Los autores reconocen el programa de esterilización canina en la UNESP-FCAV por proporcionar amablemente los ovarios. Este trabajo fue financiado por becas del FAPESP (proceso nº 2013/14293-0) y capas.

Este experimento fue realizado con los ovarios de cuatro perras mestizo donados después de la cirugía electiva en un programa de esterilización canina. Este experimento fue aprobado por el Comité de ética en el uso de animales de la UNESP-FCAV (Protocolo nº 026991/13).
1. experimental Preparación
<ol><li>Preparar o comprar 500 mL estéril de Dulbecco tamponada con fosfato solución salina (DPBS) sin calcio ni magnesio.</li>
	<li>Preparar una colagenasa stock solución mezclando 40 μg de la enzima en 1 mL de DPBS. Filtrar usando un filtro de jeringa de 0,2 μm y almacenar en alícuotas de 2 mL a-20 ° C.</li>
	<li>Preparar los medios de cultivo de glucosa baja de modificado Eagle de Dulbecco medium (DMEM) con 10% de suero fetal y 1% de los antibióticos.</li>
	<li>Recoger material estéril: tijeras y pinzas, un frasco de cultivo de tejidos, platos, tubos y pipetas de 10 mL y 5 mL.</li>
	<li>Uso de equipo de laboratorio de cultivo celular estándar: un gabinete de seguridad biológica (BSC), una incubadora de cultivo de célula fijada en 5% CO2 y 38 ° C y una centrífuga.</li>
	<li>Limpiar el BSC: con las manos enguantadas, la esterilice con el 70% EtOH, usando luces UV.</li>
</ol>2. aislamiento de células madre mesenquimales del ovario
<ol><li>Después de la cirugía, tenga los ovarios en PBS sobre hielo en un tubo cónico, hasta su llegada al laboratorio.</li>
	<li>Llenar dos platos de Petri de 100 mm con 10 mL de PBS.</li>
	<li>Quitar los ovarios del tubo y transferir a la primera placa de Petri con PBS. Lavar con mezcla de movimientos.</li>
	<li>Recuperar el ovario y lo coloca en otro plato. Con tijeras y pinzas estériles, moler el tejido en trozos muy pequeños, aproximadamente de 1 mm de tamaño.</li>
	<li>Transferir el tejido ovárico para un plato de Petri de 35 mm y añadir 2 mL de colagenasa. Picar un poco más el tejido.</li>
	<li>Coloque el plato Petri en la incubadora a 38 ° C por 3 h y sacuda suavemente el plato de Petri con movimientos circulares cada 20 minutos.</li>
	<li>Después de 3 horas, retire la placa de Petri de la incubadora.</li>
	<li>Transferir el contenido del plato a un tubo de 15 mL. Añadir 5 mL de medio de expansión.</li>
	<li>Invierta suavemente el tubo antes de la centrifugación. Centrifugar el tubo a 2100 x g durante 7 minutos quite el sobrenadante y resuspender el precipitado con 3 mL de medio de expansión.</li>
	<li>Transfiera el sedimento de una botella de T25. Coloque la botella en la incubadora con 5% CO2 a 38 ° C durante 3 horas. Nota: La parte más importante del procedimiento es cambiar los medios de comunicación después de 3 h de incubación.</li>
	<li>Quite la botella de la incubadora y retirar los medios de comunicación con el tejido restante. Añadir 3 mL de medio fresco expansión a la botella.</li>
	<li>Cambiar el medio cada 48 h y observar la botella para la confluencia celular. Nota: Los pasos principales del procedimiento pueden observarse en la figura 1.</li>
</ol>3. expansión de células madre mesenquimales del ovario
<ol><li>
Paso de la célula
	<ol>
<li>Cuando las células alcanzan confluencia, eliminar los medios de comunicación de la botella.</li>
		<li>Lave la botella con 3 mL de PBS. Retirar el PBS.</li>
		<li>Añadir 1,5 mL de tripsina y ponga la botella en la incubadora a 38 ° C durante 3 minutos golpee suavemente la botella para ayudar a las células para separar. Nota: Las células deben llegar a confluencia aproximadamente 5 - 7 d después de la chapa inicial.</li>
		<li>Añadir 3 mL de medio de expansión y transferir el contenido a un tubo cónico.</li>
		<li>Centrifugar el tubo a 2100 x g durante 7 minutos quite el sobrenadante y agregar 1 mL de medio de expansión.</li>
		<li>Homogeneizar suavemente el contenido del tubo.</li>
		<li>Tomar una alícuota de 10 μl de la muestra para realizar el conteo y vuelva a colocar el tubo con las células en la incubadora de la célula.</li>
		<li>Lugar 10 μl de la mezcla en el hemocitómetro.</li>
	</ol>
</li>
	<li>
Conteo de las células
	<ol>
<li>Uso objetivo de X 10 del microscopio y centrarse en las líneas de cuadrícula del hemocitómetro.</li>
		<li>Contar las células en cinco cuadrados pequeños (figura 2).</li>
		<li>Multiplique al número contado por 50.000 para estimar el número de células por mililitro.</li>
	</ol>
</li>
</ol>4. diferenciación de células madre mesenquimales del ovario
Nota: Se realizaron ensayos de diferenciación según las pautas establecidas en colina et al. 9.
<ol><li>Células por pozo, en triplicado, usando una placa de 4 pozos de cultivo en semillas 1.0 x 104 .</li>
	<li>Añadir 1 mL de medio de expansión.</li>
	<li>Frote suavemente el plato con movimientos circulares.</li>
	<li>Después de 24 h, sustituir los medios de cultivo con los medios de diferenciación deseables.</li>
	<li>Para osteogénico, adipogenic y diferenciación condrogénica, incuban las células con el medio de inducción de 30 d, con el reemplazo de los medios de comunicación cada 3 kits de diferenciación específicos d. fueron utilizados para cada uno de estos ensayos.</li>
	<li>Para la diferenciación de la estirpe neurogénica, incubar las células por 10 d en los medios de inducción, con el reemplazo de los medios de comunicación cada 3 días. Los medios utilizados aquí contienen DMEM baja glucosa, el ácido valproico 2 mM, 1 hidrocortisona μm, 10 μm forskoline, cloruro de potasio 5 mM, 5 μg/mL insulina e hidroxianisol butilado de 200 μm.</li>
	<li>De precursores de endodermo, incubar por 5 d en medios endodermo según instrucciones del fabricante del reactivo.</li>
	<li>Para la diferenciación del linaje de células germinales primordiales, incubar las células en los medios de inducción de 14 días, con el reemplazo de los medios de comunicación cada 3 d. Los medios utilizados aquí contienen DMEM, 10% FBS, piruvato de sodio 1 m m, 10 ng/mL LIF, aminoácidos no esenciales 1 mM, 2 mM L-glutamina, 5 insulina μg/mL, 0.1 mM β-mercaptoetanol, putrescina μm 60, 20 transferrina μg/mL, 10 ng/mL ratón factor de crecimiento epidérmico, 1 ng/mL humano factor de crecimiento básico del fibroblasto, 40 ng/mL humanos glial células factor neurotrófico derivado de línea y la penicilina de 15 mg/L.</li>
</ol>

<ol>
	<li>Gazit, Z., Pelled, G., Sheyn, D., Kimelman, N., Gazit, D., Atala, A., Lanza, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mesenchymal+stem+cells.">Mesenchymal stem cells.</a> Handbook of Stem Cells (2nd Edition). , 513-527 (2013).</li><li>Zipori, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+nature+of+stem+cells:+state+rather+than+entity.">The nature of stem cells: state rather than entity.</a> Nature Reviews Genetics. 5 (11), 873-878 (2004).</li><li>Auersperg, N., Wong, A. S., Choi, K. C., Kang, S. K., Leung, 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=Ovarian+surface+epithelium:+biology,+endocrinology,+and+pathology.">Ovarian surface epithelium: biology, endocrinology, and pathology.</a> Endocrine Reviews. 22 (2), 255-288 (2001).</li><li>Ahmed, N., Thompson, E. W., Quinn, 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=Epithelial-mesenchymal+interconversions+in+normal+ovarian+surface+epithelium+and+ovarian+carcinomas:+an+exception+to+the+norm.">Epithelial-mesenchymal interconversions in normal ovarian surface epithelium and ovarian carcinomas: an exception to the norm.</a> Journal of Cellular Physiology. 213 (3), 581-588 (2007).</li><li>Bukovsky, A., Svetlikova, M., Caudle, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oogenesis+in+cultures+derived+from+adult+human+ovaries.">Oogenesis in cultures derived from adult human ovaries.</a> Reproductive Biology and Endocrinology. 3 (1), 17 (2005).</li><li>Gong, S. 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=Embryonic+stem+cell-like+cells+established+by+culture+of+adult+ovarian+cells+in+mice.">Embryonic stem cell-like cells established by culture of adult ovarian cells in mice.</a> Fertility and Sterility. 93 (8), 2594-2601 (2010).</li><li>Johnson, J., Canning, J., Kaneko, T., Pru, J. K., Tilly, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Germline+stem+cells+and+follicular+renewal+in+the+postnatal+mammalian+ovary.">Germline stem cells and follicular renewal in the postnatal mammalian ovary.</a> Nature. 428 (6979), 145 (2004).</li><li>Deans, R. J., Moseley, A. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mesenchymal+stem+cells:+biology+and+potential+clinical+uses.">Mesenchymal stem cells: biology and potential clinical uses.</a> Experimental Hematology. 28 (8), 875-884 (2000).</li><li>Trinda Hill, A. B. T., Therrien, J., Garcia, J. M., Smith, L. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mesenchymal-like+stem+cells+in+canine+ovary+show+high+differentiation+potential.">Mesenchymal-like stem cells in canine ovary show high differentiation potential.</a> Cell Proliferation. 50 (6), 12391 (2017).</li><li>Dominici, M. L. B. 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=Minimal+criteria+for+defining+multipotent+mesenchymal+stromal+cells.+The+International+Society+for+Cellular+Therapy+position+statement.">Minimal criteria for defining multipotent mesenchymal stromal cells. The International Society for Cellular Therapy position statement.</a> Cytotherapy. 8, 4315-4317 (2006).</li><li>Friedenstein, A. J., Piatetzky-Shapiro, I. I., Petrakova, K. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Osteogenesis+in+transplants+of+bone+marrow+cells.">Osteogenesis in transplants of bone marrow cells.</a> Development. 16 (3), 381-390 (1966).</li><li>Kuznetsov, S. A., 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=Single-colony+derived+strains+of+human+marrow+stromal+fibroblasts+form+bone+after+transplantation+in+vivo.">Single-colony derived strains of human marrow stromal fibroblasts form bone after transplantation in vivo.</a> Journal of Bone and Mineral Research. 12 (9), 1335-1347 (1997).</li></ol>

Los autores no tienen nada que revelar.

Aquí proporcionamos evidencia que MSCs pueden ser aislados del tejido ovárico canino, que se considera residuos biológicos después de la ovariectomía. Debido a que muchos tipos de células pueden encontrarse en el ovario, propusimos un protocolo para seleccionar MSCs en su rápida adhesión al plástico, que selecciona con éxito células que crecieron en una monocapa con una morfología de fibroblasto-como base.
El primer informe de la derivación de MSCs de médula ósea se basa en la c...

El número de estudios publicados que enfoque en células madre ha aumentado sustancialmente en la última década, un esfuerzo de investigación que ha sido impulsado por el colectivo objetivo de descubrir terapias de Medicina Regenerativa de gran alcance. Las células madre tienen dos marcadores definición primarios: auto renovación y diferenciación. Las células madre mesenquimales son responsables de la facturación de tejido regular y tienen una capacidad más limitada de la diferenciación en comparación con las células madre embrionarias1. Recientemente, muchos estudios han demostrado una amplia gama de la diferenciación de MSCs y un tema en discusión es si existen diferencias entre las células madre embrionarias y adultas en todos los2.
El epitelio superficial de ovarium es una sin compromiso capa de células, relativamente menos diferenciada, que expresa ambos marcadores epiteliales y mesenquimales3, conservando la capacidad de diferenciarse en diferentes tipos de células en respuesta a señales ambientales4. La ubicación exacta de las células madre en el ovario no es conocida; sin embargo, se ha propuesto que bipotential progenitores en la túnica albugínea dan lugar a las células germinales5. Estudios inmunológicos han presumido que estas células tienen un origen estromal6 o se encuentra en o próximo a la superficie del ovario7. Puesto que las células madre mesenquimales expresa numerosos receptores que desempeñan un papel importante en la adherencia de la célula8, fue diseñado un experimento para probar la hipótesis de que la selección de una población de células con una rápida adhesión aislaría a una población de células claramente caracterizables como mesenquimales en la naturaleza. Recientemente, nuestro grupo reportó la derivación de MSCs del tejido ovárico basado en su capacidad de adherencia a la superficie de plástico de la placa de cultivo en las primeras 3 h de la cultura, con el fin de obtener una población purificada de células exhibe adherencia rápida9. Aquí, describimos el método desarrollado para el aislamiento de células madre mesenquimales del tejido ovárico.

<table border="0" cellpadding="0" cellspacing="0" style="width:665px;" width="666">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:221px;">DPBS&#160;</td>
			<td style="width:151px;">Thermo Fisher</td>
			<td style="width:123px;">14190144</td>
			<td style="width:171px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:221px;">Collagenase I</td>
			<td style="width:151px;">Thermo Fisher</td>
			<td style="width:123px;">17100017</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:221px;">Tissue flask</td>
			<td style="width:151px;">Corning</td>
			<td style="width:123px;">CLS3056</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:221px;">DMEM low glucose</td>
			<td style="width:151px;">Thermo Fisher</td>
			<td style="width:123px;">11054020</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:221px;">FBS</td>
			<td style="width:151px;">Thermo Fisher</td>
			<td style="width:123px;">12484-010</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:221px;">TrypLE express</td>
			<td style="width:151px;">Thermo Fisher</td>
			<td style="width:123px;">12604021</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:221px;">StemPro Adipogenesis Differentiation Kit</td>
			<td style="width:151px;">Thermo Fisher</td>
			<td style="width:123px;">A1007001</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:221px;">StemPro Chondrogenesis Differentiation Kit</td>
			<td style="width:151px;">Thermo Fisher</td>
			<td style="width:123px;">A1007101</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:221px;">StemPro Osteogenesis Differentiation Kit</td>
			<td style="width:151px;">Thermo Fisher</td>
			<td style="width:123px;">A1007201</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:221px;">STEMdiff Definitive Endoderm Kit</td>
			<td style="width:151px;">StemCell</td>
			<td style="width:123px;">5110</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:221px;">Penicillin-Streptomycin</td>
			<td style="width:151px;">Thermo Fisher</td>
			<td style="width:123px;">15070063</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:221px;">CD45</td>
			<td style="width:151px;">AbD Serotec</td>
			<td style="width:123px;">MCA 2035S</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:221px;">CD34</td>
			<td style="width:151px;">AbD Serotec</td>
			<td style="width:123px;">MCA 2411GA</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:221px;">CD90</td>
			<td style="width:151px;">AbD Serotec</td>
			<td style="width:123px;">MCA 1036G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:221px;">CD44</td>
			<td style="width:151px;">AbD Serotec</td>
			<td style="width:123px;">MCA 1041</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:221px;">Nestin</td>
			<td style="width:151px;">Milipore</td>
			<td style="width:123px;">MAB353</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:221px;">&#946;-Tubulin&#160;</td>
			<td style="width:151px;">Milipore</td>
			<td style="width:123px;">MAB1637</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:221px;">DDX4</td>
			<td style="width:151px;">Invitrogen</td>
			<td style="width:123px;">PA5 -23378</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:221px;">IgG- FITC</td>
			<td style="width:151px;">AbD Serotec</td>
			<td style="width:123px;">STAR80F</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:221px;">IgG- FITC</td>
			<td style="width:151px;">AbD Serotec</td>
			<td style="width:123px;">STAR120F</td>
			<td></td>
		</tr>
	</tbody>
</table>

derivation and differentiation of canine ovarian mesenchymal stem cells

Interés en las células madre mesenquimales (MSCs) ha aumentado en la última década debido a su facilidad de aislamiento, expansión y cultura. Recientemente, estudios han demostrado la capacidad de diferenciación amplia que poseen estas células. El ovario representa a un candidato prometedor para las terapias basadas en células debido a que es rica en MSCs y con frecuencia se descarta después de cirugías de ovariectomía como residuos biológicos. Este artículo describe los procedimientos para el aislamiento, expansión, y la diferenciación de MSCs derivado de ovario canino, sin la necesidad de clasificación celular técnicas. Este protocolo representa una herramienta importante para la medicina regenerativa debido a la amplia aplicabilidad de estas células altamente diferenciables en ensayos clínicos y aplicaciones terapéuticas.

Aislamiento de células madre mesenquimales de ovario canino:
El procedimiento de aislamiento de MSC ovárico se resume en la figura 1. Después de la cirugía, tejido picado, digestión de la colagenasa y un cambio de los medios de comunicación 3 h después del inicio de la cultura, una supuesta población de MSC con propiedades de plástico adhesivo rápidos fue ...

Watch this Scientific Journal Video about Derivation and Differentiation of Canine Ovarian Mesenchymal Stem Cells at JoVE.com

Derivation and Differentiation of Canine Ovarian Mesenchymal Stem Cells

Adjunto, describimos un método para el aislamiento, expansión y diferenciación de células madre mesenquimales de tejido ovárico canino.

Derivación y diferenciación de células mesenquimales ovárico canino

Interest in mesenchymal stem cells (MSCs) has increased over the past decade due to their ease of isolation, expansion, and culture. Recently, studies ...

El objetivo general de este procedimiento es aislar, expandir y diferenciar las ventas de tallos mesenquimales derivados del tejido ovárico canino. Este experimento se realizó con los ovarios de cuatro perros hembra mongrel donados después de una cirugía electiva en un programa de esterilización canina. Este experimento fue aprobado por el Comité de ética sobre el uso de animales de la Universidad Estatal de Sao Paulo, FVAC.Resultados representativos. Discussion.Conclusión.

derivation-differentiation-canine-ovarian-mesenchymal-stem

Research

JoVE Journal

Developmental Biology

5.6K vistas.  São Paulo State University (UNESP). El objetivo general de este procedimiento es aislar, expandir y diferenciar las ventas de tallos mesenquimales derivados del tejido ovárico canino. Este experimento se realizó con los ovarios de cuatro perros hembra mongrel donados después de una cirugía electiva en un programa de esterilización canina. Este experimento fue aprobado por el Comité de ética sobre el uso de animales de la Universidad Estatal de Sao Paulo, FVAC.Resultados representativos. Discussion.Conclusión.

Video: Derivation and Differentiation of Canine Ovarian Mesenchymal Stem Cells

Derivation of Cardiac Progenitor Cells from Embryonic Stem Cells

Cardiac progenitor cells (CPCs) have the capacity to differentiate into cardiomyocytes, smooth muscle cells (SMC), and endothelial cells and hold great promise in cell therapy against heart disease. Among various methods to isolate CPCs, differentiation of embryonic stem cell (ESC) into CPCs attracts great attention in the field since ESCs can provide unlimited cell source. As a result, numerous strategies have been developed to derive CPCs from ESCs. In this protocol, differentiation and purification of embryonic CPCs from both mouse and human ESCs is described. Due to the difficulty of using cell surface markers to isolate embryonic CPCs, ESCs are engineered with fluorescent reporters activated by CPC-specific cre recombinase expression. Thus, CPCs can be enriched by fluorescence-activated cell sorting (FACS). This protocol illustrates procedures to form embryoid bodies (EBs) from ESCs for CPC specification and enrichment. The isolated CPCs can be subsequently cultured for cardiac lineage differentiation and other biological assays. This protocol is optimized for robust and efficient derivation of CPCs from both mouse and human ESCs.

In this protocol, derivation of cardiac progenitor cells from both mouse and human embryonic stem cells will be illustrated. A major strategy in this protocol is to enrich cardiac progenitor cells with flow cytometry using fluorescent reporters engineered into the embryonic stem cell lines.

Derivación de células cardiacas progenitoras a partir de células madre embrionarias

Cardiac progenitor cells (CPCs) have the capacity to differentiate into cardiomyocytes, smooth muscle cells (SMC), and endothelial cells and hold great ...

Investigación

Biología del Desarrollo

Efficient Derivation of Retinal Pigment Epithelium Cells from Stem Cells

No cure has been discovered for age-related macular degeneration (AMD), the leading cause of vision loss in people over the age of 55. AMD is complex multifactorial disease with an unknown etiology, although it is largely thought to occur due to death or dysfunction of the retinal pigment epithelium (RPE), a monolayer of cells that underlies the retina and provides critical support for photoreceptors. RPE cell replacement strategies may hold great promise for providing therapeutic relief for a large subset of AMD patients, and RPE cells that strongly resemble primary human cells (hRPE) have been generated in multiple independent labs, including our own. In addition, the uses for iPS-RPE are not limited to cell-based therapies, but also have been used to model RPE diseases. These types of studies may not only elucidate the molecular bases of the diseases, but also serve as invaluable tools for developing and testing novel drugs. We present here an optimized protocol for directed differentiation of RPE from stem cells. Adding nicotinamide and either Activin A or IDE-1, a small molecule that mimics its effects, at specific time points, greatly enhances the yield of RPE cells. Using this technique we can derive large numbers of low passage RPE in as early as three months.

Stem cell-derived retinal pigment epithelium (RPE) cells may be used for multiple applications including cell-based therapies for retinal degeneration, disease modeling, and drug studies. Here we present a simple protocol for reproducibly deriving RPE from stem cells.

Derivación eficiente de células de la retina epitelio pigmentario de Células Madre

No cure has been discovered for age-related macular degeneration  (AMD), the leading cause of vision loss in people over the age of 55. AMD is complex ...

Derivation of Highly Purified Cardiomyocytes from Human Induced Pluripotent Stem Cells Using Small Molecule-modulated Differentiation and Subsequent Glucose Starvation

Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) have become an important cell source to address the lack of primary cardiomyocytes available for basic research and translational applications. To differentiate hiPSCs into cardiomyocytes, various protocols including embryoid body (EB)-based differentiation and growth factor induction have been developed. However, these protocols are inefficient and highly variable in their ability to generate purified cardiomyocytes. Recently, a small molecule-based protocol utilizing modulation of Wnt/&#946;-Catenin signaling was shown to promote cardiac differentiation with high efficiency. With this protocol, greater than 50%-60% of differentiated cells were cardiac troponin-positive cardiomyocytes were consistently observed. To further increase cardiomyocyte purity, the differentiated cells were subjected to glucose starvation to specifically eliminate non-cardiomyocytes based on the metabolic differences between cardiomyocytes and non-cardiomyocytes. Using this selection strategy, we consistently obtained a greater than 30% increase in the ratio of cardiomyocytes to non-cardiomyocytes in a population of differentiated cells. These highly purified cardiomyocytes should enhance the reliability of results from human iPSC-based in vitro disease modeling studies and drug screening assays.

Here, we describe a robust protocol for human cardiomyocyte derivation that combines small molecule-modulated cardiac differentiation and glucose deprivation-mediated cardiomyocyte purification, enabling production of purified cardiomyocytes for the purposes of cardiovascular disease modeling and drug screening.

Derivación de cardiomiocitos altamente purificada de células madre humanas pluripotentes inducidas Uso Pequeño Diferenciación molécula modulada y posterior glucosa inanición

Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) have become an important cell source to address the lack of primary cardiomyocytes ...

Feeder-free Derivation of Melanocytes from Human Pluripotent Stem Cells

Human pluripotent stem cells (hPSCs) represent a platform to study human development in vitro under both normal and disease conditions. Researchers can direct the differentiation of hPSCs into the cell type of interest by manipulating the culture conditions to recapitulate signals seen during development. One such cell type is the melanocyte, a pigment-producing cell of neural crest (NC) origin responsible for protecting the skin against UV irradiation. This protocol presents an extension of a currently available in vitro Neural Crest differentiation protocol from hPSCs to further differentiate NC into fully pigmented melanocytes. Melanocyte precursors can be enriched from the Neural Crest protocol via a timed exposure to activators of WNT, BMP, and EDN3 signaling under dual-SMAD-inhibition conditions. The resultant melanocyte precursors are then purified and matured into fully pigmented melanocytes by culture in a selective medium. The resultant melanocytes are fully pigmented and stain appropriately for proteins characteristic of mature melanocytes.

This work describes an in vitro differentiation protocol to produce pigmented, mature melanocytes from human pluripotent stem cells via a neural crest and melanoblast intermediate stage using a feeder-free, 25 day protocol.

Derivación sin alimentador de melanocitos partir de células madre pluripotentes

Human pluripotent stem cells (hPSCs) represent a platform to study human development in vitro under both normal and disease conditions. Researchers can ...

Epigenetic Regulation of Cardiac Differentiation of Embryonic Stem Cells and Tissues

Specific gene transcription is a key biological process that underlies cell fate decision during embryonic development. The biological process is mediated by transcription factors which bind genomic regulatory regions including enhancers and promoters of cardiac constitutive genes. DNA is wrapped around histones that are subjected to chemical modifications. Modifications of histones further lead to repressed, activated or poised gene transcription, thus bringing another level of fine tuning regulation of gene transcription. Embryonic Stem cells (ES cells) recapitulate within embryoid bodies (i.e., cell aggregates) or in 2D culture the early steps of cardiac development. They provide in principle enough material for chromatin immunoprecipitation (ChIP), a technology broadly used to identify gene regulatory regions. Furthermore, human ES cells represent a human cell model of cardiogenesis. At later stages of development, mouse embryonic tissues allow for investigating specific epigenetic landscapes required for determination of cell identity. Herein, we describe protocols of ChIP, sequential ChIP followed by PCR or ChIP-sequencing using ES cells, embryoid bodies and cardiac specific embryonic regions. These protocols allow to investigating the epigenetic regulation of cardiac gene transcription.

A fine tuning regulation of gene transcription underlies embryonic cell fate decision. Herein, we describe chromatin immunoprecipitation assays used to investigate epigenetic regulation of both cardiac differentiation of stem cells and cardiac development of mouse embryos.

La regulación epigenética de Cardíaca diferenciación de células madre embrionarias y Tejidos

Specific gene transcription is a key biological process that underlies cell fate decision during embryonic development. The biological process is mediated ...

Isolation and Expansion of Mesenchymal Stem/Stromal Cells Derived from Human Placenta Tissue

Mesenchymal stem/stromal cells (MSC) are promising candidates for use in cell-based therapies. In most cases, therapeutic response appears to be cell-dose dependent. Human term placenta is rich in MSC and is a physically large tissue that is generally discarded following birth. Placenta is an ideal starting material for the large-scale manufacture of multiple cell doses of allogeneic MSC. The placenta is a fetomaternal organ from which either fetal or maternal tissue can be isolated. This article describes the placental anatomy and procedure to dissect apart the decidua (maternal), chorionic villi (fetal), and chorionic plate (fetal) tissue. The protocol then outlines how to isolate MSC from each dissected tissue region, and provides representative analysis of expanded MSC derived from the respective tissue types. These methods are intended for pre-clinical MSC isolation, but have also been adapted for clinical manufacture of placental MSC for human therapeutic use.

Herein we describe methods for the dissection of fetal and maternal tissues from human term placenta, followed by isolation and expansion of mesenchymal stem/stromal cells (MSC) from these tissues.

Aislamiento y expansión de células madre mesenquimales / estromales derivadas de tejido de placenta humana

Mesenchymal stem/stromal cells (MSC) are promising candidates for use in cell-based therapies. In most cases, therapeutic response appears to be cell-dose ...

In Vitro Differentiation of Human Mesenchymal Stem Cells into Functional Cardiomyocyte-like Cells

Myocardial infarction and the subsequent ischemic cascade result in the extensive loss of cardiomyocytes, leading to congestive heart failure, the leading cause of mortality worldwide. Mesenchymal stem cells (MSCs) are a promising option for cell-based therapies to replace current, invasive techniques. MSCs can differentiate into mesenchymal lineages, including cardiac cell types, but complete differentiation into functional cells has not yet been achieved. Previous methods of differentiation were based on pharmacological agents or growth factors. However, more physiologically relevant strategies can also enable MSCs to undergo cardiomyogenic transformation. Here, we present a differentiation method using MSC aggregates on cardiomyocyte feeder layers to produce cardiomyocyte-like contracting cells.
Human umbilical cord perivascular cells (HUCPVCs) have been shown to have a greater differentiation potential than commonly investigated MSC types, such as bone marrow MSCs (BMSCs). As an ontogenetically younger source, we investigated the cardiomyogenic potential of first-trimester (FTM) HUCPVCs compared to older sources. FTM HUCPVCs are a novel, rich source of MSCs that retain their in utero immunoprivileged properties when cultured in vitro. Using this differentiation protocol, FTM and term HUCPVCs achieved significantly increased cardiomyogenic differentiation compared to BMSCs, as indicated by the increased expression of cardiomyocyte markers (i.e., myocyte enhancer factor 2C, cardiac troponin T, heavy chain cardiac myosin, signal regulatory protein &#945;, and connexin 43). They also maintained significantly lower immunogenicity, as demonstrated by their lower HLA-A expression and higher HLA-G expression. Applying aggregate-based differentiation, FTM HUCPVCs showed increased aggregate formation potential and generated contracting cells clusters within 1 week of co-culture on cardiac feeder layers, becoming the first MSC type to do so. 
Our results demonstrate that this differentiation strategy can effectively harness the cardiomyogenic potential of young MSCs, such as FTM HUCPVCs, and suggests that in vitro pre-differentiation could be a potential strategy to increase their regenerative efficacy in vivo.

In Vitro Differentiation of Human Mesenchymal Stem Cells into Functional Cardiomyocyte-like Cells

Here, we present a method to efficiently harness the cardiac differentiation potential of young sources of human mesenchymal stem cells in order to generate functional, contracting, cardiomyocyte-like cells in vitro.

In Vitro Diferenciación de células madre mesenquimales humanas en células funcionales en cardiomiocitos

Myocardial infarction and the subsequent ischemic cascade result in the extensive loss of cardiomyocytes, leading to congestive heart failure, the leading ...

Integration Free Derivation of Human Induced Pluripotent Stem Cells Using Laminin 521 Matrix

Xeno-free and fully defined conditions are key parameters for robust and reproducible generation of homogenous human induced pluripotent stem (hiPS) cells. Maintenance of hiPS cells on feeder cells or undefined matrices are susceptible to batch variances, pathogenic contamination and risk of immunogenicity. Utilizing the defined recombinant human laminin 521 (LN-521) matrix in combination with xeno-free and defined media formulations reduces variability and allows for the consistent generation of hiPS cells. The Sendai virus (SeV) vector is a non-integrating RNA-based system, thus circumventing concerns associated with the potential disruptive effect on genome integrity integrating vectors can have. Furthermore, these vectors have demonstrated relatively high efficiency in the reprogramming of dermal fibroblasts. In addition, enzymatic single cell passaging of cells facilitates homogeneous maintenance of hiPS cells without substantial prior experience of stem cell culture. Here we describe a protocol that has been extensively tested and developed with a focus on reproducibility and ease of use, providing a robust and practical way to generate defined and xeno-free human hiPS cells from fibroblasts.

Robust derivation of human induced pluripotent stem (hiPS) cells was achieved by using non-integrating Sendai virus (SeV) vector mediated reprogramming of dermal fibroblasts. hiPS cell maintenance and clonal expansion was performed using xeno-free and chemically defined culture conditions with recombinant human laminin 521 (LN-521) matrix and Essential E8 (E8) Medium.

Integración Derivación Gratuita de Células Madre Pluripotentes Inducidas Humanas Usando Laminin 521 Matrix

Xeno-free and fully defined conditions are key parameters for robust and reproducible generation of homogenous human induced pluripotent stem (hiPS) ...

Rapid, Directed Differentiation of Retinal Pigment Epithelial Cells from Human Embryonic or Induced Pluripotent Stem Cells

We describe a robust method to direct the differentiation of pluripotent stem cells into retinal pigment epithelial cells (RPE). The purpose of providing a detailed and thorough protocol is to clearly demonstrate each step and to make this readily available to researchers in the field. This protocol results in a homogenous layer of RPE with minimal or no manual dissection needed. The method presented here has been shown to be effective for induced pluripotent stem cells (iPSC) and human embryonic stem cells. Additionally, we describe methods for cryopreservation of intermediate cell banks that allow long-term storage. RPE generated using this protocol might be useful for iPSC disease-in-a-dish modeling or clinical application.

This protocol describes how to produce retinal pigment epithelial cells (RPE) from pluripotent stem cells. The method uses a combination of growth factors and small molecules to direct the differentiation of stem cells into immature RPE in fourteen days and mature, functional RPE after three months.

Diferenciación rápida y dirigida de las células epiteliales del pigmento retiniano de humanos embrionarios o inducidas pluripotenciales células

We describe a robust method to direct the differentiation of pluripotent stem cells into retinal pigment epithelial cells (RPE). The purpose of providing ...

Computational Analysis of the Caenorhabditis elegans Germline to Study the Distribution of Nuclei, Proteins, and the Cytoskeleton

The Caenorhabditis elegans (C. elegans) germline is used to study several biologically important processes including stem cell development, apoptosis, and chromosome dynamics. While the germline is an excellent model, the analysis is often two dimensional due to the time and labor required for three-dimensional analysis. Major readouts in such studies are the number/position of nuclei and protein distribution within the germline. Here, we present a method to perform automated analysis of the germline using confocal microscopy and computational approaches to determine the number and position of nuclei in each region of the germline. Our method also analyzes germline protein distribution that enables the three-dimensional examination of protein expression in different genetic backgrounds. Further, our study shows variations in cytoskeletal architecture in distinct regions of the germline that may accommodate specific spatial developmental requirements. Finally, our method enables automated counting of the sperm in the spermatheca of each germline. Taken together, our method enables rapid and reproducible phenotypic analysis of the C. elegans germline.

Computational Analysis of the Caenorhabditis elegans Germline to Study the Distribution of Nuclei, Proteins, and the Cytoskeleton

We present an automated method for three-dimensional reconstruction of the Caenorhabditis elegans germline. Our method determines the number and position of each nucleus within the germline and analyses germline protein distribution and cytoskeletal structure.

Análisis computacional de lo elegans de Caenorhabditis del Germline para estudiar la distribución de núcleos, proteínas y citoesqueleto

The Caenorhabditis elegans (C. elegans) germline is used to study several biologically important processes including stem cell development, apoptosis, and ...