Pleiotrophin, a paracrine regulator of hematopoietic stem cell fate

描述(申请人提供)：最近的研究表明，造血干细胞(HSCs)在骨髓(BM)中的维持和保留依赖于BM微环境或生态位内细胞的外部信号，包括血管内皮细胞(ECs)、血管周围细胞、骨线细胞、间充质基质细胞和交感神经元。然而，利基细胞单独或协同调节HSC动态平衡的确切机制尚未完全阐明。此外，骨髓抑制化疗和放射治疗后造血系统恢复所必需的控制HSC再生的机制仍然不太清楚。在这里，我们将使用创新的小鼠模型和分子技术来确定新的分泌型蛋白多营养素(PTN)在体内调节HSC命运的机制，并确定负责PTN介导的HSC池的维持和再生的BM利基细胞。HSC支持的内皮细胞的差异基因表达分析显示，PTN是一种肝素结合生长因子，在海马区正常表达，具有轴突生长因子的功能。短期培养含有PTN的小鼠骨髓HSC可使长期重新填充的HSCs扩增10倍以上，而在BM微环境中缺失PTN也导致小鼠HSC含量减少10倍。解剖学研究表明，PTN在骨髓中由VEGFR3+VEcadherin+窦内皮细胞和CXCL12+血管周细胞表达，提示PTN是HSC血管生态位中的一个独特成分。在初步研究中，PTN介导的造血干/祖细胞的体外扩增可被RAS或MEK的药物抑制剂所阻断，提示PTN通过诱导RAS/MEK信号通路介导HSC的生长。根据这些发现，我们假设PTN是BM血管壁内HSC池的旁分泌调节因子，并通过激活Ras/MEK/ERK通路来调节HSC的生长。根据这一假设，我们提出了以下具体目标：1)确定Ras/MEK/ERK通路在介导PTN对HSC的作用中的作用；2)确定哪些HSC龛细胞在体内介导PTN对HSC的维持和再生作用；3)确定PTN是否可以通过激活RAS/MEK信号通路在体外或体内扩增人HSCs。我们的长期目标是明确PTN介导HSCs自我更新和再生的机制，以促进PTN的翻译以促进人类HSCs的治疗性扩张。

DESCRIPTION (provided by applicant): Recent studies have revealed that the maintenance and retention of hematopoietic stem cells (HSCs) in the bone marrow (BM) are dependent upon extrinsic signals from cells within the BM microenvironment or niche, including vascular endothelial cells (ECs), perivascular cells, osteolineage cells, mesenchymal stromal cells and sympathetic neurons. However, the precise mechanisms through which niche cells individually or in concert regulate HSC homeostasis have yet to be fully elucidated. Furthermore, the mechanisms controlling HSC regeneration, which is necessary for the recovery of the hematopoietic system following myelosuppressive chemo- and radiotherapy, remain less well understood. Here, we will use innovative mouse models and molecular techniques to define the mechanism through which the novel secreted protein, pleiotrophin (PTN), regulates HSC fate in vivo and to identify the BM niche cells responsible for PTN-mediated maintenance and regeneration of the HSC pool. Differential gene expression analysis of HSC-supportive ECs revealed the overexpression of PTN, a heparin binding growth factor, which is normally expressed in the hippocampus and functions as a neurite outgrowth factor. Short-term culture of murine BM HSCs with PTN caused more than 10-fold expansion of long-term repopulating HSCs, whereas deletion of PTN in the BM microenvironment also caused a 10-fold deficit in HSC content in mice. Anatomic studies suggest that PTN is expressed by VEGFR3+VEcadherin+ sinusoidal ECs and CXCL12+ perivascular cells in the BM, suggesting that PTN is a unique component of the HSC vascular niche. In preliminary studies, PTN-mediated expansion of hematopoietic stem/progenitor cells in vitro was abrogated by pharmacologic inhibitors of Ras or MEK, suggesting that PTN mediates HSC growth via induction of the Ras/MEK signaling pathway. Based upon these findings, we hypothesize that PTN is a paracrine regulator of the HSC pool within the BM vascular niche and regulates HSC growth via activation of the Ras/MEK/ERK pathway. In keeping with this hypothesis, we propose the following Specific Aims: I) Determine the role of the Ras/MEK/ERK pathway in mediating PTN effects on HSCs, 2) Determine which HSC niche cells are responsible for mediating PTN effects on HSC maintenance and regeneration in vivo, and 3) Determine whether PTN can expand human HSCs ex vivo or in vivo via activation of Ras/MEK signaling pathway. Our broad, long-term objective will be to define the mechanisms through which PTN mediates the self-renewal and regeneration of HSCs to facilitate translation of PTN for the therapeutic expansion of human HSCs.