Isolering og dyrkning af Adult Zebrafisk Brain-afledte Neurosfærer
Summary
Her giver vi en reproducerbar metode til at undersøge voksen neurogenese ved anvendelse af en neurosfære assay afledt af hele hjernen eller fra enten telencephalic, tectal eller cerebellare regioner af den voksne zebrafisk hjerne. Desuden har vi beskrive, hvorledes det manipulere genekspression i zebrafisk neurosfærer.
Abstract
The zebrafish is a highly relevant model organism for understanding the cellular and molecular mechanisms involved in neurogenesis and brain regeneration in vertebrates. However, an in-depth analysis of the molecular mechanisms underlying zebrafish adult neurogenesis has been limited due to the lack of a reliable protocol for isolating and culturing neural adult stem/progenitor cells. Here we provide a reproducible method to examine adult neurogenesis using a neurosphere assay derived from zebrafish whole brain or from the telencephalon, tectum and cerebellum regions of the adult zebrafish brain. The protocol involves, first the microdissection of zebrafish adult brain, then single cell dissociation and isolation of self-renewing multipotent neural stem/progenitor cells. The entire procedure takes eight days. Additionally, we describe how to manipulate gene expression in zebrafish neurospheres, which will be particularly useful to test the role of specific signaling pathways during adult neural stem/progenitor cell proliferation and differentiation in zebrafish.
Introduction
Mammale neurale stamceller (NSC) er blevet karakteriseret in vitro ved deres evne til at vokse i frit flydende kulturer som klynger af delende celler betegnet neurosfærer 1. I nærvær af epidermal vækstfaktor (EGF) og fibroblast vækstfaktor (FGF), NSC'er opdele enten symmetrisk at generere selvfornyende NSC'er eller asymmetrisk at generere to forskellige datterceller, dvs.., En differentierende progenitorcelle og en hidtil ukendt NSC. Neurosfære kulturer er derfor en blanding af neurale stamceller / progenitorceller og mere differentierede neurale celler 2-4 NSC'er kan imidlertid skelnes fra andre neurosfære celletyper ved to specifikke egenskaber:. De udviser langvarig selvfornyelse i fri- flydende kulturer og de kan differentiere til alle neurale cellelinier (dvs. neuroner, astrocytter og oligodendrocytter) efter tilbagetrækning af vækstfaktorer og adhæsion til ekstracellulær matrix substrater. I Mammals, neurosfæren kultur-systemet var den første in vitro-system, der anvendes til at påvise tilstedeværelsen af NSC i den voksne hjerne og er fortsat det mest anvendte værktøj til at analysere spredning, selvfornyelse kapacitet og multipotency af neurale stamceller og stamceller. Selv om kugle-dannende analyser lider nogle ulemper og begrænsninger 4, denne kultur system er værdifuld for at vurdere potentialet i en celle til at opføre sig som en stamcelle, når fjernet fra dets in vivo-niche 4 og har været medvirkende til at identificere centrale regulatorer af NSC selv-fornyelse og celle skæbne beslutsomhed 5-7.
I modsætning til pattedyr, der har begrænset voksen neurogenese, zebrafisk konstitutivt producere nye neuroner langs hele hjernen akse hele deres liv. Zebrafisk voksne hjerne vises flere neurogene nicher huser neurale stamceller / progenitorceller gør zebrafisk en kraftfuld model organisme for forståelse than stamcelle aktivitet i hjernen samt de molekylære programmer, som kræves for centralnervesystemet regenerering. I løbet af de sidste 17 år, flere forskergrupper udviklet metoder til isolering og dyrkning af zebrafisk nerveceller 8,9. Disse undersøgelser havde til formål at producere embryonale neuronal og gliaceller in vitro, men ikke på at fastholde NSC'er og undersøge deres egenskaber. Selvom neurosfærer er blevet genereret i den voksne Apteronotus leptorhynchus (Brown Ghost Knifefish) 10, en neurosfære-dannende assay i zebrafisk manglede at blive etableret.
Her beskriver vi en neurosfære-dannende assay at demonstrere rolle miR-107 under zebrafisk neurogenese 11. Protokollen giver: 1) indsamling af voksne neurale stamceller / progenitorceller enten fra zebrafisk hele hjernen eller fra flere dissekerede hjernen regioner som telencephalon den Tectum, og lillehjernen; 2) dannelse af flydende og selvfornyende neurosfærer fra voksne neurale stamceller / stamceller; 3) down- og opregulering af ekspressionen af kodende gener eller små ikke-kodende RNA'er 11 i neurosfærer, for at undersøge deres rolle i proliferation og differentiering af neurale stamceller / progenitorceller.
Protocol
Representative Results
Discussion
The main aim of this protocol is to isolate and culture adult zebrafish brain-derived neurospheres for studying the cellular and molecular features of neural stem/progenitor cells. Here, we report how to select multipotent neural cells and generate the three neural cell types, i.e., astrocytes, neurons and oligodendrocytes, from the adult zebrafish brain. The protocol is highly significant since a reproducible neurosphere-forming assay had not been established in the zebrafish so far.
<p class="jove_content"…Declarações
The authors have nothing to disclose.
Acknowledgements
The authors thank Guillermina Hill-Teran and Marie-Elise Schwartz for assistance. This work was supported by grants from the National Institutes of Health (5R00HL105791 to S.N.) and from the Alzheimer (NIRP 12-259162). This work was also supported by Institut National de la Santé et de la Recherche Médicale (CFC and JLT), Agence Nationale de la Recherche (13-BSV4-0002-01 (JLT), NIH (1R01EB016629-01A1 (JLT), Connecticut Stem Cell Research Fund (13-SCA-Yale-04 (JLT).
Materials
| DPBS 1X | Life Technologies | 14190-144 | |
| DMEM/F12 1X | Life Technologies | 11330-032 | |
| L-Cysteine hydrochloride monohydrate | Sigma | C6852-25g | |
| B-27 | Life Technologies | 17504-044 | |
| N-2 | Life Technologies | 17502-048 | N-2 supplement (100x) liquid |
| HEPES | Life Technologies | 15630 | 1M |
| D-(+)-Glucose 45% | Sigma | G8769 | |
| Penicillin-streptomycin | Life Technologies | 15140-122 | |
| Fetal Bovine Serum | Life Technologies | 16000044 | |
| Human FGF-basic | Peprotech | 100-18B | |
| Human EGF | Peprotech | AF-100-15 | |
| Insulin | Sigma | I5500-50 mg | |
| DNAse | Sigma | DN25-10mg | |
| Papain | Worthington Biochemical Corporation | LS003126 | |
| Matrigel | Becton Dickinson | 356234 | |
| PFA | TCI | P0018 | |
| PBS | AmericanBio | AB11072-04000 | |
| Tricaine MS-222 | Sigma | A5040 | stock solution of 4 mg/ml. |
| Trycold gel | Sigma | TGP8 | gel pack |
| Amaxa Basic Nucleofector Kit | Lonza | VPI-1004 | |
| Trypan blue stain | Life Technologies | 15250061 |
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