title: Effective ex vivo generation of megakaryocytic cells from mobilized peripheral blood CD34(+) cells with stem cell factor and promegapoietin.
abstract: OBJECTIVE
  The additional transplantation of ex vivo-generated megakaryocytic cells might enable the clinician to ameliorate or abrogate high-dose chemotherapy-induced thrombocytopenia. Therefore, the ex vivo expansion of CD34(+) PBPC was systematically studied aiming for an optimum production of megakaryocytic cells.
  
  
  METHODS
  CD34(+) PBPC were cultured in serum-free medium comparing different (n = 23) combinations of stem cell factor (SCF) (S), IL-1beta (1), IL-3 (3), IL-6 (6), erythropoietin (EPO) (E), thrombopoietin (TPO) (T) and promegapoietin (PMP, a novel chimeric IL-3/TPO receptor agonist). Ex vivo-generated cells were assessed by flow cytometry, morphology, and progenitor cell assays.
  
  
  RESULTS
  Addition of TPO to cultures stimulated with S163E, a potent progenitor cell expansion cocktail previously described by our group, effectively induced the generation of CD61(+) cells (day 12: 31.4 +/- 7.9%). The addition of PMP tended to be more effective than TPO +/- IL-3. Whereas EPO was not required to maximize TPO- or PMP-induced megakaryocytic cell production, the use of IL-6 and IL-1beta augmented cellular expansion as well as CD61(+) cell production rates in the majority of cytokine combinations studied. Thus, the most effective CD61(+) cell expansion cocktail consisted of S163 + PMP which resulted in 65.9 +/- 3.0% CD61(+) at day 12 and an overall production of 40.7 +/- 4.5 CD61(+) cells per seeded CD34(+) PBPC. However, the basic 2-factor combination S + PMP also allowed for an effective CD61(+) cell production (day 12 CD61(+) cell production: 15.1 +/- 1.6). Moreover, maximum amplification of CFU-Meg was observed after 7 days using this two-factor cocktail (12.9 +/- 2.6-fold). The majority of CD61(+) cells generated in TPO- or PMP-based medium were low-ploidy 4N and 8N cells, and ex vivo-generated CD61(+), CD41(+), and CD42b(+) cells were mainly double positive for FACS-measured intracellular von Willebrand Factor (vWF) (76.7 +/- 3.3%, 58.8 +/- 4.4%, and 82.7 +/- 2.5%, respectively).
  
  
  CONCLUSIONS
  Taken together, this study demonstrates that megakaryocytic cells can be effectively produced ex vivo with as little as two-factors (SCF + PMP), an approach that might be favorably employed in a clinical expansion trial aiming to ameliorate high-dose chemotherapy-induced thrombocytopenia.
authors.affiliation: Departments of Internal Medicine, Hematology, Oncology, University of Tübingen, Germany.
authors.forename: K
authors.initials: K
authors.lastname: Kratz-Albers
authors.forename: S
authors.initials: S
authors.lastname: Scheding
authors.forename: R
authors.initials: R
authors.lastname: Möhle
authors.forename: H J
authors.initials: HJ
authors.lastname: Bühring
authors.forename: C M
authors.initials: CM
authors.lastname: Baum
authors.forename: J P
authors.initials: JP
authors.lastname: Mc Kearn
authors.forename: T
authors.initials: T
authors.lastname: Büchner
authors.forename: L
authors.initials: L
authors.lastname: Kanz
authors.forename: W
authors.initials: W
authors.lastname: Brugger
mesh_terms: D002454:Cell Differentiation / Q000187:drug effects; D002478:Cells, Cultured; D006023:Glycoproteins / Q000494:pharmacology*; D006133:Growth Substances / Q000494:pharmacology*; D006410:Hematopoiesis; D019650:Hematopoietic Stem Cell Mobilization; D006412:Hematopoietic Stem Cells / Q000473:pathology*; D006801:Humans; D008533:Megakaryocytes / Q000473:pathology*; D019089:Stem Cell Factor / Q000494:pharmacology*; D013926:Thrombopoietin
chemical_list: D006023:Glycoproteins; D006133:Growth Substances; D019089:Stem Cell Factor; C090367:megapoietin protein, Ovis aries; D013926:Thrombopoietin