? Embryonic Stem (ES) cells have great potential for treating a wide variety of human diseases if their differentiation can be directed to form functional organs and tissues for transplantation. It must also be shown that transplantation of in vitro produced tissues is safe. Monkey ES cells provide an invaluable model for studying the cell and molecular biology of primate ES cells, and have considerable advantages over human ES cells. ES cell lines are available from in vivo produced embryos in macaques but not in humans; these """"""""in vivo' derived cell lines can provide a database for normal cell functions and characteristics. However, most ES cell lines, in monkeys and in humans, are poorly characterized. Thorough characterization using a battery of cellular and molecular assays is needed to establish which of the available ES cell lines are most suitable for further study, and to identify additional markers of normal ES cells. We will examine and compare several existing macaque ES cell lines, obtained from both in vivo and in vitro produced embryos, for a range of cellular and molecular characteristics including mitochondrial properties, karyotype and gene expression to determine: (I) what are the characteristics of normal ES cells? (ii) How do ES cells of in vitro vs. in vivo derived embryos differ? (iii) Which characteristics of ES cells are correlated with their ability to differentiate into other cell types? (iv) How do these characteristics change during repeated passages, i.e., how stable are the various cell lines? Novel aspects of the study include evaluation of BAC-FISH probes for macaque chromosomes based on human DNA sequences, use of macaque gene micro array chips, quantitative assessment of mitochondrial properties, and direct comparison of ES cell lines generated from in vivo and in vitro produced embryos. These studies will involve collaborations with four NCRR-supported resource centers. The data obtained will help us (I) to determine which genes are important for maintaining pluripotency and the undifferentiated state; (ii) to decide which ES cell lines to use for future, in depth tissue derivation and culture media studies; (iii) to establish a panel of cellular and molecular markers of normal ES cells that can be applied to other macaque and possibly human ES cell lines. ? ?
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