Investigation the effects of vitreous humor on proliferation and dedifferentiation of differentiated NTERA2 cells / Investigar os efeitos do humor vítreo na proliferação e desdiferenciação de células NTERA2 diferenciadas

Abstract Mammals have a limited capacity to regenerate their tissues and organs. One of the mechanisms associated with natural regeneration is dedifferentiation. Several small molecules such as vitamin C and growth factors could improve reprogramming efficiency. In this study, the NTERA2-D1 (NT2) cells were induced towards differentiation (NT2-RA) with 10-5 M retinoic acid (RA) for three days and then subjected to various amounts of vitreous humor (VH). Results show that the growth rate of these cells was reduced, while this rate was partly restored upon treatment with VH (NT2-RA-VH). Cell cycle analysis with PI method also showed that the numbers of cells at the S phase of the cell cycle in these cells were increased. The levels of SSEA3 and TRA-1-81 antigens in NT2-RA were dropped but they increased in NT2- RA-VH to a level similar to the NT2 cells. The level of SSEA1 had an opposite pattern. Expression of OCT4 gene dropped after RA treatment, but it was recovered in NT2-RA-VH cells. In conclusion, we suggest VH as a potent mixture for improving the cellular reprogramming leading to dedifferentiation.

Resumo Os mamíferos têm uma capacidade limitada de regenerar seus tecidos e órgãos. Um dos mecanismos associados à regeneração natural é a desdiferenciação. Várias moléculas pequenas, como vitamina C e fatores de crescimento, podem melhorar a eficiência da reprogramação. Neste estudo, as células NTERA2-D1 (NT2) foram induzidas à diferenciação (NT2-RA) com ácido retinóico (RA) 10-5 M por três dias e depois submetidas a várias quantidades de humor vítreo (VH). Os resultados mostram que a taxa de crescimento dessas células foi reduzida, enquanto essa taxa foi parcialmente restaurada após o tratamento com VH (NT2-RA-VH). A análise do ciclo celular com o método PI também mostrou que o número de células na fase S do ciclo celular nessas células estava aumentado. Os níveis de antígenos SSEA3 e TRA-1-81 em NT2-RA diminuíram, mas aumentaram em NT2-RA-VH a um nível semelhante ao das células NT2. O nível de SSEA1 teve um padrão oposto. A expressão do gene OCT4 diminuiu após o tratamento com AR, mas foi recuperado em células NT2-RA-VH. Em conclusão, sugerimos o VH como uma mistura potente para melhorar a reprogramação celular levando à desdiferenciação.

Investigation the effects of vitreous humor on proliferation and dedifferentiation of differentiated NTERA2 cells

Abstract Mammals have a limited capacity to regenerate their tissues and organs. One of the mechanisms associated with natural regeneration is dedifferentiation. Several small molecules such as vitamin C and growth factors could improve reprogramming efficiency. In this study, the NTERA2-D1 (NT2) cells were induced towards differentiation (NT2-RA) with 10-5 M retinoic acid (RA) for three days and then subjected to various amounts of vitreous humor (VH). Results show that the growth rate of these cells was reduced, while this rate was partly restored upon treatment with VH (NT2-RA-VH). Cell cycle analysis with PI method also showed that the numbers of cells at the S phase of the cell cycle in these cells were increased. The levels of SSEA3 and TRA-1-81 antigens in NT2-RA were dropped but they increased in NT2- RA-VH to a level similar to the NT2 cells. The level of SSEA1 had an opposite pattern. Expression of OCT4 gene dropped after RA treatment, but it was recovered in NT2-RA-VH cells. In conclusion, we suggest VH as a potent mixture for improving the cellular reprogramming leading to dedifferentiation.

Resumo Os mamíferos têm uma capacidade limitada de regenerar seus tecidos e órgãos. Um dos mecanismos associados à regeneração natural é a desdiferenciação. Várias moléculas pequenas, como vitamina C e fatores de crescimento, podem melhorar a eficiência da reprogramação. Neste estudo, as células NTERA2-D1 (NT2) foram induzidas à diferenciação (NT2-RA) com ácido retinóico (RA) 10-5 M por três dias e depois submetidas a várias quantidades de humor vítreo (VH). Os resultados mostram que a taxa de crescimento dessas células foi reduzida, enquanto essa taxa foi parcialmente restaurada após o tratamento com VH (NT2-RA-VH). A análise do ciclo celular com o método PI também mostrou que o número de células na fase S do ciclo celular nessas células estava aumentado. Os níveis de antígenos SSEA3 e TRA-1-81 em NT2-RA diminuíram, mas aumentaram em NT2-RA-VH a um nível semelhante ao das células NT2. O nível de SSEA1 teve um padrão oposto. A expressão do gene OCT4 diminuiu após o tratamento com AR, mas foi recuperado em células NT2-RA-VH. Em conclusão, sugerimos o VH como uma mistura potente para melhorar a reprogramação celular levando à desdiferenciação.

Investigation the effects of vitreous humor on proliferation and dedifferentiation of differentiated NTERA2 cells.

Mammals have a limited capacity to regenerate their tissues and organs. One of the mechanisms associated with natural regeneration is dedifferentiation. Several small molecules such as vitamin C and growth factors could improve reprogramming efficiency. In this study, the NTERA2-D1 (NT2) cells were induced towards differentiation (NT2-RA) with 10-5 M retinoic acid (RA) for three days and then subjected to various amounts of vitreous humor (VH). Results show that the growth rate of these cells was reduced, while this rate was partly restored upon treatment with VH (NT2-RA-VH). Cell cycle analysis with PI method also showed that the numbers of cells at the S phase of the cell cycle in these cells were increased. The levels of SSEA3 and TRA-1-81 antigens in NT2-RA were dropped but they increased in NT2- RA-VH to a level similar to the NT2 cells. The level of SSEA1 had an opposite pattern. Expression of OCT4 gene dropped after RA treatment, but it was recovered in NT2-RA-VH cells. In conclusion, we suggest VH as a potent mixture for improving the cellular reprogramming leading to dedifferentiation.

RNAi-induced silencing in floral tissues of Petunia hybrida by agroinfiltration: a rapid assay for chalcone isomerase gene function analysis.

Variegation in flower color is commonly observed in many plant species and also occurs on Petunia (Petunia hybrida) as an ornamental plant. Variegated plants are of highly valuable in the floricultural market. Agroinfiltration is an Agrobacterium-mediated transient assay for the analysis of gene function and genetic modification in leaves, flowers and fruit tissues of various plants. Transient RNAi-induced silencing by agroinfiltration has been developed in leaves and fruits of several plant species. Here we report the establishment of a transient hairpin RNAi-induced silencing system for color modification assay in floral tissues of Petunia with different colors. chiRNAi construct was cloned into the pBI121 vector under the control of 35S promoter. Transient RNA silencing of chi in the floral tissues of Petunia was induced by delivering 530 bp chi hairpin RNAs (hpRNAs) into the petals of flowers using agroinfiltration. Impaired anthocyanin accumulation and reduction of endogenous mRNAs of the corresponding targets were observed in the infiltrated areas of the petals of four colors of Petunia. Silencing of the endogenous chi mRNAs was highly effective in reduction of chi gene and anthocyanin accumulation. This transient silencing system is a prototype for modification of the anthocyanin biosynthetic pathway in Petunia through chi gene suppression.

Comparative characteristics of mesenchymal stem cells derived from reamer-irrigator-aspirator, iliac crest bone marrow, and adipose tissue.

Mesenchymal stem cells (MSCs) have been considered promising tools for new clinical concepts in supporting cellular therapy and regenerative medicine. More recently, Ream/Irrigator/Aspirator (RIA) was introduced as a source of MSCs. In this study we compared MSCs derived from three different sources (iliac crest bone marrow (ICBM), adipose tissue (AT), and (RIA)) regarding the morphology, the success rate of isolating MSCs, colony frequency, expansion potential, osteogenic and chondrogenic differentiation capacity. MSCs were isolated from three different sources and flow cytometric analyses were performed for cell characterization. Colony-forming unit-fibroblast (CFU-F) assay and population doubling time (PDT) were evaluated for MSCs derived from three different sources and differentiation potential of RIA, ICBM-, and AT-MSCs were determined by staining. Additionally, gene expression profiles for tissue specific markers corresponding to osteogenesis and chondrogenesis were analyzed using real time polymerase chain reaction (RT-PCR). Cultured with the appropriate condition, osteogenic and chondrogenic differentiation could be confirmed in all MSC preparations. Flow cytometry analysis indicated that RIA- and AT-derived MSCs have more homogenous populations than ICBM-MSCs. A comparison of the colonogenic ability in different tissues by CFU-F assay after 10 days showed that more colonies are formed from RIA-MSCs than from ICBM-MSCs, and AT-MSCs. AT-MSCs, were dispersed with no obvious colonies. The RIA-MSCs underwent osteogenesis and chondrogenesis at a faster rate than ICBM and AT-MSCs. Direct comparisons of RIA- to ICBM- and AT-MSCs have shown the RIA-MSCs have higher differentiation toward osteoblast and chondrocytes compared to other sources of MSCs. Hence, RIA-MSCs may be recommended as a more suitable source for treating orthopedic disorders.

Hypoxia and laser enhance expression of SDF-1 in muscles cells.

Targeted homing of transplanted mesenchymal stem cells (MSCs) is a decades old discussion in regenerative medicine. It has been proved that stromal cell-derived factor-1 (SDF-1α) is a potent chemoattractant of MSCs. Therefore, different strategies have been used to increase secretion of SDF-1α in damaged tissues to elevate targeted homing of MSCs. Previous studies have revealed that increased SDF-1α expression in hypoxic necrotic tissues and also low-level laser exposure enhanced angiogenesis in injured tissues. Herein, human skeletal and cardiac muscle cells (HSKM and HCM) were treated with hypoxia and low level laser to see their effects on expression of SDF-1α and on MSCs migration towards these treated cells. The optimal treatment conditions were determined by investigating the cellular viability after treatment. Real-Time PCR and Western blot analysis were done to study the expression of SDF-1α in treated cells. Migration potential of MSCs toward hypoxic and laser treated cells was investigated via migration assay. MTT assay revealed that laser and hypoxia treatment had no effect on the viability of HCM, HSKM compared with Glioblastoma cells. Real-Time PCR showed 16- and 90-fold elevation in mRNA of SDF-1α in HSKM and HCM cells, respectively, in laser treated with 12 J/cm2 intensity. In these two groups, selected as optimal conditions, HIF-1α expression showed maximum fold changes that might be partly because of response to treatments help to SDF-1α expression. It can be concluded that hypoxia and laser treatments may recruit MSCs and applied as a useful strategy for the further targeted stem cell homing.

Adipose derived mesenchymal stem cells express keratinocyte lineage markers in a co-culture model.

Cutaneous wound healing is a complex type of biological event involving proliferation, differentiation, reprograming, trans/de-differentiation, recruitment, migration, and apoptosis of a number of cells (keratinocytes, fibroblasts, endothelial cells, nerve cells and stem cells) to regenerate a multi-layered tissue that is damaged by either internal or external factors. The exact regeneration mechanism of damaged skin is still unknown but the epithelial and other kinds of stem cells located in skin play crucial roles in the healing process. In this work, a co-culture model composed of adipose derived mesenchymal stem cells and keratinocytes was developed to understand the cellular differentiation behaviour in wound healing. Human mesenchymal stem cells were isolated from waste lipoaspirates. Keratinocytes were isolated from neonatal rats skin as well from human adult skin. Both types of cells were cultured and their culturing behaviour was observed microscopically under regular intervals of time. The identity of both cells was confirmed by flow cytometry and qRT-PCR. Cells were co-cultured under the proposed co-culturing model and the model was observed for 7, 14 and 21 days. The cellular behaviour was studied based on change in morphology, colonization, stratification, migration and expression of molecular markers. Expression of molecular markers was studied at transcriptional level and change in cellular morphology and migration capabilities was observed under the invert microscope regularly. Successfully isolated and characterized mesenchymal stem cells were found to express keratinocyte lineage markers i.e. K5, K10, K14, K18, K19 and Involucrin when co-cultured with keratinocytes after 14 and 21 days. Their expression was found to increase by increasing the time span of cell culturing. The keratinocyte colonies started to disappear after 10 days of culturing which might be due to stratification process initiated by possibly transdifferentiated stem cells. It can be concluded that mesenchymal stem cells can regenerate the damaged skin if transplanted to damaged area but for their successful differentiation and enhanced regeneration, they need a population of keratinocytes in situ which need further experiments for validation of co-culture model and its potential for being used in clinics.

Role of Berberine on molecular markers involved in migration of esophageal cancer cells.

Berberine is an isoquinoline alkaloid found in several plant species like famous chinese herb, Rhizoma coptidis which has been used locally as a strong gastrointestinal remedy for thousands of years. The inhibitory effects of berberine on tumor progression properties have been reported before. In this study, we investigated the effect of berberine on an esophageal cancer cell line, KYSE-30 with emphasis on its effects on the expression of certain chemokine receptors. The cytotoxic effect of berberine on KYSE-30 cells was analyzed by MTT assay. In vitro cell migration assay was also applied to the treated cells and the expression levels of the selected chemokine receptors (CXCR4 and CCR7) was measured at mRNA level. A retarded growth, associated with increasing concentrations of berberine, was obvious. On the other hand, the migration rate of the cells was decreased when they were treated with different concentrations of berberine and the expression levels of the two chemokine receptors, involved in the migration and metastasis of esophageal cancer cells, were decreased following the same treatments. With these results, we tend to conclude that berberine might be a proper candidate for further investigations, by targeting the chemokine receptors, and possible applications as anti-metastatic agent in cancer studies.

Investigating the effects of vitreous humour (crude extract) on growth and differentiation of rat mesenchymal stem cells (rMSCs) and human NTERA2 cells.

Two main characteristics of all types of stem cells are their potency for differentiation and self renewal capacity. There is a lot of interest to find the conditions and factors, which govern these behaviours of stem cells. It is very well documented that retinoic acid (RA) reduces growth rate by induction of cell differentiation in certain conditions and cell lines. On the other hand, hyaluronic acid (HA) is known for its growth induction on cultured cells. A natural source of HA, rabbit vitreous humour (VH), was previously shown to promote wound repair in model animals. In search for its possible mechanisms, VH extract was tested on the cultured mesenchymal stem cells and NTERA2 as human embryonal carcinoma cells in the presence of RA. Changes in some cellular and molecular markers (A2B5, Oct4, Sox2) showed that VH and possibly HA interfere with differentiating effects of RA. Therefore, this reagent may affect cell proliferation and tissue regeneration by inhibition of cell differentiation.

Mogoltacin enhances vincristine cytotoxicity in human transitional cell carcinoma (TCC) cell line.

Bladder cancer is the second common cancer of the genitourinary system throughout the world and intravesical chemotherapy is usually used to reduce tumour recurrence and progression. Human transitional cell carcinoma (TCC) is an epithelial-like adherent cell line originally established from primary bladder carcinoma. Here we report the effect of mogoltacin, a sesquiterpene coumarin from Ferula badrakema on TCC cells. Mogoltacin was isolated from the fruits of F. badrakema, using silica gel column chromatography and preparative thin layer chromatography. Mogoltacin did not have any significant cytotoxicity effect on neoplastic TCC cells at 16, 32, 64, 128, 200 and 600 microg ml(-1) concentrations. In order to analyse its combination effect, TCC cells were cultured in the presence of various combining concentrations of mogoltacin and vincristine. Cells were then observed for morphological changes (by light microscopy) and cytotoxicity using MTT assay. The effect of mogoltacin on vincristine toxicity was studied after 24, 48 and 72 h of drug administration. The results of MTT assay showed that mogoltacin can significantly enhance the cytotoxicity of vincristine and confirmed the morphological observations. Results revealed that combination of 40 microg ml(-1) vincristine with 16 microg ml(-1) mogoltacin increased the cytotoxicity of vincristine after 48 h by 32.8%.

Embryonic stem (ES) cells and embryonal carcinoma (EC) cells: opposite sides of the same coin.

Embryonal carcinoma (EC) cells are the stem cells of teratocarcinomas, and the malignant counterparts of embryonic stem (ES) cells derived from the inner cell mass of blastocyst-stage embryos, whether human or mouse. On prolonged culture in vitro, human ES cells acquire karyotypic changes that are also seen in human EC cells. They also 'adapt', proliferating faster and becoming easier to maintain with time in culture. Furthermore, when cells from such an 'adapted' culture were inoculated into a SCID (severe combined immunodeficient) mouse, we obtained a teratocarcinoma containing histologically recognizable stem cells, which grew out when the tumour was explanted into culture and exhibited properties of the starting ES cells. In these features, the 'adapted' ES cells resembled malignant EC cells. The results suggest that ES cells may develop in culture in ways that mimic changes occurring in EC cells during tumour progression.

Comparative study of lung function in Iranian factory workers exposed to silica dust.

The study compared lung function among 322 workers in pottery, ceramic, stone-cutter and stone-grinder factories in the west of the Islamic Republic of Iran. Concentrations of silica particles <2 microm were measured in the ambient air of factories. Forced expiratory volume in 1 second (FEV1) and forced vital capacity (FVC) were significantly lower in stone-grinders compared with pottery, ceramic or stone-cutter workers and a control group. No difference in lung function was found in pottery and stone-cutter workers with less than 20 years occupation compared with controls. The prevalence of respiratory symptoms in stone-grinders was higher than other workers. The concentration of silica particles of stone-grinder factories was 40-110 times higher than in ceramic and potteries factories. More attention is needed to ventilation systems and health care of stone-grinders.

Ripening-related occurrence of phosphoenolpyruvate carboxykinase in tomato fruit.

Phosphoenolpyruvate carboxykinase (PEPCK) is present in ripening tomato fruits. A cDNA encoding PEPCK was identified from a PCR-based screen of a cDNA library from ripe tomato fruit. The sequence of the tomato PEPCK cDNA and a cloned portion of the genomic DNA shows that the complete cDNA sequence contains an open reading frame encoding a peptide of 662 amino acid residues in length and predicts a polypeptide with a molecular mass of 73.5 kDa, which corresponds to that detected by western blotting. Only one PEPCK gene was identified in the tomato genome. PEPCK is shown to be present in the pericarp of ripening tomato fruits by activity measurements, western blotting and mRNA analysis. PEPCK abundance and activity both increased during fruit ripening, from an undetectable amount in immature green fruit to a high amount in ripening fruit. PEPCK mRNA, protein and activity were also detected in germinating seeds and, in lower amounts, in roots and stems of tomato. The possible role of PEPCK in the pericarp of tomato fruit during ripening is discussed.

Distribution of volatile organic compounds in ambient air of Tehran.

Transportation sources have created a major hydrocarbon pollution problem in the ambient air of Tehran. The authors used a Carbotrap tube to determine volatile organic compounds in air. Such compounds can be desorbed thermally and analyzed with gas chromatography-mass spectrometry. Samples were obtained from 8 sites in Tehran at which traffic flow varied between 500 and 2,500 vehicles/hr. A total of 54 hydrocarbons were identified in the ambient air of Tehran, and the average measured concentrations of benzene, toluene, m- and p-xylene, ethyl benzene, and o-xylene were 127.6 microg/m3, 201.1 microg/m3, 110.7 microg/m3, 58.1 microg/m3, and 57.6 microg/m3, respectively (standard deviation = 3.8-51.7 microg/m3). Emissions of individual pollutants in south Tehran exceeded those in north Tehran, and these emissions were higher during the afternoon than during the morning. The geographical parameters and the photochemical reaction also played important roles in the pollution conditions.

The HIC signalling pathway links CO2 perception to stomatal development.

Stomatal pores on the leaf surface control both the uptake of CO2 for photosynthesis and the loss of water during transpiration. Since the industrial revolution, decreases in stomatal numbers in parallel with increases in atmospheric CO2 concentration have provided evidence of plant responses to changes in CO2 levels caused by human activity. This inverse correlation between stomatal density and CO2 concentration also holds for fossil material from the past 400 million years and has provided clues to the causes of global extinction events. Here we report the identification of the Arabidopsis gene HIC (for high carbon dioxide), which encodes a negative regulator of stomatal development that responds to CO2 concentration. This gene encodes a putative 3-keto acyl coenzyme A synthase--an enzyme involved in the synthesis of very-long-chain fatty acids. Mutant hic plants exhibit up to a 42% increase in stomatal density in response to a doubling of CO2. Our results identify a gene involved in the signal transduction pathway responsible for controlling stomatal numbers at elevated CO2.

Expression of a proteasome alpha-type subunit gene during tobacco development and senescence.

Proteasomes degrade specific proteins that have been targeted for proteolysis by ubiquitination. In animals and yeast nuclear-localised proteasomes play a role in regulating the cell cycle, and other developmental processes, via control of the levels of regulatory nuclear proteins such as cyclins and transcription factors. A cDNA, NtPSA1, isolated from tobacco styles was found to have high similarity to human and yeast genes, PRCI_human and PRCI_yeast with 63.4% and 51.6% overall identity respectively. These genes are believed to encode non-catalytic alpha-type subunits of 26S proteasomes and like NtPSA1 have putative nuclear localisation signals. NtPSA1 RNA was found to accumulate to varying levels in different parts of the plant and at different developmental stages. In particular, the level of NtPSA1 RNA was high in young dividing and expanding tissues, and declined during the senescence of both leaves and flowers. These results suggest that a role of proteasomes in plant nuclei may be to regulate developmental events by controlling the levels of regulatory proteins in proliferating and developing tissues, rather than to degrade and recycle proteins during senescence.