Transient and permanent engraftment potential of murine hematopoietic stem cell subsets: differential effects of host conditioning with gamma radiation and cytotoxic drugs

Abstract

Transplant of sorted donor (B6-Gpi-1a) hematopoietic stem cell subsets and host (B6-Gpi-1b) treatment with total body irradiation (TBI) or cytotoxic drugs were compared for induction of short- and long-term engraftment in a murine chimera model of congenic bone marrow transplantation (BMT). Parallel studies on donor and host marrow were performed in vitro in long-term bone marrow cultures to determine early and late cobblestone area forming cell (CAFC) frequencies in the grafts or in the transplant recipients 1 day after conditioning. Bone marrow cells (BMC) sorted for high wheat germ agglutinin affinity (WGA ) were enriched about 30-fold for early developing CAFC (colony-forming unit-spleen [CFU-S]) but not for primitive late CAFC (pre-CFU-S). This fraction showed only temporary engraftment when transplanted in irradiated recipients. In contrast, the low affinity (WGA+) fraction were preferentially enriched (200- to 300-fold) for late developing CAFC and were very effective for providing stable long-term engraftment following transplantation. Substituting radiation for chemotherapy in the host conditioning treatment also had diverse effects on the development of bone marrow engraftment. Pretreatment with 5-fluorouracil (5-FU, 200 mg/kg) allowed a discrete wave of donor engraftment that peaked at 2 to 4 weeks and then subsided to leave mostly host cells at 10 weeks and beyond. Busulfan preparation gave over 50% engraftment at 1 month after BMT but this continued to increase to reach stable donor chimerism of 80 to 90% beyond 10 weeks. The level of long-term engraftment given by 50 mg/kg busulfan appeared similar to that induced by doses of 6 to 8 Gy TBI. The changing patterns of erythroid chimerism for each preparative agent showed a remarkable correlation with depletion of defined hematopoietic stem cell subsets as quantified using the CAFC assay at 1 day after recipient treatment. These findings collectively show that the level of depletion of host CFU-S (CAFC-10) determines the extent of early and transient repopulation, whereas the degree of pre-CFU-S (CAFC-35) depletion determines the percentage of stable chimerism. Preliminary data on bone marrow CAFC content at 9 months after busulfan and BMT revealed a long-term reduction of the stem cell pool by about 50% but with relatively minor effects on supporting bone marrow stromas. The differential cytotoxic effects on stem cell subsets in relation to subsequent donor marrow engraftment offer a systematic and mechanistic approach toward identifying more effective chemotherapeutic compounds for use in clinical BMT conditioning regimens.