Name: reprogramming primary amniotic fluid and membrane cells to pluripotency in xeno-free conditions
Uploaded: 2017-11-27T13:00:00
Description: Watch this Scientific Journal Video about Reprogramming Primary Amniotic Fluid and Membrane Cells to Pluripotency in Xeno-free Conditions at JoVE.com

Ce travail a été soutenu par le Fonds Medizinische Forschung à l’Université de Zurich, Forschungskredit de l’Université de Zurich, la NMS SCIEXCh sous ° 10.216 bourses et 12.176, The Swiss Society of Cardiology, The Swiss National Science Fondation au titre de la subvention [320030-122273] et [310030-143992], le 7e Programme-cadre, la soupape de vie, la Commission européenne au titre de la subvention [242008], la Fondation de Mayenfisch Olga, la Fondation EMDO, la subvention de démarrage 2012 de l’hôpital universitaire de Zurich, et le financement interne de l’Institut de Cancer de Mitchell.

Le protocole suit les directives institutionnelles du Comité d’éthique pour la recherche. Consentement écrit du patient a été obtenue pour utiliser le liquide amniotique pour la recherche.
Ce protocole suit les politiques de l’animalier institutionnel et le Comité d’urbanisme de l’Université de South Alabama.
1. isolement et Culture des cellules souches mésenchymateuses primaires amniotique
<ol><li>
Placage de cellules du liquides amniotiqu...

<ol>
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Pathol. 25 (4), 300-305 (2016).</li><li>Slamecka, J., 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=Non-integrating+episomal+plasmid-based+reprogramming+of+human+amniotic+fluid+stem+cells+into+induced+pluripotent+stem+cells+in+chemically+defined+conditions.">Non-integrating episomal plasmid-based reprogramming of human amniotic fluid stem cells into induced pluripotent stem cells in chemically defined conditions.</a> Cell Cycle. 15 (2), 234-249 (2016).</li><li>Jiang, G., 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+Transgene-Free+Amniotic-Fluid-Derived+Induced+Pluripotent+Stem+Cells+for+Autologous+Cell+Therapy.">Human Transgene-Free Amniotic-Fluid-Derived Induced Pluripotent Stem Cells for Autologous Cell Therapy.</a> Stem Cells Dev. 23 (21), 2613-2625 (2014).</li><li>Pipino, C., 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=Trisomy+21+mid-trimester+amniotic+fluid+induced+pluripotent+stem+cells+maintain+genetic+signatures+during+reprogramming:+implications+for+disease+modeling+and+cryobanking.">Trisomy 21 mid-trimester amniotic fluid induced pluripotent stem cells maintain genetic signatures during reprogramming: implications for disease modeling and cryobanking.</a> Cell. 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P., Piaskowski, S., Stoczynska-Fidelus, E., Rieske, P., Grzela, D. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+human+iPSCs+from+cells+of+fibroblastic+and+epithelial+origin+by+means+of+the+oriP/EBNA-1+episomal+reprogramming+system.">Generation of human iPSCs from cells of fibroblastic and epithelial origin by means of the oriP/EBNA-1 episomal reprogramming system.</a> Stem Cell Res. Ther. 6 (1), (2015).</li><li>Kang, N. -. H., 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=Potential+antitumor+therapeutic+strategies+of+human+amniotic+membrane+and+amniotic+fluid-derived+stem+cells.">Potential antitumor therapeutic strategies of human amniotic membrane and amniotic fluid-derived stem cells.</a> Cancer Gene Ther. 19 (8), 517-522 (2012).</li><li>Moschidou, D., 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=Valproic+Acid+Confers+Functional+Pluripotency+to+Human+Amniotic+Fluid+Stem+Cells+in+a+Transgene-free+Approach.">Valproic Acid Confers Functional Pluripotency to Human Amniotic Fluid Stem Cells in a Transgene-free Approach.</a> Mol. 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Protoc. 8 (2), 223-253 (2013).</li><li>Chan, E. M., 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=Live+cell+imaging+distinguishes+bona+fide+human+iPS+cells+from+partially+reprogrammed+cells.">Live cell imaging distinguishes bona fide human iPS cells from partially reprogrammed cells.</a> Nat. Biotechnol. 27 (11), 1033-1037 (2009).</li><li>Adewumi, O., 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=Characterization+of+human+embryonic+stem+cell+lines+by+the+International+Stem+Cell+Initiative.">Characterization of human embryonic stem cell lines by the International Stem Cell Initiative.</a> Nat. Biotechnol. 25 (7), 803-816 (2007).</li><li>M&#252;ller, F. -. J., 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=A+bioinformatic+assay+for+pluripotency+in+human+cells.">A bioinformatic assay for pluripotency in human cells.</a> Nature Methods. 8 (4), 315-317 (2011).</li><li>Cahan, P., Li, H., Morris, S. A., Lummertz da Rocha, E., Daley, G. Q., Collins, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CellNet:+Network+Biology+Applied+to+Stem+Cell+Engineering.">CellNet: Network Biology Applied to Stem Cell Engineering.</a> Cell. 158 (4), 903-915 (2014).</li><li>Schopperle, W. M., DeWolf, W. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+TRA-1-60+and+TRA-1-81+Human+Pluripotent+Stem+Cell+Markers+Are+Expressed+on+Podocalyxin+in+Embryonal+Carcinoma.">The TRA-1-60 and TRA-1-81 Human Pluripotent Stem Cell Markers Are Expressed on Podocalyxin in Embryonal Carcinoma.</a> STEM CELLS. 25 (3), 723-730 (2007).</li><li>Ohnishi, 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=Premature+Termination+of+Reprogramming+In+Vivo+Leads+to+Cancer+Development+through+Altered+Epigenetic+Regulation.">Premature Termination of Reprogramming In Vivo Leads to Cancer Development through Altered Epigenetic Regulation.</a> Cell. 156 (4), 663-677 (2014).</li><li>Schlaeger, T. 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La phase initiale de l’iPSC génération de cellules souches fœtales implique l’extraction de cellules source dans les tissus fœtaux, leur culture, expansion et introduction des plasmides reprogrammation épisomiques. Cette phase est suivie d’une période de culture d’environ 14 à 18 jours avant que les premières colonies entièrement reprogrammés peuvent être étendus. La phase finale est la maturation des clones iPSC. L’extraction initiale de cellules souches de membrane amniotique se faite au moyen d?...

La technologie des cellules souches pluripotentes induites (iPSC) apporte sur les thérapies de remplacement cellulaire potentielle, maladie et modélisation du développement et drogues dépistage toxicologique1,2,3. Les thérapies de remplacement est possible sur le plan conceptuel par injection de cellules, différenciées implantation tissulaire (par exemple cardiaque "patches"), in vitro, ou régénération guidée au moyen de l’ingénierie tissulaire. Liquide amniotique (AFSC) et la membrane des cellules souches (AMSC) sont une excellente source de cellules pour ces interventions soit directement4,5,6,7 ou comme une population de cellules de départ pour la reprogrammation dans la pluripotence8,9,10,11,12.
Premières approches utilisées systèmes de culture non défini ou reprogrammation méthodes nécessitant l’intégration génomique comportent des constructions9,10,11,12. Une étude plus récente a employé un milieu exempt de xeno, même si une matrice d’attachement membrane basale moins définie (BMM) a utilisé, pour générer l’iPSC de cellules épithéliales du fluides amniotique. Cependant, l’analyse de formation tératome ne figurait pas dans l’étude avec une richesse d’in vitro et les données moléculaires. Cellules épithéliales fluides amniotiques ont été trouvés pour avoir une plus grande efficacité reprogrammation environ 8 fois par rapport aux fibroblastes néonatale13. Dans une autre étude, les cellules souches mésenchymateuses du liquide amniotique se retrouvent également à être reprogrammé dans iPSC avec un beaucoup l’efficacité supérieure12.
Cellules souches pluripotentes peuvent être différenciées en représentant de tissus de tous les 3 couches de germe et donc le potentiel plus large. Les patients pédiatriques pourraient bénéficier de la récolte, la reprogrammation et l’ingénierie tissulaire de leurs cellules de tige fluides amniotiques autologues avant la naissance et cellules de membrane amniotique souches durant la période périnatale. En outre, le niveau relativement faible de la différenciation de cellules souches fœtales (inférieurs à14,de cellules souches adultes15) pourrait théoriquement aider dans la lutte contre la rétention observée de partialité épigénétique des cellules source l’iPSC16.
Nous présentons un protocole pour la reprogrammation du liquide amniotique et membranaires des cellules souches pluripotence dans défini chimiquement milieu E8 xeno-sans le recombinant vitronectine17 (VTN) à l’aide de plasmides épisomiques18. Le principal avantage des cellules du liquide et la membrane amniotique comme source de cellules pour la reprogrammation réside dans leur disponibilité avant et Pendant la période périnatale et donc cette démarche profiterait principalement recherche en génie tissulaire pédiatrique.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	
	<tbody>
		<tr >
			<td >Tumor Dissociation Kit, human</td>
			<td >Miltenyi Biotec</td>
			<td >130-095-929</td>
			<td >tissue dissociation system, reagent kit, includes tissue dissociation tubes and tissue dissociation enzymes</td>
		</tr>
		<tr >
			<td >gentleMACS Dissociator</td>
			<td >Miltenyi Biotec</td>
			<td >130-093-235</td>
			<td >tissue dissociation system, dissociator</td>
		</tr>
		<tr >
			<td >Thermo Scientific&#8482; Shandon&#8482; Disposable Scalpel No. 10, Sterile, Individually Wrapped, 5.75 (14.6cm)</td>
			<td >Thermo-Fisher</td>
			<td >3120032</td>
			<td ></td>
		</tr>
		<tr >
			<td >70 &#181;m cell strainers</td>
			<td >Corning</td>
			<td >10054-456</td>
			<td ></td>
		</tr>
		<tr >
			<td >RPMI 1640 medium</td>
			<td >Thermo-Fisher</td>
			<td >32404014&#160;</td>
			<td ></td>
		</tr>
		<tr >
			<td >rocking platform</td>
			<td >VWR</td>
			<td >40000-300</td>
			<td ></td>
		</tr>
		<tr >
			<td >50 ml centrifuge tubes</td>
			<td >Thermo-Fisher</td>
			<td >339652</td>
			<td ></td>
		</tr>
		<tr >
			<td >15 ml centrifuge tubes</td>
			<td >Thermo-Fisher</td>
			<td >339650</td>
			<td ></td>
		</tr>
		<tr >
			<td >EBM-2 basal medium</td>
			<td >Lonza</td>
			<td >CC-3156</td>
			<td >basal medium for AFMC medium</td>
		</tr>
		<tr >
			<td >FGF 2 Human (expressed in E. coli, non-glycosylated)</td>
			<td >Prospec Bio</td>
			<td >CYT-218</td>
			<td >bFGF, supplement for AFMC medium</td>
		</tr>
		<tr >
			<td >EGF Human, Pichia</td>
			<td >Prospec Bio</td>
			<td >CYT-332&#160;</td>
			<td >EGF, supplement for AFMC medium</td>
		</tr>
		<tr >
			<td >LR3 Insulin Like Growth Factor-1 Human Recombinant</td>
			<td >Prospec Bio</td>
			<td >CYT-022</td>
			<td >IGF, supplement for AFMC medium</td>
		</tr>
		<tr >
			<td >Fetal Bovine Serum, embryonic stem cell-qualified</td>
			<td >Thermo-Fisher</td>
			<td >10439024</td>
			<td >FBS</td>
		</tr>
		<tr >
			<td >Antibiotic-Antimycotic (100X)</td>
			<td >Thermo-Fisher</td>
			<td >15240062&#160;</td>
			<td >for primary AFSC/AMSC, for routine AFSC/AMSC it should not be necessary, do not use in medium for transfected cells!</td>
		</tr>
		<tr >
			<td >Accutase cell detachment solution</td>
			<td >StemCell Technologies</td>
			<td >07920</td>
			<td >cell detachment enzyme</td>
		</tr>
		<tr >
			<td >CryoStor&#8482; CS10</td>
			<td >StemCell Technologies</td>
			<td >07930</td>
			<td >complete freezing medium</td>
		</tr>
		<tr >
			<td >PBS, pH 7.4</td>
			<td >Thermo-Fisher Scientific</td>
			<td >10010023&#160;</td>
			<td ></td>
		</tr>
		<tr >
			<td >EndoFree Plasmid Maxi Kit (10)</td>
			<td >Qiagen</td>
			<td >12362</td>
			<td >for plasmid isolation</td>
		</tr>
		<tr >
			<td >pEP4 E02S EN2K</td>
			<td >Addgene</td>
			<td >20925</td>
			<td >EN2K, reprogramming factors Oct4+Sox2, Nanog+Klf4</td>
		</tr>
		<tr >
			<td >pEP4 E02S ET2K</td>
			<td >Addgene</td>
			<td >20927</td>
			<td >ET2K, reprogramming factors Oct4+Sox2, SV40LT+Klf4</td>
		</tr>
		<tr >
			<td >pCEP4-M2L</td>
			<td >Addgene</td>
			<td >20926</td>
			<td >M2L, reprogramming factors c-Myc+LIN28</td>
		</tr>
		<tr >
			<td >NanoDrop 2000c UV-Vis Spectrophotometer</td>
			<td >Thermo-Fisher</td>
			<td >ND-2000C</td>
			<td >spectrophotometer</td>
		</tr>
		<tr >
			<td >Neon&#174; Transfection System</td>
			<td >Thermo-Fisher</td>
			<td >MPK5000</td>
			<td >transfection system, components: 
			Neon pipette - transfection pipette 
			Neon device - transfection device</td>
		</tr>
		<tr >
			<td >Neon&#174; Transfection System 10 &#181;L Kit</td>
			<td >Thermo-Fisher</td>
			<td >MPK1025</td>
			<td >consumables kit for the Neon Transfection System, it contains: 
			Neon tip - transfection tip 
			Neon tube - transfection tube 
			buffer R - resuspension buffer 
			buffer E - electrolytic buffer</td>
		</tr>
		<tr >
			<td >Stemolecule&#8482; Sodium Butyrate</td>
			<td >StemGent</td>
			<td >04-0005</td>
			<td >small molecule enhancer of reprogramming</td>
		</tr>
		<tr >
			<td >TeSR-E8</td>
			<td >StemCell Technologies</td>
			<td >05940</td>
			<td >E8 medium</td>
		</tr>
		<tr >
			<td >Vitronectin XF&#8482;</td>
			<td >StemCell Technologies</td>
			<td >07180</td>
			<td >VTN, stock concentration 250 &#181;g/ml, used for coating at 1 &#181;g/cm2 in vitronectin dilution (CellAdhere) buffer</td>
		</tr>
		<tr >
			<td >CellAdhere&#8482; Dilution Buffer</td>
			<td >StemCell Technologies</td>
			<td >07183</td>
			<td >vitronectin dilution buffer</td>
		</tr>
		<tr >
			<td >UltraPure&#8482; 0.5M EDTA, pH 8.0</td>
			<td >Thermo-Fisher</td>
			<td >15575020</td>
			<td >dilute with PBS to 0.5 mM before use</td>
		</tr>
		<tr >
			<td >EVOS&#174; FL Imaging System</td>
			<td >Thermo-Fisher Scientific</td>
			<td >AMF4300</td>
			<td >LCD imaging microscope system</td>
		</tr>
		<tr >
			<td >CKX53 Inverted Microscope</td>
			<td >Olympus</td>
			<td ></td>
			<td >phase contrast cell culture microscope</td>
		</tr>
		<tr >
			<td >Pierce&#8482; 16% Formaldehyde (w/v), Methanol-free</td>
			<td >Thermo-Fisher</td>
			<td >28908</td>
			<td >dilute to 4% with PBS before use, diluted can be stored at 2-8 &#176;C for 1 week</td>
		</tr>
		<tr >
			<td >Perm Buffer III</td>
			<td >BD Biosciences</td>
			<td >558050</td>
			<td >permeabilization buffer, chill to -20 &#176;C before use</td>
		</tr>
		<tr >
			<td >Mouse IgG1, &#954; Isotype Control, Alexa Fluor&#174; 488</td>
			<td >BD Biosciences</td>
			<td >557782</td>
			<td >isotype control for Oct3/4A, Nanog</td>
		</tr>
		<tr >
			<td >Mouse IgG1, &#954; Isotype Control, Alexa Fluor&#174; 647</td>
			<td >BD Biosciences</td>
			<td >557783</td>
			<td >isotype control for Sox2</td>
		</tr>
		<tr >
			<td >Mouse anti-human Oct3/4 (Human Isoform A), Alexa Fluor&#174; 488</td>
			<td >BD Biosciences</td>
			<td >561628</td>
			<td ></td>
		</tr>
		<tr >
			<td >Mouse anti-human Nanog, Alexa Fluor&#174; 488</td>
			<td >BD Biosciences</td>
			<td >560791</td>
			<td ></td>
		</tr>
		<tr >
			<td >Mouse anti-human Sox-2, Alexa Fluor&#174; 647</td>
			<td >BD Biosciences</td>
			<td >562139</td>
			<td ></td>
		</tr>
		<tr >
			<td >Mouse IgGM, &#954; Isotype Control, Alexa Fluor&#174; 488</td>
			<td >BD Biosciences</td>
			<td >401617</td>
			<td >isotype control for TRA-1-60</td>
		</tr>
		<tr >
			<td >Mouse IgGM, &#954; Isotype Control, Alexa Fluor&#174; 647</td>
			<td >BD Biosciences</td>
			<td >401618</td>
			<td >isotype control for TRA-1-81</td>
		</tr>
		<tr >
			<td >Mouse anti-human TRA-1-60, Alexa Fluor&#174; 488</td>
			<td >BD Biosciences</td>
			<td >330613</td>
			<td ></td>
		</tr>
		<tr >
			<td >Mouse anti-human TRA-1-81, Alexa Fluor&#174; 647</td>
			<td >BD Biosciences</td>
			<td >330705</td>
			<td ></td>
		</tr>
		<tr >
			<td >Mouse IgG1, &#954; Isotype Control, Alexa Fluor&#174; 488</td>
			<td >BD Biosciences</td>
			<td >400129</td>
			<td >isotype control for SSEA-1</td>
		</tr>
		<tr >
			<td >Mouse IgG3, &#954; Isotype Control, Alexa Fluor&#174; 647</td>
			<td >BD Biosciences</td>
			<td >401321</td>
			<td >isotype control for SSEA-4</td>
		</tr>
		<tr >
			<td >Mouse anti-human SSEA-1, Alexa Fluor&#174; 488</td>
			<td >BD Biosciences</td>
			<td >323010</td>
			<td ></td>
		</tr>
		<tr >
			<td >Mouse anti-human SSEA-4, Alexa Fluor&#174; 647</td>
			<td >BD Biosciences</td>
			<td >330407</td>
			<td ></td>
		</tr>
		<tr >
			<td >Affinipure F(ab&#39;)2 Fragment Goat Anti-Mouse IgG+IgM, Alexa Fluor&#174; 488</td>
			<td >Jackson Immunoresearch</td>
			<td >115-606-068</td>
			<td >use at a dilution of 1:600 or further optimize</td>
		</tr>
		<tr >
			<td >Affinipure F(ab&#39;)2 Fragment Goat Anti-Mouse IgG+IgM, Alexa Fluor&#174; 647</td>
			<td >Jackson Immunoresearch</td>
			<td >115-546-068</td>
			<td >use at a dilution of 1:600 or further optimize</td>
		</tr>
		<tr >
			<td >DAPI</td>
			<td >Thermo-Fisher Scientific</td>
			<td >D21490</td>
			<td >stock solution 10 mM, further dilute to 1:12.000 for a working solution</td>
		</tr>
		<tr >
			<td >Corning&#174; Matrigel&#174; Growth Factor Reduced, Phenol Red-Free</td>
			<td >Corning</td>
			<td >356231</td>
			<td >basement membrane matrix (BMM)</td>
		</tr>
		<tr >
			<td >scid-beige mice, female</td>
			<td >Taconic</td>
			<td >CBSCBG-F</td>
			<td ></td>
		</tr>
		<tr >
			<td >RNeasy Plus Mini Kit (50)</td>
			<td >Qiagen</td>
			<td >74134</td>
			<td >RNA isolation kit</td>
		</tr>
		<tr >
			<td >T-25 flasks, tissue culture-treated</td>
			<td >Thermo-Fisher</td>
			<td >156367</td>
			<td ></td>
		</tr>
		<tr >
			<td >T-75 flasks, tissue culture-treated</td>
			<td >Thermo-Fisher</td>
			<td >156499</td>
			<td ></td>
		</tr>
		<tr >
			<td >Nunc&#8482; tissue-culture dish</td>
			<td >Thermo-Fisher</td>
			<td >12-567-650&#160;</td>
			<td >10 cm tissue culture dish</td>
		</tr>
		<tr >
			<td >6-well plates, tissue-culture treated</td>
			<td >Thermo-Fisher</td>
			<td >140675</td>
			<td ></td>
		</tr>
		<tr >
			<td >Neubauer counting chamber (hemacytometer)</td>
			<td >VWR</td>
			<td >15170-173</td>
			<td ></td>
		</tr>
		<tr >
			<td >Mr. Frosty&#8482; Freezing Container</td>
			<td >Thermo-Fisher</td>
			<td >5100-0001&#160;</td>
			<td >freezing container</td>
		</tr>
		<tr >
			<td >FACS tubes, Round Bottom Polystyrene Test Tube, 5ml</td>
			<td >Corning</td>
			<td >352058</td>
			<td >5 ml polystyrene tubes</td>
		</tr>
		<tr >
			<td >Eppendorf tubes, 1.5 ml</td>
			<td >Thermo-Fisher</td>
			<td >05-402-96</td>
			<td >1.5 ml microcentrifuge tubes</td>
		</tr>
		<tr >
			<td >PCR tubes, 200 &#181;l</td>
			<td >Thermo-Fisher</td>
			<td >14-222-262</td>
			<td ></td>
		</tr>
		<tr >
			<td >pipette tips, 100 to 1250 &#181;l</td>
			<td >Thermo-Fisher</td>
			<td >02-707-407</td>
			<td >narrow-bore 1 mL tips</td>
		</tr>
		<tr >
			<td >pipette tips, 5 to 300 &#181;l</td>
			<td >Thermo-Fisher</td>
			<td >02-707-410</td>
			<td ></td>
		</tr>
		<tr >
			<td >pipette tips, 0.1 to 10 &#181;l</td>
			<td >Thermo-Fisher</td>
			<td >02-707-437</td>
			<td ></td>
		</tr>
		<tr >
			<td >wide-bore pipette tips, 1000 &#181;l</td>
			<td >VWR</td>
			<td >89049-166</td>
			<td >wide-bore 1 mL tips</td>
		</tr>
		<tr >
			<td >glass Pasteur pipettes</td>
			<td >Thermo-Fisher</td>
			<td >13-678-20A</td>
			<td ></td>
		</tr>
		<tr >
			<td >ethanol, 200 proof</td>
			<td >Thermo-Fisher</td>
			<td >04-355-451</td>
			<td ></td>
		</tr>
		<tr >
			<td >vortex mixer</td>
			<td >VWR</td>
			<td >10153-842</td>
			<td ></td>
		</tr>
		<tr >
			<td >chambered coverglass, 8-well, 1.5mm borosilicate glass</td>
			<td >Thermo-Fisher</td>
			<td >155409</td>
			<td >glass-bottom confocal-grade cultureware</td>
		</tr>
		<tr >
			<td >22G needles</td>
			<td >VWR</td>
			<td >82002-366</td>
			<td ></td>
		</tr>
		<tr >
			<td >insulin syringes</td>
			<td >Thermo-Fisher</td>
			<td >22-253-260</td>
			<td ></td>
		</tr>
		<tr >
			<td >Formalin solution, neutral buffered, 10%</td>
			<td >Sigma-Aldrich</td>
			<td >HT501128-4L</td>
			<td >fixation of explanted teratomas</td>
		</tr>
		<tr >
			<td >Illumina HT-12 v4 Expression BeachChip</td>
			<td >Illumina</td>
			<td >BD-103-0204</td>
			<td >expression microarray, supported by PluriTest, discontinued by manufacturer</td>
		</tr>
		<tr >
			<td >PrimeView Human Genome U219 Array Plate</td>
			<td >Thermo-Fisher</td>
			<td >901605</td>
			<td >expression microarray (formerly Affymetrix brand), soon to be supported by PluriTest</td>
		</tr>
		<tr >
			<td >GeneChip&#8482; Human Genome U133 Plus 2.0 Array</td>
			<td >Thermo-Fisher</td>
			<td >902482</td>
			<td >expression microarray (formerly Affymetrix brand), supported by CellNet, soon to be supported by PluriTest</td>
		</tr>
		<tr >
			<td >PluriTest&#174;</td>
			<td >Coriell Institute</td>
			<td ></td>
			<td >www.pluritest.org, free service for bioinformatic assessment of pluripotency, accepts microarray data - *.idat files from HT-12 v4 platform, soon to support U133, U219 microarray and RNA sequencing data</td>
		</tr>
		<tr >
			<td >CellNet</td>
			<td >Johns Hopkins University</td>
			<td ></td>
			<td >cellnet.hms.harvard.edu, free service for bioinformatic identification of cell type, including plutipotent stem cells, based on U133 microarray data - *.cel files, soon to support RNA sequencing data</td>
		</tr>
	</tbody>
</table>

reprogramming primary amniotic fluid and membrane cells to pluripotency in xeno-free conditions

Thérapies à base cellulaire autologues obtenu un pas de plus vers la réalité avec l’introduction de cellules souches pluripotentes induites. Les cellules souches fœtales, tels que le liquide amniotique et la membrane des cellules souches mésenchymateuses, représentent un type unique de cellules non différenciées avec promesse en génie tissulaire et de reprogrammation en iPSC pour futures interventions pédiatriques et cellules souches bancaire. Le protocole présenté ici décrit une méthode optimisée pour l’extraction et la mise en culture primaire amniotique fluide et membrane de cellules souches mésenchymateuses et générant épisomiques induite par les cellules souches pluripotentes de ces cellules en culture entièrement chimiquement définie conditions utilisant la vitronectine recombinante humaine et le milieu E8. Caractérisation des nouvelles lignes en appliquant des méthodes rigoureuses – cytométrie en flux, imagerie confocale, formation de tératome et profilage transcriptionnel – est également décrite. Les lignes nouvellement générés expriment des marqueurs de cellules souches embryonnaires – Oct3/4 a, Nanog, Sox2, TRA-1-60, TRA-1-81, SSEA-4 – tout en étant négatif pour le marqueur SSEA-1. Les lignées de cellules souches forment des tératomes chez la souris scid-beige dans 6 à 8 semaines et les tératomes contiennent des tissus représentatifs de tous les trois couches de germe. Profilage transcriptionnel des lignes par envoi de données microarray expression globale à un algorithme d’évaluation de pluripotence bioinformatic réputé toutes les lignes pluripotentes et par conséquent, cette approche est une alternative intéressante à l’expérimentation animale. Les nouvelles lignes iPSC utilisable facilement en aval expériences portant sur l’optimisation de la différenciation et l’ingénierie tissulaire.

Consentement éclairé a été obtenu chez les patients avant la récolte du liquide amniotique pour des fins de tests génétiques et de consacrer une petite aliquote du liquide pour la recherche. Aucun consentement n’est requis pour l’utilisation de la membrane amniotique dans la recherche car le placenta représente les déchets médicaux. Des cellules de tige de fluide et membrane amniotique Afficher Propriétés mésenchymateuses typiques, morphologiquement leurs cellules sont fu...

Watch this Scientific Journal Video about Reprogramming Primary Amniotic Fluid and Membrane Cells to Pluripotency in Xeno-free Conditions at JoVE.com

Reprogramming Primary Amniotic Fluid and Membrane Cells to Pluripotency in Xeno-free Conditions

Ce protocole décrit la reprogrammation des primaires amniotique fluide et membrane de cellules souches mésenchymateuses sur les cellules souches pluripotentes induites en utilisant une approche épisomiques sans intégration dans des conditions totalement chimiquement définies. Les procédures d’extraction, la culture, la reprogrammation et la caractérisation des cellules souches pluripotentes induites qui en résulte par une méthodologie rigoureuse sont reprises.

Reprogrammation du liquide amniotique primaire et cellules de Membrane de pluripotence dans des Conditions sans Xeno

Autologous cell-based therapies got a step closer to reality with the introduction of induced pluripotent stem cells. Fetal stem cells, such as amniotic ...

The overall goal of this procedure is to efficiently derive induced pluripotent stem cells by means of episomal reprogramming from two different kinds of fetal stem cells. This method can help provide a source of pluripotent stem cells for tissue engineering, disease modeling, basic research, or longitudinal studies. The main advantage of this technique is that the reprogramming and the maturation of iPSCs transpires in chemically defined conditions while being available early for potential pediatric cell-based therapies.Demonstrating this procedure will be Steven McClellan, the principal investigator, and myself. Obtain placentas as soon as possible following birth. Cut nine-squared centimeter segment of the amnion, and wash it in a 50-millimeter centrifuge tube with 30 milliliters of PBS supplemented with antibiotic, antimycotic solution.Remove blood clots from the membranes, and then use a pair of fine scalpels to mince the membranes into fine pieces in a sterile 10-centimeter tissue culture dish. Use the scalpel blades to transfer the minced membrane tissue mass into one tissue dissociation tube containing 4.7 milliliters of RPMI-1640 medium. Mix in the dissociation enzymes.Mount the tube onto the tissue dissociator, and run program h_tumor_01. Then incubate the tube at 37 degrees Celsius on a rocking platform for 30 minutes. Further dilute the suspension with 35 milliliters of RPMI-1640, and apply to a 70-micron strainer placed over a 50-milliliter centrifuge tube.Centrifuge for five minutes at 200 times g at room temperature. After discarding the supernatant, resuspend the pellet in five milliliters of RPMI-1640, and count the cells using a hemocytometer. Seed the cells in cell clusters at a density of roughly 10, 000 cells per square centimeter into tissue culture flasks with freshly prepared AFMC medium supplemented with antibiotic, antimycotic solution.Harvest low passage number amniotic fluid and membrane stem cells for reprogramming from one T75 flask by adding two milliliters of cell detachment enzyme to the cell sheet. Incubate at 37 degrees Celsius for five to eight minutes. After centrifugation at 200 times g for 45 minutes, resuspend the pellet in one milliliter of PBS, and mix well to wash away serum components.Count the cells using a hemocytometer. Then adjust the cell density to 100, 000 cells per milliliter of PBS, and now quad into 1.5-milliliter microcentrifuge tubes. Place the microcentrifuge tubes in five-milliliter polystyrene tubes to allow centrifugation in a regular swing rudder, and centrifuge at 200 times g for four minutes at room temperature.After the spin, invert the tubes, and discard the supernatant into a waste container, and then perform an additional centrifugation step at 200 times g for three minutes. After the spin, use a 200-microliter pipette to carefully aspirate the remaining liquid from the tubes. Place transfection tips, tube, resuspension buffer, and electrolytic buffer into the tissue culture cabinet.Move the transfection device close so that its tube station can be placed directly into the cabinet. Fill one transfection tube with three milliliters of electrolytic buffer, and mount the tube into the station by pushing it all the way inside the slot. Take previously prepared aliquots of reprogramming plasmid solution out of the minus 80 degrees Celsius storage, and allow them to thaw at room temperature in the culture cabinet.Set the transfection parameters on the device to deliver one pulse of 40 milliseconds at 950 volts. Resuspend the pellet containing 100, 000 cells in 10 microliters of resuspension buffer. Working quickly, add 1/10 of the reprogramming plasmid solution to the resuspended cells.Mount the transfection tip onto the transfection pipette. Then aspirate the cell suspension into the transfection tip carefully avoiding formation of air bubbles. Insert the transfection pipette into the transfection tube.And press the start button on the screen at the transfection device. Wait for the screen message regarding the success of the transfection. Then remove the pipette from the tube immediately.Expel the suspension into one well of the target six-well plates. Mix in the medium from a neighboring well, and distribute the suspension equally into both wells. Repeat the transfection for each microcentrifuge tube with cells.Begin by adding 30 microliters of 0.5 millimolar EDTA, and PBS to five PCR tubes. Then load a tip onto a 10-microliter pipette set to two microliters. Hold the pipette tip at an angle at the colony edge, and carefully and gradually scrape the whole colony off the surface.Immediately aspirate the whole colony into the pipette tip, and transfer it into one of the prepared PCR tubes with EDTA. After repeating the process with four other colonies, incubate at room temperature for four to six minutes. Use a larger pipette tip to pipette each cell suspension up and down gently to break the colony down into smaller clumps.Avoid creating a single-cell suspension. Then plate the suspension directly into the target well of a recombinant vitronectin-coated 24-well plate. After repeating the process for the remaining colonies, incubate for three to six days.After four to six days, when the colonies have grown and become compact, wash the cell sheets with 300 microliters of 0.5 millimolar EDTA, and aspirate immediately. Then add another 300 microliters, and incubate at room temperature for five minutes. After aspirating the EDTA, set a one-milliliter pipette to capacity, mount to one milliliter wide bore tip, and aspirate E8 medium from the target well of a vitronectin-coated six-well plate.Squirt a stream of the medium at one well of the source plate to wash off the iPSC culture. Then transfer the suspension into the target well, and pipette up and down several times to break up the colonies into clumps of 20 to 40 cells. After plating all cells, ensure that the clumps are distributed evenly by gently rocking and shaking the plate.Then return the plate into the incubator for clump adhesion and colony growth. The amniotic fluid and membrane stem cells, which represent source cells for reprogramming display a typical mesenchymal morphology elongated and phase-bright. The cells acquire epithelial properties after they undergo the mesenchymal-to-epithelial transition, which leads to the formation of colonies with cobblestone-like cells.These colonies proliferate and create irregular cellular masses of MET cells. At the later stages of reprogramming, colonies of fully reprogrammed cells emerge, individually discernible cells with well-defined borders. These fully reprogrammed cells are present alongside MET colonies that are more numerous.A fully reprogrammed isolated mature clone is shown here. Following iPSC derivation and maturation, they can be subjected to detail characterization, and direct the differentiation into a variety of cell types. Don't forget that working with human tissues that have not been tested for the presence of pathogens can be extremely hazardous, and precautions such as personal protective equipment, and working in the biosafety cabinet should always be taken while performing this procedure.After watching this video, you should have a good understanding of how to induce pluripotency in fetal stem cells, what reagents and equipment to use, and how to routinely culture pluripotent stem cells.

Research

JoVE Journal

Developmental Biology

Stable and Efficient Genetic Modification of Cells in the Adult Mouse V-SVZ for the Analysis of Neural Stem Cell Autonomous and Non-autonomous Effects

Stable et efficace modification génétique de cellules dans le Adulte souris V-SVZ pour l&#39;analyse des cellules souches neurales autonome et effets non autonomes

Relatively quiescent somatic stem cells support life-long cell renewal in most adult tissues. Neural stem cells in the adult mammalian brain are ...

Recherche

Biologie du développement

Reprogramming Mouse Embryonic Fibroblasts with Transcription Factors to Induce a Hemogenic Program

Reprogrammation fibroblastes embryonnaires de souris avec les facteurs de transcription pour induire un programme Hemogenic

This protocol details the induction of a hemogenic program in mouse embryonic fibroblasts (MEFs) via overexpression of transcription factors (TFs). We ...

The Production of Pluripotent Stem Cells from Mouse Amniotic Fluid Cells Using a Transposon System

La production de cellules souches pluripotentes de souris amniotique Cellules Fluid Utilisation d&#39;un système de transposon

Induced pluripotent stem (iPS) cells are generated from mouse and human somatic cells by forced expression of defined transcription factors using ...

Production and Administration of Therapeutic Mesenchymal Stem/Stromal Cell (MSC) Spheroids Primed in 3-D Cultures Under Xeno-free Conditions

Production and Administration of Therapeutic Mesenchymal Stem/Stromal Cell MSC Spheroids Primed in 3-D Cultures Under Xeno-free Conditions

Production et administration de la thérapeutique mésenchymateuses souches / cellules stromales (MSC) Spheroids apprêté dans les cultures 3-D dans des conditions d&#39;Xeno-libres

Mesenchymal stem/stromal cells (MSCs) hold great promise in bioengineering and regenerative medicine. MSCs can be isolated from multiple adult tissues via ...

Conditional Reprogramming of Pediatric Human Esophageal Epithelial Cells for Use in Tissue Engineering and Disease Investigation

Reprogrammation conditionnelle de cellules Pediatric Human oesophagien épithéliales pour utilisation en génie tissulaire et recherche sur les maladies

Identifying and expanding patient-specific cells in culture for use in tissue engineering and disease investigation can be very challenging. Utilizing ...

Computational Analysis of the Caenorhabditis elegans Germline to Study the Distribution of Nuclei, Proteins, and the Cytoskeleton

Computational Analysis of the Caenorhabditis elegans Germline to Study the Distribution of Nuclei, Proteins, and the Cytoskeleton

Analyse de calcul de la lignée germinale de Caenorhabditis elegans pour étudier la Distribution des noyaux, les protéines et le cytosquelette

The Caenorhabditis elegans (C. elegans) germline is used to study several biologically important processes including stem cell development, apoptosis, and ...

Induction and Validation of Cellular Senescence in Primary Human Cells

Induction et la Validation de la sénescence cellulaire dans les cellules humaines primaires

Cellular senescence is a state of permanent cell cycle arrest activated in response to different damaging stimuli. Activation of cellular senescence is a ...

Defined Xeno-free and Feeder-free Culture Conditions for the Generation of Human iPSC-derived Retinal Cell Models

Xeno-libre défini et exempte d’engraissement des Conditions de Culture pour les modèles de cellule rétinienne Generation of Human dérivés iPSC

The production of specialized cells from pluripotent stem cells provides a powerful tool to develop new approaches for regenerative medicine. The use of ...

Intravenous and Intra-amniotic In Utero Transplantation in the Murine Model

Intravenous and Intra-amniotic In Utero Transplantation in the Murine Model

Intraveineuse amniotique et In Utero une Transplantation chez le modèle murin

In utero transplantation (IUT) is a unique and versatile mode of therapy that can be used to introduce stem cells, viral vectors, or any other substances ...

Direct Lineage Reprogramming of Adult Mouse Fibroblast to Erythroid Progenitors

Diriger la lignée reprogrammation de fibroblastes de souris adulte de progéniteurs érythroïdes

Erythroid cell commitment and differentiation proceed through activation of a lineage-restricted transcriptional network orchestrated by a group of cell ...