DS0404 - Innovation biomédicale

Preclinical validation of hepatocytes derived from pluripotent stem cells for liver biotherapy – StemHepTher

Clincal development of regenerative medicine with pluripotent stem cells for treating liver diseases

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 Liver transplantation. Engrafted hepatocytes improved the clinical outcome of several patients with inborn liver diseases. However, this cell therapy is limited by the shortage of donor liver organs. In addition, isolated hepatocytes cannot be expanded in culture with current methods.

Production of hepatocytes using an industrialisable ann cGMP process from pluripotent stem cells

There is a critical need to develop new, renewable and reliable sources of hepatocytes. The major objective of the project is use pluripotent stem cells as an unlimited in vitro source of hepatocytes. <br />The specific aims of this proposal are as follows: <br />1. To constitute a GMP-grade cell bank of hESC-derived hepatocytes fulfilling all criteria for clinical application. <br />2. To generate GMP-grade hiPSCs with validated hepatic differentiation ability. <br />3. To perform safety and efficacy studies in a relevant rat model of metabolic liver diseases (Crigler-Najjar) that we will create. <br />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. <br />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.

Existing GMP-grade hESC lines are screened for their hepatic differentiation ability using our GMP-compatible differentiation protocols consisting in induction of endoderm differentiation and then differentiation into hepatic progenitors (pStemHep). The quality of differentiation is evaluated by analysing the expression of hepatic markers (HNF4, AFP, etc..) and the loss of expression of pluripotency markers by Rt-qPCR and immunofluorescence.
Therapeutic potential of pStemHep will be assessed in the rat model Crigler-Najjar, the Gunn rat.
To have a first therapeutic valdiation, we have first set up a model of Gunn rat that is immunosuppressed by tacrolimus using human pluripotent stem cells of research grade.
The Gunn rat was immunosuppressed by daily injection of tacrolimus and were transplanted with pStemHep in the liver via the spleen. Therapeutic efficacy was evaluated by the level of serum bilirubin.

We have identified a GMP ES cell line that has the ability to differentiate into hepatocytes according to our production process.
After transplantation of pStemHep derived from research-grade pluripotent stem cells, we observed a significant decrease in bilirubinélia (30%), which is equivalent to the resuts obtained after injection of the cell gold standard, hepatocytes isolated from a human liver.

We have now established robust cell culture conditions to produce hepatocytes from simian pluripotent stem cells that we generated fro simian fibroblasts.

We have established a list of inclusion and exclusion for Crigler-Najjar patients. Nowadays, we have identifed 16 potential patients. We have participated to the creation of a registry of Crigler-Najjar patients.

Perspectives are now to produce hepatocytes from the selected GMP ES line and to evaluate their therapeutic potential for treating the Gunn rat, but also another orphan disease, the acute liver failure that we model in mice by paracetamol intoxication.
We will consitute a GMP ES bank using our conditions.
We will also label our simian hepatocytes by expressing GFP and human FIX, which will allow to follow the in vivo functionality of these hepatocytes after cell transplantation in the liver.

Patent Inserm n°EP 15 305 265.9, « Use of laminines for differentiating pluripotent stem cells into hepatic cells lineage »).

Articles:
1. Tolosa L, López S, Pareja E, Donato MT, Myara A, Nguyen TH, Castell JV, Gómez-Lechón MJ. Human neonatal hepatocyte transplantation induces long-term rescue of unconjugated hyperbilirubinemia in the Gunn rat.
Liver Transpl. 2015 Jun;21(6):801-11.
2. Ménoret S, Tesson L, Remy S, Usal C, Ouisse LH, Brusselle L, Chenouard V, Nguyen TH, David L, Anegon I. Transgenic animals and genetic engineering techniques. Nantes, France, 2-3 July, 2015. Transgenic Res. 2015 Dec;24(6):1079-85.
3. Blasimme A, Anegon I, Concordet JP, De Vos J, Dubart-Kupperschmitt A, Fellous M, Fouchet P, Frydman N, Giovannangeli C, Jouannet P, Serre JL, Steffann J, Rial-Sebbag E, Thomsen M, Cambon-Thomsen A. Genome Editing and Dialogic Responsibility: «What's in a Name?«. Am J Bioeth. 2015 Dec;15(12):54-7.
4. Hannoun Z, Steichen C*, Dianat N*, Weber A, Dubart-Kupperschmitt A. The Potential of Induced Pluripotent Stem Cell derived Hepatocytes. J Hepatol, 2016, 10.1016/j.jhep.2016.02.025, in press
5. Pourcher G, El-Kehdy H, Kanso F, Groyer-Picard MT, Gaillard M, Trassard O, Blazsek I, Agostini H, Dubart-Kupperschmitt A*, Dagher I*. Volumetric Portal Embolization: A New Concept to Improve Liver Regeneration and Hepatocyte Engraftment. Transplantation. 2016, 2016 Feb;100(2):344-54.
6. Tolosa L, Caron J, Hannoun Z, Antoni M, López S, Burks D, Castell JV, Weber A, Gomez-Lechon MJ*, Dubart-Kupperschmitt A.* Transplantation of hESC-derived hepatocytes protects mice from liver injury. Stem Cell Res Ther. 2015 Dec 12;6(1):246. doi: 10.1186/s13287-015-0227-6.
7. Dianat, N., Dubois-Pot-Schneider, H., Steichen, C., Desterke, C., Leclerc, P., Raveux, A., Combettes, L., Weber, A., Corlu, Dubart-Kupperschmitt, A.* Generation of functional cholangiocyte-like cells from human pluripotent stem cells and HepaRG cells. Hepatology 2014 60, 700-14.4.

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.

Project coordinator

Monsieur Ignacio Anegon (INSERM UMR_S 1064)

The author of this summary is the project coordinator, who is responsible for the content of this summary. The ANR declines any responsibility as for its contents.

Partner

APHP AP-HP, Hôpitaux Universitaires Paris Sud, site Antoine Béclère
University of Cambridge
Cellgenix
GOLIVERTX GOLIvER THERAPEUTICS
UMR_S 1064 INSERM UMR_S 1064
UMR_S 972 INSERM U972

Help of the ANR 766,921 euros
Beginning and duration of the scientific project: September 2014 - 48 Months

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