Purification and Characterization of Heterogeneous Pluripotent Hematopoietic Stem Cell Populations Expressing High Levels of c-kit Receptor

• Published in: Blood Vol. 82; no. 3; pp. 762 - 770
• Main Authors: Orlic, Donald, Fischer, Roxanne, Nishikawa, Shin-lchi, Nienhuis, Arthur W., Bodine, David M.
• Format: Journal Article
• Language: English
• Published: Washington, DC Elsevier Inc 01-08-1993; The Americain Society of Hematology
• Subjects: Animals; Antigens, Surface - analysis; Biological and medical sciences; Bone Marrow Cells; CD4 Antigens - analysis; Cell differentiation, maturation, development, hematopoiesis; Cell physiology; Cell Separation; Female; fms-Like Tyrosine Kinase 3; Fundamental and applied biological sciences. Psychology; Gene Expression; Hematopoietic Stem Cells - cytology; Mice; Mice, Inbred C57BL; Molecular and cellular biology; Protein-Tyrosine Kinases - genetics; Proto-Oncogene Proteins - genetics; Proto-Oncogene Proteins - metabolism; Proto-Oncogene Proteins c-kit; Receptor Protein-Tyrosine Kinases; Receptors, Cell Surface - genetics; RNA, Messenger - genetics; Vertebrata; Mammalia; Mouse; Hematopoiesis; Animal; Stem cell; Rodentia; Hematopoietic cell; Gene expression
• ISSN: 0006-4971; 1528-0020
• DOI: 10.1182/blood.V82.3.762.762

Mouse pluripotent hematopoietic stem cells (PHSC) were fractionated based on size and density using counterflow centrifugal elutriation (CCE). These heterogeneous PHSC populations were further enriched by subtraction of cells with lineage-specific markers (Lin-) followed by positive sorting for c-kit expression. The cells were characterized for their functional and biochemical properties. We defined a subpopulation of c-kit-positive cells that expressed high numbers of c-kit receptors (c-kitBR). One hundred c-kitBR cells from either low- or higher-density fractions were sufficient to repopulate the lymphohematopoietic system in WBB6F1-W/Wv (W/Wv) recipients, whereas no PHSC were found in cells with low (c-kitDULL) or no (c-kitNEG) c-kit expression. Lin- c-kitBR cells were separated into RhoDULL and RhoBR subsets based on their ability to efflux rhodamine 123 (Rho). The PHSC were concentrated in Lin- c-kitBR RhoDULL cells and the number of Lin- c-kitBR RhoBR cells correlated directly with the number of day 12 colony-forming unit-spleen (CFU-S12) in each fraction. We were not able to enrich further for PHSC using monoclonal antibodies to the cell-surface markers AA4.1 or CD4, which have been used by others to isolate PHSC. The small, low-density Lin- c-kitBR subset contained PHSC and few CFU-S12. This enabled us to assay PHSC for expression of the flk-2 gene, which encodes a tyrosine kinase receptor present on fetal liver PHSC. Purified RNA from the low-density Lin- c-kitBR subset did not contain flk-2 mRNA. We suggest that AA4.1, CD4 and flk-2 are expressed as stage-specific markers on PHSC in cell cycle.