1035. Use of Multi Color p27kip1 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
Authors: Jesper Bonde, Ryan Lahey, Phillip Herrbrich, Jan A. Nolta
Journal: Molecular Therapy (2005)

In defining ex vivo cultivation strategies for human HSC gene transduction, a key factor is the responsiveness of the most primitive cells to the 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. The major regulatory events leading to cell proliferation and differentiation occur between the G0 and G1 phases of the cell cycle, when the cell commits itself to DNA replication and both positive and negative external signals are integrated into the cell cycle. 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 p27kip1 flow cytometry assay which assesses the earliest molecular responses to a defined clinically applicable transduction protocol. Using p27kip1 as the main flow cytometric marker in combination with CD34 we developed methods to simultaneously assess the molecular events ongoing in individual human 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 p27kip1 assay is run in four color combinations with Ki67, Cyclin A, PCNA, or CD133. To evaluate whether this 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. p27kip1 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 p27kip1 expression in cells subjected to the extended protocol was between 29.8% (T=24 Hrs.) and 18.8% (T=72 Hrs.). At the same time CD34 expression increased 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 repeatedly found a small, but distinct population of p27kip1 expressing cells with up to 500 times more p27kip1 signal than the main population. We found this population to be 1.5%+/-0 0.3 (n=3) of the total cells in the cultures after 72 Hrs. Gating on this population showed no expression of the hematopoietic marker CD45 or the major stem/progenitor markers CD34, CD133, and CD117, indicating a highly quiescent cell type. In summary, the p27-based flow cytometric technique allows assessment of the cell cycle and differentiative status of individual hematopoietic stem and progenitor cells during periods of ex vivo culture.