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 taknemmelige for Dr. Magdalena Götz for hendes input under udviklingen af ​​denne protokol. Vi takker Dr. Marius Wernig (Stanford University) for generøst at give os med Sox2 kodende sekvens. Vi er også meget taknemmelige til Dr. Alexandra Lepier for virus produktion. Dette arbejde blev støttet af tilskud fra Deutsche Forschungsgemeinschaft (BE 4182/2-2) og BMBF (01GN1009A) til BB, og det bayerske stat, Ministeriet for Videnskab, forskning og kunst til MK og BBCS modtaget støtte fra binationalt SYSTHER- INREMOS Virtual Institute (tysk og slovensk forbundsministerierne for Uddannelse og Forskning) og DFG (SFB 824).

1.. Isolering og dyrkning af voksne mennesker hjerneceller Eksperimenter med humant væv bør udføres i overensstemmelse med alle relevante statslige og institutionelle regler om brug af humant materiale til forskningsformål. Den nuværende protokol blev udviklet i overensstemmelse med godkendelsen af ​​den etiske komité i det medicinske fakultet i LMU München og skriftligt informeret samtykke fra alle patienter. Denne protokol forbereder kulturer i voksent...

<ol>
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Den nuværende protokol beskriver in vitro ekspansion og berigelse af pericyt-afledte celler efter isolering fra voksent menneske hjernebarken og den efterfølgende omdannelse til iNs ved retrovirusmedieret udtryk for neurogene transskriptionsfaktorerne Sox2 og Ascl1. En sådan protokol giver et eksperimentelt in vitro-system til at studere slægt-konvertering af hjerne-resident celler til neuroner og potentielt også glia, med det mål for øje at i sidste ende omsætte denne direkte tilgang omregning...

I modsætning til omprogrammering af somatiske celler i induceret pluripotente stamceller (iPSCs), som igen er udstyret med en overflod af differentiering potentialer direkte omprogrammering sigter til lige omdannelse af en specifik celletype til en anden. Med hensyn til deres anvendelse i forbindelse med sygdom modellering og potentielle cellebaserede behandlingsformer af begge omprogrammering tilgange har særlige fordele og ulemper. Omprogrammering ind iPSCs (I) giver en næsten uendelig kilde af celler; (Ii) giver mulighed for genteknologi; (Iii) forlener med en næsten ubegrænset potentiale differentiering. Men de store ulemper ved iPSCs medføre risiko for tumorgenicitet af udifferentierede celler (teratom dannelse in vivo), og behovet for ex vivo dyrkning og efterfølgende transplantation, hvis disse celler skal anvendes til cellebaserede behandlingsformer. Omvendt er direkte afstamning-omprogrammering begrænset af lavere udbytte af de ønskede cellersom korrelerer direkte med antallet af de målrettede celler start befolkning, men har den fordel, at afstamning-omprogrammeret celler synes ikke at udvise nogen tumorigen risiko ved transplantation 1,2; desuden kan direkte omprogrammering selv opnås in situ, dvs inden for orgel, hvor disse celler ville være påkrævet, så man undgår behovet for transplantation. Med dette i tankerne, har vores laboratorium forfulgt muligheden for slægt-omprogrammering hjerne-resident celler i iNs som en ny tilgang til cellebaserede behandlingsformer af neurodegenerative sygdomme. Brain-resident celler, der kan være potentielt betragtes som cellulære mål for linjespecifikt omprogrammering omfatte forskellige typer af macroglia (astrocytter, NG2 celler og oligodendrocytter), mikroglia og mikrokar-associerede celler (endotelceller og pericytter). Vi har grundigt undersøgt in vitro omprogrammering potentiale astroglia af cerebral cortex af tidlig postnatal mus 3-5. På jagt efter tilsvarende egnede cellekilder til direkte afstamning-omprogrammering i den voksne menneskelige hjerne, stødte vi på en celle befolkning, der med held kan omprogrammeres i Ins og udstille kendetegnende for pericytes. Her beskriver vi en protokol, hvordan at høste disse celler fra voksne humane hjerne biopsier, udvide og berige disse celler in vitro, og endelig held omprogrammere en væsentlig del af disse in vitro-ekspanderede celler (i størrelsesordenen 25-30%) i ins. Omprogrammering kan opnås ved samtidig retrovirus-medieret co-ekspression af to transskriptionsfaktorer, Sox2 og ascl1. Disse PdiNs fandtes at erhverve evne til gentagen handling potentiel fyring og tjene som synaptiske mål for andre neuroner, der angiver deres evne til at integrere sig i neurale netværk. Vores protokol giver en enkel procedure til isolering og afstamning konvertering af voksne menneskelige hjerne pericytes i ins. </p&gt;

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	</tbody>
</table>

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

Direkte afstamning-omprogrammering af ikke-neuronale celler i inducerede neuroner (INS), kan give indsigt i de molekylære mekanismer, der ligger neurogenesis og muliggøre nye strategier for in vitro-modellering eller reparation af syge hjerne. Identifikation hjerne-resident non-neuronale celletyper kan underkastes direkte omdannelse til iNs kunne åbne mulighed for at iværksætte en sådan tilgang in situ, dvs inden for den beskadigede hjernevæv. Her beskriver vi en protokol udviklet i forsøg på at identificere celler fra den voksne humane hjerne, som opfylder denne forudsætning. Denne protokol omfatter: (1) dyrkning af humane celler fra den cerebrale cortex opnået fra voksne humane hjerne biopsier; (2) in vitro ekspansion (ca. kræve 2-4 uger), og karakterisering af kulturen ved immunocytokemi og flowcytometri; (3) tilsætning af fluorescens-aktiveret cellesortering (FACS) under anvendelse af anti-PDGF-receptor-β og anti-CD146-antistoffer;(4) retrovirus-medieret transduktion med neurogen transskriptionsfaktorerne Sox2 og ascl1; (5) og endelig karakterisering af de resulterende pericyt-afledte inducerede neuroner (PdiNs) efter immuncytokemi (14 dage til 8 uger efter retroviral transduktion). På dette stadium, kan iNs probes for deres elektriske egenskaber ved patch-clamp optagelse. Denne protokol giver en meget reproducerbar procedure for in vitro afstamning konvertering af hjerne-resident pericytes i funktionelle menneskelige ins.

Det første resultat af denne protokol efter med held etablere en kultur fra en prøve af voksent menneske hjernebarken består i identifikationen af ​​den cellulære sammensætning af kulturen. Immuncytokemi for celle typespecifikke proteiner afslører en betydelig grad af heterogenitet mellem kulturer afledt fra prøvemateriale fra forskellige patienter (figur 1A). Kvantificeret ved flowcytometri er der altid en væsentlig fraktion af celler, som udtrykker PDGFRβ (i gennemsnit ~ 75%, der spænder...

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 af pericyt-afledte Celler af Voksen Menneskelige hjerne til Inducerede Neuroner

Målretning hjerne-resident celler til direkte afstamning-omprogrammering giver nye perspektiver for hjernen reparation. Her beskriver vi en protokol om, hvordan man forbereder kulturer beriget med hjerne-resident pericytes fra voksent menneske hjernebarken og konvertere disse til inducerede neuroner ved retrovirusmedieret udtryk for transskriptionsfaktorerne 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

Specifikationen af ​​Telencephalic glutamaterge neuroner fra humane 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

Miljømæssige Modulationer i antallet af Midbrain dopaminneuroner i voksne 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

Udledning af Voksen humane fibroblaster og deres direkte konvertering til udvides Neural progenitorceller

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

Simpel Generation af højtydende kultur af inducerede neuroner fra menneskelige voksen hud 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

Omdannelse af humane induceret pluripotente stamceller (iPSCs) til funktionel spinal og kraniel motor neuroner brug 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

Kortsigtede fritflydende udsnit kulturer fra den voksne menneskelige hjerne

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

Migration, Chemo-Attraction, og Co-Kultur Assays for Human Stem Cell-Afledt endotelceller og GABAergic Neurons

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

Opnåelse humane mikroglia fra voksent humant hjernevæv

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

Afledning, ekspansion, kryopræservering og karakterisering af hjernemikrobielle endotelceller fra menneskeskabte pluripotente stamceller

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