Sugar-coating our way to genetically modified mesenchymal stem cells: Glycocalyx-inspired cell culture substrates that prime mesenchymal stem cells for polycation-mediated pDNA delivery.

项目总结 人骨髓间充质干细胞(HMSCs)在治疗自身免疫性疾病方面显示出良好的前景。 CER、心脏病和椎间盘退变。HMSCs通过分泌治疗药物促进组织修复- TIC生物分子，如免疫调节和促血管生成因子。基因改造在泰国很有价值-- 抑制人骨髓间充质干细胞分泌体，提高治疗效能。尽管病毒载体在基因上有效 修饰hMSCs、放大和制造方面的挑战限制了广泛的临床应用。在过多的 合成材料可以取代病毒载体，聚阳离子基因载体是最通用、规模最大的载体之一。 有能力的、经济的选择。对于聚阳离子载体在hMSCs中的高表达，我们 必须解决传递效率、细胞毒性和维持hMSC分化之间的权衡 容量。为了缓解聚阳离子介导的基因传递的瓶颈，我们将在糖胺类药物上扩增hMSCs- 模拟细胞培养底物，灵感来自于糖基化的组成和组织。我们假设- 估计碳水化合物残基的多价呈现--让人想起糖胺聚糖(GAG)-- 在hMSC-底物界面将通过隔离生长因子(GFS)介导刺激hMSC增殖 人骨髓间充质干细胞黏附和增殖。多价模拟止吐聚合物刷子将固定化GFS 高局部浓度的hMSCs，导致hMSCs比未修饰的组织增殖更快- 培养聚苯乙烯。当hMSC增殖增强时，核膜将更频繁地溶解， 促进有效载荷的核摄取，并促进聚阳离子介导的转基因表达。与生物不同- 逻辑派生的GAG，如硫酸乙酰肝素或硫酸软骨素，模拟GAG的聚合物刷子是化学- 定义明确、经济且可从一批到另一批重现，使我们能够在 底物界面性质，hMSC自我更新，以及聚阳离子介导的转基因表达。通过Synthe- 含有中性、硫化或羧化β-葡萄糖/氨基葡萄糖混合物的三元共聚聚合物刷子的施胶 残基，我们将了解硫酸盐/羧酸基序的空间分布和多价呈现 糖链残基的多少(由刷子厚度控制)直接决定了细胞的黏附、增殖和细胞命运 骨髓间充质干细胞。此外，我们将通过以下方式确定增强聚阳离子介导的基因传递的Gag模拟底物 促进hMSC核内多聚阳离子穿梭质粒(PDNA)的输入。与以前的方法不同 试图通过狭隘地专注于优化聚阳离子来提高hMSCs的转基因表达(但失败了 结构，我们采用整体概念框架，对hMSC衬底线索给予同等考虑 以及聚阳离子基因载体的分子设计。使用模拟呕吐的细胞培养底物，或者- 协调hMSC的自我更新和高效的聚阳离子介导的PDNA传递，我们将获得遗传修饰。 使用负担得起且可扩展的生物材料平台固定hMSCs。我们的研究成果可以用来降低生产效率- 治疗成本，减轻监管负担，并扩大获得hMSC疗法的途径。

PROJECT SUMMARY Human mesenchymal stem cells (hMSCs) have demonstrated promise in treating auto-immune disorders, can- cer, cardiac diseases, and intervertebral disc degeneration. hMSCs facilitate tissue repair by secreting therapeu- tic biomolecules such as immunomodulatory and pro-angiogenic factors. Genetic modification is valuable in tai- loring the hMSC secretome and boosting therapeutic potency. Although viral vectors are effective in genetically modifying hMSCs, scale-up and manufacturing challenges limit broad clinical application. Among the plethora of synthetic materials that can replace viral vectors, polycationic gene carriers are among the most versatile, scal- able, and economical options. For polycationic carriers to mediate high transgene expression in hMSCs, we must resolve the tradeoff between delivery efficiency, cellular toxicity, and maintenance of hMSC differentiation capacity. To ease bottlenecks in polycation-mediated gene delivery, we will expand hMSCs on glycosaminogly- can-mimetic cell culture substrates inspired by the composition and organization of the glycocalyx. We hypoth- esize that the multivalent presentation of carbohydrate residues—reminiscent of glycosaminoglycans (GAGs)— at hMSC–substrate interfaces will stimulate hMSC proliferation by sequestering growth factors (GFs) mediating hMSC adhesion and proliferation. Multivalent GAG-mimetic polymer brushes will present immobilized GFs to hMSCs with high local concentrations, causing hMSCs to proliferate more rapidly relative to unmodified tissue- culture polystyrene. When hMSC proliferation is enhanced, the nuclear envelope will dissolve more frequently, facilitating the nuclear uptake of payloads, and boosting polycation-mediated transgene expression. Unlike bio- logically derived GAGs such as heparan sulfate or chondroitin sulfate, GAG-mimetic polymer brushes are chem- ically defined, economical, and reproducible from batch to batch, enabling us to weave connections between substrate interfacial properties, hMSC self-renewal, and polycation-mediated transgene expression. By synthe- sizing ternary copolymer brushes bearing a mixture of neutral, sulfated, or carboxylated β-glucose/glucosamine residues, we will learn how the spatial distribution of sulfate/carboxylate motifs and the multivalent presentation of glycan residues (governed by brush thickness) directs the adhesion, proliferation, and cell fate decisions of hMSCs. Further, we will identify GAG-mimetic substrates that augment polycation-mediated gene delivery by facilitating the import of polycation-shuttled plasmids (pDNA) within hMSC nuclei. Unlike previous approaches that tried (and failed) to boost transgene expression in hMSCs by focusing narrowly on optimizing polycation structure, we embrace a holistic conceptual framework that offers equal consideration to hMSC substrate cues and the molecular design of polycationic gene carriers. Deploying GAG-mimetic cell culture substrates that or- chestrate hMSC self-renewal and efficient polycation-mediated pDNA delivery, we will obtain genetically modi- fied hMSCs using affordable and scalable biomaterial platforms. Our findings can be deployed to lower produc- tion costs, lighten regulatory burden, and broaden access to hMSC therapeutics.