Name: protocol for the differentiation of human induced pluripotent stem cells into mixed cultures of neurons and glia for neurotoxicity testing
Uploaded: 2017-06-09T14:00:00
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Description: Watch this Scientific Journal Video about Protocol for the Differentiation of Human Induced Pluripotent Stem Cells into Mixed Cultures of Neurons and Glia for Neurotoxicity Testing at JoVE.com

Forfatterne vil gerne takke Dr. Marc Peschanski (I-Stem, Évry, Frankrig) for at give IMR90-hiPSC&#39;erne; Dr. Giovanna Lazzari og Dr. Silvia Colleoni (Avantea srl, Cremona, Italien); Dr. Simone Haupt (Universitetet i Bonn, Tyskland); Dr. Tiziana Santini (Italiensk Institut for Teknologi, Rom) for at give råd om immunfluorescensfarvningsevaluering; Dr. Benedetta Accordi, Dr. Elena Rampazzo og Dr. Luca Persano (Universitetet i Padua, Italien) for deres bidrag til RPPA-analysen og antistoffvalidering. Finansiering: Dette arbejde blev støttet af det EU-finansierede projekt &quot;SCR &amp; Tox&quot; (tilskudsaftale nr. 266753).

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	<li>NRC. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Toxicity Testing in the 21st Century: A Vision and a Strategy. , (2007).</li><li>Drews, K., Jozefczuk, J., Prigione, A., Adjaye, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+induced+pluripotent+stem+cells--from+mechanisms+to+clinical+applications.">Human induced pluripotent stem cells--from mechanisms to clinical applications.</a> J Mol Med (Berl). 90 (7), 735-745 (2012).</li><li>Ho, P. J., Yen, M. L., Yet, S. F., Yen, B. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Current+applications+of+human+pluripotent+stem+cells:+possibilities+and+challenges.">Current applications of human pluripotent stem cells: possibilities and challenges.</a> Cell Transplant. 21 (5), 801-814 (2012).</li><li>Krueger, W. H., Swanson, L. C., Tanasijevic, B., Rasmussen, T. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Natural+and+artificial+routes+to+pluripotency.">Natural and artificial routes to pluripotency.</a> Int J Dev Biol. 54 (11-12), 1545-1564 (2010).</li><li>Pistollato, F., Bremer-Hoffmann, S., Healy, L., Young, L., Stacey, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Standardization+of+pluripotent+stem+cell+cultures+for+toxicity+testing.">Standardization of pluripotent stem cell cultures for toxicity testing.</a> Expert Opin Drug Metab Toxicol. 8 (2), 239-257 (2012).</li><li>Pistollato, F., 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=Development+of+a+pluripotent+stem+cell+derived+neuronal+model+to+identify+chemically+induced+pathway+perturbations+in+relation+to+neurotoxicity:+effects+of+CREB+pathway+inhibition.">Development of a pluripotent stem cell derived neuronal model to identify chemically induced pathway perturbations in relation to neurotoxicity: effects of CREB pathway inhibition.</a> Toxicol Appl Pharmacol. 280 (2), 378-388 (2014).</li><li>Zagoura, D., Canovas-Jorda, D., Pistollato, F., Bremer-Hoffmann, S., Bal-Price, A. <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+the+rotenone-induced+activation+of+the+Nrf2+pathway+in+a+neuronal+model+derived+from+human+induced+pluripotent+stem+cells.">Evaluation of the rotenone-induced activation of the Nrf2 pathway in a neuronal model derived from human induced pluripotent stem cells.</a> Neurochem Int. , (2016).</li><li>Standard operating procedure for differentiation of human induced pluripotent stem cells into post-mitotic neurons and glial cells. EURL ECVAM Available from: <a target="_blank" href="https://ecvam-dbalm.jrc.ec.europa.eu/methods-and-protocols/protocol/standard-operating-procedure-for-differentiation-of-human-induced-pluripotent-stem-cells-into-post-mitotic-neurons-and-glial-cells-%28mixed-culture%29-protocol-no.-165/key/p_1570">https://ecvam-dbalm.jrc.ec.europa.eu/methods-and-protocols/protocol/standard-operating-procedure-for-differentiation-of-human-induced-pluripotent-stem-cells-into-post-mitotic-neurons-and-glial-cells-%28mixed-culture%29-protocol-no.-165/key/p_1570</a> (2016)</li><li>Standard operating procedure for expansion of rosette-derived neural stem cells. 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Forfatterne har intet at afsløre.

Dette værk beskriver en robust og forholdsvis hurtig protokol til differentiering af IMR90-hiPSC&#39;er i postmittotiske neuroner og glialceller. Tidligere offentliggjorte neuronal differentieringsprotokoller baseret på hESC&#39;er og hiPSC&#39;er giver sædvanligvis høje procentdele af neurale forstadier 25 , 26 og et signifikant antal neuronale målceller 27 , 28 , 29 , 30 , 31 , 32 , 33 . Analogt er den her beskrevne differentieringsprotokol egnet til at danne heterogene kulturer af GABAergic, glutamatergiske og dopaminerge neuronale celler sammen med glia og en diskret andel af nestin + -celler. Tilstedeværelsen af ​​glutamatergiske (~ 35-42%) og GABAergic (~ 15-20%) neurale celler antyder atDenne kultur besidder forebrain, cortical-lignende træk og tilstedeværelsen af ​​et diskret antal dopaminerge neuroner (~ 13-20%) kan også indikere midbrainspecificitet. Endvidere kan permanensen af ​​en beskeden andel af nestin + celler vise sig egnet til undersøgelsen af ​​neurogenese og de mulige virkninger af kemikalier på NSC&#39;er, der primært er begrænset til både hippocampus og subventriculær zone (SVZ) hos forræderen 34 . Yderligere immunocytokemiske og genekspression analyser ville bidrage til bedre at definere de regionale specificiteter af de differentierede cellederivater. De to mest kritiske trin i differentieringsprotokollen beskrevet i dette dokument er: (i) opskæring af hiPSC-kolonier i homogene fragmenter (hvilket er kritisk for dannelsen af ​​EB&#39;er med homogene størrelser) og (ii) opskæring af neuroektodermale strukturer (rosetter ) Til NSC differentiering, hvilket kræver betydelig manuel færdighedOg præcision for at undgå at indsamle mesodermale og endodermale celler, som kan reducere proportionerne af neuroner og glialceller opnået ved differentiering. Det er afgørende at karakterisere fænotyperne af cellerne under ekspansion (som udifferentierede kolonier eller NSC&#39;er) og under alle differentieringsstrin. Navnlig bør gen- og proteinekspressionsprofilerne af neuronale / glialcellederivaterne vise opregulering og aktivering af neuronrelaterede signalveje, medens ekspressionen af ​​pluripotensmarkører skal nedsættes. Genereringen af ​​EB&#39;er og neuroektodermale derivater (rosettes) kan være manuelt udfordrende og udsat for variabilitet. Af denne grund udviklede vi en protokol til udvidelse af rosetteafledte NSC&#39;er og deres yderligere differentiering i neuronale / glialceller. Mulige begrænsninger af denne differentieringsprotokol er hovedsageligt (i) den relativt lave procentdel af dIfferentiated glialderivater og (ii) manglen på modne neuronale netværksfunktioner (som vist ved manglende udbrud). Desuden kan specifikke subpopulationer af astrocytter fungere som primære progenitorer eller NSC&#39;er 35 . Mens nestin / GFAP dobbelt-positive celler ikke blev observeret i denne differentierede cellekultur (data ikke vist), er det hypotetiseret, at GFAP + -cellerne i disse blandede kulturer er astrocytiske stamceller og astrocytter. Det er plausibelt, at antallet af astrocytter kan øges, og at deres morfologi bliver mere moden, som allerede angivet ved tidligere værker fra Zhangs gruppe 36 , 37 ved at forlænge differentieringstiden. I det nye testparametre for toksicitet er viden om kemisk inducerede forstyrrelser af biologiske veje yderst vigtig, når man vurderer kemisk modgang. Derfor bør in vitro testsystemer kunneRelaterer bivirkninger til forstyrrelser af signalveje, i henhold til begrebet negative bane (AOP). Som et bevis på konceptet kan rotenon bruges til at vurdere aktiveringen af ​​Nrf2-banen, som er involveret i det cellulære forsvar mod oxidativ eller elektrofil stress 38 , og oxidativ stress er en vigtig og fælles nøglebegivenhed i forskellige AOP&#39;er, der er relevante for Udviklingsmæssige og voksne neurotoksicitet 39 . Rotenon bør fremkalde aktiveringen af ​​Nrf2-vejen, hvilket kan påvises ved Nrf2-proteinkernetransplacering og øget ekspression af Nrf2-mål-enzymer, herunder NQO1 og SRXN1. Det har vist sig, at rotenon inducerer en dosisafhængig forøgelse af GFAP-proteinniveauer, hvilket indikerer astrocytaktivering 40 , 41 . Rotenon reducerer også antallet af dopaminerge (TH + ) celler, som er i overensstemmelse med previIn vitro og in vivo undersøgelser, der viser rotenonafhængig dopaminerge celledød, da denne type neuron er særligt følsom over for oxidativ stress 21 , 22 , 23 . Som konklusion er denne hiPSC-afledte neuronale og glialcellekulturmodel et værdifuldt værktøj til at vurdere de neurotoksiske virkninger af kemikalier, som fremkalder oxidativt stress, der resulterer i Nrf2-pathwayaktivering. Da denne differentieringsprotokol tillader generering af blandede kulturer af neuronale celler (GABAergic, dopaminergic og glutamatergic neurons) og astrocytter, kan det vise sig egnet til at studere krydset mellem neuroner og glia i fysiologiske og patologiske tilstande, såsom i neurodegenerative sygdomme ( Fx Parkinsons sygdom). Endvidere kan tilstedeværelsen af ​​en betydelig andel af NSC&#39;er bidrage til at vurdere de mulige virkninger af kemikalier på neurale progEnitors, som er kendt for at være det primære mål for kemisk inducerede mutationer eller virusinfektioner 42 .

US National Research Council rapport 1 forestillede sig et nyt toksicitetsprøvningsparadigm, hvor reguleringstoksicitetsprøvning ville blive forskudt fra en tilgang baseret på fænotypiske ændringer observeret hos dyr til en tilgang, der fokuserede på mekanistiske in vitro- analyser ved anvendelse af humane celler. Pluripotente stamceller (PSC) derivater kan repræsentere alternativer til cancercellemodeller, da de opnåede celler kan ligner de fysiologiske betingelser af humane væv mere og tilvejebringe mere relevante værktøjer til at studere kemisk inducerede bivirkninger. De to hovedtyper af PSC-kulturer, der er mest lovende for toksicitetstestning, er humane embryonale stamceller (hESC&#39;er) og humane inducerede pluripotente stamceller (hiPSC&#39;er), som i øjeblikket er meget udbredt inden for grundforskning og regenerativ medicin 2 , 3 . Denne ekspertise kan nu udnyttes til udvikling af en ny klasse toxicoloGiske in vitro tests med det formål at identificere de forstyrrede fysiologiske veje involveret i udviklingen af ​​bivirkninger in vivo . Testmetoder for lovgivningsmæssige sikkerhedsvurderinger baseret på hESC&#39;er vil sandsynligvis ikke blive accepteret af alle EU-medlemsstater og lande over hele verden på grund af mulige etiske bekymringer og forskellige nationale lovgivningspolitikker, der regulerer brugen af ​​embryonafledte celler. HiPSC&#39;er deler egenskaber svarende til hESCs 4 , 5 og har stort potentiale for in vitro- metoder, både til identifikation af terapeutiske mål samt for sikkerhedsvurderinger. Derudover mindsker hiPSC-teknologien begrænsningerne i en begrænset donorpulje og de etiske problemer forbundet med embryoafledte celler. En stor udfordring for hiPSC&#39;er er demonstrationen om, at disse celler reproducerbart kan danne et signifikant udvalg af toksikologisk relevante cellederivater,Med karakteristika og reaktioner, der er typiske for humane væv. Predefinerede niveauer af de markerede markører anvendes generelt til at karakterisere cellepopulationer efter differentieringsprocessen og give indsigt i stabiliteten af ​​differentieringsprocessen. Tidligere værker vurderede HiPSC&#39;ernes egnethed til at generere blandede kulturer af neuronale og glialceller og for at vurdere virkningerne af rotenon, en inhibitor af mitokondrielle respiratoriske kompleks I, ved aktiveringen af ​​Nrf2-vejen, en nøgleregulator af antioxidantforsvarsmekanismer i Mange celletyper 6 , 7 . Dette arbejde beskriver en protokol, der anvendes til differentiering af hiPSC&#39;er i blandede neuronale og gliale kulturer, idet der gives detaljer om signalveje (gen- og proteinniveau), som aktiveres ved neuronal / glialdifferentiering. Derudover viser arbejdet repræsentative resultater, der viser, hvordan detteHiPSC-afledt neuronal og glialcellemodel kan anvendes til vurdering af Nrf2-signalaktivering induceret ved akut (24-h) behandling med rotenon, hvilket muliggør vurdering af oxidativ stressinduktion. IMR90 fibroblaster blev omprogrammeret til hiPSC&#39;er ved I-Stem (Frankrig) ved viral transduktion af 2 transkriptionsfaktorer (Oct4 og Sox2) ved anvendelse af pMIG-vektorer 6 . Analoge hiPSC-modeller kan også anvendes. De nedenfor beskrevne protokoller opsummerer alle stadier af differentiering af hiPSC&#39;er til neurale stamceller (NSC&#39;er) og yderligere i blandede kulturer af postmittotiske neuroner og gliaceller (trin 1 og 2, se også EURL ECVAM DBALM-webstedet for en detaljeret beskrivelse af Protokollen) 8 . En yderligere protokol til isolering, ekspansion, kryopreservering og yderligere differentiering af NSC&#39;er i blandede neuroner og glialceller er beskrevet i trin 3 og 4 (se også EURL ECVAM DBALM viBsite for en detaljeret beskrivelse af denne protokol) 9 . Trin 5 beskriver de analyser, der kan gøres for at vurdere cellernes fænotypiske identitet under de flere faser af engagement og differentiering.

protocol for the differentiation of human induced pluripotent stem cells into mixed cultures of neurons and glia for neurotoxicity testing

Humane pluripotente stamceller kan differentiere i forskellige celletyper, som kan anvendes til humane-baserede in vitro toksicitetsanalyser. En stor fordel er, at omprogrammeringen af ​​somatiske celler til fremstilling af humane inducerede pluripotente stamceller (hiPSC&#39;er) undgår de etiske og lovgivningsmæssige problemer i forbindelse med brugen af ​​humane embryonale stamceller (hESC&#39;er). HiPSC&#39;er kan udvides og effektivt differentieres til forskellige typer neuronale og glialceller, der tjener som testsystemer til toksicitetstestning og især til vurdering af forskellige veje involveret i neurotoksicitet. Dette værk beskriver en protokol til differentiering af hiPSC&#39;er i blandede kulturer af neuronale og glialceller. Signalveje, der reguleres og / eller aktiveres ved neuronal differentiering, defineres. Disse oplysninger er kritiske for anvendelsen af ​​cellemodellen på det nye testparametre for toksicitet, hvor kemikalier vurderes ud fra deres evne til at peRturb biologiske veje. Som et bevis på konceptet blev rotenon, en inhibitor af mitokondriell respiratorisk kompleks I, anvendt til at vurdere aktiveringen af ​​Nrf2-signalvejen, en nøgleregulator af antioxidant-respons-elementet (ARE) -driven cellulær forsvarsmekanisme mod oxidativ stress .

Watch this Scientific Journal Video about Protocol for the Differentiation of Human Induced Pluripotent Stem Cells into Mixed Cultures of Neurons and Glia for Neurotoxicity Testing at JoVE.com

Protocol for the Differentiation of Human Induced Pluripotent Stem Cells into Mixed Cultures of Neurons and Glia for Neurotoxicity Testing

Humant inducerede pluripotente stamceller (hiPSC&#39;er) betragtes som et stærkt værktøj til lægemiddel- og kemisk screening og til udvikling af nye in vitro- modeller til toksicitetstestning, herunder neurotoksicitet. Her beskrives en detaljeret protokol for differentieringen af ​​hiPSC&#39;er i neuroner og glia.

Protokol for differentieringen af ​​humane inducerede pluripotente stamceller i blandede kulturer af neuroner og glia til neurotoksicitetstestning

Human pluripotent stem cells can differentiate into various cell types that can be applied to human-based in vitro toxicity assays. One major advantage is ...

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Embryonic and Induced Pluripotent Stem Cells

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Three-dimensional Quantification of Dendritic Spines from Pyramidal Neurons Derived from Human Induced Pluripotent Stem Cells

Tre-dimensionel Kvantificering af dendritiske Spines fra pyramideneuroner Afledt af menneskeskabte pluripotente stamceller

Dendritic spines are small protrusions that correspond to the post-synaptic compartments of excitatory synapses in the central nervous system. They are ...

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 ...

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

Hurtig neuronal differentiering af induceret pluripotente stamceller til måling Network Aktivitet på Micro-elektrode Arrays

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

In Vitro Differentiation of Human Pluripotent Stem Cells into Trophoblastic Cells

In Vitro Differentiation of Human Pluripotent Stem Cells into Trophoblastic Cells

In vitro differentiering af humane pluripotente stamceller til trofoblastceller

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

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

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 ...

Synaptic Microcircuit Modeling with 3D Cocultures of Astrocytes and Neurons from Human Pluripotent Stem Cells

Synaptic dataloger modellering med 3D Cocultures astrocytter og neuroner fra humane pluripotente stamceller

A barrier to our understanding of how various cell types and signals contribute to synaptic circuit function is the lack of relevant models for studying ...

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 ...

Efficient Differentiation of Human Pluripotent Stem Cells into Liver Cells

Effektiv differentiering af humane pluripotente stamceller i leverceller

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

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

I Vitro Generation af Somite derivater fra menneskelige induceret celler pluripotente stamceller

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

Protokol for differentieringen af ​​humane inducerede pluripotente stamceller i blandede kulturer af neuroner og glia til neurotoksicitetstestning

Protokol for differentieringen af humane inducerede pluripotente stamceller i blandede kulturer af neuroner og glia til neurotoksicitetstestning