Low extracellular calcium is sufficient for survival and proliferation of murine mesencephalic neural precursor cells |
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Authors: | Javorina Milosevic Franziska Juch Alexander Storch Johannes Schwarz |
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Institution: | (1) Department of Neurology, University of Leipzig, Leipzig, Germany;(2) Department of Neurology, Technical University of Dresden, Dresden, Germany;(3) Department of Neurology, Max-Bürger-Forschungszentrum, Johannisallee 30, 04103 Leipzig, Germany |
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Abstract: | Various media and Ca2+ 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 CaCl2 (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 μg total proteins in 0.1 mM Ca2+ medium vs. 316±34 μg proteins in 1 mM Ca2+ 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±2×104 NPCs/ml observed in control medium to 62±2×104 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 CaCl2 EMEM compared with 42% in 1 mM CaCl2 EMEM. Although the action of Ca2+ 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. |
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Keywords: | Neural precursor cells Neurospheres Calcium Proliferation Cell death Mouse (C57BL/6) |
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