Extensive amplification of single cells from CD34+ subpopulations in umbilical cord blood and identification of long-term culture-initiating cells present in two subsets.
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.
CRC Department of Experimental Haematology, Paterson Institute for Cancer Research, Withington, Manchester, United Kingdom.
E A
EA
de Wynter
G
G
Nadali
L H
LH
Coutinho
N G
NG
Testa
D036541:ADP-ribosyl Cyclase; D051997:ADP-ribosyl Cyclase 1; D015703:Antigens, CD; D018952:Antigens, CD34 / Q000032:analysis*; D000943:Antigens, Differentiation / Q000032:analysis; D001853:Bone Marrow / Q000528:radiation effects; D001854:Bone Marrow Cells; D002455:Cell Division / Q000187:drug effects / Q000276:immunology; D002478:Cells, Cultured / Q000166:cytology / Q000187:drug effects / Q000528:radiation effects; D016895:Culture Media, Serum-Free / Q000494:pharmacology; D005260:Female; D005312:Fetal Blood / Q000166:cytology* / Q000276:immunology; D005434:Flow Cytometry; D005455:Fluorescent Antibody Technique; D006133:Growth Substances / Q000494:pharmacology; D006684:HLA-DR Antigens / Q000032:analysis; D006410:Hematopoiesis / Q000187:drug effects / Q000276:immunology / Q000528:radiation effects; D006412:Hematopoietic Stem Cells / Q000737:chemistry / Q000166:cytology* / Q000276:immunology; D006801:Humans; D016130:Immunophenotyping; D016131:Lymphocyte Subsets / Q000737:chemistry / Q000276:immunology*; D008562:Membrane Glycoproteins; D009699:N-Glycosyl Hydrolases / Q000032:analysis; D013997:Time Factors
D015703:Antigens, CD; D018952:Antigens, CD34; D000943:Antigens, Differentiation; D016895:Culture Media, Serum-Free; D006133:Growth Substances; D006684:HLA-DR Antigens; D008562:Membrane Glycoproteins; D009699:N-Glycosyl Hydrolases; D036541:ADP-ribosyl Cyclase; C498154:CD38 protein, human; D051997:ADP-ribosyl Cyclase 1