Regeneration capability of Lin −/c-Kit +/Sca-1 + cells with or without radiation exposure for repopulation of peripheral blood in lethally irradiated mice monitored using Ly5.1 isotype on days 35, 90, and 270 after transplantation

Abstract

Hematopoietic stem cells are supposed to repopulate and maintain long-term regeneration of the recipient's bone marrow and peripheral blood. In this study, we evaluated the regeneration capability of Lin(-)/c-Kit(+)/Sca-1(+) (LKS) cells, the putative hematopoietic stem cells, after radiation exposure at graded doses, for long-term regeneration of peripheral blood in lethally irradiated recipients. LKS primitive progenitor cells, collected from the bone marrow of Ly5.1 mice that had been irradiated at graded increased doses (0.5, 1, 1.5, and 2 Gy) were transfused into lethally irradiated (9.5 Gy) Ly5.2 mice. Then, the Ly5.1 chimeric ratio in repopulated peripheral blood cells in the recipients was monitored. A reactive oxygen species (ROS)-reacting CM-H(2)DCFDA dye was used to evaluate the amount of ROS in LKS primitive progenitor cells with/without irradiation. Moreover, the amount of intracytoplasmic ROS generated after irradiation was estimated in terms of percent attenuation of cellular increase in number by the treatment with 100 microM N-acetyl-L-cysteine before irradiation. Differential regeneration capability of LKS cells irradiated at graded increased doses showed a dose-dependent suppression of regeneration of peripheral blood in the recipient mice as compared with LKS cells without radiation exposure. The amount of intracytoplasmic ROS in LKS cells was much smaller than that in mature bone marrow cells, and that of ROS in LKS increased slightly after radiation exposure, as evaluated by CM-H(2)DCFDA dye fluorescence analysis. The estimated amount of ROS generated in LKS cells after radiation exposure was different between progenitor cells for early regeneration and those for late regeneration; namely, the amount of ROS in progenitors on day 270 were estimated to be smaller than that in progenitors for day 35 or day 90. Because of the small amount of generated radiation-induced ROS calculated in terms of attenuation rate after N-acetyl-L-cysteine treatment, progenitor cells regenerating peripheral blood cells 270 days after transfusion were assumed to be anaerobic and more immature and radioresistant than those on day 35 or day 90. However, limited long-term regeneration capability (up to 270 days) of steady-state LKS cells than that of unfractionated rescue bone marrow cells suggests that LKS cells do not seem to be true hematopoietic stem cells.