Name: isolation of endothelial progenitor cells from human umbilical cord blood
Uploaded: 2017-09-14T14:00:00.000+00:00
Duration: 7 min 26 s
Description: Watch this Scientific Journal Video about Isolation of Endothelial Progenitor Cells from Human Umbilical Cord Blood at JoVE.com

Bu malzeme Grant No altında Ulusal Bilim Vakfı tarafından desteklenen çalışma üzerine kuruludur CMMI-1452943 ve Arkansas Üniversitesi onur üniversite tarafından. Biz de Arkansas Kordon Kanı Bankası kordon kanı birimleri ile bize sağlamak için kabul etmek istiyorum.

Bu araştırma Üniversitesi Arkansas kurumsal inceleme Kurulu (onay numarası 16-04-722) onayı ile gerçekleştirilmiştir. Göbek Kordon kanı birimleri sitrat fosfat dekstroz (GBM) çözümünde, Arkansas Kordon Kanı Bankası toplanmıştır ve depolama gereksinimini karşılamayan birimleri için araştırma kuruluşuna verildi. Kordon kan birimleri ortam sıcaklıklarda koleksiyonunun 24 saat içinde laboratuvara couriered.
 1. yalıtım endotelyal Progenitor Hücre kordon kanı üzerinden <ol><li>reaktifler hazırlanması.
	<ol><li>Hazırlamak endotel Bazal orta (EBM) % 10 fetal sığır serum için (FBS) ekleyerek EGM takıma 20 ng/mL İnsülin benzeri büyüme faktörü (IGF), 1 µg/mL askorbik asit, 5 ng/mL rekombinant insan epidermal büyüme faktörü (rhEGF), 22.5 µg/mL heparin ve 0,5 ng/mL vasküler endotelyal büyüme faktörü (VEGF), 10 ng/mL rekombinant insan Fibroblast büyüme faktörü B (rhFGF-B), 0.2 µg/mL hidrokortizon ve % 2 penisilin-streptomisin-glutamin ile birlikte. Kültür 0.2 µm polyethersulfone (PES) membran vakum filtre kullanarak ortamın sterilize.</li>
		<li>Hazırlama fare ben kollajen çözüm 50 µg/mL 0,02 N Asetik asit kullanarak bir konsantrasyon, kuyruk. Bu çözüm 0.2 µm şırınga filtre sterilize.</li>
		<li>Öncesi kat 6-şey tabak 2 mL hazırlanmış iri fare ile ben her şey için kollajen çözüm kuyruk. Tohum önce % 5 CO 2 ve 37 ° C en az 1 h için tutulan bir kuluçka yerleştirebilirsiniz.</li>
		<li>1:1 konsantrasyon, kordon kanı ile Hanks dengeli tuz çözüm (HBSS) yaklaşık olarak 25 mL seyreltik.</li>
		<li>Hazırlama radyo-IMMUNO yağış tahlil 150 mM Sodyum Klorür, %1 Triton X-100, % 0.5 sodyum deoxycholate, 1 mM β-gliserofosfat, % 0,1 SDS ve 50 mM Tris (pH 8.0) kullanarak (RIPA) arabellek.</li>
	</ol></li> <li>Yalıtım endotelyal progenitor hücre.
	<ol><li>Sıcak tüm hazır reaktifler yalıtım önce 37 ° C'de tutulan bir su banyosunda.</li>
		<li>Yoğunluğu degrade orta reaktif 20 mL 50 mL santrifüj tüpü ekleyin.</li>
		<li>Dikkatle katman seyreltilmiş kordon kanı pipet tüp yan duvarlarının doğru amaçlayan tarafından bu katman bozmadan yoğunluk gradient orta içeren santrifüj tüpü içinde 20 mL.</li>
		<li>Onların yoğunlukları ( şekil 1) göre kan bileşenlerinin santrifüj rotor frenler ile 800 x g oda sıcaklığında 30 dk için de centrifuging tarafından ayırın. Farklı Katmanlar rahatsız etmeyin.</li>
		<li>Plazma katmanın dikkatle bu tüp dışında diğer hücre katman pipet ucu MNC ulaşmak mümkün olana katmanları (bkz. şekil 1) rahatsız etmeden pipetting tarafından kaldırın. Kaldırılan plazma atın. 
		Not: pipet ipuçları plazma kaldırmak için kullanırken, plazma MNC katmanının (bozuklukları azaltmak için, şekil 1) küçük bir tabaka bırakın.</li>
		<li>Bir 18'lik iğne donatılmış kullanarak çok uluslu şirketler içerir buffy kat toplamak ve bir taze santrifüj tüpü transfer.</li>
		<li>Ekleyin EBM eşit bir hacmi toplanan çok uluslu şirketler. santrifüj için bu tüpler 500 x g 10 min için de 4'te ° C. atma süpernatant. 
		Not: Kırmızı hücre Pelet kırmızı kan hücreleri/eritrositler varlığı nedeniyle görünür.</li>
		<li>5 mL kırmızı kan hücreleri parçalayıcı amonyum klorür çözeltisi ekleyin. Bu tüp için 5-10 dk ara sıra sallayarak ile buz üzerinde kuluçkaya. 
		Not: çok uluslu şirketler için zararlı olabileceği kadar dikkatli olun bu adımı sırasında zaman süresini aşmayacak şekilde alınmalıdır.</li>
		<li>500 x g 4 ° C'de 10 dakika için de tüpler santrifüj kapasitesi ve süpernatant atın. Eritrositler devam ederse, kırmızı renk Pelet içinde gözlenen 1.2.8 ve 1.2.9 yükleme tamamlayana dek.</li>
		<li>Pelet hazırlanan EGM içinde resuspend ve hemasitometre çok uluslu şirketler kullanın.</li>
		<li>Aspiratı sıçan kuyruk ben (adımından 1.1.3) kolajen kaplı 6-şey plaka ve wells 1 fosfat tamponlu tuz çözeltisi (PBS) x ile üç kez yıkayın.</li>
		<li>1 x 10 7 çok uluslu şirketler her iyi bir konsantrasyon, kollajen plakasına EGM içinde resuspended MNC Pelet tohum. 37 ° C'de % 5 CO 2 kuluçka yerleştirin.</li>
		<li>Sonra 24 h, orta Aspire edin ve wells bir kez EGM ile yıkayın. Her şey için EGM orta 3 mL ekleyin ve geri 37 ° C, % 5 CO 2 kuluçka yerleştirin.</li>
		<li>7 gün boyunca her gün taze EGM orta ile plaka doldurmak. 7 gün sonra EGM orta her gün değiştirin.</li>
		<li>Bir alan parlak mikroskop kullanarak almak 6-şey plaka görüntülerini her gün endotel hücre kolonileri ilerlemesini izlemek için. Nerede koloniler ortaya onların büyüme izlemek için plaka işareti. 
		Not: Genellikle kültür görünmesini colonies için 5 ile 9 gün arasında sürer.</li>
	</ol></li> <li>Genişleme endotelyal progenitor hücre.
	<ol><li>Kat T-25 kültür şişeye fare ile kuyruk kollajen (50 µg/mL) ve yer o 37 ° C'de % 5 CO 2 kuluçka için en az 60 dk. aspiratı ve hücreleri tohum önce üç kez 1 x PBS ile T-25 hücre kültür şişeye yıka.</li>
		3 mm boyutunda 150 µL % 0.05 tripsin-EDTA çözüm her kuyuya kullanarak ulaştığınızda <li>endotel hücre koloniler trypsinize. 6-şey plaka 37 ° C, % 5 CO 2 kuluçka 2-3 dakika süreyle geri yerleştirin</li>
		<li>Ekli hücreleri çıkarmak için 6-şey plaka hafifçe dokunun. 2 mL EGM eklenir ve yaklaşık 15 mL santrifüj tüpü hücrelerde kurtarmak.</li>
		<li>Bir hemasitometre kullanarak hücreleri saymak ve ilk tohumlama yoğunluğu hesaplamak.</li>
		<li>Spin 15 mL santrifüj tüpleri 400 x g 5 dakika süreyle, hücre Pelet EGM içinde Resuspend ve T-25 şişeye ~ 500.000 hücreleri ile tohum. Bu şişeyi geçiş 1 olarak işaretlemek ve T-25 şişeye geri 37 ° C, % 5 CO 2 kuluçka yerleştirin. 
		Not: Hücreleri EBM ve DMEM büyüme EGM ile karşılaştırmak için kaplama.</li>
		<li>İçin daha fazla geçişleri, şişeye % 90 izdiham ulaştığında, adımları 1.3.1 - 1.3.5 ve hücre 10 4 hücreleri/cm 2 T-75 veya T-175 hücre kültür şişeler reseed. İzole hücreler (Adım 2 ve 3) karakterizasyonu için devam.</li>
	</ol></li></ol> 2. İzole hücreler kullanarak Western Blot karakterizasyonu <ol><li>Ekle 50-100 µL lizis, arabellek izole hücreleri ayırdetmek için genişleme adımları her geçiş. Proteinler yaklaşık 500.000 veya daha çok satır toplamak.</li>
	<li>Yukarı ve aşağı şiddetle hücreleri rüptürü ve proteinleri serbest bırakmak için pipet. Toplamak ve arabellek microcentrifuge tüp transfer.</li>
	<li>Microcentrifuge tüp 500 x g de spin ve dikkatle süpernatant yeni bir microcentrifuge tüp aktarın. Etiket ve tüpler uzun süreli depolama için-80 ° C'de depolayın.</li>
	<li>Ya Bradford kullanarak her tüpün içinde protein miktarını ölçmek &#39; s veya BCA tahlil 27 , 28 , 29.</li>
	<li>Standart prosedürler 27 , 28 , 29 kullanarak kurutma Western taşımak. İfadeye CD31, CD34, VEGFR2 ve α-SMA ait toplam protein 5 µg analiz. Veri normalleştirme için α-tübülin kullanın.</li>
</ol> 3. Dolaylı ayirt <ol><li>18-mm cam coverslips % 50 etanol içinde solüsyon içeren temizleyicide ve onları kurutun. Temiz coverslips bir 12-şey plaka gecede Biyogüvenlik mahalledeki bir UV ile onları sterilize etmek ışık bırakın.</li>
	<li>Kat coverslips ile 50 ve #181; g/kuyruk kollajen ve plaka bir 37 ° C, % 5 CO 2 kuluçka en az 60 dakika süreyle transfer mL sıçan</li>
	<li>Kollajen Aspire edin ve 1 mL 1 x PBS coverslips yıkamak için 12-şey plaka ekleyin. 1 x PBS Aspire edin ve iki kez tekrarlayın.</li>
	<li>Tohum 250.000 kültürlü EPCs her şey ve onları geri 37 ° C'de % 5 CO 2 kuluçka immunostaining taşıma önce en az 3 gün boyunca yerde.</li>
	<li>3 kez 1 x PBS ile plaka yıkayın. Her şey buz gibi metanol 1 mL ekleyin ve hücreleri düzeltmek 15 dakika oda sıcaklığında kuluçkaya.</li>
	<li>Kullanarak bir standart iletişim kuralı 27 , 28 , 29 immunostaining taşımak. Antikorlar onların uygun dilutions kullanın (Örneğin, CD31 (1:20), CD34 (1:50), α-SMA (1: 100) ve VEGFR2 (1:50)).</li>
	<li>Al epifluorescence mikroskop kullanarak immunostained coverslips görüntülerini.</li>
</ol>

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Daha önce belirtildiği gibi bir kaldırım taşı döşeli Morfoloji sahip yapışık EPCs. Bizim izole çok uluslu şirketler bir seklinde hücre koloni (Şekil 2A-2D) bir süre kültür on gün içinde bir Arnavut kaldırımlı koloni (Şekil 2E-2F) erken aşamalarında ilerledi. EPCs farklı farklı araştırma gruplarınca, endotel koloni oluşturan hücreleri5veya endotelyal progenitor hücreler<su...

Birkaç müfettişler özellikleri ve insan EPCs5,10,11,12,13potansiyelini inceledik. EPCs endotel dokusunda siteleri hipoksi, iskemi, yaralanma veya tümör oluşumu için uygun ve yeni vasküler yapılar4,14oluşumuna katkıda yeteneği olan hücreleri dolaşan olarak tanımlanabilir. Neovaskülarizasyon, doğum sonrası vasculogenesis, şeklinde gözlenen onların katılımı bir anlayış bu hücreler ve tedavi uygulamaları4,15, kullanımları Patofizyoloji yol açmış 16. bireyin EPCs sayısında kardiyovasküler patoloji9,15,16,17,18ile,19 ilişkili olduğu gösterilmiştir ,20. Diğer çalışmalar da EPCs bir vana fibroblast benzeri fenotip ayrıştırılan ve bu hücrelerin doku Mühendisliği kalp vanalar7,21için kullanılan olabilir önerdi.
EPCs yalıtmak için gerekli belirli hücre yüzey molekülleri açıkça nedeniyle İncelemeler4arasındaki tutarsızlıkları tespit edilmiştir değil. Çok uluslu şirketler yapışma maruz kalma kültür koşulları, çeşitli ile belirli bir matris için sözde EPCs olabileceğini düşündüren çeşitli gruplar1,17,22,23, sahne aldı farklı fenotipik özellikleri görüntülenemiyor. Bu özellikler phagocytotic yeteneği, Matrigel tüp oluşumu ve Dil acetylated düşük yoğunluklu lipoproteinler alımını eksikliği içerir. Yüksek klonojenik progenitör mobilizasyonu yapar ve proliferatif potansiyeli ile hangi EPCs hierarchized5olabilir iki özelliklerdir. EPCs da insan fetal akciğer fibroblastlar4ile cocultured zaman vitro tübüllerin oluşabilir. Bu hücreler endotel hücre yüzey işaretleyicileri ifade etmek ve hematopoetik işaretleri13,24,25bazılarını paylaşmak için bilinir. Fenotipleme EPCs için yaygın olarak kabul edilen olumlu ifade işaretleri vardır CD31, CD34, vasküler endotelyal büyüme faktörü reseptör 2 (VEGFR2), von Willebrand faktör (vWF), CD133, c-Kit ve vasküler endotel cadherin (VE-cadherin)4 , 18. CD90, CD45, CD14, CD115 veya alfa-düz kas aktin (α-SMA) Co hızlı hücreleri bakteri ve yetersizlik de novo insan formu için phagocytose sınırlı proliferatif potansiyel, yeteneklerini nedeniyle EPCs olmak dikkate alınmaz damarları vivo içinde4,7. Bu makalede insan göbek kordon kanı ezelî belgili tanımlık lüzum için herhangi bir hücreyi iletişim kuralları sıralama endotelyal progenitor hücre izolasyon için değiştirilmiş bir protokol özetlenmektedir. Bu yazı için biz CD31, CD34 ve VEGFR2 olumlu işaretleyicilerle α-SMA negatif göstergesi olarak olarak kullanılır.
Bu makalede, yalıtma ve göbek kordon kanı hücrelerden endotelyal progenitor hücre kullanarak sıralama kültür yöntemi büyüme faktörleri (EGM) ile desteklenmiş endotelyal büyüme orta İhtisas öneriyorum. Bu EGM vasküler endotelyal büyüme faktörü (VEGF) ve fibroblast büyüme faktörü (FGF), hayatta kalma, yayılması ve endotel hücreleri26geçiş güvenliğini artırmak içerir. Ayrıca askorbik asit, hücre Arnavut kaldırımlı Morfoloji bakımından sorumlu olan içerir; İnsülin benzeri büyüme faktörü-anjiogenik ve göçmen işlevi sağlayan 1 (IGF-1); ve heparin, büyüme faktörleri geliştirilmiş uzun vadeli istikrar içinde orta26neden olur. Endotel hücre kültür ortamına eklenen diğer büyüme faktörleri içerir takviyesi ile epidermal büyüme uyarıcı hücre çoğalması ve farklılaşma ve EGF26 hücrelere sensitizes hidrokortizon yardımcı olan faktörü (EGF), . Bu belirli büyüme orta kullanımı EPCs endotel Bazal orta (EBM) ya da Dulbecco'nın modifiye kartal orta (DMEM) göre daha yüksek sayıda verimleri göster.

<table><tbody><tr><td>&lt;strong&gt;A) For isolation and culturing&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>EGM-2 BulletKit</td><td>Lonza</td><td>CC-3162</td><td>This product comes with all the growth factors needed to make the Endothelial Growth Medium</td></tr><tr><td>Fetal Bovine Serum</td><td>Thermofisher Scientific</td><td>26140079</td><td></td></tr><tr><td>Pencillin-Streptomycin-Glutamine (100x)</td><td>Thermofisher Scientific</td><td>10378016</td><td></td></tr><tr><td>Ficoll-Paque</td><td>GE Heatlhcare</td><td>17-1440-02</td><td></td></tr><tr><td>Hank&#039;s Balanced Salt Solution</td><td>Thermofisher Scientific</td><td>14170-112</td><td></td></tr><tr><td>Ammonium Chloride</td><td>Stem Cell Technologies</td><td>7850</td><td></td></tr><tr><td>1x Phosphate Buffer Saline</td><td>Thermofisher Scientific</td><td>14190250</td><td></td></tr><tr><td>Rat Tail I Collagen</td><td>Corning</td><td>354236</td><td></td></tr><tr><td>Glacial Acetic Acid</td><td>Amresco</td><td>0714-500ML</td><td></td></tr><tr><td>0.05% Trypsin-EDTA</td><td>Thermofisher Scientific</td><td>25300054</td><td></td></tr><tr><td>HEPES buffer</td><td>Thermofisher Scientific</td><td>15630080</td><td></td></tr><tr><td>Dulbecco&#039;s Modified Eagle&#039;s Medium</td><td>Thermofisher Scientific</td><td>10566-016</td><td></td></tr><tr><td>&lt;strong&gt;B) Antibodies and cell lysates&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>CD31&amp;nbsp;</td><td>Abcam</td><td>ab28364</td><td>1:250 dilution&amp;nbsp; for Western blotting</td></tr><tr><td>CD34</td><td>Santa Cruz Biotechnology</td><td>sc-7045</td><td>1:100 dilution for Western blotting</td></tr><tr><td>&amp;alpha;-SMA</td><td>abcam</td><td>ab5694</td><td>1:100 dilution for Western blotting</td></tr><tr><td>&amp;alpha;-tubulin</td><td>abcam</td><td>ab7291</td><td>1:2,500 dilution for Western blotting</td></tr><tr><td>VEGFR2</td><td>abcam</td><td>sc504</td><td>1:100 dilution for Western blotting</td></tr><tr><td>Human umbilical vein endothelial cell lysate</td><td>Santa Cruz Biotechnology</td><td>sc24709&amp;nbsp;</td><td></td></tr><tr><td>Valve interstitial cell lysate</td><td></td><td></td><td>Primary cell line cultured from own lab and lysed with RIPA buffer</td></tr><tr><td>&lt;strong&gt;C) Western blotting and immunostaining&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>10x Tris/Glycine/SDS buffer</td><td>Biorad</td><td>161-0772</td><td>Used as running buffer</td></tr><tr><td>10x Tris/Glycine buffer</td><td>Biorad</td><td>161-0771</td><td>Used as transfer buffer</td></tr><tr><td>Immobilon-FL transfer membrane</td><td>Merck Millipore</td><td>IPFL0010</td><td>This is a PVDF transfer membrane that has 45 &amp;micro;m pore size and is mentioned in the protocol as western blot membrane</td></tr><tr><td>4x Laemmli sample buffer</td><td>Biorad</td><td>161-0747</td><td></td></tr><tr><td>2-mercaptoethanol</td><td>Biorad</td><td>161-0710</td><td></td></tr><tr><td>10% Criterion TGX precast gel</td><td>Biorad</td><td>5671033</td><td></td></tr><tr><td>Prolong Gold antifade</td><td>Thermofisher Scientific</td><td>P36930</td><td>Used for mounting immunostained coverslips for long term storage</td></tr><tr><td>Methanol</td><td>VWR Analytical</td><td>BDH1135-4LP</td><td></td></tr></tbody></table>

isolation of endothelial progenitor cells from human umbilical cord blood

Endotelyal progenitor hücreler (EPCs) periferik kan varlığı ve onun katılımı vasculogenesis ilk Ashara ve arkadaşları1tarafından rapor. Daha sonra diğerleri benzer türdeki diğer kemik iliği2,3' ten kaynaklanan EPCs varlığını belgelemiştir. Daha yakın zamanlarda, Yoder ve EPCs göbek kordon kanı türetilmiş gösterdi Ingram4,5,6kan olanlar yetişkin periferik izole göre bir daha yüksek proliferatif potansiyeli vardı. Doğum sonrası vasculogenesis dahil olmak dışında EPCs da söz doku Mühendisliği vasküler kalp kapak yapıları7,ve8oluşturmak için hücre kaynağı olarak göstermiştir. Çeşitli yalıtım protokoller bulunmaktadır, bazıları ücretli mononükleer hücreler (ÇUŞ 'un) endotel ve hematopoetik işaretleri yardımıyla yukarıda belirtildiği kaynaklardan elde edilen hücre sıralanması veya özel endotelyal büyüme ile bu ÇUŞ 'un kültür Orta veya bu teknikleri9bileşimi. Burada, biz yalıtım için bir iletişim kuralı mevcut ve kullanarak EPCs kültürünün İhtisas endotel orta immunosorting, Western Blot kullanarak izole hücrelerin karakterizasyonu tarafından takip kullanımı olmadan büyüme faktörleri ile desteklenmiş ve immunostaining.

Yalıtım ve genişleme endotelyal Progenitor Hücre: Bir şematik (şekil 1) genel Protokolü tasvir sağlanır. Farklı kan bileşeni katmanları insan göbek kordon kanı ile yoğunluk gradient Orta yoğunluk gradient Santrifüjü takip tespit edildi. Çok uluslu şirketler kollajen tedavi plakalar üzerine tohum, gün 5 ve 7 (Şekil 2A) arasında ilk koloniler akıbet gözlendi. Bu kolonil...

Watch this Scientific Journal Video about Isolation of Endothelial Progenitor Cells from Human Umbilical Cord Blood at JoVE.com

Isolation of Endothelial Progenitor Cells from Human Umbilical Cord Blood

Bu iletişim kuralının amacı göbek kordon kanı endotelyal progenitor hücrelerini izole etmektir. Bazı uygulamalar bu hücreler bir biyomarker hastaların kardiyovasküler riski ile iskemik hastalıklar, tedavi tanımlamak için kullanmayı içerir ve oluşturma doku Mühendisliği vasküler ve kalp kapak oluşturur.

Yalıtım endotelyal Progenitor Hücre insan göbek kordon kanı

The existence of endothelial progenitor cells (EPCs) in peripheral blood and its involvement in vasculogenesis was first reported by Ashara and ...

isolation-endothelial-progenitor-cells-from-human-umbilical-cord

Research

JoVE Journal

Developmental Biology

11.1K Görüntüleme.  University of Arkansas. Bu iletişim kuralının amacı göbek kordon kanı endotelyal progenitor hücrelerini izole etmektir. Bazı uygulamalar bu hücreler bir biyomarker hastaların kardiyovasküler riski ile iskemik hastalıklar, tedavi tanımlamak için kullanmayı içerir ve oluşturma doku Mühendisliği vasküler ve kalp kapak oluşturur.

Video: Isolation of Endothelial Progenitor Cells from Human Umbilical Cord Blood

Direct Induction of Human Neural Stem Cells from Peripheral Blood Hematopoietic Progenitor Cells

Human disease specific neuronal cultures are essential for generating in vitro models for human neurological diseases. However, the lack of access to primary human adult neural cultures raises unique challenges. Recent developments in induced pluripotent stem cells (iPSC) provides an alternative approach to derive neural cultures from skin fibroblasts through patient specific iPSC, but this process is labor intensive, requires special expertise and large amounts of resources, and can take several months. This prevents the wide application of this technology to the study of neurological diseases. To overcome some of these issues, we have developed a method to derive neural stem cells directly from human adult peripheral blood, bypassing the iPSC derivation process. Hematopoietic progenitor cells enriched from human adult peripheral blood were cultured in vitro and transfected with Sendai virus vectors containing transcriptional factors Sox2, Oct3/4, Klf4, and c-Myc. The transfection results in morphological changes in the cells which are further selected by using human neural progenitor medium containing basic fibroblast growth factor (bFGF) and vascular endothelial growth factor (VEGF). The resulting cells are characterized by the expression for neural stem cell markers, such as nestin and SOX2. These neural stem cells could be further differentiated to neurons, astroglia and oligodendrocytes in specified differentiation media. Using easily accessible human peripheral blood samples, this method could be used to derive neural stem cells for further differentiation to neural cells for in vitro modeling of neurological disorders and may advance studies related to the pathogenesis and treatment of those diseases.

A method was developed to directly derive human neural stem cells from hematopoietic progenitor cells enriched from peripheral blood cells.

Periferik kan Hematopoetik Progenitör Hücreleri İnsan Sinir Kök Hücre Doğrudan İndüksiyon

Human disease specific neuronal cultures are essential for generating in vitro models for human neurological diseases. However, the lack of access to ...

Gelişim Biyolojisi

Isolation of Human Umbilical Vein Endothelial Cells (HUVEC)

Angiogenesis is a complex multi-step process, where in response to angiogenic stimuli, new vessels are created from the existing vasculature. These steps include: degradation of the basement membrane, proliferation and migration (sprouting) of endothelial cells (EC) into the extracellular matrix, alignment of EC into cords, lumen formation, anastomosis, and formation of a new basement membrane. Many in vitro assays have been developed to study this process, but most only mimic certain stages of angiogenesis, and morphologically the vessels often do not resemble vessels in vivo. Here we demonstrate an optimized in vitro angiogenesis assay that utilizes human umbilical vein EC and fibroblasts. This model recapitulates all of the key early stages of angiogenesis, and importantly the vessels display patent intercellular lumens surrounded by polarized EC. Vessels can be easily observed by phase-contrast and time-lapse microscopy, and recovered in pure form for downstream applications.

Isolation of Human Umbilical Vein Endothelial Cells HUVEC

This video protocol illustrates the isolation and culture of human umbilical vein endothelial cells (HUVEC) from human umbilical cord. Once isolated these cells can be used for in vitro angiogenesis assays like the Optimized Fibrin Gel Bead Assay also demonstrated by the Hughes lab.

İnsan umbilikal ven endotel hücreleri İzolasyon (HUVEC)

Angiogenesis is a complex multi-step process, where in response to angiogenic stimuli, new vessels are created from the existing vasculature. These steps ...

Biyoloji

Isolation of Murine Embryonic Hemogenic Endothelial Cells

The specification of hemogenic endothelial cells from embryonic vascular endothelium occurs during brief developmental periods within distinct tissues, and is necessary for the emergence of definitive HSPC from the murine extra embryonic yolk sac, placenta, umbilical vessels, and the embryonic aorta-gonad-mesonephros (AGM) region. The transient nature and small size of this cell population renders its reproducible isolation for careful quantification and experimental applications technically difficult. We have established a fluorescence-activated cell sorting (FACS)-based protocol for simultaneous isolation of hemogenic endothelial cells and HSPC during their peak generation times in the yolk sac and AGM. We demonstrate methods for dissection of yolk sac and AGM tissues from mouse embryos, and we present optimized tissue digestion and antibody conjugation conditions for maximal cell survival prior to identification and retrieval via FACS. Representative FACS analysis plots are shown that identify the hemogenic endothelial cell and HSPC phenotypes, and describe a methylcellulose-based assay for evaluating their blood forming potential on a clonal level.

Hematopoietic stem and progenitor cells (HSPC) derive from specialized (hemogenic) endothelial cells during development, yet little is known about the process by which some endothelial cells specify to become blood forming. We demonstrate a flow-cytometry based method allowing simultaneous isolation of hemogenic endothelial cells and HSPC from murine embryonic tissues.

Fare Embriyonik Hemogenic endotel hücrelerinin izolasyonu

The specification of hemogenic endothelial cells from embryonic vascular endothelium occurs during brief developmental periods within distinct tissues, ...

Isolation of Human Umbilical Vein Endothelial Cells and Their Use in the Study of Neutrophil Transmigration Under Flow Conditions

Neutrophils are the most abundant type of white blood cell. They form an essential part of the innate immune system1. During acute inflammation, neutrophils are the first inflammatory cells to migrate to the site of injury. Recruitment of neutrophils to an injury site is a stepwise process that includes first, dilation of blood vessels to increase blood flow; second, microvascular structural changes and escape of plasma proteins from the bloodstream; third, rolling, adhesion and transmigration of the neutrophil across the endothelium; and fourth accumulation of neutrophils at the site of injury2,3. A wide array of in vivo and in vitro methods has evolved to enable the study of these processes4. This method focuses on neutrophil transmigration across human endothelial cells.
One popular method for examining the molecular processes involved in neutrophil transmigration utilizes human neutrophils interacting with primary human umbilical vein endothelial cells (HUVEC)5. Neutrophil isolation has been described visually elsewhere6; thus this article will show the method for isolation of HUVEC. Once isolated and grown to confluence, endothelial cells are activated resulting in the upregulation of adhesion and activation molecules. For example, activation of endothelial cells with cytokines like TNF-&alpha; results in increased E-selectin and IL-8 expression7. E-selectin mediates capture and rolling of neutrophils and IL-8 mediates activation and firm adhesion of neutrophils. After adhesion neutrophils transmigrate. Transmigration can occur paracellularly (through endothelial cell junctions) or transcellularly (through the endothelial cell itself). In most cases, these interactions occur under flow conditions found in the vasculature7,8.
The parallel plate flow chamber is a widely used system that mimics the hydrodynamic shear stresses found in vivo and enables the study of neutrophil recruitment under flow condition in vitro9,10. Several companies produce parallel plate flow chambers and each have advantages and disadvantages. If fluorescent imaging is needed, glass or an optically similar polymer needs to be used. Endothelial cells do not grow well on glass.
Here we present an easy and rapid method for phase-contrast, DIC and fluorescent imaging of neutrophil transmigration using a low volume ibidi channel slide made of a polymer that supports the rapid adhesion and growth of human endothelial cells and has optical qualities that are comparable to glass. In this method, endothelial cells were grown and stimulated in an ibidi &mu;slide. Neutrophils were introduced under flow conditions and transmigration was assessed. Fluorescent imaging of the junctions enabled real-time determination of the extent of paracellular versus transcellular transmigration.

This article first describes a procedure for isolating human endothelial cells from umbilical veins and then shows how to use these cells to examine neutrophil transmigration under flow conditions. By using a low-volume flow chamber made from a polymer with the optical characteristics of glass, live-cell fluorescent imaging of rare cell populations is also possible.

Akış Koşullarında İnsan umbilikal ven endotel hücreleri ve nötrofil Transmigration Çalışmaları Kullanılması İzolasyonu

Neutrophils are the most abundant type of white blood cell. They form an essential part of the innate immune system1. During acute inflammation, ...

İmmünoloji ve Enfeksiyon

Isolation of Precursor B-cell Subsets from Umbilical Cord Blood

Umbilical cord blood is highly enriched for hematopoietic progenitor cells at different lineage commitment stages. We have developed a protocol for isolating precursor B-cells at four different stages of differentiation. Because genes are expressed and epigenetic modifications occur in a tissue specific manner, it is vital to discriminate between tissues and cell types in order to be able to identify alterations in the genome and the epigenome that may lead to the development of disease. This method can be adapted to any type of cell present in umbilical cord blood at any stage of differentiation.	
This method comprises 4 main steps. First, mononuclear cells are separated by density centrifugation. Second, B-cells are enriched using biotin conjugated antibodies that recognize and remove non B-cells from the mononuclear cells. Third the B-cells are fluorescently labeled with cell surface protein antibodies specific to individual stages of B-cell development. Finally, the fluorescently labeled cells are sorted and individual populations are recovered. The recovered cells are of sufficient quantity and quality to be utilized in downstream nucleic acid assays.

Here we describe a protocol for isolating subsets of precursor B-cells from umbilical cord blood. A sufficient quantity and quality of nucleic acids may be extracted from the cells and used in subsequent assays utilizing DNA or RNA.

Kordon Kanı gelen Prekürsör B-hücreli altkümeleri İzolasyonu

Umbilical cord blood is highly enriched for hematopoietic progenitor cells at different lineage commitment stages. We have developed a protocol for ...

Isolation and Animal Serum Free Expansion of Human Umbilical Cord Derived Mesenchymal Stromal Cells (MSCs) and Endothelial Colony Forming Progenitor Cells (ECFCs)

The umbilical cord is a rich source for progenitor cells with high proliferative potential including mesenchymal stromal cells (also termed mesenchymal stem cells, MSCs) and endothelial colony forming progenitor cells (ECFCs). Both cell types are key players in maintaining the integrity of tissue and are probably also involved in regenerative processes and tumor formation.
To study their biology and function in a comparative manner it is important to have both cells types available from the same donor. It may also be beneficial for regenerative purposes to derive MSCs and ECFCs from the same tissue.
Because cellular therapeutics should eventually find their way from bench to bedside we established a new method to isolate and further expand progenitor cells without the use of animal protein. Pooled human platelet lysate (pHPL) replaced fetal bovine serum in all steps of our protocol to completely avoid contact of the cells to xenogeneic proteins.
This video demonstrates a methodology for the isolation and expansion of progenitor cells from one umbilical cord.
All materials and procedures will be described.

Isolation and Animal Serum Free Expansion of Human Umbilical Cord Derived Mesenchymal Stromal Cells MSCs and Endothelial Colony Forming Progenitor Cells ECFCs

This protocol describes the isolation and subsequent expansion of mesenchymal stromal cells and endothelial colony forming cells without the use of animal serum to generate autologous pairs for experimental transplantation purposes.

İnsan Göbek kordonu İzolasyon ve Hayvan Serum Genişleme Mezenkimal stromal hücreler (MKH) ve endotel Koloni Oluşturan Progenitör Hücreler (ECFCs) Türetilmiş

The umbilical cord is a rich source for progenitor cells with high proliferative potential including mesenchymal stromal cells (also termed mesenchymal ...

Pan-myeloid Differentiation of Human Cord Blood Derived CD34+ Hematopoietic Stem and Progenitor Cells

Ex vivo differentiation of human hematopoietic stem cells is a widely used model for studying hematopoiesis. The protocol described here is for cytokine induced differentiation of CD34+ hematopoietic stem and progenitor cells to the four myeloid lineage cells. CD34+ cells are isolated from human umbilical cord blood and co-cultured with MS-5 stromal cells in the presence of cytokines. Immunophenotypic characterization of the stem and progenitor cells, and the differentiated myeloid lineage cells are described. Using this protocol, CD34+ cells may be incubated with small molecules or transduced with lentiviruses to express myeloid disease mutations to investigate their impact on myeloid differentiation.

Pan-myeloid Differentiation of Human Cord Blood Derived CD34+ Hematopoietic Stem and Progenitor Cells

Here, we present a protocol for immunophenotypic characterization and cytokine induced differentiation of cord blood derived CD34+ hematopoietic stem and progenitor cells to the four myeloid lineages. The applications of this protocol include investigations on the effect of myeloid disease mutations or small molecules on myeloid differentiation of the CD34+ cells.

İnsan Kordon Kanı Kaynaklı CD34 Pan-miyeloid Farklılaşması+ Hematopoetik Kök ve Progenitor Hücreleri

Ex vivo differentiation of human hematopoietic stem cells is a widely used model for studying hematopoiesis. The protocol described here is for cytokine ...

Isolation of Endothelial Progenitor Cells from Healthy Volunteers and Their Migratory Potential Influenced by Serum Samples After Cardiac Surgery

Endothelial progenitor cells (EPCs) are recruited from the bone marrow under pathological conditions like hypoxia and are crucially involved in the neovascularization of ischemic tissues. The origin, classification and characterization of EPCs are complex; notwithstanding, two prominent sub-types of EPCs have been established: so-called &#34;early&#34; EPCs (subsequently referred to as early-EPCs) and late-outgrowth EPCs (late-EPCs). They can be classified by biological properties as well as by their appearance during in vitro culture. While &#34;early&#34; EPCs appear in less than a week after culture of peripheral blood-derived mononuclear cells in EC-specific media, late-outgrowth EPCs can be found after 2-3 weeks. Late-outgrowth EPCs have been recognized to be directly involved in neovascularization, mainly through their ability to differentiate into mature endothelial cells, whereas &#34;early&#34; EPCs express various angiogenic factors as endogenous cargo to promote angiogenesis in a paracrine manner. During myocardial ischemia/reperfusion (I/R), various factors control the homing of EPCs to regions of blood vessel formation.
Macrophage migration inhibitory factor (MIF) is a chemokine-like pro-inflammatory and ubiquitously expressed cytokine and was recently described to function as key regulator of EPCs migration at physiological concentrations1. Interestingly, MIF is stored in intracellular pools and can rapidly be released into the blood stream after several stimuli (e.g. myocardial infarction). 
This protocol describes a method for the reliable isolation and culture of early-EPCs from adult human peripheral blood based on CD34-positive selection with subsequent culture in medium containing endothelial growth factors on fibronectin-coated plates for use in in vitro migration assays against serum samples of cardiac surgical patients. Furthermore, the migratory influence of MIF on chemotaxis of EPCs compared to other well-known angiogenesis-stimulating cytokines is demonstrated.

Endothelial progenitor cells (EPCs) are crucially involved in the neovascularization of ischemic tissues. This method describes the isolation of human EPCs from peripheral blood, as well as the identification of their migratory potential against serum samples of cardiac surgical patients.

Kalp Damar Cerrahisi sonra serum Örnekleri etkilenerek Sağlıklı Gönüllüler ve Bunların Göçmen Potansiyelden Endotel Ata Hücreleri İzolasyon

Endothelial progenitor cells (EPCs) are recruited from the bone marrow under pathological conditions like hypoxia and are crucially involved in the ...

Tıp

Phenotypic and Functional Characterization of Endothelial Colony Forming Cells Derived from Human Umbilical Cord Blood

Longstanding views of new blood vessel formation via angiogenesis, vasculogenesis, and arteriogenesis have been recently reviewed1. The presence of circulating endothelial progenitor cells (EPCs) were first identified in adult human peripheral blood by Asahara et al. in 1997 2 bringing an infusion of new hypotheses and strategies for vascular regeneration and repair. EPCs are rare but normal components of circulating blood that home to sites of blood vessel formation or vascular remodeling, and facilitate either postnatal vasculogenesis, angiogenesis, or arteriogenesis largely via paracrine stimulation of existing vessel wall derived cells3. No specific marker to identify an EPC has been identified, and at present the state of the field is to understand that numerous cell types including proangiogenic hematopoietic stem and progenitor cells, circulating angiogenic cells, Tie2+ monocytes, myeloid progenitor cells, tumor associated macrophages, and M2 activated macrophages participate in stimulating the angiogenic process in a variety of preclinical animal model systems and in human subjects in numerous disease states4, 5. Endothelial colony forming cells (ECFCs) are rare circulating viable endothelial cells characterized by robust clonal proliferative potential, secondary and tertiary colony forming ability upon replating, and ability to form intrinsic in vivo vessels upon transplantation into immunodeficient mice6-8. While ECFCs have been successfully isolated from the peripheral blood of healthy adult subjects, umbilical cord blood (CB) of healthy newborn infants, and vessel wall of numerous human arterial and venous vessels 6-9, CB possesses the highest frequency of ECFCs7 that display the most robust clonal proliferative potential and form durable and functional blood vessels in vivo8, 10-13. While the derivation of ECFC from adult peripheral blood has been presented14, 15, here we describe the methodologies for the derivation, cloning, expansion, and in vitro as well as in vivo characterization of ECFCs from the human umbilical CB.

Endothelial colony forming cells (ECFCs) are circulating endothelial cells with robust clonal proliferative potential that display intrinsic in vivo vessel forming ability. Phenotypic and functional characterization of outgrowth endothelial cells derived from CB are important to identify and isolate bona fide ECFCs for potential clinical application in repairing damaged tissues.

Endotel Colony Forming Hücrelerinin Fenotipik ve Fonksiyonel Karakterizasyonu İnsan Göbek Kordonu Kanı türetilen

Longstanding views of new blood vessel formation via angiogenesis, vasculogenesis, and arteriogenesis have been recently reviewed1. The presence of ...

Ex Vivo Expansion of Hematopoietic Stem Cells from Human Umbilical Cord Blood-derived CD34+ Cells Using Valproic Acid

Umbilical cord blood (UCB) units provide an alternative source of human hematopoietic stem cells (HSCs) for patients who require allogeneic bone marrow transplantation. While UCB has several unique advantages, the limited numbers of HSCs within each UCB unit limits their use in regenerative medicine and HSC transplantation in adults. Efficient expansion of functional human HSCs can be achieved by ex vivo culturing of CD34+ cells isolated from UCBs and treated with a deacetylase inhibitor, valproic acid (VPA). The protocol detailed here describes the culture conditions and methodology to rapidly isolate CD34+ cells and expand to a high degree a pool of primitive HSCs. The expanded HSCs are capable of establishing both short-term and long-term engraftment and are able to give rise to all types of differentiated hematopoietic cells. This method also holds potential for clinical application in autologous HSC gene therapy and provides an attractive approach to overcome the loss of functional HSCs associated with gene editing.

Ex Vivo Expansion of Hematopoietic Stem Cells from Human Umbilical Cord Blood-derived CD34+ Cells Using Valproic Acid

Here, we describe the ex vivo expansion of hematopoietic stem cells from CD34+ cells derived from umbilical cord blood treated with a combination of a cytokine cocktail and VPA. This method leads to a significant degree of ex vivo expansion of primitive HSCs for either clinical or laboratory applications.

EX Vivo insan göbek kordon kanı elde edilen CD34 hematopoetik kök hücrelerden genişlemesi+ hücreleri kullanarak Valproik asit

Umbilical cord blood (UCB) units provide an alternative source of human hematopoietic stem cells (HSCs) for patients who require allogeneic bone marrow ...

Yalıtım endotelyal Progenitor Hücre insan göbek kordon kanı

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