Name: conversion of human induced pluripotent stem cells (ipscs) into functional spinal and cranial motor neurons using piggybac vectors
Uploaded: 2019-05-01T14:30:00
Description: Watch this Scientific Journal Video about Conversion of Human Induced Pluripotent Stem Cells iPSCs into Functional Spinal and Cranial Motor Neurons Using PiggyBac Vectors at JoVE.com

Forfatterne ønsker at takke Imaging Facility på Center for Life Nano Science, Istituto Italiano di tecnologia, for støtte og teknisk rådgivning. Vi er taknemmelige for medlemmer af Center for Life Nano Science for nyttige diskussioner. Dette arbejde blev delvist understøttet af et tilskud fra AriSLA (pilot tilskud 2016 "StressFUS") til AR.

1. vedligeholdelse af humane iPSCs
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Fremstilling af matrix belagte plader
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<li>Tø et 5 mL hætteglas med matrix (Se tabel over materialer) ved 4 °c om natten. De oprindelige matrixer stammer fra forskellige Stam koncentrationer, og der laves aliquoter i overensstemmelse med den fortyndingsfaktor, der er angivet på dataarket, specifikt for det enkelte parti. Det er vigtigt at holde hætteglasset og rørene iskold for at forhindre for tidlig gelerings af matricen. Der ska...

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	<li>Dimos, J. T., 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=Induced+Pluripotent+Stem+Cells+Generated+from+Patients+with+ALS+Can+Be+Differentiated+into+Motor+Neurons.">Induced Pluripotent Stem Cells Generated from Patients with ALS Can Be Differentiated into Motor Neurons.</a> Science (New York, NY). 321 (5893), 1218 (2008).</li><li>Ebert, A. D., 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=Induced+pluripotent+stem+cells+from+a+spinal+muscular+atrophy+patient.">Induced pluripotent stem cells from a spinal muscular atrophy patient.</a> Nature. 457 (7227), 277-280 (2009).</li><li>Lenzi, 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=ALS+mutant+FUS+proteins+are+recruited+into+stress+granules+in+induced+Pluripotent+Stem+Cells+(iPSCs)+derived+motoneurons.">ALS mutant FUS proteins are recruited into stress granules in induced Pluripotent Stem Cells (iPSCs) derived motoneurons.</a> Disease models &amp; mechanisms. 8 (7), 755-766 (2015).</li><li>Wijesekera, L. C., Leigh, P. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Amyotrophic+lateral+sclerosis.">Amyotrophic lateral sclerosis.</a> Orphanet journal of rare diseases. 4 (3), 1-22 (2009).</li><li>Yan, 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=Frameshift+and+novel+mutations+in+FUS+in+familial+amyotrophic+lateral+sclerosis+and+ALS/dementia.">Frameshift and novel mutations in FUS in familial amyotrophic lateral sclerosis and ALS/dementia.</a> Neurology. 75 (9), 807-814 (2010).</li><li>Boulting, G. 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=A+functionally+characterized+test+set+of+human+induced+pluripotent+stem+cells.">A functionally characterized test set of human induced pluripotent stem cells.</a> Nature biotechnology. 29 (3), 279-286 (2011).</li><li>Amoroso, M. W., 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=Accelerated+high-yield+generation+of+limb-innervating+motor+neurons+from+human+stem+cells.">Accelerated high-yield generation of limb-innervating motor neurons from human stem cells.</a> The Journal of neuroscience: the official journal of the Society for Neuroscience. 33 (2), 574-586 (2013).</li><li>Maury, Y., 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=Combinatorial+analysis+of+developmental+cues+efficiently+converts+human+pluripotent+stem+cells+into+multiple+neuronal+subtypes.">Combinatorial analysis of developmental cues efficiently converts human pluripotent stem cells into multiple neuronal subtypes.</a> Nature biotechnology. 33 (1), 89-96 (2015).</li><li>Allodi, I., 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=Differential+neuronal+vulnerability+identifies+IGF-2+as+a+protective+factor+in+ALS.">Differential neuronal vulnerability identifies IGF-2 as a protective factor in ALS.</a> Scientific reports. 6, 25960 (2016).</li><li>Kaplan, 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=Neuronal+matrix+metalloproteinase-9+is+a+determinant+of+selective+neurodegeneration.">Neuronal matrix metalloproteinase-9 is a determinant of selective neurodegeneration.</a> Neuron. 81 (2), 333-348 (2014).</li><li>Mazzoni, E. O., 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=Synergistic+binding+of+transcription+factors+to+cell-specific+enhancers+programs+motor+neuron+identity.">Synergistic binding of transcription factors to cell-specific enhancers programs motor neuron identity.</a> Nature neuroscience. 16 (9), 1219-1227 (2013).</li><li>De Santis, R., Garone, M. G., Pagani, F., de Turris, V., Di Angelantonio, S., Rosa, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+conversion+of+human+pluripotent+stem+cells+into+cranial+motor+neurons+using+a+piggyBac+vector.">Direct conversion of human pluripotent stem cells into cranial motor neurons using a piggyBac vector.</a> Stem Cell Research. 29, 189-196 (2018).</li><li>Yusa, K., Zhou, L., Li, M. A., Bradley, A., Craig, N. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+hyperactive+piggyBac+transposase+for+mammalian+applications.">A hyperactive piggyBac transposase for mammalian applications.</a> Proceedings of the National Academy of Sciences of the United States of America. 108 (4), 1531-1536 (2011).</li><li>Sances, 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=Modeling+ALS+with+motor+neurons+derived+from+human+induced+pluripotent+stem+cells.">Modeling ALS with motor neurons derived from human induced pluripotent stem cells.</a> Nature Neuroscience. 16 (4), 542-553 (2016).</li><li>Miller, J. D., 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+iPSC-based+modeling+of+late-onset+disease+via+progerin-induced+aging.">Human iPSC-based modeling of late-onset disease via progerin-induced aging.</a> Cell stem cell. 13 (6), 691-705 (2013).</li><li>Lenzi, 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+control+and+ALS+mutant+human+iPSCs+into+functional+skeletal+muscle+cells,+a+tool+for+the+study+of+neuromuscolar+diseases.">Differentiation of control and ALS mutant human iPSCs into functional skeletal muscle cells, a tool for the study of neuromuscolar diseases.</a> Stem Cell Research. 17 (1), 140-147 (2016).</li><li>Zhang, Y., 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=Rapid+Single-Step+Induction+of+Functional+Neurons+from+Human+Pluripotent+Stem+Cells.">Rapid Single-Step Induction of Functional Neurons from Human Pluripotent Stem Cells.</a> Neuron. 78 (5), 785-798 (2013).</li><li>Theka, I., 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=Rapid+generation+of+functional+dopaminergic+neurons+from+human+induced+pluripotent+stem+cells+through+a+single-step+procedure+using+cell+lineage+transcription+factors.">Rapid generation of functional dopaminergic neurons from human induced pluripotent stem cells through a single-step procedure using cell lineage transcription factors.</a> Stem cells translational medicine. 2 (6), 473-479 (2013).</li><li>Pawlowski, 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=Inducible+and+Deterministic+Forward+Programming+of+Human+Pluripotent+Stem+Cells+into+Neurons,+Skeletal+Myocytes,+and+Oligodendrocytes.">Inducible and Deterministic Forward Programming of Human Pluripotent Stem Cells into Neurons, Skeletal Myocytes, and Oligodendrocytes.</a> Stem Cell Reports. 8 (4), 803-812 (2017).</li><li>Li, X., 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=Fast+Generation+of+Functional+Subtype+Astrocytes+from+Human+Pluripotent+Stem+Cells.">Fast Generation of Functional Subtype Astrocytes from Human Pluripotent Stem Cells.</a> Stem Cell Reports. 11 (4), 998-1008 (2018).</li><li>Nehme, R., 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=Combining+NGN2+Programming+with+Developmental+Patterning+Generates+Human+Excitatory+Neurons+with+NMDAR-Mediated+Synaptic+Transmission.">Combining NGN2 Programming with Developmental Patterning Generates Human Excitatory Neurons with NMDAR-Mediated Synaptic Transmission.</a> Cell reports. 23 (8), 2509-2523 (2018).</li></ol>

Denne protokol gør det muligt effektivt at konvertere humane iPSCs til spinal og kraniel motor neuroner takket være ektopic udtryk af Lineage-specifikke transkription faktorer. Disse transgener er inducerbare af doxycyclin og stabilt integreret i genomet takket være en transposon-baseret vektor med piggyBac. I en blandet population, en eller flere eksemplarer af piggyBac vektor vil blive tilfældigt integreret i genomet af de enkelte celler, øge risikoen for genomintegritet ændringer. Desuden kan en progressiv udvæ...

Motorisk neuron (MN) degeneration spiller en årsagssammenhæng rolle i menneskelige sygdomme som Amyotrofisk lateral sklerose (ALS) og spinal muskuløs atrofi (SMA). Etablering af egnede in vitro-celle modelsystemer, der oversigter kompleksiteten af det menneskelige MN, er et vigtigt skridt i retning af udvikling af nye terapeutiske tilgange. Induceret pluripotente stamceller (ipscs), som er udstyret med bemærkelsesværdige plurilineage differentiering egenskaber, er nu blevet afledt af en række patienter ramt af motoriske neuron sygdomme1,2. Yderligere iPSC linjer transporterer patogene mutationer forbundet til MN sygdomme er blevet genereret af genredigering, begyndende fra kontrol "sund" pluripotente stamceller3. Disse linjer repræsenterer nyttige værktøjer til in vitro-sygdoms modellering og lægemiddel screening, forudsat at der findes passende metoder til differentiering af iPSC i MNs. Rationalet bag udviklingen af denne metode er at give forskersamfundet, der er interesseret i MN-sygdomme, en hurtig og effektiv differentierings protokol, der giver anledning til modne funktionelle MNs. Den første fordel ved denne metode er dens tidsramme for udførelse. Et andet relevant punkt af styrke kommer fra fjernelsen af ethvert rensningstrin. Endelig kan protokollen bruges til at generere to forskellige populationer af motoriske neuroner.
Muligheden for at generere forskellige undertyper af MNs er særlig relevant for modellering af MN-sygdomme. Ikke alle MN-undertyper er lige så sårbare i ALS og SMA, og symptomdebut i forskellige motor enheder påvirker i høj grad prognosen. I ALS, spinal debut med symptomer, der starter i øvre og nedre lemmer fører til døden i omkring 3-5 år4. Omvendt, bulbar debut, begyndende med degeneration af kranielle MNs, har en værste prognose. Desuden er andelen af bulbar debut signifikant højere hos patienter med mutationer i RNA-bindende proteiner FUS og TDP-43 end hos personer med SOD1 mutationer5. Næsten alle alternative MN-differentierings protokoller er baseret på aktiviteten af retinoinsyre (RA), som giver en spinal karakter til at differentiere ipscs6,7,8. Dette begrænser muligheden for at studere iboende faktorer, som kan være beskyttende i specifikke MN undertyper9,10.
I overensstemmelse med en tidligere arbejde i mus embryonale stamceller11, har vi for nylig vist, at i human ipscs ektopic udtryk for Ngn2, Isl1 og Lhx3 (Nil) inducerer en spinal MN identitet, mens Ngn2 og Isl1 plus Phox2a (NIP) angive kranie mns12. Vi har derfor udviklet en effektiv protokol, der fører til produktion af menneskelige MNs udstyret med funktionelle egenskaber i en 12 dages turnaround. Formålet med denne metode er at opnå, inden for en kort tidsramme og uden behov for rensning (f. eks. ved FACS), cellepopulationer stærkt beriget for MNs med spinal eller kraniel identitet.

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	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:364px;">5-Bapta</td>
			<td style="width:208px;">Sigma-Aldrich</td>
			<td style="width:143px;">A4926-1G</td>
			<td style="width:1289px;">chemicals for electrophysiological solutions</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Accutase</td>
			<td>Sigma-Aldrich</td>
			<td>A6964-100ML&#160;</td>
			<td>Cell dissociation reagent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">anti-CHAT</td>
			<td>EMD Millipore&#160;</td>
			<td>AB144P</td>
			<td>Anti-Choline Acetyltransferase. Primary antibody used in immunostaining assays. RRID: AB_2079751; Lot number: 2971003</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">anti-goat Alexa Fluor 488&#160;</td>
			<td>Thermo Fisher Scientific&#160;</td>
			<td>A11055</td>
			<td>Secondary antibody used for immonofluorescence assays. RRID: AB_2534102; Lot number: 1915848</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">anti-mouse Alexa Fluor 647</td>
			<td>Thermo Fisher Scientific&#160;</td>
			<td>A31571</td>
			<td>Secondary antibody used for immonofluorescence assays. RRID: AB_162542; Lot number: 1757130</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">anti-Oct4&#160;</td>
			<td>BD Biosciences</td>
			<td>611202</td>
			<td>Primary antibody used in immunostaining assays. RRID: AB_398736; Lot number: 5233722</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">anti-Phox2b</td>
			<td>Santa Cruz Biotechnology, Inc.</td>
			<td>sc-376997</td>
			<td>Primary antibody used in immunostaining assays. Lot number: E0117</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">anti-rabbit Alexa Fluor 594&#160;</td>
			<td>Immunological Sciences</td>
			<td>IS-20152-1</td>
			<td>Secondary antibody used for immonofluorescence assays</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">anti-TUJ1&#160;</td>
			<td>Sigma-Aldrich&#160;</td>
			<td>T2200</td>
			<td>Primary antibody used in immunostaining assays. RRID: AB_262133</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">B27</td>
			<td>Miltenyi Biotec</td>
			<td>130-093-566</td>
			<td>Serum free supplement for neuronal cell maintenance</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Bambanker</td>
			<td>Nippon Genetics</td>
			<td>NGE-BB02</td>
			<td>Cell freezing medium, used here for motor neuron progenitors</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">BDNF</td>
			<td>PreproTech</td>
			<td>450-02</td>
			<td>Brain-Derived Neurotrophic Factor</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Blasticidin</td>
			<td>Sigma-Aldrich</td>
			<td>203350</td>
			<td>Nucleoside antibiotic that inhibits protein synthesis in prokaryotes and eukaryotes</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">BSA</td>
			<td>Sigma-Aldrich</td>
			<td>A2153</td>
			<td>Bovine Serum Albumin. Blocking agent to prevent non-specific binding of antibodies in immunostaining assays</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">CaCl2</td>
			<td>Sigma-Aldrich</td>
			<td>C3881</td>
			<td>chemicals for electrophysiological solutions</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Clampex 10 software</td>
			<td>Molecular Devices</td>
			<td>Clampex 10</td>
			<td>Membrane currents recording system</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Corning Matrigel hESC-qualified Matrix</td>
			<td>Corning</td>
			<td>354277</td>
			<td style="width:1289px;">Reconstituted basement membrane preparation from the Engelbreth-Holm-Swarm (EHS) mouse sarcoma. Used for adhesion of iPSC to plastic and glass supports</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">CRYOSTEM ACF FREEZING MEDIA</td>
			<td>Biological Industries</td>
			<td>05-710-1E</td>
			<td>Freezing medium for human iPSCs</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">D-Glucose</td>
			<td>Sigma-Aldrich</td>
			<td>G5146</td>
			<td>chemicals for electrophysiological solutions</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DAPI powder</td>
			<td>Roche</td>
			<td>10236276001</td>
			<td>4&#8242;,6-diamidino-2-phenylindole. Fluorescent stain that binds to adenine&#8211;thymine rich regions in DNA used for nuclei staining in immonofluorescence assays</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DAPT</td>
			<td>AdipoGen</td>
			<td>AG-CR1-0016-M005</td>
			<td>Gamma secretase inhibitor</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dispase</td>
			<td>Gibco</td>
			<td>17105-041</td>
			<td>Reagent for gentle dissociation of human iPSCs</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DMEM/F12</td>
			<td>Sigma-Aldrich</td>
			<td>D6421-500ML</td>
			<td>Basal medium for cell culture</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Doxycycline</td>
			<td>Sigma-Aldrich</td>
			<td>D9891-1G&#160;</td>
			<td>Used to induce expression of transgenes from epB-Bsd-TT-NIL and epB-Bsd-TT-NIP vectors</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DS2U&#160;</td>
			<td>WiCell</td>
			<td>UWWC1-DS2U</td>
			<td>Commercial human iPSC line</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">E.Z.N.A Total RNA Kit&#160;</td>
			<td>Omega bio-tek</td>
			<td>R6834-02</td>
			<td>Kit for total extraction of RNA from cultured eukaryotic cells</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">GDNF</td>
			<td>PreproTech</td>
			<td>450-10</td>
			<td>Glial-Derived Neurotrophic Factor</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Gibco Episomal hiPSC Line</td>
			<td>Thermo Fisher Scientific</td>
			<td>A18945</td>
			<td>Commercial human iPSC line</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Glutamax</td>
			<td>Thermo Fisher Scientific</td>
			<td>35050038</td>
			<td>An alternative to L-glutamine with increased stability. Improves cell health.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Hepes</td>
			<td>Sigma-Aldrich</td>
			<td>H4034</td>
			<td>chemicals for electrophysiological solutions</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">iScript Reverse Transcription Supermix for RT-qPCR&#160;</td>
			<td>Bio-Rad</td>
			<td>1708841</td>
			<td>Kit for gene expression analysis using real-time qPCR</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">iTaqTM Universal SYBR Green Supermix&#160;</td>
			<td>Bio-Rad</td>
			<td>172-5121&#160;</td>
			<td>Ready-to-use reaction master mix optimized for dye-based quantitative PCR (qPCR) on any real-time PCR instrument</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">K-Gluconate</td>
			<td>Sigma-Aldrich</td>
			<td>G4500</td>
			<td>chemicals for electrophysiological solutions</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">KCl</td>
			<td>Sigma-Aldrich</td>
			<td>P9333</td>
			<td>chemicals for electrophysiological solutions</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">L-ascorbic acid</td>
			<td>LKT Laboratories</td>
			<td>A7210</td>
			<td>Used in cell culture as an antioxidant</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Laminin</td>
			<td>Sigma-Aldrich</td>
			<td>11243217001</td>
			<td>Promotes attachment and growth of neural cells in vitro</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Laser scanning confocal microscope&#160;</td>
			<td>Olympus</td>
			<td>&#160;iX83 FluoView1200</td>
			<td>Confocal microscope for acquisition of immunostaining images</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Mg-ATP</td>
			<td>Sigma-Aldrich</td>
			<td>A9187</td>
			<td>chemicals for electrophysiological solutions</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">MgCl2</td>
			<td>Sigma-Aldrich</td>
			<td>M8266</td>
			<td>chemicals for electrophysiological solutions</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Mounting Medium&#160;</td>
			<td>Ibidi</td>
			<td>50001</td>
			<td>Mounting solution used for confocal microscopy and immunofluorescence assays</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Multiclamp patch-clamp amplifier</td>
			<td>Molecular Devices</td>
			<td>700B</td>
			<td>Membrane currents recording system</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Na-GTP</td>
			<td>Sigma-Aldrich</td>
			<td>G8877</td>
			<td>chemicals for electrophysiological solutions</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">NaCl</td>
			<td>Sigma-Aldrich</td>
			<td>71376</td>
			<td>chemicals for electrophysiological solutions</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">NEAA</td>
			<td>Thermo Fisher Scientific</td>
			<td>11140035</td>
			<td>Non-Essential Amino Acids. Used as a supplement for cell culture medium, to increase cell growth and viability.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Neon 100 &#956;L Kit</td>
			<td>Thermo Fisher Scientific</td>
			<td>MPK10096</td>
			<td>Cell electroporation kit</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Neon Transfection System</td>
			<td>Thermo Fisher Scientific</td>
			<td>MPK5000</td>
			<td>Cell electroporation system</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Neurobasal Medium</td>
			<td>Thermo Fisher Scientific</td>
			<td>21103049</td>
			<td>Basal medium designed for long-term maintenance and maturation of neuronal cell populations without the need for an astrocyte feeder layer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">NutriStem-XF/FF&#160;</td>
			<td>Biological Industries</td>
			<td>05-100-1A</td>
			<td>Human iPSC culture medium</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Paraformaldehyde</td>
			<td>Electron Microscopy Sciences</td>
			<td>157-8</td>
			<td>Used for cell fixation in immunostaining assays</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PBS</td>
			<td>Sigma-Aldrich</td>
			<td>D8662-500ML</td>
			<td>Dulbecco s Phosphate Buffer Saline w Calcium w Magnesium</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PBS Ca2+/Mg2+ free</td>
			<td>Sigma-Aldrich</td>
			<td>D8537-500ML</td>
			<td>Dulbecco s Phosphate Buffer Saline w/o Calcium w/o Magnesium</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Penicillin/Streptomycin&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>P4333-100ML</td>
			<td>Penicillin/Streptomicin solution used to prevent cell culture contamination from bacteria.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">poly-ornithine</td>
			<td>Sigma-Aldrich</td>
			<td>P4957</td>
			<td>Promotes attachment and growth of neural cells in vitro</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">SU5402</td>
			<td>Sigma-Aldrich</td>
			<td>SML0443-5MG</td>
			<td>Selective inhibitor of vascular endothelial growth factor receptor 2 (VEGFR-2)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Triton X-100&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>T8787</td>
			<td>4-(1,1,3,3-Tetramethylbutyl)phenyl-polyethylene glycol,&#160;t-Octylphenoxypolyethoxyethanol, Polyethylene glycol&#160;tert-octylphenyl ether. Used for cell permeabilization in immunostaining assays</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Upright microscope</td>
			<td>Olympus</td>
			<td>BX51VI</td>
			<td>Microscope for electrophysiological recording equipped with CoolSnap Myo camera&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Y-27632&#160; (ROCK inhibitor)</td>
			<td>Enzo Life Sciences</td>
			<td>ALX-270-333-M005&#160;</td>
			<td>Cell-permeable selective inhibitor of Rho-associated, coiled-coil containing protein kinase (ROCK). Increases iPSC survival</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">&#956;-Slide 8 Well&#160;</td>
			<td>Ibidi</td>
			<td>80826</td>
			<td>Support for high&#8211;end microscopic analysis of fixed cells</td>
		</tr>
	</tbody>
</table>

conversion of human induced pluripotent stem cells (ipscs) into functional spinal and cranial motor neurons using piggybac vectors

Vi beskriver her en metode til at opnå funktionelle spinal og kraniel motoriske neuroner fra menneskeskabte pluripotente stamceller (iPSCs). Direkte omdannelse til motor neuron er opnået ved Ektopisk udtryk af alternative moduler af transkription faktorer, nemlig Ngn2, Isl1 og Lhx3 (NIL) eller Ngn2, Isl1 og Phox2a (NIP). NIL og NIP specificere henholdsvis spinal og kraniel motoriske neuron identitet. Vores protokol starter med dannelsen af modificerede iPSC linjer, hvor NIL eller NIP er stabilt integreret i genomet via en piggyBac transposon vektor. Ekspressionen af transgener induceres derefter af doxycyclin og fører i 5 dage til omdannelsen af iPSCs til MN-progenitorer. Efterfølgende modning, i 7 dage, fører til homogene populationer af spinal eller craniale MNs. Vores metode rummer flere fordele i forhold til tidligere protokoller: den er ekstremt hurtig og forenklet; Det kræver ikke virus infektion eller yderligere MN-isolation. Det gør det muligt at generere forskellige MN subpopulationer (spinal og Cranial) med en bemærkelsesværdig grad af modning, som påvist ved evnen til at brand tog af action potentialer. Desuden kan et stort antal motoriske neuroner opnås uden rensning fra blandede populationer. iPSC-afledte spinal og kraniel motor neuroner kan bruges til in vitro modellering af amyotrofisk lateral sklerose og andre neurodegenerative sygdomme i motor neuron. Homogene motoriske neuron populationer kan repræsentere en vigtig ressource for celletype specifikke Drug screeninger.

En skematisk beskrivelse af differentierings metoden er vist i figur 1. Humane iPSCs (WT I linje3) blev transficeret med EPB-BSD-TT-Nil eller EPB-BSD-TT-NIP, genererer, efter blasticidin udvælgelse, stabile og inducerbare cellelinjer12, i det følgende benævnt IPSC-Nil og IPSC-NIP, hhv. Differentierende celler blev karakteriseret for udtrykket af pluripotens markør OCT4 og den pan-neuronal markør TUJ1. Immun farvning analyse viste ensartet ...

Watch this Scientific Journal Video about Conversion of Human Induced Pluripotent Stem Cells iPSCs into Functional Spinal and Cranial Motor Neurons Using PiggyBac Vectors at JoVE.com

Conversion of Human Induced Pluripotent Stem Cells iPSCs into Functional Spinal and Cranial Motor Neurons Using PiggyBac Vectors

Denne protokol giver mulighed for hurtig og effektiv omdannelse af inducerede pluripotente stamceller til motoriske neuroner med en spinal eller kranie identitet, ved Ektopisk udtryk af transkription faktorer fra inducerble piggybac vektorer.

Omdannelse af humane induceret pluripotente stamceller (iPSCs) til funktionel spinal og kraniel motor neuroner brug PiggyBac vektorer

We describe here a method to obtain functional spinal and cranial motor neurons from human induced pluripotent stem cells (iPSCs). Direct conversion into ...

Muligheden for at generere forskellige undertyper af motoriske neuroner er særlig relevant for modellering moderne motor neuron sygdomme såsom amyotrofisk lateral sklerose. Denne metode gør det muligt at erhverve cellepopulationer, der er meget stive for motoriske neuroner med en spinal eller kraniel identitet i en kort tidsramme og uden rensning trin. Generering af humane motoriske neuroner fra inducerede pluripotente stamceller eller iPSC under forenklet kultur betingelse kan lette lægemiddelscreening for motoriske neuron sygdomme.Den inducerende udtryk for programmering transskription faktor kan bruges til at generere en anden celle typer såsom neuronal, skeletmuskulatur, og gliaceller fra en iPSC repository. For NIL og NIP iPSC linje generation, skyl den menneskelige iPSC kultur med calcium-og magnesium-fri PBS før behandling af cellerne med celle dissociation reagens i fem til 10 minutter ved 37 grader Celsius. Når cellerne er begyndt at løsne sig fra kulturbeholderen, skal du bruge en P1000-pipette til forsigtigt manuelt at adskille cellerne tre til fire gange.Cellerne overføres til et 15 milliliterrør, og det endelige volumen bringes op til 10 milliliter med frisk PBS uden calcium og magnesium til optælling. Saml en gang 10 til de sjette celler ved centrifugering og resuspend pellet i 100 mikroliter buffer R fra elektroporation kit. Der tilsættes 4,5 mikrogram transposable vektorplasma-DNA og 0,5 mikrogram af piggyBac transposaseplasma-DNA til transfektion.Transfect med celleelektroporationssystemet i henhold til producentens anvisninger med de angivne parametre. Derefter frø cellerne i humant iPSC medium suppleret med 10 micromolar ROCK-hæmmer Y-27632 i en seks millimeter matrix-belagt skål. To dage efter transfektionen tilsættes 5 mikrogram pr. milliliter blasticidin til kulturmediet, der holder cellerne i antibiotikummet i mindst syv til 10 dage for at modvirke markeringen af de celler, der ikke har integreret transgenene.Ved inkubationens afslutning opretholdes de stabilt transficerede celler som en blandet population, der består af celler med forskelligt antal transgener og forskellige integrationssteder, eller enkeltkloner isoleres. Forbered en ekstra skål, så transgenene kan udføre det effektive udtryk på et mikrogram pr. milliliter doxycyclininduktion, der skal vurderes af RT-PCR med transgenespecifikke primere til neurogenin-2 som tidligere beskrevet. På nuværende tidspunkt bør lagrene af de nye NIL- og NIP iPSC-linjer også fryses i frysemedium for humane iPSC'er.Ved differentiering af motorneuren adskilles de stabilt transficerede cellekulturer med dissociationsreagens som påvist, således at cellerne kan opsamles i et 15 milliliterrør, der indeholder fem volumener DMEM/F12-medium. Efter centrifugering, resuspenderede cellerne i humant iPSC medium suppleret med micromolar ROCK-hæmmer til optælling. Frø cellerne på matrix-belagte retter på en 6,25 gange 10 de fjerde celler per centimeter kvadreret tæthed.For de næste to dage, erstatte supernatants med frisk differentiering medium suppleret med doxycyclin. På dag to, ændre medium til neurobasal B27 medium suppleret med gamma sekretase hæmmer, vaskulære endotel vækstfaktor receptor hæmmer, og doxycylin. På dag fem skal cellerne adskilles som påvist og opsaltret de afmonterede celler i fire milliliter DMEM/F12-medium til optælling.Pipettering er vigtigt for en fuldstændig dissociation af cellerne. Frys en aliquot af de motoriske neuron stamfader i celle-frysning medium i henhold til producentens anvisninger og indsamle resten af cellerne ved centrifugering. Resuspend pellet i neuronal medium suppleret med 10 micromolar ROCK hæmmer.Frø cellerne på polyornithin laminin-belagt mikroslid understøtter med polymer dække glider på en gange 10 til den femte celler per centimeter kvadreret tæthed. På dag seks skal mediet udskiftes omhyggeligt med frisk neuronal medium uden væksthæmmer til dyrkning, indtil downstream-analysen uden at løsne cellerne fra støtteoverfladen. Et ensartet udtryk for den pluripotency markør octamer-bindende transskription faktor fire eller OTC4 kan observeres i at differentiere cellekulturer på dag nul i mangel af pan neuronal markør neuron-specifikke klasse tre beta-tubulin TUJ1 positivitet.På dag tre er et kraftigt fald i antallet af OCT4-positive celler tydeligt. Dette afspejles af udtrykket TUJ1 i en delmængde af differentiere iPSCs. På dag fem observeres der ingen ekspression af OCT4 i populationen, som viser et konsekvent udtryk for TUJ1 og en erhvervet neuronal morfologi.Immunostaining analyse syv dage efter relating af de motoriske neuron progenitor celler afslører et ensartet udtryk for TUJ1 og den modne motor neuron markør cholin acetyltransferase. iPSC NIL-afledte neuroner viser med succes spændingsafhængige natriumstrøm og spændingsafhængige kaliumstrømter. 80% af de iPSC NIL-afledte motorneuroner fastspændt i den aktuelle klemmemodalitet er i stand til at udløse spike tog, når de injiceres med en strømpust på plus 60 picoamps eller mere.Den mindste strøm, der kræves for at fremkalde gentagne fyring i mere end 50% af de registrerede celler er plus 40 picoamps med en spike tærskel på ca minus 38 millivolt og en gennemsnitlig fyring frekvens på plus 40 picoamps på omkring otte hertz, hvilket tyder på, at iPSC NIL-afledte spinal motor neuroner udviser funktionelle egenskaber typisk for modne neuroner. Spinal og kraniel motoriske neuroner kan udledes af kontrollerede iPSCs eller iPSCs transporterer patologiske mutationer, og at disse celler kan bruges som afhandling modeller eller til lægemiddelscreening. Vores metode kan bruges til at hjælpe med at forstå, hvorfor forskellige motorneurnenheder er differentieret påvirket af ALS.

Research

JoVE Journal

Neuroscience

Conversion of Human Induced Pluripotent Stem Cells (iPSCs) into Functional Spinal and Cranial Motor Neurons Using PiggyBac Vectors

Efficient Derivation of Human Neuronal Progenitors and Neurons from Pluripotent Human Embryonic Stem Cells with Small Molecule Induction

Effektiv derivering af humane Neuronal progenitorer og neuroner fra Pluripotente humane embryonale stamceller med småmolekyle Induktion

There is a large unfulfilled need for a clinically-suitable human neuronal cell source for repair or regeneration of the damaged central nervous system ...

The Specification of Telencephalic Glutamatergic Neurons from Human Pluripotent Stem Cells

Specifikationen af ​​Telencephalic glutamaterge neuroner fra humane pluripotente stamceller

Here, a stepwise procedure for efficiently generating telencephalic glutamatergic neurons from human pluripotent stem cells (PSCs) has been described. The ...

Lineage-reprogramming of Pericyte-derived Cells of the Adult Human Brain into Induced Neurons

Lineage-omprogrammering af pericyt-afledte Celler af Voksen Menneskelige hjerne til Inducerede Neuroner

Direct lineage-reprogramming of non-neuronal cells into induced neurons (iNs) may provide insights into the molecular mechanisms underlying neurogenesis ...

Directed Dopaminergic Neuron Differentiation from Human Pluripotent Stem Cells

Instrueret Dopaminerg Neuron Differentiering fra menneskelige pluripotente stamceller

Dopaminergic (DA) neurons in the substantia nigra pars compacta  (also known as A9 DA neurons) are the specific cell type that is lost in ...

Functional and Morphological Assessment of Diaphragm Innervation by Phrenic Motor Neurons

Funktionel og Morfologisk vurdering af Membran innervation af phrenic Motor neuroner

This protocol specifically focuses on tools for assessing phrenic motor neuron (PhMN) innervation of the diaphragm at both the electrophysiological and ...

Simple Generation of a High Yield Culture of Induced Neurons from Human Adult Skin Fibroblasts

Simpel Generation af højtydende kultur af inducerede neuroner fra menneskelige voksen hud fibroblaster

Induced neurons (iNs), the product of somatic cells directly converted to neurons, are a way to obtain patient-derived neurons from tissue that is easily ...

Method for High Speed Stretch Injury of Human Induced Pluripotent Stem Cell-derived Neurons in a 96-well Format

Metode for høj hastighed Stretch skade af menneskelig induceret pluripotente stamceller-afledt neuroner i en 96-brønds Format

Traumatic brain injury (TBI) is a major clinical challenge with high morbidity and mortality. Despite decades of pre-clinical research, no proven ...

Identifying Cell Surface Markers of Primary Neural Stem and Progenitor Cells by Metabolic Labeling of Sialoglycan

Identifikation af celleoverflade markører for primær neurale stamme og stamceller ved metabolisk mærkning af Sialoglycan

Neural stem and progenitor cells (NSPCs) are the cellular basis for the complex structures and functions of the brain. They are located in specialized ...

In Vivo Direct Reprogramming of Resident Glial Cells into Interneurons by Intracerebral Injection of Viral Vectors

In vivo direkte omprogrammering af residente Gliale celler til Interneuroner ved intracerebral injektion af virale vektorer

Converting resident glia in the brain into functional and subtype-specific neurons in vivo provides a step forward towards the development of alternative ...

3D Kinematic Analysis for the Functional Evaluation in the Rat Model of Sciatic Nerve Crush Injury

3D Kinematic Analyse for funktionel evaluering i rottemodel af iskiasnerven crush skade

Compared to the Sciatic Functional Index (SFI), kinematic analysis is a more reliable and sensitive method for performing functional evaluations of ...