Immune response to allogeneic hESC-derived endothelial cells

描述（由申请人提供）：本申请涉及广泛的挑战领域（11）再生医学和特定的挑战主题，11-HL-101 * 开发用于心血管、肺和血液疾病的基于细胞的疗法。鉴于其自我更新和多能性，人类胚胎干细胞（hES）和诱导多能干细胞（iPS）作为细胞替代疗法和再生医学具有很大的前景。我们最近开发了一种用于将hES细胞分化为内皮细胞谱系的2D培养系统，并证明hES细胞衍生的内皮细胞（hES-EC）可以自组装成功能性血管，这些血管在移植到免疫缺陷小鼠后自发地整合到循环系统中。然而，基于hES-EC的疗法的临床转化的关键挑战是免疫排斥。本项目的主要目标是从机制上了解同种异体hES-EC的免疫反应，并开发有效的方法来防止hES细胞衍生的内皮同种异体移植物的排斥反应。目前阻碍hES/iPS细胞及其衍生物的免疫原性和同种异体性研究的一个关键因素是缺乏合适的体内模型。我们已经开发了一种新的协议，在免疫缺陷小鼠的人免疫重建。人源化小鼠（hu-小鼠）表现出持续的多系人淋巴造血细胞再增殖和次级淋巴器官的形成，在免疫后产生强烈的抗原特异性人T细胞和抗体应答，并介导移植排斥。在这个提议中，我们将使用这个hu-小鼠模型来实现以下两个具体目标。在目的1中，我们将检查hES-EC在人小鼠中的同种异体性。该目的的目标是了解hES-EC的同种异体性，并确定不同分化阶段的hES-EC（例如，早期与晚期hES-EC）在同种异体性上不同。首先，我们将使用一系列体外试验来确定hES-EC的同种异体性。然后，我们将评估在hu小鼠中对同种异体hES-EC的体内免疫应答。我们还将评估hES-EC同种异体移植物在耗尽人T细胞或T细胞亚群的hu小鼠中的存活/排斥，以鉴定对hES-EC同种异体移植物排斥至关重要的免疫组分。目的2：通过建立混合造血嵌合体，探讨诱导hES-EC同种异体移植耐受的可能性。我们将开发用于从hES细胞体外分化可移植造血干/祖细胞的方案，并使用这些细胞在hu小鼠中诱导嵌合体和供体特异性耐受。这些研究将确定来源于用于建立造血嵌合体的相同hES细胞系的hES-EC是否可以被嵌合的hu小鼠永久接受而不需要免疫抑制。这些研究的成功完成将有助于我们了解hES-EC的同种免疫应答机制，并制定有效的策略来克服基于hES细胞的再生医学的免疫学障碍。 公共卫生相关性：免疫排斥反应是人类胚胎干细胞（hES）和诱导多能干细胞（iPS）再生医学临床转化的关键挑战。该建议旨在使用人源化小鼠模型来评估hES细胞衍生的内皮细胞的同种异体性，并开发用于控制对hES细胞衍生的内皮同种异体移植物的同种免疫应答的策略。

DESCRIPTION (provided by applicant): This application addresses broad Challenge Area (11) Regenerative Medicine and specific Challenge Topic, 11-HL-101* Develop cell-based therapies for cardiovascular, lung, and blood diseases. Given their self-renewing and pluripotent nature, human embryonic stem (hES) and induced pluripotent stem (iPS) cells hold great promise as cell replacement therapy and regenerative medicine. We recently developed a 2D culture system for differentiating hES cells into the endothelial lineage, and demonstrated that hES cell-derived endothelial cells (hES-EC) can self-assemble into functional blood vessels that spontaneously integrate into the circulatory system after transplantation into immunodeficient mice. However, a key challenge for clinical translation of hES-EC-based therapies is the immunological rejection. The major goal of this project is to mechanistically understand the immunological responses to allogeneic hES-EC, and to develop effective approaches to preventing the rejection of hES cell-derived endothelial allografts. A key factor currently impeding the study of the immunogenicity and allogenicity of hES/iPS cells and their derivatives is the lack of a suitable in vivo model. We have developed a novel protocol for human immune reconstitution in immunodeficient mice. The humanized mice (hu-mice) exhibit sustained repopulation with multilineages of human lymphohematopoietic cells and formation of secondary lymphoid organs, develop strong antigen-specific human T cell and antibody responses upon immunization, and mediate transplant rejection. In this proposal, we will use this hu-mouse model to pursue the following two specific aims. In Aim 1, we will examine the allogenicity of hES-EC in hu-mice. The goal of this aim is to understand the allogenicity of hES-EC and determine whether hES-EC at different differentiation stages (e.g., early vs. late hES-EC) differ in allogenicity. First, we will determine the allogenicity of hES-EC using a series of in vitro assays. We will then assess in vivo immune responses to allogeneic hES-EC in hu-mice. We will also assess the survival/rejection of hES-EC allografts in hu-mice that are depleted of human T cells or T cell subset(s) for identifying the immune components that are essential to hES-EC allograft rejection. In Aim 2, we will explore the possibility of inducing tolerance to hES-EC allografts in hu-mice through establishment of mixed hematopoietic chimerism. We will develop protocols for in vitro differentiation of transplantable hematopoietic stem/progenitor cells from hES cells, and with these cells to induce chimerism and donor-specific tolerance in hu-mice. These studies will determine whether hES-EC derived from the same hES cell line that is used for establishing hematopoietic chimerism can be permanently accepted by the chimeric hu-mice without the need of immunosuppression. Successful completion of the proposed studies will help us to understand the mechanisms of alloimmune responses to hES-EC and develop effective strategies to overcome the immunological hurdle to hES cell-based regenerative medicine. PUBLIC HEALTH RELEVANCE: Immunological rejection is a key challenge for clinical translation of human embryonic stem (hES) and induced pluripotent stem (iPS) cell-based regenerative medicine. This proposal aims to use a humanized mouse model to assess the allogenicity of hES cell-derived endothelial cells and to develop strategies for controlling alloimmune responses to hES cell-derived endothelial allografts.