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

候选干细胞种群的体内评估 传统上使用致命的辐照实验或 SCID/NOD-MICE探索可以检查的模型中的替代方案 人类和猴子细胞都开发了胎儿嵌合体模型 头三个月，免疫无能的胎儿胎儿已经 超声引导用人CD34+细胞移植 腹膜内注射出生时和第一次注射 12个月的生命，我们已经证明了人类的持续性嵌合 造血细胞嵌合在骨髓中最高（5-10％ CD34+细胞是外周血中的嵌合体 但是，单核部分之间的变化从0 1-4％，但是大多数 骨髓中的人CD34+细胞的凋亡是凋亡的 与 我们已经确定了假定的CD34-造血的外围血 猕猴中的干细胞种群尽管体外实验表明 这些细胞具有HSC的预期体外特征 （多能力，高度富含LTC-IC），演示恒河猴 CD34- SP细胞包含HSC需要体内实验显示 长期多素造血的其他研究人员利用 表征HSC的恒河猕猴通常检查了 致命辐照猕猴的造血重建 评估SP细胞在替代方面的植入能力 模型，我们创建了男女造血嵌合体10,000恒河 细胞从SPF供体雄性动物中纯化FACS，并 在70天的妊娠期移植到女性接受者 大约60-75天的妊娠，将SP细胞注入 将SP细胞重悬于胎儿腹膜腔中 1x106在100 ml的100毫升中受辐照（10,000个RAD）母体红细胞 使用Seru M游离PBS母体红细胞用作增加的手段 作为SP细胞的数量，tranplant的细胞的密度非常 小的细胞来源无法促成植入 选择以免混淆结果超声引导 腹膜内注射是由Tom Shipp博士进行的 马萨诸塞州总医院的产科医生/妇科医生这是 使用超声指导插入22号脊柱完成 穿过母体腹壁，子宫和胎儿腹部的针头 我们允许母亲携带怀孕直到之前 术语，这时婴儿是由剖宫产分娩的 使用PCR反应特异性进行嵌合的评估 对于y染色体，我们能够在A中实现1-10％的嵌合体 使用这种方法证明 使用A建立混合造血嵌合的可行性 胎儿移植模型，我们进行了混合淋巴细胞反应 反对捐助者和无关的第三方PBMCS四只动物 他使用Y染色体PCR展示了植入，体外表现出 对捐助者PBMC的容忍度清楚地表明了混合 已经实现了Chimerism，并为使用此问题设定了优先权 自体CTL的被动转移模型，此外 数据与鼠模型中所证明的数据一致 表明只有低水平（001％）的造血嵌合体是 为了诱导对供体PBMC的耐受性所必需的这些研究 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 该模型的子集建立应证明对 表征人类造血干细胞的新型种群