Measurement of generation‐dependent proliferation rates and death rates during mouse erythroid progenitor cell differentiation

Abstract

Herein, we describe an experimental and computational approach to perform quantitative carboxyfluorescein diacetate succinimidyl ester (CFSE) cell-division tracking in cultures of primary colony-forming unit-erythroid (CFU-E) cells, a hematopoietic progenitor cell type, which is an important target for the treatment of blood disorders and for the manufacture of red blood cells. CFSE labeling of CFU-Es isolated from mouse fetal livers was performed to examine the effects of stem cell factor (SCF) and erythropoietin (EPO) in culture. We used a dynamic model of proliferation based on the Smith-Martin representation of the cell cycle to extract proliferation rates and death rates from CFSE time-series. However, we found that to accurately represent the cell population dynamics in differentiation cultures of CFU-Es, it was necessary to develop a model with generation-specific rate parameters. The generation-specific rates of proliferation and death were extracted for six generations (G 0 -G 5 ) and they revealed that, although SCF alone or EPO alone supported similar total cell outputs in culture, stimulation with EPO resulted in significantly higher proliferation rates from G 2 to G 5 and higher death rates in G 2 , G 3 , and G 5 compared with SCF. In addition, proliferation rates tended to increase from G 1 to G 5 in cultures supplemented with EPO and EPO 1 SCF, while they remained lower and more constant across generations with SCF. The results are consistent with the notion that SCF promotes CFU-E self-renewal while EPO promotes CFU-E differentiation in culture. ' 2012 International Society for Advancement of Cytometry Key terms red blood cells; carboxyfluorescein diacetate succinimidyl ester; cell differentiation; cell division; erythropoiesis; cell division; CFSE CARBOXYFLUORESCEIN diacetate succinimidyl ester (CFSE) cell-division tracking is widely used to determine the number of divisions cells have undergone in cultures or in vivo. This flow cytometric assay is based on the redistribution and dilution of the CFSE fluorescent dye between mother and daughter cells during cell division, resulting in distinct fluorescence intensity for each cell generation (1-3). Originally developed for homogenous lymphocyte populations (1), Nordon et al. (4) extended this approach to track the divisions of primary hematopoietic progenitor cell populations that are composed of multiple cell subsets (5-7). Qualitative examination of flow cytometric histograms of CFSE fluorescence is useful but does not allow a complete interpretation of time-series experiments. However, quantitative CFSE celldivision tracking consists of using computational tools and a working model of the dynamics of cell division to extract parameters that characterize the rates of cell activation, proliferation, and death from CFSE labeling experiments (8-13).The ''transition probability'' model of the cell cycle developed by Smith and Martin (14) has been useful to develop mathematical models to extract proliferation rates from CFSE data (8-10,13,15,...

Asymmetry of Cell Division in CFSE-Based Lymphocyte Proliferation Analysis

“…These models describe the population dynamics of cells which differ in the number of completed divisions and ignore the heterogeneity of the cells within a generation with respect to the CFSE content. The immunologically relevant issues that were addressed with the models of this group include regulatory effects of IL-2 on the T cell responses (34, 35), regulation of hematopoietic stem cells cycling (36), and kinetics of mouse erythroid progenitor cell differentiation (37). …”

Appraising the suitability of succinimidyl and lipophilic fluorescent dyes to track proliferation in non-quiescent cells by dye dilution

“…The main reason for this is the inherent heterogeneity within these cells generates labelled population widths that far exceed the limits for division peak resolution. The only way to reduce this variation is to sort a narrowed population from within the peak channels of the fluorescent distribution [13][14][15][16][17][18]. However, determining the success of sort gate reduction is hampered by the fact that any measurement is always prone to error [19].…”

Mathematical modeling reveals differential effects of erythropoietin on proliferation and lineage commitment of human hematopoietic progenitors in early erythroid culture

“…In particular, the Smith-Martin model that takes into account progression of cells through the cell cycle, has been successfully used to predict and model population dynamics of in vitro erythropoiesis. [21][22][23][24][25] We have combined immunophenotyping, proliferation analysis and cytokine dependence to refine the analysis of early erythroid culture of CD34 + cells isolated from human peripheral blood (PB). Isolated PB CD34 + cells are CMP, and the appearance of detectable surface levels of the erythroid marker CD36 is the earliest identifier of progenitors restricted to the megakaryocyte/erythroid lineage.…”