Nuclei-size distributions as predictive tools of hematopoietic cell proliferation

Abstract

Current protocols for transplantation of hematopoietic stem and progenitor cells may be limited by donor-cell availability and the long time needed to restore neutrophil and platelet counts to normal levels. Ex vivo expansion of hematopoietic cells has the potential to decrease the required harvest size, and to enhance the transplant outcome, by providing greater numbers of progenitor and post-progenitor cells. However, widespread application of ex vivo expansion in the clinical setting is complicated by sample-to-sample variability in the extent and kinetics of cell expansion. For example, the lag time before active cell expansion may vary by several days and some samples may never expand under the culture conditions employed. An early determination regarding the fate of a culture would save time and resources, and would allow corrective action to be taken if desired. Furthermore, anticipation of the onset of cell cycling should prove useful in the development of culture-feeding strategies, as well as for maximizing transduction efficiency in gene-therapy protocols that employ retroviral vectors. We demonstrate that the nuclei-size distribution, which is obtained at the same time as the total nucleated cell concentration, can be used to predict the onset of cell proliferation. The formation of a second peak (with diameter > 4 microm) in the nuclei-size distribution, in addition to the smaller diameter peak (< 4 microm) present for quiescent cells, precedes total cell expansion. In particular 94% of all MNC and CD34+ cell cultures that we have observed to exhibit a second peak in the nuclei-size distribution have realized total cell expansion. Furthermore, only one of 67 observed cultures that did not exhibit the formation of a second peak realized total cell expansion. The formation of a second peak in the nuclei-size distribution is evident, either before or on the same day as the presence of a significant fraction of cells in the S-phase of the cell cycle.