Synergistic effects of mesenchymal stem cells (MSCs) isolated from bone marrow (BM), umbilical cord blood (UCB) and periosteum, and fibroblasts as mixed feeder cells (MFCs) on the expansion of hematopoietic progenitor cells (HPCs) were investigated in serum- and exogenous cytokine-free conditions. Enriched CO34(+) cells were cultured for 2 weeks over the cell lines alone, individually, or selected combinations of them. When the cells were cultured over MFCs, the maximum increase in expansion of total nucleated cells and CD34(+)/CD38(-) cells was 1573- and 128.6-fold, respectively. Furthermore, hematopoietic cytokine such as IL-6 and chemokines (e.g., IL-8, growth related oncogene (GRO), GRO-alpha, matrix metalloproteinase (MMP)-1, and MMP-3) were significantly increased in mixed feeder cells. Based on these results, MFCs can be more efficient for the ex vivo expansion of HPCs. These results strongly suggest that MFCs are more suitable for HPCs mass production. (C) 2011, The Society for Biotechnology, Japan. All rights reserved.