Use of Multi Color P27^kip1 Based Flow Cytometric Assay To Evaluate and Characterize G₀ to G₁ Cell Cycle Related Changes in Human Hematopoietic Cells after Subjection to a Clinically Applicable Ex Vivo Transduction Protocol.

In defining ex vivo cultivation strategies for human HSC gene transduction, a decisive factor to consider is the responsiveness of the most primitive cells to the employed in vitro conditions with the aim of maintaining viability without inducing terminal differentiation. Important insights have been gained into the molecular basis of cell cycle regulating mechanisms in the last few years. The major regulatory events leading to cell proliferation and differentiation occur between the G₀ and G₁ phases of the cell cycle, at which point the cell commits itself to DNA replication and both positive and negative external signals are integrated into the cell cycle. Nonetheless, evaluating the effect of an ex vivo transduction protocol with respect to this decisive junction between quiescence and cell division remains an elusive goal. Here, we present a novel p27^kip1 flow cytometry assay which assesses the earliest molecular responses to a defined clinically applicable ex vivo transduction protocol. Using p27^kip1 as the main flow cytometric marker in combination with CD34 we developed methods to simultaneously assess the molecular events ongoing in individual cord blood Lin- cells while they were cultured for 72 hours in X-Vivo 15 serum free medium supplemented with Flt3, SCF and TPO on Retronectin (RN) coated plates. The p27^kip1 assay is run in four color combinations with p21^waf, Cyclin E, Cyclin A, PCNA, or CD133. To evaluate whether the employed ex vivo protocol had the preferred effect of sustaining viability without inducing terminal differentiation we compared it to a more stimulatory extended protocol of X-Vivo 15 supplemented with SCF, Flt-3, TPO and IGF-1 on RN coated plates. p27^kip1 expression in cells subjected to the basic ex vivo transduction protocol was found to be markedly high, ranging from 74.1% (T=24 Hrs) to 68.1% (T=72 Hrs.), whereas the comparable p27^kip1 expression in cells subjected to the extended protocol was found to be between 29.8% (T=24 Hrs.) and 18.8% (T=72 Hrs.). At the same time CD34 expression rose from 53.8% (T=24 Hrs.) to 80.5% (T=72 Hrs.) in the basic protocol, while the CD34 expression on cells subjected to the extended protocol was decreased from 87.5% CD34+ (T=24 Hrs.) to 73% CD34+ (T=72 Hrs.). Finally, in cultures subjected to the 72 hr. basic ex vivo transduction protocol we have repeatedly found a small, but distinct population of p27^kip1 expressing cells with up to 500 times more p27^kip1 signal than the main population, as evaluated by fluorescence intensity. This population is 1.6% of the total cells in the cultures after 72 Hrs, and has not been observed in either the 24 or the 48 hrs. time point evaluations, nor in the cultures subjected to the extended protocol. Upon gating on this population we found no co-expression of the major stem/progenitor and haematopoietic markers CD34, CD133, CD117 or CD45, indicating a highly quiescent cell type. In summary, this technique allows assessment of the cell cycle and differentiative status of individual hematopoietic stem and progenitor cells during periods of ex vivo culture.