title: Retention of progenitor cell function in CD34+ cells purified using a novel O-sialoglycoprotease.
abstract: We previously showed that the sialoglycoprotein, CD34, which is expressed on primitive human hematopoietic progenitor cells, is cleaved by a unique glycoprotease from Pasteurella haemolytica (P.h. glycoprotease). This proteolytic enzyme specifically cleaves glycoproteins rich in O-sialoglycans. Glycoproteins containing only N-linked glycans are not cleaved. Cleavage of the CD34 antigen results in the loss of epitopes detected by five of seven CD34-designated antibodies. In this study, we investigated the role of the P.h. glycoprotease in isolating CD34+ cells from unfractionated normal human bone marrow mononuclear cells (MNCs), and determined the effect of the glycoprotease on the proliferative capacity of the progenitor-enriched fraction. CD34+ cells were isolated from MNCs using immunomagnetic beads attached via a CD34 antibody whose epitope is susceptible to removal by the cleavage with the glycoprotease. Subsequent cleavage with P.h. glycoprotease for 30 min at 37 degrees C released the CD34+ cells from the beads with a recovery of up to 78%. Using a CD34 antibody whose epitope was not removed by the glycoprotease, up to 95% of the recovered cells expressed CD34. Compared to unseparated MNCs, the CD34+ cells showed the following enrichment of committed hematopoietic progenitors, as assayed in semi-solid media: CFU-GM, 45-fold; CFU-M, 13-fold; BFU-E, 26-fold and CFU-GEMM, 81-fold. Hematopoiesis was also studied in two-stage long-term bone marrow cultures in which the CD34+ cells were co-cultured over irradiated, allogeneic adherent layers. Output of CFU-GM over a seven week period from these cultures was similar to that from control cultures with autologous adherent-cell-depleted marrow MNCs. These data suggest that the loss of O-sialo-glycosylated peptide moieties from P.h. glycoprotease-released CD34+ cells neither affects the functional capacity of committed progenitors, nor impairs the proliferation of long-term culture-generating cells. The P.h. glycoprotease can be used to facilitate the isolation and recovery of functionally competent CD34+ cells at high yield and purity, without prior removal of other adherent cells. The ability to rapidly purify CD34+ cells using this non-cytotoxic enzyme has important implications for bone marrow transplantation as well as for gene transfer studies in vitro.
authors.affiliation: Oncology Research Laboratories, Toronto Hospital, Ontario, Canada.
authors.forename: J C
authors.initials: JC
authors.lastname: Marsh
authors.forename: D R
authors.initials: DR
authors.lastname: Sutherland
authors.forename: J
authors.initials: J
authors.lastname: Davidson
authors.forename: A
authors.initials: A
authors.lastname: Mellors
authors.forename: A
authors.initials: A
authors.lastname: Keating
mesh_terms: D015703:Antigens, CD / Q000378:metabolism*; D018952:Antigens, CD34; D001854:Bone Marrow Cells; D002455:Cell Division / Q000187:drug effects; D002469:Cell Separation / Q000379:methods; D002478:Cells, Cultured; D003114:Colony-Forming Units Assay; D006412:Hematopoietic Stem Cells / Q000166:cytology / Q000187:drug effects / Q000276:immunology*; D006801:Humans; D007963:Leukocytes, Mononuclear / Q000166:cytology / Q000187:drug effects / Q000276:immunology; D008666:Metalloendopeptidases / Q000494:pharmacology*
chemical_list: D015703:Antigens, CD; D018952:Antigens, CD34; D008666:Metalloendopeptidases; C070738:O-sialoglycoprotein endopeptidase