Quantitation of murine hematopoietic stem cells using a new lympho-myeloid colony forming cell assay

We have developed a new in vitro hematopoietic stem cell assay based on the stem cell s specific ability to produce both lymphocytes and myeloid cells. Importantly, this lympho-myeloid colony forming cell assay (LM-CFC) does not require stromal cells, the growth stimulus being provided by recombinant cytokines. The assay is made possible by the identification of growth conditions permissive for the simultaneous proliferation and development of both lymphoid and myeloid cells from a single stem cell. In the initial clonal stage, test cells are cultured for 14-18 days in a serum-free, semi-solid medium supplemented with SCF, Flt-3 ligand and IL-11. During this phase the stem cells form very large colonies containing lymphoid and myeloid progenitor cells. Prior to nutrient depletion a significant number of isolated, individual colonies are transferred to separate wells where they are cultured for 7 days in conditions which encourage their maturation into mature myeloid cells (neutrophils, macrophages and megakaryocytes) and lymphocytes (B and T cells). Neutrophils and B cells are identified by functional and immunocytochemical staining, the presence of both in a single well, identifies the original parent cell as having been a lympho-myeloid colony forming cell (LM-CFC). Using this assay the frequency of LM-CFC in the femur of a normal adult mouse is approximately 0.008%, depending on age and strain. This is a number in agreement with stem cell frequencies determined by in vivo methods. Furthermore, this assay is capable of detecting LM-CFC in a quiescent population of stem cells that has long-term repopulating ability but no CFU-S8, CFU-S12 or radio-protective cells. These data strongly suggest that the LM-CFC are either identical to long-term repopulating stem cells or very closely related.