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资助的中心赠款提供的资源的许多研究子项目之一。子项目和研究者（PI）可能从另一个NIH来源获得主要资金，因此可以在其他CRISP条目中表示。所列机构为中心，不一定是研究者所在机构。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），对于细胞和基因治疗是有意义的，因为它们可以容易地从患者获得，在培养物中扩增，使用或不使用病毒进行基因工程，然后返回用于同一患者的治疗。它们之所以令人感兴趣，还因为它们会回到受损组织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，在非人类灵长类动物中进行广泛的细胞试验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病的应用显然是必不可少的。该提案的目标是：具体目标1。分离和扩大灵长类动物间充质干细胞与改进的协议，我们的实验室最近开发的分离和扩大培养的人间充质干细胞。我们已经成功地从骨髓和脂肪组织中分离出恒河猴MSCs。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MSC有许多共同的特征。实验结果表明，从恒河猴骨髓中分离的间充质干细胞（rBMSCs）和人脂肪组织中分离的间充质干细胞（hASCs）具有比从恒河猴脂肪组织中分离的间充质干细胞（rASCs）和人骨髓间充质干细胞（hBMSCs）更相似的生物学特性。体外生长动力学分析显示rBMSCs和hASCs的倍增时间较短。rBMSCs和hASCs比其他MSCs经历了更多的群体增殖。所有来源的骨髓间充质干细胞在长期培养中端粒酶活性均显著降低，但仍保持其平均端粒长度。所有MSCs均表达胚胎干细胞标志物Oct-4、雷克斯-1和Sox-2，至少传10代。MSC类型的早期群体显示出相似的多谱系分化能力。然而，只有rBMSCs和hASCs在更高的传代次数下保持更高的分化效率。来自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，结果也显示出明显的生物学差异。具体目标2。比较培养的灵长类动物MSC与人类MSC快速扩增并分化为成骨细胞、软骨细胞、脂肪细胞和神经细胞的能力。我们已经发现恒河猴细胞沿着成骨、成软骨和成脂谱系有效地经历分化沿着。人和恒河猴MSC之间的效率几乎没有区别。在神经分化方面，我们已经发现恒河猴AMSC在体外沿着该谱系分化沿着比人或骨髓来源的细胞具有更高的效率。 具体目标3。比较灵长类动物MSC与人MSC在体内在全身或颅内输注到免疫缺陷小鼠后植入多种组织的能力。这些研究目前正在进行中。我们使用立体定向递送将人和恒河猴骨髓和脂肪组织来源的MSC注射到NIH III小鼠的CNS中。我们目前正在评估这些细胞的移植和分化。 具体目标4。当最近购买的microPET仪器开始运行时，首先使用microPET实时成像检测灵长类动物细胞在免疫缺陷小鼠中的植入，然后检测细胞来源的同一灵长类动物。