Malaria remains a major cause of death and morbidity worldwide affecting 200 million people annually. Plasmodium falciparum (P. falciparum) is responsible for the majority of the 500,000 deaths attributed each year to malaria, but P. vivax, although less virulent, contributes significantly to malaria morbidity. The infection in humans is initiated by the bite of an infected mosquito which inoculates the parasite into the skin. The parasite then migrates to the liver where it interacts with non parenchymal cells (NPC) before invading the hepatocytes within which it replicates. Once mature, the parasite passes into the blood initiating the erythrocytic stage of its development which is associated with the disease symptoms and transmission. Targeting the parasite before of during its development within the liver is thus an ideal target for prophylactic approaches. But the search for novel or improved means to eliminate malaria necessitates appropriate experimental models. So far, most if not all of our basic knowledge on the parasite-host interactions during the liver stage comes from murine models of experimental malaria. Although these models have non questionable advantages, they do not recapitulate the biology of the human host cells and of the human malaria parasites. There is thus a critical need for developing novel and innovative tools, biologically more relevant to humans for the identification of novel therapeutic targets. Additionally, in vitro studies on P. falciparum or P. vivax liver stages, including screening for new antimalarial compounds, are so far routinely performed using 2D cultures of human hepatocytes within which the parasite development is not efficient and is incomplete. Moreover, these 2D systems recapitulates only partially the physiology of the host cell and do not reproduce the complex 3D architecture of the liver. Yet, it is clearly established that heterotypic cell interactions and 3D systems improve human hepatocyte functions and the predictivity of drug metabolism and toxicity assays. Finally, these 2D monocellular systems preclude any study of the interactions between the parasite and liver NPC which are, so far, largely unknown for human malaria parasites. In this context, and based on the partners extensive knowledge of human Plasmodium liver stage and human hepatic cells isolation and culture, we propose to develop new 3D organoid culture systems, including multicellular systems composed of human hepatocytes and NPC, in order to improve in vitro liver stage development, to assess the cross-talk between the parasite and the NPC and evaluate efficacy of chemotherapeutic and immunoprophylatic interventions targeting Plasmodium liver stages. Finally, implementation of this project will provide marketable systems for applied research in malaria but also in other hepatotropic pathogens.

Chronic liver diseases affect more than 500 million people worldwide. Liver transplantation is the only treatment for severe diseases, but it is limited to a small number of patients due to the shortage of organs. The implantation of bioengineered liver tissue represents a major hope. To this end, it is necessary to propose an efficient strategy for the production and freezing of tissue-engineered products that can be rapidly available at the patient's bed. We have gathered several internationally renowned academic and industrial partners with a high level of expertise in complementary fields (liver biology, human induced pluripotent stem cells (hiPSCs), organoids, encapsulation in biomaterials, and cryopreservation) to address this multidisciplinary challenge. We propose to develop protocols to freeze and thaw encapsulated organoids formed from hepatocytes, mesenchymal stem cells, and endothelial cells all derived from hiPSCs. Their cryopreservation being the major issue of this project. Different processes have already been identified and will be evaluated. Encapsulation in porous alginate beads will ensure the protection of the organoids during these phases and will facilitate future implantation in the patient. Different cellular functions will be studied in vitro during all production steps and evaluated in a preclinical mouse model. This is the first time that a project integrates all these innovations to meet the healthcare needs of the 21st century in liver regenerative medicine. Indeed, the development of cryopreservation is necessary to allow a complete characterisation of the different batches (safety/efficacy) while ensuring the rapid availability of functional and implantable encapsulated organoids in the liver.

Among the short-term tests developed to assess the mutagenicity, genotoxicity or carcinogenicity potency of chemical compounds, the micronucleus (MN) assay is largely recognized as a reliable and precise method for detecting chromosome damage. The MN assay is indeed a multi-target genotoxic endpoint, allowing the assessment of clastogenic and aneugenic events. The assay is amenable for automation and allows good extrapolation for potential limits of exposure, with easy measurement in both in vitro and in vivo experimental systems. However, classical MN assay does not provide a dynamic assessment. We propose to bring innovation in the field of tests for hazard and risk assessment of potential mutagens/carcinogens by developing a new in vitro MN assay. The GENOTRACE public/private consortium aims at monitoring the production of chromosome damage in real time, recording in parallel the transcription signal of a genotoxic stress response reporter. To achieve this aim, the project takes advantage of biotracers newly developed by Partners 1 & 2, first to label the nucleus by expressing a chromobody directed against the H2A-H2B histone complex, allowing to visualize chromatin dynamics in real-time, second to follow the DNA Damage Response (DDR) activation by expression of a p21 regulatory dependent reporter. Thus, a positive MN assay associated with either a positive or negative DDR signal will give insights regarding the MN origin, induced by clastogenic or aneugenic mechanisms respectively. These biotracers will be stably expressed into HepaRG metabolically active cells, optimized for MN assay by Partner 3, and the developed in vitro MN assay will be adapted to a medium- to high throughput straightforward readable assay, thanks to the implementation of high content screening imaging protocols and the development of an image analysis and classification-based pipeline. Overall, as our project will allow to determine and classify the genotoxic potential of substances/contaminants in vitro, and in real time, it will bring new capacity to classic genotoxic assays and may lead to progress in the prevention and/or the diagnosis of exposure to genotoxicants.

Mass-production of high-quality human hepatocytes is at the heart of an economic challenge, biotechnology and biomedical research. Indeed, the liver is the major organ for the destruction and elimination of endogenous metabolic wastes and xenobiotics (pollutants, pesticides, medical drugs). It is responsible for the metabolism of many drugs into active metabolites. However, these active metabolites, or their intermediates may induce liver toxicity. Unexpected problems of toxicity and pharmacokinetics are responsible for most failures in clinical drug development. The liver performs other vital functions such as the production of most of the serum proteins and lipids, bile production, the metabolism of food into nutrients, and glucose homeostasis (storage). Finally, it is the target organ of human specific infectious agents, such as Hepatitis C virus. Thus, there is a major interest of biopharmaceutical industries, scientists and clinicians for having human hepatocytes on demand. Studies on animal models can be misleading because the activity, specificity and intermediate metabolites produced by liver enzymes are often different from their human counterparts. Hepatocytes isolated from human liver biopsies represent the ideal source, but availability of liver biopsies is limited and hepatocytes cannot be amplified in vitro. Liver organs that are not suitable for organ transplantation are scarce and of poor quality (massive steatosis in general). This project associates the INSERM UMR 1064 unit (Nantes) and the society Biopredic (Rennes). The UMR1064 Center of Research in Transplantation and Immunology, develops a research area focuses on the induction of tolerance in transplantation and cell therapy in the Liver. Biopredic is an internationally recognized company specializing in the distribution of primary human cells and in particular human hepatocytes for research, drug development, pharmacology and toxicology. The LabCom program aims to develop a robust and reliable system capable to mass-produce human hepatocytes. The liver has an extraordinary ability to regenerate after injury and unique feature to regenerate from residual hepatocytes present in the liver. Currently, it is not possible to reconstruct in vitro the complex architecture of the liver organ capable of replicating hepatocytes. We will develop an innovative technology based on liver regeneration properties to produce rapidly and massively human hepatocytes in vivo in the rat liver, which has the ideal size as a laboratory bioreactor (1 billion hepatocytes per liver). We will develop a method for the purification of human hepatocytes from humanized livers. We will build large cell banks of cryopreserved human hepatocytes of different genotypes ready for commercialization. In addition, a second innovation of the developed technology is to provide an animal model with a humanized liver capable of modeling human liver fibrosis/ cirrhosis and respond to vaccination. Biopredic company will enrich their intellectual properties and its technological know-how, allowing it to be innovative and become a leadership in the industrial environment of a strong international competition. INSERM UMR1064 will economically develop its know-how and have a humanized liver model in rats for its research in immunology of transplantation and liver biotherapy.