Figure 2.

Limiting dilution analyses of CD133⁺-sorted hCNS-SC cells. (a) A representative limiting dilution analysis for NS-IC activity in FBr and sorted subsets. FBr (●), CD133⁻ CD34⁻ CD45⁻ (▴), and CD133⁺ CD34⁻ CD45⁻ (⧫, red line) sorted cell populations were plated in a series of limiting cell doses. FBr and CD133⁻ cells (100–10,000 cells per well) and CD133⁺ cells (10–300 cells per well) were plated into 96-well plates with a FACS–automated cell deposition unit. Cultures were carried for 6–8 weeks, and wells that did not contain neurospheres were scored as negative. A linear regression analysis was used to determine the frequency of NS-IC (20). (b) Neurospheres generated from a single-cell event. CD133⁺ CD34⁻ CD45⁻ sorted cell populations from eight different FBr samples were plated in a series of limiting cell doses into 96-well plates with a automated cell deposition unit. Results from eight different samples were combined to generate a plot of the number of cells plated vs. log percentage negative wells with SEM (error bars). Linear correlation of log percentage negative wells and number of CD133⁺ cells plated indicate a neurosphere initiated from a single-hit event. (c) NS-IC frequencies from unsorted, sorted, and cultured FBr cells. NS-IC frequency in FBr control (n = 8), CD133⁻ (n = 8), CD133⁺ (n = 8) sorted, and CD133⁺ sorted/expanded (passage 1, n = 3) cell populations was determined. NS-IC frequencies of individual samples were calculated by linear regression analysis. The average NS-IC frequency in a given population is shown alongside the bars.