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

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

is a complex surgery and carries the risks of complications inherent to surgery. Although hepatocyte transplantation is less invasive and less expensive than LT, the sources of primary hepatocytes are scarce, and these cells have weak in vitro hepatic function; for these reasons, the application of these cells for treating liver diseases is limited. 5 Moreover, only 0.1%-0.3% of primary hepatocytes migrate into host liver tissue, leading to poor therapeutic effects in vivo. 6 Dhawan et al 7 demonstrated that patients who underwent hepatocyte transplantation experienced allogeneic rejection and a decline in liver graft function within 1 year. Stem cell-based therapy has emerged as an alternative strategy to hepatocyte transplantation for improving liver function and promoting liver regeneration.
Mesenchymal stem cells (MSCs) are fibroblast-like, adherent, immunomodulatory and multipotent cells that rapidly proliferate in vitro under specific conditions. 8 They can be isolated from various tissues, including bone marrow, umbilical cord blood and adipose tissue, and can undergo hepatogenic differentiation upon culture in hepatic medium. 9 MSCs restore liver function after hepatogenic differentiation and exert immunomodulatory, anti-inflammatory, antifibrotic, antioxidative stress and antiapoptotic effects in liver cells. 10 Although MSCs exert antitumour effects via inhibition of the Wnt signalling pathway, it is worth considering that MSCs may promote tumour initiation and growth by exerting immunosuppressive and angiogenic effects in human hepatocellular carcinoma (HCC). 11 After isolation and culture in vitro, MSCs are faced with nutrient and oxygen deprivation, and external growth factors cannot maintain MSC capacities for further applications. 12 Although MSCs can undergo differentiation into various somatic cells under defined conditions in vitro, they rarely transform into target cells after transplantation. In addition, transplanted MSCs undergo apoptosis or senescence in response to the harsh microenvironment. 13 An obstacle facing MSC-based transplantation therapy is the limited number of functional stem cells available after transplantation due to the harsh microenvironment, anoikis and inflammation induced by damaged tissues or organs. 13 The acute in vivo inflammatory response effectively promotes the recruitment of progenitor cells; however, chronic inflammation significantly inhibits the recruitment and survival of local progenitor cells and implanted MSCs. 14 Thus, anti-inflammatory and paracrine mechanisms are main contributors to repairing liver tissue damage and prolonging the survival of animal models with liver injury. Mesenchymal stem cells significantly up-regulate the secretion of the anti-inflammatory cytokine interleukin (IL)-10 and decrease the production of tumour necrosis factor (TNF)-α, interferon-gamma (IFN-γ) and IL-12. 15 In addition, MSC-derived secretomes contain protein mediators such as hepatocyte growth factor (HGF), transforming growth factor (TGF)-β3, indoleamine 2,3-dioxygenase (IDO) and prostaglandin 2 (PGE2), which are important for anti-inflammatory signalling and immunoregulation. 16 Chemical agents, hypoxia, inflammatory microenvironments and gene modification can be utilized to protect MSCs against injury induced by a harsh microenvironment, thereby improving the homing capacity, survival rate and paracrine effects of MSCs in vitro and in vivo, as well as the ability of these cells to enhance liver function. [17][18][19][20] In the current review, we mainly focus on pre-treatments and the underlying mechanisms for improving the therapeutic effects of MSCs in various liver diseases ( Figure 2). In this way, pre-treated MSCs can be administered to prolong the survival of patients with end-stage liver diseases in the near future.

| THE P OTENTIAL MECHANIS MS BY WHI CH MSC ADMINIS TR ATI ON C AN TRE AT LIVER D IS E A S E S
Mesenchymal stem cells significantly reduce inflammatory factor secretion, immune cell infiltration and hepatocyte apoptosis but up-regulate antioxidant levels and energy metabolism in chemicalinduced acute liver injury. For example, MSCs reduced liver injury in d-galactosamine (d-Gal)/lipopolysaccharide (LPS)-induced acute liver failure (ALF) rats via reducing the release of inflammatory cytokines, such as IL-1β, IL-6, and TNF-α; down-regulating the nuclear factor-kappa B (NF-κB) pathway; and up-regulating the expression of haeme oxygenase-1 (HO-1). 21 MSCs inhibited the infiltration of lymphocytes, dendritic cells and Kupffer cells, further decreased the serum levels of inflammatory factors (TNF-α, interferon-γ and IL-4) and increased the serum levels of the hepatoprotective factor IL-10 in mice with concanavalin A-induced acute liver injury. 22 Meanwhile, MSCs significantly eliminated acetaminophen-induced injury and increased the survival rate of ALF mice via inhibiting cytochrome P450 activity and MAPK signalling but improving antioxidative activity. 23 Traditionally, alanine aminotransferase, aspartate aminotransferase, prothrombin time, ammonia and total bilirubin have been used as biomarkers of liver injury. Mesenchymal stem cell administration F I G U R E 1 Multiple therapies, such as LT, hepatocyte transplantation and MSC transplantation, have been developed to treat patients with end-stage liver diseases was reported to improve liver function, as shown by decreased alanine transaminase and aspartate aminotransferase expression, prothrombin time and serum ammonia, via the down-regulation of liver isoprostanes, 8-hydroxyguanosine (8-OHG) and nitrite nitrates and the maintenance of hepatic glutathione (GSH), which are protective factors that eliminate oxidative stress. Moreover, MSCs down-regulated the expression levels of TNF-α, monocyte chemoattractant protein-1 (MCP-1), IL-1β, intercellular adhesion molecule 1 (ICAM-1) and phospho-c-Jun NH2-terminal kinase (p-JNK) and increased the liver regeneration rate in acetaminophen-induced liver injury rats. 24 MSCs also attenuated hepatocyte apoptosis and accelerated the regeneration of remnant liver tissues in rats with major hepatectomy-induced ischaemia reperfusion (I/R) injury. 25 It is possible that MSCs employ redox signalling to coordinate self-renewal and differentiation or to regulate stem cell activity in response to oxidative stress. Thus, the metabolic balance is an important regulator of MSC-based regenerative medicine. In addition to inhibiting hepatocyte apoptosis, MSCs participated in maintaining metabolic balance by regulating amino acids, bile acids, sphingolipids, acylcarnitines and glycerophospholipids in liver cells to attenuate liver injury in ALF rats. 26 Liver transplantation always has a high rejection rate, and although liver tissue is a tolerogenic organ with adaptive systems, acute graft-vs-host disease is a serious and life-threatening complication of LT. 27 More recently, MSC transplantation has been recognized as a novel treatment for preventing graft rejection and treating autoimmune diseases such as graft-vs-host disease via immunomodulatory effects mediated by cell-to-cell interactions and secreted cytokines. 28 MSCs improved the prognosis of LT animals by suppressing hepatocyte apoptosis, KC apoptosis, Th1/Th17 infiltration, chemokine release and inflammatory cell infiltration. 29 In addition, transplanted MSCs inhibited allograft rejection and activated CD4+CD25+Foxp3+ Tregs to prolong the survival of LT rats. 30 Liver cirrhosis is a continuous liver injury in which quiescent hepatic stellate cells (HSCs) transform into proliferative, α-smooth muscle actin + myofibroblast-like cells that deposit collagen in liver tissue. Hepatic stellate cell activation, extracellular matrix and collagen deposition and immune cell accumulation in liver tissue result in liver fibrosis or cirrhosis in mammals. 31 MSCs effectively induced the apoptosis of HSCs and inhibited liver inflammation and collagen deposition to block hepatic fibrosis. 32,33 Patients with hepatitis C virus-induced liver fibrosis also benefited from MSC transplantation, which prompted the down-regulation of fibrotic markers and inflammatory factors and the up-regulation of anti-inflammatory factors in liver tissue. 34 On the other hand, MSCs improved the prognosis of patients with hepatitis B virus-induced liver fibrosis by up-regulating Tregs and down-regulating Th17 cells. Subsequently, MSCs increased serum TGF-β levels while decreasing the expression levels of IL-17, TNF-α and IL-6 in these fibrotic patients. 35 MSC administration significantly suppressed chemically induced HCC by inhibiting the Wnt and NF-kB signalling pathways. 36,37 In contrast, MSCs were shown to promote tumour growth and metastasis in HCC patients by supporting angiogenesis and modulating the immune response in vivo. 38 MSCs also contribute to the acceleration of HCC metastasis via the induction of epithelial-mesenchymal transition (EMT), which further contributes to shortening the overall survival of HCC patients. 39 F I G U R E 2 The underlying mechanisms of MSC pre-treatments in various liver diseases

| TR AN S PL ANTED HLC S WITH IMPROVED LIVER FUN C TI ON MAY EFFEC TIVELY PARTI CIPATE IN REPAIRING THE INJ URED LIVER
According to current studies, MSCs can transform into hepatocytes with liver-specific functions, but the immature phenotypes of these differentiated cells inhibit their application. Thus, multiple strategies Overexpression of HNF-4α significantly increased the expression levels of hepatic-specific genes, liver-enriched transcription factors and cytochrome P450 genes in hepatogenic MSCs in vitro, 19 thus transforming these MSCs into highly functional hepatocytes via activation of the Wnt/β-catenin pathway. 42 Forkhead box A2 (FOXA-2) is reported to activate liver-specific genes, including ALB and transthyretin, to promote the hepatic differentiation of MSCs.
Overexpression of HNF-4α and FOXA-2 promoted the maturation of MSC-derived HLCs and up-regulated their expression levels of ALB, urea and glucose, as well as indocyanine green uptake and cytochrome P450 activity. In addition, the transplantation of MSC-derived HLCs was deemed safe because the transplanted cells did not form tumours after 2 months. 43   and NF-κB p65 nuclear translocation to protect LO2 cells from hypoxia-reoxygenation-induced injury. 20 Heat-shock pre-treatment (HSP) significantly attenuated hydrogen peroxide (H 2 O 2 )-induced apoptosis by down-regulating Bax and cytochrome C levels and upregulating Bcl-2 levels and autophagy in MSCs. Consequently, the transplantation of MSCs exposed to HSP into I/R rats decreased serum aminotransferase levels and Suzuki scores while improving histopathology and hepatocyte proliferation. 57 Melatonin-pre-treated MSCs showed increased homing capacity to the injured liver site and significantly improved the percentage of glycogen storage while decreasing collagen and lipid accumulation in fibrotic liver tissue; this outcome stemmed from decreased expression of TGF-β1 and Bax and increased expression of matrix metalloproteinases (MMPs) and Bcl-2. 17 Furthermore, melatonin pre-conditioning significantly increased the engraftment of MSCs and attenuated liver fibrosis in rat models. 58 Pre-treatment with 10 ng/µL SDF-1α improved the homing rate of MSCs in vitro and in vivo, and intraperitoneal injection of resveratrol into rats with common bile duct ligation-induced liver cirrhosis further attenuated the common pathological changes by up-regulating sirtuin 1, CXCR-4 and MMP-9 and down-regulating p53 in the liver. 59 Although the above studies tried to improve the therapeutic effects of MSCs via multiple pathways, further studies should expand the chemical entities used to regulate MSCs for the treatment of various liver diseases. In addition, all published data focus on the effects of MSCs on I/R injury and liver fibrosis in mammals, and it is necessary to investigate more chemicals for other liver diseases, such as ALF and liver cancer.

| Hypoxic pre-treatment for MSCbased therapies for liver diseases
As the oxygen concentration seen by tissue-resident MSCs is lower than 5%, in vitro culture under hypoxic conditions may effectively mimic the in vivo microenvironment and contribute to the maintenance of MSC proliferation, differentiation, metabolic balance and other physiological processes. 18 Moreover, hypoxic pre-treatment significantly reduced cellular injury and promoted the proliferation of MSCs by maintaining energy metabolism in vitro. 60 62 On the other hand, hypoxia (1% O 2 )-pre-treated MSCs markedly increased hepatocyte proliferation by up-regulating VEGF in rats that underwent massive hepatectomy compared to normoxia-preconditioned MSCs. 63 It is worth noting that oxygen concentration will influence the stemness of MSCs in vitro and in vivo, and Hu et al 10   HLCs significantly reduced liver fibrosis and improved liver function in rats with CCl 4 -induced liver fibrosis. 66 In addition, pre-treatment with inflammatory factors also contributes to improving MSC-based therapeutic effects. Although IFN-γ and a multiple cytokine cocktail consisting of IFN-γ, TGF-β and retinoic acid had no effect on the immunomodulation of MSCs in vivo, they significantly enhanced the capacity of MSCs to inhibit the proliferation of CD4+ T cells and CD8+ T cells and the production of IFN-γ. 67 Further studies should expand the related pre-treatments to include inflammatory factors to improve MSC-based regenerative medicine for liver diseases because MSCs respond to inflammatory factors and alter their immunoregulatory capacities in vitro and in vivo.   phenotype. Various pre-treatments were proven to enhance the therapeutic effects of MSCs in acute liver injury and liver fibrosis (Table 1)

CO N FLI C T O F I NTE R E S T
The authors declare no competing financial interests.

AUTH O R S ' CO NTR I B UTI O N S
Lanjuan Li contributed to the conception of this study. Chenxia Hu and Zhongwen Wu were responsible for the literature review.
Chenxia Hu and Zhongwen Wu drafted and revised the manuscript.
All authors read and approved the final manuscript.