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 older centriole during G1/G0 phase cell cycle, while they disassemble as cells re-enter cycle at G2/M boundary. function signal hubs, by detecting and transducing extracellular signals crucial for many processes. Similar to types, all neocortical neural stem progenitor (NSPCs) have been shown harboring a PC allowing them sense transduce specific required normal cerebral cortical development. Here, we provide detailed protocols generate characterize two-dimensional (2D) three-dimensional (3D) cell-based models human induced pluripotent (hIPSCs) further dissect involvement In particular, present study biogenesis in 2D rosette-derived NSPCs including transduction Sonic Hedgehog (SHH) pathway. To take advantage organization organoids, describe simple method 3D imaging toto immunostained organoids. After optical clearing, rapid acquisition entire organoids allows detection both centrosomes on progenitors neurons whole organoid. Finally, detail procedure immunostaining clearing thick free-floating organoid sections preserving significant degree spatial information high-resolution qualitative quantitative analysis function.