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 border="1">
			<tbody>
				<tr>
					<td>Material Name</td>
					<td>Type</td>
					<td>Company</td>
					<td>Catalogue Number</td>
					<td>Comment</td>
				</tr>
				<tr>
					<td>Digital Stereotaxic Instrument w/45&deg; and 18&deg; Earbars</td>
					<td>Cranial transplantation surgical setup</td>
					<td>Leica Microsystems (previously: MyNeuroLab)</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 ...

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14.3K 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

Orthotopic Aortic Transplantation: A Rat Model to Study the Development of Chronic Vasculopathy

Research models of chronic rejection are essential to investigate pathobiological and pathophysiological processes during the development of transplant vasculopathy (TVP).
The commonly used animal model for cardiovascular chronic rejection studies is the heterotopic heart transplant model performed in laboratory rodents. This model is used widely in experiments since Ono and Lindsey (3) published their technique. To analyze the findings in the blood vessels, the heart has to be sectioned and all vessels have to be measured.
Another method to investigate chronic rejection in cardiovascular questionings is the aortic transplant model (1, 2). In the orthotopic aortic transplant model, the aorta can easily be histologically evaluated (2). The PVG-to-ACI model is especially useful for CAV studies, since acute vascular rejection is not a major confounding factor and Cyclosporin A (CsA) treatment does not prevent the development of CAV, similar to what we find in the clinical setting (4). A7-day period of CsA is required in this model to prevent acute rejection and to achieve long-term survival with the development of TVP.
This model can also be used to investigate acute cellular rejection and media necrosis in xenogeneic models (5).

This video demonstrates the orthotopic aortic transplant model as a simple model to study the development of transplant vasculopathy (TVP) in rats.

Research models of chronic rejection are essential to investigate pathobiological and pathophysiological processes during the development of transplant ...

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 ...

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 ...

Stem Cell Transplantation in an in vitro Simulated Ischemia/Reperfusion Model

Stem cell transplantation protocols are finding their way into clinical practice1,2,3. Getting better results, making the protocols more robust, and finding new sources for implantable cells are the focus of recent research4,5. Investigating the effectiveness of cell therapies is not an easy task and new tools are needed to investigate the mechanisms involved in the treatment process6. We designed an experimental protocol of ischemia/reperfusion in order to allow the observation of cellular connections and even subcellular mechanisms during ischemia/reperfusion injury and after stem cell transplantation and to evaluate the efficacy of cell therapy. H9c2 cardiomyoblast cells were placed onto cell culture plates7,8. Ischemia was simulated with 150 minutes in a glucose free medium with oxygen level below 0.5%. Then, normal media and oxygen levels were reintroduced to simulate reperfusion. After oxygen glucose deprivation, the damaged cells were treated with transplantation of labeled human bone marrow derived mesenchymal stem cells by adding them to the culture. Mesenchymal stem cells are preferred in clinical trials because they are easily accessible with minimal invasive surgery, easily expandable and autologous. After 24 hours of co-cultivation, cells were stained with calcein and ethidium-homodimer to differentiate between live and dead cells. This setup allowed us to investigate the intercellular connections using confocal fluorescent microscopy and to quantify the survival rate of postischemic cells by flow cytometry. Confocal microscopy showed the interactions of the two cell populations such as cell fusion and formation of intercellular nanotubes. Flow cytometry analysis revealed 3 clusters of damaged cells which can be plotted on a graph and analyzed statistically. These populations can be investigated separately and conclusions can be drawn on these data on the effectiveness of the simulated therapeutical approach.

Stem Cell Transplantation in an in vitro Simulated Ischemia/Reperfusion Model

We demonstrate how to set up an in vitro ischemia/reperfusion model and how to evaluate the effect of stem cell therapy on postischemic cardiac cells.

Stem cell transplantation protocols are finding their way into clinical practice1,2,3. Getting better results, making the protocols more robust, and ...

Isolation, Characterization and Comparative Differentiation of Human Dental Pulp Stem Cells Derived from Permanent Teeth by Using Two Different Methods

Developing wisdom teeth are easy-accessible source of stem cells during the adulthood which could be obtained by routine orthodontic treatments. Human pulp-derived stem cells (hDPSCs) possess high proliferation potential with multi-lineage differentiation capacity compare to the ordinary source of adult stem cells1-8; therefore, hDPSCs could be the good candidates for autologous transplantation in tissue engineering and regenerative medicine. Along with these benefits, possessing the mesenchymal stem cells (MSC) features, such as immunolodulatory effect, make hDPSCs more valuable, even in the case of allograft transplantation6,9,10. Therefore, the primary step for using this source of stem cells is to select the best protocol for isolating hDPSCs from pulp tissue. In order to achieve this goal, it is crucial to investigate the effect of various isolation conditions on different cellular behaviors, such as their common surface markers &amp; also their differentiation capacity.
Thus, here we separate human pulp tissue from impacted third molar teeth, and then used both existing protocols based on literature, for isolating hDPSCs,11-13 i.e. enzymatic dissociation of pulp tissue (DPSC-ED) or outgrowth from tissue explants (DPSC-OG). In this regards, we tried to facilitate the isolation methods by using dental diamond disk. Then, these cells characterized in terms of stromal-associated Markers (CD73, CD90, CD105 &amp; CD44), hematopoietic/endothelial Markers (CD34, CD45 &amp; CD11b), perivascular marker, like CD146 and also STRO-1. Afterwards, these two protocols were compared based on the differentiation potency into odontoblasts by both quantitative polymerase chain reaction (QPCR) &amp; Alizarin Red Staining. QPCR were used for the assessment of the expression of the mineralization-related genes (alkaline phosphatase; ALP, matrix extracellular phosphoglycoprotein; MEPE &amp; dentin sialophosphoprotein; DSPP).14

The method described isolation and characterization of human Dental Pulp Stem Cells (hDPSCs) by using either enzymatic dissociation of pulp (DPSC-ED) or direct outgrowth of stem cells from pulp tissue explants (DPSC-OG). Then followed by in vitro comparative differentiation of both types of hDPSCs into odontoblasts.

Developing wisdom teeth are easy-accessible source of stem cells during the adulthood which could be obtained by routine orthodontic treatments. Human ...

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 ...

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 ...

Normothermic Ex Vivo Kidney Perfusion for the Preservation of Kidney Grafts prior to Transplantation

Kidney transplantation has become a well-established treatment option for patients with end-stage renal failure. The persisting organ shortage remains a serious problem. Therefore, the acceptance criteria for organ donors have been extended leading to the usage of marginal kidney grafts. These marginal organs tolerate cold storage poorly resulting in increased preservation injury and higher rates of delayed graft function. To overcome the limitations of cold storage, extensive research is focused on alternative normothermic preservation methods.
Ex vivo normothermic organ perfusion is an innovative preservation technique. The first experimental and clinical trials for ex vivo lung, liver, and kidney perfusions demonstrated favorable outcomes.
In addition to the reduction of cold ischemic injury, the method of normothermic kidney storage offers the opportunity for organ assessment and repair. This manuscript provides information about kidney retrieval, organ preservation techniques, and isolated ex vivo normothermic kidney perfusion (NEVKP) in a porcine model. Surgical techniques, set up for the perfusion solution and the circuit, potential assessment options, and representative results are demonstrated.

Normothermic Ex Vivo Kidney Perfusion for the Preservation of Kidney Grafts prior to Transplantation

The severe organ shortage has resulted in increased use of marginal kidney grafts for transplantation. This has triggered interest in alternative storage methods, since marginal grafts especially tolerate cold storage poorly. The technique of normothermic ex vivo kidney perfusion (NEVKP) represents a novel preservation method for kidney grafts prior to &#160;transplantation.

Kidney transplantation has become a well-established treatment option for patients with end-stage renal failure. The persisting organ shortage remains a ...

Creation and Transplantation of an Adipose-derived Stem Cell (ASC) Sheet in a Diabetic Wound-healing Model

Artificial skin has achieved considerable therapeutic results in clinical practice. However, artificial skin treatments for wounds in diabetic patients with impeded blood flow or with large wounds might be prolonged. Cell-based therapies have appeared as a new technique for the treatment of diabetic ulcers, and cell-sheet engineering has improved the efficacy of cell transplantation. A number of reports have suggested that adipose-derived stem cells (ASCs), a type of mesenchymal stromal cell (MSC), exhibit therapeutic potential due to their relative abundance in adipose tissue and their accessibility for collection when compared to MSCs from other tissues. Therefore, ASCs appear to be a good source of stem cells for therapeutic use. In this study, ASC sheets from the epididymal adipose fat of normal Lewis rats were successfully created using temperature-responsive culture dishes and normal culture medium containing ascorbic acid. The ASC sheets were transplanted into Zucker diabetic fatty (ZDF) rats, a rat model of type 2 diabetes and obesity, that exhibit diminished wound healing. A wound was created on the posterior cranial surface, ASC sheets were transplanted into the wound, and a bilayer artificial skin was used to cover the sheets. ZDF rats that received ASC sheets had better wound healing than ZDF rats without the transplantation of ASC sheets. This approach was limited because ASC sheets are sensitive to dry conditions, requiring the maintenance of a moist wound environment. Therefore, artificial skin was used to cover the ASC sheet to prevent drying. The allogenic transplantation of ASC sheets in combination with artificial skin might also be applicable to other intractable ulcers or burns, such as those observed with peripheral arterial disease and collagen disease, and might be administered to patients who are undernourished or are using steroids. Thus, this treatment might be the first step towards improving the therapeutic options for diabetic wound healing.

Creation and Transplantation of an Adipose-derived Stem Cell ASC Sheet in a Diabetic Wound-healing Model

Adipose-derived stem cells (ASCs) are easily isolated and harvested from the fat of normal rats. ASC sheets can be created using cell-sheet engineering and can be transplanted into Zucker diabetic fatty rats exhibiting full-thickness skin defects with exposed bone and then covered with a bilayer of artificial skin.

Artificial skin has achieved considerable therapeutic results in clinical practice. However, artificial skin treatments for wounds in diabetic patients ...