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A New Assay Method for Late CFU-S Formation and Long-Term Reconstituting Activity Using a Small Number of Pluripotent Hemopoietic Stem Cells

^a First Department of Pathology, Transplantation Center, and Regeneration Research Center for Intractable Diseases, Kansai Medical University, Moriguchi City, Osaka, Japan;
^b Department of Pathology, Norman Bethune Medical University, Changchun, China

Key Words. Pluripotent hemopoietic stem cells • c-kit • CFU-S • Mouse

Susumu Ikehara, M.D., Ph.D., First Department of Pathology, Kansai Medical University, 10-15 Fumizono-cho, Moriguchi City, Osaka 570-8506, Japan. Telephone: 81-6-6993-9429; Fax: 81-6-6994-8283; e-mail: ikehara{at}takii.kmu.ac.jp

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.