We thank M. Kapitza, Margit Henry, Tamara Rotshteyn, Susan Rohani and Cornelia B&#246;ttinger for excellent technical support. This work was supported by grants from the German Research Foundation (RTG 1331) and the German Ministry for Research (BMBF).

<ol>
	<li>Liu, W. W., Deng, Y. G., Liu, Y., Gong, W. R., Deng, W. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stem+Cell+Models+for+Drug+Discovery+and+Toxicology+Studies.">Stem Cell Models for Drug Discovery and Toxicology Studies.</a> Journal of Biochemical and Molecular Toxicology. 27 (1), 17-27 (2013).</li><li>Zuba-Surma, E. K., Jozkowicz, A., Dulak, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stem+Cells+in+Pharmaceutical+Biotechnology.">Stem Cells in Pharmaceutical Biotechnology.</a> Current Pharmaceutical Biotechnology. 12 (11), 1760-1773 (2011).</li><li>Leist, M., Hartung, T., Nicotera, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+dawning+of+a+new+age+of+toxicology.">The dawning of a new age of toxicology.</a> ALTEX. 25 (2), 103-114 (2008).</li><li>Kuegler, P. B., 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=Markers+of+murine+embryonic+and+neural+stem+cells,+neurons+and+astrocytes:+reference+points+for+developmental+neurotoxicity+testing.">Markers of murine embryonic and neural stem cells, neurons and astrocytes: reference points for developmental neurotoxicity testing.</a> ALTEX. 27 (1), 17-42 (2010).</li><li>Yamada, 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=Human+oocytes+reprogram+adult+somatic+nuclei+of+a+type+1+diabetic+to+diploid+pluripotent+stem+cells.">Human oocytes reprogram adult somatic nuclei of a type 1 diabetic to diploid pluripotent stem cells.</a> Nature. , (2014).</li><li>Chambers, S. 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=Highly+efficient+neural+conversion+of+human+ES+and+iPS+cells+by+dual+inhibition+of+SMAD+signaling.">Highly efficient neural conversion of human ES and iPS cells by dual inhibition of SMAD signaling.</a> Nature Biotechnology. 27 (3), 275-280 (2009).</li><li>Takasato, 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=Directing+human+embryonic+stem+cell+differentiation+towards+a+renal+lineage+generates+a+self-organizing+kidney.">Directing human embryonic stem cell differentiation towards a renal lineage generates a self-organizing kidney.</a> Nature cell biology. 16 (1), 118-126 (2014).</li><li>Zimmer, B., 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+Developmental+Toxicants+and+Signaling+Pathways+in+a+Functional+Test+Based+on+the+Migration+of+Human+Neural+Crest+Cells.">Evaluation of Developmental Toxicants and Signaling Pathways in a Functional Test Based on the Migration of Human Neural Crest Cells.</a> Environmental Health Perspectives. 120 (8), 1116-1122 (2012).</li><li>Bosman, 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=Molecular+and+Functional+Evidence+of+HCN4+and+Caveolin-3+Interaction+During+Cardiomyocyte+Differentiation+from+Human+Embryonic+Stem+Cells.">Molecular and Functional Evidence of HCN4 and Caveolin-3 Interaction During Cardiomyocyte Differentiation from Human Embryonic Stem Cells.</a> Stem Cells and Development. 22 (11), 1717-1727 (2013).</li><li>Sartiani, L., 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=Developmental+changes+in+cardiomyocytes+differentiated+from+human+embryonic+stem+cells:+A+molecular+and+electrophysiological+approach.">Developmental changes in cardiomyocytes differentiated from human embryonic stem cells: A molecular and electrophysiological approach.</a> Stem Cells. 25 (5), 1136-1144 (2007).</li><li>Xu, X. Q., 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=Chemically+defined+medium+supporting+cardiomyocyte+differentiation+of+human+embryonic+stem+cells.">Chemically defined medium supporting cardiomyocyte differentiation of human embryonic stem cells.</a> Differentiation. 76 (9), 958-970 (2008).</li><li>Pal, R., Mamidi, M. K., Das, A. K., Bhonde, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparative+analysis+of+cardiomyocyte+differentiation+from+human+embryonic+stem+cells+under+3-D+and+2-D+culture+conditions.">Comparative analysis of cardiomyocyte differentiation from human embryonic stem cells under 3-D and 2-D culture conditions.</a> Journal of Bioscience and Bioengineering. 115 (2), 200-206 (2013).</li><li>Subramanian, 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=Spheroid+Culture+for+Enhanced+Differentiation+of+Human+Embryonic+Stem+Cells+to+Hepatocyte-Like+Cells.">Spheroid Culture for Enhanced Differentiation of Human Embryonic Stem Cells to Hepatocyte-Like Cells.</a> Stem Cells and Development. 23 (2), 124-131 (2014).</li><li>Sivertsson, L., Synnergren, J., Jensen, J., Bjorquist, P., Ingelman-Sundberg, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hepatic+Differentiation+and+Maturation+of+Human+Embryonic+Stem+Cells+Cultured+in+a+Perfused+Three-Dimensional+Bioreactor.">Hepatic Differentiation and Maturation of Human Embryonic Stem Cells Cultured in a Perfused Three-Dimensional Bioreactor.</a> Stem Cells and Development. 22 (4), 581-594 (2013).</li><li>Jagtap, S., 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=Cytosine+arabinoside+induces+ectoderm+and+inhibits+mesoderm+expression+in+human+embryonic+stem+cells+during+multilineage+differentiation.">Cytosine arabinoside induces ectoderm and inhibits mesoderm expression in human embryonic stem cells during multilineage differentiation.</a> British Journal of Pharmacology. 162 (8), 1743-1756 (2011).</li><li>Krug, A. 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+embryonic+stem+cell-derived+test+systems+for+developmental+neurotoxicity:+a+transcriptomics+approach.">Human embryonic stem cell-derived test systems for developmental neurotoxicity: a transcriptomics approach.</a> Archives of Toxicology. 87 (1), 123-143 (2013).</li><li>Leist, 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=Test+systems+of+developmental+toxicity:+state-of-the+art+and+future+perspectives.">Test systems of developmental toxicity: state-of-the art and future perspectives.</a> Archives of Toxicology. 87 (12), 2037-2042 (2013).</li><li>Itskovitz-Eldor, 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+embryonic+stem+cells+into+embryoid+bodies+compromising+the+three+embryonic+germ+layers.">Differentiation of human embryonic stem cells into embryoid bodies compromising the three embryonic germ layers.</a> Molecular medicine. 6 (2), 88-95 (2000).</li><li>Khoo, M. L. 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=Growth+and+differentiation+of+embryoid+bodies+derived+from+human+embryonic+stem+cells:+Effect+of+glucose+and+basic+fibroblast+growth+factor.">Growth and differentiation of embryoid bodies derived from human embryonic stem cells: Effect of glucose and basic fibroblast growth factor.</a> Biology of Reproduction. 73 (6), 1147-1156 (2005).</li><li>Son, M. Y., Kim, H. J., Kim, M. J., Cho, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Physical+Passaging+of+Embryoid+Bodies+Generated+from+Human+Pluripotent+Stem+Cells.">Physical Passaging of Embryoid Bodies Generated from Human Pluripotent Stem Cells.</a> Plos One. 6 (5), (2011).</li><li>Winkler, J., Sotiriadou, I., Chen, S., Hescheler, J., Sachinidis, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+potential+of+embryonic+stem+cells+combined+with+-omics+technologies+as+model+systems+for+toxicology.">The potential of embryonic stem cells combined with -omics technologies as model systems for toxicology.</a> Current medicinal chemistry. 16 (36), 4814-4827 (2009).</li><li>Gunaseeli, I., Doss, M. X., Antzelevitch, C., Hescheler, J., Sachinidis, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induced+pluripotent+stem+cells+as+a+model+for+accelerated+patient-+and+disease-specific+drug+discovery.">Induced pluripotent stem cells as a model for accelerated patient- and disease-specific drug discovery.</a> Current medicinal chemistry. 17 (8), 759-766 (2010).</li><li>Miller, M. T., Stromland, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Teratogen+update:+Thalidomide:+A+review,+with+a+focus+on+ocular+findings+and+new+potential+uses.">Teratogen update: Thalidomide: A review, with a focus on ocular findings and new potential uses.</a> Teratology. 60 (5), 306-321 (1999).</li><li>Newman, C. G. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Teratogen+Update+-+Clinical+Aspects+of+Thalidomide+Embryopathy+-+A+Continuing+Preoccupation.">Teratogen Update - Clinical Aspects of Thalidomide Embryopathy - A Continuing Preoccupation.</a> Teratology. 32 (1), 133-144 (1985).</li><li>Stern, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+assessment+of+the+teratogenic+potential+of+drugs+in+humans.">In vivo assessment of the teratogenic potential of drugs in humans.</a> Obstetrics and gynecology. 58 (5 Suppl), 3S-8S (1981).</li><li>Lynch, H. T., Reich, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diethylstilbestrol,+Genetics,+Teratogenesis,+and+Tumor+Spectrum+in+Humans.">Diethylstilbestrol, Genetics, Teratogenesis, and Tumor Spectrum in Humans.</a> Medical Hypotheses. 16 (3), 315-332 (1985).</li><li>Satoh, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Behavioral+teratology+of+mercury+and+its+compounds.">Behavioral teratology of mercury and its compounds.</a> Tohoku Journal of Experimental Medicine. 201 (1), 1-9 (2003).</li><li>Meganathan, 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+Thalidomide-Specific+Transcriptomics+and+Proteomics+Signatures+during+Differentiation+of+Human+Embryonic+Stem+Cells.">Identification of Thalidomide-Specific Transcriptomics and Proteomics Signatures during Differentiation of Human Embryonic Stem Cells.</a> Plos One. 7 (8), (2012).</li><li>Balmer, N. V., 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=Epigenetic+changes+and+disturbed+neural+development+in+a+human+embryonic+stem+cell-based+model+relating+to+the+fetal+valproate+syndrome.">Epigenetic changes and disturbed neural development in a human embryonic stem cell-based model relating to the fetal valproate syndrome.</a> Human Molecular Genetics. 21 (18), 4104-4114 (2012).</li><li>Smirnova, L., Hogberg, H. T., Leist, M., Hartung, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Developmental+neurotoxicity+-+Challenges+in+the+21st+Century+and+In+Vitro+Opportunities.">Developmental neurotoxicity - Challenges in the 21st Century and In Vitro Opportunities.</a> ALTEX. 31 (2), 129-156 (2014).</li><li>Leist, M., Efremova, L., Karreman, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Food+for+thought+...+considerations+and+guidelines+for+basic+test+method+descriptions+in+toxicology.">Food for thought ... considerations and guidelines for basic test method descriptions in toxicology.</a> ALTEX. 27 (4), 309-317 (2010).</li><li>Zimmer, B., 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=Coordinated+waves+of+gene+expression+during+neuronal+differentiation+of+embryonic+stem+cells+as+basis+for+novel+approaches+to+developmental+neurotoxicity+testing.">Coordinated waves of gene expression during neuronal differentiation of embryonic stem cells as basis for novel approaches to developmental neurotoxicity testing.</a> Cell Death and Differentiation. 18 (3), 383-395 (2011).</li><li>Kraljevic, S., Stambrook, P. J., Pavelic, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Accelerating+drug+discovery.">Accelerating drug discovery.</a> EMBO Reports. 5 (9), 837-842 (2004).</li></ol>

The authors have nothing to disclose.

Toksikolojik testler Geleneksel yaklaşımlar dolayısıyla test masraflı ve zaman alıcı hale kapsamlı hayvan çalışmaları içerir. Ayrıca, türler arası farklılıklar nedeniyle klinik öncesi hayvan güvenlik çalışmaları insanlar için uygun potansiyel ilaçların toksisitesi etkilerini tahmin etmek her zaman geçerli değildir. Insan olmayan primatlar, en öngörülebilir, halen etik güçlü ve sosyoekonomik talepleri hızla in vitro testinde hassas ve sağlam gelişen modern toplumlar tarafın...

hücrelerin çeşitli içine ayırt etmek, insan embriyonik kök hücreleri (hESC) yeteneği in vitro toksisite testleri 1, hastalık modelleme ve rejeneratif tıp 2 yeni bir dönem açtı. kök hücreler kendini çoğaltmak için kapasite, onların pluripotent devleti korumak için, ve özelleşmiş hücrelere 3,4 içine ayırt etmek için sahiptirler. HESC özellikleri, aynı zamanda, insan uyarılmış pluripotent kök hücreler (hiPSC) ya da nükleer transfer 5 tarafından üretilen hücreler gibi diğer insan pluripotent kök hücreleri, bulunan (kapasitesi tüm ana hücre tiplerine ayırt etmek için). Örneğin, birçok farklı HESC hatları hücreler 13,14 gibi nöronlar 6, böbrek hücreleri 7, nöral krest hücrelerinden 8, kardiyomiyositler 9-12 veya hepatositlere ayırt edilmiştir. Dahası, HESC kendiliğinden embriyoid organları (EBS) 19,20 her üç germ 15-18 hücre içine ayırt edebilirsiniz. Early embriyonik gelişim mikro-dizi teknolojisi kullanılarak 15 transkriptomik mRNA düzeyinde yakalandı farklı germ ile ilgili çeşitli genlerin farklı sunum-düzenlenir. Bu çabalar HESC / hiPSC ve transkriptomik analizi (inceleme için 21,22 bakınız) dayalı organa özgü toksikolojik modellerin kurulması ile sonuçlandı. Bu modeller laboratuar hayvanları hep insan güvenliği tahmin değildir kullanarak preklinik çalışmalarda olarak toksikolojik çalışmalar için laboratuvar hayvanlarının geleneksel kullanımı üzerinde avantajları var. hastalarda karşılaşılan ilaca bağlı toksisiteleri genellikle insanlar ve deney hayvanlarında arasında farklılık metabolik veya sinyal süreçleriyle ilgilidir. türler farkı insanlarda gelişimsel toksisite güvenilir erken teşhis engelledi ve bu talidomid 23,24 ve 25,26 olarak dietilstilbestrol örnek ilaçlar nedeniyle teratojenite piyasadan çekildi. Thalidomide sıçan veya farelerde herhangi gelişimsel toksisite göstermemiştir. Örneğin metil civa 27 Çevresel kimyasal maddeler, çeşitli türlerde sinir sistemi ile ilgili prenatal gelişim toksisite sonuçlandı, ancak insan belirtiler hayvanlarda modellemek zor olmuştur. Türlerin özgüllüğü sorunları sorunu çözmek için, farklı projelerde altında çalışan bilim adamları vb şüphelenilen insan toksik atıkları kullanarak embriyonik toksisite, nörotoksisite, kardiyotoksisite, hepatotoksisite ve nefrotoksisite için farklı modellerin geliştirilmesinde başlattık reprotect, ESNATS, dedektif gibi kök hücreleri dayalı insanları etkiler. Avrupa konsorsiyumu projesinde &#39;Embriyonik Kök hücre tabanlı Roman Alternatif Test Stratejileri (ESNATS)&#39; kapsamında beş test sistemleri kurulmuştur. Bir test sistemi olarak adlandırılan UKK (U niversitäts k linikum K OLN) test sistemi kısmen erken insan embriyonik gelişmeyi yakalar. Bu s olarakistem insan embriyonik H9 hücreleri üç germ (ektoderm, endoderm ve mezoderm) 15 ve germ tabakası belirli imzalar Affymetrix mikroarray platformu kullanarak profil transkriptomik tarafından esir edilmiş olarak ayrılır. Talidomid 28, valproik asit, metil cıva 16,17 veya sitosin arabinosid 15 gibi çeşitli gelişimsel toksik maddeler bu sistemde test edilmiştir ve zehirli spesifik gen imzalar elde edilmiştir. Ikinci bir test sisteminde, bu nedenle test sistemi 1 UKN1 (K onsta n = U niversity) olarak adlandırılan, H9 hücreleri 6 gün süreyle nöroektodermal projenitör hücreleri (NEP) ile ayrılır. Bu tür Pax6 ve OTX2 olarak nöral gen belirteçleri yüksek ifadesi ile belirlenir. Farklılaşma sırasında 6 gün, NEP hücreler gibi VPA, metil cıva gibi gelişimsel nöro-toksik maruz kalmıştır. Zehiri özgü de regüle transkriptomik profilleri obtai olmuşturAffymetrix mikroarray platformu 16,29 ile de tanımlanır. 21. yüzyılın toksikoloji için yeni bir vizyon test sistemleri uzun vadeli toxicant inkubasyon sonunda in vivo histopatolojik veya transcriptome değişiklikleri gibi fenotipik açıklamaları verim yapmak değil sadece öngörmektedir. Daha çok tahliller mekanistik bilgi 3. sağlamak ve bu bilgilerin sözde eşlenebilir düşündürmektedir olumsuz sonuç yolları (AOP) zararlı etkilerinden 30 için bilimsel gerekçe sağlamak. Bu tür bilgi vermek, uygulamalı test sistemleri yüksek kaliteli sağlam standart operasyon prosedürlerine göre belgelenmiş örneğin olarak, 31 kontrol edilmesi gerekiyor. Ayrıca, zaman-bağımlı değişiklikler yüksek çözünürlüklü eşlenmesi gerekir. Bu senkronize değişikliklerle 32 test sistemlerini gerektirir. Burada anlatılan UKN1 ve UKK test sistemleri bu gereksinimleri için optimize edilmiştir.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>DMEM/F-12</td>
			<td>Life Technologies</td>
			<td>11320082</td>
			<td>Dulbecco&#39;s Modified Eagle Medium:Nutrient Mixture F-12</td>
		</tr>
		<tr>
			<td>KOSR</td>
			<td>Life Technologies</td>
			<td>10828028</td>
			<td>Knockout Serum Replacement</td>
		</tr>
		<tr>
			<td>GlutaMAX</td>
			<td>Life Technologies</td>
			<td>35050061</td>
			<td>GlutaMAX supplement</td>
		</tr>
		<tr>
			<td>NEAA</td>
			<td>Life Technologies</td>
			<td>11140050</td>
			<td>MEM Nonessential Amino Acids Solution</td>
		</tr>
		<tr>
			<td>DPBS</td>
			<td>Life Technologies</td>
			<td>14190-0144</td>
			<td>Dulbecco&#39;s Phosphate-Buffered Saline, without calcium, without magnesium</td>
		</tr>
		<tr>
			<td>mTeSR medium</td>
			<td>Stemcell Technologies</td>
			<td>5850</td>
			<td></td>
		</tr>
		<tr>
			<td>Pluronic F-127</td>
			<td>Sigma</td>
			<td>P2443-250G</td>
			<td></td>
		</tr>
		<tr>
			<td>V bottom plate</td>
			<td>VWR</td>
			<td>734-0483</td>
			<td>Plate,Microwell,V BTTM,96 Well,Sterile 1 * 50 ST</td>
		</tr>
		<tr>
			<td>Vbottom plate lid</td>
			<td>VWR</td>
			<td>634-0011</td>
			<td>Lid, Microtitre plates, Cond. Ring 1 * 50 ST</td>
		</tr>
		<tr>
			<td>Pen/Strep</td>
			<td>Life Technologies</td>
			<td>15140-122</td>
			<td>Penicillin- Streptomycin, Liquid</td>
		</tr>
		<tr>
			<td>Distilled Water</td>
			<td>Life Technologies</td>
			<td>15230-089.</td>
			<td>Sterile Distilled Water</td>
		</tr>
		<tr>
			<td>Human FGF-2 (bFGF)</td>
			<td>Millipore</td>
			<td>GF003AF-100UG</td>
			<td>Fibroblast Growth Factor basic, human recombinant, animal-free</td>
		</tr>
		<tr>
			<td>Filter 0.22 &#956;m</td>
			<td>Millipore</td>
			<td>SCGPU02RE</td>
			<td>Stericup-GP, 0.22 &#956;m, polyethersulfone, 250 ml, radio- sterilized</td>
		</tr>
		<tr>
			<td>StemPro EZPassageTM Disposablte</td>
			<td>Invitrogen</td>
			<td>23181010</td>
			<td></td>
		</tr>
		<tr>
			<td>BD MatrigelTM, hESC qualified Matrix</td>
			<td>Stemcell Technologies</td>
			<td>354277</td>
			<td>5 ml vial</td>
		</tr>
		<tr>
			<td>DMSO</td>
			<td>Sigma</td>
			<td>D-2650</td>
			<td></td>
		</tr>
		<tr>
			<td>RNAlater Stabilization Solution</td>
			<td>Life Technologies</td>
			<td>AM7020</td>
			<td>It stabilizes and protect the RNA integrity in unfrozen samples.</td>
		</tr>
		<tr>
			<td>70 &#956;m Cell Strainer</td>
			<td>Becton Dickinson</td>
			<td>352350</td>
			<td>Cell strainer with 70 &#956;m Nylon mesh</td>
		</tr>
		<tr>
			<td>35 &#956;m Lid cell strainer, 5 ml tube</td>
			<td>Becton Dickinson</td>
			<td>352235</td>
			<td>5 ml polystyrene round bottom test tube, with a cell strainer cap (35 &#956;m)</td>
		</tr>
		<tr>
			<td>50 ml sterile Polypropylene tube</td>
			<td>Greiner Bio-One</td>
			<td>227261</td>
			<td>50 ml Polypropylene tube with conical bottom, Sterile</td>
		</tr>
		<tr>
			<td>T75 flask</td>
			<td>Greiner Bio-One</td>
			<td>658175</td>
			<td>CELLSTAR Filter Cap Cell Culture 75 cm2 Flasks</td>
		</tr>
		<tr>
			<td>TRIzol</td>
			<td>Life Technologies</td>
			<td>10296010</td>
			<td></td>
		</tr>
		<tr>
			<td>96 well optical bottom plates</td>
			<td>Thermo Scientific</td>
			<td>165305</td>
			<td></td>
		</tr>
		<tr>
			<td>CellTiter-Blue</td>
			<td>Promega</td>
			<td>G8081</td>
			<td></td>
		</tr>
		<tr>
			<td>Accutase</td>
			<td>PAA</td>
			<td>L11-007</td>
			<td></td>
		</tr>
		<tr>
			<td>Apotransferin</td>
			<td>Sigma-Aldrich</td>
			<td>T-2036</td>
			<td></td>
		</tr>
		<tr>
			<td>Dispase</td>
			<td>Worthington Biochemicals</td>
			<td>LS002104</td>
			<td></td>
		</tr>
		<tr>
			<td>Dorsomorphin</td>
			<td>Tocris Bioscience</td>
			<td>3093</td>
			<td></td>
		</tr>
		<tr>
			<td>EDTA</td>
			<td>Roth</td>
			<td>8043.2</td>
			<td></td>
		</tr>
		<tr>
			<td>FBS</td>
			<td>PAA</td>
			<td>A15-101</td>
			<td></td>
		</tr>
		<tr>
			<td>FGF-2</td>
			<td>R&#38;D Systems</td>
			<td>233-FB</td>
			<td></td>
		</tr>
		<tr>
			<td>Gelatine</td>
			<td>Sigma-Aldrich</td>
			<td>G1890-100G</td>
			<td></td>
		</tr>
		<tr>
			<td>Glucose</td>
			<td>Sigma-Aldrich</td>
			<td>G7021-100G</td>
			<td></td>
		</tr>
		<tr>
			<td>GlutaMAX</td>
			<td>Gibco Invitrogen</td>
			<td>35050-038</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Gibco Invitrogen</td>
			<td>15630-056</td>
			<td></td>
		</tr>
		<tr>
			<td>Insulin</td>
			<td>Sigma-Aldrich</td>
			<td>I-6634</td>
			<td></td>
		</tr>
		<tr>
			<td>Knockout DMEM</td>
			<td>Gibco Invitrogen</td>
			<td>10829-018</td>
			<td></td>
		</tr>
		<tr>
			<td>Matrigel</td>
			<td>BD Biosciences</td>
			<td>354234</td>
			<td></td>
		</tr>
		<tr>
			<td>Noggin</td>
			<td>R&#38;D Systems</td>
			<td>719-NG</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>Biochrom AG</td>
			<td>L1825</td>
			<td></td>
		</tr>
		<tr>
			<td>Progesteron</td>
			<td>Sigma-Aldrich</td>
			<td>P7556</td>
			<td></td>
		</tr>
		<tr>
			<td>Putrescine</td>
			<td>Sigma-Aldrich</td>
			<td>P-5780</td>
			<td></td>
		</tr>
		<tr>
			<td>ROCK inhibitor Y-27632</td>
			<td>Tocris Biosciences</td>
			<td>1254</td>
			<td></td>
		</tr>
		<tr>
			<td>SB431542</td>
			<td>Tocris Biosciences</td>
			<td>1614</td>
			<td></td>
		</tr>
		<tr>
			<td>SDS</td>
			<td>Bio-Rad</td>
			<td>161-0416</td>
			<td></td>
		</tr>
		<tr>
			<td>Selenium</td>
			<td>Sigma-Aldrich</td>
			<td>S-5261</td>
			<td></td>
		</tr>
		<tr>
			<td>&#946;-Mercaptoethanol</td>
			<td>Gibco Invitrogen</td>
			<td>31350-010</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>List of Kits</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>RNeasy Mini Kit (250)</td>
			<td>QIAGEN</td>
			<td>74106</td>
			<td></td>
		</tr>
		<tr>
			<td>GeneChip Hybridization, Wash, and Stain Kit</td>
			<td>Affymetrix</td>
			<td>900721, 22, 23</td>
			<td>This kit provides all reagents required for hybridization wash and staining of microarrays.</td>
		</tr>
		<tr>
			<td>Rnase-Free DNase Set</td>
			<td>QIAGEN</td>
			<td>79254</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td colspan="4">List of equipment.</td>
		</tr>
		<tr>
			<td>Inverted microscope</td>
			<td>Olympus</td>
			<td></td>
			<td>IX71</td>
		</tr>
		<tr>
			<td>Genechip Hybridisation Oven - 645</td>
			<td>Affymetrix</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Genechip Fluidics Station-450</td>
			<td>Affymetrix</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Affymetrix Gene-Chip Scanner-3000-7 G</td>
			<td>Affymetrix</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Spectramax M5</td>
			<td>Molecular Devices</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>List of softwares</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Prism 4</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Affymetrix GCOS</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Partek Genomic Suite 6.25</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Online tools for Functional annotation 
			DAVID 
			Onto-tools Intelligent Systems and Bioinformatics Laboratory</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

human pluripotent stem cell based developmental toxicity assays for chemical safety screening and systems biology data generation

-omics Teknolojiler potansiyel ilaçların in vitro toksisite testleri için yeni ufuklar açtı verimli protokoller arada çeşitli dokularda insan pluripotent kök hücreleri ayırt etmek. Bu tür tahliller için sağlam bir bilimsel temel sağlamak için, gelişim zamanı seyrine ve sistemler biyolojisi yaklaşımları ile altta yatan düzenleyici mekanizmaları üzerine sayısal bilgiler elde etmek için önemli olacaktır. İki tahlilleri dolayısıyla bu gereksinimler için buraya ayarlandı. UKK test sistemi olarak, insan embriyonik kök hücreleri (HESC) (veya başka bir pluripotent hücreler) kendiliğinden üç germ hücrelerinin üretilmesini sağlamak için, embriyoid organları 14 gün ayırt etmek için bırakılır. Bu sistem, erken insan embriyonik gelişiminin önemli adımlar özetlediği ve hücreler farklılaşma sırasında kimyasallara maruz kalırsanız o, insana özgü erken embriyonik toksisite / teratojenite tahmin edebilirsiniz. UKN1 test sistemi bir populat için ayırt hESC dayanmaktadırnöroektodermal dede iyon (NEP) 6 gün boyunca hücreler. Bu sistem, erken nöral gelişimi özetlediği ve erken gelişim nörotoksisiteyi ve kimyasallar tarafından tetiklenen epigenetik değişiklikler öngörüyor. Her iki sistem de, transcriptome mikrodizi çalışmaları ile kombinasyon halinde, toksisite biyolojik göstergeleri için uygundur. Ayrıca, sistem biyolojisi analiz için veri üretmek için kombinasyon halinde kullanılabilmektedir. Bu test sistemleri, hayvanların büyük miktarlarda gerektiren geleneksel toksikolojik çalışmalar bir çok dezavantajı vardır. test sistemleri, ilaç geliştirme ve kimyasal güvenlik değerlendirmesi için maliyetlerin azalmasına katkıda bulunabilir. Onların kombinasyonu özellikle özel nöro-gelişimsel sürecini etkileyebilir bileşikler ışık tutuyor.

UKK test sistemi Metil civa poz Sitotoksisite tahlili IC 10 bir değer metil cıva sitotoksisite için (% 10 canlılığı azalma) (Şekil 1) elde etmek üzere H9 EBS ile gerçekleştirildi. Biz de bir mikrodizi tabanlı (affymetrix platformu) biyomarker çalışması gerçekleştirdi. H9 EBS 14 gün boyunca metil civa (0.25 1 uM) maruz kalmıştır. 14. günde numuneler Trizol ile toplanmış ve RNA izole edildi. Transkripsiyonal profilleme İnsan Genom U133 art?...

Watch this Scientific Journal Video about Human Pluripotent Stem Cell Based Developmental Toxicity Assays for Chemical Safety Screening and Systems Biology Data Generation at JoVE.com

Human Pluripotent Stem Cell Based Developmental Toxicity Assays for Chemical Safety Screening and Systems Biology Data Generation

protokoller, insan embriyonik kök hücreleri ve transcriptome çalışmalarına dayalı iki in vitro gelişimsel toksisite test sistemleri (UKK ve UKN1) açıklar. test sistemleri, insan gelişimsel toksisite tehlike tahmin ve hayvan çalışmaları, maliyet ve kimyasal güvenlik testi için gerekli süreyi azaltmak için katkıda bulunabilir.

Kimyasal Güvenlik Tarama ve Sistem Biyolojisi Veri Üretimi Gelişim Toksisite Tahliller Tabanlı İnsan Pluripotent Kök Hücre

Efficient protocols to differentiate human pluripotent stem cells to various tissues in combination with -omics technologies opened up new horizons for in ...

human-pluripotent-stem-cell-based-developmental-toxicity-assays-for

Research

JoVE Journal

Developmental Biology

12.4K Görüntüleme.  University of Cologne.  protokoller, insan embriyonik kök hücreleri ve transcriptome çalışmalarına dayalı iki in vitro gelişimsel toksisite test sistemleri (UKK ve UKN1) açıklar. test sistemleri, insan gelişimsel toksisite tehlike tahmin ve hayvan çalışmaları, maliyet ve kimyasal güvenlik testi için gerekli süreyi azaltmak için katkıda bulunabilir. 

Video: Human Pluripotent Stem Cell Based Developmental Toxicity Assays for Chemical Safety Screening and Systems Biology Data Generation

Generation of Genetically Modified Organotypic Skin Cultures Using Devitalized Human Dermis

Organotypic cultures allow the reconstitution of a 3D environment critical for cell-cell contact and cell-matrix interactions which mimics the function and physiology of their in vivo tissue counterparts. This is exemplified by organotypic skin cultures which faithfully recapitulates the epidermal differentiation and stratification program. Primary human epidermal keratinocytes are genetically manipulable through retroviruses where genes can be easily overexpressed or knocked down. These genetically modified keratinocytes can then be used to regenerate human epidermis in organotypic skin cultures providing a powerful model to study genetic pathways impacting epidermal growth, differentiation, and disease progression. The protocols presented here describe methods to prepare devitalized human dermis as well as to genetically manipulate primary human keratinocytes in order to generate organotypic skin cultures. Regenerated human skin can be used in downstream applications such as gene expression profiling, immunostaining, and chromatin immunoprecipitations followed by high throughput sequencing. Thus, generation of these genetically modified organotypic skin cultures will allow the determination of genes that are critical for maintaining skin homeostasis.

The goal of this paper is to provide a comprehensive and detailed protocol on how to generate genetically modified human organotypic skin from epidermal keratinocytes and devitalized human dermis.

Güçsüz İnsan dermis kullanma Genetiği Değiştirilmiş Organotipik Cilt Kültürlerin Üretimi

Organotypic cultures allow the reconstitution of a 3D environment critical for cell-cell contact and cell-matrix interactions which mimics the function ...

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 ...

Generation of Integration-free Induced Pluripotent Stem Cells from Human Peripheral Blood Mononuclear Cells Using Episomal Vectors

Induced Pluripotent Stem Cells (iPSCs) hold great promise for disease modeling and regenerative therapies. We previously reported the use of Episomal Vectors (EV) to generate integration-free iPSCs from peripheral blood mononuclear cells (PB MNCs). The episomal vectors used are DNA plasmids incorporated with oriP and EBNA1 elements from the Epstein-Barr (EB) virus, which&#160;allow for replication and long-term retainment of plasmids in mammalian cells, respectively. With further optimization, thousands of iPSC colonies can be obtained from 1 mL of peripheral blood. Two critical factors for achieving high reprogramming efficiencies&#160;are: 1) the use of a 2A &#34;self-cleavage&#34; peptide to link OCT4 and SOX2, thus achieving equimolar expression of the two factors; 2) the use of two vectors to express MYC and KLF4 individually. Here we describe a step-by-step protocol for generating integration-free iPSCs from adult peripheral blood samples. The generated iPSCs are integration-free as residual episomal plasmids are undetectable after five passages. Although the reprogramming efficiency is comparable to that of Sendai Virus (SV) vectors, EV plasmids are considerably more economical than the commercially available SV vectors. This affordable EV reprogramming system holds potential for clinical applications in regenerative medicine and provides an approach for the direct reprogramming of PB MNCs to integration-free mesenchymal stem cells, neural stem cells, etc.

This protocol describes a detailed method for efficient generation of integration-free iPSCs from human adult peripheral blood cells. With the use of four oriP/EBNA-based episomal vectors to express the reprogramming factors, KLF4, MYC, BCL-XL, or OCT4 and SOX2, thousands of iPSC colonies can be obtained from 1 mL of peripheral blood.

Epizomal vektörlerin kullanılması insan periferik kan mononükleer hücrelerinden entegrasyon içermeyen uyarılmış pluripotent kök hücrelerin üretilmesi

Induced Pluripotent Stem Cells (iPSCs) hold great promise for disease modeling and regenerative therapies. We previously reported the use of Episomal ...

Defined and Scalable Generation of Hepatocyte-like Cells from Human Pluripotent Stem Cells

Human pluripotent stem cells (hPSCs) possess great value for biomedical research. hPSCs can be scaled and differentiated to all cell types found in the human body. The differentiation of hPSCs to human hepatocyte-like cells (HLCs) has been extensively studied, and efficient differentiation protocols have been established. The combination of extracellular matrix and biological stimuli, including growth factors, cytokines, and small molecules, have made it possible to generate HLCs that resemble primary human hepatocytes. However, the majority of procedures still employ undefined components, giving rise to batch-to-batch variation. This serves as a significant barrier to the application of the technology. To tackle this issue, we developed a defined system for hepatocyte differentiation using human recombinant laminins as extracellular matrices in combination with a serum-free differentiation process. Highly efficient hepatocyte specification was achieved, with demonstrated improvements in both HLC function and phenotype. Importantly, this system is easy to scale up using research and GMP-grade hPSC lines&#160;promising advances in cell-based modelling and therapies.

The method presented here describes a scalable and good manufacturing practice (GMP)-ready differentiation system to generate human hepatocyte-like cells from pluripotent stem cells. It serves as a cost-effective and standardized system to generate human hepatocyte-like cells for basic and applied human liver research.

Tanımlı ve hepatosit benzeri Hücrelerinin Ölçeklenebilir Nesil insan pluripotent kök hücreleri gelen

Human pluripotent stem cells (hPSCs) possess great value for biomedical research. hPSCs can be scaled and differentiated to all cell types found in the ...

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 ...

Chemical Reversion of Conventional Human Pluripotent Stem Cells to a Na&#239;ve-like State with Improved Multilineage Differentiation Potency

Na&#239;ve human pluripotent stem cells (N-hPSC) with improved functionality may have a wide impact in regenerative medicine. The goal of this protocol is to efficiently revert lineage-primed, conventional human pluripotent stem cells (hPSC) maintained on either feeder-free or feeder-dependent conditions to a na&#239;ve-like pluripotency with improved functionality. This chemical na&#239;ve reversion method employs the classical leukemia inhibitory factor (LIF), GSK3&#946;, and MEK/ERK inhibition cocktail (LIF-2i), supplemented with only a tankyrase inhibitor XAV939 (LIF-3i). LIF-3i reverts conventional hPSC to a stable pluripotent state adopting biochemical, transcriptional, and epigenetic features of the human pre-implantation epiblast. This LIF-3i method requires minimal cell culture manipulation and is highly reproducible in a broad repertoire of human embryonic stem cell (hESC) and transgene-free human induced pluripotent stem cell (hiPSC) lines. The LIF-3i method does not require a re-priming step prior to the differentiation; N-hPSC can be differentiated directly with extremely high efficiencies and maintain karyotypic and epigenomic stabilities (including at imprinted loci). To increase the universality of the method, conventional hPSC are first cultured in the LIF-3i cocktail supplemented with two additional small molecules that potentiate protein kinase A (forskolin) and sonic hedgehog (sHH) (purmorphamine) signaling (LIF-5i). This brief LIF-5i adaptation step significantly enhances the initial clonal expansion of conventional hPSC and permits them to be subsequently na&#239;ve-reverted with LIF-3i alone in bulk quantities, thus obviating the need for picking/subcloning rare N-hPSC colonies later. LIF-5i-stabilized hPSCs are subsequently maintained in LIF-3i alone without the need of anti-apoptotic molecules. Most importantly, LIF-3i reversion markedly improves the functional pluripotency of a broad repertoire of conventional hPSC by decreasing their lineage-primed gene expression and erasing the interline variability of directed differentiation commonly observed amongst independent hPSC lines. Representative characterizations of LIF-3i-reverted N-hPSC are provided, and experimental strategies for functional comparisons of isogenic hPSC in lineage-primed vs. na&#239;ve-like states are outlined.

Chemical Reversion of Conventional Human Pluripotent Stem Cells to a Na&amp;#239;ve-like State with Improved Multilineage Differentiation Potency

We present a protocol for efficient, bulk, and rapid chemical reversion of conventional lineage-primed human pluripotent stem cells (hPSC) into an epigenomically-stable na&#239;ve preimplantation epiblast-like pluripotent state. This method results in decreased lineage-primed gene expression and marked improvement in directed multilineage differentiation across a broad repertoire of conventional hPSC lines.

Geleneksel insan Pluripotent kök hücreler kimyasal Reversion için geliştirilmiş Multilineage farklılaşma potansiyeli ile saf gibi devlet

Na&#239;ve human pluripotent stem cells (N-hPSC) with improved functionality may have a wide impact in regenerative medicine. The goal of this protocol is ...

RNA-based Reprogramming of Human Primary Fibroblasts into Induced Pluripotent Stem Cells

Induced pluripotent stem cells (iPSCs) have proven to be a valuable tool to study human development and disease. Further advancing iPSCs as a regenerative therapeutic requires a safe, robust, and expedient reprogramming protocol. Here, we present a clinically relevant, step-by-step protocol for the extremely high-efficiency reprogramming of human dermal fibroblasts into iPSCs using a non-integrating approach. The core of the protocol consists of expressing pluripotency factors (SOX2, KLF4, cMYC, LIN28A, NANOG, OCT4-MyoD fusion) from in vitro transcribed messenger RNAs synthesized with modified nucleotides (modified mRNAs). The reprogramming modified mRNAs are transfected into primary fibroblasts every 48 h together with mature embryonic stem cell-specific microRNA-367/302 mimics for two weeks. The resulting iPSC colonies can then be isolated and directly expanded in feeder-free conditions. To maximize efficiency and consistency of our reprogramming protocol across fibroblast samples, we have optimized various parameters including the RNA transfection regimen, timing of transfections, culture conditions, and seeding densities. Importantly, our method generates high-quality iPSCs from most fibroblast sources, including difficult-to-reprogram diseased, aged, and/or senescent samples.

Here we describe a clinically relevant, high-efficiency, feeder-free method to reprogram human primary fibroblasts into induced pluripotent stem cells using modified mRNAs encoding reprogramming factors and mature microRNA-367/302 mimics. Also included are methods to assess reprogramming efficiency, expand clonal iPSC colonies, and confirm expression of the pluripotency marker TRA-1-60.

RNA tabanlı insan birincil fibroblastlar indüklenmiş Pluripotent kök hücre yeniden programlama

Induced pluripotent stem cells (iPSCs) have proven to be a valuable tool to study human development and disease. Further advancing iPSCs as a regenerative ...

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.