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.

Glycogen storage disease 1a (GSD1a) is a rare metabolic disease due to glucose-6 phosphatase (G6PC) deficiency and it is characterized by short fasting hypoglycemia. Since the 80’s, life expectancy of GSD1a patients has improved considerably thanks to frequent carbohydrate-rich meals and intense nutritional management. Nevertheless, these patients develop long-term chronic kidney disease, eventually leading to renal failure. Renal transplants or dialysis is the only treatment for these patients. Living with an advanced renal disease has important negative impacts on the quality of life and it represents a frequent cause of morbidity. Until recently, the molecular mechanisms involved in GSD1a nephropathy remained unclear, since the GSD1a animal models were hardly viable after weaning. Because of this, we recently developed a mouse model with a G6pc deletion specifically in the kidneys (K.G6pc-/- mice). K.G6pc-/- mice are viable and develop the same renal complications as GSD1a patients. K.G6pc-/- mice exhibit a progressive deterioration in renal functions initiated by early tubular dysfunction, later on by a filtration barrier injury, and finally by glomerulosclerosis development. Then, K.G6pc-/- mice develop renal failure and polycystic kidneys with age. The goal of this project is to improve the diagnosis and treatment of GSD1a patients, by a better understanding of the renal pathology. Using the K.G6pc-/- mice, we can: 1) decipher the molecular mechanisms responsible for the renal pathophysiology; 2) study the effect of diet on these molecular pathways, as well as the progression of the pathology; 3) identify therapeutic targets; 4) identify biomarkers specific for the early stages of the renal pathology, that will allow an early pharmacologic intervention when needed in patients; and 5) develop new gene therapy strategies, including CRISPR/cas9-based genome editing. We would also like to propose an elaboration of a register of GSD1 patients, and a creation of a bio-bank (blood and urine samples) with the agreement of the GSD1 patients registered in the “Centre de référence des maladies héréditaires du métabolisme hépatique (Paris)”, in order to follow the development of nephropathy in GSD1a patients. There are many anabolic pathways that are hyper-activated in the renal tubules due to the excess in glucose-6 phosphate. We will focus on the role of lipid accumulation (lipidomics), and their effects and consequences on the cellular defenses in K.G6pc-/- kidneys. Recently, several new early biomarkers were established in other chronic kidney diseases. We intend to test these biomarkers in K.G6pc-/- mice and in GSD1 patient samples before the microalbuminuria stage, and evaluate if we can develop a more finely-tuned diagnosis method. In order to improve and unify the nutritional advice given to patients, we will study the effect of different diets on the progression of the nephropathy and the renal metabolism as a whole. We also propose to study the effects of several pharmacological agents, alone or combined, in order to target the specific mechanisms involved in GSD1 renal pathology. Finally, the K.G6pc-/- mice are an excellent in vivo tool for testing new viral vectors for targeted gene replacement. The outcome of these studies will be translated into new therapeutic pharmacological and dietary interventions, allowing us to improve the long-term outcome and, thereby the quality of life of GSD1 patients. Identification of biomarkers that appear earlier and are more sensitive could permit a better prevention of the deterioration of renal functions in GSD1 patients.

Orthotopic liver transplantation (OLT) is the only curative treatment currently available for life threatening genetic disorders of the liver and acute liver failure. However, there is a shortage of donor livers and an increasing number of patients die waiting for a liver transplant. Our overall goal is to develop clinical application of human embryonic (hESCs) and human induced pluripotent stem cells (hiPSCs) differentiated into hepatocytes, the cells responsible for liver metabolism. The rationale of our liver-directed regenerative medicine approach is based on clinical trials showing that transplantation of allogeneic human hepatocytes is a safe therapeutic alternative to OLT. Engrafted hepatocytes improved the clinical outcome of several patients with inborn liver diseases. Between 2 to 5 billion hepatocytes must be transplanted to improve the hepatic metabolic function, which are lacking in patients. However, the supply of good quality donor livers for hepatocyte isolation is a major challenge because donor liver organs are scarce and when available prioritized for OLT. In addition, isolated hepatocytes cannot be expanded in culture with current methods. This emphasizes the critical need to develop new, renewable and reliable sources of hepatocytes. Human ESCs and hiPSCs can generate functional hepatocytes (pStemHep) in vitro that can efficiently engraft into the mouse liver. Still, the clinical application of pStemHep faces major challenges. First, dramatic differences between pluripotent stem cell lines have been observed in terms of hepatic differentiation. In addition, so far the results have been obtained with research grade hESC, which quality is insufficient to be used clinically. Thereby, the production of pStemHep from existing GMP (Good Manufacturing Practices) grade hESCs remains to be achieved and GMP-grade hiPSCs has still to be generated. Second, immunodeficient mouse with chronic or acute liver injury is currently the prevalent model for in vivo validation of human pStemHep. However, these models possess limited relevance to inherited liver diseases, thus necessitate the development of more reliable animal models. For instance, in hereditary liver diseases, hepatocytes must be engrafted in the liver and be functional in the long term whereas in acute liver failure, only transient support of hepatic metabolic functions and liver regeneration is required. Thereby, the specific aims of this proposal are as follows: 1. To constitute a GMP-grade cell bank of hESC-derived hepatocytes fulfilling all criteria for clinical application. 2. To generate GMP-grade hiPSCs with validated hepatic differentiation ability. 3. To perform safety and efficacy studies in a relevant rat model of metabolic liver diseases (Crigler-Najjar) that we will create. 4. To provide the proof-of-principle that hepatocytes differentiated from non-human primate stem cells can stably and safely engraft in the liver of recipient animals with sufficient efficacy to expect clinical benefits for transplanted patients. In this closest species to humans with respect to human liver physiology and anatomy, we will use the clinically-relevant cell transplantation procedure that we developed to obtain 5-10% of liver chimerism with 400 millions injected cells. 5. To create a database of patients in order to identify and follow up those are potentially eligible to liver cell therapy, which will accelerate the translation of StemHepTher regenerative medicine to the clinic.