Production of hepatocytes from human pluripotent stem cells for cell-based therapy
Human pluripotent stem cells (hPSCs) can self-renew indefinitely in vitro while maintaining the capacity to differentiate into diverse cell types, including hepatocytes. Thus, hPSCs allow for large-scale production of hepatocytes necessary for cell-based therapy. Importantly, cell-based therapy involving the transplantation of hepatocytes-like cells (HLCs) represent a promising alternative to liver transplantation. However, there are two major limitations that must be overcome before hPSCs derived hepatocytes can go into clinical trials. Firstly, current protocols are not compatible with Good Manufacturing Practice (GMP) as they rely on culture media which are poorly defined and contain reagents of animal origin. Secondly, HLCs derived in vitro lack the full spectrum of adult hepatocyte functionalities and expressed foetal markers indicating that they are not equivalent to primary cells. In this dissertation, I describe a method for generating a homogenous population of hepatocytes from hPSCs in conditions compatible with clinical applications. Furthermore, I show that the resulting GMP-like protocol is equally robust as research grade protocol to produce HLCs displaying key characteristics of their in vivo counterparts. Importantly, the efficacy of this new GMP protocol was validated in five different hPSCs lines, including three GMP grade human Embryonic Stem Cell lines. Finally, we have also characterized the resulting cells in vivo and demonstrated their safety after transplantation into immune deficient mice. In parallel, I have optimised our protocol of differentiation by establishing a 5 steps method that closely follows liver development. For the development of this protocol, I have screened a large number of culture conditions and identified optimum time points for each step especially for hepatoblast and foetal hepatocytes stage. Overall, the work presented in this dissertation has advanced the differentiation of hPSCs for cell-based therapy in two different ways. Firstly, it shows that production of HLCs in GMP like condition is feasible, safe and that the resulting cells could be useful for cell-based therapy in the context of liver diseases. Secondly, I have developed a new protocol with an optimised hepatoblast stage that will facilitate further clinical applications.
Hepatocytes, Cell based therapy, regenerative medicine, liver, liver differentiation, hepatocytes differentiation
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