Reprogramming By Cytosolic Extract of Human Embryonic Stem Cells Improves Dopaminergic Differentiation Potential of Human Adipose Tissue-Derived Stem Cells

The extract of pluripotent stem cells induces dedifferentiation of somatic cells with restricted plasticity. In this study, we used the extract of human embryonic stem cells (hESC) to dedifferentiate adipose tissue-derived stem cells (ADSCs) and examined the impact of this reprogramming event on dopaminergic differentiation of the cells. For this purpose, cytoplasmic extract of ESCs was prepared by repeated cycles of freezing and thawing. The plasma membrane of hADSCs was reversibly permeabilized by Streptolysin O (SLO), exposed to hESC extract and resealed by CaCl2-containing medium. As revealed by qPCR analysis, expression of OCT4, SOX2, NANOG, LIN28A and KLF4 mRNAs were downregulated in the ADSCs one week after extract incubation, while all except for KLF4 were upregulated at the end of second week. For dopaminergic differentiation, control and reprogrammed ADSCs were induced by serum-free neurobasal medium containing B27 and a cocktail of SHH, FGF8, bFGF and BDNF for 12 days. After differentiation, expression levels of some neuronal and dopaminergic-related genes, including PAX6, NESTIN, NEFL, GLI1, LMXB1, EN1, NURR1 and TH, were significantly increase in the reprogrammed ADSCs compared to the control group. As a whole, two weeks after reprogramming by ESC extract, ADSCs showed an improved dopaminergic differentiation potential. These findings suggest that the cytoplasmic extract of hESCs is containing some regulatory factors which induce the expression of pluripotency-associated markers in somatic cells and that the exposure to ESC extract may serve as a simple and rapid strategy to enhance plasticity of somaticstem cells for cell replacement therapy purposes.

Anticancer Effect of Fractions From Staphylococcus aureus and Bacillus atrophaeus on the Proliferation and Death of Human Breast Cancer Cell Line (MCF-7)

Background: Nowadays, breast cancer is known to be one of the most common cancers among women. Due to the side effects of chemotherapy and the high probability of recurrences in surgery, it is essential to identify and introduce new anticancer drugs of natural origin with fewer complications. In this regard, secondary bacterial metabolites and other microbial products have been considered. In the meantime, pathogenic and environmental bacteria have been investigated. Objective: The aim of this study is to examine the effects of the interaction between cytoplasmic extract and the cell wall of Staphylococcus aureus and Bacillus atrophaeus on the proliferation rate of human breast cancer cells. Materials and Methods: In this experimental study, cytoplasmic and cell wall extracts of bacteria were prepared. Then, SDS-PAGE was used to examine their protein contents. MCF-7 cells, as human breast cancer cells, with bacterial cytoplasmic extract and bacterial cell wall, were treated at different concentrations. Mesenchymal stem cells derived from adipose tissue were treated with different concentrations of bacterial cell wall extract. The effects of cytotoxicity were assessed by MTT assay at 24 and 48-hour intervals. The results were analyzed by SPSS. Results: The results showed that bacterial cytoplasmic extract had a concentration-dependent cytotoxic effect on cancer cells, suggesting that the increase of concentration significantly (P<0.05) increased cell death. Additionally, the bacterial cell wall extract showed a proliferative effect on cell growth (P<0.05) Conclusion: The bacterial cytoplasmic extract has a lethal effect and can, therefore, be considered as an anticancer compound in the future. This feature of the bacterium is attributed to the presence of a novel bioactive compound that can be used as an adjunct to other chemotherapy compounds. The bacterial cell wall extract, on the other hand, has cell growth-promoting components and can, therefore, be adopted as a compound for the proliferation of mesenchymal stem cells or wound healing in future studies.

Anti-proliferative effects of cell wall, cytoplasmic extract of Lactococcus lactis and nisin through down-regulation of cyclin D1 on SW480 colorectal cancer cell line

Background and Objectives: Colorectal cancer is one of the most types of cancer. Researchers have shown that lactic acid bacteria have antitumor activity. The cell wall of Lactococcus lactis, as the bacterial cytoplasmic extract and nisin can affect the proliferation of cancer cells. Since cyclin D1 plays an important role in the progression of the cell cycle, its regulation can also be a therapeutic approach. We investigated the antiproliferative effect of cell wall, cytoplasmic extract and nisin on SW480 cancer cell line and the expression level of cyclin D1 gene in treated cancer cells. Materials and Methods: SW480 cell lines were treated with different concentrations of bacterial cell wall, cytoplasmic extract and nisin. MTT test was also performed. The expression level of cyclin D1 gene was determined using Real time PCR. Data were analyzed using Graph Pad Prism software. Results: The growth rate of cancer cells treated with nisin has significantly decreased compared to the cancer cells treated by other two substances (p< 0.05). Survival rates of the cancer cells treated by nisin at a concentration of 2000 μg, cytoplasmic extract, and cell wall were 34%, 47% and 49%, respectively. Real-time PCR results showed that cyclin D1 mRNA expression has significantly decreased in nisin treated sw480 cells (P<0.05). Conclusion: The results of this study show that nisin, bacterial cytoplasmic extract, and bacterial cell wall have antiproliferative effects, which are associated with the decreased expression of cyclin D1 in SW480 cell line.

ID3018 Direct reprogramming of mouse somatic cells into pluripotent stem sells by pig germinal vesicle oocytes extract

Genomic reprogramming factors in the cytoplasm of mature oocytes could be reprogrammed somatic cell cells to totipotency cells and full-term development (cloned animals). Since then, this technique has been considered an important toot not only for animal reproduction but also for regenerative medicine, concervation of endangered species, and for study of genes function and cell biology. Moreover, in order to produce nuclear transfer embryonic stem (ntES) cells using somatic cell nuclear transfer (SCNT), the SCNT technique requires donated fresh oocytes, which raises ethical problems for production in human cloned embryo. For this reason, the use of induced pluripotent stem (iPS) cells for genomic reprogramming and for regenerative medicine is currently a hot topic in this field. However, the use of iPS cells for human therapy by the technique of retroviruses used to insert the pluripotent genes into somatic cells could cause tumors in tissues grown from the host iPS cells. Recently, we found that genomic reprogramming factors in the cytoplasm of pig germinal vesicle (GV)-stage oocytes has been shown to improve the efficiency of producing cloned mouse offspring and could reprogram pig fibroblasts to stem-like cells. In this talk, we will discuss whether pig GV cytoplasmic extract could induce pluripotent stem cells from mouse fibroblast cells (interspecies reprogramming). We first established stem-like cells from mouse fibroblast cells treated with GV oocytes extracted (gviPS cells). We demonstrated that reactivation of Oct4 promoter in mouse Oct4-GFP fibroblast cells at day 10 after treated with pig GV oocyte cytoplasmic extract and the formation of colonies is observed at 3 weeks after treatment. In addition, mouse gviPS cells reprogrammed with pig GV cytoplasmic extract can in vitro re-differentiate into neuron-like cells. Interestingly, mouse gviPS cells injected into embryos of different mouse strain could be produced chimeric mice.

Novel Nuclear Import of Vpr Promoted by Importin α Is Crucial for Human Immunodeficiency Virus Type 1 Replication in Macrophages

ABSTRACT Monocytes/macrophages are major targets of human immunodeficiency virus type 1 (HIV-1) infection. The viral preintegration complex (PIC) of HIV-1 enters the nuclei of monocyte-derived macrophages, but very little PIC migrates into the nuclei of immature monocytes. Vpr, one of the accessory gene products of HIV-1, is essential for the nuclear import of PIC in these cells, although the role of Vpr in the entry mechanism of PIC remains to be clarified. We have shown previously that Vpr is targeted to the nuclear envelope and then transported into the nucleus by importin α alone, in an importin β-independent manner. Here we demonstrate that the nuclear import of Vpr is strongly promoted by the addition of cytoplasmic extract from macrophages but not of that from monocytes and that the nuclear import activity is lost with immunodepletion of importin α from the cytoplasmic extract. Immunoblot analysis and real-time PCR demonstrate that immature monocytes express importin α at low levels, whereas the expression of three major importin α isoforms markedly increases upon their differentiation into macrophages, indicating that the expression of importin α is required for nuclear import of Vpr. Furthermore, interaction between importin α and the N-terminal α-helical domain of Vpr is indispensable, not only for the nuclear import of Vpr but also for HIV-1 replication in macrophages. This study suggests the possibility that the binding of Vpr to importin α, preceding a novel nuclear import process, is a potential target for therapeutic intervention.

p21CIP1 Controls Proliferating Cell Nuclear Antigen Level in Adult Cardiomyocytes

ABSTRACT Cell cycle withdrawal associated with terminal differentiation is responsible for the incapability of many organs to regenerate after injury. Here, we employed a cell-free system to analyze the molecular mechanisms underlying cell cycle arrest in cardiomyocytes. In this assay, incubation of S phase nuclei mixed with cytoplasmic extract of S phase cells and adult primary cardiomyocytes results in a dramatic reduction of proliferating cell nuclear antigen (PCNA) protein levels. This effect was blocked by the proteasome inhibitors MG132 and lactacystin, whereas actinomycin D and cycloheximide had no effect. Immunodepletion and addback experiments revealed that the effect of cardiomyocyte extract on PCNA protein levels is maintained by p21 but not p27. In serum-stimulated cardiomyocytes PCNA expression was reconstituted, whereas the protein level of p21 but not that of p27 was reduced. Cytoplasmic extract of serum-stimulated cardiomyocytes did not influence the PCNA protein level in S phase nuclei. Moreover, the hypertrophic effect of serum stimulation was blocked by ectopic expression of p21 and the PCNA protein level was found to be upregulated in adult cardiomyocytes derived from p21 knockout mice. Our data provide evidence that p21 regulates the PCNA protein level in adult cardiomyocytes, which has implications for cardiomyocyte growth control.