This work was supported by a Pilot and Feasibility Grants from the Indiana University School of Medicine Center (IUSM) for Excellence in Molecular Hematology (NIDDK, 5P30DK090948). Additional funding was provided by a Biomedical Enhancement Grant from IUSM. We would like to thank Dr. Karen Cowden Dahl for commenting on the manuscript.

1. embriyoidlerin Vücut (EB) Oluşumu <ol><li> Fare embriyo fibroblast (MEF&#39;ler) üzerinde muhafaza edilmiştir EKH jelatin adapte. Geçiş EKH MEFS çıkarmak için% 0.1 jelatin ile kaplanmış 6 gözlü doku kültür plakası üzerine 3 kez. <ol><li> (Tablo 1) farklılaşmamış hücreleri korumak için LİF ile ESC muhafaza ortamında hücrelerin büyümesine. Emin hücreler asla 80% izdiham aşan olun. Farklılaşması için hücrelerinden (MEF&#39;lerden çıkardıktan sonra 10 veya daha az pasajlar) düşük geçişini kullanın. </li></ol></li><li> EKH EB farklılaşması için kültürlenir önceki gün, hücreler, geçit böylece yaklaşık% 50-70 oranında konfluent ertesi gün olacaktır. Hücre pluripotensini korumak ESC bakım ortamı içinde kültürlenmesi devam edin. </li><li> 6-çukurlu levha başına: farklılaşması, aspirat ESC bakım ortamına ve günde 1 ml fosfat tamponlu salin (137 mM NaCI, 2.7 mM KCI, ve 11.9 mM fosfat tamponu, pH 7.4 PBS) ile yıkayın. <ol><li> 0.25% tryps 200 ul ekleyin/ her EDTA de ve 3 dakika süreyle 37 ° C&#39;de inkübe edin. </li><li> Farklılaşma medya 800 ul tripsin / EDTA inaktive. Pipet yukarı ve aşağı P200 ucu ile 2 ml serolojik pipet yıkmak kadar hücreleri tek bir hücre süspansiyonu içine. Medya pipet ve uç arayüzü arasında yukarı gitmez yüzden P200 ucu ucuna rahatça oturduğundan emin olun. </li><li> Pipet yukarı ve aşağı yaklaşık 5-10 kez. Pipetle sırasında baloncuklar oluşturmak için özen gösterin. </li></ol></li><li> 15 ml konik tüp hücreleri aktarın. PBS ile 10 ml sesini getirin. Oda sıcaklığında 5 dakika boyunca 315 x g&#39;de santrifüjleyin. </li><li> Süpernatantı. Lif içeren Kalan ortamı çıkarmak için 5 ml PBS ile iki kez yıkayın. Her yıkamadan için adım 1.4 olarak santrifüj. <ol><li> Farklılaşma medya (Tablo 2), 2 ml nihai hücre topağı tekrar. </li></ol></li><li> Bir hemasitometre veya hücre sayacı ile hücrelerin sayısı ve 10 cm&#39;lik bir petri plakasında 6,000-10,000 hücre / ml plaka(Non-doku kültürü tedavi). <ol><li> Her ESC hattı için ampirik tam hücre / ml belirleyin. Normalde bakteri iş veya düşük yapışma plakaları için kullanılan steril Petri tabak kullanın. NOT: En iyi sonuçlar için farklılaşma, EBS plaka takmadan süspansiyon kalır küre oluşturmalıdır. Az bağlılıkla olanları bulmak için plakaların çeşitli markaları test edin. </li><li> % 5 CO2 içeren bir 37 ° C&#39;de doku kültürü inkübatörü içinde, ayırt EKH inkübe edin. </li></ol></li></ol><table border="0" cellpadding="0" cellspacing="0"><colgroup><col /><col span="2" /><col /><col /><col /></colgroup><tbody><tr><td> Reaktif </td><td> Stok </td><td> Final Konsantrasyon </td><td> Cilt </td><td> Şirket </td><td> Katalog Numarası </td></tr><tr><td> DMEM </td><td> 1x </td><td></td><td> 410 ml </td><td> S<html> IGMA / Aldrich </td><td> D5796 </td></tr><tr><td> FBS (ES Ekranlı) </td><td> 100% </td><td> % 15 </td><td> 75 mi </td><td> Hycione </td><td> SH30070.03E </td></tr><tr><td> Gerekli olmayan Amino Asitler </td><td> 100x </td><td> 1x </td><td> 5 mi </td><td> Life Technologies </td><td> 11140 </td></tr><tr><td> L-Glutamin </td><td> 100x </td><td> 1x </td><td> 5 mi </td><td> Life Technologies </td><td> 35050 </td></tr><tr><td> Penncillin / Streptomisin </td><td> 100x </td><td> 1x </td><td> 5 mi </td><td> Life Technologies </td><td> 15070 </td></tr><tr><td> β-mersaptoetanol </td><td> 14.3 M </td><td> 114 iM </td><td> 4 ul </td><td> Sigma / Aldrich </td><td> M3148 </td></tr><tr><td> , Lösemi önleyici faktörü (LİF) </td><td> 10 7 ünite / ml </td><td> 1.000 adet / ml </td><td> 50 ul </td><td> Millipore </td><td> ESG1107 </td></tr></tbody></table>&quot;ontent&gt; Tablo 1: ESC Bakım Medya. <table border="0" cellpadding="0" cellspacing="0"><colgroup><col /><col span="2" /><col /><col span="2" /></colgroup><tbody><tr><td> Reaktif </td><td> Stok </td><td> Final Konsantrasyon </td><td> Cilt </td><td> Şirket </td><td> Katalog Numarası </td></tr><tr><td> DMEM </td><td> 1x </td><td></td><td> 410 ml </td><td> Sigma / Aldrich </td><td> D5796 </td></tr><tr><td> FBS (Kullanıcı optimum farklılaşması için Ekranlı) </td><td> 100% </td><td> % 15 </td><td> 75 mi </td><td> Hycione </td><td> SH30070.03E </td></tr><tr><td> Gerekli olmayan Amino Asitler </td><td> 100x </td><td> 1x </td><td> 5 mi </td><td> Life Technologies </td><td> 11140 </td></tr><tr><td> L-Glutamin </td><td> 100x </td><td> 1x </td><td> 5 mi </td><td> Yaşam Technolonolojiler </td><td> 35050 </td></tr><tr><td> Penncillin / Streptomisin </td><td> 100x </td><td> 1x </td><td> 5 mi </td><td> Life Technologies </td><td> 15070 </td></tr><tr><td> β-mersaptoetanol </td><td> 14.3 M </td><td> 114 iM </td><td> 4 ul </td><td> Sigma / Aldrich </td><td> M3148 </td></tr></tbody></table> Tablo 2: Farklılaşma Medya. Virüs Enfeksiyonu 2. hazırlanması EB Hücreler <ol><li> Arzu edilen gelişme aşamasında (2. gün ve 3. gün arasında), 15 ml konik bir tüp için 10 cm Petri için ikinci EBS aktarın. 5 ml PBS ile plaka yıkayın ve konik tüp yıkama ekleyin. </li><li> 5 dakika boyunca oda sıcaklığında 315 xg&#39;de EBS Pelet. 5 ml PBS ile EB hücreleri yıkayın. </li><li> Çözülen hücre ayırma çözeltisi (kolajenolitik içeren proteolitik ve enzimlerin) 1 ml ilave edilir ve OCC ile 30 dakika bir 37 ° C su banyosunda tüpleri (veya inkübatör) yerleştirmek(flicking veya hafif vorteks itibaren) asional ajitasyon. </li><li> Farklılaşma medya 1 ml ilave edilir. Pipet yukarı ve aşağı P200 pipet ucu ile 2 ml pipetle. 30-60 dakika boyunca daha 37 ° C su banyosu içinde tüp yerleştirin. </li><li> 5 dakika boyunca 315 xg&#39;de hücreleri aşağı Spin ve PBS ile hücreleri yıkayın. </li></ol> 3. Virüs Enfeksiyonu <ol><li> Farklılaşma medya 1.5 ml süspansiyon hücreleri. 6 çukurlu bir doku kültür olmayan tedavi plaka hücre süspansiyonu aktarın. </li><li> Vaktinden önce standart teknikler ile retrovirüsü hazırlayın. Viral titreye bağlı olarak hücrelere, viral süpernatanın 5-100 ul ekleyin. Bu EBS sonraki reform engeller gibi enfeksiyon geliştirmek için polybrene katmayın. </li><li> Oda sıcaklığında 90 dakika boyunca 2120 x g&#39;de santrifüj ile virüs hücreleri Spinoculate. </li></ol> 4. Asma Damlalar Viral Enfekte EB Hücreleri Farklılaşan <ol><li> I her çukuruna farklılaşma medya 2 ml ekleyinnfected EB hücre süspansiyonu. </li><li> Pipet multipipettors için steril bir tepkin madde rezervuarına enfekte İT hücre süspansiyonu, 3.5 ml. Sekiz 20 ul, 15-20 satırlar 15 cm&#39;lik Petri plakanın ters kapak üzerine damla Pipeti için multipipettor kullanın. </li><li> Petri levhasının alt yarısına steril PBS veya su ve 10 ml ilave edilir. Sonra Petri plaka üzerine düşer ve yeri ile kapağı ters çevirin. Damla odası nemlendirilmiş tutmak için baş aşağı PBS veya altında su ile kapak asılı emin olun. </li><li> % 5.0 CO2 ile 37 ° C doku kültürü inkübatörü içinde, yemekler yerleştirin. </li><li> 2 gün sonra, farklılaşma medya 4.5 ml, kapak ve yıkama üst edilerek damla asılı oluşan EBS toplar. <ol><li> Yeni bir 10 cm olmayan doku kültürü Petri plaka Transferi medya ve EBS. Farklılaşma medya ve 10 cm plaka transfer 3 ml ile bir kez daha kapağı yıkayın. </li></ol></li><li> Sitometrisi 8 olarak toplam akışı analizi için hücreler hasatfarklılaşma medya (-LIF) hücrelerin ilk transferi itibaren gün kültür. </li></ol> Akış Sitometresi Analizi 5. hazırlanması Hücreler <ol><li> 10 ml&#39;lik bir pipet ile 15 ml konik tüp hücreleri aktarın. 5 ml PBS ile plaka yıkayın ve aynı konik tüp transfer. </li><li> 315 x g Pelet hücreleri. 10 ml PBS ile pelet yıkayın. </li><li> Çözülen hücre ayırma çözeltisi 1 ml ilave edilir ve ara sıra çalkalanarak 30 dakika boyunca 37 ° C su banyosunda tüpleri. Bazı hücre yüzey antijenlerinin tespit edilmesi için, tripsin, bunun yerine kullanılabilir. </li><li> 1 ml farklılaşma medya ekleyin ve pipetlemeyin yukarı ve 2 ml pipet aşağı. Yardımcı olmak için pipet ucuna P200 ucu kullanın kırılma tek bir hücre süspansiyonu içine EB dijeste edilmiştir. <ol><li> Integral zar proteinleri, hücre yüzeyine geri dönüşüm için sırayla, 60 dakika boyunca daha 37 ° C su banyosu içinde tüp yerleştirin. </li></ol></li><li> 5 dakika boyunca 315 xg&#39;de hücrelerini pellet haline getirmek. </li><li> Hücreler, wi yıkayıninci% 0.1 Sığır Serum Albumin Fraksiyon PBS (IX PBS / BSA) içinde hazırlanmıştır V (BSA) ve belirli ile hücrelerin inkübe floresan etiketli antikorlar. </li></ol> Hematopoetik progenitörlerin 6. Sitometrisi Analizi <ol><li> 5 ml, 12 x 75 mm yuvarlak alt tüp içinde 10 6 EB türetilen hücreler yerleştirin. Tazminat sitometre ayarlarını akışını ayarlamak için, üretici talimatlarına göre tazminat boncuk kullanmayın. </li><li> 315 x g Pelet hücreleri. Tüp içinde kalan PBS / BSA çözeltisi içinde süpernatant ve tekrar süspansiyon pelet kapalı dökün. </li><li> ÇIK HPC algılamak için, flüoresan etiketli antikorlar ile hücrelerin inkübe: anti-fare CD41-PE (R Phycoerytherin) ve anti-fare CD117 (cKit) -APC / Cy7 (alofikosiyanin Siyanin 7). <ol><li> 1 antikorları seyreltin PBS / BSA içinde 100. Her bir örnek için seyreltilmiş 100 ul antikor ekleyin. Her bir satıcı, klonlama ve çok ya da antikor için antikorun doğru seyreltme belirler. Hücreleri tek tek etiketlenmesi için optimal seyreltme belirleyin. </li&gt; </ol></li><li> 30 dakika daha karanlıkta buz üzerinde örnekleri inkübe edin. </li><li> 5 dakika boyunca 315 x g&#39;de 2 ml PBS / BSA ve topak hücrelere ilave edin. </li><li> Aspire yüzenler ve 300 ul PBS veya başka bir izotonik tampon içinde tekrar süspansiyon pelet. </li><li> Bir hücre süzgeç kapağı (0.35 mcM) aracılığıyla filtre süspanse edilen hücreler, büyük hücreli agrega kaldırmak için. </li><li> Bir akış sitometresindeki hücreleri analiz. </li></ol>

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Yazarlar ifşa rakip mali çıkarlarını hiçbir var.

Yukarıda ele alındığı gibi, ilgi konusu bir indüklenebilir gen ifade ÇIK klon doksisiklin sistemler kullanılarak elde edilebilir, bununla birlikte, bu satırların kuşak zaman alıcı ve emek yoğundur. Bu protokolde tarif EBS türetilmiş ESC hazırlanan tek hücre süspansiyonu ilgi konusu bir geni ifade etmek için bir yöntemdir. Bu enfekte olmuş hücreler, daha sonra, sonraki farklılaşma incelemek asılı damla ile EBS olarak yenilenmiş. Örnekte (Şekil 3), bu farklılaşma 3. günde ...

Murine embryonic stem cells (ESCs) are pluripotent, remaining undifferentiated and self-renewing in the presence of the cytokine Leukemia Inhibitory Factor (LIF)1. Upon withdrawal of LIF they will spontaneously differentiate into 3-dimensional (3D) structures called embryoid bodies (EBs)2. The 3D architecture allows for the development of the three germ layers ectoderm, endoderm, and mesoderm, which then later give rise to mature tissue types3. ESCs are an exceptional model for elucidating the molecular mechanisms of cellular differentiation, particularly the investigation of the development of early hematopoietic progenitor cells (HPCs)4. 
Gene expression in ESCs can be manipulated by several techniques that allow for the determination of a role for individual molecules in development. One of the most common techniques is to use homologous recombination to generate ESC lines, which lack a gene of interest5,6. There are also a number of techniques that have been used to overexpress genes. The first technique used to modify gene expression in ESCs was to infect them with recombinant retroviruses7,8. The gene of interest however is often silenced as the ESCs differentiate into progenitor and mature cell types. Use of lentiviruses has been successful in limiting the silencing of virally expressed genes9. Other viral vectors used for overexpression include adenovirus and adeno-associated virus10. In addition standard transfection techniques to stably introduce expression plasmids are widely used for ESC transgene expression11. One difficulty with these systems is that often expression of a specific gene has different effects depending on its temporal expression. For example, the Smad1 protein affects the development of hematopoietic cells differently during different stages of EB development12,13. 
This problem can be circumvented by the generation of ESCs that inducibly express a gene of interest. The most common system for inducibly expressing transgenes in ESCs uses the tetracycline resistance operon from E. Coli (Escherichia coli). Several different tetracycline systems have been developed. One of the more popular strategies was developed by Kyba and colleagues14. They generated an ESC line (Ainv15), which has the reverse tetracycline transactivator gene inserted into the constitutively active ROSA26 locus. A tetracycline response element (TRE), a downstream LoxP site (locus of X-over P1 from bacteriophage P1), and a promoterless neomycin cassette were introduced into the HPRT (Hypoxanthine-guanine phosphoribosyltransferase) locus on the X chromosome. Using a CRE recombination approach a gene of interest along with a eukaryotic promoter to drive the expression of the neomycin resistance gene can be inserted into the LoxP site. Correctly targeted ESCs are isolated by G418 selection. These targeted clones then must be tested for doxycycline (or tetracycline) inducible expression of the transgene. This approach has successfully generated ESC lines that inducibly express HoxB4, Stat5, SCL, and Smad714-17. However, generation of these inducible cell lines is time consuming. Described here is a method to disaggregate ESC-derived embryoid bodies (EBs) into single cell suspensions, retrovirally infect the cell suspensions at different days of development, and then reform the EBs by hanging drop. Downstream differentiation is later evaluated by flow cytometry. In this article an example of how miRNA expression in EB-derived cells effects hematopoietic differentiation is shown. This method is useful for investigating the role of genes after specific germ layer tissue is derived.

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</table>

retroviral infection of murine embryonic stem cell derived embryoid body cells for analysis of hematopoietic differentiation

Embryonic stem cells (ESCs) are an outstanding model for elucidating the molecular mechanisms of cellular differentiation. They are especially useful for investigating the development of early hematopoietic progenitor cells (HPCs). Gene expression in ESCs can be manipulated by several techniques that allow the role for individual molecules in development to be determined. One difficulty is that expression of specific genes often has different phenotypic effects dependent on their temporal expression. This problem can be circumvented by the generation of ESCs that inducibly express a gene of interest using technology such as the doxycycline-inducible transgene system. However, generation of these inducible cell lines is costly and time consuming. Described here is a method for disaggregating ESC-derived embryoid bodies (EBs) into single cell suspensions, retrovirally infecting the cell suspensions, and then reforming the EBs by hanging drop. Downstream differentiation is then evaluated by flow cytometry. Using this protocol, it was demonstrated that exogenous expression of a microRNA gene at the beginning of ESC differentiation blocks HPC generation. However, when expressed in EB derived cells after nascent mesoderm is produced, the microRNA gene enhances hematopoietic differentiation. This method is useful for investigating the role of genes after specific germ layer tissue is derived.

RW4 jelatinize Bu çalışmalarda kullanılmıştır EKH (129X1 / SVJ fare suşundan). EBS farklılaşma 3 gün sonra izole edilmiştir. En iyi sonuçlar için EBS küresel ve yapışkan olmayan (Şekil 1A, B) olmalıdır. Virüs, ilgili gen ile birlikte GFP floresan markörü ile birlikte ifade spinoculation sonra EBS olarak asılı damla yöntemiyle ıslah edilmiştir. EBS başarılı 2.0, 2.5, ve 3.0 gün EBS hazırlanan hücre süspansiyonları ıslah edilmiştir. Ancak, EBS günden 4,0 EBS hazırla...

Watch this Scientific Journal Video about Retroviral Infection of Murine Embryonic Stem Cell Derived Embryoid Body Cells for Analysis of Hematopoietic Differentiation at JoVE.com

Retroviral Infection of Murine Embryonic Stem Cell Derived Embryoid Body Cells for Analysis of Hematopoietic Differentiation

Manipulating temporal gene expression in differentiating embryonic stem cells (ESCs) can be achieved using inducible gene systems. However, generation of these cell lines is costly and time consuming. This protocol achieves rapid expression of a transgene in differentiating ES-derived cells and subsequent analysis of downstream hematopoietic differentiation.

Kemirgen Embriyonik retroviral Enfeksiyon Hematopoetik Farklılaşma Analizi için Hücre Türetilmiş embriyoidlerin Vücut Kök Hücreler

Embryonic stem cells (ESCs) are an outstanding model for elucidating the molecular mechanisms of cellular differentiation. They are especially useful for ...

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8.4K Görüntüleme.  Harper Cancer Research Institute. Manipulating temporal gene expression in differentiating embryonic stem cells (ESCs) can be achieved using inducible gene systems. However, generation of these cell lines is costly and time consuming. This protocol achieves rapid expression of a transgene in differentiating ES-derived cells and subsequent analysis of downstream hematopoietic differentiation. 