Enrichment, Characterization, and Responsiveness of Single Primitive CD34+++ Human Umbilical Cord Blood Hematopoietic Progenitors With High Proliferative and Replating Potential

• Published in: Blood 1993-01, Vol.81 (1), p.41-48
• Main Authors: Lu, Li, Xiao, Mang, Shen, Rong-Nian, Grigsby, Susan, Broxmeyer, Hal E.
• Format: Article
• Language: English
• Subjects: Antigens, CD - analysis; Antigens, CD34; Biological and medical sciences; Cell differentiation, maturation, development, hematopoiesis; Cell Division; Cell physiology; Cell Separation; Cells, Cultured; Erythropoietin - pharmacology; Fetal Blood - cytology; Flow Cytometry; Fundamental and applied biological sciences. Psychology; Granulocyte Colony-Stimulating Factor - pharmacology; Granulocyte-Macrophage Colony-Stimulating Factor - pharmacology; Hematopoietic Cell Growth Factors - pharmacology; Hematopoietic Stem Cells - cytology; Hematopoietic Stem Cells - immunology; Humans; Immunophenotyping; Interleukin-3 - pharmacology; Molecular and cellular biology; Recombinant Proteins - pharmacology; Stem Cell Factor
• ISSN: 0006-4971; 1528-0020
• DOI: 10.1182/blood.V81.1.41.41

To characterize the growth of cord blood progenitor cells, single nonadherent, low-density, T-lymphocyte—depleted CD34+++ cells were sorted by flow cytometer with an autoclone device into single wells containing culture medium and cytokines. These cells were evaluated for proliferation and for replating ability of their progeny. This latter effect is used as a measure of self-renewal capacity. Colony formation was assessed in 1° wells containing various cytokines, alone and in combination, and single colonies deriving after 21 days in semisolid medium were replated into 2° wells in the presence of the combination of purified preparations of recombinant human steel factor (SF, a c-kit ligand), granulocyte-macrophage colony-stimulating factor (GM-CSF), granulocyte colony-stimulating factor (G-CSF), interleukin-3 (IL-3), and erythropoietin (Epo). Replating of single colonies was performed also for 3°, 4°, and 5° cultures. In the presence of serum, colony formation was observed in >66% of the wells stimulated with the combination of Epo, SF, GM-CSF, G-CSF, and IL-3, and more than 39% of the colonies formed in these 1° wells were very large in size (>2.5 mm in diameter, dense in the center, and containing >104 cells/colony). The replating efficiency of these large colonies was up to 93% with generation of subsequent colonies of very large size. Replating could be shown for up to five generations. The cells in these colonies were large, nonspecific esterase positive, and contained large amounts of cytoplasm with one or more nuclei containing several nucleoli per nucleus. Smaller colonies (1 to 2.5 mm in diameter and dense in the center) containing similar cells and making upan additional 14% of the colonies formed in 1° wells also showed extensive replating capacity, including generation of larger colonies. These colony-forming cells are likely similar to the murine macrophage high-proliferative potential colony-forming cells. The cells giving rise to these colonies are present in about eightfold higher frequency in cord blood than in adult bone marrow. These cells may at least in part be associated with the successful hematopoietic repopulating capacity of umbilical cord blood cells.