Human embryonic stem cell-derived neural crest model unveils CD55 as a cancer stem cell regulator for therapeutic targeting in MYCN-amplified neuroblastoma.

BACKGROUND: Neuroblastoma (NB) is a common childhood malignant tumor of neural crest (NC) origin with remarkable heterogeneity in outcomes. Amplification of the oncogene MYCN is strongly associated with highly malignant behaviour and poor prognosis. METHODS: This study aims to use a human embryonic stem cell (hESC)-derived NC model to identify novel downstream effectors of MYCN that can be potentially used as prognostic marker and/or therapeutic target. RESULTS: We show that MYCN-driven NB derived from human neural crest cells (hNCCs) recapitulate the pathological and molecular features of MYCN-amplified neuroblastoma (MNA-NB). By using this platform, we identify a group of 14 surface protein-encoding genes that are associated with MYCN expression level in MNA-NB. Among these genes, high CD55 expression is correlated with poor survival in MNA-NB but not in non-MNA-NB. Furthermore, CD55 promotes tumorigenesis, tumor growth, and cancer stemness in MNA-NB cell lines (MNA-NBL) through regulating the JNK pathway. Mechanistically, MYCN binds to both canonical and noncanonical E-boxes on the promoter of CD55 to regulate its transcriptional expression. Finally, neutralizing antibody targeting CD55 significantly attenuates cancer stemness, suppresses tumor growth, and improves survival exclusively in MNA-NBL-inoculated mice. CONCLUSION: MYCN shapes CD55 into a cancer stem cell regulator which represents a prognostic marker and therapeutic target of MNA-NB. The hESC-derived NC model serves as a valuable platform for investigating NB initiation and progression and developing potential therapeutic targets.

LINC01554-Mediated Glucose Metabolism Reprogramming Suppresses Tumorigenicity in Hepatocellular Carcinoma via Downregulating PKM2 Expression and Inhibiting Akt/mTOR Signaling Pathway.

Background and Aims: Cancer cells prefer aerobic glycolysis to maintain growth advantages, but the role of long non-coding RNAs (lncRNAs) in glycometabolism still remains unclear. Here we identified one cytoplasmic lncRNA LINC01554 as a significantly downregulated lncRNA in hepatocellular carcinoma (HCC) and aimed to investigate its role in cellular glucose metabolism in the development and progression of HCC. Methods: Quantitative real-time PCR was used to determine the expression level of LINC01554. Downregulation of LINC01554 by miR-365a at transcriptional level was assessed by luciferase reporter assay. Subcellular fractionation assay and RNA fluorescence in situ hybridization were performed to detect the subcellular localization of LINC01554. RNA pull-down assay, mass spectrometry, and RNA immunoprecipitation assay were used to identify the underlying molecular mechanisms. The tumor-suppressive function of LINC01554 was determined by both in vitro assay and nude mice xenograft model. Results: LINC01554 was frequently downregulated in HCC, which was significantly associated with tumor invasion (P = 0.005), tumor size (P = 0.041), tumor staging (P = 0.023) and shorter survival (P = 0.035) of HCC patients. Luciferase reporter assay unraveled that LINC01554 was negatively regulated by miR-365a. Subcellular fractionation assay and RNA FISH revealed the cytoplasmic predominance of LINC01554 in MIHA cells and HCC clinical samples. Ectopic expression of LINC01554 inhibited HCC cell growth, colony formation in soft agar, foci formation, and tumor formation in nude mice. LINC01554 promoted the ubiquitin-mediated degradation of PKM2 and inhibited Akt/mTOR signaling pathway to abolish aerobic glycolysis in HCC cells. Further study found that LINC01554-knockout could effectively reverse the tumor-suppressive effect of LINC01554. Conclusions: Our results identify LINC01554 as a novel tumor suppressor in HCC and unravel its underlying molecular mechanism in reprogramming cellular glucose metabolism. LINC01554 could possibly serve as a novel prognostic biomarker and provide the rationale for HCC therapy.

Symptoms of long-term exposure to moxa smoke in acupuncturists: a correlation analysis.

OBJECTIVE: To investigate the effects of long-term exposure to moxa smoke on acupuncturists and to consider the association between physical symptoms and gender, age, secondhand smoke (SHS) exposure, and the duration of exposure. METHODS: A self-report, web-based questionnaire was used to evaluate the effects of moxa smoke on acupuncturists. Logistic regression was used to analyze the relationship between physical symptoms and possible correlative factors. RESULTS: A total of 858 questionnaires were analyzed. The data showed that 42.42% of acupuncturists had been exposed to moxa smoke for 5-10 years. The most frequent symptom was tearing (32.98%) and the least frequent symptom was asthma (5.24%). Logistic regression analysis showed that female acupuncturists were slightly more susceptible to cough and tearing than males (cough: OR: 1.583, 95% CI: 1.079, 2.321, P = 0.019; tearing: OR: 1.519, 95% CI: 1.094, 2.108, P = 0.013). Acupuncturists aged over 25 years reported a slightly lower incidence of eye itchiness than those aged under 25 years (26-40 years: OR: 0.604, 95% CI: 0.379, 0.965, P = 0.035; over 40 years: OR: 0.330, 95% CI: 0.114, 0.958, P = 0.042). Ophthalmodynia occurred less in acupuncturists aged 26-40 years (OR: 0.591, 95% CI: 0.359, 0.970, P = 0.038) than in those younger than 25 years. The only association between prevalence of symptoms and tobacco use and SHS exposure was that smokers had a lower occurrence of phlegm in the throat (OR: 0.579, 95% CI: 0.392, 0.856, P = 0.006). Shortness of breath was less frequent in participants exposed to moxa smoke for more than 5 years (5-10 years: OR: 0.400, 95% CI: 0.204, 0.785, P = 0.008; 11-20 years: OR: 0.392, 95% CI: 0.178, 0.864, P = 0.02), but a slightly higher incidence of eye itchiness was found in those with over 20 years of moxa exposure (OR: 4.200, 95% CI: 1.344, 3.128, P = 0.014). CONCLUSION: The most frequent symptom of moxa smoke exposure in acupuncturists was tearing. The association of symptoms with age and duration of exposure to moxa smoke were rather complicated; hence, care should be taken in drawing conclusions about the safety of moxa smoke based solely on its potentially harmful ingredients.

Generation of Induced Pluripotent Stem Cells from Patients with COL3A1 Mutations and Differentiation to Smooth Muscle Cells for ECM-Surfaceome Analyses.

Use of experimentally derived induced pluripotent stem cells (iPSCs) has led to the development of cell models for differentiation, drug testing and understanding disease pathogenesis. For these models to be informative, reprogrammed cell lines need to be adequately characterized and shown to preserve all of the critical characteristics of pluripotency and differentiation. Here, we report a detailed protocol for the generation of iPSCs from human fibroblasts containing mutations in COL3A1 using a Sendai virus mediated integration-free reprogramming approach. We describe how to characterize the putative iPSCs in vivo and in vitro to ensure potency and differentiation potential. As an example of how these mutations may affect cell surface and extracellular matrix (ECM) interactions, we provide protocols for the differentiation of these cells into smooth muscle cells to illustrate how different cell types may display cell autonomous differences in collagen receptors that may affect their phenotype. These cells, when applied to mechanical model systems (see Chapter 18 by Bose et al.) facilitate an assessment of stiffness and stress-strain relationships useful for understanding how extracellular matrix dysfunction and its interactions with surface proteins contribute to disease processes.

A simple, cost-effective but highly efficient system for deriving ventricular cardiomyocytes from human pluripotent stem cells.

Self-renewable human pluripotent stem cells (hPSCs) serve as a potential unlimited ex vivo source of human cardiomyocytes (CMs) for cell-based disease modeling and therapies. Although recent advances in directed differentiation protocols have enabled more efficient derivation of hPSC-derived CMs with an efficiency of ∼50%-80% CMs and a final yield of ∼1-20 CMs per starting undifferentiated hPSC, these protocols are often not readily transferrable across lines without first optimizing multiple parameters. Further, the resultant populations are undefined for chamber specificity or heterogeneous containing mixtures of atrial, ventricular (V), and pacemaker derivatives. Here we report a highly cost-effective and reproducibly efficient system for deriving hPSC-ventricular cardiomyocytes (VCMs) from all five human embryonic stem cell (HES2, H7, and H9) and human induced PSC (hiPSC) (reprogrammed from human adult peripheral blood CD34(+) cells using nonintegrating episomal vectors) lines tested. Cardiogenic embryoid bodies could be formed by the sequential addition of BMP4, Rho kinase inhibitor, activin-A, and IWR-1. Spontaneously contracting clusters appeared as early as day 8. At day 16, up to 95% of cells were cTnT(+). Of which, 93%, 94%, 100%, 92%, and 92% of cardiac derivatives from HES2, H7, H9, and two iPSC lines, respectively, were VCMs as gauged by signature ventricular action potential and ionic currents (INa(+)/ICa,L(+)/IKr(+)/IKATP(+)); Ca(2+) transients showed positive chronotropic responses to ß-adrenergic stimulation. Our simple, cost-effective protocol required the least amounts of reagents and time compared with others. While the purity and percentage of PSC-VCMs were comparable to a recently published protocol, the present yield and efficiency with a final output of up to 70 hPSC-VCMs per hPSC was up to 5-fold higher and without the need of performing line-specific optimization. These differences were discussed. The results may lead to mass production of hPSC-VCMs in bioreactors.