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 protokoll ble utført ved hjelp av menneskelig Embryonic Stem Cell linje (hESC) H9. Denne cellelinje ble rutinemessig dyrket på mitotisk inaktiverte mus embryonale fibroblaster (MEFs) i hESC kulturmedium supplementert med bFGF og deretter dyrket i stamcelle media på 6 cm petriplater belagt med basalmembran matriks, slik som matrigel, for å bli kvitt MEFs. H9-celler fra&gt; 80% konfluente plater ble anvendt for ytterligere passasje. H9-celler dyrket i basalmembranmatrise plater bl...

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Tradisjonelle tilnærminger til toksikologisk testing bære omfattende dyreforsøk dermed gjøre testing kostbart og tidkrevende. Dessuten, på grunn av den artsforskjellene de prekliniske dyresikkerhetsstudier er ikke alltid er gyldig for å forutsi giftvirkninger av potensielle legemidler som er relevant for mennesker. Selv om ikke-menneskelige primater er mest forutsigbare, fortsatt sterk etisk og socioeconomical krav raskt øke ved moderne samfunn for å utvikle følsom og robust in vitro test Sy...

Muligheten av humane embryonale stamceller (hESC) for å skille ut ulike typer celler åpnet opp en ny æra av in vitro toksisitet testing 1, sykdom modellering og regenerativ medisin 2. Stamcellene er utstyrt med kapasitet til å kopiere seg selv, for å holde deres pluripotent tilstand, og for å skille ut spesialiserte celler 3,4. Egenskapene til hESC (evne til å differensiere til alle viktige celletyper) er også funnet i andre humane pluripotente stamceller, som menneskeskapte pluripotente stamceller (hiPSC) eller celler som genereres av kjerneoverføring 5. For eksempel har mange forskjellige hESC linjer blitt differensiert i nevroner 6, nyreceller 7, neural crest cellene 8 kardiomyocytter 9-12, eller hepatocytter som celler 13,14. Videre kan hESC spontant differensiere i celler av alle tre bakterie lag 15-18 i embryoid organer (EBS) 19,20. Early embryoutvikling er regulert av differensial uttrykk for ulike gener knyttet til de ulike bakterie lag som har blitt fanget på mRNA nivå ved transcriptomics bruker mikromatriseteknologi 15. Dette arbeidet resulterte i etableringen av organspesifikke toksikologiske modeller basert på hESC / hiPSC og transcriptomics analyse (for oversikt se 21,22). Disse modellene har fordeler fremfor den tradisjonelle bruken av forsøksdyr i toksikologiske studier, som prekliniske studier med forsøksdyr er ikke alltid logisk for menneskelig sikkerhet. Stoffet indusert toksisitet oppstått hos pasienter er ofte relatert til metabolske eller signalprosesser som skiller mellom mennesker og forsøksdyr. Arten Forskjellen har forhindret pålitelig deteksjon av tidlig utviklingstoksisitet hos mennesker, og for eksempel medikamenter som thalidomid 23,24 og 25,26 dietylstilbestrol ble trukket tilbake fra markedet på grunn av teratogenisitet. Thalidomide har ikke vist noen utviklingstoksisitet i rotter eller mus. Miljø kjemikalier som metylkvikksølv 27 resulterte i prenatal utviklingstoksisitet når det gjelder nervesystemet i en rekke arter, men humane manifestasjoner har vært vanskelig å modellere i dyr. For å løse problemet med artsspesifisitet problemer, forskere som arbeider under ulike prosjekter basert på stamceller som ReProTect, ESNATS, DETECTIVE etc. er engasjert i utvikling av ulike modeller for embryotoksisitet, nevrotoksisitet, kardiotoksisitet, levertoksisitet og nefrotoksisitet ved hjelp av menneskegiftstoffer som mistenkes å påvirke mennesker. Under det europeiske konsortiet prosjektet &quot;Embryonic Stem cellebasert Novel Alternative Testing Strategies (ESNATS)« Det er etablert fem testsystemer. En test system såkalt UKK (U niversitäts k linikum K OLN) test system delvis fanger tidlig menneskelig embryoutvikling. I denne system humane embryonale H9 celler er differensiert i tre bakterie lag (ektoderm, endoderm og mesoderm) 15 og bakterie lag spesifikke signaturer har blitt fanget av transcriptomics profil med Affymetrix microarray plattform. Ulike utviklingsgiftstoffer som thalidomid 28, valproinsyre, metylkvikksølv 16,17, eller cytosinarabinosid 15 har blitt testet i dette systemet, og toxicant bestemt gen signaturer er oppnådd. I en andre test-system, det såkalte UKN1 (U niversity av K onsta n z) testsystem 1, er H9-celler differensieres til neuroectodermal progenitorceller (NEP) i 6 dager. Dette er dokumentert av høy ekspresjon av nevrale genmarkører som Pax6 og OTX2. Under differensiering i 6 dager, har NEP celler vært utsatt for utviklings nevro-miljøgifter som VPA, metylkvikksølv. Giftstoff-spesifikke de-regulert transcriptomics profiler har vært obtainert som godt ved hjelp av Affymetrix microarray plattform 16,29. Den nye visjonen for toksikologi av det 21. århundre ser for seg at testsystemer ikke bare gi fenotypiske beskrivelser som histopatologi in vivo, eller transkriptom endringer på slutten av langsiktige toxicant inkuberingene. Det foreslår heller at analysene gir mekanistisk informasjon 3, og at denne informasjonen kan tilordnes til såkalte uønskede utfall trasé (AOP) som gir en vitenskapelig begrunnelse for skadelige effekter 30. Å gi slik informasjon, testsystemer anvendes må være høyt kvalitetskontrollert 31, som for eksempel dokumentert av robuste standarddriftsprosedyrer. Videre tidsavhengige endringer må kartlegges med høy oppløsning. Dette krever testsystemer med synkroniserte endringer 32. De UKN1 og UKK testsystemer som er beskrevet her har blitt optimalisert for disse kravene.

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		<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>
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		<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>
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		<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 for å skille menneskelige pluripotente stamceller til ulike vev i kombinasjon med -omics teknologier åpnet nye horisonter for in vitro toksisitet testing av potensielle narkotika. Å gi et solid vitenskapelig grunnlag for slike analyser, vil det være viktig å få kvantitativ informasjon om tidsforløpet av utvikling og på de underliggende reguleringsmekanismer av systembiologi tilnærminger. To analyser er derfor innstilt her for disse kravene. I UKK testsystemet, er humane embryonale stamceller (hESC) (eller andre pluripotente celler) igjen å spontant skille i 14 dager i embryoide legemer, for å tillate generering av celler av alle tre bakterie lag. Dette systemet sammenfatter viktige trinn i tidlig human embryoutvikling, og det kan forutsi human-spesifikke tidlig embryotoksisitet / teratogenisitet, hvis cellene er utsatt for kjemikalier under differensiering. Den UKN1 testsystemet er basert på hESC differensiere til en population av neuroectodermal stamfar (NEP) celler for 6 dager. Dette systemet sammenfatter tidlig neural utvikling og spår tidlig utviklings neurotoxicity og epigenetiske endringer utløst av kjemikalier. Begge systemene, i kombinasjon med transcriptome microarray undersøkelser, er egnet til å identifisere toksisitet biomarkører. Dessuten kan de anvendes i kombinasjon for å generere inngangsdata for systemer biologi analyse. Disse testsystemer har fordeler i forhold til tradisjonelle toksikologiske studier som krever store mengder av dyr. Test systemer kan bidra til en reduksjon av kostnadene for legemiddelutvikling og kjemisk sikkerhetsvurdering. Deres kombinasjon belyser spesielt på forbindelser som kan påvirke neurodevelopment spesifikt.

Metylkvikksølv eksponering i UKK testsystem Cytotoksisiteten Analysen ble utført med H9 EBS for å oppnå en IC-10-verdi (reduksjon av levedyktighet med 10%) for cytotoksisiteten av metyl-kvikksølv (figur 1). Vi har også utført en microarray basert (Affymetrix plattform) biomarkør studien. H9 EBS har vært utsatt for metylkvikksølv (0,25 og 1 mm) i 14 dager. På dag 14, har prøvene er samlet ved hjelp av TRIzol og RNA ble isolert. Transkripsjonen profileri...

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

Protokollene beskriver to in vitro utviklingstoksisitetstestsystemer (UKK og UKN1) basert på humane embryonale stamceller og transkriptom studier. De testsystemer forutsi menneskelig utviklingstoksisitet farer, og kan bidra til å redusere dyrestudier, kostnader og den tiden som kreves for kjemisk sikkerhetstesting.

Menneskelige pluripotent stamcelle Basert Utviklingstoksisitet Analyser for kjemikaliesikkerhet Screening og Systems Biology datagenerering

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

Dyrking Menneskelig Pluripotent og Neural stamceller i et lukket cellekultur System for Basic og Preklinisk Forskning

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 Utledning av Melanocytter fra Menneskelig Pluripotent 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

Storskala produksjon av cardiomyocytes fra Menneskelig Pluripotent stamceller ved hjelp av en meget reproduserbar lite molekyl-Based Differensiering 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

Generering av Induced Pluripotent stamceller fra humane melanom 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

Generasjon Integrerings--frie Utførte Pluripotent stamceller fra menneskelige perifere mononukleære blodceller Bruke 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

Definert og Scalable Generation of hepatocytter lignende celler fra menneskelige Pluripotent 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

Rask, rettet differensiering av netthinnens Pigment epitelceller fra menneskelige embryonale eller indusert Pluripotent 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

Kjemisk hjemfall til konvensjonelle menneskelige Pluripotent stamceller til en naiv-lignende tilstand med forbedret Multilineage differensiering styrke

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-baserte omprogrammering av menneskelig primære fibroblaster til indusert Pluripotent 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 ...