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.

All mice experiments and procedures in the study were approved by Case Western Reserve University&#39;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 H...

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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...

Crohn&#39;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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			<td height="21" style="height:21px;width:295px;">DMEM-LG&#160;&#160;</td>
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			<td style="width:188px;">31600-091</td>
			<td style="width:285px;"></td>
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		<tr height="21">
			<td height="21" style="height:21px;">0.25% trypsin-EDTA</td>
			<td>Gibco</td>
			<td>25-200-072</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Proteamine Sulfate</td>
			<td>Sigma Aldrich</td>
			<td>P4020-1G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">D-Luciferin</td>
			<td>Goldbio</td>
			<td>luck-500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fetal Bobine Serum</td>
			<td>Gibco</td>
			<td>26140-079</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Antibiotic-Antimycotic</td>
			<td>Gibco</td>
			<td>15240-062</td>
			<td></td>
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			<td height="21" style="height:21px;">PBS</td>
			<td>HyClone</td>
			<td>SH30256.01</td>
			<td></td>
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			<td height="21" style="height:21px;">175 cm tissue cutlure flasks</td>
			<td>Corning</td>
			<td>431080</td>
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		<tr height="21">
			<td height="21" style="height:21px;">75 cm tissue cutlure flasks</td>
			<td>Corning</td>
			<td>430720</td>
			<td></td>
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			<td height="21" style="height:21px;">Centrifuge tubes</td>
			<td>Crysalgen</td>
			<td>23-2265</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;">Tissue-Plus O.C.T. compound</td>
			<td>Fisher HealthCare</td>
			<td>4585</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cryomold Standard</td>
			<td>Tissue-Tek&#160;</td>
			<td>4557</td>
			<td></td>
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			<td height="21" style="height:21px;">Lenti-Pac HIV Expression Packaging System</td>
			<td>GeneCopoeia</td>
			<td>HPK-LvTR-20&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Povidone Ionidine swabs</td>
			<td>Medline</td>
			<td>MDS093901</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;">Hair removal cream</td>
			<td style="width:333px;">Nair</td>
			<td style="width:188px;">N/A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;">Isoflurane&#160;</td>
			<td style="width:333px;">Piramal Helathcare&#160;</td>
			<td style="width:188px;">NDC 66794 013 25</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;">Forceps&#160;</td>
			<td style="width:333px;">Fine Science Tools</td>
			<td style="width:188px;">11200-33</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;">Dissection scissors (Wagner)</td>
			<td style="width:333px;">Fine Science Tools</td>
			<td style="width:188px;">Wagner 14068-12</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;">Puralube vet ointment</td>
			<td style="width:333px;">Puralube</td>
			<td style="width:188px;">NDC 17033-211-38</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;">Aquasonic 100 ultrasound transmission gel</td>
			<td style="width:333px;">Parker laboratories</td>
			<td style="width:188px;">01 08</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Petri dish</td>
			<td>Fisher Scientific</td>
			<td>FB0875711A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">SAMP mice</td>
			<td>Cleveland Digestive Disease Research Core Centre</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">AKR mice</td>
			<td>Cleveland Digestive Disease Research Core Centre</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Vevo 770 imaging system</td>
			<td>&#160;Visual Sonics, Toronto, Canada</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">&#160;IVIS spectrum series system&#160;</td>
			<td>PerkinElmer, Waltham, MA</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Living image software&#160;</td>
			<td>CaliperLifeSciences, PerkinElmer, Waltham, MA</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Triple reporter</td>
			<td colspan="2">Kindly provided by Dr. Zheng- Hong Lee, CWRU, (citation 19)</td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

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...

Watch this Scientific Journal Video about Ultrasound-guided Intracardiac Injection of Human Mesenchymal Stem Cells to Increase Homing to the Intestine for Use in Murine Models of Experimental Inflamma

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

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 ...

The overall goal of this procedure is to use ultrasound-guided intracardiac human bone marrow derived mesenchymal stem cell or HMSC injection in a mouse model of experimental crohn's disease, to observe HMSC homing to the intestine. This method can help answer the key questions in the inflammatory bowel disease field, about the need for increased localisations of HMSC to small intestine during inflammation. The main advantage of this technique is that it facilitates a high HMSC delivery success rate with minimal mouse mobility and mortality.Generally, individuals new to this method can have trouble because multiple variables, they can affect the procedure outcome. Requiring a lot of trainee understanding of human mesenchymal stem cell biology. Demonstrating the procedures will be Paola Menghini, a post doc from our laboratory.In a specific pathogen free facility, set up the mouse echocardiogram ultrasound imaging system protocol and initialize the 30 megahertz transducer. Next, confirm the appropriate level of sedation by toe pinch and apply ointment to the animal's eyes. Use depilatory cream to remove the hair over the thorax and place the animal on the 37 degree celsius imaging table in the supine position.Tape the limbs to prevent movement during the procedure and disinfect the exposed skin of the thorax with 10%povidone iodine and 70%ethanol. Apply a thick layer of 37 degree celsius ultrasound gel to the skin and mount the transducer in the holder, adjusting the position of the transducer until the left ventricle is clearly visible in the field of view. Next, gently re-suspend 2 x 10 to the sixth transduced human mesenchymal stem cells in 150 microliters of sterile PBS to generate a homogenous cell suspension and load the cells into a one milliliter syringe equipped with a 28 gauge needle.Inject less than 150 microliters of cell suspension per mouse to avoid a volume overload and death. Secure the syringe in a syringe holder. Then, advance the syringe towards the mouse thorax using the ultrasound guidance to adjust the needle trajectory as necessary and penetrate the needle tip through the intercostal space into the left ventricle.Adequate visualization of the needle on the ultrasonographic image is critical for ensuring a proper trajectory and insertion of the needle into the left ventricle, a successful injection and the avoidance of cardiac dampening and death. The tip should be clearly visible within the left ventricle and proper placement can be further confirmed by fresh arterial blood outflow into the syringe. Apply a gentle pressure to the plunger to slowly inject the HMSC suspension over a period of two minutes.When all the cells have been injected, gently withdraw the needle and place the animal in a clean cage on a preheated pad with monitoring until full recovery. 24 hours after the injection, initialize the bioluminescence imaging system and open a new study in the in-vivo imaging software. To set the imaging parameters in the control panel, click the sequence set up.In the imaging mode, select luminescent and photograph. Set the exposure times from 0.5 seconds to 10 minutes, the bidding to medium and the f/stop to one. Set the excitation filter to block, the emission filter to open and the field of view to see for two mice.Then click on the acquisition control panel to add the sequence set up to the image wizard. Next, inject the first mouse with 300 microliters of d-luciferin. Apply ointment to the animals'eyes and transfer the mouse to the in-vivo imaging chamber.10 minutes after the d-luciferin injection, click acquire, to begin the image acquisition. After all the mice have been imaged, harvest the whole gastrointestinal tract, mesenteric lymph nodes, lungs, spleen and liver from each animal. Then image the organs as just demonstrated, for whole animal bioluminescence imaging.HMSC can be transduced with the triple reporter at a high efficiency preserving the stem cell properties and allowing their visualization in real time by bioluminescence imaging. Ex-vivo analysis of the intracardiac delivered stem cells, confirms that this root of administration facilitates HMSC homing to the inflamed small intestines of the experimental crohn's disease mice. HMSC is injected into control, inflammation free animals however, do not accumulate in the small intestine.Once mastered, this technique can be completed in less than 15 minutes per mouse if it is performed properly. While attempting this procedure, it's important to remember to use the ultrasound image to adjust the needle trajectory. A poor technique and an incorrect trajectory can lead to cardiac tamponade from hemorrhagic pericardial effusion and death.Following this procedure, other methods like histology and 3D studio microscopy can be performed to answer additional questions such as, does an increased homing of HMSC to the small intestine augment that therapeutic efficacy. This technique will allow researchers in the field of experimental IBD to explore all their mouse models like TNF trajectory, can be injected with HMSCs to enhance homing to the small intestine. After watching this video, you should have a good understanding of how to perform ultrasound guided intracardiac injection of HMSC in mice and of how to use bioluminescent imaging to analyze a bad distribution and homing.Don't forget that working with human MSCs can be hazardous and that precautions such as, using gloves and a face mask with a shield, should always be taken while performing this procedure.

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11.9K visualizações.  Case Western Reserve University. The overall goal of this procedure is to use ultrasound-guided intracardiac human bone marrow derived mesenchymal stem cell or HMSC injection in a mouse model of experimental crohn's disease, to observe HMSC homing to the intestine. This method can help answer the key questions in the inflammatory bowel disease field, about the need for increased localisations of HMSC to small intestine during inflammation. The main advantage of this technique is that it facilitates a high HMSC delivery succe...

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