BIOLOGY OF NON-HUMAN PRIMATE MARROW STROMAL CELLS

这个子项目是许多研究子项目中的一个 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得了主要资金， 因此可以在其他CRISP条目中表示。所列机构为 研究中心，而研究中心不一定是研究者所在的机构。 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。它们也是令人感兴趣的，因为它们可以找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，在非人灵长类动物中进行的细胞的广泛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。人ASC（hASC）和恒河猴BMSC（rBMSC）经历比人BMSC（hBMSC）和恒河猴ASC（rASC）显著更多的总群体增殖。所有MSC的细胞周期谱随着培养物在体外老化而改变。在P20和P30，hMSCs经历了S期细胞频率的增加。然而，来自两种来源的恒河猴MSC在P20时形成了不同的多倍体细胞群，其在P30时发展为非整倍体。骨髓间充质干细胞的核型分析显示，在恒河猴细胞在P20或P30的四倍体或非整倍体核型的发展。对早期和晚期传代的MSC的转录组的分析揭示了基因表达模式的显著改变（8.8%的基因在hBMSC与hASC中差异表达; 5.5%在rBMSC与rASC中差异表达）。随着衰老的发生，相同细胞类型中的基因表达变化要小得多（hMSCs中为0.7%，rMSCs中为0.9%）。基因本体分析显示，rASCs中蛋白质催化和pol II转录调控功能占优势，而hBMSCs中I B/NF-B级联调控功能占优势。在rASCs和hBMSCs中差异表达的基因的功能分析显示，参与细胞周期，细胞周期检查点，蛋白质泛素化和细胞凋亡的途径被改变。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ASC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。在体内比较灵长类动物MSC与人类MSC在全身或颅内输注到免疫缺陷小鼠后移植到多种组织中的能力。这些研究目前正在进行中。我们使用立体定位递送将人和恒河猴骨髓和脂肪组织来源的MSC注射到NIHIII和Twitch（受Krabbe影响的）小鼠的CNS中。我们目前正在评估这些细胞在CNS中的植入和分化。来自免疫缺陷小鼠的数据表明，细胞移植，持续长达180天，并沿着沿着神经谱系进行中度分化。目前正在收集关于Twitch小鼠研究的数据。

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. Human ASCs (hASCs) and rhesus BMSCs (rBMSCs) undergo significantly more total population doublings than human BMSCs (hBMSCs) and rhesus ASCs (rASCs). The cell cycle profile of all MSCs is altered as cultures aged in vitro. hMSCs underwent an increase in the frequency of cells in the S phase at P20 and P30. However, rhesus MSCs from both sources developed a distinct polyploid population of cells at P20, which progressed to aneuploidy by P30. Karyotype analysis of MSCs revealed the development of tetraploid or aneuploid karyotypes in the rhesus cells at P20 or P30. Analysis of the transcriptome of the MSCs from early and late passages revealed significant alterations in the patterns of gene expression (8.8% of the genes were differentially expressed in hBMSCs vs. hASCs; and 5.5% in rBMSCs vs. rASCs). Gene expression changes were much less evident within the same cell type as aging occurred (0.7% in hMSCs and 0.9% in rMSCs). Gene ontology analysis showed that functions involved in protein catabolism and regulation of pol II transcription were over-represented in rASCs, while the regulation of I¿B/NF-¿B cascade were over-represented in hBMSCs. Functional analysis of genes that were differentially expressed in rASCs and hBMSCs revealed that pathways involved in cell-cycle, cell cycle checkpoints, protein-ubiquitination, and apoptosis were altered. 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 AND Twitcher (Krabbe-affected) mice, using stereotaxic delivery. We are currently assessing engraftment and differentiation of these cells in the 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. Data on the Twitcher mouse studies is currently being collected.