Immunodeficient mouse model of stem cell plasticity

描述（申请人提供）：近年来，“干细胞可塑性”的新概念以及干细胞可用于修复受损组织的希望引起了极大的兴奋。该领域现在正在经历一个更现实和关键的调查时期，在肝脏和肌肉中的特定类型的损伤后，细胞融合已经显示出来，这两个器官都有大量的多核细胞。该领域仍然是一个非常令人兴奋的领域，在干细胞治疗组织修复可以或应该用于临床之前，还有许多问题有待回答。在我们的第一个资助期间，我们开发了必要的体内模型，现在回答有关干细胞组织修复的具体问题。我们的建议将侧重于使用这些模型来解决该领域的三个主要问题：1。哪种细胞类型最能介导肝脏和肌肉的修复？2.修复是通过融合、分化或注入的干细胞分泌血管生成因子来启动血运重建的吗？3.干细胞是如何被募集到损伤部位的，我们能否增强这一点以诱导更强大的修复？HGF是一种化学引诱物和活力因子，在受损的肝脏、心脏和骨骼肌以及其他器官中升高。它通过在缺氧损伤部位从无活性形式裂解而活化。HGF影响成肌细胞、肝卵圆细胞和造血干细胞的运动性并维持其活力。我们假设，肝细胞生长因子，激活组织损伤和缺氧，在一个梯度特异性的方式招募原始的，c-met+干细胞进入肌肉或肝脏损伤的网站。我们假设，肝细胞生长因子将维持招募的干细胞的活力，而他们是由诱导，组织特异性因子在局部微环境分化，融合，或启动血管再生，参与组织再生。为了检验这一假设，在特定目标1中，我们将定义注射到免疫缺陷小鼠中的最佳人细胞群，以诱导最稳健的招募到受损的肝脏vs.心肌。将纯化的HSC和MSC群体与ALDH hi/lin-群体（其含有HSC和内皮祖细胞）和HSC/MSC共移植进行比较。将通过从NOD/SCID/MPSVII（GUSB null）小鼠中的正常人细胞表达GUSB，结合免疫组织化学和原位杂交来实现对损伤组织的细胞追踪。在具体目标2中，我们将探讨缺氧，HGF/c-met和CXCR 4/SDF-1在招募人类祖细胞到受损肝脏和肌肉中的作用。在具体目标3中，我们将在单细胞水平上使用克隆标记来严格确定从人脐带血和骨髓中分离的单个干细胞是否可以产生肝脏或肌肉组织，以及血液。我们的总体目标是在新型免疫缺陷小鼠组织损伤模型中优化确定的人类祖细胞群体对受损肝脏和肌肉的招募，以获得更稳健的修复。

DESCRIPTION (provided by applicant): In recent years, the new concept of "stem cell plasticity", and the hope that stem cells could be used to repair damaged tissues has generated immense excitement. The field is now undergoing a more realistic and critical period of investigation, and cell fusion has been shown after specific types of damage in liver and muscle, both organs with a high number of multinucleate cells. The field is still an extremely exciting one, and many questions remain to be answered before Stem Cell Therapy for tissue repair can or should be used clinically. During our first period of funding, we developed the in vivo models necessary to now answer specific questions regarding tissue repair by stem cells. Our proposal will focus on using these models to address the three major questions in the field; 1. What cell type can best mediate repair in liver and muscle? 2. Is repair occurring through fusion, differentiation, or secretion of angiogenic factors by infused stem cells to initiate revascularization? 3. How are stem cells recruited to sites of injury and can we enhance this to induce more robust repair? HGF is a chemoattractant and viability factor elevated in injured liver, cardiac, and skeletal muscle, in addition to other organs. It is activated by cleavage from the inactive form at sites of hypoxic damage. HGF affects the motility and maintains the viability of myoblasts, hepatic oval cells, and hematopoietic stem cells. We hypothesize that HGF, activated in response to tissue injury and hypoxia, acts in a gradient-specific manner to recruit primitive, c-met+ stem cells into the site of muscle or liver injury. We hypothesize that HGF will maintain the viability of the recruited stem cells, while they are directed by inductive, tissue-specific factors in the local microenvironment to differentiate, fuse, or initiate revascularization, to participate in tissue regeneration. To test this hypothesis, in Specific Aim 1, we will define the optimal human cell population to inject into immune deficient mice to induce the most robust recruitment to damaged liver vs. cardiac muscle. Purified HSC and MSC populations will be compared to the ALDH hi/lin- population, which contains both HSC and endothelial progenitors, and to HSC/MSC co- transplantation. Cell tracking to the injured tissue will be accomplished by GUSB expression from normal human cells in NOD/SCID/MPSVII (GUSB null) mice, coupled with immunohistochemistry and in situ hybridization. In Specific Aim 2, we will explore the role of hypoxia, HGF/c-met, and CXCR4/SDF-1 in recruitment of human progenitors to injured liver and muscle. In specific aim 3, we will use clonal marking at the single cell level to rigorously determine whether individual stem cells isolated from human cord blood and bone marrow can give rise to liver or muscle tissue, in addition to blood. Our Overall Goal is to optimize recruitment of defined human progenitor populations to damaged liver and muscle in novel immune deficient mouse models of tissue injury, to obtain more robust repair.