Bu çalışma, Amerikan Kanser Derneği Araştırma Scholars Grant (RSG-12-148-01-DDC) ve CIRM Temel Biyoloji Ödülü (RB4-05779) supportedby oldu.

Insan derisi protokolü California Üniversitesi, San Diego&#39;nun Araştırma Etik Kurulu kurallarına uygun olarak gerçekleştirildi. İnsan derisi atılır cerrahi örneklerden elde edilen ya da (cilt banka Malzeme / Ekipman Tablo listelenen) deri bankalardan satın alınabilir. Deri veya verici yaşı türetilmiştir Yer deney sürece dermişte bazal membran bölgesi proteinleri (kollajen / laminin) için kritik değildir bozulmuş değildir. Güçsüz insan derisi hazırlanması 1. <ol><li> İnsan cildi aldıktan sonra, (~ 3-5 dakika) Çözülme doku kültürü kaputu doku yerleştirin. Cilt boyutuna bağlı olarak, steril bir tek 50 ml konik bir tüp ya da 125 ml şişe çözülmüş doku yerleştirin. Cilt bankadan cildin tipik boyutu 0.75 feet kare olduğunu. </li><li> 1x PBS içinde 4x penisilin ve streptomisin (pen / strep) ile% 75 tam doldurun. Kuvvetli bir şekilde çalkalanır5 dakika ve sıvı atmayın. Bu adımı 3 kez daha tekrarlayın. </li><li> Son yıkamadan sonra, yeni bir tüp / şişe doku transferi ve kabın% 75 doldurmak için taze 4x pen / strep / PBS ekleyin. 2 hafta epidermis, dermiş ayrılmasını sağlamak için, 37 ° C &#39;de, bir doku kültürü kuluçka makinesi içinde, bu karışım inkübe edin. </li><li> Steril koşullar altında, epidermis, dermis uzak soymak için forseps kullanabilir. Epidermis donör (Şekil 1A) yarış bağlı renklendirme sahip olurken dermis tamamen beyaz. İnsan dokusu ile başa çıkmak için kurumun protokolüne göre epidermis atın. </li><li> Bir tüp veya şişe dermis yerleştirilir ve (4 kez 5 dakika yıkama) karışımı içinde çalkalanarak 1 x PBS içinde seyreltilmiş 4x pen / strep yıkayın. Son yıkamadan sonra, kullanıma hazır olana değin 4 ° C&#39;de 4x pen / 1x PBS içinde seyreltilmiş strep ve mağaza yeni bir tüp / şişeye dermis aktarın. Dermis bir yıla kadar 4 ° C &#39;de muhafaza edilebilir. </li></ol&gt; 2. Genetik değiştirme İlköğretim İnsan Keratinositler Not: primer insan keratinositleri enfekte virüs üretmek için tam DMEM ortamı [DMEM +% 10 fetal bovin serumu (FBS) + pen / strep] yetiştirilen kullanımlar amfotropik Phoenix hücreleri. Bu tür gag-pol ve zarf gibi proteinleri kodlayan viral paketleme genleri sabit şekilde bir retroviral vektör ile transfekte, virüs üretimi sağlar Phoenix hücre genomu içine entegre edilir. Phoenix hücreleri yüksek verim, yüksek viral titre üretimi için izin ile transfekte edilebilir. Bu paketleme hattı üretilen virüs aynı zamanda, insan da dahil olmak üzere memeli hücreleri büyük bir çeşitlilik bulaşabilir. <ol><li> Gün 1: Transfeksiyondan bir önceki gün, tam bir DMEM ortamında oyuk başına 800,000 hücrelik bir yoğunlukta 6-çukurlu plaka içindeki tohum Phoenix hücreleri. </li><li> Gün 2: Transfeksiyon gün 3 retroviral vektörler ug oyuk başına kullanımı. 1.5 ml tüp içine retroviral vektör alikosu 3 ug. 100 mikron bir karışımını kullanınl DMEM artı vektörün her 3 ug için transfeksiyon reaktifi 6 ul. 1.5 ml bir tüp içinde 100 | il DMEM ile transfeksiyon reaktifi 6 ul karıştırın. (Kullanılan retroviral vektörler ekipman / malzeme tabloda listelenmiştir). <ol><li> 5 dakika boyunca inkübe ve retroviral vektörün 3 ug ihtiva eden ön aliquoted tüpe 106 ul karışımı ekleyin. Karıştırın ve 30 dakika boyunca oda sıcaklığında inkübe edilir. Phoenix hücrelerinin her oyuğuna, bu tüm karışım damla damla ekleyin. </li></ol></li><li> 3. Gün: Transfeksiyondan bir gün sonra, anka hücrelerinden ortamını çıkarın ve taze tam DMEM medya 2 ml ekleyin. Aynı gün, oyuk başına 75,000 hücrelik bir yoğunlukta 6-çukurlu tabak içine primer insan keratinositleri tohum. Antibiyotik, bir keratinosit, serum barındırmayan ortam (KCSFM) (pen / strep) &#39;de keratinositler tutun. NOT: Birincil insan keratinositler satın alındı. Hücreler (Malzeme / Ekipman tabloda listelenmiştir) satıcılarının çeşitli satın alınabilir. </li><li> 4. Gün: HarŞimdi viral partikülleri içeren transfekte anka hücrelerinden, medya yelek. Bir şırınga kullanılarak, 0.45 um&#39;lik bir filtreden geçirin ortamı (herhangi bir kirletici Phoenix hücreleri çıkarmak için) ve keratinositler üzerinde 2 mi yer (önceki gün / oyuk 75.000 hücre yoğunluğunda kaplanmıştır: adım 2.3). Enfeksiyon sürecine aracılık yardımcı ortama heksadimetrin bromür (5 ug / ml) eklenir. Virüs titrleri ~ 1 x 10 7 TU / ml. <ol><li> Oda sıcaklığında 1 saat boyunca 200 xg&#39;de santrifüj 6 oyuklu plakalar dönerler. Spin sonra, virüs içeren ortamı çıkartın ve KCSFM eklemeden önce 1x kez PBS ile yıkama hücreleri. </li></ol></li><li> 5. Gün: tekrar adım 2.4 gibi keratinosit aynı toplu Infect. NOT: retroviral vektör üzerinde seçilebilir bir marker olması durumunda, bir ilaç kullanan keratinositleri seçip alt analiz veya organotipik kültürlerinin gibi uygulamalarda kullanım için genişletme. </li></ol> 3. Organotipik Cilt Kültürler Kurma <ol><li> Inşa etmekOrtak laboratuvar bulunan malzeme veya kasetlerden Organotipik kaset özel bir metal imalat dükkanı aracılığıyla yapılabilir (Şekil 2A - F). 3,5 cm doku kültür plakasının bu kasetleri yapmak için, bir 3,5 cm&#39;lik bir tabak (Şekil 2A-B), kapağın merkezi bir 1,0 cm x 1,0 cm kare kaldırmak için bir koterizasyon kullanın. Steril koşullar altında bunu yapın. <ol><li> Net oje (Şekil 2C) kullanarak kaset (3.5 cm çanak kapak) alt kare mandal takın. Oje için 5 dakika kurumasını ve kare mandal (Şekil 2B) dayanan böylece kaseti ters çevirin izin verin. 6 cm çanak içine kaseti yerleştirin. NOT: Kare sanat ve zanaat mağazalar çeşitli satın alınabilir mandal. </li></ol></li><li> Keratinosit büyüme ortamı% 66 DMEM,% 22 Ham F12, 100 birim / ml penisilin / ml streptomisin,% 10 FBS, 2.67 ug / ml adenin 100 ug, 40 n oluşan (KGM) hazırlayınug / ml hidrokortizon, 10 ng / ml, kolera toksini, 4.94 / ml insülin ug, 5 ug / ml transferrin, 1.36 ng / ml triiyodo-L-tironin, 10 ng / ml EGF ve 10 ng / ml, siprofloksasin hidroklorür. Kullanıma hazır olana kadar 4 ° C&#39;de 0.22 mikron filtre ve mağaza üzerinden medyayı Filtre. </li><li> 4x pen / strep + PBS çözeltisi üzerinden dermis alın ve KGM içinde 2 kez yıkayın ve daha sonra kullanımdan önce iki gün boyunca 37 ° C&#39;de inkübe KGM. </li><li> 1,5 cm x 1,5 cm büyüklüğünde parçalar bir neşter kullanılarak dermis kesin ve sonra organotipik kaset üzerine yerleştirin. Yukarı bakacak şekilde dermis üst dermis yerleştirin. Dermis üst keratinositleri (Şekil 1B) ile temas durumuna gelir tarafında olacaktır. </li><li> (Şekil 3A) yukarı bakacak şekilde dermis üst tarafı ile organotipik kaset kare delikten üzerine önceden kesilmiş dermis yerleştirin. </li><li> Forseps kullanarak üzerinde dermis içeren tüm organotipik kaseti (Şekil 3B Çevir). Buz üzerinde hücre dışı matris çözümü çözülme. Kaset başına hücre dışı matriks solüsyonu 100 ul dışarı çekmek için bir iğne ve şırınga kullanın. Dermis (parlak tarafı) altına 5 damla ekleyin ve dermis (Şekil 3B) boyunca eşit dağılımını sağlamak için hafifçe sallayın. </li><li> Ticari ekstra hücresel matris çözümü için 5 dk tamamen (Şekil 3C) sağlamlaştırmak için izin verin. Katılaşma sonrasında geri üzerinde kaseti çevirmek için forseps kullanabilir. 6 cm çanak KGM 4 ml ekleyin. </li><li> Dermis (Şekil 3D) içeren organotipik kasetler üzerine 0,5-1.000.000 genetiği değiştirilmiş keratinositler yerleştirin. <ol><li> 5 dakika boyunca 10 cm plakalar üzerine% 0.05 tripsin 4 ml yerleştirilmesi ve tam bir DMEM ortamı, 4 ml ile söndürün ile Trypsinize keratinositler. </li><li> Hemasitometre kullanarak keratinositleri sayın ve sonra 5 dakika boyunca 200 xg&#39;de aşağı doğru döndürün. Yeniden süspanse 0,5-1000000 hücreleri 90 μ yılında (mevcut hücre sayısına bağlı olarak)ve KGM l dermis üzerine tüm hücreleri koyun. Not: Bu, yeniden oluşturulmuş epidermisin kalınlığı görülen farklılık hücre sayısı başlangıç ​​farklılıklara bağlı olmadığından emin olmak için, aynı deney içinde deri üzerinde aynı sayıda hücre yerleştirilmesi önemlidir. Dermis üzerinde az 0,5 milyon hücre yerleştirilmesi yoksul tabakalaşma ve farklılaşma yol açabilir. </li></ol></li><li> Her geçen gün organotipik kültürleri KGM ortamı değiştirin. Hasat doku dermis üzerinde keratinosit yerleştirildikten sonra 1-14 gün. Epidermis tam tabakalaşma ve farklılaşma genellikle gün 5 sonra ortaya çıkar. </li></ol>

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We have nothing to disclose.

Organotipik kültürler birçok avantaj sunan insan derisinde Genetik manipülasyon yaygın 2B kültür hücreleri yanı sıra fare modelleri okudu etmek. 2B kültürleri sağlam organ ve dokularda bulunan üç boyutlu bir hücre-hücre ve hücre-hücre dışı matris etkileşimlerine yoksundur. Son çalışmalar da 16 tümörler birincil insan derisine çok daha fazla gen ifadesi benzerlikleri gösteren 3D kültür hücreleri ile 2D ve 3D kültür cilt kanseri hücreleri arasındaki muazzam farklar bulduk. ?...

İnsan üst deri değil, aynı zamanda korumak için savunma bir birinci hattı olarak nem kaybını önlemek için sızdırmaz bir bariyer olarak hizmet eder bazal membran zone.The epidermis olarak bilinen hücre dışı bir matris boyunca yatan dermiş bağlanan bir tabakalı epitelyumdur yabancı ve zehirli maddelerden 1 vücut. Epidermisin derin katmanı bazal tabaka, epidermis 2 kalanını oluşturan farklılaşmış döl doğuran epidermal kök ve progenitör hücreler içerir. Epidermal progenitör hücrelerin ayırt olarak bunlar Spinoz tabakanın 3 olarak da bilinir farklılaşmış hücrelerin birinci tabakayı oluşturmak üzere yukarı doğru göç eder. Spinöz katmanda, hücreler daha sonra epidermis farklılaşmış katmanları fiziksel strese dayanma gücünü sağlayan keratinlerden 1 ve 10 ifadesi, açın. Spinöz tabaka hücreleri daha farklılaştırmak, onlar için epidermisin yukarı hareketplazma zarı altına monte edilir keratohyalin ve lameller granül oluşumunun yanı sıra, yapısal proteinlerin karakterize granüler tabaka m. Hücreler farklılaşma sürecinde devam ederken katmanlı granüller zengin koruyucu stratum corneum adı verilen bir lipidi 4 oluşturmak için hücreler ekstrüde edilirken, plazma membranı altından proteinleri birbirine çapraz bağlı bulunmaktadır. Epidermal büyüme ve farklılaşma etkisi ~ nüfusun 5% 20 değişiklikleri içeren hastalıklar. Bu durumda, bu işlemin mekanizmalarının anlaşılması önem taşımaktadır. Bu hastalıkların birçoğu tezahürü hücre-hücre veya hücre-matris temas etmesine bağlıdır, insanın epidermisin 3 boyutlu bir ortamda yeniden oluşturulur Organotipik kültürler 6-10 oluşturuldu. Bu yöntemler, tipik olarak, cansız insan gibi hücre dışı matrisin üzerine ekildi primer ya da dönüştürülmüş keratinositlerin kullanılmasını içerirDermiş, Matrigel veya kollajen. Epidermal büyüme ve farklılaşmasında önemli bir gen düzenleyici mekanizmaların anlaşılması, genetik olarak keratinositler 2B kültür genleri demonte veya aşın ifade etmek üzere retroviral vektörler ile işlenmesi ve sonra 3D içinde yeniden edilebilir. Bu yöntemler, neoplazi 11-21 epidermal kök ve projenitör hücre kendini yenileme ve farklılaşma gibi ilerlemesinde rol oynayan genleri karakterize etmek için yaygın olarak kullanılmaktadır. Burada, retrovirüsler kullanımı yoluyla epidermal organotipik kültürlerinin gen ekspresyonunu değiştirmek için nasıl derinlemesine bir protokolü temin edilmektedir.

<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

Organotipik kültürlerinin fonksiyonu ve in vivo doku meslektaşları fizyolojisini taklit hücre-hücre teması ve hücre-matriks etkileşimlerinin önemli bir 3 boyutlu bir ortamda tekrar oluşturulmasını sağlar. Bu sadakatle epidermal farklılaşma ve tabakalaşma programı özetlediği Organotipik cilt kültürler tarafından örneklenmiştir. Birincil insan epidermal keratinositler genleri kolayca aşırı veya aşağı çalınabilecek retrovirüslere yoluyla genetik olarak manipüle vardır. Bu genetik olarak değiştirilmiş keratinositler daha sonra genetik yollar, darbe epidermal büyüme farklılaşmasını ve hastalığın ilerlemesini araştırmak için güçlü bir model sağlanması Organotipik bir deri kültürlerinde, insan epidermisi yeniden oluşturmak için kullanılabilmektedir. Burada sunulan protokoller cansız insan dermiş hazırlanması hem de genetik olarak Organotipik bir deri kültürü oluşturmak için primer insan keratinositleri işlemek için yöntemleri tarif eder. Rejenere insan deri downstr kullanılabilirBöyle gen ifade profilleme, immün ve yüksek verimlilik sıralaması takip kromatin İmmüno olarak eam uygulamaları. Bu nedenle, bu genetik olarak değiştirilmiş organotipik cilt kültürlerin nesil cilt dengesini sağlamaya yönelik kritik genlerin belirlenmesini sağlayacaktır.

Organotipik insan cildi üreten ilk adım epidermis, dermis kaldırmaktır. 37 ° C 4x pen / strep / PBS derinin iki haftalık kuluçka epidermis (Şekil 1A), dermiş ayrılmasını sağlamak gerekir. Epidermis ve dermis ayıran zorsa o zaman bir hafta 4x kalem / strep 37 ° C / PBS de doku yerleştirin ve sonra tekrar soyulması forseps kullanarak deneyin. Devitalize dermis insan epidermis yenileyici anahtarlarından biri keratinositler dermis doğru tarafına yerleştirili...

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.

Güçsüz İnsan dermis kullanma Genetiği Değiştirilmiş Organotipik Cilt Kültürlerin Üretimi

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 Görüntüleme.  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.

POSTNATAL nöron Çalışmaları için organotipik Dilim Kültürler

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

Gelişim Biyolojisi

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.

Tavan Kültürlerde Olgun adipositlerinde ve dediferansiyonunu ile İnsan adipoz Kök Hücre Üretimi

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.

Transfeksiyon, Seçim ve İnsan uyarılmış pluripotent kök hücrelerin Colony-toplama AAVS1 Safe Harbor içine GFP geninin ile TALEN-hedefli

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.

Medüller Timik epitel hücre proliferasyonu, farklılaşması ve Promiscuous Gen İfade Destekleme 3D Organotipik Co-kültür Modeli

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.

Saç türetilmiş keratinositlerden Kullanarak Entegrasyon serbest İnsan uyarılmış pluripotent kök hücrelerin üretilmesi

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.

Epizomal vektörlerin kullanılması insan periferik kan mononükleer hücrelerinden entegrasyon içermeyen uyarılmış pluripotent kök hücrelerin üretilmesi

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.

Savunmasız Meksikalı Amerikalı Adolesanlar için Trier Değiştirilmiş Sosyal Gerilme Testi

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.

Oositlerin Mikroenjeksiyonu ile Genetiği Değiştirilmiş Farelerin Üretilmesi

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.

Oluşturma ve birincil insan lenfositi yetişkin cilt, kültür

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.

Vestibüler ve işitme duyu organları kültürleri üç boyutlu Organotypic

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