Bu yazının Toronto Genel ve Batı Vakfı ve Banting ve En İyi Diyabet Merkezi-Üniversite Sağlık Ağı Lisans Ödülü fon tarafından desteklenmiştir.

Çözümler ve Medya 1. Hazırlık NOT: steril bir ortamda hücre kültürü için tüm medya hazırlayın. Ortam yapılmış ve hemen kullanılmalıdır. Reaktif bilgilerini malzemeler Tablo l&#39;de verilmiştir. <ol><li> farklılaşma Medya <ol><li> % 1 glutamin, 2 uM CHIR 99.021, 100 ng / ml Aktivin A, 10 4 M MTG ile RPMI ortamı: Gün 0 Farklılaşma Ortam hazırlayın. </li><li> % 1 glutamin, 100 ng / ml Aktivin A, 10 4 M MTG, 5 ng / ml bFGF, 50 ug / ml askorbik asit ile RPMI ortamı: Gün 1-2 Farklılaşma ortamı hazırlayın. </li><li> % 1 glutamin,% 1 B27, 10 4 M MTG, 0.75 uM Dorsomorphin, 50 ng / ml FGF10 ile RPMI ortamı: Gün 3-5 Farklılaşma ortamı hazırlayın. </li><li> Gün 6-7 Farklılaşma ortamı hazırlayın: DMEM,% 1 glutamin,% 1 B27, 50 ug / ml askorbik asit, 50 ng / ml FGF10, 0.25 uM SANT-1, 2 uM retinoik asit, 50 ng / ml Noggin ile. NOT: RetinoikAsit geçen ortama ilave edilmesi gereken ve ortam oksidatif bozunmasını 15 önlemek için ışıktan korunmalıdır. </li><li> Günlük 8-12 Farklılaşma ortamı hazırlayın: DMEM,% 1 glutamin,% 1 B27, 50 ug / ml askorbik asit, 50 ng / ml noggin, 100 ng / ml hEGF, 10 mM nikotinamid. </li></ol></li><li> PBS içinde% 10 FBS, eksi kalsiyum ve magnezyum: FACS tamponu hazırlayın. </li></ol> 2. HESC Farklılaşma NOT: HESC, çözülmüş geçirildi ve bFGF 16 ile desteklenmiş bir KOSR tabanlı medya varlığında ışınlanmış fare embriyonik fibroblast genişletilir. Hücreler% 80-95 confluency ulaştığında hESC farklılaşma için hazırız. Bu zamanda koloniler tanımlanmış sınırlar ve bir &quot;domelike &#39;yapısı (Şekil 1A) ile büyük olmalıdır. Tüm hücre kültürü düz tabanlı,% 0.1 jelatin ile kaplanmış doku kültür levhaları muamele gerçekleştirilir. Tipik olarak, hücreler 6 yetiştirilen veyasırası ile, 2 ml veya 1 ml&#39;lik ortam hacminde 12 oyuklu plakalar. <ol><li> 0. günde, Gün 0 Farklılaşma Medya ile KOSR tabanlı medya değiştirerek farklılaşma başlar. </li><li> gün 1 ve 2, yavaşça aspire önce tek tabaka herhangi bir ölü hücreleri çıkarmak için plakayı sallayın. Gün 1-2 Farklılaşma Medya ile değiştirin. </li><li> 3 gün, flow sitometri için hasat hücreleri. Hücreler% 90&#39;dan fazla CXCR4 + / CD117 + ifade ederse, 2.4 adıma geçin. </li><li> gün 3 ve 5, nazikçe önce aspire için tek tabaka herhangi bir ölü hücreleri çıkarmak için plakayı sallayın. Gün 3-5 Farklılaşma Medya ile değiştirin. </li><li> gün 6 ve 7, Gün 6-7 Farklılaşma Medya ile değiştirin. </li><li> gün 8, 10 ve 12 üzerinde, Gün 8-12 Farklılaşma Medya ile değiştirin. </li><li> günde 13, akış hasat hücreleri sitometri PDX1 ve NKX6-1 ifadesini belirler. </li></ol> Sitometrisi Analizi 3. Hasat Hücreleri <ol><li> olan hücreleri ayırmaküreticinin protokolü ve 3 dakika süreyle 37 ° C&#39;de inkübe e göre 1x ticari tripsin çözeltisi. </li><li> 30 ul DNaz I ile 1000 ul FACS Tampon tek hücreleri ticari tripsin çözüm çıkarın ve tekrar süspansiyon </li><li> Bir mikrosantrifüj tüp 35 mikron naylon örgü hücre süzgeç ve transfer kullanarak filtre hücreleri. </li><li> 5 dakika boyunca 455 xg&#39;de Santrifüj hücreleri. Canlı veya sabit boyama için hücreleri hazırlayın. </li></ol> Akım Sitometrisi 4. Boyama <ol><li> Günde 3 canlı boyama hazırlık Akış <ol><li> 1000 ul FACS Tampon yeniden süspanse hücreleri. Aktarım 200 ul (her ikisi de lekesiz ve lekeli örnekler için), 96 oyuklu bir plaka, iki kuyulara (yaklaşık 1-3 x 10 5 hücre). </li><li> 2 dakika süreyle 931 xg&#39;de plaka santrifüj. plakayı ters çevirerek süpernatantı. </li><li> APC ve CD117: Primer-konjuge antikor, CXCR4 ile Leke PE 3 için (MALZEMELERİNİN Tablo bakınız)Oda sıcaklığında 0 dak ışıktan korunması. </li><li> 2 dakika süreyle 931 xg&#39;de plaka santrifüj. plakayı ters çevirerek süpernatantı. </li><li> 100 ul FACS Tampon yeniden süspanse örnekleri. </li><li> 2 dakika süreyle 931 xg&#39;de plaka santrifüj. plakayı ters çevirerek süpernatantı. </li><li> 1 ml mikro test tüpleri ya da 5 mL&#39;lik yuvarlak dipli bir tüpler 300-500 ul FACS tampon toplam hacimde her bir örnek ve transfer yeniden süspanse edin. </li><li> 17 akış sitometresinde Run örnekler. Numuneler hemen çalıştırmak mümkün değilse, 4 ° C&#39;de mağaza ışıktan koruyarak. </li></ol></li><li> Günde 13 sabit boyama hazırlık Akış <ol><li> 4 ° C&#39;de 24 saat süre ile, ticari sabitleme / permeabilizasyon çözeltisi içinde hücre pelletini. </li><li> 5 dakika boyunca 455 xg&#39;de santrifüjleyin. 400 ul Perm / Wash çözeltisi içinde süspanse. </li><li> Tw için numune (yaklaşık 0.5-1 x 10 6 hücre) 200 ul aktarın(IgG kontrol ve PDX1 / NKX6-1 boyama için), 96 oyuklu plakanın O kuyu. 2 dakika süreyle 931 xg&#39;de santrifüj. </li><li> Anti-PDX1 ve anti-NKX6-1 birincil veya izotop kontrol antikorları ihtiva eden 100 ul Perm / Wash çözeltisi içinde hücre pelletleri yeniden süspanse edin (Malzeme Tablo). 4 ° C&#39;de bir gece boyunca inkübe edilir. </li><li> 2 dakika süreyle 931 xg&#39;de plaka santrifüj. plakayı ters çevirerek süpernatantı. 100 ul Perm / Wash çözeltisi içinde süspanse örnekleri. </li><li> 2 dakika süreyle 931 xg&#39;de plaka santrifüj. plakayı ters çevirerek süpernatantı. </li><li> ışıktan korunan, 1 saat boyunca, oda sıcaklığında, ikincil antikorlar ihtiva eden 100 ul Perm / Wash örnekleri yeniden süspanse edin. </li><li> 2 dakika süreyle 931 xg&#39;de plaka santrifüj. plakayı ters çevirerek süpernatantı. </li><li> 100 ul Perm / Yıkama Çözeltisi yeniden süspanse örnekleri. </li><li> 2 dakika süreyle 931 xg&#39;de plaka santrifüj. plakayı ters çevirerek süpernatantı. </li><li> temsilciadım 4.2.9 ve 4.2.10 yemek. </li><li> 1 ml mikro test tüpleri ya da 5 mL&#39;lik yuvarlak dipli bir tüpler 300-500 ul FACS tampon toplam hacimde her bir örnek ve transfer yeniden süspanse edin. </li><li> 17 akış sitometresinde Run örnekler. Numuneler hemen çalıştırmak mümkün değilse, 4 ° C&#39;de mağaza ışıktan koruyarak. </li></ol></li></ol>

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Yazarlar ifşa hiçbir şey yok.

Başarıyla in vitro hPSCs gelen NKX6-1 + pankreas progenitörlerin üreten pankreatik gelişimi sırasında önemli gelişim aşamaları yöneten belirli bir sinyal yollarının doğru düzenlenmesini kapsayan hPSCs yüksek kalitede kültürleri ve yönlendirilmiş farklılaşma kullanımına dayanır. Daha önce aşağıdaki hususlar yapılmalıdır etkin NKX6-1 üretimi sağlamak için, 3 gösterildiği gibi bu protokol hPSC hatlarının çeşitli çapında NKX6-1 sağlam e...

Diyabet prevalansı artmakta ve Kanada Diyabet Derneği göre, tip 1 diyabet (T1D) 1 olan bu bireylerin% 5-10 ile, Kanada&#39;da 11 milyon kişi diyabet ya da prediyabetik olduğu tahmin edilmektedir. T1D&#39;nin Langerhans adacıklarında içinde bulunan insülin üreten β hücrelerinin yok neden olduğu bir otoimmün hastalıktır. Şu anda, T1D ile yaşayan bireylerin insülin 2 eksojen kaynakları gerektirir. insülin tedavisi gelişmelere rağmen, T1D hastalarının kan şekeri düzenleyen zor bir zaman var ve hipo ve hiperglisemi hem acı çekmeye devam devam ediyor. Bir tedavi umut verici şekilde, tip 1 diyabetin bölgesindeki normogliseminin in vivo ve in vitro 3 hem β hücreleri üretmek insülin sınırsız kaynağı oluşturmak için kullanılabilecek insan embriyonik kök hücreleri (hESC) kullanılmasıdır geri <sclass = &quot;xref&quot;&gt; 4, 5, 6, 7 kadar. HESC Farklılaşan ß-benzeri hücreler mümkün tip 2 diyabet için yeni tedavi hedeflerinin belirlenmesi için izin in vitro diyabet eğitim ve T1D&#39;nin hastalara transplantasyon için hücrelerin sağlamak için yapabiliriz. In vitro hESC insülin üreten hücrelerin üretilmesi en başarılı girişim pankreas gelişimi 4, 5 sırasında meydana gelen embriyonik olayları özetlemek etmektir. Bu doğru gelişen pankreas kilit aşamaları modellemek için farklı sinyal yollarının manipülasyonu içerir. Pankreas gelişimi CXCR4 ve CD117 (c-kit) 8, 9 sentezlenmesi ile karakterize kesin endoderm, indüksiyonu ile başlar. definit kesin düzenlemeive endoderm organizasyonu daha sonra ön-arka ve ventral-dorsal desenlendirme uğrar bağırsak tüpü, oluşumu için gereklidir. Dorsal ve ventral pankreas tomurcukları pankreas gelişimi 10 için gerekli olan pankreas ve duodenum homeobox geni (Pdx1), ifade arka foregut bölgesinden çıkmaktadır. Dorsal ve ventral tomurcukları sigorta ardından geniş epitel biçimlenme ve genişleme 11 uğrar pankreası oluşturmak için. Endokrin ve ekzokrin soy bağlılık ifade eden multipotent progenitör hücreleri (MPCs) üretimi eşlik eder, diğerlerinin yanı sıra, transkripsiyon Pdx1, Nkx6.1 ve Ptf1a 12, 13 faktörleri. Endokrin ve duktal hücreleri olacak MPCs Ptf1a ifadesini düşürürken Nkx6-1 ifade etmeye devam. Bunun aksine, ekzokrin kökenli hücreler expressio kaybedersinizNkx6-1 N ve Ptf1a ifade 12 korur. Transkripsiyon faktörü Nkx6-1, özellikle hücre kaynaşmasına karşı endokrin progenitör hücrelerin farklılaşması sırasında, pankreas gelişiminde önemli bir rol oynar. Daha önce pankreas gelişimi 14 sırasında β hücrelerinin bozulmuş oluşumuna Nkx6-1 sonuçları, silme açıklandığı gibi,. Bu nedenle, in vitro ve in vivo olarak ensülin üreten β hücrelerin üretilmesi Nkx6-1 etkin indüksiyon gerektirir. Biz son zamanlarda verimli hPSCs gelen PDX1 + / NKX6-1 + pankreas atalarıdır üretmek için bir protokol geliştirmiştir. Bu hPSC türetilen pankreas ataları bağışıklık yetersizliği olan farelere 3 transplantasyon sırasında olgun β hücreleri oluşturur. 1) kesin endoderm indüksiyon: farklılaşma protokolü 4 ayrılabilir karakteristik aşamaları2) arka foregut desenleme, 3) pankreas şartname ve 4) NKX6-1 indüksiyon. Burada yönettiği farklılaşma her adımı ayrıntılı bir açıklama sağlar.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Media and cytokines</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>1-Thioglycerol (MTG)</td>
			<td>Sigma</td>
			<td>M6145</td>
			<td></td>
		</tr>
		<tr>
			<td>Activin A</td>
			<td>R&#38;D</td>
			<td>338-AC/CF&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Ascorbic Acid</td>
			<td>Sigma</td>
			<td>A4544</td>
			<td></td>
		</tr>
		<tr>
			<td>B-27 Supplement</td>
			<td>Life Technologies</td>
			<td>12587-010&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>BD Cytofix/Cytoperm Buffer</td>
			<td>BD Bioscience</td>
			<td>554722</td>
			<td></td>
		</tr>
		<tr>
			<td>BD Perm/Wash buffer, 1x</td>
			<td>BD Bioscience</td>
			<td>554723</td>
			<td></td>
		</tr>
		<tr>
			<td>bFGF</td>
			<td>R&#38;D</td>
			<td>233-FB</td>
			<td></td>
		</tr>
		<tr>
			<td>CHIR990210</td>
			<td>Tocris</td>
			<td>4423a</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#8217;s Modified Eagle Medium (DMEM)</td>
			<td>Life Technologies</td>
			<td>11995</td>
			<td></td>
		</tr>
		<tr>
			<td>DNase I</td>
			<td>VWR</td>
			<td>80510-412&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Dorsomorphin</td>
			<td>Sigma</td>
			<td>P5499</td>
			<td></td>
		</tr>
		<tr>
			<td>EGF</td>
			<td>R&#38;D</td>
			<td>236-EG</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum (FBS)</td>
			<td>Wisent</td>
			<td>88150</td>
			<td></td>
		</tr>
		<tr>
			<td>FGF10</td>
			<td>R&#38;D</td>
			<td>345-FG</td>
			<td></td>
		</tr>
		<tr>
			<td>Gelatin from porcine skin</td>
			<td>Sigma</td>
			<td>G1890</td>
			<td></td>
		</tr>
		<tr>
			<td>Glutamine</td>
			<td>Life Technologies</td>
			<td>25030</td>
			<td></td>
		</tr>
		<tr>
			<td>Nicotinamide</td>
			<td>Sigma</td>
			<td>NO636</td>
			<td></td>
		</tr>
		<tr>
			<td>NOGGIN</td>
			<td>R&#38;D</td>
			<td>3344-NG</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin/Streptomycin</td>
			<td>Life Technologies</td>
			<td>15070-063</td>
			<td></td>
		</tr>
		<tr>
			<td>Retinoic acid</td>
			<td>Sigma</td>
			<td>R2625</td>
			<td></td>
		</tr>
		<tr>
			<td>RPMI Medium 1640</td>
			<td>Life Technologies</td>
			<td>11875</td>
			<td></td>
		</tr>
		<tr>
			<td>SANT-1</td>
			<td>Tocris</td>
			<td>1974</td>
			<td></td>
		</tr>
		<tr>
			<td>TrypLE Express Enzyme (1x), phenol red</td>
			<td>Life Technologies</td>
			<td>12605-010</td>
			<td></td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalogue Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Antibodies for flow cytometry (working dilutions)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>CD117 PE (1:100)</td>
			<td>Life Technologies</td>
			<td>CD11705</td>
			<td></td>
		</tr>
		<tr>
			<td>CXCR4 APC (1:50)</td>
			<td>BD&#160; Bioscience</td>
			<td>551966</td>
			<td></td>
		</tr>
		<tr>
			<td>Donkey Anti-Mouse IgG (H+L), Alexa Fluor 647 conjugate (1:400)</td>
			<td>Life Technologies</td>
			<td>A-31571</td>
			<td></td>
		</tr>
		<tr>
			<td>Donkey Anti-Goat IgG (H+L), Alexa Fluor 488 (1:400)&#160;</td>
			<td>Jackson ImmunoResearch Laboratories Inc.</td>
			<td>705-546-147</td>
			<td></td>
		</tr>
		<tr>
			<td>Isotype Control Mouse IgG&#160;</td>
			<td>Jackson ImmunoResearch Laboratories Inc.&#160;</td>
			<td>015-000-003</td>
			<td></td>
		</tr>
		<tr>
			<td>Isotype Control Goat IgG</td>
			<td>R&#38;D&#160;&#160;</td>
			<td>AB-108-C</td>
			<td></td>
		</tr>
		<tr>
			<td>NKX6-1 (1:2000)</td>
			<td>DSHB</td>
			<td>F55A10</td>
			<td></td>
		</tr>
		<tr>
			<td>PDX1 (1:100)</td>
			<td>R&#38;D</td>
			<td>AF2419</td>
			<td></td>
		</tr>
	</tbody>
</table>

efficient differentiation of pluripotent stem cells to nkx6-1+ pancreatic progenitors

Pluripotent kök hücreleri, kendine yenilemek ve bunların çalışma ve diyabet gelecekteki tedavisi için kullanılabilir pankreatik progenitör hücrelerinin üretimi için cazip bir kaynak yapma, birden fazla soy ayırt yeteneğine sahiptir. Bu makale, insan embriyonik kök hücreleri (hESC) pankreas progenitör hücreleri üretmek için tasarlanmış bir dört aşamalı farklılaşma protokolü özetliyor. Bu protokol, insan pluripotent kök hücre (hPSC) hatları, bir dizi uygulanabilir. pankreatik progenitör hücreleri üretmek için alınan yaklaşım doğru pankreas gelişiminin temel aşamaları modellemek için hESC ayırt etmektir. Bu Aktivin A, temel fibroblast büyüme faktörü (bFGF) ve CHIR990210 mevcudiyetinde hücrelerin kültürlenmesi ile elde edilir kesin endoderm, indüksiyonu ile başlar. Fibroblast Büyüme Faktörü 10 (FGF10) ve Dorsomorphin ile daha fazla farklılaşma ve desenlendirme arka foregut benzeyen hücreler üretir. Retin eklenmesioik asit, noggin, SANT-1 ve FGF10 pankreas endoderm karakteristik hücrelerine arka foregut hücreleri ayırır. Son olarak, epidermal büyüme faktörü (EGF) kombinasyonu, nikotinamid ve Noggin PDX1 + / NKX6-1 + hücreleri etkin bir şekilde oluşmasına sebep olur. Akım sitometri pankreas gelişimin temel aşamalarında spesifik belirteçler ifadesini teyit etmek için yapılır. Evre 4 sonunda PDX1 + / NKX6-1 + pankreas progenitörler bağışıklık yetersizliği olan farelere transplantasyonu sırasında olgun β hücreleri üretme yeteneğine sahiptir ve daha fazla in vitro insülin üreten hücreleri üretmek için ayırt edilebilir. Böylece, PDX1 verimli nesil + / NKX6-1 + pankreas ataları, bu protokolde gösterdiği gibi, bu in vitro insan pankreas gelişimini incelemek için bir platform sağlar olarak büyük önem taşımaktadır ve farklılaşma potansiyeli olan hücre kaynağı sağlar EV olabilir β hücrelerientually diyabet tedavisi için kullanılabilir.

Şekil 1A şemada gösterildiği gibi pankreas progenitörlerin verimli üretimi, farklılaşma protokolü esnasında özel sinyal molekülleri tam olarak ilave edildikten sonra farklılaşmamış hücrelerin uygun büyüme ve bakım dayanır. 0. günde, farklılaşmamış hücreler% 80-95 konfluent olmalı ve koloniler tanımlanan kenarları (Şekil 1A) sahip olmalıdır. hücre ölümü bu aşamada oldukça yaygındır beri Aşama 1 sırasında, medya ...

Watch this Scientific Journal Video about Efficient Differentiation of Pluripotent Stem Cells to NKX6-1+ Pancreatic Progenitors at JoVE.com

Efficient Differentiation of Pluripotent Stem Cells to NKX6-1+ Pancreatic Progenitors

Burada in vitro NKX6-1 + pankreas atalarıdır insan embriyonik kök hücreleri ayırt etmek için 4 aşamalı bir protokol açıklar. Bu protokol, insan pluripotent kök hücre hatları çeşitli uygulanabilir.

Pluripotent Verimli Farklılaşma NKX6-1 için Kök Hücreler<sup&gt; +</sup&gt; Pankreas Progenitörler

Pluripotent stem cells have the ability to self renew and differentiate to multiple lineages, making them an attractive source for the generation of ...

efficient-differentiation-pluripotent-stem-cells-to-nkx6-1-pancreatic

Research

JoVE Journal

Developmental Biology

Efficient Differentiation of Pluripotent Stem Cells to NKX6-1+ Pancreatic Progenitors

7.8K Görüntüleme.  University Health Network.  Burada in vitro NKX6-1 + pankreas atalarıdır insan embriyonik kök hücreleri ayırt etmek için 4 aşamalı bir protokol açıklar. Bu protokol, insan pluripotent kök hücre hatları çeşitli uygulanabilir. 

Video: Efficient Differentiation of Pluripotent Stem Cells to NKX6-1+ Pancreatic Progenitors

Directed Differentiation of Primitive and Definitive Hematopoietic Progenitors from Human Pluripotent Stem Cells

One of the major goals for regenerative medicine is the generation and maintenance of hematopoietic stem cells (HSCs) derived from human pluripotent stem cells (hPSCs). Until recently, efforts to differentiate hPSCs into HSCs have predominantly generated hematopoietic progenitors that lack HSC potential, and instead resemble yolk sac hematopoiesis.&#160;These resulting hematopoietic progenitors may have limited utility for in vitro disease modeling of various adult hematopoietic disorders, particularly those of the lymphoid lineages. However, we have recently described methods to generate erythro-myelo-lymphoid multilineage definitive hematopoietic progenitors from hPSCs using a stage-specific directed differentiation protocol, which we outline here. Through enzymatic dissociation of hPSCs on basement membrane matrix-coated plasticware, embryoid bodies (EBs) are formed. EBs are differentiated to mesoderm by recombinant BMP4, which is subsequently specified to the definitive hematopoietic program by the GSK3&#946; inhibitor, CHIR99021. Alternatively, primitive hematopoiesis is specified by the PORCN inhibitor, IWP2. Hematopoiesis is further driven through the addition of recombinant VEGF and supportive hematopoietic cytokines. The resulting hematopoietic progenitors generated using this method have the potential to be used for disease and developmental modeling, in vitro.

Here, we present human pluripotent stem cell (hPSC) culture protocols, used to differentiate hPSCs into CD34+ hematopoietic progenitors. This method uses stage-specific manipulation of canonical WNT signaling to specify cells exclusively to either the definitive or primitive hematopoietic program.

İlkel ve kesin hematopoetik ataları insan Pluripotent kök hücrelerden farklılaşma yönetmen

One of the major goals for regenerative medicine is the generation and maintenance of hematopoietic stem cells (HSCs) derived from human pluripotent stem ...

Gelişim Biyolojisi

In Vitro Differentiation of Human Pluripotent Stem Cells into Trophoblastic Cells

The placenta is the first organ to develop during embryogenesis and is required for the survival of the developing embryo. The placenta is comprised of various trophoblastic cells that differentiate from the extra-embryonic trophectoderm cells of the preimplantation blastocyst. As such, our understanding of the early differentiation events of the human placenta is limited because of ethical and legal restrictions on the isolation and manipulation of human embryogenesis. Human pluripotent stem cells (hPSCs) are a robust model system for investigating human development and can also be differentiated in vitro into trophoblastic cells that express markers of the various trophoblast cell types. Here, we present a detailed protocol for differentiating hPSCs into trophoblastic cells using bone morphogenic protein 4 and inhibitors of the Activin/Nodal signaling pathways. This protocol generates various trophoblast cell types that can be transfected with siRNAs for investigating loss-of-function phenotypes or can be infected with pathogens. Additionally, hPSCs can be genetically modified and then differentiated into trophoblast progenitors for gain-of-function analyses. This in vitro differentiation method for generating human trophoblasts starting from hPSCs overcomes the ethical and legal restrictions of working with early human embryos, and this system can be used for a variety of applications, including drug discovery and stem cell research.

In Vitro Differentiation of Human Pluripotent Stem Cells into Trophoblastic Cells

Here, we present a protocol to efficiently generate human trophoblastic cells from human pluripotent stem cells using bone morphogenic protein 4 and inhibitors of the Activin/Nodal pathways. This method is suitable for the efficient differentiation of human pluripotent stem cells and can generate large quantities of cells for genetic manipulation.

Insan pluripotent İn Vitro Farklılaşma Trofoblastik Hücreleri içine Kök Hücreler

The placenta is the first organ to develop during embryogenesis and is required for the survival of the developing embryo. The placenta is comprised of ...

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 ...

Efficient and Scalable Directed Differentiation of Clinically Compatible Corneal Limbal Epithelial Stem Cells from Human Pluripotent Stem Cells

Corneal limbal epithelial stem cells (LESCs) are responsible for continuously renewing the corneal epithelium, and thus maintaining corneal homeostasis and visual clarity. Human pluripotent stem cell (hPSC)-derived LESCs provide a promising cell source for corneal cell replacement therapy. Undefined, xenogeneic culture and differentiation conditions cause variation in research results and impede the clinical translation of hPSC-derived therapeutics. This protocol provides a reproducible and efficient method for hPSC-LESC differentiation under xeno- and feeder cell-free conditions. Firstly, monolayer culture of undifferentiated hPSC on recombinant laminin-521 (LN-521) and defined hPSC medium serves as a foundation for robust production of high-quality starting material for differentiations. Secondly, a rapid and simple hPSC-LESC differentiation method yields LESC populations in only 24 days. This method includes a four-day surface ectodermal induction in suspension with small molecules, followed by adherent culture phase on LN-521/collagen IV combination matrix in defined corneal epithelial differentiation medium. Cryostoring and extended differentiation further purifies the cell population and enables banking of the cells in large quantities for cell therapy products. The resulting high-quality hPSC-LESCs provide a potential novel treatment strategy for corneal surface reconstruction to treat limbal stem cell deficiency (LSCD).

This protocol introduces a simple two-step method for differentiating corneal limbal epithelial stem cells from human pluripotent stem cells under xeno- and feeder cell-free culture conditions. The cell culture methods presented here enable cost-efficient, large-scale production of clinical quality cells applicable to corneal cell therapy use.

Verimli ve ölçeklenebilir yönlendirilmiş farklılaşma klinik olarak uyumlu kornea Limbal epitel kök hücrelerden insan Pluripotent kök hücreler

Corneal limbal epithelial stem cells (LESCs) are responsible for continuously renewing the corneal epithelium, and thus maintaining corneal homeostasis ...

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.

Insan pluripotent kök hücrelerinin karaciğer hücrelerine verimli farklılaşma

The liver detoxifies harmful substances, secretes vital proteins, and executes key metabolic activities, thus sustaining life. Consequently, liver ...

In Vitro Generation of Somite Derivatives from Human Induced Pluripotent Stem Cells

In response to signals such as WNTs, bone morphogenetic proteins (BMPs), and sonic hedgehog (SHH) secreted from surrounding tissues, somites (SMs) give rise to multiple cell types, including the myotome (MYO), sclerotome (SCL), dermatome (D), and syndetome (SYN), which in turn develop into skeletal muscle, axial skeleton, dorsal dermis, and axial tendon/ligament, respectively. Therefore, the generation of SMs and their derivatives from human induced pluripotent stem cells (iPSCs) is critical to obtain pluripotent stem cells (PSCs) for application in regenerative medicine and for disease research in the field of orthopedic surgery. Although the induction protocols for MYO and SCL from PSCs have been previously reported by several researchers, no study has yet demonstrated the induction of SYN and D from iPSCs. Therefore, efficient induction of fully competent SMs remains a major challenge. Here, we recapitulate human SM patterning with human iPSCs in vitro by mimicking the signaling environment during chick/mouse SM development, and report on methods of systematic induction of SM derivatives (MYO, SCL, D, and SYN) from human iPSCs under chemically defined conditions through the presomitic mesoderm (PSM) and SM states. Knowledge regarding chick/mouse&#160;SM development was successfully applied to the induction of SMs with human iPSCs. This method could be a novel tool for studying human somitogenesis and patterning without the use of embryos and for cell-based therapy and disease modeling.

We present here a protocol for the differentiation of human induced pluripotent stem cells into each somite derivative (myotome, sclerotome, dermatome, and syndetome) in chemically defined conditions, which has applications in future disease modeling and cell-based therapies in orthopedic surgery.

Somite türevleri indüklenen insan nesil Vitro Pluripotent kök hücreler

In response to signals such as WNTs, bone morphogenetic proteins (BMPs), and sonic hedgehog (SHH) secreted from surrounding tissues, somites (SMs) give ...

Transient Treatment of Human Pluripotent Stem Cells with DMSO to Promote Differentiation

Despite the growing use of pluripotent stem cells (PSCs), challenges in efficiently differentiating embryonic and induced pluripotent stem cells (ESCs and iPSCs) across various lineages remain. Numerous differentiation protocols have been developed, yet variability across cell lines and low rates of differentiation impart challenges in successfully implementing these protocols. Described here is an easy and inexpensive means to enhance the differentiation capacity of PSCs. It has been previously shown that treatment of stem cells with a low concentration of dimethyl sulfoxide (DMSO) significantly increases the propensity of a variety of PSCs to differentiate to different cell types following directed differentiation. This technique has now been shown to be effective across different species (e.g., mouse, primate, and human) into multiple lineages, ranging from neurons and cortical spheroids to smooth muscle cells and hepatocytes. The DMSO pretreatment improves PSC differentiation by regulating the cell cycle and priming stem cells to be more responsive to differentiation signals. Provided here is the detailed methodology for using this simple tool as a reproducible and widely applicable means to more efficiently differentiate PSCs to any lineage of choice.

Generating differentiated cell types from human pluripotent stem cells (hPSCs) holds great therapeutic promise but remains challenging. PSCs often exhibit an inherent inability to differentiate even when stimulated with a proper set of signals. Described here is a simple tool to enhance multilineage differentiation across a variety of PSC lines.

Insan pluripotent kök hücrelerinin DMSO ile geçici tedavisi farklılaşma teşvik etmek

Despite the growing use of pluripotent stem cells (PSCs), challenges in efficiently differentiating embryonic and induced pluripotent stem cells (ESCs and ...

Assessment of Oxidative Damage in the Primary Mouse Ocular Surface Cells/Stem Cells in Response to Ultraviolet-C (UV-C) Damage

The ocular surface is subjected to regular wear and tear due to various environmental factors. Exposure to UV-C radiation constitutes an occupational health hazard. Here, we demonstrate the exposure of primary stem cells from the mouse ocular surface to UV-C radiation. Reactive oxygen species (ROS) formation is the readout of the extent of oxidative stress/damage. In an experimental in vitro setting, it is also essential to assess the percentage of dead cells generated due to oxidative stress. In this article, we will demonstrate the 2&#39;,7&#39;-Dichlorofluoresceindiacetate (DCFDA) staining of UV-C exposed mouse primary ocular surface stem cells and their quantification based on the fluorescent images of DCFDA staining. DCFDA staining directly corresponds to ROS generation. We also demonstrate the quantification of dead and live cells by simultaneous staining with propidium iodide (PI) and Hoechst 3332 respectively and the percentage of DCFDA (ROS positive) and PI positive cells.

Assessment of Oxidative Damage in the Primary Mouse Ocular Surface Cells/Stem Cells in Response to Ultraviolet-C UV-C Damage

This protocol demonstrates the simultaneous detection of reactive oxygen species (ROS), live cells, and dead cells in live primary cultures from mouse ocular surface cells. 2&#39;,7&#39;-Dichlorofluoresceindiacetate, propidium iodide, and Hoechst staining are used to assess the ROS, dead cells, and live cells, respectively, followed by imaging and analysis.

Ultraviyole-C (UV-C) Hasarına Yanıt Olarak Birincil Fare Oküler Yüzey Hücrelerinde/Kök Hücrelerinde Oksidatif Hasarın Değerlendirilmesi

The ocular surface is subjected to regular wear and tear due to various environmental factors. Exposure to UV-C radiation constitutes an occupational ...

Efficient Neural Differentiation using Single-Cell Culture of Human Embryonic Stem Cells

In vitro differentiation of human embryonic stem cells (hESCs) has transformed the ability to study human development on both biological and molecular levels and provided cells for use in regenerative applications. Standard approaches for hESC culture using colony type culture to maintain undifferentiated hESCs and embryoid body (EB) and rosette formation for differentiation into different germ layers are inefficient and time-consuming. Presented here is a single-cell culture method using hESCs instead of a colony-type culture. This method allows maintenance of the characteristic features of undifferentiated hESCs, including expression of hESC markers at levels comparable to colony type hESCs. In addition, the protocol presents an efficient method for neural progenitor cell (NPC) generation from single-cell type hESCs that produces NPCs within 1 week. These cells highly express several NPC marker genes and can differentiate into various neural cell types, including dopaminergic neurons and astrocytes. This single-cell culture system for hESCs will be useful in investigating the molecular mechanisms of these processes, studies of certain diseases, and drug discovery screens.

Presented here is a protocol for the generation of a single-cell culture of human embryonic stem cells and their subsequent differentiation into neural progenitor cells. The protocol is simple, robust, scalable, and suitable for drug screening and regenerative medicine applications.

İnsan Embriyonik Kök Hücrelerinin Tek Hücre Kültürünü Kullanarak Etkin Nöral Farklılaşma

In vitro differentiation of human embryonic stem cells (hESCs) has transformed the ability to study human development on both biological and molecular ...

Efficient Differentiation of Postganglionic Sympathetic Neurons using Human Pluripotent Stem Cells under Feeder-free and Chemically Defined Culture Conditions

Human pluripotent stem cells (hPSCs) have become a powerful tool for disease modeling and the study of human embryonic development in vitro. We previously presented a differentiation protocol for the derivation of autonomic neurons with sympathetic character that has been applied to patients with autonomic neuropathy. However, the protocol was built on Knock Out Serum Replacement (KSR) and feeder-based culture conditions, and to ensure high differentiation efficiency, cell sorting was necessary. These factors cause high variability, high cost, and low reproducibility. Moreover, mature sympathetic properties, including electrical activity, have not been verified. Here, we present an optimized protocol where PSC culture and differentiation are performed in feeder-free and chemically defined culture conditions. Genetic markers identifying trunk neural crest are identified. Further differentiation into postganglionic sympathetic neurons is achieved after 20 days without the need for cell sorting. Electrophysiological recording further shows the functional neuron identity. Firing detected from our differentiated neurons can be enhanced by nicotine and suppressed by the adrenergic receptor antagonist propranolol. Intermediate sympathetic neural progenitors in this protocol can be maintained as neural spheroids for up to 2 weeks, which allows expansion of the cultures. In sum, our updated sympathetic neuron differentiation protocol shows high differentiation efficiency, better reproducibility, more flexibility, and better neural maturation compared to the previous version. This protocol will provide researchers with the cells necessary to study human disorders that affect the autonomic nervous system.

In this protocol, we describe a stable, highly efficient differentiation strategy for the generation of postganglionic sympathetic neurons from human pluripotent stem cells. This model will make neurons available for the use of studies of multiple autonomic disorders.

Besleyicisiz ve Kimyasal Olarak Tanımlanmış Kültür Koşulları Altında İnsan Pluripotent Kök Hücreleri Kullanan Postgangliyonik Sempatik Nöronların Verimli Farklılaşması

Human pluripotent stem cells (hPSCs) have become a powerful tool for disease modeling and the study of human embryonic development in vitro. We previously ...