Secreted factors from mouse embryonic fibroblasts maintain repopulating function of single cultured hematopoietic stem cells

Hematopoietic stem cell self-renewal, proliferation, and differentiation are independently regulated by intrinsic as well as extrinsic mechanisms. We previously demonstrated that proliferation of murine hematopoietic stem cells is supported in serum-free medium supplemented with two growth factors, stem cell factor and interleukin 11. The survival of hematopoietic stem cells is additionally improved by supplementing this medium with two more growth factors, neural growth factor and collagen 1 (four growth factors) or serum-free medium conditioned by the hematopoietic stem cell-supportive stromal UG26-1B6 cells.1 Here, we describe a robust and versatile alternative source of conditioned medium from mouse embryonic fibroblasts. We found that this conditioned medium supports survival and phenotypic identity of hematopoietic stem cells, as well as cell cycle entry in single cell cultures of CD34- CD48- CD150+ Lineage- SCA1+ KIT+ cells supplemented with two growth factors. Strikingly, in comparison with cultures in serum-free medium with four growth factors, conditioned medium from mouse embryonic fibroblasts increased the numbers of proliferating clones and the number of Lineage- SCA1+ KIT+ cells, with both two and four growth factors. In addition, conditioned medium from mouse embryonic fibroblasts supported self-renewal in culture of cells with short- and long-term hematopoiesis-repopulating ability in vivo. These findings identify conditioned medium from mouse embryonic fibroblasts as a robust, alternative, serum-free source of factors to maintain self-renewal of in vivo-repopulating hematopoetic stem cells in culture.