This work has been supported by the German Cancer Research Center (DKFZ), the EU-consortium &#8220;Tolerage&#8221;, the Deutsche Forschungsgemeinschaft (SFB 938) and the Landesstiftung Baden-W&#252;rttemberg.

Bu çalışma Regierungspräsidium Karlsruhe etik komitesi tarafından onaylanmıştır. Bütün hayvanlar, Almanya Kanser Araştırma Merkezi (DKFZ), spesifik patojenden serbest durumlar altında muhafaza edildi. Yaş 1 ila 7 gün arasında değişen tüm kültür deneyleri fare yavrular için kullanılmıştır. Timustan mTECs 1. izolasyonu Not: aşağıdaki gibi bazı modifikasyonlar ile, steril koşullar altında, daha önce tarif edildiği gibi 1, aşağıdaki sindirim adımları uygulandı. <ol><li> Fare yavrular başını kesmek ve timus çıkarın. RPMI 1640 ortamı (% 5 FCS ihtiva eden) ihtiva eden bir petri plakası buz üzerinde Thymi yerleştirin. </li><li> ~ 5-30 ml RPMI ortamı ile bir yuvarlak tabanlı tüp içinde ince, küçük parçalar ve yerine Thymi kesin ve oda sıcaklığında 10 dakika süre ile bir mıknatıs kullanılarak karıştırılmıştır hafifçe. </li><li> Bundan sonra, özellikle timositleri içeren süpernatant süzün ve collagena bir yuvarlak kalan doku sırayla sindirmek37 ° C&#39;de 25 dakika her biri için kolajenaz / dispaz (0.2 mg / ml ile 1.2 U / ml nihai konsantrasyon), ardından 37 ° C&#39;de her biri için (0.2 mg / ml ve son konsantrasyonu, dağılışı 57 U / ml), 15 dakika boyunca se tip IV, Thymi kadar manyetik karıştırma ile bir su banyosu içinde Cı tamamen sindirilir. Üç Thymi ikiye 1 ml enzim kullanın. </li><li> Dokuya Pasteur pipeti ile bir kez her 7-10 dakikada bir çalkalayın. Kollajenaz / dispase fraksiyonları havuzu ve 70 mikron gazlı süzülür. </li><li> Manyetik hücreye sıralama (MACS) tarafından mTECs zenginleştirin. Daha önce, 11 tarif edilen ve Şekil 1 &#39;de gösterildiği gibi, ayırma, manyetik hücre tarafından mTECs arıtılmasını gerçekleştirin. NOT: anti-CD80-PE (16-10A1, 1 at kullanımı: 100 seyreltme) ve (1 at G8.8, kullanımı: 100 seyreltme), anti-EpCAM-bio 12 mTECs manyetik sıralama için aşağıdaki antikorları kullanılmıştır. MACS kullanarak Olgunlaşmamış ve olgun mTECs olarak tanımlandı: CD45 - EpCAM + CD80 - ve CD45 - CD80 + respectively. </li><li> Aşağıdaki (Bölüm 2.3) açıklandığı gibi MACS saflaştırma (olgunlaşmamış mTECs saflığı = 83.1 ± 6.3% ve olgun mTECs = 79,23 ± 3.42%) ardından, organotipik kültürlerinin üzerine mTECs tohum. </li><li> Alternatif olarak, CD45 MACS tükenmesi sonrasında (100 mikron memesi kullanarak) FACS sıralama mTECs aşağıdaki antikorlar kullanılarak: Anti-CD45-PerCP (30-F11, 1 at kullanımı: 100 seyreltme), anti-Ly51-FITC (6C3, 1 de kullanmak : 100 seyreltme), anti-EpCAM-Alexa647 (G8.8, 1 ile kullanımı: 500 oranında seyreltilmiş) ile 1 anti-CD80-PE (16-10A1, kullanımı: 100 seyreltme). (Gün 4): propidium iyodür (5,000 1) kullanılarak ölü hücreleri hariç. Sırasıyla EpCAM + CD80 + - CD45 - - Ly51 - EpCAM + CD80 - ve PI - CD45 - Ly51 PI: FACS kullanarak Olgunlaşmamış ve olgun mTECs olarak tanımlandı. </li></ol> 2. 3D Organotipik Co-kültürler (OTC) NOT: keratinocyt ve organotipik ekimi için 3D-dermal yapılarDaha önce tarif edildiği gibi 9,13 es hazırlandı. Tüm adımlar da hücreler, 2, 37 ° C de inkübe edildi ve% 5 bulundu. Şöyle mTECs kullanılarak OTC küçük değişiklikler ile hazırlandı. <ol><li> Insanlara özgü fibroblastlar hazırlanması Not: daha önce tarif edildiği gibi, 9, insan dermal fibroblastları de-epidermised dermişin eksplant kültürleri elde edildi. <ol><li> Kısaca, insan cildi (~ 5 cm uzunluğunda) şeritler kesildi ve termolisin, 4 ° C&#39;de, O / N (10 mM Hepes, pH 7.4 ile birlikte tuzlu su içinde 0.5 mg / ml) ile tedavi. </li><li> Bundan sonra, forseps kullanarak dermis epidermis ayırın. </li><li> İnce 10 cm&#39;lik petri plakasında küçük parçalar yerine dermis kesilir ve steril bir hava akımı altında 1-2 saat boyunca kurumaya bırakılır. DMEM% 20 FBS içeren düzenli eksplantlar tamamlayın. </li><li> Out büyüyen fibroblastlar bölmek zaman konfluent eksplantlar ileri con plakaya uymak devam emin% 0.1 tripsin kullanarak (genellikle yaklaşık 3 hafta)akıbet ardışık mermi. </li><li> Kadar DMEM 3 kat% 10 FBS aynı eksplantlardan fibroblastlar genişletin ve onları 14 cryo-korumaktadır. Her bir deney serisi için fibroblastlar aynı toplu kullanın. NOT: fibroblastlar dermal eşdeğeri set-up için ışımaya değildi. </li></ol></li><li> İskele hazırlanması <ol><li> Tam 12 iyi filtre ekler uyması için keskin bir 11 mm çaplı metal zımba kullanarak iyi sınırlı, çevrelerin içine 0.4-0.6 mm kalınlığında viskon, dokunmamış lifli malzeme (excel sayfasını destekleyen ürün detay) kesin. Sonra bir iskele olarak 12- kuyu filtre elemanı (polyester kılcal gözenek membran 3 mikron gözenek boyutu) içine yerleştirin. Steril 12 oyuklu plaka içine komple filtre setup yerleştirin. </li><li> Fibrinojen ve trombin bir kombinasyonundan oluşan cerrahi bir fibrin yapışkan, fibrin jel kiti kullanılarak hazırlanır. Fibrinogen- olarak 8 mg / ml ve 10 kit trombin bileşeni önceden seyreltinsırasıyla birim / ml. Aşağıdaki gibi, bir 12-kuyucuklu plaka tek bir oyuk için devam edin. <ol><li> Ca2 + ve Mg + 2, pH 7.0, 100 ul olmayan fibrinojen 100 ul fosfat tamponlu tuz (PBS) ile (8 mg / ml) ile seyreltilir. 270.000 fibroblastlar içeren 100 ul FCS ile 100 ul trombin (10 birim / ml) ile seyreltilir. </li><li> 2 mg / ml ve 2.5 ünite / ml trombin fibrinojen bir nihai konsantrasyonu ile sonuçlanan, (1 karışımı 1) 200 ul fibrinojen ekleme fibroblastlar iskele üzerine hücre-karışımı ihtiva eden trombin 200 ul için koyun. İyice karıştırın ve yumuşak pipetleme (Gün 1) ile iskele bütün alana eşit dağıtın. NOT: 37 ° C&#39;de 30 dakika sonra fibroblastlar kuşatan bir pıhtı, tamamen iskele iç boşlukları dolduran ve düzgün bir üst yüzey oluşturan, meydana gelmiş olacaktır. </li></ol></li></ol></li><li> MTECs ile ortak kültür <ol><li> Ön kültür için organ kültürleri daldırılmasıBir değiştirilen ortam içinde% 10 FBS, 50 ug / ml L-askorbik asit ve 1 ng / ml TGF-β1 ile DMEM, 4-5 gün süre ile her gün. </li><li> Mtec tohumlama gününde, rFAD medyumla yerine (1: 1 DMEM + DMEM / F12),% 10 FBS ile, 10 -10 M kolera toksini, 0.4 mg / ml hidrokortizon, 50 ug / ml L-askorbik asit, RANK bağı nedenle fibroblastlar tarafından salgılanan serin proteaz ile erken fibrinolizi önlenmesi (0.1 ug / ml) ve 500 birim / ml Aprotinin,. </li><li> 7-8 saat sonra, 250,000 mTECs tohumlayarak ko-kültürler-ayarlamak (tam mTECs, ya CD80 LO ve CD80 yüksek alt-gruplar), fibroblast iskele üst oyuk başına 100 ul&#39;lik bir hacim içinde. Bir Neubauer odası (gün 4) kullanarak hücreleri sayın. NOT: timüs 3D organotipik kültürler hava kaldırdı olan cilt OTC aksine medyada submersed tüm zamanlarda. </li><li> 24 saat inkübasyondan sonra, şimdi containi (adım 2.3.2) yukarıda belirtildiği gibi orta kültürleri (toplam orta değişimi) kaynağıng Aprotininin miktarlarda 250 ünite / ml (Gün 5) azalır. </li><li> MTECs proliferatif aktivitesini değerlendirmek için, kültürler tahliye edilmeden önce 4 saat OTC (yani 6.7 uM / ml, / de 10 uM) edu ekleyin. Her kültürün numunesinin veya akışla iki bölüm halinde ya sayma K14 + Edu + hücreler tarafından, mTECs çoğalma endeksleri belirleyin keratin 14. co-boyanması ile birlikte Edu Görüntüleme Kit açıklandığı gibi OTC cryo bölümlerin boyama yapın sitometri (EDU akış sitometri, Bölüm 1.7 yerine Ly51-FITC bir 1&#39;deki CDR1-PB 15 kullanın: 100 seyreltme ve 2.3.6.4). </li><li> Ko-kültür 4-7 gün sonra, RNA izolasyonu, cryo-kesit veya FACS analizi (Gün 8-11) için OTC&#39;leri ve işlemi sonlandırmak. <ol><li> Bir forseps kullanarak kuyu ucun filtreden iskele / dermal eşdeğer ayıran kültürleri sonlandırın. </li><li> Cryo-kesit için, Ekim bileşik bütün OTC embed ve li dondurmacryo-kesit önce sterlin azot buharı. Kullanılana kadar -20 ° C &#39;de, bir kriyostat ve mağaza kullanılarak 5-7 mikron kalınlığında OTC bölümleri hazırlayın. Antikorlar: (100 seyreltme oranı: 1 de GP53, kullanım), ve anti-vimentin: cryo-kesitsel OTC immüno-histokimya için, anti-keratin 14 (1000 seyreltme AF64, kullanım 1 de) kullanın. İlgili ikincil antikorlar kullanılarak dolaylı immüno histokimya boyama gerçekleştirin. </li><li> RNA izolasyonu için çözelti, 2 ml RNazsız tüpün vidalı kapak olarak (fenol ve guanidinyum tiyosiyanat ihtiva eden) Denatüre edici 1 ml. Bir neşter ile parçalar halinde tüm OTC kesin ve denatüre çözeltisi içeren tüp içine ekleyin. Mekanik bir 6.0 hızda 30 saniye boyunca iki kez FastPrep aleti ile OTC&#39;leri parçalamak, 2 dakika boyunca (örnek, bu noktadan sonra -80 ° C&#39;de muhafaza edilebilir) boyunca buz üzerinde arasında yerleştirin. -80 ° C&#39;de donduruldu durumunda, buz üzerinde eritin. 4 ° C&#39;de 10 dakika süreyle 11,500-13,000 rpm tüpler santrifüjleyin. Bir tüpe süpernatantı aktarın RT ve f de 5 dakika boyunca inkübeüretici tarafından tarif edildiği gibi asit guanidinyum tiyosiyanat-fenol-kloroform ekstraksiyonu kullanılarak ollow RNA izolasyonu protokolleri. </li><li> FACS analizi için, iskele kaldırmak ve fibrin / fibroblast / mtec jelden membran ayırın. İnce bir neşter ile jel kesilmiş ve ~ bir FACS tüpü içinde 20 dakika o sindirimi ya da tamamen manyetik karıştırma ile bir su banyosu içinde 37 ° C &#39;de kolajenaz / dispaz, 2 ml ile sindirilmiş kadar. Bir kez, bir Pasteur pipeti her 5 dakikada enzim çözeltisi çalkalayın. Tam bir sindirimden sonra, 70 um filtre üzerinden bir hücre süspansiyonu filtre; 1,7 anlatılan ve akış sitometrisi ile analiz edildiği üzere antikorlar kullanılarak tek bir hücre süspansiyonu leke. </li></ol></li></ol></li></ol>

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Yazarlar herhangi bir çıkar mali veya ticari çatışması beyan ederim.

RTOCs yanı sıra, 3D OTC TEC farklılaşma ve PGE bakım / indüksiyon diğer (i) ile karşılaştırıldığında (Tablo 1) &#39;basitleştirilmiş 3D kültürlerin&#39; kullanımı açısından çok daha üstün tarafından olmuştur - fibroblastlar yalnız iskele olmaksızın; (Ii) 2 D sistemleri kullanan -, (iii) 3T3-J2 hücreleri TEC klonlar geliştirmek olup, burada, TECS 10 ile birlikte kültürlenmiş fibroblast / besleyici hücreleri ama PGE kaybolur (iv) Matrigel veya (v) ECM bile?...

Geri kalan% 2 toplu timik stroma oluşturan çeşitli hücreler oluşur ise geliştirilmesi timositleri, timüs yaklaşık% 98 kadar olmak (yani, epitel hücreleri, dendritik hücreler, makrofajlar, B hücreleri, fibroblastlar, endotel hücreleri). Dış kortikal epitel hücreleri (cTECs) multipotent öncesi T hücreleri ve öz-MHC olumlu seçiminde kemik iliği, T hücre soyu indüksiyon yanlısı T hücrelerinin göçü temin olgunlaşmamış timositleri kısıtlı. İç medüller timik epitelyum hücreleri (mTECs) indükleyen negatif seçim ya da regülatör T hücre soyu içine sapma ya göre kendinden peptit / MHC kompleksleri için bir yüksek afiniteli TCR olan timositlerin tolerans indüksiyonu katılmaktadırlar. Merkezi tolerans indüksiyonu bağlamında, mTECs Böylece periferik öz yansıtma dokuya sınırlı bir kendi antijenlerine (TRA) çok geniş bir yelpaze ifade etmeleri açısından benzersizdir. Bu olay karışık gen ifadesi olarak adlandırılır (PGE)1,2. Çeşitli kısa vadeli 2B kültür sistemleri değişmez ilk 2 gün içinde 3-6 MHC sınıf II, FoxN1 ve Aire gibi PGE kaybı ve anahtar düzenleyici moleküllerin sonuçlandı olarak bu büyüleyici hücre tipine En güncel çalışmalar, ex vivo izole hücrelerde güveniyor . Özellikle takımlar ve timus bozulmamış 3D ağda özellikleri 2B modellerinde eksik olan, ancak belirsiz kaldı. Yeniden birikme timik organ kültürü (RTOC) sağlam bir timik mikro-7, şu ana kadar, diğer yandan, sadece 3D, bir yandan, T hücresi gelişiminin çalışma sağlar sistemi ve stromal hücre biyolojisi, olmuştur. Bununla birlikte, RTOCs zaten fetal stromal hücrelerin bilgi girişini içerir ve 5-10 günlük bir maksimum kültür dönemi tahammül bir hücre kompleks bir karışımı içeren, örneğin, bazı sınırlamaları vardır. in vitro kültür sistemlerinde indirgemeci eksikliği çalışma engellemiştirT hücre gelişimi ve timus organogenez çeşitli yönleri az değil moleküler PGE düzenlenmesi ve mTECs gelişimsel biyoloji ile ilişkisi. Deri ve timus epitel hücrelerinin yapısal kuruluşun yakın ilişkililik sayesinde, in vitro keratinositlerin farklılaşması taklit ve böylece dermal eşdeğer oluşturmak için başlangıçta geliştirilmiş olan 3D organotipik kültürü (OTC) sistemi için seçti. OTC sistem keratinositler 8,9 numaralı seribaşı olan üzerine fibrin jel, içinde sıkışıp kalırlar dermal fibroblastlar ile kaplanmış inert iskele matriks oluşur. Burada, biz saflaştırılmış mTECs ile keratinositler yerini aldı. Bu modelin temel özellikleri korurken, biz belirli parametreleri optimize edilmiş. MTECs çoğaldı kabul OTC modelinde, böylece yakından in vivo mTECs geliştirme taklit terminal farklılaşma yapıldı ve mtec kimlik ve PGE muhafaza <sup&gt; 10. Bu teknik not timus OTC aşamalı set-up sağlayan ayrıntılı bir protokol sağlar.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Pregnant C57BL/6 mice&#160;</td>
			<td>Charles River WIGA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>LS columns</td>
			<td>Miltenyi Biotec</td>
			<td>130-042-401</td>
			<td></td>
		</tr>
		<tr>
			<td>MS columns</td>
			<td>Miltenyi Biotec</td>
			<td>130-042-201</td>
			<td></td>
		</tr>
		<tr>
			<td>CD45 Microbeads, mouse</td>
			<td>Miltenyi Biotec</td>
			<td>130-052-301</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-PE Microbeads</td>
			<td>Miltenyi Biotec</td>
			<td>130-048-801</td>
			<td></td>
		</tr>
		<tr>
			<td>Streptavidin Microbeads</td>
			<td>Miltenyi Biotec</td>
			<td>130-048-101</td>
			<td></td>
		</tr>
		<tr>
			<td>EpCAM (G8.8 -Alexa 647 and -biotin)</td>
			<td>Ref. 12</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>CD80-PE antibody</td>
			<td>BD Pharmingen</td>
			<td>553769</td>
			<td></td>
		</tr>
		<tr>
			<td>CD45-PerCP antibody</td>
			<td>BD Pharmingen</td>
			<td>557235</td>
			<td></td>
		</tr>
		<tr>
			<td>Ly51-FITC antibody</td>
			<td>BD Pharmingen</td>
			<td>553160</td>
			<td></td>
		</tr>
		<tr>
			<td>CDR1-Pacific Blue</td>
			<td>Ref. 15</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Keratin 14 antibody</td>
			<td>Covance</td>
			<td>PRB-155P</td>
			<td></td>
		</tr>
		<tr>
			<td>Vimentin antibody</td>
			<td>Progen</td>
			<td>GP58</td>
			<td></td>
		</tr>
		<tr>
			<td>Cy3-conjugated AffiniPure Goat anti-Rabbit IgG (H+L)</td>
			<td>Jackson ImmunoResearch&#160;</td>
			<td>111-165-003</td>
			<td></td>
		</tr>
		<tr>
			<td>Alexa 488-conjugated AffiniPure F(ab&#39;)2 Fragment Goat anti-Guinea Pig IgG (H+L)</td>
			<td>Jackson ImmunoResearch&#160;</td>
			<td>106-546-003</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat anti-Rabbit IgG (H+L) Secondary Antibody, Alexa Fluor 488 conjugate</td>
			<td>Molecular Probes (Invitrogen GmbH)</td>
			<td>A-11008</td>
			<td></td>
		</tr>
		<tr>
			<td><a href="https://www.lifetechnologies.com/order/catalog/product/C10339">Click-iT EdU Alexa Fluor 594 Imaging Kit</a></td>
			<td>Invitrogen</td>
			<td><a href="https://www.lifetechnologies.com/order/catalog/product/C10339">C10339</a></td>
			<td></td>
		</tr>
		<tr>
			<td><a href="https://www.lifetechnologies.com/order/catalog/product/C10425">Click-iT EdU Alexa Fluor 488 Flow Cytometry Assay Kit</a></td>
			<td>Invitrogen</td>
			<td><a href="https://www.lifetechnologies.com/order/catalog/product/C10425">C10425</a></td>
			<td></td>
		</tr>
		<tr>
			<td>12-well filter inserts (thincerts)</td>
			<td>Greiner bio-one</td>
			<td>657631</td>
			<td></td>
		</tr>
		<tr>
			<td>12-well plate</td>
			<td>Greiner</td>
			<td>665180-01</td>
			<td></td>
		</tr>
		<tr>
			<td>Jettex 2005/45</td>
			<td>ORSA, Giorla Minore, Italy</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Fibrinogen TISSUECOL-Kit Immuno</td>
			<td>Baxter</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Thrombin TISSUECOL-Kit Immuno</td>
			<td>Baxter</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>Serva</td>
			<td>47302.03</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Lonza</td>
			<td>BE12-604F</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM/F12</td>
			<td>Lonza</td>
			<td>BE12-719F</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Gibco</td>
			<td>15630-049&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>FBS Gold</td>
			<td>GE Healthcare</td>
			<td>A11-151</td>
			<td></td>
		</tr>
		<tr>
			<td>Aprotinin (Trasylol)</td>
			<td>Bayer</td>
			<td>4032037</td>
			<td></td>
		</tr>
		<tr>
			<td>Cholera toxin</td>
			<td>Biomol</td>
			<td>G117</td>
			<td></td>
		</tr>
		<tr>
			<td>Hydrocortisone</td>
			<td>Seromed (Biochrom)</td>
			<td>K3520</td>
			<td></td>
		</tr>
		<tr>
			<td>L-ascorbic acid</td>
			<td>Sigma</td>
			<td>A4034</td>
			<td></td>
		</tr>
		<tr>
			<td>TGF-&#223;1</td>
			<td>Invitrogen</td>
			<td>PHG9214</td>
			<td></td>
		</tr>
		<tr>
			<td>RANKL</td>
			<td>R&#38;D systems</td>
			<td>462-TR-010</td>
			<td></td>
		</tr>
		<tr>
			<td>Thermolysin</td>
			<td>Sigma Aldrich</td>
			<td>&#160;T-7902</td>
			<td></td>
		</tr>
		<tr>
			<td>OCT Compound</td>
			<td>TissueTek</td>
			<td>4583</td>
			<td></td>
		</tr>
		<tr>
			<td>Trizol (aka. Denaturing solution - Acid guanidinium thiocyanate-phenol-chloroform extraction)</td>
			<td>Invitrogen</td>
			<td>10296028</td>
			<td></td>
		</tr>
		<tr>
			<td>FastPrep FP120</td>
			<td>Thermo Scientific</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Collagenase Type IV&#160;</td>
			<td>CellSystems</td>
			<td>LS004189</td>
			<td>0.2 mg/ml and 57U/ml final conc.</td>
		</tr>
		<tr>
			<td>Neutrale Protease (Dispase)</td>
			<td>CellSystems</td>
			<td>LS002104</td>
			<td>0.2 mg/ml and 1.2U/ml final conc.</td>
		</tr>
		<tr>
			<td>DNase I&#160;</td>
			<td>Roche</td>
			<td>11 284 932 001</td>
			<td>25 &#181;g/ml final conc.</td>
		</tr>
	</tbody>
</table>

3d organotypic co-culture model supporting medullary thymic epithelial cell proliferation, differentiation and promiscuous gene expression

Intra-timik T hücre gelişimi, çeşitli stromal hücrelerin, yani olmayan T hücrelerden oluşan karmaşık bir üç boyutlu ağ örgüsü gerektirir. Sırayla, yani T hücre reseptör repertuarı oluşturma ve seçim ardından T hücre soyu taahhüt önce çevre içine ihracat, zorunlu kontrol noktaları geçerken timositleri son derece koordineli zamansal ve mekansal için bu iskelesi travers. Bu iskele oluşturan iki ana yerleşik hücre tipleri (cTECs) kortikal ve medüller timik epitel hücreleri (mTECs) bulunmaktadır. MTECs önemli bir özelliği, bir çok dokuya sınırlı bir antijenlerin olarak adlandırılan rasgele ifadesidir. Bu dokuya sınırlı antijenler, sırasıyla kendi kendine tolerans sonuçlanan mTECs ya timik dendritik hücreler tarafından doğrudan veya dolaylı olarak timositleri olgunlaşmamış sunulmaktadır. CTECs ve mTECs gelişim yollarını ve fonksiyonlarını taklit in vitro uygun modeller şu anda lak vardırkral. Yeterli deneysel modellerin eksikliği, örneğin hala tam olarak hücresel ve moleküler düzeyde anlaşılmaktadır karışık gen ifadesinin, analiz engelliyordu. Biz ex vivo izole mTECs kültürüne 3D organotipik ko-kültür modeli uyarladı. Bu model, ilk olarak in vitro olarak, bir cilt eşdeğer üretmek üzere bir şekilde keratinosit yetiştirmek için geliştirilmiştir. , CD80 hi, Aire-pozitif mTECs, RANKL (ii) tepki ve FoxN1 (iii) sürekli ifadeye (i) çoğalması ve CD80 lo, Aire-negatif terminal farklılaşma: 3D model mtec biyoloji temel fonksiyonel özellikler korunmuş Aire ve CD80 hi mTECs doku kısıtlı genler.

Biz aslında keratinositlerde 9 in vitro uzun vadeli kültürü için geliştirilmiş olan 3D organotipik ko-kültür modeli (3D OTC) kabul etti. MACS zenginleştirilmiş mTECs (MACS zenginleştirme şeması Şekil 1 e bakınız), bir fibrin jel ve tutulu fibroblastlar içeren bir yapı iskelesi üzerine ekilmiştir. fibroblastlar in vitro mTECs destekleyen temel hücre dışı matriks (ECM) sağlarlar. MTECs hava maruz kalan in vivo ortamı (OTC set-up şeması <st...

Watch this Scientific Journal Video about 3D Organotypic Co-culture Model Supporting Medullary Thymic Epithelial Cell Proliferation, Differentiation and Promiscuous Gene Expression at JoVE.com

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

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 ...

3d-organotypic-co-culture-model-supporting-medullary-thymic

Research

JoVE Journal

Developmental Biology

10.6K Görüntüleme.  German Cancer Research Center (DKFZ). 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. 

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

Culture and Co-Culture of Mouse Ovaries and Ovarian Follicles

The mammalian ovary is composed of ovarian follicles, each follicle consisting of a single oocyte surrounded by somatic granulosa cells, enclosed together within a basement membrane. A finite pool of follicles is laid down during embryonic development, when oocytes in meiotic arrest form a close association with flattened granulosa cells, forming primordial follicles. By or shortly after birth, mammalian ovaries contain their lifetime&#8217;s supply of primordial follicles, from which point onwards there is a steady release of follicles into the growing follicular pool.
The ovary is particularly amenable to development in vitro, with follicles growing in a highly physiological manner in culture. This work describes the culture of whole neonatal ovaries containing primordial follicles, and the culture of individual ovarian follicles, a method which can support the development of follicles from an immature through to the preovulatory stage, after which their oocytes are able to undergo fertilization in vitro. The work outlined here uses culture systems to determine how the ovary is affected by exposure to external compounds. We also describe a co-culture system, which allows investigation of the interactions that occur between growing follicles and the non-growing pool of primordial follicles.

This protocol describes the primary culture/co-culture of mouse ovarian tissue, using ovaries from neonatal mice and individual ovarian follicles from prepubertal mice. The culture techniques support development in a highly physiological manner, allowing investigation of the effect of extrinsic agents on the ovary, and of interactions between ovarian follicles.

Kültür ve Co-Kültür Mouse Yumurtalıklar ve Yumurtalık Foliküller

The mammalian ovary is composed of ovarian follicles, each follicle consisting of a single oocyte surrounded by somatic granulosa cells, enclosed together ...

Gelişim Biyolojisi

Prediction and Validation of Gene Regulatory Elements Activated During Retinoic Acid Induced Embryonic Stem Cell Differentiation

Embryonic development is a multistep process involving activation and repression of many genes. Enhancer elements in the genome are known to contribute to tissue and cell-type specific regulation of gene expression during the cellular differentiation. Thus, their identification and further investigation is important in order to understand how cell fate is determined. Integration of gene expression data (e.g., microarray or RNA-seq) and results of chromatin immunoprecipitation (ChIP)-based genome-wide studies (ChIP-seq) allows large-scale identification of these regulatory regions. However, functional validation of cell-type specific enhancers requires further in vitro and in vivo experimental procedures. Here we describe how active enhancers can be identified and validated experimentally. This protocol provides a step-by-step workflow that includes: 1) identification of regulatory regions by ChIP-seq data analysis, 2) cloning and experimental validation of putative regulatory potential of the identified genomic sequences in a reporter assay, and 3) determination of enhancer activity in vivo by measuring enhancer RNA transcript level. The presented protocol is detailed enough to help anyone to set up this workflow in the lab. Importantly, the protocol can be easily adapted to and used in any cellular model system.

In this work we provide an experimental workflow of how active enhancers can be identified and experimentally validated.

Tahmin ve Gen Düzenleme Elementlerin Doğrulama Retinoik Asit Kaynaklı Embriyonik sırasında Aktif Hücre Farklılaşma Kök

Embryonic development is a multistep process involving activation and repression of many genes. Enhancer elements in the genome are known to contribute to ...

Adenoviral Gene Therapy for Diabetic Keratopathy: Effects on Wound Healing and Stem Cell Marker Expression in Human Organ-cultured Corneas and Limbal Epithelial Cells

The goal of this protocol is to describe molecular alterations in human diabetic corneas and demonstrate how they can be alleviated by adenoviral gene therapy in organ-cultured corneas. The diabetic corneal disease is a complication of diabetes with frequent abnormalities of corneal nerves and epithelial wound healing. We have also documented significantly altered expression of several putative epithelial stem cell markers in human diabetic corneas. To alleviate these changes, adenoviral gene therapy was successfully implemented using the upregulation of c-met proto-oncogene expression and/or the downregulation of proteinases matrix metalloproteinase-10 (MMP-10) and cathepsin F. This therapy accelerated wound healing in diabetic corneas even when only the limbal stem cell compartment was transduced. The best results were obtained with combined treatment. For possible patient transplantation of normalized stem cells, an example is also presented of the optimization of gene transduction in stem cell-enriched cultures using polycationic enhancers. This approach may be useful not only for the selected genes but also for the other mediators of corneal epithelial wound healing and stem cell function.

An example of adenoviral gene therapy in the human diabetic organ-cultured corneas is presented towards the normalization of delayed wound healing and markedly reduced epithelial stem cell marker expression in these corneas. It also describes the optimization of this process in stem cell-enriched limbal epithelial cultures.

Diyabetik Keratopatide için adenoviral Gen Tedavisi: İnsan Organ-kültürlü kornealar ve Limbal Epitel Hücreleri Yara İyileşmesi Üzerine Etkileri ve Hücre Marker İfade Kök

The goal of this protocol is to describe molecular alterations in human diabetic corneas and demonstrate how they can be alleviated by adenoviral gene ...

Temporal Ordering of Dynamic Expression Data from Detailed Spatial Expression Maps

During somitogenesis, pairs of epithelial somites form in a progressive manner, budding off from the anterior end of the pre-somitic mesoderm (PSM) with a strict species-specific periodicity. The periodicity of the process is regulated by a molecular oscillator, known as the "segmentation clock," acting in the PSM cells. This clock drives the oscillatory patterns of gene expression across the PSM in a posterior-anterior direction. These so-called clock genes are key components of three signaling pathways: Wnt, Notch, and fibroblast growth factor (FGF). In addition, Notch signaling is essential for synchronizing intracellular oscillations in neighboring cells. We recently gained insight into how this may be mechanistically regulated. Upon ligand activation, the Notch receptor is cleaved, releasing the intracellular domain (NICD), which moves to the nucleus and regulates gene expression. NICD is highly labile, and its phosphorylation-dependent turnover acts to restrict Notch signaling. The profile of NICD production (and degradation) in the PSM is known to be oscillatory and to resemble that of a clock gene. We recently reported that both the Notch receptor and the Delta ligand, which mediate intercellular coupling, themselves exhibit dynamic expression at both the mRNA and protein levels. In this article, we describe the sensitive detection methods and detailed image analysis tools that we used, in combination with the computational modeling that we designed, to extract and overlay expression data from distinct points in the expression cycle. This allowed us to construct a spatio-temporal picture of the dynamic expression profile for the receptor, the ligand, and the Notch target clock genes throughout an oscillation cycle. Here, we describe the protocols used to generate and culture the PSM explants, as well as the procedure to stain for the mRNA or protein. We also explain how the confocal images were subsequently analyzed and temporally ordered computationally to generate ordered sequences of clock expression snapshots, hereafter defined as "kymographs," for the visualization of the spatiotemporal expression of Delta-like1 (Dll1) and Notch1 throughout the PSM.

The segmentation clock drives oscillatory gene expression across the pre-somitic mesoderm (PSM). Dynamic Notch activity is key to this process. We use imaging and computational analyses to extract temporal dynamics from spatial expression data to demonstrate that Delta ligand and Notch receptor expression oscillate in the vertebrate PSM.

Detaylı Mekansal İfade Haritalar Dinamik İfade Verilerinin Zamansal Sipariş

During somitogenesis, pairs of epithelial somites form in a progressive manner, budding off from the anterior end of the pre-somitic mesoderm (PSM) with a ...

Rapid, Directed Differentiation of Retinal Pigment Epithelial Cells from Human Embryonic or Induced Pluripotent Stem Cells

We describe a robust method to direct the differentiation of pluripotent stem cells into retinal pigment epithelial cells (RPE). The purpose of providing a detailed and thorough protocol is to clearly demonstrate each step and to make this readily available to researchers in the field. This protocol results in a homogenous layer of RPE with minimal or no manual dissection needed. The method presented here has been shown to be effective for induced pluripotent stem cells (iPSC) and human embryonic stem cells. Additionally, we describe methods for cryopreservation of intermediate cell banks that allow long-term storage. RPE generated using this protocol might be useful for iPSC disease-in-a-dish modeling or clinical application.

This protocol describes how to produce retinal pigment epithelial cells (RPE) from pluripotent stem cells. The method uses a combination of growth factors and small molecules to direct the differentiation of stem cells into immature RPE in fourteen days and mature, functional RPE after three months.

Hızlı, yönlendirilmiş farklılaşma insan embriyonik veya İndüklenmiş Pluripotent kök hücrelerinden retina Pigment epitel hücrelerinin

We describe a robust method to direct the differentiation of pluripotent stem cells into retinal pigment epithelial cells (RPE). The purpose of providing ...

Streamlined 3D Cerebellar Differentiation Protocol with Optional 2D Modification

Reducing the complexity and cost of differentiation protocols is important for researchers. This interest fits with concerns about possible unintended effects that extrinsic patterning factors might introduce into human pluripotent stem cell (hPSC) models of brain development or pathophysiology, such as masking disease phenotype. Here, we present two cerebellar differentiation protocols for hPSCs, designed with simpler startup method, fewer patterning factors, and less material requirements than previous protocols. Recently, we developed culture procedures, which generate free-floating 3-dimensional (3D) products consistent with other brain &#34;organoid&#34; protocols, including morphologies relevant to modeling brain development such as sub/ventricular zone- and rhombic lip-like structures. The second uses an adherent, 2D monolayer procedure to complete differentiation, which is shown capable of generating functional cerebellar neurons, as products are positive for cerebellar-associated markers, and exhibit neuron-like calcium influxes. Together, these protocols offer scientists a choice of options suited to different research purposes, as well as a basic model for testing other types of streamlined neural differentiations.

We describe a simplified 3D differentiation protocol for hPSCs, using defined medium and reduced growth factors, capable of generating cell aggregates with early neuroepithelial structures and positive for cerebellar-associated markers, as well as an optional 2D modification for differentiating cells as a monolayer to generate functional neurons.

İsteğe bağlı 2D değişiklik ile akıcı 3D serebellar farklılaşma Protokolü

Reducing the complexity and cost of differentiation protocols is important for researchers. This interest fits with concerns about possible unintended ...

Clonal Analysis of Embryonic Hematopoietic Stem Cell Precursors Using Single Cell Index Sorting Combined with Endothelial Cell Niche Co-culture

The ability to study hematopoietic stem cell (HSC) genesis during embryonic development has been limited by the rarity of HSC precursors in the early embryo and the lack of assays that functionally identify the long-term multilineage engraftment potential of individual putative HSC precursors. Here, we describe methodology that enables the isolation and characterization of functionally validated HSC precursors at the single cell level. First, we utilize index sorting to catalog the precise phenotypic parameter of each individually sorted cell, using a combination of phenotypic markers to enrich for HSC precursors with additional markers for experimental analysis. Second, each index-sorted cell is co-cultured with vascular niche stroma from the aorta-gonad-mesonephros (AGM) region, which supports the maturation of non-engrafting HSC precursors to functional HSC with multilineage, long-term engraftment potential in transplantation assays. This methodology enables correlation of phenotypic properties of clonal hemogenic precursors with their functional engraftment potential or other properties such as transcriptional profile, providing a means for the detailed analysis of HSC precursor development at the single cell level.

Here we present methodology for the clonal analysis of hematopoietic stem cell precursors during murine embryonic development. We combine index sorting of single cells from the embryonic aorta-gonad-mesonephros region with endothelial cell co-culture and transplantation to characterize the phenotypic properties and engraftment potential of single hematopoietic precursors.

Embriyonik hematopoetik kök hücre öncüleri endotel hücre niş ortak kültürü ile kombine sıralama tek hücre dizini kullanarak klonal analizi

The ability to study hematopoietic stem cell (HSC) genesis during embryonic development has been limited by the rarity of HSC precursors in the early ...

Proliferation and Differentiation of Murine Myeloid Precursor 32D/G-CSF-R Cells

Understanding of the hematopoietic stem and progenitor cell biology has important implications for regenerative medicine and the treatment of hematological pathologies. Despite the most relevant data that can be acquired using in vivo models or primary cultures, the low abundance of hematopoietic stem and progenitor cells considerably restricts the pool of suitable techniques for their investigation. Therefore, the use of cell lines allows sufficient production of biological material for the performance of screenings or assays that require large cell numbers. Here we present a detailed description, readout, and interpretation of proliferation and differentiation assays which are used for the investigation of processes involved in myelopoiesis and neutrophilic differentiation. These experiments employ the 32D/G-CSF-R cytokine dependent murine myeloid cell line, which possesses the ability to proliferate in the presence of IL-3 and differentiate in G-CSF. We provide optimized protocols for handling 32D/G-CSF-R cells and discuss major pitfalls and drawbacks that might compromise the described assays and expected results. Additionally, this article contains protocols for lentiviral and retroviral production, titration, and transduction of 32D/G-CSF-R cells. We demonstrate that genetic manipulation of these cells can be employed to successfully perform functional and molecular studies, which can complement results obtained with primary hematopoietic stem and progenitor cells or in vivo models.

Here detailed protocols for culturing the murine myeloid precursor 32D/G-CSF-R cell line, performing viral infections, and carrying out proliferation and differentiation assays are presented. This cell line is suitable for studying myeloid cell development, and the role of genes of interest in myeloid cell growth and neutrophilic differentiation.

Nükleer silahların yayılmasına karşı ve fare Miyeloid habercisi 32D/G-CSF-R farklılaşma hücre

Understanding of the hematopoietic stem and progenitor cell biology has important implications for regenerative medicine and the treatment of ...

Hemogenic Reprogramming of Human Fibroblasts by Enforced Expression of Transcription Factors

The cellular and molecular mechanisms underlying specification of human hematopoietic stem cells (HSCs) remain elusive. Strategies to recapitulate human HSC emergence in vitro are required to overcome limitations in studying this complex developmental process. Here, we describe a protocol to generate hematopoietic stem and progenitor-like cells from human dermal fibroblasts employing a direct cell reprogramming approach. These cells transit through a hemogenic intermediate cell-type, resembling the endothelial-to-hematopoietic transition (EHT) characteristic of HSC specification. Fibroblasts were reprogrammed to hemogenic cells via transduction with GATA2, GFI1B and FOS transcription factors. This combination of three factors induced morphological changes, expression of hemogenic and hematopoietic markers and dynamic EHT transcriptional programs. Reprogrammed cells generate hematopoietic progeny and repopulate immunodeficient mice for three months. This protocol can be adapted towards the mechanistic dissection of the human EHT process as exemplified here by defining GATA2 targets during the early phases of reprogramming. Thus, human hemogenic reprogramming provides a simple and tractable approach to identify novel markers and regulators of human HSC emergence. In the future, faithful induction of hemogenic fate in fibroblasts may lead to the generation of patient-specific HSCs for transplantation.

This protocol demonstrates the induction of a hemogenic program in human dermal fibroblasts by enforced expression of the transcription factors GATA2, GFI1B and FOS to generate hematopoietic stem and progenitor cells.

Transkripsiyon Faktörlerinin Zorunlu İfadesi Ile İnsan Fibroblastlarının Hemojenik Yeniden Programlanması

The cellular and molecular mechanisms underlying specification of human hematopoietic stem cells (HSCs) remain elusive. Strategies to recapitulate human ...

An Ectopic Chemokine Expression Model for Testing Macrophage Recruitment In Vivo

Zebrafish are widely used in basic and biomedical research. Many zebrafish transgenic lines are currently available to label various types of cells. Owing to the transparent embryonic body of zebrafish, it is convenient for us to study the effect of one chemokine on the behavior of a certain type of cells in vivo. Here we provided a workflow to investigate the function of a chemokine on macrophage migration in vivo. We constructed a tissue-specific overexpression plasmid to overexpress IL-34 and injected the plasmid into one-cell stage transgenic fish embryos whose macrophages were specifically labeled by a fluorescent protein. We then used whole mount fluorescent in situ hybridization and immunostaining to detect the pattern of the chemokine expression and the number or location of macrophages. The injected WT embryos were raised to generate a stable transgenic line. Finally, we used confocal live imaging to directly observe macrophage behavior in the stable transgenic fish to study the function of IL-34 on macrophages in vivo.

To test the effect of a chemokine on macrophage recruitment in vivo, the whole mount in situ hybridization was used to detect the ectopic expression of the chemokine, and immunostaining was used to label macrophages. Live imaging was used for real-time observation of macrophage migration.

Vivo Makrofaj İşe Test için Bir Ektopik Kemokin İfade Modeli

Zebrafish are widely used in basic and biomedical research. Many zebrafish transgenic lines are currently available to label various types of cells. Owing ...