The current study was supported by Shanghai Innovation Funding (1402H294300) sponsored by the Science and Technology Commission of Shanghai Municipality (CN) to Dr. Wen Wang. We would like to thank Dr. Guangdong Zhou (National Tissue Engineering Center of China) for his technical assistance and scientific advice for this manuscript. We would also like to thank Mr. Huitang Xia (Shanghai Ninth People&#8217;s Hospital) for his help in animal welfare.

Procedures involving animal subjects were approved by the local Institutional Animal Care and Ethics Committee, with an effort to minimize animal suffering. The following protocol was approved by the Institutional Animal Care and Use Committee (IACUC) at Shanghai Ninth People&#8217;s Hospital affiliated to Shanghai JiaoTong University School of Medicine with the protocol number [2017]063.
1. Establishment of a Surgically-Induced Rat Knee Osteoarthritis Model
<ol>
	<li>For this surgical procedure, use 8 - 12 week old male Sprague Dawley (SD) rats with a body weight between 250 and 300 g.</li>
	<li>Anesthetize the animals with 40 mg/kg tiletamine and 40 mg/kg zolazepam via intramuscular injection.</li>
	<li>Place the animal in a left lateral position on a heated stage. Shave the right knee thoroughly with a razor. Disinfect the surgical area with 10% providone-iodine solution followed by 70% ethanol, repeat two more times and then paint with 10% providone-iodine solution and cover the non-surgical area with a surgical pad.</li>
	<li>Using a surgical scalpel, make a 2 cm incision laterally along the patellar tendon to expose the jointcapsules.</li>
	<li>Use a surgical scalpel to transect the medial collateral ligament. Reflect the meniscus toward the femur.</li>
	<li>Drip sterile 0.9% sodium chloride solution on the surface of articular cartilage during the operation to prevent the cartilage from drying out.</li>
	<li>Grab the medial meniscus with forceps and cut through the meniscus at its narrowest point from the tibial attachment with a scalpel (Figure 1A). Avoid injuring the cartilage.</li>
	<li>Close the joint capsule with a 4 - 0 absorbable suture.</li>
	<li>Monitor the surgical rats until they regain consciousness.</li>
	<li>Inject 20 mg/kg gentamicin to prevent infection and 0.05 mg/kg buprenorphine through an intramuscular route to relieve pain after surgery.</li>
	<li>Maintain the rats in a temperature-controlled animal facility under a 12 h light/dark cycle for 3 - 8 weeks to onset KOA. Animals continue to receive pain medication, including but not limited to Buprenorphine (0.05 mg/kg, i.m.) and Gentamicin (20 mg/kg, i.m.), if pain symptoms persist.</li>
</ol>
2. Histology Assessment of Rat KOA Model
<ol>
	<li>Sacrifice the rats with excessive CO2 inhalation (Add a fill rate of about 30% of the chamber volume per minute with carbon dioxide to the existing air in the chamber to make the animal unconsciousness within 2-3 min. Maintain CO2 flow for a minimum of 1 min after respiration ceases.) at indicated time points (representative results are shown at 8 weeks of KOA induction ranging from 3 to 8 weeks) after the surgery.&#160;</li>
	<li>Dissect the skin and muscle around the knee joints and cut off the knee joints with a scalpel.</li>
	<li>Fix the knee joints in 4% paraformaldehyde (PFA) for 3 days at room temperature. Afterwards, decalcify the samples in 20% ethylenediaminetetraacetic acid (EDTA) for about 4 weeks at 4 &#176;C, and change the EDTA every 3 days. 
	Caution: Paraformaldehyde is toxic, wear appropriate personal protective equipment (PPE), such as gloves and eyeglasses.</li>
	<li>Dehydrate the samples using an automatic tissue processor. Set the menu to start a flush cycle as follows: 70% ethanol for 1 h; then 80% ethanol and 95% ethanol for 1 h respectively; followed by 2 cycles of 100% ethanol for 1 h; subsequently, xylene twice for 1 h each time and paraffin 3 times at 58 &#176;C for 1 h each time. 
	Caution: Xylene is toxic, so wear appropriate PPE, such as gloves and eyeglasses. 
	NOTE: Optionally, use Coplin Jars for manual dehydration with the same set-up as the automatic tissue processor.</li>
	<li>Place the medial aspect of the intact joint face down and embed it into the paraffin block with tissue embedded into the center. 
	NOTE: Optionally, use forceps to embed the samples into the paraffin block manually.</li>
	<li>Start to cut 5 &#181;m sagittal sections from the medial margin of the joint for 30 &#181;m intervals with 2 serial sections by rotary microtome.</li>
	<li>Mount sections on glass slides and deparaffinize the slides in xylene. Between the 2 serial sections, stain one slide with Hematoxylin &#38; Eosin (H&#38;E) staining: 0.1% Hematoxylin for 15 min, 1% Acetic Acid Solution for 10 s and 0.5% Eosin for 10 min. And stain the other slide with Safranin O/Fast Green staining: 0.1% Hematoxylin for 15 min, 0.02% Fast Green for 3 min, 1% Acetic Acid Solution for 10 s and 0.1% Safranin O for 3 min. 
	Caution: Xylene is toxic, wear appropriate PPE, such as gloves, eyeglasses.</li>
	<li>Score the stained slides using Osteoarthritis Research Society International (OARSI) Histopathology Assessment System in rats6.</li>
</ol>
3. Labelling of Human Mesenchymal Stem Cells with the Fluorescent Dye DiD
<ol>
	<li>Prepare DiD cell-labeling solution in 100% ethanol at a concentration of 1 mM. Protect the cell-labeling solution from light and store it at room temperature up for 6 months.</li>
	<li>Grow haMSCs with standard cell culture procedures in Dulbecco&#39;s modified Eagle&#39;s medium (DMEM) supplemented with 1% penicillin and streptomycin and 10% fetal calf serum (FCS) in a 10 cm petri dish.</li>
	<li>Detach haMSCs with 2 mL 0.25% trypsin with 1 mM EDTA prior to around 80% confluence.</li>
	<li>Neutralize trypsin with 4 mL culture medium and spin the cells at 800 x g for 3 min at room temperature.</li>
	<li>Suspend the cells at a density of 1 x 106 cells/mL in 5 mL serum-free culture medium.</li>
	<li>Label the cells by adding 50 &#181;L DiD cell-labeling solution in 5 mL serum-free culture medium at 37 &#176;C for 50 min.</li>
	<li>Spin the labeled cells at 800 x g for 3 min.</li>
	<li>Wash the cells two more times with 5 mL phosphate buffer saline (PBS) at 800 x g for 3 min each.</li>
	<li>Check the labelled haMSCs using a fluorescent microscope with a Cy5 filter set.</li>
	<li>Count the cells with trypan blue staining and obtain 2.5 x 106 viable cells in 100 &#181;L PBS for injection.</li>
</ol>
4. In Vivo Fluorescent Imaging to Track haMSCs
<ol>
	<li>Eight weeks after the surgery (Step 1), anesthetize the KOA rats by intramuscular injection of 25 mg/kg tiletamine and 25 mg/kg zolazepam.</li>
	<li>Shave the right knee thoroughly with a razor to expose the joint area. Disinfect the joint area with 70% ethanol.</li>
	<li>Using a 26 G syringe needle, inject 2.5 x 106 labeled cells suspended in 100 &#181;L PBS at the center of the triangle area formed by the medial side of the patellar ligament, the medial femoral condyle, and the medial tibial condyle (Figure 1B).</li>
	<li>Open the imaging software and initialize the in vivo bioluminescence imaging system.</li>
	<li>Position rats in the supine position in the central stage of the imaging system and close the door of the chamber.</li>
	<li>Check the &#34;fluorescent&#34; checkbox and select fluorescent filter sets of 640 nm for excitation, and 680 nm for emission (Figure 2A).</li>
	<li>Select field of view to D. Set pixel binning to medium (8), F/Stop to 2 and exposure time to auto. Maintain the optimal exposure time consistent to obtain comparable results across the entire study (Figure 2A).</li>
	<li>Remove the animal from the stage and monitor for recovery from anesthesia. 
	NOTE: Place animal on an heating pad covered with 2-3 layers of toweling.</li>
	<li>Choose the units of radiant efficiency for the measurement of fluorescence (Figure 2B). Select regions of interest (ROI) for analysis and quantify fluorescent signals from each image (Figure 2C).</li>
	<li>Repeat the procedure of in vivo imaging for each rat sequentially to study haMSCs longitudinal retention in the joint. 
	NOTE:&#160;We suggest imaging the animals&#39; fluorescent signals by in vivo imaging system once every two days for the first week after injection and once every week until the signal cannot be detected.</li>
</ol>

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Meng Li, Ming Hao and Wen Wang are current employees and stock option holders of Cellular Biomedicine Group (Nasdaq: CBMG). The other authors declare no conflict of interest.

Safety standards and biodistribution studies of stem cell therapy are urgently needed before we can bring regenerative stem cell treatment for KOA from the bench to the bedside. However, the pathological environment of disease plays an important role in the persistence and biodistribution of transplanted haMSCs10. Recently, our group demonstrated that intra-articular injection of haMSCs persisted longer in a pathological KOA environment than did injections under normal conditions...

Knee osteoarthritis (KOA) is a degenerative disorder resulting from articular cartilage loss and progressive inflammation, which has become a major chronic disease in the elderly around the world1. However, current therapies using anti-inflammatory drugs, physical supplements, and surgical procedures may only provide temporary relief for symptomatic pain2.
Human adipose-derived mesenchymal stem cells (haMSCs) have become a promising regenerative remedy for knee osteoarthritis, owing to their multipotent differentiation potential for cartilage regeneration and immunomodulatory properties3,4. Compared with pharmacological routes to investigate mechanisms of action in vivo, tracking live haMSCs in small KOA animal models is currently instructive to establish the rationale for and feasibility of haMSC therapy prior to clinical application. For preclinical testing, medial meniscectomy (MM) destabilizes the mechanical load of the joint to induce KOA in rats, which provides a relatively feasible model with consistent reproducibility5. The onset of KOA induced by MM is earlier than anterior cruciate ligament transection alone or combined with partial medial meniscectomy6. Therefore, the long-term interactions between injected haMSCs with the pathological microenvironment of KOA are often assessed in rats induced by MM7,8.
Though the therapeutic efficacy of haMSCs has been extensively reported, relevant knowledge on the in vivo persistence of implanted haMSCs via intra-articular (IA) injection is scarce9,10. Hence, various cellular labeling methods have been developed, including immunohistology11, luciferase12, green fluorescent protein13 transfection, iron oxide labeling for magnetic resonance imaging (MRI)14, and numerous fluorescent cell dyes8,15,16. Compared with labor-intensive histology analyses, in vivo non-invasive imaging employs optical devices to detect the real-time distribution and dynamics of cells labeled with fluorescent signals10,17. For functional live cell imaging, cytocompatible fluorescent labeling is a sophisticated radioactive-free tracking technique to reveal cellular activities after stem cell transplantation18. Moreover, multicolor fluorescent lipophilic dyes possess advantages over amino-reactive hydrophilic dyes or fluorescent proteins, including their improved cell permeability and enhanced fluorescence quantum yields19.
Thus, the protocols included here utilize a red laser to excite cells labeled with lipophilic carbocyanines DiD (DilC18(5)), which is a far-red fluorescent Dil (dialkylcarbocyanines) analog20. The red-shifted excitation and emission spectra of DiD avoids autofluorescent interference and allows deep-tissue imaging over a long period of time in live animals8. This method of tracking cells in vivo labeled with DiD is valid for monitoring transplanted stem cells, such as haMSCs, in animal models, which is essential to understand and improve current stem cell regenerative therapy.

<table><tbody><tr><td>Matrx VMR animal anesthesia system</td><td>Midmark</td><td>VIP3000</td><td></td></tr><tr><td>4-0 suture</td><td>Shanghai Jinhuan</td><td>KC439</td><td></td></tr><tr><td>Razor</td><td>Pritech</td><td>LD-9987</td><td></td></tr><tr><td>Gentamicin</td><td>Zhejiang Jindakang Animal Health Product Co., Ltd.</td><td>None</td><td></td></tr><tr><td>0.9% Sodium chloride solution</td><td>Hunan Kelun Pharmaceutical Co., Ltd.</td><td>H43020455</td><td></td></tr><tr><td>Penicillin</td><td>Shanghai Kangfu chemical pharmaceutical Co., Ltd.</td><td>None</td><td></td></tr><tr><td>Buprenorphine</td><td>Tianjin Pharmaceutical Research Institute Pharmaceutical Co., Ltd.</td><td>None</td><td></td></tr><tr><td>Paraformaldehyde</td><td>Sigma-Aldrich</td><td>16005</td><td>Dilute to final concentration of 10% in PBS</td></tr><tr><td>EDTA</td><td>Sigma-Aldrich</td><td>E9884</td><td>Dilute to final concentration of 20% in PBS</td></tr><tr><td>0.1% Hematoxylin Solution, Mayer&amp;rsquo;s</td><td>Sigma-Aldrich</td><td>MHS16</td><td></td></tr><tr><td>0.5% Eosin Y solution, alcoholic</td><td>Sigma-Aldrich</td><td>HT110116</td><td></td></tr><tr><td>Safranin O</td><td>Sigma-Aldrich</td><td>S8884</td><td></td></tr><tr><td>Fast Green</td><td>Sigma-Aldrich</td><td>F7258</td><td></td></tr><tr><td>Shandon Excelsior ESTM Tissue Processor</td><td>Thermo Fisher</td><td>A78400006</td><td></td></tr><tr><td>Shandon Histocentre&amp;trade; 3 Tissue Embedding Center</td><td>Thermo Fisher</td><td>B64100010</td><td></td></tr><tr><td>Fully Automated Rotary Microtome</td><td>Leica</td><td>RM2255</td><td></td></tr><tr><td>DiD</td><td>Molecular Probes, Life&lt;br/&gt; Technologies</td><td>V-22887</td><td></td></tr><tr><td>D-MEM High Glucose</td><td>Sigma-Aldrich</td><td>D5648</td><td></td></tr><tr><td>PBS</td><td>GIBCO, Life Technologies</td><td>14190-144</td><td></td></tr><tr><td>0.25% Trypsin-EDTA</td><td>Invitrogen</td><td>25200-114</td><td></td></tr><tr><td>10 cm Petri Dish</td><td>Corning</td><td>V118877</td><td></td></tr><tr><td>Centrifuge</td><td>Beckman</td><td>Optima MAX-TL</td><td></td></tr><tr><td>Fluorescent microscope</td><td>Olympus</td><td>BX53</td><td></td></tr><tr><td>0.4% Trypan Blue solution</td><td>Sigma-Aldrich</td><td>93595</td><td></td></tr><tr><td>Titetamme</td><td>Virbac (Zoletil 50)</td><td>1000000188</td><td></td></tr><tr><td>Zolazepam</td><td>Virbac (Zoletil 50)</td><td>1000000188</td><td></td></tr><tr><td>Sterile hyposermic syringe for single use 26G</td><td>Shanghai Misawa Medical Industry</td><td>None</td><td></td></tr><tr><td>IVIS Spectrum In Vivo Imaging System</td><td>PerkinElmer</td><td>124262</td><td></td></tr><tr><td>Living Imaging 4.0 software</td><td>PerkinElmer</td><td>None</td><td></td></tr></tbody></table>

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 order to induce KOA model, MM was performed in the right knee joint of SD rats (Figure 3). Eight weeks after surgery, rats were sacrificed and the serial sections of knee joints were evaluated with both H&#38;E and Safranin O/Fast Green staining (Figure 4). For H&#38;E staining, the surface of the articular cartilage exhibited rougher borders in the surgery knees than the normal joint without surgery. For Safranin-O/Fast green...

Watch this Scientific Journal Video about In Vivo Tracking of Human Adipose-derived Mesenchymal Stem Cells in a Rat Knee Osteoarthritis Model with Fluorescent Lipophilic Membrane Dye at JoVE.com

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 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 ...

in-vivo-tracking-human-adipose-derived-mesenchymal-stem-cells-rat

Research

JoVE Journal

Developmental Biology

8.9K Views.  Cellular Biomedicine Group, China. Dit protocol beschrijft een efficiënte manier om te controleren de cel persistentie en ook menselijke adipeus afkomstige mesenchymale stamcellen (haMSCs) door far-red fluorescentie labeling in een rat knie artrose (KOA) model via intra-articulaire (IA) injectie.

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

Labeling Stem Cells with Fluorescent Dyes for non-invasive Detection with Optical Imaging

Optical imaging (OI) is an easy, fast and inexpensive tool for in vivo monitoring of new stem cell based therapies. The technique is based on ex vivo labeling of stem cells with a fluorescent dye, subsequent intravenous injection of the labeled cells and visualization of their accumulation in specific target organs or pathologies. The presented technique demonstrates how we label human mesenchymal stem cells (hMSC) by simple incubation with the lipophilic fluorescent dye DiD (C67H103CIN2O3S) and how we label human embryonic stem cells (hESC) with the FDA approved fluorescent dye Indocyanine Green (ICG). The uptake mechanism is via adherence and diffusion of the lypophilic dye across the phospholipid cell membrane bilayer. The labeling efficiency is usually improved if the cells are incubated with the dye in serum-free media as opposed to incubation in serum-containing media. Furthermore, the addition of the transfection agent Protamine Sulfate significantly improves contrast agent uptake.

This video shows techniques for labeling of human embryonic stem cells and mesenchymal stem cells with fluorescent dyes. This technique can be used for an in vivo tracking of transplanted stem cells with optical imaging and for histopathological correlations with fluorescence microscopy.

Optical imaging (OI) is an easy, fast and inexpensive tool for in vivo monitoring of new stem cell based therapies. The technique is based on ex vivo ...

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 ...

In Vivo Optical Imaging of Brain Tumors and Arthritis Using Fluorescent SapC-DOPS Nanovesicles

We describe a multi-angle rotational optical imaging (MAROI) system for in vivo monitoring of physiopathological processes labeled with a fluorescent marker. Mouse models (brain tumor and arthritis) were used to evaluate the usefulness of this method. Saposin C (SapC)-dioleoylphosphatidylserine (DOPS) nanovesicles tagged with CellVue Maroon (CVM) fluorophore were administered intravenously. Animals were then placed in the rotational holder (MARS) of the in vivo imaging system. Images were acquired in 10&#176; steps over 380&#176;. A rectangular&#160;region of interest (ROI) was placed across the full image width at the model disease site. Within the ROI, and for every image, mean fluorescence intensity was computed after background subtraction. In the mouse models studied, the labeled nanovesicles were taken up in both the orthotopic and transgenic brain tumors, and in the arthritic sites (toes and ankles). Curve analysis of the multi angle image ROIs determined the angle with the highest signal. Thus, the optimal angle for imaging each disease site was characterized. The MAROI method applied to imaging of fluorescent compounds is a noninvasive, economical, and precise tool for in vivo quantitative analysis of the disease states in the described mouse models.

In Vivo Optical Imaging of Brain Tumors and Arthritis Using Fluorescent SapC-DOPS Nanovesicles

We describe a multi-angle rotational optical imaging (MAROI) system for in vivo quantitation of a fluorescent marker delivered by saposin C (SapC)-dioleoylphosphatidylserine (DOPS) nanovesicles. Employing mouse models of cancer and arthritis, we demonstrate how the MAROI signal curve analysis can be used for the precise mapping and biological characterization of disease processes.

We describe a multi-angle rotational optical imaging (MAROI) system for in vivo monitoring of physiopathological processes labeled with a fluorescent ...

Medicine

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

Femur Window Chamber Model for In Vivo Cell Tracking in the Murine Bone Marrow

Bone marrow is a complex organ that contains various hematopoietic and non-hematopoietic cells. These cells are involved in many biological processes, including hematopoiesis, immune regulation and tumor regulation. Commonly used methods for understanding cellular actions in the bone marrow, such as histology and blood counts, provide static information rather than capturing the dynamic action of multiple cellular components in vivo. To complement the standard methods, a window chamber (WC)-based model was developed to enable serial in vivo imaging of cells and structures in the murine bone marrow. This protocol describes a surgical procedure for installing the WC in the femur, in order to facilitate long-term optical access to the femoral bone marrow. In particular, to demonstrate its experimental utility, this WC approach was used to image and track neutrophils within the vascular network of the femur, thereby providing a novel method to visualize and quantify immune cell trafficking and regulation in the bone marrow. This method can be applied to study various biological processes in the murine bone marrow, such as hematopoiesis, stem cell transplantation, and immune responses in pathological conditions, including cancer.

Femur Window Chamber Model for In Vivo Cell Tracking in the Murine Bone Marrow

The protocol describes a novel murine femur window chamber model that can be used to track movement of cells in the femoral bone marrow in vivo. Intravital multiphoton fluorescence microscopy is used to image three components of the femoral bone marrow (vasculature, collagen matrix, and neutrophils) over time.

Bone marrow is a complex organ that contains various hematopoietic and non-hematopoietic cells. These cells are involved in many biological processes, ...

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.

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.

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

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 ...

Intratracheal Instillation of Stem Cells in Term Neonatal Rats

Prolonged exposure to high concentrations of oxygen leads to inflammation and acute lung injury, which is similar to human bronchopulmonary dysplasia (BPD). In premature infants, BPD is a major complication despite early use of surfactant therapy, optimal ventilation strategies, and noninvasive positive pressure ventilation. Because pulmonary inflammation plays a crucial role in the pathogenesis of BPD, corticosteroid use is one potential treatment to prevent it. Nevertheless, systemic corticosteroid treatment is not usually recommended for preterm infants due to long-term adverse effects. Preclinical studies and human phase I clinical trials demonstrated that use of mesenchymal stromal cells (MSCs) in hyperoxia-induced lung injuries and in preterm infants is safe and feasible. Intratracheal and intravenous MSC transplantation has been shown to protect against neonatal hyperoxic lung injury. Therefore, intratracheal administration of stem cells and combined surfactant and glucocorticoid treatment has emerged as a new strategy to treat newborns with respiratory disorders. The developmental stage of rat lungs at birth is equivalent to that in human lungs at 26&#8722;28 week of gestation. Hence, newborn rats are appropriate for studying intratracheal administration to preterm infants with respiratory distress to evaluate its efficacy. This intratracheal instillation technique is a clinically viable option for delivery of stem cells and drugs into the lungs.

Described is a protocol for performing intratracheal transplantation of mesenchymal stromal cells (MSCs) through intratracheal injection in term neonatal rats. This technique is a clinically viable option for delivery of stem cells and drugs into neonatal rat lungs to evaluate their efficacy.

Prolonged exposure to high concentrations of oxygen leads to inflammation and acute lung injury, which is similar to human bronchopulmonary dysplasia ...

Visualization and Quantification of Mesenchymal Cell Adipogenic Differentiation Potential with a Lineage Specific Marker

Several dyes are currently available for use in detecting differentiation of mesenchymal cells into adipocytes. Dyes, such as Oil Red O, are cheap, easy to use and widely utilized by laboratories analyzing the adipogenic potential of mesenchymal cells. However, they are not specific to changes in gene transcription. We have developed a gene-specific differentiation assay to analyze when a mesenchymal cell has switched its fate to an adipogenic lineage. Immuno-labelling against fatty acid binding protein-4 (FABP4), a lineage-specific marker of adipogenic differentiation, enabled visualization and quantification of differentiated cells. The ability to quantify adipogenic differentiation potential of mesenchymal cells in a 96 well microplate format has promising implications for a number of applications. Hundreds of clinical trials involve the use of adult mesenchymal stromal cells and it is currently difficult to correlate therapeutic outcomes within and especially between such clinical trials. This simple high-throughput FABP4 assay provides a quantitative assay for assessing the differentiation potential of patient-derived cells and is a robust tool for comparing different isolation and expansion methods. This is particularly important given the increasing recognition of the heterogeneity of the cells being administered to patients in mesenchymal cell products. The assay also has potential utility in high throughput drug screening, particularly in obesity and pre-diabetes research.

Traditional methods of assessing adipogenic differentiation are cheap and easy to use, but are not specific to changes in gene expression. We have developed an assay to quantify mesenchymal cell differentiation into mature adipocytes using a lineage specific marker. This assay has diverse applications across basic research and clinical medicine.

Several dyes are currently available for use in detecting differentiation of mesenchymal cells into adipocytes. Dyes, such as Oil Red O, are cheap, easy ...

Intraspinal Cavity Injection of Human Mesenchymal Stem Cells and Tracking their Migration into the Rat Brain

Mesenchymal stem cells (MSCs) have been studied for the treatment of various diseases. In neurodegenerative diseases involving defects in both the brain and the spinal cord, the route of administration is very important, because MSCs must migrate to both the brain and the spinal cord. This paper describes a method for administering MSCs into the spinal canal (intraspinal cavity injection) that can target the brain and spinal cord in a rat model. One million MSCs were injected into the spinal canals of rats at the level of lumbar vertebrae 2-3. After administration, the rats were euthanized at 0, 6, and 12 h post-injection. Optical imaging and quantitative real-time polymerase chain reaction (qPCR) were used to track the injected MSCs. The results of the present study demonstrated that MSCs administered via the spinal cavity could be detected subsequently in both the brain and spinal cord at 12 h. Intraspinal cavity injection has the advantage of not requiring general anesthesia and has few side effects. However, the drawback of the low migration rate of MSCs to the brain must be overcome.

Several routes of administration can be used to deliver mesenchymal stem cells (MSCs) to the brain. In the present study, MSCs were delivered throughout the neuraxis and brain via intra-spinal cavity injection. MSCs were injected into the spinal cavities of rats, and stem cell migration was tracked and quantified.

Mesenchymal stem cells (MSCs) have been studied for the treatment of various diseases. In neurodegenerative diseases involving defects in both the brain ...

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

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