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

这项NSERC发现计划最初是在2011年资助的。它是基于通过天花板培养脂肪细胞去分化的过程。目前更新应用的总体目标是表征成熟脂肪细胞和天花板培养方法产生的成纤维细胞样细胞的去分化过程。对于即将到来的这一任期，我们提出了两个具体目标，这两个目标代表了在这笔赠款的第一个任期中产生的结果的直接和合乎逻辑的延续。 特定目的#1：研究DPP4和FAP在脂肪细胞去分化中的作用。FAP和DPP4的药理抑制剂以及从Fap缺陷小鼠分离的细胞将被用来建立和表征这些分子在天花板培养中脂肪细胞去分化中的重要性。根据初步观察，我们假设DPP4和FAP的抑制或缺失部分地损害了去分化的过程。 具体目的#2：研究转化生长因子在脂肪细胞去分化过程中的作用。药物抑制剂和从缺乏转化生长因子-1信号的小鼠模型中分离出来的细胞将被用来表征这种细胞因子对天花板培养中脂肪细胞去分化的影响。根据初步观察，我们推测转化生长因子-1是导致去分化的主要原因信号。 本提案的挑战在于描述一个可能参与调节几个器官和物种的组织细胞动力学的细胞过程。这项新的工作非常类似于第一期的拨款，将需要开发新的方法，如胶原蛋白悬浮培养，并可能使用基于阻抗的测量对细胞功能进行实时监测(已提交研究工具和仪器拨款申请)。这些技术挑战显然与工程有关。尽管我们提出了一个确实与生物科学有关的项目，但它并不是一个与健康有关的建议。我们的经验表明，脂肪细胞的去分化过程完全独立于细胞供体的特征，包括疾病状态，这大大降低了它作为健康相关参数的吸引力。我们计划的长期目标有两个：1)开发和改进培养技术，可能有助于研究脂肪细胞的生物学性质；2)产生关于去分化脂肪细胞的知识体系，这将有助于其他研究人员将去分化细胞转移到组织工程领域。

This NSERC Discovery program was initially funded in 2011. It is based on the process of adipocyte dedifferentiation through ceiling culture. The General Objective of the present renewal application is to characterize the dedifferentiation process of mature adipocytes and the fibroblast-like cells generated by the ceiling culture approach. For this coming term, we propose two specific aims that represent the direct and logical continuation of findings that were generated in the first term of this grant. Specific Aim #1: To characterize the role of DPP4 and FAP in adipocyte dedifferentiation. Pharmacological inhibitors of FAP and DPP4 as well as cells isolated from Fap-deficient mice will be used to establish and characterize the importance of these molecules in adipocyte dedifferentiation in ceiling cultures. Based on preliminary observations, we hypothesize that DPP4 and FAP inhibition or absence partially impairs the process of dedifferentiation. Specific Aim #2: To characterize the role of TGF-ß in the process of adipocyte dedifferentiation. Pharmacological inhibitors and cells isolated from mouse models deficient in TGF-ß1 signalling will be used to characterize the impact of this cytokine on adipocyte dedifferentiation in ceiling cultures. Based on preliminary observations, we hypothesize that TGF-ß1 is responsible for the main causal signal inducing dedifferentiation. The challenge of the present proposal lies in the characterization of a cell process that may be involved in the regulation of tissue cell dynamics in several organs and species. Much like in the first term of this grant, this new endeavor will require development of new approaches such as collagen suspension cultures and possibly real time monitoring of cell function using impedance-based measurements (Research Tools and Instruments grant application submitted). These technical challenges clearly relate to engineering. Although we propose a project that does, indeed, relate to biological sciences, it is not a health-related proposal. Our experience has demonstrated that the process of adipocyte dedifferentiation is quite independent from cell donor characteristics including disease state, which considerably decreases its appeal as a health-related parameter. Our program’s Long-term Objective is two-fold: 1) to develop and refine culture techniques that may be of use to study the biological nature of adipocytes; and 2) to generate a body of knowledge on dedifferentiated adipocytes that will help other investigators move dedifferentiated cells into the sphere of tissue engineering.