ESTABLISHMENT OF RHESUS BONE MARROW CHIMERAS

恒河猴骨髓嵌合体的建立

• 批准号: 6591322
• 负责人: M ROSENZWEIG
• 金额: $ 11.11万
• 依托单位: HARVARD MEDICAL SCHOOL
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-05-01 至 2003-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6591322
• 关键词: Mammalia; animal tissue; gene therapy; hematology; immunology; inflammation

In vivo evaluation of candidate stem cell populations has traditionally utilized either lethal irradiation experiments or SCID/NOD-mice To explore alternatives in a model that could examine both human and monkey cells, we have developed a fetal chimera model First trimester, immune incompetent rhesus fetuses have been transplanted with human CD34+ cells by ultrasound guided intraperitoneal injection On evaluation at birth and during the first 12 months of life, we have demonstrated persistent chimerism of human hematopoietic cells Chimerism is highest in the bone marrow (5-10% of CD34+ cells are of human phenotype) Chimerism in the peripheral blood varies between mononuclear fractions from 0 1-4% However, the majority of human CD34+ cells in the bone marrow are apoptotic This may explain the discordant level of engraftment in the bone marrow as compared to the peripheral blood We have identified a putative CD34- hematopoietic stem cell population in macaques Although in vitro experiments suggest that these cells have the expected in vitro characteristics of HSC (multipotency, highly enriched for LTC-IC), demonstration that rhesus CD34- SP cells contain HSC requires in vivo experiments showing long-term multilineage hematopoiesis Other investigators utilizing rhesus macaques to characterize HSC have typically examined hematopoietic reconstitution of lethally irradiated macaques In order to assess the engraftment capacity of the SP cells in an alternative model, we created male-female hematopoietic chimeras 10,000 rhesus SP cells were FACS purified from an SPF donor male animal and transplanted into a female recipient at 70 days of gestation At approximately 60 - 75 days of gestation, the SP cells were injected into the fetal peritoneal cavity The SP cells were resuspended in 1x106 irradiated (10,000 Rads) maternal red blood cells in 100 ml of seru m free PBS Maternal red cells were used as a means to increase the density of tranplanted cells as the number of SP cells was very small A source of cells that could not contribute to engraftment was selected so as not to confuse the results Ultrasound-guided intraperitoneal injections were carried out by Dr Tom Shipp, an obstetrician/gynecologist at Massachusetts General Hospital This was accomplished using ultrasound guidance to insert a 22 gauge spinal needle through the maternal abdominal wall, uterus and fetal abdominal wall We allowed the mother to carry the pregnancy until just prior to term, at which point the baby was delivered by cesarean section The evaluation of chimerism was performed using a PCR reaction specific for Y chromosome We have been able to achieve 1-10% chimerism in a rhesus-rhesus macaque model using this approach To demonstrate the feasibility of establishing mixed hematopoietic chimerism using a fetal transplant model, we performed mixed lym phocyte reactions against donor and an unrelated third party PBMCs Four of the animals who demonstrated engraftment using Y chromosome PCR, exhibit in vitro tolerance to donor PBMCs This demonstrates clearly that mixed chimerism has been achieved and set the precedence for using this model for the passive transfer of autologous CTL Furthermore, this data is consistent with what has been demonstrated in the murine model in showing that only a low level (0 01%) of hematopoietic chimerism is required in order to induce tolerance to donor PBMCs These studies continue in an attempt to optimise the model and determine the in vivo engraftment characteristics of various hematopoietic progenitor subsets Establishment of this model should prove useful in characterizing novel populations of human hematopoietic stem cells

对候选干细胞群体的体内评估 传统上要么使用致命的辐射实验，要么使用 SCID/NOD-小鼠在模型中探索替代方案 人类和猴子的细胞，我们已经开发出一种胎儿嵌合体模型 怀孕早期，免疫功能不全的恒河猴胎儿 超声引导下人CD34+细胞移植 出生时和第一次分娩时的腹膜内注射 12个月的生命，我们展示了人类持久的嵌合体 造血细胞在骨髓中的嵌合率最高(5%-10% CD34+细胞具有人类表型)外周血中的嵌合体 单核组分之间的差异从0.1-4%不等，然而，大多数 的人骨髓中的CD34+细胞是凋亡的，这可能解释了 骨髓中植入的不协调水平与 外周血我们已经鉴定出一种可能的CD34-造血因子 猕猴体内的干细胞群，尽管体外实验表明 这些细胞具有预期的HSC的体外特性 (多能性，高度浓缩的LTC-IC)，证明恒河猴 含有HSC的CD34-SP细胞需要体内实验证明 其他研究人员利用长期多系造血 恒河猴用来表征HSC的典型检查 致死照射猕猴的有序造血重建 以另一种方法评估SP细胞的植入能力 模型，我们创造了雄性和雌性造血嵌合体10,000只恒河猴SP 细胞是从SPF供体雄性动物中纯化的FACS和 在怀孕70天时移植到女性受者体内 大约在怀孕60-75天时，注射SP细胞 将SP细胞重新悬浮到胎儿的腹膜腔内 100毫升中1×106辐照(10,000拉兹)的母体红细胞 用血清游离PBS母体红细胞作为增加 移植细胞的密度随着SP细胞数量的增加而增加 小一个不能促进植入的细胞来源是 选择时不会混淆超声引导的结果 腹膜内注射由Tom Shipp博士进行，他是一名 马萨诸塞州综合医院的产科医生/妇科医生 使用超声引导插入22号脊柱 针刺孕妇腹壁、子宫和胎儿腹部 WALL我们允许母亲怀孕，直到 足月，此时婴儿是通过剖腹产出生的 嵌合体的评估使用了一种特定的聚合酶链式反应 对于Y染色体，我们已经能够在 用这种方法建立恒河猴-恒河猴模型 建立混合造血细胞嵌合体的可行性 胎儿移植模型，我们进行了混合淋巴细胞反应 针对捐赠者和无关第三方的PBMC中的四只动物 世卫组织使用Y染色体聚合酶链式反应证实了植入，在体外展示 对供者外周血单核细胞的耐受性清楚地表明混合 嵌合体已经实现，并为使用这一技术设定了优先顺序 自体CTL被动移植模型的研究 数据与在小鼠模型中所证明的一致 在显示只有低水平(001%)的造血细胞嵌合体是 这些研究需要诱导对供者外周血单核细胞的耐受性 继续尝试优化模型并确定体内的 不同造血祖细胞的植入特性 这个模型的子集的建立应该被证明是有用的 人类造血干细胞新群体的特征