An Enzyme- and Serum-free Neural Stem Cell Culture Model for EMT Investigation Suited for Drug Discovery

Summary

Epithelial to mesenchymal transition (EMT) allows cancers to become invasive. To investigate EMT, a neural stem cell (NSC)-based in vitro model devoid of serum and enzymes is described. This standardized system allows quantitative and qualitative assessment of cell migration, gene and protein expression. The model is suited for drug discovery.

Abstract

Epithelial to mesenchymal transition (EMT) describes the process of epithelium transdifferentiating into mesenchyme. EMT is a fundamental process during embryonic development that also commonly occurs in glioblastoma, the most frequent malignant brain tumor. EMT has also been observed in multiple carcinomas outside the brain including breast cancer, lung cancer, colon cancer, gastric cancer. EMT is centrally linked to malignancy by promoting migration, invasion and metastasis formation. The mechanisms of EMT induction are not fully understood. Here we describe an in vitro system for standardized isolation of cortical neural stem cells (NSCs) and subsequent EMT-induction. This system provides the flexibility to use either single cells or explant culture. In this system, rat or mouse embryonic forebrain NSCs are cultured in a defined medium, devoid of serum and enzymes. The NSCs expressed Olig2 and Sox10, two transcription factors observed in oligodendrocyte precursor cells (OPCs). Using this system, interactions between FGF-, BMP- and TGFβ-signaling involving Zeb1, Zeb2, and Twist2 were observed where TGFβ-activation significantly enhanced cell migration, suggesting a synergistic BMP-/TGFβ-interaction. The results point to a network of FGF-, BMP- and TGFβ-signaling to be involved in EMT induction and maintenance. This model system is relevant to investigate EMT in vitro. It is cost-efficient and shows high reproducibility. It also allows for the comparison of different compounds with respect to their migration responses (quantitative distance measurement), and high-throughput screening of compounds to inhibit or enhance EMT (qualitative measurement). The model is therefore well suited to test drug libraries for substances affecting EMT.

Introduction

During several stages of embryonic development, epithelial cells lose their strong adherence to each other (e.g., tight junctions) and acquire a migratory phenotype in a process called epithelial to mesenchymal transition (EMT)¹. EMT is required for the formation of additional cell types, such as the mesenchymal neural crest cells, a population that segregates from the neuroepithelium². EMT is not only essential during embryonic stages but also required at later stages of adult life to maintain physiological processes in the adult organism, such as wound healing³and central nervous system (CNS) regeneration in demyelinating le....

Protocol

All animal procedures followed the 'Guide for the Care and Use of Laboratory Animals' (NIH publication, 8^th edition, 2011) and were approved by the Animal Welfare Committee of Basel (Swiss Guidelines for the Care and Use of Animals). By these guidelines the animal protocol is considered of "lowest animal severity grade".

1. Preparation of Expansion Medium

Note: Work in aseptic conditions as standard for tissue culture.

1. Take two 15 ml tubes, and add 5 ml of L-glutamine-free DMEM/F12 (1:1) from a 500 ml medium bottle into each of the two 15 ml tubes. To the first 15 ml tube, add 50 mg human apo-transferrin, 50 μl....

Results

This EMT model system is based on the standardized isolation of NSCs both as single cells or as explants from a specific region of the developing neural tube, the central cortex (Figures 1 and 2). For quantitative assessment, explants were seeded right at the center of a 500 µm grid culture dish (Figure 3). Explants from the central cortex were first exposed to FGF2 for two days, followed by additional two days in different combinations o.......

Discussion

In this study a standardized system for EMT analysis utilizing NSCs is described (summarized in Supplementary Figure 3). The standardization ensures reproducibility (Table 1 and 2). The NSCs are derived from the developing cortex, a tissue that normally does not undergo EMT. This is of advantage for the analysis of early steps in EMT. Initial steps in EMT cannot be adequately studied in tumor cells that have accumulated genetic changes and may have already adopted EMT features. Moreover,.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
BMP4, rhBMP4	RnD Systems	314-BP-01M
Bovine pancreas insulin	Sigma	I1882
Boyden chamber, CytoSelect cell invasion assay	Cell Biolabs	CBA-110	24 well plate system
Cell culture dish with grid	Ibidi 500 mm dish, 35 mm	80156
CellMask Orange	Life Technologies	C10045	Plasma membrane dye, use at 1:1,000.
DAPI	LifeTechnologies	D1306	Stock at 5 mg/ml. Use at 1:10,000. Cancerogenic. Appropriate protection (gloves, coat, goggles) required.
DMEM/F12 1:1 medium bottle	Gibco Invitrogen	21331-020
FGF2, rhFGF2	RnD Systems	233-FB-01M
Fibronectine, bovine	Sigma	F4759
Glutamax supplement	Gibco Invitrogen	35050-061
Graphics software with pixel measurement feature	Fiji	fiji.sc/Fiji	version 2.0.0-rc-30/1.49s
HBSS media	Sigma	H9394
Human apo-Transferrin	Sigma	T1147	Possible lung irritant. Avoid inhalation. Use appropriate protection.
L-glutamine	Gibco Invitrogen	25030-024
Nestin, Mouse anti Nestin antibody	Genetex	GTX26142	Use at 1:100, 4% PFA fixation, Triton X100 at 0.1%
Olig2, Rabbit anti Olig2 antibody	Provided by Hirohide Takebayash	Personal stock	Use at 1:2,000, 4% PFA fixation, Triton X100 at 0.1%
Penicillin/Streptomycin/Fungizone	Gibco Invitrogen	15240-062
Podoplanin, Mouse anti Podoplanin antibody	Acris	DM3614P	Use at 1:250, 4% PFA fixation, avoid Triton X100
Poly-L-ornithine	Sigma	P3655
Putrescine	Sigma	P5780	Skin and eye irritant. Appropriate protection required.
Sodium selenite	Sigma	S5261
Sox10, Rabbit anti Sox10 antibody	Millipore Chemicon	AB5774	Use at 1:200, 4% PFA fixation, Triton X100 at 0.1%
TGFb1, rhTGFb1	RnD Systems	240-B-010
Uncoated Petri dishes	Falcon Corning	351029