Platform Session 6. Reproductive Technologies: Recent Advances in Nuclear Transfer, ICSI, Germ Cell Cryopreservation, and Embryonic Development
Sunday, July 24, 2005
3:00 PM–5:00 PM
Location: CCQ 208AB
(42) NORMALITY OF NUCLEAR TRANSFER EMBRYONIC STEM CELL LINES DERIVED FROM ADULT SOMATIC CELLS.
Wakayama, Sayaka1, 2, Senda, Sho3, Hikichi, Takafusa1, Kishigami, Satoshi1, Mizutani, Eiji1, 4, Thuan, Nguyen1, Ohta, Hiroshi1, Miyake, Masashi2, Shiota, Kunio3, Wakayama, Teruhiko1, 1 Riken, Kobe, Japan2 Kobe University, Kobe, Japan3 The University of Tokyo, Tokyo, Japan4 Tohoku University, Sendai, Japan
ABSTRACT- Nuclear transfer technology has recently been used to generate embryonic stem (ntES) cell lines from patient own somatic cells for use in regenerative medicine. We have produced more than 100 ntES cell lines from several mouse strains with a relatively high success rate, and demonstrated to be germ cell in vivo in chimera mice. However, it is still unclear whether ntES cells can be used in medicine, because there are many reports that cloned animals produced by nuclear transfer possess many abnormalities. In this study, we compared ntES cells and ES cells, derived from fertilized embryos. First we analyzed DNA methylation pattern using restriction landmark genomic scanning (RLGS) assay, and we found total 8 differences, between ntES cells and ES cells and even between ntES lines. Second, we examined the karyotypes of most of the established ntES cell lines by Giemsa staining. If there were any abnormalities found, we examined the cell lines in more detail with SKY-FISH staining. As the results indicate, most of the ntES cell lines possessed a normal karyotype in 30-80% of cells. Next, all established cell lines were analyzed for expression of ES cell specific marker, alkaline phosphate (ALP), embryoid formation (EB), SSEA-1, Oct3/4, Nanog, and the negative markers, SSEA-3 and SSEA-4. All ntES cell lines were positive for ES cell specific marker, AP, EB, SSEA-1, Oct3/4 and Nanog, whereas, they were negative for other markers, in a pattern similar to ES cells. Finally, to examine the effects of nuclear transfer, we established second generation ntES cell lines from the nuclei of first generation ntES cells and repeated nuclear transfer over 7 times, which may enhance the effect of nuclear transfer. The success rate of nuclear transfer and establishment of ntES cell lines did not decrease, even in the 8th generation. Although, RLGS profiling showed some difference between ntES and ES cell lines, we could not find any other abnormality in ntES cells. Thus we have concluded that ntES cells, like ES cells, might be useful in regenerative medicine.
KEY WORDS: ntES cell, reprograming