BIOLOGY OF NON-HUMAN PRIMATE MARROW STROMAL CELLS

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 该项目的总体目标是开发程序，使来自骨髓基质的成年干细胞可以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(MSCs)，对细胞和基因治疗感兴趣，因为它们可以很容易地从患者那里获得，在培养中扩增，使用或不使用病毒进行基因工程，然后返回治疗同一患者。它们也是令人感兴趣的，因为它们是受损组织的家园，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，几项有希望的临床试验已经启动，用于治疗儿童罕见疾病。然而，在非人类灵长类动物中进行广泛的细胞试验，显然对于目前提出的一些用于常见疾病的应用是必不可少的，例如骨质疏松症、心力衰竭、帕金森氏症、脑白质营养不良和阿尔茨海默病。该提案的目标是： 具体目的1.用我们实验室最近开发的分离和扩增人MSCs的改良方法分离和扩增灵长类MSCs。我们已经成功地从恒河猴的骨髓和脂肪组织中分离出骨髓间充质干细胞。我们对干细胞群体的体外生物学特性进行了广泛的表征。我们的数据表明，来自这两个组织的MSCs具有许多共同的特征。结果表明，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表明，rBMSCs和hASCs的倍增时间较短。RBMSCs和hASCs的群体倍增显著高于其他MSCs。所有来源的MSCs在延长培养时间后端粒酶活性显着降低，但它们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、Rex-1和Sox-2，传代至少10代。早期群体的MSCs类型表现出相似的多系分化能力。然而，只有rBMSCs和hASCs在高传代时保持了较高的分化效率。总体而言，来自这两个物种和组织来源的MSCs的体外特征显示出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。 具体目的2.比较培养中的灵长类MSCs和人类MSCs的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可以有效地沿着成骨、成软骨和成脂的方向分化。人类和恒河猴骨髓间充质干细胞的效率几乎没有区别。在神经分化方面，我们发现恒河猴ASCs在体外沿着这一谱系分化的效率高于人类或骨髓来源的细胞。 具体目的3.比较灵长类MSCs和体内人MSCs在全身或颅内注射给免疫缺陷小鼠后植入多组织的能力。这些研究目前正在进行中。我们已经将人和恒河猴骨髓和脂肪组织来源的MSCs注射到NIHIII小鼠的中枢神经系统，使用立体定向输送。我们目前正在评估这些细胞在免疫缺陷小鼠和非人灵长类中枢神经系统中的植入和分化。来自免疫缺陷小鼠的数据表明，细胞植入后可持续180天，并沿神经谱系经历适度分化。

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 ASCs 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 in immune deficient mice and nonhuman primate CNS. Data from the immune deficient mice indicate the cells engraft, persist for as long as 180 days and undergo moderate differentiation along neural lineages.