Synaptic Microcircuit Modeling with 3D Cocultures of Astrocytes and Neurons from Human Pluripotent Stem Cells

Summary

In this protocol, we aim to describe a reproducible method for combining dissociated human pluripotent stem cell derived neurons and astrocytes together into 3D sphere cocultures, maintaining these spheres in free floating conditions, and subsequently measuring synaptic circuit activity of the spheres with immunoanalysis and multielectrode array recordings.

Abstract

A barrier to our understanding of how various cell types and signals contribute to synaptic circuit function is the lack of relevant models for studying the human brain. One emerging technology to address this issue is the use of three dimensional (3D) neural cell cultures, termed 'organoids' or 'spheroids', for long term preservation of intercellular interactions including extracellular adhesion molecules. However, these culture systems are time consuming and not systematically generated. Here, we detail a method to rapidly and consistently produce 3D cocultures of neurons and astrocytes from human pluripotent stem cells. First, pre-differentiated astrocytes and neuronal progenitors are dissociated and counted. Next, cells are combined in sphere-forming dishes with a Rho-Kinase inhibitor and at specific ratios to produce spheres of reproducible size. After several weeks of culture as floating spheres, cocultures ('asteroids') are finally sectioned for immunostaining or plated upon multielectrode arrays to measure synaptic density and strength. In general, it is expected that this protocol will yield 3D neural spheres that display mature cell-type restricted markers, form functional synapses, and exhibit spontaneous synaptic network burst activity. Together, this system permits drug screening and investigations into mechanisms of disease in a more suitable model compared to monolayer cultures.

Introduction

Astrocytes are a highly abundant glial cell type within the central nervous system (CNS) with a variety of functional responsibilities beyond structural support. Through secretion of soluble synaptogenic factors and extracellular matrix (ECM) components, astrocytes aid in the establishment and clustering of mature synapses during development¹. They also play a critical role in maintaining the health and plasticity of synapses through extracellular signaling^2,3,4,5, and contribute to long-term stability of homeostatic ....

Protocol

1. Cell Culture and Reagent Preparation

NOTE: The protocols in this section are written in the order in which they appear in the differentiation protocol (section 2). See the Table of Materials for materials and catalog numbers.

1. Prepare coated plates for cell culture.
   1. Dilute extracellular matrix (ECM) coating solution with DMEM/F12 media to prepare a 1 mg/mL stock solution. Aliquot the diluted ECM stock solution into 30 conical tubes of 3 mL each and immediately store in -20 °C. For a working solution, resuspend 3 mL of ECM stock into 33 mL of pre-chilled DMEM/F12 media, bringin....

Results

When performed properly, this protocol will produce defined populations of functional cocultures of astrocytes^28,33,34 and neurons³⁵ generated from hPSCs (Figure 1A-1C), as detailed previously²⁶ and described here in steps 2.1–2.2. This stepwise procedure, with the use of microwell plates, is expected t.......

Discussion

In this protocol, we describe a systematic method for the production of 3D spheres of neural cocultures. The spheres are composed of astrocytes and neurons, which are derived independently from hPSCs. Though not the focus of this protocol, the generation of pure populations of astrocytes from hPSCs²⁸ is a critical step and can be technically challenging if performed without prior experience. This first step in the generation of these synaptic microcircuits should be performed with meticulous timin.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
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 μM
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 μg/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 μg/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 μM
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