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Identification of cell types generated through differentiation of NT2/D1 cells under

treatment with retinoic acid

Tijana Stanić

Regional Centre for Talented Youth Belgrade II, Serbia, tijana.stanic.22@gmail.com

1. Introduction

Cells derived from embryonic carcinoma have
characteristics of stem cells, which can, under certain
conditions, differentiate into different types of cells.
NT2/D1 is a cell line derived from embryonic testicular
teratocarcinoma, which resembles early neural epithelial
progenitors. Under exposure to retinoic acid these cells
differentiate into neural and glial cells. The objective of this
work was to examine the morphology and identity of cells Picture 1-untreated Picture 2 – NT2/D1 neurons
generated in the differentiation process of NT2/D1 cells. NT2/D1, alpha-tubulin after 6 weeks of RA treatment
and DAPI on fluorescence alpha-tubulin on fluorescence
micrography micrography
2. Materials and methods

NT2 cells were maintained under normal conditions and 4. Conclusion
treated with 10 µM all-trans retinoic acid. After four weeks
of differentiation, for the purpose of further analysis, NT2/D1 cell line represents a unique model system for
multilayered cellular aggregates were trypsinised and studies of human neurogenesis and expression of genes
replated. The cells were seeded on cover-slips and fixed involved in neural differentiation. With the method of
with 4% paraformaldehyde. The cell morphology was immunocytochemistry, we have determined the
analysed using the method of immunocytochemical staining morphology and identity of differentiated cells, whereas
for cytoskeletal marker alpha-tubulin, whereas neural and the huge cells, negative on both markers of differentiated
glial cell types were identified using immunostaining for neural cells, will be subject for further research.
specific markers MAP2 (for neurons) and GFAP (for glial
cells). The shape and size of cells were analysed and
recorded with phase – contrast and fluorescence 5. References
microscopy.
[1] B.D.Stefanović,V.Đorđević-Čamba,Z.Kojić,M.Bajčetić,
M.Ćetković, Integrativna neurobiologija, Mikro knjiga i
3. Results and discussion B&M, Beograd, 2003.
[2] S.J.Pleasure, C.Page, V.M.-Y.Lee, Pure, Postmitotic,
At the beginning of the treatment, NT2/D1 cells appeared Polarized Human Neurons Derived from NTera 2 Cells
as dark, undifferentiated cells of granular shape, with little Provide a System for Expressing Exogenous Proteins in
cytoplasm around a large nucleus. The cytoplasm of Terminally Differentiated Neurons, The Journal of
undifferentiated cells stained for anti-alphla-tubulin Neuroscience, 1992.
antibodies was detected with fluorescence microscopy. [3] G.Podrygajlo, M.A.Tegenge, A.Gierse, F.Paquet-
Following 4-week RA treatment, the cells formed dense Durand, S.Tan, G.Bicker, M.Stern, Cellular phenotypes of
multilayered culture. At this point, bright neuron-like cells human model neurons (NT2) after differentiation in
with axon-like processes growing in groups over large aggregate culture, Cell Tissue Res 336-439-452, 2009.
nonneural cells, were recorded. The cells which displayed [4] Peter W. Andrews, Department of Biomedical Science,
immunoreactivity for MAP2 protein were identified as Western Bank, Unievrsity of Sheffield, From
neurons. Simultaneously, glial cells incubated with anti- teratocarcinomas to embryonic stem cells, Phil. Trans. R.
GFAP antibody were detected in a small number. Also, Soc. Lond. B, 2002.
anti-alpha-tubulin antibody enabled the identification of
the third group of cells whose identity remained unknown,
as these cells did not show immunoreactivity for markers
of differentiated neural cells.