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 (approximately 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 (approximately 5%) that differentiated into CD34(-) cells. Multitype division tracking provides a detailed analysis of multipotent cell differentiation dynamics.
Ko
Kap-Hyoun
KH
Graduate School of Biomedical Engineering, Faculty of Engineering, University of New South Wales, Sydney, Australia.
Odell
Ross
R
Nordon
Robert E
RE
D000465:Algorithms; D018952:Antigens, CD34 / Q000378:metabolism; D016477:Artifacts; D002452:Cell Count; D002454:Cell Differentiation; D002455:Cell Division; D049109:Cell Proliferation; D016000:Cluster Analysis; D005312:Fetal Blood / Q000166:cytology; D005434:Flow Cytometry / Q000379:methods*; D005452:Fluoresceins / Q000378:metabolism; D005453:Fluorescence; D006801:Humans; D007700:Kinetics; D016014:Linear Models; D013234:Stem Cells / Q000166:cytology; D013388:Succinimides / Q000378:metabolism
C087165:5-(6)-carboxyfluorescein diacetate succinimidyl ester; D018952:Antigens, CD34; D005452:Fluoresceins; D013388:Succinimides