Dose Dependent Effects of STAT5 on Proliferation, Differentiation and Self Renewal of Hematopoietic Stem/Progenitor Cells.

To study the dose dependent effects of ectopically expressed STAT5A in cordblood (CB) CD34⁺ cells, we developed a tamoxifen-inducible STAT5A expression system. Constitutively active STAT5A (STAT5A1*6) was fused to the ER domain to facilitate its activation upon stimulation of the transduced cells by the addition of 4OHT. Transactivation and DNA binding studies revealed that STAT5A could be activated to different levels in a controlled way by addition of different concentrations of 4OHT. CB CD34⁺ cells were retrovirally transduced with this construct and the effects on proliferation, differentiation and self renewal was studied.

In an MS5 coculture system, we demonstrated that a high dose of 4OHT (500nM) resulted in a rapid burst of erythropoiesis and a block in myeloid differentiation (as previously demonstrated using STAT5A1*6 without an ER domain). Moderate levels of 4OHT (20nM) however, resulted in a much higher proliferation of not yet differentiated cells, ultimately resulting in a higher expansion. This higher expansion coincided with a higher frequency of early (day10) cobblestones (without 4OHT: 1,5 cobblestones per 10⁴ plated cells, 20nM 4OHT: 1750/10⁴, 500nM 4OHT:200/10⁴). RT-PCR of STAT5A1*6-ER transduced cells revealed a rapid downregulation of C/EBPalpha mRNA upon stimulation with 4OHT, with a dose response curve inversely correlated to the number of early cobblestones, suggesting that these two processes are linked.

When STAT5A1*6-ER transduced cells were cultured in methylcellulose, stimulation with increasing concentrations of 4OHT resulted in decreasing numbers of colonies (myeloid as well as erythroid). Interestingly, whereas colonies from unstimulated (control) cultures and 500nM 4OHT stimulated cultures could not be secondary plated, cultures stimulated with only very low concentrations of 4OHT (1 nM) could be secondary plated, giving rise to only myeloid colony formation. These findings demonstrate that the level of activation of STAT5 affects the proliferation and differentiation in a separate manner; low levels of STAT5A activation results in self renewal of the progenitor/stem cell pool cells, whereas high levels of STAT5A induces a rapid differentiation at the expense of proliferation and self renewal.

These data correspond well to our findings in AML cells. Downregulation of the moderate constitutive STAT5 activation in CD34⁺ AML cells by STAT5 RNAi affects especially the proliferative capacity of the AML cells. In conclusion, we hypothesise that low constitutive levels of active STAT5 might contribute to leukemic transformation.