• Asian-Australasian Journal of Animal Sciences
• /
• v.24 no.1
• /
• pp.37-44
• /
• 2011

Parthenogenetic embryonic stem (pES) cells could provide a valuable model for research into genomic imprinting and X-linked diseases. In this study, pES cell lines were established from oocytes of hybrid offspring of Kunming and 129/Sv mice, and pluripotency of pES cells was evaluated. The pES cells maintained in the undifferentiated state for more than 50 passages had normal karyotypes with XX sex chromosomes and exhibited high activities of alkaline phosphatase (AKP) and telomerase. Meanwhile, these cells expressed ES cell molecular markers SSEA-1, Oct-4, Nanog, and GDF3 but not SSEA-3 detected by immunohistochemistry and RT-PCR. The pES cells could be differentiated into various types of cells from three germ layers in vitro by analysis of embryoid bodies (EBs) with immunohistochemistry and RT-PCR, and in vivo by observation of pES cell-derived teratoma sections. Therefore, the established pES cell lines contained all features of mouse ES cells. This work provides a new strategy for isolating pES cells from Kunming mice, and the pES cell lines could be applied as the cell model in research into genomic imprinting and epigenetic regulation of Kunming mice.

• Proceedings of the KSAR Conference
• /
• 2002.06a
• /
• pp.83-83
• /
• 2002

This study was to compare the characteristics of parthenogenetic mES (P-mES) cells and in vitro fertilization mES cells. Mouse oocytes were recovered from superovulated 4wks hybrid F1 (C57BL/6xCBA/N) female mice. The oocytes were treated with 7% ethanol for 5 min and 5 ㎍/㎖ cytochalasin-B for 4 h. For IVF, the oocytes were inseminated with epididymal sperm of hybrid Fl male mice (1×10/sup 6//㎖). IVF and parthenogenetic embryos were cultured in M16 medium for 4 days. Cell number count in blastocysts was carried out differential labelling using propidium iodide (red) and bisbenzimide(blue). (omitted)

• Proceedings of the KSAR Conference
• /
• 2002.06a
• /
• pp.20-20
• /
• 2002

This study was to establish the use of parthenogenetic mouse ES (P-mES02) cells as a reproducible differentiation system for mouse cardiomyocytes. To induce differentiation, P-mES02 cells were dispersed by dissociation and the formation of ES cell aggregates in differentiation medium. After 7 days in differentiation culture, the embryoid bodies (EBs) were plated onto gelatin-coated dish. Cultures were observed daily using an inverted light microscope to determine the day of contraction onset and total duration of continuous contractile activity for each contracting focus. (omitted)

• Clinical and Experimental Reproductive Medicine
• /
• v.29 no.2
• /
• pp.129-138
• /
• 2002

Objective: This study was to compare the characteristics between parthenogenetic mES (P-mES) cells and in vitro fertilization mES cells. Materials 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 $5{\mu}g$/ml cytochalasin-B for 4 h. For IVF, oocytes were inseminated with epididymal sperm of hybrid F1 male mice ($1{times}10^6/ml$). 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, b1astocysts 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 identify ES cells, the surface markers alkaline phosphatase, SSEA-1, 3,4 and Oct4 staining were examined in rep1ated 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 (${\geq}$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 b1astocysts ($9.6{\pm}3.1,\;35.1{\pm}5.2$) were signficantly lower than those of IVF blastocysts ($19.5{\pm}4.7,\;63.2{\pm}13.0$) (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-l 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 cell 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.

• Clinical and Experimental Reproductive Medicine
• /
• v.29 no.2
• /
• pp.139-147
• /
• 2002

Objective : This study was to establish a reproducible differentiation system from the parthenogenetic mouse embryonic stem (P-mES02) cells into functional cardiomyocytes like as in vitro fertilization mouse embryonic stem (mES01) cells. Materials and Methods: To induce differentiation, P-mES02 cells were dissociated and aggregated in suspension culture environment for embryoid body (EB) formation. For differentiation into cardiomyocytes, day 4 EBs were treated with 0.75% dimethyl sulfoxide (DMSO) for another 4 days (4-/4+) and then were plated onto gelatin-coated dish. Cultured cells were observed daily using an inverted light microscope to determine the day of contraction onset and total duration of continuous contractile activity for each contracting focus. This frequency was compared with the results of DMSO not treated P-mES02 group (4-/4-) and mES01 groups (4-/4+ or 4-/4-). For confirm the generation of cardiomyocytes, beating cell masses were treated with trypsin-EDTA, dispersed cells were plated onto glass coverslips and incubated for 48 h. Attached cells were fixed using 4% paraformaldehyde and incubated with specific antibodies (Abs) to detect cardiomyocytes (anti-sarcomeric ? -actinin Ab, 1 : 100; anti-cardiac troponin I Ab, 1 : 2000) for 1 h. And the cells were finally treated with FITC or TRITC labelled 2nd Abs, respectively, then they were examined under fluorescence microscopy. Results: Rhythmically contracting areas in mES01 or P-mES02 cells were firstly appeared at 9 or 10 days after EBs plating, respectively. The highest cumulative frequency of beating EBs was not different in both treatment groups (mES01 and P-mES02, 4-/4+) with the results of 61.3 % at 13 days and 69.8% at 15 days, respectively. Also, the contracting duration of individual beating EBs was different from minimal 7 days to maximal 53 days. However, DMSO not treated groups (mES01 and P-mES02, 4-/4-) also had contracting characteristics although their frequency was a few compared to those of DMSO treated groups (6.0% and 4.0%). Cells recovered from the spontaneously contracting areas within EBs in both treated groups were stained positively with muscle specific anti-sarcomeric ? -actinin Ab and cardiac specific anti-cardiac troponin I Ab. Conclusion: This study demonstrated that the P-mES02 cell-derived cardiomyocytes displayed similarly structural properties to mES01 cell-derived cardiomyocytes and that the DMSO treatment enhanced the cardiomyocytes differentiation in vitro.

• Reproductive and Developmental Biology
• /
• v.30 no.1
• /
• pp.47-52
• /
• 2006

This study was conducted to examine whether the parthenogenetic mouse embryonic stem (P-mES) cells can differentiate into functional cardiomyocytes in vitro similar to (mES) cells. p-mES04 and IVF-derived mES03 cells were cultured by suspension culture for 4 days. The formed embryoid bodies (EBs) were treated with 0.75% dimethyl-sulfoxide (DMSO) for further 4 days (4-/4+), and then plated onto gelatin coated culture dish. The appearance of contracting cardiomyocytes from the P-mES04 and mES03 cells was examined for 30 days. The highest cumulative frequency was detected at days 13 (69.83%) and 22 (61.3%), respectively. By immunocytochemistry, beating P-mES04 cells were positively stained with muscle specific anti-sarcomeric a-actinin Ab and cardiac specific anti-cardiac troponin I Ab similar to contracted mES03 cells. When the expression of cardiac muscle-specific genes was analyzed by RT-PCR, beating P-mES04 cells were expressed cardiac specific L-type calcium channel, a1C, cardiac myosin heavy chain a, cardiac muscle heavy polypeptide $7{\beta}$, GATA binding protein 4 and atrial natriuretic factor, but not expressed skeletal muscle specific L-type calcium channel, a1S, which was similar to male adult heart cells and mES03-derived beating cardiomyocytes. The result demonstrates that the P-mES cells can be used as an alternative for the study on the characteristic analysis of in vitro cardiomyocyte differentiation from the ES cells.

• Proceedings of the KSAR Conference
• /
• 2003.06a
• /
• pp.30-30
• /
• 2003

The present study examined the developmental ability of embryonic stem (ES) cells aggregated with mouse parthenogenetic embryos. Oocytes obtained from superovulated female mouse (BCF1) were treated with 7% ethanol and 5 $\mu\textrm{g}$/$m\ell$ cytochalasin B (CB) for producing pathenotes and in vitro fertilized with fresh sperm for producing normal embryos. The reporter vector (pNeoEGFP) were inserted into ES cells (129S4/svJae) by electroporation. At the 8-cell stage, in vitro fertilized embryos and pathenotes, which the zona pellucida was removed, were co-cultured with 5~10 ES cells for 4 hr. After in vitro fertilized embryos and parthenotes aggregated with ES cells were incubated to blastocyst stage, and these blastocysts transferred into the uterus of pseudopregnant recipients. The fertilized embryos aggregated with ES cells were successfully developed to offspring, but the parthenotes aggregated with ES cells failed to develop offsprings. However, genomic DNA of ES cells was detected in the pathenogenetic fetus by polymerase chain reactions at 15 day post gestation. In this study, results indicated that parthenotes aggregated with ES cells showed possible development to fetus. In the future, this method may help to produce transgenic chimera from parthenotes aggregated with ES cells.