Name: rapid fibroblast removal from high density human embryonic stem cell cultures
Uploaded: 2012-10-28T16:00:00
Description: Watch this Scientific Journal Video about Rapid Fibroblast Removal from High Density Human Embryonic Stem Cell Cultures at JoVE.com

Dette arbejde blev finansieret af et nyt fakultet Award II fra California Institute of Regenerative Medicine (RN2-00.921-1), og en NIH-finansieret National Research Award (F32-HL104924)

1. Fremstilling af muse embryonale Feeders <ol><li> The MEF&#39;er anvendt i co-kultur af hESCs bør tidligere behandlet ved bestråling eller mitomycin-C at inhibere cellerne i at undergå deling. </li><li> To timer før MEF såning, belægge hver 60 mm plasma behandlet dyrkningsskål med 2 ml 0,05% gelatine. </li><li> Optø et hætteglas med behandlet MEF&#39;er og plade på ~ 20.000 celler / cm 2. </li><li> Tillader cellerne at adhærere natten over. </li></ol> 2. Co-seeding hESCS o...

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	<li>Bongso, A., Fong, C. Y., Ng, S. C., Ratnam, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+culture+of+inner+cell+mass+cells+from+human+blastocysts.">Isolation and culture of inner cell mass cells from human blastocysts.</a> Hum. Reprod. 9, 2110-2117 (1994).</li><li>Reubinoff, B. E., Pera, M. F., Fong, C. Y., Trounson, A., Bongso, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Embryonic+stem+cell+lines+from+human+blastocysts:+somatic+differentiation+in+vitro.">Embryonic stem cell lines from human blastocysts: somatic differentiation in vitro.</a> Nat. Biotechnol. 18, 399-404 (2000).</li><li>Thomson, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Embryonic+stem+cell+lines+derived+from+human+blastocysts.">Embryonic stem cell lines derived from human blastocysts.</a> Science. 282, 1145-1147 (1998).</li><li>Thomas, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Automated,+scalable+culture+of+human+embryonic+stem+cells+in+feeder-free+conditions.">Automated, scalable culture of human embryonic stem cells in feeder-free conditions.</a> Biotechnol Bioeng. 102, 1636-1644 (2009).</li><li>Kolhar, P., Kotamraju, V. R., Hikita, S. T., Clegg, D. O., Ruoslahti, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synthetic+surfaces+for+human+embryonic+stem+cell+culture.">Synthetic surfaces for human embryonic stem cell culture.</a> J. Biotechnol. 146, 143-146 (2010).</li><li>Hovatta, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+culture+system+using+human+foreskin+fibroblasts+as+feeder+cells+allows+production+of+human+embryonic+stem+cells.">A culture system using human foreskin fibroblasts as feeder cells allows production of human embryonic stem cells.</a> Hum. Reprod. 18, 1404-1409 (2003).</li><li>McKay, T. R. <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+cell+line+for+derivation+and+culture+of+hESc/hiPSc.">Human feeder cell line for derivation and culture of hESc/hiPSc.</a> Stem Cell Res. 7, 154-162 (2011).</li><li>Xu, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Feeder-free+growth+of+undifferentiated+human+embryonic+stem+cells.">Feeder-free growth of undifferentiated human embryonic stem cells.</a> Nat. Biotechnol. 19, 971-974 (2001).</li><li>Stein, G. S., Shi, M. J., Stencel, K., Borowski, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Thawing, Seeding, and Changing Medium of Human Embryonic Stem Cells. , (2010).</li><li>Schwartz, P. H., Brick, D. J., Nethercott, H. E., Stover, A. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Traditional+human+embryonic+stem+cell+culture.">Traditional human embryonic stem cell culture.</a> Methods Mol. Biol. 767, 107-123 .</li></ol>

Fremgangsmåden præsenteres i dette manuskript tilbyder en hurtig og mindre kostbart alternativ til at fjerne fibroblast-feeder-celler fra humane embryoniske cellekulturer. Den vellykkede fjernelse af fibroblaster er afhængig af tilstedeværelsen af ​​en tæt konfluent monolag af disse celler, der udvikler sig i længere stamcellekulturer. Efter 7-10 dage vil de voksende hESC kolonier skubbe feeder fibroblaster i en udadgående retning, genererer en stadig tæt fibroblast monolag mellem kolonier. De stærke celle-...

<table border="1"><tbody><tr><td> Navn på reagenset </td><td> Firma </td><td> Katalognummer </td><td> Kommentarer (valgfrit) </td></tr><tr><td> DDR2 </td><td> Santa Cruz </td><td> 7555 </td><td> Fibroblast Marker </td></tr><tr><td> DAPI </td><td> Calbiochem </td><td> 268.298 </td><td> Cellekerne markør </td></tr><tr><td> SSEA-4 </td><td> Millipore </td><td> MAB4304 </td><td> Humant ESC Marker </td></tr><tr><td> Oct 3/4 </td><td> Santa Cruz </td><td> 9081 </td><td> Humant ESC Marker </td></tr><tr><td> FLK-1 </td><td> BD Pharma </td><td> 555.307 </td><td> Tidlig differentieringsmarkør </td></tr><tr><td> Tra-1-81 </td><td> Acris </td><td> AM20377AF4-S </td><td> Humant ESC Marker </td></tr></tbody></table> Tabel 1. Table af specifik REAgents anvendes til immunfarvning af hESC.

rapid fibroblast removal from high density human embryonic stem cell cultures

Muse embryonale fibroblaster (MEF) blev anvendt til at etablere humane embryonale stamceller (hESCs) kulturer efter blastocyst isolering 1. Denne feeder system opretholder hESCs fra undergår spontan differentiering under celleekspansion. Men denne co-dyrkningsmetode er arbejdskrævende, kræver højt uddannet personale, og renterne lave hESC renhed 4. Mange laboratorier har forsøgt at minimere antallet af fødeceller i hESC kulturer (dvs. inkorporering matrix-coatede skåle eller andre feeder celletyper 5-8). Disse modificerede kultursystemer har vist nogle lovende, men har ikke fortrængt standard fremgangsmåde til dyrkning hESCs med mitomycin C-behandlede muse embyronic fibroblaster for at forsinke uønsket spontan differentiering af hESC kulturer. Derfor bør feeder celler anvendt i hESC ekspansion fjernes under differentiering eksperimenter. Skønt adskillige teknikker er tilgængelige til oprensning af hESC colonies (FACS, Mac, eller brug af resistente vektorer) fra foderautomater, disse teknikker er arbejdskrævende, omkostningskrævende og / eller ødelæggende for hESC. Formålet med dette projekt var at opfinde en metode til oprensning, der muliggør høst af en renere population af hESCs. Vi har observeret, at i en konfluent hESC kultur kan MEF populationen fjernes med en enkel og hurtig aspiration af MEF arket. Denne fjernelse er afhængig af flere faktorer, herunder lateral celle-til-celle-binding af MEF&#39;er, som har en lavere bindingsaffinitet til styren dyrkningsskål, og evnen hos stammen cellekolonier at skubbe fibroblasterne udad under dannelsen af ​​deres egen &quot; niche &quot;. The hESC blev derefter undersøgt for SSEA-4, Oct3 / 4 og Tra 1-81 ekspression op til 10 dage efter MEF fjernelse for at sikre opretholdelse af pluripotency. Desuden hESC kolonier var i stand til at fortsætte med at vokse fra i større formationer efter MEF fjernelse, hvilket giver et ekstra niveau af hESC ekspansion.

Watch this Scientific Journal Video about Rapid Fibroblast Removal from High Density Human Embryonic Stem Cell Cultures at JoVE.com

Rapid Fibroblast Removal from High Density Human Embryonic Stem Cell Cultures

Trods den stadige bestræbelser på at overgangen kulturer til feeder-fri betingelser, forbliver afledning og dyrkning af humane embryonale stamceller (hESC) i høj grad afhængig af co-kulturer med muse embryonale foderautomater (MEF). Her viser vi en ny metode til hurtigt at fjerne foderautomater fra hESC kulturer før eksperimenteren.

Hurtig Fibroblast fjernelse fra High Density humane embryonale stamceller Cultures

Mouse embryonic fibroblasts (MEFs) were used to establish human embryonic stem cells (hESCs) cultures after blastocyst isolation1. This feeder system ...

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