Name: human pluripotent stem cell based developmental toxicity assays for chemical safety screening and systems biology data generation
Uploaded: 2015-06-17T13:00:00
Description: 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

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).

Følgende protokol blev udført ved hjælp af humane embryonale stamceller line (embryonale) H9. Denne cellelinie var rutinemæssigt dyrket på mitotisk inaktiverede muse embryonale fibroblaster (MEF&#39;er) i hESC kulturmedie suppleret med bFGF og derefter dyrket i stamceller medier på 6 cm Petri-plader overtrukket med basalmembran matrix, såsom matrigel, for at slippe af MEF. H9 celler fra&gt; 80% sammenflydende plader blev anvendt til yderligere passage. H9-celler dyrket på basalmembran...

<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>

Traditionelle tilgange til toksikologisk testning involverer omfattende dyreforsøg dermed gøre test dyrt og tidskrævende. Som følge af de interspecies forskelle de prækliniske sikkerhedsundersøgelser undersøgelser er ikke altid gyldige til at forudsige toksikologiske effekter potentielle lægemidler er relevante for mennesker. Selv ikke-menneskelige primater er mest forudsigelige, stadig stærk etisk og socioeconomical krav hurtigt hæve ved moderne samfund for at udvikle følsomme og robust in vitro tes...

Evnen af humane embryonale stamceller (hESC) til at differentiere til forskellige typer af celler åbnet en ny æra af test in vitro toksicitet 1, sygdom modellering og regenerativ medicin 2. Stamcellerne er udstyret med evnen til at kopiere sig selv, for at holde deres pluripotente tilstand og til at differentiere til specialiserede celler 3,4. Egenskaberne af embryonale (kapacitet til at differentiere til alle større celletyper) findes også i andre humane pluripotente stamceller, såsom humane inducerede pluripotente stamceller (hiPSC) eller celler frembragt ved kernetransplantation 5. For eksempel har mange forskellige hESC linjer blevet differentieret til neuroner 6, nyreceller 7, neurale kamceller 8, cardiomyocytter 9-12 eller hepatocytter lignende celler 13,14. Desuden kan embryonale spontant differentiere til celler af alle tre kim lag 15-18 i embryoide legemer (EBS) 19,20. EArly fosterudvikling er reguleret af differentiel ekspression af forskellige gener relateret til de forskellige kim lag, der er blevet fanget på mRNA niveau ved transcriptomics hjælp microarray-teknologi 15. Disse bestræbelser har resulteret i etableringen af orgel særlige toksikologiske modeller baseret på embryonale / hiPSC og transcriptomics analyse (til gennemgang se 21,22). Disse modeller har fordele i forhold til traditionelle brug af forsøgsdyr til toksikologiske undersøgelser, som prækliniske studier anvender forsøgsdyr ikke altid kan forudsige menneskelig sikkerhed. Narkotika inducerede toksiciteter forekommer i patienter er ofte relateret til metaboliske eller signaleringsprocesser, der adskiller sig mellem mennesker og forsøgsdyr. Arten Forskellen har forhindret pålidelig tidlig påvisning af afkommet hos mennesker, og for eksempel lægemidler, såsom thalidomid 23,24 og diethylstilbestrol 25,26 blev trukket tilbage fra markedet på grund af teratogenicitet. ThaliDomide har ikke vist nogen udviklingstoksicitet hos rotter eller mus. Miljømæssige kemikalier såsom methylkviksølv 27 resulterede i prænatal udviklingstoksicitet i forhold til nervesystemet i forskellige arter, men menneskelige manifestationer have været svært at modellere i dyr. For at løse problemet med artsspecificitet spørgsmål, forskere, der arbejder under forskellige projekter baseret på stamceller som genbeskyt, ESNATS, kriminalassistent mv er engageret i udvikling af forskellige modeller for embryonale toksicitet, neurotoksicitet, kardiotoksicitet, hepatotoksicitet og nefrotoksicitet ved hjælp af humane giftstoffer mistænkes for at påvirker mennesker. Under det europæiske konsortium projektet &quot;embryonale stamceller-baserede Novel Alternative Testing Strategies (ESNATS)&quot; er blevet etableret fem testsystemer. Et testsystem den såkaldte UKK (U niversitäts k linikum K OLN) testsystem delvist fanger tidlig menneskelig fosterudvikling. I denne system humane embryonale H9 celler differentieres i tre kim lag (ectoderm, endoderm og mesoderm) 15 og kim lag specifikke underskrifter er blevet taget til fange af transcriptomics profil bruge Affymetrix microarray platform. Forskellige udviklingsmæssige giftstoffer som thalidomid 28, valproat, methyl kviksølv 16,17 eller cytosinarabinosid 15 er blevet testet i dette system, og der er opnået giftstof specifikt gen signaturer. I et andet testsystem, den såkaldte den UKN1 (U niversity K onsta n z) testsystem 1 er H9 celler differentieres til neuroectodermal stamceller (NEP) i 6 dage. Dette fremgår af høj ekspression af neurale genmarkører, såsom Pax6 og OTX2. Under differentiering i 6 dage, har NEP-celler været udsat for udviklingsmæssige neuro-giftstoffer, såsom VPA, methylkviksølv. Giftstof-specifikke deregulerede transcriptomics profiler har været obtained samt ved hjælp af Affymetrix microarray platform 16,29. Den nye vision for toksikologi det 21. århundrede, forventes testsystemer ikke kun giver fænotypiske beskrivelser ligesom histopatologi in vivo, eller transkriptom ændringer i slutningen af langsigtede toksiske inkubationer. Det foreslår snarere, at analyser giver mekanistisk information 3, og at disse oplysninger kan kortlægges såkaldte negative udfald veje (AOP), der giver en videnskabelig begrundelse for farlige virkninger 30. At give sådanne oplysninger, testsystemet anvendte nødt til at være meget kvalitet kontrolleres 31, som for eksempel dokumenteret af robuste standard drift procedurer. Desuden skal kortlægges med høj opløsning tidsafhængig ændringer. Dette kræver testsystemer med synkroniserede ændringer 32. De UKN1 og UKK testsystemer beskrevet her er blevet optimeret til disse krav.

<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

Effektive protokoller til at differentiere humane pluripotente stamceller til forskellige væv i kombination med genomenbaserede teknologier åbnet nye horisonter til test in vitro toksicitet potentielle lægemidler. At skabe et solidt videnskabeligt grundlag for sådanne analyser, vil det være vigtigt at få kvantitative oplysninger om tidsforløbet for udvikling og om de underliggende reguleringsmekanismer ved systembiologiske metoder. To analyser er derfor blevet tunet her for disse krav. I UKK testsystem, er humane embryonale stamceller (hESC) (eller andre pluripotente celler) overladt til spontant at differentiere i 14 dage i embryoide organer til at tillade generering af celler fra alle tre kim lag. Dette system rekapitulerer vigtige trin af tidlig human embryonisk udvikling, og det kan forudsige humanspecifikke tidlig embryonal / teratogenicitet, hvis cellerne udsættes for kemikalier under differentiering. Den UKN1 testsystem er baseret på embryonale differentiere til en population af neuroektodermal progenitor (NEP) celler i 6 dage. Dette system rekapitulerer tidlig neural udvikling og forudser tidlig udviklingsneurotoksicitet og epigenetiske ændringer udløst af kemikalier. Begge systemer, i kombination med transkriptom microarray studier, er egnede til at identificere toksicitet biomarkører. Endvidere kan de anvendes i kombination til at generere input data for systemer biologi analyse. Disse testsystemer har fordele i forhold til de traditionelle toksikologiske undersøgelser kræver store mængder af dyr. De testsystemer kan bidrage til en reduktion af omkostningerne til lægemiddeludvikling og evaluering kemikaliesikkerhedsvurdering. Deres kombination belyser især på forbindelser, der kan påvirke nervesystemets udvikling specifikt.

Methylkviksølv eksponering i UKK testsystem Cytotoksicitetsassayet blev udført med H9 EB&#39;er at opnå en IC 10-værdi (reduktionen af levedygtighed med 10%) for cytotoksiciteten af methyl-kviksølv (figur 1). Vi udførte også en mikroarray baseret (Affymetrix platform) biomarkør undersøgelse. H9 EB&#39;er har været udsat for methyl-kviksølv (0,25 og 1 uM) i 14 dage. På dag 14, er blevet indsamlet prøver ved anvendelse af TRIzol og RNA blev isoleret. Tr...

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

Protokollerne beskriver to in vitro udviklingsmæssige toksicitet testsystemer (UKK og UKN1) baseret på humane embryonale stamceller og transkriptom studier. Testsystemet forudser human udviklingsmæssige fare toksicitet, og kan bidrage til at reducere dyreforsøg, omkostninger og den tid, der kræves til kemisk sikkerhed test.

Menneskelig Pluripotente Stem Cell Based Udviklingstoksicitet Analyser for Kemisk Sikkerhed Screening og Systembiologi data Generation

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

Research

JoVE Journal

Developmental Biology

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

Dyrkning Menneskelig pluripotente og neurale stamceller i en Lukket Cell Culture System for Basic og Prækliniske Research

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

Feeder-fri Udledning af melanocytter fra menneskelige pluripotente stamceller

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

Storstilet produktion af cardiomyocytter fra menneskelige pluripotente stamceller Ved hjælp af en meget reproducerbar lille molekyle-Based Differentiering Protocol

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

Generation of Induced Pluripotent Stem Cells from Human Melanoma Tumor-infiltrating Lymphocytes

Generation af induceret pluripotente stamceller fra human melanoma Tumor-infiltrerende lymfocytter

Adoptive transfer of ex vivo expanded autologous tumor-infiltrating lymphocytes (TILs) can mediate durable and complete responses in significant subsets ...

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

Generation af Integration-fri induceret pluripotente stamceller fra humane perifere mononucleære blodceller Brug episomale vektorer

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

Defineret og skalerbar Generation af hepatocyt-lignende celler fra menneskelige pluripotente stamceller

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

Hurtig, instrueret differentiering af retinale Pigment epitel celler fra menneskelige embryonale eller induceret pluripotente stamceller

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

Chemical Reversion of Conventional Human Pluripotent Stem Cells to a NaÃƒÆ’Ã†'Ãƒâ€šÃ‚Â¯ve-like State with Improved Multilineage Differentiation Potency

Kemiske Reversion af konventionelle humane pluripotente stamceller til en naiv-lignende tilstand med forbedret Multilineage differentiering potens

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

RNA-baserede omprogrammering af menneskelige primære fibroblaster til induceret pluripotente stamceller

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