Module 3: Generating Glial and Mixed Cultures
Learn how to produce and characterize three major glial cell types in the brain - oligodendrocytes, astrocytes, and microglia – and how to assemble co-cultures.
Mathew Blurton-Jones, PhD
University of California, Irvine
Hiroko Nobuta, PhD
Albert Einstein College of Medicine
Jason Tchieu, PhD
Memorial Sloan Kettering Cancer Center
After reviewing Module 3, participants at all career stages should be able to:
- Outline basic methods that can be used to differentiate iPS cells into microglia and to validate the resulting phenotype.
- Identify assays that can be used to study the function of microglia in vitro and the potential use of chimeric transplantation models to study human microglia function in vivo.
- Describe growth factor-mediated human oligodendrocyte generation from iPS cells.
- Explain how to troubleshoot common experimental challenges.
- Explain the logic of directed differentiation of astrocytes from human pluripotent stem cells (hPSC’s).
- Describe some uses of astrocytes for modeling neuron/glia interactions in mixed cultures.
Module 3A: Generation and Use of iPS-Derived Microglia
In this presentation, Mathew Blurton-Jones will:
- Introduce methods to differentiate pluripotent stem cells into microglia.
- Describe approaches that can be used to validate the resulting cells.
- Describe in vitro assays that can be used to examine microglial function.
- Identify potential caveats with in vitro microglial culture.
- Explain development and use of new chimeric mouse models to study human microglial function in vivo.
Module 3B: Generation of Oligodendrocytes from iPS Cells
In this presentation, Hiroko Nobuta will introduce various methods for generating oligodendrocytes from iPS cells. Specifically, Nobuta will:
- Describe various methods of generating mouse and human oligodendrocytes and their pros and cons.
- Introduce the detailed procedure of the growth factor-mediated human oligodendrocyte generation from iPS cells.
- Provide troubleshooting tips for common problems in the growth factor-mediated human oligodendrocyte generation and functional assays.
Module 3C: Deriving Human Astrocytes from Pluripotent Stem Cells
In this presentation, Jason Tchieu will:
- Explain why human astrocytes are important.
- Provide general methods to differentiate astrocytes from PSCs, including transcription factor mediated systems.
- Describe how to stage and troubleshoot the differentiation(s).
- Introduce functional and molecular characterization.
- Introduce co-culture analysis.