Early Identification and Characterization of Connective Tissue Progenitors

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（CTPs）被定义为能够增殖和分化成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殖民地测定来估计，其中集落形成CTP通过其增殖能力和表达结缔组织表型的后代集落来检测和测量。除了测量组织样品中CTP的数量和普遍性之外，还可以基于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之间的生物学潜力差异。虽然非常有用，但传统集落形成测定的非常重要的局限性在于它们仅对初始CTP的后代（殖民地）进行直接测量。关于初始CTP的信息只能从菌落数量及其特征推断。在增殖之前，集落测定不揭示罕见的单个集落形成CTP本身的物理或生物学性质。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，该系统能够在集落形成之前对集落形成细胞（形态学、表面标志物的表达）进行系统表征。使用荧光标记方法，在铺板时标记混合的组织来源的细胞群体的感兴趣的细胞标志物。然后在6天内捕获时间推移相位对比图像，同时在大视场内形成菌落。在培养期结束时，根据已建立的标志物和菌落指标对菌落进行表征。通过反转视频，每个集落也可以追溯到识别殖民地的创始细胞，作为一个在海洋中的其他非祖细胞。使用这种方法，我们建议显着推进我们对人类CTP的性质，其多样性和亚型，其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，或选择最适合特定治疗目的的CTP。

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.