Analysis of cell differentiation by division tracking cytometry

Abstract

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 1 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 autofluorescence. Mitotic activation of cord blood CD34 1 cells by cytokines commenced after 2 days of cytokine stimulation. Mean generation number increased linearly thereafter, and it was conclusively shown that CD34 1 cells cycle slower than CD34 2 cells. Generation times for CD34 1 and CD34 2 cells were 24.7 6 0.8 h and 15.1 6 0.9 h (6SD, n 5 5), respectively. The 20-fold increase in CD341 cell numbers at Day 6 could be attributed to a high CD34 1 cell renewal rate (91% 6 2% per division). Although cultures were initiated with highly purified CD34 1 cells ($96%), CD34 2 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 1 cells ($5%) that differentiated into CD34 2 cells. Multitype division tracking provides a detailed analysis of multipotent cell differentiation dynamics. ' International Society for Analytical CytologyKey terms cell division tracking; cord blood stem cells; in vitro expansion; carboxyfluorescein diacetate succinimidyl ester LYONS and Parish were the first investigators to report serial halving of fluorescence intensity of lymphocytes stained with the vital dye CFDA-SE (carboxyfluorescein diacetate, succinimidyl ester) (1). The technique provided an important adjunct to cell cycle analysis by DNA measurement. Flow cytometry was used to identify and gate as many as eight consecutive cell cycles and so analyze the changes in expression of internal or external molecules with respect to cell generation number. The technique was originally applied to the study of lymphocyte division because of their homogeneous staining properties. By employing a simple sorting strategy, Nordon et al. (2) demonstrated that it was possible to obtain a similarly well-defined cluster pattern with other cell types that did not stain homogeneously. This technique has had an important role to play in the study hematopoietic stem cell quiescence, renewal, and differentiation (3-10).To date, the assignment of cell generation number to CFDA-SE clusters has been an empiric process that is heavily reliant o...

Stem cells of the adult cornea: From cytometric markers to therapeutic applications

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A flow cytometric assay for simultaneously measuring the proliferation and cytotoxicity of cytokine induced killer cells in combination with carboxyfluorescein succinimidyl ester (CFSE) labeling

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Ex Vivo Expansion of Haematopoietic Stem Cells to Improve Engraftment in Stem Cell Transplantation

“…Here we describe (i) a protocol for the efficient ex vivo expansion of human CD34(+) HSC isolated from UCB and (ii) a flow cytometry-based method using high-resolution division tracking to characterise the kinetics of HSC expansion (17). The stem cell expansion protocol describes the robust ex vivo proliferation of human CD34(+) cells isolated from umbilical cord blood using a combination of cytokines previously optimised by our group (17).…”

“…The stem cell expansion protocol describes the robust ex vivo proliferation of human CD34(+) cells isolated from umbilical cord blood using a combination of cytokines previously optimised by our group (17).…”