Hepatocyte cryopreservation: Is it time to change the strategy?

Liver cell transplantation presents clinical benefit in patients with inborn errors of metabolism as an alternative, or at least as a bridge, to orthotopic liver transplantation. The success of such a therapeutic approach remains limited by the quality of the transplanted cells. Cryopreservation remains the best option for long-term storage of hepatocytes, providing a permanent and sufficient cell supply. However, isolated adult hepatocytes are poorly resistant to such a process, with a significant alteration both at the morphological and functional levels. Hence, the aim of the current review is to discuss the state of the art regarding widely-used hepatocyte cryopreservation protocols, as well as the assays performed to analyse the post-thawing cell quality both in vitro and in vivo. The majority of studies agree upon the poor quality and efficiency of cryopreserved/thawed hepatocytes as compared to freshly isolated hepatocytes. Intracellular ice formation or exposure to hyperosmotic solutions remains the main phenomenon of cryopreservation process, and its effects on cell quality and cell death induction will be discussed. The increased knowledge and understanding of the cryopreservation process will lead to research strategies to improve the viability and the quality of the cell suspensions after thawing. Such strategies, such as vitrification, will be discussed with respect to their potential to significantly improve the quality of cell suspensions dedicated to liver cell-based therapies

An Optimized Protocol for Histochemical Detection of Senescence-associated Beta-galactosidase Activity in Cryopreserved Liver Tissue

Senescence-associated beta-galactosidase (SA-β-gal) activity assay is commonly used to evaluate the increased beta-galactosidase (β-gal) activity in senescent cells related to enhanced lysosomal activity. Although the optimal pH for β-gal is 4.0, this enzymatic activity has been most commonly investigated at a suboptimal pH by using histochemical reaction on fresh tissue material. In the current study, we optimized a SA-β-gal activity histochemistry protocol that can also be applied on cryopreserved hepatic tissue. This protocol was developed on livers obtained from control rats and after bile duct resection (BDR). A significant increase in β-gal liver activity was observed in BDR rats vs controls after 2 hr of staining at physiological pH 4.0 (6.98 ± 1.19% of stained/total area vs 0.38 ± 0.22; p<0.01) and after overnight staining at pH 5.8 (24.09 ± 6.88 vs 0.12 ± 0.08; p<0.01). Although we noticed that β-gal activity staining decreased with cryopreservation time (from 4 to 12 months of storage at -80C; p<0.05), the enhanced staining observed in BDR compared with controls remained detectable up to 12 months after cryopreservation (p<0.01). In conclusion, we provide an optimized protocol for SA-β-gal activity histochemical detection at physiological pH 4.0 on long-term cryopreserved liver tissue

Umbilical cord matrix stem cells maintain a stable genotype after long-term in vitro culture

Introduction: Umbilical cord matrix stem cells demonstrated interesting properties and differentiation potential towards hepatic lineage. They therefore represent a promising stem cell based treatment for human liver inborn errors of metabolism. Aims: Therapeutic use of umbilical cord matrix stem cells (UCMSC) rely on the demonstration of their stabilized properties during long-term culture. During large scale ex vivo cell expansion, cell characteristics may be altered. In this study, we investigated in vitro and in vivo genetic stability of these cells cultured up to senescence. Methods: UCMSC were isolated from the Wharton's jelly of fourteen healthy at term newborns. Cells were characterized by measuring cytoplasmic and cell surface markers expression by flow cytometry, immunofluorescence and qPCR. Cell growth, morphology and anchorage dependance was followed at each passage. Hepatic differentiation potential was assessed analyzing key hepatic metabolic functions. Long-term genotype stability was investigated by performing karyotype, telomere length, measure of telomerase activity and gene expression related to tumorogenesis. Tumorigenic potential was investigated after subcutaneous injection of 1.107 cells in an immunocompromised xenograft model. Results: Proliferative capacity was similar between cell cultures. Cells reached senescence after 27.6 ± 1.6 cumulative population doublings corresponding to a culture period of 160.9 ± 6.9 days. Cells expressed high levels (>95%) of CD73, CD90, CD105, CD44 and CD29; and maintained their original phenotype up to senescence. UCMSC were able to acquire mature hepatic metabolic functions after differentiation, nearly reaching the potential of human hepatocytes concerning urea production and Cyp3A4 activity. UCMSC remained cytogenetically stable during long-term culture. The cells did not express telomerase activity or alternative telomere lengthening mechanisms. Human telomerase reverse transcriptase expression was not detected. Levels of cell cycle related genes such as p53, p16, p21 and pRb were typically correlated with progressive cell senescence as shown by positive senescence associated beta-galactosidase staining. UCMSC did not display tumorigenic potential both in vivo or in vitro, as anchorage dependence was conserved in vitro and immunodeficient mice subcutaneously injected with UCMSC did not develop tumors. Conclusions: UCMSC is an unexhaustive, uncontroversial and easily accessible cell source. They can be expanded in vitro while maintaining a stable phenotype, genotype and differentiation capacity. UCMSCs therefore represent safe and effective candidates for liver regenerative medicine

Umbilical cord matrix stem cells: A new model to study the role of hepatogenic differentiation and its association with the susceptibility to hepatitis B virus infection

The discovery of HepaRG cell line shed light on cell differentiation state as a possible explanation of HBV liver tropism. A non-transformed cell model would be needed to better elucidate the role of hepatogenic differentiation on cell susceptibility to HBV infection. Methods: Umbilical cord matrix stem cells (UCMSCs) have been isolated by collagenase digestion of Wharton's jelly, and then cultivated, characterized and differentiated as described before (Campard, Gastroenterology 2008). To standardize infection conditions, HBV was obtained from a culture of HepAD38s, and concentrated by PEG precipitation. After infection, HBV DNA was quantified by real time qPCR using a specific TaqMan probe. cccDNA was specifically measured following digestion with a Plasmid-Safe DNase. Differentiated UCMSCs (D-UCMSCs) were compared to undifferentiated cells (UD-UCMSCs) and to primary human hepatocytes (PHH). Results: UCMSCs' mesenchymal stem cell identity was confirmed by the expression of CD90, CD73, CD105, the absence of CD45, CD133 and CD117, and by their ability to accomplish adipogenic and osteogenic differentiation. Following hepatogenic differentiation, D-UCMSC showed an increased expression of Cyp3A4, albumin and HNF4, and acquired functions typical of mature hepatocytes, such as Cyp3A4 and glucose- 6-phosphatase activity. To study HBV binding to cell membrane, cells were incubated with 2.4±1.5 x 104 IU/cell of HBV for 2 hours (h) at 4°C. After extensive washing, 7.3, 27.5 and 26.5 HBV gen.eq./cell were bound to PHH, UD-UCMSC and D-UCMSC respectively. After 1 h at 37°C, PHH, UDUCMSC and D-UCMSC were able to uptake 5%, 2.5% and 6.3% of membrane-bound HBVs, respectively. The proportion of virus uptake increased after 4 and 24 h for both PHH (12.1%, 19%) and D-UCMSC (10.8%, 25.8%), but it remained stable for UD-UCMSC (4.2%, 3.7%). Susceptibility to viral replication was studied by measuring intracellular HBV DNA at 3, 7 and 10 days post-infection (pi), as compared to 24 h pi. HBV DNA levels increased in both PHH (165%, 314%, 958%) and D-UCMSC (105%, 135% and 769%), suggesting viral replication. cccDNA was measured in D-UCMSCs 7 days pi and shown to increase 7 times as compared to 24 h pi. HBsAg and HBeAg levels in culture supernatant confirmed viral replication in D-UCMSCs. Conclusions: UCMSCs are non-transformed, easy-to-obtain, human cells that proved to be resistant to HBV infection in their undifferentiated state and to acquire susceptibility to HBV uptake and replication after hepatogenic differentiation. They constitute a new in vitro “physiological” model to study the role of differentiation on HBV-cell interactions. Note: original work - confidential

Stem cells for liver tissue repair: Current knowledge and perspectives

Stem cells from extra- or intrahepatic sources have been recently characterized and their usefulness for the generation of hepatocyte-like lineages has been demonstrated. Therefore, they are being increasingly considered for future applications in liver cell therapy. In that field, liver cell transplantation is currently regarded as a possible alternative to whole organ transplantation, while stem cells possess theoretical advantages on hepatocytes as they display higher in vitro culture performances and could be used in autologous transplant procedures. However, the current research on the hepatic fate of stem cells is still facing difficulties to demonstrate the acquisition of a full mature hepatocyte phenotype, both in vitro and in vivo. Furthermore, the lack of obvious demonstration of in vivo hepatocyte-like cell functionality remains associated to low repopulation rates obtained after current transplantation procedures. The present review focuses on the current knowledge of the stem cell potential for liver therapy. We discuss the characteristics of the principal cell candidates and the methods to demonstrate their hepatic potential in vitro and in vivo. We finally address the question of the future clinical applications of stem cells for liver tissue repair and the technical aspects that remain to be investigated