PURIFICATION OF REPOPULATING HEMATOPOIETIC-CELLS BASED ON BINDING OF BIOTINYLATED KIT-LIGAND

To characterize Kit expressing mouse bone marrow (BM) cells, and to de termine 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 detecta ble on 1-2% of BM cells after staining with biologically active biotin ylated Kit ligand (KL) or with anti-Kit antibodies (ACK-2). Compared t o unfractionated BM, the Kit(+) fractions were enriched for immature h emopoietic cells, as shown by morphological differentiation, in vitro culture, and spleen colony formation. Enrichment of colony-forming cel ls was higher in biotin-KL(+) than ACK-2(+) fractions. Colony-forming cells were not found in the Kit(-) subsets. To study the hemopoietic r epopulation capacity of the Kit(+) and Kit(-) cells, serial dilutions of the sorted fractions were transplanted into irradiated mice, and pe ripheral blood of these recipients was monitored regularly for the pre sence of donor-derived cells during al year period. Nucleated blood ce ll repopulation by male donor cells in female recipients was assessed using a Y-chromosome specific DNA probe; erythroid repopulation by nor mal donor cells in W/W-v recipients was examined flow cytometrically b y measuring the forward light scatter of donor- and host-type erythroc ytes. A 25- to 100-fold enrichment of long-term repopulating ability i n the sorted Kit(+) fractions showed that Kit(+) cells are capable of reconstitution of circulating erythrocytes and nucleated blood cells a fter 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 t hat Kit(+) cells contributed to donor-type repopulation of bone marrow , spleen and thymus. Our data demonstrate that isolation of BM cells o n the basis of Kit expression is a useful addition to the methods that are commonly applied in stem cell enrichment protocols.