Clinical trials using mesenchymal stem cells in liver diseases and inflammatory bowel diseases

Mesenchymal stem cell (MSC) therapies have been used in clinical trials in various fields. These cells are easily expanded, show low immunogenicity, can be acquired from medical waste, and have multiple functions, suggesting their potential applications in a variety of diseases, including liver disease and inflammatory bowel disease. MSCs help prepare the microenvironment, in response to inflammatory cytokines, by producing immunoregulatory factors that modulate the progression of inflammation by affecting dendritic cells, B cells, T cells, and macrophages. MSCs also produce a large amount of cytokines, chemokines, and growth factors, including exosomes that stimulate angiogenesis, prevent apoptosis, block oxidation reactions, promote remodeling of the extracellular matrix, and induce differentiation of tissue stem cells. According to ClinicalTrials.gov, more than 680 clinical trials using MSCs are registered for cell therapy of many fields including liver diseases (more than 40 trials) and inflammatory bowel diseases (more than 20 trials). In this report, we introduce background and clinical studies of MSCs in liver disease and inflammatory bowel diseases.


Background
The digestive system, which consists of the gastrointestinal tract, liver, pancreas, and biliary tree, functions in digestion, absorption, and metabolism and affects the basis of life. Various diseases, including cancer, inflammatory disease, infection, stones, and ulcers, are studied under the context of gastroenterology. While innovative drugs against Helicobacter pylori [1], hepatitis C virus [2], and inflammatory bowel disease (IBD) [3] have recently been developed, there are still unmet needs in this field, including in acute and chronic liver failure and refractory IBDs. Cell therapy may fulfill these unmet needs, and cell therapies using mesenchymal stem cells (MSCs) have become a major focus in many fields [4]. MSCs are reported to have multiple functions, especially anti-fibrosis and antiinflammatory effects are focused in acute and chronic liver failure and refractory IBDs. Furthermore, MSCs have low immunogenicity, can expand easily, and can be obtained from medical waste, suggesting their potential to expand regenerative medicine for the treatment of liver diseases and IBDs.
In this paper, we review the current status of clinical trials using autologous/allogeneic MSCs in liver diseases and IBDs.

Characteristics of MSCs
MSCs have recently received attention as potential cell sources for cell therapy due to their ease of expansion and wide range of functions. MSCs can be obtained from not only bone marrow but also medical wastes, such as adipose tissue, umbilical tissue, and dental pulp. MSCs are positive for the common markers CD73, CD90, and CD105; however, they are negative for the endothelial marker CD31 and hematopoietic marker CD45 [4][5][6][7]. The expansion of MSCs in culture is relatively easy, and under appropriate conditions, MSCs have trilineage differentiation (osteogenic, chondrogenic, and adipogenic) potential. The effects of MSCs are broadly divided into two mechanisms: (1) recruited MSCs differentiate into functional cells to replace damaged cells, permitting the treatment of bone and cartilage damage; and (2) in response to inflammatory cytokines, MSCs help prepare the microenvironment by producing immunoregulatory factors that modulate the progression of inflammation by affecting dendritic cells, B cells, T cells, and macrophages. MSCs also produce a large amount of cytokines, chemokines, and growth factors, including exosomes, which stimulate angiogenesis, prevent apoptosis, block oxidation reactions, promote remodeling of the extracellular matrix (ECM), and induce the differentiation of tissue stem cells [4,7,8]. These latter mechanisms can be applied for many diseases, including liver disease and IBSs. Some studies have reported that the effects of MSCs are determined by host conditions, such as inflammation stage and the use of immunosuppressants.
Although the behaviors of MSCs after administration have been analyzed, and some studies have shown that MSCs migrate to the injured site, MSC behaviors in humans have not been fully elucidated. Some studies have reported that MSCs disappear within a few weeks and do not remain long in the target tissue [5]. Recent studies have reported that only culture-conditioned medium or exosomes induce treatment effects, suggesting that the trophic effect is the most important effect of MSCs [9][10][11]. Another important characteristic of MSCs is that they generally have low immunogenicity. MSCs have no antigen-presenting properties and do not express major histocompatibility complex class II or costimulatory molecules; thus, injection of autologous or allogeneic MSCs has been employed in clinical studies. Allogeneic MSC therapy has the potential to expand MSC therapy to many patients [4,7].

Clinical trials using MSCs
Since MSCs can be obtained relatively easily and have multiple functions, more than 680 clinical trials are ongoing according to ClinicalTrials.gov (https://clinicaltrials.gov/); most of these studies are phase I or II trials evaluating the use of MSCs in bone/cartilage, heart, neuron, immune/autoimmune, diabetes/kidney, lung, liver, and gastrointestinal fields. These studies aim to elucidate the safety/effectiveness of MSCs in the treatment of various diseases. In liver diseases, 40 trials are registered, most of which target liver cirrhosis or acute liver diseases (Table 1) [12][13][14][15][16][17][18][19][20][21]. The MSCs used in clinical trials of the liver are derived from the bone marrow (55%), umbilical cord tissue (35%), and adipose tissue (8%). Approximately 50% of MSCs are allogeneic. Additionally, while the major administration route is the peripheral blood, approximately 40% of cases are treated via the hepatic artery, reflecting the fact that hepatologists and radiologists often use catheters to treat hepatocellular carcinoma through the hepatic artery [22,23] (Fig. 1).

Background of liver diseases
Although the liver has high regenerative capacity, acute liver damage caused by viruses, drugs, alcohol, and autoimmune diseases, or chronic liver damage caused by hepatitis B or C virus, alcohol, non-alcoholic steatohepatitis (NASH), autoimmune hepatitis, and primary biliary cholangitis often cause liver failure [34]. The liver has a variety of functions, including metabolism of protein, sugar, and fat; detoxification; production of coagulation factors; and production of bile. Thus, during liver failure, several symptoms, including jaundice, edema, ascites, hepatic encephalopathy, and increased bleeding, can appear at the same time, resulting in life-threatening disease. In addition, during liver failure caused by chronic liver disease, accumulated liver fibrosis (i.e., liver cirrhosis) can cause portal hypertension, which often induces the varices, and long-term liver damage can cause gene abnormalities, leading to liver cancers. The ultimate therapy for liver failure is liver transplantation; however, only a small portion of patients with liver failure can receive liver transplantation due to the shortage of donor organs, invasiveness of operations, and economic reasons [35]. Revolutionary treatments, such as interferon-free treatment for hepatitis C and providing information regarding the importance of the daily lifestyle to prevent alcoholic liver disease and NASH, can potentially decrease the liver diseases; however, unmet needs to treat advanced liver failure will continue.
Advanced acute liver failure and chronic liver failure (liver cirrhosis) can be good targets for cell therapy. Since 2003, Terai et al. initiated autologous bone marrow cell infusion (ABMi) therapy against decompensated liver cirrhosis and confirmed the improvement of liver fibrosis and liver function [36][37][38]. However, due to the invasiveness of liver transplantation in patients with liver failure, minimally invasive procedures using specific cells, such as MSCs and macrophages [39][40][41], are now being developed, with a focus on MSCs. In the next section, we will describe recent reported results using MSCs registered at ClinicalTrials.gov.

Effects of MSC therapy in liver disease from published papers
Animal experiments have shown that MSCs can have antiapoptotic [42] and antioxidant effects in hepatocytes [43], and antifibrotic [44,45], angiogenic [46], and immunosuppressive effects in T cells, macrophages, and dendritic cells [8]. In human clinical trials, all reports have shown that MSC injection is safe. Although the effects of cell therapy are not uniform, the majority of therapies have some beneficial effects; in contrast, in a few reports, treatment effects were not observed. For example, Kantarcioglu et al. [13] and Mohamadnejad et al. [19] injected bone marrowderived MSCs into patients with liver cirrhosis and did not observe treatment effects. However, Kharaziha et al. [14] reported phase I-II clinical trials using autologous bone marrow-derived MSCs against liver cirrhosis with a variety of etiologies, and improvement of liver function was confirmed. Jang et al. and Suk et al. [12,16] reported a pilot study and a phase II study using autologous bone marrowderived MSCs injected through the hepatic artery against alcoholic liver cirrhosis, and improvement of histological liver fibrosis and liver function was confirmed. Xu et al. [21] reported trials using autologous bone marrow-derived MSCs against hepatitis B virus-associated cirrhosis and confirmed the improvement of liver function, the decrease of Th17 cells, and the increase of regulatory T cells. Xhang et al. [17] and Wang et al. [20] reported trials using allogeneic umbilical cord-derived MSCs in patients with chronic hepatitis B having decompensated liver cirrhosis and primary biliary cirrhosis, respectively. They confirmed improvement of liver function, particularly reduced ascites and recovery of biliary enzymes, respectively. Shi et al. [15] reported a trial investigating acute or chronic liver failure associated with hepatitis B virus and confirmed that MSCs significantly increased survival rates. From these reports, MSCs appeared to improve liver function; however, additional trials are needed to confirm these effects and to elucidate the mechanisms in more detail.

Background of IBDs
IBDs are chronic inflammatory disorders, including UC and CD. The pathogenesis of IBD is thought to be highly complex due to several factors, such as environmental factors, genetic predisposition, and inflammatory abnormalities [47]. UC is characterized by inflammation of the mucosal membrane of the colon continued from the rectum. Type 2 T helper cell (Th2) cytokine profile is associated with the pathogenesis of UC. In contrast, CD is a segmental, transluminal disorder that can arise within the entire gastrointestinal tract from the mouth to the anus. Th1 cells are associated with the pathogenesis of CD [48]. Furthermore, a recent report showed that Th17 cells are present in both UC and CD. Thus, mucosal CD4+ T cells are key mediators of the driving response [49]. Macrophages that produce tumor necrosis factor (TNF)-α have also been reported to be relevant in IBD. Imbalances in other cytokines, such as interleukin (IL)-1β, IL-6, IL-8, IL-10, IL-12, IL-17, IL-23, and transforming growth factor-β (TGF-β), are also detected during diseases [48]. Recent advancements in the development of drugs for IBD include drugs targeting TNF and new candidate drugs, such as antibodies against IL-6 [50] and IL-12/23 [51][52][53], small molecules including Janus kinase inhibitors [54], antisense oligonucleotides against SMAD7 mRNA [55], and inhibitors of leukocyte trafficking to intestinal sites of inflammation [56,57]. However, some patients will fail to respond to current medical options, immunosuppressive agents, and anti-TNF biologicals. MSCs may be an effective option in these patients [9,49]. In the next section, we will describe recently reported results using MSCs registered in ClinicalTrials.gov.

Effects of MSC therapy in IBD from published papers
Eight CD trials and one UC trial have been published in ClinicalTrials.gov. Six papers describing CD are on trials treating fistula, and two papers are trials for luminal CD. Molendijk et al. [25] reported improved healing of refractory perianal fistulas using allogeneic bone marrow-derived MSCs. They administered these allogeneic MSCs locally and concluded that injection of 3 × 10 7 MSCs appeared to promote the healing of perianal fistula. Panes et al. [31] reported a phase III randomized, double-blind, parallelgroup, placebo-controlled study of complex perianal fistula using expanded allogeneic adipose-derived MSCs and confirmed the safety of the MSCs and the healing effects of MSCs on the fistula. Duijvestein et al. [32] reported a phase I study of refractory luminal CD using autologous bone marrow-derived MSCs and confirmed the safety and feasibility of MSC therapy. Forbes et al. [24] reported a phase II study using allogeneic bone marrow-derived MSCs for luminal CD refractory to biologic therapy. They administered 2 × 10 6 cells/kg weekly for 4 weeks and found that allogeneic MSCs reduced the CD activity index (CDAI) and CD endoscopic index of severity (CDEIS) scores in patients with luminal CD refractory to biologic therapy. Hu et al. [33] reported a phase I/II study for severe UC using umbilical cord-derived allogeneic MSCs by combination injection through the peripheral blood and superior mesenteric artery with a 7-day interval. They confirmed the safety of   MSCs and alleviation of diffuse and deep ulcer formation and severe inflammatory mucosa by MSCs.

Safety of the MSC therapy
MSC therapy is associated with some concerns, such as adverse events related to infusion, tumor formation during the treatment of liver cirrhosis, and long-term observations of tumor formation. Regarding adverse events related to the infusion, Lalu et al. performed a meta-analysis of the safety of MSCs in clinical trials and showed that autologous and allogeneic MSC therapies were related to transient fever but not infusion toxicity, organ system complications, infection, death, and malignancies (Table 2) [5]. Regarding tumor formation during the treatment of liver cirrhosis, Peng et al. reported that no severe adverse events or no significant differences in tumor formation were detected compared with those in the control group during autologous bone marrow-derived MSC therapy for liver cirrhosis [58]. Regarding long-term observations of tumor formation derived from MSCs, Bahr et al. reported recent autopsy data from patients in a clinical trial of graft-versus-host disease (GvHD) who received MSC therapy between 2002 and 2007 and revealed no ectopic tissues, neoplasms, or donor-derived DNA [6].

Conclusions
Many clinical trials of autologous and allogeneic MSCs have aimed to elucidate the effects and mechanisms of MSCs. MSCs can expand easily and can be obtained from medical waste, suggesting their applications in regenerative medicine for the treatment of liver diseases and IBDs.
Recently, limitations of MSCs have been reported. For example, therapeutic effects were not long term and were affected by inflammatory condition [59,60]. Thus, the results of ongoing clinical studies will be expected to provide further insights.