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Mesenchymal stromal cell therapy for liver diseases

  • Author Footnotes
    † These authors contributed equally to this work.
    Mohammed Alfaifi
    Footnotes
    † These authors contributed equally to this work.
    Affiliations
    Centre for Liver Research, Institute of Immunology and Immunotherapy, University of Birmingham, UK

    Department of Clinical Laboratory Sciences, College of Applied Medical Sciences, King Khalid University, Abha, Saudi Arabia
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  • Author Footnotes
    † These authors contributed equally to this work.
    Young Woo Eom
    Footnotes
    † These authors contributed equally to this work.
    Affiliations
    Cell Therapy and Tissue Engineering Center, Yonsei University Wonju College of Medicine, Wonju, South Korea

    Department of Internal Medicine, Yonsei University Wonju College of Medicine, Wonju, South Korea
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  • Philip N. Newsome
    Correspondence
    Corresponding authors. Addresses: National Institute for Health Research (NIHR) Birmingham Liver Biomedical Research Unit and Centre for Liver Research, University of Birmingham, Birmingham, UK (P.N. Newsome), or Department of Internal Medicine, Yonsei Univ., Wonju College of Medicine, 20 Ilsan-ro, Wonju, Gangwon-do 26426, South Korea (S.K. Baik).
    Affiliations
    Centre for Liver Research, Institute of Immunology and Immunotherapy, University of Birmingham, UK

    National Institute for Health Research Biomedical Research Centre at University Hospitals Birmingham NHS Foundation Trust and the University of Birmingham, UK

    Liver Unit, University Hospitals Birmingham NHS Foundation Trust, Birmingham, UK
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  • Soon Koo Baik
    Correspondence
    Corresponding authors. Addresses: National Institute for Health Research (NIHR) Birmingham Liver Biomedical Research Unit and Centre for Liver Research, University of Birmingham, Birmingham, UK (P.N. Newsome), or Department of Internal Medicine, Yonsei Univ., Wonju College of Medicine, 20 Ilsan-ro, Wonju, Gangwon-do 26426, South Korea (S.K. Baik).
    Affiliations
    Cell Therapy and Tissue Engineering Center, Yonsei University Wonju College of Medicine, Wonju, South Korea

    Department of Internal Medicine, Yonsei University Wonju College of Medicine, Wonju, South Korea
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  • Author Footnotes
    † These authors contributed equally to this work.
Published:February 06, 2018DOI:https://doi.org/10.1016/j.jhep.2018.01.030

      Summary

      The therapeutic potential of mesenchymal stromal cells (MSCs) in the treatment of liver fibrosis is predominantly based on their immunosuppressive properties, and their ability to secrete various trophic factors. This potential has been investigated in clinical and preclinical studies. Although the therapeutic mechanisms of MSC transplantation are still not fully characterised, accumulating evidence has revealed that various trophic factors secreted by MSCs play key therapeutic roles in regeneration by alleviating inflammation, apoptosis, and fibrosis as well as stimulating angiogenesis and tissue regeneration in damaged liver. In this review, we summarise the safety, efficacy, potential transplantation routes and therapeutic effects of MSCs in patients with liver fibrosis. We also discuss some of the key strategies to enhance the functionality of MSCs, which include sorting and/or priming with factors such as cytokines, as well as genetic engineering.

      Keywords

      Introduction

      MSCs have been proven to have potential therapeutic effects on end-stage liver disease through these immunomodulatory effects, release of antioxidants, anti-fibrotic effects, and hepatocyte-like differentiation.
      Liver disease is a major cause of mortality and morbidity that is rising globally.
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      There remain many inflammatory liver conditions for which current treatments are not effective, often patients with such conditions progress to end-stage liver disease and require liver transplantation. To prevent progression to end-stage liver disease and to treat those with advanced fibrosis, mesenchymal stromal cell (MSC) therapies have been considered and shown to have potential in such liver diseases.
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      MSCs have been shown to have beneficial effects in a range of clinical settings, including heart failure,
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      In addition, MSCs have been reported to be able to differentiate into hepatocyte-like cells, which hold promise for augmenting liver regeneration.
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      It is, therefore, timely to review the data underpinning these effects and to address the important scientific questions that remain, to establish MSC therapy for patients with liver disease.

      MSCs: definition, biology and tissue origins

      MSCs isolated and cultured in various tissues and organs have similar biological characteristics, but have been reported to show slight differences in differentiation potentials, immunomodulatory properties, and proliferative capacity.
      MSCs were initially described in the 1968 by Friedenstein,
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      and are a subtype of adult fibroblast-like cells that have the capacity for self-renewal with high proliferative ability. They can undergo tri-lineage differentiation both in vivo and in vitro down connective tissue lineages to become osteoblasts, chondrocytes and adipocytes.
      MSCs are plastic adherent cells originally identified and isolated from bone marrow, but because of their limited number (0.01 to 0.001% of total bone marrow cells)
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      umbilical cord blood (UCB),
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      amniotic fluid (AF)
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      Amniotic fluid as a rich source of mesenchymal stromal cells for transplantation therapy.
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      Human placenta-derived cells have mesenchymal stem/progenitor cell potential.
      Umbilical cord tissue (UC) has been a particularly promising source of MSCs – cells can be isolated from several compartments within UC, including umbilical vein, umbilical arteries, umbilical cord perivascular tissue, Wharton’s jelly (WJ) and sub-amniotic tissue. Furthermore, MSCs isolated from UC tissue are believed to be more primitive than other cells isolated from other tissues and are found in higher numbers, ensuring this source is gaining prominence. Notably, MSCs from different sources display similar expression profiles for MSC surface markers and similar morphological features in culture, yet they have different levels of tri-lineage differentiation potential.
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      Comparison of proliferative and multilineage differentiation potential of human mesenchymal stem cells derived from umbilical cord and bone marrow.
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      Whereas, direct comparisons of MSCs from different sources have been shown to share similar biological properties,
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      Comparing the immunomodulatory properties of bone marrow, adipose tissue, and birth-associated tissue mesenchymal stromal cells.
      other authors demonstrated differences in immunomodulatory properties between BM-MSCs, UC-MSCs, and AT-MSCs.
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      Adipose tissue-derived multipotent stromal cells have a higher immunomodulatory capacity than their bone marrow-derived counterparts.
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      MSCs from differing sources such as AT, UCB, and BM were found to express a similar pattern of surface antigens,
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      Potential mechanisms of action of MSCs in liver disease

      Mechanism of immunomodulation by MSCs

      MSCs can regulate and treat damaged tissue by regulating adaptive and innate immunity. This is accomplished through direct cell-to-cell interactions or paracrine factors secreted by MSCs.
      MSCs can modulate and repair injured tissue by modulating injurious immune responses through a range of mechanisms including direct cell-to-cell interaction or remotely by secretion of paracrine factors (Fig. 1).
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      Of note, MSCs have reduced immunogenicity because they lack expression of class II major histocompatibility (MHC) antigens when unprimed and do not express many of the molecules required for immune recognition such as CD80, CD86n and CD40.
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      Figure thumbnail gr1
      Fig. 1Modes of MSC-based therapy. MSC, mesenchymal stromal cell.

      Immunomodulatory effect of MSCs on adaptive immunity

      MSCs can inhibit the proliferation of T cells in vitro either by secreting soluble factors or by directly interacting with T-lymphocytes (Fig. 2).
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      Human bone marrow stromal cells suppress T-lymphocyte proliferation induced by cellular or nonspecific mitogenic stimuli.
      Several different molecules secreted by MSCs have been reported to have an immunomodulatory effect on T cell activities, including transforming growth factor β (TGF-β), hepatocyte growth factor (HGF),
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      and indoleamine 2,3-dioxygenase (IDO).
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      Notably, the production of these immunomodulatory molecules differs according to the source of MSCs, for example, WJ-MSCs produce higher amounts of TGF-β than BM-MSCs.
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      Figure thumbnail gr2
      Fig. 2Potential mechanisms of the MSC interactions with immune cells. MSC, mesenchymal stromal cell; NK, natural killer.
      The inflammatory environment is known to have an essential role during the interaction between MSCs and T cells, for example, the immunosuppressive capacity of MSCs is induced by treatment with a combination of cytokines (interferon [IFN]-γ, interleukin [IL]-1α, tumour necrosis factor-alpha [TNF-α], and IL-1β).
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      Mesenchymal stem cell-mediated immunosuppression occurs via concerted action of chemokines and nitric oxide.
      These cytokines can enhance some chemokines and other immune cells to easily contact the MSCs and mediate immune reactions. Another mechanism by which MSCs can suppress the proliferation of T cells is via secretion of nitric oxide (NO) which causes inhibition of STAT5 pathways.
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      MSCs have also been shown to promote the generation and development of regulatory T cells (Tregs), which can positively influence the balance of immune damage during tissue injury.
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      The induction of CD4+ CD25+ FOXP3+ Treg was mediated by secretion of TGF-β
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      and is accompanied by inhibition of the proliferation and differentiation of Th1 and Th17 helper T cells, which can further trigger activation of regulatory T cells. This mechanism was associated with an increased production of IL-10 by MSCs.
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      Mesenchymal stem cells generate a CD4+CD25+Foxp3+ regulatory T cell population during the differentiation process of Th1 and Th17 cells.
      MSCs can also inhibit the proliferation of B cells, reducing their production of immunoglobulin. Glennie et al. used CD40 and IL-4 to increase the proliferation rate of murine B cells and demonstrated that subsequent co-culture with MSCs significantly inhibited their proliferation.
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      Natural killer cells (NK) represent a critical component of the immune response against viral infections and tumour cells
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      – Sotiropoulou et al. demonstrated that MSCs reduced IL-15 secretion from IL-2 induced NK cells. This reduction was presumed to be caused by either cell-to-cell interactions or release of soluble factors such as PGE2 and TGF-β.
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      In addition, another group reported that MSCs can suppress NK cells after stimulation with IL-5.
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      In models of acute liver injury, MSCs ameliorated hepatotoxicity of NKT cells in an IDO-dependent manner, by reducing the number of IL-17 cells and stimulating FOXP3 and IL-10 through increased NK Treg numbers in the injured liver.
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      Immunomodulatory effect of MSCs in innate immunity

      Macrophages can be classified into classical pro-inflammatory macrophages (M1) or alternative macrophages (M2) that secret anti-inflammatory cytokines (Fig. 2).
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      Transcriptional regulation of macrophage polarization: enabling diversity with identity.
      MSCs have been reported to trigger polarisation of M1 toward M2 both in vivo and in vitro. This polarisation is driven by the ability of MSCs to secrete soluble factors such as interleukin (IL)-10 and IL-1Ra which have been shown to attenuate liver injury by promoting a number of M2 macrophages.
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      Allo-transplantation of mesenchymal stem cells attenuates hepatic injury through IL1Ra dependent macrophage switch in a mouse model of liver disease.
      In addition to the IL-10 mediated ability of MSCs to promote a switching of macrophages from an M1 to an M2 phenotype, MSCs can also help to promote survival of monocytes through upregulation of CCL18, which was found to indirectly mediate the ability of MSCs to induce Treg formation,
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      as demonstrated in animal models of sepsis and colitis.
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      In this study murine adipose derived MSCs significantly increased the proportion of M2-like cells by increased production of IL10 and arginase1 activities.
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      Adipose-derived mesenchymal stromal cells induce immunomodulatory macrophages which protect from experimental colitis and sepsis.
      MSCs can also regulate, and interact with, dendritic cell function by blocking differentiation of antigen presenting cells to monocytes and decreasing their expression of anti-inflammatory molecules such as IL12, TNF-α, and IFN-γ, whilst also enhancing their secretion of IL-10, which may induce regulatory T cell numbers (Fig. 2).
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      Human mesenchymal stem cells alter antigen-presenting cell maturation and induce T-cell unresponsiveness.
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      Therefore, there is now a greater recognition of the importance of the microenvironment on the immunomodulatory capacity of MSCs,
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      Mesenchymal stem cell-mediated immunosuppression occurs via concerted action of chemokines and nitric oxide.
      highlighting the need for a better understanding of the microenvironment associated with specific diseases to improve the therapeutic efficacy of MSCs.

      Anti-fibrotic activities of MSCs

      MSCs can regulate the inflammatory response and fibrosis of HSCs. Paracrine factors secreted by MSCs can inhibit HSC proliferation and ECM synthesis and can promote apoptosis of HSCs. MSCs can regulate ECM-production of HSCs by regulating the activity of inflammatory cells.
      Inflammation and fibrosis have a very close relationship in liver disease. In response to liver injury, pro-fibrotic factors such as TGF‐β, platelet‐derived growth factor (PDGF), IL-13 and IL-4, which are secreted by resident or infiltrating immune cells, play important roles in the activation and proliferation of hepatic stellate cells (HSCs), which are important cells for the production of extracellular matrix (ECM) in the liver.
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      Transforming growth factor-beta 1 induces alpha-smooth muscle actin expression in granulation tissue myofibroblasts and in quiescent and growing cultured fibroblasts.
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      Fibrotic disease and the T(H)1/T(H)2 paradigm.
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      • Shim K.Y.
      • Baik S.K.
      Diagnostic accuracy of hepatic vein arrival time performed with contrast-enhanced ultrasonography for cirrhosis: a systematic review and meta-analysis.
      Therefore, the anti-fibrotic activities of MSCs can be distinguished by their direct or indirect effects on HSCs. The indirect anti-fibrotic effects on HSCs are achieved by MSCs controlling immune cells, which subsequently inhibit the activity of HSCs, whereas the direct anti-fibrotic effects are mediated by MSCs directly inhibiting the activity of HSCs.
      Regarding the indirect anti-fibrotic effects of MSCs on HSCs, MSCs can regulate the activities of HSCs by modulating immune cell activity. MSCs can migrate towards injured sites of inflammatory reaction where they are exposed to inflammatory cytokines such as IFN-γ and IL-1β.
      • Rasmusson I.
      Immune modulation by mesenchymal stem cells.
      • Eom Y.W.
      • Shim K.Y.
      • Baik S.K.
      Mesenchymal stem cell therapy for liver fibrosis.
      These MSCs secrete various soluble mediators (e.g. NO, PGE2, IDO, IL-6, IL-10, and HLA-G), resulting in the suppression of the proliferation and activation of a variety of immune cells, as well as the induction of Treg cells.
      • Sharma R.R.
      • Pollock K.
      • Hubel A.
      • McKenna D.
      Mesenchymal stem or stromal cells: a review of clinical applications and manufacturing practices.
      Thus, suppression of immune cell activities by MSCs can also reduce fibrogenic processes and ameliorate ECM accumulation in liver disease. In particular, macrophages play a central role in both fibrosis and fibrotic resolution in the liver
      • Wynn T.A.
      • Barron L.
      Macrophages: master regulators of inflammation and fibrosis.
      • Kim G.
      • Huh J.H.
      • Lee K.J.
      • Kim M.Y.
      • Shim K.Y.
      • Baik S.K.
      Relative adrenal insufficiency in patients with cirrhosis: a systematic review and meta-analysis.
      – during hepatic fibrogenesis, pro-inflammatory M1 macrophages located near the activated hepatic myofibroblasts secrete pro-fibrogenic factors such as TGF-β, PDGF, and CCL2. This secretion leads to increased fibrogenic responses of myofibroblasts by promoting their activation, proliferation, and chemotaxis.
      • Wynn T.A.
      • Barron L.
      Macrophages: master regulators of inflammation and fibrosis.
      • Fadok V.A.
      • Bratton D.L.
      • Konowal A.
      • Freed P.W.
      • Westcott J.Y.
      • Henson P.M.
      Macrophages that have ingested apoptotic cells in vitro inhibit proinflammatory cytokine production through autocrine/paracrine mechanisms involving TGF-beta, PGE2, and PAF.
      However, macrophages co-cultured with MSCs are polarised into anti-inflammatory M2 states, which show higher phagocytic activity through increased expression of IL-10 and decreased expression of TNF-α and IL-12p40.
      • Kim J.
      • Hematti P.
      Mesenchymal stem cell-educated macrophages: a novel type of alternatively activated macrophages.
      • Maggini J.
      • Mirkin G.
      • Bognanni I.
      • Holmberg J.
      • Piazzon I.M.
      • Nepomnaschy I.
      • et al.
      Mouse bone marrow-derived mesenchymal stromal cells turn activated macrophages into a regulatory-like profile.
      These results suggest that MSCs can induce changes in the cytokine profile of activated macrophages, promoting resolution of fibrosis. PGE2 has also been reported as a major immunomodulatory molecule when MSCs are co-cultured with macrophages.
      • Nauta A.J.
      • Fibbe W.E.
      Immunomodulatory properties of mesenchymal stromal cells.
      • Keating A.
      How do mesenchymal stromal cells suppress T cells?.
      • Kang S.H.
      • Kim M.Y.
      • Baik S.K.
      Novelties in the pathophysiology and management of portal hypertension: new treatments on the horizon.
      Regarding the direct anti-fibrotic effects of MSCs on HSCs, MSCs can inhibit the proliferation and ECM production potential of HSCs, and induce apoptosis of HSCs. MSCs can secrete IL-10, HGF, TGF-β3 and TNF-α, inhibiting the proliferation of HSCs and decreasing ECM synthesis.
      • Parekkadan B.
      • van Poll D.
      • Megeed Z.
      • Kobayashi N.
      • Tilles A.W.
      • Berthiaume F.
      • et al.
      Immunomodulation of activated hepatic stellate cells by mesenchymal stem cells.
      • Wang J.
      • Bian C.
      • Liao L.
      • Zhu Y.
      • Li J.
      • Zeng L.
      • et al.
      Inhibition of hepatic stellate cells proliferation by mesenchymal stem cells and the possible mechanisms.
      TGF-β3 and HGF induce G0/G1 cell cycle arrest of HSCs, by upregulating p21Cip1 and p27Kip1 and downregulating cyclin D1, which leads to HSC growth inhibition.
      • Wang J.
      • Bian C.
      • Liao L.
      • Zhu Y.
      • Li J.
      • Zeng L.
      • et al.
      Inhibition of hepatic stellate cells proliferation by mesenchymal stem cells and the possible mechanisms.
      Similarly, neutralisation of MSC-derived TNF-α and IL-10 inhibits proliferation and ECM synthesis of activated HSCs.
      • Parekkadan B.
      • van Poll D.
      • Megeed Z.
      • Kobayashi N.
      • Tilles A.W.
      • Berthiaume F.
      • et al.
      Immunomodulation of activated hepatic stellate cells by mesenchymal stem cells.
      Moreover, MSC-derived HGF can also accelerate the rate of HSC apoptosis
      • Parekkadan B.
      • van Poll D.
      • Megeed Z.
      • Kobayashi N.
      • Tilles A.W.
      • Berthiaume F.
      • et al.
      Immunomodulation of activated hepatic stellate cells by mesenchymal stem cells.
      and MSCs cultured with HGF improve serum albumin levels and reduce liver fibrosis in rats.
      • Oyagi S.
      • Hirose M.
      • Kojima M.
      • Okuyama M.
      • Kawase M.
      • Nakamura T.
      • et al.
      Therapeutic effect of transplanting HGF-treated bone marrow mesenchymal cells into CCl4-injured rats.
      The Notch pathway is activated during direct co-culture of MSCs and HSCs through a cell–cell contact mode, significantly suppressing HSC proliferation and α-SMA expression.
      • Chen S.
      • Xu L.
      • Lin N.
      • Pan W.
      • Hu K.
      • Xu R.
      Activation of Notch1 signaling by marrow-derived mesenchymal stem cells through cell-cell contact inhibits proliferation of hepatic stellate cells.
      In liver fibrosis, activated HSCs can express the tissue inhibitors of metalloproteinase (TIMP)-1 and TIMP-2, specific inhibitors of MMP,
      • Herbst H.
      • Wege T.
      • Milani S.
      • Pellegrini G.
      • Orzechowski H.D.
      • Bechstein W.O.
      • et al.
      Tissue inhibitor of metalloproteinase-1 and -2 RNA expression in rat and human liver fibrosis.
      whereas MSCs have been reported to increase the expression of MMPs (e.g. MMP-2, -9, -13 and -14)
      • Wu Y.
      • Huang S.
      • Enhe J.
      • Ma K.
      • Yang S.
      • Sun T.
      • et al.
      Bone marrow-derived mesenchymal stem cell attenuates skin fibrosis development in mice.
      • Higashiyama R.
      • Inagaki Y.
      • Hong Y.Y.
      • Kushida M.
      • Nakao S.
      • Niioka M.
      • et al.
      Bone marrow-derived cells express matrix metalloproteinases and contribute to regression of liver fibrosis in mice.
      • Meier R.P.
      • Mahou R.
      • Morel P.
      • Meyer J.
      • Montanari E.
      • Muller Y.D.
      • et al.
      Microencapsulated human mesenchymal stem cells decrease liver fibrosis in mice.
      or decrease TIMP-1 expression,
      • Ali G.
      • Mohsin S.
      • Khan M.
      • Nasir G.A.
      • Shams S.
      • Khan S.N.
      • et al.
      Nitric oxide augments mesenchymal stem cell ability to repair liver fibrosis.
      which are generally associated with fibrosis resolution in experimental models.

      Hepatocyte-like differentiation of MSCs

      Hepatocyte transplantation is known to be suitable for the treatment of liver disease, but obtaining primary hepatocytes is not easy. Although MSC can be differentiated into hepatocytes, it is required to develop a technique that can differentiate into a fully functioning hepatocytes.
      Since hepatocytes have been reported to improve liver function and mitigate fibrosis in preclinical and clinical studies, hepatocyte transplantation has been considered an alternative to liver transplantation. Several factors influence the hepatic differentiation of MSCs. It has been reported that the treatment of MSCs with a combination of several growth factors, cytokine, and chemical compounds (i.e., HGF, fibroblast growth factor-2/-4, epidermal growth factor, oncostatin M, leukemia inhibitory factor, dexamethasone, insulin-transferrin-selenium, and/or nicotinamide) increases the expression of hepatocyte markers such as HNF-3β (FOXA1), GATA4, CK19 (KRT19), transthyretin, α-fetoprotein, albumin, and CK18 (KRT18).
      • Schwartz R.E.
      • Reyes M.
      • Koodie L.
      • Jiang Y.
      • Blackstad M.
      • Lund T.
      • et al.
      Multipotent adult progenitor cells from bone marrow differentiate into functional hepatocyte-like cells.
      In addition, when MSCs are co-cultured with liver cells
      • Lange C.
      • Bassler P.
      • Lioznov M.V.
      • Bruns H.
      • Kluth D.
      • Zander A.R.
      • et al.
      Liver-specific gene expression in mesenchymal stem cells is induced by liver cells.
      or grown by pellet culture,
      • Ong S.Y.
      • Dai H.
      • Leong K.W.
      Inducing hepatic differentiation of human mesenchymal stem cells in pellet culture.
      they can be differentiated into hepatocyte-like cells. The differentiation of MSCs into hepatocytes has been reported in rats,
      • Shu S.N.
      • Wei L.
      • Wang J.H.
      • Zhan Y.T.
      • Chen H.S.
      • Wang Y.
      Hepatic differentiation capability of rat bone marrow-derived mesenchymal stem cells and hematopoietic stem cells.
      mice,
      • Theise N.D.
      • Badve S.
      • Saxena R.
      • Henegariu O.
      • Sell S.
      • Crawford J.M.
      • et al.
      Derivation of hepatocytes from bone marrow cells in mice after radiation-induced myeloablation.
      sheep
      • Chamberlain J.
      • Yamagami T.
      • Colletti E.
      • Theise N.D.
      • Desai J.
      • Frias A.
      • et al.
      Efficient generation of human hepatocytes by the intrahepatic delivery of clonal human mesenchymal stem cells in fetal sheep.
      and humans.
      • Banas A.
      • Teratani T.
      • Yamamoto Y.
      • Tokuhara M.
      • Takeshita F.
      • Quinn G.
      • et al.
      Adipose tissue-derived mesenchymal stem cells as a source of human hepatocytes.
      Moreover, hepatic stem/progenitor cells isolated from the adult human liver have been reported to be much better at differentiating into hepatocytes than MSCs isolated from tissues other than liver.
      • Herrera M.B.
      • Bruno S.
      • Buttiglieri S.
      • Tetta C.
      • Gatti S.
      • Deregibus M.C.
      • et al.
      Isolation and characterization of a stem cell population from adult human liver.
      Several groups have also reported that MSCs differentiated into hepatocytes can help improve liver function and histopathologic grade, although they are less effective than adult hepatocytes.
      • El Baz H.
      • Demerdash Z.
      • Kamel M.
      • Atta S.
      • Salah F.
      • Hassan S.
      • et al.
      Transplant of hepatocytes, undifferentiated mesenchymal stem cells, and in vitro hepatocyte-differentiated mesenchymal stem cells in a chronic liver failure experimental model: a comparative study.
      There is still uncertainty in the literature regarding the characterisation of MSC-derived hepatocytes, which requires further evaluation, and indeed it is unclear if this will be a major means by which MSCs are utilised.

      Clinical trials using MSCs in liver disease

      MSC therapy for patients with liver disease is safe and may improve liver function. However, to improve the efficacy of MSC therapy for liver disease, preclinical and clinical studies are necessary to standardise the best delivery route of MSCs, to optimise the sufficient number of MSCs, and to elongate the survival duration of engrafted MSCs.
      Many clinical studies have been conducted on the treatment of liver disease using MSCs, focussing on clinical trial design, cell sources, injection route, patient groups, and efficacy of therapies.
      • Wang L.
      • Li J.
      • Liu H.
      • Li Y.
      • Fu J.
      • Sun Y.
      • et al.
      Pilot study of umbilical cord-derived mesenchymal stem cell transfusion in patients with primary biliary cirrhosis.
      • Zhang Z.
      • Lin H.
      • Shi M.
      • Xu R.
      • Fu J.
      • Lv J.
      • et al.
      Human umbilical cord mesenchymal stem cells improve liver function and ascites in decompensated liver cirrhosis patients.
      • Amin M.A.
      • Sabry D.
      • Rashed L.A.
      • Aref W.M.
      • el-Ghobary M.A.
      • Farhan M.S.
      • et al.
      Short-term evaluation of autologous transplantation of bone marrow-derived mesenchymal stem cells in patients with cirrhosis: Egyptian study.
      • Jang Y.O.
      • Kim Y.J.
      • Baik S.K.
      • Kim M.Y.
      • Eom Y.W.
      • Cho M.Y.
      • et al.
      Histological improvement following administration of autologous bone marrow-derived mesenchymal stem cells for alcoholic cirrhosis: a pilot study.
      • Kharaziha P.
      • Hellstrom P.M.
      • Noorinayer B.
      • Farzaneh F.
      • Aghajani K.
      • Jafari F.
      • et al.
      Improvement of liver function in liver cirrhosis patients after autologous mesenchymal stem cell injection: a phase I-II clinical trial.
      • Mohamadnejad M.
      • Alimoghaddam K.
      • Bagheri M.
      • Ashrafi M.
      • Abdollahzadeh L.
      • Akhlaghpoor S.
      • et al.
      Randomized placebo-controlled trial of mesenchymal stem cell transplantation in decompensated cirrhosis.
      • Mohamadnejad M.
      • Alimoghaddam K.
      • Mohyeddin-Bonab M.
      • Bagheri M.
      • Bashtar M.
      • Ghanaati H.
      • et al.
      Phase 1 trial of autologous bone marrow mesenchymal stem cell transplantation in patients with decompensated liver cirrhosis.
      • Salama H.
      • Zekri A.R.
      • Medhat E.
      • Al Alim S.A.
      • Ahmed O.S.
      • Bahnassy A.A.
      • et al.
      Peripheral vein infusion of autologous mesenchymal stem cells in Egyptian HCV-positive patients with end-stage liver disease.
      • Wang L.
      • Han Q.
      • Chen H.
      • Wang K.
      • Shan G.L.
      • Kong F.
      • et al.
      Allogeneic bone marrow mesenchymal stem cell transplantation in patients with UDCA-resistant primary biliary cirrhosis.
      • Amer M.E.
      • El-Sayed S.Z.
      • El-Kheir W.A.
      • Gabr H.
      • Gomaa A.A.
      • El-Noomani N.
      • et al.
      Clinical and laboratory evaluation of patients with end-stage liver cell failure injected with bone marrow-derived hepatocyte-like cells.
      • El-Ansary M.
      • Abdel-Aziz I.
      • Mogawer S.
      • Abdel-Hamid S.
      • Hammam O.
      • Teaema S.
      • et al.
      Phase II trial: undifferentiated vs. differentiated autologous mesenchymal stem cells transplantation in Egyptian patients with HCV induced liver cirrhosis.
      • Peng L.
      • Xie D.Y.
      • Lin B.L.
      • Liu J.
      • Zhu H.P.
      • Xie C.
      • et al.
      Autologous bone marrow mesenchymal stem cell transplantation in liver failure patients caused by hepatitis B: short-term and long-term outcomes.
      • Shi M.
      • Zhang Z.
      • Xu R.
      • Lin H.
      • Fu J.
      • Zou Z.
      • et al.
      Human mesenchymal stem cell transfusion is safe and improves liver function in acute-on-chronic liver failure patients.
      • Suk K.T.
      • Yoon J.H.
      • Kim M.Y.
      • Kim C.W.
      • Kim J.K.
      • Park H.
      • et al.
      Transplantation with autologous bone marrow-derived mesenchymal stem cells for alcoholic cirrhosis: Phase 2 trial.
      • Lin B.L.
      • Chen J.F.
      • Qiu W.H.
      • Wang K.W.
      • Xie D.Y.
      • Chen X.Y.
      • et al.
      Allogeneic bone marrow-derived mesenchymal stromal cells for hepatitis B virus-related acute-on-chronic liver failure: A randomized controlled trial.
      • Lanthier N.
      • Lin-Marq N.
      • Rubbia-Brandt L.
      • Clement S.
      • Goossens N.
      • Spahr L.
      Autologous bone marrow-derived cell transplantation in decompensated alcoholic liver disease: what is the impact on liver histology and gene expression patterns?.
      • El-Ansary M.
      • Mogawer S.
      • Abdel-Aziz I.
      • Abdel-Hamid S.
      Phase I trial: mesenchymal stem cells transplantation in end stage liver disease.
      Based on these viewpoints, we addressed 17 articles to summarise MSC-based therapy for liver disease from 2007 to July 15, 2017 (Table 1). Regarding study design, there was one case series, six case-control studies, five cohort studies, and five randomised clinical trials (RCTs) (Table 1). In the reported studies, a marked heterogeneity was found in injected cell dosage, stem cell source, graft type, injection route, and study design, but significant adverse effects were not reported in the included studies. These studies included patients with a variety of diseases, including acute-on-chronic liver failure (ACLF), liver failure, and cirrhosis due to alcohol, HBV, or HCV, and primary biliary cholangitis. A total of 688 patients were enrolled in the clinical studies, with a range of four patients in the case series design
      • Mohamadnejad M.
      • Alimoghaddam K.
      • Mohyeddin-Bonab M.
      • Bagheri M.
      • Bashtar M.
      • Ghanaati H.
      • et al.
      Phase 1 trial of autologous bone marrow mesenchymal stem cell transplantation in patients with decompensated liver cirrhosis.
      to 158 patients in the case-control design.
      • Peng L.
      • Xie D.Y.
      • Lin B.L.
      • Liu J.
      • Zhu H.P.
      • Xie C.
      • et al.
      Autologous bone marrow mesenchymal stem cell transplantation in liver failure patients caused by hepatitis B: short-term and long-term outcomes.
      In the clinical studies,
      • Peng L.
      • Xie D.Y.
      • Lin B.L.
      • Liu J.
      • Zhu H.P.
      • Xie C.
      • et al.
      Autologous bone marrow mesenchymal stem cell transplantation in liver failure patients caused by hepatitis B: short-term and long-term outcomes.
      BM-derived MSCs (BM-MSCs) were used in 14 studies and UC-MSCs were used in the remaining three studies. In five studies allogenic MSCs were used to treat liver disease; two were derived from BM, and three from UC.
      • Wang L.
      • Li J.
      • Liu H.
      • Li Y.
      • Fu J.
      • Sun Y.
      • et al.
      Pilot study of umbilical cord-derived mesenchymal stem cell transfusion in patients with primary biliary cirrhosis.
      • Zhang Z.
      • Lin H.
      • Shi M.
      • Xu R.
      • Fu J.
      • Lv J.
      • et al.
      Human umbilical cord mesenchymal stem cells improve liver function and ascites in decompensated liver cirrhosis patients.
      • Wang L.
      • Han Q.
      • Chen H.
      • Wang K.
      • Shan G.L.
      • Kong F.
      • et al.
      Allogeneic bone marrow mesenchymal stem cell transplantation in patients with UDCA-resistant primary biliary cirrhosis.
      • Shi M.
      • Zhang Z.
      • Xu R.
      • Lin H.
      • Fu J.
      • Zou Z.
      • et al.
      Human mesenchymal stem cell transfusion is safe and improves liver function in acute-on-chronic liver failure patients.
      • Lin B.L.
      • Chen J.F.
      • Qiu W.H.
      • Wang K.W.
      • Xie D.Y.
      • Chen X.Y.
      • et al.
      Allogeneic bone marrow-derived mesenchymal stromal cells for hepatitis B virus-related acute-on-chronic liver failure: A randomized controlled trial.
      Moreover, autologous BM-derived hepatocytes were reported to improve Child-Pugh score, model for end-stage liver disease score, fatigue scale, and performance status over controls, although no comparison was made with any undifferentiated MSC transplantation groups.
      • Amer M.E.
      • El-Sayed S.Z.
      • El-Kheir W.A.
      • Gabr H.
      • Gomaa A.A.
      • El-Noomani N.
      • et al.
      Clinical and laboratory evaluation of patients with end-stage liver cell failure injected with bone marrow-derived hepatocyte-like cells.
      However, in recent animal studies, it has been reported that undifferentiated MSCs can more effectively improve liver function than MSCs differentiated into hepatocytes.
      • El Baz H.
      • Demerdash Z.
      • Kamel M.
      • Atta S.
      • Salah F.
      • Hassan S.
      • et al.
      Transplant of hepatocytes, undifferentiated mesenchymal stem cells, and in vitro hepatocyte-differentiated mesenchymal stem cells in a chronic liver failure experimental model: a comparative study.
      Jang et al. analysed the liver function improvement after repeated MSC injections at four and eight weeks.
      • Jang Y.O.
      • Kim Y.J.
      • Baik S.K.
      • Kim M.Y.
      • Eom Y.W.
      • Cho M.Y.
      • et al.
      Histological improvement following administration of autologous bone marrow-derived mesenchymal stem cells for alcoholic cirrhosis: a pilot study.
      In pilot studies, hepatic fibrosis was found to be ameliorated or reduced in six of 11 patients (54.5%) and the Child-Pugh score improved in ten patients (90.9%).
      • Jang Y.O.
      • Kim Y.J.
      • Baik S.K.
      • Kim M.Y.
      • Eom Y.W.
      • Cho M.Y.
      • et al.
      Histological improvement following administration of autologous bone marrow-derived mesenchymal stem cells for alcoholic cirrhosis: a pilot study.
      However, in the inter-group comparison (one-time injection vs. two-time injection), two-time BM-MSC transplantation was not found to improve fibrosis over a single transplantation.
      • Suk K.T.
      • Yoon J.H.
      • Kim M.Y.
      • Kim C.W.
      • Kim J.K.
      • Park H.
      • et al.
      Transplantation with autologous bone marrow-derived mesenchymal stem cells for alcoholic cirrhosis: Phase 2 trial.
      When three studies using two injection routes were analysed separately,
      • Kharaziha P.
      • Hellstrom P.M.
      • Noorinayer B.
      • Farzaneh F.
      • Aghajani K.
      • Jafari F.
      • et al.
      Improvement of liver function in liver cirrhosis patients after autologous mesenchymal stem cell injection: a phase I-II clinical trial.
      • Amer M.E.
      • El-Sayed S.Z.
      • El-Kheir W.A.
      • Gabr H.
      • Gomaa A.A.
      • El-Noomani N.
      • et al.
      Clinical and laboratory evaluation of patients with end-stage liver cell failure injected with bone marrow-derived hepatocyte-like cells.
      • El-Ansary M.
      • Mogawer S.
      • Abdel-Aziz I.
      • Abdel-Hamid S.
      Phase I trial: mesenchymal stem cells transplantation in end stage liver disease.
      the peripheral vein (PV) was found to be most commonly used as a transplantation route in 11 cases; the hepatic artery was used in four cases, intra-splenic (IS) injection was used in three cases, intrahepatic (IH) injection in one case, and portal vein in one case. There was no difference in the efficacy of MSCs based on the route of administration (PV, IS, portal vein or IH)
      • Kharaziha P.
      • Hellstrom P.M.
      • Noorinayer B.
      • Farzaneh F.
      • Aghajani K.
      • Jafari F.
      • et al.
      Improvement of liver function in liver cirrhosis patients after autologous mesenchymal stem cell injection: a phase I-II clinical trial.
      • Amer M.E.
      • El-Sayed S.Z.
      • El-Kheir W.A.
      • Gabr H.
      • Gomaa A.A.
      • El-Noomani N.
      • et al.
      Clinical and laboratory evaluation of patients with end-stage liver cell failure injected with bone marrow-derived hepatocyte-like cells.
      • El-Ansary M.
      • Mogawer S.
      • Abdel-Aziz I.
      • Abdel-Hamid S.
      Phase I trial: mesenchymal stem cells transplantation in end stage liver disease.
      and in the incidence of HCC or mortality in hepatic failure patients with hepatitis B between the autologous MSC-infused and the control groups.
      • Peng L.
      • Xie D.Y.
      • Lin B.L.
      • Liu J.
      • Zhu H.P.
      • Xie C.
      • et al.
      Autologous bone marrow mesenchymal stem cell transplantation in liver failure patients caused by hepatitis B: short-term and long-term outcomes.
      In an efficacy analysis after MSC transplantation, 15 studies reported benefits of using MSCs, but two did not.
      Table 1Clinical studies of MSCs in chronic liver diseases.
      StudyYearDesign, F/U (month)Patient cohortSource of MSCInjection routePrimary endpointMain improvement
      Mohamadnejad et al.
      • Mohamadnejad M.
      • Alimoghaddam K.
      • Mohyeddin-Bonab M.
      • Bagheri M.
      • Bashtar M.
      • Ghanaati H.
      • et al.
      Phase 1 trial of autologous bone marrow mesenchymal stem cell transplantation in patients with decompensated liver cirrhosis.
      2007Case series

      12
      Decompensated liver cirrhosis (n = 4)Autologous BMPeripheral veinSafety and feasibilityCreatinine and MELD score
      Kharaziha et al.
      • Kharaziha P.
      • Hellstrom P.M.
      • Noorinayer B.
      • Farzaneh F.
      • Aghajani K.
      • Jafari F.
      • et al.
      Improvement of liver function in liver cirrhosis patients after autologous mesenchymal stem cell injection: a phase I-II clinical trial.
      2009Cohort

      6
      Liver cirrhosis (n = 8)Autologous BMPortal vein (n = 6) Peripheral vein (n = 2)Feasibility, safety, and efficacy (LFT and MELD score)Creatinine, prothrombin time and MELD score
      El-Ansary et al.
      • El-Ansary M.
      • Mogawer S.
      • Abdel-Aziz I.
      • Abdel-Hamid S.
      Phase I trial: mesenchymal stem cells transplantation in end stage liver disease.
      2010Case control

      6
      Decompensated liver cirrhosis due to HCV or HBV (n = 12)Autologous BMIntra-splenic (n = 6)

      Peripheral vein (n = 6)
      LFT and MELD score improvementCreatinine, prothrombin time, albumin, bilirubin and MELD score
      Amer et al.
      • Amer M.E.
      • El-Sayed S.Z.
      • El-Kheir W.A.
      • Gabr H.
      • Gomaa A.A.
      • El-Noomani N.
      • et al.
      Clinical and laboratory evaluation of patients with end-stage liver cell failure injected with bone marrow-derived hepatocyte-like cells.
      2011Case control

      6
      Decompensated liver cirrhosis due to HCV (n = 40)Autologous BMIntra-splenic (n = 10)

      Intra-hepatic (n = 10)
      Safety and short-term efficacy (LFT, MELD improvement)Ascites, peripheral oedema, albumin, MELD score, and Child-Pugh score
      Peng et al.
      • Peng L.
      • Xie D.Y.
      • Lin B.L.
      • Liu J.
      • Zhu H.P.
      • Xie C.
      • et al.
      Autologous bone marrow mesenchymal stem cell transplantation in liver failure patients caused by hepatitis B: short-term and long-term outcomes.
      2011Case control

      1, 48
      ACLF caused by HBV (n = 158)Autologous BMHepatic arteryImprovement of MELD and LFT (short term) or development of HCC and mortality (long term)Prothrombin time, albumin, bilirubin and MELD score
      El-Ansary et al.
      • El-Ansary M.
      • Abdel-Aziz I.
      • Mogawer S.
      • Abdel-Hamid S.
      • Hammam O.
      • Teaema S.
      • et al.
      Phase II trial: undifferentiated vs. differentiated autologous mesenchymal stem cells transplantation in Egyptian patients with HCV induced liver cirrhosis.
      2012Case control

      6
      Decompensated liver cirrhosis due to HCV (n = 25)Autologous BMPeripheral veinImprovement of MELD and LFTAlbumin and MELD score
      Shi et al.
      • Shi M.
      • Zhang Z.
      • Xu R.
      • Lin H.
      • Fu J.
      • Zou Z.
      • et al.
      Human mesenchymal stem cell transfusion is safe and improves liver function in acute-on-chronic liver failure patients.
      2012Case control

      12 or 18
      ACLF associated HBV (n = 43)Allogeneic UCPeripheral veinLFT and MELD improvement, adverse events, and survival ratesAlbumin, prothrombin time, bilirubin, ALT, survival rates and MELD score
      Zhang et al.
      • Zhang Z.
      • Lin H.
      • Shi M.
      • Xu R.
      • Fu J.
      • Lv J.
      • et al.
      Human umbilical cord mesenchymal stem cells improve liver function and ascites in decompensated liver cirrhosis patients.
      2012Case control

      12
      Decompensated liver cirrhosis due to HBV (n = 45)Allogeneic UCPeripheral veinSafety and efficacy (LFT and MELD)Albumin, bilirubin, MELD score and ascites
      Amin et al.
      • Amin M.A.
      • Sabry D.
      • Rashed L.A.
      • Aref W.M.
      • el-Ghobary M.A.
      • Farhan M.S.
      • et al.
      Short-term evaluation of autologous transplantation of bone marrow-derived mesenchymal stem cells in patients with cirrhosis: Egyptian study.
      2013Cohort

      6
      Post-HCV

      (n = 20)
      Autologous BMIntra-splenicSafety and efficacyAlbumin, prothrombin time, bilirubin, AST, ALT and MELD score
      Mohamadnejad et al.
      • Mohamadnejad M.
      • Alimoghaddam K.
      • Bagheri M.
      • Ashrafi M.
      • Abdollahzadeh L.
      • Akhlaghpoor S.
      • et al.
      Randomized placebo-controlled trial of mesenchymal stem cell transplantation in decompensated cirrhosis.
      2013RCT

      12
      Decompensated liver cirrhosis (n = 25)

      Autologous BMPeripheral veinSafety and efficacyNone
      Wang et al.
      • Wang L.
      • Li J.
      • Liu H.
      • Li Y.
      • Fu J.
      • Sun Y.
      • et al.
      Pilot study of umbilical cord-derived mesenchymal stem cell transfusion in patients with primary biliary cirrhosis.
      2013Cohort

      12
      UDCA-resistant PBC

      (n = 7)
      Allogeneic UCPeripheral veinSafety and efficacyAlkaline phosphatase and GGT levels
      Jang et al.
      • Jang Y.O.
      • Kim Y.J.
      • Baik S.K.
      • Kim M.Y.
      • Eom Y.W.
      • Cho M.Y.
      • et al.
      Histological improvement following administration of autologous bone marrow-derived mesenchymal stem cells for alcoholic cirrhosis: a pilot study.
      2014Cohort

      6
      Alcohol related liver cirrhosis (n = 11)Autologous BMHepatic arterySafety and efficacyMELD score and liver histology
      Salama et al.
      • Salama H.
      • Zekri A.R.
      • Medhat E.
      • Al Alim S.A.
      • Ahmed O.S.
      • Bahnassy A.A.
      • et al.
      Peripheral vein infusion of autologous mesenchymal stem cells in Egyptian HCV-positive patients with end-stage liver disease.
      2014RCT

      6
      Post-HCV end-stage liver disease (n = 40)Autologous BMPeripheral veinSafety and efficacyMELD score and Child-Pugh score
      Wang et al.
      • Wang L.
      • Han Q.
      • Chen H.
      • Wang K.
      • Shan G.L.
      • Kong F.
      • et al.
      Allogeneic bone marrow mesenchymal stem cell transplantation in patients with UDCA-resistant primary biliary cirrhosis.
      2014Cohort

      12
      UDCA-resistant PBC (n = 10)Allogeneic BMPeripheral veinSafety and efficacyALT, AST, GGT and IgM
      Suk et al.
      • Suk K.T.
      • Yoon J.H.
      • Kim M.Y.
      • Kim C.W.
      • Kim J.K.
      • Park H.
      • et al.
      Transplantation with autologous bone marrow-derived mesenchymal stem cells for alcoholic cirrhosis: Phase 2 trial.
      2016RCT

      12
      Alcohol related liver cirrhosis (n = 72)Autologous BMHepatic arterySafety and efficacyHistologic fibrosis and Child-Pugh score
      Lanthier et al.
      • Lanthier N.
      • Lin-Marq N.
      • Rubbia-Brandt L.
      • Clement S.
      • Goossens N.
      • Spahr L.
      Autologous bone marrow-derived cell transplantation in decompensated alcoholic liver disease: what is the impact on liver histology and gene expression patterns?.
      2017RCT

      1
      Decompensated alcoholic hepatitis (n = 58)Autologous BMHepatic arterySafety and efficacyNone
      Lin et al.
      • Lin B.L.
      • Chen J.F.
      • Qiu W.H.
      • Wang K.W.
      • Xie D.Y.
      • Chen X.Y.
      • et al.
      Allogeneic bone marrow-derived mesenchymal stromal cells for hepatitis B virus-related acute-on-chronic liver failure: A randomized controlled trial.
      2017RCT

      6
      ACLF associated HBV (n = 110)Allogeneic BMPeripheral veinSafety and efficacyBilirubin, MELD score and survival rates
      ACLF, acute-on-chronic liver failure; ALT, alanine aminotransferase; AST, aspartate aminotransferase; BM, bone marrow; F/U, follow-up; GGT, γ-glutamyltransferase; HBV, hepatitis B virus; HCC, hepatocellular carcinoma; HCV, hepatitis C virus; LFT, liver function test; MELD, model for end-stage liver disease; MSC, mesenchymal stromal cell; PBC, primary biliary cholangitis; RCT, randomised controlled trial; UC, umbilical cord; UDCA, ursodeoxycholic acid.
      Taken together, the results of these studies suggest that MSC treatments for patients with liver disease are safe and may improve liver function, although robust randomised clinical studies are required to gain confidence with regard to the clinical efficacy of MSCs. However, to improve the efficacy of MSC therapy for liver disease, preclinical and clinical studies are necessary to standardise the best delivery route of MSCs, to optimise the sufficient number of MSCs, and to elongate the survival duration of engrafted MSCs. Furthermore, to understand the therapeutic mechanism and fate of MSCs more clearly, it is necessary to develop a specific biomarker with low toxicity so that the transplanted MSCs can be accurately tracked.

      Future perspectives

      A wide range of efforts to improve their efficacy and organ homing, including genetic engineering, enrichment, and/or priming of MSCs, can enhance the effectiveness of treating liver disease.
      Whilst conventional unmanipulated MSCs have been the mainstay of therapeutic studies thus far, there have been extensive efforts to try and enhance their efficacy. In this section we will review some of the key strategies to enhance MSC’s efficacy, including sorting MSCs to enrich for greater functionality, priming of MSCs with factors such as cytokines and, finally, genetically engineering MSCs (Fig. 3). The main driver for these approaches is to enhance efficacy and/or organ homing, although there is also often a need to create/protect intellectual property, to generate a viable business model. Therefore, the challenge is to balance the additional costs and potential logistical/safety concerns associated with such perturbations against improvements in efficacy.
      Figure thumbnail gr3
      Fig. 3Schematic diagram illustrating the future of using modified MSCs for tissue/organ regeneration. MSC, mesenchymal stromal cell.

      MSC enrichment

      MSCs represent heterogeneous populations of cells, therefore, sorting approaches are considered to be very important for achieving homogenous populations of MSCs, resulting in enriched subsets which could crucially produce various selected populations with different therapeutic functions and open new strategies for the modification of MSCs for more beneficial effects.
      MSCs are phenotypically diverse both morphologically and functionally, thus, sorting cells based on marker expression may allow for the selection of cells with greater efficacy. This requires definition of which function is being focused on, and often markers of stemness or proliferation are reported, whereas immunomodulatory action may be the most important.
      Sorting of cells for preclinical studies is relatively straightforward and can be performed using a range of modalities including flow cell sorting, which should result in high purity yields. However, it is more challenging when such approaches are attempted in clinical practice as they need to adhere more closely to good manufacturing practice (GMP) which can restrict the modality used. Clinically approved modalities such as the CliniMACS are clinically accredited but may not result in high purities of rare populations, thus, the use of GMP fluorescence cell sorting analysis is encouraging.
      CD146+ is expressed on various cell types including endothelia cells
      • Baksh D.
      • Yao R.
      • Tuan R.S.
      Comparison of proliferative and multilineage differentiation potential of human mesenchymal stem cells derived from umbilical cord and bone marrow.
      and can contribute to biological functions such as cell migration, proliferation and differentiation.
      • Tsang W.P.
      • Shu Y.
      • Kwok P.L.
      • Zhang F.
      • Lee K.K.
      • Tang M.K.
      • et al.
      CD146+ human umbilical cord perivascular cells maintain stemness under hypoxia and as a cell source for skeletal regeneration.
      • Ulrich C.
      • Abruzzese T.
      • Maerz J.K.
      • Ruh M.
      • Amend B.
      • Benz K.
      • et al.
      Human placenta-derived CD146-positive mesenchymal stromal cells display a distinct osteogenic differentiation potential.
      CD146 expression is correlated with cellular senescence of MSCs and markedly affects the proliferation, differentiation, and stemness of hUCB-MSCs. Sorted CD146+ MSCs have delayed cellular senescence, which is mediated by regulation of Bmi-1, id1, and Twist1 expression, which can regulate the cellular senescence process.
      • Jin H.J.
      • Kwon J.H.
      • Kim M.
      • Bae Y.K.
      • Choi S.J.
      • Oh W.
      • et al.
      Downregulation of melanoma cell adhesion molecule (MCAM/CD146) accelerates cellular senescence in human umbilical cord blood-derived mesenchymal stem cells.
      This suggested that CD146+ could be a novel marker responsible for control of MSC senescence, which could be used to improve the therapeutic efficacy of MSCs.
      In a recent study, sorting MSC sub-populations based on CD73+ expression has produced cells with greater self-renewal and differentiation properties.
      • Suto E.G.
      • Mabuchi Y.
      • Suzuki N.
      • Suzuki K.
      • Ogata Y.
      • Taguchi M.
      • et al.
      Prospectively isolated mesenchymal stem/stromal cells are enriched in the CD73+ population and exhibit efficacy after transplantation.
      These sorted cells (CD73+) exhibited high levels of colony forming unit ability in contrast with an absence observed with CD73- cells.
      Another study has characterised populations of MSCs using several markers, including CD271+, known as nerve growth factor receptor and proposed as a marker of BM stromal cells, adhesion molecule (CD56), and mesenchymal stem cell antigen-1 (MSCA-1+).
      • Battula V.L.
      • Treml S.
      • Bareiss P.M.
      • Gieseke F.
      • Roelofs H.
      • de Zwart P.
      • et al.
      Isolation of functionally distinct mesenchymal stem cell subsets using antibodies against CD56, CD271, and mesenchymal stem cell antigen-1.
      Sorted dual-positive MSCA-1+ and CD56+ MSCs were reported to have twofold to fourfold greater clonal efficiency than MSCA-1+ CD56-. However, MSCA-1+ CD56- were shown to have potential ability to differentiate into adipocytes, whereas MSCA-1+ CD56+ were restricted to chondrogenic and pancreatic-like cell differentiation. Similarly, other reports indicate that enrichment of synovium-derived-MSCs using CD271 in combination with THY-1 (CD90) results in greater chondrogenic differentiation ability and colony forming potential in the CFU-F assay compared to CD271+ CD90+ BM-MSCs. Thus, this combination could be a good candidate for the isolation of MSCs from different tissue sources for cartilage regeneration.
      • Ogata Y.
      • Mabuchi Y.
      • Yoshida M.
      • Suto E.G.
      • Suzuki N.
      • Muneta T.
      • et al.
      Purified human synovium mesenchymal stem cells as a good resource for cartilage regeneration.
      Sherman et al.
      • Sherman S.E.
      • Kuljanin M.
      • Cooper T.T.
      • Putman D.M.
      • Lajoie G.A.
      • Hess D.A.
      High aldehyde dehydrogenase activity identifies a subset of human mesenchymal stromal cells with vascular regenerative potential.
      have proposed aldehyde dehydrogenase (ALDH) as a marker for MSCs with an enhanced ability to contribute to revascularisation. MSCs isolated from human bone marrow and purified into ALDHhi and ALDHlo populations had identical expression of MSC surface makers and ability to differentiate into adipocytes, osteoblasts, and chondroblasts in vitro. Notably though conditioned medium from ALDHhi MSCs was shown to promote endothelial cell expansion in vitro and enhance recruitment of endogenous vascular cells after being subcutaneously implanted into NOD/SCID mice, which was mediated by upregulation of lectin.
      • Sherman S.E.
      • Kuljanin M.
      • Cooper T.T.
      • Putman D.M.
      • Lajoie G.A.
      • Hess D.A.
      High aldehyde dehydrogenase activity identifies a subset of human mesenchymal stromal cells with vascular regenerative potential.
      Positive selection based on expression of the Stro-1 specific marker has also been proposed and such MSCs are enriched with respect to CFU-F progenitors.
      • Dennis J.E.
      • Carbillet J.P.
      • Caplan A.I.
      • Charbord P.
      The STRO-1+ marrow cell population is multipotential.
      Stro-1+ expanded MSCs were reported to have better migratory capacity in various tissues than Stro-1-.
      • Bensidhoum M.
      • Chapel A.
      • Francois S.
      • Demarquay C.
      • Mazurier C.
      • Fouillard L.
      • et al.
      Homing of in vitro expanded Stro-1- or Stro-1+ human mesenchymal stem cells into the NOD/SCID mouse and their role in supporting human CD34 cell engraftment.
      Other research groups were able to increase expression of cardiovascular-related cytokines, which can be mediated using Stro-1+ enriched MSCs.
      • Psaltis P.J.
      • Paton S.
      • See F.
      • Arthur A.
      • Martin S.
      • Itescu S.
      • et al.
      Enrichment for STRO-1 expression enhances the cardiovascular paracrine activity of human bone marrow-derived mesenchymal cell populations.
      Expression of CD200 has also been used to purify MSCs,
      • Delorme B.
      • Ringe J.
      • Gallay N.
      • Le Vern Y.
      • Kerboeuf D.
      • Jorgensen C.
      • et al.
      Specific plasma membrane protein phenotype of culture-amplified and native human bone marrow mesenchymal stem cells.
      with its expression inhibiting osteoclast formation via inhibition of RANKL signalling pathways, which consequently reduce expression of osteoclast associated genes such as tartrate resistance acid phosphatase (TRAP [ACP5]) and nuclear factor of activated T cells cytoplasmic 1 (NFATC1).
      • Varin A.
      • Pontikoglou C.
      • Labat E.
      • Deschaseaux F.
      • Sensebe L.
      CD200R/CD200 inhibits osteoclastogenesis: new mechanism of osteoclast control by mesenchymal stem cells in human.
      Another study has clearly shown that CD200+ BM-MSCs can modulate the immune response of macrophages by inhibition of TNF-α secretion when compared to CD200low BM-MSCs.
      • Pietila M.
      • Lehtonen S.
      • Tuovinen E.
      • Lahteenmaki K.
      • Laitinen S.
      • Leskela H.V.
      • et al.
      CD200 positive human mesenchymal stem cells suppress TNF-alpha secretion from CD200 receptor positive macrophage-like cells.
      Consistent with their role in immunomodulation, MSCs have been shown to drive the expression of CD200 in T cell subsets following co-culture.
      • Najar M.
      • Raicevic G.
      • Jebbawi F.
      • De Bruyn C.
      • Meuleman N.
      • Bron D.
      • et al.
      Characterization and functionality of the CD200-CD200R system during mesenchymal stromal cell interactions with T-lymphocytes.
      This upregulation was reported in both CD4+ and CD8+ T lymphocytes.
      More recently, CD362+ (Syndecan-2) has been identified as a novel marker for selecting a homogeneous population of MSCs with enhanced immunomodulatory properties (patent number WO 20131177661 A1). This marker has recently been investigated for its ability to reduce immunogenicity and enhance the immunomodulatory ability of MSCs in liver inflammation.
      • de Witte S.F.H.
      • Merino A.M.
      • Franquesa M.
      • Strini T.
      • van Zoggel J.A.A.
      • Korevaar S.S.
      • et al.
      Cytokine treatment optimises the immunotherapeutic effects of umbilical cord-derived MSC for treatment of inflammatory liver disease.
      • de Witte S.F.
      • Franquesa M.
      • Baan C.C.
      • Hoogduijn M.J.
      Toward development of imesenchymal stem cells for immunomodulatory therapy.
      Syndecan-2 was found to be expressed in haematopoietic cells and myeloid cells,
      • Teixe T.
      • Nieto-Blanco P.
      • Vilella R.
      • Engel P.
      • Reina M.
      • Espel E.
      Syndecan-2 and -4 expressed on activated primary human CD4+ lymphocytes can regulate T cell activation.
      and functionally reported to upregulate upon T cell activation and play a significant role in CD3 downregulation, through degradation of the T cell receptor (TCR).
      • Rovira-Clave X.
      • Angulo-Ibanez M.
      • Noguer O.
      • Espel E.
      • Reina M.
      Syndecan-2 can promote clearance of T-cell receptor/CD3 from the cell surface.
      These findings strongly suggest that enrichment of syndecan-2 expression in MSCs could play an essential role in immune modulation in injured tissue.
      The potential benefits of the various markers that have been used to select/enrich MSCs are detailed (Table 2).
      Table 2Reported markers for selection and purification of MSCs.
      MSC sourceSpeciesMarkers expressedPurification/selection methodsExperimental modelsTarget/mechanismRefs.
      BMHumanCD271+ and CD56+Cell sortingIn vitro
      • Increase clonogenic and proliferation potential.
      • Increase chondrocyte and pancreatic like cells differentiation.
      • Tsang W.P.
      • Shu Y.
      • Kwok P.L.
      • Zhang F.
      • Lee K.K.
      • Tang M.K.
      • et al.
      CD146+ human umbilical cord perivascular cells maintain stemness under hypoxia and as a cell source for skeletal regeneration.
      BMRatCD73+Cell sortingIn vitro

      Lewis rats
      • Enhance self-renewal and differentiation.
      • Increase engraftment.
      • Baksh D.
      • Yao R.
      • Tuan R.S.
      Comparison of proliferative and multilineage differentiation potential of human mesenchymal stem cells derived from umbilical cord and bone marrow.
      BMHumanCD200+Magnetic separationIn vitro
      • Enhance regulation of bone resorption.
      • Inhibit osteoclast formation via inhibition of RANKL signaling pathway.
      • Dennis J.E.
      • Carbillet J.P.
      • Caplan A.I.
      • Charbord P.
      The STRO-1+ marrow cell population is multipotential.
      BMHumanCD200+Cell sortingIn vitro
      • Suppress TNF-α secretion in macrophage like cells (Immunosuppressive activity).
      • Bensidhoum M.
      • Chapel A.
      • Francois S.
      • Demarquay C.
      • Mazurier C.
      • Fouillard L.
      • et al.
      Homing of in vitro expanded Stro-1- or Stro-1+ human mesenchymal stem cells into the NOD/SCID mouse and their role in supporting human CD34 cell engraftment.
      UP, AT, and BMHumanαSMA+FACSIn vitro
      • Improve MSC fate through regulation of YAP/TAZ activation.
      • Talele N.P.
      • Fradette J.
      • Davies J.E.
      • Kapus A.
      • Hinz B.
      Expression of alpha-smooth muscle actin determines the fate of mesenchymal stromal cells.
      PAMHumanCD34+FACSTAA (liver fibrosis model)
      • Reduce hepatic fibrosis and restore liver function by reduce collagen level and deactivate the hepatic stellate cells.
      • Lee P.H.
      • Tu C.T.
      • Hsiao C.C.
      • Tsai M.S.
      • Ho C.M.
      • Cheng N.C.
      • et al.
      Antifibrotic activity of human placental amnion membrane-derived CD34+ mesenchymal stem/progenitor cell transplantation in mice with thioacetamide-induced liver injury.
      BMHumanCD271+Magnetic separationIn vitro (model of wound healing)
      • Significant potential in wound healing.
      • Latifi-Pupovci H.
      • Kuci Z.
      • Wehner S.
      • Bonig H.
      • Lieberz R.
      • Klingebiel T.
      • et al.
      In vitro migration and proliferation (“wound healing”) potential of mesenchymal stromal cells generated from human CD271(+) bone marrow mononuclear cells.
      UCBHumanCD 146+FACSIn vitro
      • Reduce MSC senescence.
      • Jin H.J.
      • Kwon J.H.
      • Kim M.
      • Bae Y.K.
      • Choi S.J.
      • Oh W.
      • et al.
      Downregulation of melanoma cell adhesion molecule (MCAM/CD146) accelerates cellular senescence in human umbilical cord blood-derived mesenchymal stem cells.
      BMHumanALDHFACSIn vitro and in vivo (NOD/SCID mice)
      • Promote endothelial cell expansion.
      • Enhance recruitment of endogenous vascular cells in vivo by upregulation of lectin.
      • Psaltis P.J.
      • Paton S.
      • See F.
      • Arthur A.
      • Martin S.
      • Itescu S.
      • et al.
      Enrichment for STRO-1 expression enhances the cardiovascular paracrine activity of human bone marrow-derived mesenchymal cell populations.
      SYN and BMHumanLNGFR and THY-1FACSIn vitro
      • Greater chondrogenic differentiation ability and colony forming potential than BM-MSC.
      • Ogata Y.
      • Mabuchi Y.
      • Yoshida M.
      • Suto E.G.
      • Suzuki N.
      • Muneta T.
      • et al.
      Purified human synovium mesenchymal stem cells as a good resource for cartilage regeneration.
      UCHumanC362+ (Syndecan-2)FACSALF
      • Improve immunomodulatory properties and clonogenicity.
      • de Witte S.F.H.
      • Merino A.M.
      • Franquesa M.
      • Strini T.
      • van Zoggel J.A.A.
      • Korevaar S.S.
      • et al.
      Cytokine treatment optimises the immunotherapeutic effects of umbilical cord-derived MSC for treatment of inflammatory liver disease.
      BMHumanSTRO-1FACSIn vitro
      • Increase expression of cardiovascular relate cytokines.
      • Varin A.
      • Pontikoglou C.
      • Labat E.
      • Deschaseaux F.
      • Sensebe L.
      CD200R/CD200 inhibits osteoclastogenesis: new mechanism of osteoclast control by mesenchymal stem cells in human.
      αSMA, α-smooth muscle actin; ALDH, aldehyde dehydrogenase; ALF, acute liver failure; AT, adipose tissue; BM, bone marrow; FACS, fluorescence-activated cell sorting; LNGFR, low-affinity nerve growth factor receptor; MSC, mesenchymal stromal cell; PAM, placenta amnion membrane; RANKL, receptor activator of nuclear factor kappa-B ligand; Stro-1, stromal precursor antigen-1; SYN, synovium; TAA, thioacetamide; UC, umbilical cord; UCB, umbilical cord blood; UP, umbilical perivascular.

      MSC priming

      As with selection of MSCs, priming of cells before use is intended to enhance their biological properties for whichever clinical indication is being considered (Table 3). This may include improvements in MSC immunomodulatory effects, homing to injured organs and/or greater expansion of cells.
      Table 3Reported factors and their effect in priming of MSCs for tissue repair.
      MSC sourceMolecule nameTime of treatmentBiological functionRefs.
      Human BM

      Human AT
      IL-1β, IL-23, IL-696 h
      • Enhance secretion of TGF-β.
      • Reduce production of IL-4.
      • Exhibit significance multi-lineage. Differentiation capacity.
      • Teixe T.
      • Nieto-Blanco P.
      • Vilella R.
      • Engel P.
      • Reina M.
      • Espel E.
      Syndecan-2 and -4 expressed on activated primary human CD4+ lymphocytes can regulate T cell activation.
      Human BMIL-124 h
      • Increase production of G-CSF.
      • Increase production of IL-10.
      • Rovira-Clave X.
      • Angulo-Ibanez M.
      • Noguer O.
      • Espel E.
      • Reina M.
      Syndecan-2 can promote clearance of T-cell receptor/CD3 from the cell surface.
      Human BMIFN-γ and TNF-α24 h
      • Enhance osteogenic formation via expression of ALP.
      • Increase expression of bone matrix proteins.
      • Redondo-Castro E.
      • Cunningham C.
      • Miller J.
      • Martuscelli L.
      • Aoulad-Ali S.
      • Rothwell N.J.
      • et al.
      Interleukin-1 primes human mesenchymal stem cells towards an anti-inflammatory and pro-trophic phenotype in vitro.
      Human UCIFN-γ, TGF-β, or multiple cytokine cocktail (IFN-γ, TGF-β, and retinoic acid)72 h
      • Multiple cytokines cocktails improve the immunomodulatory properties of MSC.
      • TGF-β treated MSC increased recruitment of MSC to the liver injury in vivo.
      • de Witte S.F.H.
      • Merino A.M.
      • Franquesa M.
      • Strini T.
      • van Zoggel J.A.A.
      • Korevaar S.S.
      • et al.
      Cytokine treatment optimises the immunotherapeutic effects of umbilical cord-derived MSC for treatment of inflammatory liver disease.
      Mouse BMIFN-γ + TNF-α with IL-1712 h
      • Mediate liver injury through activation of iNOS.
      • Duijvestein M.
      • Wildenberg M.E.
      • Welling M.M.
      • Hennink S.
      • Molendijk I.
      • van Zuylen V.L.
      • et al.
      Pretreatment with interferon-gamma enhances the therapeutic activity of mesenchymal stromal cells in animal models of colitis.
      Human BMIL-1724, 48, and 120 h.
      • Induction regulatory T cells.
      • Inhibition of Th1 cytokines.
      • Enhance production of IL-6.
      • Linero I.
      • Chaparro O.
      Paracrine effect of mesenchymal stem cells derived from human adipose tissue in bone regeneration.
      Mouse BMIL-624 h
      • Improve viability of hepatocytes treated with CCL4.
      • Decreased expression of pro-apoptotic markers (BAX, caspase-3, and LDH).
      • Reduced liver fibrosis in vivo.
      • Polchert D.
      • Sobinsky J.
      • Douglas G.
      • Kidd M.
      • Moadsiri A.
      • Reina E.
      • et al.
      IFN-gamma activation of mesenchymal stem cells for treatment and prevention of graft vs. host disease.
      Mouse BMIFN-γ or (TNF-α and IL-1)Not stated
      • Increase upregulation of ICAM and VCAM.
      • Han X.
      • Yang Q.
      • Lin L.
      • Xu C.
      • Zheng C.
      • Chen X.
      • et al.
      Interleukin-17 enhances immunosuppression by mesenchymal stem cells.
      Mouse BM(IFN-γ + TNF-α + IL-1α) or (IL-1β + IFN-γ)24 h
      • Increase ability of MSC to inhibit T cell proliferations.
      • Enhance secretion of chemokines such as CXCL-9 and CXCL-10.
      • Ren G.
      • Zhang L.
      • Zhao X.
      • Xu G.
      • Zhang Y.
      • Roberts A.I.
      • et al.
      Mesenchymal stem cell-mediated immunosuppression occurs via concerted action of chemokines and nitric oxide.
      Mouse BMCXCL930 min
      • Ameliorate the adhesion of MSC to murine endothelial cells.
      • Sivanathan K.N.
      • Rojas-Canales D.M.
      • Hope C.M.
      • Krishnan R.
      • Carroll R.P.
      • Gronthos S.
      • et al.
      Interleukin-17A-induced human mesenchymal stem cells are superior modulators of immunological function.
      ALP, alkaline phosphatase; AT, adipose tissue; BAX, BCL2 associated X, apoptosis regulator; BM, bone marrow; CCl4, carbon tetrachloride; CXCL, C-X-C motif ligand; G-CSF, granulocyte-colony stimulating factor; ICAM, intercellular adhesion molecule; IFN, interferon; IL, interleukin; iNOS, inducible nitric oxide synthase; LDH, lactate dehydrogenase; MSC, mesenchymal stromal cell; TGF-β, transforming growth factor-β; TNF-α, tumour necrosis factor-α; UC, umbilical cord; VCAM, vascular cell adhesion protein.

      Enhancing immunomodulatory properties of MSCs

      Pre-treatment of MSCs with the pro-inflammatory cytokines IL-1β, IL-23 and IL-6 for 96 h
      • Pourgholaminejad A.
      • Aghdami N.
      • Baharvand H.
      • Moazzeni S.M.
      The effect of pro-inflammatory cytokines on immunophenotype, differentiation capacity and immunomodulatory functions of human mesenchymal stem cells.
      was found to enhance secretion of TGF-β and reduce production of IL-4 by MSCs, although, notably, no changes were reported in production of IFN-γ and TNF-α. In addition, cytokine-treated MSCs exhibited superior multi-lineage differentiation capacity compared to untreated MSCs, with no associated changes in their morphology. IL-1 appears to be important for pre-conditioning of MSCs, as combined treatment with IL-1α and IL-1β increases production of granulocyte-colony stimulating factor (G-CSF) and secretion of anti-inflammatory mediators such as IL-10. Moreover, microglial cells incubated with conditioned medium from IL-1 primed MSCs increase expression of anti-inflammatory cytokines such as IL10 and decrease secretion of pro-inflammatory cytokines as reported in TNF-α and IL-6.
      • Redondo-Castro E.
      • Cunningham C.
      • Miller J.
      • Martuscelli L.
      • Aoulad-Ali S.
      • Rothwell N.J.
      • et al.
      Interleukin-1 primes human mesenchymal stem cells towards an anti-inflammatory and pro-trophic phenotype in vitro.
      Duijvestein et al.
      • Duijvestein M.
      • Wildenberg M.E.
      • Welling M.M.
      • Hennink S.
      • Molendijk I.
      • van Zuylen V.L.
      • et al.
      Pretreatment with interferon-gamma enhances the therapeutic activity of mesenchymal stromal cells in animal models of colitis.
      showed that stimulating MSCs with IFN-γ enhanced the anti-inflammatory response of MSCs in an experimental animal model of colitis. In addition, IFN-γ primed MSCs were shown to significantly reduce TNF-α and IL-6 in colon homogenates, while normal MSCs had no effect. In the same model, activation of MSCs with IFN-γ further promoted immunomodulation via enhanced production of IL-17 and IL-4, which therefore inhibit Th1 and reduce T cell activation.
      • Duijvestein M.
      • Wildenberg M.E.
      • Welling M.M.
      • Hennink S.
      • Molendijk I.
      • van Zuylen V.L.
      • et al.
      Pretreatment with interferon-gamma enhances the therapeutic activity of mesenchymal stromal cells in animal models of colitis.
      Under similar conditions, pre-stimulation of BM-MSCs with IFN-γ and TNF-α stimulate production of IL-6, HGF, TGF-β.
      • Linero I.
      • Chaparro O.
      Paracrine effect of mesenchymal stem cells derived from human adipose tissue in bone regeneration.
      More interestingly, administration of MSCs pre-treated with IFN-γ could enhance survival rates of mice with GVHD, resulting in 100% survival in an in vivo GVHD model.
      • Polchert D.
      • Sobinsky J.
      • Douglas G.
      • Kidd M.
      • Moadsiri A.
      • Reina E.
      • et al.
      IFN-gamma activation of mesenchymal stem cells for treatment and prevention of graft vs. host disease.
      More recently, data from de Witte and colleagues have demonstrated that pre-treatment of UC-MSCs with different treatments such as TGF-β, IFN-γ, IFN-β or in combinations (TGF-β, IFN-γ and retinoic acid) suppress expression of CD107a on NK cells, enhancing MSC immunomodulation. In addition, MSCs treated with IFN-γ and the multiple cytokine combination were found to significantly upregulate IDO activities, which subsequently suppressed CD4 and CD8 proliferation when compared to untreated MSCs. Notably, following infusion into mice injured with a single dose of CCl4, a higher percentage of TGF-β treated MSCs homed to the injured liver (25%) compared with untreated MSCs (13%).
      • de Witte S.F.H.
      • Merino A.M.
      • Franquesa M.
      • Strini T.
      • van Zoggel J.A.A.
      • Korevaar S.S.
      • et al.
      Cytokine treatment optimises the immunotherapeutic effects of umbilical cord-derived MSC for treatment of inflammatory liver disease.
      In other liver injury studies, IL-7 treated MSCs had a superior therapeutic effect on liver injury mediated in part through increased activation of iNOS. IL-17 down-regulates gene expression of ARE/poly(U)-binding/degradation factor 1 (AUF-1) in MSCs, which is a protein known to regulate immune-related molecules
      • Han X.
      • Yang Q.
      • Lin L.
      • Xu C.
      • Zheng C.
      • Chen X.
      • et al.
      Interleukin-17 enhances immunosuppression by mesenchymal stem cells.
      and has a key role in regulating stromal cell fate.
      • Chenette D.M.
      • Cadwallader A.B.
      • Antwine T.L.
      • Larkin L.C.
      • Wang J.
      • Olwin B.B.
      • et al.
      Targeted mRNA decay by RNA binding protein AUF1 regulates adult muscle stem cell fate, promoting skeletal muscle integrity.
      Thus, a novel role of AUF1 could be enhancing the effect of IL-17 on immunosuppression. Similarly, IL-17a modified MSCs have been reported to suppress proliferation of T cells in vitro, via mechanisms such as inhibition of Th1 cytokines (IFN-γ, TNF-α, IL-10, and IL-2), enhanced production of IL-6 and induction of regulatory T cells.
      • Sivanathan K.N.
      • Rojas-Canales D.M.
      • Hope C.M.
      • Krishnan R.
      • Carroll R.P.
      • Gronthos S.
      • et al.
      Interleukin-17A-induced human mesenchymal stem cells are superior modulators of immunological function.
      IL-6 priming of MSCs infused into an acute model of CCl4 injury resulted in improved viability of isolated hepatocytes, as well as a reduction in expression of pro-apoptotic markers such as BAX, Caspase-3 and LDH activities. This finding was not observed when MSCs or IL-6 treatment were applied alone.
      • Nasir G.A.
      • Mohsin S.
      • Khan M.
      • Shams S.
      • Ali G.
      • Khan S.N.
      • et al.
      Mesenchymal stem cells and Interleukin-6 attenuate liver fibrosis in mice.
      In addition, administration of IL-6 with MSCs was found to enhance repair of liver injury in a mouse model of liver fibrosis, with reductions in fibrosis, improvements in liver synthetic function, promotion of hepatocyte survival, and decreased apoptosis in fibrotic liver.
      • Nasir G.A.
      • Mohsin S.
      • Khan M.
      • Shams S.
      • Ali G.
      • Khan S.N.
      • et al.
      Mesenchymal stem cells and Interleukin-6 attenuate liver fibrosis in mice.

      Enhancing homing of MSCs

      A study demonstrated that adhesion molecules such as intercellular adhesion molecule (ICAM) and vascular cell adhesion molecule (VCAM) can be highly expressed on MSCs following priming with a combination of IFN-γ, TNF-α and IL-1. This upregulation of expression of ICAM and VCAM led to increased recruiting of MSCs to vascular endothelium, this close contact of MSCs with immune cells could enhance the immunosuppressive properties of MSCs.
      • Ren G.
      • Zhao X.
      • Zhang L.
      • Zhang J.
      • L'Huillier A.
      • Ling W.
      • et al.
      Inflammatory cytokine-induced intercellular adhesion molecule-1 and vascular cell adhesion molecule-1 in mesenchymal stem cells are critical for immunosuppression.
      • Ren G.
      • Roberts A.I.
      • Shi Y.
      Adhesion molecules: key players in Mesenchymal stem cell-mediated immunosuppression.
      Similarly, MSCs pre-treated with IFN-γ and TNF-α can induce regulatory T cells more efficiently than non-treated MSCs. Furthermore, MSCs pre-incubated with IFN-γ and TNF-α induced secretion of CCR6 and therefore increased the adhesion of Th17 cells to MSCs, promoting the generation of regulatory T cells (FOXP3+ cells) from Th17 cells and consequently improving their immunosuppressive properties.
      • Ghannam S.
      • Pene J.
      • Moquet-Torcy G.
      • Jorgensen C.
      • Yssel H.
      Mesenchymal stem cells inhibit human Th17 cell differentiation and function and induce a T regulatory cell phenotype.
      Priming with CXCL9 has also been shown to enhance adherence of MSCs to endothelial cells and to increase spreading of MSCs on the endothelial cells, as characterised by the extension of pseudopodia in multiple directions.
      • Chamberlain G.
      • Smith H.
      • Rainger G.E.
      • Middleton J.
      Mesenchymal stem cells exhibit firm adhesion, crawling, spreading and transmigration across aortic endothelial cells: effects of chemokines and shear.
      Further characterisation of the beneficial effect of chemokines on MSC behaviour was reported in the same study using trans-well migration experiments, in which MSCs migrated across endothelial layers in the presence of chemokines such as CXCL9, CXCL16, and CXCL20, and CXCL25. Of note, no migration was observed in the presence of TNF-α alone.

      Genetic modification of MSCs (Gene editing)

      Beside enrichment and priming MSCs in vitro, transplantation of MSCs after genetic correction or modification (gene editing) represents a powerful approach for regenerative medicine (Table 4). This section will review progress with genetic engineering approaches reported with MSCs, including viral and non-viral manipulations. Viral transfection of MSCs can be achieved with several approaches including lentiviral, adenoviral and retroviral.
      • Park J.S.
      • Suryaprakash S.
      • Lao Y.H.
      • Leong K.W.
      Engineering mesenchymal stem cells for regenerative medicine and drug delivery.
      Table 4Genetically modified MSCs.
      MSC sourceExample of associated genesConditionViral vectorRepresentative biological activitiesRefs.
      Mouse BMIGF-1 overexpressionLiver cirrhosisAdenovirus
      • Ameliorate liver fibrosis by significant reduction in αSMA, collagen deposition, and TGF-β1.
      • Fiore E.J.
      • Bayo J.M.
      • Garcia M.G.
      • Malvicini M.
      • Lloyd R.
      • Piccioni F.
      • et al.
      Mesenchymal stromal cells engineered to produce IGF-I by recombinant adenovirus ameliorate liver fibrosis in mice.
      Mouse BMLet-7a knockdownIBD and GVHDsiRNA
      • Significant improvement in both models, by suppress T cell proliferation (decreased in CD3+), increase MCP-1 secretion, and enhancing expression of Fasl/Fas.
      • Yu Y.
      • Liao L.
      • Shao B.
      • Su X.
      • Shuai Y.
      • Wang H.
      • et al.
      Knockdown of MicroRNA Let-7a improves the functionality of bone marrow-derived mesenchymal stem cells in immunotherapy.
      Human BMCXCR5 overexpressionCHSLentiviral
      • Increase migration and engraftment of MSC to the site of injury.
      • Enhance immunomodulatory effects of MSC in vivo through inhibit of T cell proliferation and supress production of IFN-γ and IL-17.
      • Zhang X.
      • Huang W.
      • Chen X.
      • Lian Y.
      • Wang J.
      • Cai C.
      • et al.
      CXCR5-overexpressing mesenchymal stromal cells exhibit enhanced homing and can decrease contact hypersensitivity.
      Human BMCXCR4 overexpressionALFLentiviral
      • Enhance migration and improve liver regeneration.
      • Hamedi-Asl P.
      • Halabian R.
      • Bahmani P.
      • Mohammadipour M.
      • Mohammadzadeh M.
      • Roushandeh A.M.
      • et al.
      Adenovirus-mediated expression of the HO-1 protein within MSCs decreased cytotoxicity and inhibited apoptosis induced by oxidative stresses.
      Rat BMCXCR4 overexpressionLung injuryLentiviral
      • Improve migration and suppress inflammation of lung tissue by upregulation of IL-10 and downregulation of TNF-α.
      • Tsubokawa T.
      • Yagi K.
      • Nakanishi C.
      • Zuka M.
      • Nohara A.
      • Ino H.
      • et al.
      Impact of anti-apoptotic and anti-oxidative effects of bone marrow mesenchymal stem cells with transient overexpression of heme oxygenase-1 on myocardial ischemia.
      Rat ATmiR-27b overexpressionPartial hepatectomyMicro RNA
      • Enhance liver regeneration through reduction in ALT, TNF-α, and IL-6 in serum.
      • Reduce expression of TGF-β, MMP2, and MMP9.
      • Chen K.D.
      • Huang K.T.
      • Lin C.C.
      • Weng W.T.
      • Hsu L.W.
      • Goto S.
      • et al.
      MicroRNA-27b enhances the hepatic regenerative properties of adipose-derived mesenchymal stem cells.
      Rat MBCAMKK1 over expressionAMIsiRNA
      • Reduce scar formation and improve cardiac function in vivo.
      • Dong F.
      • Patnaik S.
      • Duan Z.H.
      • Kiedrowski M.
      • Penn M.S.
      • Mayorga M.E.
      A novel role for CAMKK1 in the regulation of the mesenchymal stem cell secretome.
      Porcine ATMMP-2 and MMP-14 knockdownIn vitrosiRNA
      • Enhance differentiation of MSC into endothelia cells by production of PECAM and V-cadherin.
      • Increase the formation of capillary like cells and Sc-LDL uptake.
      • Almalki S.G.
      • Llamas Valle Y.
      • Agrawal D.K.
      MMP-2 and MMP-14 silencing inhibits VEGFR2 cleavage and induces the differentiation of porcine adipose-derived mesenchymal stem cells to endothelial cells.
      Human BMHO-1 overexpressionIn vitroAdenovirus
      • Enhance MSC survival and resistant to oxidative stress.
      • Enhanced anti-apoptotic and anti-oxidative capabilities of MSC
      • Hamedi-Asl P.
      • Halabian R.
      • Bahmani P.
      • Mohammadipour M.
      • Mohammadzadeh M.
      • Roushandeh A.M.
      • et al.
      Adenovirus-mediated expression of the HO-1 protein within MSCs decreased cytotoxicity and inhibited apoptosis induced by oxidative stresses.
      Rat BMHO-1 overexpressionMIPlasmid
      • Enhanced anti-apoptotic and anti-oxidative properties and improved angiogenesis level.
      • Tsubokawa T.
      • Yagi K.
      • Nakanishi C.
      • Zuka M.
      • Nohara A.
      • Ino H.
      • et al.
      Impact of anti-apoptotic and anti-oxidative effects of bone marrow mesenchymal stem cells with transient overexpression of heme oxygenase-1 on myocardial ischemia.
      Human BMHGF overexpressionLiver fibrosis (DMN model)Adenovirus
      • Promote liver function and reduce liver fibrosis via significant reduction in TGF-β and PDGF-bb.
      • Kim M.D.
      • Kim S.S.
      • Cha H.Y.
      • Jang S.H.
      • Chang D.Y.
      • Kim W.
      • et al.
      Therapeutic effect of hepatocyte growth factor-secreting mesenchymal stem cells in a rat model of liver fibrosis.
      Mouse BMCOUP-TF1 knockdownStreptozocin-induced diabetic micesiRNA
      • Increase ability of BM-MSC to differentiate into IPCs.
      • Zhang T.
      • Li X.H.
      • Zhang D.B.
      • Liu X.Y.
      • Zhao F.
      • Lin X.W.
      • et al.
      Repression of COUP-TFI improves bone marrow-derived mesenchymal stem cell differentiation into insulin-producing cells.
      Rat BMAqp1 overexpressionTibia fracture ModelLentiviral
      • Enhance MSC migration in vitro and in vivo through modulation expression of FAK and β-catenin.
      • Meng F.
      • Rui Y.
      • Xu L.
      • Wan C.
      • Jiang X.
      • Li G.
      Aqp1 enhances migration of bone marrow mesenchymal stem cells through regulation of FAK and beta-catenin.
      αSMA, α-smooth muscle actin; AF, amniotic fluid; ALF, acute liver failure; ALT, alanine aminotransferase; AMI, acute myocardial infarction; Aqp1, aquaporin 1; AT, adipose tissue; BM, bone marrow; CAMKK1, calcium/calmodulin-dependent protein kinase kinase-1; CHS, contact hypersensitivity; COUP-TFI, chicken ovalbumin upstream promoter transcriptional factor I; CXCR, C-X-C motif chemokine receptor; DMN, Di-methylnitrosamine; FAK, focal adhesion kinase; GVHD, graft vs. host disease; HGF, hepatocyte growth factor; HO-1, heme oxygenase-1; IBD, inflammatory bowel disease; IGF-1, insulin growth factor like-1; IL, interleukin; IPCs, insulin producing cells; LDL, low-density lipoprotein; MI, myocardial infarction; MMPs, matrix metalloproteinases; MSC, mesenchymal stromal cell; PDGF-bb, platelet-derived growth factor-bb; PECAM, platelet endothelial cell adhesion molecule; TGF-β, transforming growth factor-β; TNF-α, tumour necrosis factor-α.
      MSCs have also been genetically modified to increase expression of CXCR4, thereby improving their homing to the injured liver and reducing liver damage.
      • Ma H.C.
      • Shi X.L.
      • Ren H.Z.
      • Yuan X.W.
      • Ding Y.T.
      Targeted migration of mesenchymal stem cells modified with CXCR4 to acute failing liver improves liver regeneration.
      Similarly, the same finding was reported in a rat model of lung injury, with increased expression of CXCR4 on MSCs resulting in enhanced hepatic migration and improvement of their immunomodulatory properties, mediated by increased production of IL-10 and reduction in TNF-α. Notably, these findings suggest that overexpression of CXCR4 not only enhanced MSCs homing but also increased their immunosuppressive effects.
      • Yang J.X.
      • Zhang N.
      • Wang H.W.
      • Gao P.
      • Yang Q.P.
      • Wen Q.P.
      CXCR4 receptor overexpression in mesenchymal stem cells facilitates treatment of acute lung injury in rats.
      Further examination of the beneficial effects of genetically modified MSCs were reported in a mouse model of liver fibrosis, following overexpression of insulin growth factor like-1 (IGF-1).
      • Sobrevals L.
      • Enguita M.
      • Quiroga J.
      • Prieto J.
      • Fortes P.
      Insulin-like growth factor I (IGF-I) expressed from an AAV1 vector leads to a complete reversion of liver cirrhosis in rats.
      After systemic administration, IGF-1 modified MSCs were able to significantly reduce the degree of fibrosis, likely through the down regulation of α-SMA, TGF-β and COL1A2 in animals treated with IGF-1 MSCs compared to animals treated with normal MSCs.
      • Fiore E.J.
      • Bayo J.M.
      • Garcia M.G.
      • Malvicini M.
      • Lloyd R.
      • Piccioni F.
      • et al.
      Mesenchymal stromal cells engineered to produce IGF-I by recombinant adenovirus ameliorate liver fibrosis in mice.
      Overexpression of HGF in MSCs was also found to reduce liver fibrosis, seemingly mediated by a reduction in TFG-β, platelet-derived growth factor-bb (PDGF-bb), and metalloprotease-14 (MMP-14).
      • Kim M.D.
      • Kim S.S.
      • Cha H.Y.
      • Jang S.H.
      • Chang D.Y.
      • Kim W.
      • et al.
      Therapeutic effect of hepatocyte growth factor-secreting mesenchymal stem cells in a rat model of liver fibrosis.
      MSCs overexpressing HGF also act on HSCs to reduce α-SMA and desmin expression, indicating that MSCs that overexpress HGF decreased both the activation and number of HSCs more than normal MSCs. This could have a therapeutic effect by preventing disease progression and fostering liver restoration.
      Another reprogramming approach showed that overexpression of miR-27b in AT derived MSCs resulted in reduction of alanine aminotransferase, aspartate aminotransferase, TNF-α, and IL-6, as well as significant suppression of TGF-β in a rat model of ischaemic liver injury.
      • Chen K.D.
      • Huang K.T.
      • Lin C.C.
      • Weng W.T.
      • Hsu L.W.
      • Goto S.
      • et al.
      MicroRNA-27b enhances the hepatic regenerative properties of adipose-derived mesenchymal stem cells.
      Moreover, these transfected cells were shown to have anti-fibrotic ability with suppression of MMP-2 and MMP-9 in liver tissue.
      Further studies linked the genetic modifications of MSCs with their capacity to undertake endothelial cell (EC) properties. For example, silencing MMP-2 and MMP-14 with endothelial growth medium can induce MSC differentiation into EC by enhancing production of endothelial markers, such as platelet and endothelial cell adhesion molecule (PECAM1) and vascular endothelial-cadherin (CDH5). These markers were increased from 4 to 15% and from 4 to 30% after silencing MMP-2 and MMP-12, respectively. This observation was in comparison with MSCs treated with endothelial growth medium only.
      • Almalki S.G.
      • Llamas Valle Y.
      • Agrawal D.K.
      MMP-2 and MMP-14 silencing inhibits VEGFR2 cleavage and induces the differentiation of porcine adipose-derived mesenchymal stem cells to endothelial cells.
      In other work, the expression level of heme oxgenase 1 (HO-1 [HMOX1]) was genetically modified in MSCs and shown to increase MSCs resistance to cell death under oxidative stress conditions and enhance their anti-apoptotic properties.
      • Hamedi-Asl P.
      • Halabian R.
      • Bahmani P.
      • Mohammadipour M.
      • Mohammadzadeh M.
      • Roushandeh A.M.
      • et al.
      Adenovirus-mediated expression of the HO-1 protein within MSCs decreased cytotoxicity and inhibited apoptosis induced by oxidative stresses.
      Moreover, more MSCs overexpressing HO-1 survived following exposure to H2O2 and hypoxia, indicating that HO-1 may shape the stress responsive and cytoprotective properties of MSCs. Notably, in the murine model of myocardial infarction, overexpression of HO-1 resulted in diminished oxidative stress and apoptosis, as well as an enhanced effect on angiogenesis. This was associated with a 2.1-fold upregulation of vascular endothelial growth factor levels compared to normal MSCs.
      • Tsubokawa T.
      • Yagi K.
      • Nakanishi C.
      • Zuka M.
      • Nohara A.
      • Ino H.
      • et al.
      Impact of anti-apoptotic and anti-oxidative effects of bone marrow mesenchymal stem cells with transient overexpression of heme oxygenase-1 on myocardial ischemia.

      Conclusions and outlook

      To increase the reliability of the clinical efficacy of MSCs, further robust randomised clinical studies are required.
      In addition, further studies are needed to determine the optimal route of delivery, sufficient number of MSCs and prolonged survival of engrafted MSCs, along with studies to increase their efficacy and organ homing.
      MSC therapy is generally regarded as a safe and potentially relevant therapeutic strategy for patients with chronic liver disease, including ACLF, liver failure, and cirrhosis due to alcohol, HBV, or HCV, and primary biliary cholangitis. However, in order for MSC therapy to be established as a clinical option for these liver diseases, further robust randomised clinical studies are required to increase the reliability of the clinical efficacy of MSCs. In addition, further studies on optimal delivery route, sufficient number of MSCs, and extension of survival of engrafted MSCs are needed to enhance the efficacy of MSC therapy. However, several concerns remain, including the low migration and fibrogenic potential of MSCs, the optimal sources, and the risk of oncogenesis and viral transmission. Whilst, conventional unmanipulated MSCs have constituted the mainstream of therapeutic clinical studies so far, there have been extensive efforts to enhance their efficacy, including enrichment and/or priming of MSCs along with genetic engineering of cells. The main driver for these approaches is to enhance efficacy and/or organ homing, although there is also often a need to create/protect intellectual property, to generate a viable business model, while also balancing the additional costs and potential safety issues against enhanced efficacy.

      Financial support

      MA is supported by a grant from the Saudi Arabian Cultural Bureau. PNN is supported by the National Institute of Health Research (NIHR) Birmingham Liver Biomedical Research Unit (BRU). The views expressed are those of the authors and not necessarily reflect those of the NHS, the NIHR or the Department of Health.
      YWE and SKB were supported by a grant of the Korea Health Technology R&D Project through the Korea Health Industry Development Institute (KHIDI), funded by the Ministry of Health & Welfare, Republic of Korea (HI15C2364, HI17C1365), and a Basic Science Research Program and a Medical Research Center Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (NRF-2017R1D1A1A02019212 and -2017R1A5A2015369, respectively).

      Conflict of interest

      The authors of this manuscript have no conflicts of interest to declare.
      Please refer to the accompanying ICMJE disclosure forms for further details.

      Authors’ contributions

      All authors contributed to the conception and writing of the manuscript.

      Supplementary data

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