Early Identification and Characterization of Connective Tissue Progenitors

结缔组织祖细胞的早期识别和表征

• 批准号: 8806193
• 负责人: George F Muschler
• 金额: $ 20.01万
• 依托单位: CLEVELAND CLINIC LERNER COM-CWRU
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-09-15 至 2016-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8806193
• 关键词: Address; Area; Aspirate substance; Back; Biologic Characteristic; Biological; Biological Assay; Biological Markers; Bone Marrow; Bone Tissue; Cartilage; Cell Count; Cell Density; Cell Proliferation; Cell Therapy; Cell Transplantation; Cell physiology; Cell surface; Cells; Cellular Morphology; Cellular biology; Characteristics; Clinical; Collection; Colony-Forming Units Assay; Connective Tissue; Data Correlations; Development; Device or Instrument Development; Devices; Diagnostic tests; Differentiation Antigens; Disease; Early identification; Embryology; Environment; Flow Cytometry; Fluorescence; Future; Growth and Development function; Health; Heterogeneity; Human; Human Characteristics; Image; In Situ; In Vitro; Individual; Kinetics; Knowledge; Label; Laboratories; Learning; Life; Location; Maintenance; Marrow; Measurement; Measures; Methods; Metric; Morphology; Natural regeneration; Nature; Pathogenesis; Pattern; Performance; Pharmaceutical Preparations; Phase; Phenotype; Population; Population Heterogeneity; Predictive Value; Prevalence; Process; Proliferating; Property; Protocols documentation; Regenerative Medicine; Reproducibility; Sampling; Sea; Source; Staining method; Stains; Stem cells; Surface; Suspension substance; Suspensions; System; Testing; Therapeutic; Time; Tissue Sample; Tissues; Variant; Video Microscopy; acronyms; base; biological systems; bone; cell motility; cell type; cellular imaging; design; drug development; imaging modality; improved; in vivo; insight; interest; novel diagnostics; progenitor; public health relevance; regenerative therapy; repaired; response; stem; stem cell population; tissue regeneration

DESCRIPTION: Cell based regenerative therapy requires the activity of stem progenitor cells for tissue regeneration. Connective tissue progenitors (CTPs) are defined as the heterogeneous population of tissue resident stem and progenitor cells that are capable of proliferating and differentiating into one or more connective tissue phenotypes. The number and heterogeneity of CTPs can be estimated using the colony formation assay, in which colony founding CTPs are detected and measured by their ability to proliferate and colony of progeny that express a connective tissue phenotype in vitro. In addition to measuring the number and prevalence of CTPs in a tissue sample, differences in biological potential between various CTPs can be assessed based on variation in cell number, distribution, morphology and expression patterns between colonies that are formed. While very useful, a very important limitation of traditional colony formation assays is that their only direct measurement is made on the progeny of the founding CTP (the colony). Information about the founding CTP can only be inferred from the number of colonies and their features. Colony assays to not reveal the physical or biological properties of the rare individual colony founding CTPs themselves, before proliferation. To address this limitation, we have developed a live cell, videomicroscopy system that enables systematic characterization of colony founding cells (morphology, expression of surface markers) before colony formation. Using multicolor labeling methods, a mixed tissue derived cell population is labeled for cell markers of interest at the time of plating. Time lapse phase contras images are then captured over 6 days, while colonies form, over a large field of view. At the end of the culture period, colonies are characterized with respect to established markers and colony metrics. By reversing the video, each colony can also be traced back to identify the colony founding cell, as one in a sea of other non-progenitors. Using this method we propose to significantly advance our understanding of the nature of human CTPs, their diversity and subtypes, their location in bone tissue and define factors that can be measured non-invasively and used to predict their biological potential, or select CTP best suited for specific therapeutic purposes.

描述：基于细胞的再生疗法需要干细胞祖细胞的活性来进行组织再生。结缔组织祖细胞（ctp）被定义为能够增殖和分化为一种或多种结缔组织表型的组织驻留干细胞和祖细胞的异质群体。ctp的数量和异质性可以使用集落形成试验来估计，其中通过其增殖能力和体外表达结缔组织表型的后代集落来检测和测量集落建立ctp。除了测量组织样本中ctp的数量和流行率外，还可以根据形成的菌落之间细胞数量、分布、形态和表达模式的变化来评估不同ctp之间生物潜力的差异。虽然非常有用，但传统的群体形成分析有一个非常重要的局限性，即它们唯一的直接测量是对创始CTP（群体）的后代进行的。关于创始CTP的信息只能从殖民地的数量及其特征中推断出来。在增殖之前，菌落分析不揭示罕见的个体菌落建立ctp本身的物理或生物特性。为了解决这一限制，我们开发了一种活细胞视频显微镜系统，可以在集落形成之前系统地表征集落形成细胞（形态学，表面标记物的表达）。使用多色标记方法，混合组织衍生细胞群在电镀时被标记为感兴趣的细胞标记物。时间推移的相位对比图像然后被捕获超过6天，而殖民地形成，在一个大的视野。在培养期结束时，根据已建立的标记和菌落指标来确定菌落的特征。通过倒转视频，每个群体也可以追溯到识别群体的创始细胞，作为其他非祖先的海洋中的一个。使用这种方法，我们建议大大提高我们对人类CTP的本质，它们的多样性和亚型，它们在骨组织中的位置的理解，并定义可以无创测量的因素，用于预测它们的生物学潜力，或选择最适合特定治疗目的的CTP。