Expression of Flt3 and c-kit during growth and maturation of human CD34+CD38- cells.
Studies of murine stem cells suggest that the cytokine receptors Flt3 and c-kit are expressed differentially on the earliest reconstitutional cells, such that Flt3 is not expressed until after stem cell activation. Much less is known about the expression of Flt3 and c-kit on primitive human cells, especially those mobilized into circulation for transplantation. In this study, early circulating precursors were analyzed for expression of Flt3 at the gene and protein levels. Flow cytometric studies showed that >90% of CD34+CD38- cells expressed Flt3 antigen (CD135). The proportion of fresh CD34+ cells expressing Flt3 decreased as CD38 staining increased. These results were confirmed by reverse transcriptase polymerase chain reaction (RT-PCR) analyses, which showed that Flt3 gene expression generally was limited to the CD34+CD38- population. Because Flt3 ligand (FL) enhances the growth and/or maintenance of primitive cells, it was important to know how long early cells retain Flt3 receptor expression in expansion culture. Both RT-PCR analyses and functional tests demonstrated that primitive cells are capable of expressing Flt3 for as long as 2 weeks in liquid medium. During the first week of culture, FL enhanced the generation of cells and progenitors without causing a loss of primitive CD34+CD38-Flt3+ cells. Flt3 expression in cell cultures was limited to precursors retaining a CD34+CD38(-/lo) phenotype. Because the most primitive human precursors are believed to express c-kit at a low level, we examined the FL responsiveness of CD34+CD38-c-kit(-/lo) cells and CD34+CD38-c-kit+ cells. CD34+CD38-c-kit(-/lo), cells constituted a small fraction (12%) of the CD34+CD38- population. Whereas both c-kit(-/lo) and c-kit+ subsets were stimulated by FL, cell expansion (p < 0.01) and colony formation (p < 0.01) were greater and maintained longer with CD34+CD38-c-kit(-/lo) cells. Furthermore, the rapid response to FL suggests that primitive CD34+CD38-c-kit(-/lo) cells express Flt3 at the time of isolation or shortly thereafter. These results demonstrate the presence of Flt3 on CD34+CD38 blood cells and suggests that Flt3 also may be present on a c-kit(-/lo) subset, among the most primitive in circulation. Flt3 is lost during maturation to committed (CD34+CD38+) lineages. Addition of FL to primitive cell cultures stimulates cell expansion while maintaining early CD34+CD38-Flt3+ precursors for at least 7 days. The possible existence of a more primitive CD34+CD38-c-kit(-/lo) Flt3(-/lo) precursor remains to be determined.
Xiao
M
M
Pacific Northwest Regional Blood Services, American Red Cross, Portland, OR 97208, USA.
Oppenlander
B K
BK
Plunkett
J M
JM
Dooley
D C
DC
D036541:ADP-ribosyl Cyclase; D051997:ADP-ribosyl Cyclase 1; D015703:Antigens, CD; D018952:Antigens, CD34 / Q000032:analysis*; D000943:Antigens, Differentiation / Q000032:analysis*; D001483:Base Sequence; D002455:Cell Division; D017931:DNA Primers; D005434:Flow Cytometry; D006412:Hematopoietic Stem Cells / Q000166:cytology / Q000276:immunology / Q000378:metabolism*; D006801:Humans; D008562:Membrane Glycoproteins; D009244:NAD+ Nucleosidase / Q000032:analysis*; D011518:Proto-Oncogene Proteins / Q000235:genetics / Q000378:metabolism*; D019009:Proto-Oncogene Proteins c-kit / Q000378:metabolism*; D012333:RNA, Messenger / Q000235:genetics; D020794:Receptor Protein-Tyrosine Kinases / Q000235:genetics / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D051941:fms-Like Tyrosine Kinase 3
D015703:Antigens, CD; D018952:Antigens, CD34; D000943:Antigens, Differentiation; D017931:DNA Primers; D008562:Membrane Glycoproteins; D011518:Proto-Oncogene Proteins; D012333:RNA, Messenger; C497970:FLT3 protein, human; D019009:Proto-Oncogene Proteins c-kit; D020794:Receptor Protein-Tyrosine Kinases; D051941:fms-Like Tyrosine Kinase 3; D036541:ADP-ribosyl Cyclase; C498154:CD38 protein, human; D009244:NAD+ Nucleosidase; D051997:ADP-ribosyl Cyclase 1