BIOLOGY OF NON-HUMAN PRIMATE MARROW STROMAL CELLS

非人灵长类动物骨髓基质细胞的生物学

• 批准号: 7348990
• 负责人: DARWIN Johnson PROCKOP
• 金额: $ 3.1万
• 依托单位: TULANE UNIVERSITY OF LOUISIANA
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-05-01 至 2007-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7348990
• 关键词: BIOLOGY; NON; HUMAN; PRIMATE; MARROW

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. The overall aim of the project is to develop procedures whereby adult stem cells from the bone marrow stroma can be used for trials of gene therapy in non-human primates. The adult stem cells, referred to as mesenchymal stem cells or marrow stromal cells (MSCs), are of interest for cell and gene therapy because they can readily be obtained from a patient, expanded in culture, genetically engineered with or without the use of viruses, and then returned for therapy of the same patient. They are also of interest because they home to damaged tissues and differentiate to replace the damaged cells in the tissues. The cells are currently being tested in many small animal models of human diseases and several promising clinical trials with the cells have been initiated in rare diseases in children. However, extensive trials of the cells in non-human primates are clearly essential for some of the currently proposed applications to common diseases such as osteoporosis, cardiac failure, Parkinsonism, leukodystrophies, and Alzheimer¿s disease. The goals of the proposal are: Specific Aim 1. Isolate and expand primate MSCs with the improved protocol our laboratory has recently developed to isolate and expand cultures of human MSCs. We have successfully isolated rhesus MSCs from both the bone marrow and adipose tissue. We have done an extensive characterization of the in vitro biologic properties of the stem cell populations. Our data indicate that the MSCs from these two tissues share many characteristics. The data indicate that MSCs isolated from rhesus bone marrow (rBMSCs) and human adipose tissue (hASCs) had more similar biologic properties than MSCs of rhesus adipose tissue (rASCs) and human bone marrow MSCs (hBMSCs). Analyses of in vitro growth kinetics revealed shorter doubling time for rBMSCs and hASCs. rBMSCs and hASCs underwent significantly more population doublings than the other MSCs. MSCs from all sources showed a marked decrease in telomerase activity over extended culture; however they maintained their mean telomere length. All of the MSCs expressed embryonic stem cell markers, Oct-4, Rex-1 and Sox-2 for at least 10 passages. Early populations of MSCs types showed similar multilineage differentiation capability. However, only the rBMSCs and hASCs retain greater differentiation efficiency at higher passages. Overall in vitro characterization of MSCs from these two species and tissue sources revealed a high level of common biologic properties. However, the results demonstrate clear biologic distinctions, as well. Specific Aim 2. Compare the primate MSCs in culture with human MSCs in their ability to expand rapidly and to differentiate into osteoblasts, chondrocytes, adipocytes, and neural cells. We have found that the rhesus cells efficiently undergo differentiation along osteogenic, chondrogenic and adipogenic lineages. The efficiency between the human and rhesus MSCs is virtually indistinguishable. In terms of neural differentiation, we have found that rhesus AMSCs differentiate along this lineage with greater efficiency in vitro than human or bone marrow derived cells. Specific Aim 3. Compare the primate MSCs to human MSCs in vivo in their ability to engraft into multiple tissues after systemic or intracranial infusion into immunodeficient mice. These studies are currently ongoing. We have injected human and rhesus bone marrow and adipose tissue derived MSCs into the CNS of NIHIII mice, using stereotaxic delivery. We are currently assessing engraftment and differentiation of these cells. Specific Aim 4. When a recently purchased microPET instrument becomes operational, assay the engraftment of the primate cells first into immunodeficient mice and then into the same primates from which the cells were derived using real-time imaging with the microPET.

该子项目是利用 NIH/NCRR 资助的中心拨款提供的资源的众多研究子项目之一。子项目和研究者 (PI) 可能已从另一个 NIH 来源获得主要资金，因此可以在其他 CRISP 条目中得到体现。列出的机构是中心的机构，不一定是研究者的机构。该项目的总体目标是开发程序，使来自骨髓基质的成体干细胞可用于非人类灵长类动物的基因治疗试验。成体干细胞，称为间充质干细胞或骨髓基质细胞（MSC），对细胞和基因治疗很有意义，因为它们可以很容易地从患者身上获得，在培养物中扩增，使用或不使用病毒进行基因工程，然后返回同一患者进行治疗。它们之所以引起人们的兴趣，还因为它们以受损组织为家，并分化以取代组织中受损的细胞。这些细胞目前正在许多人类疾病的小动物模型中进行测试，并且已经在儿童罕见疾病中启动了几项有希望的细胞临床试验。然而，对于目前提出的一些常见疾病（如骨质疏松症、心力衰竭、帕金森病、脑白质营养不良和阿尔茨海默氏病）的应用，在非人类灵长类动物中进行广泛的细胞试验显然是必要的。该提案的目标是： 具体目标 1. 使用我们实验室最近开发的用于分离和扩增人类 MSC 培养物的改进方案来分离和扩增灵长类 MSC。我们已成功从骨髓和脂肪组织中分离出恒河猴 MSC。我们对干细胞群的体外生物学特性进行了广泛的表征。我们的数据表明，这两种组织的间充质干细胞具有许多共同特征。数据表明，从恒​​河猴骨髓（rBMSCs）和人脂肪组织（hASCs）分离的MSCs比从恒河猴脂肪组织（rASCs）和人骨髓MSCs（hBMSCs）分离的MSCs具有更相似的生物学特性。体外生长动力学分析显示 rBMSC 和 hASC 的倍增时间较短。 rBMSC 和 hASC 比其他 MSC 经历了更多的群体倍增。所有来源的间充质干细胞在延长培养后端粒酶活性均显着降低；然而，他们保持了平均端粒长度。所有 MSC 均表达胚胎干细胞标记物 Oct-4、Rex-1 和 Sox-2 至少 10 代。 MSCs 类型的早期群体表现出相似的多谱系分化能力。然而，只有 rBMSC 和 hASC 在更高的传代中保持更高的分化效率。这两个物种和组织来源的 MSC 的总体体外表征揭示了高水平的共同生物学特性。然而，结果也表明了明显的生物学差异。具体目标 2. 比较培养的灵长类 MSC 与人 MSC 快速扩增和分化为成骨细胞、软骨细胞、脂肪细胞和神经细胞的能力。我们发现恒河猴细胞有效地沿着成骨、软骨和脂肪形成谱系进行分化。人类和恒河猴 MSC 的效率几乎没有区别。在神经分化方面，我们发现恒河猴 AMSC 沿该谱系分化，其体外分化效率高于人类或骨髓来源的细胞。 具体目标 3. 比较灵长类 MSC 与人 MSC 在全身或颅内输注至免疫缺陷小鼠体内后植入多个组织的能力。这些研究目前正在进行中。我们使用立体定向递送将人类和恒河猴骨髓和脂肪组织来源的间充质干细胞注射到 NIHIII 小鼠的中枢神经系统中。我们目前正在评估这些细胞的植入和分化。 具体目标 4. 当最近购买的 microPET 仪器投入使用时，首先使用 microPET 实时成像测定灵长类动物细胞在免疫缺陷小鼠中的植入情况，然后在细胞来源的相同灵长类动物中进行植入。