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Abstract

  1. Top of page
  2. Abstract
  3. COULD THE LIVER BE A TARGET FOR STEM CELL THERAPY?
  4. HOW DO ADULT STEM CELLS CONTRIBUTE TO LIVER REPAIR …?
  5. HOW DO ADULT STEM CELLS REACH THE LIVER?
  6. ADULT STEM CELLS: FRIEND OR FOE?
  7. WHAT IS THE VALUE OF ADULT STEM CELL THERAPY IN CHRONIC LIVER DISEASE?
  8. IF BONE MARROW CELLS DO NOT REPLACE DISEASED HEPATOCYTE POPULATIONS, CAN STEM CELL THERAPY WORK IN CIRRHOSIS?
  9. WHAT ARE THE ALTERNATIVES TO ADULT STEM CELLS?
  10. ADULT STEM CELL THERAPY IN LIVER DISEASE: CHALLENGES FOR THE FUTURE
  11. REFERENCES

Hematopoietic stem cells have potential in the field of regenerative medicine because of their capacity to form cells of different lineages. Bone marrow stem cells have been shown to contribute to parenchymal liver cell populations, and although this may not be functionally significant, it has sparked interest in the field of autologous stem cell infusion as a possible treatment for cirrhosis. In this review, we will examine the evidence for the contribution of bone marrow-derived cells to populations of liver cells and for the functional contribution of bone marrow-derived cells to both liver fibrosis and repair. The mechanisms by which cells are trafficked from the bone marrow to the liver are complex; the stromal derived factor-1/CXC receptor 4 axis is central to this process. There are limited data in liver injury, but we will examine findings from the bone marrow transplantation literature and discuss their relevance to liver disease. Stromal derived factor-1 also has a role in endogenous liver stem cell accumulation. Some groups have already started infusing autologous bone marrow cells into patients with cirrhosis. We will review these trials in the context of the basic science that we have discussed, and we will consider targets for investigation in the future. Liver Transpl 16:118–129, 2010. © 2010 AASLD.

COULD THE LIVER BE A TARGET FOR STEM CELL THERAPY?

  1. Top of page
  2. Abstract
  3. COULD THE LIVER BE A TARGET FOR STEM CELL THERAPY?
  4. HOW DO ADULT STEM CELLS CONTRIBUTE TO LIVER REPAIR …?
  5. HOW DO ADULT STEM CELLS REACH THE LIVER?
  6. ADULT STEM CELLS: FRIEND OR FOE?
  7. WHAT IS THE VALUE OF ADULT STEM CELL THERAPY IN CHRONIC LIVER DISEASE?
  8. IF BONE MARROW CELLS DO NOT REPLACE DISEASED HEPATOCYTE POPULATIONS, CAN STEM CELL THERAPY WORK IN CIRRHOSIS?
  9. WHAT ARE THE ALTERNATIVES TO ADULT STEM CELLS?
  10. ADULT STEM CELL THERAPY IN LIVER DISEASE: CHALLENGES FOR THE FUTURE
  11. REFERENCES

Stem cells are undifferentiated cells that are able to proliferate in an effectively unlimited fashion. There are 2 broad types of stem cells with therapeutic potential: embryonic stem (ES) cells and adult stem cells.

ES cells are derived from the early blastocyst and are able to divide and remain undifferentiated indefinitely in culture.1 They are pluripotent; that is, they are able to differentiate into any cell type within the body except for primordial germ cells.2 They are attractive targets for study in the field of regenerative medicine. However, human ES cells are associated with ethical and legal problems, as they are derived from embryos that are destroyed in the process. Alternative stem cell sources may be required. It is possible to derive ES-like cells from adult tissue by retroviral transduction. Induced pluripotent stem cells are derived from adult somatic cells (eg, skin fibroblasts) by their infection with retroviruses carrying factors that are important in maintaining the “stemness” of ES cells.3, 4 However, this work is at an early stage, and there will be concerns attached to the clinical use of retrovirally transformed cells. There are many sources of tissue-specific stem cells, including bone marrow [for both hematopoietic stem cells (HSCs) and mesenchymal stem cells (MSCs)], umbilical cord blood, placentas, fetuses, and amniotic fluid, which might be potential substrates for stem cell therapy and have been shown to exhibit plasticity,5 that is, the potential for differentiation into cells of different lineages. Taking into account ethical issues and ease of access to tissue, researchers have focused their experiments on HSCs, MSCs, and cord blood stem cells. Adult stem cells and ES cells are compared in Table 1.

Table 1. Comparison of Embryonic and Adult Stem Cells
Embryonic Stem CellsAdult Stem Cells
AdvantagesDisadvantagesAdvantagesDisadvantages
They are pluripotent: they can differentiate into any cell type (except germ cells).They come from embryos that are destroyed in the process of extraction.They come from adults. Sources of hematopoietic stem cells, for example, include bone marrow, mobilized peripheral blood, and umbilical cord blood.They are multipotent: they are able to differentiate into only a limited number of cell types.
There is the possibility of geneticLaboratory manipulation has been performed only since 1998.There is extensive experience with, for example, bone marrow transplantation for >40 years.They may not have the same capacity to multiply as embryonic stem cells.
 manipulation of embryonic stem cell lines.They are allogeneic; therefore, there are problems with immune rejection when they are transplanted.They are likely to be autologous; therefore, there will be no problems with immune rejection.They may be difficult to isolate if they are present in small quantities (especially if they are nonhematopoietic stem cells).
 There are concerns regarding malignant potential if they are transplanted: they could give rise to teratomas. They are difficult to purify to a high degree in large numbers.
 They may have nonhuman components or pathogens. All current embryonic stem cell lines were established with mouse fibroblast feeder layers. Adult stem cells are rare but are able to self-renew; progenitor cells are more common but do not self-renew and, therefore, produce fewer daughter cells. Current protocols (eg, bone marrow transplantation) include the use of progenitor cells.
 Currently, only 22 embryonic stem cell lines are eligible for US federal funding. They are difficult to expand in culture and difficult to maintain in the undifferentiated state.
 There is potential for the accumulation of genetic/epigenetic changes.  
 The limited number of cell lines currently available does not accurately represent the genetic diversity seen in populations.  

Stem cell therapy has become established in the public consciousness over recent years. Stem cells are envisaged by some as a panacea that will allow us to replace worn-out cells and organs. The most commonly mentioned therapeutic targets are the spinal cord in spinal injury and the substantia nigra in Parkinson's disease. The liver has not escaped as a potential target for stem cell therapy. Although it is able to extensively renew itself, there are well-defined situations in which repair fails in both acute and chronic liver disease. Cirrhosis-related mortality is increasing in the United Kingdom as a whole and particularly in Scotland, with a 104% increase in male cirrhosis deaths between the periods of 1987-1991 and 1997-2001.6 Paracetamol overdose remains the most common cause of acute liver failure7 despite legislative changes designed to limit the supply of paracetamol,8 at least in the United Kingdom. Liver transplantation is currently the only available therapy for many of these patients, but because of donor shortages and the implications of lifelong immunosuppression, many patients are ineligible for this. Stem cell therapy for liver disease would therefore be attractive, but is it yet a realistic option?

HSCs are the most studied adult stem cells. They are identified by CD34 immunoreactivity, among other markers. These are the cells that are used in bone marrow transplantation and are able to differentiate into all hematogenous cell types. There are also other stem cells found within the bone marrow. These are MSCs.9 They differentiate into osteoblasts, adipocytes, and other mesenchymal cell lineages. They may have a role in immune tolerance and have been used to treat graft versus host disease. Both bone marrow stem cell types can contribute to cell lineages within the liver.10, 11 In this review, we will discuss both the mechanisms by which adult stem cells may contribute to liver repair and the factors involved in stem cell trafficking that could enhance the endogenous stem cell response to liver injury.

HOW DO ADULT STEM CELLS CONTRIBUTE TO LIVER REPAIR …?

  1. Top of page
  2. Abstract
  3. COULD THE LIVER BE A TARGET FOR STEM CELL THERAPY?
  4. HOW DO ADULT STEM CELLS CONTRIBUTE TO LIVER REPAIR …?
  5. HOW DO ADULT STEM CELLS REACH THE LIVER?
  6. ADULT STEM CELLS: FRIEND OR FOE?
  7. WHAT IS THE VALUE OF ADULT STEM CELL THERAPY IN CHRONIC LIVER DISEASE?
  8. IF BONE MARROW CELLS DO NOT REPLACE DISEASED HEPATOCYTE POPULATIONS, CAN STEM CELL THERAPY WORK IN CIRRHOSIS?
  9. WHAT ARE THE ALTERNATIVES TO ADULT STEM CELLS?
  10. ADULT STEM CELL THERAPY IN LIVER DISEASE: CHALLENGES FOR THE FUTURE
  11. REFERENCES

Liver repair is felt to occur by 3 different methods. In the vast majority of cases in which hepatocyte replication is unimpaired, liver repair is the result. The classical example of this is partial hepatectomy, but recovery from carbon tetrachloride injury also occurs in this manner. In situations in which hepatocyte replication is impaired (by 2-acetylaminofluorene or retrorsine in animal models and by alcoholic liver disease or hepatitis C virus infection, among others, in humans), endogenous liver stem cells, the oval cell population, are believed to mediate repair. As detailed later, bone marrow-derived cells may have a role in liver repair when endogenous stem cells are insufficient12 (see also Fig. 1).

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Figure 1. Overview of the mobilization and destination of HSCs in acute liver injury. In acute liver injury, SDF-1, G-CSF, and other mediators are released into the peripheral blood. Within the bone marrow, there is a decrease in both SDF-1 and CXCR4 expression, which is mediated in part by an increase in neutrophil proteases. There is also an increase in HSC proliferation. As a result of this, there is an efflux of HSCs (and possibly MSCs) into the peripheral blood. Although HSCs are not initially primed to home to new niches, after a short time, CXCR4 is re-expressed, surface CD26 decreases, and the cells are able to home to new niches. Once they reach the liver, their fate may depend on the milieu that they encounter. Transdifferentiation into hepatocytes is a rare event. In a proinflammatory state, they may preferentially differentiate into myofibroblasts; in a liver in which a reparative process has been initiated, they may move into the stem cell niche and provide support to endogenous stem cell-mediated repair and resolution of fibrosis (see the text for references).

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… In Animal Models?

In 1999, Petersen and colleagues13 reported that parts of the rat liver were repopulated by cells that appeared to originate in the bone marrow under conditions of liver injury in which native hepatocytes were prevented from proliferating. Female rats that had received sex-mismatched bone marrow transplants were injured. Male DNA was initially found within the nonparenchymal cell fraction (ie, potential liver-resident stem cells) but, 13 days after injury, was also found within the hepatocyte fraction, and this implied that bone marrow-derived hepatic oval cells were differentiating into hepatocytes. Bone marrow-derived proteins were also demonstrated within biliary canaliculi and hepatic oval cells. In this model, 0.14% to 0.16% of hepatocytes showed evidence of bone marrow origins. This was a landmark publication. It added to the evidence that progenitor cells could originate outside the organ to which they might contribute. Until this time, the evidence had been restricted to skeletal muscle progenitors14 and endothelial cell progenitors.15 Since then, CD34+ cells have been shown to be antecedents for epithelial cells within the respiratory tract, small and large intestines, skin, and biliary tree.16

There have been many other reports of bone marrow derivation of hepatocytes in diverse models. The most successful model in terms of the population and functionality of bone marrow-derived hepatocytes is the mouse FAH−/− model,17 in which the mouse requires dietary supplementation with 2-(2-nitro-4-trifluoro-methylbenzyol)-1,3-cyclohexanedione to avert liver failure. After wild-type bone marrow transplantation, 4 of 9 mice survived when 2-(2-nitro-4-trifluoro-methylbenzyol)-1,3-cyclohexanedione supplementation was withdrawn after 3 weeks. At 7 months, 30% to 50% of the liver was donor-derived. This is the highest proportion by far of donor-derived hepatocytes obtained in rodent bone marrow transplantation experiments.

Models using human cells in immunocompromised mice have also demonstrated bone marrow-derived hepatocytes18 at a low frequency. At 4 weeks post-stem cell infusion, human hepatocytes were identified within the uninjured mouse liver at a rate of 0.011% of cells. It was speculated that the low frequency of transdifferentiation was due to the absence of a stimulus for proliferation as found in the FAH−/− mouse model17 or the carbon tetrachloride/2-acetylaminofluorene rat model.13 Enhanced engraftment of bone marrow-derived cells can be obtained by an injury to the liver prior to the infusion of cells. In an experiment using Gunn rats (which are deficient in bilirubin glucuronyl transferase), the engraftment efficiency of bone marrow cells infused into the portal vein was enhanced from 0.04% to 0.28% of hepatocytes by preconditioning with an ischemia-reperfusion protocol.19 This was sufficient to decrease the plasma level of unconjugated bilirubin by 35%.

… In Clinical Situations?

This phenomenon has also been demonstrated in humans. Liver biopsy samples from patients with sex-mismatched bone marrow (male donor, female recipient) or liver (female donor, male recipient) transplants were examined.20 In these patients, 5% to 40% of hepatocytes contained Y chromosomes, as did 4% to 38% of cholangiocytes. The biopsy sample showing the least liver injury contained the fewest bone marrow-derived hepatocytes and cholangiocytes, whereas the liver with the most severe injury (fibrosing cholestatic recurrent hepatitis C) contained the most (64% of hepatocytes were bone marrow-derived); this confirmed that bone marrow-derived cells are found most frequently within injured livers, although this finding has not been widely replicated.

Until April 2003, the story appeared relatively straightforward; there was a constant influx of HSCs to the liver that contributed to the oval cell population13 and then differentiated into hepatocytes13, 17, 18 and (probably) cholangiocytes20 at a very low rate. In times of stress and injury, the rate of differentiation increased somewhat so that cholangiocyte differentiation became apparent, and hepatocyte differentiation had possible functional significance. At that point, 2 studies came to light suggesting that HSCs may contribute to hepatocytes or other parenchymal liver cells by fusion rather than transdifferentiation.

… By Fusing with Native Cells?

Fusion is a process by which 2 or more cells join to form 1 or more cells, each containing DNA from the original cells. There was previous in vitro evidence of fusion of murine HSCs with ES cells21 and with cells of the monocyte/macrophage lineage.22

The FAH−/− mouse experiments were repeated.23, 24 FAH+/+ liver nodules were dissected, and the DNA was extracted.23 Surprisingly, donor-derived liver nodules contained only 12% to 48% donor DNA versus >90% donor DNA within the bone marrow and spleen. Hepatocytes from regenerative nodules expressed both donor and host alleles. Fusion also occurs in a chronic carbon tetrachloride model.25 Further questions were raised about the identity of the cells that had fused with the host hepatocytes. There was speculation that the HSCs which had been seen to fuse with ES cells in vitro were likely to be committed monocyte progenitors21; earlier reports had concerned macrophages22 capable of rescuing the FAH−/− mouse.26

The apparent bone marrow derivation of hepatic oval cells reported by Petersen et al.13 was also called into question. However, fusion is not shown in all models,18 and transdifferentiation has been demonstrated in vitro.27 The relative contribution of fusion or transdifferentiation may depend on the model and how it affects the viability of native cells. In the FAH−/− model, in which the barrier to hepatocyte replication is the absence of a factor (in this case a functioning FAH gene), the presence of donor cell components within the eventual hepatocyte is not a disadvantage. Fusion and transdifferentiation events are both extremely rare, but given the relative predominance of cells of the monocyte/macrophage lineage (in comparison with circulating HSCs), fusion is favored in this model. In models in which native cells (both hepatocytes and oval cells) are prevented from replicating because of the toxicity of, for example, retrorsine or monocrotaline,28 fusion will result in cells that cannot replicate, and transdifferentiation is favored, despite the relative scarcity of the substrate cells.

Further evidence for this comes from an in vitro study of small-airway epithelial cell injury.29 In this model, MSCs are used. The majority of MSCs that repair the epithelial monolayer do so by fusion, but a minority do so by transdifferentiation. Both can occur in vivo, but fusion or transdifferentiation may be favored according to the mechanism of injury. Bone marrow-derived cells are seen in functionally significant numbers only where there is high pressure for the replication of daughter cells.17, 28 One caveat that should be observed is that the 2 studies27, 29 with the best positive evidence for transdifferentiation (rather than an absence of evidence of fusion) used stem cells that arguably may have been reprogrammed by culturing either on plastic29 or in an intermediate host,27 and therefore this may not accurately represent the physiology.

… Without Bone Marrow Transplantation?

The studies in which animals are irradiated and bone marrow chimerism is established can be considered pathophysiological studies; they allow us to observe the innate response of bone marrow-derived cells to liver injury, but they do not amplify these responses, which are, as discussed in a review by Thorgeirsson and Grisham,30 so small as to be unlikely to be of physiological significance. As outlined in the introduction, the clinical problem with which we are faced is that, in defined situations, innate repair processes are inadequate. Chronic liver disease is not purely an immunological problem in which reprogramming of immune responses could abolish the mechanisms of injury, unlike other autoimmune diseases such as multiple sclerosis31; therefore, bone marrow transplantation will provide a new cohort of cells that, unless they have a more favorable genotype, will respond in the same way as preexisting bone marrow. It is important to consider the effects of cell infusion without myeloablation, as this can be seen as an opportunity to deliver large numbers of cells to the injured liver without the “distraction” for the cells of repopulating an empty bone marrow stem cell niche.

In a study in which cells were infused halfway through a chronic injury protocol, there was apparent transdifferentiation of infused bone marrow cells via a primitive liver cell phenotype to a mature hepatocyte phenotype.32 Plasma albumin rose from 1.62 g/dL in injured animals to 2.08 g/dL in animals that had received a bone marrow cell infusion (2.5 g/dL for uninjured animals). This was associated with the expression of albumin in 42% of hepatocytes (although only 26% were bone marrow-derived).

Similar findings were obtained in a noninjury model using analbuminemic rats. An infusion of bone marrow cells resulted in the appearance of bone marrow-derived, albumin-expressing cells.33 Bone marrow cell infusion resulted in a steady increase in serum albumin that was maintained over 8 weeks; this was unlike the greater but temporary increase seen in the same model with hepatocyte infusion. Engraftment of bone marrow-derived cells within the liver appears to be a temporary phenomenon,34 with a further study showing a decline over 150 days, but the exact timing of the infusion of cells with respect to the liver injury is not clear in this study.

Targeting the remodeling of fibrosis is equally important in the treatment of liver cirrhosis; although liver synthetic function is vital to survival, morbidity from chronic liver disease is more often associated with the complications of portal hypertension, such as variceal hemorrhage, ascites, and renal impairment. Portal hypertension is intimately related to liver fibrosis.

The infusion of bone marrow cells was associated with a significant decrease in fibrosis in a mouse model in which cells were infused partway through the fibrosis protocol.35 This was associated with a reduction of approximately a third of the hydroxyproline content of the liver in comparison with animals that had not received the infusion. Infused cells were seen in close proximity to fibrous bands, and an increase in fibrolytic enzymes was observed. No measure of portal hypertension was made in this study, and this is an important parameter for further study.

… In Acute Liver Injury?

The literature on bone marrow cell infusion in models of acute liver injury is not as extensive as the literature on chronic injury models (in particular the carbon tetrachloride model), but it is of some interest.

Returning to the analbuminemic rat as a method of demonstrating the bone marrow derivation of both cells and liver function, Zhang et al.36 examined a model of acute liver injury. In this study, animals were pretreated with retrorsine to inhibit replication of native hepatocytes. They were then subjected to partial hepatectomy with either no cell infusion or hepatocyte or bone marrow cell infusion from syngeneic rats. There was a significant survival advantage associated with bone marrow cell infusion (70%-75% versus 35% in the group with no cell infusion), whereas there was no statistical difference between the survival rates of the hepatocyte infusion group and the control group. Both cell infusion groups were associated with normal liver histology by 2 days post-injury, whereas there was still significant derangement of the control group liver histology at 28 days. However, bone marrow cell infusion was not associated with an increase in liver weight over 28 days or with albumin expression. Improvements in the liver histology and survival, therefore, do not rest on engraftment of bone marrow-derived cells in this model.

In keeping with this, in a model looking at acute liver injury with human MSC infusion, only very rare MSC-derived hepatocyte-like cells were detected, and there was no albumin expression at 4 weeks post-transplant.11 In a similar model using adipose tissue-derived MSCs, biochemical markers of liver injury improved within 24 hours.37

These apparently contradictory findings of improvement in survival and markers of injury in the absence of engraftment of bone marrow-derived cells might be explained by a study of rat acute liver injury.38 Rats were injured with D-galactosamine and then received an infusion of concentrated MSC conditioned media. This resulted in improved survival and biochemical markers of liver injury. Concentrations of proinflammatory cytokines were decreased, and there was less leukocyte infiltration. Hepatocyte apoptosis decreased by 90%, and replication was also enhanced. This could explain the speed with which improvements occur, as no donor cell adaptation to injury conditions is required, and it might provide a therapy for acute liver injury. There may be a therapeutic window with increased fibrosis observed at higher doses.

… In the Correction of Inborn Metabolic Disorders?

The FAH−/− mouse model17 is a good example of how bone marrow transplantation can correct a lethal hepatic metabolic disorder. Its efficacy is probably exaggerated in comparison with more common clinical situations, in that the underlying disorder is quickly toxic to hepatocytes, providing a very strong selective pressure for FAH+/+ hepatocytes, and the mice used as donors are syngeneic,39 thus eliminating problems with graft versus host disease and immunosuppression. However, even a low engraftment efficiency may be sufficient for a clinically significant improvement in function,40 so that a bone marrow-derived cell mass of 2.4% of the total liver cell mass is able to increase ceruloplasmin to 21% to 24% of normal levels in Long-Evans Cinnamon rats.41

Studies have focused on a number of rodent models of human metabolic liver disease, including Wilson's disease (Long-Evans Cinnamon rats),41 Crigler-Najjar syndrome (Gunn rats),19 hereditary tyrosinemia type I (FAH−/− mice),17, 39 and familial progressive intrahepatic cholestasis (spgp−/− mice),42 among others. The immune response to bone marrow cell infusion has been managed by immunosuppression with cyclosporine,19, 41 by T cell depletion of the transplanted bone marrow,39 or simply by the use of syngeneic mice,42 and the engraftment efficiency has been improved by the modification of cell surface glycoproteins41 and by ischemia/reperfusion injury to improve homing of cells to the liver.19

Clearly, bone marrow transplantation in this setting brings with it some of the disadvantages of liver and hepatocyte transplantation in the requirement for immunosuppression, the need for bone marrow donors (also a scarce resource), and the current poor engraftment efficiency in comparison with hepatocyte transplantation19; on the other hand, there are advantages because of the greater ability of bone marrow cells to be expanded ex vivo.40 It is a promising avenue for investigation.

HOW DO ADULT STEM CELLS REACH THE LIVER?

  1. Top of page
  2. Abstract
  3. COULD THE LIVER BE A TARGET FOR STEM CELL THERAPY?
  4. HOW DO ADULT STEM CELLS CONTRIBUTE TO LIVER REPAIR …?
  5. HOW DO ADULT STEM CELLS REACH THE LIVER?
  6. ADULT STEM CELLS: FRIEND OR FOE?
  7. WHAT IS THE VALUE OF ADULT STEM CELL THERAPY IN CHRONIC LIVER DISEASE?
  8. IF BONE MARROW CELLS DO NOT REPLACE DISEASED HEPATOCYTE POPULATIONS, CAN STEM CELL THERAPY WORK IN CIRRHOSIS?
  9. WHAT ARE THE ALTERNATIVES TO ADULT STEM CELLS?
  10. ADULT STEM CELL THERAPY IN LIVER DISEASE: CHALLENGES FOR THE FUTURE
  11. REFERENCES

Mobilization of HSCs into the peripheral circulation appears to be a physiological response to liver injury, the potential purpose and low efficacy of which have already been discussed. There is an increase in peripheral blood CD 34+ cells in patients with acute liver injuries such as paracetamol toxicity and alcoholic hepatitis but not in patients with chronic liver disease.43, 44 Mobilization after partial hepatectomy and small-for-size liver transplantation45, 46 is seen but not universally observed.47

Which Signals Are Involved in Mobilization from Bone Marrow?

Stromal derived factor-1 (SDF-1) is a CXC chemokine that is important in the trafficking of fetal stem cells48 and, in particular, in the homing of HSCs to bone marrow.49 CXC receptor 4 (CXCR4) is its receptor.

SDF-1 is also involved in the trafficking of adult stem cells. The blockade of CXCR4 results in an efflux of HSCs from the bone marrow.50 Peripheral blood HSCs express lower levels of CXCR4 than bone marrow HSCs, and this confirms the role of SDF-1 in maintaining them within the bone marrow niche.51 Human CD34+ cells migrate toward SDF-1 both in vivo and in vitro.52 The gradient hypothesis suggests that HSCs migrate along an SDF-1 concentration gradient. Thus, an elevation of plasma SDF-1 causes mobilization of HSCs from the bone marrow into the peripheral blood.53 There is a reversal of this gradient under some conditions of murine acute liver injury with mobilization of CD34+ cells into the peripheral circulation.54, 55 This mechanism is not exclusive to liver injury, with SDF-1 release from ischemic muscle,56 injured neural tissue,57 and lungs.58

SDF-1 is not only involved in the trafficking of cells out of the bone marrow but may also be involved in the homing of the cells to the liver. A parenchymal injection of SDF-1 results in an accumulation of HSCs even in an uninjured liver.52 Increased expression of SDF-1 within the human liver has been demonstrated in liver injuries including hepatitis C virus infection,52, 59, 60 primary biliary cirrhosis,60 autoimmune hepatitis,60 and paracetamol toxicity.44 SDF-1-positive cells are found in the biliary epithelium and in the cells of the ductular reaction. Raised serum SDF-1 levels have also been demonstrated in paracetamol toxicity, alcoholic hepatitis,43 autoimmune hepatitis, and hepatitis C virus infection.60 In hepatitis C infection, the expression of SDF-1 has been found not to correlate with the degree of inflammation present (unlike interleukin 8).59 This suggests that its function is not limited to the attraction of inflammatory cells to the site of liver injury.

Does SDF-1 Have Roles in Liver Repair That Are Independent of the Homing of Adult Stem Cells?

Little is known about the function of SDF-1 within the liver in adult life. It is necessary for the accumulation of oval cells.61, 62 The shortfall in oval cells under the conditions of SDF-1 blockade is due to an effect on cell cycling rather than apoptosis.62 The effect of SDF-1 on cell cycling in HSCs is better characterized. SDF-1 is able to trigger resting peripheral blood CD34+ cells into the cell cycle.63 SDF-1 is strongly antiapoptotic in these cells and promotes survival in an autocrine/paracrine manner. Both SDF-1 and CXCR4 are expressed by peripheral blood CD34+ cells. Under conditions that promote apoptosis, there is a release of SDF-1 with up-regulation of surface CXCR4. Survival is decreased by the addition of a specific SDF-1 blockade, and this suggests that it is mediated by the SDF-1/CXCR4 axis.63 Findings of SDF-1 and CXCR4 expression within oval cells61 suggest that a similar mechanism may be important in the liver.

ADULT STEM CELLS: FRIEND OR FOE?

  1. Top of page
  2. Abstract
  3. COULD THE LIVER BE A TARGET FOR STEM CELL THERAPY?
  4. HOW DO ADULT STEM CELLS CONTRIBUTE TO LIVER REPAIR …?
  5. HOW DO ADULT STEM CELLS REACH THE LIVER?
  6. ADULT STEM CELLS: FRIEND OR FOE?
  7. WHAT IS THE VALUE OF ADULT STEM CELL THERAPY IN CHRONIC LIVER DISEASE?
  8. IF BONE MARROW CELLS DO NOT REPLACE DISEASED HEPATOCYTE POPULATIONS, CAN STEM CELL THERAPY WORK IN CIRRHOSIS?
  9. WHAT ARE THE ALTERNATIVES TO ADULT STEM CELLS?
  10. ADULT STEM CELL THERAPY IN LIVER DISEASE: CHALLENGES FOR THE FUTURE
  11. REFERENCES

There is debate about whether HSCs contribute to the oval cell population and thus to liver repair. In a model of hepatitis B infection and cross-sex bone marrow transplantation, mice in which hepatocyte replication was suppressed were examined. Y chromosome-positive cells were seen within the liver, but these were not oval or small hepatocyte progenitor cells.64 Another group also did not find bone marrow-derived oval cells.65 They characterized the bone marrow-derived cells that they found in the liver; all expressed CD45, a leukocyte marker, but not CD90 (an oval cell and HSC marker) or CK19 (a biliary marker) and were therefore hematogenous nonstem cells. Both of these studies can be criticized on the same grounds. Bone marrow transplantation was carried out before treatment with drugs to limit hepatocyte replication (retrorsine). Retrorsine and monocrotaline (used in similar protocols by other groups) are pyrrolizidine alkaloids that are metabolized within the liver to produce alkylating agents.66 Their metabolites can be detected in the lungs, kidneys, and urine as well as the liver.66 There is evidence that monocrotaline also causes bone marrow toxicity.67 Taking this into account, Petersen's group performed bone marrow transplantation after injury with monocrotaline. They found that the timing of bone marrow transplantation with respect to monocrotaline administration was crucial in determining whether bone marrow-derived oval cells could be detected.28

Other, less welcome cell types have also been observed. In patients with cross-sex liver transplants (female into male) with significant liver fibrosis, Y chromosome-positive cells were found within fibrotic bands68; 13.9% to 45.4% of myofibroblasts in these livers were host-derived. This finding was confirmed in a female patient who had received a male bone marrow transplant and subsequently developed hepatitis C-related cirrhosis; this suggested that these cells are truly of bone marrow (rather than simply extrahepatic) origin. Examining the same phenomenon in a mouse model of fibrosis,69 researchers found that 69% of myofibroblasts were bone marrow-derived. No polyploidy was seen, and this suggests that there was no fusion. Bone marrow cells were further separated into HSCs and MSCs prior to transplantation. Y chromosome-positive cells accounted for 53% of myofibroblasts in mice receiving male MSCs versus 8.2% in mice receiving male HSCs, and this suggests that myofibroblasts are largely derived from MSCs. In another fibrosis model, only 1.06% to 3.33% of myofibroblasts were bone marrow-derived,11 whereas in a bile duct ligation model, 25% of myofibroblasts were bone marrow-derived.70 The bone marrow contribution to myofibroblasts is clearly highly variable, even within fibrosis models. This may be explained to some degree by the dose response relationship of MSC conditioned media with the resolution of injury and fibrosis.38 Different injuries and severities of injury may result in differing MSC mobilization, homing, and secretion of cytokines.

Fibrosis phenotypes can be altered by changes in the types of cells infused.69 With bone marrow from mice that express a form of collagen resistant to degradation and that develop a characteristically severe form of fibrosis, it is possible to induce severe fibrosis in wild-type mice. Manipulation of bone marrow in order to influence fibrosis phenotypes may be a target for future investigation.

Hepatocellular carcinomas are believed to be derived from liver resident stem cells rather than mature hepatocytes.71, 72 However, despite attempts with 2 different rodent carcinogenesis models,62, 73 bone marrow-derived hepatocellular carcinoma has not been demonstrated. Nevertheless, bone marrow-derived activation of the hepatic stem cell compartment remains of concern.

WHAT IS THE VALUE OF ADULT STEM CELL THERAPY IN CHRONIC LIVER DISEASE?

  1. Top of page
  2. Abstract
  3. COULD THE LIVER BE A TARGET FOR STEM CELL THERAPY?
  4. HOW DO ADULT STEM CELLS CONTRIBUTE TO LIVER REPAIR …?
  5. HOW DO ADULT STEM CELLS REACH THE LIVER?
  6. ADULT STEM CELLS: FRIEND OR FOE?
  7. WHAT IS THE VALUE OF ADULT STEM CELL THERAPY IN CHRONIC LIVER DISEASE?
  8. IF BONE MARROW CELLS DO NOT REPLACE DISEASED HEPATOCYTE POPULATIONS, CAN STEM CELL THERAPY WORK IN CIRRHOSIS?
  9. WHAT ARE THE ALTERNATIVES TO ADULT STEM CELLS?
  10. ADULT STEM CELL THERAPY IN LIVER DISEASE: CHALLENGES FOR THE FUTURE
  11. REFERENCES

The epidemiology of chronic liver disease has driven forward a number of trials of autologous stem cell therapy. Many are still at a pilot stage and are therefore unrandomized and uncontrolled, but they show some interesting results that require confirmation.

One study looked at patients who required expansion of the left lobe of the liver prior to resection of neoplastic lesions in the right lobe. Selective embolization of the right lobe was performed, and autologous CD133+ cells were infused into the left portal vein in those patients in the treatment arm.74 Growth of the left lobe was measured; resection of the right lobe was carried out when it was felt that the remaining liver volume was sufficient to sustain life. Patients who received stem cells showed an increased rate of growth of the left lobe. All the patients in the treatment group survived, whereas all those in the control group died. Mobilization of CD133+ cells has been reported after partial hepatectomy.46 The authors suggested that a direct infusion of cells into the portal vein may increase the efficiency of homing of cells to the liver and thus improve regeneration. Direct infusion may be required in this model, as there is no injury to the targeted lobe, and homing signals might be expected to originate in the injured part.

Several groups have used this and the animal data to go on to use autologous bone marrow cells as a treatment for cirrhosis. Most of the studies that have been published to date are uncontrolled. A variety of infusion routes have been used, with some using direct portal vein or hepatic artery infusion75–78 and others using peripheral stem cell infusion.79 An animal study has suggested that the route may not be important.36 Increases in hepatocyte proliferation,79 endothelial cells,80 and alpha-fetoprotein expression79 have been shown on biopsy. There is a trend toward decreased serum fibrosis markers,76, 79 and an increase in serum vascular endothelial growth factor was seen in 1 patient.80 Improvements have been seen in bilirubin75, 78 and albumin75, 79 and in Model for End-Stage Liver Disease80 and Child-Pugh scores.78–80 Complications of cirrhosis79, 80 are diminished. In 1 of the studies,80 it is possible that these improvements were due to abstinence from alcohol, although in others, injury appeared to be ongoing. These studies represent a total of 33 patients, and the largest groups are those of Habib78 and Sakaida.79 The latter group also published in abstract form their ongoing experience with another 7 patients.81

Another trial has compared patients with hepatitis B-related cirrhosis.82 In this study, a total of 40 subjects were randomized to receive granulocyte colony stimulating factor (G-CSF) alone or G-CSF with leukapheresis and re-infusion of peripheral blood monocytes into the hepatic artery. There was 6-month follow-up. No antiviral therapy was used. There was no change in the hepatitis B DNA level in either group, and this made an immunomodulatory role of the infused monocytes less likely. There were significant improvements in the serum albumin and prothrombin time in both groups, with a greater improvement in albumin in the group that had received the cell infusion. The Child-Pugh score also improved in both groups, but this improvement was sustained only in the cell infusion group. The data are clouded by the routine use of albumin infusions as a treatment for ascites; in the group that had received the cell infusion, 13 patients became independent of albumin versus 2 in the group receiving G-CSF alone. This trial offers relatively prolonged follow-up with sustained improvements in the albumin, prothrombin time, and Child Pugh score in the cell infusion group.

G-CSF has been used in acute liver failure. To patients with fulminant hepatic failure by paracetamol overdose,83 G-CSF was administered. The numbers were small (6 patients per group), and there was no effect of the dose of G-CSF on survival. In another study of patients with alcoholic hepatitis on a background of alcohol-related cirrhosis, the authors found an increase in proliferation among the ductular hepatocytes seen at biopsy in comparison with both the baseline and control. G-CSF appeared to be safe in this group, but there was somewhat disappointing mobilization of CD34+ cells attributed to splenic sequestration, and the study was too small to make any comment regarding survival or efficacy as a treatment.84 It may not be necessary to collect and re-infuse CD34+ cells in order to gain benefit.

It is difficult to draw conclusions from the aforementioned studies. The use of autologous HSCs to treat human liver injury is still in its infancy, although there are some promising early data. It is not clear at present whether the route of infusion is important or whether mobilization with G-CSF without leukapheresis would suffice. It will also be important to understand more about the mechanisms involved. Is there an improvement in liver architecture that ameliorates portal hypertension? Is there additional proliferation of hepatocytes? Perhaps more importantly, we should question whether existing protocols cause any improvement in liver function in larger controlled and adequately powered trials (see also Table 2 and Fig. 2).

Table 2. Considerations for the Design of Clinical Trials of Hematopoietic Stem Cells in Chronic Liver Disease
  1. Abbreviations: G-CSF, granulocyte colony stimulating factor; HCV, hepatitis C virus; MELD, Model for End-Stage Liver Disease.

Case identificationPatients with (initially) the same etiology of disease
Patients with a similar stage of disease by recognized scoring systems
Cells used for infusionMobilized peripheral blood
Iliac crest bone marrow cells
Route of infusionNo infusion (eg, G-CSF mobilization without leukapheresis)
Peripheral veins
Portal vein
Hepatic artery
TimingWhen injury is ongoing (eg, untreated HCV or ongoing alcohol abuse)
In patients without ongoing inflammation and injury
Markers of improvementRobust recognized scoring systems such as MELD and Child-Pugh scores
Robust and blinded randomization with appropriate controls
Studies must be appropriately powered.
Pathophysiological considerations: What is the fate of the cells?Consider cell tracking with radionuclides or magnetic nanoparticles if CD34+ cell function is not inhibited.
Consider liver biopsy.
thumbnail image

Figure 2. Considerations in the design of trials of the infusion of autologous stem cells in liver disease.

Download figure to PowerPoint

This question has been asked in the field of therapeutic use of HSCs in the context of myocardial infarction. Three similar studies have been published,85–87 showing modest improvements that may be short-lived,88 with reports suggesting that there is no difference between treated patients and controls at 18 months.

IF BONE MARROW CELLS DO NOT REPLACE DISEASED HEPATOCYTE POPULATIONS, CAN STEM CELL THERAPY WORK IN CIRRHOSIS?

  1. Top of page
  2. Abstract
  3. COULD THE LIVER BE A TARGET FOR STEM CELL THERAPY?
  4. HOW DO ADULT STEM CELLS CONTRIBUTE TO LIVER REPAIR …?
  5. HOW DO ADULT STEM CELLS REACH THE LIVER?
  6. ADULT STEM CELLS: FRIEND OR FOE?
  7. WHAT IS THE VALUE OF ADULT STEM CELL THERAPY IN CHRONIC LIVER DISEASE?
  8. IF BONE MARROW CELLS DO NOT REPLACE DISEASED HEPATOCYTE POPULATIONS, CAN STEM CELL THERAPY WORK IN CIRRHOSIS?
  9. WHAT ARE THE ALTERNATIVES TO ADULT STEM CELLS?
  10. ADULT STEM CELL THERAPY IN LIVER DISEASE: CHALLENGES FOR THE FUTURE
  11. REFERENCES

As discussed previously, the contribution of bone marrow-derived cells to hepatocyte populations is minimal,13, 18 except in very specialized circumstances.17 Although bone marrow-derived cells have been detected in the human liver, their contribution to functional hepatocytes may be of minor importance. However, they have the potential to contribute significantly to fibrosis. In addition to this, it could be argued that simply replacing “worn-out” hepatocytes in a patient with an ongoing liver injury (eg, chronic hepatitis C) provides cannon fodder for the virus and neither changes the underlying pathology nor ameliorates the architectural disturbances that are responsible for portal hypertension and its life-limiting sequelae.

The studies that have shown the best functional improvement are those in which bone marrow has been infused into an unirradiated recipient in the context of ongoing liver injury.32, 33, 35 This description might also apply to a large proportion of the patients involved in clinical trials of stem cell therapy. In animal studies, an improvement in serum albumin32, 33 has been shown, although there are doubts about the longevity of the effect34; perhaps repeated infusions are required. A decrease in fibrosis has also been seen35; thus, there may be resolution of portal hypertension. How are these effects mediated?

Until these questions can be answered, stem cell therapy should be used in the context of clinical trials, which should be carried out in close collaboration with basic scientists so that hypotheses generated by either human or animal experience can be adequately tested. It seems likely that the major role for stem cell therapy will be as a bridge to transplantation or as a way of maintaining those patients who are not eligible for transplantation with repeated infusions with the aim of stabilizing their liver condition. With better understanding of the physiology, it may be that cells will be differentiated in vitro to macrophages or hepatocyte precursors with the aim of supplying cells to the correct location within the liver in order to influence remodeling.

WHAT ARE THE ALTERNATIVES TO ADULT STEM CELLS?

  1. Top of page
  2. Abstract
  3. COULD THE LIVER BE A TARGET FOR STEM CELL THERAPY?
  4. HOW DO ADULT STEM CELLS CONTRIBUTE TO LIVER REPAIR …?
  5. HOW DO ADULT STEM CELLS REACH THE LIVER?
  6. ADULT STEM CELLS: FRIEND OR FOE?
  7. WHAT IS THE VALUE OF ADULT STEM CELL THERAPY IN CHRONIC LIVER DISEASE?
  8. IF BONE MARROW CELLS DO NOT REPLACE DISEASED HEPATOCYTE POPULATIONS, CAN STEM CELL THERAPY WORK IN CIRRHOSIS?
  9. WHAT ARE THE ALTERNATIVES TO ADULT STEM CELLS?
  10. ADULT STEM CELL THERAPY IN LIVER DISEASE: CHALLENGES FOR THE FUTURE
  11. REFERENCES

Although the use of HSCs in liver disease would not simply be aimed at the replacement of native hepatocytes, it may be useful to consider alternative strategies to promote this. If the replacement of hepatocytes is the aim of cellular therapy in this group of patients, might it not be simpler to use human hepatocytes, which could be cryopreserved89, 90 and used on demand? Hepatocyte transplantation carries with it some of the disadvantages of whole liver transplantation, particularly the need for immunosuppression. If the requirement for additional hepatocyte mass is likely to be temporary, then it might be possible to withdraw immunosuppression and allow transplanted cells to be cleared by the immune system. However, this field remains underdeveloped with many technical problems to be solved before this could be considered a routine procedure.91, 92

ADULT STEM CELL THERAPY IN LIVER DISEASE: CHALLENGES FOR THE FUTURE

  1. Top of page
  2. Abstract
  3. COULD THE LIVER BE A TARGET FOR STEM CELL THERAPY?
  4. HOW DO ADULT STEM CELLS CONTRIBUTE TO LIVER REPAIR …?
  5. HOW DO ADULT STEM CELLS REACH THE LIVER?
  6. ADULT STEM CELLS: FRIEND OR FOE?
  7. WHAT IS THE VALUE OF ADULT STEM CELL THERAPY IN CHRONIC LIVER DISEASE?
  8. IF BONE MARROW CELLS DO NOT REPLACE DISEASED HEPATOCYTE POPULATIONS, CAN STEM CELL THERAPY WORK IN CIRRHOSIS?
  9. WHAT ARE THE ALTERNATIVES TO ADULT STEM CELLS?
  10. ADULT STEM CELL THERAPY IN LIVER DISEASE: CHALLENGES FOR THE FUTURE
  11. REFERENCES

There is a requirement for new therapies for both acute and chronic liver disease. Pathophysiological studies have shown evidence of bone marrow derivation of hepatocytes, but except in specific models, the frequency of this event is too low to be functionally significant. The finding of bone marrow-derived oval cells is highly dependent on the model used, and it is likely that the next 10 years will see adjustments of models to produce a better understanding of the physiology. Bone marrow cells influence fibrosis phenotypes; the development of techniques for ex vivo manipulation of MSCs will be helpful in remodeling the cirrhotic liver. Albumin expression and fibrosis improve in animal models in which cells are infused without myeloablation, but further research focusing on remodeling of fibrosis and portal hypertension during HSC infusion is required.

Bone marrow transplantation is effective in the correction of inborn errors of metabolism in animal models, but the translation of this into the clinical setting will progress only if problems of immunosuppression and poor engraftment efficiency are overcome. Identification of the humeral factors improving survival in animal models of acute liver injury will be identified, and new therapies will be developed.

There have been many studies using autologous bone marrow cell infusion in patients with cirrhosis. Optimization and randomized controlled testing of these protocols are needed, and once the mechanism of action is more fully understood, it is likely that new therapies suitable for use outside the tertiary referral center will emerge.

REFERENCES

  1. Top of page
  2. Abstract
  3. COULD THE LIVER BE A TARGET FOR STEM CELL THERAPY?
  4. HOW DO ADULT STEM CELLS CONTRIBUTE TO LIVER REPAIR …?
  5. HOW DO ADULT STEM CELLS REACH THE LIVER?
  6. ADULT STEM CELLS: FRIEND OR FOE?
  7. WHAT IS THE VALUE OF ADULT STEM CELL THERAPY IN CHRONIC LIVER DISEASE?
  8. IF BONE MARROW CELLS DO NOT REPLACE DISEASED HEPATOCYTE POPULATIONS, CAN STEM CELL THERAPY WORK IN CIRRHOSIS?
  9. WHAT ARE THE ALTERNATIVES TO ADULT STEM CELLS?
  10. ADULT STEM CELL THERAPY IN LIVER DISEASE: CHALLENGES FOR THE FUTURE
  11. REFERENCES
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