Name: 2d and 3d human induced pluripotent stem cell-based models to dissect primary cilium involvement during neocortical development
Uploaded: 2022-03-25T12:30:00
Description: 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

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>
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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. 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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. 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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 ...

Research

JoVE Journal

Developmental Biology

Human Pluripotent Stem Cell Based Developmental Toxicity Assays for Chemical Safety Screening and Systems Biology Data Generation

Efficient protocols to differentiate human pluripotent stem cells to various tissues in combination with -omics technologies opened up new horizons for in ...

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

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

Cultivate Primary Nasal Epithelial Cells from Children and Reprogram into Induced Pluripotent Stem Cells

Nasal epithelial cells (NECs) are the part of the airways that respond to air pollutants and are the first cells infected with respiratory viruses. They ...

Differentiating Chondrocytes from Peripheral Blood-derived Human Induced Pluripotent Stem Cells

In this study, we used peripheral blood cells (PBCs) as seed cells to produce chondrocytes via induced pluripotent stem cells (iPSCs) in an ...

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

We describe a robust method to direct the differentiation of pluripotent stem cells into retinal pigment epithelial cells (RPE). The purpose of providing ...

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

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

Microfluidic Assay for the Assessment of Leukocyte Adhesion to Human Induced Pluripotent Stem Cell-derived Endothelial Cells (hiPSC-ECs)

Microfluidic Assay for the Assessment of Leukocyte Adhesion to Human Induced Pluripotent Stem Cell-derived Endothelial Cells hiPSC-ECs

Endothelial cells (ECs) are essential for the regulation of inflammatory responses by either limiting or facilitating leukocyte recruitment into affected ...

RNA-based Reprogramming of Human Primary Fibroblasts into Induced Pluripotent Stem Cells

Induced pluripotent stem cells (iPSCs) have proven to be a valuable tool to study human development and disease. Further advancing iPSCs as a regenerative ...

Generation of 3D Skin Organoid from Cord Blood-derived Induced Pluripotent Stem Cells

The skin is the body&#8217;s largest organ and has many functions. The skin acts as a physical barrier and protector of the body and regulates bodily ...

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

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