Module 2: Neuronal Differentiation
Learn how to produce and characterize human neurons derived from induced pluripotent stem cells (IPSCs).
Gabriele Ciceri, PhD
Memorial Sloan Kettering Cancer Center
Nan Yang, PhD
Icahn School of Medicine at Mount Sinai
Zhiping Pang, PhD
Rutgers Robert Wood Johnson Medical School
After reviewing Module 2, participants at all career stages should be able to:
- Describe techniques and methods used to define human neurons derived from human iPS cells.
- Summarize the rationale of directed neuronal differentiation from human pluripotent stem cells.
- Describe how to apply the use of small molecules/morphogens to generate major neuron types.
- Introduce step-wise quality control criteria to assure the generation of the desired neuron type and troubleshoot the differentiations.
- Explain the principles for direct lineage conversion.
- Identify the key techniques used to directly generate human neurons from iPS cells by transiently expressing transcription factors.
- Describe how to establish long-term culture for disease modeling.
Module 2A: Directing the Differentiation of hPSCs Toward Neuronal Identities
In this presentation, Gabriele Ciceri will introduce experimental strategies to generate neurons from human pluripotent stem cells via directed differentiation. Specifically, Ciceri will:
- Describe general principles of neuronal specification in vivo and their application to in vitro differentiation protocols.
- Introduce how to mimic development in a dish.
- Introduce paradigms for neural induction (PNS vs. CNS).
- Describe specification of regional identity through integration of multiple patterning signals and generation of specific neuron type.
- Explain how to control for lineage progression.
- Describe validation of directed differentiations.
Module 2B: Direct Lineage Conversions in Disease Modeling
In this presentation, Nan Yang will:
- Introduce lineage conversion, a few principles on identifying transcription factors for different neuronal subtypes, and the application of the method.
- Describe detailed procedure of generating induced neuronal cells from human iPS cells and create neuron & astroglia co-cultures.
- Share troubleshooting tips for common problems.
Module 2C: Morphological and Functional Analysis of Human Neurons
In this presentation, Zhiping Pang will introduce parameters for defining human neurons derived from iPSCs. Specifically, Pang will:
- Describe morphological features of human neurons (immature to mature characteristics).
- Introduce morphological and biochemical techniques to characterize human neurons.
- Provide information about functional characterization of human neurons, including electrophysiology (cover intrinsic neuronal membrane property; active membrane property; synapse transmission) and calcium imaging (network activity and contributions of synaptic transmission).