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: 7562264
• 负责人: VINCENT FRANCESCO LARUSSA
• 金额: $ 3.02万
• 依托单位: TULANE UNIVERSITY OF LOUISIANA
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-05-01 至 2008-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7562264
• 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; 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; 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 (developed from January 06'  July 06'). 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. 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 are using 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. This year, we have confirmed and expanded 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, in the spring of 05', 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 in year 5 (06'). To this end, this past year, we have obtained 2 monkeys and performed 2 individual autologous stem cell harvests primed with GCSF and SCF. The stem cells have been labeled and cryopreserved and ready for transplant.

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。 该项目的目的是研究来自骨髓的干细胞是否可以再生肝组织。 我们已经取得了良好的进展，对我们的第三年  2005年的5项基准如下所述。 基准1.干细胞分离和T细胞净化 我们的干细胞移植研究是在非常高的风险，移植物抗宿主病，移植物抗宿主病，移植物排斥反应。 为了防止GVHD和/或女性受体对干细胞移植物的排斥，我们现在已经通过另外的净化步骤进一步改进了干细胞移植物的工程化，以消除污染的免疫活性T细胞和其它抗原呈递细胞，从而工程化由最原始的或形态学上不可区分的干细胞群体或“原始细胞”组成的干细胞移植物。细胞，因为这种细胞群被认为代表了能够多器官移植的最稀有/多能干细胞（从2006年1月开发的）。  06年7月）。 基准2：真实的 时间定量PCR 将猕猴性别决定区Y（SRY）基因的272 bp片段克隆到pCR Ⅱ-TOPO质粒中。通过测序确认质粒插入物，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，以确定给定细胞样品（雄性和雌性细胞混合物）中的绝对SRY阳性细胞数（雄性细胞）。 我们将用FAM/塔姆拉染料标记引物和探针，并在Applied Biosystems 7700系统检测器上运行样品。 基准3：“Y”染色体SRY探针与CD 34和肝、肺和肾的其他组织标志物的组合FISH 我们的目标是建立一个肝再生和多器官移植的干细胞移植模型。 我们正在使用“Y”染色体作为“干细胞跟踪”的标记，并确定“Y+”干细胞在女性受体的各种组织和器官中的植入。 我们使用联合荧光免疫杂交技术，FISH。 去年，我们仅在肝脏中成功检测了干细胞因子受体CD 117（ckit）。 今年，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CD 34抗体与CD 117和CK 8/18组合的不同克隆，证实并扩展了去年在肝脏切片中的研究。 基准4：初步研究：大规模GCSF和SCF致敏的骨髓收获物、PKH荧光标记和PKH标记的单核细胞的冷冻保存用于自体移植由于最初在2005年春天提出的与无关的错配异基因干细胞移植相关的高死亡率和发病率风险，TNPRC的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的整个移植方案，包括放射，免疫抑制方案等，以及在最初的主要研究中提出的利用无关的同种异体干细胞的部分肝切除术。 初步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，这些猴子将在第5年（06 '）移植错配的异基因干细胞。 为此，在过去的一年里，我们获得了2只猴子，并进行了2次用GCSF和SCF引发的自体干细胞收获。 干细胞已经被标记并冷冻保存，准备移植。