Name: differentiation, maintenance, and analysis of human retinal pigment epithelium cells: a disease-in-a-dish model for best1 mutations
Uploaded: 2018-08-24T12:30:00
Description: Watch this Scientific Journal Video about Differentiation, Maintenance, and Analysis of Human Retinal Pigment Epithelium Cells: A Disease-in-a-dish Model for BEST1 Mutations at JoVE.com

Dette projekt blev finansieret af NIH tilskud EY025290, GM127652 og University of Rochester start-up finansiering.

1. differentiering af hPSC til ÅV
<ol><li>Vedligeholde og passage hPSCs som tidligere beskrevet18. Bemærk: Alle celler (herunder hPSCs og hPSC-RPEs) er vokset i 37 ° C i 5% CO2 i hele varigheden af protokollerne vækst og differentiering.</li>
	<li>
Forud for differentiering, opdele sammenflydende hPSCs til præ-coatede 6-godt plader.
	<ol>
<li>Coat plader, tø basalmembranen matrix på isen for ca 1 h, suspendere liquidized matrix i 4 ° C DMEM medium på en 1:...

<ol>
	<li>Allikmets, R., 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=Evaluation+of+the+Best+disease+gene+in+patients+with+age-related+macular+degeneration+and+other+maculopathies.">Evaluation of the Best disease gene in patients with age-related macular degeneration and other maculopathies.</a> Hum Genet. 104 (6), 449-453 (1999).</li><li>Burgess, R., 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=Biallelic+mutation+of+BEST1+causes+a+distinct+retinopathy+in+humans.">Biallelic mutation of BEST1 causes a distinct retinopathy in humans.</a> Am J Hum Genet. 82 (1), 19-31 (2008).</li><li>Davidson, A. 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=Missense+mutations+in+a+retinal+pigment+epithelium+protein,+bestrophin-1,+cause+retinitis+pigmentosa.">Missense mutations in a retinal pigment epithelium protein, bestrophin-1, cause retinitis pigmentosa.</a> Am J Hum Genet. 85 (5), 581-592 (2009).</li><li>Kramer, F., 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=Mutations+in+the+VMD2+gene+are+associated+with+juvenile-onset+vitelliform+macular+dystrophy+(Best+disease)+and+adult+vitelliform+macular+dystrophy+but+not+age-related+macular+degeneration.">Mutations in the VMD2 gene are associated with juvenile-onset vitelliform macular dystrophy (Best disease) and adult vitelliform macular dystrophy but not age-related macular degeneration.</a> Eur J Hum Genet. 8 (4), 286-292 (2000).</li><li>Marquardt, 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=Mutations+in+a+novel+gene,+VMD2,+encoding+a+protein+of+unknown+properties+cause+juvenile-onset+vitelliform+macular+dystrophy+(Best's+disease).">Mutations in a novel gene, VMD2, encoding a protein of unknown properties cause juvenile-onset vitelliform macular dystrophy (Best's disease).</a> Hum Mol Genet. 7 (9), 1517-1525 (1998).</li><li>Petrukhin, K., 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=Identification+of+the+gene+responsible+for+Best+macular+dystrophy.">Identification of the gene responsible for Best macular dystrophy.</a> Nat Genet. 19 (3), 241-247 (1998).</li><li>Yardley, 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=Mutations+of+VMD2+splicing+regulators+cause+nanophthalmos+and+autosomal+dominant+vitreoretinochoroidopathy+(ADVIRC).">Mutations of VMD2 splicing regulators cause nanophthalmos and autosomal dominant vitreoretinochoroidopathy (ADVIRC).</a> Invest Ophthalmol Vis Sci. 45 (10), 3683-3689 (2004).</li><li>Yang, T., Justus, S., Li, Y., Tsang, S. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=BEST1:+the+Best+Target+for+Gene+and+Cell+Therapies.">BEST1: the Best Target for Gene and Cell Therapies.</a> Mol Ther. 23 (12), 1805-1809 (2015).</li><li>Marmorstein, A. D., 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=Bestrophin,+the+product+of+the+Best+vitelliform+macular+dystrophy+gene+(VMD2),+localizes+to+the+basolateral+plasma+membrane+of+the+retinal+pigment+epithelium.">Bestrophin, the product of the Best vitelliform macular dystrophy gene (VMD2), localizes to the basolateral plasma membrane of the retinal pigment epithelium.</a> Proc Natl Acad Sci U S A. 97 (23), 12758-12763 (2000).</li><li>Boon, C. 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=The+spectrum+of+ocular+phenotypes+caused+by+mutations+in+the+BEST1+gene.">The spectrum of ocular phenotypes caused by mutations in the BEST1 gene.</a> Prog Retin Eye Res. 28 (3), 187-205 (2009).</li><li>Marmorstein, A. D., Cross, H. E., Peachey, N. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+roles+of+bestrophins+in+ocular+epithelia.">Functional roles of bestrophins in ocular epithelia.</a> Prog Retin Eye Res. 28 (3), 206-226 (2009).</li><li>Fujii, S., Gallemore, R. P., Hughes, B. A., Steinberg, R. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+evidence+for+a+basolateral+membrane+Cl-+conductance+in+toad+retinal+pigment+epithelium.">Direct evidence for a basolateral membrane Cl- conductance in toad retinal pigment epithelium.</a> Am J Physiol. 262, C374-C383 (1992).</li><li>Gallemore, R. P., Steinberg, R. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+DIDS+on+the+chick+retinal+pigment+epithelium.+II.+Mechanism+of+the+light+peak+and+other+responses+originating+at+the+basal+membrane.">Effects of DIDS on the chick retinal pigment epithelium. II. Mechanism of the light peak and other responses originating at the basal membrane.</a> J Neurosci. 9 (6), 1977-1984 (1989).</li><li>Gallemore, R. P., Steinberg, R. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Light-evoked+modulation+of+basolateral+membrane+Cl-+conductance+in+chick+retinal+pigment+epithelium:+the+light+peak+and+fast+oscillation.">Light-evoked modulation of basolateral membrane Cl- conductance in chick retinal pigment epithelium: the light peak and fast oscillation.</a> J Neurophysiol. 70 (4), 1669-1680 (1993).</li><li>Li, Y., Nguyen, H. V., Tsang, S. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Skin+Biopsy+and+Patient-Specific+Stem+Cell+Lines.">Skin Biopsy and Patient-Specific Stem Cell Lines.</a> Methods Mol Biol. 1353, 77-88 (2016).</li><li>Milenkovic, 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=Bestrophin+1+is+indispensable+for+volume+regulation+in+human+retinal+pigment+epithelium+cells.">Bestrophin 1 is indispensable for volume regulation in human retinal pigment epithelium cells.</a> Proc Natl Acad Sci U S A. 112 (20), E2630-E2639 (2015).</li><li>Moshfegh, 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=BESTROPHIN1+mutations+cause+defective+chloride+conductance+in+patient+stem+cell-derived+RPE.">BESTROPHIN1 mutations cause defective chloride conductance in patient stem cell-derived RPE.</a> Hum Mol Genet. 25 (13), 2672-2680 (2016).</li><li>Li, 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=Patient-specific+mutations+impair+BESTROPHIN1's+essential+role+in+mediating+Ca2+-dependent+Cl-+currents+in+human+RPE.">Patient-specific mutations impair BESTROPHIN1's essential role in mediating Ca2+-dependent Cl- currents in human RPE.</a> Elife. 6, (2017).</li><li>Marmorstein, L. 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=The+light+peak+of+the+electroretinogram+is+dependent+on+voltage-gated+calcium+channels+and+antagonized+by+bestrophin+(best-1).">The light peak of the electroretinogram is dependent on voltage-gated calcium channels and antagonized by bestrophin (best-1).</a> J Gen Physiol. 127 (5), 577-589 (2006).</li><li>Volland, S., Esteve-Rudd, J., Hoo, J., Yee, C., Williams, D. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+comparison+of+some+organizational+characteristics+of+the+mouse+central+retina+and+the+human+macula.">A comparison of some organizational characteristics of the mouse central retina and the human macula.</a> PLoS One. 10 (4), e0125631 (2015).</li><li>Kamao, 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=Characterization+of+human+induced+pluripotent+stem+cell-derived+retinal+pigment+epithelium+cell+sheets+aiming+for+clinical+application.">Characterization of human induced pluripotent stem cell-derived retinal pigment epithelium cell sheets aiming for clinical application.</a> Stem Cell Reports. 2 (2), 205-218 (2014).</li><li>Idelson, 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=Directed+differentiation+of+human+embryonic+stem+cells+into+functional+retinal+pigment+epithelium+cells.">Directed differentiation of human embryonic stem cells into functional retinal pigment epithelium cells.</a> Cell Stem Cell. 5 (4), 396-408 (2009).</li><li>Mandai, 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=Autologous+Induced+Stem-Cell-Derived+Retinal+Cells+for+Macular+Degeneration.">Autologous Induced Stem-Cell-Derived Retinal Cells for Macular Degeneration.</a> N Engl J Med. 376 (11), 1038-1046 (2017).</li><li>Maminishkis, 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=Confluent+monolayers+of+cultured+human+fetal+retinal+pigment+epithelium+exhibit+morphology+and+physiology+of+native+tissue.">Confluent monolayers of cultured human fetal retinal pigment epithelium exhibit morphology and physiology of native tissue.</a> Invest Ophthalmol Vis Sci. 47 (8), 3612-3624 (2006).</li><li>Maruotti, 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=A+simple+and+scalable+process+for+the+differentiation+of+retinal+pigment+epithelium+from+human+pluripotent+stem+cells.">A simple and scalable process for the differentiation of retinal pigment epithelium from human pluripotent stem cells.</a> Stem Cells Transl Med. 2 (5), 341-354 (2013).</li><li>Yang, T., He, L. L., Chen, M., Fang, K., Colecraft, H. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bio-inspired+voltage-dependent+calcium+channel+blockers.">Bio-inspired voltage-dependent calcium channel blockers.</a> Nat Commun. 4, 2540 (2013).</li><li>Yang, T., Hendrickson, W. A., Colecraft, H. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preassociated+apocalmodulin+mediates+Ca2+-dependent+sensitization+of+activation+and+inactivation+of+TMEM16A/16B+Ca2+-gated+Cl-+channels.">Preassociated apocalmodulin mediates Ca2+-dependent sensitization of activation and inactivation of TMEM16A/16B Ca2+-gated Cl- channels.</a> Proc Natl Acad Sci U S A. 111 (51), 18213-18218 (2014).</li><li>Yang, 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=Structure+and+selectivity+in+bestrophin+ion+channels.">Structure and selectivity in bestrophin ion channels.</a> Science. 346 (6207), 355-359 (2014).</li><li>Yang, T., Puckerin, A., Colecraft, H. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distinct+RGK+GTPases+differentially+use+alpha1-+and+auxiliary+beta-binding-dependent+mechanisms+to+inhibit+CaV1.2/CaV2.2+channels.">Distinct RGK GTPases differentially use alpha1- and auxiliary beta-binding-dependent mechanisms to inhibit CaV1.2/CaV2.2 channels.</a> PLoS One. 7 (5), e37079 (2012).</li><li>Yang, T., Suhail, Y., Dalton, S., Kernan, T., Colecraft, H. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetically+encoded+molecules+for+inducibly+inactivating+CaV+channels.">Genetically encoded molecules for inducibly inactivating CaV channels.</a> Nat Chem Biol. 3 (12), 795-804 (2007).</li><li>Yang, T., Xu, X., Kernan, T., Wu, V., Colecraft, H. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rem,+a+member+of+the+RGK+GTPases,+inhibits+recombinant+CaV1.2+channels+using+multiple+mechanisms+that+require+distinct+conformations+of+the+GTPase.">Rem, a member of the RGK GTPases, inhibits recombinant CaV1.2 channels using multiple mechanisms that require distinct conformations of the GTPase.</a> J Physiol. 588 (Pt 10), 1665-1681 (2010).</li><li>Gong, 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=Differentiation+of+Human+Protein-Induced+Pluripotent+Stem+Cells+toward+a+Retinal+Pigment+Epithelial+Cell+Fate.">Differentiation of Human Protein-Induced Pluripotent Stem Cells toward a Retinal Pigment Epithelial Cell Fate.</a> PLoS One. 10 (11), e0143272 (2015).</li><li>Zhang, 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=ATP+activates+bestrophin+ion+channels+through+direct+interaction.">ATP activates bestrophin ion channels through direct interaction.</a> Nat Commun. 9 (1), 3126 (2018).</li></ol>

Den vigtigste procedure til sygdom i parabol tilgang er at skelne hPSCs med en sygdomsfremkaldende mutation til den korrekte celle afstamning, som er ÅV for BEST1. Således, efter hver differentiering eksperiment, de deraf følgende hPSC-RPE celler skal omhyggeligt valideres for deres modne status af ÅV-specifikke morfologier og protein markører16,17,18. For at minimere klonede artefakt, bør flere hiPSC kloner fra d...

Det er blevet dokumenteret at mindst fem retinal degenerative sygdomme er forårsaget af genetiske mutationer i BEST1 genet1,2,3,4,5,6 , 7 , 8, med antallet af rapporterede mutationer allerede over 200 og stadig stigende. Disse BEST1-associerede sygdomme, også kendt som bestrophinopathies, forårsage progressiv synstab og endda blindhed, og der er i øjeblikket ingen effektive behandlinger. Protein produkt af BEST1, nemlig BESTROPHIN1 (BEST1), er en Ca2 +-aktiverede Cl- kanal (CaCC) specifikt kommer til udtryk i den retinale pigment epitel (ÅV) øjne5,6, 8,9. Vigtigere er, en klinisk Fænotypen af BEST1-associerede sygdomme er den reducerede visuelle reaktion på lys stimuli, kaldet light peak (LP) målt i electrooculogram10,11; LP menes at være medieret af en CaCC ÅV12,13,14. For at bedre forstå de patologiske mekanismer af BEST1 mutationer og arbejde hen imod potentielle terapier er det vigtigt at studere mutant BEST1 kanaler endogent udtrykt i ÅV menneskeceller.
Men, at opnå ÅV celler direkte fra live patienter er meget upraktisk. Selvom indfødte ÅV celler kan høstes fra biopsier af menneskelige kadavere og fostre, begrænser vanskelig adgang til disse kilder væsentligt videnskabelig forskning. Derfor er det afgørende at have alternative ÅV kilder end menneskers øjne. Dette opkald er blevet besvaret af de seneste fremskridt i stamceller teknikker, som funktionelle ÅV celler kan nu differentieres fra humane pluripotente stamceller (hPSCs), herunder embryonale stamceller (hESCs) og induceret pluripotente stamceller (hiPSCs), sidstnævnte der genereres af omprogrammering primære hud fibroblaster fra donorer16,17,18. Vigtigere, sikrer selvfornyelse og pluripotency af hPSCs en pålidelig kilde for at generere RPEs, mens patienten-specificiteten af hiPSCs og genomisk ændring potentiale af hESCs (f.eks.af CRISPR) tilbyder en alsidig sygdom i skål model for ønskede BEST1 mutationer.
hPSC-RPE har flere fordele sammenlignet med mus ÅV modeller: 1) BEST1 knockout mus ikke vise nogen retinal abnormitet19, at øge muligheden for forskellige genetiske kravet om BEST1 i ÅV mellem mus og mennesker; 2) kun 3% af ÅV menneskeceller er binucleate, i modsætning til 35% i mus20; 3) hiPSC-RPE potentiates autolog transplantation i klinisk behandling af retinal lidelser21. Ikke desto mindre dyremodeller er stadig nødvendig for at studere ÅV fysiologi og patologi i et levende system, og det muterede potentiale i hiPSC ikke blive overset.
Proceduren her beskriver en nyttig og moderat enkle hPSC til ÅV differentiering protokol, der kan bruges til forskning og kliniske formål. Denne protokol bruger nicotinamid (vitamin B3) til at forøge differentiering af hPSCs til neurale væv, som yderligere er foranlediget til at differentiere i ÅV ved behandling med activin-A. Nicotinamid behandling har vist sig at øge antallet af pigmenterede celler (et tegn på differentiering af ÅV), muligvis af formildende apoptotiske aktivitet at skelne celler22. De resulterende hPSC-RPE celler vise samme nøgle markører, brostensbelagte morfologi og cellulære funktioner som indfødte menneskelige ÅV celler22. Således i forskning omgivelser er de resulterende hPSC-RPE celler egnet til downstream funktionelle analyser herunder immunoblotting, immunfarvning og hele-celle patch clamp, som detaljeret eksperimentelle procedurer er også til rådighed. Klinisk, har ÅV celler fremstillet af stamceller vist stort potentiale for transplantation behandling af makuladegeneration i både dyreforsøg og human forsøg23.

<table>
	<tbody>
		<tr>
			<td>Knock-Out (KO) DMEM</td>
			<td>ThermoFisher</td>
			<td>10829018</td>
			<td></td>
		</tr>
		<tr>
			<td>KO serum replacement</td>
			<td>ThermoFisher</td>
			<td>10829028</td>
			<td></td>
		</tr>
		<tr>
			<td>Nonessential amino acids</td>
			<td>ThermoFisher</td>
			<td>11140050</td>
			<td></td>
		</tr>
		<tr>
			<td>Glutamine</td>
			<td>ThermoFisher</td>
			<td>35050061</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-streptomycin</td>
			<td>ThermoFisher</td>
			<td>10378016</td>
			<td></td>
		</tr>
		<tr>
			<td>Nicotinamide</td>
			<td>Sigma-Aldrich</td>
			<td>N0636</td>
			<td></td>
		</tr>
		<tr>
			<td>Human activin-A</td>
			<td>PeproTech</td>
			<td>120-14</td>
			<td></td>
		</tr>
		<tr>
			<td>MEM (a modification)</td>
			<td>Sigma-Aldrich</td>
			<td>M4526</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>VWR</td>
			<td>97068-085</td>
			<td></td>
		</tr>
		<tr>
			<td>N1 supplement</td>
			<td>Sigma-Aldrich</td>
			<td>N6530</td>
			<td></td>
		</tr>
		<tr>
			<td>Glutamine-penicillin-streptomycin</td>
			<td>Sigma-Aldrich</td>
			<td>G1146</td>
			<td></td>
		</tr>
		<tr>
			<td>Nonessential amino acids</td>
			<td>Sigma-Aldrich</td>
			<td>M7156</td>
			<td></td>
		</tr>
		<tr>
			<td>Taurine</td>
			<td>Sigma-Aldrich</td>
			<td>T0625</td>
			<td></td>
		</tr>
		<tr>
			<td>Hydrocortisone</td>
			<td>Sigma-Aldrich</td>
			<td>H0386</td>
			<td></td>
		</tr>
		<tr>
			<td>Triiodo-thyronin</td>
			<td>Sigma-Aldrich</td>
			<td>T5516</td>
			<td></td>
		</tr>
		<tr>
			<td>mTeSR-1 medium</td>
			<td>Stemcell Technologies</td>
			<td>5850</td>
			<td></td>
		</tr>
		<tr>
			<td>Matrigel</td>
			<td>Corning</td>
			<td>356230</td>
			<td></td>
		</tr>
		<tr>
			<td>Collagenase</td>
			<td>Gibco</td>
			<td>17104019</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin</td>
			<td>VWR</td>
			<td>45000-664</td>
			<td></td>
		</tr>
		<tr>
			<td>M-PER mammalian protein extraction reagent</td>
			<td>Pierce</td>
			<td>78501</td>
			<td></td>
		</tr>
		<tr>
			<td>proteinase inhibitor cocktail</td>
			<td>Sigma-Aldrich</td>
			<td>4693159001</td>
			<td></td>
		</tr>
		<tr>
			<td>RPE65 antibody</td>
			<td>Novus Biologicals</td>
			<td>NB100-355</td>
			<td></td>
		</tr>
		<tr>
			<td>CRALBP antibody</td>
			<td>Abcam</td>
			<td>ab15051</td>
			<td></td>
		</tr>
		<tr>
			<td>BEST1 antibody</td>
			<td>Novus Biologicals</td>
			<td>NB300-164</td>
			<td></td>
		</tr>
		<tr>
			<td>Beta Actin antibody</td>
			<td>ThermoFisher</td>
			<td>MA5-15739</td>
			<td></td>
		</tr>
		<tr>
			<td>Alexa Fluor 488-conjugated donkey anti-mouse IgG</td>
			<td>ThermoFisher</td>
			<td>A-21202</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat anti-mouse IgG</td>
			<td>ThermoFisher</td>
			<td>SA5-35521</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat anti-Rabbit IgG</td>
			<td>LI-COR Biosciences</td>
			<td>926-68071</td>
			<td></td>
		</tr>
		<tr>
			<td>Hoechst 33342</td>
			<td>ThermoFisher</td>
			<td>62249</td>
			<td></td>
		</tr>
		<tr>
			<td>HEKA EPC10 patch clamp amplifier</td>
			<td>Warner Instruments</td>
			<td>895000</td>
			<td></td>
		</tr>
		<tr>
			<td>Patchmaster</td>
			<td>Warner Instruments</td>
			<td>895040</td>
			<td></td>
		</tr>
	</tbody>
</table>

differentiation, maintenance, and analysis of human retinal pigment epithelium cells: a disease-in-a-dish model for best1 mutations

Selv om over 200 genetiske mutationer i det menneskelige gen, BEST1 er blevet identificeret og knyttet til retinal degenerative sygdomme, fortsat de patologiske mekanismer undvigende hovedsagelig på grund af manglen på en god i vivo model til at studere BEST1 og dens mutationer fysiologiske betingelser. BEST1 koder en ion-kanal, nemlig BESTROPHIN1 (BEST1), som fungerer i retinale pigment epitel (ÅV); imidlertid udgør meget begrænset adgang til native ÅV menneskeceller en stor udfordring for videnskabelig forskning. Denne protokol beskriver hvordan man kan generere menneskelige RPEs forsynet med BEST1 sygdomsforårsagende mutationer af inducerede differentiering fra humane pluripotente stamceller (hPSCs). Som hPSCs er selvstændige vedvarende, denne fremgangsmåde gør det muligt for forskere at have en konstant kilde til hPSC-RPEs for forskellige eksperimentelle analyser, såsom immunoblotting, immunofluorescens og patch clamp, og dermed giver en meget kraftfuld sygdom i skål model for BEST1-forbundet retinal betingelser. Denne strategi kan især anvendes til at studere ÅV (patho) fysiologi og andre gener af interesse indbygget udtrykt i ÅV.

Den teknisk mest udfordrende skridt er den manuelle isolation, som sigter mod at opnå en høj renhed af en differentieret P0 hPSC-RPE befolkning. Efter en vellykket isolation, > 90% celler i P0 befolkning vil vokse og modnes til at vise signatur ÅV morfologier (figur 1 c). Eksistensen af en mindre del af ikke-ÅV eller umodne ÅV celler i P0 befolkning er næsten uundgåeligt, men vil ikke forstyrre de downstream eksperiment...

Watch this Scientific Journal Video about Differentiation, Maintenance, and Analysis of Human Retinal Pigment Epithelium Cells: A Disease-in-a-dish Model for BEST1 Mutations at JoVE.com

Differentiation, Maintenance, and Analysis of Human Retinal Pigment Epithelium Cells: A Disease-in-a-dish Model for BEST1 Mutations

Differentiering, vedligeholdelse og analyse af menneskers retinale Pigment epitel celler: en sygdom i skål Model for BEST1 mutationer

Her præsenterer vi en protokol for at differentiere retinale pigment epitel (ÅV) celler fra humane pluripotente stamceller forsynet med patienten-afledte mutationer. De mutante cellelinjer kan anvendes til funktionelle analyser herunder immunoblotting, immunofluorescens og patch klemme. Denne sygdom i parabol fremgangsmåde omgår vanskeligheden ved at opnå indfødte ÅV menneskeceller.

Although over 200 genetic mutations in the human BEST1 gene have been identified and linked to retinal degenerative diseases, the pathological mechanisms ...

This method can help answer key questions in the ion-channel related retinal diseases field about the molecular mechanisms of BEST1 mutations and pathology of bestrophinopathies. The main advantage of this technique is that it circumvents the difficulty of directly obtaining RPE cells from humans. The implications of this technique extend toward the therapy of bestrophinopathies, including Best disease, as the RPE cells can be used for both research purposes and cell replacement therapies.When the hPSC culture reaches confluency, replace the culture medium with four milliliters of differentiation medium per well of a six-well plate. After two weeks of culture, supplement the differentiation medium with 100 nanograms per milliliter of human activin-A, changing the supplemented media three times a week for another 14 days. On day 29, switch to non-supplemented differentiation medium for another eight to 10 weeks until pigmented RPE cell clusters appear.To isolate the pigmented RPE cells, first hold individual nine-inch glass Pasteur pipettes horizontally over the flame of a Bunsen burner, about two-thirds down the length of the thin part of the pipette. When the heated glass turns soft, quickly pull the two ends apart to form a microscraper-like tip at the new end of the pipette. Spray the pulled pipettes with 70%ethanol and let them air dry in a cell culture hood.When the pipettes are ready, wash each well of the differentiation culture with PBS and add one milliliter of 0.05%trypsin supplemented with one unit per microliter of collagenase to each well. After 20 to 30 minutes at 37 degrees Celsius, stop the digestion with two milliliters of 37-degrees Celsius RPE medium per well of a six-well plate and place the plate onto a microscope stage. Using a pulled pipette, gently tap the pigmented RPE clusters.As soon as the cells detach from the bottom of the well, use a 20-microliter micropipette to transfer dissociated cells into individual wells of a new cell culture plate containing RPE medium. When approximately 5, 000 RPE cells have been added to each well of a 12-well plate, bring the final volume up to two milliliters with fresh RPE medium and culture the isolated hPSC-RPE cells for another six to eight weeks until a monolayer of pigmented and cobblestone-shaped mature RPE cells is observed. 24 to 72 hours before the patch clamp, wash the pigmented monolayer cells with two milliliters of PBS, then, after removing the PBS, add one milliliter of 0.05%trypsin plus one unit per microliter of collagenase to the cells in each well of a 12-well plate for an eight-minute incubation period at 37 degrees Celsius.When the cells begin to detach, use a one-milliliter micropipette to gently wash the cells from the bottom of the well and pool the cell suspension into a 15-milliliter conical tube. Add one milliliter of 37-degrees Celsius trypsin-collagenase solution to the well. Incubate for another eight minutes at 37 degrees Celsius, then wash any remaining cells from the bottom of the well and combine the cell suspension to the same 15-milliliter conical tube.Incubate the cells within a 37-degrees Celsius dry bath for eight minutes, then use a one-milliliter micropipette to gently triturate the cells 10 to 15 times. After incubating and triturating the cells two more times, add five milliliters of 37-degrees Celsius RPE medium to the single-cell suspension and pellet the cells by centrifugation. Resuspend the pellet in the appropriate volume for seeding onto pre-coated plates with cover slips at 10 to 20%confluency and perform whole-cell patch clamp according to standard protocols.After successful isolation, greater than 90%of the cells in the passage zero population should mature to display the signature RPE cell morphology. With the hPSC-RPE-based disease-in-a-dish approach, each patient-specific BEST1 mutation can be comprehensively characterized for its protein expression, membrane trafficking, and ion channel function to obtain critical information for elucidating RPE pathological mechanisms. It should be noted that some BEST1 mutations might affect its protein expression, so other well-established RPE markers should also be checked.Human pluripotent stem RPE cell differentiation takes three to six months, depending on the human pluripotent stem cell line and mutation type, and isolation of the differentiated human pluripotent cells can be completed in two hours if it is performed properly. While attempting to split the cells for the patch clamp, it is important to remember to triturate the cells very gently to avoid excess cell death. Following this procedure, other methods like 3D RPE cell culture can be performed to answer additional questions about how ion transport on the apical and basal membranes of RPE cells is affected by BEST1 mutations.After its development, this technique paved the way for the researchers in the field of retinal degenerative diseases to explore the physiological phenotypes and pathological mechanisms of patient-derived RPE cells. After watching this video, you should have a good understanding of how to differentiate pluripotent stem cells into retinal pigment epithelium cells and to prepare the RPE cells for whole-cell patch clamp.

Research

JoVE Journal

Genetics

Differentiation, Maintenance, and Analysis of Human Retinal Pigment Epithelium Cells: A Disease-in-a-dish Model for BEST1 Mutations

Analysis of Cap-binding Proteins in Human Cells Exposed to Physiological Oxygen Conditions

Analyse af Cap-bindende proteiner i Human celler udsat for Fysiologiske iltforhold

Translational control is a focal point of gene regulation, especially during periods of cellular stress. Cap-dependent translation via the eIF4F complex ...

Genome Editing and Directed Differentiation of hPSCs for Interrogating Lineage Determinants in Human Pancreatic Development

Genome Redigering og Directed Differentiering af hPSCs spørgekriterierne Lineage Determinanter i Human pancreas Development

Interrogating gene function in self-renewing or differentiating human pluripotent stem cells (hPSCs) offers a valuable platform towards understanding ...

Immunostaining for DNA Modifications: Computational Analysis of Confocal Images

Immunfarvning for DNA ændringer: beregningsmæssige analyse af Konfokal billeder

For several decades, 5-methylcytosine (5mC) has been thought to be the only DNA modification with a functional significance in metazoans. The discovery of ...

Highly Efficient Gene Disruption of Murine and Human Hematopoietic Progenitor Cells by CRISPR/Cas9

Højeffektive gen afbrydelse af Murine og menneskelige hæmatopoietisk stamceller ved CRISPR/Cas9

Advances in the hematopoietic stem cell (HSCs) field have been aided by methods to genetically engineer primary progenitor cells as well as animal models. ...

Single Droplet Digital Polymerase Chain Reaction for Comprehensive and Simultaneous Detection of Mutations in Hotspot Regions

Enkelt dråbe Digital Polymerase Chain Reaction for omfattende og samtidige påvisning af mutationer i Hotspot regioner

Droplet digital polymerase chain reaction (ddPCR) is a highly sensitive quantitative polymerase chain reaction (PCR) method based on sample fractionation ...

Endogenous Protein Tagging in Human Induced Pluripotent Stem Cells Using CRISPR/Cas9

Endogene Protein Tagging i menneskelige induceret pluripotente stamceller ved hjælp af CRISPR/Cas9

A protocol is presented for generating human induced pluripotent stem cells (hiPSCs) that express endogenous proteins fused to in-frame&#160;N- or ...

CRISPR-mediated Genome Editing of the Human Fungal Pathogen Candida albicans

CRISPR-mediated Genome Editing of the Human Fungal Pathogen Candida albicans

CRISPR-medieret genom-redigering af menneskelige svampe patogen Candida albicans

This method describes the efficient CRISPR mediated genome editing of the diploid human fungal pathogen Candida albicans. CRISPR-mediated genome editing ...

Digital PCR-based Competitive Index for High-throughput Analysis of Fitness in Salmonella

Digital PCR-based Competitive Index for High-throughput Analysis of Fitness in Salmonella

Digitalt PCR-baseret konkurrencemæssigt indeks til analyse af egnethed til salmonella med høj gennemløb

A competitive index is a common method used to assess bacterial fitness and/or virulence. The utility of this approach is exemplified by its ease to ...

Single-cell RNA Sequencing and Analysis of Human Pancreatic Islets

Enkelt celle-RNA-sekvensering og analyse af humane bugspytkirtel-øer

Pancreatic islets comprise of endocrine cells with distinctive hormone expression patterns. The endocrine cells show functional differences in response to ...

Identifying Mutations by High Resolution Melting in a TILLING Population of Rice

Identificering af mutationer ved høj opløsning smelter i en TILLING population af ris

Target Induced Local Lesions In Genomes (TILLING) is a strategy of reverse genetics for the high-throughput screening of induced mutations. However, the ...