Fig. 5. The phenomenon of re-sphering is density dependent and the re-sphered cells resemble the primary Myc cells. (A) In 96-well plates (in 150 µl of 2% FCS per 25 mm² well, one plate per experiment), no re-sphering was seen if less than 200 Myc NPCs were plated per well. Also, the percentage of re-sphering did not increase by adding of more cells after 400 cells per well or by time. The numbers shown represent averages of the three counted time points. (40–100 cells per well 0%, n=5; 200–300 cells per well 0.3%, s.d.=0.4, n=4; 400–600 cells per well 2.9%, s.d.=1.9, n=3; 700–900 cells per well 1.5%, s.d.=1.5, n=6; 4000 cells per well 2.2%, n=1). (B) A western blot of primary and re-sphered Myc neurosphere populations shows that the level of Myc expression was approximately the same in both populations and thus was not increased in the re-sphered population. Staining for nucleolin verifies the same result. Staining for β-actin shows that the amount of the cell lysate per lane is similar. (C) When re-sphered single Myc neurospheres after 4 weeks of differentiation in FCS were changed back to FGF and EGF and expanded into a NPC population, the proportion of self-renewing cells increased from the initial 22.5% to 29% but the difference is not statistically significant (s.d.=9,7, n=10, Student's t-test P=0,1). (D) The re-sphered Myc cells proliferated fast but the increase in cell number is not statistically significant (re-sphered Myc 8100, s.d.=638, n=3, Student's t-test P=0.0531). (E) Staining and FACS analysis show no significant change in Bmi-1- or nestin-positive cells. (Re-sphered Myc nestin 83.1%, s.d.=2.2, n=3 and Bmi-1 83.9%, s.d.=3.5, n=3.) (F) Re-sphered Myc NPCs can differentiate. Redifferentiated re-sphered Myc NPCs again attached to the culture well and expressed neuronal and astrocyte markers TUJ-1 and GFAP after 4 days culture in 2% FCS.