Single-cell image analysis to assess ABC-transporter-mediated efflux in highly purified hematopoietic progenitors.
BACKGROUND
Normal and malignant hematopoietic stem cells are characterized by their capacity to actively extrude fluorescent dyes. The contribution of different ATP-binding cassette (ABC) transporters to this phenomenon is largely unknown due to the small stem cell numbers limiting the use of standard methods to assess functional efflux.


METHODS
We used epifluorescence microscopy (EFM) in combination with single-cell image analysis to study ABC-transporter-mediated efflux in highly purified, viable, CD34+CD38- cells sorted on an adhesive biolayer. P-glycoprotein and multidrug-resistant protein (MRP)-mediated efflux were quantitated using fluorescent substrates (rhodamine-123 and calcein acetoxymethyl ester [calcein-AM]) and specific inhibitors (verapamil and probenecid, respectively).


RESULTS
The feasibility, sensitivity, and reproducibility of rhodamine-123 efflux quantitation using single-cell EFM was shown in cell lines and compared with standard flow cytometric assessment. P-glycoprotein-mediated transport was higher in CD34+CD38- cells than in more differentiated progenitors (mean efflux index = 2.24 +/- 0.35 and 1.14 +/- 0.11, respectively; P = 0.01). P-glycoprotein-mediated transport was the main determinant of the rhodamine "dull" phenotype of these cells. In addition, significant MRP-mediated efflux was demonstrated in CD34+CD38- and CD38+ cells (mean efflux index = 1.42 +/- 0.19 and 1.28 +/- 0.18, respectively).


CONCLUSIONS
The described method is a valuable tool for assessing ABC-transporter-mediated efflux in highly purified single cells. Both P-glycoprotein and MRP-mediated efflux are present in human CD34+CD38- hematopoietic stem cells.
Raaijmakers
H G P
HG
Department of Hematology, University Medical Center Nijmegen, Nijmegen, The Netherlands. h.raaijmakers@chl.umcn.nl
Van Den Bosch
G
G
Boezeman
J
J
De Witte
T
T
Raymakers
R A P
RA
D036541:ADP-ribosyl Cyclase / Q000378:metabolism; D051997:ADP-ribosyl Cyclase 1; D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000378:metabolism; D018528:ATP-Binding Cassette Transporters / Q000378:metabolism*; D015703:Antigens, CD / Q000378:metabolism; D018952:Antigens, CD34 / Q000378:metabolism; D001681:Biological Assay / Q000379:methods*; D005434:Flow Cytometry; D005456:Fluorescent Dyes / Q000378:metabolism; D006412:Hematopoietic Stem Cells / Q000187:drug effects / Q000378:metabolism* / Q000473:pathology; D006801:Humans; D019044:Image Cytometry / Q000295:instrumentation / Q000379:methods*; D007091:Image Processing, Computer-Assisted; D019169:Jurkat Cells; D008562:Membrane Glycoproteins; D008856:Microscopy, Fluorescence; D027425:ATP-Binding Cassette, Sub-Family C Proteins / Q000378:metabolism; D009101:Multiple Myeloma / Q000378:metabolism / Q000473:pathology; D011339:Probenecid / Q000494:pharmacology; D015203:Reproducibility of Results; D014700:Verapamil / Q000494:pharmacology
D036541:ADP-ribosyl Cyclase; D051997:ADP-ribosyl Cyclase 1; D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D018528:ATP-Binding Cassette Transporters; D015703:Antigens, CD; D018952:Antigens, CD34; D005456:Fluorescent Dyes; D008562:Membrane Glycoproteins; D027425:ATP-Binding Cassette, Sub-Family C Proteins; D011339:Probenecid; D014700:Verapamil; C498154:CD38 protein, human