Name: isolation and culture of neural crest stem cells from human hair follicles
Uploaded: 2013-04-06T14:00:00
Description: Watch this Scientific Journal Video about Isolation and Culture of Neural Crest Stem Cells from Human Hair Follicles at JoVE.com

Dette arbejde understøttes af NIH tilskud R01AR054593 og R01AR054593-S1 til Xu.

1. Fremstilling af vævskulturplader <ol><li> Coat hver brønd med tilstrækkelig Poly-D-Lysin (PDL) til at dække bunden af ​​brønden. Tillade pladerne at tørre i stinkskab. </li><li> Efter at brøndene er tørre, skylles med sterilt vand, og der opsuges. Tillade pladerne at tørre i stinkskab. </li><li> Når de er tørre, pels med fibronectin (der blev opløst i BioWhittaker vand natten over ved 37 ° C ved en koncentration på 1 mg i 6 ml). </li><li> Tilføj NCSC medium [95 ml DMEM/F12, 1 ml Penn / Strep (P /...

<ol>
	<li>Cotsarelis, G., Cheng, S. Z., Dong, G., Sun, T. T., Lavker, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Existence+of+slow-cycling+limbal+epithelial+basal+cells+that+can+be+preferentially+stimulated+to+proliferate:+implications+on+epithelial+stem+cells.">Existence of slow-cycling limbal epithelial basal cells that can be preferentially stimulated to proliferate: implications on epithelial stem cells.</a> Cell. 57, 201-209 (1989).</li><li>Cotsarelis, G., Sun, T. T., Lavker, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Label-retaining+cells+reside+in+the+bulge+area+of+pilosebaceous+unit:+implications+for+follicular+stem+cells,+hair+cycle,+and+skin+carcinogenesis.">Label-retaining cells reside in the bulge area of pilosebaceous unit: implications for follicular stem cells, hair cycle, and skin carcinogenesis.</a> Cell. 61, 1329-1337 (1990).</li><li>Tanimura, 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=Hair+follicle+stem+cells+provide+a+functional+niche+for+melanocyte+stem+cells.">Hair follicle stem cells provide a functional niche for melanocyte stem cells.</a> Cell Stem Cell. 8, 177-187 (2011).</li><li>Yu, 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=Isolation+of+a+novel+population+of+multipotent+adult+stem+cells+from+human+hair+follicles.">Isolation of a novel population of multipotent adult stem cells from human hair follicles.</a> Am. J. Pathol. 168, 1879-1888 (2006).</li><li>Yu, H., Kumar, S. M., Kossenkov, A. V., Showe, L., Xu, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stem+cells+with+neural+crest+characteristics+derived+from+the+bulge+region+of+cultured+human+hair+follicles.">Stem cells with neural crest characteristics derived from the bulge region of cultured human hair follicles.</a> J. Invest. Dermatol. 130, 1227-1236 (2010).</li><li>Wong, C. 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=Neural+crest-derived+cells+with+stem+cell+features+can+be+traced+back+to+multiple+lineages+in+the+adult+skin.">Neural crest-derived cells with stem cell features can be traced back to multiple lineages in the adult skin.</a> J. Cell Biol. 175, 1005-1015 (2006).</li><li>Sieber-Blum, M., Grim, M., Hu, Y. F., Szeder, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pluripotent+neural+crest+stem+cells+in+the+adult+hair+follicle.">Pluripotent neural crest stem cells in the adult hair follicle.</a> Dev. Dyn. 231, 258-269 (2004).</li><li>Amoh, 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=Implanted+hair+follicle+stem+cells+form+Schwann+cells+that+support+repair+of+severed+peripheral+nerves.">Implanted hair follicle stem cells form Schwann cells that support repair of severed peripheral nerves.</a> Proc. Natl. Acad. Sci. U.S.A. 102, 17734-17738 (2005).</li><li>Koliatsos, V. E., Xu, L., Yan, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+stem+cell+grafts+as+therapies+for+motor+neuron+disease.">Human stem cell grafts as therapies for motor neuron disease.</a> Expert Opin. Biol. Ther. 8, 137-141 (2008).</li><li>McKenzie, I. A., Biernaskie, J., Toma, J. G., Midha, R., Miller, F. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Skin-derived+precursors+generate+myelinating+Schwann+cells+for+the+injured+and+dysmyelinated+nervous+system.">Skin-derived precursors generate myelinating Schwann cells for the injured and dysmyelinated nervous system.</a> J. Neurosci. 26, 6651-6660 (2006).</li><li>LeDouarin, N. M., Kalcheim, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The Neural Crest. , (1999).</li><li>Tumbar, T., 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=Defining+the+epithelial+stem+cell+niche+in+skin.">Defining the epithelial stem cell niche in skin.</a> Science. 303, 359-363 (2004).</li><li>Roh, 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=Multi-potentiality+of+a+new+immortalized+epithelial+stem+cell+line+derived+from+human+hair+follicles.">Multi-potentiality of a new immortalized epithelial stem cell line derived from human hair follicles.</a> In Vitro Cell Dev. Biol. Anim. 44, 236-244 (2008).</li></ol>

De celleisolering og kultur beskrevne metoder er reproducerbare og robust. Vi har genereret NCSCs fra snesevis af enkeltpersoner på tværs af en bred aldersgruppe. Selv om det er bedst at behandle vævet lige efter tissue høst, fandt vi, at hovedbunden væv kan opbevares i medier på is natten over transport med minimal indvirkning på cellelevedygtighed. Det er vigtigt at behandle hovedbunden væv med antibiotika og bruge aseptisk teknik under hårsækken isolation for at undgå potentiel...

<table border="1">
 <tbody>
 <tr>
 <td>Name of the reagent</td>
 <td>Company</td>
 <td>Catalogue number</td>
 </tr>
 <tr>
 <td>DMEM</td>
 <td>Invitrogen</td>
 <td>11965-092</td>
 </tr>
 <tr>
 <td>DMEM/F12</td>
 <td>Invitrogen</td>
 <td>11330-32</td>
 </tr>
 <tr>
 <td>Heat-inactivated FBS</td>
 <td>Hyclone</td>
 <td>SH30071.03</td>
 </tr>
 <tr>
 <td>B27 supplement</td>
 <td>Invitrogen</td>
 <td>17504044</td>
 </tr>
 <tr>
 <td>N2 supplement</td>
 <td>Invitrogen</td>
 <td>17502048</td>
 </tr>
 <tr>
 <td>bFGF</td>
 <td>Invitrogen</td>
 <td>PHG0026</td>
 </tr>
 <tr>
 <td>EGF</td>
 <td>R&amp;D system</td>
 <td>236-EG-01M</td>
 </tr>
 <tr>
 <td>IGF-I</td>
 <td>R&amp;D system</td>
 <td>291-G1-050</td>
 </tr>
 <tr>
 <td>0.05% Trypsin/EDTA</td>
 <td>Invitrogen</td>
 <td>25300-054</td>
 </tr>
 <tr>
 <td>Dispase</td>
 <td>Invitrogen</td>
 <td>17105041</td>
 </tr>
 <tr>
 <td>Penicillin-Streptomycin</td>
 <td>Invitrogen</td>
 <td>15070063</td>
 </tr>
 </tbody> 
</table>
Table 1.

isolation and culture of neural crest stem cells from human hair follicles

Hårsækkene undergår livslang vækst og hår cyklus er en velkontrolleret proces, der involverer stamcelleproliferation og hviletilstand. Hår bule er en velkarakteriseret niche for voksne stamceller 1. Dette segment af det ydre root sheath indeholder en række forskellige typer stamceller, herunder epitelceller stamceller 2, melanocyt stamceller 3 og crista neuralis ligesom stamceller 4-7. Hårsækkene repræsenterer en tilgængelig og rig kilde til forskellige typer af humane stamceller. Vi og andre har isoleret neural crest stamceller (NCSCs) fra humane føtale og voksne hårsækkene 4,5. Disse humane stamceller er label-fastholde celler og er i stand til selvfornyelse gennem asymmetrisk celledeling in vitro. De udtrykker umodne neural crest cellemarkører, men ikke differentieringsmarkører. Vores udtryk profilering undersøgelse viste, at de har et lignende genekspressionsmønster med murin hud umodne neurale crest cells. De udviser klonal multipotency, der kan give anledning til myogene, melanocytisk, og neuronale cellelinier efter in vitro klonal enkelt cellekultur. Differentierede celler ikke kun erhverve slægt-specifikke markører, men også demonstrere relevante funktioner i ex vivo-betingelser. Desuden viser disse NCSCs differentieringspotentiale mod mesenchymale slægter. Differentierede neuronale celler kan forblive i musehjerne og fastholde neuronale differentieringsmarkører. Det er blevet vist, at hårsækken afledt NCSCs kan hjælpe nerve genvækst, og de ​​forbedrer motorisk funktion hos mus transplanteret med disse stamceller efter transecting rygmarvsskade 8. Endvidere har perifere nerver blevet repareret med stamceller transplantater 9 og implantation af hud-afledte precursorceller støder op til knust ischiadicus nerver har resulteret i remyelinisering 10. Derfor har de hårsækken / hud afledte NCSCs allerede vist lovende resultater for regenerative terapi i prækliniske modeller. Somatiske celler omprogrammering til induceret pluripotente stamceller (iPS) celler har vist et enormt potentiale for regenerativ medicin. Men der er stadig mange problemer med iPS celler, især langtidseffekten af ​​onkogen / virus integration og potentiel tumorgenicitet af pluripotente stamceller ikke er blevet korrekt behandlet. Der er stadig mange forhindringer, der skal overvindes, før iPS-celler kan bruges til regenerativ medicin. Henviser til voksne stamceller vides at være sikre, og de er blevet anvendt klinisk i mange år, såsom knoglemarvstransplantation. Mange patienter har allerede nydt godt af den behandling. Autologe adulte stamceller stadig foretrukne celler til transplantation. Derfor er de let tilgængelige og kan udvides voksne stamceller i huden / håret hårsække er en værdifuld kilde til regenerativ medicin.

Watch this Scientific Journal Video about Isolation and Culture of Neural Crest Stem Cells from Human Hair Follicles at JoVE.com

Isolation and Culture of Neural Crest Stem Cells from Human Hair Follicles

Denne artikel præsenterer en robust protokol for isolering og dyrkning af neurale crest stamceller fra menneskelige hårsækkene.

Isolering og Kultur neuralkam stamceller fra menneskelige hårsække

Hair follicles undergo lifelong growth and hair cycle is a well-controlled process involving stem cell proliferation and quiescence. Hair bulge is a ...

Research

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