Department of Neurosurgery, University of Maryland School of Medicine, Baltimore, MD 21201, USA.
Department of Biomedical Engineering, The Johns Hopkins School of Medicine, Baltimore, MD 21205, USA; Translational Cell and Tissue Engineering Center, The Johns Hopkins School of Medicine, Baltimore, MD 21231, USA.
Biomater Adv. 2022 Mar;134:112675. doi: 10.1016/j.msec.2022.112675. Epub 2022 Jan 21.
This study sets the stage for the therapeutic use of AcManNTProp, an N-acetylmannosamine (ManNAc) analog that installs thiol-modified sialoglycans onto the surfaces of human neural stem cells (hNSC). First, we compared hNSC adhesion to the extracellular matrix (ECM) proteins laminin, fibronectin, and collagen and found preferential adhesion and concomitant changes to cell morphology and cell spreading for AcManNTProp-treated cells to laminin, compared to fibronectin where there was a modest response, and collagen where there was no observable increase. PCR array transcript analysis identified several classes of cell adhesion molecules that responded to combined AcManNTProp treatment and hNSC adhesion to laminin. Of these, we focused on integrin α6β1 expression, which was most strongly upregulated in analog-treated cells incubated on laminin. We also characterized downstream responses including vinculin display as well as the phosphorylation of focal adhesion kinase (FAK) and extracellular signal-related kinase (ERK). In these experiments, AcManNTProp more strongly induced all tested biological endpoints compared to AcManNTGc, showing that the single methylene unit that structurally separates the two analogs finely tunes biological responses. Together, the concerted modulation of multiple pro-regenerative activities through AcManNTProp treatment, in concert with crosstalk with ECM components, lays a foundation for using our metabolic glycoengineering approach to treat neurological disorders by favorably modulating endpoints that contribute to the viability of transplanted NSCs.
这项研究为 AcManNTProp 的治疗用途奠定了基础，AcManNTProp 是一种 N-乙酰甘露糖胺 (ManNAc) 类似物，可将巯基修饰的唾液酸化糖链安装到人神经干细胞 (hNSC) 的表面。首先，我们比较了 hNSC 对细胞外基质 (ECM) 蛋白层粘连蛋白、纤连蛋白和胶原蛋白的粘附，发现与纤连蛋白相比，巯基修饰的唾液酸化糖链修饰的 hNSC 对层粘连蛋白具有优先粘附作用，并且伴随着细胞形态和细胞铺展的变化，而纤连蛋白的反应较弱，胶原蛋白则没有明显的增加。PCR 阵列转录分析鉴定了几类细胞粘附分子，它们对 AcManNTProp 联合处理和 hNSC 粘附到层粘连蛋白的反应。在这些分子中，我们重点关注整合素 α6β1 的表达，它在模拟处理的细胞中被强烈上调，并在层粘连蛋白上孵育。我们还对下游反应进行了特征描述，包括粘着斑蛋白（vinculin）的显示以及粘着斑激酶（FAK）和细胞外信号相关激酶（ERK）的磷酸化。在这些实验中，与 AcManNTGc 相比，AcManNTProp 更强烈地诱导了所有测试的生物学终点，表明两个类似物之间结构上分离的单个亚甲基单元精细地调节了生物学反应。总之，通过 AcManNTProp 处理协同调节多种促再生活性，与细胞外基质成分的串扰一起，为我们通过代谢糖基工程方法治疗神经紊乱奠定了基础，通过有利地调节有助于移植 NSCs 存活的终点。
