Ji Yeon Ahn

β-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/β‐catenin signalling molecules are important and should be tightly regulated for uterine function. To investigate the role of β‐catenin signalling in adenomyosis, the expression of β‐catenin was examined. Nuclear and cytoplasmic β‐catenin expression was significantly higher in epithelial cells of human adenomyosis compared to control endometrium. To determine whether constitutive activation of β‐catenin in the murine uterus leads to development of adenomyosis, mice that expressed a dominant stabilized β‐catenin in the uterus were used by crossing PR‐Cre mice with Ctnnb1f(ex3)/+ mice. Uteri of PRcre/+ Ctnnb1f(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 PRcre/+ Ctnnb1f(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 PRcre/+ Ctnnb1f(ex3)/+ mice but not in control mice. Vimentin and COUP‐TFII, mesenchymal cell markers, were expressed in some epithelial cells of PRcre/+ Ctnnb1f(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 β‐catenin contributes to adenomyosis development through the induction of EMT. Copyright

A feeder-free, defined three-dimensional polyethylene glycol-based extracellular matrix niche for culture of human embryonic stem cells.

We report optimization of a serum- and feeder-free, three-dimensional (3D) niche created with a synthetic polyethylene glycol (PEG)-based extracellular matrix for self-renewal of human embryonic stem cells (hESCs). Three hESC lines (H9, H1 and Novo) were cultured in hydrogels of different mechanical properties, and cellular morphology and activity were compared to culture in feeder-free or feeder-containing two-dimensional (2D) niches. Significant effects of PEG concentration (5, 7.5, 10, 12.5 or 15%) and vinyl sulfone-functionalized PEG multiarm number (3, 4 or 8) on hESC morphology were detected in the H9 line. Cell growth was maximal with an 8 multiarm architecture of any PEG concentration, which yielded the highest expression of stemness-related genes. Alkaline phosphatase activity in cultured H9 cells was similar between the optimized feeder-free 3D and the feeder-containing 2D systems. However, increased expression of the KLF4, CDH1, TERT, SOX2, and UTF1 genes and expression of pluripotency-specific SSEA-4, Oct3/4, Nanog, Tra-1-60 and Tra-1-81 were detected in the 3D-cultured hESC clumps. H1 and Novo cell lines also expanded in the optimized 3D system, which maintain stemness properties. Although different proliferation activities were detected among three lines, the difference was decreased after the 3D culture. These results demonstrate that chemically defined, acellular niches created using PEG-based hydrogels have the potential to support hESC self-renewal. Modulation of 3D properties can create various models for cell transformation and differentiation.

Extracellular Signal-Regulated Kinase 1/2 Signaling Pathway Is Required for Endometrial Decidualization in Mice and Human

Decidualization is a crucial change required for successful embryo implantation and the maintenance of pregnancy. During this process, endometrial stromal cells differentiate into decidual cells in response to the ovarian steroid hormones of early pregnancy. Extracellular signal-regulated protein kinases 1 and 2 (ERK1/2) are known to regulate cell proliferation and apoptosis in multiple cell types, including uterine endometrial cells. Aberrant activation of ERK1/2 has recently been implicated in the pathological processes of endometriosis and endometrial cancer. However, the function of ERK1/2 signaling during implantation and decidualization is still unknown. To determine the role and regulation of ERK1/2 signaling during implantation and decidualization, we examine ERK1/2 signaling in the mouse uterus during early pregnancy using immunostaining and qPCR. Interestingly, levels of phospho-ERK1/2 were highest within decidual cells located at the implantation sites. Expression levels of ERK1/2 target genes were also significantly higher at implantation sites, when compared to either inter-implantation sites. To determine if ERK1/2 signaling is also important during human endometrial decidualization, we examined levels of phospho-ERK1/2 in cultured human endometrial stromal cells during in vitro decidualization. Following treatment with a well-established decidualization-inducing steroidogenic cocktail, levels of phospho-ERK1/2 were markedly increased. Treatment with the ERK1/2 inhibitor, U0126, significantly decreased the expression of the known decidualization marker genes, IGFBP1 and PRL as well as inhibited the induction of known ERK1/2 target genes; FOS, MSK1, STAT1, and STAT3. Interestingly, the phosphorylation level of CCAAT/ enhancer binding protein β (C/EBPβ), a protein previously shown to be critical for decidualization, was significantly reduced in this model. These results suggest that ERK1/2 signaling is required for successful decidualization in mice as well as human endometrial stromal cells and implicates C/EBPβ as a downstream target of ERK1/2.

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.—Lee, S. T., Gong, S. P., Yum, K. E., Lee, E. J., Lee, C. H., Choi, J. H., Kim, D. Y., Han, H., Kim, K.‐S., Hysolli, E., Ahn, J. Y., Park, I.‐H., Han, J. Y., Jeong, J.‐W., Lim, J. M. Transformation of somatic cells into stem cell‐like cells under a stromal niche. FASEB J. 27, 2644‐2656 (2013). www.fasebj.org

Mass Production of Early-Stage Bone-Marrow-Derived Mesenchymal Stem Cells of Rat Using Gelatin-Coated Matrix

Although preparation of early-stage bone-marrow-derived mesenchymal stem cells (BM-MSCs) is critical for successful cell transplantation therapy, no culture system offers a sufficient number of early-stage BM-MSCs for cell transplantation. Accordingly, we developed a culture system capable of producing a large number of early-stage BM-MSCs by using gelatin-coated matrix. The greatest retrieval and proliferation rates of the earliest-stage rat BM-MSCs were detected in bone-marrow-derived cells cultured on 1% (wt/v) gelatin-coated matrix, which showed significantly greater colony forming unit-fibroblast number, diameter, and total cell number. Moreover, continuous culture of the earliest-stage BM-MSCs on 1% (wt/v) gelatin-coated matrix resulted in a maximum of 21.2 ± 2.7 fold increase in the cumulative total number of early-stage BM-MSCs at passage 5. BM-MSCs generated in large quantities due to a reduced doubling time and an increased yield of cell population in S/G2/M phase showed typical fibroblast-like morphology and no significant differences in BM-MSC-related surface marker expression and differentiation potential, except for an increased ratio of differentiation into a neurogenic lineage. The use of gelatin-coated matrix in the retrieval and culture of BM-MSCs contributes greatly to the effective isolation and mass production of early-stage BM-MSCs.

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 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 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.

Culture of preantral follicles in poly(ethylene) glycol‐based, three‐dimensional hydrogel: a relationship between swelling ratio and follicular developments

This study was undertaken to examine how the softness of poly(ethylene) glycol (PEG)‐based hydrogels, creating a three‐dimensional (3D) microenvironment, influences the in vitro growth of mouse ovarian follicles. Early secondary, preantral follicles of 2u2009week‐old mice were cultured in a crosslinked four‐arm PEG hydrogel. The hydrogel swelling ratio, which relates to softness, was modified within the range 25.7–15.5 by increasing the reactive PEG concentration in the precursor solution from 5% to 15% w/v, but it did not influence follicular growth to form the pseudoantrum (60–80%; pu2009=u20090.76). Significant (pu2009<u20090.04) model effects, however, were detected in the maturation and developmental competence of the follicle‐derived oocytes. A swelling ratio ofu2009>u200921.4 yielded better oocyte maturation than other levels, while the highest competence to develop pronuclear and blastocyst formation was detected at 20.6. In conclusion, gel softness, as reflected in swelling ratio, was one of the essential factors for supporting folliculogenesis in vivo within a hydrogel‐based, 3D microenvironment.

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 100u2009ng/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. Mol. Reprod. Dev. 80: 725–733, 2013.