Generation and enrichment of cerebellar GABAergic interneurons from human induced pluripotent stem cells and intracellular calcium measurements

Federica Pilotto; Rim Diab; Zahraa Al Qassab; Smita Saxena

doi:10.1016/j.xpro.2024.102936

Generation and enrichment of cerebellar GABAergic interneurons from human induced pluripotent stem cells and intracellular calcium measurements

STAR Protoc. 2024 May 10;5(2):102936. doi: 10.1016/j.xpro.2024.102936. Online ahead of print.

Authors

Federica Pilotto¹, Rim Diab², Zahraa Al Qassab², Smita Saxena³

Affiliations

¹ Institut Neuromyogenè, Inserm Lyon, Lyon, France; CNRS Université Claude Bernard Lyon I, Lyon, France.
² Department of Neurology, Inselspital University Hospital, Bern, Switzerland; Department for BioMedical Research, University of Bern, Bern, Switzerland.
³ Department of Neurology, Inselspital University Hospital, Bern, Switzerland; Department for BioMedical Research, University of Bern, Bern, Switzerland; Department of Physical Medicine & Rehabilitation, University of Missouri, Columbia, MO, USA; NextGen Precision Health, University of Missouri, Columbia, MO, USA. Electronic address: smitasaxena@missouri.edu.

Abstract

GABAergic interneurons are inhibitory neurons of the CNS, playing a fundamental role in neural circuitry and activity. Here, we provide a robust protocol for the successful enrichment of human cerebellar GABAergic interneurons from human induced pluripotent stem cells (iPSCs) and measuring intracellular calcium transients. We describe in detail steps for culturing iPSCs; generating embryoid bodies; and differentiating and enriching for cerebellar GABAergic neurons (cGNs), with precise steps for their molecular characterization. We then detail the procedure for adeno-associated virus-mediated transduction of cGNs with genetically encoded calcium indicators, followed by intracellular calcium imaging and analyses. For complete details on the use and execution of this protocol, please refer to Pilotto et al.¹.

Keywords: Cell Biology; Neuroscience; Stem Cells.