Jin Hee Eum

Human Somatic Cell Nuclear Transfer Using Adult Cells

Derivation of patient-specific human pluripotent stem cells via somatic cell nuclear transfer (SCNT) has the potential for applications in a range of therapeutic contexts. However, successful SCNT with human cells has proved challenging to achieve, and thus far has only been reported with fetal or infant somatic cells. In this study, we describe the application of a recently developed methodology for the generation of human ESCs via SCNT using dermal fibroblasts from 35- and 75-year-old males. Our study therefore demonstrates the applicability of SCNT for adult human cells and supports further investigation of SCNT as a strategy for regenerative medicine.

Recombinant human phospholipase C zeta 1 induces intracellular calcium oscillations and oocyte activation in mouse and human oocytes

BACKGROUND Oocyte activation is a crucial step that comprises the release of the oocyte from meiotic arrest, pronuclear formation and subsequent embryo development. Oocytes are activated by repetitive increases in the intracellular concentration of free Ca(2+), [Ca(2+)](i) oscillations, which are triggered during fertilization by the introduction of the sperm-specific phospholipase C zeta 1 (PLCZ1). Recent studies have shown that sperm from patients lacking expression of PLCZ1 or expressing mutant forms of PLCZ1 fail to induce [Ca(2+)](i) oscillations or oocyte activation. We first purified recombinant human PLCZ1 (hPLCZ1) protein and evaluated its [Ca(2+)](i) oscillation activity in mouse and human oocytes with the view to investigate its application in the clinic for assisted oocytes activation in lieu of chemical agents. METHODS Recombinant hPLCZ1 was synthesized using the Escherichia coli system, and subjected to immunoblot analysis with anti-PLCZ1 and anti-His tag antibodies. [Ca(2+)](i) oscillations by microinjection of recombinant hPLCZ1 into mouse or human oocytes were examined by [Ca(2+)](i) monitoring with Fluo 4. Ploidy of the oocytes with recombinant hPLCZ1 injection was confirmed with fluorescence in situ hybridization. RESULTS A band of 68 kDa on recombinant protein was detected with both antibodies. Injection of recombinant hPLCZ1 induced [Ca(2+)](i) oscillations in a dose-dependent manner in both mouse and human oocytes. These oscillations, which closely resembled those initiated by the sperm upon fertilization, triggered activation and cleavage in oocytes of both species, although further development of the mice embryos was low. U73122, a PLC inhibitor, blocked the ability of hPLCZ1 to initiate oscillations. Microinjection of recombinant hPLCZ1 into ICSI-failed human oocytes rescued fertilization failure in five of eight attempts. CONCLUSIONS Repeated [Ca(2+)](i) oscillations and oocyte activation were induced in mouse and human oocytes by microinjection of recombinant hPLCZ1 synthesized in E. Coli. Injection of recombinant protein could thus provide a biological solution for inducing artificial activation of oocytes.

Histone Demethylase Expression Enhances Human Somatic Cell Nuclear Transfer Efficiency and Promotes Derivation of Pluripotent Stem Cells.

The extremely low efficiency of human embryonic stem cell (hESC) derivation using somatic cell nuclear transfer (SCNT) limits its potential application. Blastocyst formation from human SCNT embryos occurs at a low rate and with only some oocyte donors. We previously showed in mice that reduction of histone H3 lysine 9 trimethylation (H3K9me3) through ectopic expression of the H3K9me3 demethylase Kdm4d greatly improves SCNT embryo development. Here we show that overexpression of a related H3K9me3 demethylase KDM4A improves human SCNT, and that, as in mice, H3K9me3 in the human somatic cell genome is an SCNT reprogramming barrier. Overexpression of KDM4A significantly improves the blastocyst formation rate in human SCNT embryos by facilitating transcriptional reprogramming, allowing efficient derivation of SCNT-derived ESCs using adult Age-related Macular Degeneration (AMD) patient somatic nuclei donors. This conserved mechanistic insight has potential applications for improving SCNT in a variety of contexts, including regenerative medicine.

Long-term liquid nitrogen vapor storage of mouse embryos cryopreserved using vitrification or slow cooling

OBJECTIVE To store embryos in LN(2) vapor, which eliminates direct contact with LN(2), is presented as an alternative method to protect embryos cryopreserved by vitrification or slow cooling from LN(2)-borne pathogens. DESIGN Basic animal research. SETTING University-affiliated hospital. ANIMAL(S) Two-cell mouse embryos collected from superovulated ICR mice. INTERVENTION(S) Embryos cryopreserved by vitrification or slow cooling were stored in LN(2) or LN(2) vapor and then thawed after 1 week, 1 month, or 6 months. Thawed embryos cultured to the blastocyst stage in vitro were evaluated or transferred into the uterus of foster mothers. MAIN OUTCOME MEASURE(S) Survival, apoptosis, and in vitro and in vivo development of freeze-thawed mouse embryos as a function of cryopreservation and cryostorage methods were determined. RESULT(S) After short- and long-term storage of vitrified and slow-cooled embryos, embryonic survival and development rate were the same for embryos stored in LN(2) vapor and in direct contact with LN(2). Cell numbers and apoptosis frequency were similarly indistinguishable between the two groups after storage of embryos for 6 months, and there were no differences in delivery rate or litter size after ET. CONCLUSION(S) The present study shows that embryos stored in LN(2) vapor retain full developmental potential. This storage system thus represents a useful alternative for safe and effective long-term storage of embryos that avoids direct contact with LN(2).

Stem cell factor/c-Kit signaling in in vitro cultures supports early mouse embryonic development by accelerating proliferation via a mechanism involving Akt-downstream genes

PurposeStem cell factor (SCF)/c-Kit regulates the proliferation and survival of germ cells or stem cells; however, little is known about the role of SCF/c-Kit in pre-implantation embryo development.MethodsUsing exogenous SCF supplementation and c-Kit siRNA injection, we investigated the role and mechanism of SCF/c-Kit in pre-implantation mouse embryos.ResultsAddition of soluble SCF to the culture medium improved blastocyst formation. c-Kit gene silencing reduced the rate of blastocyst formation and delayed embryonic development. The number of proliferating cells in c-Kit gene-silenced blastocysts decreased, whereas the number of apoptotic cells in blastocysts obtained from both experimental and the control groups was not affected. RT-PCR, immunostaining and western blotting revealed that proliferation-related Akt downstream targets were substantially affected by c-Kit gene silencing.ConclusionSCF/c-Kit signaling through Akt downstream targets is likely involved in mediating the cleavage and proliferation of blastomeres during mouse pre-implantation embryogenesis.

Clinical outcomes of single versus double blastocyst transfer in fresh and vitrified-warmed cycles

Objective Assisted reproductive technology has been associated with an increase in multiple pregnancies. The most effective strategy for reducing multiple pregnancies is single embryo transfer. Beginning in October 2015, the National Supporting Program for Infertility in South Korea has limited the number of embryos that can be transferred per in vitro fertilization (IVF) cycle depending on the patients age. However, little is known regarding the effect of age and number of transferred embryos on the clinical outcomes of Korean patients. Thus, this study was performed to evaluate the effect of the number of transferred blastocysts on clinical outcomes. Methods This study was carried out in the Fertility Center of CHA Gangnam Medical Center from January 2013 to December 2014. The clinical outcomes of 514 women who underwent the transfer of one or two blastocysts on day 5 after IVF and of 721 women who underwent the transfer of one or two vitrified-warmed blastocysts were analyzed retrospectively. Results For both fresh and vitrified-warmed cycles, the clinical pregnancy rate and live birth or ongoing pregnancy rate were not significantly different between patients who underwent elective single blastocyst transfer (eSBT) and patients who underwent double blastocyst transfer (DBT), regardless of age. However, the multiple pregnancy rate was significantly lower in the eSBT group than in the DBT group. Conclusion The clinical outcomes of eSBT and DBT were equivalent, but eSBT had a lower risk of multiple pregnancy and is, therefore, the best option.

An efficient SCNT technology for the establishment of personalized and public human pluripotent stem cell banks

Although three different research groups have reported successful derivations of human somatic cell nuclear transfer-derived embryonic stem cell (SCNT-ESC) lines using fetal, neonatal and adult fibroblasts, the extremely poor development of cloned embryos has hindered its potential applications in regenerative medicine. Recently, however, our group discovered that the severe methylation of lysine 9 in Histone H3 in a human somatic cell genome was a major SCNT reprogramming barrier, and the overexpression of KDM4A, a H3K9me3 demethylase, significantly improved the blastocyst formation of SCNT embryos. In particular, by applying this new approach, we were able to produce multiple SCNT-ES cell lines using oocytes obtained from donors whose eggs previously failed to develop to the blastocyst stage. Moreover, the success rate was closer to 25%, which is comparable to that of IVF embryos, so that our new human SCNT method seems to be a practical approach to establishing a pluripotent stem cell bank for the general public as well as for individual patients. [BMB Reports 2016; 49(4): 197-198]

Predictive value of antral follicle count and serum anti-Müllerian hormone: Which is better for live birth prediction in patients aged over 40 with their first IVF treatment?

OBJECTIVE To evaluate clinical utility of antral follicle count (AFC) and anti-Müllerian hormone (AMH) in predicting in vitro fertilization (IVF) outcomes among the patients over 40 years old in their first IVF cycles. STUDY DESIGN Total 219 patients aged 40 or older who underwent their first IVF with gonadotropin-releasing hormone antagonist protocol from January 2013 to September 2014 in CHA Gangnam fertility center were retrospectively analyzed. AFC and serum samples were measured prior to IVF treatment. The main outcomes were clinical pregnancy rate and live birth. RESULTS 36 out of 219 patients achieved clinical pregnancy (16.4%) and 27 out of 219 patients delivered (12.3%). The receiver operating characteristic curve analysis to predict clinical pregnancy showed that both age and AFC equally had higher accuracy by area under the curve (AUC = 0.657, P < 0.01) than serum AMH (AUC 0.613, P = 0.03). The optimum cut-off value of age was ≤41 and that of AFC was >3 to predict clinical pregnancy. For the prediction of live birth, AFC had the highest accuracy (AUC 0.698, P < 0.01), followed by age (AUC 0.674, P < 0.01) and the number of total retrieved oocytes (AUC 0.620, P = 0.02). The optimum cut-off value of age was ≤41, that of AFC was >3 and that of the number of total retrieved oocytes were >6. With multivariate regression analysis, age and AMH were significantly correlated with clinical pregnancy (age, odds ratio [OR] 0.53, P < 0.01; AMH, OR 1.31, P = 0.04), whereas age and AFC were association with live birth significantly (age, OR 0.41, P < 0.01; AFC, OR 1.10, P = 0.02). CONCLUSION In patients aged over 40, AFC and AMH were shown to be good biomarkers for the prediction of clinical pregnancy and live birth. Although AMH was positively correlated with clinical pregnancy and had no association with live birth, the predictive value of AFC and AMH were similar for both clinical pregnancy and live birth. To predict the live birth, age ≤41, AFC >3 and total retrieved oocytes >6 appeared to be meaningful. This study demonstrated the significance of AMH and AFC as predictors of clinical pregnancy and live birth for old aged women at their first IVF cycle with gonadotropin-releasing hormone antagonist protocol.

Prematuration Culture with Phosphodiesterase Inhibitors After Vitrification May Induce Recovery of Mitochondrial Activity in Vitrified Mouse Immature Oocytes

This study investigates the possible causes for low development of blastocysts in vitrified immature oocytes by evaluating the changes of mitochondrial membrane potential and reactive oxygen species (ROS) production and finds a recovery mechanism for these conditions in vitrified immature oocytes. To recover from the cryoinjury, we cultured vitrified immature oocytes in milrinone containing medium for 1, 3, and 5 hours and then extended the culture for oocyte maturation. There was no difference in in vitro maturation and fertilization rate between fresh and vitrified/warmed oocytes. However, the development rate of blastocysts in vitrified/warmed oocytes was significantly lower than that in fresh oocytes (p < 0.05). The development rate of blastocysts was recovered if these oocytes were cultured for 3 hours in milrinone. Vitrified/warmed oocytes incubated in milrinone for 0 and 1 hour showed a significantly higher level of ROS (p < 0.05) and a significantly lower mitochondrial membrane potential (p < 0.05) than fresh oocytes. However, there was no significant difference (p > 0.05) between vitrified oocytes incubated in milrinone for 3 hours and fresh oocytes in terms of ROS level and mitochondrial membrane potential. In conclusion, alteration of highly polarized mitochondria distribution in vitrified oocytes may have an effect on mitochondrial activity, including ROS production during fertilization and further development. Preincubation in milrinone before in vitro maturation of immature vitrified/warmed oocytes may help the redistribution of highly polarized mitochondrial inner membrane potential and in reducing ROS and enhance the further embryonic development after fertilization.

Characterization of Tetraploid Somatic Cell Nuclear Transfer-Derived Human Embryonic Stem Cells

ABSTRACT Polyploidy is occurred by the process of endomitosis or cell fusion and usually represent terminally differentiated stage. Their effects on the developmental process were mainly investigated in the amphibian and fishes, and only observed in some rodents as mammalian model. Recently, we have established tetraploidy somatic cell nuclear transfer-derived human embryonic stem cells (SCNT-hESCs) and examined whether it could be available as a research model for the polyploidy cells existed in the human tissues. Two tetraploid hESC lines were artificially acquired by reintroduction of remained 1st polar body during the establishment of SCNT-hESC using MII oocytes obtained from female donors and dermal fibroblasts (DFB) from a 35-year-old adult male. These tetraploid SCNT-hESC lines (CHA-NT1 and CHA-NT3) were identified by the cytogenetic genotyping (91, XXXY,-6, t[2:6] / 92,XXXY,-12,+20) and have shown of indefinite proliferation, but slow speed when compared to euploid SCNT-hESCs. Using the eight Short Tendem Repeat (STR) markers, it was confirmed that both CHA-NT1 and CHA-NT3 lines contain both nuclear and oocyte donor genotypes. These hESCs expressed pluripotency markers and their embryoid bodies (EB) also expressed markers of the three embryonic germ layers and formed teratoma after transplantation into immune deficient mice. This study showed that tetraploidy does not affect the activities of proliferation and differentiation in SCNT-hESC. Therefore, tetraploid hESC lines established after SCNT procedure could be differentiated into various types of cells and could be an useful model for the study of the polyploidy cells in the tissues.