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Cell therapeutic applications based on induced pluripotent stem cells (iPSCs) appear highly promising and challenging at the same time. Good manufacturing practice (GMP) regulations impose necessary yet demanding requirements for quality and consistency when manufacturing iPSCs and their differentiated progeny. Given the scarcity of accessible GMP iPSC lines, we have established a corresponding production workflow to generate the first set of compliant cell banks. Hence, these lines met a comprehensive set of release specifications and, for instance, displayed a low overall mutation load reflecting their neonatal origin, cord blood. Based on these iPSC lines, we have furthermore developed a set of GMP-compatible workflows enabling improved gene targeting at strongly enhanced efficiencies and directed differentiation into critical cell types: A new protocol for the generation of retinal pigment epithelium (RPE) features a high degree of simplicity and efficiency. Mesenchymal stromal cells (MSCs) derived from iPSCs displayed outstanding expansion capacity. A fully optimized cardiomyocyte differentiation protocol was characterized by a particularly high batch-to-batch consistency at purities above 95%. Finally, we introduce a universal immune cell induction platform that converts iPSCs into multipotent precursor cells. These hematopoietic precursors could selectively be stimulated to become macrophages, T cells, or natural killer (NK) cells. A switch in culture conditions upon NK-cell differentiation induced a several thousand-fold expansion, which opens up perspectives for upscaling this key cell type in a feeder cell-independent approach. Taken together, these cell lines and improved manipulation platforms will have broad utility in cell therapy as well as in basic research.
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Hyaluronic acid is composed of repeating sugar units, glucuronic acid and N-acetylglucosamine, which are often associated with increased tumor progression. Urtica dioica agglutinin is a potential component that exhibits a high affinity for binding to N-acetylglucosamine. This study aimed to investigate U. dioica Agglutinin's potential to inhibit the proliferation and migration of prostate cancer cells with high expression of hyaluronic acid through molecular docking and in vitro studies. The expression of hyaluronan synthase genes in prostate tissue and cell lines was checked by an in silico study, and the interaction between hyaluronic acid with both CD44 transmembrane glycoprotein and U. dioica agglutinin was analyzed through molecular docking. U. dioica Agglutinin's effect on cell viability (neutral red uptake assay), migration (scratch wound healing assays), and both CD44 and Nanog expression (quantitative real-time polymerase chain reaction) were assessed in vitro. The results showed that in prostate cancer cell lines, the PC3 cell line has the highest expression of hyaluronan synthase genes. U. dioica agglutinin exhibits an interaction of six specific residues on CD44 compared to hyaluronic acid's singular residue. While U. dioica agglutinin alone effectively reduced cell viability and wound closer (≥ 150 µg/mL), combining it with hyaluronic acid significantly shifted the effective concentration to a higher dose (≥ 350 µg/mL). These results, together with low Nanog and high CD44 gene expression, suggest that U. dioica agglutinin may impair the CD44-HA pathway in PC3 cells. This possibility is supported by U. dioica Agglutinin's ability to compete with hyaluronic acid for binding to CD44. Based on this, U. dioica agglutinin as a plant lectin shows promise in inhibiting cancer proliferation and migration by targeting its dependence on hyaluronic acid.
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Movimento Celular , Proliferação de Células , Receptores de Hialuronatos , Ácido Hialurônico , Neoplasias da Próstata , Urtica dioica , Humanos , Ácido Hialurônico/farmacologia , Masculino , Neoplasias da Próstata/tratamento farmacológico , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Urtica dioica/química , Linhagem Celular Tumoral , Receptores de Hialuronatos/metabolismo , Simulação de Acoplamento Molecular , Sobrevivência Celular/efeitos dos fármacos , Aglutininas/farmacologia , Hialuronan Sintases/metabolismo , Células PC-3RESUMO
Colorectal cancer is the third most common and the second deadliest cancer worldwide. To date, colorectal cancer becomes one of the most important challenges of the health system in many countries. Since the clinical symptoms of this cancer appear in the final stages of the disease and there is a significant golden time between the formation of polyps and the onset of cancer, early diagnosis can play a significant role in reducing mortality. Today, in addition to colonoscopy, minimally invasive methods such as liquid biopsy have received much attention. The treatment of this complex disease has been mostly based on traditional treatments including surgery, radiotherapy, and chemotherapy; the high mortality rate indicates a lack of success for current treatment methods. Moreover, disease recurrence is another problem of traditional treatments. Recently, new approaches such as targeted therapy, immunotherapy, and nanomedicine have opened new doors for cancer treatment, some of which have already entered the market, and many methods have shown promising results in clinical trials. The success of immunotherapy in the treatment of refractory disease, the introduction of these methods into neoadjuvant therapy, and the successful results in tumor shrinkage without surgery have made immunotherapy a tough competitor for conventional treatments. It seems that the combination of those methods with such targeted therapies will go through promising changes in the future of colorectal cancer treatment.
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Neoplasias Colorretais , Humanos , Neoplasias Colorretais/terapia , Neoplasias Colorretais/diagnóstico , Neoplasias Colorretais/patologia , Carcinogênese , Imunoterapia/métodos , AnimaisRESUMO
BACKGROUND: All cell types express long non-coding RNAs (lncRNAs), which have the potential to play a role in carcinogenesis by altering the levels of their expression. Squamous cell carcinoma of the esophagus (ESCC) is a deadly disease with a poor prognosis and a high frequency of lymphatic metastases. Understanding the functional role and signaling pathways of two neighboring lncRNAs, CCAT1 and PVT1, in this oncogene's pathogenesis may help us determine ESCC. Furthermore, it is still unclear whether these lncRNAs are linked to the clinicopathological characteristics of patients with ESCC. METHODS: For this study, we used biopsy from the Imam Khomeini Cancer Institute's tumor bank in Tehran, Iran to obtain 40 ESCC tumor samples and their normal margin counterparts. The expression levels of the CCAT1, PVT1, and c-MYC genes were assessed using quantitative Real-Time RT-PCR. Additionally, demographic data and clinical-pathologic characteristics, such as tumor grade, tumor stage, lymph node, and metastasis, were taken into consideration. Graphpad prism version 8 was used for bioinformatics analyses. RESULTS: Comparing ESCC tissues to non-tumor tissues, we found significant upregulation of PVT1, CCAT1, and c-MYC. Patients with ESCC who had increased PVT1 expression also had higher rates of advanced stage and lymph node metastasis, whereas increased CCAT1 expression was only linked to advanced stage and wasn't associated with lymph node metastasis. In predicting ESCC, CCAT1 (p < 0.05) was found to be an important factor. Overall survival was reduced by c-MYC and PVT1 overexpression (p < 0.001), according to Kaplan-Meier analysis. PVT1, CCAT1, and c-MYC were found to interact with 23 miRNAs with high and medium score classes, as shown in a bioinformatics study. We summarized the experimentally proven interactions between c-MYC, PVT1, and CCAT1 and other miRNAs, lncRNAs, and proteins. CONCLUSION: This is the first report that CCAT1, PVT1 and c-MYC have been found to be up-regulated simultaneously in ESCC. It is possible that these genes may be involved in ESCC as a result of these findings. Therefore, as consequence, more research is needed to determine whether or not these lncRNAs play an oncogenic role in ESCC development and progression, as well as the regulatory mechanisms that control them.
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Neoplasias Esofágicas , Carcinoma de Células Escamosas do Esôfago , MicroRNAs , Proteínas Proto-Oncogênicas c-myc , RNA Longo não Codificante , Humanos , Biomarcadores Tumorais/genética , Neoplasias Esofágicas/patologia , Carcinoma de Células Escamosas do Esôfago/genética , Carcinoma de Células Escamosas do Esôfago/patologia , Regulação Neoplásica da Expressão Gênica , Genes myc , Irã (Geográfico) , Metástase Linfática , Oncogenes , Prognóstico , RNA Longo não Codificante/genética , RNA Longo não Codificante/metabolismo , Regulação para CimaRESUMO
In this study, we put forth a new deep neural network framework to predict flow behavior in a coronary arterial network with different properties in the presence of any abnormality like stenosis. An artificial neural network (ANN) model is trained using synthetic data so that it can predict the pressure and velocity within the arterial network. The data required to train the neural network were obtained from the CFD analysis of several geometries of arteries with specific features in ABAQUS software. The proposed approach precisely predicts the hemodynamic behavior of the blood flow. The average accuracy of the pressure prediction was 98.7%, and the average velocity magnitude accuracy was 93.2%. Our model can also be used to predict fractional flow reserve (FFR), which is one of the main indices to determine the severity of stenosis, and our model predicts this index successfully based on the artery features.
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Estenose Coronária , Reserva Fracionada de Fluxo Miocárdico , Humanos , Vasos Coronários/diagnóstico por imagem , Reserva Fracionada de Fluxo Miocárdico/fisiologia , Constrição Patológica , Estenose Coronária/diagnóstico por imagem , Hemodinâmica , Aprendizado de Máquina , Angiografia Coronária , Valor Preditivo dos TestesRESUMO
Background: Today, numerous antimicrobial and anticancer properties have been reported for plant lectins due to their ability to bind to carbohydrates. The Urtica dioica agglutinin (UDA lectin) is a monomeric, small, and low molecular weight glycoprotein. It has attracted the attention of many researchers for identification, treatment, and other clinical purposes. Objectives: The aim of this study is the optimization of the chitin affinity chromatography based on Sepharose 4B (CNBr-activated Sepharose 4B) for the rapid purification of UDA lectin from Urtica dioica rhizome. Materials and Methods: The chitin ligands were dissolved in 40% Trichloroacetic acid and attached to Sepharose 4B according to the Amersham-Biosciences instructions. The attachment of the ligand to the Sepharose 4B beads was investigated by Fourier transform infrared (FTIR) spectroscopy. An acidic crude extract of nettle rhizome passes from chromatographic columns in two sizes with dimensions: 24 x 0.51 cm and 8.44 x 0.86 cm. Quantity and quality of purified lectin were calculated by the Bradford microplate method: SDS-PAGE gel electrophoresis and human erythrocyte cell (RBC) hemagglutination, respectively. Results: The analysis of FTIR spectrograms showed that major changes were observed in the fingerprint regions. Besides, due to the dissolution of Sepharose 4B and chitin in the aqueous phase, this difference was not significant in the Imine and Nitrile regions. On the other hand, the comparative results of purification chromatograms showed that increasing the column length causes a smaller half-width and increases the length of the purified peak. Also, it leads to high-quality purified UDA lectin, with a molecular weight of almost 12.5 kDa in gel electrophoresis. Hemagglutination activity on trypsinized red blood cells was displayed, and agglutination of purified UDA lectin started at least at 300 µg.mL-1 concentration. Conclusion: According to our findings, we suggested that dissolving chitin in the polar solvent of Trichloroacetic acid, using Sepharose 4B as the beads of a matrix, and increasing the column length might lead to a decrease in the half-width of the peak. These can increase the purity and concentration of purified UDA lectin, and speed up the purification process. These findings could be used by researchers to accelerate the purification of UDA lectin in other studies, dealing with drug delivery systems, ELISA techniques, and cell growth.
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Iron oxide nanoparticle (IONPs) have become a subject of interest in various biomedical fields due to their magnetism and biocompatibility. They can be utilized as heat mediators in magnetic hyperthermia (MHT) or as contrast media in magnetic resonance imaging (MRI), and ultrasound (US). In addition, their high drug-loading capacity enabled them to be therapeutic agent transporters for malignancy treatment. Hence, smartening them allows for an intelligent controlled drug release (CDR) and targeted drug delivery (TDD). Smart magnetic nanoparticles (SMNPs) can overcome the impediments faced by classical chemo-treatment strategies, since they can be navigated and release drug via external or internal stimuli. Recently, they have been synchronized with other modalities, e.g., MRI, MHT, US, and for dual/multimodal theranostic applications in a single platform. Herein, we provide an overview of the attributes of MNPs for cancer theranostic application, fabrication procedures, surface coatings, targeting approaches, and recent advancement of SMNPs. Even though MNPs feature numerous privileges over chemotherapy agents, obstacles remain in clinical usage. This review in particular covers the clinical predicaments faced by SMNPs and future research scopes in the field of SMNPs for cancer theranostics.
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BACKGROUND: Loci controlling DNA double-strand breaks (DSBs) repair play an important role in defending against the harmful health effects of benzene, toluene, ethylbenzene, and xylene (BTEX), but their gene variants may alter their repair capacity. The aim of the current study was to determine the relationship of functional polymorphisms ATM-rs228589 A>T, WRN-rs1800392 G>T and H2AX-rs7759 A>G in DBS repair loci with the abnormal hematological indices in workers who exposed to BTEXs. METHODS: We included 141 cases with one or more abnormal hematological parameters, who had been occupationally exposed to BTEX chemicals and 152 controls with a similar exposure condition but without any abnormal hematological parameters. Atmospheric concentrations of BTEXs were measured and whole blood samples were taken from the participants to determine hematologic parameters and SNP genotyping. RESULTS: Results showed that T allele of ATM-rs228589 and G allele of H2AX-rs7759 had a higher frequency in cases than controls (p = 0.012 and p = 0.001, respectively). Also, AT and TT genotypes of ATM-rs228589 and AG and GG genotypes of H2AX-rs7759 were higher in cases compared to controls. The AT and TT genotypes of ATM-rs228589 have significant associations with a risk of hematological abnormalities in the codominant (AT vs. AA, p = 0.018), dominant (AT + TT vs. AA, p = 0.010) and overdominant (AT vs. AA + TT, p = 0.037) models. The GG and AG genotypes of H2AX-rs7759 were in relation with increased risk of abnormal hematological indices under codominant (GA vs. AA, p = 0.009 & GG vs. AA, p = 0.005), dominant (AG + GG vs. AA, p = 0.001), and recessive (GG vs. AA + AG, p = 0.025) models. CONCLUSIONS: These observations may help to understand the mechanisms of BTEX hematotoxicity and identify useful biomarkers of risk assessment for workers exposed to BTEX.
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Benzeno , Xilenos , Proteínas Mutadas de Ataxia Telangiectasia/genética , Benzeno/efeitos adversos , Derivados de Benzeno , Predisposição Genética para Doença/genética , Histonas , Humanos , Polimorfismo de Nucleotídeo Único/genética , ToluenoRESUMO
Introduction: The microenvironment of solid tumors such as breast cancer is heterogeneous and complex, containing different types of cell, namely, cancer stem cells and immune cells. We previously reported the immunoregulatory behavior of the human immune cell in a solid tumor microenvironment-like culture under serum starvation stress for 96 h. Here, we examined the effect of this culture-derived solution on breast cancer development in rats. Method: Ninety-six-hour starved PBMCs supernatant (96 h-SPS) was collected after culturing human PBMCs for 96 h under serum starvation condition. Breast cancer stem cells, LA7 cell line, was used for in vitro study by analyzing gene expression status and performing cytotoxicity, proliferation, scratch wound healing assays, followed by in vivo tumor induction in three groups of mature female Sprague Dawley rats. Animals were treated with 96 h-SPS or RPMI and normal saline as control, n = 6 for each group. After biochemical analysis of iron, lactate, and pH levels in the dissected tumors, Ki67 antigen expression, angiogenesis, and necrosis evaluation were carried out. Metabolic-related gene expression was assessed using RT-qPCR. Moreover, 96 h-SPS composition was discovered by Nano-LC-ESI-MS/MS. Results: 96 h-SPS solution reduced the LA7 cell viability, proliferation, and migration and Gch1 and Spr genes expression in vitro (p< 0.05), whereas stemness gene Oct4 was upregulated (p< 0.01). The intracellular lactate was significantly decreased in the 96 h-SPS treated group (p = 0.007). In this group, Gch1 and Spr were significantly downregulated (p< 0.05), whereas the Sox2 and Oct4 expression was not changed significantly. The number of vessels and mitosis (Ki67+ cells) in the 96 h-SPS-treated group was significantly reduced (p = 0.024). The increased rate of necrosis in this group was statistically significant (p = 0.04). Last, proteomics analysis revealed candidate effectors' components of 96 h-SPS solution. Conclusion: 96 h-SPS solution may help to prevent cancer stem cell mediated tumor development. This phenomenon could be mediated through direct cytotoxic effects, inhibition of cell proliferation and migration in association with reduction in Gch1 and Spr genes expression, angiogenesis and mitosis rate, and necrosis augmentation. The preliminary data obtained from the present study need to be investigated on a larger scale and can be used as a pilot for further studies on the biology of cancer development.
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Neoplasias da Mama , Animais , Feminino , Ratos , Neoplasias da Mama/patologia , Antígeno Ki-67/metabolismo , Leucócitos Mononucleares/metabolismo , Necrose/patologia , Células-Tronco Neoplásicas/metabolismo , Ratos Sprague-Dawley , Espectrometria de Massas em Tandem , Microambiente TumoralRESUMO
Bone-marrow-derived mesenchymal stem cells (BM-MSCs) are one of the most widely studied postnatal stem cell populations and are considered to utilize more frequently in cell-based therapy and cancer. These types of stem cells can undergo multilineage differentiation including blood cells, cardiac cells, and osteogenic cells differentiation, thus providing an alternative source of mesenchymal stem cells (MSCs) for tissue engineering and personalized medicine. Despite the ability to reprogram human adult somatic cells to induced pluripotent stem cells (iPSCs) in culture which provided a great opportunity and opened the new door for establishing the in vitro disease modeling and generating an unlimited source for cell base therapy, using MSCs for regeneration purposes still have a great chance to cure diseases. In this review, we discuss the important issues in MSCs biology including the origin and functions of MSCs and their application for craniofacial and periodontal tissue regeneration, discuss the potential and clinical applications of this type of stem cells in differentiation to maxillofacial bone and cartilage in vitro, and address important future hopes and challenges in this field.
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Gastric cancer is the leading cause of cancer-related mortality worldwide. Given the importance of gastric cancer in public health, identifying biomarkers associated with disease onset is an important part of precision medicine. The hedgehog signaling pathway is considered as one of the most significant widespread pathways of intracellular signaling in the early events of embryonic development. This pathway contributes also to the maintenance of pluripotency of cancer stem cells pluripotency. In this study, we analyzed the expression levels of sonic hedgehog (Shh) signaling pathway genes IHH, BOC, RAB23a and their regulatory miRNAs including MIR-195-5p, MIR-509-3-5p, MIR-6738-3p in gastric cancer patients. In addition, the impact of infection status on the expression level of those genes and their regulatory miRNAs was investigated. One hundred samples taken from 50 gastric cancer patients (50 tumoral tissues and their adjacent non-tumoral counterparts) were included in this study. There was a significant difference in all studied genes and miRNAs in tumoral tissues in comparison with their adjacent non-tumoral counterparts. The lower expression of IHH, BOC, RAB23, miR-195-5p, and miR-6738-3p was significantly associated with more advanced cancer stage. Additionally, IHH upregulation was significantly associated with CMV infection (P < 0.001). Also, receiver operating characteristic (ROC) curve analysis indicated that mir-195 was significantly related to several clinicopathological features including tumor stage, grade, age, gender, and infection status of gastric cancer and can be considered as a potential diagnostic biomarker for gastric cancer. This study confirms the important role of Shh signaling pathway genes in gastric cancer tumorigenesis and their potential as novel molecular biomarkers and therapeutic targets.
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Proteínas Hedgehog/genética , Imunoglobulina G/genética , MicroRNAs/genética , Receptores de Superfície Celular/genética , Neoplasias Gástricas/genética , Neoplasias Gástricas/patologia , Proteínas rab de Ligação ao GTP/genética , Idoso , Biomarcadores Tumorais/genética , Feminino , Regulação Neoplásica da Expressão Gênica , Proteínas Hedgehog/metabolismo , Humanos , Imunoglobulina G/metabolismo , Masculino , MicroRNAs/metabolismo , Pessoa de Meia-Idade , Estadiamento de Neoplasias , Curva ROC , Receptores de Superfície Celular/metabolismo , Transdução de Sinais/genética , Neoplasias Gástricas/virologia , Proteínas rab de Ligação ao GTP/metabolismoRESUMO
Brugada syndrome (BrS) is a rare hereditary arrhythmia syndrome that increases an individual's risk for sudden cardiac death (SCD) due to ventricular fibrillation. This disorder is regarded as a notable cause of death in individuals aged less than 40 years, responsible for up to 40% of sudden deaths in cases without structural heart disease, and is reported to be an endemic in Asian countries. Mutations in SCN5A are found in approximately 30% of patients with Brugada syndrome. This study aimed to investigate mutations in the SCN5A gene in a group of Iranian Brugada syndrome patients. Nine probands (n = 9, male, mean age = 39) diagnosed with Brugada syndrome were enrolled in this study. Exon 2 to 29 were amplified by PCR and subjected to direct sequencing. Eight in silico prediction tools were used to anticipate the effects of non-synonymous variants. Seven known polymorphisms and 2 previously reported disease-causing mutations, including H558R and G1406R, were found in the studied cases. Twenty novel variants were identified: 15 missense, 2 frameshift, 2 synonymous, and one nonsense variants. In silico tools predicted 11 non-synonymous variants to have damaging effects, whereas frameshift and nonsense variants were considered inherently pathogenic. The novel variants identified in this study, alongside previously reported mutations, are highly likely to be the cause of the Brugada syndrome phenotype observed in the patient group. Further analysis is required to understand the physiological effects caused by these variants.
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Síndrome de Brugada , Canal de Sódio Disparado por Voltagem NAV1.5 , Adulto , Síndrome de Brugada/genética , Humanos , Irã (Geográfico) , Masculino , Mutação , Canal de Sódio Disparado por Voltagem NAV1.5/genética , FenótipoRESUMO
BACKGROUND: The present study aimed to investigate and compare the effect of starved fibroblast culture supernatant (SFS), DMEM and normal saline alone or along with LA7 on dexamethasone-treated immunosuppressed Wistar rats. METHODS: After the isolation of fibroblasts from the fresh foreskin of children, it was cultured in serum-free DMEM, and the supernatant collected after 16 hours (16h-SFS). This solution and the other treatments were injected subcutaneously into the rats from each group once daily for 14 days. The liver, intestine and lung histology along with blood cellular and biochemical characteristics were studied. RESULTS: The results showed that dexamethasone as immunosuppressant reduced the body weight. The histological change in the liver was mild fibrosis induced by LA7+16h-SFS. Also, among the different blood cellular and biochemical indices measured, the eosinophil percentage in the 16h-SFS treated rats , glucose levels in the 16h-SFS+LA7 group and triglyceride concentrations in the 16h-SFS group were changed (p<0.05). CONCLUSION: This study showed that the secretions of starved fibroblasts especially that combined with LA7 cancer stem cells could induce some minor histological and biochemical changes in immunosuppressed rats, and also it opened a new window for subsequent investigations on unknown mechanisms related to this work.
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Down-regulation of stemness genes expression is important in differentiation therapy against cancer stem cells (CSCs). The aim of this study was to evaluate the Oct4 , Sox2, Nanog, and C-myc expression in rat breast cancer stem cells (LA7) which treated with human ovarian follicular fluid (FF), replicative senescent fibroblast culture supernatant (P14), and 16 h serum starved fibroblast supernatant (16 h-SFS). The cells were exposed to these biological fluids for 24 h, 72 h, and 7 days. Stem-loop RT-qPCR assay was used to quantify the expression of above mentioned genes. Results showed that FF had the least cytotoxic effect on the LA7 cells. Except for Nanog gene, exposure of LA7 cell line to 16 h-SFS and P14 decreased significantly expression of the three other genes after 24 h (P < 0.05). Nanog and Sox2 genes expression was also decreased in LA7 cells which have been already treated with FF for 24 h. Moreover, compared to the control solution, the expression of Oct4 increased significantly after 7 days exposure to FF (P < 0.05). Annexin V-PE /7-AAD-, acridine orange/ethidium bromide staining and doubling time assays revealed apoptosis and necrosis induction by these biological fluids in LA7 cells. Moreover, in an in vitro model of metastasis assay, i.e., scratch test, these fluids exhibited anti-LA7 migration activity which culminated in 16 h-SFS treated cells. Generally, this study showed that FF, 16 h-SFS, and P14 have positive effects on down-regulation of Nanog, Oct4, Sox2 and C-myc expression, and consequently can increase the differentiation of breast cancer stem cells. For the first time, this study provided some evidence indicating that some biological fluids have potential to differentiate the CSCs, show anti- survival, growth-, and cell migration activity.
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Líquidos Corporais/fisiologia , Regulação Neoplásica da Expressão Gênica , Neoplasias Mamárias Animais/genética , Células-Tronco Neoplásicas , Fatores de Transcrição/genética , Animais , Diferenciação Celular/genética , Linhagem Celular Tumoral , Proliferação de Células , Sobrevivência Celular , Meios de Cultura/farmacologia , Regulação para Baixo , Feminino , Líquido Folicular/fisiologia , Genes myc , Humanos , Proteína Homeobox Nanog/genética , Células-Tronco Neoplásicas/patologia , Fator 3 de Transcrição de Octâmero/genética , Ratos , Reação em Cadeia da Polimerase em Tempo Real , Fatores de Transcrição SOXB1/genéticaRESUMO
Heart development is a complex process, tightly regulated by numerous molecular mechanisms. Key components of the regulatory network underlying heart development are transcription factors (TFs) and microRNAs (miRNAs), yet limited investigation of the role of miRNAs in heart development has taken place. Here, we report the first parallel genome-wide profiling of polyadenylated RNAs and miRNAs in a developing murine heart. These data enable us to identify dynamic activation or repression of numerous biological processes and signaling pathways. More than 200 miRNAs and 25 long non-coding RNAs were differentially expressed during embryonic heart development compared to the mature heart; most of these had not been previously associated with cardiogenesis. Integrative analysis of expression data and potential regulatory interactions suggested 28 miRNAs as novel regulators of embryonic heart development, representing a considerable expansion of the current repertoire of known cardiac miRNAs. To facilitate follow-up investigations, we constructed HeartMiR (http://heartmir.sysbiolab.eu), an open access database and interactive visualization tool for the study of gene regulation by miRNAs during heart development.
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Pluripotent cells emanate from the inner cell mass (ICM) of the blastocyst and when cultivated under optimal conditions immortalize as embryonic stem cells (ESCs). The fundamental mechanism underlying ESC derivation has, however, remained elusive. Recently, we have devised a highly efficient approach for establishing ESCs, through inhibition of the MEK and TGF-ß pathways. This regimen provides a platform for dissecting the molecular mechanism of ESC derivation. Via temporal gene expression analysis, we reveal key genes involved in the ICM to ESC transition. We found that DNA methyltransferases play a pivotal role in efficient ESC generation. We further observed a tight correlation between ESCs and preimplantation epiblast cell-related genes and noticed that fundamental events such as epithelial-to-mesenchymal transition blockage play a key role in launching the ESC self-renewal program. Our study provides a time course transcriptional resource highlighting the dynamics of the gene regulatory network during the ICM to ESC transition.
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Diferenciação Celular , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/metabolismo , Transição Epitelial-Mesenquimal , Animais , Biomarcadores , Massa Celular Interna do Blastocisto/citologia , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/genética , Metilação de DNA , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Transição Epitelial-Mesenquimal/genética , Perfilação da Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento , Camundongos , Interferência de RNA , TranscriptomaRESUMO
The role of striatin interacting protein 2 (Strip2) in differentiation of embryonic stem cells (ESCs) is still under debate. Strip2-silenced murine (KD) ESCs were differentiated for 4, 8, 12, and 16 days. We show that Strip2 is distributed in the perinucleus or nuclei of wild-type (WT) undifferentiated ESCs, but is localized in high-density nuclear bodies in differentiated cells. CellNet analysis of microarray gene expression data for the KD and scrambled control (SCR) embryoid bodies (EBs), as well as immunostainings of key pluripotent factors, demonstrated that differentiation of KD ESCs is repressed. This occurs even in 16-day-old EBs, which possessed a high tumorigenic potential. Correlated with very high expression levels of epigenetic regulator genes, Hat1 and Dnmt3, enzymatic activities of the histone acetyltransferase type B (Hat1) and DNA (cytosine-5)-methyltransferase 3 beta (Dnmt3b) were higher in differentiated 16-day-old KD EBs than in SCR or WT EBs. The expression levels of let-7, 290, and 302 microRNA families were opposed in KD ESCs, while KD EBs had levels comparable to WT and SCR ESCs during differentiation. Strip2 is critical for the regular differentiation of ESCs. Moreover, Strip2 deficient ESCs showed a dysregulation of epigenetic regulators and microRNAs regulating pluripotency.
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It has previously been reported that mouse epiblast stem cell (EpiSC) lines comprise heterogeneous cell populations that are functionally equivalent to cells of either early- or late-stage postimplantation development. So far, the establishment of the embryonic stem cell (ESC) pluripotency gene regulatory network through the widely known chemical inhibition of MEK and GSK3beta has been impractical in late-stage EpiSCs. Here, we show that chemical inhibition of casein kinase 1alpha (CK1alpha) induces the conversion of recalcitrant late-stage EpiSCs into ESC pluripotency. CK1alpha inhibition directly results in the simultaneous activation of the WNT signaling pathway, together with inhibition of the TGFbeta/SMAD2 signaling pathway, mediating the rewiring of the gene regulatory network in favor of an ESC-like state. Our findings uncover a molecular mechanism that links CK1alpha to ESC pluripotency through the direct modulation of WNT and TGFbeta signaling.