Enrichment, characterization, and responsiveness of single primitive CD34 human umbilical cord blood hematopoietic progenitors with high proliferative and replating potential

Abstract

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.

Granulocyte colony‐stimulating factor‐mobilized peripheral blood CD34+ cells from children contain the same levels of long‐term culture‐initiating cells producing the same numbers of colony‐forming cells as those from adults, but display greater in vitro monocyte/macrophage potential

“…We observed a similar profile of the distribution of myeloid CFCs derived from LTC-ICs, with an age-related decrease in the proportion of CFU-M-derived LTC-ICs. The propensity to produce monocyte/macrophage lineage cells in culture has already been underlined in cord blood (Lu et al, 1993) and in fetal liver (Nicolini et al, 1999). This propensity does not seem be owing only to accessory cells (Muller et al, 1996;Fietz et al, 1999), but also to intrinsic programming because of its ontogenically related intensity detected from the LTC-IC stage, and shown for the first time in this study.…”

Transduction of recombinant human erythropoietin receptor cDNA into daughter progenitors derived from single CD343+ cord blood cells changes the differentiation profile of daughter progenitors

“…For the purposes of this study, we did not distinguish among CFU-GM colonies containing monocytes, macrophages, and/or different types of granulocytes. HPP-CFC colonies contain mainly or entirely cells of the macrophage lineage and have not in any of our previous studies [9] contained any erythroid cells, even after staining for erythroid cells. BFU-E colonies contained only hemoglobinized red cells, and CFU-GEMM colonies contained hemoglobinized red cells mixed with other cell types such as monocytes, macrophages, granulocytes, and to a lesser extent, megakaryocytes.…”

“…Semi-solid culture medium contained Iscove's modified Dulbecco's medium (IMDM, GIBCO-BRL, Gaithersburg, MD), 1% methylcellulose, 30% fetal bovine serum (FBS, Hyclone Laboratory, Logan, UT), and 0.1 mM hemin (Eastman Kodack Co., Rochester, NY) [9][10][11] and the same concentrations of growth factor used in the prestimulation studies noted above. Cells were incubated under humidified conditions at 37°C, 5% CO 2 and lowered (5%) O 2 for determination after 14 days of granulocyte-macrophage (CFU-GM), multipotential (CFU-GEMM), and BFU-E-colonies or after 21 days for high proliferative potential-colony forming cell (HPP-CFC) colonies.…”

Enhancing engraftment of cord blood cells via insight into the biology of stem/progenitor cell function

“…Cord blood (CB) has served as a transplantable source of hematopoietic stem cells (HSCs) and hematopoietic progenitor cells (HPCs) to treat malignant and nonmalignant disorders since our initial laboratory, [1][2][3][4][5][6][7][8][9] and clinical studies, [10][11][12][13][14] began over 20 years ago. There have now been over 25,000 CB transplants performed worldwide 15 since our initial clinical report of a child with Fanconi anemia who was successfully treated with CB from his human leukocyte antigen (HLA)-matched sister.…”