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

Summary

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.

Abstract

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.

Introduction

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 vertebrates^1,2. 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....

Protocol

1. Generation of 2D hIPS cell-based models of neocortical development

1. Neural rosette formation
   1. Start with hIPSC cultures harboring large regular colonies, exhibiting less than 10% differentiation and no more than 80% confluency.
   2. Rinse the hIPSCs with 2 mL of PBS.
   3. Add 2 mL of NSPC induction medium supplemented with the Rock inhibitor (NIM + 10 µM of Y-27632).
   4. Manually dissect each hIPSC colony from one 35 mm dish using a needle to cut each colony precisely in horizontal and vertical directions to create a checker-board pattern dividing each colony into equal clusters.
   ....

Results

2D hIPS cell-based models to study primary cilium biogenesis and function
The protocol detailed here has been adapted from previously published studies^20,21,22. 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 .......

Discussion

PC are now regarded as key organelles regulating crucial steps during normal cerebral cortical development^18,19,31 including NSPC expansion and commitment^8,9,10,11,12 as well as neuronal migration^13,14 and syn.......

Materials

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