Rat dental pulp stem cells: isolation and phenotypic characterization method aiming bone tissue bioengineering

ABSTRACT: Dental pulp stem cells (DPSC) have been showing a considerable potential for regenerative medicine. Pulps were collected from lower incisors (n=2) through direct access of the tooth pulp chamber. The isolated cells were cultured in alfa-MEM 10% FBS, in standard culture conditions. At the third passage, DPSC were characterized by flow cytometry (MHCI, CD54, CD73, CD90, CD45, CD11 and CD34); RT-PCR for Nanog gene; and their differentiation capacity in osteogenic, adipogenic and chondrogenic cell lines. Isolated cells exhibited adhesion capacity to plastic; fusiform morphology, and 80% confluence reached in approximately 3 days. These cells have also revealed positive expression for CD54, CD73 and CD90 markers; and negative expression for CD11, CD34 and CD45. Nanog expression was detected by RT-PCR, expected for a mesenchymal stem cell profile. DPSC chondrogenic differentiation was confirmed by positive staining in Alcian Blue; lipidic droplets stained with oil red confirmed their capacity to differentiate in adipogenic fate; while mineralized beads, stained with alizarin red, confirmed their differentiation in osteogenic phenotype. These results indicate the viability of the isolation and expansion of rat DPSC following this method, and osteogenic differentiation potential opens new perspectives for in vivo studies and the use of these cells in cellular therapies and tissue bioengineering, aiming bone repair.

Poros Nucleares y Función Celular en Epitelio Mamario / Nuclear Pores and Cellular Function

El presente artículo tiene como objetivo central evidenciar la interesante relación que se establece entre la función celular y el número de poros nucleares, relación que modula el activo intercambio nucleo-citoplasmatico en distintas etapas del ciclo celular de la estirpe HC11.

The main objective of this article is related to the study of different existing relationships between cellular function and the number of nuclear pores in order to explain the amount of nuclear-cytoplasmatic exchange through HC11 cell cycle stages.

Isolamento e caracterização de células-tronco obtidas de corações de camundongos adultos / Isolation and characterization of stem cells from hearts of adult mice

O uso de células-tronco representa uma alternativa para o tratamento das doenças que acometem o coração, devido à capacidade que essas células indiferenciadas têm de preservar sua própria população e de se diferenciar em células dos diversos tecidos, incluindo o cardíaco. Nesse trabalho comparamos as características de células-tronco isoladas a partir do tecido cardíaco e da medula óssea de camundongos transgênicos para a proteína fluorescente verde (GFP). As células-tronco cardíacas e da medula óssea apresentaram característica morfológica fibroblastóide e imunofenotípica de células-tronco mesenquimais, com alta expressão dos marcadores CD44, CD90, CD73, Sca-1 e baixa expressão dos marcadores de células hematopoiéticas. A análise citogenética revelou um cariótipo poliplóide a partir da terceira passagem das células-tronco isoladas do coração e da medula-óssea. A capacidade de diferenciação em vários tipos celulares, tais como adipócitos, osteócitos e condrócitos, também foi avaliada nas células-tronco de ambas as fontes. Tanto as células-tronco isoladas do coração como da medula óssea foram capazes de se diferenciar nessas três linhagens. Quando estimuladas com 5’azacitidina para testar o potencial cardiomiogênico das células isoladas do coração e da medula óssea, apenas as células-tronco cardíacas passaram a expressar alguns marcadores de cardiomiócitos, tais como troponina T cardíaca e GATA-4...

Stem cells are undifferentiated cells with the ability of self-renewal and differentiation into different cell types, with the potential to treat heart diseases. In the present study we compared the characteristics of stem cells isolated from the heart to bone marrow stem cells, both obtained from EGFP transgeneic mice. Cardiac and bone marrow stem cells presented fibroblastic morphology and an immunophenotype compatible with mesenchymal stem cells – high expression of CD44, CD90, CD73, Sca-1 and low expression of of hematopoietic lineage markers. Cytogenetic analysis demonstrated polyploid karyotypes after the third passage of the stem cells isolated from heart and bone marrow. Both bone marrow and heart stem cells were able to differentiate into adipocytes, osteocytes and chondrocytes. In order to test the potential of differentiation into cardiomyocytes, cells were stimulated with 5’azacytidine and only cardiac stem cells expressed heart-specific markers: cardiac T troponin and GATA-4...

Human bone marrow-derived mesenchymal stem cells differentiate into retinal cells in vitro / 中国药理学通报

Aim To study the differentiation of human bone marrow-derived mesenchymal stem cells ( HM-SCs) into retinal cells in vitro. Methods HMSCs were isolated from human bone marrow after Ficoll den-sity gradient centrifugation. The adherent cells after at least 5 passages were used for study. Immunopheno-type of the cells was analysed by flow cytometer, and cellular differentiation was identified by immunofluores-cence labeling technique. Results The target cells derived from human bone marrow adhered to the plate with fibroblastic-like morphology, whose surface mark-ers were similar to mesenchymal stem cells. Major cells were positive for CD90 , CD44 , CD147 , while they were all negative for CD34, CD45, HLA-DR. In the differentiation study, HMSCs cultured in induced me-dium can differentiate into nestin ( neural stem cell ) -positive cell, GFAP ( glial fibrillary acidic protein ) -positive glial cells and retina-specific neurons express-ing Rhodopsin with CD90 ( mesenchymal stem cells )-negative. Conclusion HMSCs have the ability to dif-ferentiate into retinal neural cells in vitro.

El factor de crecimiento epidérmico y la diferenciación celular del epitelio mamario / Epidermal growth factor and mammary epithelial differentiation

El objetivo de este artículo es presentar una revisión relativa a evidenciar las características generales, estructura y funcionalidad de los factores de crecimiento con especial énfasis en precisar el rol que ejerce el factor de crecimiento epidérmico (EGF) como agente generador del proceso de diferenciación celular en el epitelio mamario.

The objective of this article is to present a review referred to general characteristics, such as structure and functionality of the growth factors, particularly those related to the Epidermal Growth Factor (EGF), as a responsible generator agent of cell differentiation at the mammary epithelial level.

Organogénesis del sistema digestivo del pez Pterophylum scalare (Perciformes: Cichlidae)

Organogenesis of the digestive system of the fish Pterophylum scalare (Perciformes: Cichlidae). There is little knowledge on the development of the angelfish Pterophyllum scalare (Liechtenstein 1823), a species of economical and biological value for inland water ecosystems. We recorded net development time of each organogenetic stage, cumulative time and characteristic structure differentiation for each stage. We found eight organogenetic stages for the digestive system, between the gastrula and the total re-adsorption of the vitelin sack. The total time for the organogenetic development of the digestive system was 119 hours and 44 minutes. Rev. Biol. Trop. 56 (4): 1857-1870. Epub 2008 December 12.

Debido a la poca información sobre el desarrollo de sistemas orgánicos en el pez Pterophylum scalare (Liechtenstein 1823) estudiamos yiempo neto de desarrollo de cada estadio de la organogénesis, tiempo acumulado y diferenciación de estructuras características de cada estadio. Se obtuvo un total de 8 estadios en la organogénesis del sistema digestivo, comprendidos entre la gástrula y la reabsorción total del saco vitelino. La duración de la organogénesis del sistema digestivo fue de 119 horas 44 minutos.

Análise dos genes diferencialmente expressos durante a osteodiferenciação induzida por proteínas morfogenéticas de osso (BMP2 e BMP7) em células C2C12 e super-expressão de rhBMP2 e rhBMP7 em células de mamíferos / Analysis of differentially expressed genes during osteodifferentiation induced by bone morphogenetic proteins (BMP2 e BMP7) of C2C12 cells and overexpression of rhBMP2 and rhBMP7 in mammalian cells

As BMPs (Bone Morphogenetic Proteins) são membros da superfamília de proteínas TGF-β (Transforming Growth Factor β ), regulam o crescimento e diferenciação de vários tipos celulares em diversos tecidos, e algumas delas desempenham um papel crítico na diferenciação de células de origem mesenquimal em osteoblastos. Particularmente, rhBMP2 e rhBMP7, promovem osteoindução tanto /"in vitro/" como /"in vivo,/" sendo, ambas as proteínas utilizadas terapeuticamente em Ortopedia/Odontologia para reparo ósseo. A expressão diferencial de genes durante a osteodiferenciação de células C2C12 induzida por rhBMP2 e rhBMP7, foi analisada através de microarranjos de DNA, selecionando 31 genes, dos quais 24 foram validados por qPCR, 13 dos quais são relacionados à transcrição, quatro associados a algumas vias de sinalização celular e sete associados à matriz extracelular. Análise funcional destes genes permitirá conhecer, com maiores detalhes, os eventos moleculares que ocorrem durante a diferenciação osteoblástica de células C2C12 induzida por rhBMPs. Em paralelo, foi perseguida a super-expressão de rhBMP2 e rhBMP7 em células HEK293T, demonstrando-se a atividade de rhBMP7, induzindo osteodiferenciação /"in vitro/" e formação de osso /"in vivo/", demonstrando a viabilidade do objetivo de se produzir estas proteínas para futura aplicação como biofármacos no Brasil.

The BMPs (Bone Morphogenetic Proteins) are members of the TGF-β (Transforming Growth Factor β) superfamily of proteins, regulate growth and differentiation of various cell types in various tissues, and some play a critical role in differentiation of mesenchymal cells into osteoblasts. Particularly, rhBMP2 and rhBMP7, promote osteoinduction /"in vitro/" and /"in vivo/" and both proteins are used therapeutically in Orthopedics and Dentistry. The differential expression of genes during osteodifferentiation induced by rhBMP2 and rhBMP7 in C2C12 cells was analyzed through DNA microarrays, allowing the selection of 31 genes, of which 24 were validated by qPCR, 13 of which are related to transcription, four associated with cell signaling pathways and seven are associated with the extracellular matrix. Subsequent functional analysis of these genes should reveal more details on the molecular events which take place during C2C12 cells osteoblastic differentiation induced by rhBMPs In paralel, rhBMPs 2 and 7 were overexpressed in HEK293T cells and BMP7 activity to induce osteodifferentiation /"in vitro/" and bone formation /"in vivo/" was demonstrated, reinforcing the viability of our objective to produce these proteins for future application as biopharmaceuticals in Brazil

Induction of neural stem cells into oligodendrocytes / 重庆医科大学学报

Objective:To induce neural stem cells(NSCs)differentiating into oligodendrocytes in vitro and provide experiment foundation for NSCs-derived oligodendrocytes.Methods:The primary NSCs were isolated from endbrain and midbrain of neonatal rats,and then the cells were cultured in vitro.NSCs were induced by thyroid hormone(T3)and(or)insulin,and the effect of T3 and insulin was analysed by comparing the proliferation and the differentiation rate of NSCs and by measuring the prominency of GalC-positive cells.Results:NSCs were induced into oligodendrocytes by thyroid hormone and/or insulin,and different concentration of T3 and/or insulin had different effects on differentiation.Conclusion:Thyroid hormone and insulin can induce NSCs to differentiate mainly into oligodendrocyte and promote the proliferation and maturation of oligodendrocytes.The effect is more outstanding when 40ng/ml T3 is used in combination with 25?g/ml insulin.

Progress in The Functional Identification of Neural Stem Cell/Neural Precursor Differentiation / 生物化学与生物物理进展

The differentiation of neural stem cells/neural precursors (NSCs/NPs) is a hot spot in neurobiological research. It used to identify their differentiation degree only by morphologic appearances. The functional characteristics, such as electrical properties of cellular membrane and ion channel activities, are drawing more and more attention with the development of patch clamp technique. It was summarized the recent progress in the study of NSCs/NPs functional differentiation using patch clamp, some existing problems and research perspectives were suggested.

A analysis of the proteome of HL60 cell line induced by a new steroidal drug as a monocytic inducer / 中国药理学通报

Aim The potentiality as a differentiation inducer of the new steroidal drug(NSC67657) had been studied.Then the expression differences of protein between treated and untreated HL60 cell line could be analysed.Method Firstly,the expression patterns of C/EBP alpha gene and protein were observed between treated and untreated HL60 cell line.Then the proliferation of HL60 cell line could be investigated by MTT.At the same time cellular chemical staining could be employed to investigate which direction HL60 cell line would be induced by NSC67657.Then the flow cytometry(FCM) could be employed to detect the profile of differentiation of HL60 cell line induced in different time and at different drug concentrations,by which the most suitable drug concentration and inducing time could be found. Following that,the information of cellular cycle and ultramicrostructure could be analysed by FCM and electronmicro scope,by which whether the apoptosis had happened or not under the drug treatment could also be found. Finally,the protein of these two group HL60 cell lines could be separated by modified two-dimensional electrophoresis (2-DE).Results The expression of C/EBP alpha gene and protein could be promoted under the treatment of NSC67657.Then the proliferation of HL60 cell line was inhibited significantly.From cellular chemical staining,the monocytic differentiation could be easily found and the perfect inducing time and drug concentration were defined as 10 ?mol?L-1NSC67657 and constantly inducing HL60 cell line within 5 days.The cellular number of G0~G1 was increased and hardly any apoptosis which might be happened during drug inducing ability could be seen.The protein of HL60 cell lines were separated by modified 2-DE technology.Then there were 14 protein spots which could only be found in the differentiated gels,on the other hand,20 protein spots could only be found in the undifferentiated gels,which would have been analyzed by MALDI-TOF.Conclusions HL60 cell line could be induced to monocyte by NSC67657 which could also stimulate the C/EBP? in the early stage.2-DE could separate the protein directly which expressed differentially,from which some proteins essential in cellular differentiation might be found.

STUDY ON INDUCTION AND DIFFERENTIATION OF NEURAL STEM CELLS DERIVED FROM ADULT HUMAN BONE MARROW / 解放军医学杂志

To explore the feasibility and validity of isolated neural stem cells(NSC) derived from adult human bone marrow, the adult human bone marrow stromal cells, harvested by density gradient centrifugation following an ilium puncture, were cultured in "Cytokines NSC medium" for ascertaining the optimal survival conditions in vitro . Proliferation of NSCs was evaluated by formation of cell clones. Antibodies against Nestin, NSE and GFAP were chosen for identifying NSCs, neurons and glial cells, respectively. The involved cytokines in culture included GDNF(20ng/ml), LIF(10ng/ml)and RA(0 5?g/ml). Among the adult human bone marrow stromal cells, there were some NSCs with rough and large cytoplasmic granules proliferating rapidly into islets shaped cellular spheres with positive Nestin, a specific antigen on the fetal neural epithelium. After separating the cellular spheres into single cells and then plating them, we found the islets shaped cellular spheres appeared again. Following differentiation of the NSCs spheres, some of them formed small buds,which then developed further into long projects connecting each other. Most of the cells with long projects showed positive NSE or GFAP. It is possible that adult human bone marrow stromal cells could be induced into NSCs under certain experimental conditions. Also it implied the feasibility and validity that the adult human bone marrow might be used as the seed cells of the neural stem cells.

EXPERIMENT ON CULTURE, IDENTIFICATION OF NEURAL STEM CELL FROM SD RATS / 解放军医学杂志

To explore the feasibility of culture and identification of neural stem cells (NSCs) from ependyma, subventricular zone and cotex of fetal and adult rat respectively, and to make the basis for treatment of degenerative diseases with NSCs transplantation, all the seed cells derived from ependyma, subventricular zone and cotex were isolated from both fetal and adult SD rat, respectively. They were then cultured, induced to differentiation and subcultured continuously in a "CYTOKINE NSCs culture medium". Identification was carried out using Nestin, NSE and GFAP antibodies for differentiated NSCs, neuron and neuroglials, respectively. The seed cells from these four locations proliferated rapidly under some corresponding conditions, and formed "neurologic spheres", which consisted of many cells and expressed Nestin antigen. After continuous culture and subculture, NSCs might divide and proliferate further. Some NSCs buds developed processes and formed nerve fibers further, while the soma enlarged into the cells with "long processes", which connected or crisscrossed with each other, and were confirmed as neurons and neuroglias by immunocytochemistry. Seed cells from fetal rats might generate more NSCs than those from adult rats, and those from ependyma and subventricular zone produced more NSCs than those from cortex. There was no special morphological difference between ependyma NSCs and cortex NSCs. It is suggested that NSCs existed not only in ependyma and cotex of fetal SD rat, but also in the subventricular zone and cotex of adult SD rat. Fetal rat nerve tissue possesses much more NSCs than adult one.

Axon Formation Follows Dendritic Differentiation in the Cultured Ventral Spinal Cord Neurons of Rat / 대한해부학회지

To understand the early cellular differentiation of neurons, we studied the differentiation of ventral spinal cord (VSC) neurons in culture. Immunofluorescence techniques with myelin associated protein 2 (MAP2) and phosphorylated neurofilament heavy chain were used with phase contrast microscopy. VSC neurons were best grown and differentiated on the coverslips coated with polyethylenimine or poly-L-Lysine. During 3 days of culture, VSC neurons changed from a round cell with no neurites to multipolar neurons with an axon and dendrites. The differentiating VSC neurons could be classified into 4 types based on the shape and length of processes. The process with axonal character, that is MAP2 negative and phosphorylated neurofilament positive, was first identified at the tip of dendritic process when one or more processes grew out. Our results suggest that the formation of an axon in VSC neurons may follow the formation of dendrites.