The therapeutic potential of bone marrow-derived mesenchymal stem cells on hepatic cirrhosis

Hepatic cirrhosis is the end-stage of chronic liver diseases. The majority of patients with hepatic cirrhosis die from life-threatening complications occurring at their earlier ages. Liver transplantation has been the most effective treatment for these patients. Since liver transplantation is critically limited by the shortage of available donor livers, searching for an effective alternative therapy has attracted great interest in preclinical studies. The transplantation of autologous bone marrow-derived mesenchymal stem cells holds great potential for treating hepatic cirrhosis. Mesenchymal stem cells can differentiate to hepatocytes, stimulate the regeneration of endogenous parenchymal cells, and enhance fibrous matrix degradation. Experimental and clinical studies have shown promising beneficial effects. This review is intended to translate the bench study results to the patients' bedside. The potential interventions of mesenchymal stem cells on cirrhosis are illustrated in terms of the cellular and molecular mechanisms of hepatic fibrogenesis.     

Functional units in rainbow trout (Salmo gairdneri, Richardson) liver: III. Morphometric analysis of parenchyma, stroma, and component cell types

Hepatic stroma and parenchyma with its component cell types were quantitatively described in adult male and female actively-spawning 5-year-old rainbow trout (Salmo gairdneri, Richardson). Point-count morphometry of glycol methacrylate sections estimated volume compartments for stroma and parenchyma. Veins composed 85% of the stroma while arteries and bile ducts occupied approximately 6-7% each. Parenchyma accounted for 95% of hepatic volume. Point-count morphometry of transmission electron micrographs estimated volume compartments as well as numerical and surface density measurements for parenchymal components. Within the hepatic parenchymal compartment, hepatocytes occupied 85% and showed significant sex differences. Female hepatocytes were significantly more numerous but were smaller, only 60% of the volume of male hepatocytes. Since hepatocyte nuclear volume was equal in both sexes, differences were due to reduced cytoplasmic volume in females. Perisinusoidal macrophages of females occupied larger volumes of their respective parenchymal compartments, and their larger mean cytoplasmic volumes suggested activation. Biliary epithelial cells of preductules and ductules were numerous. Ratios of numerical density of hepatocytes to biliary epithelial cells were consistent with a tubular arrangement of hepatocytes. Factors possibly mediating the sexual dimorphism are discussed.     

The growth of preneoplastic hepatocytes in the spleen of recipient mice]

Hepatocytes and the fraction of non parenchymal cells enriched with oval cells were extracted from the preneoplastic mouse liver at the stage of hyperplastic node formation and implanted into the spleen. In 14-16 months after the transplantation, multiple islets of hepatocytes which replaced up to 25% of the spleen cut area, were found in 57% (4 of 7) and 22% (8 of 36) of recipients respectively. The hepatocytes formed 2-3-cell bulks or solid masses organized into multicellular trabecules, and expressed biliary capillary antigen, albumin and transferrin. In the inoculation of nonparenchymal cell fraction, the growth of hepatic tissue in the spleen depended on the magnitude of hepatocyte admixture to be undetectable in absence of hepatocytes in the donor suspension. The growth of hepatic tissue in spleen was observed following the injection of a small number (3 x 10(-4)-6 x 10(-5)) of live hepatocytes. This fact evidences an extremely high clonogenic potency of clonogenic potency of preneoplastic hepatocytes.     

Zonal expression of hepatocytic marker enzymes during liver repopulation

Hepatocytes are metabolically specialised cells displaying distinctive gene expression patterns within the liver lobule. Here, we investigate whether pre-cultured adult rat hepatocytes adopt periportal and pericentral enzyme expression following their transplantation into the regenerating rat liver. Isolated primary rat hepatocytes, representing a mixture of both periportal and pericentral origin, lost expression of carbamoyl phosphate synthetase I (CPS I) and cytochrome P450 subtype 2B1 (CYP2B1) in culture as shown by immunofluorescence and Western blot analysis. Accordingly, urea synthesis and CYP2B1 enzyme activity decreased. Hepatocytes from DPPIV (CD26) wild type rats were cultured for 4 and 7 days, and then transplanted into the livers of CD26 deficient rats following prior treatment with retrorsine and partial hepatectomy to drive selective donor cell proliferation. CD26 positive donor cells engrafted in the periportal regions and grew in clusters expanding into the parenchyma as time proceeded. Ten weeks after transplantation, cells derived from donors surrounding the portal veins expressed CPS I, but not CYP2B1. The reverse was true for CD26 positive cells in close proximity to the central veins displaying immunoreactivity to CYP2B1, but no longer to CPS I. Hepatocytes lose their specific marker enzyme expression in culture. After transplantation, donor hepatocytes proliferate in the host parenchyma whilst acquiring the position-specific enzyme expression of the surrounding periportal and pericentral host hepatocytes. These results indicate the high degree of plasticity of gene expression in hepatocytes subjected to a change in microenvironment.     

Microbiochemical approach to liver cell heterogeneity around terminal hepatic venules

Using lyophilized cryostat sections of liver the activities of alanine aminotransferase, lactate dehydrogenase, and pyruvate kinase were measured using a Lowry technique in the first layer of hepatocytes adjacent to terminal hepatic venules and in the residual parenchymal of the perivenous zone of the acinus in normally fed adult male Wistar rats. Alanine aminotransferase was homogeneously distributed in the two areas measured (ratio hepatocytes adjacent to terminal hepatic venules/residual parenchyma of the perivenous zone: 1.05). Enzyme activities of the lactate dehydrogenase were significantly lower in the hepatocytes adjacent to terminal hepatic venules (ratio: 0.65) and those of the pyruvate kinase significantly higher (ratio: 1.12) than in the residual parenchyma of the perivenous zone indicating liver cell heterogeneity in this zone of the liver acinus.     

骨髓干细胞移植治疗肝硬化的基础与临床研究现状

肝硬化是一种临床常见的肝病良性终末期表现。目前临床上尚缺乏有效的治疗措施。肝脏移植是最理想的治疗方法,但受供体肝脏来源限制,且费用昂贵。近年来开展的自体骨髓干细胞(BMSCs)移植治疗,为肝硬化的治疗带来了新的希望。BMSCs主要包括造型血干细胞和间充质干细胞,其具有可塑性,体外通过生长因子,体内利用特定微环境均可诱导BMSCs分化为肝前体细胞和成熟肝细胞,并明显改善肝功能。从动物实验到临床研究亦表明,BMSCs具有来源丰富、费用低廉、损伤小、自体移植不栓塞、无排斥反应等优点,为治疗肝病带来了新思路,有望成为生物人工肝的细胞来源。本文就BMSCs移植治疗肝硬化的研究现状,尤其是移植途径以及在肝脏内定居、迁移和分化机制的示踪观察方法和存在的问题作一综述,以期为从事肝病研究的同仁提供参考依据。通过对BMSCs移植从基础研究及临床应用的最新进展的描述,展示BMSCs在肝硬化治疗方面良好的治疗前景。     

A 3-dimensional presentation of the functional liver unit]

The reconstruction of the liver parenchyma of a golden hamster after poisoning with allyl formate is described. Allyl formate primarily destroys the periportal areas and leads, following the microvascularisation of the liver parenchyma, to a necrosis of the hepatocytes progressing towards the terminal hepatic venule. The still intact parenchymal zones can be characterized by the positive PAS reaction. In this study the preterminal and the terminal portal branches as well as zone 3, situated in the vasculatory periphery, were reconstructed. By this method, a three-dimensional presentation of the acinar functional zones was possible for the first time.     

The role of HGF on invasive properties and repopulation potential of human fetal hepatic progenitor cells

The success of hepatocyte transplantation has been limited by the low efficiency of transplanted cell integration into liver parenchyma. Human fetal hepatic progenitor cells (hepatoblasts) engraft more effectively than adult hepatocytes in mouse livers. However, the signals required for their integration are not yet fully understood. We investigated the role of HGF on the migration and invasive ability of human hepatic progenitors in vitro and in vivo.Hepatoblasts were isolated from the livers of human fetuses between 10 and 12 weeks of gestation. Their invasive ability was assessed in the presence or absence of HGF. These cells were also transplanted into immunodeficient mice and analyzed by immunohistochemistry.In contrast to TNF-alpha, HGF increased the motogenesis and invasiveness of hepatoblasts, but not of human adult hepatocytes, via phosphorylation of extracellular signal-regulated kinase (ERK) 1/2. The invasive ability of human hepatoblasts correlated with the expression and secretion of matrix metalloproteinases (MMPs). Hepatoblasts stimulated with HGF prior transplantation into newborn mice migrated from the portal area into the hepatic parenchyma.Conclusions: In contrast to adult hepatocytes, hepatoblasts display invasive ability that can be modulated by HGF in vitro and in vivo.     

Allotransplantation in utero and immortalization of primate fetal hepatocytes

We are developing cell therapy approaches on non-human primates as a preclinical model for the treatment of hepatic metabolic diseases. In foetuses, the tissues, including liver, are in expansion, which should facilitate hepatocytes engraftment, and the immune system becomes fully mature only after birth. We have set out conditions for isolation of fetal hepatocytes from macaca mulatta at the end of the 2nd trimester of gestation (90-100 days), their cryopreservation and retroviral transduction. Two different routes of administration of hepatocytes were evaluated: the umbilical vein which was deleterious for the foetuses, and the intraparenchymatous injection which was well tolerated by the animals. Administration of hepatocytes into the hepatic parenchyma resulted in microchimerism and allogenic cells were visualized 9 days after transplantation. Another approach has been to immortalize simian foetal hepatocytes using a retroviral vector expressing SV40 Large T flanked by lox sites. A cell line has been established for 2 years, which is not tumorigenic when injected subcutaneously into nude mice and display characteristics of bipotent hepatoblasts, precursors of hepatocytes and biliary cells. After orthotopic transplantation into nude mice via the portal vein, these cells expressed albumin until the sacrifice of the animals (17 days). The next steps will be to define conditions for transplantation of retrovirally transduced fetal primary and/or immortalized hepatocytes into young foetuses (60 days of gestation) and post-natally.     

Cell turnover in the repopulated rat liver: distinct lineages for hepatocytes and the biliary epithelium

The dynamics of cell renewal in the normal adult liver remains an unresolved issue. We investigate the possible contribution of a common biliary precursor cell pool to hepatocyte turnover in the chimeric long-term repopulated rat liver. The retrorsine (RS)-based model of massive liver repopulation was used. Animals not expressing the CD26 marker (CD26^-) were injected with RS, followed by transplantation of 2 million syngeneic hepatocytes isolated from a normal CD26-expressing donor. Extensive (80-90 %) replacement of resident parenchymal cells was observed at 1 year post-transplantation and persisted at 2 years, as expected. A panel of specific markers, including cytokeratin 7, OV6, EpCAM, claudin 7 and α-fetoprotein, was employed to locate the in situ putative progenitor and/or biliary epithelial cells in the stably repopulated liver. No overlap was observed between any of these markers and the CD26 tag identifying transplanted cells. Exposure to RS was not inhibitory to the putative progenitor and/or biliary epithelial cells, nor did we observe any evidence of cell fusion between these cells and the transplanted cell population. Given the long-term (>2 years) stability of the donor cell phenotype in this model of liver repopulation, the present findings suggest that hepatocyte turnover in the repopulated liver is fuelled by a cell lineage distinct from that of the biliary epithelium and relies largely on the differentiated parenchymal cell population. These results support the solid biological foundation of liver repopulation strategies based on the transplantation of isolated hepatocytes.     

The involvement of parenchymal,Kupffer and endothelial liver cells in the hepatic uptake of intravenously injected liposomes effects of lanthanum and gadolinium salts

¹²⁵I-labeled albumin or poly(vinyl pyrrolidone) encapsulated in intermediate size multilamellar or unilamellar liposomes with 30–40% of cholesterol were injected intravenously into rats. In other experiments liposomes containing phosphatidyl[Me-¹⁴C]choline were injected. 1 h after injection parenchymal or non-parenchymal cells were isolated. Non-parenchymal cells were separated by elutriation centrifugation into a Kupffer cell fraction and an endothelial cell fraction. From the measurements of radioactivities in the various cell fractions it was concluded that the liposomes are almost exclusively taken up by the Kupffer cells. Endothelial cells did not contribute at all and hepatocytes only to a very low extent to total hepatic uptake of the ¹²⁵I-labels. Of the ¹⁴C-label, which orginates from the phosphatidylcholine moiety of the liposomes, much larger proportions were recovered in the hepatocytes. A time-dependence study suggested that besides the involvement of phosphatidylcholine exchange between liposomes and high density lipoprotein, a process of intercellular transfer of lipid label from Kupffer cells to the hepatocytes may be involved in this phenomenon. Lanthanum or gadolinium salts, which effectively block Kupffer cell activity, failed to accomplish an increase in the fraction of liposomal material recovered in the parenchymal cells. This is compatible with the notion that liposomes of the type used in these experiments have no, or at most very limited, access to the liver parenchyma following their intravenous administration to rats.     

The road to regenerative liver therapies: The triumphs,trials and tribulations

The liver is one of the few organs that possess a high capacity to regenerate after liver failure or liver damage. The parenchymal cells of the liver, hepatocytes, contribute to the majority of the regeneration process. Thus, hepatocyte transplantation presents an alternative method to treating liver damage. However, shortage of hepatocytes and difficulties in maintaining primary hepatocytes still remain key obstacles that researchers must overcome before hepatocyte transplantation can be used in clinical practice. The unique properties of pluripotent stem cells (PSCs) and induced pluripotent stem cells (iPSCs) have provided an alternative approach to generating enough functional hepatocytes for cellular therapy. In this review, we will present a brief overview on the current state of hepatocyte differentiation from PSCs and iPSCs. Studies of liver regenerative processes using different cell sources (adult liver stem cells, hepatoblasts, hepatic progenitor cells, etc.) will be described in detail as well as how this knowledge can be applied towards optimizing culture conditions for the maintenance and differentiation of these cells towards hepatocytes. As the outlook of stem cell-derived therapy begins to look more plausible, researchers will need to address the challenges we must overcome in order to translate stem cell research to clinical applications.     

Characterization and cell cycle kinetics of hepatocyte populations isolated from adult liver tissue by a nonenzymatic procedure

Suspensions of liver cells enriched in lobular parenchymal hepatocytes were isolated from adult mouse hepatic tissue by nonenzymatic dispersion in chelating buffer and sedimentation of the released cells at unit g. Single cell suspensions so obtained were suitable for flow cytometric measurements of hepatic ploidy class distributions. The more quickly sedimenting cell population consisted of 88% albumin/transferrin-positive epithelial hepatocytes, the nuclei of which were bimodally distributed with respect to RNA content. This dual G1 population was observed in 2C DNA content liver nuclei prepared by several methods and appears to be a general cytochemical characteristic of adult liver parenchymal cells.     

Effects of genetically modified human skin fibroblasts,stably overexpressing hepatocyte growth factor,on hepatic functions of cocultured C3A cells

Diseases leading to terminal hepatic failure are among the most common causes of death worldwide. Transplant of the whole organ is the only effective method to cure liver failure. Unfortunately, this treatment option is not available universally due to the serious shortage of donors. Thus, alternative methods have been developed that are aimed at prolonging the life of patients, including hepatic cells transplantation and bridging therapy based on hybrid bioartificial liver devices. Parenchymal liver cells are highly differentiated and perform many complex functions, such as detoxification and protein synthesis. Unfortunately, isolated hepatocytes display a rapid decline in viability and liver‐specific functions. A number of methods have been developed to maintain hepatocytes in their highly differentiated state in vitro, amongst them the most promising being 3D growth scaffolds and decellularized tissues or coculture with other cell types required for the heterotypic cell‐cell interactions. Here we present a novel approach to the hepatic cells culture based on the feeder layer cells genetically modified using lentiviral vector to stably produce additional amounts of hepatocyte growth factor and show the positive influence of these coculture conditions on the preservation of the hepatic functions of the liver parenchymal cells' model—C3A cells.     

Isolation of primary human hepatocytes from normal and diseased liver tissue: a one hundred liver experience

Successful and consistent isolation of primary human hepatocytes remains a challenge for both cell-based therapeutics/transplantation and laboratory research. Several centres around the world have extensive experience in the isolation of human hepatocytes from non-diseased livers obtained from donor liver surplus to surgical requirement or at hepatic resection for tumours. These livers are an important but limited source of cells for therapy or research. The capacity to isolate cells from diseased liver tissue removed at transplantation would substantially increase availability of cells for research. However no studies comparing the outcome of human hepatocytes isolation from diseased and non-diseased livers presently exist. Here we report our experience isolating human hepatocytes from organ donors, non-diseased resected liver and cirrhotic tissue. We report the cell yields and functional qualities of cells isolated from the different types of liver and demonstrate that a single rigorous protocol allows the routine harvest of good quality primary hepatocytes from the most commonly accessible human liver tissue samples.     

In vivo cholesteryl ester selective uptake of mildly and standardly oxidized LDL occurs by both parenchymal and nonparenchymal mouse hepatic cells but SR-BI is only responsible for standardly oxidized LDL selective uptake by nonparenchymal cells

In blood circulation, low density lipoproteins (LDL) can undergo modification, such as oxidation, and become key factors in the development of atherosclerosis. Although the liver is the major organ involved in the elimination of oxidized LDL (oxLDL), the identity of the receptor(s) involved remains to be defined. Our work aims to clarify the role of the scavenger receptor class B type I (SR-BI) in the hepatic metabolism of mildly and standardly oxLDL as well as the relative contribution of parenchymal (hepatocytes) and nonparenchymal liver cells with a special emphasis on CE-selective uptake. The association of native LDL and mildly or standardly oxLDL labeled either in proteins or in cholesteryl esters (CE) was measured on primary cultures of mouse hepatocytes from normal and SR-BI knock-out (KO) mice. These in vitro assays demonstrated that hepatocytes are able to mediate CE-selective uptake from both LDL and oxLDL and that SR-BI KO hepatocytes have a 60% reduced ability to selectively take CE from LDL but not towards mildly or standardly oxLDL. When lipoproteins were injected in the mouse inferior vena cava, parenchymal and nonparenchymal liver cells accumulated more CE than proteins from native, mildly and standardly oxLDL, indicating that selective uptake of CE from these lipoproteins occurs in vivo in these two cell types. The parenchymal cells contribute near 90% of the LDL-CE selective uptake and SR-BI for 60% of this pathway. Nonparenchymal cells capture mainly standardly oxLDL while parenchymal and nonparenchymal cells equally take up mildly oxLDL. An 82% reduction of standardly oxLDL-CE selective uptake by the nonparenchymal cells of SR-BI KO mice allowed emphasizing the contribution of SR-BI in hepatic metabolism of standardly oxLDL. However, SR-BI is not responsible for mildly oxLDL metabolism. Thus, SR-BI is involved in LDL- and standardly oxLDL-CE selective uptake in parenchymal and nonparenchymal cells, respectively.     

Stem cells, cell transplantation and liver repopulation

Liver transplantation is currently the only therapeutic option for patients with end-stage chronic liver disease and for severe acute liver failure. Because of limited donor availability, attention has been focused on the possibility to restore liver mass and function through cell transplantation. Stem cells are a promising source for liver repopulation after cell transplantation, but whether or not the adult mammalian liver contains hepatic stem cells is highly controversial. Part of the problem is that proliferation of mature adult hepatocytes is sufficient to regenerate the liver after two-thirds partial hepatectomy or acute toxic liver injury and participation of stem cells is not required. However, under conditions in which hepatocyte proliferation is blocked, undifferentiated epithelial cells in the periportal areas, called "oval cells", proliferate, differentiate into hepatocytes and restore liver mass. These cells are referred to as facultative liver stem cells, but they do not repopulate the normal liver after their transplantation. In contrast, epithelial cells isolated from the early fetal liver can effectively repopulate the normal liver, but they are already traversing the hepatic lineage and may not be true stem cells. Mesenchymal stem cells and embryonic stem cells can be induced to differentiate along the hepatic lineage in culture, but at present these cells are inefficient in repopulating the liver. This review will characterize these various cell types and compare the properties of these cells and the conditions under which they do or do not repopulate the liver following their transplantation.     

Evidence that the nonparenchymal cells of the liver are the principal source of cholesteryl ester transfer protein in primates

Previous studies showed that 90% or more of the cholesteryl ester transfer protein (CETP) mRNA is contained in the liver of cynomolgus monkeys. The purpose of this study was to determine if the parenchymal cells (hepatocytes) were the hepatic cell type that contained that mRNA. The parenchymal and nonparenchymal cells were separated by standard methods, and the CETP, apoA-I, apoB, and apoE mRNA content of the preparation determined at each step in the purification process. ApoA-I and apoB are produced only in the parenchymal cells; apoE is produced by both cell types. The mRNA measurements showed that the CETP mRNA: apoA-I mRNA and the CETP mRNA: apoB mRNA ratios were more than 2500-fold greater in the nonparenchymal cell preparation than in the starting material, and that the purified parenchymal cell fraction was virtually devoid of CETP mRNA. In situ hybridization studies showed that, whereas the apoA-I mRNA signal was evenly distributed over the tissue section, the CETP mRNA signal was associated with the hepatic sinusoids, suggesting that it was the hepatic sinusoidal cells that were principally responsible for the high CETP mRNA levels in the liver. We conclude that the nonparenchymal cells are the principal source of CETP in the cynomolgus monkey.     

ELECTRON-OPAQUE, LIPID-CONTAINING BODIES IN MOUSE LIVER AT EARLY INTERVALS AFTER PARTIAL HEPATECTOMY AND SHAM OPERATION

The appearance and distribution of electron-opaque, lipid-containing bodies have been studied in liver of adult male mice of the C3H strain. The mice were either partially hepatectomized or sham-operated, and the liver was fixed in Veronal acetate-buffered 2 per cent osmium tetroxide at various postoperative intervals (10, 20, 40, 60, and 120 minutes). Normal, non-operated mice served as controls. As early as 10 minutes after both sham operation and partial hepatectomy, lipid-containing bodies have been observed, not only in the cytoplasm of hepatic parenchymal cells, but also in the space of Disse. At the very early postoperative intervals studied, minute lipid bodies are repeatedly found to be more numerous in the space of Disse than at later intervals. It is suggested that the lipid-containing bodies enter the parenchymal cell from the circulation. At the cell membrane, numerous invaginations, each containing a lipid body, have been observed; this suggests that the lipid bodies enter the hepatic parenchymal cells by the process of pinocytosis.The fact that only hepatic parenchymal cells contain the lipid bodies, whereas von Kupffer, endothelial lining, and Ito's fat-storing cells do not, may indicate a specific lipid mobilization response on the part of the cells of the hepatic parenchyma.