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Fibrin gel is appropriate for maintaining quiescence characteristics in primary culture of mouse hepatic stellate cells. Embedded culture of hepatic stellate cells in fibrin gel simulates in vivo cell morphology. Stiffness and adhesion molecules of fibrin gel play a crucial role in the hepatic stellate cell’s primary culture.

Background and Aim: Hepatic stellate cells (HSCs) activation, a critical event in liver fibrosis, has been recently shown to be related to autophagy. Determine whether chloroquine (CQ) could affect (i) the activation of HSC in vivo and (ii) the hepatic damage in a mice acute liver injury model. Methods: The acute liver injury was induced in BALB/c mice by carbon tetrachloride (CCl4 group); 24 h before and after CCl4 administration animals were treated by CQ (CCl4 + CQ group). As control, mice treated by olive oil were considered. After 48 h from CCl4/olive oil administration, blood samples, liver tissues, and HSCs were harvested for analysis. Results: In vivo, CQ attenuates CCl4-induced acute liver damage as evidenced by (i) the reduction of liver enlargement, (ii) the reduction of liver swelling and necrosis also supported by a certain decrease of circulating transaminases level, and (iii) the reduction of liver fibrosis evaluated by collagen deposition and α-sma protein expression. In HSCs isolated from CQ treated group, we observed the inhibition of autophagy proved by the increase in p62 protein and the decrease of lc3 protein. In addition, CQ reduced the expression of the HSCs activation markers α-sma/collagen-I and down-regulated the expression of the proliferative marker ki67. Conclusion: The autophagy attenuation exerted by CQ together with the reduction of the expression of the proliferation marker in HSCs can lessen the acute liver damage potentially opening the way to novel therapeutic approaches for hepatic fibrosis.

Introduction: Biliary atresia (BA) is the leading cause of liver fibrosis and failure in neonates with surgical jaundice, leading to poor outcome. Clinical and animal studies showing that granulocyte colony-stimulating factor (GCSF) treatment could improve liver fibrosis and cirrhosis suggest that GCSF may be offered as a low-cost intervention to improve the course of BA. This study aims to test the hypothesis that 10 µg/kg/day x 5 days of GCSF could improve liver function, reduce molecular pro-fibrotic markers and decrease liver fibrosis in a mouse model of bile duct ligation (BDL). Methods: Balb/c mice underwent Sham surgery, or BDL for seven days followed by subcutaneous GCSF administration at 10 µg/kg/day for five consecutive days. Twelve days post-operation, blood samples were taken from the facial vein for leukocyte/neutrophil count and for measurement of serum enzymatic activities. The median lobe of the liver was acquired for total RNA and protein extraction. Moreover, the median liver lobe was used for hematoxylin-eosin staining, sirius red staining, and for visualization by immunohistochemistry (IHC). Results: Twelve days post-operation, GCSF-treated bile duct-ligated (BDL) mice had a higher survival rate than that of placebo-treated mice (hazard ratio=1.88, p=0.084). The GCSF-treated mice had diminished liver serum transaminase activities (AST: 228.92 ± 222.67 vs. 313.46 ± 164.80 IU/L; ALP: 573.24 ± 177.89 IU/L vs. 471.75 ± 117.92 IU/L). GCSF treatment also reduced fibrosis with down-regulation of expression of pro-fibrotic markers including TGF-β1 (-2.61-fold mRNA), α-SMA (-2.46-fold mRNA; -1.88-fold protein, p

Liver fibrosis (LF) mortality rate is approximately 2 million per year. Irrespective of the etiology of LF, a key element in its development is the transition of hepatic stellate cells (HSCs) from a quiescent phenotype to a myofibroblast-like cell with the production of fibrotic proteins. It is necessary to define optimal isolation and culturing conditions for good HSCs yield and proper phenotype preservation for studying the activation of HSCs in vitro. In the present study, the optimal conditions of HSC isolation and culture were examined to maintain the HSC’s undifferentiated phenotype. HSCs were isolated from Balb/c mice liver using Nycodenz, 8, 9.6, and 11%. The efficiency of the isolation procedure was evaluated by cell counting and purity determination by flow cytometry. Quiescent HSCs were cultured in test media supplemented with different combinations of fetal bovine serum (FBS), glutamine (GLN), vitamin A (vitA), insulin, and glucose. The cells were assessed at days 3 and 7 of culture by evaluating the morphology, proliferation using cell counting kit-8, lipid storage using Oil Red O (ORO) staining, expression of a-smooth muscle actin, collagen I, and lecithin-retinol acyltransferase by qRT-PCR and immunocytochemistry (ICC). The results showed that Nycodenz, at 9.6%, yielded the best purity and quantity of HSCs. Maintenance of HSC undifferentiated phenotype was achieved optimizing culturing conditions (serum-free Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with glucose (100 mg/dl), GLN (0.5 mM), vitA (100 μM), and insulin (50 ng/ml)) with a certain degree of proliferation allowing their perpetuation in culture. In conclusion, we have defined optimal conditions for HSCs isolation and culture.

Background: Transplantation of adipose-derived stem cells (ADSCs) is a potential therapy for a variety of liver diseases. Previous studies have shown that ADSC-based therapy is promising for liver fibrosis treatment. Recently, many publications have suggested that pretreating ADSCs with growth factors before transplantation can elevate the effectiveness of the therapy. Therefore, we hypothesize that human ADSCs (hADSCs) pretreated with platelet-rich plasma (PRP) and hepatocyte growth factor (HGF), compared to ADSCs alone, would accelerate the treatment effects on liver fibrosis in mice. Methods: The hADSCs were cultured solely with conventional media, or with HGF (human recombinant; 20 ng/ml), or with HGF and PRP (from healthy human blood, 10%), concomitantly added to the medium for 7 days before transplantation. Eight-week-old male mice were treated with CCl4 (1 ml/kg) for 11 weeks to induce liver fibrosis. The mice were then subsequently divided into: 1) Placebo group (PBS injection); 2) ADSCs/HGF+PRP (5x105HGF and PRP pre-treated cells/mice); 3) ADSCs/HGF (5x105HGF pre-treated cells/mice) and 4) ADSCs (5x105non-pretreated cells/mice). Results: Seven days post-transplantation, the alanine transaminase (ALT) level in the placebo was notably elevated (143.10±14.96 IU/L), compared to ALT levels of ADSCs-, ADSCs/HGF+PR-, and ADSCs/HGF-transplanted mice, which showed an improvement (67.94±18.57 IU/L, 49.44±7.56 IU/L, and 57.93±5.75 IU/L, respectively). The procollagen-α1 and alpha-smooth muscle actin (α-SMA) mRNA levels were significantly down-regulated in the ADSCs-transplanted group compared to those of the placebo. Importantly, these levels were lower in ADSCs/HGF+PR (procollagen-α1: 75.64±45.89; α-SMA: 36.17±36.09) than those of ADSCs/HGF (procollagen-α1: 212.8±84.35; α-SMA: 52.41±7.93) and ADSCs only (procollagen-α1: 310.50 ± 55.36; α-SMA: 184.70±14.06). Stem cell transplantation also improved histological index, reducing inflammation and collagen/necrotic structure accumulation. However, there were no statistical differences between three ADSCs treatment groups after 14 days after transplantation. Conclusion: Pre-treatment with PRP and HGF for 7 days enhanced the efficacy of ADSCs in alleviating liver fibrosis in vivo.

Mesenchymal stem cells (MSCs) are now an attractive source of cell therapy. We test the hypothesis that autologous adipose-derived mesenchymal stem cells ameliorate liver cirrhosis in mice. In this study, male Swiss mice were treated orally with olive oil or CCl4 for 11 weeks (3 times/week). Mouse adipose-derived stem cells (mADSCs) from adipose tissue of mice injuried by CCl4 for 3 weeks were cultured prior to transfer by tail vein injection. Hepatocyte-enriched markers were characterized using q-RT-PCR and MSC markers, hepatocyte-enriched proteins, and fibrosis were evaluated by flow cytometry and/or immunohistochemistry. Mice were divided into 4 groups (n = 10 each group): (1) normal, (2) cirrhotic, (3) cirrhotic/PBS, (4) cirrhotic/mADSCs (5 × 105 cells/mice). A separate set of mADSCs was labeled with CFDA to investigate cell homing. The results of in vitro evaluation on mADSCs showed that these cells are highly expression variety of hepatic-related genes such as Hgf, Alb, Ck18, Ck19, Cyp1a1, Afp, MUC1, Ldl receptor and strongly expression of Cyp1a1 and Hgf markers. Dose of 5 × 105 cells/animal improved AST/ALT/bilirubin/albumin index after 7 days of injection (p < 0.05); significantly down-regulate gene expression of TGF-beta 1 (14 fold less), procollagen (3 fold less) and nt5e (8 fold less), (p < 0.05). 100% mice improved histology index (HAI modified) and diminished the accumulation of collagen fibers after 21 days compared to 33.3% cirrhotic/PBS. mADSCs “home” to the liver tissue after 21 days.

Introduction: To date, there have been many studies indicating the positive effects of stem cells on treating liver cirrhosis. In this study, we used umbilical cord blood-derived mesenchymal stem cells (UCB-MSCs) for treatment in a mouse model of liver cirrhosis. Specifically, we determined and compared the effectiveness of two methods of MSC injection (tail vein versus portal vein). Methods: Liver cirrhosis in male Swiss mice (of age approximately 11 weeks or under) was induced by administration of carbon tetrachloride (CCl4; 1 ml/kg). One million UCB-MSCs were then transplanted into cirrhotic mice via the portal vein or tail vein. After 21 days, blood samples were collected for measurement of transaminase, bilirubin and albumin. The expression of fibrosis-associated genes, specifically procollagen – alpha 1 and integrin – beta1, were assessed using quantitative RT-PCR. The histopathology of the specimens was also evaluated using hematoxylin/eosin, Masson trichrome staining, and immunohistochemistry using collagen type 1 and alpha-SMA antibodies. Results: After 21 days, cirrhotic mice treated with UCB-MSCs showed recovery of bilirubin index, increase of liver albumin synthesis, inhibition of fibrosis-related gene expression (e.g. procollagen – alpha 1 and integrin – beta1), and remodeling of liver histology. From comparison of the different routes of transplantation, UCB-portal route was significantly more effective than UCB-tail route at reducing aspartate transaminase (AST) activity and bilirubin index (P

Background: Stem cell therapy in liver cirrhosis treatment is attracting the attention of the scientific community. Adipose tissue-derived mesenchymal stem cells are a potential source of cells because they have self-renewal, high proliferation, and differentiation into a variety of cell types, including hepatocytes as potential cell sources for cirrhosis treatment. Platelet-rich plasma (PRP) growth factors contribute to regeneration and wound healing. We test the hypothesis that PRP co-administration enhances MSC treatment for mouse cirrhosis. Method: Male Swiss mice were treated orally with olive oil or CCl4 for 11 weeks. PRP was obtained from healthy mice. Mouse adipose-derived stem cells (mADSCs) from adipose tissue of 3 weeks CCl4 mice were cultured for three passages (P3-mADSCs) before the transfer by tail vein injection with or without PRP into 11 weeks CCl4 mice. Mice were divided into six groups (n=10 each group). 1) normal, 2) cirrhotic, 3) cirrhotic /PBS; 4) cirrhotic/PRP (0.2 ml/mice with PRP from healthy mice), 5) cirrhotic/mADSCs (5 x 105 cells/mice), and 6) cirrhotic/mADSC-PRP. Result: mADSCs were highly positive for CD44, CD90, and CD105. Relative to liver cells, P3-mADSCs highly expressed Alb, Ck18, Ck19, Tnf, c-met, Cyp1a1, Afp, Muc1, Ldl receptor. mADSCs were strongly positive for Cyp1a1 (98.21±1.57%) and Hgf (95.55±3.11%); moderately positive for alfa-fetoprotein (45.99±2.08%), Aat (44.43±7.79%), Alb (57.81±8.49%) and differentiated into hepatocyte-like cells under induction medium. After transplantation, CFDA-transplanted cells into CCl4-treated mice were found in the liver at day 21 st. Compared to mADSCs, mADSCs and PRP co-treatment most effectively improved serum AST/ALT/bilirubin and albumin levels in day seven cirrhotic mice (p

Background: Adipose-derived stem cells (ADSCs) have the potential therapeutic impact on the liver fibrosis. Hepatocyte growth factor (HGF) is pivotal for damage repair with its anti-apoptotic, anti-fibrotic and cell migration-promoting effect. In this study, the therapy with HGF-pretreated hADSCs was expected to enhance the therapeutic effect in the amelioration of liver fibrosis compared with untreated hADSCs. Method: HGF-hADSCs were prepared by culturing hADSCs for seven days in the medium added HGF. HGF-hADSCs transplantation was performed to investigate the therapeutic effect of these cells on carbon tetrachloride (CCl4)-induced liver fibrosis in a mouse model. Results: After seven days and fourteen days of cell transfusion, HGF-hADSCs ameliorated the liver fibrosis. The results showed the attenuation of the liver injury (ALT level), the down-regulation of procollagen-1 (7 days, 3-fold; 14 days, 6.7-fold) and alpha -smooth muscle actin (alpha-SMA) expression (7 days, 10-fold; 14 days, 2-fold), and the histological improvement. Notably, there was the significant difference in the procollagen-1 between HGF-hADSCs and untreated hADSCs groups. Thus, the therapy with HGF-hADSCs was more efficient in the liver fibrosis treatment compared with untreated hADSCs. Conclusion: HGF pretreated hADSCs have a potential therapy in the treatment of liver fibrosis

Background: Because of their ease of isolation, high proliferation capacity in vitro, as well as their ability to differentiate into liver cells, Adipose-derived stem cell (ADSCs), are considered to be a promised candidate for liver disease treatment, including liver cirrhosis treatment. Recent studies show that hepatocyte growth factor (HGF) can stimulate ADSC to migrate to the injured areas and platelet rich plasma (PRP) can increase the stemness of ADSCs. Method: In this study, we cultured ADSCs in the medium supplemented 20 ng/ml and 10% PRP; then transplanted them into the cirrhosis mouse model. After 11 weeks of CCl4 induction (1ml/kg, three times per week), mice were divided into 3 groups: (1) PBS group which were injected 0.2 ml PBS; (2) ADSC group which were transplanted 5x105 ADSCs non-pretreated with HGF and PRP; (3) ADSC/HGF+PRP group which were transplanted 5x105 ADSCs pretreated with HGF and PRP via the tail vein. We evaluated the effectiveness of the therapeutic at the day 7th and 14th after transplantation. Results: The results show that the transplantation of ADSC pretreated with HGF and PRP after seven days improves the body weight (increase 4.673%); decreases the ALT level (p < 0.05), the total leukocyte number (p < 0.05) and the expression of Pro-collagen (decrease 4.1 times) as well as α-SMA (decrease 5.1 times), in comparison to the ADSC group. ADSCs pretreated with HGF and PRP also help to improve the liver tissue structure. Conclusion: The therapy using ADSCs pretreated with HGF and PRP is considered to be a promising therapy.

Background: Up to date, there have been some studies indicating positive effects of stem cells on treating the liver cirrhosis. In this study, we compared the effectiveness of two methods in which mesenchymal stem cells harvested from umbilical cord blood (UCB-MSCs) were transfused either via portal or tail veins to the mouse models of liver cirrhosis. Methods: Liver cirrhosis was induced by CCl4 (1 ml/kg) on male Swiss mice within 11 weeks, followed by administration of 106 UCB-MSCs via the portal or tail vein. After 21 days, blood samples were collected for measuring transaminase, bilirubin and albumin activities. The expression of fibrosis-associated genes, specifically procollagen – alpha 1 and integrin – beta1, were assessed using qRT-PCR. The histopathology was also evaluated using hematoxylin/eosin, Masson trichrome staining and immunohistochemistry with collagen type 1 and alpha-SMA antibody. Results: UCB-MSCs transplantation significantly improved post-21 days of treatment in the liver fibrosis mice as compared with placebo group. Notably, UCB-MSCs transferred through portal veins revealed a more positive effect than via tail veins as indicated by the improvement in the biochemical indexes, fibrosis-related genes expression, and liver histopathology. Conclusion: The UCB-MSCs therapy proved to be a promising method for treating the liver cirrhosis. The method of delivering stem cells through portal vein was more effective than through tail vein

Background: The application of mesenchymal stem cell (MSC) therapy in liver fibrosis treatment has been increasingly investigated in recent years. MSCs obtained from a variety of sources (e.g. bone marrow, umbilical cord blood and adipose tissue) have been studied and have achieved remarkable results. In this study, we compared the effects of adipose-derived mesenchymal stem cells (AD-MSC) transplantation with bone marrow-derived mesenchymal stem cell (BM-MSC) transplantation in a mouse model of liver fibrosis, induced by carbon tetrachloride (CCl4). Methods: Eight-week old mice were treated with CCl4 for 11 weeks to induce liver fibrosis then 5x105 cells were transplanted into mice via the tail vein. Results: After 21 days of transplantation, the results showed that the stem cell treated groups ameliorated better than the placebo group. MSC treated groups showed reduced AST and ALT levels, down-regulated expression of extracellular matrix (ECM) genes, and improved liver histopathology. Both sources of MSCs (bone marrow and adipose tissue) were effective in the mouse model of liver fibrosis. Conclusion: Our results also indicated that AD-MSC transplantation in mice accelerated liver regeneration better than BM-MSC transplantation.

International audience; no abstract

Noise and hearing ability profiles were determined in a textile factory in Vietnam. Noise mapping done in the weaving section showed that the noise levels exceeded the Vietnamese standard of 90 dBA by as much as 9 dBA in some areas. Audiometric tests performed on 69 female workers from the weaving section revealed that workers with more than 10 years of noise exposure had the worst hearing threshold levels at 1,000 and 4,000 Hz. Similar findings were observed for workers greater than 35 years old. The 4,000 Hz notch, suggestive of exposure to intense noise, was noted in the audiograms of 26 subjects.