A simple method for the preparation of quiescent, long-term repopulating stem cells

Adult murine hematopoietic stem cells are obtained from the bone marrow of normal mice or alternatively following treatment with a single injection of 5-fluorouracil (5-FU). These sources contain many developmentally diverse progenitor cells that are often problematic in having characteristics and behavior similar to stem cells. Current methods for removing these cells are time consuming, have low yields and the populations of primitive cells obtained are still heterogeneous. We have developed a simple, inexpensive method for preparing an ideal population of quiescent stem cells, in which virtually all of the non-stem cell progenitors have been removed. This population of cells does not contain any CFU-S8, CFU-S12 or CFC that proliferate in response to individual growth factors such as IL-3 and GM-CSF. However, a combination of growth factors used with a new lympho-myeloid CFC, stem cell assay reveals that >80% of the in vitro colony forming cells are lympho-myeloid stem cells. When FACS sorted, these cells have a Sca-1+Lin−Thy-1loc-kit− phenotype, just one of them being capable of generating as many as 2×108 progeny. Furthermore, the cells are able to fully repopulate the hematopoietic system and maintain the long-term survival of irradiated mice, but only when co-injected with other cells that have short-term repopulating, radio-protective ability. The preparation of this cell population is based on the principle that stem cells proliferate less frequently than other progenitors. While the traditional single injection of 5-FU removes some of the non-stem cell progenitors, not all are in cycle during this short period of exposure. However, multiple i.p. injections of 5-FU (200 mg/kg), each separated by 24 hr results in a cell population virtually completely ablated of non-stem cell progenitors, yet still containing many of the quiescent lympho-myeloid stem cells.