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Analysis of cell differentiation by division tracking cytometry
Author(s) -
Ko KapHyoun,
Odell Ross,
Nordon Robert E.
Publication year - 2007
Publication title -
cytometry part a
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.316
H-Index - 90
eISSN - 1552-4930
pISSN - 1552-4922
DOI - 10.1002/cyto.a.20437
Subject(s) - cd34 , cell division , microbiology and biotechnology , haematopoiesis , cord blood , biology , stem cell , cell growth , mitosis , population , cell cycle , cell , immunology , biochemistry , medicine , environmental health
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