Optimizing Assays of Human Stem Cells in Bone Marrow

描述（由申请人提供）：在整个生命过程中，所有组织均由干细胞底层系统维持，通过生成新细胞不断更新组织。这些系统涉及一系列分层阶段，起源于罕见的上游干细胞群，并通过更多的下游祖细胞群进展。衰老与组织健康、外观和修复能力的变化相关（并且可能由组织健康、外观和修复能力的变化来定义），这些变化不可避免地与干细胞数量和/或其下游动力学（例如激活、自我更新、增殖、迁移、分化和存活）的潜在变化相关。 骨髓与衰老尤其相关。没有任何组织会随着年龄的增长而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 (HSC) 支持血细胞和免疫系统的形成和维持。间充质干细胞 (MSC) 和下游结缔组织祖细胞 (CTP) 支持骨骼、软骨、脂肪、纤维组织和骨骼肌的生成。此外，即使骨髓中数量较少的干细胞库（例如多能成体祖细胞（MAPC）、侧群（SP）细胞）也已被证明能够对多个胚层组织做出贡献，包括肝脏、大脑、肺和肠道。这种多维性质使骨髓对于理解干细胞系统和这些系统中与年龄相关的变化至关重要，这些变化有助于保持健康和疾病的功能调节。然而，由于与采样偏差、血液细胞稀释和污染相关的较大误差，通过抽吸采集和取样骨髓来源细胞的传统方法不太适合干细胞群的定量分析。此外，抽吸破坏了与干细胞功能相关的信息，这些信息是由干细胞和祖细胞群的分布和组织水平组织特征提供的。 该提案将使用基于细胞的集成建模策略来解决缺陷，以实现对干细胞群的系统评估，并提供最佳执行和解释从抽吸样本和小直径骨芯活检中获得的干细胞测定所需的客观信息。 160 名接受选择性髋关节和膝关节置换术的患者（年龄 30 至 90 岁）将提供评估广泛年龄范围的骨骼和骨髓样本的机会。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集落测定、LTClC测定、端粒酶和端粒长度测定、骨和骨髓的定量组织形态计量学以及病理评估。

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.