Name: microrna expression profiles of human ips cells, retinal pigment epithelium derived from ips, and fetal retinal pigment epithelium
Uploaded: 2014-06-24T13:00:00
Description: Watch this Scientific Journal Video about MicroRNA Expression Profiles of Human iPS Cells, Retinal Pigment Epithelium Derived From iPS, and Fetal Retinal Pigment Epithelium at JoVE.com

Udtalelserne eller påstande, der er indeholdt heri, er de private synspunkter forfatternes og skal ikke opfattes som tjenestemand eller som afspejler de synspunkter, Department of the Army eller det amerikanske forsvarsministerium. Denne forskning blev udført, mens forfatterne Whitney A. Greene, Alberto Muñiz og Ramesh R. Kaini afholdt en National Research Council postdoc Associateship på USAISR. Microarray analyser blev udført af Greehey Børns Cancer Research Institute Microarray Core Facility og Bioinformatik Institut på UT Health Science Center i San Antonio. Dette arbejde blev støttet af US Army Klinisk Rehabiliterende Medicin Research Program (CRMRP) og militære operationelle Medicine Research Program (MOMRP).

1.. Udarbejdelse af Kultur Reagenser og dyrkningsplader <ol><li> mTeSR1 medier: Forbered mTeSR1 medier i overensstemmelse med producentens anvisninger. Optøs 100 ml mTeSR1 5x supplement natten over ved 4 ° C. Tilsæt 100 ml mTeSR1 5x supplement til 400 ml mTeSR1 basale medier og bland godt. Dette medie er stabilt ved 4 ° C i op til 2 uger og ved -20 ° C i 6 måneder. </li><li> Differentiering medier: Forbered differentiering medier i DMEM/F12 til opnåelse af 0,1 mM β-mercaptoethanol, 0,1 mM ikke-essentielle amin...

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Afslutningsvis denne rapport beskriver de metoder, der anvendes til kultur iPS celler, iPS-RPE og føtal RPE. RPE stammer fra iPS er morfologisk og funktionelt ligner føtal RPE. IPS-RPE udtrykker også karakteristiske RPE gener, herunder RPE65, CRALBP, PEDF og LRAT 16.. For yderligere at karakterisere disse celler blev RNA ekstraheret og anvendt til at udføre microarray analyse for at identificere differentielt udtrykte miRNA. Analyse af signalintensiteter afslørede, at det største antal differentielt udt...

Stamceller har evnen til at replikere uden begrænsning og potentialet til at differentiere til enhver somatiske celletype. Udviklingen af teknikker til at omprogrammere somatiske celler i pluripotente stamceller har fremkaldt stor begejstring i forskerkredse og blandt klinikere, som fremkomsten af personlige vævsregenerering er på horisonten 1.. Induceret pluripotente stamceller (iPS-celler) udviser de samme funktioner i ubegrænset replikation potentiale og pluripotency som embryonale stamceller (ES) celler, mens omgå de etiske dilemmaer, der er forbundet med økonomiske og sociale råd. Desuden vil patientens stamceller ikke stimulerer et immunrespons, hvilket øger sandsynligheden for vellykket terapeutiske anvendelser 2-3. Under bestemte dyrkningsbetingelser har iPS celler blevet vist at differentiere til flere forskellige celletyper in vitro, herunder cardiomyocytter, neuroner, pancreas-beta-celler, hepatocytter og retinal pigment epithelium (RPE) 4-12. RPE er en specialiseret lag af pigmenterede epitelceller placeret på bagsiden af ​​nethinden, der udfører en række funktioner, der er afgørende for visuel sundhed og funktion, såsom absorption af falsk lys, fagocytose af fotoreceptor ydre segmenter, og behandling af retinoider til fremstilling af visuel chromophor. Dysfunktion af RPE grund af skader eller sygdom påvirker dybt fotoreceptor sundhed og visuel funktion som det fremgår af blindende sygdomme, som er resultatet af underliggende RPE patologi, såsom aldersrelateret makulær degeneration (AMD), Stargardts sygdom og retinitis pigmentosa (RP) 13.. Virkelig effektive behandlinger, der kan genoprette vision ikke er blevet nået, og udskiftning af syge RPE med sund RPE kan være den bedste mulighed for at forhindre tab af synet 14-15. RPE afledt iPS (iPS-RPE) er en mulig kilde til celler til at erstatte den beskadigede RPE. iPS-RPE udtrykker kendetegntic RPE proteiner LRAT, CRALBP, PEDF og RPE65; viser den klassiske stærkt pigmenteret sekskantede RPE morfologi; og udfører RPE funktioner såsom fagocytose, retinoid behandling, og sekretion af 11 - cis retinal 5,16. Men før iPS-RPE kan bruges terapeutisk, IPS-RPE skal være grundigt karakteriseret. Forståelse af de faktorer, der styrer RPE differentiering er nødvendig for at forbedre udbyttet og renheden af ​​de celler, der skal anvendes til kliniske anvendelser. Celledifferentiering er resultatet af stærkt reguleret genekspression. Epigenetisk ombygning af genomet og koordinering af transkriptionsfaktorer er nødvendige for celle skæbne beslutninger, der opstår under differentiering og udvikling 17.. Regulering af besked RNA (mRNA) oversættelse af microRNA (miRNA) præsenterer endnu et niveau af regulering, der påvirker celle skæbne 1. MiRNA er korte, ~ 22 nt, længder af nukleotider, der enten undertrykke oversættelsebinding til 3&#39;UTR af mRNA eller målrette mRNA for nedbrydning. MiRNA er blevet påvist i næsten alle væv og til dato har over 2.000 unikke menneskelige microRNA er blevet registreret i miRBase databasen. Da miRNA kræver kun delvis komplementaritet til at binde til target mRNA, kan en enkelt miRNA potentielt binder til ti eller hundrede mål, og vice versa, dvs en enkelt mRNA kan målrettes ved flere forskellige miRNA. Denne promiskuøs binding egenskab dramatisk øger niveauet af kompleksitet regulering samt sværhedsgraden bestemme funktionerne enkelte miRNA og den rolle hver spiller i cellulære funktioner 18-21. Ikke desto mindre har undersøgelser vist, at miRNA raffinerer genekspression under differentiering ved at påvirke DNA methylering status 17. I en undersøgelse mere specifik RPE blev miR-204/211 vist sig at fremme epitel fænotype af RPE 22. En anden gruppe analyserede miRNA profilenaf RPE under differentiering fra ES-celler og afslørede forskellige sæt miRNA udtrykkes under differentieringsprocessen 23. Faktisk kan miRNA profiler utvetydigt at skelne celletyper, herunder ES-celler, precursorceller og terminalt differentierede celler 24,25. Over 250 miRNA er udtrykt i nethinden. Baseret på disse studier, vi hypotesen, at miRNA spiller en vigtig rolle under differentieringen af ​​RPE fra iPS. Formålet med denne rapport er at beskrive de protokoller for differentiering af RPE fra IMR90-4 iPS celler, samling af RNA til analyse ved microarray og analyse af microarray data til at identificere miRNA, der udtrykkes forskelligt blandt tre celletyper, iPS celler IPS-RPE og føtal RPE. Totalt RNA blev ekstraheret fra kulturer af hver celletype og hybridiseret til en miRNA microarray indeholdende prober specifikke for 1.205 miRNAer og 144 virale miRNA. Microarray resultater blev sammenlignd for at bestemme, hvilke miRNA blev differentielt udtrykt blandt de forskellige celletyper. miRNA med 2 gange eller mere fold ændring i ekspressionen blev udvalgt til yderligere analyse. En miRNA analyse software program der blev brugt til at identificere potentielle mål for de differentielt udtrykte miRNA og skabe mobilnetværk reguleret af de udvalgte miRNA.

<table border="0" cellpadding="0" cellspacing="0" width="676">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:235px;">mTeSR1 media + 5X supplement</td>
			<td style="width:156px;">Stem Cell Technologies</td>
			<td style="width:119px;">5850</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DMEM/F12</td>
			<td>Life Technologies</td>
			<td>11330-032</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">2-Mercaptoethanol</td>
			<td style="width:156px;">Sigma</td>
			<td style="width:119px;">M-7154</td>
			<td></td>
		</tr>
		<tr height="18">
			<td height="18" style="height:18px;width:235px;">Non essential amino acids</td>
			<td style="width:156px;">Hyclone(Fisher)</td>
			<td>SH30853.01</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:235px;">Knockout serum replacement</td>
			<td style="width:156px;">Life Technologies</td>
			<td style="width:119px;">10828-028</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:235px;">Gentamicin&#160;</td>
			<td style="width:156px;">Life Technologies</td>
			<td style="width:119px;">15750-060</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:235px;">MEM media</td>
			<td style="width:156px;">Life Technologies</td>
			<td style="width:119px;">10370-021</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:235px;">N1 supplement</td>
			<td style="width:156px;">Sigma</td>
			<td style="width:119px;">N-6530-5ML</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:235px;">Taurine</td>
			<td style="width:156px;">Sigma</td>
			<td style="width:119px;">T-8691-25G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:235px;">Hydrocortisone</td>
			<td style="width:156px;">Sigma</td>
			<td style="width:119px;">H0888-1G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:235px;">Fetal bovine serum</td>
			<td style="width:156px;">Hyclone(Fisher)</td>
			<td>SH3008803HI</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Triiodo-l-thyronine sodium salt</td>
			<td style="width:156px;">Sigma</td>
			<td style="width:119px;">T6397</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:235px;">Sodium hydroxide</td>
			<td style="width:156px;">Sigma</td>
			<td style="width:119px;">S5881</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fetal RPE media RTEGM kit</td>
			<td style="width:156px;">Life Technologies</td>
			<td style="width:119px;">195406</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:235px;">Dispase</td>
			<td style="width:156px;">Life Technologies</td>
			<td>17105-041</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:235px;">Matrigel</td>
			<td style="width:156px;">BD Biosciences</td>
			<td style="width:119px;">354277</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:235px;">Phosphate buffered saline</td>
			<td style="width:156px;">Hyclone(Fisher)</td>
			<td>10010-023</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:235px;">Trypsin</td>
			<td style="width:156px;">Hyclone(Fisher)</td>
			<td style="width:119px;">25200-072</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:235px;">Miltenyi Biotec washing buffer</td>
			<td style="width:156px;">StemGent</td>
			<td style="width:119px;">130-092-987</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:235px;">Miltenyi Biotec rinsing buffer</td>
			<td style="width:156px;">StemGent</td>
			<td style="width:119px;">130-092-222</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:235px;">Anti-TRA-1-60 microbead kit</td>
			<td style="width:156px;">StemGent</td>
			<td style="width:119px;">130-095-816</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:235px;">Miltenyi Biotec cell sorter column</td>
			<td style="width:156px;">StemGent</td>
			<td style="width:119px;">130-021-101</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:235px;">RNeasy micro kit</td>
			<td style="width:156px;">Qiagen</td>
			<td style="width:119px;">74004</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:235px;">QIA shredder</td>
			<td style="width:156px;">Qiagen</td>
			<td style="width:119px;">79654</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:235px;">RNA 6000 Pico LabChip kit</td>
			<td style="width:156px;">Agilent</td>
			<td style="width:119px;">G2938-90046</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:235px;">Human miRNA microarray v16</td>
			<td style="width:156px;">Agilent</td>
			<td style="width:119px;">G4471A</td>
			<td></td>
		</tr>
	</tbody>
</table>

microrna expression profiles of human ips cells, retinal pigment epithelium derived from ips, and fetal retinal pigment epithelium

Formålet med denne rapport er at beskrive de protokoller til sammenligning af microRNA (miRNA) profiler af human induceret pluripotente stamceller (iPS-celler), retinal pigment epitel (RPE) afledt af humane iPS celler (iPS-RPE), og føtal RPE. Protokollerne omfatter indsamling af RNA til analyse ved microarray og analyse af microarray data til at identificere miRNA, der udtrykkes forskelligt blandt tre celletyper. Metoderne til dyrkning af iPS celler og føtale RPE er forklaret. Den protokol, der anvendes til differentiering af RPE fra human iPS er også beskrevet. RNA-ekstraktion teknik beskriver vi blev udvalgt til at tillade maksimal genvinding af meget små RNA til anvendelse i en miRNA microarray. Endelig cellulære vej og netværk analyse af microarray data forklaret. Disse teknikker vil lette sammenligningen af ​​de miRNA profiler af tre forskellige celletyper.

iPS-celler (figur 1a) blev dyrket i differentiering betingelser for at inducere differentiering i iPS-RPE celler. IPS-RPE udstillet klassiske RPE fænotype af sekskantede pigmenteret Cellemorfologien (figur 1B) ligner føtal RPE (figur 1C). For bedre at forstå den rolle, som miRNA kan spille i løbet af processen af ​​differentiering fra iPS til iPS-RPE blev microarray analyse af miRNA-ekspression udført. Total RNA blev opsamlet fra iP...

Watch this Scientific Journal Video about MicroRNA Expression Profiles of Human iPS Cells, Retinal Pigment Epithelium Derived From iPS, and Fetal Retinal Pigment Epithelium at JoVE.com

MicroRNA Expression Profiles of Human iPS Cells, Retinal Pigment Epithelium Derived From iPS, and Fetal Retinal Pigment Epithelium

MicroRNA (miRNA) profiler af human induceret pluripotente stamceller (iPS-celler), retinal pigment epitel (RPE) fra humant induceret pluripotente stamceller (iPS) celler (iPS-RPE), og føtal RPE, blev sammenlignet.

MicroRNA Expression Profiler af menneskelige iPS celler, retinale pigmentepitel arvet fra iPS og Føtal retinale pigmentepitel

The objective of this report is to describe the protocols for comparing the microRNA (miRNA) profiles of human induced-pluripotent stem (iPS) cells, ...

Research

JoVE Journal

Biology

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Subretinal Transplantation af humane embryonale stamceller afledt retinal Pigment epitel celler ind i en stor-eyed Model af geografisk atrofi

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Nitroreductase/Metronidazole-Mediated Ablation and a MATLAB Platform RpEGEN for Studying Regeneration of the Zebrafish Retinal Pigment Epithelium

Nitroreductase/metronidazolmedieret ablation og en MATLAB-platform (RpEGEN) til undersøgelse af regenerering af zebrafiskens retinale pigmentepitel

The retinal pigment epithelium (RPE) resides at the back of the eye and performs functions essential for maintaining the health and integrity of adjacent ...

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The retinal pigment epithelium (RPE) is a monolayer of polarized pigmented epithelial cells, located between the choroid and neuroretina in the retina. ...

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Isolering af primære muse retinale pigmenterede epitelceller

The retinal pigmented epithelium (RPE) layer lies immediately behind the photoreceptors and harbors a complex metabolic system that plays several critical ...

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Effektiv og konsistent generering af retinal pigmentepitel / choroid flatmounts fra menneskelige øjne til histologisk analyse

The retinal pigment epithelium (RPE) and retina are functionally and structurally connected tissues that work together to regulate light perception and ...

Real-Time Analysis of Bioenergetics in Primary Human Retinal Pigment Epithelial Cells Using High-Resolution Respirometry

Realtidsanalyse af bioenergetik i primære humane retinale pigmentepitelceller ved hjælp af respirometri med høj opløsning

Metabolic dysfunction of retinal pigment epithelial cells (RPE) is a key pathogenic driver of retinal diseases such as age-related macular degeneration ...