MSC-derived exosomes attenuate hepatic fibrosis in primary sclerosing cholangitis through inhibition of Th17 differentiation

Primary sclerosing cholangitis(PSC)is an autoimmune cholangiopathy characterized by chronic inflammation of the biliary epithelium and periductal fibrosis,with no curative treatment available,and liver transplantation is inevitable for end-stage patients.Human placentalmesenchymal stem cell(hpMSC)-derived exosomes have demonstrated the ability to prevent fibrosis,inhibit collagen production and possess immunomodulatory properties in autoimmune liver disease.Here,we prepared hpMSC-derived exosomes(Exo^(MSC))and further investigated the anti-fibrotic effects and detailed mechanism on PSC based on Mdr2^(−/−)mice and multicellular organoids established from PSC patients.The results showed that Exo^(MSC) ameliorated liver fibrosis in Mdr2^(−/−)mice with significant collagen reduction in the preductal area where Th17 differentiation was inhibited as demonstrated by RNAseq analysis,and the percentage of CD4+IL-17A+T cells was reduced both in Exo^(MSC)-treated Mdr2^(−/−)mice(Mdr2^(−/−)-Exo)in vivo and Exo^(MSC)-treated Th17 differentiation progressed in vitro.Furthermore,Exo^(MSC) improved the hypersecretory phenotype and intercellular interactions in the hepatic Th17 microenvironment by regulating PERK/CHOP signaling as supported by multicellular organoids.Thus,our data demonstrate the antifibrosis effect of Exo^(MSC) in PSC disease by inhibiting Th17 differentiation,and ameliorating the Th17-induced microenvironment,indicating the promising potential therapeutic role of Exo^(MSC) in liver fibrosis of PSC or Th17-related diseases.

Dynamic changes of key metabolites during liver fibrosis in rats

BACKGROUND Fibrosis is the single most important predictor of significant morbidity and mortality in patients with chronic liver disease.Established non-invasive tests for monitoring fibrosis are lacking,and new biomarkers of liver fibrosis and function are needed.AIM To depict the process of liver fibrosis and look for novel biomarkers for diagnosis and monitoring fibrosis progression.METHODS CCl4 was used to establish the rat liver fibrosis model.Liver fibrosis process was measured by liver chemical tests,liver histopathology,and Masson’s trichrome staining.The expression levels of two fibrotic markers includingα-smooth muscle actin and transforming growth factorβ1 were assessed using immunohistochemistry and real-time polymerase chain reaction.Dynamic changes in metabolic profiles and biomarker concentrations in rat serum during liver fibrosis progression were investigated using ultra-performance liquid chromatography coupled to quadrupole time-of-flight mass spectrometry.The discriminatory capability of potential biomarkers was evaluated by receiver operating characteristic(ROC)curve analysis.RESULTS To investigate the dynamic changes of metabolites during the process of liver fibrosis,sera from control and fibrosis model rats based on pathological results were analyzed at five different time points.We investigated the association of liver fibrosis with 21 metabolites including hydroxyethyl glycine,L-threonine,indoleacrylic acid,β-muricholic acid(β-MCA),cervonoyl ethanolamide(CEA),phosphatidylcholines,and lysophosphatidylcholines.Two metabolites,CEA andβ-MCA,differed significantly in the fibrosis model rats compared to controls(P<0.05)and showed prognostic value for fibrosis.ROC curve analyses performed to calculate the area under the curve(AUC)revealed that CEA andβ-MCA differed significantly in the fibrosis group compared to controls with AUC values exceeding 0.8,and can clearly differentiate early stage from late stage fibrosis or cirrhosis.CONCLUSION This study identified two novel biomarkers of fibrosis,CEA andβ-MCA,which were effective for diagnosing fibrosis in an animal model.

Hypoxia-inducible factor-1α–mediated upregulation of CD99 promotes the proliferation of placental mesenchymal stem cells by regulating ERK1/2

BACKGROUND As human placenta-derived mesenchymal stem cells(hP-MSCs)exist in a physiologically hypoxic microenvironment,various studies have focused on the influence of hypoxia.However,the underlying mechanisms remain to be further explored.AIM The aim was to reveal the possible mechanisms by which hypoxia enhances the proliferation of hP-MSCs.METHODS A hypoxic cell incubator(2.5%O2)was used to mimic a hypoxic microenvironment.Cell counting kit-8 and 5-ethynyl-20-deoxyuridine incorporation assays were used to assay the proliferation of hP-MSCs.The cell cycle was profiled by flow cytometry.Transcriptome profiling of hP-MSCs under hypoxia was performed by RNA sequencing.CD99 mRNA expression was assayed by reverse transcription-polymerase chain reaction.Small interfering RNA-mediated hypoxia-inducible factor 1α(HIF-1α)or CD99 knockdown of hP-MSCs,luciferase reporter assays,and the ERK1/2 signaling inhibitor PD98059 were used in the mechanistic analysis.Protein expression was assayed by western blotting;immunofluorescence assays were conducted to evaluate changes in expression levels.RESULTS Hypoxia enhanced hP-MSC proliferation,increased the expression of cyclin E1,cyclin-dependent kinase 2,and cyclin A2,and decreased the expression of p21.Under hypoxia,CD99 expression was increased by HIF-1α.CD99-specific small interfering RNA or the ERK1/2 signaling inhibitor PD98059 abrogated the hypoxia-induced increase in cell proliferation.CONCLUSION Hypoxia promoted hP-MSCs proliferation in a manner dependent on CD99 regulation of the MAPK/ERK signaling pathway in vitro.

Mesenchymal stem cells from the human umbilical cord ameliorate fulminant hepatic failure and increase survival in mice

BACKGROUND：Cell therapy has been promising for various diseases.We investigated whether transplantation of human umbilical cord mesenchymal stem cells（h UCMSCs）has any therapeutic effects on D-galactosamine/lipopolysaccharide（Gal N/LPS）-induced fulminant hepatic failure in mice.METHODS：h UCMSCs isolated from human umbilical cord were cultured and transplanted via the tail vein into severe combined immune deficiency mice with Gal N/LPS-induced fulminant hepatic failure.After transplantation,the localization and differentiation of h UCMSCs in the injured livers were investigated by immunohistochemical and genetic analy- ses. The recovery of the injured livers was evaluated histologi- cally. The survival rate of experimental animals was analyzed by the Kaplan-Meier method and log-rank test. RESULTS： hUCMSCs expressed high levels of CD29, CD73, CD13, CD105 and CD90, but did not express CD31, CD79b, CD133, CD34, and CD45. Cultured hUCMSCs displayed adip- ogenic and osteogenic differentiation potential. Hematoxylin and eosin staining revealed that transplantation of hUCMSCs reduced hepatic necrosis and promoted liver regeneration. Transplantation of hUCMSCs prolonged the survival rate of mice with fulminant hepatic failure. Polymerase chain reaction for human alu sequences showed the presence of human cells in mouse livers. Positive staining for human albumin, human alpha-fetoprotein and human cytokeratin 18 suggested the for- mation of hUCMSCs-derived hepatocyte-like cells in vivo.CONCLUSIONS： hUCMSC was a potential candidate for stem cell based therapies. After transplantation, hUCMSCs partially repaired hepatic damage induced by GalN/LPS in mice. hUC- MSCs engrafted into the injured liver and differentiated into hepatocyte-like cells.

Assessment of biological functions for C3A cells interacting with adverse environments of liver failure plasma

Background:For its better differentiated hepatocyte phenotype,C3A cell line has been utilized in bioar-tificial liver system.However,up to now,there are only a few of studies working at the metabolic alter-nations of C3A cells under the culture conditions with liver failure plasma,which mainly focus on car-bohydrate metabolism,total protein synthesis and ureagenesis.In this study,we investigated the effects of acute liver failure plasma on the growth and biological functions of C3A cells,especially on CYP450 enzymes.Methods:C3A cells were treated with fresh DMEM medium containing 10%FBS,fresh DMEM medium containing 10%normal plasma and acute liver failure plasma,respectively.After incubation,the C3A cells were assessed for cell viabilities,lactate dehydrogenase leakage,gene transcription,protein levels,albu-min secretion,ammonia metabolism and CYP450 enzyme activities.Results:Cell viabilities decreased 15%,and lactate dehydrogenase leakage had 1.3-fold elevation in acute liver failure plasma group.Gene transcription exhibited up-regulation,down-regulation or stability for different hepatic genes.In contrast,protein expression levels for several CYP450 enzymes kept constant,while the CYP450 enzyme activities decreased or remained stable.Albumin secretion reduced about 48%,and ammonia accumulation increased approximately 41%.Conclusions:C3A cells cultured with acute liver failure plasma showed mild inhibition of cell viabilities,reduction of albumin secretion,and increase of ammonia accumulation.Furthermore,CYP450 enzymes demonstrated various alterations on gene transcription,protein expression and enzyme activities.

Long non-coding RNA SNHG16 promotes human placenta-derived mesenchymal stem cell proliferation capacity through the PI3K/AKT pathway under hypoxia

BACKGROUND The effect of hypoxia on mesenchymal stem cells(MSCs)is an emerging topic in MSC biology.Although long non-coding RNAs(lncRNAs)and messenger RNAs(mRNAs)are reported to play a critical role in regulating the biological characteristics of MSCs,their specific expression and co-expression profiles in human placenta-derived MSCs(hP-MSCs)under hypoxia and the underlying mechanisms of lncRNAs in hP-MSC biology are unknown.AIM To reveal the specific expression profiles of lncRNAs in hP-MSCs under hypoxia and initially explored the possible mechanism of lncRNAs on hP-MSC biology.METHODS Here,we used a multigas incubator(92.5%N_(2),5%CO_(2),and 2.5%O_(2))to mimic the potential of hP-MSCs.RNA sequencing technology was applied to identify the exact expression profiles of lncRNAs and mRNAs under hypoxia.RESULTS We identified 289 differentially expressed lncRNAs and 240 differentially expressed mRNAs between the hypoxia and normoxia groups.Among them,the lncRNA SNHG16 was upregulated under hypoxia,which was also validated by reverse transcription-polymerase chain reaction.SNHG16 was confirmed to affect hP-MSC proliferation rates using a SNHG16 knockdown model.SNHG16 overexpression could significantly enhance the proliferation capacity of hP-MSCs,activate the PI3K/AKT pathway,and upregulate the expression of cell cycle-related proteins.CONCLUSION Our results revealed the specific expression characteristics of lncRNAs and mRNAs in hypoxiacultured hP-MSCs and that lncRNA SNHG16 can promote hP-MSC proliferation through the PI3K/AKT pathway.

Human Placental Mesenchymal Stem Cells Relieve Primary Sclerosing Cholangitis via Upregulation of TGR5 in Mdr2^(-/-)Mice and Human Intrahepatic Cholangiocyte Organoid Models

Primary sclerosing cholangitis(PSC)is a biliary disease accompanied by chronic inflammation of the liver and biliary stricture.Mesenchymal stem cells(MSCs)are used to treat liver diseases because of their immune regulation and regeneration-promoting functions.This study was performed to explore the therapeutic potential of human placental MSCs(hP-MSCs)in PSC through the Takeda G protein-coupled receptor 5(TGR5)receptor pathway.Liver tissues were collected from patients with PSC and healthy donors(n=4)for RNA sequencing and intrahepatic cholangiocyte organoid construction.hP-MSCs were injected via the tail vein into Mdr2^(-/-),bile duct ligation(BDL),and 3,5-diethoxycarbonyl-1,4-dihydrocollidine(DDC)mouse models or co-cultured with organoids to confirm their therapeutic effect on biliary cholangitis.Changes in bile acid metabolic profile were analyzed by liquid chromatography/tandem mass spectrometry(LC-MS/MS).Compared with healthy controls,liver tissues and intrahepatic cholangiocyte organoids from PSC patients were characterized by inflammation and cholestasis,and marked downregulation of bile acid receptor TGR5 expression.hP-MSC treatment apparently reduced the inflammation,cholestasis,and fibrosis in Mdr2^(-/-),BDL,and DDC model mice.By activating the phosphatidylinositol 3 kinase/extracellular signal-regulated protein kinase pathway,hP-MSC treatment promoted the proliferation of cholangiocytes,and affected the transcription of downstream nuclear factorκB through regulation of the binding of TGR5 and Pellino3,thereby affecting the cholangiocyte inflammatory phenotype.

Immunomodulatory and Antiviral Therapy Improved Functional Cure Rate in CHB Patients with High HBsAg Level Experienced NA

Background and Aims:A functional cure,or hepatitis B virus(HBV)surface antigen(HBsAg)loss,is difficult to achieve in patients with hepatitis B virus e antigen(HBeAg)-positive chronic hepatitis B.The HBV vaccine and granulocyte-macrophage colony-stimulating factor(GM-CSF)have been reported to help reduce HBsAg levels and promote HBsAg loss.In this prospective randomized trial,we evaluated HBsAg loss in patients receiving pegylated interferon α2b(PEGIFN-α2b)and tenofovir disoproxil fumarate(TDF),with and without GM-CSF and HBV vaccination.Methods:A total of 287 patients with HBeAg positive chronic hepati-tis B and seroconversion after nucleot(s)ide analog treat-ment were assigned randomly to three treatment groups for 48 weeks,TDF alone(control),PEGIFN-α2b+TDF,and PEGIFN-α2b+TDF+GM-CSF+HBV vaccine.The prima-ry endpoints were the proportions of patients with HBsAg loss and seroconversion at 48 and 72 weeks.Resu/ts:The cumulative HBsAg loss rates in the control,PEGIFN-α2b+TDF,and PEGIFN-α2b+TDF+GM-CSF+HBV vaccine groups at week 48 were 0.0%,28.3%,and 41.1%,respec-tively.The cumulative HBsAg seroconversion rates in these groups at week 48 were 0.0%,21.7%,and 33.9%,respec-tively.Multivariate regression analysis showed that GM-CSF use plus HBV vaccination was significantly associated with HBsAg loss(p=0.017)and seroconversion(p=0.030).Con-clusions:In patients with HBeAg-positive chronic hepatitis B and seroconversion after nucleot(s)ide analog treatment,immunomodulatory/antiviral treatment regimens effective-ly improved HBsAg loss,and the regimen including GM-CSF and HBV vaccination was most effective.

Prognostic Nomogram for Patients with Hepatitis E Virus-related Acute Liver Failure:A Multicenter Study in China

Background and Aims:Timely and effective assessment scoring systems for predicting the mortality of patients with hepatitis E virus-related acute liver failure(HEV-ALF)are urgently needed.The present study aimed to establish an effective nomogram for predicting the mortality of HEV-ALF patients.Methods:The nomogram was based on a cross-sectional set of 404 HEV-ALF patients who were identified and enrolled from a cohort of 650 patients with liver failure.To compare the performance with that of the model for end-stage liver disease(MELD)scoring and CLIF-Consortiumacute-on-chronic liver failure score(CLIF-C-ACLFs)models,we assessed the predictive accuracy of the nomogram using the concordance index(C-index),and its discriminative ability using time-dependent receiver operating characteristics(td-ROC)analysis,respectively.Results:Multivariate logistic regression analysis of the development set carried out to predict mortality revealed that γ-glutamyl transpeptidase,albumin,total bilirubin,urea nitrogen,creatinine,international normalized ratio,and neutrophil-to-lymphocyte ratio were independent factors,all of which were incorporated into the new nomogram to predict the mortality of HEV-ALF patients.The area under the curve of this nomogram for mortality prediction was 0.671(95%confidence interval:0.602-0.740),which was higher than that of the MELD and CLIF-C-ACLFs models.Moreover,the td-ROC and decision curves analysis showed that both discriminative ability and threshold probabilities of the nomogram were superior to those of the MELD and CLIF-C-ACLFs models.A similar trend was observed in the validation set.Conclusions:The novel nomogram is an accurate and efficient mortality prediction method for HEV-ALF patients.