The dynamics of genome size and GC contents evolution in genus Nicotiana / A dinâmica do tamanho do genoma e a evolução dos conteúdos de GC no gênero Nicotiana

Hybridization and Polyploidization are most common of the phenomenon observed in plants, especially in the genus Nicotiana leading to the duplication of genome. Although genomic changes associated with these events has been studied at various levels but the genome size and GC content variation is less understood because of absence of sufficient genomic data. In this study the flow cytometry technique was used to uncover the genome size and GC contents of 46 Nicotiana species and we compared the genomic changes associated with the hybridization events along evolutionary time scale. The genome size among Nicotiana species varied between 3.28 pg and 11.88 pg whereas GC contents varied between 37.22% and 51.25%. The tetraploid species in genus Nicotiana including section Polydiclae, Repandae, Nicotiana, Rustica and Sauveolentes revealed both up and downsizing in their genome sizes when compared to the sum of genomes of their ancestral species. The genome sizes of three homoploid hybrids were found near their ancestral species. Loss of large genome sequence was observed in the evolutionary more aged species (>10 Myr) as compared to the recently evolved one’s (<0.2 Myr). The GC contents were found homogenous with a mean difference of 2.46% among the Nicotiana species. It is concluded that genome size change appeared in either direction whereas the GC contents were found more homogenous in genus Nicotiana.

A hibridização e a poliploidização são os fenômenos mais comuns observados em plantas, principalmente no gênero Nicotiana, levando à duplicação do genoma. Embora as mudanças genômicas associadas a esses eventos tenham sido estudadas em vários níveis, o tamanho do genoma e a variação do conteúdo de GC são menos compreendidos devido à ausência de dados genômicos suficientes. Neste estudo, a técnica de citometria de fluxo foi usada para descobrir o tamanho do genoma e o conteúdo de GC de 46 espécies de Nicotiana, e comparamos as mudanças genômicas associadas aos eventos de hibridização ao longo da escala de tempo evolutiva. O tamanho do genoma entre as espécies de Nicotiana variou entre 3,28 pg e 11,88 pg, enquanto os conteúdos de GC variaramentre 37,22% e 51,25%. As espécies tetraploides do gênero Nicotiana, incluindo as seções Polydiclae, Repandae, Nicotiana, Rustica e Sauveolentes, revelaram aumento e redução do tamanho do genoma quando comparados à soma dos genomas de suas espécies ancestrais. Os tamanhos do genoma de três híbridos homoploides foram encontrados perto de suas espécies ancestrais. A perda da grande sequência do genoma foi observada nas espécies evolutivas mais velhas (> 10 Myr) em comparação com as que evoluíram recentemente (< 0,2 Myr). Os teores de GC foram homogêneos com diferença média de 2,46% entre as espécies de Nicotiana. Conclui-se que a mudança no tamanho do genoma apareceu em ambas as direções, enquanto os conteúdos de GC foram encontrados mais homogêneos no gênero Nicotiana.

Aislación y caracterización de las células epiteliales del amnios / Isolation and characterization of amniotic ephitelial cells

RESUMEN: Las células epiteliales del amnios (hAECs) son células madre pluripotenciales; tienen capacidad de diferenciarse en células de las tres capas embrionarias. Como tales, se utilizan en algunas terapias regenerativas en medicina. Este estudio tiene por objetivo describir un protocolo de aislación de las células epiteliales del amnios (hAECs) a partir de placentas humanas de partos por cesárea, así como su caracterización y comportamiento in vitro. Se aislaron hAECs de 20 placentas de partos por cesárea con un protocolo optimizado. Se caracterizaron las células mediante citometría de flujo, microscopia óptica y de fluorescencia, y se evaluó la proliferación de las células mediante MTT a los 1, 3, 5 y 7 días con y sin β-mercaptoetanol en el medio de cultivo. El análisis histológico del amnios mostró un desprendimiento prácticamente completo de las células después de la segunda digestión del amnios. El promedio de células obtenidas fue de 10.97 millones de células por gramo de amnios. Las hAECs mostraron una proliferación limitada, la cual no fue favorecida por la adición de β-mercaptoetanol en el cultivo. Se observó un cambio de morfología espontanea de epitelial a mesenquimal después del cuarto pasaje. Las células epiteliales del amnios pueden ser aisladas con un protocolo simple y efectivo, sin embargo, presentan escasa capacidad proliferativa. Bajo las condiciones de este estudio, la adición de β-mercaptoetanol no favorece la capacidad proliferativa de las células.

SUMMARY: human amnion epithelial cells (hAECs) are pluripotent stem cells; they have the ability to differentiate into cells of the three embryonic layers, and are used in various regenerative therapies in medicine. This study aims to describe a protocol for the isolation of amnion epithelial cells (hAECs) from human placentas from cesarean delivery, as well as their characterization and culture conditions in vitro. hAECs were isolated from 20 cesarean delivery placentas with an optimized protocol. The cells were characterized by flow cytometry, light and fluorescence microscopy, and the proliferation of the cells was evaluated by MTT at 1, 3, 5 and 7 days with and without β-mercaptoethanol in the culture medium. Histological analysis of the amnion showed a practically complete detachment of the cells of the underlying membrane after the second digestion. The average number of cells obtained was 10.97 million cells per amnion. The hAECs perform a limited proliferation rate, which was not favored by the addition of β-mercaptoethanol in the culture. A spontaneous morphology change from epithelial to mesenchymal morphology is exhibited after the fourth passage. The epithelial cells of the amnion can be isolated with a simple and effective protocol, however, they present little proliferative capacity. Under the conditions of this study, the addition of β-mercaptoethanol does not favor the proliferation of the cells.

Optimizing the recovery of peripheral blood mononuclear cells trapped in leukoreduction filters - A comparison study

Abstract Introduction The isolation of captured peripheral blood mononuclear cells (PBMNCs) from leukoreduction filters (LRFs) can be of great importance in terms of bringing the lost cells back into use. Objective The aim of this study was to evaluate various methods based on their potential to recover the peripheral blood cells from LRFs with a focus on mononuclear cells (MNCs). Method For cell isolation from LRFs, three distinct methods (back-flushing, direct and vacuum pump) were compared through the calculation of the yield of isolated MNCs. The viability of extracted cells was determined by the flow cytometry technique. Moreover, the recovered MNCs were characterized regarding the presence of blood stem cell purification. The cell culture, microscopic observation, and immunophenotyping were employed to characterize the blood stem cells (hematopoietic, mesenchymal and progenitor endothelial stem cells). Results The yield of isolation obtained in the back-flushing, direct and vacuum pump methods were 17.7 ± 1.28, 17.3 ± 0.96 and 21.2 ± 0.90 percent, respectively. Although the highest potential for total blood cell recovery belonged to the vacuum pump method, the lowest cell viability (85.73 ± 4.84%) was observed in this method. However, the isolation process of the back-flushing and direct methods had less effect on cell viability. The characterization of the isolated MNCs displayed that the dominant positive phenotype was for CD34/CD45, indicating hematopoietic stem cells. In addition, the endothelial stem/progenitor cells were significantly detected as CD31/CD133 positive cells. Conclusion According to our results and considering the safety and efficiency potential of each of the applied methods, the back-flushing in comparison with the other methods can be considered a suitable procedure for MNC isolation from LRFs.

Identification of new human mesenchymal stem cell biomarker by aptamers / Identificação de novos biomarcadores de células tronco mesenquimais de origem humana por meio de aptâmeros

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

Continuous purification and culture of rat type 1 and type 2 alveolar epithelial cells by magnetic cell sorting / 中华创伤杂志(英文版)

PURPOSE@#The incidence of acute lung injury (ALI) in severe trauma patients is 48% and the mortality rate following acute respiratory distress syndrome evolved from ALI is up to 68.5%. Alveolar epithelial type 1 cells (AEC1s) and type 2 cells (AEC2s) are the key cells in the repair of injured lungs as well as fetal lung development. Therefore, the purification and culture of AEC1s and AEC2s play an important role in the research of repair and regeneration of lung tissue.@*METHODS@#Sprague-Dawley rats (3-4 weeks, 120-150 g) were purchased for experiment. Dispase and DNase I were jointly used to digest lung tissue to obtain a single-cell suspension of whole lung cells, and then magnetic bead cell sorting was performed to isolate T1α positive cells as AEC1s from the single-cell suspension by using polyclonal rabbit anti-T1a (a specific AEC1s membrane protein) antibodies combined with anti-rabbit IgG microbeads. Afterwards, alveolar epithelial cell membrane marker protein EpCAM was designed as a key label to sort AEC2s from the remaining T1α-neg cells by another positive immunomagnetic selection using monoclonal mouse anti-EpCAM antibodies and anti-mouse IgG microbeads. Cell purity was identified by immunofluorescence staining and flow cytometry.@*RESULTS@#The purity of AEC1s and AEC2s was 88.3% ± 3.8% and 92.6% ± 2.7%, respectively. The cell growth was observed as follows: AEC1s stretched within the 12-16 h, but the cells proliferated slowly; while AEC2s began to stretch after 24 h and proliferated rapidly from the 2nd day and began to differentiate after 3 days.@*CONCLUSION@#AEC1s and AEC2s sorted by this method have high purity and good viability. Therefore, our method provides a new approach for the isolation and culture of AEC1s and AEC2s as well as a new strategy for the research of lung repair and regeneration.

Papila Apical Dentaria como fuente de Células Madre: Relato del Caso / Papila Apical Dentária como Fonte de Células Tronco: Relato de Caso / Dental Apical Papila Stem Cell: Case Report

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.

Impact of cell harvesting methods on detection of cell surface proteins and apoptotic markers

Assays based on the flow cytometry technique allow a convenient analysis of multiple cellular parameters; however, their results should be interpreted cautiously due to a strong impact of confounding factors. Different techniques in cell culturing such as either enzymatic or mechanic detachment of adherent cells can heavily influence the structure of the cell membrane or presence of the surface antigens leading to strong false positive signals, and finally, substantial experimental bias. The aim of our study was to assess and compare the impact of cell harvesting methods (both enzymatic and non-enzymatic) on the apoptosis process and on the surface antigen cytometric analyses. We found significant differences in the quality of analysis in terms of the amount of detected surface markers determined by the detachment method. Our results demonstrated clearly how important it is to carefully choose the appropriate detachment method and may help to avoid mistakes in experiment planning. In conclusion, we recommend to adjust the detachment method to the type of analyzed markers (surface antigens or translocated phosphatidylserine).

Establecimiento e implementación de un protocolo simplificado de expansión y cultivo de Células Madre de Pulpa Dental Humana (DPSCh) / Estabelecimento e implementação de um protocolo simplificado para expansão e cultura de células-tronco da polpa dentária humana (DPSCh) / Establishing and implementing a simplified protocol for the expansion and culture of human dental pulp stem cells (hDPSCs)

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.

Comparison of protein precipitation methods for sample preparation prior to proteomic analysis of Chinese hamster ovary cell homogenates

BACKGROUND: Chinese hamster ovary (CHO) cells are the workhorse for obtaining recombinant proteins. Proteomic studies of these cells intend to understand cell biology and obtain more productive and robust cell lines for therapeutic protein production in the pharmaceutical industry. Because of the great importance of precipitation methods for the processing of samples in proteomics, the acetone, methanol-chloroform (M/C), and trichloroacetic acid (TCA)-acetone protocols were compared for CHO cells in terms of protein recovery, band pattern resolution, and presence on SDS-PAGE. RESULTS: Higher recovery and similar band profile with cellular homogenates were obtained using acetone precipitation with ultrasonic bath cycles (104.18 ± 2.67%) or NaOH addition (103.12 ± 5.74%), compared to the other two protocols tested. TCA-acetone precipitates were difficult to solubilize, which negatively influenced recovery percentage (77.91 ± 8.79%) and band presence. M/C with ultrasonic homogenization showed an intermediate recovery between the other two protocols (94.22 ± 4.86%) without affecting protein pattern on SDS-PAGE. These precipitation methods affected the recovery of low MW proteins (< 15 kDa). CONCLUSIONS: These results help in the processing of samples of CHO cells for their proteomic study by means of an easily accessible, fast protocol, with an almost complete recovery of cellular proteins and the capture of the original complexity of the cellular composition. Acetone protocol could be incorporated to sample-preparation workflows in a straightforward manner and can probably be applied to other mammalian cell lines as well.

Procedimiento para el cultivo e identificación de células madre obtenidas de lipoaspirado humano con fines de investigación / Procedure for culture and identification of stem cells from human lipoaspirate for research purposes

RESUMEN Las células madre humanas nacen con la creación de la vida misma y algunas de estas permanecen durante toda la vida. Por consiguiente, se pueden hallar en tejidos adultos y utilizarlas para investigaciones a nivel básico y aplicado. Actualmente, en nuestro país existe un creciente interés en el estudio y aplicación de células madre; sin embargo, existe poco conocimiento acerca del procedimiento para su identificación. Es por ello que este artículo tiene como objetivo dar a conocer, desde un punto de vista práctico, un procedimiento para el cultivo e identificación de células madre/estromales obtenidas de lipoaspirado humano (Adipose Stem Cells) con fines de investigación, el cual incluye la caracterización a nivel de inmunofenotipo, el potencial de diferenciación celular, la expresión génica y el control de calidad del cultivo celular, que sirva de apoyo para los profesionales de la comunidad científica peruana que deseen desarrollar esta línea de investigación.

ABSTRACT Human stem cells are born with the creation of life itself and some of them remain throughout life. Therefore, they can be found in adult tissues and used for basic and applied research. Currently, in our country there is a growing interest in the study and application of stem cells; however, little is known about the identification procedure. For this reason, this study aims to present, from a practical point of view, a procedure for the culture and identification of stem/stromal cells obtained from human lipoaspirate (Adipose Stem Cells), for research purposes. This procedure includes the immunophenotype characterization, cell differentiation potential, gene expression and cell culture quality control; and will serve as support for Peruvian scientific community professionals who wish to develop this line of research.

Phenotype changes of oral epithelial stem cells after in vitro culture

Abstract The aim of our study was to isolate populations of keratinocyte stem cells based on the expression of cell surface markers and to investigate whether the culture could affect their phenotype. keratinocytes from human oral mucosa were sorted based on the expression of the epithelial stem cell markers p75NTR and CD71. We also examined the co-expression of other epithelial stem markers such as integrins β1 and α6 and their stem cell-like proprieties in in vitro assays. Three passages after being sorted by MACS, more than 93% of the p75NTR+ve cells lost the expression of p75NTR, while 5.46% of the p75NTR-ve gained it. Within the small population of the p75NTR+ve cells, 88% co-expressed other epithelial stem cell markers such as integrins β1 and α6, while only 28% of p75NTR-ve cells co-expressed these markers. These results were confirmed by sorting cells by FACS. Additionally, when double staining was used for sorting cells, 99% of the p75NTR+veCD71-ve and 33% of the p75NTR-veCD71+ve cells expressed both integrins, but just one week after culture, only 1.74% of the p75NTR+veCD71-ve cells still expressed p75NTR and only 0.32% still expressed CD71. Similar results were obtained when co-culturing p75NTR+ve and p75NTR-ve populations before analysis. Our results suggest that phenotype changes may be part of an intrinsic cellular mechanism to conserve levels of protein expression as they may found in the human body. In addition, in vitro culture may not offer ideal conditions for epithelial stem cell maintenance due to phenotype changes under standard culture conditions.

Characterization of Decellularized Human Pericardium for Tissue Engineering and Regenerative Medicine Applications / Caracterização do Pericárdio Humano Descelularizado para Engenharia de Tecidos e Aplicações de Medicina Regenerativa

Abstract Background: Pericardium tissue allograft can be used for surgical repair in several procedures. One of the tissue engineering strategies is the process of decellularization. This process decreases immunogenic response, but it may modify the natural extracellular matrix composition and behavior. Objective: The aim of this study was to evaluate the effectiveness of cell removal, maintenance of extracellular matrix properties and mechanical integrity of decellularized human pericardium using a low concentration solution of sodium dodecyl sulfate. Methods: Decellularization was performed with sodium dodecyl sulfate and ethylenediaminetetraacetic acid. Histological analysis, DNA quantification, evaluation of glycosaminoglycans and collagen were performed. Biomechanical assay was performed using tensile test to compare the decellularization effects on tissue properties of tensile strength, elongation and elastic modulus. P < 0.05 was considered significant. Results: There was reduction in visible nuclei present in pericardium tissue after decellularization, but it retained collagen and elastin bundles similar to fresh pericardium. The DNA contents of the decellularized pericardium were significantly reduced to less than 511.23 ± 120.4 ng per mg of dry weight (p < 0.001). The biomechanical assay showed no significant difference for fresh or decellularized tissue. Conclusion: The decellularization process reduces cell content as well as extracellular matrix components without changing its biomechanical properties.

Resumo Fundameto: O enxerto de pericárdio pode ser usado em muitos procedimentos de correção cirúrgica. Uma das estratégias da engenharia tecidual é o processo de descelularização. No entanto, embora esse processo diminua a resposta imunogênica, a descelularização pode modificar tanto o comportamento como a composição da matriz extracelular natural. Objetivos: Avaliar a eficácia da descelularização usando baixa concentração de dodecil sulfato de sódio na remoção celular, na manutenção das propriedades da matriz extracelular e na integridade mecânica do pericárdio humano descelularizado. Métodos: A descelularização foi realizada com dodecil sulfato de sódio e ácido etilenodiamino tetra-acético. Foi realizada análise histológica, quantificação de DNA, e avaliação de glicosaminoglicanos e colágeno. O estudo biomecânico foi conduzido pelo teste de tração para comparar os efeitos da descelularização sobre as propriedades teciduais de resistência à tração, alongamento e módulo de elasticidade. Foi considerado um valor de p < 0,05 como estatisticamente significativo. Resultados: Observou-se uma redução na quantidade de núcleos presentes no pericárdio após a descelularização, apesar de manter quantidades similares de feixes de elastina e de colágeno. As concentrações de DNA do pericárdio descelularizado foram significativamente reduzidas para menos que 511,23 ± 120,4 ng por mg de peso seco (p < 0,001). O teste biomecânico não apontou diferenças entre os tecidos fresco e descelularizado. Conclusão: A descelularização reduziu a concentração de células bem como os componentes da matriz extracelular sem afetar suas propriedades biomecânicas.

Decellularization of human umbilical arteries / Descelularización de las arterias umbilicales humanas

SUMMARY: Arterial obstruction in small diameter (<6 mm) vessels are many times treated with grafts, however autologous aren't always available and synthetic have a high rate of complications. Decellularization of umbilical arteries may provide a solution, but the ideal method is debatable. We compare effectiveness between SDS and Triton X-100. Umbilical cords obtained from full term pregnancies with normal development and no evident complications in the newborn, were micro-dissected within 12 h and stored in phosphate buffered saline without freezing. Arteries were then processed for decellularization using 0.1 % and 1 % SDS, and 1 % Triton X100 protocols. Evaluation of cellular and nuclear material, collagen fibers, elastic fibers, and glycosoaminoglycans of the extracellular matrix (ECM) were evaluated as well as morphometric analysis under histological and immunohistochemical techniques. Triton X-100 was ineffective, preserving nuclear remains identified by immunofluorescence, had the most notable damage to elastic fibers, and decrease in collagen. SDS effectively eliminated the nuclei and had a less decrease in elastic fibers and collagen. Laminin was preserved in all groups. No significant differences were identified in luminal diameters; however the middle layer decreased due to decellularization of muscle cells. In conclusion, 0.1 % SDS decellularization was the most effective in eliminating cells and preserving the main components of the ECM.

RESUMEN: La obstrucción arterial en vasos de pequeño diámetro (<6 mm) se trata muchas veces con injertos, sin embargo, los autólogos no siempre están disponibles y los sintéticos tienen una alta tasa de complicaciones. La descelularización de las arterias umbilicales puede proporcionar una solución, pero el método ideal es discutible. Comparamos la efectividad entre los métodos SDS y Triton X-100. Cordones umbilicales obtenidos a partir de embarazos a término con evolución normal y sin complicaciones evidentes del recién nacido, se microdiseccionaron en 12 horas y se almacenaron en solución salina con fosfato sin congelación. Las arterias se procesaron luego para la descelularización usando los protocolos de SDS al 0,1 % y 1 %, y Triton X-100 al 1 %. Se realizó la evaluación de material celular y nuclear, fibras de colágeno, fibras elásticas y glucosoaminoglicanos de la matriz extracelular (MEC), así como el análisis morfométrico bajo técnicas histológicas e inmunohistoquímicas. Triton X-100 fue ineficaz, conservando los restos nucleares identificados por inmunofluorescencia, tuvo el daño más notable a las fibras elásticas y la disminución del colágeno. SDS efectivamente eliminó los núcleos y tuvo una disminución menor en las fibras elásticas y el colágeno. Laminina fue preservado en todos los grupos. No se identificaron diferencias significativas en los diámetros luminales; sin embargo, la capa media disminuyó debido a la descelularización de las células musculares. la descelularización con SDS al 0,1 % fue la más efectiva para eliminar células y preservar los principales componentes de la MEC.

Methods for mammalian single cell research - a review / 生物工程学报

Basic research in life science and medicine has dug into single cell level in recent years. Single-cell analysis offers to understand life from diverse perspectives and is used to profile cell heterogeneity to investigate mechanism of diseases. Single cell technologies have also found applications in forensic medicine and clinical reproductive medicine, while the techniques are rapidly evolving and have become more and more sophisticated. In this article, we reviewed various single cell isolation techniques and their pros and cons, including manual cell picking, laser capture microdissection and microfluidics, as well as analysis methods for DNA, RNA and protein in single cell. In addition, we summarized major up-to-date single cell research achievements and their potential applications.

Separation and forensic identification of sperm from cell mixtures using anti-hLCN6 monoclonal antibody coupled magnetic beads / 生物工程学报

Human lipocalin 6 (hLCN6) is an epididymis-specific secretory protein. It binds to sperm and plays important role in sperm maturation. To explore the feasibility for isolating spermatozoa from mixed cells using anti-hLCN6 monoclonal antibody-conjugated immunomagnetic beads (anti-hLCN6 IMBs) and establish a new method for the separation of sperms from mixed stains, 2 sets of 30 cases of cell mixture suspensions and stains containing different proportions of sperm and epithelial cells were prepared. Biotin-labeled anti-hLCN6 monoclonal antibody (mAb) was incubated with the cell mixtures, and the spermatozoa were then isolated with avidin-coated IMBs. Sperm DNA was extracted and analyzed by PCR-STR typing. Differential lysis was also conducted to compare the effect of the two different isolation methods. The dissociation constant (Kd) of anti-hLCN6 mAb was 3.47×10⁻⁹ mol/L measured by ELISA. Western blotting and immunofluorescence assays showed that hLCN6 was detectable on sperm cells and mainly located on the post-acrosomal region of the sperm head, but not in epithelial cells. Anti-hLCN6 IMBs could capture and separate the sperm cells successfully. Microscopic observation showed that the IMBs could bind to the head of sperm specifically. The success rate of STR typing (more than 13 STR loci, RFU>200) was 90% when the number of sperm cells was 10³/mL and 100% when the sperm cells number was equal to or more than 10⁴/mL. When the number of sperm cells was 10³/mL, 10⁴/mL and 10⁵/mL in mixed stain samples, the success rate of STR typing were 40%, 90% and 100%, respectively. Taken together, the anti-hLCN6 immunomagnetic beads (IMB) method described here could be effective for the isolation of sperm from mixed cells, and the success rate was higher than that of the traditional differential lysis strategy. IMB sorting is a simple and efficient method for the separation of sperms from sperm and epithelial cell mixture, and can be utilized as a supplementary method for forensic mixture samples analysis in sexual assault cases.

A modified protocol of mouse hippocampal primary microglia culture by using manual dissociation, magnetic activated cell sorting and TIC medium / 生理学报

In this study, we improved the culture method of mouse hippocampal primary microglia to obtain hippocampal ramified microglia with high activity and purity, which were resemble to the resting status of normal microglia in healthy brain in vivo. Hippocampal tissue was excised from 2-4-week-old SPF C57BL/6J mice and cut into pieces after PBS perfusion, and then manually dissociated into the single-cell suspension by using Miltenyi Biotec's Adult Brain Dissociation Kit. The tissue fragments such as myelin in the supernatant were removed by debris removal solution in the kit. The cell suspension was incubated with CD11b immunomagnetic beads for 15 min at 4 °C. To obtain high-purity microglia, we used two consecutive cell-sorting steps by magnetic activated cell sorting (MACS). After centrifugation, the cells were resuspended and seeded in a 24-well culture plate. The primary microglia were cultured with complete medium (CM) or TIC medium (a serum-free medium with TGF-β, IL-34 and cholesterol as the main nutritional components) for 4 days, and then were used for further experiments. The results showed that: (1) The cell viability was (56.03 ± 2.10)% by manual dissociation of hippocampus; (2) Compared with immunopanning, two-step MACS sorting allowed for efficient enrichment of microglia with higher purity of (86.20 ± 0.68)%; (3) After being incubated in TIC medium for 4 d, microglia exhibited branching, quiescent morphology; (4) The results from qRT-PCR assay showed that the levels of TNF-α, IL-1β and CCL2 mRNA in TIC cultured-microglia were similar to freshly isolated microglia, while those were much higher in CM cultured-microglia after incubation for 4 d and 7 d (P < 0.05). Taken together, compared to the conventional approaches, this modified protocol of mouse hippocampal primary microglia culture by using MACS and TIC medium enables the increased yield and purity of microglia in the quiescent state, which is similar to normal ramified microglia in healthy brain in vivo.

Establishment of STO Cell Lines Expressing Green Fluorescent Protein and Mouse Leukemia Inhibitory Factor / 中国实验血液学杂志

OBJECTIVE@#To establish the STO cell lines expressing green fluorescent protein (GFP) and mouse leukemia inhibitory factor (LIF) , and try to culture the mouse embryonic stem cells (mESCs) by using the established STO-GFP-mLIF cells as the feeder layer.@*METHODS@#The lentiviral particles containing GFP and mLIF and puromycin-resistance gene were constructed and transduced into STO cell lines. The cell lines stably expressing GFP and mLIF genes were screened out. The expression level of the inserted exogenous LIF gene was tested by Western blot and ELISA. The STO-GFP-mLIF cells were treated with different concentrations of mitomycin C (5, 10, 15, 20 µg/ml) for different time (1.5, 2.5, 3, 3.5 hours) to prepare feeder layers and the cell proliferation level on feeder layer was observed. Mouse embryonic stem cells were cultured on mitomycin C-treated feeder layer and the growth of cell colonies was observed.@*RESULTS@#The expression level of LIF protein in STO-GFP-mLIF cells was up-regulated, as compared with STO cells (P＜0.05). It was confirmed that the optimal concentration and time for inhibiting the proliferetion of STO-GFP-mLIF cells by mitomycin C were 10 µg/ml and 3 hours respectively. The observation also found that the embryonic stem cells could develop into typic "birdnest" shaped stem cell colony on mitomycin C-treated feeder layer.@*CONCLUSION@#The stable STO cell lines effectively expressing green fluorescent protein and mouse leukemia inhibitory factor have been established successfully, which can maintain the undifferentiated state of mouse embryonic stem cells.

Citometría de flujo en reticulocitos de sangre periférica como indicador de inestabilidad cromosómica en pacientes con gliomas de alto grado / Flow cytometry in peripheral blood reticulocytes as a marker of chromosome instability in highgrade glioma patients

Resumen Introducción. La cuantificación de la inestabilidad cromosómica es un parámetro importante para evaluar la genotoxicidad y la radiosensibilidad. Las técnicas convencionales requieren cultivos celulares o laboriosos análisis microscópicos de cromosomas o núcleos. La citometría de flujo en reticulocitos ha surgido como una alternativa para los estudios in vivo, ya que reduce los tiempos de análisis e incrementa hasta en 20 veces el número de células analizables. Objetivos. Estandarizar los parámetros de citometría de flujo requeridos para seleccionar y cuantificar reticulocitos micronucleados (RET-MN) a partir de muestras de sangre periférica, y cuantificar la frecuencia de esta subpoblación anormal como medida de inestabilidad citogenética en sendas poblaciones de voluntarios sanos (n=25) y pacientes (n=25) recién diagnosticados con gliomas de alto grado antes de iniciar el tratamiento. Materiales y métodos. Las células sanguíneas se marcaron con anti-CD71-PE para reticulocitos, anti- CD61-FITC para la exclusión de plaquetas y yoduro de propidio para detectar el ADN en reticulocitos. La fracción celular MN-RETCD71+ se seleccionó y se cuantificó con un citómetro de flujo automático. Resultados. Se describió detalladamente la estandarización de los parámetros citométricos, con énfasis en la selección y la cuantificación de la subpoblación celular MN-RETCD71+. Se establecieron los niveles basales de MN-RETCD71+ en la población de control y en los pacientes se encontró un incremento de 5,2 veces antes de iniciar el tratamiento (p<0,05). Conclusión. Los resultados evidenciaron la utilidad de la citometría de flujo acoplada a la marcación de las células RETCD71+ como método eficiente para cuantificar la inestabilidad cromosómica in vivo. Se sugieren posibles razones del incremento de micronúcleos en células RETCD71+ de pacientes con gliomas.

Abstract Introduction: The quantification of chromosomal instability is an important parameter to assess genotoxicity and radiosensitivity. Most conventional techniques require cell cultures or laborious microscopic analyses of chromosomes or nuclei. However, a flow cytometry that selects the reticulocytes has been developed as an alternative for in vivo studies, which expedites the analytical procedures and increases up to 20 times the number of target cells to be analyzed. Objectives: To standardize the flow cytometry parameters for selecting and quantifying the micronucleated reticulocytesCD71+ (MN-RET) from freshly drawn peripheral blood and to quantify the frequency of this abnormal cell subpopulation as a measure of cytogenetic instability in populations of healthy volunteers (n =25), and patients (n=25), recently diagnosed with high-grade gliomas before the onset of treatment. Materials and methods: Blood cells were methanol-fixed and labeled with anti-CD-71-PE for reticulocytes, antiCD-61-FITC for platelet exclusion, and propidium iodide for DNA detection in reticulocytes. The MN-RETCD71+ cell fraction was selected and quantified with an automatic flow cytometer. Results: The standardization of cytometry parameters was described in detail, emphasizing the selection and quantification of the MN-RETCD71+ cellular fraction. The micronuclei basal level was established in healthy controls. In patients, a 5.2-fold increase before the onset of treatment was observed (p <0.05). Conclusion: The data showed the usefulness of flow cytometry coupled with anti-CD-71-PE and anti- CD-61-FITC labeling in circulating reticulocytes as an efficient and high resolution method to quantify chromosome instability in vivo. Finally, possible reasons for the higher average of micronuclei in RETCD71+ cells from untreated high-grade glioma patients were discussed.

Evaluating the use of fluorescence-based flow cytometry assay for dengue diagnosis using peripheral blood mononuclear cells

Abstract INTRODUCTION: Dengue virus (DENV) is the most important arthropod-borne viral disease worldwide with an estimated 50 million infections occurring each year. METHODS: In this study, we present a flow cytometry assay (FACS) for diagnosing DENV, and compare its results with those of the non-structural protein 1 (NS1) immunochromatographic assay and reverse transcriptase polymerase chain reaction (RT-PCR). RESULTS: All three assays identified 29.1% (39/134) of the patients as dengue-positive. The FACS approach and real-time RT-PCR detected the DENV in 39 and 44 samples, respectively. On the other hand, the immunochromatographic assay detected the NS1 protein in 40.1% (56/134) of the patients. The Cohen's kappa coefficient analysis revealed a substantial agreement among the three methods. CONCLUSIONS: The FACS approach may be a useful alternative for dengue diagnosis and can be implemented in public and private laboratories.

Neuroprotective effects of oligodendrocyte precursor cells on white matter damage in preterm infants / 中国当代儿科杂志

White matter damage, characterized by demyelination due to the damage of oligodendrocyte precursor cells (OPCs), is the most common type of brain damage in preterm infants. Survivors are often subject to long-term neurodevelopmental sequelae because of the lack of effective treatment. In recent years, it has been found that cell transplantation has the potential for the treatment of white matter damage. OPCs are frequently used cells in cell transplantation therapy. With abilities of migration and myelinization, OPCs are the best seed cells for the treatment of white matter damage. Several studies have found that OPCs may not only replace impaired cells to reconstruct the structure and function of white matter, but also inhibit neuronal apoptosis, promote the proliferation of endogenous neural stem cells, and enhance the repairment of the blood-brain barrier. However, the clinical application of OPC transplantation therapy faces many challenges, such as the effectiveness, risk of tumorigenesis and immune rejection. With reference to these studies, this article reviewed the development of myelination, the obtainment of OPCs, the therapeutic mechanism as well as application research, and analyzed the current challenges of OPC transplantation, in order to provide a new direction for clinical treatment of white matter damage in preterm infants.