TMEM180 contributes to SW480 human colorectal cancer cell proliferation through intra-cellular metabolic pathways.

TMEM180, a novel colon cancer-specific protein with a 12-transmembrane topology, is upregulated at low oxygen. Previously, we established a humanized monoclonal antibody against TMEM180 aimed at clinical trials. Prior to such trials, it is necessary to clarify the function of TMEM180 in cancer. To compare SW480 human colon cancer cells and their TMEM180-knockdown derivatives, we analyzed proliferation and oxygen consumption, and also performed phosphorylation proteomics, metabolomics, and next-generation sequencing (NGS). The preliminary results revealed that TMEM180 appeared to promote the growth of colon cancer but had almost no effect on oxygen consumption or expression of phosphorylated proteins. By contrast, glycolysis differed dramatically between SW480 and TMEM180-knockdown cells. The NGS analysis revealed that TMEM180 promotes enzyme expression in nitric oxide (NO) synthesis system, suggesting that it promotes glucose and glutamine metabolism, thereby contributing to cancer growth. Overall, the results of this study warrant further basic studies of TMEM180 molecule.

Optimized conditions for the supplementation of human-induced pluripotent stem cell cultures with a GSK-3 inhibitor during embryoid body formation with the aim of inducing differentiation into mesodermal and cardiac lineage.

We optimized the conditions for the differentiation of human induced pluripotent stem cells (hiPSCs) into mesoderm lineage-committed cells by supplementing the cultures with CHIR, a selective GSK-3 inhibitor, during embryoid body (EB) formation. In vitro treatment with 4 µM CHIR during the late 2 days of a 4-day suspension culture period was most effective at promoting mesodermal differentiation. The resulting EBs showed a significant increase in the expression levels of mesoderm-associated genes (WNT3A, T, DKK1, GATA4, FOXC1, and MESP1) and a maintenance of OCT3/4 and NANOG expressions. Upon subsequent differentiation into a cardiac cell lineage, these EBs were shown to generate contractile cardiomyocytes. When shortening the CHIR treatment period to 1 day, the resulting EBs showed reduced expression of mesoderm-associated genes in comparison to the 2-day CHIR treatment. In particular, the expression level of FOXC1 in the 1-day CHIR-treated EBs was much lower than that of the 2-day CHIR-treated EBs. When the treatment period with CHIR was extended to 4 days, the resulting EBs presented significantly reduced expression of WNT3A, OCT3/4, and NANOG upon CHIR concentrations above 4 µM. Similarly, when CHIR treatment was conducted after the formation of EBs, the effectiveness of the GSK-3 inhibitor was reduced compared to a treatment performed during EB formation. Our results indicate that spatiotemporal constraints associated with EB formation, i.e., three-dimensional structuration and cell development in EBs, should be taken into account when designing EB formation-based differentiation protocol involving CHIR treatment.

Optimization of the treatment conditions with glycogen synthase kinase-3 inhibitor towards enhancing the proliferation of human induced pluripotent stem cells while maintaining an undifferentiated state under feeder-free conditions.

The small molecule inhibitor CHIR99021 (CHIR) is well known as a selective glycogen synthase kinase-3 inhibitor. The purpose of our study was to optimize the conditions of CHIR supplementation that will enhance the proliferation of human induced pluripotent stem cells (hiPSCs) while maintaining their undifferentiated state under feeder-free conditions in adherent cultures for 4 days. Our results revealed that both of the timing and concentration of CHIR affected cell behaviors of hiPSCs, such as colony formation, cell proliferation, and differentiation. The addition of 1-3 µM CHIR to hiPSCs cultures in the late 2-day period of a 4-day cultivation was effective in enhancing cell proliferation. Treatment with 3 µM CHIR significantly enhanced cell proliferation, but led to differentiation of hiPSCs when the treatment was carried out over 4 days. Treatment with higher concentration of CHIR was also conducive to deviating hiPSCs from their undifferentiated state. Treatment with 10 µM CHIR led to decreased expression of pluripotency-associated genes and increased level of mesoderm marker genes, but failed to provided any growth-promoting effect. Interestingly, when treatment with 1 µM CHIR was confined to the late 2-day period of a 4-day cultivation, cell proliferation was enhanced without detectable deviation from the undifferentiated state as evidenced by the expression levels of pluripotency-associated genes, e.g., OCT3/4, NANOG, SOX2, and REX1. Repeated use of 1 µM CHIR in subcultures provided no adverse effect on the proliferation of hiPSCs. Our results indicated that carefully designed CHIR treatment allows for enhanced proliferation of hiPSCs.

Effective Rho-associated protein kinase inhibitor treatment to dissociate human iPS cells for suspension culture to form embryoid body-like cell aggregates.

Treatment conditions using Y-27632 in the preparation of cell suspension of dissociated human pluripotent stem cells (hiPSCs) were investigated in the context of embryoid body (EB)-like cell aggregates. The effectiveness of a pretreatment with Y-27632 before cell dissociation and that of a Y-27632 treatment during cell dissociation were investigated from the viewpoint of simplicity and robustness. The duration of Y-27632 treatment in the preparation process affected the circularity and agglomeration of dissociated hiPSCs. A single application of pretreatment failed to prevent the onset of blebbing. However, a pretreatment promoted the agglomeration of dissociated hiPSCs when combined with the addition of Y-27632 to cell suspension. Our results indicate that pretreatment enhances the agglomeration potential of dissociated hiPSCs. When cell dissociation was performed in the presence of Y-27632, dissociated hiPSCs possessed the highest circularity and significant agglomerating property. It was shown that treatment with Y-27632 during cell dissociation is a simple and robust method to prepare dissociated hiPSCs for suspension culture to form EB-like cell aggregates.

Effects of hanging drop culture conditions on embryoid body formation and neuronal cell differentiation using mouse embryonic stem cells: optimization of culture conditions for the formation of well-controlled embryoid bodies.

Hanging drop (HD) cultures were carried out with a drop volume of either 20 or 30 µl. An incubation period of 3 days was determined to be appropriate for the formation of well-controlled embryoid bodies (EBs), and the initial cell number was identified as the most critical factor in the growth and neuronal cell differentiation of EBs.

Effect of glucose concentration during embryoid body (EB) formation from mouse embryonic stem cells on EB growth and cell differentiation.

Embryoid body (EB) formation is an important step in the differentiation of pluripotent stem cells. Although glucose concentration is physiologically maintained at 5.5mM (low glucose; LG) in vivo, a medium containing 25 mM glucose (high glucose; HG) has been widely used for forming EBs in vitro. In this study, we investigated the effect of glucose concentration during EB formation from mouse embryonic stem (ES) cells on EB growth and cell differentiation. EBs were formed under various glucose concentrations: 40, 25, 5.5, and 0mM. Cells aggregated to form EBs regardless of glucose concentration, but 0mM glucose was not appropriate for supporting EB growth as compared with 25 mM glucose. The EBs that formed in the presence of 5.5mM glucose (LG-EBs) were similar both in terms of appearance and decreased expression levels of undifferentiated-ES-cell-marker genes to the EBs that formed in the presence of 25 mM glucose (HG-EBs). However, there was a difference in the propensity for cell differentiation between LG-EBs and HG-EBs. In directed differentiation cultures of EBs into cardiomyocytes and neuronal cells, the HG-EBs more efficiently generated beating cardiac muscle, and the LG-EBs more specifically generated ßIII-tubulin-positive cells. These findings demonstrate that the high-glucose (25 mM) condition was not necessary for EB formation in mouse ES cells, whereas the glucose concentration during EB formation affects the propensity for cell differentiation in the attachment cultures of formed EBs. The physiological low-glucose (5.5mM) condition was suitable for forming EBs directed toward neuronal cell differentiation in mouse ES cells.