CAUSES OF CHIRROSIS

INTRODUCTION

LIVER CIRRHOSIS

Cirrhosis is a complication of many liver diseases that is characterized by abnormal structure and function of the liver. The diseases that lead to cirrhosis do so because they injure and kill liver cells, and the inflammation and repair that is associated with the dying liver cells causes scar tissue to form. The liver cells that do not die multiply in an attempt to replace the cells that have died. This results in clusters of newly-formed liver cells (regenerative nodules) within the scar tissue. There are many causes of cirrhosis; they include chemicals (such as alcohol, fat, and certain medications), viruses toxic metals (such as iron and copper that accumulate in the liver as a result of genetic diseases), and autoimmune liver disease in which the body's immune system attacks the liver.

Cirrhosis refers to scarring of the liver. Scar tissue forms because of injury or long-term disease. It replaces healthy tissue.

Scar tissue cannot do what healthy liver tissue does—make protein, help fight infections, clean the blood, help digest food, and store energy for when you need it. Scar tissue also blocks the normal flow of blood through the liver. Too much scar tissue means that your liver cannot work properly. To live, you need a liver that works.

Cirrhosis can be life-threatening, but it can also be controlled if treated early.

CAUSES OF CHIRROSIS:

Cirrhosis has many causes, including

• alcohol abuse (alcoholic liver disease)
• chronic viral hepatitis (hepatitis B, C, or D)
• autoimmune hepatitis, which is destruction of liver cells by the body’s immune system
• nonalcoholic fatty liver disease or nonalcoholic steatohepatitis (NASH), which is fat deposits and inflammation in the liver
• some drugs, toxins, and infections
• blocked bile ducts, the tubes that carry bile from the liver
• some inherited diseases such as
• hemochromatosis (HEE-moh-KROH-muh-TOH-sus), a disease that occurs when the body absorbs too much iron and stores the excess iron in the liver, pancreas, and other organs
• Wilson disease, which is caused by the buildup of too much copper in the liver
• protoporphyria (PROH-toh-pour-FEAR-ee-uh), a disorder that affects the skin, bone marrow, and liver

Sometimes the cause of cirrhosis remains unknown even after a thorough medical examination.

INTRODUCTION

Most liver diseases lead to hepatocyte dysfunction with the possibility of eventual organ failure. The replacement of diseased hepatocytes and the stimulation of endogenous or exogenous regeneration by stem cells are the main aims of liver-directed

Bone marrow appears to contain three types of stem cell populations hematopoietic (hscs)endothelial progenitor and mesenchymal .the hscs produce all types of blood cells in the body .endothelial flat cells that line the blood and lymphatic vessels ,the heart,and various other body cavities .mesenchymal cells,a network of cells derived from the embronic mesoderm ,give rise to connective tissues and other parts of the cardiovascular system .each of these kinds of stemcells can be isolated from adult marrow and apper to have the potential for multiple lineges of sifferentation

Bone marrow differentiation

The transdifferentiation of BMSC into hepatic cells invivo was described in rats[3,17], mice[7] and humans[8,18]. Thishas brought new hope to cell therapy using autologous bone marrow . MSC can easily be obtained following a simple bone marrowaspiration procedure, and may be subsequently culturedand expanded in vitro without losing their stem cellpotential, making them an attractive target for cell therapy

THERAPETIC APPLICATIONS OF BONE MARROW –DERIVED STEM CELLS IN LIVER TRANSPLANTATION FOR END –STAGE LIVER DISEASES

Bone marrow-derived stem cells (BMDSCs) are capable of differentiating into hepatocyte-like cells and contribute to liver injury repair. The microenvironment of liver injury caused by rejection, ischemia/reperfusion, loss of liver mass, recurrence of HCV and “small-for-size syndrome� after LT can attract a variety of bone marrow-derived stem cell population to the peripheral circulation and then migration to the injury liver to promote the hepatic function restoration. Additionally, BMDSCs can also take part in the functional regeneration of living donor liver after LDLT. This participation in liver regeneration may be associated to the interaction between SDF-1and its receptor CXCR4, involving HGF, IL-8, MMP9, and VEGF/VEGFR-2. BMDSC with its bio-characteristics could maintain the allograft tolerance from different angles and in different ways.

ADIPOSE STEM CELLS:

Adipose tissue-derived mesenchymal stem cells as a source of human hepatocytes

Recent observations indicate that several stem cells can differentiate into hepatocytes; thus, cell-based therapy is a potential alternative to liver transplantation. The goal of the present study was to examine the in vitro hepatic differentiation potential of adipose tissue-derived mesenchymal stem cells (AT-MSCs). We used AT-MSCs from different age patients and found that, after incubation with specific growth factors (hepatocyte growth factor [HGF], fibroblast growth factor [FGF1], FGF4) the CD105(+) fraction of AT-MSCs exhibited high hepatic differentiation ability in an adherent monoculture condition. CD105(+) AT-MSC-derived hepatocyte-like cells revealed several liver-specific markers and functions, such as albumin production, low-density lipoprotein uptake, and ammonia detoxification. More importantly, CD105(+) AT-MSC-derived hepatocyte-like cells, after transplantation into mice incorporated into the parenchyma of the liver..

Stem cells are a population possessing 1) self-renewal capacity, 2) long-term viability, and 3) multilineage potential. The multilineage potential of embryonic stem cells and adult stem cells from the bone marrow has been characterized extensively. Although embryonic stem cell potential is enormous, many ethical and political issues accompany their use. Therefore, adult stem cells from the bone marrow stroma (i.e., mesenchymal stem cells, MSCs) have been proposed as an alternative source. Originally identified as a source of osteoprogenitor cells, MSCs differentiate into adipocytes, chondrocytes, osteoblasts, and myoblasts in vitro

Adipose tissue differentiation

Adipose tissue provides an abundant and accessible source of adult stem termed adipose tissue-derived stem cells (ADSC) ADSC differentiation into tissues of nonmesodermal origin, an initial effort has been made to differentiate ADSC into hepatocytes, endocrine pancreatic cells, neurons, cardiomyocytes, hepatocytes, and endothelial/vascular cells

ADSC treated with HGF, oncostatin M (OSM), and dimethyl sulfoxide have the potential to develop a hepatocyte-like phenotype expressing albumin and alpha-fetoprotein. HGF is a potent mitogen that acts via the HGF receptor c-Met, a transmembrane protein with an intracellular tyrosine kinase domain. HGF plays an important role in liver regeneration and embryonic development. OSM is a member of the IL-6 cytokine family regulating hepatocyte differentiation.

OSM is a member of the IL-6 cytokine family regulating hepatocyte differentiation

Expanding on these in vitro data, intravenously injected ADSC show integration into the liver in mice, an effect that can be enhanced after partial hepatectomy that promotes liver regeneration

Comparative between Bone marrow and Adpiose stem cells:

In one study comparing bone marrow-derived mesenchymal stem cells (BMMSC) and lipoaspirate-derived ADSC [28] from the same patient, no significant differences were observed regarding the yield of adherent stromal cells, growth kinetics, cell senescence, multilineage differentiation capacity, or gene transduction efficiency. Metabolic characteristics and fat cell viability seem not to differ when comparing standard liposuction with syringe aspiration, and no unique combination of preparation or harvesting techniques has appeared superior to date [25] cell therapy

ADSC compared with MSC from other sources possessed the longest culture period and the highest proliferation capacity[23]. Thus, adipose tissue may be an ideal source of large amounts of autologous stemcells attainable by a less invasive method than BMSC

” In addition, a comparative study of the hepatogenic differentiation potential in vitro of both types of MSC has not yet been performed”.

Based on these previous fi ndings, the aim of this study was to investigate and compare the hepatic differentiation of human MSC from bone marrow (BMSC) and adipose tissue (ADSC) obtained from healthy donors. To this end, these cells were isolated and cultured under similar prohepatogenic conditions to those of liver development to define the different capacities of hepatic differentiation of the two subsets in vitro

Mesenchymal stem cells (MSCs) isolated from bone marrow (BM), cartilage, and adipose tissue (AT) possess the capacity for self-renewal and the potential for multilineage differentiation, and are therefore perceived as attractive sources of stem cells for cell therapy. However, MSCs from these different sources have different characteristics. We compared MSCs of adult Sprague Dawley rats derived from these three sources in terms of their immunophenotypic characterization, proliferation capacity, differentiation ability, expression of angiogenic cytokines, and anti-apoptotic ability. According to growth curve, cell cycle, and telomerase activity analyses, MSCs derived from adipose tissue (AT-MSCs) possess the highest proliferation potential, followed by MSCs derived from BM and cartilage (BM-MSCs and C-MSCs). In terms of multilineage differentiation, MSCs from all three sources displayed osteogenic, adipogenic, and chondrogenic differentiation potential. The result of realtime RT-PCR indicated that these cells all expressed angiogenic cytokines, with some differences in expression level. Flow cytometry and MTT analysis showed that C-MSCs possess the highest resistance toward hydrogen peroxide -induced apoptosis, while AT-MSCs exhibited high tolerance to serum deprivation-induced apoptosis. Both AT and cartilage are attractive alternatives to BM as sources for isolating MSCs, but these differences must be considered when choosing a stem cell source for clinical application.

Mesenchymal stem cells (MSCs) represent a promising tool for new clinical concepts in supporting cellular therapy. Bone marrow (BM) was the first source reported to contain MSCs. However, for clinical use, BM may be detrimental due to the highly invasive donation procedure and the decline in MSC number and differentiation potential with increasing age. More recently, umbilical cord blood (UCB), attainable by a less invasive method, was introduced as an alternative source for MSCs. Another promising source is adipose tissue (AT). We compared MSCs derived from these sources regarding morphology, the success rate of isolating MSCs, colony frequency, expansion potential, multiple differentiation capacity, and immune phenotype. No significant differences concerning the morphology and immune phenotype of the MSCs derived from these sources were obvious. Differences could be observed concerning the success rate of isolating MSCs, which was 100% for BM and AT, but only 63% for UCB. The colony frequency was lowest in UCB, whereas it was highest in AT. However, UCB-MSCs could be cultured longest and showed the highest proliferation capacity, whereas BM-MSCs possessed the shortest culture period and the lowest proliferation capacity. Most strikingly, UCB-MSCs showed no adipogenic differentiation capacity, in contrast to BM- and AT-MSCs. Both UCB and AT are attractive alternatives to BM in isolating MSC: AT as it contains MSCs at the highest frequency and UCB as it seems to be expandable to higher numbers

Distinctions between PLA and MSC Population

Analysis of PLA cells and MSCs in this study has identified many similarities between the two populations, lending support to the theory that stem cells can be found within adipose tissue. However, these similarities may also indicate that PLA cells are simply an MSC population located within the adipose compartment, perhaps the result of infiltration of MSCs from the peripheral blood supply. However, we do no believe this to be the case. First, the presence of MSCs in the peripheral blood is controversial. Moreover, if present within the peripheral blood, the number of MSCs within the bone marrow stroma is extremely low (~1 MSC per 10⁵ stromal cells; Rikard et al., 1994 ; Bruder et al., 1997 ; Pittenger et al., 1999 ) and is likely to be even lower in the peripheral blood. This low level is unlikely to give the relatively high levels of differentiation observed in this study. Second, we have observed several distinctions between PLA and MSC populations that suggest they are similar, but not identical, cell types: 1) Preliminary results on PLA cells indicate that sera screening is not necessary for their expansion and differentiation (Zuk et al., 2001 ), a requirement for MSCs (Lennon et al., 1996 ). 2) MSCs did not undergo chondrogenic or myogenic differentiation under the conditions used in this study, suggesting distinctions in differentiation capacities and/or kinetics. 3) Immunofluorescence analysis identified differences in CD marker profile between PLA and MSC populations. In contrast to MSCs, expression of CD106 was not observed on PLA cells, whereas PLA cells were found to express CD49d. 4) Distinctions between PLA and MSC populations may also extend to the gene level. For example, osteocalcin expression was restricted to PLA samples induced specifically with VD. Although treatment of MSCs with VD also induced OC expression, expression of this gene was also observed in dexamethasone-treated and non-induced MSCs, albeit at lower levels (our unpublished data; online Figure S5). In addition, PLA cells and MSCs exhibited distinctions in BMP-2 and dlx5 expression, both of which were found in induced MSCs only. Because dlx5 and BMP2 are known to mediate expression of multiple osteogenic genes, it is possible that PLA and MSC populations differ in their regulation of the osteogenic differentiation pathway. Taken together, these differences may indicate that adipose tissue contains stem cells, distinct from those found in the bone marrow stroma. However, the possibility that PLA cells are a clonal variant of circulating MSCs cannot be ruled out.

Adipose tissue, like bone marrow, is derived from the mesenchyme and contains a supportive stroma that is easily isolated. Based on this, adipose tissue may represent a source of stem cells that could have far-reaching effects on several fields. We have previously identified a putative stem cell population within human lipoaspirates (Zuk et al., 2001 ). This cell population, called processed lipoaspirate (PLA) cells, can be isolated from adipose tissue in significant numbers and exhibits stable growth and proliferation kinetics in culture. Moreover, PLA cells, like MSCs, differentiate in vitro toward the osteogenic, adipogenic, myogenic, and chondrogenic lineages when treated with established lineage-specific factors. The multilineage differentiation capacity of PLA cells led us to speculate that a population of multipotent stem cells, comparable with MSCs, can be isolated from human adipose tissue.

To confirm whether PLA cells represent a stem cell population, we conducted an extensive molecular and biochemical characterization of the PLA population and several clonal isolates, termed adipose-derived stem cells (ADSCs). PLA cells expressed several CD marker antigens similar to those observed on MSC controls. Induction of PLA cells and clones toward multiple mesodermal lineages resulted in the expression of several lineage-specific genes and proteins similar to those observed in induced MSC controls and lineage-committed precursor cell lines. Moreover, established biochemical assays confirmed lineage-specific metabolic activity in induced PLA populations. In addition to mesodermal capacity, PLA cells and clones differentiated into putative neurogenic cells exhibiting a neuronal-like morphology and expressing several proteins consistent with the neuronal phenotype. Finally, PLA cells exhibited unique characteristics distinct from that seen in MSCs, including differences in CD marker and gene expression profiles. In conclusion, the results presented in this study suggest that adipose tissue may be an additional source of unique, pluripotent stem cells with multi-germline potential.

Cell Culture and Differentiation

PLA cells were obtained from raw human lipoaspirates and cultured as described in a previous study (Zuk et al., 2001 ). Briefly, raw lipoaspirates were washed extensively with sterile phosphate-buffered saline (PBS) to remove contaminating debris and red blood cells. Washed aspirates were treated with 0.075% collagenase (type I; Sigma-Aldrich, St. Louis, MO) in PBS for 30 min at 37°C with gentle agitation. The collagenase was inactivated with an equal volume of DMEM/10% fetal bovine serum (FBS) and the infranatant centrifuged for 10 min at low speed. The cellular pellet was resuspended in DMEM/10% FBS and filtered through a 100-μm mesh filter to remove debris. The filtrate was centrifuged as detailed above and plated onto conventional tissue culture plates in control medium (Table 1). Normal human osteoblasts (NHOst), normal human chondrocytes from the knee (NHCK), and a population of MSCs from human bone marrow were purchased from Clonetics (Walkersville, MD) and maintained in commercial medium. The murine 3T3-L1 preadipocyte cell line (Green and Meuth, 1974 ) was obtained from American Type Culture Collection (Manassas, VA). NHOst, PLA cells, and 3T3-L1 cells were treated with mesenchymal lineage-specific media as outlined in Table 1. MSCs were induced using commercial control medium supplemented with the growth factors outlined in Table 1. NHOst and NHCK cells were induced using commercially available induction media (Clonetics).

PLA Cells Are Phenotypically Similar to MSCs

Characterization of MSCs has been performed using the expression of cell-specific proteins and CD markers (Bruder et al., 1998b ; Conget and Minguell, 1999 ; Pittenger et al., 1999 ). Like MSCs, PLA cells expressed CD29, CD44, CD71, CD90, CD105/SH2, and SH3 and were absent for CD31, CD34, and CD45 expression (online Figure S1). Moreover, flow cytometry on PLA cells confirmed the expression of CD13, whereas no expression of CD14, 16, 56, 62e, or 104 was detected (Table 2). These results demonstrate that similar CD complements are expressed on both PLA cells and MSCs. However, distinctions in two CD markers were observed: PLA cells were positive for CD49d and negative for CD106, whereas the opposite was observed on MSCs. Expression of CD106 has been confirmed in the bone marrow stroma and, specifically, MSCs (Levesque et al., 2001 ) where it is functionally associated with hematopoiesis. The lack of CD106 on PLA cells is consistent with the localization of these cells to a non-hematopoietic tissue.

Liver transplantation is the primary treatment for various end-stage hepatic diseases but is hindered by the lack of donor organs and by complications associated with rejection and immunosuppression. There is increasing evidence to suggest the bone marrow is a transplantable source of hepatic progenitors. We previously reported that multipotent bone marrow-derived mesenchymal stem cells differentiate into functional hepatocyte-like cells with almost 100% induction frequency under defined conditions, suggesting the potential for clinical applications. The aim of this study was to critically analyze the various parameters governing the success of bone marrow-derived mesenchymal stem cell-based therapy for treatment of liver diseases.

METHODS: Lethal fulminant hepatic failure in nonobese diabetic severe combined immunodeficient mice was induced by carbon tetrachloride gavage. Mesenchymal stem cell-derived hepatocytes and mesenchymal stem cells were then intrasplenically or intravenously transplanted at different doses.

RESULTS: BMSC and ADSC exhibited a fibroblastic morphology that changed to a polygonal shape when cells differentiated. Expression of stem cell marker Thy1 decreased in differentiated ADSC and BMSC. However, the expression of the hepatic markers, albumin and CYPs increased to a similar extent in differentiated BMSC and ADSC. Hepatic gene activation could be attributed to increased liver-enriched transcription factors (C/EBPβ and HNF4α), as demonstrated by adenoviral expression vectors.

CONCLUSION: Mesenchymal stem cells can be induced to hepatogenic transdifferentiation in vitro . ADSCs have a similar hepatogenic differentiation potential to BMSC, but a longer culture period and higher proliferation capacity. Therefore, adipose tissue may be an ideal source of large amounts of autologous stem cells, and may become an alternative for hepatocyte regeneration, liver cell trans transplantation or preclinical drug testing.

Adipose tissue is a source of multipotent stem cells that can be easily isolated, selected, and induced into mature, transplantable hepatocytes. The fact that they are easy to procure ex vivo in large numbers makes them an attractive tool for clinical studies in the context of establishing an alternative therapy for liver dysfunction.

Bone marrow-derived mesenchymal stem cells can effectively rescue experimental liver failure and contribute to liver regeneration and offer a potentially alternative therapy to organ transplantation for treatment of liver diseases.