Isolation of pluripotent hemopoietic stem cells and clonable precursor cells of erythrocytes, granulocytes, macrophages and megakaryocytes from mouse bone marrow.

Overview

Murine pluripotent hematopoietic stem cells and precursor cells with restricted commitment to erythrocytes, granulocytes and macrophages as well as megakaryocytes have been purified 30- to 50-fold from mouse bone marrow cells. Purification was achieved by a three-step procedure. Bone marrow cell populations free of erythroid cells and lymphocytes were obtained by culturing the cells for several weeks. Macrophages and adherent polymorphic neutrophils (PMN) were removed by adherence to plastic. The remainder of the PMN along with more primitive granulocytes (but not promyelocytes and some monocytoid cells) were removed either by neutral density centrifugation or by differential centrifugation after rosette formation with sheep erythrocytes coated with immunoglobulin (EA rosettes). The remaining population of marrow-derived cells contained 40-66% blast cells, 20-35% promyelocytes and 5-10% other cells (usually PMNs and monocytes). Using cloning techniques to detect immature hemopoietic cells, this population contained 15-35% granulocyte-macrophage progenitor cells, approximately 0.2% erythroid burst-forming cells, approximately 0.1% megakaryocyte progenitor cells and 1-3% pluripotent stem cells (based on seeding efficiency 0.06).