Purification and analysis of rat hematopoietic stem cells by flow cytometry

Abstract

The monoclonal antibody OX7 recognizes an epitope expressed on the Thy‐1 glycoprotein, OX22 recognizes the high molecular weight forms(s) on leukocyte common antigen, and W3/13 recognized determinants found on certain sialoglycoproteins. Recently, the rat colony‐forming unit spleen (CFU‐S) was characterized as being OX7 upper 20% positive (OX7u20%), OX22 negative (OX22−), and W3/13 weakly positive (W3/13+). In the present study these observations have been extended to include the hematopoietic stem cell (HSC). Rat marow cells were incubated with allophycocyanine‐OX7 Fab' (APC‐OX7 Fab') and phycoerythrin (B‐OX22) Fab' (PhyB‐OX22 Fab'). The cells were sorted with a FACS‐ii instrument by using a Krypton laser tuned to the 530 nm spectral line for phycobiliprotein excitation. It was found that marrow cells capable of protecting lethally irradiated Lewis rats (9.5 Gy total body radiation, 0.4 Gy/min Co60) had the phenotype OX7u20%, OX22−. The percentage of cells in the marrow with this phenotype was found to be 0.34 ± 0.01 (mean ± S.E.). Three thousand of these cells were required to rescue 50% of lethally irradiated recipients (30‐d survival), while the number of unsorted bone marrow cells required was 1.05 × 106. Thus, a 350‐fold purification of the HSC was realized. Although CFU‐S copurified with HSC, purification of only 105‐fold was obtained. This might indicate that purified HSC have a reduced capacity to generate splenic hematopoietic colonies. The OX7u20%, OX22−‐enriched HSC population could be further divided into W3/13 dimand W3/13+ subpopulations by three‐parameter immunofluorescence analysis with the use of a new optical bench arrangement.

Isolation of rat bone marrow mast lineage cells using Thy 1.1 and rat stem cell factor

“…In Figure 2a, a typical profile of staining with OX-7 shows that 42.8 ? 6.3% (n = 6) of rat bone marrow cells reacted positively for Thy 1.1, confirming previous observations (Goldschneider et al, 1978;McCarthy et al, 1987;van den Brink et al, 1991). We isolated three separate subpopulations (Fig.…”

Marrow frequency of rat long-term repopulating cells: evidence that marrow hematopoietic stem cell concentration may be inversely proportional to species body weight

“…23 For the rat, 7 Gy was also found to be satisfactory. Further evidence that rejection did not take place across minor histocompatible barriers was that: (1) allogeneic NBr marrow was found more effective for protection of lethally irradiated Lewis rats than syngeneic Lewis marrow cells, 13 (2) NBr marrow usually generates 1.5 times greater numbers of 12-day CFU-s in Lewis recipients than does Lewis marrow (data not shown), and (3) NBr marrow can be serially transplanted, at near limiting dilution, through sublethally irradiated recipients with 10 or more cell doublings, which is similar to that observed in mice using retrovirally tagged cells. 24 Taken as a whole, there is no evidence for rejection of NBr marrow by histocompatible Lewis recipients conditioned with radiation doses of 7 Gy.…”

Immunology and Hematology

“…NK activity of large granular lymphocytes is upregulated by IFN or IL-2 in rats (Henney et al, 1981). Rat CFU-S could be characterized as OX7 upper 20% positive, W3/13 lower 50% positive, and OX22 negative (McCarthy et al, 1985(McCarthy et al, , 1987. As a result of the remarkable development of multiparameter cell-sorting techniques, major progress has been made in biological and/or medical research on cell surface marker expression on HSC and progenitor cells.…”