Los autores agradecen a Xiu-Ping Wang ayuda inicial con culturas DESC. Los autores son financiados en parte por becas y subvenciones de los Institutos Nacionales de Salud (K99-DE022059 a AHJ, K12-GM081266 a MGC, K08-DE022377-02 a OH y R01-DE021420 a ODK).

1. Dissect Menores Hemi-mandíbulas de Adultos Mouse <ol><li> Antes de realizar este protocolo, obtener la aprobación institucional necesario y asegúrese de cumplir con todas las directrices de cuidado de animales. La eutanasia a los animales utilizando el estándar aprobado por el IACUC procedimientos. Para este trabajo se utilizó asfixia de CO 2 seguido por dislocación cervical. <ol><li> OPCIONAL: Cabeza separada del resto del cuerpo, utilizando una hoja de afeitar. </li></ol></li><li> Quite la piel del labio inferior hasta el cuello. </li><li> Usando un bisturí, hacer una incisión entre los dos incisivos inferiores, que están conectados libremente. Al aplicar presión, la mandíbula se dividirá en dos mitades. </li><li> Cuña El escalpelo entre el cóndilo de la mandíbula y la articulación mandibular temporal y cortar junto con el músculo de la mandíbula para separar la mandíbula. </li><li> Quite todo el músculo, tendón y ligamento hasta sólo queda el hueso. </li></ol> 2. Aislar el Incisivo <ol><li> Usando un bisturí # 15, Afeitarse cuidadosamente el hueso en un ángulo de 45 ° desde el extremo distal hasta el extremo proximal de la región membranosa. Use la punta del bisturí para recoger la basura cualquier hueso remanente incluyendo los fragmentos de hueso en el borde. Poco a poco empiezan a recoger lejos en la cortical lingual de la mandíbula, empezando justo debajo del 3 er molar. </li><li> Continúe hasta que el incisivo es limpio, trabajando cuidadosamente hacia el área que contiene el CL. </li><li> Retire el conjunto de nervios alveolar inferior. </li><li> Haga un corte limpio para quitar primero el hueso y los tejidos proximal a la CL. </li><li> Haga un segundo corte distal con el bisturí, más o menos por debajo del 2 º molar. La región de los incisivos proximal se diseca a cabo mediante la inserción de la escalpelo entre el hueso y la superficie medial del incisivo proximal. Esto debe hacerse cuidadosamente, con el fin de no romper el tejido. El movimiento de la incisión es proximal-distal. PASO ALTERNATIVA: Quite la mayor cantidad de hueso como sea posible a lo largo de la zona superior de la incisor. Una vez que el área se limpia, quite cuidadosamente todo incisivo usando un tamaño de 5 pinzas para manejar la porción de esmalte más cercana a la región CL. </li><li> Coloque el tejido diseccionado (ya sea CL aislado como en la Figura 3E, o todo incisivo como en alternativa Paso 2.5) en 2% de colagenasa en 1X PBS durante 3-4 horas a 4 ° C en una placa de baja adherencia. Para 1-10 incisivos, utilice 100 l por pocillo en una placa de 12 pocillos. Por más de 10 incisivos, utilice 500 l por pocillo en una placa de 6 pocillos de baja adherencia. </li></ol> 3. Microdissect el labial Cervical Loop <ol><li> Retire región CL de la colagenasa y lugar en los medios de comunicación DMEM/F12 frío. </li><li> Tire suavemente en el extremo inferior de la CL utilizando ya sea el tamaño de 5 fórceps o una jeringa de insulina (1 cc, 28 G ½); el mesénquima se vendrá abajo mientras que el epitelio se mantiene intacta. El CL y el epitelio adyacente que se extiende lateralmente como una estructura &quot;de ala&quot; serán visibles. </li><li> Eliminarla yema apical del epitelio adyacente al hacer una incisión en forma de V a cada lado, y colocar inmediatamente en DMEM/F12 frío en hielo. </li><li> Repita el procedimiento para todas las cartas circulares, recoge en 1,5 ml tubo de microcentrífuga. </li><li> Centrifugar el tubo de microcentrífuga con muestras, extraer los medios de comunicación, reemplace con una solución de 100 células destacamento l. </li><li> Incubar los tejidos en solución de desprendimiento de las células durante 30 min a 37 ° C. </li><li> Disociar los tejidos para producir una suspensión de células pipeteando suavemente hacia arriba y el uso de un bajo coeficiente de adherencia de puntas de pipeta 1.000 l abajo. Las células también se pueden colocar a través de un filtro de células estéril para lograr la disociación. </li><li> Contar las células con un hemocitómetro, placa de plástico de cultivo de tejido u otro material deseado. </li></ol> 4. Cultivo primario de DESCs <ol><li> Las células de la placa en una placa de 6 pocillos a 1-6 x 10 4 células / ml en medio DESC, que consta de DMEM/F12 complementado con proteína recombinante EGF de ratón a una concentración de 20 ng / ml, la proteína recombinante FGF a una concentración de 25 ng / ml, 1X suplemento B27, y solución de 1% de antibiótico (penicilina, 100 U / ml, estreptomicina, 50 mg / ml). </li><li> Crecer células sembradas en una placa de 6 pocillos a 37 º C en 5% de CO 2 en 1 ml de medio DESC. No moleste a los cultivos iniciales durante 5 días después de la siembra para permitir la adhesión celular y la formación de colonias. </li><li> Para el primer cambio de los medios de comunicación, reemplace la mitad del volumen (500 l) de los medios tradicionales con la mitad del volumen (500 l) de los nuevos medios de comunicación. Compruebe colonias bajo el microscopio al cambiar el soporte. </li><li> Después del primer cambio de los medios de comunicación, a seguir cambiando los medios de comunicación (1-2 ml) cada 2 días. Compruebe colonias bajo el microscopio al cambiar el soporte. </li></ol> 5. DESCs Passage <ol><li> Medios Aspirar y lavar las células 3 veces con PBS precalentado. </li><li> Añadir precalentado 0,25% de tripsina-EDTA en solución salina y se incuba a 37 ° C durante 10-15 minutos para los DESCs separar. </li><li> Añadir media DESC para neutralizar la tripsina, la pipeta suavemente a lo largo de la placa de cultivo de tejidos para recoger las células, y el lugar en un tubo cónico de 15 ml. </li><li> Centrifugar las células a 800 xg durante 2 min. </li></ol>

<ol>
	<li>Sharpe, P. T., Young, C. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Test-tube+teeth..">Test-tube teeth..</a> Sci Am. 293, 34-41 (2005).</li><li>D'Souza, R. N., Klein, O. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Unraveling+the+molecular+mechanisms+that+lead+to+supernumerary+teeth+in+mice+and+men:+Current+concepts+and+novel+approaches.">Unraveling the molecular mechanisms that lead to supernumerary teeth in mice and men: Current concepts and novel approaches.</a> Cells Tissues Organs. 186, 60-69 (2007).</li><li>Jernvall, J., Thesleff, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tooth+shape+formation+and+tooth+renewal:+evolving+with+the+same+signals.">Tooth shape formation and tooth renewal: evolving with the same signals.</a> Development. 139, 3487-3497 (2012).</li><li>Yen, A. H., Yelick, 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=Dental+Tissue+Regeneration+A+Mini-Review.">Dental Tissue Regeneration A Mini-Review.</a> Gerontology. 57, 85-94 (2011).</li><li>Jheon, A. H., Seidel, K., Biehs, B., Klein, O. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=From+molecules+to+mastication:+the+development+and+evolution+of+tooth+development.">From molecules to mastication: the development and evolution of tooth development.</a> WIREs Dev Biol. 2, 165-182 (2013).</li><li>Wang, X. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+integrated+gene+regulatory+network+controls+stem+cell+proliferation+in+teeth.">An integrated gene regulatory network controls stem cell proliferation in teeth.</a> PLoS Biol. 5, (2007).</li><li>Harada, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Localization+of+putative+stem+cells+in+dental+epithelium+and+their+association+with+Notch+and+FGF+signaling.">Localization of putative stem cells in dental epithelium and their association with Notch and FGF signaling.</a> J Cell Biol. 147, 105-120 (1999).</li><li>Juuri, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sox2++stem+cells+contribute+to+all+epithelial+lineages+of+the+tooth+via+Sfrp5++progenitors.">Sox2+ stem cells contribute to all epithelial lineages of the tooth via Sfrp5+ progenitors.</a> Dev Cell. 23, 317-328 (2012).</li><li>Seidel, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hedgehog+signaling+regulates+the+generation+of+ameloblast+progenitors+in+the+continuously+growing+mouse+incisor.">Hedgehog signaling regulates the generation of ameloblast progenitors in the continuously growing mouse incisor.</a> Development. 137, 3753-3761 (2010).</li><li>Smith, C. E., Warshawsky, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+renewal+in+the+enamel+organ+and+the+odontoblast+layer+of+the+rat+incisor+as+followed+by+radioautography+using+3H-thymidine.">Cellular renewal in the enamel organ and the odontoblast layer of the rat incisor as followed by radioautography using 3H-thymidine.</a> Anat Rec. 183, 523-561 (1975).</li><li>Smith, C. E., Warshawsky, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+analysis+of+cell+turnover+in+the+enamel+organ+of+the+rat+incisor.+Evidence+for+ameloblast+death+immediately+after+enamel+matrix+secretion.">Quantitative analysis of cell turnover in the enamel organ of the rat incisor. Evidence for ameloblast death immediately after enamel matrix secretion.</a> Anat Rec. 187, 63-98 (1977).</li><li>Parsa, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Signaling+by+FGFR2b+controls+the+regenerative+capacity+of+adult+mouse+incisors.">Signaling by FGFR2b controls the regenerative capacity of adult mouse incisors.</a> Development. 137, 3743-3752 (2010).</li><li>Chang, J. Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Self-renewal+and+multilineage+differentiation+of+mouse+dental+epithelial+stem+cells.">Self-renewal and multilineage differentiation of mouse dental epithelial stem cells.</a> Stem Cell Res. 11, 990-1002 (2013).</li><li>Biehs, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bmi1+represses+Ink4a/Arf+and+Hox+genes+to+regulate+stem+cells+in+the+rodent+incisor.">Bmi1 represses Ink4a/Arf and Hox genes to regulate stem cells in the rodent incisor.</a> Nat Cell Biol. 15, 846-852 (2013).</li><li>Hwang, W. S., Tonna, E. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Autoradiographic+Analysis+of+Labeling+Indices+and+Migration+Rates+of+Cellular+Component+of+Mouse+Incisors+Using+Tritiated+Thymidine+(H3tdr).">Autoradiographic Analysis of Labeling Indices and Migration Rates of Cellular Component of Mouse Incisors Using Tritiated Thymidine (H3tdr).</a> J Dent Res. 44, 42-53 (1965).</li><li>Li, C. Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=E-cadherin+regulates+the+behavior+and+fate+of+epithelial+stem+cells+and+their+progeny+in+the+mouse+incisor.">E-cadherin regulates the behavior and fate of epithelial stem cells and their progeny in the mouse incisor.</a> Dev Biol. 366, 357-366 (2012).</li><li>Smith, C. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+and+chemical+events+during+enamel+maturation.">Cellular and chemical events during enamel maturation.</a> Crit Rev Oral Biol Med. 9, 128-161 (1998).</li><li>Lapthanasupkul, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ring1a/b+polycomb+proteins+regulate+the+mesenchymal+stem+cell+niche+in+continuously+growing+incisors.">Ring1a/b polycomb proteins regulate the mesenchymal stem cell niche in continuously growing incisors.</a> Dev Biol. 367, 140-153 (2012).</li><li>Chavez, M. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+dental+epithelial+stem+cells+from+the+mouse+incisor+with+two-dimensional+and+three-dimensional+platforms.">Characterization of dental epithelial stem cells from the mouse incisor with two-dimensional and three-dimensional platforms.</a> Tissue Eng Part C Methods. 19, 15-24 (2013).</li><li>Kawano, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Establishment+of+dental+epithelial+cell+line+(HAT-7)+and+the+cell+differentiation+dependent+on+Notch+signaling+pathway.">Establishment of dental epithelial cell line (HAT-7) and the cell differentiation dependent on Notch signaling pathway.</a> Connect Tissue Res. 43, 409-412 (2002).</li><li>Briggaman, R. A., Abele, D. C., Harris, S. R., Wheeler, C. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+and+characterization+of+a+viable+suspension+of+postembryonic+human+epidermal+cells.">Preparation and characterization of a viable suspension of postembryonic human epidermal cells.</a> J Invest Dermatol. 48, 159-168 (1967).</li><li>Fusenig, N. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+cultivation+of+epidermal+cells+from+embryonic+mouse+skin.">Isolation and cultivation of epidermal cells from embryonic mouse skin.</a> Naturwissenschaften. 58, 421 (1971).</li><li>Fusenig, N. E., Worst, P. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mouse+epidermal+cell+cultures.+I.+Isolation+and+cultivation+of+epidermal+cells+from+adult+mouse+skin.">Mouse epidermal cell cultures. I. Isolation and cultivation of epidermal cells from adult mouse skin.</a> J Invest Dermatol. 63, 187-193 (1974).</li><li>Rheinwald, J. G., Green, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Serial+cultivation+of+strains+of+human+epidermal+keratinocytes:+the+formation+of+keratinizing+colonies+from+single+cells.">Serial cultivation of strains of human epidermal keratinocytes: the formation of keratinizing colonies from single cells.</a> Cell. 6, 331-343 (1975).</li><li>Barrandon, Y., Green, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Three+clonal+types+of+keratinocyte+with+different+capacities+for+multiplication.">Three clonal types of keratinocyte with different capacities for multiplication.</a> Proc Natl Acad Sci U S A. 84, 2302-2306 (1987).</li><li>Blanpain, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Self-renewal,+multipotency,+and+the+existence+of+two+cell+populations+within+an+epithelial+stem+cell+niche.">Self-renewal, multipotency, and the existence of two cell populations within an epithelial stem cell niche.</a> Cell. 118, 635-648 (2004).</li><li>Toma, J. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+multipotent+adult+stem+cells+from+the+dermis+of+mammalian+skin.">Isolation of multipotent adult stem cells from the dermis of mammalian skin.</a> Nat Cell Biol. 3, 778-784 (2001).</li><li>Nowak, J. A., 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=Isolation+and+culture+of+epithelial+stem+cells.">Isolation and culture of epithelial stem cells.</a> Methods Mol Biol. 482, 215-232 (2009).</li><li>Rheinwald, J. G., Green, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epidermal+growth+factor+and+the+multiplication+of+cultured+human+epidermal+keratinocytes.">Epidermal growth factor and the multiplication of cultured human epidermal keratinocytes.</a> Nature. 265, 421-424 (1977).</li><li>Brewer, G. J., Torricelli, J. R., Evege, E. K., Price, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimized+survival+of+hippocampal+neurons+in+B27-supplemented+Neurobasal,+a+new+serum-free+medium+combination.">Optimized survival of hippocampal neurons in B27-supplemented Neurobasal, a new serum-free medium combination.</a> J Neurosci Res. 35, 567-576 (1993).</li><li>Lesuisse, C., Martin, L. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long-term+culture+of+mouse+cortical+neurons+as+a+model+for+neuronal+development,+aging,+and+death.">Long-term culture of mouse cortical neurons as a model for neuronal development, aging, and death.</a> J Neurobiol. 51, 9-23 (2002).</li><li>Felszeghy, S., Suomalainen, M., Thesleff, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Notch+signalling+is+required+for+the+survival+of+epithelial+stem+cells+in+the+continuously+growing+mouse+incisor.">Notch signalling is required for the survival of epithelial stem cells in the continuously growing mouse incisor.</a> Differentiation. 80, 241-248 (2010).</li><li>Fujimori, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Wnt/beta-catenin+signaling+in+the+dental+mesenchyme+regulates+incisor+development+by+regulating+Bmp4.">Wnt/beta-catenin signaling in the dental mesenchyme regulates incisor development by regulating Bmp4.</a> Dev Biol. 348, 97-106 (2010).</li><li>Klein, O. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+FGF+signaling+loop+sustains+the+generation+of+differentiated+progeny+from+stem+cells+in+mouse+incisors.">An FGF signaling loop sustains the generation of differentiated progeny from stem cells in mouse incisors.</a> Development. 135, 377-385 (2008).</li></ol>

Los autores no tienen conflictos de intereses a revelar.

Las células epiteliales fueron primero cultivadas con éxito hace más de 40 años 21-24, y más recientemente, el aislamiento con éxito de células madre epiteliales 25-27 ha avanzado nuestro conocimiento de la biología epitelial. Se presenta un protocolo para aislar el DESCs del incisivo ratón adulto, una población relativamente poco estudiado de las células madre que tiene el potencial de producir importantes conocimientos sobre la biología y la formación del esmalte dental. Este protoco...

Una de las características únicas de los vertebrados es la evolución de los dientes. El diente se ha convertido en un importante sistema modelo para muchas áreas de investigación, como las vías moleculares y especializaciones morfológicas relevantes a este órgano se han investigado desde varias perspectivas, incluso por los biólogos del desarrollo y evolución 5. Más recientemente, el campo de la medicina regenerativa ha comenzado a obtener información valiosa utilizando el diente como modelo. En particular, el descubrimiento de las células madre epiteliales dentales ha sido un importante avance 6-13. Todos los roedores tienen incisivos de crecimiento continuo cuyo crecimiento es impulsado por las células madre, por lo que estos dientes un modelo de sistema accesible para estudiar la regulación de células madre adultas. Experimentos de etiquetado en la década de 1970 10,11, seguido de linaje genético experimentos de rastreo 8,9,12,14, han demostrado que DESCs residen en la región proximal del incisivo. El vástagoprogenie de células en el compartimiento epitelial en el lado labial movimiento fuera del nicho putativo, conocido como el bucle de cuello uterino labial (CL), y contribuir a una población de células llamadas de tránsito de amplificación de células (TA) (Figura 1). Específicamente, DESCs residen en el epitelio externo del esmalte (OEE) y retículo estrellado (SR) 8,9,14, y el epitelio interno del esmalte (IEE) da lugar a las células de AT que progresan a través de un número limitado de ciclos celulares y luego mueven distalmente a lo largo de la longitud del incisivo (Figura 1). Los ameloblastos de diferenciación en el incisivo ratón continúan moviéndose distalmente a lo largo del incisivo a un ritmo notable de aproximadamente 350 micras en un solo día 15,16. Mientras se mueven, las células se diferencian en ameloblastos maduros y células estrato intermedio (SI). Después de depositar el espesor completo de la matriz del esmalte, muchos de los ameloblastos someterse a la apoptosis, y las células restantes se encogen en tamaño y regulan maduración del esmalte &lt;sup&gt; 17. El linaje de los otros tipos de células en el labial CL, como el SR, es menos clara, y los datos con respecto a las células madre en el mesénquima de 18 años y en la lingual CL sólo están comenzando a emerger. Utilizando el modelo de ratón incisivo, un número de grupos han trabajado para dilucidar las vías genéticas y los procesos biológicos de células que participan en la renovación de órganos basado en células madre natural. Sin embargo, el labial CL contiene un número relativamente pequeño de células, estimada en aproximadamente el 5,000 por incisivo ratón, lo que hace que el trabajo con células primarias desafiante. Por lo tanto, se han hecho esfuerzos para la cultura y expandir estas células in vitro 6,16,19,20 con el fin de abrir nuevas puertas a los enfoques experimentales que no son alcanzables in vivo. El estudio más reciente ha demostrado que estas células pueden tanto auto-renovarse y diferenciarse en células que expresan amelogenina cuando se cultivan 13. A continuación, describimos y demostrar un método para the cultura fiable y consistente de las células de la labial ratón CL.

<table border="0" cellpadding="0" cellspacing="0" width="584">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Forceps, size 5</td>
			<td style="width:83px;">Fine Science Tools</td>
			<td style="width:119px;">11251-30</td>
			<td style="width:167px;">FST by Dumont, Dumoxel,&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Forceps Straight, fine, 3.5</td>
			<td style="width:83px;">Roboz</td>
			<td style="width:119px;">RS5070</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Razorblades</td>
			<td style="width:83px;">Electron Microscopy Services</td>
			<td style="width:119px;">#71960</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Scalpel Handle No.3</td>
			<td style="width:83px;">VWR</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Feather Blade No. 15</td>
			<td style="width:83px;">Electron Microscopy Services</td>
			<td style="width:119px;">#72044-15</td>
			<td>Surgical Stainless Steel</td>
		</tr>
		<tr height="126">
			<td height="126" style="height:126px;width:216px;">Collagenase Type-1 filtered</td>
			<td style="width:83px;">Worthington Biochemical Corporation</td>
			<td style="width:119px;">#4214</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Insulin syringe</td>
			<td style="width:83px;">BD</td>
			<td style="width:119px;">#329424</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Accumax</td>
			<td style="width:83px;">EMD Millipore</td>
			<td style="width:119px;">#SCR006</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">DMEM/F12</td>
			<td style="width:83px;">Gibco</td>
			<td style="width:119px;">11320-033</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">EGF</td>
			<td style="width:83px;">R&#38;D</td>
			<td style="width:119px;">2028-EG-200</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">FGF2</td>
			<td style="width:83px;">R&#38;D</td>
			<td style="width:119px;">233-FB-025</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">B27 Supplement</td>
			<td style="width:83px;">Gibco</td>
			<td style="width:119px;">10889-038</td>
			<td></td>
		</tr>
	</tbody>
</table>

isolation and culture of dental epithelial stem cells from the adult mouse incisor

La comprensión de los mecanismos celulares y moleculares que subyacen a la regeneración de dientes y la renovación se ha convertido en un tema de gran interés 1-4, y el incisivo ratón proporciona un modelo para estos procesos. Este órgano notable crece continuamente durante toda la vida del animal y genera todos los tipos de células necesarios de piscinas activas de las células madre adultas alojados en el labial (hacia el labio) y lingual (hacia la lengua) (CL) regiones cervical bucle. Sólo las células madre dentales de la labial CL dan lugar a ameloblastos que generan esmalte, la cubierta externa de los dientes, en la superficie labial. Esta formación del esmalte asimétrica permite a la abrasión en la punta de los incisivos, y progenitores y células madre en el incisivo proximal asegurar que los tejidos dentales se reponen constantemente. La capacidad para aislar y cultivar estas células progenitoras o madre in vitro permite su expansión y abre las puertas a numerosos experimentos no alcanzables in vivo, tales como High prueba de rendimiento de los factores de regulación potencial de células madre. A continuación, describimos y demostrar un método fiable y consistente para las células de cultivo de la CL labial del incisivo ratón.

El ratón hemimandíbula comprende un continuo crecimiento de los incisivos y molares tres raíces (Figura 1A). Todos los dientes se componen de la dentina y el esmalte, los dos componentes mineralizados de la corona del diente (Figuras 1a y 1a &#39;). Las casas incisivos dos nichos de células madre llaman la CL labial y lingual CL, y el esmalte se forma exclusivamente en el lado labial (Figura 1A &#39;). DESCs son responsables de la formación del esm...

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

El continuo crecimiento de los incisivos ratón proporciona un modelo para el estudio de la renovación de los tejidos dentales a partir de células madre epiteliales dentales (DESCs). Un sistema sólido para la obtención de manera consistente y confiable de estas células de la incisivo y expandir in vitro se informó aquí.

Aislamiento y cultivo de células madre epiteliales dental del adulto Ratón 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 vistas.  University of California, San Francisco.  El continuo crecimiento de los incisivos ratón proporciona un modelo para el estudio de la renovación de los tejidos dentales a partir de células madre epiteliales dentales (DESCs). Un sistema sólido para la obtención de manera consistente y confiable de estas células de la incisivo y expandir in vitro se informó aquí. 

Video: 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.

Aislamiento de células madre de la retina del ojo del ratón

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 ...

Investigación

Biología

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.

Aislamiento y cultivo de células madre adultas epiteliales de la piel 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.

Generación de ratones derivados de células madre pluripotentes inducidas

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.

Aislamiento y cultivo de fibras individuales y sus células satélite del músculo esquelético adulto

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 Aislamiento de Primaria cultivos 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.

Inducción y Análisis de epitelial a mesenquimal de transición

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.

Aislamiento y cultivo de queratinocitos de ratón primario de piel de ratón neonatal y 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.

Aislamiento, caracterización y diferenciación de células cardiacas desde el corazón de ratón 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.

Aislamiento simultáneo y cultura de los miocitos atriales, los miocitos ventriculares y los no miocitos de un corazón de ratón 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.

Aislamiento y cultivo de queratinocitos orales primarios del paladar del ratón adulto

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