A new culture and quantitation system for megakaryocyte growth using cord blood CD34+ cells and the GPIIb/IIIa marker.

A new culture and quantitation system has been established for growth of megakaryocyte-lineage cells from human progenitor cells. CD34+ progenitor cells were enriched from umbilical cord blood using an avidin-biotin immunoadsorption process. These cells were preincubated in bulk liquid culture for 3 to 4 days in the presence of the growth factors interleukin-3 (IL-3) and IL-6. The cells were then washed and seeded at 5000 cells/well in 96-well plates that contained a variety of test samples. The plates were incubated for 7 days, and the cells were then washed, transferred to ELISA plates, and fixed. Megakaryocyte growth was determined by an ELISA for the platelet glycoprotein (GP) IIb/IIIa, an abundant membrane protein found on cells committed to the megakaryocyte lineage. The growth factor IL-3 was found to produce a very strong signal in this assay. The addition of granulocyte-macrophage colony-stimulating factor (GM-CSF), IL-6, stem cell factor (SCF), or leukemia inhibitory factor (LIF) to low levels of IL-3 also stimulated megakaryocyte growth, as measured by IIb/IIIa expression. Plasma from patients with aplastic anemia was also stimulatory in this assay, and showed marked synergy with IL-3. This progenitor cell culture system, due to its judicious use of progenitor cells and an automated, 96-well quantitation method, allows for screening large numbers of test samples and multiple combinations and concentrations of growth factors.