Low extracellular calcium is sufficient for survival and proliferation of murine mesencephalic neural precursor cells.
Various media and Ca(2+) concentrations are employed to culture neural progenitor cells (NPCs). We have therefore explored the effects of extracellular calcium concentrations on the survival, proliferation, spontaneous apoptosis and self-renewal capacity of mesencephalic NPCs grown adherently and as free-floating neurospheres. We employed EMEM supplemented with various concentrations of extracellular CaCl(2) (0.1-1 mM). Raising the calcium concentration from 0.1 mM to 0.6 mM resulted in an increased number of NPCs growing as a monolayer and increased the protein yield of cells growing in neurospheres (24+/-3 mug total proteins in 0.1 mM Ca(2+) medium vs. 316+/-34 mug proteins in 1 mM Ca(2+) medium). Concentrations more than 0.6 mM did not result in a further improvement of proliferation or survival. Elimination of calcium from our control medium by 1 mM EGTA resulted in a decrease in cell number from 82+/-2x10(4) NPCs/ml observed in control medium to 62+/-2x10(4) NPCs/ml observed in calcium-free media. Protein yield dropped significantly in calcium-free media, accompanied by the decreased expression of the proliferation marker PCNA and the pro-survival marker Bcl-2. Two weeks of expansion as neurospheres caused spontaneous cell death in more than 90% of NPCs grown in 0.1 mM CaCl(2) EMEM compared with 42% in 1 mM CaCl(2) EMEM. Although the action of Ca(2+) on NPCs appears to be complex, the presented data strongly suggest that extracellular calcium plays a crucial role in the maintenance of NPCs in a healthy and proliferating state; physiological concentrations (>1.0 mM) are not required, a concentration of 0.5 mM being adequate for cell maintenance.[1]References
- Low extracellular calcium is sufficient for survival and proliferation of murine mesencephalic neural precursor cells. Milosevic, J., Juch, F., Storch, A., Schwarz, J. Cell Tissue Res. (2006) [Pubmed]
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