Effect of cold atmospheric plasma on changing of biomolecular structures involved in apoptosis pathways of melanoma cancer.

BACKGROUND: Cold atmospheric plasma (CAP), is a technology based on non-thermal ionized gas that is used for cancer therapy in research. We evaluated the effect of CAP on malignant melanoma cancer cell line (B16) in comparison with normal cells (L929). METHODS: The effect of CAP on the cytotoxicity of B16 and L929 cell lines was assayed by the MTT method and inverted microscopy. The induction of apoptosis in cells was evaluated using a fluorescence microscope. FTIR monitored the CAP effect in biomacromolecules changes in these cell lines. QPCR assayed gene expression of BAX, BCL-2, and Caspase-3 (CASP-3). RESULTS: The results of the MTT test showed CAP has a cytotoxic effect on the B16 cancer cell line more than L929 normal cells (p < 0.0001). The results of invert and fluorescence microscopy showed CAP-induced apoptotic morphology on cancerous cells. FTIR spectroscopy indicated CAP changes biomacromolecules structure. Evaluation of gene expression showed CAP increased BAX and CASP-3 gene expression. Also, it decreased BCL-2 gene expression. CONCLUSIONS: Taken together, CAP may change biomacromolecule structures involved in apoptosis pathways, decrease proliferation and induce apoptosis in cancer cells.

Enhanced yield of cholinergic neurons from induced pluripotent stem cells (iPSC): A two-step induction protocol.

BACKGROUND: Cholinergic neurons, a type of neurons found in central nervous system, play a vital role in muscle movement and activities. Cholinergic neurons degeneration is the main pathological symptom of neurodegenerative diseases. Among a variety of stem cells, iPSCs have emerged as a promising candidate for transplantation to improve the repair of neuronal lesion sites. However, the establishment of an appropriate induction method to yield large numbers of cholinergic neurons has yet to be determined. Here, we studied the differentiation potential of iPSCs to generate cholinergic neurons by developing a new optimized differentiation protocol. METHODS: The iPSCs were harvested on 6-well matrigel-coated plate and incubated with serum­free DMEM/F12 with 2 % B27 supplement, 20 ng/ml the basic fibroblast growth factor and 20 ng/ml epidermal growth factor for 48 hours. Then, the pre-induced cells were treated in neuronal induction medium supplemented with all-trans retinoic acid, sonic hedgehog, 100 ng/ml glial-derived neurotrophic factor and 200 ng/ml brain-derived neurotrophic factor for 7 days. Cell viability during induction stages was tested by MTT assay. Differentiated cells were evaluated with crystal violet staining, immunocytochemistry and real­time PCR. RESULTS: Our results showed that the survival rate of iPSCs leveled out and was similar to that in the control group following the differentiation process. Immunochemistry results revealed that the expression of ChAT was observed in cells in both pre­induction and induction stages with a significantly higher expression level at the induction stage as compared to the pre-induction stage. However, none of these markers was expressed in the iPSCs. Cresyl violet staining confirmed the neuronal phenotype of differentiated cells. The induction group significantly expressed the higher levels of Islet1, Olig2 and HB9, whereas pluripotency markers including those of Oct4 and Nestin plunged. CONCLUSION: Our investigation represents a highly efficient protocol for iPSCs differentiation toward cholinergic neurons which could be used for further preclinical transplantation studies (Tab. 1, Fig. 5, Ref. 35). Text in PDF www.elis.sk Keywords: induced pluripotent stem cells, cholinergic neurons, neurotrophic factors, induction protocol, preclinical transplantation.

Matrigel enhances differentiation of human adipose tissue-derived stem cells into dopaminergic neuron.

BACKGROUND: Therapy based stem cells have offered a novel therapeutic approach for the improvement of neurodegenerative diseases, specially Parkinson. Hence, developing a well-established culture model with appropriate stem cells is extremely crucial in regenerative engineering to provide efficient targeted cells. Human adult mesenchymal stem cells derived from adipose tissue (hADSCs) have emerged as a promising source of stem cells due to their unique potentials of self-renewal and differentiation into other stem cells. The purpose of this study was to investigate the differentiation capacity of hADSCs into dopaminergic and neuron-like cells in the 3D culture plate (Matrigel). METHODS AND MATERIALS: hADSCs were obtained from adipose tissues of patients and then characterized morphologically with flowcytometry. Isolated cells were harvested to perform differentiation on Matrigel and tissue culture plate (TCP) supplemented with induction factors. The survival rate of cells during neural induction was monitored by MTT. The expression of specific cell markers was analyzed by QRT-PCR and immunocytochemistry on days 2, 8 and 14. The level of released dopamine was measured using HPLC technique. RESULTS: Matrigel had a positive effect on maintaining cell growth compared to those on TCP. Moreover, the number of TH and MAPII positive cells is substantially higher in Matrigel than in TCP. Sox2 and Nestin had a prominent expression in hADSCs within the first days of differentiation. The gene expression of neural markers such as TH, Nurr1, LMX1A and DAT was detected and increased after day 8. Moreover, the dopamine released in the cell harvested on Matrigel was greater than those seeded on TCP. CONCLUSIONS: Overall, hADSCs could generate dopaminergic cells, which suggest its strong capability to serve as a tool for Parkinson disease model in the regenerative medicine.