Current understanding of mesenchymal stem cells in liver diseases

Liver diseases caused by various factors have become a significant threat to public health worldwide. Liver transplantation has been considered as the only effective treatment for end-stage liver diseases; however, it is limited by the shortage of donor organs, postoperative complications, long-term immunosuppression, and high cost of treatment. Thus, it is not available for all patients. Recently, mesenchymal stem cells (MSCs) transplantation has been extensively explored for repairing hepatic injury in various liver diseases. MSCs are multipotent adult progenitor cells originated from the embryonic mesoderm, and can be found in mesenchymal tissues including the bone marrow, umbilical cord blood, adipose tissue, liver, lung, and others. Although the precise mechanisms of MSC transplantation remain mysterious, MSCs have been demonstrated to be able to prevent the progression of liver injury and improve liver function. MSCs can self-renew by dividing, migrating to injury sites and differentiating into multiple cell types including hepatocytes. Additionally, MSCs have immune-modulatory properties and release paracrine soluble factors. Indeed, the safety and effectiveness of MSC therapy for liver diseases have been demonstrated in animals. However, pre-clinical and clinical trials are largely required to confirm its safety and efficacy before large scale clinical application. In this review, we will explore the molecular mechanisms underlying therapeutic effects of MSCs on liver diseases. We also summarize clinical advances in MSC-based therapies.     

Therapeutic potential of bone marrow stem cells for liver diseases

Stem cells of the bone marrow, including hematopoietic stem cells (HSC), mesenchymal stem cells (MSC) and hepatic progenitors were reported to give rise to hepatocytes by both transdifferentiation and cellular fusion. Transdifferentiation was observed without liver damage although significant numbers of stem cell derived hepatocytes were not described. Cellular fusion was demonstrated in the presence of a proliferation stimulus in conjunction with impaired intrinsic liver regeneration capacity. Here, we review potential therapeutic applications of stem cell derived hepatocytes depending on how they emerge. Stem cells turning into hepatocytes by transdifferentiation introduce new functioning liver cells into a diseased organ, which can support intrinsic liver regeneration or bridge the time gap until a definitive treatment is available. When cellular fusion is the mechanism behind stem cell plasticity, however, no new cells emerge in the first place, whereas new genetic material is introduced. The fusion cell thereby acquires a selective advantage over resident hepatocytes allowing for extensive proliferation and liver repopulation. Therefore genetic deficiencies might be the predominant target for cell fusion therapies. We conclude that transdifferentiation and cellular fusion might be powerful tools for the therapy of liver diseases in the future and we propose the introduction of artificial cell fusion as well as stem cell differentiation as therapeutic options.     

Differentiation of human adipose stromal cells into hepatic lineage in vitro and in vivo

Embryonic stem cells (ES cells), bone marrow-derived mesenchymal stem cells, umbilical cord blood-derived mesenchymal stem cells, and hepatic stem cells in liver have been known as a useful source that can induce to differentiate into hepatocytes. In this study, we examined whether human adipose tissue-derived stromal cells (hADSC) can differentiate into hepatic lineage in vitro. hADSC, that were induced to differentiate into hepatocyte-like cells by the treatment of HGF and OSM, had morphology similar to hepatocytes. Addition of DMSO enhanced differentiation into hepatocytes. RT-PCR and immunocytochemical analysis showed that hADSC express albumin and alpha-fetoprotein during differentiation. Differentiated hADSC showed LDL uptake and production of urea. Additionally, transplanted hADSC to CCl4-injured SCID mouse model were able to be differentiated into hepatocytes and they expressed albumin in vivo. Mesenchymal stem cells isolated from human adipose tissue are immunocompatible and are easily isolated. Therefore, hADSC may become an alternative source to hepatocyte regeneration or liver cell transplantation.     

How important is differentiation in the therapeutic effect of mesenchymal stromal cells in liver disease?

Background aimsThe protocols for differentiation of hepatocyte-like cells (HLCs) from mesenchymal stromal cells (MSCs) have been well established. Previous data have shown that MSCs and their derived HLCs were able to engraft injured liver and alleviate injuries induced by carbon tetrachloride. The goal of the current study was to determine the differences of MSCs and their derived HLCs in terms of therapeutic functions in liver diseases.MethodsAfter hepatic differentiation of umbilical cord–derived MSCs in vitro, we detected both MSC and HLC expressions of adhesion molecules and chemokine receptor CXCR4 by flow cytometry; immunosuppressive potential and hepatocyte growth factor expression were determined by means of enzyme-linked immunosorbent assay. We compared the therapeutic effect for fulminant hepatic failure in a mouse model.ResultsMSC-derived-HLCs expressed lower levels of hepatocyte growth factor, accompanied by impaired immunosuppression in comparison with MSCs. Furthermore, undifferentiated MSCs showed rescuing potentials superior to those in HLCs for the treatment of fulminant hepatic failure.ConclusionsAfter differentiation, HLCs lost several major properties in comparison with undifferentiated MSCs, which are beneficial for their application in liver diseases. Undifferentiated MSCs may be more appropriate than are HLCs for the treatment of liver diseases.     

成体干细胞向肝细胞分化

成体干细胞跨越胚层限制分化为其他胚层来源的细胞,对揭示不同胚层细胞间相互分化的生物学意义和机制具有重要学术价值,并可以为临床细胞移植治疗开辟新的途径,从而成为当前研究的热点之一。综述了近年来肝源性卵圆细胞、成肝细胞、骨髓源干细胞和其他成体干细胞跨越分化为肝细胞的研究现状与进展,以及卵圆细胞、成肝细胞等的分离鉴定,表面标志、生物学特征和跨越分化机制,并对成体干细胞在肝脏疾病细胞治疗上的应用前景作了展望。     

间充质干细胞对免疫细胞的抑制作用及其机制

间充质干细胞是一群来源于发育早期中胚层的具有自我更新和多向分化潜能的干细胞,具有分化为脂肪细胞、肝细胞、成骨细胞、软骨细胞、神经细胞等多种细胞的能力.近年来的相关研究表明,间充质干细胞具有低免疫原性,它可以通过抑制淋巴细胞的增殖、抑制抗原呈递细胞分化成熟及功能发挥、抑制细胞毒性T淋巴细胞的形成、增加调节性T细胞比例等多种途径发挥免疫调节作用,从而成为移植领域、各种退行性和衰竭性疑难病症的替代治疗的研究热点.本文就间充质干细胞对免疫细胞的抑制作用及其机制的研究进展进行综述.     

The application of umbilical cord-derived MSCs in cardiovascular diseases

Transplantation of stem cells is a promising, emerging treatment for cardiovascular diseases in the modern era. Mesenchymal stem cells (MSCs) derived from the umbilical cord are one of the most promising cell sources because of their capacity for differentiation into cardiomyocytes, endothelial cells and vascular smooth muscle cells in vitro/in vivo. In addition, umbilical cord-derived MSCs (UC-MSCs) secrete many effective molecules regulating apoptosis, fibrosis and neovascularization. Another important and specific characteristic of UC-MSCs is their low immunogenicity and immunomodulatory properties. However, the application of UC-MSCs still faces some challenges, such as low survivability and tissue retention in a harmful disease environment. Gene engineering and pharmacological studies have been implemented to overcome these difficulties. In this review, we summarize the differentiation ability, secretion function, immunoregulatory properties and preclinical/clinical studies of UC-MSCs, highlighting the advantages of UC-MSCs for the treatment of cardiovascular diseases.     

Induction of hepatocyte‐like cells from human umbilical cord‐derived mesenchymal stem cells by defined microRNAs

Generating functional hepatocyte‐like cells (HLCs) from mesenchymal stem cells (MSCs) is of great urgency for bio‐artificial liver support system (BALSS). Previously, we obtained HLCs from human umbilical cord‐derived MSCs by overexpressing seven microRNAs (HLC‐7) and characterized their liver functions in vitro and in vivo. Here, we aimed to screen out the optimal miRNA candidates for hepatic differentiation. We sequentially removed individual miRNAs from the pool and examined the effect of transfection with remainder using RT‐PCR, periodic acid—Schiff (PAS) staining and low‐density lipoprotein (LDL) uptake assays and by assessing their function in liver injury models. Surprisingly, miR‐30a and miR‐1290 were dispensable for hepatic differentiation. The remaining five miRNAs (miR‐122, miR‐148a, miR‐424, miR‐542‐5p and miR‐1246) are essential for this process, because omitting any one from the five‐miRNA combination prevented hepatic trans‐differentiation. We found that HLCs trans‐differentiated from five microRNAs (HLC‐5) expressed high level of hepatic markers and functioned similar to hepatocytes. Intravenous transplantation of HLC‐5 into nude mice with CCl₄‐induced fulminant liver failure and acute liver injury not only improved serum parameters and their liver histology, but also improved survival rate of mice in severe hepatic failure. These data indicated that HLC‐5 functioned similar to HLC‐7 in vitro and in vivo, which have been shown to resemble hepatocytes. Instead of using seven‐miRNA combination, a simplified five‐miRNA combination can be used to obtain functional HLCs in only 7 days. Our study demonstrated an optimized and efficient method for generating functional MSC‐derived HLCs that may serve as an attractive cell alternative for BALSS.     

Pluripotent plasticity of stem cells and liver repopulation

Different types of stem cells have a role in liver regeneration or fibrous repair during and after several liver diseases. Otherwise, the origin of hepatic and/or extra‐hepatic stem cells in reactive liver repopulation is under controversy. The ability of the human body to self‐repair and replace the cells and tissues of some organs is often evident. It has been estimated that complete renewal of liver tissue takes place in about a year. Replacement of lost liver tissues is accomplished by proliferation of mature hepatocytes, hepatic oval stem cells differentiation, and sinusoidal cells as support. Hepatic oval cells display a distinct phenotype and have been shown to be a bipotential progenitor of two types of epithelial cells found in the liver, hepatocytes, and bile ductular cells. In gastroenterology and hepatology, the first attempts to translate stem cell basic research into novel therapeutic strategies have been made for the treatment of several disorders, such as inflammatory bowel diseases, diabetes mellitus, celiachy, and acute or chronic hepatopaties. In the future, pluripotent plasticity of stem cells will open a variety of clinical application strategies for the treatment of tissue injuries, degenerated organs. The promise of liver stem cells lie in their potential to provide a continuous and readily available source of liver cells that can be used for gene therapy, cell transplant, bio‐artificial liver‐assisted devices, drug toxicology testing, and use as an in vitro model to understand the developmental biology of the liver. Copyright © 2010 John Wiley & Sons, Ltd.     

Insulin-like growth factor 1 (IGF-I) improves hepatic differentiation of human bone marrow-derived mesenchymal stem cells

The ability of MSCs (mesenchymal stem cells) to differentiate between other cell types makes these cells an attractive therapeutic tool for cell transplantation. This project was designed to improve transdifferentiation of human MSCs into liver cells using IGF-I (insulin-like growth factor 1) which, despite its important role in liver development, has not been used for in vitro hepatic differentiation. In the present study, the MSCs derived from healthy human bone marrow samples were cultured and characterized by immunophenotyping and differentiation potential into osteoblast and adipocytes. Transdifferentiation into hepatocyte-like cells was performed in the presence/absence of IGF-I in combination with predefined hepatic differentiation cocktail. To evaluate transdifferentiation, morphological features, immuno-cytochemical staining of specific biological markers and hepatic functions were assessed. Morphological assessment and evaluation of glycogen content, albumin and AFP (α-feto protein) expression as well as albumin and urea secretion revealed statistically significant difference between experimental groups compared with the control. Morphology and function (albumin secretion) of IGF-I-treated cells were significantly better than IGF-I-free experimental group. To the best of our knowledge, our study is the first to demonstrate that the combination of IGF-I with the predefined hepatic differentiation cocktail will significantly improve the morphological features of the differentiated cells and albumin secretion.     

Mesenchymal stem cells: biology and clinical potential in type 1 diabetes therapy

Mesenchymal stem cells (MSCs) can be derived from adult bone marrow, fat and several foetal tissues. In vitro , MSCs have the capacity to differentiate into multiple mesodermal and non-mesodermal cell lineages. Besides, MSCs possess immunosuppressive effects by modulating the immune function of the major cell populations involved in alloantigen recognition and elimination. The intriguing biology of MSCs makes them strong candidates for cell-based therapy against various human diseases. Type 1 diabetes is caused by a cell-mediated autoimmune destruction of pancreatic β-cells. While insulin replacement remains the cornerstone treatment for type 1 diabetes, the transplantation of pancreatic islets of Langerhans provides a cure for this disorder. And yet, islet transplantation is limited by the lack of donor pancreas. Generation of insulin-producing cells (IPCs) from MSCs represents an attractive alternative. On the one hand, MSCs from pancreas, bone marrow, adipose tissue, umbilical cord blood and cord tissue have the potential to differentiate into IPCs by genetic modification and/or defined culture conditions In vitro . On the other hand, MSCs are able to serve as a cellular vehicle for the expression of human insulin gene. Moreover, protein transduction technology could offer a novel approach for generating IPCs from stem cells including MSCs. In this review, we first summarize the current knowledge on the biological characterization of MSCs. Next, we consider MSCs as surrogate β-cell source for islet transplantation, and present some basic requirements for these replacement cells. Finally, MSCs-mediated therapeutic neovascularization in type 1 diabetes is discussed.     

Prolongation of liver-specific function for primary hepatocytes maintenance in 3D printed architectures

Isolated primary hepatocytes from the liver are very similar to in vivo native liver hepatocytes, but they have the disadvantage of a limited lifespan in 2D culture. Although a sandwich culture and 3D organoids with mesenchymal stem cells (MSCs) as an attractive assistant cell source to extend lifespan can be used, it cannot fully reproduce the in vivo architecture. Moreover, long-term 3D culture leads to cell death because of hypoxic stress. Therefore, to overcome the drawback of 2D and 3D organoids, we try to use a 3D printing technique using alginate hydrogels with primary hepatocytes and MSCs. The viability of isolated hepatocytes was more than 90%, and the cells remained alive for 7 days without morphological changes in the 3D hepatic architecture with MSCs. Compared to a 2D system, the expression level of functional hepatic genes and proteins was higher for up to 7 days in the 3D hepatic architecture. These results suggest that both the 3D bio-printing technique and paracrine molecules secreted by MSCs supported long-term culture of hepatocytes without morphological changes. Thus, this technique allows for widespread expansion of cells while forming multicellular aggregates, may be applied to drug screening and could be an efficient method for developing an artificial liver.     

Human umbilical cord mesenchymal stem cell-derived extracellular vesicles: A novel therapeutic paradigm

Mesenchymal stem cells (MSCs) have been revealed to hold great potential for the development of new treatment approaches for various diseases. However, the clinical use of these cells is limited due to their tumorigenic effects. The therapeutic benefits of MSCs are largely dependent on paracrine factors including extracellular vesicles (EVs). EVs are nano-sized bilayer membrane structures containing lipids, microRNAs and proteins which play key roles in cell-to-cell communications. Because of their lower immunogenicity, tumorigenicity, and easier management, EVs have emerged as a new promising alternative to whole-cell therapy. Therefore, this paper reviews current preclinical studies on the use of EVs derived from human umbilical cord MSCs (hucMSCs) as a therapeutic approach in treatment of several diseases including neurological, cardiovascular, liver, kidney, and bone diseases as well as the cutaneous wound, inflammatory bowel disease, cancers, infertility, and other disorders.     

Human mesenchymal stem cells - current trends and future prospective

Stem cells are cells specialized cell, capable of renewing themselves through cell division and can differentiate into multi-lineage cells. These cells are categorized as embryonic stem cells (ESCs), induced pluripotent stem cells (iPSCs) and adult stem cells. Mesenchymal stem cells (MSCs) are adult stem cells which can be isolated from human and animal sources. Human MSCs (hMSCs) are the non-haematopoietic, multipotent stem cells with the capacity to differentiate into mesodermal lineage such as osteocytes, adipocytes and chondrocytes as well ectodermal (neurocytes) and endodermal lineages (hepatocytes). MSCs express cell surface markers like cluster of differentiation (CD)29, CD44, CD73, CD90, CD105 and lack the expression of CD14, CD34, CD45 and HLA (human leucocyte antigen)-DR. hMSCs for the first time were reported in the bone marrow and till now they have been isolated from various tissues, including adipose tissue, amniotic fluid, endometrium, dental tissues, umbilical cord and Wharton''s jelly which harbours potential MSCs. hMSCs have been cultured long-term in specific media without any severe abnormalities. Furthermore, MSCs have immunomodulatory features, secrete cytokines and immune-receptors which regulate the microenvironment in the host tissue. Multilineage potential, immunomodulation and secretion of anti-inflammatory molecules makes MSCs an effective tool in the treatment of chronic diseases. In the present review, we have highlighted recent research findings in the area of hMSCs sources, expression of cell surface markers, long-term in vitro culturing, in vitro differentiation potential, immunomodulatory features, its homing capacity, banking and cryopreservation, its application in the treatment of chronic diseases and its use in clinical trials.     

Signalling pathways involved in the process of mesenchymal stem cells differentiating into hepatocytes

End‐stage liver disease can be the termination of acute or chronic liver diseases, with manifestations of liver failure; transplantation is currently an effective treatment for these. However, transplantation is severely limited due to the serious lack of donors, expense, graft rejection and requirement of long‐term immunosuppression. Mesenchymal stem cells (MSCs) have attracted considerable attention as therapeutic tools as they can be obtained with relative ease and expanded in culture, along with features of self‐renewal and multidirectional differentiation. Many scientific groups have sought to use MSCs differentiating into functional hepatocytes to be used in cell transplantation with liver tissue engineering to repair diseased organs. In most of the literature, hepatocyte differentiation refers to use of various additional growth factors and cytokines, such as hepatocyte growth factor (HGF), fibroblast growth factor (FGF), epidermal growth factor (EGF), oncostatin M (OSM) and more, and most are involved in signalling pathway regulation and cell–cell/cell–matrix interactions. Signalling pathways have been shown to play critical roles in embryonic development, tumourigenesis, tumour progression, apoptosis and cell‐fate determination. However, mechanisms of MSCs differentiating into hepatocytes, particularly signalling pathways involved, have not as yet been completely illustrated. In this review, we have focused on progress of signalling pathways associated with mesenchymal stem cells differentiating into hepatocytes along with the stepwise differentiation procedure.     

Human Umbilical Cord Mesenchymal Stem Cells and Derived Hepatocyte-Like Cells Exhibit Similar Therapeutic Effects on an Acute Liver Failure Mouse Model

Mesenchymal stem cells (MSCs) have exhibited therapeutic effects in multiple animal models so that are promising liver substitute for transplantation treatment of end-stage liver diseases. However, it has been shown that over-manipulation of these cells increased their tumorigenic potential, and that reducing the in vitro culture time could minimize the risk. In this study, we used a D-galactosamine plus lipopolysaccharide (Gal/LPS)-induced acute liver failure mouse model, which caused death of about 50% of the mice with necrosis of more than 50% hepatocytes, to compare the therapeutic effects of human umbilical cord MSCs (hUCMSCs) before and after induction of differentiation into hepatocyte (i-Heps). Induction of hUCMSCs to become i-Heps was achieved by treatment of the cells with a group of growth factors within 4 weeks. The resulted i-Heps exhibited a panel of human hepatocyte biomarkers including cytokeratin (hCK-18), α-fetoprotein (hAFP), albumin (hALB), and hepatocyte-specific functions glycogen storage and urea metabolism. We demonstrated that transplantation of both cell types through tail vein injection rescued almost all of the Gal/LPS-intoxicated mice. Although both cell types exhibited similar ability in homing at the mouse livers, the populations of the hUCMSCs-derived cells, as judged by expressing hAFP, hCK-18 and human hepatocyte growth factor (hHGF), were small. These observations let us to conclude that the hUCMSCs was as effective as the i-Heps in treatment of the mouse acute liver failure, and that the therapeutic effects of hUCMSCs were mediated largely via stimulation of host hepatocyte regeneration, and that delivery of the cells through intravenous injection was effective.     

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

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