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1.
São Paulo; s.n; s.n; 2022. 116 p. tab, graf.
Thesis in English | LILACS | ID: biblio-1378343

ABSTRACT

Stem cells are undifferentiated cells that can be distinguished from others by their ability to self-renew and to differentiate into new specific cell types. Mesenchymal stem cells (MSC) are adult stem cells that can be obtained from different sources, such as adipose tissue, bone marrow, dental pulp, and umbilical cord. They can either replicate, originating new identical cells, or differentiate into cells of mesodermal origin and from other germ layers. MSC have been studied as new tools for regenerative therapy. Although encouraging results have been demonstrated, MSC-based therapies still face a great barrier: the difficulty of isolating these cells from heterogeneous environments. MSC are currently characterized by immunolabelling through a set of multiple surface membrane markers, including CD29, CD73, CD90 and CD105, which are also expressed by other cell types. Hence, the present work aimed to identify new specific biomarkers for the characterization of human MSC using DNA aptamers produced by the SELEX (Systematic Evolution of Ligands by EXponential Enrichment) technique. Our results showed that MSC from different origins bound to DNA candidate aptamers, that is, DNA or RNA oligonucleotides selected from random libraries that bind specifically to biological targets. Aptamer-bound MSC could be isolated by fluorescenceactivated cell sorting (FACS) procedures, enhancing the induction of differentiation into specific phenotypes (chondrocytes, osteocytes and adipocytes) when compared to the whole MSC population. Flow cytometry analyses revealed that candidate aptamers bound to 50% of the MSC population from dental pulp and did not present significant binding rates to human fibroblasts or lymphocytes, both used as negative control. Moreover, immunofluorescence images and confocal analyses revealed staining of MSC by aptamers localized in the surfacemembrane of these cells. The results also showed internal staining of human monocytes by our investigated aptamers. A non-specific control aptamer (CNTR APT) obtained from the random pool was then utilized to compare the specificity of the aptamers bound to the analyzed non-apoptotic cells, showing no staining for MSC. However, 40% of the monocytes bound to the CNTR APT. Normalized data based on the cells bound to candidate aptamers compared to those bound to the CNTR APT, revealed a 10 to 16-fold higher binding rate for MSC against 2-fold for monocytes. Despite its low specificity, monocyte-aptamer binding occurs probably due to the expression of shared markers with MSC, since monocytes are derived from hematopoietic stem cells and are important for the immune system ability to internalize/phagocyte external molecules. Given that, we performed a pull-down assay followed by mass spectrometry analysis to detect which MSC-specific protein or other target epitope not coexpressed by monocytes or the CNTR APT would bind to the candidate aptamer. Distinguishing between MSC and monocyte epitopes is important, as both cells are involved in immunomodulatory effects after MSC transplantations. ADAM17 was found to be a target of the APT10, emerging as a possible biomarker of MSC, since its involvement in the inhibition of the TGF signaling cascade, which is responsible for the differentiation of MSC. Thus, MSC with a higher stemness profile should overexpress the protein ADAM17, which presents a catalytic site with affinity to APT10. Another target of Apt 10 is VAMP3, belonging to a transmembrane protein complex that is involved in endocytosis and exocytosis processes during immune and inflammatory responses. Overall, proteins identified as targets of APT10 may be cell surface MSC biomarkers, with importance for MSC-based cell and immune therapies


Células tronco são células indiferenciadas que podem ser distinguidas de outros tipos celulares por meio da habilidade de se auto renovarem e de se diferenciarem em novos tipos celulares. Células tronco mesenquimais (MSC) são células tronco adultas encontradas em diferentes tecidos como tecido adiposo, polpa de dente e cordão umbilical. Estas células podem se autodividir em células idênticas ou se diferenciarem em células de origem mesodermal. Estas células têm sido estudadas em novas aplicações que envolvem terapia regenerativas. Embora resultados encorajadores tenham sido demonstrados, terapias que utilizam MSC ainda encontram uma grande barreira: a dificuldade no isolamento destas células a partir de um ambiente heterogêneo. MSC são caracterizadas por populações positivas em ensaios de imunomarcação para os epítopos membranares CD29, CD73, CD90 e CD105, presentes também em outros tipos celulares. Assim, o presente trabalho tem o objetivo de identificar novos biomarcadores de MSC de origem humana, utilizando aptâmeros de DNA produzidos pela técnica SELEX (Systematic Evolution of Ligands by EXponential Enrichment) como ferramenta. Nossos resultados mostraram que MSC de diferentes origens ligam-se a aptâmeros (oligonucleotídeos de DNA ou RNA que atuam como ligantes específicos de alvos moleculares) de DNA candidatos que atuam no isolamento de MSC por meio da técnica FACS de separação celular, promovendo uma maior indução de diferenciação em células específicas (condrócitos, osteócitos e adipócitos) comparada com a população total de MSC. Análises de citometria de fluxo mostraram que os aptâmeros candidatos se ligam a 50% das MSC de polpa de dente e não apresentam taxa de ligação significante para fibroblastos e linfócitos de origem humana - utilizados como controles negativo. Além domais, imagens de imunofluorescência e confocal mostraram ligação na superfície da membrana de MSC e a marcação interna de monócitos a estes aptâmeros. Portanto, um aptâmero controle (CNTR APT) foi utilizado para comparar a especificidade dos aptâmeros ligados a células viáveis, mostrando a não ligação deste aptâmero a MSC. Porém, 40% da população de monócitos ligou-se ao CNTR APT. Uma normalização baseada na comparação entre as taxas de ligação entre células ligadas com aptâmeros candidatos e o aptâmero controle gerou uma taxa de especificidade entre 10-16 vezes maior para MSC contra 2,5 vezes para os monócitos. Deste modo, embora os resultados tenham mostrado uma taxa de ligação entre monócitos e aptâmeros, as MSC ligadas aos aptâmeros candidatos possuem uma maior taxa de especificidade devido a uma maior presença de antígenos que são expressos em ambas as células. Um ensaio de Pull Down seguido de espectrometria de massas foi utilizado para a identificação de biomarcadores que se ligariam aos aptâmeros candidatos, e que não seriam co-expressos por monócitos e por antígenos ligados ao aptâmero controle. Deste modo, a proteína ADAM17 foi identificada nas amostras de APT10 ligadas às MSC. Tal proteína está relacionada à inibição de uma cascata de sinalização da família de proteínas TGF, responsável pela diferenciação de MSC. Assim, MSC com maior potencial tronco deveriam expressar ADAM17 em maior quantidade. Tal proteína apresenta um sítio catalítico que demonstra interagir com o APT10, de acordo com predição Docking entre proteína e DNA. Foi identificada também, a proteína VAMP3, que pertence a um complexo proteico transmembranar responsável pelos processos de endocitose e exocitose, e que podem ter um papel importante na liberação de citocinas e outras moléculas relacionadas às respostas imune e inflamatórias. Deste modo, o APT10 identificou proteínas importantes que devem estar relacionas com a melhora de imunoterapias que utilizam MSC


Subject(s)
Stem Cells , Biomarkers/analysis , SELEX Aptamer Technique/instrumentation , Mesenchymal Stem Cells/classification , ADAM17 Protein/pharmacology , Patient Isolation , Mass Spectrometry/methods , Staining and Labeling/methods , Transplantation/adverse effects , Umbilical Cord , DNA/agonists , Transforming Growth Factors/agonists , Cell Separation/instrumentation , Cytokines/adverse effects , Adipocytes/metabolism , Chondrocytes/classification , Scientists for Health and Research for Development , Adult Stem Cells/classification , Fibroblasts/chemistry , Flow Cytometry/instrumentation , Germ Layers , Antigens/adverse effects
2.
Sci Rep ; 11(1): 3063, 2021 02 04.
Article in English | MEDLINE | ID: mdl-33542256

ABSTRACT

Articular chondral lesions, caused either by trauma or chronic cartilage diseases such as osteoarthritis, present very low ability to self-regenerate. Thus, their current management is basically symptomatic, progressing very often to invasive procedures or even arthroplasties. The use of amniotic fluid stem cells (AFSCs), due to their multipotentiality and plasticity, associated with scaffolds, is a promising alternative for the reconstruction of articular cartilage. Therefore, this study aimed to investigate the chondrogenic potential of AFSCs in a micromass system (high-density cell culture) under insulin-like growth factor 1 (IGF-1) stimuli, as well as to look at their potential to differentiate directly when cultured in a porous chitosan-xanthan (CX) scaffold. The experiments were performed with a CD117 positive cell population, with expression of markers (CD117, SSEA-4, Oct-4 and NANOG), selected from AFSCs, after immunomagnetic separation. The cells were cultured in both a micromass system and directly in the scaffold, in the presence of IGF-1. Differentiation to chondrocytes was confirmed by histology and by using immunohistochemistry. The construct cell-scaffold was also analyzed by scanning electron microscopy (SEM). The results demonstrated the chondrogenic potential of AFSCs cultivated directly in CX scaffolds and also in the micromass system. Such findings support and stimulate future studies using these constructs in osteoarthritic animal models.


Subject(s)
Adult Stem Cells/cytology , Cartilage, Articular/drug effects , Chondrogenesis/genetics , Osteoarthritis/genetics , Tissue Scaffolds/chemistry , Adult Stem Cells/transplantation , Amniotic Fluid/cytology , Cartilage, Articular/growth & development , Cartilage, Articular/ultrastructure , Cell Culture Techniques , Cell Differentiation/drug effects , Chitosan/pharmacology , Gene Expression Regulation, Developmental/drug effects , Humans , Insulin-Like Growth Factor I/pharmacology , Microscopy, Electron, Scanning , Osteoarthritis/pathology , Osteoarthritis/therapy , Polysaccharides, Bacterial/pharmacology , Proto-Oncogene Proteins c-kit/genetics , Tissue Engineering/methods
3.
Odontoestomatol ; 23(38): e207, 2021. graf
Article in Spanish | LILACS, BNUY-Odon, BNUY | ID: biblio-1340273

ABSTRACT

Resumen Objetivos: Establecer e implementar un protocolo simplificado de extracción, aislamiento primario y cultivo de células madre derivadas de la pulpa dental humana (DPSCh). Analizar cuantitativamente y cualitativamente las células aisladas. Metodología: 10 terceros molares sanos donados por pacientes que concurrieron a la Facultad de Odontología, UdelaR y otorgaron su consentimiento escrito fueron procesados antes de las 48 hs. Se realizó la fractura de la pieza para la obtención del tejido pulpar y se procesó por el método explante. Se analizó viabilidad celular y expresión de marcadores por citometría de flujo en pasajes 4 y 12 y se corroboró mediante inmunocitoquímica. Resultados: Las células obtenidas presentaron una vitalidad mayor al 90% en todos los pasajes, observándose una morfología característica y expresión de marcadores de células madre mesenquimales CD90, C105, CD73, CD29 y 166 mediante citometría de flujo en ambos pasajes. Conclusiones: Se logró establecer un protocolo de aislamiento y expansión celular, con alta tasa de éxito de una población de DPSCh.


Resumo Objetivos: Estabelecer e implementar um protocolo simplificado para a extração, isolamento primário e cultura de células-tronco da polpa dentária humana (DPSCh). Analise as células isoladas quantitativa e qualitativamente. Metodologia: 10 terceiros molares saudáveis ​​doados por pacientes que frequentaram a Faculdade de Odontologia UdelaR e deram consentimento por escrito foram processados ​​antes de 48 horas. A fratura da peça foi realizada para obtenção do tecido pulpar e processada pelo método do explante. A viabilidade celular e a expressão do marcador foram analisadas por citometría de fluxo nas passagens 4 e 12 e confirmadas por inmunocitoquímica. Resultados: As células obtidas apresentaram viabilidade superior a 90% em todas as passagens, observando uma morfologia característica e expressão dos marcadores de células-tronco mesenquimais CD90, C105, CD73, CD29 e 166 por citometría de fluxo em ambas as passagens. Conclusões: Foi possível estabelecer um protocolo de isolamento celular, com alta taxa de sucesso e segurança para isolar o DPSCh.


Abstract Objectives: To establish and implement a simplified protocol for the extraction, primary isolation, and culture of human dental pulp stem cells (hDPSCs). To analyze the isolated cells quantitatively and qualitatively. Methodology: Ten healthy third molars were donated by patients who attended the School of Dentistry, UdelaR, and gave their written consent. The teeth were processed within 48 hours. The teeth were sectioned to obtain the pulp tissue and processed with the explant method. Cell viability and marker expression were analyzed by flow cytometry at passages 4 and 12 and verified by immunocytochemistry. Results: The cells obtained had a vitality greater than 90% in all passages. We found the characteristic morphology and the expression of CD90, C105, CD73, CD29 and 166 mesenchymal stem cell markers by flow cytometry in both passages. Conclusion: It was possible to establish a cell isolation protocol that is highly successful and safe to isolate hDPSC.


Subject(s)
Humans , Male , Female , Adolescent , Adult , Young Adult , Cell Separation , Cell Culture Techniques/methods , Dental Pulp/cytology , Cell Proliferation , Adult Stem Cells , Cell Survival , Mesenchymal Stem Cells , Flow Cytometry , Molar/cytology
4.
Rev. Fac. Odontol. (B.Aires) ; 36(83): 67-74, 2021. ilus
Article in Spanish | LILACS | ID: biblio-1343747

ABSTRACT

El presente trabajo de investigación tiene como objetivo principal el aislar, expandir y caracterizar inmunofenotípicamente células madre mesenquimales de la pulpa dental humana, según los criterios mínimos propuestos por The International Society for Cellular Therapy (ISCT), como así también establecer la puesta a punto de las técnicas y protocolos de procedimientos para tal fin. Los cultivos fueron permanentemente monitoreados mediante microscopio invertido con contraste de fase y la inmunotipificación fue realizada por citometría de flujo (AU)


Subject(s)
Humans , Male , Female , Tissue Engineering , Dental Pulp , Adult Stem Cells , Mesenchymal Stem Cells , Phenotype , Argentina , Schools, Dental , Cell Culture Techniques , Regenerative Medicine
5.
Braz. dent. sci ; 24(3): 1-8, 2021. tab
Article in English | BBO - Dentistry , LILACS | ID: biblio-1281295

ABSTRACT

Objective: To evaluate the level of awareness and attitude among dental practitioners regarding the use of stem cells in dentistry and to determine their knowledge of ethical concerns related to the recent therapy. Material and Methods: A cross-sectional survey-based study was conducted at Taibah University Dental College and Hospital. Medina and at governmental and private dental clinics at the western region of Saudi Arabia. Responses of dental practitioners who completed the survey were recorded between March 2019 and July 2019 without containing any personal identifiers. Level of awareness and attitude and knowledge about ethical issues in relation to stem cell therapy was established. Results: A total of 214 male and female dental practitioners participated in this study and the majority were registered at the Saudi Commission for Health Specialists 128 (59.8%). Dental consultants reported the highest percentage of awareness about dental stem cells (96%, p= 0.005), whereas general dental practitioners (56.2%, p= 0.005) and specialists (52%, p= 0.005), respectively had a lower percentage. When ethical concerns were determined, dental consultants had the highest percentage of knowledge (56%, p= 0.005), whereas dental practitioners (71.2%, p= 0.005) with < 5 years of experience (69.1, p= 0.002) lacked information about related ethical issues. Conclusion: Ways to increase stem cell awareness among dental practitioners in this study recommended including stem cell topics in the dental curriculum and organizing frequent seminars and conferences on this subject. (AU)


Objetivo: Avaliar o nível de consciência e a atitude dos dentistas em relação ao uso de células-tronco na odontologia, e determinar o conhecimento desses profissionais sobre as questões éticas relacionadas à terapia recente. Material e Métodos: Um estudo transversal baseado em pesquisa foi conduzido na Faculdade e Hospital de Odontologia da Universidade Taibah de Medina e em clínicas odontológicas governamentais e privadas na região oeste da Arábia Saudita. As respostas dos dentistas que responderam à pesquisa foram registradas entre março de 2019 e julho de 2019, sem conter nenhum identificador pessoal. Foi estabelecido o nível de consciência, atitude e conhecimento sobre questões éticas em relação à terapia com células-tronco. Resultados: Um total de 214 dentistas do sexo masculino e feminino participaram deste estudo, sendo 128 (59,8%) desses cadastrada na Comissão Saudita de Especialistas em Saúde. Os consultores em odontologia relataram o maior percentual de conhecimento sobre as células-tronco dentárias (96%, p = 0,005), enquanto os dentistas gerais (56,2%, p = 0,005) e especialistas (52%, p = 0,005) tiveram um percentual menor. Quando as questões éticas foram determinadas, os consultores em odontologia tiveram o maior percentual de conhecimento (56%, p = 0,005), enquanto os dentistas (71,2%, p = 0,005) com menos de 5 anos de experiência (69,1%, p = 0,002) tinham menos informações sobre questões éticas relacionadas. Conclusão: Formas de aumentar a conscientização sobre as células-tronco entre os dentistas deste estudo incluem tópicos sobre células-tronco no currículo de odontologia, além de frequentemente organizar seminários e conferências sobre o assunto. (AU)


Subject(s)
Humans , Male , Female , Knowledge , Dentists , Ethics , Adult Stem Cells
6.
Rev. odontopediatr. latinoam ; 11(1): 419145, 2021. ilus
Article in Spanish | LILACS, COLNAL | ID: biblio-1417006

ABSTRACT

Las células madre mesenquimales (MSCs) son células madre adultas que tienen la capacidad de diferenciarse en varios tipos de células. Una rica fuente de células madre mesenquimales puede ser obtenida de tejidos dentales como la papila apical. El objetivo de este trabajo fue extraer las células madre de la papila apical de terceros molares humano para mirar su viabilidad de usarse en la práctica clínica. La de separación celular con Stro-1 y estudio de cinéticas fueron realizados. Como resultado, esas células, presentaron alta tasa de proliferación, formación de colonias y fácil acceso. Concluimos que el uso de células madre obtenidas de diente puede ser una buena alternativa por ser de fácil acceso, alta viabilidad celular y expresión positiva para marcadores celulares mesenquimales. La regeneración tisular o la formación de estructuras craneofaciales constituyen el futuro de la medicina regenerativa, ofreciendo una posibilidad para el tratamiento de malformaciones congénitas, traumas y otras enfermedades.


As células tronco mesenquimais (CTMs) são células-tronco adultas que têm a capacidade de diferenciar em vários tipos de células. Uma rica fonte de células tronco mesenquimais pode ser obtida de tecidos dentais como a papila apical. O objetivo deste trabalho foi extrair as células tronco da papila apical de terceiros molares humano para ver sua viabilidade de ser usado na prática clinica. Teste separação celular com Stro-1 e estudo de cinéticas foram realizados. Como resultado essas células apresentaram alta taxa de proliferação, formação de colônias e de fácil acesso. Concluímos que o uso de células tronco obtidas de dente pode ser uma boa alternativa por ser fácil acesso, alta viabilidade celular e expressão positiva para marcadores celulares mesenquimais. A regeneração tecidual ou a formação de novo de estruturas craniofaciais é o futuro da medicina regenerativa, oferecendo uma possibilidade para tratamento de malformações congênitas, traumas e outras doenças.


Mesenchymal stem cells (MSCs) are adult stem cells that have the ability to diffe-rentiate into various cell types. A rich source of mesenchymal stem cells can be obtained from dental tissues such as the apical papilla. The objective of this work was to extract stem cells from the apical papilla of third human molars to see its feasibility to be used in clinical practice. Testing of cell separation with Stro-1 and study of kinetics were performed. As a result, these cells had a high rate of pro-liferation, formation of colonies and easy access. We conclude that the use of stem cells obtained from teeth can be a good alternative because they are easy to ac-cess, high cell viability and positive ex-pression for mesenchymal cell markers. Tissue regeneration or new formation of craniofacial structures is the future of re-generative medicine, offering a possibili-ty for treatment of congenital malforma-tions, traumas and other diseases.


Subject(s)
Humans , Female , Adult , Regeneration , Molar , Preceptorship , Congenital Abnormalities , Cell Separation , Regenerative Medicine , Adult Stem Cells , Mesenchymal Stem Cells
7.
Biosci Rep ; 40(7)2020 07 31.
Article in English | MEDLINE | ID: mdl-32667622

ABSTRACT

BACKGROUND: Skin wounds continue to be a global health problem. Several cellular therapy protocols have been used to improve and accelerate skin wound healing. Here, we evaluated the effect of transplantation of mesenchymal stromal cells (MSC) on the wound re-epithelialization process and its possible relationship with the presence of epithelial progenitor cells (EPC) and the expression of growth factors. METHODS: An experimental wound model was developed in C57BL/6 mice. Human MSCs seeded on collagen membranes (CM) were implanted on wounds. As controls, animals with wounds without treatment or treated with CM were established. Histological and immunohistochemical (IH) studies were performed at day 3 post-treatment to detect early skin wound changes associated with the presence of EPC expressing Lgr6 and CD34 markers and the expression of keratinocyte growth factor (KGF) and basic fibroblast growth factor (bFGF). RESULTS: MSC transplantation enhanced skin wound re-epithelialization, as compared with controls. It was associated with an increase in Lgr6+ and CD34+ cells and the expression of KGF and bFGF in the wound bed. CONCLUSION: Our results show that cutaneous wound healing induced by MSC is associated with an increase in EPC and growth factors. These preclinical results support the possible clinical use of MSC to treat cutaneous wounds.


Subject(s)
Mesenchymal Stem Cell Transplantation , Re-Epithelialization/physiology , Skin/injuries , Adult Stem Cells/metabolism , Animals , Antigens, CD34/metabolism , Disease Models, Animal , Epithelial Cells/metabolism , Fibroblast Growth Factor 2/metabolism , Fibroblast Growth Factor 7/metabolism , Healthy Volunteers , Humans , Male , Mice , Primary Cell Culture , Receptors, G-Protein-Coupled/metabolism , Skin/cytology , Skin/metabolism
8.
Anim. Reprod. ; 17(3): e20200044, 2020. ilus
Article in English | VETINDEX | ID: vti-26831

ABSTRACT

The creation of a genetic resource bank of avian species aims to prevent the decline and fragmentation of wild bird populations, which in turn lead to the loss of genetic diversity and, in more serious cases, the extinction of the most threatened species. In order for the collected genetic material to be stored in a bank and useful when necessary, it is essential to improve the technique ensuring its effectiveness. Thus, our study used feather follicle cells from the domestic gallus species to standardize the technique of cell culture and subsequent cryopreservation. This study aimed to establish a protocol, in vitro, of isolation and primary culture of somatic cells derived from the feather follicle, with the purpose of establishing a cell lineage, and evaluate its viability for the biobank formation. Developing feathers of gallus domesticus were collected at 12, 21 and 34 days of age. The feathers were morphologically analyzed and then we selected the region of the calamus due to the presence of pulp for cell culture and cryopreservation. The results showed that it is possible to find cells with distinct morphology; cells in elliptical shape with central nucleus also in elliptical shape, cells with shape and round nucleus, cells compatible with the fibers of the barbules, cell agglomerates and cells adhered to the bottom of the plate with fibroblastatoid shape. After 24 hours of culture there was the presence of primary culture with 80% of confluence and after cryopreservation the average viability after freezing was 68.8%, with cellular morphologies being maintained. Therefore, we proved the isolation of somatic cells from the follicle of birds feathers, suggesting that this is a source of great value, viable and effective for obtaining biological material for the elaboration of a biobank.(AU)


Subject(s)
Animals , Chickens/genetics , Chick Embryo , Feathers , Adult Stem Cells
9.
Anim. Reprod. (Online) ; 17(3): e20200044, 2020. ilus
Article in English | VETINDEX | ID: biblio-1461530

ABSTRACT

The creation of a genetic resource bank of avian species aims to prevent the decline and fragmentation of wild bird populations, which in turn lead to the loss of genetic diversity and, in more serious cases, the extinction of the most threatened species. In order for the collected genetic material to be stored in a bank and useful when necessary, it is essential to improve the technique ensuring its effectiveness. Thus, our study used feather follicle cells from the domestic gallus species to standardize the technique of cell culture and subsequent cryopreservation. This study aimed to establish a protocol, in vitro, of isolation and primary culture of somatic cells derived from the feather follicle, with the purpose of establishing a cell lineage, and evaluate its viability for the biobank formation. Developing feathers of gallus domesticus were collected at 12, 21 and 34 days of age. The feathers were morphologically analyzed and then we selected the region of the calamus due to the presence of pulp for cell culture and cryopreservation. The results showed that it is possible to find cells with distinct morphology; cells in elliptical shape with central nucleus also in elliptical shape, cells with shape and round nucleus, cells compatible with the fibers of the barbules, cell agglomerates and cells adhered to the bottom of the plate with fibroblastatoid shape. After 24 hours of culture there was the presence of primary culture with 80% of confluence and after cryopreservation the average viability after freezing was 68.8%, with cellular morphologies being maintained. Therefore, we proved the isolation of somatic cells from the follicle of bird’s feathers, suggesting that this is a source of great value, viable and effective for obtaining biological material for the elaboration of a biobank.


Subject(s)
Animals , Chick Embryo , Chickens/genetics , Feathers , Adult Stem Cells
10.
Braz. dent. sci ; 23(1): 1-10, 2020. tab, ilus
Article in English | LILACS, BBO - Dentistry | ID: biblio-1049185

ABSTRACT

Objective: The aim of this study was to evaluate the impact on the isolation and characterization of stem cells from pulp tissues obtained through rotary instrumentation techniques compared to the manual technique. Material and Methods: Thirty permanent teeth were included, 15 of which were instrumented with rotational technique (Protaper SX) and other 15 with manual technique. Cells obtained were characterized by flow cytometry and proliferation was evaluated by the MTT assay. The plasticity was evaluated for adipogenic, osteogenic and odontogenic differentiations. Results: Cells isolated from the pulp of permanent teeth, by manual techniques, presented fibroblast morphology and were able to differentiate successfully. All lineages expressed CD29, CD73, CD90, CD105, CD146, CD166 and were negative for CD31, CD34 and CD45. MTT assay showing significantly increased proliferation of hDPSCs in 5 and 7 days of the culture. Conclusions: The present study demonstrated that manual instrumentation technique is one of the best candidates to harvest dental pulp tissue as the dental stem cell source due to ability effective expanded with less tissue invasion. The technique of rotational instrumentation proved to be very harmful to the tissues of the dental pulp, and we can't obtain cells using this technique. (AU)


Objetivo: O objetivo deste estudo foi avaliar o impacto no isolamento e caracterização de células-tronco de tecidos pulpares obtidos por meio de técnicas de instrumentação rotatória em comparação à técnica manual. Material e Métodos: Trinta dentes permanentes foram incluídos, 15 dos quais foram instrumentados com técnica mecanizada (Protaper SX) e outros 15 com técnica manual. As células obtidas foram caracterizadas por citometria de fluxo e a proliferação foi avaliada pelo ensaio MTT. A plasticidade foi avaliada quanto às diferenciações adipogênica, osteogênica e odontogênica. Resultados: células isoladas da polpa de dentes permanentes, por técnicas manuais, apresentaram morfologia de fibroblastos e foram capazes de se diferenciar com sucesso. Todas as linhagens expressaram CD29, CD73, CD90, CD105, CD146, CD166 e foram negativas para CD31, CD34 e CD45. O teste de MTT mostrou proliferação significativamente aumentada de hDPSCs em 5 e 7 dias da cultura. Conclusões: O presente estudo demonstrou que a técnica de instrumentação manual é um dos melhores candidatos para a colheita de tecido pulpar como fonte de células tronco dentárias devido à boa capacidade de proliferação celular com menor invasão tecidual. A técnica de instrumentação rotatória provou ser muito prejudicial para os tecidos da polpa dentária, e não possibilitou obter células. (AU)


Subject(s)
Humans , Adolescent , Middle Aged , Pulpectomy , Endodontics , Adult Stem Cells
11.
Stem Cell Rev Rep ; 15(4): 506-518, 2019 08.
Article in English | MEDLINE | ID: mdl-31140074

ABSTRACT

The lack of clear regulations for the use of veterinary stem cells has triggered the commercialization of unproven experimental therapies for companion animal diseases. Adult stem cells have complex biological characteristics that are directly related to the therapeutic application, but several questions remain to be answered. In order to regulate the use of these cells, well-conducted, controlled scientific studies that generate high-quality data should be performed, in order to assess the efficacy and safety of the intended treatment. This paper discusses the scientific challenges of mesenchymal stem cell therapy in veterinary regenerative medicine, and reviews published trials of adipose-tissue-derived stem cells in companion animal diseases that spontaneously occur.


Subject(s)
Adult Stem Cells/metabolism , Animal Diseases , Mesenchymal Stem Cell Transplantation , Mesenchymal Stem Cells , Adult Stem Cells/pathology , Animal Diseases/metabolism , Animal Diseases/pathology , Animal Diseases/therapy , Animals , Mesenchymal Stem Cells/metabolism , Mesenchymal Stem Cells/pathology , Regenerative Medicine , Veterinary Medicine
12.
J Chem Neuroanat ; 98: 117-123, 2019 07.
Article in English | MEDLINE | ID: mdl-31047946

ABSTRACT

In the adult hippocampus new neurons are generated in the dentate gyrus from neural progenitor cells. Adult-born neurons integrate into the hippocampal circuitry and contribute to hippocampal function. PSD95 is a major postsynaptic scaffold protein that is crucial for morphological maturation and synaptic development of hippocampal neurons. Here we study the function of PSD95 in adult hippocampal neurogenesis by downregulating PSD95 expression in newborn cells using retroviral-mediated RNA interference. Retroviruses coding for a control shRNA or an shRNA targeting PSD95 (shPSD95) were stereotaxically injected into the dorsal dentate gyrus of 2-month-old C57BL/6 mice. PSD95 knockdown did not affect neuronal differentiation of newborn cells into neurons, or migration of newborn neurons into the granule cell layer. Morphological analysis revealed that newborn neurons expressing shPSD95 showed increased dendritic length and increased number of high-order dendrites. Concomitantly, dendrites from shPSD95-expressing newborn granule neurons showed a reduction in the density of dendritic spines. These results suggest that PSD95 is required for proper dendritic and spine maturation of adult-born neurons, but not for early stages of neurogenesis in the hippocampus.


Subject(s)
Disks Large Homolog 4 Protein/metabolism , Hippocampus/cytology , Neural Stem Cells/cytology , Neurogenesis/physiology , Neurons/cytology , Adult Stem Cells/cytology , Adult Stem Cells/metabolism , Animals , Cell Differentiation/physiology , Cell Movement/physiology , Hippocampus/metabolism , Mice , Mice, Inbred C57BL , Neural Stem Cells/metabolism , Neurons/metabolism
13.
Adv Exp Med Biol ; 1123: 1-3, 2019.
Article in English | MEDLINE | ID: mdl-31016591

ABSTRACT

Adult endogenous stem cells are crucial to maintain organ homeostasis due to their particular capacity to originate more specialized cell populations in a coordinated manner based on the body necessity. Extensive studies in a variety of tissues have highlighted the importance of stem cells for the functioning of our organism, including the skin, intestine, stomach, skeletal muscle, bone marrow, and others. Although significant progress has been made in our understanding of stem cell biology, our knowledge about these cells still remains limited due to their complexity and their dynamics. The advancement of our knowledge on these essential cells will have substantial implications in our understanding of tissue homeostasis and disease. Importantly, not all stem cells are alike even within the same tissue. They differ in their cell cycle status, surface marker expression, response to various extrinsic molecules, and distinct lineage outputs after transplant. The expanding literature which backs heterogeneity within stem cells is presently of great interest and brings questions as how stem cell subpopulations are generated, why they exist, and whether stem cells heterogeneity influences disease progression or therapy options. In more recent years, the combination of fluorescent and confocal microscopy with genetic state-of-art techniques, such as fate lineage tracking and single-cell RNA sequencing, enabled remarkable advance in the discovery of multiple novel essential functions for stem cell subpopulations in health and disease, before unexpected. This book provides an overview on our knowledge of stem cell subtypes in different organs under physiological and pathological conditions and discusses the possible origins and consequences of stem cells heterogeneity. This book's initial title was Stem Cells Heterogeneity. However, due to the current great interest in this topic, we were able to assemble more chapters than would fit in one book, covering stem cell biology under distinct circumstances. Therefore, the book was subdivided into three volumes entitled: Stem Cells Heterogeneity-Novel Concepts, Stem Cells Heterogeneity in Different Organs, and Stem Cells Heterogeneity in Cancer. Here, we offer a selected compilation of comprehensive chapters on what we know so far about heterogeneity within stem cells. More than 30 chapters written by scientists in the field outline our present knowledge on stem cells heterogeneity.


Subject(s)
Adult Stem Cells/cytology , Cell Cycle , Homeostasis , Humans , Membrane Proteins
14.
Einstein (Sao Paulo) ; 16(4): eRB4587, 2018 Nov 29.
Article in English, Portuguese | MEDLINE | ID: mdl-30517369

ABSTRACT

The biology of stem cells is one of the most dynamic and promising fields of the biological sciences, since it is the basis for the development of organisms. Its biological complexity demands efforts from several lines of research aimed mainly at its therapeutic use. Nanotechnology has been emerging as a new field of study, which shows great potential in the treatment of various diseases. This new area of health has been called "Nanomedicine" or "Bionanotechnology", which can be applied in Medicine by transport and drug delivery systems, robotic tools to be used in diagnostic and surgical processes, nanobiomaterials, gene therapies, nanobiomedical devices, among others. Because stem cells and Nanotechnology are two areas of extremely promising science, a new field of study, called "stem cell Nanotechnology", has gradually emerged. In this, Nanotechnology is used to help the stem cells apply their therapeutic potential in the treatment, cure, and repair of the damaged tissues, in an effective and safe way. In this way, stem cell Nanotechnology has generated great interest, since it may result in significant contributions to Regenerative Medicine and tissue engineering. The present work aims to present the state-of-the-art regarding its therapeutic use in Human Medicine.


Subject(s)
Adult Stem Cells , Multipotent Stem Cells , Nanotechnology/methods , Regenerative Medicine/methods , Bone Diseases/therapy , Cardiovascular Diseases/therapy , Humans , Nanomedicine/methods , Nanostructures/therapeutic use , Neoplasms/therapy , Nervous System Diseases/therapy , Tissue Engineering/methods
15.
Neuron ; 99(3): 425-427, 2018 08 08.
Article in English | MEDLINE | ID: mdl-30092210

ABSTRACT

The pathways that convert neural stem cells (NSCs) into functional neurons in the adult hippocampus are tightly regulated. In this issue of Neuron, Yeh et al. (2018) demonstrate that the activity of dentate mossy cells determines the balance between quiescence and activation of NSCs.


Subject(s)
Adult Stem Cells , Neural Stem Cells , Adult , Cell Differentiation , Hippocampus , Humans , Mossy Fibers, Hippocampal , Neurogenesis
16.
Int J Mol Sci ; 19(5)2018 Apr 25.
Article in English | MEDLINE | ID: mdl-29693604

ABSTRACT

Top-down tissue engineering aims to produce functional tissues using biomaterials as scaffolds, thus providing cues for cell proliferation and differentiation. Conversely, the bottom-up approach aims to precondition cells to form modular tissues units (building-blocks) represented by spheroids. In spheroid culture, adult stem cells are responsible for their extracellular matrix synthesis, re-creating structures at the tissue level. Spheroids from adult stem cells can be considered as organoids, since stem cells recapitulate differentiation pathways and also represent a promising approach for identifying new molecular targets (biomarkers) for diagnosis and therapy. Currently, spheroids can be used for scaffold-free (developmental engineering) or scaffold-based approaches. The scaffold promotes better spatial organization of individual spheroids and provides a defined geometry for their 3D assembly in larger and complex tissues. Furthermore, spheroids exhibit potent angiogenic and vasculogenic capacity and serve as efficient vascularization units in porous scaffolds for bone tissue engineering. An automated combinatorial approach that integrates spheroids into scaffolds is starting to be investigated for macro-scale tissue biofabrication.


Subject(s)
Adult Stem Cells/cytology , Bone and Bones/cytology , Cartilage/cytology , Spheroids, Cellular/cytology , Tissue Engineering/methods , Tissue Scaffolds/chemistry , Adult Stem Cells/physiology , Animals , Cell Proliferation , Humans , Nanofibers/chemistry , Spheroids, Cellular/physiology
17.
R. bras. Reprod. Anim. ; 42(1): 3-8, jan.-mar. 2018. tab
Article in Portuguese | VETINDEX | ID: vti-736326

ABSTRACT

A clonagem por transferência nuclear de células somáticas consiste em uma atraente ferramenta para a conservação e multiplicação de espécies. A eficiência desta biotécnica depende da obtenção e seleção de células doadoras de núcleo derivadas da pele de indivíduos de interesse. Em alguns mamíferos encontrados em regiões de difícil acesso ou distantes de laboratórios especializados, o armazenamento a 4°C de tecidos somáticos da pele seria uma alternativa para a conservação do material genético desses animais. Contudo, o emprego desta técnica depende de alguns fatores, como os períodos e as condições de armazenamento a 4°C das amostras, os quais podem influenciar na recuperação das células após cultivo in vitro dos tecidos. Em mamíferos domésticos, estudos têm mostrado variações quanto ao período de estocagem e a presença de meio nutritivo. Já em mamíferos silvestres, apenas são relatados o uso da refrigeração como ferramenta de transporte em curto prazo. Assim, o objetivo desta revisão é apresentar as diferentes condições de armazenamento a 4°C de tecidos somáticos, evidenciando a importância dessa técnica para a conservação da biodiversidade.(AU)


Cloning by somatic cell nuclear transfer is an attractive tool for conservation and multiplication of species. The efficiency of this biotechnique depends on the obtaining and selection of nucleus donor cells derived from the skin of individuals of interest. In some mammals found in regions difficult to access or distant from specialized laboratories, the storage at 4°C of somatic tissues of the skin would be an alternative for the conservation of the genetic material of these animals. Nevertheless, the use of this technique depends on some factors, such as periods and storage conditions at 4°C of the samples, which may influence the recovery of the cells after tissue culture in vitro. In domestic mammals, studies have shown variations regarding the period of storage and the presence of nutrient medium. Already, in wild mammals, only is related the use of refrigeration as transportation tool in the short term. Thus, the aim of this review is to present the different conditions of storage at 4°C of somatic tissues, evidencing the importance of this technique for the conservation of biodiversity.(AU)


Subject(s)
Animals , Adult Stem Cells/cytology , Product Storage , Refrigeration , Good Distribution Practices , Quality Control , Cloning, Organism/veterinary , In Vitro Techniques , Mammals , Skin
18.
Rev. bras. reprod. anim ; 42(1): 3-8, jan.-mar. 2018. tab
Article in Portuguese | VETINDEX | ID: biblio-1492503

ABSTRACT

A clonagem por transferência nuclear de células somáticas consiste em uma atraente ferramenta para a conservação e multiplicação de espécies. A eficiência desta biotécnica depende da obtenção e seleção de células doadoras de núcleo derivadas da pele de indivíduos de interesse. Em alguns mamíferos encontrados em regiões de difícil acesso ou distantes de laboratórios especializados, o armazenamento a 4°C de tecidos somáticos da pele seria uma alternativa para a conservação do material genético desses animais. Contudo, o emprego desta técnica depende de alguns fatores, como os períodos e as condições de armazenamento a 4°C das amostras, os quais podem influenciar na recuperação das células após cultivo in vitro dos tecidos. Em mamíferos domésticos, estudos têm mostrado variações quanto ao período de estocagem e a presença de meio nutritivo. Já em mamíferos silvestres, apenas são relatados o uso da refrigeração como ferramenta de transporte em curto prazo. Assim, o objetivo desta revisão é apresentar as diferentes condições de armazenamento a 4°C de tecidos somáticos, evidenciando a importância dessa técnica para a conservação da biodiversidade.


Cloning by somatic cell nuclear transfer is an attractive tool for conservation and multiplication of species. The efficiency of this biotechnique depends on the obtaining and selection of nucleus donor cells derived from the skin of individuals of interest. In some mammals found in regions difficult to access or distant from specialized laboratories, the storage at 4°C of somatic tissues of the skin would be an alternative for the conservation of the genetic material of these animals. Nevertheless, the use of this technique depends on some factors, such as periods and storage conditions at 4°C of the samples, which may influence the recovery of the cells after tissue culture in vitro. In domestic mammals, studies have shown variations regarding the period of storage and the presence of nutrient medium. Already, in wild mammals, only is related the use of refrigeration as transportation tool in the short term. Thus, the aim of this review is to present the different conditions of storage at 4°C of somatic tissues, evidencing the importance of this technique for the conservation of biodiversity.


Subject(s)
Animals , Product Storage , Good Distribution Practices , Quality Control , Adult Stem Cells/cytology , Refrigeration , Cloning, Organism/veterinary , Mammals , Skin , In Vitro Techniques
19.
Ci. Rural ; 48(3): 1-4, 2018.
Article in English | VETINDEX | ID: vti-733658

ABSTRACT

The aim of this study was to evaluate the use of mesenchymal stem cells (MSC) in the treatment of myonecrosis induced by Bothrops alternatus venom in rats. Seventy-five male adult Wistar rats were divided into three experimental groups. G1 and G2 were injected in the gastrocnemius muscle with 120g of B. alternatus venom, while G3 received 200L of PBS only. Three days after the venom injection, 12 rats from G1 were treated with 5.0 x 106 MSC in PBS, whereas G2 and G3 rats received PBS. Every three days, blood and muscle samples of five animals from each group were taken for serum biochemical and pathological analyses. Histological examinations showed more intense muscle lesions following MSC treatment, characterized by disorganization and loss of muscle fibers, with focal necrosis and inflammatory infiltration by mononuclear cells. In conclusion, the use of MSC for the treatment of local damage caused by inoculation of B. alternatus venom impaired muscle regeneration and interfered in the healing process.(AU)


O objetivo do presente trabalho foi avaliar a utilização das células tronco mesenquimais (MSC) no tratamento da mionecrose induzida pelo veneno de Bothrops alternatus em ratos. 75 ratos Wistar adultos foram distribuídos em três grupos experimentais. G1 e G2 receberam 120g de veneno de B. alternatus, enquanto o G3 recebeu apenas 200L de PBS. Três dias após a administração do veneno, os ratos do grupo G1 foram tratados com 5.0 x 106 MSC, enquanto G2 e G3 receberam exclusivamente PBS. A cada três dias, amostras de sangue e tecido muscular foram coletadas de cinco animais de cada grupo para avaliação bioquímica sérica e patológica, respectivamente. A análise histológica revelou lesão muscular mais intensa após a aplicação das MSC, caracterizada pela desorganização e perdas das fibras musculares, com necrose focal e infiltrado inflamatório mononuclear. É possível concluir que a utilização das MSC para o tratamento da lesão local causada pela inoculação do veneno B. alternatus deteriorou a regeneração muscular e interferiu com o processo de cicatrização.(AU)


Subject(s)
Animals , Rats , Adult Stem Cells , Snake Venoms , Snake Bites/therapy , Muscles/injuries , Rats, Wistar/injuries , Bothrops , Necrosis
20.
Repert. med. cir ; 27(3): 145-154, 2018. tab.
Article in English, Spanish | LILACS, COLNAL | ID: biblio-981916

ABSTRACT

Desde su descripción inicial, hace ya más de 40 años, las células madre mesenquimales (MSC) fueron reconocidas como una importante alternativa para el manejo de enfermedades caracterizadas por la pérdida aguda o crónica de tejido, gracias a su capacidad de proliferación y diferenciación, lo cual les permitiría sustituir las células perdidas y de esta forma recuperar la estructura y función. Cada vez es más abundante la evidencia que sugiere el potencial de estas células para el manejo de un amplio grupo de enfermedades, al menos en modelos experimentales preclínicos. No obstante, esta capacidad no ha podido refrendarse contundente y consistentemente en ensayos clínicos. Con la presente revisión, se pretende presentar una visión del estado actual del desarrollo conceptual en torno a las capacidades terapéuticas de las MSC y un análisis crítico de algunos de los factores, que han impedido que estas sean una opción terapéutica usable en la práctica clínica diaria


Since they were initially identified more than 40 years ago, mesenchymal stem cells (MSCs) were recognized as an important therapeutic alternative for diseases characterized by acute or chronic loss of tissue, thanks to their proliferation and differentiation ability, which would allow the replacement of lost cells and their structural and functional recuperation. Evidence suggesting the therapeutic potential of these cells for a vast group of diseases increases day by day, at least in preclinical experimental models. However, clinical trials have been unable to obtain consistent and categorical results to demonstrate this capacity. This review aims to provide, an overview on the current status of the conceptual development on the therapeutic properties of MSCs, and a critical analysis of some factors which have hindered the application of this therapeutic option in daily clinical practice.


Subject(s)
Humans , Stem Cells , Cell Differentiation , Adult Stem Cells , Mesenchymal Stem Cells
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