Open Access Highly Accessed Research article

Successful elimination of non-neural cells and unachievable elimination of glial cells by means of commonly used cell culture manipulations during differentiation of GFAP and SOX2 positive neural progenitors (NHA) to neuronal cells

Monika Witusik1, Sylwester Piaskowski1, Krystyna Hulas-Bigoszewska1, Magdalena Zakrzewska1, Sylwia M Gresner1, S Ausim Azizi2, Barbara Krynska2, Pawel P Liberski1 and Piotr Rieske1*

Author Affiliations

1 Medical University of Lodz, Department of Molecular Pathology and Neuropathology, 8/10 Czechoslowacka str. 92-216 Lodz, Poland

2 Department of Neurology, Temple University School of Medicine, 3401 N Broad St. 558 Parkinson Pavilion, Philadelphia, PA 19140, USA

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BMC Biotechnology 2008, 8:56  doi:10.1186/1472-6750-8-56

Published: 19 July 2008

Abstract

Background

Although extensive research has been performed to control differentiation of neural stem cells – still, the response of those cells to diverse cell culture conditions often appears to be random and difficult to predict. To this end, we strived to obtain stabilized protocol of NHA cells differentiation – allowing for an increase in percentage yield of neuronal cells.

Results

Uncommitted GFAP and SOX2 positive neural progenitors – so-called, Normal Human Astrocytes (NHA) were differentiated in different environmental conditions to: only neural cells consisted of neuronal [MAP2+, GFAP-] and glial [GFAP+, MAP2-] population, non-neural cells [CD44+, VIMENTIN+, FIBRONECTIN+, MAP2-, GFAP-, S100β-, SOX2-], or mixture of neural and non-neural cells.

In spite of successfully increasing the percentage yield of glial and neuronal vs. non-neural cells by means of environmental changes, we were not able to increase significantly the percentage of neuronal (GABA-ergic and catecholaminergic) over glial cells under several different cell culture testing conditions. Supplementing serum-free medium with several growth factors (SHH, bFGF, GDNF) did not radically change the ratio between neuronal and glial cells – i.e., 1,1:1 in medium without growth factors and 1,4:1 in medium with GDNF, respectively.

Conclusion

We suggest that biotechnologists attempting to enrich in vitro neural cell cultures in one type of cells – such as that required for transplantology purposes, should consider the strong limiting influence of intrinsic factors upon extracellular factors commonly tested in cell culture conditions.