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Abstract
Introduction
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Developmental Biology

Synaptic dataloger modellering med 3D Cocultures astrocytter og neuroner fra humane pluripotente stamceller

Published: August 16, 2018
doi:
10.3791/58034
, ,
1Center for Neuroregeneration, Department of Neurosurgery,Houston Methodist Research Institute

Summary

I denne protokol, vi har til formål at beskrive en reproducerbar metode til at kombinere dissocieres humane pluripotente stamceller afledt neuroner og astrocytter sammen i 3D sfære cocultures, vedligeholdelse af disse områder i frie flydende betingelser, og efterfølgende måle synaptic kredsløb aktivitet af kugler med immunoanalysis og multielectrode array optagelser.

Abstract

En hindring for vores forståelse af, hvordan forskellige celletyper og signaler bidrage til synaptic kredsløb funktion er manglen på relevante modeller for at studere den menneskelige hjerne. En ny teknologi til at løse dette problem er brugen af tre-dimensionelle (3D) neurale cellekulturer, kaldes ‘organoids’ eller ‘spheroids’, for langsigtet præservering af intercellulære interaktioner herunder ekstracellulære adhæsionsmolekyler. Men systemerne kultur er tidskrævende og ikke systematisk genereret. Her, vi beskriver en metode til hurtigt og konsekvent producere 3D cocultures af neuroner og astrocytter fra humane pluripotente stamceller. Første, forhånd differentieret astrocytter og neuronal ophav er adskilt og tælles. Næste, celler i kugle-dannende kombineret retter med en Rho-Kinase inhibitor og på specifikke nøgletal til at producere kugler af reproducerbare størrelse. Efter flere ugers kultur som flydende kugler, er cocultures (‘asteroider’) Endelig sectioned for immunfarvning eller forgyldt ved multielectrode arrays til at måle synaptic tæthed og styrke. Generelt forventes det, at denne protokol vil give 3D neurale kugler, der vise modne celletype begrænset markører, danne funktionelle synapser og udstille spontan synaptic netværk burst aktivitet. Sammen, tillader dette system stof screening og undersøgelser mekanismer af sygdom i en mere velegnet model i forhold til éncellelag kulturer.

Introduction

Astrocytter er en meget rigelige glial celletype inden for det centrale nervesystem (CNS) med en lang række funktionelle ansvar uden for strukturstøtte. Gennem udskillelsen af opløselige synaptogenic faktorer og ekstracellulære matrix (ECM) komponenter, astrocytter støtte i etableringen og klynger af modne synapser under udvikling1. De også spiller en afgørende rolle i at bevare sundhed og plasticitet i synapserne gennem ekstracellulære signaling2,3,4,5, og bidrage til stabilitet på lang sigt af homeostatiske miljøer ved at regulere ekstracellulære kalium samt glutamat og udskillelsen af energi substrater og ATP6,7,8. Endelig, de kan bidrage til neurotransmission ved at påvirke extrasynaptic strømninger9, og kan indirekte påvirke aktivitet gennem andre celletyper såsom fremme af myelination10. Vigtigere, fordi abnormitet eller dysfunktion af astrocytter kan føre til mange udviklingsforstyrrelser syndromer og voksen neuropatologiske, er der et indlysende behov for at medtage astrocytter sammen med neuroner i manipuleret neurale netværk i rækkefølge for en forbedret model af den endogene hjernen miljø. En integreret kendetegner astrocytter er deres evne til formen dynamisk samspil med neuronal synapser1,11,12. I mangel af glia danner neuroner et begrænset antal synapser, der generelt mangler også funktionelle modenhed13.

Menneskelige astrocytter vise morfologiske, transcriptional og funktionelle egenskaber — såsom øget størrelsen og kompleksiteten af forgrenede, samt artsspecifikke gener – der ikke er sammenfattet i gnavere12,14, 15. Som et resultat, studerer udnytte menneskelige pluripotente stamceller (hPSC)-afledte neurale celler har blive bredt accepteret som en mulighed for at undersøge CNS-relaterede sygdomme i vitro samtidig udvikle nye behandlingsformer, skade modeller og kultur paradigmer16 ,17. Derudover tillader hPSCs undersøgelse af menneskelige synapse dannelsen og funktionen uden behov for primære væv18,19.

En hindring for vores forståelse af, hvordan forskellige celletyper og signaler bidrage til synaptic kredsløb funktion er manglen på relevante modeller af den menneskelige hjerne. Der er behov for en passende platform til at sammenfatte sine synaptic netværk med high fidelity og reproducerbarhed. For nylig har interesse er opstået i produktion af 3D kultur systemer (bredt kendt som ‘organoids,’ ‘spheroids’ eller ‘mini hjerner’)20 model komplekse tredimensionale (3D) strukturer på cellulære og makro niveau. 3D kultur systemer bevarer ECM og celle-celle interaktioner, der er normalt fraværende eller begrænset under typiske 2D coculture paradigmer21,22. En overflod af teknikker findes for dyrkning 3D neurale spheroids23,24,25; men mange kræver langvarige kultur perioder (måneder til år) til spontan udvikling og lag bevarelse, med brugeren udviser meget lidt kontrol over output.

Her viser vi en systematisk metode til hurtigt og konsekvent bioengineer neurale interaktioner blandt flere celletyper (pre differentieret neuroner og astrocytter) afledt af hPSCs ved at samle celler i område cocultures (‘asteroider’)26 at sammenfatte menneske-specifikke morfologiske kompleksiteter i 3D. Denne high-density neurale systemet genererer jævnt spredt neurale undertyper, der tager på ældre ejendomme over tid og kan screenet eller analyseres i en høj overførselshastighed måde. Vi demonstrere for første gang at menneskelige astrocytter fremkalde synaptic netværksaktivitet brast i disse 3D cocultures. Derudover er denne protokol let at tilpasse til at generere områder af forskellige størrelser, til at udnytte cellerne angivet til forskellige regionale identiteter i CNS, og til at studere samspillet mellem flere andre celletyper som ønsket.

Protocol

1. celle kultur og reagens Bemærk: Protokoller i dette afsnit er skrevet i den rækkefølge som de vises i differentiering protokol (afsnit 2). Se Tabel af materialer til materialer og katalog numre. Forberede overtrukne plader til cellekultur. Fortynd ekstracellulære matrix (ECM) belægning løsning med DMEM/F12 medier til at forberede en 1 mg/mL stamopløsning. Alikvot fortyndet ECM lagerfører løsning til 30 koniske rør af 3 mL og u…

Representative Results

Når udført korrekt, vil denne protokol producere definerede populationer af funktionelle cocultures astrocytter28,33,34 og neuroner35 genereret fra hPSCs (figur 1A-1C), som detaljerede tidligere26 og beskrevet her i trin 2.1-2.2. Denne trinvis procedure, med brug af microwell plader, forventes at give…

Discussion

I denne protokol beskriver vi en systematisk metode til fremstilling af 3D kugler af neurale cocultures. Områderne består af astrocytter og neuroner, som er afledt uafhængigt af hPSCs. Dog ikke i fokus i denne protokol, generation af ren populationer af astrocytter fra hPSCs28 er et kritisk skridt og kan være en teknisk udfordring, hvis udføres uden forudgående erfaring. Dette første skridt i generation af disse synaptic mikrokredsløb bør udføres med minutiøse tidtagning og sans for det…

Disclosures

The authors have nothing to disclose.

Acknowledgements

Vi vil gerne takke Dr. Erik Ullian (UCSF) for intellektuelle bidrag til udformningen af disse procedurer, Dr. Michael Ward (NIH) for teknisk rådgivning om iNeuron differentiering, og Saba Barlas for foreløbige billedanalyse.

Materials

6 well plate Fisher Scientific 08-772-1B
15 ml conical tubes Olympus Plastics 28-101
Accutase Sigma A6964-100ML Detachment solution
AggreWell plate Stemcell Technologies 34850
Anti-Adherence Rinsing Solution Stemcell Technologies 7010 Prevent cell adhesion to microwell plates
Anti/anti Thermofisher 15240062
B27 Thermofisher 17504044 Media Supplement
BrainPhys neuronal medium Stemcell Technologies 5790 Neurophysiological basal medium alternative
Circular glass coverslips Neuvitro GG-12-oz
Cryostor CS10 Stemcell Technologies 7930 Cryopreservation medium with 10% DMSO
DMEM/F12 Thermofisher 10565-042 With GlutaMAX supplement
DMH-1 Stemcell Technologies 73634 HAZARD: Toxic if swallowed. Working concentration: 2 uM
Donkey serum Lampire Biological Laboratories 7332100 Working concentration: 5% in primary blocking buffer, 1% in secondary blocking buffer
Doxycycline Hydrochloride (Dox) Sigma D3072-1ml HAZARD: Toxic for pregnant women. Working concentration: 2 ug/mL
Epidermal growth factor (EGF) Peprotech AF-100-15 Working concentration: 10 ng/mL
Fibroblast growth factor-2 (FGF) Peprotech 100-18B Working concentration: 10 ng/mL
Fluoromount-G mounting solution Southern Biotech 0100-01
Glass slides Fisherbrand 22-037-246
Goat serum Lampire Biological Laboratories 7332500 Working concentration: 5% in primary blocking buffer, 1% in secondary blocking buffer
Hemacytometer or automatic cell counter Life Technologies AMQAX1000
Heparin Sigma H3149-50KU Working concentration: 2 mg/mL
Magnetic plate DLAB 8030170200
Matrigel membrane matrix Corning 354230 ECM coating solution. Working concentration: 80 ug/ml. Prepare on ice and ensure that pipettes, tubes, and media are pre-chilled.
MEA 2100 System Multichannel Systems MEA2100
Mounting solution
N2 Thermofisher 17502048 Media Supplement
OCT Tissue-Tek 4583 Tissue embedding solution for cryosectioning
Pap Pen (Aqua Hold) Scientific Device Laboratory 9804-02
Paraformaldehyde (PFA) Acros Organics 169650025 HAZARD: Toxic if inhaled. Working concentration: 4% in PBS
Phosphate buffered saline (PBS) Stemcell Technologies CA008-300
Poly-l-ornithine (PLO) Sigma P3655-100MG Working concentration: 0.5 mg/mL
Rectangular glass cover slips Fisherfinest Premium Superslip 12-545-88
ReLeSR Stemcell Technologies 5872 Detachment and passaging reagent
Rho-Kinase Inhibitor Y27632- (Y) Tocris 1254 Working concentration: 10 uM
SB431542 Stemcell Technologies 72234 Working concentration: 2 uM
Spinner flasks Fisher Scientific 4500-125
Sucrose Fisher Chemical S5-3 Working concentration: 20% or 30% in PBS
T25 Culture Flask Olympus Plastics 25-207 Vented caps
T75 Culture Flask Olympus Plastics 25-209 Vented caps
Terg-A-zyme Sigma Z273287-1EA Detergent. Working concentration: 1%
TeSR-E8 basal medium Stemcell Technologies 5940 Human pluripotent stem cell (hPSC) medium
TeSR-E8 supplements Stemcell Technologies 5940 Supplements for human pluripotent stem cell medium
TritonX-100 Sigma X100-500ML Detergent for cell permeabilization. Working concentration: 0.25% in blocking buffer
Trypan blue Invitrogen T10282
Antibodies
AlexaFluor 488 Thermofisher A-11029 Secondary antibody
AlexaFluor 594 Thermofisher A-11037 Secondary antibody
Ezrin Thermofisher MA5-13862 Primary antibody; astrocytes perisynaptic
GFAP Chemicon MAB360 Primary antibody; astrocytes
GFP Aves GFP-1020 Primary antibody; astrocytes
Glt1 Gift from Dr. Jeffrey Rothstein n/a Primary antibody; astrocytes
Homer Synaptic Systems 160 011 Primary antibody; neurons, post-synaptic
MAP2 Synaptic Systems 188 004 Primary antibody; neurons
PSD95 Abcam ab2723 Primary antibody; neurons, post-synaptic
S100 Abcam ab868 Primary antibody; astrocytes
Synapsin 1 Synaptic Systems 106 103 Primary antibody; neurons, pre-synaptic
TuJ1/β3-tubulin (TUBB3) Covance MMS-435P Primary antibody; neurons
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Tags

Synaptic Microcircuit3D CoculturesAstrocytesNeuronsHuman Pluripotent Stem CellsNeural Cell TypesSynaptic Circuit FormationNeuroscienceOrganoid TechnologiesDrug DevelopmentNeuroregenerationNeural ProgenitorsAstrocyte ProgenitorsAstrocyte SubtypesNeural InductionNeural MediumRho-kinase InhibitorECM-coated PlatesCell AggregationCell DissociationCell Identity ConfirmationCryo-preservation
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Cvetkovic, C., Basu, N., Krencik, R. Synaptic Microcircuit Modeling with 3D Cocultures of Astrocytes and Neurons from Human Pluripotent Stem Cells. J. Vis. Exp. (138), e58034, doi:10.3791/58034 (2018).
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