iTEC as a new experimental system for TEC biology

iTEC 作为 TEC 生物学的新实验系统

• 批准号: 10493405
• 负责人: Nancy R Manley
• 金额: $ 18.88万
• 依托单位: UNIVERSITY OF GEORGIA
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-09-22 至 2024-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10493405
• 关键词: Address; Adoption; Autologous; Biology; Cell Cycle; Cell Differentiation process; Cell Proliferation; Cell physiology; Cells; Cellular biology; Coculture Techniques; Complex; Coupled; Down-Regulation; Embryo; Endoderm; Epithelial; Epithelial Cell Proliferation; Fibroblasts; Future; Generations; Genetic; Goals; In Vitro; MHC Class II Genes; Maintenance; Methods; Modification; Molecular; Mus; Organ; Organ Size; Organogenesis; Pathway interactions; Process; Protocols documentation; Role; Signal Transduction; System; T cell differentiation; T-Cell Development; T-Lymphocyte; Testing; Therapeutic; Thymic epithelial cell; Thymus Gland; Transplantation; Up-Regulation; Work; adaptive immune response; age related; cell type; design; directed differentiation; dosage; epithelial stem cell; experimental study; fetal; improved; in vivo; induced pluripotent stem cell; notch protein; novel; organoid transplantation; overexpression; postnatal; programs; response; single-cell RNA sequencing; stem cells; thymocyte; tool; transcription factor; two-dimensional

Project Summary/Abstract The thymus is the key organ required for T cell generation and the formation of an adaptive immune response. One key cell type, endoderm-derived thymic epithelium, is required both for all thymus functions and to orchestrate the assembly and differentiation of all other cell types within the thymus. The genetic pathways underlying the specification and differentiation of these thymic epithelial cells (TECs) are still poorly understood. However, a single transcription factor, FOXN1, is known to control multiple key aspects of TEC proliferation, differentiation, and maintenance in both the fetal and postnatal thymus. Work from our lab and others has shown that Foxn1 acts differentially in TEC subsets and is incredibly dosage-sensitive, and that its expression in TEC progenitors is sufficient to drive most if not all of the TEC differentiation program. Because of this central role, Foxn1 is a key target in ongoing efforts to generate TEC by the directed differentiation of induced pluripotent stem cells (iPSCs). However, many questions remain about the precise FOXN1 functions in TEC differentiation and proliferation. While at least some of the FOXN1 targets required for TEC function are known, key questions remain unanswered, including the molecular pathways that establish TEC identity and initiate Foxn1 expression, and how diverse levels of FOXN1 in different TEC subsets differentially control TEC biology. We were part of a collaborative team that showed that enforced expression of FOXN1 in murine embryonic fibroblasts (MEFs) is sufficient to convert MEFs into functional TEC. These “induced TECs” (iTECs) can, upon transplantation, direct the assembly of a fully functional thymus organ that supports development of T cells in vivo. iTECs also show promise in promoting differentiation of immature thymocytes into single positive T cells in 2-dimensional culture. iTECs may thus provide a novel tool for long-term goals of generating autologous TEC in vitro that could be used to generate organoids for transplant, or for in vitro generation of T cells for therapeutic purposes. More broadly, the field of thymus biology lacks a viable in vitro culture system for studying the molecular requirements for TEC biology and differentiation, or TEC-thymocyte interactions that direct T cell development and selection. iTECs thus could provide a useful in vitro system for studying both FOXN1 function and the genetic pathways that control TEC differentiation and function. This proposal is designed to address key aspects of iTEC generation that limit its broader adoption as an experimental system. We propose three specific aims focused on improving the control of iTEC differentiation and proliferation that will allow us to develop this method for broad experimental applications: 1) Foxn1 dosage sensitivity during iTEC generation; 2) mechanisms to promote mTEC differentiation in iTEC cultures; and 3) MHCII expression and iTEC proliferation. Successful completion of the proposed experiments will substantially improve iTEC generation and function, with the goal of establishing iTECs as into a much-needed in vitro experimental system with broad utility for studying FOXN1 function and TEC biology, T cell differentiation, and TEC-thymocyte interactions.

项目摘要/摘要 胸腺是T细胞产生所需的关键器官，并且是自适应免疫响应的形成。 所有胸腺功能都需要一种关键的细胞类型，内胚层衍生的胸腺上皮，既需要 策划胸腺内所有其他细胞类型的组装和分化。遗传途径 这些胸腺上皮细胞（TEC）的规范和分化的基础仍然很少了解。 但是，已知一个单个转录因子FOXN1控制TEC增殖的多个关键方面， 胎儿和产后胸腺的分化和维持。我们实验室和其他人的工作表明 FOXN1在TEC子集中有不同的作用，并且对剂量敏感，并且其表达在TEC中 祖细胞足以推动大多数TEC分化计划（如果不是全部）。由于这种核心角色， FOXN1是通过诱导多能的指示分化来生成TEC的持续努力的关键目标 干细胞（IPSC）。但是，关于TEC差异化的精确FOXN1功能仍然存在许多问题 和增殖。虽然至少已经知道了一些FOXN1目标，但关键问题 保持未得到答复，包括建立TEC身份并启动FOXN1表达的分子途径， 以及不同TEC子集中FOXN1水平的多样化对TEC生物学的控制。我们是一个 协作团队表明，在鼠类胚胎成纤维细胞（MEFS）中强制执行FOXN1的表达是 足以将MEF转换为功能性TEC。这些“诱导的TEC”（ITEC）可以直接直接 支持体内T细胞发育的功能性胸腺器官的组装。 ITEC也显示 在二维培养中促进未成熟胸腺细胞分化为单个阳性T细胞的有望。 因此，ITEC可以为在体外生成自体TEC的长期目标提供新颖的工具，可以使用 生成用于移植的器官，或用于治疗目的的体外产生T细胞。更广泛地 胸腺生物学领域缺乏可行的体外培养系统，用于研究TEC的分子需求 生物学和分化，或指向T细胞发育和选择的TEC-胞胞菌相互作用。 itecs 因此，可以为研究FOXN1功能和遗传途径提供有用的体外系统 控制TEC分化和功能。该建议旨在解决ITEC生成的关键方面 这限制了其作为实验系统的广泛采用。我们提出了三个专注于改进的特定目标 ITEC分化和增殖的控制，这将使我们能够开发此方法进行广泛的实验 应用：1）ITEC生成期间FOXN1剂量敏感性； 2）促进MTEC的机制 ITEC文化的分化； 3）MHCII表达和ITEC增殖。成功完成 提出的实验将大大改善ITEC的生成和功能，目的是建立 ITEC作为一个急需的体外实验系统，具有广泛的实用性，用于研究FOXN1功能和 TEC生物学，T细胞分化和TEC-胞胞菌相互作用。