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.
As measured by the long-term repopulating cell (LTRC) assay, only a few hematopoietic stem cells (HSCs) or perhaps a single HSC are required to totally repopulate the lymphohematopoietic tissues of lethally irradiated mice, cats, and humans, raising the question as to why large mammals require more marrow cells to either rescue them from lethal irradiation or establish a long-term hematopoietic graft than do small mammals. An explanation might be that HSC marrow frequency across species is not constant, but decreases as species body weight increases. This hypothesis was tested by comparing the LTRC marrow concentration of mice to that of rats. Specifically, histocompatible AKR/J Thy 1.1 marrow was transferred to 7-Gy irradiated C3H/ HeN, Thy 1.2 mice, and histocompatible Norway Black marrow (NBr), RT 7.2 marrow was transferred to 7-Gy irradiated RT 7.1 Lewis rats. The recipients were scored for successful grafts 6 to 20 weeks later.
In the present study, population sizes of high self‐renewal potential stem cells, i.e. colony forming units (CFU), and low self‐renewal potential stem cells, i.e. transient endogenous colony forming units (TE‐CFU) in Sl/Sld mice and their normal congenic littermates were measured and compared. By correcting for differences in the seeding efficiency ‘f’, it was possible to demonstrate that Sl/Sld mice suffer a deficiency of both stem cell populations. Therefore, it is concluded that the defective stromal tissue of the Sl/Sld mouse does not support normal size stem cell populations. However, as noted in the discussion, it remains an open question as to whether the defective stromal tissue supports normal erythroid differentiation at the stem cell level.
A new assay is described that measures the numbers (n) of endogenous long-term repopulating cells (eLTRC) surviving sublethal irradiation. The assay is based on analysis of variances of Pgk-1a phenotypes within groups of sublethally irradiated Pgk-1a/b mice. When ln n is plotted as a function of dose, the radiosensitivity (D0) is the negative reciprocal of the slope and the y-intercept is N or total numbers of eLTRC per mouse. The assay is unique in that N is an absolute value not requiring correction for seeding efficiency, or f. From two independent estimates, total numbers of eLTRC were determined to be 8,700 or 11,900 cells per mouse and D0 was found to be 0.82 or 0.83 Gy. If eLTRC are, in fact, the long-term repopulating cells (LTRC) defined by classical transfer assay, then LTRC can home to the marrow with nearly unit efficiency, only one to two LTRC are required for a mouse to survive a radiation LD50/30, and LTRC possess nearly unlimited self-renewal potential.
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