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

Abstract

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.

Introduction

Mesenchymal stem cells
Under disease conditions, MSCs secrete disease-specific therapeutic substances via paracrine actions¹ that have been reported to regulate immune responses, restore damaged tissues, and remove toxic substances². Therefore, MSC therapy is considered more effective than single-target therapy in treating multifactorial diseases such as Alzheimer's disease and sarcopenia^3,4,5,6. Additionally, in contrast to pharmaceuticals, MSCs have a homing effect, moving to the region of the damaged tissue by recognizing inflammatory cytokines or chemokines in the body^7,8. Unfortunately, only a subset of the cells reach the damaged area, and the viability of MSCs decreases during migration^9,10,11,12. Thus, to maximize the therapeutic efficacy of MSCs, it is necessary to deliver viable cells to the target site. Therefore, when administering MSCs, it is important to choose the proper route of administration, based on the nature of the target disease.

Injection route
There are numerous routes by which therapeutic agents are administered to patients. The most common methods are intravenous injection into the systemic circulation, oral administration, and subcutaneous or intramuscular injection. In neurodegenerative diseases, the main obstacle in delivering therapeutic agents to the brain is the blood-brain barrier (BBB). The BBB protects the brain from external pathogens by means of tight junctions between blood vessels and the brain parenchyma^13,14. However, the BBB also paradoxically prevents therapeutic agents from entering the brain parenchyma. Therefore, passage through the BBB is the main hurdle in the development of brain disease therapies^15,16. Intracerebral injection is performed to overcome this drawback by injecting target substances directly into the brain through surgical operation^17,18,19. However, the side effects of surgical interventions should be considered, especially as the needle damages neuronal cells during the procedure.

Intraspinal cavity administration
Intrathecal administration-the administration of drugs into the spinal canal or subarachnoid space-delivers drugs to the brain or neuraxis through the cerebrospinal fluid (CSF) and is a viable alternative to intracerebral injection. Intrathecal injections can be subdivided according to the injection site: lateral ventricle, cisterna magna, and spinal cavity. All three routes allow drugs or cells to disperse throughout the CSF into the brain and spinal cord. Drug delivery to the brain may be more efficient in the case of intracerebroventricular and intra-cisterna magna injections because the agent is injected close to the brain. However, intraspinal cavity injection has the advantages of not requiring general anesthesia or surgery for inserting an intraventricular reservoir, being generally safe²⁰, and can be repeatedly performed if necessary.

The purpose of this study was to validate intraspinal cavity administration as a means of delivering MSCs to both the brain and spinal cord. First, the intraspinal cavity was established in a rat model. Next, MSCs were labeled with a lipophilic tracer, DiD (DiIC18(5); 1,1-dioctadecyl-3,3,3,3- tetramethylindodicarbocyanine, 4-chlorobenzenesulfonate salt), to evaluate the efficiency of stem cell migration to the spinal cord and brain. Ex vivo optical imaging was performed to assess cell dispersion. This simple protocol can be performed without surgical intervention and may be used for the purpose of not only administering stem cells, but also pharmaceuticals, antibodies, contrast media, and other substances intended to be delivered to the spinal cord or brain.

Protocol

NOTE: This study was approved by the Institutional Animal Care and Use Committee (Approval number: 20170125001, Date: January 25, 2017) of the Samsung Biomedical Research Institute (SBRI) at Samsung Medical Center. As an accredited facility of the Association for Assessment and Accreditation of Laboratory Animal Care International, the SBRI acts in accordance with the guidelines set forth by the Institute of Laboratory Animal Resources.

1. Preparation of human Wharton's jelly-derived MSCs

1. Cultivation of human Wharton's jelly-derived mesenchymal stem cells (WJ-MSCs)
   1. Thaw a vial of human WJ-MSCs quickly in a 37 °C water bath. Transfer the WJ-MSCs to a 50 mL conical tube, and add growth medium at a volume at least 10 x that of the cells (v/v). Pipet up and down to suspend the cells.
   2. Centrifuge at 300 × g for 5 min. Carefully discard the supernatant, and resuspend the cells.
      NOTE: Be careful not to discard the cell pellet.
   3. Seed WJ-MSCs in T175 flasks at a density of 5,000-6,000 cells/cm². Incubate WJ-MSCs in a 37 °C CO₂ incubator. Change the growth medium every 72 h until WJ-MSCs reach 80-90% confluency.
      ​NOTE: Generally, it takes 3-4 days for the MSCs to reach 80-90% confluency.
2. Subcultivation of human WJ-MSCs
   1. Discard the growth medium, and wash the cells with 10 mL of phosphate-buffered saline (PBS). Remove the PBS, and add 5 mL of 0.25% trypsin-disodium ethylenediaminetetraacetic acid (EDTA) (see the Table of Materials). Incubate the cells at 37 °C in a CO₂ incubator for 3 min until the WJ-MSCs detach from the culture flask.
   2. Add 5 mL of growth medium containing 10% fetal bovine serum to neutralize the 0.25% trypsin-EDTA. Collect the cell mixture and transfer it to a 50 mL conical tube. Wash the cell culture flask with 10 mL of growth medium, and collect the cells in a 50 mL tube using a sterile serological pipet.
   3. Centrifuge the cell mixture at 300 × g for 5 min. Discard the supernatant, resuspend the cells in 10 mL of growth media, and count the number of WJ-MSCs.
      ​CAUTION: Be careful not to discard the cell pellet.
   4. Seed WJ-MSCs at a density of 4,000-6,000 cells/cm², depending on the experiment.
3. Labeling WJ-MSCs with DiD dye and the preparation of WJ-MSCs for intraspinal cavity injection
   NOTE: The DiD dye-labeling procedure was performed following manufacturer's instructions.
   1. Detach WJ-MSCs when they reach 80% confluency, using the procedure mentioned above. Suspend WJ-MSCs at a density of 1 × 10⁶/mL in phenol-red-free minimum essential medium (MEM) α without serum.
   2. Add 5 µL of DiD labeling solution per 1 mL of cell suspension; mix gently with pipetting.
   3. Incubate for 15 min at 37 °C; centrifuge the cell suspension at 300 × g for 5 min.
   4. Remove the supernatant, and resuspend the WJ-MSCs in phenol-red-free MEM α at a density of 1 × 10⁶/0.2 mL.

2. Intraspinal cavity injection of WJ-MSCs

1. Preparation for intraspinal cavity injection
   1. Anesthetize 6-week-old Sprague-Dawley rats with 5% isoflurane; then, maintain anesthesia with 2% isoflurane throughout the surgical procedure.
      NOTE: Optimize the anesthetic conditions before starting the experiment.
   2. Shave the surgical area using an electric shaver for small animals.
      ​NOTE: The electric shaver can be replaced with a manual razor and shaving gel.
   3. Disinfect the surgical area using povidone-iodine. Create a 3 cm incision in the skin with a surgical blade. Resect the remaining skin and muscle tissue using a surgical blade and scissors. Reveal the spinous processes at lumbar 2-3 (L2-3).
2. Injection of DiD-labeled WJ-MSCs via the intraspinal cavity
   1. Place the rat in a prone position. Flex the rat's spine appropriately to widen the distance between the adjacent spinous processes, using sufficient amounts of paper tissue or other materials that can aid in maintaining the appropriate position.
   2. Fill a 1 mL syringe with 0.2 mL of DiD-labeled WJ-MSCs. Place a 23 G syringe-needle combination vertically between the spinous processes of L2 and L3, and insert the needle until it touches the vertebral body.
   3. When the needle touches the vertebral body, retract it by approximately 0.5 cm, placing the tip of the needle in the spinal canal. Tilt the syringe, and place the tip of the needle such that it points toward the rostral direction. Inject WJ-MSCs into the spinal cavity over a 1 min period.
      ​NOTE: The speed of injection should be optimized in advance.
   4. After injection, completely remove the syringe from the spinal canal. Suture the incision, and then disinfect the surgical site using povidone-iodine.
3. Post-procedure treatment
   1. Stabilize and restrain the rat to prevent any movement, placing it upside-down at a 45° angle for 15 min, while it is still under anesthesia. After 15 min, discontinue anesthesia, and wait for the rat to rouse.

3. Evaluation of intraspinal cavity injection

1. Euthanasia of the rats and isolation of the brain and spinal cord at 0, 6, and 12 h post-injection
   1. Anesthetize the experimental animals with 5% isoflurane; maintain anesthesia with 2% isoflurane during PBS perfusion.
   2. Make an incision below the diaphragm using surgical scissors. Open the incision with forceps, and cut the rib cage rostrally to expose the heart.
   3. Make a small hole in the right atrium, and insert a butterfly needle into the left ventricle. Perfuse 100 mL of cold PBS into the left ventricle for 4-5 min, until the liver is cleared of blood.
   4. After perfusion, make a long incision on the backside from the head to the tail using a surgical blade along the longitudinal plane. Isolate the remaining brain and the whole spine using surgical scissors, forceps, and a bone cutter. Remove the remaining ribs, connected bones, and flesh.
2. Ex vivo DiD fluorescent optical imaging
   1. Place the isolated tissues in the chamber of the optical imaging device.
   2. Set the parameters as follows: emission, 700 nm; excitation, 605 nm; and exposure time, 2 seconds, as photons per second per centimeter squared per steradian (p/s/cm²/sr). Capture the optical images.
      NOTE: All images should be acquired with identical illumination settings (lamp voltage, filters, f/stop, field of view, and binning).
   3. Draw three rectangular regions of interest (ROIs) of equivalent size for the spinal cord and one circle ROI for the brain using the drawing tool. Measure the fluorescent intensities of the ROIs .
3. Extraction of genomic DNA (gDNA) from the spinal cord and brain tissue
   1. Remove the skull and spine carefully using surgical forceps, scissors, and a rongeur.
   2. Harvest the brain and spinal cord from the skull and spine. Cut the spinal cord into three pieces (cervical, thoracic, and lumbar).
      ​NOTE: The harvested tissues must be stored at -80 °C if they are not analyzed immediately.
   3. Grind the tissues using a pre-chilled mortar, pestle, and liquid nitrogen. Extract gDNA using commercial products, following the manufacturer's instructions.
4. Quantitative real-time polymerase chain reaction (qPCR)
   1. Quantify the amount of gDNA in each sample using a spectrophotometer.
   2. Perform qPCR using 10 ng of gDNA per sample and human Arthrobacter luteus (ALU) primers^12,21.
   3. Calculate the exact number of WJ-MSCs in the samples using the ΔΔC_T method²².

Representative Results

To evaluate the efficacy of intraspinal cavity injection of MSCs, DiD-labeled MSCs were used in the present study. Before injecting MSCs into the spinal cavity, the labeling efficacy was assessed in vitro using optical imaging and fluorescence microscopy (Figure 1). After staining the MSCs with the DiD labeling reagent using the procedure described in protocol section 3.1, optical images were taken of the culture plates on which DiD-labeled MSCs were seeded (Figure 1A). DiD-labeled MSCs (+DiD) are shown in red; naïve MSCs (-DiD), which were not labeled with DiD dye, did not show a positive signal. This result was confirmed using fluorescence microscopy (Figure 1B). MSCs were also stained with 4′,6-diamidino-2-phenylindole (DAPI) to visualize their shape. Naïve MSCs (-DiD) appeared blue, indicating staining with DAPI or nuclear staining, but did not show red color, which is associated with DiD staining. In contrast, DiD-labeled MSCs (+DiD) showed both blue and red colors, indicating that the DiD labeling method had been successful.

Figure 1: DiD labeling of MSCs in vitro. (A) The DiD-labeled MSCs were seeded in a 6-well culture plate, and optical imaging was performed. (B) Images were taken using fluorescence microscopy. The MSC nuclei are indicated by blue (DAPI), and the incorporated DiD is localized in the cytosol of +DiD cells (red). Scale bars = 500 µm. This figure has been modified from Kim et al.¹². Abbreviations: -DiD = naïve MSCs, +DiD = MSCs labeled with the DiD reagent; MSC = mesenchymal stem cell; DiD = DiIC18(5); 1,1-dioctadecyl-3,3,3,3- tetramethylindodicarbocyanine, 4-chlorobenzenesulfonate salt; DAPI = 4′,6-diamidino-2-phenylindole. Please click here to view a larger version of this figure.

Next, the method of intraspinal cavity injection was evaluated. To evaluate and optimize the intraspinal cavity injection procedure, trypan blue dye was used instead of DiD-labeled MSCs, as described in protocol section 2 (Figure 2). Trypan blue dye was injected, and the rat was euthanized immediately. The spinal cord tissue harvested from the rat was cut into three pieces transversely, and the brain was cut into coronal sections. It was found that the injected trypan blue dye had stained the spinal cord tissues (lumbar, thoracic, and cervical cords). Moreover, both the inferior and superior sides of the brain were stained blue. However, the trypan blue dye did not penetrate the lateral ventricle of the brain. These results indicated that this method of intraspinal cavity injection was successful.

Figure 2: Confirmation of intraspinal cavity injection in a rat model. The trypan blue dye was injected into the spinal cavity of a test rat. After injection, the rat was euthanized, and a necropsy was performed. The trypan blue dye injected via the spinal canal migrated to and stained both the spinal cord (lumbar, thoracic, and cervical) and the brain blue. Scale bars = 1 cm. This figure has been modified from Kim et al.¹². Please click here to view a larger version of this figure.

Using the protocol successfully optimized as described above, DiD-labeled MSCs were injected via the intraspinal cavity in rats (Figure 3). The rats were euthanized 0, 6, and 12 h post-injection, and ex vivo optical imaging was performed (Figure 3A). Compared with signals in the control (no-injection) animals, high and condensed positive signals were detected in the lumbar spinal cords of rats euthanized immediately post-injection (0 h). At 6 h post-injection, the positive signal was dispersed throughout the lumbar spinal cord. Finally, high-positive signals were observed in the lumbar and cervical cord regions and even in the brain at 12 h post-injection. The signal intensity of the optical images was quantified at each time-point using image analysis software (Figure 3B). A significant increase in signal intensity was identified in the lumbar spinal cord at 0 h post-injection and in the brain at 12 h post-injection.

Figure 3: Optical imaging analysis to evaluate the efficacy of intraspinal cavity injection. (A) After injecting the MSCs into the spinal cavity, the rats were euthanized at the following time points: 0, 6, and 12 h post-injection, after which ex vivo optical imaging was performed. The positive signal is shown as yellow-red color. (B) From the images, the signal intensities were quantified using software. Equivalent regions of interest were drawn for each experimental group. Data are presented as the mean ± standard error of mean (SEM). *p < 0.05. This figure has been modified from Kim et al.¹². Abbreviations: MSCs= mesenchymal stem cells; CTL = control. Please click here to view a larger version of this figure.

A previous study reported that the human ALU sequence can be used as a quantitative marker for measuring the number of human origin cells, such as neural stem cells and MSCs, in the context of xenograft transplantation^9,11,12. Following the guidelines from the previous study, qPCR analysis using a human-specific ALU primer was used in the present study to evaluate the in vivo distribution and migration of DiD-labeled MSCs (Figure 4). After amplification of the ALU sequence, the PCR product was separated on an agarose gel and visualized (Figure 4A). Compared with the control sample (the gDNA extracted from the brains of rats in the no-injection group), the ALU sequence was highly amplified in the 12-h sample (gDNA extracted from the brains of rats at 12 h post-injection). However, in both samples, mouse Gapdh was highly amplified, indicating that human origin cells, especially the human MSCs used in this study, were present only in the 12-h group sample. Next, the distribution of MSCs in the brain and other organs (heart, lung, liver, spleen, and kidney) was confirmed using the same method (Figure 4B). Similar to the results of optical imaging, a significant number of MSCs were detected only in the brain at 12 h post-injection. The other samples showed variance in the ratio of ALU to Gapdh, but all the signals from the samples, except the brain at 12 h post-injection, were all under the limit of detection, which implies that no significant amplification occurred.

Figure 4: Quantitative real-time polymerase chain reaction to confirm the distribution and migration of MSCs. (A) The amplified PCR products of brain genomic DNA extracted from the control and 12-h groups were visualized on an agarose gel. ALU is a human-specific primer, and mouse Gapdh primer was used for normalization. The amplified PCR products are shown as intense white bands. (B) qPCR analysis was performed, and the ratio of ALU to Gapdh in each sample was calculated. The red dashed line indicates the limit of detection in real-time PCR analysis. Data are presented as the mean ± standard error of mean (SEM). *p < 0.05. This figure has been modified from Kim et al.¹². Abbreviations: ALU = Arthrobacter luteus; PCR = polymerase chain reaction; qPCR = quantitative real-time PCR; MSCs = mesenchymal stem cells; Gapdh = glyceraldehyde 3-phosphate dehydrogenase. Please click here to view a larger version of this figure.

The results of the present study demonstrate that MSCs delivered via intraspinal cavity injection were distributed in the lumbar region at all time-points and migrated to the brain at 12 h post-injection.

Discussion

The optimal route of administration for treatment with MSCs should be chosen depending on the target disease, the patient's condition, and the type of drug to be delivered. In cell therapies, including MSC therapy, direct injection of stem cells into the brain or intrathecally via the CSF must be considered as the cells cannot pass through the BBB¹⁹. Intraspinal cavity injection is relatively non-invasive and does not cause neuronal damage in the brain, unlike intracerebroventricular injections, and is associated with a low risk of side effects²⁰. Accessing CSF via a lumbar puncture is a procedure that can be performed very easily at clinical sites. Therefore, it is not difficult to administer stem cells, drugs, contrast agents, or other substances to patients via intraspinal cavity injections^23,24,25. In contrast, intracerebroventricular injections, which require surgical intervention, are more complicated. However, for experimental animals, injection into the intraspinal cavity is more difficult than administering agents directly into the brain. This is because rodents, including mice and rats, are very small compared to humans²⁶.

In the case of intraspinal cavity administration, the needle must be inserted between the spinous processes. Compared with the gaps in humans, the gaps between the bones in a rat are very narrow, making access difficult. To overcome this, the smallest needle, ideally a 23 G needle, should be used. Although a 26 G needle can be used, such a thin needle can bend easily. The size of the needle can be adjusted based on the age of the experimental animal. Additionally, to facilitate intraspinal cavity administration in a rat model, it is necessary to flex the spine, widening the gap between spinous processes, making it easier for the needle to access the space between the spinous processes. The injection site and direction of the needle can also be adjusted as needed. The gaps between cervical spinous processes are relatively wider than those between lumbar spinous processes. However, if the injection is performed near the cervical or thoracic portions of the spinal cord, incorrect placement of the needle may cause serious spinal cord damage, including paralysis of the lower limbs of the experimental animal or patient.

Therefore, when selecting an upper spinal cord region, care must be taken to prevent damage to the spinal cord and nerves. In humans, the spinal cord ends at L1-2, where the cauda equina starts. The cauda equina is a bundle of lumbar and sacral nerves; therefore, the lumbar spine is a relatively safer location for injection than the cervical or thoracic spine. Therefore, only the lumbar spine, particularly the region under L2 where the cauda equina starts, is recommended for intraspinal cavity injection. Based on this consideration, the lumbar spine under L2 was selected for the present study to minimize spinal nerve damage. To track the stem cells delivered to the spinal cord and brain, a DiD reagent was used to label the WJ-MSCs, which were visible under a fluorescence microscope and in in vitro and ex vivo optical imaging experiments (Figure 1 and Figure 3). Unlabeled WJ-MSCs did not show any positive DiD fluorescence in vivo in the control group (no-injection). These results indicate that the lipophilic DiD dye can be used as a tracking agent for stem cell therapy. Currently, many different agents have been developed to track transplanted stem cells^27,28,29. These tracking reagents can be adjusted based on the equipment used for evaluation, such as magnetic resonance imaging, computed tomography, and optical imaging. A previous study reported the use of iron nanoparticles to track MSCs delivered via intracerebroventricular injection into the brain^9,29. Thus, various metallic nanoparticles and lipophilic agents, such as DiD, can be used for in vivo and ex vivo stem cell tracking.

To evaluate the migration and distribution of WJ-MSCs delivered via intraspinal cavity injection, qPCR analysis was performed with an ALU primer. The primary objective of the present study was to optimize the method of intraspinal cavity administration and evaluate its efficacy. Therefore, analysis methods were selected for tracking and quantifying the overall distribution and migration of WJ-MSCs throughout the brain and spinal cord at various time-points. For this reason, optical imaging was performed with the brain and spinal cord still connected. The whole brain or spinal cord (cervical, thoracic, and lumbar) was ground up, and the exact numbers of WJ-MSCs in those tissues were calculated via qPCR analysis with an ALU primer. The human-specific primer ALU has been reported to have high sensitivity and specificity for detecting human origin cells among rodent cells²¹. Additionally, the Ministry of Food and Drug Safety in Korea recommends using human ALU primers to evaluate the biodistribution of stem cells as part of the preclinical data collected for investigational new drug approval. To identify the exact location of WJ-MSCs migrating toward the brain and spinal cord at different time-points, immunohistochemical staining (IHC) should be performed. However, IHC was not performed here, which is a limitation of this study.

The euthanasia time-points should also be appropriately selected. The speed of stem cell migration toward the brain and the distribution pattern throughout the neuraxis depend on the delivered substances and the state of the experimental animals or patients. It is important to determine the physiological and chemical characteristics of injected materials. Various factors such as size, mass, lipophilicity, and half-life can affect the time required to migrate to the brain and disperse throughout the entire central nervous system (CNS). Therefore, an appropriate euthanasia time-point must be established in accordance with the properties of the substance being administered. Moreover, the physical state of the test subject is also important. In both patients and diseased animal models, there are many substances, such as inflammatory cytokines and target epitopes, that can attract therapeutic agents (stem cells, immune cells, and antibody drugs) toward lesion sites. Therefore, it will take less time for WJ-MSCs to reach the brain if a CNS disease model is used. In the present study using a wild-type rat model, three different time-points (0, 6, and 12 h) were selected. The experimental animals in the 0 h group were euthanized immediately after stem cell injection, and WJ-MSCs were detected only in the lumbar spinal cord around the injection site. In contrast, WJ-MSCs were observed in the brains and cervical spinal cords of rats in the 12 h group, indicating that it took a minimum of 12 h for the WJ-MSCs to migrate to the brain and cervical cord in a wild-type rat model. Theoretically, additional WJ-MSCs can migrate to the brain as time progresses, but this was not evaluated or proven in the present study.

The intraspinal cavity administration of MSCs has the disadvantage of low efficiency of delivery to the brain compared with that of intracerebroventricular or intraparenchymal administration¹². The first reason for this is the distance from the administration site to the brain, and the second reason pertains to CSF flow. As CSF is produced in the choroid plexus located in the lateral ventricle of the brain, CSF flows from the lateral ventricle to the spinal cord³⁰. Therefore, in this study, the rats were placed in an upside-down position at a 45° angle for 15 min to aid the migration of MSCs to the brain. A greater angle or longer wait time may promote increased migration of MSCs to the brain. Additionally, the volume, speed, and dosage of the injection can be modified to achieve more efficient delivery to the brain and spinal cord. The present study introduces a process by which WJ-MSCs can be administered via the intraspinal cavity at L2-3 and evaluated the migration and distribution patterns of the stem cells at 0, 6, and 12 h post-injection in a rat model. Although only a small number of WJ-MSCs delivered via the intraspinal cavity route moved to the rat brain in the present study, this number can be increased by adjusting several variables. The preclinical data provided in the present study can be considered as scientific basis for the clinical use of intraspinal cavity injection of stem cell therapy, immunotherapy, and other curative substances.

Materials

Name	Company	Catalog Number	Comments
0.25% Trypsin-EDTA	Gibco-invitrogen	25200114	Cell culture
Fetal bovine serum	biowest	S1520	Culture medium supplement
gentamicin	Gibco-invitrogen	15710-072	Culture medium supplement
Gentra Puregene Tissue Kit	QIAGEN	158689	gDNA isolation
MEM, no glutamine, no phenol red	Gibco	51200038	WJ-MSC fomulation for injection
Miminum Essential Medium alpha	Gibco-invitrogen	12571063	WJ-MSC culture medium
Power SYBR Green PCR Master Mix	Applied Biosystems	4368577	quantitative real time PCR reagent
QuantStudio 6 Flex Real-Time PCR System	Thermo fisher	4485694	quantitative real time PCR
trypan blue	Gibco	15250061	Injection
Vybrant DiD Cell-Labeling Solution	invitrogen	V22887	Stem cell labeling solution
Xenogen IVIS Spectrum system	Perkin Elmer	124262	Optical imaging device