Post-differentiation Replating of Human Pluripotent Stem Cell-derived Neurons for High-content Screening of Neuritogenesis and Synapse Maturation

Summary

This protocol describes a detailed procedure for resuspending and culturing human stem cell derived neurons that were previously differentiated from neural progenitors in vitro for multiple weeks. The procedure facilitates imaging-based assays of neurites, synapses, and late-expressing neuronal markers in a format compatible with light microscopy and high-content screening.

Abstract

Neurons differentiated in two-dimensional culture from human pluripotent stem-cell-derived neural progenitor cells (NPCs) represent a powerful model system to explore disease mechanisms and carry out high content screening (HCS) to interrogate compound libraries or identify gene mutation phenotypes. However, with human cells the transition from NPC to functional, mature neuron requires several weeks. Synapses typically start to form after 3 weeks of differentiation in monolayer culture, and several neuron-specific proteins, for example the later expressing pan-neuronal marker NeuN, or the layer 5/6 cerebral cortical neuron marker CTIP2, begin to express around 4-5 weeks post-differentiation. This lengthy differentiation time can be incompatible with optimal culture conditions used for small volume, multi-well HCS platforms. Among the many challenges are the need for well-adhered, uniformly distributed cells with minimal cell clustering, and culture procedures that foster long-term viability and functional synapse maturation. One approach is to differentiate neurons in a large volume format, then replate them at a later time point in HCS-compatible multi-wells. Some main challenges when using this replating approach concern reproducibility and cell viability, due to the stressful disruption of the dendritic and axonal network. Here we demonstrate a detailed and reliable procedure for enzymatically resuspending human induced pluripotent stem cell (hiPSC)-derived neurons after their differentiation for 4-8 weeks in a large-volume format, transferring them to 384-well microtiter plates, and culturing them for a further 1-3 weeks with excellent cell survival. This replating of human neurons not only allows the study of synapse assembly and maturation within two weeks from replating, but also enables studies of neurite regeneration and growth cone characteristics. We provide examples of scalable assays for neuritogenesis and synaptogenesis using a 384-well platform.

Introduction

Human pluripotent stem cell (hiPSC)-derived neurons are increasingly relevant in the areas of basic research, drug development, and regenerative medicine. Workflows and procedures to optimize their culture and maintenance, and improve the efficiency of differentiation into specific neuronal subtypes, are evolving rapidly^1,2. To improve the utility and cost-effectiveness of human stem cell-derived neurons as model systems amenable to high-content analyses in drug discovery and target validation, it is useful to decrease the culturing time required to generate mature, functional neurons, while retaining maximum ....

Protocol

1. Differentiation Period Prior to Replating

1. Differentiate neurons on 10 cm dishes, using a protocol of choice^7,8 until neurons have formed a thick network with their processes and express not only early neuronal markers such as MAP2 or TuJ1, but also late markers such as NeuN.
2. Change half the medium of choice every 4 days during the neuronal differentiation process.
   NOTE: More extensive or frequent medium changes dilute essential trophic factors, and could disfavor maturation.
   1. For the iPSC-derived WT126 neurons, use the following post-differentiation c....

Results

The replating of hiPSCs-derived neurons that have been differentiated for multiple weeks offers several advantages. However, detaching and replating differentiated neurons that have long, interconnected dendrites and axons (Figure 1A) can result in a high fraction of irreversibly damaged neurons.

As described in the protocol section, we used incubation with a proteolytic enzyme to detach the neurons from the substrate. Typically, due to their thic.......

Discussion

We have demonstrated a straight-forward procedure for the resuspension and replating of human neuronal cultures that optimizes viability, differentiation success, and subcellular imaging in a manner that is compatible with high content screening platforms, and other assays relevant to drug discovery. Figure 6 illustrates the overall workflow and examples of such applications.

Although here we focused on hiPSC-derived neurons that are directed toward a cortical neu.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Post Replating Media
L-Ascorbic Acid	Sigma	A4403	Add 1 mL of 200 mM stock to 1 L of N2B27 media
Dibutyryl-cAMP	Sigma	D0627	Add 1 µM
Human BDNF	Peprotech	450-02	10 ng/ml final concentration
B27 (50x)	Thermofisher Scientific	17504044	Add 20 mL to 1 L N2B27 media
DMEM/F12 with Glutamax	Thermofisher Scientific	31331093	Add N2 and distribute in 50 mL conicals; parafilm wrap lids
Human GDNF	Peprotech	450-10	10 ng/mL final concentration
Glutamax	Thermofisher Scientific	35050038	Add 10 ml to 1 L N2B27 media; glutamine supplement
Mouse Laminin	Sigma	P3655-10mg	Add 100 µL to 50 mL N2B27
MEM Nonessential Amino Acids	Thermofisher Scientific	11140035	Add 5 mL to 1 L N2B27 media
N2 (100x) Supplement	Life Technologies	17502048	Add 5 mL to 500 mL media
Neurobasal A Media	Thermofisher Scientific	10888022	Combine with DMEM/F12 to generate N2B27 media for CVB wt cells; neural basal A media
Neurobasal Media	Thermofisher Scientific	21103049	for WT126 cells; neural basal media
SM1 Supplement	StemCell Technologies	5711	Add 1:50 to media
Sodium bicarbonate	Thermofisher Scientific	25080-094	Add 10 mL to 1 L N2B27 media
Plate Preparation
10 cm Tissue Culture Dishes	Fisher Scientific	08772-E	Plastic TC-treated dishes
6-well Tissue Culture Dishes	Thomas Scientific	1194Y80	NEST plates
Mouse Laminin	Life Technologies	23017-015	Add 1:400 on plastic
Poly-Ornithine	Sigma	P3655-10mg	Add 1:1,000 on plastic
UltraPure Distilled Water	Life Technologies	10977-015	To dilute Poly-L-Ornithine
Replating Reagents
100 mM Cell Strainer	Corning	431752	Sterile, individually wrapped
384-well plate, uncoated	PerkinElmer	6007550	Coat with PLO and Laminin
DPBS	Life Technologies	14190144	Dulbecco's phosphate-buffered saline
Poly-D-Lysine-Precoated 384-well Plates	PerkinElmer	6057500	Rinse before coating with laminin
StemPro Accutase	Life Technologies	A1110501	Apply 1 mL/10 cm plate for 30-45 min; proteolytic enzyme
Fixation Materials
37% Formaldehyde	Fisher Scientific	F79-1	Dissolved in PBS
Sucrose	Fisher Scientific	S5-12	0.8 g per 10 mL of fixative
Immunostaining Materials
Alexa Fluor 488 Goat anti-mouse	Invitrogen	A-11001	secondary antibody
Alexa Fluor 568 Goat anti-chicken	Invitrogen	A-11041	secondary antibody
Alexa Fluor 647 Goat anti-chicken	Invitrogen	A-21449	secondary antibody
Alexa Fluor 561 Goat anti-rat	Invitrogen	A-11077	secondary antibody
DAPI	Biotium	40043	visualizes DNA
Mouse antibody against b3-tubulin (TuJ-1)	Neuromics	MO15013	early stage neuronal marker
Rat antibody against CTIP2	Abcam	ab18465	layer 5/6 cortical neurons
Chicken antibody against MAP2	LifeSpan Biosciences	LS-B290	early stage neuronal marker
Chicken antibody against NeuN	Millipore	ABN91	late stage neuronal marker
Rabbit antibody against MAP2	Shelley Halpain	N/A	early stage neuronal marker
Mouse antibody against PSD-95	Sigma	P-246	post-synaptic marker
Rabbit antibody against Synapsin 1	Millipore	AB1543	pre-synaptic marker
Bovine serum albumin (BSA)	GE Healthcare Life Sciences	SH30574.02	10% in PBS for blocking
Titon X-100	Sigma	9002931	Dilute to 0.2% on PBS for permeabilization
Viability Markers
Vivafix 649/660	Biorad	135-1118	cell death marker
Calcium Imaging
Name of Reagent/ Equipment	Company	Catalog Number	Comments/Description
AAV8-syn-jGCAMP7f-WPRE	THE SALK INSTITUTE, GT3 Core Facility	N/A	calcium reporter in a viral delivery system
hiPSC-derived NPCs
WT 126 (Y2610)	Gage lab	N/A	Marchetto et al., 2010
CVB WT24	Yeo and Goldstein labs	N/A	unpublished