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1.
Cryobiology ; 71(1): 151-60, 2015 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-25641609

RESUMO

BACKGROUND: The therapeutic use of human embryonic stem cells (hESCs) is dependent on an efficient cryopreservation protocol for long-term storage. The aim of this study was to determine whether the combination of three cryoprotecting reagents using two freezing systems might improve hESC recovery rates with maintenance of hESC pluripotency properties for potential cell therapy application. METHODS: Recovery rates of hESC colonies which were frozen in three cryoprotective solutions: Me2SO/HES/SR medium, Defined-medium® and Me2SO/SFB in medium solution were evaluated in ultra-slow programmable freezing system (USPF) and a slow-rate freezing system (SRF). The hESC pluripotency properties after freezing-thawing were evaluated. RESULTS: We estimated the distribution frequency of survival colonies and observed that independent of the freezing system used (USPF or SRF) the best results were obtained with Me2SO/HES/SR as cryopreservation medium. We showed a significant hESC recovery colonies rate after thawing in Me2SO/HES/SR medium were 3.88 and 2.9 in USPF and SRF, respectively. The recovery colonies rate with Defined-medium® were 1.05 and 1.07 however in classical Me2SO medium were 0.5 and 0.86 in USPF and SRF, respectively. We showed significant difference between Me2SO/HES/SR medium×Defined-medium® and between Me2SO/HES/SR medium×Me2SO medium, for two cryopreservation systems (P<0.05). CONCLUSION: We developed an in house protocol using the combination of Me2SO/HES/SR medium and ultra-slow programmable freezing system which resulted in hESC colonies that remain undifferentiated, maintain their in vitro and in vivo pluripotency properties and genetic stability. This approach may be suitable for cell therapy studies.


Assuntos
Criopreservação/métodos , Dimetil Sulfóxido/farmacologia , Células-Tronco Embrionárias Humanas/efeitos dos fármacos , Células-Tronco Embrionárias Humanas/fisiologia , Derivados de Hidroxietil Amido/farmacologia , Substitutos do Plasma/farmacologia , Sobrevivência Celular/efeitos dos fármacos , Terapia Baseada em Transplante de Células e Tecidos/métodos , Crioprotetores/farmacologia , Meios de Cultura Livres de Soro/farmacologia , Congelamento , Células-Tronco Embrionárias Humanas/citologia , Humanos , Células-Tronco Pluripotentes/citologia
2.
BMC Genomics ; 11 Suppl 5: S2, 2010 Dec 22.
Artigo em Inglês | MEDLINE | ID: mdl-21210968

RESUMO

BACKGROUND: The thymus is a central lymphoid organ, in which bone marrow-derived T cell precursors undergo a complex process of maturation. Developing thymocytes interact with thymic microenvironment in a defined spatial order. A component of thymic microenvironment, the thymic epithelial cells, is crucial for the maturation of T-lymphocytes through cell-cell contact, cell matrix interactions and secretory of cytokines/chemokines. There is evidence that extracellular matrix molecules play a fundamental role in guiding differentiating thymocytes in both cortical and medullary regions of the thymic lobules. The interaction between the integrin α5ß1 (CD49e/CD29; VLA-5) and fibronectin is relevant for thymocyte adhesion and migration within the thymic tissue. Our previous results have shown that adhesion of thymocytes to cultured TEC line is enhanced in the presence of fibronectin, and can be blocked with anti-VLA-5 antibody. RESULTS: Herein, we studied the role of CD49e expressed by the human thymic epithelium. For this purpose we knocked down the CD49e by means of RNA interference. This procedure resulted in the modulation of more than 100 genes, some of them coding for other proteins also involved in adhesion of thymocytes; others related to signaling pathways triggered after integrin activation, or even involved in the control of F-actin stress fiber formation. Functionally, we demonstrated that disruption of VLA-5 in human TEC by CD49e-siRNA-induced gene knockdown decreased the ability of TEC to promote thymocyte adhesion. Such a decrease comprised all CD4/CD8-defined thymocyte subsets. CONCLUSION: Conceptually, our findings unravel the complexity of gene regulation, as regards key genes involved in the heterocellular cell adhesion between developing thymocytes and the major component of the thymic microenvironment, an interaction that is a mandatory event for proper intrathymic T cell differentiation.


Assuntos
Adesão Celular/fisiologia , Células Epiteliais/metabolismo , Regulação da Expressão Gênica/genética , Integrina alfa5/genética , Linfócitos T/fisiologia , Timo/citologia , Adesão Celular/genética , Diferenciação Celular/fisiologia , Movimento Celular/fisiologia , Fibronectinas/metabolismo , Técnicas de Silenciamento de Genes , Humanos , Integrina alfa5/metabolismo , Interferência de RNA
3.
J Immunol Res ; 2014: 159247, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-24829926

RESUMO

Qa-2 and Qa-1 are murine nonclassical MHC class I molecules involved in the modulation of immune responses by interacting with T CD8(+) and NK cell inhibitory receptors. During thymic education, the Aire gene imposes the expression of thousands of tissue-related antigens in the thymic medulla, permitting the negative selection events. Aiming to characterize the transcriptional profiles of nonclassical MHC class I genes in spatial-temporal association with the Aire expression, we evaluated the gene expression of H2-Q7(Qa-2), H2-T23(Qa-1), H2-Q10(Qa-10), and Aire during fetal and postnatal development of thymus and other tissues. In the thymus, H2-Q7(Qa-2) transcripts were detected at high levels throughout development and were positively correlated with Aire expression during fetal ages. H2-Q7(Qa-2) and H2-T23(Qa-1) showed distinct expression patterns with gradual increasing levels according to age in most tissues analyzed. H2-Q10(Qa-10) was preferentially expressed by the liver. The Aire transcriptional profile showed increased levels during the fetal period and was detectable in postnatal ages in the thymus. Overall, nonclassical MHC class I genes started to be expressed early during the ontogeny. Their levels varied according to age, tissue, and mouse strain analyzed. This differential expression may contribute to the distinct patterns of mouse susceptibility/resistance to infectious and noninfectious disorders.


Assuntos
Regulação da Expressão Gênica , Antígenos de Histocompatibilidade Classe I/genética , Timo/metabolismo , Fatores de Transcrição/genética , Transcriptoma , Animais , Perfilação da Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento , Antígenos H-2/genética , Antígenos H-2/imunologia , Antígenos de Histocompatibilidade Classe I/imunologia , Tecido Linfoide/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Especificidade de Órgãos/genética , Timo/imunologia , Proteína AIRE
4.
Virology ; 449: 190-9, 2014 Jan 20.
Artigo em Inglês | MEDLINE | ID: mdl-24418552

RESUMO

The typical characteristics of mesenchymal stem cells (MSCs) can be affected by inflammatory microenvironment; however, the exact contribution of HTLV-1 to MSC dysfunction remains to be elucidated. In this study, we demonstrated that MSC cell surface molecules VCAM-1 and ICAM-1 are upregulated by contact with HTLV-1, and HLA-DR was most highly expressed in MSCs co-cultured with MT2 cells. The expression levels of VCAM-1 and HLA-DR were increased in MSCs cultured in the presence of PBMCs isolated from HTLV-1-infected symptomatic individuals compared with those cultured with cells from asymptomatic infected individuals or healthy subjects. HTLV-1 does not impair the MSC differentiation process into osteocytes and adipocytes. In addition, MSCs were efficiently infected with HTLV-1 in vitro through direct contact with HTLV-1-infected cells; however, cell-free virus particles were not capable of causing infection. In summary, HTLV-1 can alter MSC function, and this mechanism may contribute to the pathogenesis of this viral infection.


Assuntos
Infecções por HTLV-I/virologia , Vírus Linfotrópico T Tipo 1 Humano/fisiologia , Células-Tronco Mesenquimais/virologia , Diferenciação Celular , Células Cultivadas , Infecções por HTLV-I/genética , Infecções por HTLV-I/imunologia , Infecções por HTLV-I/fisiopatologia , Humanos , Molécula 1 de Adesão Intercelular/genética , Molécula 1 de Adesão Intercelular/imunologia , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/imunologia , Fenótipo , Molécula 1 de Adesão de Célula Vascular/genética , Molécula 1 de Adesão de Célula Vascular/imunologia
5.
J Virol Methods ; 173(1): 92-8, 2011 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-21277903

RESUMO

Since the discovery of RNAi technology, several functional genomic and disease therapy studies have been conducted using this technique in the field of oncology and virology. RNAi-based antiviral therapies are being studied for the treatment of retroviruses such as HIV-1. These studies include the silencing of regulatory, infectivity and structural genes. The HTLV-1 structural genes are responsible for the synthesis of proteins involved in the entry, assembly and release of particles during viral infection. To examine the possibility of silencing HTLV-1 genes gag and env by RNA interference technology, these genes were cloned into reporter plasmids. These vectors expressed the target mRNAs fused to EGFP reporter genes. Three small interference RNAs (siRNAs) corresponding to gag and three corresponding to env were designed to analyze the effect of silencing by RNAi technology. The plasmids and siRNAs were co-transfected into HEK 293 cells. The results demonstrated that the expression of the HTLV-1 gag and env genes decreased significantly in vitro. Thus, siRNAs can be used to inhibit HTLV-1 structural genes in transformed cells, which could provide a tool for clarifying the roles of HTLV-1 structural genes, as well as a therapy for this infection.


Assuntos
Produtos do Gene env/antagonistas & inibidores , Produtos do Gene gag/antagonistas & inibidores , Inativação Gênica , Vírus Linfotrópico T Tipo 1 Humano/genética , RNA Interferente Pequeno/genética , Fusão Gênica Artificial , Produtos do Gene env/genética , Produtos do Gene gag/genética , Genes Reporter , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Células HEK293 , Humanos
6.
Stem Cells Dev ; 20(1): 169-80, 2011 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-20504151

RESUMO

Reprogramming of somatic cells to pluripotency promises to boost cellular therapy. Most instances of direct reprogramming have been achieved by forced expression of defined exogenous factors using multiple viral vectors. The most used 4 transcription factors, octamer-binding transcription factor 4 (OCT4), (sex determining region Y)-box 2 (SOX2), Kruppel-like factor 4 (KLF4), and v-myc myelocytomatosis viral oncogene homolog (C-MYC), can induce pluripotency in mouse and human fibroblasts. Here, we report that forced expression of a new combination of transcription factors (T-cell leukemia/lymphoma protein 1A [TCL-1A], C-MYC, and SOX2) is sufficient to promote the reprogramming of human fibroblasts into pluripotent cells. These 3-factor pluripotent cells are similar to human embryonic stem cells in morphology, in the ability to differentiate into cells of the 3 embryonic layers, and at the level of global gene expression. Induced pluripotent human cells generated by a combination of other factors will be of great help for the understanding of reprogramming pathways. This, in turn, will allow us to better control cell-fate and apply this knowledge to cell therapy.


Assuntos
Reprogramação Celular/genética , Fibroblastos/metabolismo , Técnicas de Transferência de Genes , Lentivirus/genética , Proteínas Proto-Oncogênicas c-myc/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Fatores de Transcrição SOXB1/metabolismo , Diferenciação Celular/genética , DNA/genética , Derme/citologia , Corpos Embrioides/citologia , Corpos Embrioides/metabolismo , Fibroblastos/citologia , Imunofluorescência , Perfilação da Expressão Gênica , Genoma Humano/genética , Humanos , Cinética , Fator 4 Semelhante a Kruppel , Modelos Biológicos , Células-Tronco Pluripotentes/citologia , Células-Tronco Pluripotentes/metabolismo
7.
Ann N Y Acad Sci ; 1150: 282-9, 2008 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-19120314

RESUMO

The MHC region (6p21) aggregates the major genes that contribute to susceptibility to type 1 diabetes (T1D). Three additional relevant susceptibility regions mapped on chromosomes 1p13 (PTPN22), 2q33 (CTLA-4), and 11p15 (insulin) have also been described by linkage studies. To evaluate the contribution of these susceptibility regions and the chromosomes that house these regions, we performed a large-scale differential gene expression on lymphomononuclear cells of recently diagnosed T1D patients, pinpointing relevant modulated genes clustered in these regions and their respective chromosomes. A total of 4608 cDNAs from the IMAGE library were spotted onto glass slides using robotic technology. Statistical analysis was carried out using the SAM program, and data regarding gene location and biological function were obtained at the SOURCE, NCBI, and FATIGO programs. Three induced genes were observed spanning around the MHC region (6p21-6p23), and seven modulated genes (5 repressed and 2 repressed) were seen spanning around the 6q21-24 region. Additional modulated genes were observed in and around the 1p13, 2q33, and 11p15 regions. Overall, modulated genes in these regions were primarily associated with cellular metabolism, transcription factors and signaling transduction. The differential gene expression characterization may identify new genes potentially involved with diabetes pathogenesis.


Assuntos
Diabetes Mellitus Tipo 1/genética , Perfilação da Expressão Gênica , Predisposição Genética para Doença/genética , Análise de Sequência com Séries de Oligonucleotídeos , Adolescente , Estudos de Casos e Controles , Criança , Pré-Escolar , Mapeamento Cromossômico , Cromossomos Humanos Par 11 , Cromossomos Humanos Par 6 , Análise por Conglomerados , Feminino , Frequência do Gene , Genes MHC da Classe II , Humanos , Masculino
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