Alterations of proliferative and differentiation potentials of human embryonic stem cells during long-term culture

Human embryonic stem cells (hESCs) are considered to be able to stably maintain their characteristics in vitro for prolonged periods, but we had previously encountered changes in proliferative ability and differentiation potential during extended culture of hESCs. Therefore, we investigated the proliferative ability and differentiation potential of hESCs during long-term culture. The hESCs, SNUhES3, were used to analyze population-doubling time, proliferation rate and differentiation potential. We classified hESCs into three groups according to culture period. Ten colonies of hESCs for each group were daily measured colony area and population-doubling time was assessed by the changes of colony area. Proliferation rate of hESCs was measured by 5-bromo-2'-deoxyuridine (BrdU) assay and telomerase activity. To evaluate differentiation potentials for hESCs, expression levels of undifferentiated and/or differentiated hESCs markers were examined by FACS, RT-PCR and immunostaining. Population-doubling time of early passage hESCs was longer than those of middle or late passage. Proliferative ability of hESCs was accelerated depending on culture periods. Cellular morphologies and the expression level of each three germ layer markers were obviously different from each passage of reattached embryoid bodies (EBs) after spontaneous differentiation. Differentiated cells of late passage expressed higher levels of undifferentiated markers such as Oct4 and SSEA4 than those of early and middle passage. But differentiated cells of early and middle passage expressed higher level of differentiated state markers, Nestin (ectoderm), Brachyury (mesoderm), HNF3beta (endoderm). From these results, it can be inferred that hESCs show higher proliferative abilities and reduced differentiation potentials as the passage number increased. Therefore, we conclude that early passage hESCs could be more suitable than middle and late passage hESCs in differentiation studies.

Establishment of Mouse Embryonic Stem Cell-like Cells from In Vitro Fertilized Embryos / 대한불임학회지

OBJECTIVE: In order to acquire the technique for the establishment of human embryonic stem cells (ESC) derived from the human frozen-thawed embryos produced in IVF-ET program, this study was performed to establish mouse ESC derived from the in vitro fertilized embryos. MATERIALS AND METHODS: After F1 hybrid (C57BL x CBA) female mice were superovulated with PMSG and hCG treatment, their oocytes were retrieved and inseminated, and the fertilized embryos were cultured for 96-120 hours until the expected stages of blastocysts were obtained. To isolate the inner cell mass (ICM), either the blastocysts were treated with immunosurgery, or the whole embryos were cultured for 4 days. Isolated ICMs were then cultured onto STO feeder cell layer, and the resultant ICM colonies were subcultured with trypsin-EDTA treatment. During the subculture process, ESC-like cell colonies were observed with phase contrast microscopy. To identify ESC in the subcultured ESC-like cell colonies, alkaline phosphatase activity and Oct-4 (octamer-binding transcription factor-4) expression were examined by immunohistochemistry and RT-PCR, respectively. To examine the spontaneous differentiation, ESC-like cell colonies were cultured without STO feeder cell layer and leukemia inhibitory factor (LIF). RESULTS: Seven ESC-like cell lines were established from ICMs isolated from the in vitro fertilized embryos. According to the developmental stage, the growth of ICMs isolated from the expanded blastocysts was significantly better than that of ICMs isolated from the hatched blastocysts (80.3% vs. 58.7%, p<0.05). ESC-like cell colonies were only obtained from ICMs of expanded blastocysts. However, the ICMs isolated from the embryos treated with immunosurgery were poorly grown and frequently differentiated during the culture process. The established ESC-like cell colonies were positively stained with alkaline phosphatase and expressed Oct-4, and their morphology resembled that observed in the previously reported mouse ESC. In addition, following the extended in vitro culture process, they maintained their expression of cell surface markers characteristic of the pluripotent stem cells such as alkaline phosphatase and Oct-4. When cultured without STO feeder cell layer and LIF, they were spontaneously differentiated into the various types of cells. CONCLUSION: The findings of this study suggest that the establishment of mouse ESC can be successfully derived from the in vitro fertilized embryos. The established ESC-like cells expressed the cell surface markers characteristic of the pluripotent stem cells and spontaneously differentiated into the various types of cells.

The Study on Vitrification and Ultrarapid Thawing of Human Embryonic Stem Cells / 대한불임학회지

OBJECTIVE: This study was carried out to establish the effectiveness of the vitrification method and the optimal cryoprotectants in the cryopreservation of human embryonic stem cells (ESC). MATERIALS AND METHODS: Human ESC clumps established at Seoul National University Hospital (SNUhES 1) were cryopreserved with the vitrification method using the EM grid. EDS and EFS40 were used as vitrification solutions. RESULTS: Between the EDS and EFS40 groups, there was no significant difference in the recovery rate after cryopreservation of human ESC. The formation rates of ESC colonies in the vitrified groups were significantly lower than those in the control ESC group (p<0.05, p<0.05). In addition, the formation rate of ESC colonies in the EDS group was significantly higher than that in the EFS40 group (p<0.05). The ESC colonies in the vitrified groups were significantly smaller after culture duration of 2 and 4 days, respectively, compared with the control ESC group (p<0.01, p<0.05). However, these effects could be reduced to nonsignificant level by the additional culture of ESC colonies. The vitrified human ESC retained the properties of pluripotent cells, including the expression of cell surface markers for the undifferentiated cells such as alkaline phosphatase and SSEA-4 (stage-specific embryonic antigen-4), and the expression of transcription factor Oct-4 (octamer-binding transcription factor-4), and the normal karyotype. CONCLUSION: The vitrification method using the EM grid and EDS solution was confirmed to be very effective for the cryopreservation of human ESC.

Influence of GnRH Agonist and Neural Antagonists on Stress-blockade of LH and Prolactin Surges Induced by 17 beta-estradiol in Ovariectomized Rats

In our previous study, we demonstrated that immobilization stress blocked estrogen-induced luteinizing hormone(LH) surge possibly by inhibiting the synthesis and release of gonadotropin-releasing hormone (GnRH) at the hypothalamic level and by blocking estrogen-induced prolactin (PRL) surge by increasing the synthesis of dopamine receptor at the pituitary level in ovariectomized rats. The present study was performed to determine whether immobilization stress affects pituitary LH responsiveness to GnRH, and whether endogenous opioid peptide (EOP) and dopamine systems are involved in blocking LH and PRL surges during immobilization stress. Immobilization stress was found to inhibit basal LH release and to completely abolish LH surge. However, the intravenous application of GnRH agonist completely restored immobilization-blocked LH surge and basal LH release. Treatment with naloxone did not exert any effect on immobilization-blocked LH surge but increased basal LH release during immobilization stress. Pimozide did not affect immobilization-blocked LH surge or basal LH release. Naloxone also decreased immobilization-induced basal PRL release, but had no effect on immobilization-blocked PRL surge. Immobilization-increased basal PRL levels were augmented by pimozide treatment and immobilization-blocked PRL surge was dramatically restored by pimozide. We conclude that immobilization stress does not impair pituitary LH response to GnRH, and that the immobilization stress-induced blockage of LH surge is probably not mediated by either the opioidergic or the dopaminergic system. However, immobilization-blockade of PRL surge may be partly mediated by the dopaminergic system.

Regulation of FSH Gene Expression and Release in Cultured Rat Anterior Pituitary Cells / 대한내분비학회지

BACKGROUND: FSH is a heterodimeric glycoprotein and is composed of alpha and beta subunits. alpha subunit is common to FSH and LH, while an unique beta subunit determines the biological specificity of each hormone. The synthesis of beta subunit is the primary rate-limiting step in the synthesis of each hormone. Although FSH plays a pivotal role in folliculogenesis and ovulation, very little studies have been performed on the regulation of FSH beta gene expression. Therefore, the present study attempted to examine the effect of GnRH or activin on the expression of FSH beta mRNA as well as FSH release and signaling pathway involved in their actions. METHODS: The primary cultures of rat anterior pituitary were used for this study. To determine FSH beta mRNA levels, northern blotting method was used. The concentration of FSH in the culture medium was evaluated by using a specific radioimmunoassay for rat FSH. RESULTS: PMA, an activator of PKC, increased FSH beta mRNA levels and FSH release, whereas forskolin, an activator of adenylate cyclase, showed no effect. The application of GnRH augmented FSH release, but not FSH beta mRNA levels. However, the administration of activin increased FSH beta mRNA levels as well as FSH release. Staurosporine, an inhibitor of PKC, suppressed activin-induced increment of FSH beta mRNA levels and FSH release. CONCLUSION: The present study demonstrated that activin rather than GnRH is a major regulator for FSH beta mRNA expression, and suggest that PKC-dependent pathway is also involved in the action of activin on the expression of FSH beta mRNA and FSH release.

Regulation of Estrogen Receptor mRNA in Rat Anterior Pituitary Gland / 대한내분비학회지

The estrogen receptor (ER) is present in a wide variety of mammalian tissues and is required for the physiological responses of estrogen, including estrogen-induced tissue-specific changes in gene expression. But most of our knowledge on the regulation of ER mRNA levels comes from in vivo steroid replacement experiments or cancer cell lines that express the ER. Thus the present study was attempted to determine 1) the anterior pituitary ER mRNA levels during rat estrous cycle 2) if estradiol itself directly modulates the ER mRNA levels in cultured rat anterior pituitary using RT-PCR method. In rats with 4 day estrous cycle, the ER mRNA levels in anterior pituitary gland reached to maximum at proestrus 11:00h just before serum estradiol concentration showed the highest. From then, the ER mRNA levels gradually declined during the rest of the proestrus. On the other hands, in cultured rat anterior pituitary cells, the ER mRNA levels were significantly decreased by the treatment of estradiol. These results indicate that the surge of estradiol was proceeded by the increase in pituitary ER mRNA levels during the proestrus and in cultured anterior pituitary cells, estrogen might be involved in the down-regulation of the ER mRNA levels.