Alpha 4 integrin deficiency in human CD34+ cells engenders a precocious erythroid differentiation ,but inhibits enucleation.

The functional impact of integrin expression in erythropoiesis has been previously emphasized through its decisive influence on erythroid cell-microenvironmental (matrix and cellular) interactions especially under conditions of stress. Beyond that in several in vitro studies the relationship between the two erythroid integrins, a4 and a5, has been incongruous in terms of a proliferative support, either synergistic or antagonistic, whereas a dominant influence of a4 integrin on terminal erythropoiesis in vitro and in vivo has been consistently emphasized. However, the specific cellular and molecular details of this effect have not been defined, especially for human cells. In the present study we have cultured human CD34+ progenitor cells with induced deficiency of a4 integrin (shRNAa4) under erythroid differentiation conditions, in which expanded erythroid progenitor cells are directed to terminal erythroid maturation stages in the absence of any microenvironmental influence. Our data documented that early proliferative expansion in cells lacking a4 expression is significantly limited, but, although erythroid differentiation can proceed normally to terminal stages, their enucleation is drastically impaired. This novel aspect of a4 integrin participation in the enucleation process in vitro resonates on the lack of in vivo enucleation of primitive erythroid cells lacking any integrin expression but affecting adult cells only under stress conditions. CD34+ hematopoietic progenitor cells from three different donors were treated with shRNA specific to either a4 integrin, b1 integrin, a scrambled control, or no virus as an additional control. All samples were harvested at the same day of erythroid culture in biological triplicate. In total, 12 samples were processed: 3 shRNA for a4 + 3 shRNA for b1 + 3 shRNA for scrambled control + 3 WT no transduction.