This work was supported by NIH NINDS grant R01 NS074388 581 (C.L. Limoli), California Institute for Regenerative Medicine (CIRM) Grant RS1-00413 (C.L.L.), CIRM Training Grant TG2- 0115 (M.M.A.), and a CIRM Grant to JOVE in support of the video documentation.

Our experimental plan is schematically diagrammed in Figure 1.
1. Growth and preparation of human neural stem cells (NSCs) for transplantation
<ol>
	<li>The EnStem-A cell line (EMD Millipore) was used in this study. NSCs are routinely validated by the manufacturer for high levels of expression of the multipotent markers nestin and Sox2, and low-level expression of the pluripotent marker Oct-4, along with the ability to differentiate into multiple neuronal phenotypes and to maintain a normal karyotype after multiple passages. Under our growth conditions, these NSCs displayed abundant expression of Sox2 and nestin and retained their ability to differentiate, as described previously1. NSCs were grown on poly-L-ornithine (20 &mu;g/ml) and laminin (5 &mu;g/ml) coated tissue culture treated flasks. Cells were grown in neural expansion medium (Millipore) supplemented with L-glutamine (2 mM) and basic fibroblast growth factor (bFGF; 20 ng/ml). Adherent monolayers of NSCs were passaged every other day (1:2) with accutase as the dissociation agent1. For transplantation studies, NSCs were used at passages below 10.</li>
	<li>NSCs were labeled with 5-bromo-2'-deoxyuridine (BrdU) by supplementing the growth media with 4 &mu;M BrdU for three days prior to transplantation. To verify the BrdU labeling index (i.e. percentage of BrdU positive cells), cells were plated on chamber slides and processed for BrdU detection using standard immunocytochemical approaches. Cells utilized for transplantation routinely exhibit labeling indices in excess of 90%2. Alternatively, transplanted human stem cells were detected using the human specific nuclear antigen marker (HuNu)2.</li>
	<li>On the day of transplantation, accutase and neural expansion media were pre-warmed in a 37&deg;C water bath. Cells were placed in incubation medium (neural expansion media containing 10 &mu;M of Y-27632) for one hour. Y-27632 (ROCK inhibitor, EMD-Calbiochem) was used to improve the survival of NSCs post-transplantation.</li>
	<li>After one hour, incubation media was removed and cells were treated with accutase for 5 minutes. After this brief treatment add an equal volume of neural expansion media to neutralize the accutase and strain the cells through a 70 &mu;M cell strainer.</li>
	<li>Count the cells with a haemocytometer and prepare 1.0 x 105 live NSCs per microliter in injection medium (neural expansion media containing 10 &mu;M of Y-27632, 40 ng/ml bFGF, 20 ng/ml BDNF).</li>
	<li>Cells were stored in neural expansion media (as described in 1.5) and kept on ice until the time of transplantation, and should be used within 6h to minimize cell death by prolonged cold-storage.</li>
</ol>
2. Radiotherapy - treatment planning and irradiation
<ol>
	<li>Sedated athymic nude rat(s) were placed in a laboratory MRI scanner in the prone position with the skull at the superior end (head first). This 3 tesla scanner is designed for small animal scans. It can provide exquisite soft-tissue contrast with high spatial resolution cross-sectional (axial) images. The scan focused on the skull region and a set of 22 T2 E60 weighted images with 0.8 mm image thickness were generated. These images provide necessary information to identify the left and right hippocampi within the rat's brain.</li>
	<li>Following the MRI scan (24h), animals were sedated [anesthesia cocktail (ketamine, 30 mg/kg, xylazine, 2.5 mg/kg and acepromazine, 1 mg/kg)] and placed in a radiation oncology CT scanner where another cross-sectional (axial) image volume was generated. The rat was placed in the same or close to the same position as used for the MRI scan and the CT unit was set to scan the skull region. A study of 106 CT images with 0.8 mm image thickness was generated which became the CT treatment planning data.</li>
	<li>Treatment Planning
	<ol class="lalpha">
		<li>The MRI and CT image data were transferred to the ECLIPSE (Varian Medical Systems, Palo Alto, CA) treatment planning software via DICOM (Digital-Imaging and Communications in Medicine). ECLIPSE is a sophisticated and commercially available software used in radiation oncology to model and design the best radiation treatment plan for a patient. It incorporates organ density information that is extracted from the CT image data and used to calculate the radiation dose distribution within the body.</li>
		<li>Cover the animal using a small surgical drape with only the head exposed. Position the animal head firmly within the stereotaxic frame (Benchmark Digital, Leica-myNeurolab) by inserting ear bars into the external auditory meatus. Take extreme care when sliding the tip of the ear bar into the ear canal. Place the incisor bar by hooking the rat's upper incisors and adjusting the bar height to the standard reference point, then tighten nose clamp.</li>
		<li>Center the position of the head between the ear bars with minimal lateral movement (&plusmn; 4 mm) to achieve stereotaxic zero.</li>
		<li>The next step in the process is to determine the irradiation technique to be used. Intensity-modulated radiation therapy (IMRT) and volumetrically-modulated arc therapy (VMAT) in the form of RapidArc are 2 high-precision irradiation techniques that use a 6 MV photon beam to deliver a radiation dose3-6. The difference between these techniques is that IMRT delivers a dose using several static trajectories each corresponding to a different direction but converging at the target volume more suitable for extremely small target volumes. RapidArc on the other hand, delivers a dose dynamically through one or more arcs focused on the target region (Fig. 4).</li>
		<li>Regardless of delivery technique, information on target dose, target conformity and dose constraints to critical organs have to be entered in the dose optimization window in ECLIPSE. This information along with organ volumes is used to tailor the dose distribution, which is achieved by continually varying the beam attenuation during irradiation.</li>
		<li>Once a plan was generated, ECLIPSE provides the calculated dose superimposed to the axial, coronal and sagittal images (Fig. 5) as well as in dose-volume histogram (DVH) format (Fig. 6). It is at this point when the plan must be evaluated to determine if it is suitable for delivery or needs to be improved.</li>
	</ol>
	</li>
	<li>Irradiation Process
	<ol>
		<li>Once a plan was approved for delivery, digitally-reconstructed radiograph (DRR) images were generated from the CT treatment planning data in ECLIPSE. These are orthogonal images weighted on bone density to highlight the skull and other bony landmarks. Thereafter, treatment plan and DRRs are sent to the treatment delivery computer system via DICOM.</li>
		<li>The delivery system controls a Varian Trilogy Linear Accelerator normally used for radiotherapy of humans. The accelerator is equipped with 120 computer controlled thin multileaf collimators (MLC) and a diagnostic quality x-ray imaging system included within the on-board imaging (OBI) system. This delivery system uploads the ECLIPSE-specific information and reconfigures the collimator parameters in the accelerator for delivering the planned dose.</li>
		<li>At this point the rat was prepared for irradiation, sedated and covered with a paper blanket to keep it warm. After a few minutes, the rat, with its blanket but with the skull exposed, was placed on the treatment table in the same position used to generate the CT scan. This is a critical step because position accuracy is directly correlated to accuracy in dose delivery.</li>
		<li>Once the rat was positioned properly on the treatment table, a set of orthogonal X-ray images is taken using the Trilogy's OBI system. These are high resolution images that were subsequently fused to the ECLIPSE generated DRRs using the OBI image fusion software.</li>
		<li>The X-ray images and DRRs were digitally matched using software that provide information on table position shifts that must be made to achieve co-registration of the images (Fig. 7). After reviewing this information and verifying that the resultant shifts do not inadvertently lead into any collisions between the accelerator and the treatment table, the computer applies the shifts and the treatment table moves automatically to the desired position.</li>
		<li>At this point, irradiation commences. To deliver a 10 Gy dose with a 6-field IMRT plan, the beam-on time is approximately 10 minutes while a two-arc RapidArc takes ~3 minutes. After treatment delivery is complete, the rat was removed from the treatment room and allowed to recover in a holding cage kept on a heating pad.</li>
	</ol>
	</li>
</ol>
3. Stereotaxic surgery for intra-hippocampal transplantation of human stem cells
<ol>
	<li>Animals husbandry and surgical preparation
	<ol class="lalpha">
		<li>For this study, we employed two month old ATN rats acquired from National Cancer Institute (strain 0N01 Cr:NIH-rnu). Animals were kept in sterile cages and maintained in a temperature- and light controlled barrier-A environment with 12-h/12-h light/dark cycle. Rats were provided autoclaved food and water ad libitum and care was taken to prevent eye infection by cleaning eyes every week with Vetropolycin eye ointment (Western Medical Supply, Arcadia, CA).</li>
		<li>For stereotaxic surgery, rats were anesthetized with an i.p. injection of anesthesia cocktail (ketamine, 30 mg/kg, xylazine, 2.5 mg/kg and acepromazine, 1 mg/kg). Complete anesthesia was induced after 10-15 minutes and sedation was monitored using palpable reflex (toe pinch). If required during surgery, 15-20% of the original dose may be given to maintain sufficient anesthesia.</li>
		<li>Remove fur from head using electric clippers. Approximately 200% of surgery area should be shaved to prevent infection by hairs. Clean shaved part using providone/iodine (3 times) followed by 70% alcohol (3 times) before incision.</li>
	</ol>
	</li>
	<li>Stereotaxic surgery
	<ol class="lalpha">
		<li>Stereotaxic instruments (digital monitor and frame), micromotor drill, dry bed sterilizer and controllers should be kept in a Laminar flow hood to prevent possible infection during surgery procedure in ATN rats. A stereotaxic stage with an embedded heating pad can be used to keep the animals warm during surgery. We used 'Kimberly-Clark Safeskin Purple Nitrile Sterile Exam Gloves' thoughout the surgery. These gloves are individually packed in sterile bag (Cat. no. 55093). In addition, surgeon used 70% alcohol spray to sterilize hands during surgery.</li>
		<li>Cover the animal using a small surgical drape with only the head exposed. Position the animal head firmly within the stereotaxic frame (Benchmark Digital, Leica-myNeurolab) by inserting ear bars into the external auditory meatus. Take extreme care when sliding the tip of the ear bar into the ear canal. Place the incisor bar by hooking the rat's upper incisors and adjusting the bar height to the standard reference point, then tighten nose clamp.</li>
		<li>Center the position of the head between the ear bars with minimal lateral movement (&plusmn; 4 mm) to achieve stereotaxic zero.</li>
		<li>Following these positioning steps, apply lubricating eye ointment to prevent drying and protect them from possible iodine or alcohol spills. Use this ointment at least 2 times during surgery.</li>
		<li>Make midline skin incision (2 cm) along scalp using sterile scalpel, and clean using sterile cotton tipped applicator. Avoid damage in caudal and neck muscles areas.</li>
		<li>By using dissecting retractor, hold the periosteum open and clear soft tissue using cotton tipped applicator dipped in 1% hydrogen peroxide (made in PBS). To stop bleeding, maintain firm pressure for at least 1 min using cotton tipped applicator or gauze. Apply firm scraping motion to clean skull and dabbing motion for skin until skull sutures, bregma and lambda were visible.</li>
		<li>Precise stereotaxic coordinates, referenced to the bregma, were determined using the rat brain atlas7. Stem cell transplantation was done at four distinct sites for each hemisphere using the following stereotaxic coordinates:
		<ol class="lroman">
			<li>Anterio-posterior (AP) 3.0 mm from bregma, medio-lateral (ML) 1.8mm from midline, and dorso-ventral (DV) 3.2mm from the surface of the brain.</li>
			<li>AP, 3.6 mm; ML, 2.5 mm; DV, 3.2 mm</li>
			<li>AP, 4.2 mm; ML, 3.2 mm; DV, 3.2mm</li>
			<li>AP, 4.2 mm; ML, 3.2 mm; DV, 3.2mm</li>
		</ol>
		</li>
		<li>Once the bregma has been identified, all three coordinates (AP, ML and DV) should be zeroed on the digital display/controller (on the digital stereotaxic frame). Then proceed to mark the precise transplantation sites (using the foregoing coordinates) with a fine point marker pen attached to a small probe holder on the frame.</li>
		<li>Drill a 0.35mm hole (no. 1/4 dental burr, carbide vanadium drill bits) through the skull using the foot pedal controlled micromotor drill (Leica-myNeurolab). Take care to prevent damage to the dura membrane. If bleeding occurs during drilling, apply firm pressure using cotton tipped applicator. Do not apply alcohol or iodine at the drill site, as this will cause irritation to the animal.</li>
		<li>Rats received bilateral intra-hippocampal injections of a suspension of NSCs injected in a maximum volume of 1 &mu;L using a 5 &mu;L Hamilton microsyringe (30 gauge). The precise number of cells within this volume will vary according to experimental specifics. To accomplish this, the microsyringe was attached to a small probe holder on the stereotaxic frame. Carefully insert the tip of the needle in the skull until reaching the surface of the brain (i.e. meninges). At this point, the DV coordinate should be zeroed on the digital display/controller, then gently insert the needle to the desired depth (DV, 3.2 mm). Wait 1 min before initiating any injection of cells.</li>
		<li>Inject 0.25 &mu;L volume/min (slow release) using a timer. Once a total of 1 &mu;L is injected, wait 8 min before retracting needle from transplantation site. After this time, slowly retract the needle from the transplantation site (0.5 mm/min) to prevent capillary reflux of the injection contents back through the needle track.</li>
		<li>Follow steps 3.2(j-k) for the remaining transplantation sites (4 per hemisphere).</li>
		<li>Remove the skin retractor from the skull and use blunt tweezers to gently pull back retracted skin and apply 4-5 sterile stainless clips using Autoclips surgical suture applier (Leica-myNeurolab).</li>
		<li>After removing the animal from the frame, inject with analgesic (Buprenorphin, 0.1 mg/kg, s.c., every 12h) and Lactated Ringer's solution (5 mL/250 g adult rat, s.c.).</li>
		<li>Place the animal back in its cage that should be kept on a heating pad. Monitor the animal until it becomes conscious before returning to its holding room. Place some moistened pellets (animal chow) and transgel in a separate petri dish in each animal cage. Alternatively, DietGel Recovery (ClearH2O, Portland, ME) can be used as a source of water and nutrients.</li>
		<li>Monitor animals during their recovery time and apply analgesia (buprenorphine 0.02 mg/kg (every 12h for 2 days). Check for signs of pain, distress, reddening or infection of surgery site. Apply providone/iodine or neosporin ointment once daily (till 2-3 days) to prevent possible infection. If any signs of infection, pain or distress persist following analgesic or antibiotic treatment within 12 hours of surgery, consult veterinarian or animal care staff for further assistance.</li>
	</ol>
	</li>
</ol>
4. Representative results:
<img alt="Figure 1" src="/files/ftp_upload/3107/3107fig1.jpg" /> 
Figure 1. Schematic representation of our experimental plan.
<img alt="Figure 2" src="/files/ftp_upload/3107/3107fig2.jpg" /> 
Figure 2. Fusion of CT (bony anatomy) and MRI (soft-tissue) images within the ECLIPSE software. Matching of axial, coronal, and sagittal sections allow for the co-registry of the critical anatomical features of the rat brain derived from each imaging modality.
<img alt="Figure 3" src="/files/ftp_upload/3107/3107fig3.jpg" /> 
Figure 3. Contoured hippocampal regions of the brain. Subsequent to image fusion, hippocampi and brain excluding hippocampi are identified and contoured axially defining specific volumetric regions for these organs. These regions provide anatomical and volume information necessary for adjusting the dose distribution to the desired target(s).
<img alt="Figure 4" src="/files/ftp_upload/3107/3107fig4.jpg" /> 
Figure 4. High precision irradiation options using intensity-modulated radiation therapy (IMRT) or volumetrically-modulated arc therapy (VMAT) in the form of RapidArc. These techniques deliver 6 MV photon beams either as multiple static trajectories that can converge on very small target volumes (IMRT), or dynamically as one or more arcs focused on the target region (RapidArc).
<img alt="Figure 5" src="/files/ftp_upload/3107/3107fig5.jpg" /> 
Figure 5. ECLIPSE calculated doses superimposed to the axial images. Doses shown are for single or dual hippocampi irradiation treatment plans.
<img alt="Figure 6" src="/files/ftp_upload/3107/3107fig6.jpg" /> 
Figure 6. ECLIPSE calculated dose-volume histogram. Data contrasts the percentage of the irradiated versus the non-irradiated hippocampal volume under the single hippocampus treatment plan shown in Fig. 5.
<img alt="Figure 7" src="/files/ftp_upload/3107/3107fig7.jpg" /> 
Figure 7. Image guided rat positioning for radiotherapy. Orthogonal digitally-reconstructed radiograph (DRR) images generated from the CT treatment planning data in ECLIPSE are fused to orthogonal x-ray images of the rat on the treatment table taken with the Trilogy's on-board imaging (OBI) system. The DRRs are weighted on bone density that highlights the skull and other bony landmarks which facilitate co-registration with the OBI images. Matching of these sets provide treatment table position shifts that must be made to achieve the co-registration of the CT and x-ray images.
<img alt="Figure 8" src="/files/ftp_upload/3107/3107fig8.jpg" /> 
Figure 8. Location of transplanted NSCs following stereotaxic surgery. At 1-month post-transplantation, animals were perfused, brains were sectioned and stained with BrdU (to detect transplanted NSCs) and counter stained with hematoxylin. The needle track (Nt, red line), indicates the injection trajectory that deposited NSCs at the transplant-release site (Tr), just below the corpus callosum (CC) and above the CA1. Transplanted NSCs showed extensive migration from Tr throughout the host hippocampus (dentate gyrus, DG; dentate hilus, DH; CA1 and CA3 subfields; magnification x4). Scale bar, 200 &mu;m.

<ol>
	<li>Acharya, M. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Consequences+of+ionizing+radiation-induced+damage+in+human+neural+stem+cells.">Consequences of ionizing radiation-induced damage in human neural stem cells.</a> Free. Radic. Biol. Med. , (2010).</li><li>Acharya, M. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rescue+of+radiation-induced+cognitive+impairment+through+cranial+transplantation+of+human+embryonic+stem+cells.">Rescue of radiation-induced cognitive impairment through cranial transplantation of human embryonic stem cells.</a> Proc. Natl. Acad. Sci. U. S. A. 106, 19150-19155 (2009).</li><li>Boyer, A., Khan, F. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ch.+11.">Ch. 11.</a> Treatment planning in Radiation Oncology. , (2007).</li><li>Lawson, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intracranial+application+of+IMRT+based+radiosurgery+to+treat+multiple+or+large+irregular+lesions+and+verification+of+infra-red+frameless+localization+system.">Intracranial application of IMRT based radiosurgery to treat multiple or large irregular lesions and verification of infra-red frameless localization system.</a> J. Neurooncol. 97, 59-66 (2010).</li><li>Ling, C. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Commissioning+and+quality+assurance+of+RapidArc+radiotherapy+delivery+system.">Commissioning and quality assurance of RapidArc radiotherapy delivery system.</a> Int. J. Radiat. Oncol. Biol. Phys. 72, 575-581 (2008).</li><li>Otto, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Volumetric+modulated+arc+therapy:+IMRT+in+a+single+gantry+arc.">Volumetric modulated arc therapy: IMRT in a single gantry arc.</a> Med. Phys. 35, 310-317 (2008).</li><li>Paxinos, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The Rat Brain in stereotaxic coordinates. , (2007).</li><li>Hess, D. C., Borlongan, C. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell-based+therapy+in+ischemic+stroke.">Cell-based therapy in ischemic stroke.</a> Expert. Rev. Neurother. 8, 1193-1201 (2008).</li><li>Hefferan, M. P., Johe, K., Feldman, E. L., Lunn, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimization+of+immunosuppressive+therapy+for+spinal+grafting+of+human+spinal+stem+cells+in+a+rat+model+of+ALS.">Optimization of immunosuppressive therapy for spinal grafting of human spinal stem cells in a rat model of ALS.</a> Cell. Transplant. , (2011).</li></ol>

No conflicts of interest declared.

Considerable research is underway exploring the myriad of ways that stem cells can be used clinically to restore normal functions to damaged, aged and diseased tissues8. The eventual realization of these efforts will require a detailed understanding of the behavior of engrafted cells within unique microenvironments that are distinct from undamaged normal tissue. Our work has demonstrated that within the irradiated tissue bed, cranially engrafted NSCs can functionally restore cognition, where they survive, migrate and differentiate along neural and glial lineages2. Precisely how these cells mediate recovery of cognition is uncertain at present, but does depend on conducting a series of carefully controlled experimental procedures in a reproducible manner. We have detailed these critical procedures here in efforts to expedite the translational potential of stem cell therapies for ameliorating adverse cognitive effects associated with the clinical management of brain and other forms of cancer. Additional considerations that are likely to have a significant impact of the quality of data are highlighted below.
Transplantation based therapies depend on stem cells as the critical reagent, and accordingly, care must be exercised to properly characterize cultures, maintain sterility and use matched passage numbers for reliability of results. Transplanted human stem cells were labeled with BrdU prior to surgery, to provide one means for tracking them in vivo. Under our experimental conditions, transplanted NSCs did not undergo extensive proliferation, so that dilution of the BrdU label was not problematic. Alternatively, transplanted human stem cells can be distinguished from host cells by immunostaining for human specific markers such as nuclear matrix protein (h-NUC or hNUMA)9 or human-specific nuclear antigen (HuNu)2. Human stem cells could also be labeled with a variety of fluorescent markers to facilitate their identification within the host brain.
Attention to irradiation parameters define precise dose delivery techniques, and those described here model current clinical practices in radiation oncology. Reproducible transplantation of stem cells in the brain is also critical, and is accomplished using a digital stereotaxic instrument. The capability to precisely micromanipulate a micro-syringe for stem cell implantation in small brain structures like the hippocampus greatly reduces human error. With this apparatus, NSCs were transplanted in four distinct sites spanning the anterior to posterior regions of the rat hippocampus. Dorso-ventral (DV) coordinates were determined based on experience with athymic nude (ATN) rats such that surgical procedures would not cause damage to the hippocampal formation2. A coronal section through the rat brain reveals key structures of the hippocampal formation including the dentate gyrus (DG), the dentate hilus (DH) and CA1 and CA3 subfields (Fig. 8). Transplanted NSCs, introduced dorsally from the visible needle tract (Nt, redline), are visualized as darkly staining (brown) cells deposited at the transplant-release site (Tr) just below the corpus callosum (CC), that subsequently migrate throughout the septo-temporal axis of the hippocampus.

<table><tbody><tr><td>Digital Stereotaxic Instrument w/45&amp;deg; and 18&amp;deg; Earbars</td><td>Cranial transplantation surgical setup</td><td>Leica Microsystems</td><td>39463501</td><td>Useful accessories: Stage with heater and variable current source (to maintain body temperature during surgery)</td></tr></tbody></table>

stem cell transplantation strategies for the restoration of cognitive dysfunction caused by cranial radiotherapy

Radiotherapy often provides the only clinical recourse for those afflicted with primary or metastatic brain tumors. While beneficial, cranial irradiation can induce a progressive and debilitating decline in cognition that may, in part, be caused by the depletion of neural stem cells. Given the increased survival of patients diagnosed with brain cancer, quality of life in terms of cognitive health has become an increasing concern, especially in the absence of any satisfactory long-term treatments.
To address this serious health concern we have used stem cell replacement as a strategy to combat radiation-induced cognitive decline. Our model utilizes athymic nude rats subjected to cranial irradiation. The ionizing radiation is delivered as either whole brain or as a highly focused beam to the hippocampus via linear accelerator (LINAC) based stereotaxic radiosurgery. Two days following irradiation, human neural stem cells (hNSCs) were stereotaxically transplanted into the hippocampus. Rats were then assessed for changes in cognition, grafted cell survival and for the expression of differentiation-specific markers 1 and 4-months after irradiation. Our cognitive testing paradigms have demonstrated that animals engrafted with hNSCs exhibit significant improvements in cognitive function. Unbiased stereology reveals significant survival (10-40%) of the engrafted cells at 1 and 4-months after transplantation, dependent on the amount and type of cells grafted. Engrafted cells migrate extensively, differentiate along glial and neuronal lineages, and express a range of immature and mature phenotypic markers.
Our data demonstrate direct cognitive benefits derived from engrafted human stem cells, suggesting that this procedure may one day afford a promising strategy for the long-term functional restoration of cognition in individuals subjected to cranial radiotherapy. To promote the dissemination of the critical procedures necessary to replicate and extend our studies, we have provided written and visual documentation of several key steps in our experimental plan, with an emphasis on stereotaxic radiosurgey and transplantation.

Watch this Scientific Journal Video about Stem Cell Transplantation Strategies for the Restoration of Cognitive Dysfunction Caused by Cranial Radiotherapy at JoVE.com

Stem Cell Transplantation Strategies for the Restoration of Cognitive Dysfunction Caused by Cranial Radiotherapy

Brain tumor patients routinely undergo cranial radiotherapy, and while beneficial, this treatment often results in debilitating cognitive dysfunction. This serious unresolved problem has at present, no clinical recourse, and has driven our efforts to devise stem cell based therapies for the recovery of radiation-induced cognitive decrements.

Radiotherapy often provides the only clinical recourse for those afflicted with primary or metastatic brain tumors. While beneficial, cranial irradiation ...

stem-cell-transplantation-strategies-for-restoration-cognitive

Research

JoVE Journal

Medicine

14.4K Views.  University of California Irvine. Brain tumor patients routinely undergo cranial radiotherapy, and while beneficial, this treatment often results in debilitating cognitive dysfunction. This serious unresolved problem has at present, no clinical recourse, and has driven our efforts to devise stem cell based therapies for the recovery of radiation-induced cognitive decrements.

Video: Stem Cell Transplantation Strategies for the Restoration of Cognitive Dysfunction Caused by Cranial Radiotherapy

A Protocol for Rapid Post-mortem Cell Culture of Diffuse Intrinsic Pontine Glioma (DIPG)

Diffuse Intrinsic Pontine Glioma (DIPG) is a childhood brainstem tumor that carries a universally fatal prognosis. Because surgical resection is not a viable treatment strategy and biopsy is not routinely performed, the availability of patient samples for research is limited. Consequently, efforts to study this disease have been challenged by a paucity of faithful disease models. To address this need, we describe here a protocol for the rapid processing of post-mortem autopsy tissue samples in order to generate durable patient-derived cell culture models that can be used in in vitro assays or in vivo orthotopic xenograft experiments. These models can be used to screen for potential drug targets and to study fundamental pathobiological processes within DIPG. This protocol can further be extended to analyze and isolate tumor and microenvironmental cells using Fluorescence-activated Cell Sorting (FACS), which enables subsequent analysis of gene expression, protein expression, or epigenetic modifications of DNA at the bulk cell or single cell level. Finally, this protocol can also be adapted to generate patient-derived cultures for other central nervous system tumors.

A Protocol for Rapid Post-mortem Cell Culture of Diffuse Intrinsic Pontine Glioma DIPG

This protocol describes a method for the rapid processing of post-mortem diffuse intrinsic pontine glioma samples for the establishment of patient-derived cell culture models or direct characterization of tumor and microenvironmental cells.

Diffuse Intrinsic Pontine Glioma (DIPG) is a childhood brainstem tumor that carries a universally fatal prognosis. Because surgical resection is not a ...

Cancer Research

Intraspinal Cell Transplantation for Targeting Cervical Ventral Horn in Amyotrophic Lateral Sclerosis and Traumatic Spinal Cord Injury

Respiratory compromise due to phrenic motor neuron loss is a debilitating consequence of a large proportion of human traumatic spinal cord injury (SCI) cases 1 and is the ultimate cause of death in patients with the motor neuron disorder, amyotrophic laterals sclerosis (ALS) 2.
ALS is a devastating neurological disorder that is characterized by relatively rapid degeneration of upper and lower motor neurons. Patients ultimately succumb to the disease on average 2-5 years following diagnosis because of respiratory paralysis due to loss of phrenic motor neuron innnervation of the diaphragm 3. The vast majority of cases are sporadic, while 10% are of the familial form. Approximately twenty percent of familial cases are linked to various point mutations in the Cu/Zn superoxide dismutase 1 (SOD1) gene on chromosome 21 4. Transgenic mice 4,5 and rats 6 carrying mutant human SOD1 genes (G93A, G37R, G86R, G85R) have been generated, and, despite the existence of other animal models of motor neuron loss, are currently the most highly used models of the disease.
Spinal cord injury (SCI) is a heterogeneous set of conditions resulting from physical trauma to the spinal cord, with functional outcome varying according to the type, location and severity of the injury 7. Nevertheless, approximately half of human SCI cases affect cervical regions, resulting in debilitating respiratory dysfunction due to phrenic motor neuron loss and injury to descending bulbospinal respiratory axons 1. A number of animal models of SCI have been developed, with the most commonly used and clinically-relevant being the contusion 8.
Transplantation of various classes of neural precursor cells (NPCs) is a promising therapeutic strategy for treatment of traumatic CNS injuries and neurodegeneration, including ALS and SCI, because of the ability to replace lost or dysfunctional CNS cell types, provide neuroprotection, and deliver gene factors of interest 9.
Animal models of both ALS and SCI can model many clinically-relevant aspects of these diseases, including phrenic motor neuron loss and consequent respiratory compromise 10,11. In order to evaluate the efficacy of NPC-based strategies on respiratory function in these animal models of ALS and SCI, cellular interventions must be specifically directed to regions containing therapeutically relevant targets such as phrenic motor neurons. We provide a detailed protocol for multi-segmental, intraspinal transplantation of NPCs into the cervical spinal cord ventral gray matter of neurodegenerative models such as SOD1G93A mice and rats, as well as spinal cord injured rats and mice 11.

Neural precursor transplantation is a promising strategy for protecting and/or replacing lost/dysfunctional cervical phrenic motor neurons in spinal cord injury (SCI) and the motor neuron disorder, amyotrophic laterals sclerosis (ALS). We provide a protocol for cell delivery to cervical spinal cord ventral horn in rodent models of ALS and SCI.

Respiratory compromise due to phrenic motor neuron loss is a debilitating consequence of a large proportion of human traumatic spinal cord injury (SCI) ...

Quantifying Cognitive Decrements Caused by Cranial Radiotherapy

With the exception of survival, cognitive impairment stemming from the clinical management of cancer is a major factor dictating therapeutic outcome. For many patients afflicted with CNS and non-CNS malignancies, radiotherapy and chemotherapy offer the best options for disease control. These treatments however come at a cost, and nearly all cancer survivors (~11 million in the US alone as of 2006) incur some risk for developing cognitive dysfunction, with the most severe cases found in patients subjected to cranial radiotherapy (~200,000/yr) for the control of primary and metastatic brain tumors1. Particularly problematic are pediatric cases, whose long-term survival plagued with marked cognitive decrements results in significant socioeconomic burdens2. To date, there are still no satisfactory solutions to this significant clinical problem.
We have addressed this serious health concern using transplanted stem cells to combat radiation-induced cognitive decline in athymic rats subjected to cranial irradiation3. Details of the stereotaxic irradiation and the in vitro culturing and transplantation of human neural stem cells (hNSCs) can be found in our companion paper (Acharya et al., JoVE reference). Following irradiation and transplantation surgery, rats are then assessed for changes in cognition, grafted cell survival and expression of differentiation-specific markers 1 and 4-months after irradiation. To critically evaluate the success or failure of any potential intervention designed to ameliorate radiation-induced cognitive sequelae, a rigorous series of quantitative cognitive tasks must be performed. To accomplish this, we subject our animals to a suite of cognitive testing paradigms including novel place recognition, water maze, elevated plus maze and fear conditioning, in order to quantify hippocampal and non-hippocampal learning and memory. We have demonstrated the utility of these tests for quantifying specific types of cognitive decrements in irradiated animals, and used them to show that animals engrafted with hNSCs exhibit significant improvements in cognitive function3.
The cognitive benefits derived from engrafted human stem cells suggest that similar strategies may one day provide much needed clinical recourse to cancer survivors suffering from impaired cognition. Accordingly, we have provided written and visual documentation of the critical steps used in our cognitive testing paradigms to facilitate the translation of our promising results into the clinic.

Cognitive impairment resulting from the radiotherapeutic management of brain tumors represents a clinically intractable condition that adversely impacts quality of life. The capability to critically evaluate potential interventions for ameliorating radiation-induced cognitive decrements ultimately depends on the capability to undertake rigorous quantitative assessments of cognition.

With the exception of survival, cognitive impairment stemming from the clinical management of cancer is a major factor dictating therapeutic outcome. For ...

Controlled Cortical Impact Model of Mouse Brain Injury with Therapeutic Transplantation of Human Induced Pluripotent Stem Cell-Derived Neural Cells

Traumatic brain injury (TBI) is a leading cause of morbidity and mortality worldwide. Disease pathology due to TBI progresses from the primary mechanical insult to secondary injury processes, including apoptosis and inflammation. Animal modeling has been valuable in the search to unravel injury mechanisms and evaluate potential neuroprotective therapies. This protocol describes the controlled cortical impact (CCI) model of focal, open-head TBI. Specifically, parameters for producing a mild unilateral cortical injury are described. Behavioral consequences of CCI are analyzed using the adhesive tape removal test of bilateral sensorimotor integration. Regarding experimental therapy for TBI pathology, this protocol also illustrates a process for transplanting cultured cells into the brain. Neural cell cultures derived from human induced pluripotent stem cells (hiPSCs) were chosen for their potential to show superior functional restoration in human TBI patients. Chronic survival of hiPSCs in the host mouse brain tissue is detected using a modified DAB immunohistochemical process.

This protocol demonstrates methodologies for a mouse model of open-skull traumatic brain injury and transplantation of cultured human induced pluripotent stem cell-derived cells into the injury site. Behavioral and histologic tests of outcomes from these procedures are also described in brief.

Traumatic brain injury (TBI) is a leading cause of morbidity and mortality worldwide. Disease pathology due to TBI progresses from the primary mechanical ...

Developmental Biology

Intracranial Cannula Implantation for Serial Locoregional Chimeric Antigen Receptor (CAR) T Cell Infusions in Mice

Pediatric CNS tumors are responsible for the majority of cancer-related deaths in children and have poor prognoses, despite advancements in chemotherapy and radiotherapy. As many tumors lack efficacious treatments, there is a crucial need to develop more promising therapeutic options, such as immunotherapies; the use of chimeric antigen receptor (CAR) T cell therapy&#160;directed against CNS tumors is of particular interest. Cell surface targets such as B7-H3, IL13RA2, and the disialoganglioside GD2 are highly expressed on the surface of several pediatric and adult CNS tumors,&#160;raising the opportunity to use CAR T cell therapy against these and other surface targets. To evaluate the repeated locoregional delivery of CAR T cells in preclinical murine models, an indwelling catheter system that recapitulates indwelling catheters currently being used in human clinical trials was established. Unlike stereotactic delivery, the indwelling catheter system allows for repeated dosing without the use of multiple surgeries. This protocol describes the intratumoral placement of a fixed guide cannula that has been used to successfully test serial CAR T cell infusions in orthotopic murine models of pediatric brain tumors. Following orthotopic injection and engraftment of the tumor cells in mice, intratumoral placement of a fixed guide cannula is completed on a stereotactic apparatus and secured with screws and acrylic resin. Treatment cannulas are then inserted through the fixed guide cannula for repeated CAR T cell delivery. Stereotactic placement of the guide cannula can be adjusted to deliver CAR T cells directly into the lateral ventricle or other locations in the brain. This platform offers a reliable mechanism for the preclinical testing of repeated intracranial infusions of CAR T cells and other novel therapeutics for these devastating pediatric tumors.

Intracranial Cannula Implantation for Serial Locoregional Chimeric Antigen Receptor CAR T Cell Infusions in Mice

Central nervous system (CNS) tumors are the leading cause of cancer-related death in children, and locoregional immune-based therapies are increasingly being tested for patients in clinical trials. This protocol describes methods for locoregional cannula implantation in mice for the preclinical evaluation of immunotherapeutic infusions targeting CNS tumors.

Pediatric CNS tumors are responsible for the majority of cancer-related deaths in children and have poor prognoses, despite advancements in chemotherapy ...

Neural Stem Cell Transplantation in Experimental Contusive Model of Spinal Cord Injury

Spinal cord injury is a devastating clinical condition, characterized by a complex of neurological dysfunctions. Animal models of spinal cord injury can be used both to investigate the biological responses to injury and to test potential therapies. Contusion or compression injury delivered to the surgically exposed spinal cord are the most widely used models of the pathology. In this report the experimental contusion is performed by using the Infinite Horizon (IH) Impactor device, which allows the creation of a reproducible injury animal model through definition of specific injury parameters. Stem cell transplantation is commonly considered a potentially useful strategy for curing this debilitating condition. Numerous studies have evaluated the effects of transplanting a variety of stem cells. Here we demonstrate an adapted method for spinal cord injury followed by tail vein injection of cells in CD1 mice. In short, we provide procedures for: i) cell labeling with a vital tracer, ii) pre-operative care of mice, iii) execution of a contusive spinal cord injury, and iv) intravenous administration of post mortem neural precursors. This contusion model can be utilized to evaluate the efficacy and safety of stem cell transplantation in a regenerative medicine approach.

Spinal cord injury is a traumatic condition that causes severe morbidity and high mortality. In this work we describe in detail a contusion model of spinal cord injury in mice followed by a transplantation of neural stem cells.

Spinal cord injury is a devastating clinical condition, characterized by a complex of neurological dysfunctions. Animal models of spinal cord injury can ...

Stereotactic Adoptive Transfer of Cytotoxic Immune Cells in Murine Models of Orthotopic Human Glioblastoma Multiforme Xenografts

Glioblastoma multiforme (GBM), the most frequent and aggressive primary brain cancer in adults, is generally associated with a poor prognosis, and scarce efficient therapies have been proposed over the last decade. Among the promising candidates for designing novel therapeutic strategies, cellular immunotherapies have been targeted to eliminate highly invasive and chemo-radioresistant tumor cells, likely involved in a rapid and fatal relapse of this cancer. Thus, administration(s) of allogeneic GBM-reactive immune cell effectors, such as human V&#978;9V&#948;2 T lymphocytes, in the vicinity of the tumor would represents a unique opportunity to deliver efficient and highly concentrated therapeutic agents directly into the site of brain malignancies. Here, we present a protocol for the preparation and the stereotaxic administration of allogeneic human lymphocytes in immunodeficient mice carrying orthotopic human primary brain tumors. This study provides a preclinical proof-of-concept for both the feasibility and the antitumor efficacy of these cellular immunotherapies that rely on stereotactic injections of allogeneic human lymphocytes within intrabrain tumor beds.

Here, we present a protocol for the preparation and the stereotaxic administration of allogeneic human lymphocytes in immunodeficient mice carrying orthotopic human primary brain tumors. This study provides a proof-of-concept for both the feasibility and the antitumor efficacy of intrabrain-delivered cellular immunotherapies.

Glioblastoma multiforme (GBM), the most frequent and aggressive primary brain cancer in adults, is generally associated with a poor prognosis, and scarce ...

Immunology and Infection

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.

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 ...

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 ...

Intracerebral Transplantation and In Vivo Bioluminescence Tracking of Human Neural Progenitor Cells in the Mouse Brain

Cell therapy has long been an emerging treatment paradigm in experimental neurobiology. However, cell transplantation studies often rely on end-point measurements and can therefore only evaluate longitudinal changes of cell migration and survival to a limited extent. This paper provides a reliable, minimally invasive protocol to transplant and longitudinally track neural progenitor cells (NPCs) in the adult mouse brain. Before transplantation, cells are transduced with a lentiviral vector comprising a bioluminescent (firefly-luciferase) and fluorescent (green fluorescent protein [GFP]) reporter. The NPCs are transplanted into the right cortical hemisphere using stereotaxic injections in the sensorimotor cortex. Following transplantation, grafted cells were detected through the intact skull for up to five weeks (at days 0, 3, 14, 21, 35) with a resolution limit of 6,000 cells using in vivo bioluminescence imaging. Subsequently, the transplanted cells are identified in histological brain sections and further characterized with immunofluorescence. Thus, this protocol provides a valuable tool to transplant, track, quantify, and characterize cells in the mouse brain.

Intracerebral Transplantation and In Vivo Bioluminescence Tracking of Human Neural Progenitor Cells in the Mouse Brain

We describe the intraparenchymal transplantation of human neural progenitor cells transduced with a dual reporter vector expressing luciferase-green fluorescent protein (GFP) in the mouse brain. After transplantation, the luciferase signal is repeatedly measured using in vivo bioluminescence and GFP-expressing grafted cells identified in brain sections using fluorescence microscopy.

Cell therapy has long been an emerging treatment paradigm in experimental neurobiology. However, cell transplantation studies often rely on end-point ...

Stem Cell Transplantation Strategies for the Restoration of Cognitive Dysfunction Caused by Cranial Radiotherapy

Podsumowanie

Przeglądaj więcej filmów

Rozdziały w tym wideo

A Protocol for Rapid Post-mortem Cell Culture of Diffuse Intrinsic Pontine Glioma (DIPG)

Intraspinal Cell Transplantation for Targeting Cervical Ventral Horn in Amyotrophic Lateral Sclerosis and Traumatic Spinal Cord Injury

Quantifying Cognitive Decrements Caused by Cranial Radiotherapy

Controlled Cortical Impact Model of Mouse Brain Injury with Therapeutic Transplantation of Human Induced Pluripotent Stem Cell-Derived Neural Cells

Intracranial Cannula Implantation for Serial Locoregional Chimeric Antigen Receptor (CAR) T Cell Infusions in Mice

Neural Stem Cell Transplantation in Experimental Contusive Model of Spinal Cord Injury

Stereotactic Adoptive Transfer of Cytotoxic Immune Cells in Murine Models of Orthotopic Human Glioblastoma Multiforme Xenografts

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

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

Intracerebral Transplantation and In Vivo Bioluminescence Tracking of Human Neural Progenitor Cells in the Mouse Brain