Expression of a foreign protein in human megakaryocytes and platelets by retrovirally mediated gene transfer

Recent progress in the culture of human megakaryocytes (MKs) has led to the capacity to produce platelets in vitro. This capability enables investigat ion into the possibility of modifying platelet structure and/or function by genetically altering the MK. To this end, a cDNA for the murine CD9 (mCD9) cell surface protein was introduced into MK progenitors by retrovirally me diated gene transfer and subsequently detected in cultured MKs with a monoc lonal antibody (MoAb) that specifically recognizes the murine protein. CD34 + human peripheral blood or marrow progenitors, enriched by immunomagnetic bead selection, were cultured for 5 days in the presence of growth factors, including stem cell factor and thrombopoietin, to induct MK progenitors in to the cell cycle. The stimulated cells were then cocultured with the mCD9 retroviral producer cell line for 3 days, followed by culture in serum-depl eted medium for 3 to 7 additional days. Flow cytometry analysis using the a nti-CD9 MoAb and TAB, a MoAb recognizing human GPIIb, revealed that a large proportion (40-100%) of the MKs expressed mCD9. To ascertain whether these cells were capable of producing mCD9+ platelets, flow cytometry analysis w as performed at a time when proplatelets were observed in the culture. mCD9 was detected in up to 59% of the TAB+ platelet-sized particles. Because de teriorating MKs can produce platelet-sized particles in vitro, experiments were performed to determine whether mCD9+ TABS particles were functionally active. Addition of phorbol myristate acetate resulted in the redistributio n of P-selectin (CD62) from the alpha granule to the platelet surface as de tected by MoAbs S12 and G5 in three-color flow cytometry analyses. These st udies showed that up to 76% of the mCD9+ TABS particles were functionally a ctive. The data show that retrovirally mediated gene transfer is a viable a pproach for genetically altering MK progenitors, resulting in platelets tha t express heterologous proteins. (C) 1999 International Society for Experim ental Hematology. Published by Elsevier Science Inc.