Contreras and Watts Laboratuvarları ve Dr. William Raphael. Bu deneyler NHLBI F31 HL128035-01 (doku sindirim protokol standardizasyonu), NHLBI 5R01HL117847-02 ve 2P01HL070687-11A1 (hayvanlar) ve NHLBI 5R01HL117847-02 (hücre izolasyonu ve kültür) tarafından desteklenmiştir.

Bu yazıda açıklanan tüm prosedürler, Michigan Devlet Üniversitesi&#39;nin Kurumsal Hayvan Bakımı ve Kullanım Komitesi (IACUC) tarafından oluşturulan yönergeleri izlemektedir. Tüm tamponlar ve medyalar ışığa karşı korunmalıdır. 1. Tamponların, Ortamların ve Aletlerin Hazırlanması <ol><li> 135 mM sodyum klorür, 5 mM potasyum klorür, 1 mM magnezyum sülfat, 0.4 mM potasyum fosfat dibazik, 5.5 mM glukoz,% 1 antibiyotik / antimikotik (10,000 ünite / mL penisilin, 10,000 μg) Krebs Ringer Bikarbonat Tamponlanmış çözeltiyi (KRBB) hazırlayın. / ML streptomisin, 25 ug / mL amfoterisin B) ve 10 mM HEPES (pH = 7.4). Bu solüsyon steril tutulduğunda ve 4 ° C&#39;de 3 hafta boyunca stabildir. </li><li> Kollajenaz Tip 1 Solüsyonu hazırlayın: KRBB&#39;de% 1 sığır Serum Albümini (BSA) içeren 1 mg / mL. Bu solüsyon 37 ° C&#39;de tutulmalı ve 4 saat boyunca stabildir. </li><li> Eritrosit Lizis Tamponu Çözeltisi Hazırlayın: 154 mM amonyum klorür, 10 mM potasyum bikarbonat, Ve 0.1 mM EDTA. Bir aya kadar 4 ° C&#39;de tutun. </li><li> MCS Engelleme Tamponu Hazırlayın: DMEM / F12 Ortam tabanı,% 10 Fetal Sığır Serumu (FBS),% 5 normal eşek serum, 40 uL / ​​mL F (ab) Parç편 Eşek Anti-Rat IgG. Bir aya kadar 4 ° C&#39;de tutun. </li><li> MCS Tampon Çözeltisi Hazırlayın: Fosfat Tamponlu Tuz (PBS, pH = 7.5),% 0.5 BSA ve 2 mM EDTA. Bir aya kadar 4 ° C&#39;de tutun. De-gaz çözeltisi, bir cam kap içerisinde 37 ° C&#39;ye ısıtılarak ve daha sonra 15 saniye süreyle vakum uygulanarak hazırlanır. Kullanılmayan tamponlu mühürlü bırakın. </li><li> Dulbecco&#39;s-Modifiye Kartal Ortamı (DMEM): F12,% 15 Fetal Buzağı Serumu (FCS),% 1 antibiyotik / antimikotik (10,000 ünite / mL penisilin, 10,000 μg / mL streptomisin, 25 ug / ML amfoterisin B), 44.05 mM sodyum bikarbonat, 100 uM askorbik asit, 33 uM biyotin, 17 uM pantotenat, 2 mM L-glutamin ve 20 mM HEPES. Steril olun ve 4 ° C&#39;de 2 hafta kadar tutun. </li><li> APC &amp;Lösemi inhibe edici faktör (10 ng / mL), trombosit türevli büyüme faktörü BB (10 ng / mL) ve bazik fibroblast büyüme faktörü (10 ng / mL) dahil olmak üzere ilave büyüme faktörleri olan Basal Medya 5 ng / mL). Steril olun ve 4 ° C&#39;de 2 hafta kadar tutun. </li><li> APC İndüksiyon Ortamı hazırlayın:% 10 FBS, 2.5 μg / mL insülin, 0.5 mM 2-izobütil-1-metilaksantin (IBMX), 1 uM deksametazon ve 200 pM T3 (triiodotironin tiroid hormonu) ile APC Ortamı. Steril olun ve 4 ° C&#39;de 2 hafta kadar tutun. </li></ol> 2. Adiposit Progenitörlerin İzolasyonu <ol><li> Sıçan, kurumsal kurallara göre anestezi altına alınız. Sıçanı sırt üstü yatma durumuna getirin. Anestezi derinliğini bir ayak parmak ucu ve bu acı uyarıya refleks tepkisi kaybı ile teyit edin. NOT: Bu protokol, intraperitoneal bir enjeksiyon yoluyla 10 haftalık Sprague Dawley sıçanları ve 70 mg / kg pentobarbital kullanmaktadır. </li><li> Dikey orta hat kesi wGöğüs bölgesinde sternum boyunca makas ve perineal alana. Karın boşluğuna erişin ve üst mezenterik arteri, küçük mezenterik dirençli damarları (mPVAT) ve torasik aortayı (aPVAT) ortaya çıkarın. <ol><li> Mezenterya ve aortaya olan tüm bağlantıları kesip hayvanlardan damarları çıkarın. Bir disseksiyon mikroskopu ve PVDF&#39;yi izole etmek için KRBB ile dolu Petri kabı kullanarak PVAT&#39;yi ayırın. </li><li> Bu deneyde, bir PVAT olmayan yağ deposunu temsil etmek için gonadal (GON) adipozu toplayın. İzole edilmiş yağ pedlerini KRBB&#39;de buz üzerinde 10 mM HEPES (pH = 7.4) ile yerleştirin. </li><li> Biyogüvenlikli bir kaputta yaklaşık 50 mg doku, 1 mL kollajenaz tip I solüsyonu ile 1.7 mL&#39;lik bir tüpe aktarılır ve doku makası ile kıyılır (1-3 mm&#39;lik parçalar). </li></ol></li><li> 1 saat rotisserie inkübatör (veya bir orbital çalkalayıcı ile inkübatör) 37 ° C&#39;de inkübe ederek numuneleri sindirin. Biyogüvenlikli bir davlumbazda sindirilmiş malzemeyi 100 ve 40<html> Μm hücre süzücüleri 50 mL&#39;lik bir tüp içine yerleştirin. Elde edilen süzüntü, 4 ° C&#39;de 10 dakika, 300 x g&#39;de santrifüjleyin. NOT: Buradan itibaren protokoldeki tüm basamaklar, hücreleri steril tutmak için biyogüvenlikli bir başlıkta gerçekleştirilmelidir. </li><li> Süpernatantı boşaltın ve 1 mL Eritrosit Lizis Tampon Çözeltisi içindeki SVF hücrelerini içeren topakları tekrar süspansiyon haline getirin ve 1.7 mL mikrofüj tüpüne aktarın. Işıktan korunan oda sıcaklığında 5 dakika inkübe edin ve 300 x g&#39;de 5 ° C&#39;de 4 ° C&#39;de santrifüjleyin. </li><li> Süpernatantı boşaltın ve kalan hücre peletini SVF Basal Media&#39;da tekrar süspanse edin. Trypan Blue Solution ile canlı hücreleri saymak için 20 μL alt numune toplayın. NOT: İzole edilebilen SVF hücrelerinin sayısı siteye göre değişir. Mg doku başına hasat edilen ortalama SVF sayısı: aPVAT = 5.0 ± 2.0x103, mPVAT = 1.04 ± 0.62x104, GON = 2.4 ± 1.2x105. </li></ol> 3. Manyetik olarak aktive olan Hücre Ayırma Not: APC&#39;yi, tüm adımları 4 ° C&#39;de gerçekleştirerek CD34 ve PDGFRα hücre yüzey markerlerine dayanan SVF&#39;den ayırın. <ol><li> Hücreleri 300 x g&#39;de 5 dakika döndürün. Süpernatantı boşaltın ve hücre peletini 1 x 10 6 hücre / mL&#39;de MCS Engelleme Tamponuna tekrar süspanse edin ve 20 dakika inkübe edin. NOT: 1 x 10 6 - 2 x 10 8 hücre / mL hücre süspansiyonları etkili bir şekilde ayrılabilir. </li><li> 5 uL FITC konjuge Fare anti-CD34 (1 μg / 1 x 10 6 hücre) ile 30 dakika süreyle inkübe hücreler ° C. Hücreleri 300 xg ve 4 ° C&#39;de 5 dakika döndürün. <ol><li> CD34 + ve CD34 - hücrelerini ayırmak için karanlıkta 4 ° C&#39;de 5 dakika süreyle 4 μL anti-FITC mikro-boncukları ve 96 uL MCS tamponu (toplam hacim 100 μL) ile inkübe edin. </li></ol></li><li> Manyetik ayırıcıyı standa takın ve MultiSort (MS) Kolonunu, kolon kanatları öne gelecek şekilde ayırıcıya yerleştirin. 5 mL yerleştirinToplama tüpünü üst boru tutucudaki MS sütununun altına yerleştirin. </li><li> MS Sütununu, MCS Tampon Çözeltisi ile durulayarak hazırlayın. Sütun üstüne 500 μL MCS Tamponu uygulayın ve arabellek geçmesine izin verin. Atık suyu atın ve toplama tüpünü değiştirin. </li><li> Hazırlanan MS sütununa antikor etiketli hücre süspansiyonu yükleyin. Etiketsiz hücreler içeren akışı toplayın. </li><li> MS sütununu 500 μL gazı alınmış MCS Tamponu 3x ile yıkayın. Etiketsiz hücreleri toplayın ve bir önceki aşamadaki akışla birleşin. </li><li> MS Sütununu manyetik ayırıcıdan çıkarın ve yeni bir toplama tüpüne yerleştirin. MS sütununa 1 mL MCS Tamponu pipetleyin. Manyetik olarak etiketlenmiş hücrelerle fraksiyonu hemen sık sık, fakat yavaşça sütun içerisine fazla gaz girmemesi için kolonla birlikte verilen pistonu uygulayarak boşaltın. NOT: Yalıtılmış hücre sayıları bölgeye göre değişir. SVF popülasyonundan izole edilmiş ortalama CD34 + APC sayısıMg doku: aPVAT = 2.6 ± 0.43x102, mPVAT = 9.6 ± 1.4x102, GON = 1.3 ± 0.22x103. </li><li> Hücreleri 5 dakika 300 × g ve 4 ° C&#39;de döndürün. 10 μL 1: 200 çözelti / 1 x 10 6 hücre tavşan anti-PDGFRα toplanan CD34 + fraksiyonu 4 ° C&#39;de 30 dakika inkübe edin. <ol><li> 300 xg ve 4 ° C&#39;de 5 dakika boyunca hücreleri tekrar santrifüjleyin. Etiketli hücreleri, izole etmek için 3.3-3.7 adımlarını tekrarlayarak 4 uL anti-tavşan IgG mikro-boncukları ve 96 uL MCS tamponu ile inkübe edin. NOT: Yalıtılmış hücre sayıları bölgeye göre değişir. Mg doku başına CD34 + popülasyondan izole edilen PDGFRα + APC&#39;nin ortalama sayıları: aPVAT = 2.4 ± 0.64, mPVAT = 8.4 ± 2.4, GON = 10.4 ± 1.9, ki bu daha önce izole edilen popülasyonun% 0.5-10&#39;udur. </li></ol></li></ol> 4. Hücre Kültürü ve Adipogenez İndüksiyonu <ol><li> SVF&#39;yi kültür edinVe APC&#39;yi 6-Well doku kültürü plakalarında bazal medyumda değiştirerek her 2 günde bir değiştirin. 3 seri pasajdan sonra, 8, 24, 48 ve 96 saatte ve 24 oyuklu plakalarda 50,000 hücre / çukurda değerlendirilen çoğalma tahlilleri için siyah 96-oyuklu doku kültürü plakalarını 1x102 hücre / oyuk ile veya Nitel ve nicel adipogenez analizleri için 48-Well doku kültürü plakalarında 10,000 hücre / çukur. </li><li> Konfüzyon sonrası 48 saat ve kemik morfojenik protein 4 (3.3 nmol / L) ile endüksiyon öncesi APC bazal medyasını, farklılaşma için 10 olarak belirtildiği gibi ilave edin. Hücreleri inkübe etmek için APC İndüksiyon Ortamını kullanarak 48 saatlik% 100 konfluans (gün 0) sonrasında hücreleri indükleyin. </li><li> 48 saat sonra, IBMX ve deksametazon içermeyen APC İndüksiyon ortamında hücreleri muhafaza etmek için medyayı değiştirin; medyanın her 48 saatte bir değiştirilmesi ile 14 gün boyunca. </li><li> FACS tarafından onaylanan MCS izolasyonu. <ol><li> 50 μL (50.000 hücre) alt örnek manyetik olarak ayırınAted hücreler ve FACS çözeltisi ile yıkayın. </li><li> Hücreleri santrifüjleyin ve PDGFRα + hücrelerini etiketlemek için, eşek anti-tavşan IgG Dylight 405&#39;in 1: 1000 çözeltisinden 100 uL&#39;ye tekrar süspanse edin. Işıktan ve 4 ° C&#39;de 30 dakika inkübe edin. </li><li> Hücreleri yıkayın ve bir akış sitometresinde 488 nm (FITC) ve 405 nm (Dylight 405) filtreler kullanarak analiz için zaman kadar 200 uL% 2 formaldehid solüsyonuna tekrar süspanse edin. NOT: Kültürlenmiş hücrelerin lipid birikimi, lipofilik bir adipogenezis flüoresans tahlilini kullanarak, flüoresanı ölçen bir mikroplate okuyucu kullanılarak ve lipid birikimi için kalibratörler olarak preadipositleri kullanarak niceliksel olarak değerlendirilir. Lipid birikimi ayrıca nitel olarak lipid boyası lekelenmesi ve bir kamera ile donatılmış ters mikroskop üzerinde gerçekleştirilen görüntüleme ile ölçülür ve lipid gözlemlenebilen veya içermeyen toplam hücrelerin yüzdesini oluşturur. 485 nm&#39;de uyarılma ve 572&#39;de emisyon ile flüoresanı ölçebilen herhangi bir plaka okuyucuNm, analiz için olduğu kadar dijital görüntüleri yakalayabilen bir kameraya sahip herhangi bir mikroskop için de uygundur. </li></ol></li></ol>

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Yazarların açıklayacak bir şeyi yok.

Bu deneyin odak noktası PVAT depolarından APC&#39;nin izolasyonu, genleşmesi ve adipogenik indüksiyonudur. Burada yüzey işaretleri CD34 ve PDGFRα ifade hücrelerin tanımlanması dayalı APC izolasyonu için bir protokol sunuyoruz. Bu yüzey proteinleri daha önce APC üzerinde yüksek proliferasyon oranları ve çeşitli yağ depoları 14,15&#39;te beyaz veya kahverengi adipositleri ayırma potansiyeli ile tanımlandı. Bu belirteçlere dayalı hücreleri seçerek, seçilen PVAT&#39;de gözlemlenen adiposit feno...

Perivasküler Adipoz Doku (PVAT), kan damarlarına yakın olması nedeniyle damar sistemi fonksiyonu 1&#39;de önemli parakrin sinyal bileşenidir. Bu yağ dokusunun genleşmesi, 2 , 3 mevcut Adiposit Progenitör Hücrelerinin (APC) fenotipine bağlıdır. Primer adipositler kırılgan, yüzen ve aralıklı olduğundan, yağ dokularından hücrelerin izolasyonu zordur. Bazı izolasyon teknikleri aynı zamanda hücrelerin bütünlüğünü koruyan bir protokolün önemini vurgulayan, inflamatuvar protein sentezini arttırarak ve adipogenik gen ekspresyonunu azaltarak hücre fenotipini ve morfolojisini değiştirebilir. Birincil hücrelerin kültürü ve spesifik preadiposit alt popülasyonları, in vivo büyümeye indirgemeci bir yaklaşım sağlar ve eşdeğer hücresel genetik yapıyı korur 5 , ancak tiYaşlanma, ya da yaşlanma 6 ile bozulma nedeniyle bu hücrelerle beni sınırlıdır. Deri altı ve omental depolar da dahil olmak üzere çeşitli yağ depolardan preadipositler, spesifik anatomik bölgelerden hücrelerin toplanmasının önemini vurgulayan proliferasyon 7&#39;deki farklılıkları da gösterir. PVAT dışı beyaz yağ depolarından öncül hücreler önceki çalışmalar 7 , 8 , 9&#39;da karakterize edilmiştir, ancak PVAT APC fenotipleri hakkında daha az bilgi bilinmektedir. Burada açıklanan teknikler, morfolojileri, yaşayabilirlikleri ve çoğalması ve farklılaşması potansiyeli üzerinde asgari düzeyde etkisi olan spesifik ve tanımlanmış APC popülasyonlarının analiz edilmesine izin verir. Manyetik olarak aktive olan Hücre Ayırma (MCS), hücrenin değiştirilmeden boncukların çözünmesi nedeniyle kültür gibi müteakip uygulamalara uygundur. MCS de ekonomiktir ve bir kez antikor koniSantitler standart hale getirildiğinden, akış sitometrisi deneylerine olan ihtiyaç azdır. PVAT öncülleri ile yapılan in vitro çalışmalar, bu primer hücrelerin sahip olabileceği potansiyelin bir görüntüsünü de verebilir.

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expansion and adipogenesis induction of adipocyte progenitors from perivascular adipose tissue isolated by magnetic activated cell sorting

Parakrin sinyal vasıtasıyla vasküler fonksiyonların büyük düzenleyicisi olan perivasküler yağ dokusunun (PVAT) genişlemesi, obezite sırasında hipertansiyonun gelişimi ile doğrudan ilişkilidir. Hipertrofi ve hiperplazinin derecesi, depo yer, cinsiyet ve mevcut Adiposit Progenitör Hücre (APC) fenotiplerinin türüne bağlıdır. Son 10 yılda APC ve preadipositler izolasyonu için kullanılan teknikler, spesifik hücre yüzeyi belirteçlerine dayalı olarak tek tek hücrelerin tanımlanabileceği doğruluğunu büyük ölçüde geliştirmiştir. Bununla birlikte, APC ve adipositlerin izolasyonu, özellikle hücrelerin hücre kültürü uygulamaları için muhafaza edilmesi gerekiyorsa, hücrenin kırılganlığından dolayı bir meydan okuma olabilir. Manyetik olarak aktive olan Hücre Ayırma ( MCS), yağ dokusunun ağırlık birimi başına daha fazla canlı APC&#39;yi izole etmek için bir yöntem sağlar. MCS tarafından hasat edilen APC, preapeni genişletmek için in vitro protokoller için kullanılabilirDipositleri ve büyüme faktörü kokteyllerini kullanarak hücreler tarafından tutulan üretken ve adipojenik potansiyelin analizine izin vererek adipositlere ayırdık. Bu deney genişleme sırasında kardiyovasküler hastalığın gelişiminde kilit rol oynayan aortik ve mezenterik PVAT depoları üzerine odaklanmıştır. Bu protokoller, APC&#39;nin tanımlanmış bir popülasyonunu izole etmek, genişletmek ve ayırmak için kullanılan yöntemleri tanımlamaktadır. Bu MCS protokolü, hücrenin sınıflandırılmasına minimum ekipman veya eğitimle ihtiyaç duyulan herhangi bir deneyde izolasyonun kullanılmasını sağlar. Bu teknikler, hücre yüzeyi markörlerinin varlığına dayanan spesifik hücre popülasyonlarının işlevselliğini belirlemek için ileri deneylere yardımcı olabilir.

Preadipositlerin proliferatif kapasitesi ve adiposit öncüllerinin adipojenik potansiyeli, in vitro 11&#39;de muhafaza edilen özelliklerdir. İzole SVF&#39;nin ve APC&#39;nin, erkek sıçanlardan aPVAT, mPVAT ve GON&#39;dan in vitro proliferasyonu, kantitatif bir DNA testi kullanılarak kaplamadan 8, 24, 48 ve 96 saat sonra değerlendirildi. Aynı bölgedeki SVF hücrelerine kıyasla 96 saat daha az proliferasyona sahip olan bir PVA&#3...

Watch this Scientific Journal Video about Expansion and Adipogenesis Induction of Adipocyte Progenitors from Perivascular Adipose Tissue Isolated by Magnetic Activated Cell Sorting at JoVE.com

Expansion and Adipogenesis Induction of Adipocyte Progenitors from Perivascular Adipose Tissue Isolated by Magnetic Activated Cell Sorting

Burada Manyetik Aktif Hücre Ayırma (MCS) kullanarak Perivasküler Adipoz Doku (PVAT) Adiposit Progenitör Hücre (APC) popülasyonlarının izolasyonu için bir yöntem rapor. Bu yöntem, floresan-Aktive Hücre Ayırma (FACS) ile karşılaştırıldığında, yağ dokusunun gramı başına APC&#39;nin artan bir izolasyonuna izin verir.

Manyetik Aktive Hücre Ayırma ile İzole Edilen Perivasküler Adipoz Doku&#39;ndan Adiposit Progenitörlerinin Genleşmesi ve Adipogenez İndüksiyonu

Expansion of Perivascular Adipose Tissue (PVAT), a major regulator of vascular function through paracrine signaling, is directly related to the ...

expansion-adipogenesis-induction-adipocyte-progenitors-from

Research

JoVE Journal

Developmental Biology

9.4K Görüntüleme.  Michigan State University.  Burada Manyetik Aktif Hücre Ayırma (MCS) kullanarak Perivasküler Adipoz Doku (PVAT) Adiposit Progenitör Hücre (APC) popülasyonlarının izolasyonu için bir yöntem rapor. Bu yöntem, floresan-Aktive Hücre Ayırma (FACS) ile karşılaştırıldığında, yağ dokusunun gramı başına APC'nin artan bir izolasyonuna izin verir. 

Video: Expansion and Adipogenesis Induction of Adipocyte Progenitors from Perivascular Adipose Tissue Isolated by Magnetic Activated Cell Sorting

Ex Vivo Culture of Pharyngeal Arches to Study Heart and Muscle Progenitors and Their Niche

The pharyngeal mesoderm of developing embryos contributes to broad regions of head and heart musculature. We have developed a novel method to study head and heart progenitor cell development with pharyngeal arches (also known as branchial arches) ex vivo. Using this method, we have recently described that the second pharyngeal arch contains self-renewing heart progenitors and serves as a microenvironment for expansion of the progenitors during mouse heart development. The progenitor cells remain undifferentiated and expansive inside the arch, but quickly become functional cardiomyocytes as they migrate out of the arch. We also reported that first pharyngeal arch contains muscle progenitors giving rise to myotubes after leaving the arch. Here, we demonstrate the procedure for the dissection and ex vivo culture of first and second pharyngeal arches from developing mouse embryos. The method enables one to study head and heart progenitor/muscle development, including cardiomyocyte and myotube formation in detail ex vivo.

Ex Vivo Culture of Pharyngeal Arches to Study Heart and Muscle Progenitors and Their Niche

Here, we present a protocol to culture pharyngeal arches to study the biology of heart and muscle progenitor cells and their microenvironment.

Faringeal Arches Ex Vivo Kültür Kalp ve kas atalarıdır ve Bunların Niche Eğitim için

The pharyngeal mesoderm of developing embryos contributes to broad regions of head and heart musculature. We have developed a novel method to study head ...

Gelişim Biyolojisi

Isolation and Differentiation of Adipose-Derived Stem Cells from Porcine Subcutaneous Adipose Tissues

Obesity is an unconstrained worldwide epidemic. Unraveling molecular controls in adipose tissue development holds promise to treat obesity or diabetes. Although numerous immortalized adipogenic cell lines have been established, adipose-derived stem cells from the stromal vascular fraction of subcutaneous white adipose tissues provide a reliable cellular system ex vivo much closer to adipose development in vivo. Pig adipose-derived stem cells (pADSC) are isolated from 7- to 9-day old piglets. The dorsal white fat depot of porcine subcutaneous adipose tissues is sliced, minced and collagenase digested. These pADSC exhibit strong potential to differentiate into adipocytes. Moreover, the pADSC also possess multipotency, assessed by selective stem cell markers, to differentiate into various mesenchymal cell types including adipocytes, osteocytes, and chondrocytes. These pADSC can be used for clarification of molecular switches in regulating classical adipocyte differentiation or in direction to other mesenchymal cell types of mesodermal origin. Furthermore, extended lineages into cells of ectodermal and endodermal origin have recently been achieved. Therefore, pADSC derived in this protocol provide an abundant and assessable source of adult mesenchymal stem cells with full multipotency for studying adipose development and application to tissue engineering of regenerative medicine.

This protocol describes the isolation of pig adipose-derived stem cells (pADSC) from subcutaneous adipose tissues with examination of multipotency. The multipotent pADSC are used to delineate processes of adipocyte differentiation and study transdifferentiation into multiple cell lineages of mesodermal mesenchyme or further lineages of ectoderm and endoderm for regenerative studies.

Domuz Subkutan yağ dokularındaki İzolasyonu ve Yağ-Türetilmiş Kök Hücre Farklılaşması

Obesity is an unconstrained worldwide epidemic. Unraveling molecular controls in adipose tissue development holds promise to treat obesity or diabetes. ...

Isolation and Expansion of Mesenchymal Stem/Stromal Cells Derived from Human Placenta Tissue

Mesenchymal stem/stromal cells (MSC) are promising candidates for use in cell-based therapies. In most cases, therapeutic response appears to be cell-dose dependent. Human term placenta is rich in MSC and is a physically large tissue that is generally discarded following birth. Placenta is an ideal starting material for the large-scale manufacture of multiple cell doses of allogeneic MSC. The placenta is a fetomaternal organ from which either fetal or maternal tissue can be isolated. This article describes the placental anatomy and procedure to dissect apart the decidua (maternal), chorionic villi (fetal), and chorionic plate (fetal) tissue. The protocol then outlines how to isolate MSC from each dissected tissue region, and provides representative analysis of expanded MSC derived from the respective tissue types. These methods are intended for pre-clinical MSC isolation, but have also been adapted for clinical manufacture of placental MSC for human therapeutic use.

Herein we describe methods for the dissection of fetal and maternal tissues from human term placenta, followed by isolation and expansion of mesenchymal stem/stromal cells (MSC) from these tissues.

İnsan Plasenta Doku türetilen İzolasyon ve mezenkimal kök Genişleme / Stromal hücreler

Mesenchymal stem/stromal cells (MSC) are promising candidates for use in cell-based therapies. In most cases, therapeutic response appears to be cell-dose ...

Isolation of Perivascular Multipotent Precursor Cell Populations from Human Cardiac Tissue

Multipotent mesenchymal stem/stromal cells (MSC) were conventionally isolated, through their plastic adherence, from primary tissue digests whilst their anatomical tissue location remained unclear. The recent discovery of defined perivascular and MSC cell marker expression by perivascular cells in multiple tissues by our group and other researchers has provided an opportunity to prospectively isolate and purify specific homogenous subpopulations of multipotent perivascular precursor cells. We have previously demonstrated the use of fluorescent activated cell sorting (FACS) to purify microvascular CD146+CD34- pericytes and vascular CD34+CD146- adventitial cells from human skeletal muscle. Herein we describe a method to simultaneously isolate these two perivascular cell subsets from human myocardium by FACS, based on the expression of a defined set of cell surface markers for positive and negative selections. This method thus makes available two specific subpopulations of multipotent cardiac MSC-like precursor cells for use in basic research and/or therapeutic investigations.

Human cardiac tissue harbours multipotent perivascular precursor cell populations that may be suitable for myocardial regeneration. The technique described here allows for the simultaneous isolation and purification of two multipotent stromal cell populations associated with native blood vessels, i.e. CD146+CD34- pericytes and CD34+CD146- adventitial cells, from the human myocardium.

İnsan Kardiyak Doku perivasküler Multipotent Öncü hücre popülasyonlarının izolasyonu

Multipotent mesenchymal stem/stromal cells (MSC) were conventionally isolated, through their plastic adherence, from primary tissue digests whilst their ...

Induction of Acute Skeletal Muscle Regeneration by Cardiotoxin Injection

Skeletal muscle regeneration is a physiological process that occurs in adult skeletal muscles in response to injury or disease. Acute injury-induced skeletal muscle regeneration is a widely used, powerful model system to study the events involved in muscle regeneration as well as the mechanisms and different players. Indeed, a detailed knowledge of this process is essential for a better understanding of the pathological conditions that lead to skeletal muscle degeneration, and it aids in identifying new targeted therapeutic strategies. The present work describes a detailed and reproducible protocol to induce acute skeletal muscle regeneration in mice through a single intramuscular injection of cardiotoxin (CTX). CTX belongs to the family of snake venom toxins and causes myolysis of myofibers, which eventually triggers the regeneration events. The dynamics of skeletal muscle regeneration is evaluated by histological analysis of muscle sections. The protocol also illustrates the experimental procedures for dissecting, freezing, and cutting the Tibialis Anterior muscle, as well as the routine Hematoxylin &#38; Eosin staining that is widely used for subsequent morphological and morphometric analysis.

This manuscript describes a detailed protocol to induce acute skeletal muscle regeneration in adult mice and subsequent manipulations of the muscles, such as dissection, freezing, cutting, routine staining, and myofiber cross-sectional area analysis.

Cardiotoxin Enjeksiyonu ile Akut İskelet Kası Regeneration indüksiyon

Skeletal muscle regeneration is a physiological process that occurs in adult skeletal muscles in response to injury or disease. Acute injury-induced ...

Chondrogenic Differentiation Induction of Adipose-derived Stem Cells by Centrifugal Gravity

Impaired cartilage cannot heal naturally. Currently, the most advanced therapy for defects in cartilage is the transplantation of chondrocytes differentiated from stem cells using cytokines. Unfortunately, cytokine-induced chondrogenic differentiation is costly, time-consuming, and associated with a high risk of contamination during in vitro differentiation. However, biomechanical stimuli also serve as crucial regulatory factors for chondrogenesis. For example, mechanical stress can induce chondrogenic differentiation of stem cells, suggesting a potential therapeutic approach for the repair of impaired cartilage. In this study, we demonstrated that centrifugal gravity (CG, 2,400 &#215; g), a mechanical stress easily applied by centrifugation, induced the upregulation of sex determining region Y (SRY)-box 9 (SOX9) in adipose-derived stem cells (ASCs), causing them to express chondrogenic phenotypes. The centrifuged ASCs expressed higher levels of chondrogenic differentiation markers, such as aggrecan (ACAN), collagen type 2 alpha 1 (COL2A1), and collagen type 1 (COL1), but lower levels of collagen type 10 (COL10), a marker of hypertrophic chondrocytes. In addition, chondrogenic aggregate formation, a prerequisite for chondrogenesis, was observed in centrifuged ASCs.

Mechanical stress can induce the chondrogenic differentiation of stem cells, providing a potential therapeutic approach for the repair of impaired cartilage. We present a protocol to induce the chondrogenic differentiation of adipose-derived stem cells (ASCs) using centrifugal gravity (CG). CG-induced upregulation of SOX9 results in the development of chondrogenic phenotypes.

Santrifüj Yerçekimi ile adipoz kaynaklı Kök Hücrelerin kondrojenik Farklılaşma İndüksiyon

Impaired cartilage cannot heal naturally. Currently, the most advanced therapy for defects in cartilage is the transplantation of chondrocytes ...

Identification Of Erythromyeloid Progenitors And Their Progeny In The Mouse Embryo By Flow Cytometry

Macrophages are professional phagocytes from the innate arm of the immune system. In steady-state, sessile macrophages are found in adult tissues where they act as front line sentinels of infection and tissue damage. While other immune cells are continuously renewed from hematopoietic stem and progenitor cells (HSPC) located in the bone marrow, a lineage of macrophages, known as resident macrophages, have been shown to be self-maintained in tissues without input from bone marrow HSPCs. This lineage is exemplified by microglia in the brain, Kupffer cells in the liver and Langerhans cells in the epidermis among others. The intestinal and colon lamina propria are the only adult tissues devoid of HSPC-independent resident macrophages. Recent investigations have identified that resident macrophages originate from the extra-embryonic yolk sac hematopoiesis from progenitor(s) distinct from fetal hematopoietic stem cells (HSC). Among yolk sac definitive hematopoiesis, erythromyeloid progenitors (EMP) give rise both to erythroid and myeloid cells, in particular resident macrophages. EMP are only generated within the yolk sac between E8.5 and E10.5 days of development and they migrate to the fetal liver as early as circulation is connected, where they expand and differentiate until at least E16.5. Their progeny includes erythrocytes, macrophages, neutrophils and mast cells but only EMP-derived macrophages persist until adulthood in tissues. The transient nature of EMP emergence and the temporal overlap with HSC generation renders the analysis of these progenitors difficult. We have established a tamoxifen-inducible fate mapping protocol based on expression of the macrophage cytokine receptor Csf1r promoter to characterize EMP and EMP-derived cells in vivo by flow cytometry.

While infiltrating macrophages are continuously recruited to adult tissues from circulating precursors, resident macrophages seed their tissue during development, where they are maintained without further input from progenitors. The progenitors for resident macrophages were recently identified. Here, we present methods for the genetic fate mapping of the resident macrophage progenitors.

Akım Sitometresi ile Fare Embriyosunda Eritromiyeloid Progenitörlerin Ve Döllerinin Belirlenmesi

Macrophages are professional phagocytes from the innate arm of the immune system. In steady-state, sessile macrophages are found in adult tissues where ...

Whole-cell Patch-clamp Recordings of Isolated Primary Epithelial Cells from the Epididymis

The epididymis is an essential organ for sperm maturation and reproductive health. The epididymal epithelium consists of intricately connected cell types that are distinct not only in molecular and morphological features but also in physiological properties. These differences reflect their diverse functions, which together establish the necessary microenvironment for the post-testicular sperm development in the epididymal lumen. The understanding of the biophysical properties of the epididymal epithelial cells is critical for revealing their functions in sperm and reproductive health, under both physiological and pathophysiological conditions. While their functional properties have yet to be fully elucidated, the epididymal epithelial cells can be studied using the patch-clamp technique, a tool for measuring the cellular events and the membrane properties of single cells. Here, we describe the methods of cell isolation and whole-cell patch-clamp recording to measure the electrical properties of primary dissociated epithelial cells from the rat cauda epididymides.

We present a protocol that combines cell isolation and whole-cell patch-clamp recording to measure the electrical properties of the primary dissociated epithelial cells from the rat cauda epididymides. This protocol allows for investigation of the functional properties of primary epididymal epithelial cells to further elucidate the physiological role of the epididymis.

Epididimden İzole Edilmiş Primer Epitelyal Hücrelerin Tüm Hücreli Yama-Kelepçesi Kayıtları

The epididymis is an essential organ for sperm maturation and reproductive health. The epididymal epithelium consists of intricately connected cell types ...

Generation of Scaffold-free, Three-dimensional Insulin Expressing Pancreatoids from Mouse Pancreatic Progenitors In Vitro

The pancreas is a complex organ composed of many different cell types that work together to regulate blood glucose homeostasis and digestion. These cell types include enzyme-secreting acinar cells, an arborized ductal system responsible for the transportation of enzymes to the gut, and hormone-producing endocrine cells. 
Endocrine beta-cells are the sole cell type in the body that produce insulin to lower blood glucose levels. Diabetes, a disease characterized by a loss or the dysfunction of beta-cells, is reaching epidemic proportions. Thus, it is essential to establish protocols to investigate beta-cell development that can be used for screening purposes to derive the drug and cell-based therapeutics. While the experimental investigation of mouse development is essential, in vivo studies are laborious and time-consuming. Cultured cells provide a more convenient platform for screening; however, they are unable to maintain the cellular diversity, architectural organization, and cellular interactions found in vivo. Thus, it is essential to develop new tools to investigate pancreatic organogenesis and physiology.
Pancreatic epithelial cells develop in the close association with mesenchyme from the onset of organogenesis as cells organize and differentiate into the complex, physiologically competent adult organ. The pancreatic mesenchyme provides important signals for the endocrine development, many of which are not well understood yet, thus difficult to recapitulate during the in vitro culture. Here, we describe a protocol to culture three-dimensional, cellular complex mouse organoids that retain mesenchyme, termed pancreatoids. The e10.5 murine pancreatic bud is dissected, dissociated, and cultured in a scaffold-free environment. These floating cells self-assemble with mesenchyme enveloping the developing pancreatoid and a robust number of endocrine beta-cells developing along with the acinar and the duct cells. This system can be used to study the cell fate determination, structural organization, and morphogenesis, cell-cell interactions during organogenesis, or for the drug, small molecule, or genetic screening.

Generation of Scaffold-free, Three-dimensional Insulin Expressing Pancreatoids from Mouse Pancreatic Progenitors In Vitro

Here, we present a protocol to generate insulin expressing 3D murine pancreatoids from free-floating e10.5 dissociated pancreatic progenitors and the associated mesenchyme.

İskele-free, üç boyutlu insülin ifade Pancreatoids fare pankreas ataları Vitro gelen nesil

The pancreas is a complex organ composed of many different cell types that work together to regulate blood glucose homeostasis and digestion. These cell ...

Efficient Generation of Pancreas/Duodenum Homeobox Protein 1+ Posterior Foregut/Pancreatic Progenitors from hPSCs in Adhesion Cultures

Human pluripotent stem cell (hPSC)-derived pancreatic cells are a promising cell source for regenerative medicine and a platform to study human developmental processes. Stepwise directed differentiation that recapitulates developmental processes is one of the major ways to generate pancreatic cells including pancreas/duodenum homeobox protein 1+ (PDX1+) pancreatic progenitor cells. Conventional protocols initiate the differentiation with small colonies shortly after the passage. However, in the state of colonies or aggregates, cells are prone to heterogeneities, which might hamper the differentiation to PDX1+ cells. Here, we present a detailed protocol to differentiate hPSCs into PDX1+ cells. The protocol consists of four steps and initiates the differentiation by seeding dissociated single cells. The induction of SOX17+ definitive endoderm cells was followed by the expression of two primitive gut tube markers, HNF1&#946; and HNF4&#945;, and eventual differentiation into PDX1+ cells. The present protocol provides easy handling and may improve and stabilize the differentiation efficiency of some hPSC lines that were previously found to differentiate inefficiently into endodermal lineages or PDX1+ cells.

Efficient Generation of Pancreas/Duodenum Homeobox Protein 1+ Posterior Foregut/Pancreatic Progenitors from hPSCs in Adhesion Cultures

Here, we present a detailed protocol to differentiate human pluripotent stem cells (hPSCs) into pancreas/duodenum homeobox protein 1+ (PDX1+) cells for the generation of pancreatic lineages based on the non-colony type monolayer growth of dissociated single cells. This method is suitable for producing homogenous hPSC-derived cells, genetic manipulation and screening.

Manyetik Aktive Hücre Ayırma ile İzole Edilen Perivasküler Adipoz Doku'ndan Adiposit Progenitörlerinin Genleşmesi ve Adipogenez İndüksiyonu

Özet

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Faringeal Arches Ex Vivo Kültür Kalp ve kas atalarıdır ve Bunların Niche Eğitim için

Domuz Subkutan yağ dokularındaki İzolasyonu ve Yağ-Türetilmiş Kök Hücre Farklılaşması

İnsan Plasenta Doku türetilen İzolasyon ve mezenkimal kök Genişleme / Stromal hücreler

İnsan Kardiyak Doku perivasküler Multipotent Öncü hücre popülasyonlarının izolasyonu

Cardiotoxin Enjeksiyonu ile Akut İskelet Kası Regeneration indüksiyon

Santrifüj Yerçekimi ile adipoz kaynaklı Kök Hücrelerin kondrojenik Farklılaşma İndüksiyon

Akım Sitometresi ile Fare Embriyosunda Eritromiyeloid Progenitörlerin Ve Döllerinin Belirlenmesi

Epididimden İzole Edilmiş Primer Epitelyal Hücrelerin Tüm Hücreli Yama-Kelepçesi Kayıtları

İskele-free, üç boyutlu insülin ifade Pancreatoids fare pankreas ataları Vitro gelen nesil

Verimli üretimi, pankreas/oniki parmak bağırsağı Homeobox Protein 1⁺ Posterior Foregut/pankreas ataları hPSCs yapışma kültürlerde üzerinden

Manyetik Aktive Hücre Ayırma ile İzole Edilen Perivasküler Adipoz Doku'ndan Adiposit Progenitörlerinin Genleşmesi ve Adipogenez İndüksiyonu

Özet

Daha Fazla Video Keşfet

Bu videodaki bölümler

Faringeal Arches Ex Vivo Kültür Kalp ve kas atalarıdır ve Bunların Niche Eğitim için

Domuz Subkutan yağ dokularındaki İzolasyonu ve Yağ-Türetilmiş Kök Hücre Farklılaşması

İnsan Plasenta Doku türetilen İzolasyon ve mezenkimal kök Genişleme / Stromal hücreler

İnsan Kardiyak Doku perivasküler Multipotent Öncü hücre popülasyonlarının izolasyonu

Cardiotoxin Enjeksiyonu ile Akut İskelet Kası Regeneration indüksiyon

Santrifüj Yerçekimi ile adipoz kaynaklı Kök Hücrelerin kondrojenik Farklılaşma İndüksiyon

Akım Sitometresi ile Fare Embriyosunda Eritromiyeloid Progenitörlerin Ve Döllerinin Belirlenmesi

Epididimden İzole Edilmiş Primer Epitelyal Hücrelerin Tüm Hücreli Yama-Kelepçesi Kayıtları

İskele-free, üç boyutlu insülin ifade Pancreatoids fare pankreas ataları Vitro gelen nesil

Verimli üretimi, pankreas/oniki parmak bağırsağı Homeobox Protein 1+ Posterior Foregut/pankreas ataları hPSCs yapışma kültürlerde üzerinden

Verimli üretimi, pankreas/oniki parmak bağırsağı Homeobox Protein 1⁺ Posterior Foregut/pankreas ataları hPSCs yapışma kültürlerde üzerinden