In these studies, we present a simple and reproducible method for generating functional human hepatocytes from pluripotent ES cells. Although successful methods for hepatic differentiation of hES cells have been described, none have generated cells with function adequate for clinical use 8, 12, 28–30
. We have not determined whether co-culture with liver non-parenchymal cells might increase the efficiency of hepatic differentiation, as has been described for mouse ES cell differentiation 31
. Hepatic differentiation, greater than the 18–26% described here, would be desirable, but the simplicity of the protocol may facilitate clinical application and the eventual scaling up that will be required to generate ES-derived hepatocytes in numbers that could be used in patients.
The protocol we describe incorporates a step involving generating EBs. However, based on the work of D’Amour et al 32
, we have performed a number of additional real-time PCR and albumin and urea production experiments that show essentially identical hepatocyte-specific differentiation whether or not the EB formation step is included in the differentiation program. In fact, there is more extensive and earlier loss of Oct 3/4, Nanog, Sox7, and AFP when the EB step is removed (Supplementary Figure 3
). In addition, our ability to induce differentiation using KSR instead of serum allows removal of animal products from the process, an important consideration for clinical application.
A critically important component of the differentiation protocol relates to enrichment for ES-derived hepatocytes. ASGPR expression is unique for liver cells 33
. While 55% of differentiated cells expressed albumin by immunohistochemistry, significantly fewer cells expressed ASGPR, indicating that enrichment based on ASGPR expression may be more selective than sorting based on previously-described gene transfer techniques using reporter genes driven from liver-specific promoters. The present strategy does not depend on transduction efficiency for selecting a relatively homogeneous population of cells. While we employed flow sorting for enrichment, this technique is relatively time consuming and can lead to significant cell injury. In addition, its efficacy can be affected by sorting speed, which can seriously affect the yield and viability of the differentiated ES cells recovered and allow recovery of unwanted cells. The ASGPR-based sorting approach outlined, however, would also be amenable to enrichment by magnetic sorting 34
or panning on antibody coated plates. Such strategies might be more efficient than flow sorting since the steps can be performed repeatedly for enhanced selection, and would result in significantly less damage to the recovered cells. In addition, they might be faster and more appropriate for large scale high-throughput enrichment than flow cytometry.
As demonstrated in our studies, tumor risk remains an issue that must continue to be addressed. It appears that neither the number of cells that can be transplanted into immune deficient mice nor the length of time transplanted rodents can be followed will be adequate to unequivocally determine whether cell preparations are safe for clinical use. Large scale studies, performed in non human primates using frozen stocks of differentiated cells, when possible, may be needed for such an analysis. While ES cell-derived hepatocytes may not be immediately useful for transplantation therapies, they are likely to find early application for the study of human drug metabolism and drug discovery. Mature human livers express important drug metabolizing enzymes 35
, some of which are inducible following exposure to phenobarbital or (BNF) 23, 24
. Our studies indicate that ES-derived hepatocytes express human CYP genes at levels near those of adult human hepatocytes, and that prior exposure to BNF results in robust induction in the metabolism of ethoxyresorufin, a known substrate for CYP 1A1/2, by both ES-derived hepatocytes and normal human liver cells. Our studies also show that ES-derived hepatocytes and normal human hepatocytes convert testosterone to 6-beta-hydroxytestosterone, a specific measure of CYP 3A4-mediated metabolism, to a similar degree in culture. While prior exposure to phenobarbital did not increase the metabolism of testosterone in ES-derived cells, as occurs with normal human hepatocytes (data not shown), the CYP metabolic activity demonstrated by ES-derived cells is substantial. It is possible that further maturation of CYP functional activity may require differentiation on a more physiologic extracellular matrix or interaction with other cells.
While we have examined the use of hES cells in this series of experiments, successful generation of hepatocytes from precursors derived from individual patients 36
could lead to the development of individualized patient-specific drug regimens and might eventually be employed to circumvent the need for life-long immune suppression following hepatocyte transplantation. In summary, these studies provide a foundation for efficient development of functional human hepatocytes from hES cells. Further studies will be needed to determine whether the differentiation protocol and enrichment strategy outlined can be scaled for use in patients and can be modified to eliminate the risk of contaminating cells and risk of tumor formation following transplantation. Application of ES-derived hepatocytes for the study of human drug metabolism and drug discovery, however, may soon be possible.