Abstract
Background We have shown previously that fibronectin (FN) together with megakaryocyte (Mk) growth and development factor (MGDF) enhanced generation of Mk progenitors determined by colony-forming unit (CFU)-Mk assay. MGDF can activate β1-integrins on progenitor cells and increase binding to FN. We studied the role of β1-integrin–tetraspanin complexes by which FN may enhance megakaryopoiesis.
Methods Cord blood CD34+ cells were cultured for up to 8 days in serum-free medium containing IL-3, IL-6 and SCF with or without MGDF in the presence or absence of FN. Immunofluorescence flow cytometry was used to monitor changes in β1-integrin–tetraspanin complexes. CFU-Mk assay was used to assess Mk commitment.
Results The cocktail of cytokines irrespective of the presence of MGDF altered the percentage expression of β1-integrins CD49d and CD49e on CD34+ cells. CD49d expression fell initially (98% to 15%) and then rose to 75%, whereas CD49e progressively increased over the 8 days of culture, from 5.4% to 22%. However, with the addition of FN, similar changes in the expression of β1-integrins were observed but the expression was maintained at a higher level. MGDF and FN increased expression of tetraspanin molecules, CD63 and CD151, as well as their co-expression with the β1-integrins on both the CD34+ and CD34− cells (e.g. and increase from 0% to 80% co-expression of CD49d and CD151 on CD34+). Blocking of β1-integrins or the tetraspanin CD151 with the respective MAb reduced Mk progenitor generation in a stromal cell model.
Discussion FN enhanced Mk progenitor generation through modulation of β1-integrin–tetraspanin complexes, such as CD151/CD49d.