Ex vivo expansion is critical in facilitating the application of hematopoietic/progenitor stem cells (HSPC5) for regenerative therapies. Wnt signaling is implicated in the expansion and self-renewal maintenance of HSPC5. However, a reasonable method to regulate Wnt signaling in ex vivo cultures to achieve robust expansion of HSPC5 has not yet been investigated. Here, cord blood-derived CD34(+) cells were cultured with the activator of Wnt signaling 6-bromoindirubin-3'-oxime (BIO) under the following conditions: vehicle control (group A); BIO was added to the culture on days 0, 4, and 7 (group B); and BIO was added to the culture on days 0 and 7 (group C). Initial BIO treatment promoted the expansion of CD34(+) cells on day 4. However, BIO supplementation on days 0 and 4 in group B attenuated HSPC expansion on day 7, while enhancing the multilineage commit potential and secondary expansion ability of expanded CD34(+) cells. Based on this finding, an optimized BIO feeding strategy (group C) was proposed to support substantial expansion of HSPCs. After 10 days of culture, the expansion fold of CD34(+) cells was 28.70 +/- 0A6-folds, which was significantly higher than group A (16.20 +/- 0.72-folds, p < 0.05). Moreover, the optimized BIO feeding strategy achieved increased primitive HSPC expansion without the loss of biological functions. Mechanistically, the optimized BIO feeding strategy avoided the excessive activation of Wnt observed in group B while maintaining a moderate level of intracellular B-catenin. These results provide an experimental and theoretical basis for Wnt regulation in ex vivo culture process and a potential strategy to expand HSPCs for transplantation. (C) 2020, The Society for Biotechnology, Japan. All rights reserved.