“In our manuscript we provide the first demonstration of directed differentiation toward an endoderm lineage, specifically hepatocytes.”

The ability of a healthy human liver to regenerate is gradually lost in chronic liver disease. The standard treatment for advanced degenerating or inherited metabolic liver diseases (such as cirrhosis and Criggler-Najjar syndrome) has been liver transplantation, but this procedure is limited by the availability of donor tissue. Recently, hepatocyte (liver cell) transplantation has been used to provide liver function when donor organs are unavailable. Unfortunately, primary hepatocytes are sourced from cadaveric tissues, resulting in considerable variability between samples and insufficient supply. In addition, human hepatocytes are also valuable tools for assessing toxicity of new drug candidates, a critical step in drug discovery and drug development. However, the utility of human hepatocytes in either clinical or pharmaceutical applications is limited by: 1) their availability, 2) variability from donor to donor, 3) limited proliferation, and 4) decline in hepatic functions after in vitro culture of these cells. Therefore, there is considerable need for a reproducible, consistent source of functional cells.

Embryonic stem cells may provide another potential source of hepatocytes given their unlimited proliferative and pluripotent differentiative capacity. The isolation of human embryonic stem cells (hESCs) may provide a solution to this problem. First isolated in 1998, hESCs appear to have an unlimited proliferative capacity and can differentiate into derivatives of all three germ layers; ectoderm, mesoderm, and endoderm. These cell lines are generated by removing the inner cell mass (ICM) from preimplantation blastocysts and growing these cells for prolonged periods of time. hESCs have been maintained in vitro for over 250 population doublings and show remarkable phenotypic and karyotypic stability. Therefore, hESCs may serve as an appropriate cell source for cell replacement strategies and drug screening. However, the use of hESC cells for cell replacement requires careful study of the undifferentiated hESCs and their differentiated progeny. The generation of cell populations for cell therapies and drug screening will require: 1) the identification of an appropriate cell population, 2) thorough characterization of this population, 3) optimization of the differentiation process(es), 4) optimization of the transplantation, methodologies, 5) demonstration of efficacy in animal models after transplantation and 6) long term safety assessment after transplantation.

To date, hESCs have been shown to differentiate into a variety of cell lineages, such as neural cells, cardiomyocytes, hematopoietic cells, and endothelial cells, but no reports have demonstrated the generation of hepatocytes from hESCs in vitro. In our manuscript we provide the first demonstration of directed differentiation toward an endoderm lineage, specifically hepatocytes. Our data show that hESCs have the capacity to differentiate into cells resembling hepatocytes using at least two differentiation protocols. We describe the generation of an enriched cell population which express markers consistent with hepatocytes and show that this population has functional enzymatic activity similar to hepatocytes. hESC-derived hepatocyte-like cells express appropriate molecular and biochemical markers, such as albumin, AAT, and glycogen storage, but lack the expression of the more immature hepatic marker, AFP. The cells also show inducible cytochrome P450 activity, consistent with a functional drug metabolism phenotype. Although these populations show many similarities to primary human hepatocytes, we did not demonstrate equivalence to mature hepatocytes, indicating that more optimization may be necessary. This work represents a very important first step toward the use of hESCs in cell therapies and drug screening.

In summary, we have successfully derived differentiated cells with hepatocyte features from hESC cell cultures that have been maintained for more than 40 passages (approximately 200 population doublings). Because hESCs are stable and have remarkable proliferative capacity, they provide an abundant, uniform, and reproducible source of cells as starting material. Combined with our direct differentiation protocol that enables large scale production, hESC-derived hepatocyte-like cells will prove valuable for a variety of applications, such as toxicity testing, drug screening, and therapeutic transplantation.