Name: in vivo imaging and tracking of technetium-99m labeled bone marrow mesenchymal stem cells in equine tendinopathy
Uploaded: 2015-12-09T13:00:00.000+00:00
Duration: 7 min 52 s
Description: 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

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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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>Technetium&lt;sup&gt;99m&lt;/sup&gt;&amp;nbsp;</td><td></td><td></td><td>Please enquire with local ionisation radiation supplier in accordance with legal requirements.&amp;nbsp; The isotope must be used within 2 hr of elution from the molybdenum-99 generator</td></tr><tr><td>Ceretec - Hexamethylpropyleneamine oxime (HMPAO)&amp;nbsp;</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>18 G and 19 G Needles</td><td>Terumo Medical</td><td>NN-1838R (18 G);&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp; NN1938R (19 G)</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

Research

JoVE Journal

Medicine

9.7K Views.  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.

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

Labeling hESCs and hMSCs with Iron Oxide Nanoparticles for Non-Invasive in vivo Tracking with MR Imaging

In recent years, stem cell research has led to a better understanding of developmental biology, various diseases and its potential impact on regenerative medicine. A non-invasive method to monitor the transplanted stem cells repeatedly in vivo would greatly enhance our ability to understand the mechanisms that control stem cell death and identify trophic factors and signaling pathways that improve stem cell engraftment. MR imaging has been proven to be an effective tool for the in vivo depiction of stem cells with near microscopic anatomical resolution. In order to detect stem cells with MR, the cells have to be labeled with cell specific MR contrast agents. For this purpose, iron oxide nanoparticles, such as superparamagnetic iron oxide particles (SPIO), are applied, because of their high sensitivity for cell detection and their excellent biocompatibility. SPIO particles are composed of an iron oxide core and a dextran, carboxydextran or starch coat, and function by creating local field inhomogeneities, that cause a decreased signal on T2-weighted MR images. This presentation will demonstrate techniques for labeling of stem cells with clinically applicable MR contrast agents for subsequent non-invasive in vivo tracking of the labeled cells with MR imaging.

For the evaluation of new stem cell therapies it is important to non-invasively track the injected cells in vivo. This video will show you how to label human mesenchymal and embryonic stem cells with iron oxide based contrast agents in vivo for subsequent MR imaging in vivo.

In recent years, stem cell research has led to a better understanding of developmental biology, various diseases and its potential impact on regenerative ...

Biology

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 ...

In Vivo Tracking of Human Adipose-derived Mesenchymal Stem Cells in a Rat Knee Osteoarthritis Model with Fluorescent Lipophilic Membrane Dye

In order to support the clinical application of human adipose-derived mesenchymal stem cell (haMSC) therapy for knee osteoarthritis (KOA), we examined the efficacy of cell persistence and biodistribution of haMSCs in animal models. We demonstrated a method to label the cell membrane of haMSCs with lipophilic fluorescent dye. Subsequently, intra-articular injection of the labeled cells in rats with surgically induced KOA was monitored dynamically by an in vivo imaging system. We employed the lipophilic carbocyanines DiD (DilC18 (5)), a far-red fluorescent Dil (dialkylcarbocyanines) analog, which utilized a red laser to avoid excitation of the natural green autofluorescence from surrounding tissues. Furthermore, the red-shifted emission spectra of DiD allowed deep-tissue imaging in live animals and the labeling procedure caused no cytotoxic effects or functional damage to haMSCs. This approach has been shown to be an efficient tracking method for haMSCs in a rat KOA model. The application of this method could also be used to determine the optimal administration route and dosage of MSCs from other sources in pre-clinical studies.

In Vivo Tracking of Human Adipose-derived Mesenchymal Stem Cells in a Rat Knee Osteoarthritis Model with Fluorescent Lipophilic Membrane Dye

This protocol describes an efficient way to monitor the cell persistence and biodistribution of human adipose-derived mesenchymal stem cells (haMSCs) by far-red fluorescence labeling in a rat knee osteoarthritis (KOA) model via intra-articular (IA) injection.

In order to support the clinical application of human adipose-derived mesenchymal stem cell (haMSC) therapy for knee osteoarthritis (KOA), we examined the ...

Developmental Biology

Tracking Dynamics of Muscle Engraftment in Small Animals by In Vivo Fluorescent Imaging

Muscular dystrophies are a group of degenerative muscle diseases characterized by progressive loss of contractile muscle cells. Currently, there is no curative treatment available. Recent advances in stem cell biology have generated new hopes for the development of effective cell based therapies to treat these diseases. Transplantation of various types of stem cells labeled with fluorescent proteins into muscles of dystrophic animal models has been used broadly in the field. A non-invasive technique with the capability to track the transplanted cell fate longitudinally can further our ability to evaluate muscle engraftment by transplanted cells more accurately and efficiently.
In the present study, we demonstrate that in vivo fluorescence imaging is a sensitive and reliable method for tracking transplanted GFP (Green Fluorescent Protein)-labeled cells in mouse skeletal muscles. Despite the concern about background due to the use of an external light necessary for excitation of fluorescent protein, we found that by using either nude mouse or eliminating hair with hair removal reagents much of this problem is eliminated. Using a CCD camera, the fluorescent signal can be detected in the tibialis anterior (TA) muscle after injection of 5 x 105 cells from either GFP transgenic mice or eGFP transduced myoblast culture. For more superficial muscles such as the extensor digitorum longus (EDL), injection of fewer cells produces a detectable signal. Signal intensity can be measured and quantified as the number of emitted photons per second in a region of interest (ROI). Since the acquired images show clear boundaries demarcating the engrafted area, the size of the ROI can be measured as well. If the legs are positioned consistently every time, the changes in total number of photons per second per muscle and the size of the ROI reflect the changes in the number of engrafted cells and the size of the engrafted area. Therefore the changes in the same muscle over time are quantifiable. In vivo fluorescent imaging technique has been used primarily to track the growth of tumorogenic cells, our study shows that it is a powerful tool that enables us to track the fate of transplanted stem cells.

Tracking Dynamics of Muscle Engraftment in Small Animals by In Vivo Fluorescent Imaging

We describe an in vivo fluorescence imaging protocol to monitor muscle regeneration by GFP-labeled myoblasts after transplantation into skeletal muscles of both healthy and dystrophic mice. This protocol can be adapted to study muscle regeneration by transplantation of other types of cells and in other muscular conditions as well.

Muscular dystrophies are a group of degenerative muscle diseases characterized by progressive loss of contractile muscle cells. Currently, there is no ...

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 ...

Tracking Mouse Bone Marrow Monocytes In Vivo

Real time multiphoton imaging provides a great opportunity to study cell trafficking and cell-to-cell interactions in their physiological 3-dimensionnal environment. Biological activities of immune cells mainly rely on their motility capacities. Blood monocytes have short half-life in the bloodstream; they originate in the bone marrow and are constitutively released from it. In inflammatory condition, this process is enhanced, leading to blood monocytosis and subsequent infiltration of the peripheral inflammatory tissues. Identifying the biomechanical events controlling monocyte trafficking from the bone marrow towards the vascular network is an important step to understand monocyte physiopathological relevance. We performed in vivo time-lapse imaging by two-photon microscopy of the skull bone marrow of the Csf1r-Gal4VP16/UAS-ECFP (MacBlue) mouse. The MacBlue mouse expresses the fluorescent reporters enhanced cyan fluorescent protein (ECFP) under the control of a myeloid specific promoter 1, in combination with vascular network labelling. We describe how this approach enables the tracking of individual medullar monocytes in real time to further quantify the migratory behaviour within the bone marrow parenchyma and the vasculature, as well as cell-to-cell interactions. This approach provides novel insights into the biology of the bone marrow monocyte subsets and allows to further address how these cells can be influenced in specific pathological conditions.

Tracking Mouse Bone Marrow Monocytes In Vivo

Monocytes are key regulators of innate immunity and play a critical role in the renewal of the peripheral mononuclear phagocytic system and in case of inflammation. This manuscript describes the procedure of real time imaging of the mouse calvaria bone marrow to study the monocyte mobilisation mechanism.

Real time multiphoton imaging provides a great opportunity to study cell trafficking and cell-to-cell interactions in their physiological 3-dimensionnal ...

Immunology and Infection

Real-Time Imaging of CCL5-Induced Migration of Periosteal Skeletal Stem Cells in Mice

Periosteal skeletal stem cells (P-SSCs) are essential for lifelong bone maintenance and repair, making them an ideal focus for the development of therapies to enhance fracture healing.&#160; Periosteal cells rapidly migrate to an injury to supply new chondrocytes and osteoblasts for fracture healing. Traditionally, the efficacy of a cytokine to induce cell migration has only been conducted in vitro by performing a transwell or scratch assay. With advancements in intravital microscopy using multiphoton excitation, it was recently discovered that 1) P-SSCs express the migratory gene CCR5 and 2) treatment with the CCR5 ligand known as CCL5 improves fracture healing and the migration of P-SSCs in response to CCL5. These results have been captured in real-time. Described here is a protocol to visualize P-SSC migration from the calvarial suture skeletal stem cell (SSC) niche towards an injury after treatment with CCL5. The protocol details the construction of a mouse restraint and imaging mount, surgical preparation of the mouse calvaria, induction of a calvaria defect, and acquisition of time-lapse imaging.

This protocol describes the detection of CCL5-mediated periosteal skeletal stem cell migration in real-time using live animal intravital microscopy.

Periosteal skeletal stem cells (P-SSCs) are essential for lifelong bone maintenance and repair, making them an ideal focus for the development of ...

Ultrasound-guided Intracardiac Injection of Human Mesenchymal Stem Cells to Increase Homing to the Intestine for Use in Murine Models of Experimental Inflammatory Bowel Diseases

Crohn&#39;s disease (CD) is a common chronic inflammatory disease of the small and large intestines. Murine and human mesenchymal stem cells (MSCs) have immunosuppressive potential and have been shown to suppress inflammation in mouse models of intestinal inflammation, even though the route of administration can limit their homing and effectiveness 1,3,4,5. Local application of MSCs to colonic injury models has shown greater efficacy at ameliorating inflammation in the colon. However, there is paucity of data on techniques to enhance the localization of human bone marrow-derived MSCs (hMSCs) to the small intestine, the site of inflammation in the SAMP-1/YitFc (SAMP) model of experimental Crohn&#39;s disease. This work describes a novel technique for the ultrasound-guided intracardiac injection of hMSCs in SAMP mice, a well-characterized spontaneous model of chronic intestinal inflammation. Sex- and age-matched, inflammation-free AKR/J (AKR) mice were used as controls. To analyze the biodistribution and the localization, hMSCs were transduced with a lentivirus containing a triple reporter. The triple reporter consisted of firefly luciferase (fl), for bioluminescent imaging; monomeric red fluorescent protein (mrfp), for cell sorting; and truncated herpes simplex virus thymidine kinase (ttk), for positron emission tomography (PET) imaging. The results of this study show that 24 h after the intracardiac administration, hMSCs localize in the small intestine of SAMP mice as opposed to inflammation-free AKR mice. This novel, ultrasound-guided injection of hMSCs in the left ventricle of SAMP mice ensures a high success rate of cell delivery, allowing for the rapid recovery of mice with minimal morbidity and mortality. This technique could be a useful method for the enhanced localization of MSCs in other models of small-intestinal inflammation, such as TNF&#916;RE6. Future studies will determine if the increased localization of hMSCs by intra-arterial delivery can lead to increased therapeutic efficacy.

Murine studies in models of colonic inflammation have demonstrated that a small percentage (1 - 5%) of mesenchymal stem cells (MSC) injected intravenously or intraperitoneally home to the inflamed colon1,2. This study shows that ultrasound-guided intracardiac injections of MSCs result in increased localization to the intestine.

Crohn&#39;s disease (CD) is a common chronic inflammatory disease of the small and large intestines. Murine and human mesenchymal stem cells (MSCs) have ...

Tracking Superparamagnetic Iron Oxide-labeled Mesenchymal Stem Cells using MRI after Intranasal Delivery in a Traumatic Brain Injury Murine Model

Stem cell-based therapies for brain injuries, such as traumatic brain injury (TBI), are a promising approach for clinical trials. However, technical hurdles such as invasive cell delivery and tracking with low transplantation efficiency remain challenges in translational stem-based therapy. This article describes an emerging technique for stem cell labeling and tracking based on the labeling of the mesenchymal stem cells (MSCs) with superparamagnetic iron oxide (SPIO)&#160;nanoparticles, as well as intranasal delivery of the labeled MSCs. These nanoparticles are fluorescein isothiocyanate (FITC)-embedded and safe to label the MSCs, which are subsequently delivered to the brains of TBI-induced mice by the intranasal route. They are then tracked non-invasively in vivo by real-time magnetic resonance imaging (MRI). Important advantages of this technique that combines SPIO for cell labeling and intranasal delivery include (1) non-invasive, in vivo MSC tracking after delivery for long tracking periods, (2) the possibility of multiple dosing regimens due to the non-invasive route of MSC delivery, and (3) possible applications to humans, owing to the safety of SPIO, non-invasive nature of the cell-tracking method by MRI, and route of administration.

Presented here is a protocol for non-invasive mesenchymal stem cell (MSC) delivery and tracking in a mouse model of traumatic brain injury. Superparamagnetic iron oxide&#160;nanoparticles are employed as a magnetic resonance imaging (MRI) probe for MSC labeling and non-invasive in vivo tracking following intranasal delivery using real-time MRI.

Stem cell-based therapies for brain injuries, such as traumatic brain injury (TBI), are a promising approach for clinical trials. However, technical ...

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) maintain the turnover of all mature blood cell lineages. The coordination of the complex signals leading to specific HSC fates relies upon the interaction between HSCs and the intricate bone marrow microenvironment, which is still poorly understood[1-2].
We describe how by combining a newly developed specimen holder for stable animal positioning with multi-step confocal and two-photon in vivo imaging techniques, it is possible to obtain high-resolution 3D stacks containing HSPCs and their surrounding niches and to monitor them over time through multi-point time-lapse imaging. High definition imaging allows detecting ex vivo labeled hematopoietic stem and progenitor cells (HSPCs) residing within the bone marrow. Moreover, multi-point time-lapse 3D imaging, obtained with faster acquisition settings, provides accurate information about HSPC movement and the reciprocal interactions between HSPCs and stroma cells.
Tracking of HSPCs in relation to GFP positive osteoblastic cells is shown as an exemplary application of this method. This technique can be utilized to track any appropriately labeled hematopoietic or stromal cell of interest within the mouse calvarium bone marrow space.

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

The nature of the interactions between hematopoietic stem and progenitor cells (HSPCs) and bone marrow niches is poorly understood. Custom hardware modifications and a multi-step acquisition protocol allow the use of two-photon and confocal microscopy to image ex vivo labeled HSPCs homed within bone marrow areas, tracking interactions and movement.

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) ...

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

Summary

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Chapters in this video

Labeling hESCs and hMSCs with Iron Oxide Nanoparticles for Non-Invasive in vivo Tracking with MR Imaging

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

In Vivo Tracking of Human Adipose-derived Mesenchymal Stem Cells in a Rat Knee Osteoarthritis Model with Fluorescent Lipophilic Membrane Dye

Tracking Dynamics of Muscle Engraftment in Small Animals by In Vivo Fluorescent Imaging

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

Tracking Mouse Bone Marrow Monocytes In Vivo

Real-Time Imaging of CCL5-Induced Migration of Periosteal Skeletal Stem Cells in Mice

Ultrasound-guided Intracardiac Injection of Human Mesenchymal Stem Cells to Increase Homing to the Intestine for Use in Murine Models of Experimental Inflammatory Bowel Diseases

Tracking Superparamagnetic Iron Oxide-labeled Mesenchymal Stem Cells using MRI after Intranasal Delivery in a Traumatic Brain Injury Murine Model

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow