We have previously shown that engagement of the integrins VLA-4 and VLA-5 to the fibronectin fragment CH-296 in combination with cytokines sustained the capacity of cultured human CD34+ cells to undergo hematopoiesis in immunodeficient mice for 7 to 12 months, whereas this capacity was rapidly lost in cells cultured in suspension with the same cytokines. In the current study, we assessed the molecular pathways that might explain the loss of long-term engraftment capacity in cells cultured in suspension. Although the cell cycle profile was similar between cells cultured in suspension versus on fibronectin, levels of cell death were higher in the suspended cultures. While the CDK inhibitors p27Kip1 and p57Kip2 were present at equal levels in cells from both cultures, low levels of p21Cip1 were detectable only in the cytoplasmic compartment of cells cultured in suspension. Cytoplasmic location of p21Cip1 has been linked to monocytic differentiation. The levels of c-myb and GATA-2, transcription factors associated with stem cell maintenance, were higher in cells cultured on fibronectin as compared with suspension. In contrast, the levels of PU.1, which is induced during myeloid differentiation, were higher in cells cultured in suspension. There were no significant differences in surface expression of CD34 on the cells after culture, but total CD34 protein, assessed by immunoblotting, was significantly higher in cells cultured on fibronectin. Our data suggest that, in the presence of cytokines, the engagement of VLA-4 and VLA-5 integrins to the fibronectin fragment CH-296 preserves the expression of specific transcription factors associated with primitive stem cell maintenance. In contrast, a lack of integrin engagement leads to the induction of cellular markers associated with myeloid differentiation.
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