Heat Shock Effects on Human Mesenchymal Stem Cell Differentiation in Hydrogel

描述(申请人提供)：结合干细胞的组织工程是再生医学的一个新兴领域，有望修复因疾病、衰老和创伤而受损的组织和器官。为了提高干细胞的治疗利用率，重要的是研究如何提高干细胞增殖和分化的质量和数量，从而增加细胞生长到所需类型的组织或结构的机会。温度是一个重要的物理因素，对生长过程的影响仅次于氧气供应。更具体地说，体内研究表明，局部透热可以刺激骨形成。此外，热休克蛋白(HSPs)的上调及其在胚胎发育中的重要作用也得到了广泛的认识。在组织损伤后的炎症过程中，局部或全身温度升高是常见的，并可能在组织再生中起重要作用。然而，温度诱导骨细胞分化以及胚胎发育过程中热休克蛋白过度表达的机制尚不清楚。温度升高是否会影响成体干细胞的募集和分化也是未知的。尽管人们对其他因素的影响进行了深入的研究，但关于热效应对干细胞分化的影响的研究还很少。因此，PI建议对热休克对间充质干细胞(MSC)增殖和分化的影响进行全面的研究，以验证我们的假设--温和的温度升高可能促进MSC的分化和成熟。这项为期两年的试点项目的具体目标是：(1)利用合成和自组装的多肽水凝胶-PuraMatrix建立优化的三维培养条件，用于人间充质干细胞(HMSC)的增殖；使用经典的分化诱导剂，比较三维PuraMatrix培养和传统二维成骨培养或三维颗粒培养成骨和成软骨的hMSC分化；(2)在优化的三维培养环境中，探讨热休克对hMSC增殖和向成骨细胞和软骨细胞分化的动态影响，并利用针对HSP70和HSP27的siRNAs研究热休克蛋白(HSPs)在热休克诱导hMSC分化中的作用。这项热休克对人骨髓间充质干细胞向骨和软骨细胞分化的初步研究的预期意义是：(A)促进人骨髓间充质干细胞向成骨细胞/软骨细胞的增殖和分化；(B)提高成熟度，使分化后的细胞能够像成人细胞一样有效地发挥作用；(C)提供三维体外MSC培养模型，最大限度地模拟体内分化环境；以及(D)为设计临床适用于完全分化的干细胞用于生物器官/细胞移植的热处理方案提供基础科学数据。 公共卫生相关性：这项初步研究可能导致一种新的策略，使用热处理来提高从间充质干细胞分化出的成骨细胞(骨细胞)和软骨细胞(软骨细胞)的成熟水平。随着成熟度的提高，分化细胞在组织生成中更好地发挥与成体细胞相似的功能，用于修复受损或老化的器官。此外，这项试点提案的授予将有助于在纽约城市学院生物医学工程(BME)系建立长期干细胞研究。

DESCRIPTION (provided by applicant): Tissue engineering combined with stem cells, an emerging field of regenerative medicine, holds promise for the restoration of tissues and organs damaged by diseases, aging, and trauma. In order to improve the therapeutic usage of stem cells, it is important to investigate ways to enhance the qualitative and quantitative extent of stem cell proliferation and differentiation, and thus increase the chances for cells to grow into desired types of tissues or structures. Temperature is an important physical factor only second to oxygen supply in its influence on growth processes in general. More specifically, in vivo study has shown that bone formation is stimulated by local diathermy. In addition, the upregulation of heat shock proteins (HSPs) and their important roles in embryonic development have been widely recognized. Local or systemic temperature elevation is common during inflammation after tissue injury and may have an important role in tissue regeneration. However, the mechanisms for temperature-induced bone cell differentiation as well as overexpression of HSPs during embryonic development are unclear. Whether or not elevated temperature affects adult stem cell recruitment and differentiation is also unknown. Although intensive investigations have been conducted on the influences of other factors, few studies have been done on thermal effects on stem cell differentiation. Therefore, the PI proposes to conduct a comprehensive study on heat shock effects on mesenchymal stem cell (MSC) proliferation and differentiation to investigate our hypothesis - that mild temperature increase may facilitate MSC differentiation and maturation. The specific aims for this two-year pilot project are: (1) to establish an optimized 3D culture condition for human mesenchymal stem cell (hMSC) proliferation using a synthetic and self-assembling peptide hydrogel - PuraMatrix, and to evaluate hMSC differentiation on 3D PuraMatrix culture versus conventional 2D culture for osteogenesis or 3D pellet culture for chondrogenesis, using classic differentiation inducers; and (2) to explore dynamic heat-shock effects on hMSC proliferation and differentiation into osteoblasts and chondrocytes in the optimized 3D culture environment, and to investigate the role of heat shock proteins (HSPs) in heat shock effects on hMSC differentiation using siRNAs targeting HSP70 and HSP27. The expected significances of this pilot study of heat shock effects on hMSC differentiation into bone and cartilage cells are: (a) to facilitate hMSC proliferation and differentiation into osteoblasts/chondrocytes; (b) to improve maturature so that differentiated cells can function as efficiently as adult cells; (c) to provide a 3D in vitro MSC culture model to maximally mimic the in vivo differentiation environment; and (d) to provide basic scientific data for the design of a thermal treatment protocol that is clinically applicable using fully differentiated stem cells in bioartifical organ/cell transplantation. PUBLIC HEALTH RELEVANCE: This pilot study may lead to a new strategy that uses thermal treatments to enhance the maturature levels of osteoblasts (bone cells) and chondrocytes (cartilage cells) differentiated from mesenchymal stem cells. With increased maturature, differentiated cells are better to perform functions similar to adult cells in tissue generation for the repair of injured or aged organs. In addition, the award of this pilot proposal will help to establish long term stem cell research in the Biomedical Engineering (BME) Department at the City College of New York (CCNY).