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

候选干细胞群的体内评估 传统上利用致命辐射实验或 SCID/NOD-小鼠 探索可以检查的模型中的替代方案 人类和猴子细胞，我们开发了胎儿嵌合体模型 妊娠早期，免疫功能低下的恒河猴胎儿已被 通过超声引导移植人类 CD34+ 细胞 腹腔注射 在出生时和第一次出生时进行评估 12个月的生命中，我们展示了人类持久的嵌合状态 造血细胞嵌合现象在骨髓中最高（5-10% CD34+细胞具有人类表型）外周血中的嵌合现象 单核分数在 0 1-4% 之间变化，但是，大多数 骨髓中的人类 CD34+ 细胞凋亡 这可以解释 与骨髓移植相比，骨髓移植水平不一致 外周血中我们已经鉴定出一种假定的 CD34- 造血细胞 猕猴的干细胞群 尽管体外实验表明 这些细胞具有预期的 HSC 体外特征 （多能性，高度富集 LTC-IC），证明恒河猴 CD34-SP细胞含有HSC需要体内实验证明 长期多系造血 其他研究人员利用 恒河猴表征 HSC 通常会进行检查 受致命辐射的猕猴的造血重建 评估 SP 细胞在替代方案中的植入能力 模型中，我们创建了雄性-雌性造血嵌合体 10,000 只恒河猴 SP 细胞是从 SPF 供体雄性动物中通过 FACS 纯化的，并且 移植到妊娠 70 天的女性接受者体内 大约在妊娠 60 - 75 天时，注射 SP 细胞 将SP细胞重悬于胎儿腹膜腔 1x106 经辐照（10,000 拉德）母体红细胞，溶于 100 毫升 使用无血清 PBS 母体红细胞作为增加 由于SP细胞的数量非常多，所以移植细胞的密度 小 无法促进植入的细胞来源是 选择以免混淆结果 超声引导 腹腔注射是由 Tom Shipp 博士进行的，他是一名 马萨诸塞州总医院的妇产科医生 这是 使用超声波引导插入 22 号脊柱完成 针穿过孕妇腹壁、子宫和胎儿腹部 wall 我们允许母亲怀孕直到临产前 足月，此时婴儿通过剖腹产分娩 使用特定的 PCR 反应进行嵌合状态的评估 对于 Y 染色体，我们已经能够实现 1-10% 的嵌合 使用这种方法的恒河猴模型来演示 使用a建立混合造血嵌合体的可行性 胎儿移植模型，我们进行混合淋巴细胞反应 针对捐赠者和不相关的第三方 PBMCs 四只动物 他们使用 Y 染色体 PCR 证明了植入，并在体外展示 对供体 PBMC 的耐受性 这清楚地表明，混合 嵌合现象已经实现，并为使用该技术奠定了先例 自体 CTL 被动转移模型 此外， 数据与小鼠模型中所证实的一致 表明只有低水平 (0 01%) 的造血嵌合 为了诱导对供体 PBMC 的耐受性，这些研究是必需的 继续尝试优化模型并确定体内 各种造血祖细胞的植入特征 该模型的建立应该被证明是有用的 表征人类造血干细胞的新群体