Yazarlar, bu araştırmayı desteklemek için NIH aşağıdaki hibe kabul etmek istiyorum: K08DK082783 (AR), P30DK56465 (BTS), U01HL099993 (BTS), T32HL00715036 (SKS) ve K12HL0806406 (SKS); ve JP McCarthy Vakfı (AR).

Fred Hutchinson Kanser Araştırma Merkezi Kurumsal Değerlendirme Kurulları ve Çocuklar &quot;ın Philadelphia Hastanesi tarafından onaylanan, yazılı izin, periferik kan örneği alınmadan önce hastalardan elde edilmiştir. Tüm kurumsal düzenlemeler gözlendi. MEF nesil dahil olmak üzere tüm hayvan deneyleri ve teratom oluşumu Kurumsal Hayvan Bakım ve Kullanım Kurulu tarafından kabul edildi. PBMC&#39;lerin ve eritroblast Genişleme 1. Ficoll Ayırma - Gündüz 0 <ol><li> Dulbecco Fosfat Tamponlu Salin (DPBS) ile 1: Çevresel kan 1 seyreltin. Bir 15 ml&#39;lik yuvarlak dipli bir polistiren bir tüpte, dikkatli bir şekilde oda sıcaklığında, Ficoll-Hypaque 3 ml&#39;lik seyreltilmiş kan 7 ml katman. </li><li> 30 dakika boyunca 400 x g&#39;de santrifüjleyin. </li><li> PBMC&#39;ler plazma ve bulanık beyaz bir tabaka halinde görülmektedir Ficoll-Hypaque arasındaki arayüz yerleşmiş olacaktır. Steril bir transfer pipet kullanarak, PB toplamakBir 15 ml konik tüp içine MH&#39;si. DPBS kullanarak 10 ml ses getirmek. </li><li> Hemasitometre, PBMC&#39;Ierde saymak. </li><li> 5 dakika boyunca 300 xg&#39;de ayrı 15 ml konik tüp ve santrifüj içine Pipet 2 × 10 6 PBMC&#39;leri. % 90 fetal sığır serumu (FBS) /% 10 dimetil sülfoksit (DMSO) içinde 2 x 10 6 PBMC / ml lik bir konsantrasyonda kalan hücrelerin ve dondurularak aşağı doğru dönerler. </li><li> Genişletme maddesi hazırlayın: QBSF-60 serum barındırmayan ortam hücre faktörü (ışıktan koruma) + Penisilin / streptomisin (% 1) + askorbik asit (50 ng / ml) + kök (SCF, 50 ng / ml) + interlökin 3 (IL- 3, 10 ng / ml) + eritropoietin (EPO, 2 U / ml) +, insülin benzeri büyüme faktörü 1 (IGF-1, 40 ng / ml) + deksametazon (1 uM, ışıktan korumak, taze bir kısım çözülme her medya ) değiştirebilirsiniz. </li><li> Taze genleşme maddesi, 2 ml içinde süspanse 2 x 10 6 PBMC 12 çukurlu bir doku kültür plakasının bir oyuğuna yerleştirmek ve 5 olan bir atmosfere sahip 37 ° C nemlendirilmiş inkübatör% O2,% 5 CO2 ve% 90 N2. </li><li> Gün 3 ve 6. günde, ortamı değiştirin. <ol><li> Hücreleri toplamak ve kalan hücrelerin toplanması için QBSF-60, 2 ml ile iyice yıkayın. </li><li> 5 dakika boyunca 300 xg&#39;de hücre süspansiyonu santrifüj ve süpernatant aspire. </li><li> Yeni Genişleme Orta 2 ml hücreleri tekrar ve aynı 12 plaka onları geri. </li></ol></li></ol> Nucleofection tarafından 2. yeniden programlanması Hücreler - Gün 9 <ol><li> Steril bir 1.5 mi Eppendorf tüpüne (Tablo 1) yeniden programlama plazmidleri Pipet. </li><li> Steril 1.5 ml&#39;lik Eppendorf tüpü içine Supplement (imalatçısı tarafından sağlanan) ve pipet ile nucleofection çözelti karıştırın. </li><li> Pipet 2, bir nemlendirilmiş 37 ° C inkübatör içinde 12 oyuklu plaka ve yer arasında bir oyuğuna genişletme maddesi (% 5 O2,% 5 CO2 ve% 90 N2) önce hücreler ilave edilmeden dengeleme için mi. </li><li> Toplamakkültürlenmiş hücreler kalan hücrelerin toplanması için QBSF-60, 2 ml ile iyice yıkayın ve. </li><li> 5 dakika boyunca 300 xg&#39;de hücreleri Santrifüj ve süpernatant aspire. </li><li> DPBS 5 ml yeniden süspansiyon haline getirilmesini ve 5 dakika boyunca 300 x g&#39;de santrifüj ile hücreler yıkayın. </li><li> Süpernatant aspire. </li><li> Üreten kabarcıkları önlemek için özen, nucleofection solüsyonu + Supplement 100 ul hücre pelletini. </li><li> Aşağı bir kez yeniden programlama plazmidler, pipet ve içeren tüp içine hücre süspansiyonu pipetle ve nucleofection küvetinin dibine örnek pipetle. </li><li> Nucleofection makinenin içine küvet ve çalıştırmak Programı T-019 yerleştirin. </li><li> Nucleofection küvetin içine (Adım 2.3 dengelenmesi plaka) prewarmed Expansion Orta 100 ul aktarın. </li><li> Hemen önceden ısıtılmış Genişleme Orta kuyuya tüm örnek ve mevduat toplamak. Beyaz, yüzen ölü hücre artıkları toplama kaçının. </li&gt; <li> Büyüme için, nemlendirilmiş 37 ° C inkübatör (% 5 O2,% 5 CO2 ve% 90 N2) altında levhayı yerleştirin. </li></ol> 3. Kaplama Besleyici Hücreler - Gün 10 ya da 11 <ol><li> 10. günde, levha besleyici hücreler. <ol><li> Jelatin ile kaplayın, bir 6 oyuklu doku kültürü plakası. <ol><li> Pipet, plakanın her oyuğuna Hank Tamponlu Tuz Çözeltisi (HBSS) içinde 1 M,% 0.1 jelatin. </li><li> En az 5 dakika süreyle, nemlendirilmiş 37 ° C inkübatör plaka koyun. </li><li> Kullanmadan hemen önce, plakadan jelatin solüsyonu aspire. </li></ol></li><li> Ön ısıtması yapılan MEF Media (Dulbecco Modified Eagle Medium (DMEM) + FBS (% 10) + Penisilin / streptomisin (% 1)), 10 ml 3,000 cGy ile ışımaya 2 x 10 6 fare embriyonik fibroblastlar bir şişe çözülme. </li><li> 5 dakika boyunca 300 xg&#39;de hücreleri Santrifüj ve süpernatant aspire. 12 MEF Medya ml ve pipet 2 ml hücreleri tekrar süspansiyon ijelatin kaplı 6 gözlü bir plakanın her bir oyuğuna nGoogle&#39;a. Ortalama 12 olana kadar, bir nemlendirilmiş 37 ° C inkübatör (% 5 O2,% 5 CO2 ve% 90 N2) altında levhayı yerleştirin. </li></ol></li></ol> 4. Besleyici Hücreleri Hücreler Kaplama ve Orta değiştirme - Gün 12 <ol><li> Bir adet 15 ml konik tüp nucleofected hücreleri toplamak ve QBSF-60 kuyu ile 2 ml yıkayarak kalan hücreleri toplamak. 5 dakika boyunca 300 x g&#39;de santrifüjleyin. </li><li> Hazırlama yeniden programlanması Orta: Iscove Modifiye Dulbecco ortamı (IMDM) içinde + FBS (% 10) + hayvansal olmayan L-glutamin (1 mM) + esansiyel olmayan amino asitler (NEAA,% 1) + Penisilin / streptomisin (% 1) + β P-mersaptoetanol (0.1 mM) + temel fibroblast büyüme faktörü + Askorbik asit (bFGF besleme, 10 ng / ml taze ilave edilmiş) (50 ug / ml, besleme taze ilave edildi). </li><li> Süpernatant aspire ve yeniden programlanması Orta 12 ml hücreleri tekrar süspansiyon. Hücre süspansiyonunun oyuk başına Pipet 2 ml &#39;si içerisinebesleyici hücreler 6 oyuklu plaka (ve Bölüm 3&#39;te hazırlandı). Oda sıcaklığında 30 dakika için 50 x g&#39;de plaka santrifüjleyin. </li><li> Büyüme için, nemlendirilmiş 37 ° C inkübatör (% 5 O2,% 5 CO2 ve% 90 N2) altında levhayı yerleştirin. IPSC koloniler (1-2 hafta) görünmeye başlaması kadar yeniden programlanması Medium her gün değiştirin. </li><li> HESC Orta hazırlayın: DMEM / F12 1: 1 + nakavt Serum Replacement (% 20) + penisilin / streptomisin (% 1) + MEM&#39;dir (% 1) + hayvansal olmayan L-glutamin (1 mM) + sodyum piruvat (1%) + sodyum bikarbonat (% 0.12) + β-mersaptoetanol (0.1 mM) +, bFGF (besleme taze ilave edilmiş 10 ng / ml) </li><li> HDAC inhibitörleri hazırlayın: (besleme taze eklendi, 100 iM) sodyum bütirat, suberanilohydroxamic asit (SAHA, 200 nM, beslenme taze eklendi). IPSC koloniler görünür kez, her gün değişen orta, HESC Orta + HDAC inhibitörleri ile hücrelerini besleyerek başlar. Not: Sodyum butirat plastik borular üzerine adsorbe küçük bir molekül olduğu, t&#39;ninherefore 4 ° C&#39;de bir borosilikat cam tüp içinde saklayın. </li></ol> 5. Yalıtımlı iPSC Klonlar <ol><li> IPSC koloni (20-25 hücre) izole edilmesi için yeterince büyük olan bir kez, bir P20 pipet ile çekme. </li><li> HDAC inhibitörleri hazırlayın: sodyum butirat (beslenme taze eklendi, 100 M), SAHA (200 nM, beslenme taze eklendi) </li><li> HESC Orta + HDAC inhibitörleri + Klonlama ve Kurtarma eki içeren bireysel 35 mm yemekleri iMEFs üzerine izole koloniler yerleştirin. </li><li> Gün koloniler toplandıktan sonra, HESC Orta + HDAC inhibitörleri orta değiştirmek. </li><li> IPSC koloniler geçiş için hazır olana kadar her gün orta değiştirin. </li><li> Koloniler pasajda 2-3 stabilize bir kez ortamdan HDAC inhibitörleri çıkarın. </li></ol>

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Yazarlar hiçbir rakip mali çıkarlarının olmadığını beyan ederim.

Bu protokolü kullanarak başarılı iPSC nesil için, dikkat edilmesi gereken birkaç önemli uyarılar vardır. Sapmalar hedef progenitör hücre popülasyonunun verimsiz stimülasyonu ve iPSC nesil daha düşük bir verimliliğe neden olabileceğinden erythroblast genişletme aşamasında, ortam değiştirme programı sıkı, takip edilmelidir. Her ortam değişimi ile taze deksametazon ile yeni genişleme orta yapmak önemlidir; ve QBSF-60 taban ortamı deksametazon ve depolama sırasında ışıktan korunmalıdır....

iPSCs pluripotense genlerin en az sayıda ektopik ekspresyonu ile somatik dokularından elde edilmektedir. Bu teknik, ilk olarak yüksek bir pluripotent durumda 1 olarak ifade edilmiştir Oct4, Sox2, Klf4 ve c-myc insan fibroblastları, retroviral kalıt ile gösterilmiştir. Bu geçici ifade &quot;yeniden programlama faktörleri&quot; insan embriyonik kök hücreleri 2 hedef hücrenin epigenetik peyzaj ve gen ifadesi profili benzer değiştirebilir. Oluşturulan kez, iPSCs potansiyel ileri soruşturma için herhangi bir doku tipinin içine ayırt edilebilir. Böylece, rejeneratif tıpta, hastalık modelleme ve gen terapisi uygulamalarında kullanılmak için umut vermektedir. Ancak, entegre virüs ile genomu bozan endojen gen ifadesinin, etkisi hücresel fenotip değiştirme potansiyeline sahiptir, ve sonuçta bilimsel sonuçlarını taraflı. Ayrıca, rastgele viral entegrasyonlar zararlı hücresel e yol açabilirmalign transformasyon 3 veya onkojenik transgenlerin 4 yeniden ifade olasılığı dahil olmak üzere fektleri. Gelecekteki klinik uygulamalar, entegre iPSC nesil gerektirir. Ekstra kromozomal dairesel DNA molekülleri epizomları, maliyet etkin, entegrasyon-ücretsiz iPSCs 5 üretmek için bir strateji sunar. Tablo 1 &#39;de gösterilen epizomal vektörlerin kombinasyonu yeniden programlama Oct4, Sox2, Klf4 MYCL ve LIN28A faktörleri ifade eder. PCXLE_hOCT3 / 4-shp53-F Plazmid aynı zamanda hücre yeniden programlama 6 geliştirmek için TP53 geçici bastırılması için bir p53 shRNA içerir. Çoğaltma eksik pCXWB-EBNA1 vektör EBNA1 ifade 7 geçici bir artışa sağlayarak yeniden programlama faktörleri büyütülmesine ve artan yeniden programlama verimliliğini teşvik. PCXLE_EGFP plazmid deterjanları amacıyla nucleofection karışımına ilave edilebilirhücre tasnif uygulamalar için transfeksiyon verimliliğinin veya rmining. P CXWB-EBNA1 dışında, bu protokolde kullanılan epizomal plasmidleri konakçı hücrenin, 8 bölünme sırasında replikasyonu ve epizom bölümü vasıta olan viral replikasyon ve EBNA1 gen, Epstein-Barr virüsü kopyalama kaynağını kullanır. Epizomları kendiliğinden ardışık iPSC genişleme 7 ile kaybolur. Alt-klonlama ve EGFP ifade kaybı anlaşılacağı epizomal vektör kaybı ile iPSCs karakterizasyonu, gelecekte yapılacak klinik uygulamalarda tamamen entegrasyon bilgilerini iPSCs yol açabilir. IPSC nesil işleminden dolayı soyundan bir genin bastırılması ve pluripotense ilişkili genlerin reaktivasyonu. Gen ekspresyonunun düzenlenmesi hedef transkripsiyon faktörleri için DNA&#39;nın ve kromatin modifikasyon, düzenleyici DNA elemanları, RNA polimeraz erişimi, çekirdek içinde birden fazla seviyede gerçekleşirgenleri yer alır. Küresel kromatin modifikasyonları ile epigenetik manzara Tadilat yeniden ifade için önemli bir bileşeni Pluripotency genetik program olduğunu. Gen ekspresyonunun düzenlenmesinde önemli olan bir özel kromatin modifikasyon histon-DNA bobinin azalma gerilim üzerinden hedef genlere erişim sağlar, özellikle lizin kalıntıları ile histonların asetilasyon, bir. HDAC inhibitörleri, bağlı asetillenmiş durumunu 11 desteklenmesi için muhtemelen iPSC yeniden programlama ve HESC kendini yenileme 9,10 artırmak için gösterilmiştir küçük moleküllerdir. Entegre lentivirüsler 12 kullanan önceki bir yayında uyarlanan Aşağıda tarif edilen protokol, periferal kan kullanılarak epizomlar ve HDAC inhibitörleri optimize iPSC üretimi için bir adım-adım bir yöntem sağlar. Burada kullanılan adı geçen HDAC inhibitörü konsantrasyonları Ware tarafından tarif edilenler yarısı olan, ve ark., 9 ve rutin standart üzerinde tam Yeniden programlanan iPSC kolonideki 2 kat artışa yol açmıştırHDAC inhibitörleri olmadan episomal yeniden programlama protokolleri. Yeniden programlama Bu düzeyde Lentiviral yöntemleri ile dikkat verimliliği ile eşit olduğunu. Bu protokolü kullanarak, verimli yaş 87 yıl gibi eski kişilerden iPSC yarattı.

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		<tr height="21">
			<td height="21" style="height:21px;width:329px;">Name of Reagent/ Equipment</td>
			<td style="width:151px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:503px;">Comments/Description</td>
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			<td height="21" style="height:21px;width:329px;">Ficoll-Paque PLUS</td>
			<td style="width:151px;">Fisher Scientific</td>
			<td style="width:119px;">45-001-750</td>
			<td>Store at 4&#176;C. Warm to room temperature before use.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">DPBS</td>
			<td style="width:151px;">Life Technologies</td>
			<td style="width:119px;">14190-250</td>
			<td>Store at 4&#176;C.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">RPMI 1640</td>
			<td style="width:151px;">Life Technologies</td>
			<td style="width:119px;">11875-093</td>
			<td>Store at 4&#176;C.</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:329px;">fetal bovine serum (FBS)</td>
			<td style="width:151px;">Life Technologies</td>
			<td style="width:119px;">10437028</td>
			<td>Store at -20&#176;C until needed. Thaw, aliquot, store at 4&#176;C.</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:329px;">dimethyl sulfoxide (DMSO)</td>
			<td style="width:151px;">Sigma-Aldrich</td>
			<td style="width:119px;">154938</td>
			<td>Store at room temperature.</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:329px;">QBSF-60 serum-free medium</td>
			<td style="width:151px;">Fisher Scientific</td>
			<td style="width:119px;">50-983-234</td>
			<td>Store at 4&#176;C.</td>
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			<td height="21" style="height:21px;width:329px;">penicillin/streptomycin</td>
			<td style="width:151px;">Life Technologies</td>
			<td style="width:119px;">15140122</td>
			<td>Store at 4&#176;C.</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:329px;">Cell Line Nucleofector Kit V</td>
			<td style="width:151px;">Lonza</td>
			<td style="width:119px;">VCA-1003</td>
			<td>Store at 4&#176;C.</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:329px;">2% gelatin solution</td>
			<td style="width:151px;">Sigma-Aldrich</td>
			<td style="width:119px;">G1393</td>
			<td>Store at 4&#176;C, liquify in 37&#176;C water bath before use.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">Hank&#39;s Balanced Saline Solution (HBSS)</td>
			<td style="width:151px;">Life Technologies</td>
			<td style="width:119px;">14175-103</td>
			<td>Store at 4&#176;C.</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:329px;">Dulbecco&#39;s Modified Eagle Medium (DMEM)</td>
			<td style="width:151px;">Life Technologies</td>
			<td style="width:119px;">11965-092</td>
			<td>Store at 4&#176;C.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">Iscove&#39;s Modified Dulbecco&#39;s Medium (IMDM)</td>
			<td style="width:151px;">Life Technologies</td>
			<td style="width:119px;">12440-061</td>
			<td>Store at 4&#176;C.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">L-glutamine, 200 mM</td>
			<td style="width:151px;">Life Technologies</td>
			<td style="width:119px;">25030-081</td>
			<td>Aliquot, freeze at -20&#176;C.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">non-essential amino acids (NEAA), 100X</td>
			<td style="width:151px;">Life Technologies</td>
			<td style="width:119px;">11140-050</td>
			<td>Store at 4&#176;C, away from light.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">DMEM/Ham&#39;s F12, 1:1</td>
			<td style="width:151px;">Fisher Scientific</td>
			<td style="width:119px;">SH30023.02</td>
			<td>Store at 4&#176;C.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">KnockOut Serum Replacement</td>
			<td style="width:151px;">Life Technologies</td>
			<td style="width:119px;">10828-028</td>
			<td>Aliquot, freeze at -20&#176;C.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">sodium pyruvate, 100 mM</td>
			<td style="width:151px;">Life Technologies</td>
			<td style="width:119px;">11360-070</td>
			<td>Store at 4&#176;C.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">sodium bicarbonate, 7.5%</td>
			<td style="width:151px;">Life Technologies</td>
			<td style="width:119px;">25080094</td>
			<td>Store at 4&#176;C.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">basic fibroblast growth factor (bFGF)</td>
			<td style="width:151px;">Life Technologies</td>
			<td style="width:119px;">PHG0263</td>
			<td>Make 10 mg/mL stock in DPBS, aliquot, freeze at -20&#176;C. Once thawed, store at 4&#176;C.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">sodium butyrate&#160;</td>
			<td style="width:151px;">Sigma-Aldrich</td>
			<td style="width:119px;">B5887-1G</td>
			<td>Make 2000X stock (400 mM) in DPBS, aliquot, freeze at -20&#176;C. Once thawed, store at 4&#176;C.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">hES Cell Cloning &#38; Recovery Supplement</td>
			<td style="width:151px;">Stemgent</td>
			<td style="width:119px;">01-0014-500</td>
			<td>Store at -20&#176;C until needed. Once thawed, store at 4&#176;C.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">ESC-qualified BD matrigel</td>
			<td style="width:151px;">BD Biosciences</td>
			<td style="width:119px;">35-4277</td>
			<td>Thaw overnight on ice at 4&#176;C, aliquot into pre-chilled tubes using pre-chilled pipette tips. Store at -20&#176;C until needed. Thaw at 4&#176;C, use immediately.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:329px;">StemSpan SFEM&#160;</td>
			<td style="width:151px;">STEMCELL Technologies</td>
			<td style="width:119px;">9650</td>
			<td>Aliquot, freeze at -20&#176;C.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">ascorbic acid, powdered</td>
			<td style="width:151px;">Sigma-Aldrich</td>
			<td style="width:119px;">A4403-100MG</td>
			<td>Make 5 mg/mL stock in DPBS, sterile filter, store at 4&#176;C.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">recombinant human stem cell factor (SCF)</td>
			<td style="width:151px;">R &#38; D Systems</td>
			<td style="width:119px;">255-SC-010</td>
			<td>Make 100 &#956;g/mL stock in SFEM, aliquot, freeze at -20&#176;C. Once thawed, store at 4&#176;C.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">recombinant human interleukin 3 (IL-3)</td>
			<td style="width:151px;">R &#38; D Systems</td>
			<td style="width:119px;">203-IL-010</td>
			<td>Make 100 &#956;g/mL stock in SFEM, aliquot, freeze at -20&#176;C. Once thawed, store at 4&#176;C.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">erythropoietin (EPO)</td>
			<td style="width:151px;">R &#38; D Systems</td>
			<td style="width:119px;">287-TC-500</td>
			<td>Make 1000 U/mL stock in SFEM, aliquot, freeze at -20&#176;C. Once thawed, store at 4&#176;C.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:329px;">recombinant human insulin-like growth factor 1 (IGF-1)</td>
			<td style="width:151px;">R &#38; D Systems</td>
			<td style="width:119px;">291-G1-200</td>
			<td>Make 100 &#956;g/mL stock in SFEM, aliquot, freeze at -20&#176;C. Once thawed, store at 4&#176;C.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">&#946;-mercaptoethanol</td>
			<td style="width:151px;">Sigma-Aldrich</td>
			<td style="width:119px;">M7522</td>
			<td>Make 1000X stock (100 mM) in DPBS.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">dexamethasone</td>
			<td style="width:151px;">Sigma-Aldrich</td>
			<td style="width:119px;">D4902-25MG</td>
			<td>Make 50X stock (50 &#956;M) in DPBS, sterile filter, store at 4&#176;C.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">SAHA (vorinostat)</td>
			<td style="width:151px;">Cayman Chemical</td>
			<td style="width:119px;">149647-78-9</td>
			<td>Make 2000X stock (400 mM) in DPBS, aliquot, freeze at -20&#176;C. Once thawed, store at 4&#176;C.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">12 well tissue culture plate</td>
			<td style="width:151px;">Fisher Scientific</td>
			<td style="width:119px;">08-772-29</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">15 mL conical tube</td>
			<td style="width:151px;">Sarstedt</td>
			<td style="width:119px;">62553002</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">1.5 mL Eppendorf tube</td>
			<td style="width:151px;">Fisher Scientific</td>
			<td style="width:119px;">05-408-129</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">6 well tissue culture plate</td>
			<td style="width:151px;">Fisher Scientific</td>
			<td style="width:119px;">08-772-1B</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">35 mm tisue culture plates</td>
			<td style="width:151px;">BD Biosciences</td>
			<td style="width:119px;">353001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">10 mL disposable serological pipettes</td>
			<td style="width:151px;">Fisher Scientific</td>
			<td style="width:119px;">13-675-20</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">5 mL disposable serological pipettes</td>
			<td style="width:151px;">Fisher Scientific</td>
			<td style="width:119px;">13-675-22</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">2 mL disposable serological pipettes</td>
			<td style="width:151px;">Fisher Scientific</td>
			<td style="width:119px;">13-675-17</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">20 &#956;L pipette tips, barrier tips</td>
			<td style="width:151px;">Genessee</td>
			<td style="width:119px;">24-404</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">glass Pasteur pipettes</td>
			<td style="width:151px;">Fisher Scientific</td>
			<td style="width:119px;">13-678-20D</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">pipette aid</td>
			<td style="width:151px;">Fisher Scientific</td>
			<td style="width:119px;">13-681-15</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">pCXLE-hOCT3/4-shp53-F</td>
			<td style="width:151px;">Addgene</td>
			<td style="width:119px;">27077</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">pCXLE-hSK</td>
			<td style="width:151px;">Addgene</td>
			<td style="width:119px;">27078</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">pCXLE-hUL</td>
			<td style="width:151px;">Addgene</td>
			<td style="width:119px;">27080</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">pCXLE-EGFP</td>
			<td style="width:151px;">Addgene</td>
			<td style="width:119px;">27082</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">pCXWB-EBNA1</td>
			<td style="width:151px;">Addgene</td>
			<td style="width:119px;">37624</td>
			<td></td>
		</tr>
	</tbody>
</table>

efficient ips cell generation from blood using episomes and hdac inhibitors

Bu yazıda verimli episomal plazmitlerini ve histon deasetilaz (HDAC) inhibitörleri kullanılarak periferik kandan entegrasyonu-özgür insan uyarılmış pluripotent kök hücrelerini (iPSCs) oluşturmak için bir protokol göstermektedir. Bu yaklaşımın avantajı şunlardır: (1) bir kaynak malzeme olarak periferal kan minimal bir miktarda kullanımı; (2) yeniden programlama vektörleri nonintegrating; (3) vektörü bilgilerini iPSCs üretilmesi için ekonomik bir yöntem; (4) tek bir transfeksiyon; ve (5) küçük moleküllerin kullanımı epigenetik yeniden programlama kolaylaştırmak için. Tekrar programlanması için oldukça müsait bir yüksek proliferatif eritrosit projenitör hücre popülasyonu elde etmek için kısa bir süre, periferal kan mononükleer hücreleri (PBMC), rutin flebotomi örnekleri izole edilir ve tanımlanmamış başka büyüme faktörleri içinde kültürlenmiştir. Nonintegrating, Oct4, Sox2, Klf4 MYCL, LIN28A ve p53 kısa saç tokası (sh) RNA&#39;yı ifade bulaşıcı olmayan epizomal plasmidleri introdu vardırTek nucleofection ile türetilmiş eritroblast içine ced. Yeşil floresan proteini gelişmiş sentezleyen bir epizom (eGFP) olarak kotransfeksiyon transfekte hücrelerin kolay tanımlama sağlar. Epstein-Barr çekirdek antijeni 1 (EBNA1) ifade eden ayrı bir replikasyon-eksikli Plazmid aynı zamanda epizomal proteinlerin artan ekspresyonu için, reaksiyon karışımına ilave edilir. Transfekte edilmiş hücreler daha sonra, sürekli tekrar programlanması için ışınlanan fare embriyonik fibroblastlar (iMEFs) bir tabaka üzerine kaplanır. En kısa iPSC gibi koloniler nucleofection sonra yaklaşık on iki gün sonra ortaya çıktıkça, HDAC inhibitörleri epigenetik yenileme kolaylaştırmak için ortama ilave edilir. Biz, HDAC inhibitörlerinin dahil rutin olarak 2 kat kadar tam olarak yeniden programlanması iPSC kolonilerin oluşturulmasına arttırdığını bulduk. IPSC koloniler tipik insan embriyonik kök hücre (HESC) morfoloji kez, yavaşça sürekli büyüme ve genişleme için bireysel Imef kaplı doku kültürü plakalarına aktarılır.

Üç gün nucleofection sonra iMEFs üzerine nucleofected hücreleri kaplama önce başarılı nucleofection etkinliği eGFP için floresan mikroskobu ile tahmin edilmelidir. 1 EGFP ifade toplam hücre nüfusunun yaklaşık% 5-10, tipik Nucleofection deneyi göstermektedir. Yeniden programlanan iPSC koloniler nucleofection yaklaşık iki hafta sonra görünmeye başlayacaktır. Koloniler, iyi tanımlanmış sınırlara sahip, genel olarak dairesel olan ve y?...

Watch this Scientific Journal Video about Efficient iPS Cell Generation from Blood Using Episomes and HDAC Inhibitors at JoVE.com

Efficient iPS Cell Generation from Blood Using Episomes and HDAC Inhibitors

Burada bir episomuna göre yeniden programlama strateji ve histon deasetilaz inhibitörleri kullanılarak periferal kandan alınan insan uyarılmış pluripotent kök hücrelerin üretilmesi için bir protokol açıklar.

Epizomlar ve HDAC inhibitörleri kullanarak Blood Verimli iPS Hücre Üretimi

This manuscript illustrates a protocol for efficiently creating integration-free human induced pluripotent stem cells (iPSCs) from peripheral blood using ...

efficient-ips-cell-generation-from-blood-using-episomes-hdac

Research

JoVE Journal

Biology

12.7K Görüntüleme.  Fred Hutchinson Cancer Research Center.  Burada bir episomuna göre yeniden programlama strateji ve histon deasetilaz inhibitörleri kullanılarak periferal kandan alınan insan uyarılmış pluripotent kök hücrelerin üretilmesi için bir protokol açıklar. 

Video: Efficient iPS Cell Generation from Blood Using Episomes and HDAC Inhibitors

Blood Collection from the American Horseshoe Crab, Limulus Polyphemus

The horseshoe crab has the best-characterized immune system of any long-lived invertebrate. The study of immunity in horseshoe crabs has been facilitated by the ease in collecting large volumes of blood and from the simplicity of the blood. Horseshoe crabs show only a single cell type in the general circulation, the granular amebocyte. The plasma has the salt content of sea water and only three abundant proteins, hemocyanin, the respiratory protein, the C-reactive proteins, which function in the cytolytic destruction of foreign cells, including bacterial cells, and &alpha;2-macroglobulin, which inhibits the proteases of invading pathogens. Blood is collected by direct cardiac puncture under conditions that minimize contamination by lipopolysaccharide (a.k.a., endotoxin, LPS), a product of the Gram-negative bacteria. A large animal can yield 200 - 400 mL of blood. For the study of the plasma, blood cells are immediately removed from the plasma by centrifugation and the plasma can then be fractionated into its constituent proteins. The blood cells are conveniently studied microscopically by collecting small volumes of blood into LPS-free isotonic saline (0.5 M NaCl) under conditions that permit direct microscopic examination by placing one of more LPS-free coverglasses on the culture dish surface, then mounting those coverglasses in simple observation chambers following cell attachment. A second preparation for direct observation is to collect 3 - 5 mL of blood in a LPS-free embryo dish and then explanting fragments of aggregated amebocytes to a chamber that sandwiches the tissue between a slide and a coverglass. In this preparation, the motile amebocytes migrate onto the coverglass surface, where they can readily be observed. The blood clotting system involves aggregation of amebocytes and the formation of an extracellular clot of a protein, coagulin, which is released from the secretory granules of the blood cells. Biochemical analysis of washed blood cells requires that aggregation and degranulation does not occur, which can be accomplished by collecting blood into 0.1 volumes of 2% Tween-20, 0.5 M LPS-free NaCl, followed by centrifugation of the cells and washing with 0.5 M NaCl.

The American horseshoe crab, Limulus polyphemus, is arguably the most convenient source for large quantities of blood of any invertebrate. The blood is simple in composition, with only one cell-type in the general circulation, the granular amebocyte, and only three abundant proteins in the plasma, hemocyanin, the C-reactive proteins, and &alpha;2-macroglobulin. Blood is collected from the heart and the blood cells and plasma are separated by centrifugation.

Amerikan Horseshoe Yengeç, Limulus Polyphemus&#39;un Kan Toplama

The horseshoe crab has the best-characterized immune system of any long-lived invertebrate.  The study of immunity in horseshoe crabs has been facilitated ...

Biyoloji

Generation of Single-Cell Suspensions from Mouse Neural Tissue

Within the nervous system, hundreds of neuronal and glial cell types have been described. Each specific cell type in the brain or spinal cord has a repertoire of cell surface molecules, or molecular determinants, through which it can be identified and characterized. Currently, robust cell identification and separation technologies require single-cell preparations to be generated while simultaneously limiting cell death and destruction of characteristic surface protein. The gentleMACS Dissociator, when used in combination with trypsin or papain-based dissociation kits, can effectively and gently dissociate brain tissue while preserving antigen epitopes and limiting cell loss. Standardized preparation of single-cell suspensions is achieved using C Tubes and optimized, preset gentleMACS Programs. Once generated, single-cell suspensions can be treated with monoclonal conjugates like Anti-Prominin-1 MicroBeads, which identify neural progenitors, or purified further using Myelin Removal Beads.

Dissociating cells from specific tissue types requires specific parameters for tissue aggitation to obtain a high volume of viable, culturable cells. The Miltenyi gentleMACS Dissociator optimizes this task with a simple, practical protocol. In this publication the use of this apparatus on nerual tissue is explained.

Fare Sinir Doku Hücre Tek Cezalar Üretimi

Within the nervous system, hundreds of neuronal and glial cell types have been described. Each specific cell type in the brain or spinal cord has a ...

Isolation and Culture of Pulmonary Endothelial Cells from Neonatal Mice

Endothelial cells provide a useful research model in many areas of vascular biology. Since its first isolation 1, human umbilical vein endothelial cells (HUVECs) have shown to be convenient, easy to obtain and culture, and thus are the most widely studied endothelial cells. However, for research focused on processes like angiogenesis, permeability or many others, microvascular endothelial cells (ECs) are a much more physiologically relevant model to study 2. Furthermore, ECs isolated from knockout mice provide a useful tool for analysis of protein function ex vivo. Several approaches to isolate and culture microvascular ECs of different origin have been reported to date 3-7, but consistent isolation and culture of pure ECs is still a major technical problem in many laboratories. Here, we provide a step-by-step protocol on a reliable and relatively simple method of isolating and culturing mouse lung endothelial cells (MLECs). In this approach, lung tissue obtained from 6- to 8-day old pups is first cut into pieces, digested with collagenase/dispase (C/D) solution and dispersed mechanically into single-cell suspension. MLECS are purified from cell suspension using positive selection with anti-PECAM-1 antibody conjugated to Dynabeads using a Magnetic Particle Concentrator (MPC). Such purified cells are cultured on gelatin-coated tissue culture (TC) dishes until they become confluent. At that point, cells are further purified using Dynabeads coupled to anti-ICAM-2 antibody. MLECs obtained with this protocol exhibit a cobblestone phenotype, as visualized by phase-contrast light microscopy, and their endothelial phenotype has been confirmed using FACS analysis with anti-VE-cadherin 8 and anti-VEGFR2 9 antibodies and immunofluorescent staining of VE-cadherin. In our hands, this two-step isolation procedure consistently and reliably yields a pure population of MLECs, which can be further cultured. This method will enable researchers to take advantage of the growing number of knockout and transgenic mice to directly correlate in vivo studies with results of in vitro experiments performed on isolated MLECs and thus help to reveal molecular mechanisms of vascular phenotypes observed in vivo.

Here, we describe a protocol for isolation and culture of murine pulmonary endothelial cells. This method comprises mechanic and enzymatic lung tissue dissociation as well as a 2-step purification process using anti-PECAM-1 and anti-ICAM-2 antibodies conjugated to magnetic beads, which produces a pure endothelial cell population of mostly microvascular origin.

Yenidoğan Fareler Pulmoner Endotel Hücreleri İzolasyon ve Kültür

Endothelial cells provide a useful research model in many areas of vascular biology. Since its first isolation 1, human umbilical vein endothelial cells ...

Generation of Human Induced Pluripotent Stem Cells from Peripheral Blood Using the STEMCCA Lentiviral Vector

Through the ectopic expression of four transcription factors, Oct4, Klf4, Sox2 and cMyc, human somatic cells can be converted to a pluripotent state, generating so-called induced pluripotent stem cells (iPSCs)1-4. Patient-specific iPSCs lack the ethical concerns that surround embryonic stem cells (ESCs) and would bypass possible immune rejection. Thus, iPSCs have attracted considerable attention for disease modeling studies, the screening of pharmacological compounds, and regenerative therapies5.
We have shown the generation of transgene-free human iPSCs from patients with different lung diseases using a single excisable polycistronic lentiviral Stem Cell Cassette (STEMCCA) encoding the Yamanaka factors6. These iPSC lines were generated from skin fibroblasts, the most common cell type used for reprogramming. Normally, obtaining fibroblasts requires a skin punch biopsy followed by expansion of the cells in culture for a few passages. Importantly, a number of groups have reported the reprogramming of human peripheral blood cells into iPSCs7-9. In one study, a Tet inducible version of the STEMCCA vector was employed9, which required the blood cells to be simultaneously infected with a constitutively active lentivirus encoding the reverse tetracycline transactivator. In contrast to fibroblasts, peripheral blood cells can be collected via minimally invasive procedures, greatly reducing the discomfort and distress of the patient. A simple and effective protocol for reprogramming blood cells using a constitutive single excisable vector may accelerate the application of iPSC technology by making it accessible to a broader research community. Furthermore, reprogramming of peripheral blood cells allows for the generation of iPSCs from individuals in which skin biopsies should be avoided (i.e. aberrant scarring) or due to pre-existing disease conditions preventing access to punch biopsies.
Here we demonstrate a protocol for the generation of human iPSCs from peripheral blood mononuclear cells (PBMCs) using a single floxed-excisable lentiviral vector constitutively expressing the 4 factors. Freshly collected or thawed PBMCs are expanded for 9 days as described10,11 in medium containing ascorbic acid, SCF, IGF-1, IL-3 and EPO before being transduced with the STEMCCA lentivirus. Cells are then plated onto MEFs and ESC-like colonies can be visualized two weeks after infection. Finally, selected clones are expanded and tested for the expression of the pluripotency markers SSEA-4, Tra-1-60 and Tra-1-81. This protocol is simple, robust and highly consistent, providing a reliable methodology for the generation of human iPSCs from readily accessible 4 ml of blood.

Here we show a simple and effective protocol for the generation of human iPSCs from 3-4 ml of peripheral blood using a single lentiviral reprogramming vector. Reprogramming of readily available blood cells promises to accelerate the utilization of iPSC technology by making it accessible to a broader research community.

STEMCCA lentiviral Vektör Kullanma Çevresel Blood İnsan İsteyerek Pluripotent Kök Hücre Üretimi

Through the ectopic expression of four transcription factors, Oct4, Klf4, Sox2 and cMyc, human somatic cells can be converted to a pluripotent state, ...

Rapid and Efficient Generation of Neurons from Human Pluripotent Stem Cells in a Multititre Plate Format

Existing protocols for the generation of neurons from human pluripotent stem cells (hPSCs) are often tedious in that they are multistep procedures involving the isolation and expansion of neural precursor cells, prior to terminal differentiation. In comparison to these time-consuming approaches, we have recently found that combined inhibition of three signaling pathways, TGF&beta;/SMAD2, BMP/SMAD1, and FGF/ERK, promotes rapid induction of neuroectoderm from hPSCs, followed by immediate differentiation into functional neurons. Here, we have adapted our procedure to a novel multititre plate format, to further enhance its reproducibility and to make it compatible with mid-throughput applications. It comprises four days of neuroectoderm formation in floating spheres (embryoid bodies), followed by a further four days of differentiation into neurons under adherent conditions. Most cells obtained with this protocol appear to be bipolar sensory neurons. Moreover, the procedure is highly efficient, does not require particular expert skills, and is based on a simple chemically defined medium with cost-efficient small molecules. Due to these features, the procedure may serve as a useful platform for further functional investigation as well as for cell-based screening approaches requiring human sensory neurons or neurons of any type.

Protocols for neuronal differentiation of pluripotent human stem cells (hPSCs) are often time-consuming and require substantial cell culture skills. Here, we have adapted a small molecule-based differentiation procedure to a multititre plate format, allowing simple, rapid, and efficient generation of human neurons in a controlled manner.

Bir Multititre Plaka Biçimi İnsan Pluripotent Kök Hücreler Nöron Hızlı ve Verimli Üretim

Existing protocols for the generation of neurons from human pluripotent stem cells (hPSCs) are often tedious in that they are  multistep procedures ...

Isometric and Eccentric Force Generation Assessment of Skeletal Muscles Isolated from Murine Models of Muscular Dystrophies

Critical to the evaluation of potential therapeutics for muscular disease are sensitive and reproducible physiological assessments of muscle function. Because many pre-clinical trials rely on mouse models for these diseases, isolated muscle function has become one of the standards for Go/NoGo decisions in moving drug candidates forward into patients. We will demonstrate the preparation of the extensor digitorum longus (EDL) and diaphragm muscles for functional testing, which are the predominant muscles utilized for these studies. The EDL muscle geometry is ideal for isolated muscle preparations, with two easily accessible tendons, and a small size that can be supported by superfusion in a bath. The diaphragm exhibits profound progressive pathology in dystrophic animals, and can serve as a platform for evaluating many potential therapies countering fibrosis, and promoting myofiber stability. Protocols for routine testing, including isometric and eccentric contractions, will be shown. Isometric force provides assessment of strength, and eccentric contractions help to evaluate sarcolemma stability, which is disrupted in many types of muscular dystrophies. Comparisons of the expected results between muscles from wildtype and dystrophic muscles will also be provided. These measures can complement morphological and biochemical measurements of tissue homeostasis, as well as whole animal assessments of muscle function.

Muscle function measurements contribute to the evaluation of potential therapeutics for muscle pathology, as well as to the determination of mechanisms underlying physiology of this tissue. We will demonstrate the preparation of the extensor digitorum longus and diaphragm muscles for functional testing. Protocols for isometric and eccentric contractions will be shown, as well as differences in results between dystrophic muscles, representing a pathological state, and wildtype muscles.

İskelet Kası İzometrik ve Eksantrik Kuvvet Oluşturma Değerlendirme İzole Müsküler Distrofisi arasında murin Models

Critical to the evaluation of potential  therapeutics for muscular disease are sensitive and reproducible physiological  assessments of muscle function. ...

Rapid and Efficient Zebrafish Genotyping Using PCR with High-resolution Melt Analysis

Zebrafish is a powerful vertebrate model system for studying development, modeling disease, and performing drug screening. Recently a variety of genetic tools have been introduced, including multiple strategies for inducing mutations and generating transgenic lines. However, large-scale screening is limited by traditional genotyping methods, which are time-consuming and labor-intensive. Here we describe a technique to analyze zebrafish genotypes by PCR combined with high-resolution melting analysis (HRMA).&#160;This approach is rapid, sensitive, and inexpensive, with lower risk of contamination artifacts.&#160;Genotyping by PCR with HRMA can be used for embryos or adult fish, including in high-throughput screening protocols.

PCR combined with high-resolution melt analysis (HRMA) is demonstrated as a rapid and efficient method to genotype zebrafish.

Yüksek çözünürlüklü Melt Analizi ile PCR kullanarak hızlı ve verimli Zebra Genotiplemesi

Zebrafish is a powerful vertebrate model system for studying development, modeling disease, and performing drug screening. Recently a variety of genetic ...

Isolation of Blood-vessel-derived Multipotent Precursors from Human Skeletal Muscle

Since the discovery of mesenchymal stem/stromal cells (MSCs), the native identity and localization&#160;of MSCs have been obscured by their retrospective isolation in culture. Recently, using fluorescence-activated cell sorting (FACS), we and other researchers prospectively identified and purified three subpopulations of multipotent precursor cells associated with the vasculature of human skeletal muscle. These three cell populations: myogenic endothelial cells (MECs), pericytes (PCs), and adventitial cells (ACs), are localized respectively to the three structural layers of blood vessels: intima, media, and adventitia. All of these human blood-vessel-derived stem cell (hBVSC) populations not only express classic MSC markers but also possess mesodermal developmental potentials similar to typical MSCs. Previously, MECs, PCs, and ACs have been isolated through distinct protocols and subsequently characterized in separate studies. The current isolation protocol, through modifications to the isolation process and adjustments in the selective cell surface markers, allows us to simultaneously purify all three hBVSC subpopulations by FACS from a single human muscle biopsy. This new method will not only streamline the isolation of multiple BVSC subpopulations but also facilitate future clinical applications of hBVSCs for distinct therapeutic purposes.

Blood vessels within human skeletal muscle harbor several multi-lineage precursor populations that are ideal for regenerative applications. This isolation method allows simultaneous purification of three multipotent precursor cell populations respectively from three structural layers of blood vessels: myogenic endothelial cells from intima, pericytes from media, and adventitial cells from adventitia.

İnsan İskelet Kası Kan-damar-türevli Multipotent Öncüleri İzolasyonu

Since the discovery of mesenchymal stem/stromal cells (MSCs), the native identity and localization&#160;of MSCs have been obscured by their retrospective ...

Efficient Generation Human Induced Pluripotent Stem Cells from Human Somatic Cells with Sendai-virus

A few years ago, the establishment of human induced pluripotent stem cells (iPSCs) ushered in a new era in biomedicine. Potential uses of human iPSCs include modeling pathogenesis of human genetic diseases, autologous cell therapy after gene correction, and personalized drug screening by providing a source of patient-specific and symptom relevant cells. However, there are several hurdles to overcome, such as eliminating the remaining reprogramming factor transgene expression after human iPSCs production. More importantly, residual transgene expression in undifferentiated human iPSCs could hamper proper differentiations and misguide the interpretation of disease-relevant in vitro phenotypes. With this reason, integration-free and/or transgene-free human iPSCs have been developed using several methods, such as adenovirus, the piggyBac system, minicircle vector, episomal vectors, direct protein delivery and synthesized mRNA. However, efficiency of reprogramming using integration-free methods is quite low in most cases.
Here, we present a method to isolate human iPSCs by using Sendai-virus (RNA virus) based reprogramming system. This reprogramming method shows consistent results and high efficiency in cost-effective manner.

Here, we present our established method to reprogram human somatic cells into transgene-free human iPSCs with Sendai virus, which shows consistent outcome and enhanced efficiency.

Sendai-virüs ile İnsan Somatik Verimli Üretimi İnsan uyarılmış pluripotent kök hücrelerin

A few years ago, the establishment of human induced pluripotent stem cells (iPSCs) ushered in a new era in biomedicine. Potential uses of human iPSCs ...

MicroRNA Expression Profiles of Human iPS Cells, Retinal Pigment Epithelium Derived From iPS, and Fetal Retinal Pigment Epithelium

The objective of this report is to describe the protocols for comparing the microRNA (miRNA) profiles of human induced-pluripotent stem (iPS) cells, retinal pigment epithelium (RPE) derived from human iPS cells (iPS-RPE), and fetal RPE. The protocols include collection of RNA for analysis by microarray, and the analysis of microarray data to identify miRNAs that are differentially expressed among three cell types. The methods for culture of iPS cells and fetal RPE are explained. The protocol used for differentiation of RPE from human iPS is also described. The RNA extraction technique we describe was selected to allow maximal recovery of very small RNA for use in a miRNA microarray. Finally, cellular pathway and network analysis of microarray data is explained. These techniques will facilitate the comparison of the miRNA profiles of three different cell types.

The microRNA (miRNA) profiles of human induced-pluripotent stem (iPS) cells, retinal pigment epithelium (RPE) derived from human induced-pluripotent stem (iPS) cells (iPS-RPE), and fetal RPE, were compared.

İnsan iPS Hücreleri MicroRNA İfade Profilleri, iPS Kaynaklanan Retina pigment epiteli ve Fetal Retina pigment epiteli

The objective of this report is to describe the protocols for comparing the microRNA (miRNA) profiles of human induced-pluripotent stem (iPS) cells, ...