CD34(+) CELL EXPANSION AND EXPRESSION OF LINEAGE MARKERS DURING LIQUID CULTURE OF HUMAN PROGENITOR CELLS

A 96-well-based suspension culture system for human hematopoietic progenitor cells has been developed to monitor the commitment and differentiation of CD34(+) cells to specific lineages and the maintenance and expansion of CD34(+) cells in vitro. The expression of maturation and lineage markers on the cells in culture was measured by enzyme-linked immunosorbent assay (ELISA). CD34(+) cells were isolated from umbilical cord blood and fetal liver (90% purity) and were grown in liquid culture in 96-well plates for 10 days. The cells were then fixed with a glutaraldehyde-paraformaldehyde mixture, attaching the cells firmly to the plastic. An ELISA was performed, using appropriate primary antibodies directed against cell surface markers. The expression of four different lineage markers was measured: CD14 (monocyte), CD15 (neutrophil), platelet glycoprotein (GP) IIb/IIIa (CD41a, megakaryocyte) and glycophorin A (erythroid). The two-growth factor combination of interleukin 3 (IL-3) and stem cell factor (SCF) stimulated expression of CD14, CD15 and GP IIb/IIIa. Lineage-restricted growth factors such as erythropoietin (EPO), in combination with SCF, stimulated expression of glycophorin A. The three-factor combination of IL-3, SCF and EPO stimulated expression of all four lineage markers. Other multiple growth factor combinations all stimulated myeloid and megakaryocyte growth, as measured by ELISA and flow cytometry, but erythroid growth was present only when EPO was included in the growth factor mixture. In serum-free medium or plasma-containing medium, CD14 expression was markedly reduced, whereas glycophorin A expression was greatly elevated in serum-free medium. The expression of CD34 was also measured over time in culture and was low hut detectable and slightly increased over the culture period. CD34 expression was generally higher with fetal liver cells than with cord blood cells. This study demonstrates the detection of CD34 and lineage markers by ELISA as an objective, high-capacity method of characterizing progenitor cell growth.