Protocol for live imaging of axonal transport in iPSC-derived iNeurons

STAR Protoc. 2025 Mar 21;6(1):103556. doi: 10.1016/j.xpro.2024.103556. Epub 2025 Jan 12.

Abstract

Studies of human induced pluripotent stem cell (iPSC)-derived neurons promise important insights into neurodegenerative diseases. Here, we present a protocol for live imaging of axonal transport in glutamatergic iPSC-derived neurons (iNeurons). We describe steps for the differentiation of iPSCs into iNeurons via PiggyBac-mediated neurogenin 2 (NGN2) delivery, iNeuron culture and transfection, and the acquisition and analysis of time-lapse images. Our protocol is optimized for the widely available catalog of KOLF2.1J iPSCs with mutations relevant to neurodegenerative diseases but is also applicable to other iPSC lines. For complete details on the use and execution of this protocol, please refer to Dou et al.1,2.

Keywords: cell biology; microscopy; stem cells.

MeSH terms

  • Axonal Transport* / physiology
  • Basic Helix-Loop-Helix Transcription Factors / genetics
  • Basic Helix-Loop-Helix Transcription Factors / metabolism
  • Cell Differentiation
  • Humans
  • Induced Pluripotent Stem Cells* / cytology
  • Induced Pluripotent Stem Cells* / metabolism
  • Nerve Tissue Proteins / genetics
  • Nerve Tissue Proteins / metabolism
  • Neurons* / cytology
  • Neurons* / metabolism
  • Time-Lapse Imaging / methods

Substances

  • Nerve Tissue Proteins
  • Basic Helix-Loop-Helix Transcription Factors
  • NEUROG2 protein, human