A PRIMATE MODEL FOR LIVER REGENERATION BY MARROW-DERIVED STEM CELLS

利用骨髓干细胞进行肝脏再生的灵长类动物模型

• 批准号: 7716202
• 负责人: VINCENT FRANCESCO LARUSSA
• 金额: $ 2.36万
• 依托单位: TULANE UNIVERSITY OF LOUISIANA
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-07-21 至 2009-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7716202
• 关键词: Allogenic; Antibodies; Antigen-Presenting Cells; Autologous; Autologous Transplantation; Base Pairing; Benchmarking; Blast Cell; Bone Marrow; Bone Marrow Stem Cell; C-KIT Gene; CD34 gene; CSF3 gene; Cell Count; Cell Separation; Cells; Computer Retrieval of Information on Scientific Projects Database; Condition; Cryopreservation; Data; Dyes; Engineering; Engraftment; Female; Fluorescence; Funding; Genes; Goals; Graft Rejection; Grant; Harvest; Immunocompromised Host; Immunosuppressive Agents; Individual; Institution; Kidney; Label; Liver; Liver Regeneration; Lung; Macaca mulatta; Marrow; Modeling; Monkeys; Mononuclear; Morbidity - disease rate; Natural regeneration; Organ; Paraffin Embedding; Partial Hepatectomy; Pilot Projects; Plasmids; Pluripotent Stem Cells; Polymerase Chain Reaction; Population; Primates; Proto-Oncogene Protein c-kit; Protocols documentation; Publishing; Radiation; Reaction; Research; Research Personnel; Resources; Reverse Transcriptase Polymerase Chain Reaction; Risk; Running; Sampling; Source; Standards of Weights and Measures; Stem cell transplant; Stem cells; System; T-Lymphocyte; Testing; Time; Tissues; Transplantation; Treatment Protocols; United States National Institutes of Health; Y Chromosome; animal care; detector; graft vs host disease; human SOX1 protein; human SOX11 protein; human SOX12 protein; human SOX13 protein; human SOX14 protein; human SOX15 protein; human SOX17 protein; human SOX18 protein; human SOX21 protein; human SOX4 protein; human SOX5 protein; human SOX6 protein; human SOX7 protein; human SOX8 protein; male; mortality; mouse Sox5 protein; mouse Sox7 protein; prevent; purge; sex

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 goal of this project is to investigate whether stem cells from bone marrow can regenerate liver tissue. We have made good progress against our year 3  5 benchmarks in 2005 as described below. Benchmark 1. Stem Cell Isolation and T-cell Purging Our stem cell transplantation studies are at very high risk for graft versus host disease, GVHD, and graft rejection. To prevent GVHD and/or rejection of the stem cell graft by the female recipients, we have now further modified the engineering of the stem cell grafts by an additional purging step in order to eliminate the contaminating immuno-competent T cells and other antigen presenting cells to engineer a stem cell graft consisting of the most primitive or morphologically indistinguishable stem cell populations or "blast" cells as this cell population is believed to represent the most rare/pluripotent stem cells capable of multiorgan engraftment. Benchmark 2: Real Time Quantitative PCR The 272 base pair portion of the macaca mulatta sex determing region Y(SRY) gene was cloned into a p CR II-TOPO plasmid. The plasmid insert was confirmed by sequencing and PCR was performed using the primer set that will be used for the RT-PCR. This plasmid will be used to create the RT-PCR standard curve, allowing the determination of the absolute SRY positive cells number (male cells) in a given cell sample(male and female cells mixture). We are going to label the primer and probe with FAM/TAMRA dye and run the samples on Applied Biosystems 7700 System Detector. The data generated from this study has been demonstrated to effectively detect Y chromosome containing cells and has been published. Benchmark 3: Combined FISH for the "Y" chromosome SRY probe with CD34 and other tissue markers for liver, lung and kidney Our goal is to establish a stem cell transplantation model for liver regeneration and multi-organ engraftment. We have used the "Y" chromosome as a marker for "stem cell tracking" and determining the engraftment of "Y+" stem cells in various tissues and organs of female recipients. We are using combined fluorescence immuno-hybridization, FISH. Last year we were only successful in detecting the stem cell factor receptor, CD117(ckit), in liver only. Over the past year, we have confirmed and expanded on last years studies in liver sections by optimized the reaction conditions and further testing different clones of CD34 antibodies in combination with, CD117 and CK8/18 on other rhesus monkey paraffin embedded tissues including lungs, kidney, and gut. Benchmark 4: Pilot Study: large scale GCSF and SCF primed bone marrow harvests, PKH-fluorescence labeling, and cryopreservation of PKH labeled mononuclear cells for autologous transplantation. Due to the high risk of mortality and morbidity associated with unrelated mismatched allogeneic stem cell transplantation originally proposed, the Institutional Animal Care Users Committee (IACUC) at the TNPRC, has recommended and approved a "Pilot" Study utilizing autologous stem cells in combination with all of the entire transplant protocols including radiation, immunosuppressive regimens, etc and partial hepatectomy proposed in the original main study utilizing unrelated allogeneic stem cells. The results from the Pilot study will allow us to safely manage immunocompromised monkeys that will be transplanted with mismatched allogeneic stem cells. To this end, this past year, we have performed individual autologous stem cell harvests primed with GCSF and SCF. The stem cells have been labeled and cryopreserved and ready for transplant.

该子项目是利用该技术的众多研究子项目之一 资源由 NIH/NCRR 资助的中心拨款提供。子项目和 研究者 (PI) 可能已从 NIH 的另一个来源获得主要资金， 因此可以在其他 CRISP 条目中表示。列出的机构是 对于中心来说，它不一定是研究者的机构。 该项目的目标是研究骨髓干细胞是否可以再生肝组织。 如下所述，与 2005 年第 3-5 年基准相比，我们取得了良好进展。 基准 1. 干细胞分离和 T 细胞清除 我们的干细胞移植研究存在移植物抗宿主病、GVHD 和移植物排斥的极高风险。为了防止 GVHD 和/或女性受者对干细胞移植物的排斥，我们现在通过额外的净化步骤进一步修改了干细胞移植物的工程，以消除污染的免疫活性 T 细胞和其他抗原呈递细胞，以工程化由最原始或形态上难以区分的干细胞群体或“母细胞”组成的干细胞移植物，因为该细胞群体被认为代表了最原始的或形态上无法区分的干细胞群体或“母细胞”。 能够进行多器官移植的稀有/多能干细胞。 基准2：实时定量PCR 将猕猴性别决定区Y(SRY)基因的272个碱基对部分克隆到p CR II-TOPO质粒中。通过测序确认质粒插入，并使用将用于 RT-PCR 的引物组进行 PCR。该质粒将用于创建 RT-PCR 标准曲线，从而确定给定细胞样本（雄性和雌性细胞混合物）中 SRY 阳性细胞的绝对数量（雄性细胞）。 我们将用 FAM/TAMRA 染料标记引物和探针，并在 Applied Biosystems 7700 系统检测器上运行样品。这项研究产生的数据已被证明可以有效检测含有 Y 染色体的细胞，并已发表。 基准3：“Y”染色体SRY探针与CD34和其他肝、肺、肾组织标志物的组合FISH 我们的目标是建立肝再生和多器官移植的干细胞移植模型。我们使用“Y”染色体作为“干细胞追踪”的标记，并确定“Y+”干细胞在女性受者的各种组织和器官中的植入情况。 我们正在使用联合荧光免疫杂交，FISH。 去年我们仅在肝脏中成功检测到干细胞因子受体 CD117(ckit)。在过去的一年里，我们通过优化反应条件并在其他恒河猴石蜡包埋组织（包括肺、肾和肠道）上进一步测试 CD34 抗体的不同克隆与 CD117 和 CK8/18 的组合，确认并扩展了去年肝切片研究的结果。 基准 4：试点研究：大规模 GCSF 和 SCF 引发的骨髓收获、PKH 荧光标记以及 PKH 标记单核细胞的冷冻保存用于自体移植。 由于最初提出的不相关的不匹配同种异体干细胞移植相关的死亡率和发病率较高，TNPRC 机构动物护理用户委员会 (IACUC) 建议并批准了一项“试点”研究，利用自体干细胞结合所有整个移植方案，包括放射、免疫抑制方案等以及最初提出的部分肝切除术。 利用不相关的同种异体干细胞的研究。试点研究的结果将使我们能够安全地管理免疫功能低下的猴子，这些猴子将被移植不匹配的同种异体干细胞。 为此，去年，我们进行了用 GCSF 和 SCF 引发的个体自体干细胞收获。 干细胞已被标记并冷冻保存，准备移植。