Questo lavoro è stato supportedby l&#39;American Cancer Society Research Scholars Grant (RSG-12-148-01-DDC) e il Premio Biologia CIRM di base (RB4-05779).

Il protocollo pelle umana è stata effettuata in conformità con le linee guida della University of California, Ricerca Comitato Etico di San Diego. La pelle umana può essere ottenuto da campioni chirurgici scartati o comprato da banche della pelle (pelle banca è elencato nella tabella Materiali / Equipment). La posizione in cui la pelle è realizzata o età del donatore non è critico per l&#39;esperimento finché le proteine ​​membrana basale (collagene / laminina) nel derma non sono degradati. 1. Preparazione di devitalizzate derma umano <ol><li> Alla ricezione di pelle umana, posizionare il tessuto nella cappa coltura tissutale per scongelare (~ 3-5 min). A seconda delle dimensioni della pelle, posizionare il tessuto scongelato in un monouso tubo da 50 ml o 125 ml sterile bottiglia. La dimensione tipica della pelle da parte della banca pelle è di 0,75 mq. </li><li> Riempire il serbatoio pieno al 75% con 4x la penicillina e streptomicina (penna / strep) in PBS 1x. Agitare energicamente per5 min e smaltire il liquido. Ripetere questa operazione altre 3 volte. </li><li> Dopo l&#39;ultimo lavaggio, trasferire il tessuto in una nuova provetta / bottiglia e aggiungere 4x fresco pen / strep / PBS per riempire 75% del contenitore. Incubare questa miscela in un incubatore di coltura tissutale a 37 ° C per 2 settimane per consentire la separazione del derma dell&#39;epidermide. </li><li> In condizioni sterili, utilizzare una pinza a staccarsi l&#39;epidermide dal derma. Il derma è completamente bianco, mentre l&#39;epidermide avranno colorazione a seconda della razza del donatore (Figura 1A). Eliminare l&#39;epidermide secondo il protocollo dell&#39;istituzione per affrontare con i tessuti umani. </li><li> Posizionare il derma in un tubo o bottiglia e lavarlo in 4x penna / strep diluito in PBS 1x con vigorosi tremanti (5 min lavaggi per 4 volte). Dopo l&#39;ultimo lavaggio, trasferire il derma in una nuova provetta / bottiglia in 4x penna / strep diluito in PBS 1x e conservare a 4 ° C fino al momento dell&#39;uso. Derma possono essere conservati a 4 ° C per un massimo di un anno. </li></ol&gt; 2. modificare geneticamente cheratinociti umani primari NOTA: utilizzare le cellule phoenix amphotropic cresciute nei media DMEM completo [DMEM + 10% siero bovino fetale (FBS) + pen / strep] per la produzione di virus per infettare cheratinociti umani primari. Geni imballaggio virali che codificano proteine ​​come gag-pol e busta sono stabilmente integrati nel genoma della cellula Phoenix che consente la produzione di virus quando trasfettate con un vettore retrovirale. Phoenix cellule possono essere transfettate con alta efficienza che consente un&#39;elevata produzione titolo virale. Virus prodotto da questa linea di confezionamento può infettare una grande varietà di cellule di mammiferi compreso umana. <ol><li> 1 ° giorno: Il giorno prima trasfezione, le cellule fenice seme in un 6-pozzetti ad una densità di 800.000 cellule per pozzetto nei media DMEM completo. </li><li> Giorno 2: Il giorno della transfezione, utilizzare 3 mg di vettori retrovirali per pozzetto. Aliquotare 3 mg di vettore retrovirale in una provetta da 1,5 ml. Utilizzare un mix di 100 μ; l DMEM più 6 ml di reagente di trasfezione per ogni 3 mg di vettore. Mescolare 6 ml di reagente di trasfezione con 100 microlitri DMEM in una provetta da 1,5 ml. (I vettori retrovirali che vengono utilizzati sono elencati nella tabella attrezzature / materiali). <ol><li> Incubare per 5 minuti e aggiungere il composto di 106 microlitri nel tubo pre-aliquotati contenente 3 mg di vettore retrovirale. Mescolare e incubare a temperatura ambiente per 30 min. Aggiungere questa intera miscela gocce a ciascun pozzetto di cellule Phoenix. </li></ol></li><li> 3 ° giorno: Il giorno dopo la trasfezione, rimuovere il supporto dalle cellule phoenix e aggiungere 2 ml di fresco supporto completo DMEM. Lo stesso giorno, seminare cheratinociti umani primari in 6 pozzetti ad una densità di 75.000 cellule per pozzetto. Mantenere i cheratinociti in un mezzo di siero cheratinociti gratuito (KCSFM) con antibiotici (penna / strep). NOTA: cheratinociti umani primari sono stati acquistati. Le cellule possono essere acquistati da una varietà di fornitori (elencati nella tabella Materiali / Equipment). </li><li> 4 ° giorno: Harconferire al supporto dalle cellule trasfettate fenice, che ora contiene particelle virali. Far passare il materiale attraverso un filtro da 0,45 micron con una siringa (per rimuovere tutte le cellule fenice contaminanti) e posizionare 2 ml sulle cheratinociti (placcato a 75.000 cellule / pozzetto del giorno precedente: vedere il punto 2.3). Aggiungere hexadimethrine bromuro (5 mg / ml) per i media per aiutare a mediare il processo di infezione. Titoli virali sono ~ 1 x 10 7 TU / ml. <ol><li> Spin piastre da 6 pozzetti in una centrifuga a 200 xg per 1 ora a RT. Dopo lo spin, rimuovere i supporti contenenti virus e lavare le cellule una volta con PBS 1x prima di aggiungere KCSFM. </li></ol></li><li> 5 ° giorno: infettare la stessa partita di cheratinociti di nuovo come al punto 2.4. NOTA: Selezionare i cheratinociti usando un farmaco, se vi è un marcatore sul vettore retrovirale ed espandere per l&#39;uso in analisi a valle o applicazioni come colture organotipiche. </li></ol> 3. Configurazione Culture pelle organotipiche <ol><li> Costruire ilcassetta organotipica da materiali comuni che si trovano in laboratorio o le cassette può essere personalizzato attraverso un negozio di fabbricazione di metallo (Figura 2A - F). Per rendere queste cassette da una piastra di coltura tissutale 3.5 cm, utilizzare un bisturi per rimuovere un 1,0 cm x 1,0 cm quadrato dal centro del coperchio di un piatto 3.5 cm (Figura 2A-B). Fate questo in condizioni di sterilità. <ol><li> Attaccare pioli quadrati sul fondo della cassetta (coperchio di 3.5 cm piatto) utilizzando smalto trasparente (Figura 2C). Lasciare 5 minuti per lo smalto si asciughi e capovolgere la cassetta sopra in modo che poggi sui pioli quadrati (Figura 2D). Posizionare la cassetta in un piatto 6 centimetri. NOTA: Piazza pioli possono essere acquistati da una varietà di arti e botteghe artigiane. </li></ol></li><li> Preparare terreno di crescita dei cheratinociti (KGM) composto da 66% DMEM, il 22% F12 Ham, 100 unità / ml di penicillina, 100 mg / ml di streptomicina, il 10% FBS, 2,67 mg / ml di adenina, 40 ng / ml idrocortisone, 10 ng tossina colerica ml /, 4,94 mg / ml di insulina, 5 mg / ml transferrina, 1.36 ng / ml triiodo-L-tironina, 10 ng / ml EGF, e 10 ug / ml cloridrato ciprofloxacina. Filtrare il mezzo attraverso un filtro e conservare 0,22 micron a 4 ° C fino al momento dell&#39;uso. </li><li> Prendere il derma dalla 4x pen / strep + soluzione PBS e lavare 2 volte in KGM e incubare in KGM a 37 ° C per due giorni prima dell&#39;uso. </li><li> Tagliare il derma con un bisturi a 1,5 cm x 1,5 cm pezzi di dimensioni e quindi posizionare sul vassoio organotipica. Posizionare il derma con la parte superiore del derma rivolte verso l&#39;alto. La parte superiore del derma sarà il lato che viene a contatto con i cheratinociti (Figura 1B). </li><li> Posizionare il derma pretagliati sul foro quadrato della cassetta organotipiche con il lato superiore del derma rivolti verso l&#39;alto (Figura 3A). </li><li> Capovolgere l&#39;intera cassetta organotipica contenente oltre il derma con pinze (Figura 3B). Scongelare la soluzione matrice extracellulare sul ghiaccio. Usare un ago e la siringa a tirar fuori 100 ml di soluzione di matrice extracellulare per cassetta. Aggiungere 5 gocce al fondo del derma (lato lucido) e agitare leggermente per assicurare una distribuzione derma (Figura 3B). </li><li> Lasciare 5 minuti per la soluzione matrice extracellulare commerciale a solidificare completamente (Figura 3C). Dopo la solidificazione, utilizzare pinze per capovolgere il cassetto sopra. Aggiungere 4 ml di KGM al piatto 6 cm. </li><li> Posizionare 0,5-1 milioni di cheratinociti geneticamente modificati sulle cassette organotipiche contenenti il derma (Figura 3D). <ol><li> Trypsinize cheratinociti mettendo 4 ml di 0,05% tripsina su piastre di 10 cm per 5 minuti e spegnere con 4 ml di mezzi DMEM completo. </li><li> Contare i cheratinociti utilizzando un emocitometro e poi spin down a 200 xg per 5 min. Risospendere 0.5-1 milioni cellule (a seconda del numero di cellule disponibili) in 90 μl di KGM e posizionare tutte le cellule sulle derma. NOTA: È importante posizionare lo stesso numero di celle sul derma nello stesso esperimento per garantire che eventuali differenze rilevate spessore dell&#39;epidermide rigenerata non è dovuta a differenze nel numero iniziale di cellule. Posizionamento di meno di 0,5 milioni di cellule sul derma può portare a poveri stratificazione e la differenziazione. </li></ol></li><li> Modificare i media KGM sulle culture organotipiche ogni altro giorno. Tessuto raccolto 1-14 giorni dopo il posizionamento dei cheratinociti sulle derma. Stratificazione e la differenziazione completa dell&#39;epidermide avviene di solito dopo giorno 5. </li></ol>

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We have nothing to disclose.

La manipolazione genetica in pelle umana colture organotipiche offrono molti vantaggi a comunemente studiato 2D cellule in coltura e modelli di mouse. Culture 2D mancano i tre cellula-cellula e cellula-matrice extracellulare interazioni dimensionali si trovano in tessuti e organi intatti. Recenti studi hanno anche trovato enormi differenze tra le colture di cellule tumorali della pelle 2D e 3D con le cellule in coltura 3D che mostra molto di più somiglianze di espressione genica per la pelle umana primaria tumori ...

L&#39;epidermide umana è un epitelio stratificato che si collega al derma sottostante attraverso una matrice extracellulare noto come l&#39;epidermide zone.The membrana basale non serve solo come una barriera impermeabile per evitare la perdita di umidità, ma anche come una prima linea di difesa per proteggere la corpo dalle sostanze estranee e tossiche 1. Lo strato basale, che è lo strato più profondo dell&#39;epidermide, contiene le cellule staminali epidermiche e progenitrici che danno luogo alla progenie differenziata che formano il resto dell&#39;epidermide 2. Come le cellule progenitrici epidermiche differenziano migrano verso l&#39;alto per formare il primo strato di cellule differenziate note come strato spinoso 3. Nello strato spinoso, cellule attivano l&#39;espressione di cheratine 1 e 10, che quindi fornisce la forza di resistere allo stress fisico per gli strati differenziati dell&#39;epidermide. Come le cellule strato spinoso ulteriormente differenziano, si muovono verso l&#39;alto nell&#39;epidermide a perm strato granulare che è caratterizzata dalla formazione di keratohyalin e lamellari granuli così come proteine ​​strutturali che vengono assemblati sotto la membrana plasmatica. Poiché le cellule procedere nel processo di differenziazione, le proteine ​​sotto la membrana plasmatica sono trasversali collegati tra loro, mentre i granuli vengono estrusi lamellari dalle cellule per formare una barriera lipidica ricco chiamato strato corneo 4. Malattie che coinvolgono alterazioni nella crescita epidermico e impatto differenziazione ~ 20% della popolazione 5. Così, la comprensione dei meccanismi di questo processo è di grande importanza. Dal momento che la manifestazione di molte di queste malattie è subordinato cellula-cellula o cellula-matrice contatto, colture organotipiche dove l&#39;epidermide umana è ricostituito in un ambiente 3D sono stati creati 6-10. Questi metodi tipicamente comportano l&#39;uso di cheratinociti primari o trasformati seminate su matrice extracellulare come devitalizzati umanaderma, Matrigel o collagene. Per comprendere gene meccanismi di regolamentazione che sono importanti nella crescita e differenziazione epidermica, cheratinociti possono essere geneticamente manipolati tramite vettori retrovirali per atterramento o iperespressione di geni nella cultura 2D e poi ricostituito in 3D. Questi metodi sono stati ampiamente utilizzati per caratterizzare i geni coinvolti nella epidermica staminali e cellule progenitrici auto-rinnovamento e la differenziazione, nonché la progressione di neoplasia 11-21. Qui, è previsto un protocollo approfondito su come modificare l&#39;espressione genica in colture organotipiche epidermiche attraverso l&#39;uso di 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

Colture organotipiche permettono la ricostituzione di un ambiente 3D critico per interazioni di contatto cellula-cellula e cellula-matrice che imita la funzione e la fisiologia dei loro omologhi dei tessuti in vivo. Questo è esemplificato da culture pelle organotipiche che riassume fedelmente la differenziazione epidermica e il programma di stratificazione. Cheratinociti epidermici primari umani sono geneticamente manipolabile attraverso retrovirus in cui i geni possono essere facilmente overexpressed o abbattuti. Questi cheratinociti geneticamente modificati possono poi essere utilizzati per rigenerare epidermide umana in colture organotipiche della pelle che forniscono un modello potente per studiare percorsi genetici di crescita epidermico impattante, la differenziazione e la progressione della malattia. I protocolli qui presentati descrivono metodi per preparare derma umano devitalizzati nonché manipolare geneticamente cheratinociti umani primari per generare culture organotipiche pelle. Pelle umana rigenerata può essere utilizzato in downstrapplicazioni EAM come il profilo di espressione genica, immunostaining, e immunoprecipitazioni cromatina seguiti da sequenziamento ad alta produttività. Così, la generazione di queste culture organotipiche pelle geneticamente modificati consentirà la determinazione di geni cruciali per mantenere l&#39;omeostasi della pelle.

Il primo passo nella generazione di pelle umana organotipica è rimuovere l&#39;epidermide dal derma. I due settimana incubazione della pelle a 37 ° C in 4x pen / strep / PBS dovrebbe consentire la separazione del derma dall&#39;epidermide (Figura 1A). Se la separazione dell&#39;epidermide e del derma è difficile posizionare il tessuto a 37 ° C a 4x penna / strep / PBS per un&#39;altra settimana e poi provare di nuovo peeling con pinze. Una delle chiavi per rigenerare il ...

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.

Generazione di culture geneticamente modificate pelle organotipiche Uso devitalizzate derma umano

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

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Research

JoVE Journal

Developmental Biology

11.1K Views.  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.

Colture organotipiche Slice di studi di post-natale neurogenesi

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

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Biologia dello sviluppo

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.

Generazione di cellule staminali adiposo umano attraverso dedifferenziazione di adipociti maturi in Culture soffitto

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, selezione, e Colonia-picking di cellule staminali umane pluripotenti indotte TALEN-mirati con Gene GFP nel 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 organotipica Co-cultura Modello Sostenere midollare timica epiteliale proliferazione, differenziazione e promiscua genica

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.

Generazione di cellule staminali Integrazione-libero umano pluripotenti indotte Utilizzando capelli derivati ​​da cheratinociti

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.

Generazione di integrazione-liberi cellule staminali pluripotenti indotte dalle cellule mononucleari di sangue periferico umano Utilizzando episomiale Vettori

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.

Un test di stress sociale modificato di Trier per adolescenti americani messicani vulnerabili

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.

Generazione di topi modificati geneticamente attraverso la microinjection of oocytes

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.

Generazione e la coltura dei Keratinocytes umani primari da pelle adulta

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.

Colture organotipiche tridimensionale degli organi sensoriali uditivi e vestibolari

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