Differential Expression of Hematopoietic Genes in Murine Embryonic E14 Stem Cells upon G-CSF and IL-3 Stimulation.

Introduction:

G-CSF is used widely in humans as in stem cell mobilization protocols for clinical transplantation. Even though it’s mechanisms of action on differentiated cells are well described, it’s effects on stem cells are widely unknown. IL-3 acts as a survival factor without differentiational effects. The presence of G-CSF and IL-3 receptors on embryonic stem cells have been described. We here use murine embryonic E14 stem cells (ESC) to study cellular and molecular events induced by G-CSF and IL-3 during early hematopoietic development.

Methods:

Cells were precultured in standard medium (DMEM, 20% FCS) for 6 days and then kept as embryoid bodies (EBs) for 0, 5, 10, 15, 20, 25, 30 days either in standard or medium continuously supplemented with G-CSF (50 ng/ml) and/or IL-3 (10 ng/ml). Suspended cells were analyzed for CD 90 (thy1) positivity by flow cytometry. CD13 stained cells of embryoid bodies were examined by fluorescent microscopy. cDNA was analyzed for PU.1, GATA-2, Flk-1, SCL, Epo, HoxB4, LMO2, LMO4 and cyclin D1 activation by Taqman PCR.

Results:

First hematopoietic specific CD90 positivity is found on embryoid bodies at day 6+5, either with or without cytokine stimulation. There is a marked increase in hematopoietic development at day 6+10 consisting in 5% CD90 positivity declining at later time points in unstimulated EBs. Interestingly the fraction of CD90 positive cells could not be augmented by G-CSF alone. Upon IL-3 stimulation the fraction of CD90 positive EBs increased twofolded as compared to unstimulated controls. This remained for the entire duration until day 6+30. Double stimulation with IL-3 and G-CSF abrogated this stimulatory effect. We selected a number of genes of interest in hematopoietic development based on published gene expression profiling studies. Under G-CSF stimulation there is a continuous minor upregulation of some of these genes. Interestingly, under IL-3 stimulation most of these genes (GATA-2, SCL, PU.1, Epo and HoxB4) are markedly downregulated while CD90 positive cell fractions are stimulated. This downregulation is most prominent at day 6+10 and 6+15.

Conclusion:

G-CSF activates key genes of hematopoietic development in E14 ESCs without causing differentiation. IL-3 activation of hematopoiesis in EBs parallels downregulation of important genes known as activators of adult hematopoiesis. This could mean that causally determined effects of growth factors may differ at early and committed progenitor cell levels.