Adipocyte dedifferentiation in the ceiling culture system: regulators of cell matrix remodelling

这个NSERC发现计划最初于2011年获得资助。2015年提交的更新提案获得了一年的资助。本申请提供了一个修订后的项目，考虑了评审员提出的建设性意见。**我们的总体目标是使用天花板培养方法进一步表征成熟脂肪细胞向成纤维细胞样细胞的去分化过程，重点关注这一现象的原因。提出的实验代表了直接的和逻辑的延伸，在第一个任期内获得的补助金的结果。具体而言，以下描述的项目遵循我们开发的六孔板，非缺氧天花板培养方法和延时显微镜，以及我们发现的基质重塑因子，如转化生长因子β（TGF β），成纤维细胞活化蛋白α（FAP）和二肽基肽酶IV（DPP 4）在脂肪细胞去分化过程中的潜在作用。我们提出了四个具体目标，每个目标代表一个具体项目。*具体目标1：为了解决在天花板培养物中进行去分化的分离成熟脂肪细胞中可能存在的污染间充质细胞问题的不确定性。我们将从一种在成熟脂肪细胞中特异性过表达GFP的小鼠品系中分离成熟脂肪细胞，并在体外监测去分化。我们还将测试细胞悬液洗涤、细胞悬液离心和细胞增殖抑制剂的影响。**具体目标#2：表征TGF-β在脂肪细胞去分化过程中的作用。我们将从TGF-β 1信号传导缺陷的小鼠品系中分离成熟脂肪细胞，并在体外监测细胞去分化。TGF-β 1也将与药理学抑制剂联合使用。**具体目标#3：表征FAP在脂肪细胞去分化过程中的作用。FAP的药理学抑制剂以及从Fap缺陷小鼠分离的脂肪细胞将用于建立和表征这种酶在天花板培养物中去分化的重要性。具体目标#4：表征DPP4在脂肪细胞去分化过程中的作用。DPP4的药理学抑制剂以及从Dpp4缺陷小鼠中分离的脂肪细胞将用于建立和表征这种酶在天花板培养物中脂肪细胞去分化中的重要性。* * 在这一领域工作的大多数研究小组主要关注去分化过程产生的细胞，而不是转化本身。我们建议，更好地了解这个过程的驱动程序或抑制剂，沿着改进的培养技术，将大大促进去分化脂肪细胞在组织工程领域的应用。该技术的发展将有助于HQP培训，并为脂肪细胞生物学研究提供新的模型。

This NSERC Discovery program was initially funded in 2011. The renewal proposal submitted in 2015 was awarded one year of funding. The present application offers a revised project taking into account the constructive comments made by Reviewers.******Our General Objective is to further characterize the dedifferentiation process of mature adipocytes toward fibroblast-like cells using the ceiling culture approach, with a focus on causal agents of this phenomenon. The experiments proposed represent a direct and logical extension of the results obtained in the first term of the grant. Specifically, the projects described below follow our development of a six-well plate, non-hypoxic ceiling culture approach and time-lapse microscopy, as well as our discoveries on the potential role of matrix-remodelling factors such as Transforming growth factor β (TGFβ), Fibroblast-activation protein α (FAP) and Dipeptidyl peptidase IV (DPP4) in the process of adipocyte dedifferentiation. We propose four Specific Aims, each representing a specific project.******Specific Aim #1: To address uncertainty regarding the issue of contaminating mesenchymal cells that may be present with isolated mature adipocytes undergoing dedifferentiation in ceiling cultures. We will isolate mature adipocytes from a mouse strain overexpressing GFP specifically in mature adipocytes, and monitor dedifferentiation in vitro. We will also test the impact of cell suspension washing, cell suspension centrifugation and inhibitors of cell proliferation.******Specific Aim #2: To characterize the role of TGF-β in the process of adipocyte dedifferentiation. We will isolate mature adipocytes from a mouse strain deficient in TGF-β1 signalling and monitor cell dedifferentiation in vitro. TGF-β1 will also be used in conjunction with pharmacological inhibitors.******Specific Aim #3: To characterize the role of FAP in the process of adipocyte dedifferentiation. Pharmacological inhibitors of FAP as well as adipocytes isolated from Fap-deficient mice will be used to establish and characterize the importance of this enzyme for dedifferentiation in ceiling cultures.******Specific Aim #4: To characterize the role of DPP4 in the process of adipocyte dedifferentiation. Pharmacological inhibitors of DPP4 as well as adipocytes isolated from Dpp4-deificent mice will be used to establish and characterize the importance of this enzyme in adipocyte dedifferentiation in ceiling cultures. ******Most research groups working in this area focus mainly on the cells generated by the dedifferentiation process, not on the transformation itself. We propose that a better understanding of the drivers or inhibitors of this process, along with improved culture techniques, would greatly facilitate the use of dedifferentiated adipocytes in the tissue engineering realm. The techniques developed will contribute to HQP training and provide new models for studies on adipocyte biology.