Cell cycle distribution of primitive haematopoietic cells stimulated in vitro and in vivo

Abstract

A novel approach is used to study the proliferating behaviour of primitive haematopoietic cell populations in response to different stimuli. A mathematical model based on the average proportion of apoptotic, dividing and quiescent cells in primitive haematopoietic cell populations is developed to describe the mitotic history of 5- (and 6-) carboxyfluorescein diacetate succinimidyl ester-labelled cells. The cell cycle distributions in different cytokine-supplemented cultures of primitive human and mouse bone marrow cells are determined and compared with those found in vivo. The results indicate that a combination of flt-3 ligand, Steel factor and interleukin-11 or hyper-interleukin-6 provide a level of mitogenic stimulation similar to that existing in vivo after a myeloablative radiation dose. The comparison of the cell cycle distribution obtained for different cultures of human bone marrow CD34(+)(45RA/71)(-) cells demonstrates that the addition of flt-3 ligand in these cultures decreases apoptosis significantly but does not reduce quiescence. In addition, in vivo and in vitro, it was found that more than 3 days of stimulation are required to recruit a maximum number of quiescent cells into active cell cycle. These kinetics of cell cycle activation are found to be similar to those identified for the haematopoietic stem cells compartment in the same cultures. This mathematical analysis provides a useful tool for the development of haematopoietic stem cell culture processes for clinical applications.

Analysis of cell differentiation by division tracking cytometry

“…The linear regression model [Eq. (10)] fitted the data well (r 5 0.99). Dilution factor and auto-fluorescence were estimated to be 0.544 6 0.006 (6SE) and 0.364 6 1.20, respectively.…”

“…10 Calculate generation gates by cubic spline interpolation between cluster regression lines (see Figure 1). 11…”

“…Mononuclear cells were resuspended in PBS with 1 mg/ml bovine serum albumin at 10 7 cells/ml and a small volume of stock solution of CFDA-SE was added to the cell suspension to make up a final concentration of 2.5 lM CFDA-SE. Cells were incubated for 37°C for 10 min.…”

“…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)(4)(5)(6)(7)(8)(9)(10).…”

Estimating Lymphocyte Division and Death Rates from CFSE Data

“…Most of the earlier work on modeling CFSE dilution has been done with models resembling the Smith and Martin (1973) model with an explicit time delay for the S, G2, and M phases of the cell cycle (Nordon et al, 1999;Bernard et al, 2003;Pilyugin et al, 2003;De Boer and Perelson, 2005;Ganusov et al, 2005), or with other models allowing for an explicit time delay (Zhang et al, 2001;Deenick et al, 2003;Leon et al, 2004). CFSE data have also been interpreted by fitting the data to models that do not explicitly incorporate a minimum cell cycle length, i.e., autonomous ODE models where the times to division and death are exponentially distributed (Veiga-Fernandes et al, 2000;Revy et al, 2001).…”