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应用于临床，特别是对于肌肉和软骨等难以修复的组织。这个通过定向分化编码实现可再生细胞和类器官（RECODE）项目的目标是了解成人干细胞如何转化为软骨细胞（软骨中的细胞）。最终，这些工程细胞可用于修复创伤性和衰弱性软骨损伤。 该项目将产生有关基于干细胞的软骨形成调节因子的新知识，可用于开发新的再生策略。在组织功能需要长期稳定性的情况下，该结果也将广泛适用于组织再生领域。这项工作为培训本科生和研究生科学家和工程师团队提供了一个独特的机会，以增强将生物学基础研究转化为复杂的工程系统的方法。该 RECODE 项目旨在采用创新方法，将生物物理技术、数据引导算法和合成基因电路工程相结合，对控制成人间充质干细胞 (hMSC) 分化为软骨细胞的驱动因素进行细胞状态特异性扰动。具体来说，该项目重点关注驱动 hMSC 衍生软骨细胞 (hMdCh) 表型成熟为稳定关节软骨细胞或肥大软骨细胞的条件。将使用高通量方法动态且非破坏性地测量分泌组和新生组织不断变化的硬度来监测生物物理条件对长期 hMdCh 表型的影响。通过将 RNA 和 microRNA 测序以及全基因组染色质结构数据与表型转变的分泌标记物相关联，可以识别与表型转变相关的调控途径。将生成表型转变的数学模型，以查明在特定环境条件下驱动表型变化的转录和翻译调节因子。 灵活的合成基因电路平台将用于设计基因电路，同时激活和/或抑制维持关节或肥大软骨细胞表型的基因组调节因子。最后，将测试这些基因电路维持 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，为合成基因回路的设计提供信息，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项目以及分子和细胞生物科学部门的系统和合成生物学项目共同资助。该奖项反映了 NSF 的法定使命通过使用基金会的智力价值和更广泛的影响审查标准进行评估，并被认为值得支持。

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.