HEMATOPOIESIS Long-Term Ex Vivo Maintenance and Expansion of Transplantable Human Hematopoietic Stem Cells

We have developed a stromal-based in vitro culture system that facilitates ex vivo expansion of transplantable CD341 thy-11 cells using long-term hematopoietic reconstitution in severe combined immunodeficient-human (SCID-hu) mice as an in vivo assay for transplantable human hematopoietic stem cells (HSCs). The addition of leukemia inhibitory factor (LIF) to purified CD341 thy-11 cells on AC6.21 stroma, a murine bone marrow–derived stromal cell line, caused expansion of cells with CD341 thy-11 phenotype. Addition of other cytokines, including interleukin-3 (IL-3), IL-6, granulocytemacrophage colony-stimulating factor, and stem cell factor, to LIF in the cultures caused a 150-fold expansion of cells retaining the CD341 thy-11 phenotype. The ex vivo–expanded CD341 thy-11 cells gave rise to multilineage differentiation, including myeloid, T, and B cells, when transplanted into SCID-hu mice. Both murine LIF (cannot bind to human LIF receptor) and human LIF caused expansion of human CD341 thy-11 cells in vitro, suggesting action through the murine stroma. Furthermore, another human HSC candidate, CD341 CD382 cells, shows a similar pattern of proliferative response. This suggests that ex vivo expansion of transplantable human stem cells under this in vitro culture system is a general phenomenon and not just specific for CD341 thy-11 cells. r 1999 by The American Society of Hematology.