Modeling Human Cerebellar Development In Vitro in 2D Structure

J Vis Exp. 2022 Sep 16;(187). doi: 10.3791/64462.


The precise and timely development of the cerebellum is crucial not only for accurate motor coordination and balance but also for cognition. In addition, disruption in cerebellar development has been implicated in many neurodevelopmental disorders, including autism, attention deficit-hyperactivity disorder (ADHD), and schizophrenia. Investigations of cerebellar development in humans have previously only been possible through post-mortem studies or neuroimaging, yet these methods are not sufficient for understanding the molecular and cellular changes occurring in vivo during early development, which is when many neurodevelopmental disorders originate. The emergence of techniques to generate human-induced pluripotent stem cells (iPSCs) from somatic cells and the ability to further re-differentiate iPSCs into neurons have paved the way for in vitro modeling of early brain development. The present study provides simplified steps toward generating cerebellar cells for applications that require a 2-dimensional (2D) monolayer structure. Cerebellar cells representing early developmental stages are derived from human iPSCs via the following steps: first, embryoid bodies are made in 3-dimensional (3D) culture, then they are treated with FGF2 and insulin to promote cerebellar fate specification, and finally, they are terminally differentiated as a monolayer on poly-l-ornithine (PLO)/laminin-coated substrates. At 35 days of differentiation, iPSC-derived cerebellar cell cultures express cerebellar markers including ATOH1, PTF1α, PAX6, and KIRREL2, suggesting that this protocol generates glutamatergic and GABAergic cerebellar neuronal precursors, as well as Purkinje cell progenitors. Moreover, the differentiated cells show distinct neuronal morphology and are positive for immunofluorescence markers of neuronal identity such as TUBB3. These cells express axonal guidance molecules, including semaphorin-4C, plexin-B2, and neuropilin-1, and could serve as a model for investigating the molecular mechanisms of neurite outgrowth and synaptic connectivity. This method generates human cerebellar neurons useful for downstream applications, including gene expression, physiological, and morphological studies requiring 2D monolayer formats.

Publication types

  • Video-Audio Media
  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Cell Differentiation / genetics
  • Cerebellum
  • Fibroblast Growth Factor 2 / metabolism
  • GABAergic Neurons / metabolism
  • Humans
  • Induced Pluripotent Stem Cells*
  • Insulins* / metabolism
  • Laminin / metabolism
  • Neuropilin-1 / metabolism
  • Semaphorins* / metabolism


  • Insulins
  • Laminin
  • Semaphorins
  • Fibroblast Growth Factor 2
  • Neuropilin-1