Diese Arbeit war supportedby der American Cancer Society Forschung Gelehrte Grant (RSG-12-148-01-DDC) und dem CIRM Grund Biologie Award (RB4-05779).

Die menschliche Haut-Protokoll wurde in Übereinstimmung mit den Richtlinien der University of California, Forschungsethikkommission von San Diego durchgeführt. Die menschliche Haut kann aus gebrauchten chirurgischen Proben erhalten oder von Hautbanken kauften (Hautbank in der Material / Ausrüstung Tabelle aufgeführt sind) werden. Der Ort, wo die Haut vor oder Alter des Spenders abgeleitet ist nicht kritisch für das Experiment, solange die Basalmembran-Proteinen (Kollagen / Laminin) in der Dermis nicht verschlechtert werden. 1. Herstellung von devitalisierten Menschen Dermis <ol><li> Auf der menschlichen Haut empfangen, legen Sie das Gewebe in der Gewebekultur Haube zu tauen (~ 3-5 min). Abhängig von der Größe der Haut die aufgetauten Gewebe in einem sterilen Wegwerf 50 ml konischen Röhrchen oder 125 ml Flasche. Die typische Größe der Haut von der Haut Bank 0,75 Quadratmetern. </li><li> Füllen Sie den Behälter zu 75% voll mit 4x Penicillin und Streptomycin (Pen / Strep) in 1x PBS. Kräftig schütteln5 Minuten und entsorgen Sie die Flüssigkeit. Wiederholen Sie diesen Schritt 3 weitere Male. </li><li> Nach dem letzten Wasch, übertragen das Gewebe in ein neues Röhrchen / Flasche und fügen frische 4x Pen / Strep / PBS auf 75% des Behälters zu füllen. Inkubieren dieser Mischung in einem Gewebekultur-Inkubator bei 37 ° C für 2 Wochen, um die Trennung der Dermis von der Epidermis zu ermöglichen. </li><li> Unter sterilen Bedingungen verwenden Pinzette von der Dermis abziehen die Epidermis. Die Dermis vollständig weiß ist, während die Epidermis wird Färbung in Abhängigkeit von der Rasse des Spenders (1A) haben. Entsorgen Sie die Haut gemäß dem Protokoll des Instituts für den Umgang mit menschlichem Gewebe. </li><li> Legen Sie die Dermis in einer Tube oder Flasche und waschen Sie ihn in 4x Pen / Strep in 1x PBS unter kräftigem Schütteln verdünnt (5 min wäscht 4 mal). Nach dem letzten Wasch, übertragen Sie die Dermis in ein neues Röhrchen / Flasche in 4x Pen / Strep in 1x PBS verdünnt und bei 4 ° C bis zur Verwendung. Dermis können bei 4 ° C für bis zu einem Jahr gelagert werden. </li></ol&gt; 2. genetische Modifizierung primären humanen Keratinozyten HINWEIS: Verwenden Sie amphotrope phoenix Zellen in vollständigem DMEM media [DMEM + 10% fötalem Rinderserum (FBS) + Pen / Strep] gewachsen, um Viren zu produzieren, um primäre menschliche Keratinozyten zu infizieren. Virale Verpackungs Gene, die Proteine ​​wie gag-pol und Umschlag kodieren, stabil in das Zellgenom Phoenix die für die Virusproduktion ermöglicht, wenn sie mit einem retroviralen Vektor transfiziert integriert. Phoenix-Zellen mit hoher Effizienz unter Berücksichtigung hoher Virustiter Produktions transfiziert werden. Virus von dieser Verpackungslinie produziert kann auch infizieren eine Vielzahl von Säugetierzellen einschließlich menschlicher. <ol><li> Tag 1: Am Tag vor der Transfektion seed Phoenix-Zellen in einer Platte mit 6 Vertiefungen in einer Dichte von 800.000 Zellen pro Vertiefung in komplettem DMEM Medium. </li><li> Tag 2: Tag der Transfektion, verwenden Sie 3 ug von retroviralen Vektoren pro Vertiefung. Aliquot 3 ug retroviralen Vektor in einem 1,5-ml-Röhrchen. Verwenden Sie eine Mischung von 100 μ; l DMEM plus 6 ul Transfektionsreagenz für jeweils 3 ug Vektor. MIX 6 ul Transfektionsreagenz mit 100 ul DMEM in einem 1,5-ml-Röhrchen. (Die retroviralen Vektoren, die verwendet werden, sind in der Geräte / Materialien Tabelle aufgeführt). <ol><li> Inkubation für 5 min und fügen Sie die 106 &amp; mgr; l-Gemisch in die Pre-aliquotierten Röhrchen mit dem 3 ug retroviralen Vektor. Mischen und Inkubation bei RT für 30 min. Fügen Sie diese gesamte Mischung tropfenweise zu jeder Vertiefung der Phoenix-Zellen. </li></ol></li><li> 3. Tag: Der Tag nach der Transfektion, entfernen Sie die Medien aus den Phoenix-Zellen und 2 ml frisches vollständiges DMEM-Medium. Am selben Tag, Samen primärer menschlicher Keratinozyten in 6-Well-Platten in einer Dichte von 75.000 Zellen pro Vertiefung. Pflegen Sie die Keratinozyten in einer Keratinozyten-serumfreiem Medium (KCSFM) mit Antibiotika (Pen / Strep). HINWEIS: Primäre menschliche Keratinozyten wurden gekauft. Zellen können aus einer Vielzahl von Anbietern (in der Material / Ausrüstung Tabelle aufgeführt) erworben werden. </li><li> Tag 4: HarWeste das Medium aus den transfizierten phoenix-Zellen, die jetzt enthält Viruspartikel. Führen Sie das Material durch ein 0,45 um-Filter mit einer Spritze (um alle verunreinigenden phoenix Zellen zu entfernen) und legen Sie 2 ml auf die Keratinozyten (bei 75.000 Zellen / gut am Vortag: siehe Schritt 2.3). In Hexadimethrinbromid (5 ug / ml) zu den Medien zu helfen, vermitteln die Infektionsprozess. Virustiter sind ~ 1 x 10 &amp; sup7; TU / ml. <ol><li> Drehen die Platten mit 6 Vertiefungen in einer Zentrifuge bei 200 × g für 1 h bei RT. Nach dem Spin, entfernen Sie die Medien-Virus enthält, und waschen Sie die Zellen einmal mit 1x PBS vor der Zugabe KCSFM. </li></ol></li><li> Tag 5: Infizieren derselben Charge von Keratinozyten wieder wie in Schritt 2.4. HINWEIS: Wählen Sie die Keratinozyten mit einem Medikament, wenn es einen selektierbaren Marker auf dem retroviralen Vektor und erweitern für den Einsatz in nachgelagerten Analyse oder Anwendungen wie organotypischen Kulturen. </li></ol> 3. Einrichten Organotypische Hautkulturen <ol><li> Bauen Sie dieorganotypischen Kassette aus gängigen Materialien im Labor festgestellt oder die Kassetten können benutzerdefinierte durch einen Metall Stanzerei gemacht werden (2A - F). Um diese Kassetten von einem 3,5 cm-Gewebekulturplatte zu machen, verwenden Sie ein Brenneisen, ein 1,0 cm x 1,0 cm im Quadrat aus der Mitte des Deckels aus einem 3,5 cm-Schale (2A-B) zu entfernen. Tun Sie dies unter sterilen Bedingungen. <ol><li> Bringen quadratischen Zapfen an der Unterseite der Kassette (Deckel 3,5 cm Schale) mit klarem Nagellack (2C). Lassen Sie 5 Minuten für den Nagellack zu trocknen und drehen Sie die Kassette auf, so dass es auf den quadratischen Zapfen (2D) ruht. Setzen Sie die Kassette in einen 6 cm Schale. HINWEIS: Square Pegs können aus einer Vielzahl von Kunsthandwerk-Läden gekauft werden. </li></ol></li><li> Vorbereitung Keratinozyten-Wachstumsmedium (KGM) von 66% DMEM, 22% Ham-F12, 100 Einheiten / ml Penicillin, 100 ug / ml Streptomycin, 10% FBS, 2,67 ug / ml Adenin, 40 n bestehtg / ml Hydrocortison, 10 ng / ml Cholera-Toxin, 4,94 &amp; mgr; g / ml Insulin, 5 ug / ml Transferrin, 1,36 ng / ml Triiod-L-thyronin, 10 ng / ml EGF und 10 ug / ml Ciprofloxacin-Hydrochlorid. Filtern Sie die Medien durch ein 0,22 um-Filter und bei 4 ° C bis zur Verwendung. </li><li> Nehmen die Dermis aus dem 4x Pen / Strep + PBS-Lösung und wäscht 2 mal in KGM und dann in KGM bei 37 ° C für zwei Tage vor der Verwendung. </li><li> Schneiden Sie die Lederhaut mit einem Skalpell bis 1,5 cm x 1,5 cm große Stücke schneiden und dann auf den organotypischen Kassette zu platzieren. Platzieren der Dermis mit der Oberseite der Dermis nach oben zeigt. Der obere Teil der Dermis wird die Seite, die in Kontakt mit den Keratinozyten (1B) kommt. </li><li> Legen Sie die vorgeschnittenen Lederhaut auf die quadratische Öffnung des organotypischen Kassette mit der Oberseite der Dermis nach oben (3A). </li><li> Klappen Sie den gesamten organotypischen Kassette, die die Lederhaut mehr als mit einer Pinzette (3B). Tauen die extrazelluläre Matrix-Lösung auf Eis. Verwenden Sie eine Nadel und Spritze zu ziehen 100 ul der extrazellulären Matrix-Lösung pro Kassette. 5 Tropfen auf den Boden der Dermis (glänzende Seite) und schütteln Sie leicht, um eine gleichmäßige Verteilung in der gesamten Dermis (3B) zu gewährleisten. </li><li> Lassen Sie 5 Minuten für die kommerzielle extrazellulären Matrix-Lösung vollständig zu verfestigen (3C). Nach dem Erstarren verwenden Pinzette, um die Kassette wieder umdrehen. 4 ml KGM an die 6 cm Schale. </li><li> Platzieren ,5-1.000.000 gentechnisch veränderten Keratinozyten auf die organotypische Kassetten, die die Lederhaut (Figur 3D). <ol><li> Trypsinize Keratinozyten, indem 4 ml von 0,05% Trypsin auf 10 cm-Platten für 5 min und schrecke mit 4 ml vollständigem DMEM-Medium. </li><li> Zählen Sie die Keratinozyten mit einer Zählkammer und dann drehen bei 200 xg für 5 min nach unten. Resuspendieren 0.5-1 Millionen Zellen (in Abhängigkeit von der Anzahl der verfügbaren Zellen) in 90 μl KGM und legen Sie alle Zellen auf der Dermis. Hinweis: Es ist wichtig, dieselbe Anzahl von Zellen auf die Lederhaut in dem gleichen Experiment zu platzieren, um sicherzustellen, daß die Unterschiede in der Dicke der regenerierten Epidermis gesehen nicht aufgrund von Unterschieden im Ausgangszellzahl. Platzieren von weniger als 0,5 Millionen Zellen auf Dermis kann zu schlechter Schichtung und Differenzierung führen. </li></ol></li><li> Ändern KGM Medien auf die organotypische Kulturen jeden zweiten Tag. Erntegewebe 1-14 Tage nach der Platzierung der Keratinozyten auf der Dermis. Voll Schichtung und Differenzierung der Epidermis tritt in der Regel nach Tag 5. </li></ol>

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Genmanipulation in der menschlichen Haut organotypischen Kulturen bieten viele Vorteile häufig gelernten 2D kultivierten Zellen als auch Mausmodellen. 2D Kulturen fehlen die drei Zell-Zell und Zell-extrazellulären Matrix-Interaktionen dimensional in intakten Geweben und Organen gefunden. Jüngste Studien haben auch festgestellt, enorme Unterschiede zwischen 2D- und 3D-kultivierten Hautkrebszellen mit den 3D-kultivierten Zellen, die viel mehr Genexpression Ähnlichkeiten mit primären humanen Tumoren der Haut 16.<...

Die menschliche Epidermis ist eine geschichtete Epithel, die zu der darunter liegenden Dermis durch eine extrazelluläre Matrix, wie die Basalmembran Zone Epidermis nicht nur als Barriere, um den Verlust von Feuchtigkeit zu verhindern, sondern auch als erste Verteidigungslinie, um die zu schützen dient bekannt verbindet Körper vor fremden und toxische Stoffe 1. Die basale Schicht, die der tiefsten Schicht der Epidermis ist, enthält die epidermalen Stammzellen und Vorläuferzellen, die zu dem differenzierten Nachkommen, die den Rest der Oberhaut 2 zu bilden ergeben. Der epidermale Stammzellen differenzieren sie sich nach oben zu wandern, um die erste Schicht von differenzierten Zellen wie spinosus Schicht 3 bekannt ist. Im Stratum spinosum, drehen Zellen auf die Expression der Keratine 1 und 10, die dann liefern die Kraft, körperliche Belastung für den differenzierten Schichten der Epidermis zu widerstehen. Da die Dornfortsätze Schicht-Zellen weiter zu differenzieren, nach oben in der Epidermis, um sie zu bewegenm die Körnerschicht, die durch die Bildung von Keratohyalin und lamellarem Granulate sowie Strukturproteine, die unterhalb der Plasmamembran montiert sind charakterisiert ist. Da die Zellen in dem Differenzierungsprozess fortzufahren, die Proteine ​​unter der Plasmamembran sind Quer miteinander verbunden, während die plättchenförmigen Körnchen werden aus den Zellen extrudiert, um eine lipidreiche Barriere genannt Stratum corneum 4 bilden. Erkrankungen, die Störungen der epidermalen Wachstums und der Differenzierung Schlag ~ 20% der Bevölkerung 5 einzubeziehen. Somit Verständnis der Mechanismen bei diesem Verfahren ist von großer Bedeutung. Da Manifestation viele dieser Krankheiten abhängig Zell-Zell- oder Zell-Matrix-Kontakt haben organotypischen Kulturen, in denen die menschliche Epidermis in einer 3D Umgebung rekonstituiert 6-10 erstellt. Diese Verfahren beinhalten typischerweise die Verwendung von primären oder transformierte Keratinozyten auf der extrazellulären Matrix ausgesät wie devitalisierten menschlichenDermis, Matrigel oder Kollagen. Gen-Regulationsmechanismen, die in den epidermalen Wachstums und der Differenzierung wichtig zu verstehen, können Keratinozyten genetisch durch retrovirale Vektoren manipuliert den Zuschlag oder überexprimieren Gene in 2D-Kultur und dann in 3D rekonstituiert. Diese Methoden sind sehr häufig benutzt worden, um Gene in epidermalen Stammzellen und Progenitorzellen Selbsterneuerung und Differenzierung sowie Progression Neoplasie 11-21 beteiligt charakterisieren. Hier eine eingehende Protokoll, wie die Genexpression in der Epidermis organotypischen Kulturen durch den Einsatz von Retroviren zu ändern ist.

<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

Organotypischen Kulturen erlauben die Rekonstruktion eines 3D-Umgebung kritisch für Zell-Zell-Kontakt und Zell-Matrix-Wechselwirkungen, die die Funktion und Physiologie ihrer in vivo Gewebegegen nachahmt. Dies wird durch organotypische Hautkulturen, die treu rekapituliert die epidermale Differenzierung und Stratifizierung Programm veranschaulicht. Primären humanen epidermalen Keratinozyten sind genetisch manipulierbar durch Retroviren, wo Gene lassen sich überexprimiert oder abgerissen werden. Diese genetisch veränderte Keratinozyten kann dann verwendet werden, um die menschliche Epidermis in organotypischen Hautkulturen Bereitstellung einer leistungsfähiges Modell zu genetischen Signalwege zu untersuchen aufprall epidermalen Wachstums, Differenzierung und Fortschreiten der Krankheit zu regenerieren. Die hier vorgestellten Protokolle beschreiben Verfahren zur devitalized menschlichen Dermis vorzubereiten sowie genetisch zu manipulieren primären humanen Keratinozyten, um organotypischen Hautkulturen zu erzeugen. Regeneriert die menschliche Haut kann in downstr verwendet werdeneam Anwendungen wie Genexpressionsanalysen, Immunfärbung und Chromatin-Immunpräzipitation, gefolgt von Hochdurchsatz-Sequenzierung. So wird Generation dieser gentechnisch veränderten organotypischen Hautkulturen der Bestimmung von Genen, die für die Aufrechterhaltung der Homöostase der Haut sind zu ermöglichen.

Der erste Schritt bei der Erzeugung von organotypischen menschliche Haut, um die Epidermis von der Dermis zu entfernen. Der zweiwöchigen Inkubation der Haut bei 37 ° C in 4 x Pen / Strep / PBS sollte die Trennung der Dermis von der Epidermis (1A) zu erlauben. Wenn die Trennung der Epidermis und Dermis ist schwierig, dann das Gewebe bei 37 ° C in 4x Pen / Strep / PBS für eine Woche und dann noch einmal versuchen Peeling mit einer Pinzette. Einer der Schlüssel zum Regener...

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.

Herstellung von genetisch veränderten Organotypische Hautkulturen Mit devitalisierten Menschen Dermis

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 Aufrufe.  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. 

Serie: 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.

Organotypischen Kulturen für Studien der postnatale Neurogenese

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

Forschung

Entwicklungsbiologie

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.

Generation of Human Fettgewebestammzellen durch Dedifferenzierung der Reife Adipozyten in Decken Kulturen

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.

Transfektion, Selektion und Colony-Picking für Menschen induzierten pluripotenten Stammzellen mit einem GFP-Gen in die AAVS1 Safe Harbor TALEN ziel

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 Organotypische Cokulturmodell Unterstützung Mark Thymusepithel Zellproliferation, Differenzierung und Genexpression Promiscuous

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.

Generation der Integration freie Menschen induzierten pluripotenten Stammzellen Mit Haarstamm Keratinozyten

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.

Die Erzeugung von Integration freien induzierte pluripotente Stammzellen aus menschlichen peripheren einkernigen Blutzellen Mit Episomale Vektoren

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.

Ein Modified Trier Social Stress Test für anfällige mexikanische amerikanische Jugendliche

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.

Erzeugung von genetisch modifizierten Mäusen durch die Mikroinjektion von Oozyten

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.

Generation und Kultivierung der primären menschlichen Keratinozyten von Erwachsenen

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.

Dreidimensionale organotypischen Kulturen der vestibulären und akustische Sinnesorgane

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