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Enrichment, characterization, and responsiveness of single primitive CD34
human umbilical cord blood hematopoietic progenitors with high
proliferative and replating potential
L Lu, M Xiao, RN Shen, S Grigsby and HE Broxmeyer
Department of Medicine (Hematology/Oncology), Indiana University School of
Medicine, Indianapolis 46202-5121.
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 degree
wells containing various cytokines, alone and in combination, and single
colonies deriving after 21 days in semisolid medium were replated into 2
degree 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 degrees, 4 degrees, and 5 degrees
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 degree wells
were very large in size (> 2.5 mm in diameter, dense in the center, and
containing > 10(4) 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 up an additional 14% of
the colonies formed in 1 degree 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.
Volume 81,
Issue 1,
pp. 41-48,
01/01/1993
Copyright © 1993 by The American Society of Hematology
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