EVALUATION OF THE POTENCY OF RHESUS MONKEY STERM CELLS

本实验的目的是确定胚胎干细胞在恒河猴体内的发育潜力。这种对发育潜力的测量被认为对于在临床上使用胚胎干细胞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)记录猕猴胚胎干细胞在体外嵌合胚胎植入前发育过程中的参与情况。将转β-半乳糖苷酶报告基因的ES细胞注射到二倍体4-8细胞期胚胎中，或将未转染的ES细胞注射到4-8细胞期四倍体胚胎中，将产生嵌合胚胎。通过β-半乳糖苷酶活性或通过荧光原位杂交(FISH)的倍性测定，将在单个细胞中证实ES细胞后代是否参与了体外嵌合胚泡的ICM和滋养外胚层。在实现这一目标后，工作重点将集中在2)确定ES细胞后代对嵌合体恒河猴特定细胞谱系的贡献。将未转基因的ES细胞注射到正常的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、经宫颈的程序转移到同步的代谢物中。胚胎干细胞衍生物在胚胎、新生儿或幼猴体内的组织分布将通过遗传分析、β-半乳糖苷酶活性或FISH倍性测定来确定。这项研究将加强干细胞作为一个模型生物系统，增加关于目前实验室中现有的8个独立的恒河猴ES细胞系的ES细胞发展潜力的具体新信息。

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.