Effects of recombinant human thrombopoietin alone and in combination with erythropoietin and early-acting cytokines on human mobilized purified CD34(+) progenitor cells cultured in serum-depleted medium

The effects of recombinant thrombopoietin (TPO) alone and in combination with erythropoietin (EPO) and early-acting cytokines such as interleukin 3 (IL-3), stem cell factor (SCF) and GM-CSF on highly purified mobilized human CD34(+) progenitor cells were studied in a serum-depleted culture system, Eight leukapheresis samples were cultured for seven days and analyzed; aliquots sere replated and re-evaluated on day 12. Three-color bow cytometry was used together with morphologic analysis to determine proliferation and megakaryocytic or erythroid maturation, TPO alone was sufficient for cell survival and proliferation in serum-depleted medium, In the absence of other growth factors, almost all CD34(+) cells differentiated along the megakaryocytic pathway within 12 days, Concomitantly, the progenitor cells gradually acquired the morphologic features of mature megakaryocytes. After exposure to TPO for one week, 50% of the cells still expressed CD34; by day 12 the remaining CD34(+) cells (11%) were all coexpressing CD41. TPO alone did not support proliferation of glycophorin-A-positive cells. The addition of TPO to early-acting cytokines (EPO, GM-CSP, SCF and/or IL-3) not only increased the overall megakaryocyte expansion, but also generated a different maturation pattern of the CD41(+) megakaryocyte progenitors, It further doubled the number of erythroid cells and c-kit(+) cells in the second week of culture. Interestingly, the overall number of CD34(+) cells was increased about fivefold when TPO was added to the early-acting cytokines, with a marked expansion of the CD34(+)/CD41(+) and CD34(+)/CD117(+) subpopulations, TPO can augment the pool of committed progenitors, thereby increasing the number of its own target cells and the number of EPO-responsive cells, These properties make TPO an interesting cytokine for the ex vivo expansion of human progenitor cells.