Expression of cell cycle-related genes with cytokine-induced cell cycle progression of primitive hematopoietic stem cells - PubMed (original) (raw)

Peter J Quesenberry et al. Stem Cells Dev. 2010 Apr.

Abstract

Primitive marrow lineage-negative rhodamine low and Hoechst low (LRH) stem cells isolated on the basis of quiescence respond to the cytokines thrombopoietin, FLT3L, and steel factor by synchronously progressing through cell cycle. We have now profiled the mRNA expression, as determined by real-time RT-PCR, of 47 hematopoietic or cell cycle-related genes, focusing on the variations in the cell cycle regulators with cycle transit. LRH stem cells, at isolation, showed expression of all interrogated genes, but at relatively low levels. In our studies, there was a good deal of consistency with regard to cell cycle regulatory genes involved in the G1/S progression point of LRH murine stem cells. The observed pattern of expression of cyclin A2 is consistent with actions at these phases of cell cycle. Minimal elevations were seen at 16 h with higher elevations at 24, 32, 40, and 48 h times encompassing S, G2, and M phases. CDK2 expression pattern was also consistent with a role in G1/S transition with a modest elevation at 24 h and more substantial elevation at 32 h. The observed pattern of expression of cyclin F mRNA with marked elevations at 16-40 h was also consistent with actions in S and G2 phases. Cyclin D1 expression pattern was less consistent with its known role in G1 progression. The alterations in multiple other cell cycle regulators were consistent with previous information obtained in other cell systems. The cycle regulatory mechanics appears to be preserved across broad ranges of cell types.

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Figures

FIG. 1.

FIG. 1.

Endogenous control through cell cycle. Average cycle threshold (CT) values for β2-microglobulin are displayed as the rhodamine low and Hoechst low (LRH) stem cells are samples at various points in cell cycle transit. Error bars represent the standard error between experiments where the number of experiments (n) is indicated for each time point.

FIG. 2.

FIG. 2.

Absolute gene expression at isolation. Individual ΔCT values, which have been corrected with the endogenous control cycle threshold (CT) value, are expressed at isolation (0 h in culture).

FIG. 3.

FIG. 3.

Mapping cyclin gene expression. Fold differences relative to 0-h control in rhodamine low and Hoechst low (LRH) stem cells cultured in TPO, FLT3, and steel factor. Number of experiments (n) is 2 for all values except 32-h samples (n = 6): cyclin D1; cyclin A1; cyclin A2; 40-h samples (n = 4); 48-h samples (n = 3). Statistical significance by Student's t_-test: *P ≤ 0.05; †_P < 0.01; ‡P ≤ 0.005.

FIG. 4.

FIG. 4.

Normalized gene expression of rhodamine low and Hoechst low (LRH) stem cells through cycle. Cells cultured in TPO, FLT3, SCF, and analyzed at 0 through 32 h. Gene expression is represented as fold differences relative to the 0-h control. Variations in gene expression are shown with cycle transit. Statistical significance by Student's _t_-test: *P ≤ 0.05.

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