Extracellular laminin regulates hematopoietic potential of pluripotent stem cells through integrin β1-ILK-β-catenin-JUN axis
Extracellular laminin regulates hematopoietic potential of pluripotent stem cells through integrin β1-ILK-β-catenin-JUN axis
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DOI：
10.1016/j.scr.2021.102287
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发表时间：
2021-04-02
影响因子：
1.2
通讯作者：
Eto, Koji
中科院分区：
文献类型：
--
作者：
Yuzuriha, Akinori;Nakamura, Sou;Eto, Koji
重组基质已经使得能够进行人多能干细胞（hPSC）的无饲养细胞维持培养，层粘连蛋白511-E8片段（LM 511-E8）被广泛使用。然而，我们在本文中报道了维持在LM 511-E8上的hPSC抵抗分化为多能造血祖细胞（HPC），这与维持在LM 421-E8或LM 121-E8上的hPSC不同。与LM 511-E8相比，后两种LM-E8与hPSC的结合较弱，并激活了经典的Wnt/β-连环蛋白信号通路。此外，细胞外LM-E8依赖的优先造血与整合素β 1（ITGB 1）和下游整合素连接蛋白激酶（ILK）、β-连环蛋白和磷酸化JUN的较高表达相关。因此，较低的LM 511-E8包被浓度或添加Wnt/β-连环蛋白信号激活剂CHIR 99021有助于较高的HPC产量。相反，通过抑制剂或mRNA敲低抑制ILK、Wnt或JNK抑制HPC产量。这些发现表明，细胞外层粘连蛋白支架通过在未分化阶段激活ITGB 1-ILK-β-连环蛋白-JUN轴来调节hPSC的造血分化潜能。最后，低浓度的LM 511-E8和改进的hPSC-sac方法的组合，该方法将bFGF、SB 431542和肝素添加到常规方法中，使得能够获得更高的HPC产量和更高的确定性造血速率，这表明通常从hPSC获得分化的造血细胞的有用方案。
Recombinant matrices have enabled feeder cell-free maintenance cultures of human pluripotent stem cells (hPSCs), with laminin 511-E8 fragment (LM511-E8) being widely used. However, we herein report that hPSCs maintained on LM511-E8 resist differentiating to multipotent hematopoietic progenitor cells (HPCs), unlike hPSCs maintained on LM421-E8 or LM121-E8. The latter two LM-E8s bound weakly to hPSCs compared with LM511-E8 and activated the canonical Wnt/beta-catenin signaling pathway. Moreover, the extracellular LM-E8-dependent preferential hematopoiesis was associated with a higher expression of integrin beta 1 (ITGB1) and downstream integrin-linked protein kinase (ILK), beta-catenin and phosphorylated JUN. Accordingly, the lower coating concentration of LM511-E8 or addition of a Wnt/beta-catenin signaling activator, CHIR99021, facilitated higher HPC yield. In contrast, the inhibition of ILK, Wnt or JNK by inhibitors or mRNA knockdown suppressed the HPC yield. These findings suggest that extracellular laminin scaffolds modulate the hematopoietic differentiation potential of hPSCs by activating the ITGB1-ILK-beta-catenin-JUN axis at the undifferentiated stage. Finally, the combination of low-concentrated LM511-E8 and a revised hPSC-sac method, which adds bFGF, SB431542 and heparin to the conventional method, enabled a higher yield of HPCs and higher rate for definitive hematopoiesis, suggesting a useful protocol for obtaining differentiated hematopoietic cells from hPSCs in general.