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 / 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.

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.

Three novel Bacillus strains from a traditional lacto-fermented pickle as potential probiotics.

AIMS: Bacillus probiotics recently gained attention due to the production of resistant cells. The in vitro probiotic potentials and safety assessment were evaluated for three Bacillus strains obtained from traditional pickle. METHODS AND RESULTS: Three bacterial strains designated as 437F, 1630F and 1020G were isolated from a traditional pickle and identified as members of the genus Bacillus. The novel strains showed high acid and bile tolerance. They exhibited antagonistic activity against various pathogens. Antioxidant activity, auto- and co-aggregation ability as well as their surface hydrophobicity and attachment capacity to the Caco-2 cells were in the range of other well-known probiotic strains. They were susceptible to various antibiotics. The enterotoxin (HBl and NHe), cytotoxin (Cytk1) and emetic (Ces) genes were not detected based on PCR assay. They were not toxic against HT-29 cells. CONCLUSION: With respect to their characteristics and safety aspect, these Bacillus species may have potential to consider as probiotics for animal and/or human applications. SIGNIFICANCE AND IMPACT OF THE STUDY: Nondairy-fermented foods are interesting sources for isolation of novel probiotics. Identification of novel Bacillus strains with remarkable probiotic potentials would increase their contribution in food/feed and pharmaceutical industries.

A novel red pigment from marine Arthrobacter sp. G20 with specific anticancer activity.

AIMS: Bacterial pigments are promising compounds in the prevention and treatment of various cancers. In the current study, the antioxidant, cytotoxic and antimicrobial effects of a red pigment obtained from a marine bacterial strain were investigated. METHODS AND RESULTS: Optimization of the pigment production by the marine strain was conducted using the one-factor-at-a-time approach. Chemical identification of the pigment was achieved by UV-visible, FTIR and HPLC analyses. The biological activities of the pigment were evaluated by DPPH, MTT and microbroth dilution assays. The strain was identified as Arthrobacter, and its pigment was related to carotenoids. The EC50 antioxidant activity of the pigment was evaluated as 4·5 mg ml-1 . It showed moderate anticancer effects on an oesophageal cancer cell line, KYSE30, while no inhibition was observed on normal HDF (human dermal fibroblasts) cells. The pigment had no antibacterial effects on the four tested strains. CONCLUSION: The antitumour activity of a carotenoid-related pigment from Arthrobacter sp. was reported for the first time. SIGNIFICANCE AND IMPACT OF THE STUDY: Marine environments are interesting sources for the identification of novel bioproducts. The identification of carotenoid pigments from marine bacteria with remarkable antioxidant and anticancer activities would result in better insights into the potential pharmaceutical applications of carotenoids and marine environments.

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%.

Knocking-down the expression of nucleostemin significantly decreases rate of proliferation of rat bone marrow stromal stem cells in an apparently p53-independent manner.

OBJECTIVES: Nucleostemin (NS) is a recently identified GTP-binding protein, predominantly expressed in embryonic and adult stem cells but not in terminally differentiated cells. NS is expressed in bone marrow-derived mesenchymal stem cells, and its expression ceases upon induction of neural differentiation. The major aim of this study was to determine whether down-regulation of NS expression acts as a promoter, or otherwise as a by-product of differentiation and senescence processes. MATERIALS AND METHODS: We used RNA interference protocols to specifically knock down NS in rat bone marrow-derived stromal stem cells. Changes in rate of proliferation and cell cycle profile after knocking-down of NS were measured. In addition, changes in expression of associated genes were studied by semiquantitative RT-PCR, Western blotting and immunocytochemistery. RESULTS: Knocked-down expression of NS caused a significant decrease in the rate of cell proliferation with concomitant shutting off of expression of cyclin D1 and survivin, two other well-known regulators of cell proliferation. Interestingly, we noticed no obvious changes in expression level of p21, the main effector of p53 for its cell cycle repressing function. CONCLUSION: Our findings revealed a master role for NS in promoting proliferation of rat bone marrow-derived stromal stem cells. Moreover, we suggest that despite previous proposals, the cell cycle arrest/inhibitory role of NS is unlikely to be related to its proposed property of interaction with p53.

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.

A positive selection vector combining tetracycline resistance that eliminates the need for bacterial plating.

We describe here the construction of a plasmid that combines positive selection with tetracycline resistance. The vector comprises a modified version of the gene encoding the cytosine-specific DNA methyltransferase MspI and a modified form of the pBR322 tetA(C) gene. The combination of these two genes facilitates the selection of recombinant plasmids in broth cultures, thereby eliminating the need for bacterial plating.