Este trabalho foi supportedby Sociedade Americana de Câncer Research Scholars Grant (RSG-12-148-01-DDC) eo Prêmio Biologia CIRM Básico (RB4-05779).

O protocolo de pele humana foi realizada em conformidade com as diretrizes da Universidade da Califórnia, o Comitê de Ética em Pesquisa do San Diego. A pele humana pode ser obtido a partir de amostras cirúrgicas descartados ou comprados a partir de bancos de pele (pele banco está listado na Tabela de Materiais / Equipamentos). A localização, onde a pele é derivado a partir de ou a idade do dador não é crítica para a experiência, enquanto as proteínas da membrana basal (zona de colagénio / laminina) na derme não são degradados. 1. Preparação de desvitalizados derme humana <ol><li> Ao receber a pele humana, colocar o tecido no capuz de cultura de tecidos de descongelar (~ 3-5 min). Dependendo do tamanho da pele, colocar o tecido descongelado num descartável tubo de 50 ml estéril ou frasco de 125 ml. O tamanho típico da pele a partir do banco de pele é de 0,75 pés quadrados. </li><li> Encha o depósito de 75% completo com penicilina e estreptomicina 4x (pen / strep) em 1x PBS. Agite vigorosamente durante5 min e descarte do líquido. Repita este passo mais 3 vezes. </li><li> Após a última lavagem, transferir o tecido para um novo tubo / frasco e adicionar 4x fresco Pen / Strep / PBS para encher 75% do recipiente. Incubar esta mistura numa incubadora de cultura de tecidos a 37 ° C durante 2 semanas para permitir a separação da derme da epiderme. </li><li> Sob condições estéreis, utilizar uma pinça para descascar a epiderme da derme. A derme é completamente branco enquanto a epiderme terá coloração, dependendo da raça do doador (Figura 1A). Descarte a epiderme acordo com o protocolo da instituição para lidar com o tecido humano. </li><li> Coloque a derme em um tubo ou garrafa e lavá-lo em 4x pen / strep diluído em 1x PBS com agitação forte (5 mínimo de lavagens por 4 vezes). Após a última lavagem, transferir a derme para um novo tubo / frasco em 4x pen / strep diluído em 1x PBS e armazenar a 4 ° C até que esteja pronto para uso. Derme pode ser armazenado a 4 ° C durante até um ano. </li></ol&gt; 2. modificar geneticamente Primária queratinócitos humanos NOTA: Use pilhas phoenix anfotrï¿½icas cultivadas em meio DMEM completo [DMEM + 10% de soro fetal bovino (FBS) + pen / strep] para produzir o vírus para infectar queratinócitos humanos primários. Genes de empacotamento virais que codificam proteínas tais como gag-pol e do envelope são integrados de forma estável no genoma da célula Phoenix que permite a produção de vírus quando transfectada com um vector retroviral. Phoenix células podem ser transfectadas com alta eficiência, permitindo a produção em alta título viral. Vírus produzido a partir desta linha de embalagem também pode infectar uma grande variedade de células de mamíferos incluindo o ser humano. <ol><li> Dia 1: No dia antes da transfecção, as células Phoenix semente em uma placa de 6 poços a uma densidade de 800.000 células por poço em meio DMEM completo. </li><li> Dia 2: O dia da transfecção, 3 ug de utilizar vectores retrovirais por poço. Aliquota de 3 ug de vector retroviral para um tubo de 1,5 ml. Use uma mistura de 100 μ; l DMEM mais 6 ul de reagente de transfecção para cada 3 ug de vector. Misturar 6 ul de reagente de transfecção com 100 ul de DMEM em um tubo de 1,5 ml. (Os vectores retrovirais que são usados ​​estão indicados na tabela do equipamento / materiais). <ol><li> Incubar durante 5 min e adicionar 106 uL da mistura para dentro do tubo de pré-alíquota contendo a 3 ug de vector retroviral. Misturar e incubar à TA durante 30 min. Adicionar gota a gota toda esta mistura a cada poço das células Phoenix. </li></ol></li><li> Dia 3: O dia após a transfecção, remova a mídia a partir das células phoenix e adicionar 2 ml de meio DMEM completo fresco. No mesmo dia, semear queratinócitos humanos primários em placas de 6 poços a uma densidade de 75.000 células por poço. Manter os queratinócitos em um meio de soro livre de queratinócitos (KCSFM) com antibióticos (pen / strep). NOTA: queratinócitos humanos primários foram comprados. As células podem ser adquiridos a partir de uma variedade de fornecedores (listada na tabela de material / Equipamento). </li><li> Dia 4: Harcolete a mídia a partir das células transfectadas phoenix, que agora contém partículas virais. Passe a mídia através de um filtro de 0,45 um, utilizando uma seringa (para remover quaisquer células phoenix contaminantes) e coloque 2 ml para os queratinócitos (banhados em 75.000 células / poço no dia anterior: ver passo 2.3). Adiciona-se brometo de hexadimetrina (5 ug / ml) para os meios para ajudar a mediar o processo de infecção. Títulos virais são de 1 x 10 ~ 7 TU / mL. <ol><li> Rodar as placas de 6 poços numa centrífuga a 200 xg durante 1 h à TA. Após a rotação, remova a mídia que contém vírus e lavar as células uma vez com 1x PBS antes de adicionar KCSFM. </li></ol></li><li> Dia 5: infectar o mesmo lote de queratinócitos de novo como no passo 2.4. NOTA: Escolha os queratinócitos usando um fármaco se houver um marcador seleccionável no vector retroviral e expandir para uso em análise ou aplicações, tais como as culturas organotípicas de jusante. </li></ol> 3. Configurando culturas de pele organotï¿½icas <ol><li> Construir ocassete organotípico a partir de materiais comuns encontrados no laboratório ou as cassetes podem ser feitos através de uma oficina de fabricação de metal (Figura 2A - F). Para fazer com que essas cassetes de 3,5 cm de uma placa de cultura de tecido, utilizar um cautério para remover um 1,0 cm x 1,0 cm quadrados do centro da tampa de uma 3,5 cm de prato (Figura 2A-B). Faça isso em condições estéreis. <ol><li> Anexar pinos quadrados para a parte inferior da cassete (tampa de 3,5 cm de placa) utilizando claro unha polonês (Figura 2C). Permitir 5 min para a unha polonês para secar e virar a cassete de forma que ele está descansando sobre os pinos quadrados (Figura 2D). Coloque a cassete em um disco de 6 cm. NOTA: Praça estacas podem ser comprados a partir de uma variedade de lojas de artesanato. </li></ol></li><li> Preparar o meio de crescimento de queratinócitos (KGM) composta de 66% de DMEM, 22% de F12 de Ham, 100 unidades / ml de penicilina, 100 ug / ml de estreptomicina, 10% de FBS, 2,67 ug / ml de adenina, 40 Ng / ml de hidrocortisona, 10 ng de toxina da cólera ml /, 4,94 ug / ml de insulina, 5 ug / ml de transferrina, 1,36 ng / ml triiodo-L-tironina, 10 ng / ml de EGF, e 10 ug / ml de cloridrato de ciprofloxacina. Filtra-se a meios de comunicação através de um filtro de 0,22? M e armazenamento a 4 ° C até estar pronto para usar. </li><li> Aqui a derme para fora da 4x Pen / Strep + solução de PBS e lavar 2 vezes em KGM e em seguida incuba-se a 37 KGM ° C durante dois dias antes da utilização. </li><li> Corte a derme utilizando um bisturi para 1,5 cm x 1,5 cm pedaços de tamanho e, em seguida, coloque-a cassete organotípico. Coloque a derme com a parte superior da derme virados para cima. A parte superior da derme será o lado que entra em contacto com os queratinócitos (Figura 1B). </li><li> Coloque a derme pré-cortadas para o furo quadrado da cassete organotípicas com o lado superior da derme voltadas para cima (Figura 3A). </li><li> Virar toda a cassete organotípico contendo a derme mais usando uma pinça (Figura 3B). Descongelar a solução de matriz extracelular em gelo. Use uma agulha e seringa para tirar 100 ml de solução de matriz extracelular por cassete. Adicione 5 gotas para o fundo da derme (lado brilhante) e agitar ligeiramente para assegurar uma distribuição uniforme da derme (Figura 3B). </li><li> Permitir 5 min para a solução comercial de matriz extra-celular para solidificar completamente (Figura 3C). Após a solidificação, utilize uma pinça para rodar a fita para trás sobre. Adicionar 4 ml de KGM com o disco de 6 cm. </li><li> Coloque 0,5-1 milhões de queratinócitos geneticamente modificados para as cassetes organotï¿½icas contendo a derme (Figura 3D). <ol><li> Tripsinizar queratinócitos por colocar 4 ml de tripsina a 0,05% em placas de 10 cm durante 5 minutos e diluiu-se com 4 ml de meio DMEM completo. </li><li> Contagem dos queratinócitos utilizando um hemocitómetro e, em seguida girar a 200 xg durante 5 min. Ressuspender as células 0,5-1 milhões (dependendo do número de células disponíveis) em 90 μl de KGM e colocar todas as células para a derme. NOTA: É importante colocar o mesmo número de células na derme dentro da mesma experiência para assegurar que quaisquer diferenças observadas na espessura da epiderme regenerada não é devido a diferenças no número de células de partida. Colocando menos de 0,5 milhões de células em derme pode levar a uma má estratificação e diferenciação. </li></ol></li><li> Mudar meios KGM sobre as culturas organotípicas de todos os outros dias. Tecido colheita 1-14 dias após a colocação dos queratinócitos na derme. Estratificação completa e diferenciação da epiderme geralmente ocorre após o dia 5. </li></ol>

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We have nothing to disclose.

A manipulação genética na pele humana culturas organotípicas oferecem muitas vantagens para comumente estudada 2D células cultivadas, bem como modelos de mouse. Culturas 2D faltam os três célula-célula e célula-matriz extracelular interacções dimensionais encontradas em tecidos e órgãos intactos. Estudos recentes também têm encontrado enormes diferenças entre as células de câncer de pele cultivadas em 2D e 3D com as células cultivadas em 3D que mostram muito mais semelhanças de expressão do gene par...

A epiderme humana é um epitélio estratificado que se conecta à derme subjacente através de uma matriz extracelular conhecida como a epiderme zone.The membrana basal não somente serve como uma barreira impermeável para impedir a perda de humidade, mas também como uma primeira linha de defesa para proteger o corpo de substâncias estranhas e tóxicas 1. A camada basal, que é a camada mais profunda da epiderme, contém as células estaminais e progenitoras da epiderme que dão origem a progenia diferenciada para que formam o resto da epiderme 2. Como as células progenitoras diferenciam epidérmicas que migram para cima para formar a primeira camada de células diferenciadas conhecidos como a camada espinhosa 3. Na camada espinhosa, as células ligar a expressão de queratinas 1 e 10, que, em seguida, fornecer a força necessária para suportar o esforço físico para as camadas diferenciadas da epiderme. À medida que as células da camada espinhosa diferenciar ainda mais, que se movem para cima na epiderme para am a camada granular, que é caracterizada pela formação de querato-hialina e grânulos lamelares, bem como proteínas estruturais que são montados por baixo da membrana de plasma. Como as células em prosseguir o processo de diferenciação, as proteínas abaixo da membrana plasmática estão transversal ligados uns aos outros, enquanto os grânulos lamelares são extrudadas a partir das células de modo a formar uma barreira de lípidos ricos chamada estrato córneo 4. As doenças que envolvem alterações no crescimento epidérmico e impacto diferenciação ~ 20% da população 5. Assim, o entendimento dos mecanismos deste processo é de grande importância. Desde manifestação de muitas destas doenças é contingente após a célula-célula ou célula-matriz de contacto, as culturas organotípicas de onde a epiderme humana é reconstituída num ambiente 3D foram criadas 6-10. Estes métodos envolvem tipicamente a utilização de queratinócitos primários ou transformadas semeadas em matriz extracelular, tais como humano desvitalizadoderme, Matrigel, ou colagénio. Para compreender os mecanismos regulatórios dos genes que são importantes no crescimento e na diferenciação da epiderme, os queratinócitos podem ser geneticamente manipulados através de vectores retrovirais para knockdown ou sobre-expressar genes em cultura e em seguida reconstituída 2D em 3D. Estes métodos têm sido amplamente utilizados para caracterizar genes envolvidos na haste epidérmica de células progenitoras e de auto-renovação e diferenciação, bem como a progressão para neoplasia 11-21. Aqui, um protocolo em profundidade sobre como alterar a expressão genética em culturas organotípicas epidérmicas através da utilização de retrovírus é fornecido.

<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

Culturas organotípicas permitir a reconstituição de um ambiente 3D crítica para interacções de contacto célula-célula e célula-matriz que imita a fisiologia e função dos seus homólogos de tecido in vivo. Isto é exemplificado por culturas de pele organotï¿½icas que recapitula fielmente a diferenciação epidérmica e programa de estratificação. Queratinócitos epidérmicos humanos primários são geneticamente manipuláveis ​​através de retrovírus onde os genes podem ser facilmente sobre-expressos ou derrubado. Estes queratinócitos geneticamente modificados podem então ser usadas para regenerar epiderme humana em culturas organotípicas de pele proporcionando um poderoso modelo para estudar vias genéticas crescimento epidérmico de impacto, a diferenciação e a progressão da doença. Os protocolos aqui apresentados descrevem métodos para preparar derme humana desvitalizados, bem como manipular geneticamente queratinócitos humanos primários, a fim de gerar culturas organotípicas de pele. Pele humana regenerado pode ser usado em downstream aplicações, tais como perfil de expressão gênica, immunostaining, e imunoprecipitações cromatina seguido de seqüenciamento de alta taxa de transferência. Assim, a geração destas culturas organotípicas de pele geneticamente modificados permite a determinação dos genes que são essenciais para a manutenção da homeostase da pele.

O primeiro passo na geração de pele humana organotípica é remover a epiderme da derme. A incubação de duas semana da pele a 37 ° C em 4x Pen / Strep / PBS deve permitir a separação da derme da epiderme (Figura 1A). Se a separação da epiderme e da derme, em seguida, é difícil colocar o tecido a 37 ° C em 4x Pen / Strep / PBS durante uma semana e, em seguida, tente novamente casca utilizando fórceps. Uma das chaves para a regeneração da epiderme humana na derm...

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.

Geração de Culturas Geneticamente Modificados Pele organotï¿½icas Usando desvitalizado derme 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 visualizações.  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. 

Vídeo: 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.

Cultures Organotípicas fatia de Estudos de Pós-natal Neurogênese

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

Biologia do Desenvolvimento

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.

Geração de adiposo humano Stem Cells através Dedifferentiation de adipócitos maduros em Culturas de teto

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.

Transfection, seleção e Colony-picking de induzidas pelo homem pluripotentes Células-Tronco TALEN-alvo com um gene GFP no AAVS1 Safe Harbor

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 Organotípicas Co-cultura Modelo de Apoio medular Thymic Epithelial proliferação, diferenciação e Expression Promiscuous Gene

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.

Geração de Integração Induced-livre Human células estaminais pluripotentes usando o cabelo derivado queratinócitos

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.

Geração de Integração-livres induzidas células-tronco pluripotentes a partir de células mononucleares de sangue periférico humano Usando epissï¿½ica Vectors

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.

Um Teste de Stress Social Modificado de Trier para Vulneráveis ​​Mexicano-Adolescentes

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.

Geração de ratos geneticamente modificados através da microinjecção de oócitos

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.

Geração e cultivo de queratinócitos humanos primários da pele de 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 tridimensional de órgãos sensoriais auditivas e vestibulares

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