Extensive amplification of single cells from CD34+ subpopulations in umbilical cord blood and identification of long-term culture-initiating cells present in two subsets.

Abstract

CD34+ cord blood cells were isolated with immunomagnetic beads and fractionated by fluorescence-activated cell sorting (FACS) into three subpopulations: CD34+38+DR+, CD34+38-DR+ and CD34+38-DR-, using antibodies specific for these cell surface markers. Cells from each of the three subsets were plated as single cells in serum-free medium supplemented with a combination of growth factor and individual cells were monitored for proliferation and the capacity to form colony-forming cells. Single cells from the CD34+38+DR+ subset showed the lowest expansion capacity, generating up to 1.1 x 10(6) cells at five weeks, while individual cells from both the CD34+38-DR+ and 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 different 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 liquid culture, but this rapidly declined. Generation of lineage-committed colony-forming cells was better sustained in the CD34+38-DR- population and continued for up to six weeks in culture. Overall, the generation of colony-forming cells declined with time in culture, although the cell numbers continued to expand. However, when the same populations were plated on irradiated bone marrow stroma, both the CD34+38-DR+ and the CD34+38-DR- cells were capable of producing granulocytemacrophage colony-forming cells (GM-CFCs) for 10 to 12 weeks. As hemopoiesis was sustained for almost three months, it appears that these populations were significantly enriched in long-term culture-initiating cells (LTC-ICs). Although both populations generated GM-CFCs, the CD34+38-DR- cells sustained production of higher numbers of colony-forming cells than the CD34+38-DR+ population. These results demonstrate that cells from cord blood can be efficiently monitored at the single-cell level for proliferation, expansion and colony-forming capacity. Furthermore, at least two populations of LTC-ICs can be distinguished in cord blood CD34+38- cells by the differential expression of the HLA-DR antigen.