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 ...

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

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 vitro toxicity testing of potential drugs. To provide a solid scientific basis for such assays, it will be important to gain quantitative information on the time course of development and on the underlying regulatory mechanisms by systems biology approaches. Two assays have therefore been tuned here for these requirements. In the UKK test system, human embryonic stem cells (hESC) (or other pluripotent cells) are left to spontaneously differentiate for 14 days in embryoid bodies, to allow generation of cells of all three germ layers. This system recapitulates key steps of early human embryonic development, and it can predict human-specific early embryonic toxicity/teratogenicity, if cells are exposed to chemicals during differentiation. The UKN1 test system is based on hESC differentiating to a population of neuroectodermal progenitor (NEP) cells for 6 days. This system recapitulates early neural development and predicts early developmental neurotoxicity and epigenetic changes triggered by chemicals. Both systems, in combination with transcriptome microarray studies, are suitable for identifying toxicity biomarkers. Moreover, they may be used in combination to generate input data for systems biology analysis. These test systems have advantages over the traditional toxicological studies requiring large amounts of animals. The test systems may contribute to a reduction of the costs for drug development and chemical safety evaluation. Their combination sheds light especially on compounds that may influence neurodevelopment specifically.

The protocols describe two in vitro developmental toxicity test systems (UKK and UKN1) based on human embryonic stem cells and transcriptome studies. The test systems predict human developmental toxicity hazard, and may contribute to reduce animal studies, costs and the time required for chemical safety testing.

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 distributed along the dendrites. Their morphology is largely dependent on neuronal activity, and they are dynamic. Dendritic spines express glutamatergic receptors (AMPA and NMDA receptors) on their surface and at the levels of postsynaptic densities. Each spine allows the neuron to control its state and local activity independently. Spine morphologies have been extensively studied in glutamatergic pyramidal cells of the brain cortex, using both in vivo approaches and neuronal cultures obtained from rodent tissues. Neuropathological conditions can be associated to altered spine induction and maturation, as shown in rodent cultured neurons and one-dimensional quantitative analysis 1. The present study describes a protocol for the 3D quantitative analysis of spine morphologies using human cortical neurons derived from neural stem cells (late cortical progenitors). These cells were initially obtained from induced&#160;pluripotent stem cells. This protocol allows the analysis of spine morphologies at different culture periods, and with possible comparison between induced&#160;pluripotent stem cells obtained from control individuals with those obtained from patients with psychiatric diseases.

Dendritic spines of pyramidal neurons are the sites of most excitatory synapses in mammalian brain cortex. This method describes a 3D quantitative analysis of spine morphologies in human cortical pyramidal glutamatergic neurons derived from induced&#160;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 are also involved in many airway diseases through their innate immune response and interaction with immune and airway stromal cells. NECs are of particular interest for studies in children due to their accessibility during clinical visits. Human induced pluripotent stem cells (iPSCs) have been generated from multiple cell types and are a powerful tool for modeling human development and disease, as well as for their potential applications in regenerative medicine. This is the first protocol to lay out methods for successful generation of iPSCs from NECs derived from pediatric participants for research purposes. It describes how to obtain nasal epithelial cells from children, how to generate primary NEC cultures from these samples, and how to reprogram primary NECs into well-characterized iPSCs. Nasal mucosa samples are useful in epidemiological studies related to the effects of air pollution in children, and provide an important tool for studying airway disease. Primary nasal cells and iPSCs derived from them can be a tool for providing unlimited material for patient-specific research in diverse areas of airway epithelial biology, including asthma and COPD research.

This publication demonstrates methods for successful sampling and culture of nasal epithelial mucosa from children, and reprogramming these cells to induced Pluripotent Stem Cells (iPSCs).

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 integration-free method. Following embryoid body (EB) formation and fibroblastic cell expansion, the iPSCs are induced for chondrogenic differentiation for 21 days under serum-free and xeno-free conditions. After chondrocyte induction, the phenotypes of the cells are evaluated by morphological, immunohistochemical, and biochemical analyses, as well as by the quantitative real-time PCR examination of chondrogenic differentiation markers. The chondrogenic pellets show positive alcian blue and toluidine blue staining. The immunohistochemistry of collagen II and X staining is also positive. The sulfated glycosaminoglycan (sGAG) content and the chondrogenic differentiation markers COLLAGEN 2 (COL2), COLLAGEN 10 (COL10), SOX9, and AGGRECAN are significantly upregulated in chondrogenic pellets compared to hiPSCs and fibroblastic cells. These results suggest that PBCs can be used as seed cells to generate iPSCs for cartilage repair, which is patient-specific and cost-effective.

We present a protocol to generate a chondrogenic lineage from human peripheral blood (PB) via induced pluripotent stem cells (iPSCs) using an integration-free method, which includes embryoid body (EB) formation, fibroblastic cells expansion, and chondrogenic induction.

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 a detailed and thorough protocol is to clearly demonstrate each step and to make this readily available to researchers in the field. This protocol results in a homogenous layer of RPE with minimal or no manual dissection needed. The method presented here has been shown to be effective for induced pluripotent stem cells (iPSC) and human embryonic stem cells. Additionally, we describe methods for cryopreservation of intermediate cell banks that allow long-term storage. RPE generated using this protocol might be useful for iPSC disease-in-a-dish modeling or clinical application.

This protocol describes how to produce retinal pigment epithelial cells (RPE) from pluripotent stem cells. The method uses a combination of growth factors and small molecules to direct the differentiation of stem cells into immature RPE in fourteen days and mature, functional RPE after three months.

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

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 tissues via a well-characterized cascade of pro-adhesive receptors which are upregulated on the leukocyte cell surface upon the inflammatory trigger. Inflammatory responses differ between individuals in the population and the genetic background can contribute to these differences. Human induced pluripotent stem cells (hiPSCs) have been shown to be a reliable source of ECs (hiPSC-ECs), thus representing an unlimited source of cells that capture the genetic identity and any genetic variants or mutations of the donor. hiPSC-ECs can therefore be used for modeling inflammatory responses in donor-specific cells. Inflammatory responses can be modeled by determining leukocyte adhesion to the hiPSC-ECs under physiological flow. This step-by-step protocol provides a detailed description of the experimental setup and data analysis for the assessment of inflammatory responses in hiPSC-ECs and the analysis of leukocyte adhesion under physiological flow.

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

This step-by-step protocol provides a detailed description of the experimental setup and data analysis for the assessment of inflammatory responses in hiPSC-ECs and the analysis of leukocyte adhesion under physiological flow.

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 therapeutic requires a safe, robust, and expedient reprogramming protocol. Here, we present a clinically relevant, step-by-step protocol for the extremely high-efficiency reprogramming of human dermal fibroblasts into iPSCs using a non-integrating approach. The core of the protocol consists of expressing pluripotency factors (SOX2, KLF4, cMYC, LIN28A, NANOG, OCT4-MyoD fusion) from in vitro transcribed messenger RNAs synthesized with modified nucleotides (modified mRNAs). The reprogramming modified mRNAs are transfected into primary fibroblasts every 48 h together with mature embryonic stem cell-specific microRNA-367/302 mimics for two weeks. The resulting iPSC colonies can then be isolated and directly expanded in feeder-free conditions. To maximize efficiency and consistency of our reprogramming protocol across fibroblast samples, we have optimized various parameters including the RNA transfection regimen, timing of transfections, culture conditions, and seeding densities. Importantly, our method generates high-quality iPSCs from most fibroblast sources, including difficult-to-reprogram diseased, aged, and/or senescent samples.

Here we describe a clinically relevant, high-efficiency, feeder-free method to reprogram human primary fibroblasts into induced pluripotent stem cells using modified mRNAs encoding reprogramming factors and mature microRNA-367/302 mimics. Also included are methods to assess reprogramming efficiency, expand clonal iPSC colonies, and confirm expression of the pluripotency marker TRA-1-60.

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 functions. Biomimetics is the imitation of the models, systems, and elements of nature for the purpose of solving complex human problems1. Skin biomimetics is a useful tool for in vitro disease research and in vivo regenerative medicine. Human induced pluripotent stem cells (iPSCs) have the characteristic of unlimited proliferation and the ability of differentiation to three germ layers. Human iPSCs are generated from various primary cells, such as blood cells, keratinocytes, fibroblasts, and more. Among them, cord blood mononuclear cells (CBMCs) have emerged as an alternative cell source from the perspective of allogeneic regenerative medicine. CBMCs are useful in regenerative medicine because human leukocyte antigen (HLA) typing is essential to the cell banking system. We provide a method for the differentiation of CBMC-iPSCs into keratinocytes and fibroblasts and for generation of a 3D skin organoid. CBMC-iPSC-derived keratinocytes and fibroblasts have characteristics similar to a primary cell line. The 3D skin organoids are generated by overlaying an epidermal layer onto a dermal layer. By transplanting this 3D skin organoid, a humanized mice model is generated. This study shows that a 3D human iPSC-derived skin organoid may be a novel, alternative tool for dermatologic research in vitro and in vivo.

We propose a protocol that shows how to differentiate induced pluripotent stem cell-derived keratinocytes and fibroblasts and generate a 3D skin organoid, using these keratinocytes and fibroblasts. This protocol contains an additional step of generating a humanized mice model. The technique presented here will improve dermatologic research.

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 rise to multiple cell types, including the myotome (MYO), sclerotome (SCL), dermatome (D), and syndetome (SYN), which in turn develop into skeletal muscle, axial skeleton, dorsal dermis, and axial tendon/ligament, respectively. Therefore, the generation of SMs and their derivatives from human induced pluripotent stem cells (iPSCs) is critical to obtain pluripotent stem cells (PSCs) for application in regenerative medicine and for disease research in the field of orthopedic surgery. Although the induction protocols for MYO and SCL from PSCs have been previously reported by several researchers, no study has yet demonstrated the induction of SYN and D from iPSCs. Therefore, efficient induction of fully competent SMs remains a major challenge. Here, we recapitulate human SM patterning with human iPSCs in vitro by mimicking the signaling environment during chick/mouse SM development, and report on methods of systematic induction of SM derivatives (MYO, SCL, D, and SYN) from human iPSCs under chemically defined conditions through the presomitic mesoderm (PSM) and SM states. Knowledge regarding chick/mouse&#160;SM development was successfully applied to the induction of SMs with human iPSCs. This method could be a novel tool for studying human somitogenesis and patterning without the use of embryos and for cell-based therapy and disease modeling.

We present here a protocol for the differentiation of human induced pluripotent stem cells into each somite derivative (myotome, sclerotome, dermatome, and syndetome) in chemically defined conditions, which has applications in future disease modeling and cell-based therapies in orthopedic surgery.

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

Preparation of Small RNA Libraries for Sequencing from Early Mouse Embryos

MicroRNAs (miRNAs) are important for the complex regulation of cell fate decisions and developmental timing. In vivo studies of the contribution of miRNAs during early development are technically challenging due to the limiting cell number. Moreover, many approaches require a miRNA of interest to be defined in assays such as northern blotting, microarray, and qPCR. Therefore, the expression of many miRNAs and their isoforms have not been studied during early development. Here, we demonstrate a protocol for small RNA sequencing of sorted cells from early mouse embryos to enable relatively unbiased profiling of miRNAs in early populations of neural crest cells. We overcome the challenges of low cell input and size selection during library preparation using amplification and gel-based purification. We identify embryonic age as a variable accounting for variation between replicates and stage-matched mouse embryos must be used to accurately profile miRNAs in biological replicates. Our results suggest that this method can be broadly applied to profile the expression of miRNAs from other lineages of cells. In summary, this protocol can be used to study how miRNAs regulate developmental programs in different cell lineages of the early mouse embryo.

We describe a technique for profiling microRNAs in early mouse embryos. This protocol overcomes the challenge of low cell input and small RNA enrichment. This assay can be used to analyze changes in miRNA expression over time in different cell lineages of the early mouse embryo.

MicroRNAs (miRNAs) are important for the complex regulation of cell fate decisions and developmental timing. In vivo studies of the contribution of miRNAs ...