Department of Chemistry and Biotechnology, Graduate School of Engineering, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo, 113-8656, Japan.
Division of Stem Cell Therapy, Center for Stem Cell Biology and Regenerative Medicine, Institute of Medical Science, University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo, 108-8639, Japan.
Stem Cell Rev Rep. 2018 Feb;14(1):101-109. doi: 10.1007/s12015-017-9768-7.
Hematopoietic stem cells (HSCs) are a valuable resource in transplantation medicine. Cytokines are often used to culture HSCs aiming at better clinical outcomes through enhancement of HSC reconstitution capability. Roles for each signal molecule downstream of receptors in HSCs, however, remain puzzling due to complexity of the cytokine-signaling network. Engineered receptors that are non-responsive to endogenous cytokines represent an attractive tool for dissection of signaling events. We here tested a previously developed chimeric receptor (CR) system in primary murine HSCs, target cells that are indispensable for analysis of stem cell activity. Each CR contains tyrosine motifs that enable selective activation of signal molecules located downstream of the c-Mpl receptor upon stimulation by an artificial ligand. Signaling through a control CR with a wild-type c-Mpl cytoplasmic tail sufficed to enhance HSC proliferation and colony formation in cooperation with stem cell factor (SCF). Among a series of CRs, only one compatible with selective Stat5 activation showed similar positive effects. The HSCs maintained ex vivo in these environments retained long-term reconstitution ability following transplantation. This ability was also demonstrated in secondary recipients, indicating effective transmission of stem cell-supportive signals into HSCs via these artificial CRs during culture. Selective activation of Stat5 through CR ex vivo favored preservation of lymphoid potential in long-term reconstituting HSCs, but not of myeloid potential, exemplifying possible dissection of signals downstream of c-Mpl. These CR systems therefore offer a useful tool to scrutinize complex signaling pathways in HSCs.
造血干细胞（HSCs）是移植医学中的宝贵资源。细胞因子常被用于培养 HSCs，以期通过增强 HSC 重建能力来获得更好的临床结果。然而，由于细胞因子信号网络的复杂性，受体下游的每个信号分子在 HSCs 中的作用仍然令人费解。对天然细胞因子无反应的工程化受体代表了用于解析信号事件的一种有吸引力的工具。我们在这里测试了先前开发的嵌合受体（CR）系统在原代小鼠 HSCs 中的作用，HSCs 是分析干细胞活性不可或缺的靶细胞。每个 CR 都包含酪氨酸基序，使其能够在受到人工配体刺激时选择性激活位于 c-Mpl 受体下游的信号分子。通过与干细胞因子（SCF）合作，具有野生型 c-Mpl 细胞质尾巴的对照 CR 的信号传导足以增强 HSC 的增殖和集落形成。在一系列 CR 中，只有一种与选择性 Stat5 激活兼容的 CR 表现出类似的积极效果。在这些环境中体外维持的 HSCs 在移植后保持长期重建能力。这种能力在次级受体中也得到了证明，表明在培养过程中，这些人工 CR 通过有效的信号传递将支持干细胞的信号传递到 HSCs 中。通过 CR 体外选择性激活 Stat5 有利于长期重建的 HSCs 中保留淋巴细胞潜力，但不保留髓系潜力，这例证了 c-Mpl 下游信号的可能解析。因此，这些 CR 系统为深入研究 HSCs 中的复杂信号通路提供了有用的工具。
