In vitro recovery of FIX clotting activity as a marker of highly functional hepatocytes in a hemophilia B iPSC model.

Pluripotent stem cell-derived hepatocytes differentiated in monolayer culture are known to have more foetal than adult hepatocyte characteristics. If numerous studies tend to show that this immature phenotype might not necessarily be an obstacle to their use in transplantation, other applications such as drug screening, toxicological studies or bioartificial livers are reliant on hepatocyte functionality and require full differentiation of hepatocytes. New technologies have been used to improve the differentiation process in recent years, usually evaluated by measuring the albumin production and CYP450 activity. Here we used the complex production and most importantly the activity of the coagulation factor IX (FIX) produced by mature hepatocytes to assess the differentiation of Hemophilia B patient's induced pluripotent stem cells (iPSCs) in both monolayer culture and organoids. Indeed, Hemophilia B (HB) is an X-linked monogenic disease due to an impaired activity of FIX synthesized by hepatocytes in the liver. We have developed an in vitro model of HB hepatocytes using iPSCs generated from fibroblasts of a severe HB patient. We used CRISPR/Cas9 technology to target the genomic insertion of an F9 mini-gene bearing the Padua mutation to enhance FIX activity. Non-corrected and corrected iPSCs were differentiated into hepatocytes under both 2D and 3D differentiation protocols and deciphered the production of active FIX in vitro. Functional FIX, whose post-translational modifications only occur in fully mature hepatocytes, was only produced in corrected iPSCs differentiated in organoids. Finally, we assessed the therapeutic efficacy of this approach in vivo using a mouse model of HB. Immunohistochemistry analyses indicated a good cell engraftment and the FIX activity detected in the plasma of transplanted animals confirmed rescue of the bleeding phenotype.