A new assay method for late CFU-S formation and long-term reconstituting activity using a small number of pluripotent hemopoietic stem cells.

We have previously reported that Lin-/CD71-/MHC class Ihigh/c-kit<low bone marrow cells (c-kit<low cells) are pluripotent hemopoietic stem cells (P-HSCs), since they have the capacity to self-renew for at least 2 years in mice and differentiate into all hemopoietic lineage cells over the long term when serial bone marrow transplantation is carried out using 500 c-kit<low cells. In addition, we have found that the c-kit<low cells do not form colony-forming units-spleen (CFU-S) on days 8 to 14 but form late CFU-S (after 16 days). In the present study, to confirm that c-kit<low cells are truly P-HSCs, we examine whether a few (< or = 50) c-kit<low cells can form late CFU-S and reconstitute lethally irradiated recipients. We have established a new method to rescue lethally irradiated mice by transplantation of a few cells so that they survive for more than 16 days: 0.2 ml of 20 Gy-irradiated peripheral blood (PB) was injected into the recipients every 3 days. All the mice that had been transplanted with 25 or 50 c-kit<low cells alone died within 12 days, and no CFU-S were detected in their spleens. However, when 25 or 50 c-kit<low cells were injected and 0.2 ml of 20 Gy-irradiated PB was injected every 3 days, the recipients survived, and a small number of CFU-S were detected after 16 days. About 40% of the recipients injected with 50 c-kit<low cells and about 15% of those injected with 25 c-kit<low cells survived for more than 6 months. Moreover, donor-derived multilineage cells were detected in all the hematolymphoid organs of the recipient mice. This new assay method using a small number of cells would be of great advantage for clarifying which cells are truly P-HSCs.