Establishment of a piglet model for peritoneal metastasis of ovarian cancer.

BACKGROUND: A piglet model for peritoneal metastasis (PM) of ovarian cancer was developed. It will contribute to establishing innovative chemotherapeutical and surgical strategies without any limitation on rodent models. METHODS: A total of 12 four- to five-week-old piglets of 7 to 8 kg were used. Two phases of ovarian cancer cell injections were performed with laparoscopic surgery. In phase I trial, 5.0 × 106 SK-OV-3 cells in 0.1 ml suspension were inoculated into the omentum, peritoneum, and uterine horns of two piglets twice with a one-week interval. In the phase II trial, 5.0 × 106 SNU-008 cells in 0.1 ml suspension were injected only into uterine horns within the same time frame because tumor implantation after inoculation of SK-OV-3 cells was not observed at the omentum or peritoneum in the phase I trial. Modified peritoneal cancer index (PCI) score was used to monitor tumorigenesis up to 4 weeks after inoculation. Tumor tissues disseminated in the peritoneum 4 weeks after injection were used for histological examination with hematoxylin and eosin (H&E) and paired-box gene 8 (PAX-8) staining. RESULTS: In the phase I trial, two piglets showed PM with modified PCI scores of 5 and 4 at 3 weeks after the first inoculation, which increased to 14 and 15 after 4 weeks, respectively. In the phase II trial, PM was detected in eight of ten piglets, which showed modified PCI scores of 6 to 12 at 4 weeks after the first inoculation. The overall incidence of PM from the total of 12 piglets after inoculation was 75%. Immunohistochemical H&E and PAX-8 staining confirmed metastatic tumors. CONCLUSIONS: This study provides strong evidence that piglets can be employed as a model for PM by inoculating ovarian cancer cell lines from humans. Using two cell lines, the PM rate is 75%.

Asp149 and Asp152 in chicken and human ANP32A play an essential role in the interaction with influenza viral polymerase.

The acidic nuclear phosphoprotein 32 family member A (ANP32A) is a cellular host factor that determines the host tropism of the viral polymerase (vPol) of avian influenza viruses (AIVs). Compared with human ANP32A (hANP32A), chicken ANP32A contains an additional 33 amino acid residues (176-208) duplicated from amino acid residues 149-175 (27 residues), suggesting that these residues could be involved in increasing vPol activity by strengthening interactions between ANP32A and vPol. However, the molecular interactions and functional roles of the 27 residues within hANP32A during AIV vPol activity remain unclear. Here, we examined the functional role of 27 residues of hANP32A based on comparisons with other human (h) ANP32 family members. It was notable that unlike hANP32A and hANP32B, hANP32C could not support vPol activity or replication of AIVs, despite the fact that hANP32C shares a higher sequence identity with hANP32A than hANP32B. Pairwise comparison between hANP32A and hANP32C revealed that Asp149 (D149) and Asp152 (D152) are involved in hydrogen bonding and electrostatic interactions, respectively, which support vPol activity. Mutation of these residues reduced the interaction between hANP32A and vPol. Finally, we demonstrated that precise substitution of the identified residues within chicken ANP32A via homology-directed repair using the CRISPR/Cas9 system resulted in a marked reduction of viral replication in chicken cells. These results increase our understanding of ANP32A function and may facilitate the development of AIV-resistant chickens via precise modification of residues within ANP32A.

Multi-Path and Group-Loss-Based Network for Speech Emotion Recognition in Multi-Domain Datasets.

Speech emotion recognition (SER) is a natural method of recognizing individual emotions in everyday life. To distribute SER models to real-world applications, some key challenges must be overcome, such as the lack of datasets tagged with emotion labels and the weak generalization of the SER model for an unseen target domain. This study proposes a multi-path and group-loss-based network (MPGLN) for SER to support multi-domain adaptation. The proposed model includes a bidirectional long short-term memory-based temporal feature generator and a transferred feature extractor from the pre-trained VGG-like audio classification model (VGGish), and it learns simultaneously based on multiple losses according to the association of emotion labels in the discrete and dimensional models. For the evaluation of the MPGLN SER as applied to multi-cultural domain datasets, the Korean Emotional Speech Database (KESD), including KESDy18 and KESDy19, is constructed, and the English-speaking Interactive Emotional Dyadic Motion Capture database (IEMOCAP) is used. The evaluation of multi-domain adaptation and domain generalization showed 3.7% and 3.5% improvements, respectively, of the F1 score when comparing the performance of MPGLN SER with a baseline SER model that uses a temporal feature generator. We show that the MPGLN SER efficiently supports multi-domain adaptation and reinforces model generalization.

Host-Specific Restriction of Avian Influenza Virus Caused by Differential Dynamics of ANP32 Family Members.

BACKGROUND: Influenza viruses must utilize host factors to complete their lifecycle. Species-specific differences in host factors between birds and mammals mean that avian influenza viruses (AIVs) replicate well in avian hosts but not in human hosts. Acidic nuclear phosphoprotein 32 family member A (ANP32A) has been identified as the host restriction factor for the viral polymerase (vPol) activity of AIVs. The ANP32A belongs to the conserved ANP32 family, the functional roles of which during viral replication remain unclear. METHODS: In this study, we targeted chicken ANP32A using clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9-mediated genome editing to examine the functional roles of ANP32A and other members of the ANP32 family. RESULTS: We showed that chicken ANP32A only, not ANP32B and ANP32E, plays a pivotal role in supporting vPol activity of AIVs. Furthermore, we found that the human ANP32C, ANP32D, and ANP32E have suppressive effects on vPol activity in contrast to human ANP32A and ANP32B. CONCLUSIONS: Chicken and human ANP32 family members had different effects on vPol activity, suggesting that species-specific vPol activity of AIVs could be caused by the differential functions and overall competency of ANP32 family members.

Alterations in intracellular Ca2+ levels in human endometrial stromal cells after decidualization.

Calcium (Ca2+) is an important element for many physiological functions of the uterus, including embryo implantation. Here, we investigated the possible involvement of altered intracellular Ca2+ levels in decidualization in human endometrial stromal cells (hEMSCs). hEMSCs showed high levels of mesenchymal stem cell marker expression (CD73, CD90, and CD105) and did not express markers of hematopoietic progenitor cells (CD31, CD34, CD45, and HLA-DR). Decidualization is a process of ovarian steroid-induced endometrial stromal cell proliferation and differentiation. Several types of ion channels, which are regulated by the ovarian hormones progesterone and estradiol, as well as growth factors, are important for endometrial receptivity and embryo implantation. The combined application of progesterone (1 µM medroxyprogesterone acetate) and cyclic AMP (0.5 mM) for 6 days not only elevated inositol 1,4,5-triphosphate receptor (IP3R)-mediated Ca2+ release and IP3R expression, it also promoted ORAI and STIM expression as well as cyclopiazonic acid-induced Ca2+ release. Finally, intracellular Ca2+ levels and ion channel gene expression influenced hEMSC proliferation. These results suggest that cytosolic Ca2+ dynamics, mediated by specific ion channels, serve as an important step in the decidualization of hEMSCs.

G-protein coupled receptor 64 (GPR64) acts as a tumor suppressor in endometrial cancer.

BACKGROUND: Endometrial cancer is the most common gynecological cancer. G-protein coupled receptor 64 (GPR64) belongs to a family of adhesion GPCRs and plays an important role in male fertility. However, the function of GPR64 has not been studied in endometrial cancer. Our objective is to investigate the role of GPR64 in endometrial cancer. METHODS: We examined the levels of GPR64 in human endometrioid endometrial carcinoma by immunohistochemistry analysis. To determine a tumor suppressor role of GPR64 in endometrial cancer, we used a siRNA loss of function approach in human endometrial adenocarcinoma cell lines. RESULTS: GPR64 levels were remarkably lower in 10 of 21 (47.62%) of endometrial carcinoma samples compared to control. Depletion of GPR64 by siRNA transfection revealed an increase of colony formation ability, cell proliferation, cell migration, and invasion activity in Ishikawa and HEC1A cells. The expression of Connexin 43 (Cx43), a member of the large family of gap junction proteins, was reduced through activation of AMP-activated protein kinase (AMPK) in Ishikawa cells with GPR64-deficicy. CONCLUSIONS: These results suggest that GPR64 plays an important tumor suppressor role in endometrial cancer.

RBPJ mediates uterine repair in the mouse and is reduced in women with recurrent pregnancy loss.

Unexplained recurrent pregnancy loss (uRPL) is associated with repeated embryo loss and endometrial repair with elevated endometrial expression of inflammatory cytokines, including IFN-γ. Notch signaling through its transcription factor recombination signal binding protein Jκ (RBPJ) regulates mechanisms including the immune response and repair after tissue injury. Initially, null mutation of RBPJ in the mouse uterus ( Pgrcre/+Rbpjf/f; Rbpj c-KO) results in subfertility, but we have found that these mice become infertile after pregnancy as a result of dysfunctional postpartum uterine repair, including delayed endometrial epithelial and myometrial regeneration. RNA sequencing of postpartum uterine repair sites revealed global up-regulation of inflammatory pathways, including IFN signaling. Consistent with elevated IFN-γ, macrophages were recruited and polarized toward an M1-cytotoxic phenotype, which is associated with preventing repair and promoting further tissue injury. Through embryo transfer experiments, we show that dysfunctional postpartum repair directly impairs future embryo implantation in Rbpj c-KO mice. Last, we clinically correlated our findings from the Rbpj c-KO mouse in women diagnosed with uRPL. Reduced RBPJ in women with uRPL was associated with increased levels of IFN-γ. The data, taken together, indicate that RBPJ regulates inflammation during endometrial repair, which is essential for future pregnancy potential, and its dysregulation may serve as an unidentified contributor to uRPL in women.-Strug, M. R., Su, R.-W., Kim, T. H., Mauriello, A., Ticconi, C., Lessey, B. A., Young, S. L., Lim, J. M., Jeong, J.-W., Fazleabas, A. T. RBPJ mediates uterine repair in the mouse and is reduced in women with recurrent pregnancy loss.

The importance of post-thaw subculture for standardizing cellular activity of fresh or cryopreserved mouse embryonic stem cells.

OBJECTIVE: Remarkable difference in cellular activity was found between early and late subpassaged embryonic stem cell (ESCs) lines, which can be created by subtle changes in cell manipulation protocol. This study subsequently examined whether post-thaw subculture of early subpassaged ESC lines could further affect the activity of the ESCs. METHODS: Fresh (as a control treatment) or cryopreserved F1 hybrid (B6CBAF1) early ESC lines (C57BL/6xCBA) of the 4 (P4) or the 19 passage (P19) were subcultured once, twice or six times under the same condition. The post-thaw survival of the ESCs was monitored after the post-treatment subculture and the ability of cell proliferation, reactive oxygen species (ROS) generation, apoptosis and mitochondrial ATP synthesis was subsequently examined. RESULTS: Regardless of the subculture number, P19 ESCs showed better (p<0.05) doubling time and less ATP production than P4 ESCs and such difference was not influenced by fresh or cryopreservation. The difference between P4 and P19 ESC lines became decreased as the post-treatment subculture was increased and the six times subculture eliminated such difference. Similarly, transient but prominent difference in ROS production and apoptotic cell number was detected between P4 and P19 ESCs only at the 1st subculture after treatment, but no statistical differences between two ESC lines was detected in other observations. CONCLUSION: The results of this study suggest that post-thaw subculture of ESCs under the same environment is recommended for standardizing their cellular activity. The activity of cell proliferation ability and ATP synthesis can be used as parameters for quality control of ESCs.

Electrically tunable binary phase Fresnel lens based on a dielectric elastomer actuator.

We propose and demonstrate an all-solid-state tunable binary phase Fresnel lens with electrically controllable focal length. The lens is composed of a binary phase Fresnel zone plate, a circular acrylic frame, and a dielectric elastomer (DE) actuator which is made of a thin DE layer and two compliant electrodes using silver nanowires. Under electric potential, the actuator produces in-plane deformation in a radial direction that can compress the Fresnel zones. The electrically-induced deformation compresses the Fresnel zones to be contracted as high as 9.1% and changes the focal length, getting shorter from 20.0 cm to 14.5 cm. The measured change in the focal length of the fabricated lens is consistent with the result estimated from numerical simulation.

Curcumin induces ER stress-mediated apoptosis through selective generation of reactive oxygen species in cervical cancer cells.

Prolonged accumulation of misfolded or unfolded proteins caused by cellular stress, including oxidative stress, induces endoplasmic reticulum stress, which then activates an unfolded protein response (UPR). ER stress is usually maintained at higher levels in cancer cells as compared to normal cells due to altered metabolism in cancer. Here, we investigated whether curcumin is ER stress-mediated apoptosis in cervical cancer cells, and ROS increased by curcumin are involved in the process as an upstream contributor. Curcumin inhibited proliferation of cervical cancer cells (C33A, CaSki, HeLa, and ME180) and induced apoptotic cell death. Curcumin activated ER-resident UPR sensors, such as PERK, IRE-1α, and ATF6, and their downstream-signaling proteins in cervical cancer cells, but not in normal epithelial cells and peripheral blood mononuclear cells (PBMCs). CHOP, a key factor involved in ER stress-mediated apoptosis, was also activated by curcumin. CHOP decreased the ratio of anti-apoptotic protein Bcl-2 to pro-apoptotic protein Bax expression, and subsequently increased the apoptotic population of cervical cancer cells. Furthermore, curcumin elevated levels of intracellular reactive oxygen species (ROS) in cervical cancer cells, but not in normal epithelial cells. Scavenging ROS resulted in inhibition of ER stress and partially restored cell viability in curcumin-treated cancer cells. Collectively, these observations show that curcumin promotes ER stress-mediated apoptosis in cervical cancer cells through increase of cell type-specific ROS generation. Therefore, modulation of these differential responses to curcumin between normal and cervical cancer cells could be an effective therapeutic strategy without adverse effects on normal cells.

Gene expression profiles of human subcutaneous and visceral adipose-derived stem cells.

Subcutaneous and visceral adipose tissues show a different risk effect on metabolic disorders because they have distinct cellular properties. We isolated stem cells from the separate human adipose tissues to investigate that subcutaneous and visceral fat depots have metabolic differences. Adipose-derived stem cells (ASCs) were characterized by immunophenotype and differentiation potentials into adipogenic, osteogenic, and chondrogenic lineages. Although subcutaneous and visceral ASCs (S-ASC and V-ASC) express same surface markers (CD31- , CD34- , CD45- , CD73+ , CD90+ , and CD105+ ) and have differentiation potentials, S-ASCs had higher capacity to proliferate and to differentiate into adipogenic lineage than V-ASCs. Next, we identified that S-ASC and V-ASC were genetically distinct based on microarray analysis. Among a total of 810 genes detected in ASCs of both depots, the differentially expressed genes were involved in energy and lipid metabolism. These data show the existence of the intrinsic difference between S-ASC and V-ASC and suggest the differences of anatomically separated adipose tissue. On the basis of the differentially expressed gene profiles between S-ASC and V-ASC, we suggested significant evidence that adipose tissues originating from different anatomic regions are distinguished at the level of the undifferentiated stem cells such as mature adipocytes. V-ASCs had the upregulated clusters of genes related to lipid biosynthesis and metabolism. By contrast, S-ASCs highly expressed genes involved in DNA-dependent transcription, contributing to proliferation. We provide further insights for ASCs with the different origins to understand fat accumulation and distribution and a possibility of ASCs as a therapeutic target against metabolic disorders or cancer.

Development of a chemically defined in vitro culture system to effectively stimulate the proliferation of adult human dermal fibroblasts.

Despite the fact that dermal fibroblasts are a practical model for research related to cell physiology and cell therapy, an in vitro culture system excluding serum, which complicates standardization and specificity and induces variability and unwanted effects, does not exist. We tried to establish a CDCS that supports effective proliferation of aHDFs. KDMEM supplemented with 5% (v/v) KSR, 12 ng/ml bFGF, 5 ng/ml EGF and 1 µg/ml hydrocortisone supported sufficient proliferation of aHDFs for 1 week. However, aHDF proliferation was decreased greatly after subculture. This problem could be overcome by culturing aHDFs in CDCM in culture plates coated with 10 µg/ml FN. Long-term culture of aHDFs was achieved using CDCM and FN-coated culture plates for 7 weeks. The optimized CDCS increased the proliferation of aHDFs significantly, without any increase in the senescence rate or alteration in morphology of aHDFs, despite long-term culture. In conclusion, we established a CDCS that improved proliferation of aHDFs while inhibiting cellular senescence. The CDCS will contribute to advances in various future research related to clinical skin regeneration.

Transformation of somatic cells into stem cell-like cells under a stromal niche.

To study the genomic plasticity of somatic cells without ectopic genetic manipulation, we cultured mouse fibroblasts with ovarian cells, embryonic fibroblasts of different strains, and parthenogenetic embryonic stem cells (ESCs). Of 41 trials, cell aggregation resembling nascent ESC colony from inner cell mass was detected in 9 cases (22%), and 6 cases (67%) yielded fibroblast-derived colonies with ESC morphology. Cells used in coculture provided the critical (P=0.0061) inducing factor for the aggregation. These colony-forming fibroblasts (CFFs) showed similar characteristics to those in ESCs and induced pluripotent stem cells (iPSCs), including pluripotency gene expression, in vitro differentiation, and teratoma formation. Furthermore, CFFs produced somatic chimera, although none showed germline chimerism. CFFs had a tetraploid-like karyotype, and their imprinting patterns differed from parthenogenetic ESCs, thereby confirming their nongermline transmissibility. We observed dysregulation of cell cycle-related proteins, as well as both homologous and heterologous recombination of genomic single-nucleotide polymorphisms in CFFs. Our observations provide information on somatic cell plasticity, resulting in stemness or tumorigenesis, regardless of colony-forming cell progenitors in the fibroblast population. The plasticity of somatic genomes under environmental influences, as well as acquisition of pluripotency by cell fusion, is also implicated.

ß-Catenin activation contributes to the pathogenesis of adenomyosis through epithelial-mesenchymal transition.

Adenomyosis is defined by the presence of endometrial glands and stroma within the myometrium. Despite its frequent occurrence, the precise aetiology and physiopathology of adenomyosis is still unknown. WNT/ß-catenin signalling molecules are important and should be tightly regulated for uterine function. To investigate the role of ß-catenin signalling in adenomyosis, the expression of ß-catenin was examined. Nuclear and cytoplasmic ß-catenin expression was significantly higher in epithelial cells of human adenomyosis compared to control endometrium. To determine whether constitutive activation of ß-catenin in the murine uterus leads to development of adenomyosis, mice that expressed a dominant stabilized ß-catenin in the uterus were used by crossing PR-Cre mice with Ctnnb1(f(ex3)/+) mice. Uteri of PR(cre) (/+) Ctnnb1(f(ex3)/+) mice displayed an abnormal irregular structure and highly active proliferation in the myometrium, and subsequently developed adenomyosis. Interestingly, the expression of E-cadherin was repressed in epithelial cells of PR(cre) (/+) Ctnnb1(f(ex3)/+) mice compared to control mice. Repression of E-cadherin is one of the hallmarks of epithelial-mesenchymal transition (EMT). The expression of SNAIL and ZEB1 was observed in some epithelial cells of the uterus in PR(cre) (/+) Ctnnb1(f(ex3)/+) mice but not in control mice. Vimentin and COUP-TFII, mesenchymal cell markers, were expressed in some epithelial cells of PR(cre) (/+) Ctnnb1(f(ex3)/+) mice. In human adenomyosis, the expression of E-cadherin was decreased in epithelial cells compared to control endometrium, while CD10, an endometrial stromal marker, was expressed in some epithelial cells of human adenomyosis. These results suggest that abnormal activation of ß-catenin contributes to adenomyosis development through the induction of EMT.

Progesterone induces expression of Lrp2 in the murine uterus.

Progesterone (P4) and progesterone receptor (PR) have important functions in uterine environment. In previous studies, using high density DNA microarray analysis, we identified low density lipoprotein receptor-related protein 2 (Lrp2) is one of the genes upregulated by P4 and PR. In present studies, we examined the expression of Lrp2 through real-time PCR, in situ hybridization and immunohistochemistry by P4-PR response. Lrp2 mRNA transcript was significantly increased after P4 treatment in the luminal and glandular epithelium of the wild-type mice. However, Lrp2 expression was not observed in the progesterone receptor knock out (PRKO) mice treated with P4. The expression of Lrp2 expression is not regulated by estrogen. During early pregnancy, the expression of Lrp2 was detected at 2.5 dpc and then significantly increased at 3.5 dpc in luminal and glandular epithelium. These results suggest that Lrp2 is a novel target gene by P4 and PR.

Generation of priming mesenchymal stem cells with enhanced potential to differentiate into specific cell lineages using extracellular matrix proteins.

Poor understanding of the differentiation of mesenchymal stem cells (MSCs) has resulted in a low differentiation yield, and has hindered their application in medicine. As a solution, priming MSCs sensitive to signaling, thus stimulating differentiation into a specific cell lineage, may improve the differentiation yield. To demonstrate this, priming MSCs were produced by using a gelatin matrix for the isolation of primary MSCs from bone-marrow-derived primary cells. Subsequently, cellular characteristics and sensitivity to specific differentiation signals were analyzed at passage five. Compared to non-priming MSCs, priming MSCs showed no significant differences in cellular characteristics, but demonstrated a significant increase in sensitivity to neurogenic differentiation signals. These results demonstrate that generation of priming MSCs by specific extracellular signaling increases the rate of differentiation into a cell-specific lineage.

Development of a serum-free defined system employing growth factors for preantral follicle culture.

This study was conducted to evaluate if mouse preantral follicles can yield developmentally competent oocytes following culture in serum-free, defined medium. Donor follicles were obtained from 14-day-old B6CBAF1 mice, and cultured in α-MEM-Glutamax medium. The replacement of fetal bovine serum with knockout serum replacement (KSR) did not significantly reduce follicle growth or oocyte maturation in vitro, although it significantly reduced the development of oocytes after activation. Regardless of the replacement medium, follicle growth was not influenced by the addition of leukemia inhibitory factor (LIF). The addition of 100 ng/ml stem cell factor (SCF) to the KSR-supplemented serum-free medium significantly stimulated follicle development, which further improved blastocyst formation after oocyte activation. On Day 3 of culture, a significant increase in Bmp7 expression was detected in the SCF-containing medium compared with the serum-containing medium, whereas Gdf9 and Amh were increased in the serum-containing medium. A significant increase in estradiol production was detected under serum-free conditions, but minimal progesterone secretion was detected throughout the culture period. In conclusion, serum-free media can be used to optimize ovarian follicle cultures, and the addition of SCF is beneficial for deriving developmentally competent oocytes through follicle culture.

Development of Polyethylene Glycol-based Hydrogels Optimized for In Vitro 3D Culture of HepG2 Hepatocarcinoma Cells.

BACKGROUND/AIM: We report an in vitro three-dimensional (3D) culture system optimized for the growth of HepG2 hepatocarcinoma cells. MATERIALS AND METHODS: The 3D culture system was fabricated based on polyethylene glycol (PEG)-based hydrogels; their mechanical strength was controlled by differences in the arm number and concentration of PEG-vinylsulfone. Moreover, cellular growth was evaluated after culturing HepG2 cells in PEG-based hydrogels with various mechanical strengths. RESULTS: HepG2 cell culture in the 3D PEG-based hydrogels induced the formation of spherical colonies. Moreover, the highest number of spherical colonies formed from HepG2 cells at the single-cell level, and the formation of spherical colonies with a uniform size was observed in HepG2 cells cultured in 5% (w/v) 8-arm PEG-based hydrogels. CONCLUSION: 5% (w/v) 8-arm PEG-based hydrogels may be developed as a 3D culture system optimized for stimulating the in vitro growth of HepG2 cells.

Replacement of mouse embryonic fibroblasts with bone marrow stromal cells for use in establishing and maintaining embryonic stem cells in mice.

We have investigated the use of BMSC (bone marrow stromal cell) as a feeder cell for improving culture efficiency of ESC (embryonic stem cell). B6CBAF1 blastocysts or ESC stored after their establishment were seeded on to a feeder layer of either SCA-1+/CD45-/CD11b- BMSC or MEF (mouse embryonic fibroblast). Feeder cell activity in promoting ESC establishment from the blastocysts and in supporting ESC maintenance did not differ significantly between BMSC and MEF feeders. However, the highest efficiency of colony formation after culturing of inner cell mass cells of blastocysts was observed with the BMSC line that secreted the largest amount of LIF (leukaemia inhibitory factor). Exogenous LIF was essential for the ESC establishment on BMSC feeder, but not for ESC maintenance. Neither change in stem cell-specific gene expression nor increase in stem cell aneuploidy was detected after the use of BMSC feeder. We conclude that BMSC can be utilized as the feeder of ESC, which improves culture efficiency.

Generation of viable embryos and embryonic stem cell-like cells from cultured primary follicles in mice.

Primary follicles retrieved from B6CBAF1 prepubertal mice were cultured in a stepwise manner in an alpha-minimum essential medium-based medium to generate viable embryos and embryonic stem cell (ESC)-like cells. A significant increase in follicle growth and oocyte maturation accompanied by increased secretion of 17beta-estradiol and progesterone was achieved by exposing primary follicles to 100 or 200 mIU of follicle-stimulating hormone (FSH) during culture. More oocytes developed into blastocysts following in vitro fertilization (IVF) or parthenogenetic activation after culture with 200 mIU of FSH during the entire culture period than with 100 mIU. Eleven ESC-like cell lines, consisting of four heterozygotic and seven homozygotic phenotypes, were established from 25 trials of primary follicle culture combined with IVF or parthenogenetic activation. In conclusion, primary follicles can potentially yield developmentally competent oocytes, which produce viable embryos and ESC-like cell lines following in vitro manipulation. We suggest a method to utilize immature follicles, which are most abundant in ovaries, to improve reproductive efficiency and for use in regenerative medicine.