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Experimental Hematology Mar 2000The additional transplantation of ex vivo-generated megakaryocytic cells might enable the clinician to ameliorate or abrogate high-dose chemotherapy-induced...
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.
MATERIALS AND 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.
Topics: Cell Differentiation; Cells, Cultured; Glycoproteins; Growth Substances; Hematopoiesis; Hematopoietic Stem Cell Mobilization; Hematopoietic Stem Cells; Humans; Megakaryocytes; Stem Cell Factor; Thrombopoietin
PubMed: 10720698
DOI: 10.1016/s0301-472x(99)00152-6 -
Experimental Hematology Oct 2001The signaling pathways induced by promegapoietin (PMP), a family of chimeric growth factors that activate the human IL-3 and c-Mpl receptors, were investigated.
OBJECTIVE
The signaling pathways induced by promegapoietin (PMP), a family of chimeric growth factors that activate the human IL-3 and c-Mpl receptors, were investigated.
METHODS
The biological activity of PMP was examined by receptor binding, cell proliferation, ex vivo expansion of hematopoietic progenitor cells, and in vivo production of platelets. The activation of signaling pathways was examined by Western blot and Northern blot analyses.
RESULTS
Two PMP molecules, PMP-1 and PMP-1a, induced proliferation of cells expressing the IL-3 receptor, c-Mpl, or both receptors and bound to the IL-3 receptor and c-Mpl with high affinity. Ex vivo expansion assays using human bone marrow CD34(+) cells suggested that PMP-1 induced greater total cellular expansion as well as expansion of CD41(+) megakaryocytic precursor cells than IL-3 or c-Mpl ligand alone. Subcutaneous administration of 50 microg/kg of PMP-1 for 10 days to rhesus monkeys resulted in increased platelet production in vivo from a baseline of 357 +/- 45 x 10(3) cells/mL to 1376 +/- 151 x 10(3) cell/mL. PMP-1 induced phosphorylation of the beta(c) subunit of IL-3 receptor and c-Mpl, JAK2, and STAT5b, but not STAT3. PMP-1 induced greater expression of Pim-1, c-Myc, and cyclin D2 than did either an IL-3 receptor agonist or c-Mpl receptor agonist alone. The magnitude of induction of early response genes was similar for PMP and the coaddition of IL-3 receptor agonist and c-Mpl receptor agonist.
CONCLUSION
PMP combines the biological activities of IL-3 and c-Mpl ligand in a single molecule that can simultaneously activate signaling pathways induced by both these ligands.
Topics: Amino Acid Sequence; Animals; Blood Platelets; Bone Marrow Cells; Cell Division; Cell Line; Cells, Cultured; Cloning, Molecular; DNA-Binding Proteins; Female; Growth Substances; Hematopoietic Stem Cells; Humans; Interleukin-3; Janus Kinase 2; Macaca mulatta; Megakaryocytes; Milk Proteins; Molecular Sequence Data; Neoplasm Proteins; Phosphorylation; Phosphotyrosine; Protein-Tyrosine Kinases; Proto-Oncogene Proteins; Receptors, Cytokine; Receptors, Interleukin-3; Receptors, Thrombopoietin; Recombinant Fusion Proteins; STAT5 Transcription Factor; Signal Transduction; Thrombopoietin; Trans-Activators; Transfection
PubMed: 11602319
DOI: 10.1016/s0301-472x(01)00694-4