Analysis of cell differentiation by division tracking cytometry

We propose a quantitative method to characterize growth and differentiation dynamics of multipotent cells from time series carboxyfluorescein diacetate, succinimidyl ester (CFDA-SE) division tracking data. The dynamics of cell proliferation and differentiation was measured by combining (CFDA-SE) division tracking with phenotypic analysis. We define division tracking population statistics such as precursor cell frequency, generation time and renewal rate that characterize growth of various phenotypes in a heterogeneous culture system. This method is illustrated by study of the divisional recruitment of cord blood CD34+ cells by hematopoietic growth factors. The technical issue of assigning the correct generation number to cells was addressed by employing high-resolution division tracking methodology and daily histogram analysis. We also quantified division-tracking artifacts such as CFDA-SE degeneration and cellular auto-fluorescence. Mitotic activation of cord blood CD34+ cells by cytokines commenced after 2 days of cytokine stimulation. Mean generation number increased linearly thereafter, and it was conclusively shown that CD34+ cells cycle slower than CD34− cells. Generation times for CD34+ and CD34− cells were 24.7 ± 0.8 h and 15.1 ± 0.9 h (±SD, n = 5), respectively. The 20-fold increase in CD34+ cell numbers at Day 6 could be attributed to a high CD34+ cell renewal rate (91% ± 2% per division). Although cultures were initiated with highly purified CD34+ cells (∼96%), CD34− numbers had expanded rapidly by Day 6. This rapid expansion could be explained by their short generation time as well as a small fraction of CD34+ cells (∼5%) that differentiated into CD34− cells. Multitype division tracking provides a detailed analysis of multipotent cell differentiation dynamics. © 2007 International Society for Analytical Cytology