Gonadal efficacy of Thymus quinquecostatus Celakovski: Regulation of testosterone levels in aging mouse models.

Late-onset hypogonadism (LOH) is an age-related disease in men characterized by decreased testosterone levels with symptoms such as decreased libido, erectile dysfunction, and depression. Thymus quinquecostatus Celakovski (TQC) is a plant used as a volatile oil in traditional medicine, and its bioactive compounds have anti-inflammatory potential. Based on this knowledge, the present study aimed to investigate the effects of TQC extract (TE) on LOH in TM3 Leydig cells and in an in vivo aging mouse model. The aqueous extract of T. quinquecostatus Celakovski (12.5, 25, and 50 µg/mL concentrations) was used to measure parameters such as cell viability, testosterone level, body weight, and gene expression, via in vivo studies. Interestingly, TE increased testosterone levels in TM3 cells in a dose-dependent manner without affecting cell viability. Furthermore, TE significantly increased the expression of genes involved in the cytochrome P450 family (Cyp11a1, Cyp17a1, Cyp19a1, and Srd5a2), which regulate testosterone biosynthesis. In aging mouse models, TE increased testosterone levels without affecting body weight and testicular tissue weight tissue of an aging animal group. In addition, the high-dose TE-treated group (50 mg/kg) showed significantly increased expression of the cytochrome p450 enzymes, similar to the in vitro results. Furthermore, HPLC-MS analysis confirmed the presence of caffeic acid and rosmarinic acid as bioactive compounds in TE. Thus, the results obtained in the present study confirmed that TQC and its bioactive compounds can be used for LOH treatment to enhance testosterone production.

Anti-Wrinkle and Skin Moisture Efficacy of 7-MEGATM: A Randomized, Double-Blind, Placebo Comparative Clinical Trial.

7-MEGATM is a food product made from purified Alaska pollack fish oil containing palmitoleic acid (16:1), commonly referred to as omega-7. We sought to quantitatively evaluate whether this substance inhibits skin aging. A total of 101 middle-aged females were randomly allocated to the intervention (N = 50) or placebo group (N = 51). Each participant was advised to take either 500 mg of 7-MEGATM or a placebo twice daily for 12 weeks. The primary outcomes were the degree of improvement in wrinkles and the degree of moisture filling after consumption for 12 weeks compared to baseline. The secondary outcomes were improvement in skin wrinkles; moisture changes at 4 and 8 weeks from baseline; changes in transdermal water loss, skin elasticity, the melanin index, the erythema index, and the Global Photo Damage Score. We found a significant improvement in skin wrinkles and elasticity at 12 weeks in the 7-MEGATM-consuming group compared to that in the placebo group; skin moisture, elasticity, and the melanin index were also improved. No supplement-related adverse reactions were observed and 7-MEGATM was identified as safe. 7-MEGATM was effective for human skin function in terms of wrinkles, moisture, elasticity, and melanin production and may be useful as a skin nutritional supplement.

Assessment of phthalate exposure at a fire site in Korean firefighters.

To determine phthalate exposure in 32 firefighters, the concentrations of urinary phthalate metabolites, immediately (exposure day) and three weeks (control day) after fire suppression, were compared. Mono-(2-ethyl-5-carboxypentyl) phthalate, mono-(2-ethyl-5-hydroxyhexyl) phthalate, mono-(2-ethyl-5-oxohexyl) phthalate, mono-n-butyl phthalate (MBP), mono-n-benzyl phthalate (MBzP), and total phthalates (∑phthalates) levels, and creatinine-adjusted levels of MBP, MBzP, and ∑phthalates were significantly higher on exposure day than on control day. Phthalate concentration was significantly higher in firefighters who performed the fire extinguishing tasks (geometric mean [GM], 149.9 µg/L) than in those who performed other tasks (GM 70.8 µg/L) (p = .012). The GM concentration of firefighters who were active ≤ 50 m from the fire was 119.0 µg/L, and 37.6 µg/L for those who were > 50 m away (p = .012). The GM concentration was significantly different (p = .039) in firefighters with subjective symptoms after fire suppression (151.9 µg/L) compared to those without symptoms (81.6 µg/L). This study showed that firefighters were exposed to phthalate.

Glial cell proteome using targeted quantitative methods for potential multi-diagnostic biomarkers.

Glioblastoma is one of the most malignant primary brain cancer. Despite surgical resection with modern technology followed by chemo-radiation therapy with temozolomide, resistance to the treatment and recurrence is common due to its aggressive and infiltrating nature of the tumor with high proliferation index. The median survival time of the patients with glioblastomas is less than 15 months. Till now there has been no report of molecular target specific for glioblastomas. Early diagnosis and development of molecular target specific for glioblastomas are essential for longer survival of the patients with glioblastomas. Development of biomarkers specific for glioblastomas is most important for early diagnosis, estimation of the prognosis, and molecular target therapy of glioblastomas. To that end, in this study, we have conducted a comprehensive proteome study using primary cells and tissues from patients with glioblastoma. In the discovery stage, we have identified 7429 glioblastoma-specific proteins, where 476 proteins were quantitated using Tandem Mass Tag (TMT) method; 228 and 248 proteins showed up and down-regulated pattern, respectively. In the validation stage (20 selected target proteins), we developed quantitative targeted method (MRM: Multiple reaction monitoring) using stable isotope standards (SIS) peptide. In this study, five proteins (CCT3, PCMT1, TKT, TOMM34, UBA1) showed the significantly different protein levels (t-test: p value ≤ 0.05, AUC ≥ 0.7) between control and cancer groups and the result of multiplex assay using logistic regression showed the 5-marker panel showed better sensitivity (0.80 and 0.90), specificity (0.92 and 1.00), error rate (10 and 2%), and AUC value (0.94 and 0.98) than the best single marker (TOMM34) in primary cells and tissues, respectively. Although we acknowledge that the model requires further validation in a large sample size, the 5 protein marker panel can be used as baseline data for the discovery of novel biomarkers of the glioblastoma.

Glomerular filtration barrier modeling on a chip with tunable basement membrane deposition and 3D cultured podocytes.

In vitro investigation of a glomerular filtration barrier (GFB) remains difficult because of the inability to mimic its specialized structure, although various kidney diseases are characterized by GFB dysfunction. Here, the development of a microfluidic model that replicates the physiology of the GFB has been achieved by tunable glomerular basement membrane (gBM) deposition and 3D co-culture of podocytes with glomerular endothelial cells (gECs). By precisely controlling the thickness of the gBM, our model successfully reproduced the biphasic response of the GFB, where variations in gBM thickness influence barrier properties. Moreover, this microscale proximity of gECs and podocytes facilitated their dynamic crosstalk, which is essential for maintaining the integrity and function of the GFB. We observed that addition of gBM and podocytes enhanced barrier function of gECs by inducing up-regulation of gEC's tight junctions synergistically, and moreover, found an ultrastructure of gECs-gBM-podocytes' foot process contacting each other by confocal and TEM imaging. The dynamic interaction of gECs and podocytes played a significant role in the response to drug-induced injury and the regulation of barrier properties. Nephrotoxic injury simulated in our model helped to elucidate that the over-production of vascular endothelial growth factor A from the injured podocytes mediates GFB impairment. We believe that our GFB model can provide a valuable tool for mechanistic studies such as investigating GFB biology, comprehending disease mechanisms, and evaluating potential therapeutic approaches in a controlled and physiologically relevant environment.

Association between sleep quality and type of shift work in Korean firefighters.

Background: Shift work that interferes with normal sleep patterns, is known to be a cause of sleep disturbance and has been studied through various occupational groups. However, it is not known which shift type is better for sleep health. Methods: This study included 568 firefighters. Sleep quality was evaluated using Pittsburgh Sleep Quality Index. Sleep quality was categorized into 2 groups; good quality (≤ 5 points) and poor quality (≥ 6 points). Demographic variables, depression, anxiety, type of shift, and job were collected by self-reported questionnaires. The χ2 test, t-test, and multiple logistic regression analysis were used to evaluate the effect of shift type on the sleep quality of firefighters. Results: Three hundred thirty-seven firefighters (59.3%) have poor sleep quality. Compared to day workers, the odds ratios (ORs) of poor sleep quality were 2.169 (95% confidence interval: 1.137-4.134) in 6-day cycle, 2.161 (1.150-4.062) in 9-day cycle, 1.805 (1.087-2.997) in 21-day cycle, and 1.485 (0.718-3.069) in 3-day cycle. The ORs of poor sleep quality were 1.697(1.021-2.823) in fire suppression and 2.325 (1.213-4.455) in emergency medical service compared to administration. Conclusions: All shift work type except for the 3-day cycle was associated with poor sleep quality compared to day work.

Microfluidic model for in vitro acute Toxoplasma gondii infection and transendothelial migration.

The protozoan parasite Toxoplasma gondii (T. gondii) causes one of the most common human zoonotic diseases and infects approximately one-third of the global population. T. gondii infects nearly every cell type and causes severe symptoms in susceptible populations. In previous laboratory animal studies, T. gondii movement and transmission were not analyzed in real time. In a three-dimensional (3D) microfluidic assay, we successfully supported the complex lytic cycle of T. gondii in situ by generating a stable microvasculature. The physiology of the T. gondii-infected microvasculature was monitored in order to investigate the growth, paracellular and transcellular migration, and transmission of T. gondii, as well as the efficacy of T. gondii drugs.

Recapitulated Crosstalk between Cerebral Metastatic Lung Cancer Cells and Brain Perivascular Tumor Microenvironment in a Microfluidic Co-Culture Chip.

Non-small cell lung carcinoma (NSCLC), which affects the brain, is fatal and resistant to anti-cancer therapies. Despite innate, distinct characteristics of the brain from other organs, the underlying delicate crosstalk between brain metastatic NSCLC (BM-NSCLC) cells and brain tumor microenvironment (bTME) associated with tumor evolution remains elusive. Here, a novel 3D microfluidic tri-culture platform is proposed for recapitulating positive feedback from BM-NSCLC and astrocytes and brain-specific endothelial cells, two major players in bTME. Advanced imaging and quantitative functional assessment of the 3D tri-culture model enable real-time live imaging of cell viability and separate analyses of genomic/molecular/secretome from each subset. Susceptibility of multiple patient-derived BM-NSCLCs to representative targeted agents is altered and secretion of serpin E1, interleukin-8, and secreted phosphoprotein 1, which are associated with tumor aggressiveness and poor clinical outcome, is increased in tri-culture. Notably, multiple signaling pathways involved in inflammatory responses, nuclear factor kappa-light-chain-enhancer of activated B cells, and cancer metastasis are activated in BM-NSCLC through interaction with two bTME cell types. This novel platform offers a tool to elucidate potential molecular targets and for effective anti-cancer therapy targeting the crosstalk between metastatic cancer cells and adjacent components of bTME.

Microfluidic one-directional interstitial flow generation from cancer to cancer associated fibroblast.

Tumors, unlike normal tissue, have vascular anomalies and create interstitial flow (IF), which allows soluble substances from cancer cells to be transported directionally toward the tumor stroma. In the stroma, IF activates fibroblasts. Cancer-associated fibroblasts (CAFs) are formed from stimulated cells and aid cancer growth. A microfluidic device was designed to generate a one-directional flow of a small volume mimicking IF from donor cells to recipient at steady-state conditions only based on the medium evaporation from reservoirs with different diameter. The IF carried substances from donor cells, which stimulated the activation of fibroblasts on the receiving side, as well as their migration and stellate formation. Matrix metallopeptidases 9 and 14 as well as CAF markers such as fibroblast activation protein alpha, vimentin, and alpha-smooth muscle actin are abundantly expressed in the migrating fibroblasts. The created platform mimicked one-directional delivery in tumor stroma. This will allow researchers to investigate how cancer cells activate and differentiate stromal cells. STATEMENT OF SIGNIFICANCE: We show how to provide continuous one-directional interstitial flow (IF) in a microfluidic device without using any power source and instrumentation. This microfluidic technology was used to simulate the tumor microenvironment. Fibroblasts in the tumor stroma are activated and migrated toward cancer cells, as recapitulated by co-culture of cancer cells as donor and fibroblasts as recipient under the one-directional IF. We believe that soluble substances from cancerous cells delivered by the one-directional IF efficiently regulated the development of cancer-associated fibroblasts (CAFs),  as shown by increasing roundness and decreased circularity, taking on a stellate morphology, and by enhanced invasion into a type I collagen hydrogel. Migrating fibroblasts into the hydrogel had significant levels of MMP-9, MMP-14, FAP, vimentin, and αSMA, all of which are CAF markers, bearing a capacity to form hot stroma affecting tumor malignancy.

Relationship between job rotation and work-related low back pain: a cross-sectional study using data from the fifth Korean working conditions survey.

BACKGROUND: Job rotation was introduced in various industries as a strategic form of work for improving workers' job skills and health management. This study aims to examine the relationship between job rotation and work-related low back pain (LBP), one of the typical work-related musculoskeletal symptoms of Korean workers. METHODS: We conducted this study using the data of the 5th Korean Working Conditions Survey (KWCS). As the subject of this study, 27,163 wage workers were selected, and classified into three groups according to occupational type (white-collar, service and sales, and blue-collar). In this study, job rotation means to change the work-related activities with other colleagues periodically and work-related LBP was defined as whether there was work-related LBP in the last 12 months. Chi-square test and logistic regression were used to analyze the relationship between job rotation and work-related LBP. RESULTS: Out of 27,163 workers, 2,421 (8.9%) answered that they had job rotation and 2,281 (8.4%) answered that they experienced work-related LBP. According to the results from logistic regression, job rotation was significantly associated with low prevalence of work-related LBP among blue-collar workers (odds ratio [OR]: 0.71, 95% confidence interval [CI]: 0.58-0.88), whereas no significant relationship was observed among white-collar, service and sales groups. In addition, the negative association between job rotation and work-related LBP among blue-collar workers was more pronounced when exposed to ergonomic risk factors (uncomfortable posture OR: 0.79, 95% CI: 0.64-0.98; heavy work OR: 0.74, 95% CI: 0.57-0.96; repetitive work OR: 0.74, 95% CI: 0.60-0.92). CONCLUSIONS: Job rotation was associated with low prevalence of work-related LBP among workers in the blue-collar occupational group in Korea. It is necessary to evaluate the effect of job rotation by occupational type and introduce an appropriate method of job rotation to reduce workers' work-related musculoskeletal symptoms.

Microfluidic Reconstitution of Tumor Microenvironment for Nanomedical Applications.

Nanoparticles have an extensive range of diagnostic and therapeutic applications in cancer treatment. However, their current clinical translation is slow, mainly due to the failure to develop preclinical evaluation techniques that can draw similar conclusions to clinical outcomes by adequately mimicking nanoparticle behavior in complicated tumor microenvironments (TMEs). Microfluidic methods offer significant advantages over conventional in vitro methods to resolve these challenges by recapitulating physiological cues of the TME such as the extracellular matrix, shear stress, interstitial flow, soluble factors, oxygen, and nutrient gradients. The methods are capable of de-coupling microenvironmental features, spatiotemporal controlling of experimental sequences, and high throughput readouts in situ. This progress report highlights the recent achievements of microfluidic models to reconstitute the physiological microenvironment, especially for nanomedical tools for cancer treatment.

Psychosocial factors affecting sleep quality of pre-employed firefighters: a cross-sectional study.

BACKGROUND: There have been no health-related studies of pre-employed firefighters without firefighter-specific job-related factors (FSJRF). This study aimed to evaluate the sleep quality of pre-employed firefighters and to examine the relationship between sleep quality and psychosocial factors. METHODS: We conducted a self-report questionnaire survey for 602 pre-employed firefighters at 3 Fire Service Academies after brief lecture about sleep. Sleep quality and psychosocial variables such as depression, anxiety, stress and social support were evaluated. The independent 2 sample t-test, χ2 test and multiple logistic regression analysis were used to evaluate the effect of the variables on the sleep quality of pre-employed firefighters. RESULTS: Among a total of 602 people, 347 (57.6%) had good sleep quality and 255 (42.4%) had poor sleep quality. Pittsburgh Sleep Quality Index score of them was 3.29 ± 1.41) and 7.87 ± 2.20), respectively. 24 (4.0%) were evaluated to have insomnia by Insomnia Severity Index. Logistic regression analyses showed that the depression (adjusted odds ratio [aOR]: 5.940, 95% confidence interval [CI]: 3.124-11.292), anxiety (aOR: 4.233, 95% CI: 2.138-8.381), stress (aOR: 2.880, 95% CI: 1.915-4.330) and social support (aOR: 0.959, 95% CI: 0.939-0.980) have a significant effect on sleep quality after adjusted by sex, age, smoking status, drinking status, caffeine intake, past shift working and circadian rhythm type. CONCLUSIONS: Depression, anxiety, stress and social support were associated with sleep quality among pre-employed firefighters. Repeated follow-up studies of pre-employed firefighters are needed to further assess their change of sleep quality and identify the FSJRF that may affect the sleep quality of firefighters.

Evaluation of Cell-Penetrating Peptides Using Microfluidic In Vitro 3D Brain Endothelial Barrier.

In drug delivery to the human brain, blood vessels are a significant hurdle because they restrict the entry of most solutes to protect brain. To overcome this hurdle, an in vitro 3D model for brain endothelial barrier is developed using a microfluidic device with hydrogel providing a 3D extracellular matrix scaffold. Using the model, peptides known to utilize receptor-mediated transcytosis are verified, which has been one of the most promising mechanisms for brain-specific penetration. The cytotoxicity and cellular damage to the peptide are investigated and the receptor-mediated transcytosis and brain endothelial specific penetrating abilities of the peptides in a quantitative manner are demonstrated. As a preclinical test, applying the quantification assays conducted in this study are suggested, including the penetrating ability, cytotoxicity, endothelial damage, and receptor specificity. Using this microfluidic device as an in vitro platform for evaluating various brain targeting drugs and drug carrier candidates is also proposed.

Relationship between shift work and liver enzymes: a cross-sectional study based on the Korea National Health and Examination Survey (2007-2015).

BACKGROUND: Shift work has well-known adverse effects on health. However, few studies have investigated the relationship between shift work and hepatic disorders. This study aimed to evaluate the association between shift work and abnormal level of liver enzymes. METHODS: The aggregated data from the 2007-2009, 2010-2012, and 2013-2015 cycles of the Korea National Health and Nutrition Examination Survey was used for this study. The χ2 test and multiple logistic regression analysis were used to assess relationship between shift work and abnormal level of liver enzymes stratified by gender. RESULTS: The odds ratio (OR) of abnormal serum level of alanine aminotransferase (abnormal ALT) in female shift workers was higher with 1.31 (95% confidence interval: 1.00-1.71) compared with day workers after adjusting for covariates. After dividing into subgroups of the shift work pattern, the ORs of abnormal liver enzymes for each pattern compared with day work were not significantly higher. CONCLUSIONS: This study provides limited support for the hypothesis that shift work is related to liver enzyme abnormalities, but offers some evidence in favor of the idea that shift work affects female workers more than males on abnormal ALT. Further studies are needed to define the relationship between shift work and abnormal liver enzymes to be carried out as well as the gender difference in the association.

Graphene-oxide quenching-based molecular beacon imaging of exosome-mediated transfer of neurogenic miR-193a on microfluidic platform.

Graphene-oxide (GO) quenching-based molecular beacon was developed for rapid and sensitive detection of RNAs in living cells and tissues. Here, we applied GO quenching-based molecular beacon sensor to visualize neurogenic miR-193a levels delivered via exosomes during cell-non-autonomous neurogenesis in neural progenitor cells on a microfluidic platform. Exosomal transport was visualized using CD63-RFP plasmid vector, and FAM-labeled peptide nucleic acid (PNA) probe for the miR-193 sequence was designed to detect endogenous miR-193 expression. Fluorescence signals of FAM-PNA193a-GO were recovered in dibutyryl-cAMP-induced F11 cells, resulting from increased expression of miR-193a after neuronal differentiation. We observed delivery of miR-193a-containing exosomes released from differentiated donor F11 cells to recipient undifferentiated F11 cells. Fluorescence recovery was evident in exosome-stimulated recipient individual F11 cells in the microfluidic system. We propose molecular beacon imaging using PNA-GO complex for visualization of individual cellular expression of mature microRNAs. This system reveals the precise spatial localization and temporal sequences of mature miRNAs by intercellular exosomal delivery of messages for processes such as cell-non-autonomous neurogenesis.

Metabolic syndrome status over 2 years predicts incident chronic kidney disease in mid-life adults: a 10-year prospective cohort study.

We investigated whether changes in MetS status over two years modify the 10-year risk of CKD and proteinuria. A prospective cohort study was conducted in 7,251 subjects without CKD at baseline. We categorized subjects according to MetS status over two years: non-MetS (no MetS at either visit), intermittent MetS (positive for MetS at one assessment), and persistent MetS (positive for MetS at two assessments). The hazard ratio (HR) of new-onset CKD over 10-year was calculated using Cox models. During the 10-year follow-up period, 923 (12.7%) developed CKD. Compared to the non-MetS group, the fully adjusted HR for new-onset CKD was the highest in the persistent MetS group (HR, 1.53; 95% CI, 1.23-1.90), followed by the intermittent MetS group (HR, 1.29; 95% CI, 1.04-1.59) (P for trend <0.001). The HR for developing proteinuria was 1.79 (95% CI, 1.15-2.79) in the persistent MetS group and 0.70 (95% CI, 0.42-1.19) in the intermittent MetS group when the non-MetS group was considered as the reference group. Temporal changes in MetS status over two years influenced the 10-year risk of incident CKD and proteinuria. Our findings suggest that monitoring and strictly controlling MetS are important in preventing renal function decline.

Convective exosome-tracing microfluidics for analysis of cell-non-autonomous neurogenesis.

The effective role of exosome delivering neurogenic microRNA (miRNA) enables to induce efficient differentiation process during neurogenesis. The microfludic system capable of visualizing the exosomal behavior such as secretion, migration, and uptake of individual exosomes can be used as a robust technique to understand the exosome-mediated change of cellular behavior. Here, we developed the exosome-tracing microfluidic system to visualize exosomal transport carrying the neurogenic miRNA from leading to neighboring cells, and found a new mode of exosome-mediated cell-non-autonomous neurogenesis. The miR-193a facilitated neurogenesis in F11 cells by blocking proliferation-related target genes. In addition to time-lapse live-cell imaging using microfluidics visualized the convective transport of exosomes from differentiated to undifferentiated cells. Individual exosomes containing miR-193a from differentiated donor cells were taken up by undifferentiated cells to lead them to neurogenesis. Induction of anti-miR-193a was sufficient to block neurogenesis in F11 cells. Inhibition of the exosomal production by manumycin-A and treatment of anti-miR-193a in the differentiated donor cells failed to induce neurogenesis in undifferentiated recipient cells. These findings indicate that exosomes of neural progenitors and neurogenic miRNA within these exosomes propagate cell-non-autonomous differentiation to neighboring progenitors, to delineate the roles of exosome mediating neurogenesis of population of homologous neural progenitor cells.

In vivo bioluminescence imaging for leptomeningeal dissemination of medulloblastoma in mouse models.

BACKGROUND: The primary cause of treatment failure in medulloblastomas (MB) is the development of leptomeningeal dissemination (seeding). For translational research on MB seeding, one of the major challenges is the development of reliable experimental models that simulate the seeding and growth characteristics of MBs. To overcome this obstacle, we improved an experimental mouse model by intracisternal inoculation of human MB cells and monitoring with in vivo live images. METHODS: Human MB cells (UW426, D283 and MED8A) were transfected with a firefly luciferase gene and a Thy1.1 (CD90.1) marker linked with IRES under the control of the CMV promoter in a retroviral DNA backbone (effLuc). The MB-effLuc cells were injected into the cisterna magna using an intrathecal catheter, and bioluminescence images were captured. We performed histopathological analysis to confirm the extent of tumor seeding. RESULTS: The luciferase activity of MB-effLuc cells displayed a gradually increasing pattern, which correlated with a quantitative luminometric assay. Live imaging showed that the MB-effLuc cells were diffusely distributed in the cervical spinal cord and the lumbosacral area. All mice injected with UW426-effLuc, D283-effLuc and MED8A-effLuc died within 51 days. The median survival was 22, 41 and 12 days after injection of 1.2 × 10(6) UW426-effLuc, D283-effLuc and MED8A-effLuc cells, respectively. The histopathological studies revealed that the MB-effLuc cells spread extensively and diffusely along the leptomeninges of the brain and spinal cord, forming tumor cell-coated layers. The tumor cells in the subarachnoid space expressed a human nuclei marker and Ki-67. Compared with the intracerebellar injection method in which the subfrontal area and distal spinal cord were spared by tumor cell seeding in some mice, the intracisternal injection model more closely resembled the widespread leptomeningeal seeding observed in MB patients. CONCLUSION: The results and described method are valuable resources for further translational research to overcome MB seeding.

Fluorescence imaging of in vivo miR-124a-induced neurogenesis of neuronal progenitor cells using neuron-specific reporters.

BACKGROUND: Facilitation of the differentiation of the stem cells toward neuronal lineage is crucial for enhancing the differentiation efficacy of grafted stem cells for the possible treatment of neurodegenerative disorders. MicroRNA124a (miR-124a) has been considered as a neuronal lineage regulator, possessing the capability to activate neuronal differentiation. In this study, using a neuronal promoter-based reporter and live-cell fluorescence imaging, we visualized in vitro and in vivo the enhanced neuronal differentiation of neuronal progenitor cells with miR-124a overproduction. METHODS: The neuron specific alpha1 tubulin promoter-driven RFP reporter (pTa1-RFP) was used to trace the miR-124a-induced neuronal differentiation in live cell condition. MiR-124a or miR-scramble in 10 % glucose buffer was mixed with in vivo-jetPEITM and in vivo fluorescence images were obtained daily using Maestro spectral fluorescent imager. RESULTS: Neurite outgrowth was clearly seen in F11 cells after miR-124a transfection, and immunofluorescence staining showed increase of Tuj1 and NF at 48 hours. When pTa1-RFP-transfected F11 cells were implanted simultaneously with miR-124a into the nude mice, gradually increasing reporter signals and morphological changes indicated neuronal differentiation for 48 hours in live cells in vitro. The miR-124a-treated F11 cells showed higher reporter signals on in vivo fluorescence imaging than miR-scramble-treated cells, which were verified by ex vivo confirmation of Tuj1 and NF expression. CONCLUSIONS: These results indicated that neuronal reporter-based neurogenesis imaging can be used for monitoring miR-124a acting as neuronal activator when miRNA was injected in in vivo PEI-coated form for miRNA-mediated regenerative therapy.