Differentiation of human teratocarcinoma cell line into motor neurons
2009
The aim of this project was to develop cell culture techniques
for the directed differentiation of the human teratocarcinoma
cell line (Ntera-2) into postmitotic motoneurons. Immunocytochemical
and electrophysiological methods were used to characterize the cellular
properties of the differentiated neurons in vitro. To investigate
integration, differentiation and maturation of the NT2 cells in an in vivo
system, they have been transplanted into the nervous system of chick embryos.
Upon treatment with RA, and a proliferation step in
free-floating cell aggregates, NT2 precursor cells can be induced to
differentiate into postmitotic neurons (NT2N)
within one month. NT2N cells showed neuronal morphology and immunoreactivity to a number of neuronal markers
including cytoskeletal proteins such as β- III-tubulin, microtubule-associated protein 2, and phosphorylated tau. Moreover,
neural processes displayed a punctate immunoreactivity for synapsin
and synaptotagmin. Using the outside-out
patch-clamp configuration, the kinetics of currents elicited by a rapid
application of the amino acid neurotransmitters glutamate and GABA were
measured. Spontaneous postsynaptic currents could be detected in glia free cell cultures, indicating the formation of
functional synapses. Differentiated NT2 neurons when cultured in vitro,
are a heterogeneous population displaying glutamatergic
markers, serotonin, gamma-amino-butyric acid, and its synthesizing enzyme glutamic acid decarboxylase immunoreactivity. Subpopulations respond to the
application of nitric oxide donors with the synthesis of cGMP.
A major subset shows immunoreactivity to the
cholinergic markers choline acetyl-transferase (ChAT), vesicular
acetylcholine transporter and the nonphosphorylated form of neurofilament
H, all indicative of motor neurons. In co-cultures of NT2 neurons with mouse myotubes, morphological structures resembling
neuromuscular junctions were found that stain for ChAT,
synapsin and α-bungarotoxin.
Human NT2 neurons have been successfully
transplanted by many groups into the nervous tissue of adult experimental
animals and a limited number of human neurological patients. For the first
time, a study of integration and differentiation of various NT2 cells in the
cells in the environment of the chick embryo was conducted. I have injected
NT2 precursor cells (NT2), NT2 pretreated with
retinoic acid (NT2+RA), NT2 pretreated with
retinoic acid and Sonic hedgehog (NT2+RA+Shh), and NT2 neurons (NT2N) into
the spinal cord and brain. Monitoring of integration, differentiation and
outgrowth ratio between transplanted cell types was done after 3-5 days of
incubation by morphological analysis and staining for β-III-tubulin. The spinal cord appears to facilitated neurite extension more than the brain. In addition,
extending neurites of spinal grafts were often
approaching the ventral roots, thus resembling motoneuronal
projections. In summary, NT2 neurons and pretreated
NT2 cells transplanted into chick embryos, may serve as a unique model for
the in vivo study of human neuronal cells development.
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