Lin^-Sca-1⁺c-Kit^lowCD48⁺CD71⁺ Cells Are the Engine of Bone Marrow Regeneration

The hematopoietic tissue is the most suitable tissue for studies into the biology of regeneration. We examined bone marrow regeneration starting from a very low number of repopulating cells.

Mice were irradiated by a sublethal dose of 6 Gy and recovery of hematopoiesis was examined in its three basic compartments: stem and progenitor cells, precursors of blood cells, and peripheral blood between days 8 – 90 following irradiation. The irradiation reduced existing pools of progenitors and stem cells to ̴0.1%. We focused on the pools of stem cells and progenitors that were evaluated by several assays.

A novel assay based on engraftment of a defined number of transplanted competitors revealed delayed filling of the niches for long-term repopulating cells (LTRCs; stem cells) as compared to filling of the niches for short-term repopulating cells (STRCs; progenitors) by endogenous STRCs and LTRCs generated by cells surviving irradiation. This indicated a very slow recovery of the pool of stem cells, lagging after recovery of the pool of progenitors. This was confirmed by a traditional competitive transplantation assay, as well as by another novel assay that measured the capacity of bone marrow to establish pools of stem cells and progenitors in lethally irradiated mice. Direct assays of progenitors by the spleen colony (CFU-S) and in vitro CFC-GM assays indicated a low frequency of the clonogenic cells in regenerating bone marrow. Productive hematopoiesis was re-established about 10 days after irradiation in presence of very low pools of progenitors and stem cells.

Next we examined numbers of immature hematopoietic cells by flow cytometry focused at lineage negative (Lin^-), Sca-1 positive (Sca-1⁺), c-Kit⁺ cells (LSK cells), characterized further by means of the CD150, CD48 and CD71 antigenic markers. In absolute terms, only the population of LSK CD150⁺CD48⁺ recovered and overshot normal values after day 12 following irradiation. In relative terms, the LSK CD48⁺ cells (CD150⁺ and CD150^-) represented more than 99% of all LSK cells two weeks after irradiation. Also in relative terms, the LSK CD150⁺CD48^- cells, which are highly enriched for hematopoietic stem cells in normal bone marrow, recovered by day 30 and became relatively abundant later on. Another striking change consisted in initial lack and later severe scarcity of cells with the phenotype of LSK CD150^-CD48^-. These cells, serving as multipotent progenitors in normal bone marrow thus remained severely depleted in regenerating bone marrow during the whole follow-up period. LSK cells were mostly negative for CD71 (transferrin receptor) in normal bone marrow but became highly positive for this marker between days 10 – 20 after irradiation.

In conclusion, our semi-quantitative evaluation of regenerating hematopoiesis reveals its highly peculiar features. Those include an early recovery of blood cell production, followed by significant expansion of the Lin^-Sca-1⁺c-Kit^lowCD48⁺CD71⁺ cells in presence of a highly reduced pool of hematopoietic stem cells.