Nous remercions Yoshiko Miyake, Sayaka Kanaami, Chihana Fujita, Miho Yamaguchi, Natsuko Henmi, et Rei Ohno de l&#39;École de médecine de l&#39;Université de Keio pour l&#39;assistance technique. Ce travail a été financé en partie par un programme de soutien à la R &amp; D Systems pour accélérer l&#39;utilisation pratique des résultats de la recherche en santé, et le Programme de route pour la réalisation de la médecine régénérative.

1. Préparer les lymphocytes T activés humain <ol><li> Recueillir 10 ml héparinés sang total d&#39;un donneur par ponction veineuse. <ol><li> Obtenir le consentement éclairé des donneurs à l&#39;avance de la ponction veineuse. Ponction veineuse doit être effectué par du personnel qualifié et formé. </li><li> Manipulez les échantillons sanguins obtenus avec du matériel stérile et des lignes directrices en matière de biosécurité appropriées suivantes. </li></ol></li><li> Diluer 10 ml de sang entier héparinisé avec 10 ml D-PBS (-). </li><li> Mettre 15 ml de solution de Ficoll (PREMIUM Ficoll-Paque) dans un nouveau tube conique de 50 ml. </li><li> Couche de la solution de sang de 20 ml préparée à l&#39;étape 1.2 sur la solution de Ficoll à l&#39;étape 1.3. En utilisant une pipette électrique, laissez solution de sang préparé à l&#39;étape 1.2 courir lentement le long de la paroi intérieure du tube. Veillez à ne pas mélanger la solution de sang et de solution de Ficoll. </li><li> Centrifuger à 400 g pendant 30 min à température ambiante. Réglez la vitesse de décélération centrifuge pour &quot;ralentir&quot;. Remarque: Après centrifugation, une fine blanchecouche émerge entre une couche de plasma laiteuse supérieure et une couche de Ficoll inférieure claire. Cette mince couche blanche contient des cellules mononucléaires. </li><li> Transférer la couche de cellules mononucléées dans un nouveau tube conique de 50 ml avec une pipette de 1 ml. Soyez prudent de ne pas inclure solution de Ficoll. </li><li> Ajouter D-PBS (-) pour les cellules mononucléaires à un volume total de 10 ml, et centrifuger à 200 g pendant 5 min à température ambiante. </li><li> Jeter le surnageant. Ajouter 10 ml de D-PBS (-) pour les cellules mononucléaires, et centrifuger à 200 g pendant 5 min à température ambiante. </li><li> Jeter le surnageant, ajouter 1 ml de milieu KBM502 aux cellules mononucléaires recueillies, et de compter les cellules en utilisant un hémocytomètre. Plus de 5 x 10 6 cellules mononucléaires peuvent être obtenus à partir de 10 ml du sang total hépariné avec une séparation appropriée à l&#39;aide de Ficoll. </li><li> Préparer une solution anti-CD3 humain anticorps à une concentration de 10 pg / ml dans du D-PBS (-). Ajouter la solution d&#39;anticorps anti-CD3 humain à une plaque de 6 puits, faire tremper les vaguesas de chaque puits et incuber le plat à 37 ° C dans un incubateur à CO 2 de 5% pendant au moins 30 min. </li><li> Retirer la solution d&#39;anticorps anti-CD3 humain à partir de chaque puits et laver une fois avec du D-PBS (-) juste avant l&#39;ensemencement des cellules. </li><li> Des cellules mononucléaires de semences qui ont été préparés à l&#39;étape 1.9, à une densité de 2,5 x 10 5 cellules / cm 2 sur les CD3 anti-humain de l&#39;anticorps enduit des plaques à 6 puits dans du milieu KBM502. Incuber à 37 ° C dans un 5 incubateur -CO 2% pendant 3-7 jours sans changement de milieu jusqu&#39;à ce que les cellules atteignent la confluence T. Cellules T dans cette période sont les deux cellules en suspension et adhérents. </li></ol> 2. Infect T cellules humaines avec SeV Vecteurs <ol><li> Après la culture 3-7 jours, recueillir les cellules T activées avec un milieu existant par pipetage. Les transférer à un tube conique de 15 ml et centrifuger à 200 g pendant 5 min à température ambiante. </li><li> Retirer le surnageant et ajouter 1 ml de milieu de KBM502 frais. Compter les cellules et la plaque 1.5 x 10 6 cellules (dans 2 ml de milieu frais KBM502) dans chaque puits d&#39;une plaque à 6 puits revêtues d&#39;anticorps anti-CD3. </li><li> Décongeler les solutions SeV de OCT3 / 4-SEV / TSΔF, SOX2-SEV / TSΔF, KLF4-SEV / TSΔF, et c-MYC (HNL) -sev / TS15ΔF sur la glace. </li><li> Ajouter les solutions contenant OCT3 SeV / 4-SeV / TSΔF, SOX2-SeV / TSΔF, KLF4-SeV / TSΔF, et c-MYC (HNL) -sev / TS15ΔF individuellement dans les puits, chacun à une multiplicité d&#39;infection (MOI) 10. Incuber à 37 ° C dans un 5 incubateur -CO 2% pour 24 une autre heure. </li></ol> 3. Retirez Vecteurs SEV de la T cellules humaines <ol><li> 24 h après l&#39;infection, les cellules infectées recueillir avec une pipette. Si il ya des cellules adhérentes, elles se détachent facilement les pipetage à plusieurs reprises de haut en bas. Les transférer à un tube conique de 15 ml, et centrifuger à 200 g pendant 5 min à température ambiante. </li><li> Retirer le surnageant contenant les vecteurs SEV, et ajouter 2 ml de milieu de KBM502 frais. Cellules replate dans each des puits. Incuber à 37 ° C dans un 5 incubateur -CO 2% pour une heure 24 supplémentaire. </li></ol> 4. Les cellules réensemencer sur une couche de Matrigel <ol><li> Préparer la solution de la matrice de la membrane basale par le gel de la matrice de la décongélation (s&#39;il vous plaît voir la liste des matériaux pour la marque spécifique utilisée dans cette étude) au 4 ° C et la dissolution de 0,2 ml dans 10 ml de milieu DMEM / F12. Remarque: gel de Matrix (voir s&#39;il vous plaît la Liste des matériaux pour la marque spécifique utilisée dans cette étude) besoin de dégeler O / N à 4 ° C pour être complètement dégelé. Gardez-le sur la glace jusqu&#39;à ce que juste avant l&#39;utilisation. </li><li> Planche 5 ml de solution de matrice de la membrane basale dans des plats par boîte de 100 mm et de laisser à température ambiante pendant au moins 30 min avant l&#39;ensemencement des cellules. Au moins deux plats de 100 mm sont nécessaires pour une expérience. </li><li> Recueillir des cellules SeV-infectés par le pipetage, les transférer dans un tube conique de 15 ml, et centrifuger à 200 g pendant 5 min à température ambiante. </li><li> Retirer le surnageant et ajouter 1 ml de milieu de KBM502 frais. Comptez le nombre de CElls en utilisant un hémocytomètre, retirez la solution sous-sol de la matrice de la membrane des plats de 100 mm préparé à l&#39;étape 4.2 et la plaque 1 × 10 5 et 1 × 10 6 cellules (dans 10 ml de milieu de mTeSR frais) sur un 100-mm membrane basale matrice revêtu de la vaisselle. Enfin, utilisez la plaque dans laquelle les colonies sont facilement ramassés. </li><li> Incuber le plat à 37 ° C dans un 5 incubateur% -CO 2 H / N. </li><li> Changer le support à 10 ml chair moyen mTeSR chaque autre jour. Remarque: Environ 20-30 jours après l&#39;infection, les colonies qui ressemblent étroitement à des cellules souches embryonnaires (CSE) apparaîtra. </li></ol> 5. Développer T CSPi cellulaires dérivées <ol><li> Préparer la solution de la matrice de la membrane basale par le gel de la matrice de la décongélation (s&#39;il vous plaît voir la liste des matériaux pour la marque spécifique utilisée dans cette étude) au 4 ° C et la dissolution de 0,2 ml dans 10 ml de milieu DMEM / F12. </li><li> Planche 1 ml de la solution de matrice de membrane basale par puits dans une plaque à 6 puits et laisser à température ambiante pendant aumoins 30 min avant prélèvement de colonies. </li><li> Remplir une plaque de 96 puits avec 20 pi de milieu mTeSR par puits. </li><li> Sélectionnez colonies qui ressemblent CES sous le stéréomicroscope aide d&#39;une pipette de 20 pi. Remarque: Les colonies de CES et CSPi sont rond et plat comme dans la figure 1B. </li><li> Transfert colonies sélectionnées dans la plaque de 96 puits rempli avec un milieu mTeSR à l&#39;étape 5.3. </li><li> Ajouter 200 pi de milieu mTeSR à chaque puits et soigneusement la pipette de haut en bas pour briser la colonie en petites touffes sous le stéréomicroscope aide d&#39;une pipette de 200 pi. Retirer la solution de la matrice de la membrane basale du bien préparé en 5.2 et de mettre chaque colonie dans un puits de la matrice revêtu membrane basale plaque de 6 puits avec 2 ml de milieu mTeSR par puits. </li><li> Changer le support à 2 ml chair moyen mTeSR tous les autres jours. </li></ol> 6. Maintenir T CSPi cellulaires dérivées Les iPSCs dérivés de cellules T peuvent être maintenuset stockée en utilisant les mêmes techniques que pour les CSPi humaines et CES de l&#39;homme. Une fois colonies iPSC grandissent, développez-les dans de grands plats en répétant la même procédure. <ol><li> Changer le support mTeSR tous les 1-2 jours. </li><li> Lorsque les colonies deviennent confluentes tous les 5-7 jours, passage des cellules en utilisant une solution de dissociation pour les CSPi humaines et CES de l&#39;homme conformément aux instructions du fabricant. </li></ol>

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Les auteurs ne sont pas des intérêts concurrents ou à divulguer contradictoires.

We describe a protocol for generating iPSCs from human peripheral T cells in serum-free and feeder-free conditions using a combination of matrigel, mTeSR medium, and SeV vectors. For clinical applications of iPSCs, it is important to have a protocol for stably generating iPSCs and a less-invasive method for cell sampling. Although generating iPSCs with the combination of matrigel and mTeSR medium showed lower reprogramming efficiency than that with knockout serum replacement (KSR) medium and feeder-cells, this combination achieves stable generation of iPSCs from donors16. Stable iPSC generation and less-invasive cell sampling has the advantage of being able to increase the number of donors for iPSC generation. 
SeV vectors, a minus-strand RNA virus, is not integrated into the host genome. Therefore the risks of tumorigenesis can be avoided at the step of reprogramming factor induction20-24. Additionally, the feeder-free conditions, which use a combination of defined culture medium and matrigel instead of a feeder layer, make it possible to minimize the potential risks of exposure to unknown exogenous factors. The fusion of these techniques provides a less invasive and safer iPSC technology for regenerative medicine16.
Important steps in this protocol are the step of activating human T cells (Step 1) and infecting them with SeV vectors (Step 2). When no ESC-like colony is obtained in the culture dish at an optimal time after SeV infection, the following should be considered. First, the confluency of the mononuclear cells before T cell activation may not be appropriate because optimal activation of T cells is critical for SeV infection25,26. Too high a density of mononuclear cells leads to cell death, interfering with the proper activation of T cells. Too low a density of mononuclear cells also disturbs the proper activation and proliferation of T cells. Therefore, the confluency of mononuclear cells should be checked and adjusted accordingly. Second, the dosage of SeV vectors may not be sufficient. The induction efficiency of iPSC colonies depends on the dosage of the virus6. If no ESC-like colony is observed after SeV infection, the option of increasing the virus dosage up to an MOI of 15-20 should be considered. If signs of iPSC colony differentiation are observed, the frequency at which medium is changed may be increased up to every other day, or to every day when colonies are larger. 
The limitation in this protocol consists of this method not being virus free. Although SeV for cell reprogramming is commercially available with ease, users need to prepare equipment according to the appropriate biosafety level. As another method for generating integration-free iPSCs, episomal vectors have been used until now27-29. Although episomal vectors can be used with equipment with a lower level of biosafety, episomal vectors might insert into the host genome at extremely low rates. Therefore, additional checks are required to confirm the disappearance of transgenes, as when using SeV.
There is another limitation in that this technique is not absolutely free from animal-derived products. Some substrates, such as matrigel, anti-CD3 mAb, dissociation solution and SeV solution are derived from animal products and are therefore associated with a risk of transferring xenogeneic pathogens. However, the reduction of animal-derived substrates in the culture system is meaningful for the clinical application of iPSC technology due to the lower risk.

CSPi ont attiré une attention considérable en tant que source d&#39;inauguration de cellules pour la médecine régénérative 1-3. À ce jour, diverses méthodes pour générer des CSPi ont été rapportés 4,5. Parmi ceux-ci, CSPi générées à partir de cellules T humaines ont été d&#39;un intérêt particulier en raison de la méthode moins invasive de l&#39;échantillonnage de la cellule 6-8. En outre, iPSCs dérivée de cellules T contient le gène réarrangements du récepteur des cellules T (TCR) et sont donc une source de cellules T spécifiques d&#39;antigène 9,10. Par conséquent, la génération de cellules T dérivées CSPi est utile pour progresser en toute sécurité la médecine régénérative. Cette méthode est basée sur le concept de la réduction des risques de pathogénicité imprévisible. Pour l&#39;application clinique sûre des CSPi il est important de réduire le risque d&#39;exposition à des agents pathogènes 11. Auparavant, de nombreux systèmes de culture de cellules souches pluripotentes, les cellules nourricières et de sérum bovin foetal ont été utilisés comme réactif essentielS 12. Cependant, l&#39;élimination de ces deux réactifs du système de culture est préférable pour la production iPSC à réduire les risques de pathogénicité imprévisible. En outre, ce procédé présente l&#39;avantage d&#39;éviter l&#39;échantillonnage de cellules invasive de patients et laborieux préparation de cellules nourricières. Parce que les lymphocytes T dérivés CSPi ont déjà été utilisés avec succès dans la recherche de la maladie 13,14, cette méthode est également applicable et utile pour générer des CSPi maladies spécifiques de patients. Parmi les méthodes de reprogrammation de cellules T, en utilisant le vecteur virus de Sendai (SeV) comme un véhicule génique est une méthode qui peut générer des CSPi avec un rendement élevé 7,16. En outre, parce SeV est un virus à ARN simple brin et n&#39;a pas besoin d&#39;une phase d&#39;ADN pour la réplication, son utilisation dans la production iPSC évite la rupture du génome de l&#39;hôte 17-19. Par conséquent, nous avons établi des protocoles pour générer CSPi de cellules périphériques T humains dans le sérum-frEE et les conditions de libre-alimentation utilisant une combinaison de matrigel, moyen mTeSR et SeV vecteurs.

<table border="0">
	<tbody>
		<tr>
			<td>Ficoll-Paque PREMIUM</td>
			<td>GE Healthcare</td>
			<td>17-5442-02</td>
			<td></td>
		</tr>
		<tr>
			<td>Purified NA/LE mouse anti-human CD3</td>
			<td>BD Pharmingen</td>
			<td>555336</td>
			<td></td>
		</tr>
		<tr>
			<td>KBM502 medium</td>
			<td>KOHJIN BIO</td>
			<td>16025020</td>
			<td>Warm in 37 &#8451; water bath before use</td>
		</tr>
		<tr>
			<td>Bovine albumin fraction V solution</td>
			<td>Gibco</td>
			<td>15260-037</td>
			<td></td>
		</tr>
		<tr>
			<td>BD Matrigel Matrix Growth Factor Reduced</td>
			<td>BD Biosciences</td>
			<td>354230</td>
			<td>Thaw completely at 4&#8451; overnight and dilute it 50 times with Dulbecco&#39;s Modified Eagle&#39;s Medium before coating culture dishes</td>
		</tr>
		<tr>
			<td>mTeSR1 medium kit</td>
			<td>STEM CELL</td>
			<td>5850</td>
			<td>Warm at room temperature before use</td>
		</tr>
		<tr>
			<td>Dissociation Solution</td>
			<td>ReproCELL</td>
			<td>RCHETP002</td>
			<td></td>
		</tr>
		<tr>
			<td>D-PBS(&#8211;)</td>
			<td>Wako</td>
			<td>045-29795</td>
			<td></td>
		</tr>
		<tr>
			<td>SeV Vector kit CytoTune-iPS ver.1.0</td>
			<td>DNAVEC</td>
			<td>DV-0303c</td>
			<td>Thaw on ice before use</td>
		</tr>
		<tr>
			<td>100-mm tissue culture dish</td>
			<td>Falcon</td>
			<td>353003</td>
			<td></td>
		</tr>
		<tr>
			<td>96-well tissue culture plate</td>
			<td>Falcon</td>
			<td>353078</td>
			<td></td>
		</tr>
		<tr>
			<td>6-well tissue culture plate</td>
			<td>Falcon</td>
			<td>353046</td>
			<td></td>
		</tr>
		<tr>
			<td>15ml Centrifuge Tube</td>
			<td>Greiner Bio-One&#160;</td>
			<td>188271</td>
			<td></td>
		</tr>
		<tr>
			<td>50ml Centrifuge Tube</td>
			<td>Greiner Bio-One&#160;</td>
			<td>227261</td>
			<td></td>
		</tr>
	</tbody>
</table>

generation of induced pluripotent stem cells from human peripheral t cells using sendai virus in feeder-free conditions

Récemment, CSPi ont attiré l&#39;attention comme une nouvelle source de cellules pour des thérapies régénératrices. Bien que la méthode initiale pour CSPi générant invoqué fibroblastes dermiques obtenus par biopsie invasive et l&#39;insertion génomique rétroviral de transgènes, il ya eu beaucoup d&#39;efforts pour éviter ces inconvénients. Cellules périphériques humaines de T sont une source cellulaire unique pour générer des CSPi. iPSCs dérivées de cellules T contiennent des réarrangements du récepteur des cellules T (TCR) des gènes et sont une source de cellules T spécifiques d&#39;antigène. En outre, le réarrangement T du récepteur de la cellule dans le génome a le potentiel pour marquer lignées cellulaires individuelles et la distinction entre les cellules transplantées et donateurs. Pour l&#39;application clinique de iPSCs sûr, il est important de réduire au minimum le risque d&#39;exposer iPSCs nouvellement générées à des agents nocifs. Bien que les cellules de sérum fœtal bovin et d&#39;alimentation ont été essentielles pour la culture de cellules souches pluripotentes, il est préférable de les retirer du système de culture pour réduire le risquede pathogénicité imprévisible. Pour y remédier, nous avons établi un protocole pour générer CSPi de cellules périphériques T humains en utilisant le virus Sendai afin de réduire le risque d&#39;exposition à des agents pathogènes CSPi indéfinis. Bien que le virus de Sendai manipulation nécessite un équipement avec le niveau de biosécurité appropriées, Sendai virus infecte les cellules T activées sans insertion du génome, mais avec une grande efficacité. Dans ce protocole, nous démontrons la génération de iPSCs à partir de cellules T périphériques humaines dans des conditions exemptes d&#39;alimentation-en utilisant une combinaison de la culture des cellules T activées et le virus Sendai.

<html> En utilisant ce protocole, les utilisateurs sont en mesure de générer des CSPi de cellules périphériques T humains de manière stable. génération iPSC à partir de cellules avec SeV et matrigel T a montré environ 0,002% -. 0,005% de l&#39;efficacité de la reprogrammation cellulaire entre plusieurs cas de donateurs et SeV ont pas été détectés après plusieurs passages 16 Figure 1A montre un schéma du protocole pour générer des cellules T-dérivé CSPi dans des conditions exemptes de cellules nourricières utilisant matrigel et moyennes mTeSR. Environ 20-30 jours après l&#39;infection par SEV, colonies iPSC sont reconnus par leur ESC colonie comme la morphologie (figure 1B). Immunofluorescence a révélé l&#39;expression de marqueurs de cellules pluripotentes typiques (NANOG, OCT3 / 4, SSEA4, TRA-1-60, et TRA-1-81) dans T CSPi dérivés de cellules généré dans des conditions sans cellules nourricières (figure 1C). &quot;Width =« 700 &quot;/&gt; Figure 1. (A): Un schéma du protocole pour la reprogrammation des cellules T dans des conditions sans cellules nourricières dans cette étude. (B): Une colonie iPSC ESC comme typique le jour 27 après le prélèvement de sang dans des conditions sans alimentation. (C): ALP coloration et immunofluorescence pour les marqueurs de pluripotence et de surface (NANOG, OCT3 / 4, SSEA4, TRA-1-60, et TRA-1-81) dans T CSPi dérivés de cellules produites dans des conditions sans alimentation. <a href="https://www.jove.com/files/ftp_upload/53225/53225fig1highres.jpg" target="_blank">S&#39;il vous plaît cliquez ici pour voir une version plus grande de cette figure.</a>

Watch this Scientific Journal Video about Generation of Induced Pluripotent Stem Cells from Human Peripheral T Cells Using Sendai Virus in Feeder-free Conditions at JoVE.com

Generation of Induced Pluripotent Stem Cells from Human Peripheral T Cells Using Sendai Virus in Feeder-free Conditions

This protocol describes how to generate induced pluripotent stem cells (iPSCs) from human peripheral T cells in feeder-free conditions using a combination of matrigel and Sendai virus vectors containing reprogramming factors.

Génération de cellules souches pluripotentes induites à partir de cellules T périphériques humains à l&#39;aide de virus Sendai dans des conditions libres Feeder-

Recently, iPSCs have attracted attention as a new source of cells for regenerative therapies. Although the initial method for generating iPSCs relied on ...

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11.9K vues.  Keio University School of Medicine. This protocol describes how to generate induced pluripotent stem cells (iPSCs) from human peripheral T cells in feeder-free conditions using a combination of matrigel and Sendai virus vectors containing reprogramming factors.

Vidéo: Generation of Induced Pluripotent Stem Cells from Human Peripheral T Cells Using Sendai Virus in Feeder-free Conditions

Generation of Induced Pluripotent Stem Cells from Frozen Buffy Coats using Non-integrating Episomal Plasmids

Somatic cells can be reprogrammed into induced pluripotent stem cells (iPSCs) by forcing the expression of four transcription factors (Oct-4, Sox-2, Klf-4, and c-Myc), typically expressed by human embryonic stem cells (hESCs). Due to their similarity with hESCs, iPSCs have become an important tool for potential patient-specific regenerative medicine, avoiding ethical issues associated with hESCs. In order to obtain cells suitable for clinical application, transgene-free iPSCs need to be generated to avoid transgene reactivation, altered gene expression and misguided differentiation. Moreover, a highly efficient and inexpensive reprogramming method is necessary to derive sufficient iPSCs for therapeutic purposes. Given this need, an efficient non-integrating episomal plasmid approach is the preferable choice for iPSC derivation. Currently the most common cell type used for reprogramming purposes are fibroblasts, the isolation of which requires tissue biopsy, an invasive surgical procedure for the patient. Therefore, human peripheral blood represents the most accessible and least invasive tissue for iPSC generation.
In this study, a cost-effective and viral-free protocol using non-integrating episomal plasmids is reported for the generation of iPSCs from human peripheral blood mononuclear cells (PBMNCs) obtained from frozen buffy coats after whole blood centrifugation and without density gradient separation.

Induced pluripotent stem cells (iPSCs) represent a source of patient-specific tissues for clinical applications and basic research. Here, we present a detailed protocol to reprogram human peripheral blood mononuclear cells (PBMNCs) obtained from frozen buffy coats into viral-free iPSCs using non-integrating episomal plasmids.

Génération de cellules souches pluripotentes induites à partir de Frozen Buffy Manteaux en utilisant non-intégration épisomique plasmides

Somatic cells can be reprogrammed into induced pluripotent stem cells (iPSCs) by forcing the expression of four transcription factors (Oct-4, Sox-2, ...

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Biologie du développement

Generation of Integration-free Human Induced Pluripotent Stem Cells Using Hair-derived Keratinocytes

Recent advances in reprogramming allow us to turn somatic cells into human induced pluripotent stem cells (hiPSCs). Disease modeling using patient-specific hiPSCs allows the study of the underlying mechanism for pathogenesis, also providing a platform for the development of in vitro drug screening and gene therapy to improve treatment options. The promising potential of hiPSCs for regenerative medicine is also evident from the increasing number of publications (>7000) on iPSCs in recent years. Various cell types from distinct lineages have been successfully used for hiPSC generation, including skin fibroblasts, hematopoietic cells and epidermal keratinocytes. While skin biopsies and blood collection are routinely performed in many labs as a source of somatic cells for the generation of hiPSCs, the collection and subsequent derivation of hair keratinocytes are less commonly used. Hair-derived keratinocytes represent a non-invasive approach to obtain cell samples from patients. Here we outline a simple non-invasive method for the derivation of keratinocytes from plucked hair. We also provide instructions for maintenance of keratinocytes and subsequent reprogramming to generate integration-free hiPSC using episomal vectors.

This manuscript provides a step-by-step procedure for the derivation and maintenance of human keratinocytes from plucked hair and subsequent generation of integration-free human induced pluripotent stem cells (hiPSCs) by episomal vectors.

Génération de l&#39;intégration sans anthropiques cellules souches pluripotentes utilisant des kératinocytes cheveux dérivés

Recent advances in reprogramming allow us to turn somatic cells into human induced pluripotent stem cells (hiPSCs). Disease modeling using ...

Feeder-free Derivation of Melanocytes from Human Pluripotent Stem Cells

Human pluripotent stem cells (hPSCs) represent a platform to study human development in vitro under both normal and disease conditions. Researchers can direct the differentiation of hPSCs into the cell type of interest by manipulating the culture conditions to recapitulate signals seen during development. One such cell type is the melanocyte, a pigment-producing cell of neural crest (NC) origin responsible for protecting the skin against UV irradiation. This protocol presents an extension of a currently available in vitro Neural Crest differentiation protocol from hPSCs to further differentiate NC into fully pigmented melanocytes. Melanocyte precursors can be enriched from the Neural Crest protocol via a timed exposure to activators of WNT, BMP, and EDN3 signaling under dual-SMAD-inhibition conditions. The resultant melanocyte precursors are then purified and matured into fully pigmented melanocytes by culture in a selective medium. The resultant melanocytes are fully pigmented and stain appropriately for proteins characteristic of mature melanocytes.

This work describes an in vitro differentiation protocol to produce pigmented, mature melanocytes from human pluripotent stem cells via a neural crest and melanoblast intermediate stage using a feeder-free, 25 day protocol.

Dérivation libre Feeder-de mélanocytes de pluripotentes humaines Cellules souches

Human pluripotent stem cells (hPSCs) represent a platform to study human development in vitro under both normal and disease conditions. Researchers can ...

Generation of Induced Pluripotent Stem Cells from Human Melanoma Tumor-infiltrating Lymphocytes

Adoptive transfer of ex vivo expanded autologous tumor-infiltrating lymphocytes (TILs) can mediate durable and complete responses in significant subsets of patients with metastatic melanoma. Major obstacles of this approach are the reduced viability of transferred T cells, caused by telomere shortening, and the limited number of TILs obtained from patients. Less-differentiated T cells with long telomeres would be an ideal T cell subset for adoptive T cell therapy;however, generating large numbers of these less-differentiated T cells is problematic. This limitation of adoptive T cell therapy can be theoretically overcome by using induced pluripotent stem cells (iPSCs) that self-renew, maintain pluripotency, have elongated telomeres, and provide an unlimited source of autologous T cells for immunotherapy. Here, we present a protocol to generate iPSCs using Sendai virus vectors for the transduction of reprogramming factors into TILs. This protocol generates fully reprogrammed, vector-free clones. These TIL-derived iPSCs might be able to generate less-differentiated patient- and tumor-specific T cells for adoptive T cell therapy.

The goal of this protocol is to show the protocol for reprogramming melanoma tumor-infiltrating lymphocytes into induced pluripotent stem cells.

Génération de Induced cellules souches pluripotentes à partir de mélanome humain Lymphocytes tumorales infiltrant

Adoptive transfer of ex vivo expanded autologous tumor-infiltrating lymphocytes (TILs) can mediate durable and complete responses in significant subsets ...

Induced Pluripotent Stem Cell Generation from Blood Cells Using Sendai Virus and Centrifugation

The recent development of human induced pluripotent stem cells (hiPSCs) proved that mature somatic cells can return to an undifferentiated, pluripotent state. Now, reprogramming is done with various types of adult somatic cells: keratinocytes, urine cells, fibroblasts, etc. Early experiments were usually done with dermal fibroblasts. However, this required an invasive surgical procedure to obtain fibroblasts from the patients. Therefore, suspension cells, such as blood and urine cells, were considered ideal for reprogramming because of the convenience of obtaining the primary cells. Here, we report an efficient protocol for iPSC generation from peripheral blood mononuclear cells (PBMCs). By plating the transduced PBMCs serially to a new, matrix-coated plate using centrifugation, this protocol can easily provide iPSC colonies. This method is also applicable to umbilical cord blood mononuclear cells (CBMCs). This study presents a simple and efficient protocol for the reprogramming of PBMCs and CBMCs.

We propose a protocol for reprogramming peripheral blood mononuclear cells (PBMCs) into induced pluripotent stem cells (iPSCs). By plating the transduced blood cells onto matrix-coated plates with centrifugation, iPSCs are successfully induced from floating cells. This technique suggests a simple and effective reprogramming protocol for cells such as PBMCs and CBMCs.

Induite cellules souches pluripotentes génération de cellules sanguines utilisant Sendai Virus et Centrifugation

The recent development of human induced pluripotent stem cells (hiPSCs) proved that mature somatic cells can return to an undifferentiated, pluripotent ...

Generation of Integration-free Induced Pluripotent Stem Cells from Human Peripheral Blood Mononuclear Cells Using Episomal Vectors

Induced Pluripotent Stem Cells (iPSCs) hold great promise for disease modeling and regenerative therapies. We previously reported the use of Episomal Vectors (EV) to generate integration-free iPSCs from peripheral blood mononuclear cells (PB MNCs). The episomal vectors used are DNA plasmids incorporated with oriP and EBNA1 elements from the Epstein-Barr (EB) virus, which&#160;allow for replication and long-term retainment of plasmids in mammalian cells, respectively. With further optimization, thousands of iPSC colonies can be obtained from 1 mL of peripheral blood. Two critical factors for achieving high reprogramming efficiencies&#160;are: 1) the use of a 2A &#34;self-cleavage&#34; peptide to link OCT4 and SOX2, thus achieving equimolar expression of the two factors; 2) the use of two vectors to express MYC and KLF4 individually. Here we describe a step-by-step protocol for generating integration-free iPSCs from adult peripheral blood samples. The generated iPSCs are integration-free as residual episomal plasmids are undetectable after five passages. Although the reprogramming efficiency is comparable to that of Sendai Virus (SV) vectors, EV plasmids are considerably more economical than the commercially available SV vectors. This affordable EV reprogramming system holds potential for clinical applications in regenerative medicine and provides an approach for the direct reprogramming of PB MNCs to integration-free mesenchymal stem cells, neural stem cells, etc.

This protocol describes a detailed method for efficient generation of integration-free iPSCs from human adult peripheral blood cells. With the use of four oriP/EBNA-based episomal vectors to express the reprogramming factors, KLF4, MYC, BCL-XL, or OCT4 and SOX2, thousands of iPSC colonies can be obtained from 1 mL of peripheral blood.

Génération d&#39;Intégration-libres induits des cellules souches pluripotentes à partir de cellules mononucléaires sang périphérique humain utilisant épisomique Vecteurs

Induced Pluripotent Stem Cells (iPSCs) hold great promise for disease modeling and regenerative therapies. We previously reported the use of Episomal ...

Defined and Scalable Generation of Hepatocyte-like Cells from Human Pluripotent Stem Cells

Human pluripotent stem cells (hPSCs) possess great value for biomedical research. hPSCs can be scaled and differentiated to all cell types found in the human body. The differentiation of hPSCs to human hepatocyte-like cells (HLCs) has been extensively studied, and efficient differentiation protocols have been established. The combination of extracellular matrix and biological stimuli, including growth factors, cytokines, and small molecules, have made it possible to generate HLCs that resemble primary human hepatocytes. However, the majority of procedures still employ undefined components, giving rise to batch-to-batch variation. This serves as a significant barrier to the application of the technology. To tackle this issue, we developed a defined system for hepatocyte differentiation using human recombinant laminins as extracellular matrices in combination with a serum-free differentiation process. Highly efficient hepatocyte specification was achieved, with demonstrated improvements in both HLC function and phenotype. Importantly, this system is easy to scale up using research and GMP-grade hPSC lines&#160;promising advances in cell-based modelling and therapies.

The method presented here describes a scalable and good manufacturing practice (GMP)-ready differentiation system to generate human hepatocyte-like cells from pluripotent stem cells. It serves as a cost-effective and standardized system to generate human hepatocyte-like cells for basic and applied human liver research.

Définition et génération évolutive des cellules hépatocytes-like de pluripotentes humaines Cellules souches

Human pluripotent stem cells (hPSCs) possess great value for biomedical research. hPSCs can be scaled and differentiated to all cell types found in the ...

Differentiating Chondrocytes from Peripheral Blood-derived Human Induced Pluripotent Stem Cells

In this study, we used peripheral blood cells (PBCs) as seed cells to produce chondrocytes via induced pluripotent stem cells (iPSCs) in an integration-free method. Following embryoid body (EB) formation and fibroblastic cell expansion, the iPSCs are induced for chondrogenic differentiation for 21 days under serum-free and xeno-free conditions. After chondrocyte induction, the phenotypes of the cells are evaluated by morphological, immunohistochemical, and biochemical analyses, as well as by the quantitative real-time PCR examination of chondrogenic differentiation markers. The chondrogenic pellets show positive alcian blue and toluidine blue staining. The immunohistochemistry of collagen II and X staining is also positive. The sulfated glycosaminoglycan (sGAG) content and the chondrogenic differentiation markers COLLAGEN 2 (COL2), COLLAGEN 10 (COL10), SOX9, and AGGRECAN are significantly upregulated in chondrogenic pellets compared to hiPSCs and fibroblastic cells. These results suggest that PBCs can be used as seed cells to generate iPSCs for cartilage repair, which is patient-specific and cost-effective.

We present a protocol to generate a chondrogenic lineage from human peripheral blood (PB) via induced pluripotent stem cells (iPSCs) using an integration-free method, which includes embryoid body (EB) formation, fibroblastic cells expansion, and chondrogenic induction.

Différenciation des chondrocytes provenant des cellules souches pluripotentes induites par le sang périphérique dérivé du sang

In this study, we used peripheral blood cells (PBCs) as seed cells to produce chondrocytes via induced pluripotent stem cells (iPSCs) in an ...

Generation of Standardized and Reproducible Forebrain-type Cerebral Organoids from Human Induced Pluripotent Stem Cells

The human cortex is highly expanded and exhibits a complex structure with specific functional areas, providing higher brain function, such as cognition. Efforts to study human cerebral cortex development have been limited by the availability of model systems. Translating results from rodent studies to the human system is restricted by species differences and studies on human primary tissues are hampered by a lack of tissue availability as well as ethical concerns. Recent development in human pluripotent stem cell (PSC) technology include the generation of three-dimensional (3D) self-organizing organotypic culture systems, which mimic to a certain extent human-specific brain development in vitro. Currently, various protocols are available for the generation of either whole brain or brain-region specific organoids. The method for the generation of homogeneous and reproducible forebrain-type organoids from induced PSC (iPSC), which we previously established and describe here, combines the intrinsic ability of PSC to self-organize with guided differentiation towards the anterior neuroectodermal lineage and matrix embedding to support the formation of a continuous neuroepithelium. More specifically, this protocol involves: (1) the generation of iPSC aggregates, including the conversion of iPSC colonies to a confluent monolayer culture; (2) the induction of anterior neuroectoderm; (3) the embedding of neuroectodermal aggregates in a matrix scaffold; (4) the generation of forebrain-type organoids from neuroectodermal aggregates; and (5) the fixation and validation of forebrain-type organoids. As such, this protocol provides an easily applicable system for the generation of standardized and reproducible iPSC-derived cortical tissue structures in vitro.

Cerebral organoids represent a new model system to investigate early human brain development in vitro. This article provides the detailed methodology to efficiently generate homogeneous dorsal forebrain-type organoids from human induced pluripotent stem cells including critical characterization and validation steps.

Génération de type prosencéphale standardisées et reproductibles organoïdes cérébrale de l’homme induites par les cellules souches pluripotentes

The human cortex is highly expanded and exhibits a complex structure with specific functional areas, providing higher brain function, such as cognition. ...

Generation of 3D Skin Organoid from Cord Blood-derived Induced Pluripotent Stem Cells

The skin is the body&#8217;s largest organ and has many functions. The skin acts as a physical barrier and protector of the body and regulates bodily functions. Biomimetics is the imitation of the models, systems, and elements of nature for the purpose of solving complex human problems1. Skin biomimetics is a useful tool for in vitro disease research and in vivo regenerative medicine. Human induced pluripotent stem cells (iPSCs) have the characteristic of unlimited proliferation and the ability of differentiation to three germ layers. Human iPSCs are generated from various primary cells, such as blood cells, keratinocytes, fibroblasts, and more. Among them, cord blood mononuclear cells (CBMCs) have emerged as an alternative cell source from the perspective of allogeneic regenerative medicine. CBMCs are useful in regenerative medicine because human leukocyte antigen (HLA) typing is essential to the cell banking system. We provide a method for the differentiation of CBMC-iPSCs into keratinocytes and fibroblasts and for generation of a 3D skin organoid. CBMC-iPSC-derived keratinocytes and fibroblasts have characteristics similar to a primary cell line. The 3D skin organoids are generated by overlaying an epidermal layer onto a dermal layer. By transplanting this 3D skin organoid, a humanized mice model is generated. This study shows that a 3D human iPSC-derived skin organoid may be a novel, alternative tool for dermatologic research in vitro and in vivo.

We propose a protocol that shows how to differentiate induced pluripotent stem cell-derived keratinocytes and fibroblasts and generate a 3D skin organoid, using these keratinocytes and fibroblasts. This protocol contains an additional step of generating a humanized mice model. The technique presented here will improve dermatologic research.

Génération de peau 3D Organoïde de dérivés du sang de cordon induite par les cellules souches pluripotentes

The skin is the body&#8217;s largest organ and has many functions. The skin acts as a physical barrier and protector of the body and regulates bodily ...