Cell Journal (Yakhteh)

Number 12, 2022

705-714
Differentiation of Human Adipose-Derived Mesenchymal Stromal/Stem Cells into Insulin-Producing Cells with A Single Tet-Off Lentiviral Vector System

Authors: Hiroyuki Moriyama; Mariko Moriyama; Toshiyuki Ozawa; Daisuke Tsuruta; Takao Hayakawa

Abstract

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Objective: Human adipose-derived mesenchymal stromal/stem cells (hASC) constitute an attractive source of stem cells for cell-based therapies in regenerative medicine and tissue engineering as they are easy to acquire from lipoaspirate, expansion, and genetic modification ex vivo. The combination of Pdx-1, MafA, and NeuroD1 has been indicated to possess the ability to reprogram various types of cells into insulin-producing cells. The aim of this study is to investigate whether MafA and NeuroD1 would cooperate with Pdx-1 in the differentiation of hASC into insulin-producing cells. Materials and Methods: In this experimental study, we generated polycistronic expression vectors expressing Pdx1 and MafA/NeuroD1 with a reporter from a human EF-1α promoter using 2A peptides in a single tet-off lentiviral vector system. Briefly, hASC were transduced with the lentiviral vectors and allowed to differentiate into insulin-producing cells in vitro and in vivo. Thereafter, RNA expression, dithizone staining, and immunofluorescent analysis were conducted. Results: Cleaved transcriptional factors from a single tet-off lentiviral vector were functionally equivalent to their native proteins and strictly regulated by doxycycline (Dox). Insulin gene expression in hASC transduced with Pdx1, Pdx1/ MafA, and Pdx1/NeuroD1 in differentiation medium were successfully increased by 1.89 ± 0.39, 4.81 ± 0.98, 5.51 ± 0.63, respectively, compared to venus-transduced, control hASC. These cells could form dithizone-positive cell clusters in vitro and were found to express insulin in vivo. Conclusion: Using our single tet-off lentiviral vector system, Pdx-1 and MafA/NeuroD1 could be simultaneously expressed in the absence of Dox. Further, this system allowed the differentiation of hASC into insulin-producing cells.

715-722
Assessment of Adiponectin and Sperm Function Parameters in Obese and Non-Obese: A Comprehensive Study

Authors: Marziyeh Pooladi; Mohamadreza Sharifi; Gholamreza Dashti

Abstract

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Objective The role of adiponectin in sperm function is inconclusive and there is a paucity of evidence. Obesity shows an ambiguous influence on sperm motility, and male subfertility. The aim of this study was to compare the role of adiponectin and sperm functional parameters among obese and non-obese men. Materials and Methods In this comprehensive study, 64 male patients were included, and were classified as non-obese [body mass index (BMI)< 24.9 kg/m2, n=32] and obese (BMI >25 kg/m2, n=32) groups. Sperm analysis, was conducted using World Health Organization (WHO) 2010 standards. Real-time polymerase chain reaction (PCR) and enzyme-linked immunosorbent assay (ELISA) were used for the analysis of adiponectin gene expression and protein levels, respectively. Sperm viability was assessed using the 3-(4, 5-dimethylthiazol-2-yl)-2, 5- diphenyl tetrazolium bromide (MTT test), Acridine orange (AO) test was utilized to detect DNA denaturation, and sperm chromatin dispersion (SCD) technique was used to investigate the fragmentation of DNA. Results In obese men, adiponectin gene expression (P<0.0001) and protein levels (P<0.001) were significantly lower compared to the non-obese group. Additionally, sperm motility, was significantly lower in the obese group. The rapid progressive (RP) motility was less in obese men in comparison to the non-obese group (P<0.001). Sperm count and morphology were not significantly different in the two groups. DNA denaturation and DNA fragmentation were significantly more frequent in the obese group than in non-obese men (P<0.05) and (P<0.01), respectively. The obese men showed significantly lower sperm viability compared to the non-obese group (P<0.05). Conclusion This study showed no significant correlation between the evaluated variables (sperm parameter, sperm viability, DNA fragmentation and integrity), and obesity in men. Based on these results, adiponectin may potentially play positive role in sperm function for acquiring fertility.

723-731
Mutant Allele of CD44 (rs8193C>T) and Pum2 Regulatory Element as A Prognosis Factor of Prostate Neoplasms: A Case-Control and In Silico Studies

Authors: Mohammadkazem Heydari; Abasalt Hosseinzadeh Colagar; Emadoddin Moudi

Abstract

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Objective Expression of CD44 variant 6 (CD44v6) as a homing-associated cell adhesion molecule (HCAM), has proved to change most cancer cells. Aim of the study is the effect of mutant allele of CD44 (rs8193C>T) and Pum2 regulatory element as a prognosis factor of prostate neoplasms: a case-control and in silico studies in the Mazandaran province-Iran. Materials and Methods In a case-control study, CD44-rs8193C>T genotyping of the 420 prostate neoplasms (210 benign prostatic hyperplasia (BPH) patients and 210 prostate cancer patients) and 150 healthy samples are performed by the touchdown polymerase chain reaction with confronting two-pair primers (PCR-CTPP) method. The T mutant allele effects on the mRNA structure and cell pathways were also investigated in silico methods. Results Our results showed that the increase of T mutant allele frequency was significantly associated with BPH compared with prostate cancer. Furthermore, results showed TT genotype was significantly associated with BPH [odds ratio (OR)=0.572 and P=0.015], and also influenced the CD44v6 transcript secondary structure, miRNA binding, and regulatory element-binding site for Pum2 protein. Attachment of Pum2 to standard CD44 transcript may lead to transcript isoform-switching and shift-expression to a variety of CD44 isoforms, which can trigger some of the cell signaling pathways, such as Nanog-Stat, PKC-Nanog, and PKC-Twist. Conclusion Based on this, the presence of the T mutant allele of CD44 (rs8193C>T) in the populations may create a regulatory element-binding site for Pum2. So, it could be known as a prognosis factor and prediction of prostate neoplasms. However, more comprehensive studies in different populations (with various ethnicities and large population sizes), and also CD44v6 gene expression studies in protein and transcript levels are required to confirm our data.

732-740
CTRP1 Aggravates Cardiac Fibrosis by Regulating The NOX2/P38 Pathway in Macrophages

Authors: Chenyu Li; Shaozhen Ying; Xiaolin Wu; Tongjian Zhu; Qing Zhou; Yue Zhang; Yongsheng Liu; Rui Zhu; He Hu

Abstract

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Objective C1q/TNF-related proteins 1 (CTRP1) is a recently identified adiponectin associated with obesity-linked disorders and adverse cardiovascular events. The effect of CTRP1 on cardiac fibrosis has not yet been fully elucidated; thus, we aimed to explore this association. Materials and Methods In this experimental study, a mouse model of cardiac fibrosis was established by administering isoproterenol (ISO) (subcutaneously injecting 10 mg/kg/day for 3 days and then 5 mg/kg/day for 11 days). Mice were also injected with recombinant CTRP1 protein (200 μg/kg) 14 days after the final ISO administration. Adult mouse fibroblasts were isolated and stimulated with transforming growth factor (TGF) β1, followed by treatment with recombinant CTRP1. Primary bone marrow-derived macrophages were isolated from C57BL/6J mice and treated with recombinant CTRP1 as well. Results CTRP1 level was increased in mouse plasma and heart tissue 2 weeks after ISO injection. Our findings indicated that recombinant CTRP1 injection aggravated ISO-induced cardiac fibrosis and dysfunction. However, recombinant CTRP1 did not alter TGFβ1-induced fibroblast proliferation and activation or collagen transcription. Recombinant CTRP1 exacerbated ISO-induced macrophage infiltration and inflammatory response. We determined that macrophages treated with recombinant CTRP1 showed increased pro-inflammatory cytokine release. Fibroblasts co-cultured with macrophages treated with recombinant CTRP1 showed increased proliferation and collagen transcription. We also found that CTRP1 upregulated the NADPH oxidase 2 (NOX2)/p38 pathway in macrophages. When we inhibited p38 signaling, the pro-inflammatory effect of CTRP1 on macrophages was counteracted. Fibroblasts co-cultured with macrophages treated with a p38 inhibitor also showed limited proliferation and collagen transcription. Conclusion: Cardiac fibrosis was aggravated with the activation of the NOX2/p38 pathway in macrophages after CTRP1 treatment.

741-747
Left Ventricular Geometry and Angiogenesis Improvement in Rat Chronic Ischemic Cardiomyopathy following Injection of Encapsulated Mesenchymal Stem Cells

Authors: Negar Karimi Hajishoreh; Nafiseh Baheiraei; Nasim Naderi; Mojdeh Salehnia; Mehdi Razavi

Abstract

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Objective Injection of hydrogel and cells into myocardial infarction (MI) patients is one of the emerging treatment techniques, however, it has some limitations such as a lack of electromechanical properties and neovascularization. We investigated the therapeutic potential of new electroactive hydrogel [reduced graphene oxide (rGO)/Alginate (ALG)] encapsulated human bone marrow mesenchymal stem cells (BMSCs). Materials and Methods The experimental study involved ligating the left anterior descending coronary artery (LAD) in rat models of chronic ischemic cardiomyopathy. Echocardiograms were analyzed at 4 and 8 weeks after MI treatment. In the eighth week after injection in the heart, the rats were sacrificed. Histological and immunohistochemical analyses were performed using Hematoxylin and Eosin (H&E) staining, Masson’s trichrome staining and anti-CD31 antibody to analyze tissue structure and detect neovascularization. Results In comparison to the control and other treatment groups, MSCs encapsulated in rGO-ALG showed significant improvements in fractional shortening (FS), ejection fraction (EF), wall thickness and internal diameters (P<0.05). The morphological observation showed several small blood vessels formed around the transplantation site in all treated groups especially in the MSC-ALG-rGO group 8 weeks after the transplantation. Also, Masson’s trichrome staining indicated an increased amount of collagen fibers in rGO-ALG-MSC. Microvessel density was significantly higher using MSC-ALG-rGO compared to controls (P<0.01). Conclusion This study demonstrates that intramyocardial injection of rGO/ALG, a bio-electroactive hydrogel, is safe for increasing LV function, neovascularization, and adjusting electrical characteristics following MI. The results confirm ALG promising capability as a natural therapeutic for cardiac regeneration.

748-756
Impact of Intraventricular Human Adipose-Derived Stem Cells Transplantation with Pregnenolone Treatment on Remyelination of Corpus Callosum in A Rat Model of Multiple Sclerosis

Authors: Mohammad Mardani; Rasoul Ganji; Nazem Ghasemi; Mohammad Kazemi; Shahnaz Razavi

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Objective Multiple sclerosis (MS) is known as a nerve tissue disorder, which causes demyelination of central nervous system (CNS) fibers. Cell-based treatment is a novel strategy for the treatment of demyelinating diseases such as MS. Adipose-derived stem cells (ADSCs) have neuroprotective and neuroregenerative effects and pregnenolone as a neurosteroid has remarkable roles in neurogenesis. We intend to examine the impact of intraventricular transplantation of human ADSCs and systemic injection of pregnenolone on the remyelination of a rat model cuprizone-induced demyelination. Materials and Methods This experimental study was performed on 36 male Wistar rats that received a regular diet and a cuprizone diet for 3 weeks for M.S. induction. Through lipoaspirate surgery, human-ADSCs (hADSCs) were obtained from a patient. Six groups of rats (n=6): healthy, MS, sham, pregnenolone injection, ADSCs transplantation, and pregnenolone injection/ADSCs transplantation were included in this study. For assessment of remyelination, transmission electron microscopy (TEM), immunohistochemistry staining, real-time reverse transcription-polymerase chain reaction (RT-PCR), and enzyme-linked immunosorbent assay (ELISA) were performed. Results TEM outcomes revealed an increase in the thickness of the fibers myelin in the treatment groups (P<0.05). We also observed a significant upregulation of MBP, PDGFR-α, and MOG after treatment with hADSCs and pregnenolone compared to other study groups (P<0.001). These results were confirmed by immunostaining analysis. Moreover, there was no significant difference between the ADSCs/pregnenolone group and the control group regarding the level of MBP, A2B5, and MOG proteins in ELISA. Conclusion Our data implied that the remyelination and cell recovery were more improved by intraventricular ADSCs transplantation and pregnenolone injection after inducing a rat model of MS.

757-763
Aberrant DNA Methylation Status and mRNA Expression Level of SMG1 Gene in Chronic Myeloid Leukemia: A Case-Control Study

Authors: Tahereh Hojjatipour; Mahsa Sohani; Amirhosein Maali; Sharbano Rostami; Mehdi Azad

Abstract

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Objective Chronic myeloid leukemia (CML) is a myeloproliferative malignancy with different stages. Aberrant epigenetic modifications, such as DNA methylation, have been introduced as a signature for diverse cancers which also plays a crucial role in CML pathogenesis and development. Suppressor with morphogenetic effect on genitalia (SMG1) gene recently has been brought to the spotlight as a potent tumor suppressor gene that can be suppressed by tumors for further progress. The present study aims to investigate SMG1 status in CML patients. Materials and Methods In this case-control study, peripheral blood from 30 patients with different phases of CML [new case (N)=10, complete molecular remission (CMR)=10, blastic phase (BP)=10] and 10 healthy subjects were collected. Methylation status and expression level of SMG1 gene promoter was assessed by methylation-specific polymerase chain reaction (MSP) and quantitative reverse-transcription PCR, respectively. Results MSP results of SMG1 gene promotor in the new case group were methylated (60% methylated, 30% hemimethylated and 10% unmethylated). All CMR and control group patients were unmethylated in the SMG1 gene promoter. In the BP group, methylated SMG1 promoter was seen (50% of patients had a methylated status and 50% had hemimethylated status). In comparison with the healthy subjects, expression level of SMG1 in the new case group was decreased (P<0.01); in the CMR group and BP-CML groups, it was increased (P<0.05). No significant correlation between patients’ hematological features and SMG1 methylation was seen. Conclusion Our results demonstrated that aberrant methylation of SMG1 occurred in CML patients and it had a significant association with SMG1 expression. SMG1 gene promoter showed diverse methylated status and subsequent expression levels in different phases of CML. These findings suggested possible participation of SMG1 suppression in the CML pathogenesis.

764-773
Generation and Differentiation of Induced Pluripotent Stem Cells from Mononuclear Cells in An Age-Related Macular Degeneration Patient

Authors: Tongmiao Wang; Jingwen Liu; Jianhua Chen; Bo Qin

Abstract

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Objective We aimed to generate induced pluripotent stem cells (iPSCs)-derived retinal pigmented epithelium (RPE) cells from peripheral blood mononuclear cells (PBMCs) and age-related macular degeneration (AMD) patient to provide potential cell sources for both basic scientific research and clinical application. Materials and Methods In this experimental study, PBMCs were isolated from the whole blood of a 70-year-old female patient with AMD and reprogrammed into iPSCs by transfection of Sendai virus that contained Yamanaka factors (OCT4, SOX2, KLF4, and c-MYC). Flow cytometry, real-time quantitative polymerase chain reaction (qPCR), karyotype analysis, embryoid body (EB) formation, and teratoma detection were performed to confirm that AMD-iPSCs exhibited full pluripotency and maintained a normal karyotype after reprogramming. AMD-iPSCs were induced into RPE cells by stepwise induced differentiation and specific markers of RPE cells examined by immunofluorescence and flow cytometry. Results The iPSC colonies started to form on three weeks post-infection. AMD-iPSCs exhibited typical morphology including roundness, a large nucleus, sparse cytoplasm, and conspicuous nucleoli. QPCR data showed that AMDiPSCs expressed pluripotency markers (endo-OCT4, endo-SOX2, NANOG and REX1). Flow cytometry indicated 99.7% of generated iPSCs was TRA-1-60 positive. Methylation sequencing showed that the regions of OCT4 and NANOG promoter were demethylated in iPSCs. EBs and teratomas formation assay showed that iPSCs had strong differentiation potential and pluripotency. After a series of inductions with differentiation mediums, a monolayer of AMDiPSC-RPE cells was observed on day 50. The AMD-iPSC-RPEs highly expressed specific RPE markers (MITF, ZO-1, Bestrophin, and PMEL17). Conclusion A high quality iPSCs could be established from the PBMCs obtained from elderly AMD patient. The AMDiPSC displayed complete pluripotency, enabling for scientific study, disease modeling, pharmacological testing, and therapeutic applications in personalized medicine. Collectively, we successfully differentiated the iPSCs into RPE with native RPE characteristics, which might provide potential regenerative treatments for AMD patients.

774-778
Androgen Receptor Blockade Using Enzalutamide Suppresses Long Non-Coding RNA ARLNC1 in Prostate Cancer Cells

Authors: Günel Huseynova; Emre Özgür; Sema Bilgiç Gazioğlu; Ebru Esin Yörüker; Ugur Gezer

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Prostate cancer (PCa) is a common malignant disease with high mortality rates that develops and progresses in an androgen-dependent way. In recent years, RNA sequencing enabled identification of many PCa-related long noncoding RNAs including androgen receptor-regulated long non-coding RNA 1 (ARLNC1) and prostate cancer-associated transcript 1 (PCAT1). In the present study, our goal was to illuminate expression changes of ARLNC1 and PCAT1 in the context of androgen stimulation or androgen receptor (AR) blockade with respect to AR expression status. In this experimental study, LNCaP cells and higher AR-expressing LNCaP-AR++ cells were used as cell models. Cells were treated with dihydrotestosterone (DHT) as an androgen stimulator and/or enzalutamide as an AR inhibitor. Cell viability was assessed using annexin V and propidium iodide (PI) staining in flow cytometry. Androgen stimulation prompted baseline ARLNC1 levels by 53.5-fold in the LNCaP cells (P=0.01) and by 25-fold in the LNCAP-AR+ cells (P=0.18). AR inhibition by enzalutamide reduced baseline ARLNC1 in LNCaP-AR++ cells by 2-fold (P=0.01), but to a lesser extent in LNCaP cells. Co-treatment of cells with DHT and enzalutamide led to a remarkable decrease in the DHT effect on ARLNC1 expression. No specific effect of androgen stimulation or AR blockade on PCAT1 expression was detected. Our results revealed that the extent of induction of ARLNC1 by androgen is modulated by receptor expression status. In addition, we determined that AR blockade, via enzalutamide, effectively suppresses ARLNC1 both at baseline and after induction by DHT.

779-781
Inhibition of miR-200b Promotes Angiogenesis in Endothelial Cells by Activating The Notch Pathway

Authors: Tie-Ying Qiu; Jin Huang; Li-Ping Wang; Bi-Song Zhu

Abstract

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In this article which was published in Cell J, Vol 23, No 1, Spring 2021, on pages 51-60, the authors discovered that Figures 1B, 2D, 2F, 5B, and 5D some errors that occurred accidentally during figure organization in this article. The figures below have been corrected. The authors would like to apologies for any inconvenience caused.