Evidence for a common progenitor of epithelial and mesenchymal components of the liver

Tissues of the adult organism maintain the homeostasis and respond to injury by means of progenitor/stem cell compartments capable to give rise to appropriate progeny. In organs composed by histotypes of different embryological origins (e.g. the liver), the tissue turnover may in theory involve different stem/precursor cells able to respond coordinately to physiological or pathological stimuli. In the liver, a progenitor cell compartment, giving rise to hepatocytes and cholangiocytes, can be activated by chronic injury inhibiting hepatocyte proliferation. The precursor compartment guaranteeing turnover of hepatic stellate cells (HSCs) (perisinusoidal cells implicated with the origin of the liver fibrosis) in adult organ is yet unveiled. We show here that epithelial and mesenchymal liver cells (hepatocytes and HSCs) may arise from a common progenitor. Sca+ murine progenitor cells were found to coexpress markers of epithelial and mesenchymal lineages and to give rise, within few generations, to cells that segregate the lineage-specific markers into two distinct subpopulations. Notably, these progenitor cells, clonally derived, when transplanted in healthy livers, were found to generate epithelial and mesenchymal liver-specific derivatives (i.e. hepatocytes and HSCs) properly integrated in the liver architecture. These evidences suggest the existence of a ‘bona fide'' organ-specific meso-endodermal precursor cell, thus profoundly modifying current models of adult progenitor commitment believed, so far, to be lineage-restricted. Heterotopic transplantations, which confirm the dual differentiation potentiality of those cells, indicates as tissue local cues are necessary to drive a full hepatic differentiation. These data provide first evidences for an adult stem/precursor cell capable to differentiate in both parenchymal and non-parenchymal organ-specific components and candidate the liver as the instructive site for the reservoir compartment of HSC precursors as yet non-localized in the adult.     

Stimulative effect of non-parenchymal liver cells on ability of tyrosine aminotransferase induction in hepatocytes

Hepatocytes and non-parenchymal liver cells were isolated from adult rat liver and co-cultured for 48 hours as a monolayer on polystyrene culture dishes. The ability of tyrosine aminotransferase (TAT) induction in hepatocytes was examined in the presence of dexamethasone and dibutyryl cAMP. Non-parenchymal cells greatly enhance the ability of TAT induction of hepatocytes. A soluble factor with molecular weight of more than 10,000 is responsible for this enhancement, because conditioned medium prepared from non-parenchymal cells is also stimulatory. Non-parenchymal cells restored the ability in hepatocytes damaged with the addition of D-galactosamine. Conditioned medium prepared from non-parenchymal cells treated with D-galactosamine had higher activity of enhancement than the medium from normal cells. The soluble factor might be released in response to some signal of injury. Hepatocytes and non-parenchymal cells were immobilized within Ca-alginate, and although immobilized hepatocytes rapidly lost the ability to induce TAT, hepatocytes co-immobilized with non-parenchymal cells maintained the ability during 4 days of culture. These results indicated that non-parenchymal liver cells, as well as hepatocytes, could be used to construct a bioartificial liver support system.     

Culture of bovine hepatocytes: a non-perfusion technique for cell isolation

In this work we have studied the isolation and culture of mature bovine hepatocytes on plastic dishes without exogenous matrix. The liver has been disaggregated in a collagenase solution instead of undergoing a perfusion step. After a few days in culture, the plates showed several clusters of different cell types. Although the average yield was 1.60±0.57×10⁸ viable liver cells per gram of tissue, these cultures were formed by non-parenchymal cells and only very few or none by parenchymal cells. In these cultures, actin structures used as a marker for Stellate (Ito) cells have been visualized by immunocytochemical techniques. In order to increase the proportion of parenchymal cells a centrifugation on Percoll, which separates cell sub-populations, has been introduced. Though the yield was lower than in the previous method, these pre-purified cultures were only composed of hepatocytes. It has been shown that these cells exhibited albumin synthesis, which is a specific hepatocytes function. In addition, these cultures were capable of producing metabolites of 7-ethoxycoumarin at a higher rate than non purified cell cultures. Therefore this simplified procedure for the isolation and culture of functional and viable hepatocytes may be applied for in vitro studies in bovine.     

Pre‐treatments enhance the therapeutic effects of mesenchymal stem cells in liver diseases

Liver diseases caused by viral infection, alcohol abuse and metabolic disorders can progress to end‐stage liver failure, liver cirrhosis and liver cancer, which are a growing cause of death worldwide. Although liver transplantation and hepatocyte transplantation are useful strategies to promote liver regeneration, they are limited by scarce sources of organs and hepatocytes. Mesenchymal stem cells (MSCs) restore liver injury after hepatogenic differentiation and exert immunomodulatory, anti‐inflammatory, antifibrotic, antioxidative stress and antiapoptotic effects on liver cells in vivo. After isolation and culture in vitro, MSCs are faced with nutrient and oxygen deprivation, and external growth factors maintain MSC capacities for further applications. In addition, MSCs are placed in a harsh microenvironment, and anoikis and inflammation after transplantation in vivo significantly decrease their regenerative capacity. Pre‐treatment with chemical agents, hypoxia, an inflammatory microenvironment and gene modification can protect MSCs against injury, and pre‐treated MSCs show improved hepatogenic differentiation, homing capacity, survival and paracrine effects in vitro and in vivo in regard to attenuating liver injury. In this review, we mainly focus on pre‐treatments and the underlying mechanisms for improving the therapeutic effects of MSCs in various liver diseases. Thus, we provide evidence for the development of MSC‐based cell therapy to prevent acute or chronic liver injury. Mesenchymal stem cells have potential as a therapeutic to prolong the survival of patients with end‐stage liver diseases in the near future.     

Mesenchymal stem cells support hepatocyte function in engineered liver grafts

Recent studies suggest that organ decellularization is a promising approach to facilitate the clinical application of regenerative therapy by providing a platform for organ engineering. This unique strategy uses native matrices to act as a reservoir for the functional cells which may show therapeutic potential when implanted into the body. Appropriate cell sources for artificial livers have been debated for some time. The desired cell type in artificial livers is primary hepatocytes, but in addition, other supportive cells may facilitate this stem cell technology. In this context, the use of mesenchymal stem cells (MSC) is an option meeting the criteria for therapeutic organ engineering. Ideally, supportive cells are required to (1) reduce the hepatic cell mass needed in an engineered liver by enhancing hepatocyte function, (2) modulate hepatic regeneration in a paracrine fashion or by direct contact, and (3) enhance the preservability of parenchymal cells during storage. Here, we describe enhanced hepatic function achieved using a strategy of sequential infusion of cells and illustrate the advantages of co-cultivating bone marrow-derived MSCs with primary hepatocytes in the engineered whole-liver scaffold. These co-recellularized liver scaffolds colonized by MSCs and hepatocytes were transplanted into live animals. After blood flow was established, we show that expression of adhesion molecules and proangiogenic factors was upregulated in the graft.     

Mesenchymal stem cells support hepatocyte function in engineered liver grafts

Recent studies suggest that organ decellularization is a promising approach to facilitate the clinical application of regenerative therapy by providing a platform for organ engineering. This unique strategy uses native matrices to act as a reservoir for the functional cells which may show therapeutic potential when implanted into the body. Appropriate cell sources for artificial livers have been debated for some time. The desired cell type in artificial livers is primary hepatocytes, but in addition, other supportive cells may facilitate this stem cell technology. In this context, the use of mesenchymal stem cells (MSC) is an option meeting the criteria for therapeutic organ engineering. Ideally, supportive cells are required to (1) reduce the hepatic cell mass needed in an engineered liver by enhancing hepatocyte function, (2) modulate hepatic regeneration in a paracrine fashion or by direct contact, and (3) enhance the preservability of parenchymal cells during storage. Here, we describe enhanced hepatic function achieved using a strategy of sequential infusion of cells and illustrate the advantages of co-cultivating bone marrow-derived MSCs with primary hepatocytes in the engineered whole-liver scaffold. These co-recellularized liver scaffolds colonized by MSCs and hepatocytes were transplanted into live animals. After blood flow was established, we show that expression of adhesion molecules and proangiogenic factors was upregulated in the graft.     

Isoenzyme patterns in parenchymal and non-parenchymal cells isolated from regenerating and regenerated rat liver

Parenchymal and non-parenchymal cells were isolated from adult rat liver that had been fully regenerated after a 70% partial hepatectomy. The characteristics of the parenchymal cell preparations from regenerated rat liver indicated that they were a homogeneous population and comparable with parenchymal cells isolated from intact liver. The parenchymal cells from regenerated adult rat liver contain glucokinase, hexokinase, pyruvate kinase type I and aldolase B. The non-parenchymal cells contain hexokinase, pyruvate kinase type III and aldolase B. When cells were isolated at different times of the day from rats on controlled feeding schedules, variation of tyrosine aminotransferase activity and liver glycogen content were observed in the parenchymal cells in keeping with the reported diurnal oscillations found in whole liver extracts. When parenchymal cells were isolated from rats 48 and 72h after partial hepatectomy, different isoenzyme patterns were observed. These cells appeared to synthesize pyruvate kinase type III, a function that was assigned previously to non-parenchymal cells or to foetal rat liver hepatocytes.     

Oval cell proliferation in p16INK4a expressing mouse liver is triggered by chronic growth stimuli.

Terminal differentiation requires molecules also involved in aging such as the cell cycle inhibitor p16(INK4a).Like other organs, the adult liver represents a quiescent organ with terminal differentiated cells, hepatocytes and cholangiocytes. These cells retain the ability to proliferate in response to liver injury or reduction of liver mass. However, under conditions which prevent mitotic activation of hepatocytes, regeneration can occur instead from facultative hepatic stem cells.For therapeutic application a non-toxic activation of this stem cell compartment is required. We have established transgenic mice with conditional overexpression of the cell cycle inhibitor p16(INK4a) in hepatocytes and have provoked and examined oval cell activation in adult liver in response to a range of proliferative stimuli.We could show that the liver specific expression of p16(INK4a) leads to a faster differentiation of hepatocytes and an activation of oval cells already in postnatal mice without negative consequences on liver function.     

Stem/progenitor cells in liver injury repair and regeneration

Morbidity and mortality from cirrhosis is increasing rapidly in the world. Currently, orthotopic liver transplantation is the only definitive therapeutic option. However, its clinical use is limited, because of poor long‐term graft survival, donor organ shortage and high costs associated with the procedure. Stem cell replacement strategies are therefore being investigated as an attractive alternative approach to liver repair and regeneration. In this review we discuss recent preclinical and clinical investigations that explore the therapeutic potential of stem cells in repair of liver injuries. Several types of stem cells. including embryonic stem cells, haematopoietic stem cells and mesenchymal stem cells, can be induced to differentiate into hepatocyte‐like cells by defined culture conditions in vitro. Stem cell transplantation has been shown to significantly improve liver function and increase animal survival in experimentally‐induced liver‐injury models. Moreover, several pilot clinical studies have reported encouraging therapeutic effects in patients treated with stem cells. Although there remain many unresolved issues, the available data support the notion that stem cell technology may lead to the development of effective clinical modalities for human liver diseases.     

Pluripotent plasticity of stem cells and liver repopulation

Different types of stem cells have a role in liver regeneration or fibrous repair during and after several liver diseases. Otherwise, the origin of hepatic and/or extra‐hepatic stem cells in reactive liver repopulation is under controversy. The ability of the human body to self‐repair and replace the cells and tissues of some organs is often evident. It has been estimated that complete renewal of liver tissue takes place in about a year. Replacement of lost liver tissues is accomplished by proliferation of mature hepatocytes, hepatic oval stem cells differentiation, and sinusoidal cells as support. Hepatic oval cells display a distinct phenotype and have been shown to be a bipotential progenitor of two types of epithelial cells found in the liver, hepatocytes, and bile ductular cells. In gastroenterology and hepatology, the first attempts to translate stem cell basic research into novel therapeutic strategies have been made for the treatment of several disorders, such as inflammatory bowel diseases, diabetes mellitus, celiachy, and acute or chronic hepatopaties. In the future, pluripotent plasticity of stem cells will open a variety of clinical application strategies for the treatment of tissue injuries, degenerated organs. The promise of liver stem cells lie in their potential to provide a continuous and readily available source of liver cells that can be used for gene therapy, cell transplant, bio‐artificial liver‐assisted devices, drug toxicology testing, and use as an in vitro model to understand the developmental biology of the liver. Copyright © 2010 John Wiley & Sons, Ltd.     

Clonal mesenchymal stem cells derived from human bone marrow can differentiate into hepatocyte‐like cells in injured livers of SCID mice

There is increasing evidence that human mesenchymal stem cells (hMSCs) can be a valuable, transplantable source of hepatocytes. Most of the hMSCs preparations used in these studies were likely heterogeneous cell populations, isolated by adherence to plastic surfaces or by density gradient centrifugation. Therefore, the participation of other unknown trace cell populations cannot be rigorously discounted. Here we report the isolation and establishment of a cloned human MSC line (chMSC) from human bone marrow primary culture, through which we confirmed the hepatic differentiation capability of authentic hMSCs. chMSCs expressed markers of mesenchymal cells, but not markers of hematopoietic stem cells. In vitro, chMSCs can differentiate into either mesenchymal cells or cells exhibiting hepatocyte‐like phenotypes. When transplanted intrasplentically into carbon tetrachloride‐injured livers of SCID mice, EGFP‐tagged chMSCs engrafted into the host liver parenchyma, exhibited typical hepatocyte morphology, form a three‐dimensional architecture, and differentiate into hepatocyte‐like cells expressing human albumin and α‐1‐anti‐trypsin. By confocal microscopy, ultrafine intercellular nanotubular structures were visible between adjacent transplanted and host hepatocytes. We postulate that these structures may assist in the phenotype conversion of chMSCs, possibly by exchange of cytoplasmic components between native hepatocytes and transplanted cells. Thus, a clonal pure population of hMSCs, which can be expanded in culture, may have potential as a cellular source for substitution damaged cells in hepatic injury. J. Cell. Biochem. 108: 693–704, 2009. © 2009 Wiley‐Liss, Inc.     

脐带间充质干细胞移植对慢性试验性肝损伤的治疗作用

目的观察脐带间充质干细胞（UC-MSC）对慢性实验性肝损伤的治疗作用并探讨其分子生物学机理。方法 50只7周龄的NOD/SCID小鼠注射四氯化碳（CCL4）制备慢性肝损伤模型后,应用随机数字表的方法随机将实验小鼠随机分成2组：模型组（25只）和UC-MSC移植组（25只）。UC-MSC移植组通过尾静脉注射移植1×106 UC-MSC,模型组注射同样体积的PBS。分别于移植后1、2、3和4周收集肝组织,应用免疫组织化学,RT-PCR和Western blot的方法分析细胞移植前后肝组织的病理生理学特征的变化。采用t检验和方差分析进行统计学分析。结果 UC-MSC移植治疗后肝组织表达人肝细胞特异性AFP,Alb,和内皮细胞特异性CD31,Flk-1。细胞移植4周后v WF标记的血管密度明显增加,同时伴有部分的肝功能改善,谷丙转氨酶（ALT）水平从（55.71±11.33）U/L减至（36.75±12.80）U/L（P〈0.05）。此外,本研究结果表明UC-MSC分泌几种重要的生长因子HGF,FGF-2,VEGF,和VEGF受体通过旁分泌的途径发挥肝组织修复的功能。结论在CCL4诱导的慢性肝损伤模型肝组织,人UC-MSC可以分化成肝细胞样细胞和内皮细胞样细胞,同时旁分泌多种细胞生长因子修复损伤的肝细胞,并伴有肝功能的改善。认为UCMSC移植或许成为将来肝脏损伤疾病一个重要的治疗选择。     

The role of hepatocytes and oval cells in liver regeneration and repopulation

The liver has the unique capacity to regulate its growth and mass. In rodents and humans, it grows rapidly after resection of more than 50% of its mass. This growth process, as well as that following acute chemical injury is known as liver regeneration, although growth takes place by compensatory hyperplasia rather than true regeneration. In addition to hepatocytes and non-parenchymal cells, the liver contains intra-hepatic "stem" cells which can generate a transit compartment of precursors named oval cells. Liver regeneration after partial hepatectomy does not involve intra or extra-hepatic (hemopoietic) stem cells but depends on the proliferation of hepatocytes. Transplantation and repopulation experiments have demonstrated that hepatocytes, which are highly differentiated and long-lived cells, have a remarkable capacity for multiple rounds of replication. In this article, we review some aspects of the regulation of hepatocyte proliferation as well as the interrelationships between hepatocytes and oval cells in different liver growth processes. We conclude that in the liver, normally quiescent differentiated cells replicate rapidly after tissue resection, while intra-hepatic precursor cells (oval cells) proliferate and generate lineage only in situations in which hepatocyte proliferation is blocked or delayed. Although bone marrow stem cells can generate oval cells and hepatocytes, transdifferentiation is very rare and inefficient.     

The uptake and subsequent loss of beryllium by rat liver parenchymal and non-parenchymal cells after the intravenous administration of particulate and soluble forms

A cell isolation technique has been used to study the uptake and subsequent loss of beryllium (Be) by rat liver after intravenous administration of non-lethal doses of either particulate beryllium phosphate or the more hepatotoxic soluble BeSO₄. It has been shown that beryllium phosphate is removed from the blood predominantly by the non-parenchymal (sinusoidal) cells of the liver and to a lesser extent more slowly by the parenchymal cells. After 24 h when the parenchymal cells have reached maximal Be content there has been a 50% loss of Be from the non-parenchymal cells and a similar loss from whole liver which is reflected in an increased level of Be in the blood. The Be count of non-parenchymal cells subsequently decreases much more slowly in a manner similar to that of the parenchymal cells, both being only halved during the following week. Within 24–48 h some redistribution of Be to the spleen occurs and it is suggested that this in part may be the result of Kupffer cell death. In splenectomized animals a high proportion of this redistributed Be appears to be retaken up by the liver mainly by the parenchymal cell population. After administration of BeSO₄, which is known to form beryllium phosphate in plasma, a greater proportion of the Be is taken up slowly by the parenchymal cells and no redistribution of Be to the spleen is observed. It is suggested that this behaviour is related primarily to the smaller size and nature of the beryllium phosphate particles formed in plasma under these conditions. The rate of loss of Be from both the parenchymal and non-parenchymal cells is similar to that measured in beryllium phosphate treated animals. It has been estimated that liver cell death is produced when the cell content exceeds 2–3 nmol Be/10⁶ cells although parenchymal cells appear to be more sensitive to Be derived from BeSO₄ than preformed beryllium phosphate.     

Different types of mitochondria in parenchymal and non-parenchymal rat-liver cells.

1. Intact and pure parenchymal and non-parenchymal cells were isolated from rat liver. The specific activities of several mitochondrial enzymes were determined in both parenchymal and non-parenchymal cell homogenates to characterize the mitochondria in these liver cell types. 2.In general the activities of mitochondrial enzymes were lower in non-parenchymal liver cells than in parenchymal cells. The specific activity of pyruvate carboxylase in non-parenchymal cells expressed as the percentage of that in parenchymal cells was onlu 2% for glutamate dehydrogenase 4.3% and for cytochrome c oxidase 79.4%. Monoamine oxidase, as an exception, has an equal specific activity in both cell types. 3. The activity ratio of pyruvate carboxylase at 10 mM pyruvate over 0.1 mM pyruvate is 3.35 for parenchymal cells and 1.50 for non-parenchymal cells. This indicates that non-parenchymal liver cells only contain the high affinity form of pyruvate carboxylase in contrast to parenchymal cells. 4. The ratio of glycerol-3-phosphate cytochrome c reductase over succinate cytochrome c reductase activity differs from parenchymal (0.01) and non-parenchymal cells (0.10). This might indicate that the glycerol-3-phosphate shuttle, which is important for the transport of reduction equivalents for cytosol to mitochondria is relatively more active in non-parenchymal cells than in parenchymal cells. 5. The activity pattern of mitochondrial enzymes in parenchymal and non-parenchymal cell homogenates indicates that these cell types contain different types of mitochondria. The presence of these different cell types in liver will therefore contribute to the heterogeneity of isolated rat liver mitochondria in which the mitochondria from non-parenchymal cells might be considered as "non-gluconeogenic".     

In vivo characteristics of a specific recognition site for LDL on non-parenchymal rat liver cells which differs from the 17 alpha-ethinyl estradiol-induced LDL receptor on parenchymal liver cells

Chemical modification of lysine or arginine residues of apolipoprotein B-100 in human low-density lipoprotein (LDL) with respectively reductive methylation (Me-LDL) or cyclohexanedione treatment (CHD-LDL) was applied to determine the role of these amino acids in LDL recognition by the various liver cell types. The cell association of native human LDL, Me-LDL and CHD-LDL to parenchymal and non-parenchymal cells was determined in vivo by isolating the various cell types 30 min after intravenous injection of the lipoproteins. In order to prevent degradation or release of cell-bound apolipoproteins during cell dissociation and purification, a low-temperature (8 degrees C) liver perfusion and cell isolation procedure was performed. It was found that reductive methylation of LDL inhibits the association of LDL to both parenchymal and non-parenchymal cells, indicating that lysine residues are important for recognition of LDL by both these cell types. In contrast, cyclohexanedione treatment of LDL did not influence the cell association of LDL to non-parenchymal cells. 17 alpha-Ethinyl estradiol treatment selectively increases the cell association of LDL by parenchymal cells (16-fold), leaving the non-parenchymal cell association uninfluenced. The increased cell-association of LDL to parenchymal cells is almost completely blocked by cyclohexanedione treatment of LDL (by 81%) or by methylation of LDL (by 97%). These data indicate that the arginine residues in LDL are not important for the recognition of LDL by non-parenchymal cells, whereas for the cell association of LDL to the estrogen-stimulated binding site on parenchymal cells both arginine and lysine residues are essential. The in vivo cell association of CHD-LDL or native LDL to non-parenchymal cells was lowered to the level of Me-LDL by ethyl oleate treatment of the rats, while no effect of ethyl oleate on parenchymal cells was noticed. These data suggest that the specific site for LDL on non-parenchymal cells, which need lysine residues on LDL for recognition, can be down-regulated by ethyl oleate treatment. The LDL, internalized by non-parenchymal cells, is effectively degraded. This degradation occurs at least partly in the lysosomes. It is suggested that the unique recognition site for LDL on non-parenchymal cells may be quantitatively important for serum LDL catabolism.     

Stability of hexokinases A, B and C and N-acetylglucosamine kinase in liver cells isolated from rats submitted to diabetes and several dietary conditions.

The effect of dietary and hormonal variations on the specific activities of hexokinase isoenzymes, N-acetylglucosamine kinase and pyruvate kinase isoenzymes in parenchymal and non-parenchymal liver cells was studied. Hexokinase D was markedly decreased in hepatocytes from animals fasted or fed on the carbohydrate-free diet as well as from diabetic rats, attaining a constant low level of about 17% of normal values. Pyruvate kinase L was also diminished in hepatocytes under the same experimental conditions. In contrast, the three high-affinity hexokinase isoenzymes A, B and C remained without variation in total amount or in their relative proportions in hepatocytes and non-parenchymal liver cells isolated from animals under the various conditions studied. N-Acetylglucosamine kinase activities also did not change either in parenchymal or in non-parenchymal liver cells under all conditions. The results are discussed in relation to the significance of N-acetylglucosamine kinase and the various hexokinase isoenzymes for the phosphorylation of glucose after dietary and hormonal manipulations.     

The perspectives of treatment of liver insufficiency by stem cells

The insufficiency of liver functions remains one of the major causes of death. The liver transplantation is the most effective method for treating severe liver diseases. The shortage of donor organs and high risk of graft rejection are the main problems for liver transplantation. Stem cells and isolated hepatocytes are the alternative means for repopulating liver after various injuries instead of liver transplantation. This review analyses achievements in therapy of liver insufficiency by means of stem cells in model experiments on animals as well as in clinical practice and also perspectives of employment of stem cells for treatment of liver insufficiency.