Quantification of Hematopoietic Progenitor Cells

RBCs from PB were lysed with BD Pharm Lyse buffer (BD Biosciences). Nucleated cells were subsequently washed twice and used for CFU-GM colonies. Briefly, cells were resuspended in human methylcellulose base media provided by the manufacturer (R&D Systems, Inc., Minneapolis, MN, USA), supplemented with 25 ng/ml recombinant murine granulocyte macrophage colony-stimulating factor (mGM-CSF) and 10 ng/ml recombinant murine interleukin 3 (mIL-3; Millipore, Billerica, MA, USA). Cultures were incubated for 7 days, at which time they were scored for the number of CFU-GM colonies under an inverted microscope. To evaluate the number of clonogenic progenitor cells, BMMNCs were supplemented with erythropoietin (5 U/ml; Stem Cell Tech., Vancouver, BC, Canada) plus stem cell factor (5 ng/ml) and resuspended in methylcellulose base medium (for determining the number of burst-forming units-erythroid), supplemented with thrombopoietin (100 ng/ml) plus murine interleukin 3 (10 ng/ml), and the resuspended plasma clots used for determining the number of CFU-megakaryocytes. The CFU-GM assay was performed as above. Cultures were incubated for 7 days (37°C, 95% humidity, and 5% CO₂), at which time they were scored under an inverted microscope for the number of each type of colony, as described^{17 (link),19 (link)}.