Name: 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
Uploaded: 2017-09-01T12:30:00
Description: 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

Maneesh Dave understøttes af Department of Defense grant PR141774 og Crohns og Colitis Foundation of America karriere udvikling Award. Laboratoriet af Fabio Cominelli understøttes af NIH tilskud DK042191 (FC), DK055812 (FC), DK091222 (FC) og DK07948 (FC). Laboratoriet for Arnold Caplan er delvist understøttet af L. David og E. Virginia Baldwin Foundation. De finansielle organer spillede ingen rolle i undersøgelse analyse eller skrivning af manuskriptet. Dens indhold er udelukkende ansvarlig for forfatterne.

alle mus eksperimenter og procedurer i undersøgelsen blev godkendt ved Case Western Reserve University &#39; s institutionelle Animal Care og brug udvalget. Procedurerne, der blev gennemført i tilknytning til vurdering og akkreditering af laboratorier dyrs pleje (AAALAC) godkendt facilitet. BM var indsugning under et universitet sygehuse institutionelle Review Board-godkendt protokollen på stamcelle core facilitet ved Case Western Reserve University efter informeret samtykke.
 1. kultur og t...

<ol>
	<li>Duijvestein, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pretreatment+with+interferon-gamma+enhances+the+therapeutic+activity+of+mesenchymal+stromal+cells+in+animal+models+of+colitis.">Pretreatment with interferon-gamma enhances the therapeutic activity of mesenchymal stromal cells in animal models of colitis.</a> Stem Cells. 29 (10), 1549-1558 (2011).</li><li>Sala, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mesenchymal+Stem+Cells+Reduce+Colitis+in+Mice+via+Release+of+TSG6,+Independently+of+Their+Localization+to+the+Intestine.">Mesenchymal Stem Cells Reduce Colitis in Mice via Release of TSG6, Independently of Their Localization to the Intestine.</a> Gastroenterology. 149 (1), 163-176 (2015).</li><li>Gonzalez, M. A., Gonzalez-Rey, E., Rico, L., Buscher, D., Delgado, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adipose-derived+mesenchymal+stem+cells+alleviate+experimental+colitis+by+inhibiting+inflammatory+and+autoimmune+responses.">Adipose-derived mesenchymal stem cells alleviate experimental colitis by inhibiting inflammatory and autoimmune responses.</a> Gastroenterology. 136 (3), 978-989 (2009).</li><li>Dave, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stem+cells+for+murine+interstitial+cells+of+cajal+suppress+cellular+immunity+and+colitis+via+prostaglandin+e2+secretion.">Stem cells for murine interstitial cells of cajal suppress cellular immunity and colitis via prostaglandin e2 secretion.</a> Gastroenterology. 148 (5), 978-990 (2015).</li><li>Manieri, N. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mucosally+transplanted+mesenchymal+stem+cells+stimulate+intestinal+healing+by+promoting+angiogenesis.">Mucosally transplanted mesenchymal stem cells stimulate intestinal healing by promoting angiogenesis.</a> J. Clin. Invest. 125 (9), 3606-3618 (2015).</li><li>Kontoyiannis, D., Pasparakis, M., Pizarro, T. T., Cominelli, F., Kollias, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Impaired+on/off+regulation+of+TNF+biosynthesis+in+mice+lacking+TNF+AU-rich+elements:+implications+for+joint+and+gut-associated+immunopathologies.">Impaired on/off regulation of TNF biosynthesis in mice lacking TNF AU-rich elements: implications for joint and gut-associated immunopathologies.</a> Immunity. 10 (3), 387-398 (1999).</li><li>Abraham, C., Cho, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inflammatory+bowel+disease.">Inflammatory bowel disease.</a> N. Engl. J. Med. 361 (21), 2066-2078 (2009).</li><li>Dave, M., Papadakis, K. A., Faubion, W. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunology+of+inflammatory+bowel+disease+and+molecular+targets+for+biologics.">Immunology of inflammatory bowel disease and molecular targets for biologics.</a> Gastroenterol. Clin. North Am. 43 (3), 405-424 (2014).</li><li>Dave, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mesenchymal+Stem+Cell+Therapy+for+Inflammatory+Bowel+Disease:+A+Systematic+Review+and+Meta-analysis.">Mesenchymal Stem Cell Therapy for Inflammatory Bowel Disease: A Systematic Review and Meta-analysis.</a> Inflamm. Bowel Dis. 21 (11), 2696-2707 (2015).</li><li>Panes, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expanded+allogeneic+adipose-derived+mesenchymal+stem+cells+(Cx601)+for+complex+perianal+fistulas+in+Crohn's+disease:+a+phase+3+randomised,+double-blind+controlled+trial.">Expanded allogeneic adipose-derived mesenchymal stem cells (Cx601) for complex perianal fistulas in Crohn's disease: a phase 3 randomised, double-blind controlled trial.</a> Lancet. 388 (10051), (2016).</li><li>Lee, R. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intravenous+hMSCs+improve+myocardial+infarction+in+mice+because+cells+embolized+in+lung+are+activated+to+secrete+the+anti-inflammatory+protein+TSG-6.">Intravenous hMSCs improve myocardial infarction in mice because cells embolized in lung are activated to secrete the anti-inflammatory protein TSG-6.</a> Cell Stem Cell. 5 (1), 54-63 (2009).</li><li>Gao, J., Dennis, J. E., Muzic, R. F., Lundberg, M., Caplan, A. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+dynamic+in+vivo+distribution+of+bone+marrow-derived+mesenchymal+stem+cells+after+infusion.">The dynamic in vivo distribution of bone marrow-derived mesenchymal stem cells after infusion.</a> Cells Tissues Organs. 169 (1), 12-20 (2001).</li><li>English, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+of+mesenchymal+stromal+cell+immunomodulation.">Mechanisms of mesenchymal stromal cell immunomodulation.</a> Immunol Cell Biol. 91 (1), 19-26 (2013).</li><li>Pizarro, T. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SAMP1/YitFc+mouse+strain:+a+spontaneous+model+of+Crohn's+disease-like+ileitis.">SAMP1/YitFc mouse strain: a spontaneous model of Crohn's disease-like ileitis.</a> Inflamm. Bowel Dis. 17 (12), 2566-2584 (2011).</li><li>Lennon, D. P., Caplan, A. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+human+marrow-derived+mesenchymal+stem+cells.">Isolation of human marrow-derived mesenchymal stem cells.</a> Exp. Hematol. 34 (11), 1604-1605 (2006).</li><li>Lin, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Serial+transplantation+and+long-term+engraftment+of+intra-arterially+delivered+clonally+derived+mesenchymal+stem+cells+to+injured+bone+marrow.">Serial transplantation and long-term engraftment of intra-arterially delivered clonally derived mesenchymal stem cells to injured bone marrow.</a> Mol. Ther. 22 (1), 160-168 (2014).</li><li>Dennis, J. E., Caplan, A. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Porous+ceramic+vehicles+for+rat-marrow-derived+(Rattus+norvegicus)+osteogenic+cell+delivery:+effects+of+pre-treatment+with+fibronectin+or+laminin.">Porous ceramic vehicles for rat-marrow-derived (Rattus norvegicus) osteogenic cell delivery: effects of pre-treatment with fibronectin or laminin.</a> J. Oral Implantol. 19 (2), 106-115 (1993).</li><li>Lin, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+lentiviral+transduction+of+human+mesenchymal+stem+cells+that+preserves+proliferation+and+differentiation+capabilities.">Efficient lentiviral transduction of human mesenchymal stem cells that preserves proliferation and differentiation capabilities.</a> Stem Cells Transl. Med. 1 (12), 886-897 (2012).</li><li>Love, Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+of+mesenchymal+stem+cell+transplant+by+bioluminescence+and+PET.">Imaging of mesenchymal stem cell transplant by bioluminescence and PET.</a> PET. J. Nucl. Med. 48 (12), 2011-2020 (2007).</li><li>Haynesworth, S. E., Goshima, J., Goldberg, V. M., Caplan, A. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+cells+with+osteogenic+potential+from+human+marrow.">Characterization of cells with osteogenic potential from human marrow.</a> Bone. 13 (1), 81-88 (1992).</li><li>Ishizaka, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intra-arterial+cell+transplantation+provides+timing-dependent+cell+distribution+and+functional+recovery+after+stroke.">Intra-arterial cell transplantation provides timing-dependent cell distribution and functional recovery after stroke.</a> Stroke. 44 (3), 720-726 (2013).</li><li>Schenk, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Monocyte+chemotactic+protein-3+is+a+myocardial+mesenchymal+stem+cell+homing+factor.">Monocyte chemotactic protein-3 is a myocardial mesenchymal stem cell homing factor.</a> Stem Cells. 25 (1), 245-251 (2007).</li><li>Hayashi, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Topical+implantation+of+mesenchymal+stem+cells+has+beneficial+effects+on+healing+of+experimental+colitis+in+rats.">Topical implantation of mesenchymal stem cells has beneficial effects on healing of experimental colitis in rats.</a> J. Pharmacol. Exp. Ther. 326 (2), 523-531 (2008).</li><li>Kodani, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flexible+colonoscopy+in+mice+to+evaluate+the+severity+of+colitis+and+colorectal+tumors+using+a+validated+endoscopic+scoring+system.">Flexible colonoscopy in mice to evaluate the severity of colitis and colorectal tumors using a validated endoscopic scoring system.</a> J. Vis. Exp. (80), e50843 (2013).</li></ol>

Denne undersøgelse beskriver en ny teknik for ultralyd-styrede intracardiac injektion af hMSCs i en lille-tarm musemodel af eksperimenterende CD. Denne teknik har meget høj sats for overlevelse og succes, som bane af nålen kan justeres baseret på real-time, høj opløsning billeder af musen venstre hjertekammer, leveret af ultralyd. Fordelen ved levering til venstre hjertekammer er, at hMSCs fordeles derefter intra-arterially og omgå venøs cirkulation, således at man undgår sammenlægning af cellerne i lungerne. ...

Crohns sygdom (CD) er en fælles kronisk inflammatorisk sygdom i små og store tarme og menes at skyldes en uhensigtsmæssig reaktion i immunsystemet vært til tarm mikrober7,8. Nylige undersøgelser har vist, at både murine og menneskelige mesenchymale til stamceller (msc) kan undertrykke betændelse i musemodeller tarmbetændelse1,3,4,5. Der er flere igangværende kliniske forsøg, som bruger menneskelige MSCs stammer fra knoglemarven eller fedtvæv til behandling af patienter med inflammatorisk tarmsygdom (IBD), som omfatter CD9. To ruter for MSC terapi har været brugt i disse kliniske forsøg: en indebærer den systemiske infusion (dvs. intravenøst) af MSCs luminale IBD (herunder CD), og den anden omfatter lokaliseret anvendelse/injektion af stamceller i fistlen tarmkanalen af patienter med endetarmsåbning CD. I en nylig meta-analyse af MSC terapi for IBD, systemisk (dvs., intravenøs) MSC terapi for luminale IBD (herunder CD) blev effektiv i op til 40% (95% CI: 7-79%) af patienter, der henviser til, at effekten var meget højere, på 61% (95% CI: 36-85%), når MSCs blev sprøjtet lokalt ind den syge CD fistel9. En nylig fase III multicenter randomiseret placebo kontrollerede forsøg af allogene adipøst stamceller injiceres direkte i perianal fistel CD patienter viste statistisk signifikant klinisk og radiologiske tegn på endetarmsåbning fistler healing, bekræfter resultaterne af meta-analyse10. Årsagerne til den lave effekten af MSC terapi gives intravenøst luminale CD er blevet utilstrækkeligt undersøgt, men en af grundene kan være den utilstrækkelige homing af MSC'er til stedet for betændelse. Murine undersøgelser i modeller af Colon betændelse har vist, at kun en lille procentdel af MSCs (1-5%) injiceres intravenøst nå betændte tyktarmen; de resterende MSCs er filtreret af lungerne (first-pass effekt)1,2 ,5,11,12. Flere murine forskningsundersøgelser har derfor brugt intraperitoneal ruten (i.p.) for MSC administration i dyremodeller for colitis4. De har imidlertid også vist, at kun en lille brøkdel af celler nå og indpode tyktarmen og at effekten er relateret til udskillelsen af opløselige paracrine faktorer, såsom tumor nekrose faktor-inducerbar gen 6 protein (TSG-6)2. MSC mekanisme af immunosuppression og healing indebærer en flerstrenget tilgang, der involverer paracrine; celle nærhed-uafhængige faktorer, som GTS-6; og celle nærhed-afhængige faktorer, ligesom programmeret død-ligand 1 (PD-L1); eller takkede 1. Derfor, MSC lokalisering til webstedet af betændelse kan resultere i øget effektivitet9,13. Faktisk viste en nylig undersøgelse, at MSC'er direkte implanteret i stedet for Colon skade resulterede i healing ved at udskille angiogenese-fremme vaskulær endotel vækstfaktor (VEGF). På den anden side blev en minimal helbredende effekt bemærket efter intravenøs injektion5. For at øge deres lokalisering til webstedet af betændelse (dvs., tyndtarmen i SAMP mus), blev denne ultralydsvejledt intracardiac injektionsteknik for MSC administration i venstre hjertekammer udviklet. Image-vejledt injektion sikrer en præcis indsprøjtning, hvilket fører til en højere sats for succes og at faldt sygelighed og dødelighed. Derudover leverer indsprøjtning af MSCs i venstre hjertekammer dem til arteriel omsætning, hvor de kan nå de betændte tyndtarmen før at blive fanget i lungerne.
I denne undersøgelse, blev menneskelige knoglemarv-afledte MSCs (hMSCs) brugt til injektion i den SAMP-1/YitFc (SAMP) murine model CD14. STEMPLET er en vel karakteriseret spontan murine model af kronisk inflammation, der udvikler små tarmbetændelse med næsten 100% penetrans14. Inflammation udvikler sig som reaktion på mikrofloraen i mangel af enhver kemisk, immunologisk eller genetisk manipulation og ligner menneskelige CD11. Sex - og alder-matchede inflammation-free RANDIS/J (RANDIS) mus, Børnesikring mus af SAMP, anvendtes i denne undersøgelse.
HMSCs var isoleret og udvidet i laboratorium fra knoglemarv (BM) prøver fra normal, uidentificerede donorer efter informeret samtykke ved hjælp af validerede og tidligere offentliggjorte protokoller15,16. Efter isolering og ekspansion, MSC evne i osteogenic, adipogenic, og chondrogenic differentiering blev evalueret i laboratoriet af flere assays15. Den osteogenic funktionel analyse blev udført af implanterer keramiske terninger af hydroxyapatit/tricalciumphosphat fosfat matrix indeholdende hMSCs subkutant i immunkompromitterede CB17-Prkdc SCID mus17. Cube analysen viser osteogenesis og chondrogenesis potentielle og betragtes som den ultimative test til at vurdere individuelle MSC præparater17. For at visualisere hMSCs i vivo efter injektion, blev lentivirus brugt til at transduce hMSCs med tredobbelt reporter gen konstruktion, der består af firefly luciferase (fl), monomere rødt fluorescerende proteiner (mRFP) og herpes simplex viral thymidine kinase (ttk ), drevet af en modificeret myeloproliferative sarkom virus (mnd) promotor18. Firefly luciferase i tredobbelt reporter er et enzym der dækfjer injiceres luciferin til oxyluciferin i hMSCs og producerer fotoner/hvidt lys. Dette registreres af følsomme charge - sammen enhed (CCD) kamera (bioluminescens) i en i vivo optisk tænkelig ordning, muliggører visualisering af levende hMSCs i mus. Bioluminescens imaging (BLI) er en følsom teknik, der kan bruges seriefremstillede til sporing af celler og for ex vivo analyse. Brug af en stærk mnd promotor drev kontinuerlig udtryk for triple fusion reporter gen konstruktion og giver mulighed for billeddannelse af injicerede hMSCs for mere end 16 uger19. HMSCs er vanskeligt at transduce og har en lav transduktion effektivitet. Bruger en optimeret protokol, hMSCs transduktion effektivitet er blevet forbedret og transgen udtryk blev udvidet18. Ved hjælp af mRFP udtryk, (en af generne, tredobbelt reporter) på flowcytometri, blev evnen til at transduce hMSCs med en høj virkningsgrad på op til 83% demonstreret. Differentiering assays og i vivo cube assays demonstreret den transduced hMSCs evne til at differentiere i chondrocytter, adipocytter og osteocytes17.

<table border="0" cellpadding="0" cellspacing="0" width="1100">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;">DMEM-LG&#160;&#160;</td>
			<td style="width:333px;">Gibco</td>
			<td style="width:188px;">31600-091</td>
			<td style="width:285px;"></td>
		</tr>
		<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>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PBS</td>
			<td>HyClone</td>
			<td>SH30256.01</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">175 cm tissue cutlure flasks</td>
			<td>Corning</td>
			<td>431080</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">75 cm tissue cutlure flasks</td>
			<td>Corning</td>
			<td>430720</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Centrifuge tubes</td>
			<td>Crysalgen</td>
			<td>23-2265</td>
			<td></td>
		</tr>
		<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>
		</tr>
		<tr height="21">
			<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

Crohns sygdom (CD) er en fælles kronisk inflammatorisk sygdom i små og store tarmene. Murine og menneskelige mesenchymale til stamceller (msc) har immunosuppressive potentiale og har vist at undertrykke betændelse i musemodeller af tarmbetændelse, selvom indgiftsmåde kan begrænse deres målsøgende og effektivitet 1 , 3 , 4 , 5. lokal anvendelse af MSC'er til Colon skade modeller har vist større effekt på mildne betændelse i tyktarmen. Men der er mangel på data om teknikker til at forbedre lokalisering af menneskelige knoglemarv-afledte MSCs (hMSCs) til tyndtarmen, site af betændelse i SAMP-1/YitFc (SAMP) modellen af eksperimentelle Crohns sygdom. Dette arbejde beskriver en ny teknik for ultralyd-styrede intracardiac injektion af hMSCs i SAMP mus, en vel karakteriseret spontan model af kronisk tarmbetændelse. Køn og alder-matchede, inflammation-free RANDIS/J (RANDIS) mus blev brugt som kontrol. For at analysere biodistributionen og lokalisering, var hMSCs transduced med en lentivirus, der indeholder en tredobbelt reporter. Den tredobbelte reporter bestod af firefly luciferase (fl), for en bioluminescerende imaging; monomere rødt fluorescerende proteiner (mrfp), til celle sortering; og afkortet herpes simplex virus thymidine kinase (ttk), for positron emission tomografi (PET) imaging. Resultaterne af denne undersøgelse viser at 24 h efter intracardiac administration, hMSCs lokalisere i tyndtarmen af SAMP mus i modsætning til inflammation-free RANDIS mus. Denne roman, ultralydsvejledt injektion af hMSCs i venstre hjertekammer af SAMP mus sikrer en høj succesrate på celle levering, giver mulighed for hurtig inddrivelse af mus med minimal sygelighed og dødelighed. Denne teknik kunne være en nyttig metode til forbedret lokaliseringen af MSCs i andre modeller af små-tarm inflammation, såsom TNFΔRE6. Fremtidige undersøgelser vil bestemme, hvis den øgede lokalisering af hMSCs af intraarteriel levering kan føre til øget terapeutiske virkning.

Figur 1 viser, at hMSCs kan være transduced med den tredobbelte reporter på en høj effektivitet, bevare deres stamcelle egenskaber. Transduced hMSCs kan visualiseres i realtid af BLI (figur 1 c, figur 3). Denne roman, ultralydsvejledt injektion af hMSCs i venstre hjertekammer af SAMP mus sikrer en meget høj succesrate af injektion, giver mulighed for hurtig inddrivelse af musene med minimal sygel...

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 undersøgelser i modeller af Colon betændelse har vist, at en lille procentdel (1-5%) af mesenchymale stamceller (MSC) injiceres intravenøst eller intraperitoneal hjem til den betændte kolon1,2. Denne undersøgelse viser, at ultralydsvejledt intracardiac injektioner af MSCs resultere i øget lokalisering til tarmen.

Ultralydsvejledt Intracardiac injektion af humane mesenkymale stamceller til at øge Homing til tarmen til brug i Murine modeller af eksperimentelle inflammatoriske tarmsygdomme

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.

Research

JoVE Journal

Medicine

The Measurement and Treatment of Suppression in Amblyopia

Måling og behandling af Suppression i Amblyopi

Amblyopia, a developmental disorder of the visual cortex, is one of the leading causes of visual dysfunction in the working age population. Current ...

The Use of Primary Human Fibroblasts for Monitoring Mitochondrial Phenotypes in the Field of Parkinson's Disease

Anvendelsen af ​​primære humane fibroblaster til overvågning Mitochondriale fænotyper i Field af Parkinsons sygdom

Parkinson's disease (PD) is the second most  common movement disorder and affects 1% of people over the age of 60 1. Because ageing is  the most important ...

Subretinal Injection of Gene Therapy Vectors and Stem Cells in the Perinatal Mouse Eye

Subretinal injektion af genterapivektorer og stamceller i mus Eye

The loss of sight affects approximately 3.4 million people in the United States and is expected to increase in the upcoming years.1 Recently, gene therapy ...

DNBS/TNBS Colitis Models: Providing Insights Into Inflammatory Bowel Disease and Effects of Dietary Fat

DNBS / TNBS Colitis modeller: giver indsigt i inflammatorisk tarmsygdom og Effekter af kosten fedt

Inflammatory Bowel Diseases (IBD), including Crohn&#39;s Disease and Ulcerative Colitis, have long been associated with a genetic basis, and more recently ...

Ultrasound-guided Transthoracic Intramyocardial Injection in Mice

Ultralydsvejledt Transtorakal intramyocardial Injektion i mus

Murine models of cardiovascular disease are important for investigating pathophysiological mechanisms and exploring potential regenerative therapies. ...

Studying Pancreatic Cancer Stem Cell Characteristics for Developing New Treatment Strategies

Studere i bugspytkirtlen Kræft stamceller Karakteristik for udvikling af nye behandlingsstrategier

Pancreatic ductal adenocarcinoma (PDAC) contains a subset of exclusively tumorigenic cancer stem cells (CSCs) which have been shown to drive tumor ...

Injection of Syngeneic Murine Melanoma Cells to Determine Their Metastatic Potential in the Lungs

Injektion af Syngene Murine melanomaceller at bestemme deres metastatiske potentiale i lungerne

Approximately 90% of human cancer deaths are linked to metastasis.  Despite the prevalence and relative harm of metastasis, therapeutics for treatment or ...

Standardiseret Tuina-manipulation til knæartrose hos rotter

Tuina Manipulation to Reduce Inflammation and Cartilage Loss in Knee Osteoarthritis Rats

Forfatter Spotlight: Forståelse af mekanismen for Tuina-manipulation hos rotter 

Tuina manipulation for at reducere betændelse og brusk tab i knæ slidgigt rotter

Clinical trials suggest that Tuina manipulation is effective in treating knee osteoarthritis (KOA), while further studies are required to discover its ...