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: 10373479
• 负责人: Nancy R Manley
• 金额: $ 22.65万
• 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 2023-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10373479
• 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; 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.

项目概要/摘要 胸腺是 T 细胞生成和适应性免疫反应形成所需的关键器官。 一种关键细胞类型，即内胚层来源的胸腺上皮，是所有胸腺功能和 协调胸腺内所有其他细胞类型的组装和分化。 这些胸腺上皮细胞（TEC）的规格和分化的背后仍然知之甚少。 然而，已知单一转录因子 FOXN1 可以控制 TEC 增殖的多个关键方面， 我们实验室和其他人的工作已经表明胎儿和出生后胸腺的分化和维持。 Foxn1 在 TEC 亚群中的作用不同，并且对剂量非常敏感，并且它在 TEC 中的表达 由于这一核心作用，祖细胞足以驱动大多数（如果不是全部）TEC 分化计划。 Foxn1 是通过诱导多能定向分化产生 TEC 的持续努力的关键目标 然而，关于 FOXN1 在 TEC 分化中的精确功能仍然存在许多问题。 虽然 TEC 功能所需的至少一些 FOXN1 靶标是已知的，但关键问题是。 仍然没有答案，包括建立 TEC 身份和启动 Foxn1 表达的分子途径， 以及不同 TEC 亚群中不同水平的 FOXN1 如何差异性地控制 TEC 生物学。 合作团队表明，FOXN1 在小鼠胚胎成纤维细胞 (MEF) 中的强制表达是 足以将 MEF 转化为功能性 TEC，这些“诱导 TEC”（iTEC）可以在移植后直接发挥作用。 还显示了支持体内 T 细胞发育的功能齐全的胸腺器官的组装。 有望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。 因此，iTEC 可能为实现体外生成自体 TEC 的长期目标提供一种新的工具 生成用于移植的类器官，或更广泛地说，用于体外生成 T 细胞。 胸腺生物学领域缺乏可行的体外培养系统来研究 TEC 的分子需求 生物学和分化，或指导 T 细胞发育和选择的 TEC-胸腺细胞相互作用。 因此可以提供一个有用的体外系统来研究 FOXN1 功能和遗传途径 控制 TEC 的分化和功能 该提案旨在解决 iTEC 生成的关键问题。 这限制了其作为实验系统的更广泛采用，我们提出了三个旨在改进的具体目标。 iTEC 分化和增殖的控制将使我们能够开发这种方法进行广泛的实验 应用：1) iTEC 生成过程中的 Foxn1 剂量敏感性；2) 促进 mTEC 的机制； iTEC 培养物中的分化；以及 3) MHCII 表达和 iTEC 增殖的成功完成。 拟议的实验将大大改善 iTEC 的生成和功能，目标是建立 iTEC 已成为急需的体外实验系统，具有广泛的用途，可用于研究 FOXN1 功能和 TEC 生物学、T 细胞分化和 TEC-胸腺细胞相互作用。