1. Enriched Neuronal Cultures Enable More Accurate Assessment of
Receptor Expression and Enhance Growth of Neuronal Processes
Lizeth Martinez, See Yang, and Kiowa Bower
Department of Natural Sciences and Mathematics, Dominican University of California, San
Rafael, CA 94901
Abstract
We are studying the changes in receptor expression levels as embryonic stem cells (ESCs) differentiate into neurons by adding AraC ,a
selective inhibitor of DNA synthesis, and inducing them in a monolayer fashion. We can identify which cells in our culture are neurons by using
MAP2 as a neuronal marker . Our results indicate that this protocol yields higher percentages of neurons and thus more reliable results from our
gene-expression studies. Because reduced glial cell proliferation leaves more space for the growth of the developing neurons, we examined the
potential effect of the new conditions on neuron morphology. Using immunocytochemistry to visualize extent of axon and dendrite growth, we
found that the new conditions led to increased numbers of these neuronal processes.
Background
•Neuroreceptors are membrane proteins
that are activated by chemical signals from
other cells, and are most abundant at the
synapses between neurons.
•These receptors are critical for neuron
communication and some play a role in
neuronal differentiation during the
embryonic development of the nervous
system.
•P19 embroynal carcinoma cells are an
effective model system for investigating
neuronal differentiation because their
development, morphology, and
biochemistry are similar to in vivo
differentiation of neurons.
Materials and Methods
•The ESCs are grown in a growth media
with 5% fetal bovine serum
•The cells become induced by retionic
acid in a monolayer method using a T75
confluent flask. Induction occurs for 4
days.
•Induced cells are seeded on 12 well
plates with glass slides, coated with
proteins.
•AraC is added after 3 days of seeding.
Results
•Visible changes in cell morphology after
1 day
•Cells appear dead & lysed but some cell
components remain attached
•Immunocytochemistry
DNA (Nucleus) MAP 2
Synapsin Merged
Day of drug addition 1 day after addition of drug
Conclusions
•Mitotic inhibitors are effective
Validity of receptor expression
studies
•Monolayer induction better than EB
protocol
Easier methodology
Consistency of neuron
populations
More numerous dendrites
•Potentially useful for synaptogenesis
studies
•Identified expression changes in
several neuroreceptors