EXTENSIVE AMPLIFICATION OF SINGLE CELLS FROM CD34(-CORD BLOOD AND IDENTIFICATION OF LONG-TERM CULTURE-INITIATING CELLS PRESENT IN 2 SUBSETS() SUBPOPULATIONS IN UMBILICAL)

CD34(+) cord blood cells were isolated with immunomagnetic beads and f ractionated by fluorescence-activated cell sorting (FAGS) into three s ubpopulations: CD34(+)38(+)DR(+), CD34(+)38(-)DR(+) and CD34(+)38(-)DR (-), using antibodies specific for these cell surface markers, Cells f rom each of the three subsets were plated as single cells in serum-fre e medium supplemented with a combination of growth factor and individu al cells were monitored for proliferation and the capacity to form col ony-forming cells, Single cells from the CD34(+)38(+)DR(+) subset show ed the lowest expansion capacity, generating up to 1.1 x 10(6) cells a t five weeks, while individual cells from both the CD34(+)38(-)DR(+) a nd CD34(+)38(-)DR(-) subsets could be expanded up to 1.8 x 10(6) and 9 .2 x 10(6) cells, respectively, over a period of six weeks, The differ ent subpopulations also generated colony-forming cells which gave rise to erythroid, myeloid and erythroid/myeloid colonies, CD34(+)38(-)DR( +) cells generated large numbers of colonies within two weeks in liqui d culture, but this rapidly declined, Generation of lineage-committed colony-forming cells was better sustained in the CD34(+)38(-)DR(-) pop ulation and continued for up to six weeks in culture. Overall, the gen eration of colony-forming cells declined with time in culture, althoug h the cell numbers continued to expand, How;el er, when the same popul ations were plated on irradiated bone marrow stroma, both the CD34(+)3 8(-)DR(+) and the CD34(+)38(-)DR(-) cells were capable of producing gr anulocyte-macrophage colony-forming cells (GM-CFCs) for 10 to 12 weeks , As hemopoiesis was sustained for almost three months, it appears tha t these populations were significantly enriched in long-term culture-i nitiating cells (LTC-ICs), Although both populations generated GM-CFCs , the CD34(+)38(-)DR(-) cells sustained production of higher numbers o f colony-forming cells than the CD34(+)38(-)DR(-) population, These re sults demonstrate that cells from cord blood can be efficiently monito red at the single-cell level for proliferation, expansion and colony-f orming capacity, Furthermore, at least two populations of LTC-ICs can be distinguished in cord blood CD34(+)38(-) cells by the differential expression of the KLA-DR antigen.