Effect of 5-fluorouracil on “primitive” hematopoietic stem cells that reconstitute whole erythropoiesis of genetically anemic W/Wv mice

Abstract

The potential to reconstitute the whole erythropoiesis of a genetically anemic (WB X C57BL/6)F1-W/Wv (WBB6F1-W/Wv) mouse for at least 8 weeks was compared between 5-fluorouracil (5FU)-treated and nontreated bone marrow cells. C57BL/6-Pgk-1b/Pgk-1a female mice, in which each stem cell had either A-type or B-type phosphoglycerate kinase (PGK) owing to the random inactivation of one of two X chromosomes, were used as donors. As a marker of the reconstitution, electrophoretic pattern of hemoglobin was used. The concentration of the stem cells that reconstitute the whole erythropoiesis of WBB6F1-W/Wv mouse was higher in the marrow of donors that had received an injection of 5FU two days previously (two-day 5FU-treated) than in the marrow of nontreated donors. In the marrow of four-day 5FU-treated mice, however, the concentration was comparable to that of nontreated mice. The PGK electrophoretic pattern of WBB6F1-W/Wv mice reconstituted by nontreated marrow cells was comparable to the PGK pattern of WBB6F1-W/Wv mice reconstituted by four-day 5FU-treated marrow cells. Thus, a single stem cell with extensive proliferative potential rather than multiple spleen colony-forming units appeared to be responsible for the erythropoietic reconstitution in the transplantation of nontreated healthy marrow cells as well as 5FU-treated marrow cells.

In vitro proliferation of primitive hemopoietic stem cells supported by stromal cells: evidence for the presence of a mechanism(s) other than that involving c-kit receptor and its ligand.

“…It is well established that a single stem cell can give rise to cells of all lymphohemopoietic lineages (2)(3)(4). Such stem cells have been operationally defined and assayed by their capacity for long-term repopulation of lymphohemopoietic cells after injection of them into either lethally irradiated or genetically anemic WBB6F1-W/W ~ mice (5)(6)(7)(8). These stem cells are physically separable from the majority of the cells that form macroscopic colonies in the spleens of lethally irradiated mice (spleen colony-forming units [CFU-S] 1) (9-11).…”

“…Therefore, the concentration of the primitive hemopoietic stem cells in the starting bone marrow cell population is calculated to be 6.8/106 ceils. Nakano et al (7) injected varying numbers of bone marrow cells freshly isolated from C57BL/6 mice into WBB6F1-W/W ~ mice and estimated by limiting dilution analysis the concentration of the primitive hemopoietic stem cells to be 9/106 bone marrow cells. By comparison of these estima-tions, ,u75% of the primitive hemopoietic stem cells are supposed to have sustained their proliferative capacity during 6 wk of the coculture with PA6 cells, showing that PA6 cells can provide a fairly suitable microenvironment for the proliferation of primitive hemopoietic stem ceils.…”

“…However, hemopoietic progenitor cells capable of forming multilineage colonies in methylcdkilose cultures are strongly suggested to be in Go state for a variable period after inoculation, and, when triggered into cell cycle, they proliferate at relatively constant doubling rates (36). Nakano et al (7) reported that hemopoietic stem cells capable of reconstituting in vivo hemopoiesis are not in S phase. It appears to take only 4-5 d for the colonies in our colony assay system to reach macroscopic size, since population doubling time of the cells within the colonies was 7-8 h (H. Kodama, unpublished observations).…”

Genetic control of the frequency of hematopoietic stem cells in mice: mapping of a candidate locus to chromosome 1.

“…How stem cells decide whether to differentiate and how the pool of stem cells is maintained is not well understood. The incidence of stem cells has been estimated to be about 1/104 to 1/105 cell in marrow both in mouse and man (1)(2)(3)(4)(5)(6)(7)(8). Stem cell levels in mice are relatively stable in individuals of a given strain of mice (1-5; for review see reference 9).…”

Haemopoietic stem cells: their heterogeneity and regulation

“…That pre-CFU-S are the major marrow repopulating cells has received support from experiments demonstrating that 5-fluorouracil, a drug that preferentially spares pre-CFU-S , also spares cells responsible for long-term myeloid and lymphoid repopulating in a competitive repopulation assay (Lerner & Harrison 1990). Furthermore, it has been well established that the repopulating abilities of transplanted haemopoietic cells are all less than expected from assays of their CFU-S content (Micklem et al 1975;Harrison & Astle 1982;Kretchmar & Conover 1970) and estimates of pluripotential stem cell numbers are 10-100 times lower than predicted from CFU-S concentrations (Boggs et al 1982;Micklem et al 1987;Harrison et al 1988;Nakano et al 1989). In radiation chimeras where the donor and host cells are readily distinguished, the numbers and dispersion of repopulating clones can be estimated using binomial statistics.…”