Name: hypoxic preconditioning of marrow-derived progenitor cells as a source for the generation of mature schwann cells
Uploaded: 2017-06-14T13:00:00
Description: Watch this Scientific Journal Video about Hypoxic Preconditioning of Marrow-derived Progenitor Cells As a Source for the Generation of Mature Schwann Cells at JoVE.com

Forfatterne vil gjerne anerkjenne Dr. Nai-Sum Wong for å gi hypoxiekammerapparatet og fru Alice Lui for den tekniske støtten.

Alle prosedyrer som involverer dyr ble utført i henhold til NIHs retningslinjer for pleie og bruk av laboratoriedyr og godkjent av komiteen for bruk av levende dyr for undervisning og forskning, Li Ka Shing fakultet for medisin, universitetet i Hong Kong. Humane beinmargeprøver ble oppnådd fra iliackampen av sunne givere etter å ha oppnådd informert samtykke. Protokoller ble godkjent av Institutional Review Board, University of Hong Kong. 1. Fremstilling av rotte-MSC-kulturer <ol><li>...

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	<li>Wiliams, R. R., Bunge, M. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Schwann+cell+transplantation:+a+repair+strategy+for+spinal+cord+injury?.">Schwann cell transplantation: a repair strategy for spinal cord injury?.</a> Prog Brain Res. 201, 295-312 (2012).</li><li>Kanno, H., Pearse, D. D., Ozawa, H., Itoi, E., Bunge, M. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Schwann+cell+transplantation+for+spinal+cord+injury+repair:+its+significant+therapeutic+potential+and+prospectus.">Schwann cell transplantation for spinal cord injury repair: its significant therapeutic potential and prospectus.</a> Rev Neurosci. 26 (2), 121-128 (2015).</li><li>Lindvall, O., Kokaia, Z. <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+in+human+neurodegenerative+disorders--time+for+clinical+translation?.">Stem cells in human neurodegenerative disorders--time for clinical translation?.</a> J Clin Invest. 120 (1), 29-40 (2010).</li><li>Terzic, 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=Directed+Differentiation+of+Oligodendrocyte+Progenitor+Cells+From+Mouse+Induced+Pluripotent+Stem+Cells.">Directed Differentiation of Oligodendrocyte Progenitor Cells From Mouse Induced Pluripotent Stem Cells.</a> Cell Transplant. 25 (2), 411-424 (2016).</li><li>Takashima, 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=Neuroepithelial+cells+supply+an+initial+transient+wave+of+MSC+differentiation.">Neuroepithelial cells supply an initial transient wave of MSC differentiation.</a> Cell. 129 (7), 1377-1388 (2007).</li><li>Felling, R. 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=Neural+stem/progenitor+cells+participate+in+the+regenerative+response+to+perinatal+hypoxia/ischemia.">Neural stem/progenitor cells participate in the regenerative response to perinatal hypoxia/ischemia.</a> J Neurosci. 26 (16), 4359-4369 (2006).</li><li>Shea, G. K., Tsui, A. Y., Chan, Y. S., Shum, D. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bone+marrow-derived+Schwann+cells+achieve+fate+commitment--a+prerequisite+for+remyelination+therapy.">Bone marrow-derived Schwann cells achieve fate commitment--a prerequisite for remyelination therapy.</a> Exp Neurol. 224 (2), 448-458 (2010).</li><li>Jessen, K. R., Mirsky, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+origin+and+development+of+glial+cells+in+peripheral+nerves.">The origin and development of glial cells in peripheral nerves.</a> Nat Rev Neurosci. 6 (9), 671-682 (2005).</li><li>Mung, K. 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=Rapid+and+efficient+generation+of+neural+progenitors+from+adult+bone+marrow+stromal+cells+by+hypoxic+preconditioning.">Rapid and efficient generation of neural progenitors from adult bone marrow stromal cells by hypoxic preconditioning.</a> Stem Cell Res Ther. 7 (1), 146 (2016).</li><li>Ao, Q., 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+regeneration+of+transected+sciatic+nerves+of+adult+rats+using+chitosan+nerve+conduits+seeded+with+bone+marrow+stromal+cell-derived+Schwann+cells.">The regeneration of transected sciatic nerves of adult rats using chitosan nerve conduits seeded with bone marrow stromal cell-derived Schwann cells.</a> Biomaterials. 32 (3), 787-796 (2011).</li><li>Tondreau, 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=Isolation+of+BM+mesenchymal+stem+cells+by+plastic+adhesion+or+negative+selection:+phenotype,+proliferation+kinetics+and+differentiation+potential.">Isolation of BM mesenchymal stem cells by plastic adhesion or negative selection: phenotype, proliferation kinetics and differentiation potential.</a> Cytotherapy. 6 (4), 372-379 (2004).</li><li>Baksh, D., Song, L., Tuan, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adult+mesenchymal+stem+cells:+characterization,+differentiation,+and+application+in+cell+and+gene+therapy.">Adult mesenchymal stem cells: characterization, differentiation, and application in cell and gene therapy.</a> J Cell Mol Med. 8 (3), 301-316 (2004).</li><li>Jirsova, K., Sodaar, P., Mandys, V., Bar, P. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cold+jet:+a+method+to+obtain+pure+Schwann+cell+cultures+without+the+need+for+cytotoxic,+apoptosis-inducing+drug+treatment.">Cold jet: a method to obtain pure Schwann cell cultures without the need for cytotoxic, apoptosis-inducing drug treatment.</a> J. Neurosci. Methods. 78 (1-2), 133-137 (1997).</li></ol>

Det er viktig å opprettholde &quot;stivhet&quot; av MSC før anrikning av nevrale progenitorer via hypoksisk forbehandling og neurosfærekultur. Fra vår erfaring kan multipotente MSCs pålitelig identifiseres av deres langstrakte fibroblastlignende morfologi. I motsetning til dette, har MSC som har vedtatt en mer flatt, firkantet morfologi, med fremtredende cytoskeletale stressfibre, ikke lett å vedta nevrale cellefeller og bør kasseres. Generelt bruker vi ikke MSCer med passasjerer større enn åtte. For å bevare ...

Transplantasjonen av nevrale progenitorer og deres derivater demonstrerer løfte som en behandlingsstrategi etter traumatisk nerveskade 1 , 2 og nevrogenerasjon 3 , 4 . Før klinisk bruk er det viktig å sikre: i) en metode for tilgang og utvidelse av en autolog kilde til stamme / stamceller og ii) et middel for å lede dem til relevante modne celletyper 3 . Vår interesse for celleterapi for ryggmargenskade førte oss til å søke en robust, autolog cellekilde av nevrale progenitorer fra voksenvev. En subpopulasjon av MSCs stammer fra nevralkampen og er lett tilgjengelig fra marvhulen. Disse cellene er nevrale progenitorer som kan generere nevroner og glia 5 . Dyrmodeller av cerebral iskemi viser at hypoksi fremmer progresjonen<html> Iferasjon og multipotens av nevrale progenitorer i hjernen 6 . Dette var grunnlaget for å utnytte hypoksisk forkjøling som et middel til å ekspandere på margenavledede nevrale progenitorer. Transplantasjonen av Schwann-celler i den skadede ryggraden fremmer regenerering 2 . SCLC kan genereres fra MSCs ved hjelp av tilskudd med gliogene faktorer ( dvs. p-heregulin, bFGF og PDGF-AA), men demonstrerer fenotypisk ustabilitet. Ved tilbaketrekking av vekstfaktorer vender de tilbake til en fibroblastlignende fenotype 7 . Fenotypisk ustabilitet er uønsket ved celletransplantasjon på grunn av risikoen for avvikende differensiering og karsinogenese. Som Schwann-celleprecursorer er assosiert med axonbunter innenfor den embryonale perifere nerve 8 , ble vi ført til coculture-SCLCer med rensede embryonale DRG-neuroner 7 ,Ass = &quot;xref&quot;&gt; 9. Resulterende modne Schwann-celler er skjebneforpliktet og demonstrerer funksjon in vitro 7 , 9 og in vivo 10 . Vår protokoll for berikelse av nevrale progenitorer fra MSC er enkel og effektiv og resulterer i en økning i celle nummer for etterfølgende analyser. Avledningen av skjebnebegrensede Schwann-celler via coculture-plattformen gjør det mulig å studere glialdifferensiering og for generering av stabile og funksjonelle Schwann-celler for potensiell klinisk anvendelse.

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			<td>Sigmaaldrich</td>
			<td>P7886-1G</td>
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			<td>15140-122</td>
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			<td>TPP</td>
			<td>92006</td>
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			<td height="20" style="height:20px;">UltraLow 6-well plate for non-adherent culture</td>
			<td>Corning</td>
			<td>3471</td>
			<td>Used for neural progenitor enrichment</td>
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			<td height="20" style="height:20px;">anti-human CD90(Thy-1)</td>
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			<td>555593</td>
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			<td height="20" style="height:20px;">anti-human CD73</td>
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			<td>550256</td>
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			<td height="20" style="height:20px;">anti-human/rat STRO-1</td>
			<td>R&#38;D Systems</td>
			<td>MAB1038</td>
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			<td height="20" style="height:20px;">anti-human nestin</td>
			<td>R&#38;D Systems</td>
			<td>MAB1259</td>
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			<td height="20" style="height:20px;">anti-human CD45</td>
			<td>BD Biosciences</td>
			<td>555480</td>
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		<tr height="20">
			<td height="20" style="height:20px;">anti-rat CD90(Thy-1)</td>
			<td>BD Biosciences</td>
			<td>554895</td>
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		<tr height="20">
			<td height="20" style="height:20px;">anti-rat CD73</td>
			<td>BD Biosciences</td>
			<td>551123</td>
			<td></td>
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		<tr height="20">
			<td height="20" style="height:20px;">anti-rat nestin</td>
			<td>BD Biosciences</td>
			<td>MAB1259</td>
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		<tr height="20">
			<td height="20" style="height:20px;">anti-rat CD45</td>
			<td>BD Biosciences</td>
			<td>554875</td>
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			<td height="20" style="height:20px;">Anti-S100&#946;</td>
			<td>Dako</td>
			<td>Z031101</td>
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			<td height="21" style="height:21px;">Anti-p75</td>
			<td>Millipore</td>
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			<td height="20" style="height:20px;">Anti-GFAP</td>
			<td>Sigmaaldrich</td>
			<td>G3893</td>
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			<td height="20" style="height:20px;">Anti-Class III-beta tubulin (Tuj-1)</td>
			<td>Covance</td>
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hypoxic preconditioning of marrow-derived progenitor cells as a source for the generation of mature schwann cells

Dette manuskriptet beskriver et middel for å berike for nevrale progenitorer fra marvstromcelle (MSC) -populasjonen og deretter å henvise dem til den modne Schwann-cellens skjebne. Vi utsatte rotte og humane MSC til forbigående hypoksiske tilstander (1% oksygen i 16 timer) etterfulgt av ekspansjon som neurosfærer på lavt bindingsunderlag med epidermal vekstfaktor (EGF) / basisk fibroblastvekstfaktor (bFGF) tilskudd. Neurosfærer ble sådd på poly-D-lysin / laminin-belagt vevskulturplast og dyrket i en gliogen cocktail inneholdende β-heregulin, bFGF og blodplate-avledet vekstfaktor (PDGF) for å danne Schwann-cellelignende celler (SCLCs). SCLCs ble rettet til skjebneforpliktelse via kokkultur i 2 uker med rensede dorsalrotganglia (DRG) -neuroner oppnådd fra E14-15-gravid Sprague Dawley-rotter. Eldre Schwann-celler viser utholdenhet i S100p / p75-uttrykk og kan danne myelin-segmenter. Celler generert på denne måten har potensial aPlikasjoner i autolog celletransplantasjon som følge av ryggmargsskade, samt i sykdomsmodellering.

En oversikt over hovedstadiene i protokollen er illustrert i figur 1 . I sammendraget velges rotte- og humane MSCer for å vedheve vevskulturplast. Utvidede MSCs er forkjølt med hypoksi og er så gjenstand for neurosfæredannende tilstander. Neurosfærer er belagt og tillatt å skille seg inn i SCLC. SCLCs blir kultivert med rensede DRG-neuroner for å generere skjebnebegrensede Schwann-celler. M...

Watch this Scientific Journal Video about Hypoxic Preconditioning of Marrow-derived Progenitor Cells As a Source for the Generation of Mature Schwann Cells at JoVE.com

Hypoxic Preconditioning of Marrow-derived Progenitor Cells As a Source for the Generation of Mature Schwann Cells

Marvstromceller (MSCs) med nevrale potensial finnes i benmarg. Vår protokoll berikker denne populasjonen av celler via hypoksisk forbehandling og deretter dirigerer dem til å bli modne Schwann-celler.

Hypoksisk forbehandling av margenavledede progenitorceller som kilde for generering av modne Schwann-celler

This manuscript describes a means to enrich for neural progenitors from the marrow stromal cell (MSC) population and thereafter to direct them to the ...

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