Development of this protocol was supported by the Advancing a Healthier Wisconsin and the National Center for Advancing Translational Sciences, National Institutes of Health, through Grant Number 8UL1TR000055. Its contents are solely the responsibility of the authors and do not necessarily represent the official views of the NIH.

NOT: Hastalar ve / veya veliler çalışmayı onayladı bir Kurumsal Değerlendirme Kurulu katılmak için bilgilendirilmiş onam vermelidir. 1. Tamponlar ve Medya Hazırlıklar <ol><li> Yıkama tamponu: yıkama tamponu 100 ml hazırlamak fosfat tamponlu salin (PBS) ile 99 ml 100x pen / strep çözeltisi (Streptomisin 100 U / ml penisilin + 100 ug / ml), 1 ml ekleyin. </li><li> İdrar Progenitor Hücre (UPC) Orta: UPC orta hazırlamak keratinosit Serum Ücretsiz (KSF) Orta + Progenitor Hücre Orta eşit hacimlerde karıştırmak için. <ol><li> KSF Orta: epidermal büyüme faktörü 5 ng / ml (EGF), büyükbaş hayvan hipofız bezi ekstraktı, 50 ng / ml (BPB), kolera toksini, 30 ng / ml, ve pen / strep çözeltisi (100 u ilave keratinosit ortamı hazırlamak için / ml penisilin, 500 ml, KSF ortam için, 100 ug / ml streptomisin). </li><li> Progenitor Hücre Orta: FBS, 0.4 ug / ml hidrokortizon, 0.1 Ham-F12 medya dörtte biri ile DMEM dörtte üçü ekleyin ve% 10 ile eknM, kolera toksini, 5 ng / ml insülin, 1.8 x 10 -4 M adenin / ml transferin, 2 x 10 -9 M triiyodo tironin, 10 ng / ml EGF ve pen / strep solüsyonu 5 ug. </li></ol></li><li> embriyonik kök Ortamı:% 20 KO-serum yerine (K-SR), 1x MEM esansiyel olmayan amino asitler, 2 mM L-glutamin, 100 uM β-merkaptoetanol, 20 ng / ml bFGF ve pen / strep ekleyin 400 mi DMEM / F12 ortamı. </li></ol> 2. İdrar Numune Toplama <ol><li> Idrar yeterli miktarda (~ 30-40 ml) tahsil edilebilir olmasını sağlamak için idrar toplama 30 dakika önce sıvıları içmek hasta söyleyin. </li><li> Hasta ve / veya onların veliler aşağıdaki öğeleri içeren bir idrar toplama kiti ver: (1) steril ya da temiz yakalamak idrar örneği almak için nasıl yazılı talimatlar, (2) nemli bir anti-bakteriyel tuvaletler, ve (3) 100 ml steril örnek toplama fincan. Örnek toplamak için hasta söyleyin. </li><li> Hemen buz üzerinde idrar örnekleri yer ve laborant transferBir soğutucu tory. Hemen hücre izolasyonu için idrar işleyin. Bir gecikme kaçınılmaz ise, hücre canlılığı kaybıyla kadar 4 saat boyunca buz üzerinde numune saklamak. </li></ol> 3. İzolasyon ve İdrar Hücreleri Genişleme NOT: Bir BSL2 Biyolojik Güvenlik Kabinesi steril koşullar altında aşağıdaki adımları uygulayın. <ol><li> Steril pipetler kullanarak, transferi idrar örnekleri 50-ml santrifüj tüpleri steril alanlar. Oda sıcaklığında 10 dakika boyunca 400 x g&#39;de santrifüjleyin. </li><li> Tüp içinde 1 ml bırakarak süpernatant aspire; Hücre pelet rahatsız etmemek için dikkatli olun. </li><li> Süspanse yıkama tamponu 7 ml birden çok tüpünden elde edilen taneler bir araya ve oda sıcaklığında 10 dakika boyunca 400 x g&#39;de tekrar santrifüj ve. </li><li> Dikkatle hücre pelet ve süpernatant ~ 0,2-0,5 ml bırakarak, süpernatant aspire. İdrar Progenitor Hücre (UPC) orta 2-3 ml süspanse ve un 4-6 kuyulara süspansiyon transferikaplanmış 24 oyuklu plaka. </li><li> 72 saat sonra, göz başına taze UPC ortamı, 0.5 ml kültür ek ve 2-3 gün sonra ortam değiştirin. plaka takmak gerekmez eritrosit ve skuamöz hücreler orta değişikliklerle silinecektir. </li><li> 4-6 gün sonra kültür izleyin. NOT: Küçük 2-4 hücre kolonileri görünmeye başlayacak. Hücreler yuvarlak veya tip I veya renal epitel (RE) hücrelerin sırasıyla 3 tip II temsil eden uzamış olacaktır. </li><li> Hücreler görünür bir kez, onlar hızlı bir genişleme fazı uğrayacak beri her 2-3 günde orta değiştirin. </li><li> Hücreler% 80-90, kaplama sonra yaklaşık 9-15 gün ulaştıklarında, daha da genişlemesi için 24 plaka 2-4 kuyuları üzerine hücreleri (15,000-18,000 hücre / cm 2) ayırmak ve geçit. Hücre geçişi 1 (P1) olarak bu işaretleyin. </li><li> Akım sitometri analizi yoluyla soy özellikleri için idrar hücreleri karakterize immunohistochemnik boyama ya da ters transkriptaz-polimeraz zincir reaksiyonu yoluyla (RT-PCR). </li><li> Uzun süreli dondurulup saklanma (DMEM% 10 serum ve% 10 DMSO ile desteklenmiş) izole idrar hücreleri (bölüm 4) reprogram veya Orta Donma onları aşağı dondurma. </li></ol> Sendai Virüs Kullanılması 4. İdrar Hücre yeniden programlanması NOT: transfekte maddeler işlenirken doğru kullanımı ve PPE (kişisel koruyucu ekipman) kullanılması tavsiye edilir. Bir BSL2 Biyolojik Güvenlik Kabinesi steril koşullar altında aşağıdaki adımları uygulayın. ajan ve / veya transfekte hücreleri transfekte uygun atılması, çevre ve sağlık tehlikeleri riskini önlemek için tavsiye edilir. Aşağıda ayrıntılı olarak idrar hücre yeniden programlanması için, üreticinin besleyici bağımlı protokol değişiklikleri ile Sendai yeniden programlanması Kit kullanın. <ol><li> Sendai virüsü kullanarak yüksek verim yeniden programlama sağlamak için, kullanım hızlı olan idrar hücrelerinin 1-5 geçişlerily bölünmesi. Hücreler de çoğaltmak veya yaşlanınca yoksa onlar verimli reprogram olmayacak gibi, onları atın. </li><li> Tohum, 6 gözlü bir plakanın iki kuyu içinde oyuk başına 60,000 hücre. Mark Day -2 olarak bu. Hücre tohumlama yoğunluğunu ayarlayın (5 x 04-09 Ekim x 10 4 hücre kuyu başına) Günün 0% 80-90 ulaşmak için gerektiği gibi. </li><li> Gün 0 (hücreleri kaplama sonra 48 saat), dört OSKM faktörlerin her içeren Sendai yeniden programlama vektörleri (SeV) hazırlamak. Önceden ısıtılmış UPC ortamı, 1 ml için vektörlerin her ekleyin. Yeniden programlama idrar hücreleri için yeterli olan 1-1.5 MOI (5-7,5 x 10 5 UKÜ) kullanın. Orta aspire ve yavaş yavaş idrar hücrelerinin bir kuyuya 1 ml UPC + Sev ekleyin. Transdüksiyon negatif kontrol olarak ikinci kuyu kullanın. </li><li> Gün 1, taze UPC ortam ile UPC + SeV orta yerine. Bazı hücre ölümü nedeniyle viral sitotoksisite 24 saat sonra görülür. </li><li> Reprogrammed Clon verimliliğine bağlıE oluşumları ve kompakt morfolojisi, günde 6 ya da 7 kadar günlük orta değişen tutmak. </li><li> Önce kalıt aktarımlı hücrelerin (gün 5 veya 6), Mitomisin-C (3 st için 10 ug / ml), 0.1 5 x 10 4 hücre / cm2, yoğunluğu tedavi-MEF&#39;ler kaplama ile MEF besleyici plakalar hazırlamak ayrışması için bir günlük % jelatin 100 mm 2 kültür yemekleri kaplı. </li><li> Ertesi gün (gün 6 ya da 7),% 0.25 tripsin ile hücreleri ayırmak UPC orta ve plaka 5 x 10 4 hücreleri tekrar süspansiyon - 2 x 10 5 hücre / 100 mm2 MEF besleyici plaka. </li><li> Ertesi gün, embriyonik kök ortamına geçiş ve ortam her gün değiştirin. Dönüştürülmüş hücreler izlemek için bir mikroskop altında hücre dikkate alınmalıdır. NOT: Hücreler karakteristik arnavut morfolojisi ve sitoplazmik oranı yüksek çekirdeğe sahip (12-18 gün sonrası transdüksiyon) ile klonal agregatlar oluştururlar. </li><li> 2-3 hafta içinde transdüksiyon sonra, koloniler almak ve klon için yeni plakalar transferal genişleme. </li><li> 37 ° C&#39;de 1 saat süre ile TRA-1-81 antikoru ile inkübe edilmesi ile iPSC klonların seçilmesi için canlı hücre boyama gerçekleştirme C CO CO2 inkübatör içinde 37 ° C&#39;de 1 saat boyunca özel bir floresan boya konjuge sekonder antikor ile karşı boyama ile 2 kuluçka takip etti. </li><li> Bir 26 G 1½ inç iğne kullanın küçük eşit büyüklükte parçalar halinde büyük TRA-1-81 pozitif iPSC klonlar (klon başına 4-6 adet) çapraz yumurtadan. </li><li> Çekme ve transfer Matrigel her çukuruna (10 ug / cm2) birden çok klonun 10-20 çapraz kesik parça embriyonik kök ortamı ile 24 yuvalı plaka -kaplı bir steril pipet ucu kullanın. NOT: Tek bir kuyuya ayrı ayrı kaplama bir tek klon gelen adet genişletmek veya pluripotensini korumak değil. </li><li> Programlanması klonlar Matrigel yüzeyine bağlı sonra iPSC ortamına hes 24-48 saat değiştirin. </li><li> Matrigel kaplı plakalar, manuel SCRA üzerinde bakım sırasındape-off tamamen yeniden programlanması ve pluripotent distrofik iPSC (MD-iPSC) klonları için zenginleştirmek için bir pipet-ucu kullanarak herhangi bir farklılaşmış hücre veya kontamine MEF besleyici hücreler. </li><li> Tek tek klonlar, ~ 100 um boyutlara ulaşmış sonra, 3 dakika boyunca 0.48 mM EDTA (etilendiamintetraasetik asit) çözeltisi ile ayrıştırma ve iPSC ortamda süspansiyon haline getirilmiş, MD-iPSCs küçük hücre toplulukları kaplama ile klonlar genişletme (5 uM Y27632 Rho Kinaz ile takviye önleyicisi ) taze matrigel (10 ug / cm 2) 12 plaka kaplama. Günlük iPSC orta değiştirin. </li><li> Her bir klonun tam yeniden programlama OSKM genler ve Pluripotency belirteçlerinin (Oct3 / 4 ve TRA-1-81) immünohistokimyasal boyama her iki gen ekspresyon analizi ile tespit edilebilir. Pluripotency potansiyeli titiz bir değerlendirme tamamen reprogrammed iPSC hatları üç germ içine ayırt edebilirsiniz onaylamak için istihdam edilmelidir. Bu embriyoit gövdesi (EB) oluşumu ve farklılaştırma denemesinde veya ter yoluyla gerçekleştirilebilmektedirAtoma formasyonu deneyi. </li></ol>

<ol>
	<li>Guan, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dystrophin-deficient+cardiomyocytes+derived+from+human+urine:+New+biologic+reagents+for+drug+discovery.">Dystrophin-deficient cardiomyocytes derived from human urine: New biologic reagents for drug discovery.</a> Stem Cell Research. 12 (2), 467-480 (2014).</li><li>Dick, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exon+Skipping+and+Gene+Transfer+Restore+Dystrophin+Expression+in+Human+Induced+Pluripotent+Stem+Cells-Cardiomyocytes+Harboring+DMD+Mutations.">Exon Skipping and Gene Transfer Restore Dystrophin Expression in Human Induced Pluripotent Stem Cells-Cardiomyocytes Harboring DMD Mutations.</a> Stem Cells Dev. 22 (20), 2714-2724 (2013).</li><li>Zhou, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+human+induced+pluripotent+stem+cells+from+urine+samples.">Generation of human induced pluripotent stem cells from urine samples.</a> Nat Protoc. 7 (12), 2080-2089 (2012).</li><li>Bellin, M., Marchetto, M. C., Gage, F. H., Mummery, C. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induced+pluripotent+stem+cells:+the+new+patient.">Induced pluripotent stem cells: the new patient.</a> Nat Rev Mol Cell Biol. 13 (11), 713-726 (2012).</li><li>Carvajal-Vergara, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Patient-specific+induced+pluripotent+stem-cell-derived+models+of+LEOPARD+syndrome.">Patient-specific induced pluripotent stem-cell-derived models of LEOPARD syndrome.</a> Nature. 465 (7299), 808-812 (2010).</li><li>Sun, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Patient-Specific+Induced+Pluripotent+Stem+Cells+as+a+Model+for+Familial+Dilated+Cardiomyopathy.">Patient-Specific Induced Pluripotent Stem Cells as a Model for Familial Dilated Cardiomyopathy.</a> Science Translational Medicine. 4 (130), 130ra147 (2012).</li><li>Zhang, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Urine+derived+cells+are+a+potential+source+for+urological+tissue+reconstruction.">Urine derived cells are a potential source for urological tissue reconstruction.</a> J Urol. 180 (5), 2226-2233 (2008).</li><li>Bayart, E., Cohen-Haguenauer, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Technological+overview+of+iPS+induction+from+human+adult+somatic+cells.">Technological overview of iPS induction from human adult somatic cells.</a> Curr Gene Ther. 13 (2), 73-92 (2013).</li><li>Malik, N., Rao, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+review+of+the+methods+for+human+iPSC+derivation.">A review of the methods for human iPSC derivation.</a> Methods Mol Biol. 997, 23-33 (2013).</li><li>Nakanishi, M., Otsu, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+Sendai+virus+vectors+and+their+potential+applications+in+gene+therapy+and+regenerative+medicine.">Development of Sendai virus vectors and their potential applications in gene therapy and regenerative medicine.</a> Curr. Gene Ther. 12 (5), 410-416 (2012).</li><li>Gonz&#225;lez-Ram&#237;rez, R., Morales-L&#225;zaro, S. L., Tapia-Ram&#237;rez, V., Mornet, D., Cisneros, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nuclear+and+nuclear+envelope+localization+of+dystrophin+Dp71+and+dystrophin-associated+proteins+(DAPs)+in+the+C2C12+muscle+cells:+DAPs+nuclear+localization+is+modulated+during+myogenesis.">Nuclear and nuclear envelope localization of dystrophin Dp71 and dystrophin-associated proteins (DAPs) in the C2C12 muscle cells: DAPs nuclear localization is modulated during myogenesis.</a> J Cell Biochem. 105 (3), 735-745 (2008).</li><li>Villarreal-Silva, M., Centeno-Cruz, F., Su&#225;rez-S&#225;nchez, R., Garrido, E., Cisneros, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Knockdown+of+dystrophin+Dp71+impairs+PC12+cells+cycle:+localization+in+the+spindle+and+cytokinesis+structures+implies+a+role+for+Dp71+in+cell+division.">Knockdown of dystrophin Dp71 impairs PC12 cells cycle: localization in the spindle and cytokinesis structures implies a role for Dp71 in cell division.</a> PloS One. 6 (8), e23504 (2011).</li></ol>

The authors declare that they have no competing financial interests.

IPSCs kullanarak kardiyovasküler hastalıklar Modelleme genetik katkı 4-6 anlamak için ortak bir yaklaşım haline gelmektedir. Hastaların hücre örnekleri elde bazıları beklenmedik zorluklar, özellikle küçük çocuklar, böyle bir idrar toplama gibi bir non-invaziv bir yaklaşım seçeneği sunarak önlenebilir. Bu genç hasta popülasyonunda, bu yeniden programlama için yeterli idrar hücreleri elde etmek için yeterli idrar hacmini toplamak genellikle zordur. Sıvıları idrar toplama öncesinde...

Kardiyomiyopati Duchenne ve Becker kas distrofisi (MD) olan hastalarda ölüm ikinci önde gelen nedenidir. X&#39;e bağlı distrofin genindeki mutasyonlar 1 ortaya karşın: 3.500 erkek doğum, çok az ilerici kalp kası hasarına yol açan moleküler ve hücresel olaylar hakkında bilinmektedir. Müsküler distrofi hastalardan elde edilen insan uyarılmış pluripotent kök hücreleri altta yatan hastalık mekanizmaları incelemek ve ilaç 1,2 tarama için kullanılacak yeni bir araç olarak ortaya çıkmıştır. cilt biyopsisi veya kan örneklerinin beklenen rahatsızlık çalışması katılım için onay vermek, genç hastalar ve / veya onların veliler caydırmak olabilir. İdrar numuneleri yeniden programlama yöntemleri iyileştirilebilir olan somatik hücre non-invaziv bir kaynağıdır. Son zamanlarda, kas distrofisinin, hastalardan toplanan idrar hücreler kültürlendi olabileceğini göstermiştir ve verimli bir şekilde Yamanaka faktörleri ile retroviral transdüksiyonu ile iPSCs (içine yeniden programlanabilir adresOct3 / 4, Sox2 Klf4 ve c-Myc; OSKM) 1. retroviral gen aktarımı dezavantajı konak kromozom içine yeniden programlanması genlerin rastgele bütünleşmesidir. Bu sınırlamayı aşmak için, biz idrar hücre yeniden programlama olmayan entegre Sendai virüsü kullandık. Bu protokol daha sonra çalışma için kardiyomiyositlerde ya da diğer hücre tiplerine ayırt edilebilir kas distrofisi hastalardan elde edilen idrar hücrelerinin Sendai virüsü yeniden programlama göstermektedir. Bu protokol, aynı zamanda diğer hasta belirli hastalıklar için adapte edilebilir.

<table border="0" cellpadding="0" cellspacing="0" width="1143">
	<tbody>
		<tr height="21">
			<td colspan="4" height="21" style="height:21px;width:1144px;">List of Materials</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Name of Reagent/Material/Equipment</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">4 Oz. Specimen Cup with Lid</td>
			<td>STL Medical Supply</td>
			<td>M9AMSAS340</td>
			<td>For Urine Sample Collection</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">15 mL BD-Falcon Tubes</td>
			<td>Fisher Scientific</td>
			<td>352097</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">50 mL BD-Falcon Tubes</td>
			<td>Fisher Scientific</td>
			<td>352098</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Round Glass Coverslips</td>
			<td>Fisher Scientific</td>
			<td>12-545-81</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">CytoTune-iPS Sendai Reprogramming Kit</td>
			<td>Life Technologies</td>
			<td>A1378-001</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">PBS, pH 7.4</td>
			<td>Life Technologies</td>
			<td>10010-023</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Pen-Strep w/o Glutamine&#160;</td>
			<td>Life Technologies</td>
			<td>15140-122</td>
			<td>Warm in 37 &#176;C water bath before use</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">RPMI Medium 1640</td>
			<td>Life Technologies</td>
			<td>11875-093</td>
			<td>Warm in 37 &#176;C water bath before use</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">B-27 Supplement w/Insulin (50x)</td>
			<td>Life Technologies</td>
			<td>17504-044</td>
			<td>Warm in 37 &#176;C water bath before use</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">B-27 Supplement w/o Insulin (50x)</td>
			<td>Life Technologies</td>
			<td>0050129SA</td>
			<td>Warm in 37 &#176;C water bath before use</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">DMEM/F12 (1:1)</td>
			<td>Life Technologies</td>
			<td>11330-032</td>
			<td>Warm in 37 &#176;C water bath before use</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Versene, 1:5000&#160;</td>
			<td>Life Technologies</td>
			<td>15040066</td>
			<td>Warm in 37 &#176;C water bath before use</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">bFGF (10 &#181;g)</td>
			<td>Life Technologies</td>
			<td>13256-029</td>
			<td>Warm in 37 &#176;C water bath before use</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Cell Counter Cartridges</td>
			<td>Life Technologies</td>
			<td>C10228</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Knockout Serum (500 mL Bottle)</td>
			<td>Life Technologies</td>
			<td>10828-028</td>
			<td>Warm in 37 &#176;C water bath before use</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Recovery Cell Culture Freezing Medium</td>
			<td>Life Technologies</td>
			<td>12648-010</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Keratinocyte-SFM (1X), Liquid</td>
			<td>Life Technologies</td>
			<td>17005-042</td>
			<td>Warm in 37 &#176;C water bath before use</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">DMEM, High Glucose, Glutamax</td>
			<td>Life Technologies</td>
			<td>10566-016</td>
			<td>Warm in 37 &#176;C water bath before use</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Ham&#39;s F-12 Nutrient Mix&#160;</td>
			<td>Life Technologies</td>
			<td>11765-054</td>
			<td>Warm in 37 &#176;C water bath before use</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">EGF Recombinant Human Protein, Liquid Form</td>
			<td>Life Technologies</td>
			<td>PHG0311L</td>
			<td>Warm in 37 &#176;C water bath before use</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Insulin, Human Recombinant, Zinc Soln</td>
			<td>Life Technologies</td>
			<td>12585-014</td>
			<td>Warm in 37 &#176;C water bath before use</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">TeSR-E8</td>
			<td>Stem Cell Technologies</td>
			<td>5940</td>
			<td>Warm in 37 &#176;C water bath before use</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">ROCK Inhibitor (Y-27632)</td>
			<td>Selleck</td>
			<td>S1049</td>
			<td>Warm in 37 &#176;C water bath before use</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Matrigel</td>
			<td>BD Biosciences</td>
			<td>354277</td>
			<td>hESC Qualified Matrix, LVED Free</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">RNeasy Mini Kit&#160;</td>
			<td>Qiagen</td>
			<td>74104</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">iScript cDNA Synthesis Kit</td>
			<td>Bio-Rad</td>
			<td>170-8890</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">DreamTaq Green PCR Master Mix (2X)</td>
			<td>Thermo-Scientific</td>
			<td>K1081</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">FBS-Qualified</td>
			<td>Sigma Aldrich</td>
			<td>F6178</td>
			<td>Warm in 37 &#176;C water bath before use</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">ADENINE BIOREAGENT</td>
			<td>Sigma Aldrich</td>
			<td>A2786-5G</td>
			<td>Warm in 37 &#176;C water bath before use</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">CHOLERA TOXIN FROM VIBRIO CHOLERAE</td>
			<td>Sigma Aldrich</td>
			<td>C8052-.5MG</td>
			<td>Warm in 37 &#176;C water bath before use</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">HYDROCORTISONE</td>
			<td>Sigma Aldrich</td>
			<td>H0888-1G</td>
			<td>Warm in 37 &#176;C water bath before use</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">HOLO-TRANSFERRIN FROM HUMAN</td>
			<td>Sigma Aldrich</td>
			<td>T0665-50MG</td>
			<td>Warm in 37 &#176;C water bath before use</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">3,3&#39;,5-TRIIODO-L-THYRONINE SODIUM SALT&#160;</td>
			<td>Sigma Aldrich</td>
			<td>T6397-100MG</td>
			<td>Warm in 37 &#176;C water bath before use</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">DAPI&#160;</td>
			<td>Santa Cruz Biotechnology</td>
			<td>SC-3598</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Fluoromount Aquous mounting medium</td>
			<td>Sigma Aldrich</td>
			<td>F4680</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">16% Paraformaldehyde</td>
			<td>Alfa-Aesar</td>
			<td>43368</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;"></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td colspan="4" height="21" style="height:21px;">List of Antibodies</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Name of Reagent/Material/Equipment</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Mouse anti CD44 - labelled with FITC&#160;</td>
			<td>BD Biosciences</td>
			<td>560977</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Mouse anti CD146 - labelled with PE</td>
			<td>BD Biosciences</td>
			<td>561013</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Mouse anti CD73 - labelled with PE</td>
			<td>BD Biosciences</td>
			<td>561014</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Mouse anti-&#945;-smooth muscle actin</td>
			<td>Sigma Aldrich</td>
			<td>A2547</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Mouse anti-Vimentin</td>
			<td>Abcam</td>
			<td>ab8978-100</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Mouse Anti - TRA-1-81</td>
			<td>Life Technologies</td>
			<td>411100</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Rabbit anti Oct 3/4</td>
			<td>Santa Cruz Biotechnology</td>
			<td>SC-9081</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:415px;">Mouse Anti Dystrophin</td>
			<td>Leica Biosystems</td>
			<td>NCL-DYSB</td>
			<td></td>
		</tr>
	</tbody>
</table>

generation of induced pluripotent stem cells from muscular dystrophy patients: efficient integration-free reprogramming of urine derived cells

Distrofik kardiyomiyopati müsküler distrofi bir tam olarak anlaşılamamıştır sonucudur. Kas distrofisi olan hastalarda gelen uyarılmış pluripotent kök hücrelerin (iPSCs) üretilmesi, in vitro bir hastalık modeli sistemleri için çok değerli bir hücre kaynağıdır ve ilaç tarama çalışmaları için kullanılabilir. Hasta türetilmiş idrar hücreleri distrofik kardiyomiyopati 1 modellemek için uyarılmış pluripotent kök hücrelerin içine başarılı bir yeniden programlama kullanılmıştır. Vektör sistemlerinin entegre güvenlik endişelerini hitaben, biz Yamanaka transdüksiyon müsküler distrofi hastalardan alınan idrar hücrelerine faktörler olmayan bir entegre Sendai virüsü vektörü kullanılarak bir protokol mevcut. Bu protokol, tamamen 2-3 hafta içinde klonlar yeniden programlanan üretir. pluripotent hücreler geçit-13 vektör-ücretsizdir. Bu distrofik iPSCs kardiyomiyositlerde ayrılmakta ve hastalık mekanizmalarını tam ve ilaç taraması için çalışma halinde kullanılabilir.

(Sırasıyla, 97,37%, 97.09%, ve% 97.3; Şekil 1A ve 1B) insan idrarından izole en projenitör hücreleri, CD44, CD73 ve CD146 gibi epitelyal progenitör ve perisit işaretleri için pozitiftir. Bu hücreler, aynı zamanda, alfa-düz kas aktin ve vimentin (Şekil 1B) gibi diğer mezenşimal ifade edilen işaretçileri değiştirmemiştir. RT-PCR analizi sitokeratin-7 zayıf sentezleme olduğu kültürlerin hücre oluşan karışık bir popülasyonda kanıt sağlar (CK-...

Watch this Scientific Journal Video about Generation of Induced Pluripotent Stem Cells from Muscular Dystrophy Patients: Efficient Integration-free Reprogramming of Urine Derived Cells at JoVE.com

Generation of Induced Pluripotent Stem Cells from Muscular Dystrophy Patients: Efficient Integration-free Reprogramming of Urine Derived Cells

This protocol entails detailed procedures for isolation of urine derived cells from muscular dystrophy patients; their efficient and rapid reprogramming through Sendai virus transduction.

Musküler Distrofi Hastalarda gelen uyarılmış pluripotent kök hücrelerin oluşturulması: İdrar Türetilmiş Hücre Verimli Entegrasyonu-ücretsiz yeniden programlanması

Dystrophic cardiomyopathy is a poorly understood consequence of muscular dystrophy. Generating induced Pluripotent Stem Cells (iPSCs) from patients with ...

generation-induced-pluripotent-stem-cells-from-muscular-dystrophy

Research

JoVE Journal

Medicine

11.1K Görüntüleme.  Medical College of Wisconsin. This protocol entails detailed procedures for isolation of urine derived cells from muscular dystrophy patients; their efficient and rapid reprogramming through Sendai virus transduction. 

Video: Generation of Induced Pluripotent Stem Cells from Muscular Dystrophy Patients: Efficient Integration-free Reprogramming of Urine Derived Cells

Isolation, Characterization and Comparative Differentiation of Human Dental Pulp Stem Cells Derived from Permanent Teeth by Using Two Different Methods

Developing wisdom teeth are easy-accessible source of stem cells during the adulthood which could be obtained by routine orthodontic treatments. Human pulp-derived stem cells (hDPSCs) possess high proliferation potential with multi-lineage differentiation capacity compare to the ordinary source of adult stem cells1-8; therefore, hDPSCs could be the good candidates for autologous transplantation in tissue engineering and regenerative medicine. Along with these benefits, possessing the mesenchymal stem cells (MSC) features, such as immunolodulatory effect, make hDPSCs more valuable, even in the case of allograft transplantation6,9,10. Therefore, the primary step for using this source of stem cells is to select the best protocol for isolating hDPSCs from pulp tissue. In order to achieve this goal, it is crucial to investigate the effect of various isolation conditions on different cellular behaviors, such as their common surface markers &amp; also their differentiation capacity.
Thus, here we separate human pulp tissue from impacted third molar teeth, and then used both existing protocols based on literature, for isolating hDPSCs,11-13 i.e. enzymatic dissociation of pulp tissue (DPSC-ED) or outgrowth from tissue explants (DPSC-OG). In this regards, we tried to facilitate the isolation methods by using dental diamond disk. Then, these cells characterized in terms of stromal-associated Markers (CD73, CD90, CD105 &amp; CD44), hematopoietic/endothelial Markers (CD34, CD45 &amp; CD11b), perivascular marker, like CD146 and also STRO-1. Afterwards, these two protocols were compared based on the differentiation potency into odontoblasts by both quantitative polymerase chain reaction (QPCR) &amp; Alizarin Red Staining. QPCR were used for the assessment of the expression of the mineralization-related genes (alkaline phosphatase; ALP, matrix extracellular phosphoglycoprotein; MEPE &amp; dentin sialophosphoprotein; DSPP).14

The method described isolation and characterization of human Dental Pulp Stem Cells (hDPSCs) by using either enzymatic dissociation of pulp (DPSC-ED) or direct outgrowth of stem cells from pulp tissue explants (DPSC-OG). Then followed by in vitro comparative differentiation of both types of hDPSCs into odontoblasts.

İnsan Diş Pulp Kök Hücre İzolasyonu, Karakterizasyonu ve Karşılaştırmalı Farklılaşma İki Farklı Yöntemler Kullanılarak Daimi Dişler türetilen

Developing wisdom teeth are easy-accessible source of stem cells during the adulthood which could be obtained by routine orthodontic treatments. Human ...

Tıp

Patient Derived Cell Culture and Isolation of CD133+ Putative Cancer Stem Cells from Melanoma

Despite improved treatments options for melanoma available today, patients with advanced malignant melanoma still have a poor prognosis for progression-free and overall survival. Therefore, translational research needs to provide further molecular evidence to improve targeted therapies for malignant melanomas. In the past, oncogenic mechanisms related to melanoma were extensively studied in established cell lines. On the way to more personalized treatment regimens based on individual genetic profiles, we propose to use patient-derived cell lines instead of generic cell lines. Together with high quality clinical data, especially on patient follow-up, these cells will be instrumental to better understand the molecular mechanisms behind melanoma progression.
Here, we report the establishment of primary melanoma cultures from dissected fresh tumor tissue. This procedure includes mincing and dissociation of the tissue into single cells, removal of contaminations with erythrocytes and fibroblasts as well as primary culture and reliable verification of the cells' melanoma origin.
Recent reports revealed that melanomas, like the majority of tumors, harbor a small subpopulation of cancer stem cells (CSCs), which seem to exclusively fuel tumor initiation and progression towards the metastatic state. One of the key markers for CSC identification and isolation in melanoma is CD133. To isolate CD133+ CSCs from primary melanoma cultures, we have modified and optimized the Magnetic-Activated Cell Sorting (MACS) procedure from Miltenyi resulting in high sorting purity and viability of CD133+ CSCs and CD133- bulk, which can be cultivated and functionally analyzed thereafter.

Patient Derived Cell Culture and Isolation of CD133+ Putative Cancer Stem Cells from Melanoma

This article describes the preparation of freshly obtained melanoma tissue into primary cell cultures, and how to remove contaminations of erythrocytes and fibroblasts from the tumor cells. Finally, we describe how CD133+ putative melanoma stem cells are sorted from the CD133- bulk using Magnetic Activated Cell Sorting (MACS).

Hasta Türetilen Hücre Kültürü ve CD133 İzolasyonu<sup&gt; +</supMelanom dan&gt; sağlayan olası Kanser Kök Hücreleri

Despite improved treatments options for melanoma available today, patients with advanced malignant melanoma still have a poor prognosis for ...

Studying Pancreatic Cancer Stem Cell Characteristics for Developing New Treatment Strategies

Pancreatic ductal adenocarcinoma (PDAC) contains a subset of exclusively tumorigenic cancer stem cells (CSCs) which have been shown to drive tumor initiation, metastasis and resistance to radio- and chemotherapy. Here we describe a specific methodology for culturing primary human pancreatic CSCs as tumor spheres in anchorage-independent conditions. Cells are grown in serum-free, non-adherent conditions in order to enrich for CSCs while their more differentiated progenies do not survive and proliferate during the initial phase following seeding of single cells. This assay can be used to estimate the percentage of CSCs present in a population of tumor cells. Both size (which can range from 35 to 250 micrometers) and number of tumor spheres formed represents CSC activity harbored in either bulk populations of cultured cancer cells or freshly harvested and digested tumors 1,2. Using this assay, we recently found that metformin selectively ablates pancreatic CSCs; a finding that was subsequently further corroborated by demonstrating diminished expression of pluripotency-associated genes/surface markers and reduced in vivo tumorigenicity of metformin-treated cells. As the final step for preclinical development we treated mice bearing established tumors with metformin and found significantly prolonged survival. Clinical studies testing the use of metformin in patients with PDAC are currently underway (e.g., NCT01210911, NCT01167738, and NCT01488552). Mechanistically, we found that metformin induces a fatal energy crisis in CSCs by enhancing reactive oxygen species (ROS) production and reducing mitochondrial transmembrane potential. In contrast, non-CSCs were not eliminated by metformin treatment, but rather underwent reversible cell cycle arrest. Therefore, our study serves as a successful example for the potential of in vitro sphere formation as a screening tool to identify compounds that potentially target CSCs, but this technique will require further in vitro and in vivo validation to eliminate false discoveries.

Pancreatic cancer stem cells (CSCs) can be expanded in vitro using the anchorage-independent sphere culture technique, which represents a powerful tool to study CSC biology and can serve as the first step to develop novel CSC-targeting therapies. Here the methodology for expanding, analyzing and targeting of pancreatic CSCs is provided.

Pankreas Kanseri Yeni Tedavi Stratejileri Geliştirme Hücre Özellikleri Kök incelenmesi

Pancreatic ductal adenocarcinoma (PDAC) contains a subset of exclusively tumorigenic cancer stem cells (CSCs) which have been shown to drive tumor ...

Generation of Prostate Cancer Patient Derived Xenograft Models from Circulating Tumor Cells

Patient derived xenograft (PDX) models are gaining popularity in cancer research and are used for preclinical drug evaluation, biomarker identification, biologic studies, and personalized medicine strategies. Circulating tumor cells (CTC) play a critical role in tumor metastasis and have been isolated from patients with several tumor types. Recently, CTCs have been used to generate PDX experimental models of breast and prostate cancer. This manuscript details the method for the generation of prostate cancer PDX models from CTCs developed by our group. Advantages of this method over conventional PDX models include independence from surgical sample collection and generating experimental models at various disease stages. Density gradient centrifugation followed by red blood cell lysis and flow cytometry depletion of CD45 positive mononuclear cells is used to enrich CTCs from peripheral blood samples collected from patients with metastatic disease. The CTCs are then injected into immunocompromised mice; subsequently generated xenografts can be used for functional studies or harvested for molecular characterization. The primary limitation of this method is the negative selection method used for CTC enrichment. Despite this limitation, the generation of PDX models from CTCs provides a novel experimental model to be applied to prostate cancer research.

This manuscript details a method used to generate prostate cancer patient derived xenografts (PDXs) from circulating tumor cells (CTCs). The generation of PDX models from CTCs provides an alternative experimental model to study prostate cancer; the most commonly diagnosed tumor and a frequent cause of death from cancer in men.

Prostat Kanseri Hasta Üretimi Tümör Hücreleri Sirkülasyon gelen Xenograftlarında Modeller Türetilmiş

Patient derived xenograft (PDX) models are gaining popularity in cancer research and are used for preclinical drug evaluation, biomarker identification, ...

Modeling Myotonic Dystrophy 1 in C2C12 Myoblast Cells

Myotonic dystrophy 1 (DM1) is a common form of muscular dystrophy. Although several animal models have been established for DM1, myoblast cell models are still important because they offer an efficient cellular alternative for studying cellular and molecular events. Though C2C12 myoblast cells have been widely used to study myogenesis, resistance to gene transfection, or viral transduction, hinders research in C2C12 cells. Here, we describe an optimized protocol that includes daily maintenance, transfection and transduction procedures to introduce genes into C2C12 myoblasts and the induction of myocyte differentiation. Collectively, these procedures enable best transfection/transduction efficiencies, as well as consistent differentiation outcomes. The protocol described in establishing DM1 myoblast cell models would benefit the study of myotonic dystrophy, as well as other muscular diseases.

In this protocol, we present the procedures in establishing myotonic dystrophy 1 myoblast models, including optimized C2C12 cell maintenance, gene transfection/transduction, and myocyte differentiation.

C2C12 Miyoblast Hücrelerde Myotonik Distrofi 1 Modelleme

Myotonic dystrophy 1 (DM1) is a common form of muscular dystrophy. Although several animal models have been established for DM1, myoblast cell models are ...

Rapid and Efficient Generation of Recombinant Human Pluripotent Stem Cells by Recombinase-mediated Cassette Exchange in the AAVS1 Locus

Even with the revolution of gene-targeting technologies led by CRISPR-Cas9, genetic modification of human pluripotent stem cells&#160;(hPSCs)&#160;is still time consuming. Comparative studies that use recombinant lines with transgenes integrated into safe harbor loci could benefit from approaches that use site-specific targeted recombinases, like Cre or FLPe, which are more rapid and less prone to off-target effects. Such methods have been described, although they do not significantly outperform gene targeting in most aspects. Using Zinc-finger nucleases, we previously created a master cell line in the AAVS1 locus of hPSCs&#160;that contains a GFP-Hygromycin-tk expressing cassette, flanked by heterotypic FRT sequences. Here, we describe the procedures to perform FLPe recombinase-mediated cassette exchange (RMCE) using this line. The master cell line is transfected with a RMCE donor vector, which contains a promoterless Puromycin resistance, and with FLPe recombinase. Application of both a positive (Puromycin) and negative (FIAU) selection program leads to the selection of RMCE without random integrations. RMCE generates fully characterized pluripotent polyclonal transgenic lines in 15 d&#160;with 100% efficiency. Despite the recently described limitations of the AAVS1 locus, the ease of the system paves the way for hPSC transgenesis in isogenic settings, is necessary for comparative studies, and enables semi-high-throughput genetic screens for gain/loss of function analysis that would otherwise be highly time consuming.

Rapid and Efficient Generation of Recombinant Human Pluripotent Stem Cells by Recombinase-mediated Cassette Exchange in the AAVS1 Locus

Here we report a rapid and efficient gene editing method based on RMCE in the AAVS1 locus of human Pluripotent Stem Cells (hPSCs) that improves upon previously described systems. Using this technique, isogenic lines can be rapidly and reliably generated for proper comparative studies, facilitating transgenesis-mediated research with hPSCs.

içinde Recombinase aracılı Kaset Borsası tarafından Rekombinant insan pluripotent kök hücreleri hızlı ve verimli Nesil<em&gt; AAVS1</em&gt; Odağı

Even with the revolution of gene-targeting technologies led by CRISPR-Cas9, genetic modification of human pluripotent stem cells&#160;(hPSCs)&#160;is ...

Forskolin-induced Swelling in Intestinal Organoids: An In Vitro Assay for Assessing Drug Response in Cystic Fibrosis Patients

Recently-developed cystic fibrosis transmembrane conductance regulator (CFTR)-modulating drugs correct surface expression and/or function of the mutant CFTR channel in subjects with cystic fibrosis (CF). Identification of subjects that may benefit from these drugs is challenging because of the extensive heterogeneity of CFTR mutations, as well as other unknown factors that contribute to individual drug efficacy. Here, we describe a simple and relatively rapid assay for measuring individual CFTR function and response to CFTR modulators in vitro. Three dimensional (3D) epithelial organoids are grown from rectal biopsies in standard organoid medium. Once established, the organoids can be bio-banked for future analysis. For the assay, 30-80 organoids are seeded in 96-well plates in basement membrane matrix and are then exposed to drugs. One day later, the organoids are stained with calcein green, and forskolin-induced swelling is monitored by confocal live cell microscopy at 37 &#176;C. Forskolin-induced swelling is fully CFTR-dependent and is sufficiently sensitive and precise to allow for discrimination between the drug responses of individuals with different and even identical CFTR mutations. In vitro swell responses correlate with the clinical response to therapy. This assay provides a cost-effective approach for the identification of drug-responsive individuals, independent of their CFTR mutations. It may also be instrumental in the development of future CFTR modulators.

Forskolin-induced Swelling in Intestinal Organoids: An In Vitro Assay for Assessing Drug Response in Cystic Fibrosis Patients

This protocol describes an assay for measuring CFTR function and CFTR modulator responses in cultured tissue from subjects with cystic fibrosis (CF). Biopsy-derived intestinal organoids swell in a cAMP-driven fashion, a response that is defective (or strongly reduced) in CF organoids and can be restored by exposure to CFTR modulators.

Forskolin kaynaklı bağırsak Organoids şişme: Bir<em&gt; İn Vitro</em&gt; Deney Kistik Fibrozis Hastalarında İlaç Tepki değerlendirilmesi için

Recently-developed cystic fibrosis transmembrane conductance regulator (CFTR)-modulating drugs correct surface expression and/or function of the mutant ...

Evaluation of Exon Inclusion Induced by Splice Switching Antisense Oligonucleotides in SMA Patient Fibroblasts

Spinal muscular atrophy (SMA), a lethal neurological disease caused by the loss of SMN1, presents a unique case in the field of antisense oligonucleotide (AON)-mediated therapy. While SMN1 mutations are responsible for the disease, AONs targeting intronic splice silencer (ISS) sites in SMN2, including FDA-approved nusinersen, have been shown to restore SMN expression and ameliorate the symptoms. Currently, many studies involving AON therapy for SMA focus on investigating novel AON chemistries targeting SMN2 that may be more effective and less toxic than nusinersen. Here, we describe a protocol for in vitro evaluation of exon inclusion using lipotransfection of AONs followed by reverse transcription polymerase chain reaction (RT-PCR), quantitative polymerase chain reaction (qPCR), and Western blotting. This method can be employed for various types of AON chemistries. Using this method, we demonstrate that AONs composed of alternating locked nucleic acids (LNAs) and DNA nucleotides (LNA/DNA mixmers) lead to efficient SMN2 exon inclusion and restoration of SMN protein at a very low concentration, and therefore, LNA/DNA mixmer-based antisense oligonucleotides may be an attractive therapeutic strategy to treat splicing defects caused by genetic diseases. The in vitro evaluation method described here is fast, easy, and sensitive enough for the testing of various novel AONs.

Various antisense oligonucleotides (AONs) have been shown to induce exon inclusion (splice modulation) and rescue SMN expression for spinal muscular atrophy (SMA). Here, we describe a protocol for AON lipotransfection to induce exon inclusion in the SMN2 gene and the evaluation methods to determine the efficacy in SMA patient fibroblasts.

Tarafından indüklenen Exon dahil değerlendirilmesi Splice SMA hasta fibroblastlar anahtarlama antianlamlı Oligonucleotides

Spinal muscular atrophy (SMA), a lethal neurological disease caused by the loss of SMN1, presents a unique case in the field of antisense oligonucleotide ...

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

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

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

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

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

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

Single Myofiber Isolation and Culture from a Murine Model of Emery-Dreifuss Muscular Dystrophy in Early Post-Natal Development

Autosomal dominant Emery-Dreifuss muscular dystrophy (EDMD) is caused by mutations in the LMNA gene, which encodes the A-type nuclear lamins, intermediate filament proteins that sustain the nuclear envelope and the components of the nucleoplasm. We recently reported that muscle wasting in EDMD can be ascribed to intrinsic epigenetic dysfunctions affecting muscle (satellite) stem cells regenerative capacity. Isolation and culture of single myofibers is one of the most physiological ex-vivo approaches to monitor satellite cells behavior within their niche, as they remain between the basal lamina surrounding the fiber and the sarcolemma. Therefore, it represents an invaluable experimental paradigm to study satellite cells from a variety of murine models. Here, we describe a re-adapted method to isolate intact and viable single myofibers from post-natal hindlimb muscles (Tibialis Anterior, Extensor Digitorum Longus, Gastrocnemius and Soleus). Following this protocol, we were able to study satellite cells from Lamin &#916;8-11 -/- mice, a severe EDMD murine model, at only 19 days after birth.
We detail the isolation procedure, as well as the culture conditions for obtaining a good amount of myofibers and their associated satellite-cells-derived progeny. When cultured in growth-factors rich medium, satellite cells derived from wild type mice activate, proliferate, and eventually differentiate or undergo self-renewal. In homozygous Lamin &#916;8-11 -/- mutant mice these capabilities are severely impaired.
This technique, if strictly followed, allows to study all processes linked to the myofiber-associated satellite cell even in early post-natal developmental stages and in fragile muscles.

Here, we propose a method to efficiently obtain single muscle fibers at early post-natal developmental stages from homozygous mutant Lamin &#916;8-11 mouse model, a very severe model for Emery-Dreifuss muscular dystrophy (EDMD).

Erken Doğum Sonrası Gelişimde Emery-Dreifuss Musküler Distrofi'nin Murine Modelinden Tek Miyofiber İzolasyon ve Kültür

Autosomal dominant Emery-Dreifuss muscular dystrophy (EDMD) is caused by mutations in the LMNA gene, which encodes the A-type nuclear lamins, intermediate ...