3-D Optical Tracking of Bone Marrow Derived Skin Stem Cells

该奖项由2009年《美国复苏和再投资法案》(公法第111-5条)资助0852658博帕特皮肤是我们最大的器官，在体液平衡、体温调节、免疫监测和自我修复方面发挥关键作用。疾病和/或人体大部分部位的丧失？S的皮肤可能会导致残疾，并可能危及生命，在美国和世界各地都是一个主要的健康问题。干细胞在修复和再生包括皮肤在内的许多组织中发挥着关键作用。因此，阐明干细胞在皮肤中扮演的角色不仅对我们对干细胞动力学的基本理解有重大影响，而且对我们治疗皮肤病、在医疗应用中取代皮肤或在老龄化人口中恢复皮肤活力具有重要影响。光学成像技术的最新进展提供了前所未有的高时空成像分辨率的组合，现在可以用于可视化正常皮肤内皮肤干细胞的复杂三维(3-D)动力学，以及对皮肤损伤和皮肤替换的响应，例如移植工程皮肤替换后。这一提议的智力价值首次通过先进的光学生物医学成像方法表现出来，用于阐明体内和工程皮肤移植物中皮肤干细胞的复杂动力学。这项研究的假设是，光学相干和多光子显微镜在一个集成的平台上，可以唯一地跟踪和量化自体和同种异体工程化皮肤移植物内和周围不同的动态三维干细胞行为。随着最近发现位于毛囊隆起区的皮肤干细胞生态位，这些干细胞在从骨髓迁移到皮肤龛以及在响应皮肤损伤和疾病的过程中的动态行为引起了许多问题。因此，这个项目在四个方面具有智力价值。首先，将利用一种能够同时进行光学相干和多光子显微镜的先进集成显微镜来独特地显示体内皮肤内干细胞的结构和功能关系。其次，该项目研究并纵向成像3-D皮肤干细胞的迁移模式和动态。这将从根本上洞察它们在维持皮肤功能和健康方面所起的作用。第三，将调查由放置自体皮肤移植物(皮肤穿孔活检)引起的皮肤损伤的影响，提供对愈合反应中干细胞动力学的洞察。第四，该项目将纵向成像移植同种异体工程化皮肤结构后体内干细胞和组织的反应，有助于理解皮肤干细胞如何与生物宿主内的工程组织移植物相互作用。更多定量成像技术的发展和应用来分析单细胞或细胞群体水平的动力学将进一步深入了解皮肤干细胞的作用，并最终为需要皮肤移植的人类疾病的治疗提供更好的方法。综上所述，该项目在这四个领域都是新颖的，使用这些先进的成像技术进行这些研究对干细胞生物学和组织工程领域具有变革性。考虑到更广泛的范围，该项目的结果可能会对干细胞在体内动态行为的基础和临床理解产生重大而广泛的影响。这个项目解决了干细胞生物学和组织工程学中的主要挑战：如何在体内、三维以及高度分散的工程组织和自然组织中随时间纵向显示和跟踪细胞和小细胞群体。该项目将把最先进的研究与教育元素相结合，以促进发现和促进教学、培训和学习。除研究生外，本科生还将完成与这项工作相关的论文。这些学生以及博士后研究员和研究科学家将在光学、图像处理、细胞和组织培养、生物学以及临床前模型的使用方面培养终身职业技能。包括妇女和少数群体在内的代表性不足的群体将成为研究机会的目标，并将举行年度实验室和校园范围的开放参观活动，以外联K-12和社区群体。该项目的成果和图像数据库将通过我们的教育网站、地方和国家会议以及领先的科学、工程和医学出版物广泛传播。

This award is funded under the American Recovery and Reinvestment Act of 2009 (Public Law 111-5)0852658 Boppart Skin is our largest organ, serving critical roles in fluid homeostasis, thermoregulation, immune surveillance, and self-healing. Disease and/or the loss of major portions of a human?s skin can be disabling and potentially life threatening, and is a major health problem in the U.S. and throughout the world. Stem cells play a critical role in repairing and regenerating many tissues, including the skin. Elucidating the roles that stem cells play in the skin will therefore have a significant impact on not only our fundamental understanding of stem cell dynamics, but also on our treatment of skin diseases, for replacing skin in medical applications, or in rejuvenating skin in our aging population. Recent advances in optical imaging techniques offer an unprecedented combination of high spatiotemporal imaging resolution that can now be applied to visualizing the complex three-dimensional (3-D) dynamics of skin stem cells within normal skin, and in response to skin injury and skin replacement, such as after grafting engineered skin replacements. The intellectual merit of this proposal is represented for the first time by an advanced optical biomedical imaging approach for elucidating the complex dynamics of skin stem cells in vivo and within engineered skin grafts. The hypothesis of this research is that optical coherence and multi-photon microscopy, in an integrated platform, can uniquely track and quantify the different dynamic 3-D in vivo stem cell behaviors in and around autologous and allogeneic engineered skin grafts. With the recent discovery of the skin stem cell niche located within the bulge region of hair follicles, many questions arise as to the dynamic behavior of these stem cells as they migrate from the bone marrow and into the skin niche, as well as in and out of the niche in response to skin injury and disease. This project therefore has intellectual merit in four areas. First, an advanced integrated microscope capable of simultaneous optical coherence and multi-photon microscopy will be utilized to uniquely visualize the structural and functional relationships of stem cells within in vivo skin. Second, this project investigates and longitudinally images in 3-D the migration patterns and dynamics of skin stem cells. This will provide fundamental insight into the role they play in maintaining the function and health of skin. Third, the effects of skin injury, induced by the placement of an autologous skin graft (skin punch biopsy), will be investigated, providing insight into the stem cell dynamics in the healing response. Fourth, this project will longitudinally image the stem cell and tissue responses in vivo following the grafting of allogeneic engineered skin constructs, contributing significantly to the understanding of how skin stem cells interact with engineered tissue grafts within biological hosts. The development and application of more quantitative imaging techniques to analyze the dynamics at the single-cell or cell-population levels will provide further insight into the ability to understand the role of skin stem cells, and ultimately provide a better approach for the treatment of human pathologies that require skin grafting. Taken together, this project is novel in each of these four areas, and the use of these advanced imaging techniques to carry out these investigations is transformative for the fields of stem cell biology and tissue engineering.Considering the broader scope, the outcome of this project is likely to have a significant and broad impact on both the fundamental and clinical understanding on how stem cells behave dynamically in vivo. This project addresses major challenges in stem cell biology and tissue engineering: how to visualize and track cells and small populations of cells in vivo, in 3-D, and longitudinally over time in highly-scattering engineered and natural tissues.This project will integrate state-of-the-art research with educational elements to advance discovery and promote teaching, training, and learning. Undergraduate students, in addition to graduate students, will complete theses related to this work. These students, as well as post-doctoral fellows and research scientists, will develop lifelong career skills in optics, image processing, cell and tissue culture and biology, and the use of pre-clinical models. Under-represented groups including women and minorities will be targeted for research opportunities, and annual laboratory and campus-wide open-house events will be held for outreach to K-12 and community groups. The results and image databases from this project will be disseminated widely through our educational website, local and national conferences, and leading scientific, engineering, and medical publications.