277 ESTABLISHMENT OF EMBRYONIC STEM CELL LINES DERIVED FROM A EGFP-TRANSGENIC MOUSE AND THEIR SURVIVAL IN THE COCHLEA OF C57BL/6 MICE

Abstract

Embryonic stem (ES) cells isolated from inner cell mass cells of blastocyst-stage embryos are capable of differentiating into various cell lineages. Transplantation of these cells may potentially be a treatment for many degenerative diseases. Such cell therapy has often been tested using allografts of ES cells in mice. However, it has been difficult to locate transplanted ES cells and to avoid the rejection of allogeneic ES cells by the host. The aims of this study were to establish ES cell lines ubiquitously expressing enhanced green fluorescent protein (EGFP) and to test survival of ES cells in allografts into the cochlea of inbred C57BL/6 mice. Nine hatched blastocysts collected from a C57BL/6-green mouse that ubiquitously expresses transgene EGFP were plated onto an inactivated STO feeder layer. Two putative ES-like colonies were obtained from the plated blastocysts, and repeated subculture of these colonies produced two cell lines expressing EGFP. The cell lines possessed typical characteristics of ES cells, including densely packed colonies of the cells with prominent nucleoli, a high nuclear-cytoplasmic ratio, and high alkaline phosphatase activity. In suspension culture, these cells formed simple and cystic embryoid bodies. Undifferentiated EGFP-transgenic ES cells (10⁶ cells per mouse) were injected into the cochlea of five C57BL/6 mice deafened by gentamycin treatment. Although no behavioral changes were noticed until four weeks after the transplantation, histological study revealed that grafted cells survived in the scala media of all injected mice. Incorporation of the cells expressing EGFP into the host was found along the auditory nerve fibers close to the organ of Corti. Such incorporation was also discovered in the area of the spiral ganglion neurons, cochlear sensory epithelia, and stria vascularis. Morphology and size of the cells varied depending on their sites of incorporation. The results from the present study demonstrate that, due to their survival in transplantation without allogeneic rejection as well as ubiquitous and stable expression of EGFP, ES cells from an EGFP-transgenic mouse may be a useful means of studying cell therapy.