Rapid Detection of Neurodevelopmental Phenotypes in Human Neural Precursor Cells (NPCs)

Summary

Neurodevelopmental processes such as proliferation, migration, and neurite outgrowth are often perturbed in neuropsychiatric diseases. Thus, we present protocols to rapidly and reproducibly assess these neurodevelopmental processes in human iPSC-derived NPCs. These protocols also allow the assessment of the effects of relevant growth factors and therapeutics on NPC development.

Abstract

Human brain development proceeds through a series of precisely orchestrated processes, with earlier stages distinguished by proliferation, migration, and neurite outgrowth; and later stages characterized by axon/dendrite outgrowth and synapse formation. In neurodevelopmental disorders, often one or more of these processes are disrupted, leading to abnormalities in brain formation and function. With the advent of human induced pluripotent stem cell (hiPSC) technology, researchers now have an abundant supply of human cells that can be differentiated into virtually any cell type, including neurons. These cells can be used to study both normal brain development and disease pathogenesis. A number of protocols using hiPSCs to model neuropsychiatric disease use terminally differentiated neurons or use 3D culture systems termed organoids. While these methods have proven invaluable in studying human disease pathogenesis, there are some drawbacks. Differentiation of hiPSCs into neurons and generation of organoids are lengthy and costly processes that can impact the number of experiments and variables that can be assessed. In addition, while post-mitotic neurons and organoids allow the study of disease-related processes, including dendrite outgrowth and synaptogenesis, they preclude the study of earlier processes like proliferation and migration. In neurodevelopmental disorders, such as autism, abundant genetic and post-mortem evidence indicates defects in early developmental processes. Neural precursor cells (NPCs), a highly proliferative cell population, may be a suitable model in which to ask questions about ontogenetic processes and disease initiation. We now extend methodologies learned from studying development in mouse and rat cortical cultures to human NPCs. The use of NPCs allows us to investigate disease-related phenotypes and define how different variables (e.g., growth factors, drugs) impact developmental processes including proliferation, migration, and differentiation in only a few days. Ultimately, this toolset can be used in a reproducible and high-throughput manner to identify disease-specific mechanisms and phenotypes in neurodevelopmental disorders.

Introduction

The use of simpler organisms and mouse models has elucidated the mechanisms of basic brain development as well as disease pathogenesis. Despite these advances, the etiology of many neuropsychiatric disorders remains elusive because not all findings in simpler organisms are directly relevant to complex aspects of human disease. Further, the greater complexity of the human brain often makes it difficult to model human development and disorders in animals. With the evolution and progress of human induced pluripotent stem cells (hiPSCs) technology, somatic cells can be reprogrammed into stem cells and then differentiated into neuronal cells to study human disease. Advance....

Protocol

1. Safety Procedures and Biosafety Cabinet Maintenance

1. Biosafety Level-2 (BSL-2) Safety Procedures
   1. Follow the institution's guidelines on working with BSL-2 materials. Dispose of BSL-2 materials according to the institution's practices. Indicate rooms and equipment that are used for BSL-2 materials. Wear all personal protective equipment (PPE), including lab coat and gloves.
2. Biosafety Cabinet Maintenance
   1. Use a biosafety cabinet certified for use of BSL-2 level materials.
   2. Turn on the UV-light in the biosafety cabinet for at least 15 min, and then spray surfa....

Results

One goal of these studies is to define the proliferative activity of the NPCs, that is, an increase in cell numbers. This is achieved by assessing DNA synthesis of the total cell population, a high-throughput approach that measures the incorporation of radioactive tracer tritiated thymidine into cell extracts, and reflects all cells engaged in S-phase, whether they are synthesizing for 5 minutes or the entire two hours. Additionally, these assays allow the determination of the proportion .......

Discussion

The protocols presented here illustrate quick and simple methods to study fundamental neurodevelopmental processes and test growth factors and drugs using hiPSC-derived neural precursor cells. hiPSC technology has revolutionized the study of the pathogenesis of neurodevelopmental diseases by providing us with unprecedented access to live human neuronal cells from affected individuals. Indeed, there have been numerous hiPSC studies of neurodevelopmental disorders including Rett Syndrome, Timothy Syndrome, Fragile-X syndro.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
PSC Neural Induction Medium: Protocol Link: https://goo.gl/euub7a	ThermoFischer Scientific	A1647801	This is a kit that consists of Neurobasal (NB) medium and a 50x Neural Induction Supplement (NIS). The NIS is used to make 1X Neural Induction Medium and 100% Expansion Medium
Advanced DMEM/F12 Medium	ThermoFischer Scientific	12634-010	Component of 100% Expansion Medium
Neurobasal Medium	ThermoFischer Scientific	21103049	Component of both NIM and 100% Expansion Medium
hESC-qualified Matrigel	Corning	354277	hESC-qualified extracellular matrix-mimic gel (ECM-mimic gel)
Y-27632 (2HCl), 1 mg	Stem Cell Technologies	72302	ROCK inhibitor
6 well plates	Corning	COR-3506	Polystyrene plates used for NPC maintenance and for Neurosphere Migration Assay
24 well plates	ThermoFischer Scientific	2021-05	Polystyrene plates: Used for NPC DNA Synthesis Assay
35 mm dishes	ThermoFischer Scientific	2021-01	Polystyrene plates: Used for NPC S-Phase Entry and Neurite Assay
Natural Mouse Laminin	Invitrogen	23017-015	Substrate for coating plates: Used for NPC DNA Synthesis, S-Phase Entry, and Cell Number Assays
Fibronectin	Sigma	F1141	Substrate for coating plates: Used for Neurite Assay
Poly-D-Lysine	Sigma	P0899	Substrate for coating plates
Penicillin/Streptomycin	ThermoFischer Scientific	15140122	Antibiotic, component of NIM, 100% Expansion and 30% Expansion Media
StemPro Accutase	Gibco	A11105-01	1X Cell Detachment Solution
2.5% Trypsin (10X)	Gibco	15090-046	10X enzymatic solution
0.5 M EDTA	ThermoFischer Scientific	AM9261	used in trypsin solution for lifting cells for DNA synthesis assay
tritiated [3H]-thymidine	PerkinElmer	NET027E001	Radioactive tritium, thymidine
Fisherbrand 7 mL HDPE Scintillation Vials	Fisherbrand	03-337-1	Vials for liquid scintillation counting
EcoLite(+)	MP Biomedicals	0188247501	Liquid scintillation cocktail
LS 6500 multi-purpose liquid scintillation counter	Beckman Coulter	8043-30-1194	Liquid Scintillation Counter
Skatron Semi-automactic Cell Harvester Type 11019	Molecular Devices & Skatron Instruments, Inc.		Semi-automatic cell harvester
Click-iT EdU Alexa Fluor® 488 Imaging Kit	ThermoFisher Scientific	C10337	EdU and staining kit for S-Phase Entry Assay
Trypan Blue Solution, 0.4%	ThermoFisher Scientific	15250061	Assessing viability of cells
Grade GF/C filter paper	GE Healthcare Life Sciences, Whatman	1822-849	Glass fiber filter paper
Human Basic FGF-2	Peprotech	100-18B	growth factor
Pituitary Adenylate Cyclase Activating Polypeptide (PACAP-38)	BACHEM	H-8430	neuropeptide