Keywords

13.1 Introduction

It is well known that chronic liver diseases including viral infection (hepatitis B virus, hepatitis C virus), autoimmune hepatitis (AIH), primary biliary cholangitis (PBC), nonalcoholic steatohepatitis (NASH), alcoholic hepatitis (AH), and some others may lead to the development of liver cirrhosis and ultimately end-stage liver failure [1]. Although the causes of liver cirrhosis differ according to area or county, no life-saving treatment for this condition other than liver transplantation currently exists. A recent etiological survey has confirmed that HBV infection, HCV infection, alcoholic liver disease, and NASH are the leading causes of liver failure [1]. However, the development of effective antiviral treatments has changed the clinical situation. It is estimated that the incidence of HCV-related cirrhosis will decrease dramatically within the next few decades [2]. Also, the introduction of effective nucleos(t)ide analogues for HBV infection has successfully suppressed the replication of HBV, resulting in clinically significant and durable suppression of hepatic fibrosis and inflammation in the long term. Thus, the current major causes of liver cirrhosis are remaining liver diseases such as NASH, for which no fundamental treatments have been established. Moreover, there are still many patients with established liver failure who continuously suffer from complications such as ascites, hepatic encephalopathy, spontaneous bacterial peritonitis, and ultimately hepatocellular carcinoma (HCC) [1]. Currently, liver transplantation, either cadaveric or from living donors, is recognized as the only option for end-stage liver disease. However, its use is limited by a shortage of donors, a high incidence of surgical complications, and high medical costs. In this situation, the development of medical therapies other than liver transplantation would be desirable [3]. The present strategy of medical therapy for liver cirrhosis is (1) resolution of hepatic fibrosis, (2) recovery of hepatic function (both synthetic and metabolic), and (3) reducing the incidence of complications [4]. Recently, various anti-fibrotic drugs have been investigated in clinical trials, including the apoptosis signal-regulating kinase 1 (ASK1) inhibitor, selonsertib [5]. To reverse the decline in the synthetic function of the liver, several nutritional therapies such as branched-chain amino acid (BCAA) supplementation have been applied, although their effects have been proven only for patients with comparative reversal of decompensated cirrhosis. Furthermore, artificial liver support including extracorporeal xenogeneic hepatocyte-based approaches has demonstrated limited effects in patients with chronic liver failure. As a consequence, these forms of artificial liver support are merely regarded as temporary bridging therapies to liver transplantation. Moreover, no rational approach has been established for prevention of liver cirrhosis in patients with end-stage liver diseases. Against this background, the development and introduction of novel therapies for end-stage liver diseases would seem to be desirable. Among them, cell-based therapy has been regarded as very promising. The purpose of cell therapy is for grafted cells to migrate to damaged organs and participate in tissue recovery. For this purpose, cell therapy would seem to be a more effective approach than the use of artificial extracorporeal devices for hepatic disease. The specific characteristics of the liver, such as its ample blood supply, a marked capacity for regeneration, and comparatively easy access from the body surface, are all amenable to the development of novel forms of cellular therapy for intractable end-stage liver disease. For example, as access routes for cellular infusion, transplanted cells can be injected peripherally, intra-arterially (via the hepatic artery), or via the portal vein. However, when considering the possible complications of cell therapy, administration of cells via a peripheral vein may decrease the risk of such complications. In this review, we discuss the current status of cell-based therapy for end-stage liver diseases.

13.2 Stem Cells

Stem cells are known to have various specific abilities such as self-renewal and differentiation. There are several types of stem cells in mammals, and embryonic stem (ES) cells are the most prototypic. However, due to their limited accessibility and ethical issues, the clinical application of ES cells has a number of specific hurdles. Another type of stem cell is the mesenchymal stem cell (MSC). The application of MSCs as a source of cell therapy has been investigated worldwide for numerous conditions. MSCs have the advantage of easy accessibility; they can be obtained even from medical waste tissues such as adipose tissue, umbilical tissue, and dental pulp. Another type of stem cell is hematopoietic stem cells, which are reported to differentiate into hepatocyte-like cells under certain conditions [6]. A number of studies using animal models and some human investigational trials have described their application for hepatic regenerative therapy [7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28]. Unfortunately, however, most of those studies were hampered by significant bias [29]. Table 13.1 summarizes the major clinical trials of cell therapy for liver cirrhosis. The majority of cellular sources have been autologous bone marrow or allogenic umbilical cord. Clinical trials of this form of cell therapy have obtained data based on laboratory tests (albumin, total bilirubin, aspartate aminotransferase (AST), alanine aminotransferase (ALT), etc.), residual hepatic function (Child-Pugh score), MELD (model for end-stage liver disease) score, clinical symptoms (hepatic encephalopathy, anemia, edema, ascites fluid), and occasionally histological evaluation. In these clinical trials, umbilical cord MSCs were administered via a peripheral vein, and the number of cells infused was usually around 5.0 × 106 cells per kg body weight, being given three times at 4-week intervals. Some of the studies reported an improvement in the serum levels of albumin and total bilirubin, a decrease of the MELD score, or an improvement of clinical symptoms such as ascites at the end of the observation period [25, 29]. Clinical trials using bone marrow-derived MSCs as the cell source have made use of autologous bone marrow and administration via various routes such as the hepatic artery, portal vein, peripheral veins, or intrahepatic vessels. The number of transplanted cells in those studies ranged between 3.4 × 108 and 0.75 × 106/patient [29]. Although some studies reported an improvement of surrogate markers, the results were not consistent [20, 29, 30]. A few explorative clinical studies of MSC administration resulted in partial improvement of hepatic reserve in patients with alcoholic cirrhosis [22, 31]. However, a recent well-conducted randomized trial concluded that there was no beneficial effect of MSC administration combined with administration of granulocyte colony-stimulating factor [32]. This disappointing result further emphasizes the need for novel cell-based therapies for chronic liver failure [33]. At least, we need to summarize the reasons for this trial failure in scientific views. This includes the fundamental questions such as the candidacy of MSC as the cellular source toward the organ like the liver, which is one of the largest organs consisting mammalian body.

Table 13.1 Summary of clinical trial regarding cell therapy for liver cirrhosis
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13.3 Future Novel Cellular Therapies Including Muse Cell Administration Toward Chronic Liver Failures (Fig. 13.1)

Current forms of cellular therapy require harvesting of MSCs from bone marrow and a certain period of time to prepare a sufficient number of pure cells, which limits the clinical application of this approach, especially in emergency cases such as acute liver failure. ES cells were initially reported as the potential cell source for administration. However, tumorigenicity and ethical issues for using fertilized egg are major barriers for feasibility of ES cells, and there are numerous issues to be overcome before reaching clinical trials with this cell source. Inducible pluripotent stem (iPS) cells have been engineered to overcome these difficulties and can theoretically differentiate into various types of cells, tissues, and organs [18, 34,35,36]. Although this approach is reported safe so far, there are still significant concerns about the artificial introduction of exogenous genes such as retroviral vectors [37]. Since the long-term efficacy and safety of iPS cell administration have not been proved, we need to be very careful about its clinical application as a standard form of care.

Fig. 13.1
figure 1

Current concept of cell-based therapy for chronic liver failure

The major possible cellular source was either mesenchymal stem cells (MSCs), Muse cells, or iPS cells. The former two cells were essentially isolated from bone marrow, whereas the latter could be transformed by genetic modification. Muse cells could be subspecialized population of MSCs (see text). Although these concepts have been proposed, none of these have proven their efficacy in phase II clinical trials in chronic liver failure

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Multilineage-differentiating stress-enduring (Muse) cells are a form of mesenchymal stem cell with several of the novel characteristics of non-tumorigenic pluripotent characters [38, 39]. The previous reports demonstrated the capabilities of pluripotent differential abilities of Muse cells into liver-constituting cells such as hepatocytes [6, 40,41,42]. As Muse cells are able to recognize the sites of tissue damage/injury, thus contributing to tissue repair and promoting the improvement of organ function, their application to cellular therapy has naturally attracted attention. Besides differentiation capability, Muse cells also have other technical advantages over traditional MSCs. Muse cells were reported to home specifically into damaged tissue after intravenous injection and keep engrafted as tissue-specific cells for a longer period over several months, while majority of MSCs other than Muse cells, namely, non-Muse MSCs, basically do not home into damaged tissue nor they engraft as differentiated cells in the tissue [43]. The most recent study revealed that the sphingosine-1-phosphate is the major migratory factor of Muse cells, which will in turn be utilized for more efficient future isolation methods [44]. Interestingly, this humoral factor has been reported to be important by independent researchers [13]. Moreover, there is no need for Muse cells to introduce exogenous genes for acquiring pluripotency, which is essential for iPS cells, because Muse cells are already pluripotent. Besides safety profiles, Muse cells have superiority over MSC, ES cells, or iPS cells; without any prior gene introductions, Muse cells can selectively home into damaged tissue and efficiently replenish tissue-specific cells by intravenous injection. Exploiting this property, Muse cell administration has been reportedly effective in animal models with cerebral infarction, nephropathy, myocardial infarction, and liver resection [45,46,47,48,49]. The recent report by Iseki et al. used the Muse cell administration in mice model of liver cirrhosis [46]. In this report, the authors demonstrated that intravenously injected Muse cells have been recruited selectively to the liver and not to other organs. Moreover, Muse cells spontaneously differentiated in the damaged liver tissue into hepatocyte marker-positive cells without fusing with host hepatocytes [46]. These differentiated cells express major hepatocyte markers such as HepPar-1, albumin, and ant1-trypsin. They also expressed cytochrome (CPY) 1A2, an enzyme for detoxication, and glucose-6-phosphatase, an enzyme for glyocolysis, as representative markers of hepatocytes. As a consequence, the elevation of serum albumin levels and the decrease of total bilirubin levels were delivered by intravenous administration of Muse cells [46]. Surprisingly, even hepatic fibrosis has been improved in this animal model of Muse cell administration [46]. One of the explanations for the fibrolytic activities of Muse cells is the production of matrix metalloproteinases (MMPs) [40]. Since the liver is an organ playing a significant immunological role, it is capable of inducing transplantation-related immunological tolerance. Therefore, the liver could be a better target for Muse cell administration than other solid organs.

For organ repair after specific forms of injury, stem cells need to contribute to the replenishment of tissue-specific cells that are actually functional in situ. Muse cell administration in a mouse model of partial hepatectomy has shown that Muse cells differentiate spontaneously into major liver components, including hepatocytes, cholangiocytes, sinusoidal endothelial cells, and Kupffer cells [47]. Not only do Muse cells have the ability to home to and accumulate in damaged organs, they can also contribute to the resolution of inflammation and fibrosis [47]. Thus, based on the results obtained from small-animal models, Muse cell treatment appears to have promising as a novel regenerative treatment for liver cirrhosis. The safety of MSC administration therapy has been reported by several clinical trials [30]. Muse cells are a subpopulation of MSC and thus are expected to be safe. As for the efficacy, since the liver is the largest human organ, high efficacy for homing into the damaged liver and for engraftment as functional hepatocytes is key point for outcomes, which should be estimated in large-animal models (i.e., swine, etc.). Establishment of suitable models involving large animals with chronic liver failure and fibrosis will help to clarify a life-size efficacy and safety of Muse cell administration, leading in turn to human clinical trials. In this viewpoint, the most recent press release announcing the launch of clinical trials of Muse cell administration to evaluate its efficacy and safety in acute myocardial infarction has given a definite conviction of the application of this fascinating cell administration toward liver diseases in the near future [50].