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.

NOTE: Les patients et / ou leurs tuteurs doivent donner un consentement éclairé à participer à un Institutional Review Board étude approuvée. 1. Tampons et préparations des médias <ol><li> tampon de lavage: Pour préparer 100 ml de tampon de lavage, ajouter 1 ml de stylo 100x / Solution streptococcique (100 U / ml de pénicilline + 100 pg / ml de streptomycine) à 99 ml de solution saline tamponnée au phosphate (PBS). </li><li> Progenitor urinaire cellulaire (UPC) Medium: Pour préparer le milieu UPC, mélanger des volumes égaux de kératinocytes Free Serum (KSF) Medium + de cellules souches moyen. <ol><li> KSF Medium: Pour préparer le milieu de kératinocytes, ajouter 5 ng / ml de facteur de croissance épidermique (EGF), 50 ng / ml d&#39;extrait pituitaire bovin (BPE), 30 ng / ml de toxine cholérique, et la solution pen / strep (100 U / ml de pénicilline, 100 ug / ml de streptomycine) à 500 ml de milieu KSF. </li><li> Moyen de cellules souches: Ajouter trois quarts de DMEM avec un quart des médias Ham-F12 et de compléter avec 10% de FBS, 0,4 pg / ml hydrocortisone, 0,1nM toxine du choléra, 5 ng / ml d&#39;insuline, 1,8 x 10 -4 M d&#39;adénine, 5 ug / ml de transferrine, 2 x 10 -9 M triiodo thyronine, 10 ng / ml d&#39;EGF, et pen / strep solution. </li></ol></li><li> hES moyenne: 20% Ajouter remplacement KO-sérum (M-SR), 1x MEM acides aminés non essentiels, 2 mM de L-glutamine, 100 uM de β-mercaptoéthanol, 20 ng / ml de bFGF et pen / strep à 400 ml de DMEM / F12. </li></ol> 2. Prélèvement d&#39;échantillons d&#39;urine <ol><li> Demander aux patients de boire des liquides 30 min avant la collecte d&#39;urine pour se assurer qu&#39;une quantité adéquate (~ 30-40 ml) d&#39;urine peut être collectée. </li><li> Donner aux patients et / ou leurs tuteurs un kit de collecte d&#39;urine contenant les éléments suivants: (1) des instructions écrites sur la façon d&#39;obtenir l&#39;échantillon captures d&#39;urine stérile ou propre, (2) toilettes anti-bactériennes humides, et (3) un 100 ml stérile collecte de spécimens tasse. Demander aux patients pour recueillir l&#39;échantillon. </li><li> Placer immédiatement les échantillons d&#39;urine sur la glace et transfert à l&#39;laboraTory dans une glacière. Traiter l&#39;urine pour l&#39;isolement cellulaire immédiatement. Si un retard est inévitable, stocker les échantillons sur la glace jusqu&#39;à 4 heures avec une perte minimale de la viabilité cellulaire. </li></ol> 3. Isolement et expansion des cellules d&#39;urine REMARQUE: Effectuez les étapes suivantes dans des conditions stériles dans un Cabinet de sécurité biologique BSL2. <ol><li> L&#39;aide de pipettes stériles, les échantillons d&#39;urine pour le transfert stériles tubes de centrifugation de 50 ml. Centrifuger à 400 g pendant 10 min à température ambiante. </li><li> Aspirer le surnageant laissant 1 ml dans le tube; attention à ne pas perturber le culot cellulaire. </li><li> Remettre en suspension et de combiner les pellets à partir de tubes multiples dans 7 ml de tampon de lavage et répéter centrifugation à 400 g pendant 10 min à température ambiante. </li><li> Aspirer soigneusement le surnageant, en laissant le culot de cellules et de ~ 0,2 à 0,5 ml de surnageant. Remettre en suspension dans 2-3 ml de Progenitor urinaire cellulaire (UPC) à moyen et à transférer la suspension dans 4-6 puits d&#39;une nonplaque de 24 puits revêtue. </li><li> Après 72 h, compléter la culture avec 0,5 ml de milieu frais UPC par puits et changer moyenne tous les 2-3 jours après. Les érythrocytes et des cellules squameuses qui ne se attachent pas à la plaque seront enlevés avec les moyennes changements. </li><li> Surveiller la culture après 4-6 jours. Remarque: Les petits 2-4 colonies de cellules vont commencer à apparaître. Les cellules seront arrondies ou allongées qui représentent de type I ou II de l&#39;épithélium rénal (RE) des cellules respectivement 3. </li><li> Changer le support tous les 2-3 jours car une fois les cellules apparaissent, ils seront soumis à une phase d&#39;expansion rapide. </li><li> Une fois que les cellules atteignent 80-90% de confluence, autour de 9-15 jours après placage, et le passage dissocier les cellules (15,000-18,000 cellules / cm 2) sur 2-4 puits de plaque à 24 puits pour poursuivre son expansion. Marquer comme ce passage d&#39;une cellule (P1). </li><li> Caractériser les cellules d&#39;urine pour les spécifications de la lignée à travers l&#39;analyse de cytométrie en flux, immunohistochemcoloration ical ou par réaction en chaîne de polymérase-transcriptase inverse (RT-PCR). </li><li> Reprogrammer des cellules isolées d&#39;urine (section 4) ou congelez-les dans un milieu de congélation (DMEM complété avec 10% de sérum et 10% de DMSO) pour cryoconservation à long terme. </li></ol> 4. urine cellulaire Reprogrammation Utilisation Sendai Virus NOTE: La manipulation et l&#39;utilisation des EPI (équipements de protection individuelle) est recommandée lors de la manipulation des agents de transfection. Effectuez les étapes suivantes dans des conditions stériles dans un Cabinet de sécurité biologique BSL2. L&#39;élimination appropriée des transfection de cellules de l&#39;agent et / ou transfectées est recommandé d&#39;éviter tout risque de risques environnementaux et sanitaires. Pour l&#39;urine cellules reprogrammation, utiliser une reprogrammation Kit Sendai avec des modifications du protocole d&#39;alimentation dépendant du fabricant tel que détaillé ci-dessous. <ol><li> Pour assurer la reprogrammation de haute efficacité utilisant le virus Sendai, l&#39;utilisation des passages 1-5 de cellules d&#39;urine qui sont rapideLy divisant. Si les cellules ne se répliquent pas bien ou deviennent sénescentes, jetez-les car ils ne seront pas reprogrammer efficacement. </li><li> Seed 60 000 cellules par puits dans deux puits d&#39;une plaque à 6 puits. Marquer ce que la Journée -2. Régler la densité d&#39;ensemencement des cellules (5 x oct 4 à 9 x 10 4 cellules par puits) comme nécessaire pour atteindre 80 à 90% de confluence au jour 0. </li><li> Le jour 0 (48 h après étalement des cellules), préparer les vecteurs de reprogrammation Sendai (DVE) contenant chacun des quatre facteurs de OSKM. Ajouter chacun des vecteurs à 1 ml de milieu préchauffé UPC. Utiliser une MOI de 1 à 1,5 (de 5 à 7,5 x 10 5 CIU), qui est suffisante pour la reprogrammation des cellules d&#39;urine. Aspirer le milieu et ajouter lentement 1 ml de la UPC + SeV à une bien des cellules d&#39;urine. Utilisez la seconde ainsi que la transduction contrôle négatif. </li><li> Le Jour 1, remplacer le milieu UPC + SeV avec le milieu UPC frais. Certains mort cellulaire est observée après 24 h en raison de la cytotoxicité virale. </li><li> Selon efficacité de Clon reprogramméformations e et la morphologie compacte, ne cessent de changer le milieu tous les jours jusqu&#39;au jour 6 ou 7. </li><li> Un jour avant la dissociation des cellules transduites (jour 5 ou 6), préparer des plaques d&#39;alimentation MEF par placage mitomycine C (10 ug / ml pendant 3 heures)-MEF traitées à une densité de 5 x 10 4 cellules / cm 2, sur 0,1 % de gélatine enduite 100 mm 2 plats de culture. </li><li> Le lendemain (jour 6 ou 7), dissocier les cellules avec 0,25% de trypsine, remettre en suspension les cellules dans du milieu UPC et de la plaque 5 x octobre 4-2 x 10 5 cellules / mm 2 100 MEF plaque d&#39;alimentation. </li><li> Le lendemain, passer à moyen hES et de changer le milieu tous les jours. Respecter les cellules sous un microscope pour surveiller cellules transformées. REMARQUE: Les cellules forment des agrégats clonales et une morphologie caractéristique pavée et ayant noyau supérieur rapport cytoplasmique (12-18 jours après transduction). </li><li> Dans les deux à trois semaines après la transduction, prélever des colonies et de les transférer sur de nouvelles plaques pour clonextension al. </li><li> Effectuer la coloration de cellules vivantes pour la sélection de clones iPSC en incubant des cellules avec TRA-1-81 anticorps pendant 1 heure à 37 ° C dans un incubateur CO 2 suivie d&#39;une contre-coloration avec un colorant fluorescent spécifique conjugué anticorps secondaire pendant 1 heure à 37 ° C dans un incubateur CO 2. </li><li> Utilisez un G 1½ aiguille 26 pouces de contre-éclore les grands TRA-1-81 clones positifs iPSC en petits morceaux de taille égale (4-6 pièces par clone). </li><li> Utiliser une pipette à pointe stérile pour ramasser et le transfert de 10 à 20 pièces hachurées de multiples clones dans chaque puits de Matrigel (10 ug / cm 2) doté d&#39;un revêtement plaque à 24 puits avec du milieu hES. REMARQUE: Pièces d&#39;un clone unique plaqué individuellement dans un seul puits ne étendre ou de maintenir la pluripotence. </li><li> Changer de hES à moyen iPSC 24-48 heures après les clones reprogrammées sont attachés à la surface Matrigel. </li><li> Lors de l&#39;entretien sur des plaques enduites de Matrigel, SCRA manuellementpe-off des cellules différenciées ou contaminantes cellules nourricières MEF aide d&#39;une pipette à pointe pour enrichir iPSC (MD-iPSC) clones dystrophiques entièrement reprogrammées et pluripotentes. </li><li> Après clones individuels ont atteint ~ 100 taille um, d&#39;élargir les clones en dissociant avec mM d&#39;EDTA 0,48 (acide éthylènediaminetétraacétique) solution pendant 3 min et le placage de petits agrégats de cellules de MD-iPSCs en suspension dans du milieu iPSC (additionné de 5 uM Y27632, la Rho kinase inhibiteur ) sur matrigel frais (10 ug / cm 2) revêtu 12 plaque bien. Changer le milieu iPSC quotidienne. </li><li> La reprogrammation complète de chaque clone peut être déterminée à la fois par analyse d&#39;expression génique de gènes de OSKM et la coloration immunohistochimique de marqueurs de la pluripotence (Oct3 / 4 et TRA-1-81). Une évaluation rigoureuse du potentiel de la pluripotence devrait être utilisée pour confirmer lignes iPSC entièrement reprogrammées peuvent se différencier en trois feuillets embryonnaires. Ceci peut être réalisé soit en corps embryoïdes (EB) la formation et de la différenciation ou d&#39;un dosage teratoma formation dosage. </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.

Modélisation des maladies cardiovasculaires à l&#39;aide CSPi devient une approche commune pour comprendre la contribution génétique 4-6. Certaines difficultés imprévues d&#39;obtenir des échantillons de cellules de patients, en particulier les jeunes enfants, peuvent être évités en offrant la possibilité d&#39;une approche non invasive comme une collection d&#39;urine. Dans cette jeune population de patients, il est souvent difficile de recueillir un volume d&#39;urine suffisante pour produire des...

La cardiomyopathie est la deuxième principale cause de décès chez les patients atteints de dystrophie musculaire de Duchenne et de Becker (MD). Bien que des mutations dans le gène de la dystrophine liée à l&#39;X se produisent dans une: 3500 naissances masculines, on sait très peu sur les événements moléculaires et cellulaires conduisant à une dégradation progressive du muscle cardiaque. cellules souches pluripotentes humaines induites provenant de patients atteints de dystrophie musculaire ont émergé comme un nouvel outil pour étudier les mécanismes de la maladie sous-jacents et à utiliser pour le dépistage de drogues 1,2. Le malaise prévue de biopsies de la peau ou des échantillons de sang peut dissuader les jeunes patients et / ou leurs tuteurs de donner son consentement pour la participation à l&#39;étude. Les échantillons d&#39;urine sont une source non invasive de cellules somatiques qui sont amendable des procédés de reprogrammation. Nous avons récemment montré que les cellules d&#39;urine prélevés de patients atteints de dystrophie musculaire peuvent être cultivées et efficacement reprogrammé en utilisant CSPi transduction rétrovirale avec les facteurs Yamanaka (Oct3 / 4, Sox2, Klf4, et c-Myc; OSKM) 1. L&#39;inconvénient de délivrance de gène rétroviral est l&#39;intégration aléatoire de gènes de reprogrammation dans les chromosomes de l&#39;hôte. Pour surmonter cette limitation, nous avons utilisé le virus Sendai non-intégration pour la reprogrammation des cellules d&#39;urine. Ce protocole détaille le virus Sendai reprogrammation des cellules isolées urine de patients atteints de dystrophie musculaire qui peut ensuite être différenciées en cardiomyocytes ou d&#39;autres types de cellules pour une étude plus approfondie. Ce protocole peut également être adapté à d&#39;autres maladies spécifiques des patients.

<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

Cardiomyopathie dystrophique est une conséquence mal compris de la dystrophie musculaire. Générer des cellules souches pluripotentes induites (CISP) provenant de patients atteints de dystrophie musculaire est une source précieuse cellulaire pour des systèmes de modèles in vitro de la maladie à l&#39;intérieur et peut être utilisé pour des études de criblage de médicaments. Les cellules d&#39;urine provenant de patients ont été utilisés dans une reprogrammation réussie dans les cellules souches pluripotentes induites afin de modéliser une cardiomyopathie dystrophique. Répondre aux préoccupations de l&#39;intégration des systèmes de vecteurs de sécurité, nous présentons un protocole utilisant un vecteur de virus Sendai non-intégration pour la transduction de Yamanaka facteurs dans les cellules d&#39;urine prélevés chez des patients atteints de dystrophie musculaire. Ce protocole génère entièrement reprogrammé clones dans les 2-3 semaines. Les cellules pluripotentes sont indemne de vecteurs par passage-13. Ces CSPi dystrophiques peuvent être différenciées en cardiomyocytes et utilisés soit pour étudier les mécanismes de la maladie ou pour le dépistage de la drogue.

La plupart des cellules souches isolées à partir d&#39;urine humaine sont positives pour les marqueurs progénitrices et péricytes uroépithéliales tels que CD44, CD73, et CD146 (97,37%, 97,09% et 97,3%, respectivement: la figure 1A et 1B). Ces cellules ont également exprimé d&#39;autres marqueurs mésenchymateuses tels que l&#39;alpha-actine musculaire lisse et vimentine (figure 1B). Analyse RT-PCR donne la preuve d&#39;une population mixte de cellules dans les c...

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.

Génération de cellules souches pluripotentes induites à partir de Dystrophie musculaire patients: efficace Reprogrammation libre-intégration de l&#39;urine cellules dérivées

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 vues.  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. 

Vidéo: 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.

Différenciation isolement, caractérisation et comparative de l&#39;homme dentaires cellules souches de pulpe Dérivé de dents permanentes à l&#39;aide de deux méthodes différentes

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

Recherche

Médecine

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).

Patient Culture cellulaire dérivée et isolement de CD133<sup&gt; +</sup&gt; Putatifs cellules souches du cancer du mélanome

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.

Etudier le cancer du pancréas Stem Cell Caractéristiques pour le développement de nouvelles stratégies de traitement

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.

Génération de cancer de la prostate des patients Dérivé modèles de xénogreffe de cellules tumorales circulantes

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.

Modélisation de la dystrophie myotonique 1 dans les cellules C2C12 myoblastes

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.

Génération rapide et efficace des Recombinant pluripotentes humaines cellules souches par recombinase médiée Cassette Exchange dans le<em&gt; AAVS1</em&gt; Locus

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.

Forskoline induite par Gonflement dans Intestinal organites: Un<em&gt; In vitro</em&gt; Essai pour évaluer la réponse des médicaments dans les patients atteints de fibrose kystique

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.

Évaluation de l’Inclusion d’Exon induite par Splice commutation oligonucléotides antisens de fibroblastes de patients de SMA

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.

Expansion ex Vivo de cellules souches hématopoïétiques CD34 de dérivés du sang de cordon ombilical humain+ des cellules à l’aide de l’acide valproïque

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).

Isolement et culture myofiber uniques à partir d’un modèle murin de la dystrophie musculaire Emery-Dreifuss dans le développement post-natal précoce

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