Department of Chemistry and Biotechnology, School of Engineering, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8656, Japan.
J Biotechnol. 2013 Dec;168(4):659-65. doi: 10.1016/j.jbiotec.2013.09.012. Epub 2013 Sep 23.
The technique to expand hematopoietic stem cells (HSCs) ex vivo is eagerly anticipated to secure an enough amount of HSCs for clinical applications. Previously we developed a scFv-thrombopoietin receptor (c-Mpl) chimera, named S-Mpl, which can transduce a proliferation signal in HSCs in response to a cognate antigen. However, a remaining concern of the S-Mpl chimera may be the magnitude of the cellular expansion level driven by this molecule, which was significantly less than that mediated by endogenous wild-type c-Mpl. In this study, we engineered a tyrosine motif located in the intracellular domain of S-Mpl based on a top-down approach in order to change the signaling properties of the chimera. The truncated mutant (trunc.) and an amino-acid substitution mutant (Q to L) of S-Mpl were constructed to investigate the ability of these mutants to expand HSCs. The result showed that the truncated and Q to L mutants gave higher and considerably lower number of the cells than unmodified S-Mpl, respectively. The proliferation level through the truncated mutant was even higher than that of non-transduced HSCs with the stimulation of a native cytokine, thrombopoietin. Moreover, we analyzed the signaling properties of the S-Mpl mutants in detail using a pro-B cell line Ba/F3. The data indicated that the STAT3 and STAT5 activation levels through the truncated mutant increased, whereas activation of the Q to L mutant was inhibited by a negative regulator of intracellular signaling, SHP-1. This is the first demonstration that a non-natural artificial mutant of a cytokine receptor is effective for ex vivo expansion of hematopoietic cells compared with a native cytokine receptor.
体外扩增造血干细胞（HSCs）的技术备受期待，以确保有足够数量的 HSCs 用于临床应用。此前，我们开发了一种 scFv-血小板生成素受体（c-Mpl）嵌合体，命名为 S-Mpl，它可以在 HSCs 中传递增殖信号，以响应同源抗原。然而，S-Mpl 嵌合体的一个遗留问题可能是该分子驱动的细胞扩增水平的幅度，这显著小于内源性野生型 c-Mpl 介导的水平。在这项研究中，我们基于自上而下的方法对位于 S-Mpl 细胞内结构域中的酪氨酸基序进行了工程改造，以改变嵌合体的信号转导特性。构建了 S-Mpl 的截断突变体（trunc.）和氨基酸取代突变体（Q 到 L），以研究这些突变体扩增 HSCs 的能力。结果表明，截断和 Q 到 L 突变体分别比未修饰的 S-Mpl 产生更多和显著更少的细胞。通过截断突变体的增殖水平甚至高于未转导的 HSCs 在天然细胞因子血小板生成素刺激下的增殖水平。此外，我们使用前 B 细胞系 Ba/F3 详细分析了 S-Mpl 突变体的信号转导特性。数据表明，通过截断突变体的 STAT3 和 STAT5 激活水平增加，而 Q 到 L 突变体的激活被细胞内信号负调节剂 SHP-1 抑制。这是首次证明与天然细胞因子受体相比，细胞因子受体的非天然人工突变体可有效用于体外扩增造血细胞。
