Immunological acceptance of embryonic stem cell-derived cardiomyocytes by imitating fetomaternal tolerance

当今移植领域最紧迫的问题是越来越缺乏合适的供体器官和组织。器官移植的一种可能的替代方法是细胞治疗，其目的是替换、修复或增强患病器官或受损组织的生物学功能。因为它们直接来自于它们将被用于的同一患者，所以它们不会受到免疫排斥。然而，成体干细胞的功效仍在争论中，特别是对于心肌再生。植入率低或不存在，通过旁分泌机制的作用是短暂的。实际上，心肌没有真正的再生。我们和其他人坚信，多能干细胞，如人类胚胎干细胞（hES）细胞最终将是实现真实的组织再生所必需的。然而，hES细胞不能是患者特异性的，而是同种异体的，并且不可避免地诱导免疫排斥。细胞移植物（与实体器官相反）非常适合于移植前的体外基因工程。因此，我们建议表征hES细胞在分化过程中和移植后的免疫表型（目的1），以产生低免疫原性hES细胞诱导耐受性（目的2），并描述异种免疫反应相比，hES细胞在体内。首先单独采用从母胎耐受性调整的策略，以（1）防止T细胞介导的hES细胞裂解，（2）吸引TcB，和（3）防止巨噬细胞和NK细胞介导的hES细胞裂解，并在靶细胞特异性免疫测定中进行测试。随后，将组合三种策略以产生低免疫原性hES细胞系，并且将在同种异体人源化小鼠模型中验证最有希望的致耐受性组合。最后，我们的目标是产生低免疫原性hES细胞衍生的心肌细胞，并在心肌梗死模型中测试其治疗潜力（目的3）。将低免疫原性hES细胞衍生的心肌细胞移植到经历永久性LAD结扎的人源化小鼠的缺血边缘区中。长期细胞存活和心脏功能的改善将使用多种尖端成像方式（BLI，回声，microCT）进行评估。本研究中获得的信息将对指导将来使用hES细胞作为人类疾病的临床治疗具有重要意义。

Todays most urgent problem in transplantation is the increasing shortage of suitable donor organs and tissues. One possible alternative to organ transplantation may be cell therapy, whereby the aim is to replace, repair, or enhance the biological function of the diseased organ or damaged tissue.Autologous, patient-specific adult stem cells are currently under investigation. Because they are derived directly from the same patient they will be used in, they are not subject to immune rejection. However, the efficacy of adult stem cells is still under debate, especially for myocardial regeneration. Engraftment is low or absent and the effect via paracrine mechanisms transient. Indeed, there is no actual regeneration of myocardium. We and others firmly believe that pluripotent stem cells, such as human embryonic stem (hES) cells will eventually be necessary to achieve real tissue regeneration. hES cells, however, cannot be patient-specific, but are allogeneic and inevitably induce immune rejection. Cellular grafts (in contrast to solid organs) lend themselves exceptionally well to pre-transplantation in vitro genetic engineering. We therefore propose to characterize the immunophenotype of hES cells during differentiation and after transplantation (aim 1), to generate hypo-immunogenic hES cells that induce tolerance (aim 2) and to describe xenogeneic immune responses in comparison to hES cells in vivo. The strategies adapted from fetomaternal tolerance to (1) prevent T cell mediated hES cell lysis, to (2) attract Tregs, and to (3) prevent macrophage and NK cell mediated hES cell lysis will first be employed individually and tested in target-cell specific immune assays. Later, the three strategies will be combined to generate a hypo-immunogenic hES cell line and the most promising tolerogenic combination will be validated in an allogeneic, humanized mouse model. Finally, we are aiming to generate hypo-immunogenic hES cell-derived cardiomyocytes and test their therapeutic potential in a myocardial infarction model (aim 3). Hypo-immunogenic hES cell-derived cardiomyocytes will be transplanted into the ischemic border zone of humanized mice that underwent permanent LAD ligation. Long-term cell survival and improvement of cardiac function will be assessed using multiple cutting edge imaging modalities (BLI, echo, microCT).The information gained in this study will be important in guiding the use of hES cells as a clinical treatment for human diseases in the future.