Collaborative Research: Bioengineering Thymus Organoids Towards Generation Of Humanized Mice Models

合作研究：生物工程胸腺类器官以产生人源化小鼠模型

• 批准号: 1804728
• 负责人: Yong Fan
• 金额: $ 30万
• 依托单位: Allegheny-Singer Research Institute
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-12-01 至 2021-11-30
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=1804728&HistoricalAwards=false
• 关键词: Collaborative; Research; Bioengineering; Thymus; Organoids

Humanized mice (aka, mice with absent or limited immune responses that are transplanted with human immune cells, tissues and stem cells, etc.) are widely used as preclinical models to develop and evaluate novel therapies without putting the patients at risk. Over the past two decades, substantial progress has been made in developing humanized mouse models for modeling various human diseases (e.g., infectious diseases and cancer). Despite all of these successes, replicating human immune responses in humanized mouse models proves to be challenging. A major hurdle is the mouse host's lack of a human thymus gland -- the tissue that is responsible for the generation of T cells, which play such a key role in the human immune system. Human T cells developed in the mouse thymus are defective in engaging other human immune cells and fail to mount proper immune responses. The goal of this research project is to use human pluripotent stem cells (stem cells that can develop into numerous cell types) to generate an artificial thymus that can be used as a substitute to reconstruct the human immune system in humanized mice. Two key technologies will be developed and optimized: 1) novel biomaterial intervention methods to generate thymic cells from human pluripotent stem cells; and 2) innovative bioengineering processes to construct a functional artificial thymus. Successful replication of human immune responses in humanized mice with artificial thymus transplants will have tremendous scientific and translational impacts, including: 1) as a powerful tool to evaluate and optimize the effectiveness of immunotherapy; 2) to further understand disease cause; and 3) to develop novel immune interventions that are ready for clinical translation. This cross-disciplinary project will provide an excellent opportunity for minority women and individuals from underrepresented groups to participate in research and educational activities. The project is a culmination of diverse engineering techniques, encompassing biomaterials, stem cells and immune engineering. Hence, it will provide the participants with well-rounded training opportunities. Collaborative educational and outreach efforts include a joint summer internship program for underrepresented minority undergraduate students and development of course modules in Immune-engineering and Stem Cell Bioengineering that will be incorporated into online educational resources for K-12 students and teachers.The goal of this project is to generate a humanized mouse model by implanting bioengineered human thymus organoids in CD34+ hematopoietic stem cell (HSC) engrafted NOD-scid.Il2rgnull (NSG) mice in order to promote human T cell development and adaptive immune responses. Preliminary studies have demonstrated the feasibility of establishing normal immune function in athymic mice by reconstructing a decellularized thymus with primary murine thymic epithelial cells (TECs). This project will take the next step by generating human thymus organoids from human pluripotent stem cells (hPSCs) and then using these organoids to produce a humanized mouse model. The Research Plan is organized under three aims. The first aim is to generate functional thymus organoids with hPSC-derived TECs. The hPSCs will be differentiated into TECs while encapsulated in alginate cultures. The derived hPSC-TEPCs will then be used to reconstruct decellularized thymus scaffolds. The functionality of thymus organoid constructs will be characterized in-vitro (to determine gene markers of mature TECS and early T-Cell development) and in-vivo (under the kidney capsules of nude mice). Ex vivo studies will be performed to test the functional efficiency of thymus organoid generated from hPSC-TEPCs to support ex-vivo T cell development. The second aim is to engineer compartmental organization of TECs (cortical (cTECS) and medullary (mTECS) regions) within the bioengineered thymus organoids. The hESC-TEPCs will be segregated into cTEC and mTEC spheroids that will be used to reconstruct the thymus organoid with the goal of reestablishing both cell-cell contact and compartmental organization. The 3D cellular organization, with reestablished cell-cell and cell-ECM communication, is expected to enhance long-term survival and function of the TECs within the thymic scaffold and to improve the selection and differentiation of subpopulations of T-cells. The third aim is to generate humanized mouse models with bioengineered thymus organoids. Humanized mice will be generated by transplanting cord blood CD34+ HSCs into myelosupressive agent preconditioned NSG mice, which are subsequently transplanted with thymus organoids bioengineered from hESC-derived TEPCs. Development of multiple hematopoietic lineages, especially the development of CD4+ and CD8+ T cells, will be characterized and the function of the cells will be examined. The models will be followed for 24 weeks post reconstruction. Successful development of a long lasting, functional human immune system in a mouse model will be advantageous for studying human immune responses and for developing proper therapeutics for cancer, infectious diseases, and autoimmune disorders.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.

人源化小鼠（aka，移植了人免疫细胞、组织和干细胞等的免疫应答缺失或有限的小鼠）被广泛用作临床前模型，以开发和评估新的疗法，而不会使患者处于危险之中。在过去的二十年中，在开发用于模拟各种人类疾病（例如，传染病和癌症）。尽管取得了所有这些成功，但在人源化小鼠模型中复制人类免疫应答被证明是具有挑战性的。一个主要的障碍是小鼠宿主缺乏人类胸腺--负责产生T细胞的组织，T细胞在人类免疫系统中起着关键作用。在小鼠胸腺中发育的人类T细胞在参与其他人类免疫细胞方面存在缺陷，并且无法产生适当的免疫反应。该研究项目的目标是利用人类多能干细胞（可发育成多种细胞类型的干细胞）生成人工胸腺，该人工胸腺可用作替代品，在人源化小鼠中重建人类免疫系统。将开发和优化两项关键技术：1）从人类多能干细胞产生胸腺细胞的新型生物材料干预方法; 2）构建功能性人工胸腺的创新生物工程方法。人工胸腺移植在人源化小鼠中成功复制人类免疫应答将产生巨大的科学和转化影响，包括：1）作为评估和优化免疫治疗有效性的有力工具; 2）进一步了解疾病原因; 3）开发可用于临床转化的新型免疫干预措施。这个跨学科项目将为少数族裔妇女和代表性不足群体的个人提供参与研究和教育活动的绝佳机会。该项目是多种工程技术的顶峰，包括生物材料，干细胞和免疫工程。因此，它将为参与者提供全面的培训机会。合作的教育和外展工作包括为代表性不足的少数民族本科生提供联合暑期实习计划，以及开发免疫工程和干细胞生物工程课程模块，这些课程模块将纳入K-12学生和教师的在线教育资源。该项目的目标是通过将生物工程改造的人类胸腺类器官植入CD 34+造血干细胞（HSC）移植的NOD-scid. Il 2 rgnull（NSG）小鼠，以促进人T细胞发育和适应性免疫应答。 初步研究表明，通过用原代小鼠胸腺上皮细胞（TEC）重建脱细胞胸腺，在无胸腺小鼠中建立正常免疫功能是可行的。该项目的下一步将是从人类多能干细胞（hPSC）中产生人类胸腺类器官，然后使用这些类器官产生人源化小鼠模型。 研究计划是根据三个目标组织的。 第一个目的是用hPSC衍生的TEC产生功能性胸腺类器官。hPSC将分化成TEC，同时包封在藻酸盐培养物中。然后将衍生的hPSC-TEPC用于重建脱细胞胸腺支架。胸腺类器官构建体的功能将在体外（以确定成熟TECS和早期T细胞发育的基因标志物）和体内（在裸鼠的肾包膜下）表征。将进行离体研究以测试由hPSC-TEPC产生的胸腺类器官支持离体T细胞发育的功能效率。第二个目标是在生物工程化的胸腺类器官内设计TEC（皮质（cTECS）和髓质（mTECS）区域）的隔室组织。将hESC-TEPC分离成cTEC和mTEC球状体，其将用于重建胸腺类器官，目的是重建细胞-细胞接触和隔室组织。 具有重建的细胞-细胞和细胞-ECM通信的3D细胞组织预计将增强胸腺支架内TEC的长期存活和功能，并改善T细胞亚群的选择和分化。第三个目标是用生物工程胸腺类器官产生人源化小鼠模型。将通过将脐带血CD 34 + HSC移植到骨髓抑制剂预处理的NSG小鼠中来产生人源化小鼠，随后将其移植有从hESC衍生的TEPC生物工程化的胸腺类器官。将表征多种造血谱系的发育，特别是CD 4+和CD 8 + T细胞的发育，并检查细胞的功能。重建后将对模型进行24周随访。在小鼠模型中成功开发持久的、功能性的人类免疫系统将有利于研究人类免疫反应，并为癌症、传染病和自身免疫性疾病开发适当的治疗方法。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。

项目成果

De novo construction of T cell compartment in humanized mice engrafted with iPSC-derived thymus organoids

• DOI: 10.1038/s41592-022-01583-3
• 发表时间: 2022-09-05
• 期刊: NATURE METHODS
• 影响因子: 48
• 作者: Zeleniak, Ann;Wiegand, Connor;Fan, Yong
• 通讯作者: Fan, Yong