The authors would like to acknowledge funding from the Horserace Betting Levy Board U.K. (grant number 721) and VetCell BioScience Ltd, U.K. and by Consejer&#236;a de Innovaci&#242;n, Ciencia y Empresa, Junta de Andaluc&#236;a, Spain.

The cases described herein were performed following Ethical permission granted by the Animal Ethics and Welfare Committee of the Colegio de Veterinarios de Malaga, Spain, and the Royal Veterinary College, North Mymms, U.K. The procedures used on the horses are based on approved protocols that are used in clinic on horses receiving stem cell based therapies which includes sedation, bone-marrow aspiration, intra-tendinous injection, regional perfusion, intravenous injection, post-procedural treatment and pain management and monitoring after implantation.
1. Key Arrangements to be Made in Advance
<ol>
	<li>Seek institutional ethics and animal welfare approval and adhere to local legal requirements prior to starting work.</li>
	<li>Seek institutional approval to work with ionizing radiation as dictated by local and legal regulations including the appropriate level of protection of personnel and the environment and the disposal of contaminated waste.</li>
	<li>Use the following inclusion criteria: horses present with a non-traumatic overstrain injury (tendinopathy or desmopathy) to the superficial flexor digital tendon of the forelimb and lesions resulting in a defect within the tendon and the defect is surrounded by intact tendon and/or paratenon. 
	Note: The examination and diagnosis for such injuries and the clinical preparation of the horses for cell implantation has been detailed elsewhere6,15. 
	Note: The isolation and expansion in culture of BMMSC derived from the bone marrow of horses has been detailed previously6,16. The current protocol for injecting BMMSC for SDFT injuries in the horse uses 10 million cells per ml4 of autologous bone marrow supernatant (BMS)16.</li>
	<li>Use cells that have undergone no more than 4 passages for in vivo implantation to avoid potential cellular phenotype or genetic alterations associated with high passage number cells.</li>
	<li>Deliver the required number of cells in the BMS (within 24 hr in a chill box at 4 &#8211; 8 oC) to the veterinary clinic where the cell implantation is to be performed. 
	Note: The clinical use of mesenchymal stem cells in bone marrow supernatant is a patented procedure owned by ReCellerate Inc (U.S.A.) and its use may require authorization. 
	Note: Tc-99m is a gamma emitter (140 keV) with a relatively short half-life (6.0058 hr, therefore nearly 94% of it decays to its stable form of 99Tc in 24 hr). The gamma energy makes it suitable for detection by gamma cameras (scintigraphy) and the short half-life results in very limited radiation exposure time to both the horse and the animal handling personnel. This protocol describes the on-site labeling of cells using Tc-99m-HMPAO [HMPAO labeled with Tc-99m (as sodium pertechnetate)].</li>
	<li>Order the Tc-99m fresh from the supplier such that it is delivered and used for labeling the cells within 2 hr of elution from the Tc-99m generator. Ensure that the Tc-99m is supplied as 1 GBq in a 1 ml solution (supplier&#8217;s standard buffer).</li>
	<li>Use all buffers at RT. Perform the labeling procedure, implantation of the cells and imaging (gamma scintigraphy) in an isotope containment room with all equipment cleaned with alcohol wipes prior to the start of work.</li>
</ol>
2. Preparation of the Horse and Ultrasonography of the Forelimb Tendon

<ol>
	<li>Record ultrasound scans at first admission for injury (when bone marrow is aspirated) and then at the time point when radiolabeled cells are injected.</li>
	<li>Perform ultrasonography and cell implantation with the horse bearing weight evenly in a stall and under standing sedation (rather than general anesthesia). Administer sedation using a mixture of detomidine HCl and butorphanol tartrate, each at 0.01 to 0.02 mg/kg IV. 
	Note: Recovery is rapid and post-surgical treatment requires no special considerations.&#160;</li>
	<li>After perineural anesthesia is applied (step 2.6) in the affected limb, keep the horse at the station under sedation to reduce movement of the horse during the treatment. Visually check the appropriate level of sedation judged from the lowered head position and the demeanour of the horse and that the horse is comfortable during cell injection and the movements of the gamma camera during the acquisition of scintigraphic images. 
	Note: It is not necessary to apply vet ointment on eyes (to prevent dryness) because under sedation blinking is not affected and the horse is able to maintain hydration of the eyes. The monitoring after the implantation of the cells is non-invasive (ultrasound and gamma scintigraphy).</li>
	<li>Clip closely the hair overlying the tendon (with horse hair clippers) then clean the clipped area using a combination of a chlorhexidine surgical scrub and finally alcohol.</li>
	<li>Apply acoustic coupling gel to the area and scan methodically to record scans of the complete palmar metacarpal region, sequentially labeled levels 1 - 717, with a linear transducer (12 MHz or similar) to obtain serial on-incidence transverse and longitudinal images. Store the images digitally so that the cross-sectional area and the maximal injury zone4 can be obtained with the aid of image analysis software.</li>
	<li>Prepare the area overlying the palmar nerves immediately distal to the carpus for aseptic injection of local anesthetic to ensure complete desensitization of the skin overlying the tendon and the superficial digital flexor tendon. Inject 2 ml of mepivicaine both subcutaneously and adjacent to the palmar nerves (in their sub-fascial location) between the flexor tendons and the suspensory ligament immediately distal to the carpus on both sides of the limb.</li>
	<li>Once the preliminary scan has been performed prepare the implantation site by scrubbing the area with a chlorhexidine surgical scrub solution for at least 5 min and finally alcohol with soaked gauze. Perform all subsequent steps in an aseptic fashion.</li>
</ol>
3. Tc-99m-labeling of Equine Mesenchymal Stem Cells
Note: The cell labeling steps can be performed during ultrasonography of the forelimb as this will minimize isotope decay between cell labeling and implantation of labeled cells into the tendon.
<ol>
	<li>Remove the BMS from the cells as it interferes with the uptake of isotope. To do this, retrieve the vial of BMMSC (10 million cells in 1 ml of BMS) from the chill box and pellet the cells in a microcentrifuge at 350 x g for 5 min at RT. Carefully remove the supernatant with an 18G or 19G needle (attached to a 2 ml syringe), leaving a small drop ( &#8804;0.1 ml) in the tube to aid cell resuspension. Save the supernatant (the BMS) in a sterile microcentrifuge tube and keep on ice for later re-use.</li>
	<li>Resuspend the cells in the residual droplet of supernatant by flicking the tube gently with fingers (rather than vortex) and leave the tube in a rack at RT. Ensure the cells are completely resuspended and that there are no visible cell clumps.</li>
	<li>Prepare the Tc-99m as follows. Transfer 1 ml of technetium pertechnetate into a vial of HMPAO using a 1 ml syringe and needle. Mix gently but thoroughly and leave for 5 min behind lead shielding. Note: This step can be done while the cells are spinning in step 2.1.</li>
	<li>Add all of the Tc-99m-HMPAO mixture using a 1 ml syringe and needle to the cells and mix gently. Incubate for 30 min at RT behind lead shielding.</li>
	<li>For the next steps, use a forceps (or a piece of tissue paper) to open the lid of the microcentrifuge tube to minimize isotope contamination of the (gloved) thumb and subsequently other equipment. Use a 2 ml syringe and 21 G needle to add or remove wash buffer. 
	Note: The use of a field isotope monitor is encouraged to monitor contamination of surfaces and equipment.</li>
	<li>Add 1 ml PBS to the Tc-99m-HMPAO-cell mixture, close the lid, mix gently and spin to pellet the cells as in step 3.1.</li>
	<li>Carefully remove the PBS to a new tube (save this behind lead shielding and label as W1). Resuspend the cell pellet in 1 ml of fresh PBS. Centrifuge the tube and remove the supernatant as above (save this and label as W2).</li>
	<li>Calculate the efficiency of labeling by measuring the counts in W1 and W2 tubes and in the cell pellet. Do this by placing each tubes in a Well isotope dose calibrator instrument and recording the radioactivity as counts per minute (CPM). Calculate the labeling efficiency as (Radioactivity as CPM of cells) / (Radioactivity of cells + supernatant) x 100.</li>
	<li>Resuspend the cells thoroughly in the residual PBS and then add the BMS saved from step 3.1. The cells are ready for intralesional implantation.</li>
</ol>
4. Implantation of Tc-99m-HMPAO-labeled Cells
<ol>
	<li>Intralesional implantation into the tendon under ultrasound guidance
	<ol>
		<li>Slowly introduce a 1.5 or 2 inch (50 mm) 20 G needle into the maximum injury zone of the lesion in a longitudinal orientation with the ultrasound transducer, covered in a sterile drape, in line with the needle so that the length of the needle can be identified ultrasonographically and the tip if the needle is accurately located in the center of the lesion.</li>
		<li>Using appropriate shielding, collect the cells into a 2 ml syringe (Luer-lock, to minimize the risk of external contamination and in a lead-shielded syringe cover) using an 20 G needle. Discard the needle being careful not to lose any fluid. Now attach the syringe with the labeled cells onto the needle pre-located in the tendon.</li>
		<li>Place the ultrasound probe under the needle so that the needle tract in the tissue is clearly visible. Inject the cells at a slow but steady pace into the lesion. Ensure that there is no resistance to injection. If this is encountered carefully reposition the needle under ultrasound guidance. Check that the fluid ejecting into the lesion is visible on the ultrasound image as anechoic fluid containing hyperechogenic air bubbles.</li>
		<li>Withdraw the needle and immediately place pressure over the needle hole to prevent loss of injected cells and to minimize the spread of isotope over the external surface of the limb. Immediately dress the limb with one layer of autoadhesive bandage. The horse is now ready for gamma scintigraphy. Perform this immediately. 
		Note: After injecting the cells into the tendon, acquire a count of the empty syringe and needle to assess possible direct loss of cells that may remain in the syringe.</li>
	</ol>
	</li>
	<li>Regional perfusion of Tc-99m-HMPAO-labeled cells
	<ol>
		<li>Follow step 2.4.</li>
		<li>Apply a 100 mm rubber tourniquet bandage on the proximal metacarpus with two 100 mm cotton roll gauze bandage positioned medially and laterally.</li>
		<li>Aseptically prepare the skin over the lateral palmar digital vein at the level of the lateral proximal sesamoid bone by scrubbing the area with a chlorhexidine surgical scrub solution for at least 5 min and finally with gauze soaked in alcohol.&#160; Perform all subsequent steps in an aseptic fashion.</li>
		<li>Prepare a 100 mm extension set with a 21 G x &#190;&#8221; (0.8 x 19 mm) butterfly needle for venipuncture by filling with heparinized sterile saline and then introduce the needle into the lateral palmar digital vein. Once the vein is entered blood will partially fill the extension set. At the Luer lock connector attach a Luer lock rubber cap for facilitating subsequent injection of labeled cells.</li>
		<li>Dilute the MSCs in 19 ml of PBS by collecting the cells in a 20 ml syringe preloaded with the PBS.&#160; Apply the cells via the rubber cup using an 18 G (1.2 x 40 mm) hypodermic needle at a slow but steady rate (30 &#8211; 40 sec).&#160; Flush the catheter with 1 ml of PBS preloaded in a syringe.</li>
		<li>Remove the needle and immediately apply pressure at the skin over the needle hole with a layer of five 4 x 4 cotton gauzes. Then place a light bandage with elastic adhesive over the proximal sesamoid area&#160;and leave the tourniquet in place for 30 min after injection.</li>
		<li>Release the tourniquet after 30 min. The horse is now ready for gamma scintigraphy. 
		Note: After injecting the cells, acquire a count of the empty syringe and needle to assess possible direct loss of cells that may remain in the syringe.</li>
	</ol>
	</li>
</ol>
5. Gamma Scintigraphy
<ol>
	<li>Scintigraphy images
	<ol>
		<li>Acquire planar scintigraphy images with a gamma camera (set at 140 KeV photoelectric peak using a 20% symmetrical window) equipped with a low energy, parallel hole collimator.
		<ol>
			<li>Obtain all images after time 0 on standing horses sedated as in step 2.2. After the time 0 scintigram, replace the light bandage over the implantation site with a padded bandage. Remove this bandage prior to each scintigraphy exam.</li>
			<li>Acquire static images for 60 sec each (256 by 256 matrix) using the processing software with the &#8220;acquire&#8221; &#8211; &#8220;study selection&#8221; - &#8220;bone static&#8221; options. It is essential that the horse does not move during the image acquisition period because the static mode option does not have a module for correction of movement.</li>
			<li>For intralesional injection, obtain lateral images from the lesion area from the carpus to the distal extremity, the equivalent area of the contralateral limb, the left lung field and the left thyroid. During the acquisition of forelimbs images, position a lead screen between the legs to avoid imaging of the limb contralateral. Acquire the images at 5 min after injection (time 0), and then at 1, 3, 6, 12, 24, 36 and 48 hr.</li>
			<li>For regional perfusion, obtain lateral and dorsal gamma images as for 5.1.1.3. Obtain images 5 min after tourniquet release. This will be time 0. It can be useful to image the limb immediately after injecting the cells to assess counts prior to tourniquet release. Obtain gamma images at 1, 3, 6, 12, 24, 36 and 48 hr after administration of the cells.</li>
		</ol>
		</li>
		<li>After the images have been acquired, process the images manually using the &#8220;processing&#8221; option of the software with options selected for &#8220;Sokoloff&#8221; color and &#8220;Invert&#8221; color. Next select the &#8220;region of interest&#8221; (ROI) and &#8220;ellipse&#8221; as this allows reshaping of the selection mask and movement over the lesion area. Obtain the counts over the ROI by choosing the &#8220;add to statistics&#8221; option. Ensure that identically sized regions of interest (ROI) are recorded for each animal at the different time-points.</li>
		<li>Next, correct the counts for the predicted decay of the technetium at each time point. Use the following equation to correct for decay: A = Aoe-(0.693t/T1/2), where Ao is the initial activity of the isotope, A is the decay corrected radioactivity at time zero, t is the elapsed time and T1/2 is the half-life of the isotope. Then express the corrected counts at each time point as a percentage of the ROI at time 0 to work out the percentage of cells remaining, using the following formula:</li>
	</ol>
	</li>
</ol>
%cells remaining (t) = 100 &#8211; {[(predicted decay(t) &#8211; decay(t))/ predicted decay(t)]}&#160;x 100
decay(t) = ROI(t) / ROI(0) x 100.

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V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mesenchymal+stem+cells+and+insulin-like+growth+factor-I+gene-enhanced+mesenchymal+stem+cells+improve+structural+aspects+of+healing+in+equine+flexor+digitorum+superficialis+tendons.">Mesenchymal stem cells and insulin-like growth factor-I gene-enhanced mesenchymal stem cells improve structural aspects of healing in equine flexor digitorum superficialis tendons.</a> Journal of Orthopaedic Research. 27, 1392-1398 (2009).</li><li>Smith, R. 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Inflammation/Infection Taskgroup of the European Association of Nuclear Medicine.</a> European Journal of Nuclear Medicine and Molecular Imaging. 37, 842-848 (2010).</li><li>Trela, J. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Scintigraphic+comparison+of+intra-arterial+injection+and+distal+intravenous+regional+limb+perfusion+for+administration+of+mesenchymal+stem+cells+to+the+equine+foot.">Scintigraphic comparison of intra-arterial injection and distal intravenous regional limb perfusion for administration of mesenchymal stem cells to the equine foot.</a> Equine Veterinary Journal. 46, 479-483 (2014).</li><li>Barbash, I. 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JD and RKS are Scientific Advisory Board members to ReCellerate Inc.

In addition to bone marrow, stem cells isolated from sources such as adipose tissue are suitable for labeling with this protocol. Furthermore, cells from a frozen state may be revived and expanded in culture to the desired numbers for labeling studies12.
A critical factor that determines the efficiency of labeling the BMMSC is the time between elution of the Tc-99m from the molybdenum generator at the radiopharmacy, preparation of Tc-99m-HMPAO and use of the radiopharmaceutical in t...

Regenerative strategies for the repair of diseased or damaged tissues are based on multipotent stem cells derived from a variety of tissue and implanted into the affected area. Recent advances in the application of autologous BMMSC for the treatment of tendon and ligament injuries in the horse have shown improved outcome measures in both experimental1-5 and clinical studies6. The horse is a particularly attractive model for assessing the efficacy of stem cell related treatments because it suffers from age and overstrain related injuries to the tendons of the distal forelimb, it is an athletic animal, and it is a large, facilitating bone marrow recovery and accurate implantation. Tendon injuries heal naturally with fibrosis but the healed tendon is functionally inferior7 and has a high risk of re-injury8. The superficial digital flexor tendon (SDFT) is most commonly affected as it has evolved to act as an elastic energy store and experiences high loading stresses to achieve energy efficient and high speed locomotion. Restoring function after injury is therefore critical. These injuries are similar to those affecting the Achilles tendon in humans which performs a similar function9. There are no good treatment options to treat or achieve good repair for such injuries, therefore cell-based regenerative strategies offer an attractive opportunity to improve outcomes and to reduce re-injury.
In most studies 5 &#8211; 20 million autologous BMMSC are injected directly into the lesion which usually occur within the core of the tendon body which therefore acts as a receptacle for the cells. The fate of the cells once injected is not clear and different cell labeling methods to track the cells have been described recently. Cells labeled with a fluorescence tag were shown to survive only in relatively small numbers (&#60;10%)10,11. Fluorescence labels necessitate tissue extraction and sectioning for histological analysis which is time consuming and does not readily facilitate temporal analysis in a large animal model or in clinical cases. In more recent work we have used the radioisotope 99mTc to label cells and follow their fate by gamma scintigraphy1. This method allows rapid comparisons to be made between different routes of cell delivery, including intralesional, intravenous via the jugular vein1 or regional perfusion via intra-arterial12 or intravenous1,12 injections. The persistence and distribution of cells can then be imaged by gamma scintigraphy of various organs. This has demonstrated that only 24% of the cells injected intralesionally remained in the lesion by 24 hr1 and this is supported by another study using experimentally created lesions and using the same radiolabel5. Furthermore, the cells show limited ability to home into tendon lesions when delivered by regional perfusion or intravenously but are dispersed into the lungs by the latter routes4.
BMMSC labeled with iron nanoparticles is an alternative method to track cells implanted into forelimb tendons13. Although iron nanoparticle labeled cells allow cell tracking in vivo by MRI, temporal studies in a large animal are limited by the number of times anesthesia can be administered at each time point for performing the MRI scans. Furthermore, iron nanoparticles are hypointense on MRI which limits the information on the migration of labeled cells into the tendon body. Other radioisotopes that can be used include Indium-111 but this suffers the disadvantage of a longer half-life than Tc-99m (2.8 days vs 6.0 hr) and higher gamma ray emission energy. In addition, cell viability has been reported to be reduced when labeled with Indium-11114. Tc-99m, on the other hand, is routinely used in both equine and human nuclear medicine to label peripheral blood mononuclear cells and follow their distribution in vivo by scintigraphy. It can be relatively easily taken up by cells using HMPAO as a linker molecule to bind the technetium, as Tc-99m-HMPAO, to cells. Tc-99m-HMPAO labeled BMMSC show good viability and can proliferate in vitro4. This protocol details the labeling and tracking of equine autologous BMMSC implanted into naturally occurring lesions in the forelimb SDFT.
It is important to note that the protocol is intended only to be used as a research tool. Its use as a clinical therapeutic modality is not recommended as the effect of the radiolabel on cellular phenotype has not been fully elucidated.

<table>
	<tbody>
		<tr>
			<td>Technetium99m&#160;</td>
			<td></td>
			<td></td>
			<td>Please enquire with local ionisation radiation supplier in accordance with legal requirements.&#160; The isotope must be used within 2 h of elution from the molybdenum-99 generator</td>
		</tr>
		<tr>
			<td>Ceretec - Hexamethylpropyleneamine oxime (HMPAO)&#160;</td>
			<td>GE HealthCare</td>
			<td></td>
			<td>Please enquire directly with GE HealthCare</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Microfuge, Minispin/Minispin Plus</td>
			<td>Ependorf</td>
			<td>22620100</td>
			<td></td>
		</tr>
		<tr>
			<td>18G and 19G Needles</td>
			<td>Terumo Medical</td>
			<td>NN-1838R (18G);&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; NN1938R (19G)</td>
			<td></td>
		</tr>
		<tr>
			<td>Syringes 1 mL and 2 mL</td>
			<td>Scientific Laboratory Supplies Ltd</td>
			<td>SYR6200 (1 mL); SYR6003 (2 mL)</td>
			<td></td>
		</tr>
		<tr>
			<td>Microcentrifuge tubes 1.5 mL</td>
			<td>Greiner Bio-One Ltd</td>
			<td>616201</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS - Phosphate-Buffered Saline</td>
			<td>LifeTechnologies</td>
			<td>14190</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile Gauze Swabs</td>
			<td>Shermond Ltd</td>
			<td>UNG602</td>
			<td></td>
		</tr>
		<tr>
			<td>CoflexVet self adhering bandage</td>
			<td>Andover Healthcare, Inc.</td>
			<td>3540RB-018</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultrasound imaging software</td>
			<td>Scion Image, Scion Corporation, USA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>MicasXplus Scintigram processing software</td>
			<td>Bartec Technologies Ltd</td>
			<td></td>
			<td>http://www.bartectechnologies.com/veterinaryscintigraphy.html</td>
		</tr>
		<tr>
			<td>Field isotope counter for monitoring isotope</td>
			<td>John Caunt U.K.</td>
			<td>GMS1800a</td>
			<td>http://www.johncaunt.com/</td>
		</tr>
		<tr>
			<td>Well counter for isotope measurements, dose calibrator</td>
			<td>Capintec Southern Scientific</td>
			<td>CRC-25R</td>
			<td></td>
		</tr>
	</tbody>
</table>

in vivo imaging and tracking of technetium-99m labeled bone marrow mesenchymal stem cells in equine tendinopathy

Recent advances in the application of bone marrow mesenchymal stem cells (BMMSC) for the treatment of tendon and ligament injuries in the horse suggest improved outcome measures in both experimental and clinical studies. Although the BMMSC are implanted into the tendon lesion in large numbers (usually 10 - 20 million cells), only a relatively small number survive (&#60;10%) although these can persist for up to 5 months after implantation. This appears to be a common observation in other species where BMMSC have been implanted into other tissues and it is important to understand when this loss occurs, how many survive the initial implantation process and whether the cells are cleared into other organs. Tracking the fate of the cells can be achieved by radiolabeling the BMMSC prior to implantation which allows non-invasive in vivo imaging of cell location and quantification of cell numbers.
This protocol describes a cell labeling procedure that uses Technetium-99m (Tc-99m), and tracking of these cells following implantation into injured flexor tendons in horses. Tc-99m is a short-lived (t1/2 of 6.01 hr) isotope that emits gamma rays and can be internalized by cells in the presence of the lipophilic compound hexamethylpropyleneamine oxime (HMPAO). These properties make it ideal for use in nuclear medicine clinics for the diagnosis of many different diseases. The fate of the labeled cells can be followed in the short term (up to 36 hr) by gamma scintigraphy to quantify both the number of cells retained in the lesion and distribution of the cells into lungs, thyroid and other organs. This technique is adapted from the labeling of blood leukocytes and could be utilized to image implanted BMMSC in other organs.

Tc-99m-HMPAO incorporation into BMMSs does not adversely affect their ability to adhere to tissue culture plastic and while they show proliferation ability to form monolayers (Figure 1) we have not fully determined whether proliferation rates or other cellular phenotypes are affected. Their morphology is similar to unlabeled cells with a typical spindle shape. The cellular labeling efficiency (i.e., uptake of label) typically varies from about 1.5% to 25%. The main reason for the low labeling ef...

Watch this Scientific Journal Video about In Vivo Imaging and Tracking of Technetium-99m Labeled Bone Marrow Mesenchymal Stem Cells in Equine Tendinopathy at JoVE.com

In Vivo Imaging and Tracking of Technetium-99m Labeled Bone Marrow Mesenchymal Stem Cells in Equine Tendinopathy

This protocol describes the radiolabeling of equine mesenchymal stem cells and their implantation into tendon injuries in the horse in order to determine cell survival and tissue distribution using gamma scintigraphy.

In Vivo Imaging and Tracking of Technetium-99m Labeled Bone Marrow Mesenchymal Stem Cells in Equine Tendinopathy

Recent advances in the application of bone marrow mesenchymal stem cells (BMMSC) for the treatment of tendon and ligament injuries in the horse suggest ...

in-vivo-imaging-tracking-technetium-99m-labeled-bone-marrow

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Medicine

9.7K visualizações.  The Royal Veterinary College. This protocol describes the radiolabeling of equine mesenchymal stem cells and their implantation into tendon injuries in the horse in order to determine cell survival and tissue distribution using gamma scintigraphy.

Vídeo: In Vivo Imaging and Tracking of Technetium-99m Labeled Bone Marrow Mesenchymal Stem Cells in Equine Tendinopathy

Multi-modal Imaging of Angiogenesis in a Nude Rat Model of Breast Cancer Bone Metastasis Using Magnetic Resonance Imaging, Volumetric Computed Tomography and Ultrasound

Angiogenesis is an essential feature of cancer growth and metastasis formation. In bone metastasis, angiogenic factors are pivotal for tumor cell proliferation in the bone marrow cavity as well as for interaction of tumor and bone cells resulting in local bone destruction. Our aim was to develop a model of experimental bone metastasis that allows in vivo assessment of angiogenesis in skeletal lesions using non-invasive imaging techniques.
For this purpose, we injected 105 MDA-MB-231 human breast cancer cells into the superficial epigastric artery, which precludes the growth of metastases in body areas other than the respective hind leg1. Following 25-30 days after tumor cell inoculation, site-specific bone metastases develop, restricted to the distal femur, proximal tibia and proximal fibula1. Morphological and functional aspects of angiogenesis can be investigated longitudinally in bone metastases using magnetic resonance imaging (MRI), volumetric computed tomography (VCT) and ultrasound (US).
MRI displays morphologic information on the soft tissue part of bone metastases that is initially confined to the bone marrow cavity and subsequently exceeds cortical bone while progressing. Using dynamic contrast-enhanced MRI (DCE-MRI) functional data including regional blood volume, perfusion and vessel permeability can be obtained and quantified2-4. Bone destruction is captured in high resolution using morphological VCT imaging. Complementary to MRI findings, osteolytic lesions can be located adjacent to sites of intramedullary tumor growth. After contrast agent application, VCT angiography reveals the macrovessel architecture in bone metastases in high resolution, and DCE-VCT enables insight in the microcirculation of these lesions5,6. US is applicable to assess morphological and functional features from skeletal lesions due to local osteolysis of cortical bone. Using B-mode and Doppler techniques, structure and perfusion of the soft tissue metastases can be evaluated, respectively. DCE-US allows for real-time imaging of vascularization in bone metastases after injection of microbubbles7.
In conclusion, in a model of site-specific breast cancer bone metastases multi-modal imaging techniques including MRI, VCT and US offer complementary information on morphology and functional parameters of angiogenesis in these skeletal lesions.

In the pathogenesis of bone metastasis, angiogenesis is a crucial process and therefore represents a target for imaging and therapy. Here, we present a rat model of site-specific breast cancer bone metastasis and describe strategies to non-invasively image angiogenesis in vivo using magnetic resonance imaging, volumetric computed tomography and ultrasound.

Angiogenesis is an essential feature of cancer growth and metastasis formation. In bone metastasis, angiogenic factors are pivotal for tumor cell ...

Pre-clinical Evaluation of Tyrosine Kinase Inhibitors for Treatment of Acute Leukemia

Receptor tyrosine kinases have been implicated in the development and progression of many cancers, including both leukemia and solid tumors, and are attractive druggable therapeutic targets. Here we describe an efficient four-step strategy for pre-clinical evaluation of tyrosine kinase inhibitors (TKIs) in the treatment of acute leukemia. Initially, western blot analysis is used to confirm target inhibition in cultured leukemia cells. Functional activity is then evaluated using clonogenic assays in methylcellulose or soft agar cultures. Experimental compounds that demonstrate activity in cell culture assays are evaluated in vivo using NOD-SCID-gamma (NSG) mice transplanted orthotopically with human leukemia cell lines. Initial in vivo pharmacodynamic studies evaluate target inhibition in leukemic blasts isolated from the bone marrow. This approach is used to determine the dose and schedule of administration required for effective target inhibition. Subsequent studies evaluate the efficacy of the TKIs in vivo using luciferase expressing leukemia cells, thereby allowing for non-invasive bioluminescent monitoring of leukemia burden and assessment of therapeutic response using an in vivo bioluminescence imaging system. This strategy has been effective for evaluation of TKIs in vitro and in vivo and can be applied for identification of molecularly-targeted agents with therapeutic potential or for direct comparison and prioritization of multiple compounds.

Receptor tyrosine kinases are ectopically expressed in many cancers and have been identified as therapeutic targets in acute leukemia. This manuscript describes an efficient strategy for pre-clinical evaluation of tyrosine kinase inhibitors for the treatment of acute leukemia.

Receptor tyrosine kinases have been implicated in the development and progression of many cancers, including both leukemia and solid tumors, and are ...

In vivo 19F MRI for Cell Tracking

In vivo 19F MRI allows quantitative cell tracking without the use of ionizing radiation. It is a noninvasive technique that can be applied to humans. Here, we describe a general protocol for cell labeling, imaging, and image processing. The technique is applicable to various cell types and animal models, although here we focus on a typical mouse model for tracking murine immune cells. The most important issues for cell labeling are described, as these are relevant to all models. Similarly, key imaging parameters are listed, although the details will vary depending on the MRI system and the individual setup. Finally, we include an image processing protocol for quantification. Variations for this, and other parts of the protocol, are assessed in the Discussion section. Based on the detailed procedure described here, the user will need to adapt the protocol for each specific cell type, cell label, animal model, and imaging setup. Note that the protocol can also be adapted for human use, as long as clinical restrictions are met.

In vivo 19F MRI for Cell Tracking

We describe a general protocol for in vivo cell tracking using MRI in a mouse model with ex vivo labeled cells. A typical protocol for cell labeling, image acquisition processing and quantification is included.

In vivo 19F MRI allows quantitative cell tracking without the use of ionizing radiation. It is a noninvasive technique that can be applied to humans. ...

Sequential In vivo Imaging of Osteogenic Stem/Progenitor Cells During Fracture Repair

Bone turns over continuously and is highly regenerative following injury. Osteogenic stem/progenitor cells have long been hypothesized to exist, but in vivo demonstration of such cells has only recently been attained. Here, in vivo imaging techniques to investigate the role of endogenous osteogenic stem/progenitor cells (OSPCs) and their progeny in bone repair are provided. Using osteo-lineage cell tracing models and intravital imaging of induced microfractures in calvarial bone, OSPCs can be directly observed during the first few days after injury, in which critical events in the early repair process occur. Injury sites can be sequentially imaged revealing that OSPCs relocate to the injury, increase in number and differentiate into bone forming osteoblasts. These methods offer a means of investigating the role of stem cell-intrinsic and extrinsic molecular regulators for bone regeneration and repair.

Sequential In vivo Imaging of Osteogenic Stem/Progenitor Cells During Fracture Repair

Quantitative measurement of bone progenitor function in fracture healing requires high resolution serial imaging technology. Here, protocols are provided for using intravital microscopy and osteo-lineage tracking to sequentially image and quantify the migration, proliferation and differentiation of endogenous osteogenic stem/progenitor cells in the process of repairing bone fracture.

Bone turns over continuously and is highly regenerative following injury. Osteogenic stem/progenitor cells have long been hypothesized to exist, but in ...

Femoral Bone Marrow Aspiration in Live Mice

Serial sampling of the cellular composition of bone marrow (BM) is a routine procedure critical to clinical hematology. This protocol describes a detailed step-by-step technical procedure for an analogous procedure in live mice which allows for serial characterization of cells present in the BM. This procedure facilitates studies aimed to detect the presence of exogenously administered cells within the BM of mice as would be done in xenograft studies for instance. Moreover, this procedure allows for the retrieval and characterization of cells enriched in the BM such as hematopoietic stem and progenitor cells (HSPCs) without sacrifice of mice. Given that the cellular composition of peripheral blood is not necessarily reflective of proportions and types of stem and progenitor cells present in the marrow, procedures which provide access to this compartment without requiring termination of the mice are very helpful. The use of femoral bone marrow aspiration is illustrated here for cytological analysis of marrow cells, flow cytometric characterization of the hematopoietic stem/progenitor compartment, and culture of sorted HSPCs obtained by femoral BM aspiration compared with conventional marrow harvest.

This protocol describes a procedure for serial sampling of femoral bone marrow (BM) without requiring the sacrifice of mice. This procedure facilitates longitudinal studies of the BM composition of mice over time and provides serial access to cells within the BM for ex vivo and transplantation studies.

Serial sampling of the cellular composition of bone marrow (BM) is a routine procedure critical to clinical hematology. This protocol describes a detailed ...

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 ...

Isolation and Cellular Phenotyping of Mesenchymal Stem Cells Derived from Synovial Fluid and Bone Marrow of Minipigs

Mesenchymal stem cells (MSCs) have been established after isolation from various tissue sources, including bone marrow and synovial fluid. Recently, synovial-fluid-derived MSCs were reported to have multi-lineage differentiation potential and immunomodulatory features, which indicates that these cells can be used for tissue engineering and systemic treatments. This study presents a protocol for simple and non-invasive isolation of MSCs derived from the bone marrow and synovial fluid of minipigs to analyze cell surface markers for cell phenotyping and in vitro culturing. Using sexually mature six-month-old minipigs, bone marrow was extracted from the iliac crest bone using a bone marrow extractor, and the synovial fluid was aspirated from the femorotibial joint. Procedures for the collection of samples from both sources were non-invasive. The protocols for effective isolation of MSCs from harvested cell sources and for creating in vitro culture conditions to expand stable MSCs from minipigs and the application of systemic autologous treatments are provided. For cell phenotyping, the cell surface markers of both cells were analyzed using flow cytometry. In the results, the MSCs were isolated from the synovial fluid of the minipigs and showed that synovial-fluid-derived MSCs have a similar morphology and cell phenotype to bone-marrow-derived MSCs. Therefore, non-invasively obtained synovial fluid is a valuable source of MSCs.

A protocol establishing mesenchymal stem cells (MSCs) isolated from the synovial fluid and bone marrow of minipigs and non-invasively collected using syringe aspiration is presented. Cellular phenotyping was performed using flow cytometry after cell isolation and in vitro cultivation of bone marrow and synovial fluids derived from MSCs.

Mesenchymal stem cells (MSCs) have been established after isolation from various tissue sources, including bone marrow and synovial fluid. Recently, ...

Depletion of Mouse Cells from Human Tumor Xenografts Significantly Improves Downstream Analysis of Target Cells

The use of in vitro cell line models for cancer research has been a useful tool. However, it has been shown that these models fail to reliably mimic patient tumors in different assays1. Human tumor xenografts represent the gold standard with respect to tumor biology, drug discovery, and metastasis research 2-4. Tumor xenografts can be derived from different types of material like tumor cell lines, tumor tissue from primary patient tumors4 or serially transplanted tumors. When propagated in vivo, xenografted tissue is infiltrated and vascularized by cells of mouse origin. Multiple factors such as the tumor entity, the origin of xenografted material, growth rate and region of transplantation influence the composition and the amount of mouse cells present in tumor xenografts. However, even when these factors are kept constant, the degree of mouse cell contamination is highly variable.
Contaminating mouse cells significantly impair downstream analyses of human tumor xenografts. As mouse fibroblasts show high plating efficacies and proliferation rates, they tend to overgrow cultures of human tumor cells, especially slowly proliferating subpopulations. Mouse cell derived DNA, mRNA, and protein components can bias downstream gene expression analysis, next-generation sequencing, as well as proteome analysis 5. To overcome these limitations, we have developed a fast and easy method to isolate untouched human tumor cells from xenografted tumor tissue. This procedure is based on the comprehensive depletion of cells of mouse origin by combining automated tissue dissociation with the benchtop tissue dissociator and magnetic cell sorting. Here, we demonstrate that human target cells can be can be obtained with purities higher than 96% within less than 20 min independent of the tumor type.

Human tumor xenografts are vascularized and infiltrated by cells of mouse origin during the growth phase in vivo. To circumvent the bias caused by these contaminating cells during downstream analysis, we developed a method allowing for comprehensive depletion of all mouse cells by magnetic cell sorting.

The use of in vitro cell line models for cancer research has been a useful tool. However, it has been shown that these models fail to reliably mimic ...

Nerve Stimulator-guided Injection of Autologous Stem Cells Near the Equine Left Recurrent Laryngeal Nerve

Recurrent laryngeal neuropathy (RLN) commonly affects horses and is characterized by abnormal respiratory sounds and exercise intolerance. The recurrent laryngeal nerve shows lesions of demyelination. The benefit of applying stem cells to demyelinated nerves has been demonstrated in various animal models. The aim of the study was to test the feasibility and safety of a peri-neuronal injection of autologous muscle-derived mesenchymal stem cells to the left recurrent laryngeal nerve in healthy horses by using an electrical nerve stimulator.
Muscle-derived stems cell are obtained from five healthy Standardbred horses by sampling 20 mg of muscle tissue with a semi-automatic 14 G biopsy needle from the triceps muscle. Movements of the larynx are monitored via upper-airway video endoscopy. The left recurrent laryngeal nerve is approached with an insulated nerve block needle. Nerve stimulation is applied, starting at 2 mA, and the successful abduction of the left arytenoid is monitored. The stimulation intensity is reduced progressively. When a loss of the motor response is observed at 0.5 mA, 107 autologous muscle-derived stem cells are injected. Two examiners, who are blinded to the time point, score the laryngeal function of the horses prior to the treatment and at day 1, day 7, and day 28 after the injection of the cells. In a sixth horse, 1 mL of 2% lidocaine is injected to further confirm the correct positioning of the needle. This leads to a temporary paralysis of the left arytenoid cartilage.
This study proves that the recurrent laryngeal nerve can be approached with the help of an electrical nerve stimulator and that the electrical stimulation of the nerve is well tolerated by the horses. No modification of the laryngeal function was observed in any of the horses after the injection of the stem cells. Further studies should be conducted to describe the effects of a peri-neuronal injection of autologous muscle-derived mesenchymal stem cells to horses suffering from RLN.

Here, we present a protocol to inject autologous muscle-derived stem cells in the proximity to the recurrent laryngeal nerve with the help of an electrical nerve stimulator. This novel technique may become useful for the treatment of equine recurrent laryngeal neuropathy.

Recurrent laryngeal neuropathy (RLN) commonly affects horses and is characterized by abnormal respiratory sounds and exercise intolerance. The recurrent ...

Quantitative [18F]-Naf-PET-MRI Analysis for the Evaluation of Dynamic Bone Turnover in a Patient with Facetogenic Low Back Pain

Imaging techniques that reflect dynamic bone turnover may aid in characterizing a wide range of bone pathologies. Bone is a dynamic tissue undergoing continuous remodeling with the competing activity of osteoblasts, which produce the new bone matrix, and osteoclasts, whose function is to eliminate mineralized bone. [18F]-NaF is a positron emission tomography (PET) radiotracer that enables visualization of bone metabolism. [18F]-NaF is chemically absorbed into hydroxyapatite in the bone matrix by osteoblasts and can thus noninvasively detect osteoblastic activity, which is occult to conventional imaging techniques. Kinetic modeling of dynamic [18F]-NaF-PET data provides detailed quantitative measures of bone metabolism. Conventional semi-quantitative PET data, which utilizes standardized uptake values (SUVs) as a measure of radiotracer activity, is referred to as a static technique due to its snapshot of tracer uptake in time.&#160; Kinetic modeling, however, utilizes dynamic image data where tracer levels are continuously acquired providing tracer uptake temporal resolution. From the kinetic modeling of dynamic data, quantitative values like blood flow and metabolic rate (i.e., potentially informative metrics of tracer dynamics) can be extracted, all with respect to the measured activity in the image data. When combined with dual modality PET-MRI, region-specific kinetic data can be correlated with anatomically registered high-resolution structural and pathologic information afforded by MRI. The goal of this methodological manuscript is to outline detailed techniques for performing and analyzing dynamic [18F]-NaF-PET-MRI data. The lumbar facet joint is a common site of degenerative arthritis disease and a common cause for axial low back pain.&#160; Recent studies suggest [18F]-NaF-PET may serve as a useful biomarker of painful facetogenic disease.&#160; The human lumbar facet joint will, therefore, be used as a prototypical region of interest for dynamic [18F]-NaF-PET-MRI analysis in this manuscript.

Quantitative [18F]-Naf-PET-MRI Analysis for the Evaluation of Dynamic Bone Turnover in a Patient with Facetogenic Low Back Pain

Imaging techniques that reflect dynamic bone turnover may aid in characterizing a wide range of bone pathologies. We present detailed methodologies for performing and analyzing dynamic [18F]-NaF-PET-MRI data in a patient with facetogenic low back pain using the lumbar facet joints as a prototypical region of interest.

Imaging techniques that reflect dynamic bone turnover may aid in characterizing a wide range of bone pathologies. Bone is a dynamic tissue undergoing ...