Name: modeling astrocytoma pathogenesis in vitro and in vivo using cortical astrocytes or neural stem cells from conditional, genetically engineered mice
Uploaded: 2014-08-12T15:00:00
Description: Watch this Scientific Journal Video about Modeling Astrocytoma Pathogenesis In Vitro and In Vivo Using Cortical Astrocytes or Neural Stem Cells from Conditional, Genetically Engineered Mice at JoVE.co

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.

All animal studies were approved by the University of North Carolina Institutional Animal Care and Use Committee.
1. Culturing Cortical Astrocytes from Neonatal Mice
<ol>
	<li>Preparation
	<ol>
		<li>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.</li>
		<li>Add 1 ml&#160;of HBSS to a 60 mm tis...

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...

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&#160;(NSC), and genetically engineered mice (GEM)10-14. We developed an alternative, non-germline GEM (nGEM) model15 utilizing primary brain cells &#8211; cortical astrocytes and NSC &#8211; 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 &#62;94% C57/Bl6 background with floxed RB pathway - Rb1loxP/loxP, or TgGZT121 &#8211; and floxed RTK/RAS/PI3K pathway - Nf1loxP/loxP, KrasG12D, PtenloxP/loxP&#160;&#8211; 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.

<table border="0" cellpadding="0" cellspacing="0" width="784">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:241px;">Name of Material/ Equipment</td>
			<td style="width:191px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:233px;">Comments/Description</td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:241px;">Dulbecco&#39;s Modified Eagle Medium (DMEM) (1X)</td>
			<td style="width:191px;">Invitrogen</td>
			<td style="width:119px;">11995065</td>
			<td style="width:233px;">DMEM can also be purchased from other suppliers including GIBCOSigma and Cellgro</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:241px;">Fetal Bovine Serum, Regular</td>
			<td style="width:191px;">Cellgro</td>
			<td style="width:119px;">35-010-CV</td>
			<td style="width:233px;"></td>
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		<tr height="20">
			<td height="20" style="height:20px;width:241px;">Penicillin-Streptomycin</td>
			<td style="width:191px;">Invitrogen</td>
			<td style="width:119px;">15140122</td>
			<td style="width:233px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:241px;">Sharp-Pointed Dissecting Scissors</td>
			<td style="width:191px;">Fisher Scientific</td>
			<td style="width:119px;">08-395</td>
			<td style="width:233px;"></td>
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			<td height="40" style="height:40px;width:241px;">Cartilage Thumb Forceps, Curved</td>
			<td style="width:191px;">Fisher Scientific</td>
			<td style="width:119px;">1631</td>
			<td style="width:233px;"></td>
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		<tr height="40">
			<td height="40" style="height:40px;width:241px;">Miltex 17-301 Style 1 Jeweler Style Forceps, Fine, 4</td>
			<td style="width:191px;">Miltex</td>
			<td style="width:119px;">17-301</td>
			<td style="width:233px;"></td>
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			<td height="20" style="height:20px;width:241px;">Ethanol (200 proof)</td>
			<td style="width:191px;">Decon Labs</td>
			<td style="width:119px;">2710</td>
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			<td height="20" style="height:20px;width:241px;">Razor Blades</td>
			<td style="width:191px;">VWR</td>
			<td style="width:119px;">55411-050</td>
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			<td height="40" style="height:40px;width:241px;">Hanks&#39; Balanced Salt Solution (HBSS) (1X), liquid</td>
			<td style="width:191px;">Invitrogen</td>
			<td style="width:119px;">14175-095</td>
			<td style="width:233px;"></td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:241px;">TrypLE Express (1X), Phenol Red</td>
			<td style="width:191px;">Invitrogen</td>
			<td style="width:119px;">12605-010</td>
			<td style="width:233px;">Other Trypsin solutions are also suitable for cortical astrocyte havest and culture</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:241px;">CULTURE DISH, 60 x 15 mm</td>
			<td style="width:191px;">Thomas Scientific</td>
			<td style="width:119px;">9380H77</td>
			<td style="width:233px;"></td>
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		<tr height="20">
			<td height="20" style="height:20px;width:241px;">15 ml tubes</td>
			<td style="width:191px;">BD Biosciences</td>
			<td style="width:119px;">352096</td>
			<td style="width:233px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:241px;">50 ml tubes</td>
			<td style="width:191px;">BD Biosciences</td>
			<td style="width:119px;">352070</td>
			<td style="width:233px;"></td>
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		<tr height="60">
			<td height="60" style="height:60px;width:241px;">Adenovirus stock</td>
			<td style="width:191px;">Gene Transfer Vector Core, U. Iowa</td>
			<td style="width:119px;">Ad5CMVCre</td>
			<td style="width:233px;">Store in 5 &#181;l aliquots at -80 C. Hazardous.&#160; Use Bsl2 safetyy&#160; precautions</td>
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		<tr height="26">
			<td height="26" style="height:26px;width:241px;">Sodium sulfate (Na2SO4)&#160;</td>
			<td style="width:191px;">Sigma-Aldrich</td>
			<td style="width:119px;">238597</td>
			<td style="width:233px;"></td>
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		<tr height="26">
			<td height="26" style="height:26px;width:241px;">Potassium sulfate (K2SO4)</td>
			<td style="width:191px;">Sigma-Aldrich</td>
			<td style="width:119px;">221325</td>
			<td style="width:233px;"></td>
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			<td height="26" style="height:26px;width:241px;">Magnesium chloride (MgCl2)</td>
			<td style="width:191px;">Sigma-Aldrich</td>
			<td style="width:119px;">M8266</td>
			<td style="width:233px;"></td>
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		<tr height="26">
			<td height="26" style="height:26px;width:241px;">Calcium chloride (CaCl2)</td>
			<td style="width:191px;">Sigma-Aldrich</td>
			<td style="width:119px;">746495</td>
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			<td height="20" style="height:20px;width:241px;">HEPES potassium salt</td>
			<td style="width:191px;">Sigma-Aldrich</td>
			<td style="width:119px;">H0527</td>
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			<td height="20" style="height:20px;width:241px;">D-(+)- Glucose</td>
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			<td height="20" style="height:20px;width:241px;">Phenol Red</td>
			<td style="width:191px;">Sigma-Aldrich</td>
			<td style="width:119px;">P3532</td>
			<td style="width:233px;"></td>
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			<td height="20" style="height:20px;width:241px;">Sodium hydroxide (NaOH)</td>
			<td style="width:191px;">Sigma-Aldrich</td>
			<td style="width:119px;">S5881</td>
			<td style="width:233px;"></td>
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			<td height="20" style="height:20px;width:241px;">Papain</td>
			<td style="width:191px;">Worthington</td>
			<td style="width:119px;">LS003127</td>
			<td style="width:233px;"></td>
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			<td height="20" style="height:20px;width:241px;">L-Cysteine-HCl</td>
			<td style="width:191px;">Sigma-Aldrich</td>
			<td style="width:119px;">C1276</td>
			<td style="width:233px;"></td>
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		<tr height="20">
			<td height="20" style="height:20px;width:241px;">Syringe filter (0.22 &#181;m pore size)</td>
			<td style="width:191px;">Millipore</td>
			<td style="width:119px;">SLGP033NS</td>
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			<td height="80" style="height:80px;width:241px;">Neurocult proliferation kit, mouse</td>
			<td style="width:191px;">Stemcell Technologies</td>
			<td style="width:119px;">5702</td>
			<td style="width:233px;">This kit contains the NeuroCult NSC Basal Medium and NeuroCult NSC supplement needed for NSC culture</td>
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			<td height="20" style="height:20px;width:241px;">0.2% Heparin solution</td>
			<td style="width:191px;">Stemcell Technologies</td>
			<td style="width:119px;">7980</td>
			<td style="width:233px;"></td>
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		<tr height="20">
			<td height="20" style="height:20px;width:241px;">EGF&#160;</td>
			<td style="width:191px;">Invitrogen</td>
			<td style="width:119px;">PMG8041</td>
			<td style="width:233px;"></td>
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		<tr height="20">
			<td height="20" style="height:20px;width:241px;">bFGF&#160;</td>
			<td style="width:191px;">Invitrogen</td>
			<td style="width:119px;">PHG0261</td>
			<td style="width:233px;"></td>
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		<tr height="40">
			<td height="40" style="height:40px;width:241px;">Hanks&#39; Balanced Salt Solution (HBSS) (10X)</td>
			<td style="width:191px;">Invitrogen</td>
			<td style="width:119px;">14185-052</td>
			<td style="width:233px;"></td>
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		<tr height="26">
			<td height="26" style="height:26px;width:241px;">Magnesium sulfate (MgSO4)</td>
			<td style="width:191px;">Sigma-Aldrich</td>
			<td style="width:119px;">M7506</td>
			<td style="width:233px;"></td>
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		<tr height="26">
			<td height="26" style="height:26px;width:241px;">Sodium bicarbonate (NaHCO3)</td>
			<td style="width:191px;">Sigma-Aldrich</td>
			<td style="width:119px;">S5761</td>
			<td style="width:233px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:241px;">E-64</td>
			<td style="width:191px;">Sigma-Aldrich</td>
			<td style="width:119px;">E3132</td>
			<td style="width:233px;">Make 10 mM stock in DMSO, store at -20 &#176;C</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:241px;">6-well plates</td>
			<td style="width:191px;">Fisher Scientific</td>
			<td style="width:119px;">07-200-83</td>
			<td style="width:233px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:241px;">Cell strainer (40 &#181;m pore size)</td>
			<td style="width:191px;">Corning</td>
			<td style="width:119px;">352340</td>
			<td style="width:233px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:241px;">Stem cell dissociation solution</td>
			<td style="width:191px;">Stemcell Technologies</td>
			<td style="width:119px;">5707</td>
			<td style="width:233px;">Alternatively, use gentle enzyme solutions such as Accutase</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:241px;">Methyl cellulose 15 cP</td>
			<td style="width:191px;">Sigma-Aldrich</td>
			<td style="width:119px;">M7027</td>
			<td style="width:233px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:241px;">Dulbecco&#39;s Modified Eagle&#39;s Media 2X</td>
			<td style="width:191px;">Millipore</td>
			<td style="width:119px;">SLM-202-B</td>
			<td style="width:233px;">For making 5% methyl cellulose solution</td>
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		<tr height="40">
			<td height="40" style="height:40px;width:241px;">1.7mL Snap Cap Microcentrifuge Tube</td>
			<td style="width:191px;">Corning</td>
			<td align="right" style="width:119px;">3620</td>
			<td style="width:233px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:241px;">Hamilton syringe, 250 &#181;l LT no needle</td>
			<td style="width:191px;">Fisher Scientific</td>
			<td style="width:119px;">14-815-92</td>
			<td style="width:233px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:241px;">PB600-1 Antigen Dispenser</td>
			<td style="width:191px;">Hamilton</td>
			<td style="width:119px;">&#160;83700</td>
			<td style="width:233px;"></td>
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		<tr height="20">
			<td height="20" style="height:20px;width:241px;">Disposable 18 ga needles&#160;</td>
			<td style="width:191px;">Fisher Scientific</td>
			<td style="width:119px;">NC9015638&#160;</td>
			<td style="width:233px;"></td>
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			<td height="20" style="height:20px;width:241px;">27 ga 1/2&#34; luer tip needle</td>
			<td style="width:191px;">Fisher Scientific</td>
			<td style="width:119px;">14-826-48</td>
			<td style="width:233px;"></td>
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			<td height="20" style="height:20px;width:241px;">2,2,2-Tribromoethanol (Avertin)</td>
			<td style="width:191px;">Sigma-Aldrich</td>
			<td style="width:119px;">T48402</td>
			<td style="width:233px;"></td>
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			<td height="20" style="height:20px;width:241px;">Betadine</td>
			<td style="width:191px;">Fisher Scientific</td>
			<td style="width:119px;">NC9386574</td>
			<td style="width:233px;"></td>
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		<tr height="20">
			<td height="20" style="height:20px;width:241px;">Puralube Opthalmic Ointment</td>
			<td style="width:191px;">Fisher Scientific</td>
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			<td style="width:233px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:241px;">&#160;Model 900 Stereotaxic frame</td>
			<td style="width:191px;">Kopf Instruments</td>
			<td style="width:119px;"></td>
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		<tr height="20">
			<td height="20" style="height:20px;width:241px;">VETBOND</td>
			<td style="width:191px;">Fisher Scientific</td>
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			<td style="width:233px;">Tissue adhesive&#160;</td>
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		<tr height="20">
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		<tr height="60">
			<td height="60" style="height:60px;width:241px;">CellTiter 96 AQueous One Solution Cell Proliferation Assay (MTS)</td>
			<td style="width:191px;">Promega</td>
			<td style="width:119px;">G3580</td>
			<td style="width:233px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:241px;">Anti-Sox2</td>
			<td style="width:191px;">Millipore</td>
			<td style="width:119px;">AB5603</td>
			<td style="width:233px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:241px;">Polyclonal Rabbit Anti-Glial Fibrillary Acidic Protein (GFAP)</td>
			<td style="width:191px;">Dako</td>
			<td style="width:119px;">Z0334&#160;</td>
			<td style="width:233px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:241px;">IVIS Kinetic</td>
			<td style="width:191px;">PerkinElmer</td>
			<td style="width:119px;"></td>
			<td style="width:233px;">For in vivo imaging</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:241px;">D-Luciferin - K+ Salt Bioluminescent Substrate</td>
			<td style="width:191px;">PerkinElmer</td>
			<td style="width:119px;">122796</td>
			<td style="width:233px;">For in vivo bioluminescence imaging</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:241px;">EdU Imaging Kit</td>
			<td style="width:191px;">Invitrogen</td>
			<td style="width:119px;">C10340</td>
			<td style="width:233px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:241px;">MSCV Luciferase PGK-hygro</td>
			<td style="width:191px;">Addgene</td>
			<td style="width:119px;">18782</td>
			<td></td>
		</tr>
	</tbody>
</table>

modeling astrocytoma pathogenesis in vitro and in vivo using cortical astrocytes or neural stem cells from conditional, genetically engineered mice

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.

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

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.

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

Current astrocytoma models are limited in their ability to define the roles of oncogenic mutations in specific brain cell types during disease ...

modeling-astrocytoma-pathogenesis-vitro-vivo-using-cortical

Research

JoVE Journal

Neuroscience

Generation of Neural Stem Cells from Discarded Human Fetal Cortical Tissue

Neural stem cells (NSCs) reside along the ventricular zone neuroepithelium during the development of the cortical plate. These early progenitors ultimately give rise to intermediate progenitors and later, the various neuronal and glial cell subtypes that form the cerebral cortex. The capacity to generate and expand human NSCs (so called neurospheres) from discarded normal fetal tissue provides a means with which to directly study the functional aspects of normal human NSC development 1-5. This approach can also be directed toward the generation of NSCs from known neurological disorders, thereby affording the opportunity to identify disease processes that alter progenitor proliferation, migration and differentiation 6-9. We have focused on identifying pathological mechanisms in human Down syndrome NSCs that might contribute to the accelerated Alzheimer's disease phenotype 10,11. Neither in vivo nor in vitro mouse models can replicate the identical repertoire of genes located on human chromosome 21. 
Here we use a simple and reliable method to isolate Down syndrome NSCs from aborted human fetal cortices and grow them in culture. The methodology provides specific aspects of harvesting the tissue, dissection with limited anatomical landmarks, cell sorting, plating and passaging of human NSCs. We also provide some basic protocols for inducing differentiation of human NSCs into more selective cell subtypes.

A simple and reliable method on isolation and culture of neural stem cells from discarded human fetal cortical tissue is described. Cultures derived from known human neurological disorders can be used for characterization of pathological cellular and molecular processes, as well as provide a platform to assess pharmacological efficacy.

Neural stem cells (NSCs) reside along the ventricular zone neuroepithelium during the development of the cortical plate. These early progenitors ...

Modeling Neural Immune Signaling of Episodic and Chronic Migraine Using Spreading Depression In Vitro

Migraine and its transformation to chronic migraine are healthcare burdens in need of improved treatment options. We seek to define how neural immune signaling modulates the susceptibility to migraine, modeled in vitro using spreading depression (SD), as a means to develop novel therapeutic targets for episodic and chronic migraine. SD is the likely cause of migraine aura and migraine pain. It is a paroxysmal loss of neuronal function triggered by initially increased neuronal activity, which slowly propagates within susceptible brain regions. Normal brain function is exquisitely sensitive to, and relies on, coincident low-level immune signaling. Thus, neural immune signaling likely affects electrical activity of SD, and therefore migraine. Pain perception studies of SD in whole animals are fraught with difficulties, but whole animals are well suited to examine systems biology aspects of migraine since SD activates trigeminal nociceptive pathways. However, whole animal studies alone cannot be used to decipher the cellular and neural circuit mechanisms of SD. Instead, in vitro preparations where environmental conditions can be controlled are necessary. Here, it is important to recognize limitations of acute slices and distinct advantages of hippocampal slice cultures. Acute brain slices cannot reveal subtle changes in immune signaling since preparing the slices alone triggers: pro-inflammatory changes that last days, epileptiform behavior due to high levels of oxygen tension needed to vitalize the slices, and irreversible cell injury at anoxic slice centers. 
In contrast, we examine immune signaling in mature hippocampal slice cultures since the cultures closely parallel their in vivo counterpart with mature trisynaptic function; show quiescent astrocytes, microglia, and cytokine levels; and SD is easily induced in an unanesthetized preparation. Furthermore, the slices are long-lived and SD can be induced on consecutive days without injury, making this preparation the sole means to-date capable of modeling the neuroimmune consequences of chronic SD, and thus perhaps chronic migraine. We use electrophysiological techniques and non-invasive imaging to measure neuronal cell and circuit functions coincident with SD. Neural immune gene expression variables are measured with qPCR screening, qPCR arrays, and, importantly, use of cDNA preamplification for detection of ultra-low level targets such as interferon-gamma using whole, regional, or specific cell enhanced (via laser dissection microscopy) sampling. Cytokine cascade signaling is further assessed with multiplexed phosphoprotein related targets with gene expression and phosphoprotein changes confirmed via cell-specific immunostaining. Pharmacological and siRNA strategies are used to mimic and modulate SD immune signaling.

Modeling Neural Immune Signaling of Episodic and Chronic Migraine Using Spreading Depression In Vitro

Migraine and its transformation to chronic migraine are immense healthcare burdens in need of improved treatment options. We seek to define how neural immune signaling modulates the susceptibility to migraine, modeled in vitro using spreading depression in hippocampal slice cultures, as a means to develop novel therapeutic targets.

Migraine and its transformation to chronic migraine are healthcare burdens in need of improved treatment options.  We seek to define how neural immune ...

Functional Neuroimaging Using Ultrasonic Blood-brain Barrier Disruption and Manganese-enhanced MRI

Although mice are the dominant model system for studying the genetic and molecular underpinnings of neuroscience, functional neuroimaging in mice remains technically challenging. One approach, Activation-Induced Manganese-enhanced MRI (AIM MRI), has been used successfully to map neuronal activity in rodents 1-5. In AIM MRI, Mn2+ acts a calcium analog and accumulates in depolarized neurons 6,7. Because Mn2+ shortens the T1 tissue property, regions of elevated neuronal activity will enhance in MRI. Furthermore, Mn2+ clears slowly from the activated regions; therefore, stimulation can be performed outside the magnet prior to imaging, enabling greater experimental flexibility. However, because Mn2+ does not readily cross the blood-brain barrier (BBB), the need to open the BBB has limited the use of AIM MRI, especially in mice.
One tool for opening the BBB is ultrasound. Though potentially damaging, if ultrasound is administered in combination with gas-filled microbubbles (i.e., ultrasound contrast agents), the acoustic pressure required for BBB opening is considerably lower. This combination of ultrasound and microbubbles can be used to reliably open the BBB without causing tissue damage 8-11.
Here, a method is presented for performing AIM MRI by using microbubbles and ultrasound to open the BBB. After an intravenous injection of perflutren microbubbles, an unfocused pulsed ultrasound beam is applied to the shaved mouse head for 3 minutes. For simplicity, we refer to this technique of BBB Opening with Microbubbles and UltraSound as BOMUS 12. Using BOMUS to open the BBB throughout both cerebral hemispheres, manganese is administered to the whole mouse brain. After experimental stimulation of the lightly sedated mice, AIM MRI is used to map the neuronal response.
To demonstrate this approach, herein BOMUS and AIM MRI are used to map unilateral mechanical stimulation of the vibrissae in lightly sedated mice 13. Because BOMUS can open the BBB throughout both hemispheres, the unstimulated side of the brain is used to control for nonspecific background stimulation. The resultant 3D activation map agrees well with published representations of the vibrissae regions of the barrel field cortex 14. The ultrasonic opening of the BBB is fast, noninvasive, and reversible; and thus this approach is suitable for high-throughput and/or longitudinal studies in awake mice.

A technique is described for broadly opening the blood-brain barrier in the mouse using microbubbles and ultrasound. Using this technique, manganese can be administered to the mouse brain. Because manganese is an MRI contrast agent that accumulates in depolarized neurons, this approach enables imaging of neuronal activity.

Although mice are the dominant model system for studying the genetic and molecular underpinnings of neuroscience, functional neuroimaging in mice remains ...

Expansion of Embryonic and Adult Neural Stem Cells by In Utero Electroporation or Viral Stereotaxic Injection

Somatic stem cells can divide to generate additional stem cells (expansion) or more differentiated cell types (differentiation), which is fundamental for tissue formation during embryonic development and tissue homeostasis during adulthood 1. Currently, great efforts are invested towards controlling the switch of somatic stem cells from expansion to differentiation because this is thought to be fundamental for developing novel strategies for regenerative medicine 1,2. However, a major challenge in the study and use of somatic stem cell is that their expansion has been proven very difficult to control.
Here we describe a system that allows the control of neural stem/progenitor cell (altogether referred to as NSC) expansion in the mouse embryonic cortex or the adult hippocampus by manipulating the expression of the cdk4/cyclinD1 complex, a major regulator of the G1 phase of the cell cycle and somatic stem cell differentiation 3,4. Specifically, two different approaches are described by which the cdk4/cyclinD1 complex is overexpressed in NSC in vivo. By the first approach, overexpression of the cell cycle regulators is obtained by injecting plasmids encoding for cdk4/cyclinD1 in the lumen of the mouse telencephalon followed by in utero electroporation to deliver them to NSC of the lateral cortex, thus, triggering episomal expression of the transgenes 5-8. By the second approach, highly concentrated HIV-derived viruses are stereotaxically injected in the dentate gyrus of the adult mouse hippocampus, thus, triggering constitutive expression of the cell cycle regulators after integration of the viral construct in the genome of infected cells 9. Both approaches, whose basic principles were already described by other video protocols 10-14, were here optimized to i) reduce tissue damage, ii) target wide portions of very specific brain regions, iii) obtain high numbers of manipulated cells within each region, and iv) trigger high expression levels of the transgenes within each cell. Transient overexpression of the transgenes using the two approaches is obtained by different means i.e. by natural dilution of the electroporated plasmids due to cell division or tamoxifen administration in Cre-expressing NSC infected with viruses carrying cdk4/cyclinD1 flanked by loxP sites, respectively 9,15.
These methods provide a very powerful platform to acutely and tissue-specifically manipulate the expression of any gene in the mouse brain. In particular, by manipulating the expression of the cdk4/cyclinD1 complex, our system allows the temporal control of NSC expansion and their switch to differentiation, thus, ultimately increasing the number of neurons generated in the mammalian brain. Our approach may be critically important for basic research and using somatic stem cells for therapy of the mammalian central nervous system while providing a better understanding of i) stem cell contribution to tissue formation during development, ii) tissue homeostasis during adulthood, iii) the role of adult neurogenesis in cognitive functions, and perhaps, iv) better using somatic stem cells in models of neurodegenerative diseases.

Expansion of Embryonic and Adult Neural Stem Cells by In Utero Electroporation or Viral Stereotaxic Injection

Controlling the expansion of somatic stem cells is a major factor hampering their study and use in therapy. Here we describe a system to temporally control neural stem cells expansion during development and adulthood, which can be used to increase the number of neurons generated in the mouse brain.

Somatic stem cells can divide to generate additional stem cells (expansion) or more differentiated cell types (differentiation), which is fundamental for ...

Isolation and Culture of Neural Crest Cells from Embryonic Murine Neural Tube

The embryonic neural crest (NC) is a multipotent progenitor population that originates at the dorsal aspect of the neural tube, undergoes an epithelial to mesenchymal transition (EMT) and migrates throughout the embryo, giving rise to diverse cell types 1-3. NC also has the unique ability to influence the differentiation and maturation of target organs4-6. When explanted in vitro, NC progenitors undergo self-renewal, migrate and differentiate into a variety of tissue types including neurons, glia, smooth muscle cells, cartilage and bone. 
NC multipotency was first described from explants of the avian neural tube7-9. In vitro isolation of NC cells facilitates the study of NC dynamics including proliferation, migration, and multipotency. Further work in the avian and rat systems demonstrated that explanted NC cells retain their NC potential when transplanted back into the embryo10-13. Because these inherent cellular properties are preserved in explanted NC progenitors, the neural tube explant assay provides an attractive option for studying the NC in vitro. 
To attain a better understanding of the mammalian NC, many methods have been employed to isolate NC populations. NC-derived progenitors can be cultured from post-migratory locations in both the embryo and adult to study the dynamics of post-migratory NC progenitors11,14-20, however isolation of NC progenitors as they emigrate from the neural tube provides optimal preservation of NC cell potential and migratory properties13,21,22. Some protocols employ fluorescence activated cell sorting (FACS) to isolate a NC population enriched for particular progenitors11,13,14,17. However, when starting with early stage embryos, cell numbers adequate for analyses are difficult to obtain with FACS, complicating the isolation of early NC populations from individual embryos. Here, we describe an approach that does not rely on FACS and results in an approximately 96% pure NC population based on a Wnt1-Cre activated lineage reporter23. 
The method presented here is adapted from protocols optimized for the culture of rat NC11,13. The advantages of this protocol compared to previous methods are that 1) the cells are not grown on a feeder layer, 2) FACS is not required to obtain a relatively pure NC population, 3) premigratory NC cells are isolated and 4) results are easily quantified. Furthermore, this protocol can be used for isolation of NC from any mutant mouse model, facilitating the study of NC characteristics with different genetic manipulations. The limitation of this approach is that the NC is removed from the context of the embryo, which is known to influence the survival, migration and differentiation of the NC2,24-28.

Isolation of embryonic neural crest from the neural tube facilitates the use of in vitro methods for studying migration, self-renewal, and multipotency of neural crest.

The embryonic neural crest (NC) is a multipotent progenitor population that originates at the dorsal aspect of the neural tube, undergoes an epithelial to ...

In Vivo Two-photon Imaging Of Experience-dependent Molecular Changes In Cortical Neurons

The brain's ability to change in response to experience is essential for healthy brain function, and abnormalities in this process contribute to a variety of brain disorders1,2. To better understand the mechanisms by which brain circuits react to an animal's experience requires the ability to monitor the experience-dependent molecular changes in a given set of neurons, over a prolonged period of time, in the live animal. While experience and associated neural activity is known to trigger gene expression changes in neurons1,2, most of the methods to detect such changes do not allow repeated observation of the same neurons over multiple days or do not have sufficient resolution to observe individual neurons3,4. Here, we describe a method that combines in vivo two-photon microscopy with a genetically encoded fluorescent reporter to track experience-dependent gene expression changes in individual cortical neurons over the course of day-to-day experience. 
One of the well-established experience-dependent genes is Activity-regulated cytoskeletal associated protein (Arc)5,6. The transcription of Arc is rapidly and highly induced by intensified neuronal activity3, and its protein product regulates the endocytosis of glutamate receptors and long-term synaptic plasticity7. The expression of Arc has been widely used as a molecular marker to map neuronal circuits involved in specific behaviors3. In most of those studies, Arc expression was detected by in situ hybridization or immunohistochemistry in fixed brain sections. Although those methods revealed that the expression of Arc was localized to a subset of excitatory neurons after behavioral experience, how the cellular patterns of Arc expression might change with multiple episodes of repeated or distinctive experiences over days was not investigated. 
In vivo two-photon microscopy offers a powerful way to examine experience-dependent cellular changes in the living brain8,9. To enable the examination of Arc expression in live neurons by two-photon microscopy, we previously generated a knock-in mouse line in which a GFP reporter is placed under the control of the endogenous Arc promoter10. This protocol describes the surgical preparations and imaging procedures for tracking experience-dependent Arc-GFP expression patterns in neuronal ensembles in the live animal. In this method, chronic cranial windows were first implanted in Arc-GFP mice over the cortical regions of interest. Those animals were then repeatedly imaged by two-photon microscopy after desired behavioral paradigms over the course of several days. This method may be generally applicable to animals carrying other fluorescent reporters of experience-dependent molecular changes4.

In Vivo Two-photon Imaging Of Experience-dependent Molecular Changes In Cortical Neurons

Experience-dependent molecular changes in neurons are essential for the brain's ability to adapt in response to behavioral challenges. An in vivo two-photon imaging method is described here that allows the tracking of such molecular changes in individual cortical neurons through genetically encoded reporters.

The brain's ability to change in response to experience is essential for healthy brain function, and abnormalities in this process contribute to a variety ...

Imaging Intracellular Ca2+ Signals in Striatal Astrocytes from Adult Mice Using Genetically-encoded Calcium Indicators

Astrocytes display spontaneous intracellular Ca2+ concentration fluctuations ([Ca2+]i) and in several settings respond to neuronal excitation with enhanced [Ca2+]i signals. It has been proposed that astrocytes in turn regulate neurons and blood vessels through calcium-dependent mechanisms, such as the release of signaling molecules. However, [Ca2+]i imaging in entire astrocytes has only recently become feasible with genetically encoded calcium indicators (GECIs) such as the GCaMP series. The use of GECIs in astrocytes now provides opportunities to study astrocyte [Ca2+]i signals in detail within model microcircuits such as the striatum, which is the largest nucleus of the basal ganglia. In the present report, detailed surgical methods to express GECIs in astrocytes in vivo, and confocal imaging approaches to record [Ca2+]i signals in striatal astrocytes in situ, are described. We highlight precautions, necessary controls and tests to determine if GECI expression is selective for astrocytes and to evaluate signs of overt astrocyte reactivity. We also describe brain slice and imaging conditions in detail that permit reliable [Ca2+]i imaging in striatal astrocytes in situ. The use of these approaches revealed the entire territories of single striatal astrocytes and spontaneous [Ca2+]i signals within their somata, branches and branchlets. The further use and expansion of these approaches in the striatum will allow for the detailed study of astrocyte [Ca2+]i signals in the striatal microcircuitry.

Imaging Intracellular Ca2+ Signals in Striatal Astrocytes from Adult Mice Using Genetically-encoded Calcium Indicators

The properties and functions of astrocyte intracellular Ca2+ signals in the striatum remain incompletely explored. We describe methods to express genetically encoded calcium indicators in striatal astrocytes using adeno-associated viruses of serotype 2/5 (AAV2/5), as well as procedures to reliably image Ca2+ signals within striatal astrocytes in situ.

Astrocytes display spontaneous intracellular Ca2+ concentration fluctuations ([Ca2+]i) and in several settings respond to neuronal excitation with ...

In Vivo Two-photon Imaging of Cortical Neurons in Neonatal Mice

Two-photon imaging is a powerful tool for the in vivo analysis of neuronal circuits in the mammalian brain. However, a limited number of in vivo imaging methods exist for examining the brain tissue of live newborn mammals. Herein we summarize a protocol for imaging individual cortical neurons in living neonatal mice. This protocol includes the following two methodologies: (1) the Supernova system for sparse and bright labeling of cortical neurons in the developing brain, and (2) a surgical procedure for the fragile neonatal skull. This protocol allows the observation of temporal changes of individual cortical neurites during neonatal stages with a high signal-to-noise ratio. Labeled cell-specific gene silencing and knockout can also be achieved by combining the Supernova with RNA interference and CRISPR/Cas9 gene editing systems. This protocol can, thus, be used for analyzing the developmental dynamics of cortical neurons, molecular mechanisms that control the neuronal dynamics, and changes in neuronal dynamics in disease models.

In Vivo Two-photon Imaging of Cortical Neurons in Neonatal Mice

We present an in vivo two-photon imaging protocol for imaging the cerebral cortex of neonatal mice. This method is suitable for analyzing the developmental dynamics of cortical neurons, the molecular mechanisms that control the neuronal dynamics, and the changes in neuronal dynamics in disease models.

Two-photon imaging is a powerful tool for the in vivo analysis of neuronal circuits in the mammalian brain. However, a limited number of in vivo imaging ...

The Detection of 5-Hydroxymethylcytosine in Neural Stem Cells and Brains of Mice

Multiple&#160;DNA modifications have been identified in the mammalian genome. Of that, 5-methylcytosine and 5-hydroxymethylcytosine-mediated epigenetic mechanisms have been intensively studied. 5-hydroxymethylcytosine displays dynamic features during embryonic and postnatal development of the brain, plays a regulatory function in gene expression, and is involved in multiple neurological disorders. Here, we describe the detailed methods including immunofluorescence staining and&#160;DNA dot-blot to detect 5-hydroxymethylcytosine in cultured cells and brain tissues of mouse.

Here, we present a protocol to detect 5-hydroxymethylcytosine in cells and brain tissues, utilizing immunofluorescence staining and DNA dot-blot methods.

Multiple&#160;DNA modifications have been identified in the mammalian genome. Of that, 5-methylcytosine and 5-hydroxymethylcytosine-mediated epigenetic ...

Neuronal Differentiation from Mouse Embryonic Stem Cells In vitro

The neural differentiation of mouse embryonic stem cells (mESCs) is a potential tool for elucidating the key mechanisms involved in neurogenesis and potentially aid in regenerative medicine. Here, we established an efficient and low cost method for neuronal differentiation from mESCs in vitro, using the strategy of combinatorial screening. Under the conditions defined here, the 2-day embryoid body formation + 6-day retinoic acid induction protocol permits fast and efficient differentiation from mESCs into neural precursor cells (NPCs), as seen by the formation of well-stacked and neurite-like A2lox and 129 derivatives that are Nestin positive. The healthy state of embryoid bodies and the timepoint at which retinoic acid (RA) is applied, as well as the RA concentrations, are critical in the process. In the subsequent differentiation from NPCs into neurons, N2B27 medium II (supplemented by Neurobasal medium) could better support the long term maintenance and maturation of neuronal cells. The presented method is highly efficiency, low cost and easy to operate, and can be a powerful tool for neurobiology and developmental biology research.

Here, we established a low cost and easy to operate method that directs fast and efficient differentiation from embryonic stem cells into neurons. This method is suitable for popularization among laboratories and can be a useful tool for neurological research.

The neural differentiation of mouse embryonic stem cells (mESCs) is a potential tool for elucidating the key mechanisms involved in neurogenesis and ...