Bu proje NIH hibe EY025290, GM127652 ve Rochester başlangıç finansman Üniversitesi tarafından finanse edildi.

1. hPSC RPE için farklılaşma
<ol><li>Korumak ve geçit hPSCs daha önce18açıklandığı gibi. Not: tüm hücreleri (hPSCs ve hPSC-RPEs dahil olmak üzere) %5 CO2 büyüme ve farklılaşma protokolleri süresi boyunca 37 ° C'de yetiştirilen.</li>
	<li>
Farklılaşma önce önceden kaplanmış 6-şey plakaları için konfluent hPSCs bölme.
	<ol>
<li>Tabak kat, membran matris yaklaşık 1 h için buz çözme, 1:50, 4 ° C DMEM orta liquidized matris askıya için seyreltme, her şey için 800 µL kaplama karışımı ekleyin ve en az 1 h 37 ° C'de için kuluçkaya</li>
		<li>Kaplama tamamlandığında karışım Aspire edin ve hemen 1.5-2 mL orta kuyu ekleyin.</li>
		<li>HPSCs eski kültür plakalar üzerinden kaldırmak için PBS artı 0,5 mM EDTA, oda sıcaklığında 5 min için hücrelerle kuluçkaya, çözüm Aspire edin ve kültür orta ile küçük kümeleri hücrelerde resuspend.</li>
		<li>~ % 20 confluency Yeni tabaklar içinde Tohum hücreleri. Not:-20 ° C'de 100 µL aliquots membran matris depolamak ve bir 6-şey plaka kat için bir aliquot kullanın. Kaplama süre 4 h 37 ° C'de veya gecede oda sıcaklığında genişletilebilir. Askıya alma ve hPSCs 3-5 hücreler yerine tek hücreleri küçük kümeleri tohum önemlidir.</li>
	</ol>
</li>
	<li>Hücreler için tam confluency büyüdüğün zaman, farklılaşma Orta (4 mL/de) ile kültür orta yerine (Tablo 1, aktivin olmadan-A). Kültür 14 gün, Orta (4 mL/de) değiştirme 3 x / hafta. Not: HPSC plaka yoğunluğu çiftler yaklaşık her 24 h önemli hücre ölümü farklılaşma yordamı başladıktan sonra bekleniyor. Toplamları ölü hücreleri ve enkaz Wells orta değişiklikler sırasında çıplak gözle görülebilir.</li>
	<li>Gün 15 gün 28, farklılaşma orta 100 ng/mL insan aktivin-A (Tablo 1, insan aktivin-A) ile ek. Orta (4 mL/de) değiştirin 3 x / hafta.</li>
	<li>Gün 29 (Tablo 1, insan aktivin-A olmadan) başlangıç aktivin-A takviyesi durdurmak. Hücre farklılaşması ortamda 8 – 10 pigmente hPSC-RPE kümeleri (Şekil 1A-B) görününceye hafta kültür devam edin. Not: Pigmentli hücreleri kümeleri olarak bir hücre kültür plaka üzerinde küçük karanlık noktalar (Şekil 1A-B, üst) çıplak gözle görülebilir. İmza Arnavut kaldırımı şeklinde bireysel pigmentli hücreleri 20 X mikroskop (Şekil 1A-B, alt) altında görülebilir.</li>
</ol>2. izolasyon ve farklılaştırılmış RPE hücrelerinin kültür
<ol><li>Farklılaşma orta kaldırmak, yıkama bir kez PBS ile % 0.05 tripsin artı 1 U/µL her kuyuya collagenase 1 mL ekleyin ve 20-30 dk. Kaldır tripsin/collagenase 37 ° C'de kuluçkaya ve yavaşça önceden ısıtılmış RPE orta 6-şey plaka her kuyuya 2 mL ekleyin (< C0 > tablo 2). Not: ters bir mikroskop tripsin/collagenase tedavi öncesi poligonal, hPSC-RPE hücrelerinin morfolojisi izlemek için kullanın ve sonra yeterli sindirim yuvarlak haline.</li>
	<li>
Pasteur pipetler çekme cam bir Bunsen burner kullanın.
	<ol>
<li>İki elle, uzun bir (9 inç) Pasteur pipet yatay bu yüzden her iki ucunda ve Bunsen burner yukarıda tutun yaklaşık 2/3 pipet ince parçası uzunluğu aşağı.</li>
		<li>Hızlı bir şekilde cam alev yumuşak olur sonra öyle ki mikro kazıyıcı gibi bahşiş pipet (Şekil 2) yeni sonunda oluşan iki ucu ayrı, çek.</li>
		<li>Birden çok hücre"Kazıma aletleri" oluşturmak için birkaç kere tekrar edin. Sterilizasyon için % 70 etanol ile çekti pipetler sprey ve onları hücre kültür mahallede kuruması.</li>
	</ol>
</li>
	<li>
Mikroskop altında bir mikro "hücre kazıyıcı" kullanmak yavaşça plaka kümelerden pigmente hPSC-RPE ayırmak için.
	<ol>
<li>Hafifçe dokunun (değil kazımak) doğrudan pigmentli hücreleri üzerinde hangi float kadar kültür orta bir kez onlar kuyunun dibinden ayırmak.</li>
		<li>Hemen 20 µL micropipette yavaşça Disosiye hPSC-RPE hücrelerinin emiş ve önceden kaplanmış bir 6-şey plakasına RPE orta24ile aktarmak için kullanın.</li>
		<li>Her iyi bir 12-şey tabak adımları 2.3.1–2.3.2 birden çok kez, gerektiğinde tekrar ederek ön kaplamalı ~ 5 x 103 hücre (20 X mikroskop altında gözlemlediği gibi ~ %10 confluency) toplamak. Orta son hacmi 2 mL getir. Not: Disosiye hücreleri ortamda yüzen ama hala yerel olarak konsantre kalır bu Disosiye hPSC-RPE önlemek için yüzen gelen hücreler, hareket plakaların ayrılma işlemi sırasında kaçının.</li>
	</ol>
</li>
	<li>Kültür yeni izole hPSC RPE hücreleri (P0) 12-şey plakaları RPE ortamda başka bir 6-8 hafta pigmentli monolayer (Şekil 1 c) oluşturmak izin vermek için.</li>
	<li>Orta 3 değiştirmek x / hafta. Orta değiştirirken, eski orta yaklaşık 1/3 hacmi her kuyuya bırakın ve taze, önceden ısıtılmış RPE orta hacmi 2/3 ekleyin. Bu aşamada, her şey bir 12-şey plaka RPE Orta 2 mL kullanmak (böylece, orta 1.3 mL her zaman değişimi). Not: P0 hPSC-RPE hücrelerinin monolayer kubbeler, hücrelerin aktif olarak sıvı taşınması ve sıkı kavşak25tarafından demirlemiş düşündüren oluşabilir. Bu nedenle, kubbenin oluşumu olgun RPE durumunun bir göstergesidir.</li>
</ol>3. RPE kader doğrulama Immunoblotting tarafından
<ol><li>HPSC-RPE hücre lysate memeli protein ayıklama tampon İndinavir inhibitörü ile birlikte kullanarak kokteyl yapmak. 30 dk aralıklarla 13.000 x g 4 ° C'de undissolved hücre artıkları atmak için 15 dk için de önce buz lysate cepten kuluçkaya. Toplam protein konsantrasyonu lysate ölçmek.</li>
	<li>40 denatüre µg toplam protein Laemmli SDS örnek arabelleği 75 ° C'de 10 dk. ayırmak için protein örnekleri 15 dk ve sonra 150 V boya açık ulaşır kadar 90 V sabit voltaj, % 10 Tris-glisin SDS-sayfa jel üzerinde alt jel.</li>
	<li>Proteinler nitroselüloz membran 100 V 1 h için % 10 metanol ile takıma önceden soğutulmuş Tris-glisin arabellekte üzerine aktarın.</li>
	<li>Tris arabelleğe alınmış serum %0,1 non-iyonik deterjan (TBST) ve % 5 yağsız Kuru süt oda sıcaklığında 1 h için membran engelleyin.</li>
	<li>Membran gecede 4 ° C'de engelleme arabellekte seyreltilmiş birincil antikorlar ile kuluçkaya. RPE belirli işaret proteinler aşağıdaki gibi hedefleme antikorlar seyreltik: RPE65, 1:1, 000; CRALBP, 1:1, 000; BEST1, 1:1, 000. β-aktin, β-aktin yükleme denetimi olarak algılamaya 1:10,000 karşı antikor sulandırmak.</li>
	<li>Membran TBST 3 kez, 5 min her yıkama için ile ile yıkayın.</li>
	<li>Fareyle fluorophore konjuge keçi Anti-IgG seyreltilmiş ikincil antikor 1:10,000 oda sıcaklığında 1 h için engelleme arabellekte membran kuluçkaya.</li>
	<li>Membran TBST 4 kez, 10 min her yıkama için ile ile yıkayın.</li>
	<li>Bir kızılötesi görüntüleme sistemi (Şekil 3) kullanarak proteinler algılamak.</li>
</ol>4. BEST1 hücre altı yerelleştirme Immunostaining tarafından kontrol
<ol><li>HPSC-RPE hücrelerinin PBS 2 mL ile bir kez yıkama ve % 4 paraformaldehyde 45 dakika oda sıcaklığında 2 mL fix.</li>
	<li>PBS 2 mL ile iki kez yıkama ve PBS 2 mL % 0,1 iyonik olmayan yüzey aktif ve % 2 eşek serum spesifik olmayan bağlayıcı siteleri engellemek 45 dk ile hücrelerde kuluçkaya.</li>
	<li>BEST1 antikor (1: 200 seyreltme) Oda sıcaklığında 2 h için hücrelerle kuluçkaya.</li>
	<li>PBS 3 kez 2 mL hücrelerle yıkayın. İle kuluçkaya fluorophore Birleşik oda sıcaklığında 1 h için ikincil IgG (1:1, 000).</li>
	<li>İlişkisiz ikincil antikor kaldırmak için PBS 3 kez 2 mL ile yıkayın.</li>
	<li>Lekeli hücreleri confocal mikroskobu (Şekil 4) gözlemlemek.</li>
</ol>5. kayıt Ca2 +-hPSC-RPE tarafından bütün hücreli yama kelepçe bağımlı Cl- akım
<ol><li>24-72 saat önce yama kelepçe, bölünmüş bir tam konfluent de (12-şey plaka) üzerinde olgun P0 hPSC-RPE hücrelerinin (Arnavut kaldırımı şeklinde ve Pigmentli). Orta, yıkama bir kez PBS ile Aspire edin ve 1 mL % 0.05 tripsin artı 1 U/µL collagenase kuyunun içine ekleyin. Not: önceden tripsin/collagenase kullanmadan önce 37 ° c sıcak.</li>
	<li>8 dk. 37 ° C'de kuluçkaya. Not: Bu adımdan sonra hala yapışık ve bağlı hPSC-RPE hücrelerdir.</li>
	<li>1 mL micropipette hücreleri plaka dışına hafifçe yıkamak için kullanın ve sindirim karışımı hücrelerde 15 mL konik tüp aktarın. Önceden ısıtılmış tripsin/collagenase kalan hücreleri toplamak ve onları 15 mL konik tüp içine birleştirmek için 1 mL ile iyi yıkayın. Not: HPSC-RPE hücrelerinin bu noktada hala büyük kümeleri vardır. Dinç pipetting tarafından hücre kümeleri kırmaya çalışmayın. Bazı kümeleri 1 mL ucu iç duvara yapışabilir.</li>
	<li>8 dk. 37 ° C kuru banyo konik tüp kuluçkaya.</li>
	<li>1 mL micropipette hafifçe alçalarak pipet için 10-15 kat sindirim karışımı kullanın. Not: Hücre kümeleri çok daha küçük ama hala görünür hale gelir. Dinç pipetting kaçının.</li>
	<li>Önceki iki adımı yineleyin. Not: Çoğu hücreleri tek hücre süspansiyon pipetting sonra olmalıdır. Kabul edilebilir bazı küçük hücre kümeleri olabilir. İsteğe bağlı: bir ek 5 min nazik pipetting tarafından takip için 37 ° C'de konik tüp kuluçkaya. Tripsin/collagenase 37 ° C'de toplam zaman 30 dk (8 + 8 + 8 + 5 = 29) aşmaması gerekir.</li>
	<li>Önceden ısıtılmış RPE orta 5 mL sindirilir hücreleri içeren 15 mL konik tüp ekleyin. 200 x g hücreleri toplamak 5 min için de spin. Ön kaplamalı 35 mm yemekleri, 10-%20 confluency yeniden askıya alma ve tohum hücreleri (Örneğin, % 100 de % 10 confluency için beş 35 mm yemekleri için 12-şey tabakta konfluent). Not: her zaman, güçlü pipetting, kaçının hangi aralıklarla sonra belirgin karanlık hücre artıkları süpernatant içinde gösterdiği gibi önemli hücre ölümüne neden olabilir. İyi ayrılmış tek hücreli tohum yama kelepçe kayıt için gereklidir.</li>
	<li>Bütün hücre yama gerçekleştirmek18,26,27,28,29,30,31daha önce açıklandığı gibi kelepçe. Aileden adım potansiyelleri olan geçerli izleri elde (-100 + 100 için mV holding potansiyel 0 dan mV) (Şekil 5). Not: İç ve dış çözümleri tarifleri Tablo 3 ve Tablo 4, sırasıyla açıklanmıştır.</li>
</ol>

<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>

Yazarlar ifşa gerek yok.

En önemli hastalık içinde yemek yaklaşım için hPSCs RPE BEST1için olduğunu doğru hücre lineage bir hastalığa neden olan mutasyona ile ayırt etmek için işlemdir. Böylece, her farklılaşma deneyden sonra elde edilen hPSC-RPE hücrelerinin dikkatle olgun durumları için RPE özel türleri Morfoloji ve protein işaretleri16,17,18tarafından doğrulanması. Klonal artifakı en aza indirmek için birden çok...

En az beş Retina Dejeneratif hastalıkları BEST1 gen1,2,3,4,5,6 genetik mutasyonlar neden olduğu belgelenmiş oldu , 7 , 8, bildirilen mutasyonlar zaten üzerinde 200 ve hala artan sayıda. Bu BEST1-ilişkili hastalıklar, olarak da bilinen bestrophinopathies, neden ilerleyici görme kaybı ve hatta körlüğe ve şu anda hiçbir etkili tedaviler vardır. Protein ürünü olan BEST1, yani BESTROPHIN1 (BEST1), bu bir Ca2 +-özellikle retina pigment epiteli gözler5,6, içinde (RPE) ifade aktif Cl- kanal (CaCC) 8,9. Önemlisi, BEST1, bir klinik fenotip-ilişkili hastalıklar yanıttır azaltılmış görsel ışık tepe (LP) electrooculogram10,11'; ölçülen denilen hafif uyaranlara LP RPE12,13,14CaCC tarafından aracılı inanılıyor. BEST1 mutasyonlar patolojik mekanizmaları daha iyi anlamak için ve potansiyel tedaviler doğru çalışması için mutant BEST1 kanalları endogenously ifade insan RPE hücrelerinde çalışma esastır.
RPE alma hücreleri doğrudan doğruya--dan canlı hastalar ise son derece pratik. Her ne kadar yerel RPE hücrelerinin insan kadavra ve fetusa biyopsi--dan hasat edilebilir, bu kaynakları için zor erişilebilirlik önemli ölçüde bilimsel araştırma sınırlar. Bu nedenle, insan gözü dışında alternatif RPE kaynaklar için önemlidir. Fonksiyonel RPE hücrelerinin şimdi insan pluripotent kök embriyonik kök hücreler (hESCs) de dahil olmak üzere hücre (hPSCs), Ayrıştırılan ve pluripotent kök hücreler (hiPSCs), ikinci indüklenen gibi bu çağrı kök hücre teknikleri, son gelişmeler tarafından yanıtlandı Birincil deri fibroblastlar bağış16,17,18yeniden programlama tarafından oluşturulan. Önemlisi, kendini yenileme ve pluripotency hPSCs, RPEs, hiPSCs hasta özgüllük ve hESCs (Örneğin, CRISPR tarafından) potansiyelini genomik değişiklik için çok yönlü bir çanak içinde hastalık modeli sunarken oluşturmak için güvenilir bir kaynak sağlamak İstenen BEST1 mutasyonlar.
hPSC-RPE fareler RPE modelleri birçok avantajı vardır: 1) BEST1 nakavt fareler fareler ve insanlar; arasında BEST1 RPE içinde farklı genetik gereksinimi olasılığını yükseltmek herhangi bir retina anormallik19, görüntü yok 2) yalnızca %3 insan RPE hücrelerinin binucleate, fareler%2035 aksine; 3) hiPSC-RPE potentiates klinik olarak otolog transplantasyon Retina tedavisinde bozuklukları21. Yine de hayvan modelleri hala canlı bir sistem içinde RPE fizyolojisi ve patoloji eğitimi için vazgeçilmezdir ve hiPSC oncogenic potansiyelini göz ardı edemem.
Araştırma ve klinik amaçlar için kullanılabilir RPE farklılaşma protokolü için kullanışlı ve orta derecede basit hPSC burada ile ilgili yordamı açıklamaktadır. HPSCs için daha fazla RPE ayırt etmek için tedavi aktivin-a. tarafından indüklenen olduğu sinir dokusu, farklılaşma çoğaltmak için nikotinamid (B3 vitamini) bu iletişim kuralını kullanır Nikotinamid tedavi pigmentli hücreleri (bir işareti farklılaşma RPE içine), sayısını artırmak için muhtemelen apoptotik hücreler22ayırt faaliyet inceltiyorum tarafından gösterilmiştir. Elde edilen hPSC-RPE hücreler aynı anahtar işaretleri, Arnavut kaldırımı Morfoloji ve hücresel işlevselliği yerel insan RPE hücrelerinin22görüntüler. Böylece, bir araştırma ortamda elde edilen hPSC-RPE hücrelerinin immunoblotting, immunostaining ve bütün hücreli yama kelepçe, kendisi için de ayrıntılı deneysel yordamlar sağlanmaktadır gibi aşağı akım fonksiyonel analizleri için uygundur. Klinik olarak, kök hücrelerden türetilmiş RPE hücrelerinin glokom tedavisinde nakli için büyük bir potansiyel hayvan çalışmaları ve insan denemeler23göstermiştir.

<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

Genetik mutasyonların 200'den fazla insan BEST1 gen tespit ve Retina dejeneratif hastalıklara bağlı olsa da, patolojik mekanizmaları özellikle BEST1 çalışmak için iyi vivo içinde manken eksikliği nedeniyle zor kalır ve onun mutasyonlar fizyolojik koşullar altında. BEST1 kodlar bir iyon kanalı, yani BESTROPHIN1 (BEST1), hangi işlevleri retina pigment epiteli (RPE); Ancak, yerel insan RPE hücrelerinin için son derece sınırlı erişilebilirlik bilimsel araştırmalar için büyük bir sorun temsil eder. Bu iletişim kuralı, insan RPEs insan pluripotent kök hücreler (hPSCs) üzerinden indüklenen farklılaşma tarafından BEST1 hastalığa neden olan mutasyonlar taşıyan oluşturmak açıklar. HPSCs kendi kendine yenilenebilir olduğu gibi bu yaklaşım araştırmacılar hPSC-RPEs immunoblotting, ayirt ve yama kelepçe, gibi çeşitli deneysel analizleri için sabit bir kaynağı olmasını sağlar ve böylece için çok güçlü bir çanak içinde hastalık modeli sağlar BEST1-Retina koşulları ilişkili. Özellikle, bu strateji RPE (patho) Fizyoloji ve diğer genler özgün olarak içinde RPE ifade ilgi çalışmaya uygulanır.

Farklılaştırılmış P0 hPSC-RPE popülasyondan alınmış bir yüksek saflıkta elde amaçlamaktadır el ile yalıtım teknik açıdan en zor adımdır. Sonra başarılı bir yalıtım > P0 popülasyonda % 90 hücreleri büyüyecek ve imza RPE türleri morfoloji (Şekil 1 c) görüntülemek için olgun. RPE sigara veya P0 nüfus olgunlaşmamış RPE hücrelerinde küçük bir bölümünü varlığını neredeyse kaçınıl...

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

Farklılaşma, bakım ve insan Retina Pigment epitel hücre Analizi: BEST1 mutasyonlar için bir tabak içinde hastalık modeli

Burada retina pigment epiteli (RPE) hücreleri insan pluripotent kök hücre hasta kaynaklı mutasyonlar taşıyan ayırt etmek için bir iletişim kuralı mevcut. Mutant hücre hatları immunoblotting, ayirt ve yama kelepçe gibi fonksiyonel analizleri için kullanılabilir. Bu hastalık içinde yemek yaklaşım yerel insan RPE hücrelerinin alma zorluğu kaçınmanızı sağlar.

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

differentiation-maintenance-analysis-human-retinal-pigment-epithelium

Research

JoVE Journal

Genetics

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

6.7K Görüntüleme.  University of Rochester, School of Medicine and Dentistry. Burada retina pigment epiteli (RPE) hücreleri insan pluripotent kök hücre hasta kaynaklı mutasyonlar taşıyan ayırt etmek için bir iletişim kuralı mevcut. Mutant hücre hatları immunoblotting, ayirt ve yama kelepçe gibi fonksiyonel analizleri için kullanılabilir. Bu hastalık içinde yemek yaklaşım yerel insan RPE hücrelerinin alma zorluğu kaçınmanızı sağlar.

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

Efficient Derivation of Retinal Pigment Epithelium Cells from Stem Cells

No cure has been discovered for age-related macular degeneration (AMD), the leading cause of vision loss in people over the age of 55. AMD is complex multifactorial disease with an unknown etiology, although it is largely thought to occur due to death or dysfunction of the retinal pigment epithelium (RPE), a monolayer of cells that underlies the retina and provides critical support for photoreceptors. RPE cell replacement strategies may hold great promise for providing therapeutic relief for a large subset of AMD patients, and RPE cells that strongly resemble primary human cells (hRPE) have been generated in multiple independent labs, including our own. In addition, the uses for iPS-RPE are not limited to cell-based therapies, but also have been used to model RPE diseases. These types of studies may not only elucidate the molecular bases of the diseases, but also serve as invaluable tools for developing and testing novel drugs. We present here an optimized protocol for directed differentiation of RPE from stem cells. Adding nicotinamide and either Activin A or IDE-1, a small molecule that mimics its effects, at specific time points, greatly enhances the yield of RPE cells. Using this technique we can derive large numbers of low passage RPE in as early as three months.

Stem cell-derived retinal pigment epithelium (RPE) cells may be used for multiple applications including cell-based therapies for retinal degeneration, disease modeling, and drug studies. Here we present a simple protocol for reproducibly deriving RPE from stem cells.

Kök Hücreleri Retina pigment epiteli hücrelerinin Verimli türetilmesi

No cure has been discovered for age-related macular degeneration  (AMD), the leading cause of vision loss in people over the age of 55. AMD is complex ...

Gelişim Biyolojisi

Experimental Models for Study of Retinal Pigment Epithelial Physiology and Pathophysiology

We have developed a cell culture procedure that can produce large quantities of confluent monolayers of primary human fetal retinal pigment epithelium (hfRPE) cultures with morphological, physiological and genetic characteristics of native human RPE. These hfRPE cell cultures exhibit heavy pigmentation, and electron microscopy show extensive apical membrane microvilli. The junctional complexes were identified with immunofluorescence labeling of various tight junction proteins. Epithelial polarity and function of these easily reproducible primary cultures closely resemble previously studied mammalian models of native RPE, including human. These results were extended by the development of therapeutic interventions in several animal models of human eye disease. We have focused on strategies for the removal of abnormal fluid accumulation in the retina or subretinal space. The extracellular subretinal space separates the photoreceptor outer segments and the apical membrane of the RPE and is critical for maintenance of retinal attachments and a whole host of RPE/retina interactions.

We provide a reproducible method for culturing confluent monolayers of human fetal retinal pigment epithelial cells (hfRPE) cells that exhibit morphology, physiology, polarity, and protein and gene expression patterns of adult native tissue. This work has been extended to an animal model of several eye diseases.

Retina Pigment Epitel Fizyoloji ve Patofizyoloji Çalışma Deneysel Modeller

We have developed a cell culture procedure that can produce large quantities of confluent monolayers of primary human fetal retinal pigment epithelium ...

Nörobilim

Defined Xeno-free and Feeder-free Culture Conditions for the Generation of Human iPSC-derived Retinal Cell Models

The production of specialized cells from pluripotent stem cells provides a powerful tool to develop new approaches for regenerative medicine. The use of human-induced pluripotent stem cells (iPSCs) is particularly attractive for neurodegenerative disease studies, including retinal dystrophies, where iPSC-derived retinal cell models mark a major step forward to understand and fight blindness. In this paper, we describe a simple and scalable protocol to generate, mature, and cryopreserve retinal organoids. Based on medium changing, the main advantage of this method is to avoid multiple and time-consuming steps commonly required in a guided differentiation of iPSCs. Mimicking the early phases of retinal development by successive changes of defined media on adherent human iPSC cultures, this protocol allows the simultaneous generation of self-forming neuroretinal structures and retinal pigmented epithelial (RPE) cells in a reproducible and efficient manner in 4 weeks. These structures containing retinal progenitor cells (RPCs) can be easily isolated for further maturation in a floating culture condition enabling the differentiation of RPCs into the seven retinal cell types present in the adult human retina. Additionally, we describe quick methods for the cryopreservation of retinal organoids and RPE cells for long-term storage. Combined together, the methods described here will be useful to produce and bank human iPSC-derived retinal cells or tissues for both basic and clinical research.

The production of specialized retinal cells from pluripotent stem cells is a turning point in the development of stem cell-based therapy for retinal diseases. The present paper describes a simple method for an efficient generation of retinal organoids and retinal pigmented epithelium for basic, translational, and clinical research.

Tanımlanmış Xeno-alerjik ve besleyici-Alerjik kültür koşulları üretimi insan IPSC kaynaklı Retina hücre modelleri için

The production of specialized cells from pluripotent stem cells provides a powerful tool to develop new approaches for regenerative medicine. The use of ...

Generation of Human Patient iPSC-derived Retinal Organoids to Model Retinitis Pigmentosa

Retinitis pigmentosa (RP) is a rare and inherited retinal degenerative disease with a prevalence of approximately 1/4,000 people worldwide. The majority of RP patients have progressive photoreceptor degeneration leading to peripheral vision loss, night blindness, and finally, total blindness. To date, thousands of mutations in more than 90 genes have been reported to be associated with RP. Currently, there are few animal models available for all the affected genes and different types of mutations, which largely hampers the deciphering of the mechanisms underlying the gene/mutation pathology and limits treatment and drug development. Patient induced pluripotent stem cell (iPSC)-derived 3D retinal organoids (ROs) have provided a better system to model the human early-onset disease than cells and animals. In order to study RP, those patient-derived 3D retinal organoids were utilized to recapitulate the clinical phenotypes of RP. In the RP patient-derived ROs, Rhodopsin mislocalization was clearly displayed. Compared with other animal models, patient iPSC-derived retinal organoid models more closely recapitulated RP features and represent an ideal approach for investigating the disease pathogenesis and for drug development.

In this protocol, retinitis pigmentosa patient induced pluripotent stem cell (iPSC)-derived 3D retinal organoids were generated. Those organoids successfully recapitulated some clinical phenotypes of the retinitis pigmentosa disease.

Retinitis pigmentosa (RP) is a rare and inherited retinal degenerative disease with a prevalence of approximately 1/4,000 people worldwide. The majority ...

Generation of Retinal Organoids from Healthy and Retinal Disease-Specific Human-Induced Pluripotent Stem Cells

Pluripotent stem cells can generate complex tissue organoids that are useful for in vitro disease modeling studies and for developing regenerative therapies. This protocol describes a simpler, robust, and stepwise method of generating retinal organoids in a hybrid culture system consisting of adherent monolayer cultures during the first 4 weeks of retinal differentiation till the emergence of distinct, self-organized eye field primordial clusters (EFPs). Further, the doughnut-shaped, circular, and translucent neuro-retinal islands within each EFP are manually picked and cultured under suspension using non-adherent culture dishes in a retinal differentiation medium for 1-2 weeks to generate multilayered 3D optic cups (OC-1M). These immature retinal organoids contain PAX6+ and ChX10+ proliferating, multipotent retinal precursors. The precursor cells are linearly self-assembled within the organoids and appear as distinct radial striations. At 4 weeks after suspension culture, the retinal progenitors undergo post-mitotic arrest and lineage differentiation to form mature retinal organoids (OC-2M). The photoreceptor lineage committed precursors develop within the outermost layers of retinal organoids. These CRX+ and RCVRN+ photoreceptor cells morphologically mature to display inner segment-like extensions. This method can be adopted for generating retinal organoids using human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs). All steps and procedures are clearly explained and demonstrated to ensure replicability and for wider applications in basic science and translational research.

This protocol describes an efficient method of differentiating hiPSCs into eye field clusters and generating neuro-retinal organoids using simplified culture conditions involving both adherent and suspension culture systems. Other ocular cell types, such as the RPE and corneal epithelium, can also be isolated from mature eye fields in retinal cultures.

Sağlıklı ve Retina Hastalığına Özgü İnsan Kaynaklı Pluripotent Kök Hücrelerden Retinal Organoidlerin Üretimi

Pluripotent stem cells can generate complex tissue organoids that are useful for in vitro disease modeling studies and for developing regenerative ...

Toxicity Screens in Human Retinal Organoids for Pharmaceutical Discovery

Organoids provide a promising platform to study disease mechanism and treatments, directly in the context of human tissue with the versatility and throughput of cell culture. Mature human retinal organoids are utilized to screen potential pharmaceutical treatments for the age-related retinal degenerative disease macular telangiectasia type 2 (MacTel).
We have recently shown that MacTel can be caused by elevated levels of an atypical lipid species, deoxysphingolipids (deoxySLs). These lipids are toxic to the retina and may drive the photoreceptor loss that occurs in MacTel patients. To screen drugs for their ability to prevent deoxySL photoreceptor toxicity, we generated human retinal organoids from a non-MacTel induced pluripotent stem cell (iPSC) line and matured them to a post-mitotic age where they develop all of the neuronal lineage-derived cells of the retina, including functionally mature photoreceptors. The retinal organoids were treated with a deoxySL metabolite and apoptosis was measured within the photoreceptor layer using immunohistochemistry. Using this toxicity model, pharmacological compounds that prevent deoxySL-induced photoreceptor death were screened. Using a targeted candidate approach, we determined that fenofibrate, a drug commonly prescribed for the treatment of high cholesterol and triglycerides, can also prevent deoxySL toxicity in the cells of the retina.
The toxicity screen successfully identified an FDA-approved drug that can prevent photoreceptor death. This is a directly actionable finding owing to the highly disease-relevant model tested. This platform can be easily modified to test any number of metabolic stressors and potential pharmacological interventions for future treatment discovery in retinal diseases.

Here we present a step-by-step protocol to generate mature human retinal organoids and utilize them in a photoreceptor toxicity assay to identify pharmaceutical candidates for the age-related retinal degenerative disease macular telangiectasia type 2 (MacTel).

Farmasötik Keşif için İnsan Retinal Organoidlerinde Toksisite Taramaları

Organoids provide a promising platform to study disease mechanism and treatments, directly in the context of human tissue with the versatility and ...

Biyomühendislik

Characterization of a Novel Human Organotypic Retinal Culture Technique

Previous human organotypic retinal culture (HORC) models have utilized detached retinas; however, without the structural support conferred by retinal pigment epithelium-choroid (RPE-choroid) and sclera, the integrity of the fragile retina can easily be compromised. The aim of this study was to develop a novel HORC model that contains the retina, RPE-choroid and sclera to maintain retinal integrity when culturing retinal explants.
After cutting circumferentially along the limbus to remove iris and lens, four deep incisions were made to flatten the eyecup. In contrast to previous HORC protocols, a trephine was used to cut through not only the retina but also the RPE-choroid and sclera. The resultant triple-layered explants were cultured for 72 h. Hematoxylin and Eosin staining (H&#38;E) was used to assess anatomical structures and retinal explants were further characterized by immunohistochemistry (IHC) for apoptosis, M&#252;ller cell integrity and retinal inflammation. To confirm the possibility of disease induction, explants were exposed to high glucose (HG) and pro-inflammatory cytokines (Cyt), to mimic diabetic retinopathy (DR). The Luminex magnetic bead assay was used to measure DR-related cytokines released into the culture medium.
H&#38;E staining revealed distinct retinal lamellae and compact nuclei in retinal explants with the underlying RPE-choroid and sclera, while retinas without the underlying structures exhibited reduced thickness and severe nuclei loss. IHC results indicated absence of apoptosis and retinal inflammation as well as preserved M&#252;ller cell integrity. The Luminex&#160;assays showed significantly increased secretion of DR-associated pro-inflammatory cytokines in retinal explants exposed to HG + Cyt relative to baseline levels at 24 h.
We successfully developed and characterized a novel HORC protocol in which retinal integrity was preserved without apoptosis or retinal inflammation. Moreover, the induced secretion of DR-associated pro-inflammatory biomarkers when exposing retinal explants to HG + Cyt suggests that this model could be used for clinically translatable retinal disease studies.

This study aims to develop a novel human organotypic retinal culture (HORC) model that prevents compromising retinal integrity during explant handling. This is achieved by culturing the retina with the overlying vitreous and the underlying retinal pigment&#160;epithelium-choroid (RPE-choroid) and sclera.

Yeni bir İnsan Organotipik Retina Kültürü Tekniğinin Karakterizasyonu

Previous human organotypic retinal culture (HORC) models have utilized detached retinas; however, without the structural support conferred by retinal ...

Tıp

Isolation, Culture, and Genetic Engineering of Mammalian Primary Pigment Epithelial Cells for Non-Viral Gene Therapy

Age-related macular degeneration (AMD) is the most frequent cause of blindness in patients &#62;60 years, affecting ~30 million people worldwide. AMD is a multifactorial disease influenced by environmental and genetic factors, which lead to functional impairment of the retina due to retinal pigment epithelial (RPE) cell degeneration followed by photoreceptor degradation. An ideal treatment would include the transplantation of healthy RPE cells secreting neuroprotective factors to prevent RPE cell death and photoreceptor degeneration. Due to the functional and genetic similarities and the possibility of a less invasive biopsy, the transplantation of iris pigment epithelial (IPE) cells was proposed as a substitute for the degenerated RPE. Secretion of neuroprotective factors by a low number of subretinally-transplanted cells can be achieved by Sleeping Beauty (SB100X) transposon-mediated transfection with genes coding for the pigment epithelium-derived factor (PEDF) and/or the granulocyte macrophage-colony stimulating factor (GM-CSF). We established the isolation, culture, and SB100X-mediated transfection of RPE and IPE cells from various species including rodents, pigs, and cattle. Globes are explanted and the cornea and lens are removed to access the iris and the retina. Using a custom-made spatula, IPE cells are removed from the isolated iris. To harvest RPE cells, a trypsin incubation may be required, depending on the species. Then, using RPE-customized spatula, cells are suspended in medium. After seeding, cells are monitored twice per week and, after reaching confluence, transfected by electroporation. Gene integration, expression, protein secretion, and function were confirmed by qPCR, WB, ELISA, immunofluorescence, and functional assays. Depending on the species, 30,000-5 million (RPE) and 10,000-1.5 million (IPE) cells can be isolated per eye. Genetically modified cells show significant PEDF/GM-CSF overexpression with the capacity to reduce oxidative stress and offers a flexible system for ex vivo&#160;analyses and in vivo studies transferable to humans to develop ocular gene therapy approaches.

Here, a protocol to isolate and transfect primary iris and retinal pigment epithelial cells from various mammals (mice, rat, rabbit, pig, and bovine) is presented. The method is ideally suited to study ocular gene therapy approaches in various set-ups for ex vivo&#160;analyses and&#160;in vivo studies transferable to humans.

Viral Olmayan Gen Tedavisi için Memeli Primer Pigment Epitel Hücrelerinin İzolasyonu, Kültürü ve Genetik Mühendisliği

Age-related macular degeneration (AMD) is the most frequent cause of blindness in patients &#62;60 years, affecting ~30 million people worldwide. AMD is a ...

Biyoloji

Efficient Dissection and Culture of Primary Mouse Retinal Pigment Epithelial Cells

Eye disorders affect millions of people worldwide, but the limited availability of human tissues hinders their study. Mouse models are powerful tools to understand the pathophysiology of ocular diseases because of their similarities with human anatomy and physiology. Alterations in the retinal pigment epithelium (RPE), including changes in morphology and function, are common features shared by many ocular disorders. However, successful isolation and culture of primary mouse RPE cells is very challenging. This paper is an updated audiovisual version of the protocol previously published by Fernandez-Godino et al. in 2016 to efficiently isolate and culture primary mouse RPE cells. This method is highly reproducible and results in robust cultures of highly polarized and pigmented RPE monolayers that can be maintained for several weeks on Transwells. This model opens new avenues for the study of the molecular and cellular mechanisms underlying eye diseases. Moreover, it provides a platform to test therapeutic approaches that can be used to treat important eye diseases with unmet medical needs, including inherited retinal disorders and macular degenerations.

This protocol, which was originally reported by Fernandez-Godino et al. in 20161, describes a method to efficiently isolate and culture mouse RPE cells, which form a functional and polarized RPE monolayer within one week on Transwell plates. The procedure takes approximately 3 hours.

Birincil Fare Retina Pigment Epitel Hücrelerinin Verimli Diseksiyonu ve Kültürü

Eye disorders affect millions of people worldwide, but the limited availability of human tissues hinders their study. Mouse models are powerful tools to ...

<meta charset="utf8"></head><body>Kemirgen Gözbebeklerinde Retina Kalınlığı ve Ganglion Hücre Tabakası Sayılarının Değerlendirilmesi

Preparation and Analysis of Histological Slides of Rat and Mouse Eyeballs to Evaluate the Retina

A rodent eyeball is a powerful tool for researching the pathomechanisms of many ophthalmic diseases, such as glaucoma, hypertensive retinopathy, and many more. Preclinical experiments enable researchers to examine the efficacy of novel drugs, develop new methods of treatment, or seek new pathomechanisms involved in the disease's onset or progression. A histological examination provides a lot of information necessary to assess the effects of the conducted experiments and can reveal degeneration, tissue remodeling, infiltration, and many other pathologies. In clinical research, there is rarely any chance of obtaining eye tissue suitable for a histological examination, which is why researchers should take advantage of the opportunity offered by the examination of eyeballs from rodents. This manuscript presents a protocol for the histological preparation of rodent eyeballs' sections. The procedure is presented for the eyeballs of mice and rats and has the following steps: (i) harvesting the eyeball, (ii) preserving the eyeball for further analysis, (iii) processing the tissue in paraffin, (iv) preparing slides, (v) staining with hematoxylin and eosin, (vi) assessing the tissue under a light microscope. With the proposed method, the retina can be easily visualized and assessed in detail.

<meta charset="utf8"></head><body>Retinayı değerlendirmek için sıçan ve fare gözbebeklerinin histolojik slaytlarının hazırlanması ve analizi

This manuscript presents a protocol for the histological preparation of slides from rodent eyeballs. With the proposed method, the inner retina can be easily visualized and assessed under a light microscope. The procedure is presented for the eyeballs of mice and rats.

Retinayı değerlendirmek için sıçan ve fare gözbebeklerinin histolojik slaytlarının hazırlanması ve analizi

A rodent eyeball is a powerful tool for researching the pathomechanisms of many ophthalmic diseases, such as glaucoma, hypertensive retinopathy, and many ...