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Method Article

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

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DOI:

10.3791/55367

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September 1st, 2017

 ,  ,  ,  ,  ,  ,  , 
1Division of Gastroenterology and Liver Disease, University Hospitals, Digestive Health Research Institute, Case Western Reserve University, 2Case Cardiovascular Research Institute, School of Medicine, Case Western Reserve University, 3Department of Medicine, Harrington Discovery Institute, Harrington Heart and Vascular Institute, University Hospitals Case Medical Center, 4Department of Radiology, University Hospitals Case Medical Center, 5Department of Biology - Skeletal Research Center, Case Western Reserve University

In This Article

Summary

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.

Abstract

Crohn'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'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 TNFΔRE6. Future studies will determine if the increased localization of hMSCs by intra-arterial delivery can lead to increased therapeutic efficacy.

Introduction

Crohn's disease (CD) is a common chronic inflammatory disease of the small and large intestines and is thought to result from an inappropriate response of the host immune system to intestinal microbes7,8. Recent studies have shown that both murine and human mesenchymal stem cells (MSCs) can suppress inflammation in mouse models of intestinal inflammation1,3,4,5. There are multiple ongoing clinical trials that use human MSCs derived from bone marrow or adipose tissue to treat patients with inflammatory bowel disease (IBD), which includes CD9. Two routes for MSC therapy have been used in these clinical trials: one involves the systemic infusion (i.e., intravenously) of MSCs for luminal IBD (including CD), and the other involves the localized application/injection of stem cells in the fistula tract of patients with perianal CD. In a recent meta-analysis of MSC therapy for IBD, systemic (i.e., intravenous) MSC therapy for luminal IBD (including CD) was efficacious in up to 40% (95% CI: 7 - 79%) of patients, whereas the efficacy was much higher, at 61% (95% CI: 36 - 85%), when the MSCs were injected locally into the diseased CD fistula9. A recent phase III multicenter randomized placebo controlled trial of allogeneic adipose stem cells injected directly into the perianal fistula of CD patients showed statistically significant clinical and radiological evidence of perianal fistulae healing, corroborating the findings of the meta-analysis10. The reasons for the low efficacy of MSC therapy given intravenously for luminal CD has been inadequately investigated, but one reason may be the inadequate homing of MSCs to the site of inflammation. Murine studies in models of colonic inflammation have demonstrated that only a small percentage of MSCs (1 - 5%) injected intravenously reach the inflamed colon; the remaining MSCs are filtered by the lungs (first-pass effect)1,2,5,11,12. Multiple murine research studies have therefore used the intraperitoneal route (i.p.) for MSC administration in animal models of colitis4. However, they have also demonstrated that only a small fraction of cells reach and engraft the colon and that the efficacy is related to the secretion of soluble paracrine factors, like tumor necrosis factor-inducible gene 6 protein (TSG-6)2. The MSC mechanism of immunosuppression and healing involves a multipronged approach that involves paracrine; cell proximity-independent factors, like TSG-6; and cell proximity-dependent factors, like programmed death-ligand 1 (PD-L1); or Jagged 1. Therefore, MSC localization to the site of inflammation may result in increased efficacy9,13. In fact, a recent study showed that MSCs directly implanted at the site of colonic injury resulted in healing by secreting angiogenesis-promoting vascular endothelial growth factor (VEGF). On the other hand, a minimal healing effect was noted after intravenous injection5. To increase their localization to the site of inflammation (i.e., the small intestine in SAMP mice), this ultrasound-guided intracardiac injection technique for MSC administration in the left ventricle was developed. Image-guided injection ensures an accurate injection, which leads to a higher rate of success and to decreased morbidity and mortality rates. Moreover, the injection of MSCs into the left ventricle delivers them to arterial circulation, where they can reach the inflamed small intestine before becoming trapped in the lungs.

In this study, human bone marrow-derived MSCs (hMSCs) were used for injection in the SAMP-1/YitFc (SAMP) murine model of CD14. SAMP is a well-characterized spontaneous murine model of chronic inflammation that develops small-intestinal inflammation with nearly 100% penetrance14. The inflammation develops in response to microflora in the absence of any chemical, immunological, or genetic manipulation and closely resembles human CD11. Sex- and age-matched inflammation-free AKR/J (AKR) mice, the parental control mice of SAMP, were used in this study.

The hMSCs were isolated and expanded in the laboratory from bone marrow (BM) samples obtained from normal, unidentified donors after informed consent using validated and previously published protocols15,16. After isolation and expansion, the MSC ability in osteogenic, adipogenic, and chondrogenic differentiation was evaluated in the laboratory by multiple assays15. The osteogenic functional assay was performed by implanting ceramic cubes of hydroxyapatite/tricalcium phosphate matrix containing hMSCs subcutaneously in immunocompromised CB17-Prkdc SCID mice17. The cube assay demonstrates osteogenesis and chondrogenesis potential and is considered the ultimate test for evaluating individual MSC preparations17. To visualize hMSCs in vivo after injection, lentivirus was used to transduce hMSCs with triple reporter gene construct that consists of firefly luciferase (fl), monomeric red fluorescent protein (mRFP), and herpes simplex viral thymidine kinase (ttk), driven by a modified myeloproliferative sarcoma virus (mnd) promoter18. The firefly luciferase in the triple reporter is an enzyme that coverts injected luciferin to oxyluciferin in hMSCs and produces photons/white light. This is detected by the sensitive charge-coupled device (CCD) camera (bioluminescence) in an in vivo optical imaging system, enabling the visualization of live hMSCs in mice. Bioluminescence imaging (BLI) is a sensitive technique that can be used serially for tracking cells and for ex vivo analysis. The use of a strong mnd promoter drives the continuous expression of the triple fusion reporter gene construct and allows for the imaging of injected hMSCs for more than 16 weeks19. The hMSCs are difficult to transduce and have a low transduction efficiency. Using an optimized protocol, the hMSCs transduction efficiency was improved and the transgene expression was enhanced18. Using mRFP expression (one of the triple reporter genes) on flow cytometry, the ability to transduce hMSCs with a high efficiency of up to 83% was demonstrated. Differentiation assays and in vivo cube assays demonstrated the ability of the transduced hMSCs to differentiate into chondrocytes, adipocytes, and osteocytes17.

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Protocol

All mice experiments and procedures in the study were approved by Case Western Reserve University's Institutional Animal Care and Use Committee. The procedures were conducted in Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC) accredited facility. The BM was aspirated under a University Hospitals Institutional Review Board-approved protocol at the stem cell core facility at Case Western Reserve University after informed consent.

1. Culture and Transduction of Human Bone Marrow-derived Mesenchymal Stem Cells (hMSCs)

NOTE: The isolation of hMSCs is described in detail in previous publications15,20.

  1. Culture isolated hMSCs in Dulbecco's Modified Eagle Medium-low glucose (DMEM-LG) medium supplemented with 10% fetal bovine serum (FBS). Culture the cells at 37 °C, 95% humidity, and 5% CO2 in T175 flasks with 25 mL of growth medium. Replace the medium every 3 - 4 days. On day 14, perform the primary passage of hMSCs.
    1. When the cells reach 80% confluence, completely remove the old medium and gently wash the cells with 5 mL of sterile phosphate-buffered saline (PBS; for 75-cm tissue culture flasks).
    2. Remove the PBS from the flask and add 4 mL of 0.25% trypsin-ethylenediaminetetraacetic acid (EDTA). Place the flask back in the incubator at 37 °C for 5 - 10 min. Keep the time of exposure as brief as possible.
    3. When the majority of cells have become well-rounded or have detached from the culture dish, stop the reaction by adding a volume of bovine calf serum equal to half the volume of trypsin.
    4. Transfer the cell suspension to an appropriate-size centrifuge tube. Centrifuge the cells at 400 x g for 5 min and then carefully remove the supernatant.
    5. Resuspend the cells in 5 mL of medium (DMEM-LG + 10% FBS) and count the cells with a hemocytometer.
      NOTE: At this point, the cells can be subcultured or transduced with triple reporter.
  2. Perform the transduction of hMSCs with triple reporter.
    1. Seed 1.5 x 106 cells in one T175 flask. Culture the cells in 25 mL of growth medium at 37 °C, 95%, humidity and 5% CO2. Wait 24 h for the cells to attach.
    2. Make a virus cocktail in 15-mL tubes, as described in reference 18.
      1. Thaw lentivirus at 37 °C in a water bath. Add 160 µL of a 5 mg/mL proteamine sulfate solution in Dulbecco's Modified Eagle Medium (DMEM) to 8 mL of culture medium (DMEM-LG + 10% FBS) for a final concentration of 100 µg/mL. Vortex for 3 s. Add a virus multiplicity of infection (MOI) of 5 (1st time thawed).
    3. Remove the medium from the cell culture flask (step 1.2.1) and add the virus cocktail to the cells. Incubate for 10 h at 37 °C, 95% humidity, and 5% CO2.
  3. Add 4 mL of 100 µg/mL proteamine sulfate in 4 mL of DMEM-LG + 10% FBS and incubate for an additional 14 h.
  4. Stop the transduction after 24 h by replacing with 25 mL of medium (DMEM-LG + 10% FBS).
  5. Confirm transduction efficiency by evaluating mRFP expression by flow cytometry18.
    NOTE: Here, hMSCs that were transduced with over 65% efficiency were used.
  6. Expand the cells by replacing with 25 mL of fresh medium (DMEM-LG + 10% FBS) twice a week. Culture the cells at 37 °C, 95%, humidity and 5% CO2. Passage cells after they reach 80% confluence.
  7. Once the desired number of cells is reached, perform trypsinization per steps 1.1.2 - 1.1.4.
  8. Count the cells and suspend them in sterile PBS (1x) in the final concentration of 1 x 106 cells/75 µL. Keep the cells on ice for transport to the ultrasound machine. For experiments, use hMSCs from passage numbers 2 - 5.

2. Ultrasound-guided Intracardiac Injection of hMSCs into the Left Ventricle

NOTE: Use SAMP1/YitFc with established small-intestinal inflammation and age- and gender-matched AKR/J mice for the experiments. Maintain the SAMP and AKR mice under specific pathogen-free conditions, feed them standard laboratory chow, and keep them on 12-h light/dark cycles. Here, the ultrasound-guided cardiac injections were performed in a dedicated pathogen-free cardiac ultrasound room located in the CWRU animal research facility that was sanitized with a disinfectant and rinsed with 70% alcohol. Laboratory research personnel must wear personal protective equipment, including gowns, face masks with eye shields, and sterile gloves during the intracardiac injections. The body heat of the mouse must be maintained for the duration of the procedure.

  1. Set up the ultrasound imaging system before anesthetizing the mouse. For this protocol, use the ultrasound machine designed for mouse echocardiogram.
    1. Turn on the power and initialize the transducer (30 MHz) to set up the new study.
  2. Anesthetize the mouse in an induction chamber using 3 - 4% isoflurane in 100% oxygen at a rate of 0.2 - 0.5 L/min. Maintain the animal for the whole procedure with 2% isoflurane in 100% oxygen via a nose cone. Confirm proper anesthetization prior to performing imaging by pinching the toe, rolling the mouse, and observing the absence of movement.
    NOTE: Ophthalmic ointment should be applied to the eyes following the induction of anesthesia to prevent corneal drying. Transfer the anesthetized mice to the imaging machines (i.e., bioluminescence imaging system and ultrasound), where they will receive a maintenance dose of anesthetic isoflurane. Determine the anesthesia start time and the depth of anesthesia by observing a lack of reaction to pinches on the tail and foot pad every 10 min for the entire duration of anesthesia.
  3. Set the temperature of the imaging table and ultrasound gel to 37 °C.
  4. Completely remove the fur over the thorax area of the anesthetized mouse using hair removal cream.
  5. Place the mouse on the imaging table in supine position and secure both the upper and lower limbs with adhesive tape to avoid body movement during the procedure. Use an electrocardiogram monitor during the procedure for all mice.
  6. Clean the skin of the thorax area using a 10% povidone/iodine swab followed by a 70% ethanol swab. Apply a thick layer of gel to the thorax area of the mouse.
  7. Mount the transducer in the holder and adjust its position until the left ventricle is clearly visible on the field of view.
  8. Load 150 µL of transduced hMSC cell suspension (2 x 106 hMSCs in 150 µL of sterile PBS) into a 1-mL syringe with a 28-G needle and secure the syringe to the appropriate holder. To visualize pulsating blood, keep a small air column in the syringe. To avoid clumping, suspend the cells before the injection.
  9. Advance the syringe towards the mouse thorax, adjust the needle trajectory using the ultrasound guidance, and enter the left ventricle.
  10. Following the guidance of the ultrasound imaging, penetrate the syringe needle through the intercostal space and into the left ventricle of the mouse.
    NOTE: The syringe needle tip should be clearly visible in the left ventricle on the ultrasonographic image. Proper placement should be further confirmed by fresh arterial blood outflow into the syringe. These are signs of a successful insertion.
  11. Inject the hMSC cell suspension very slowly over 2 min, applying gentle pressure.
  12. Following the injection of suspended cells, gently withdraw the needle, clean off the ultrasound gel, and release the mouse from the tape restraints.
  13. Place the animal in a new, clean cage with a preheated pad.
    NOTE: After the completion of the procedure, a warm environment should be provided to the mice until recovery. Over-heating should be avoided, since peripheral vasodilation compromises the recovery. The mice should be monitored closely and kept isolated from other mice until their full recovery, indicated by their ability to maintain sternal recumbency.
  14. Monitor the animal until they achieve complete recovery from the anesthesia and daily until the end of the experiment.

3. Bioluminescence (BLI) Imaging of hMSCs

  1. Image the mice 24 h after the intracardiac injection in the bioluminescence imaging system. Start the in vivo imaging software. Initialize the imaging system and open a new study.
    1. In the control panel, set the imaging parameters by clicking "sequence setup." In the imaging mode, select "luminescence" and "photograph." Set the exposure times from 0.5 s to 10 min. Set the binning to "medium" and the F/stop to "1." Set the excitation filter to "block" and the emission filter to "open." Set the field of view to "C" for two mice. Add the sequence setup to the image wizard by clicking on the acquisition control panel.
  2. Switch on the oxygen pump and set the isoflurane to 2.5% in both the induction chamber and the nose cone inside the machine.
  3. Inject the mouse i.p. with 300 µL of D-Luciferin (12.5 mg/mL).
  4. Place the mouse in the anesthesia induction chamber.
    1. After 2 - 3 min, transfer the mouse into the in vivo imaging chamber, with its head in the nose cone on the anesthesia manifold. Apply optical ointment to protect the eyes during imaging. To image multiple mice, use black light baffle to prevent the reflection of light onto adjacent mice.
  5. Click the "acquire" button to start the image acquisition 10 min after D-Luciferin injection.
  6. Repeat steps 3.3 - 3.5 to image more mice.
  7. After the in vivo image acquisition, euthanize the mice by CO2 inhalation followed by cervical dislocation, to confirm death. Perform the ex vivo analysis.
    1. If the ex vivo analysis is performed after 20 min, repeat the luciferin injection (step 3.3) before sacrificing the mice; the BLI signal may decrease 20 min after the luciferin injection.
  8. Dissect the mice to remove the whole gastrointestinal tract (i.e., from the stomach to the rectum), mesenteric lymph node (MLN), lungs, spleen, and liver. Place them in petri dishes using forceps and scissors2,4.
  9. Place the petri dish containing the explanted organs in the imaging chamber and acquire BLI images per steps 3.1 - 3.6.
  10. After the image acquisition, transfer the explanted organs to specimen molds containing optimal cutting temperature (OCT) compound. Cryofreeze the molds at -80 °C using dry ice. Perform immunohistochemistry using anti-luciferase antibody on frozen sections to confirm the presence of hMSCs16.
  11. For the quantification of light intensity, use the region of interest (ROI) tool in the tool palette16,19. Select the ROI frame and move it to region of interest on the image. Click "ROI measurement" and save the data in SEQ file format. Export the data as a .txt file and use statistical software to perform basic statistical tests for quantification16.

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Results

Figure 1 shows that hMSCs can be transduced with the triple reporter at a high efficiency, preserving their stem cell properties. Transduced hMSCs can be visualized in real time by BLI (Figure 1C, Figure 3). This novel, ultrasound-guided injection of hMSCs into the left ventricle of SAMP mice ensures a very high success rate of injection, allowing for the rapid recovery of the mice with minimal morbi...

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Discussion

This study describes a novel technique for the ultrasound-guided intracardiac injection of hMSCs in a small-intestinal mouse model of experimental CD. This technique has very high rate of survival and success, as the trajectory of the needle can be adjusted based on real-time, high-resolution images of the mouse left ventricle, provided by the ultrasound. The advantage of delivery to the left ventricle is that hMSCs are then distributed intra-arterially and bypass venous circulation, thus avoiding the aggregation of cell...

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Disclosures

The authors have nothing to disclose.

Acknowledgements

Maneesh Dave is supported by Department of Defense grant PR141774 and the Crohn's and Colitis Foundation of America Career Development Award. The laboratory of Fabio Cominelli is supported by NIH grants DK042191 (F.C.), DK055812 (F.C.), DK091222 (F.C.), and DK07948 (F.C.). The laboratory of Arnold Caplan is supported in part by the L. David and E. Virginia Baldwin Foundation. The funding agencies had no role in the study analysis or writing of the manuscript. Its contents are solely the responsibility of the authors.

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Materials

List of materials used in this article
NameCompanyCatalog NumberComments
DMEM-LG  Gibco31600-091
0.25% trypsin-EDTAGibco25-200-072
Proteamine SulfateSigma AldrichP4020-1G
D-LuciferinGoldbioluck-500
Fetal Bobine SerumGibco26140-079
Antibiotic-AntimycoticGibco15240-062
PBSHyCloneSH30256.01
175 cm tissue cutlure flasksCorning431080
75 cm tissue cutlure flasksCorning430720
Centrifuge tubesCrysalgen23-2265
Tissue-Plus O.C.T. compoundFisher HealthCare4585
Cryomold StandardTissue-Tek 4557
Lenti-Pac HIV Expression Packaging SystemGeneCopoeiaHPK-LvTR-20 
Povidone Ionidine swabsMedlineMDS093901
Hair removal creamNairN/A
Isoflurane Piramal Helathcare NDC 66794 013 25
Forceps Fine Science Tools11200-33
Dissection scissors (Wagner)Fine Science ToolsWagner 14068-12
Puralube vet ointmentPuralubeNDC 17033-211-38
Aquasonic 100 ultrasound transmission gelParker laboratories01 08
Petri dishFisher ScientificFB0875711A
SAMP miceCleveland Digestive Disease Research Core Centre
AKR miceCleveland Digestive Disease Research Core Centre
Vevo 770 imaging system Visual Sonics, Toronto, Canada
 IVIS spectrum series system PerkinElmer, Waltham, MA
Living image software CaliperLifeSciences, PerkinElmer, Waltham, MA
Triple reporterKindly provided by Dr. Zheng- Hong Lee, CWRU, (citation 19)

References

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