A multifactorial analysis of umbilical cord blood, adult bone marrow and mobilized peripheral blood progenitors using the improved ML-IC assay.
OBJECTIVE
Assays that can evaluate the potential of individual human hematopoietic stem cells (HSC) are still lacking. We previously developed the myeloid-lymphoid initiating cell (ML-IC) assay that enumerates single CD34(+) cells that generate long-term culture-initiating (LTC-IC) and NK-initiating (NK-IC) daughter cells, or single primitive progenitors with multilineage potential. When transplanted in vivo, umbilical cord blood (UCB) has greater repopulating ability than bone marrow (BM) or mobilized peripheral blood (MPB). Whether the greater in vivo repopulating ability is due to an increased frequency of HSC in UCB and generative potential of UCB, BM, and MPB CD34(+) cells is not known.


METHODS
Single UCB, BM, and MPB CD34(+)CD38(-)Lin(-) or CD34(+)CD38(-)CD33(-) cells were plated in ML-IC assay and after 2 to 4 weeks, progeny was evaluated for frequency and generative potential of ML-IC. We also tested whether the ML-IC assay could be used to define if increased numbers of primitive progenitors generated by different cytokines in expansion cultures are mediated by recruitment of quiescent cells or by increasing their generative potential.


RESULTS
The frequency of ML-IC in BM, UCB, and MPB was similar, but the generative potential of UCB ML-IC was significantly higher. Substitution of Flt3-L, SCF, and IL-7 with Flt3-L and thrombopoietin significantly increased the generative potential of ML-IC, whereas Flt3-L, SCF, and hyper-IL-6 increased both ML-IC frequency and generative potential.


CONCLUSIONS
The ML-IC assay demonstrates that the greater repopulating ability of UCB is due to the higher generative ability of HSC in UCB. Furthermore, the ML-IC assay can discriminate between cytokine-mediated expansion of hematopoietic progenitors by enhancing generation of immature daughter cells or by recruiting otherwise quiescent cells.
Theunissen
Koen
K
Stem Cell Biology Program, Department of Medicine and Cancer Center, University of Minnesota Medical School, Minneapolis, Minn., USA.
Verfaillie
Catherine M
CM
D000328:Adult; D015703:Antigens, CD / Q000097:blood; D018952:Antigens, CD34 / Q000097:blood; D001854:Bone Marrow Cells / Q000166:cytology*; D002455:Cell Division; D002470:Cell Survival; D003602:Cytotoxicity, Immunologic; D005163:Factor Analysis, Statistical; D005312:Fetal Blood / Q000166:cytology*; D019650:Hematopoietic Stem Cell Mobilization; D006801:Humans; D007231:Infant, Newborn; D007694:Killer Cells, Natural / Q000276:immunology; D033581:Stem Cell Transplantation
D015703:Antigens, CD; D018952:Antigens, CD34