Bu çalışma Det sutable Forskningsråd / Sundhed og Sygdom Bir Genetik Pilot ödülü NCCR Frontiers, Juvenil Diyabet Araştırma Vakfı Hibe 41-2009-775 ve Grant 12-126875 tarafından sırayla finanse edildi. Yazarlar video çekimi sahipliği için Spagnoli laboratuvar teşekkür ederim.

Bu protokol kemirgen E10.5 türetilen pankreas organoids epitel hücreleri pankreas ayrışmış büyümeyi hedeflemektedir. Protokol hayvan deney için etik kurul onayı gerektirir. E10.5 fare embriyoları Dorsal Pankreas Bud 1. Diseksiyon <ol><li> Bir makas ile karın açmak, embriyonik gün (E) 10.5 aşımına hamile fareler kurban, iki rahim boynuzları çıkarın ve soğuk fosfat tamponu (PBS) veya Dulbeccol ile dolu bir 10 cm Petri kabındaki koyun modifiye temel, orta (DMEM) buz üzerinde tutuldu. Hücre ekimi için kurban gelen toplam deney hücre zarar görmesini önlemek için, 60-90 dakika içinde yapılır. </li><li> Küçük bir makas kullanarak tek tek embriyo bölümler halinde rahim ayırın. Gibi soğuk PBS ile bir 35 mm Petri kabı, bir embriyo transfer ve üstten aydınlatma ile bir mikroskop altında görselleştirmek. Diseksiyon forseps ile çevreleyen kas, desidua ve yumurta sarısı sa kaldırmakc ve embriyo maruz. Lütfen soğuk DMEM ortama embriyo yerleştirin. Embriyolar kolayca 3 ml&#39;lik plastik transferi pipet / damlalık kullanarak transfer edilebilir. Devam etmeden önce benzer bir şekilde, her bir embriyonun izole edin. </li><li> PBS içinde temiz bir 35 mm Petri kabı içine bir embriyo yerleştirin. <ol><li> Ön ayakları kaldırmak için ince forseps (0.05 mm genişlik) kullanın. Yavaşça omurilik bölgesinden sindirim sistemi (Şekil 1) ayırmak için açılış forseps yerleştirin. İSTEĞE BAĞLI: daha rahat, mide görmek aşağı sarısı sap altında kalp ve kuyruk bölgeye, embriyonun üst vücut kaldırmak için. </li><li> Mide, karaciğer ve bağırsak bulun. Forseps kullanılarak, bağırsak mideden mide-bağırsak yolu izole etmek ve (Şekil 1), buz üzerinde, soğuk DMEM içine yerleştirin. Dorsal pankreatik bud mide dorsal, posterior (Şekil 1) takılır. </li><li> Bir simi her bireyin gastrointestinal izoleDevam etmeden önce lar şekilde. Embriyoların genotip gerekiyorsa, diseksiyon zamanda kuyruk toplar ve buz soğuk DMEM ile 24 kuyucuklu bir plaka içerisinde farklı bir kuyu içinde her bir mide-bağırsak yolu tutmak. </li></ol></li><li> Soğuk PBS ile temiz bir 35 mm Petri tabağına yerleştirin, bir mide-bağırsak yolu. Şimdi diseksiyon mikroskop altında dorsal pankreas tomurcuk görselleştirmek ve teşrih parlak alanda aşağıdan aydınlatma kullanın. Bu koşullar hacimlerinin görselleştirme optimize eder. Elektrolitik-bilenmiş tungsten iğne veya 20 G şırınga iğneleri kullanarak, epiteli etrafında mümkün olduğunca az mezenşimin ile pankreas tomurcuğu (Şekil 1) izole. <ol><li> 2-3 dakika boyunca, bir soğuk dispase çözeltisi (1.25 mg / ml) ihtiva eden bir petri çanağı içine izole edilmiş tomurcuk aktarın. Bu noktadan itibaren, transfer en uygun bir ağızdan-kontrol edilen esnek bir boru bağlanmış alev çekilmiş 50 ul bir cam kılcal ile yapılabilir. Alternatif olarak, ancak daha ritomurcuğu kaybetme sk, uygun plastik ipuçları ile 10 ul otomatik pipet (Pipetman) kullanın. </li><li> Mikroskopik kontrol altında pankreas tomurcuk aspirasyon ve fırlamasını gerçekleştirin. Geri PBS içinde pankreas tomurcuk koydu. Bundan başka, cam kılcal (Şekil 1) kullanılarak, iğnelerin ve yumuşak aspirasyon ile mezenşimin gelen izole edilmiş pankreas tomurcuk temizleyin. </li><li> Tüm mezenşimin çıkarıldığında, soğuk PBS içinde pankreatik tomurcuk durulama; (60-iyi mini tepsi soğuk DMEM 10 ul ile dolu) bireysel kuyularda soğuk DMEM&#39;ye her tomurcuk transfer. Bu doku çok yapışkan yapar, dispase yılında mezenşimi kaldırmak için önemli değildir. </li></ol></li></ol> Dağılmış hücreler 2. Kaplama ve Kültür <ol><li> Durulama için 10 ul PBS ile dolu 60-iyi mini kasetlerinden konik kuyuların içine alev-kapiller çekti cam ile tüm embriyolar disseke epitelyaya aktarın. <ol><li> Tripsin% 0.05 a 10 ul transfer tomurcuknd bu 4 dakika süreyle 37 ° C&#39;de inkübe edin. 10 ul DMEM +% 10 fetal dana serumu (FCS) ile bir kuyuya tomurcuk aktararak tripsin etkisiz hale getirirler. </li><li> Bir pipet çektirmesi ile çekilmiş ince kılcal damar yoluyla aspirasyon ile hücre süspansiyonu ayrışır. Bu hücreleri ayırmak için yukarı ve aşağı pipetleme sırasında bu aşamada kabarcıkları önlemek için önemlidir. Pankreas organoids optimum 5-15 hücre küçük gruplar başlar ve bu nedenle kısmi ayrılma (Şekil 1) kullanılması tavsiye edilir. </li></ol></li><li> Tek tek işleme nedeniyle farklılıkları en aza indirmek amacıyla bir Eppendorf tüpü içine birkaç embriyolarından hücreleri havuzda toplayın. , 1:3 oranında Matrigel soğutulmuş hücre süspansiyonu seyreltin. Kısım 96 oyuklu bir plaka, 8 ul / oyuk veya görüntüleme için optimize edilmiş bir plaka içinde (aşağıya bakınız) için bu karışımı ile gerçekleştirilmektedir. </li><li> Matrigel kalınlaştırmak için izin veren, 5 dakika boyunca 37 ° C&#39;de inkübe edin. Seçim ortamı 70 ul ile kuyu doldurun (organoid veya küre, sekmesine bakınles 1 ve 2) ve 37 ° C de% 5 CO2 ve% 95 hava içeren nemlendirilmiş bir ortamda bırakmak </li><li> Her 4. günde orta yerine. Günlük büyüyen pankreas koloniler izlemek ve görüntüleme işlemini belgelemek. </li><li> Daha önce rapor edildiği gibi, 28 çıkar küçük moleküller ya da proteinler, girişim deneyleri için bu aşamada ortamına ilave edilebilir. </li></ol> Tablo 1: organoid ortamı. <table border="0" cellpadding="0" cellspacing="0"><tbody><tr><td> Malzeme Adı </td><td> Hazır Konsantrasyon </td><td> Son ortam içinde konsantre </td><td> Stokunun hacmi </td></tr><tr><td> Penisilin-Streptomisin </td><td> % 100 </td><td> % 1 </td><td> 50 ul </td></tr><tr><td> Nakavt Serum değiştirme (ek) </td><td> % 100 </td><td> % 10. </td><td> 500 ul </td></tr><tr><td> 2-merkaptoetanol </td><td> 14.3 M </td&gt; <td> 0.1 mM </td><td> 1 ul </td></tr><tr><td> Forbol Miristat Asetat (PMA) </td><td> 16 iM </td><td> 16 nM </td><td> 5 ul </td></tr><tr><td> Y-27632 (ROCK inhibitörü) </td><td> 50 mM </td><td> 10 uM </td><td> 1 ul </td></tr><tr><td> EGF </td><td> 50 ug / ml </td><td> 25 ng / ml </td><td> 2.5 ul </td></tr><tr><td> Rekombinant İnsan 1 R-spondin </td><td> 250 ug / ml </td><td> 500 ng / ml </td><td> 10 ul </td></tr><tr><td> - Ya da - </td><td></td><td></td><td></td></tr><tr><td> Rekombinant Fare R-1 spondin </td><td> 250 ug / ml </td><td> 500 ng / ml </td><td> 10 ul </td></tr><tr><td> Rekombinant İnsan FGF1 (aFGF) </td><td> 100 ug / ml </td><td> 25 ug / ml </td><td> 1.25 ul </td></tr><tr><td> Heparin (Liquemin) </td><td> 2500 U / ml </td><td> 2.5 U / ml </td><td> 2 ul </td></tr><tr><td> Recombinant İnsan FGF10 </td><td> 100 ug / ml </td><td> 100 ng / ml </td><td> 5 ul </td></tr><tr><td> DMEM/F-12 </td><td></td><td></td><td> 4,412.25 ul </td></tr><tr><td> Toplam </td><td></td><td></td><td> 5000 ul </td></tr></tbody></table> Tablo 2: Küre ortamı. <table border="0" cellpadding="0" cellspacing="0"><tbody><tr><td> Malzeme Adı </td><td> Hazır Konsantrasyon </td><td> Son ortam içinde konsantre </td><td> Stokunun hacmi </td></tr><tr><td> Penisilin-Streptomisin </td><td> % 100 </td><td> % 1 </td><td> 50 ul </td></tr><tr><td> B27 x50 (ek) </td><td> % 100 </td><td> % 10. </td><td> 100 ul </td></tr><tr><td> Rekombinant İnsan FGF2 (bFGF) </td><td> 100 ug / ml </td><td> 64 ng / ml </td><td> 3.2 ul </td></tr><tr><td> Y-27632 (ROCK inhibitörü) </td><td> 50 mM </td><td> 10 uM </td><td> 1 ul </td></tr><tr><td> DMEM/F-12 </td><td></td><td></td><td> 4845,8 ul </td></tr><tr><td> Toplam </td><td></td><td></td><td> 5000 ul </td></tr></tbody></table> Organoid Geliştirme İlerlemesi 3. Görüntüleme <ol><li> Görüntü organoids günlük veya zaman atlamalı mikroskopi bir floresan time-lapse mikroskop kullanılarak. Zaman atlamalı görüntüleme için, bir XY (Z) otomatik ters floresan mikroskop kullanın. </li><li> 4-çukurlu plakalara veya 2-5 ml ortam ile doldurulmuş olan cam-tabanlı plakalar içerisinde 3 ul&#39;lik Deposit küçük damlacıklar halinde. 10x uzun mesafe hedefi ile görüntü. Not: floresan izleyiciler ifade eden transjenik farenin kullanılabilir. Örneğin Movie 1 gösterileri Pdx1-Ngn3-ER TM-ires-nGFP + farelerden 4 organoids ilk genişleme. Nükleer GFP tek nesneleri ancak benzer ilkeler zar floresan ile hücreleri izlemek için uygulanabilir olarak hücreleri izlemek için sağlar bumT / mG fareler 29 gibi. </li><li> Odak sürükleniyor önlemek ve elle tanımlanmış pozisyonlarda 3 veya daha fazla gün için 1 resim / saat almak için yazılım kontrol otomasyon ayarlamak için tohum hücreleri sonra time-lapse görüntüleme 3 saat başlayın. Her bir konumu için, floresan markör ifade raporlama, diferansiyel girişim kontrast (DIC) görüntü hem de GFP sinyali elde. </li></ol> Histoloji için Organoids 4. Recovery <ol><li> Buz üzerinde 96 oyuklu plaka koyun ve buz gibi soğuk PBS ile değiştirilmesi, orta çıkarın. Bu kısmen Matrigel depolimerize. </li><li> Yavaşça genel yapısını bozmayacak amacıyla 1.000 ul ucu kullanarak çevredeki Matrigel kaldırarak, her organoid aspire. Buz gibi soğuk PBS ile bir kuyuya her organoid aktarın. Buz üzerinde plaka tutun. Matrigel Doğrudan tespit de mümkündür. </li><li> ,% 4 paraformaldehit (PFA) içerisinde 15 dakika boyunca organoid saptamak sukroz içinde dondurularak saklanabilmesi ve jelatin içine gömmek. Her Süreçorganoid önce (Johansson et al., 2007) tarif edildiği gibi 4 cryosectioning ve histoloji için. </li></ol> PCR ve Biyokimya Organoids 5. Recovery <ol><li> Buz üzerinde 96 oyuklu plaka yerleştirin ve orta çıkarın. Hücresel RNA stabilize etmek ve korumak için oyuk başına RNAlater 60 ul ekle. </li><li> Kısmen buz üzerinde depolymerizing her iyi mekanik olarak jel bozar. Genel yapısını bozabilir veya 200 ul ucu ile mekanik jel bozarak (Matrigel ile) tüm iyileşmez için 1.000 ul uç kullanarak bireysel organoids kurtarmak ya. Bir RNAse serbest olmayan yapışkan Eppendorf tüpüne da içerik aktarmak için 1.000 ul ucu kullanın buz üzerinde tutulmalıdır. </li><li> 60 ul RNAlater ile kuyu yıkayın ve aynı Eppendorf tüpüne kalan içerik eklemek. </li><li> 4 ° C &#39;de 500-1000 x g&#39;de 5 dakika boyunca tüpleri Spin </li><li> Sadece 20-30 ul bırakarak, süpernatant kaldırmakbirlikte pelet ile tüp içinde RNAlater. Biyokimya için, mümkün olduğu kadar kuru numune saklayın. </li><li> Hemen RNAlater örnekleri dondurma etmeyin; 4 ° CO / N de mağaza (RNAlater iyice dokuya nüfuz sağlamak için). Doku, uzun süreli depolama için -20 ° C &#39;de muhafaza edilebilir ve daha sonra, doku bozulması ve RNA küçük miktarlarda çıkarılması için işlenebilir. </li></ol>

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A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cerebral+organoids+model+human+brain+development+and+microcephaly.">Cerebral organoids model human brain development and microcephaly.</a> Nature. 10, (2013).</li><li>Greggio, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Artificial+three-dimensional+niches+deconstruct+pancreas+development+in+vitro.">Artificial three-dimensional niches deconstruct pancreas development in vitro.</a> Development. 140, 4452-4462 (2013).</li><li>Muzumdar, M. D., Tasic, B., Miyamichi, K., Li, L., Luo, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+global+double-fluorescent+Cre+reporter+mouse.">A global double-fluorescent Cre reporter mouse.</a> Genesis. 45, 593-605 (2007).</li></ol>

The authors have nothing to disclose.

In vitro fonksiyonel beta hücrelerinin büyük ölçekli üretim hala 1 etkisizdir. Bu zorlu bağlamda, gelişim biyolojisi çalışma fonksiyonel beta hücrelerinin farklılaşması için gerekli olan tam sinyalleri deşifre yardımcı olabilir. Bu protokol, in vitro embriyo pankreas progenitörlerin bakım, genişleme ve farklılaşması için izin verir. Bu, diğer endokrin hormon eş bildirilmemektedir insülin üreten beta hücrelerinin oluşumunu içeren, Pdx1 yüksek düzeyde, insül...

Organ kültür vivo araştırmalarda karmaşık ama son derece alakalı arasındaki boşlukları ve hücre çizgi modellerin uygun ama yaklaşık simülasyon köprü kullanışlı bir model sağlar. Pankreas durumunda, endokrin ve ekzokrin hücreleri taklit hücre çizgileri bulunmaktadır transforme edilir, ancak pankreas progenitörlerin mükemmel eşdeğer herhangi bir hücre hattı yoktur. Yetişkin Bütün pankreas kültüre olamaz; izole edilmiş endokrin adacık hücresi çoğalması olmadan birkaç hafta muhafaza edilebilir ve doku dilimleri birkaç saat 5 için in vitro olarak tutulabilir. Embriyonik pankreas kültürü yaygın gelişimini incelemek için sadece kullanılır olmuştur, ama aynı zamanda 8 işler görüntüye, epitelyal-mezenkimal etkileşimleri 4,6,7 araştırmak veya kimyasal onlarla 9 ile müdahale. İki organ kültürü yöntemleri ağırlıklı olarak kullanılır: İlk conve fibronektin kaplı plakalar 2, üzerinde pankreas tomurcukları kültürleme oluşurGörüntüleme amacıyla nient; İkinci seçenek kültürüne en morfogenetiğine korur hava-sıvı arayüzü 3,4 de filtrelerde organ olduğunu. Çok faydalı olmasına rağmen, bu yöntemler, düzleşme belli bir dereceye kadar yol; normal gelişimi ile karşılaştırıldığında ve başlangıç ​​popülasyonu pankreas hücreleri ve mezenkimal hücreler her türlü içeren karmaşık olduğu gibi progenitörlerin genişleme çok sınırlıdır. Kültür yeteneği ve dağınık birincil hücreleri genişletmek soy ilişkileri incelemek ve izole hücre tiplerinin 10 içsel özelliklerini ortaya çıkarmak için değerlidir. Sugiyama ve ark. 11. pankreas atalarıdır ve besleyici katmanlar kültürde 3-5 gündür bazı fonksiyonel karakterleri korunur endokrin atalarıdır korumak olabilir. Atalarıdır doğası / kök hücrelerin rağmen neurospheres 12 ve 13 mammospheres benzer Pancreatospheres, yetişkin adacık ve duktal hücrelerinden genişletilmiştirBu küreler oluşturmak olduğu açık değildir. Buna ek olarak, fizyolojik gelişimi ile aksine, bazı pancreatospheres nöronlar 14,15 ihtiva etmiştir. Küreler yakın zamanda embriyonik pankreas atalarıdır 16,17 ve iyi progenitör genişleme ve sonraki farklılaşma ile pankreata 18 yenileyici üretilir ama morfogenetiğine özetlemek başarısız olduğu görülmüştür. Minyatür organları içine kendi kendini düzenleyebilir ve genellikle dağınık tanımlanan hücrelerden 3 boyutlu modeller son gelişti ve bu bağırsak 19,20, 21, mide, karaciğer 22, prostat 23 ve nefes borusu gibi birden fazla organların gelişimi veya yetişkin devir taklit adres 24. Optik bardak 25, 26 ya da bağırsak, beyin 27 olduğu gibi, bazı durumlarda, gelişme morfolojisi ve farklılaşması, ES hücrelerinden in 3D değinmeyecek edilmiştir. Burada, biz desOnlar farklılaştırmak ve öz-örgütlenmeyi bir 3D Matrigel iskele ayrışmış multipotent pankreas atalarıdır genişletmek için bir yöntem cribe.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Penicillin-Streptomycin</td>
			<td>Gibco</td>
			<td>15070-063</td>
			<td>Stock keept at -20&#176;C</td>
		</tr>
		<tr>
			<td>KnockOut Serum replacement (supplement)</td>
			<td>Gibco</td>
			<td>10828-028</td>
			<td>Stock keept at -20&#176;C</td>
		</tr>
		<tr>
			<td>2-mercaptoethanol</td>
			<td>Sigma Aldrich</td>
			<td>3148-25ML</td>
			<td>Stock keept at 4&#176;C</td>
		</tr>
		<tr>
			<td>Phorbol Myristate Acetate (PMA)</td>
			<td>Calbiotech</td>
			<td>524400-1MG</td>
			<td>Stock keept at -20&#176;C</td>
		</tr>
		<tr>
			<td>Y-27632 (ROCK inhibitor)</td>
			<td>Sigma Aldrich</td>
			<td>ab120129</td>
			<td>Stock keept at -20&#176;C- Attention! Stability/source is a frequent source of problems</td>
		</tr>
		<tr>
			<td>EGF</td>
			<td>Sigma Aldrich</td>
			<td>E9644-2MG</td>
			<td>Stock keept at -80&#176;C</td>
		</tr>
		<tr>
			<td>Recombinant Human R-spondin 1</td>
			<td>R&#38;D</td>
			<td>4645-RS-025/CF</td>
			<td>Stock keept at -80&#176;C</td>
		</tr>
		<tr>
			<td>&#160;- or -&#160;</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Recombinant Mouse R-spondin 1</td>
			<td>R&#38;D</td>
			<td>3474-RS-050</td>
			<td>Stock keept at -80&#176;C</td>
			<td></td>
		</tr>
		<tr>
			<td>Recombinant Human FGF1 (aFGF)</td>
			<td>R&#38;D</td>
			<td>232-FA-025</td>
			<td>Stock keept at -80&#176;C- do not include to increase beta cell production</td>
		</tr>
		<tr>
			<td>Heparin (Liquemin)</td>
			<td>Drossapharm</td>
			<td></td>
			<td>Stock keept at 4&#176;C</td>
		</tr>
		<tr>
			<td>Recombinant Human FGF10</td>
			<td>R&#38;D</td>
			<td>345-FG-025</td>
			<td>Stock keept at -80&#176;C</td>
		</tr>
		<tr>
			<td>DMEM/F-12</td>
			<td>Gibco</td>
			<td>21331-020</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin</td>
			<td>Gibco</td>
			<td>15070-063</td>
			<td>Stock keept at -20&#176;C</td>
		</tr>
		<tr>
			<td>B27 x50 (supplement)</td>
			<td>Gibco</td>
			<td>17504-044</td>
			<td>Stock keept at -20&#176;C</td>
		</tr>
		<tr>
			<td>Recombinant Human FGF2 (bFGF)</td>
			<td>R&#38;D</td>
			<td>233-FB-025</td>
			<td>Stock keept at -80&#176;C</td>
		</tr>
		<tr>
			<td>Y-27632 (ROCK inhibitor)</td>
			<td>Sigma Aldrich</td>
			<td>ab120129</td>
			<td>Stock keept at -20&#176;C- Attention! Stability/source is a frequent source of problems</td>
		</tr>
		<tr>
			<td>DMEM/F-12</td>
			<td>Gibco</td>
			<td>21331-020</td>
			<td></td>
		</tr>
		<tr>
			<td>Matrigel</td>
			<td>Corning</td>
			<td>356231</td>
			<td>Stock keept at -20&#176;C</td>
		</tr>
		<tr>
			<td>Trypsin 0.05%</td>
			<td>Gibco</td>
			<td>25300-054</td>
			<td>Stock keept at 4&#176;C</td>
		</tr>
		<tr>
			<td>RNAlater - RNA stabilizing reagent</td>
			<td>Qiagen</td>
			<td>76104</td>
			<td>Store at room temperature</td>
		</tr>
		<tr>
			<td>Dispase&#160;</td>
			<td>Sigma Aldrich</td>
			<td>D4818-2MG</td>
			<td>Stock keept at -20&#176;C</td>
		</tr>
		<tr>
			<td>BSA for reconstitution</td>
			<td>Milipore</td>
			<td><a href="http://www.millipore.com/catalogue/item/81-068-3">81-068</a></td>
			<td>For reconstituition of cytokines&#160; - Stock keept at -20&#176;C</td>
		</tr>
		<tr>
			<td>Fetal calf serum (FCS)</td>
			<td>Gibco</td>
			<td>16141079</td>
			<td>Stock keept at -20&#176;C</td>
		</tr>
		<tr>
			<td>60 well MicroWell trays</td>
			<td>Sigma Aldrich</td>
			<td>M0815-100EA</td>
			<td></td>
		</tr>
		<tr>
			<td>4-well plates</td>
			<td>Thermo Scientific</td>
			<td>176740</td>
			<td></td>
		</tr>
		<tr>
			<td>95-well plates F bottom</td>
			<td>Greiner Bio</td>
			<td>6555180</td>
			<td></td>
		</tr>
		<tr>
			<td>Glas bottom plates</td>
			<td>Ibidi</td>
			<td>81158</td>
			<td></td>
		</tr>
		<tr>
			<td>Disposal micropittes</td>
			<td>Blaubrand</td>
			<td>708745</td>
			<td></td>
		</tr>
	</tbody>
</table>

in vitro pancreas organogenesis from dispersed mouse embryonic progenitors

The pancreas is an essential organ that regulates glucose homeostasis and secretes digestive enzymes. Research on pancreas embryogenesis has led to the development of protocols to produce pancreatic cells from stem cells 1. The whole embryonic organ can be cultured at multiple stages of development 2-4. These culture methods have been useful to test drugs and to image developmental processes. However the expansion of the organ is very limited and morphogenesis is not faithfully recapitulated since the organ flattens. 
We propose three-dimensional (3D) culture conditions that enable the efficient expansion of dissociated mouse embryonic pancreatic progenitors. By manipulating the composition of the culture medium it is possible to generate either hollow spheres, mainly composed of pancreatic progenitors expanding in their initial state, or, complex organoids which progress to more mature expanding progenitors and differentiate into endocrine, acinar and ductal cells and which spontaneously self-organize to resemble the embryonic pancreas. 
We show here that the in vitro process recapitulates many aspects of natural pancreas development. This culture system is suitable to investigate how cells cooperate to form an organ by reducing its initial complexity to few progenitors. It is a model that reproduces the 3D architecture of the pancreas and that is therefore useful to study morphogenesis, including polarization of epithelial structures and branching. It is also appropriate to assess the response to mechanical cues of the niche such as stiffness and the effects on cell&#180;s tensegrity.

3D Matrigel&#39;de ayrışmış ve seribaşı E10.5 dorsal pankreas atalarıdır pankreas gelişimini özetlemek. Atalarıdır en kolay floresan gazetecilere takip edilebilir. Bizim durumumuzda (Şekil 2) Neurog3 4 etkinleştirmeden tamoksifen yokluğunda ve böylece Pdx1 promoteri (Pdx1-Ngn3-ER TM-nGFP) (Film 1) tarafından kontrol edilen bir nükleer GFP proteini eksprese eden transgenik bir fare kullanılmıştır. Organoid orta ile...

Watch this Scientific Journal Video about In Vitro Pancreas Organogenesis from Dispersed Mouse Embryonic Progenitors at JoVE.com

In Vitro Pancreas Organogenesis from Dispersed Mouse Embryonic Progenitors

Bu protokol açıklanan üç boyutlu kültür yöntemi dallı bir organ haline onların önemli genişleme, farklılaşma ve morfojenezinin dahil dağınık embriyonik fare pankreas atalarıdır, gelen pankreas gelişimini özetlediği. Bu yöntem, görüntüleme, niş fonksiyonel müdahale ve manipülasyon için uygun değildir.

Dağınık Fare Embriyonik progenitörlerin gelen Vitro Pankreas organogeneziste

The pancreas is an essential organ that regulates glucose homeostasis and secretes digestive enzymes. Research on pancreas embryogenesis has led to the ...

in-vitro-pancreas-organogenesis-from-dispersed-mouse-embryonic

Research

JoVE Journal

Biology

In Vitro Pancreas Organogenesis from Dispersed Mouse Embryonic Progenitors

13.7K Görüntüleme.  Swiss Institute for Experimental Cancer Research.  Bu protokol açıklanan üç boyutlu kültür yöntemi dallı bir organ haline onların önemli genişleme, farklılaşma ve morfojenezinin dahil dağınık embriyonik fare pankreas atalarıdır, gelen pankreas gelişimini özetlediği. Bu yöntem, görüntüleme, niş fonksiyonel müdahale ve manipülasyon için uygun değildir. 

Video: In Vitro Pancreas Organogenesis from Dispersed Mouse Embryonic Progenitors

In vitro Differentiation of Mouse Embryonic Stem (mES) Cells Using the Hanging Drop Method

Stem cells have the remarkable potential to develop into many different cell types. When a stem cell divides, each new cell has the potential to either remain a stem cell or become another type of cell with a more specialized function, This promising of science is leading scientists to investigate the possibility of cell-based therapies to treat disease.

When culture in suspension without antidifferentiation factors, embryonic stem cells spontaneously differentiate and form three-dimensional multicellular aggregates. These cell aggregates are called embryoid bodies(EB). Hanging drop culture is a widely used EB formation induction method. The rounded bottom of hanging drop allows the aggregation of ES cells which can provide mES cells a good environment for forming EBs. The number of ES cells aggregatied in a hanging drop can be controlled by varying the number of cells in the initial cell suspension to be hung as a drop from the lid of Petri dish. Using this method we can reproducibly form homogeneous EBs from a predetermined number of ES cells. 

In vitro Differentiation of Mouse Embryonic Stem mES Cells Using the Hanging Drop Method

This video demonstrates how to conduct in vitro differentiation of mouse embryonic stem cells to embryoid bodies using the hanging drop method.

Asma Bırak Yöntemiyle Fare Embriyonik Kök (MES) Hücreler in vitro Farklılaşma

Stem cells have the remarkable potential to develop into many different cell types. When a stem cell divides, each new cell has the potential to either ...

Biyoloji

Single-cell Suction Recordings from Mouse Cone Photoreceptors

Rod and cone photoreceptors in the retina are responsible for light detection. In darkness, cyclic nucleotide-gated (CNG) channels in the outer segment are open and allow cations to flow steadily inwards across the membrane, depolarizing the cell. Light exposure triggers the closure of the CNG channels, blocks the inward cation current flow, and thus results in cell hyperpolarization. Based on the polarity of photoreceptors, a suction recording method was developed in 1970s that, unlike the classic patch-clamp technique, does not require penetrating the plasma membrane 1. Drawing the outer segment into a tightly-fitting glass pipette filled with extracellular solution allows recording the current changes in individual cells upon test-flash exposure. However, this well-established "outer-segment-in (OS-in)" suction recording is not suitable for mouse cone recordings, because of the low percentage of cones in the mouse retina (3%) and the difficulties in identifying the cone outer segments. Recently, an inner-segment-in (IS-in) recording configuration was developed to draw the inner segment/nuclear region of the photoreceptor into the recording pipette 2,3. In this video, we will show how to record from individual mouse cone photoresponses using single-cell suction electrode.

We will show how to record flash responses from single mouse cones using a suction electrode.

Tek hücreli fare Koni fotoreseptörlerin Emiş Kayıtlar

Rod and cone photoreceptors in the retina are responsible for light detection. In darkness, cyclic nucleotide-gated (CNG) channels in the outer segment ...

In vivo Imaging and Therapeutic Treatments in an Orthotopic Mouse Model of Ovarian Cancer

Human cancer and response to therapy is better represented in orthotopic animal models. This paper describes the development of an orthotopic mouse model of ovarian cancer, treatment of cancer via oral delivery of drugs, and monitoring of tumor cell behavior in response to drug treatment in real time using in vivo imaging system. In this orthotopic model, ovarian tumor cells expressing luciferase are applied topically by injecting them directly into the mouse bursa where each ovary is enclosed. Upon injection of D-luciferin, a substrate of firefly luciferase, luciferase-expressing cells generate bioluminescence signals. This signal is detected by the in vivo imaging system and allows for a non-invasive means of monitoring tumor growth, distribution, and regression in individual animals. Drug administration via oral gavage allows for a maximum dosing volume of 10 mL/kg body weight to be delivered directly to the stomach and closely resembles delivery of drugs in clinical treatments. Therefore, techniques described here, development of an orthotopic mouse model of ovarian cancer, oral delivery of drugs, and in vivo imaging, are useful for better understanding of human ovarian cancer and treatment and will improve targeting this disease.

In vivo Imaging and Therapeutic Treatments in an Orthotopic Mouse Model of Ovarian Cancer

Orthotopic animal models of ovarian cancer replicate better human disease and therefore enhance our understanding of cancer progression and tumor response to therapy. A mouse model receives an intrabursal injection of luciferase-expressing ovarian tumor cells. Treatment is administered via oral gavage. Tumor growth is monitored by in vivo imaging system.

In vivo Görüntüleme ve Yumurtalık Kanseri Ortotopik Fare Modeli Tedavi Tedaviler

Human cancer and response to therapy is better represented in orthotopic animal models.  This paper describes the development of an orthotopic mouse model ...

Purification of Progenitors from Skeletal Muscle

Skeletal muscle contains multiple progenitor populations of distinct embryonic origins and developmental potential. Myogenic progenitors, usually residing in a "satellite cell position" between the myofiber plasma membrane and the laminin-rich basement membrane that ensheaths it, are self-renewing cells that are solely committed to the myogenic lineage1,2. We have recently described a second class of vessel associated progenitors that can generate myofibroblasts and white adipocytes, which responds to damage by efficiently entering proliferation and provides trophic support to myogenic cells during tissue regeneration3,4. One of the most trusted assays to determine the developmental and regenerative potential of a given cell population relies on their isolation and transplantation5-7. To this end we have optimized protocols for their purification by flow cytometry from enzymatically dissociated muscle, which we will outline in this article. The populations obtained using this method will contain either myogenic or fibro/adipogenic colony forming cells: no other cell types are capable of expanding in vitro or surviving in vivo delivery. However, when these populations are used immediately after the sort for molecular analysis (e.g qRT-PCR) one must keep in mind that the freshly sorted subsets may contain other contaminant cells that lack the ability of forming colonies or engrafting recipients.

Method for the enzymatic dissociation, surface labeling and purification by flow cytometry of fibro/adipogenic and myogenic progenitors from murine skeletal muscle.

İskelet Kası progenitörlerin saflaştırılması

Skeletal muscle contains multiple progenitor populations of distinct embryonic origins and developmental potential. Myogenic progenitors, usually residing ...

A System for ex vivo Culturing of Embryonic Pancreas

The pancreas controls vital functions of our body, including the production of digestive enzymes and regulation of blood sugar levels1. Although in the past decade many studies have contributed to a solid foundation for understanding pancreatic organogenesis, important gaps persist in our knowledge of early pancreas formation2. A complete understanding of these early events will provide insight into the development of this organ, but also into incurable diseases that target the pancreas, such as diabetes or pancreatic cancer. Finally, this information will generate a blueprint for developing cell-replacement therapies in the context of diabetes.
 During embryogenesis, the pancreas originates from distinct embryonic outgrowths of the dorsal and ventral foregut endoderm at embryonic day (E) 9.5 in the mouse embryo3,4. Both outgrowths evaginate into the surrounding mesenchyme as solid epithelial buds, which undergo proliferation, branching and differentiation to generate a fully mature organ2,5,6. Recent evidences have suggested that growth and differentiation of pancreatic cell lineages, including the insulin-producing &beta;-cells, depends on proper tissue-architecture, epithelial remodeling and cell positioning within the branching pancreatic epithelium7,8. However, how branching morphogenesis occurs and is coordinated with proliferation and differentiation in the pancreas is largely unknown. This is in part due to the fact that current knowledge about these developmental processes has relied almost exclusively on analysis of fixed specimens, while morphogenetic events are highly dynamic.
 Here, we report a method for dissecting and culturing mouse embryonic pancreatic buds ex vivo on glass bottom dishes, which allow direct visualization of the developing pancreas (Figure 1). This culture system is ideally devised for confocal laser scanning microscopy and, in particular, live-cell imaging. Pancreatic explants can be prepared not only from wild-type mouse embryos, but also from genetically engineered mouse strains (e.g. transgenic or knockout), allowing real-time studies of mutant phenotypes. Moreover, this ex vivo culture system is valuable to study the effects of chemical compounds on pancreatic development, enabling to obtain quantitative data about proliferation and growth, elongation, branching, tubulogenesis and differentiation. In conclusion, the development of an ex vivo pancreatic explant culture method combined with high-resolution imaging provides a strong platform for observing morphogenetic and differentiation events as they occur within the developing mouse embryo.

A System for ex vivo Culturing of Embryonic Pancreas

Here, we describe a method for isolation, culture and manipulation of mouse embryonic pancreas. This represents an excellent ex vivo system for studying various aspects of pancreatic development, including morphogenesis, differentiation and growth. Pancreatic bud explants can be cultured for several days and used in a range of different applications, including whole-mount immunofluorescence and live imaging.

Için bir sistem<em&gt; Ex vivo</emEmbriyonik Pankreas&gt; ekimi

The pancreas controls vital functions of our body,  including the production of digestive enzymes and regulation of blood sugar  levels1. Although in the ...

Generation of Mice Derived from Induced Pluripotent Stem Cells

The production of induced pluripotent stem cells (iPSCs) from somatic cells provides a means to create valuable tools for basic research and may also produce a source of patient-matched cells for regenerative therapies. iPSCs may be generated using multiple protocols and derived from multiple cell sources. Once generated, iPSCs are tested using a variety of assays including immunostaining for pluripotency markers, generation of three germ layers in embryoid bodies and teratomas, comparisons of gene expression with embryonic stem cells (ESCs) and production of chimeric mice with or without germline contribution2. Importantly, iPSC lines that pass these tests still vary in their capacity to produce different differentiated cell types2. This has made it difficult to establish which iPSC derivation protocols, donor cell sources or selection methods are most useful for different applications.
The most stringent test of whether a stem cell line has sufficient developmental potential to generate all tissues required for survival of an organism (termed full pluripotency) is tetraploid embryo complementation (TEC)3-5. Technically, TEC involves electrofusion of two-cell embryos to generate tetraploid (4n) one-cell embryos that can be cultured in vitro to the blastocyst stage6. Diploid (2n) pluripotent stem cells (e.g. ESCs or iPSCs) are then injected into the blastocoel cavity of the tetraploid blastocyst and transferred to a recipient female for gestation (see Figure 1). The tetraploid component of the complemented embryo contributes almost exclusively to the extraembryonic tissues (placenta, yolk sac), whereas the diploid cells constitute the embryo proper, resulting in a fetus derived entirely from the injected stem cell line.
Recently, we reported the derivation of iPSC lines that reproducibly generate adult mice via TEC1. These iPSC lines give rise to viable pups with efficiencies of 5-13%, which is comparable to ESCs3,4,7 and higher than that reported for most other iPSC lines8-12. These reports show that direct reprogramming can produce fully pluripotent iPSCs that match ESCs in their developmental potential and efficiency of generating pups in TEC tests. At present, it is not clear what distinguishes between fully pluripotent iPSCs and less potent lines13-15. Nor is it clear which reprogramming methods will produce these lines with the highest efficiency. Here we describe one method that produces fully pluripotent iPSCs and "all- iPSC" mice, which may be helpful for investigators wishing to compare the pluripotency of iPSC lines or establish the equivalence of different reprogramming methods.

Generating induced pluripotent stem cell (iPSC) lines produces lines of differing developmental potential even when they pass standard tests for pluripotency. Here we describe a protocol to produce mice derived entirely from iPSCs, which defines the iPSC lines as possessing full pluripotency1.

İsteyerek Pluripotent Kök Hücrelerinin Elde Edilen Fare Üretimi

The production of induced pluripotent stem cells (iPSCs) from somatic cells provides a means to create valuable tools for basic research and may also ...

Cell Labeling and Injection in Developing Embryonic Mouse Hearts

Testing the fate of embryonic or pluripotent stem cell-derivatives in in vitro protocols has led to controversial outcomes that do not necessarily reflect their in vivo potential. Preferably, these cells should be placed in a proper embryonic environment in order to acquire their definite phenotype. Furthermore, cell lineage tracing studies in the mouse after labeling cells with dyes or retroviral vectors has remained mostly limited to early stage mouse embryos with still poorly developed organs. To overcome these limitations, we designed standard and ultrasound-mediated microinjection protocols to inject various agents in targeted regions of the heart in mouse embryos at E9.5 and later stages of development. &#160;Embryonic explant or embryos are then cultured or left to further develop in utero. These agents include fluorescent dyes, virus, shRNAs, or stem cell-derived progenitor cells. Our approaches allow for preservation of the function of the organ while monitoring migration and fate of labeled and/or injected cells. These technologies can be extended to other organs and will be very helpful to address key biological questions in biology of development.

We describe a series of methods to inject dyes, DNA vectors, virus, and cells in order to monitor both cell fate and phenotype of endogenous and grafted cells derived from embryonic or pluripotent cells within mouse embryos at embryonic day (E)9.5 and later stages of development.

Embriyonik Fare Kalpler Gelişmekte Hücre Etiketleme ve Enjeksiyon

Testing the fate of embryonic or pluripotent stem cell-derivatives in in vitro protocols has led to controversial outcomes that do not necessarily reflect ...

RNA Isolation from Mouse Pancreas: A Ribonuclease-rich Tissue

Isolation of high-quality RNA from ribonuclease-rich tissue such as mouse pancreas presents a challenge. As a primary function of the pancreas is to aid in digestion, mouse pancreas may contain as much a 75 mg of ribonuclease. We report modifications of standard phenol/guanidine thiocyanate lysis reagent protocols to isolate RNA from mouse pancreas. Guanidine thiocyanate is a strong protein denaturant and will effectively disrupt the activity of ribonuclease under most conditions. However, critical modifications to standard protocols are necessary to successfully isolate RNA from ribonuclease-rich tissues. Key steps include a high lysis reagent to tissue ratio, removal of undigested tissue prior to phase separation and inclusion of a ribonuclease inhibitor to the RNA solution. Using these and other modifications, we routinely isolate RNA with RNA Integrity Number (RIN) greater than 7. The isolated RNA is of suitable quality for routine gene expression analysis. Adaptation of this protocol to isolate RNA from ribonuclease rich tissues besides the pancreas should be readily achievable.

We report a procedure to isolate RNA with high integrity from the ribonuclease rich mouse pancreas.

Fare pankreasından alınan RNA İzolasyonu: A Ribonuclease zengin Doku

Isolation of high-quality RNA from ribonuclease-rich tissue such as mouse pancreas presents a challenge.  As a primary function of the pancreas is to aid ...

Protein Isolation from the Developing Embryonic Mouse Heart Valve Region

Western blot analysis is a commonly employed technique for detecting and quantifying protein levels. However, for small tissue samples, this analysis method may not be sufficiently sensitive to detect a protein of interest. To overcome these difficulties, we examined protocols for obtaining protein from adult human cardiac valves and modified these protocols for the developing early embryonic mouse counterparts. In brief, the mouse embryonic aortic valve regions, including the aortic valve and surrounding aortic wall, are collected in the minimal possible volume of a Tris-based lysis buffer with protease inhibitors. If required based on the breeding strategy, embryos are genotyped prior to pooling four embryonic aortic valve regions for homogenization. After homogenization, an SDS-based sample buffer is used to denature the sample for running on an SDS-PAGE gel and subsequent western blot analysis. Although the protein concentration remains too low to quantify using spectrophotometric protein quantification assays and have sample remaining for subsequent analyses, this technique can be used to successfully detect and semi-quantify phosphorylated proteins via western blot from pooled samples of four embryonic day 13.5 mouse aortic valve regions, each of which yields approximately 1 &#956;g of protein. This technique will be of benefit for studying cell signaling pathway activation and protein expression levels during early embryonic mouse valve development.

The analysis of protein expression in young embryonic mouse valves has been hampered by the limited tissue available. This manuscript provides a protocol for preparing protein from developing embryonic mouse valve regions for western blot analysis.

Gelişmekte Embriyonik Fare Kalp Vana Bölgesi&#39;nde Protein İzolasyonu

Western blot analysis is a commonly employed technique for detecting and quantifying protein levels. However, for small tissue samples, this analysis ...

Procedure to Evaluate the Efficiency of Flocculants for the Removal of Dispersed Particles from Plant Extracts

Plants are important to humans not only because they provide commodities such as food, feed and raw materials, but increasingly because they can be used as manufacturing platforms for added-value products such as biopharmaceuticals. In both cases, liquid plant extracts may need to be clarified to remove particulates. Optimal clarification reduces the costs of filtration and centrifugation by increasing capacity and longevity. This can be achieved by introducing charged polymers known as flocculants, which cross-link dispersed particles to facilitate solid-liquid separation. There are no mechanistic flocculation models for complex mixtures such as plant extracts so empirical models are used instead. Here a design-of-experiments procedure is described that allows the rapid screening of different flocculants, optimizing the clarification of plant extracts and significantly reducing turbidity. The resulting predictive models allow the identification of robust process conditions and sets of polymers with complementary properties, e.g. effective flocculation in extracts with specific conductivities. The results presented for tobacco leaf extracts can easily be adapted to other plant species or tissues and will thus facilitate the development of more cost-effective downstream processes for commodities and plant-derived pharmaceuticals.

The design-of-experiments procedure presented here allows the evaluation of different flocculants in terms of their ability to aggregate dispersed particles in plant extracts, thus reducing turbidity and the costs of downstream processing.

Prosedür bitki özleri gelen Dağınık Parçacıkların çıkarması için Flokülantlar Verimliliği değerlendirin

Plants are important to humans not only because they provide commodities such as food, feed and raw materials, but increasingly because they can be used ...