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

Autor: Sophie Thomas, Nathalie Lefort, Nadia Bahi-Buisson, Tania Attié-Bitach, Sophie Saunier, Corinne Lebreton, Sébastien Dupichaud, Cécile Deleschaux, Clémantine Dimartino, Camille Maillard, Nicolas Goudin, Julie Pernelle, Sarah Farcy, Damelys Calderon, Céline Banal, Marie Michael, Lucile Boutaud
Přispěvatelé: Imagine - Institut des maladies génétiques (IHU) (Imagine - U1163), Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Paris Cité (UPCité), Structure Fédérative de Recherche Necker (SFR Necker - UMS 3633 / US24), Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Université Paris Cité (UPCité), CHU Necker - Enfants Malades [AP-HP], Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP), Service de neurologie pédiatrique [CHU Necker], Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-CHU Necker - Enfants Malades [AP-HP], ANR-17-CE16-0003,CentrosomeCiliaMicroceph,Mécanismes physiopathologiques des microcéphalies Rôle du centrosome et du cil primaire dans le développement du cortex cérébral(2017), ANR-16-CE16-0011,DYNEINOPATHY,Mecanismes moléculaires et cellulaires des dyneinopathies(2016), ANR-19-CE16-0002,ATOMy,Axonal Transport in SMALED, a Motor Neuron Disease(2019), ANR-10-IAHU-0001,Imagine,Institut Hospitalo-Universitaire Imagine(2010), Université Paris Cité, Equipe HAL, Mécanismes physiopathologiques des microcéphalies Rôle du centrosome et du cil primaire dans le développement du cortex cérébral - - CentrosomeCiliaMicroceph2017 - ANR-17-CE16-0003 - AAPG2017 - VALID, Mecanismes moléculaires et cellulaires des dyneinopathies - - DYNEINOPATHY2016 - ANR-16-CE16-0011 - AAPG2016 - VALID, Axonal Transport in SMALED, a Motor Neuron Disease - - ATOMy2019 - ANR-19-CE16-0002 - AAPG2019 - VALID, Instituts Hospitalo-Universitaires - Institut Hospitalo-Universitaire Imagine - - Imagine2010 - ANR-10-IAHU-0001 - IAHU - VALID
Jazyk: angličtina
Rok vydání: 2022
Předmět:
Zdroj: Journal of visualized experiments : JoVE
Journal of visualized experiments : JoVE, 2022, 181, ⟨10.3791/62667⟩
ISSN: 1940-087X
DOI: 10.3791/62667⟩
Popis: International audience; 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.
Databáze: OpenAIRE
Externí odkaz: