Korean J Fertil Steril.  2002 Jun;29(2):129-138.

Parthenogenetic Mouse Embryonic Stem Cells have Similar Characteristics to In Vitro Fertilization mES Cells

• Park SP
• Kim EY
• Lee KS
• Lee YJ
• Shin HA
• Min HJ
• Lee HT
• Chung KS
• Lim JH

Abstract

OBJECTIVE
This study was to compare the characteristics between parthenogenetic mES (P-mES) cells and in vitro fertilization mES cells. MATERIAL AND METHODS: Mouse oocytes were recovered from superovulated 4 wks hybrid F1 (C57BL/6xCBA/N) female mice. For parthenogenetic activation, oocytes were treated with 7% ethanol for 5 min and 5microgram/ml cytochalasin-B for 4 h. For IVF, oocytes were inseminated with epididymal perm of hybrid F1 male mice(1x106/ml). IVF and parthenogenetic embryos were cultured in M16 medium for 4 days. Cell number count of blastocysts in those two groups was taken by differential labelling using propidium iodide (red) and bisbenzimide (blue). To establish ES cells, blastocysts in IVF and parthenogenetic groups were treated by immunosurgery and recovered inner cell mass (ICM) cells were cultured in LIF added ES culture medium. To identity ES cells, the surface markers alkaline phosphatase, SSEA-1, 3, 4 and Oct4 staining were examined in replated ICM colonies. Chromosome numbers in P-mES and mES were checked. Also, in vitro differentiation potential of P-mES and mES was examined.
RESULTS
Although the cleavage rate (> or =2-cell) was not different between IVF (76.3%) and parthenogenetic group (67.0%), in vitro development rate was significantly low in parthenogenetic group (24.0%) than IVF group (68.4%) (p<0.05). Cell number count of ICM and total cell in parthenogenetic blastocysts (9.6+/-3.1, 35.1+/-5.2) were significantly lower than those of IVF blastocysts (19.5+/-4.7, 63.2+/-13.0) (p<0.05). Through the serial treatment procedure such as immunosurgery, plating of ICM and colony formation, two ICM colonies in IVF group (mES, 10.0%) and three ICM colonies (P-mES, 42.9%) in parthenogenetic group were able to culture for extended duration (25 and 20 passages, respectively). Using surface markers, alkaline phosphatase, SSEA-1 and Oct4 in P-mES and mES colony were positively stained. The number of chromosome was normal in ES colony from two groups. Also, in vitro neural and cardiac differentiation derived from mES or P-mES cells was confirmed.
CONCLUSION
This study suggested that P-mES cells can be successfully established and that those cell lines have similar characteristics to mES cells.

MeSH Terms

Alkaline Phosphatase
Animals
Antigens, CD15
Bisbenzimidazole
Blastocyst
Cell Count
Cell Line
Embryonic Stem Cells*
Embryonic Structures
Ethanol
Female
Fertilization in Vitro*
Humans
Male
Mice*
Oocytes
Propidium
Alkaline Phosphatase
Antigens, CD15
Bisbenzimidazole
Ethanol
Propidium