Hepatic Stellate Cells Improve Engraftment of Human Primary Hepatocytes: A Preclinical Transplantation Study in an Animal Model.

Human hepatocytes are used for liver cell therapy, but the small number of engrafting cells limits the benefit of cell transplantation. We tested whether cotransplantation of hepatocytes with hepatic stellate cells (HSCs) could improve hepatocyte engraftment in vivo. Human primary hepatocytes were transplanted into SCID mice either alone or in a mixture with HSCs (quiescent or after culture activation) or LX-2 cells (ratio 20:1). Four weeks after transplantation into mouse livers, human albumin-positive (huAlb(+)) hepatocytes were found scattered. When cotransplanted in a mixture with HSCs or LX-2 cells, huAlb(+) hepatocytes formed clusters and were more numerous occupying 2- to 5.9-fold more surface on the tissue section than in livers transplanted with hepatocytes alone. Increased huAlb mRNA expression in livers transplanted with the cell mixtures confirmed those results. The presence of HSCs increased the number of hepatocytes entrapped in the host liver at an early time point posttransplantation but not their proliferation in situ as assessed by cumulative incorporation of BrdU. Importantly, 4 weeks posttransplantation, we found no accumulation of αSMA(+)-activated HSCs or collagen deposition. To follow the fate of transplanted HSCs, HSCs derived from GFP(+) mice were injected into GFP(-) littermates: 17 h posttransplant, GFP(+) HSCs were found in the sinusoids, without proliferating or actively producing ECM; they were undetectable at later time points. Coculture with HSCs improved the number of adherent hepatocytes, with best attachment obtained when hepatocytes were seeded in contact with activated HSCs. In vivo, cotransplantation of hepatocytes with HSCs into a healthy liver recipient does not generate fibrosis, but significantly improves the engraftment of hepatocytes, probably by ameliorating cell homing.

Adult human liver mesenchymal stem/progenitor cells participate in mouse liver regeneration after hepatectomy.

The advances in stem cell science have promoted research on their use in liver regenerative medicine. Beyond the demonstration of their ability to display metabolic functions in vitro, candidate cells should demonstrate achievable in situ differentiation and ability to participate to liver repopulation. In this work, we studied the in vivo behavior of adult liver mesenchymal stem/progenitor cells (ADHLSCs) after transplantation into immunodeficient mice. The kinetics of engraftment and in situ hepatogenic differentiation were analyzed. Response of transplanted ADHLSCs to regenerative stimulus was also evaluated. Nondifferentiated ADHLSCs were intrasplenically transplanted into SCID mice. Efficiency of transplantation was evaluated at the level of engraftment and in situ differentiation using immunohistochemistry, in situ hybridization, and RT-PCR. After bromodeoxyuridine (BrdU) implantation, proliferation of transplanted ADHLSCs in response to 20% hepatectomy was assessed using immunohistochemistry. We demonstrated that ADHLSC engraftment in the SCID mouse liver was low but remained stable up to 60 days posttransplantation, when albumin (ALB) immunopositive ADHLSCs were still detected and organized as clusters. Coexpression of ornithine transcarbamylase (OTC) demonstrated ADHLSC in situ differentiation mostly near the hepatic portal vein. After 20% hepatectomy on 1 month transplanted mice, the percentage of BrdU and human ALB immunopositive ADHLSCs increased from 3 to 28 days post-BrdU implantation to reach 31.3 ± 5.4% of the total analyzed human cells. In the current study, we demonstrate that transplanted ADHLSCs are able to differentiate in the non preconditioned SCID mouse liver mainly in the periportal area. In response to partial hepatectomy, integrated ADHLSCs proliferate and participate to recipient mouse liver regeneration.

Adult-derived human liver progenitor cells in long-term culture maintain appropriate gatekeeper mechanisms against transformation.

The use of human liver progenitor cells in the development of clinical cell therapy depends on their constant availability and unaltered properties during culture. The present study investigates the effects of long-term in vitro culture on the specific characteristics of these cells and on their genetic stability. Adult-derived human liver progenitor cells (ADHLPCs) were isolated from 12 donors and cultured until senescence and cell death. Cells were analyzed at different time points for their phenotype stability and differentiation potential. In addition, growth characteristics, chromosomal karyotype, telomere maintenance mechanisms, and activity of cell cycle-related genes were studied. Finally, their in vivo tumorigenicity was investigated in a xenograft assay. The long-term culture of ADHLPCs revealed a variable proliferation capacity. Cells maintained their original phenotype and acquired hepatocyte-like metabolic functions after differentiation. Eight of the 12 cell populations grew fast (doubling time of 6.3 days) during a limited time period (mean, 116.2 days), and mainly presented normal cytogenetic features. The four other cell cultures presented an early decline in growth potential (doubling time of 28.6 days) and premature senescence. Chromosomal alterations were detected in three of four cultures at passage 6. Cytogenetic anomalies were not correlated with tumorigenic potential in vitro or in vivo, and expression of cell cycle-related genes was appropriately upregulated, inducing senescence. Although chromosomal anomalies may occur in long-term cell cultures, neither transformation nor alteration of their characteristics was noted during in vitro expansion. All ADHLPCs reached senescence and growth arrest. Presenescent ADHLPCs might therefore be considered as a suitable source for liver-based cell therapy.

In vitro differentiated adult human liver progenitor cells display mature hepatic metabolic functions: a potential tool for in vitro pharmacotoxicological testing.

The potential use of stem/progenitor cells as alternative cell sources to mature hepatocytes remains basically dependent on their ability to exhibit some, if not all, the metabolic liver functions. In the current study, four major liver functions were investigated in adult derived human liver stem/progenitor cell (ADHLSCs) populations submitted to in vitro hepatogenic differentiation: gluconeogenesis, ammonia detoxification, and activity of phase I and phase II drug-metabolizing enzymes. These acquired hepatic activities were compared to those of primary adult human hepatocytes, the standard reference. Amino acid content was also investigated after hepatogenic differentiation. Differentiated ADHLSCs display higher de novo synthesis of glucose correlated to an increased activity of glucose-6 phosphatase and mRNA expression of key related enzymes. Differentiated ADHLSCs are also able to metabolize ammonium chloride and to produce urea. This was correlated to an increase in the mRNA expression of relevant key enzymes such arginase. With respect to drug metabolism, differentiated ADHLSCs express mRNAs of all the major cytochromes investigated, among which the CYP3A4 isoform (the most important drug-metabolizing enzyme). Such increased expression is correlated to an enhanced phase I activity as independently demonstrated using fluorescence-based assays. Phase II enzyme activity and amino acid levels also show a significant enhancement in differentiated ADHLSCs. The current study, according to data independently obtained in different labs, demonstrates that in vitro differentiated ADHLSCs are able to display advanced liver metabolic functions supporting the possibility to develop them as potential alternatives to primary hepatocytes for in vitro settings.

Adult-derived human liver mesenchymal-like cells as a potential progenitor reservoir of hepatocytes?

It is currently accepted that adult tissues may develop and maintain their own stem cell pools. Because of their higher safety profile, adult stem cells may represent an ideal candidate cell source to be used for liver cell therapies. We therefore evaluated the differentiation potential of mesenchymal-like cells isolated from adult human livers. Mesenchymal-like cells were isolated from enzymatically digested adult human liver and expanded in vitro. Cell characterization was performed using flow cytometry, RT-PCR, and immunofluorescence, whereas the differentiation potential was evaluated both in vitro after incubation with specific media and in vivo after intrasplenic transplantation of uPA(+/+)-SCID and SCID mice. Adult-derived human liver mesenchymal-like cells expressed both hepatic and mesenchymal markers among which albumin, CYP3A4, vimentin, and alpha-smooth muscle actin. In vitro differentiation studies demonstrated that these mesenchymal-like cells are preferentially determined to differentiate into hepatocyte-like cells. Ten weeks following intrasplenic transplantation into uPA(+/+)-SCID mice, recipient livers showed the presence of human hepatocytic cell nodules positive for human albumin, prealbumin, and alpha-fetoprotein. In SCID transplanted liver mice, human hepatocyte-like cells were mostly found near vascular structures 56 days posttransplantation. In conclusion, the ability of isolated adult-derived liver mesenchymal stem-like cells to proliferate and differentiate into hepatocyte-like cells both in vitro and in vivo leads to propose them as an attractive expandable cell source for stem cell therapy in human liver diseases.

Epithelial cells with hepatobiliary phenotype: is it another stem cell candidate for healthy adult human liver?

AIM: To investigate the presence and role of liver epithelial cells in the healthy human adult liver. METHODS: Fifteen days after human hepatocyte primary culture, epithelial like cells emerged and started proliferating. Cell colonies were isolated and subcultured for more than 160 d under specific culture conditions. Cells were analyzed for each passage using immunofluorescence, flow cytometry and reverse transcription-polymerase chain reaction (RT-PCR). RESULTS: Flow cytometry analysis demonstrated that liver epithelial cells expressed common markers for hepatic and stem cells such as CD90, CD44 and CD29 but were negative for CD34 and CD117. Using immunofluorescence we demonstrated that liver epithelial cells expressed not only immature (alpha-fetoprotein) but also differentiated hepatocyte (albumin and CK-18) and biliary markers (CK-7 and 19), whereas they were negative for OV-6. RT-PCR analysis confirmed immunofluorescence data and revealed that liver epithelial cells did not express mature hepatocyte markers such as CYP2B6, CYP3A4 and tyrosine amino-transferase. Purified liver epithelial cells were transplanted into SCID mice. One month after transplantation, albumin positive cell foci were detected in the recipient mouse parenchyma. CONCLUSION: According to their immature and bipotential phenotype, liver epithelial cells might represent a pool of precursors in the healthy human adult liver other than oval cells.