Manipulating HGF signaling reshapes the cirrhotic liver niche and fills a therapeutic gap in regeneration mediated by transplanted stem cells.

Long-term stem cell survival in the cirrhotic liver niche to maintain therapeutic efficacy has not been achieved. In a well-defined diethylnitrosamine (DEN)-induced liver fibrosis/cirrhosis animal model, we previously showed that liver-resident stem/progenitor cells (MLpvNG2+ cells) or immune cells have improved survival in the fibrotic liver environment but died via apoptosis in the cirrhotic liver environment, and increased levels of hepatocyte growth factor (HGF) mediated this cell death. We tested the hypothesis that inhibiting HGF signaling during the cirrhotic phase could keep the cells alive. We used adeno-associated virus (AAV) vectors designed to silence the c-Met (HGF-only receptor) gene or a neutralizing antibody (anti-cMet-Ab) to block the c-Met protein in the DEN-induced liver cirrhosis mouse model transplanted with MLpvNG2+ cells between weeks 6 and 7 after DEN administration, which is the junction of liver fibrosis and cirrhosis at the site where most intrahepatic stem cells move toward apoptosis. After 4 weeks of treatment, the transplanted MLpvNG2+ cells survived better in c-Met-deficient mice than in wild-type mice, and cell activity was similar to that of the mice that received MLpvNG2+ cells at 5 weeks after DEN administration (liver fibrosis phase when most of these cells proliferated). Mechanistically, a lack of c-Met signaling remodeled the cirrhotic environment, which favored transplanted MLpvNG2+ cell expansion to differentiation into mature hepatocytes and initiate endogenous regeneration by promoting mature host hepatocyte generation and mediating functional improvements. Therapeutically, c-Met-mediated regeneration can be mimicked by anti-cMet-Ab to interfere functions, which is a potential drug for cell-based treatment of liver fibrosis/cirrhosis.

Effectiveness of mild ovarian stimulation versus GnRH agonist protocol in women undergoing assisted reproductive technology: a meta-analysis.

OBJECTIVE: our meta-analysis was conducted to evaluate the effectiveness of the mild ovulation induction protocol using CC/gonadotropin/GnRH antagonist compared to the conventional GnRH agonist protocol in women undergoing ART. METHOD: Six electronic databases were searched from their date of establishment until August 2016. Outcomes in our analysis were calculated in terms of relative risk (RR) and weighted mean differences (WMD) and standard mean differences (SMD) with 95% confidence intervals (CI) using random effect models or fixed effect models. RESULTS: Six prospective controlled clinical trials with 1543 women comparing the clinical impacts of the two protocols were included. The synthesized results suggested a significant reduction in the quantity of gonadotropins (SMD: -1.96, 95% CI: -2.28 to 1.64, I2 = 78.5%), the incidence of OHSS (RR: 0.16, 95% CI 0.03-0.86, I2 = 0%) and an increase in the cycle cancelation rate (RR: 1.46, 95% CI 1.05-2.03, I2 = 89.4%). While no evidence of statistically significant differences between the groups existed in the other clinical outcomes. CONCLUSION: This study suggested that the probable benefits of the mild protocol, including its less costs and safer process without reducing the overall IVF treatment success rates, seemed to make it a better treatment option. Larger sample prospective trials evaluating live birth, clinical pregnancy, OHSS, multiple pregnancy incidence and so on were desired to establish.

New advances of NG2-expressing cell subset in marrow mesenchymal stem cells as novel therapeutic tools for liver fibrosis/cirrhosis.

BACKGROUND: Bone marrow-derived mesenchymal stem cell (BMMSC)-based therapy has become a major focus for treating liver fibrosis/cirrhosis. However, although these cell therapies promote the treatment of this disease, the heterogeneity of BMMSCs, which causes insufficient efficacy during clinical trials, has not been addressed. In this study, we describe a novel Percoll-Plate-Wait procedure (PPWP) for the isolation of an active cell subset from BMMSC cultures that was characterized by the expression of neuroglial antigen 2 (NG2/BMMSCs). METHODS: By using the key method of PPWP and other classical biological techniques we compared NG2/BMMSCs with parental BMMSCs in biological and functional characteristics within a well-defined diethylnitrosamine (DEN)-induced liver fibrosis/cirrhosis injury male C57BL/6 mouse model also in a culture system. Of note, the pathological alterations in the model is quite similar to humans'. RESULTS: The NG2/BMMSCs revealed more advantages compared to parentalBMMSCs. They exhibited greater proliferation potential than parental BMMSCs, as indicated by Ki-67 immunofluorescence (IF) staining. Moreover, higher expression of SSEA-3 (a marker specific for embryonic stem cells) was detected in NG2/BMMSCs than in parental BMMSCs, which suggested that the "stemness" of NG2/BMMSCs was greater than that of parental BMMSCs. In vivo studies revealed that an injection of NG2/BMMSCs into mice with ongoing DEN-induced liver fibrotic/cirrhotic injury enhanced repair and functional recovery to a greater extent than in mice treated with parental BMMSCs. These effects were associated with the ability of NG2/BMMSCs to differentiate into bile duct cells (BDCs). In particular, we discovered for the first time that NG2/BMMSCs exhibit unique characteristics that differ from those of parental BMMSCs in terms of producing liver sinusoidal endothelial cells (LSECs) to reconstruct injured blood vessels and sinusoidal structures in the diseased livers, which are important for initiating hepatocyte regeneration. This unique potential may also suggest that NG2/BMMSCs could be an novel off-liver progenitor of LSECs. Ex vivo studies revealed that the NG2/BMMSCs exhibited a similar trend to that of their in vivo in terms of functional differentiation responding to the DEN-diseased injured liver cues. Additionally, the obvious core role of NG2/BMMSCs in supporting the functions of BMMSCs in bile duct repair and BDC-mediated hepatocyte regeneration might also be a novel finding. CONCLUSIONS: Overall, the PPWP-isolated NG2/BMMSCs could be a novel effective cell subset with increased purity to serve as a new therapeutic tool for enhancing treatment efficacy of BMMSCs and special seed cell source (BDCs, LSECs) also for bioliver engineering.

A route planning for oil sample transportation based on improved A* algorithm.

The traditional A* algorithm suffers from issues such as sharp turning points in the path, weak directional guidance during the search, and a large number of computed nodes. To address these problems, a modified approach called the Directional Search A* algorithm along with a path smoothing technique has been proposed. Firstly, the Directional Search A* algorithm introduces an angle constraint condition through the evaluation function. By converting sharp turns into obtuse angles, the path turning points become smoother. This approach reduces the occurrence of sharp turns in the path, resulting in improved path smoothness. Secondly, the algorithm enhances the distance function to strengthen the directional guidance during the path search. By optimizing the distance function, the algorithm tends to prefer directions that lead towards the target, which helps reduce the search space and shorten the overall path planning time. Additionally, the algorithm removes redundant nodes along the path, resulting in a more concise path representation. Lastly, the algorithm proposes an improved step size adjustment method to optimize the number of path nodes obtained. By appropriately adjusting the step size, the algorithm further reduces the number of nodes, leading to improved path planning efficiency. By applying these methods together, the Directional Search A* algorithm effectively addresses the limitations of the traditional A* algorithm and produces smoother and more efficient path planning results. Simulation experiments comparing the modified A* algorithm with the traditional A* algorithm were conducted using MATLAB. The experimental results demonstrate that the improved A* algorithm can generate shorter paths, with reduced planning time and smoother trajectories. This indicates that the Directional Search A* algorithm, incorporating the angle constraint condition in the evaluation function and the direction-guided strategy, outperforms the traditional A* algorithm in path planning and provides better solutions to the existing issues.

Hepatocyte growth factor-mediated apoptosis mechanisms of cytotoxic CD8+ T cells in normal and cirrhotic livers.

Intrahepatic stem/progenitor cells and cytotoxic CD8+ T cells (CD8+ T cells) in the cirrhotic liver undergo apoptosis, which potentially facilitates progression to cancer. Here, we report that hepatocyte growth factor (HGF) signaling plays an important role in promoting normal and damaged liver CD8+ T cell Fas-mediated apoptosis through its only receptor, c-Met. In addition to binding with HGF, c-Met also binds to Fas to form a complex. Using a diethylnitrosamine (DEN)-induced liver fibrosis/cirrhosis mouse model, immunostaining, and terminal deoxynucleotidyl transferase (TdT) dUTP nick-end labeling (TUNEL) staining, we found that HGF secretion was significantly higher at 10 weeks post-DEN, the liver cirrhotic phase (LCP), than at 3 weeks post-DEN, the liver fibrotic phase (LFP). Correspondingly, differences in CD8+ T cell proliferation and apoptosis were noted between the two phases. Interestingly, staining and TUNEL assays revealed lower smooth muscle actin (α-SMA)+ cell apoptosis, a marker for hepatic stellate cells (HSCs), in the LFP group than in the LCP group, which suggested a beneficial correlation among HGF, CD8+ T cells and HSCs in improving the fibrotic load during damaged liver repair. In cultures, when met different concentrations of recombinant HGF (rHGF), phytohemagglutinin (PHA)-stimulated naive mouse splenic CD8+ T cells (pn-msCD8+ T cells) responded differently; as increases in rHGF increased were associated with decreases in the clonal numbers of pn-msCD8+ T cells, and when the rHGF dose was greater than 200 ng/mL, the clonal numbers significantly decreased. In the presence of 400 ng/mL rHGF, the death-inducing signaling complex (DISC) can be directly activated in both nsCD8+ T cells and healthy human peripheral blood CD8+ T cells (hp-CD8+ T cells), as indicated by recruitment of FADD and caspase-8 because DISC forms via the recruitment of FADD and caspase-8, among others. These findings suggest that Fas-mediated apoptosis, may also indicate a regulatory role of HGF signaling in hepatic homeostasis.

Comparative effects of hepatocyte growth factor and tacrolimus on acute liver allograft early tolerance.

Allostimulated CD8+ T cells (aCD8+ T cells), as the main mediators of acute liver rejection (ARJ), are hyposensitive to apoptosis due to the inactivation of death receptor FAS-mediated pathways and fail to allow tolerance induction, eventually leading to acute graft rejection. Although tacrolimus (FK506), the most commonly used immunosuppressant (IS) in the clinic, allows tolerance induction, its use is limited because its target immune cells are unknown and it is associated with increased incidences of malignancy, infection, and nephrotoxicity, which substantially impact long-term liver transplantation (LTx) outcomes. The dark agouti (DA)-to-Lewis rat LTx model is a well-known ARJ model and was hence chosen for the present study. We show that both hepatocyte growth factor (HGF) (cHGF, containing the main form of promoting HGF production) and recombinant HGF (h-rHGF) exert immunoregulatory effects mainly on allogeneic aCD8+ T cell suppression through FAS-mediated apoptotic pathways by inhibiting cMet to FAS antagonism and Fas trimerization, leading to acute tolerance induction. We also showed that such inhibition can be abrogated by treatment with neutralizing antibodies against cMet (HGF-only receptor). In contrast, we did not observe these effects in rats treated with FK506. However, we observed that the effect of anti-rejection by FK506 was mainly on allostimulated CD4+ T cell (aCD4+ T cell) suppression and regulatory T cell (Treg) promotion, in contrast to the mechanism of HGF. In addition, the protective mechanism of HGF in FK506-mediated nephrotoxicity was addressed. Therefore, HGF as a tolerance inducer, whether used in combination with FK506 or as monotherapy, may have good clinical value. Additional roles of these T-cell subpopulations in other biological systems and studies in these fields will also be meaningful.

Hepatocyte growth factor mediates a novel form of hepatic stem/progenitor cell-induced tolerance in a rat xenogeneic liver rejection model.

We have previously identified novel neural/glial antigen 2-expressing hepatic stem/progenitor cells (NG2+ HSPs) that are beneficial for tissue repair by inhibiting the immune cell response. In this in vivo study, we investigated the use of hepatocyte growth factor (HGF)-secreting NG2+ HSPs as a tolerogen in the well-established Syrian golden hamster (SGH) to Lewis (LEW) xenogeneic rat acute liver rejection (ARJ) model. Liver and blood cells were collected for histology and functional analyses using immunofluorescence staining, western blot, ELISA, and TUNEL assays. All recipient rats were randomly divided into 5 groups (n = 14 rats/group) and treated with: (1) ARJ + PBS: (2) ARJ + NG2: tail vein injection of NG2+ HSPs; (3) ARJ + tacrolimus (FK506, oral administration); (4) ARJ + an anti-cMet functional blocking antibody (a-cMet-Ab, I.V) 24 h before the injection of NG2+ HSPs; (5) ARJ + cHGF (clinically used HGF). LEW to LEW syngeneic rats were considered "normal" (n = 14, namely Syn). Significantly prolonged mean survival times (MSTs) and improved graft functions were observed after NG2+ HSP transplantation. An anti-cMet Ab significantly blocked the effect of NG2+ HSPs, suggesting that the effects were likely associated with HGF secreted from NG2+ HSPs. Notably, when intravenously injected into the xenogeneic rat model, the injected cHGF not only prolonged the MST of recipient rats but also increased the number of TUNEL-expressing xenoreactive cytotoxic T lymphocytes (CD8+ T cells). Based on these results, HGF-secreting NG2+ HSPs may specifically target recipient CD8+ T cells by inducing their apoptosis.

Comparison of the biological and functional characteristics of mesenchymal stem cells from intrahepatic and identical bone marrow.

In our privious work, our reseach group characterized a population of hepatic-sourced mesenchymal stem cells (MSCs) called MLpvNG2+ cells. In the present study, we compared the biological and functional characteristics of naïve MLpvNG2 cells with identical bone marrow-derived MSCs (niBM-MSCs) using in vitro (conditioned media) and in vivo (a well-set diethylnitrosamine (DEN)-induced liver fibrotic/cirrhotic murine model) procedures. The intrahepatic-sourced mesodermal MLpvNG2+ cells exhibited some biological characteristics (e.g., a set of surface markers) similar to those of extrahepatic niBM-MSCs. In responsed to signals of pathological conditions, such as singals of fibrotic/cirrhotic liver, MLpvNG2+ cells showed higher survival and favored differentiation into ALB(+) and G6Pc(+) hepatocytes, whereas niBM-MSCs predominantly differentiated into CK/KRT19(+) cholangiocytes. We identified C/EBPα/ß expression as a biological characteristic differentiating these two populations of MSCs, wherein MLpvNG2+ cells are likely regulated by C/EBPß transcriptional signaling, whereas niBM-MSCs are likely controlled by C/EBPα transcriptional signaling. Notably, although C/EBPα and C/EBPß transcriptional signaling regulate hepatocyte and cholangiocyte fate, respectively, the expression of these proteins in MLpvNG2+ cells is, to our knowledge, reported for the first time in the present study. We used anti-C/EBP neutralizing antibodies (Abs) both in vitro and in vivo to determine the functional characteristics of these proteins. We conclude that the biological characteristics of these two populations of MSCs depend on their differential C/EBPα/ß expression patterns.

Survival of endogenous hepatic stem/progenitor cells in liver tissues during liver cirrhosis.

AIMS: This study addressed whether endogenous hepatic stem/progenitor (HSP) cells survival were related to the injured signals during liver cirrhosis. MATERIAL AND METHODS: Liver cirrhosis was induced in C57BL/6 mice by administering diethylnitrosamine (DEN) in drinking water. Hematoxylin-eosin staining and Masson's trichrome staining were used to identify infiltrative cells and connective tissues, respectively. The inflammatory activity grade and fibrotic stage, represented as G and S respectively, and evaluated by the International Simplified Grading and Staging System (ISGSS). Endogenous HSP cells (Ng2+HSP) were identified by immunofluorescence staining with an anti-neuro/glial antigen 2 (Ng2) antibody. All data were analyzed using SPSS 22.0 and GraphPad Prism 6 and Student's t-test was performed to determine statistical significance. p-values < 0.05 were considered statistically significant. KEY FINDINGS: All mice administered oral DEN developed liver fibrosis, liver cirrhosis and hepatocellular carcinoma (HCC). During the fibrosis period, observed a positive correlation of survival of endogenous HSP (Ng2+HSP) cells with inflammatory activity. As the disease developed into the cirrhotic stage, when lost correlation of endogenous HSP cells with inflammatory activity, revealed a dramatically reduced survival rate of endogenous HSP (Ng2+HSP) cells. SIGNIFICANCE: The DEN-induced liver cirrhosis could develop into three time zone of fibrosis, cirrhosis and cancer, and the histological patterns in the model are similar to those described in humans. Dramatic survival and less apoptosis of endogenous HSP (Ng2+HSP) cells was within fibrotic state, where inflammation signals is strong, indicating such time zone (1-6 weeks) during liver cirrhosis is important for mobilizing endogenous HSP-based regeneration.

Data on a novel liver bioscaffold (rDLS) generated from regenerative liver with activated extracellular matrix for functional liver regeneration.

The data presented in this article are related to the original research article entitled "A novel bioscaffold with naturally-occurring extracellular matrix promotes hepatocyte survival and vessel patency in mouse models of heterologous transplantation" (Yang et al., in press) [1]. This article describes a decellularized liver scaffold (DLS) that derived from partial hepatectomy liver (rDLS) which supported primary hepatocyte survival and promoted blood patency, as compared with a conventional scaffold that generated from naïve liver (nDLS). Analysis by immunochemistry and scanning electron microscope (SEM) showed that the vessel network and extracellular matrix (ECM) components were similar to the nDLS. The rDLS could prevent blood clotting after transplanted it in vivo, identified by immunofluorescence staining for the integrin (αIIb, α4) expression and liver transplantation models (mice, pigs) a formed well-blood petency liver lobules. These data indicate that the novel scaffold (rDLS) with naturally-occurring "activated ECM" that may be useful for the implantation in vivo of a bioengineered organoid that is able to exert function long term without clotting in future clinic.

A novel bioscaffold with naturally-occurring extracellular matrix promotes hepatocyte survival and vessel patency in mouse models of heterologous transplantation.

BACKGROUND: Naïve decellularized liver scaffold (nDLS)-based tissue engineering has been impaired by the lack of a suitable extracellular matrix (ECM) to provide "active micro-environmental" support. AIM: The present study aimed to examine whether a novel, regenerative DLS (rDLS) with an active ECM improves primary hepatocyte survival and prevents thrombosis. METHODS: rDLS was obtained from a 30-55% partial hepatectomy that was maintained in vivo for 3-5 days and then perfused with detergent in vitro. Compared to nDLS generated from normal livers, rDLS possesses bioactive molecules due to the regenerative period in vivo. Primary mouse hepatocyte survival was evaluated by staining for Ki-67 and Trypan blue exclusion. Thrombosis was assessed by immunohistochemistry and ex vivo diluted whole-blood perfusion. Hemocompatibility was determined by near-infrared laser-Doppler flowmetry and heterotopic transplantation. RESULTS: After recellularization, rDLS contained more Ki-67-positive primary hepatocytes than nDLS. rDLS had a higher oxygen saturation and blood flow velocity and a lower expression of integrin αIIb and α4 than nDLS. Tumor necrosis factor-α, hepatocyte growth factor, interleukin-10, interleukin-6 and interleukin-1ß were highly expressed throughout the rDLS, whereas expression of collagen-I, collagen-IV and thrombopoietin were lower in rDLS than in nDLS. Improved blood vessel patency was observed in rDLS both in vitro and in vivo. The results in mice were confirmed in large animals (pigs). CONCLUSION: rDLS is an effective DLS with an "active microenvironment" that supports primary hepatocyte survival and promotes blood vessel patency. This is the first study to demonstrate a rDLS with a blood microvessel network that promotes hepatocyte survival and resists thrombosis.