Yazarlar plasental doku sağlamak için Soon-Cheol Hong (MD, Ph.D., Doçent, Kadın Hastalıkları ve Doğum Anabilim Dalı, Medical College Kore Üniversitesi) teşekkür etmek istiyorum. Bu eser Kore Ulusal Araştırma Vakfı (UÇK), Kore Cumhuriyeti hibe (R1211902) tarafından kısmen desteklenmiştir.

Etik bildirimi: İnsan plasenta çalışması Kore Üniversitesi (AN09085-001) insan araştırma için Kurumsal Değerlendirme Kurulu onayı ile, ileriye dönük olarak. Tüm deneyler Kore Üniversitesi Tıp Merkezi&#39;nde Temiz Germ-Alerjik Oda tesis yapıldı. deneysel tasarım ve hPSCs kullanarak prosedürleri Kore Üniversitesi Tıp Merkezi Kurumsal Değerlendirme Kurulu (AN12277-003) tarafından onaylanmıştır. Araçların, Kültür Medya ve Yemekleri 1. Hazırlık <ol><li> % 0.1 jelatin ile kaplayın tüm kültür yemekleri. Otoklav fosfat tamponlu tuz (PBS) ve forseps. Birden fazla 15 dakika £ 15 psi altında 121 ° C &#39;de, cerrahi makas, mikrosantrifüj tüpleri, gazlı bez sterilize edin. </li><li> Hazırlanan önceden ısıtılmış% 0.25 tripsin-etilen amin tetra-asetik asit (tripsin-EDTA) ve önceden ısıtılmış, süzülmüş kültür ortamı (DMEM)% 20 fetal sığır serumu (FBS), 100 U / ml penisilin içeren ve 100 ug / ml streptomycprosedürleri başlamadan önce. </li></ol> Insan plasentasından 2. Hücre İzolasyonu <ol><li> Yazılı onam alındıktan sonra gebelik 7-32 haftalık normal veya terapötik kürtaj geçiren sağlıklı gebe kadınların sezaryen doğumdan sonra plasenta dokusu, edinin. </li><li> Plasenta obstetrik uzman cerrahi ayrı insan plasenta koryonik plakaları (HPC) var; 20 dakika boyunca 37 ° C&#39;de% 0.25 tripsin-EDTA, bu inkübe, ve daha sonra 5 ml PBS ile yıkayın. </li><li> Makas kullanarak HPC gelen koryonik villus izole edin; villus kıyma ve 1ml PBS ile onları üç kez yıkayın. Ufalamadan sonra bir pipet kullanarak bir mikrofüj tüpü içine koryonik villi yer; aşağı yukarı pipetleme ve 500 ul PBS ile numuneler yıkayın ve 3 dakika boyunca 120 xg için tüpler santrifüj. Önceden ısıtılmış kültür ortamında 500 ul iki kez dokuları yıkayın. </li><li> 37 ° C &#39;de, jelatin-kaplanmış plakalar üzerinde ortam içinde kültür hücreleri,% 5 CO2 içinde </sub&gt; ve% 95 altında nem. Ön kaplama, 37 ° C&#39;de 1 saat süre ile,% 0.1 jelatin ile, tüm plakalar. Fibroblast-benzeri hücrelerin koloniler oluşturur ve yaklaşık 5-7 gün, aynı ortam içinde kültür başladıktan sonra yapışkan bir büyüme olur. </li><li> Üçüncü pasaj kadar her 2-3 günde bir orta değiştirin. Kültür sırasında, kültür ortamı içinde yüzen hücre birikintilerinin ayrılmasıyla; büyüyen plasental fibroblastlar plaka eklenecektir. Kültür daha sonra 10. geçide HPC kadar elde edilen ve fibroblast-benzeri hücreler tripsinizasyon ve mitomisin-C (10 ug / ml) tedavi sonrası HESC kültürleri için hasat. </li><li> Hisse senedi örnekleri olarak HPC dondurmadan önce, HPC genellikle (M. fermentans, M. hyorhinis, M. Arginin, M mikoplazma dahil plasentayı, enfekte patojenler ile kontamine olmadığını teyit etmek için ters transkripsiyon-polimeraz zincir reaksiyonu (RT-PCR) kullanımı . Orale, M. salivarium, M. hominis, M. pulmonis, M. arthritidis, M. neurolyticum, M. hyopneumoniae,ve M. capricolum) ve bakteriyel türler, Ureaplasma urealyticum, bir mikoplazma belirleme kiti kullanılarak. <ol><li> RT-PCR gerçekleştirmek için üreticinin talimatlarına uyun kitte sağlanan tüm primerler ve karışımları kullanmak. Mycoplasma tespiti için 36 gün süre ile kültive edilmiştir hücre süpernatantlar kullanın. PCR karışımı, bu örneklerin 3 ul ekleyin ve 30 saniye, 2 dakika için 55 ° C ve 1 dakika süre ile 72 ° C&#39;de her biri 94 ° C kapsayan 35 çevrim için 1 st PCR prosedürü uygulayın. </li><li> Dikkatle PCR karışımı 1 st PCR ürünü, 0.5 ul ve daha önce olduğu gibi aynı şartlar kullanılarak 30 çevrim için 2. PCR prosedürü uygulayın. % 1 agaroz jel elektroforez işlemi aracılığı ile amplifiye ürünlerin analiz; jeller 1 ve 2. PCR ürünlerinin her birinin 10 ul geçerlidir. </li></ol></li><li> HPC patojenler ile kontamine olduğu tespit edilmiştir, bu ortadan kaldırmaküreticinin talimatlarına göre, bir antibiyotik kombinasyonu (BM-siklin) kullanılarak gerçekleştirilmiştir. 3 gün BM-siklin 1 10 ug / ml ihtiva eden yeni ortam ekleyin ve daha sonra başka bir 4 gün için 5 | ig / ml BM-siklin 2 ile hücreleri tedavi. Iki kez bu döngüyü tekrarlayın ve sonra tekrar Mycoplasma kirlenme kontrol edin. </li></ol> 3. Hasat İnsan Plasenta türetilmiş hücreler Orta (hPCCM) Klima <ol><li> Kirlenme hariç sonra, bağlanmış hücreler (85-90% konfluansa tedavi (1.5 x 10 7 hücre / 2 saat ve 30 dakika boyunca mitomisin-C (10 ug / mi) ile bir şişe) ve 10 ml hücreler üç kez yıkandı PBS ile yıkandı. 24 saat, mitomisin-C olmayan önceden ısıtılmış ortam ile inkübe hücreleri. </li><li> Bir gün tedaviden sonra, 10 mi ortam içinde hücrelerin inkübe (DMEM-F12,% 20 nakavt serum Değiştirilmesi [KOSR] ile takviye edilmiş, 0.1 mM β-merkaptoetanol,% 1 NEAA ve% 1 penisilin-streptomisin). 15 ml konik bir tüp içinde 24 saat inkübasyon, hasat CM sonra ve0.22 um şırınga filtresi kullanılarak cm filtre. </li><li> Yeni orta ve inkübasyon bir 10 ml ekleyin. 1 hafta boyunca her gün kültürlerden tüm süpernatantlar toplayın ve -80 ° C&#39;de hasat aracı madde dondurularak. Tekrarlanan donma-erime döngülerinden kaçınmak. </li></ol> 4. Kültür İnsan Embriyonik Kök Hücreleri (hESC) ve Pluripotent Kök Hücre İndüksiyon (iPSCs) <ol><li> İnsan embriyonik kök hücre hatları elde etmek, (sırasıyla, isim WA01 ve WA09 altında NIH HESC kayıt defterinde listelenen) H1 ve H9 hücreleri ve uyarılmış pluripotent kök hücre-iPSC-1: iPS (sünnet derisi) -1 ve iPSC-2 (IISH1i- WiCell Araştırma Enstitüsü (Madison, WI, ABD) BM). Hücreler, üreticinin talimatları izlenerek çözündürüldükten ve kültüre olmalıdır. </li><li> Kontrol grubu için, 37 ° C &#39;de yaygın olarak kullanılan besleyici içermeyen ve serum içermeyen tanımlanmış bir kültür ortamı içinde Bazal Membran Matrisi-kaplı kaplar üzerinde ve% 5 CO2 kültür hücreleri. Başlangıçta, hPSCs 80-100 kümeleri tohum in 35 mm yemekleri. 70-80% izdiham ve rutin geçiş hücreleri kadar bu yemekleri hücreleri büyümek alt kültüre, her 5-6 günde bir, mekanik veya enzimatik yöntemler (Dispase) kullanarak. Ortam ile hücreler iki kez yıkanır ve 1 arasında bir oranda bunların plaka: 4 arasındadır. Her gün taze ortam ile orta değiştirin. </li><li> Deney grupları için, yemekler hPCCM kültür hücreleri ya mekanik ya da enzimatik olarak,% 0.1 jelatin ile ön-kaplanmış ve bir kez 5 günde, rutin ile gerçekleşen pasajlanmasını da yerine getirir. 6-1: 10: 1 aralığı içinde oranlarında plakası hücreleri. hPSCs transferden sonra 2 gün içinde jelatin ekleyin. Günlük taze ortam ile orta değiştirin. </li></ol> İnsan Pluripotent Kök Hücre 5. Karakterizasyonu Not: alkalin fosfataz boyama, İmmünositokimya, kantitatif gerçek zamanlı PCR (qRT-PCR) ve western blotting gibi çeşitli yöntemler kullanılarak hPSCs karakterize eder. <ol><li> İmmünfloresan boyama <ol><li> Immunofluorescenc içinE boyama, kültür hPSCs% 4, 10 dakika boyunca (ağırlık / hacim) paraformaldehid ile 8 oyuklu kayıcı odaları hücreleri gidermek; Daha sonra, (v / v) Triton-X100, 15 dakika boyunca, ve% 0.1 içeren PBS içinde% 3 (h / h), normal at serumu ile 1 saat boyunca bloke edilir (h / h),% 0.1 ile hücrelerin geçirgen Tween 20. Her adım arasında 5 dakika boyunca iki kez PBS yıkayın. </li><li> 4 ° C &#39;de ve sekonder antikor ile O / N: Primer antikor (1000 1 Ekim-4, SSEA4, TRA-1-81, TRA-1-60 seyreltme) ile inkübe hücreleri. </li><li> Montaj önce, karanlık bir ortamda 5 dakika boyunca 4 &#39;, 6-diamidino-2-fenilindol (DAPI) ile hücrelerin inkübe edin. Yıkama hücresi sıkı karanlıkta her kuru hücrelerin tamamen 5 dakika için üç kez. Floresan montaj ortamına hücreleri korumak ve flöresanlı bir mikroskop altında gözleyin. </li></ol></li><li> Mah-PCR <ol><li> Üreticinin talimatlarına göre bir RNeasy Mini kiti kullanılarak hücrelerden toplam RNA izole ve Nano Bırak Spektrofotometre kullanılarak ekstre RNA ölçmek. </li&gt; <li> 20 ul reaksiyon karışımı ihtiva eden oligo toplam RNA 2 ug ekleyerek cDNA sentez (dT) primerleri ve Superscript II ters transkriptaz; Bu prosedürde üreticinin talimatlarına uyun. </li><li> IQ SYBR Green qPCR Master Mix ve Ek Tablo S1 tarif edilen primer sekanslar kullanılarak, bir Bio-Rad iCycler iQ sistemi ile sentezlenen cDNA yı çoğaltmak. Gliseraldehid-3-fosfat dehidrojenaz (GAPDH) kişilerce ilgi genlerinin döngüsü eşik değerlerini normalize. </li></ol></li><li> Alkalen fosfataz (AP) boyama <ol><li> Üreticinin protokolüne uygun olarak, ES hücre Karakterizasyonu Kiti kullanılarak AP aktivitesi algılar. </li><li> 5. günde, ortam aspire ve 12 dakika boyunca PBS içinde% 4 paraformaldehit ile hPSCs düzeltin. Overfix etmeyin; daha uzun bir süre, 2 dakika için hücrelerin sabitlenmesi alkalin fosfataz inaktivasyonu ile sonuçlanacaktır. Fiksatif aspire ve 1x Durulama Tampon ile durulayın. Yapamazkuyu adımları arasında kurumasını bekleyin. </li><li> Her çukurun (35 mm çanak oyuk başına 1 mi) karşılamak için yeterli boyama solüsyonu ekleyin. 15 dakika boyunca oda sıcaklığında karanlıkta inkübe edin. </li><li> İnkübasyondan sonra, 1x Durulama Tamponu ile bulaşıkları yıkama. 1x PBS ile Kapak hücreleri kurumasını önlemek ve Olympus mikroskop kullanılarak boyanan hücrelerin incelenmesi ve görüntü için. </li></ol></li><li> Western Blot <ol><li> Daha önce 9&#39;da tarif edildiği gibi Western blot analizi yapın. Kısaca, hücre lizatları içindeki protein yoğunluklarının belirlenmesi. Sodyum dodesil sülfat-poliakrilamid jel elektroforezi (SDS-PAGE) jel ile ilgili protein eşit miktarlarda gidermek ve poliviniliden flüorür (PVDF) membran üzerine proteinleri aktarın. </li><li> Leke Blok ve 4 ° C&#39;de farklı primer antikorlar ile G / Ç N o prob; Daha sonra 1 saat HRP-konjuge sekonder antikor ile leke inkübe edin. 10 dakika inkubasyon arasındaki her zaman için membranı üç kez yıkayın. Bir ECL reaktifi kullanılarak sinyalleri algılar. </li&gt; </ol></li><li> Kısa tekrar dizisi (STR) analizi <ol><li> hPSCs aynı pasajda de deney ve kontrol grupları toplandı. Genomik DNA üreticinin talimatlarına göre, bir DNA Mikro kiti kullanılarak ekstre edilmiştir. </li><li> Üreticinin talimatlarına ve kapiler elektroforez göre AmpF / STR PCR Amplifikasyonu kiti kullanılarak 16 farklı genetik lokus için genomik DNA&#39;yı büyütmek Genetik Analiz cihazı kullanılarak gerçekleştirildi. </li></ol></li></ol>

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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=Basic+fibroblast+growth+factor+support+of+human+embryonic+stem+cell+self-renewal.">Basic fibroblast growth factor support of human embryonic stem cell self-renewal.</a> Stem Cells. 24 (3), 568-574 (2006).</li><li>Park, 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=Human+feeder+cells+can+support+the+undifferentiated+growth+of+human+and+mouse+embryonic+stem+cells+using+their+own+basic+fibroblast+growth+factors.">Human feeder cells can support the undifferentiated growth of human and mouse embryonic stem cells using their own basic fibroblast growth factors.</a> Stem Cells Dev. 20 (11), 1901-1910 (2011).</li><li>Park, 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+efficacy+of+human+placenta+as+a+source+of+the+universal+feeder+in+human+and+mouse+pluripotent+stem+cell+culture.">The efficacy of human placenta as a source of the universal feeder in human and mouse pluripotent stem cell culture.</a> Cell Reprogram. 12 (3), 315-328 (2010).</li><li>Park, 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=Undifferentiated+propagation+of+the+human+embryonic+stem+cell+lines,+H1+and+HSF6,+on+human+placenta-derived+feeder+cells+without+basic+fibroblast+growth+factor+supplementation.">Undifferentiated propagation of the human embryonic stem cell lines, H1 and HSF6, on human placenta-derived feeder cells without basic fibroblast growth factor supplementation.</a> Stem Cells Dev. 19 (11), 1713-1722 (2010).</li><li>Seok, 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=Human+placenta-derived+feeders+support+prolonged+undifferentiated+propagation+of+a+human+embryonic+stem+cell+line,+SNUhES3:+comparison+with+human+bone+marrow-derived+feeders.">Human placenta-derived feeders support prolonged undifferentiated propagation of a human embryonic stem cell line, SNUhES3: comparison with human bone marrow-derived feeders.</a> Stem Cells Dev. 16 (3), 421-428 (2007).</li><li>Levenstein, M. 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=Basic+fibroblast+growth+factor+support+of+human+embryonic+stem+cell+self-renewal.">Basic fibroblast growth factor support of human embryonic stem cell self-renewal.</a> Stem Cells. 24 (3), 568-574 (2006).</li><li>Xu, R. H., Peck, R. M., Li, D. S., Feng, X., Ludwig, T., Thomson, J. 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=Basic+FGF+and+suppression+of+BMP+signaling+sustain+undifferentiated+proliferation+of+human+ES+cells.">Basic FGF and suppression of BMP signaling sustain undifferentiated proliferation of human ES cells.</a> Nat Methods. 2 (3), 185-190 (2005).</li><li>Mahmood, A., Harkness, L., Schrøder, H. D., Abdallah, B. M., Kassem, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enhanced+differentiation+of+stem+cells+mesenchymal+progenitors+by+inhibition+of+TGF-beta/activin/nodal+using+SB-431542.">Enhanced differentiation of stem cells mesenchymal progenitors by inhibition of TGF-beta/activin/nodal using SB-431542.</a> J Bone Miner Res. 25 (6), 1216-1233 (2010).</li></ol>

Yazarlar herhangi bir çıkar çatışmaları gösterir.

Bu model, örneğin bFGF veya insülin gibi eksojen rekombinant büyüme faktörlerinin ilavesi olmadan insanlaştınlmış besleyici içermeyen kültür koşullarında özelliklerini muhafaza eden bir insanlaştırılmış mikroçevresinin hPSCs manipülasyonu sağlarken başarıyla hPSCs yaymak için geliştirilmiştir. Ekzojen bFGF takviyesi yaygındır ve Matrigel veya laminin gibi alt tabakalarının uygulanması hPSCs 14 besleyici içermeyen kültür için gereklidir. Bu pro...

The goal of this protocol is the propagation of human pluripotent stem cells (hPSCs) including human embryonic stem cells (hESCs) and human induced pluripotent stem cells (iPSCs) in fully humanized ex vivo feeder-free conditions without requiring additional exogenous supplementation and synthetic substrates. To date, the development of ex vivo hPSC culture models, that enable the introduction of culture products to the clinic, has been a major concern in stem cell research. Specifically, two critical problems need to be addressed. First, a humanized ex vivo culture system for the propagation of hPSCs that obviates the risk of contamination by animal cell products is needed. Second, a feeder-free culture model is needed to facilitate easy and economic mass production of hPSCs. For therapeutic applications of hPSCs, it is necessary to identify the factors that regulate their self-renewal and differentiation.
Since Xu et al. initially reported the feasibility of using conditioned media (CM) derived from mouse embryonic fibroblasts (MEF) to grow hESCs on Matrigel1, many studies have examined optimal ex vivo propagation methods for hPSCs2-4. However, an ideal humanized ex vivo feeder-free hPSC propagation system has not been developed because current methods require additional exogenous substrates including bFGF and insulin, well-known hPSC-sustaining factors, in culture media5-7. Moreover, synthetic substrata such as Matrigel or laminin are used in all feeder-free cultures.
The rationale behind the development and use of this protocol is based on our previous studies showing that human placenta chorion cells excellently support the propagation of hPSCs without bFGF supplementation8-11. This protocol has a number of advantages including its simplicity with respect to handling and its cost-effectiveness, and it is a near-perfect humanized culture that enables hPSC propagation without exogenous synthetic substrates. The application of human placenta-derived CM (hPCCM) for hPSCs involves 3 steps. First, chorion cells are isolated from the human placenta and cultured. Second, hPCCM is produced from cultured cells. Third, hPSCs are cultured using hPCCM and their characteristics are confirmed. 
This protocol will facilitate clinical applications of hPSCs and studies of the mechanisms of hPSC proliferation and attachment. In this paper, the protocol for the successful propagation of hPSCs in hPCCM on a gelatin-coated dish is presented.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Mitomycin-C</td>
			<td>Sigma-Aldrich Corporation</td>
			<td>M4287</td>
			<td>10ug/ml</td>
		</tr>
		<tr>
			<td>Mycoplasma detection kit</td>
			<td>TaKaRa Bio Inc.</td>
			<td>#6601</td>
			<td></td>
		</tr>
		<tr>
			<td>Matrigel</td>
			<td>BD Biosciences</td>
			<td>#354277</td>
			<td></td>
		</tr>
		<tr>
			<td>mTeSR1</td>
			<td>STEMCELL Technologies Inc.</td>
			<td>#05850</td>
			<td></td>
		</tr>
		<tr>
			<td>Dispase</td>
			<td>Worthington Biochemical Corporation</td>
			<td>LS02100</td>
			<td>1mg/ml</td>
		</tr>
		<tr>
			<td>Gelatin</td>
			<td>Sigma-Aldrich Corporation</td>
			<td>G2500</td>
			<td>0.10%</td>
		</tr>
		<tr>
			<td>BM-Cyclin</td>
			<td>Roche</td>
			<td>799 050</td>
			<td>10ug/ml</td>
		</tr>
		<tr>
			<td>RNeasy mini kit&#160;</td>
			<td>Qiagen</td>
			<td>74104</td>
			<td></td>
		</tr>
		<tr>
			<td>Nano Drop Spectrophotometer</td>
			<td>Thermo Fisher Scientific Inc</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>iQ SYBR Green qPCR Master Mix&#160;</td>
			<td>Bio-Rad Laboratories</td>
			<td>#170-8882AP</td>
			<td></td>
		</tr>
		<tr>
			<td>ES Cell Characterization kit</td>
			<td>Chemicon International, Inc.,</td>
			<td>SCR001</td>
			<td></td>
		</tr>
		<tr>
			<td>Power cDNA Synthesis Kit&#160;</td>
			<td>iNtRON Biotechnology</td>
			<td>25011</td>
			<td></td>
		</tr>
		<tr>
			<td>QIAamp&#174; DNA Micro kit</td>
			<td>Qiagen</td>
			<td>56304</td>
			<td></td>
		</tr>
		<tr>
			<td>AmpF/STR&#174; Identifiler&#174; PCR Amplification kit</td>
			<td>Applied Biosystems Inc.</td>
			<td>4322288</td>
			<td></td>
		</tr>
		<tr>
			<td>Applied Biosystems&#174; 3130xl Genetic Analyzer&#160;</td>
			<td>Applied Biosystems Inc.</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

a novel culture model for human pluripotent stem cell propagation on gelatin in placenta-conditioned media

The propagation of human pluripotent stem cells (hPSCs) in conditioned medium derived from human cells in feeder-free culture conditions has been of interest. Nevertheless, an ideal humanized ex vivo feeder-free propagation method for hPSCs has not been developed; currently, additional exogenous substrates including basic fibroblast growth factor (bFGF), a master hPSC-sustaining factor, is added to all of culture media and synthetic substrata such as Matrigel or laminin are used in all feeder-free cultures. Recently, our group developed a simple and efficient protocol for the propagation of hPSCs using only conditioned media derived from the human placenta on a gelatin-coated dish without additional exogenous supplementation or synthetic substrata specific to hPSCs. This protocol has not been reported previously and might enable researchers to propagate hPSCs efficiently in humanized culture conditions. Additionally, this model obviates hPSC contamination risks by animal products such as viruses or unknown proteins. Furthermore, this system facilitates easy mass production of hPSCs using the gelatin coating, which is simple to handle, dramatically decreases the overall costs of ex vivo hPSC maintenance.

Bu hPSC yayılım yöntemin bir avantajı insan hücrelerinden salgılanan bileşenleri kullanmasıdır. Protokolde en çok ciritical aşama insan plasentası türetilen hücrelerin izolasyonu ve kültürü. Bu gerektirir ve HPC plaka villus kesin bir kısmından doğru diseksiyon. 1 insan plasenta kaynaklı hücre izolasyonu için prosedürü göstermektedir. Bu işlem, basit ve hücrelerin izolasyonu için uygun olan ve kolay 1 saat içerisinde gerçekleştirilebilir. Şekil 2...

Watch this Scientific Journal Video about A Novel Culture Model for Human Pluripotent Stem Cell Propagation on Gelatin in Placenta-conditioned Media at JoVE.com

A Novel Culture Model for Human Pluripotent Stem Cell Propagation on Gelatin in Placenta-conditioned Media

This protocol provides a simple and efficient way to propagate human pluripotent stem cells (hPSCs) using only conditioned media derived from the human placenta in a gelatin-coated dish without additional exogenous supplementation or hPSC-specific synthetic substrata.

A Novel Kültür Modeli İnsan Pluripotent için Plasenta klimalı Medyada Jelatin üzerinde hücre çoğalma Kök

The propagation of human pluripotent stem cells (hPSCs) in conditioned medium derived from human cells in feeder-free culture conditions has been of ...

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Research

JoVE Journal

Developmental Biology

7.8K Görüntüleme.  Graduate School of Medicine, Korea University. This protocol provides a simple and efficient way to propagate human pluripotent stem cells (hPSCs) using only conditioned media derived from the human placenta in a gelatin-coated dish without additional exogenous supplementation or hPSC-specific synthetic substrata.

Video: A Novel Culture Model for Human Pluripotent Stem Cell Propagation on Gelatin in Placenta-conditioned Media

Alginate Encapsulation of Pluripotent Stem Cells Using a Co-axial Nozzle

Pluripotent stem cells (PS cells) are the focus of intense research due to their role in regenerative medicine and drug screening. However, the development of a mass culture system would be required for using PS cells in these applications. Suspension culture is one promising culture method for the mass production of PS cells, although some issues such as controlling aggregation and limiting shear stress from the culture medium are still unsolved. In order to solve these problems, we developed a method of calcium alginate (Alg-Ca) encapsulation using a co-axial nozzle. This method can control the size of the capsules easily by co-flowing N2 gas. The controllable capsule diameter must be larger than 500 &#181;m because too high a flow rate of N2 gas causes the breakdown of droplets and thus heterogeneous-sized capsules. Moreover, a low concentration of Alg-Na and CaCl2 causes non-spherical capsules. Although an Alg-Ca capsule without a coating of Alg-PLL easily dissolves enabling the collection of cells, they can also potentially leak out from capsules lacking an Alg-PLL coating. Indeed, an alginate-PLL coating can prevent cellular leakage but is also hard to break. This technology can be used to research the stem cell niche as well as the mass production of PS cells because encapsulation can modify the micro-environment surrounding cells including the extracellular matrix and the concentration of secreted factors.

We established a method of encapsulating pluripotent stem cells (PS cells) into alginate hydrogel capsules using a co-axial nozzle. This prevents cells from aggregating excessively and limits the shear stress experienced by cells in suspension culture. The technique is applicable to the mass production of PS cells as well as research on stem cell niche.

Bir Koaksiyel Memesini kullanma Pluripotent Kök Hücre Aljinat Encapsulation

Pluripotent stem cells (PS cells) are the focus of intense research due to their role in regenerative medicine and drug screening.  However, the ...

Gelişim Biyolojisi

Culturing Human Pluripotent and Neural Stem Cells in an Enclosed Cell Culture System for Basic and Preclinical Research

This paper describes how to use a custom manufactured, commercially available enclosed cell culture system for basic and preclinical research. Biosafety cabinets (BSCs) and incubators have long been the standard for culturing and expanding cell lines for basic and preclinical research. However, as the focus of many stem cell laboratories shifts from basic research to clinical translation, additional requirements are needed of the cell culturing system. All processes must be well documented and have exceptional requirements for sterility and reproducibility. In traditional incubators, gas concentrations and temperatures widely fluctuate anytime the cells are removed for feeding, passaging, or other manipulations. Such interruptions contribute to an environment that is not the standard for cGMP and GLP guidelines. These interruptions must be minimized especially when cells are utilized for therapeutic purposes. The motivation to move from the standard BSC and incubator system to a closed system is that such interruptions can be made negligible. Closed systems provide a work space to feed and manipulate cell cultures and maintain them in a controlled environment where temperature and gas concentrations are consistent. This way, pluripotent and multipotent stem cells can be maintained at optimum health from the moment of their derivation all the way to their eventual use in therapy.

Here is a protocol to grow pluripotent stem cells (PSC) and neural stem cells (NSC) in an enclosed cell culture system that permits maximum sterility and reproducibility, replacing the traditional biosafety cabinet and incubator. This equipment meets clinical good manufacturing practice (cGMP) and clinical good lab practice (cGLP) guidelines.

Temel ve Klinik öncesi Araştırma bir Kapalı Hücre Kültürü Sistemi İnsan Pluripotent ve Sinir Kök Hücre Kültürleme

This paper describes how to use a custom manufactured, commercially available enclosed cell culture system for basic and preclinical research. Biosafety ...

Feeder-free Derivation of Melanocytes from Human Pluripotent Stem Cells

Human pluripotent stem cells (hPSCs) represent a platform to study human development in vitro under both normal and disease conditions. Researchers can direct the differentiation of hPSCs into the cell type of interest by manipulating the culture conditions to recapitulate signals seen during development. One such cell type is the melanocyte, a pigment-producing cell of neural crest (NC) origin responsible for protecting the skin against UV irradiation. This protocol presents an extension of a currently available in vitro Neural Crest differentiation protocol from hPSCs to further differentiate NC into fully pigmented melanocytes. Melanocyte precursors can be enriched from the Neural Crest protocol via a timed exposure to activators of WNT, BMP, and EDN3 signaling under dual-SMAD-inhibition conditions. The resultant melanocyte precursors are then purified and matured into fully pigmented melanocytes by culture in a selective medium. The resultant melanocytes are fully pigmented and stain appropriately for proteins characteristic of mature melanocytes.

This work describes an in vitro differentiation protocol to produce pigmented, mature melanocytes from human pluripotent stem cells via a neural crest and melanoblast intermediate stage using a feeder-free, 25 day protocol.

Insan pluripotent kök hücreleri gelen melanosit besleyici içermeyen türetilmesi

Human pluripotent stem cells (hPSCs) represent a platform to study human development in vitro under both normal and disease conditions. Researchers can ...

Large-Scale Production of Cardiomyocytes from Human Pluripotent Stem Cells Using a Highly Reproducible Small Molecule-Based Differentiation Protocol

Maximizing the benefit of human pluripotent stem cells (hPSCs) for research, disease modeling, pharmaceutical and clinical applications requires robust methods for the large-scale production of functional cell types, including cardiomyocytes. Here we demonstrate that the temporal manipulation of WNT, TGF-&#946;, and SHH signaling pathways leads to highly efficient cardiomyocyte differentiation of single-cell passaged hPSC lines in both static suspension and stirred suspension bioreactor systems. Employing this strategy resulted in ~ 100% beating spheroids, consistently containing &#62; 80% cardiac troponin T-positive cells after 15 days of culture, validated in multiple hPSC lines. We also report on a variation of this protocol for use with cell lines not currently adapted to single-cell passaging, the success of which has been verified in 42 hPSC lines. Cardiomyocytes generated using these protocols express lineage-specific markers and show expected electrophysiological functionalities. Our protocol presents a simple, efficient and robust platform for the large-scale production of human cardiomyocytes.

Here, we present a robust, fast and scalable cardiomyocyte differentiation protocol for human pluripotent stem cells (hPSCs). Cardiomyocytes derived using this large-scale method can provide sufficient cell numbers for their effective use in human cardiovascular disease modeling, high-throughput drug screening, and potentially clinical applications.

Oldukça Tekrarlanabilir Küçük Molekül Tabanlı Farklılaşma Protokolü kullanarak insan pluripotent kök hücreleri gelen kardiyomiyositlerde Büyük Ölçekli Üretim

Maximizing the benefit of human pluripotent stem cells (hPSCs) for research, disease modeling, pharmaceutical and clinical applications requires robust ...

Rapid Neuronal Differentiation of Induced Pluripotent Stem Cells for Measuring Network Activity on Micro-electrode Arrays

Neurons derived from human induced Pluripotent Stem Cells (hiPSCs) provide a promising new tool for studying neurological disorders. In the past decade, many protocols for differentiating hiPSCs into neurons have been developed. However, these protocols are often slow with high variability, low reproducibility, and low efficiency. In addition, the neurons obtained with these protocols are often immature and lack adequate functional activity both at the single-cell and network levels unless the neurons are cultured for several months. Partially due to these limitations, the functional properties of hiPSC-derived neuronal networks are still not well characterized. Here, we adapt a recently published protocol that describes production of human neurons from hiPSCs by forced expression of the transcription factor neurogenin-212. This protocol is rapid (yielding mature neurons within 3 weeks) and efficient, with nearly 100% conversion efficiency of transduced cells (&#62;95% of DAPI-positive cells are MAP2 positive). Furthermore, the protocol yields a homogeneous population of excitatory neurons that would allow the investigation of cell-type specific contributions to neurological disorders. We modified the original protocol by generating stably transduced hiPSC cells, giving us explicit control over the total number of neurons. These cells are then used to generate hiPSC-derived neuronal networks on micro-electrode arrays. In this way, the spontaneous electrophysiological activity of hiPSC-derived neuronal networks can be measured and characterized, while retaining interexperimental consistency in terms of cell density. The presented protocol is broadly applicable, especially for mechanistic and pharmacological studies on human neuronal networks.

We modify and implement a previously published protocol describing the rapid, reproducible, and efficient differentiation of human&#160;induced&#160;Pluripotent Stem Cells (hiPSCs) into excitatory cortical neurons12. Specifically, our modification allows for control of neuronal cell density and use on micro-electrode arrays to measure electrophysiological properties at the network level.

Mikro-elektrot dizileri Ağ Aktivite Ölçüm uyarılmış pluripotent kök hücrelerin Rapid Nöronal Farklılaşma

Neurons derived from human induced Pluripotent Stem Cells (hiPSCs) provide a promising new tool for studying neurological disorders. In the past decade, ...

A Novel Mammary Fat Pad Transplantation Technique to Visualize the Vessel Generation of Vascular Endothelial Stem Cells

Endothelial cells (ECs) are the fundamental building blocks of the vascular architecture and mediate vascular growth and remodeling to ensure proper vessel development and homeostasis. However, studies on endothelial lineage hierarchy remain elusive due to the lack of tools to gain access as well as to directly evaluate their behavior in vivo. To address this shortcoming, a new tissue model to study angiogenesis using the mammary fat pad has been developed. The mammary gland develops mostly in the postnatal stages, including puberty and pregnancy, during which robust epithelium proliferation is accompanied by extensive vascular remodeling. Mammary fat pads provide space, matrix, and rich angiogenic stimuli from the growing mammary epithelium. Furthermore, mammary fat pads are located outside the peritoneal cavity, making them an easily accessible grafting site for assessing the angiogenic potential of exogenous cells. This work also describes an efficient tracing approach using fluorescent reporter mice to specifically label the targeted population of vascular endothelial stem cells (VESCs) in vivo. This lineage tracing method, coupled with subsequent tissue whole-mount microscopy, enable the direct visualization of targeted cells and their descendants, through which the proliferation capability can be quantified and the differentiation commitment can be fate-mapped. Using these methods, a population of bipotent protein C receptor (Procr) expressing VESCs has recently been identified in multiple vascular systems. Procr+ VESCs, giving rise to both new ECs and pericytes, actively contribute to angiogenesis during development, homeostasis, and injury repair. Overall, this manuscript describes a new mammary fat pad transplantation and in vivo lineage tracing techniques that can be used to evaluate the stem cell properties of VESCs.

This work demonstrates a novel approach to assess the proliferation, differentiation, and vessel-forming potential of vascular endothelial stem cells (VESCs) through mammary fat pad transplantation followed by whole-mount tissue preparation for microscopic observation. A lineage tracing strategy to investigate the behavior of VESCs in vivo is also presented.

Vasküler Endotelyal Kök Hücrelerin Damar Oluşumunu Görselleştirmek İçin Yeni Bir Göğüs Göğsü Transplantasyon Tekniği

Endothelial cells (ECs) are the fundamental building blocks of the vascular architecture and mediate vascular growth and remodeling to ensure proper ...

Rapid, Directed Differentiation of Retinal Pigment Epithelial Cells from Human Embryonic or Induced Pluripotent Stem Cells

We describe a robust method to direct the differentiation of pluripotent stem cells into retinal pigment epithelial cells (RPE). The purpose of providing a detailed and thorough protocol is to clearly demonstrate each step and to make this readily available to researchers in the field. This protocol results in a homogenous layer of RPE with minimal or no manual dissection needed. The method presented here has been shown to be effective for induced pluripotent stem cells (iPSC) and human embryonic stem cells. Additionally, we describe methods for cryopreservation of intermediate cell banks that allow long-term storage. RPE generated using this protocol might be useful for iPSC disease-in-a-dish modeling or clinical application.

This protocol describes how to produce retinal pigment epithelial cells (RPE) from pluripotent stem cells. The method uses a combination of growth factors and small molecules to direct the differentiation of stem cells into immature RPE in fourteen days and mature, functional RPE after three months.

Hızlı, yönlendirilmiş farklılaşma insan embriyonik veya İndüklenmiş Pluripotent kök hücrelerinden retina Pigment epitel hücrelerinin

We describe a robust method to direct the differentiation of pluripotent stem cells into retinal pigment epithelial cells (RPE). The purpose of providing ...

Using Human Induced Pluripotent Stem Cell-derived Hepatocyte-like Cells for Drug Discovery

The ability to differentiate human induced pluripotent stem cells (iPSCs) into hepatocyte-like cells (HLCs) provides new opportunities to study inborn errors in hepatic metabolism. However, to provide a platform that supports the identification of small molecules that can potentially be used to treat liver disease, the procedure requires a culture format that is compatible with screening thousands of compounds. Here, we describe a protocol using completely defined culture conditions, which allow the reproducible differentiation of human iPSCs to hepatocyte-like cells in 96-well tissue culture plates. We also provide an example of using the platform to screen compounds for their ability to lower Apolipoprotein B (APOB) produced from iPSC-derived hepatocytes generated from a familial hypercholesterolemia patient. The availability of a platform that is compatible with drug discovery should allow researchers to identify novel therapeutics for diseases that affect the liver.

The protocol presented here describes a platform for identifying small molecules for the treatment of liver disease. A step-by-step description is presented detailing how to differentiate iPSCs into cells with hepatocyte characteristics in 96-well plates, and to use the cells to screen for small molecules with potential therapeutic activity.

İnsan kullanarak Pluripotent kök hücre kaynaklı hepatosit benzeri hücreler indüklenen ilaç bulma için

The ability to differentiate human induced pluripotent stem cells (iPSCs) into hepatocyte-like cells (HLCs) provides new opportunities to study inborn ...

A Familial Hypercholesterolemia Human Liver Chimeric Mouse Model Using Induced Pluripotent Stem Cell-derived Hepatocytes

Familial hypercholesterolemia (FH) is mostly caused by low-density lipoprotein receptor (LDLR) mutations and results in an increased risk of early-onset cardiovascular disease due to marked elevation of LDL cholesterol (LDL-C) in blood. Statins are the first line of lipid-lowering drugs for treating FH and other types of hypercholesterolemia, but new approaches are emerging, in particular PCSK9 antibodies, which are now being tested in clinical trials. To explore novel therapeutic approaches for FH, either new drugs or new formulations, we need appropriate in vivo models. However, differences in the lipid metabolic profiles compared to humans are a key problem of the available animal models of FH. To address this issue, we have generated a human liver chimeric mouse model using FH induced pluripotent stem cell (iPSC)-derived hepatocytes (iHeps). We used Ldlr-/-/Rag2-/-/Il2rg-/- (LRG) mice to avoid immune rejection of transplanted human cells and to assess the effect of LDLR-deficient iHeps in an LDLR null background. Transplanted FH iHeps could repopulate 5-10% of the LRG mouse liver based on human albumin staining. Moreover, the engrafted iHeps responded to lipid-lowering drugs and recapitulated clinical observations of increased efficacy of PCSK9 antibodies compared to statins. Our human liver chimeric model could thus be useful for preclinical testing of new therapies to FH. Using the same protocol, similar human liver chimeric mice for other FH genetic variants, or mutations corresponding to other inherited liver diseases, may also be generated.

Here, we present a protocol to generate a human liver chimeric mouse model of familial hypercholesterolemia using human induced pluripotent stem cell-derived hepatocytes. This is a valuable model for testing new therapies for hypercholesterolemia.

İndüklenmiş Pluripotent kök hücre kaynaklı tetkikine kullanarak ailesel hiperkolesterolemi insan karaciğer Chimeric fare modeli

Familial hypercholesterolemia (FH) is mostly caused by low-density lipoprotein receptor (LDLR) mutations and results in an increased risk of early-onset ...

Efficient Differentiation of Human Pluripotent Stem Cells into Liver Cells

The liver detoxifies harmful substances, secretes vital proteins, and executes key metabolic activities, thus sustaining life. Consequently, liver failure&#8212;which can be caused by chronic alcohol intake, hepatitis, acute poisoning, or other insults&#8212;is a severe condition that can culminate in bleeding, jaundice, coma, and eventually death. However, approaches to treat liver failure, as well as studies of liver function and disease, have been stymied in part by the lack of a plentiful supply of human liver cells. To this end, this protocol details the efficient differentiation of human pluripotent stem cells (hPSCs) into hepatocyte-like cells, guided by a developmental roadmap that describes how liver fate is specified across six consecutive differentiation steps. By manipulating developmental signaling pathways to promote liver differentiation and to explicitly suppress the formation of unwanted cell fates, this method efficiently generates populations of human liver bud progenitors and hepatocyte-like cells by days 6 and 18 of PSC differentiation, respectively. This is achieved through the temporally-precise control of developmental signaling pathways, exerted by small molecules and growth factors in a serum-free culture medium. Differentiation in this system occurs in monolayers and yields hepatocyte-like cells that express characteristic hepatocyte enzymes and have the ability to engraft a mouse model of chronic liver failure. The ability to efficiently generate large numbers of human liver cells in vitro has ramifications for treatment of liver failure, for drug screening, and for mechanistic studies of liver disease.

This protocol details a monolayer, serum-free method to efficiently generate hepatocyte-like cells from human pluripotent stem cells (hPSCs) in 18 days. This entails six steps as hPSCs sequentially differentiate into intermediate cell-types such as the primitive streak, definitive endoderm, posterior foregut and liver bud progenitors before forming hepatocyte-like cells.