Efficient expansion of mouse hematopoietic stem cells ex vivo by membrane anchored Angptl2
Efficient expansion of mouse hematopoietic stem cells ex vivo by membrane anchored Angptl2
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通过膜锚定 Angptl2 离体高效扩增小鼠造血干细胞
DOI：
10.1016/j.bbrc.2022.05.067
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发表时间：
2022
影响因子：
3.1
通讯作者：
Zhigang Lu
中科院分区：
文献类型：
--
作者：
Jie Yang;Guanghui Shen;Jun Cao;Jinlan Zhang;Yunqing Gu;Xinyu Zhang;Xiaoyi Jiang;Min Luo;Zhigang Lu
造血干细胞（HSC）移植是治疗多种血液和免疫疾病的重要手段。已经进行了许多努力来实现可获得的离体HSC扩增。我们先前显示血管生成素样蛋白2（Angptl 2）结合并激活免疫抑制受体人白细胞免疫球蛋白（IG）样受体B2（LILRB 2）以支持HSC的扩增。然而，可溶性Angptl 2是不稳定的，并且下游信号传导将被配体结合触发的受体内吞作用减弱，从而损害Angptl 2扩增HSC的潜力。我们提出膜锚定的Angptl 2将克服这些限制。在本研究中，我们通过在C端添加跨膜结构域或在N端添加锚序列，构建了C端和N端锚定膜Angptl 2（Cm-Angptl 2和Nm-Angptl 2）。两种形式的Angptl 2均在饲养细胞表面显示出高效表达。Nm-Angptl 2而不是Cm-Angptl 2诱导LILRB 2报告基因的有效活化，表明Angptl 2 C末端的纤连蛋白（FBN）结构域对于刺激LILRB 2信号传导是必需的。与可溶性Angptl 2相比，Nm-Angptl 2显示出更高的激活LILRB 2报告基因的活性，并且通过移植和有限稀释测定来促进小鼠HSC的扩增。我们的研究揭示了FBN结构域对于Angptl 2激活LILRB 2的重要性，并证明Nm-Angptl 2在LILRB 2激活和HSC扩增中具有比可溶性蛋白更高的活性，提供了探索配体诱导受体信号传导模式的策略，以及体外扩增HSC的优化方法。
Hematopoietic stem cell (HSC) transplantation represents an important curative therapy for numerous hematological and immune diseases. Many efforts have been applied to achieve attainable ex vivo HSC expansion. We previously showed that angiopoietin-like proteins 2 (Angptl2) binds and activates the immune inhibitory receptor human leukocyte immunoglobulin (Ig)-like receptor B2 (LILRB2) to support the expansion of HSC. However, soluble Angptl2 is unstable and the downstream signaling would be attenuated by ligand-binding triggered receptor endocytosis, compromising the potential of Angptl2 to expand HSCs. We proposed that membrane anchored Angptl2 will overcome these limitations. In this study, we constructed the C-terminal and N-terminal anchored membrane Angptl2 (Cm-Angptl2 and Nm-Angptl2) by adding a transmembrane domain at the C-terminal or an anchor sequence at the N-terminal respectively. Both forms of Angptl2 showed efficient expression on the surface of feeder cells. Nm-Angptl2, but not Cm-Angptl2, induces a potent activation of LILRB2 reporter, indicating the fibronectin (FBN) domain at the C-terminus of Angptl2 is essential to stimulate LILRB2 signaling. Compared to soluble Angptl2, Nm-Angptl2 displays higher activities to activate LILRB2 reporter, and to promote the expansion of mouse HSCs as determined by transplantation and limiting dilution assay. Our study revealed the importance of FBN domain for Angptl2 to activate LILRB2 and demonstrated that Nm-Angptl2 have enhanced activities than the soluble protein in LILRB2 activation and HSC expansion, providing a strategy to explore the mode of ligand induced receptor signaling, and an optimized approach to expand HSCs ex vivo.