• 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)

• Proceedings of the KSAR Conference
• /
• 2003.06a
• /
• pp.80-80
• /
• 2003

Huntington's disease (HD) is an autosomal dominant neurodegenerative disorder characterized by motor, cognitive, and psychiatric symptoms, accompanied by marked cell death in the striatum and cortex. Stereotaxic injection of quinolinic acid (QA) into striatum results in a degeneration of GABAergic neurons and exhibits abnormal motor behaviors typical of the illness. The objective of this study was carried out to obtain basic information about whether parthenogenetic mouse embryonic stem (PmES) cells are suitable for cell replacement therapy of HD. To establish PmES cell lines, hybrid F1 (C57BL/6xCBA/N) mouse oocytes were treated with 7% ethanol for 5 min and cytochalasin-B for 4 hr to initiate spontaneous cleavage. Thus established PmES cells were induced to differentiate using bFGF (20ng/ml) followed by selection of neuronal precursor cells for 8 days in N2 medium. After selection, cells were expanded at the presence of bFGF (20 ng/ml) for another 6 days, then a final differentiation step in N2 medium for 7 days. To establish recipient animal models of HD, young adult mice (7 weeks age ICR mice) were lesioned unilaterally with a stereotaxic injection of QA (60 nM) into the striatum and the rotational behavior of the animals was tested using apomorphine (0.1mg/kg, IP) 7 days after the induction of lesion. Animals rotating more than 120 turns per hour were selected and the differentiated PmES cells (1$\times$10$^4$cells/ul) were implanted into striatum. Four weeks after the graft, immunohistochemical studies revealed the presence of cells reactive to anti-NeuN antibody. However, only a slight improvement of motor behavior was observed. By Nissl staining, cell mass resembling tumor was found at the graft site and near cortex which may explain the slight behavioral improvement. Detailed experiment on cell viability, differentiation and migration explanted in vivo is currently being studied.

• 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.

• 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.

• Reproductive and Developmental Biology
• /
• v.35 no.1
• /
• pp.123-129
• /
• 2011

The pig has been considered to serve as an appropriate model of human disease. Therefore, establishment of porcine embryonic stem cell lines is important. The purpose of the present study was to further work in this direction. We produced porcine parthenogenetic embryos, and separately aggregated two of each of two-cell ($2{\times}2$), four-cell ($2{\times}4$), and eight-cell ($2{\times}8$) embryos derived by parthenogenesis. After culture for 4 days, the developmental ability of the aggregates and total blastocyst cell numbers were evaluated. The percentage of blastocysts was significantly higher in both $2{\times}4$- and $2{\times}8$-aggregated embryos ($58.3{\pm}1.9%$ and $37.2{\pm}2.8%$, respectively) than in the control or $2{\times}2$-aggregated embryos ($23.6{\pm}1.1%$ and $12.5{\pm}2.4%$, respectively). Total blastocyst cell numbers were increased in the $2{\times}4$- and $2{\times}8$-aggregated embryos (by $44{\pm}3.0%$ and $45{\pm}3.3%$, respectively) compared with those of control or $2{\times}2$-aggregated embryos ($30.5{\pm}2.1%$ and $30.7{\pm}2.6%$, respectively; p<0.05). The levels of mRNA encoding Oct-4 were higher in both the $2{\times}4$- and $2{\times}8$-aggregated embryos than in the control. When blastocysts derived from $2{\times}4$- aggregated embryos or intact normal embryos were cultured on mouse embryonic fibroblast feeder cells to obtain porcine stem cells, blastocysts from aggregated embryos formed colonies that were better in shape compared with those derived from intact blastocysts. Together, the data show that aggregation of porcine embryos not only improves blastocyst quality but also serves as an efficient procedure by which porcine embryonic stem cells can become established.

• Journal of Embryo Transfer
• /
• v.27 no.3
• /
• pp.171-174
• /
• 2012

Various small molecules can be used to control major signaling pathways to enhance stemness and inhibit differentiation in murine embryonic stem cell (mESC) culture. Small molecules inhibiting the fibroblast growth factor (FGF)/ERK pathway can preserve pluripotent cells from stimulation of differentiation. In this study, we aimed to evaluate the effect of pluripotin (SC-1), an inhibitor of the FGF/ERK pathway, on the colony formation of outgrowing presumptive mESCs. After plating the zona pellucida-free blastocyst on the feeder layer, attached cell clumps was cultured with SC-1 until the endpoint of the experiment at passage 10. In this experiment, when the number of colonies was counted at passage 3, SC-1-treated group showed 3.4 fold more mESC colonies when compared with control group. However, after passage 4, there was no stimulating effect of SC-1 on the colony formation. In conclusion, SC-1 treatment can be used to promote mESC generation by increasing the number of early mESC colonies.

• Reproductive and Developmental Biology
• /
• v.36 no.1
• /
• pp.33-37
• /
• 2012

Differential capacity of the parthenogenetic embryonic stem cells (PESCs) is still under controversy and the mechanisms of its neural induction are yet poorly understood. Here we demonstrated neural lineage induction of PESCs by addition of insulin-like growth factor-2 (Igf2), which is an important factor for embryo organ development and a paternally expressed imprinting gene. Murine PESCs were aggregated to embryoid bodies (EBs) by suspension culture under the leukemia inhibitory factor-free condition for 4 days. To test the effect of exogenous Igf2, 30 ng/ml of Igf2 was supplemented to EBs induction medium. Then neural induction was carried out with serum-free medium containing insulin, transferrin, selenium, and fibronectin complex (ITSFn) for 12 days. Normal murine embryonic stem cells derived from fertilized embryos (ESCs) were used as the control group. Neural potential of differentiated PESCs and ESCs were analyzed by immunofluorescent labeling and real-time PCR assay (Nestin, neural progenitor marker; Tuj1, neuronal cell marker; GFAP, glial cell marker). The differentiated cells from both ESC and PESC showed heterogeneous population of Nestin, Tuj1, and GFAP positive cells. In terms of the level of gene expression, PESC showed 4 times higher level of GFAP expression than ESCs. After exposure to Igf2, the expression level of GFAP decreased both in derivatives of PESCs and ESCs. Interestingly, the expression level of $Tuj1$ increased only in ESCs, not in PESCs. The results show that IGF2 is a positive effector for suppressing over-expressed glial differentiation during neural induction of PESCs and for promoting neuronal differentiation of ESCs, while exogenous Igf2 could not accelerate the neuronal differentiation of PESCs. Although exogenous Igf2 promotes neuronal differentiation of normal ESCs, expression of endogenous $Igf2$ may be critical for initiating neuronal differentiation of pluripotent stem cells. The findings may contribute to understanding of the relationship between imprinting mechanism and neural differentiation and its application to neural tissue repair in the future.