Understanding and Using Variation in Source Materials for MSC Fabrication

项目摘要 该提案的总体目标是推进干细胞生物学、基于细胞的诊断和细胞领域 使用系统分离特定干细胞的新途径进行细胞治疗和药物开发的来源 来自肌肉骨骼结缔组织中多个小生境的细胞和祖细胞亚型 “基于绩效的选择”（PBS）。我们的目标是为患者、临床医生和研究人员提供 知识和严格的和可复制的工具和标准，他们需要，以确定和获得最好的 用于再生和关节保存疗法、药物发现和基础研究的可能细胞。 间充质基质细胞（MSC）来源于在小鼠中建立结缔组织祖细胞（CTPs）的集落。 骨和其它组织对于细胞治疗具有广泛的价值。然而，批次间MSC变化 代表了生物医学研究界正在进行的一个深刻的“痛苦”点，这限制了临床研究的进展。 基于MSC的产品的评估和性能。临床治疗需要高水平的可重复性， 再现性最大限度地减少变异对于推进新兴的细胞疗法领域至关重要。 我们假设，这种变化的一个很大的来源是基于MSC制造的传统方法的失败。 在“竞争性扩增”（CE）中，用户只需将来自组织来源的所有细胞放入培养物中， 竞争。不对包含或排除的单元格进行选择。用户只是接受一个结果， 可以通过初始培养物中集落形成细胞的类型和混合的随机变化来预先确定。 该提案整合了几个独特的技术平台，以实现快速有效的进展， 克服这些限制，具体而言： 1)用于自动化大视场（LFOV）（“Google Earth”）成像和图像分析的标准化机器人 来鉴定和测量CTP及其后代。 2)延时视频显微镜，可连续跟踪每个CTP衍生菌落的形成 从电镀开始。反转视频使我们能够第一次看到并描述殖民地的特征 创建CTP，因为它们在从我们的身体中移除后立即存在，并且在它们分裂之前。 3)自动化机器人平台（Cell X™），其使得能够选择（“挑选”）单个CTP衍生物， 克隆以及自动化处理，这是需要扩大他们的后代“免提”，使 比目前的手工方法具有更高的精度和可重复性。 这一提议建立了关于现金转拨方案在生物潜力方面差异很大的知识。利用这些工具，我们将 确定MSC生产中应包括或不应包括的菌落建立CTP的属性 然后提供使用这些知识来实现高质量和高度可重复的结果的方法。

Project Summary The Broad Aim of this proposal is to advance the field of stem cell biology, cell-based diagnostics, and cell sourcing for cell therapy and drug development using a new pathway for systematic isolation of specific stem cell and progenitor cell subtypes from the plurality of niches in musculoskeletal connective tissues that we refer to as “Performance-Based Selection” (PBS). Our goal is to provide patients, clinicians and researchers with the knowledge and rigorous and reproducible tools and standards that they need, to identify and obtain the best possible cells for use in regenerative and joint preservation therapies, drug discovery and basic research. Mesenchymal Stromal Cells (MSCs) derived from colony founding Connective Tissue Progenitors (CTPs) in bone and other tissues are widely valuable for cellular therapy. However, batch to batch MSC variation represents a profound point of ongoing “pain” in the biomedical research community that is limiting the clinical evaluation and performance of MSC-based products. Clinical therapies demand high levels of repeatability and reproducibility. Minimizing variation is essential to advancing the emerging field of cellular therapies. We hypothesize that a large source of this variation is the failure of traditional methods of MSC fabrication based on “Competitive Expansion” (CE), where the user simply puts all cells from tissue source into culture, where they compete. No choice is made regarding cells to include or exclude. The user simply accepts an outcome that may be predetermined by random variation in the type and mix of colony founding cells in the initial culture. This proposal integrates several unique technology platforms to enable rapid and effective progress in overcoming these limitations, specifically: 1) Standardized robotics for automated large-field-of-view (LFOV) (“Google Earth”) imaging and image analysis to identify and measure CTPs and their progeny. 2) Time Lapse Videomicroscopy that enables continuous tracking of the formation of each CTP-derived colony from the time of plating. Reversing the video enables us, for the first time, to see and to characterize colony founding CTPs as they exist immediately after removal from our bodies and before they divide. 3) An automated robotic platform (Cell X™) that enables both the selection (“Picking”) of individual CTP-derived clones as well as the automated processing that is needed to expand their progeny “hands free”, enabling vastly greater precision and reproducibility than current manual methods. This proposal builds the knowledge that CTPs vary widely in biological potential. Using these tools, we will determine the attributes of colony founding CTPs that should or should not be included in MSC manufacturing and then provide the means to use this knowledge to achieve high quality and highly reproducible outcomes.