Functional dissection of human A-MSC vs BM-MSC phenotype

描述(由申请人提供)： 来自啮齿动物和人骨髓的间充质干细胞(BM-MSC)可以分化为肌肉、骨、软骨、脂肪和成纤维细胞。我们最近鉴定了具有类似分化能力的人类细胞，这些细胞是从更容易获得和丰富的来源--人类脂肪组织中分离出来的。这些人脂肪来源的间充质干细胞(AMSC)在培养过程中扩增迅速，很容易被逆转录病毒载体转导，并在免疫缺陷小鼠体内植入包括脑在内的多种组织。然而，像BM-MSC一样，AMSC群体中最原始的细胞的表型尚不清楚，我们在体外和体内研究的快速生长的细胞板可能是相当不同的。本研究的一个主要目的是在体外和体内研究最原始的AMSC和BM-MSC群体的表型特征和功能。我们已经在BM-MSC和AMSC中发现了低水平表达肝细胞生长因子(HGF)受体c-MET的细胞亚群。HGF是一种化学诱导剂和活性因子，在许多组织中损伤部位表达上调，包括肌肉、肝脏、肠道和肺，以及心肌和骨骼肌。肝细胞生长因子影响许多原始细胞类型的运动能力并维持其活性。我们假设，局部分泌的HGF是从循环中招募干细胞进入损伤部位以介导修复的主要因素，最原始和最多功能的MSC将表达HCF的受体c-met。因此，我们将通过从脂肪组织和骨髓中分离人C-MET/CD45-细胞来开始我们的AMSC和BM-MSC分室的表型解剖。我们将进一步将人类CD45-/e-MET和CD45-/c-MET-群体细分为表达和不表达CD105和CD133的子集。在特定的目标1中，我们将测试候选的多潜能间充质干细胞群体在体外向肌肉、骨、软骨和脂肪分化的能力，以证明其多能性。在特定目标2中，我们将研究在肌肉、胰腺、肝脏和脑的慢性和急性损伤状态下，AMSC和BM-MSC亚群在体内归巢到免疫缺陷小鼠的特定组织中。在这一目标中，我们将专门寻找不仅能回到受损的小鼠组织中，而且还能以强大的方式介导修复的人MSC群体。最后，在特定的目标3中，我们将在体外诱导候选的多潜能人MSC群体进入周期，用逆转录病毒载体标记它们，并在体外和体内分化后进行克隆整合分析。这一最终目标将从骨髓和脂肪组织中严格鉴定人类MSC的亚群，这些亚群可以分化为多个谱系的后代。这项研究的总体目标是系统地剖析人类间充质干细胞分化的层次结构，并开发模型来分析每个亚群在组织修复中的表现，以便最好地利用这些有希望的细胞进行安全和合理的再生治疗。

DESCRIPTION (provided by applicant): Mesenchymal stem cells from rodent and human bone marrow (BM-MSC) can differentiate into muscle, bone, cartilage, fat, and fibroblast. We have recently characterized human cells with a similar differentiative capacity, which are isolated from a more accessible and plentiful source; human adipose tissue. These human adipose-derived mesenchymal stem cells (AMSC) expand rapidly in culture, are easily transduced by retroviral vectors, and home into multiple tissues in immune deficient mice, including brain. However, like BM-MSC, the phenotype of the most primitive cells in the AMSC population is not known, and the rapidly growing plates of cells that we have studied in vitro and in vivo are likely quite heterogeneous. A major goal of the proposed studies is to phenotypically characterize and to define the function of the most primitive populations of AMSC and BM-MSC, in vitro and in vivo. We have identified a subpopulation of cells in both BM-MSC and AMSC that expresses low levels of c-met, the receptor for Hepatocyte Growth Factor (HGF). HGF is a chemoattractant and viability factor that is upregulated at the site of injury in many tissues, including muscle, liver, intestine, and lung, plus cardiac and skeletal muscle. HGF affects the motility and maintains the viability of many primitive cell types. We hypothesize that HGF, secreted locally in response to tissue injury, is a major factor in recruiting stem cells from the circulation into the site of injury to mediate repair, and that the most primitive and pluripotent MSC will express c-met, the receptor for HCF. We will therefore begin our phenotypic dissection of the AMSC and BM-MSC compartments by isolating human C-met+/CD45- cells from adipose tissue and bone marrow. We will further subfractionate the human CD45-/e-met+ and CD45-/c-met- populations into subsets that do and do not express CD105, and CD133. In Specific Aim 1, we will test the candidate pluripotent MSC populations for their capacity to differentiate to muscle, bone, cartilage, and fat in vitro to demonstrate their multipotency. In Specific Aim 2, we will examine the in vivo homing of the AMSC and BM-MSC subpopulations into specific tissues of immune deficient mice, during chronic and acute injury states in muscle, pancreas, liver, and brain. In this aim, we will specifically seek populations of human MSC that can not only home into the injured murine tissues, but those that can also mediate repair in a robust manner. Finally, in Specific Aim 3, we will induce the candidate pluripotent human MSC populations into cycle in vitro, mark them with retroviral vectors, and perform clonal integration analysis studies following in vitro and in vivo differentiation. This final aim will stringently identify the subpopulations of human MSC from bone marrow and adipose tissue which can differentiate into progeny of multiple lineages. The overall goal of the proposed studies is to systematically dissect the hierarchy of human MSC differentiation and to develop models to analyze the performance of each subpopulation in tissue repair so that these promising cells can be best harnessed for safe and rational regenerative therapies.