Cardiac differentiation is modulated by anti-apoptotic signals in murine embryonic stem cells

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.

Septation and shortening of outflow tract in embryonic mouse heart involve changes in cardiomyocyte phenotype and α-SMA positive cells in the endocardium

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

CREG accelerates vasculogenesis in mouse embryonic stem sellsderived embryonic bodies

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.

TGF-β receptors in mouse ES-5 cells and their differentiated derivatives

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.

TRPC3 is required for the survival, pluripotency and neural differentiation of mouse embryonic stem cells(mESCs)

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.

Derivation of Haploid Neurons from Mouse Androgenetic Haploid Embryonic Stem Cells

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].

Incorporation of a histone mutant with H3K56 site substitution perturbs the replication machinery in mouse embryonic stem cells

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.

Propofol inhibits neuronal differentiation of mouse embryonic stem cells in vitro

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.

Role of circadian gene Clock during differentiation of mouse pluripotent stem cells

Biological rhythms controlled by the circadian clock are absent in embryonic stem cells （ESCs）. However, they start to develop during the differentiation of pluripotent ESCs to downstream cells. Conversely, biological rhythms in adult somatic cells disappear when they are reprogrammed into induced pluripotent stem cells （iPSCs）. These studies indicated that the development of biological rhythms in ESCs might be closely associated with the maintenance and differentiation of ESCs. The core circadian gene Clock is essential for regulation of biological rhythms. Its role in the development of biological rhythms of ESCs is totally unknown. Here, we used CRISPR/CAS9-mediated genetic editing techniques, to completely knock out the Clock expression in mouse ESCs. By AP, teratoma formation, quantitative real-time PCR and Immunofluorescent staining, we did not find any dif- ference between Clock knockout mESCs and wild type mESCs in morphology and pluripotent capability under the pluripotent state. In brief, these data indicated Clock did not influence the maintaining of pluripotent state. However, they exhibited decreased proliferation and increased apoptosis. Furthermore, the biological rhythms failed to develop in Clock knockout mESCs after spontaneous differentiation, which indicated that there was no compensational factor in most peripheral tissues as described in mice models before （DeBruyne et ah, 2007b）. After spontaneous differentiation, loss of CLOCK protein due to Clock gene silencing induced spontaneous differentiation of mESCs, indicating an exit from the pluripotent state, or its differentiating ability. Our findings indicate that the core circadian gene Clock may be essential during normal mESCs differentiation by regulating mESCs proliferation, apoptosis and activity.

Fibroblasts upregulate expression of adhesion molecules and promote lymphocyte retention in 3D fibroin/gelatin scaffolds

Bioengineered scaffolds are crucial components in artificial tissue construction.In general,these scaffolds provide inert three-dimensional(3D)surfaces supporting cell growth.However,some scaffolds can affect the phenotype of cultured cells,especially,adherent stromal cells,such as fibroblasts.Here we report on unique properties of 3D fibroin/gelatin materials,which may rapidly induce expression of adhesion molecules,such as ICAM-1 and VCAM-1,in cultured primary murine embryonic fibroblasts(MEFs).In contrast,two-dimensional(2D)fibroin/gelatin films did not show significant effects on gene expression profiles in fibroblasts as compared to 3D culture conditions.Interestingly,TNF expression was induced in MEFs cultured in 3D fibroin/gelatin scaffolds,while genetic or pharmacological TNF ablation resulted in diminished ICAM-1 and VCAM-1 expression by these cells.Using selective MAPK inhibitors,we uncovered critical contribution of JNK to 3D-induced upregulation of these adhesion molecules.Moreover,we observed ICAM-1/VCAM-1-dependent adhesion of lymphocytes to fibroblasts cultured in 3D fibroin/gelatin scaffolds,but not on 2D fibroin/gelatin films,suggesting functional reprogramming in stromal cells,when exposed to 3D environment.Finally,we observed significant infiltration of lymphocytes into 3D fibroin/gelatin,but not into collagen scaffolds in vivo upon subcapsular kidney implantation in mice.Together our data highlight the important features of fibroin/gelatin scaffolds,when they are produced as 3D sponges rather than 2D films,which should be considered when using these materials for tissue engineering.