Purification and analysis of rat hematopoietic stem cells by flow cytometry

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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.