Vol. 20 No. 3 September 2015

DOI: 10.1515/cmble-2015-0019 Volume 20 (2015) pp 351-373
Title TRANSCRIPTIONAL PROFILING OF BOVINE MUSCLE-DERIVED SATELLITE CELLS DURING DIFFERENTIATION IN VITRO BY HIGH THROUGHPUT RNA SEQUENCING
Authors Hui Li Tong1, Hong Yan Yin1, Wei Wei Zhang1, Qian Hu1, Shu Feng Li1, Yun Qin Yan1,* and Guang Peng Li
Abstract In this study, we utilized high throughput RNA sequencing to obtain a comprehensive gene expression profile of muscle-derived satellite cells (MDSCs) upon induction of differentiation. MDSCs were cultured in vitro and RNA was extracted for sequencing prior to differentiation (MDSC-P), and again during the early and late differentiation (MDSC-D1, and MDSC-D3, respectively) stages. Sequence tags were assembled and analyzed by digital gene expression profile to screen for differentially expressed genes, Gene Ontology annotation, and pathway enrichment analysis. Quantitative real-time PCR was used to confirm the results of RNA sequencing. Our results indicate that certain of genes were changed during skeletal muscle cell development, cell cycle progression, and cell metabolism during differentiation of bovine MDSCs. Furthermore, we identified certain genes that could be used as novel candidates for future research of muscle development. Additionally, the sequencing results indicated that lipid metabolism might be the predominant cellular process that occurs during MDSC differentiation.
Keywords MDSC, Development, Differentiation, RNA sequencing, Gene expression analysis, Differentially expressed genes, Gene Ontology analysis, Pathway enrichment analysis, Cell cycle progression, Lipid metabolism
Address and Contact Information 1The Laboratory of Cell and Developmental Biology, Northeast Agricultural University, Harbin, Heilongjiang 150030, China,
2The Key Laboratory of Mammal Reproductive Biology and Biotechnology Ministry of Education, Inner Mongolia University, Hohhot 010021, China
* Author for correspondence. e-mail: yanyunqin@sohu.com, phone: 86-0451-55190846

DOI: 10.1515/cmble-2015-0022 Volume 20 (2015) pp 374-384
Title INHIBITION OF CEA RELEASE FROM EPITHELIAL CELLS BY LIPID A OF GRAM-NEGATIVE BACTERIA
Authors Fakhraddin Naghibalhossaini1,2,*, Khatere Sayadi1, Hajar Jaberie1, Abdollah Bazargani3, Ebrahim Eftekhar1,4 and Massood Hosseinzadeh5
Abstract A number of bacterial species, both pathogenic and non-pathogenic, use the human CEACAM family membersas receptors for internalization into epithelial cells. The GPI-linked CEA and CEACAM6 might play a role in the innate immune defense, protecting the colon from microbial invasion. Previous studies showed that CEA is released from epithelial cells by an endogenous GPI-PLD enzyme. GPI-PLD activity was reported to be inhibited by several synthetic and natural forms of lipid A. We hypothesized that CEA engagement by Gram-negative bacteria might attenuate CEA release from epithelial cells and that this might facilitate bacterial colonization. We tested the hypothesis by examining the effect of Escherichia coli on CEA release from colorectal cancer cells in a co-culture experiment. A subconfluent monolayer culture of colorectal cancer cells (LS-180, Caco-2 and HT29/219) was incubated with E. coli. While there was a significant reduction in CEA secretion from LS-180 and HT29/219 cells, we found only a small reduction of CEA shedding from Caco-2 cells compared to the level from the untreated control cells. Furthermore, lipid A treatment of LS-180 cells inhibited CEA release from the cells in a dose-dependent manner. Western blot analysis of total lysates showed that CEA expression levels in cells co-cultured with bacteria did not differ from those in untreated control cells. These results suggest that lipid A of Gram-negative bacteria might play a role in preventing the release of CEA from mucosal surfaces and promote mucosal colonization by bacteria.
Keywords CEA, Gram-negative bacteria, Lipid A, Innate immunity, GPI-PLD, Adhesin, CEACAM, Epithelial cell, Co-culture, GPI
Address and Contact Information 1Department of Biochemistry, Shiraz University of Medical Sciences, School of Medicine, Shiraz, Iran,
2Autoimmune Research Center, Shiraz University of Medical Sciences, School of Medicine, Shiraz, Iran,
3Department of Bacteriology, Shiraz University of Medical Sciences, School of Medicine, Shiraz, Iran,
4Current address: Food and Cosmetic Health Research Center, Hormozgan University of Medical Sciences, Bandar Abbas, Iran,
5Colorectal Research Center,Shiraz University of Medical Sciences, Shiraz, Iran
* Author for correspondence. Email: fakhr addin.naghibalhossaini@mail.mcgill.ca; phone: 98-71-32303029; fax: 98-71-32303029

DOI: 10.1515/cmble-2015-0023 Volume 20 (2015) pp 385-403
Title NEUROTROPHIN-3 MAY CONTRIBUTE TO NEURONAL DIFFERENTIATION OF MESENCHYMAL STEM CELLS THROUGH THE ACTIVATION OF THE BONE MORPHOGENETIC PROTEIN PATHWAY
Authors Lei Li1,*, Yunpeng Li1 and Hongkun Jiang2,*
Abstract We investigated whether neurotrophin-3 (NT-3) can promote differentiation of mouse bone mesenchymal stem cells (MSCs) into neurons via the bone morphogenetic protein pathway. MSCs were prepared from rat bone marrow and either transfected with pIRES2-EGFP or pIRES2-EGFP-NT-3 or treated with bone morphogenetic protein 4. The pIRES2-EGFP-NT-3-transfected MSCs further underwent noggin treatment or siRNA-mediated knockout of the TrkC gene or were left untreated. Immunofluorescence staining, real-time PCR and Western blot analyses were performed to evaluate the transcription and expression of neural-specific genes and BMP-Smad signaling. MSCs were efficiently transduced by the NT-3 gene via pIRES2-EGFP vectors. pIRES2-EGFP-NT-3 could initiate the transcription and expression of neural-specific genes, including nestin, NSE and MAP-2, and stimulate BMP-Smad signaling. The transcription and expression of neural-specific genes and BMP-Smad signaling were significantly suppressed by siRNA-mediated knockdown of the TrkC gene of MSCs.These findings suggest that the BMP signaling pathway may be a key regulatory point in NT-3-transfected neuronal differentiation of MSCs. The BMP and neurotrophin pathways contribute to a tightly regulated signaling network that directs the precise connections between neuronal differentiation of MSCs and their targets.
Keywords Neurotrophin-3, Mesenchymal stem cells, Bone morphogenetic protein, Neuronal differentiation, Drosophila mothers against decapentaplegic protein, Smad, Noggin, Tyrosine protein kinases C, Electrophysiological analyses, Calcium imaging
Address and Contact Information 1Department of Orthopedics, Shengjing Hospital of China Medical University, Shenyang 110004, China,
2Department of Pediatrics, First Affiliated Hospital of China Medical University, Shenyang 110001, China
* Authors for correspondence. Lei Li – Email: lilei169@163.com; phone: +86-24-9661533212. Hong-Kun Jiang – Email: jianghongkun88@126.com; phone: +86-24-83282003; fax: +86-24-8328 2645

DOI: 10.1515/cmble-2015-0024 Volume 20 (2015) pp 404-417
Title IN VITRO AND IN VIVO ANALYSIS OF HUMAN FIBROBLAST REPROGRAMMING AND MULTIPOTENCY
Authors Rongqing Pang1, Xiangqing Zhu1, Jia Geng2, Yongyun Zhang3, Qiang Wang1, Jie He1, Jinxiang Wang1, Guangxu Zhu1, Fu Xiong4, Cheng Zhang4, Guangping Ruan1,* and Xinghua Pan1,*
Abstract Multipotent stem cells have potential therapeutic roles in the treatment of Duchenne muscular dystrophy (DMD). However, the limited access to stem cell sources restricts their clinical application. To address this issue, we established a simple in vitro epigenetic reprogramming technique in which skin fibroblasts are induced to dedifferentiate into multipotent cells. In this study, human fibroblasts were isolated from circumcised adult foreskin and were reprogrammed by co-culture for 72 h with fish oocyte extract (FOE) in serum-free medium. The cells were then observed and analyzed by immunofluorescence staining, flow cytometry and in vitro differentiation assays. Then FOE-treated human fibroblasts were transplanted by tail vein injection into irradiated mdx mice, an animal model of DMD. Two months after injection, the therapeutic effects of FOE-treated fibroblasts on mdx skeletal muscle were evaluated by serum creatine kinase (CK) activity measurements and by immunostaining and RT-PCR of human dystrophin expression. The results indicated that the reprogrammed fibroblasts expressed higher levels of the pluripotent antigen markers SSEA-4, Nanog and Oct-4, and were able to differentiate in vitro into adipogenic cells, osteoblastic cells, and myotube-like cells. Tail vein injection of FOE-treated fibroblasts into irradiated mdx mice slightly reduced serum CK activity and the percentage of centrally nucleated myofibers two months after cell transplantation. Furthermore, we confirmed human dystrophin protein and mRNA expression in mdx mouse skeletal muscle. These data demonstrated that FOE-treated fibroblasts were multipotent and could integrate into mdx mouse myofibers through the vasculature.
Keywords Multipotent stem cells, Fibroblasts, Dystrophin, Mdx mice, Duchenne muscular dystrophy, Fish oocyte extract, Reprogramming, Serum-free medium, Differentiation, Therapy
Address and Contact Information 1Stem Cell Engineering Laboratory of Yunnan Province, Kunming General Hospital of Chengdu Military Command, Kunming, 650032, P. R. China,
2Neurology Department, The First Affiliated Hospital, Kunming Medical University, Kunming, P. R. China,
3Center for Experiment, Yunnan Agricultural University, Kunming, 650201, P. R. China,
4Department of Neurology, The First Affiliated Hospital, Sun Yat-sen University, Guangzhou, P. R. China
*Authors for correspondence. Guang-Ping Ruan, email: 370164778@qq.com, phone: 087164774771; Xing-Hua Pan, email: ynkmry@163.com, phone: 087164774773

DOI: 10.1515/cmble-2015-0012 Volume 20 (2015) pp 418-447
Title THE PHARMACOLOGICAL FEATURES OF BILIRUBIN: THE QUESTION OF THE CENTURY
Authors Farhana Zahir1,*, Gulam Rabbani2, Rizwan Hasan Khan2,*, Shamim J Rizvi1, Mohammad Sarwar Jamal3 and Adel M. Abuzenadah4
Abstract This review looks at the toxicity and metabolism of bilirubin in terms of its pharmacological potential. Its role has gained importance as more research has revealed the functional significance and interrelationship between the gasotransmitters nitric oxide and carbon monoxide. The biological actions of bilirubin have mostly been characterized in the high micromolar range where toxic effects occur. However, it could also prove to be an important cytoprotector for brain tissue, which is inherently less equipped for antioxidant defense. Plasma bilirubin levels negatively correlate to a number of disease states. Higher levels of bilirubin that are still within the normal range provide a protective effect to the body. The effects on various disorders could be tested using controlled pharmacological upregulation of the molecule with animal models. At nanomolar concentrations, considerable benefits have been obtained when the molecule was delivered pharmacologically under in vitro or in vivo test conditions, particularly in neurodegenerative disorders and after tissue or organ transplantation. The induction of heme oxygenase-1 (HMOX-1) via the activation of nuclear factor erythroid 2-related factor or the use of bile pigments in the harvesting of diseased tissue are novel applications, and like every new therapy, should be used with caution. HMOX-1 is tissue specific, and in exceptional states, such as schizophrenia and specific types of renal disorder, the same therapy may have disastrous effects.
Keywords Antioxidant, Bilirubin, Cardioprotectant, Heme oxigenase, Neuroprotectant, Neonatal hyperbilirubinimia
Address and Contact Information 1Interdisciplinary Brain Research Centre, Jawaharlal Nehru Medical College, Aligarh Muslim University, Aligarh-202002, India,
2Interdisciplinary Biotechnology Unit, Aligarh Muslim University, Aligarh-202002, India,
3King Fahd Medical Research Center, King Abdulaziz University, PO Box 80216, Jeddah 21589, Saudi Arabia,
4KACST Technology Innovation Center in Personalized Medcine, King Abdulaziz University, PO Box 80216, Jeddah 21589, Saudi Arabia
* Authors for correspondence. Farhana Zahir – Email: farhanazahir@gmail.com, Rizwan Hasan Khan – Email: rizwanhkhan@hotmail.com

DOI: 10.1515/cmble-2015-0025 Volume 20 (2015) pp 448-465
Title EVALUATION OF THE EXPRESSIONS PATTERN OF miR-10b, 21, 200c, 373 and 520c TO FIND THE CORRELATION BETWEEN EPITHELIAL-TO-MESENCHYMAL TRANSITION AND MELANOMA STEM CELL POTENTIAL IN ISOLATED CANCER STEM CELLS
Authors Motahareh Rajabi Fomeshi1,2, Marzieh Ebrahimi2,*, Seyed Javad Mowla3, Pardis Khosravani2, Javad Firouzi2 and Hamid Khayatzadeh3
Abstract Small non-coding RNAs named microRNAs (miRNAs) modulate some functions and signaling pathways in skin epithelial cells and melanocytes. They also function as oncogenes or tumor suppressors in malignancies and tumor metastasis. We investigated the expression patterns of miRNAs, including miR-10b, 21, 200c, 373 and 520c, which regulate epithelial-to-mesenchymal transition (EMT) and metastasis in isolated cancer stem cells (CSCs) and non-CSCs. Six melanoma cell lines were tested for the expressions of stem cell markers. Melanoma stem cells were enriched via fluorescence-activated cell sorting (FACS) using the CD133 cell surface marker or spheroid culture. They were then characterized based on colony and sphere formation, and the expressions of stemness and EMT regulator genes and their invasion potential were assessed using real-time qRT-PCR and invasion assay. Our esults indicate that cells enriched via sphere formation expressed all the stemness-related genes and had an enhanced number of colonies, spheres and invaded cells compared to cells enriched using the CD133 cell surface marker. Moreover, miRNAs controlling metastasis increased in the melanospheres. This may be related to the involvement of CSCs in the metastatic process. However, this must be further confirmed through the application of knockdown experiments. The results show that sphere formation is a useful method for enriching melanoma stem cells. Melanospheres were found to upregulate miR-10b, 21, 200c, 373 and 520c, so we suggest that they may control both metastasis and stemness potential.
Keywords MicroRNA, Melanoma stem cell, Epithelial-to-mesenchymal transition, Melanosphere, Expression profiling, CD133, ABCG2, Invasion, Colony formation, Cancer stem cell, Metastasis
Address and Contact Information 1Department of Developmental Biology, University of Science and Culture, ACECR, Tehran, Iran,
2Department of Stem Cells and Developmental Biology at Cell Science Research Center, Royan Institute for Stem Cell Biology and Technology, ACECR, Tehran, Iran,
3Molecular Genetics Department, Faculty of Biological Sciences, Tarbiat Modares University, Tehran, Iran
* Author for correspondence. Email: mebrahimi@royaninstitute.org; phone: +98 21 22306485; fax: +98 21 23562507

DOI: 10.1515/cmble-2015-0026 Volume 20 (2015) pp 466-474
Title EFFECTS OF NEURITIN ON THE MIGRATION, SENESCENCE AND PROLIFERATION OF HUMAN BONE MARROW MESENCHYMAL STEM CELLS
Authors Xuhui Wang1,§, Chunyan Liu1,§, Fen Xu1, Lijuan Cui1, Siwei Tan2, Rong Chen2, Lei Yang2,* and Jin Huang1,*
Abstract Neuritin is a neurotrophic factor associated with neuroplasticity. Most studies on neuritin focus on the nervous system; however, there has been no comprehensive evaluation of neuritin in non-neuronal cells. In this study, we screened 11 cell lines and found that neuritin was not expressed in bone marrow-derived mesenchymal stem cells (BMSCs). Neuritin-expressing BMSCs were obtained by transfection. In the neuritin-expressing BMSC model, we observed significantly greater cell migration and improved anti-senescence protection, in addition to reduced proliferation and viability. In conclusion, neuritin not only plays an important role in the nervous system but also has an effect on the migration, senescence, proliferation, and viability of stem cells. This study provides a theoretical basis for understanding the function of neuritin.
Keywords Neuritin, Migration, Senescence, Proliferation, Cell viability, BMSCs, Non-neuronal cells, Wound scratch, β-galactosidase, Cell count
Address and Contact Information 1The Key Laboratory of Xinjiang Endemic & Ethnic Diseases and Department of Biochemistry, Shihezi University School of Medicine, Shihezi, Xinjiang, P. R. China,
2Hangzhou Normal University, School of Medicine, Hangzhou, Zhejiang, P. R. China
§ These authors contributed equally to this study
* Authors for correspondence. Jin Huang, email: huangjin623@163.com, Lei Yang, email: 20080009@hznu.edu.cn

DOI: 10.1515/cmble-2015-0027 Volume 20 (2015) pp 475-487
Title LIPOXIN A4 METHYL ESTER ALLEVIATES VASCULAR COGNITION IMPAIRMENT BY REGULATING THE EXPRESSION OF PROTEINS RELATED TO AUTOPHAGY AND ER STRESS IN THE RAT HIPPOCAMPUS
Authors Yanqiu Jia1,§, Wei Jin1,§, Yining Xiao2, Yanhong Dong1, Tianjun Wang1, Mingyue Fan1, Jing Xu1, Nan Meng1, Ling Li1 and Peiyuan Lv1,*
Abstract Since autophagy and endoplasmic reticulum stress mechanisms are involved in some neurodegenerative and cerebral vascular diseases, we suspected that similar mechanisms might participate in vascular cognitive impairments induced by chronic cerebral hypoperfusion. Lipoxin A4 methyl ester (LXA4 ME) is an inflammation inhibitor that exhibits potent protective effects in experimental stroke models. In an earlier study, we found that LXA4 ME improved cognitive deficit in a rat model of vascular cognitive impairment created using bilateral common carotid artery ligation (BCCAL) and two-vessel occlusion (2VO). In this study, LXA4 ME treatment of 2VO rats improved brain morphological defects. We found that LXA4 ME reduced the expression of some autophagy- and ERS-related factors in the hippocampus of 2VO rats, namely C/EBP homologous protein, beclin1 and the ratio of microtubule-associated protein light chain 3 II (LC3-II) to LC3-I. By contrast, LXA4 ME upregulated the protein expression of phospho-mTOR, total-mTOR, glucose-regulated protein 78 and spliced and unspliced X-box binding protein-1 mRNA. Differential protein regulation by LXA4 ME might underlie its ability to protect cognition after chronic cerebral hypoperfusion.
Keywords Lipoxin A4 methyl ester, Hippocampus, Chronic cerebral hypoperfusion, Autophagy, Endoplasmic reticulum stress, Unfolded protein response
Address and Contact Information 1Department of Neurology, Hebei General Hospital, 348th Heping West Road, Shijiazhuang, 050051, P. R. China,
2Department of Neurology, Hebei Medical University, 361th Zhongshan East Road, Shijiazhuang, 050017, P. R. China
§ Authors contributed equally to this paper
* Author for correspondence. Email: peiyuanlu@163.com, phone: +86 311 85988906

DOI: 10.1515/cmble-2015-0028 Volume 20 (2015) pp 488-509
Title BIOMEDICAL AND AGRICULTURAL APPLICATIONS OF ENERGY DISPERSIVE X-RAY SPECTROSCOPY IN ELECTRON MICROSCOPY
Authors Elżbieta Wyroba*, Szymon Suski, Karolina Miller and Rafał Bartosiewicz
Abstract Energy dispersive X-ray spectroscopy (EDS) in electron microscopy has been widely used in many research areas since it provides precise information on the chemical composition of subcellular structures that may be correlated with their high resolution images. In EDS the characteristic X-rays typical of each element are analyzed and the new detectors – an example of which we describe – allow for setting precisely the area of measurements and acquiring signals as a point analysis, as a linescan or in the image format of the desired area. Mapping of the elements requires stringent methods of sample preparation to prevent redistribution/loss of the elements as well as elimination of the risk of overlapping spectra. Both qualitative and quantitative analyses may be performed at a low probe current suitable for thin biological samples. Descriptions of preparation techniques, drawbacks and precautions necessary to obtain reliable results are provided, including data on standards, effects of specimen roughness and quantification. Data on EPMA application in different fields of biomedical and agricultural studies are reviewed. In this review we refer to recent EDS/EPMA applications in medical diagnostics, studies on air pollution and agrochemicals as well as on plant models used to monitor the environment.
Keywords EDS, Electron microscopy, Overlapping spectra, Sample preparation, X-ray, Standards, EPMA
Address and Contact Information Laboratory of Electron Microscopy, Nencki Institute of Experimental Biology, The Polish Academy of Sciences, 3 Pasteur Street, 02-093 Warsaw, Poland
* Author for correspondence. Phone: 48-22 5892357, Fax: 48-228225342, e-mail: e.wyroba@nencki.gov.pl

DOI: 10.1515/cmble-2015-0029 Volume 20 (2015) pp 510-533
Title THE EFFECT OF THE BIOACTIVE SPHINGOLIPIDS S1P AND C1P ON MULTIPOTENT STROMAL CELLS – NEW OPPORTUNITIES IN REGENERATIVE MEDICINE
Authors Krzysztof Marycz1,2,*, Agnieszka Śmieszek1,2, Marta Jeleń2, Klaudia Chrząstek2, Jakub Grzesiak2 and Justyna Meissner2
Abstract Sphingosine-1-phosphate (S1P) and ceramide-1-phosphate (C1P) belong to a family of bioactive sphingolipids that act as important extracellular signaling molecules and chemoattractants. This study investigated the influence of S1P and C1P on the morphology, proliferation activity and osteogenic properties of rat multipotent stromal cells derived from bone marrow (BMSCs) and subcutaneous adipose tissue (ASCs). We show that S1P and C1P can influence mesenchymal stem cells (MSCs), each in a different manner. S1P stimulation promoted the formation of cellular aggregates of BMSCs and ASCs, while C1P had an effect on the regular growth pattern and expanded intercellular connections, thereby increasing the proliferative activity. Although osteogenic differentiation of MSCs was enhanced by the addition of S1P, the effectiveness of osteoblast differentiation was more evident in BMSCs, particularly when biochemical and molecular marker levels were considered. The results of the functional osteogenic differentiation assay, which includes an evaluation of the efficiency of extracellular matrix mineralization (SEM-EDX), revealed the formation of numerous mineral aggregates in BMSC cultures stimulated with S1P. Our data demonstrated that in an appropriate combination, the bioactive sphingolipids S1P and C1P may find wide application in regenerative medicine, particularly in bone regeneration with the use of MSCs.
Keywords Mesenchymal stem cells, Sphingolipids, Sphingosine-1-phosphate, Ceramide-1-phosphate, Osteogenic differentiation, Regenerative medicine, Bone marrow stem cells, Adipose stem cells
Address and Contact Information 1Wrocław Research Centre EIT+, Stabłowicka 147, 54-066 Wrocław, Poland,
2University of Environmental and Life Sciences Wroclaw, Faculty of Biology, Kożuchowska 5b, 50-631 Wrocław, Poland
* Author for correspondence. Email: krzysztof.marycz@up.wroc.pl