1. Preparação Regent <ol><li> Buffer: 1% (w / v) de soro albumina bovina em solução salina balanceada de Hank </li><li> Enzimas reconstituir. Enzima hialuronidase: 40mg / mL, dissolvido em tampão. Colagenase II: 23mg / mL, dissolvido em tampão. Utilizar soluções recentemente enzima preparado fresco para cada isolamento. Quando dissolvido loja, a 4 ° C até o uso para a digestão. </li><li> 50 mM de cloreto de cálcio em água destilada. Filtro de esterilização através de um filtro tamanho 0,2 uM poros. </li><li> Glândulas salivares do rato meio de cultura (MSG): DMEM: F12 com penicilina (100 UI / mL), estreptomicina (100 mg / mL), Glutamax (2 mM), fator de crescimento epidérmico-2 (20 ng / mL), fator de crescimento de fibroblastos -2 (20 ng / mL), N2 suplemento (1%), a insulina (10 mcg / mL) e dexametasona (1 mM). </li></ol> 2. A digestão enzimática do tecido mecânico e <ol><li> Pesar as glândulas salivares dissecadas. </li><li> Pique glândulas em uma polpa homogênea usando uma tesoura esterilizada dissecção curvas em uma pequena placa de Petri. </li><li> Coletar tecido picada em 14 mL tubos, usando 1mL de tampão por tecido submandibular 80mg. Lave os pratos de Petri limpa de tecido usando algumas das buffer. </li><li> Adicionar outro 1 mL de tampão por 80 mg de tecido, seguido de 25 mL de solução de enzima colagenase II, 25 mL de solução de enzima hialuronidase e 250 mL de solução de cloreto de cálcio por 80 mg de tecido. Se trabalhar com grandes quantidades de tecido, as etapas 2,4-2,9 pode ser realizada em frascos de cultura de tecidos T25 por conveniência. </li><li> Incubar em banho-maria agitando-se a 37 ° C por 20 minutos. Remova os tubos e triturar com uma pipeta para misturar completamente enzima através do tecido novo. </li><li> Substituir em banho-maria por mais 20 minutos. </li><li> Coletar tecido por centrifugação a 400 xg, por 8 minutos. Elimine o sobrenadante. </li><li> Ressuspender em 2 mL de tampão para cada tecido de 80 mg, e repita enzima ea adição de cloreto de cálcio como acima. Incubar 20 minutos em banho-maria agitando. Remova os tubos e triturar com uma pipeta para misturar enzimas completamente. </li><li> Incubar por 20 minutos finais em tremendo banho de água. Coletar células por centrifugação, tal como acima, elimine o sobrenadante. </li></ol> 3. Etapas de lavagem <ol><li> Ressuspender cada 80 mg de tecido em 2 mL de tampão e pipetar para lavar tecidos livres de enzimas. </li><li> Centrífuga como antes para coletar. Elimine o sobrenadante. </li><li> Repita lavagem usando um tampão por 80 mL de tecido mg. Centrífuga para coletar, elimine o sobrenadante. </li></ol> 4. Filtragem <ol><li> Ressuspender solução tecido em 1 mL de tampão por 80 mg de tecido. </li><li> Adicionar solução para filtro de 100 mm de poro de tamanho colocado mais de 50 tubos Falcon mL. Não aplique mais de 3 mL de solução de tecido picada por coluna, como filtros podem ficar bloqueados. Permitir a vazar. Remover o material filtrado pendurado na parte de baixo do filtro, com uma pipeta, e adicionar ao filtrado. </li><li> Use seringa com agulha de calibre 26 para tomar a partir de 50 mL filtrado tubos e aplicar filtros para 50 mm tamanho dos poros, em 5 tubos mL. Permitem filtrar, auxiliando soltando tampas, se necessário. </li><li> Tubos de centrífuga como antes para coletar. Elimine o sobrenadante. </li></ol> 5. Revestimento e Médio <ol><li> Combine todos os pellets em um volume. Contagem usando contador de células automatizado ou hemocitómetro. </li><li> Células da placa com uma densidade de 0,4 x 10 6 células por poço, de 12 bem-placa, ou 2,67 x 10 6 células por frasco de cultura de tecidos T25. Adicionar um meio MSG mL em cada poço ou 6 ml para cada frasco T25. </li><li> Incubar a 37 ° C. Esferas devem ser claramente visível de dia 2. </li></ol> 6. Resultados representativos: Depois de 2-3 dias em cultura, pequenos agregados de células (salispheres) será evidente nas culturas. Salispheres continuará a crescer em tamanho ao longo de um período de 10 dias em cultura. Representante fase de imagens de microscopia de contraste de salispheres são mostrados na Figura 1. Células em proliferação expressando as proteínas de células-tronco associada marcador pode ser isolado de tais esferas, otimamente entre os dias de isolamento pós 3-5, e são capazes de diferenciação em funcionais, saliva células acinares produzindo. <img src="/files/ftp_upload/2484/2484fig1.jpg" alt="Figura 1" /> Figura 1. Salisphere formação in vitro. Após a digestão mecânica e enzimática utilizando o protocolo atual, esferas de tamanho crescente pode ser encontrado nas culturas flutuante. Os painéis são representativos fase de imagens de microscopia de contraste de esferas de dias 0 (A), 4 (B), 7 (C) e 10 (D). Barra de escala = 50 mm.

<ol>
	<li>Vissink, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oral+Sequelae+of+Head+and+Neck+Radiotherapy.">Oral Sequelae of Head and Neck Radiotherapy.</a> Crit Rev Oral Biol Med. 14, 199-212 (2003).</li><li>Nape&amp;ntilde, a. s., J, J., Brennan, M. T., Fox, P. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diagnosis+and+treatment+of+xerostomia+(dry+mouth).">Diagnosis and treatment of xerostomia (dry mouth).</a> Odontology. 97, 76-83 (2009).</li><li>Denny, P. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Parenchymal+cell+proliferation+and+mechanisms+for+maintenance+of+granular+duct+and+acinar+cell+populations+in+adult+male+mouse+submandibular+gland.">Parenchymal cell proliferation and mechanisms for maintenance of granular duct and acinar cell populations in adult male mouse submandibular gland.</a> 235, 475-485 (2005).</li><li>Man, Y. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Persistence+of+a+perinatal+cellular+phenotype+in+submandibular+glands+of+adult+rat.">Persistence of a perinatal cellular phenotype in submandibular glands of adult rat.</a> J. Histochem. Cytochem. 43, 1203-1215 (1995).</li><li>Cotroneo, E., Proctor, G. B., Carpenter, G. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regeneration+of+acinar+cells+following+ligation+of+rat+submandibular+gland+retraces+the+embryonic-perinatal+pathway+of+cytodifferentiation.">Regeneration of acinar cells following ligation of rat submandibular gland retraces the embryonic-perinatal pathway of cytodifferentiation.</a> Differentiation. 79, 120-130 (2010).</li><li>Eirew, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+method+for+quantifying+normal+human+mammary+epithelial+stem+cells+with+in+vivo+regenerative+ability.">A method for quantifying normal human mammary epithelial stem cells with in vivo regenerative ability.</a> Nat Med. 14, 1384-1389 (2008).</li><li>Gorjup, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glandular+tissue+from+human+pancreas+and+salivary+gland+yields+similar+stem+cell+populations.">Glandular tissue from human pancreas and salivary gland yields similar stem cell populations.</a> European Journal of Cell Biology. 88, 409-421 (2009).</li><li>Alonso, L., Fuchs, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stem+cells+of+the+skin+epithelium.">Stem cells of the skin epithelium.</a> Proc Natl Acad Sci U S A. 100, 11830-11835 (2003).</li><li>Lombaert, I. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rescue+of+salivary+gland+function+after+stem+cell+transplantation+in+irradiated+glands.">Rescue of salivary gland function after stem cell transplantation in irradiated glands.</a> Plos One. 3, e2063-e2063 (2008).</li><li>Coppes, R. P., Goot, A. v. a. n. d. e. r., Lombaert, I. M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stem+Cell+Therapy+to+Reduce+Radiation-Induced+Normal+Tissue+Damage.">Stem Cell Therapy to Reduce Radiation-Induced Normal Tissue Damage.</a> Seminars in Radiation Oncology. 19, 112-121 (2009).</li></ol>

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O método de cultura de tecidos descrito aqui representa um protocolo reprodutível para o isolamento de células-tronco contêm salispheres das glândulas salivares de ratos. Estudos utilizando células isoladas desta forma têm destacado a capacidade regenerativa das células da glândula salivar-tronco 9. Transplante de uma centena de células + c-Kit derivado do salispheres induzida a recuperação funcional das glândulas salivares de ratos irradiados. Estes dados são estimulantes e fornecer um ponto de partida para a investigação de células-tronco para terapia baseada xerostomia. Muitos caminhos ainda precisam ser exploradas no entanto, incluindo a plena perfil de expressão do marcador de proteína das células-tronco, a capacidade das glândulas submandibulares de resgatar a função das glândulas salivares parótidas irradiadas e vice-versa, ea caracterização da suposta in vivo nicho de células-tronco de as células. Em última análise, a tradução deste protocolo para amostras de tecido humano eo potencial subseqüente para o tratamento da xerostomia em pacientes humanos utilizando células isoladas é a aplicação mais empolgante do método descrito.

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		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Hyaluronidase</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>H3506</td>
<td>Store at &#x2013; 20 &deg;C</td>
</tr>
<tr>
<td>Collagenase II</td>
<td>&nbsp;</td>
<td>Gibco</td>
<td>17101-015</td>
<td>Store at 4 &deg;C</td>
</tr>
<tr>
<td>Epidermal Growth Factor-2</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>E9644</td>
<td>Make a stock of 10 &mu;g / mL in phosphate buffered saline (PBS). Store at &#x2013; 20 &deg;C in single use aliquots.</td>
</tr>
<tr>
<td>Fibroblast Growth Factor-2</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>F0291</td>
<td>Make stock of 25 &mu;g / mL in PBS. Store at &#x2013; 20 &deg;C in single use aliquots.</td>
</tr>
<tr>
<td>N2 supplement</td>
<td>&nbsp;</td>
<td>Gibco</td>
<td>17502-048</td>
<td>As manufacturer instructions.</td>
</tr>
<tr>
<td>Insulin</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>I6634</td>
<td>Make stock of 2 mg / mL in tap water. Adjust water to pH 2-3 using glacial acetic acid prior to dissolving.</td>
</tr>
<tr>
<td>Dexamethasone</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>D4902</td>
<td>&nbsp;</td>
<tr>
<td>100 &mu;M pore-size sterile cell strainers</td>
<td>&nbsp;</td>
<td>BD Falcon</td>
<td>352360</td>
<td>&nbsp;</td>
<tr>
<td>Polystyrene round-bottomed tubes with cell strainer caps (50 &mu;M pore size)</td>
<td>&nbsp;</td>
<td>BD Falcon</td>
<td>352235</td>
<td>&nbsp;</td>		</tbody>
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isolation of mouse salivary gland stem cells

Madura glândulas salivares de origem humana e do rato compreendem um mínimo de cinco tipos de células, cada qual facilita a produção e excreção de saliva na cavidade oral. Células acinares serosas e as mucosas são as proteínas e fábricas que produzem muco da glândula, respectivamente, e representam a origem da produção de saliva. Uma vez sintetizados, os vários enzimática e outros componentes proteicas de saliva são secretados por uma série de células epiteliais ductais rolamentos do tipo morfologia, até que a eventual expulsão da saliva através de um duto grande para a cavidade da boca. A composição da saliva também é modificado pelas células ductais durante este processo. Na manifestação de doenças como a síndrome de Sjögren, e em algumas situações clínicas, tais como o tratamento de radioterapia para câncer de cabeça e pescoço, a produção de saliva pelas glândulas é drasticamente reduzido 1,2. A xerostomia resultante, uma sensação subjetiva de boca seca, afeta não só a capacidade do paciente de engolir e falar, mas também incentiva o desenvolvimento de cárie dentária e pode ser socialmente debilitante para o sofredor. A restauração da produção de saliva nas condições acima mencionadas clínica, portanto, representa uma necessidade não atendida clínica, e, como tal, vários estudos têm demonstrado a capacidade de regeneração das glândulas salivares 3-5. Mais para o isolamento de células-tronco como as populações de células de vários tecidos dentro do mouse e corpos humanos 6-8, temos mostrado usando o método descrito que as células-tronco isoladas das glândulas salivares do mouse pode ser usado para resgatar a produção de saliva na salivares irradiadas glândulas 9,10. Esta descoberta abre o caminho para o desenvolvimento de células-tronco terapias baseadas no tratamento de condições xerostomic em seres humanos, e também para a exploração da glândula salivar como um microambiente contendo células multipotentes com capacidades de auto-renovação.

Watch this Scientific Journal Video about Isolation of Mouse Salivary Gland Stem Cells at JoVE.com

Isolation of Mouse Salivary Gland Stem Cells

Um protocolo otimizado para o isolamento de células-tronco a partir da glândula salivar do mouse é descrito. O método emprega digestão enzimática e mecânica, e permite o isolamento de salispheres contendo células com características de células-tronco.

Isolamento de Células-Tronco do rato das Glândulas Salivares

Mature salivary glands of both human and mouse origin comprise a minimum of five cell types, each of which facilitates the production and excretion of ...

isolation-of-mouse-salivary-gland-stem-cells

Research

JoVE Journal

Biology

23.5K visualizações.  University Medical Center Groningen, University of Groningen. Um protocolo otimizado para o isolamento de células-tronco a partir da glândula salivar do mouse é descrito. O método emprega digestão enzimática e mecânica, e permite o isolamento de salispheres contendo células com características de células-tronco.

Vídeo: Isolation of Mouse Salivary Gland Stem Cells

Isolation and Transplantation of Hematopoietic Stem Cells (HSCs)

Isolation and Transplantation of Hematopoietic Stem Cells HSCs

Isolamento e Transplante de Células-Tronco Hematopoéticas (HSCs)

Biologia

Isolation and Derivation of Mouse Embryonic Germinal Cells

The ability of embryonic germinal cells (EG) to differentiate into primordial germinal cells (PGCs) and later into gametes during early developmental stages is a perfect model to address our hypothesis about cancer and infertility. This protocol shows how to isolate primordial germinal cells from developing gonads in 10.5-11.5 days post coitum (dpc) mouse embryos.  Developing gonadal ridges from mouse embryos (C57BL6J) were dissociated by mechanical disruption with collagenase, then plated in a mouse embryo fibroblast feeder layer (MEF-CF1) that was previously mitotically inactivated with mitomycin C in the presence of knockout media and supplemented with Leukemia Inhibitor Factor (LIF), basic Fibroblast Growth Factor (bFGF), and Stem Cell Factor (SCF).  Using these optimized methods for PCG identification, isolation, and establishment of culture conditions permits long term cultures of EG cells for more than 40 days.  The embryonic germinal cell lines showed embryonic phenotype and expression of common used markers of the pluripotent state.  Isolation and derivation of germinal cells in culture provide a tool to understand their development in vitro and offer the opportunity to monitor cumulative damage at genetic and epigenetic levels after exposure to oxidative stress.

The ability of embryonic germinal cells to differentiate into primordial germinal cells during early development stages is a perfect model to address our hypothesis about cancer and infertility. This protocol shows how to isolate primordial germinal cells from developing gonads in 10.5-11.5 days post coitum mouse embryos.

Isolamento e derivação de células embrionárias de rato Germinal

The ability of embryonic germinal cells (EG) to differentiate into primordial germinal cells (PGCs) and later into gametes during early developmental ...

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 ...

Isolation &#38; Characterization of Hoechstlow CD45negative Mouse Lung Mesenchymal Stem Cells

Tissue resident mesenchymal stem cells (MSC) are important regulators of tissue repair or regeneration, fibrosis, inflammation, angiogenesis and tumor formation. Taken together these studies suggest that resident lung MSC play a role during pulmonary tissue homeostasis, injury and repair during diseases such as pulmonary fibrosis (PF) and arterial hypertension (PAH). Here we describe a technology to define a population of resident lung MSC. The definition of this population in vivo pulmonary tissue using a define set of markers facilitates the repeated isolation of a well-characterized stem cell population by flow cytometry and the study of a specific cell type and function.

Isolation &amp; Characterization of Hoechstlow CD45negative Mouse Lung Mesenchymal Stem Cells

In this article we demonstrate the isolation of murine resident lung mesenchymal stem cells (lung MSC), their expansion, characterization and analysis of immunomodulatory properties.

Isolamento &amp; Caracterização da Hoechst Baixa CD45 Negativo Mouse Células-Tronco Mesenquimais de pulmão

Tissue resident mesenchymal stem cells (MSC) are important regulators of tissue repair or regeneration, fibrosis, inflammation, angiogenesis and tumor ...

Isolation of Basal Cells and Submucosal Gland Duct Cells from Mouse Trachea

The large airways are directly in contact with the environment and therefore susceptible to injury from toxins and infectious agents that we breath in 1. The large airways therefore require an efficient repair mechanism to protect our bodies. This repair process occurs from stem cells in the airways and isolating these stem cells from the airways is important for understanding the mechanisms of repair and regeneration. It is also important for understanding abnormal repair that can lead to airway diseases 2. The goal of this method is to isolate a novel stem cell population from the mouse tracheal submucosal gland ducts and to place these cells in in vitro and in vivo model systems to identify the mechanisms of repair and regeneration of the submucosal glands 3. This production shows methods that can be used to isolate and assay the duct and basal stem cells from the large airways 3.This will allow us to study diseases of the airway, such as cystic fibrosis, asthma and chronic obstructive pulmonary disease. Currently, there are no methods for isolation of submucosal gland duct cells and there are no in vivo models to study the regeneration of submucosal glands.

Here we demonstrate our protocol for isolation of basal and submucosal gland duct cells from mouse tracheas. We also demonstrate the method of injecting stem cells into the dorsal mouse fat pad to create an in vivo model of submucosal gland regeneration.

Isolamento de células basais e células Duct submucosa Glândulas de Rato Traquéia

The large airways are directly in contact with the environment and therefore susceptible to injury from toxins and infectious agents that we breath in 1. ...

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 ...

Genetic Modification and Recombination of Salivary Gland Organ Cultures

Branching morphogenesis occurs during the development of many organs, and the embryonic mouse submandibular gland (SMG) is a classical model for the study of branching morphogenesis. In the developing SMG, this process involves iterative steps of epithelial bud and duct formation, to ultimately give rise to a complex branched network of acini and ducts, which serve to produce and modify/transport the saliva, respectively, into the oral cavity1-3. The epithelial-associated basement membrane and aspects of the mesenchymal compartment, including the mesenchyme cells, growth factors and the extracellular matrix, produced by these cells, are critical to the branching mechanism, although how the cellular and molecular events are coordinated remains poorly understood 4. The study of the molecular mechanisms driving epithelial morphogenesis advances our understanding of developmental mechanisms and provides insight into possible regenerative medicine approaches. Such studies have been hampered due to the lack of effective methods for genetic manipulation of the salivary epithelium. Currently, adenoviral transduction represents the most effective method for targeting epithelial cells in adult glands in vivo5. However, in embryonic explants, dense mesenchyme and the basement membrane surrounding the epithelial cells impedes viral access to the epithelial cells. If the mesenchyme is removed, the epithelium can be transfected using adenoviruses, and epithelial rudiments can resume branching morphogenesis in the presence of Matrigel or laminin-1116,7. Mesenchyme-free epithelial rudiment growth also requires additional supplementation with soluble growth factors and does not fully recapitulate branching morphogenesis as it occurs in intact glands8. Here we describe a technique which facilitates adenoviral transduction of epithelial cells and culture of the transfected epithelium with associated mesenchyme. Following microdissection of the embryonic SMGs, removal of the mesenchyme, and viral infection of the epithelium with a GFP-containing adenovirus, we show that the epithelium spontaneously recombines with uninfected mesenchyme, recapitulating intact SMG glandular structure and branching morphogenesis. The genetically modified epithelial cell population can be easily monitored using standard fluorescence microscopy methods, if fluorescently-tagged adenoviral constructs are used. The tissue recombination method described here is currently the most effective and accessible method for transfection of epithelial cells with a wild-type or mutant vector within a complex 3D tissue construct that does not require generation of transgenic animals.

A technique to genetically manipulate epithelial cells within whole ex vivo cultured embryonic mouse submandibular glands (SMGs) using viral gene transfer is described. This method takes advantage of the innate ability of SMG epithelium and mesenchyme to spontaneously recombine after separation and infection of epithelial rudiments with adenoviral vectors.

Modificação genética e recombinação de culturas de órgãos de glândula salivar

Branching  morphogenesis occurs during the development of many organs, and the embryonic  mouse submandibular gland (SMG) is a classical model for the ...

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, Culture, and Transplantation of Muscle Satellite Cells

Muscle satellite cells are a stem cell population required for postnatal skeletal muscle development and regeneration, accounting for 2-5% of sublaminal nuclei in muscle fibers. In adult muscle, satellite cells are normally mitotically quiescent. Following injury, however, satellite cells initiate cellular proliferation to produce myoblasts, their progenies, to mediate the regeneration of muscle. Transplantation of satellite cell-derived myoblasts has been widely studied as a possible therapy for several regenerative diseases including muscular dystrophy, heart failure, and urological dysfunction. Myoblast transplantation into dystrophic skeletal muscle, infarcted heart, and dysfunctioning urinary ducts has shown that engrafted myoblasts can differentiate into muscle fibers in the host tissues and display partial functional improvement in these diseases. Therefore, the development of efficient purification methods of quiescent satellite cells from skeletal muscle, as well as the establishment of satellite cell-derived myoblast cultures and transplantation methods for myoblasts, are essential for understanding the molecular mechanisms behind satellite cell self-renewal, activation, and differentiation. Additionally, the development of cell-based therapies for muscular dystrophy and other regenerative diseases are also dependent upon these factors.

However, current prospective purification methods of quiescent satellite cells require the use of expensive fluorescence-activated cell sorting (FACS) machines. Here, we present a new method for the rapid, economical, and reliable purification of quiescent satellite cells from adult mouse skeletal muscle by enzymatic dissociation followed by magnetic-activated cell sorting (MACS). Following isolation of pure quiescent satellite cells, these cells can be cultured to obtain large numbers of myoblasts after several passages. These freshly isolated quiescent satellite cells or ex vivo expanded myoblasts can be transplanted into cardiotoxin (CTX)-induced regenerating mouse skeletal muscle to examine the contribution of donor-derived cells to regenerating muscle fibers, as well as to satellite cell compartments for the examination of self-renewal activities.

The isolation and culture of a pure population of quiescent satellite cells, a muscle stem cell population, is essential to the understanding of muscle stem cell biology and regeneration, as well as stem cell transplantation for therapies in muscular dystrophy and other degenerative diseases.&#160;

Isolamento, cultura e transplante de células musculares satélite

Muscle satellite cells are a stem cell population required for postnatal skeletal muscle development and regeneration, accounting for 2-5% of sublaminal ...

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

Understanding the cellular and molecular mechanisms that underlie tooth regeneration and renewal has become a topic of great interest1-4, and the mouse incisor provides a model for these processes. This remarkable organ grows continuously throughout the animal&#39;s life and generates all the necessary cell types from active pools of adult stem cells housed in the labial (toward the lip) and lingual (toward the tongue) cervical loop (CL) regions. Only the dental stem cells from the labial CL give rise to ameloblasts that generate enamel, the outer covering of teeth, on the labial surface. This asymmetric enamel formation allows abrasion at the incisor tip, and progenitors and stem cells in the proximal incisor ensure that the dental tissues are constantly replenished. The ability to isolate and grow these progenitor or stem cells in vitro allows their expansion and opens doors to numerous experiments not achievable in vivo, such as high throughput testing of potential stem cell regulatory factors. Here, we describe and demonstrate a reliable and consistent method to culture cells from the labial CL of the mouse incisor.

The continuously growing mouse incisor provides a model for studying renewal of dental tissues from dental epithelial stem cells (DESCs). A robust system for consistently and reliably obtaining these cells from the incisor and expanding them in vitro is reported here.

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 ...