EVALUATION OF THE POTENCY OF RHESUS MONKEY STERM CELLS

本实验的目的是确定恒河猴胚胎干（ES）细胞的发育潜力。这种发育潜力的测量被认为对于临床上使用ES细胞来修复或替换受损或患病的组织或器官的长期目标至关重要。具体目标包括：1）记录恒河猴ES细胞参与体外嵌合胚胎植入前发育。通过将用β-半乳糖苷酶报告基因构建体转染的ES细胞注射到二倍体4-8细胞期胚胎中或通过将未转染的ES细胞注射到4-8细胞期四倍体胚胎中来产生嵌合胚胎。将通过β-半乳糖苷酶活性或通过使用荧光原位杂交（FISH）测定倍性，在单个细胞中证实ES细胞后代参与体外嵌合囊胚的ICM和滋养外胚层。在实现这一目标之后，努力将集中在2）确定ES细胞后代对嵌合恒河猴中特定细胞谱系的贡献。通过将未转染的ES细胞注射到正常二倍体胚胎中产生的嵌合体胚胎，以及根据特定目标1产生的嵌合体，将通过非手术的transcerebrovascular程序转移到同步的替代物中。将通过遗传分析、β-半乳糖苷酶活性或倍性FISH测定来确定胚胎、新生或婴儿猴中ES细胞衍生物的组织分布。这项研究将增强干细胞作为一个模型生物系统，增加特定的新信息的胚胎干细胞的发育潜力，从8个独立的恒河猴胚胎干细胞系目前在这个实验室。

The objective of this experimentation is to determine the developmental potential of embryonic stem (ES) cells in the rhesus monkey. Such a measure of developmental potential is considered essential to the long term objective of using ES cells clinically, to repair or replace damaged or diseased tissues or organs. Specific aims include 1) documentation of the participation of rhesus monkey ES cells in the preimplantation development of chimeric embryos in vitro. Chimeric embryos will be produced by injecting ES cells, transfected with a beta-galactosidase reporter gene construct, into diploid 4-8 cell stage embryos or by injecting non-transfected, ES cells into 4-8 cell stage, tetraploid embryos. The participation of ES cell progeny in the ICM and trophectoderm of the resulted chimeric blastocysts in vitro will be confirmed in individual cells by beta-galactosidase activity or by the determination of ploidy using fluorescent In situ hybridization (FISH). Following achievement of this aim, efforts will focus on 2) a determination of the contribution of ES cell progeny to specific cell lineages in chimeric rhesus monkeys. Chimeric embryos produced by injection of nontransfected ES cells into normal diploid embryos, as well as chimeras produced under specific aim 1, will be transferred into synchronized surrogates by a non-surgical, transcervical procedure. The tissue distribution of ES cell derivatives in the resultant fetal, neonatal or infant monkeys will be determined by genetic analysis, beta-galactosidase activity or by FISH determination of ploidy. This research will enhance stem cells as a model biological system adding specific new information on the developmental potential of ES cells from 8 separate rhesus monkey ES cell lines currently available in this laboratory.