Human hepatobiliary progenitor cells as new candidates for liver cell therapy

Guillaume Rommelaere, Kris Gellynck, Tuba Baran, Sarah Snykers, Luca Falciola, Philippe Stordeur, Mustapha Najimi, Giuseppe Mazza, Etienne Sokal

Background: Orthotopic liver transplantation is the only treatment for patients with end-stage liver diseases. However, this methodology is limited due to a shortage of organ donors and liver cell therapy has been suggested as an alternative treatment. Injection of hepatocytes has been tested but this method suffers from practical limitations (difficulty in expansion and cryopreservation). As a solution to this problem, we isolated human hepatobiliary progenitor cells (H2Stem) from unsuitable livers for transplantation. H2Stem proliferate in culture, show high hepatic functionality in vitro and engraft in vivo in pre-clinical models.

Methods: Human livers segments were dissociated using a two-step perfusion method and the parenchymal cell fraction isolated by low speed centrifugations. When put in adherent culture, a population of epithelial-like proliferating cells develops from the liver cell suspensions and grows for several passages in vitro. The expression profile of hepatobiliary stem cells was determined pre- and post-differentiation by a full transcriptomic analysis and confirmed by immunostaining and flow cytometry. In addition, the hepatic functionality of this cell population was evaluated after in vitro differentiation in 2D as well as in 3D cultures. Lastly, their ability to repopulate and differentiate in damaged livers was assessed by the infusion in FRG mice models.

Results: Gene expression analysis and flow cytometry analysis indicated that the proliferative cell population isolated from human livers expresses both biliary and hepatic markers. After in vitro differentiation (2D or 3D), H2Stem cells present high CYP3A4 activities and the ability to secrete hepatic markers such as albumin or alpha-1-antitrypsin. Notably, H2Stem cells are able to repopulate livers from the immunodeficient FRG mice after intra-splenic injection. Engrafted islets stain negatively for CK19 and positively for albumin, alpha-1-antitrypsin, ornithine transcarbamylase and arginase. Furthermore, human albumin and alpha-1-Antitrypsin are detected in the blood of infused animals thus confirming in vivo differentiation.

Conclusion: In this study hepatobiliary progenitor cells isolated from human livers present an ability to grow in culture and to engraft in livers of FRG mice as well as showing hepatic functionality in vivo. Developments are currently ongoing to further expand H2Stem stem cells in vitro under GMP-like conditions before evaluating safety and efficacy in patients affected by liver diseases.