Pediatric Clinical Research Center "Romeo and Enrica Invernizzi", L. Sacco Department of Biomedical and Clinical Sciences, University of Milano, Milano, 20157, Italy.
Department of Chemistry, Materials and Chemical Engineering "Giulio Natta", Politecnico di Milano, Milano, 20133, Italy.
Nanotheranostics. 2021 Jan 1;5(1):8-26. doi: 10.7150/ntno.50633. eCollection 2021.
Stem Cells (SCs) show a great potential in therapeutics for restoring and regenerating native tissues. The clinical translation of SCs therapies is currently hindered by the inability to expand SCs in large therapeutic dosages, while maintaining their safety and potency. The use of biomaterials allows for the generation of active biophysical signals for directing SCs fate through 3D micro-scaffolds, such as the one named "Nichoid", fabricated with two-photon laser polymerization with a spatial resolution of 100 nm. The aims of this study were: i) to investigate the proliferation, differentiation and stemness properties of neural precursor cells (NPCs) following their cultivation inside the Nichoid micro-scaffold; ii) to assess the therapeutic effect and safety of NPCs cultivated in the Nichoid in a preclinical experimental model of Parkinson's Disease (PD). Nichoids were fabricated by two photon laser polymerization onto circular glass coverslips using a home-made SZ2080 photoresist. NPCs were grown inside the Nichoid for 7 days, counted and characterized with RNA-Seq, Real Time PCR analysis, immunofluorescence and Western Blot. Then, NPCs were transplanted in a murine experimental model of PD, in which parkinsonism was induced by the intraperitoneal administration of the neurotoxin MPTP in C57/bl mice. The efficacy of engrafted Nichoid-expanded NPCs was evaluated by means of specific behavioral tests and, after animal sacrifice, with immunohistochemical studies in brain slices. NPCs grown inside the Nichoid show a significantly higher cell viability and proliferation than in standard culture conditions in suspension. Furthermore, we report the mechanical conditioning of NPCs in 3D micro-scaffolds, showing a significant increase in the expression of pluripotency genes. We also report that such mechanical reprogramming of NPCs produces an enhanced therapeutic effect in the model of PD. Recovery of PD symptoms was significantly increased when animals were treated with Nichoid-grown NPCs, and this is accompanied by the recovery of dopaminergic markers expression in the striatum of PD affected mice. SCs demonstrated an increase in pluripotency potential when expanded inside the Nichoid, without the need of any genetic modification of cells, showing great promise for large-scale production of safe and functional cell therapies to be used in multiple clinical applications.
干细胞（SCs）在修复和再生原生组织的治疗中具有巨大的潜力。然而，SCs 疗法的临床转化目前受到限制，因为无法在大治疗剂量下扩增干细胞，同时保持其安全性和效力。生物材料的使用允许通过 3D 微支架产生用于指导干细胞命运的主动生物物理信号，例如使用双光子激光聚合技术制造的具有 100nm 空间分辨率的“Nichoid”。本研究的目的是：i）研究神经前体细胞（NPCs）在 Nichoid 微支架内培养后的增殖、分化和干细胞特性；ii）评估在帕金森病（PD）的临床前实验模型中培养的 NPCs 在 Nichoid 中的治疗效果和安全性。Nichoids 通过双光子激光聚合技术在圆形玻璃盖玻片上使用自制的 SZ2080 光致抗蚀剂制造。将 NPCs 在 Nichoid 内培养 7 天，通过 RNA-Seq、实时 PCR 分析、免疫荧光和 Western Blot 进行计数和表征。然后，将 NPCs 移植到 C57/bl 小鼠的 PD 实验模型中，通过腹腔内给予神经毒素 MPTP 诱导帕金森病。通过特定的行为测试和动物处死时的脑切片免疫组织化学研究评估植入的 Nichoid 扩增 NPCs 的疗效。在 Nichoid 内生长的 NPCs 比在悬浮培养中的标准培养条件下具有更高的细胞活力和增殖。此外，我们报告了 NPCs 在 3D 微支架中的机械调节，显示出多能性基因表达的显著增加。我们还报告说，NPCs 的这种机械重编程在 PD 模型中产生了增强的治疗效果。当用 Nichoid 生长的 NPCs 治疗时，PD 症状的恢复明显增加，并且伴随着 PD 受影响小鼠纹状体中多巴胺能标志物表达的恢复。当在 Nichoid 内扩增时，SCs 显示出增加的多能性潜力，而无需对细胞进行任何基因修饰，这为大规模生产安全有效的细胞疗法以用于多种临床应用提供了巨大的希望。
