The use of hepatocyte transplantation as a clinical alternative to whole organ transplant has been limited by the lack of sufficient numbers of functionally proficient cells. Stem cell derived hepatocytes have been proposed as an alternative source of cells for transplantation. Several research groups have established protocols to differentiate various stem cell types into definitive endoderm, and then into cells with hepatocyte characteristics (14
). These reports describe varying degrees of success, and researchers are still confronted with ethical issues related to the use of stem cells derived from human embryos or fetuses. Placenta-derived stem cells are isolated from a tissue that is normally discarded after a live birth. Moreover, they retain characteristics of ESCs, thus representing a novel source of cells for clinical application. It is commonly accepted that ESCs need to differentiate to definitive endoderm prior to further differentiation to endoderm-derived cell types (36
). Activin-A, a member of the TGFβ family, can have different effects on stem cells, depending on their source and stage of differentiation. Several investigators reported that endodermal differentiation of some ESC lines is enhanced by exposure to Activin-A (29
) while recent work from other groups suggests that Activin/Nodal signaling might inhibit the early stages of ESC differentiation in vitro
, by playing a key role in maintaining an undifferentiated state (38
In these studies, hAECs did not express endoderm markers after treatment with Activin-A, but rather upregulated the expression of stem cell genes (). Also, Activin-A did not improve long term hepatic differentiation of hAECs, suggesting that this regulatory protein is not required for endoderm differentiation of hAECs (Supplemental Figure 1
The idea that the liver microenvironment may be critical for the induction of hepatic differentiation has been supported by the results obtained co-culturing ESCs with different hepatic cell types (i.e. hepatocytes, stellate cells) (41
).. Co-culture with mouse hepatocytes improved hepatic differentiation of hAECs (), which were shown to express markers of mature hepatocytes along with metabolically active and inducible CYP3A enzymes (). Co-culture with mouse hepatocytes is a difficult and inconvenient way to induce hepatic differentiation of hAECs. We surmised that hepatocyte conditioned media might provide the same inductive influence in a protocol more easily standardized. Unfortunately, no strong hepatic inductive effect in gene expression was observed with human hepatocyte conditioned media (data not shown) which suggests that interaction with neighboring cells enhances hepatic commitment of hAECs.
When cell to cell interaction is lost, basement membrane matrix proteins are critical to the maintenance of a differentiated state in primary human hepatocytes (26
). Extracellular matrices (ECM) were utilized as a substrate for differentiation of hAECs (). Interestingly, matrigel, a commercial matrix preparation which is known to enhance and maintain differentiation of adult hepatocytes, was ineffective at inducing hepatic differentiation of hAE cells. However, L-ECM was shown to strongly induce expression of mature hepatocyte marker genes and activities ( and ). Differentiated hAECs were able to metabolize Ammonia, 17-OHPC and possessed inducible CYP3A and 1A enzymes (). The in vitro
results suggest that the presence of extracellular matrix proteins together with a cocktail of growth factors, cytokines and hormones are required for proficient differentiation of hAECs into hepatocyte-like cells.
Although these results are promising, the expression levels of hepatocyte genes of in vitro
differentiated hAECs were closer to those of fetal cells rather than adult hepatocytes (supplemental table 2
). Although expression is low, hAECs expressed genes characteristic of adult human liver. CYP 3A4 and 1A2 are typically expressed in adult hepatocytes, while 3A7 and 1A1 are more highly expressed in fetal cells (42
). Hepatocyte-like cells derived from hAECs also metabolized drugs in a manner similar to fetal human hepatocytes, as shown by the metabolism of 17-OHPC (). A characteristic of some of the CYP and phase II enzymes is that their expression can be induced by prior exposure to prototypical inducing agents (31
). Differentiated hAECs expressed CYP 3A7, 1A1 and UGT1A1 which were induced by exposure to PB or BNF (); however, in the current studies, CYP 3A4 and 1A2 were not responsive to the treatment unless the hAECs were co-cultured with mouse hepatocytes () suggesting that the liver microenvironment significantly contributes to the hepatic induction of the hAECs. Extremely important in the interpretation of these results with the mouse co-culture experiments is the observation that prior exposure of the hAECs cocultures to Rif induced the metabolism of TE to the 6-β-hydroxy metabolite, a standard assay for human CYP3A4 (31
). Since Rif is a specific inducer of human CYP3A4 with little or no activity toward mouse CYP3A genes/activities (32
), these results clearly indicate that the CYP3A metabolic activity observed in these experiments results from the human cells present in the cultures. Another compound, PB, which induces both the mouse and human CYP3A genes shows a moderate induction of metabolic activity in the cultures of mouse alone, and a more robust activity when the human cells are present. These results strongly suggest that the difference in metabolic activity between the hAEC/mouse co-cultures and the cultures with only mouse cells can be attributed to the induction of CYP3A4 in the hAECs.
Given only the partial differentiation of hAECs to hepatocyte-like cells, in vitro
, and the strong inductive influence of the mouse hepatocyte co-culture experiments, we examined the fate of the cells following transplantation into mouse liver. Profiling of genes normally expressed in adult human liver, with PCR primers that are specific for human transcripts, revealed a mature level of expression of 23 out of 24 genes examined () in the hAECs in mouse liver at 6 months following transplantation. These results suggest that the hAECs can differentiate to mature hepatocyte-like cells following transplantation, in vivo
. In support of this hypothesis, hAECs transplants were recently shown to be effective for the correction of the serum amino acid and brain neurochemical imbalances normally observed in a mouse model of Maple Syrup Urine Disease (43
The well characterized Retrorsine-based model of liver repopulation was used for the in vivo
studies described above (35
); however only low levels of repopulation with human cells were observed (<3%). RS treatment is known to be less effective on mice than on rats. In order to test the ability of amniotic cells to engraft and replicate in the liver, a syngenic rat model was used (35
). RS-treated DPPIV−
rats were transplanted with DPPIV+
, but otherwise syngenic rAECs. Large clusters of DPPIV+, rAEC-derived hepatocyte-like cells were observed, indicating that rAECs were able to engraft and incorporate into the parenchyma to form cells with morphology typical of mature hepatocytes. These cells were positive for Albumin, CYP2E1 and 3A1 (). Based on the results obtained in the in vivo
studies, we believe that the liver microenvironment itself strongly induces hepatocyte differentiation of amniotic epithelial cells. This study demonstrates that amniotic epithelial cells differentiate, in vitro
, into hepatocyte-like cells with characteristics of fetal hepatocytes while in vivo
they mature into cells with a gene expression profile comparable to adult hepatocytes. Genes involved in metabolic liver disease such as OTC, A1AT and UGT1A1 and BSEP, were highly expressed in hAECs after transplantation. We suggest that hAECs represent a non controversial source of cells for liver-based regenerative medicine.