Murine Embryonic Stem Cell-Derived Hepatic Progenitor Cells Engraft in Recipient Livers with Limited Capacity of Liver Tissue Formation

Abstract

Directed endodermal differentiation of murine embryonic stem (ES) cells gives rise to a subset of cells with a hepatic phenotype. Such ES cell-derived hepatic progenitor cells (ES-HPC) can acquire features of hepatocytes in vitro, but fail to form substantial hepatocyte clusters in vivo. In this study, we investigated whether this is due to inefficient engraftment or an immature phenotype of ES-HPC. ES cells engrafted into recipient livers of NOD/SCID mice with a similar efficacy as adult hepatocytes after 28 days. Because transplanted unpurified ES-HPC formed teratomas in the spleen and liver, we applied an albumin promoter/enhancer-driven reporter system to purify ES-HPC by cell sorting. RT-PCR analyses for hepatocyte-specific genes showed that the cells exhibited a hepatic phenotype, lacking the expression of the pluripotency marker Oct4, comparable to cells of day 11.5 embryos. Sorted ES-HPC derived from beta-galactosidase transgenic ES cells were injected into fumaryl-acetoacetate-deficient (FAH(-/-)) SCID mice and analyzed after 8 to 12 weeks. Staining with X-gal solution revealed the presence of engrafted cells throughout the liver. However, immunostaining for the FAH protein indicated hepatocyte formation at a very low frequency, without evidence for large hepatocyte cluster formation. In conclusion, the limited repopulation capacity of ES-HPC is not caused by a failure of primary engraftment, but may be due to an immature hepatic phenotype of the transplanted ES-HPC.

Repopulation Efficiencies of Adult Hepatocytes, Fetal Liver Progenitor Cells, and Embryonic Stem Cell-Derived Hepatic Cells in Albumin-Promoter-Enhancer Urokinase-Type Plasminogen Activator Mice

“…38 For hepatic differentiation, 600 ESCs were cultivated in hanging drops (20 l), as described previously to form embryoid bodies. On day 5, embryoid bodies were plated onto gelatinized dishes in culture medium II (IMDM; Invitrogen) supplemented with 20% FCS, 450 mol/L ␣-monothioglycerol (Sigma) and all additives except leukemia inhibitory factor.…”

“…38 Cells, 5 ϫ 10 5 , (viability Ͼ85% by trypan blue exclusion) were transplanted via the intrasplenic route into the liver of C57BL/6 mice. After 24 hours, 48 hours, 14 days, and 28 days, the livers (n ϭ 4 for each time point) were harvested and native fluorescence microscopy was performed on cryo-sections (10 m) to detect and count PKH26 positive cells.…”

Induced pluripotent stem cell–derived hepatocytes have the functional and proliferative capabilities needed for liver regeneration in mice

“…However, only cells that have the same functional and proliferative capabilities as normal adult hepatocytes are effective in liver repopulation. Cells that exhibit incomplete differentiation and impaired proliferation, a prominent example being hepatocytelike cells generated from ES cells with current in vitro differentiation proto cols, fail to restore liver function in FAHdeficient mice or similar liver repopulation models (16)(17)(18).…”

Cell Transplantation: A Possible Alternative to Orthotopic Liver Transplant (OLT)

“…review by Banas et al 2007) but few have determined the cells significantly contribute to improved liver function and regeneration. Induction rates remain low regardless of the method used, and general hepatic function of the cells, even once transplanted, were very limited when compared to mature hepatocytes (Sharma et al, 2008). However, a successful report described ESCs demonstrating liver function able to overcome liver damage in mice (Heo et al, 2006).…”