This work was supported by grants from the Agence Nationale de la Recherche (ANR) to S.T. (ANR-17-CE16-0003-01) and N.B.B. (ANR-16-CE16-0011 and ANR-19-CE16-0002-01). LB is supported by the ANR under Investissements d&#39;avenir program (ANR-10-IAHU-01) and the Fondation Bettencourt Schueller (MD-PhD program). The Imagine Institute is supported by state funding from the ANR under the Investissements d&#39;avenir program (ANR-10-IAHU-01, CrossLab projects) and as part of the second Investissements d&#39;Avenir program (ANR-17-RHUS-0002).

1. Generation of 2D hIPS cell-based models of neocortical development
<ol>
	<li>Neural rosette formation
	<ol>
		<li>Start with hIPSC cultures harboring large regular colonies, exhibiting less than 10% differentiation and no more than 80% confluency.</li>
		<li>Rinse the hIPSCs with 2 mL of PBS.</li>
		<li>Add 2 mL of NSPC induction medium supplemented with the Rock inhibitor (NIM + 10 &#181;M of Y-27632).</li>
		<li>Manually dissect each hIPSC colony from one 35 mm dish using a needle...

<ol>
	<li>Huangfu, 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=Hedgehog+signalling+in+the+mouse+requires+intraflagellar+transport+proteins.">Hedgehog signalling in the mouse requires intraflagellar transport proteins.</a> Nature. 426 (6962), 83-87 (2003).</li><li>Goetz, S. C., Anderson, K. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+primary+cilium:+a+signalling+centre+during+vertebrate+development.">The primary cilium: a signalling centre during vertebrate development.</a> Nature Reviews. Genetics. 11 (5), 331-344 (2010).</li><li>Hansen, D. V., Lui, J. H., Parker, P. R. L., Kriegstein, A. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neurogenic+radial+glia+in+the+outer+subventricular+zone+of+human+neocortex.">Neurogenic radial glia in the outer subventricular zone of human neocortex.</a> Nature. 464 (7288), 554-561 (2010).</li><li>Lui, J. H., Hansen, D. V., Kriegstein, A. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+and+evolution+of+the+human+neocortex.">Development and evolution of the human neocortex.</a> Cell. 146 (1), 18-36 (2011).</li><li>Nonaka-Kinoshita, 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=Regulation+of+cerebral+cortex+size+and+folding+by+expansion+of+basal+progenitors.">Regulation of cerebral cortex size and folding by expansion of basal progenitors.</a> The EMBO Journal. 32 (13), 1817-1828 (2013).</li><li>Taverna, E., G&#246;tz, M., Huttner, W. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+cell+biology+of+neurogenesis:+toward+an+understanding+of+the+development+and+evolution+of+the+neocortex.">The cell biology of neurogenesis: toward an understanding of the development and evolution of the neocortex.</a> Annual Review of Cell and Developmental Biology. 30, 465-502 (2014).</li><li>Fern&#225;ndez, V., Llinares-Benadero, C., Borrell, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cerebral+cortex+expansion+and+folding:+what+have+we+learned.">Cerebral cortex expansion and folding: what have we learned.</a> The EMBO Journal. 35 (10), 1021-1044 (2016).</li><li>Spear, P. C., Erickson, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Apical+movement+during+interkinetic+nuclear+migration+is+a+two-step+process.">Apical movement during interkinetic nuclear migration is a two-step process.</a> Developmental Biology. 370 (1), 33-41 (2012).</li><li>Wilsch-Br&#228;uninger, M., Florio, M., Huttner, W. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neocortex+expansion+in+development+and+evolution+-+from+cell+biology+to+single+genes.">Neocortex expansion in development and evolution - from cell biology to single genes.</a> Current Opinion in Neurobiology. 39, 122-132 (2016).</li><li>Anderson, C. T., Stearns, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Centriole+age+underlies+asynchronous+primary+cilium+growth+in+mammalian+cells.">Centriole age underlies asynchronous primary cilium growth in mammalian cells.</a> Current Biology: CB. 19 (17), 1498-1502 (2009).</li><li>Paridaen, J. T. M. L., Wilsch-Br&#228;uninger, M., Huttner, W. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Asymmetric+inheritance+of+centrosome-associated+primary+cilium+membrane+directs+ciliogenesis+after+cell+division.">Asymmetric inheritance of centrosome-associated primary cilium membrane directs ciliogenesis after cell division.</a> Cell. 155 (2), 333-344 (2013).</li><li>Gabriel, E., 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=CPAP+promotes+timely+cilium+disassembly+to+maintain+neural+progenitor+pool.">CPAP promotes timely cilium disassembly to maintain neural progenitor pool.</a> The EMBO Journal. 35 (8), 803-819 (2016).</li><li>Higginbotham, H., 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=Arl13b-regulated+cilia+activities+are+essential+for+polarized+radial+glial+scaffold+formation.">Arl13b-regulated cilia activities are essential for polarized radial glial scaffold formation.</a> Nature Neuroscience. 16 (8), 1000-1007 (2013).</li><li>Baudoin, J. -. P., 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=Tangentially+migrating+neurons+assemble+a+primary+cilium+that+promotes+their+reorientation+to+the+cortical+plate.">Tangentially migrating neurons assemble a primary cilium that promotes their reorientation to the cortical plate.</a> Neuron. 76 (6), 1108-1122 (2012).</li><li>Higginbotham, H., 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=Arl13b+in+primary+cilia+regulates+the+migration+and+placement+of+interneurons+in+the+developing+cerebral+cortex.">Arl13b in primary cilia regulates the migration and placement of interneurons in the developing cerebral cortex.</a> Developmental Cell. 23 (5), 925-938 (2012).</li><li>Kumamoto, N., 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+role+for+primary+cilia+in+glutamatergic+synaptic+integration+of+adult-born+neurons.">A role for primary cilia in glutamatergic synaptic integration of adult-born neurons.</a> Nature Neuroscience. 15 (3), 399-405 (2012).</li><li>Guadiana, S. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Arborization+of+dendrites+by+developing+neocortical+neurons+is+dependent+on+primary+cilia+and+type+3+adenylyl+cyclase.">Arborization of dendrites by developing neocortical neurons is dependent on primary cilia and type 3 adenylyl cyclase.</a> The Journal of Neuroscience: The Official Journal of the Society for Neuroscience. 33 (6), 2626-2638 (2013).</li><li>Thomas, S., Boutaud, L., Reilly, M. L., Benmerah, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cilia+in+hereditary+cerebral+anomalies.">Cilia in hereditary cerebral anomalies.</a> Biology of the Cell. 111 (9), 217-231 (2019).</li><li>Hasenpusch-Theil, K., Theil, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+multifaceted+roles+of+primary+cilia+in+the+development+of+the+cerebral+cortex.">The multifaceted roles of primary cilia in the development of the cerebral cortex.</a> Frontiers in Cell and Developmental Biology. 9, 630161 (2021).</li><li>Shi, Y., Kirwan, P., Livesey, F. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Directed+differentiation+of+human+pluripotent+stem+cells+to+cerebral+cortex+neurons+and+neural+networks.">Directed differentiation of human pluripotent stem cells to cerebral cortex neurons and neural networks.</a> Nature Protocols. 7 (10), 1836-1846 (2012).</li><li>Boissart, C., 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+from+human+pluripotent+stem+cells+of+cortical+neurons+of+the+superficial+layers+amenable+to+psychiatric+disease+modeling+and+high-throughput+drug+screening.">Differentiation from human pluripotent stem cells of cortical neurons of the superficial layers amenable to psychiatric disease modeling and high-throughput drug screening.</a> Translational Psychiatry. 3, 294 (2013).</li><li>Chambers, S. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Highly+efficient+neural+conversion+of+human+ES+and+iPS+cells+by+dual+inhibition+of+SMAD+signaling.">Highly efficient neural conversion of human ES and iPS cells by dual inhibition of SMAD signaling.</a> Nature Biotechnology. 27 (3), 275-280 (2009).</li><li>Lancaster, M. A., Knoblich, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+cerebral+organoids+from+human+pluripotent+stem+cells.">Generation of cerebral organoids from human pluripotent stem cells.</a> Nature Protocols. 9 (10), 2329-2340 (2014).</li><li>Qian, 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=Generation+of+human+brain+region-specific+organoids+using+a+miniaturized+spinning+bioreactor.">Generation of human brain region-specific organoids using a miniaturized spinning bioreactor.</a> Nature Protocols. 13 (3), 565-580 (2018).</li><li>Krefft, O., Jabali, A., Iefremova, V., Koch, P., Ladewig, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+standardized+and+reproducible+forebrain-type+cerebral+organoids+from+human+induced+pluripotent+stem+cells.">Generation of standardized and reproducible forebrain-type cerebral organoids from human induced pluripotent stem cells.</a> Journal of Visualized Experiments: JoVE. (131), (2018).</li><li>Kadoshima, 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=Self-organization+of+axial+polarity,+inside-out+layer+pattern,+and+species-specific+progenitor+dynamics+in+human+ES+cell-derived+neocortex.">Self-organization of axial polarity, inside-out layer pattern, and species-specific progenitor dynamics in human ES cell-derived neocortex.</a> Proceedings of the National Academy of Sciences of the United States of America. 110 (50), 20284-20289 (2013).</li><li>Topol, A., Tran, N. N., Brennand, K. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+guide+to+generating+and+using+hiPSC+derived+NPCs+for+the+study+of+neurological+diseases.">A guide to generating and using hiPSC derived NPCs for the study of neurological diseases.</a> Journal of Visualized Experiments: JoVE. (96), e52495 (2015).</li><li>Berg, 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=ilastik:+interactive+machine+learning+for+(bio)image+analysis.">ilastik: interactive machine learning for (bio)image analysis.</a> Nature Methods. 16 (12), 1226-1232 (2019).</li><li>Hansen, J. N., 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=Multifocal+imaging+for+precise,+label-free+tracking+of+fast+biological+processes+in+3D.">Multifocal imaging for precise, label-free tracking of fast biological processes in 3D.</a> bioRxiv. , (2020).</li><li>Pa&#537;ca, S. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+rise+of+three-dimensional+human+brain+cultures.">The rise of three-dimensional human brain cultures.</a> Nature. 553 (7689), 437-445 (2018).</li><li>Andreu-Cervera, A., Catala, M., Schneider-Maunoury, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cilia,+ciliopathies+and+hedgehog-related+forebrain+developmental+disorders.">Cilia, ciliopathies and hedgehog-related forebrain developmental disorders.</a> Neurobiology of Disease. 150, 105236 (2021).</li><li>Christensen, S. T., Morthorst, S. K., Mogensen, J. B., Pedersen, L. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Primary+cilia+and+coordination+of+Receptor+Tyrosine+Kinase+(RTK)+and+Transforming+Growth+Factor+&#946;+(TGF-&#946;)+signaling.">Primary cilia and coordination of Receptor Tyrosine Kinase (RTK) and Transforming Growth Factor &#946; (TGF-&#946;) signaling.</a> Cold Spring Harbor Perspectives in Biology. 9 (6), (2017).</li><li>Wheway, G., Nazlamova, L., Hancock, J. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Signaling+through+the+primary+cilium.">Signaling through the primary cilium.</a> Frontiers in Cell and Developmental Biology. 6, 8 (2018).</li><li>Sivitilli, A. 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=Robust+production+of+uniform+human+cerebral+organoids+from+pluripotent+stem+cells.">Robust production of uniform human cerebral organoids from pluripotent stem cells.</a> Life Science Alliance. 3 (5), (2020).</li><li>Quelennec, E., 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=Generation+of+two+induced+pluripotent+stem+cell+lines+IMAGINi004-A+and+IMAGINi005-A+from+healthy+donors.">Generation of two induced pluripotent stem cell lines IMAGINi004-A and IMAGINi005-A from healthy donors.</a> Stem Cell Research. 48, 101959 (2020).</li><li>Belle, 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=Tridimensional+visualization+and+analysis+of+early+human+development.">Tridimensional visualization and analysis of early human development.</a> Cell. 169 (1), 161-173 (2017).</li><li>Vigouroux, R. J., Belle, M., Ch&#233;dotal, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuroscience+in+the+third+dimension:+shedding+new+light+on+the+brain+with+tissue+clearing.">Neuroscience in the third dimension: shedding new light on the brain with tissue clearing.</a> Molecular Brain. 10 (1), 33 (2017).</li><li>Lallemant, L., Lebreton, C., Garfa-Traor&#233;, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+different+clearing+and+acquisition+methods+for+3D+imaging+of+murine+intestinal+organoids.">Comparison of different clearing and acquisition methods for 3D imaging of murine intestinal organoids.</a> Journal of Biological Methods. 7 (4), 141 (2020).</li><li>Aoyagi, Y., Kawakami, R., Osanai, H., Hibi, T., Nemoto, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+rapid+optical+clearing+protocol+using+2,2'-thiodiethanol+for+microscopic+observation+of+fixed+mouse+brain.">A rapid optical clearing protocol using 2,2'-thiodiethanol for microscopic observation of fixed mouse brain.</a> PloS One. 10 (1), 0116280 (2015).</li></ol>

The authors declare no conflicts of interest.

PC are now regarded as key organelles regulating crucial steps during normal cerebral cortical development18,19,31 including NSPC expansion and commitment8,9,10,11,12 as well as neuronal migration13,14 and syn...

Primary cilia (PC)&#160;are microtubule-based organelles that sense and transduce a plethora of chemical and mechanical cues from the extracellular environment. In particular, PC is the central organelle for the transduction of the Hedgehog signaling pathway in vertebrates1,2. While most neural cells have long been shown harboring a PC, the contribution of this organelle in shaping the central nervous system has long been undervalued.&#160;Studies on neocortical development have led to the discovery of multiple neural stem and progenitor cells (NSPCs), all harboring a PC, the location of which has been proposed to be crucial for progenitor fate determination3,4,5,6,7. PC has been shown crucial for cell mechanisms that are required for normal cerebral cortical development, including NSPC expansion and commitment8,9,10,11,12 as well as apicobasal polarity of radial glial scaffold supporting neuronal migration13. In addition, PC are required during interneurons tangential migration to the cortical plate14,15. Finally, a role for the PC has been proposed in the establishment of synaptic connections of neurons in the cerebral cortex16,17. Altogether, these findings argue for a crucial role of PC at major steps of cerebral cortical development18,19 and raise the need to investigate their involvement in the pathological mechanisms underlying anomalies of cerebral cortical development.
Recent studies have largely improved our understanding of important cellular and molecular differences between cortical development in human and animal models, emphasizing the need to develop human model systems.&#160;In this view, human induced pluripotent stem cells (hIPSCs) represent a promising approach to study disease pathogenesis in a relevant genetic and cellular context. Adherent two-dimensional (2D) cell-based models or neural rosettes contain NSPCs similar to those seen in the developing cerebral cortex, which become organized into rosette-shaped structures showing correct apicobasal polarity20,21,22. Furthermore, the three-dimensional (3D) culture system allows the generation of dorsal forebrain organoids that recapitulate many features of human cerebral cortical development23,24,25,26. Those two complementary cell-based modeling approaches offer exciting perspectives to dissect the involvement of PC during normal and pathological development of the cerebral cortex.
Here, we provide detailed protocols for the generation and characterization of neural rosettes and derived NSPCs as well as dorsal forebrain organoids. We also provide detailed protocols to analyze the biogenesis and function of PC present on NSPCs by testing the transduction of the Sonic Hedgehog pathway and analyzing the dynamics of crucial molecules involved in this pathway. To take advantage of the 3D organization of the cerebral organoids, we also set up a simple and cost-effective method for 3D imaging of in toto immunostained cerebral organoids allowing rapid acquisition, thanks to a light sheet microscope, of the entire organoid, with high resolution enabling to visualize PC on all types of neocortical progenitors and neurons of the whole organoid. Finally, we adapted immunohistochemistry on 150 &#181;m free-floating sections with subsequent clearing and acquisition using resonant scanning confocal microscope allowing high-resolution image acquisition, which is required for the detailed analysis of PC biogenesis and function. Specifically, 3D-imaging software allows 3D-reconstruction of PC with subsequent analysis of morphological parameters including length, number, and orientation of PC as well as signal intensity measurement of ciliary components along the axoneme.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>2-Mercapto&#233;thanol (50 mM)</td>
			<td>ThermoFisher Scientific</td>
			<td>31350010</td>
			<td></td>
		</tr>
		<tr>
			<td>6-well Clear Flat Bottom Ultra-Low Attachment Multiple Well Plates</td>
			<td>Corning</td>
			<td>3471</td>
			<td></td>
		</tr>
		<tr>
			<td>96-well Clear Round Bottom Ultra-Low Attachment Microplate</td>
			<td>Corning</td>
			<td>7007</td>
			<td></td>
		</tr>
		<tr>
			<td>B-27 Supplement (50X), minus vitamin A</td>
			<td>ThermoFisher Scientific</td>
			<td>12587010</td>
			<td></td>
		</tr>
		<tr>
			<td>B-27 Supplement (50X), serum free</td>
			<td>ThermoFisher Scientific</td>
			<td>17504044</td>
			<td></td>
		</tr>
		<tr>
			<td>CellAdhere Dilution Buffer</td>
			<td>StemCell Technologies</td>
			<td>7183</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM/F-12, Glutamax</td>
			<td>ThermoFisher Scientific</td>
			<td>31331028</td>
			<td></td>
		</tr>
		<tr>
			<td>DMSO</td>
			<td>ATCC</td>
			<td>4-X</td>
			<td></td>
		</tr>
		<tr>
			<td>Dorsomorphin</td>
			<td>StemCell Technologies</td>
			<td>72102</td>
			<td></td>
		</tr>
		<tr>
			<td>Easy Grip 35 10mm</td>
			<td>Falcon</td>
			<td>353001</td>
			<td></td>
		</tr>
		<tr>
			<td>EDTA</td>
			<td>ThermoFisher Scientific</td>
			<td>15575020</td>
			<td></td>
		</tr>
		<tr>
			<td>EGF , 25&#181;g</td>
			<td>Thermofischer</td>
			<td>PHG0315</td>
			<td></td>
		</tr>
		<tr>
			<td>FGF2 , 25&#181;g</td>
			<td>Thermofischer</td>
			<td>PHG0264</td>
			<td></td>
		</tr>
		<tr>
			<td>Gentle Cell Dissociation Reagent</td>
			<td>StemCell Technologies</td>
			<td>7174</td>
			<td></td>
		</tr>
		<tr>
			<td>Insulin</td>
			<td>ThermoFisher Scientific</td>
			<td>12585014</td>
			<td></td>
		</tr>
		<tr>
			<td>KnockOut Serum</td>
			<td>ThermoFisher Scientific</td>
			<td>10828028</td>
			<td></td>
		</tr>
		<tr>
			<td>Laminin (1mg)</td>
			<td>Thermofischer</td>
			<td>23017015</td>
			<td></td>
		</tr>
		<tr>
			<td>LDN193189</td>
			<td>StemCell Technologies</td>
			<td>72147</td>
			<td></td>
		</tr>
		<tr>
			<td>Matrigel Growth Factor Reduced</td>
			<td>Corning</td>
			<td>354230</td>
			<td></td>
		</tr>
		<tr>
			<td>MEM Non-Essential Amino Acids Solution (100X)</td>
			<td>ThermoFisher Scientific</td>
			<td>11140050</td>
			<td></td>
		</tr>
		<tr>
			<td>Mowiol 4-88</td>
			<td>Sigma Aldrich</td>
			<td>81381-250G</td>
			<td></td>
		</tr>
		<tr>
			<td>mTeSR1</td>
			<td>StemCell Technologies</td>
			<td>85850</td>
			<td></td>
		</tr>
		<tr>
			<td>Neural Basal Medium</td>
			<td>Thermofischer</td>
			<td>21103049</td>
			<td></td>
		</tr>
		<tr>
			<td>Orbital shaker</td>
			<td>Dutscher</td>
			<td>995002</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>ThermoFisher Scientific</td>
			<td>14190094</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin (10,000 U/mL)</td>
			<td>ThermoFisher Scientific</td>
			<td>15140122</td>
			<td></td>
		</tr>
		<tr>
			<td>PFA 32%</td>
			<td>Electron Microscopy Sciences</td>
			<td>15714</td>
			<td></td>
		</tr>
		<tr>
			<td>Poly-L-Ornithine (PO)</td>
			<td>Sigma</td>
			<td>P4957</td>
			<td></td>
		</tr>
		<tr>
			<td>Recombinant human BDNF 10 &#181;g</td>
			<td>Stem Cell Technologies</td>
			<td>78005</td>
			<td></td>
		</tr>
		<tr>
			<td>Recombinant Human FGF-basic</td>
			<td>Peprotech</td>
			<td>100-18B</td>
			<td></td>
		</tr>
		<tr>
			<td>rSHH</td>
			<td>R&#38;D Systems</td>
			<td>8908-SH</td>
			<td></td>
		</tr>
		<tr>
			<td>SAG</td>
			<td>Santa Cruz</td>
			<td>Sc-202814</td>
			<td></td>
		</tr>
		<tr>
			<td>SB431542</td>
			<td>StemCell Technologies</td>
			<td>72232</td>
			<td></td>
		</tr>
		<tr>
			<td>Stembeads FGF2</td>
			<td>StemCulture</td>
			<td>SB500</td>
			<td></td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>Sigma Aldrich</td>
			<td>S7903-250G</td>
			<td></td>
		</tr>
		<tr>
			<td>Superfrost Plus Adhesion Slides</td>
			<td>Thermo Scientific</td>
			<td>J1800AMNZ</td>
			<td></td>
		</tr>
		<tr>
			<td>Suppl&#233;ment N2- (100X)</td>
			<td>ThermoFisher Scientific</td>
			<td>17502048</td>
			<td></td>
		</tr>
		<tr>
			<td>TDE 2,2&#8217;-Thiodiethanol</td>
			<td>Sigma Aldrich</td>
			<td>166782-500G</td>
			<td></td>
		</tr>
		<tr>
			<td>Vitronectin</td>
			<td>StemCell Technologies</td>
			<td>7180</td>
			<td></td>
		</tr>
		<tr>
			<td>Y-27632</td>
			<td>StemCell Technologies</td>
			<td>72304</td>
			<td></td>
		</tr>
		<tr>
			<td>Primary Antibodies</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>ARL13B</td>
			<td>Abcam</td>
			<td>Ab136648</td>
			<td>1/200e</td>
		</tr>
		<tr>
			<td>ARL13B</td>
			<td>Proteintech</td>
			<td>17711-1-AP</td>
			<td>1/500e</td>
		</tr>
		<tr>
			<td>CTIP2</td>
			<td>Abcam</td>
			<td>Ab18465</td>
			<td>1/500e</td>
		</tr>
		<tr>
			<td>GLI2</td>
			<td>R&#38;D Systems</td>
			<td>AF3526</td>
			<td>1/100</td>
		</tr>
		<tr>
			<td>GPR161</td>
			<td>Proteintech</td>
			<td>13398-1-AP</td>
			<td>1/100</td>
		</tr>
		<tr>
			<td>N-Cadherin</td>
			<td>BD Transduction Lab</td>
			<td>610921</td>
			<td>1/500e</td>
		</tr>
		<tr>
			<td>P-Vimentin</td>
			<td>MBL</td>
			<td>D076-3</td>
			<td>1/500e</td>
		</tr>
		<tr>
			<td>PAX6</td>
			<td>Biolegend</td>
			<td>PRB-278P</td>
			<td>1/200e</td>
		</tr>
		<tr>
			<td>PCNT</td>
			<td>Abcam</td>
			<td>Ab4448</td>
			<td>1/1000e</td>
		</tr>
		<tr>
			<td>S0X2</td>
			<td>R&#38;D Systems</td>
			<td>MAB2018</td>
			<td>1/200e</td>
		</tr>
		<tr>
			<td>SATB2</td>
			<td>Abcam</td>
			<td>Ab51502</td>
			<td>1/200e</td>
		</tr>
		<tr>
			<td>TBR2</td>
			<td>Abcam</td>
			<td>Ab216870</td>
			<td>1/400e</td>
		</tr>
		<tr>
			<td>TPX2</td>
			<td>NovusBio</td>
			<td>NB500-179</td>
			<td>1/500e</td>
		</tr>
		<tr>
			<td>&#947;TUBULIN</td>
			<td>Sigma Aldrich</td>
			<td>T6557</td>
			<td>1/500e</td>
		</tr>
		<tr>
			<td>Secondary Antibodies</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Donkey anti-rabbit AF488</td>
			<td>ThermoFisher Scientific</td>
			<td>A21206</td>
			<td>1/500e</td>
		</tr>
		<tr>
			<td>Goat anti-mouse AF555</td>
			<td>ThermoFisher Scientific</td>
			<td>A21422</td>
			<td>1/500e</td>
		</tr>
		<tr>
			<td>Goat anti-mouse AF647</td>
			<td>ThermoFisher Scientific</td>
			<td>A21236</td>
			<td>1/500e</td>
		</tr>
		<tr>
			<td>Goat anti-rat AF555</td>
			<td>ThermoFisher Scientific</td>
			<td>A21434</td>
			<td>1/500e</td>
		</tr>
	</tbody>
</table>

2d and 3d human induced pluripotent stem cell-based models to dissect primary cilium involvement during neocortical development

Primary cilia (PC) are non-motile dynamic microtubule-based organelles that protrude from the surface of most mammalian cells. They emerge from the older centriole during the G1/G0 phase of the cell cycle, while they disassemble as the cells re-enter the cell cycle at the G2/M phase boundary. They function as signal hubs, by detecting and transducing extracellular signals crucial for many cell processes. Similar to most cell types, all neocortical neural stem and progenitor cells (NSPCs) have been shown harboring a PC allowing them to sense and transduce specific signals required for the normal cerebral cortical development. Here, we provide detailed protocols to generate and characterize two-dimensional (2D) and three-dimensional (3D) cell-based models from human induced pluripotent stem cells (hIPSCs) to further dissect the involvement of PC during neocortical development. In particular, we present protocols to study the PC biogenesis and function in 2D neural rosette-derived NSPCs including the transduction of the Sonic Hedgehog (SHH) pathway. To take advantage of the three-dimensional (3D) organization of cerebral organoids, we describe a simple method for 3D imaging of in toto immunostained cerebral organoids. After optical clearing, rapid acquisition of entire organoids allows detection of both centrosomes and PC on neocortical progenitors and neurons of the whole organoid. Finally, we detail the procedure for immunostaining and clearing of thick free-floating organoid sections preserving a significant degree of 3D spatial information and allowing for the high-resolution acquisition required for the detailed qualitative and quantitative analysis of PC biogenesis and function.

2D hIPS cell-based models to study primary cilium biogenesis and function 
The protocol detailed here has been adapted from previously published studies20,21,22. This protocol allows the generation of neural rosette structures that contain neocortical progenitors and neurons similar to those seen in the developing neocortex. Detailed validation can be performed by conventional immunostaining analysis using ...

Watch this Scientific Journal Video about 2D and 3D Human Induced Pluripotent Stem Cell-Based Models to Dissect Primary Cilium Involvement during Neocortical Development at JoVE.com

2D and 3D Human Induced Pluripotent Stem Cell-Based Models to Dissect Primary Cilium Involvement during Neocortical Development

We present detailed protocols for the generation and characterization of 2D and 3D human induced pluripotent stem cell (hIPSC)-based models of neocortical development as well as complementary methodologies enabling qualitative and quantitative analysis of primary cilium (PC) biogenesis and function.

Primary cilia (PC) are non-motile dynamic microtubule-based organelles that protrude from the surface of most mammalian cells. They emerge from the older ...

The overall goal of the procedure we detail here is to dissect involvement of primary cilium during neocortical development. Primary cilia have been shown crucial for many steps of neocortical development, including progenitor cell expansion, fate determination, as well as neuronal migration and maturation. To further dissect cell involvement during neocortical development, we set up relevant tools by taking advantage of emerging 2D and 3D iPS cell-based models of neocortical development.In other words, neural rosettes and dorsal forebrain organoids. 3D iPS cell-based models begin with embryoid body formation. Adhesion of said embryoid bodies, allow generation of 2D neural rosette structures, while free-floating culture of such EBs allow generation of dorsal forebrain organoids.The protocol we set up to generate cerebral organoids is based on previous published protocols using the gagging method by adding pre-patterning factors to specifically generate dorsal forebrain organoids by using inhibitors of the activin nodal and BMP pathway, also known as dual SMAD inhibition. To take advantage of the 3D organization of the organoids, we set up a simple and fast method, allowing in total immunolabeling, clearing, and lighted acquisition of the entire organoid with high resolution. And to focus on primary cilium biogenesis and functions, we adapted immunohistochemistry on free-floating sections cut with a vibratome.After immunostaining and clearing of the sections, we acquired the images using resonant scanning confocal microscopy. Start with iPS cell cultures harboring large regular colonies, exhibiting less than 10%differentiation and are no more than 80%compliant. Manually dissect each iPS cell colony using a needle, allowing to catch precisely each colony in horizontal and vertical directions to create a checkerboard pattern, dividing each colony into equal clusters.Detach the colony using a cell scrapper and transfer them onto an ultra low attachment culture plate. Let them float overnight in the incubator so that they can form embryoid bodies. The next day, turn the medium, taking care not to aspirate the embryoid bodies, and transfer them into Poly-L-ornithine Laminine-coated dishes until the formation of neural rosettes.That takes approximately 12 to 14 days. Daily refresh induction, medium, and check under microscope for formation of neural rosette structures. From this step, neural rosettes can be expanded, differentiated, and processed for immunostaining analysis or disassociated to get isolated neural stem and progenital cell cultures.First of all, start with iPS cultures harboring large regular colonies exhibiting less than 10%differentiation and which have been passaged almost once on the monolayer. On day zero, allow iPS cell to form embryoid bodies in EB medium containing low concentration of fJ2 and a high concentration of ROCK inhibitor required for hiiPS cell survival to avoid disturbing EB formation incubator plates for three days without medium change. On day three, EBs should measure approximately 400 micrometers and show regular borders.If past criteria rich for most EBs, change the medium with induction medium one containing dual SMAD inhibitors certainly to brightening up the contra DBs by best six to seven, indicating neural tomaly concession. And they turn other DBs into gross-factor reduced basement membrane metrics. From this step always use sterile scissors to get the opening of the peptides to avoid damaging of the organoids.First transfer about 15 organoids to a clinical tube. Let the EB settle and remove the medium at 50 microliters of induction medium two and transfer the organoids to a microsensitive tube containing 100 microliters of matrix and homogenized by pipetting up and down. Spread the mixture onto the center of a well of a neutral attachment plate.And finally, space EBs so that they do not touch each other to avoid them to fuse. Let the matter solidify in the incubator for 45 minutes, add induction medium two to each well and incubate in the incubator. Refresh induction medium two every other day.On day 17, disassociate the basement membrane matrix manually by pipetting up and down 10 times with five ml pipettes. Incubate the organoid plate in the translation medium one under agitation to improve nutritional absorption. Importantly, use a different incubator for stationary culture and culture on orbital shaker to avoid any vibrations detrimental for adherent iPS cell growth.After fixation, permeabilization, blocking and incubation with primary and secondary antibodies takes 10 days, including washing steps. The clearing method we favored relies on TDE, a glycol derivative. For optimal clearing, intubate the organoid in TDE solution with gradually-increased concentration for 24 hours each.For organoid embedding, use custom main molding system made from the one ml syringe, which tip is cut off using a scalpel. Low melting point Agarose is recommended, as its melting temperature is only approximately 60 degrees Celsius. Prepare 4%low melting Agarose in 60%TDE solution and let it to cool just about 37 degrees Celsius in a water base pre-organoid embedding.Put in the syringe 600 microliters of the gel solution using the plunger, then position the sample and fill in the syringe with 400 microliters of the gel solution. Let the gel polymerize, and finally, the sample is mounted ideally within the gel and can be easily pushed out within the lighted chamber to position the organoid in front of the objective. Lighted fluorescence microscopy is ideal for first imaging of such clear organoid.We've reduced photo imaging and weighed sub-cellular resolution. Here, we used a 20x objective immerged in 80%TDE solution added in the sample chamber to allow to accurately adjust to the refractive index of our clearing method. The largest sample order has been designed to accommodate a one-milliliter syringe that can be inserted from the top with the plunger that can be operated once the sample order is mounted to position the organoid in front of the objective.The acquisition of an entire organoid takes approximately 5 to 10 minutes. For immuno staining on free-floating sections, collect and fix your organoids in four-person part overnight at four degrees Celsius. Embed the organoids into 4%low-melting Agarose and section by using a vibratome to obtain 150 micrometer of six sections transferred in PBS solution, using a paintbrush to avoid damaging them.After permeabilization and blocking, incubate the free-floating sections with primary antibody overnight at four degrees Celsius and under agitation. Wash the sections carefully before incubation with secondary antibody for two hours at room temperature and under agitation. Finally, for optimal clearing, incubate the section in TDE solution with gradually increased concentration for one hour and 30 in 60%TDE and then in 80%TDE overnight at room temperature.Store the section in 80%TDE and TLAC solution at four degrees Celsius. Mount free-floating section in a cell chamber, endeavoring to maintain the sample in 80%TDE solution and designed to adapt on a motorized stage of clinical microscopes. First, put one round cover slip in the chamber and transfer carefully the clear protection using a paint brush.Fill the chamber with 80%TDE solution and then add two standard cover slips plus one second round cover slip and a Silicon seal. Finally, screw on the screwing ring of the chamber to perfectly seal the system. Nightsheet, as well as resonance acquisitions are processed thanks to a software enabling 3D utilization and an analysis of the entire immunostain sample.Such software allows you to rapidly open huge data we generate to make easily snapshots and animations. It allowed to move the sample in different orientation and we can generate 2D views of the image thanks to a 2D slicer tool in different orientation, X, Y and Y, Zed, for example. In addition, such software allows automatic detection of both centrosome and primary cilia, enabling to quantify the number in pathological versus control conditions.It also allows 3D reconstitution of primary cilia for precise measurement of the sites. The protocol we described for 2D iPS cell-based modeling of new cortical development allows generation of neural rosette structures that contain new cortical progenitors and neurons similar to those seen in the developing neocortex. Detailed validation can be performed by conventional immunostaining analysis.Applicable progenitors should be double-stained with us to impact sensitivities while intermediate progenitors are revealed by TBI2 staining and early bone neocortical neurons by CTIP2 positive staining. Such neural rosette-like structures model interkinetic nuclear migration of radical progenitors that can be visualized by immunostaining with antibodies raised against phospho vimentin that stain mototic nuclei and TBX2 that sustains the mitotic spindle. Finally, ciliagenesis can be analyzed by immunostaining with antibodies raised against pericentric, that stains the back body of primary cilia and are of certain means that stains the axiom.On such rosette structure, primary cilia extracted from the apical poly Fabri-Kal progenitors and densely above the central lumen of each ventriculite region while they are also protruding from CTIP2-positive neurons. Dissociation of adenoneural rosettes allows to generate isolated neural stem and progenitor cell cultures that are highly useful to analyze primary cilium biogenesis and function. In particular, we detail the procedure to test the induction of the sonic drug pathway after treatment with recommended sonic drug or osmosin agonists.Subsequent RTPCR SA tests the induction of sonica drug target genes, such as GLI1 and PTCH1, and immunofluorescence analysis tests the dynamics of key sonic drug components along the primary cilium that get be quantified, thanks to open source tools, ilastik and CiliaQ. Different ciliary parameters, including length and orientation, are investigated but also intensity of primary cilium components are shown here for GLI2 and GPR161 that, respectively, accumuulate or exit from the primary cilium in response to recommended sonica drug treatment. The protocol we detail for out 3D iPS cell-based modeling of neocortical development allows generation of homogeneous cerebral organoid with dorsal forebrain identity that can be characterized either by conventional immunostaining analysis or by woman staining, as shown here, with optimal penetration of the antibodies raised, again, N-cadherin, PAX6, and CTIP2 that stain respectively neuronal cadherin, in pink, apical progenitors in the ventricular zone like region, in green, and all newborn neocortical neurons, located in the cortical plate like region.Here is a second movie of the whole immunostain organoid with antibodies raised again, CTIP2, in red, possibly maintain in pink, and TPX2, in green, allowing to test several characteristics of neocortical progenitors, such as, interkinetic nuclear migration of Fabri-Kal progenitors. Interestingly, phospho vimentin positive progenitors are also observed in the supraventricular zone like region and harbor a unique positive process extending to the positive surface, reminiscent of outer radial glial progenitors and illustrating the high resolution of this method. Here is a movie of an immunostain, free-floating section, acquired thanks to a resonance-scanning microscope showing primary cilia detected with pericentrin antibody that's stained the back of the body, in green, and ARLT3B antibodies that stain the axiom, in pink.Primary cilia extend from all neuronal stem and progenitor cells, in particular, at the epical surface of the epical progenitors but also in neocortical neurons of the pre-plate like region. Finally 2D neuron rosettes and isolated stem and progenitor cells recapitulate several aspects of cerebral cortical development and represent complementary, useful tools to test ciliary biogenesis and function. The protocol we detect here allowed us to successfully generate dorsal forebrain organoids from this distinct iPS cell lines that give rise to homogeneous results, ensuring the robustness of this procedure.The protocols we set up for immunostaining, clearing, and imaging of entire organoids or free-floating sections are simple, fast, and cost-effective. Combining such cell-based models and 3D imaging analysis on iPS cells, generated either by reprogramming ciliary perturbation cells or by using CRISPR 9 technology to specifically edit ciliary genes, should allow significant progress in the understanding of the contribution of the primary cilium during normal and pathological development of the cerebral cortex.

2d-3d-human-induced-pluripotent-stem-cell-based-models-to-dissect

Research

JoVE Journal

Developmental Biology

3.7K ビュー.  Université de Paris. ここで詳述する手順の全体的な目標は、新皮質発生中の一次繊毛の関与を解剖することです。原発性繊毛は、前駆細胞の増殖、運命の決定、ならびにニューロンの移動および成熟を含む、新皮質発達の多くのステップにとって重要であることが示されている。新皮質発生中の細胞関与をさらに解剖するために、我々は新皮質発生の新興2Dおよび3D iPS細胞ベースのモデルを...