In many multicellular organisms, mature gametes originate from primordial germ cells (PGCs). Impr... more In many multicellular organisms, mature gametes originate from primordial germ cells (PGCs). Improvements in the culture of PGCs are important not only for developmental biology research, but also for preserving endangered species, and for genome editing and transgenic animal technologies. SMAD2/3 appear to be powerful regulators of gene expression; however, their potential positive impact on the regulation of PGC proliferation has not been taken into consideration. Here, the effect of TGF‐β signaling as the upstream activator of SMAD2/3 transcription factors was evaluated on chicken PGCs' proliferation. For this, chicken PGCs at stages 26−28 Hamburger−Hamilton were obtained from the embryonic gonadal regions and cultured on different feeders or feeder‐free substrates. The results showed that TGF‐β signaling agonists (IDE1 and Activin‐A) improved PGC proliferation to some extent while treatment with SB431542, the antagonist of TGF‐β, disrupted PGCs' proliferation. However, t...
Objective: Any damage to the optic nerve can potentially lead to degeneration of non-regenerating... more Objective: Any damage to the optic nerve can potentially lead to degeneration of non-regenerating axons and ultimately death of retinal ganglion cells (RGCs) that in most cases, are not curable by surgery or medication. Neuroprotective functions of different types of stem cells in the nervous system have been evaluated in many studies investigating the effectiveness of these cells in various retinal disease models. Neural progenitor cells (NPCs) secrete an assortment of trophic factors that are vital to the protection of the visual system. We aimed to assess the therapeutic potentials of NPCs in an ONC mouse model. Materials and methods: In this experimental study, NPCs were produced using noggin and retinoic acid from human embryonic stem cells (hESCs). Fifty mice were divided into the following three groups: i. Intact , ii. Vehicle [optic nerve crush+Hank's balanced salt solution (HBSS)], and iii. Treatment (optic nerve crush+NPCs). The visual behavior of the mice was examined using the Visual Cliff test, and in terms of RGC numbers, they were assessed by Brn3a immunostaining and retrograde tracing using DiI injection. Results: Intravenous injection of 50,000 NPCs through visual cliff did not produce any visual improvement. However, our data suggest that the RGCs protection was more than two-times in NPCs compared to the vehicle group as examined by Brn3a staining and retrograde tracing. Conclusion: Our study indicated that intravenous injection of NPCs could protect RGCs probably mediated by trophic factors. Due to this ability and good manufacturing practices (GMP) grade production feasibility, NPCs may be used for optic nerve protection.
Cardiomyopathies are a group of common heart disorders that affect numerous people worldwide. Lef... more Cardiomyopathies are a group of common heart disorders that affect numerous people worldwide. Left ventricular non-compaction (LVNC) is a structural disorder of the ventricular wall, categorized as a type of cardiomyopathy that mostly caused by genetic disorders. Genetic variations are underlying causes of developmental deformation of the heart wall and the resultant contractile insufficiency. Here, we investigated a family with several affected members exhibiting LVNC phenotype. By whole-exome sequencing (WES) of three affected members, we identified a novel heterozygous missense variant (c.1963C>A:p.Leu655Met) in the gene encoding myosin heavy chain 7 (MYH7). This gene is evolutionary conserved among different organisms. We identified MYH7 as a highly enriched myosin, compared to other types of myosin heavy chains, in skeletal and cardiac muscles. Furthermore, MYH7 was among a few classes of MYH in mouse heart that highly expresses from early embryonic to adult stages. In silic...
Additional file 3: Figure S3. Morphology and histology of the spinal cord injury (SCI) in s contu... more Additional file 3: Figure S3. Morphology and histology of the spinal cord injury (SCI) in s contusion model. (A) The adult male Wistar rat's spinal cord was exposed at T10. Through a dorsal midline incision, a T9–11 laminectomy was performed and the spinal cord was exposed. A contusion injury was made using a standardized weight-drop injury device (NYU impactor) as indicated by hemorrhage. (B) Hematoxylin and eosin (H&E) staining of the rat injured spinal cord sections at one week post-injury. The lesion core and glial scar are shown in the longitudinal section. (C) Transverse images show Gfap-immunoreactive astrocytes at the injured, caudal, and rostral sites. Note that the expression of Gfap was higher around the injured site relative to the caudal and rostral sites. (D) Double immunostaining for fibronectin (green) and Gfap (red) in a longitudinal spinal cord section one week post-injury in the rat injured spinal cord. The lesion site was filled with a fibronectin positive ma...
Additional file 2: Figure S2. The validation of inducible vector. (A) Immunostaining for astrocyt... more Additional file 2: Figure S2. The validation of inducible vector. (A) Immunostaining for astrocyte and neural stem cell (NSC) markers in transfected astrocytes. Transduction was performed with an empty vector under two conditions: Astrocytes in astrocyte medium without DOX (A) and astrocytes in induction medium (IM) and DOX (AD). In addition, astrocytes were transfected by the Zfp521 vector in the present and the absence of Dox in IM (AZD and AZ, respectively). Nuclei were counterstained with DAPI. (B) Quantification of the immunostainings for astrocytes and NSC markers in transfected astrocytes. Data are shown as mean ± SD of three biological replicates. Data were analyzed by ANOVA and Mann-Whitney U test as post hoc. ***: p
Additional file 1: Figure S1. Isolation and characterization of adult rat brain astrocytes. (A) P... more Additional file 1: Figure S1. Isolation and characterization of adult rat brain astrocytes. (A) Phase-contrast images of astrocytes. (B) RT-PCR analysis of astrocytes and neural stem cell (NSC) markers. (C) qRT-PCR analysis of astrocytes and NSC markers. Data were normalized against GAPDH and presented relative to the expression of each indicated gene in the astrocytes. (D) Immunostaining images for astrocyte markers. Nuclei were counterstained with DAPI. (E) Quantification of the immunostainings for astrocytes and NSC markers. Data in C and E are shown as mean ± SD of three biological replicates. ND: Not detected. The number of counted cells is presented in Additional file 4: Table S3.
Additional file 4: Table S1. List of antibodies used for immunostaining. Table S2.Primer sequence... more Additional file 4: Table S1. List of antibodies used for immunostaining. Table S2.Primer sequences used for quantitative real-time PCR (qRT-PCR). Table S3. The number of cells in each immunostaining analysis.
Aims: Shigellosis is caused by Shigella species. Considering the high frequency of morbidity and ... more Aims: Shigellosis is caused by Shigella species. Considering the high frequency of morbidity and mortality reports and antibiotics resistance, designing and producing a vaccine against this disease is one of the goals of World Health Organization. Invasion Plasmid Antigen especially IpaC and IpaB are the major Shigella virulence agents and are also vaccine candidates. The aim of this study was to express ipaC gene which is of the virulence factors in order to investigate its immunogenicity. Materials & Methods: In this study, ipaC gene was obtained from NCBI gene bank and primers were designed. After genome extraction from Shigella dysenteriae, it was used as template for PCR amplification. The amplified ipaC gene by PCR was cloned into pTZ57R and sub-cloned into the expression vector pET-28a(+). E. coli BL21(DE3)plysS was transformed by recombinant vector pET-28a(+)/ipaC and expression of the recombinant ipaC was investigated by IPTG induction and SDS-PAGE electrophoresis. Results:...
Objective Dual inhibition of mitogen-activated protein kinase (MAPK) kinase (also known as MEK) a... more Objective Dual inhibition of mitogen-activated protein kinase (MAPK) kinase (also known as MEK) and transforming growth factor β (TGFβ) type I receptors by PD0325901 and SB431542, known as R2i has been introduced as a highly efficient approach to the generation of mouse embryonic stem cells (ESC). In the present study, we investigated the molecular mechanisms underlying ESC derivation in the R2i condition. Materials and Methods In this experimental study, zona-free whole E3.5 blastocysts were seeded on mouse embryonic fibroblast (MEF) feeder cells in both R2i and serum conventional media. The isolated inner cell mass (ICM), ESCs and the ICM-outgrowths were collected on days 3, 5 and 7 post-blastocyst culture for quantitative real time- polymerase chain reaction (qRT-PCR) analysis as well as to assess the DNA methylation status at the time points during the transition from ICM to ESC. Results qRT-PCR revealed a significantly higher expression of the pluripotency-related genes (Oct4, ...
Objective Systemic sclerosis (SSc) is a connective tissue disease associated with vascular damage... more Objective Systemic sclerosis (SSc) is a connective tissue disease associated with vascular damage and multi organ fibrotic changes with unknown pathogenesis. Most SSc patients suffer from defective angiogenesis/vasculogenesis and cardiac conditions leading to high mortality rates. We aimed to investigate the cardiovascular phenotype of SSc by cardiogenic differentiation of SSc induced pluripotent stem cells (iPSC). Materials and Methods In this experimental study, we generated iPSC from two diffuse SSc patients, followed by successful differentiation into endothelial cells (ECs) and cardiomyocytes (CMs). Results SSc-derived EC (SSc-EC) expressed KDR, a nearly EC marker, similar to healthy control-EC (C1-EC). After sorting and culturing KDR+ cells, the resulting EC expressed CD31, a late endothelial marker, but vascular endothelial (VE)-cadherin expression markedly dropped resulting in a functional defect as reflected in tube formation failure of SSc-EC. Interestingly, upregulation o...
Objective Limb regeneration mediated by blastema cells (BlCs) in mammals is limited to the digit ... more Objective Limb regeneration mediated by blastema cells (BlCs) in mammals is limited to the digit tips of neonates. Due to the lack of access to BlCs in adults and the difficulty in isolating and expanding BlCs from neonates, the use of a cellular population with similar features of BlCs would be a valuable strategy to direct a non-regenerative wound towards regeneration. In this study, we have initially isolated and cultured BlCs, and explored their characteristics in vitro. Next, we compared the capability of bone marrow-derived mesenchymal stem cells (BM-MSCs) as an alternative accessible cell source to BlCs for regeneration of appendages. Materials and Methods In this experimental study, BM-MSCs were isolated from BM and we obtained BlCs from the neonatal regenerating digit tip of C57B/6 mice. The cells were characterized for expressions of cell surface markers by flow cytometry. Quantitative-reverse transcription polymerase chain reaction (qRT-PCR) and lineage-specific staining ...
Objective Genetic modification of human embryonic stem cells (hESCs) is critical for their extens... more Objective Genetic modification of human embryonic stem cells (hESCs) is critical for their extensive use as a fundamental tool for cell therapy and basic research. Despite the fact that various methods such as lipofection and electroporation have been applied to transfer the gene of interest (GOI) into the target cell line, however, there are few re- ports that compare all parameters, which influence transfection efficiency. In this study, we examine all parameters that affect the efficiency of electroporation and lipofection for transient and long-term gene expression in three different cell lines to introduce the best method and determinant factor. Materials and Methods In this experimental study, both electroporation and lipofection approaches were employed for genetic modification. pCAG-EGFP was applied for tran- sient expression of green fluorescent protein in two genetically different hESC lines, Roy- an H5 (XX) and Royan H6 (XY), as well as human foreskin fibroblasts (hFF). F...
Highlights • A soluble form of mouse Neuropilin1 (msNRP1) has been cloned in a lentiviral vector ... more Highlights • A soluble form of mouse Neuropilin1 (msNRP1) has been cloned in a lentiviral vector • msNRP1 was successfully secreted by the transduced HEK293T in the supernatant • msNRP1-containing supernatant inhibited growth cone collapse activity of Semaphorin 3A Plain Language Summary Multiple Sclerosis (MS) is an inflammatory disease of the central nervous system. In MS, axons of neuronal cells become demyelinated, and remyelination process is also defective. In fact, oligodendrocyte precursor cells must survive, proliferate, and migrate towards demyelinated neurons and differentiate into mature oligodendrocytes to produce myelin and repair the neurons. Semaphorin 3A (Sema 3A) is a molecule that reduce the remyelination by preventing oligodendrocyte precursor cells to proliferate, migrate towards lesions, and differentiate into mature oligodendrocyte. We successfully produced mouse soluble Neuropilin 1 (msNRP1), that can bind Sema 3A and inhibit its activity. Then, the msNRP1 was used inhibit the activity of Sema 3A. Neural cells of dorsal root ganglia was cultured in presence of Sema 3A with and without msNRP1. We observed that when msNRP1 was added to the neuronal cells culture, the growth cones were able to form. On the other hand, the growth cones collapsed when msNRP1 was not added to the cultures. This finding is important in terms of MS treatment. Remyelination is defective in MS patients, and axons are progressively demyelinated. Finding a molecule that can prevent demyelination and enhance remyelination will be of great importance for MS treatment and improvement of their condition.
Background Spinal cord injury (SCI) results in glial scar formation and irreversible neuronal los... more Background Spinal cord injury (SCI) results in glial scar formation and irreversible neuronal loss, which finally leads to functional impairments and long-term disability. Our previous studies have demonstrated that the ectopic expression of Zfp521 reprograms fibroblasts and astrocytes into induced neural stem cells (iNSCs). However, it remains unclear whether treatment with Zfp521 also affects endogenous astrocytes, thus promoting further functional recovery following SCI. Methods Rat astrocytes were transdifferentiated into neural stem cells in vitro by ZFP521 or Sox2. Then, ZFP521 was applied to the spinal cord injury site of a rat. Transduction, real-time PCR, immunohistofluorescence, and function assessments were performed at 6 weeks post-transduction to evaluate improvement and in vivo lineage reprogramming of astrocytes. Results Here, we show that Zfp521 is more efficient in reprogramming cultured astrocytes compared with Sox2. In the injured spinal cord of an adult rat, resi...
This study aims to prepare intermediate mesoderm-like cells from mouse embryonic fibroblasts (MEF... more This study aims to prepare intermediate mesoderm-like cells from mouse embryonic fibroblasts (MEFs). In the first step, intermediate mesoderm-like cells (IMLCs) and renal epithelial-like cells (RELCs) were extracted from mouse embryonic stem cells (mESCs) in a specified media that contained two small molecules, CHIR99021 and TTNPB, along with growth factors, FGF9and BMP7. Then, MEFs were directly converted into IM by genes for the pluripotency factors, which encode the transcription factors; Oct4, Sox2, Klf4, and c-Myc (OSKM). These unstable intermediate cells were quickly encouraged to form IM with the assistance of CHIR99021 and TTNPB. The results showed that exogenous expression of OSKM factors for four days was adequate to generate partially reprogrammed cells (SSEA1 + /Nanog-). Real-time PCR and immunocytochemistry analysis confirmed the presence of the MEF-derived IMs. This study introduced a method for mESCs differentiation to RELCs followed by MEF conversion in an attempt to generate IM by circumventing pluripotency.
Pluripotent cells appear to be in a transient state during early development. These cells have th... more Pluripotent cells appear to be in a transient state during early development. These cells have the capability to transition into embryonic stem cells (ESCs). It has been reported that mouse pluripotent cells cultivated in chemically defined media sustain the ground state of pluripotency. Because the epigenetic pattern of pluripotent cells reflects their environment, culture under different conditions causes epigenetic changes, which could lead to genomic instability. This study focused on the DNA methylation pattern of repetitive elements (REs) and their activation levels under two ground‐state conditions and assessed the genomic integrity of ESCs. We measured the methylation and expression level of REs in different media. The results indicated that although the ground‐state conditions show higher REs activity, they did not lead to DNA damage; therefore, the level of genomic instability is lower under the ground‐state compared with the conventional condition. Our results indicated t...
Amputation of the proximal region in mammals is not followed by regeneration because blastema cel... more Amputation of the proximal region in mammals is not followed by regeneration because blastema cells (BCs) and expression of regenerative genes such as Msh homeobox (Msx) genes are absent in this animal group. The lack of BCs and positional information in other cells are therefore the main obstacle to therapeutic approaches for limb regeneration. Hence, this study aimed to create blastema- like cells (BlCs) by overexpressing Msx1 and Msx2 genes in mouse bone marrow- derived mesenchymal stem cells (mBMSCs) to regenerate a proximally amputated digit tip. We transduced mBMSCs with Msx1 and Msx2 genes and compared osteogenic activity and expression levels of several Msx-regulated genes (Bmp4, Fgf8, and keratin 14 (K14)) in BlCs groups including MSX1, MSX 2, and MSX1/2 (in a 1:1 ratio) with those in mBMSCs and BCs in vitro and in vivo following injection into the amputation site. We found that Msx gene overexpression increased expression of specific blastemal markers and enhanced prolifer...
Journal of Shahrekord University of Medical Sciences, Jun 15, 2012
Background and aims: Shigella dysenteriae is one of the most important pathogens which in spite o... more Background and aims: Shigella dysenteriae is one of the most important pathogens which in spite of many attempts vaccine preparation, extended researches are still in the way, Transport and surface expression of the invasion plasmid antigens (IpaD proteins) have essential role in the pathogenicity of Shigella spp. IpaD has been one of the most important proteins for Shigella vaccine candidate. Studies have shown that Nterminal region of this protein has a key role in the pathogen city and invasion. This study was done to evaluate the optimization of N-terminal region of Ipad in order to increase the production of recombinant protein. Methods: In this experimental labortary study, desired region of IpaD cloned in vector pET-28a (+). For confirming cloning procedure ,standard tests were performed. The effect of IPTG concentration, temperature & induction times on the level of protein expression were evaluated by SDS-PAGE, qualitatively. The gels were evaluated with 2-D gel analysis software (Melanie 7). The recombinant protein was extracted by Urea & eventually purificated with affinity chromatography column. Results : SDS-PAGE analysis showed that approximately the same amount of recombinant protein is expressed at different times, but software analysis proved that the optimized condition for the expression of recombinant protein was in the final concentration of 0.7 mM of IPTG, 37˚C and 3 hours induction. Conclusion: According to the results every protein has its own expression after the homogenization process, and the temperature and the cells induction time length are more effective in the amount of protein production.
Cholera toxin B subunit (CtxB) is a homopantameric, nontoxic subunit of cholera toxin that is res... more Cholera toxin B subunit (CtxB) is a homopantameric, nontoxic subunit of cholera toxin that is responsible for its binding to the cell and has been known as a mucosal adjuvant for vaccines that could increase homoral and mocusal immunity response. In this work, the CtxB gene was fused to the StxB gene from Shigella dysenteriae type I a vaccine antigen candidate against this pathogen, by a nonfurin linker then ligated with pGEM vector and subcloned in the pET28a(+) as an expression vector . The CtxB-StxB fusion protein was expressed in Escherichia coli, and purified by a Ni-NTA resin column, then detected molecular weight and immunogenicity by SDS-PAGE and Western-blot. StxB has low molecular weight, so immune response against it is low, while CtxB is a potent mucosal adjuvant. In this method, the CtxB-StxB fusion protein was expressed in Escherichia coli in order to use its natural adjuvanticity of the CtxB which will enhance immune response against StxB, as well as, it will produce ...
In many multicellular organisms, mature gametes originate from primordial germ cells (PGCs). Impr... more In many multicellular organisms, mature gametes originate from primordial germ cells (PGCs). Improvements in the culture of PGCs are important not only for developmental biology research, but also for preserving endangered species, and for genome editing and transgenic animal technologies. SMAD2/3 appear to be powerful regulators of gene expression; however, their potential positive impact on the regulation of PGC proliferation has not been taken into consideration. Here, the effect of TGF‐β signaling as the upstream activator of SMAD2/3 transcription factors was evaluated on chicken PGCs' proliferation. For this, chicken PGCs at stages 26−28 Hamburger−Hamilton were obtained from the embryonic gonadal regions and cultured on different feeders or feeder‐free substrates. The results showed that TGF‐β signaling agonists (IDE1 and Activin‐A) improved PGC proliferation to some extent while treatment with SB431542, the antagonist of TGF‐β, disrupted PGCs' proliferation. However, t...
Objective: Any damage to the optic nerve can potentially lead to degeneration of non-regenerating... more Objective: Any damage to the optic nerve can potentially lead to degeneration of non-regenerating axons and ultimately death of retinal ganglion cells (RGCs) that in most cases, are not curable by surgery or medication. Neuroprotective functions of different types of stem cells in the nervous system have been evaluated in many studies investigating the effectiveness of these cells in various retinal disease models. Neural progenitor cells (NPCs) secrete an assortment of trophic factors that are vital to the protection of the visual system. We aimed to assess the therapeutic potentials of NPCs in an ONC mouse model. Materials and methods: In this experimental study, NPCs were produced using noggin and retinoic acid from human embryonic stem cells (hESCs). Fifty mice were divided into the following three groups: i. Intact , ii. Vehicle [optic nerve crush+Hank's balanced salt solution (HBSS)], and iii. Treatment (optic nerve crush+NPCs). The visual behavior of the mice was examined using the Visual Cliff test, and in terms of RGC numbers, they were assessed by Brn3a immunostaining and retrograde tracing using DiI injection. Results: Intravenous injection of 50,000 NPCs through visual cliff did not produce any visual improvement. However, our data suggest that the RGCs protection was more than two-times in NPCs compared to the vehicle group as examined by Brn3a staining and retrograde tracing. Conclusion: Our study indicated that intravenous injection of NPCs could protect RGCs probably mediated by trophic factors. Due to this ability and good manufacturing practices (GMP) grade production feasibility, NPCs may be used for optic nerve protection.
Cardiomyopathies are a group of common heart disorders that affect numerous people worldwide. Lef... more Cardiomyopathies are a group of common heart disorders that affect numerous people worldwide. Left ventricular non-compaction (LVNC) is a structural disorder of the ventricular wall, categorized as a type of cardiomyopathy that mostly caused by genetic disorders. Genetic variations are underlying causes of developmental deformation of the heart wall and the resultant contractile insufficiency. Here, we investigated a family with several affected members exhibiting LVNC phenotype. By whole-exome sequencing (WES) of three affected members, we identified a novel heterozygous missense variant (c.1963C>A:p.Leu655Met) in the gene encoding myosin heavy chain 7 (MYH7). This gene is evolutionary conserved among different organisms. We identified MYH7 as a highly enriched myosin, compared to other types of myosin heavy chains, in skeletal and cardiac muscles. Furthermore, MYH7 was among a few classes of MYH in mouse heart that highly expresses from early embryonic to adult stages. In silic...
Additional file 3: Figure S3. Morphology and histology of the spinal cord injury (SCI) in s contu... more Additional file 3: Figure S3. Morphology and histology of the spinal cord injury (SCI) in s contusion model. (A) The adult male Wistar rat's spinal cord was exposed at T10. Through a dorsal midline incision, a T9–11 laminectomy was performed and the spinal cord was exposed. A contusion injury was made using a standardized weight-drop injury device (NYU impactor) as indicated by hemorrhage. (B) Hematoxylin and eosin (H&E) staining of the rat injured spinal cord sections at one week post-injury. The lesion core and glial scar are shown in the longitudinal section. (C) Transverse images show Gfap-immunoreactive astrocytes at the injured, caudal, and rostral sites. Note that the expression of Gfap was higher around the injured site relative to the caudal and rostral sites. (D) Double immunostaining for fibronectin (green) and Gfap (red) in a longitudinal spinal cord section one week post-injury in the rat injured spinal cord. The lesion site was filled with a fibronectin positive ma...
Additional file 2: Figure S2. The validation of inducible vector. (A) Immunostaining for astrocyt... more Additional file 2: Figure S2. The validation of inducible vector. (A) Immunostaining for astrocyte and neural stem cell (NSC) markers in transfected astrocytes. Transduction was performed with an empty vector under two conditions: Astrocytes in astrocyte medium without DOX (A) and astrocytes in induction medium (IM) and DOX (AD). In addition, astrocytes were transfected by the Zfp521 vector in the present and the absence of Dox in IM (AZD and AZ, respectively). Nuclei were counterstained with DAPI. (B) Quantification of the immunostainings for astrocytes and NSC markers in transfected astrocytes. Data are shown as mean ± SD of three biological replicates. Data were analyzed by ANOVA and Mann-Whitney U test as post hoc. ***: p
Additional file 1: Figure S1. Isolation and characterization of adult rat brain astrocytes. (A) P... more Additional file 1: Figure S1. Isolation and characterization of adult rat brain astrocytes. (A) Phase-contrast images of astrocytes. (B) RT-PCR analysis of astrocytes and neural stem cell (NSC) markers. (C) qRT-PCR analysis of astrocytes and NSC markers. Data were normalized against GAPDH and presented relative to the expression of each indicated gene in the astrocytes. (D) Immunostaining images for astrocyte markers. Nuclei were counterstained with DAPI. (E) Quantification of the immunostainings for astrocytes and NSC markers. Data in C and E are shown as mean ± SD of three biological replicates. ND: Not detected. The number of counted cells is presented in Additional file 4: Table S3.
Additional file 4: Table S1. List of antibodies used for immunostaining. Table S2.Primer sequence... more Additional file 4: Table S1. List of antibodies used for immunostaining. Table S2.Primer sequences used for quantitative real-time PCR (qRT-PCR). Table S3. The number of cells in each immunostaining analysis.
Aims: Shigellosis is caused by Shigella species. Considering the high frequency of morbidity and ... more Aims: Shigellosis is caused by Shigella species. Considering the high frequency of morbidity and mortality reports and antibiotics resistance, designing and producing a vaccine against this disease is one of the goals of World Health Organization. Invasion Plasmid Antigen especially IpaC and IpaB are the major Shigella virulence agents and are also vaccine candidates. The aim of this study was to express ipaC gene which is of the virulence factors in order to investigate its immunogenicity. Materials & Methods: In this study, ipaC gene was obtained from NCBI gene bank and primers were designed. After genome extraction from Shigella dysenteriae, it was used as template for PCR amplification. The amplified ipaC gene by PCR was cloned into pTZ57R and sub-cloned into the expression vector pET-28a(+). E. coli BL21(DE3)plysS was transformed by recombinant vector pET-28a(+)/ipaC and expression of the recombinant ipaC was investigated by IPTG induction and SDS-PAGE electrophoresis. Results:...
Objective Dual inhibition of mitogen-activated protein kinase (MAPK) kinase (also known as MEK) a... more Objective Dual inhibition of mitogen-activated protein kinase (MAPK) kinase (also known as MEK) and transforming growth factor β (TGFβ) type I receptors by PD0325901 and SB431542, known as R2i has been introduced as a highly efficient approach to the generation of mouse embryonic stem cells (ESC). In the present study, we investigated the molecular mechanisms underlying ESC derivation in the R2i condition. Materials and Methods In this experimental study, zona-free whole E3.5 blastocysts were seeded on mouse embryonic fibroblast (MEF) feeder cells in both R2i and serum conventional media. The isolated inner cell mass (ICM), ESCs and the ICM-outgrowths were collected on days 3, 5 and 7 post-blastocyst culture for quantitative real time- polymerase chain reaction (qRT-PCR) analysis as well as to assess the DNA methylation status at the time points during the transition from ICM to ESC. Results qRT-PCR revealed a significantly higher expression of the pluripotency-related genes (Oct4, ...
Objective Systemic sclerosis (SSc) is a connective tissue disease associated with vascular damage... more Objective Systemic sclerosis (SSc) is a connective tissue disease associated with vascular damage and multi organ fibrotic changes with unknown pathogenesis. Most SSc patients suffer from defective angiogenesis/vasculogenesis and cardiac conditions leading to high mortality rates. We aimed to investigate the cardiovascular phenotype of SSc by cardiogenic differentiation of SSc induced pluripotent stem cells (iPSC). Materials and Methods In this experimental study, we generated iPSC from two diffuse SSc patients, followed by successful differentiation into endothelial cells (ECs) and cardiomyocytes (CMs). Results SSc-derived EC (SSc-EC) expressed KDR, a nearly EC marker, similar to healthy control-EC (C1-EC). After sorting and culturing KDR+ cells, the resulting EC expressed CD31, a late endothelial marker, but vascular endothelial (VE)-cadherin expression markedly dropped resulting in a functional defect as reflected in tube formation failure of SSc-EC. Interestingly, upregulation o...
Objective Limb regeneration mediated by blastema cells (BlCs) in mammals is limited to the digit ... more Objective Limb regeneration mediated by blastema cells (BlCs) in mammals is limited to the digit tips of neonates. Due to the lack of access to BlCs in adults and the difficulty in isolating and expanding BlCs from neonates, the use of a cellular population with similar features of BlCs would be a valuable strategy to direct a non-regenerative wound towards regeneration. In this study, we have initially isolated and cultured BlCs, and explored their characteristics in vitro. Next, we compared the capability of bone marrow-derived mesenchymal stem cells (BM-MSCs) as an alternative accessible cell source to BlCs for regeneration of appendages. Materials and Methods In this experimental study, BM-MSCs were isolated from BM and we obtained BlCs from the neonatal regenerating digit tip of C57B/6 mice. The cells were characterized for expressions of cell surface markers by flow cytometry. Quantitative-reverse transcription polymerase chain reaction (qRT-PCR) and lineage-specific staining ...
Objective Genetic modification of human embryonic stem cells (hESCs) is critical for their extens... more Objective Genetic modification of human embryonic stem cells (hESCs) is critical for their extensive use as a fundamental tool for cell therapy and basic research. Despite the fact that various methods such as lipofection and electroporation have been applied to transfer the gene of interest (GOI) into the target cell line, however, there are few re- ports that compare all parameters, which influence transfection efficiency. In this study, we examine all parameters that affect the efficiency of electroporation and lipofection for transient and long-term gene expression in three different cell lines to introduce the best method and determinant factor. Materials and Methods In this experimental study, both electroporation and lipofection approaches were employed for genetic modification. pCAG-EGFP was applied for tran- sient expression of green fluorescent protein in two genetically different hESC lines, Roy- an H5 (XX) and Royan H6 (XY), as well as human foreskin fibroblasts (hFF). F...
Highlights • A soluble form of mouse Neuropilin1 (msNRP1) has been cloned in a lentiviral vector ... more Highlights • A soluble form of mouse Neuropilin1 (msNRP1) has been cloned in a lentiviral vector • msNRP1 was successfully secreted by the transduced HEK293T in the supernatant • msNRP1-containing supernatant inhibited growth cone collapse activity of Semaphorin 3A Plain Language Summary Multiple Sclerosis (MS) is an inflammatory disease of the central nervous system. In MS, axons of neuronal cells become demyelinated, and remyelination process is also defective. In fact, oligodendrocyte precursor cells must survive, proliferate, and migrate towards demyelinated neurons and differentiate into mature oligodendrocytes to produce myelin and repair the neurons. Semaphorin 3A (Sema 3A) is a molecule that reduce the remyelination by preventing oligodendrocyte precursor cells to proliferate, migrate towards lesions, and differentiate into mature oligodendrocyte. We successfully produced mouse soluble Neuropilin 1 (msNRP1), that can bind Sema 3A and inhibit its activity. Then, the msNRP1 was used inhibit the activity of Sema 3A. Neural cells of dorsal root ganglia was cultured in presence of Sema 3A with and without msNRP1. We observed that when msNRP1 was added to the neuronal cells culture, the growth cones were able to form. On the other hand, the growth cones collapsed when msNRP1 was not added to the cultures. This finding is important in terms of MS treatment. Remyelination is defective in MS patients, and axons are progressively demyelinated. Finding a molecule that can prevent demyelination and enhance remyelination will be of great importance for MS treatment and improvement of their condition.
Background Spinal cord injury (SCI) results in glial scar formation and irreversible neuronal los... more Background Spinal cord injury (SCI) results in glial scar formation and irreversible neuronal loss, which finally leads to functional impairments and long-term disability. Our previous studies have demonstrated that the ectopic expression of Zfp521 reprograms fibroblasts and astrocytes into induced neural stem cells (iNSCs). However, it remains unclear whether treatment with Zfp521 also affects endogenous astrocytes, thus promoting further functional recovery following SCI. Methods Rat astrocytes were transdifferentiated into neural stem cells in vitro by ZFP521 or Sox2. Then, ZFP521 was applied to the spinal cord injury site of a rat. Transduction, real-time PCR, immunohistofluorescence, and function assessments were performed at 6 weeks post-transduction to evaluate improvement and in vivo lineage reprogramming of astrocytes. Results Here, we show that Zfp521 is more efficient in reprogramming cultured astrocytes compared with Sox2. In the injured spinal cord of an adult rat, resi...
This study aims to prepare intermediate mesoderm-like cells from mouse embryonic fibroblasts (MEF... more This study aims to prepare intermediate mesoderm-like cells from mouse embryonic fibroblasts (MEFs). In the first step, intermediate mesoderm-like cells (IMLCs) and renal epithelial-like cells (RELCs) were extracted from mouse embryonic stem cells (mESCs) in a specified media that contained two small molecules, CHIR99021 and TTNPB, along with growth factors, FGF9and BMP7. Then, MEFs were directly converted into IM by genes for the pluripotency factors, which encode the transcription factors; Oct4, Sox2, Klf4, and c-Myc (OSKM). These unstable intermediate cells were quickly encouraged to form IM with the assistance of CHIR99021 and TTNPB. The results showed that exogenous expression of OSKM factors for four days was adequate to generate partially reprogrammed cells (SSEA1 + /Nanog-). Real-time PCR and immunocytochemistry analysis confirmed the presence of the MEF-derived IMs. This study introduced a method for mESCs differentiation to RELCs followed by MEF conversion in an attempt to generate IM by circumventing pluripotency.
Pluripotent cells appear to be in a transient state during early development. These cells have th... more Pluripotent cells appear to be in a transient state during early development. These cells have the capability to transition into embryonic stem cells (ESCs). It has been reported that mouse pluripotent cells cultivated in chemically defined media sustain the ground state of pluripotency. Because the epigenetic pattern of pluripotent cells reflects their environment, culture under different conditions causes epigenetic changes, which could lead to genomic instability. This study focused on the DNA methylation pattern of repetitive elements (REs) and their activation levels under two ground‐state conditions and assessed the genomic integrity of ESCs. We measured the methylation and expression level of REs in different media. The results indicated that although the ground‐state conditions show higher REs activity, they did not lead to DNA damage; therefore, the level of genomic instability is lower under the ground‐state compared with the conventional condition. Our results indicated t...
Amputation of the proximal region in mammals is not followed by regeneration because blastema cel... more Amputation of the proximal region in mammals is not followed by regeneration because blastema cells (BCs) and expression of regenerative genes such as Msh homeobox (Msx) genes are absent in this animal group. The lack of BCs and positional information in other cells are therefore the main obstacle to therapeutic approaches for limb regeneration. Hence, this study aimed to create blastema- like cells (BlCs) by overexpressing Msx1 and Msx2 genes in mouse bone marrow- derived mesenchymal stem cells (mBMSCs) to regenerate a proximally amputated digit tip. We transduced mBMSCs with Msx1 and Msx2 genes and compared osteogenic activity and expression levels of several Msx-regulated genes (Bmp4, Fgf8, and keratin 14 (K14)) in BlCs groups including MSX1, MSX 2, and MSX1/2 (in a 1:1 ratio) with those in mBMSCs and BCs in vitro and in vivo following injection into the amputation site. We found that Msx gene overexpression increased expression of specific blastemal markers and enhanced prolifer...
Journal of Shahrekord University of Medical Sciences, Jun 15, 2012
Background and aims: Shigella dysenteriae is one of the most important pathogens which in spite o... more Background and aims: Shigella dysenteriae is one of the most important pathogens which in spite of many attempts vaccine preparation, extended researches are still in the way, Transport and surface expression of the invasion plasmid antigens (IpaD proteins) have essential role in the pathogenicity of Shigella spp. IpaD has been one of the most important proteins for Shigella vaccine candidate. Studies have shown that Nterminal region of this protein has a key role in the pathogen city and invasion. This study was done to evaluate the optimization of N-terminal region of Ipad in order to increase the production of recombinant protein. Methods: In this experimental labortary study, desired region of IpaD cloned in vector pET-28a (+). For confirming cloning procedure ,standard tests were performed. The effect of IPTG concentration, temperature & induction times on the level of protein expression were evaluated by SDS-PAGE, qualitatively. The gels were evaluated with 2-D gel analysis software (Melanie 7). The recombinant protein was extracted by Urea & eventually purificated with affinity chromatography column. Results : SDS-PAGE analysis showed that approximately the same amount of recombinant protein is expressed at different times, but software analysis proved that the optimized condition for the expression of recombinant protein was in the final concentration of 0.7 mM of IPTG, 37˚C and 3 hours induction. Conclusion: According to the results every protein has its own expression after the homogenization process, and the temperature and the cells induction time length are more effective in the amount of protein production.
Cholera toxin B subunit (CtxB) is a homopantameric, nontoxic subunit of cholera toxin that is res... more Cholera toxin B subunit (CtxB) is a homopantameric, nontoxic subunit of cholera toxin that is responsible for its binding to the cell and has been known as a mucosal adjuvant for vaccines that could increase homoral and mocusal immunity response. In this work, the CtxB gene was fused to the StxB gene from Shigella dysenteriae type I a vaccine antigen candidate against this pathogen, by a nonfurin linker then ligated with pGEM vector and subcloned in the pET28a(+) as an expression vector . The CtxB-StxB fusion protein was expressed in Escherichia coli, and purified by a Ni-NTA resin column, then detected molecular weight and immunogenicity by SDS-PAGE and Western-blot. StxB has low molecular weight, so immune response against it is low, while CtxB is a potent mucosal adjuvant. In this method, the CtxB-StxB fusion protein was expressed in Escherichia coli in order to use its natural adjuvanticity of the CtxB which will enhance immune response against StxB, as well as, it will produce ...
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Papers by Mahdi Hesaraki