Os autores agradecem Xiu-Ping Wang ajuda inicial com culturas desconto. Os autores são financiados em parte por bolsas de estudo e bolsas de Institutos Nacionais de Saúde (K99-DE022059 a AHJ, K12-GM081266 para MGC, K08-DE022377-02 a OH, e R01-DE021420 para ODK).

1. Dissecar mais baixos hemi-mandíbulas de rato adulto <ol><li> Antes de realizar este protocolo, obter aprovação institucional necessário e não deixe de cumprir com todas as orientações de cuidados de animais. Eutanásia animal utilizando padrão IACUC procedimentos aprovados. Para este trabalho asfixia com CO2 foi usado seguido por deslocação cervical. <ol><li> OPCIONAL: cabeça separada do resto do corpo usando uma lâmina de barbear. </li></ol></li><li> Retire a pele do lábio inferior para o decote. </li><li> Usando um bisturi, fazer uma incisão entre os dois incisivos inferiores, que são vagamente conectados. Mediante a aplicação de pressão, a mandíbula será dividida em duas metades. </li><li> Wedge o bisturi entre o côndilo da mandíbula e da articulação mandibular temporal e cortar ao lado do músculo da mandíbula para separar a mandíbula. </li><li> Remova todo músculo, tendão, ligamento e até que apenas o osso permanece. </li></ol> 2. Isolar o Incisivo <ol><li> Usando um bisturi n º 15, Cuidadosamente raspar o osso em um ângulo de 45 ° a partir da extremidade distal para a extremidade proximal da região membranoso. Utilize a ponta do bisturi para escolher qualquer distância osso remanescente, incluindo os fragmentos de osso no bordo. Lentamente começam a escolher afastado na placa cortical lingual da mandíbula, começando logo abaixo do 3 º molar. </li><li> Continue até que o incisivo é limpo, trabalhando com cuidado para a área que contém o CL. </li><li> Remover pacote do nervo alveolar inferior. </li><li> Faça um corte limpo primeiro remover o osso e tecidos proximal ao CL. </li><li> Faça um segundo corte distal com o bisturi, aproximadamente abaixo do 2 º molar. A região proximal incisivo é então dissecadas para fora através da inserção do bisturi entre o osso e a superfície média do incisivo proximal. Isto deve ser feito com cuidado, de modo a não rasgar o tecido. O movimento é incisão proximal-distal. PASSO ALTERNATIVA: Remover o máximo possível de osso ao longo da zona superior da incisor. Uma vez que a área é limpa, remova cuidadosamente incisivo inteiro usando um tamanho de 5 fórceps para lidar com a parte de esmalte mais próximo a região CL. </li><li> Colocar o tecido dissecado (ou CL isolado como na Figura 3E, ou incisivo inteira como no Passo alternativa 2.5) em 2% de colagenase em 1X PBS durante 3-4 horas a 4 ° C em uma placa de baixa aderência. Para 1-10 incisivos, utilizar 100 ul por poço de uma placa de 12 poços. Para maior do que 10 incisivos, utilizar 500 ul por poço de uma placa de 6 poços aderência baixa. </li></ol> 3. Microdissect the Loop Cervical Labial <ol><li> Remover região CL da colagenase e coloque em mídia DMEM/F12 frio. </li><li> Puxe na extremidade inferior do CL usando tamanho 5 fórceps ou uma seringa de insulina (1 cc, 28 L C); mesênquima vai desmoronar enquanto o epitélio permanece intacta. O CL eo epitélio adjacente que se estende lateralmente como uma estrutura de &quot;asa-like&quot; serão visíveis. </li><li> Removera gema apical do epitélio adjacente, fazendo uma incisão em forma de V de cada lado, e coloque imediatamente em DMEM/F12 frio no gelo. </li><li> Repita o procedimento para todos os CLs, recolher em 1,5 ml tubo de microcentrífuga. </li><li> Gire para baixo tubo de microcentrífuga com exemplares, ao remover a mídia, substitua por 100 solução desprendimento de células mL. </li><li> Incubar os tecidos em solução de desprendimento de células durante 30 min a 37 ° C. </li><li> Dissociar os tecidos para produzir uma suspensão única célula cuidado pipetando para cima e para baixo usando um 1000 mL de baixa aderência ponteira. As células também podem ser colocadas através de um coador de células esterilizado para conseguir a dissociação. </li><li> Contar as células com um hemocitómetro, na placa de cultura de tecidos de plástico ou outro material desejado. </li></ol> 4. Cultura primária de DESCs <ol><li> Células da placa em uma placa de 6 poços a 1-6 x 10 4 células / ml em meios DESC, a qual consiste em DMEM/F12 suplementado com proteína recombinante de rato de EGF numa concentração de 20 ng / ml, a proteína recombinante de FGF, a uma concentração de 25 ng / ml, suplemento B27 1X e solução de 1% de antibióticos (penicilina, 100 U / ml, estreptomicina 50 ug / ml). </li><li> Cultivar células plaqueadas numa placa de 6 poços, a 37 ° C em 5% de CO 2 em 1 ml de meio de desconto. Não perturbe culturas iniciais, durante 5 dias após o plaqueamento para permitir a adesão celular e formação de colônias. </li><li> Pela primeira mudança mídia, substitua metade do volume (500 mL) de velha mídia com metade do volume (500 mL) de novas mídias. Confira as colônias sob o microscópio quando mudar de mídia. </li><li> Após a primeira mudança de mídia, continuam a mudar mídia (1-2 ml) a cada 2 dias. Confira as colônias sob o microscópio quando mudar de mídia. </li></ol> 5. DESCs Passage <ol><li> Mídia Aspirar e lavar as células 3x com pré-aquecido PBS. </li><li> Adicionar pré-aquecida de 0,25% de Tripsina-EDTA em solução salina e incubar a 37 ° C durante 10-15 minutos para os DESCs para separar. </li><li> Adicionar mídia DESC para neutralizar a tripsina, Pipeta suavemente ao longo do tecido placa de cultura para recolher as células, e coloque em um tubo de 15 ml. </li><li> Gire as células a 800 xg por 2 min. </li></ol>

<ol>
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Os autores não têm conflitos de interesse de divulgar.

As células epiteliais foram primeiro cultivadas com sucesso ao longo de 40 anos, 21-24, e, mais recentemente, o isolamento bem sucedido de células estaminais epiteliais 25-27 avançou nosso conhecimento da biologia epitelial. Relata-se um protocolo para isolar o DESCs do incisivo do rato adulto, uma população relativamente pouco estudado de células-tronco que tem o potencial para produzir insights importantes sobre a biologia dental e formação do esmalte. Este protocolo foi inicialmente base...

Uma das características únicas dos vertebrados é a evolução dos dentes. O dente tornou-se um sistema modelo importante para muitas áreas de pesquisa, como as vias moleculares e especializações morfológicas relevantes a este órgão foram investigados a partir de várias perspectivas, inclusive no desenvolvimento e evolutivos biólogos 5. Mais recentemente, no campo da medicina regenerativa começou a obter informações valiosas usando o dente como um modelo. Em particular, a descoberta de células estaminais epiteliais dental tem sido um importante avanço 6-13. Todos os roedores possuem dentes incisivos de crescimento contínuo, cujo crescimento é alimentado por células-tronco, tornando estes dentes um sistema modelo acessível para estudar regulação de células estaminais adultas. Experimentos de marcação na década de 1970 10,11, seguido por linhagem genética experimentos de rastreamento 8,9,12,14, demonstraram que DESCs residem na região proximal do incisivo. A hasteprogénie celular no compartimento epitelial em movimento lateral vestibular para fora do nicho putativo, conhecido como o ciclo cervical labial (CL), e contribui para uma população de células chamadas células (TA)-amplificação de trânsito (Figura 1). Especificamente, DESCs residem no epitélio externo do esmalte (OEE) e retículo estrelado (SR) 8,9,14, eo epitélio interno do esmalte (IEE) dá origem às células TA que progridem através de um número limitado de ciclos de células e, em seguida, mover distalmente ao longo do comprimento do incisivo (Figura 1). Os ameloblastos diferenciação na incisivo rato continuar a mover-se distalmente ao longo do incisivo a uma taxa excepcional de cerca de 350 mM de um único dia 15,16. Como eles se movem, as células se diferenciam em ameloblastos maduros e estrato intermediário células (SI). Depois de depositar a espessura total da matriz do esmalte, muitos dos ameloblastos sofrem apoptose, e as restantes células diminuem de tamanho e regular a maturação do esmalte &lt;sup&gt; 17. A linhagem de outros tipos de células no CL vestibular, tal como o SR, é menos claro, e dados em relação às células estaminais do mesênquima e 18 da CL lingual só agora começam a surgir. Utilizando o modelo de ratinho incisivo, um certo número de grupos têm trabalhado para elucidar as vias genéticas e os processos biológicos envolvidos na renovação das células estaminais naturais com base em células de órgãos. No entanto, o CL labial contém um número relativamente pequeno de células, que se estima ser de cerca de 5,000 por incisivo do rato, o que facilita o trabalho com células primárias desafiando. Por conseguinte, têm sido feitos esforços para a cultura e expandir as células in vitro 6,16,19,20, a fim de abrir as portas para novos abordagens experimentais que não são atingíveis in vivo. O estudo mais recente mostrou que estas células podem tanto a auto-renovação e diferenciação em células que expressam amelogenina quando cultivadas 13. Aqui, descrevemos e demonstrar um método para poe cultura confiável e consistente de células do rato labial CL.

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			<td height="63" style="height:63px;width:216px;">Forceps, size 5</td>
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isolation and culture of dental epithelial stem cells from the adult mouse incisor

Compreender os mecanismos celulares e moleculares subjacentes à regeneração do dente e renovação tornou-se um tema de grande interesse 1-4, eo incisivo rato fornece um modelo para estes processos. Este órgão notável cresce continuamente ao longo da vida do animal e gera todos os tipos de células necessárias de grupos ativos de células-tronco adultas alojados no vestibular (em direção ao lábio) e lingual (em direção à língua) regiões (CL) alça cervical. Apenas as células estaminais dentárias da CL labial dar origem a ameloblastos que geram o esmalte, o revestimento externo dos dentes, na superfície labial. Esta formação do esmalte assimétrica permite abrasão na ponta incisivo e progenitores e células-tronco no incisivo proximal garantir que os tecidos dentários são constantemente reabastecidos. A capacidade de isolar e cultivar essas células progenitoras ou tronco in vitro permite a sua expansão e abre as portas para inúmeras experiências não alcançáveis ​​in vivo, tais como htestes de rendimento igh de fatores de regulação de células-tronco em potencial. Aqui, descrevemos e demonstrar um método confiável e consistente para células de cultura do CL labial do incisivo mouse.

O mouse hemi-mandíbula é composto por um contínuo crescimento incisivo e três molares enraizadas (Figura 1A). Todos os dentes consistem de dentina e esmalte, as duas componentes mineralizados da coroa do dente (Figuras 1A e 1A &#39;). As casas incisivos dois nichos de células estaminais chamado o CL CL labial e lingual, e esmalte é formada exclusivamente no lado labial (Figura 1A). DESCs são responsáveis ​​pela formação do esmalte dos dent...

Watch this Scientific Journal Video about Isolation and Culture of Dental Epithelial Stem Cells from the Adult Mouse Incisor at JoVE.com

Isolation and Culture of Dental Epithelial Stem Cells from the Adult Mouse Incisor

O contínuo crescimento incisivo do rato é um modelo para o estudo de renovação dos tecidos dentários a partir de células-tronco do epitélio dental (DESCs). Um sistema robusto para consistente e confiável a obtenção destas células a partir do incisivo e expandi-las in vitro é relatado aqui.

Isolamento e Cultura da Dental epiteliais células-tronco do Adulto Rato Incisivo

Understanding the cellular and molecular mechanisms that underlie tooth regeneration and renewal has become a topic of great interest1-4, and the mouse ...

isolation-culture-dental-epithelial-stem-cells-from-adult-mouse

Research

JoVE Journal

Biology

15.1K visualizações.  University of California, San Francisco.  O contínuo crescimento incisivo do rato é um modelo para o estudo de renovação dos tecidos dentários a partir de células-tronco do epitélio dental (DESCs). Um sistema robusto para consistente e confiável a obtenção destas células a partir do incisivo e expandi-las in vitro é relatado aqui. 

Vídeo: Isolation and Culture of Dental Epithelial Stem Cells from the Adult Mouse Incisor

Isolation of Retinal Stem Cells from the Mouse Eye

The adult mouse retinal stem cell (RSC) is a rare quiescent cell found within the ciliary epithelium (CE) of the mammalian eye1,2,3. The CE is made up of non-pigmented inner and pigmented outer cell layers, and the clonal RSC colonies that arise from a single pigmented cell from the CE are made up of both pigmented and non-pigmented cells which can be differentiated to form all the cell types of the neural retina and the RPE. There is some controversy about whether all the cells within the spheres all contain at least some pigment4; however the cells are still capable of forming the different cell types found within the neural retina1-3. In some species, such as amphibians and fish, their eyes are capable of regeneration after injury5, however; the mammalian eye shows no such regenerative properties. We seek to identify the stem cell in vivo and to understand the mechanisms that keep the mammalian retinal stem cells quiescent6-8, even after injury as well as using them as a potential source of cells to help repair physical or genetic models of eye injury through transplantation9-12. Here we describe how to isolate the ciliary epithelial cells from the mouse eye and grow them in culture in order to form the clonal retinal stem cell spheres. Since there are no known markers of the stem cell in vivo, these spheres are the only known way to prospectively identify the stem cell population within the ciliary epithelium of the eye.

In this video, we will demonstrate how to isolate retinal stem cells from the ciliary epithelium of the mouse eye and grow them in culture to form clonal retinal spheres. The spheres that are isolated possess the cardinal properties of stem cells: self-renewal and multipotentiality.

Isolamento de Células-Tronco da retina do olho do rato

The adult mouse retinal stem cell (RSC) is a rare quiescent cell found within the ciliary epithelium (CE) of the mammalian eye1,2,3. The CE is made up of ...

Biologia

Isolation and Culture of Adult Epithelial Stem Cells from Human Skin

The homeostasis of all self-renewing tissues is dependent on adult stem cells. As undifferentiated stem cells undergo asymmetric divisions, they generate daughter cells that retain the stem cell phenotype and transit-amplifying cells (TA cells) that migrate from the stem cell niche, undergo rapid proliferation and terminally differentiate to repopulate the tissue.
Epithelial stem cells have been identified in the epidermis, hair follicle, and intestine as cells with a high in vitro proliferative potential and as slow-cycling label-retaining cells in vivo 1-3. Adult, tissue-specific stem cells are responsible for the regeneration of the tissues in which they reside during normal physiologic turnover as well as during times of stress 4-5. Moreover, stem cells are generally considered to be multi-potent, possessing the capacity to give rise to multiple cell types within the tissue 6. For example, rodent hair follicle stem cells can generate epidermis, sebaceous glands, and hair follicles 7-9. We have shown that stem cells from the human hair follicle bulge region exhibit multi-potentiality 10.
Stem cells have become a valuable tool in biomedical research, due to their utility as an in vitro system for studying developmental biology, differentiation, tumorigenesis and for their possible therapeutic utility. It is likely that adult epithelial stem cells will be useful in the treatment of diseases such as ectodermal dysplasias, monilethrix, Netherton syndrome, Menkes disease, hereditary epidermolysis bullosa and alopecias 11-13. Additionally, other skin problems such as burn wounds, chronic wounds and ulcers will benefit from stem cell related therapies 14,15. Given the potential for reprogramming of adult cells into a pluripotent state (iPS cells)16,17, the readily accessible and expandable adult stem cells in human skin may provide a valuable source of cells for induction and downstream therapy for a wide range of disease including diabetes and Parkinson's disease.

A rapid, robust way of isolating viable adult epithelial stem cells from human skin is described. The method utilizes enzymatic digestion of skin collagen matrix , followed by plucking of hair follicles and isolation of single cell suspensions or tissue fragments for cell culture.

Isolamento e Cultura de Células-Tronco Adultas epitelial da pele humana

The homeostasis of all self-renewing tissues is dependent on adult stem cells. As undifferentiated stem cells undergo asymmetric divisions, they generate ...

Generation of Mice Derived from Induced Pluripotent Stem Cells

The production of induced pluripotent stem cells (iPSCs) from somatic cells provides a means to create valuable tools for basic research and may also produce a source of patient-matched cells for regenerative therapies. iPSCs may be generated using multiple protocols and derived from multiple cell sources. Once generated, iPSCs are tested using a variety of assays including immunostaining for pluripotency markers, generation of three germ layers in embryoid bodies and teratomas, comparisons of gene expression with embryonic stem cells (ESCs) and production of chimeric mice with or without germline contribution2. Importantly, iPSC lines that pass these tests still vary in their capacity to produce different differentiated cell types2. This has made it difficult to establish which iPSC derivation protocols, donor cell sources or selection methods are most useful for different applications.
The most stringent test of whether a stem cell line has sufficient developmental potential to generate all tissues required for survival of an organism (termed full pluripotency) is tetraploid embryo complementation (TEC)3-5. Technically, TEC involves electrofusion of two-cell embryos to generate tetraploid (4n) one-cell embryos that can be cultured in vitro to the blastocyst stage6. Diploid (2n) pluripotent stem cells (e.g. ESCs or iPSCs) are then injected into the blastocoel cavity of the tetraploid blastocyst and transferred to a recipient female for gestation (see Figure 1). The tetraploid component of the complemented embryo contributes almost exclusively to the extraembryonic tissues (placenta, yolk sac), whereas the diploid cells constitute the embryo proper, resulting in a fetus derived entirely from the injected stem cell line.
Recently, we reported the derivation of iPSC lines that reproducibly generate adult mice via TEC1. These iPSC lines give rise to viable pups with efficiencies of 5-13%, which is comparable to ESCs3,4,7 and higher than that reported for most other iPSC lines8-12. These reports show that direct reprogramming can produce fully pluripotent iPSCs that match ESCs in their developmental potential and efficiency of generating pups in TEC tests. At present, it is not clear what distinguishes between fully pluripotent iPSCs and less potent lines13-15. Nor is it clear which reprogramming methods will produce these lines with the highest efficiency. Here we describe one method that produces fully pluripotent iPSCs and "all- iPSC" mice, which may be helpful for investigators wishing to compare the pluripotency of iPSC lines or establish the equivalence of different reprogramming methods.

Generating induced pluripotent stem cell (iPSC) lines produces lines of differing developmental potential even when they pass standard tests for pluripotency. Here we describe a protocol to produce mice derived entirely from iPSCs, which defines the iPSC lines as possessing full pluripotency1.

Geração de camundongos derivadas de células-tronco pluripotentes induzidas

The production of induced pluripotent stem cells (iPSCs) from somatic cells provides a means to create valuable tools for basic research and may also ...

Isolation and Culture of Individual Myofibers and their Satellite Cells from Adult Skeletal Muscle

Muscle regeneration in the adult is performed by resident stem cells called satellite cells. Satellite cells are defined by their position between the basal lamina and the sarcolemma of each myofiber. Current knowledge of their behavior heavily relies on the use of the single myofiber isolation protocol. In 1985, Bischoff described a protocol to isolate single live fibers from the Flexor Digitorum Brevis (FDB) of adult rats with the goal to create an in vitro system in which the physical association between the myofiber and its stem cells is preserved 1. In 1995, Rosenblattmodified the Bischoff protocol such that myofibers are singly picked and handled separately after collagenase digestion instead of being isolated by gravity sedimentation 2, 3. The Rosenblatt or Bischoff protocol has since been adapted to different muscles, age or conditions 3-6. The single myofiber isolation technique is an indispensable tool due its unique advantages. First, in the single myofiber protocol, satellite cells are maintained beneath the basal lamina. This is a unique feature of the protocol as other techniques such as Fluorescence Activated Cell Sorting require chemical and mechanical tissue dissociation 7. Although the myofiber culture system cannot substitute for in vivo studies, it does offer an excellent platform to address relevant biological properties of muscle stem cells. Single myofibers can be cultured in standard plating conditions or in floating conditions. Satellite cells on floating myofibers are subjected to virtually no other influence than the myofiber environment. Substrate stiffness and coating have been shown to influence satellite cells' ability to regenerate muscles 8, 9 so being able to control each of these factors independently allows discrimination between niche-dependent and -independent responses. Different concentrations of serum have also been shown to have an effect on the transition from quiescence to activation. To preserve the quiescence state of its associated satellite cells, fibers should be kept in low serum medium 1-3. This is particularly useful when studying genes involved in the quiescence state. In serum rich medium, satellite cells quickly activate, proliferate, migrate and differentiate, thus mimicking the in vivo regenerative process 1-3. The system can be used to perform a variety of assays such as the testing of chemical inhibitors; ectopic expression of genes by virus delivery; oligonucleotide based gene knock-down or live imaging. This video article describes the protocol currently used in our laboratory to isolate single myofibers from the Extensor Digitorum Longus (EDL) muscle of adult mice (6-8 weeks old).

Isolation and culture of myofibers is the gold standard in vitro system to study the transition of satellite cells through quiescence, activation and differentiation. Importantly, the single myofiber culture system preserves the myofiber/stem cell association, which is an essential component of the muscle stem cell niche.

Isolamento e Cultura de miofibras individuais e suas células satélites do músculo esquelético

Muscle  regeneration in the adult is performed by resident stem cells called satellite  cells. Satellite cells are defined by  their position between the ...

Na&iuml;ve Adult Stem Cells Isolation from Primary Human Fibroblast Cultures

Over the last decade, several adult stem cell populations have been identified in human skin 1-4. The isolation of multipotent adult dermal precursors was first reported by Miller F. D laboratory 5, 6. These early studies described a multipotent precursor cell population from adult mammalian dermis 5. These cells--termed SKPs, for skin-derived precursors-- were isolated and expanded from rodent and human skin and differentiated into both neural and mesodermal progeny, including cell types never found in skin, such as neurons 5. Immunocytochemical studies on cultured SKPs revealed that cells expressed vimentin and nestin, an intermediate filament protein expressed in neural and skeletal muscle precursors, in addition to fibronectin and multipotent stem cell markers 6. Until now, the adult stem cells population SKPs have been isolated from freshly collected mammalian skin biopsies.
Recently, we have established and reported that a population of skin derived precursor cells could remain present in primary fibroblast cultures established from skin biopsies 7. The assumption that a few somatic stem cells might reside in primary fibroblast cultures at early population doublings was based upon the following observations: (1) SKPs and primary fibroblast cultures are derived from the dermis, and therefore a small number of SKP cells could remain present in primary dermal fibroblast cultures and (2) primary fibroblast cultures grown from frozen aliquots that have been subjected to unfavorable temperature during storage or transfer contained a small number of cells that remained viable 7. These rare cells were able to expand and could be passaged several times. This observation suggested that a small number of cells with high proliferation potency and resistance to stress were present in human fibroblast cultures 7.
We took advantage of these findings to establish a protocol for rapid isolation of adult stem cells from primary fibroblast cultures that are readily available from tissue banks around the world (Figure 1). This method has important significance as it allows the isolation of precursor cells when skin samples are not accessible while fibroblast cultures may be available from tissue banks, thus, opening new opportunities to dissect the molecular mechanisms underlying rare genetic diseases as well as modeling diseases in a dish.

Na&amp;#239;ve Adult Stem Cells Isolation from Primary Human Fibroblast Cultures

We report a method to isolate na&#239;ve multipotent skin-derived precursor (SKP) cells from primary human fibroblast cultures. We show that these SKPs derived from fibroblast cultures share similar stem cell properties to the ones derived directly from human skin biopsies. These cells express the neural crest marker, nestin, in addition to the multipotent markers such as OCT4 and Nanog.

Naïve Adult Stem Cells isolamento de culturas primárias de fibroblastos humanos

Over the last decade, several adult stem cell  populations have been identified in human skin 1-4.  The isolation of multipotent adult dermal precursors ...

Induction and Analysis of Epithelial to Mesenchymal Transition

Epithelial to mesenchymal transition (EMT) is essential for proper morphogenesis during development. Misregulation of this process has been implicated as a key event in fibrosis and the progression of carcinomas to a metastatic state. Understanding the processes that underlie EMT is imperative for the early diagnosis and clinical control of these disease states. Reliable induction of EMT in vitro is a useful tool for drug discovery as well as to identify common gene expression signatures for diagnostic purposes. Here we demonstrate a straightforward method for the induction of EMT in a variety of cell types. Methods for the analysis of cells pre- and post-EMT induction by immunocytochemistry are also included. Additionally, we demonstrate the effectiveness of this method through antibody-based array analysis and migration/invasion assays.

A straightforward method is described for the induction of epithelial to mesenchymal transition (EMT) in a variety of cell types. Methods for the analysis of cells in EMT states by immunocytochemistry are included.

Indução e Análise de epitelial para mesenquimal Transição

Epithelial  to mesenchymal transition (EMT) is essential for proper morphogenesis during  development. Misregulation of this  process has been implicated ...

Isolation and Culture of Primary Mouse Keratinocytes from Neonatal and Adult Mouse Skin

The keratinocyte (KC) is the predominant cell type in the epidermis, the outermost layer of the skin. Epidermal KCs play a critical role in providing skin defense by forming an intact skin barrier against environmental insults, such as UVB irradiation or pathogens, and also by initiating an inflammatory response upon those insults. Here we describe methods to isolate KCs from neonatal mouse skin and from adult mouse tail skin. We also describe culturing conditions using defined growth supplements (dGS) in comparison to chelexed fetal bovine serum (cFBS). Functionally, we show that both neonatal and adult KCs are highly responsive to high calcium-induced terminal differentiation, tight junction formation and stratification. Additionally, cultured adult KCs are susceptible to UVB-triggered cell death and can release large amounts of TNF upon UVB irradiation. Together, the methods described here will be useful to researchers for the setup of in vitro models to study epidermal biology in the neonatal mouse and/or the adult mouse.

Epidermal keratinocytes form a functional skin barrier and are positioned at the front line of host defense against external environmental insults. Here we describe methods for isolation and primary culture of epidermal keratinocytes from neonatal and adult mouse skin, and induction of terminal differentiation and UVB-triggered inflammatory response from keratinocytes.

Isolamento e Cultura de Queratinócitos de Rato Primário de Pele de Rato Neonatal e Adulto

The keratinocyte (KC) is the predominant cell type in the epidermis, the outermost layer of the skin. Epidermal KCs play a critical role in providing skin ...

Isolation, Characterization, and Differentiation of Cardiac Stem Cells from the Adult Mouse Heart

Myocardial infarction (MI) is a leading cause of morbidity and mortality around the world. A major goal of regenerative medicine is to replenish the dead myocardium after MI. Although several strategies have been used to regenerate myocardium, stem cell therapy remains a major approach to replenish the dead myocardium of an MI heart. Accumulating evidence suggests the presence of resident cardiac stem cells (CSCs) in the adult heart and their endocrine and/or paracrine effects on cardiac regeneration. However, CSC isolation and their characterization and differentiation toward myocardial cells, especially cardiomyocytes, remains a technical challenge. In the present study, we provided a simple method for the isolation, characterization, and differentiation of CSCs from the adult mouse heart. Here, we describe a density gradient method for the isolation of CSCs, where the heart is digested by a 0.2% collagenase II solution. To characterize the isolated CSCs, we evaluated the expression of CSCs/cardiac markers Sca-1, NKX2-5, and GATA4, and pluripotency/stemness markers OCT4, SOX2, and Nanog. We also determined the proliferation potential of isolated CSCs by culturing them in a Petri dish and assessing the expression of the proliferation marker Ki-67. For evaluating the differentiation potential of CSCs, we selected seven- to ten-days cultured CSCs. We transferred them to a new plate with a cardiomyocyte differentiation medium. They are incubated in a cell culture incubator for 12 days, while the differentiation medium is changed every three days. The differentiated CSCs express cardiomyocyte-specific markers: actinin and troponin I. Thus, CSCs isolated with this protocol have stemness and cardiac markers, and they have a potential for proliferation and differentiation toward cardiomyocyte lineage.

The overall goal of this article is to standardize the protocol for the isolation, characterization, and differentiation of cardiac stem cells (CSCs) from the adult mouse heart. Here, we describe a density gradient centrifugation method to isolate murine CSCs and elaborated methods for CSC culture, proliferation, and differentiation into cardiomyocytes.

Isolamento, caracterização e diferenciação de células-tronco cardíacas do coração de rato adulto

Myocardial infarction (MI) is a leading cause of morbidity and mortality around the world. A major goal of regenerative medicine is to replenish the dead ...

Simultaneous Isolation and Culture of Atrial Myocytes, Ventricular Myocytes, and Non-Myocytes from an Adult Mouse Heart

The isolation and culturing of cardiac myocytes from mice has been essential for furthering the understanding of cardiac physiology and pathophysiology. While isolating myocytes from neonatal mouse hearts is relatively straightforward, myocytes from the adult murine heart are preferred. This is because compared to neonatal cells, adult myocytes more accurately recapitulate cell function as it occurs in the adult heart in vivo. However, it is technically difficult to isolate adult mouse cardiac myocytes in the necessary quantities and viability, which contributes to an experimental impasse. Furthermore, published procedures are specific for the isolation of either atrial or ventricular myocytes at the expense of atrial and ventricular non-myocyte cells. Described here is a detailed method for isolating both atrial and ventricular cardiac myocytes, along with atrial and ventricular non-myocytes, simultaneously from a single mouse heart. Also provided are the details for optimal cell-specific culturing methods, which enhance cell viability and function. This protocol aims not only to expedite the process of adult murine cardiac cell isolation, but also to increase the yield and viability of cells for investigations of atrial and ventricular cardiac cells.

A method is described for the simultaneous isolation of myocytes and non-myocytes from both the atria and ventricles of a single adult mouse heart. This protocol results in consistent yields of highly viable cardiac myocytes and non-myocytes and details optimal cell-specific culture conditions for phenotyping and in vitro analysis.

Isolamento simultâneo e cultura de miócitos atrial, miócitos ventriculares e não-miócitos de um coração de rato adulto

The isolation and culturing of cardiac myocytes from mice has been essential for furthering the understanding of cardiac physiology and pathophysiology. ...

Isolation and Culture of Primary Oral Keratinocytes from the Adult Mouse Palate

For years, most studies involving keratinocytes have been conducted using human and mouse skin epidermal keratinocytes. Recently, oral keratinocytes have attracted attention because of their unique function and characteristics. They maintain the homeostasis of the oral epithelium and serve as resources for applications in regenerative therapies. However, in vitro studies that use oral primary keratinocytes from adult mice have been limited due to the lack of an efficient and well-established culture protocol. Here, oral primary keratinocytes were isolated from the palate tissues of adult mice and cultured in a commercial low-calcium medium supplemented with a chelexed-serum. Under these conditions, keratinocytes were maintained in a proliferative or stem cell-like state, and their differentiation was inhibited even after increased passages. Marker expression analysis showed that the cultured oral keratinocytes expressed the basal cell markers p63, K14, and &#945;6-integrin and were negative for the differentiation marker K13 and the fibroblast marker PDGFR&#945;. This method produced viable and culturable cells suitable for downstream applications in the study of oral epithelial stem cell functions in vitro.

The present protocol describes the isolation and culture of oral keratinocytes derived from the adult mouse palate. An evaluation method using immunostaining is also reported.

Isolamento e Cultura de Queratinócitos Orais Primários do Paladar do Rato Adulto

For years, most studies involving keratinocytes have been conducted using human and mouse skin epidermal keratinocytes. Recently, oral keratinocytes have ...