Stromal Cell Mimic for Hematopoietic Stem Cell Expansion

描述(由申请人提供)： 造血干细胞(HSCs)可分化为所有血细胞系。造血干细胞的体外扩增将极大地促进病毒疾病(如艾滋病毒)和遗传性血液疾病的细胞和基因治疗，并将改善成人脐带血移植的前景。然而，培养中的HSC分裂与分化为潜能降低的细胞有关。这与体内持续的HSC扩增形成了对比，并导致了干细胞“小众”支持骨髓自我更新的假说。这个利基包括与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黏附分子(CAM)配体和细胞因子的表达，以模拟利基环境。HSC表达许多CaM和细胞因子受体，因此很可能必须模仿利基的多个方面才能显著增强HSC的自我更新。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。一个动态的仿膜表面将被用来允许CAM配体和细胞因子重新定位以匹配HSCs上的CAM和受体的位置，同时保持定位于培养表面。脂联素(FN)的3个结合域将被合成类似的脂联肽，据报道它能促进HSC的扩增。不同的肽-脂组合将被用来评估FN结构域在以下方面的单独和协同作用：(1)通过离心法进行细胞黏附，(2)通过带有磷酸化特异性抗体的免疫印迹进行整合素调节的信号转导，(3)通过共聚焦显微镜进行CAM聚集，(4)通过流式细胞术进行细胞分裂跟踪和HSC标记CD34和Thy-1的细胞表面表达的自我更新，以及(5)通过祖细胞分析和流式细胞术分化为主要的造血细胞谱系，用于谱系特异性抗原的表达，脂联的乙酰肝素模拟物，它在支持基质细胞系扩增HSC方面起着至关重要的作用，还将合成一种模拟血小板生成素的多肽，它是HSC存活和扩增的关键细胞因子。为了进一步加强HSC的扩展，将对这些化合物进行单独和相互结合以及FN结构域模拟的评估。

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.