Human iPSC-derived hepatocyte-like cells support Plasmodium liver-stage infection in vitro.
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ABSTRACT:
SUBMITTER: Ng S
PROVIDER: S-EPMC4375936 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Plasmodium parasites infect hepatocytes of their mammalian hosts and undergo obligate liver stage development. The specific host cell attributes that are important for liver infection remain largely unknown. Several host signalling pathways are perturbed in infected hepatocytes, some of which are important in the generation of hepatocyte polyploidy. To test the functional consequence of polyploidy on liver infection, we infected hepatocytes with the rodent malaria parasite Plasmodium yoelii both in vitro and in vivo and examined the ploidy of infected and uninfected hepatocytes by flow cytometry. In both hepatoma cell lines and in the mouse liver, the fraction of polyploid cells was higher in the infected cell population than in the uninfected cell population. When the data were reanalysed by comparing the extent of Plasmodium infection within each ploidy subset, we found that infection rates were elevated in more highly polyploid cells and lower in diploid cells. Furthermore, we found that the parasite's preference for host cells with high ploidy is conserved among rodent malaria species and the human malaria parasite Plasmodium falciparum. This parasite preference for host cells of high ploidy cannot be explained by differences in hepatocyte size or DNA replication. We conclude that Plasmodium preferentially infects and develops in polyploid hepatocytes.
| S-EPMC4008336 | biostudies-literature
Project description:The liver is the first destination of malaria parasites in humans. After reaching the liver by the blood stream, Plasmodium sporozoites cross the liver sinusoid epithelium, enter and exit several hepatocytes, and eventually invade a final hepatocyte host cell. At present, the mechanism of hepatocyte invasion is only partially understood, presenting a key research gap with opportunities for the development of new therapeutics. Recently, human EphA2, a membrane-bound receptor tyrosine kinase, was implicated in hepatocyte infection by the human malaria parasite Plasmodium falciparum and the rodent parasite Plasmodium yoelii, but its role is not known for Plasmodium vivax, a major human parasite whose liver infection poses a specific challenge for malaria treatment and elimination. In this study, the role of EphA2 in P. vivax infection was investigated. It was found that surface expression of several recombinant fragments of EphA2 enhanced the parasite infection rate, thus establishing its role in P. vivax infection. Furthermore, a new permanent cell line (EphA2Extra-HC04) expressing the whole extracellular domain of EphA2 was generated. This cell line supports a higher rate of P. vivax infection and is a valuable tool for P. vivax liver-stage research.
| S-EPMC9746569 | biostudies-literature
Project description:Plasmodium parasites are highly selective when infecting hepatocytes and induce many changes within the host cell upon infection. While several host cell factors have been identified that are important for liver infection, our understanding of what facilitates the maintenance of infection remains incomplete. Here, we describe a role for phosphorylated ribosomal protein S6 (Ser235/236) (p-RPS6) in Plasmodium yoelii-infected hepatocytes. Blocking RPS6 phosphorylation prior to infection decreases the number of liver stage parasites within 24 h. Infected hepatocytes exhibit elevated levels of p-RPS6 while simultaneously abrogating the induction of phosphorylation of RPS6 in response to insulin stimulation. This is in contrast with the regulation of p-RPS6 by Toxoplasma gondii, which elevates levels of p-RPS6 after infection but does not alter the response to insulin. Our data support a model in which RPS6 phosphorylation is uncoupled from canonical regulators in Plasmodium-infected hepatocytes and is relied on by the parasite to maintain infection.
| S-EPMC6447308 | biostudies-literature
Project description:In vitro human liver models are essential for drug screening, disease modeling, and cell-based therapies. Induced pluripotent stem cell (iPSC)-derived hepatocyte-like cells (iHeps) mitigate sourcing limitations of primary human hepatocytes (PHHs) and enable precision medicine; however, current protocols yield iHeps with very low differentiated functions. The composition and stiffness of liver's extracellular matrix (ECM) cooperatively regulate hepatic phenotype in vivo, but such effects on iHeps remain unelucidated. Here, we utilized ECM microarrays and high content imaging to assess human iHep attachment and functions on ten major liver ECM proteins in single and two-way combinations robotically spotted onto polyacrylamide gels of liver-like stiffnesses; microarray findings were validated using hydrogel-conjugated multiwell plates. Collagen-IV supported higher iHep attachment than collagen-I over 2 weeks on 1 kPa, while laminin and its combinations with collagen-III, fibronectin, tenascin C, or hyaluronic acid led to both high iHep attachment and differentiated functions; laminin and its combination with tenascin or fibronectin led to similar albumin expression in iHeps and PHHs. Additionally, several collagen-IV-, laminin-, fibronectin-, and collagen-V-containing combinations on 1 kPa led to similar or higher CYP3A4 staining in iHeps than PHHs. Lastly, collagen-I or -III mixed with laminin, collagen-IV mixed with lumican, and collagen-V mixed with fibronectin led to high and stable functional output (albumin/urea secretions; CYP1A2/2C9/3A4 activities) in iHep cultures versus declining PHH numbers/functions for 3 weeks within multiwell plates containing 1 kPa hydrogels. Ultimately, these platforms can help elucidate ECM's role in liver diseases and serve as building blocks of engineered tissues for applications. STATEMENT OF SIGNIFICANCE: We utilized high-throughput extracellular matrix (ECM) microarrays and high content imaging to assess the attachment and differentiated functions of iPSC-derived human hepatocyte-like cells (iHep) on major liver ECM protein combinations spotted onto polyacrylamide gels of liver-like stiffnesses. We observed that iHep responses are regulated in unexpected ways via the cooperation between ECM stiffness and protein composition. Using this approach, we induced mature functions in iHeps on substrates of physiological stiffness and select ECM coatings at higher levels over 3+ weeks than analogous primary human hepatocyte cultures, which is useful for building platforms for drug screening, disease modeling, and regenerative medicine.
| S-EPMC9869484 | biostudies-literature
Project description:During liver development, hepatoblasts and liver non-parenchymal cells (NPCs) such as liver sinusoidal endothelial cells (LSECs) and hepatic stellate cells (HSCs) constitute the liver bud where they proliferate and differentiate. Accordingly, we reasoned that liver NPCs would support the maturation of hepatocytes derived from human induced pluripotent stem cells (hiPSCs), which usually exhibit limited functions. We found that the transforming growth factor β and Rho signaling pathways, respectively, regulated the proliferation and maturation of LSEC and HSC progenitors isolated from mouse fetal livers. Based on these results, we have established culture systems to generate LSECs and HSCs from hiPSCs. These hiPSC-derived NPCs exhibited distinctive phenotypes and promoted self-renewal of hiPSC-derived liver progenitor cells (LPCs) over the long term in the two-dimensional culture system without exogenous cytokines and hepatic maturation of hiPSC-derived LPCs. Thus, a functional human liver model can be constructed in vitro from the LPCs, LSECs, and HSCs derived from hiPSCs.
| S-EPMC5549957 | biostudies-literature
Project description:Eradication of malaria is difficult because of the ability of hypnozoite, the dormant liver-stage form of Plasmodium vivax, to cause relapse in patients. Research efforts to better understand the biology of P. vivax hypnozoite and design relapse prevention strategies have been hampered by the lack of a robust and reliable model for in vitro culture of liver-stage parasites. Although the HC-04 hepatoma cell line is used for culturing liver-stage forms of Plasmodium, these cells proliferate unrestrictedly and detach from the culture dish after several days, which limits their usefulness in a long-term hypnozoite assay.A novel immortalized hepatocyte-like cell line (imHC) was evaluated for the capability to support P. vivax sporozoite infection. First, expression of basic hepatocyte markers and all major malaria sporozoite-associated host receptors in imHC was investigated. Next, in vitro hepatocyte infectivity and intracellular development of sporozoites in imHC were determined using an indirect immunofluorescence assay. Cytochrome P450 isotype activity was also measured to determine the ability of imHC to metabolize drugs. Finally, the anti-liver-stage agent primaquine was used to test this model for a drug sensitivity assay.imHCs maintained major hepatic functions and expressed the essential factors CD81, SR-BI and EphA2, which are required for host entry and development of the parasite in the liver. imHCs could be maintained long-term in a monolayer without overgrowth and thus served as a good, supportive substrate for the invasion and growth of P. vivax liver stages, including hypnozoites. The observed high drug metabolism activity and potent responses in liver-stage parasites to primaquine highlight the potential use of this imHC model for antimalarial drug screening.imHCs, which maintain a hepatocyte phenotype and drug-metabolizing enzyme expression, constitute an alternative host for in vitro Plasmodium liver-stage studies, particularly those addressing the biology of P. vivax hypnozoite. They potentially offer a novel, robust model for screening drugs against liver-stage parasites.
| S-EPMC5785895 | biostudies-other
Project description:BackgroundThe sodium taurocholate cotransporting polypeptide (NTCP) is a functional receptor for hepatitis B virus (HBV). NTCP-reconstituted human hepatoma cells support HBV infection, but the infection is suboptimal and no apparent HBV spread has been observed in this system.ResultsWe found that NTCP-reconstituted HepG2 cells were highly susceptible to HBV infection after cells were cultured in a commercial human inducible pluripotent stem cell (iPSC)-derived hepatocyte maintenance medium (HMM). The enhanced HBV infection coincided with increased NTCP expression, and was observed in six different clones of HepG2-NTCP cells. Promoter assays indicated that HMM activated the cytomegalovirus immediate-early (IE) promoter that drives the NTCP expression in the HepG2-NTCP cells. RNA-Seq analysis revealed that HMM upregulated multiple metabolic pathways. Despite highly upregulated NTCP expression by HMM, no obvious HBV spread was observed even in the presence of PEG 8000.ConclusionsOur data suggest that this particular medium could be used to enhance HBV infection in NTCP-reconstituted hepatocytes in vitro.
| S-EPMC8256212 | biostudies-literature
Project description:Alcohol consumption has long been a global problem affecting human health, and has been found to influence both fetal and adult liver functions. However, how alcohol affects human liver development and liver progenitor cells remains largely unknown. Here, we used human induced pluripotent stem cells (iPSCs) as a model to examine the effects of alcohol, on multi-stage hepatic cells including hepatic progenitors, early and mature hepatocyte-like cells derived from human iPSCs. While alcohol has little effect on endoderm development from iPSCs, it reduces formation of hepatic progenitor cells during early hepatic specification. The proliferative activities of early and mature hepatocyte-like cells are significantly decreased after alcohol exposure. Importantly, at a mature stage of hepatocyte-like cells, alcohol treatment increases two liver progenitor subsets, causes oxidative mitochondrial injury and results in liver disease phenotypes (i.e., steatosis and hepatocellular carcinoma associated markers) in a dose dependent manner. Some of the phenotypes were significantly improved by antioxidant treatment. This report suggests that fetal alcohol exposure may impair generation of hepatic progenitors at early stage of hepatic specification and decrease proliferation of fetal hepatocytes; meanwhile alcohol injury in post-natal or mature stage human liver may contribute to disease phenotypes. This human iPSC model of alcohol-induced liver injury can be highly valuable for investigating alcoholic injury in the fetus as well as understanding the pathogenesis and ultimately developing effective treatment for alcoholic liver disease in adults.
| S-EPMC4997049 | biostudies-literature
Project description:Plasmodium vivax is the most widely distributed human malaria parasite. Despite its importance, both clinical research and basic research have been hampered by lack of a convenient in vitro culture system, in part due to the parasite's infection preference of reticulocytes rather than mature erythrocytes. The use of reticulocyte-producing hematopoietic stem cell culture has been proposed for the maintenance of the parasite, but good numbers of reticulocytes and P. vivax parasites sufficient for practical use in research have been difficult to produce from this system. Here, we report an improved method of hematopoietic stem cell culture for P. vivax infection, which requires less time and produces higher or equivalent percentage of reticulocytes than previously reported systems. Reticulocytes were cultured from cryopreserved erythroblasts that were frozen after 8day-cultivation of purified CD34+ cells from human umbilical cord blood. This method of production allowed the recovery of reticulocytes in a shorter time than with continuous stem cell culture. We obtained a relatively high percentage of peak reticulocyte production by using co-cultivation with a mouse stromal cell line. Using P. vivax mature stage parasites obtained from infected Aotus monkeys, we observed substantial numbers (up to 0.8% of the total number of the cells) of newly invaded reticulocytes 24h after initial cultivation. The addition of fresh reticulocytes after 48h culture, however, did not result in significant increase of second cycle reticulocyte invasion. Assays of invasion inhibition with specific antibodies were successful with this system, demonstrating potential for study of biological processes as well as the conditions necessary for long-term maintenance of P. vivax in vitro.
| S-EPMC3943572 | biostudies-literature
Project description:Familial hypercholesterolemia (FH) patients suffer from excessively high levels of Low Density Lipoprotein Cholesterol (LDL-C), which can cause severe cardiovascular disease. Statins, bile acid sequestrants, PCSK9 inhibitors, and cholesterol absorption inhibitors are all inefficient at treating FH patients with homozygous LDLR gene mutations (hoFH). Drugs approved for hoFH treatment control lipoprotein production by regulating steady-state Apolipoprotein B (apoB) levels. Unfortunately, these drugs have side effects including accumulation of liver triglycerides, hepatic steatosis, and elevated liver enzyme levels. To identify safer compounds, we used an iPSC-derived hepatocyte platform to screen a structurally representative set of 10,000 small molecules from a proprietary library of 130,000 compounds. The screen revealed molecules that could reduce the secretion of apoB from cultured hepatocytes and from humanized livers in mice. These small molecules are highly effective, do not cause abnormal lipid accumulation, and share a chemical structure that is distinct from any known cholesterol lowering drug.
| S-EPMC10125972 | biostudies-literature