Name: conditional reprogramming of pediatric human esophageal epithelial cells for use in tissue engineering and disease investigation
Uploaded: 2017-03-22T13:00:00
Description: Watch this Scientific Journal Video about Conditional Reprogramming of Pediatric Human Esophageal Epithelial Cells for Use in Tissue Engineering and Disease Investigation at JoVE.com

We would like to acknowledge Connecticut Children's Medical Center Strategic Research Funding for supporting this work.

Esophageal biopsier blev opnået efter informeret samtykke blev opnået fra forældre / værger for de pædiatriske patienter og i overensstemmelse med den institutionelle Review Board (IRB # 13-094). 1. sterilisering af instrumenter og gelatineopløsning <ol><li> Autoklave pincet, barberblade og sakse forud for håndtering af væv for at forhindre forurening. </li><li> For at gøre 200 ml 0,1% gelatineopløsning, kombinere 200 ml destilleret vand med 0,2 g gelatine. Autoklave og afkøles før brug. </li></ol>...

<ol>
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De vigtigste skridt for at isolere og udvide esophageal epitelceller fra patient biopsier er: 1) i tilstrækkelig grad at dissociere biopsi væv med minimal celledød; 2) at sikre ROCK inhibitor sættes til cellekulturmediet ved hver udskiftning af mediet; 3) Brug ikke flere feeder celler end anbefalet; 4) opretholde en ren aseptisk kultur; og 5) passage celler lige før nå sammenløb. På grund af de patientrelaterede forskelle i biopsiprøver opnået for betinget omprogrammering, er det r...

Esophageal tissue engineering og eosinofil esophagitis (EoE) har været i fokus for forskning i mange laboratorier i det seneste årti. Medfødte defekter, såsom esophageal atresia, ses hos ca. 1 i 4000 levendefødte, hvilket resulterer i ufuldstændig udvikling af spiserøret fører til manglende evne til at spise 1. Forekomsten og udbredelsen af EoE har været stigende lige siden identifikation af sygdommen enhed i 1993. Forekomsten af EoE varierede fra 0,7 til 10 / 100.000 per person-år, og forekomsten varierede fra 0,2 til 43 / 100.000 2. En ny attraktiv kirurgisk tilgang til behandling af dyb kløft esophageal atresia består i at generere vævskonstruktioner til implantation udnytte patientens egne celler. Disse celler sammen med syntetisk stillads vil generere et autologt konstrukt, der ikke kræver immunsuppression. Nogle grupper er allerede begyndt at undersøge ose af stamceller-lignende celler til esophageal vævsmanipulering 3 samt anvendelse af native øsofageale epitelceller at genbefolke slimhinden 4 - 7. Sygdomme, som er til stede i spiserøret af pædiatriske patienter er ofte svære at diagnosticere eller studere uden indgriben. Desuden udnytter dyremodeller eller in vitro udødeliggjort cellelinje modeller for pædiatriske sygdomme som EoE omfatter ikke den præcise sygdom patogenese eller patientspecifikke 8 forskelle. Derfor, evnen til at studere en patients sygdom proces in vitro med henblik på at identificere specifikke sygdomstilstande udløser antigener, evaluere underliggende mekanismer og undersøge lægemiddelbehandlinger ville være hidtil ukendte og give klinikere med oplysninger, der kan støtte i patientbehandlingen. Der har været mange autologe eller patientspecifikke celletyper, der er blevet foreslået til anvendelse i tissue teknik og forskning i menneskers sygdomme patogenese. Imidlertid har nogle af disse celletyper er begrænset i deres evne til at generere nok celler af en specifik fænotype til frø et stort stillads eller udføre high throughput in vitro-undersøgelser. Anvendelsen af pluripotente eller multipotente stamceller har været emnet for megen forskning diskussion imidlertid begrænsninger og mangler til anvendelse af disse celler er blevet godt beskrevet 9. Brugen af ​​humane embryonale stamceller er meget omdiskuteret og præsenterer mange etiske spørgsmål. Vigtigst er disse celler danner teratomer, som svarer til en tumor, hvis de ikke skelnes fra deres pluripotent tilstand før leverer dem ind i en levende vært 10. Endvidere ville anvendelse af embryonale stamceller ikke være patient-specifikke og kan fremkalde en allogen reaktion og behovet for immunsuppression 10. Inducerede pluripotente stamceller (iPSCs) er pluripotente celler, der kanvære afledt af en patientens egne celler. Somatiske celler, såsom hudceller, kan induceres til en pluripotent tilstand under anvendelse af en række integrative og ikke-integrative teknikker. Disse celler så tjene som en patient-specifikke celle kilder til tissue engineering eller sygdom undersøgelse. Integrationen af uønsket genetisk materiale i disse celler er en bekymring mange har beskrevet og selv hvis sekvenser er fuldstændigt fjernet iPSCs synes at bevare en epigenetisk &quot;hukommelse&quot; mod celletype, hvorfra de blev afledt 11. Disse celler vil også danne teratomer in vivo hvis ikke differentieres før transplantation 11. Mange differentieringsfaktorer protokoller er blevet undersøgt med fokus på epiteliale slægter 12, 13, 14, er det imidlertid meget vigtigt at bemærke, at de celletyper, der følger i slutningen af differentiering ikke homogene og oun besidder en brøkdel af celletypen af ​​interesse. Dette resulterer i lavt udbytte og behovet for at oprense den ønskede celletype. Selvom iPSCs er en potentiel patient-specifikke cellekilde, at processen opnå en celletype af interesse for enten vævskonstruktion eller sygdom undersøgelse er meget ineffektiv. Humane epitelceller med succes er blevet isoleret fra en række både syge og ikke sygdomsangrebne væv i den menneskelige krop, herunder: lunge 15, bryst 16, tyndtarm 17, colon 18, blære 19 og spiserøret 20. Det er vigtigt at bemærke, at humane primære celler har et endeligt antal passager, hvor fænotypen opretholdes 21, 22. Det betyder desværre, at antallet af celler er nødvendige for sygdom undersøgelse eller til såning en manipuleret stilladstil implantation kan ikke opnås. Derfor er der behov nye teknikker til at udvide patientens celler og samtidig bibeholde en epitelial fænotype. Betinget omprogrammering af normale og kræft epitelceller udnytte feeder celler og ROCK inhibitor blev beskrevet i 2012 af Liu et al. 2 3. Denne teknik blev anvendt til at udvide cancerøse epitelceller opnået fra biopsier af prostata og brystkræft ved hjælp bestrålede feeder-celler, ROCK inhibitor og betinget omprogrammering medium. Målet var at generere nok celler til in vitro-assays, såsom screening narkotika. Denne teknik er i stand til at udvide epitelceller ubestemt tid med &quot;omprogrammering&quot; disse celler til en stam- eller progenitor-lignende tilstand, som er meget proliferativ. Det er blevet påvist, at disse celler er ikke-tumorigene og ikke har evnen til at danne teratomer 23, 24. Endvidere er der ingenkromosomafvigelser eller genetiske manipulationer var til stede efter passage af disse celler i kultur ved hjælp af denne teknik 23, 24. Vigtigst er disse celler kun i stand til at differentiere til den native celletype af interesse. Derfor er denne teknik giver et stort reservoir af patientspecifikke epitelceller for sygdom undersøgelse eller tissue engineering uden behov for immortalisering. Opnåelse epithelvæv fra et specifikt organ for at studere sygdomsprocesser er ofte begrænsede og ikke altid mulig på grund af patientens risiko. For de patienter, der lider esophageal sygdom eller defekter, endoskopisk biopsi hentning er en minimalt invasiv fremgangsmåde til opnåelse epitelvæv, der kan adskilles og betinget omprogrammeres til at give et ubestemt cellekilde, der er specifik til slimhinden i denne patientens spiserør. Dette tillader derefter for in vitro-undersøgelseraf epitelcellerne at evaluere sygdomsprocesser og en skærm til potentielle lægemidler. En sygdomsproces der i høj grad kunne drage fordel af denne fremgangsmåde er eosinofil oesophagitis som er blevet beskrevet som allergisk sygdom i spiserøret 8. Allergi test samt terapeutiske metoder kan evalueres in vitro ved hjælp af patientens egne epitelceller og disse data kan derefter overført til den behandlende læge til at udvikle individualiserede behandling planer. Teknikken med betingede omprogrammering sammenholdt med opnåelse endoskopiske biopsier fra pædiatriske patienter giver mulighed for at udvide normale øsofageale epitelceller ubestemt tid fra enhver patient. Denne celle kilde kunne derfor gået sammen med naturlige eller syntetiske stilladser til at give en patient-specifikke kirurgisk mulighed for mangler, sygdom eller traumer. Under en ubestemt celle nummer ville hjælpe ingeniør esophageal konstruktioner, der besidder en helt reseededlumen med esophageal epitelceller med henblik på at hjælpe med at fremme regenerering af de resterende celletyper.

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			<td height="21" style="height:21px;width:385px;">Primocin</td>
			<td style="width:172px;">InVivogen</td>
			<td style="width:119px;">ant-pm2</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:385px;">Isopentane</td>
			<td style="width:172px;">Sigma Aldrich</td>
			<td style="width:119px;">277258-1L</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:385px;">Gelatin From Porcine Skin</td>
			<td style="width:172px;">Sigma Aldrich</td>
			<td style="width:119px;">G1890-100G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:385px;">DMEM</td>
			<td style="width:172px;">Thermofisher Scientific</td>
			<td style="width:119px;">11965092</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:385px;">Cryomold</td>
			<td style="width:172px;">TissueTek</td>
			<td style="width:119px;">4565</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:385px;">Cryomatrix OCT</td>
			<td style="width:172px;">Thermofisher Scientific</td>
			<td style="width:119px;">6769006</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:385px;">15ml Conical Tubes</td>
			<td style="width:172px;">Denville Scientific</td>
			<td style="width:119px;">C1017-p</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:385px;">Complete Keratinocyte Serum Free Medium</td>
			<td style="width:172px;">Thermofisher Scientific</td>
			<td style="width:119px;">10724011</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:385px;">Penicillin Streptomycin</td>
			<td style="width:172px;">Thermofisher Scientific</td>
			<td style="width:119px;">15140122</td>
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			<td height="21" style="height:21px;width:385px;">Glutamax</td>
			<td style="width:172px;">Thermofisher Scientific</td>
			<td style="width:119px;">35050061</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:385px;">Insulin Solution</td>
			<td style="width:172px;">Sigma Aldrich</td>
			<td style="width:119px;">I9278-5ml</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:385px;">Human Epidermal Growth Factor (EGF)</td>
			<td style="width:172px;">Peprotech</td>
			<td style="width:119px;">AF-100-15</td>
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		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:385px;">ROCK Inhibitor (Y-27632)</td>
			<td style="width:172px;">Fisher Scientific</td>
			<td style="width:119px;">125410</td>
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		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:385px;">F-12 Medium&#160;</td>
			<td style="width:172px;">Thermofisher Scientific</td>
			<td style="width:119px;">11765054</td>
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		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:385px;">Fetal Bovine Serum</td>
			<td style="width:172px;">Denville Scientific</td>
			<td style="width:119px;">FB5001</td>
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		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:385px;">Dispase</td>
			<td style="width:172px;">Thermofisher Scientific</td>
			<td style="width:119px;">17105041</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:385px;">0.05% Trypsin-EDTA</td>
			<td style="width:172px;">Thermofisher Scientific</td>
			<td style="width:119px;">25300062</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:385px;">0.25% Trypsin-EDTA</td>
			<td style="width:172px;">Thermofisher Scientific</td>
			<td style="width:119px;">25200072</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:385px;">100mm Dishes</td>
			<td style="width:172px;">Denville Scientific</td>
			<td style="width:119px;">T1110-20</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:385px;">150mm Dishes</td>
			<td style="width:172px;">Denville Scientific</td>
			<td>T1115</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:385px;">50ml Conicals</td>
			<td style="width:172px;">Denville Scientific</td>
			<td style="width:119px;">&#160; C1062-9&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:385px;">Phosphate Buffered Saline Tablets</td>
			<td style="width:172px;">Fisher Scientific</td>
			<td style="width:119px;">BP2944-100</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:385px;">5ml Pipettes</td>
			<td style="width:172px;">Fisher Scientific</td>
			<td>1367811D</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:385px;">10ml Pipettes</td>
			<td style="width:172px;">Fisher Scientific</td>
			<td>1367811E</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:385px;">25ml Pipettes</td>
			<td style="width:172px;">Fisher Scientific</td>
			<td>1367811</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:385px;">9&#34; Pasteur Pipettes</td>
			<td style="width:172px;">Fisher Scientific</td>
			<td style="width:119px;">13-678-20D</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:385px;">NIH 3T3 Cells</td>
			<td style="width:172px;">ATCC</td>
			<td style="width:119px;">CRL1658</td>
			<td></td>
		</tr>
	</tbody>
</table>

conditional reprogramming of pediatric human esophageal epithelial cells for use in tissue engineering and disease investigation

Identifying and expanding patient-specific cells in culture for use in tissue engineering and disease investigation can be very challenging. Utilizing various types of stem cells to derive cell types of interest is often costly, time consuming and highly inefficient. Furthermore, undesired cell types must be removed prior to using this cell source, which requires another step in the process. In order to obtain enough esophageal epithelial cells to engineer the lumen of an esophageal construct or to screen therapeutic approaches for treating esophageal disease, native esophageal epithelial cells must be expanded without altering their gene expression or phenotype. Conditional reprogramming of esophageal epithelial tissue offers a promising approach to expanding patient-specific esophageal epithelial cells. Furthermore, these cells do not need to be sorted or purified and will return to a mature epithelial state after removing them from conditional reprogramming culture. This technique has been described in many cancer screening studies and allows for indefinite expansion of these cells over multiple passages. The ability to perform esophageal screening assays would help revolutionize the treatment of pediatric esophageal diseases like eosinophilic esophagitis by identifying the trigger mechanism causing the patient's symptoms. For those patients who suffer from congenital defect, disease or injury of the esophagus, this cell source could be used as a means to seed a synthetic construct for implantation to repair or replace the affected region.

Et resumé af de vigtigste skridt i at isolere esophageal epitelceller fra patient biopsier er opsummeret i figur 1. Kolonier af epitelceller vil dannes i ca. 4-5 dage og vil være omgivet af fibroblast fødeceller (figur 2A). Da disse kolonier udvide de vil fusionere med andre kolonier at danne større kolonier (figur 2B). Når kulturerne er blevet 70% sammenflydende, de skal udvides (figur 2C). For at sikre at fibrobla...

Watch this Scientific Journal Video about Conditional Reprogramming of Pediatric Human Esophageal Epithelial Cells for Use in Tissue Engineering and Disease Investigation at JoVE.com

Conditional Reprogramming of Pediatric Human Esophageal Epithelial Cells for Use in Tissue Engineering and Disease Investigation

Udvidelse af humane pædiatriske øsofageale epitelceller anvender betinget omprogrammering giver undersøgere med en patient-specifik population af celler, der kan anvendes til manipulering esophageal konstruktioner til autolog implantation til behandling defekter eller skade og tjene som et reservoir til terapeutiske screeningsassays.

Betinget Omprogrammering af Pediatric Menneskelig Esophageal epitelceller til brug i Tissue Engineering og Sygdom Investigation

Identifying and expanding patient-specific cells in culture for use in tissue engineering and disease investigation can be very challenging. Utilizing ...

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