Method Article

Modeling Astrocytoma Pathogenesis In Vitro and In Vivo Using Cortical Astrocytes or Neural Stem Cells from Conditional, Genetically Engineered Mice

DOI:

10.3791/51763

⸱

August 12th, 2014

 ,  ,  ,  ,  ,  ,  ,  , 
1Department of Pathology and Laboratory Medicine, University of North Carolina School of Medicine, 2Lineberger Comprehensive Cancer Center, University of North Carolina School of Medicine, 3Division of Neuropathology, Department of Pathology and Laboratory Medicine, University of North Carolina School of Medicine, 4Curriculum in Genetics and Molecular Biology, University of North Carolina School of Medicine, 5Biological and Biomedical Sciences Program, University of North Carolina School of Medicine, 6Department of Radiation Oncology, Emory University School of Medicine, 7Department of Neurology, Neurosciences Center, University of North Carolina School of Medicine

In This Article

Summary

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Phenotypically wild-type astrocytes and neural stem cells harvested from mice engineered with floxed, conditional oncogenic alleles and transformed via viral Cre-mediated recombination can be used to model astrocytoma pathogenesis in vitro and in vivo by orthotopic injection of transformed cells into brains of syngeneic, immune-competent littermates.

Abstract

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Current astrocytoma models are limited in their ability to define the roles of oncogenic mutations in specific brain cell types during disease pathogenesis and their utility for preclinical drug development. In order to design a better model system for these applications, phenotypically wild-type cortical astrocytes and neural stem cells (NSC) from conditional, genetically engineered mice (GEM) that harbor various combinations of floxed oncogenic alleles were harvested and grown in culture. Genetic recombination was induced in vitro using adenoviral Cre-mediated recombination, resulting in expression of mutated oncogenes and deletion of tumor suppressor genes. The phenotypic consequences of these mutations were defined by measuring proliferation, transformation, and drug response in vitro. Orthotopic allograft models, whereby transformed cells are stereotactically injected into the brains of immune-competent, syngeneic littermates, were developed to define the role of oncogenic mutations and cell type on tumorigenesis in vivo. Unlike most established human glioblastoma cell line xenografts, injection of transformed GEM-derived cortical astrocytes into the brains of immune-competent littermates produced astrocytomas, including the most aggressive subtype, glioblastoma, that recapitulated the histopathological hallmarks of human astrocytomas, including diffuse invasion of normal brain parenchyma. Bioluminescence imaging of orthotopic allografts from transformed astrocytes engineered to express luciferase was utilized to monitor in vivo tumor growth over time. Thus, astrocytoma models using astrocytes and NSC harvested from GEM with conditional oncogenic alleles provide an integrated system to study the genetics and cell biology of astrocytoma pathogenesis in vitro and in vivo and may be useful in preclinical drug development for these devastating diseases.

Introduction

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Astrocytomas are the most common primary brain tumor and glioblastoma (GBM), a grade IV astrocytoma, is the most common and aggressive subtype with a median survival of 12-15 months1,2. Invasion of diffuse astrocytomas, particularly GBM, precludes complete surgical resection, limits the effectiveness of adjuvant therapies, and inevitably leads to post-treatment recurrence3. Patients initially present either with de novo (primary) GBM or with lower grade astrocytomas that inevitably progresses to (secondary) GBM4. GBM is genomically heterogeneous and characterized by mutually exclusive and co-occurring mutations in genes that govern three core signaling pathways: the G1/S (Rb) cell cycle checkpoint, receptor tyrosine kinase (RTK), and TP53 pathways5-7. GBM consists of four genomic subtypes with distinct expression profiles that resemble different brain cell types, suggesting that GBM subtype is influenced by its cell of origin6,8,9. Better astrocytoma models are required to define the role of specific combinations of mutations in particular cell types during astrocytoma pathogenesis. Leveraging these models for more efficient preclinical drug development will ultimately help improve patient outcomes. Current astrocytoma models include established human cell lines, patient derived xenografts (PDX), genetically modified normal human astrocytes and neural stem cells (NSC), and genetically engineered mice (GEM)10-14. We developed an alternative, non-germline GEM (nGEM) model15 utilizing primary brain cells – cortical astrocytes and NSC – harvested from GEM harboring various combinations of floxed oncogenic alleles. The goal was to generate astrocytoma models with genetically defined cells that could be phenotypically characterized both in vitro and in vivo and potentially utilized for preclinical drug development in immune-competent mice.

Established human cell lines are the most commonly used model of astrocytoma pathogenesis and drug response in vitro and in vivo. They are technically straight forward, widely available, and have defined kinetics and tumorigenicity upon orthotopic xenografting in immunodeficient mice10,11,16-18. Their disadvantages include the inability to generate established cell lines from low-grade astrocytomas, limiting study only to high-grade astrocytomas; lack of a defined cell of origin; the presence of complex genomic abnormalities, often with genomic profiles that differ markedly from the original patient sample; and susceptibility to phenotypic and genotypic drift during serial culture in serum11,17,19-22. The phenotypic consequences of individual oncogenic mutations in established human GBM cell lines can be masked by the multitude of abnormalities that are actually present, which often precludes elucidation of direct genotype-phenotype consequences.

PDX are generated through subcutaneous passage of patient-isolated astrocytoma cells in immunodeficient mice or through their culture as non-adherent spheroids in defined, serum-free medium prior to orthotopic injection into the brains of immunodeficient mice12,23. PDX more accurately maintain the genomic landscape of human astrocytomas, but similar to established human cell lines, the phenotypic effect of individual oncogenic mutations can be masked due to their genomic complexity19,24. To define the phenotypic consequences of specific oncogenic mutations, particularly in response to novel therapies, panels of established human cell lines or PDX are frequently utilized to establish genotype-phenotype correlations, show generalizability, and minimize the likelihood of cell line-specific effects. While PDX accurately recapitulate the histopathological hallmarks of human astrocytomas, including invasion, orthotopic xenografts of established human cell lines generally do not21,23,25. Additionally, normal human astrocytes and NSC have been genetically-engineered with defined oncogenic mutations to model astrocytoma tumorigenesis in vitro and in vivo13,14,26. These cells lack the genomic complexity of established human cell lines and PDX and accurately recapitulate human astrocytoma histopathology, but require xenografting in immunodeficient rodents in vivo. Because all human cell models require immunodeficient rodent hosts to prevent immune-mediated xenograft rejection, these models fail to recapitulate the native tumor-stroma interactions of a syngeneic system and lack an intact immune system, limiting preclinical investigation of stroma-targeted and immune-modulatory therapies10,11.

GEM permit examination of the phenotypic consequences of predetermined combinations of oncogenic mutations in vivo during in situ tumorigenesis. Whereas non-conditional GEM have mutations within all tissues throughout development, conditional GEM have floxed oncogenic alleles that enable targeting of mutations by restricting Cre-mediated recombination to specific cell types through use of cell type-specific promoters10,11,15,18. Conditional astrocytoma GEM have been utilized to elucidate the functional roles of oncogenic mutations in distinct cell types within an intact brain11. The preclinical utility of in situ gliomagenesis using conditional GEM is limited by a number of factors including 1) the lack of an in vitro correlate, 2) difficulty in generating large cohorts of mice with complex genotypes, 3) long latency of in situ tumor development, 4) and stochastic tumor progression. Because in situ tumorigenesis lacks a corresponding in vitro model, drug testing in vitro cannot be performed with conventional conditional GEM models. In contrast to other cancers, conditional GEM models of astrocytomas are rarely induced by single oncogenic mutations11. Thus, complex breeding schemes are required to generate conditional GEM with multiple oncogenic mutations. Moreover, astrocytoma initiation occurs with variable penetrance after a long latency period in these models, while progression to high-grade astrocytomas generally occurs in a non-uniform, stochastic manner and ultimately gives rise to tumors with complex genomic landscapes and rapid growth kinetics27,28. The variable penetrance and stochastic nature of malignant progression in conditional GEM models requires that individual mice be screened by radiographic imaging to detect the presence and location of high-grade astrocytomas before their enrollment in preclinical drug trials. Taken together, these limitations hinder the generation and testing of the large cohorts of conditional GEM required for preclinical drug testing.

The RCAS-tva GEM system, which utilizes avian retroviral (RCAS) vectors to infect GEM engineered to express the viral receptor (tva) on specific neural cell types, has been extensively utilized to model astrocytoma tumorigenesis11. In contrast to conditional GEM, this model system enables introduction of multiple oncogenic mutations in specific cells types without the requirement for complex breeding schemes. However, it is limited by variable penetrance, the requirement for actively dividing cells to achieve viral integration, and the random insertion of transgenes into the host genome29.

Non-germline GEM (nGEM) models, which utilize cells harvested from GEM, are becoming increasingly important because they overcome many of the limitations of other model systems15. The role of initiating cell type and co-occurring mutations in astrocytoma pathogenesis are difficult to determine using established human GBM cell lines or PDX because they are derived from end-stage tumors that have accumulated extensive genetic mutations in undefined cell types during the course of malignant progression. In contrast, all grades of astrocytomas can be modeled using nGEM by inducing defined genetic mutations within specific purified brain cell types11,30. Thus, the influence of specific genetic mutations and cell type on cellular and molecular phenotypes can be determined in vitro and in vivo. Similar to established human GBM cell lines, initial in vitro drug testing using nGEM can be used to prioritize drugs for in vivo testing utilizing the same cells. Tumorigenesis in vivo can then be determined by allografting nGEM cells orthotopically into the brains of immune-competent syngeneic littermates30. These orthotopic allograft models therefore permit in vivo testing not only of conventional cytotoxic and targeted therapies, but immune-modulatory and stroma-targeted therapies as well. Finally, the role of the microenvironment on tumor initiation and progression can be determined by comparing results between nGEM and conventional GEM models using the same mutations in the same cell types.

We and others have developed astrocytoma nGEM using primary cells - astrocytes, NSC, or oligodendrocyte precursor cells (OPC) - harvested from GEM30-34. The rationale behind the development of an astrocytoma nGEM was to create a model to determine the phenotypic consequences of oncogenic mutations in specific cell types that could potentially be used for preclinical drug testing in vitro and in vivo in immune-competent animals. We harvested phenotypically WT cortical astrocytes and NSC from non-Cre expressing, conditional GEM maintained on a >94% C57/Bl6 background with floxed RB pathway - Rb1loxP/loxP, or TgGZT121 – and floxed RTK/RAS/PI3K pathway - Nf1loxP/loxP, KrasG12D, PtenloxP/loxP – genes in various combinations35-39. We induced genetic recombination in vitro using adenoviral vectors encoding Cre recombinase. Because cortical astrocyte harvests contain a mixture of cell types, we used Ad5GFAPCre vectors or dominant oncogenic transgenes, such as TgGZT121 driven from the human GFAP promoter, to enrich for GFAP+ cortical astrocytes in these cultures. We defined the phenotypic consequences of G1/S (Rb), MAPK, and PI3K pathway mutations in cortical astrocytes and NSC in vitro and in vivo. MAPK and PI3K pathway-activated G1/S-defective astrocytes molecularly mimicked human proneural GBM and, upon orthotopic injection, formed tumors in a pre-defined location with uniform growth kinetics, short latencies, and the histopathological hallmarks of human GBM30. Longitudinal monitoring of tumor growth in vivo aids preclinical drug testing through normalization of treatment cohorts and quantitative analysis of tumor growth in response to treatment40. We determined tumor growth kinetics by longitudinal bioluminescence imaging of mice injected with luciferase expressing cortical astrocytes. Therefore, cortical astrocytes and NSC derived from conditional GEM provide a tractable model system for definition of functional consequences of astrocytoma-associated mutations and a potential model system for preclinical drug development.

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Protocol

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All animal studies were approved by the University of North Carolina Institutional Animal Care and Use Committee.

1. Culturing Cortical Astrocytes from Neonatal Mice

  1. Preparation
    1. Crumple 2-3 delicate task tissues and place into the bottom of a flask containing 70% ethanol. Place dissecting scissors, curved forceps and 2 pairs of straight micro forceps into this flask. The tissues are used to avoid bending the micro forceps.
    2. Add 1 ml of HBSS to a 60 mm tissue culture dish. Prepare a separate dish for each animal and maintain dishes on ice.
    3. Anesthetize animals per institutional regulations.
    4. Warm 7 ml of DMEM with 10% fetal bovine serum and 1% penicillin-streptomycin (complete DMEM) for each animal in a 37 °C water bath. NOTE: For 5 mice, steps 1.1.1-1.1.4 will take ~15 min.
  2. Tissue Harvest
    1. Sacrifice neonatal mouse (day 1-3) per institutional regulations.
    2. Remove dissecting scissors from ethanol, allow them to drip dry, and make a sagittal cut in the skin over the cranium from the spinal cord to the nose.
    3. Make a cut in the cranium along the sagittal suture, starting from the spinal cord and extending past the olfactory bulbs. Take care to keep the scissor tips near the inner surface of the cranium to minimize brain tissue damage.
    4. Using curved forceps, gently peel each hemisphere of the cranium laterally away from the brain.
    5. Using straight micro forceps, gently pinch away the dorsal portion of each cortical hemisphere and place it into the tissue culture dish containing HBSS. Take care to avoid the cerebellum and olfactory bulb. Do not take any tissue below the corpus callosum.
    6. Using a dissecting microscope and 2 pairs of micro forceps, gently remove the meninges from each cortical hemisphere. Return the tissue culture dish to ice until beginning tissue homogenization.
    7. Repeat steps 1.2.1 through 1.2.6 for each additional animal. NOTE: For 5 mice, steps 1.2.1-1.2.7 will take ~30 min.
  3. Tissue Homogenization
    1. Using a clean razor blade, finely dice the cortical hemispheres and move the plate to a tissue culture hood.
    2. Transfer the diced tissue into a sterile 15 ml conical tube. Wash the plate with 1 ml of ice cold HBSS and transfer to the tube containing tissue.
    3. Wait until tissue settles to the bottom of the tube, then carefully remove excess HBSS using a pipette.
    4. Add 2 ml of room temperature trypsin/EDTA. Pipette ~10x with a 1 ml pipette to further dissociate cells.
    5. Incubate the cell suspension at 37 °C for 15 - 20 min. Carefully mix by inversion every 5 min.
    6. Add 3 ml of complete DMEM to inhibit trypsin. Pipette ~10x with a 1 ml pipette to mix the solution.
    7. Centrifuge at 200 x g for 5 min at room temperature.
    8. Remove the supernatant with a 1 ml pipette. Do not aspirate the medium because the pellet may be loose.
    9. Resuspend the pellet in 4 ml of 37 °C complete DMEM. Transfer the cell suspension to a 75 cm screw top tissue culture flask. NOTE: For 5 mice, steps 1.3.1-1.3.9 will take ~50 min.
    10. Approximately 16 hr after astrocyte harvest, wash the flask with 4 ml of 37 °C HBSS, then add 4 ml of 37 °C complete DMEM. Maintain the cortical astrocytes at 37 °C in 5% CO2. NOTE: The wash step is important for removal of non-adherent cells and debris from the culture dish.
    11. When the culture is ~95% confluent, shake overnight at 37 °C in 5% CO2, remove the media containing the detached cells, then wash the flask with 4 ml of 37 °C HBSS. Add 4 ml of 37 °C complete DMEM and maintain cells at 37 °C in 5% CO2. NOTE: This step is important to enrich cultures for cortical astrocytes, as contaminating microglia and oligodendrocyte progenitor cells detach with this procedure and are removed from the culture41.
    12. Repeat steps 1.3.1-1.3.11 for each animal.
  4. Induction of Genetic Recombination
    1. 24 hr after completing step 1.3.11, replace the medium with 2 ml of 37 °C complete DMEM. Add 1 ml of 37 °C SCM containing 1 µl of >1010 pfu/ml of Ad5CMVCre or Ad5GFAPCre. Incubate for 6 hr at 32 °C in 5% CO2.CAUTION: Use BSL2 safety precautions when handling recombinant adenovirus.
    2. Remove the virus containing media and add 37 °C complete DMEM without virus to the cortical astrocytes. CAUTION: Use BSL2 safety precautions when handling recombinant adenoviruses and discard the virus-containing media per institutional regulations. NOTE: For 5 astrocyte cultures, steps 1.4.1 - 1.4.3 will take ~15 min excluding the 6 hr incubation.
    3. Expand cortical astrocyte cultures for in vitro and in vivo characterization. NOTE: Determine the presence of mutations following Cre-recombination by PCR with primers specific for the recombined gene or by immunoblots for either the specific mutated protein or its signaling consequences. The time required for phenotypic stabilization of cortical astrocyte cultures after Cre-recombination will be dependent on the mutations utilized and must be determined empirically (Figure 2).

2. Culturing Neural Stem Cells from Neonatal Mice

  1. Preparation
    1. Before starting the dissection, prepare 4 ml digestion solution per brain by adding 3.1 mg papain and 1.3 mg cysteine to 4 ml dissociation medium - 98 mM Na2SO4, 30 mM K2SO4, 5.8 mM MgCl2, 0.25 mM CaCl2, 1 mM HEPES (from 1 M HEPES pH 7.4 stock) 20 mM glucose, 0.001% Phenol red, and 0.125 mM NaOH. Addition of papain and cysteine to the dissociation medium will turn the solution yellow.
    2. Incubate in 37 °C water bath for 15 min. Mix periodically by inversion.
    3. Add 0.1 M NaOH drop wise until digestion solution returns to light red color.
    4. In a tissue culture hood, sterile filter the digestion solution using a syringe filter (0.22 µm pore size). Keep the digestion solution on ice.
    5. Prepare 50 ml of Stem Cell Medium (SCM) by mixing 44.5 ml NSC Basal Medium, 5 ml NSC supplement, 0.5 ml penicillin-streptomycin, 50 µl 0.2% heparin, 10 µl 100 µg/ml EGF, and 10 µl 100 µg/ml bFGF. SCM is stable for 1 week when stored at 4 °C.
    6. Prepare complete HBSS (cHBSS) by mixing 50 ml of 10x HBSS, 1.25 ml 1 M HEPES (pH 7.4), 15 ml 1 M D-glucose, 5 ml 100 mM CaCl2, 5 ml 100 mM MgSO4, and 2 ml 1 M NaHCO3. Bring the total volume to 500 ml with ddH2O.
    7. Filter sterilize cHBSS with a 0.2 µm pore size filter and store at 4 °C.
    8. Crumple 2-3 delicate task tissues and place into the bottom of a flask containing 70% ethanol. Place dissecting scissors, curved forceps, and 2 pairs of straight micro forceps into this flask. The tissues are used to avoid bending the micro forceps. NOTE: For 5 mice, steps 2.1.1-2.1.8 will take ~45 min.
  2. Tissue Harvest
    1. Sacrifice neonatal (day 1-3) mice per institutional regulations.
    2. Remove dissecting scissors from the 70% ethanol, allow them to drip dry, and make a sagittal cut in the skin over the cranium from the spinal cord to the nose.
    3. Make a cut in the cranium along the sagittal suture, starting from the spinal cord and extending past the olfactory bulbs. Take care to keep the scissor tips near the inner surface of the cranium to minimize brain tissue damage.
    4. Using curved forceps, gently peel each hemisphere of the cranium laterally away from the brain.
    5. Gently remove the entire brain and place in ice cold cHBSS.
    6. Using a dissection microscope, carefully remove meninges from the brain with fine dissection tools.
    7. Place the brain on its bottom surface and remove cerebellum/hindbrain and olfactory bulb/frontal cortex with vertical (coronal) cuts with a size 11 scalpel.
    8. Rotate the remaining brain by 90° to place it on the caudal portion, thus generating a coronal view of the brain. Locate the lateral ventricles.
    9. Cut out a cubical section containing the subventricular zone (SVZ).
    10. Transfer SVZ-containing tissue to a 60 mm tissue culture dish with fresh ice cold cHBSS and mince tissue with size 11 scalpel.
    11. Transfer the minced tissue into a sterile 15 ml tube. Place the tube on ice.
    12. Wash the petri dish with 1-2 ml ice cold cHBSS. Transfer the wash solution to the tube containing tissue.
    13. Repeat steps 2.2.1-2.2.12 with additional neonatal mice if necessary. Transfer all 15 ml tubes to a tissue culture hood for the remainder of the protocol. NOTE: For 5 mice, steps 2.2.1 - 2.2.13 will take ~45 min.
  3. Tissue Homogenization
    1. Place the digestion solution (prepared in step 2.1.1) in a 37 °C water bath for 5 min. Also, warm 2 ml SCM per brain in a 37 °C water bath.
    2. Carefully remove the supernatant of the minced tissue with a 1 ml pipette. Do not aspirate.
    3. Add 2 ml 37 °C digestion solution per 15 ml tube and mix carefully by inversion.
    4. Incubate in 37 °C water bath for 15 min. Mix by inversion every 5 min.
    5. Add another 2 ml of 37 °C digestion solution to each tube and repeat step 2.3.4.
    6. Allow the cells to settle to the bottom of the tube by gravity, then remove the supernatant from digested tissue using a 1 ml pipette.
    7. Add 2 ml of 10 µM E-64 diluted in cHBSS and mix carefully by inversion.
    8. Incubate for 3 min at room temperature.
    9. Allow the cells to settle to the bottom of the tube by gravity, then remove the supernatant from digested tissue.
    10. Add 1 ml of 37 °C cHBSS and homogenize with a 1 ml pipette tip (~20 strokes). Avoid injecting air bubbles during tissue homogenization.
    11. Pass the tissue homogenate through a 40 µm pore size cell strainer, then rinse the strainer with 5 ml of 37 °C cHBSS.
    12. Centrifuge the tissue homogenate at 125 x g for 5 min.
    13. Remove 95% of supernatant with a 1 ml pipette without disturbing the cell pellet. Carefully resuspend cell pellet in 200 µl 37 °C SCM with a 200 µl pipette tip.
    14. Add 1.8 ml 37 °C SCM and transfer the cell suspension to a well of a sterile 6 well plate. NOTE: For 5 mice, steps 2.3.1-2.3.14 will take ~75 min.
  4. Neural Stem Cell Culture
    1. Replenish the medium after 3 days by adding another 2 ml 37 °C SCM.
    2. After 7 days, transfer the neurospheres to a 15 ml tube and let the neurospheres settle by gravity for >5 min.
    3. Remove the supernatant and add 2 ml 37 °C SCM.
    4. Transfer the neurospheres to a new well of a 6 well plate.
    5. Add 2 ml of 37 °C SCM every 3-4 days, and change medium completely every 7 days.
    6. If neurospheres are > 100 µm in diameter, carefully dissociate with stem cell dissociation solution and replate the NSC at the desired cell density.
  5. Induction of Genetic Recombination
    1. Add 1 ml of 37 °C SCM containing 1 µl of >1010 pfu/ml of Ad5CMVCre or Ad5GFAPCre to the 2 ml of SCM currently on the cells. CAUTION: Use BSL2 safety precautions when handling recombinant adenovirus.
    2. Incubate for 6 hr at 32 °C in 5% CO2.
    3. Transfer SCM with neurospheres into a 15 ml tube and let spheres settle by gravity for 5 min.
    4. Remove the supernatant first with a 1 ml pipette, then with a 200 µl pipette. CAUTION: Use BSL2 safety precautions when handling recombinant adenoviruses and discard the virus-containing media per institutional regulations.
    5. Add 2 ml of 37 °C SCM without virus to the NSC, then transfer to a 6 well plate. NOTE: For 5 NSC cultures, steps 2.5.1-2.5.5 will take ~15 min excluding the 6 hr incubation.
    6. The next day, repeat steps 2.5.1-2.5.5, followed by neurosphere passaging when necessary. The spheres can now be expanded for 2-3 passages before in vitro characterization and orthotopic injection in mouse brains in vivo. NOTE: Determine the presence of mutations following Cre-recombination by PCR with primers specific for the recombined gene or by immunoblots for either the specific mutated protein or its signaling consequences. The time required for phenotypic stabilization of NSC cultures after Cre-recombination will be dependent on the mutations utilized and must be determined empirically (Figures 2 and 3).

3. Orthotopic Injection of Recombined Cells into the Brain of Recipient Iimmunocompetent Mice

  1. Preparation of 5% Methyl Cellulose
    1. Dissolve 5 g of 15 cPs methyl cellulose in deionized water to a final volume of 50 ml in a 100 ml screw cap bottle. Add the powder slowly while stirring at 4 °C to prevent clumping. It can take up to 24 hr of stirring at 4 °C for all the methyl cellulose to enter solution.
    2. Weigh the bottle and record the weight.
    3. Autoclave the 5% methyl cellulose solution. Once autoclaved, the methyl cellulose will become a semisolid gel.
    4. Weigh the bottle and calculate the weight lost during autoclaving.
    5. Aseptically add 1 ml of sterile water for every gram of weight lost.
    6. Place on ice and add 50 ml of ice cold 2x DMEM.
    7. Mix overnight using a magnetic stirrer at 4 °C. Use sterile technique to divide the 5% methyl cellulose solution into 20 ml aliquots. Store aliquots at 4 °C for up to six months or until the 2x DMEM expires. NOTE: Preparation of 5% methyl cellulose solution in steps 3.1.1-3.1.7 will take ~2.5 days.
  2. Preparation of Cortical Astrocytes for Injection
    1. Harvest cortical astrocytes when ~90% confluent.
    2. To harvest, aspirate complete DMEM and wash plates with an equal volume of 37 °C HBSS.
    3. Add enough trypsin to cover the plate. Gently tilt and tap the plate until cells start to detach.
    4. Inactivate trypsin with an equal volume of 37 °C complete DMEM.
    5. Transfer cells to a 50 ml conical tube and wash the plate with the same volume of 37 °C complete DMEM used in 3.2.4.
    6. Transfer wash media to the tube containing cells. Centrifuge at 200 x g for 3 min.
    7. Aspirate supernatant and resuspend cells in 1 ml of 37 °C complete DMEM.
    8. Count the cell suspension using a hemocytometer or another appropriate cell counter to determine number of cells/µl.
    9. Transfer the desired number of cells to a new 15 ml conical tube. Centrifuge at 200 x g for 3 min.
    10. Resuspend cells in 800 µl of 37 °C complete DMEM. Determine the volume of the cell pellet (total volume of cell suspension – 800 µl). NOTE: It is essential to achieve an accurate cell concentration for injection. Thus, the volume of the cell pellet must be determined in this step in order to calculate the volume of 5% methyl cellulose to add in step 3.2.12.
    11. Transfer cells to a sterile 1.5 ml microcentrifuge tube and then centrifuge at 200 x g for 3 min.
    12. Aspirate supernatant from the cell pellet and resuspend the cortical astrocytes in the appropriate volume of ice-cold 5% methyl cellulose (desired total volume – volume of the cell pellet) to obtain the desired number of cells/µl.
    13. Place cells on ice until injection.
    14. Place a 250 µl glass syringe into a Repeating Antigen Dispenser then place a blunt 18 G needle onto the syringe.
    15. Withdraw the plunger slowly with the needle in the cortical astrocyte suspension; there will be an air bubble above the cells that must be removed, since air bubbles will compress and diminish the volume actually injected into the brain.
    16. Hold the tip of the needle just above the cortical astrocyte suspension and push the plunger in quickly. The air bubble will move faster than the cell suspension and will be mostly expelled.
    17. Repeat step 3.2.16 until all air bubbles are removed from the needle.
    18. Fill the syringe to about ~200 µl, then hold the needle up and pull the plunger back until it stops. Little air bubbles can be removed by holding the needle tip up and rapidly pushing the plunger in about 50 µl then slowly withdrawing to full capacity.
    19. Keep the tip upright and repeat step 3.2.18 4-5x.
    20. Discard the 18 gauge needle and fit a 27 G needle onto the syringe.
    21. Press the button on the antigen dispenser until fluid is expelled from the needle. NOTE: For 1 astrocyte cell line, steps 3.2.1-3.2.21 will take ~60 min.
  3. Preparation of NSC for Injection
    1. When neurospheres are > 100 µm in diameter, transfer to 15 ml tubes and let the neurospheres settle by gravity for >5 min.
    2. Remove the supernatant and dissociate the neurospheres with a gentle dissociation reagent, such as stem cell dissociation solution or accutase according to the manufacturer’s instructions or with %0.05 trypsin-EDTA as described42.
    3. Inhibit the dissociation reagents according to the manufacturer’s instructions without exposing the NSC to serum. Centrifuge at 100 x g for 5 min.
    4. Aspirate supernatant, then resuspend the cells in 1 ml of 37 °C cHBSS to generate a single cell suspension.
    5. Count the cells using a hemocytometer or another appropriate cell counter to determine the number of cells/µl.
    6. Transfer the desired number of NSC to a new 15 ml conical tube. Centrifuge at 100 x g for 5 min.
    7. Resuspend cells in 800 µl of 37 °C HBSS. Determine the volume of the cell pellet (total volume of cell suspension – 800 µl). NOTE: It is essential to achieve an accurate cell concentration for injection. Thus, the volume of the cell pellet must be determined in this step in order to calculate the volume of 5% methyl cellulose to add in step 3.3.9.
    8. Transfer the cells to a sterile 1.5 ml microcentrifuge tube and then centrifuge at 100 x g for 5 min.
    9. Aspirate the supernatant and finish preparing the cells for orthotopic injection as in 3.2.12-3.2.21. NOTE: For 1 NSC cell line, steps 3.3.1-3.3.9 will take ~60 min.
  4. Preparation of Mouse for Injection
    1. Anesthetize the recipient animal via a I.P. injection of 250 mg/kg Avertin (2,2,2 Tribromoethanol) or 100 mg/kg ketamine plus 10 mg/kg xylazine, per institutional IACUC guidelines. NOTE: Utilized herein are mice at 3-6 months of age as allograft hosts. Mice at different ages may be utilized to investigate the microenvironmental impact of developmental brain age on tumorigenesis.
    2. Shave the scalp over the incision site with clippers or scissors.
    3. Sterilize the surgical site with 3 alternating swabs of 70% ethanol and betadine.
    4. Apply ophthalmic ointment to the eyes to prevent any damage due to loss of blink reflex.
    5. Assess depth of anesthesia by pedal withdrawal (toe pinch) reflex. NOTE: For 5 mice, steps 3.4.1-3.4.5 will take ~15 min.
  5. Orthotopic Implantation
    1. Secure the animal in the stereotaxic frame.
    2. Make an incision over the sagittal suture approximately 0.5 cm long between the ears and eyes.
    3. Locate the intersection of the coronal and sagittal sutures (Bregma), as well as the intersection of the lambdoid and sagittal sutures (Lambda). Ensure that Bregma and Lambda are in the same horizontal plane.
    4. Attach the syringe containing the cell suspension and repeating antigen dispenser to the stereotaxic frame over the head. Bring the tip of the 27 G needle into contact with Bregma. This is the origin that will be used for the manipulation of three dimensional coordinates.
    5. Raise the needle slightly off the skull surface and move 2 mm lateral and 1 mm rostral from Bregma.
    6. Lower the needle carefully through the skull to its destination 4 mm ventral from Bregma. NOTE: We utilized these stereotactic coordinates to target the basal ganglia. Different stereotactic coordinates may be utilized to investigate the microenvironmental impact of different brain regions on tumorigenesis.
    7. Inject 5 µl of the cell suspension by activating the repeating antigen dispenser one time. Leave the needle in place for 2 min to allow intracranial pressure to equilibrate.
    8. Withdraw the needle slowly over a period of 30 sec. Use a cotton swab to apply pressure to any bleeding that may occur.
    9. Approximate the wound edges and close the incision using tissue adhesive. Administer 20 µl of Lidocaine subcutaneously at the incision site. NOTE: For 5 mice, steps 3.5.1-3.5.9 will take ~50 min. The UNC IACUC approved the postoperative use of local anesthesia only. Consultation with local institutional IACUC is recommended regarding requirements for postoperative analgesic use after this procedure.
  6. Post-Surgical Care
    1. Place the animals in a clean, warm cage to recover. Animals should not be placed directly into bedding, as accidental aspiration may occur.
    2. Observe the animals for resumption of normal behavior such as grooming, eating, and defecation. NOTE: Resumption of normal behavior can take ~60 min.

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Results

$$\rightleftharpoonup{xx}$$ $$\longleftharp{xx}$$, $$\longrightharp{xx}$$,

We developed an nGEM model system with cortical astrocytes and NSC harvested from neonatal GEM harboring floxed conditional oncogenic alleles that can be phenotypically characterized in vitro and in vivo (Figure 1). In order to investigate the consequences of oncogenic mutations specifically in cortical astrocytes in vitro, it is critical to first enrich for astrocytes. Cortical astrocyte harvests contain a mixture of microglia, astrocytes, oligodendrocytes, OPC, and neurons, b...

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Discussion

$$\rightleftharpoonup{xx}$$ $$\longleftharp{xx}$$, $$\longrightharp{xx}$$,

The most critical steps to ensure proper harvest and culture of cortical astrocytes are 1) to excise the cortex without taking tissue below the corpus callosum, 2) to remove the meninges, 3) to thoroughly dissociate the cells, and 4) to enrich for GFAP+ astrocytes. Although we used mechanical (shaking) and genetic (restriction of genetic recombination with an Ad5GFAPCre vector or utilization of a dominant transforming transgene (TgGZT121) under GFAP promoter control) metho...

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Disclosures

$$\rightleftharpoonup{xx}$$ $$\longleftharp{xx}$$, $$\longrightharp{xx}$$,

The authors have nothing to disclose.

Acknowledgements

$$\rightleftharpoonup{xx}$$ $$\longleftharp{xx}$$, $$\longrightharp{xx}$$,

CRM is a Damon Runyon-Genentech Clinical Investigator. This work was supported, in part, by grants to CRM from the Damon Runyon Cancer Research Foundation (CI-45-09), Department of Defense (W81XWH-09-2-0042), and University of North Carolina University Cancer Research Fund (UCRF). The authors wish to thank Daniel Roth for mouse husbandry assistance. The authors also wish to thank Hannah Chae, Carter McCormick, Demi Canoutas, Stephanie Gillette, and Susannah Krom for tissue culture and immunofluorescence assistance.

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Materials

List of materials used in this article
NameCompanyCatalog NumberComments
Dulbecco's Modified Eagle Medium (DMEM) (1X)Invitrogen11995065DMEM can also be purchased from other suppliers including GIBCOSigma and Cellgro
Fetal Bovine Serum, RegularCellgro35-010-CV
Penicillin-StreptomycinInvitrogen15140122
Sharp-Pointed Dissecting ScissorsFisher Scientific08-395
Cartilage Thumb Forceps, CurvedFisher Scientific1631
Miltex 17-301 Style 1 Jeweler Style Forceps, Fine, 4Miltex17-301
Ethanol (200 proof)Decon Labs2710
Razor BladesVWR55411-050
Hanks' Balanced Salt Solution (HBSS) (1x), liquidInvitrogen14175-095
TrypLE Express (1x), Phenol RedInvitrogen12605-010Other trypsin solutions are also suitable for cortical astrocyte havest and culture
Culture Dish, 60 x 15 mmThomas Scientific9380H77
15 ml TubesBD Biosciences352096
50 ml TubesBD Biosciences352070
Adenovirus stockGene Transfer Vector Core, U. IowaAd5CMVCreStore in 5 µl aliquots at -80 °C. Hazardous. Use Bsl2 safety precautions
Sodium sulfate (Na2SO4) Sigma-Aldrich238597
Potassium sulfate (K2SO4)Sigma-Aldrich221325
Magnesium chloride (MgCl2)Sigma-AldrichM8266
Calcium chloride (CaCl2)Sigma-Aldrich746495
HEPES potassium saltSigma-AldrichH0527
D-(+)- GlucoseSigma-AldrichG8270 
Phenol RedSigma-AldrichP3532
Sodium hydroxide (NaOH)Sigma-AldrichS5881
PapainWorthingtonLS003127
L-Cysteine-HClSigma-AldrichC1276
Syringe filter (0.22 µm pore size)MilliporeSLGP033NS
Neurocult proliferation kit, mouseStemcell Technologies5702This kit contains the NeuroCult NSC Basal Medium and NeuroCult NSC supplement needed for NSC culture
0.2% Heparin solutionStemcell Technologies7980
EGF InvitrogenPMG8041
bFGF InvitrogenPHG0261
Hanks' Balanced Salt Solution (HBSS) (10x)Invitrogen14185-052
Magnesium sulfate (MgSO4)Sigma-AldrichM7506
Sodium bicarbonate (NaHCO3)Sigma-AldrichS5761
E-64Sigma-AldrichE3132Make 10 mM stock in DMSO, store at -20 °C
6-well PlatesFisher Scientific07-200-83
Cell strainer (40 µm pore size)Corning352340
Stem cell dissociation solutionStemcell Technologies5707Alternatively, use gentle enzyme solutions such as Accutase
Methyl cellulose 15 cPSigma-AldrichM7027
Dulbecco's Modified Eagle's Media 2xMilliporeSLM-202-BFor making 5% methyl cellulose solution
1.7 ml Snap Cap Microcentrifuge TubeCorning3620
Hamilton syringe, 250 µl LT no needleFisher Scientific14-815-92
PB600-1 Antigen DispenserHamilton 83700
Disposable 18 G needlesFisher ScientificNC9015638
27 ga 1/2" luer tip needleFisher Scientific14-826-48
2,2,2-Tribromoethanol (Avertin)Sigma-AldrichT48402
BetadineFisher ScientificNC9386574
Puralube Opthalmic OintmentFisher ScientificNC9689910
Model 900 Stereotaxic frameKopf Instruments
VETBONDFisher ScientificNC9259532 Tissue adhesive 
Lidocaine ShopMedVetRXLIDO-EPI
CellTiter 96 AQueous One Solution Cell Proliferation Assay (MTS)PromegaG3580
Anti-Sox2MilliporeAB5603
Polyclonal Rabbit Anti-Glial Fibrillary Acidic Protein (GFAP)DakoZ0334 
IVIS KineticPerkinElmerFor in vivo imaging
D-Luciferin - K+ Salt Bioluminescent SubstratePerkinElmer122796For in vivo bioluminescence imaging
EdU Imaging KitInvitrogenC10340
MSCV Luciferase PGK-hygroAddgene18782

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Astrocytoma ModelingConditional MiceAdenoviral CreOrthotopic AllograftBioluminescence ImagingGenetic RecombinationTumorigenesis AssayDrug Response Testing

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