c‐kit <low Pluripotent Hemopoietic Stem Cells Form CFU‐S on Day 16

Using Ly5 congenic mice, we characterized the early differentiation step of pluripotent hemopoietic stem cells. Lineage − (Lin − )/CD71 − cells in the bone marrow cells were separated into major histocompatibility complex (MHC) class I high /c‐kit low and MHC class I high /c‐kit <low populations from C57BL/6 Ly5.1 male mice. These two populations (1,000 cells) were transplanted into lethally irradiated (5.5 Gy × 2) C57BL/6 Ly5.2 female mice. Colony‐forming unit in spleen (CFU‐S) assays were carried out on days 10, 12, 14, 16, and 20. In the mice that received c‐kit low cells, CFU‐S were first detected on day 12, and the CFU‐S counts gradually increased. In contrast, no visible colony was detected until day 14 in the mice that received c‐kit <low cells; CFU‐S were first observed on day 16. Donor‐derived (Ly5.1 + ) cells, such as B cells, T cells, and myeloid cells, were detected by fluorescence‐activated cell sorter analyses, and donor‐derived erythroid cells were detected by polymerase chain reaction analyses using Y‐chromosome‐specific primers. Donor‐derived cells in the recipients of c‐kit low cells were detected in the spleen, bone marrow, and peripheral blood on day 12 after transplantation, while they were detected on day 16 in the mice that received c‐kit <low cells. Therefore, c‐kit <low cells have the capacity not only to form CFU‐S on day 16 but also to reconstitute the recipients with donor‐derived hematolymphoid cells 16 days after transplantation.