Therapeutic potential of mesenchymal stem cells overexpressing human forkhead box A2 gene in the regeneration of damaged liver tissues

Background and Aim: Although a liver transplantation is considered to be the only effective long‐term treatment in many cases of liver diseases, it is limited by a lack of donor organs and immune rejection. As an autologous stem cell approach, this study was conducted to assess whether forkhead box A2 (Foxa2) gene overexpression in bone marrow‐derived mesenchymal stem cells (MSC) could protect the liver from hepatic diseases by stimulating tissue regeneration after cell transplantation. Methods: Rat MSC (rMSC) were isolated, characterized, and induced to hepatocytes that expressed liver‐specific markers. Four different treatments (control [phosphate‐buffered saline], rMSC alone, rMSC/pIRES–enhanced green fluorescent protein (EGFP) vector, and rMSC/pIRES–EGFP/human Foxa2) were injected into the spleen of carbon tetrachloride‐injured rats. Biochemical and histological analyses on days 30, 60, and 90 post‐transplantation were performed to evaluate the therapeutic capacities of MSC overexpressing hFoxa2. Results: rMSC transfected with hFoxa2 were induced into hepatogenic linage and expressed several liver‐specific genes, such as, Foxa2, α‐fetoprotein, cytokeratin‐18, hepatocyte nuclear factor‐1α, and hepatocyte growth factor. A group of animals treated with MSC/hFoxa2 showed significant recovery of liver‐specific enzyme expressions to normal levels at the end of the study (90 days). Furthermore, when compared to the fibrotic areas of the samples treated with MSC alone or MSC/vector, the fibrotic area of the samples treated with rMSC/hFoxa2 for 90 days significantly decreased, until they were completely gone. Conclusions: Human Foxa2 efficiently promoted the incorporation of MSC into liver grafts, suggesting that hFoxa2 genes could be used for the structural or functional recovery of damaged liver cells.     

Adipose‐derived stem cells: A candidate for liver regeneration

The scarcity of donor livers and the impracticality of hepatocyte transplantation represent the biggest obstacles for the treatment of liver failure. Adipose‐derived stem cells, with their ability to differentiate into the hepatic lineage, provide a reliable alternative cell source with clear ethical and practical advantages. Moreover, adipose‐derived stem cells can effectively repair liver damage by the dominant indirect pattern and increase the number of hepatocytes by the secondary direct pattern. In recent years, the development of the indirect pattern, which mainly includes immunomodulatory and trophic effects, has become a hot topic in the field of cell engineering. Therefore, adipose‐derived stem cells are considered to be ideal therapeutic stem cells for human liver regeneration. In this article, we reviewed the advantages of adipose‐derived stem cells in liver regeneration, and explore their underlying mechanisms.     

Adipose may actively delay progression of NAFLD by releasing tumor‐suppressing,anti‐fibrotic miR‐122 into circulation

Nonalcoholic fatty liver disease (NAFLD) is the most common liver pathology. Here we propose tissue‐cooperative, homeostatic model of NAFLD. During early stages of NAFLD the intrahepatic production of miR‐122 falls, while the secretion of miRNA‐containing exosomes by adipose increases. Bloodstream carries exosome to the liver, where their miRNA cargo is released to regulate their intrahepatic targets. When the deterioration of adipose catches up with the failing hepatic parenchyma, the external supply of liver‐supporting miRNAs gradually tapers off, leading to the fibrotic decompensation of the liver and an increase in hepatic carcinogenesis. This model may explain paradoxical observations of the disease‐associated decrease in intrahepatic production of certain miRNAs with an increase in their levels in serum. Infusions of miR‐122 and, possibly, some other miRNAs may be efficient for preventing NAFLD‐associated hepatocellular carcinoma. The best candidates for exosome‐wrapped miRNA producer are adipose tissue‐derived mesenchymal stem cells (MSCs), known for their capacity to shed large amounts of exosomes into the media. Notably, MSC‐derived exosomes with no specific loading are already tested in patients with liver fibrosis. Carrier exosomes may be co‐manufactured along with their cargo. Exosome‐delivered miRNA cocktails may augment functioning of human organs suffering from a variety of chronic diseases.     

Insight into the development of obesity: functional alterations of adipose‐derived mesenchymal stem cells

Obesity is associated with a variety of disorders including cardiovascular diseases, diabetes mellitus and cancer. Obesity changes the composition and structure of adipose tissue, linked to pro‐inflammatory environment, endocrine/metabolic dysfunction, insulin resistance and oxidative stress. Adipose‐derived mesenchymal stem cells (ASCs) have multiple functions like cell renewal, spontaneous repair and homeostasis in adipose tissue. In this review article, we have summarized the recent data highlighting that ASCs in obesity are defective in various functionalities and properties including differentiation, angiogenesis, motility, multipotent state, metabolism and immunomodulation. Inflammatory milieu, hypoxia and abnormal metabolites in obese tissue are crucial for impairing the functions of ASCs. Further work is required to explore the precise molecular mechanisms underlying its alterations and impairments. Based on these data, we suggest that deregulated ASCs, possibly also other mesenchymal stem cells, are important in promoting the development of obesity. Restoration of ASCs/mesenchymal stem cells might be an additional strategy to combat obesity and its associated diseases.     

Effect of human umbilical cord blood‐derived mesenchymal stem cells in a cirrhotic rat model

Background/Aim: Cirrhosis is a long‐term consequence of chronic hepatic injury and no effective therapy is currently available for this disease. Recent reports have shown that the mesenchymal stem cells (MSCs) have the capacity to differentiate into hepatocytes, and umbilical cord blood is a rich source of MSCs. Hence, we investigated the effect of infusing of human umbilical cord blood‐derived MSCs (HMSCs) in carbon tetrachloride (CCl₄)‐induced cirrhosis in a rat model. Methods: The effect of HMSCs on cirrhosis was evaluated using haematoxylin and eosin and Masson's trichrome staining. To evaluate cirrhosis‐related factors, we measured protein and mRNA expression of transforming growth factor β₁ (TGF‐β₁), collagen type I and α‐smooth muscle actin (α‐SMA). Results: Histological findings showed that liver fibrosis in rats was alleviated by HMSCs infusion. Interestingly, CM‐DiI‐labelled HMSCs expressed the hepatocyte‐specific markers, human albumin and α‐fetoprotein. Infusion of HMSCs significantly inhibited TGF‐β₁, collagen type I and α‐SMA expressions in CCl₄‐induced cirrhotic rats. Conclusion: Our results showed that HMSCs infusion could improve liver fibrosis in rats with CCl₄‐induced cirrhosis, raising the possibility for clinical use of HMSCs in the treatment of cirrhosis.     

Melatonin prevents cadmium‐induced bone damage: First evidence on an improved osteogenic/adipogenic differentiation balance of mesenchymal stem cells as underlying mechanism

Melatonin (MLT) plays a role in preserving bone health, a function that may depend on homeostatic effects on both mature osteoblasts and mesenchymal stem cells (MSCs) of the bone tissue. In this study, these functions of MLT have been investigated in rat bone (femur) and in human adipose MSC (hMSC) during chronic exposure to low‐grade cadmium (Cd) toxicity, a serious public health concern. The in vivo findings demonstrate that MLT protects against Cd‐induced bone metabolism disruption and accumulation of bone marrow adipocytes, a cue of impaired osteogenic potential of skeletal MSC niches. This latter symptom was recapitulated in hMSCs in which Cd toxicity stimulated adipogenic differentiation. MLT was found to rescue, at least in part, the osteogenic differentiation properties of these cells. This study reports on a new bone cytoprotection function of MLT pertinent to Cd toxicity and its interfering effect on skeletal MSC differentiation properties that is worth investigating for its possible impact on human bone pathophysiology.     

大鼠脂肪间充质干细胞在部分肝切除模型中向肝细胞分化的研究

目的探讨脂肪间充质干细胞（ADMSCs）在部分肝切除模型中向肝细胞的分化。方法从大鼠脂肪组织中分离出干细胞，并进行体外扩增、传代，取第2代ADMSCs用PKH26标记，制作部分肝切除模型，将标记细胞经门静脉自体植入体内。2周后切下肝脏制成冰冻切片，荧光显微镜下观察标记细胞在肝脏的定位，进行免疫荧光染色检测标记细胞白蛋白的表达。结果从脂肪组织中分离出的ADMSCs能在体外大量扩增，PKH26标记后细胞在荧光显微镜下发红色荧光，细胞标记率约95％；荧光显微镜下可见肝脏冰冻切片中散在分布红色标记细胞，免疫荧光染色显示大多数标记细胞白蛋白染色阳性。结论 大鼠脂肪间充质干细胞在肝再生环境中能向肝细胞分化，有可能在肝部分切除后参与肝再生。     

Differentiation of hepatocytoid cell induced from whole-bone-marrow method isolated rat myeloid mesenchymal stem cells

AIM: To explore the expansion and differentiation of hepatocytoid cell induced from myeloid mesenchymal stem cell (MSC) in vitro, in order to find suitable resource of hepatocytes for bioartificial liver or liver transplantation. METHODS: The rat myeloid MSC was isolated and divided into three groups which were cultured by Frieden-steion method, and then were induced by culture fluid, culture fluid plus cholestatic serum and culture fluid plus hepatocyte growth factor (HGF), respectively. Hepatocytoid cell as well as expression of CK18 and AFP was observed by immunohistochemistry. RESULTS: After the induction for 21 d, hepatocytoid cell was observed, and its expression of CK18 and AFP was detected by immunohistochemistry in MSC cultured with cholestatic serum. Furthermore, on the 35th d, albumin mRNA was expressed in the cell, suggesting the inducing effect was similar to that by HGF. CONCLUSION: Rat myeloid MSC can differentiate into hepatocyte lineage under appropriate condition. This method is easy to operate.     

Gelatin-Based Hydrogels Promote Chondrogenic Differentiation of Human Adipose Tissue-Derived Mesenchymal Stem Cells In Vitro

Due to the weak regeneration potential of cartilage, there is a high clinical incidence of articular joint disease, leading to a strong demand for cartilaginous tissue surrogates. The aim of this study was to evaluate a gelatin-based hydrogel for its suitability to support chondrogenic differentiation of human mesenchymal stem cells. Gelatin-based hydrogels are biodegradable, show high biocompatibility, and offer possibilities to introduce functional groups and/or ligands. In order to prove their chondrogenesis-supporting potential, a hydrogel film was developed and compared with standard cell culture polystyrene regarding the differentiation behavior of human mesenchymal stem cells. Cellular basis for this study were human adipose tissue-derived mesenchymal stem cells, which exhibit differentiation potential along the adipogenic, osteogenic and chondrogenic lineage. The results obtained show a promotive effect of gelatin-based hydrogels on chondrogenic differentiation of mesenchymal stem cells in vitro and therefore encourage subsequent in vivo studies.     

Rapid hepatic fate specification of adipose-derived stem cells and their therapeutic potential for liver failure

Background and Aim:  Multipotential mesenchymal stem cells (MSC), present in many organs and tissues, represent an attractive tool for the establishment of a successful stem cell-based therapy in the field of regeneration medicine. Adipose tissue mesenchymal stem cells (AT-MSC), known as adipose-derived stem cells (ASC) are especially attractive in the context of future clinical applications because of their high accessibility and minimal invasiveness during the procedure to obtain them. The goal of the present study was to induce human ASC into functional hepatocytes in vitro within a very short period of time and to check their therapeutic potential in vivo .
Methods:  In vitro generated ASC-derived hepatocytes were checked for hepatocyte-specific markers and functions. Afterwards, they were transplanted into nude mice with liver injury. Twenty-four hours after transplantation, biochemical parameters were evaluated in blood serum.
Results:  We have shown here that ASC can be differentiated into hepatocytes within 13 days and can reach the functional properties of primary human hepatocytes. After transplantation into mice with acute liver failure, ASC-derived hepatocytes can restore such liver functions as ammonia and purine metabolism. Markers of liver injury, alanine aminotransferase, aspartate aminotransferase, as well as ammonia, were decreased after ASC-derived hepatocyte transplantation.
Conclusions:  Our data highlight the properties of ASC as having a special affinity for hepatocyte differentiation in vitro and liver regeneration in vivo . Thus, ASC may be a superior choice for the establishment of a therapy for injured liver.     

Persistence of functional hepatocyte‐like cells in immune‐compromised mice

Background: Human embryonic stem cells (hESCs) can be efficiently differentiated to hepatocyte‐like cells (HLCs) in vitro and demonstrate many of the functions and gene expression found in the adult liver. Aims: In this study, we assess the therapeutic value of HLCs in long‐term cell‐based therapies in vivo. Methods: hESC‐derived HLCs were injected into the spleen of acutely injured NODscid^IL‐2Rγnull mice and analysed at various time points post‐transplantation up to 3 months. Results: Large clusters of human cells engrafted in the spleen after 3 days and had expanded considerably by 31 days. At these time points, we identified human cells expressing parenchymal hepatocyte markers, exhibiting biliary duct‐like structures and expressing myofibroblast markers. Three months after transplantation, we could detect human HLCs that were positive for albumin and CK18 by immunostaining and human DNA by fluorescent in situ hybridisation. Moreover, we could detect secretion of human serum albumin by enzyme‐linked immunoabsorbant assay. Conclusions: We observed the persistence, engraftment and function of HLCs in vivo up to 3 months post‐translation; however, all murine recipients developed large splenic and liver tumours that contained endodermal and mesodermal cell types. Although our studies demonstrate that hESC‐derived HLCs have the potential to play an important role in cell‐based therapies, current methodologies and transplantation strategies require substantial refinement before they can be deployed safely.     

人脂肪源性间充质干细胞的分离培养与鉴定

目的探索人脂肪间充质干细胞(ADMSCs)分离、培养的方法,以证明人脂肪组织中含有多向分化潜能的ADMSCs,为临床应用提供实验基础。方法采用Ⅰ型胶原酶消化法及贴壁法分离培养腹部手术患者皮下脂肪组织,取第3代细胞,以磷酸盐缓冲生理盐水为空白对照,进行CD13、CD34、CD44、CD45、CD105、HLA-DR、Ⅷ因子表面抗原的免疫细胞化学鉴定。结果人脂肪中含有大量长梭形细胞,免疫化学鉴定显示与空白对照相比较CD44、CD13、CD105抗原呈阳性反应,其余标志物CD45、CD34、HLA-DR、Ⅷ因子抗原均为阴性反应。结论初步证明了人脂肪组织中含有间充质干细胞,为今后ADMSCs的分离培养提供了更简单有效的方法。     

脂肪间充质干细胞向肝细胞横向分化的研究

目的探讨脂肪源性间充质干细胞（AMSC）向肝细胞横向分化的可能性。方法胶原酶消化脂肪组织,贴壁培养,体外扩增后以流式细胞仪鉴定其表面标志。取扩增3代的AMSC分为2组,诱导分化组在含有2％FBS的DMEM-F12培养基中加入肝细胞生长因子20 ng/ml和成纤维细胞生长因子4 10 ng/ml、1×ITS和地塞米松0．1μmol/L,培养14 d；空白对照组则不加任何细胞因子。RT-PCR检测诱导分化过程中肝细胞核因子1、GATA4等基因转录水平的变化。2周后,采用流式细胞术检测AFP和Alb阳性细胞在两组细胞中的比例,检测肝细胞特异性细胞角蛋白（CK） 18、CK19的表达。结果分离、培养的AMSC呈成纤维细胞样生长,可以稳定传代。流式细胞术检测结果显示第3代脂肪间充质干细胞高表达表面CD29、CD44；不表达CD34、CD45。RT-PCR检测诱导5、8、11、14 d的细胞,显示有肝细胞特异性转录因子GATA4和肝细胞核因子1A基因的表达,并随时间延长而逐渐增多。流式细胞术检测诱导14 d的细胞,发现30．0％的细胞表达Alb,17．8％细胞表达AFP,双阳性的细胞为6．9％；免疫荧光检测发现诱导细胞表达CK18、CK19。空白对照组脂肪间充质干细胞则未见上述各项变化。结论在低血清培养体系中,采用细胞因子联合诱导,显示脂肪间充质细胞在体外能定向分化为肝细胞样细胞。     

Differentiation of hepatocytoid cell induced from whole-bone-marrow method isolated rat myeloid mesenchymal stem cells

AIM: To explore the expansion and differentiation of hepatocytoid cell induced from myeloid mesenchymal stem cell (MSC)in vitro, in order to find suitable resource of hepatocytes for bioartificial liver or liver transplantation. METHODS: The rat myeloid MSC was isolated and divided into three groups which were cultured by Frieden steion method, and then were induced by culture fluid, culture fluid plus cholestatic serum and culture fluid plus hepatocyte growth factor (HGF), respectively. Hepatocytoid cell as well as expression of CK18 and AFP was observed by immunohistochemistry. RESULTS: After the induction for 21 d, hepatocytoid cell was observed, and its expression of CK18 and AFP was detected by immunohistochemistry in MSC cultured withcholestatic serum. Furthermore, on the 35th d, albumin mRNA was expressed in the cell, suggesting the inducing effect was similar to that by HGF. CONCLUSION: Rat myeloid MSC can differentiate into hepatocyte lineage under appropriate condition. This method is easy to operate.     

Biochemical and molecular characterization of hepatocyte-like cells derived from human bone marrow mesenchymal stem cells on a novel three-dimensional biocompatible nanofibrous scaffold

Background:  There is significant interest in using nanofibers in tissue engineering from stem cells. The transdifferentiation of mesenchymal stem cells into the hepatic lineage in a nanofibrous structure has not been reported. In this study, a three dimensional nanofibrous scaffold is introduced for differentiation of human bone marrow derived mesenchymal stem cells (hBMSCs) into hepatocytes.
Methods:  A scaffold composed of Poly (ε-caprolactone), collagen and polyethersulfone was fabricated by the electrospinning technique. After characterization of isolated hBMSCs, the performance of the cells on the scaffold was evaluated by Scanning Electron Microscopy (SEM) and MTT assay. Cytological, molecular and biochemical markers were measured to confirm differentiation potential of hBMSCs into hepatocytes.
Results:  The isolated cells possessed the basic properties of mesenchymal stem cells (MSCs). Based on scanning electron microscope (SEM) analysis and MTT assay, it was shown that the cells adhere, penetrate and proliferate on the nanofibers. Cultured cells on the nanofibers differentiated into hepatocyte-like cells and expressed hepatocyte specific markers such as albumin, α-fetoprotein, cytokeratin-18, cytokeratin-19 and cytochrome P450 3A4 at mRNA levels. Appearance of a considerable number of albumin-positive cells cultivated on the scaffold (47 ± 4%) as compared to the two-dimensional culture system (28 ± 6%) indicates the supporting role of the scaffold. The efficiency of the cells to produce albumin, urea, transferrin, serum glutamic pyruvic transaminase and serum oxaloacetate aminotransferase in hepatocytes on the scaffold further attest to the functionality of the cells.
Conclusion:  The data presented in this study show that the engineered nanofibrous scaffold is a conductive matrix which supports and enhances MSC development into functional hepatocyte-like cells.     

Liver-specific gene expression in mesenchymal stem cells is induced by liver cells

AIM: The origin of putative liver cells from distinct bone marrow stem cells, e.g. hematopoietic stem cells or multipotent adult progenitor cells was found in recent in vitro studies. Cell culture experiments revealed a key role of growth factors for the induction of liver-specific genes in stem cell cultures. We investigated the potential of rat mesenchymal stem cells (MSC) from bone marrow to differentiate into hepatocytic cells in vitro. Furthermore, we assessed the influence of cocultured liver cells on induction of liver-specific gene expression. METHODS: Mesenchymal stem cells were marked with green fluorescent protein (GFP) by retroviral gene transduction. Clonal marked MSC were either cultured under liver stimulating conditions using fibronectin-coated culture dishes and medium supplemented with SCF, HGF, EGF, and FGF-4 alone, or in presence of freshly isolated rat liver cells. Cells in cocultures were harvested and GFP+ or GFP-cells were separated using fluorescence activated cell sorting. RT-PCR analysis for the stem cell marker Thy1 and the hepatocytic markers CK-18, albumin, CK-19, and AFP was performed in the different cell populations. RESULTS: Under the specified culture conditions, rat MSC cocultured with liver cells expressed albumin-, CK-18, CK-19, and AFP-RNA over 3 weeks, whereas MSC cultured alone did not show liver specific gene expression. CONCLUSION: The results indicate that (1) rat MSC from bone marrow can differentiate towards hepatocytic lineage in vitro, and (2) that the microenvironment plays a decisive role for the induction of hepatic differentiation of rMSC.