The goal of this proposed project is to establish the infrastructure to develop, characterize, and share useful human embryonic stem cell lines from the group of derivations designated CY12, CY30, CY40, CY51, CY81, CY82, CY91, CY92, and CY10 on the NIH Human Embryonic Stem Cell Registry. These cells meet the federal criteria for funding. The cells are at a very early stage of development. They were frozen at early passage, and exist now as frozen specimens; there is one vial of CY12 and of CY92, but there are at least two, and as many as 6 frozen vials of the others. The cells have been passaged up to 5 times, and have given rise to alkaline phosphatase-positive colonies. Since the cells are part of a limited resource, the first aim is a cautious approach to developing conditions that allow survival of the cells. The other 3 aims are to characterize the cells and provide them to researchers. Specific Aim 1: Establish culture conditions for recovery, expansion, and production of clonal lines of early stage human derivations. Using established hES cell lines, we will evaluate and improve if necessary the co-culture methods used for the initial derivation of the cells. Specific Aim 2: Establish cell lines from early stage human derivations and characterize them by standard criteria. We will use the methods developed in Aim l to recover cells from the derivations, and use the standard criteria for identifying the cells as ES cells. Specific Aim 3: Genotype and phenotype the hES cell lines. We will use microsatellite genotyping and a simple SNP screen to obtain genetic information about the cells. We will phenotype the cells by limited microarray analysis similar to that done for mouse ES cells, and confirm and extend the microarray results with quantitative RTPCR. Specific Aim 4: Expand hES cell lines and provide them to other laboratories. We plan to distribute the cells to other researchers and share our expertise in order to help in establishing uniform standards.
Thesaurus Terms: cell proliferation, growth media, human embryonic stem cell line, tissue /cell culture genotype, phenotype SCID mouse, biotechnology, immunocytochemistry, microarray technology, polymerase chain reaction, single nucleotide polymorphism
