Gene cloning and sequence analysis of the RPL29 gene and its effect on lipogenesis in goat intramuscular adipocytes / 生物工程学报

The aim of this study was to clone the goat RPL29 gene and analyze its effect on lipogenesis in intramuscular adipocytes. Using Jianzhou big-eared goats as the object, the goat RPL29 gene was cloned by reverse transcription-polymerase chain reaction (RT-PCR), the gene structure and expressed protein sequence were analyzed by bioinformatics, and the mRNA expression levels of RPL29 in various tissues and different differentiation stages of intramuscular adipocytes of goats were detected by quantitative real-time PCR (qRT-PCR). The RPL29 overexpression vector pEGFP-N1-RPL29 constructed by gene recombination was used to transfect into goat intramuscular preadipocytes and induce differentiation. Subsequently, the effect of overexpression of RPL29 on fat droplet accumulation was revealed morphologically by oil red O and Bodipy staining, and changes in the expression levels of genes related to lipid metabolism were detected by qRT-PCR. The results showed that the length of the goat RPL29 was 507 bp, including a coding sequence (CDS) region of 471 bp which encodes 156 amino acid residues. It is a positively charged and stable hydrophilic protein mainly distributed in the nucleus of cells. Tissue expression profiling showed that the expression level of this gene was much higher in subcutaneous adipose tissue and inter-abdominal adipose tissue of goats than in other tissues (P < 0.05). The temporal expression profile showed that the gene was expressed at the highest level at 84 h of differentiation in goat intramuscular adipocytes, which was highly significantly higher than that in the undifferentiated period (P < 0.01). Overexpression of RPL29 promoted lipid accumulation in intramuscular adipocytes, and the optical density values of oil red O staining were significantly increased (P < 0.05). In addition, overexpression of RPL29 was followed by a highly significant increase in ATGL and ACC gene expression (P < 0.01) and a significant increase in FASN gene expression (P < 0.05). In conclusion, the goat RPL29 may promote intra-muscular adipocyte deposition in goats by up-regulating FASN, ACC and ATGL.

Cloning, identification and functional analysis of the goat transcription factor c-fos / 生物工程学报

C-fos is a transcription factor that plays an important role in cell proliferation, differentiation and tumor formation. The aim of this study was to clone the goat c-fos gene, clarify its biological characteristics, and further reveal its regulatory role in the differentiation of goat subcutaneous adipocytes. We cloned the c-fos gene from subcutaneous adipose tissue of Jianzhou big-eared goats by reverse transcription-polymerase chain reaction (RT-PCR) and analyzed its biological characteristics. Using real-time quantitative PCR (qPCR), we detected the expression of c-fos gene in the heart, liver, spleen, lung, kidney, subcutaneous fat, longissimus dorsi and subcutaneous adipocytes of goat upon induced differentiation for 0 h to 120 h. The goat overexpression vector pEGFP-c-fos was constructed and transfected into the subcutaneous preadipocytes for induced differentiation. The morphological changes of lipid droplet accumulation were observed by oil red O staining and bodipy staining. Furthermore, qPCR was used to test the relative mRNA level of the c-fos overexpression on adipogenic differentiation marker genes. The results showed that the cloned goat c-fos gene was 1 477 bp in length, in which the coding sequence was 1 143 bp, encoding a protein of 380 amino acids. Protein structure analysis showed that goat FOS protein has a basic leucine zipper structure, and subcellular localization prediction suggested that it was mainly distributed in the nucleus. The relative expression level of c-fos was higher in the subcutaneous adipose tissue of goats (P < 0.05), and the expression level of c-fos was significantly increased upon induced differentiation of subcutaneous preadipocyte for 48 h (P < 0.01). Overexpression of c-fos significantly inhibited the lipid droplets formation in goat subcutaneous adipocytes, significantly decreased the relative expression levels of the AP2 and C/EBPβ lipogenic marker genes (P < 0.01). Moreover, AP2 and C/EBPβ promoter are predicted to have multiple binding sites. In conclusion, the results indicated that c-fos gene was a negative regulatory factor of subcutaneous adipocyte differentiation in goats, and it might regulate the expression of AP2 and C/EBPβ gene expression.

DNA methylation analysis identifies key transcription factors involved in mesenchymal stem cell osteogenic differentiation

BACKGROUND: Knowledge about regulating transcription factors (TFs) for osteoblastogenesis from mesenchymal stem cells (MSCs) is limited. Therefore, we investigated the relationship between genomic regions subject to DNA-methylation changes during osteoblastogenesis and the TFs known to directly interact with these regulatory regions. RESULTS: The genome-wide DNA-methylation signature of MSCs differentiated to osteoblasts and adipocytes was determined using the Illumina HumanMethylation450 BeadChip array. During adipogenesis no CpGs passed our test for significant methylation changes. Oppositely, during osteoblastogenesis we identified 2462 differently significantly methylated CpGs (adj. p < 0.05). These resided outside of CpGs islands and were significantly enriched in enhancer regions. We confirmed the correlation between DNA-methylation and gene expression. Accordingly, we developed a bioinformatic tool to analyse differentially methylated regions and the TFs interacting with them. By overlaying our osteoblastogenesis differentially methylated regions with ENCODE TF ChIP-seq data we obtained a set of candidate TFs associated to DNA-methylation changes. Among them, ZEB1 TF was highly related with DNA-methylation. Using RNA interference, we confirmed that ZEB1, and ZEB2, played a key role in adipogenesis and osteoblastogenesis processes. For clinical relevance, ZEB1 mRNA expression in human bone samples was evaluated. This expression positively correlated with weight, body mass index, and PPARγ expression. CONCLUSIONS: In this work we describe an osteoblastogenesis-associated DNA-methylation profile and, using these data, validate a novel computational tool to identify key TFs associated to age-related disease processes. By means of this tool we identified and confirmed ZEB TFs as mediators involved in the MSCs differentiation to osteoblasts and adipocytes, and obesity-related bone adiposity.

circ_0003204 regulates the osteogenic differentiation of human adipose-derived stem cells via miR-370-3p/HDAC4 axis / 国际口腔科学杂志·英文版

Human adipose-derived stem cells (hASCs) are a promising cell type for bone tissue regeneration. Circular RNAs (circRNAs) have been shown to play a critical role in regulating various cell differentiation and involve in mesenchymal stem cell osteogenesis. However, how circRNAs regulate hASCs in osteogenesis is still unclear. Herein, we found circ_0003204 was significantly downregulated during osteogenic differentiation of hASCs. Knockdown of circ_0003204 by siRNA or overexpression by lentivirus confirmed circ_0003204 could negatively regulate the osteogenic differentiation of hASCs. We performed dual-luciferase reporting assay and rescue experiments to verify circ_0003204 regulated osteogenic differentiation via sponging miR-370-3p. We predicted and confirmed that miR-370-3p had targets in the 3'-UTR of HDAC4 mRNA. The following rescue experiments indicated that circ_0003204 regulated the osteogenic differentiation of hASCs via miR-370-3p/HDAC4 axis. Subsequent in vivo experiments showed the silencing of circ_0003204 increased the bone formation and promoted the expression of osteogenic-related proteins in a mouse bone defect model, while overexpression of circ_0003204 inhibited bone defect repair. Our findings indicated that circ_0003204 might be a promising target to promote the efficacy of hASCs in repairing bone defects.

Quantifying the state of cell differentiation based on the gene networks entropy / 生物工程学报

Studies of cellular dynamic processes have shown that cells undergo state changes during dynamic processes, controlled mainly by the expression of genes within the cell. With the development of high-throughput sequencing technologies, the availability of large amounts of gene expression data enables the acquisition of true gene expression information of cells at the single-cell level. However, most existing research methods require the use of information beyond gene expression, thus introducing additional complexity and uncertainty. In addition, the prevalence of dropout events hampers the study of cellular dynamics. To this end, we propose an approach named gene interaction network entropy (GINE) to quantify the state of cell differentiation as a means of studying cellular dynamics. Specifically, by constructing a cell-specific network based on the association between genes through the stability of the network, and defining the GINE, the unstable gene expression data is converted into a relatively stable GINE. This method has no additional complexity or uncertainty, and at the same time circumvents the effects of dropout events to a certain extent, allowing for a more reliable characterization of biological processes such as cell fate. This method was applied to study two single-cell RNA-seq datasets, head and neck squamous cell carcinoma and chronic myeloid leukaemia. The GINE method not only effectively distinguishes malignant cells from benign cells and differentiates between different periods of differentiation, but also effectively reflects the disease efficacy process, demonstrating the potential of using GINE to study cellular dynamics. The method aims to explore the dynamic information at the level of single cell disorganization and thus to study the dynamics of biological system processes. The results of this study may provide scientific recommendations for research on cell differentiation, tracking cancer development, and the process of disease response to drugs.

The role of retinoic acid in the commitment to meiosis / 亚洲男科学杂志(英文版)

Male meiosis is a complex process whereby spermatocytes undergo cell division to form haploid cells. This review focuses on the role of retinoic acid (RA) in meiosis, as well as several processes regulated by RA before cell entry into meiosis that are critical for proper meiotic entry and completion. Here, we discuss RA metabolism in the testis as well as the roles of stimulated by retinoic acid gene 8 (STRA8) and MEIOSIN, which are responsive to RA and are critical for meiosis. We assert that transcriptional regulation in the spermatogonia is critical for successful meiosis.

Melatonin has a stimulatory effect on osteoblasts by upregulating COL-I and OPN expression/secretion / Melatonina tem efeito estimulador em osteoblastos, pela regulação positiva da expressão/secreção de COL-I e OPN

ABSTRACT Melatonin (MLT) is a potential signaling molecule in the homeostasis of bone metabolism and may be an important mediator of bone formation and stimulation. The aim of this in vitro study was to evaluate the effect of MLT on the viability, mRNA/protein expression and mineralization of pre-osteoblastic cells. The concentrations 5, 2.5, 1, 0.1 and 0.01 mM MLT were tested on pre-osteoblastic cells (MC3T3) compared to control (no MLT), evaluating proliferation and cell viability (C50), gene expression (RT-PCR) and secretion (ELISA) of COL-I and OPN at 24h, 48h and 72h, and the formation of mineral nodules (alizarin red and fast red) after 10 days of treatment. MLT at 5 and 2.5 mM proved to be cytotoxic (C50), so only 0.01, 0.1 and 1 mM were used for the subsequent analyses. OPN mRNA expression increased with MLT at 0.1 mM - 1 mM, which was followed by increased secretion of OPN both at 24h and 72h compared to the remaining groups (p <0.05). COL-I mRNA and COL-1 secretion followed the same pattern as OPN at 0.1 mM MLT at 72h of treatment (p <0.05). Regarding mineralization, all MLT doses (except 1mM) caused an increase (p <0.05) in the formation of mineral nodules compared to the control. Melatonin at 0.01mM - 1mM had a stimulatory effect on osteoblasts by upregulating COL-I and OPN expression/ secretion and mineralization, thereby fostering osteogenesis.

RESUMO A melatonina (MLT) é uma molécula potencial de sinalização na homeostase do metabolismo ósseo e pode ser um importante mediador da formação e estimulação óssea. O objetivo deste estudo in vitro foi avaliar o efeito da MLT na viabilidade, na expressão do mRNA da proteína e mineralização de células préosteoblásticas. As concentrações de MLT 5, 2,5, 1, 0,1 e 0,01 mM foram testadas em células pré-osteoblásticas da linhagem MC3T3 em comparação ao controle (sem MLT), avaliando a proliferação e a viabilidade celular (C50), expressão gênica (rtPCR) e secreção (Elisa) de Colágeno tipo 1 (COL-I) e osteopontina (OPN) às 24, 48 e 72 horas, além da formação de nódulos minerais por meio do teste vermelho de Alizarina fast red após 10 dias de tratamento. MLT a 5 e 2,5 mM provou ser tóxico (C50). Portanto, as concentrações de 0,01, 0,1 e 1 mM foram utilizadas para as análises subsequentes. A expressão do mRNA da OPN aumentou com MLT a 0,1 mM-1mM, seguida pela secreção aumentada de OPN às 24 e 72 horas em comparação aos demais grupos (p<0,05). O mRNA de COL-I e a secreção de COL-I seguiram o mesmo padrão do OPN a 0,1 mM de MLT em 72 horas de tratamento (p<0,05). Em relação à mineralização, todas as doses de MLT (exceto 1mM) causaram aumento (p<0,05) na formação de nódulos minerais em comparação ao controle. A MLT na concentração entre 0,01mM a 1 mM teve um efeito estimulador sobre os osteoblastos, ao regular positivamente a expressão e secreção de COL-I e OPN, além da mineralização, favorecendo a osteogênese.

Smad1/5 signal transduction regulates the ameloblastic differentiation of induced pluripotent stem cells

Abstract Induced pluripotent stem (iPS) cells could be induced into ameloblast-like cells by ameloblasts serum-free conditioned medium (ASF-CM), and bone morphogenetic proteins (BMPs) might be essential during the regulation of this process. The present study investigates the signal transduction that regulates the ameloblastic differentiation of iPS cells induced by ASF-CM. Mouse iPS cells were characterized and then cultured for 14 days in epithelial cell medium (control) or ASF-CM. Bone morphogenetic protein receptor II (BMPR-II) siRNA, inhibitor of Smad1/5 phosphorylation activated by activin receptor-like kinase (ALK) receptors, and inhibitors of mitogen-activated protein kinases (MAPKs) phosphorylation were used to treat the iPS cells in combination with ASF-CM. Real-time PCR, western blotting, and immunofluorescent staining were used to evaluate the expressions of ameloblast markers ameloblastin, enamelin, and cytokeratin-14. BMPR-II gene and protein levels increased markedly in ASF-CM-treated iPS cells compared with the controls, while the mRNA levels of Bmpr-Ia and Bmpr-Ib were similar between the ASF-CM and control groups. ASF-CM stimulation significantly increased the gene and protein expression of ameloblastin, enamelin and cytokeratin-14, and phosphorylated SMAD1/5, p38 MAPK, and ERK1/2 MAPK compared with the controls. Knockdown of BMPR-II and inhibition of Smad1/5 phosphorylation both could significantly reverse the increased expression of ameloblastin, enamelin, and cytokeratin-14 induced by ASF-CM, while neither inhibition of p38 nor ERK1/2 phosphorylation had significant reversing effects. We conclude that smad1/5 signaling transduction, activated by ALK receptors, regulates the ameloblastic differentiation of iPS cells induced by ameloblast-conditioned medium.

Smad1/5 signal transduction regulates the ameloblastic differentiation of induced pluripotent stem cells

Abstract Induced pluripotent stem (iPS) cells could be induced into ameloblast-like cells by ameloblasts serum-free conditioned medium (ASF-CM), and bone morphogenetic proteins (BMPs) might be essential during the regulation of this process. The present study investigates the signal transduction that regulates the ameloblastic differentiation of iPS cells induced by ASF-CM. Mouse iPS cells were characterized and then cultured for 14 days in epithelial cell medium (control) or ASF-CM. Bone morphogenetic protein receptor II (BMPR-II) siRNA, inhibitor of Smad1/5 phosphorylation activated by activin receptor-like kinase (ALK) receptors, and inhibitors of mitogen-activated protein kinases (MAPKs) phosphorylation were used to treat the iPS cells in combination with ASF-CM. Real-time PCR, western blotting, and immunofluorescent staining were used to evaluate the expressions of ameloblast markers ameloblastin, enamelin, and cytokeratin-14. BMPR-II gene and protein levels increased markedly in ASF-CM-treated iPS cells compared with the controls, while the mRNA levels of Bmpr-Ia and Bmpr-Ib were similar between the ASF-CM and control groups. ASF-CM stimulation significantly increased the gene and protein expression of ameloblastin, enamelin and cytokeratin-14, and phosphorylated SMAD1/5, p38 MAPK, and ERK1/2 MAPK compared with the controls. Knockdown of BMPR-II and inhibition of Smad1/5 phosphorylation both could significantly reverse the increased expression of ameloblastin, enamelin, and cytokeratin-14 induced by ASF-CM, while neither inhibition of p38 nor ERK1/2 phosphorylation had significant reversing effects. We conclude that smad1/5 signaling transduction, activated by ALK receptors, regulates the ameloblastic differentiation of iPS cells induced by ameloblast-conditioned medium.

Proliferation and differentiation of mouse spermatogonial stem cells on a three-dimensional surface composed of PCL/gel nanofibers / Proliferación y diferenciación de células madre espermatogónicas de ratón en una superficie tridimensional compuesta de nanofibras PCL / gel

Spermatogonial stem cells (SSCs) have self-renewal and differentiation capacity essential for sperm production throughout the male reproductive life. The electrospun polycaprolactone/gelatin (PCL/Gel) nanofibrous scaffold mimics important features of the extracellular matrix (ECM), which can provide a promising technique for the proliferation and differentiation of SSCs in vitro. The goal of the present study was to investigate the effects of PCL/Gel nanofibrous scaffold on the propagation and differentiation of neonate mouse SSCs (mSSCs). mSSCs were enzymatically isolated, and the cells were purified by differential plating method and seeded on scaffold. After 2 weeks, viability, colony number and diameter, and expression of specific spermatogonial cell genes were investigated. After mSSCs propagation, the cells were cultivated in a differentiation medium on the scaffold for another 2 weeks, and differentiating cells were analyzed by real-time PCR. The number of mSSC colony (P<0.01) and expression levels of specific spermatogonial genes Plzf and Inga6 (P<0.01) and also differentiation genes c-Kit, Tp1 and Ptm1 (P<0.05) were higher in scaffold group compared with control during the culture period. We conclude that mSSCs can be expanded and can differentiate toward spermatid cells on PCL/Gel nanofibrous scaffold with improved developmental parameters.

Las células madre espermatogónicas (CME) tienen capacidad de auto renovación y diferenciación esenciales para la producción de esperma a lo largo de la vida reproductiva masculina. El «scaffold¼ nanofibroso de policaprolactona / gelatina (PCL / Gel) electrohilado imita características importantes de la matriz extracelular (MEC), que puede proporcionar una técnica prometedora para la proliferación y diferenciación de CME in vitro. El objetivo del presente estudio fue investigar los efectos del «scaffold¼ nanofibroso PCL / Gel en la propagación y diferenciación de CME de ratones neonatos (mSSC). Los mSSC se aislaron enzimáticamente y las células se purificaron mediante un método de siembra diferencial y se sembraron en un «scaffold¼. Después de 2 semanas, se investigaron la viabilidad, el número y el diámetro de las colonias y la expresión de genes específicos de células espermatogónicas. Después de la propagación de mSSC, las células se cultivaron en un medio de diferenciación en el «scaffold¼ durante otras 2 semanas, y las células se analizaron mediante PCR en tiempo real. El número de colonias mSSC (P <0,01) y los niveles de expresión de los genes espermatogónicos específicos Plzf e Inga6 (P <0,01) y también los genes de diferenciación c-Kit, Tp1 y Ptm1 (P <0,05) fueron mayores en el grupo de «scaffold¼ en comparación con el control durante el período de cultivo. Concluimos que los mSSC pueden expandirse y diferenciarse en células espermátidas en un «scaffold¼ de nanofibras PCL / Gel con parámetros de desarrollo mejorados.

Desarrollo neuroembriológico: el camino desde la proliferación hasta la perfección / Neuronal Embryonic Development: A Pathway from Proliferation to Perfection

El desarrollo neurológico humano requiere una serie de pasos que permitan orientar, regular y diferenciar los diversos componentes cerebrales, para así garantizar, de una manera bastante precisa, la correcta organización y funcionamiento de las estructuras neuronales. La neurogénesis está clásicamente dividida en cuatro etapas consecutivas: proliferación, migración, diferenciación y maduración. En los humanos, estas ocurren desde la tercera semana de gestación hasta la vida adulta y precisan de un complejo grupo de paquetes genéticos, así como de algunos factores asociados, que se han ido descubriendo gracias a los avances en la biología molecular. El artículo es una revisión acerca del desarrollo neuroembriológico humano y los componentes genéticos más relevantes encontrados en la literatura.

The human neuronal development requires a number of concrete steps which lead to orientation, regulation and differentiation of several brain components. They must be done to guarantee, in a very precise way, the correct organization and functioning of the neuronal structures. Neurogenesis is commonly divided into four consecutive stages: proliferation, migration, differentiation and maturation. In humans, those stages take place since the third week of prenatal Iife until the adult Iife. They also require a complex group of genetic packs and associated molecular factors, most of which have been recen tly discovered by the molecular biology technology. A review was made about the human neuronal and embryological development and the most relevant genetic components described by the literature so far.

Small activating RNA induces myogenic differentiation of rat adipose-derived stem cells by upregulating MyoD

ABSTRACTPurpose:RNA activation (RNAa) is a mechanism of gene activation triggered by promoter-targeted small double stranded RNAs (dsRNAs), also known as small activating RNAs (saRNAs). Myogenic regulatory factor MyoD is regarded as the master activator of myogenic differentiation cascade by binding to enhancer of muscle specific genes. Stress urinary incontinence (SUI) is a condition primarily resulted from urethral sphincter deficiency. It is thus expected that by promoting differentiation of adipose-derived stem cells (ADSCs) into myoblasts by activating MyoD gene through RNAa may offer benefits to SUI.Materials and Methods:Rats ADSCs were isolated, proliferated in vitro, and identified by flow cytometry. Purified ADSCs were then transfected with a MyoD saRNA or control transfected. Real-time polymerase chain reaction (RT-PCR) and western blotting were used to detect MyoD mRNA and protein expression, respectively. Immunocytochemical staining was applied to determine the expression of desmin protein in transfected cells. Cell viability was measured by using CellTiter 96® AQueous One Solution Cell Proliferation Assay kit.Results:Transfection of a MyoD saRNA (dsMyoD) into ADSCs significantly induced the expression of MyoD at both the mRNA and protein levels, and inhibited cell proliferation. Desmin protein expression was detected in dsMyoD treated ADSCs 2 weeks later.Conclusion:Our findings show that RNAa mediated overexpression of MyoD can promote transdifferentiation of ADSCs into myoblasts and may help treat stress urinary incontinence (SUI)–a condition primarily resulted from urethral sphincter deficiency.

Early surgical complications after gastric by-pass: a literature review / Complicações cirúrgicas precoces após bypass gástrico: revisão da literatura

INTRODUCTION: Gastric bypass is today the most frequently performed bariatric procedure,but, despite of it, several complications can occur with varied morbimortality. Probably all bariatric surgeons know these complications, but, as bariatric surgery continues to spread, general surgeon must be familiarized to it and its management. Gastric bypass complications can be divided into two groups: early and late complications, taking into account the two weeks period after the surgery. This paper will focus the early ones. METHOD: Literature review was carried out using Medline/PubMed, Cochrane Library, SciELO, and additional information on institutional sites of interest crossing the headings: gastric bypass AND complications; follow-up studies AND complications; postoperative complications AND anastomosis, Roux-en-Y; obesity AND postoperative complications. Search language was English. RESULTS: There were selected 26 studies that matched the headings. Early complications included: anastomotic or staple line leaks, gastrointestinal bleeding, intestinal obstruction and incorrect Roux limb reconstruction. CONCLUSION: Knowledge on strategies on how to reduce the risk and incidence of complications must be acquired, and every surgeon must be familiar with these complications in order to achieve an earlier recognition and perform the best intervention. .

INTRODUÇÃO: O bypass gástrico é hoje o procedimento bariátrico mais realizado, mas, apesar disso, várias complicações podem ocorrer com variada morbimortalidade. Provavelmente todos os cirurgiões bariátricos conhecem essas complicações, mas como a cirurgia bariátrica continua a se espalhar, o cirurgião geral deve estar familiarizado com essas complicações e seu manuseio. As complicações do bypass gástrico podem ser divididas em dois grupos: as precoces e tardias, tendo em conta o período de duas semanas após a operação. Este artigo irá focar as precoces. MÉTODO: Foi realizada revisão da literatura utilizando as bases Medline/PubMed, Cochrane Library, SciELO, e informações adicionais sobre sites institucionais de interesse cruzando os descritores: bypass gástrico AND complicações; seguimento AND complicações; complicações pós-operatórias AND anastomose, Roux-en-Y; obesidade AND complicações pós-operatórias. A língua usada para a busca foi o inglês. RESULTADOS: Foram selecionados 26 artigos que combinavam com os descritores. As complicações imediatas foram: fístula na linha de grampeamento, sangramento gastrointestinal, obstrução intestinal e reconstrução incorreta da alça em Roux. CONCLUSÃO: O conhecimento sobre as estratégias de como reduzir o risco e incidência das complicações deve ser adquirido ao longo do tempo, e cada cirurgião deve estar familiarizado com essas complicações, a fim de reconhecê-las precocemente e realizar a melhor intervenção. .

Functional disruption of human leukocyte antigen II in human embryonic stem cell

BACKGROUND: Theoretically human embryonic stem cells (hESCs) have the capacity to self-renew and differentiate into all human cell types. Therefore, the greatest promise of hESCs-based therapy is to replace the damaged tissues of patients suffering from traumatic or degenerative diseases by the exact same type of cells derived from hESCs. Allo-graft immune rejection is one of the obstacles for hESCs-based clinical applications. Human leukocyte antigen (HLA) II leads to CD4+ T cells-mediated allograft rejection. Hence, we focus on optimizing hESCs for clinic application through gene modification. RESULTS: Transcription activator-like effector nucleases (TALENs) were used to target MHC class II transactivator (CIITA) in hESCs efficiently. CIITA(-/-)hESCs did not show any difference in the differentiation potential and self-renewal capacity. Dendritic cells (DCs) derived from CIITA(-/-)hESCs expressed CD83 and CD86 but without the constitutive HLA II. Fibroblasts derived from CIITA(-/-)hESCs were powerless in IFN-γ inducible expression of HLA II. CONCLUSION: We generated HLA II defected hESCs via deleting CIITA, a master regulator of constitutive and IFN-γ inducible expression of HLA II genes. CIITA(-/-)hESCs can differentiate into tissue cells with non-HLA II expression. It's promising that CIITA(-/-)hESCs-derived cells could be used in cell therapy (e.g., T cells and DCs) and escape the attack of receptors' CD4+ T cells, which are the main effector cells of cellular immunity in allograft.

A Gene and Neural Stem Cell Therapy Platform Based on Neuronal Cell Type-Inducible Gene Overexpression

PURPOSE: Spinal cord injury (SCI) is associated with permanent neurological damage, and treatment thereof with a single modality often does not provide sufficient therapeutic outcomes. Therefore, a strategy that combines two or more techniques might show better therapeutic effects. MATERIALS AND METHODS: In this study, we designed a combined treatment strategy based on neural stem cells (NSCs) introduced via a neuronal cell type-inducible transgene expression system (NSE::) controlled by a neuron-specific enolase (NSE) promoter to maximize therapeutic efficiency and neuronal differentiation. The luciferase gene was chosen to confirm whether this combined system was working properly prior to using a therapeutic gene. The luciferase expression levels of NSCs introduced via the neuronal cell type-inducible luciferase expression system (NSE::Luci) or via a general luciferase expressing system (SV::Luci) were measured and compared in vitro and in vivo. RESULTS: NSCs introduced via the neuronal cell type-inducible luciferase expressing system (NSE::Luci-NSCs) showed a high level of luciferase expression, compared to NSCs introduced via a general luciferase expressing system (SV::Luci-NSCs). Interestingly, the luciferase expression level of NSE::Luci-NSCs increased greatly after differentiation into neurons. CONCLUSION: We demonstrated that a neuronal cell type-inducible gene expression system is suitable for introducing NSCs in combined treatment strategies. We suggest that the proposed strategy may be a promising tool for the treatment of neurodegenerative disorders, including SCI.

Analyses of the TCR repertoire of MHC class II-restricted innate CD4+ T cells

Analysis of the T-cell receptor (TCR) repertoire of innate CD4+ T cells selected by major histocompatibility complex (MHC) class II-dependent thymocyte-thymocyte (T-T) interaction (T-T CD4+ T cells) is essential for predicting the characteristics of the antigens that bind to these T cells and for distinguishing T-T CD4+ T cells from other types of innate T cells. Using the TCRmini Tg mouse model, we show that the repertoire of TCRalpha chains in T-T CD4+ T cells was extremely diverse, in contrast to the repertoires previously described for other types of innate T cells. The TCRalpha chain sequences significantly overlapped between T-T CD4+ T cells and conventional CD4+ T cells in the thymus and spleen. However, the diversity of the TCRalpha repertoire of T-T CD4+ T cells seemed to be restricted compared with that of conventional CD4+ T cells. Interestingly, the frequency of the parental OT-II TCRalpha chains was significantly reduced in the process of T-T interaction. This diverse and shifted repertoire in T-T CD4+ T cells has biological relevance in terms of defense against diverse pathogens and a possible regulatory role during peripheral T-T interaction.

Delivery of the Sox9 gene promotes chondrogenic differentiation of human umbilical cord blood-derived mesenchymal stem cells in an in vitro model

SRY-related high-mobility-group box 9 (Sox9) gene is a cartilage-specific transcription factor that plays essential roles in chondrocyte differentiation and cartilage formation. The aim of this study was to investigate the feasibility of genetic delivery of Sox9 to enhance chondrogenic differentiation of human umbilical cord blood-derived mesenchymal stem cells (hUC-MSCs). After they were isolated from human umbilical cord blood within 24 h after delivery of neonates, hUC-MSCs were untreated or transfected with a human Sox9-expressing plasmid or an empty vector. The cells were assessed for morphology and chondrogenic differentiation. The isolated cells with a fibroblast-like morphology in monolayer culture were positive for the MSC markers CD44, CD105, CD73, and CD90, but negative for the differentiation markers CD34, CD45, CD19, CD14, or major histocompatibility complex class II. Sox9 overexpression induced accumulation of sulfated proteoglycans, without altering the cellular morphology. Immunocytochemistry demonstrated that genetic delivery of Sox9 markedly enhanced the expression of aggrecan and type II collagen in hUC-MSCs compared with empty vector-transfected counterparts. Reverse transcription-polymerase chain reaction analysis further confirmed the elevation of aggrecan and type II collagen at the mRNA level in Sox9-transfected cells. Taken together, short-term Sox9 overexpression facilitates chondrogenesis of hUC-MSCs and may thus have potential implications in cartilage tissue engineering.

Peptide: N- glycanase is expressed in prestalk cells and plays a role in the differentiation of prespore cells during development of Dictyostelium discoideum.

Peptide: N- glycanase (PNGase) enzyme is found throughout eukaryotes and plays an important role in the misfolded glycoprotein degradation pathway. This communication reports the expression patterns of the pngase transcript (as studied by the analysis of β- galactosidase reporter driven by the putative pngase promoter) and protein (as studied by the analysis of β- galactosidase reporter expressed under the putative pngase promoter as a fusion with the pngase ORF) during development and further elucidated the developmental defects of the cells lacking PNGase (png-). The results show that the DdPNGase is an essential protein expressed throughout development and β- galactosidase activity was present in the anterior part of the slug. In structures derived from a null mutant for pngase, the prestalk A and AO patterning was expanded and covered a large section of the prespore region of the slugs. When developed as chimeras with wild type, the png- cells preferentially populate the prestalk/stalk region. When the mutants were mixed in higher ratios, they also tend to form the prespore/spore cells. The results emphasize that the DdPNGase has an essential role during development and the mutants have defects in a system that changes the physiological dynamics in the prespore cells. DdPNGase play a role in development both during aggregation and in the differentiation of prespore cells.

Utilização de células-tronco mesenquimais em procedimentos visando o crescimento ósseo maxilomandibular û revisão de literatura / Use of mesenchymal stem cells in procedures for maxilo-mandibular bone growth û literature review

As células-tronco mesenquimais são uma população de células adultas obtidas através da medula óssea, sangue do cordão umbilical, e estão cada vez mais sendo utilizadas para tratamentos regenerativos e pesquisas de engenharia tecidual nas mais diversas áreas da medicina, isto devido ao fato de essas células serem multipotenciais, que conseguem se diferenciar em quase todos os tipos de tecidos humanos, com exceção da placenta e anexos embrionários. Quando coletadas são criopreservadas a temperaturas abaixo de 180°C para manterem o seu potencial de proliferação e diferenciação osteogênica in vitro. Elas possuem capacidade de se autoproliferar e se diferenciar ao longo de várias linhagens, incluindo osso, cartilagem, tecido adiposo, células musculares e neurais. O objetivo deste artigo é revisar a literatura sobre a capacidade que as células-tronco possuem em se diferenciar e proporcionar o crescimento ósseo através de seu potencial osteogênico (contendo células formadoras de osso), osteoindutor (contendo substâncias osteoindutoras) e osteocondutor (servem como uma base para formação de osso). Uma variedade de opiniões a respeito do tipo de materiais que deve ser utilizado para aplicações clínicas em defeitos ósseos torna ampla a discussão sobre resultados de traumas e ressecções de tumores ósseos. Para isso, os biomateriais fornecem uma excelente base para o crescimento ósseo quando combinado com um aspirado de concentrado de medula óssea. Apesar de estar comprovado cientificamente que as células estromais podem formar ossos, ainda não há estudos clínicos suficientes.

The mesenchymal stem cells are a population of mature cells obtained from bone marrow and umbilical cord blood. These cells are being widely used on regenerative treatment and tissue engineering research in several areas of medicine. That is occurring due to the fact that these cells are multipotent, being able to differentiate themselves into almost every type of human tissues, with the exception of the placenta and embryonic annexes. When collected these cells, are cryopreserved at temperatures below -180°C to preserve their potential for proliferation and osteogenic differentiation in vitro. They also have the ability to self-proliferate and differentiate along various cell lineages, including bone, cartilage, adipose tissue, muscle, and nerve cells. The aim of this study was to review the literature on the ability that mesenchymal cells have to differentiate themselves and provide bone growth through its osteogenic potential (containing bone-forming cells), osteoinductivity potential (containing osteoinductive substances) and osteoconductive (serve as a basis for bone formation). A variety of opinions about the type of materials that should be used for clinical application in bone defects amplifies the discussion around trauma and bone tumors resections.Biomaterials provide an excellent foundation for new bone growth when combined with an aspirated bone marrow concentrate. Although, it has been scientifically proven that stromal cells can form new bones, there are not enough clinical studies about it.

Mecanismos de ação da bradicinina na diferenciação neural in vitro / Mechanisms of bradykinin in neural differentiation

Durante o desenvolvimento do sistema nervoso, as células têm a tarefa de proliferar, migrar, diferenciar, morrer ou amadurecer de modo altamente preciso para formar estruturas complexas. Tal precisão é alcançada em decorrência da interação perfeita entre as células que se comunicam por meio de mensageiros químicos no ambiente extracelular. Nesse contexto, nosso grupo tem reportado o envolvimento da bradicinina (BK) em processos do desenvolvimento neural. Recentemente, observou-se que a BK desempenha um papel importante na determinação do destino neural, favorecendo a neurogênese em detrimento da gliogênese em diversos modelos de diferenciação, além de potencializar a migração celular observada no modelo de neuroesferas de rato (Trujillo et al, 2012). Essas descobertas motivaram, como objetivo geral dessa tese, a investigação dos mecanismos subjacentes à BK que determinam seus efeitos. Dessa forma, o principal modelo de diferenciação utilizado foi as células precursoras neurais (CPNs) isoladas do telencéfalo de embriões de camundongos. Estas células proliferam na presença dos fatores de crescimento (GFs) EGF + FGF2, mantendo-se multipotentes e formando as neuroesferas, ao passo que migram e diferenciam em neurônios e glias pela remoção desses GFs, com boa proximidade aos eventos do desenvolvimento do cortex in vivo. Como resultados do presente trabalho, observou-se, inicialmente, que a BK também influencia efetivamente na diferenciação neural no modelo de CPNs murinas. Ao término da diferenciação, observou-se que esta cinina favoreceu a migração e promoveu o enriquecimento neuronal, evidenciado pelo aumento da expressão das proteínas ß3-Tubulina e MAP2. Constatou-se também, que se observa uma baixa taxa de proliferação ao término da diferenciação na presença de BK (Trujillo et al, 2012), em consequência da grande proporção de neurônios em cultura estimulada por esta cinina. Esta relação causal foi evidenciada pelo ensaio de incorporação de EdU e concomitante imuno-detecção dos marcadores ß3-Tubulina, GFAP e Nestina. Fatores que promovem a neurogênese podem promovê-la suprimindo a proliferação celular em CPNs indiferenciadas, mais especificamente, alongando a fase G1 do ciclo celular que resulta na divisão de diferenciação. Assim, investigou-se também se a BK influencia nesse processo. Análises por citometria de fluxo demonstraram que esta cinina suprimiu a proliferação estimulada pelos GFs, levando ao acúmulo de células na fase G1 do ciclo celular. Esse acúmulo não provém do bloqueio do ciclo, uma vez que se observam grandes proporções de células nas fases subsequentes à G1, indicando que essa fase foi apenas prolongada pela BK e, assim, corroboraria no favorecimento da neurogênese. Outra face dos mecanismos adjacentes à BK para seus efeitos na diferenciação neural se refere às vias de sinalização disparadas por esta cinina. Observou-se que a BK induz a produção de AMPc por intermédio de proteínas G sensíveis à toxina pertussis (TP) (provavelmente através da subunidade ßγ de proteínas Gi) e promove a mobilização de cálcio dos estoques intracelulares, evidenciando o envolvimento da família de proteínas Gq. Esses resultados sugerem que o receptor B2 de cinina acopla-se tanto às proteínas Gi quanto às proteínas Gq em CPNs. A exposição dessas células à BK também ativou as vias da PI3K/Akt e da MAPK p38, mas não influenciou na ativação de STAT3 e JNK. Destaca-se o potencial da rota da MAPK ERK como uma das principais cascatas responsáveis por decodificar sinais de mensageiros externos em respostas celulares. O tratamento com BK em CPNs ativou a ERK por tempo prolongado e estimulou sua translocação para o núcleo. O efeito de BK na glio- e neurogênese de CPNs foi dependente da atividade de ERK, porque o bloqueio farmacológico dessa enzima impediu esse efeito de BK. Por outro lado, o favorecimento da migração induzido por esta cinina foi dependente da atividade da p38, enquanto, o seu efeito antiproliferativo foi condicionado à atividade das suas duas MAPKs, ERK e p38. Além disso, a via da PI3K/Akt ativada por BK não influenciou nos três eventos avaliados. Finalmente, utilizou-se nessa tese uma abordagem reducionista da diferenciação, porém amplamente utilizada por estudos mecanísticos de neurogênese, as células PC12. Assim, observou-se que a BK também ativa a ERK por tempo prolongado e com translocação nuclear, sendo que tal forma de ativação dessa quinase é proposta na literatura como necessária e suficiente para induzir a neurogênese dessas células. Demonstrou-se ainda que o bloqueio apenas da ativação sustentada de ERK, pela inibição das atividades das PKCs clássicas, impede o favorecimento da neurogênese por BK em células PC12. Juntos, esses resultados contribuem para elucidação dos mecanismos de ação da BK na regulação da diferenciação neural, colaborando para melhor entender esse processo e prevendo possíveis aplicações em terapias de reparo neuronal em pacientes com doenças, por exemplo, de Parkinson, Alzheimer, Esclerose Múltipla e lesões isquêmicas

During CNS development cells perform the task of proliferating, migrating, differentiating, dying or maturing in highly accurate patterns. Such accuracy is reached as a result of the perfect interaction among the cells that constantly communicate with each other through cell-cell contact or through chemical messengers present in the extracellular medium. In this context, our group has reported the involvement of bradykinin (BK) in neural differentiation of stem cell models (Trujillo et al, 2012). Recently, it has been observed that BK plays an important role in determining neural destination, favoring neurogenesis over gliogenesis in several models of differentiation, besides potentializing cell migration observed in the model of rat neurospheres. These discoveries have motivated, as the general objective of this thesis, the investigation of the mechanisms underlying BK-promoted effects on neural differentiation using neural precursor cells (NPCs) isolated from the telencephalon of mice embryos. These cells proliferate in the presence of growth factors (GFs) EGF + FGF2, remaining multipotent and forming neurospheres, while they migrate and differentiate in neurons and glias following removal of these GFs, resembling in simplified conditions events of the development of the cortex in vivo. As results of the present thesis, it was initially observed that BK also effectively influences neural differentiation fate of the mouse NPC model. This kinin favored migration and promoted neuronal enrichment, evidenced by increased expression of ß3-Tubulin and MAP2 marker proteins. Moreover, proliferation rates were largely decreased following differentiation in the presence of BK (Trujillo et al, 2012), due to the large proportion of neurons in the culture stimulated by this kinin. This causal relation was evidenced by the EdU incorporation assay and the concomitant immunodetection rates of ß3- Tubulin, GFAP and Nestin markers. Factors which promote neurogenesis can promote it by suppressing cell proliferation in undifferentiated NPCs, more specifically, prolonging the G1 phase of the cell cycle that result in the division of differentiation. Thus, it was further investigated whether BK influences this process. Flow cytometry analyses showed that this kinin suppressed the proliferation stimulated by GFs, resulting in the accumulation of cells in the G1 phase of the cell cycle. This accumulation is not caused by a cycle block, since wide proportions of cells are observed in phases subsequent to the G1, indicating that this phase was only prolonged by BK, thus corroborating for favoring neurogenesis. Another aspect of the mechanisms adjacent to BK for its effects on neural differentiation refers to the signaling pathways triggered by this kinin. Here, we show that the kinin B2 receptor couples to both Gi and Gq proteins in NPCs. BK induced the production of intracellular cAMP by activation of G proteins sensitive to pertussis toxin (PT) (probably through ßγ subunit of Gi proteins) and promoted the mobilization of calcium from intracellular stocks, demonstrating the involvement of YM-254890-sensitive Gq proteins. Exposure of these cells to BK also activated PI3K/Akt and MAPK p38 pathways, but did not affect the activation of STAT3 and JNK. It is important to note the potential MAPK-ERK route as one of the main cascades responsible for decoding signals from external messengers into cellular responses. NPC treatment with BK activated ERK for prolonged time and stimulated its translocation into the nucleus. The effect of BK on glio- and neurogenesis of NPCs depended plainly on ERK activity, because the pharmacological blockade of this enzyme prevented the BK-exerted effects. On the other hand, the favoring of migration induced by this kinin was dependent on p38 activity, while its antiproliferative effect was conditioned to the activity of both the MAPKs ERK and p38. In addition, the PI3K/Akt pathway activated by BK did not affect any of the three evaluated events. Finally, we used in this thesis a reductionist approach of differentiation based on the use of PC12 cells, which has been widely used for mechanistic studies of neurogenesis. Thus, it was observed that BK also activated ERK for prolonged time and with nuclear translocation, considering that such form of kinase activation is proposed in the literature as necessary and sufficient to induce neurogenesis in these cells. This study also demonstrated that blockade only of the sustained ERK activation, through the inhibition of the activity of classic PKCs, prevents the favoring of neurogenesis by BK in PC12 cells. Together, these results compose novel mechanisms of action of BK on events of neural development in vitro, contributing to the better understanding of this process and foreseeing possible applications in the future for neuronal repair strategies