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, s uch 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, e specially those mobilized into circulation for transplantation. In this stu dy, early circulating precursors were analyzed for expression of Flt3 at th e gene and protein levels. Flow cytometric studies shelved that >90% of CD3 4(+)CD38(-) cells expressed Flt3 antigen (CD135), The proportion of fresh C D34(+) cells expressing Flt3 decreased as CD38 staining increased. These re sults were confirmed by reverse transcriptase polymerase chain reaction (RT -FCR) analyses, which showed that Flt3 gene expression generally was limite d to the CD34(+)CD38(-) population, Because Flt3 ligand (FL) enhances the g rowth and/or maintenance of primitive cells, it was important to know how l ong 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 progenit ors without causing a loss of primitive CD34(+)CD38(-)Flt3(+) cells. Flt3 e xpression in cell cultures was limited to precursors retaining a CD34(+)CD3 8(-/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-k it(-/lo) cells constituted a small fraction (12%) of the CD34(+)CD38(-) pop ulation. Whereas both c-kit(-/lo) and c-kit(+) subsets were stimulated by F I,, cell expansion (p < 0.01) and colony formation (p < 0.01) were greater and maintained longer with CD34(+)CD38(-)c-kit(-/lo) cells, Furthermore, th e rapid response to FL suggests that primitive CD34(+)CD38(-)c-kit(-/lo) ce lls express Flt3 at the time of isolation or shortly thereafter, These resu lts demonstrate the presence of Flt3 on CD34(+)CD38(-) blood cells and sugg ests 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 he determined. (C) 1999 Interna tional Society for Experimental Hematology, Published by Elsevier Science I nc.