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Neurosciences

Differentiering af musen embryonale stamceller i kortikale Interneuron prækursorer

Published: December 03, 2017
doi:
10.3791/56358
,
1Neuroscience Graduate Group, Perelman School of Medicine,University of Pennsylvania, 2Department of Psychiatry, Children’s Hospital of Philadelphia,University of Pennsylvania School of Medicine

Summary

Denne protokol beskriver en metode til at generere kortikale interneuron ophav og post mitotiske interneuron prækursorer fra mus embryonale stamceller ved hjælp af en modificeret embryoid krop til éncellelag metode. Disse stamfaderen/prækursorer kan være brugt i vitro fluorescently sorteret og transplanteret ind i neonatal neocortex for at studere skæbne bestemmelse eller anvendes til terapeutiske formål.

Abstract

GABAergic kortikale interneurons er en heterogen population af celler, der spiller en kritisk rolle i regulere produktionen af excitatoriske pyramideformet neuroner samt synkronisering udgange pyramideformet neuron ensembler. Underskud i interneuron funktion har været impliceret i en bred vifte af neuropsykiatriske lidelser, herunder skizofreni, autisme og epilepsi. Afledning af kortikale interneurons fra embryonale stamceller ikke kun giver mulighed for undersøgelse af deres udvikling og funktion, men giver indsigt i de molekylære mekanismer patogenesen af kortikale interneuron-relaterede lidelser. Interneurons har også den bemærkelsesværdige evne til at overleve, overføre og integrere i vært kortikale kredsløb efter transplantation, hvilket gør dem ideelle kandidater til brug i celle-baserede behandlinger. Her præsenterer vi en skalerbar, højeffektive, modificerede embryoid krop til éncellelag metode for fastsættelse af Nkx2.1-udtrykker interneuron ophav og deres afkom fra mus embryonale stamceller (mESCs). Bruger en Nkx2.1::mCherry:Lhx6::GFP dobbelt reporter mESC linje, kan Nkx2.1 ophav eller deres Lhx6-udtrykker post mitotiske afkom isoleres via fluorescens-aktiveret celle sortering (FACS) og efterfølgende brugt i en række downstream applikationer. Vi leverer også metoder til at berige for parvalbumin (PV) og somatostatin (SST) interneuron undergrupper, som kan være nyttige til at studere aspekter af skæbne bestemmelse eller anvendelse i terapeutiske anvendelsesmuligheder, der ville drage fordel af interneuron undergruppe-beriget transplantationscentre.

Introduction

Både mus og mennesker, omtrent halvdelen af alle kortikale hæmmende interneurons (CIns) stammer senest en forbigående subkortikale struktur kendt som den mediale ganglionære eminence (MGE), hvor neuroepithelial stamfaderen til CIns og andre neuronal og glial undergrupper express transkriptionsfaktor Nkx2.11,2. CIn undergrupper eller undertyper er defineret ved krydsende morfologiske, neurokemiske, elektrofysiologiske og connectivity egenskaber3,4. Den MGE-afledte CIns kan inddeles i hovedsageligt ikke-overlappende undergrupper baseret på deres udtryk for enten PV eller SST, udtryk for som korrelerer med særlig elektrofysiologiske og connectivity tendenser5. Dysfunktion af interneurons, navnlig i PV undergruppe, har været involveret i flere neuropsykiatriske lidelser og sygdomme6,7. Det overordnede mål med denne metode er at fremstille stamceller-afledt mitotiske ophav og vandrende prækursorer beriget til enten PV eller SST CIn skæbne for at studere kortikale interneuron biologi og til brug i celle-baserede behandlinger.

Vi har udviklet en skalerbar, yderst effektiv metode til afledning af Nkx2.1-udtrykker interneuron ophav og deres afkom fra mESCs. Ved hjælp af en Nkx2.1::mCherry:Lhx6::GFP dobbelt reporter mESC linie8, Nkx2.1 ophav eller deres Lhx6-udtrykker post mitotiske afkom kan isoleres via FACS og efterfølgende brugt i en række downstream applikationer. Ved at manipulere en række signaling veje, varighed af kultur og tilstand af neurogenese, kan vi få millioner af fluorescently mærket interneuron prækursorer velegnet til et væld af downstream applikationer.

Selv om flere andre metoder til at generere MGE-lignende ophav fra mESCs9,10,11,12,13,14, vores metode, som bygger på Wnt antagonist XAV-939, er især effektive til at generere Foxg1/Nkx2.1 Co udtrykker telencephalic ophav. Derudover forbedrer mulighed for at vælge til interneuron ophav eller deres post mitotiske Lhx6-udtrykker afkom via vores dual reporter system, høj grad evnen til at generere forskellige ophav og deres afkom.

Protocol

Bemærk: Den dobbelte reporter mESC linje er beskrevet i denne protokol er tilgængelige på anmodning (sande@mail.med.upenn.edu). 1. medier forberedelse Bemærk: Varm alle medier til 37 ° C før brug i cellekultur. Musen embryonale Fibroblast (MEF) medier (for at forberede 500 mL) Der tilsættes 50 mL føtalt bovint serum (FBS) til 449 mL Dulbeccos modificerede Eagle’s Medium (DMEM) og filtreres gennem et 500 mL 0,22 µm pore …

Representative Results

Den protokol, der er beskrevet i denne hvidbog er en modificeret udgave af vores publicerede protokoller15,16,17 og er blevet optimeret til brug sammen med vores Nkx2.1::mCherry:Lhx6::GFP dual-reporter mESC linje. Ved at tilføje Wnt hæmmer XAV-939 fra DD0-5, kombineret med re plating på DD8, opnå vi robuste Nkx2.1 induktion, hvori op mod 50% af alle DAPI + kerner i kultur er også Nkx2.1 at u…

Discussion

Mens denne metode er meget effektiv til mønster J1-afledte mESCs (SCRC-1010), har vi oplevet svingende held med andre mESC linjer og klonede isolater. For eksempel, Foxg1::venus mESCs (EB3-stammer; Danjo et al. 13) reagerer dårligt til denne protokol og Foxg1 induktion af DD12 er typisk om 1-2%. Af årsager, vi ikke helt forstår, producerer en anden Nkx2.1::mCherry:Lhx6::GFP dual reporter klon (såkaldte JQ59), der blev isoleret samtidigt som den linje, der er beskrevet i denne protoko…

Divulgations

The authors have nothing to disclose.

Acknowledgements

Vi er taknemmelige for Qing Xu for at udvikle Nkx2.1::mCherry:Lhx6:GFP dual reporter mESC linje som Jennifer Tyson, Asif Maroof og Tim Petros for deres tidlige arbejde på at udvikle denne protokol. Vi takker også CHOP flow flowcytometri kerne for teknisk bistand. Dette arbejde blev støttet af en NIH R01 MH066912 (SA) og F30 MH105045-02 (DT).

Materials

Bottle-top vacuum filter system Corning CLS430769
Test Tube with Cell Strainer Snap Cap ThermoFisher Corning 352235
Mouse embryonic fibroblasts (CF-1 MEF IRR 7M) MTI-Globalstem GSC-6101G 1 vial of 7M MEFs is sufficient for four 10-cm TC plates. References: 29,35
FBS Atlanta Biologicals S11150H
Primocin Invivogen Ant-pm-2 Also known as antimicrobial agent. Do not filter with base media — add after filtration. References: 9,11,36,37
N2 supplement-B Stemcell Technologies 7156 Do not filter with base media — add after filtration
Glutamax (100x) ThermoFisher 35050061 Also known as L-alanine-L-glutamine. References: 9,11,38,39
KnockOut Serum Replacement (KSR) ThermoFisher 10828028 Also known as serum-free medium supplement. References: 9,11
L-glutamine (100x) ThermoFisher 25030081
MEM-NEAA (100x) ThermoFisher 11140050
2-Mercaptoethanol ThermoFisher 21985023
KnockOut DMEM ThermoFisher 10829018 Also known as non-glutamine containing DMEM. References: 9,11
Hyclone FBS VWR 82013-578 Also known as stem cell grade FBS. References: 9,11
Tissue culture treated dish (10cm) BD Falcon 353003
Non-adherent sterile petri dish (10cm) VWR 25384-342
Leukemia inhibitory factor (mLIF) Chemicon ESG1107 Do not freeze, store at 4'C. References: 9,11
DMEM/F12 ThermoFisher 11330032
0.1% Gelatin Solution ATCC ATCC PCS-999-027
Laminin Sigma L2020
Poly-L-lysine Sigma P6282
Trypsin-EDTA (0.05%) ThermoFisher 25300054
Accutase ThermoFisher A1110501 Also known as non-trypsin containing cell dissociation reagent. References: 9,11
RQ1 RNase-Free DNase Promega M610A
LDN-193189 Stemgent 04-0074 Resuspend in DMSO and store at -80'C in single use aliquots
XAV939 Stemgent 04-0046 Resuspend in DMSO and store at -80'C in single use aliquots
rhFGF-2 R&D Systems 233-FB Resuspend in PBS with 0.1% BSA and store at -80'C in single use aliquots
rhIGF-2 R&D Systems 291-G1 Resuspend in PBS with 0.1% BSA and store at -80'C in single use aliquots
ROCK inhibitor (Y-27632) Tocris 1254 Resuspend in DMSO and store at -80'C in single use aliquots
Smoothened agonist (SAG) Millipore 566660-1MG Resuspend in H20 and store at -80'C in single use aliquots
rm Sonic Hedgehog/SHH R&D Systems 464-SH-025 Resuspend in PBS with 0.1% BSA and store at -80'C in single use aliquots
PKCζ Pseudosubstrate Inhibitor, Myristoylated EMD Millipore 539624 Resuspend in H20 and store at -80'C in single use aliquots
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Tags

Keywords: Mouse Embryonic Stem CellsCortical Interneuron ProgenitorsNkx2.1ParvalbuminSomatostatinMEF Feeder CellsTrypsin-EDTAGelatin-coated PlatesKSR-N2 MediaEmbryoid Bodies
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Tischfield, D. J., Anderson, S. A. Differentiation of Mouse Embryonic Stem Cells into Cortical Interneuron Precursors. J. Vis. Exp. (130), e56358, doi:10.3791/56358 (2017).
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