State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, Shanghai 200237, PR China.
State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, Shanghai 200237, PR China.
J Biosci Bioeng. 2021 Feb;131(2):190-197. doi: 10.1016/j.jbiosc.2020.09.020. Epub 2020 Oct 27.
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.
体外扩增对于促进造血/祖细胞（HSPC）在再生疗法中的应用至关重要。Wnt 信号通路参与 HSPC 的扩增和自我更新维持。然而，尚未研究出一种合理的方法来调节 HSPC 体外培养中的 Wnt 信号，以实现 HSPC 的有效扩增。在此，我们采用 Wnt 信号激活剂 6-溴靛红-3'-肟（BIO）对脐血来源的 CD34 细胞进行培养，实验设置如下：对照组（A 组）；在第 0、4 和 7 天添加 BIO（B 组）；在第 0 和 7 天添加 BIO（C 组）。初步的 BIO 处理可促进第 4 天 CD34 细胞的扩增。然而，B 组中 BIO 在第 0 和 4 天的补充作用减弱了第 7 天 HSPC 的扩增，同时增强了扩增 CD34 细胞的多谱系定向潜能和二次扩增能力。基于这一发现，我们提出了一种优化的 BIO 喂养策略（C 组），以支持 HSPC 的大量扩增。经过 10 天的培养，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 喂养策略实现了原始 HSPC 的扩增，而不会丧失其生物学功能。从机制上讲，优化的 BIO 喂养策略避免了 B 组中观察到的 Wnt 过度激活，同时保持了中等水平的细胞内β-连环蛋白。这些结果为 HSPC 体外培养过程中的 Wnt 调节提供了实验和理论基础，也为 HSPC 移植的扩增提供了一种潜在策略。
