High TXNIP expression accelerates the migration and invasion of the GDM placenta trophoblast.

INTRODUCTION: Our previous study has proofed the glucose sensitive gene-thioredoxin-interacting protein (TXNIP) expression was up in the placenta of the patients with gestational diabetes mellitus (GDM), but the pathological mechanisms underlying abnormal TXNIP expression in the placenta of patients with GDM is completely unclear and additional investigations are required to explain the findings we have observed. In the present study, we simulated the high TXNIP expression via introducing the Tet-On "switch" in vitro, approximate to its expression level in the real world, to explore the following consequence of the abnormal TXNIP. METHODS: The expression and localization of TXNIP in the placenta of GDM patients and the health control was investigated via immunofluorescent staining, western blot and RT-qPCR. Overexpression of TXNIP was achieved through transfecting Tet-on system to the human trophoblastic cell line-HTR-8/Svneo cell. TXNIP knockout was obtained via CRISPR-Cas9 method. The cell phenotype was observed via IncuCyte Imaging System and flow cytometry. The mechanism was explored via western blot and RT-qPCR. RESULTS: The expression level of TXNIP in the GDM placenta was nearly 2-3 times higher than that in the control. The TXNIP located at trophoblastic cells of the placenta. When the expression of TXNIP was upregulated, the migration and invasion of the cells accelerated, but cell apoptosis and proliferation did not changed compared with the control group. Furthermore, the size of the TetTXNIP cells became larger, and the expression level of Vimentin and p-STAT3 increased in the TetTXNIP cells. All the changes mentioned above were opposite in the TXNIP-KO cells. CONCLUSIONS: Abnormal expression of TXNIP might be related to the impairment of the GDM placental function, affecting the migration and invasion of the placental trophoblast cells through STAT3 and Vimentin related pathway; thus, TXNIP might be the potential therapeutic target for repairing the placental dysfunction deficient in GDM patients.

Fgf21 knockout mice generated using CRISPR/Cas9 reveal genetic alterations that may affect hair growth.

OBJECTIVE: To investigate fibroblast growth factor 21 (Fgf21) alterations that may affect hair growth and the underlying molecular mechanisms by constructing Fgf21 global knockout (KO) mice using microinjection-mediated CRISPR/Cas9. RESULTS: Following genomic DNA sequencing, we identified 18 mice carrying Ffg21 mutations among the total 63 offspring mice obtained by injecting 340 embryos, which yielded a mutation rate of 28.6 percent. Of these 18 mice, three had both alleles knocked out and 15 were monoallelic KO mice. Compared with the wild-type (WT) mice, the phenotypic analysis showed that the litter size of Fgf21 KO mice significantly reduced (p < 0.05), but physiological indexes of the birth weight, gender rate, body weight (0-8 week) and body weight of adult male and female were no significant difference (p > 0.05). Compared to WT mice, physiological anatomy indicated that the morphological characters of vital organs in Fgf21 KO mice were normal. Depilation experiments demonstrated that compared to the WT mice, the hair regrowth speed was reduced in the Fgf21 KO mice. The number of hair shafts in these mice considerably decreased, as indicated by the tissue sample analyses. Real-time quantitative PCR showed that Erk and Akt expression in the KO mice was significantly decreased (P < 0.05), whereas western blotting demonstrated that the expression of Erk and Akt proteins and their phosphorylation levels in KO mice decreased at different rates (P < 0.05). CONCLUSIONS: Fgf21 was shown to affect hair follicle development and growth cycle, which may be associated with Pi3k/Akt and Mapk/Erk signaling pathways.

Asymmetricity Between Sister Cells of Pluripotent Stem Cells at the Onset of Differentiation.

Various somatic stem cells divide asymmetrically; however, it is not known whether embryonic stem cells (ESCs) divide symmetrically or asymmetrically, not only while maintaining an undifferentiated state but also at the onset of differentiation. In this study, we observed single ESCs using time-lapse imaging and compared sister cell pairs derived from the same mother cell in either the maintenance or differentiation medium. Mouse ESCs were cultured on E-cadherin-coated glass-based dishes, which allowed us to trace single cells. The undifferentiated cell state was detected by green fluorescent protein (GFP) expression driven by the Nanog promoter, which is active only in undifferentiated cells. Cell population analysis using flow cytometry showed that the peak width indicating distribution of GFP expression broadened when cells were transferred to the differentiation medium compared to when they were in the maintenance medium. This finding suggested that the population of ESCs became more heterogeneous at the onset of differentiation. Using single-cell analysis by time-lapse imaging, we found that although the total survival ratio decreased by changing to differentiation medium, the one-live-one-dead ratio of sister cell pairs was smaller compared with randomly chosen non-sister cell pairs, defined as an unsynchronized cell pair control, in both media. This result suggested that sister cell pairs were more positively synchronized with each other compared to non-sister cell pairs. The differences in interdivision time (the time interval between mother cell division and the subsequent cell division) between sister cells was smaller than that between non-sister cell pairs in both media, suggesting that sister cells divided synchronously. Although the difference in Nanog-GFP intensity between sister cells was smaller than that between non-sister cells in the maintenance medium, it was the same in differentiation medium, suggesting asymmetrical Nanog-GFP intensity. These data suggested that ESCs may divide asymmetrically at the onset of differentiation resulting in heterogeneity.

The differentiation and isolation of mouse embryonic stem cells toward hepatocytes using galactose-carrying substrata.

A simple culture system to achieve the differentiation of embryonic stem (ES) cells toward hepatocytes with high efficiency is crucial in providing a cell source for the medical application. In this study, we report the effect of a matrix-dependent enrichment of ES cell-derived hepatocytes using immobilized poly(N-p-vinylbenzyl-4-O-ß-D-galactopyranosyl-D-gluconamide) (PVLA) with E-cadherin-IgG Fc (E-cad-Fc) as a galactose-carrying substratum. PVLA and E-cad-Fc were confirmed to be stably co-adsorbed onto polystyrene surface by quartz crystal microbalance (QCM). We showed that the E-cad-Fc/PVLA hybrid substratum was efficient in culturing primary hepatocytes and maintaining liver functions, on which the undifferentiated ES cells also maintained high proliferative capability. Furthermore, ES cell-derived hepatocytes on this hybrid matrix expressed elevated level of liver specific genes and functions together with early expression of definitive hepatocyte marker, asialoglycoprotein receptor (ASGPR). Finally, we isolated a high percentage of cells (about 60%) with ASGPR expression after re-seeding onto PVLA-coated surface, and observed the elimination of the poorly differentiated cells (Gata6(+) and Sox17(+)) and the ones toward another cell lineage (brachyury(+) and Pdx1(+)). The system uses a glycopolymer as an extracellular substratum for isolation and enrichment of ES cell-derived hepatocytes with adequate homogeneity and functionality.

The effect of recombinant E-cadherin substratum on the differentiation of endoderm-derived hepatocyte-like cells from embryonic stem cells.

Generation of specific lineages of cells from embryonic stem (ES) cells is pre-requisite to use these cells in pre-clinical applications. Here, we developed a recombinant E-cadherin substratum for generation of hepatic progenitor populations at single cell level. This artificial acellular feeder layer supports the stepwise differentiation of ES cells to cells with characteristics of definitive endoderm, hepatic progenitor cells, and finally cells with phenotypic and functional characteristics of hepatocytes. The efficient differentiation of hepatic endoderm cells (approximately 55%) together with the absence of neuroectoderm and mesoderm markers suggests the selective induction of endoderm differentiation. The co-expression of E-cahderin and alpha-fetoprotein (approximately 98%) suggests the important role of E-cadherin as a surface marker for the enrichment of hepatic progenitor cells. With extensive expansion, approximately 92% albumin expressing cells can be achieved without any enzymatic stress and cell sorting. Furthermore, these mouse ES cell-derived hepatocyte-like cells showed higher morphological similarities to primary hepatocytes. In conclusion, we demonstrated that E-cadherin substratum can guide differentiation of ES cells into endoderm-derived hepatocyte-like cells. This recombinant extracellular matrix could be effectively used as an in vitro model for studying the mechanisms of early stages of liver development even at single cell level.

Self-organization of the compositional gradient structure in hyaluronic acid and poly(N-isopropylacrylamide) blend film.

A compositional gradient structure in hyaluronic acid (HA) and poly(N-isopropylacrylamide) (PIPAAm) blend film was self-organized from a homogeneous aqueous solution in a plasma-treated polystyrene dish (PTPSD), and the formation mechanisms of the gradient structure were studied by casting the same solution on PTPSD and a non-treated polystyrene dish (NTPSD) under ambient and vacuum conditions. The formation of a compositional gradient structure in HA/PIPAAm blend film was confirmed by scanning electron microscopy, energy dispersive X-ray (EDX) mapping analysis and step-scan photoacoustic Fourier transformed infrared spectroscopy (PAS-FT-IR) measurements. The EDX mapping measurements for Na element revealed that the HA component gradually decreases from the dish-side to the air-side of the film cast on PTPSD, while for the film cast on NTPSD no such obvious change was observed on the cross-section. Further studies on the films prepared on PTPSD and NPTPSD under ambient and vacuum conditions demonstrated that the hydrophilic interaction and the solvent evaporation rate were the most significant factors leading to the formation of a compositional gradient structure in the HA/PIPAAm blend system.

Artificial extracellular matrix for embryonic stem cell cultures: a new frontier of nanobiomaterials.

Nanobiomaterials can play a central role in regenerative medicine and tissue engineering by facilitating cellular behavior and function, such as those where extracellular matrices (ECMs) direct embryonic stem (ES) cell morphogenesis, proliferation, differentiation and apoptosis. However, controlling ES cell proliferation and differentiation using matrices from natural sources is still challenging due to complex and heterogeneous culture conditions. Moreover, the systemic investigation of the regulation of self-renewal and differentiation to lineage specific cells depends on the use of defined and stress-free culture conditions. Both goals can be achieved by the development of biomaterial design targeting ECM or growth factors for ES cell culture. This targeted application will benefit from expansion of ES cells for transplantation, as well as the production of a specific differentiated cell type either by controlling the differentiation in a very specific pathway or by elimination of undesirable cell types.

Safety evaluation of surgical materials by cytotoxicity testing.

The cytotoxicity of three kinds of commercially available absorbable hemostats [oxidized cellulose (Surgicel, gauze and cotton types), microfibrillar collagen (Avitene), and cotton-type collagen (Integran)] and one adhesion barrier [sodium hyaluronate and carboxymethyl-cellulose membrane (Seprafilm)] were comparatively assessed by a colony assay using V79 cells and a minimum essential medium (MEM) elution assay in combination with a neutral red assay using L929 cells. Strong cytotoxicity was detected for Surgicel by both the MEM elution assay and the colony assay. For Avitene, both methods revealed weak cytotoxicity. For Seprafilm, no cytotoxicity was detected by the MEM elution assay, while a moderate degree of cytotoxicity was observed in the colony assay. For Integran cytotoxicity was not detected by either the MEM elution or the colony assay. The results of the different methods showed some inconsistency in terms of the degree of cytotoxicity of the materials. It is proposed that the combination of two or more sensitive cytotoxicity testing methods for the evaluation of biomaterials is necessary to avoid false-negative results for biomaterials at the preclinical stage. Furthermore, investigation of the correlation between the cytotoxicity and the extraction period of the surgical materials is helpful for predicting the effect of prolonged in vivo use of biomaterials on surrounding cells, tissues, and organs.