Functional dissection of human adipose and bone marrow-derived MSC phenotypes

我们描述了人脂肪来源的间充质干细胞（A-MSC）的体外和体内特性，并将其与骨髓间充质干细胞（BM-MSC）进行了比较。我们了解到MSC以低水平进入免疫缺陷小鼠的多个组织，但BM-MSC和A-MSC从血液中迁移到缺氧组织损伤区域的能力要强得多，我们正在继续确定其中的机制。我们解剖了假定的标记物和表型，并了解到表面蛋白的改变取决于微环境因素，如氧浓度和配体结合。我们的假设是MSC的“组织修复亚群”由高度适应性的细胞组成，这些细胞通过改变细胞表面受体表达、整合素、MMPS和其他与迁移、保留和存活相关的蛋白质，准备对伤口或炎症微环境做出快速反应。MSC是“护理人员”，通过组织分泌分泌旁分泌因子，启动内源性修复和血运重建的级联反应。如果他们能够对环境做出反应并迅速适应，这是有益的。但是，这种性质使得分离MSC用于组织修复变得困难。因此，最原始的间充质干细胞区室的表型，以允许从骨髓或脂肪组织中分离，仍然没有很好地定义，仍然是资助申请的目标。我们假设，非生理性的标准间充质干细胞培养条件正在将间充质干细胞分化为与最大组织修复能力不相容的状态。目前的资助范围缩小，目标是在标准培养和更多生理条件下进行减法和继续检查MSC表型，目标是前瞻性地分离和潜在地扩大原始种群，这些原始种群保留形成软骨、骨骼、脂肪和/或损伤组织的能力。我们将比较在标准条件下扩增到更生理条件下的MSC的表型和功能，并与预期分离的ALDH hi/CD45-/CD31-MSC群体进行比较。我们将使用克隆整合标记和新的异种移植模型来确定允许将最多数量的原始人类间充质干细胞募集到组织损伤区域的条件，并更好地定义这个有趣的成体干细胞群体的多能性和自我更新。

We have characterized the in vitro and in vivo characteristics of human adipose-derived mesenchymal stem cells (A-MSC), and compared them to marrow MSC (BM-MSC). We learned that MSC lodge at low levels into multiple tissues in immune deficient mice, but that both BM-MSC and A-MSC migrate from the bloodstream into areas of hypoxic tissue damage much more robustly, and we are continuing to define the mechanisms involved. We have dissected putative markers and phenotypes, and learned that surface proteins are altered dependent upon microenvironmental factors such as oxygen concentration and ligand binding. Our hypothesis is that the “tissue-repairing subset” of MSC is composed of highly adaptable cells that are poised to respond rapidly to the wound or inflammatory microenvironment by altering cell surface receptor expression, integrins, MMPS, and other proteins related to migration, retention, and survival. MSC are “paramedics”, navigating through tissue to secrete paracrine factors that initiate cascades of endogenous repair and revascularization. It is beneficial if they are able to respond to the environment and to adapt very rapidly. But, this quality makes it difficult to prospectively isolate MSC for tissue repair. Thus, the phenotype of the most primitive MSC compartment, to allow prospective isolation from marrow or adipose tissue, is still not well defined and remains a goal of the grant application. We hypothesize that standard MSC culture conditions, which are non-physiological, are differentiating MSC to states that are not compatible with maximal tissue repair capacity. The goal of the current grant, reduced in scope is to subfractionate and to continue to examine MSC phenotype, in standard culture and in more physiological conditions, with the goal of prospectively isolating and potentially expanding primitive populations which retain the ability to form cartilage, bone, and fat and/or to home to damaged tissues. We will compare the phenotype and function of MSC expanded under standard conditions to more physiologic conditions, in comparison to prospectively isolated ALDH hi/CD45-/CD31-MSC populations. We will use clonal integration marking and novel xenotransplantation models to define conditions that allow recruitment of the highest numbers of primitive human MSC to areas of tissue damage, and to better define multipotency and self-renewal in this interesting adult stem cell population.