IDENTIFICATION OF CD34(- ENGRAFTMENT OF CD34(+)THY-1(+)LIN(-) STEM-CELLS IN FETAL SHEEP() SUBSETS AFTER GLYCOPROTEASE SELECTION )

Epitopes on the CD34 molecule detected by some CD34 antibodies can be cleaved by a unique glycoprotease from Pasteurella haemolytica, which cleaves only glycoproteins rich in O-linked glycans. A method to isola te CD34(+) cells from adult bone marrow was developed subsequently, in which CD34(+) cells were isolated in high purity and yield following immunomagnetic bead selection and detachment with the glycoprotease. U sing a variety of other cell-surface markers shown here to be insensit ive to glycoprotease, committed progenitors of T lymphoid, B lymphoid, monomyeloid, megakaryoblastic, or erythroid lineages could be identif ied. Significantly, candidate hematopoietic stem cells (HSC) that are contained within a CD34(+)Lin(-) (CD2(-), CD14(-), CD15(-), CD16(-), C D19(-)) (or CD34(+)CD38(-)) subset expressing the Thy-1 antigen (CDw90 ), c-kit receptor (CD117), and CDw109 but lacking expression of CD71 a nd HLA-DR antigens also were detected. Functionally distinct subsets o f glycoprotease-selected CD34(+) cells were identified and subfraction ated using flow cytometry and fluorescence-activated cell sorting (FAG S). These subsets included candidate HSCs expressing the CD34(+)Thy-1( +)Lin(-) phenotype, which were sorted from a CD34(+) fraction of a mob ilized peripheral blood (MPB) sample. In a fetal sheep model, when CD3 4(+)Thy-1(+)Lin(-) cells were injected intraperitoneally, they were ca pable of homing to the marrow, where they generated long-term multilin eage hematopoiesis and maintained human CD34(+) cells, indicating that candidate HSC subsets of CD34(+) cells selected with this highly spec ific enzyme were capable of engraftment in vivo. The ability to identi fy and purify virtually any phenotypically defined subset of glycoprot ease-selected CD34(+) stem/progenitor cells should facilitate the stud y of hematopoiesis in vitro and in animal models in vivo as well as th e development of novel genetic techniques for the correction of specif ic blood cell disorders in humans.