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 støttes av Forsvarsdepartementet grant PR141774 og Crohns og kolitt Foundation of America karriere Development Award. Laboratoriet av Fabio Cominelli støttes av NIH gir DK042191 (FC), DK055812 (FC), DK091222 (FC) og DK07948 (FC). Laboratoriet av Arnold Caplan støttes delvis av L. David og E. Virginia Baldwin Foundation. Virkemiddelapparat ikke hadde noen rolle i studien analyse eller skriving av manuskriptet. Innholdet er ansvar forfatterne.

alle mus eksperimenter og prosedyrer i studien ble godkjent ved Case Western Reserve University &#39; s institusjonelle Animal Care og bruk komiteen. Prosedyrene ble gjennomført i foreningen for vurdering og akkreditering av laboratoriet dyr omsorg (AAALAC) akkreditert anlegget. BM var pustende under en University sykehus institusjonelle Review Board-godkjent protokoll på stamcelleforskningen kjernen anlegget ved Case Western Reserve University etter samtykke.
 1. kultur og signaltransduksjon...

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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. 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Denne studien beskriver en ny teknikk for ultralyd-guidede intracardiac injeksjon av hMSCs i en liten-tarm musemodell av eksperimentelle CD. Denne teknikken har meget høy rate av overlevelse og suksess, som banen nålen kan bli justert basert på sanntid, høyoppløselige bilder av musen venstre ventrikkel, levert av ultralyd. Fordelen med levering til venstre ventrikkel er at hMSCs fordeles deretter intra-arterially og omkjøringsvei venøs sirkulasjon, dermed unngår aggregering av celler i lungene. Tidligere brukte a...

Crohns sykdom (CD) er en vanlig kronisk inflammatorisk sykdom av små og store tarmen og er antatt å skyldes en upassende reaksjon av immunsystemet vert intestinal mikrober7,8. Nyere studier har vist at både murine og menneskelige stamceller (MSCs) kan undertrykke betennelse i musen modeller intestinale betennelsen1,3,4,5. Det er flere pågående kliniske forsøk som bruker menneskelige MSCs avledet fra benmarg eller fettvev for å behandle pasienter med inflammatorisk tarmsykdom (IBD), som inkluderer CD9. To ruter for MSC terapi har blitt brukt i disse kliniske forsøk: en innebærer systemisk infusjon (dvs. intravenøst) av MSCs for luminal IBD (inkludert CD), og den andre innebærer lokaliserte søknad/injeksjon av stamceller i fistel skrift av pasienter med perianale CD. I en nylig meta-analyse av MSC terapi for IBD systemisk (dvs. intravenøs) MSC terapi for luminal IBD (inkludert CD) var effektiv i opp til 40% (95% CI: 7-79%) pasienter, mens effekten var mye høyere, 61% (95% CI: 36-85%), når MSCs ble injisert lokalt i syke CD fistel9. En siste fase III multisenter randomisert placebo kontrollerte studie av allogene adipose stamceller injiseres direkte til perianale fistel CD pasienter viste statistisk signifikant klinisk og radiologiske tegn til perianale fistulae healing, bekrefter funnene i meta-analyse10. Årsakene til lav effekten av MSC terapi gitt intravenøst for luminal CD har blitt mangelfullt undersøkt, men en grunn kan være utilstrekkelig homing av MSCs til området av betennelse. Murine studier i modeller av colonic betennelse har vist at bare en liten prosentandel av MSCs (1-5%) injiseres intravenøst når betent tykktarmen; de gjenværende MSCs er filtrert av lungene (første-pass effekt)1,2 ,5,11,12. Flere murine undersøkelser har derfor brukt intraperitoneal ruten (IP) for MSC administrasjon i dyremodeller kolitt4. Men har de også vist at bare en liten brøkdel av cellene nå og engraft kolon og at effekten er knyttet til utskillelsen av løselig paracrine faktorer, som tumor nekrose faktor-induserbart genet 6 protein (TSG-6)2. MSC mekanisme immunsuppresjon og helbredelse innebærer en multipronged tilnærming som innebærer paracrine; cellen nærhet-uavhengige faktorer, som TSG-6; og nærhet-avhengige faktorer, som programmert død-ligand 1 (PD-L1); eller Jagged 1. MSC lokalisering til området av betennelse kan derfor føre til økt effekt9,13. Faktisk viste en fersk studie at MSCs direkte implantert på stedet av colonic skade resulterte i healing av sekresjon angiogenese-fremme vaskulær endotelial vekstfaktor (VEGF). På den annen side, ble en minimal helbredende effekt bemerket etter intravenøs injeksjon5. For å øke deres lokalisering til området av betennelse (dvs. tynntarmen i SAMP mus), ble denne ultralyd-guidede intracardiac injeksjon teknikken for MSC administrasjon i venstre ventrikkel utviklet. Bilde-guidede injeksjon sikrer en nøyaktig injeksjon, noe som fører til en høyere rate av suksess og til redusert sykelighet og dødelighet. Videre leverer injeksjon av MSCs i venstre ventrikkel dem til arterial sirkulasjon, der de kan nå betent tynntarmen før bli fanget i lungene.
I denne studien var menneskelige Ben margtransplantasjon-avledet MSCs (hMSCs) brukes for injeksjon i SAMP-1/YitFc (SAMP) murine modellen CD14. SAMP er en godt karakterisert spontan murine modell av kronisk betennelse som utvikler små-tarm betennelse med nesten 100% penetrance14. Betennelse utvikler svar mikroflora i fravær av kjemiske, immunologiske eller genetisk manipulasjon og ligner menneskelige CD11. Kjønn og alder-matchet betennelse-free AKR/J (AKR) mus, foreldrekontroll musene av SAMP, ble brukt i denne studien.
HMSCs ble isolert og utvidet i laboratoriet fra benmargen (BM) prøver innhentet fra normal, uidentifisert givere etter samtykke bruker godkjent og publisert tidligere protokoller15,16. Etter isolasjon og utvidelse, MSC evne i osteogenic, adipogenic, og chondrogenic differensiering ble vurdert i laboratoriet av flere analyser15. Osteogenic funksjonelle analysen ble utført av implanting keramiske kuber av hydroxyapatite/tricalcium fosfat matrise som inneholder hMSCs subcutaneously i immunsupprimerte CB17-Prkdc-SCID mus17. Cube analysen er viser osteogenesis og chondrogenesis potensielle og den ultimate testen for å vurdere personlige MSC forberedelser17. For å visualisere hMSCs i vivo etter injeksjon, ble lentivirus brukt til å transduce hMSCs med trippel reporter genet konstruere som består av firefly luciferase (fl), monomerisk rød fluorescerende protein (mRFP) og herpes simplex virus thymidine kinase (ttk ), drevet av en modifisert myeloproliferative sarkomspesialitet virus (mnd) promoter18. Firefly luciferase i trippel reporteren er et enzym som konverterer injisert luciferin til oxyluciferin i hMSCs og produserer fotoner/hvitt lys. Dette oppdages av sensitive kostnader - sammen enhet (CCD) kameraet (bioluminescens) i en i vivo optisk tenkelig system, muliggjør visualisering av live hMSCs i mus. Bioluminescens imaging (BLI) er en følsom teknikk som kan brukes serielt for sporing celler og ex vivo analyse. Bruk av en sterk mnd promoter kjører kontinuerlig uttrykk for trippel fusion reporter genet Konstruer og tillater avbilding av injisert hMSCs i mer enn 16 uker19. HMSCs er vanskelig å transduce og har en lav signaltransduksjon effektivitet. Bruker en optimalisert protokollen, hMSCs signaltransduksjon effektiviteten ble forbedret og transgene uttrykket ble forbedret18. Bruker mRFP uttrykk (en av trippel reporter genene) på flowcytometri, ble muligheten til å transduce hMSCs med høy effektivitet på opptil 83% demonstrert. Differensiering analyser og i vivo kube analyser vist evne til den transduced hMSCs å skille ut chondrocytes, adipocytter og osteocytes17.

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			<td height="21" style="height:21px;">0.25% trypsin-EDTA</td>
			<td>Gibco</td>
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			<td height="20" style="height:20px;">Proteamine Sulfate</td>
			<td>Sigma Aldrich</td>
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			<td>Goldbio</td>
			<td>luck-500</td>
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			<td height="21" style="height:21px;">Fetal Bobine Serum</td>
			<td>Gibco</td>
			<td>26140-079</td>
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			<td>Gibco</td>
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			<td>Corning</td>
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			<td height="21" style="height:21px;">75 cm tissue cutlure flasks</td>
			<td>Corning</td>
			<td>430720</td>
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			<td>Crysalgen</td>
			<td>23-2265</td>
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			<td height="21" style="height:21px;">Tissue-Plus O.C.T. compound</td>
			<td>Fisher HealthCare</td>
			<td>4585</td>
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			<td>Tissue-Tek&#160;</td>
			<td>4557</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>
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			<td height="21" style="height:21px;width:295px;">Hair removal cream</td>
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			<td style="width:188px;">N/A</td>
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			<td height="21" style="height:21px;width:295px;">Isoflurane&#160;</td>
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			<td height="21" style="height:21px;width:295px;">Forceps&#160;</td>
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			<td style="width:188px;">11200-33</td>
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			<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>
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		<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>
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			<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>
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			<td height="20" style="height:20px;">Petri dish</td>
			<td>Fisher Scientific</td>
			<td>FB0875711A</td>
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			<td height="21" style="height:21px;">SAMP mice</td>
			<td>Cleveland Digestive Disease Research Core Centre</td>
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			<td height="21" style="height:21px;">AKR mice</td>
			<td>Cleveland Digestive Disease Research Core Centre</td>
			<td></td>
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			<td height="21" style="height:21px;">Vevo 770 imaging system</td>
			<td>&#160;Visual Sonics, Toronto, Canada</td>
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		</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 sykdom (CD) er en vanlig kronisk inflammatorisk sykdom av små og store tarmen. Murine og menneskelige stamceller (MSCs) har suppressive potensial og har vist seg å undertrykke betennelse i musen modeller av tarm betennelse, selv om inntaksmetoden kan begrense deres homing og effektiviteten 1 , 3 , 4 , 5. lokal anvendelse av MSCs colonic skade modeller har vist større effekt på ameliorating betennelse i tykktarmen. Men er det mangelen på data om teknikker for å forbedre lokaliseringen av menneskelig bein margtransplantasjon-avledet MSCs (hMSCs) til tynntarm, området av betennelse i SAMP-1/YitFc (SAMP) modell av eksperimentelle Crohns sykdom. Dette verket beskriver en ny teknikk for ultralyd-guidede intracardiac injeksjon av hMSCs i SAMP mus, et godt karakterisert spontan modell av kronisk intestinal inflammasjon. Kjønn og alder-samsvar, betennelse-free AKR/J (AKR) mus ble brukt som kontroller. For å analysere biodistribution og lokaliseringen, ble hMSCs transduced med en lentivirus som inneholder en trippel reporter. Trippel reporteren besto av firefly luciferase (fl), for bioluminescent avbildning; monomerisk rød fluorescerende protein (mrfp), i celle sortering; og avkortede herpes simplex virus thymidine kinase (ttk), fantes et positron utslipp stengte (PET) tenkelig. Resultatene av denne studien viser at 24 timer etter den intracardiac administrasjonen, hMSCs lokalisere i tynntarmen SAMP mus i motsetning til betennelse-free AKR mus. Denne romanen, ultralyd-guidede injeksjon av hMSCs i venstre ventrikkel SAMP mus sikrer en høy suksessrate på cellen levering, slik at for rask utvinning av mus med minimal sykelighet og dødelighet. Denne teknikken kan være en nyttig metode for den forbedrede lokaliseringen av MSCs i andre modeller av små-tarm betennelse, for eksempel TNFΔRE6. Fremtidige studier vil avgjøre hvis den økte lokaliseringen av hMSCs av intra arteriell levering kan føre til økt terapeutiske effekten.

Figur 1 viser at hMSCs kan være transduced med trippel reporter på en høy effektivitet, bevare stamcelleforskningen egenskapene. Transduced hMSCs kan visualiseres i sanntid BLI (figur 1 c, Figur 3). Denne romanen, ultralyd-guidede injeksjon av hMSCs i venstre ventrikkel SAMP mus sikrer en meget høy suksessrate på injeksjon, slik at for rask utvinning av mus med minimal sykelighet og dødelighet ...

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 studier i modeller av colonic betennelse har vist at en liten prosentandel (1-5%) av mesenchymal stamceller (MSC) injiseres intravenøst eller intraperitoneally hjem til betent kolon1,2. Denne studien viser at ultralyd-guidede intracardiac injeksjoner av MSCs resultere i økt lokalisering til tarmen.

Ultralyd-guidede Intracardiac injeksjon for menneskelige stamceller å øke Homing til tarmen for bruk i Murine modeller av eksperimentelle inflammatorisk tarm sykdommer

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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