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Lin28A is an RNA-binding protein that controls mammalian development and maintenance of the pluripotency of embryonic stem cells (ESCs) via regulating the processing of the microRNA let-7. Lin28A is highly expressed in ESCs, and ectopic expression of this protein facilitates reprogramming of somatic cells to induced pluripotent stem cells. However, the mechanisms underlying the post-translational regulation of Lin28A protein stability in ESCs remain unclear. In the present study, we identified Kap1 (KRAB-associated protein 1) as a novel Lin28A-binding protein using affinity purification and mass spectrometry. Kap1 specifically interacted with the N-terminal region of Lin28A through its coiled-coil domain. Kap1 overexpression significantly attenuated Lin28A ubiquitination and increased its stability. However, small interfering RNA-mediated knockdown of Kap1 promoted the ubiquitination of Lin28A, leading to its proteasomal degradation. Trim71, an E3 ubiquitin ligase, induced Lin28A degradation and Kap1 knockdown accelerated the Trim71-dependent degradation of Lin28A. Mutation of the lysine 177 residue of Lin28A to arginine abrogated the ubiquitination and degradation of Lin28A which were accelerated by Kap1 silencing. Moreover, Kap1 overexpression led to the accumulation of Lin28A in the cytoplasm, but not in the nucleus, and reduced the levels of let-7 subtypes. These results suggest that Kap1 plays a key role in regulation of the stability of Lin28A by modulating the Trim71-mediated ubiquitination and subsequent degradation of Lin28A, thus playing a pivotal role in the regulation of ESC self-renewal and pluripotency.

Chronic neuropathic pain is caused by dysfunction of the peripheral nerves associated with the somatosensory system. Mesenchymal stem cells (MSCs) have attracted attention as promising cell therapeutics for chronic pain; however, their clinical application has been hampered by the poor in vivo survival and low therapeutic efficacy of transplanted cells. Increasing evidence suggests enhanced therapeutic efficacy of spheroids formed by three-dimensional culture of MSCs. In the present study, we established a neuropathic pain murine model by inducing a chronic constriction injury through ligation of the right sciatic nerve and measured the therapeutic effects and survival efficacy of spheroids. Monolayer-cultured and spheroids were transplanted into the gastrocnemius muscle close to the damaged sciatic nerve. Transplantation of spheroids alleviated chronic pain more potently and exhibited prolonged in vivo survival compared to monolayer-cultured cells. Moreover, spheroids significantly reduced macrophage infiltration into the injured tissues. Interestingly, the expression of mouse-origin genes associated with inflammatory responses, Ccl11/Eotaxin, interleukin 1A, tumor necrosis factor B, and tumor necrosis factor, was significantly attenuated by the administration of spheroids compared to that of monolayer. These results suggest that MSC spheroids exhibit enhanced in vivo survival after cell transplantation and reduced the host inflammatory response through the regulation of main chronic inflammatory response-related genes.

NANOG, a stemness-associated transcription factor, is highly expressed in many cancers and plays a critical role in regulating tumorigenicity. Transformation/transcription domain-associated protein (TRRAP) has been reported to stimulate the tumorigenic potential of cancer cells and induce the gene transcription of NANOG. This study aimed to investigate the role of the TRRAP-NANOG signaling pathway in the tumorigenicity of cancer stem cells. We found that TRRAP overexpression specifically increases NANOG protein stability by interfering with NANOG ubiquitination mediated by FBXW8, an E3 ubiquitin ligase. Mapping of NANOG-binding sites using deletion mutants of TRRAP revealed that a domain of TRRAP (amino acids 1898–2400) is responsible for binding to NANOG and that the overexpression of this TRRAP domain abrogated the FBXW8-mediated ubiquitination of NANOG. TRRAP knockdown decreased the expression of CD44, a cancer stem cell marker, and increased the expression of P53, a tumor suppressor gene, in HCT-15 colon cancer cells. TRRAP depletion attenuated spheroid-forming ability and cisplatin resistance in HCT-15 cells, which could be rescued by NANOG overexpression. Furthermore, TRRAP knockdown significantly reduced tumor growth in a murine xenograft transplantation model, which could be reversed by NANOG overexpression. Together, these results suggest that TRRAP plays a pivotal role in the regulation of the tumorigenic potential of colon cancer cells by modulating NANOG protein stability.

OCT4 (also known as Oct3 and Oct3/4), which is encoded by Pou5f1, is expressed in early embryonic cells and plays an important role in early development, pluripotency maintenance, and self-renewal of embryonic stem cells. It also regulates the reprogramming of somatic cells into induced pluripotent stem cells. Several OCT4-binding proteins, including SOX2 and NANOG, reportedly regulate gene transcription in stem cells. An increasing number of evidence suggests that not only gene transcription but also post-translational modifications of OCT4 play a pivotal role in regulating the expression and activity of OCT4. For instance, ubiquitination and sumoylation have been reported to regulate OCT4 protein stability. In addition, the phosphorylation of Ser347 in OCT4 also stabilizes the OCT4 protein level. Recently, we identified KAP1 as an OCT4-binding protein and reported the KAP1-mediated regulation of OCT4 protein stability. KAP1 overexpression led to an increased proliferation of mouse embryonic stem cells and promoted the reprogramming of somatic cells resulting in induced pluripotent stem cells. In this review, we discuss how the protein stability and function of OCT4 are regulated by protein-protein interaction in stem cells.

Oct4 plays a crucial role in the regulation of self-renewal of embryonic stem cells (ESCs) and reprogramming of somatic cells to induced pluripotent stem cells. However, the molecular mechanisms underlying posttranslational regulation and protein stability of Oct4 remain unclear. Using affinity purification and mass spectrometry analysis, we identified Kap1 as an Oct4-binding protein. Silencing of Kap1 reduced the protein levels of Oct4 in ESCs, whereas the overexpression of Kap1 stimulated the levels of Oct4. In addition, Kap1 overexpression stimulated the self-renewal of ESCs and attenuated the spontaneous differentiation of ESCs in response to LIF withdrawal. Kap1 overexpression increased the stability of Oct4 by inhibiting the Itch-mediated ubiquitination of Oct4. Silencing of Kap1 augmented Itch-mediated ubiquitination and inhibited the stability of Oct4. We identified the lysine 133 (K133) residue in Oct4 as a ubiquitination site responsible for the Kap1-Itch-dependent regulation of Oct4 stability. Preventing ubiquitination at the lysine residue by mutation to arginine augmented the reprogramming of mouse embryonic fibroblasts to induced pluripotent stem cells. These results suggest that Kap1 plays a crucial role in the regulation of the pluripotency of ESCs and somatic cell reprogramming by preventing Itch-mediated ubiquitination and the subsequent degradation of Oct4.

Background Endothelial cells have been shown to mediate angiogenesis in ischemic injury sites and contribute to the repair of damaged tissues. However, the treatment of ischemic disease requires a significant number of endothelial cells, which are difficult to isolate from patients. Embryonic stem cells have been considered a potential source of therapeutic cells due to their unlimited self-renewal and pluripotent properties. With regard to vascular development, Notch1 has been established as a key regulator of the specification of arterial endothelial cells. Methods Using a doxycycline-induced expression system of the intracellular domain of Notch1, we explored the role of Notch1 in the differentiation of embryonic stem cells to arterial endothelial cells. The therapeutic effect of the arterial endothelial cells was investigated in a murine hindlimb ischemia model. The blood perfusion rate in the ischemic limb was determined by laser Doppler perfusion imaging, and vasculogenesis was quantified using immunocytochemistry. Results Induced expression of the intracellular domain of Notch1 increased the levels of endothelial markers, such as CD31 and VE-cadherin, in differentiated endothelial cells. Induction of intracellular domain of Notch1 stimulated expression of the arterial-type endothelial cell markers (Nrp1 and Ephrin B2), but not the venous-type endothelial cell markers (Nrp2 and Coup-TFII). In addition, overexpression of intracellular domain of Notch1 resulted in increased expression of CXCR4, a chemokine receptor involved in vascular development. Induction of intracellular domain of Notch1 increased endothelial tube formation and migration of differentiated endothelial cells. Intramuscular administration of Notch1-induced arterial endothelial cells was more effective than administration of the control endothelial cells in restoring the blood flow in an ischemic hindlimb mouse model. Transplantation of Notch1-induced arterial endothelial cells augmented the number of blood vessels and incorporation of endothelial cells into newly formed blood vessels. Conclusions These results suggest that Notch1 promotes endothelial maturation and arterial specification during the differentiation of embryonic stem cells to endothelial cells and increases the angiogenic potential of endothelial cells. Electronic supplementary material The online version of this article (10.1186/s13287-018-0945-7) contains supplementary material, which is available to authorized users.

A hydrogenated amorphous silicon thin-film transistor (a-Si:H TFT) source driver for e-paper was designed with a latch and bootstrap selection circuit. The source driver was optimized by simulation and verified with measurements after circuit fabrication. The output waveforms of the conventional and proposed a-Si:H source drivers were compared. The proposed a-Si:H TFT source driver provided a shorter rise time and higher output voltages than the conventional one.

In this study, we investigated the effects of high-energy electron beam irradiation (HEEBI) on the device properties of indium-gallium-zinc-oxide (IGZO)-based transparent thin film transistors (TTFTs). The developed TTFTs had a top gate structure, which used IGZO and Al2O3 films for the active layer and the gate dielectric, respectively. The developed TTFTs were treated with HEEBI in air at RT at an electron beam energy of 0.8 MeV and a dose of 1 × 1014 electrons/cm2. Without the HEEBI treatment, the devices operated in depletion mode with a threshold voltage (V th ) of −11.25 V, a field-effect mobility (µ FE ) of 8.71 cm2/Vs, an on-off ratio (I on/off ) of 1.3 × 108 and a sub-threshold slope (SS) of 0.3 V/decade. A huge positive-shifted V th of −1 V, a very high µ FE of 420 cm2/Vs, a high I on/off of 6.1 × 108, and a lower SS of 0.25 V/decade were achieved for the HEEBI-treated devices, suggesting that the device characteristics of the developed TTFTs were significantly improved by the HEEBI treatment. The best device characteristics, which include I on/off of 8.1 × 108, SS of 0.25 V/decade, V th of +1 V, µ FE of 8.8 cm2/Vs, and operation in the enhancement mode without aging, were obtained for the samples that had been annealed after HEEBI treatment. On the basis of the experimental results, we believe that HEEBI treatment can be crucial to develop IGZO-based TFTs with high performance and long-term reliability.

We explored the effects of high-energy electron beam irradiation (HEEBI) on the gate-bias and light stabilities of indium-gallium-zinc-oxide (IGZO)-based transparent thin film transistors (TTFTs). The developed TTFTs had a top gate structure, which used IGZO and Al2O3 films for the active layer and the gate dielectric, respectively. The developed TTFTs were treated with HEEBI in air at room temperature at an electron beam energy of 0.8 MeV and a dose of 1 × 1014 electrons/cm2. Without the gate bias and/or light stresses, the HEEBI-treated devices showed a positive threshold voltage (Vth) shift (+ΔVth), suggesting that acceptor defects might have been generated by HEEBI treatment near the valence band edge. The HEEBI-treated devices also exhibited a lower +ΔVth, a higher negative Vth shift (−ΔVth), and a much lower −ΔVth under positive gate bias, negative gate bias, and light stresses compared to those for the HEEBI-untreated devices, respectively. These Vth instabilities were observed without significant change in the sub-threshold slope, indicating that charge trapping in the gate dielectric and/or at the active layer/dielectric interface was the dominant mechanism of the device instability.

Zinc oxide (ZnO)-based thin-film transistors (TFTs) with top gate structures were produced. Radio-frequency-magnetron sputtering was used to deposit indium tin oxide for both the source and the drain electrodes and n-type undoped ZnO at high oxygen partial pressures for the active layer. Direct-current-magnetron sputtering and plasma enhanced chemical vapor deposition were used to deposit Al for the gate electrode and the SiN gate dielectric, respectively. The devices operated in the enhancement mode with a threshold voltage, mobility, on-off ratio and sub-threshold slope of 9 V, 0.05 cm2/Vs, ∼5 × 105, and 1.3 V/decade, respectively.

Under white light illumination, amorphous indium–gallium–zinc oxide (a-IGZO)-based thin-film transistors (TFTs) showed a large negative shift of threshold voltage of more than -15 V depending on the process conditions. We investigated the influences of both gate bias and white light illumination on device properties of IGZO-based TFTs untreated and treated with high-energy electron beam irradiation (HEEBI). The TFTs were treated with HEEBI in air at room temperature (RT), electron beam energy of 0.8 MeV, and a dose of 1×1014 electrons/cm2. The HEEBI-treated TFTs showed an improved stability under negative bias illumination stress (NBIS) and positive bias illumination stress (PBIS) compared with non-HEEBI-treated TFTs, suggesting that the acceptor-like defects might be generated by HEEBI treatment near the valence band edge.