Alterations in embryonic neural stem cells play crucial roles in the pathogenesis of amyotrophic lateral sclerosis. We hypothesized that embryonic neural stem cells from SOD1G93A individuals might be more susceptible ...Alterations in embryonic neural stem cells play crucial roles in the pathogenesis of amyotrophic lateral sclerosis. We hypothesized that embryonic neural stem cells from SOD1G93A individuals might be more susceptible to oxidative injury, resulting in a propensity for neurodegeneration at later stages. In this study, embryonic neural stem cells obtained from human superoxide dis- mutase 1 mutant (SOD1G93A) and wild-type (SOD1wv) mouse models were exposed to H202. We assayed cell viability with mitochondrial succinic dehydrogenase colorimetric reagent, and measured cell apoptosis by flow cytometry. Moreover, we evaluated the expression of the adenos- ine monophosphate-activated protein kinase (AMPK) ct-subunit, paired box 3 (Pax3) protein, and p53 in western blot analyses. Compared with SOD1wr cells, SOD1~93A embryonic neural stem cells were more likely to undergo H202-induced apoptosis. Phosphorylation of AMPKct in SOD1G93A cells was higher than that in SOD1wr cells. Pax3 expression was inversely correlated with the phosphorylation levels of AMPKct. p53 protein levels were also correlated with AMPKct phosphorylation levels. Compound C, an inhibitor of AMPKa, attenuated the effects of H20~. These results suggest that embryonic neural stem cells from SOD1C93A mice are more susceptible to apoptosis in the presence of oxidative stress compared with those from wild-type controls, and the effects are mainly mediated by Pax3 and p53 in the AMPKa pathway.展开更多
Objective To invest the efficient method which can culture and induce embryonic stem cells to neuroeyte in vitro. Methods Isolate the blastula o f 3.5 d from BALB/c species mouse. Culture the cells from inner cell ma...Objective To invest the efficient method which can culture and induce embryonic stem cells to neuroeyte in vitro. Methods Isolate the blastula o f 3.5 d from BALB/c species mouse. Culture the cells from inner cell mass (inner cell mass, ICM) which were isolated by mechanical method on the mouse embryonic fibroblaste cell (MEF) feeder layer or 0.1% gelatin coated dishes. The stem ceils were identified by characterized morphology, alkaline phosphatase stain, differential potency in vivo and immunoehemistry stain. The isolated cells were differentiated by serial induction method that mimicking the intrinsic developmental process of the neural system. Results The isolated cells were positive for alkaline phosphatatse and SSEA-1 ( stage specific embryonic antigen 1 ). Moreover they were identified pluripotent by differentiation in vivo. Therefore the isolated ceils presented the characters of ESCs. Then the isolated cells were able to differentiate into neuroeytes in vitro. Conclusion Mouse embryonic stem ceils isolation, culture and differentiation system has been established.展开更多
Background Cellular repressor of ElA-stimulated genes(CREG) is homeostatic modulated gene,which regulate a number of cellular processes,including cell differentiation, motility and survival.Previous studies have demon...Background Cellular repressor of ElA-stimulated genes(CREG) is homeostatic modulated gene,which regulate a number of cellular processes,including cell differentiation, motility and survival.Previous studies have demonstrated that CREG was expressed in all three germ layers,suggesting that it might act as a vital regulator during embryonic developing.The aim of the present study was to investigate the role of CREG in an embryonic stem cell(ESC) differentiation model that recapitulates the developmental steps of vasculogenesis.Methods The ES cells were stably transfected either pCXN2-FLAG-CREG-IRES-EGFP plasmid or pDS1- shRNA-CREG plasmid to produce the CREG+/ES cells and CREG-siRNA/ES cells,respectively.Vasculogenesis was detected by whole mount immunostainings for CD31.Dil labeled acLDL staining assay was used to detect branching pseudopods in cultures in Matrigel.Real-time PCR and Western blot analysis were employed to determine expressions of VEGF and Flk-1.Results CREG +/ES-derived embryoid bodies(EBs) were found to form spontaneously a primitive vascular network after 6 days of differentiation.In contrast, wildtype EBs exhibit theirs vasculogenesis until 13 days of differentiation by whole mount immunostainings for CD31. CREG +/EBs developed more rapidly branching pseudopods at 9 days compared with that of wildtype EBs by Dil labeled acLDL staining assay.In contrast,CREG-siRNA/ES exhibits an undifferentiated morphogenesis associated with an increase in apoptotic cells in spite of being derived from LIF and feeder layers.Administration of CREG-siRNA/ES cells with recombinant CREG protein rescued the phenomena that CREG boosted vasculogenesis in a dose-dependent fasion. Mechanically,Real-time PCR and Western blot analysis revealed the expressions both VEGF and Flk-1 significantly in- creased in CREG+/EBs.Moreover,after treatment of CREG+ /EBs with neurtralizing antibody against VEGF,the rapid vasculogenesis was significantly repressed.Conclusions Our data strongely demonstrate that CREG play a pivotal role in accelerating vasculogenesis in development of ES cells. VEGF,as its important downstream effector,mediated this bio-function.展开更多
The histological observation was experimentally conducted on in vitro cultured mouse embryonic myocardium cells and myocardiumoid cell mass. The mouse embryo tissue were cultured and regular pulsatile myocardiumoid ti...The histological observation was experimentally conducted on in vitro cultured mouse embryonic myocardium cells and myocardiumoid cell mass. The mouse embryo tissue were cultured and regular pulsatile myocardiumoid tissue could be found. During in vitro culture, the myofilament bundles in the cell were gradually increasing and strongly connectted each other with embryonic age and there were loose muscle fibers initially and intercalated discs were close to each other. The lose myofilament bundles were developed in muscle fibers with age and the distance between intercalated discs was enlarged. There were myofilamentoid structure in inactive cells and filament peripherily.展开更多
By radioreceptor binding studies with iodinated TGF-β1, it has been shown that an undifferentiated ES-5 cell expresses approximately 3270 receptors with a dissociation constant Kd=130pM, but after the induction of di...By radioreceptor binding studies with iodinated TGF-β1, it has been shown that an undifferentiated ES-5 cell expresses approximately 3270 receptors with a dissociation constant Kd=130pM, but after the induction of differenti-ation by retinoic acid and dBcAMP, the receptor number of a differentiated RA-ES-5 cell was increased about 80% and the Kd was also increased to 370 pM. Furthermore,more direct evidence supporting the expression of TGF-βtype Ⅰand type Ⅱ receptors in both ES-5 and RA-ES-5 cells has come from dot blot hybridization of cellular mRNA with cDNA probes for type Ⅰ and type Ⅱ recep-tors. Meanwhile, mRNA expression level of types Ⅰ and Ⅱreceptors in RA-ES-5 cells were higher than that in ES-5 cells. Down regulation of TGF-β receptors with a signifi-cant decrease in the rate of cell proliferation in both cells, was found by employing a pretreatment with neutralizing antibody to TGF-β1. The possible role of receptors for TGF-β in cen differentiation is discussed here.展开更多
Background Studies on human, rat and chicken embryos have demonstrated that during the period of outflow tract septation, retraction of the distal myocardial margin of the outflow tract from the junction with aortic ...Background Studies on human, rat and chicken embryos have demonstrated that during the period of outflow tract septation, retraction of the distal myocardial margin of the outflow tract from the junction with aortic sac to the level of semilunar valves leads to the shortening of the myocardial tract. However, the mechanism is not clear. So we investigated the mechanism of outflow tract shortening and remodeling and the spatio-temporal distribution pattern of α-SMA positive cells in the outflow tract cushion during septation of the outflow tract in the embryonic mouse heart Methods Serial sections of mouse embryos from embryonic day 9 (ED 9) to embryonic day 16 (ED 16) were stained with monoclonal antibodies against α-SCA, α-SMA, or desmin, while apoptosis was assessed using the terminal deoxyribonucleotidy transferase-mediated dUTP-digoxigenin nick-end labeling (TUNEL) assay Results Between ED 11 and ED 12, the cardiomyocytes in the distal portion of the outflow tract were observed losing their myocardial phenotype without going into apoptosis, suggesting that trans-differentiation of cardiomyocytes into the cell components of the free walls of the intrapericardial ascending aorta and pulmonary trunk The accumulation of α-SMA positive cells in the cardiac jelly began on ED 10 and participated in the ridge fusion and septation of the outflow tract Fusion of the distal ridges resulted in the formation of the facing walls of the intrapericardial ascending aorta and pulmonary trunk Fusion of the proximal ridges was accompanied by the accumulation of α-SMA positive cells into a characteristic central whorl, in which cell apoptosis could be observed Subsequent myocardialization resulted in the formation of the partition between the subaortic and subpulmonary vestibules Conclusions The shortening of the embryonic heart outflow tract in mice may result not from apoptosis, but from the trans-differentiation of cells with cardiomyocyte phenotype in the distal portion of the outflow tract into the cell components of the free walls of the intrapericardial ascending aorta and pulmonary trunk The primary roles of α-SMA positive cells in the septation and remodeling of the outflow tract may assure proper fusion of the outflow ridges and form the facing walls of the intrapericardial ascending aorta and pulmonary trunk展开更多
The aim of this study was to investigate the role of selenoprotein M(SelM)in endoplasmic reticulum stress and apoptosis in nickel-exposed mouse hearts and to explore the detoxifying effects of melatonin.At 21 d after ...The aim of this study was to investigate the role of selenoprotein M(SelM)in endoplasmic reticulum stress and apoptosis in nickel-exposed mouse hearts and to explore the detoxifying effects of melatonin.At 21 d after intraperitoneal injection of nickel chloride(NiCl_(2))and/or melatonin into male wild-type(WT)and SelM knockout(KO)C57BL/6J mice,NiCl_(2)was found to induce changes in the microstructure and ultrastructure of the hearts of both WT and SelM KO mice,which were caused by oxidative stress,endoplasmic reticulum stress,and apoptosis,as evidenced by decreases in malondialdehyde(MDA)content and total antioxidant capacity(T-AOC)activity.Changes in the messenger RNA(mRNA)and protein expression of genes related to endoplasmic reticulum stress(activating transcription factor 4(ATF4),inositol-requiring protein 1(IRE1),c-Jun N-terminal kinase(JNK),and C/EBP homologous protein(CHOP))and apoptosis(B-cell lymphoma-2(Bcl-2),Bcl-2-associated X protein(Bax),Caspase-3,Caspase-9,and Caspase-12)were also observed.Notably,the observed damage was worse in SelM KO mice.Furthermore,melatonin alleviated the heart injury caused by NiCl_(2)in WT mice but could not exert a good protective effect in the heart of SelM KO mice.Overall,the findings suggested that the antioxidant capacity of SelM,as well as its modulation of endoplasmic reticulum stress and apoptosis,plays important roles in nickel-induced heart injury.展开更多
BACKGROUND Embryonic stem cells(ESCs)serve as a crucial ex vivo model,representing epiblast cells derived from the inner cell mass of blastocyst-stage embryos.ESCs exhibit a unique combination of self-renewal potency,...BACKGROUND Embryonic stem cells(ESCs)serve as a crucial ex vivo model,representing epiblast cells derived from the inner cell mass of blastocyst-stage embryos.ESCs exhibit a unique combination of self-renewal potency,unlimited proliferation,and pluripotency.The latter is evident by the ability of the isolated cells to differ-entiate spontaneously into multiple cell lineages,representing the three primary embryonic germ layers.Multiple regulatory networks guide ESCs,directing their self-renewal and lineage-specific differentiation.Apoptosis,or programmed cell death,emerges as a key event involved in sculpting and forming various organs and structures ensuring proper embryonic development.How-ever,the molecular mechanisms underlying the dynamic interplay between diffe-rentiation and apoptosis remain poorly understood.AIM To investigate the regulatory impact of apoptosis on the early differentiation of ESCs into cardiac cells,using mouse ESC(mESC)models-mESC-B-cell lym-phoma 2(BCL-2),mESC-PIM-2,and mESC-metallothionein-1(MET-1)-which overexpress the anti-apoptotic genes Bcl-2,Pim-2,and Met-1,respectively.METHODS mESC-T2(wild-type),mESC-BCL-2,mESC-PIM-2,and mESC-MET-1 have been used to assess the effect of potentiated apoptotic signals on cardiac differentiation.The hanging drop method was adopted to generate embryoid bodies(EBs)and induce terminal differentiation of mESCs.The size of the generated EBs was measured in each condition compared to the wild type.At the functional level,the percentage of cardiac differentiation was measured by calculating the number of beating cardiomyocytes in the manipulated mESCs compared to the control.At the molecular level,quantitative reverse transcription-polymerase chain reaction was used to assess the mRNA expression of three cardiac markers:Troponin T,GATA4,and NKX2.5.Additionally,troponin T protein expression was evaluated through immunofluorescence and western blot assays.RESULTS Our findings showed that the upregulation of Bcl-2,Pim-2,and Met-1 genes led to a reduction in the size of the EBs derived from the manipulated mESCs,in comparison with their wild-type counterpart.Additionally,a decrease in the count of beating cardiomyocytes among differentiated cells was observed.Furthermore,the mRNA expression of three cardiac markers-troponin T,GATA4,and NKX2.5-was diminished in mESCs overexpressing the three anti-apoptotic genes compared to the control cell line.Moreover,the overexpression of the anti-apoptotic genes resulted in a reduction in troponin T protein expression.CONCLUSION Our findings revealed that the upregulation of Bcl-2,Pim-2,and Met-1 genes altered cardiac differentiation,providing insight into the intricate interplay between apoptosis and ESC fate determination.展开更多
Dear Editor,Haploid embryonic stem cells(ha ESCs)hold great potential for genetic screening and the analysis of recessive phenotypes.Several studies have recently reported the generation of mammalian ha ESCs through...Dear Editor,Haploid embryonic stem cells(ha ESCs)hold great potential for genetic screening and the analysis of recessive phenotypes.Several studies have recently reported the generation of mammalian ha ESCs through gamete manipulation,and evaluated the benefits of using them for studying functional genomics in different mammals[1–4].展开更多
Background: Outflow tract (OFT) septation defects are a common cause of congenital heart disease. Numerous studies have focused on the septation mechanism of the OFT, but have reported inconsistent conclusions. Thi...Background: Outflow tract (OFT) septation defects are a common cause of congenital heart disease. Numerous studies have focused on the septation mechanism of the OFT, but have reported inconsistent conclusions. This study, therefore, aimed to investigate the septation of the aortic sac and the OFT in the early embryonic human heart. Methods: Serial sections of 27 human embryonic hearts from Carnegie stage (CS) 10 to CS19 were immunohistochemically stained with antibodies against α-smooth muscle actin (α-SMA) and myosin heavy chain. Results: At CS10-CS11, the OFT wail was an exclusively myocardial structure that was continuous with the aortic sac at the margin of the pericardial cavity. From CS13 onward, the OFT was divided into nonmyocardial and myocardial portions. The cushion formed gradually, and its distal border with the OFT myocardium was consistently maintained. The aortic sac between the fourth and sixth aortic arch arteries was degenerated. At CS16, the α-SMA-positive aortopulmonary septum formed and fused with the two OFT cushions, thus septating the nonmyocardial portion of the OFT into two arteries. At this stage, the cushions were not fused. At CS19, the bilateral cushions were fused to septate the myocardial portion of the OFT. Conclusions: Data suggest that the OFT cushion is formed before the aortopulmonary septum is formed. Thus, the OFT cushion is not derived from the aortopuhnonary septum. In addition, the nonmyocardial part of the OFT is septated into the aorta and pulmonary trunk by the aortopulmonary septum, while the main part of the cushion fuses and septates the myocardial portion of the OFT.展开更多
Propofol (2, 6-diisopropylphenol) is a general intravenous anesthetic which plays roles in the central neural system by binding GABAA receptors (GABAARs) and enhancing the chloride channels of the neurons.1 Previo...Propofol (2, 6-diisopropylphenol) is a general intravenous anesthetic which plays roles in the central neural system by binding GABAA receptors (GABAARs) and enhancing the chloride channels of the neurons.1 Previous studies mainly focused on the effects of anesthetics on mature neurons, but little attention was paid to their role in early neural differentiation or neural stem cells. Therefore, in the present study, we choose the widely used mouse embryonic cells (ES) cells as the model to investigate the potential effect ofpropofol on neuronal differentiation.展开更多
The different cell types in an animal are often considered to be specified by combinations of transcription factors,and defined by marker gene expression.This paradigm is challenged,however,in stem cell research and a...The different cell types in an animal are often considered to be specified by combinations of transcription factors,and defined by marker gene expression.This paradigm is challenged,however,in stem cell research and application.Using a mouse embryonic stem cell(mESC) culture system,here we show that the expression level of many key stem cell marker genes/transcription factors such as Oct4,Sox2 and Nanog failed to monitor cell status transition during mESC differentiation.On the other hand,the response patterns of cell signalling network to external stimuli,as monitored by the dynamics of protein phosphorylation,changed dramatically.Our results also suggest that an irreversible alternation in the cell signalling network precedes the adjustment of transcription factor levels.This is consistent with the notion that signal transduction events regulate cell fate specification.We propose that interrogating a cell signalling network can assess the cell property more precisely,and provide a sensitive measurement for the early events in cell fate transition.We wish to bring attention to the potential problem of cell identification using a few marker genes,and suggest a novel methodology to address this issue.展开更多
Transient receptor potential canonical subfamily member 3(TRPC3) is known to be important for neural development and the formation of neuronal networks. Here, we investigated the role of TRPC3 in undifferentiated mous...Transient receptor potential canonical subfamily member 3(TRPC3) is known to be important for neural development and the formation of neuronal networks. Here, we investigated the role of TRPC3 in undifferentiated mouse embryonic stem cells(mESCs) and during the differentiation of mESCs into neurons. CRISPR/Cas9-mediated knockout(KO) of TRPC3 induced apoptosis and the disruption of mitochondrial membrane potential both in undifferentiated mESCs and in those undergoing neural differentiation. In addition, TRPC3 KO impaired the pluripotency of mESCs. TRPC3 KO also dramatically repressed the neural differentiation of mESCs by inhibiting the expression of markers for neural progenitors, neurons, astrocytes and oligodendrocytes.Taken together, our new data demonstrate an important function of TRPC3 with regards to the survival, pluripotency and neural differentiation of mESCs.展开更多
Sense mutations in several conserved modifiable sites of histone H3 have been found to be strongly correlated with multiple tissuespecific clinical cancers.These clinical site mutants acquire a distinctively new epige...Sense mutations in several conserved modifiable sites of histone H3 have been found to be strongly correlated with multiple tissuespecific clinical cancers.These clinical site mutants acquire a distinctively new epigenetic role and mediate cancer evolution.In this study,we mimicked histone H3 at the 56th lysine(H3K56)mutant incorporation in mouse embryonic stem cells(mESCs)by lentivirus-mediated ectopic expression and analyzed the effects on replication and epigenetic regulation.The data show that two types of H3K56 mutants,namely H3 lysine 56-to-methionine(H3K56M)and H3 lysine 56-to-alanine(H3K56A),promote replication by recruiting more minichromosome maintenance complex component 3 and checkpoint kinase 1 onto chromatin compared with wild-type histone H3 and other site substitution mutants.Under this condition,the frequency of genomic copy number gain in H3K56M and H3K56A cells globally increases,especially in the Mycl1 region,a known molecular marker frequently occurring in multiple malignant cancers.Additionally,we found the disruption of H3K56 acetylation distribution in the copy-gain regions,which indicates a probable epigenetic mechanism of H3K56M and H3K56A.We then identified that H3K56M and H3K56A can trigger a potential adaptation to transcription;genes involved in the mitogen-activated protein kinase pathway are partially upregulated,whereas genes associated with intrinsic apoptotic function show obvious downregulation.The final outcome of ectopic H3K56M and H3K56A incorporation in mESCs is an enhanced ability to form carcinomas.This work indicates that H3K56 site conservation and proper modification play important roles in harmonizing the function of the replication machinery in mESCs.展开更多
Nanog is a novel transcription factor specifically expressed in mouse embryonic stem cells (mES cells). It has been reported that Nanog plays an essential role in maintaining multi-potency of ES cells. The expression ...Nanog is a novel transcription factor specifically expressed in mouse embryonic stem cells (mES cells). It has been reported that Nanog plays an essential role in maintaining multi-potency of ES cells. The expression of Nanog is very sensitive to ES cells differentiation, making Nanog one of the best markers to indicate the status of ES cells. In this study, we developed an efficient method to construct Nanog promoter driven EGFP reporter system based on the BAC homologous recombination. We further generated a Nanog-EGFP reporter mES cell line. This reporter mES cell line exhibited features similar to those of normal mES cells, and the EGFP reporter efficiently reflected the expression of Nanog, indi- cating the differentiation status of mES cells. We achieved a reliable experimental reporter system to research self-renewal and differentiation of mES cells. The system could facilitate research on culture system of mES cells and researches on the expression and regulation of Nanog and other related fac- tors in mES cells.展开更多
基金supported by a grant from the National Natural Sciences Foundation of China,No.81030019
文摘Alterations in embryonic neural stem cells play crucial roles in the pathogenesis of amyotrophic lateral sclerosis. We hypothesized that embryonic neural stem cells from SOD1G93A individuals might be more susceptible to oxidative injury, resulting in a propensity for neurodegeneration at later stages. In this study, embryonic neural stem cells obtained from human superoxide dis- mutase 1 mutant (SOD1G93A) and wild-type (SOD1wv) mouse models were exposed to H202. We assayed cell viability with mitochondrial succinic dehydrogenase colorimetric reagent, and measured cell apoptosis by flow cytometry. Moreover, we evaluated the expression of the adenos- ine monophosphate-activated protein kinase (AMPK) ct-subunit, paired box 3 (Pax3) protein, and p53 in western blot analyses. Compared with SOD1wr cells, SOD1~93A embryonic neural stem cells were more likely to undergo H202-induced apoptosis. Phosphorylation of AMPKct in SOD1G93A cells was higher than that in SOD1wr cells. Pax3 expression was inversely correlated with the phosphorylation levels of AMPKct. p53 protein levels were also correlated with AMPKct phosphorylation levels. Compound C, an inhibitor of AMPKa, attenuated the effects of H20~. These results suggest that embryonic neural stem cells from SOD1C93A mice are more susceptible to apoptosis in the presence of oxidative stress compared with those from wild-type controls, and the effects are mainly mediated by Pax3 and p53 in the AMPKa pathway.
基金This work was supported by China National Programs of High and New Technology Development ( 863 ) ( 2002AA216101 ) National Natural Science Foundation of China (30300110).
文摘Objective To invest the efficient method which can culture and induce embryonic stem cells to neuroeyte in vitro. Methods Isolate the blastula o f 3.5 d from BALB/c species mouse. Culture the cells from inner cell mass (inner cell mass, ICM) which were isolated by mechanical method on the mouse embryonic fibroblaste cell (MEF) feeder layer or 0.1% gelatin coated dishes. The stem ceils were identified by characterized morphology, alkaline phosphatase stain, differential potency in vivo and immunoehemistry stain. The isolated cells were differentiated by serial induction method that mimicking the intrinsic developmental process of the neural system. Results The isolated cells were positive for alkaline phosphatatse and SSEA-1 ( stage specific embryonic antigen 1 ). Moreover they were identified pluripotent by differentiation in vivo. Therefore the isolated ceils presented the characters of ESCs. Then the isolated cells were able to differentiate into neuroeytes in vitro. Conclusion Mouse embryonic stem ceils isolation, culture and differentiation system has been established.
文摘Background Cellular repressor of ElA-stimulated genes(CREG) is homeostatic modulated gene,which regulate a number of cellular processes,including cell differentiation, motility and survival.Previous studies have demonstrated that CREG was expressed in all three germ layers,suggesting that it might act as a vital regulator during embryonic developing.The aim of the present study was to investigate the role of CREG in an embryonic stem cell(ESC) differentiation model that recapitulates the developmental steps of vasculogenesis.Methods The ES cells were stably transfected either pCXN2-FLAG-CREG-IRES-EGFP plasmid or pDS1- shRNA-CREG plasmid to produce the CREG+/ES cells and CREG-siRNA/ES cells,respectively.Vasculogenesis was detected by whole mount immunostainings for CD31.Dil labeled acLDL staining assay was used to detect branching pseudopods in cultures in Matrigel.Real-time PCR and Western blot analysis were employed to determine expressions of VEGF and Flk-1.Results CREG +/ES-derived embryoid bodies(EBs) were found to form spontaneously a primitive vascular network after 6 days of differentiation.In contrast, wildtype EBs exhibit theirs vasculogenesis until 13 days of differentiation by whole mount immunostainings for CD31. CREG +/EBs developed more rapidly branching pseudopods at 9 days compared with that of wildtype EBs by Dil labeled acLDL staining assay.In contrast,CREG-siRNA/ES exhibits an undifferentiated morphogenesis associated with an increase in apoptotic cells in spite of being derived from LIF and feeder layers.Administration of CREG-siRNA/ES cells with recombinant CREG protein rescued the phenomena that CREG boosted vasculogenesis in a dose-dependent fasion. Mechanically,Real-time PCR and Western blot analysis revealed the expressions both VEGF and Flk-1 significantly in- creased in CREG+/EBs.Moreover,after treatment of CREG+ /EBs with neurtralizing antibody against VEGF,the rapid vasculogenesis was significantly repressed.Conclusions Our data strongely demonstrate that CREG play a pivotal role in accelerating vasculogenesis in development of ES cells. VEGF,as its important downstream effector,mediated this bio-function.
文摘The histological observation was experimentally conducted on in vitro cultured mouse embryonic myocardium cells and myocardiumoid cell mass. The mouse embryo tissue were cultured and regular pulsatile myocardiumoid tissue could be found. During in vitro culture, the myofilament bundles in the cell were gradually increasing and strongly connectted each other with embryonic age and there were loose muscle fibers initially and intercalated discs were close to each other. The lose myofilament bundles were developed in muscle fibers with age and the distance between intercalated discs was enlarged. There were myofilamentoid structure in inactive cells and filament peripherily.
文摘By radioreceptor binding studies with iodinated TGF-β1, it has been shown that an undifferentiated ES-5 cell expresses approximately 3270 receptors with a dissociation constant Kd=130pM, but after the induction of differenti-ation by retinoic acid and dBcAMP, the receptor number of a differentiated RA-ES-5 cell was increased about 80% and the Kd was also increased to 370 pM. Furthermore,more direct evidence supporting the expression of TGF-βtype Ⅰand type Ⅱ receptors in both ES-5 and RA-ES-5 cells has come from dot blot hybridization of cellular mRNA with cDNA probes for type Ⅰ and type Ⅱ recep-tors. Meanwhile, mRNA expression level of types Ⅰ and Ⅱreceptors in RA-ES-5 cells were higher than that in ES-5 cells. Down regulation of TGF-β receptors with a signifi-cant decrease in the rate of cell proliferation in both cells, was found by employing a pretreatment with neutralizing antibody to TGF-β1. The possible role of receptors for TGF-β in cen differentiation is discussed here.
基金ThisworkwassupportedbytheScientificResearchFoundationforReturnedOverseasChineseScholars,StateEducationMinistryandEducationMinistryofShanxiProvince (No 9845 )
文摘Background Studies on human, rat and chicken embryos have demonstrated that during the period of outflow tract septation, retraction of the distal myocardial margin of the outflow tract from the junction with aortic sac to the level of semilunar valves leads to the shortening of the myocardial tract. However, the mechanism is not clear. So we investigated the mechanism of outflow tract shortening and remodeling and the spatio-temporal distribution pattern of α-SMA positive cells in the outflow tract cushion during septation of the outflow tract in the embryonic mouse heart Methods Serial sections of mouse embryos from embryonic day 9 (ED 9) to embryonic day 16 (ED 16) were stained with monoclonal antibodies against α-SCA, α-SMA, or desmin, while apoptosis was assessed using the terminal deoxyribonucleotidy transferase-mediated dUTP-digoxigenin nick-end labeling (TUNEL) assay Results Between ED 11 and ED 12, the cardiomyocytes in the distal portion of the outflow tract were observed losing their myocardial phenotype without going into apoptosis, suggesting that trans-differentiation of cardiomyocytes into the cell components of the free walls of the intrapericardial ascending aorta and pulmonary trunk The accumulation of α-SMA positive cells in the cardiac jelly began on ED 10 and participated in the ridge fusion and septation of the outflow tract Fusion of the distal ridges resulted in the formation of the facing walls of the intrapericardial ascending aorta and pulmonary trunk Fusion of the proximal ridges was accompanied by the accumulation of α-SMA positive cells into a characteristic central whorl, in which cell apoptosis could be observed Subsequent myocardialization resulted in the formation of the partition between the subaortic and subpulmonary vestibules Conclusions The shortening of the embryonic heart outflow tract in mice may result not from apoptosis, but from the trans-differentiation of cells with cardiomyocyte phenotype in the distal portion of the outflow tract into the cell components of the free walls of the intrapericardial ascending aorta and pulmonary trunk The primary roles of α-SMA positive cells in the septation and remodeling of the outflow tract may assure proper fusion of the outflow ridges and form the facing walls of the intrapericardial ascending aorta and pulmonary trunk
基金supported by the Heilongjiang Provincial Natural Science Foundation for Outstanding Youth(No.YQ2021C021),China。
文摘The aim of this study was to investigate the role of selenoprotein M(SelM)in endoplasmic reticulum stress and apoptosis in nickel-exposed mouse hearts and to explore the detoxifying effects of melatonin.At 21 d after intraperitoneal injection of nickel chloride(NiCl_(2))and/or melatonin into male wild-type(WT)and SelM knockout(KO)C57BL/6J mice,NiCl_(2)was found to induce changes in the microstructure and ultrastructure of the hearts of both WT and SelM KO mice,which were caused by oxidative stress,endoplasmic reticulum stress,and apoptosis,as evidenced by decreases in malondialdehyde(MDA)content and total antioxidant capacity(T-AOC)activity.Changes in the messenger RNA(mRNA)and protein expression of genes related to endoplasmic reticulum stress(activating transcription factor 4(ATF4),inositol-requiring protein 1(IRE1),c-Jun N-terminal kinase(JNK),and C/EBP homologous protein(CHOP))and apoptosis(B-cell lymphoma-2(Bcl-2),Bcl-2-associated X protein(Bax),Caspase-3,Caspase-9,and Caspase-12)were also observed.Notably,the observed damage was worse in SelM KO mice.Furthermore,melatonin alleviated the heart injury caused by NiCl_(2)in WT mice but could not exert a good protective effect in the heart of SelM KO mice.Overall,the findings suggested that the antioxidant capacity of SelM,as well as its modulation of endoplasmic reticulum stress and apoptosis,plays important roles in nickel-induced heart injury.
基金Supported by the National Council for Scientific Research in Lebanon,CNRS-L.
文摘BACKGROUND Embryonic stem cells(ESCs)serve as a crucial ex vivo model,representing epiblast cells derived from the inner cell mass of blastocyst-stage embryos.ESCs exhibit a unique combination of self-renewal potency,unlimited proliferation,and pluripotency.The latter is evident by the ability of the isolated cells to differ-entiate spontaneously into multiple cell lineages,representing the three primary embryonic germ layers.Multiple regulatory networks guide ESCs,directing their self-renewal and lineage-specific differentiation.Apoptosis,or programmed cell death,emerges as a key event involved in sculpting and forming various organs and structures ensuring proper embryonic development.How-ever,the molecular mechanisms underlying the dynamic interplay between diffe-rentiation and apoptosis remain poorly understood.AIM To investigate the regulatory impact of apoptosis on the early differentiation of ESCs into cardiac cells,using mouse ESC(mESC)models-mESC-B-cell lym-phoma 2(BCL-2),mESC-PIM-2,and mESC-metallothionein-1(MET-1)-which overexpress the anti-apoptotic genes Bcl-2,Pim-2,and Met-1,respectively.METHODS mESC-T2(wild-type),mESC-BCL-2,mESC-PIM-2,and mESC-MET-1 have been used to assess the effect of potentiated apoptotic signals on cardiac differentiation.The hanging drop method was adopted to generate embryoid bodies(EBs)and induce terminal differentiation of mESCs.The size of the generated EBs was measured in each condition compared to the wild type.At the functional level,the percentage of cardiac differentiation was measured by calculating the number of beating cardiomyocytes in the manipulated mESCs compared to the control.At the molecular level,quantitative reverse transcription-polymerase chain reaction was used to assess the mRNA expression of three cardiac markers:Troponin T,GATA4,and NKX2.5.Additionally,troponin T protein expression was evaluated through immunofluorescence and western blot assays.RESULTS Our findings showed that the upregulation of Bcl-2,Pim-2,and Met-1 genes led to a reduction in the size of the EBs derived from the manipulated mESCs,in comparison with their wild-type counterpart.Additionally,a decrease in the count of beating cardiomyocytes among differentiated cells was observed.Furthermore,the mRNA expression of three cardiac markers-troponin T,GATA4,and NKX2.5-was diminished in mESCs overexpressing the three anti-apoptotic genes compared to the control cell line.Moreover,the overexpression of the anti-apoptotic genes resulted in a reduction in troponin T protein expression.CONCLUSION Our findings revealed that the upregulation of Bcl-2,Pim-2,and Met-1 genes altered cardiac differentiation,providing insight into the intricate interplay between apoptosis and ESC fate determination.
基金supported by the Strategic Priority Research Program of the Chinese Academy of Sciences(XDA01010201)the National Key Basic Research and Development Program of China(2015CB964500 and 2014CB964804)the National Natural Science Foundation of China(91219303,31430058,and 31401261)
文摘Dear Editor,Haploid embryonic stem cells(ha ESCs)hold great potential for genetic screening and the analysis of recessive phenotypes.Several studies have recently reported the generation of mammalian ha ESCs through gamete manipulation,and evaluated the benefits of using them for studying functional genomics in different mammals[1–4].
文摘Background: Outflow tract (OFT) septation defects are a common cause of congenital heart disease. Numerous studies have focused on the septation mechanism of the OFT, but have reported inconsistent conclusions. This study, therefore, aimed to investigate the septation of the aortic sac and the OFT in the early embryonic human heart. Methods: Serial sections of 27 human embryonic hearts from Carnegie stage (CS) 10 to CS19 were immunohistochemically stained with antibodies against α-smooth muscle actin (α-SMA) and myosin heavy chain. Results: At CS10-CS11, the OFT wail was an exclusively myocardial structure that was continuous with the aortic sac at the margin of the pericardial cavity. From CS13 onward, the OFT was divided into nonmyocardial and myocardial portions. The cushion formed gradually, and its distal border with the OFT myocardium was consistently maintained. The aortic sac between the fourth and sixth aortic arch arteries was degenerated. At CS16, the α-SMA-positive aortopulmonary septum formed and fused with the two OFT cushions, thus septating the nonmyocardial portion of the OFT into two arteries. At this stage, the cushions were not fused. At CS19, the bilateral cushions were fused to septate the myocardial portion of the OFT. Conclusions: Data suggest that the OFT cushion is formed before the aortopulmonary septum is formed. Thus, the OFT cushion is not derived from the aortopuhnonary septum. In addition, the nonmyocardial part of the OFT is septated into the aorta and pulmonary trunk by the aortopulmonary septum, while the main part of the cushion fuses and septates the myocardial portion of the OFT.
文摘Propofol (2, 6-diisopropylphenol) is a general intravenous anesthetic which plays roles in the central neural system by binding GABAA receptors (GABAARs) and enhancing the chloride channels of the neurons.1 Previous studies mainly focused on the effects of anesthetics on mature neurons, but little attention was paid to their role in early neural differentiation or neural stem cells. Therefore, in the present study, we choose the widely used mouse embryonic cells (ES) cells as the model to investigate the potential effect ofpropofol on neuronal differentiation.
基金supported by the National Institutes of Health through the NIH Roadmap for Nanomedicine (PN2 EY018228)a Research Project Grant R01 EY015417 (YL) ZY was partially supported by a CIRM postdoc fellowship
文摘The different cell types in an animal are often considered to be specified by combinations of transcription factors,and defined by marker gene expression.This paradigm is challenged,however,in stem cell research and application.Using a mouse embryonic stem cell(mESC) culture system,here we show that the expression level of many key stem cell marker genes/transcription factors such as Oct4,Sox2 and Nanog failed to monitor cell status transition during mESC differentiation.On the other hand,the response patterns of cell signalling network to external stimuli,as monitored by the dynamics of protein phosphorylation,changed dramatically.Our results also suggest that an irreversible alternation in the cell signalling network precedes the adjustment of transcription factor levels.This is consistent with the notion that signal transduction events regulate cell fate specification.We propose that interrogating a cell signalling network can assess the cell property more precisely,and provide a sensitive measurement for the early events in cell fate transition.We wish to bring attention to the potential problem of cell identification using a few marker genes,and suggest a novel methodology to address this issue.
基金supported by the Hong Kong Research Grants Council(RGC)General Research Fund awards(662113,16101714,16100115)the ANR/RGC joint research scheme award(AHKUST601/13)+1 种基金the Hong Kong Theme-based Research Scheme award(T13-706/11-1)the Hong Kong Innovation and Technology Commission(ITCPD/17-9)
文摘Transient receptor potential canonical subfamily member 3(TRPC3) is known to be important for neural development and the formation of neuronal networks. Here, we investigated the role of TRPC3 in undifferentiated mouse embryonic stem cells(mESCs) and during the differentiation of mESCs into neurons. CRISPR/Cas9-mediated knockout(KO) of TRPC3 induced apoptosis and the disruption of mitochondrial membrane potential both in undifferentiated mESCs and in those undergoing neural differentiation. In addition, TRPC3 KO impaired the pluripotency of mESCs. TRPC3 KO also dramatically repressed the neural differentiation of mESCs by inhibiting the expression of markers for neural progenitors, neurons, astrocytes and oligodendrocytes.Taken together, our new data demonstrate an important function of TRPC3 with regards to the survival, pluripotency and neural differentiation of mESCs.
基金supported by grants from the National Key Research and Development Program of China(2017YFA0103301)the National Natural Science Foundation of China(81972743)China Postdoctoral Science Foundation(2020M671205).
文摘Sense mutations in several conserved modifiable sites of histone H3 have been found to be strongly correlated with multiple tissuespecific clinical cancers.These clinical site mutants acquire a distinctively new epigenetic role and mediate cancer evolution.In this study,we mimicked histone H3 at the 56th lysine(H3K56)mutant incorporation in mouse embryonic stem cells(mESCs)by lentivirus-mediated ectopic expression and analyzed the effects on replication and epigenetic regulation.The data show that two types of H3K56 mutants,namely H3 lysine 56-to-methionine(H3K56M)and H3 lysine 56-to-alanine(H3K56A),promote replication by recruiting more minichromosome maintenance complex component 3 and checkpoint kinase 1 onto chromatin compared with wild-type histone H3 and other site substitution mutants.Under this condition,the frequency of genomic copy number gain in H3K56M and H3K56A cells globally increases,especially in the Mycl1 region,a known molecular marker frequently occurring in multiple malignant cancers.Additionally,we found the disruption of H3K56 acetylation distribution in the copy-gain regions,which indicates a probable epigenetic mechanism of H3K56M and H3K56A.We then identified that H3K56M and H3K56A can trigger a potential adaptation to transcription;genes involved in the mitogen-activated protein kinase pathway are partially upregulated,whereas genes associated with intrinsic apoptotic function show obvious downregulation.The final outcome of ectopic H3K56M and H3K56A incorporation in mESCs is an enhanced ability to form carcinomas.This work indicates that H3K56 site conservation and proper modification play important roles in harmonizing the function of the replication machinery in mESCs.
基金Supported by the National Natural Science Foundation of China (Grant No. 30421004)
文摘Nanog is a novel transcription factor specifically expressed in mouse embryonic stem cells (mES cells). It has been reported that Nanog plays an essential role in maintaining multi-potency of ES cells. The expression of Nanog is very sensitive to ES cells differentiation, making Nanog one of the best markers to indicate the status of ES cells. In this study, we developed an efficient method to construct Nanog promoter driven EGFP reporter system based on the BAC homologous recombination. We further generated a Nanog-EGFP reporter mES cell line. This reporter mES cell line exhibited features similar to those of normal mES cells, and the EGFP reporter efficiently reflected the expression of Nanog, indi- cating the differentiation status of mES cells. We achieved a reliable experimental reporter system to research self-renewal and differentiation of mES cells. The system could facilitate research on culture system of mES cells and researches on the expression and regulation of Nanog and other related fac- tors in mES cells.