Early Identification and Characterization of Connective Tissue Progenitors

结缔组织祖细胞的早期识别和表征

基本信息

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
项目类别：
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

项目摘要

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）被定义为组织驻留干细胞和祖细胞的异质群体，它们能够增殖并分化成一种或多种结缔组织表型。 CTP 的数量和异质性可以使用集落形成测定来估计，其中通过其在体外增殖和表达结缔组织表型的后代集落的能力来检测和测量集落建立 CTP。除了测量组织样品中 CTP 的数量和普遍性之外，还可以根据形成的集落之间的细胞数量、分布、形态和表达模式的变化来评估各种 CTP 之间的生物潜力差异。虽然非常有用，但传统集落形成测定的一个非常重要的限制是它们唯一的直接测量是对创始 CTP 的后代（集落）进行的。有关创始 CTP 的信息只能从菌落数量及其特征来推断。集落测定不会揭示在增殖前建立 CTP 的稀有单个集落本身的物理或生物学特性。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，能够在集落形成之前对集落形成细胞进行系统表征（形态学、表面标记物的表达）。使用多色标记方法，在铺板时对混合组织来源的细胞群进行感兴趣的细胞标记物标记。然后在 6 天的时间里捕捉延时相位对比图像，同时在大视野中形成菌落。在培养期结束时，根据已建立的标记和菌落指标对菌落进行表征。通过倒转视频，还可以追溯每个菌落以识别菌落创建细胞，就像其他非祖细胞海洋中的细胞一样。我们建议使用这种方法显着增进我们对人类 CTP 的性质、其多样性和亚型、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潜力的因素，或选择最适合的 CTP用于特定的治疗目的。