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Extensive Amplification of Single Cells from CD34⁺ Subpopulations in Umbilical Cord Blood and Identification of Long-Term Culture-Initiating Cells Present in Two Subsets

CRC Department of Experimental Haematology, Paterson Institute for Cancer Research, Withington, Manchester, United Kingdom

Key Words. CD34⁺ subsets • Amplification • Long-term culture-initiating cells • Cord blood • Colony-forming cells • Primitive cells

Correspondence: Dr. E. A. de Wynter, CRC Department of Experimental Haematology, Paterson Institute for Cancer Research,Withington, Manchester, M20 9BX, United Kingdom.

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 10⁶ 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 10⁶ and 9.2 x 10⁶ 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 granulocyte-macrophage 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.

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