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 I high /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 (≤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.