Bu çalışmada hibe Karolinska Enstitüsü, görme engelliler, Edwin Jordan Vakfı Oftamolojik araştırma, İsveçli göz Vakfı, Kral Gustav V Foundation, ARMEC için Crown Princess Margareta'nın Vakfı tarafından desteklenmiştir Lindeberg Vakfı ve Cronqvist Vakfı.

Aşağıdaki iletişim kuralı Karolinska Instituet hayvan bakımı kuralları izler. Yeni Zelanda albino tavşan (Tablo reçetesi) kullanarak tüm hayvan deneyleri bölgesel hayvan Etik Komitesi (Stockholms Norra Djurförsöksetiska Nämnd) tarafından onaylanmış olan (izin: dnr 56/15). HESC (dnr 2011/745-31/3) kullanımı ve transfer ve hESC-RPE (dnr 2013/813-31/2) manipülasyon da İsveçli mevzuat ve Karolinska Institutet düzenlemelere uygun şekilde ve bölgesel insan Etik Komitesi (tarafından onaylandı Regionala Etikprövningsnämnden ben Stockholm).
1. sodyum iyodat (NaIO3) subretinal enjeksiyon büyük gözlü hayvan modeli içine
<ol><li>35 mg/kg ketamin karışımı ile uyluk kas içi yönetiminde ve tuz 30 G şırınga kullanarak 5 mg/kg xylazine hayvanları anestezi ve öğrenciler topikal göz damlası % 0.75 cyclopentolate ve % 2.5 phenylephrine bir karışımını kullanarak genişletmek. Uygun anesthetization doğruladı Eğer hayvan arka bacağını zor bir tutam tepki vermez.</li>
	<li>Cerrahi mikroskop altında tavşan ile baş cerrah (şekil 1A) karşı karşıya koyun. Bir kapak Retraktörü göz kapakları ve nictitans membran bulaşma riskini en aza indirmek için bir steril bezle kaldırmak için kullanın. Kullanımı dengeli tuz solüsyonu (BSS) altında anestezi sırasında her iki gözde kuruluk önlemek için.</li>
	<li>
Mikrocerrahi, 2 bağlantı noktasını kullan (veya isteğe bağlı 3-bağlantı noktası) için 25 G transvitreal pars plana tekniği ile mikrocerrahi aletleri (şekil 1B) ekleme için Vanalı sigara trocars. Çok işlevli vitrektomi makine bir infüzyon kanül, endoillumination, vitrector ve endolaser bağlanmak için bağlantı noktası yok. Subretinal enjeksiyonlar için sadece endoillumination 2-liman set-up yeterli kılan, zorunludur. Endoillumation üst sol trokar yerleştirebilir ve üst sağ trokar subretinal enjeksiyon kanül için kullanabilirsiniz.
	<ol>
<li>3-port kurulum için daha düşük zamansal trokar BSS infüzyon kanül için kullanın. (Örneğin vitrector) isteğe bağlı aygıtlar olup olmadığını kullanılan, bunları üst sağ trokar ekleyin.</li>
		<li>Üst 2 trocars 1-2 mm kapmak ve ekleme sitesi örten konjonktiva yerinden pençeli forseps kullanarak limbus ekleyin.</li>
		<li>Trocars eklenen transsclerally 30-45 ° limbus paralel açı ve trokar ucunu ortasına kadar devam emin olun. Sonra 90 ° trokar dönüp gözün arka kutbunda amaçlayan göz içine ilerlemek. Bu yordam sclerotomies üzerinden ameliyat sonrası kaçağı önlemek ve aynı zamanda endoftalmi (Yani, bakteriyel enfeksiyon göz) riskini azaltmak. Ayrıca şekil 2B trokar pozisyonlar için bkz.</li>
		<li>Tek kişi kullanımlı düz kontak lens kornea göz ve Kontakt lens (şekil 1A) arasında iletişim jel olarak sentetik gözyaşları ile retina görselleştirmek için koymak.</li>
		<li>Bir uzatma tüpü (yardımcı tarafından işletilen) bağlı bir 1 mL şırınga ve bir 38 G polytip kanül (cerrah tarafından işletilen) içine NaIO3 500 µL çizin.</li>
		<li>Endoillumination sonda aracılığıyla üst sol trokar ve enjeksiyon kanül aracılığıyla üst sağ trokar yerleştirin ve optik sinir başı altındaki alanda yönelik retina doğru vitreus uzayda kanül ilerlemek.</li>
		<li>Odak bir beyazlatma görünene kadar yavaş yavaş retina dokunmak kanül ucu izin. Enjeksiyon kendisi subretinal teslimat için izin retina, nüfuz. Bu kanama neden olabilir beri retina, nüfuz kanül izin vermeyin.</li>
		<li>50 µL NaIO3 subretinally 5 s dönemde enjekte et. Doğal göğüs arası uçak retina ve alttaki koroid arasında olduğundan, açıkça görülebilen bir yarı saydam bleb enjeksiyon sırasında yavaş yavaş oluşturmalıdır.</li>
		<li>Enjeksiyon sırasında yavaş yavaş iğne geri çek, ama belgili tanımlık uç reflü en aza indirmek için bleb içinde korunur emin olun.</li>
		<li>Sonra endoillumation ve enjeksiyon kanül kaldırılması, pençeli forseps kullanarak trocars kaldırın ve 30 için hafif basınç uygulamak için belgili tanımlık uç veya künt sonuna forseps kullanarak kendinden contalı dikiş daha az sclerotomies s.</li>
	</ol>
</li>
	<li>Post-surgically 10 mL subkutan su kaybı önlemek için tuz verin. Ameliyat sonrası topikal steroid veya antibiyotik vermek değil. Ağrı kesici için ameliyattan sonra 0.5 mL buprenorfin 0.3 mg/ml subkutan sonra cerrahi, hem de gün ver.</li>
	<li>Onları birkaç saniye için öncelikle % 70 etanol, ikincisi % 45 etanol içinde çeker tarafından tüm aletleri kullanımdan sonra yıkama ve son olarak içinde distile su. Onları düzgün bir kağıt havlu ile kurulayın.</li>
	<li>Onlar yeterli kendine gelmek ve onları tek tek encaged yere kadar hayvan katılmak. (Örneğin, immünhistokimya amaçlar için) gerekirse, hayvanlar 100 mg/kg Fentobarbital intravenöz enjeksiyon tarafından ötenazi ( Tablo malzemelerigörmek).</li>
	<li>HESC-RPE hücrelerinin nakli ile devam etmek için 7 gün bekleyin.</li>
</ol>2. subretinal nakli hayvanlarda tedavi hESC-RPE hücrelerinin
<ol><li>1-2 mm den daha düşük zamansal kadranda limbus hESC-RPE nakli önce 1 hafta eklenen 30 g enjeksiyon iğne kullanarak imzalat hayvanlarda intravitreal triamsinolon, 2 mg (100 µL) yönetmek ve 3 ayda yeniden yönetmek. İpucu bir objektif dokunmatik önlemek için göz arka direğe doğru işaret etmek emin olun.</li>
	<li>1daha önce açıklandığı gibi bir hESC-RPE monolayer kültür.</li>
	<li>24-şey Konfluent hESC-RPE monolayer hücre farklılaşması medya (bkz. Tablo reçetesi) kaldırın ve her yıkama PBS 500 µL Ca2 + ve Mg2 +olmadan ile iyi. Bir kez daha, bu eylem 2 yıkama toplam için yineleyin.</li>
	<li>Süpernatant atmak, tripsin iyi başına 500 µL eklemek ve 37 ° C'de 12 dk için kuluçkaya</li>
	<li>Plaka eğilme ve tripsin (hücreleri plakasına bağlı kalmalıdır) dikkatli bir şekilde çıkarın. Taze 37 ° C prewarmed farklılaşma medya 800 µL hücrelerde nazik pipetting veya hatta bir tek hücre süspansiyon elde etmek için gerekirse, kazıma toplamak. Not: Hücre kümeleri gözlenen bir 40 µm hücre süzgeç kullanın.</li>
	<li>% 0,2 kullanarak hemasitometre odasında hücreleri hesaplama Trypan mavi, üreticinin yönergelerine göre.</li>
	<li>Farklılaşma medya ve santrifüj hücreleri, oda sıcaklığında, 300 x g 5 min için 5 mL ekleyin.</li>
	<li>Süpernatant atın ve Pelet taze filtre sterilize PBS 1000 hücre/µL son bir konsantrasyon (bir 25 mm şırınga filtreden geçirilen) içinde resuspend.</li>
	<li>Aliquot önceki hücre süspansiyon 600 µL aliquots ve buz kadar ve ameliyat sırasında üzerinde tutun.</li>
	<li>Tuz 30 G şırınga kullanarak 35 mg/kg ketamin ve 5 mg/kg xylazine karışımı ile uyluk kas içi yönetim tarafından hayvanların anestezi. Öğrenciler topikal göz damlası % 0.75 cyclopentolate ve % 2.5 phenylephrine amix kullanarak genişletmek. Uygun anesthetization doğruladı Eğer hayvan arka bacağını zor bir tutam tepki vermez.</li>
	<li>Tavşan ile baş cerrah bakacak şekilde yerleştirin. Bir kapak Retraktörü göz kapakları ve nictitans membran bulaşma riskini en aza indirmek için bir steril bezle kaldırmak için kullanın. BSS kuruluk anestezi sırasında her iki gözde altında engellemek için kullanın.</li>
	<li>
Mikrocerrahi, 2 bağlantı noktasını kullan (veya isteğe bağlı 3-bağlantı noktası) için 25 G transvitreal pars plana tekniği ile trocars adım 1.3 ve Şekil 2' de açıklandığı gibi aynı pozisyonda yerleştirilir. Önceki sclerotomies görünür durumdaysa, trokar ekleme sadece bitişik gerçekleştirmek ancak ameliyat sonrası kaçak riskini en aza indirmek için bunlar aracılığıyla.
	<ol>
<li>Tek kişi kullanımlı düz kontak lens kornea göz ve Kontakt lens (şekil 1A) arasında iletişim jel olarak sentetik gözyaşları ile retina görselleştirmek için koymak.</li>
		<li>(Bkz: adım 1.3.5 ve 1.3.6) konumlandırma uygun ucu sonra hafifçe karışık hESC-RPE süspansiyon (50.000 hücreleri), 50 µL subretinally enjekte. Karakteristik "metal" endo-aydınlatma refleks tarafından seçkin NaIO ön işleme3 alanının için amaç. Neurosensory retina kolayca açıkça görünür bleb oluşturma ayrı olmalıdır. Tavşan/sonra steril H2O arada iğneyle kullanın iğne nedeniyle hücre tıkanmasını önlemek için floş kümeler. Değişikliği ne zaman tıkanma iğneyi fark ettim.</li>
		<li>Enjeksiyon sırasında yavaş iğne geri çek ama ucu reflü en aza indirmek için bleb içinde korunur emin olun.</li>
		<li>Sonra endoillumation ve enjeksiyon kanül kaldırılması, pençeli forseps kullanarak trocars kaldırın ve 30 için hafif basınç uygulamak için belgili tanımlık uç veya künt sonuna forseps kullanarak kendinden contalı dikiş daha az sclerotomies s.</li>
	</ol>
</li>
	<li>Post-surgically 10 mL subkutan su kaybı önlemek için tuz verin. Ameliyat sonrası topikal steroid veya antibiyotik vermek değil. Ağrı kesici için ameliyattan sonra 0.5 mL buprenorfin 0.3 mg/ml subkutan sonra cerrahi, hem de gün ver.</li>
	<li>Onları birkaç saniye, öncelikle % 70 etanol, ikincisi % 45 etanol içinde çeker tarafından tüm aletleri kullanımdan sonra yıkama ve son olarak içinde distile su. Onları düzgün bir kağıt havlu ile kurulayın.</li>
	<li>Onlar yeterli kendine gelmek ve onları tek tek encaged yere kadar hayvan katılmak. (Örneğin immünhistokimya amaçlar için), gerekirse 100 mg/kg Fentobarbital intravenöz enjeksiyon tarafından hayvanlara ötenazi.</li>
</ol>3. in Vivo retina ve Subretinal görüntüleme
<ol><li>
Spektral etki alanı optik Koherens tomografi (SD-Ekim) aygıt ile birlikte verilen yazılımı kullanın (kesitsel b-taranmasını üreticinin yönergelerine uygun tedavi hayvanlar elde etmek için malzemeler tablo bkz:). Görüntü bulanık önlemek için her 30-60 s topikal serum temizlenerek kornea nemli tutmak emin olun.
	<ol>
<li>Engelsiz bir yol enstrüman ışık kaynağından tavşan retina elde etmek için ayarlanabilir bir Mount (bkz. Adım 1.1 ve 2.10) imzalat ve öğrenci büyümüş hayvanlar koyun.</li>
		<li>En az 3 OCT taramaları ile eşzamanlı kızılötesi confocal tarama lazer ophthalmoscopy enjekte alanının üst, orta ve alt kısmını temsil eden (IR-cSLO) yansıma referans görüntü elde.</li>
		<li>Tr-yüz cSLO mavi, yeşil, kızılötesi ve çok renkli yansıma (Yani, birden çok eşzamanlı lazer renk), sırasıyla lazer ile fundus görüntü elde.</li>
		<li>Mavi ışık autofluorescence (BAF) SD-Ekim cihazın mavi ışık lazer özelliği kullanarak çekim.</li>
	</ol>
</li>
</ol>

<ol>
	<li>Plaza Reyes, 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=Xeno-Free+and+Defined+Human+Embryonic+Stem+Cell-Derived+Retinal+Pigment+Epithelial+Cells+Functionally+Integrate+in+a+Large-Eyed+Preclinical+Model.">Xeno-Free and Defined Human Embryonic Stem Cell-Derived Retinal Pigment Epithelial Cells Functionally Integrate in a Large-Eyed Preclinical Model.</a> Stem Cell Rep. 6 (1), 9-17 (2016).</li><li>Bartuma, H., 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=In+Vivo+Imaging+of+Subretinal+Bleb-Induced+Outer+Retinal+Degeneration+in+the+Rabbit.">In Vivo Imaging of Subretinal Bleb-Induced Outer Retinal Degeneration in the Rabbit.</a> Invest Ophthalmol Vis Sci. 56 (4), 2423-2430 (2015).</li><li>Petrus-Reurer, S., 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=Integration+of+Subretinal+Suspension+Transplants+of+Human+Embryonic+Stem+Cell-Derived+Retinal+Pigment+Epithelial+Cells+in+a+Large-Eyed+Model+of+Geographic+Atrophy.">Integration of Subretinal Suspension Transplants of Human Embryonic Stem Cell-Derived Retinal Pigment Epithelial Cells in a Large-Eyed Model of Geographic Atrophy.</a> Invest Ophthalmol Vis Sci. 58 (2), 1314-1322 (2017).</li><li>Carido, M., 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=Characterization+of+a+mouse+model+with+complete+RPE+loss+and+its+use+for+RPE+cell+transplantation.">Characterization of a mouse model with complete RPE loss and its use for RPE cell transplantation.</a> Invest Ophthalmol Vis Sci. 55 (8), 5431-5444 (2014).</li><li>Lund, R. D., 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=Human+embryonic+stem+cell-derived+cells+rescue+visual+function+in+dystrophic+RCS+rats.">Human embryonic stem cell-derived cells rescue visual function in dystrophic RCS rats.</a> Cloning Stem Cells. 8 (3), 189-199 (2006).</li><li>Vugler, 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=Elucidating+the+phenomenon+of+HESC-derived+RPE:+anatomy+of+cell+genesis,+expansion+and+retinal+transplantation.">Elucidating the phenomenon of HESC-derived RPE: anatomy of cell genesis, expansion and retinal transplantation.</a> Exp Neurol. 214 (2), 347-361 (2008).</li><li>Schwartz, S. D., 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=Embryonic+stem+cell+trials+for+macular+degeneration:+a+preliminary+report.">Embryonic stem cell trials for macular degeneration: a preliminary report.</a> Lancet. 379 (9817), 713-720 (2012).</li><li>Hughes, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+schematic+eye+for+the+rabbit.">A schematic eye for the rabbit.</a> Vision Res. 12 (1), 123-138 (1972).</li><li>Blanch, R. J., Ahmed, Z., Berry, M., Scott, R. A., Logan, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Animal+models+of+retinal+injury.">Animal models of retinal injury.</a> Invest Ophthalmol Vis Sci. 53 (6), 2913-2920 (2012).</li><li>Nork, T. M., 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=Functional+and+anatomic+consequences+of+subretinal+dosing+in+the+cynomolgus+macaque.">Functional and anatomic consequences of subretinal dosing in the cynomolgus macaque.</a> Arch Ophthalmol. 130 (1), 65-75 (2012).</li></ol>

Yazarlar rakip çıkarları ya da çakışan ilgi alanları yok.

Bu protokol için hESC-RPE Tümleştirme vivo içinde değerlendirmek için büyük gözlü manken GA ve preklinik kullanımı nesil açıklanmıştır.
Rejeneratif terapileri için çeviri GA ve ilgili hastalıklar için klinik7içine, geliştirmek ve sadakatle nakli ve görüntüleme için klinik yöntemleri yakalama yöntemleri optimize etmek önemlidir. Tavşan bu açıdan çekici olduğunu: göz içi cerrahi ve kullanımını standart görüntüleme izni n...

Bu iletişim kuralı bir transplante hESC-RPE subretinal uzayda entegrasyonunu değerlendirilmesi sağlar coğrafi Atrofi (GA) büyük gözlü preklinik model nesil açıklar. HESC1, yanı sıra dış retina hasar ve GA benzeri fenotip RPE hücrelerinin zenginleştirilmiş, saf ve fonksiyonel bir nüfusun üretim göstermek 3 son yayınlarda ayrıntılı olarak açıklanan yöntemleri kullanılmıştır fizyolojik tuzlu Çözümleri (Yani, BSS ve PBS) veya tavşan göz2,3NaIO3 subretinal enjeksiyonla indüklenen. Biz daha fazla alt retinal süspansiyon nakli hESC-RPE, kapsamlı işlevsel monolayers photoreceptor kurtarma kapasitesi2formu gösterdi.
Çeşitli yararları GA manken hastalığı nesil tavşan göz kullanımına eşlik. İlk olarak, boyutu tavşan göz hangi yetişkin bir insan gözü hacmi % 70 's, klinik olarak anlamlı nakli rutin olarak küçük kemirgen gözler (1000 hücre/µL vs 50.000 hücre/µL)4 ' te kullanılan çok daha bir hücre yoğunluğu kullanarak verir , 5. ikinci olarak, kemirgenler genellikle transscleral retina bariyeri ödün vermez ve potansiyel bir enflamatuar yanıt ve mümkün ret6tetikler koroid aracılığıyla ameliyatta. İki faktör birlikte multilayering ve Transplante hücrelerinin ve kesintiye yerli retina mendil transplante hücrelerde genel zayıf bir entegrasyon topaklanma neden olabilir. Ancak, büyük gözlü tavşan modeli bir cerrahi teknik klinik bir ayarı için aynı araçları ile performans sağlar. Üçüncü olarak, büyük gözlü manken de görüntüleme ve Transplante hücrelerin ve üstteki retina ile zaman1,2,3izleme yüksek çözünürlüklü vivo içinde verir. Böylece, kemirgenler normal ve hastalıklı retina ve alt retinal uzay araştırma ilgi olan herkes için cazip bir alternatif olması gereken bir klinik olarak ilgili ve maliyet-etkin preklinik modeli açıklar.

<table >
	<tbody>
		<tr >
			<td >NutriStem hESC XF differentiation medium &#8211;bFGF and &#8211;TGFb</td>
			<td >Biological Industries</td>
			<td >06-5100-01-1A</td>
			<td ></td>
		</tr>
		<tr >
			<td >TrypLE Select 1x</td>
			<td >Gibco, ThermoFisher Scientific Corp</td>
			<td >12563-011</td>
			<td ></td>
		</tr>
		<tr >
			<td >PBS without Ca2+ and Mg2+</td>
			<td >Gibco, ThermoFisher Scientific Corp</td>
			<td >14190-094</td>
			<td></td>
		</tr>
		<tr >
			<td >Cell strainer 40 &#956;m Nylon</td>
			<td >VWR</td>
			<td >732-2757</td>
			<td ></td>
		</tr>
		<tr >
			<td >Needle 30g 0.5&#8217;&#8217;; 0.3 x 13mm</td>
			<td >BD Microlance</td>
			<td >304827</td>
			<td ></td>
		</tr>
		<tr >
			<td >Acrodisc 25mm Syringe Filter</td>
			<td >Acrodisc</td>
			<td >PN4612</td>
			<td></td>
		</tr>
		<tr >
			<td >0.4 % trypan blue</td>
			<td >ThermoFisher Scientific Corp</td>
			<td >15250061</td>
			<td >Use at 0.2%</td>
		</tr>
		<tr >
			<td >NaIO3</td>
			<td >Sigma-Aldrich Corp</td>
			<td>S4007</td>
			<td ></td>
		</tr>
		<tr >
			<td >BSS</td>
			<td >Alcon Nordic A/S</td>
			<td >65079550</td>
			<td ></td>
		</tr>
		<tr >
			<td >70% Ethanol</td>
			<td >Solveco AB</td>
			<td>1047</td>
			<td></td>
		</tr>
		<tr >
			<td >Ketaminol, 100 mg/mL</td>
			<td >Intervet, Boxmeer</td>
			<td >511519</td>
			<td >Use 35 mg/kg ketamine</td>
		</tr>
		<tr >
			<td >Rompun vet, 20 mg/mL</td>
			<td >Bayer Animal Health</td>
			<td >22545</td>
			<td >Use 5 mg/kg xylazine</td>
		</tr>
		<tr >
			<td >Triescence, 40 mg/mL</td>
			<td >Alcon Nordic A/S</td>
			<td >412915</td>
			<td >2 mg intraviterial</td>
		</tr>
		<tr >
			<td >Cyklopentolat-phenylephrine, 0.75% + 2.5%</td>
			<td >APL</td>
			<td >321968</td>
			<td >Use 1 drop in each eye</td>
		</tr>
		<tr >
			<td >Viscotears</td>
			<td>Laboratoires Th&#233;a</td>
			<td >597562</td>
			<td ></td>
		</tr>
		<tr >
			<td >Topical saline</td>
			<td >Apotea AB</td>
			<td>7053249369080</td>
			<td></td>
		</tr>
		<tr >
			<td >Allfatal vet. 100 mg/mL</td>
			<td >Omnidea</td>
			<td>77168</td>
			<td >Use 100 mg/mL pentobarbital</td>
		</tr>
		<tr >
			<td >Extension tube (Hammer)</td>
			<td >MedOne Surgical Inc</td>
			<td >3223</td>
			<td ></td>
		</tr>
		<tr >
			<td >25G/38G polytip subretinal cannula</td>
			<td >MedOne Surgical Inc</td>
			<td >3219</td>
			<td >25G/38G</td>
		</tr>
		<tr >
			<td >Single Use Flat Lens</td>
			<td >Volk</td>
			<td >#VWFD10</td>
			<td ></td>
		</tr>
		<tr >
			<td >Barraquer Colibri lid retractor</td>
			<td >AgnTho&#39;s AB</td>
			<td >42-020-030</td>
			<td></td>
		</tr>
		<tr >
			<td >Non-valved trocars</td>
			<td >Alcon Nordic A/S</td>
			<td>8065751448</td>
			<td></td>
		</tr>
		<tr >
			<td >Clawed forceps</td>
			<td >Bausch &#38; Lomb Nordic AB</td>
			<td >ET1811</td>
			<td></td>
		</tr>
		<tr >
			<td >Alcon Accurus 400VS Vitrectomy machine</td>
			<td >Alcon Nordic A/S</td>
			<td >8065740238</td>
			<td ></td>
		</tr>
		<tr >
			<td >Accurus 25+ Gauge Vitrectomy TotalL Plus Pak</td>
			<td >Alcon Nordic A/S</td>
			<td >8065751493</td>
			<td ></td>
		</tr>
		<tr >
			<td >SD-OCT device</td>
			<td>Heidelberg Engineering</td>
			<td>Spectralis HRA+OCT</td>
			<td>Use Heidelberg Eye Explorer version 1.9.10.0</td>
		</tr>
		<tr >
			<td >24 well plates</td>
			<td >Sarstedt</td>
			<td >83.3922</td>
			<td></td>
		</tr>
		<tr >
			<td >Neubauer hemocytometer</td>
			<td >VWR</td>
			<td >631-0925</td>
			<td></td>
		</tr>
		<tr >
			<td >New Zealand albino rabbits</td>
			<td>Lidk&#246;pings Rabbit Farm, Sweden</td>
			<td ></td>
			<td></td>
		</tr>
		<tr >
			<td >hESC-RPE cells</td>
			<td ></td>
			<td ></td>
			<td>See reference number 1</td>
		</tr>
	</tbody>
</table>

subretinal transplantation of human embryonic stem cell derived-retinal pigment epithelial cells into a large-eyed model of geographic atrophy

Coğrafi Atrofi (GA), Kuru yaşa bağlı makula dejenerasyonu geç aşamasında kaybı hayati retina yapıları (örneğin, photoreceptors) sonraki dejenerasyonu uzaklaşan retina pigment epitel (RPE) katmanın, karakterize neden şiddetli bozukluğu. Benzer şekilde, RPE kaybı ve görme keskinliği azalma görülür uzun süreli takip kadar intravitreal Anti-vasküler endotelyal büyüme faktörü (VEGF) tedavi gören hastaların gelişmiş ıslak yaşa bağlı makula dejenerasyonu (AMD) ile. Bu nedenle, bir yandan, bu verimli bir şekilde RPE hücrelerinin replasman tedavisi hizmet verebilir bir sınırsız kaynak türetmek için esastır. Öte yandan, davranış ve entegrasyon-in hastalık gerektiren bir modeli türetilmiş hücrelerde cerrahi değerlendirmek önemlidir ve görüntüleme yöntemleri olarak bu insanlarda uygulanan mümkün olduğunca yakın. İnsan embriyonik kök hücre değerlendirilmesi retina pigment epitel hücreleri (hESC-RPE) türetilmiş için burada, GA preklinik bir model nesil açıklayan önceki yayınlarımızı dayalı ayrıntılı bir protokol albino tavşan göz kullanarak içinde sağlamak bir Klinik ayarı. Farklılaşmış hESC-RPE nakledilen saf gözler veya gözleri NaIO3-GA gibi retina dejenerasyonu 25 G transvitreal pars plana tekniği kullanarak indüklenen. Değerlendirme dejenere ve Transplante bölgelerinden multimodal yüksek çözünürlüklü non-invaziv gerçek zamanlı görüntüleme tarafından gerçekleştirilir.

BAF, IR-cSLO ve SD-Ekim normal albino tavşan retinanın temsilcisi vivo içinde görüntülerini Şekil 2' de gösterilmiştir. SD-Ekim enstrüman tarafından yakalanan ışık yansıması farklı düzeylerinin farklı retina katmanlarla unutmayın.
Şekil 1A ve şekil 1Balt retinal blebs oluşturmak için kurulum resimli: 3 troca...

Watch this Scientific Journal Video about Subretinal Transplantation of Human Embryonic Stem Cell Derived-retinal Pigment Epithelial Cells into a Large-eyed Model of Geographic Atrophy at JoVE.com

Subretinal Transplantation of Human Embryonic Stem Cell Derived-retinal Pigment Epithelial Cells into a Large-eyed Model of Geographic Atrophy

Büyük gözlü manken coğrafi Atrofi, içine insan embriyonik kök hücre elde edilen retina Pigment epitel hücrelerinin subretinal nakli

Retina pigment epitel hücreleri hücre-replasman tedavisi için Kuru yaşa bağlı makula dejenerasyonu gelişmiş form olarak hizmet verebilir. Bu iletişim kuralı coğrafi Atrofi büyük gözlü modeli ve hastalık bu modeline insan embriyonik kök hücre kaynaklı retina pigment epitel hücrelerinin nakli subretinal nesil açıklar.

Geographic atrophy (GA), the late stage of dry age-related macular degeneration is characterized by loss of the retinal pigment epithelial (RPE) layer, ...

subretinal-transplantation-human-embryonic-stem-cell-derived-retinal

Research

JoVE Journal

Biology

9.9K Görüntüleme.  Karolinska Institutet. Retina pigment epitel hücreleri hücre-replasman tedavisi için Kuru yaşa bağlı makula dejenerasyonu gelişmiş form olarak hizmet verebilir. Bu iletişim kuralı coğrafi Atrofi büyük gözlü modeli ve hastalık bu modeline insan embriyonik kök hücre kaynaklı retina pigment epitel hücrelerinin nakli subretinal nesil açıklar.

Video: Subretinal Transplantation of Human Embryonic Stem Cell Derived-retinal Pigment Epithelial Cells into a Large-eyed Model of Geographic Atrophy

Propagation of Human Embryonic Stem (ES) Cells

Propagation of Human Embryonic Stem ES Cells

Yeah, so I'm Lauren Deon. I'm in charge of the Human Amgen stem cell co facility at MGH. Yeah, so I'm Lauren, I'm in charge of the Human AM stem cell co facility at MGH.So I've been working with human Amgen stem cells for years now since I was a postdoc in George De Lab before, from four, four years. And so right now we have two cell lines, H one and H nine, which are both from Y cell. And so the way we are expanding or maintaining these cells in culture is by, at least the way we are splitting these cells is by using collagenous.So what I usually do is for the first few passage after throwing the cells, I use a technical, the picking colonies. So in this case, I can check under the microscope which colonies are good and differentiate and nice shape, and I will collect only these colonies and I will cut them in few pieces and put them back in, in a fresh flat. And the, the colonies that look kind of differentiate it are just left them alone.The other way to pass the cells is by using trypsin. So I don't use this technique here. I think collagenase is a little bit safer because human being stem cells don't like to be individualized or they don't like to be alone.So if you do trypsin, if you use trypsin for a little bit too long, if you use it for like three to four minutes, which is the way we use strips in for other type of cells, human stem cells will be individualized and most of them will die or just differentiate or will not attach to the myth. So the idea is you have to keep the cells as clumps. You don't want them to be alone in the in, in the flask.So using collagenase, the advantage is to keep cells as plants. Using chipsy, you just select for cells that will survive this very, very harsh process of g opsonization. So these cells are usually very highly proliferative.So you get, you can adapt your cells to this tripsin process. The problem is that you have cells that they behave differently, they go much faster. And one risk is that you can get some commercial abnormality to, to this, to this very high division process.So you say that ization can cause selection of cells in culture for most proliferative cells.Right. More ative and probably also higher survival rates than other cells. So after proliferation, basically culture after proliferation in tripsin, after several passages become a different culture, these different properties.So it's, it's hard to figure out if they are exactly identical. If you do the test, the usual test, let's say you check markers, regular markers of end differentiated cells like OC four, nano one 60 SC four C, C3, all this molecule might still be expressed. If you do TER formation, this cells might still differentiate and be formed like al like normal cells.If you look at the karyotype, you might have some abnormality, but you might still have a normal karyotype. The, the, the selective advantage might be due to just a mutation that you cannot see by doing a karyotype. So the fact that they grow much faster is by itself I think a little bit worser.And if we want to use these cells for transplantation at some point, I would definitely not use this type of cells.Okay. So would you like to tell me what are the main technical problems with using this technique for, for passages of cells? So using S, yes.It's just a little bit longer and also it's, since the cells are not going as fast, it's just much longer to get a high number of cells to do some experiment. So it's just less convenient I would say. Would you like to tell me about few words about the goals of your experiment?How do you use CS cells? Okay, so my main project here is to get endothelial cells from human AB stem cells. And the idea is to be able to make blood.And we are working a lot with tissue bioengineering labs and one of their goal is to make tissue in vitro. But one of the limitation to this is the lack of blood vessels going through this tissues. So if we can bring endothelial cells or put endothelial cells in this tissue, we would have better growth of this tissue.So the idea few sources of cells are known for endothelial cells like cold blood or pressure blood, but it would be interesting to have another source of cells like human years. And the advantage is since human years can be expanded and most infinitely, you could modify these cells, make some inducible system and be able to use it. Then in your final product, your material cells, in this case.

İnsan Embriyonik Kök Hücre (ES) yayılması

Biyoloji

Isolation and Transplantation of Hematopoietic Stem Cells (HSCs)

Isolation and Transplantation of Hematopoietic Stem Cells HSCs

I am Christina Ceso. I am a postdoc in David's Den lab and I work on the hematopoietic stem cells. Today we are going to purify long-term recuperating hematopoietic stem cells and we'll transplant them into a mouse.We start by spraying the mice with alcohol so that first of all, the mind starts sterilized and also the hair doesn't interfere with the dissection. We use PBS with 2%serum for the dissection. I cut the back skin and I pull the skin apart in order to expose the tissue.In order to isolate the tibia, I hold the tibia with tweezers. I cut the tendon on top of the knee and I cut the muscles below the knee and after that I cut through the knee so that the TBI is not connected to the rest of the limb anymore and I pull in order to isolate it. I then hold the TB upside down and with the scissors I separate the foot from the tibia In order to obtain the femur.I cut the muscles around the femur and then I dislocate the femur from the hip. In order to isolate the hip, I cut the muscle around the hip bone on the side of the tail of the mouse and then I pull the hip away from the body of the mouth In order to isolate the spine. I cut through the tail as close as I can to the beginning of the back and then I cut on the two sides of the spine and below the spine, trying to stay as close as possible to the spine so that the peritoneum is not cut and the intestine is left untouched.A first round of cleaning of the bones is done with a scalpel, which is used to scrape the bone, the muscles away from the bone. The scalpel is good to clean the femurs, the hip and the spine. In order to clean the tibia, I hold them with forceps and I use the forceps to remove as much tissue as possible.A second round of cleaning is done using clean wipe to completely clean the bones outta the muscle, and I use the F wipe to clean the tibia, the femurs and the hips. So this is how bones look. After cleaning, I prefer 50 amount tubes with filters on top and I transfer the bones into the mortar.I add the section media and I crush the bones ask of the first round of crashing of the bones. I piped the mixture in order to release as many styles as possible into solution and reduce clumps. I transferred the mixture on the tubes through the filters and this is what the bone fragments look like after the first round of crushing.So I add more dissection medium to the bone fragments and I flash them again. I transferred the seline mixture to the tubes through the filters and at this point the remaining bone fragments are white and there is no bone marrow left. The tubes are filled with PBS 2%serum and in order to have them all with the same weight so that they're balanced in the centrifuge, but also in order to wash the filters so that we obtain as many cells as possible, I then centrifuge the cell mixture and after centrifugation, the palt includes the bone marrow cells.I aspirate the medium and I move back and forward the tube on the rack so that the pelle is becomes loose and fewer clamps are formed. When I add medium, at this point I add one ml of medium per tube and I ics since I'm sorting hematopoietic stem cells. Later on, I'm going to store an ALI output of total bone marrow sizes at this point, which I will use later for the fact single color control.I add lineage antibody cocktail. I ordered the samples to mix them well and I invaded four degrees for 15 minutes. In the meantime, I start preparing the columns for the lineage division.I put the columns on the magnet and I prepare Ian tubes under the columns. I also prepared the gas PBS using aster flip filter and attaching it to the vacuum so that all the bubbles of air move in the top part of the filter. I then add three ml of the gas DBS to each column in order for the column to become wet.After the incubation with the antibodies, I add medium to the cell samples and I centrifuge them in order to remove the excess of antibodies. After the centrifugation, the stain cells are in the pallet. I aspirate the medium and I suspend the pallet in one mile of the gas PBSI Add the strip tab beans, I mix the cells and I incubate for 15 minutes at four degrees.After the incubation, I put new elucian tubes under the columns. I add two ml of PBS to each tube with the cells and I transferred the cell mixture to the column through a 40 micron filter. I wash the tubes with more medium in order to collect all the cells remaining and I apply the remaining medium through the filters to the columns.The progenitor cells lineage low are alluded into the collection views. I pulled the luted samples and I sent tofu them. So after the centrifugation, the depleted cells are in a white pate.I aspirate the medium and re suspend the cells. I store an adequate of the depleted cells and I add the antibodies to the cell mixture for the sorting and also to all the single color controls. The antibodies are incubated in that with the size for 15 minutes in the fridge.After the ation, I add medium to the cells and and centrifuge them to remove the ex excess of antibodies. After the centrifugation, the cells are in the pt, I aspirate the media that I suspend the cells and I transfer the cells into fox tubes with a blue filter. At this point, the cells are ready to be sorted.I place, I place the mouse under a heating lamp for a few minutes so that the veins dilate and they are more easily seen. When preparing the syringe, you have to be careful to eliminate all the bubbles from the, the mixture that is in the syringe and then you bend the needle at 90 degrees. I put the mouse under a glass cup with a weight on top so that it can run away.I sterilize the tail with the alcohol pad so that the site of injection is sterile, but also it's easier to identify the vein. If you look at the tail of the mouse from the top in the middle and top of the tail, there is an artery while on the two sides there are the two veins and we inject the cells in one of the two veins. I insert the needle into the vein and I inject following injection.It is to wait for a couple of seconds before removing the needle, the needle, and after that I clean the side of injection. This is a representation of the syringe with the ben needle bent at 90 degrees with the knee, the at this ankle compared to the tail, which makes the injection the easiest. When you injecting the tail of the mouse, you have to keep in mind that the tail vein is extremely superficial, so really this is the orientation of the needle compared to the tail.And it looks like you will just keep sliding on top of the tail, but actually you will get into the tail then. I am Christina Ceso. I am a post in David Kaden lab, and I work on the hematopoietic stem cells.I usually use for animals to start with, and I had more variability at the beginning, but once you become consistent with your methods, I consistently get about 50, 000 to 60, 000 stem cells out for mice. I did my PhD in London with Dr.Fiona Wat working on derma stem cells. After that, I just wanted to have a wider knowledge of adult stem cells in various tissues, and so I decided to move to David Kaden lab to work on hematopoietic stem cells, mainly because the things that are known about hematopoietic stem cells are the things that are not known for the epidermic stem cells and a number of assays that can be done with epi.Epidermal stem cells are not really done yet with the hematopoietic stem cells, and so at this point I'm trying to put together the, the knowledge from the two different fields and ultimately I'm interested in the study of the mechanism to regulate adult tissue sensors in general, so not just the epidermis or the stem cells, but also different tissues because there are, there are similar mechanism that is, it's always the same genes that are involved in the regulation that just interact with each other in different ways, and it's just interesting to understand what makes each stem cell be a stem, but then ultimately a stem cell in a different tissue. So giving rise to a different protein.

İzolasyon ve Hematopoetik Kök Hücre Transplantasyonu (HKH&#39;lerin)

Derivation of Hematopoietic Stem Cells from Murine Embryonic Stem Cells

A stem cell is defined as a cell with the capacity to both self-renew and generate multiple differentiated progeny.  Embryonic stem cells (ESC) are derived from the blastocyst of the early embryo and are pluripotent in differentiative ability.  Their vast differentiative potential has made them the focus of much research centered on deducing how to coax them to generate clinically useful cell types.  The successful derivation of hematopoietic stem cells (HSC) from mouse ESC has recently been accomplished and can be visualized in this video protocol.  HSC, arguably the most clinically exploited cell population, are used to treat a myriad of hematopoietic malignancies and disorders.  However, many patients that might benefit from HSC therapy lack access to suitable donors.  ESC could provide an alternative source of HSC for these patients.  The following protocol establishes a baseline from which ESC-HSC can be studied and inform efforts to isolate HSC from human ESC.  In this protocol, ESC are differentiated as embryoid bodies (EBs) for 6 days in commercially available serum pre-screened for optimal hematopoietic differentiation.  EBs are then dissociated and infected with retroviral HoxB4.  Infected EB-derived cells are plated on OP9 stroma, a bone marrow stromal cell line derived from the calvaria of  M-CSF-/- mice, and co-cultured in the presence of hematopoiesis promoting cytokines for ten days.  During this co-culture, the infected cells expand greatly, resulting in the generation a heterogeneous pool of 100s of millions of cells.  These cells can then be used to rescue and reconstitute lethally irradiated mice.

This protocol details the derivation of transplantable hematopoietic stem cells from mouse embryonic stem cells (ESC) and their subsequent injection into lethally irradiated recipient mice. Briefly, ESC are differentiated as embryoid bodies, which are then infected with retroviral HoxB4 and co-cultured with OP9 stromal cells and hematopoietic cytokines.

Murin Embriyonik Kök Hücre Hematopoetik Kök Hücre türetilmesi

A stem cell is defined as a cell with the capacity to both self-renew and generate multiple differentiated progeny.  Embryonic stem cells (ESC) are ...

Tracheotomy: A Method for Transplantation of Stem Cells to the Lung

Lung disease is a leading cause of death and likely to become an epidemic given increases in pollution and smoking worldwide. Advances in stem cell therapy may alleviate many of the symptoms associated with lung disease and induce alveolar repair in adults. Concurrent with the ongoing search for stem cells applicable for human treatment, precise delivery and homing (to the site of disease) must be reassured for successful therapy. Here, I report that stem cells can safely be instilled via the trachea opening a non-stop route to the lung. This method involves a skin incision, caudal insertion of a cannula into and along the tracheal lumen, and injection of a stem cell vehicle mixture into airways of the lung. A broad range of media solutions and stabilizers can be instilled via tracheotomy, resulting in the ability to deliver a wider range of cell types. With alveolar epithelium confining these cells to the lumen, lung expansion and negative pressure during inhalation may also assist in stem cell integration. Tracheal delivery of stem cells, with a quick uptake and the ability to handle a large range of treatments, could accelerate the development of cell-based therapies, opening new avenues for treatment of lung disease.

Significant breakthroughs in stem cell identification are continuously being made. To translate these discoveries, however, novel methods for cellular delivery must be devised. Here I report that the airways provide a safe route for stem cell transplantation to the lungs.

Trakeotomi: Akciğer Kök Hücre Nakli Bir Yöntem

Lung disease is a leading cause of death and likely to become an epidemic given increases in pollution and smoking worldwide. Advances in stem cell ...

Derivation of Human Embryonic Stem Cells by Immunosurgery

The ability of human embryonic stem cells to self-renew and differentiate into all cell types of 
the body suggests that they hold great promise for both medical applications and as a research 
tool for addressing fundamental questions in development and disease. Here, we provide a 
concise, step-by-step protocol for the derivation of human embryonic stem cells from embryos 
by immunosurgical isolation of the inner cell mass. 


The ability of human embryonic stem cells to self-renew and differentiate into all cell types of 
the body suggests that they hold great promise for both medical applications and as a research 
tool for addressing fundamental questions in development and disease. Here, we provide a 
concise, step-by-step protocol for the derivation of human embryonic stem cells from embryos 
by immunosurgical isolation of the inner cell mass.

Immunosurgery İnsan Embriyonik Kök Hücreleri türetilmesi

The ability of human embryonic stem cells to self-renew and differentiate into all cell types of 
the body suggests that they hold great promise for both ...

Generation of Human CD40-activated B cells

CD40-activated B cells (CD40-B cells) have been identified as an alternative source of immuno-stimulatory antigen-presenting cells (APC) for cancer immunotherapy 1-3. Compared to Dendritic cells (DCs), the best characterized APC, CD40-B cells have several distinct biological and technical properties. Similar to DCs, B cells show an increased expression of MHC and co-stimulatory molecules (Fig.1b), exhibit a strong migratory capacity and present antigen presentation efficiently to T cells, after stimulation with interleukin-4 and CD40 ligand (CD40L). However, in contrast to immature or mature DCs, CD40-B cells express the full lymph node homing triad consisting of CD62L, CCR7/CXCR4, and leukocyte function antigen-1 (LFA1, CD11a/CD18), necessary for homing to secondary lymphoid organs (Fig.1a) 3. CD40-B cells can be generated without difficulties from very small amounts of peripheral blood which can be further expanded in vitro to very large amounts of highly-pure CD40-B cells (&gt;109 cells per patient) from healthy donors as well as cancer patients (Fig.1c,d) 1,4. 
In this protocol we demonstrate how to obtain fully activated CD40-B cells from human PBMC. Key molecules for the cell culture are CD40 ligand, interleukin-4 (IL-4) and cyclosporin A (CsA), which are replenished in a 3-4 day culture cycle. For laboratory purposes CD40-stimulation is provided by NIH/3T3 cells expressing recombinant human CD40 ligand (tCD40L NIH/3T3) 5. To avoid contamination with non-transfected cells, expression of the human CD40 ligand on the transfectants has to be checked regularly (Fig.2). 
After 14 days CD40-B cell cultures consist of more than 95% pure B cells and an expansion of CD40-B cells over 65 days is frequently possible without any loss of function 1, 4. CD40-B cells efficiently take up, process and present antigens to T cells 6. They do not only prime na&#970;ve, but also expand memory T cells 7,8. CD40-activated B cells can be used to study B-cell activation, differentiation and function. Moreover, they represent a promising tool for therapeutic or preventive vaccination against tumors 9.

In this video we present the ex vivo generation and expansion of human CD40-activated B cells (CD40-B) from peripheral blood mononuclear cells (PBMC) by stimulation with CD40 ligand and interleukin-4.

İnsan CD40 aktive B hücreleri üretilmesi

CD40-activated B cells (CD40-B cells) have been identified as an alternative source of immuno-stimulatory antigen-presenting cells (APC) for cancer ...

Differentiation of Embryonic Stem Cells into Oligodendrocyte Precursors

Oligodendrocytes are the myelinating cells of the central nervous system. For regenerative cell therapy in demyelinating diseases, there is significant interest in deriving a pure population of lineage-committed oligodendrocyte precursor cells (OPCs) for transplantation. OPCs are characterized by the activity of the transcription factor Olig2 and surface expression of a proteoglycan NG2. Using the GFP-Olig2 (G-Olig2) mouse embryonic stem cell (mESC) reporter line, we optimized conditions for the differentiation of mESCs into GFP+Olig2+NG2+ OPCs. In our protocol, we first describe the generation of embryoid bodies (EBs) from mESCs. Second, we describe treatment of mESC-derived EBs with small molecules: (1) retinoic acid (RA) and (2) a sonic hedgehog (Shh) agonist purmorphamine (Pur) under defined culture conditions to direct EB differentiation into the oligodendroglial lineage. By this approach, OPCs can be obtained with high efficiency (&gt;80%) in a time period of 30 days. Cells derived from mESCs in this protocol are phenotypically similar to OPCs derived from primary tissue culture. The mESC-derived OPCs do not show the spiking property described for a subpopulation of brain OPCs in situ. To study this electrophysiological property, we describe the generation of spiking mESC-derived OPCs by ectopically expressing NaV1.2 subunit. The spiking and nonspiking cells obtained from this protocol will help advance functional studies on the two subpopulations of OPCs.

We describe a small molecule-based protocol for differentiation of mouse embryonic stem cells into oligodendrocyte precursor cells (OPCs). This protocol generates Olig2+NG2+ OPCs with high efficiency by 30 days of differentiation. We also describe a method to generate "spiking" OPCs that can fire action potentials.

Embriyonik Kök Hücreler farklılaşma oligodendrosit Öncüleri

Oligodendrocytes are the myelinating cells of the central nervous system. For regenerative cell therapy in demyelinating diseases, there is significant ...

Fate Mapping of Human Embryonic Stem Cells by Teratoma Formation 

Human embryonic stem cells (hESCs) have an unlimited capacity for self-renewal, and the ability to differentiate into cells derived from all three embryonic germ layers (1). Directed differentiation of hESCs into specific cell types has generated much interest in the field of regenerative medicine (e.g., (2-5)), and methods for determining the in vivo fate of selected or manipulated hESCs are essential to this endeavor. We have adapted a highly efficient teratoma formation assay for this purpose. A small number of specifically selected hESCs is mixed with undifferentiated wild type hESCs and Phaseolus vulgaris lectin to form a cell pellet. This is grafted beneath the kidney capsule in an immunodeficient mouse. As few as 2.5 x 105 hESCs are needed to form a 16 cm3 teratoma within 8-12 weeks. The fate of the originally selected hESCs can then be determined by immunohistochemistry. This method provides a valuable tool for characterizing tissue-specific reagents for cell-based therapy.

Fate Mapping of Human Embryonic Stem Cells by Teratoma Formation

Directed differentiation of hESCs into specific cells has generated much interest in regenerative medicine. We provide a concise, step-by-step protocol for determining the in vivo fate of selected hESCs that provides a valuable tool for characterizing tissue-specific reagents for cell-based therapy.

Teratom Oluşumu İnsan Embriyonik Kök Hücre Kader Haritalama

Human embryonic stem cells (hESCs) have an unlimited capacity for self-renewal, and the ability to differentiate into cells derived from all three ...

Xenotransplantation of Human Stem Cells into the Chicken Embryo

The chicken embryo is a classical animal model for studying normal embryonic and fetal development and for xenotransplantation experiments to study the behavior of cells in a standardized in vivo environment. The main advantages of the chicken embryo include low cost, high accessibility, ease of surgical manipulation and lack of a fully developed immune system. Xenotransplantation into chicken embryos can provide valuable information about cell proliferation, differentiation and behavior, the responses of cells to signals in defined embryonic tissue niches, and tumorigenic potential. Transplanting cells into chicken embryos can also be a step towards transplantation experiments in other animal models. Recently the chicken embryo has been used to evaluate the neurogenic potential of human stem and progenitor cells following implantation into neural anlage1-6. In this video we document the entire procedure for transplanting human stem cells into the developing central nervous system of the chicken embryo. The procedure starts with incubation of fertilized eggs until embryos of the desired age have developed. The eggshell is then opened, and the embryo contrasted by injecting dye between the embryo and the yolk. Small lesions are made in the neural tube using microsurgery, creating a regenerative site for cell deposition that promotes subsequent integration into the host tissue. We demonstrate injections of human stem cells into such lesions made in the part of the neural tube that forms the hindbrain and the spinal cord, and into the lumen of the part of the neural tube that forms the brain. Systemic injections into extraembryonic veins and arteries are also demonstrated as an alternative way to deliver cells to vascularized tissues including the central nervous system. Finally we show how to remove the embryo from the egg after several days of further development and how to dissect the spinal cord free for subsequent physiological, histological or biochemical analyses.

In this paper we present a method for transplanting human stem cells into various regions of the central nervous system of the chicken embryo. This provides an in vivo model for assessing the proliferation and differentiation of various types of human stem cells in embryonic tissue environments.

Tavuk embriyo haline İnsan Kök Hücre Xenotransplantation

The chicken embryo is a classical animal model for studying normal embryonic and fetal development and for xenotransplantation experiments to study the ...

Efficient Derivation of Human Cardiac Precursors and Cardiomyocytes from Pluripotent Human Embryonic Stem Cells with Small Molecule Induction

To date, the lack of a suitable human cardiac cell source has been the major setback in regenerating the human myocardium, either by cell-based transplantation or by cardiac tissue engineering1-3. Cardiomyocytes become terminally-differentiated soon after birth and lose their ability to proliferate. There is no evidence that stem/progenitor cells derived from other sources, such as the bone marrow or the cord blood, are able to give rise to the contractile heart muscle cells following transplantation into the heart1-3. The need to regenerate or repair the damaged heart muscle has not been met by adult stem cell therapy, either endogenous or via cell delivery1-3. The genetically stable human embryonic stem cells (hESCs) have unlimited expansion ability and unrestricted plasticity, proffering a pluripotent reservoir for in vitro derivation of large supplies of human somatic cells that are restricted to the lineage in need of repair and regeneration4,5. Due to the prevalence of cardiovascular disease worldwide and acute shortage of donor organs, there is intense interest in developing hESC-based therapies as an alternative approach. However, how to channel the wide differentiation potential of pluripotent hESCs efficiently and predictably to a desired phenotype has been a major challenge for both developmental study and clinical translation. Conventional approaches rely on multi-lineage inclination of pluripotent cells through spontaneous germ layer differentiation, resulting in inefficient and uncontrollable lineage-commitment that is often followed by phenotypic heterogeneity and instability, hence, a high risk of tumorigenicity6-8 (see a schematic in Fig. 1A). In addition, undefined foreign/animal biological supplements and/or feeders that have typically been used for the isolation, expansion, and differentiation of hESCs may make direct use of such cell-specialized grafts in patients problematic9-11. To overcome these obstacles, we have resolved the elements of a defined culture system necessary and sufficient for sustaining the epiblast pluripotence of hESCs, serving as a platform for de novo derivation of clinically-suitable hESCs and effectively directing such hESCs uniformly towards clinically-relevant lineages by small molecules12 (see a schematic in Fig. 1B). After screening a variety of small molecules and growth factors, we found that such defined conditions rendered nicotinamide (NAM) sufficient to induce the specification of cardiomesoderm direct from pluripotent hESCs that further progressed to cardioblasts that generated human beating cardiomyocytes with high efficiency (Fig. 2). We defined conditions for induction of cardioblasts direct from pluripotent hESCs without an intervening multi-lineage embryoid body stage, enabling well-controlled efficient derivation of a large supply of human cardiac cells across the spectrum of developmental stages for cell-based therapeutics.

We have established a protocol for induction of cardioblasts direct from pluripotent human embryonic stem cells maintained under defined conditions with small molecules, which enables derivation of a large supply of human cardiac progenitors and functional cardiomyocytes for cardiovascular repair.