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

The authors wish to thank the Imaging Facility at Center for Life Nano Science, Istituto Italiano di Tecnologia, for support and technical advice. We are grateful to members of the Center for Life Nano Science for helpful discussion. This work was partially supported by a grant from AriSLA (pilot grant 2016 &#34;StressFUS&#34;) to AR.

1. Maintenance of Human iPSCs
<ol>
	<li>Preparation of matrix-coated plates
	<ol>
		<li>Thaw one 5 mL vial of matrix (see Table of Materials) at 4 &#176;C overnight. The original matrix stocks come at different stock concentrations and aliquots are made according to the dilution factor indicated on the datasheet, specific for the individual lot. It is important to keep the vial and tubes ice cold to prevent premature gelling of the matrix. Dispense matrix into aliquots in pre-chilled c...

<ol>
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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. 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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>

This protocol allows to efficiently convert human iPSCs into spinal and cranial motor neurons thanks to the ectopic expression of lineage-specific transcription factors. These transgenes are inducible by doxycycline and stably integrated in the genome thanks to a piggyBac transposon-based vector. In a mixed population, one or several copies of the piggyBac vector will be randomly integrated into the genome of individual cells, increasing the risk of genome integrity alterations. Moreover, a progressive selection of iPSC ...

Motor neuron (MN) degeneration plays a causative role in human diseases such as Amyotrophic Lateral Sclerosis (ALS) and Spinal Muscular Atrophy (SMA). Establishing suitable in vitro cell model systems that recapitulate the complexity of the human MN is an important step towards the development of new therapeutic approaches. Induced pluripotent stem cells (iPSCs), which are endowed with remarkable plurilineage differentiation properties, have now been derived from a number of patients affected by motor neuron diseases1,2. Additional iPSC lines carrying pathogenic mutations associated to MN diseases have been generated by gene editing, starting from control &#34;healthy&#34;&#160;pluripotent stem cells3. These lines represent useful tools for in vitro disease modeling and drug screening, provided that appropriate methods for iPSC differentiation into MNs are available. The rationale behind the development of this method is to provide the scientific community interested in MN diseases with a fast and efficient differentiation protocol giving rise to mature functional MNs. The first advantage of this method is its timeframe of execution. Another relevant point of strength comes from the elimination of any purification step. Finally, the protocol can be used to generate two distinct populations of motor neurons.
The possibility of generating different subtypes of MNs is particularly relevant for modeling of MN diseases. Not all MN subtypes are equally vulnerable in ALS and SMA and the onset of symptoms in different motor units greatly influences the prognosis. In ALS, spinal onset with symptoms starting in upper and lower limbs leads to death in about 3-5&#8239;years4. Conversely, bulbar onset, starting with degeneration of cranial MNs, has a worst prognosis. Moreover, the percentage of bulbar onset is significantly higher in patients with mutations in the RNA-binding proteins FUS and TDP-43 than in individuals with SOD1 mutations5. Almost the totality of alternative MN differentiation protocols relies on the activity of retinoic acid (RA), which confer a spinal character to differentiating iPSCs6,7,8. This limits the possibility of studying intrinsic factors, which could be protective in specific MN subtypes9,10.
Consistent with a previous work in mouse embryonic stem cells11, we have recently shown that in human iPSCs ectopic expression of Ngn2, Isl1 and Lhx3 (NIL) induces a spinal MN identity, while Ngn2 and Isl1 plus Phox2a (NIP) specify cranial MNs12. We have hence developed an efficient protocol, leading to the production of human MNs endowed with functional properties in a 12 days turnaround. The purpose of this method is to obtain, in a short time frame and without the need for purification (e.g., by FACS), cell populations highly enriched for MNs with spinal or cranial identity.

<table border="0" cellpadding="0" cellspacing="0" style="width:2004px;" width="2004">
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	</colgroup>
	<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

We describe here a method to obtain functional spinal and cranial motor neurons from human induced pluripotent stem cells (iPSCs). Direct conversion into motor neuron is obtained by ectopic expression of alternative modules of transcription factors, namely Ngn2, Isl1 and Lhx3 (NIL) or Ngn2, Isl1 and Phox2a (NIP). NIL and NIP specify, respectively, spinal and cranial motor neuron identity. Our protocol starts with the generation of modified iPSC lines in which NIL or NIP are stably integrated in the genome via a piggyBac transposon vector. Expression of the transgenes is then induced by doxycycline and leads, in 5 days, to the conversion of iPSCs into MN progenitors. Subsequent maturation, for 7 days, leads to homogeneous populations of spinal or cranial MNs. Our method holds several advantages over previous protocols: it is extremely rapid and simplified; it does not require viral infection or further MN isolation; it allows generating different MN subpopulations (spinal and cranial) with a remarkable degree of maturation, as demonstrated by the ability to fire trains of action potentials. Moreover, a large number of motor neurons can be obtained without purification from mixed populations. iPSC-derived spinal and cranial motor neurons can be used for in vitro modeling of Amyotrophic Lateral Sclerosis and other neurodegenerative diseases of the motor neuron. Homogeneous motor neuron populations might represent an important resource for cell type specific drug screenings.

A schematic description of the differentiation method is shown in Figure 1. Human iPSCs (WT I line3) were transfected with epB-Bsd-TT-NIL or epB-Bsd-TT-NIP, generating, upon blasticidin selection, stable and inducible cell lines12, hereafter referred to as iPSC-NIL and iPSC-NIP, respectively. Differentiating cells were characterized for the expression of the pluripotency marker OCT4 and the pan-neuronal marker TUJ1. Immunostaining analysis show...

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

This protocol allows rapid and efficient conversion of induced pluripotent stem cells into motor neurons with a spinal or cranial identity, by ectopic expression of transcription factors from inducible piggyBac vectors.

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

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

The possibility of generating different subtypes of motor neurons is particularly relevant for modeling modern motor neuron diseases such as amyotrophic lateral sclerosis. This method allows the acquisition of cell populations that are highly rigid for motor neurons with a spinal or cranial identity in a short time frame and without the purification step. The generation of human motor neurons from induced pluripotent stem cells or iPSC under simplified culture condition may facilitate drug screening for motor neuron diseases.The inducible expression of programming transcription factor can be used to generate another cell types such as neuronal, skeletal muscle, and glial cells from an iPSC repository. For NIL and NIP iPSC line generation, rinse the human iPSC culture with calcium-and magnesium-free PBS before treating the cells with cell dissociation reagent for five to 10 minutes at 37 degrees Celsius. When the cells have begun to detach from the culture container, use a P1000 pipette to gently manually dissociate the cells three to four times.Transfer the cells to a 15 milliliter tube and bring the final volume up to 10 milliliters with fresh PBS without calcium and magnesium for counting. Collect one times 10 to the sixth cells by centrifugation and resuspend the pellet in 100 microliters of buffer R from the electroporation kit. Add 4.5 micrograms of transposable vector plasma DNA and 0.5 micrograms of the piggyBac transposase plasma DNA for transfection.Transfect with the cell electroporation system according to the manufacturer's instructions with the indicated parameters. Then, seed the cells in human iPSC medium supplemented with 10 micromolar ROCK inhibitor Y-27632 in a six millimeter matrix-coated dish. Two days after the transfection, add 5 micrograms per milliliter of blasticidin to the culture medium keeping the cells in the antibiotic for at least seven to 10 days to counterselect the cells that have not integrated the transgenes.At the end of the incubation, maintain the stably transfected cells as a mixed population composed of cells with different numbers of transgenes and different integration sites or isolate single clones. Prepare an additional dish to allow the effective expression of the transgenes upon one microgram per milliliter of doxycycline induction to be assessed by RT-PCR with transgene-specific primers for neurogenin-2 as previously described. At this stage, stocks of the novel NIL and NIP iPSC lines should also be frozen in freezing medium for human iPSCs.For motor neuron differentiation, dissociate the stably transfected cell cultures with dissociation reagent as demonstrated to allow the cells to be collected in a 15 milliliter tube containing five volumes of DMEM/F12 medium. After centrifugation, resuspend the cells in human iPSC medium supplemented with micromolar ROCK inhibitor for counting. Seed the cells on matrix-coated dishes at a 6.25 times 10 the fourth cells per centimeter squared density.For the next two days, replace the supernatants with fresh differentiation medium supplemented with doxycycline. On day two, change the medium to neurobasal B27 medium supplemented with gamma secretase inhibitor, vascular endothelial growth factor receptor inhibitor, and doxycyline. On day five, dissociate the cells as demonstrated and resuspend the detached cells in four milliliters of DMEM/F12 medium for counting.Pipetting is important for a complete dissociation of the cells. Freeze an aliquot of the motor neuron progenitors in cell-freezing medium according to the manufacturer's instructions and collect the rest of the cells by centrifugation. Resuspend the pellet in neuronal medium supplemented with 10 micromolar ROCK inhibitor.Seed the cells on polyornithine laminin-coated microslide supports with polymer cover slips at a one times 10 to the fifth cells per centimeter squared density. On day six, carefully replace the medium with fresh neuronal medium without inhibitor for culture until downstream analysis without detaching the cells from the support surface. A uniform expression of the pluripotency marker octamer-binding transcription factor four or OTC4 can be observed in differentiating cell cultures on day zero in the absence of pan neuronal marker neuron-specific class three beta-tubulin TUJ1 positivity.On day three, a strong decrease in the number of OCT4-positive cells is evident. That is mirrored by the expression of TUJ1 in a subset of differentiating iPSCs. At day five, no expression of OCT4 is observed in the population which shows a consistent expression of TUJ1 and an acquired neuronal morphology.Immunostaining analysis seven days post replating of the motor neuron progenitor cells reveals a uniform expression of TUJ1 and the mature motor neuron marker choline acetyltransferase. iPSC NIL-derived neurons successfully display voltage-dependent sodium currents and voltage-dependent potassium currents. 80%of the iPSC NIL-derived motor neurons clamped in current clamp modality are able to trigger spike trains when injected with a current pulse of plus 60 picoamps or more.The minimum current required to elicit repetitive firing in more than 50%of the recorded cells is plus 40 picoamps with a spike threshold of approximately minus 38 millivolts and an average firing frequency at plus 40 picoamps of about eight hertz, suggesting that iPSC NIL-derived spinal motor neurons exhibit functional properties typical of mature neurons. Spinal and cranial motor neurons can be derived from controlled iPSCs or iPSCs carrying pathological mutations and that these cells can be used as thesis models or for drug screening. Our method can be used to help in understanding why different motor neuron units are differentially affected by ALS.

Research

JoVE Journal

Neuroscience

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

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

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

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

Feeder-free Derivation of Neural Crest Progenitor Cells from Human Pluripotent Stem Cells

Human pluripotent stem cells (hPSCs) have great potential for studying human embryonic development, for modeling human diseases in the dish and as a ...

Directed Dopaminergic Neuron Differentiation from Human Pluripotent Stem Cells

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

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

Derivation of Adult Human Fibroblasts and their Direct Conversion into Expandable Neural Progenitor Cells

Generation of&#160;induced pluripotent stem cell (iPSCs) from adult skin fibroblasts and subsequent differentiation into somatic cells provides ...

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

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

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