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* These authors contributed equally
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 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.
Primary cilia (PC) 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. 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 propose....
1. Generation of 2D hIPS cell-based models of neocortical development
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 .......
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.......
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'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'avenir program (ANR-10-IAHU-01, CrossLab projects) and as part of the second Investissements d'Avenir program (ANR-17-RHUS-0002).
....Name | Company | Catalog Number | Comments |
2-Mercaptoéthanol (50 mM) | ThermoFisher Scientific | 31350010 | |
6-well Clear Flat Bottom Ultra-Low Attachment Multiple Well Plates | Corning | 3471 | |
96-well Clear Round Bottom Ultra-Low Attachment Microplate | Corning | 7007 | |
B-27 Supplement (50X), minus vitamin A | ThermoFisher Scientific | 12587010 | |
B-27 Supplement (50X), serum free | ThermoFisher Scientific | 17504044 | |
CellAdhere Dilution Buffer | StemCell Technologies | 7183 | |
DMEM/F-12, Glutamax | ThermoFisher Scientific | 31331028 | |
DMSO | ATCC | 4-X | |
Dorsomorphin | StemCell Technologies | 72102 | |
Easy Grip 35 10mm | Falcon | 353001 | |
EDTA | ThermoFisher Scientific | 15575020 | |
EGF , 25µg | Thermofischer | PHG0315 | |
FGF2 , 25µg | Thermofischer | PHG0264 | |
Gentle Cell Dissociation Reagent | StemCell Technologies | 7174 | |
Insulin | ThermoFisher Scientific | 12585014 | |
KnockOut Serum | ThermoFisher Scientific | 10828028 | |
Laminin (1mg) | Thermofischer | 23017015 | |
LDN193189 | StemCell Technologies | 72147 | |
Matrigel Growth Factor Reduced | Corning | 354230 | |
MEM Non-Essential Amino Acids Solution (100X) | ThermoFisher Scientific | 11140050 | |
Mowiol 4-88 | Sigma Aldrich | 81381-250G | |
mTeSR1 | StemCell Technologies | 85850 | |
Neural Basal Medium | Thermofischer | 21103049 | |
Orbital shaker | Dutscher | 995002 | |
PBS | ThermoFisher Scientific | 14190094 | |
Penicillin-Streptomycin (10,000 U/mL) | ThermoFisher Scientific | 15140122 | |
PFA 32% | Electron Microscopy Sciences | 15714 | |
Poly-L-Ornithine (PO) | Sigma | P4957 | |
Recombinant human BDNF 10 µg | Stem Cell Technologies | 78005 | |
Recombinant Human FGF-basic | Peprotech | 100-18B | |
rSHH | R&D Systems | 8908-SH | |
SAG | Santa Cruz | Sc-202814 | |
SB431542 | StemCell Technologies | 72232 | |
Stembeads FGF2 | StemCulture | SB500 | |
Sucrose | Sigma Aldrich | S7903-250G | |
Superfrost Plus Adhesion Slides | Thermo Scientific | J1800AMNZ | |
Supplément N2- (100X) | ThermoFisher Scientific | 17502048 | |
TDE 2,2’-Thiodiethanol | Sigma Aldrich | 166782-500G | |
Vitronectin | StemCell Technologies | 7180 | |
Y-27632 | StemCell Technologies | 72304 | |
Primary Antibodies | |||
ARL13B | Abcam | Ab136648 | 1/200e |
ARL13B | Proteintech | 17711-1-AP | 1/500e |
CTIP2 | Abcam | Ab18465 | 1/500e |
GLI2 | R&D Systems | AF3526 | 1/100 |
GPR161 | Proteintech | 13398-1-AP | 1/100 |
N-Cadherin | BD Transduction Lab | 610921 | 1/500e |
P-Vimentin | MBL | D076-3 | 1/500e |
PAX6 | Biolegend | PRB-278P | 1/200e |
PCNT | Abcam | Ab4448 | 1/1000e |
S0X2 | R&D Systems | MAB2018 | 1/200e |
SATB2 | Abcam | Ab51502 | 1/200e |
TBR2 | Abcam | Ab216870 | 1/400e |
TPX2 | NovusBio | NB500-179 | 1/500e |
γTUBULIN | Sigma Aldrich | T6557 | 1/500e |
Secondary Antibodies | |||
Donkey anti-rabbit AF488 | ThermoFisher Scientific | A21206 | 1/500e |
Goat anti-mouse AF555 | ThermoFisher Scientific | A21422 | 1/500e |
Goat anti-mouse AF647 | ThermoFisher Scientific | A21236 | 1/500e |
Goat anti-rat AF555 | ThermoFisher Scientific | A21434 | 1/500e |
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