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.

Effects of cumulus cells on the in vitro cytoplasmic maturation of immature oocytes in pigs.

The exposure of cumulus cells to nuclear matured oocytes can be regulated through the forced delay of nuclear maturation or the alteration of in vitro maturation (IVM) time in cumulus-oocyte complexes (COCs). However, to date, no evidence has been presented for the enhancement of cytoplasmic maturation by them, indicating irrelevance of cumulus cells in cytoplasmic maturation. Therefore, in order to identify the requirement of cumulus cells in achieving the cytoplasmic maturation of immature oocytes, this study investigated the effects of cumulus cells on the in vitro cytoplasmic maturation of oocytes within COCs derived from porcine medium antral follicles (MAFs) post-the completion of nuclear maturation. For these, with IVM of COCs for 44 h (control), cumulus cell-free oocytes with completed nuclear maturation were in-vitro-matured additionally for 0, 6, or 12 h, and then a variety of factors representing the cytoplasmic maturation of oocytes were analyzed and compared. As the results, the IVM of COCs for 32 h showed complete nuclear maturation and incomplete cytoplasmic maturation. Moreover, after the removal of cumulus cells from COCs with the completion of nuclear maturation, IVM for an additional 6 or 12 h resulted in significant increases in the size of the perivitelline space, the proportion of oocytes with a normal intracellular mitochondrial distribution and a normal round first polar body, and the preimplantation development into the 2-cell and blastocyst stages after parthenogenetic activation. Simultaneously, they showed significant reduction in the level of intracellular reactive oxygen species and no significant differences in the total number of blastocysts. Furthermore, oocytes obtained by this approach did not significantly differ from control oocytes produced by IVM of COCs for 44 h. Our results demonstrate that the cumulus cells enclosing COCs derived from porcine MAFs are not essential for the completion of cytoplasmic maturation after complete nuclear maturation by COCs.

Effect of Human Peripheral Blood Mononuclear Cells on Mouse Endometrial Cell Proliferation: A Potential Therapeutics for Endometrial Regeneration.

OBJECTIVES: The persistently thin endometrium is a major cause of repeated implantation failure; however, there is no definite treatment for it yet. This study aimed to confirm the potential of human peripheral blood mononuclear cells (hPBMCs) as a therapeutic agent for endometrial regeneration. DESIGN: An experimental study was carried out. PARTICIPANTS/MATERIALS, SETTING, METHODS: To assess the in vitro effect of hPBMC, the human primary endometrial epithelial cell lines SNU-685 and SNU-1077 were co-cultured with or without 1 × 105 hPBMCs for 24 h. To evaluate the in vivo effect, either 1 × 105 hPBMCs in PBS or PBS alone were injected into the left uterine horn of nonobese diabetic-severe combined immune-deficient mice, and the right untreated uterine horn was used as control. RESULTS: Co-culture with hPBMCs stimulated significant proliferation in both SNU-685 and SNU-1077 cell lines (p = 0.002 and 0.044, respectively). Moreover, treatment with hPBMCs significantly increased the thickness in all parts of the endometrium compared with that in the untreated control uterine horn (proximal: 1.69 ± 0.19 vs. 1.00 ± 0.10, p = 0.009; middle: 1.51 ± 0.14 vs. 1.00 ± 0.12, p = 0.010; distal: 1.72 ± 0.22 vs. 1.00 ± 0.12, p = 0.003, respectively). Compared with the PBS injection group, the hPBMC injection group had significantly thickened endometrium in the middle (p = 0.036) and distal segments (p = 0.002) of the uterine horn. Immunohistochemical analysis revealed the presence of exogenously injected hPBMCs in the uterus of recipient mice. hPBMC-recipient mice had cyclic uterus with normal histology in the endometrium. LIMITATIONS: hPBMCs were not applied directly to a mouse model with thin endometrium, so further study is needed. CONCLUSION: The beneficial effect of hPBMCs on endometrium may suggest their clinical feasibility for the safe treatment of infertile patients with persistently thin endometrium.

Endothelin-1 enhances the regenerative capability of human bone marrow-derived mesenchymal stem cells in a sciatic nerve injury mouse model.

We expanded the application of endothelin-1 (EDN1) by treating human mesenchymal stem cell (hMSC) organotypic spinal cord slice cultures with EDN1. EDN1-treated hMSCs significantly enhanced neuronal outgrowth. The underlying mechanism of this effect was evaluated via whole-genome methylation. EDN1 increased whole-genome demethylation and euchromatin. To observe demethylation downstream of EDN1, deaminases and glycosylases were screened, and APOBEC1 was found to cause global demethylation and OCT4 gene activation. The sequence of methyl-CpG-binding domain showed similar patterns between EDN1- and APOBEC1-induced demethylation. SWI/SNF-related, matrix-associated, actin-dependent regulator of chromatin subfamily A member 4 (SMARC A4) and SMARC subfamily D, member 2 (SMARC D2) were screened via methyl-CpG-binding domain sequencing as a modulator in response to EDN1. Chromatin immunoprecipitation of the H3K9me3, H3K27me3, and H3K4me4 binding sequences on the APOBEC1 promoter was analyzed following treatment with or without siSMARC A4 or siSMARC D2. The results suggested that SMARC A4 and SMARC D2 induced a transition from H3K9me3 to H3K4me3 in the APOBEC1 promoter region following EDN1 treatment. Correlations between EDN1 pathways and therapeutic efficacy in hBM-MSCs were determined in a sciatic nerve injury mouse model. Thus, EDN1 may be a useful novel-concept bioactive peptide and biomaterial component for improving hMSC regenerative capability.

The native form of follicle-stimulating hormone is essential for the growth of mouse preantral follicles in vitro.

To assess whether the follicle-stimulating hormone (FSH) subunits observed in patients with gonadotroph adenomas (GA) can cause infertility, the effects of subunits and heterodimeric FSH on the in vitro follicle development were evaluated in mice. The partial forms of FSH in follicle culture did not induce development into pseudoantral follicles, whereas follicles cultured with native FSH developed into pseudoantral follicles and produced mature metaphase II oocyte. Therefore, intact FSH is needed for folliculogenesis, implying that production of FSH with a partial structure in GA may result in infertility.

Hepatic stellate cell-specific knockout of transcriptional intermediary factor 1γ aggravates liver fibrosis.

Transforming growth factor ß (TGFß) is a crucial factor in fibrosis, and transcriptional intermediary factor 1γ (TIF1γ) is a negative regulator of the TGFß pathway; however, its role in liver fibrosis is unknown. In this study, mesenchymal stem cells derived from human embryonic stem cells (hE-MSCs) that secrete hepatocyte growth factor (HGF) were used to observe the repair of thioacetamide (TAA)-induced liver fibrosis. Our results showed that TIF1γ was significantly decreased in LX2 cells when exposed to TGFß1. Such decrease of TIF1γ was significantly prevented by co-culture with hE-MSCs. Interaction of TIF1γ with SMAD2/3 and binding to the promoter of the α-smooth muscle gene (αSMA) suppressed αSMA expression. Phosphorylation of cAMP response element-binding protein (CREB) and binding on the TIF1γ promoter region induced TIF1γ expression. Furthermore, hepatic stellate cell-specific TIF1γ-knockout mice showed aggravation of liver fibrosis. In conclusion, loss of TIF1γ aggravates fibrosis, suggesting that a strategy to maintain TIF1γ during liver injury would be a promising therapeutic approach to prevent or reverse liver fibrosis.

Human Embryonic Stem Cells-Derived Mesenchymal Stem Cells Reduce the Symptom of Psoriasis in Imiquimod-Induced Skin Model.

BACKGROUND: Mesenchymal stem cells (MSCs) can be used for a wide range of therapeutic applications because of not only their differentiation potential but also their ability to secrete bioactive factors. Recently, several studies have suggested the use of human embryonic stem cell-derived MSCs (hE-MSCs) as an alternative for regenerative cellular therapy due to mass production of MSCs from a single donor. METHODS: We generated hE-MSCs from embryonic stem cell lines, SNUhES3, and analyzed immune properties of these cells. Also, we evaluated the in-vivo therapeutic potential of hE-MSCs in immune-mediated inflammatory skin disease. RESULTS: The cell showed the suppression of immunity associated with allogenic peripheral blood mononuclear cells in mixed lymphocyte response assay. We also detected that cytokines and growth factor related to the immune response were secreted from these cells. To assessed the in-vivo therapeutic potential of hE-MSCs in immune-mediated inflammatory skin disease, we used imiquimod (IMQ)-induced skin psoriasis mouse model. The score of clinical skin was significantly reduced in the hE-MSCs treated group compared with control IMQ group. In histological analysis, the IMQ-induced epidermal thickness was significantly decreased by hE-MSCs treatment. It was correlated with splenomegaly induced by IMQ which was also improved in the hE-MSCs. Moreover, IMQ-induced inflammatory cytokines; Th1 cytokines (TNF-α, IFN-α, IFN-γ,and IL-27) and Th17 cytokines (IL-17A and IL-23), in the serum and skin showed marked inhibition by hE-MSCs. CONCLUSION: These results suggested that hE-MSCs have a potency of immune modulation in psoriasis, which might be the key factor for the improved psoriasis.

Hepatocyte Growth Factor Improves the Therapeutic Efficacy of Human Bone Marrow Mesenchymal Stem Cells via RAD51.

Human embryonic stem cell-derived mesenchymal stem cells (hE-MSCs) have greater proliferative capacity than other human mesenchymal stem cells (hMSCs), suggesting that they may have wider applications in regenerative cellular therapy. In this study, to uncover the anti-senescence mechanism in hE-MSCs, we compared hE-MSCs with adult bone marrow (hBM-MSCs) and found that hepatocyte growth factor (HGF) was more abundantly expressed in hE-MSCs than in hBM-MSCs and that it induced the transcription of RAD51 and facilitated its SUMOylation at K70. RAD51 induction/modification by HGF not only increased telomere length but also increased mtDNA replication, leading to increased ATP generation. Moreover, HGF-treated hBM-MSCs showed significantly better therapeutic efficacy than naive hBM-MSCs. Together, the data suggest that the RAD51-mediated effects of HGF prevent hMSC senescence by promoting telomere lengthening and inducing mtDNA replication and function, which opens the prospect of developing novel therapies for liver disease.

Development of Au nanowire injector system to deliver plasmid into mouse embryo.

In this data article, we developed a Au nanowire injector (Au NWI) for directly delivering plasmid into the 1-cell stage of the mouse embryos designed to successfully attach and detach the plasmid on the Au NWI, highly minimizing physical and chemical damage on the embryos. This data presents that a Au NWI system does not induce detrimental damages on development of embryos and efficiently express the green fluorescence protein in vitro. The data provided herein is in association with the research article related to reduce the occurrence of mosaicism by a Au NWI," Suppressing Mosaicism by Au Nanowire Injector-driven Direct Delivery of Plasmids into Mouse Embryos" (Park et al., 2017 [1]).

Suppressing mosaicism by Au nanowire injector-driven direct delivery of plasmids into mouse embryos.

Transgenic animals have become key tools in a variety of biomedical research areas. However, microinjection commonly used for producing transgenic animals has several challenges such as physical and chemical damage to the embryos due to microinjector with buffer, and low transgene integration rates with frequent mosaicism. Here, we report direct delivery of plasmids into mouse embryos using a Au nanowire injector (NWI) that significantly improved transgene integration efficiency and suppressed mosaicism. The Au NWI could deliver plasmid into the pronucleus (PN) of a mouse zygote without buffer and rapidly release it with electric pulse. Because zygote, which is a fertilized 1-cell stage embryo, has two physical barriers (cytoplasmic membrane and zona pellucida), direct delivery of plasmids into PN of zygote is more difficult than into a normal cell type. To penetrate the two physical barriers with minimal disruption of the embryo, we optimized the diameter and length of Au NWI. The mosaicism is more reduced in the Au NWI injected embryos than in micropipette injected embryos, which was determined by the expression of transgene in a blastocyst stage. We suggest that Au NWI can increase the efficiency of gene delivery into zygote with suppressed mosaicism and become a useful alternative.