Cell Journal (Yakhteh)

132-137

Authors: Wenli Zhang, Shaohua Liu, Weichen Zhang, Wei Hu, Min Jiang, Amin Tamadon, Yi Feng

Objective: Passive CLARITY is a whole-tissue clearing protocol, based on sodium dodecyl sulfate (SDS) clearing, for imaging intact tissue containing transgenic or immunolabeled fluorescent proteins. In this study, we present an improved passive CLARITY protocol with efficient immunolabeling without the need for electrophoresis or complex instrumentation. Materials and Methods: In this experimental study, after perfusion of C57BL/6N mice with phosphate-buffered saline (PBS) and then with acrylamide-paraformaldehyde (PFA), the quadriceps femoris muscle was removed. The muscle samples were post-fixed and degassed to initiate polymerization. After removing the excess hydrogel around the muscle, lipids were washed out with the passive CLARITY technique. The transparent whole intact muscles were labeled for vessel and neuron markers, and then imaged by confocal microscopy. Three-dimensional images were reconstructed to present the muscle tissue architecture. Results: We established a simple clearing protocol using wild type mouse muscle and labeling of vasculatures and neurons. Imaging the fluorescent signal was achieved by protein fixation, adjusting the pH of the SDS solution and using an optimum temperature (37˚C) for tissue clearing, all of which contributed to the superiority of our protocol. Conclusion: We conclude that this passive CLARITY protocol can be successfully applied to three-dimensional cellular and whole muscle imaging in mice, and will facilitate structural analyses and connectomics of large assemblies of muscle cells, vessels and neurons in the context of three-dimensional systems.

150-156

Authors: Maedeh Rouigari, Moein Dehbashi, Kamran Ghaedi, meraj pourhossein

Objective: For the first time, we used molecular signaling pathway enrichment analysis to determine possible involvement of miR-126 and IRS-1 in neurotrophin pathway. Materials and Methods: In this prospective study, validated and predicted targets (targetome) of miR-126 were collected following searching miRtarbase (http://mirtarbase.mbc.nctu.edu.tw/) and miRWalk 2.0 databases, respectively. Then, approximate expression of miR-126 targeting in Glioma tissue was examined using UniGene database (http://www.ncbi. nlm.nih.gov/unigene). In silico molecular pathway enrichment analysis was carried out by DAVID 6.7 database (http://david. abcc.ncifcrf.gov/) to explore which signaling pathway is related to miR-126 targeting and how miR-126 attributes to glioma development. Results: MiR-126 exerts a variety of functions in cancer pathogenesis via suppression of expression of target gene including PI3K, KRAS, EGFL7, IRS-1 and VEGF. Our bioinformatic studies implementing DAVID database, showed the involvement of miR-126 target genes in several signaling pathways including cancer pathogenesis, neurotrophin functions, Glioma formation, insulin function, focal adhesion production, chemokine synthesis and secretion and regulation of the actin cytoskeleton. Conclusion: Taken together, we concluded that miR-126 enhances the formation of glioma cancer stem cell probably via down regulation of IRS-1 in neurotrophin signaling pathway.

168-176

Authors: Mehrafarin Fesharaki, Shahnaz Razavi, Laleh Ghasemi-Mobarakeh, Mohaddeseh Behjati, Reyhaneh Yarahmadian, Mohammad Kazemi, Hossein Hejazi

Objective: This study aimed to isolate and culture SADS cells, investigate their neurogenic capacity and evaluate their application for nerve tissue engineering. Materials and Methods: In this experimental study, SADS cells were isolated from human adipose tissue. After 7-day treatment of SADS cells with insulin, indomethacin and isobutylmethylxanthine, neurogenic differentiation of SADS cells was investigated. During this study, Poly (ε-caprolactone) (PCL) and PCL/gelatin nanofibrous scaffolds were fabricated using electrospinning and subsequently nanofibrous scaffolds were coated with platelet-rich plasma (PRP). SADS cells were also seeded on nanofibrous scaffolds and neurogentic differentiation of these cells on nanofibers was also evaluated. Effect of PRP on proliferation and differentiation of SADS cells on scaffolds was also studied. Results: Our results showed that after 7-day treatment of SADS cells with insulin, indomethacin and isobutylmethylxanthine, SADS cells expressed markers characteristic of neural cells such as nestin and neuron specific nuclear protein (NEUN) (as early neuronal markers) as well as microtubule-associated protein 2 (MAP2) and neuronal microtubule-associated (TAU) (as mature neuronal markers) while mature astrocyte maker (GFAP) was not expressed. MTT assay and SEM results showed that incorporation of gelatin and PRP into the structure of nanofibrous scaffolds has a significant positive influence on the bioactivity of scaffolds. Our results also showed neurogentic differentiation of SADS cells on scaffolds. Conclusion: Our results demonstrated that SADS cells have potential to differentiate into early and mature progenitor neurons, in vitro. PCL/gelatin/PRP was found to be a promising substrate for proliferation of SADS cells and differentiation of these cells into neural cells which make these scaffolds a candidate for further in vivo experiments and suggest their application for nerve tissue engineering.

183-187

Authors: Alireza Abdanipour, Behnaz Shahsavandi, Mohsen Alipour, Hadi Feizi

Objective: Ghrelin is a peptide which has a proliferative and antiapoptotic effect in many cells including bone marrow stromal cells (BMSCs). Homeobox protein B4 (HOXB4) is a transcription factor involved in stem cell regeneration and survival. The aim of the study was to find out the efect of ghrelin on Hoxb4 expression in BMSCs. Materials and Methods: In this experimental study, rat BMSCs were cultivated in Dulbecco’s Modified Eagle Medium (DMEM). Passage three BMSCs were treated with ghrelin 100 μM for 48 hours. Real-time polymerase chain reaction (PCR) was carried out from the untreated BMSCs (B), BMSCs treated with 125 μM H2O2 (BH), BMSCs treated with 100 μM ghrelin then 125 μM H2O2 (BGH) and BMSCs treated with 100 μM ghrelin (BG) groups. For immunofluorescence, cells were incubated with an anti-HOXB4 monoclonal antibody. Primary antibodies were visualized using the Fluorescein isothiocyanate (FITC) method. All data are presented as mean ± SEM and P<0.05 was considered as statistical significant. Results: Hoxb4 expression significantly increased in the BG compared with BH and BGH groups. Furthermore, 100 μM ghrelin, increased the mean of HOXB4 positive immunoreactive cells compared to the BH group. Conclusion: Ghrelin probably enhances proliferation and viability of BMSCs through Hoxb4 upregulation. However, the signaling pathway and other biological outcomes of this effect should be elucidated in different stem cells.

195-203

Authors: Mohammad Houshmand, Mozhde Nakhlestani Hagh, Masoud Soleimani, Amir Ali Hamidieh, Saeed Abroun, Mahin Nikougoftar Zarif

Objective: In order to clarify the role of microRNAs (miRNA) in megakaryocyte differentiation, we ran a microRNA microarray experiment to measure the expression level of 961 human miRNA in megakaryocytes differentiated from human umbilical cord blood CD133+ cells. Materials and Methods: In this experimental study, human CD133+ hematopoietic stem cells were collected from three human umbilical cord blood (UCB) samples, and then differentiated to the megakaryocytic lineage and characterized by flow cytometry, CFU-assay and ploidy analysis. Subsequently, microarray analysis was undertaken followed by quantitative polymerase chain reaction (qPCR) to validate differentially expressed miRNA identified in the microarray analysis. Results: A total of 10 and 14 miRNAs were upregulated (e.g. miR-1246 and miR-148-a) and down-regulated (e.g. miR- 551b and miR-10a) respectively during megakaryocyte differentiation, all of which were confirmed by qPCR. Analysis of targets of these miRNA showed that the majority of targets are transcription factors involved in megakaryopoiesis. Conclusion: We conclude that miRNA play an important role in megakaryocyte differentiation and may be used as targets to change the rate of differentiation and further our understanding of the biology of megakaryocyte commitment.

211-219

Authors: Mugdha Kunte, Krutika Desai

Objective: Considering the bioactivities exhibited by microalgae, the effect of protein extract of Chlorella minutissimma (CP extract) was investigated on the expression of human matrix metalloproteinases-1 (MMP-1) in the breast cancer cell line MDA-MB231, and that of MMP-2 and -9 in hepatocellular cancer cell line HepG2 at different expression levels. The study aimed identification and analysis of inhibitory activity of microalgal components extracted from Chlorella minutissima against human MMPs. Materials and Methods: In this experimental study, we analysed the effect of Chlorella extracts on MMP-1, -2, and -9 expression at various levels. Gelatin zymography was performed to study the inhibitory effect of Chlorella exracts on human gelatinases at the activity level, followed by western blotting to analyse the expression of all three MMPs at the protein level. The similar effect at the mRNA level along with the probable mechanism underlying inhibition of MMPs was assessed using real-time polymerase chain reaction (PCR). Results: The results reveal that the treatment with CP extract decreased the mRNA expression of MMP-1, MMP-2, and MMP-9 by 0.26-, 0.29-, and 0.40-fold, respectively, at 20 μg/ml concentration as well as inhibited the activity of MMP-2 and MMP-9 by 37.56 and 42.64%, respectively, at 15 μg/ml concentration. Additionally, upregulated mRNA expression of tissue inhibitor of metalloproteinases-3 (TIMP-3) by 1.68-fold was seen in HepG2 cells at 20 μg/ml concentration treatment group. However, CP extract did not induce any change in the mRNA expression of the TIMP-1, -2 and -4 in HepG2 and TIMP-1, -2, -3 and -4 in MDA-MB231 cells. Activator protein-1 (AP-1)-dependent c-Jun-mediated transcriptional regulation of MMP-1, -2, and -9 was also studied to elucidate the appropriate mechanism involved in the inhibition of MMPs. Conclusion: The CP extract successfully inhibited MMP-1, -2, and -9 at different expression levels through TIMP-3 upregulation and c-Jun downregulation.

231-243

Authors: Rohiyeh Souldouzi, Mazdak Razi, Ali Shalizar Jalali, Ghader Jalilzadeh-Amin, Saeedeh Amani

Objective: Pulegone (PGN) is a monoterpene ketone, whose metabolites exert several cytotoxic effects in various tissues. The present study was conducted in order to evaluate the (R)-(+) PGN-induced alterations in ovarian aromatization, proto-oncogenes and estrogen receptorα ( ERα) and ERβ receptors expressions. Materials and Methods: In this experimental study, mature albino mice were divided into experimental (received 25 mg/kg, 50 mg/kg and 100 mg/kg PGN, orally for 35 days) and control (received 2% solution of Tween 80 as a PGN solvent, orally) groups. The mRNA levels of Erα, Erβ, p53, Bcl-2, and cytochrome p450 (Cyp19) as well as ovarian angiogenesis were analyzed through reverse transcription polymerase chain reaction and immunohistochemical techniques, respectively. Moreover, apoptosis of follicular cells, serum estrogen and progesterone levels and mRNA damage were investigated via using terminal transferase and biotin-16-dUTP staining, electrochemilunescence and fluorescent microscopy methods, respectively. Results: The PGN reduced Erα, Erβ and Cyp19 expression at 50 mg/kg and 100 mg/kg doses, while significantly elevating p53 and reducing Bcl-2 expression. Finally, PGN impaired ovarian angiogenesis, increased apoptosis, elevated follicular atresia and reduced serum levels of estrogen and progesterone. Conclusion: Chronic exposure to PGN (50 mg/kg and 100 mg/kg), severely affects ovarian aromatization, protooncogenes mRNA levels and expression of ERs.

250-258

Authors: Mehdi Najar, Mohammad Fayyad-Kazan, Gordana Raicevic, Hussein Fayyad-Kazan, Nathalie Meuleman, Dominique Bron, Laurence Lagneaux

Objective: We aimed at characterizing the transcription profiles of immunological receptors associated with the biology of mesenchymal stromal cells (MSCs). Materials and Methods: In this experimental study, quantitative real time-polymerase chain reaction (qRTPCR) was performed to establish the transcription profiles of advanced glycation end-products (RAGE) receptor, C-type lectin receptors (CLRs, including DECTIN-1, DECTIN-2 and MINCLE), leukotriene B4 (LTB4) receptors (BLT1 and BLT2) and cysteinyl leukotrienes (CysLTs) receptors (CYSLTR1 and CYSLTR2) in distinct populations of MSCs grown under basic or inflammatory conditions. Results: MSCs derived from adipose tissue (AT), foreskin (FSK), Wharton’s jelly (WJ) and bone marrow (BM) exhibited significantly different transcription levels for these genes. Interestingly, these transcription profiles substantially changed following exposure of MSCs to inflammatory signals. Conclusion: Collectively, for the first time, our data highlights that MSCs depending on their tissue-source, present several relevant receptors potentially involved in the regulation of inflammatory and immunological responses. Understanding the roles of these receptors within MSCs immunobiology will incontestably improve the efficiency of utilization of MSCs during cell-based therapies.

267-277

Authors: Mohammad Hassan Naseri, Hoda Madani, Seyed Hossein Ahmadi Tafti, Maryam MoshkaniFarahani, Davood Kazemi-Saleh, Hossein Hosseinnejad, Saeid Hosseini, Sepideh Hekmat, Zargham Hossein Ahmadi, Majid Dehghani, Alireza Saadat, Soura Mardpour, Seyedeh-Esmat Hosseini, Maryam Esmaeilzadeh, Hakimeh Sadeghian, Gholamreza Bahoush, Ali Bassi, Ahmad Amin, Roghayeh Fazeli, Yaser Sharafi, Leila Arab, Mansour Movahhed, Saeid Davaran, Narges Ramezanzadeh, Azam Kouhkan, Ali Hezavehei, Mehrnaz Namiri, Fahimeh Kashfi, Ali Akhlaghi, Fattah Sotoodehnejadnematalahi, Ahmad VosoughDizaji, Hamid Gourabi, Naeema Syedi, Abdolhosein Shahverdi, Hossein Baharvand, Nasser Aghdami

Objective: The regenerative potential of bone marrow-derived mononuclear cells (MNCs) and CD133+ stem cells in the heart varies in terms of their pro-angiogenic effects. This phase II/III, multicenter and double-blind trial is designed to compare the functional effects of intramyocardial autologous transplantation of both cell types and placebo in patients with recent myocardial infarction (RMI) post-coronary artery bypass graft. Materials and Methods: This was a phase II/III, randomized, double-blind, placebo-controlled trial COMPARE CPM-RMI (CD133, Placebo, MNCs - recent myocardial infarction) conducted in accordance with the Declaration of Helsinki that assessed the safety and efficacy of CD133 and MNCs compared to placebo in patients with RMI. We randomly assigned 77 eligible RMI patients selected from 5 hospitals to receive CD133+ cells, MNC, or a placebo. Patients underwent gated single photon emission computed tomography assessments at 6 and 18 months post-intramyocardial transplantation. We tested the normally distributed efficacy outcomes with a mixed analysis of variance model that used the entire data set of baseline and between-group comparisons as well as within subject (time) and group×time interaction terms. Results: There were no related serious adverse events reported. The intramyocardial transplantation of both cell types increased left ventricular ejection fraction by 9% [95% confidence intervals (CI): 2.14% to 15.78%, P=0.01] and improved decreased systolic wall thickening by -3.7 (95% CI: -7.07 to -0.42, P=0.03). The CD133 group showed significantly decreased non-viable segments by 75% (P=0.001) compared to the placebo and 60% (P=0.01) compared to the MNC group. We observed this improvement at both the 6- and 18-month time points. Conclusion: Intramyocardial injections of CD133+ cells or MNCs appeared to be safe and efficient with superiority of CD133+ cells for patients with RMI. Although the sample size precluded a definitive statement about clinical outcomes, these results have provided the basis for larger studies to confirm definitive evidence about the efficacy of these cell types (Registration Number: NCT01167751).

284-289

Authors: Farah Talebi, Farideh Ghanbari Mardasi, Javad Mohammadi Asl, Ali Lashgari, Friedun farhadi

Bardet-Biedl syndrome (BBS) is a pleiotropic and multisystemic disorder characterized by rod-cone dystrophy, polydactyly, learning difficulties, renal abnormalities, obesity and hypogonadism. This disorder is genetically heterogeneous. Until now, a total of nineteen genes have been identified for BBS whose mutations explain more than 80% of diagnosed cases. Recently, the development of next generation sequencing (NGS) technology has accelerated mutation screening of target genes, resulting in lower cost and less time consumption. Here, we screened the most common BBS genes (BBS1-BBS13) using NGS in an Iranian family of a proposita displaying symptoms of BBS. Among the 18 mutations identified in the proposita, one (BBS12 c.56T>G and BBS12 c.1156C>T) was novel. This compound heterozygosity was confirmed by Sanger sequencing in the proposita and her parents. Although our data were presented as a case report, however, we suggest a new probable genetic mechanism other than the conventional autosomal recessive inheritance of BBS. Additionally, given that in some Iranian provinces, like Khuzestan, consanguineous marriages are common, designing mutational panels for genetic diseases is strongly recommended, especially for those with an autosomal recessive inheritance pattern.

293-293

Authors: Oranous Bashti, Mehrdad Noruzinia, Masoud Garshasbi, Morteza Abtahi

This article published in Cell J (Yakhteh), Vol 20, No 1, Apr-Jun 2018, on pages 84-89, the labels of columns in "Figure 3B" was changed. The correct one is presented below.