A new culture and quantitation system for megakaryocyte growth using cord blood CD34+ cells and the GPIIb/IIIa marker.
A new culture and quantitation system has been established for growth of megakaryocyte-lineage cells from human progenitor cells. CD34+ progenitor cells were enriched from umbilical cord blood using an avidin-biotin immunoadsorption process. These cells were preincubated in bulk liquid culture for 3 to 4 days in the presence of the growth factors interleukin-3 (IL-3) and IL-6. The cells were then washed and seeded at 5000 cells/well in 96-well plates that contained a variety of test samples. The plates were incubated for 7 days, and the cells were then washed, transferred to ELISA plates, and fixed. Megakaryocyte growth was determined by an ELISA for the platelet glycoprotein (GP) IIb/IIIa, an abundant membrane protein found on cells committed to the megakaryocyte lineage. The growth factor IL-3 was found to produce a very strong signal in this assay. The addition of granulocyte-macrophage colony-stimulating factor (GM-CSF), IL-6, stem cell factor (SCF), or leukemia inhibitory factor (LIF) to low levels of IL-3 also stimulated megakaryocyte growth, as measured by IIb/IIIa expression. Plasma from patients with aplastic anemia was also stimulatory in this assay, and showed marked synergy with IL-3. This progenitor cell culture system, due to its judicious use of progenitor cells and an automated, 96-well quantitation method, allows for screening large numbers of test samples and multiple combinations and concentrations of growth factors.
Warren
M K
MK
Department of Hematology, Otsuka America Pharmaceutical, Inc., Rockville, MD 20850.
Guertin
M
M
Rudzinski
I
I
Seidman
M M
MM
D015703:Antigens, CD / Q000097:blood*; D018952:Antigens, CD34; D015415:Biomarkers / Q000097:blood; D002455:Cell Division / Q000502:physiology; D002478:Cells, Cultured; D004797:Enzyme-Linked Immunosorbent Assay; D005312:Fetal Blood / Q000166:cytology; D006133:Growth Substances / Q000494:pharmacology; D006412:Hematopoietic Stem Cells / Q000166:cytology* / Q000276:immunology; D006801:Humans; D008533:Megakaryocytes / Q000737:chemistry / Q000166:cytology*; D010980:Platelet Membrane Glycoproteins / Q000032:analysis*
D015703:Antigens, CD; D018952:Antigens, CD34; D015415:Biomarkers; D006133:Growth Substances; D010980:Platelet Membrane Glycoproteins