Stem cell-mediated reversal of thymic involution in premature aging models

描述(申请人提供)：老年人发病和死亡的一个主要原因是感染，包括常见的病毒感染，如流感和呼吸道合胞病毒(RSV)，健康年轻人的免疫系统很容易与之抗争。虽然与年龄相关的免疫下降可能有多种原因，但一个关键的问题是胸腺退化，这会导致新T细胞(最近的胸腺移民，RTE)的产生减少。我们和其他人的研究表明，与年轻的胸腺不同，衰老的标志是胸腺退缩，这是由于胸腺上皮细胞(TEC)的丧失，胸腺上皮细胞产生胸腺祖细胞扩张和分化所需的信号，特别是IL-7和Kit配体(KL)。使用按时间顺序老化的小鼠和Klotho早衰模型，我们已经证明，胸腺退化可以通过细胞因子(角质细胞生长因子)介导的TEC的扩张和维持而逆转，从而导致T细胞产生、外周T淋巴细胞数量和免疫反应性的正常化。KGF的作用是暂时的，大约持续3周，但重复给药可以维持恢复的胸腺微结构和T细胞产生。这种瞬时效应表明，KGF反应的TEC前体细胞继续快速衰老。在衰老过程中恢复胸腺功能的另一种方法是将干细胞来源的TEC前体细胞移植到老龄小鼠体内。在正常胚胎发育过程中，TEC由内胚祖细胞发育而来。DiGeorge综合征(DGS)是一种常见的儿童畸形综合征，包括由于TEC发育失败而导致的免疫缺陷。T-box基因TBX1位于DGS关键区，其靶向突变导致小鼠出现DGS表型，包括肌无力。我们已经能够从小鼠胚胎干细胞(MESC)中获得具有功能的TEC。用激活素A刺激骨髓间充质干细胞产生稳定的内胚层，然后用慢病毒转导TBX1基因。将确定的内胚层细胞(DE-TBX1)与胚胎胸腺间充质细胞共培养。获得的细胞表达TEC的几个标记，并在胸腺重聚集实验中具有在体外诱导T细胞分化的能力。在拟议的研究中，将在Klotho早衰小鼠模型中测试mESC来源的TEC样细胞的能力，以确定它们是否可以移植并支持体内胸腺生成。我们假设1)mESC来源的TEC将概括正常的TEC个体发育；2)mESC来源的TEC前体细胞可以移植到Klotho小鼠早衰模型中并纠正胸腺退缩；3)mESC来源的TEC将保持其本体年龄，例如它们的基因表达特征将是“年轻”的TEC，而不是老年TEC。这些研究将提供有关mESC来源的TEC的产生、可移植性和在纠正年龄相关性胸腺退缩中的有效性的重要信息。与公共卫生相关：T淋巴细胞是免疫系统的细胞，控制身体对感染的反应。胸腺在生命早期制造T淋巴细胞，但随着年龄的增长，T淋巴细胞的产生显著减少。这些研究将研究如何利用胚胎干细胞生成胸腺上皮细胞，将胸腺上皮细胞移植到经历胸腺过早老化的小鼠体内，以再生胸腺并恢复制造T淋巴细胞的能力。

DESCRIPTION (provided by applicant): A major cause of morbidity and mortality in the aged is infections, including common viral infections such as influenza and respiratory syncytial virus (RSV), that the immune system of healthy young individuals has little difficulty combating. While there are likely to be multiple causes of age-related immune decline, a key problem is thymic involution, which results in decreased production of new T cells (recent thymic emigrants, RTE). Work by ourselves and others has demonstrated that in contrast to the young thymus, aging is marked by thymic involution due to the loss of thymic epithelial cells (TEC), which produce the signals needed in the thymus for progenitor cell expansion and differentiation, notably IL-7 and Kit ligand (KL). Using both chronologically aged mice and the Klotho premature aging models, we have shown that thymic involution can be reversed by cytokine-mediated (keratinocyte growth factor) expansion and maintenance of TEC, resulting in normalization of T-cell production, peripheral T lymphocyte numbers and of immune responsiveness. The effects of KGF are transient, lasting approximately 3 weeks, but repeated dosing can maintain the restored thymic microarchitecture and T-cell production. The transient effects suggest that KGF-responsive TEC progenitors continue to rapidly senesce. An alternative approach to restoring thymic function in aging is to transplant stem-cell derived TEC progenitors into aged mice. During normal embryogenesis, TEC develop from endodermal progenitors. The DiGeorge syndrome (DGS) is a common pediatric malformation complex that includes immune deficiency due to developmental failure of TEC. The T- box gene Tbx1 is located in the DGS critical region, and targeted mutation of Tbx1 results in a DGS phenotype in mice, including athymia. We have been able to derive functional TEC from murine embryonic stem cells (mESC). Definitive endoderm was generated by Activin A stimulation of mESC, and the cells were then lentivirally transduced with the Tbx1 gene. The definitive endoderm-Tbx1 cells (DE-Tbx1) were then co-cultured with embryonic thymic mesenchymal cells. The resultant cells express several markers of TEC, and have the ability to induce T-cell differentiation in vitro in thymic re-aggregation assays. In the proposed studies, the ability of the mESC-derived TEC-like cells will be tested in the Klotho premature aging mouse model to determine whether they can engraft and support in vivo thymopoiesis. We hypothesize that 1) mESC-derived TEC will recapitulate normal TEC ontogeny; 2) that mESC derived TEC progenitors can engraft in the Klotho murine model of premature aging and correct thymic involution; 3) mESC-derived TEC will maintain their ontological age, e.g., their gene expression signature will be that of "young" TEC, rather than aged TEC. The studies will provide essential information regarding the generation, transplantability and efficacy of mESC-derived TEC in the correction of age-related thymic involution. PUBLIC HEALTH RELEVANCE: T lymphocytes are cells of the immune system that control the body's response to infection. The thymus makes T lymphocytes in early life, but production of T lymphocytes profoundly decreases during aging. The studies will examine how embryonic stem cells can be used to generate thymic epithelial cells, which can be transplanted into mice that undergo premature thymic aging to regenerate the thymus and restore the ability to make T lymphocytes.