Name: progenitor-derived oligodendrocyte culture system from human fetal brain
Uploaded: 2012-12-20T11:00:00
Description: Watch this Scientific Journal Video about Progenitor-derived Oligodendrocyte Culture System from Human Fetal Brain at JoVE.com

Denne forskningen ble støttet av egenutført Research Program ved National Institutes of Health, NINDS. Forfatterne ønsker å takke alle medlemmene av Laboratory of Molecular Medicine og nevrovitenskap, Rick Dreyfuss for å hjelpe med mikroskopi og Pamela C. sikting for å hjelpe med redigering.

Merk: For rutine dyrkning av nevrale progenitor og oligodendrocytic avstamning celler, er inkubering utført ved 37 ° C i en fuktet 5% CO 2 atmosfære. Hver 2 dager, blir mediet erstattet med 50 til 100% av friskt medium hvis kulturen er 40-70% konfluent. På tidspunktet for nær sammenflyting, er kulturene passaged på 2-2.5e6/T75 kolbe vanligvis på en ukeplan. 1. Klargjøre Coated Flask <ol><li> Å tilberede belagte kolber fortynne 5 mg av poly-D-lysin (PDL)...

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Denne protokollen beskriver hvordan man utlede fosterets oligodendrocytes fra primære humane nevrale stamceller og karakterisere deres fenotype med både flowcytometri og immunfluorescens flekker. Utvidelsen og vekst av nevrale stamceller fra foster CNS har vært veldig godt beskrevet 1-4. Imidlertid, å skaffe tilstrekkelige menneskelige oligodendrocytes for in vitro eksperimentering fortsatt vanskelig, selv om det er mulig å identifisere og isolere glial forløpere fra voksen human hvit ...

<table border="1"><tbody><tr><td> Navnet på reagensen </td><td> Selskapet </td><td> Katalognummer </td><td> Endelig konsentrasjon </td></tr><tr><td> DME / HAMS F12 01:01 </td><td> Omega Forsker </td><td> DM-251 </td><td> 1X </td></tr><tr><td> Bovine Albumin </td><td> Sigma </td><td> A9418 </td><td> 1% </td></tr><tr><td> Gentamicin </td><td> Quality Biologicals </td><td> 120-098-031 </td><td> 50 ug / ml </td></tr><tr><td> L-Glutamin </td><td> Quality Biologicals </td><td> 118-084-061 </td><td> 2 mM </td></tr><tr><td> T3 </td><td> Sigma </td><td> T2877 </td><td> 3 nM </td></tr><tr><td> N2 komponenter </td><td> Gibco BRL </td><td> 17502 </td><td> 1:100 </td></tr><tr><td> NT-3 </td><td> PeproTech Inc </td><td> 450-03 </td><td> 2 ng / ml </td></tr><tr><td> Shh </td><td> R &amp; D System </td><td> 1314-SH/CF </td><td> 2 ng / ml </td></tr><tr><td> bFGF </td><td> PeproTech Inc </td><td> 100-18B </td><td> 20 ng / ml </td></tr><tr><td> PDGF-AA </td><td> PeproTech Inc </td><td> 100-13A </td><td> 10 ng / ml </td></tr><tr><td> PDL </td><td> Sigma </td><td> P6407 </td><td> 50 ug / m </td></tr><tr><td> PFA </td><td> Elektronmikroskopi Sciences </td><td> 15712 </td><td> 2% </td></tr><tr><td> Trypsin </td><td> Quality Biologicals </td><td> 118-087-721 </td><td></td></tr><tr><td> Papain </td><td> Worthington </td><td> LK003178 </td><td> 20 U / ml </td></tr><tr><td> DNase hetteglass </td><td> Worthington </td><td> LK003172 </td><td> 0,005% </td></tr><tr><td> EBSS </td><td> Worthington </td><td> LK003188 </td><td></td></tr><tr><td> Forleng Gold medDAPI </td><td> Invitrogen </td><td> P36931 </td><td></td></tr><tr><td></td><td></td><td></td><td> Tabell 1. Reagenser. </td></tr><tr><td></td><td></td><td></td><td><table border="1"><tbody><tr><td> Primær Abs (alle på 1 ug / ml) </td><td></td><td> Arter isotype </td><td> Kilde </td><td> Sekundær Abs </td></tr><tr><td colspan="5"> Flowcytometri </td></tr><tr><td> A2B5-Biotin </td><td> Mus </td><td> IgM </td><td> Gave J. Nielson </td><td> Streptavidin PETR, Invitrogen, CA </td></tr><tr><td> O4FITC </td><td> Mus </td><td> IgM </td><td> Gave fra J. Nielson </td><td></td></tr><tr><td> A2B5 </td><td> Mus </td><td> IgM </td><td> Millipore, MA </td><td> gαm IgM-FITC, Invitrogen, CA </td></tr><tr><td> O4 </td><td> Mus </td><td> IgM </td><td> Millipore, MA </td><td> gαm IgM-FITC, Invitrogen, CA </td></tr><tr><td> GalC </td><td> Mus </td><td> IgG 3 </td><td> Millipore, MA </td><td> gαm IgG 3-PE, Sør Biotech, AL </td></tr><tr><td> MBP </td><td> Kylling </td><td> IGY </td><td> Millipore, MA </td><td> dαck IGY-AMCA, Jackson immunologiske., PA </td></tr><tr><td> Nestin </td><td> Mus </td><td> IgG 1 </td><td> Messam et al. 2000 28 </td><td> gαm IgG 1-PECy5, Invitrogen, CA </td></tr><tr><td> GFAP </td><td> Kanin </td><td> IgG </td><td> Millipore, MA </td><td> gαrb IgG-PE, Jackson immunologiske, PA </td></tr><tr><td> βIII tubulin </td><td> Mus </td><td> IgG 2 </td><td> Covance, CA </td><td> gαm IgG 2 a-PETR, Invitrogen, CA </td></tr><tr><td colspan="6"> Immunocytokjemi </td></tr><tr><td> βIII tubulin (1:1,500) </td><td> Mus </td><td> IgG 2a </td><td> Covance, CA </td><td> gαm IgG 2a-FITC, Invitrogen, CA (1:1,000) </td></tr><tr><td> GFAP (1:1,000) </td><td> Kanin </td><td> IgG </td><td> Millipore, MA </td><td> gαrb IgG-FITC, Jackson immunologiske, PA (1:500) </td></tr><tr><td> MBP (1:50) </td><td> Kylling </td><td> IGY </td><td> Millipore, MA </td><td> dαck IGY-FITC, Jackson immunologiske, PA (1:100) </td></tr><tr><td> O4 (1:100) </td><td> Mus </td><td> IgM </td><td> Millipore, MA </td><td> gαm IgM-AF546, Invitrogen, CA (1: 100) </td></tr><tr><td> GalC (1:10) </td><td> Kanin </td><td> IgG </td><td> Millipore, MA </td><td> gαm IgM-AF750, Invitrogen, CA (1:100) </td></tr></tbody></table> . Tabell 2 antistoffer (Abs) brukes til flowcytometri og immunocytokjemi analyser Antistoff konjugater: PE, phycoerythrin, PETR, phycoerythrin Texas Red, AMCA, amino-metyl-kumarinliknende acetat, Cy, cyanine, FITC, fluorescein isothiocyanate Ig:. Immunglobulin. . AF: Alexa Fluor; gαm: geit anti-mus, gαrb: geit anti-kanin; dαck: esel anti-kylling. </td></tr></tbody></table>

progenitor-derived oligodendrocyte culture system from human fetal brain

Differensiering av menneskelige nevrale stamceller i neuronal og glial celletyper tilbyr en modell for å studere og sammenligne molekylær regulering av neural celle avstamning utvikling. In vitro utvidelse av nevrale stamceller fra foster CNS vev har blitt godt karakterisert. Til tross for identifisering og isolering av glial stamfedre fra voksent menneske sub-kortikal hvit materie og utvikling av ulike kultur betingelser for direkte differensiering av fosterets nevrale stamceller i myelin produserende oligodendrocytes, anskaffe tilstrekkelige menneskelige oligodendrocytes for in vitro eksperimentering er fortsatt vanskelig. Differensiering av galactocerebroside + (GalC) og O4 + oligodendrocyte forløper eller stamceller (OPC) fra nevrale forløper celler er rapportert ved hjelp andre trimester fosterets hjerne. Men disse cellene ikke sprer i fravær av støtte celler inkludert astrocytes og nerveceller, og er tapt raskt over tid i kultur. Behovet fortsatt for en kultur-system for å produsere celler av oligodendrocyte avstamning egnet for in vitro forsøk. Dyrkning av primære humane oligodendrocytes kunne, for eksempel, være en nyttig modell for å studere patogenesen av neurotropisk smittestoffer som menneskelige polyomavirus, JCV at in vivo infiserer disse cellene. Disse dyrkede celler kunne også gi modeller av andre demyeliniserende sykdommer i det sentrale nervesystemet (CNS). Primær, human fosterets hjerne-avledet, multipotential nevrale stamceller sprer in vitro og samtidig opprettholde evnen til å differensiere i nevroner (stamfar-avledet nevroner, PDN) og astrocytes (stamfar-avledet astrocytes, PDA) Denne studien viser at nevrale stamceller kan induseres å skille gjennom mange av de stadier av oligodendrocytic avstamning utvikling (stamfar-avledet oligodendrocytes, PUD). Vi kultur nevrale stamceller i DMEM-F12 serum-frie medier supiverksatt med grunnleggende fibroblast vekstfaktor (bFGF), blodplate avledet vekstfaktor (PDGF-AA), Sonic pinnsvin (SHH), neurotrop faktor 3 (NT-3), N-2 og trijodtyronin (T3). Dyrkede celler passaged på 2.5e6 celler per 75cm kolber omtrent hvert sju dager. Ved hjelp av disse forhold, flertallet av celler i kultur opprettholde en morfologi preget av få prosesser og uttrykke markører av pre-oligodendrocyte celler, for eksempel A2B5 og O-4. Når vi fjerner de fire vekstfaktorer (GF) (bFGF, PDGF-AA, Shh, NT-3) og legge kondisjonert medium fra PDN, cellene begynner å anskaffe flere prosesser og uttrykke markører spesifikke for oligodendrocyte differensiering, for eksempel GalC og myelin grunnleggende protein (MBP). Vi utførte fenotypisk karakterisering ved hjelp av flerfarget flowcytometri å identifisere unike markører for oligodendrocyte.

Det er svært viktig å starte differensiering prosessen fra en 70% -80% confluent nevrale stamceller kultur (figur 1A). Mange celler vil dø ut etter å endre kulturen medium fra stamfar til oligo medium siden det omfatter bestemte vekstfaktorer. Dette indikerer at veksten av nevrale stamceller ikke forpliktet til en oligodendrocytic fenotype ikke vil bli støttet av det nye mediet (figur 1B). Inkubering i Oligo medium + GF for en uke ga en mellomliggende kultur oppviser en smal, bipol...

Watch this Scientific Journal Video about Progenitor-derived Oligodendrocyte Culture System from Human Fetal Brain at JoVE.com

Progenitor-derived Oligodendrocyte Culture System from Human Fetal Brain

Primær, menneskelige hjernens-deriverte, multipotential stamceller sprer<em&gt; In vitro</em&gt; Mens opprettholde evnen til å differensiere til neuroner og astrocytes. Dette arbeidet viser at nevrale stamceller kan bli overtalt til å skille gjennom stadier av oligodendrocytic avstamning av condition med utvalgte vekstfaktorer.

Stamfar-avledet Oligodendrocyte Kultur System fra Human fosterets hjerne

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Understanding blood-brain barrier function under physiological and pathophysiological conditions is critical for the development of new therapeutic ...

Migration, Chemo-Attraction, and Co-Culture Assays for Human Stem Cell-Derived Endothelial Cells and GABAergic Neurons

Migrasjon, kjemo-attraksjon og co-kultur analyser for menneskelige stamcelle-avledet endotelceller og GABAergic Nevroner

Role of brain vasculature in nervous system development and etiology of brain disorders is increasingly gaining attention. Our recent studies have ...

Measuring and Manipulating Functionally Specific Neural Pathways in the Human Motor System with Transcranial Magnetic Stimulation

Måling og manipulering funksjonelt spesifikke nevrale veier i det menneskelige motorsystemet med transkraniell magnetisk stimulering

Understanding interactions between brain areas is important for the study of goal-directed behavior. Functional neuroimaging of brain connectivity has ...

Generation of Oligodendrocytes and Oligodendrocyte-Conditioned Medium for Co-Culture Experiments

Generasjon oligodendrocytes og oligodendrocyte-conditioned medium for co-kultur eksperimenter

In the central nervous system, oligodendrocytes are well-known for their role in axon myelination, that accelerates the propagation of action potentials ...