ESTABLISHMENT OF RHESUS BONE MARROW CHIMERAS

候选干细胞群的体内评价具有 传统上利用致死辐射实验， SCID/NOD小鼠为了在一个可以检查 人类和猴子的细胞，我们已经开发了一个胎儿嵌合体模型， 在怀孕的头三个月，免疫缺陷的恒河猴胎儿 超声引导下移植人CD 34+细胞 腹腔注射在出生时和第一次评估期间 12个月的生命，我们已经证明了人类的持续嵌合体 造血细胞嵌合体在骨髓中最高（造血细胞的5-10%）。 CD 34+细胞具有人类表型）外周血中的嵌合体 单核级分之间的变化为0.1 -4%。 骨髓中的人CD 34+细胞是凋亡的，这可能解释了 骨髓中的植入水平不一致， 外周血我们已经确定了一个假定的CD 34造血 尽管体外实验表明， 这些细胞具有HSC的预期体外特征， （多能性，LTC-IC高度富集），证明恒河猴 含有HSC的CD 34- SP细胞需要体内实验来证明 长期多系造血其他研究者使用 恒河猴表征HSC通常检查 造血重建的致死辐射猕猴 为了评估SP细胞在替代方案中的植入能力， 模型，我们创建了10，000只恒河猴SP的雄性-雌性造血嵌合体 从SPF供体雄性动物中FACS纯化细胞， 在妊娠70天时移植到女性受体中。 在妊娠约60 - 75天时， 将SP细胞重悬于 100 ml中的1x 106个辐照（10，000 Rads）母体红细胞 使用无血清PBS母体红细胞作为增加 移植的细胞密度与SP细胞的数量非常接近， 小不能有助于植入的细胞来源是 选择，以免混淆结果超声引导 腹膜内注射由Tom Shipp博士进行， 马萨诸塞州总医院的妇产科医生 使用超声引导插入22号脊柱 针穿过母体腹壁、子宫和胎儿腹部 我们允许母亲怀孕，直到 足月，在这一点上，婴儿是通过剖宫产分娩的。 使用特异性PCR反应进行嵌合性评价。 对于Y染色体，我们已经能够实现1-10%的嵌合体， 恒河猴-恒河猴模型使用这种方法 用免疫荧光法建立混合造血嵌合体的可行性 胎儿移植模型，我们进行了混合淋巴细胞反应， 针对供体和无关的第三方PBMC 使用Y染色体PCR证明植入的患者， 对供体PBMC的耐受性这清楚地表明， 嵌合体已经实现，并为使用这种方法设定了优先顺序。 自体CTL被动转移的模型此外， 数据与在鼠模型中所证实的一致 在显示只有低水平（0.01%）的造血嵌合体， 为了诱导对供体PBMC的耐受性，这些研究 继续尝试优化模型并确定体内 不同造血祖细胞植入特性 该模型的建立将证明在以下方面是有用的： 表征新的人造血干细胞群体

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