Hematopoietic Stem Cell Differentiation and Commitment

描述(申请人提供)：成体干细胞存在于许多组织中，并在一生中补充这些组织的细胞成分。最近，越来越多的证据表明，至少在某些条件下，成体干细胞的后代可以在其来源组织之外产生细胞。在我们的实验室里，我们在肌肉中的干细胞产生血液和骨髓中的干细胞产生肌肉的背景下，研究了这种所谓的干细胞“可塑性”。肌肉中产生血液的干细胞是起源于造血的，是上一个资助期的重点。我们最近还发现，造血干细胞(HSC)生成肌肉的能力很可能是由于髓系细胞直接整合到再生肌肉中所致。在这里，以肝再生为模型，我们建议更详细地研究这种非固有细胞掺入的机制，以期从长远的角度提高其治疗应用的效率。在第一个目标中，我们将明确鉴定能够参与肝细胞生成的主要造血细胞类型。我们将利用cre-lox谱系标记策略以及细胞移植到遗传缺陷的肝脏模型中来评估血肝转换。我们还将使用多个供体和受体标记物来确定在该模型中是否存在非融合性肝再生机制的证据。在第二个目标中，我们将检查是否可以在两种不同的肝再生模式下通过融合依赖或独立机制发生血-肝转换。这将揭示这一治疗策略对不同类型的肝病和再生模式的适用性。最后，在第三个目标中，我们将通过a)在没有骨髓移植的情况下提供细胞时诱导融合，b)使用可诱导的病毒融合蛋白提高融合效率，以及c)研究融合时发生的核重新编程事件，通过使用表达标记的组织特异性蛋白的BAC转基因来研究血-肝转换的分子需求。总之，这些研究将提高我们对控制异型细胞融合的因素以及核重新编程改变细胞特性的步骤的理解。这些见解将是迈向干细胞疗法治疗应用的关键。

DESCRIPTION (provided by applicant): Adult stem cells are present in many tissues and replenish the cellular constituents of those tissues throughout life. Recently, evidence has accumulated that at least under some conditions, progeny of adult stem cells can generate cells outside their tissue of origin. In our laboratory, we have examined this so-called stem cell "plasticity" in the context of generation of blood by stem cells in the muscle, and generation of muscle by stem cells from bone marrow. Stem cells in muscle that generate blood are hematopoietic in origin, and were the focus of the previous funding period. We have also recently shown that the ability of hematopoietic stem cells (HSC) to generate muscle is most likely due to the direct incorporation of myeloid cells into regenerating muscle. Here, using liver regeneration as a model, we propose to examine the mechanisms of this non-autochthonous cell incorporation in much more detail, with the long-term view of improving the efficiency for its therapeutic use. In the first Aim, we will definitively identify the major hematopoietic cell types capable of participating in generation of hepatocytes. We will utilize cre-lox lineage marking strategies as well as cell transplantation into a genetically deficient liver model to assess hematohepatic conversion. We will also determine whether there is any evidence for a non-fusion hepatic regeneration mechanism in this model using multiple donor and recipient markers. In the second Aim, we will examine whether hemato-hepatic conversion can occur in the context of two distinct models of liver regeneration through either fusion-dependent or -independent mechanisms. This will reveal the applicability of this therapeutic strategy to different types of liver disease and modes of regeneration. Finally, in the third Aim, we will study the molecular requirements for hemato-hepatic conversion by a) Inducing fusion when cells are given in the absence of bone marrow transplantation, b) Increasing the efficiency of fusion using inducible viral fusion proteins, and c) Studying the nuclear reprogramming events occurring upon fusion using BAC-transgenics expressing tagged tissue-specific proteins. Together, these studies will improve our understanding of the factors that govern heterotypic cell fusion as well as the steps in the reprogramming of nuclei to alter cell identity. These insights will be essential for moving towards therapeutic applications of stem cell-based therapies.