P Han1, X H Guo, C J Story. 1. Department of Haematology, Women's and Children's Hospital, North Adelaide, South Australia, Australia.
Abstract
BACKGROUND: Megakaryopoiesis occurs as a result of a complex interaction between hemopoietic cells, stromal cells, hemopoietic growth factors and extracellular matrix (ECM). Megakaryocyte growth and development factor (MGDF) or thrombopoietin is the main growth factor that induces megakaryocyte commitment. The role of adhesion proteins in the generation of megakaryocytic progenitors has not been well studied. METHODS: We isolated CD34(+) cells from umbilical cord (UC) blood and cultured them in serum-free medium containing IL-3, IL-6, SCF and MGDF, with or without fibronectin. Cultures were sampled at Days 4 and 8 for cell count, immunophenotyping by flow cytometry, and megakaryocyte colony-forming units (CFU-Mk) assay. RESULTS: Fibronectin in synergy with MGDF increased both total and CD34(+) cell count. Immunophenotyping of the CD34(+) and CD34(-) cells showed that the percentage expression of CD61 and CD41a on CD34(-) cells was increased by fibronectin compared with CD34(+) cells by Day 8 of culture. The CFU-Mk assay confirmed that fibronectin increased generation of megakaryocytic progenitors by 2.4-fold by Day 8 of culture, but the absolute number was less than that of Day 4 culture. The cells in the CFU-Mk colonies derived from culture containing fibronectin were larger than those from cytokine-only culture. CONCLUSION: It was concluded that the addition of fibronectin to the culture system enhanced MGDF, induced ex vivo expansion of megakaryocytic progenitors from CD34(+) cells, as well as increasing their maturation towards later stages of megakaryocyte differentiation.
BACKGROUND: Megakaryopoiesis occurs as a result of a complex interaction between hemopoietic cells, stromal cells, hemopoietic growth factors and extracellular matrix (ECM). Megakaryocyte growth and development factor (MGDF) or thrombopoietin is the main growth factor that induces megakaryocyte commitment. The role of adhesion proteins in the generation of megakaryocytic progenitors has not been well studied. METHODS: We isolated CD34(+) cells from umbilical cord (UC) blood and cultured them in serum-free medium containing IL-3, IL-6, SCF and MGDF, with or without fibronectin. Cultures were sampled at Days 4 and 8 for cell count, immunophenotyping by flow cytometry, and megakaryocyte colony-forming units (CFU-Mk) assay. RESULTS:Fibronectin in synergy with MGDF increased both total and CD34(+) cell count. Immunophenotyping of the CD34(+) and CD34(-) cells showed that the percentage expression of CD61 and CD41a on CD34(-) cells was increased by fibronectin compared with CD34(+) cells by Day 8 of culture. The CFU-Mk assay confirmed that fibronectin increased generation of megakaryocytic progenitors by 2.4-fold by Day 8 of culture, but the absolute number was less than that of Day 4 culture. The cells in the CFU-Mk colonies derived from culture containing fibronectin were larger than those from cytokine-only culture. CONCLUSION: It was concluded that the addition of fibronectin to the culture system enhanced MGDF, induced ex vivo expansion of megakaryocytic progenitors from CD34(+) cells, as well as increasing their maturation towards later stages of megakaryocyte differentiation.