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*开发心血管、肺和血液疾病的基于细胞的疗法。人类胚胎干细胞(HES)和诱导多能干细胞(IPS)具有自我更新和多潜能的特性，在细胞替代治疗和再生医学中具有广阔的应用前景。我们最近开发了一种将HES细胞分化为内皮细胞的2D培养系统，并证明了HES细胞来源的内皮细胞(HES-EC)在移植到免疫缺陷小鼠体内后，可以自组装成具有功能的血管，并自发整合到循环系统中。然而，基于HES-EC的疗法临床翻译的一个关键挑战是免疫排斥。该项目的主要目标是从机制上了解同种异体HES-EC的免疫学反应，并开发有效的方法来预防HES细胞来源的同种异体内皮细胞移植的排斥反应。目前阻碍HES/iPS细胞及其衍生物免疫原性和同种异体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方案，用于在免疫缺陷小鼠中重建人类免疫。人源化小鼠(HU小鼠)表现出持续的多系人淋巴造血细胞再繁殖和次级淋巴器官的形成，免疫后产生强烈的抗原特异性T细胞和抗体反应，并介导移植排斥反应。在这项建议中，我们将利用这种胡鼠模型来追求以下两个具体目标。在目标1中，我们将检测HES-EC在HU-小鼠体内的同种异体。这一目的是为了了解HES-EC的同种异体，并确定不同分化阶段的HES-EC(如早期和晚期HES-EC)在同种异体活性上是否存在差异。首先，我们将通过一系列的体外检测来确定HES-EC的同种异体。然后，我们将评估同种异体HES-EC在HU-小鼠体内的免疫反应。我们还将评估人T细胞或T细胞亚群缺失的HES-EC同种异体移植鼠的存活/排斥反应(S)，以确定对HES-EC同种异体移植排斥反应至关重要的免疫成分。在目的2中，我们将探索通过建立混合造血嵌合体来诱导HES-EC同种异体移植耐受的可能性。我们将开发从HES细胞体外分化可移植的造血干/祖细胞的方案，并与这些细胞一起在HU-小鼠中诱导嵌合体和供体特异性耐受。这些研究将确定来自用于建立造血嵌合体的同一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的同种异体免疫反应的机制，并开发有效的策略来克服HES细胞再生医学的免疫学障碍。 公共卫生相关性：免疫排斥是人类胚胎干细胞(HES)和诱导多能干细胞(IPS)再生医学临床翻译的关键挑战。这项建议旨在使用人源化的小鼠模型来评估HES细胞来源的内皮细胞的同种异体反应，并开发控制HES细胞来源的同种异体内皮细胞移植的同种免疫反应的策略。

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.