Stromal Cell Mimic for Hematopoietic Stem Cell Expansion

描述（由申请人提供）： 造血干细胞（HSCs）分化为所有的血细胞谱系。HSC的离体扩增将极大地促进用于病毒性疾病（例如，艾滋病毒）和遗传性血液疾病，并将改善成人脐带血移植的前景。然而，培养物中的HSC分裂与分化成具有降低的潜能的细胞相关。这与HSC在体内的持续扩增形成对比，并导致了干细胞“小生境”支持骨髓中自我更新的假设。小生境包括基质和细胞相关的细胞因子，以及与基质细胞的直接接触。将开发一种培养表面，用于控制基质细胞表达的细胞粘附分子（CAM）配体和细胞因子的呈递，以模拟小生境。HSC表达许多CAM和细胞因子受体，因此很可能必须模拟小生境的多个方面以显著增强HSC自我更新。在静态表面上以适当的分布呈现多个CAM配体和细胞因子是有问题的。动态膜模拟表面将用于允许CAM配体和细胞因子自身重新定向以匹配HSC上CAM和受体的位置，同时保持定位于培养表面。将针对纤维连接蛋白（Fn）的3个结合结构域合成脂质连接肽模拟物，已报道其增强HSC扩增。肽-脂质的不同组合将用于评价Fn结构域对以下各项的单独和协同作用：（1）通过离心测定的细胞粘附，（2）通过用磷酸特异性抗体的免疫印迹的整联蛋白调节的信号转导，（3）通过共聚焦显微镜的CAM聚类，（4）通过流式细胞术进行HSC自体再生，用于细胞分裂追踪和HSC标志物CD 34和Thy-1的细胞表面表达，和（5）通过祖细胞测定和流式细胞术分化成主要的造血细胞谱系，用于谱系特异性抗原表达，硫酸乙酰肝素的脂质连接模拟物，其在基质细胞系支持HSC扩增中起重要作用，还将合成血小板生成素的肽模拟物，血小板生成素是HSC存活和扩增的关键细胞因子。这些化合物将单独和彼此组合以及与Fn结构域模拟物组合进行评估，以进一步增强HSC扩增。

DESCRIPTION (provided by applicant): Hematopoietic stem cells (HSCs) differentiate into all of the blood cell lineages. Ex vivo expansion of HSCs would greatly facilitate cell and gene therapies for viral diseases (e.g., HIV) and genetic blood disorders, and would improve the prospects for umbilical cord blood transplants in adults. However, HSC division in culture is associated with differentiation into cells with decreased potential. This contrasts with sustained HSC expansion in vivo, and led to the hypothesis that a stem cell "niche" supports self-renewal in the bone marrow. The niche includes matrix- and cell-associated cytokines, as well as direct contact with stromal cells. A culture surface will be developed for controlled presentation of cell adhesion molecule (CAM) ligands and cytokines that are expressed by stromal cells in order to mimic the niche. HSCs express many CAMs and cytokine receptors, so it is likely that multiple aspects of the niche will have to be mimicked to substantially enhance HSC self-renewal. Presenting multiple CAM ligands and cytokines in the proper distribution is problematic on a static surface. A dynamic membrane-mimetic surface will be used to allow CAM ligands and cytokines to reorient themselves to match the location of CAMs and receptors on HSCs, while remaining localized to the culture surface. Lipid-linked peptide mimics will be synthesized for the 3 binding domains offibronectin (Fn), which has been reported to enhance HSC expansion. Different combinations of the peptide-lipids will be used to evaluate individual and synergistic effects of the Fn domains on: (1) cell adhesion via a centrifugation assay, (2) integrin-modulated signal transduction via immunoblots with phospho-specific antibodies, (3) CAM clustering via confocal microscopy, (4) HSC selfrenewal via flow cytometry for cell division tracking and cell-surface expression of the HSC markers CD34 and Thy-1, and (5) differentiation into the major hematopoietic cell lineages via progenitor cell assays and flow cytometry for lineage-specific antigen expression, Lipid-linked mimics of heparan sulfate, which plays an essential role in the support of HSC expansion by stromal cell lines, and a peptide mimic of thrombopoietin, which is a key cytokine for HSC survival and expansion, will also be synthesized. These compounds will be evaluated, alone and in combination with each other and the Fn domain mimics, in order to further enhance HSC expansion.