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

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Abstract
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 were replated and re‐evaluated on day 12. Three‐color flow 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‐CSF, 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.