Regulating the Quality and Potency of Stem Cells with Biophysical Cues from Dynamic Nanofibrous Hydrogels for Therapeutic Purposes

项目摘要/摘要 人骨髓间充质干细胞(HMSCs)被认为是同种异体治疗的来源。 各种疾病。由于需求呈指数级增长，因此需要新的战略来生产 强大的hMSCs，服务于不同的患者群体。目前，传统的平面培养和生物反应器是 用作放大生产方法。然而，这些并不是专门为hMSCs扩展量身定做的。 它们可能改变细胞表型和分泌组，影响临床疗效。进一步研究以了解 为了实现可重复生产，需要基质力学在hMSC扩张中的作用。数不胜数 支架替代品复制了天然细胞外基质(ECM)的几个特征。然而，它的 在调节关键的细胞过程中起着基础性作用的动力学机制还不够充分。 还没学过。此外，大多数体外基质是静态的和超生理学僵硬的。静电衬底具有 为产生高细胞数提供了实质性的好处；然而，hMSCs已被证明保留了 机械信息，限制了治疗能力。为了解决这个问题，这项拟议的研究试图 研究动态细胞-基质相互作用和纳米地形信号在免疫调节中的作用 使用包裹在动态水凝胶中的电纺纤维复合材料的hMSCs的潜力，与 假设这种复合生物材料将促进hMSCs的高产量，具有相关的治疗价值， 同时消除了传统细胞培养系统所报道的限制。K99期间将重点放在 设计和表征动态纳米纤维水凝胶复合材料，推动我建立 它们调节细胞质量和效力的机制具有相关的治疗价值 (在R00阶段)。在目标1中，我们将使用透明质酸开发动态纳米纤维系统。 水凝胶网络通过动态共价肼键交联能捕获ECM的粘弹性 纸巾。四个变量，包括不同密度的电纺胶原纳米纤维的包封率， 纤维直径、纤维长度和水凝胶的应力松弛时间将以此为目标进行表征。 促进人骨髓间充质干细胞的活性和增殖。在目标2中，hMSCs细胞质量和潜能将通过以下方式进行评估 测定水凝胶参数对细胞分泌活性的影响。免疫调节特性将是 通过量化共培养中的淋巴细胞抑制以及hMSC表面标志的表达来评估。 还将评估hMSCs的分化能力。在目标3中，将生物物理 纳米纤维水凝胶的参数对hMSC分泌活性的影响将通过检测细胞粘附性来探讨 蛋白质和转录因子或机械提示传感器的激活。总而言之，拟议的研究 将带来新的见解来生产具有高治疗价值的hMSCs，这将使新的培养基质成为可能 实现对可重复性和细胞质量的控制，以服务于不同的患者群体。

Project Summary/Abstract Human mesenchymal stem cells (hMSCs) are considered a source for allogeneic therapies to treat diverse diseases. Due to the exponential increase in demand, there is a need for new strategies to produce potent hMSCs to serve diverse patient populations. Currently, conventional planar culture and bioreactors are used as scale-up manufacturing methods. However, these are not specifically tailored for hMSCs expansion. They may alter the cell phenotype and secretome, affecting clinical effectiveness. Further studies to understand the role of substrate mechanics on hMSC expansion are required to achieve reproducible production. Numerous scaffolding alternatives replicate several characteristics of the native extracellular matrix (ECM). However, its dynamic mechanics, which plays a fundamental role in regulating crucial cellular processes, has not been amply studied yet. Furthermore, most in-vitro substrates are static and supraphysiologically stiff. Static substrates have offered a substantial benefit for generating high cell numbers; however, hMSCs have been shown to retain mechanical information, limiting therapeutic capabilities. To address this problem, this proposed research seeks to investigate the role of dynamic cell-matrix interactions and nano-topographical cues on the immunomodulatory potential of hMSCs using a composite of electrospun-fibers encapsulated in a dynamic hydrogel, with the hypothesis that this composite biomaterial will promote high hMSCs production with relevant therapeutic value, while eliminating the limitations reported for the conventional cell culture systems. The K99 period will focus on engineering and characterizing the dynamic nanofibrous hydrogel composites to propel me toward establishing the mechanisms by which they modulate cell quality and potency attributes with relevant therapeutic value (during the R00 phase). In Aim 1, we will develop the dynamic nanofibrous system using a hyaluronic acid hydrogel network crosslinked via dynamic covalent hydrazone bonds that capture the viscoelasticity of ECM in tissues. Four variables, including the encapsulation of the electrospun collagen nanofibers at various densities, fiber diameter, fiber length, and the stress relaxation timescale of the hydrogel will be characterized in this aim to promote hMSC viability and proliferation. In Aim 2, hMSCs cell quality and potency will be assessed by measuring the effect of hydrogel parameters on cellular secretory activity. Immunomodulatory properties will be evaluated by quantifying lymphocyte suppression in co-culture, as well as expression of hMSC surface markers. The capacity of the hMSCs to differentiate will also be assessed. In aim 3, the mechanism linking the biophysical parameters of the nanofibrous hydrogel to hMSC secretory activity will be probed by examining cell adhesive proteins and the activation of transcription factors or sensors of mechanical cues. In sum, the proposed research will lead to new insights to produce hMSCs with high therapeutic value, which will enable new culture substrates that achieve control in reproducibility and cell quality to serve diverse patient populations.