title: Producing megakaryocytes from a human peripheral blood source.
abstract: Cultured megakaryocytes could prove useful in the study of human diseases, but it is difficult to produce sufficient numbers for study. We describe and evaluate the use of an expansion process to develop mature megakaryocytes from peripheral blood-derived human hematopoietic stem and progenitor cells (HSPCs).
  
  
  
  HSPCs (CD34+) were isolated from peripheral blood by positive selection and expanded using an optimal CD34+ expansion supplement. We evaluated megakaryocyte growth, maturation, and morphology in response to thrombopoietin (TPO) stimulation using flow cytometry and electron microscopy. TPO demonstrated a dose-dependent stimulatory effect on both megakaryocyte number and maturation.
  
  
  
  From 90 to 120 mL of unmanipulated peripheral blood, we isolated a mean of 1.5 × 10(5) HSPCs (1.5 × 10(3) cells/mL of whole blood). HSPCs expanded nine-fold after a 4-day culture using an expansion supplement. Expanded cells were cultured for an additional 8 days with TPO (20 ng/mL), which resulted in a 2.9-fold increase in megakaryocytic cells where 83% of live cells expressed CD41a+, a marker of megakaryocyte commitment, and 50% expressed CD42b+, a marker for megakaryocyte maturation. The expanded HSPCs responded to TPO stimulation to yield more than 1.0 × 10(6) megakaryocytes. This cell number was sufficient for morphologic studies that demonstrated these expanded HSPCs produced mature polyploid megakaryocytes capable of forming proplatelet extensions.
  
  
  
  Peripheral blood HSPCs can be expanded and differentiated into functional, mature megakaryocytes, a finding that supports the use of this process to study inherent platelet (PLT) production disorders as well as study factors that impair normal PLT production.
authors.affiliation: Department of Biochemistry and Biomedical Sciences, McMaster University, Hamilton, Ontario.
authors.forename: Nikola
authors.initials: N
authors.lastname: Ivetic
authors.affiliation: Department of Medicine, Michael G. DeGroote School of Medicine, McMaster University, Hamilton, Ontario.
authors.forename: Ishac
authors.initials: I
authors.lastname: Nazi
authors.affiliation: Department of Medicine, Michael G. DeGroote School of Medicine, McMaster University, Hamilton, Ontario.
authors.forename: Nadia
authors.initials: N
authors.lastname: Karim
authors.affiliation: Department of Medicine, Michael G. DeGroote School of Medicine, McMaster University, Hamilton, Ontario.
authors.forename: Rumi
authors.initials: R
authors.lastname: Clare
authors.affiliation: Department of Medicine, Michael G. DeGroote School of Medicine, McMaster University, Hamilton, Ontario.
authors.forename: James W
authors.initials: JW
authors.lastname: Smith
authors.affiliation: Department of Medicine, Michael G. DeGroote School of Medicine, McMaster University, Hamilton, Ontario.|Department of Pathology and Molecular Medicine, McMaster University.
authors.forename: Jane C
authors.initials: JC
authors.lastname: Moore
authors.affiliation: Department of Biochemistry and Biomedical Sciences, McMaster University, Hamilton, Ontario.
authors.forename: Kristin J
authors.initials: KJ
authors.lastname: Hope
authors.affiliation: Department of Medicine, Michael G. DeGroote School of Medicine, McMaster University, Hamilton, Ontario.
authors.forename: John G
authors.initials: JG
authors.lastname: Kelton
authors.affiliation: Department of Medicine, Michael G. DeGroote School of Medicine, McMaster University, Hamilton, Ontario.|Canadian Blood Services, Hamilton, Ontario, Canada.
authors.forename: Donald M
authors.initials: DM
authors.lastname: Arnold
mesh_terms: D018952:Antigens, CD34 / Q000032:analysis; D018929:Cell Culture Techniques; D002454:Cell Differentiation; D049109:Cell Proliferation; D002478:Cells, Cultured; D006801:Humans; D008533:Megakaryocytes / Q000166:cytology*; D000072916:Peripheral Blood Stem Cells / Q000166:cytology*; D019038:Platelet Glycoprotein GPIb-IX Complex / Q000032:analysis; D034061:Thrombopoiesis / Q000187:drug effects*; D013926:Thrombopoietin / Q000494:pharmacology; D013997:Time Factors
chemical_list: D018952:Antigens, CD34; D019038:Platelet Glycoprotein GPIb-IX Complex; D013926:Thrombopoietin