Dynamic deformylation of 5-formylcytosine and decarboxylation of 5-carboxylcytosine during differentiation of mouse embryonic stem cells into mouse neurons

Regulation of cell fate requires the establishment and erasure of 5-methylcytosine(5mC) in genomic DNA.The formation of 5mC is achieved by DNA cytosine methyltransferases(DNMTs),whereas the removal of5mC can be accomplished by various pathways.Aside from ten-eleven translocation(TET)-mediated oxidation of 5mC followed by thymine DNA glycosylase(TDG)-initiated base excision repair(BER),the direct deformylation of 5-formylcytosine(5fC) and decarboxylation of 5-carboxylcytosine(5caC) have also been discovered as the novel DNA demethylation pathways.Although these novel demethylation pathways have been identified in stem cells and somatic cells,their precise roles in regulating cell fate remain unclear.Here,we differentiate mouse embryonic stem cells(mESCs) into mouse embryoid bodies(mEBs),followed by further differentiation into mouse neural stem cells(mNSCs) and finally into mouse neurons(mNeurons).During this sequential differentiation process,we employ probe molecules,namely2'-fluorinated 5-formylcytidine(F-5fC) and 2'-fluorinated 5-carboxyldeoxycytidine(F-5caC),for metabolic labeling.The results of mass spectrometry(MS) analysis demonstrate the deformylation and decarboxylation activities are progressively decreased and increased respectively during differentiation process,and this opposite demethylation tendency is not associated with DNMTs and TETs.

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.

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.

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.

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.

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.