We would like to thank Patrick C. Lee, Cyril Ramathal, and Saravanan Karumbayaram (SK) for their assistance in performing the iPSC derivation and characterization experiments; Aaron Cooper for performing the iPSC analysis experiments; Vittorio Sebastiano and Renee A. Reijo Pera for directing the initial reprogramming efforts; SK, William E. Lowry, Jerome A. Zack, and Donald B. Kohn for directing the establishment of the UCLA GMP facilities permitting the conversion and characterization of clinical-grade iPSCs; Gustavo Mostoslavsky for providing us with the STEMCCA polycistronic reprogramming vector. This work is based on a research collaboration with Fibrocell Science and the Clinical Investigations for Dermal Mesenchymally Obtained Derivatives (CIDMOD) Initiative to generate safe personalized cellular therapeutics. This work was supported by funding from the Eli and Edythe Broad Center of Regenerative Medicine and Stem Cell Research at UCLA, The Phelps Family Foundation, Fibrocell Science, Inc., and the UCLA CTSI Scholar&#8217;s Award to JAB.

NOTE: Cette méthode a été utilisée dans la recherche présentée dans la crainte et al 10.. 1. Reprogrammation adulte humain fibroblastes dermiques avec STEMCCA <ol><li> Fibroblastes dermiques adulte Thaw humaines (HUF), passage 4 ou inférieures, dans un bain d&#39;eau à 37 ° pendant 2 min, en prenant soin de ne pas submerger le haut du flacon avec de l&#39;eau. </li><li> Placez la pâte de 1 ml de fibroblastes humains dans une fiole conique de 15 ml et placer lentement 4 ml de milieu HUF standard, pré-chauffé à 37 ° C, dans un mode goutte à goutte, pour diluer sur le diméthylsulfoxyde (DMSO) et remettre en suspension les cellules. </li><li> Centrifuger le flacon à 200 xg pendant 10 min à température ambiante. Aspirer le surnageant et remettre en suspension les cellules dans un volume adéquat de médias pour créer une suspension de 100 000 cellules / ml, puis ajouter 2 ml dans un puits d&#39;une plaque à six puits, pré-revêtu pendant 20 minutes avec 0,2% de gélatine. REMARQUE: Une sœur bien avec un nombre égal de cellules par ligne et l&#39;état doit être ensemencée for le comptage / de multiplicité d&#39;infection (MOI) de calcul cellule. </li><li> Rock the côté de la plaque à l&#39;autre et d&#39;avant en arrière pour répartir les cellules. Incuber pendant une nuit à 37 ° C, 5% de CO2 dans un incubateur humidifié. </li><li> Le jour de transduction, d&#39;obtenir le nombre de cellules (s) de la sœur bien (s) environ 24 heures après l&#39;étalement de HUF initiale. </li><li> Dégeler 2 x 10 8 TU, ou équivalent, concentré lentiviral (STEMCCA) et diluer à 1 x 10 8 TU / ml en HUF médias. </li><li> Transduction de cellules à un rapport 10-MOI en HUF médias supplémenté avec 8 pg / ml agent de transfection. Mélanger uniformément transduites puits et incuber pendant une nuit à 37 ° C, 5% de CO2 dans un incubateur humidifié. </li><li> Environ 24 heures après transduction lentivirale, basculer les médias HUF de cellules souches pluripotentes humaines (CFP) milieu standard. </li><li> Le jour 2 à 6, changer de support quotidienne avec le milieu CFP humaine. </li><li> Au jour 6, plaque deux 0,2% revêtues de gélatine plaques de 10 cm de1,5 à 1,75 x 10 6 MEF provenant de souris CF1 en HUF médias 12. </li><li> Au jour 7, en faisant attention de ne pas centrifuger plus de 100 XG, l&#39;utilisation la température ambiante de la trypsine pour décoller les jours 7 fibroblastes reprogrammés d&#39;un puits de la plaque à six puits et le passage à un rapport 01:16, en étalant toutes les cellules de façon égale entre deux plaques de 10 cm, avec les médias CFP humain frais, qui ont été plaqué avec MEF la veille. Distribuer des agrégats de cellules dans un mouvement en spirale dans le sens horaire et le lieu dans un 37 ° C, 5% de CO 2 incubateur humidifié nuit. </li><li> Environ 48 heures après l&#39;ensemencement des cellules reprogrammées sur MEF et tous les jours par la suite, changer les médias CFP humains passés avec des milieux frais. </li><li> Après 3 à 4 semaines, prélever des colonies individuelles avec typique ESC comme la morphologie avec une aiguille de calibre 21 et de sous-cloner les dans une plaque de 24 puits en plaçant environ 8 à 10 morceaux individuels de colonies spécifiques dans des puits individuels dans un 0,2% de gélatine-couché plaque de 24 puits de pré-amorçageed avec FAE provenant de souris CF1 en milieu CFP humaine. NOTE: Après l&#39;expansion des colonies cueillies, les colonies doivent être caractérisée comme pluripotentes par la formation de corps embryoïdes et l&#39;analyse de l&#39;expression des marqueurs de pluripotence au niveau de la protéine. </li></ol> 2. vecteur d&#39;intégration Analyse du site et STEMCCA Excision <ol><li> Analyser le site d&#39;intégration STEMCCA par linéaire non restrictive amplification par PCR comme décrit 10. </li><li> Après dosage pour assurer une intégration dans une zone souhaitée du génome, accise STEMCCA par aspiration hors l&#39;ancien milieu ESC humaine. Ensuite, mélanger 45 pi de virus concentré adéno-Cre-PuroR avec 8 pg / ml agent de transfection dans 3 ml de médias CFP standards pendant 24 heures. </li><li> Après la période d&#39;incubation de 24 h, aspirer le surnageant virale mixte et laver les cellules deux fois avec les médias de la CFP humaines. Placez médias CFP humains frais le long avec 2 ug / ml de puromycine pour une période de cinq jours, en changeant les médias esprit quotidienneh médias et antibiotiques frais. </li><li> Après 5 jours, les colonies restantes sous-clone avec une aiguille de calibre 21 sur 0,2% puits revêtues de gélatine, dans une plaque à 12 puits pré-enduits avec MEF dérivés de CF1mice et étendre comme indiqué ci-dessus. NOTE: Après une dilatation suffisante pour obtenir un stock congelé, conversion en conditions libre-alimentation est nécessaire. Attendez au moins 95% des colonies de cellules souches mort comme la plupart des cellules ne seront pas transduites avec succès et succomberont à l&#39;antibiotique au cours de la période de 5 jours d&#39;incubation. Bien adéno-Cre-PuroR intègre à un rendement de 0,001 à 1% dans les chromosomes de l&#39;hôte de cellules infectées, reexposing un échantillon de colonies facteur libre à la puromycine pour assurer 100% de mort cellulaire confirmera pas d&#39;intégration se est produite 13. </li><li> Dégeler une fiole de pré-aliquoté matrice de membrane basale sur de la glace, sur une période d&#39;environ 2 heures (les recommandations du fabricant) jusqu&#39;à ce que la matrice est un liquide et diluer jusqu&#39;à une concentration finale de 1:30 dans un milieu basal froid. Coau nombre désiré de puits dans une plaque à six puits avec 1 ml par puits. Laisser reposer à la température ambiante pendant 1 heure. </li><li> Croix-Hatch (en utilisant une aiguille de calibre 18, ou un verre ou en plastique &quot;pointe&quot;) au moins 20 à 30 colonies d&#39;un puits préalablement revêtu d&#39;MEF. Soyez prudent de réduire MEF édifiante et transférer de la plaque. <ol><li> Générer le dispositif hachures par un certain nombre d&#39;approches différentes, provenant du chauffage plus compliqué, tirant, et la finition d&#39;une pipette Pasteur en verre sur une flamme nue, à la simple utilisation d&#39;une pointe de pipette en plastique stérile ou, comme dans notre cas, 21- et / ou de calibre 18 aiguilles. </li></ol></li><li> Aspirer la matrice de l&#39;enduit bien après incubation. </li><li> Retirer les morceaux de colonies par grattant délicatement la vieille plaque avec une pipette de 200 pi et placer soigneusement dans 3 ml de médias combinés frais 1 dans la plaque de matrice revêtu. Incuber pendant une nuit dans un 37 ° C, 5% de CO 2 incubateur humidifié. Changer les médias 48 heures après repiquage. </li><li> Extraire l&#39;ADN génomique de hiPSCs soumettre excisées et sans alimentation converties, à plus de 80% de confluence, en utilisant des kits d&#39;extraction d&#39;ADN commercial. </li><li> Analyser pour l&#39;excision correcte avec des amorces spécifiques pour les intégrations exogènes du lentivirus STEMCCA: gDNA-Hendo-MycS-avant, 5&#39;-acgagcacaagctcacctct-3 &#39;; gDNA-hWPRE-inverse, 5&#39;-tcagcaaacacagtgcacacc-3 &#39;. Reconstituer amorces lyophilisées avec de l&#39;eau de qualité PCR à une solution stock de 10 uM. </li><li> Exécuter une PCR avec le protocole en cinq étapes suivantes: dénaturation initiale à 95 ° C pendant 3 min; 35 cycles de chacun de ce qui suit: dénaturation à 98 ° C pendant 20 s, annelage d&#39;amorce à 62 ° C pendant 15 secondes, et extension à 72 ° C pendant 15 s; suivie d&#39;une extension finale unique de cycle à 72 ° C pendant 3 min. </li><li> Ajouter un gel d&#39;agarose à 3% en pesant 1,5 g d&#39;agarose et en le plaçant dans 50 ml de tampon 1 x Tris-acétate-EDTA. Micro-ondes jusqu&#39;à agarose est dissoute et le lieu dans un gel d&#39;ADN tache à la pRoper dilution, mélanger et distribuer volume correct dans la cassette de gel se solidifier pendant 1 heure à température ambiante. </li><li> Charge 12 pi de produit de PCR a mélangé avec 3 ul de colorant de charge en un gel d&#39;agarose à 3%. Exécutez le gel pendant 30 min à 80 V. </li><li> Visualisez de la lumière ultraviolette pour l&#39;absence d&#39;une bande indiquant excision correcte. </li></ol> 3. La quantification des différences d&#39;expression génétique à l&#39;aide de PCR quantitative de pré et post-hiPSCs excisées <ol><li> Extraire l&#39;ARN total de hiPSCs soumettre excisées et sans alimentation converties en utilisant les kits d&#39;extraction d&#39;ARN commerciales. </li><li> Reverse-transcrire jusqu&#39;à 1 ug d&#39;ARN total en utilisant des kits commerciaux en conformité avec les instructions du fabricant. Utilisez-oligo ancrée (dT) 18 et amorces hexamères aléatoires. REMARQUE: Assurez-vous que le protocole PCR est adapté pour les transcriptions de gènes de plus de ou moins de 4 ko. Amorces spécifiques devront être prises pour i selon gène STEMCCA origine intégrénto. </li><li> Reconstituer l&#39;amorce lyophilisée avec de l&#39;eau de qualité PCR à une solution stock de 20 uM. </li><li> Utilisation de 5 ng par échantillon 20 ul de la réaction qui se compose de sonde UPL 10 uM, 2x LightCycler 480 Probes Master, et 20 uM des amorces directe et inverse. </li></ol> 4. La conversion en conditions de qualité GMP <ol><li> Le premier jour de la transition les hiPSCs soumettre excisées plus aux conditions de qualité GMP, faire un mélange des médias composé de CFP humaine combinée médias 1 (médias combinée 1) et grade clinique bonnes pratiques de fabrication (BPF) médias conforme (médias combinés 2 ) dans un rapport 80:20. </li><li> Aspirer les anciens médias combinés 1 et 3 ml placer des nouveaux médias combinés 1 et 2, préchauffé, le ratio 80:20. Changer de support jour pendant 3 jours avec ce rapport spécifique. La place des cellules de nouveau dans un 37 ° C, 5% de CO 2 incubateur. </li><li> Le jour 4, assurez les médias combinée 1 et 2 à un ratio 50:50. Aspirer l&#39;ancien milieu et REPLACe avec les médias préchauffé, combinés avec les médias 1 et 2 à la proportion 50:50. Changer de support pour 3 jours avec ce rapport spécifique. La place des cellules de nouveau dans un 37 ° C, 5% de CO 2 incubateur. </li><li> Au jour 7, faire les médias combinée 1 et 2 à un ratio 20:80. Aspirer l&#39;ancien milieu et remplacer avec les médias préchauffées, combinés avec les médias 1 et 2 au rapport 20:80. Changer de support pour 3 jours avec ce rapport spécifique. La place des cellules de nouveau dans un 37 ° C, 5% de CO 2 incubateur. </li><li> Au jour 10, rendre les médias combinés 1 et 2 à un 0: rapport 100. Aspirer l&#39;ancien milieu et remplacer avec les médias pré-chauffé, avec les médias combinés 1 et 2 au 0: rapport 100. Changer les médias tous les jours à ce rapport spécifique. Les cellules sont maintenant dans un milieu exempt de xéno-complètement défini. La place des cellules de nouveau dans un 37 ° C, 5% de CO 2 incubateur. REMARQUE: Pendant ce processus de conversion, des passages de routine avec une aiguille de calibre 18 est nécessaire pour maintenir la taille des colonies et la densité correcte. Plan sur la passaging cellules toutes les 4 à 5 jours, sur la base de la taille des colonies, la confluence, et la différenciation. </li><li> Revêtez un puits dans une plaque à six puits avec un substrat sans xéno-défini comme recommandé par le fabricant. </li><li> Passage mécaniquement une confluence ainsi, déjà converti aux conditions de médias Xeno-libre, d&#39;une plaque à six puits avec une aiguille de calibre 18. Surtout, placer les nouveaux médias (médias combinés 2) sur les cellules avec un inhibiteur de ROCK 1x pendant 1 heure, avant repiquage d&#39;effectuer une pré-état les médias. </li><li> Aspirer la solution de substrat synthétique au large de la nouvelle plaque que les cellules sont transférés. Retirez tous les médias contenant les amas de cellules souches de la vieille plaque et transférer à la nouvelle plaque. </li><li> La place des cellules de nouveau dans un 37 ° C, 5% de CO 2 incubateur pour deux jours avec un minimum de perturbation physique (idéalement plaques, une fois dans l&#39;incubateur, ne sont pas directement touchés en aucune façon) pour permettre la fixation maximale. Note: Les cellules nécessiteront des changements quotidiens des médias. Passagin continueg tous les 4 jours seront importants. En utilisant une aiguille de calibre 21, passage que les parties de colonies avec la morphologie optimale ESC-like (rapport nucléocytoplasmique élevé). Cela permettra d&#39;assurer la sélection pour les colonies de hiPSC appropriées qui poussent bien et finalement donneront colonies homogènes. Temps global à la dérivation de l&#39;ESC comme colonies se situe entre 15 à 20 jours après le changement initial combiné de médias. </li><li> Geler les bas hiPSCs post-excisé de qualité GMP d&#39;abord quadrillé toutes les colonies en utilisant une aiguille de calibre 18. Lever toutes les pièces en utilisant un large ul pipette 200 et transférer tous les médias contenant les cellules à un 15 ml flacon et centrifuger conique à 200 g pendant 5 min à 4 ° C. </li><li> Bien que les cellules sont centrifugation, faire un support de gel consistant en des volumes égaux de deux supports combinés à froid et un milieu de congélation avec une concentration finale en DMSO de 7,5%. </li><li> Après que les cellules sont culottées, Aspirer ancien milieu et goutte à goutte re-suspendre le culot avec les médias de congélation. Immédiatement transfert dans des flacons de gel et mis dans un congélateur à -80 ° C. </li></ol> 5. hiPSCs Caractérisation GMP grade post-excisées <ol><li> Pour assurer une bonne expression de marqueurs de pluripotence post-conversion, utilisez QPCR de quantifier l&#39;expression des marqueurs de pluripotence clés (SOX2, OCT4 et NANOG) en utilisant les amorces énumérées dans le tableau 1 avec les mêmes étapes que mentionné dans l&#39;étape 3. méthodologies de QPCR suivre la mêmes étapes énumérées à l&#39;étape 3. </li><li> Utilisation cytométrie en flux pour détecter l&#39;acide sialique non humain Neu5Gc (l&#39;acide -glycolylneuraminic N) conformément aux conditions standards listés dans la trousse comme précédemment publiée 10. </li><li> Lavez délicatement les plaques de culture cellulaire avec des cellules en utilisant 1x phosphate salin (PBS). </li><li> Dissocier colonies en utilisant 1x réactif de dissociation pendant 5 min à température ambiante. </li><li> Pendant le temps d&#39;incubation de 5 minutes de l&#39;étape 5.4, de préparer la solution de blocage (fourni danskit) en faisant un 0,5% d&#39;agent de blocage dans du PBS glacé. </li><li> Étiqueter l&#39;ensemble des tubes pour cytométrie en flux analyse suivante: Unstained; 4 &#39;, 6-diamidino-2-phénylindole (DAPI) DAPI; anticorps de contrôle 1: 200; L&#39;anticorps primaire 1: 200; Anticorps secondaire 1: 200 d&#39;âne anti-Poulet IgG (H + L); MEF échantillon; Cellules post-excisée échantillon sur la matrice; et les cellules sur substrat synthétique échantillon post-excisée. </li><li> Secouant doucement agrégats de cellules de l&#39;étape 5.4 en ajoutant 2 ml de solution de blocage et transférer le contenu dans un tube conique de 15 ml. Centrifuger à 80 g pendant 5 min à 4 ° C. </li><li> Laver les cellules deux fois avec une solution de blocage et centrifuger comme indiqué dans l&#39;étape 5.7. </li><li> Remettre en suspension doucement environ 1 x 10 6 cellules dans 100 ul de solution de blocage avec le volume de dilution correspondant de chaque anticorps. Incuber avec doux balancement à 4 ° C pendant 1 heure. </li><li> Répétez lavages trois fois à l&#39;étape 5.8. </li><li> Remettre en suspension le culot cellulaire dans 400 pi de diluant Buffer (dans le kit) avec 1: 100 de DAPI pour les tubes 2, 6, 7, et 8 comme indiqué dans l&#39;étape 5.6. </li><li> cellules souches à travers une passoire 40 uM et traversent cytomètre de flux. </li></ol>

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James A. Byrne (JAB) and Agustin Vega-Crespo receive research funding from Fibrocell Science, Inc. JAB is a scientific consultant for Fibrocell Science, Inc. No other authors have any competing interests to disclose.

Nous décrivons une méthode consistant à dériver hiPSCs libre-facteur et de les rendre cliniquement significative par la conversion de ces cellules dans des conditions de qualité GMP de différenciation cellulaire en aval dans les futurs thérapeutique humaine. Bien que ce protocole est largement applicable à une variété de types de cellules, nous avons choisi de reprogrammer les fibroblastes dermiques humains, en raison de la facilité d&#39;extraction du patient et de leur applicabilité à la thérapeutique hu...

Adult human cells have been shown to be capable of undergoing epigenetic remodeling and reprogramming, as a result of lentiviral-based expression of four key transcription factors4,5. An important advancement in the reprogramming field was the use of a single excisable lentiviral stem cell cassette (STEMCCA), which housed all four reprogramming transcription factors that allowed a precise stoichiometric ratio of protein expression6. Additionally, when transduced in specific multiplicity of infection ranges, STEMCCA can lead to predominantly single genomic integration events during the reprogramming process7. The introduction of an excisable version of STEMCCA, which utilizes Cre/loxP technology followed by excision of the reprogramming vector after derivation of the stem cell line, enabled factor-free human induced pluripotent stem cell (hiPSC) lines to be derived8. Additionally, in order to enhance therapeutic applications of hiPSCs, a novel, quick, and readily applicable methodology for good manufacturing practice (GMP)-grade cell line conversion, from xeno-containing to xeno-free conditions, needed to be implemented. Here, we discuss a relevant methodology that more precisely assesses integrated gene expression differences, specifically when integrated into an intron, and clinical-grade cell conversion into putative GMP conditions. 
Previous research has used only relatively insensitive microarray transcriptional analysis to analyze gene expression differences in integrated genes after STEMCCA transduction3,9. Here, we introduce the methodology of sensitive quantitative polymerase chain reaction (PCR) analysis, to further examine integrated gene expression differences. Importantly, current safe-harbor criteria discard hiPSCs that have genes with viral integrations, thus limiting the applicability of these cells for downstream human cellular therapeutics9. We propose that the status quo may change with the use of fully characterized and transgene-free intronically reprogrammed hiPSCs. Additionally, we introduce a robust GMP-grade cell conversion protocol that can be readily applied to a variety of different cell types, which were originally derived under xeno-containing conditions10. This provides significant opportunities for the development of future cell reprogramming experiments, which require clinical-grade conditions to maintain human therapeutic relevance. 
These methodologies provide a foundation upon which current safe-harbor criteria may be expanded to include characterized STEMCCA reprogrammed hiPSC lines that maintain a normal gene expression profile after STEMCCA excision from the integrated intron. Also, full conversion into clinical-grade conditions, free from non-human animal antigens, will help to incorporate many more cell types, which have previously been reprogrammed and characterized only in xeno-containing conditions. These methodologies combined, are persuasive grounds for the US Food and Drug Administration (FDA) to consider expanding their limited approval from human embryonic stem cell (ESC)-based therapeutics to hiPSC-based therapeutics11. 
We recently detailed the derivation of a factor-free hiPSC line that was fully characterized and converted into putative clinical-grade conditions10. Here, we detail the protocol for hiPSC derivation by utilizing the STEMCCA lentivirus. These stem cells then undergo an excision process followed by gene expression characterization. Finally, the hiPSCs are converted over into GMP-grade conditions by a slow conversion methodology.

<table border="0" cellpadding="0" cellspacing="0" width="521">
	<tbody>
		<tr height="22">
			<td height="22" style="height:22px;width:140px;">Name</td>
			<td style="width:111px;">Company</td>
			<td style="width:136px;">Catalog Number</td>
			<td style="width:135px;">Comments</td>
		</tr>
		<tr height="21">
			<td colspan="4" height="21" style="height:21px;width:521px;">Media and reagents</td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:140px;">DMEM/F12 (basal media)</td>
			<td style="width:111px;">Invitrogen (Carlsbad, CA, USA)</td>
			<td style="width:136px;">11330057</td>
			<td style="width:135px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:140px;">Fetal bovine serum</td>
			<td style="width:111px;">Invitrogen</td>
			<td style="width:136px;">16000044</td>
			<td style="width:135px;"></td>
		</tr>
		<tr height="106">
			<td height="106" style="height:106px;width:140px;">Minimum essential medium (MEM) non-essential amino acids (NEAA), 100x</td>
			<td style="width:111px;">Invitrogen</td>
			<td style="width:136px;">11140050</td>
			<td style="width:135px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:140px;">Glutamax, 100x</td>
			<td style="width:111px;">Invitrogen</td>
			<td style="width:136px;">35050-061</td>
			<td style="width:135px;"></td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:140px;">PenStrep, penicillin-streptomycin, 100x</td>
			<td style="width:111px;">Invitrogen</td>
			<td style="width:136px;">15140-122</td>
			<td style="width:135px;">Thaw at 4 &#176;C, aliquot and store at &#8722;20 &#176;C</td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:140px;">Knockout serum replacement</td>
			<td style="width:111px;">Invitrogen</td>
			<td style="width:136px;">10828028</td>
			<td style="width:135px;">Thaw at 4 &#176;C, aliquot and store at &#8722;20 &#176;C</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:140px;">Trypsin/EDTA, 0.5%</td>
			<td style="width:111px;">Invitrogen</td>
			<td style="width:136px;">15400-054</td>
			<td style="width:135px;">Dilute stock out to 0.05% in 1x PBS</td>
		</tr>
		<tr height="127">
			<td height="127" style="height:127px;width:140px;">Basic fibroblast growth factor</td>
			<td style="width:111px;">GlobalStem (Rockville, MD, USA)</td>
			<td style="width:136px;">GSR-2001</td>
			<td style="width:135px;">Reconstitute to 10 &#181;g/ml stock in 0.1% bovine serum albumin dissolved in 1x PBS and store at &#8722;80 &#176;C</td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:140px;">&#946;-mercaptoethanol</td>
			<td style="width:111px;">Millipore (Billerica, MA, USA)</td>
			<td style="width:136px;">ES-007-E</td>
			<td></td>
		</tr>
		<tr height="127">
			<td height="127" style="height:127px;width:140px;">Matrigel (basement membrane matrix)</td>
			<td style="width:111px;">BD Biosciences (San Jose, CA, USA)</td>
			<td style="width:136px;">356231</td>
			<td style="width:135px;">Dilute stock Matrigel vial with 10 ml of DMEM/F12 while on ice for a 1:2 dilution. Aliquot and store at &#8722;20 &#176;C</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:140px;">CELLstart (Synthetic Substrate)</td>
			<td style="width:111px;">Invitrogen</td>
			<td style="width:136px;">A1014201</td>
			<td style="width:135px;"></td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:140px;">Stemmolecule Y27632</td>
			<td style="width:111px;">Stemgent (Cambridge, MA, USA)</td>
			<td style="width:136px;">04-0012-02</td>
			<td style="width:135px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:140px;">Puromycin</td>
			<td style="width:111px;">Invitrogen</td>
			<td style="width:136px;">A1113802</td>
			<td style="width:135px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:140px;">LightCycler 480 Probes Master</td>
			<td style="width:111px;">Roche (Basel, Switzerland)</td>
			<td style="width:136px;">4707494001</td>
			<td style="width:135px;"></td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:140px;">ProFreeze-CDM Medium/freezing medium</td>
			<td style="width:111px;">Lonza (Basel, Switzerland)</td>
			<td style="width:136px;">12-769E</td>
			<td style="width:135px;"></td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:140px;">Dimethyl sulfoxide</td>
			<td style="width:111px;">Sigma-Aldrich (St. Louis, MO, USA)</td>
			<td style="width:136px;">D8418</td>
			<td style="width:135px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:140px;">PBS</td>
			<td style="width:111px;">Invitrogen</td>
			<td style="width:136px;">14190-250</td>
			<td style="width:135px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:140px;">100 BP DNA Ladder</td>
			<td style="width:111px;">Invitrogen</td>
			<td style="width:136px;">15628019</td>
			<td style="width:135px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:140px;">SYBR Safe DNA Gel Stain 10000x</td>
			<td style="width:111px;">Invitrogen</td>
			<td style="width:136px;">S33102</td>
			<td style="width:135px;"></td>
		</tr>
		<tr height="89">
			<td height="89" style="height:89px;width:140px;">Agarose</td>
			<td style="width:111px;">Bio-Rad Laboratories, Inc. (Hercules, CA, USA)</td>
			<td style="width:136px;">161-3101</td>
			<td style="width:135px;"></td>
		</tr>
		<tr height="85">
			<td height="85" style="height:85px;width:140px;">Gelatin, from porcine skin</td>
			<td style="width:111px;">Sigma-Aldrich</td>
			<td style="width:136px;">G1890-100G</td>
			<td style="width:135px;">Make stock at 0.2% in PBS, autoclave and store at room temperature</td>
		</tr>
		<tr height="127">
			<td height="127" style="height:127px;width:140px;">mTeSR1</td>
			<td style="width:111px;">StemCell Technologies (Vancouver, BC, Canada)</td>
			<td style="width:136px;">5850</td>
			<td style="width:135px;">Combine Supplement 5X with the basic medium, aliquot and store at 4 &#176;C for up to 2 weeks.&#160;</td>
		</tr>
		<tr height="85">
			<td height="85" style="height:85px;width:140px;">Stemedia NutriStem XF/FF Culture Medium</td>
			<td style="width:111px;">Stemgent</td>
			<td style="width:136px;">05-100-1A</td>
			<td style="width:135px;">Thaw at 4 &#176;C O/N, aliquot and store at 4 &#176;C for up to 2 weeks.</td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:140px;">Primocin</td>
			<td style="width:111px;">InvivoGen (San Diego, CA, USA)</td>
			<td style="width:136px;">ant-pm-1</td>
			<td style="width:135px;"></td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:140px;">Accutase (Dissociation Reagent)</td>
			<td style="width:111px;">Invitrogen</td>
			<td style="width:136px;">A1110501</td>
			<td style="width:135px;"></td>
		</tr>
		<tr height="85">
			<td height="85" style="height:85px;width:140px;">Donkey anti-Chicken IgG AlexaFluor 488</td>
			<td style="width:111px;">Jackson ImmunoResearch (West Grove, PA, USA)</td>
			<td style="width:136px;">703-546-155</td>
			<td style="width:135px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:140px;">Polybrene/transfection agent</td>
			<td style="width:111px;">Millipore</td>
			<td style="width:136px;">TR-1003-G</td>
			<td style="width:135px;"></td>
		</tr>
		<tr height="22">
			<td colspan="4" height="22" style="height:22px;width:521px;">Plasticware</td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:140px;">12-well plates</td>
			<td style="width:111px;">VWR (West Chester, PA, USA)</td>
			<td style="width:136px;">29442-038</td>
			<td style="width:135px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:140px;">6-well plates</td>
			<td style="width:111px;">VWR</td>
			<td style="width:136px;">29442-042</td>
			<td style="width:135px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">10-cm plates</td>
			<td style="width:111px;">Sigma-Aldrich</td>
			<td style="width:136px;">Z688819</td>
			<td style="width:135px;"></td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:140px;">18-gauge needle</td>
			<td style="width:111px;">Fisher Scientific (Pittsburgh, PA, USA)</td>
			<td style="width:136px;">148265D</td>
			<td style="width:135px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:140px;">21-gauge needle</td>
			<td style="width:111px;">Fisher Scientific</td>
			<td style="width:136px;">14-829-10D</td>
			<td style="width:135px;"></td>
		</tr>
		<tr height="22">
			<td colspan="4" height="22" style="height:22px;width:521px;">Equipment</td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:140px;">BD LSRII Flow Cytometer</td>
			<td style="width:111px;">KSystem by Nikon (Tokyo, Japan)</td>
			<td style="width:136px;"></td>
			<td style="width:135px;"></td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:140px;">BD FACSDiva Version 6.1.3 Software</td>
			<td style="width:111px;">BD Biosciences</td>
			<td style="width:136px;"></td>
			<td></td>
		</tr>
		<tr height="22">
			<td colspan="4" height="22" style="height:22px;width:521px;">Kits</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:140px;">PureLink Genomic DNA Mini Kit</td>
			<td style="width:111px;">Invitrogen</td>
			<td style="width:136px;">K182000</td>
			<td style="width:135px;"></td>
		</tr>
		<tr height="85">
			<td height="85" style="height:85px;width:140px;">KAPA HiFi Hotstart ReadyMix PCR Kit</td>
			<td style="width:111px;">KAPA Biosystems (Wilmington, MA, USA)</td>
			<td style="width:136px;">KK2601</td>
			<td style="width:135px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:140px;">High Pure RNA Isolation Kit</td>
			<td style="width:111px;">Roche</td>
			<td style="width:136px;">11828665001</td>
			<td style="width:135px;"></td>
		</tr>
		<tr height="66">
			<td height="66" style="height:66px;width:140px;">Transcriptor First Strand cDNA Synthesis Kit</td>
			<td style="width:111px;">Roche</td>
			<td style="width:136px;">4379012001</td>
			<td style="width:135px;"></td>
		</tr>
		<tr height="54">
			<td height="54" style="height:54px;width:140px;">Sialix anti-Neu5Gc Basic Pack Kit</td>
			<td style="width:111px;">Sialix (Newton, MA, USA)</td>
			<td style="width:136px;">Basic Pack</td>
			<td style="width:135px;"></td>
		</tr>
		<tr height="54">
			<td height="54" style="height:54px;width:140px;">Media</td>
			<td style="width:111px;"></td>
			<td style="width:136px;"></td>
			<td style="width:135px;"></td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:140px;">Combined media 1</td>
			<td style="width:111px;">StemCell Technologies and Stemgent</td>
			<td style="width:136px;"></td>
			<td style="width:135px;">Consists of equal parts mTeSR1 and Nutristem</td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:140px;">Combined media 2</td>
			<td style="width:111px;">StemCell Technologies and Stemgent</td>
			<td style="width:136px;"></td>
			<td style="width:135px;">Consists of equal parts TeSR2 and Nutristem</td>
		</tr>
		<tr height="190">
			<td height="190" style="height:190px;width:140px;">HUF Media</td>
			<td style="width:111px;"></td>
			<td style="width:136px;"></td>
			<td style="width:135px;">Dulbecco&#8217;s modified Eagle&#8217;s medium/F12 [DMEM/F12] supplemented with 10% fetal bovine serum, 1x non-essential amino acids, 1x Glutamax, and 1x Primocin</td>
		</tr>
		<tr height="253">
			<td height="253" style="height:253px;width:140px;">Human Pluripotent Stem Cell Media</td>
			<td style="width:111px;"></td>
			<td style="width:136px;"></td>
			<td style="width:135px;">DMEM/F12 supplemented with 20% knockout serum replacement, 1x Glutamax, 1x non-essential amino acids, 1x Primocin, 1x &#946;-mercaptoethanol, and 10 ng/ml basic fibroblast growth factor</td>
		</tr>
		<tr height="22">
			<td colspan="2" height="22" style="height:22px;">DMEM/F12, Dulbecco&#8217;s modified Eagle&#8217;s medium/F12; PBS, phosphate-buffered saline.</td>
			<td style="width:136px;"></td>
			<td style="width:135px;"></td>
		</tr>
	</tbody>
</table>

derivation and characterization of a transgene-free human induced pluripotent stem cell line and conversion into defined clinical-grade conditions

Anthropiques cellules souches pluripotentes (de hiPSCs) peuvent être générés avec des méthodologies de reprogrammation base lentivirus. Toutefois, des traces de gènes potentiellement oncogènes restants dans les régions activement transcrits du génome, limitent leur potentiel pour une utilisation dans des applications thérapeutiques humaines 1. En outre, les antigènes non-humains issus de la reprogrammation de cellules souches ou la différenciation en dérivés thérapeutiques pertinentes empêchent ces hiPSCs d&#39;être utilisé dans un contexte clinique humain 2. Dans cette vidéo, nous vous présentons une procédure pour reprogrammer et analyser hiPSCs gratuitement facteur-libres de transgènes exogènes. Ces hiPSCs peuvent ensuite être analysés pour des anomalies d&#39;expression génique spécifique dans l&#39;intron contenant le lentivirus. Cette analyse peut être effectuée en utilisant sensible réaction en chaîne par polymérase quantitative (PCR), qui a un avantage sur les techniques moins sensibles précédemment utilisé pour détecter l&#39;expression du gène 3 différences. La conversion totale ende qualité clinique bonnes pratiques de fabrication (BPF) des conditions, permet pertinence clinique humaine. Notre protocole offre une autre méthode-à condition que les critères actuels exemptées seront étendre et inclure caractérisés dérivés de libre-facteur-base-hiPSC pour des applications à des fins thérapeutiques humains découlant hiPSCs de qualité GMP, ce qui devrait éliminer tout risque d&#39;immunogénicité due à des antigènes non-humains. Ce protocole est largement applicable aux cellules reprogrammées lentiviraux de tout type et fournit une méthode reproductible pour convertir cellules reprogrammées dans des conditions de qualité GMP.

Nous présentons un protocole pour dériver hiPSCs gratuitement facteur-qualité clinique en utilisant l&#39;approche de reprogrammation base-lentiviral STEMCCA. Figure 1A montre une image représentative de trois lignes hiPSC pré-excisée différents, après la reprogrammation à l&#39;approche STEMCCA sur une couche de MEF. Le principal avantage de l&#39;approche de reprogrammation STEMCCA réside dans le succès de reprogrammation cohérente atteint par plusieurs scientifiques, dans les différents ...

Watch this Scientific Journal Video about Derivation and Characterization of a Transgene-free Human Induced Pluripotent Stem Cell Line and Conversion into Defined Clinical-grade Conditions at JoVE.com

Derivation and Characterization of a Transgene-free Human Induced Pluripotent Stem Cell Line and Conversion into Defined Clinical-grade Conditions

We describe a protocol for deriving lentiviral-based reprogrammed and characterized factor-free human induced pluripotent stem cells and conversion into putative clinical-grade conditions.

Préparation et caractérisation d&#39;un libre-Transgene humain souches pluripotentes induites lignée cellulaire et la conversion en des conditions définies de qualité clinique

Human induced pluripotent stem cells (hiPSCs) can be generated with lentiviral-based reprogramming methodologies. However, traces of potentially oncogenic ...

derivation-characterization-transgene-free-human-induced-pluripotent

Research

JoVE Journal

Biology

7.8K vues.  University of California, Los Angeles (UCLA). We describe a protocol for deriving lentiviral-based reprogrammed and characterized factor-free human induced pluripotent stem cells and conversion into putative clinical-grade conditions. 

Vidéo: Derivation and Characterization of a Transgene-free Human Induced Pluripotent Stem Cell Line and Conversion into Defined Clinical-grade Conditions

Derivation of Human Embryonic Stem Cells by Immunosurgery

The ability of human embryonic stem cells to self-renew and differentiate into all cell types of 
the body suggests that they hold great promise for both medical applications and as a research 
tool for addressing fundamental questions in development and disease. Here, we provide a 
concise, step-by-step protocol for the derivation of human embryonic stem cells from embryos 
by immunosurgical isolation of the inner cell mass. 


The ability of human embryonic stem cells to self-renew and differentiate into all cell types of 
the body suggests that they hold great promise for both medical applications and as a research 
tool for addressing fundamental questions in development and disease. Here, we provide a 
concise, step-by-step protocol for the derivation of human embryonic stem cells from embryos 
by immunosurgical isolation of the inner cell mass.

Dérivation des cellules souches embryonnaires humaines par Immunosurgery

The ability of human embryonic stem cells to self-renew and differentiate into all cell types of 
the body suggests that they hold great promise for both ...

Recherche

Biologie

Isolation and Derivation of Mouse Embryonic Germinal Cells

The ability of embryonic germinal cells (EG) to differentiate into primordial germinal cells (PGCs) and later into gametes during early developmental stages is a perfect model to address our hypothesis about cancer and infertility. This protocol shows how to isolate primordial germinal cells from developing gonads in 10.5-11.5 days post coitum (dpc) mouse embryos.  Developing gonadal ridges from mouse embryos (C57BL6J) were dissociated by mechanical disruption with collagenase, then plated in a mouse embryo fibroblast feeder layer (MEF-CF1) that was previously mitotically inactivated with mitomycin C in the presence of knockout media and supplemented with Leukemia Inhibitor Factor (LIF), basic Fibroblast Growth Factor (bFGF), and Stem Cell Factor (SCF).  Using these optimized methods for PCG identification, isolation, and establishment of culture conditions permits long term cultures of EG cells for more than 40 days.  The embryonic germinal cell lines showed embryonic phenotype and expression of common used markers of the pluripotent state.  Isolation and derivation of germinal cells in culture provide a tool to understand their development in vitro and offer the opportunity to monitor cumulative damage at genetic and epigenetic levels after exposure to oxidative stress.

The ability of embryonic germinal cells to differentiate into primordial germinal cells during early development stages is a perfect model to address our hypothesis about cancer and infertility. This protocol shows how to isolate primordial germinal cells from developing gonads in 10.5-11.5 days post coitum mouse embryos.

L&#39;isolement et la dérivation de cellules germinales embryonnaires de souris

The ability of embryonic germinal cells (EG) to differentiate into primordial germinal cells (PGCs) and later into gametes during early developmental ...

Transfecting and Nucleofecting Human Induced Pluripotent Stem Cells

Genetic modification is continuing to be an essential tool in studying stem cell biology and in setting forth potential clinical applications of human embryonic stem cells (HESCs)1. While improvements in several gene delivery methods have been described2-9, transfection remains a capricious process for HESCs, and has not yet been reported in human induced pluripotent stem cells (iPSCs). In this video, we demonstrate how our lab routinely transfects and nucleofects human iPSCs using plasmid with an enhanced green fluorescence protein (eGFP) reporter. Human iPSCs are adapted and maintained as feeder-free cultures to eliminate the possibility of feeder cell transfection and to allow efficient selection of stable transgenic iPSC clones following transfection. For nucleofection, human iPSCs are pre-treated with ROCK inhibitor11, trypsinized into small clumps of cells, nucleofected and replated on feeders in feeder cell-conditioned medium to enhance cell recovery. Transgene-expressing human iPSCs can be obtained after 6 hours. Antibiotic selection is applied after 24 hours and stable transgenic lines appear within 1 week. Our protocol is robust and reproducible for human iPSC lines without altering pluripotency of these cells.

Despite recent advancements in genetic modification, transfection of human embryonic stem cells (HESCs) remains a capricious process. To our knowledge, systematic and efficient methods to transfect human induced pluripotent stem cells (iPSCs) have not been reported. Here, we describe robust protocols to efficiently transfect and nucleofect human iPSCs.

Transfection et Nucleofecting homme cellules souches pluripotentes induites

Genetic modification is continuing to be an essential tool in studying stem cell biology and in setting forth potential clinical applications of human ...

Efficient Derivation of Human Cardiac Precursors and Cardiomyocytes from Pluripotent Human Embryonic Stem Cells with Small Molecule Induction

To date, the lack of a suitable human cardiac cell source has been the major setback in regenerating the human myocardium, either by cell-based transplantation or by cardiac tissue engineering1-3. Cardiomyocytes become terminally-differentiated soon after birth and lose their ability to proliferate. There is no evidence that stem/progenitor cells derived from other sources, such as the bone marrow or the cord blood, are able to give rise to the contractile heart muscle cells following transplantation into the heart1-3. The need to regenerate or repair the damaged heart muscle has not been met by adult stem cell therapy, either endogenous or via cell delivery1-3. The genetically stable human embryonic stem cells (hESCs) have unlimited expansion ability and unrestricted plasticity, proffering a pluripotent reservoir for in vitro derivation of large supplies of human somatic cells that are restricted to the lineage in need of repair and regeneration4,5. Due to the prevalence of cardiovascular disease worldwide and acute shortage of donor organs, there is intense interest in developing hESC-based therapies as an alternative approach. However, how to channel the wide differentiation potential of pluripotent hESCs efficiently and predictably to a desired phenotype has been a major challenge for both developmental study and clinical translation. Conventional approaches rely on multi-lineage inclination of pluripotent cells through spontaneous germ layer differentiation, resulting in inefficient and uncontrollable lineage-commitment that is often followed by phenotypic heterogeneity and instability, hence, a high risk of tumorigenicity6-8 (see a schematic in Fig. 1A). In addition, undefined foreign/animal biological supplements and/or feeders that have typically been used for the isolation, expansion, and differentiation of hESCs may make direct use of such cell-specialized grafts in patients problematic9-11. To overcome these obstacles, we have resolved the elements of a defined culture system necessary and sufficient for sustaining the epiblast pluripotence of hESCs, serving as a platform for de novo derivation of clinically-suitable hESCs and effectively directing such hESCs uniformly towards clinically-relevant lineages by small molecules12 (see a schematic in Fig. 1B). After screening a variety of small molecules and growth factors, we found that such defined conditions rendered nicotinamide (NAM) sufficient to induce the specification of cardiomesoderm direct from pluripotent hESCs that further progressed to cardioblasts that generated human beating cardiomyocytes with high efficiency (Fig. 2). We defined conditions for induction of cardioblasts direct from pluripotent hESCs without an intervening multi-lineage embryoid body stage, enabling well-controlled efficient derivation of a large supply of human cardiac cells across the spectrum of developmental stages for cell-based therapeutics.

We have established a protocol for induction of cardioblasts direct from pluripotent human embryonic stem cells maintained under defined conditions with small molecules, which enables derivation of a large supply of human cardiac progenitors and functional cardiomyocytes for cardiovascular repair.

Dérivation efficace des ressources humaines précurseurs cardiaques et les cardiomyocytes à partir pluripotentes cellules souches embryonnaires humaines à l&#39;induction de petites molécules

To date, the lack of a suitable human cardiac cell source has been the major setback in regenerating the human myocardium, either by cell-based ...

Selecting and Isolating Colonies of Human Induced Pluripotent Stem Cells Reprogrammed from Adult Fibroblasts

Herein we present a protocol of reprogramming human adult fibroblasts into human induced pluripotent stem cells (hiPSC) using retroviral vectors encoding Oct3/4, Sox2, Klf4 and c-myc (OSKM) in the presence of sodium butyrate 1-3. We used this method to reprogram late passage (&gt;p10) human adult fibroblasts derived from Friedreich's ataxia patient (GM03665, Coriell Repository). The reprogramming approach includes highly efficient transduction protocol using repetitive centrifugation of fibroblasts in the presence of virus-containing media. The reprogrammed hiPSC colonies were identified using live immunostaining for Tra-1-81, a surface marker of pluripotent cells, separated from non-reprogrammed fibroblasts and manually passaged 4,5. These hiPSC were then transferred to Matrigel plates and grown in feeder-free conditions, directly from the reprogramming plate. Starting from the first passage, hiPSC colonies demonstrate characteristic hES-like morphology. Using this protocol more than 70% of selected colonies can be successfully expanded and established into cell lines. The established hiPSC lines displayed characteristic pluripotency markers including surface markers TRA-1-60 and SSEA-4, as well as nuclear markers Oct3/4, Sox2 and Nanog. 
The protocol presented here has been established and tested using adult fibroblasts obtained from Friedreich's ataxia patients and control individuals 6, human newborn fibroblasts, as well as human keratinocytes.

We present a protocol for efficient reprogramming of human somatic cells into human induced pluripotent stem cells (hiPSC) using retroviral vectors encoding Oct3/4, Sox2, Klf4 and c-myc (OSKM) and identification of correctly reprogrammed hiPSC by live staining with Tra-1-81 antibody.

Colonies sélectionner et isoler de l&#39;homme cellules souches pluripotentes induites à partir de fibroblastes adultes Reprogrammed

Herein we present a protocol of reprogramming human adult fibroblasts into human induced pluripotent stem cells (hiPSC) using retroviral vectors encoding ...

Reprogramming Human Somatic Cells into Induced Pluripotent Stem Cells (iPSCs) Using Retroviral Vector with GFP

Human embryonic stem cells (hESCs) are pluripotent and an invaluable cellular sources for in vitro disease modeling and regenerative medicine1. It has been previously shown that human somatic cells can be reprogrammed to pluripotency by ectopic expression of four transcription factors (Oct4, Sox2, Klf4 and Myc) and become induced pluripotent stem cells (iPSCs)2-4 . Like hESCs, human iPSCs are pluripotent and a potential source for autologous cells. Here we describe the protocol to reprogram human fibroblast cells with the four reprogramming factors cloned into GFP-containing retroviral backbone4. Using the following protocol, we generate human iPSCs in 3-4 weeks under human ESC culture condition. Human iPSC colonies closely resemble hESCs in morphology and display the loss of GFP fluorescence as a result of retroviral transgene silencing. iPSC colonies isolated mechanically under a fluorescence microscope behave in a similar fashion as hESCs. In these cells, we detect the expression of multiple pluripotency genes and surface markers.

Reprogramming Human Somatic Cells into Induced Pluripotent Stem Cells iPSCs Using Retroviral Vector with GFP

A method to generate human induced pluripotent stem cells (iPSCs) via retrovirus-mediated ectopic expression of OCT4, SOX2, KLF4 and MYC is described. A practical way to identify human iPSC colonies based on GFP expression is also discussed.

Reprogrammation des cellules somatiques humaines dans cellules souches pluripotentes induites (CISP) Utilisation vecteur rétroviral avec la GFP

Human embryonic stem cells (hESCs) are pluripotent and an invaluable cellular sources for in vitro disease modeling and regenerative medicine1. It has ...

Generation of Human Cardiomyocytes: A Differentiation Protocol from Feeder-free Human Induced Pluripotent Stem Cells

In order to investigate the events driving heart development and to determine the molecular mechanisms leading to myocardial diseases in humans, it is essential first to generate functional human cardiomyocytes (CMs). The use of these cells in drug discovery and toxicology studies would also be highly beneficial, allowing new pharmacological molecules for the treatment of cardiac disorders to be validated pre-clinically on cells of human origin. Of the possible sources of CMs, induced pluripotent stem (iPS) cells are among the most promising, as they can be derived directly from readily accessible patient tissue and possess an intrinsic capacity to give rise to all cell types of the body 1. Several methods have been proposed for differentiating iPS cells into CMs, ranging from the classical embryoid bodies (EBs) aggregation approach to chemically defined protocols 2,3. In this article we propose an EBs-based protocol and show how this method can be employed to efficiently generate functional CM-like cells from feeder-free iPS cells.

Pluripotent stem cells, either embryonic or induced pluripotent stem (iPS) cells, constitute a valuable source of human differentiated cells, including cardiomyocytes. Here, we will focus on cardiac induction of iPS cells, showing how to use them to obtain functional human cardiomyocytes through an embryoid bodies-based protocol.

Génération de cardiomyocytes humains: Un protocole de différenciation des induits des cellules souches pluripotentes humaines chargeur sans

In order to investigate the events driving heart development and to determine the molecular mechanisms leading to myocardial diseases in humans, it is ...

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

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

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

Génération efficace d&#39;origine humaine souches pluripotentes à partir de cellules somatiques humaines avec Sendai-virus

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

High Efficiency Differentiation of Human Pluripotent Stem Cells to Cardiomyocytes and Characterization by Flow Cytometry

There is an urgent need to develop approaches for repairing the damaged heart, discovering new therapeutic drugs that do not have toxic effects on the heart, and improving strategies to accurately model heart disease. The potential of exploiting human induced pluripotent stem cell (hiPSC) technology to generate cardiac muscle &#8220;in a dish&#8221; for these applications continues to generate high enthusiasm. In recent years, the ability to efficiently generate cardiomyogenic cells from human pluripotent stem cells (hPSCs) has greatly improved, offering us new opportunities to model very early stages of human cardiac development not otherwise accessible. In contrast to many previous methods, the cardiomyocyte differentiation protocol described here does not require cell aggregation or the addition of Activin A or BMP4 and robustly generates cultures of cells that are highly positive for cardiac troponin I and T (TNNI3, TNNT2), iroquois-class homeodomain protein IRX-4 (IRX4), myosin regulatory light chain 2, ventricular/cardiac muscle isoform (MLC2v) and myosin regulatory light chain 2, atrial isoform (MLC2a) by day 10 across all human embryonic stem cell (hESC) and hiPSC lines tested to date. Cells can be passaged and maintained for more than 90 days in culture. The strategy is technically simple to implement and cost-effective. Characterization of cardiomyocytes derived from pluripotent cells often includes the analysis of reference markers, both at the mRNA and protein level. For protein analysis, flow cytometry is a powerful analytical tool for assessing quality of cells in culture and determining subpopulation homogeneity. However, technical variation in sample preparation can significantly affect quality of flow cytometry data. Thus, standardization of staining protocols should facilitate comparisons among various differentiation strategies. Accordingly, optimized staining protocols for the analysis of IRX4, MLC2v, MLC2a, TNNI3, and TNNT2 by flow cytometry are described.

The article describes the detailed methodology to efficiently differentiate human pluripotent stem cells into cardiomyocytes by selectively modulating the Wnt pathway, followed by flow cytometry analysis of reference markers to assess homogeneity and identity of the population.

Haut différenciation de l&#39;efficacité de pluripotentes humaines des cellules souches à des cardiomyocytes et caractérisation par cytométrie en flux

There is an urgent need to develop approaches for repairing the damaged heart, discovering new therapeutic drugs that do not have toxic effects on the ...

Extraction, Labeling, and Purification of Lineage-Specific Cells from Human Antral Follicles

The activation, growth, development, and maturation of oocytes is a complex process that is coordinated not just between multiple cell types of the ovary but also across multiple points of control within the hypothalamic/pituitary/ovarian circuit. Within the ovary, multiple specialized cell types grow in close association with the oocyte within the ovarian follicles. The biology of these cells has been well described at the later stages, when they are easily recovered as byproducts of assisted reproductive treatments. However, the in-depth analysis of small antral follicles isolated directly from the ovary is not commonly carried out due to the scarcity of human ovarian tissue and the limited access to the ovary in patients undergoing assisted reproductive treatments. 
These methods for processing whole ovaries for the cryopreservation of cortical strips with the concurrent identification/isolation of ovary resident cells enable the high-resolution analysis of the early stages of antral follicle development. We demonstrate protocols for isolating discrete cell types by treating antral follicles enzymatically and separating the granulosa, theca, endothelial, hematopoietic, and stromal cells. The isolation of cells from the antral follicles at various sizes and developmental stages enables the comprehensive analysis of the cellular and molecular mechanisms that drive follicle growth and ovarian physiology and provides a source of viable cells that can be cultured in vitro to recapitulate the follicle microenvironment.

Here, we present protocols for the identification and purification of ovarian cells from antral follicles. We elaborate on methods for processing whole ovaries for the cryopreservation of cortical strips while also harvesting intact antral follicles that are treated enzymatically to liberate multiple follicle resident cell types, including granulosa, theca, endothelial, hematopoietic, and stromal cells.

Extraction, marquage et purification de cellules spécifiques à la lignée à partir de follicules antraux humains

The activation, growth, development, and maturation of oocytes is a complex process that is coordinated not just between multiple cell types of the ovary ...