Cryopreserved cGMP-compliant human pluripotent stem cells-derived immature hepatic progenitors rescue mice from acute liver failure

Abstract

Liver transplantation remains the only curative treatment for end-stage liver diseases. Unfortunately, there is a drastic organ donor shortage. Hepatocyte transplantation emerged as a viable alternative to liver transplantation. In light of their unique expansion capabilities and their potency to be driven towards a chosen cell fate, pluripotent stem cells (PSC) are extensively studied as an unlimited cell source of hepatocytes for cell therapy. It has been previously shown that freshly prepared hepatocyte-like cells can cure mice from acute and chronic liver failures and restore liver functions. In this study, we generated human PSC-derived immature hepatic progenitors (GStemHep) using current good manufacturing practice (cGMP) compliant conditions from PSC amplification, hepatic differentiation to cell cryopreservation. These GStemHep cells present an immature hepatic phenotype (alpha-fetoprotein positive, albumin negative), secrete hepatocyte growth factor (HGF) and do not express MHC type I or II. The therapeutic potential of GStemHep was assessed in two clinically relevant models of acute liver failure. A single dose of thawed GStemHep rescue mice from sudden death caused by acetaminophen and thioacetamide-induced acute live failure, both in immunodeficient and immunocompetent animals in absence of immunosuppression. The mode of action was studied by several analytical methods including unbiased proteomic analyses. The swiftness of the therapeutic effect suggests a paracrine mechanism of action of GStemHep leading to a rapid reduction of inflammation and a rapid cytoprotective effect. Therapeutic biological effects were observed as soon as 3 hours post-cell transplantation with reduction in serum transaminases and in liver necrosis. Mode of action of GStemHep relies on alleviation of inhibition factors of liver regeneration, increase in proliferation- promoting factors and decrease liver inflammation. In conclusion, we generated cGMP-compliant human PSC-derived immature hepatic progenitors that were highly effective in treating acute liver failure. This is also the first report highlighting that human allogeneic cells could be used as cryopreserved cells and in absence of immunosuppression for a human PSC-based regenerative medicine of acute liver injuries.

Poly(ethylene oxide)/Gelatin-Based Biphasic Photocrosslinkable Hydrogels of Tunable Morphology for Hepatic Progenitor Cell Encapsulation

“…PSCs were differentiated into hepatic progenitors (GStemHep) and cryopreserved according to the previously described protocol. 45 For the experiments, hepatic progenitor cells were thawed at 37 °C, pelleted by low-speed centrifugation, and resuspended in RPMI/B27 medium (Life Technologies). Cell count and viability were evaluated using NC-200 NucleoCounter (ChemoMetec).…”