Low extracellular calcium is sufficient for survival and proliferation of murine mesencephalic neural precursor cells.
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 microg total proteins in 0.1 mM Ca2+ medium vs. 316+/-34 microg 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 x 10(4) NPCs/ml observed in control medium to 62+/-2 x 10(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 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.
Milosevic
Javorina
J
Department of Neurology, University of Leipzig, Leipzig, Germany. javorina.milosevic@medizin.uni-leipzig.de
Juch
Franziska
F
Storch
Alexander
A
Schwarz
Johannes
J
D000818:Animals; D002118:Calcium / Q000378:metabolism*; D018929:Cell Culture Techniques; D049109:Cell Proliferation; D002470:Cell Survival; D002478:Cells, Cultured; D045604:Extracellular Fluid / Q000378:metabolism*; D008636:Mesencephalon / Q000166:cytology*; D051379:Mice; D008810:Mice, Inbred C57BL; D039902:Multipotent Stem Cells / Q000166:cytology*; D009474:Neurons / Q000737:chemistry* / Q000166:cytology
D002118:Calcium