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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

Institute for Med. Oncology and Hematology, Med. Clinic 5, Nürnberg City Hospital, Nürnberg, Germany

Key Words. Thrombopoietin • Human • Progenitor cells • Megakaryocytes • Lineage specificity • Maturation • Serum-free culture

Dr. Josef Birkmann, Inst. f. Med. Onkologie und Hämatologie, Med. Klinik 5, Flurstrasse 17, D-90340 Nürnberg, Germany.

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.

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