoid of Plasmodium 18S rRNA amplification. The phylogenetic trees indicate a close relationship with Wolbachia strains in subgroup B. CONCLUSION: To the best of our knowledge, the data presented herein provide the first molecular evidence of a Wolbachia strain in An. minimus, hereinafter named wAnmi, in a low-malaria transmission area in the Umphang Valley of western Thailand. Further biological characterization is required to examine its potential for malaria transmission control in the field.

Generation of human liver organoids from pluripotent stem cell-derived hepatic endoderms.

BACKGROUND: The use of a personalized liver organoid derived from human-induced pluripotent stem cells (HuiPSCs) is advancing the use of in vitro disease models for the design of specific, effective therapies for individuals. Collecting patient peripheral blood cells for HuiPSC generation is preferable because it is less invasive; however, the capability of blood cell-derived HuiPSCs for hepatic differentiation and liver organoid formation remains uncertain. Moreover, the currently available methods for liver organoid formation require a multistep process of cell differentiation or a combination of hepatic endodermal, endothelial and mesenchymal cells, which is a major hurdle for the application of personalized liver organoids in high-throughput testing of drug toxicity and safety. To demonstrate the capability of blood cell-derived HuiPSCs for liver organoid formation without support from endothelial and mesenchymal cells. METHODS: The peripheral blood-derived HuiPSCs first differentiated into hepatic endoderm (HE) in two-dimensional (2D) culture on Matrigel-coated plates under hypoxia for 10 days. The HE was then collected and cultured in 3D culture using 50% Matrigel under ambient oxygen. The maturation of hepatocytes was further induced by adding hepatocyte growth medium containing HGF and oncostatin M on top of the 3D culture and incubating the culture for an additional 12-17 days. The function of the liver organoids was assessed using expression analysis of hepatocyte-specific gene and proteins. Albumin (ALB) synthesis, glycogen and lipid storage, and metabolism of indocyanine were evaluated. The spatial distribution of albumin was examined using immunofluorescence and confocal microscopy. RESULTS: CD34+ hematopoietic cell-derived HuiPSCs were capable of differentiating into definitive endoderm expressing SOX17 and FOXA2, hepatic endoderm expressing FOXA2, hepatoblasts expressing AFP and hepatocytes expressing ALB. On day 25 of the 2D culture, cells expressed SOX17, FOXA2, AFP and ALB, indicating the presence of cellular heterogeneity. In contrast, the hepatic endoderm spontaneously formed a spherical, hollow structure in a 3D culture of 50% Matrigel, whereas hepatoblasts and hepatocytes could not form. Microscopic observation showed a single layer of polygonal-shaped cells arranged in a 3D structure. The hepatic endoderm-derived organoid synthesis ALB at a higher level than the 2D culture but did not express definitive endoderm-specific SOX17, indicating the greater maturity of the hepatocytes in the liver organoids. Confocal microscopic images and quantitative ELISA confirmed albumin synthesis in the cytoplasm of the liver organoid and its secretion. Overall, 3D culture of the hepatic endoderm is a relatively fast, simple, and less laborious way to generate liver organoids from HuiPSCs that is more physiologically relevant than 2D culture.

A novel method to purify neutrophils enables functional analysis of zebrafish hematopoiesis.

Zebrafish is a useful model to study vertebrate hematopoiesis, but lack of antibodies to zebrafish proteins has limited purification of hematopoietic cells. Here, we purified neutrophils from larval and adult zebrafish using the lectin Phaseolus vulgaris erythroagglutinin (PHA-E) and DRAQ5, a DNA-staining fluorescent dye. In adult kidney marrow, we purified neutrophil-like PHA-E4low DRAQ5low cells, which neutrophil-type granules. Specifically, at 96-hr post-fertilization, we sorted large-sized cells from larvae using forward scatter and found that they consisted of PHA-Elow DRAQ5low populations. These cells had myeloperoxidase activity, were Sudan Black B-positive and expressed high levels of neutrophil-specific (csf3r and mpx) mRNAs, all neutrophil characteristics. Using this method, we conducted functional analysis suggesting that zyxin (Zyx) plays a role in neutrophil generation in zebrafish larvae. Overall, PHA-E and DRAQ5-based flow cytometry serves as a tool to purify zebrafish neutrophils.

Paracrine CCL17 and CCL22 signaling regulates hematopoietic stem/progenitor cell migration and retention in mouse fetal liver.

Fetal liver (FL) is the major embryonic hematopoietic organ and a site where circulating hematopoietic stem/progenitor cells (HSPCs) reside. However, HSPC migration/retention mechanisms in FL remain unclear. A chemokine screen revealed that the CCR4 ligands CCL17 and CCL22 are highly expressed in mouse embryonic day (E) 12.5 FL. Flow cytometric analysis confirmed CCR4 expression in FL HSPCs. To identify sources of CCL17 and CCL22, we fractionated FL into various cell types and found that Ccl17 and Ccl22 were predominantly expressed in HPCs/matured HCs. In vitro cell migration analysis confirmed enhanced HSPC migration in the presence of HPCs/matured HCs. Furthermore, exo-utero injection of anti-CCR4 neutralizing antibody into pregnant mice significantly reduced the number of FL HSPCs in embryos. These data demonstrate a paracrine mechanism by which HSPC migration/retention is regulated by CCL17 and CCL22 secreted from HPCs or matured HCs in FL.

A simple monochromatic flow cytometric assay for assessment of intraerythrocytic development of Plasmodium falciparum.

BACKGROUND: Gold standard microscopic examination of Plasmodium falciparum intraerythrocytic stage remains an important process for staging and enumerating parasitized erythrocytes in culture; however, microscopy is laborious and its accuracy is dependent upon the skill of the examiner. METHODS: In this study, ViSafe Green (VSG), which is a nucleic acid-binding fluorescent dye, was used for assessing in vitro development of P. falciparum using flow cytometry. RESULTS: Fluorescence intensity of VSG was found to depend on the developmental stage of parasites. Specifically, multiple-nuclei-containing schizonts were observed in the VSGhigh population, and growing trophozoites and ring-shaped forms were observed in the VSGintermediate and VSGlow populations. The efficacy of VSG-based assay was found to be comparable to the microscopic examination method, and it demonstrated an ability to detect as low as 0.001% of the parasitaemia estimated by Giemsa staining. Moreover, when applying VSG for anti-malarial drug test, it was able to observe the growth inhibitory effect of dihydroartemisinin, the front-line drug for malaria therapy. CONCLUSIONS: Taken together, the results of this study suggest the VSG-based flow cytometric assay to be a simple and reliable assay for assessing P. falciparum malaria development in vitro.