Differentiation Of P19 Cells Research Articles

Mechanical stimulation promotes the maturation of cardiomyocyte-like cells from P19 cells and the function in a mouse model of myocardial infarction.

In this study, we aimed to promote the maturation of cardiomyocytes-like cells by mechanical stimulation, and evaluate their therapeutic potential against myocardial infarction. The cyclic tensile strain was used to induce the maturation of cardsiomyocyte-like cells from P19 cells in vitro. Western blot and qPCR assays were performed to examine protein and gene expression, respectively. High-resolution respirometry was used to assay cell function. The induced cells were then evaluated for their therapeutic effect. In vitro, we observed cyclic tensile strain induced P19 cell differentiation into cardiomyocyte-like cells, as indicated by the increased expression of cardiomyocyte maturation-related genes such as Myh6, Myl2, and Gja1. Furthermore, cyclic tensile strain increased the antioxidant capacity of cardiomyocytes by upregulating the expression Sirt1, a gene important for P19 maturation into cardiomyocyte-like cells. High-resolution respirometry analysis of P19 cells following cyclic tensile strain showed enhanced metabolic function. In vivo, stimulated P19 cells enhanced cardiac function in a mouse model of myocardial infarction, and these mice showed decreased infarction-related biomarkers. The current study demonstrates a simple yet effective mean to induce the maturation of P19 cells into cardiomyocyte-like cells, with a promising therapeutic potential for the treatment of myocardial infarction.

Pcgf5: An important regulatory factor in early embryonic neural induction

Polycomb group RING finger (PCGF) proteins, a crucial subunits of the Polycomb complex, plays an important role in regulating gene expression, embryonic development, and cell fate determination. In our research, we investigated Pcgf5, one of the six PCGF homologs, and its impact on the differentiation of P19 cells into neural stem cells. Our findings revealed that knockdown of Pcgf5 resulted in a significant decrease in the expression levels of the neuronal markers Sox2, Zfp521, and Pax6, while the expression levels of the pluripotent markers Oct4 and Nanog increased. Conversely, Pcgf5 overexpression upregulated the expression of Sox2 and Pax6, while downregulating the expression of Oct4 and Nanog. Additionally, our analysis revealed that Pcgf5 suppresses Wnt3 expression via the activation of Notch1/Hes1, and ultimately governs the differentiation fate of neural stem cells. To further validate our findings, we conducted in vivo experiments in zebrafish. We found that knockdown of pcgf5a using morpholino resulted in the downregulated expression of neurodevelopmental genes such as sox2, sox3, and foxg1 in zebrafish embryos. Consequently, these changes led to neurodevelopmental defects. In conclusion, our study highlights the important role of Pcgf5 in neural induction and the determination of neural cell fate.

A unique poly(A) tail profile uncovers the stability and translational activation of TOP transcripts during neuronal differentiation

Cell differentiation is associated with global changes in translational activity. Here, we characterize how mRNA poly(A) tail processing supports this dynamic. We observe that decreased translation during neuronal differentiation of P19 cells correlates with the downregulation of 5'-terminal oligopyrimidine (TOP) transcripts which encode the translational machinery. Despite their downregulation, TOP transcripts remain highly stable and show increased translation as cells differentiate. Changes in TOP mRNA metabolism are reflected by their accumulation with poly(A) tails ∼60-nucleotide (nt) long. The dynamic changes in poly(A) processing can be partially recapitulated by depleting LARP1 or activating the mTOR pathway in undifferentiated cells. Although mTOR-induced accumulation of TOP mRNAs with tails ∼60-nt long does not trigger differentiation, it is associated with reduced proliferation of neuronal progenitors. We propose that while TOP mRNAs are transcriptionally silenced, their post-transcriptional regulation mediated by a specific poly(A) processing ensures an adequate supply of ribosomes to complete differentiation.

Functionalized Exosomes Promote Neural Differentiation of P19 Cells by Activating the Wnt Signaling Pathway.

Global population growth and population aging continue to accelerate, and lead to a further increase in the risk of neurological diseases worldwide. Extracellular vesicles secreted by mesenchymal stem cells carry many proteins, lipids, and genetic material that mediate cell-to-cell communication and improve therapeutic outcomes for neurological disorders. Stem cells from human exfoliated deciduous teeth are considered a suitable cell source for tissue regeneration, which exerts therapeutic effects via the secretion of exosomes. This study was performed to assess the effect of functionalized exosomes on neural differentiation of embryonic carcinoma cell line P19. We stimulated stem cells from human exfoliated deciduous teeth with the glycogen synthase kinase-3β inhibitor TWS119 and then extracted its exosomes. P19 cells were induced to differentiate using functionalized exosomes, and the biological functions and involved signaling pathways of differentially expressed genes were analyzed by RNA-sequencing. Immunofluorescence techniques detected neuronal specific markers. It was found that TWS119 activated the Wnt signaling pathway in stem cells from human exfoliated deciduous teeth. RNA-sequencing showed that upregulated differentially expressed genes in the functionalized exosome-treated group were responsible for cell differentiation, neurofilament, and structural constituent of the synapse. Kyoto Encyclopedia of Genes and Genomes enrichment analysis revealed that the functionalized exosome-treated group activated the Wnt signaling pathway. Immunofluorescence showed that functionalized exosomes induced neurite outgrowth in P19 cells. Our results demonstrated that functionalized exosomes promoted neural differentiation of P19 cells by activating the Wnt signaling pathway.

Enhanced neural differentiation by applying electrical stimulation utilizing conductive and antioxidant alginate-polypyrrole/poly-l-lysine hydrogels

The challenge of restoration from neurodegenerative disorder requires effective solutions. To enhance the healing efficiencies, scaffolds with antioxidant activities, electroconductivity, and versatile features to encourage neuronal differentiation are potentially useful. Herein, polypyrrole-alginate (Alg-PPy) copolymer was used to design antioxidant and electroconductive hydrogels through the chemical oxidation radical polymerization method. The hydrogels have antioxidant effects to combat oxidative stress in nerve damage thanks to the introduction of PPy. Additionally, poly-l-lysine (PLL) provided these hydrogels with a great differentiation ability of stem cells. The morphology, porosity, swelling ratio, antioxidant activity, rheological behavior, and conductive characteristics of these hydrogels were precisely adjusted by altering the amount of PPy. Characterization of hydrogels showed appropriate electrical conductivity and antioxidant activity for neural tissue applications. Cytocompatibility, live/dead assays, and Annexin V/PI staining by flow cytometry using P19 cells confirmed the excellent cytocompatibility and cell protective effect under ROS microenvironment of these hydrogels in both normal and oxidative conditions. The neural marker investigation in the induction of electrical impulses was assessed through RT-PCR and immunofluorescence assay, demonstrating the differentiation of P19 cells to neurons cultured in these scaffolds. In summary, the antioxidant and electroconductive Alg-PPy/PLL hydrogels demonstrated excellent potential as promising scaffolds for treating neurodegenerative disorders.

Exosomes induce neurogenesis of pluripotent P19 cells

Exosomes play a role in tissue/organ development and differentiation. Retinoic acid induces differentiation of P19 cells (UD-P19) to P19 neurons (P19N) that behave like cortical neurons and express characteristic neuronal genes such as NMDA receptor subunits. Here we report P19N exosome-mediated differentiation of UD-P19 to P19N. Both UD-P19 and P19N released exosomes with characteristic exosome morphology, size, and common protein markers. P19N internalized significantly higher number of Dil-P19N exosomes as compared to UD-P19 with accumulation in the perinuclear region. Continuous exposure of UD-P19 to P19N exosomes for six days induced formation of small-sized embryoid bodies that differentiated into MAP2-/GluN2B-positive neurons recapitulating RA-induction of neurogenesis. Incubation with UD-P19 exosomes for six days did not affect UD-P19. Small RNA-seq identified enrichment of P19N exosomes with pro-neurogenic non-coding RNAs (ncRNAs) such as miR-9, let-7, MALAT1 and depleted with ncRNAs involved in maintenance of stem cell characteristics. UD-P19 exosomes were rich with ncRNAs required for maintenance of stemness. P19N exosomes provide an alternative method to genetic modifications for cellular differentiation of neurons. Our novel findings on exosomes-mediated differentiation of UD-P19 to P19 neurons provide tools to study pathways directing neuron development/differentiation and develop novel therapeutic strategies in neuroscience.Graphical

Identification of differential microRNAs and messenger RNAs resulting from ASXL transcriptional regulator 3 knockdown during during heart development

ABSTRACT Congenital heart disease (CHD) is the most common birth defect. Although ASXL transcriptional regulator 3 (ASXL3) has been reported to cause hereditary CHD, ASXL3-mediated mechanisms in heart development remain unclear. In this study, we used dimethyl sulfoxide (DMSO) to induce differentiation in P19 cells, observed cell morphology using light microscopy after ASXL3 knockdown, and determined the levels of associated myocardial cell markers using reverse transcription-quantitative polymerase chain reaction and western blotting. Subsequently, we used microRNA sequencing, messenger RNA (mRNA) sequencing, and bioinformatics to initially identify the possible mechanisms through which ASXL3-related microRNAs and mRNAs affect heart development. The results indicated that DMSO induced P19 cell differentiation, which could be inhibited by ASXL3 knockdown. We screened 1214 and 1652 differentially expressed microRNAs and mRNAs, respectively, through ASXL3 knockdown and sequencing; these differentially expressed miRNAs were largely enriched in PI3K-Akt, mitogen-activated protein kinase, and Rap1 signaling pathways. Additionally, 11 miRNAs associated with heart development were selected through a literature review. Our analysis indicated the involvement of mmu-miR-323-3p in P19 cell differentiation through the PI3K-Akt pathway. In conclusion, ASXL3 may be involved in the regulation of heart development. This comprehensive study of differentially expressed microRNAs and mRNAs through ASXL3 knockdown in P19 cells provides new insights that may aid the prevention and treatment of CHD.

Prdm12 regulates inhibitory neuron differentiation in mouse embryonal carcinoma cells.

The epigenetic regulatory system significant influences the fate determination of cells during developmental processes. Prdm12 is a transcriptional regulator that modulates gene expression epigenetically. The Prdm12 gene has been shown to be expressed in neural tissues, specifically during development, but its detailed function is not fully understood. This study investigated the function of the Prdm12 gene in P19 mouse embryonic tumor cells as a model for neural differentiation. A decrease in the expression of neuron-specific genes and the alterations of dendrites and axons morphology was confirmed in Prdm12-knockout P19 cells. In addition, almost no astrocytes were generated in Prdm12-knockout P19 cells. Comprehensive gene expression analysis revealed that there was a reduction in the expression of the inhibitory neuron-specific genes Gad1/2 and Glyt2, but not the excitatory neuron-specific gene VGLUT2, in Prdm12-knockout P19 cells. Furthermore, the expression of inhibitory neuron-related factors, Ptf1a, Dbx1, and Gsx1/2, decreased in Prdm12-knockout P19 cells. Gene expression analysis also revealed that the Ptf1a, Hic1, and Foxa1 genes were candidate targets of Prdm12 during neurogenesis. These results suggest that Prdm12 regulates the differentiation of inhibitory neurons and astrocytes by controlling the expression of these genes during the neural differentiation of P19 cells.

NRF2 activation protects against valproic acid-induced disruption of neurogenesis in P19 cells

Valproic acid (VPA) is a commonly prescribed antiepileptic drug that causes fetal valproate syndrome (FVS) in developing embryos exposed to it. Symptoms of FVS include neural tube defects (NTDs), musculoskeletal abnormalities, and neurodevelopmental difficulties. One proposed mechanism of VPA-induced developmental toxicity is via oxidative stress, defined as the disruption of redox-sensitive cell signaling. We propose that redox imbalances caused by VPA exposure result in improper cellular differentiation that may contribute to FVS. In undifferentiated P19 mouse embryonal carcinoma cells treated with VPA, glutathione disulfide (GSSG) concentrations were higher and the glutathione (GSH)/GSSG redox potential (Eh) was more oxidizing compared to vehicle-treated control cells, both of which are indications of potential intracellular oxidative stress. Interestingly, VPA had no effect on GSH or GSSG levels in differentiated P19 neurons. Undifferentiated cells pretreated with 3H-1,2-dithiole-3-thione (D3T), an inducer of the nuclear factor erythroid 2-related factor 2 (NRF2) antioxidant response that combats cellular redox disruption, were protected from VPA-induced alterations to the GSH/GSSG system. To assess differential periods of susceptibility, P19 cells were exposed to VPA at various time points during their neuronal differentiation. Cells exposed to VPA early in the differentiation process did not undergo normal neurogenesis as measured by POU domain, class 5, transcription factor 1 (OCT4) and tubulin beta-3 chain (βIII-tubulin), markers of cell stemness and neuronal differentiation, respectively. Neurogenesis was improved with D3T pretreatments prior to VPA exposure. Furthermore, differentiating P19 cells treated with VPA exhibited increased protein oxidation that was diminished with D3T pretreatment. These findings demonstrate that VPA inhibits neurogenesis and propose NRF2-mediated redox homeostasis as a means to promote normal neuronal differentiation, thereby potentially decreasing the prevalence of FVS outcomes.

Valproic acid promotes SOD2 acetylation: a potential mechanism of valproic acid-induced oxidative stress in developing systems

Valproic acid (VPA) is an antiepileptic, bipolar, and migraine medication, which is associated with embryonic dysmorphology, more specifically neural tube defects (NTDs), if taken while pregnant. One mechanism by which VPA may cause NTDs is through oxidative stress that cause disruption of cell signaling. However, mechanisms of VPA-induced oxidative stress are not fully understood. Since VPA is a deacetylase inhibitor, we propose that VPA promotes mitochondrial superoxide dismutase-2 (SOD2) acetylation, decreasing SOD2 activity and increasing oxidant levels. Using the pluripotent embryonal carcinoma cell line, P19, VPA effects were evaluated in undifferentiated and neurodifferentiated cells. VPA treatments increased oxidant levels, oxidized the glutathione (GSH)/glutathione disulfide (GSSG) redox couple, and decreased total SOD and SOD2 activity in undifferentiated P19 cells but not in differentiated P19 cells. VPA caused a specific increase in mitochondrial oxidants in undifferentiated P19 cells, VPA did not alter respirometry measurements. Immunoblot analyses demonstrated that VPA increased acetylation of SOD2 at lysine68 (AcK68 SOD2) in undifferentiated P19 cells but not in differentiated P19 cells. Pretreatments with the Nrf2 inducer, dithiol-3-thione (D3T), in undifferentiated P19 cells prevented increased oxidant levels, GSH/GSSG redox oxidation and restored total SOD and SOD2 activity, correlating with a decrease in AcK68 SOD2 levels. In embryos, VPA decreased total SOD and SOD2 activity and increased levels of AcK68 SOD2, and D3T pretreatments prevented VPA effects, increasing total SOD and SOD2 activity and lowering levels of AcK68 SOD2. These data demonstrate a potential, contributing oxidizing mechanism by which VPA incites teratogenesis in developing systems. Moreover, these data also suggest that Nrf2 interventions may serve as a means to protect developmental signaling and inhibit VPA-induced malformations.

Fabrication of Graphene Oxide Aerogel to Repair Neural Tissue

: The neural tissue engineering has been designed as a subset of tissue engineering for treating congenital malformations and accident injuries, particularly for individuals requiring tissue grafting. Such transplants, usually performed as autografting, can often not meet the requirements of an effective scaffold used in nerve tissue engineering. A novel neural tissue scaffold was introduced here to solve the problem concerning the reduced graphene oxide. The three-dimensional graphene oxide in the neural canal restricts the formation of fibroglandular tissues and facilities neural stem cell proliferation and growth. In these techniques, graphene oxide aerogel was initially made. Then, the freeze-drying process was used to fix the geometry of reduced graphene oxide hydrogels prepared using graphene oxide dispersion and ethylenediamine and gain aerogels. The X-ray diffraction patterns, FTIR and morphological related to samples were examined, followed by conducting in-vitro micropropagation and 4, 6-diamidino-2-phenylindol (DAPI) staining in fibroblast and P19 cultures. The results from immunofluorescence staining demonstrated the neural differentiation of P19 cells. It can be concluded that most cells attached to and differentiated on the scaffold surface and axons can penetrate randomly through them. Finally, the three-dimensional graphene oxide was proposed as an ideal alternative to be used in neural tissue engineering.

Mechanical stretch promotes antioxidant responses and cardiomyogenic differentiation in P19 cells

Accumulating evidence has suggested that mechanical stimuli play a crucial role in regulating the lineage-specific differentiation of stem cells through fine-tuning redox balance. We aimed to investigate the effects of cyclic tensile strain (CTS) on the expression of antioxidant enzymes and cardiac-specific genes in P19 cells, a widely characterized tool for cardiac differentiation research. A stretching device was applied to generate different magnitude and duration of cyclic strains on P19 cells. The messenger RNA and protein levels of targeted genes were determined by real-time polymerase chain reaction and Western blot assays, respectively. Proper magnitude and duration of cognitivestimulationtherapy (CST) stimulation substantially enhanced the expression of both antioxidant enzymes and cardiac-specific genes in P19 cells. Sirtuin 1 (SIRT1) played an essential role in the CTS-induced cardiomyogenic differentiation of P19, as evidenced by changes in the expression of antioxidant enzymes and cardiac-specific genes. Mechanical loading promoted the cardiomyogenic differentiation of P19 cells. SIRT1 was involved in CST-mediated P19 differentiation, implying that SIRT1 might serve as an important target for developing methods to promote cardiomyogenic differentiation of stem cells.

Purα regulates the induction of Znf179 transcription during neuronal differentiation

Development of the mammalian central nervous system is an important process, which is accomplished through precise regulations of many different genes. Zinc finger protein 179 (Znf179) is one of the essential genes that plays a critical role in neuronal differentiation. In our previous study, Znf179 knockout mice displayed brain malformation and impaired brain functions. We have also previously shown that Znf179 involves in cell cycle regulation, but the regulatory mechanism of Znf179 expression is not yet fully characterized. Herein, we identified that Purα is an essential factor for the promotor activity of Znf179. We also showed concurrent expression of Znf179 and Purα during neuronal differentiation. We also found that overexpression of Purα increased Znf179 expression in neuronal differentiated P19 cells. Through its direct binding to Znf179, as shown using DAPA, Purα upregulates Znf179 expression, suggesting that Purα is important for the regulation of Znf179 expression during neuronal differentiation. Our data indicated that Purα is involved in the transcriptional regulation of Znf179 gene during neuronal differentiation, and is indispensable during the brain development.

All-trans-retinoid acid induces the differentiation of P19 cells into neurons involved in the PI3K/Akt/GSK3β signaling pathway.

The pluripotent mouse embryonal carcinoma cell line P19 is widely used as a model for research on all-trans-retinoid acid (RA)-induced neuronal differentiation; however, the signaling pathways involved in this process remain unclear. This study aimed to reveal the molecular mechanism underlying the RA-induced neuronal differentiation of P19 cells. Real-time quantitative polymerase chain reaction and Western blot analysis were used to determine the expression of neuronal-specific markers, whereas flow cytometry was used to analyze cell cycle and cell apoptosis. The expression profiles of messenger RNAs (mRNAs) in RA-induced neuronal differentiation of P19 cells were analyzed using high-throughput sequencing, and the functions of differentially expressed mRNAs (DEMs) were determined by bioinformatics analysis. RA induced an increase in both class III β-tubulin (TUBB3) and neurofilament medium (NEFM) mRNA expression, indicating that RA successfully induces neuronal differentiation of P19 cells. Cell apoptosis was not affected; however, cell proliferation decreased. We found 4117 DEMs, which were enriched in the phosphoinositide 3-kinase/protein kinase B (PI3K/Akt) signaling pathway, Wnt signaling pathway, and cell cycle. Particularly, a few DEMs could be identified in the PI3K/Akt signaling pathway networks, such as PI3K, Akt, glycogen synthase kinase-3β (GSK3β), cyclin-dependent kinase 4 (CDK4), P21, and Bax. RA significantly increased the protein expression of PI3K, Akt, phosphorylated Akt, GSK3β, phosphorylated GSK3β, CDK4, and P21, but it reduced Bax protein expression. The Akt inhibitor affected the increase of TUBB3 and NEFM mRNA expression in RA-induced P19 cells. The molecular mechanism underlying the RA-induced neuronal differentiation of P19 cells is potentially involved in the PI3K/Akt/GSK3β signaling pathway. The decreased cell proliferation ability of neuronally differentiated P19 cells could be associated with the expression of cell cycle proteins.

TAK1/Map3k7 enhances differentiation of cardiogenic endoderm from mouse embryonic stem cells

Specification of the primary heart field in mouse embryos requires signaling from the anterior visceral endoderm (AVE). The nature of these signals is not known. We hypothesized that the TGFβ-activated kinase (TAK1/Map3k7) may act as a cardiogenic factor, based on its expression in heart-inducing endoderm and its requirement for cardiac differentiation of p19 cells. To test this, mouse embryonic stem (ES) cells overexpressing Map3k7 were isolated and differentiated as embryoid bodies (EBs). Map3k7-overexpressing EBs showed increased expression of AVE markers but interestingly, showed little effect on mesoderm formation and had no impact on overall cardiomyocyte formation. To test whether the pronounced expansion of endoderm masks an expansion of cardiac lineages, chimeric EBs were made consisting of Map3k7-overexpressing ES and wild type ES cells harboring a cardiac reporter transgene, MHCα::GFP, allowing cardiac differentiation to be assessed specifically in wild type ES cells. Wild type ES cells co-cultured with Map3k7-overexpressing cells had a 4-fold increase in expression of the cardiac reporter, supporting the hypothesis that Map3k7 increases the formation of cardiogenic endoderm. To further examine the role of Map3k7 in early lineage specification, other endodermal markers were examined. Interestingly, markers that are expressed in both the VE and later in gut development were expanded, whereas transcripts that specifically mark the early definitive (streak-derived) endoderm (DE) were not. To determine if Map3k7 is necessary for endoderm differentiation, EBs were grown in the presence of the Map3k7 specific inhibitor 5Z-7-oxozeaenol. Endoderm differentiation was dramatically decreased in these cells. Western blot analysis showed that known downstream targets of Map3k7 (Jnk, Nemo-like kinase (NLK) and p38 MAPK) were all inhibited. By contrast, transcripts for another TGFβ target, Sonic Hedgehog (Shh) were markedly upregulated, as were transcripts for Gli2 (but not Gli1 and Gli3). Together these data support the hypothesis that Map3k7 governs the formation, or proliferation of cardiogenic endoderm.

Yokukansan, a Kampo medicine, enhances the level of neuronal lineage markers in differentiated P19 embryonic carcinoma cells

Yokukansan (YKS), a traditional Japanese Kampo medicine, affects neurological and psychiatric disorders. It ameliorates hippocampal neurogenesis in animals. However, its effect on neuronal cell differentiation remains unclear. Therefore, we investigated the effects of YKS on pluripotent P19 embryonic carcinoma cells as neuronal differentiation model cells.Western blotting and immunocytochemistry revealed that 10 μg/mL YKS treatment during embryoid body formation or neuronal differentiation increased the expression of the neuronal stem cell marker, Nestin, by 1.9-fold and 1.7-fold, respectively, and of the mature neuron marker, NeuN, by 1.5-fold and 1.4-fold, respectively.We examined the effect of YKS on intracellular signaling pathways in P19 cells and found significant elevation in phospho-PDK1 and phospho-mTOR expression (1.1-fold and 1.2-fold, respectively). Therefore, we investigated the effect of PDK1 and mTOR inhibitors on the level of neuronal lineage markers. We found that the mTOR inhibitor significantly abolished the YKS effect on the level of neuronal lineage markers.Moreover, to identify the target(s) of YKS, antibody array analysis that simultaneously detects 16 phosphorylated proteins was performed. YKS significantly upregulated 10 phosphorylated proteins including PDK1, Akt, AMPK, PRAS40, mTOR, p70 S6 kinase, GSK-3α, Bad and ERK1/2 under cell proliferation conditions. These results suggest that YKS simultaneously activates multiple signaling pathways.Thus, we concluded that YKS enhances the level of neuronal lineage markers in differentiated P19 cells, however it does not induce neuronal differentiation. Furthermore, mTOR is the predominant mediator of the YKS effect on these cells.

Ceramide: a novel inducer for neural tube defects.

Circulating plasma ceramides, a class of bioactive sphingolipids, are elevated in metabolic disorders, including obesity. Infants of women with these disorders are at 2- to 3-fold greater risk for developing a neural tube defect (NTD). This study aimed to test the effects of embryonic exposure to C2-ceramides (C2) during neural tube closure. Preliminary data shows an increase in NTDs in chick embryos after C2 exposure, and addresses potential mechanisms. Cell and embryo models were used to examine redox shifts after ceramide exposure. While undifferentiated P19 cells were resistant to ceramide exposure, neuronally differentiated P19 cells exhibited an oxidizing shift. Consistent with these observations, GSH E h curves revealed a shift to a more oxidized state in C2 treated embryos without increasing apoptosis or changing Pax3 expression, however cell proliferation was lower. Neural tube defects were observed in 45% of chick embryos exposed to C2, compared to 12% in control embryos. C2 exposure during critical developmental stages increased the frequency of NTDs in the avian model. Increased ROS generation in cell culture, along with the more oxidative GSH E h profiles of C2 exposed cells and embryos, support a model wherein ceramide affects neural tube closure via altered tissue redox environments.

Induction of Tyrosine Hydroxylase Gene Expression in Embryonal Carcinoma Stem Cells Using a Natural Tissue-Specific Inducer.

A large number of studies have focused on the generation of dopaminergic neurons from pluripotent cells. Differentiation of stem cells into distinct cell types is influenced by tissue-specific microenvironment. Since, central nervous system undergoes further development during postnatal life, in the present study neonatal rat brain tissue extract (NRBE) was applied to direct the differentiation of embryonal carcinoma stem cell line, P19 into dopaminergic (DA) phenotypes. Additionally, a neuroprotective drug, deprenyl was used alone or in combination with the extract. Results from morphological, immunofluorescence, and qPCR analyses showed that during a period of one to three weeks, a large percentage of stem cells were differentiated into neural cells. The results also indicated the greater effect of NRBE on the differentiation of the cells into tyrosine hydroxylase-expressing cells. MS analysis of NRBE showed the enrichment of gene ontology terms related to cell differentiation and neurogenesis. Network analysis of the studied genes and some DA markers resulted in the suggestion of potential regulatory candidates such as AVP, ACHE, LHFPL5, and DLK1 genes. In conclusion, NRBE as a natural native inducer was apparently able to simulate the brain microenvironment and support neural differentiation of P19 cells.

Role of lncRNA uc.457 in the differentiation and maturation of cardiomyocytes.

Congenital heart disease (CHD) is the most common type of birth defect, and the leading cause of fetal mortality. The long noncoding RNA (lncRNA) uc.457 is differentially expressed in cardiac tissue from patients with a ventricular septal defect; however, its role in cardiac development and CHD remains unknown. In the present study, the role of uc.457 in the differentiation and maturation of cardiomyocytes was investigated. Bioinformatics approaches were employed to analyze putative transcription factor (TF) regulation, histone modifications and the biological functions of uc.457. Subsequently, uc.457 overexpression and small interfering RNA‑mediated knockdown were performed to evaluate the functional role of the lncRNA in the dimethyl sulfoxide‑induced differentiation of P19 cells into cardiomyocytes. Bioinformatics analyses predicted that uc.457 binds to TFs associated with cardiomyocyte growth and cardiac development. Cell Counting Kit‑8 assays demonstrated that uc.457 overexpression inhibited cell proliferation, whereas knockdown of uc.457 enhanced the proliferation of differentiating cardiomyocytes. Additionally, reverse transcription‑quantitative polymerase chain reaction and western blot analyses revealed that overexpression of uc.457 suppressed the mRNA and protein expression of histone cell cycle regulation defective homolog A, natriuretic peptide A, cardiac muscle troponin T and myocyte‑specific enhancer factor 2C. Collectively, the results indicated that overexpression of uc.457 inhibited the differentiation and proliferation of cardiomyocytes, suggesting that dysregulated uc.457 expression may be associated with CHD.

Effects of long non-coding RNA uc.245 on cardiomyocyte-like differentiation in P19 cells via FOG2

Each year, cardiac diseases may cause a high morbidity and mortality worldwide. Long non-coding RNAs (lncRNAs) that contained ultra-conserved elements (UCEs) may play important roles on cardiomyocytes differentiation. Further investigations underlying mechanisms of lncRNA-UC regulating embryonic heart development are necessary.In this study, we investigated the effects of lnc-uc.245 on proliferation, migration, apoptosis, and cardiomyocyte-like differentiation in P19 cells with DMSO stimulation, and hypothesized that lnc-uc.245 would influence cardiomyocytes differentiation via FOG2. Lentiviral vectors of pGPU6/GFP/Neo-uc.245 and pGPU6/GFP/Neo-shRNA-uc.245 were respectively transfected into P19 cells to overexpress or silence uc.245. MTT assay, Annexin V-FITC/PI double-staining, scratch test and transwell assay were performed and the results showed that uc.245 overexpression could significantly suppress P19 cell proliferation, migration, cardiomyocyte-like differentiation but promote cell apoptosis. Contrarily, sh-uc.245 treatment caused the opposite changes. Uc.245 overexpression obviously downregulated the expression of cardiomyogenic-specific molecular markers (cTnI, ANP, α-MHC, Nkx2.5, GATA4, MEF2C) but remarkably upregulated the expression of FOG2. Subsequently, we transfected the recombinant vectors loaded FOG2 or shRRNA-FOG2 into P19 cells to further address the functional significance of FOG2 in uc.245-regulated cardiomyocyte-like differentiation. Interestingly, we found that overexpressing of FOG2 promoted cell proliferation, migration, and inhibited apoptosis both in uc.245 overexpressed and silenced P19 cells, especially in uc.245 silenced cell line. In addition, sh-FOG2 promoted cardiomyocyte-like differentiation and upregulated the expression of cardiomyogenic-specific markers at the gene and protein levels both in uc.245 overexpressed and silenced P19 cells. Similarly, this upregulation effect of sh-FOG2 was more obvious after uc.245 silencing.These findings suggest that FOG2 is a key mediator during uc.245-regulated differentiation of P19 cells into cardiomyocytes. It is expected that lnc-uc.245/FOG2 will become a promising therapeutic target for cardiac diseases.