RESUMEN

Klotho, as an antiaging protein, is involved in the maintenance and differentiation of neuronal or glial cells and, therefore, has been noticed as a potential therapeutic target for neurodegenerative disorders. Expression of Klotho has been examined in different cells and organs, however, our information about the developmental pattern of this protein during differentiation of mesenchymal stem cells (MSCs) into neuron-like cells is limited. In this study, we conducted neural differentiation of mouse bone marrow-derived-MSCs and monitored the expression of Klotho together with selected neuron-specific genes at messenger RNA (mRNA) on days 7 and 14 of differentiation using quantitative real-time PCR. In addition, Klotho status at protein level was evaluated by immunocytochemistry. The results showed a significant change in the morphology of MSCs towards neuron-like cells. These changes were observed with progressive growth and formation of cell connections towards the formation of a chain of neuron-like cells which occurred in the second week of differentiation. Morphological changes were associated with a significant increase in the expression of neuron-specific genes like pax-6, neuN and, neurofilaments (NfL). Likewise, there was an increased expression of Klotho mRNA, and accumulation of Klotho protein in neuronal cell bodies, during the cellular differentiation of MSCs. These findings provided new evidence that neuronal differentiation from the MSCs is associated with increased expression of Klotho. These data may provide insight into the importance of Klotho protein in stem cell differentiation and regeneration in response to cell death in the central nervous system.

Asunto(s)

Médula Ósea , Células Madre Mesenquimatosas , Ratones , Animales , Neuronas/metabolismo , Diferenciación Celular/genética , Inmunohistoquímica , Células Madre Mesenquimatosas/metabolismo , Células de la Médula Ósea , Células Cultivadas

RESUMEN

CXCR4 plays an important role in colorectal cancer (CRC) development and metastasis. Some previous studies have indicated CXCR4 as a therapeutic target in cancer. CXCR4 is known as a direct target of miR-146a. The present study aimed to investigate how exogenous induction of miR-146a affects CXCR4 gene and protein expression and also proliferation, apoptosis and migration of CRC cells. Transfection of Caco-2 and SW480 cells by a synthetic miR-146a mimic led to downregulation of CXCR4 expression at both gene and protein levels. It also downregulated expression of several miR-146a targets, including GSK3B, IRAK1, TRAF6, AKT2, SMAD4, EGFR and NFKB1, mostly in SW480 cells. Overexpression of miR-146a resulted in a partial cell cycle arrest in the both cell lines, while the apoptotic rate was also decreased. In regards to epithelial-mesenchymal transition factors, VIM was downregulated in the both cell lines, but SNAI1 was upregulated in Caco-2 cells. The wound closure assay showed a reduction in cell migration in SW480 cells, but an opposite effect was detected in Caco-2 cells following transfection with miR-146a mimic. Therefore, our results are indicating that overexpression of miR-146a, despite downregulation of oncogenic CXCR4, may not lead to a universal tumor suppressive effect in all CRC cells, and this is possibly due to differences in miR-146a effects on signaling pathways in each cell type. Selection of miR-146a for tumor suppression requires enough details regarding the signaling profile of cancer cells otherwise it may produce unexpected outcome.

Asunto(s)

Movimiento Celular/genética , Neoplasias Colorrectales/genética , Neoplasias Colorrectales/patología , Regulación Neoplásica de la Expresión Génica , MicroARNs/metabolismo , Receptores CXCR4/genética , Apoptosis/genética , Ciclo Celular/genética , Puntos de Control del Ciclo Celular/genética , Línea Celular Tumoral , Proliferación Celular/genética , Supervivencia Celular/genética , Ciclina D1/genética , Ciclina D1/metabolismo , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/genética , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/metabolismo , Regulación hacia Abajo/genética , Transición Epitelial-Mesenquimal/genética , Receptores ErbB/metabolismo , Humanos , MicroARNs/genética , FN-kappa B/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Receptores CXCR4/metabolismo , Transducción de Señal , Receptores Toll-Like/metabolismo , Cicatrización de Heridas/genética

RESUMEN

Directing the fate of mesenchymal stem cells (MSCs) to dopaminergic neurons has great importance in both biomedical studies and cell therapy of Parkinson's disease. We recently generated dopamine-secreting cells from human adipose tissue-derived stem cells (hADSCs) by exposing the cells to a growth factor cocktail composed of SHH, bFGF, FGF8 and BDNF in low-serum condition. In the current study, we induced the cells by the same dopaminergic inducing cocktail in serum-free B27-supplemented Neurobasal medium. ADSCs differentiated in both conditions expressed several neuronal and dopaminergic markers. However, there were higher gene expression levels under the serum-free condition. Higher levels of TUJ1 and TH proteins were also detected in the cells exposed to the dopaminergic-inducing cocktail under serum-free Neurobasal condition. TH protein was expressed in about 28% and 60% of the cells differentiated in the low-serum and serum-free Neurobasal media, respectively. Moreover, the cells exposed to the dopaminergic-inducing cocktail in the serum-free Neurobasal condition released a more significant amount of dopamine in response to KCl-induced depolarization. Altogether, these findings show a greater efficiency of the serum-free Neurobasal condition for growth factor-directed differentiation of hADSCs to functional dopamine-secreting cells which may be valuable for transplantation therapy of Parkinson's disease in future.

Asunto(s)

Tejido Adiposo/citología , Neuronas Dopaminérgicas/citología , Células Madre/citología , Adulto , Diferenciación Celular , Células Cultivadas , Medio de Cultivo Libre de Suero , Femenino , Humanos , Persona de Mediana Edad

RESUMEN

Previous studies have identified the heart as a source and a target tissue for oxytocin and relaxin hormones. These hormones play important roles in the regulation of cardiovascular function and repair of ischemic heart injury. In the current study, we examined the impact of oxytocin and relaxin on the development of cardiomyocytes from mesenchymal stem cells. For this purpose, mouse adipose tissue-derived stem cells (ADSCs) were treated with different concentrations of oxytocin or relaxin for 4 days. Three weeks after initiation of cardiac induction, differentiated ADSCs expressed cardiac-specific genes, Gata4, Mef2c, Nkx2.5, Tbx5, α- and ß-Mhc, Mlc2v, Mlc2a and Anp, and cardiac proteins including connexin 43, desmin and α-actinin. 10 -7 M oxytocin and 50 ng/mL relaxin induced the maximum upregulation in the expression of cardiac markers. A combination of oxytocin and relaxin induced cardiomyocyte differentiation more potently than the individual factors. In our experiment, oxytocin-relaxin combination increased the population of cardiac troponin I-expressing cells to 6.84% as compared with 2.36% for the untreated ADSCs, 3.7% for oxytocin treatment and 3.41% for relaxin treatment groups. In summary, the results of this study indicated that oxytocin and relaxin hormones individually and in combination can improve cardiac differentiation of ADSCs, and treatment of the ADSCs and possibly other mesenchymal stem cells with these hormones may enhance their cardiogenic differentiation and survival after transplantation into the ischemic heart tissue.

Asunto(s)

Tejido Adiposo/citología , Biomarcadores/metabolismo , Diferenciación Celular/efectos de los fármacos , Miocitos Cardíacos/citología , Oxitocina/farmacología , Relaxina/farmacología , Células Madre/citología , Tejido Adiposo/efectos de los fármacos , Tejido Adiposo/metabolismo , Animales , Células Cultivadas , Ratones , Miocitos Cardíacos/efectos de los fármacos , Miocitos Cardíacos/metabolismo , Organogénesis , Oxitócicos/farmacología , Células Madre/efectos de los fármacos , Células Madre/metabolismo

RESUMEN

Several studies have demonstrated the differentiation of human adipose tissue-derived stem cells (hADSCs) to neuronal and glial phenotypes, but directing the fate of these cells toward dopaminergic neurons has not been frequently reported. The aim of this study was to investigate dopaminergic specification of hADSCs in vitro. ADSCs were isolated from subcutaneous abdominal adipose tissue and were characterized. For dopaminergic differentiation, a cocktail of sonic hedgehog, fibroblast growth factor 8, basic fibroblast growth factor, and brain-derived neurotrophic factor were used under a low serum condition. As the control group, the ADSCs were cultured under the same low serum condition without the dopaminergic cocktail. At the end of differentiation period, the cells expressed neuron-specific markers, NES, NSE, and NEFL, and dopaminergic markers, EN1, NURR1, PITX3, VMAT2, TH, and GIRK2 genes. TH, NURR1, and EN1 mRNAs were upregulated in the dopaminergic group compared with the control group. NEFL and TH proteins were also expressed in the differentiated cells. A total of 27.9% of the cells differentiated in dopaminergic induction medium showed positive staining for TH protein. Based on reversed-phase high-performance liquid chromatography analysis, the differentiated cells released a significant amount of dopamine in response to KCl-induced depolarization. In conclusion, results of this study indicate that hADSCs can be induced by a growth factor cocktail to produce dopamine secreting cells with possible applications for future cell replacement therapy of Parkinson's disease.

Asunto(s)

Tejido Adiposo/citología , Dopamina/metabolismo , Células Madre/citología , Adulto , Biomarcadores/metabolismo , Diferenciación Celular/genética , Separación Celular , Neuronas Dopaminérgicas/citología , Neuronas Dopaminérgicas/metabolismo , Femenino , Regulación de la Expresión Génica , Humanos , Persona de Mediana Edad , Células Madre/metabolismo

RESUMEN

Adipose tissue-derived stem cells (ADSCs) are capable of multipotential differentiation and express several angiogenic, anti-apoptotic and immunomodulatory markers. These features make adipose tissue as a promising source of stem cells for regenerative medicine. However, for efficient translational use, culture-induced changes in the gene expression profile and resistance of the ADSCs to ischemic environment should be taken into consideration. We compared the expression of some clinically important markers between the unpassaged and third-passaged ADSCs by RT-PCR, qPCR and flow cytometry. Our results demonstrated that the embryonic stem cell (ESC)-specific markers were expressed in the unpassaged ADSCs but were downregulated after three passages. The expression of stemness-related genes, TGFB and FGF2, was upregulated while FGF4 and LIF were downregulated after three passages. The expression of angiogenic genes in the third-passaged ADSCs was higher than the unpassaged cells. Epithelial-mesenchymal transition (EMT) markers were either expressed in the third-passaged ADSCs or significantly upregulated after three passages. In contrast, cell cycle inhibitors, CDKN1A and TP53, were downregulated with early subcultures. The unpassaged and third-passaged ADSCs showed nearly similar resistance to oxidative stress, hypoxia and serum deprivation. In conclusion, the primary cultures of human adipose tissue contain a subpopulation of cells expressing ESC-specific genes and proteins, but the expression of these pluripotency markers subsides rapidly in standard mesenchymal stem cell culture medium. The expression of angiogenic and EMT markers also varies with early subcultures. Altogether, early-passaged ADSCs may be better choices for transplantation therapy of injured tissues, especially after ischemic conditions.

RESUMEN

OBJECTIVES: In some previous studies, the extract of embryonic carcinoma cells (ECCs) and embryonic stem cells (ESCs) have been used to reprogram somatic cells to more dedifferentiated state. The aim of this study was to investigate the effect of mouse ESCs extract on the expression of some pluripotency markers in human adipose tissue-derived stem cells (ADSCs). MATERIALS AND METHODS: Human ADSCs were isolated from subcutaneous abdominal adipose tissue and characterized by flow cytometric analysis for the expression of some mesenchymal stem cell markers and adipogenic and osteogenic differentiation. Frequent freeze-thaw technique was used to prepare cytoplasmic extract of ESCs. Plasma membranes of the ADSCs were reversibly permeabilized by streptolysin-O (SLO). Then the permeabilized ADSCs were incubated with the ESC extract and cultured in resealing medium. After reprogramming, the expression of some pluripotency genes was evaluated by RT-PCR and quantitative real-time PCR (qPCR) analyses. RESULTS: Third-passaged ADSCs showed a fibroblast-like morphology and expressed mesenchymal stem cell markers. They also showed adipogenic and osteogenic differentiation potential. QPCR analysis revealed a significant upregulation in the expression of some pluripotency genes including OCT4, SOX2, NANOG, REX1 and ESG1 in the reprogrammed ADSCs compared to the control group. CONCLUSION: These findings showed that mouse ESC extract can be used to induce reprogramming of human ADSCs. In fact, this method is applicable for reprogramming of human adult stem cells to a more pluripotent sate and may have a potential in regenerative medicine.

RESUMEN

The miR-302 family is one of the main groups of microRNAs, which are highly expressed in embryonic stem cells (ESCs). Previous reports have indicated that miR-302 can reduce the proliferation rate of some cancer cells while compromising on their oncogenic potential at the same time without having the same effect on normal somatic cells. In this study we aimed to further investigate the role of the miR-302 cluster in multiple cancer signaling pathways using A-375 melanoma and HT-29 colorectal cancer cells. Our results indicate that the miR-302 cluster has the potential to modulate oncogenic properties of cancer cells through inhibition of proliferation, angiogenesis and invasion, and through reversal of the epithelial-to-mesenchymal transition (EMT) in these cells. We showed for the first time that overexpression of miR-302 cluster sensitized A-375 and HT-29 cells to hypoxia and also to the selective BRAF inhibitor vemurafenib. MiR-302 is a pleiotropically acting miRNA family which may have significant implications in controlling cancer progression and invasion. It acts through a reprogramming process, which has a global effect on a multitude of cellular pathways and events. We propose that reprogramming of cancer cells by epigenetic factors, especially miRNAs might provide an efficient tool for controlling cancer and especially for those with more invasive nature.

Asunto(s)

Neoplasias del Colon/patología , Melanoma/patología , MicroARNs/genética , Apoptosis/efectos de los fármacos , Apoptosis/genética , Adhesión Celular/efectos de los fármacos , Adhesión Celular/genética , Células Madre Embrionarias/efectos de los fármacos , Células Madre Embrionarias/metabolismo , Células HT29 , Humanos , Indoles/farmacología , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Sistema de Señalización de MAP Quinasas/genética , Invasividad Neoplásica , Metástasis de la Neoplasia , Neovascularización Patológica/genética , Sulfonamidas/farmacología , Hipoxia Tumoral/efectos de los fármacos , Hipoxia Tumoral/genética , Regulación hacia Arriba/efectos de los fármacos , Regulación hacia Arriba/genética , Vemurafenib

RESUMEN

Chlorpyrifos (CPF) is the most commonly used organophosphorus insecticide which causes neurodevelopmental toxicity. So far, animals have been used as ideal models for neurotoxicity studies, but working with animals is very expensive, laborious, and ethically challenging. This has encouraged researchers to seek alternatives. During recent years, several studies have reported successful differentiation of embryonic and adult stem cells to neurons. This has provided an excellent model for neurotoxicologic studies. In this study, neural differentiation of mouse adipose tissue-derived stem cells (ADSCs) was used as an in vitro model for investigation of CPF neurotoxicity. For this purpose, mouse ADSCs were cultured in a medium containing knockout serum replacement and were treated with different concentrations of CPF at several stages of differentiation. Cytotoxic effect of CPF and the expression of neuron-specific genes and proteins were studied in the differentiating ADSCs. Furthermore, the activity of acetylcholinesterase was assessed by Ellman assay at different stages of differentiation. This study showed that up to 500 µM CPF did not alter viability of the undifferentiated ADSCs, whereas viability of the differentiating cells decreased with 500 µM CPF. CPF upregulated the expression of some neuron-specific genes and seemed to decrease the number of ß-tubulin III and MAP2 proteins-expressing cells. There was no detectable acetylcholine esterase activity in differentiated ADSCs. In summary, it was shown that CPF treatment can decrease the viability of ADSC-derived neurons and dysregulate the expression of some neuronal markers through acetylcholinesterase-independent mechanisms. © 2015 Wiley Periodicals, Inc. Environ Toxicol 31: 1510-1519, 2016.

Asunto(s)

Tejido Adiposo/citología , Cloropirifos/toxicidad , Insecticidas/toxicidad , Neuronas/efectos de los fármacos , Síndromes de Neurotoxicidad/etiología , Células Madre/efectos de los fármacos , Acetilcolinesterasa/metabolismo , Animales , Diferenciación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Ratones , Neuronas/citología , ARN Mensajero/análisis , Células Madre/enzimología , Células Madre/fisiología

RESUMEN

Differentiation of embryonic stem (ES) cells is a heterogeneous process which is influenced by different parameters, including growth and differentiation factors. The aim of the present study was to investigate the effect of bone morphogenetic protein-4 (BMP4) signaling on differentiation of mouse ES cells to endodermal lineages. For this purpose, differentiation of the ES cells was induced by embryoid body (EB) formation through hanging drop method. During the suspension stage, EBs were treated with BMP4 in a medium containing either fetal bovine serum (FBS) or knockout serum replacement (KoSR). After plating, EBs showed differentiation to a heterogeneous population of specialized cell types. Two weeks after plating, all the experimental groups expressed three germ layer markers and some primitive and definitive endoderm-specific genes. Quantitative real-time PCR analysis showed higher expression levels of Sox17, Pdx1, Cdx2 and Villin mRNAs in the KoSR plus BMP4 condition and higher Gata4 and Afp expression levels in the FBS plus BMP4 condition. Formation of visceral endoderm and derivatives of definitive endoderm was detected in the BMP4 treated EBs. In conclusion, we demonstrated that both BMP4 signaling and serum composition have significant roles in differentiation of mouse ES cells towards endodermal lineages.

RESUMEN

Natriuretic peptide precursor-A (NPPA) is an early and specific marker for functional myocardium of the embryonic heart. NPPA gene encodes for a precursor of atrial natriuretic peptide (ANP). So far, three alternatively spliced variants have been reported for NPPA in human. In mouse, no alternatively spliced transcript of NPPA has been reported. In the current study, we investigated the expression of NPPA gene during cardiac differentiation of mouse adipose-tissue-derived stem cells (ADSCs) and embryonic stem (ES) cells. As revealed by reverse-transcription polymerase chain reaction analysis, 2-week-differentiated cells expressed some cardiac-specific makers, including ANP. Three additional intron-retained splice variants of NPPA were also detected during cardiac differentiation of the ADSCs and ES cells. In addition, we detected three intron-retained splice variants of NPPA in 8.5-day mouse embryonic heart. In the mature cardiomyocytes of 1-week-old mice, only the correctly spliced isoform of NPPA gene was expressed. Freshly isolated stromal vascular fraction also expressed one intron-retained isoform of NPPA gene. In conclusion, our findings have provided evidence for the expression of intron-retained splices of NPPA mRNA during the early stages of mouse cardiogenesis as well as in the mouse adipose tissue.

Asunto(s)

Expresión Génica , Miocardio/metabolismo , Péptido Natriurético Tipo-C/metabolismo , Precursores de Proteínas/metabolismo , Grasa Abdominal/citología , Células Madre Adultas/fisiología , Animales , Factor Natriurético Atrial , Secuencia de Bases , Diferenciación Celular , Técnicas de Cocultivo , Células Madre Embrionarias/fisiología , Regulación del Desarrollo de la Expresión Génica , Corazón/embriología , Corazón/crecimiento & desarrollo , Ratones , Datos de Secuencia Molecular , Contracción Miocárdica , Miocardio/citología , Miocitos Cardíacos/metabolismo , Péptido Natriurético Tipo-C/genética , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Precursores de Proteínas/genética , Sitios de Empalme de ARN , Troponina I/metabolismo

RESUMEN

To investigate the effect of BMP4 on cardiomyocyte differentiation of adipose tissue-derived stem cells (ADSCs), mouse ADSCs were treated with different concentrations of BMP4 in media containing fetal bovine serum (FBS) or Knockout™ Serum Replacement (KoSR). 3 weeks after cardiac induction, differentiated ADSCs expressed some cardiac-specific genes and proteins. BMP4 treatment upregulated the expression of cardiac transcription factors. In both FBS and KoSR-supplemented media, lower concentrations of BMP4 had a positive effect on the expression of MLC2A gene, while MLC2V was more expressed with higher concentrations of BMP4. BMP4 treatment in KoSR supplemented medium was more efficient for cardiac induction. Supplementation of culture media with insulin-transferrin-selenium improved the expression of MLC2A gene. The results of this study indicated that BMP4 is important for cardiac differentiation of the ADSCs. However, BMP4 was not enough for structural and functional maturation of the ADSC-derived cardiomyocytes.

Asunto(s)

Proteína Morfogenética Ósea 4/metabolismo , Diferenciación Celular/efectos de los fármacos , Células Madre/efectos de los fármacos , Células Madre/fisiología , Animales , Células Cultivadas , Medios de Cultivo/química , Perfilación de la Expresión Génica , Humanos , Ratones , Cadenas Ligeras de Miosina/análisis , Cadenas Ligeras de Miosina/genética

RESUMEN

The expression pattern of pluripotency markers in adipose tissue-derived stem cells (ADSCs) is a subject of controversy. Moreover, there is no data about the signaling molecules that regulate these markers in ADSCs. In the present study, we studied the roles of leukemia inhibitory factor (LIF) and miR-302 in this regard. Freshly isolated mouse ADSCs expressed hematopoietic, mesenchymal, and pluripotency markers. One day after plating, ADSCs expressed OCT4 and Sox2 proteins. After three passages, the expression of hematopoietic and pluripotency markers decreased, while the expression of mesenchymal stem cell markers exhibited a striking rise. Both supplementation of culture media with LIF and transfection of the ADSCs with miR-302 family upregulated the expression levels of OCT4, Nanog, and Sox2 mRNAs. These findings showed that mouse adipose tissue contains a population of cells with molecular resemblance to embryonic stem cells, and LIF and miR-302 family positively affect the expression of pluripotency markers.

Asunto(s)

Tejido Adiposo/metabolismo , Biomarcadores/metabolismo , Factor Inhibidor de Leucemia/genética , Células Madre Mesenquimatosas/metabolismo , MicroARNs/genética , Regulación hacia Arriba/genética , Animales , Factor Inhibidor de Leucemia/metabolismo , Ratones , Ratones Endogámicos BALB C , MicroARNs/metabolismo , Factor 3 de Transcripción de Unión a Octámeros/genética , Factor 3 de Transcripción de Unión a Octámeros/metabolismo , ARN Mensajero/genética , Factores de Transcripción SOXB1/genética , Factores de Transcripción SOXB1/metabolismo

RESUMEN

The neurohypophyseal hormone oxytocin plays a role in stimulation of neurogenesis in the adult brain. However, the exact role of oxytocin in neural development is still not well understood. In the present study, we evaluated the effect of oxytocin on neural differentiation of mouse adipose tissue-derived stem cells (ADSCs). For this purpose, ADSCs were cultured in a medium containing Knockout™ Serum Replacement (KoSR) and treated with different concentrations of oxytocin at the first or eighth day of differentiation. Two weeks after neural induction, ADSCs expressed several early and late neuron-specific genes and proteins. MTT assay and cell cycle analysis revealed a stimulatory effect of oxytocin on viability and proliferation of differentiating ADSCs. As detected by quantitative real-time PCR, treatment of the ADSCs with low concentrations of oxytocin induced neurogenesis. Oxytocin treatment also upregulated the expression of oxytocin receptor mRNA. These results demonstrated for the first time that oxytocin treatment can promote neural differentiation of the ADSCs in a dose-dependent and time-dependent manner. Oxytocin has a significant role in neurogenesis, and this may have implications in regeneration of adult neurons.

Asunto(s)

Tejido Adiposo/citología , Células-Madre Neurales/citología , Células-Madre Neurales/efectos de los fármacos , Neurogénesis/efectos de los fármacos , Oxitocina/farmacología , Animales , Diferenciación Celular , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Ratones , Oxitocina/metabolismo , Fosfopiruvato Hidratasa/metabolismo , Antígeno Nuclear de Célula en Proliferación/metabolismo , Receptores de Oxitocina/metabolismo

RESUMEN

Neural differentiation of embryonic and adult stem cells has been reported previously. Several studies have used different proportions of serum or a cocktail of growth and differentiation factors for this purpose. In the present study, we examined neural differentiation of mouse embryonic stem (ES) cells in KoSR-containing media. We also investigated neural differentiation of mouse adipose tissue-derived stem cells (ADSCs) in a medium containing KoSR, a synthetic serum replacement, and compared it with neural differentiation in low-serum condition. Meanwhile, effect of ß-ME on neural differentiation was investigated in both conditions. As revealed by RT-PCR and immunocytochemistry analyses, KoSR-containing medium induced neural differentiation of mouse ES cells. Moreover, under the culture conditions we tested, ADSCs were differentiated to neuron-like cells and expressed some neuronal markers. Low concentration of ß-ME improved neuron-like differentiation of the ADSCs in the 4% FBS-supplemented medium, while addition of ß-ME in KoSR condition decreased neural differentiation. KoSR-containing medium without any additional factor improved generation of neuron-like cells, upregulated the expression of mature neuronal markers and led to the formation of cytoplasmic processes. In summary, our findings are indicating that mouse embryonic and mesenchymal stem cells are capable of neural development in KoSR-containing media.

Asunto(s)

Tejido Adiposo/citología , Diferenciación Celular/efectos de los fármacos , Medio de Cultivo Libre de Suero/farmacología , Células Madre Embrionarias/fisiología , Mercaptoetanol/farmacología , Células Madre Mesenquimatosas/fisiología , Neuronas/citología , Animales , Biomarcadores/metabolismo , Línea Celular , Regulación de la Expresión Génica/efectos de los fármacos , Ratones , Ratones Endogámicos C57BL

RESUMEN

MicroRNA-34 family has anti-proliferative and apoptotic roles. Recent studies have shown that p53 upregulates miR-34 family leading to direct repression of several key oncogenes. Inactivation of miR-34a has been reported in multiple types of malignancies including breast cancer. The critical role of miR-34a in p53-mediated cell cycle arrest and apoptosis invokes studies focusing on the specific role of miR-34a dysregulation in carcinogenesis. While presence of p53 mutations has frequently been described in breast cancer, still most of the breast tumors do not show any variation in the p53 coding sequence or protein expression. Therefore, it is important to clarify possible involvement of other mediators of p53 pathway in breast cancer. In this study, expression of mature miR-34a in breast tumors with wild-type p53 was investigated in order to find any correlation between dysregulation of miR-34a expression and breast cancer. In about 40 % of the wild-type p53 samples, miR-34a was significantly downregulated. Neither hypermethylation of the miR-34a promoter nor genetic variations of the p53-binding site were detected in tumor samples with downregulated miR-34a. This study has provided evidence that miR-34a expression can be affected in a significant proportion of breast tumors independent of p53. Furthermore, downregulation of miR-34a was significantly associated with metastasis, while there was a significant correlation between upregulation of miR-34a and non-metastatic condition indicating a protective role for miR-34a against more invasive disease. Knowledge of miR-34a status may provide additional useful information regarding the nature of breast tumors, especially when p53 testing does not show any aberration.

Asunto(s)

Neoplasias de la Mama/genética , Regulación Neoplásica de la Expresión Génica/genética , MicroARNs/biosíntesis , Invasividad Neoplásica/genética , Proteína p53 Supresora de Tumor/genética , Adulto , Anciano , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Metilación de ADN , Análisis Mutacional de ADN , Regulación hacia Abajo , Femenino , Humanos , Inmunohistoquímica , MicroARNs/genética , Persona de Mediana Edad , Mutación , Regiones Promotoras Genéticas/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa

RESUMEN

Stainless steel (SS) is one of the most applicable materials in fabrication of cardiac implants. The aim of this study is to investigate the effect of atomic structure of polycrystalline stainless steel on the response of adipose tissue-derived stem cells (ADSCs). Samples are prepared from differently processed extruded rod and rolled sheet of 316L SS having different crystallographic structure. X-ray diffraction analysis indicated (200) and (111) orientations with distinct volume fractions in the specimens. Morphology and ADSCs behavior including adhesion, proliferation and differentiation are assessed. The expression of cardiac specific protein (cardiac troponin I) and genes of differentiating cardiomyocytes is analyzed by immunofluorescence and RT-PCR. The number of attached and grown cells on the rod sample is higher than the sheet sample also the scanning electron microscopy (SEM) analysis of ADSCs grown on the samples demonstrates higher cell density and spreading pattern on the surface of rod sample. In differentiated ADSCs on the rod sample the expression of all genes except ANF are detectable, while on the sheet sample only the MEF2C and ß-MHC are expressed. This study shows that the cellular response is influenced by the crystal structure of the substrate therefore; the skill to alter the structure of substrate may lend itself to engineer a biomaterial which could be suitable for differentiation of stem cells into a definite lineage.

Asunto(s)

Tejido Adiposo/citología , Acero Inoxidable/farmacología , Células Madre/citología , Células Madre/efectos de los fármacos , Animales , Adhesión Celular/efectos de los fármacos , Recuento de Células , Diferenciación Celular/efectos de los fármacos , Diferenciación Celular/genética , Proliferación Celular/efectos de los fármacos , Forma de la Célula/efectos de los fármacos , Regulación de la Expresión Génica/efectos de los fármacos , Ratones , Ratones Endogámicos BALB C , Microscopía de Fuerza Atómica , Miocardio/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Células Madre/metabolismo , Células Madre/ultraestructura , Difracción de Rayos X

RESUMEN

Differentiation of embryonic stem cell (ESC)-derived embryoid bodies (EBs) is a heterogeneous process. ESCs can differentiate in vitro into different cell types including beating cardiomyocytes. The main aim of the present study was to develop an improved preparation method for scanning electron microscopic study of ESC-derived cardiac bundles and to investigate the fine structural characteristics of mouse ESCs-derived cardiomyocytes using electron microscopy. The mouse ESCs differentiation was induced by EBs' development through hanging drop, suspension and plating stages. Cardiomyocytes appeared in the EBs' outgrowth as beating clusters that grew in size and formed thick branching bundles gradually. Cardiac bundles showed cross striation even when they were observed under an inverted microscope. They showed a positive immunostaining for cardiac troponin I and α-actinin. Transmission and scanning electron microscopy (TEM & SEM) were used to study the structural characteristics of ESC-derived cardiomyocytes. Three weeks after plating, differentiated EBs showed a superficial layer of compact fibrous ECM that made detailed observation of cardiac bundles impossible. We tried several preparation methods to remove unwanted cells and fibers, and finally we revealed the branching bundles of cardiomyocytes. In TEM study, most cardiomyocytes showed parallel arrays of myofibrils with a mature sarcomeric organization marked by H-bands, M-lines and numerous T-tubules. Cardiomyocytes were connected to each other by intercalated discs composed of numerous gap junctions and fascia adherences.

RESUMEN

Bone marrow and adipose tissue have provided two suitable sources of mesenchymal stem cells. Although previous studies have confirmed close similarities between bone marrow-derived stem cells (BM-MSCs) and adipose tissue-derived stem cells (ADSCs), the molecular phenotype of ADSCs is still poorly identified. In the present study, mouse ADSCs were isolated from the inguinal fat pad of 12-14 weeks old mice. Freshly isolated and three passaged ADSCs were analyzed for the expression of OCT4, Sca-1, c-kit and CD34 by RT-PCR. Three passaged ADSCs were analyzed by flow cytometry for the presence of CD11b, CD45, CD31, CD29 and CD44. Moreover, cardiogenic, adipogenic and neurogenic differentiation of ADSCs were induced in vitro. Freshly isolated ADSCs showed the expression of OCT4, Sca-1, c-kit and CD34, and two days cultured ADSCs were positively immunostained with anti-OCT4 monoclonal antibody. After three passages, the expression of OCT4, c-kit and CD34 eliminated, while the expression of Sca-1 showed a striking enhancement. These cells were identified positive for CD29 and CD44 markers, and they showed the lack of CD45 and CD31 expression. Three passaged ADSCs were differentiated to adipocyte-, cardiomyocyte- and neuron-like cells that were identified based on the positive staining with Sudan black, anti-cardiac troponin I antibody and anti-map-2 antibody, respectively. In conclusion, adipose tissue contains a stem cell population that seems to be a good multipotential cell candidate for the future cell replacement therapy.

Asunto(s)

Tejido Adiposo/citología , Diferenciación Celular , Separación Celular/métodos , Células Madre Multipotentes/citología , Animales , Biomarcadores/metabolismo , Células Cultivadas , Células Endoteliales/citología , Células Endoteliales/metabolismo , Citometría de Flujo , Regulación de la Expresión Génica , Células Madre Hematopoyéticas/citología , Células Madre Hematopoyéticas/metabolismo , Ratones , Ratones Endogámicos BALB C , Células Madre Multipotentes/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Células del Estroma/citología , Células del Estroma/metabolismo

RESUMEN

Cell based-gene delivery has provided an important therapeutic strategy for different disorders in the recent years. This strategy is based on the transplantation of genetically modified cells to express specific genes and to target the delivery of therapeutic factors, especially for the treatment of cancers and neurological, immunological, cardiovascular and heamatopoietic disorders. Although, preliminary reports are encouraging, and experimental studies indicate functionally and structurally improvements in the animal models of different disorders, universal application of this strategy for human diseases requires more evidence. There are a number of parameters that need to be evaluated, including the optimal cell source, the most effective gene/genes to be delivered, the optimal vector and method of gene delivery into the cells and the most efficient route for the delivery of genetically modified cells into the patient. Also, some obstacles have to be overcome, including the safety and usefulness of the approaches and the stability of the improvements. Here, recent studies concerning with the cell-based gene delivery for spinal cord injury and some neurodegenerative disorders such as amyotrophic lateral sclerosis, Parkinson's disease and Alzheimer's disease are briefly reviewed, and their exciting consequences are discussed.

Asunto(s)

Trasplante de Células , Terapia Genética , Enfermedades Neurodegenerativas/terapia , Traumatismos de la Médula Espinal/terapia , Células Madre/metabolismo , Animales , Citoprotección , Modelos Animales de Enfermedad , Vectores Genéticos/genética , Humanos , Factores de Crecimiento Nervioso/genética , Factores de Crecimiento Nervioso/metabolismo , Enfermedades Neurodegenerativas/patología , Enfermedades Neurodegenerativas/fisiopatología , Neuronas/patología , Recuperación de la Función , Retroviridae/genética , Traumatismos de la Médula Espinal/patología , Traumatismos de la Médula Espinal/fisiopatología , Células Madre/patología , Transgenes/genética