RECODE: Non-invasive cell patterning and monitoring to generate data-guided computational models that inform synthetic gene circuit-guided cartilage development

开发基于干细胞的再生策略的努力尚未在临床上使用，特别是对于难以修复的组织，如肌肉和软骨。这个通过定向分化编码（RECODE）项目的可再生细胞和类器官的目标是了解成人干细胞如何转化为软骨细胞，软骨细胞。最终，这些工程细胞可用于修复创伤性和衰弱性软骨损伤。 该项目将产生关于基于干细胞的软骨形成的调节剂的新知识，可用于开发新的再生策略。研究结果也将广泛适用于组织功能需要长期稳定性的组织再生领域。这项工作提供了一个独特的机会，培养本科生和研究生科学家和工程师团队的方法，提高生物学的基础研究转化为复杂的工程系统。这个RECODE项目旨在采用创新的方法，结合联合收割机生物物理技术，数据引导算法，和细胞状态的合成基因电路工程-控制成人间充质干细胞（hMSC）分化为软骨细胞的驱动因子的特异性扰动。具体而言，该项目的重点是驱动hMSC衍生的软骨细胞（hMdCh）表型成熟为稳定的关节软骨软骨细胞或肥大软骨细胞的条件。将使用高通量方法监测生物物理条件对长期hMdCh表型的影响，以动态地和非破坏性地测量新生组织的分泌组和进化刚度。将通过将RNA和microRNA测序和全基因组染色质结构数据与表型转换的分泌组标志物相关联来鉴定与表型转换相关的调控途径。将生成表型转换的数学模型，以确定在特定环境条件下驱动表型变化的转录和翻译调节因子。 一个灵活的合成基因电路平台将用于工程基因电路，同时激活和/或抑制基因组调节剂，发现保持关节或肥大软骨细胞表型。最后，将测试这些基因回路维持hMdCh表型和产生功能性软骨组织的功效。这项工作的成果将展示数据驱动模型的实用性，为合成基因电路的设计提供信息，以改善组织再生策略。该RECODE项目由化学，生物工程，环境和运输系统部门的工程生物学和健康集群，土木，机械和制造创新部门的生物力学和机械生物学计划，该奖项反映了NSF的法定使命，并通过使用基金会的知识价值和更广泛的影响审查标准进行评估，被认为值得支持。

Efforts to develop stem-cell based regeneration strategies have yet to be clinically usable especially for difficult to repair tissues like muscle and cartilage. The goal of this Reproducible Cells and Organoids via Directed-Differentiation Encoding (RECODE) project is to understand how adult human stem cells transform into chondrocytes, the cells in cartilage. Ultimately, these engineered cells could be used to repair traumatic and debilitating cartilage injuries. This project will generate new knowledge about the regulators of stem cell-based cartilage formation that can be used to develop new regeneration strategies. The results will also be broadly applicable to the tissue regeneration field in cases where tissue functionality requires long-term stability. This work offers a unique opportunity to train teams of undergraduate and graduate scientists and engineers in methods that enhance the translation of fundamental studies of biology into sophisticated engineering systems.This RECODE project aims to employ innovative methods that combine biophysical techniques, data-guided algorithms, and synthetic gene circuit engineering for the cell state–specific perturbation of the drivers controlling differentiation of adult human mesenchymal stem cells (hMSCs) into chondrocytes. Specifically, this project focuses on conditions that drive maturation of the hMSC derived chondrocyte (hMdCh) phenotype into either stable articular cartilage chondrocytes or hypertrophic chondrocytes. The influence of the biophysical conditions on long-term hMdCh phenotype will be monitored using high-throughput methods to dynamically and non-destructively measure the secretome and the evolving stiffness of the nascent tissues. The regulatory pathways associated with transitions in phenotype will be identified by correlating RNA- and microRNA-sequencing and genome-wide chromatin architectural data with the secretomic markers of phenotype transitions. A mathematical model of phenotype transitions will be generated to pinpoint transcriptional and translational regulators that drive phenotype changes under specific environmental conditions. A flexible synthetic gene circuit platform will be used to engineer gene circuits that simultaneously activate and/or repress the genomic regulators found to maintain articular or hypertrophic chondrocyte phenotypes. Finally, the efficacy of these gene circuits to maintain hMdCh phenotype and produce functional cartilage tissues will be tested. The outcomes of this work will demonstrate the utility of data-driven models to inform the design of synthetic gene circuits to improve tissue regeneration strategies.This RECODE project is jointly funded by the Engineering Biology and Health Cluster in the Division of Chemical, Bioengineering, Environmental, and Transport Systems, the Biomechanics and Mechanobiology Program in the Division of Civil, Mechanical, and Manufacturing Innovation, and the Systems and Synthetic Biology Program in the Division of Molecular and Cellular Biosciences.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.