Myeloid-lymphoid initiating cells (ML-IC) are highly enriched in the rhodamine-c-kit(+)CD33(-)CD38(-) fraction of umbilical cord CD34(+) cells.
OBJECTIVE
The study of hematopoietic stem cells (HSC) is limited by lack of specific markers for HSC. Rhodamine 123 (Rho) is one of the substrates of P-glycoprotein (Pgp), and the presence of active Pgp can be shown by the efflux of Rho. Rho can also be used to measure the mitochondrial transmembrane potential (energy state) of a cell. We reasoned that selection of hematopoietic progenitors using a combination of Rho efflux and phenotypic markers might be superior to use of phenotypic markers alone.


METHODS
We used the myeloid-lymphoid initiating cell (ML-IC) assay as functional measure of primitive progenitors. Umbilical cord blood CD34(+)CD33(-)CD38(-), CD34(+)CD33(-)CD38(-)Rho(-), and CD34(+)CD33(-)CD38(-)Rho(-)c-kit(+) cells were sorted singly onto AFT024 feeders to assess their capacity to become ML-IC.


RESULTS
The frequency of ML-IC in CD34(+)CD33(-)CD38(-)Rho(-) cells was significantly higher (15 +/- 0.4%) than that in CD34(+)CD33(-)CD38(-) cells (6.2 +/- 0.9%, p < 0.05). However, the frequency of long-term culture-initiating cells (LTC-IC) (17 +/- 3% vs 12 +/- 1.5%) and natural killer culture-initiating cells (NK-IC) (25 +/- 3% vs 20 +/- 4%) was similar in the two populations. Following the treatment of CD34(+)CD33(-)CD38(-)Rho(-) cells with verapamil, which blocks Pgp function, no increase in ML-IC was detected compared with CD34(+)CD33(-)CD38(-) cells (6 +/- 0.7%), suggesting that differences in the energy state, which is reflected by Rho staining after verapamil treatment, cannot be used as a criterion to identify human HSC. Further selection of CD34(+)CD33(-)CD38(-)Rho(-) cells based on expression of c-kit significantly increased the frequency of ML-IC, LTC-IC and NK-IC by 1.75-, 1.3-, and 1.8-fold, respectively.


CONCLUSIONS
Combining the function of Pgp and phenotypic features of hematopoietic progenitors enriches the frequency of cord blood ML-IC to greater than 25%. Use of such enriched populations will allow us to characterize the biological behavior of human HSC.
Liu
Hsingjin
H
Division of Hematology, Oncology and Transplantation, Department of Medicine, University of Minnesota, Minneapolis 55455, USA.
Verfaillie
Catherine M
CM
D036541:ADP-ribosyl Cyclase; D051997:ADP-ribosyl Cyclase 1; D000818:Animals; D017304:Annexin A5 / Q000032:analysis; D015703:Antigens, CD / Q000032:analysis*; D018952:Antigens, CD34 / Q000032:analysis*; D000943:Antigens, Differentiation / Q000032:analysis*; D015214:Antigens, Differentiation, Myelomonocytic / Q000032:analysis*; D017209:Apoptosis; D015415:Biomarkers / Q000032:analysis; D002453:Cell Cycle; D002454:Cell Differentiation; D002478:Cells, Cultured; D005312:Fetal Blood / Q000166:cytology*; D006412:Hematopoietic Stem Cells / Q000166:cytology*; D006801:Humans; D016130:Immunophenotyping; D007231:Infant, Newborn; D007951:Leukemia, Myeloid / Q000097:blood; D008562:Membrane Glycoproteins; D051379:Mice; D027425:ATP-Binding Cassette, Sub-Family C Proteins / Q000032:analysis; D009244:NAD+ Nucleosidase / Q000032:analysis*; D019009:Proto-Oncogene Proteins c-kit / Q000032:analysis*; D012235:Rhodamines / Q000493:pharmacokinetics; D063268:Sialic Acid Binding Ig-like Lectin 3; D017154:Stromal Cells / Q000166:cytology
D036541:ADP-ribosyl Cyclase; D051997:ADP-ribosyl Cyclase 1; D017304:Annexin A5; D015703:Antigens, CD; D018952:Antigens, CD34; D000943:Antigens, Differentiation; D015214:Antigens, Differentiation, Myelomonocytic; D015415:Biomarkers; D008562:Membrane Glycoproteins; D027425:ATP-Binding Cassette, Sub-Family C Proteins; D009244:NAD+ Nucleosidase; D019009:Proto-Oncogene Proteins c-kit; D012235:Rhodamines; D063268:Sialic Acid Binding Ig-like Lectin 3; C571163:CD33 protein, human; C571165:Cd33 protein, mouse; C498154:CD38 protein, human; C498155:Cd38 protein, mouse