Optimizing Assays of Human Stem Cells in Bone Marrow

描述(申请人提供)：在整个生命过程中，所有组织都由干细胞的基本系统维持，这些干细胞系统通过新细胞的生成不断更新组织。这些系统包括一系列分级阶段，起源于稀有的上游干细胞群体，并通过更多的下游祖细胞群体进行。衰老与组织健康、外观和修复能力的变化有关(也可能由其定义)，这些变化不可避免地与干细胞数量和/或其下游动力学(如激活、自我更新、增殖、迁移、分化和存活)相关。 骨髓与衰老特别相关。没有一种组织会随着年龄发生如此深刻的变化。此外，骨髓是多个干细胞库的所在地。造血干细胞(HSCs)支持血细胞和免疫系统的形成和维持。间充质干细胞(MSCs)和下游结缔组织前体细胞(CTPs)支持骨、软骨、脂肪、纤维组织和骨骼肌的生成。此外，骨髓中数量更少的干细胞库(如多能成体祖细胞(MAPC)、侧群(SP)细胞)被证明能够促进多个生殖层的组织，包括肝、脑、肺和肠道。这种多维度的性质使骨髓在理解干细胞系统以及这些系统中有助于保持健康和疾病功能适应的与年龄相关的变化方面处于核心重要地位。然而，传统的抽吸法采集和采样骨髓来源的细胞，由于采样偏差和稀释以及与血液细胞的污染有关的大误差，不太适合干细胞数量的定量分析。此外，抽吸破坏了与干细胞功能有关的信息，这些信息是由干细胞和祖细胞群体的分布和组织水平组织提供的。 这项建议将使用综合的基于细胞的建模策略来解决不足之处，以实现对干细胞群体的系统评估，并提供必要的客观信息，以最佳地执行和解释从抽吸样本和小直径骨核活检获得的干细胞分析。160名接受择期髋关节和膝关节置换术的患者(年龄在30岁到90岁之间)将有机会评估广泛年龄范围的骨骼和骨髓样本。每个样本都将采用综合的多学科分析策略，包括：荧光激活细胞分选(FACS)和细胞分数分析、CTP集落分析、LTClC分析、端粒酶和端粒长度分析、骨和骨髓的定量组织形态计量学和病理学评估。

DESCRIPTION (provided by applicant): Throughout life all tissues are maintained by underlying systems of stem cells that continually renew tissues through the generation of new Cells. These systems involve a series of hierarchical stages, originating with a rare population of upstream stem cells, and progressing through more numerous populations of downstream progenitor cells. Aging is associated with (and possibly defined by) changes in tissue health, appearance, and capacity for repair that are inevitably associated with underlying changes in the number of stem cells and/or their downstream kinetics (e.g. activation, self renewal, proliferation, migration, differentiation, and survival). Bone marrow is particularly relevant to aging. No tissue undergoes such profound change with age. Moreover, bone marrow is the home of multiple stem cell pools. Hematopoetic stem cells (HSCs) support the formation and maintenance of blood cells and the immune system. Mesenchymal Stem Cells (MSCs) and downstream connective tissue progenitors (CTPs), support the generation of bone, cartilage, fat, fibrous tissue, and skeletal muscle. Furthermore, even less numerous stem cell pools in bone marrow (e.g. multipotent adult progenitor cells (MAPCs), side population (SP) cells) have been shown to be capable of contributing to tissues of multiple germ layers, including liver, brain, lung and gut. This multidimensional nature places bone marrow in a position of central importance for understanding stem cell systems and the age related changes in these systems that contribute to preservation of health and functional accommodation to disease. However, conventional methods for harvest and sampling of bone marrow derived cells by aspiration is not well suited for quantitative assay of the stem cell population, due to large errors associated with sampling bias and dilution and contamination with cells from blood. Furthermore, aspiration disrupts information related to stem cell function that is provided by features of distribution and tissue level organization of stem cell and progenitor populations. This proposal will address deficiencies using an integrated cell-based modeling strategy to enable the systematic assessment of stem cell populations, and provide objective information necessary to optimally perform and interpret stem cell assays available from aspirate samples and small diameter bone core biopsies. 160 patients undergoing elective hip and knee arthroplasty (age 30 to 90), will provide the opportunity to assess bone and marrow samples from a broad age range. An integrated multidisciplinary analysis strategy will be applied to each sample, including: fluorescence activated cell sorting (FACS) and cell fraction analysis, CTP colony assay, LTClC assay, assay of telomerase and telomere length, quantitative histomorphometry of bone and marrow and pathological assessment.