The expansion of megakaryocyte progenitors from CD34+-enriched mobilized peripheral blood stem cells is inhibited by Flt3-L.
This study aimed to determine the optimal growth factor combination for expansion of megakaryocyte (Mk) progenitors with clonogenic potential from CD34+-enriched mobilized peripheral blood stem cells (PBSC). Mobilized PBSC were monocyte depleted and CD34+ enriched, then cultured with various combinations of interleukin-3 (IL-3), IL-6, IL-11, Flt3 ligand (Flt3-L), stem cell factor (SCF), granulocyte-macrophage colonystimulating factor (GM-CSF), and erythropoietin (EPO), using a 2(7-3) IV fractional factorial design. Expansion of Mk committed progenitors (CD41+) and primitive precursors (CD61+ CD34+) was determined using FACS and colony-forming assays. Amplification of Mk progenitor production was attributed to IL-3 (p < 0.002), SCF (p < 0.001), and GM-CSF (p < 0.05). Flt3-L inhibited the production of total CD61+ cells (p < 0.05), CD61+CD34+ cells (p < 0.03), and total CD41a+ cells (p < 0.01). Addition of Flt3-L to the optimum growth factor combination of megakaryocyte growth and development factor (MGDF), SCF, IL-3, and GM-CSF caused the greatest increase in total nucleated cells but reduced Mk progenitor expansion. There was also a 20% reduction in Mk+ colonies from cells expanded in the presence of Flt3-L. Factorial analysis identified the optimal combination of growth factors required to expand Mk precursors with clonogenic potential. The addition of Flt3-L to the optimal combination of MGDF, SCF, IL-3, and GM-CSF reduced both the fold expansion of Mk progenitors and Mk colony numbers.
Department of Clinical Haematology, St. George Hospital, and University of New South Wales, Sydney, Australia.
J
J
Case
C
C
Hicks
A
A
Trickett
Y L
YL
Kwan
A
A
Manoharan
D018952:Antigens, CD34 / Q000096:biosynthesis* / Q000097:blood; D002454:Cell Differentiation / Q000502:physiology*; D049109:Cell Proliferation; D002478:Cells, Cultured; D002999:Clone Cells; D003114:Colony-Forming Units Assay; D005163:Factor Analysis, Statistical; D016178:Granulocyte-Macrophage Colony-Stimulating Factor / Q000097:blood / Q000502:physiology; D006131:Growth Inhibitors / Q000097:blood / Q000502:physiology*; D019650:Hematopoietic Stem Cell Mobilization / Q000706:statistics & numerical data; D006412:Hematopoietic Stem Cells / Q000166:cytology / Q000378:metabolism / Q000502:physiology*; D006801:Humans; D007377:Interleukin-3 / Q000097:blood / Q000502:physiology; D008024:Ligands; D008533:Megakaryocytes / Q000166:cytology / Q000378:metabolism* / Q000502:physiology; D008565:Membrane Proteins / Q000097:blood / Q000502:physiology*; D019089:Stem Cell Factor / Q000097:blood / Q000502:physiology; D013926:Thrombopoietin / Q000097:blood / Q000502:physiology
D018952:Antigens, CD34; D006131:Growth Inhibitors; C508596:IL3 protein, human; D007377:Interleukin-3; D008024:Ligands; D008565:Membrane Proteins; D019089:Stem Cell Factor; C084881:flt3 ligand protein; D016178:Granulocyte-Macrophage Colony-Stimulating Factor; D013926:Thrombopoietin