Les auteurs tiennent à remercier Soon-Cheol Hong (MD, Ph.D., professeur agrégé, Département d&#39;obstétrique et de gynécologie, Collège médical de l&#39;Université de Corée) pour fournir le tissu placentaire. Ce travail a été financé en partie par des subventions (R1211902) de la National Research Foundation de Corée (NRF), République de Corée.

Déclaration éthique: L&#39;étude de placenta humain a été réalisée de manière prospective, avec l&#39;approbation de l&#39;Institutional Review Board pour la recherche humaine de l&#39;Université de Corée (AN09085-001). Toutes les expériences ont été réalisées dans une salle blanche Germ-gratuit au Centre médical de l&#39;Université de Corée. La conception et les procédures utilisant hPSCs expérimentale ont été approuvés par l&#39;Institutional Review Board de l&#39;University Medical Center Corée (AN12277-003). 1. Préparation des Instruments, Culture Media, et Plats <ol><li> Manteau de toutes les boîtes de culture avec 0,1% de gélatine. Autoclave une solution saline tamponnée au phosphate (PBS) et une pince. Stériliser ciseaux chirurgicaux, des microtubes, et de la gaze à 121 ° C sous £ 15 psi pendant plus de 15 min. </li><li> 0,25% de trypsine-éthylène amine tétra-acétique, préparé préchauffé (trypsine-EDTA) et support préchauffé culture filtré (DMEM) contenant 20% de sérum bovin fœtal (FBS), 100 U / ml de pénicilline et 100 ug / ml streptomycavant de commencer les procédures. </li></ol> 2. Isolement cellulaire à partir de placenta humain <ol><li> Obtenir le tissu placentaire, après la livraison de césarienne, de femmes enceintes en bonne santé qui subissent un avortement thérapeutique normal ou au 7-32 semaines de gestation après l&#39;obtention du consentement éclairé écrit. </li><li> Avoir le spécialiste obstétriques placenta humain plaques choriales chirurgicalement séparées (CAH) du placenta; incuber dans ces 0,25% de trypsine-EDTA à 37 ° C pendant 20 min, puis laver avec 5 ml de PBS. </li><li> Isoler villosités choriales du CAH l&#39;aide de ciseaux; émincer les villosités et les laver trois fois avec 1 ml de PBS. Après hachage, placer les villosités choriales dans un tube de centrifugeuse en utilisant une pointe de pipette; laver les échantillons avec 500 pi de PBS par pipetage de haut en bas, et centrifuger les tubes pour 120 g pendant 3 min. Laver deux fois les tissus avec 500 ul de milieu de culture préchauffé. </li><li> les cellules dans du milieu de culture sur des plaques revêtues de gélatine à 37 ° C, dans 5% de CO 2 </sub&gt; et sous 95% d&#39;humidité. Pré-manteau toutes les plaques avec 0,1% de gélatine pendant 1 heure à 37 ° C. Les colonies de cellules de fibroblastes se formeront et la croissance adhérente environ 5-7 jours après le début de la culture dans le même milieu. </li><li> Remplacez le milieu tous les 2-3 jours jusqu&#39;à ce que le troisième passage. Au cours de la culture, l&#39;enlèvement des débris de cellules flottant dans le milieu de culture; fibroblastes placentaires croissance vont attacher à la plaque. Culture des cellules de fibroblastes provenant de l&#39;HPC à la 10 ème passage, puis les récolter pour les cultures de CSEh après trypsinisation et la mitomycine-C (10 pg / ml) de traitement. </li><li> Avant la congélation CAH que des échantillons d&#39;achat d&#39;actions, utiliser transcription inverse-réaction de polymérisation en chaîne (RT-PCR) pour confirmer que BHP ne sont pas contaminés par des agents pathogènes qui infectent couramment le placenta, y compris les mycoplasmes (M. fermentans, M. hyorhinis, M. Arginine, M . orale, M. salivarium, M. hominis, M. pulmonis, M. arthritidis, M. neurolyticum, M. hyopneumoniae,et M. capricolum) et les espèces bactériennes, Ureaplasma urealyticum, en utilisant un kit de détection de mycoplasme. <ol><li> Suivez les instructions du fabricant pour effectuer RT-PCR, utiliser toutes les amorces et des mélanges fournis dans le kit. Utilisez les surnageants des cellules qui ont été cultivées pendant 36 jours pour la détection des mycoplasmes. Ajouter 3 ul de ces échantillons pour les mélanges de PCR et effectuer la procédure 1 er PCR pendant 35 cycles, chacun consistant en 94 ° C pendant 30 sec, 55 ° C pendant 2 min, et 72 ° C pendant 1 min. </li><li> Ajouter avec précaution 0,5 ul du produit de PCR 1 er au mélange PCR et effectuer la procédure 2 ème PCR pendant 30 cycles en utilisant les mêmes conditions que précédemment. Analyser les produits amplifiés par électrophorèse sur gel d&#39;agarose à 1%; appliquer 10 pi de chacun des 1 er et 2 ème produits PCR pour les gels. </li></ol></li><li> Si CAH sont trouvés d&#39;être contaminés par des agents pathogènes, les élimineren utilisant une combinaison d&#39;antibiotiques (BM-cycline), en suivant les instructions du fabricant. Ajouter un nouveau milieu contenant 10 pg / ml de BM-cycline 1 pendant 3 jours, puis traiter les cellules avec 5 ug / ml BM-cycline 2 pendant 4 jours. Répétez ce cycle deux fois, puis vérifier la contamination par des mycoplasmes nouveau. </li></ol> 3. Les cellules de placenta humain récolte dérivés milieu conditionné (hPCCM) <ol><li> Après l&#39;exclusion de la contamination, de traiter les cellules attachées (85-90% de confluence; (1,5 × 10 7 cellules / fiole) avec de la mitomycine-C (10 ug / ml) pendant 2 heures et 30 min, et lavé les cellules trois fois avec 10 ml PBS. incuber les cellules avec un milieu préchauffé sans mitomycine-C pendant 24 heures. </li><li> Un jour après le traitement, incuber les cellules dans 10 ml de milieu (DMEM-F12 supplémenté avec 20% de sérum knock-out remplacement [KOSR], 0,1 mM de β-mercaptoéthanol, 1% de NEAA et 1% de pénicilline-streptomycine). Après 24 heures d&#39;incubation, la récolte CM dans un tube conique de 15 ml etfiltrage du CM à l&#39;aide d&#39;un filtre à seringue de 0,22 um. </li><li> Ajouter encore 10 ml de nouveau milieu et l&#39;incubation. Collecter toutes les surnageants des cultures chaque jour pendant 1 semaine et geler le milieu récolté à -80 ° C. D&#39;éviter les cycles de gel-dégel. </li></ol> 4. culture des droits de cellules souches embryonnaires (CSEh) et l&#39;induction de cellules souches pluripotentes (CISP) <ol><li> Obtenir des lignées de cellules souches embryonnaires humaines, les cellules H1 et H9 (énumérés dans le registre NIH CSEh sous le nom WA01 et WA09, respectivement) et induit-pluripotentes cellules souches iPSC-1: iPS (prépuce) -1 et iPSC-2 (IISH1i- BM) de l&#39;Institut de recherche WiCell (Madison, WI, USA). Les cellules doivent être décongelés et mis en culture selon les instructions du fabricant. </li><li> Pour le groupe de contrôle, des cellules de culture sur la vaisselle membrane basale Matrix-revêtus largement utilisé dans le milieu de culture défini sans sérum sans chargeur et à 37 ° C et à 5% de CO 2. Initialement, semences 80-100 touffes de hPSCs in 35 mm plats. Cultiver les cellules dans ces plats jusqu&#39;à 70-80% de cellules de passage de confluence et de routine sous-culture une fois tous les 5-6 jours, en utilisant des méthodes mécaniques ou enzymatiques (Dispase). Laver les cellules deux fois avec du milieu de pré eux dans un rapport de 1: 4. Remplacez le milieu avec un milieu frais tous les jours. </li><li> Pour les groupes expérimentaux, des cellules en culture dans hPCCM sur des plats pré-revêtues avec 0,1% de gélatine et d&#39;effectuer des passages de routine une fois tous les 5 jours, soit mécaniquement, soit par voie enzymatique. cellules de la plaque à des rapports dans la plage de 1: 6-1: 10. hPSCs attachent à la gélatine dans les 2 jours après le transfert. Remplacez le support avec un milieu frais tous les jours. </li></ol> 5. Caractérisation des pluripotentes humaines Cellules souches NOTE: Caractériser hPSCs utilisant plusieurs méthodes, telles que alcaline phosphatase coloration, immunocytochimie, quantitative PCR en temps réel (qRT-PCR) et Western blot. <ol><li> Immunofluorescence <ol><li> Pour immunofluorescence coloration, hPSCs de culture et fixer les cellules dans 8 puits chambres de diapositives avec 4% (p / v) de paraformaldehyde pendant 10 min; ensuite, les cellules perméabilisées avec 0,1% (v / v) de Triton-X100 pendant 15 min, et de bloquer les cellules pendant 1 heure avec 3% (v / v) de sérum de cheval normal dans du PBS contenant 0,1% (v / v) de Tween 20. Laver PBS deux fois pendant 5 min entre chaque étape. </li><li> Incuber les cellules avec l&#39;anticorps primaire (OCT-4, SSEA4, TRA-1-81, TRA-1-60, dilution de 1: 1000) O / N à 4 ° C et avec l&#39;anticorps secondaire. </li><li> Avant le montage, incuber les cellules avec 4 &#39;, 6-diamidino-2-phénylindole (DAPI) pendant 5 minutes dans l&#39;obscurité. Laver les cellules rigoureusement trois fois pendant 5 min chacun et secs cellules complètement, dans l&#39;obscurité. Préserver les cellules de la fluorescence milieu de montage et de les observer sous un microscope à fluorescence. </li></ol></li><li> qRT-PCR <ol><li> Isoler l&#39;ARN total à partir de cellules en utilisant un kit RNeasy selon les spécifications du fabricant et de quantifier l&#39;ARN extrait à l&#39;aide d&#39;un spectrophotomètre Goutte Nano. </li&gt; <li> La synthèse d&#39;ADNc par addition de 2 pg de l&#39;ARN total à un oligonucléotide contenant 20 ul de mélange réactionnel (dT) et des amorces de transcriptase inverse Superscript II; suivre les instructions du fabricant dans cette procédure. </li><li> Amplifier l&#39;ADNc synthétisé avec un système de iQ iCycler Bio-Rad, en utilisant iQ qPCR SYBR Green Master Mix et les séquences d&#39;amorces décrites dans le tableau supplémentaire S1. Normaliser les valeurs de seuil de cycle des gènes d&#39;intérêt à ceux de la glycéraldéhyde-3-phosphate déshydrogénase (GAPDH). </li></ol></li><li> Phosphatase alcaline (AP) coloration <ol><li> Détecter l&#39;activité AP en utilisant la caractérisation de cellules ES Kit, selon le protocole du fabricant. </li><li> Au jour 5, aspirer les médias et fixer les hPSCs avec 4% de paraformaldehyde dans PBS pendant 12 min. Ne pas overfix; fixation des cellules pendant plus de 2 min donnera lieu à l&#39;inactivation de la phosphatase alcaline. Aspirer le fixateur et rincer à l&#39;1x Rinse Buffer. Ne paslaisser sécher les puits entre les étapes. </li><li> Ajouter une solution de coloration assez pour couvrir chaque puits (1 ml par puits d&#39;une boîte de 35 mm). Incuber les plaques dans l&#39;obscurité à température ambiante pendant 15 min. </li><li> Après incubation, laver la vaisselle avec 1x Rinse Buffer. cellules de couverture avec 1x PBS pour éviter le dessèchement et examinent et l&#39;image les cellules colorées en utilisant un microscope Olympus. </li></ol></li><li> Western Blot <ol><li> Effectuer une analyse western blot comme décrit précédemment dans 9. En bref, déterminer les concentrations protéiques dans les lysats cellulaires. Résoudre des quantités égales de protéine à une électrophorèse sur gel de sulfate-polyacrylamide (SDS-PAGE) de gel de dodécylsulfate de sodium et le transfert des protéines sur un polyfluorure de vinylidène (PVDF). </li><li> Bloquer le transfert et sonder O / N avec des anticorps primaires différentes à 4 ° C; incuber ensuite l&#39;blot avec des anticorps secondaires conjugués à HRP pendant 1 heure. Laver la membrane trois fois pendant 10 min à chaque fois entre les incubations. Détecter des signaux à l&#39;aide d&#39;un réactif ECL. </li&gt; </ol></li><li> Court Répétez Tandem (STR) Analyse <ol><li> hPSCs ont été récoltées dans les groupes témoins et expérimentaux dans le même passage. L&#39;ADN génomique a été extrait en utilisant un kit Micro ADN, selon les instructions du fabricant. </li><li> Amplifier l&#39;ADN génomique pour 16 loci génétiques différents en utilisant le kit d&#39;amplification PCR ampf / STR, selon les spécifications du fabricant et électrophorèse capillaire a été réalisée en utilisant un analyseur génétique. </li></ol></li></ol>

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E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Basic+fibroblast+growth+factor+support+of+human+embryonic+stem+cell+self-renewal.">Basic fibroblast growth factor support of human embryonic stem cell self-renewal.</a> Stem Cells. 24 (3), 568-574 (2006).</li><li>Park, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+feeder+cells+can+support+the+undifferentiated+growth+of+human+and+mouse+embryonic+stem+cells+using+their+own+basic+fibroblast+growth+factors.">Human feeder cells can support the undifferentiated growth of human and mouse embryonic stem cells using their own basic fibroblast growth factors.</a> Stem Cells Dev. 20 (11), 1901-1910 (2011).</li><li>Park, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+efficacy+of+human+placenta+as+a+source+of+the+universal+feeder+in+human+and+mouse+pluripotent+stem+cell+culture.">The efficacy of human placenta as a source of the universal feeder in human and mouse pluripotent stem cell culture.</a> Cell Reprogram. 12 (3), 315-328 (2010).</li><li>Park, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Undifferentiated+propagation+of+the+human+embryonic+stem+cell+lines,+H1+and+HSF6,+on+human+placenta-derived+feeder+cells+without+basic+fibroblast+growth+factor+supplementation.">Undifferentiated propagation of the human embryonic stem cell lines, H1 and HSF6, on human placenta-derived feeder cells without basic fibroblast growth factor supplementation.</a> Stem Cells Dev. 19 (11), 1713-1722 (2010).</li><li>Seok, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+placenta-derived+feeders+support+prolonged+undifferentiated+propagation+of+a+human+embryonic+stem+cell+line,+SNUhES3:+comparison+with+human+bone+marrow-derived+feeders.">Human placenta-derived feeders support prolonged undifferentiated propagation of a human embryonic stem cell line, SNUhES3: comparison with human bone marrow-derived feeders.</a> Stem Cells Dev. 16 (3), 421-428 (2007).</li><li>Levenstein, M. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Basic+fibroblast+growth+factor+support+of+human+embryonic+stem+cell+self-renewal.">Basic fibroblast growth factor support of human embryonic stem cell self-renewal.</a> Stem Cells. 24 (3), 568-574 (2006).</li><li>Xu, R. H., Peck, R. M., Li, D. S., Feng, X., Ludwig, T., Thomson, J. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Basic+FGF+and+suppression+of+BMP+signaling+sustain+undifferentiated+proliferation+of+human+ES+cells.">Basic FGF and suppression of BMP signaling sustain undifferentiated proliferation of human ES cells.</a> Nat Methods. 2 (3), 185-190 (2005).</li><li>Mahmood, A., Harkness, L., Schrøder, H. D., Abdallah, B. 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Les auteurs indiquent aucun conflit d&#39;intérêt.

Ce modèle a été mis au point pour se propager avec succès hPSCs, tout en conservant leurs caractéristiques, dans des conditions de culture sans cellules nourricières humanisés sans l&#39;addition de facteurs de croissance recombinants exogènes tels que bFGF ou de l&#39;insuline, ce qui permet la manipulation de hPSCs dans un micro-environnement humanisé. Exogène supplémentation bFGF est commun et l&#39;application de substrats tels que Matrigel ou laminine est essentiel pour la culture libre-chargeur de hPSCs...

The goal of this protocol is the propagation of human pluripotent stem cells (hPSCs) including human embryonic stem cells (hESCs) and human induced pluripotent stem cells (iPSCs) in fully humanized ex vivo feeder-free conditions without requiring additional exogenous supplementation and synthetic substrates. To date, the development of ex vivo hPSC culture models, that enable the introduction of culture products to the clinic, has been a major concern in stem cell research. Specifically, two critical problems need to be addressed. First, a humanized ex vivo culture system for the propagation of hPSCs that obviates the risk of contamination by animal cell products is needed. Second, a feeder-free culture model is needed to facilitate easy and economic mass production of hPSCs. For therapeutic applications of hPSCs, it is necessary to identify the factors that regulate their self-renewal and differentiation.
Since Xu et al. initially reported the feasibility of using conditioned media (CM) derived from mouse embryonic fibroblasts (MEF) to grow hESCs on Matrigel1, many studies have examined optimal ex vivo propagation methods for hPSCs2-4. However, an ideal humanized ex vivo feeder-free hPSC propagation system has not been developed because current methods require additional exogenous substrates including bFGF and insulin, well-known hPSC-sustaining factors, in culture media5-7. Moreover, synthetic substrata such as Matrigel or laminin are used in all feeder-free cultures.
The rationale behind the development and use of this protocol is based on our previous studies showing that human placenta chorion cells excellently support the propagation of hPSCs without bFGF supplementation8-11. This protocol has a number of advantages including its simplicity with respect to handling and its cost-effectiveness, and it is a near-perfect humanized culture that enables hPSC propagation without exogenous synthetic substrates. The application of human placenta-derived CM (hPCCM) for hPSCs involves 3 steps. First, chorion cells are isolated from the human placenta and cultured. Second, hPCCM is produced from cultured cells. Third, hPSCs are cultured using hPCCM and their characteristics are confirmed. 
This protocol will facilitate clinical applications of hPSCs and studies of the mechanisms of hPSC proliferation and attachment. In this paper, the protocol for the successful propagation of hPSCs in hPCCM on a gelatin-coated dish is presented.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Mitomycin-C</td>
			<td>Sigma-Aldrich Corporation</td>
			<td>M4287</td>
			<td>10ug/ml</td>
		</tr>
		<tr>
			<td>Mycoplasma detection kit</td>
			<td>TaKaRa Bio Inc.</td>
			<td>#6601</td>
			<td></td>
		</tr>
		<tr>
			<td>Matrigel</td>
			<td>BD Biosciences</td>
			<td>#354277</td>
			<td></td>
		</tr>
		<tr>
			<td>mTeSR1</td>
			<td>STEMCELL Technologies Inc.</td>
			<td>#05850</td>
			<td></td>
		</tr>
		<tr>
			<td>Dispase</td>
			<td>Worthington Biochemical Corporation</td>
			<td>LS02100</td>
			<td>1mg/ml</td>
		</tr>
		<tr>
			<td>Gelatin</td>
			<td>Sigma-Aldrich Corporation</td>
			<td>G2500</td>
			<td>0.10%</td>
		</tr>
		<tr>
			<td>BM-Cyclin</td>
			<td>Roche</td>
			<td>799 050</td>
			<td>10ug/ml</td>
		</tr>
		<tr>
			<td>RNeasy mini kit&#160;</td>
			<td>Qiagen</td>
			<td>74104</td>
			<td></td>
		</tr>
		<tr>
			<td>Nano Drop Spectrophotometer</td>
			<td>Thermo Fisher Scientific Inc</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>iQ SYBR Green qPCR Master Mix&#160;</td>
			<td>Bio-Rad Laboratories</td>
			<td>#170-8882AP</td>
			<td></td>
		</tr>
		<tr>
			<td>ES Cell Characterization kit</td>
			<td>Chemicon International, Inc.,</td>
			<td>SCR001</td>
			<td></td>
		</tr>
		<tr>
			<td>Power cDNA Synthesis Kit&#160;</td>
			<td>iNtRON Biotechnology</td>
			<td>25011</td>
			<td></td>
		</tr>
		<tr>
			<td>QIAamp&#174; DNA Micro kit</td>
			<td>Qiagen</td>
			<td>56304</td>
			<td></td>
		</tr>
		<tr>
			<td>AmpF/STR&#174; Identifiler&#174; PCR Amplification kit</td>
			<td>Applied Biosystems Inc.</td>
			<td>4322288</td>
			<td></td>
		</tr>
		<tr>
			<td>Applied Biosystems&#174; 3130xl Genetic Analyzer&#160;</td>
			<td>Applied Biosystems Inc.</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

a novel culture model for human pluripotent stem cell propagation on gelatin in placenta-conditioned media

The propagation of human pluripotent stem cells (hPSCs) in conditioned medium derived from human cells in feeder-free culture conditions has been of interest. Nevertheless, an ideal humanized ex vivo feeder-free propagation method for hPSCs has not been developed; currently, additional exogenous substrates including basic fibroblast growth factor (bFGF), a master hPSC-sustaining factor, is added to all of culture media and synthetic substrata such as Matrigel or laminin are used in all feeder-free cultures. Recently, our group developed a simple and efficient protocol for the propagation of hPSCs using only conditioned media derived from the human placenta on a gelatin-coated dish without additional exogenous supplementation or synthetic substrata specific to hPSCs. This protocol has not been reported previously and might enable researchers to propagate hPSCs efficiently in humanized culture conditions. Additionally, this model obviates hPSC contamination risks by animal products such as viruses or unknown proteins. Furthermore, this system facilitates easy mass production of hPSCs using the gelatin coating, which is simple to handle, dramatically decreases the overall costs of ex vivo hPSC maintenance.

Un avantage de cette méthode de propagation de HPSC est qu&#39;elle utilise des composants sécrétées à partir de cellules humaines. Une étape la plus ciritical dans le protocole est l&#39;isolement et la culture de cellules placentaires humaines dérivées. Cela exige et la dissection précise d&#39;une partie précise de HPC plaque villosités. La figure 1 montre la procédure pour l&#39;isolement cellulaire dérivée du placenta humain. Ce procédé est simple et commode pour l&#39;isolement de ...

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A Novel Culture Model for Human Pluripotent Stem Cell Propagation on Gelatin in Placenta-conditioned Media

This protocol provides a simple and efficient way to propagate human pluripotent stem cells (hPSCs) using only conditioned media derived from the human placenta in a gelatin-coated dish without additional exogenous supplementation or hPSC-specific synthetic substrata.

A Novel Culture Modèle de cellules souches pluripotentes humaines Propagation de gélatine dans les médias Placenta conditionné

The propagation of human pluripotent stem cells (hPSCs) in conditioned medium derived from human cells in feeder-free culture conditions has been of ...

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Research

JoVE Journal

Developmental Biology

7.8K vues.  Graduate School of Medicine, Korea University. This protocol provides a simple and efficient way to propagate human pluripotent stem cells (hPSCs) using only conditioned media derived from the human placenta in a gelatin-coated dish without additional exogenous supplementation or hPSC-specific synthetic substrata.

Vidéo: A Novel Culture Model for Human Pluripotent Stem Cell Propagation on Gelatin in Placenta-conditioned Media

Alginate Encapsulation of Pluripotent Stem Cells Using a Co-axial Nozzle

Pluripotent stem cells (PS cells) are the focus of intense research due to their role in regenerative medicine and drug screening. However, the development of a mass culture system would be required for using PS cells in these applications. Suspension culture is one promising culture method for the mass production of PS cells, although some issues such as controlling aggregation and limiting shear stress from the culture medium are still unsolved. In order to solve these problems, we developed a method of calcium alginate (Alg-Ca) encapsulation using a co-axial nozzle. This method can control the size of the capsules easily by co-flowing N2 gas. The controllable capsule diameter must be larger than 500 &#181;m because too high a flow rate of N2 gas causes the breakdown of droplets and thus heterogeneous-sized capsules. Moreover, a low concentration of Alg-Na and CaCl2 causes non-spherical capsules. Although an Alg-Ca capsule without a coating of Alg-PLL easily dissolves enabling the collection of cells, they can also potentially leak out from capsules lacking an Alg-PLL coating. Indeed, an alginate-PLL coating can prevent cellular leakage but is also hard to break. This technology can be used to research the stem cell niche as well as the mass production of PS cells because encapsulation can modify the micro-environment surrounding cells including the extracellular matrix and the concentration of secreted factors.

We established a method of encapsulating pluripotent stem cells (PS cells) into alginate hydrogel capsules using a co-axial nozzle. This prevents cells from aggregating excessively and limits the shear stress experienced by cells in suspension culture. The technique is applicable to the mass production of PS cells as well as research on stem cell niche.

Encapsulation alginate de cellules souches pluripotentes en utilisant une buse de co-axiale

Pluripotent stem cells (PS cells) are the focus of intense research due to their role in regenerative medicine and drug screening.  However, the ...

Recherche

Biologie du développement

Culturing Human Pluripotent and Neural Stem Cells in an Enclosed Cell Culture System for Basic and Preclinical Research

This paper describes how to use a custom manufactured, commercially available enclosed cell culture system for basic and preclinical research. Biosafety cabinets (BSCs) and incubators have long been the standard for culturing and expanding cell lines for basic and preclinical research. However, as the focus of many stem cell laboratories shifts from basic research to clinical translation, additional requirements are needed of the cell culturing system. All processes must be well documented and have exceptional requirements for sterility and reproducibility. In traditional incubators, gas concentrations and temperatures widely fluctuate anytime the cells are removed for feeding, passaging, or other manipulations. Such interruptions contribute to an environment that is not the standard for cGMP and GLP guidelines. These interruptions must be minimized especially when cells are utilized for therapeutic purposes. The motivation to move from the standard BSC and incubator system to a closed system is that such interruptions can be made negligible. Closed systems provide a work space to feed and manipulate cell cultures and maintain them in a controlled environment where temperature and gas concentrations are consistent. This way, pluripotent and multipotent stem cells can be maintained at optimum health from the moment of their derivation all the way to their eventual use in therapy.

Here is a protocol to grow pluripotent stem cells (PSC) and neural stem cells (NSC) in an enclosed cell culture system that permits maximum sterility and reproducibility, replacing the traditional biosafety cabinet and incubator. This equipment meets clinical good manufacturing practice (cGMP) and clinical good lab practice (cGLP) guidelines.

La culture pluripotentes humaines et cellules souches neurales dans un système de culture cellulaire clos pour la recherche fondamentale et préclinique

This paper describes how to use a custom manufactured, commercially available enclosed cell culture system for basic and preclinical research. Biosafety ...

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 ...

Large-Scale Production of Cardiomyocytes from Human Pluripotent Stem Cells Using a Highly Reproducible Small Molecule-Based Differentiation Protocol

Maximizing the benefit of human pluripotent stem cells (hPSCs) for research, disease modeling, pharmaceutical and clinical applications requires robust methods for the large-scale production of functional cell types, including cardiomyocytes. Here we demonstrate that the temporal manipulation of WNT, TGF-&#946;, and SHH signaling pathways leads to highly efficient cardiomyocyte differentiation of single-cell passaged hPSC lines in both static suspension and stirred suspension bioreactor systems. Employing this strategy resulted in ~ 100% beating spheroids, consistently containing &#62; 80% cardiac troponin T-positive cells after 15 days of culture, validated in multiple hPSC lines. We also report on a variation of this protocol for use with cell lines not currently adapted to single-cell passaging, the success of which has been verified in 42 hPSC lines. Cardiomyocytes generated using these protocols express lineage-specific markers and show expected electrophysiological functionalities. Our protocol presents a simple, efficient and robust platform for the large-scale production of human cardiomyocytes.

Here, we present a robust, fast and scalable cardiomyocyte differentiation protocol for human pluripotent stem cells (hPSCs). Cardiomyocytes derived using this large-scale method can provide sufficient cell numbers for their effective use in human cardiovascular disease modeling, high-throughput drug screening, and potentially clinical applications.

Production à grande échelle de cardiomyocytes à partir pluripotentes humaines cellules souches en utilisant un petit protocole Différenciation Molecule-Based hautement reproductible

Maximizing the benefit of human pluripotent stem cells (hPSCs) for research, disease modeling, pharmaceutical and clinical applications requires robust ...

Rapid Neuronal Differentiation of Induced Pluripotent Stem Cells for Measuring Network Activity on Micro-electrode Arrays

Neurons derived from human induced Pluripotent Stem Cells (hiPSCs) provide a promising new tool for studying neurological disorders. In the past decade, many protocols for differentiating hiPSCs into neurons have been developed. However, these protocols are often slow with high variability, low reproducibility, and low efficiency. In addition, the neurons obtained with these protocols are often immature and lack adequate functional activity both at the single-cell and network levels unless the neurons are cultured for several months. Partially due to these limitations, the functional properties of hiPSC-derived neuronal networks are still not well characterized. Here, we adapt a recently published protocol that describes production of human neurons from hiPSCs by forced expression of the transcription factor neurogenin-212. This protocol is rapid (yielding mature neurons within 3 weeks) and efficient, with nearly 100% conversion efficiency of transduced cells (&#62;95% of DAPI-positive cells are MAP2 positive). Furthermore, the protocol yields a homogeneous population of excitatory neurons that would allow the investigation of cell-type specific contributions to neurological disorders. We modified the original protocol by generating stably transduced hiPSC cells, giving us explicit control over the total number of neurons. These cells are then used to generate hiPSC-derived neuronal networks on micro-electrode arrays. In this way, the spontaneous electrophysiological activity of hiPSC-derived neuronal networks can be measured and characterized, while retaining interexperimental consistency in terms of cell density. The presented protocol is broadly applicable, especially for mechanistic and pharmacological studies on human neuronal networks.

We modify and implement a previously published protocol describing the rapid, reproducible, and efficient differentiation of human&#160;induced&#160;Pluripotent Stem Cells (hiPSCs) into excitatory cortical neurons12. Specifically, our modification allows for control of neuronal cell density and use on micro-electrode arrays to measure electrophysiological properties at the network level.

Rapid Neuronal Différenciation des Induced cellules souches pluripotentes pour mesurer l&#39;activité réseau sur Arrays Micro-électrode

Neurons derived from human induced Pluripotent Stem Cells (hiPSCs) provide a promising new tool for studying neurological disorders. In the past decade, ...

A Novel Mammary Fat Pad Transplantation Technique to Visualize the Vessel Generation of Vascular Endothelial Stem Cells

Endothelial cells (ECs) are the fundamental building blocks of the vascular architecture and mediate vascular growth and remodeling to ensure proper vessel development and homeostasis. However, studies on endothelial lineage hierarchy remain elusive due to the lack of tools to gain access as well as to directly evaluate their behavior in vivo. To address this shortcoming, a new tissue model to study angiogenesis using the mammary fat pad has been developed. The mammary gland develops mostly in the postnatal stages, including puberty and pregnancy, during which robust epithelium proliferation is accompanied by extensive vascular remodeling. Mammary fat pads provide space, matrix, and rich angiogenic stimuli from the growing mammary epithelium. Furthermore, mammary fat pads are located outside the peritoneal cavity, making them an easily accessible grafting site for assessing the angiogenic potential of exogenous cells. This work also describes an efficient tracing approach using fluorescent reporter mice to specifically label the targeted population of vascular endothelial stem cells (VESCs) in vivo. This lineage tracing method, coupled with subsequent tissue whole-mount microscopy, enable the direct visualization of targeted cells and their descendants, through which the proliferation capability can be quantified and the differentiation commitment can be fate-mapped. Using these methods, a population of bipotent protein C receptor (Procr) expressing VESCs has recently been identified in multiple vascular systems. Procr+ VESCs, giving rise to both new ECs and pericytes, actively contribute to angiogenesis during development, homeostasis, and injury repair. Overall, this manuscript describes a new mammary fat pad transplantation and in vivo lineage tracing techniques that can be used to evaluate the stem cell properties of VESCs.

This work demonstrates a novel approach to assess the proliferation, differentiation, and vessel-forming potential of vascular endothelial stem cells (VESCs) through mammary fat pad transplantation followed by whole-mount tissue preparation for microscopic observation. A lineage tracing strategy to investigate the behavior of VESCs in vivo is also presented.

Une nouvelle technique de transplantation de graisse mammaire mammaire pour visualiser la génération de vaisseaux de cellules souches endothéliales vasculaires

Endothelial cells (ECs) are the fundamental building blocks of the vascular architecture and mediate vascular growth and remodeling to ensure proper ...

Rapid, Directed Differentiation of Retinal Pigment Epithelial Cells from Human Embryonic or Induced Pluripotent Stem Cells

We describe a robust method to direct the differentiation of pluripotent stem cells into retinal pigment epithelial cells (RPE). The purpose of providing a detailed and thorough protocol is to clearly demonstrate each step and to make this readily available to researchers in the field. This protocol results in a homogenous layer of RPE with minimal or no manual dissection needed. The method presented here has been shown to be effective for induced pluripotent stem cells (iPSC) and human embryonic stem cells. Additionally, we describe methods for cryopreservation of intermediate cell banks that allow long-term storage. RPE generated using this protocol might be useful for iPSC disease-in-a-dish modeling or clinical application.

This protocol describes how to produce retinal pigment epithelial cells (RPE) from pluripotent stem cells. The method uses a combination of growth factors and small molecules to direct the differentiation of stem cells into immature RPE in fourteen days and mature, functional RPE after three months.

Rapide, réalisée la différenciation des cellules épithéliales pigmentaire rétinien de cellules souches pluripotentes humaines embryonnaires ou induit

We describe a robust method to direct the differentiation of pluripotent stem cells into retinal pigment epithelial cells (RPE). The purpose of providing ...

Using Human Induced Pluripotent Stem Cell-derived Hepatocyte-like Cells for Drug Discovery

The ability to differentiate human induced pluripotent stem cells (iPSCs) into hepatocyte-like cells (HLCs) provides new opportunities to study inborn errors in hepatic metabolism. However, to provide a platform that supports the identification of small molecules that can potentially be used to treat liver disease, the procedure requires a culture format that is compatible with screening thousands of compounds. Here, we describe a protocol using completely defined culture conditions, which allow the reproducible differentiation of human iPSCs to hepatocyte-like cells in 96-well tissue culture plates. We also provide an example of using the platform to screen compounds for their ability to lower Apolipoprotein B (APOB) produced from iPSC-derived hepatocytes generated from a familial hypercholesterolemia patient. The availability of a platform that is compatible with drug discovery should allow researchers to identify novel therapeutics for diseases that affect the liver.

The protocol presented here describes a platform for identifying small molecules for the treatment of liver disease. A step-by-step description is presented detailing how to differentiate iPSCs into cells with hepatocyte characteristics in 96-well plates, and to use the cells to screen for small molecules with potential therapeutic activity.

À l’aide de l’homme induit des hépatocytes-comme dérivé de cellules souches pluripotentes for Drug Discovery

The ability to differentiate human induced pluripotent stem cells (iPSCs) into hepatocyte-like cells (HLCs) provides new opportunities to study inborn ...

A Familial Hypercholesterolemia Human Liver Chimeric Mouse Model Using Induced Pluripotent Stem Cell-derived Hepatocytes

Familial hypercholesterolemia (FH) is mostly caused by low-density lipoprotein receptor (LDLR) mutations and results in an increased risk of early-onset cardiovascular disease due to marked elevation of LDL cholesterol (LDL-C) in blood. Statins are the first line of lipid-lowering drugs for treating FH and other types of hypercholesterolemia, but new approaches are emerging, in particular PCSK9 antibodies, which are now being tested in clinical trials. To explore novel therapeutic approaches for FH, either new drugs or new formulations, we need appropriate in vivo models. However, differences in the lipid metabolic profiles compared to humans are a key problem of the available animal models of FH. To address this issue, we have generated a human liver chimeric mouse model using FH induced pluripotent stem cell (iPSC)-derived hepatocytes (iHeps). We used Ldlr-/-/Rag2-/-/Il2rg-/- (LRG) mice to avoid immune rejection of transplanted human cells and to assess the effect of LDLR-deficient iHeps in an LDLR null background. Transplanted FH iHeps could repopulate 5-10% of the LRG mouse liver based on human albumin staining. Moreover, the engrafted iHeps responded to lipid-lowering drugs and recapitulated clinical observations of increased efficacy of PCSK9 antibodies compared to statins. Our human liver chimeric model could thus be useful for preclinical testing of new therapies to FH. Using the same protocol, similar human liver chimeric mice for other FH genetic variants, or mutations corresponding to other inherited liver diseases, may also be generated.

Here, we present a protocol to generate a human liver chimeric mouse model of familial hypercholesterolemia using human induced pluripotent stem cell-derived hepatocytes. This is a valuable model for testing new therapies for hypercholesterolemia.

Un modèle de souris chimériques du foie humain de l’hypercholestérolémie utilisant des hépatocytes de dérivés de cellules souches pluripotentes induites

Familial hypercholesterolemia (FH) is mostly caused by low-density lipoprotein receptor (LDLR) mutations and results in an increased risk of early-onset ...

Efficient Differentiation of Human Pluripotent Stem Cells into Liver Cells

The liver detoxifies harmful substances, secretes vital proteins, and executes key metabolic activities, thus sustaining life. Consequently, liver failure&#8212;which can be caused by chronic alcohol intake, hepatitis, acute poisoning, or other insults&#8212;is a severe condition that can culminate in bleeding, jaundice, coma, and eventually death. However, approaches to treat liver failure, as well as studies of liver function and disease, have been stymied in part by the lack of a plentiful supply of human liver cells. To this end, this protocol details the efficient differentiation of human pluripotent stem cells (hPSCs) into hepatocyte-like cells, guided by a developmental roadmap that describes how liver fate is specified across six consecutive differentiation steps. By manipulating developmental signaling pathways to promote liver differentiation and to explicitly suppress the formation of unwanted cell fates, this method efficiently generates populations of human liver bud progenitors and hepatocyte-like cells by days 6 and 18 of PSC differentiation, respectively. This is achieved through the temporally-precise control of developmental signaling pathways, exerted by small molecules and growth factors in a serum-free culture medium. Differentiation in this system occurs in monolayers and yields hepatocyte-like cells that express characteristic hepatocyte enzymes and have the ability to engraft a mouse model of chronic liver failure. The ability to efficiently generate large numbers of human liver cells in vitro has ramifications for treatment of liver failure, for drug screening, and for mechanistic studies of liver disease.

This protocol details a monolayer, serum-free method to efficiently generate hepatocyte-like cells from human pluripotent stem cells (hPSCs) in 18 days. This entails six steps as hPSCs sequentially differentiate into intermediate cell-types such as the primitive streak, definitive endoderm, posterior foregut and liver bud progenitors before forming hepatocyte-like cells.