Name: lineage-reprogramming of pericyte-derived cells of the adult human brain into induced neurons
Uploaded: 2014-05-12T21:45:00
Description: Watch this Scientific Journal Video about Lineage-reprogramming of Pericyte-derived Cells of the Adult Human Brain into Induced Neurons at JoVE.com

Vi er takknemlige til Dr. Magdalena Götz for hennes innspill under utviklingen av denne protokollen. Vi takker Dr. Marius Wernig (Stanford University) for sjenerøst å gi oss med Sox2 koding sekvensen. Vi er også svært takknemlige til Dr. Alexandra Lepier for virusproduksjon. Dette arbeidet ble støttet med tilskudd av Deutsche Forschungsgemeinschaft (BE 4182/2-2) og BMBF (01GN1009A) til BB, og den bayerske stats Ministry of Sciences, forskning og kunst til MK og BBCS fått støtte fra binational SYSTHER- INREMOS Virtual Institute (tysk og slovensk Federal Ministries Utdannings-og forskningsdepartementet) og DFG (SFB 824).

En. Isolering og dyrking av voksent menneske hjerneceller Forsøk med menneskelig vev skal utføres i samsvar med alle relevante statlige og institusjonelle regler om bruk av humant materiale for forskningsformål. Den nåværende protokollen ble utviklet i samsvar med godkjenning av etisk komité ved det medisinske fakultet ved LMU München og skriftlig informert samtykke fra alle pasienter. Denne protokollen av forbereder kulturer av menneske voksen cerebral cort...

<ol>
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Den nåværende protokollen beskriver in vitro utvidelse og berikelse av pericyte-avledet celler følgende isolasjon fra voksent menneske cerebral cortex og senere konvertering til moduler ved retrovirus-mediert uttrykk for nevrogen transkripsjonsfaktorene Sox2 og Ascl1. En slik protokoll gir en eksperimentell in vitro system for å studere avstamning-konvertering av hjerne-bosatt celler i nevroner og potensielt også gliaceller, med målet i tankene til slutt oversette dette direkte konvertering tiln?...

I motsetning til omprogrammering av somatiske celler i indusert pluripotente stamceller (iPSCs), som i sin tur er utstyrt med en mengde differensiering potensialer, sikter direkte reprogrammering for direkte omdannelse av en spesifikk celletype til en annen. Med hensyn til deres anvendelse i sammenheng med sykdommen modellering og eventuelle celle-baserte terapier, både omprogrammering tilnærminger har spesielle fordeler og ulemper. Omprogrammering inn iPSCs (i) gir en tilnærmet uendelig kilde av celler; (Ii) gir mulighet for genteknologi; (Iii) endows med en nesten ubegrenset differensiering potensial. Imidlertid store ulemper ved iPSCs medføre risiko for tumorgenisitet av udifferensierte celler (teratom dannelse in vivo), og behovet for ex vivo dyrking og påfølgende transplantasjon hvis disse celler skal anvendes for celle-baserte terapier. Omvendt, er direkte lineage-omprogrammering er begrenset av den nedre utbyttet av de ønskede cellenesom korrelerer direkte med antallet av de målrettede celler i utgangs befolkningen, men har den fordel at lineage-omprogrammeres cellene ser ut til å oppvise ingen tumorigen risiko ved transplantasjon 1,2; Videre kan direkte reprogrammering også bli oppnådd in situ, dvs. inne i organet hvor disse celler ville være nødvendig, for derved å unngå behovet for transplantasjon. Med dette i bakhodet, har vår lab forfulgt muligheten for avstamning-omprogrammere hjernen-resident celler inn ins som en ny tilnærming mot celle-baserte terapier av nevrodegenerative sykdommer. Hjerne-resident celler som kan være potensielt anses som cellulære mål for avstamning-omprogrammering omfatter ulike typer macroglia (astrocytes, NG2 celler og oligodendrocytes), microglia, og microvessel-assosiert celler (endotelceller og pericytes). Vi har grundig studert in vitro omprogrammering potensialet astroglia av cerebral cortex av tidlig postnatal mus 3-5. På jakt etter tilsvarende egnede celle kilder for direkte avstamning-omprogrammering i den voksne menneskehjernen, møtte vi en cellepopulasjon som kan være vellykket omprogrammeres til ins og utstilling kjennetegnene ved pericytes. Her beskriver vi en protokoll for å høste slike celler fra voksne humane hjernebiopsi, for å utvide og berike disse cellene in vitro, og til slutt med hell omprogrammere en vesentlig del av disse in vitro-ekspanderte celler (i området fra 25 til 30%) i INS. Omprogrammering kan oppnås ved samtidig retrovirus-mediert co-uttrykk for to transkripsjonsfaktorer, Sox2 og ascl1. Disse PdiNs ble funnet å tilegne seg evnen av repeterende handling potensial avfyring og å tjene som synaptiske mål for andre nerveceller som indikerer deres evne til å integrere inn i nevrale nettverk. Vår protokollen gir en enkel prosedyre for isolering og avstamning konvertering av voksne menneskelige hjerne pericytes inn ins. </p&gt;

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	</tbody>
</table>

lineage-reprogramming of pericyte-derived cells of the adult human brain into induced neurons

Direkte avstamning-omprogrammering av ikke-nevronale celler til indusert nevroner (INS) kan gi innsikt i de molekylære mekanismene bak neurogenesis og aktivere nye strategier for in vitro modellering eller reparere den syke hjernen. Identifisere hjerne-resident ikke-nevronale celletyper mottagelig for direkte konvertering til ins kan gi rom for å lansere en slik tilnærming i situ, dvs innen skadet hjernevev. Her beskriver vi en protokoll utviklet i forsøket på å identifisere celler avledet fra den voksne menneskehjernen som oppfyller denne forutsetningen. Denne protokoll omfatter: (1) dyrking av humane celler fra den cerebrale cortex erholdt fra voksne humane hjernebiopsi; (2) in vitro-ekspansjon (ca. kreve 2-4 uker) og karakterisering av kulturen ved immunocytokjemi og flowcytometri; (3) anrikning av fluorescens-aktivert cellesortering (FACS) under anvendelse av anti-PDGF-reseptor-β-og anti-CD146 antistoff;(4) retrovirus-mediert transduksjon med nevrogen transkripsjonsfaktorer Sox2 og ascl1; (5) og til slutt karakterisering av de resulterende pericyte-avledet indusert nevroner (PdiNs) etter immunocytokjemi (14 dager til 8 uker etter retroviral transduksjon). På dette stadiet, kan Ins bli analysert for deres elektriske egenskaper av patch-clamp opptak. Denne protokollen gir en svært reproduserbar prosedyre for in vitro avstamning konvertering av hjerne-resident pericytes i funksjonelle menneskelige ins.

Det første resultat av denne protokollen etter vellykket etablering av en kultur fra en prøve av human voksen hjerne cortex består i identifikasjon av den cellulære sammensetning av kulturen. Immunocytochemistry for celletypespesifikke proteiner som viser en betydelig grad av heterogenitet mellom kulturer avledet fra prøven med forskjellige pasienter (Figur 1a). Som kvantifisert ved strømningscytometri er det alltid en betydelig fraksjon av celler som uttrykker PDGFRβ (i gjennomsnitt ~ 75%, fra 3...

Watch this Scientific Journal Video about Lineage-reprogramming of Pericyte-derived Cells of the Adult Human Brain into Induced Neurons at JoVE.com

Lineage-reprogramming of Pericyte-derived Cells of the Adult Human Brain into Induced Neurons

Lineage-omprogrammering av Pericyte-deriverte celler av den voksne Human Brain inn Utførte Nerveceller

Målretting hjerne-resident celler for direkte avstamning-omprogrammering gir nye perspektiver for hjernen reparasjon. Her beskriver vi en protokoll for hvordan du kan forberede kulturer beriket for hjerne-resident pericytes fra voksent menneske cerebral cortex og konvertere disse til induserte nevroner av retrovirus-mediert uttrykk for transkripsjonsfaktorene Sox2 og Ascl1.

Direct lineage-reprogramming of non-neuronal cells into induced neurons (iNs) may provide insights into the molecular mechanisms underlying neurogenesis ...

Research

JoVE Journal

Neuroscience

Studying the Integration of Adult-born Neurons

Neurogenesis occurs in adult mammalian brains in the sub-ventricular zone (SVZ) of the lateral ventricle and in the sub-granular zone (SGZ) of the ...

The Specification of Telencephalic Glutamatergic Neurons from Human Pluripotent Stem Cells

Spesifikasjonen av Telencephalic glutamatergic Neurons fra menneskelige pluripotente stamceller

Here, a stepwise procedure for efficiently generating telencephalic glutamatergic neurons from human pluripotent stem cells (PSCs) has been described. The ...

Environmental Modulations of the Number of Midbrain Dopamine Neurons in Adult Mice

Miljø modulasjoner av antall midthjernen dopaminneuroner i voksen Mus

Long-lasting changes in the brain or &#8216;brain plasticity&#8217; underlie adaptive behavior and brain repair following disease or injury. Furthermore, ...

Derivation of Adult Human Fibroblasts and their Direct Conversion into Expandable Neural Progenitor Cells

Avledning av voksent menneske fibroblaster og deres direkte konvertering til Utvid Neural stamceller

Generation of&#160;induced pluripotent stem cell (iPSCs) from adult skin fibroblasts and subsequent differentiation into somatic cells provides ...

Simple Generation of a High Yield Culture of Induced Neurons from Human Adult Skin Fibroblasts

Enkel generasjon av en høy avkastning kultur indusert neurons fra menneskelige voksne huden fibroblaster

Induced neurons (iNs), the product of somatic cells directly converted to neurons, are a way to obtain patient-derived neurons from tissue that is easily ...

Conversion of Human Induced Pluripotent Stem Cells (iPSCs) into Functional Spinal and Cranial Motor Neurons Using PiggyBac Vectors

Conversion of Human Induced Pluripotent Stem Cells iPSCs into Functional Spinal and Cranial Motor Neurons Using PiggyBac Vectors

Konvertering av Human indusert pluripotent Stem Cells (iPSCs) i funksjonell spinal og skallen motor neurons bruke PiggyBac vektorer

We describe here a method to obtain functional spinal and cranial motor neurons from human induced pluripotent stem cells (iPSCs). Direct conversion into ...

Short-Term Free-Floating Slice Cultures from the Adult Human Brain

Kortsiktige fritt flytende Slice kulturer fra Adult Human Brain

Organotypic, or slice cultures, have been widely employed to model aspects of the central nervous system functioning in vitro. Despite the potential of ...

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 ...

Obtaining Human Microglia from Adult Human Brain Tissue

Få humanmikroglia fra voksent menneskelig hjernevev

Microglia are resident innate immune cells of the central nervous system (CNS). Microglia play a critical role during development, in maintaining ...

Derivation, Expansion, Cryopreservation and Characterization of Brain Microvascular Endothelial Cells from Human Induced Pluripotent Stem Cells

Avledning, utvidelse, kryopreservasjon og karakterisering av mikrovaskulære endotelceller fra humane induserte pluripotente stamceller

Brain microvascular endothelial cells (BMECs) can be differentiated from human induced pluripotent stem cells (iPSCs) to develop ex vivo cellular models ...