• 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)

• 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
• /
• 2004.06a
• /
• pp.272-272
• /
• 2004

The objective of this study is to test whether human embryonic stem cells expressing Nurr1 (Nurr1-transfected hES cells) could be expressed TH according to neuronal differentiation. As an effort to direct differentiation of hES (MB03 registered in NIH) cells to dopamine-producing neuronal cells, Nurr1 was transfected using conventional transfection protocol into MB03 cell and examined the expression of tyrosine hydroxylase (TH) after differentiation induced by retinoic acid (RA) and ascorbic acid (AA). (omitted)

• Proceedings of the KSAR Conference
• /
• 2004.06a
• /
• pp.273-273
• /
• 2004

This study was to examine in vitro neural cell differentiation pattern of the genetically modified human embryonic stem cells expressing tyrosine hydroxylase (TH). Human embryonic stem (hES, MB03) cell was transfected with cDNAs cording for TH. Successful transfection was confirmed by western immunoblotting. (omitted)

• Proceedings of the KSAR Conference
• /
• 2004.06a
• /
• pp.276-276
• /
• 2004

Pluripotent stem cells have been generated from two embryonic sources. ES cells are generated from ICM of blastocyst stage embryos, and embryonic germ (EG) cells are generated from primordial germ cells (PGCs). Both ES and EG cells are pluripotent and present important characteristics such as high levels of alkaline phosphatase (AP) activity, multi-cellular colony formation, normal and stable karyotypes, continuously passaging ability, and the capability of differentiation into all three embryonic germ layers. (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)

• Animal cells and systems
• /
• v.13 no.2
• /
• pp.235-245
• /
• 2009

Embryonic stem (ES) cells have a capability to generate all types of cells. However, the mechanism by which ES cells differentiate into specific cell is still unclear. Using microarray technology, the differentiation process in mouse embryonic stem cells was characterized by temporal gene expression changes of mouse ES cells during differentiation in a monolayer culture. A large number of genes were differentially regulated from 1 day to 14 days, and less number of genes were differentially expressed from 14 days to 28 days. The number of up-regulated genes was linearly increased throughout the 28 days of in vitro differentiation, while the number of down-regulated genes reached the plateau from 14 days to 28 days. Most differentially expressed genes were functionally classified into transcriptional regulation, development, extra cellular matrix (ECM),cytoskeleton organization, cytokines, receptors, RNA processing, DNA replication, chromatin assembly, proliferation and apoptosis related genes. While genes encoding ECM proteins were up-regulated, most of the genes related to proliferation, chromatin assembly, DNA replication, RNA processing, and cytoskeleton organization were down-regulated at 14 days. Genes known to be associated with embryo development or transcriptional regulation were differentially expressed mostly after 14 days of differentiation. These results indicate that the altered expression of ECM genes constitute an early event during the spontaneous differentiation, followed by the inhibition of proliferation and lineage specification. Our study might identify useful time-points for applying selective treatments for directed differentiation of mouse ES cells.

• BMB Reports
• /
• v.44 no.3
• /
• pp.176-181
• /
• 2011

Inhibiting histone deacetylase (HDAC) activity modulates the epigenetic status of cells, resulting in an alteration of gene expression and cellular function. Here, we investigated the effects of HDAC inhibitors on mouse embryonic stem (ES) cells. The HDAC inhibitors trichostatin A, suberoylanilide hydroxamic acid, sodium butyrate, and valproic acid induced early differentiation of mouse ES cells and triggered induction of heat-shock protein (HSP)70. In contrast, class III HDAC inhibitors failed to induce differentiation or HSP70 expression. Transcriptional upregulation of HSP70 was confirmed by mRNA expression analysis, an inhibitor study, and chromatin immunoprecipitation. HSP70 induction was dependent on the SAPK/JNK, p38, and PI3K/Akt pathways. Differentiation and induction of HSP70 by a subset of HDAC inhibitors was also examined in human ES cells, which suggests that the phenomenon generally occurs in ES cells. A better understanding of the effects of HDAC inhibitors may give more insight into their application in stem cell biology.

• Reproductive and Developmental Biology
• /
• v.31 no.4
• /
• pp.221-226
• /
• 2007

Embryonic stem (ES) cells are known to have an infinite proliferation and pluripotency that are associated with complex processes. The objective of this study was to examine expression of genes differentially regulated during differentiation of human ES cells by suppression subtractive hybridization (SSH). Human ES cells were induced to differentiate into neural precursor cells via embryoid body. Neural precursor cells were isolated physically based on morphological criteria. Immunocytochemical analysis showed expression of pax6 in neural precursor cells, confirming that the isolated cells were neural precursor cells. Undifferentiated human ES cells and neural precursor cells were subject to the SSH. TPX2 (Targeting Protein for Xklp2 (Xenopus centrosomal kinesin-like protein 2)) was identified, cloned and analyzed during differentiation of human ES cells into neural lineages. Expression of TPX2 was gradually down-regulated in embryoid bodies and neural precursor cells relative to undifferentiated ES cells. Targeting Protein for Xklp2 has been shown to be involved in cell division by interaction with microtubule development in cancer cells. Taken together, result of this study suggests that TPX2 may be involved in proliferation and differentiation of human ES cells.

• Development and Reproduction
• /
• v.3 no.1
• /
• pp.45-51
• /
• 1999

It is crucial to remove trophectoderm (TE) cells of blastocysts for an efficient isolation of pluripotent embryonic stem (ES)-like cells from bovine blastocysts. We evaluated the effectiveness of chemosurgery using calcium ionophore A23l87 (CIPA) by investigating the viability and pluripotency of ES-like cell lines isolated from in vitro-produced bovine blastocysts after CIPA treatment. The blastocysts treated with 50 $\mu$M CIPA for 25 min colonized most efficiently (51% of blastocysts) and developed to ES-like cell lines through 10 passages (4.8% of blastocysts) among CIPA-treated groups with different concentration and duration. In comparison with CIPA-untreated blastocysts, the colonization rate and overall viability of the CIPA-treated blastocysts were five times higher, suggesting that CIPA treatment condition defined in this study was highly efficient for establishing ES-like cell lines without apparent toxicity of CIPA. We evaluated in vitro pluripotency of the established three ES-like cell lines by examining alkaline phosphatase (AP) activity, capability of embryoid body formation, and chromosomal euploidity of the cells. Our cells showed a heterogeneous AP activity similarly to other reports. The cells were able to form simple embryoid bodies during suspension culture and majority of them showed a normal chromosome number of 60, the euploid chromosomal complement of bovine Therefore, our data suggest that CIPA treatment can be safely used for an efficient isolation of ES-like cell lines from bovine blastocysts.