Name: intranasal delivery of therapeutic stem cells to glioblastoma in a mouse model
Uploaded: 2017-06-04T15:00:00
Description: Watch this Scientific Journal Video about Intranasal Delivery of Therapeutic Stem Cells to Glioblastoma in a Mouse Model at JoVE.com

This work was supported by NIH R01NS087990 (MSL, IVB).

All animal procedures must be approved by the Institutional Animal Care and Use Committee (IACUC) or equivalent. If there is any uncertainty regarding the specific procedures described herein, do not proceed. Clarify with the institution&#39;s IACUC and a designated veterinary staff member.
1. Ensuring Sterility of Cultured Cells and Follow Principles of Aseptic Techniques
<ol>
	<li>Follow the standard good laboratory practice in all cell culture procedures and IACUC requirements for cell te...

<ol>
	<li>Shah, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stem+cell-based+therapies+for+tumors+in+the+brain:+are+we+there+yet?.">Stem cell-based therapies for tumors in the brain: are we there yet?.</a> Neuro Oncol. 18 (8), 1066-1078 (2016).</li><li>Balyasnikova, I. V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intranasal+delivery+of+mesenchymal+stem+cells+significantly+extends+survival+of+irradiated+mice+with+experimental+brain+tumors.">Intranasal delivery of mesenchymal stem cells significantly extends survival of irradiated mice with experimental brain tumors.</a> Mol Ther. 22 (1), 140-148 (2014).</li><li>Reitz, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intranasal+delivery+of+neural+stem/progenitor+cells:+a+noninvasive+passage+to+target+intracerebral+glioma.">Intranasal delivery of neural stem/progenitor cells: a noninvasive passage to target intracerebral glioma.</a> Stem Cells Transl Med. 1 (12), 866-873 (2012).</li><li>Dey, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intranasal+Oncolytic+Virotherapy+with+CXCR4-Enhanced+Stem+Cells+Extends+Survival+in+Mouse+Model+of+Glioma.">Intranasal Oncolytic Virotherapy with CXCR4-Enhanced Stem Cells Extends Survival in Mouse Model of Glioma.</a> Stem Cell Reports. 7 (3), 471-482 (2016).</li><li>Ahmed, A. U., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+preclinical+evaluation+of+neural+stem+cell-based+cell+carrier+for+targeted+antiglioma+oncolytic+virotherapy.">A preclinical evaluation of neural stem cell-based cell carrier for targeted antiglioma oncolytic virotherapy.</a> J Natl Cancer Inst. 105 (13), 968-977 (2013).</li><li>Kim, S. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+neural+stem+cells+target+experimental+intracranial+medulloblastoma+and+deliver+a+therapeutic+gene+leading+to+tumor+regression.">Human neural stem cells target experimental intracranial medulloblastoma and deliver a therapeutic gene leading to tumor regression.</a> Clin Cancer Res. 12 (18), 5550-5556 (2006).</li><li>Lee, D. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeting+rat+brainstem+glioma+using+human+neural+stem+cells+and+human+mesenchymal+stem+cells.">Targeting rat brainstem glioma using human neural stem cells and human mesenchymal stem cells.</a> Clin Cancer Res. 15 (15), 4925-4934 (2009).</li><li>Lesniak, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeted+therapy+for+malignant+glioma:+neural+stem+cells.">Targeted therapy for malignant glioma: neural stem cells.</a> Expert Rev Neurother. 6 (1), 1-3 (2006).</li><li>Robinson, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=GLPs+and+the+Importance+of+Standard+Operating+Procedures.">GLPs and the Importance of Standard Operating Procedures.</a> BioPharm International. 16 (8), (2003).</li><li>World Health Organization on behalf of the Special Programme for Research and Training in Tropical Diseases. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Handbook: Good Laboratory Practice (GLP). , (2009).</li><li>NIH. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Number: NOT-OD-16-011. Implementing Rigor and Transparency) in NIH &amp; AHRQ Research Grant Applications. , (2015).</li><li>Wakimoto, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Maintenance+of+primary+tumor+phenotype+and+genotype+in+glioblastoma+stem+cells.">Maintenance of primary tumor phenotype and genotype in glioblastoma stem cells.</a> Neuro Oncol. 14 (2), 132-144 (2012).</li><li>Cheng, S. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamic+In+Vivo+SPECT+Imaging+of+Neural+Stem+Cells+Functionalized+with+Radiolabeled+Nanoparticles+for+Tracking+of+Glioblastoma.">Dynamic In Vivo SPECT Imaging of Neural Stem Cells Functionalized with Radiolabeled Nanoparticles for Tracking of Glioblastoma.</a> J Nucl Med. 57 (2), 279-284 (2016).</li><li>Pritchett-Corning, K. R., Luo, Y., Mulder, G. B., White, W. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Principles+of+rodent+surgery+for+the+new+surgeon.">Principles of rodent surgery for the new surgeon.</a> J Vis Exp. (47), (2011).</li><li>Clark, A. J., Fakurnejad, S., Ma, Q., Hashizume, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bioluminescence+Imaging+of+an+Immunocompetent+Animal+Model+for+Glioblastoma.">Bioluminescence Imaging of an Immunocompetent Animal Model for Glioblastoma.</a> J Vis Exp. (107), (2016).</li><li>Ulasov, I. V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Survivin-driven+and+fiber-modified+oncolytic+adenovirus+exhibits+potent+antitumor+activity+in+established+intracranial+glioma.">Survivin-driven and fiber-modified oncolytic adenovirus exhibits potent antitumor activity in established intracranial glioma.</a> Hum Gene Ther. 18 (7), 589-602 (2007).</li><li>Danielyan, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intranasal+delivery+of+cells+to+the+brain.">Intranasal delivery of cells to the brain.</a> Eur J Cell Biol. 88 (6), 315-324 (2009).</li><li>Dhuria, S. V., Hanson, L. R., Frey, W. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intranasal+delivery+to+the+central+nervous+system:+mechanisms+and+experimental+considerations.">Intranasal delivery to the central nervous system: mechanisms and experimental considerations.</a> J Pharm Sci. 99 (4), 1654-1673 (2010).</li><li>Gross, E. A., Swenberg, J. A., Fields, S., Popp, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparative+morphometry+of+the+nasal+cavity+in+rats+and+mice.">Comparative morphometry of the nasal cavity in rats and mice.</a> J Anat. 135 (Pt 1), 83-88 (1982).</li><li>Marieb, E. N., Hoehn, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Human Anatomy &amp; Physiology. , (2007).</li></ol>

Although the intranasal route of drug delivery has been widely explored for small molecules, nanomedicines, and protein compounds alike18, the application of therapeutic stem cells for intranasal brain tumor targeting is very new in the spectrum of brain tumor therapeutics under development2,3,4. There are intrinsic complexities involved regarding the behaviors of stem cells in the nasal cavity, and molec...

Low toxicity, low immunogenicity, and intrinsic brain tumor tropism of human stem cells are attractive traits for the delivery of therapeutics vehicles1. Novel stem cell-based therapeutics for malignant brain tumors are promising innovations developed in recent years, and the intranasal adaptation of this therapeutic strategy represents a leap towards clinical translation, in that non-invasive and repeated administration might dramatically reduce the barrier for patient applications and may be adaptable for out-patient services without general anesthesia or lengthy in-patient service associated with invasive surgical procedures1,2,3,4.
We and others have pioneered the intranasal route of stem cell delivery to brain tumors and have laid the ground work for some of the basic principles of translational research using mouse xenograft models2,3,4, as well as investigated the migration of stem cells in vivo via magnetic resonance imaging (MRI) reagent carriers2. Through these pilot explorations, we have accumulated substantial experience and gained insight on how to best construct a robust pre-clinical evaluation strategy using well-established patient-derived xenograft (PDX) mouse models of malignant glioma, while maintaining the investigative resolution to examine the nuanced mechanistic details of the sophisticated biological phenomena of the intranasal brain entry of therapeutic stem cells delivered to the nasal cavity. Here, we describe the principles of a standardized operating protocol to demonstrate the current state of experimental investigations using a well-established human neural stem cell line HB1.F3.CD5,6,7,8, which is readily modifiable to adapt to specific tumor models or strategies using human stem cells as therapeutic carriers.

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			<td height="21" style="height:21px;width:236px;">Stereotaxic frame</td>
			<td style="width:221px;">Kopf Instruments</td>
			<td style="width:119px;">Model 900</td>
			<td style="width:199px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:236px;">Hypoxic Cell Culture Incubator</td>
			<td style="width:221px;">ThermoFisher Scientific</td>
			<td style="width:119px;">VIOS 160i</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:236px;">Cell culture supplies (Plastics)</td>
			<td style="width:221px;">ThermoFisher Scientific</td>
			<td style="width:119px;">Varies</td>
			<td>Replaceable with any source</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:236px;">Legend Micro 21R Refrigerated Microcentrifuge</td>
			<td style="width:221px;">ThermoFisher Scientific</td>
			<td style="width:119px;">75002490</td>
			<td>Replaceable with any source</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:236px;">Bench centrifuge Sorvall ST16R&#160;</td>
			<td style="width:221px;">ThermoFisher Scientific</td>
			<td style="width:119px;">75004240</td>
			<td>Replaceable with any source</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:23px;width:236px;">Micro syringe 702N 25&#181;l (22S/2&#34;/2)</td>
			<td style="width:221px;">Hamilton Company</td>
			<td style="width:119px;">80400</td>
			<td>Flat tip</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:236px;">Sample Tray for Irradiator</td>
			<td style="width:221px;">Best Theratronics</td>
			<td style="width:119px;">A13826</td>
			<td style="width:199px;">To set up mice protection with lead shield</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:236px;">Leica DMi8 Microscope</td>
			<td style="width:221px;">Leica Microsystem</td>
			<td style="width:119px;"></td>
			<td>Custom setup</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:236px;">Leica CM1860 UV cryostat</td>
			<td style="width:221px;">Leica Microsystem</td>
			<td style="width:119px;"></td>
			<td>Custom setup</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:236px;">Exel International Insulin Syringe</td>
			<td style="width:221px;">ThermoFisher Scientific</td>
			<td style="width:119px;">14-841-31</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:236px;">Corning Phosphate Buffer Saline</td>
			<td style="width:221px;">Corning Cellgro/ThermoFisher</td>
			<td style="width:119px;">21-031-CV</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:21px;width:236px;">Dulbecco&#39;s Modified Eagle Medium&#160;</td>
			<td style="width:221px;">Corning Cellgro/ThermoFisher</td>
			<td style="width:119px;">11965-084</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:236px;">Trypsin 0.05%</td>
			<td style="width:221px;">Corning Cellgro/ThermoFisher</td>
			<td style="width:119px;">25300054</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:236px;">Hyaluronidase from bovine testes</td>
			<td style="width:221px;">MilliporeSigma</td>
			<td style="width:119px;">H3506</td>
			<td></td>
		</tr>
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intranasal delivery of therapeutic stem cells to glioblastoma in a mouse model

The intrinsic tropism towards brain malignancies renders stem cells as promising carriers of therapeutic agents against malignant tumors. The delivery of therapeutic stem cells via the intranasal route is a recently discovered alternative strategy, with strong potential for clinical translation, due to its non-invasive nature compared to intracranial implantation or delivery via systemic routes. The lack of blood brain barrier further strengthens the therapeutic potential of stem cells undergoing intranasal brain entry. This article summarizes the essential techniques utilized in our studies and outlines the basic principles of intranasal strategy for stem cell delivery using a mouse model of intracranial glioma xenografts. We demonstrate the optimized procedures that generate consistent and reproducible results with specific predetermined experimental parameters and offer guidelines for streamlined work flow that ensure efficient execution and reliable experimental outcome. The article is designed to serve as a baseline for further experimental customization based on hypothesis, stem cell types, or tumor specifics.

Both hypoxic pre-treatment (Figure 4A)4 and CXCR4 overexpression (Figures 4B and 4C)4 significantly upregulate the cell membrane presence of CXCR4 receptors as demonstrated by flow cytometry. The tumor tropism demonstrated by NSCs (blue arrows), is shown in the tumor tissue histology (red circle). The presence of MPIO-labeled (Figure 4D) stem cells ...

Watch this Scientific Journal Video about Intranasal Delivery of Therapeutic Stem Cells to Glioblastoma in a Mouse Model at JoVE.com

Intranasal Delivery of Therapeutic Stem Cells to Glioblastoma in a Mouse Model

Stem cells are promising therapeutic carriers to treat brain tumors due to their intrinsic tumor tropism. Non-invasive intranasal stem cell delivery bypasses the blood brain barrier and demonstrates strong potential for clinical translation. This article summarizes the basic principles of intranasal stem cell delivery in a mouse model of glioma.

The intrinsic tropism towards brain malignancies renders stem cells as promising carriers of therapeutic agents against malignant tumors. The delivery of ...

The overall goal of this protocol is to deliver therapeutic stem cells to the brain intranasally in a preclinical mouse model of glioblastoma. This method can help answer key questions in neuro-oncology field about whether therapeutic stem cells treatment can achieve a robust, therapeutic effect in pre-clinical brain tumor model. The main advantage of this technique is that intranasal administration facilitates the delivery of non-invasive, repeatable brain tumor treatment that bipasses the obstructive blood-brain barrier.This technique is not limited to glioblastoma. It can be broadly extended toward therapy of other brain malignancies and brain disorders. My colleagues, Dou Yu, a research assistant professor, Gina Li, a research technologist will be demonstrating this procedure.Begin by culturing the glioma cells of interest from frozen stock at one times 10 to the six cells per T 75 square centimeter culture flask in 15 milliliters of serum supplemented or serum-free medium as appropriate in a humidified, carbon-dioxide-supplemented cell culture incubator. When the cultures have reached 80 percent confluency, wash the culture with calcium and magnesium-free PBS. Then, dissociate the cells with point zero five percent trypsin at room temperature.Then tap the flask to detach the cells from the bottom on the container. Immediately neutralize the enzymes with eight to nine milliliters of serum-containing medium. Use serological pipettes to transfer the resulting cell suspension into conical tubes.Collect the cells by centrifugation. Followed by two washes in 10 milliliters of PBS each. We suspend the pellets in sterile saline at a five to 20 times 10 to the fifth cells per two point five microliters concentration and transfer the cells into a sterile micro-centrifuge tube on ice.After confirming the appropriate level of sedation by toe pinch, mount an approximately six-week old, immuno-compromised mouse onto an ethanol-sterilized stereotaxic frame in the appropriate head position. Put the inserted Hamilton micro-syringe as perpendicular to the surface of the skull around the burr hole. Use the stereotaxic frame knobs to position the flat tip of the micro-syringe until it is just at the opening of the burr hole then slowly lower the syringe until the needle reaches three millimeters below the dura surface.Retract the syringe zero point five millimeters to maintain the needle tip at two point five millimeters below the dura and inject two point five microliters of pre-loaded glioma cells over a one minute period. Leave the needle in position for another one to two minutes before slowly withdrawing the syringe over the course of one minute to minimize the cell backflow. Apply a sterile bone wax to avoid extra-cranial tumor add growth and use seven millimeter stainless steel wound clips to close the wound.Then deliver one milliliter of 37 degree Celsius sterile lactated ringer solution by subcutaneous injection and place the animal in a recovery chamber positioned halfway onto a heating pad with monitoring until full recovery. Seven to eight days post glioma cell implantation, preform head-only exposure to the radiation path. Then deliver two grades of irradiation to the animals everyday for five consecutive days, assessing the tumors by bioluminescence imaging after the last irradiation dose.For human neural stem cell genetic modification, when the stem cell culture reaches 75 to 80 percent confluency, adhering CXCR4 encoded antivirus at five viral particles per cell in polybrene directly into the medium for eight to 16 hours of culture. At the end of the incubation, replace the transduction medium with fresh blasticidin supplemented medium for positive selection of the transduced cells. Harvest the cells by trypsin digestion as demonstrated.Validate the level of CXCR4 expression by flow cytometry using anti-CXCR4 anti-body and matching isotope anti-body controls according to standard flow cytometric analysis protocols. Then suspend the cells in sterile saline for intranasal delivery. For hypoxic preconditioning of the neuro cells, when the culture reaches 90 percent confluency, transfer the flask to a humidified carbon dioxide incubator supplied with one percent oxygen.After 24 hours, harvest the cells by trypsin digestion. Suspend the cells in sterile saline for intranasal delivery. To load human neural stem cells with oncolytic virus, infect the stem cells with 50 conditional replicative adenovirus viral particles per cell, according to standard adenovirus transfection protocols, for two hours at room temperature with periodic tapping.Then wash the cells three times with fresh medium to remove any of the unbound viral particles and suspend the cells in sterile saline for intransal delivery. When the cells have been modified as experimentally appropriate, place the anesthetized, tumor-bearing animals in the supine position on a clean drape over a heat pad and place a padded pillow under the heads to maintain the nostrils at an upright angle. The pillow material should be either alcohol-wipe-able plastic or disposable rolled up paper towels to avoid cross-animal contamination.When the mice are in position, use a micro-pipette to dispense two microliters of the experimental human neuro stem cell population of interest into each nostril of the first mouse. Visually confirm an aspiration of the droplet before applying the next volume of cells. Repeat the delivery to each mouse in turn.Then wait five minutes and apply the next round of cells. When all four rounds of cells have been administered, place the animals in a recovery chamber with monitoring until full recovery. Both hypoxic pre-treatment and CXCR4 overexpression significantly upregulate the cell membrane presence of CXCR4 receptors as demonstrated by flow cytometry.The presence of micron-sized paramagnetic ion oxide labeled stem cells in the tumor can be confirmed via Prussian Blue Staining. The tumor tropism demonstrated by neural stem cells can be visualized by histological analysis of the tumor tissue. Tissue analysis also reveals that head-only irradiation induces a reduction in tumor volume as well as SDF-1 upregulation in tumor and surrounding tissues.Confirming that CXCR4 receptor upregulation in neural stem cells, we facilitate tumor tropism after irradiation. Survival analyses indicate that although irradiation alone is beneficial for the survival of tumor-bearing animals, additional benefits can be achieved using hypoxic neural stem cells loaded with irradiated oncolytic virus over control-irradiated, non-hypoxic stem cells loaded with oncolytic virus. Significant survival benefits are also observed with CXCR4 transduced neural stem cells after oncolytic viral loading and irradiation.Further, both hypoxic neural stem cells and CXCR4 transduced neural stem cells exhibit a significantly enhanced tumor-targeted delivery of oncolytic virus particles as evidenced by viral hexon protein staining. Once mastered, this intranasal delivery technique can be completed in one hour if it's performed properly. While attempting this procedure, it's important to remember to follow all the steps in a timely manner exactly as described.This technique allows the exploration of therapeutic potential stem cells for broad spectrum brain disorders in pre-clinical rodent models. After watching this video, you should have a good understanding of how to perform consistent and effective intranasal therapeutic stem cell delivery for the targeting of mouse brain tumors. Don't forget that working with human cells and viruses can be hazardous and that precautions, such as using a-subject techniques and proper personal protection equipment, should always be taken when performing these procedures.

intranasal-delivery-therapeutic-stem-cells-to-glioblastoma-mouse

Research

JoVE Journal

Medicine

A Mouse Model of in Utero Transplantation

The transplantation of stem cells and viruses in utero has tremendous potential for treating congenital disorders in the human fetus. For example, in utero transplantation (IUT) of hematopoietic stem cells has been used to successfully treat patients with severe combined immunodeficiency.1,2 In several other conditions, however, IUT has been attempted without success.3 Given these mixed results, the availability of an efficient non-human model to study the biological sequelae of stem cell transplantation and gene therapy is critical to advance this field. We and others have used the mouse model of IUT to study factors affecting successful engraftment of in utero transplanted hematopoietic stem cells in both wild-type mice4-7 and those with genetic diseases.8,9 The fetal environment also offers considerable advantages for the success of in utero gene therapy. For example, the delivery of adenoviral10, adeno-associated viral10, retroviral11, and lentiviral vectors12,13 into the fetus has resulted in the transduction of multiple organs distant from the site of injection with long-term gene expression. in utero gene therapy may therefore be considered as a possible treatment strategy for single gene disorders such as muscular dystrophy or cystic fibrosis. Another potential advantage of IUT is the ability to induce immune tolerance to a specific antigen. As seen in mice with hemophilia, the introduction of Factor IX early in development results in tolerance to this protein.14 
In addition to its use in investigating potential human therapies, the mouse model of IUT can be a powerful tool to study basic questions in developmental and stem cell biology. For example, one can deliver various small molecules to induce or inhibit specific gene expression at defined gestational stages and manipulate developmental pathways. The impact of these alterations can be assessed at various timepoints after the initial transplantation. Furthermore, one can transplant pluripotent or lineage specific progenitor cells into the fetal environment to study stem cell differentiation in a non-irradiated and unperturbed host environment.
The mouse model of IUT has already provided numerous insights within the fields of immunology, and developmental and stem cell biology. In this video-based protocol, we describe a step-by-step approach to performing IUT in mouse fetuses and outline the critical steps and potential pitfalls of this technique.

A Mouse Model of in Utero Transplantation

The mouse model of in utero transplantation is a versatile tool that can be used to study the potential clinical applications of stem cell transplantation and gene therapy in the fetus. In this protocol, we present a general approach to performing this technique

The transplantation of stem cells and viruses in utero has tremendous potential for treating congenital disorders in the human fetus.  For example, in ...

Stereotactic Intracranial Implantation and In vivo Bioluminescent Imaging of Tumor Xenografts in a Mouse Model System of Glioblastoma Multiforme

Glioblastoma multiforme (GBM) is a high-grade primary brain cancer with a median survival of only 14.6 months in humans despite standard tri-modality treatment consisting of surgical resection, post-operative radiation therapy and temozolomide chemotherapy 1. New therapeutic approaches are clearly needed to improve patient survival and quality of life. The development of more effective treatment strategies would be aided by animal models of GBM that recapitulate human disease yet allow serial imaging to monitor tumor growth and treatment response. In this paper, we describe our technique for the precise stereotactic implantation of bio-imageable GBM cancer cells into the brains of nude mice resulting in tumor xenografts that recapitulate key clinical features of GBM 2. This method yields tumors that are reproducible and are located in precise anatomic locations while allowing in vivo bioluminescent imaging to serially monitor intracranial xenograft growth and response to treatments 3-5. This method is also well-tolerated by the animals with low perioperative morbidity and mortality.

Stereotactic Intracranial Implantation and In vivo Bioluminescent Imaging of Tumor Xenografts in a Mouse Model System of Glioblastoma Multiforme

We describe an integrated method for the precise, stereotactic implantation of human glioblastoma multiforme cells into the brains of nude mice and subsequent serial in vivo imaging to monitor growth and response to treatment of the resultant xenografts.

Glioblastoma multiforme (GBM) is a high-grade primary brain cancer with a median survival of only 14.6 months in humans despite standard tri-modality ...

Intranasal Administration of CNS Therapeutics to Awake Mice

Intranasal administration is a method of delivering therapeutic agents to the central nervous system (CNS). It is non-invasive and allows large molecules that do not cross the blood-brain barrier access to the CNS. Drugs are directly targeted to the CNS with intranasal delivery, reducing systemic exposure and thus unwanted systemic side effects1. Delivery from the nose to the CNS occurs within minutes along both the olfactory and trigeminal neural pathways via an extracellular route and does not require drug to bind to any receptor or axonal transport2. Intranasal delivery is a widely publicized method and is currently being used in human clinical trials3.
Intranasal delivery of drugs in animal models allows for initial evaluation of pharmacokinetic distribution and efficacy. With mice, it is possible to administer drugs to awake (non-anesthetized) animals on a regular basis using a specialized intranasal grip. Awake delivery is beneficial because it allows for long-term chronic dosing without anesthesia, it takes less time than with anesthesia, and can be learned and done by many people so that teams of technicians can dose large numbers of mice in short periods. Efficacy of therapeutics administered intranasally in this way to mice has been demonstrated in a number of studies including insulin in diabetic mouse models 4-6 and deferoxamine in Alzheimer's mouse models. 7,8
The intranasal grip for mice can be learned, but is not easy and requires practice, skill, and a precise grip to effectively deliver drug to the brain and avoid drainage to the lung and stomach. Mice are restrained by hand using a modified scruff in the non-dominant hand with the neck held parallel to the floor, while drug is delivered with a pipettor using the dominant hand. It usually takes 3-4 weeks of acclimating to handling before mice can be held with this grip without a stress response. We have prepared this JoVE video to make this intranasal delivery technique more accessible.

A method to intranasally administer drugs to awake mice for the purpose of targeting the brain is described. This method allows for repeat dosing over long periods using intranasal administration of drug without anesthesia, and nose-to-brain delivery with minimal systemic exposure.

Intranasal administration is a method of delivering  therapeutic agents to the central nervous system (CNS). It is  non-invasive and allows large ...

High Throughput Characterization of Adult Stem Cells Engineered for Delivery of Therapeutic Factors for Neuroprotective Strategies

Mesenchymal stem cells (MSCs) derived from bone marrow are a powerful cellular resource and have been used in numerous studies as potential candidates to develop strategies for treating a variety of diseases. The purpose of this study was to develop and characterize MSCs as cellular vehicles engineered for delivery of therapeutic factors as part of a neuroprotective strategy for rescuing the damaged or diseased nervous system. In this study we used mouse MSCs that were genetically modified using lentiviral vectors, which encoded brain-derived neurotrophic factor (BDNF) or glial cell-derived neurotrophic factor (GDNF), together with green fluorescent protein (GFP). 
Before proceeding with in vivo transplant studies it was important to characterize the engineered cells to determine whether or not the genetic modification altered aspects of normal cell behavior. Different culture substrates were examined for their ability to support cell adhesion, proliferation, survival, and cell migration of the four subpopulations of engineered MSCs. High content screening (HCS) was conducted and image analysis performed. 
Substrates examined included: poly-L-lysine, fibronectin, collagen type I, laminin, entactin-collagen IV-laminin (ECL). Ki67 immunolabeling was used to investigate cell proliferation and Propidium Iodide staining was used to investigate cell viability. Time-lapse imaging was conducted using a transmitted light/environmental chamber system on the high content screening system.
Our results demonstrated that the different subpopulations of the genetically modified MSCs displayed similar behaviors that were in general comparable to that of the original, non-modified MSCs. The influence of different culture substrates on cell growth and cell migration was not dramatically different between groups comparing the different MSC subtypes, as well as culture substrates. 
This study provides an experimental strategy to rapidly characterize engineered stem cells and their behaviors before their application in long-term in vivo transplant studies for nervous system rescue and repair.

This study describes an experimental platform to rapidly characterize engineered stem cells and their behaviors before their application in long-term in vivo transplant studies for nervous system rescue and repair.

Mesenchymal stem cells (MSCs) derived from bone marrow are a powerful cellular resource and have been used in numerous studies as potential candidates to ...

Studying Pancreatic Cancer Stem Cell Characteristics for Developing New Treatment Strategies

Pancreatic ductal adenocarcinoma (PDAC) contains a subset of exclusively tumorigenic cancer stem cells (CSCs) which have been shown to drive tumor initiation, metastasis and resistance to radio- and chemotherapy. Here we describe a specific methodology for culturing primary human pancreatic CSCs as tumor spheres in anchorage-independent conditions. Cells are grown in serum-free, non-adherent conditions in order to enrich for CSCs while their more differentiated progenies do not survive and proliferate during the initial phase following seeding of single cells. This assay can be used to estimate the percentage of CSCs present in a population of tumor cells. Both size (which can range from 35 to 250 micrometers) and number of tumor spheres formed represents CSC activity harbored in either bulk populations of cultured cancer cells or freshly harvested and digested tumors 1,2. Using this assay, we recently found that metformin selectively ablates pancreatic CSCs; a finding that was subsequently further corroborated by demonstrating diminished expression of pluripotency-associated genes/surface markers and reduced in vivo tumorigenicity of metformin-treated cells. As the final step for preclinical development we treated mice bearing established tumors with metformin and found significantly prolonged survival. Clinical studies testing the use of metformin in patients with PDAC are currently underway (e.g., NCT01210911, NCT01167738, and NCT01488552). Mechanistically, we found that metformin induces a fatal energy crisis in CSCs by enhancing reactive oxygen species (ROS) production and reducing mitochondrial transmembrane potential. In contrast, non-CSCs were not eliminated by metformin treatment, but rather underwent reversible cell cycle arrest. Therefore, our study serves as a successful example for the potential of in vitro sphere formation as a screening tool to identify compounds that potentially target CSCs, but this technique will require further in vitro and in vivo validation to eliminate false discoveries.

Pancreatic cancer stem cells (CSCs) can be expanded in vitro using the anchorage-independent sphere culture technique, which represents a powerful tool to study CSC biology and can serve as the first step to develop novel CSC-targeting therapies. Here the methodology for expanding, analyzing and targeting of pancreatic CSCs is provided.

Pancreatic ductal adenocarcinoma (PDAC) contains a subset of exclusively tumorigenic cancer stem cells (CSCs) which have been shown to drive tumor ...

A Neonatal Mouse Spinal Cord Compression Injury Model

Spinal cord injury (SCI) typically causes devastating neurological deficits, particularly through damage to fibers descending from the brain to the spinal cord. A major current area of research is focused on the mechanisms of adaptive plasticity that underlie spontaneous or induced functional recovery following SCI. Spontaneous functional recovery is reported to be greater early in life, raising interesting questions about how adaptive plasticity changes as the spinal cord develops. To facilitate investigation of this dynamic, we have developed a SCI model in the neonatal mouse. The model has relevance for pediatric SCI, which is too little studied. Because neural plasticity in the adult involves some of the same mechanisms as neural plasticity in early life1, this model may potentially have some relevance also for adult SCI. Here we describe the entire procedure for generating a reproducible spinal cord compression (SCC) injury in the neonatal mouse as early as postnatal (P) day 1. SCC is achieved by performing a laminectomy at a given spinal level (here described at thoracic levels 9-11) and then using a modified Yasargil aneurysm mini-clip to rapidly compress and decompress the spinal cord. As previously described, the injured neonatal mice can be tested for behavioral deficits or sacrificed for ex vivo physiological analysis of synaptic connectivity using electrophysiological and high-throughput optical recording techniques1. Earlier and ongoing studies using behavioral and physiological assessment have demonstrated a dramatic, acute impairment of hindlimb motility followed by a complete functional recovery within 2 weeks, and the first evidence of changes in functional circuitry at the level of identified descending synaptic connections1.

This article describes a method for generating a reproducible spinal cord compression injury (SCI) in the neonatal mouse. The model provides an advantageous platform for studying mechanisms of adaptive plasticity that underlie spontaneous functional recovery.

Spinal cord injury (SCI) typically causes devastating neurological deficits, particularly through damage to fibers descending from the brain to the spinal ...

Vein Interposition Model: A Suitable Model to Study Bypass Graft Patency

Bypass grafting is an established treatment method for coronary artery disease. Graft patency continues to be the Achilles heel of saphenous vein grafts. Research models for bypass graft failure are essential for a better understanding of pathobiological and pathophysiological processes during graft patency loss. Large animal models, such as pigs or sheep, resemble human anatomical structures but require special facilities and equipment. This video describes a rat vein interposition model to investigate vein graft patency loss. Rats are inexpensive and easy to handle. Compared to mouse models, the convenient size of rats permits better operability and enables a sufficient amount of material to be obtained for further diverse analysis. In brief, the inferior epigastric vein of a donor rat is harvested and used to replace a segment of the femoral artery. Anastomosis is conducted via single stitches and sealed with fibrin glue. Graft patency can be monitored non-invasively using duplex sonography. Myointimal hyperplasia, which is the main cause for graft patency loss, develops progressively over time and can be calculated from histological cross sections.

This video demonstrates a model to study the development of myointimal hyperplasia after venous interposition surgery in rats.

Bypass grafting is an established treatment method for coronary artery disease. Graft patency continues to be the Achilles heel of saphenous vein grafts. ...

Image-Guided Resection of Glioblastoma and Intracranial Implantation of Therapeutic Stem Cell-seeded Scaffolds

Glioblastoma (GBM), the most common and aggressive primary brain cancer, carries a life expectancy of 12-15 months. The short life expectancy is due in part to the inability of the current treatment, consisting of surgical resection followed by radiation and chemotherapy, to eliminate invasive tumor foci. Treatment of these foci may be improved with tumoricidal human mesenchymal stem cells (MSCs). MSCs exhibit potent tumor tropism and can be engineered to express therapeutic proteins that kill tumor cells. Advancements in preclinical models indicate that surgical resection induces premature MSC loss and reduces therapeutic efficacy. Efficacy of MSC treatment can be improved by seeding MSCs on a biodegradable poly(lactic acid) (PLA) scaffold. MSC delivery into the surgical resection cavity on a PLA scaffold restores cell retention, persistence, and tumor killing. To study the effects of MSC-seeded PLA implantation on GBM, an accurate preclinical model is needed. Here we provide a preclinical surgical protocol for image-guided tumor resection of GBM in immune-deficient mice followed by MSC-seeded scaffold implantation. MSCs are engineered with lentiviral constructs to constitutively express and secrete therapeutic TNF&#945;-related apoptosis-inducing ligand (TRAIL) as well as green fluorescent protein (GFP) to allow fluorescent tracking. Similarly, the U87 tumor cells are engineered to express mCherry and firefly luciferase, providing dual fluorescent/luminescent tracking. While currently used for investigating stem cell mediated delivery of therapeutics, this protocol could be modified to investigate the impact of surgical resection on other GBM interventions.

Tumor-seeking therapeutic mesenchymal stem cells (MSCs) show promise as a treatment for invasive glioblastoma. Optimal transplantation involves delivery of MSCs into the tumor resection cavity on scaffolds. Here, preclinical techniques to study MSC treatment of glioblastoma are provided including: image-guided tumor resection; implantation of MSC-seeded scaffolds; and postoperative therapy tracking.

Glioblastoma (GBM), the most common and aggressive primary brain cancer, carries a life expectancy of 12-15 months. The short life expectancy is due in ...

Isometric Contractility Measurement of the Mouse Mesenteric Artery Using Wire Myography

The wire myograph technique is used to assess the contractility of vascular smooth muscles in response to depolarization, GPCR agonists/inhibitors and drugs. It is widely used in many studies on the physiological functions of vascular smooth muscle, the pathogenesis of vascular diseases such as hypertension, and the development of smooth muscle relaxant drugs. The mouse is a widely used model animal with a large pool of disease models and genetically modified strains. We introduced this method to measure the isometric contraction of mouse mesenteric artery in detail. A 1.4-mm segment of mouse mesenteric resistance artery was isolated and mounted on a myograph chamber by passing two steel wires through its lumen. After equilibration and normalization steps, the vessel segment was potentiated by a high-K+ solution twice prior to the contraction assay. As an example of the application of this method in drug development, we measured the relaxant effect of a novel natural substance, neoliensinine, isolated from a Chinese herb, embryos of the lotus seed (Nelumbo nucifera Gaertn.) on mouse mesenteric arteries. The vessel segments mounted on the myograph chamber were stimulated with a high-K+ solution. When the force tension reached a stable sustained phase, cumulative doses of neoliensinine were added to the chamber. We found that neoliensinine had a dose-dependent relaxant effect on smooth muscle contraction, thus suggesting that it bears potential activity against hypertension. In addition, as the vessel segment can survive at least 4 hours after mounting and maintain contractility induced by the high-K+ solution for many times, we suggest that the wire myograph system may be used for the time-consuming process of drug screening.

The wire myograph technique is used to investigate vascular smooth muscle functions and screen new drugs. We report a detailed protocol for measuring the isometric contractility of the mouse mesenteric artery and for screening new relaxants of vascular smooth muscle.

The wire myograph technique is used to assess the contractility of vascular smooth muscles in response to depolarization, GPCR agonists/inhibitors and ...

Novel Methods for Intranasal Administration Under Inhalation Anesthesia to Evaluate Nose-to-Brain Drug Delivery

Intranasal administration has been reported to be a potential pathway for nose-to-brain delivery of therapeutic agents that circumvents the blood-brain barrier. However, there have been few reports regarding not only the quantitative analysis but also optimal administration conditions and dosing regimens for investigations of nose-to-brain delivery. The limited progress in research on nose-to-brain pathway mechanisms using rodents represents a significant impediment in terms of designing nose-to-brain delivery systems for candidate drugs.
To gain some headway in this regard, we developed and evaluated two novel methods of stable intranasal administration under inhalation anesthesia for experimental animals. We also describe a method for the evaluation of drug distribution levels in the brain via the nose-to-brain pathway using radio-labeled [14C]-inulin (molecular weight: 5,000) as a model substrate of water-soluble macromolecules.
Initially, we developed a pipette-based intranasal administration protocol using temporarily openable masks, which enabled us to perform reliable administration to animals under stable anesthesia. Using this system, [14C]-inulin could be delivered to the brain with little experimental error.
We subsequently developed an intranasal administration protocol entailing reverse cannulation from the airway side through the esophagus, which was developed to minimize the effects of mucociliary clearance (MC). This technique led to significantly higher levels of [14C]-inulin, which was quantitatively detected in the olfactory bulb, cerebrum, and medulla oblongata, than the pipette method. This appears to be because retention of the drug solution in the nasal cavity was substantially increased by active administration using a syringe pump in a direction opposite to the MC into the nasal cavity.
In conclusion, the two methods of intranasal administration developed in this study can be expected to be extremely useful techniques for evaluating pharmacokinetics in rodents. The reverse cannulation method, in particular, could be useful for evaluating the full potential of nose-to-brain delivery of drug candidates.

Here, we describe two novel methods of stable intranasal administration under inhalation anesthesia with minimal physical stress for experimental animals. We also describe a method for quantitative evaluation of drug distribution levels in the brain via the nose-to-brain pathway using radiolabeled [14C]-inulin as a model substrate of water-soluble macromolecules.

Intranasal administration has been reported to be a potential pathway for nose-to-brain delivery of therapeutic agents that circumvents the blood-brain ...