Understanding and Using Variation in Source Materials for MSC Fabrication

项目摘要 这项建议的广泛目标是推进干细胞生物学、基于细胞的诊断和细胞 利用系统分离特定干细胞的新途径为细胞治疗和药物开发提供资源 我们所指的肌肉骨骼结缔组织中多个壁龛中的细胞和祖细胞亚型 改为“绩效选拔”(PBS)。我们的目标是为患者、临床医生和研究人员提供 他们需要的知识和严格的、可重复使用的工具和标准，以确定和获得最佳 可能用于再生和联合保存疗法、药物发现和基础研究的细胞。 骨髓间充质干细胞(MSCs)来源的集落形成结缔组织前体细胞(CTPs) 骨和其他组织对细胞治疗具有广泛的价值。然而，批次到批次的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 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和 再现性。最大限度地减少变异对于推进细胞疗法这一新兴领域至关重要。 我们假设，这种差异的一个很大来源是基于MSC制造的传统方法的失败 在“竞争性扩增”(CE)中，使用者只需将来自组织来源的所有细胞放入培养中，在那里他们 竞争。对于要包括或排除的单元格，不会做出任何选择。用户简单地接受可以 是由最初培养的集落形成细胞的类型和混合的随机变化决定的。 该提案集成了几个独特的技术平台，以实现快速有效地在 克服这些限制，特别是： 1)用于自动大视场(LFOV)(“Google Earth”)成像和图像分析的标准化机器人技术 识别和测量CTP及其后代。 2)时间推移视频显微镜，能够连续跟踪每个CTP衍生的菌落的形成 从电镀的时候起。倒转视频使我们第一次能够看到并描述殖民地 创建CTP，因为它们在从我们的身体移除后立即存在，并且在它们分裂之前。 3)自动化机器人平台(CELL X™)，该平台既能选择(“挑选”)由CTP衍生的个体 克隆以及扩展其后代所需的自动化处理，从而实现 比目前的手工方法有更高的精确度和重复性。 这一建议建立了这样一种知识，即CTP在生物潜力上差异很大。使用这些工具，我们将 确定应该或不应该包括在MSC制造中的菌落创建CTP的属性 然后提供利用这些知识实现高质量和高度可重复性的结果的手段。

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.