Te Liu

Human amniotic epithelial stem cells (HuAECs) exhibit pluripotent characteristics, which are similar to those of embryonic stem cells, and can differentiate into various adult tissues and cells through directed induction. However, in culture, HuAECs tend to lose their pluripotency, and their directed differentiation capability declines with increasing passage number. The stem cell pluripotency factor octamer‑binding protein 4 (Oct4) is an important transcription factor that promotes stem cell self‑proliferation and maintains their pluripotency. Previous studies have demonstrated that WW domain containing E3 ubiquitin protein ligase 2 (WWP2) negatively regulates Oct4 expression and stem cell pluripotency. Therefore, the present study aimed to investigate the regulation of WWP2 by microRNAs (miRs), and to evaluate the expression of the downstream factor Oct4 and the maintenance of HuAEC pluripotency. Bioinformatics analysis identified a complementary binding site for miR‑32 in the 3'untranslated region of the WWP2 gene, thus suggesting that it may be a target gene of miR‑32. Post‑infection of HuAECs with a vector overexpressing miR‑32, the endogenous expression of WWP2 was significantly decreased, whereas Oct4 expression was significantly increased. Furthermore, miR‑32‑infected cells differentiated into β islet‑like cells by directed induction. The results indicated that after induction, HuAECs overexpressing miR‑32 also overexpressed the biomarkers of β islet‑like cells. In addition, the ability to secrete insulin was markedly enhanced in response to glucose stimulation, in cells overexpressing miR‑32. In conclusion, the present study suggested that miR‑32 may effectively inhibit WWP2 expression in HuAECs and promote Oct4 overexpression to maintain their pluripotency.

Since the Japanese scientist Shinya Yamanaka used a viral vector to transfer the combination of 4 factors into differentiated somatic cells and reprogramed them to obtain similar embryonic stem cells and induced pluripotent stem cells (iPSCs), it provided one integrative method for studying many medical fields. Patient-derived iPSCs have provided an opportunity to study human diseases for which no suitable model systems are available. iPSC technology has since become a major breakthrough in the field of stem cell research. With the continuous development of iPSC technology and the continuous improvement of technical levels, excellent advances have become more and more common in the basic research and medical fields of life sciences. This article reviews the development history, clinical application, and problems and prospects of iPSC, and focuses on the application of iPSC in neurological diseases.

The present study has reported a novel method for producing induced pluripotent stem (iPS) cells. Primary human amniotic epithelial cells (HuAECs) were isolated from the amniotic membranes of pregnant women who received Cesarean sections. These cells were infected with retroviruses carrying octamer‑binding transcription factor 4 (Oct4), (sex determining region Y)‑box 2 (Sox2) and Yes‑associated protein (Yap) (OSY). Following in vitro culture for ~14 days, epithelial‑like HuAECs exhibited several iPS clone‑like cell colonies (OSY‑iPS). These cell clones presented positive alkaline phosphatase features and expressed high levels of embryonic stem cell‑like markers (Nanog homeobox, Sox2, Oct4, reduced expression protein 1, and SSES3/4). Additionally, epigenetic analysis results indicated that the methylation of CpG islands on endogenous Oct4 and Sox2 promoters was reduced in OSY‑iPS cells. Furthermore, the majority of the histone H3 at lysine 9 sites that interacted with the Oct4 and Sox2 promoters were acetylated, suggesting that the transcription activities of the above two transcription factors significantly increased. In vivo and in vitro induced differentiation experiments demonstrated that OSY‑iPS could develop into embryoid bodies in vitro, and express numerous cellular markers in the three germ layers. Furthermore, OSY‑iPS could form teratomas in immunodeficient mice. The pathological detection results suggest that these teratomas contain numerous types of cells from the three germ layers. However, the results from the quantitative polymerase chain reaction and western blot analyses suggest that the Hippo‑Yap signaling pathway was significantly activated in OSY‑iPS cells. In conclusion, a novel method for iPS induction was established in the present study. HuAECs were successfully induced to reprogram iPS cells through the introduction of OSY to activate the Hippo‑Yap signaling pathway.

Aims: The aim of this study was to explore the efficacy of Jian-Pi Lian-Se Tang (JPLST), a formula of traditional Chinese medicine for the treatment of salivation and frequent nighttime urination (FNU) in patients with amyotrophic lateral sclerosis (ALS). Method: Forty-eight patients with ALS suffering from salivation and/or FNU who were outpatients or inpatients of Shuguang Hospital were included into the study and divided into 2 groups as follows: the JPLST group (n = 24) included patients who were additionally treated with JPLST, and the control group (n = 24) included patients who were treated by routine Western medicine (for 6 weeks). The changes in the quantity of salivation/sialorrhea (QS) (in mL) and in FNU as the primary result and the total scores of the Amyloid Lateral Sclerosis Function Rating Scale (ALSFRS) and of the Epworth Sleepiness Scale (ESS) as the secondary result were used to evaluate the clinical efficacy for both groups. Results: No significant differences were found for the baseline of QS, FNU, and the scores of the ESS and of the ALSFRS before and after treatment for both groups. At the end of the 6th week, 2 patients in the JPLST group and 4 patients in the control group withdrew from the study. QS and FNU were much less severe in the JPLST group than before treatment, and the ESS scores were improved, too. The ALSFRS did not show any significant differences in both groups compared with before treatment at the end of week 6. No side effects were found for both groups by laboratory tests. Conclusion: The much improved parameters of QS, FNU, and ESS for the JPLST group indicate that JPLST may be a potential additional treatment for salivation and FNU in ALS patients. Large-scale multicenter double-blind randomized-control studies are needed to verify the effectiveness of JPLST in improving salivation and FNU in patients with ALS.

Glioma stem cells (GSCs) are a special subpopulation of glioma cells that are key to the sensitivity of tumors to treatments and to the possibility of tumor recurrence. Identifying new strategies that inhibit the growth of GSCs are therefore important for developing novel therapies for glioblastoma multiforme (GBM). In this study, CD133+ human glioma stem cells were isolated and cultured. Magnetic nanoparticles were used to mediate the expression of siRNAs targeting the HOTAIR (si-HOTAIR) sequence in human gliomas. Effect of downregulation of HOTAIR expression on proliferation, invasion and in vivo tumorigenicity of human GSCs and underlying molecular mechanisms were further evaluated. The results of the MTT assay and flow cytometric analysis showed that downregulation of HOTAIR expression inhibited cell proliferation and induced cell cycle arrest. Transwell assays demonstrated that downregulation of HOTAIR expression resulted in a decrease in the invasive capability of GSCs. Moreover, magnetic nanoparticle-mediated low expression of HOTAIR effectively reduced the tumorigenic capacity of glioma stem cells in vivo. In addition, the results of qRT-PCR and western blot analysis demonstrated that downregulation of HOTAIR expression significantly increased the expression of PDCD4 in GSCs, in addition to reducing the expression of CCND1 and CDK4. An in-depth mechanistic analysis showed that downregulation of HOTAIR expression reduced the recruitment of downstream molecules, EZH2 and LSD1, thereby activating the expression of PDCD4 at the transcriptional level. In conclusion, downregulation of HOTAIR expression effectively promoted the expression of PDCD4, thereby inhibiting the proliferation, invasion and in vivo tumorigenicity of human GSCs.

Glioma stem cells belong to a special subpopulation of glioma cells that are characterized by strong proliferation, invasion and drug resistance capabilities. Magnetic nanoparticles are nanoscale biological materials with magnetic properties. In this study, CD133⁺ primary glioma stem cells were isolated from patients and cultured. Then, magnetic nanoparticles were used to mediate the transfection and expression of a microRNA-374a overexpression plasmid in the glioma stem cells. Transmission electron microscopy detected the presence of significant magnetic nanoparticle substances within the CD133⁺ glioma stem cells after transfection. The qRT-PCR and Northern blot results showed that the magnetic nanoparticles could be used to achieve the transfection of the microRNA-374a overexpression plasmid into glioma stem cells and the efficient expression of mature microRNA-374a. The MTT and flow cytometry results showed that the proliferation inhibition rate was significantly higher in cells from the microRNA-374a transfection group than in cells from the microRNA-mut transfection group; additionally, the former cells presented significant cell cycle arrest. The Transwell experiments confirmed that the overexpression of microRNA-374a could significantly reduce the invasiveness of CD133⁺ glioma stem cells. Moreover, the high expression of microRNA-374a mediated by the magnetic nanoparticles effectively reduced the tumourigenicity of CD133⁺ glioma stem cells in nude mice. The luciferase assays revealed that mature microRNA-374a fragments could bind to the 3'UTR of Neuritin (NRN1), thereby interfering with Neuritin mRNA expression. The qRT-PCR and Western blotting results showed that the overexpression of microRNA-374a significantly reduced the expression of genes such as NRN1, CCND1, CDK4 and Ki67 in glioma stem cells. Thus, magnetic nanoparticles can efficiently mediate the transfection and expression of microRNA expression plasmids in mammalian cells. The overexpression of microRNA-374a can effectively silence NRN1 expression, thereby inhibiting the proliferation, invasion and in vivo tumourigenicity of human glioma stem cells.

Oxidative stress and ageing are important factors contributing to the pathogenesis of Alzheimer's disease (AD), which is associated with neuronal damage and β‑amyloid (Aβ) deposition. The p66shc adaptor protein is important for the regulation of oxidative stress and ageing. In the present study, SH‑SY5Y human neuroblastoma cells were treated with anisomycin in order to establish a cell model of oxidative stress‑induced neuronal damage. The results from quantitative polymerase chain reaction, enzyme‑linked immunosorbent assay and western blotting demonstrated that anisomycin was able to stimulate the secretion of Aβ1‑42 from SH‑SY5Y cells and upregulate the expression levels of p66shc, which was associated with concomitant damage to SH‑SY5Y cells. In addition, the protective effects of various small molecule compounds with antioxidant properties against neuronal damage were evaluated. Notably, treatment of SH‑SY5Y cells with gallic acid was associated with significant downregulation of p66shc protein expression levels, reduced anisomycin‑induced secretion of Aβ1‑42, and increased superoxide dismutase activity and acetylcholine secretion levels. The results of the present study suggested that anisomycin is able to promote oxidative neuronal damage by inducing the secretion of Aβ1‑42 from neurons and increasing the neuronal expression of p66shc, and this damage may be attenuated by treatment with gallic acid. Therefore, gallic acid and similar small molecule compounds may be considered for the alleviation of neuronal oxidative stress injury in patients with AD.

ROR functions as a ceRNA to regulate Nanog expression by sponging miR-145 and predicts poor prognosis in pancreatic cancer

Isoflurane is widely used in anaesthesia for surgical operations. However, whether it elicits unwanted side effects, particularly in neuronal cells, remains to be fully elucidated. The Lkb1-p53-p21 signalling pathway is able to modulate neuronal self‑renewal and proliferation. Furthermore, the suppression of Lkb1‑dependent p21 induction leads to apoptosis. In the present study, whether Lkb1‑p53‑p21 signalling is involved in the response to isoflurane was investigated. A comparison of mouse primary, wild‑type neural stem cells (WT NSCs) with the p53‑/‑ NSC cell line, NE‑4C, revealed that isoflurane inhibited proliferation in a dose‑, a time‑ and a p53‑dependent manner. However, flow cytometric analysis revealed that the concentration of isoflurane which caused 50% inhibition (the IC50 value) induced cell cycle arrest in WT NSCs. Furthermore, the protein expression levels of LKB1, p53 and p21 were increased, although those of nestin and survivin decreased, following treatment of WT NSCs with isoflurane. On the other hand, isoflurane induced the phosphorylation of Ser15 in p53 in WT NSCs, which was associated with p53 binding to the p21 promoter, and consequentially, the transcriptional activation of p21. All these events were abrogated in NE‑4C cells. Taken together, the present study has demonstrated that isoflurane suppresses the self-renewal of normal mouse NSCs by activating the Lkb1-p53-p21 signalling pathway.