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 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.
Birkmann
J
J
Institute for Med. Oncology and Hematology, Med. Clinic 5, Nürnberg City Hospital, Germany.
Oez
S
S
Smetak
M
M
Kaiser
G
G
Kappauf
H
H
Gallmeier
W M
WM
D018952:Antigens, CD34 / Q000097:blood; D015415:Biomarkers / Q000097:blood; D002455:Cell Division / Q000187:drug effects; D002465:Cell Movement; D002478:Cells, Cultured; D016895:Culture Media, Serum-Free; D004357:Drug Synergism; D004359:Drug Therapy, Combination; D004920:Erythropoiesis / Q000187:drug effects; D004921:Erythropoietin / Q000494:pharmacology*; D016178:Granulocyte-Macrophage Colony-Stimulating Factor / Q000494:pharmacology; D006410:Hematopoiesis / Q000187:drug effects; D006412:Hematopoietic Stem Cells / Q000166:cytology* / Q000276:immunology*; D006801:Humans; D007377:Interleukin-3 / Q000494:pharmacology; D008533:Megakaryocytes / Q000166:cytology; D019039:Platelet Glycoprotein GPIIb-IIIa Complex / Q000032:analysis; D011994:Recombinant Proteins / Q000494:pharmacology; D019089:Stem Cell Factor / Q000494:pharmacology; D013926:Thrombopoietin / Q000494:pharmacology*
D018952:Antigens, CD34; D015415:Biomarkers; D016895:Culture Media, Serum-Free; D007377:Interleukin-3; D019039:Platelet Glycoprotein GPIIb-IIIa Complex; D011994:Recombinant Proteins; D019089:Stem Cell Factor; D004921:Erythropoietin; D016178:Granulocyte-Macrophage Colony-Stimulating Factor; D013926:Thrombopoietin