MAR 24, 2020 11:00 AM PDT

3-D Organoid Models for Differentiation of PSC-Derived Dopaminergic Neurons

Speaker

Abstract
DATE:   March 24, 2020
TIME:   11:00am PT, 2:00pm ET
 
 
While in vitro cell culture has long been used to study neurological diseases, researchers have come to the realization that 2-D systems do not accurately model the complex cell interactions and self-organizing architecture of the brain. Neuroscientists are increasingly turning to 3-D organoid systems that include a greater diversity of cell types and more closely mimic brain development in vivo.
 
We have generated organoids rich in midbrain dopaminergic (DA) neurons, the specific cells which degenerate in Parkinson's Disease (PD). Starting with suspension culture of pluripotent stem cells, we passage into low-attachment 96-well U-bottom plates to begin differentiation of uniformly-sized spheroids. A low concentration of extracellular matrix (ECM) molecules are added during neural specification to enhance the organoid architecture, while improving throughput relative to ECM encapsulation methods. The ECM speeds dopaminergic maturation as indicated by Neuromelanin production. Multielectrode array (MEA) recording of replated spheroids shows spontaneous burst activity in as little as five weeks, followed by gradual refinement toward coordinated rhythmic bursting. To model Parkinson's Disease in vitro we introduced the PD-related SNCA A30P mutation into a Cas9-expressing iPSC line. We have generated midbrain organoids from these lines and are evaluating the sensitivity of mutant and wildtype DA neurons to induced oxidative stress. The specific effects on DA neurons are assayed by antibody detection of active caspase in wholemount or cryosectioned organoids and evaluated via High-Content Analysis (HCA).
 
In this work we have produced midbrain organoids with functional DA neurons while reducing our time and effort. In combination with more accessible and higher-throughput 3-D assays, we hope this may contribute toward a more illuminating in vitro PD model.
 
Learning Objectives:
  • Discover the advantages of 3-D culture for directed neuronal differentiation of PSC
  • Learn how to modify early specification steps to expedite neuronal maturation
  • Understand the constraints that must be balanaced to adapt 2-D differentiation protocols to 3-D
 
 
 
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