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 gerne anerkende Dr. Nai-Sum Wong for at levere hypoxiakammerapparatet og fru Alice Lui til den tekniske support.

Alle procedurer involveret dyr blev udført i nøje overensstemmelse med NIH Guide for pleje og brug af laboratoriedyr og godkendt af Udvalget for Anvendelse af Levende Dyr til Undervisning og Forskning, Li Ka Shing Medicinsk Fakultet, Hongkong Universitet. Humane knoglemarvsprøver blev opnået fra iliac crest af sunde donorer efter at have opnået informeret samtykke. Protokoller blev godkendt af Institutional Review Board, University of Hong Kong. 1. Fremstilling af rotte-MSC-kulturer <...

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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. 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Det er vigtigt at bevare MSC&#39;ernes stivhed forud for berigelsen af ​​neurale progenitorer via hypoxisk forkonditionering og neurosfærekultur. Ud fra vores erfaring kan multipotente MSC pålideligt identificeres af deres langstrakte fibroblastlignende morfologi. I modsætning hertil kan MSC&#39;er, som har vedtaget en mere udpladet, firkantet morfologi med fremtrædende cytoskeletale stressfibre, ikke let vedtage neurale cellefates og bør kasseres. Generelt bruger vi ikke MSC&#39;er med passage numre større en...

Transplantationen af ​​neurale progenitorer og deres derivater demonstrerer løfte som en behandlingsstrategi efter traumatisk nerveskade 1 , 2 og neurodegenerering 3 , 4 . Før klinisk anvendelse er det vigtigt at sikre: i) en metode til at få adgang til og udvide en autolog kilde til stamceller / stamceller og ii) et middel til at lede dem til relevante modne celletyper 3 . Vores interesse for celleterapi for rygmarvsskade fik os til at søge en robust autolog cellekilde af neurale progenitorer fra voksne væv. En subpopulering af MSC&#39;er stammer fra neuralkammen og er let tilgængelig fra marvhulen. Disse celler er neurale progenitorer, der kan generere neuroner og glia 5 . Dyrmodeller af cerebral iskæmi viser, at hypoxi fremmer prolinen<html> Iferation og multipotens af neurale progenitorer i hjernen 6 . Dette var grundlaget for at udnytte hypoxisk forkonditionering som et middel til at ekspandere på margenafledte neurale progenitorer. Transplantationen af ​​Schwann-celler i den skadede rygmarv fremmer regenerering 2 . SCLC&#39;er kan genereres fra MSC&#39;er ved hjælp af supplering med gliogene faktorer ( dvs. P-Heregulin, bFGF og PDGF-AA), men demonstrerer fænotypisk ustabilitet. Ved tilbagetrækning af vækstfaktorer vender de tilbage til en fibroblastlignende fænotype 7 . Fænotypisk ustabilitet er uønsket ved celletransplantation på grund af risikoen for afvigende differentiering og carcinogenese. Da Schwann-celleprecursorer er associeret med axonbundler inden for den embryonale perifere nerve 8 , blev vi ført til coculture-SCLC&#39;er med oprensede embryonale DRG-neuroner 7 ,Ass = &quot;xref&quot;&gt; 9. Resulterende modne Schwann-celler er skæbneforpligtede og demonstrerer funktion in vitro 7 , 9 og in vivo 10 . Vores protokol til berigelse af neurale progenitorer fra MSC&#39;er er enkel og effektiv og resulterer i en stigning i celleantal til efterfølgende analyser. Afledningen af ​​skæbnebegyndte Schwann-celler via coculture-platformen muliggør undersøgelse af glialdifferentiering og generering af stabile og funktionelle Schwann-celler til potentiel klinisk anvendelse.

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			<td height="20" style="height:20px;">anti-human/rat STRO-1</td>
			<td>R&#38;D Systems</td>
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			<td height="20" style="height:20px;">anti-human nestin</td>
			<td>R&#38;D Systems</td>
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			<td height="20" style="height:20px;">anti-human CD45</td>
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			<td height="20" style="height:20px;">anti-rat CD90(Thy-1)</td>
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			<td>554895</td>
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			<td height="20" style="height:20px;">anti-rat CD73</td>
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			<td>551123</td>
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		<tr height="20">
			<td height="20" style="height:20px;">anti-rat nestin</td>
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			<td>MAB1259</td>
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			<td height="20" style="height:20px;">anti-rat CD45</td>
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			<td>554875</td>
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			<td height="20" style="height:20px;">Anti-S100&#946;</td>
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hypoxic preconditioning of marrow-derived progenitor cells as a source for the generation of mature schwann cells

Dette manuskript beskriver et middel til at berige for neurale progenitorer fra marvstromcelle (MSC) populationen og derefter henvise dem til den modne Schwann-cellefate. Vi udsatte rotte- og humane MSC&#39;er til forbigående hypoxiske tilstande (1% ilt i 16 timer) efterfulgt af ekspansion som neurospherer på sublimat med lav tilhæftning med supplerende epidermal vækstfaktor (EGF) / basisk fibroblastvækstfaktor (bFGF). Neurospheres blev podet på poly-D-lysin / laminin-coatet vævskulturplast og dyrket i en gliogen cocktail indeholdende p-heregulin, bFGF og blodpladeafledt vækstfaktor (PDGF) til dannelse af Schwann-cellelignende celler (SCLC&#39;er). SCLC&#39;er blev rettet mod skæbneforpligtelse via dyrkultur i 2 uger med rensede dorsalrotganglia (DRG) neuroner opnået fra E14-15 gravid Sprague Dawley rotter. Modne Schwann-celler viser persistens i S100p / p75-ekspression og kan danne myelin-segmenter. Celler dannet på denne måde har potentiale aPlikationer i autolog celletransplantation efter rygmarvsskade såvel som i sygdomsmodellering.

En oversigt over nøglefaser i vores protokol er illustreret i figur 1 . Sammenfattende udvælges rotte- og humane MSC&#39;er ved vedhæftning til vævskulturplastik. Udvidede MSC&#39;er er forbehandlet med hypoxi og udsættes derefter for neurosfæredannende tilstande. Neurospheres er belagt og tilladt at differentiere i SCLC&#39;er. SCLC&#39;er kultiveres med oprensede DRG-neuroner for at generere skæbnebegyndte Schwann-celler. <p class="jove_content"...

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

Marvstromaceller (MSC&#39;er) med neurale potentialer findes inden for knoglemarven. Vores protokol beriger denne population af celler via hypoxisk forkonditionering og derefter leder dem til at blive modne Schwann-celler.

Hypoksisk forbehandling af marv-afledte progenitorceller som en kilde til generering af 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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