The complex roles of m^(6)A modifications in neural stem cell proliferation, differentiation, and self-renewal and implications for memory and neurodegenerative diseases

N6-methyladenosine(m^(6)A), the most prevalent and conserved RNA modification in eukaryotic cells, profoundly influences virtually all aspects of mRNA metabolism. mRNA plays crucial roles in neural stem cell genesis and neural regeneration, where it is highly concentrated and actively involved in these processes. Changes in m^(6)A modification levels and the expression levels of related enzymatic proteins can lead to neurological dysfunction and contribute to the development of neurological diseases. Furthermore, the proliferation and differentiation of neural stem cells, as well as nerve regeneration, are intimately linked to memory function and neurodegenerative diseases. This paper presents a comprehensive review of the roles of m^(6)A in neural stem cell proliferation, differentiation, and self-renewal, as well as its implications in memory and neurodegenerative diseases. m^(6)A has demonstrated divergent effects on the proliferation and differentiation of neural stem cells. These observed contradictions may arise from the time-specific nature of m^(6)A and its differential impact on neural stem cells across various stages of development. Similarly, the diverse effects of m^(6)A on distinct types of memory could be attributed to the involvement of specific brain regions in memory formation and recall. Inconsistencies in m^(6)A levels across different models of neurodegenerative disease, particularly Alzheimer's disease and Parkinson's disease, suggest that these disparities are linked to variations in the affected brain regions. Notably, the opposing changes in m^(6)A levels observed in Parkinson's disease models exposed to manganese compared to normal Parkinson's disease models further underscore the complexity of m^(6)A's role in neurodegenerative processes. The roles of m^(6)A in neural stem cell proliferation, differentiation, and self-renewal, and its implications in memory and neurodegenerative diseases, appear contradictory. These inconsistencies may be attributed to the timespecific nature of m^(6)A and its varying effects on distinct brain regions and in different environments.

Maintenance of stem cell self-renewal by sex chromosomal zincfinger transcription factors

In this Editorial review,we would like to focus on a very recent discovery showing the global autosomal gene regulation by Y-and inactivated X-chromosomal transcription factors,zinc finger gene on the Y chromosome(ZFY)and zinc finger protein X-linked(ZFX).ZFX and ZFY are both zinc-finger proteins that encode general transcription factors abundant in hematopoietic and embryonic stem cells.Although both proteins are homologs,interestingly,the regulation of self-renewal by these transcriptional factors is almost exclusive to ZFX.This fact implies that there are some differential roles between ZFX and ZFY in regulating the maintenance of self-renewal activity in stem cells.Besides the maintenance of stemness,ZFX overexpression or mutations may be linked to certain cancers.Although cancers and stem cells are double-edged swords,there is no study showing the link between ZFX activity and the telomere.Thus,stemness or cancers with ZFX may be linked to other molecules,such as Oct4,Sox2,Klf4,and others.Based on very recent studies and a few lines of evidence in the past decade,it appears that the ZFX is linked to the canonical Wnt signaling,which is one possible mechanism to explain the role of ZFX in the self-renewal of stem cells.

Glutathione metabolism is essential for self-renewal and chemoresistance of pancreatic cancer stem cells

BACKGROUND Cellular metabolism regulates stemness in health and disease.A reduced redox state is essential for self-renewal of normal and cancer stem cells(CSCs).However,while stem cells rely on glycolysis,different CSCs,including pancreatic CSCs,favor mitochondrial metabolism as their dominant energy-producing pathway.This suggests that powerful antioxidant networks must be in place to detoxify mitochondrial reactive oxygen species(ROS)and maintain stemness in oxidative CSCs.Since glutathione metabolism is critical for normal stem cell function and CSCs from breast,liver and gastric cancer show increased glutathione content,we hypothesized that pancreatic CSCs also rely on this pathway for ROS detoxification.AIM To investigate the role of glutathione metabolism in pancreatic CSCs.METHODS Primary pancreatic cancer cells of patient-derived xenografts(PDXs)were cultured in adherent or CSC-enriching sphere conditions to determine the role of glutathione metabolism in stemness.Real-time polymerase chain reaction(PCR)was used to validate RNAseq results involving glutathione metabolism genes in adherent vs spheres,as well as the expression of pluripotency-related genes following treatment.Public TCGA and GTEx RNAseq data from pancreatic cancer vs normal tissue samples were analyzed using the webserver GEPIA2.The glutathione-sensitive fluorescent probe monochlorobimane was used to determine glutathione content by fluorimetry or flow cytometry.Pharmacological inhibitors of glutathione synthesis and recycling[buthionine-sulfoximine(BSO)and 6-Aminonicotinamide(6-AN),respectively]were used to investigate the impact of glutathione depletion on CSC-enriched cultures.Staining with propidium iodide(cell cycle),Annexin-V(apoptosis)and CD133(CSC content)were determined by flow cytometry.Self-renewal was assessed by sphere formation assay and response to gemcitabine treatment was used as a readout for chemoresistance.RESULTS Analysis of our previously published RNAseq dataset E-MTAB-3808 revealed upregulation of genes involved in the KEGG(Kyoto Encyclopedia of Genes and Genomes)Pathway Glutathione Metabolism in CSC-enriched cultures compared to their differentiated counterparts.Consistently,in pancreatic cancer patient samples the expression of most of these up-regulated genes positively correlated with a stemness signature defined by NANOG,KLF4,SOX2 and OCT4 expression(P<10-5).Moreover,3 of the upregulated genes(MGST1,GPX8,GCCT)were associated with reduced disease-free survival in patients[Hazard ratio(HR)2.2-2.5;P=0.03-0.0054],suggesting a critical role for this pathway in pancreatic cancer progression.CSC-enriched sphere cultures also showed increased expression of different glutathione metabolism-related genes,as well as enhanced glutathione content in its reduced form(GSH).Glutathione depletion with BSO induced cell cycle arrest and apoptosis in spheres,and diminished the expression of stemness genes.Moreover,treatment with either BSO or the glutathione recycling inhibitor 6-AN inhibited self-renewal and the expression of the CSC marker CD133.GSH content in spheres positively correlated with intrinsic resistance to gemcitabine treatment in different PDXs r=0.96,P=5.8×1011).Additionally,CD133+cells accumulated GSH in response to gemcitabine,which was abrogated by BSO treatment(P<0.05).Combined treatment with BSO and gemcitabine-induced apoptosis in CD133+cells to levels comparable to CD133-cells and significantly diminished self-renewal(P<0.05),suggesting that chemoresistance of CSCs is partially dependent on GSH metabolism.CONCLUSION Our data suggest that pancreatic CSCs depend on glutathione metabolism.Pharmacological targeting of this pathway showed that high GSH content is essential to maintain CSC functionality in terms of self-renewal and chemoresistance.

Correlation between amino acid metabolism and self-renewal of cancer stem cells: Perspectives in cancer therapy

Cancer stem cells(CSCs)possess self-renewal and differentiation potential,which may be related to recurrence,metastasis,and radiochemotherapy resistance during tumor treatment.Understanding the mechanisms via which CSCs maintain self-renewal may reveal new therapeutic targets for attenuating CSC resistance and extending patient life-span.Recent studies have shown that amino acid metabolism plays an important role in maintaining the self-renewal of CSCs and is involved in regulating their tumorigenicity characteristics.This review summarizes the relationship between CSCs and amino acid metabolism,and discusses the possible mechanisms by which amino acid metabolism regulates CSC characteristics particularly self-renewal,survival and stemness.The ultimate goal is to identify new targets and research directions for elimination of CSCs.

Macrophage populations and self-renewal:Changing the paradigm

The origin of macrophages has been considered since several decades to be a continuum from bone marrow(BM) to tissue via monocytes as precursors. The development of new tools such as genetic lineage tracing,parabiosis and BM chimeras changed the paradigm of macrophage origin. In steady state,most resident macrophages are of embryonic origin,whereas a monocyte origin remains prominent in pathological conditions. The findings of a proliferation of mature macrophages will oblige us to reappraise the relationship between proliferation and differentiation in macrophages. This review is based on the recent explosion of high impact articles on macrophage biology. It summarizes new data on the origin of macrophages and their selfrenewal potential in steady states. While monocytes are required for intestinal macrophage development,the microglia is independent of monocyte influx and skin macrophages provide an excellent model of the balance between monocyte input and self-renewal. In addition,macrophage proliferation requires intrinsic and extrinsic factors including growth factors and cytokines. It also analyzes the impact of this new paradigm in human diseases such as athrosclerosis,cancer,infectious diseases and neurodegenerative diseases. In atherosclerosis,the finding of macrophage proliferation within the lesions will change our understanding of disease pathophysiology,this new paradigm may have therapeutical impact in the future.

TRIM27 maintains gut homeostasis by promoting intestinal stem cell self-renewal

Dysregulation of gut homeostasis is associated with irritable bowel syndrome(IBS),a chronic functional gastrointestinal disorder affecting approximately 11.2%of the global population.The poorly understood pathogenesis of IBS has impeded its treatment.Here,we report that the E3 ubiquitin ligase tripartite motif-containing 27(TRIM27)is weakly expressed in IBS but highly expressed in inflammatory bowel disease(IBD),a frequent chronic organic gastrointestinal disorder.Accordingly,knockout of Trim27 in mice causes spontaneously occurring IBS-like symptoms,including increased visceral hyperalgesia and abnormal stool features,as observed in IBS patients.Mechanistically,TRIM27 stabilizesβ-catenin and thus activates Wnt/β-catenin signaling to promote intestinal stem cell(ISC)self-renewal.Consistent with these findings,Trim27 deficiency disrupts organoid formation,which is rescued by reintroducing TRIM27 orβ-catenin.Furthermore,Wnt/β-catenin signaling activator treatment ameliorates IBS symptoms by promoting ISC self-renewal.Taken together,these data indicate that TRIM27 is critical for maintaining gut homeostasis,suggesting that targeting the TRIM27/Wnt/β-catenin axis could be a potential treatment strategy for IBS.Our study also indicates that TRIM27 might serve as a potential biomarker for differentiating IBS from IBD.

Regulation of embryonic stem cell self-renewal and differentiation by TGF-β family signaling

Embryonic stem (ES) cells are characterized by their ability to indefinitely self-renew and potential to differentiate into all the cell lineages of the body. ES cells are considered to have potential applications in regenerative medicine. In particular, the emergence of an ES cell analogue-induced pluripotent stem (iPS) cells via somatic cell reprogramming by co-expressing a limited number of critical stemness-related transcriptional factors has solved the problem of obtaining patient-specific pluripotent cells, encouraging researchers to develop more specific and functional cell lineages from ES or iPS cells for broad therapeutic applications. ES cell fate choice is delicately controlled by a core transcriptional network, epigenetic modification profiles and complex signaling cascades both intrinsically and extrinsically. Of these signals, transforming growth factor β (TGF-β) family members, including TGF-β, bone morphogenetic protein (BMP), Activin and Nodal, have been reported to influence cell self-renewal and a broad spectrum of lineage differentiation in ES cells, in accordance with the key roles of TGF-β family signaling in early embryo development. In this review, the roles of TGF-β family signals in coordinating ES cell fate determination are summarized.

The Hippo pathway regulates stem cell proliferation,self-renewal,and differentiation

Stem cells and progenitor cells are the cells of origin for multi-cellular organisms and organs.They play key roles during development and their dysregulation gives rise to human diseases such as cancer.The recent de-velopment of induced pluripotent stem cell(iPSC)technology which converts somatic cells to stem-like cells holds great promise for regenerative medicine.Nevertheless,the understanding of proliferation,dif-ferentiation,and self-renewal of stem cells and or-gan-specific progenitor cells is far from clear.Recently,the Hippo pathway was demonstrated to play important roles in these processes.The Hippo pathway is a newly established signaling pathway with critical functions in limiting organ size and suppressing tumorigenesis.This pathway was first found to inhibit cell proliferation and promote apoptosis,therefore regulating cell num-ber and organ size in both Drosophila and mammals.However,in several organs,disturbance of the pathway leads to specific expansion of the progenitor cell com-partment and manipulation of the pathway in embryonic stem cells strongly affects their self-renewal and dif-ferentiation.In this review,we summarize current ob-servations on roles of the Hippo pathway in different types of stem cells and discuss how these findings changed our view on the Hippo pathway in organ de-velopment and tumorigenesis.

Icariin Promotes Self-Renewal of Neural Stem Cells:An Involvement of Extracellular Regulated Kinase Signaling Pathway

Objective： To investigate the effects and underlying molecular mechanisms of icariin （ICA） on self-renewal and differentiation of neural stem cells （NSCs）. Methods： NSCs were derived from forebrains of mice embryos by mechanical dissociation into single cell suspension. The self-renewal of NSCs was measured by neurosphere formation assay. The proliferation of NSCs was detected by water-soluble tetrazolium （WST） and 5-ethynyl-2＇-deoxyuridine （EdU） incorporation assay. Protein expression of neuron-specific marker tubulin-βⅢ（TuJ1） and astrocyte-specific marker glial fibrillary acidic protein （GFAP） were measured by immunofluorescence and Western blotting. Using microarray, the differentially expressed genes （DEGs） were screened between NSCs with or without ICA treatment. The signaling pathways enriched by these DEGs and their role in mediating effects of ICA were analyzed. Results： ICA significantly promoted neurosphere formation of NSCs cultured in growth protocol in a dose-dependent manner and achieved the maximum effects at 100 nmol/L. ICA also increased optical absorbance value and EdU incorporation into nuclei of NSCs. ICA had no significant effects on the percentage of TuJ1 or GFAP-positive cells, and TuJ1 or GFAP protein expression in NSCs cultured in differentiation protocol. A total of 478 genes were found to be differentially regulated. Among signaling pathways significantly enriched by DEGs, mitogen activated protein kinase （MAPK） pathway was of interest. Blockade of extracellular signal-regulated kinase （ERK）/MAPK, other than p38/MAPK subfamily pathway partially abolished effects of ICA on neurosphere formation and EdU incorporation of NSCs. Conclusion： ICA can promote the self- renewal of NSCs at least partially through ERK/MAPK signaling pathway.

Extrinsic and intrinsic factors controlling spermatogonial stem cell self-renewal and differentiation

Spermatogonial stem cells （SSCs）, the stem cells responsible for male fertility, are one of a small number of cells with the abilities of both self-renewal and generation of large numbers of haploid cells. Technology improvements, most importantly, transplantation assays and in vitro culture systems have greatly expanded our understanding of SSC self-renewal and differentiation. Many important molecules crucial for the balance between self-renewal and differentiation have been recently identified although the exact mechanism（s） remain largely undefined. In this review, we give a brief introduction to SSCs, and then focus on extrinsic and intrinsic factors controlling SSCs self-renewal and differentiation.

rtcisE2F promotes the self-renewal and metastasis of liver tumorinitiating cells via N^(6)-methyladenosine-dependent E2F3/E2F6 mRNA stability

Liver cancer is highly heterogeneous,and the tumor tissue harbors a variety of cell types.Liver tumor initiating cells(TICs)well contribute to tumor heterogeneity and account for tumor initiation and metastasis,but the molecular mechanisms of liver TIC self-renewal are elusive.Here,we identified a functional read-through rt-circRNA,termed rtcisE2F,that is highly expressed in liver cancer and liver TICs.rtcisE2F plays essential roles in the self-renewal and activities of liver TICs.rtcisE2F targets E2F6 and E2F3 mRNAs,attenuates mRNA turnover,and increases E2F6/E2F3 expression.Mechanistically,rtcisE2F functions as a scaffold of N^(6)-methyladenosine(m^(6)A)reader IGF2BP2 and E2F6/E2F3 mRNA.rtcisE2F promotes the association of E2F6/E2F3 mRNAs with IGF2BP2,and inhibits their association with another m^(6)A reader,YTHDF2.IGF2BP2 inhibits E2F6/E2F3 mRNA decay,whereas YTHDF2 promotes E2F6/E2F3 mRNA decay.By switching m^(6)A readers,rtcisE2F enhances E2F6/E2F3 mRNA stability.E2F6 and E2F3 are both required for liver TIC self-renewal and Wnt/β-catenin activation,and inhibition of these pathways is a potential strategy for preventing liver tumorigenesis and metastasis.In conclusion,the rtcisE2F-IGF2BP2/YTHDF2-E2F6/E2F3-Wnt/β-catenin axis drives liver TIC self-renewal and initiates liver tumorigenesis and metastasis,and may provide a strategy to eliminate liver TICs.

A chromatin modulator sustains self-renewal and enables differentiation of postnatal neural stem and progenitor cells

It remains unknown whether H3K4 methylation,an epigenetic modification associated with gene activation,regulates fate determination of the postnatal neural stem and progenitor cells(NSPCs).By inactivating the Dpy30 subunit of the major H3K4 methyltransferase complexes in specific regions of mouse brain,we demonstrate a crucial role of efficient H3K4 methylation in maintaining both the self-renewal and differentiation capacity of postnatal NSPCs.Dpy30 deficiency disrupts development of hippocampus and especially the dentate gyrus and subventricular zone,the major regions for postnatal NSC activities.Dpy30 is indispensable for sustaining the self-renewal and proliferation of NSPCs in a cell-intrinsic manner and also enables the differentiation of mouse and human neural progenitor cells to neuronal and glial lineages.Dpy30 directly regulates H3K4 methylation and the induction of several genes critical in neurogenesis.These findings link a prominent epigenetic mechanism of gene expression to the fundamental properties of NSPCs and may have implications in neurodevelopmental disorders.

The molecular mechanism of embryonic stem cell pluripotency and self-renewal

The self-renewal and pluripotency of embryonic stem cells (ESCs) is regulated by a network, which consists of a series of cell factors in microenviroments, a chain of transcription factors and certain signal conduction pathways. This article reviews recent progress in this field to elucidate the mecha-nism involved.

Transcriptome Analysis Identifies SenZfp536,a Sense LncRNA that Suppresses Self-renewal of Cortical Neural Progenitors

Long non-coding RNAs(lncRNAs)regulate transcription to control development and homeostasis in a variety of tissues and organs.However,their roles in the development of the cerebral cortex have not been well elucidated.Here,a bioinformatics pipeline was applied to delineate the dynamic expression and potential cis-regulating effects of mouse lncRNAs using transcriptome data from 8 embryonic time points and sub-regions of the developing cerebral cortex.We further characterized a sense lncRNA,SenZfp536,which is transcribed downstream of and partially overlaps with the protein-coding gene Zfp536.Both SenZfp536 and Zfp536 were predominantly expressed in the proliferative zone of the developing cortex.Zfp536 was cis-regulated by SenZfp536,which facilitates looping between the promoter of Zfp536 and the genomic region that transcribes SenZfp536.Surprisingly,knocking down or activating the expression of SenZfp536 increased or compromised the proliferation of cortical neural progenitor cells(NPCs),respectively.Finally,overexpressing Zfp536 in cortical NPCs reversed the enhanced proliferation of cortical NPCs caused by SenZfp536 knockdown.The study deepens our understanding of how lncRNAs regulate the propagation of cortical NPCs through cis-regulatory mechanisms.

Self-Renewal Consortium Blockchain Based on Proof of Rest and Strong Smart Contracts

Focusing on the business alliance scenario in blockchains,this paper proposes a new consensus mechanism named proof of rest(PoR)and strong smart contracts.The block structure and logic of PoR consensus are described.And a consortium blockchain system supporting strong smart contracts is designed.We modify the difficulty value algorithm based on proof of work(PoW)and add adjustable parameters.The longer a node rests after creating a block,the less difficult it is to create another new block,hence the term PoR.The penalty for slack nodes,the joining and quitting of nodes,and the adjustment of the expected block creation time can all be accomplished using the strong smart contracts,so the consortium blockchain can realize self-renewal.

Spermatogonial Stem Cell Self-renewal and Differentiation

Mammalian spermatogenesis is a complicated and precisely controlled process that requires spermatogonial stem cells(SSCs).SSCs maintain the stem cell pool,balance self-renewal–commitment with differentiation,and produce millions of sperm daily.Self-renewal and differentiation are controlled by intrinsic factors within SSCs and extrinsic factors from the"niche."In this review,we discuss the biology of SSCs and the factors regulating their self-renewal and differentiation.

Screening for self-renewal factors by a combination of mRNA and CGH microarray in human embryonic stem cells

Human embryonic stem cells(hESCs)undergo self-renewal while maintaining pluripotency.However,the molecular mechanism that demonstrates how these cells maintain their undifferentiated state and how they self-renew is poorly understood.Here,we characterized an aneuploidy H1 hESC subline(named H1T)using karyotyping and comparative genomic hybridization(CGH)microarray.Because the H1T hESC line displays a self-renewal advantage while maintaining an undiffer-entiated state,we speculated that the expression patterns of specific genes which are related to pluripotency or differentiation were altered;therefore,we attempted to screen for molecules that are propitious for maintenance of stemness by performing a combination of mRNA and CGH microarray analysis which compared the aneuploidy H1T hESC subline versus the euploid H1 hESC line.It is discovered that some genes are up-regulated in H1T hESC subline such as TBX2 and Wnt3,while some are downregulated,for example,Fbxo7 and HMG2L1.Ourfindings should fascilitate the study of the complex signaling network which maintains hESC pluripotency and function.

Interleukin-12 supports in vitro self-renewal of long-term hematopoietic stem cells

Hematopoietic stem cells(HSCs)self-renew or differentiate through division.Cytokines are essential for inducing HSC division,but the optimal cytokine combination to control self-renewal of HSC in vitro remains unclear.In this study,we compared the effects of interleukin-12(IL-12)and thrombopoietin(TPO)in combination with stem cell factor(SCF)on in vitro self-renewal of HSCs.Single-cell assays were used to overcome the heterogeneity issue of HSCs,and serum-free conditions were newly established to permit reproduction of data.In single-cell cultures,CD150^(+)CD48^(-)CD41^(-)CD34^(-)c-Kit^(+)Sca-1^(+)lineage^(-)SCs divided significantly more slowly in the presence of SCF+IL-12 compared with cells in the presence of SCF+TPO.Serial transplantation of cells from bulk and clonal cultures revealed that TPO was more effective than IL-12 at supporting in vitro self-renewal of short-term(<6 months)HSCs,resulting in a monophasic reconstitution wave formation,whereas IL-12 was more effective than TPO at supporting the in vitro selfrenewal of long-term(>6 months)HSCs,resulting in a biphasic reconstitution wave formation.The control of division rate in HSCs appeared to be crucial for preventing the loss of self-renewal potential from their in vitro culture.

Vitamin C maintaining bone marrow mesenchymal stem cell self-renewal

Background Bone marrow mesenchymal stem cells （BMSCs） can be isolated and cultured to many passages However, Stem cells including BMSCs quickly undergo senescence in culture. The cell senescence and multidirectional differentiation have hampered producing BMSCs in quantity with their undifferentiated state. In this study we report a natural compound, vitamin C （Vc）, maintains BMSCs stem property. Methods Human BMSCs were isolated from bone marrow and purified by 1.073 g/mL density gradient centrifugation. 50 ng/mL Vc were added to BMSCs for different time point. Flowcytometry was used to detect cell surface markers of BMSCs with or without Vc treatment. BMSCs proliferation was analyzed by MTF assay. PCR（polymerase chain reaction） and real-time PCR were used for detecting c-kit, nanog, and Oct-4 genes expression levels. DNA methyltransferase （Dnmt） 1 and Dnmt3b levels were also detected by real-time PCR. Results Flowcytometry showed that after Vc treatment for 6 h, the surface markers of BMSCs were almost unchanged. Vc increased the proliferation activity of BMSCs from 6h to 24 h. PCR showed the expression of c-kit, nanog, and oct-4 genes were obviously increased c-kit, nanog, and oct-4 in Vc treated group than control group at 12 h. Real-time PCR showed that the level of genes were unregulated from 6h to 12h compared with control group. Vc also increased Dnmt3b but not Dnmtl gene expression. Conclusions Our results showed Vc acts at least accelerates BMSCs proliferation and maintains stem cell property. In our study BMSCs generation and provided additional insights into the we highlighted a method of improving the speed of mechanistic basis of preventing BMSCs senescence

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.