Effects And Epigenetic Mechanisms Of Culture Dimensional Changes On Hepatogenic Differentiation Of Mesenchymal Stem Cells

Introduction:Hepatocyte-like cells(HLCs)differentiated from mesenchymal stem cells for liver cell transplantation is considered an effective alternative treatment for liver diseases.Different culture dimensions can affect the induction efficacy of human adipose derived mesenchymal stem cells(hADMSCs)hepatogenic differentiation.Three-dimensional cell spheroids culture is regarded as a culture model more similar to the microenvironment in vivo,which not only enhances stemness of stem cells,but also enhances the phenotypes and functions of hepatocyte-like cells.Therefore,it is necessary to explore effects of different culture dimensions and dimensional transformations on hADMSCs differentiated into hepatocyte-like cells.Histone acetylation plays an important role in stem cell differentiation.Histone H3 lysine 56 acetylation(H3K56ac)modification can make DNA more relaxed and activate downstream gene transcription.Studies have shown that H3K56 acetylation regulates the expression of pluripotent transcription factors in human embryonic stem cells,and p300 can regulate H3K56 acetylation,thereby activating gene transcription by chromatin assembly.Our previous studies have found that compared to 2D differentiated HLCs,the transcription level of ALB in 3D differentiation showed significant changes.Histone acetylation regulates gene activation transcription in epigenetics.Therefore,we performed acetyl-proteomics assay on HLCs derived from both in 2D and 3D differentiation.The results showed that the level of H3K56 acetylation was significantly higher in 3D differentiation compared to 2D differentiation.However,whether different culture dimensions and dimensional transformations activate the ALB gene transcription in HLCs via p300-mediated H3K56 acetylation and their potential molecular mechanisms,which have not been reported thus far.The main focus of this study is to investigate the effects of different culture dimensions and dimensional transformations on hADMSCs differentiated into HLCs,and further explore the related epigenetic mechanisms involving p300-mediated H3K56 acetylation in HLCs under different culture dimensions and dimensional transformations.The drug screening platform and tissue engineering application were used to investigate the effect of dimensional transformations on hADMSCs differentiated into HLCs,and provide a new strategy for hADMSCs differentiated into HLCs and new technical supports and application prospects for hepatocytes drug screening and transplantation therapy.Part 1:Phenotypic changes and epigenetic mechanisms of hADMSCs differentiated into hepatocyte-like cells under different culture dimensionsObjective:To investigate the epigenetic mechanisms involved in the phenotypic changes of hADMSCs differentiated into HLCs under different culture dimensions.Methods:1.Effects of different culture dimensions on hepatic differentiation of hADMSCs(1)Isolation and identification of hADMSCs: hADMSCs were extracted from human adipose tissue using type I collagenase digestion.Cell morphology was observed under a microscope,and the expression of marker proteins of hADMSCs were detected by flow cytometry.Alizarin red and Oil red O staining were used to assess the osteogenic and adipogenic differentiation of hADMSCs,respectively.(2)Expression of pluripotency transcription factors of hADMSCs under different culture dimensions: 3D cell spheroids were constructed using agarose microwells.RT-qPCR and Western blot were used to detect the expression of pluripotency transcription factors Sox2,Oct4,and Nanog.(3)hADMSCs differentiated into HLCs under different culture dimensions: Cell morphology was observed under a microscope during the induction process.Live/Dead staining was used to detect cell viability after induction.RT-qPCR,Western blot and immunofluorescence analysis were used to assess mRNA and protein expression level of liver-specific genes.ELISA and quantitative colorimetric method were used to measure the secretion of albumin and urea,respectively.Periodic acid-Schiff(PAS)staining was used to detect glycogen synthesis.2.Epigenetic mechanisms underlying the effects of different culture dimensions on hADMSCs hepatogenic differentiation(1)Proteomic analysis of acetylation modifications in HLCs differentiated from hADMSCs under different culture dimensions.Differentially expressed acetylation-modified proteins will be screened,and bioinformatics analysis methods were used to annotate protein functions and enrichment analysis.(2)Validation of the differential expression of the target protein H3K56 ac using Western blot and immunofluorescence assays.The enrichment efficiency of H3K56 ac in the promoter region of ALB was detected by chromatin immunoprecipitation quantitative PCR(ChIP-qPCR).(3)The role of p300 in hADMSCs hepatogenic differentiation under different culture dimensions: A-485 was used to inhibit the effect of p300 in hepatic differentiation.The experiment was divided into six groups: 2D-DMSO,2D-A-485,2D-CTB,3D-DMSO,3D-A-485,and 3D-CTB.RT-qPCR was used to detect mRNA expression,and Western blot and immunofluorescence analysis were used to detect the protein expression levels of ALB,AFP,H3K56 ac,Sox2,and Oct4.ELISA and colorimetry methods were used to measure the secretion of albumin and urea,respectively.PAS staining was used to detect glycogen synthesis.Results:1.Effects of different culture dimensions on hADMSCs hepatic differentiation(1)Successful extraction of hADMSCs: cells exhibited spindle-shaped fibroblast-like morphology with a vortex arrangement and rapid proliferation;The expression of mesenchymal surface markers CD44,CD90 and CD105 was positive,while the expression of hematopoietic stem cell markers CD34,CD45 and human leukocyte antigen class II HLA-DR was negative;cells could undergo osteogenic and adipogenic differentiation,meeting the phenotypic characteristics of mesenchymal stem cells.(2)Three-dimensional culture significantly increased the mRNA and protein levels of Sox2,Oct4,and Nanog in hADMSCs.(3)After 21 days of hepatic differentiation,the cell morphology changed from fibrous spindle-shape to polygonal shape,and both 2D and 3D-induced HLCs exhibited high cell viability;compared with 2D induction,3D induction significantly upregulated the mRNA expression of liver-related genes ALB,CK18,CYP1A2,and CYP3A4 and the protein expression of ALB,and significantly decreased the mRNA and protein expression of AFP;3D induction significantly increased the secretion of albumin and urea,and increased the intracellular glycogen synthesis.2.Epigenetic mechanism of different culture dimensions on hepatocyte-like cells differentiated from hADMSCs(1)According to the results of acetyl-proteomics analysis,significantly upregulated acetylation of H3K56 and acetyltransferase p300,involved in gene transcription activation and acetyltransferase p300,were identified.There was a direct protein interaction between H3K56 ac and p300.(2)Compared to 2D-HLCs,3D-HLCs exhibited a significantly increase in acetylation level of H3K56,upregulated ALB protein expression,and downregulated the AFP protein expression.The H3K56 acetylation level on the ALB gene promoter region was significantly increased in 3D-HLCs.(3)The role of p300 in hADMSCs hepatic differentiation under different culture dimensions:(1)For 2D differentiation: Compared to DMSO normal group,the p300 inhibitor A-485 significantly inhibited the polygonal cell morphology transformation from hADMSCs to HLCs,while the p300 activator CTB promoted cell morphology transformation to polygonal shape;A-485 significantly decreased the protein expression of ALB and the acetylation level of H3K56,and increased the protein expression of Sox2,while CTB significantly up-regulated the acetylation level of H3K56 and down-regulated the protein expression of ALB and AFP;A-485 significantly inhibited the secretion of albumin and urea,and decreased the intracellular glycogen synthesis,while CTB significantly increased the secretion of urea.(2)For 3D induction: Compared to DMSO group,A-485 significantly decreased the protein expression of ALB and the acetylation level of H3K56,and increased the protein expression of Sox2,while CTB did not increase the acetylation level of H3K56 and the protein expression level of ALB;A-485 significantly inhibited the secretion of albumin and urea,while CTB significantly increased the secretion of albumin and urea.Conclusion:hADMSCs were successfully differentiated into HLCs under different culture dimensions.Three-dimensional differentiation enhanced the phenotypes and functions of HLCs,the mechanism involved p300-mediated H3K56 acetylation,which activated ALB transcription,then promoted hADMSCs hepatic differentiation.Part 2: Phenotypic changes and epigenetic mechanisms of hADMSCs differentiated into hepatocyte-like cells after culture dimensional transformationObjective:To investigate the potential epigenetic mechanisms of hADMSCs differentiated into hepatocyte-like cell after culture dimensional transformation.Methods:1.Effects of culture dimensional transformation on hepatic differentiation of hADMSCsThe experiment was divided into four groups: hADMSCs differentiated into HLCs under different culture dimensions(2D,3D)were subjected to culture dimensional transformation.The 2D differentiated cells were transferred to 2D culture(2D-2D)and 3D culture(2D-3D),respectively,and the 3D differentiated cells were transferred to 2D culture(3D-2D)and 3D culture(3D-3D),respectively.RT-qPCR was performed to detect the mRNA expression of liver-related genes ALB,AFP,HNF-4a,CYP1A2,and CYP3A4.Western blot and IF were used to detect the protein expression of ALB,AFP,Sox2,and Oct4.ELISA and quantitative colorimetry assays were performed to measure the levels of albumin and urea in the culture supernatant,respectively.PAS staining was used to assess glycogen synthesis.2.Epigenetic mechanism of culture dimensional transformation on hADMSCs differentiated into hepatocyte-like cells(1)Western blot analysis was used to detect the level of protein expression of p300 and the acetylation level of H3K56 in the four groups of HLCs after culture dimensional transformation.(2)Effects of p300 on the 2D-3D transformed HLCs: The experiment was divided into three groups: 2D-3D-DMSO,2D-3D-A-485,and 2D-3D-CTB.RT-qPCR was performed to detect the mRNA expression of liver-specific genes ALB,CYP1A2,and CYP3A4.Western blot and IF were used to detect the protein expression of ALB,AFP,Sox2,Nanog and the acetylation level of H3K56.ELISA and quantitative colorimetry assays were performed to measure the levels of albumin and urea secretion in the culture supernatant.Results:1.Effects of culture dimensional transformation on hADMSCs hepatogenic differentiationCompared to the other three groups,the 2D-3D group showed significant up-regulation of mRNA expression of ALB,HNF-4a,CYP1A2 and CYP3A4,and up-regulation in the ALB protein expression.It showed significant down-regulation of mRNA and protein expression of AFP,and significant downregulation the levels of Sox2 and Oct4 protein.2D-3D group also significantly increased the production of albumin and urea,and stimulated glycogen synthesis.2.Epigenetic mechanism of culture dimensional transformation on hADMSCs differentiated into hepatocyte-like cells(1)Compared to the other three groups after culture dimensional transformation,the 2D-3D group showed a significant increase in p300 protein expression and H3K56 acetylation level.(2)Compared to the 2D-3D-DMSO group,2D-3D-A-485 significantly decreased the mRNA levels of ALB,CYP1A2 and CYP3A4,significantly down-regulated the protein expression of ALB and the acetylation level of H3K56,and up-regulated AFP protein expression.Furthermore,it also significantly decreased albumin and urea levels.CTB did not show a significant upregulation of ALB protein expression and H3K56 acetylation level,nor did it increase the levels of albumin and urea.Conclusion:Culture dimensional transformation of 2D-3D increased the liver-related gene and protein expressions of HLCs,and enhanced the hepatic function.Culture dimensional transformation of 2D-3D promoted the maturation of HLCs and thus promoted hADMSCs differentiated into HLCs.The mechanism may be related to p300-mediated H3K56 acetylation regulation.Part 3.Application study of hADMSCs differentiated into hepatocyte-like cells after culture dimensional transformationObjective:To investigate the application effects of hepatocyte-like cells differentiated from hADMSCs after culture dimensional transformation using a drug screening platform and tissue engineering,and to verify the promotion of hADMSCs differentiated into hepatocyte-like cells by 2D-3D culture dimensional transformation.Methods:1.Drug screening platform study(1)Drug toxicity: Four groups of HLCs after culture dimensional transformation were treated with different concentrations of acetaminophen and amiodarone,respectively.After 48 hours of incubation,the Cell Titer-Glo(?) 3D Cell Viability Assay was used to measure the luminescence values of each well.(2)Drug metabolism: The four groups were treated with the CYP3A4 enzyme inducer,rifampicin(25 μM),for 48 hours.Then,testosterone(100 μM),a CYP3A4 enzyme substrate,was added for another 48 hours.The mRNA expression level of CYP3A4 in the four groups was determined using RT-qPCR.P450-Glo TM CYP450 Assay Kit was used to determine the CYP3A4 enzyme activity.HPLC analysis was used to measure the concentrations of testosterone and 6β-hydroxytestosterone in the cell supernatant of the four groups.2.Tissue engineering study(1)Construction of double-channel sodium alginate filaments and scaffolds: 3D coaxial bioprinting technology was used to print double-channel sodium alginate filaments and scaffolds,and their parameters were characterized using optical microscopy and scanning electron microscopy.(2)Perfusion of loaded cells in double-channel sodium alginate filaments and scaffolds: Live/Dead staining was used to evaluate cell activity in the filaments and scaffolds.CCK8 analysis was used to detect the proliferation of cells inside the filaments.ELISA was used to detect the level of albumin and urea in the filament loaded with HepG2 cells,and the level of insulin loaded with Min6 cells.IF method was used to detect the protein expression of ALB in the filament loaded with HepG2 cells and the protein expression of PDX-1 in the filament loaded with Min6 cells.(3)Perfusion of double-channel filament to simulate the interaction between Min6 and HepG2: 1% type I Collagen/Min6(Min6 group)and 1% Na-Alg/HepG2(HepG2 group)were respectively injected into one channel of the double-channel filament as the control group.1% type I Collagen/Min6 and 1% Na-Alg/HepG2 were both injected into one channel of the double-channel filament as the experimental group(Min6+HepG2 group).After 24 hours of perfusion with high glucose culture medium,ELISA was used to measure the insulin content in the effluent at different time points,and the glucose and pyruvate at each time point were determined by glucose and pyruvate assay kits.After perfusion,the uptake of glucose by the cells in each group was evaluate using a 2-NBDG uptake experiment.(4)Dual-channel filament perfusion validated the effect of culture dimensional transformation on hADMSCs differentiated into HLCs: 2D-3D and 3D-3D were respectively mixed with 1% Na-Alg and seeded into dual-channel filament for static and dynamic perfusion culture for 48 hours.Western blot was used to detect the protein expression of ALB and AFP in each group of cells,and ELISA and quantitative colorimetry were used to measure the levels of albumin and urea in the culture effluent.Results:1.Drug screening platform study(1)Compared with the L02 and HepG2 positive control groups,only the IC50 values of acetaminophen and amiodarone in the 2D-3D group were similar to the IC50 values of L02 or HepG2 in 3D.(2)Compared with the other three groups,the 2D-3D group significantly increased the mRNA expression and enzyme activity of CYP3A4.The remaining testosterone percentages in the culture media of each group were 9.1±0.75% for 2D-3D,39.2±1.07% for 2D-2D,55.3±1.2% for 3D-2D,and 12.9±0.81% for 3D-3D,indicating that the 2D-3D group had the strongest ability to metabolize testosterone,significantly higher than the other three groups,which indirectly indicated that the 2D-3D group had the strongest CYP3A4 enzyme activity.2.Tissue engineering study(1)The flow test confirmed that the coaxially printed double-channel sodium alginate filaments were hollow structures.One channel of the double-channel sodium alginate filament seeded with cell-laden hydrogels,and the other channel was perfused with medium,and two channels were separated by an interval wall of alginate,whose thickness(50 μm)is beneficial to supplement nutrients via perfusion.(2)HepG2 cells encapsulated with 1% Na-Alg hydrogel and seeded into double-channel filament showed significantly increased cell viability,ALB protein expression,and secretion functions of albumin and urea in the perfusion compared to the non-perfusion.Similarly,Min6 cells encapsulated with 1% type I collagen hydrogel and seeded into double-channel filament also showed significantly increased cell viability and PDX-1 protein expression in the perfused group compared to the non-perfused group.When 1% Na-Alg/HepG2 was seeded in an 8-layer cubic double-channel scaffolds,the cell viability gradually decreased with the decrease of nutrients and oxygen in the perfusion medium.(3)In simulation experiment of Min6 and HepG2 interaction,the insulin level in the Min6+HepG2 group was significantly lower than that in the Min6 group,and the glucose level in the Min6+HepG2 group was significantly lower than that in the Min6 group or HepG2 group.The glucose uptake ability of HepG2 cells in the Min6+HepG2 group was significantly higher than that in the Min6 group and HepG2 group.(4)In the double-channel alginate filament with and without perfusion,the ALB protein expression of the 2D-3D hepatocyte-like cells was significantly higher than that of the 3D-3D group,and the AFP protein expression was significantly lower than that of the 3D-3D group.The levels of albumin and urea in the 2D-3D group were significantly higher than those in the 3D-3D group.Compared to the non-perfusion 2D-3D group,the perfusion 2D-3D group significantly decreased the protein expression of AFP and increased levels of albumin and urea.Conclusion:(1)Drug toxicity and drug metabolism experiments have confirmed that the 2D-3D culture dimensional transformation promoted the hepatic differentiation of hADMSCs.(2)Dual-channel sodium alginate filaments with biocompatibility,valid perfusion,and available were successfully constructed,and the dual-channel sodium alginate filament dynamic culture model further validated the promotion of hepatogenic differentiation of hADMSCs through the 2D-3D culture dimensional transformation.