Este trabajo fue supportedby la Sociedad Americana del Cáncer de Investigación estudiosos Grant (RSG-12-148-01-DDC) y el Premio de Biología CIRM Básica (RB4-05779).

El protocolo de la piel humana se realizó de acuerdo con las directrices de la Universidad de California, Comité de Ética de Investigación de San Diego. La piel humana se puede obtener a partir de muestras quirúrgicas desechadas o comprado a los bancos de la piel (banco de piel aparece en la Tabla de materiales / Equipo). La ubicación donde la piel se deriva de o la edad del donante no es crítico para el experimento, siempre y cuando las proteínas de la membrana basal (colágeno / laminina) en la dermis no se degradan. 1. Preparación de desvitalizados Dermis Humanos <ol><li> Tras la recepción de la piel humana, colocar el tejido en la campana de cultivo de tejido para descongelar (~ 3-5 min). Dependiendo del tamaño de la piel, colocar el tejido descongelado en un tubo estéril desechable de 50 ml o 125 ml cónica botella. El tamaño típico de la piel desde el banco de piel es de 0,75 pies cuadrados. </li><li> Llene el recipiente de 75% de su capacidad con penicilina y estreptomicina 4x (penicilina / estreptomicina) en 1x PBS. Agitar vigorosamente durante5 min y deseche el líquido. Repita este paso 3 veces más. </li><li> Después del último lavado, transferir el tejido a un nuevo tubo / botella y añadir 4x fresco pen / strep / PBS para llenar 75% del contenedor. Incubar esta mezcla en una incubadora de cultivo de tejidos a 37 ° C durante 2 semanas para permitir la separación de la dermis de la epidermis. </li><li> En condiciones estériles, el uso de fórceps para despegar la epidermis de la dermis. La dermis es completamente blanco, mientras que la epidermis tendrán coloración en función de la carrera del donante (Figura 1A). Deseche la epidermis, según el protocolo de la institución para hacer frente a los tejidos humanos. </li><li> Coloque la dermis en un tubo o botella y lavarlo en 4x pen / strep diluido en PBS 1x con agitación vigorosa (5 min lavados por 4 veces). Después del último lavado, transferir la dermis a un nuevo tubo / botella en 4x pen / strep diluido en PBS 1x y se almacena a 4 ° C hasta su utilización. Dermis pueden almacenarse a 4 ° C durante un máximo de un año. </li></ol&gt; 2. Modificar genéticamente primaria queratinocitos humanos NOTA: Use las células phoenix anfotrópicos cultivadas en medio DMEM completo [DMEM + 10% de suero bovino fetal (FBS) + penicilina / estreptomicina] para producir virus para infectar los queratinocitos humanos primarios. Genes de empaquetamiento virales que codifican proteínas tales como gag-pol y el sobre se integran de manera estable en el genoma de la célula phoenix que permite la producción de virus cuando se transfecta con un vector retroviral. Células Phoenix se pueden transfectar con una alta eficiencia permitiendo para alta producción de título viral. Virus producido a partir de esta línea de envasado también puede infectar una gran variedad de células de mamífero incluyendo humanos. <ol><li> Día 1: El día antes de la transfección, las células Phoenix semillas en una placa de 6 pocillos a una densidad de 800.000 células por pocillo en medio DMEM completo. </li><li> Día 2: El día de la transfección, utilice 3 g de vectores retrovirales por pocillo. Alícuota 3 g de vector retroviral en un tubo de 1,5 ml. Use una mezcla de 100 μ; l DMEM más de 6 l de reactivo de transfección por cada 3 g de vector. Mezclar 6 l de reactivo de transfección con 100 l DMEM en un tubo de 1,5 ml. (Los vectores retrovirales que se utilizan se enumeran en la tabla de equipos / materiales). <ol><li> Incubar durante 5 min y añadir la mezcla de 106 l en el tubo de pre-alícuota que contiene el 3 g de vector retroviral. Mezclar e incubar a temperatura ambiente durante 30 min. Añadir gota a gota toda esta mezcla a cada pocillo de las células Phoenix. </li></ol></li><li> Día 3: El día después de la transfección, retire el medio de las células Phoenix y añadir 2 ml de medio DMEM fresca completa. El mismo día, la semilla queratinocitos humanos primarios en placas de 6 pocillos a una densidad de 75.000 células por pocillo. Mantener los queratinocitos en un medio de suero de queratinocitos libre (KCSFM) con antibióticos (penicilina / estreptomicina). NOTA: los queratinocitos humanos primarios fueron adquiridos. Las células se pueden comprar en una variedad de proveedores (que aparece en la tabla de materiales / Equipo). </li><li> Día 4: Harconferir los medios de comunicación de las células transfectadas phoenix, que ahora contiene partículas virales. Pase los medios de comunicación a través de un filtro de 0,45 micras con una jeringa (para eliminar cualquier célula phoenix contaminantes) y coloque 2 ml en los queratinocitos (chapados a 75.000 células / pocillo el día anterior: véase el paso 2.3). Añadir bromuro de hexadimetrina (5 g / ml) a los medios para ayudar a mediar en el proceso de infección. Los títulos virales son ~ 1 x 10 7 TU / ml. <ol><li> Haga girar las placas de 6 pocillos en una centrifugadora a 200 xg durante 1 hora a temperatura ambiente. Después de la escisión, retire el soporte que contiene el virus y lavar las células una vez con PBS 1x antes de añadir KCSFM. </li></ol></li><li> Día 5: infectar el mismo lote de queratinocitos de nuevo como en el paso 2.4. NOTA: Seleccionar los queratinocitos utilizando un fármaco si hay un marcador seleccionable en el vector retroviral y expandir para su uso en análisis de aguas abajo o aplicaciones tales como cultivos organotípicos. </li></ol> 3. Configuración de las Culturas de la piel organotípicos <ol><li> Construir elcasete organotípico a partir de materiales comunes que se encuentran en el laboratorio o las cintas puede ser por encargo a través de un taller de fabricación de metal (Figura 2 A - F). Para hacer estos cassettes de una placa de cultivo tisular 3,5 cm, utilizar un cauterio para eliminar un cuadrado de 1,0 cm x 1,0 cm desde el centro de la tapa de una placa de 3,5 cm (Figura 2A-B). Haga esto en condiciones estériles. <ol><li> Adjuntar clavijas cuadradas a la parte inferior de la casete (tapa de 3,5 cm plato) usando esmalte de uñas transparente (Figura 2C). Permitir 5 min para el esmalte de uñas se seque y voltear la casete de manera que está descansando sobre las clavijas cuadradas (Figura 2D). Coloque el casete en un plato de 6 cm. NOTA: Plaza clavijas se pueden comprar en una variedad de tiendas de artesanías. </li></ol></li><li> Preparar medio de crecimiento de queratinocitos (KGM) compuesta de 66% de DMEM, 22% F12 de Ham, 100 unidades / ml de penicilina, 100 mg / ml de estreptomicina, 10% de FBS, 2,67 g / ml adenina, 40 ng / ml de hidrocortisona, 10 la toxina del cólera ng / ml, 4,94 mg / ml de insulina, 5 mg / ml de transferrina, 1,36 ng / ml triyodo-L-tironina, 10 ng / ml EGF, y 10 mg / ml de hidrocloruro de ciprofloxacino. Filtra los medios de comunicación a través de un filtro y tienda de 0,22 micras a 4 ° C hasta su utilización. </li><li> Tome la dermis de la solución PBS 4x penicilina / estreptomicina + y lavar 2 veces en KGM y luego incubar en KGM a 37 ° C durante dos días antes de su uso. </li><li> Cortar la dermis utilizando un bisturí para 1,5 cm x 1,5 cm trozos de tamaño y luego se coloca en el cassette organotípico. Coloque la dermis con la parte superior de la dermis hacia arriba. La parte superior de la dermis será el lado que entra en contacto con los queratinocitos (Figura 1B). </li><li> Coloque la dermis pre-cortadas en el orificio cuadrado de la casete organotípicos con el lado superior de la dermis hacia arriba (Figura 3A). </li><li> Voltear todo el casete organotípico contiene la dermis sobre el uso de fórceps (Figura 3B). Descongele la solución de la matriz extracelular en el hielo. Use una aguja y una jeringa para extraer 100 l de solución de matriz extracelular por casete. Añadir 5 gotas a la parte inferior de la dermis (lado brillante) y agitar ligeramente para asegurar una distribución uniforme por toda la dermis (Figura 3B). </li><li> Permitir a 5 min de la solución comercial matriz extracelular para solidificar completamente (Figura 3C). Después de la solidificación, utilizar pinzas para voltear el casete hacia atrás. Añadir 4 ml de KGM al plato 6 cm. </li><li> Coloque 0,5-1 millones de queratinocitos modificados genéticamente en los cassettes que contienen organotípicos la dermis (Figura 3D). <ol><li> Trypsinize queratinocitos mediante la colocación de 4 ml de tripsina al 0,05% en placas de 10 cm durante 5 min y se inactiva con 4 ml de medio DMEM completo. </li><li> Contar los queratinocitos utilizando un hemocitómetro y luego girar hacia abajo a 200 xg durante 5 min. Resuspender las células 0,5-1.000.000 (dependiendo del número de células disponibles) en 90 μl de KGM y colocar todas las células en la dermis. NOTA: Es importante colocar el mismo número de células en la dermis dentro del mismo experimento para garantizar que las diferencias observadas en espesor de la epidermis regenerada no es debido a diferencias en el número de células de partida. Colocación de menos de 0,5 millones de células de la dermis puede conducir a una mala estratificación y diferenciación. </li></ol></li><li> Cambie medios KGM en los cultivos organotípicos cada dos días. Tejido Cosecha 1-14 días después de la colocación de los queratinocitos en la dermis. Estratificación completa y la diferenciación de la epidermis por lo general se produce después del día 5. </li></ol>

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We have nothing to disclose.

La manipulación genética en la piel humana cultivos organotípicos ofrecen muchas ventajas para estudió comúnmente 2D células cultivadas, así como modelos de ratón. Culturas 2D carecen de las tres interacciones célula-matriz celular y extracelular de las células dimensionales que se encuentran en tejidos y órganos intactos. Estudios recientes también han encontrado enormes diferencias entre las células de cáncer de piel cultivadas en 2D y 3D con las células cultivadas en 3D que muestran mucho más similitu...

La epidermis humana es un epitelio estratificado que se conecta a la dermis subyacente a través de una matriz extracelular conocida como la epidermis terraza, plancha membrana basal no sólo sirve como una barrera impermeable para evitar la pérdida de humedad, sino también como una primera línea de defensa para proteger la cuerpo de sustancias extrañas y tóxicos 1. La capa basal, que es la capa más profunda de la epidermis, contiene las células madre epidérmicas y progenitoras que dan lugar a la progenie diferenciada que forman el resto de la epidermis 2. Como las células progenitoras se diferencian epidérmicas que migran hacia arriba para formar la primera capa de células diferenciadas conocida como la capa espinosa 3. En la capa espinosa, las células se convierten en la expresión de las queratinas 1 y 10, que a continuación proporciona la fuerza para soportar el estrés físico para las capas diferenciadas de la epidermis. Como las células de la capa espinosas diferenciar aún más, se mueven hacia arriba en la epidermis a FORm la capa granular, que se caracteriza por la formación de gránulos de queratohialina y laminares, así como proteínas estructurales que se ensamblan por debajo de la membrana plasmática. Como las células proceden en el proceso de diferenciación, las proteínas por debajo de la membrana plasmática son cruz vinculadas entre sí, mientras que los gránulos lamelares son extruidos a partir de las células para formar un lípido barrera rica llama el estrato córneo 4. Enfermedades que implican alteraciones en crecimiento epidérmico y el impacto diferenciación ~ 20% de la población 5. Por lo tanto, la comprensión de los mecanismos de este proceso es de gran importancia. Desde manifestación de muchas de estas enfermedades está supeditada a la célula-célula o célula-matriz de contacto, cultivos organotípicos donde la epidermis humana se reconstituye en un entorno 3D se han creado 6-10. Estos métodos implican típicamente el uso de queratinocitos primarios o transformadas sembradas sobre la matriz extracelular tales como humano desvitalizadodermis, Matrigel, o colágeno. Para entender los mecanismos de regulación de genes que son importantes en crecimiento epidérmico y la diferenciación, los queratinocitos se pueden manipular genéticamente a través de vectores retrovirales para derribar o sobreexpresar genes en cultivo 2D y luego reconstituirse en 3D. Estos métodos se han utilizado ampliamente para caracterizar genes implicados en la madre epidérmicas y células progenitoras auto-renovación y diferenciación, así como la progresión a neoplasia 11-21. Aquí, se proporciona un protocolo en profundidad sobre cómo alterar la expresión de genes en cultivos organotípicos epidérmicas a través del uso de los retrovirus.

<table>
	<tbody>
		<tr>
			<td>Human skin</td>
			<td>New York Firefighters Skin Bank</td>
			<td><a href="http://www.cornellsurgery.org/pro/services/burn-surgery/skin-bank.html">http://www.cornellsurgery.org/pro/services/burn-surgery/skin-bank.html</a></td>
		</tr>
		<tr>
			<td>PEN/STREP</td>
			<td>GIBCO</td>
			<td>15140-122</td>
		</tr>
		<tr>
			<td>amphotropic phoenix cell lines</td>
			<td>ATCC</td>
			<td>CRL-3213</td>
		</tr>
		<tr>
			<td>FUGENE 6 transfection reagent</td>
			<td>Promega</td>
			<td>E2691</td>
		</tr>
		<tr>
			<td>Keratinocyte Media (KCSFM)</td>
			<td>Life Technologies</td>
			<td>17005042</td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>GIBCO</td>
			<td>11995</td>
		</tr>
		<tr>
			<td>Ham&#39;s F12</td>
			<td>Cambrex</td>
			<td>12-615F</td>
		</tr>
		<tr>
			<td>FBS</td>
			<td>GIBCO</td>
			<td>10437-028</td>
		</tr>
		<tr>
			<td>Adenine</td>
			<td>Sigma</td>
			<td>A-9795</td>
		</tr>
		<tr>
			<td>Cholera Toxin</td>
			<td>Sigma</td>
			<td>&#160;C-8052</td>
		</tr>
		<tr>
			<td>Hydrocortisone</td>
			<td>Calbiochem</td>
			<td>3896</td>
		</tr>
		<tr>
			<td>Insulin</td>
			<td>Sigma</td>
			<td>I-1882</td>
		</tr>
		<tr>
			<td>EGF</td>
			<td>Invitrogen</td>
			<td>13247-051</td>
		</tr>
		<tr>
			<td>Transferrin&#160;</td>
			<td>Sigma</td>
			<td>T-0665</td>
		</tr>
		<tr>
			<td>Ciprofloxacin Hydrochloride</td>
			<td>Serologicals</td>
			<td>89-001-1</td>
		</tr>
		<tr>
			<td>cautery</td>
			<td>Bovie Medical Corporation</td>
			<td>AA01</td>
		</tr>
		<tr>
			<td>Matrigel</td>
			<td>Corning</td>
			<td>354234</td>
		</tr>
		<tr>
			<td>Keratin 1 antibody</td>
			<td>Biolegend</td>
			<td>PRB-149P</td>
		</tr>
		<tr>
			<td>square pegs</td>
			<td>Arts and crafts stores</td>
			<td></td>
		</tr>
		<tr>
			<td>human neonatal keratinocytes</td>
			<td>ATCC</td>
			<td>PCS-200-010</td>
		</tr>
		<tr>
			<td>human neonatal keratinocytes</td>
			<td>Cell Applications</td>
			<td>102K-05n</td>
		</tr>
		<tr>
			<td>MSCV retroviral vector</td>
			<td>Clontech</td>
			<td>634401</td>
		</tr>
		<tr>
			<td>LZRS retroviral vector</td>
			<td>Addgene</td>
			<td></td>
		</tr>
		<tr>
			<td>pSuper.Retro.Puro Retroviral vector</td>
			<td>Oligoengine</td>
			<td>VEC-PRT-0002&#160;</td>
		</tr>
		<tr>
			<td>hexadimethrine bromide&#160;</td>
			<td>Sigma</td>
			<td>H9268-5G</td>
		</tr>
	</tbody>
</table>

generation of genetically modified organotypic skin cultures using devitalized human dermis

Los cultivos organotípicos permiten la reconstitución de un entorno 3D crítico para contacto célula-célula y célula-matriz interacciones que imita la función y la fisiología de sus homólogos de tejidos in vivo. Esto se ejemplifica con cultivos de piel organotípicos que recapitula fielmente la diferenciación epidérmica y el programa de la estratificación. Queratinocitos epidérmicos humanos primarios son genéticamente manipulable mediante retrovirus donde los genes pueden ser fácilmente sobreexpresan o derribados. Estos queratinocitos modificados genéticamente se pueden utilizar para regenerar la epidermis humana en cultivos de piel organotípicos proporcionan un poderoso modelo para estudiar vías genéticas de crecimiento epidérmico de impacto, la diferenciación y la progresión de la enfermedad. Los protocolos presentados aquí describen métodos para preparar dermis humana desvitalizados, así como para manipular genéticamente los queratinocitos humanos primarios a fin de generar cultivos de piel organotípicos. La piel humana regenerada se puede utilizar en downstream aplicaciones tales como perfiles de expresión génica, la inmunotinción y inmunoprecipitaciones cromatina seguido de secuenciación de alto rendimiento. Por lo tanto, la generación de estos cultivos de piel organotípicos modificados genéticamente permitirá la determinación de los genes que son críticos para el mantenimiento de la homeostasis de la piel.

El primer paso en la generación de la piel humana organotípicos es eliminar la epidermis de la dermis. La incubación de dos semanas de la piel en 37 ° C en 4x pen / strep / PBS debería permitir la separación de la dermis de la epidermis (Figura 1A). Si es difícil separar la epidermis y la dermis luego colocar el tejido a 37 ° C en 4x penicilina / estreptomicina / PBS durante una semana y luego tratar de pelar de nuevo utilizando fórceps. Una de las claves para la re...

Watch this Scientific Journal Video about Generation of Genetically Modified Organotypic Skin Cultures Using Devitalized Human Dermis at JoVE.com

Generation of Genetically Modified Organotypic Skin Cultures Using Devitalized Human Dermis

The goal of this paper is to provide a comprehensive and detailed protocol on how to generate genetically modified human organotypic skin from epidermal keratinocytes and devitalized human dermis.

Generación de Genéticamente Modificados cultivos de piel organotípicos Uso Desvitalizada Dermis Humana

Organotypic cultures allow the reconstitution of a 3D environment critical for cell-cell contact and cell-matrix interactions which mimics the function ...

generation-genetically-modified-organotypic-skin-cultures-using

Research

JoVE Journal

Developmental Biology

11.1K vistas.  UCSD Stem Cell Program, University of California, San Diego. The goal of this paper is to provide a comprehensive and detailed protocol on how to generate genetically modified human organotypic skin from epidermal keratinocytes and devitalized human dermis. 

Video: Generation of Genetically Modified Organotypic Skin Cultures Using Devitalized Human Dermis

Organotypic Slice Cultures for Studies of Postnatal Neurogenesis

Here we describe a technique for studying hippocampal postnatal neurogenesis in the rodent brain using the organotypic slice culture technique. This method maintains the characteristic topographical morphology of the hippocampus while allowing direct application of pharmacological agents to the developing hippocampal dentate gyrus. Additionally, slice cultures can be maintained for up to 4 weeks and thus, allow one to study the maturation process of newborn granule neurons. Slice cultures allow for efficient pharmacological manipulation of hippocampal slices while excluding complex variables such as uncertainties related to the deep anatomic location of the hippocampus as well as the blood brain barrier. For these reasons, we sought to optimize organotypic slice cultures specifically for postnatal neurogenesis research.

Here we describe a technique for studying hippocampal postnatal neurogenesis using the organotypic slice culture technique. This method allows for in vitro manipulation of adult neurogenesis and allows for the direct application of pharmacological agents to the cultured hippocampus.

Culturas organotípicos rebanada de Estudios de Postnatal neurogénesis

Here we describe a technique for studying hippocampal postnatal neurogenesis in the rodent brain using the organotypic slice culture technique. This ...

Investigación

Biología del Desarrollo

Generation of Human Adipose Stem Cells through Dedifferentiation of Mature Adipocytes in Ceiling Cultures

Mature adipocytes have been shown to reverse their phenotype into fibroblast-like cells in vitro through a technique called ceiling culture. Mature adipocytes can also be isolated from fresh adipose tissue for depot-specific characterization of their function and metabolic properties. Here, we describe a well-established protocol to isolate mature adipocytes from adipose tissues using collagenase digestion, and subsequent steps to perform ceiling cultures. Briefly, adipose tissues are incubated in a Krebs-Ringer-Henseleit buffer containing collagenase to disrupt tissue matrix. Floating mature adipocytes are collected on the top surface of the buffer. Mature cells are plated in a T25-flask completely filled with media and incubated upside down for a week. An alternative 6-well plate culture approach allows the characterization of adipocytes undergoing dedifferentiation. Adipocyte morphology drastically changes over time of culture. Immunofluorescence can be easily performed on slides cultivated in 6-well plates as demonstrated by FABP4 immunofluorescence staining. FABP4 protein is present in mature adipocytes but down-regulated through dedifferentiation of fat cells. Mature adipocyte dedifferentiation may represent a new avenue for cell therapy and tissue engineering.

Mature adipocytes may represent an abundant source of stem cells through dedifferentiation, which leads to a homogenous population of fibroblast-like cells. Collagenase digestion is used to isolate mature adipocytes from human fat. The goal of our protocol is to obtain multipotent, dedifferentiated fat cells from human mature adipocytes.

Generación de células madre adiposas Humano a través Desdiferenciación de adipocitos maduros en culturas de techo

Mature adipocytes have been shown to reverse their phenotype into fibroblast-like cells in vitro through a technique called ceiling culture. Mature ...

Transfection, Selection, and Colony-picking of Human Induced Pluripotent Stem Cells TALEN-targeted with a GFP Gene into the AAVS1 Safe Harbor

Targeted transgene addition can provide persistent gene expression while circumventing the gene silencing and insertional mutagenesis caused by viral vector mediated random integration. This protocol describes a universal and efficient transgene targeted addition platform in human iPSCs based on utilization of validated open-source TALENs and a gene-trap-like donor to deliver transgenes into a safe harbor locus. Importantly, effective gene editing is rate-limited by the delivery efficiency of gene editing vectors. Therefore, this protocol first focuses on preparation of iPSCs for transfection to achieve high nuclear delivery efficiency. When iPSCs are dissociated into single cells using a gentle-cell dissociation reagent and transfected using an optimized program, >50% cells can be induced to take up the large gene editing vectors. Because the AAVS1 locus is located in the intron of an active gene (PPP1R12C), a splicing acceptor (SA)-linked puromycin resistant gene (PAC) was used to select targeted iPSCs while excluding random integration-only and untransfected cells. This strategy greatly increases the chance of obtaining targeted clones, and can be used in other active gene targeting experiments as well. Two weeks after puromycin selection at the dose adjusted for the specific iPSC line, clones are ready to be picked by manual dissection of large, isolated colonies into smaller pieces that are transferred to fresh medium in a smaller well for further expansion and genetic and functional screening. One can follow this protocol to readily obtain multiple GFP reporter iPSC lines that are useful for in vivo and in vitro imaging and cell isolation.

TALEN-mediated gene editing at the safe harbor AAVS1 locus enables high-efficiency transgene addition in human iPSCs. This protocol describes the procedures for preparing iPSCs for TALEN and donor vector delivery, transfecting iPSCs, and selecting and isolating iPSC clones to achieve targeted integration of a GFP gene to generate reporter lines.

La transfección, selección y Colonia-cosecha de células madre humanas pluripotentes inducidas TALEN dirigidos con un gen GFP en el puerto seguro AAVS1

Targeted transgene addition can provide persistent gene expression while circumventing the gene silencing and insertional mutagenesis caused by viral ...

3D Organotypic Co-culture Model Supporting Medullary Thymic Epithelial Cell Proliferation, Differentiation and Promiscuous Gene Expression

Intra-thymic T cell development requires an intricate three-dimensional meshwork composed of various stromal cells, i.e., non-T cells. Thymocytes traverse this scaffold in a highly coordinated temporal and spatial order while sequentially passing obligatory check points, i.e., T cell lineage commitment, followed by T cell receptor repertoire generation and selection prior to their export into the periphery. The two major resident cell types forming this scaffold are cortical (cTECs) and medullary thymic epithelial cells (mTECs). A key feature of mTECs is the so-called promiscuous expression of numerous tissue-restricted antigens. These tissue-restricted antigens are presented to immature thymocytes directly or indirectly by mTECs or thymic dendritic cells, respectively resulting in self-tolerance.
Suitable in vitro models emulating the developmental pathways and functions of cTECs and mTECs are currently lacking. This lack of adequate experimental models has for instance hampered the analysis of promiscuous gene expression, which is still poorly understood at the cellular and molecular level. We adapted a 3D organotypic co-culture model to culture ex vivo isolated mTECs. This model was originally devised to cultivate keratinocytes in such a way as to generate a skin equivalent in vitro. The 3D model preserved key functional features of mTEC biology: (i) proliferation and terminal differentiation of CD80lo, Aire-negative into CD80hi, Aire-positive mTECs, (ii) responsiveness to RANKL, and (iii) sustained expression of FoxN1, Aire and tissue-restricted genes in CD80hi mTECs.

Studying medullary thymic epithelial cells in vitro has been largely unsuccessful, as current 2D culture systems do not mimic the in vivo scenario. The 3D culture system described herein - a modified skin organotypic culture model - has proven superior in recapitulating mTEC proliferation, differentiation and maintenance of promiscuous gene expression.

3D organotípicos Co-cultura de apoyo medular tímica epitelial proliferación celular, la diferenciación y la Expresión Génica Promiscuo Modelo

Intra-thymic T cell development requires an intricate three-dimensional meshwork composed of various stromal cells, i.e., non-T cells. Thymocytes traverse ...

Generation of Integration-free Human Induced Pluripotent Stem Cells Using Hair-derived Keratinocytes

Recent advances in reprogramming allow us to turn somatic cells into human induced pluripotent stem cells (hiPSCs). Disease modeling using patient-specific hiPSCs allows the study of the underlying mechanism for pathogenesis, also providing a platform for the development of in vitro drug screening and gene therapy to improve treatment options. The promising potential of hiPSCs for regenerative medicine is also evident from the increasing number of publications (>7000) on iPSCs in recent years. Various cell types from distinct lineages have been successfully used for hiPSC generation, including skin fibroblasts, hematopoietic cells and epidermal keratinocytes. While skin biopsies and blood collection are routinely performed in many labs as a source of somatic cells for the generation of hiPSCs, the collection and subsequent derivation of hair keratinocytes are less commonly used. Hair-derived keratinocytes represent a non-invasive approach to obtain cell samples from patients. Here we outline a simple non-invasive method for the derivation of keratinocytes from plucked hair. We also provide instructions for maintenance of keratinocytes and subsequent reprogramming to generate integration-free hiPSC using episomal vectors.

This manuscript provides a step-by-step procedure for the derivation and maintenance of human keratinocytes from plucked hair and subsequent generation of integration-free human induced pluripotent stem cells (hiPSCs) by episomal vectors.

Generación de células madre Integración de libre Humanos pluripotentes inducidas Usando derivada de pelo queratinocitos

Recent advances in reprogramming allow us to turn somatic cells into human induced pluripotent stem cells (hiPSCs). Disease modeling using ...

Generation of Integration-free Induced Pluripotent Stem Cells from Human Peripheral Blood Mononuclear Cells Using Episomal Vectors

Induced Pluripotent Stem Cells (iPSCs) hold great promise for disease modeling and regenerative therapies. We previously reported the use of Episomal Vectors (EV) to generate integration-free iPSCs from peripheral blood mononuclear cells (PB MNCs). The episomal vectors used are DNA plasmids incorporated with oriP and EBNA1 elements from the Epstein-Barr (EB) virus, which&#160;allow for replication and long-term retainment of plasmids in mammalian cells, respectively. With further optimization, thousands of iPSC colonies can be obtained from 1 mL of peripheral blood. Two critical factors for achieving high reprogramming efficiencies&#160;are: 1) the use of a 2A &#34;self-cleavage&#34; peptide to link OCT4 and SOX2, thus achieving equimolar expression of the two factors; 2) the use of two vectors to express MYC and KLF4 individually. Here we describe a step-by-step protocol for generating integration-free iPSCs from adult peripheral blood samples. The generated iPSCs are integration-free as residual episomal plasmids are undetectable after five passages. Although the reprogramming efficiency is comparable to that of Sendai Virus (SV) vectors, EV plasmids are considerably more economical than the commercially available SV vectors. This affordable EV reprogramming system holds potential for clinical applications in regenerative medicine and provides an approach for the direct reprogramming of PB MNCs to integration-free mesenchymal stem cells, neural stem cells, etc.

This protocol describes a detailed method for efficient generation of integration-free iPSCs from human adult peripheral blood cells. With the use of four oriP/EBNA-based episomal vectors to express the reprogramming factors, KLF4, MYC, BCL-XL, or OCT4 and SOX2, thousands of iPSC colonies can be obtained from 1 mL of peripheral blood.

Generación de Integración libres de células madre pluripotentes inducidas a partir de células mononucleares de sangre periférica humana Utilizando vectores episomales

Induced Pluripotent Stem Cells (iPSCs) hold great promise for disease modeling and regenerative therapies. We previously reported the use of Episomal ...

A Modified Trier Social Stress Test for Vulnerable Mexican American Adolescents

The Trier Social Stress Test (TSST) is a well validated and widely used social stressor that has been shown to induce a 2-4 fold increase in cortisol, the biological output produced by the Hypothalamic-Pituitary-Adrenal (HPA) axis in humans. While studies have explored how modifications to the TSST influence stress responsivity, few studies have created a modified TSST appropriate for vulnerable youth that elicits a significant cortisol stress response. Thus, the current study sought to modify or adjust the TSST in a culturally sensitive way for a vulnerable sample of 14 year-old adolescents. The present study took place within the context of a longitudinal birth cohort study of Mexican American families in California called the Center for the Health Assessment of Mothers and Children of Salinas (CHAMACOS). The CHAMACOS sample was optimal to test the effectiveness of a modified culturally appropriate TSST, as it is comprised of Mexican American youth, who are often excluded from research. These youths also have experienced significant early life adversity. Example modifications included timed prompts, alternative math tasks, use of same-ethnicity peers as confederates, debriefing immediately after the conclusion of the TSST, and using an unknown youth examiner to administer the debrief. Saliva samples were collected at baseline (after a resting phase), and then again at 15, 30, and 45 min post-TSST onset to assess cortisol concentration. A pilot study of 50 participants (50% female) have been analyzed for cortisol reaction to the TSST. Results confirmed that this modified version of the TSST was successful at eliciting a significant cortisol reaction, with a wide range of variability likely due to individual differences. Goals for modifications and ethnicity considerations are discussed. This study provides the foundation for future research to utilize a modified TSST with vulnerable youth.

Here, we present a protocol that provoked cortisol reactivity in a vulnerable adolescent Mexican American sample utilizing a modified version of the Trier Social Stress Test (TSST). Saliva samples were collected at baseline, 15, 30, and 45 min post-TSST onset. Future research could utilize this modified TSST with vulnerable youth.

Una Prueba de Estrés Social Modificada de Trier para Adolescentes Mexicanos Vulnerables

The Trier Social Stress Test (TSST) is a well validated and widely used social stressor that has been shown to induce a 2-4 fold increase in cortisol, the ...

Generation of Genetically Modified Mice through the Microinjection of Oocytes

The use of genetically modified mice has significantly contributed to studies on both physiological and pathological in vivo processes. The pronuclear injection of DNA expression constructs into fertilized oocytes remains the most commonly used technique to generate transgenic mice for overexpression. With the introduction of CRISPR technology for gene targeting, pronuclear injection into fertilized oocytes has been extended to the generation of both knockout and knockin mice. This work describes the preparation of DNA for injection and the generation of CRISPR guides for gene targeting, with a particular emphasis on quality control. The genotyping procedures required for the identification of potential founders are critical. Innovative genotyping strategies that take advantage of the "multiplexing" capabilities of CRISPR are presented herein. Surgical procedures are also outlined. Together, the steps of the protocol will allow for the generation of genetically modified mice and for the subsequent establishment of mouse colonies for a plethora of research fields, including immunology, neuroscience, cancer, physiology, development, and others.

The microinjection of mouse oocytes is commonly used for both classic transgenesis (i.e., the random integration of transgenes) and CRISPR-mediated gene targeting. This protocol reviews the latest developments in microinjection, with a particular emphasis on quality control and genotyping strategies.

Generación de ratones modificados genéticamente a través de la microinyección de ovocitos

The use of genetically modified mice has significantly contributed to studies on both physiological and pathological in vivo processes. The pronuclear ...

Generation and Culturing of Primary Human Keratinocytes from Adult Skin

The main function of keratinocytes is to provide the structural integrity of the epidermis, thereby maintaining a mechanical barrier to the outside world. In addition, keratinocytes play an essential role in the initiation, maintenance, and regulation of epidermal immune responses by being part of the innate immune system responding to antigenic stimuli in a fast, nonspecific manner. Here, we describe a protocol for isolation of primary human keratinocytes from adult skin, and demonstrate that these cells respond to calcium-induced terminal differentiation, as measured by an increased expression of the differentiation marker involucrin. In addition, we show that the isolated keratinocytes are responsive to IL-1&#946;-induced activation of intracellular signaling pathways as measured by the activation of the p38 MAPK pathway. Taken together, we describe a method for isolation and culturing of primary human keratinocytes from adult skin. Because the keratinocytes are the predominant cell type in the epidermis, this method is useful to study molecular mechanisms in cutaneous biology in vitro.

The human skin acts as a first line of defense against the external environment. We present a method for isolating primary human keratinocytes from adult skin. These isolated keratinocytes are useful in numerous experimental setups, and are a highly suitable model for studying molecular mechanisms in cutaneous biology in vitro.

Generación y cultivo de Keratinocytes humanos primarios de piel adulto

The main function of keratinocytes is to provide the structural integrity of the epidermis, thereby maintaining a mechanical barrier to the outside world. ...

Three-dimensional Organotypic Cultures of Vestibular and Auditory Sensory Organs

The sensory organs of the inner ear are challenging to study in mammals due to their inaccessibility to experimental manipulation and optical observation. Moreover, although existing culture techniques allow biochemical perturbations, these methods do not provide a means to study the effects of mechanical force and tissue stiffness during development of the inner ear sensory organs. Here we describe a method for three-dimensional organotypic culture of the intact murine utricle and cochlea that overcomes these limitations. The technique for adjustment of a three-dimensional matrix stiffness described here permits manipulation of the elastic force opposing tissue growth. This method can therefore be used to study the role of mechanical forces during inner ear development. Additionally, the cultures permit virus-mediated gene delivery, which can be used for gain- and loss-of-function experiments. This culture method preserves innate hair cells and supporting cells and serves as a potentially superior alternative to the traditional two-dimensional culture of vestibular and auditory sensory organs.

Three-dimensional organotypic cultures of the murine utricle and cochlea in optically clear collagen I gels preserve innate tissue morphology, allow for mechanical stimulation through adjustment of matrix stiffness, and permit virus-mediated gene delivery.

Culturas Organotypic tridimensionales de órganos sensoriales vestibulares y auditivas

The sensory organs of the inner ear are challenging to study in mammals due to their inaccessibility to experimental manipulation and optical observation. ...