Integrin engagement on fibronectin modulates the protein levels of c-myb, CD34, P21, and P130 in primary human hematopoietic progenitors

Interaction between stem cells and stroma or fibronectin (FN CH-296) during ex vivo culture is critical in maintaining the long-term engraftment potentiality of human hematopoietic stem cells in immune deficient bnx mice (Dao et al., 1998). In the current study, we examined molecular pathways by which integrin engagement may alter the pluripotentiality of primitive hematopoietic progenitors. Bone marrow and cord blood CD34+ progenitors were cultured with IL-3, IL-6, and SCF for 2,3, or 7 days on BSA- or FN-coated plates. The majority of cells on BSA plates remained in suspension throughout the three time points. After 2 days on FN plates, more than 80% of the cells (FN-Ad) spread firmly; however, by 7 days, nearly half of the cells were nonadherent (FN-NA). Subcellular fractionation followed by immunoblotting for c-myb, GATA-2, CD34, p130, and p21 were performed. After 2 days culture, BSA cells showed a detectable level of p21, a protein that has been linked to differentiation. After 7 days in culture, there was a decrease in the levels of nuclear c-myb and CD34 protein in BSA cells as well as FN-NA cells, in comparison to FN-Ad cells. In addition, FN-Ad cells showed a markedly higher expression of an Rb-related G0 protein, p130. Our data suggests the following conclusions: 1. Engagement of integrins on FN maintains expression of c-myb and CD34 which are both present in early primitive hematopoietic progenitors and 2. Stimulation with growth factors on Fn induces cell division, generating two populations of cells which differ at the molecular levels.