Immunomodulation of activated hepatic stellate cells by mesenchymal stem cells

Bone marrow-derived mesenchymal stem cells (MSCs) have been reported to prevent the development of liver fibrosis in a number of pre-clinical studies. Marked changes in liver histopathology and serological markers of liver function have been observed without a clear understanding of the therapeutic mechanism by which stem cells act. We sought to determine if MSCs could modulate the activity of resident liver cells, specifically hepatic stellate cells (SCs) by paracrine mechanisms using indirect cocultures. Indirect coculture of MSCs and activated SCs led to a significant decrease in collagen deposition and proliferation, while inducing apoptosis of activated SCs. The molecular mechanisms underlying the modulation of SC activity by MSCs were examined. IL-6 secretion from activated SCs induced IL-10 secretion from MSCs, suggesting a dynamic response of MSCs to the SCs in the microenvironment. Blockade of MSC-derived IL-10 and TNF-alpha abolished the inhibitory effects of MSCs on SC proliferation and collagen synthesis. In addition, release of HGF by MSCs was responsible for the marked induction of apoptosis in SCs as determined by antibody-neutralization studies. These findings demonstrate that MSCs can modulate the function of activated SCs via paracrine mechanisms provide a plausible explanation for the protective role of MSCs in liver inflammation and fibrosis, which may also be relevant to other models of tissue fibrosis.     

MiR‐122 modification enhances the therapeutic efficacy of adipose tissue‐derived mesenchymal stem cells against liver fibrosis

Mesenchymal stem cell (MSC) transplantation alone may be insufficient for treatment of liver fibrosis because of complicated histopathological changes in the liver. Given that miR‐122 plays an essential role in liver fibrosis by negatively regulating the proliferation and transactivation of hepatic stellate cells (HSCs), this study investigated whether miR‐122 modification can improve the therapeutic efficacy of adipose tissue‐derived MSCs in treating liver fibrosis. MiR‐122‐modified AMSCs (AMSC‐122) were constructed through lentivirus‐mediated transfer of pre‐miR‐122. MiR‐122‐modified AMSCs expressed high level of miR‐122, while they retained their phenotype and differentiation potential as naïve AMSCs. AMSC‐122 more effectively suppressed the proliferation of and collagen maturation in HSCs than scramble miRNA‐modified AMSCs. In addition, AMSC‐derived exosomes mediated the miR‐122 communication between AMSCs and HSCs, further affecting the expression levels of miR‐122 target genes, such as insulin‐like growth factor receptor 1 (IGF1R), Cyclin G(1) (CCNG1) and prolyl‐4‐hydroxylase α1 (P4HA1), which are involved in proliferation of and collagen maturation in HSCs. Moreover, miR‐122 modification enhanced the therapeutic efficacy of AMSCs in the treatment of carbon tetrachloride (CCl₄)‐induced liver fibrosis by suppressing the activation of HSCs and alleviating collagen deposition. Results demonstrate that miR‐122 modification improves the therapeutic efficacy of AMSCs through exosome‐mediated miR‐122 communication; thus, miR‐122 modification is a new potential strategy for treatment of liver fibrosis.     

Implications of hedgehog signaling antagonists for cancer therapy

The hedgehog (Hh) pathway, initially discovered in Drosophila by two Nobel laureates, Dr. Eric Wieschaus and Dr. Christiane Nusslein-Volhard, is a major regulator for cell differentiation, tissue polarity and cell proliferation. Studies from many laboratories, including ours, reveal activation of this pathway in most basal cell carcinomas and in approximately 30% of extracutaneous human cancers, including medulloblastomas, gastrointestinal, lung, breast and prostate cancers. Thus, it is believed that targeted inhibition of Hh signaling may be effective in treating and preventing many types of human cancers. Even more exciting is the discovery and synthesis of specific signaling antagonists for the Hh pathway, which have significant clinical implications in novel cancer therapeutics. This review discusses the major advances in the current understanding of Hh signaling activation in different types of human cancers, the molecular basis of Hh signaling activation, the major antagonists for Hh signaling inhibition and their potential clinical application in human cancer therapy.     

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.     

Sonic hedgehog enhances the proliferation and osteogenic differentiation of bone marrow-derived mesenchymal stem cells

MSCs (mesenchymal stem cells) may be promising seed cells for tissue regeneration because of their self-renewal and multi-differentiation potential. Shh (sonic hedgehog) is involved in the skeletal formation during embryo development and skeletal regeneration. However, how Shh regulates the biological characteristics of BM-MSCs (bone marrow-derived MSCs) is poorly understood. We have investigated the effect of rShh-N (recombinant N-terminal Shh) on the proliferation and osteogenic differentiation of rBM-MSCs (rat BM-MSCs) in vitro. rBM-MSCs were treated with rShh-N at concentrations up to 200 ng/ml. Proliferation and colony-forming ability of rBM-MSCs were increased in a dose-dependent manner. rShh-N increased the ratio of cells in S and G2/M phase, as well as the number of Ki-67+ cells. In addition, ALP (alkaline phosphatase) activity and matrix mineralization were enhanced by 200 ng/ml rShh-N. Real-time PCR showed that rShh-N (200 ng/ml) up-regulated the expression of genes encoding Cbfa-1 (core-binding factor α1), osteocalcin, ALP and collagen type I in rBM-MSCs. This information reveals some potential of rShh-N in the therapeutics of bone-related diseases.     

Mesenchymal stem cells regulate the proliferation and differentiation of neural stem cells through Notch signaling

The effects of mesenchymal stem cells (MSCs) on proliferation and cell fate determination of neural stem cells (NSCs) have been investigated. NSCs were co-cultured with MSCs or NIH3T3 cells using an in vitro transwell system. After 4 days, immunofluorescence staining showed that the number of cells positive for the cell proliferation antigen, ki-67, in neurospheres in MSCs was greater than in NIH3T3 cells. In some experiments, the top-layers of MSCs and NIH3T3 cells were removed to induce NSCs differentiation. Seven days after initiating differentiation, the levels of the neuronal marker, NSE, were higher in NSCs in MSCs co-culture group, and those of glial fibrillary acidic protein (GFAP) were lower, compared with NIH3T3 cells co-culture group. These were confirmed by immunofluorescence. The role of the Notch signaling pathway analyzed with the specific inhibitor, DAPT, and by examining the expression of Notch-related genes using RT-PCR showed that after co-culturing with MSCs for 24 h, NSCs expressed much higher levels of ki-67, Notch1, and Hes1 than did NSCs co-cultured with NIH3T3 cells. Treatment with DAPT decreased ki-67, Notch1 and Hes1 expression in NCSs, and increased Mash1 expression. The data indicate that the interactions between MSCs and NSCs promote NSCs proliferation and are involved in specifying neuronal fate, mediated in part by Notch signaling.     

The interaction between mesenchymal stem cells and steroids during inflammation

Mesenchymal stem cells (MSCs) are believed to exert their regenerative effects through differentiation and modulation of inflammatory responses. However, the relationship between the severity of inflammation and stem cell-mediated tissue repair has not been formally investigated. In this study, we applied different concentrations of dexamethasone (Dex) to anti-CD3-activated splenocyte cultured with or without MSCs. As expected, Dex exhibited a classical dose-dependent inhibition of T-cell proliferation. Surprisingly, although MSCs also blocked T-cell proliferation, the presence of Dex unexpectedly showed a dose-dependent reversion of T-cell proliferation. This effect of Dex was found to be exerted through interfering STAT1 phosphorylation-prompted expression of inducible nitric oxide synthase (iNOS). Interestingly, inflammation-induced chemokines in MSCs was unaffected. To test the role of inflammation severity in stem cell-mediated tissue repair, we employed mice with carbon tetrachloride-induced advanced liver fibrosis and found that although MSCs alone were effective, concurrent administration of Dex abrogated the therapeutic effects of MSCs on fibrin deposition, serum levels of bilirubin, albumin, and aminotransferases, as well as T-lymphocyte infiltration, especially IFN-γ⁺CD4⁺ and IL-17A⁺CD4⁺T cells. Likewise, iNOS^−/− MSCs, which produce chemokines but not nitric oxide under inflammatory conditions, are ineffective in treating advanced liver fibrosis. Therefore, inflammation has a critical role in MSC-mediated tissue repair. In addition, concomitant application of MSCs with steroids should be avoided.     

Pancreatic cancer cells activate CCL5 expression in mesenchymal stromal cells through the insulin-like growth factor-I pathway

Mesenchymal stromal cells (MSCs) have a critical role in cancer progression and metastasis. Despite extensive studies of the physiological responses in cancer cells, the molecular mechanisms regulating gene expression in MSCs by cancer cells remain undefined. Here we demonstrate that CC chemokine ligand 5 (CCL5) expression was increased in MSCs co-cultured with pancreatic cancer cells (PCCs), and this activation was dependent on extracellular insulin-like growth factor (IGF-I). Moreover, CCL5 induction in MSCs was required for the activation of IGF-I pathway in PCCs. These results reveal a link between the IGF-I pathway in PCCs and CCL5 pathway in MSCs through the interaction of those cells.     

Autophagy inhibition via Becn1 downregulation improves the mesenchymal stem cells antifibrotic potential in experimental liver fibrosis

Liver fibrosis (LF) is the result of a vicious cycle between inflammation-induced chronic hepatocyte injury and persistent activation of hepatic stellate cells (HSCs). Mesenchymal stem cell (MSC)-based therapy may represent a potential remedy for treatment of LF. However, the fate of transplanted MSCs in LF remains largely unknown. In the present study, the fate and antifibrotic effect of MSCs were explored in a LF model induced by CCl₄ in mouse. Additionally, MSCs were stimulated in vitro with LF-associated factors, tumor necrosis factor-α (TNF-α), interferon-γ (IFN-γ), and transforming growth factor-β1 (TGF-β1), to mimic the LF microenvironment. We unveiled that MSCs exhibited autophagy in response to the LF microenvironment through Becn1 upregulation both in vivo and in vitro. However, autophagy suppression induced by Becn1 knockdown in MSCs resulted in enhanced antifibrotic effects on LF. The improved antifibrotic potential of MSCs may be attributable to their inhibitory effects on T lymphocyte infiltration, HSCs proliferation, as well as production of TNF-α, IFN-γ, and TGF-β1, which may be partially mediated by elevated paracrine secretion of PTGS2/PGE₂. Thus, autophagy manipulation in MSCs may be a novel strategy to promote their antifibrotic efficacy.     

Effects of FGF21‐secreting adipose‐derived stem cells in thioacetamide‐induced hepatic fibrosis

Mesenchymal stem cells (MSCs) have been investigated to treat liver diseases, but the efficiency of MSCs to treat chronic liver diseases is conflicting. FGF21 can reduce inflammation and fibrosis. We established FGF21‐secreting adipose derived stem cells (FGF21_ADSCs) to enhance the effects of ADSCs and transplanted them into thioacetamide (TAA)‐induced liver fibrosis mice via the tail vein. Transplantation of FGF21_ADSCs significantly improved liver fibrosis by decreasing serum hyaluronic acid and reducing the expression of fibrosis‐related factors such as α‐smooth muscle actin (α‐SMA), collagen and tissue inhibitor of metalloproteinase‐1 (TIMP‐1) compared with the Empty_ADSCs by inhibition of p‐JNK, NF‐κB and p‐Smad2/3 signalling. α‐lactoalbumin (LA) and lactotransferrin (LTF), secretory factors produced from FGF21_ADSCs inhibited TGF‐β1‐induced expression of α‐SMA and collagen in LX‐2 cells. These results suggest that transplantation of FGF21_ADSCs inhibited liver fibrosis more effectively than Empty_ADSCs, possibly via secretion of α‐LA and LTF.     

Hepatogenic differentiation of mesenchymal stem cells in a rat model of thioacetamide-induced liver cirrhosis

Implantation of bone-marrow-derived MSCs (mesenchymal stem cells) has emerged as a potential treatment modality for liver failure, but in vivo differentiation of MSCs into functioning hepatocytes and its therapeutic effects have not yet been determined. We investigated MSC differentiation process in a rat model of TAA (thioacetamide)-induced liver cirrhosis. Male Sprague-Dawley rats were administered 0.04% TAA-containing water for 8 weeks, MSCs were injected into the spleen for transsplenic migration into the liver, and liver tissues were examined over 3 weeks. Ingestion of TAA for 8 weeks induced micronodular liver cirrhosis in 93% of rats. Injected MSCs were diffusely engrafted in the liver parenchyma, differentiated into CK19 (cytokeratin 19)- and thy1-positive oval cells and later into albumin-producing hepatocyte-like cells. MSC engraftment rate per slice was measured as 1.0-1.6%. MSC injection resulted in apoptosis of hepatic stellate cells and resultant resolution of fibrosis, but did not cause apoptosis of hepatocytes. Injection of MSCs treated with HGF (hepatocyte growth factor) in vitro for 2 weeks, which became CD90-negative and CK18-positive, resulted in chronological advancement of hepatogenic cellular differentiation by 2 weeks and decrease in anti-fibrotic activity. Early differentiation of MSCs to progenitor oval cells and hepatocytes results in various therapeutic effects, including repair of damaged hepatocytes, intracellular glycogen restoration and resolution of fibrosis. Thus, these results support that the in vivo hepatogenic differentiation of MSCs is related to the beneficial effects of MSCs rather than the differentiated hepatocytes themselves.     

The hedgehog pathway in nonalcoholic fatty liver disease

Nonalcoholic fatty liver disease (NAFLD) encompasses a spectrum of obesity-associated liver diseases and it has become the major cause of cirrhosis in the Western world. The high prevalence of NAFLD-associated advanced liver disease reflects both the high prevalence of obesity-related fatty liver (hepatic steatosis) and the lack of specific treatments to prevent hepatic steatosis from progressing to more serious forms of liver damage, including nonalcoholic steatohepatitis (NASH), cirrhosis, and primary liver cancer. The pathogenesis of NAFLD is complex, and not fully understood. However, compelling evidence demonstrates that dysregulation of the hedgehog (Hh) pathway is involved in both the pathogenesis of hepatic steatosis and the progression from hepatic steatosis to more serious forms of liver damage. Inhibiting hedgehog signaling enhances hepatic steatosis, a condition which seldom results in liver-related morbidity or mortality. In contrast, excessive Hh pathway activation promotes development of NASH, cirrhosis, and primary liver cancer, the major causes of liver-related deaths. Thus, suppressing excessive Hh pathway activity is a potential approach to prevent progressive liver damage in NAFLD. Various pharmacologic agents that inhibit Hh signaling are available and approved for cancer therapeutics; more are being developed to optimize the benefits and minimize the risks of inhibiting this pathway. In this review we will describe the Hh pathway, summarize the evidence for its role in NAFLD evolution, and discuss the potential role for Hh pathway inhibitors as therapies to prevent NASH, cirrhosis and liver cancer.     

Hedgehog信号通路与脂肪细胞发育育

TGFβ、Wnt、FGF和Hedgehog（Hh)等信号通路是参与胚胎发育的关键信号通路.从果蝇到人类,Hh信号通路广泛存在并高度保守,在多种器官的发育过程中发挥重要作用. 脂肪细胞发育的过程包括多潜能干细胞向前脂肪细胞定向和脂肪细胞终末分化两个阶段.近年来,Hh信号通路在脂肪细胞发育过程中的作用逐渐成为研究热点.越来越多的研究表明,Hh信号通路抑制脂肪细胞发育.本文将对Hh信号通路抑制脂肪细胞发育的作用以及其发挥作用的阶段进行综述,并分析将该信号通路作为靶点治疗肥胖症及相关疾病的可行性.     

Improved differentiation of umbilical cord blood-derived mesenchymal stem cells into insulin-producing cells by PDX-1 mRNA transfection

Numerous studies have sought to identify diabetes mellitus treatment strategies with fewer side effects. Mesenchymal stem cell (MSC) therapy was previously considered as a promising therapy; however, it requires the cells to be trans-differentiated into cells of the pancreatic-endocrine lineage before transplantation. Previous studies have shown that PDX-1 expression can facilitate MSC differentiation into insulin-producing cells (IPCs), but the methods employed to date use viral or DNA-based tools to express PDX-1, with the associated risks of insertional mutation and immunogenicity. Thus, this study aimed to establish a new method to induce PDX-1 expression in MSCs by mRNA transfection. MSCs were isolated from human umbilical cord blood and expanded in vitro, with stemness confirmed by surface markers and multipotentiality. MSCs were transfected with PDX-1 mRNA by nucleofection and chemically induced to differentiate into IPCs (combinatorial group). This IPC differentiation was then compared with that of untransfected chemically induced cells (inducer group) and uninduced cells (control group). We found that PDX-1 mRNA transfection significantly improved the differentiation of MSCs into IPCs, with 8.3±2.5% IPCs in the combinatorial group, 3.21±2.11% in the inducer group and 0% in the control. Cells in the combinatorial group also strongly expressed several genes related to beta cells (Pdx-1, Ngn3, Nkx6.1 and insulin) and could produce C-peptide in the cytoplasm and insulin in the supernatant, which was dependent on the extracellular glucose concentration. These results indicate that PDX-1 mRNA may offer a promising approach to produce safe IPCs for clinical diabetes mellitus treatment.     

Salvianolic acid B‐induced microRNA‐152 inhibits liver fibrosis by attenuating DNMT1‐mediated Patched1 methylation

Epithelial‐mesenchymal transition (EMT) was reported to be involved in the activation of hepatic stellate cells (HSCs), contributing to the development of liver fibrosis. Epithelial‐mesenchymal transition can be promoted by the Hedgehog (Hh) pathway. Patched1 (PTCH1), a negative regulatory factor of the Hh signalling pathway, was down‐regulated during liver fibrosis and associated with its hypermethylation status. MicroRNAs (miRNAs) are reported to play a critical role in the control of various HSCs functions. However, miRNA‐mediated epigenetic regulations in EMT during liver fibrosis are seldom studied. In this study, Salvianolic acid B (Sal B) suppressed the activation of HSCs in CCl₄‐treated mice and mouse primary HSCs, leading to inhibition of cell proliferation, type I collagen and alpha‐smooth muscle actin. We demonstrated that the antifibrotic effects caused by Sal B were, at least in part, via inhibition of EMT and the Hh pathway. In particular, up‐regulation of PTCH1 was associated with decreased DNA methylation level after Sal B treatment. Accordingly, DNA methyltransferase 1 (DNMT1) was attenuated by Sal B in vivo and in vitro. The knockdown of DNMT1 in Sal B‐treated HSCs enhanced PTCH1 expression and its demethylation level. Interestingly, increased miR‐152 in Sal B‐treated cells was responsible for the hypomethylation of PTCH1 by Sal B. As confirmed by the luciferase activity assay, DNMT1 was a direct target of miR‐152. Further studies showed that the miR‐152 inhibitor reversed Sal B‐mediated PTCH1 up‐regulation and DNMT1 down‐regulation. Collectively, miR‐152 induced by Sal B, contributed to DNMT1 down‐regulation and epigenetically regulated PTCH1, resulting in the inhibition of EMT in liver fibrosis.     

Hedgehog signaling controls homeostasis of adult intestinal smooth muscle

The Hedgehog (Hh) pathway plays multiple patterning roles during development of the mammalian gastrointestinal tract, but its role in adult gut function has not been extensively examined. Here we show that chronic reduction in the combined epithelial Indian (Ihh) and Sonic (Shh) hedgehog signal leads to mislocalization of intestinal subepithelial myofibroblasts, loss of smooth muscle in villus cores and muscularis mucosa as well as crypt hyperplasia. In contrast, chronic over-expression of Ihh in the intestinal epithelium leads to progressive expansion of villus smooth muscle, but does not result in reduced epithelial proliferation. Together, these mouse models show that smooth muscle populations in the adult intestinal lamina propria are highly sensitive to the level of Hh ligand. We demonstrate further that Hh ligand drives smooth muscle differentiation in primary intestinal mesenchyme cultures and that cell-autonomous Hh signal transduction in C3H10T1/2 cells activates the smooth muscle master regulator Myocardin (Myocd) and induces smooth muscle differentiation. The rapid kinetics of Myocd activation by Hh ligands as well as the presence of an unusual concentration of Gli sties in this gene suggest that regulation of Myocd by Hh might be direct. Thus, these data indicate that Hh is a critical regulator of adult intestinal smooth muscle homeostasis and suggest an important link between Hh signaling and Myocd activation. Moreover, the data support the idea that lowered Hh signals promote crypt expansion and increased epithelial cell proliferation, but indicate that chronically increased Hh ligand levels do not dampen crypt proliferation as previously proposed.     

Change in hepatocyte growth factor concentration promote mesenchymal stem cell-mediated osteogenic regeneration

Mesenchymal stem cells (MSCs) play a crucial role in tissue repair by secretion of tissue nutrient factors such as hepatocyte growth factor (HGF). However, studies examining the effects of HGF on the proliferation and differentiation of MSCs used different concentrations of HGF and reported conflicting conclusions. This study aimed to determine the mechanisms by which different concentrations of HGF regulate MSC proliferation and osteogenic differentiation, and validate the mechanism in an animal model of early stage avascular necrosis of femoral head (ANFH). Our results demonstrate that a low concentration of HGF (20 ng/ml) preferentially promotes MSC osteogenic differentiation through increased c-Met expression and phosphorylation, Akt pathway activation, and increased expression of p27, Runx2 and Osterix. In contrast, a high concentration of HGF (100 ng/ml) strongly induced proliferation by inducing strong activation of the ERK1/2 signalling pathway. As validated by animal experiments, high localized expression of HGF achieved by transplantation of HGF transgenic MSCs into ANFH rabbits increased the number of MSCs. Subsequently, 2 weeks after transplantation, HGF levels decreased and MSCs differentiated into osteoblasts and resulted in efficient tissue repair. Our results demonstrate that sequential concentration changes in HGF control the proliferation and osteogenic differentiation of MSCs in vivo. This phenomenon can be exploited therapeutically to induce bone regeneration and, in turn, improve the efficacy of pharmacological intervention for ANFH treatment.     

Trefoil factor 2 secreted from damaged hepatocytes activates hepatic stellate cells to induce fibrogenesis

Liver fibrosis is a common characteristic of chronic liver diseases. The activation of hepatic stellate cells (HSCs) plays a key role in fibrogenesis in response to liver injury, yet the mechanism by which damaged hepatocytes modulate the activation of HSCs is poorly understood. Our previous studies have established that liver-specific deletion of O-GlcNAc transferase (OGT)leads to hepatocyte necroptosis and spontaneous fibrosis. Here, we report that OGT-deficient hepatocytes secrete trefoil factor 2 (TFF2) that activates HSCs and contributes to the fibrogenic process. The expression and secretion of TFF2 are induced in OGT-deficient hepatocytes but not in WT hepatocytes. TFF2 activates the platelet-derived growth factor receptor beta signaling pathway that promotes the proliferation and migration of primary HSCs. TFF2 protein expression is elevated in mice with carbon tetrachloride-induced liver injury. These findings identify TFF2 as a novel factor that mediates intercellular signaling between hepatocytes and HSCs and suggest a role of the hepatic OGT–TFF2 axis in the process of fibrogenesis.