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前体细胞的活性来进行组织再生。结缔组织祖细胞(CTPs)是指能够增殖和分化为一种或多种结缔组织表型的异质性组织干细胞和祖细胞群体。CTPs的数量和异质性可以用克隆形成实验来估计，在该实验中，建立CTPs的克隆是通过它们的增殖能力和表达结缔组织表型的子代的克隆来检测和测量的。除了测量组织样本中CTPs的数量和流行程度外，还可以根据所形成的克隆之间的细胞数量、分布、形态和表达模式的变化来评估不同CTPs之间的生物潜力差异。虽然非常有用，但传统的集落形成试验的一个非常重要的局限性是，它们唯一的直接测量是对创始CTP(集落)的后代进行的。关于建立CTP的信息只能从菌落的数量和它们的特征中推断出来。菌落分析不揭示在增殖之前建立CTP本身的稀有个体菌落的物理或生物学特性。为了解决这一局限性，我们开发了一种活细胞视频显微镜系统，该系统能够在克隆形成之前对克隆形成细胞(形态、表面标记的表达)进行系统的表征。使用多色标记方法，在培养时标记混合组织来源的细胞群以寻找感兴趣的细胞标记。然后在6天内捕获时间推移相位相反的图像，同时在大视野内形成蜂群。在培养期末，根据已建立的标记和菌落指标来确定菌落的特征。通过反转视频，还可以追溯到每个殖民地，以识别殖民地创建细胞，即其他非祖细胞海洋中的一个。使用这种方法，我们建议显著提高我们对人类CTP的性质、它们的多样性和亚型、它们在骨组织中的位置的理解，并定义可以非侵入性测量并用于预测其生物学潜力的因素，或者选择最适合特定治疗目的的CTP。