HGF Induces Robust Human Thymopoiesis in Mice

描述（由申请人提供）：在过去的十年中，使用免疫缺陷小鼠作为人类造血干细胞移植的受体极大地改善了干细胞研究。研究人造血干细胞分化为大多数血细胞谱系的模型的开发已经完成，并且系统已经优化。然而，研究人类血细胞的一个特定谱系T淋巴细胞发育的强大系统尚未完成。最具重现性的体外系统，允许从纯化的干细胞发育人类T细胞;胎儿胸腺器官培养，需要精确定时交配的小鼠育种，然后仔细解剖产生的第15天胚胎。当成功时，该技术产生非常低数量的人类T细胞。scid/hu thy/liv小鼠是目前可用于研究体内人类T细胞发育的最佳系统，它需要获得人类胎儿组织并在小鼠中完成存活手术，因此许多实验室都无法使用。不幸的是，T细胞的发展并没有发生在最流行的小鼠异种移植受体，NOD/SCID小鼠。在该小鼠中，移植的人干细胞和祖细胞强烈且快速地转向B细胞分化，并且没有观察到人T细胞。即使是成熟的人T细胞在移植到NOD/SCID小鼠后也会迅速清除。 尽管现有系统存在缺陷，但科学界不断努力改进这些模型，因为研究小鼠中大量人类T细胞产生的能力将对人类健康产生巨大影响。延迟的免疫重建是干细胞移植中的一个主要问题，每年导致许多人死于移植后感染。一个研究干细胞移植后T细胞从纯化的人类干细胞重建的小鼠系统可以允许解剖可以帮助移植患者更快地发展功能性免疫系统的因素。迄今为止，缺乏不需要手术技巧的可重复和有效的T细胞发育测定一直是干细胞领域的主要障碍。 我们已经发现，共同移植的人间充质干细胞工程表达肝细胞生长因子（HGF）与纯化的人干细胞引起大量的人T淋巴细胞的发展和扩增NOD/SCID/B2 M无效小鼠。我们假设人类“胸腺生成”在所有组织中进行，其中分泌HGF的MSC与干细胞共定位。为了支持这一理论，MSC/HGF/B2 M小鼠在其脾脏和肝脏以及胸腺中具有大量的双阳性CD 4 +/CD 8+人细胞，但骨髓中没有。这些是人类MSC在小鼠中归巢的位点。从我们最初的观察，T细胞发育似乎模仿正常的胸腺生成，除了循环中的成熟CD 4+和CD 8+细胞外，器官中还产生大量的双阳性（CD 4 +/CD 8+）人T细胞。MSC/HGF/HSC共移植小鼠形成了我们迄今为止在异种移植领域观察到的最稳健的体内T细胞发育系统。本申请提出了研究以改进该系统，理解它，并进一步开发它，使其成为任何希望在体内研究从造血干细胞发育人T细胞的研究者的有价值的资源。

DESCRIPTION (provided by applicant): Over the past decade, the use of immune deficient mice as recipients for human hematopoietic stem cell transplantation has dramatically improved stem cell research. Development of models to study the differentiation of human hematopoietic stem cells into most blood cell lineages has been accomplished and the systems have been optimized. However, robust systems to study development of one particular lineage of human blood cell, the T lymphocyte, have not yet been accomplished. The most reproducible in vitro system that allows human T cell development from purified stem cells; the fetal thymic organ culture, requires precise timed mating of murine breeders and then careful dissection of the resulting day 15 embryos. When successful, the technique produces very low numbers of human T cells. The best system currently available to study human T cell development in vivo, the scid/hu thy/liv mouse, requires procurement of human fetal tissues and accomplishment of survival surgery in mice, so is not available to many laboratories. Unfortunately, T cell development does not occur in the most popular murine xenograft recipient, the NOD/SCID mouse. In this mouse, transplanted human stem and progenitor cells are strongly and rapidly diverted toward B cell differentiation, and no human T cells are observed. Even mature human T cells are rapidly cleared following transplantation into NOD/SCID mice. In spite of the drawbacks to the existing systems, the scientific community constantly strives to improve these models because the ability to study production of large numbers of human T cells in mice would have an enormous impact on human health care. Delayed immune reconstitution is a major problem in stem cell transplantation, resulting in many deaths from post-transplant infection each year. A murine system to study post-stem cell transplant T cell reconstitution from purified human stem cells could allow dissection of the factors that could help transplant patients develop a functional immune system much more rapidly. The lack of a reproducible and effective T cell development assay that does not require surgical skill has been a major stumbling block for the stem cell field to date. We have discovered that co-transplantation of human mesenchymal stem cells engineered to express hepatocyte growth factor (HGF) with purified human stem cells causes massive human T lymphocyte development and expansion in NOD/SCID/B2M null mice. We hypothesize that human "thymopoiesis" is ongoing in all tissues where the HGF-secreting MSC co-localize with the stem cells. In support of this theory, the MSC/HGF/B2M mice have large numbers of double positive CD4+/CD8+ human cells in their spleen and liver, as well as thymus, but not bone marrow. These are the sites of human MSC homing in the mice. From our initial observations, T cell development appears to mimic normal thymopoiesis, with large numbers of double positive (CD4+/CD8+) human T cells produced in the organs, in addition to mature CD4+ and CD8+ cells in the circulation. The MSC/HGF/HSC cotransplanted mice form the most robust in vivo T cell development system that we have observed in the xenograft field to date. The current application proposes studies to refine this system, to understand it, and to develop it further to be a valuable resource for any investigator that wishes to study development of human T cells from hematopoietic stem cells in vivo.