Purification of repopulating hemopoietic cells based on binding of biotinylated Kit ligand.

To characterize Kit expressing mouse bone marrow (BM) cells, and to determine their contribution to short- and long-term repopulation of the hemopoietic system of irradiated recipients, we have purified Kit+ BM cells by flow cytometry. A high level of Kit expression was detectable on 1-2% of BM cells after staining with biologically active biotinylated Kit ligand (KL) or with anti-Kit antibodies (ACK-2). Compared to unfractionated BM, the Kit+ fractions were enriched for immature hemopoietic cells, as shown by morphological differentiation, in vitro culture, and spleen colony formation. Enrichment of colony-forming cells was higher in biotin-KL+ than ACK-2+ fractions. Colony-forming cells were not found in the Kit- subsets. To study the hemopoietic repopulation capacity of the Kit+ and Kit- cells, serial dilutions of the sorted fractions were transplanted into irradiated mice, and peripheral blood of these recipients was monitored regularly for the presence of donor-derived cells during a 1 year period. Nucleated blood cell repopulation by male donor cells in female recipients was assessed using a Y-chromosome specific DNA probe; erythroid repopulation by normal donor cells in W/Wv recipients was examined flow cytometrically by measuring the forward light scatter of donor- and host-type erythrocytes. A 25- to 100-fold enrichment of long-term repopulating ability in the sorted Kit+ fractions showed that Kit+ cells are capable of reconstitution of circulating erythrocytes and nucleated blood cells after BM transplantation. Transient repopulation of the red blood cell lineage was observed after transplantation of Kit- cells. Detection of donor-derived nucleated cells 1 year after transplantation showed that Kit+ cells contributed to donor-type repopulation of bone marrow, spleen and thymus. Our data demonstrate that isolation of BM cells on the basis of Kit expression is a useful addition to the methods that are commonly applied in stem cell enrichment protocols.