Dual Lentiviral Transduction To Generate Mutant Neural Stem Cells For Glioma Research

Summary

This protocol describes the generation of mutant murine neural stem cells (NSCs) for glioma research using sequential lentiviral transductions. Oncogenic mutations are first introduced into NSCs, followed by a bicistronic lentivirus containing reporter genes. After dual antibiotic selection, mutant NSCs enable real-time imaging of tumor growth and treatment response.

Abstract

Gliomas are the most prevalent primary brain tumors in adults, and murine modeling suggests that they may arise from oncogenic mutations in NSCs or early differentiating progenitors. Orthotopic transplantation models derived from mutant NSCs thus have the potential to yield valuable preclinical information about glioma pathogenesis, outcomes, and treatment response. These models require the introduction of an oncogenic mutation into NSCs, preferentially coupled with reporters to aid in the determination of transfection/transduction efficiency and to monitor tumor growth in vivo. This protocol outlines a dual transduction-based methodology for generating mutant NSCs suitable for in vivo tumor monitoring. In this technique, isolated NSCs are first transduced with lentiviruses that contain a gene of interest (oncogenic mutation or green fluorescent protein [GFP] control) in addition to a blasticidin-resistance element. Following blasticidin selection, cells are secondarily transduced with a bicistronic lentiviral vector expressing luciferase and red fluorescent protein (RFP), as well as a puromycin-resistance element. After puromycin selection, cells can be expanded for proliferation assays, orthotopic transplantation, or other downstream applications. This dual transduction approach allows for estimation of transduction efficiency at both infection stages as well as monitoring in vivo tumor establishment, growth, and response to therapy using noninvasive bioluminescent imaging (BLI). This system represents a useful tool for glioma research, merging the biological relevance of adult neural stem cell-derived tumor modeling with the practical benefit of real-time imaging capability, ultimately enhancing the understanding of glioma biology and the advancement of new therapeutic approaches.

Introduction

Gliomas are the most prevalent form of primary brain tumors affecting adults, constituting approximately 80% of all malignant brain neoplasms in this population¹. The majority of these are high-grade gliomas², which are typically very invasive and carry a poor prognosis^3,4 despite significant advances in neurosurgical techniques, radiation therapy, and chemotherapeutic approaches⁵. The high mortality rate in patients afflicted by these tumors highlights the need for ongoing research into glioma formation and progression.

Protocol

This study was conducted in accordance with the ethical standards and regulatory guidelines established by Washington University in St. Louis. For this protocol, the cortex of adult mice was micro-dissected as per the Brossier laboratory’s previously described technique²⁴ and used to isolate primary murine NSCs for subsequent expansion in non-adherent culture according to established protocol^24,25,26. All reagents used in culture are either supplied in sterile format or passed through a 0.2 µM membrane filter to ensure sterility. The reagent....

Results

Adult NSCs were isolated from the cortex of adult C57BL/6 mice and grown in non-adherent culture³⁹, resulting in the formation of primary neurospheres (20 – 250 µm) within 7 to 10 days (Figure 1). Cells were then expanded in vitro. Optimization of blasticidin selection was then performed in untransduced NSCs (Step 3). After 24 h of selection, concentrations at or above 1000 ng/mL (1 µg/mL) blasticidin resulted in reduced neurosphere size (

Discussion

This two-step lentiviral transduction method successfully generated genetically modified NSCs suitable for oncology research. Here, effective integration of the BRAF V600E oncogenic mutation (found in a subset of adult high-grade gliomas and used here as a proof of concept) or GFP control with dual reporter systems for bioluminescence imaging (luciferase) and fluorescence microscopy detection (RFP) was illustrated. Successive transduction at a constant MOI of 20, together with a double antibiotic selection schem.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
β-mercaptoethanol (BME)	Sigma	M3148-25ML	Use inside biological safety cabinet. Make 50 mM stocks by adding 35 mL BME to 10 mL low-glucose DMEM, sterile filter and aliquot into 500 mL aliquots for use in media preparation.
Anti-mouse IgG, HRP-linked Antibody	Cell Signal Technology	7076	Used at 1:2000 dilution in 1 % Non-fat milk
B-27 Supplement (50X),	Gibco	17504-044	At first use, thaw and make 1ml aliquots for future use; store aliquots at -20 °C. Keep supplements on ice after thawing until ready to add to media
Blasticidin S HCl (10 mg/mL)	Gibco	A1113903	Make aliquots of the 10 mg/mL stock; place in 200 mL small tubes and store at  -20 °C until use.
Bovine Serum Albumin (BSA)	Millipore-Sigma	A1470-10G	Used as 10% BSA weight/volume in 1x PBS. BSA is sticky; make sure to take care to weigh it properly and add an appropriate volume of diluent to make the exact solution.
Cell Strainer (40 µm)	Midsci	40ICS	Pre-sterilized and individually wrapped. Must be opened inside the biological safety cabinet. Do not touch with bare hands
D-Luciferin, Potassium Salt	Gold Biotechnology	eLUCK-100	Make 15 mg/mL working solution (diluted in molecular grade water) and sterile filter before use. Prepare fresh stock each time and add directly to the media to achieve final working concentration of 150 mg/mL (e.g. 5 mL working solution per 500 mL culture)
DMEM, Low-Glucose	Gibco	11885-084	Used to make base NSC medium
Epidermal Growth Factor (EGF)	Corning	354001	Supplied as a sterile lyophilized powder; to reconstitute 1000X stock, add 4.95 mL PBS and 50 µL 10% BSA (in 1x PBS) to 100 µg lyophilized EGF powder, and pipette to combine.  Make 500 µL or smaller aliquots in small tubes and store at -20° C until use for complete NSC media and NSC feeding supplement. Avoid repeat freeze-thaws.
Fibroblast Growth Factor, Basic (FGF)	R&D systems	233-FB	Supplied as a sterile lyophilized powder; to reconstitute 1000X stock, add 1.24 mL PBS and 12.5  µL 10% BSA (in 1x PBS) to 25 µg lyophilized FGF powder, and pipette to combine.  Make 500 µL or smaller aliquots in small tubes and store at -20° C until use for complete NSC media and NSC feeding supplement. Avoid repeat freeze-thaws.
Glucose, 45%	Corning	25-037-CI	Supplied as sterile solution. Do not open outside of the biological safety cabinet
HA-Tag (C29F4) Rabbit mAb	Cell Signal Technology	3724	Used at 1: 1000 dilution in 3 % Bovine serum albumin (Western blotting, to verify HA-tag expression)
HBSS, 10x	Gibco	14185-052	Supplied as sterile solution. Do not open outside of the biological safety cabinet
HEPES, 1 M	Corning	25-060-CI	Supplied as sterile solution. Do not open outside of the biological safety cabinet
IVIS 50	PerkinElmer		The instrument should be powered on 30 min before use to allow the camera to cool to optimal temperature. Living Image software (version 4.3.1) is used for quantification.
Lentivirus-Luciferase (Firefly)-2A-RFP	AMSBio	LVP324-PBS	LVP must be aliquoted and stored at -80 °C refrigerator
L-Glutamine, 200 mM	Corning	25-005-CI	Supplied as sterile solution. Do not open outside of the biological safety cabinet
Molecular-Grade Water	Corning	46-000-CM	Supplied as sterile solution. Do not open outside of the biological safety cabinet
N-2 Supplement (100x)	Gibco	17502-048	At first use, thaw and make 1ml aliquots for future use; store aliquots at -20 °C. Keep supplements on ice after thawing until ready to add to media
Nestin (E5C7I) Rabbit mAb	Cell Signal Technology	89529	Used at 1: 1000 dilution in 3 % Bovine serum albumin (Western blotting, to verify NSC marker expression)
Neurobasal Media	Gibco	21103-049	Used to make base NSC medium
Penicillin-Streptomycin (10,000 U/mL)	Gibco	15140-122	Supplied as sterile solution; aliquot and store at -20°C until use.
Phosphate Buffered Saline, 1x (PBS)	Corning	MT21040CV	Supplied as sterile solution. Do not open outside of the biological safety cabinet
Protease/Phosphatase inhibitor	Cell Signal Technology	5872S	Must be stored at -20 °C
Puromycin Dihydrochloride	Gibco	A1113803	Make aliquots of the 10mg/ml stock; place in 200 mL small tubes and store at  -20 °C until use.
RIPA Lysis Buffer, 10x	Millipore-Sigma	20-188	Store at -80 °C and make aliquots of 1x in sterile double distilled water
Serum-Free Cell Freezing Medium	Bambanker (Nippon Genetics)	CS-04-001/BB02	Dilute cells in 1ml freezing medium per cryovial to store NSCs. After addition of freezing medium to cells, cells can be placed at -80 °C for long-term storage; there is no need for slow cooling or storage in liquid N2.
Sodium Bicarbonate (7.5%)	Corning	25-035-CI	Supplied as sterile solution. Do not open outside of the biological safety cabinet
Sox2 (D9B8N) Rabbit mAb	Cell Signal Technology	23064	Used at 1: 1000 dilution in 3 % Bovine serum albumin (Western blotting, to verify NSC marker expression)
Trypan Blue	Sigma Life Science	T8154-100ML	Add 10 mL trypan blue with with 10 mL cell solution for trypan blue exclusion
Trypsin-EDTA (0.05%)	Gibco	25300-054	Aliquot and keep frozen at -20 °C until use. When thawed, it should be warmed to room temperature for 1h rather than placing in bead bath. Residual trypsin can be stored at 4 °C for one week.
Ultra-low attachment multiple well plate (6-well)	Corning	CLS3471	Supplied sterile; open inside hood only.
Ultra-low attachment multiple well plate (24-well)	Corning	CLS3473	Supplied sterile; open inside hood only.
Ultra-low attachment T25 flasks	Corning	CL4616	Supplied sterile; open inside hood only.