Name: human pluripotent stem cell culture on polyvinyl alcohol-co-itaconic acid hydrogels with varying stiffness under xeno-free conditions
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Cette recherche a été partiellement prises en charge par le ministère de la Science et technologie, Taiwan sous concession numéros 106-2119-M-008-003, 105-2119-M-008-006 et 104-2221-E-008-107-MY3. Cette recherche a été également pris en charge par le projet de l’hôpital de Landseed de Taiwan (NCU-LSH-105-A-001). Une subvention pour la recherche scientifique (numéro 15K 06591) depuis le ministère de l’éducation, Culture, Sports, Science et technologie du Japon est également reconnue. A. Higuchi aimerait remercier pour le programme de partenariat scientifique International (ISPP-0062) Vice Rectorat pour les études supérieures et postdoctorales, Université du Roi Saoud, Riyadh 11451, Royaume d’Arabie saoudite.

Les expériences dans cette étude ont été approuvées par les comités d’éthique de l’hôpital de Landseed de Taiwan (IRB-13-05) et l’Université nationale centrale. Toutes les expériences ont été menées conformément aux règlements et lignes directrices gouvernementales et institutionnelles pertinentes et applicables au cours de cette étude.
1. solution et préparation des médias
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Purification de polymère
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<li>Purifier P-IA avec groupe acide c...

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	<li>Higuchi, A., Ling, Q. D., Chang, Y., Hsu, S. T., Umezawa, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Physical+cues+of+biomaterials+guide+stem+cell+differentiation+fate.">Physical cues of biomaterials guide stem cell differentiation fate.</a> Chem. Rev. 113, 3297-3328 (2013).</li><li>Higuchi, 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=Physical+cues+of+cell+culture+materials+lead+the+direction+of+differentiation+lineages+of+pluripotent+stem+cells.">Physical cues of cell culture materials lead the direction of differentiation lineages of pluripotent stem cells.</a> J. Mater. Chem. B. 3, 8032-8058 (2015).</li><li>Wen, J. 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S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assaying+stem+cell+mechanobiology+on+microfabricated+elastomeric+substrates+with+geometrically+modulated+rigidity.">Assaying stem cell mechanobiology on microfabricated elastomeric substrates with geometrically modulated rigidity.</a> Nat. Protoc. 6, 187-213 (2011).</li><li>Shih, Y. R. V., Tseng, K. F., Lai, H. Y., Lin, C. H., Lee, O. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Matrix+Stiffness+Regulation+of+Integrin-Mediated+Mechanotransduction+During+Osteogenic+Differentiation+of+Human+Mesenchymal+Stem+Cells.">Matrix Stiffness Regulation of Integrin-Mediated Mechanotransduction During Osteogenic Differentiation of Human Mesenchymal Stem Cells.</a> J. Bone Miner. 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J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+substrate+stiffness+and+cell-cell+contact+on+mesenchymal+stem+cell+differentiation.">Effects of substrate stiffness and cell-cell contact on mesenchymal stem cell differentiation.</a> Biomaterials. 98, 184-191 (2016).</li><li>Witkowska-Zimny, 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=Effect+of+substrate+stiffness+on+differentiation+of+umbilical+cord+stem+cells.">Effect of substrate stiffness on differentiation of umbilical cord stem cells.</a> Acta Biochim. Pol. 59 (2), 261-264 (2012).</li><li>Witkowska-Zimny, 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=Effect+of+substrate+stiffness+on+the+osteogenic+differentiation+of+bone+marrow+stem+cells+and+bone-derived+cells.">Effect of substrate stiffness on the osteogenic differentiation of bone marrow stem cells and bone-derived cells.</a> Cell Biol. Int. 37 (6), 608-616 (2013).</li><li>Macri-Pellizzeri, L., 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=Substrate+stiffness+and+composition+specifically+direct+differentiation+of+induced+pluripotent+stem+cells.">Substrate stiffness and composition specifically direct differentiation of induced pluripotent stem cells.</a> Tissue Eng. Part A. 21 (9-10), 1633-1641 (2015).</li><li>Cozzolino, A. 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=Modulating+the+Substrate+Stiffness+to+Manipulate+Differentiation+of+Resident+Liver+Stem+Cells+and+to+Improve+the+Differentiation+State+of+Hepatocytes.">Modulating the Substrate Stiffness to Manipulate Differentiation of Resident Liver Stem Cells and to Improve the Differentiation State of Hepatocytes.</a> Stem Cells Int. , 5481493 (2016).</li><li>Mattei, G., Ferretti, C., Tirella, A., Ahluwalia, A., Mattioli-Belmonte, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Decoupling+the+role+of+stiffness+from+other+hydroxyapatite+signalling+cues+in+periosteal+derived+stem+cell+differentiation.">Decoupling the role of stiffness from other hydroxyapatite signalling cues in periosteal derived stem cell differentiation.</a> Sci. Rep. 5, 10778 (2015).</li><li>Zouani, O. F., Kalisky, J., Ibarboure, E., Durrieu, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+BMP-2+from+matrices+of+different+stiffnesses+for+the+modulation+of+stem+cell+fate.">Effect of BMP-2 from matrices of different stiffnesses for the modulation of stem cell fate.</a> Biomaterials. 34 (9), 2157-2166 (2013).</li><li>Ye, K., Cao, L., Li, S., Yu, L., Ding, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interplay+of+Matrix+Stiffness+and+Cell-Cell+Contact+in+Regulating+Differentiation+of+Stem+Cells.">Interplay of Matrix Stiffness and Cell-Cell Contact in Regulating Differentiation of Stem Cells.</a> ACS Appl. Mater. Interfaces. 8 (34), 21903-21913 (2016).</li><li>Hogrebe, N. J., Gooch, K. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+influence+of+culture+dimensionality+on+human+mesenchymal+stem+cell+differentiation+at+various+matrix+stiffnesses+using+a+fibrous+self-assembling+peptide+hydrogel.">Direct influence of culture dimensionality on human mesenchymal stem cell differentiation at various matrix stiffnesses using a fibrous self-assembling peptide hydrogel.</a> J. Biomed. Mater. Res. A. 104 (9), 2356-2368 (2016).</li><li>Olivares-Navarrete, R., 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=Substrate+Stiffness+Controls+Osteoblastic+and+Chondrocytic+Differentiation+of+Mesenchymal+Stem+Cells+without+Exogenous+Stimuli.">Substrate Stiffness Controls Osteoblastic and Chondrocytic Differentiation of Mesenchymal Stem Cells without Exogenous Stimuli.</a> PLoS One. 12, e0170312 (2017).</li><li>Kumar, S. S., 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+combined+influence+of+substrate+elasticity+and+surface-grafted+molecules+on+the+ex+vivo+expansion+of+hematopoietic+stem+and+progenitor+cells.">The combined influence of substrate elasticity and surface-grafted molecules on the ex vivo expansion of hematopoietic stem and progenitor cells.</a> Biomaterials. 34, 7632-7644 (2013).</li><li>Higuchi, A., Iijima, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DSC+investigation+of+the+states+of+water+in+poly(vinyl+alcohol)+membranes.">DSC investigation of the states of water in poly(vinyl alcohol) membranes.</a> Polymer. 26, 1207-1211 (1985).</li><li>Higuchi, A., Iijima, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DSC+investigation+of+the+states+of+water+in+poly(vinyl+alcohol-co-itaconic+acid)+membranes.">DSC investigation of the states of water in poly(vinyl alcohol-co-itaconic acid) membranes.</a> Polymer. 26, 1833-1837 (1985).</li><li>Higuchi, 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=Long-term+xeno-free+culture+of+human+pluripotent+stem+cells+on+hydrogels+with+optimal+elasticity.">Long-term xeno-free culture of human pluripotent stem cells on hydrogels with optimal elasticity.</a> Sci Rep. 5, 18136 (2015).</li><li>Wang, P. 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=Pluripotency+maintenance+of+amniotic+fluid-derived+stem+cells+cultured+on+biomaterials.">Pluripotency maintenance of amniotic fluid-derived stem cells cultured on biomaterials.</a> J Mater Chem B. 3, 3858-3869 (2015).</li><li>Muduli, S., 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=Proliferation+and+osteogenic+differentiation+of+amniotic+fluid-derived+stem+cells.">Proliferation and osteogenic differentiation of amniotic fluid-derived stem cells.</a> J Mater Chem B. 5, 5345-5354 (2017).</li><li>Muduli, S., 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=Stem+cell+culture+on+polyvinyl+alcohol+hydrogels+having+different+elasticity+and+immobilized+with+ECM-derived+oligopeptides.">Stem cell culture on polyvinyl alcohol hydrogels having different elasticity and immobilized with ECM-derived oligopeptides.</a> JPoly Eng. 37, 647 (2017).</li><li>Chen, Y. 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=Xeno-free+culture+of+human+pluripotent+stem+cells+on+oligopeptide-grafted+hydrogels+with+various+molecular+designs.">Xeno-free culture of human pluripotent stem cells on oligopeptide-grafted hydrogels with various molecular designs.</a> Sci Rep. 7, 45146 (2017).</li><li>Higuchi, A., Ling, Q. D., Ko, Y. A., Chang, Y., Umezawa, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biomaterials+for+the+feeder-free+culture+of+human+embryonic+stem+cells+and+induced+pluripotent+stem+cells.">Biomaterials for the feeder-free culture of human embryonic stem cells and induced pluripotent stem cells.</a> Chem Rev. 111 (5), 3021-3035 (2011).</li><li>Higuchi, 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=Design+of+polymeric+materials+for+culturing+human+pluripotent+stem+cells:+Progress+toward+feeder-free+and+xeno-free+culturing.">Design of polymeric materials for culturing human pluripotent stem cells: Progress toward feeder-free and xeno-free culturing.</a> Prog Polym Sci. 39, 1348-1374 (2014).</li><li>Higuchi, 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=Stem+cell+therapies+for+myocardial+infarction+in+clinical+trials:+bioengineering+and+biomaterial+aspects.">Stem cell therapies for myocardial infarction in clinical trials: bioengineering and biomaterial aspects.</a> Lab Invest. 97, 1167-1179 (2017).</li><li>Higuchi, 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=Stem+cell+therapies+for+reversing+vision+loss.">Stem cell therapies for reversing vision loss.</a> Trends Biotechnol. 35, 1102-1117 (2017).</li></ol>

P-IA-oligoECM et P-IA-ECM hydrogels avec raideur variable ont été développées pour l’expansion à long terme de ES humain et CSPi conservant leur pluripotence pour plus de dix passages dans des conditions sans xeno, ainsi que pour la culture de cellules humaines de l’AFS, cellules d’annonces, et les cellules souches hématopoïétiques25,28,32. P-IA hydrogels immobilisées avec oligoECM sont un excellent candidat pour ...

Le sort de la différenciation des cellules souches dans une lignée spécifique des cellules et la prolifération à long terme des cellules souches, cellules surtout humaines pluripotentes induites (iPS) et les cellules souches embryonnaires humaines (ES), est connu pour être régulée par des inhibiteurs, facteurs de croissance, et / ou de petites molécules bioactives dans les milieux de culture. Récemment, les repères physiques des biomatériaux, particulièrement la rigidité des biomatériaux de culture cellulaire, ont été reconnues comme un facteur important, guider le destin des cellules souches la prolifération et la différenciation1,2, 3,4,5,6. Par conséquent, plusieurs chercheurs ont commencé à enquêter sur le sort des cellules souches, qui sont cultivées sur des hydrogels, sur la différenciation, principalement à l’aide de polyacrylamide hydrogels avec raideur variable.
La rigidité des biomatériaux peut contrôler des adhérences focales, la morphologie cellulaire, phénotype cellulaire et l’adhérence de cellules souches, en particulier dans la culture à deux dimensions (2d)1,2,3,5. Mécano-détection des biomatériaux par les cellules souches est généralement contrôlée par adhérence focale signalisation via intégrines. NMMIIA, myosine non musculaire contractilité IIA-dépendante du cytosquelette d’actine joue un rôle essentiel dans le processus de mechanosensing des cellules souches en 2-D cell culture systèmes3,4,5, 7,8,9,10,11.
Engler et ses collègues ont développé une notion intéressante que les cellules souches adultes, comme la moelle osseuse des cellules de tige (BMS) cultivées sur les biomatériaux de culture cellulaire avec une rigidité similaire à celle des tissus spécifiques, ont tendance à se différencier en cellules provenaient de 5de tissus spécifiques. BMS cellules incubées sur polyacrylamide doux 2D hydrogels recouverts de collagène de type I (avec une rigidité comparable à celle des tissus cérébraux) dans les médias d’expansion ont été spontanément induites à se différencier en premières lignées de neurone, tandis que BMS cellules cultivées sur hydrogels avec une rigidité similaire à celle des tissus musculaires ou osseux collagénique trouvées pour induire la différenciation en premières lignées des myocytes et les ostéoblastes, respectivement, du 2D polyacrylamide hydrogels3,5. Plusieurs chercheurs ont étudié le devenir de cellules souches de la différenciation cultivés sur polyacrylamide hydrogels immobilisées avec le collagène de type I12,13,14,15,16 , 17 , 18 , 19 , 20 , 21. Toutefois, il convient de mentionner que certains contradictoires rapports1,18,22,23,24 existent pour la célèbre idée suggérée par Engler et al. 5 c’est parce qu’idée5 de Engler a été développé uniquement sur polyacrylamide hydrogels et leurs résultats provenir des caractéristiques particulières de ce biomatériau (polyacrylamide) et pas uniquement à partir de la file d’attente physique (rigidité) de ce biomatériau. Par conséquent, il est important de développer un autre type d’hydrogel, dont la rigidité peut être contrôlée par réticulation de l’hydrogel. À cette fin, les hydrogels bioinert ont été développés, qui ont été préparés de polyvinyle alcool-co-itaconique acide (P-IA) avec une rigidité différente, qui était contrôlée par le degré de réticulation avec une évolution réticulation temps25, 26 , 27 , 28 , 29 , 30 , 31 , 32. les cellules souches peuvent être cultivées sur ici P-IA hydrogels, ainsi que P-IA hydrogels greffés avec des matrices extracellulaires (ECMs) et oligopeptides. Dans une précédente étude25des cellules souches hématopoïétiques humaines (hHSCs) du sang de cordon ombilical ont été cultivés sur P-IA hydrogels avec des valeurs de rigidité différente allant d’un 3 kPa à 30 kPa module de stockage où la fibronectine ou un oligopeptide dérivé la fibronectine (CS1, EILDVPST) a été greffée sur les hydrogels P-IA. Haute ex vivo expansion pli de hHSCs a été observée chez les hydrogels P-IA greffées avec CS1 ou la fibronectine, qui affiche une rigidité intermédiaire allant de 12 kPa à 30 kPa25.
IPS humaines et cellules d’ES ne peut pas être cultivées sur culture de tissus classiques polystyrène (TCP) plats33,34 parce que ES humains et cellules iPS nécessitent une fixation spécifique aux SEGDA, tels que la vitronectine ou laminine pour maintenir leur pluripotence au cours de la culture à long terme. Par conséquent, plusieurs structures d’oligopeptide-greffés P-IA hydrogels présentant des caractéristiques de rigidité optimale ont été conçus et préparés dans les formations d’une seule chaîne, une chaîne unique avec un segment commun, une double chaîne avec un segment commun et un type ramifié 32de chaîne. Oligopeptide séquences ont été sélectionnés dans les domaines de liaison intégrine et glycosaminoglycanes d’ECMs. Les hydrogels P-IA greffés avec les oligopeptides dérivés de vitronectine avec une double chaîne ou un segment commun, d’un indice de stockage à environ 25 kPa, prise en charge de la culture à long terme de ES humains et de cellules iPS pour plus de 12 passages sous xeno-libre et chimique 32de conditions définies. Le segment mixte et double chaîne de molécules d’adhérence cellulaire sur les hydrogels a facilité la prolifération et la pluripotence des humains ES et iPS cellules32. Ici, un protocole pour la préparation des hydrogels P-IA (avec un module de stockage de 10 kPa à 30 kPa, ce qui a été mesurée dans des conditions humides dans l’air) greffées avec et sans les oligopeptides ou ECMs est décrite. Comment la culture et le passage de plusieurs cellules souches (y compris les cellules souches fluides amniotiques, adipeux dérivées de cellules souches, les cellules ES humaines et cellules iPS humaines) s’affiche.

<table>
	<tbody>
		<tr>
			<td>GTPGPQGIAGQRGVV</td>
			<td>PH Japan</td>
			<td>none</td>
			<td>Specification: Oligopeptide 
			Abbreviation: Cyclic RGD, cRGD</td>
		</tr>
		<tr>
			<td>GACRGDCLGA</td>
			<td>PH Japan</td>
			<td>none</td>
			<td>Specification: Oligopeptide 
			Abbreviation: FN1</td>
		</tr>
		<tr>
			<td>KGGAVTGRGDSPASS</td>
			<td>PH Japan</td>
			<td>none</td>
			<td>Specification: Oligopeptide 
			Abbreviation: CS1</td>
		</tr>
		<tr>
			<td>EILDVPST</td>
			<td>PH Japan</td>
			<td>none</td>
			<td>Specification: Oligopeptide 
			Abbreviation: VN1</td>
		</tr>
		<tr>
			<td>KGGPQVTRGDVFTMP</td>
			<td>PH Japan</td>
			<td>none</td>
			<td>Specification: Oligopeptide 
			Abbreviation: HBP1</td>
		</tr>
		<tr>
			<td>GKKQRFRHRNRKG</td>
			<td>PH Japan</td>
			<td>none</td>
			<td>Specification: Oligopeptide 
			Abbreviation: HBP2C</td>
		</tr>
		<tr>
			<td>CGGGKKQRFRHRNRKG</td>
			<td>PH Japan</td>
			<td>none</td>
			<td>Specification: Oligopeptide 
			Abbreviation: VN1G</td>
		</tr>
		<tr>
			<td>GGGGKGGPQVTRGDVFTMP</td>
			<td>PH Japan</td>
			<td>none</td>
			<td>Specification: Oligopeptide 
			Abbreviation: VN2C</td>
		</tr>
		<tr>
			<td>GCGGKGGPQVTRGDVFTMP</td>
			<td>PH Japan</td>
			<td>none</td>
			<td>Specification: Extracellular matrix 
			Abbreviation: rVN</td>
		</tr>
		<tr>
			<td>Vitronectin</td>
			<td>Thermo Fisher scientific</td>
			<td>A14700</td>
			<td>Specification: Extracellular matrix 
			Abbreviation: FN</td>
		</tr>
		<tr>
			<td>Fibronectin</td>
			<td>Sigma-Aldrich</td>
			<td>F2006</td>
			<td>Specification: Commercially available coating material 
			Abbreviation: Synthemax II</td>
		</tr>
		<tr>
			<td>Synthemax II</td>
			<td>Corning</td>
			<td>3535</td>
			<td>Specification: Polymer</td>
		</tr>
		<tr>
			<td>Polyvinylalcohol-co-itaconic acid</td>
			<td>Japan VAM &#38; Poval</td>
			<td>AF-17</td>
			<td>Specification: Chemical</td>
		</tr>
		<tr>
			<td>Glutaraldehyde</td>
			<td>Sigma-Aldrich</td>
			<td>G5882</td>
			<td>Specification: Chemical</td>
		</tr>
		<tr>
			<td>Na2SO4</td>
			<td>Sigma-Aldrich</td>
			<td>239313</td>
			<td>Specification: Chemical</td>
		</tr>
		<tr>
			<td>H2SO4</td>
			<td>Sigma-Aldrich</td>
			<td>339741</td>
			<td>Specification: Chemical 
			Abbreviation: EDC</td>
		</tr>
		<tr>
			<td>N-(3-Dimethylaminopropyl)-N&#39;-ethylcarbodiimide hydrochloride</td>
			<td>Sigma-Aldrich</td>
			<td>E7750</td>
			<td>Specification: Chemical 
			Abbreviation: NHS</td>
		</tr>
		<tr>
			<td>N-hydroxysuccinimide</td>
			<td>Sigma-Aldrich</td>
			<td>56480</td>
			<td>Specification: Chemical</td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>Sigma-Aldrich</td>
			<td>P6148</td>
			<td>Specification: Chemical</td>
		</tr>
		<tr>
			<td>Triton-X100</td>
			<td>Sigma-Aldrich</td>
			<td>T8787</td>
			<td>Specification: Cell culture consumable</td>
		</tr>
		<tr>
			<td>Cell scraper</td>
			<td>Corning</td>
			<td>3008</td>
			<td>Specification: Cell culture consumable</td>
		</tr>
		<tr>
			<td>Dispase II</td>
			<td>Sigma-Aldrich</td>
			<td>SI-D4693</td>
			<td>Specification: Cell culture medium</td>
		</tr>
		<tr>
			<td>Essential 6</td>
			<td>Thermo Fisher scientific</td>
			<td>A1516401</td>
			<td>Specification: Cell culture medium</td>
		</tr>
		<tr>
			<td>Essential 8</td>
			<td>Thermo Fisher scientific</td>
			<td>A1517001</td>
			<td>Specification: Cell culture medium</td>
		</tr>
		<tr>
			<td>DMEM/F12</td>
			<td>Thermo Fisher scientific</td>
			<td>11330-032</td>
			<td>Specification: Cell culture medium</td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Thermo Fisher scientific</td>
			<td>12800-017</td>
			<td>Specification: Cell culture medium</td>
		</tr>
		<tr>
			<td>MCDB 201</td>
			<td>Sigma-Aldrich</td>
			<td>M6770</td>
			<td>Specification: ES cell</td>
		</tr>
		<tr>
			<td>Human ES cell</td>
			<td>WiCell Research Institute, Inc..</td>
			<td>WA09</td>
			<td>Specification: iPS cell</td>
		</tr>
		<tr>
			<td>Human iPS cell</td>
			<td>Riken Cell Bank</td>
			<td>HS0077</td>
			<td>Specification: 35 mm 
			Abbreviation: TCP</td>
		</tr>
		<tr>
			<td>TCP dish</td>
			<td>Corning</td>
			<td>353001</td>
			<td>Specification: 60 mm 
			Abbreviation: TCP</td>
		</tr>
		<tr>
			<td>TCP dish</td>
			<td>Corning</td>
			<td>353002</td>
			<td>Specification: 24 well dish</td>
		</tr>
		<tr>
			<td>24 well dish</td>
			<td>Corning</td>
			<td>353047</td>
			<td>Specification: Blocking agent</td>
		</tr>
		<tr>
			<td>Bovine serum albumin</td>
			<td>Sigma-Aldrich</td>
			<td>A8806</td>
			<td>Specification: Detection reagent</td>
		</tr>
		<tr>
			<td>Alkaline phosphatase live stain</td>
			<td>Thermo Fisher scientific</td>
			<td>A14353</td>
			<td>Specification: Detection reagent</td>
		</tr>
		<tr>
			<td>Hematoxylin &#38; eosin</td>
			<td>Sigma-Aldrich</td>
			<td>1.05175</td>
			<td>Specification: EB formation dish</td>
		</tr>
		<tr>
			<td>6-well ultralow attachment dish</td>
			<td>Corning</td>
			<td>3471</td>
			<td>Specification: Coating material</td>
		</tr>
		<tr>
			<td>gelatin</td>
			<td>Sigma-Aldrich</td>
			<td>G9391</td>
			<td>Specification: Coating material</td>
		</tr>
		<tr>
			<td>Matrigel</td>
			<td>Corning</td>
			<td>354230</td>
			<td>Specification: Mice</td>
		</tr>
		<tr>
			<td>NOD-SCID mice</td>
			<td>National Applied Research Laboratories</td>
			<td>None</td>
			<td>Specification: Serum</td>
		</tr>
		<tr>
			<td>Fetal bovine serum</td>
			<td>Biological Industries</td>
			<td>04-001-1A</td>
			<td>Specification: Antibiotic</td>
		</tr>
		<tr>
			<td>antimycotic antibiotic</td>
			<td>Thermo Fisher scientific</td>
			<td>15240-062</td>
			<td>Specification: Antibody</td>
		</tr>
		<tr>
			<td>Antibody for Nanog</td>
			<td>Invitrogen</td>
			<td>MA1-017</td>
			<td>Specification: Antibody</td>
		</tr>
		<tr>
			<td>Antibody for SSEA4</td>
			<td>Abcam</td>
			<td>ab16287</td>
			<td>Specification: Antibody</td>
		</tr>
		<tr>
			<td>Antibody for OCT3/4</td>
			<td>Invitrogen</td>
			<td>PA5-27438</td>
			<td>Specification: Antibody</td>
		</tr>
		<tr>
			<td>Antibody for Sox2</td>
			<td>Invitrogen</td>
			<td>48-1400</td>
			<td>Specification: Antibody</td>
		</tr>
		<tr>
			<td>Antibody for Smooth Muscle Actin</td>
			<td>Invitrogen</td>
			<td>PA5-19465</td>
			<td>Specification: Antibody</td>
		</tr>
		<tr>
			<td>Antibody for AFP</td>
			<td>Invitrogen</td>
			<td>PA5-21004</td>
			<td>Specification: Antibody</td>
		</tr>
		<tr>
			<td>Antibody GFAP</td>
			<td>Invitrogen</td>
			<td>MA5-15086</td>
			<td>Specification: Antibody</td>
		</tr>
		<tr>
			<td>Alexa Fluor 555 &#8211; conjugated Goat anti-Mouse antibody</td>
			<td>Invitrogen</td>
			<td>A-21422</td>
			<td>Specification: Antibody</td>
		</tr>
		<tr>
			<td>Alexa Fluor 488 &#8211; conjugated Goat anti-Rabbit antibody</td>
			<td>Invitrogen</td>
			<td>A-11008</td>
			<td>Specification: Antibody</td>
		</tr>
	</tbody>
</table>

human pluripotent stem cell culture on polyvinyl alcohol-co-itaconic acid hydrogels with varying stiffness under xeno-free conditions

L’effet d’indices physiques, comme la rigidité des biomatériaux sur la prolifération et la différenciation des cellules souches, a été étudié par plusieurs chercheurs. Toutefois, la plupart de ces chercheurs ont utilisé des hydrogels de polyacrylamide pour culture de cellules souches dans leurs études. Par conséquent, leurs résultats sont controversés car ces résultats pourraient provenir de la spécificité de la polyacrylamide et non à partir de la file d’attente physique (rigidité) des biomatériaux. Nous décrivons ici un protocole pour la préparation des hydrogels, qui ne reposent pas sur polyacrylamide, où les différentes souches, les cellules dont les cellules souches embryonnaires humaines (ES) et humaines pluripotentes induites des cellules souches (iPS), peuvent être cultivées. Hydrogels avec raideur variable ont été préparés à partir bioinert, polyvinyl alcool-co-itaconique acide (P-IA), avec raideur, contrôlé par le degré de réticulation en changeant de temps de réticulation. Les hydrogels de P-IA greffées avec et sans les oligopeptides provenant de la matrice extracellulaire a été étudiées comme une future plate-forme de la culture de cellules souches et différenciation. La culture et le passage de cellules de tige fluides amniotique, adipeux dérivées de cellules souches, les cellules ES humaines et cellules iPS humaines est décrit en détail ici. Les hydrogels oligopeptide P-IA ont montré des performances supérieures, qui ont été induits par leurs propriétés de rigidité. Ce protocole indique la synthèse de ce biomatériau, manipulation de leur surface, ainsi que de contrôler les propriétés de rigidité et enfin, leur impact sur les cellules souches sort en utilisant des conditions de culture libre xeno. Se fondant sur des études récentes, ces substrats mis à jour le peuvent agir comme des futures plateformes pour soutenir et diriger le destin de diverses cellules souches ligne à liaisons différentes ; et encore, de se régénérer et de restaurer les fonctions de l’organe perdu ou un tissu.

P-IA hydrogels greffés avec oligopeptide dérivé ECM (oligoECM) ou ECM avec des élasticités différentes ont été préparés en suivant le schéma réactionnel, comme on le voit à la Figure 1 a, à l’aide de différents types d’oligoECM (Figure 1 b). Les élasticités de l’hydrogel étaient régies par l’intensité de la réticulation appliquée (temps) (Figure 1). P-IA hydrogels greff?...

Watch this Scientific Journal Video about Human Pluripotent Stem Cell Culture on Polyvinyl Alcohol-Co-Itaconic Acid Hydrogels with Varying Stiffness Under Xeno-Free Conditions at JoVE.com

Human Pluripotent Stem Cell Culture on Polyvinyl Alcohol-Co-Itaconic Acid Hydrogels with Varying Stiffness Under Xeno-Free Conditions

Un protocole est présenté afin de préparer le polyvinyliques alcool-co-itaconique acides hydrogels avec raideur variable, qui ont été greffés avec et sans les oligopeptides, pour étudier l’effet de la rigidité des biomatériaux sur la différenciation et la prolifération des cellules souches. La rigidité des hydrogels était contrôlée par le temps de réticulation.

Culture de cellules souches pluripotentes humaines sur Polyvinyl alcool-Co-itaconique acides Hydrogels avec raideur variable dans des Conditions sans Xeno

The effect of physical cues, such as the stiffness of biomaterials on the proliferation and differentiation of stem cells, has been investigated by ...

The overall goal of this experiment is to prepare either unmodified or oligopeptide or extra-cellular matrix-grafted polyvinyl alcohol-co-itaconic acid hydrogels with the optimal elasticity for culturing and passaging stem cells. This method can help answer key questions in the stem cell research field about long term content of stem cells under xeno-free conditions. The main advantage of this technique is that it provides a platform to investigate a different hydrogel elasticity on stem cell differentiation into specific lineages.Demonstrating the procedure will be me, Jia-Sin Yang and Yeh-Chia Tseng, graduate students from Professor Higuchi's laboratory. To begin the procedure, place 20 grams of P-IA and 200 milliliters of ethanol in a 500 milliliter conical flask. Stir the mixture for 24 to 30 hours, replacing the ethanol every eight to ten hours.Collect the purified P-IA on Buchner funnel. Dry the P-IA under vacuum at room temperature for 24 hours. Next, over the course of 15 minutes, slowly dissolve 15 milligrams of purified P-IA in 100 milliliters of ultra-pure water.Heat the solution to above 60 degrees Celsius and stir for one hour. Confirm that the solution has become clear before proceeding. Then, turn off the heat.Allow the P-IA solution to cool to room temperature on the cooling hot plate while stirring. Continue stirring the solution at room temperature for 48 hours. Then, leave the P-IA solution undisturbed at room temperature for 20 to 24 hours to ensure that visible air bubbles are not present in the solution.Next, dispense one milliliter of the P-IA solution into each of eight 35 milliliter tissue culture-treated dishes. Dry the dishes in an oven at 45 degrees Celsius for two days to produce P-IA films in the dishes. Next, add to 10 milliliters of ultra-pure water 100 microliters of a high-purity aqueous 25%glutaraldehyde solution, two grams of sodium sulfate and 100 microliters of sulfuric acid.Stir the mixture well to obtain the crosslinking solution. Gently add one milliliter of the crosslinking solution to each dish, being careful not to damage the fragile P-IA films. Leave each film immersed in the crosslinking solution for half, one, two, four, six, twelve, twenty four or forty eight hours to obtain the hydrogels.Rinse each hydrogel with one milliliter of ultra-pure water after its crosslinking period. Sterilize each hydrogel by immersion in an aqueous 75%ethanol solution for one minute. Rinse each sterilized hydrogel with ultrapure water six times.Cover the hydrogels in fresh ultra-pure water, place the lids on the dishes and store the hydrogel dishes in a clean area until ready for activation and grafting. Next, activate each sterilized hydrogel by immersion in one milliliter of an aqueous solution containing 10 milligrams per milliliter each of EDC and NHS, either for one hour at 37 degrees Celsius or four hours at four degrees Celsius. Then, rinse each activated hydrogel with one milliliter of Ph 7.2 phosphate-buffered saline three times.Immerse each hydrogel in one milliliter of a solution of either oligopeptides or ECMs in PBS for 24 hours at four degrees Celsius. Finally, wash each hydrogel with one milliliter of ultra-pure water three times after grafting is complete. Use standard method to culture human ES or IPS cells on a membrane matrix in six centimeter culture dishes until near confluent.Then, incubate the cells in DMMF-12 supplemented with two milligrams per milliliter of dispase-2 at 37 degrees Celsius for eight to ten minutes. Rinse the cells twice with DMMF-12. Add two milliliters of DMMF-12 to each culture dish and attach weekly adherent colonies with the pipette or cell scraper.Collect the cells in 15 milliliter centrifuge tubes and centrifuge the cells at 160 times G for five minutes at 37 degrees Celsius. Discard the supernatant and re-suspend the cells in one milliliter of xeno-free eight component medium. Count the cell density in the suspension.Then, obtain culture dishes or multi-well plates containing sterilized P-IA hydrogels grafted with either oligopeptides or ECMs. Load the appropriate volumes of the cell suspension onto the hydrogels. Passage the cells on grafted P-IA hydrogels until 80%confluent the desired number of times.To begin the immuno-staining process for a 24 well plate of passaged human ES or IPS cells on grafted hydrogels, add 0.5 milliliters of four per cent paraformaldahyde by volume to each well. Incubate the cells at four degrees Celsius for 15 minutes to fix the cells. Aspirate the paraformeldehyde solution from the wells.Add one milliliter of PBS to each well and aspirate the PBS. Perform this PBS rinse three times in total. Then, add 0.5 milliliters of 0.3%octoxynol 9 by volume in PBS to each well.Incubate for 30 minutes at room temperature. Aspirate the octoxynol 9 solution. Add 300 microliters of two per cent BSA and PBS to each well and incubate for another 30 minutes at room temperature.Aspirate the two per cent BSA solution from the cells and add the primary antibodies. Incubate the cells for one day at four degrees Celsius. Then, aspirate the primary antibody solutions and wash the cells with 300 microliters of 05 per cent polysorbate 20 in PBS three times.Add 300 microliters of secondary antibodies specific to the primary IDG subtype in two per cent BSA to each well. Incubate the cells for one hour at room temperature in the absence of light. Finally, wash the cells with 300 microliters of 05 per cent polysorbate 20 and PBS three times in the dark.Image the stained cells with fluorescence microscopy. P-IA hydrogels crosslinked for 24 hours and grafted with vitronectin-derived oligopeptides, supported cultures of human ES and IPS cells for at least 10 to 20 passages. Human ESLs cultured on commercially available coated dishes were found to be more easily differentiated at the edges of the colonies.Differentiated cells were not observed on the hydrogels, indicating that human ESLs maintained their pluripotency. Pluripotency proteins were satisfactorily expressed on human ES cells cultured on hydrogels grafted with three different Vitronectin-derived oliogopeptide designs for ten passages. Satisfactory expression was also observed on recomibinent Vitronectin-coated dishes.Similar results were observed for human IPS cells. Both human ES and IPS cells differentiated into cells derived from all three germ layers after cultivation on P-IA hydrogels and on recombinant Vitronectin-coated dishes for at least ten passages. Human ES cells cultivated on hydrogels grafted with Vitronectin-derived oligopeptides were injected into NOD mice.The harvested teratomas exhibited cells originating from all three germ layers indicating that pluripotency is maintained in vivo. Following this procedure, methods like media screening can be performed to answer additional questions like the effect of calcium arterial and stem cell differentiation and proliferation. After this development, this technique paved the way for researchers in the field of stem cell co-division to explore tissue or organ regeneration in neurodegenerative medicine.Once mastered, this technique can be done in four days if it's performed properly. After watching this video, you should have a good understanding of how to prepare P-IA hydrogel dishes, both with and without ECM. While attempting this procedure, remember to set up a clean environment for preparing P-IA hydrogel dishes.Don't forget that working with ethanol, hot water and the sulfuric acid can be extremely hazardous. The appropriate lab coat, gloves and the protective glasses should always be worn while performing this procedure.

Research

JoVE Journal

Bioengineering

Development, Expansion, and In vivo Monitoring of Human NK Cells from Human Embryonic Stem Cells (hESCs) and Induced Pluripotent Stem Cells (iPSCs)

Development, Expansion, and In vivo Monitoring of Human NK Cells from Human Embryonic Stem Cells hESCs and Induced Pluripotent Stem Cells iPSCs

Le développement, l&#39;expansion et<em&gt; In vivo</em&gt; Suivi des cellules NK humaines à partir de cellules souches embryonnaires humaines (CSEh) et et induit des cellules souches pluripotentes (CISP)

We present a method for deriving natural killer (NK) cells from undifferentiated hESCs and iPSCs using a feeder-free approach. This method gives rise to ...

Recherche

Bio-ingénierie

Tunable Hydrogels from Pulmonary Extracellular Matrix for 3D Cell Culture

Accordable hydrogels de matrice extracellulaire pulmonaire pour la 3D Culture Cellulaire

Here we present a method for establishing multiple component cell culture hydrogels for in vitro lung cell culture. Beginning with healthy en bloc lung ...

Hyaluronic-Acid Based Hydrogels for 3-Dimensional Culture of Patient-Derived Glioblastoma Cells

L’acide hyaluronique basée Hydrogels pour 3-Dimensional Culture de cellules de glioblastome dérivé de Patient

Glioblastoma (GBM) is the most common, yet most lethal, central nervous system cancer. In recent years, many studies have focused on how the extracellular ...

Guided Differentiation of Mature Kidney Podocytes from Human Induced Pluripotent Stem Cells Under Chemically Defined Conditions

Différenciation guidée des podocytes rénaux matures à partir de cellules souches pluripotentes induites par l’homme dans des conditions chimiquement définies

Kidney disease affects more than 10% of the global population and costs billions of dollars in federal expenditures. The most severe forms of kidney ...

Controlled Strain of 3D Hydrogels under Live Microscopy Imaging

Souche contrôlée d’hydrogels 3D sous imagerie par microscopie dynamique

External forces are an important factor in tissue formation, development, and maintenance. The effects of these forces are often studied using specialized ...

Single-Cell Optical Action Potential Measurement in Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes

Mesure du potentiel d’action optique unicellulaire dans les cardiomyocytes dérivés de cellules souches pluripotentes induites par l’homme

Conventional intracellular microelectrode techniques to quantify cardiomyocyte electrophysiology are extremely complex, labor intensive, and typically ...

A Culture Method to Maintain Quiescent Human Hematopoietic Stem Cells

Une méthode de culture pour maintenir les cellules souches hématopoïétiques humaines quiescentes

Human hematopoietic stem cells (HSCs), like other mammalian HSCs, maintain lifelong hematopoiesis in the bone marrow. HSCs remain quiescent in vivo, ...

Microfluidic Fabrication of Core-Shell Microcapsules carrying Human Pluripotent Stem Cell Spheroids

Fabrication microfluidique de microcapsules noyau-coquille transportant des sphéroïdes de cellules souches pluripotentes humaines

Three-dimensional (3D) or spheroid cultures of human pluripotent stem cells (hPSCs) offer the benefits of improved differentiation outcomes and ...

Isogenic Kidney Glomerulus Chip Engineered from Human Induced Pluripotent Stem Cells

Puce de glomérule rénal isogénique conçue à partir de cellules souches pluripotentes induites par l’homme

Chronic kidney disease (CKD) affects 15% of the U.S. adult population, but the establishment of targeted therapies has been limited by the lack of ...

Generation of Human Blood Vessel Organoids from Pluripotent Stem Cells

Génération d’organoïdes de vaisseaux sanguins humains à partir de cellules souches pluripotentes

An organoid is defined as an engineered multicellular in vitro tissue that mimics its corresponding in vivo organ such that it can be used to study ...