Chemically-defined and scalable culture system for intestinal stem cells derived from human intestinal organoids.

Three-dimensional human intestinal organoids (hIO) are widely used as a platform for biological and biomedical research. However, reproducibility and challenges for large-scale expansion limit their applicability. Here, we establish a human intestinal stem cell (ISC) culture method expanded under feeder-free and fully defined conditions through selective enrichment of ISC populations (ISC3D-hIO) within hIO derived from human pluripotent stem cells. The intrinsic self-organisation property of ISC3D-hIO, combined with air-liquid interface culture in a minimally defined medium, forces ISC3D-hIO to differentiate into the intestinal epithelium with cellular diversity, villus-like structure, and barrier integrity. Notably, ISC3D-hIO is an ideal cell source for gene editing to study ISC biology and transplantation for intestinal diseases. We demonstrate the intestinal epithelium differentiated from ISC3D-hIO as a model system to study severe acute respiratory syndrome coronavirus 2 viral infection. ISC3D-hIO culture technology provides a biological tool for use in regenerative medicine and disease modelling.

The down-regulation of melanogenesis via MITF and FOXO1 signaling pathways in SIRT1 knockout cells using CRISPR/Cas9 system.

Hair graying is processed by the inactivation of tyrosinase caused by the accumulation of oxidative stress and a decrease in the number of melanocytes. Therefore, the purpose of this study was to investigate the effect of SIRT1 gene knockout using the CRISPR/Cas9 system on the protein and gene expressions related to melanogenesis. In this study, the mutation in the SIRT1 knockout(KO) gene was verified by T7EI assay and Sanger DNA sequencing. Furthermore, the expression levels of SIRT1 protein and gene in KO cells were remarkably decreased compared with normal cells. Therefore, the SIRT1 gene KO cell line was successfully established for further study. The KO cells also increased SA-ß-galactosidase and decreased melanin production and the scavenging activity of hydrogen peroxide. In particular, the down-regulation of p38 and c-kit as well as the up-regulation of ERK resulted in the inactivation of MITF in the KO cells. Thus, KO cells reduced the expressions of Tyrosinase, Tyrosine hydroxylase, TRP-1 and TRP-2 through the negative modulation of MITF. Furthermore, SIRT1 gene KO cells negatively modulated antioxidant proteins such as Catalase, MnSOD, MsrA and MsrB3 through FOXO1 and Keap1. Therefore, it is suggested that SIRT1 could play a positive role in melanogenesis via MITF and FOXO1.

Rapid and sensitive detection of melanin using glutathione conjugated gold nanocluster based fluorescence quenching assay.

In the present study, a rapid, facile, and highly sensitive assay based on glutathione conjugated gold nanocluster (GSH-AuNCs) is developed for the detection of melanin. The analysis of melanin which is linked to several diseases is crucial. The current methods for melanin estimation are complex and long, thus demands an alternative technology. In general, melanin exhibits photoactive properties, thus, it might have fluorescence quenching properties through the phenomenon of fluorescence resonance energy transfer. To verify our assumption, we have developed the fluorescence quenching assay based on gold nanocluster and melanin interaction. As a result, under the optimized condition, the developed quenching assay demonstrated the high selectivity and sensitivity toward melanin with a limit of detection and correlation coefficient of 0.060 µg/mL and 0.993, respectively. Moreover, the whole process represented the rapid assay time of 30 min to complete. To validate the performance of our assay on real samples, B16F1 cells lysate, and hair samples were tested that provided satisfactory results. Therefore, we believe that our assay due to good sensitivity and short assay time could be beneficial for the clinical diagnosis of melanin in the future study.

Tomatidine inhibits cell invasion through the negative modulation of gelatinase and inactivation of p38 and ERK.

BACKGROUND: Despite of the most effective surgical removal of malignant tumors, metastasis makes cancer treatment difficult. The studies on natural compounds to inhibit this metastasis have been actively performed until now. However, the effect of tomatidine on metastasis remains unclear. METHOD: The effect of tomatidine on antioxidative activity was measured with DPPH radical assay and reducing power assay. After treatment with tomatidine, the viability of human fibrosarcoma cells (HT1080 cells) was evaluated with MTT assay. The effect of tomatidine on the inhibition of matrix metalloproteinase-2 (MMP-2) and MMP-9, gelatinases related to metastasis, was analyzed using gelatin zymography, western blot and immunofluorescence staining. Cell invasion assay was used to investigate anti-metastasis activity of tomatidine. RESULT: Tomatidine showed no DPPH radical scavenging effect and showed 8% of reduction power at 8 µM. Furthermore, tomatidine below 8 µM showed more than 80% of cell viability in MTT assay. The inhibition of tomatidine on MMP-2 activity and its protein expression levels were observed by gelatin zymography, western blot and immunofluorescence. It was observed that tomatidine inhibited not only p38 and ERK but also cell invasion. CONCLUSION: Above results suggest that tomatidine could use as a potential candidate for cancer prevention and metastasis through the inhibitory effect on gelatinase.