Premalignant Alteration Assessment In Liver-like Tissue Derived From Embryonic Stem Cells By Aristolochic Acid I Exposure

Part ?.Establishment of premalignant alteration assessment model during the differentiation of embryoid bodies into liver-like tissue with AAI exposureObjective:Investigating whether the differentiation process of embryonic stem(ES)cells-derived embryoid bodies(EBs)into liver-like tissue with aristolochic acid I(AAI)exposure could be a reliable in vitro model for predicting the hepatic premalignant alterations and looking into the early pathological alterations of chemical-induced hepatic carcinogenesis.Methods:ES cells were cultivated in hanging drops to form EBs for 5 days.On the 5th day(D 5+0),EBs were plated into culture plates and cultured for another 18 days to generate mature liver-like tissue.At D 5+18,immunofluorescence staining assay was applied to identify in situ expression of liver-specific biomarkers such as albumin(ALB),hepatocyte nuclear factor 4?(HNF4?),?1-antitrypsin(AAT).Hematoxylin and eosin(H&E)staining was used to identify the structure phenotype of liver-like tissue.qRT-PCR was used to detect the mRNA expression levels of liver-specific functional genes.ALB secretion,Periodic Acid-Schiff(PAS)staining,Indocyanine Green(ICG)uptake and urea production assays were used to investigate functional characterizations of mature liver-like tissue.For AAI treatment,the concentrations of AAI to be incubated with EBs were determined by MTT assay.EBs were cultured from D 5+0 to D 5+18 with AAI incubation.On D 5+18,flow cytometry was used to detect the differentiation ratio of hepatocyte-like cells.ELISA was used to detect the levels of ALB,alanine transaminase(ALT),aspartate aminotransferase(AST),interleukin 6(IL-6)and a-fetoprotein(AFP)in the culture supernatant.Immunofluorescence staining assay was applied to identify the co-expression of ALB with IL-6,signaling transducer and activator of transcription-3(STAT3),p-STAT3,AFP,oncogenic transcription factor Myc(c-Myc)and oncofetal RNA-binding protein Lin28 homolog B(Lin28B).Western Blot was used to detect the expression of c-Myc,Lin28B,AFP,signaling pathways of IL-6/STAT3,transcription factor nuclear factor ?B(NF-?B),mitogen-activated protein kinase(MAPK)and activator protein 1(AP-1)in the liver-like tissue.For in vivo premalignant evaluation,the mature liver-like tissues were digested into single cell suspension and injected subcutaneously into the back of mice.H&E staining was used to examine the composition of the xenografts excised from the mice.Immunofluorescence was applied to identify the expressions of ALB with AFP,cytokeratin 7(K7)and K19.Western Blot and qRT-PCR were used to detect the expressions of genes and proteins related to hepatic premalignancy.Results:1.Identifications of the mature liver-like tissue derived from ES cells:(1)morphological characterizations:hepatocytes displayed the typical bi-nuclear phenotype and stained positive for ALB with HNF4?,AAT,PECAM-1 and K19;H&E staining showed the typical sinusoid-like structures of mature liver-like tissue;(2)functional characterizations:qRT-PCR results showed that the expression levels of liver-specific genes in the liver-like tissue such as Aat,G6p,Cyp1a2,Cyp2e1,Cyp7a1 and Ugt1a6 were similar with those of mouse adult livers;the hepatocyte-like cells showed stable secretion levels of ALB,urea production,PAS staining and ICG uptake.2.The establishment of in vitro premalignant evaluation system based on the differentiation process of ES cells-derived EBs into liver-like tissue:(1)At D 5+18,flow cytometry,immunofluorescence and ELISA results showed that 1.25?M and 2.50 pM AAI did not affect the differentiation ratio of hepatocyte-like cells,ALB secretion level and the sinusoid-like structures of liver-like tissue.(2)ELISA results showed that compared to DMSO,ALT,AST,IL-6 and AFP levels in the supernatant with AAI treatment were significantly increased,suggesting that the Kupffer-like cells in the liver-like tissue were activated and secreted large amounts of IL-6.Immunofluorescence staining revealed that c-Myc and Lin28B were co-expressed with ALB in several hepatocyte-like cells in response to AAI treatment,indicating that it was hepatocyte-like cells that underwent premalignant transformation.Furthermore,AFP and octamer-binding transcription factor 4(Oct4)were also co-expressed with ALB in cytoplasm of a few hepatocyte-like cells by AAI exposure,suggesting that AAI triggered the dedifferentiation of hepatocyte-like cells in the mature liver-like tissue.(3)Western Blot results showed that IL-6/STAT3 pathway proteins,phosphorylation of extracellular signal-regulated kinase 1/2(p-ERKl/2)and AP-1 were significantly increased by AAI treatment,suggesting that AAI can activate IL-6/STAT3,MAPK signaling pathways and AP-1 to induce hepatic inflammation and increase the risk of precancerous lesions.(4)In the in vivo evaluation,liver-like tissues treated with DMSO or AAI can form xenografts with similar size at the indicated time.The ratio of survived mice in both groups was also similar.The ratio of xenografts with HCC-like phenotypic sections robustly increased,and reached 54%(13/24)in AAI xenografts compared with DMSO xenografts(1/23).H&E staining showed that compared to DMSO xenografts,uniform area of hepatocyte-like cells with malignant nuclei appeared in the xenografts growing from AAI-exposed EBs.The cells exhibited bigger and deep-dyed nuclei,arranging in disorder,pseudolobule or micro vascular invasion.Moreover,compared with DMSO,the teratomas in AAI xenografts showed the much smaller size.Immunofluorescence staining revealed that xenografts in the AAI group with malignant phenotypic cells co-expressed ALB with AFP,K7 and K19 respectively compared with DMSO xenografts.The co-expression ratios were 37.50%,54.17%and 50.00%respectively.Conclusions:AAI induced inflammatory response with precancerous lesions in the differentiation process of EBs into liver-like tissue derived from murine ES cells.This in vitro premalignant assessment model could provide a set of correlate and evaluative biomarkers and allow evaluating either hepatic premalignant alterations or anti-hepatic canceration activity of chemicals on the different segments of hepatocarcinogenicity.Part ?.Establishment of premalignant alteration assessment model in the mature liver-like tissue with AAI exposureObjective:Investigating whether the ES cells-derived mature liver-like tissue by aristolochic acid I(AAI)exposure could be a reliable in vitro model for predicting the hepatic premalignant alterations and looking into the early pathological alterations of chemical-induced hepatic carcinogenesis.Methods:ES cells were cultivated in hanging drops to form EBs for 5 days.On the 5th day(D 5+0),EBs were cultured for 18 days to generate mature liver-like tissue.For AAI treatment,the concentrations of AAI to be incubated with mature liver-like tissue were determined by MTT assay.On the D 5+18,the mature liver-like tissue was incubated with indicated concentrations of AAI for 10 days(from D 23+0 to D 23+10).On the D 23+10,ELISA was used to detect the secretion levels of ALB and IL-6 in the culture supernatant.Immunofluorescence staining was applied to identify the co-expression of ALB with p-STAT3,AFP,c-Myc,Lin28B and NF-?B(p65,p50).Results:ELISA and immunofluorescence results showed that 1.25 and 2.50 ?M AAI did not significantly downregulate expression and secretion of ALB;5.00 and 10.00 ?M AAI significantly downregulate secretion of ALB from D 23+8 and D 23+6 respectively compared with DMSO.10.00 ?M AAI even induced apoptosis of some hepatocyte-like cells.1.25,2.50,5.00 and 10.00 ?M AAI could significantly upregulate secretion level of IL-6;when incubated with 10.00 ?M AAI,secretion of IL-6 increased from D 23+2 to D 23+4 but decreased from D 23+4 to D 23+10.Collectively,5.00 ?M AAI was used for further investigations.Immunofluorescence results showed that 5.00 ?M AAI triggered expression of c-Myc,Lin28B,AFP and NF-?B(p65)in a few hepatocyte-like cells,indicating that premalignant alterations could also be induced when the mature liver-like tissue was incubated with AAI.Conclusions:AAI induced inflammatory response with precancerous lesions in the mature liver-like tissue derived from ES cells.This in vitro premalignant assessment model could allow evaluating hepatic premalignant alterations induced by chemicals on the different segments of hepatocarcinogenicity.Addendum:Evaluation in anti-hepatic canceration activity of Asiatic acid and Schisandrin B on AAI-induced premalignant transformation during the differentiation process of ES cells-derived EBs into liver-like tissueObjective:To explore whether Asiatic acid(AA)and Schisandrin B(Sch B)possessed the potential anti-hepatic canceration activity against AAI-induced inflammation and premalignant lesions during the differentiation process of mature liver-like tissue from ES cells-derived EBs.Methods:The concentrations of AA and Sch B to be incubated with EBs were measured by MTT assay.On the D 5+0,2.5?M AAI and appropriate concentrations of AA or Sch B were incubated with EBs for 18 days.At terminal of differentiation,flow cytometry was used to detect the differentiation ratio of hepatocyte-like cells.ELISA was used to detect the secretion levels of ALB,ALT,IL-6 and AFP in the culture supernatant.Immunofluorescence staining assay was utilized to detect the co-expression of ALB with IL-6,c-Myc,Lin28B and p-STAT3.Western Blot was used to detect the expressions of c-Myc,Lin28B,AFP and inflammation-related proteins and signaling pathways.Then the mature liver-like tissues treated with DMSO,AAI,and AAI+AA respectively were digested into single cell suspension and injected subcutaneously into the back of mice.H&E staining was used to examine the composition of the xenografts excised from the mice.Immunofluorescence assay was applied to identify the expressions of ALB with AFP,K7 and K19.Results:(1)ELISA results showed that the levels of ALT and IL-6 in the supernatant were significantly downregulated with 1.00 ?M AA and 5.0,10.0 ?M Sch B compared to those only with AAI.AFP level in the supernatant was significantly downregulated when treated with 1.00?M AA and 2.5 ?M AAI in contrast to AAI only treatment.However,Sch B could not downregulate AFP level in the culture supernatant.(2)Western Blot and immunofluorescence results showed that 1.00 ?M AA could significantly downregulate the expression of c-Myc,Lin28B and AFP triggered by AAI,but Sch B could not downregulate the expression of AFP,c-Myc and Lin28B induced by AAI;AA and Sch B could significantly downregulate the expression of IL-6/STAT3,p-ERK1/2 and c-Fos induced by AAI,and 10.0 ?M Sch B could significantly upregulate the expression of Nrf2.(3)During the in vivo evaluatio,H&E staining showed that the number of HCC-like tissue phenotypes in the AAI xenografts(11/23)was significantly higher than that in the DMSO xenografts(1/22),while there was no significant difference in the number of HCC-like tissue phenotypes between AAI+AA(10/23)and AAI xenografts.Immunofluorescence results showed compared with the DMSO xenografts,the co-staining ratio of ALB with AFP,K7 and K19 increased to 43.48%,69.57%and 65.22%respectively in AAI xenografts.In the AAI+AA xenografts,the ratio of ALB+AFP staining was significantly reduced to 17.39%,while the co-staining ratios of ALB with K7 and K19 did not significantly change.Conclusion:AA probably had the potential anti-hepatic canceration activity against the in vitro inflammation and precancerous lesions induced by AAI in differentiation process from ES cells-derived EBs to mature liver-like tissue.Sch B probably had the potential anti-inflammatory activity against AAI in vitro,but had no effects on the premalignant alterations triggered by AAI treatment.