Ex vivo expansion is critical in facilitating the application of hematopoietic/progenitor stem cells (HSPCs) for regenerative therapies. Wnt signaling is implicated in the expansion and self-renewal maintenance of HSPCs. However, a reasonable method to regulate Wnt signaling in ex vivo cultures to achieve robust expansion of HSPCs 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 ± 0.46-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 β-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.

离体扩增对于促进将造血干细胞/祖干细胞（HSPC）应用于再生疗法至关重要。Wnt信号与HSPC的扩展和自我更新维护有关。但是，尚未研究一种调节离体培养物中Wnt信号传导以实现HSPC稳定扩增的合理方法。在此，在以下条件下，用Wnt信号的6-溴代靛红素-3'-肟（BIO）的活化剂培养脐血来源的CD34 ⁺细胞。在第0、4和7天（B组）将BIO加入培养物中；在第0天和第7天（C组）将BIO添加到培养物中。最初的BIO治疗促进了CD34 ⁺的扩增在第4天的细胞中。然而，B组在第0天和第4天的BIO补充在第7天减弱了HSPC的扩增，同时增强了已扩增CD34 ⁺细胞的多谱系提交潜能和二次扩增能力。基于此发现，提出了一种优化的BIO喂养策略（C组）以支持HSPC的大幅扩展。培养10天后，CD34 ⁺细胞的扩增倍数为28.70±0.46倍，明显高于A组（16.20±0.72倍，p<0.05）。此外，优化的BIO喂养策略在不损失生物学功能的情况下实现了原始HSPC扩展的增加。从机理上讲，优化的BIO喂养策略避免了在B组中观察到的Wnt过度活化，同时维持了中等水平的细胞内β-catenin水平。这些结果为离体培养过程中Wnt调控提供了实验和理论基础，并为扩大HSPCs移植提供了潜在的策略。