The octamer-binding transcription factor 4 (Oct3/4) is a master gene in the transcriptional regulatory network of pluripotent cells. Repression of Oct3/4 in embryonic stem cells (ESCs) is associated with cell differentiation and loss of pluripotency, whereas forced overexpression in cooperation with other transcriptional factors, such as Nanog, Sox2, and Lin28, can reprogram somatic cells back into pluripotent cells, termed induced pluripotent stem cells (iPSCs). However, random integration and potential tumorigenic transformation caused by viral transduction limit the clinical application of iPSCs. By performing a cell-based high throughput screening (HTS) campaign, we identified several potential small molecules as inducers of Oct3/4 expression. Here we report a lead structure ethyl 2-((4-chlorophenyl)amino)-thiazole-4-carboxylate, termed O4I2, showing high activity in enforcing Oct3/4 expression. On the basis of chemical expansion, we further identified derivatives having increased activities toward Oct3/4 induction. Thus, O4I2 and its derivatives should provide a new class of small molecules suitable for iPSC generation.

Reprogramming somatic cells into induced-pluripotent cells (iPSCs) provides new access to all somatic cell types for clinical application without any ethical controversy arising from the use of embryonic stem cells (ESCs). Established protocols for iPSCs generation based on viral transduction with defined factors are limited by low efficiency and the risk of genetic abnormality. Several small molecules have been reported as replacements for defined transcriptional factors, but a chemical able to replace Oct3/4 allowing the generation of human iPSCs is still unavailable. Using a cell-based High Throughput Screening (HTS) campaign, we identified that 2-[4-[(4-methoxyphenyl)methoxy]phenyl]acetonitrile (1), termed O4I1, enhanced Oct3/4 expression. Structural verification and modification by chemical synthesis showed that O4I1 and its derivatives not only promoted expression and stabilization of Oct3/4 but also enhanced its transcriptional activity in diverse human somatic cells, implying the possible benefit from using this class of compounds in regenerative medicine.

PurposeOct4 was reported to be one of the most important pluripotency transcription factors in the biology of stem cells including cancer stem cells, and progressed malignant cells. Here we report the investigation of gene expression control of Oct4 by selected human microRNAs and the physiological effect of Oct4 silencing in invasive cancer cells.Methods and resultsHigh throughput luciferase activity assay revealed the microRNA-299-3p to be the most effective in reducing gene expression of Oct4, which was confirmed by Western blot analysis and Oct4 promoter activity in a target luciferase assay. Furthermore, it could be demonstrated that downregulation of Oct4 by microRNAs-299-3p in breast cancer and fibrosarcoma cells lead to a decreased invasiveness in a microfluidic chip assay. Additionally, microRNA-299-3p causes apoptosis in cancer cells. Comparison with Oct4 specific siRNA transfection confirmed that this effect is primary due to the blockade of Oct4 expression.ConclusionThe results suggest that microRNA-299-3p is an interesting target for potential clinical use. It may be able to decrease invasive behaviour of carcinoma cells; or even kill these cells by causing apoptosis.

The increasing prevalence of cartilage destruction during arthritis has entailed an intensified amount for in vitro cartilage models to analyze pathophysiological processes and to screen for antirheumatic drugs. Tissue engineering offers the opportunity to establish highly organized 3D cell cultures facilitating the formation of in vitro models that reflect the human situation. We report the comparison of porcine chondrocyte pellet and alginate bead cultures as model systems for human cartilage and the further development into a human system that was applied in an arthritis model. In porcine pellet and alginate cultures, formation of cartilage matrix similar to human matrix was verified by histology and PCR. As alginate beads could be cultivated batch-wise in one well of a multiwell plate, we further developed this setting into a human system. In contrast, each pellet had to be cultivated individually in one well of a multiwell plate, which is time consuming. Following stimulation of human chondrocyte alginate cultures with conditioned media from human synovial fibroblasts derived from arthritis patients, microarray analysis verified the induction of genes related to cartilage destruction (like MMP10, -12) and inflammation (like IL6, -8 and chemokines). Several genes are coding for proteins that are members of inflammatory and catabolic pathways. Belonging to the most affected pathways, we identified the focal adhesion, cytokine-cytokine receptor interaction, ECM-receptor signalling, Jak-STAT signalling, and toll-like receptor signalling pathways, all relevant in arthritis. Therefore, we demonstrate that engineered cartilage of porcine and human origin represents a powerful in vitro model for cartilage in vivo.