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Objective: The role of epigenetic in regulating of the gene expression profile the embryo has been documented. MicroRNAs [miRNAs] are one of these epigenetic mechanisms. Twins are valuable models in determining the relative contributions of genetics and the environment. In this study, we compared differences in the expression levels of 44 miRNAs in hematopoietic stem cells [HSCs] of identical twins to that of fraternal twins as a controls
Materials and Methods: In this experimental study, CD133+ HSCs were isolated from cord blood of identical and fraternal twins via magnetic-activated cell sorting [MACS]. Variation in of gene expression levels of 44 miRNAs were evaluated using quantitative reverse transcription-polymerase chain reaction [qRT-PCR]
Results: Significant differences in expression were observed in both fraternal and identical twins to varying degrees, but variations alteration in expression of the miRNAs were higher in fraternal twins
Conclusion: Identical twins had a positive correlation in miRNA expression, while the correlation was not statistically significant in fraternal twins. Altogether, more differences in miRNA expression level in fraternal twins can be attributed to the both genetics and the intrauterine environment. The contribution of the intrauterine environment and genetics to miRNAs expression in HSCs was estimated 8 and 92%, respectively. By comparing of miRNA expression in identical and fraternal twins and identification of their target genes and biological pathways, it could be possible to estimate the effects of genetics and the environment on a number of biological pathways
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Objective: In this study, we describe an efficient approach for stable knockdown of adenosine kinase [ADK] using lentiviral system, in an astrocytoma cell line and in human Wharton's jelly mesenchymal stem cells [hWJMSCs]. These sources of stem cells besides having multilineage differentiation potential and immunomodulatory activities, are easily available in unlimited numbers, do not raise ethical concerns and are attractive for gene manipulation and cell-based gene therapy
Materials and Methods: In this experimental study, we targeted adenosine kinase mRNA at 3' and performed coding sequences using eight miR-based expressing cassettes of anti-ADK short hairpin RNA [shRNAs]. First, these cassettes with scrambled control sequences were cloned into expressing lentiviral pGIPZ vector. Quantitative real time-polymerase chain reaction [qRT-PCR] was used to screen multi-cassettes anti-ADK miR-shRNAs in stably transduced U-251 MG cell line and measuring ADK gene expression at mRNA level. Extracted WJMSCs were characterized using flow cytometry for expressing mesenchymal specific marker [CD44+] and lack of expression of hematopoietic lineage marker [CD45-]. Then, the lentiviral vector that expressed the most efficient anti-ADK miR-shRNA, was employed to stably transduce WJMSCs
Results: Transfection of anti-ADK miR-shRNAs in HEK293T cells using CaPO4 method showed high efficiency. We successfully transduced U-251 cell line by recombinant lentiviruses and screened eight cassettes of anti-ADK miR- shRNAs in stably transduced U-251 MG cell line by qRT-PCR. RNAi-mediated down-regulation of ADK by lentiviral system indicated up to 95% down-regulation of ADK. Following lentiviral transduction of WJMSCs with anti-ADK miR- shRNA expression cassette, we also implicated, down-regulation of ADK up to 95% by qRT-PCR and confirmed it by western blot analysis at the protein level
Conclusion: Our findings indicate efficient usage of shRNA cassette for ADK knockdown. Engineered WJMSCs with genome editing methods like CRISPR/cas9 or more safe viral systems such as adeno-associated vectors [AAV] might be an attractive source in cell-based gene therapy and may have therapeutic potential for epilepsy
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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
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Background: myocardial infarction is the main cause of death worldwide. Angiogenesis, a promising new therapy for the treatment of diffuse coronary artery disease, shows a poor response to conventional revascularization techniques. This study focused on improving myocardial function using endothelial cells [ECs] and mesenchymal stem cells [MSCs] in a sheep animal model
Methods: acute myocardial infarction was induced in 18 sheep [12 treated cases and 6 controls]. Autologous MSCs and ECs were injected in the infarcted area and the border zone. Two months after transplantation, echocardiography, electron microscopy, and immunohistochemistry were performed
Results: echocardiography in both MSC and EC groups revealed a significant improvement in the ejection fraction compared with the control group [p value < 0.05]. Vascular density, estimated by antibodies against the von Willebrand factor and smooth muscle actin, increased in both study groups. The pattern of vascularity in the MSC and EC groups was diffused. The electron microscopic evaluation of the infracted areas revealed cardiomyocytes in variable stages of development in the border zone in both EC and MSC groups
Conclusion: both ECs and MSCs were able to promote angiogenesis and improve cardiac function. Presumably, MSCs differentiate into ECs and cause angiogenesis as it occurs for ECs
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Background: Islet transplantation could be an ideal alternative treatment to insulin therapy for type 1 diabetes Mellitus [T1DM]. This clinical and experimental field requires a model that covers problems such as requiring a large number of functional and viable islets, the optimal transplantation site, and the prevention of islet dispersion. Hence, the methods of choice for isolation of functional islets and transplantation are crucial
Methods: The present study has introduced an experimental model that overcomes some critical issues in islet transplantation, including in situ pancreas perfusion by digestive enzymes through common bile duct. In comparison with conventional methods, we inflated the pancreas in Petri dishes with only 1 ml collagenase type XI solution, which was followed by hand-picking isolation or Ficoll gradient separation to purify the islets. Then we used a hydrogel composite in which the islets were embedded and transplanted into the peritoneal cavity of the streptozotocin-induced diabetic C57BL/6 mice
Results: As compared to the yield of the classical methods, in our modified technique, the mean yield of isolation was about 130-200 viable islets/mouse pancreas. In vitro glucosemediated insulin secretion assay indicated an appropriate response in isolated islets. In addition, data from in vivo experiments revealed that the allograft remarkably maintained blood glucose levels under 400 mg/dl and hydrogel composite prevents the passage of immune cells
Conclusion: In the model presented here, the rapid islet isolation technique and the application of biomimetic hydrogel wrapping of islets could facilitate islet transplantation procedures
Subject(s)
Animals, Laboratory , Biomimetic Materials , Biomimetics , Hydrogels , Diabetes Mellitus, Type 1 , Mice , Diabetes Mellitus, Experimental , StreptozocinABSTRACT
Background: The Definitive Endoderm [DE] differentiation using the undefined media and non-human feeders can cause contaminations in the generated cells for therapeutic applications. Therefore, generating safer and more appropriate DE cells is needed. This study compared five different methods to establish an appropriate method for inducing an efficient DE differentiation from Human Induced Pluripotent Stem Cells [hiPSCs] on an appropriate feeder in a more defined medium
Methods: Human Induced Pluripotent Stem Cells [hiPSCs] were cultured on inactivated feeders. Passaged hiPSCs, without feeder, were incubated for three days with Activin-A and different endodermal differentiation media including 1-FBS, 2-B27, 3- ITS and albumin fraction-V, 4-B27 and ITS and 5-like the third medium. The feeder cells in the first four methods were Mouse Embryonic Fibroblasts [MEFs] and in the fifth method were human adult bone marrow Mesenchymal Stem Cells [hMSCs]. DE markers FOXA2, SOX17 and CXCR4 and also pluripotency marker OCT4 were evaluated using qRT-PCR, as well as FOXA2 by the immunocytochemistry
Results: QRT-PCR analysis showed that after three days, the expression levels of DE and pluripotency markers in the differentiated hiPSCs among all five groups did not have any significant differences. Similarly, the immunocytochemistry analysis demonstrated that the differentiated hiPSCs expressed FOXA2, with no significant differences
Conclusion: Despite this similarity in the results, the third differentiation medium has more defined and cost effective components. Furthermore, hMSC, a human feeder, is safer than MEF. Therefore, the fifth method is preferable among other DE differentiation methods and can serve as a fundamental method helping the development of regenerative medicine
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Today the regulatory role of microRNAs [miRs] is well characterized in many diverse cellular processes. MiR-based regulation is categorized under epigenetic regulatory mechanisms. These small non-coding RNAs participate in producing and maturing erythrocytes, expressing hematopoietic factors and regulating expression of globin genes by post-transcriptional gene silencing. The changes in expression of miRs [miR-144/-320/-451/-503] in thalassemic/sickle cells compared with normal erythrocytes may cause clinical severity. According to the suppressive effects of certain miRs [miR-15a/-16-1/-23a/-26b/-27a/-451] on a number of transcription factors [myeloblastosis oncogene [MYB], B-cell lymphoma 11A [BCL11A], GATA1, Krüppel-like factor 3 [KLF3] and specificity protein 1 [Sp1]] during beta globin gene expression, It has been possible to increasing ? globin gene expression and fetal hemoglobin [HbF] production. Therefore, this strategy can be used as a novel therapy in infusing HbF and improving clinical complications of patients with hemoglobinopathies
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Objective: Gastric cancer [GC] is widely associated with chronic inflammation. The pro inflammatory microenvironment provides conditions that disrupt stem/progenitor cell proliferation and differentiation. The signal transducer and activator of transcrip-tion-3 [STAT3] signaling pathway is involved in inflammation and also contributes to the maintenance of embryonic stem cell [ESCs] pluripotency. Here, we have investi-gated the activation status of STAT3 in GC stem-like cells [GCSLCs]
Materials and Methods: In this experimental research, CSLCs derived from the human GC cell line MKN-45 and patient specimens, through spheroid body formation, characterized and then assayed for the STAT3 transcription factor expression in mRNA and protein level further to its activation
Results: Spheroid cells showed higher potential for spheroid formation than the parental cells. Furthemore, stemness genes NANOG, c-MYC and SOX-2 were over expressed in spheroids of MKN-45 and in patient samples. In MKN-45 spheroid cells, epithelial mesenchymal transition [EMT] related markers CDH2, SNAIL2, TWIST and VIMENTIN were upregulated [P<0.05], but we observed no change in expression of the E-cadherin epithelial marker. These cells exhibited more resistance to docetaxel [DTX] when compared with parental cells [P<0.05] according to the MTS assay. Although immunostaining and Western blotting showed expression of the STAT3 protein in both spheroids and parents, the mRNA level of STAT3 in spheroids was higher than the parents. Nuclear translocation of STAT3 was accompanied by more intensive phospho-STAT3 [p-STAT3] in spheroid structures relative to the parent cells according to flow cytometry analysis [P<0.05]
Conclusion: The present findings point to STAT3 over activation in GCSLCs. Complementary experiments are required to extend the role of STAT3 in stemness features and invasion properties of GCSCs and to consider the STAT3 pathway for CSC targeted therapy
ABSTRACT
Objective: MicroRNAs [miRNAs] are small endogenous non-coding regulatory RNAs that control mRNAs post-transcriptionally. Several mouse stem cells miRNAs are cloned differentially regulated in different hematopoietic lineages, suggesting their possible role in hematopoietic lineage differentiation. Recent studies have shown that specific miRNAs such as Mir-451 have key roles in erythropoiesis
Materials and Methods: In this experimental study, murine embryonic stem cells [mESCs] were infected with lentiviruses containing pCDH-Mir-451. Erythroid differentiation was assessed based on the expression level of transcriptional factors [Gata-1, Klf-1, Epor] and hemoglobin chains [alpha, beta, gamma, epsilon and zeta] genes using quantitative reverse transcriptase-polymerase chain reaction [qRT-PCR] and presence of erythroid surface antigens [TER-119 and CD235a] using flow cytometery. Colony-forming unit [CFU] assay was also on days 14thand 21thafter transduction
Results: Mature Mir-451 expression level increased by 3.434-fold relative to the untreated mESCs on day 4 after transduction [P<0.001]. Mir-451 up-regulation correlated with the induction of transcriptional factor [Gata-1, Klf-1, Epor] and hemoglobin chain [alpha, beta, gamma, epsilon and zeta] genes in mESCs [P<0.001] and also showed a strong correlation with presence of CD235a and Ter- 119 markers in these cells [13.084and 13.327-fold increse, respectively] [P<0.05]. Moreover, mESCs treated with pCDH-Mir-451 showed a significant raise in CFU-erythroid [CFU-E] colonies [5.2-fold] compared with untreated control group [P<0.05]
Conclusion: Our results showed that Mir-451 up-regulation strongly induces erythroid differentiation and maturation of mESCs. Overexpression of Mir-451 may have the potential to produce artificial red blood cells [RBCs] without the presence of any stimulatory cytokines
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Objective: Treatment and repair of neurodegenerative diseases such as brain tumors, spinal cord injuries, and functional disorders, including Alzheimer's disease, are challenging problems. A common treatment approach for such disorders involves the use of mesenchymal stem cells [MSCs] as an alternative cell source to replace injured cells. However, use of these cells in hosts may potentially cause adverse outcomes such as tumorigenesis and uncontrolled differentiation. In attempt to generate mesenchymal derived neural cells, we have infected MSCs with recombinant lentiviruses that expressed nerve growth factor [NGF] and assessed their neural lineage genes
Materials and Methods: In this experimental study, we cloned the NGF gene sequence into a helper dependent lentiviral vector that contained the green fluorescent protein [GFP] gene. The recombinant vector was amplified in DH5 bacterial cells. Recombinant viruses were generated in the human embryonic kidney 293 [HEK-293] packaging cell line with the helper vectors and analyzed under fluorescent microscopy. Bone marrow mesenchymal cells were infected by recombinant viruses for three days followed by assessment of neural differentiation. We evaluated expression of NGF through measurement of the NGF protein in culture medium by ELISA; neural specific genes were quantified by real-time polymerase chain reaction [PCR]
Results: We observed neural morphological changes after three days. Quantitative PCR showed that expressions of NESTIN, glial derived neurotrophic factor [GDNF], glial fibrillary acidic protein [GFAP] and Microtubule-associated protein 2 [MAP2] genes increased following induction of NGF overexpression, whereas expressions of endogenous NGF and brain derived neural growth factor [BDNF] genes reduced
Conclusion: Ectopic expression of NGF can induce neurogenesis in MSCs. Direct injection of MSCs may cause tumorigenesis and an undesirable outcome. Therefore an alternative choice to overcome this obstacle may be the utilization of differentiated neural stem cells
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Background: in this study, we compared the effect of ibuprofen [IB] while incorporating by Poly Lactic-co-Glycolic Acid [PLGA] nanofiber on expression of adhesion molecules ICAM-1 and VCAM-1 in a mice adhesion model
Materials and Methods: using an adhesion model were induced in mice, PLGA-IB and PLGA membranes and IB were sutured between the abdominal wall and peritoneum after surgical operation to reveal the best membrane for prevention of postoperative adhesion bands by comparison of ICAM-1 and VCAM-1 expression
Results: compared with other groups, PLGA-IB showed a greater ability to reduce ICAM-1 and VCAM-1 expression
Conclusion: these results suggested that in considering the FDA approved polymers, PLGA-IB could be introduced as a potential candidate for prevention of abdominal post-surgery inflammation and adhesion band formation after surgeries
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Objective: receptor activator of nuclear factor-kappa B ligand [RANKL] appears to be an osteoclast-activating factor, bearing an important role in the pathogenesis of multiple myeloma. Some studies demonstrated that U-266 myeloma cell line and primary myeloma cells expressed RANK and RANKL. It had been reported that the expression of myeloid and monocytoid markers was increased by co-culturing myeloma cells with hematopoietic stem cells [HSCs]. This study also attempted to show the molecular mechanism of RANK and RANKL on differentiation capability of human cord blood HSC to osteoclast, as well as expression of calcitonin receptor [CTR] on cord blood HSC surface
Materials and Methods: in this experimental study, CD133[+] hematopoietic stem cells were isolated from umbilical cord blood and cultured in the presence of macrophage colony-stimulating factor [M-CSF] and RANKL. Osteoclast differentiation was characterized by using tartrate-resistant acid phosphatase [TRAP] staining, giemsa staining, immunophenotyping, and reverse transcription-polymerase chain reaction [RT-PCR] assay for specific genes
Results: hematopoietic stem cells expressed RANK before and after differentiation into osteoclast. Compared to control group, flow cytometric results showed an increased expression of RANK after differentiation. Expression of CTR mRNA showed TRAP reaction was positive in some differentiated cells, including osteoclast cells
Conclusion: presence of RANKL and M-CSF in bone marrow could induce HSCs differentiation into osteoclast
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Objective: micro-RNAs [miRNAs] are a class of posttranscriptional regulators that play crucial roles in various biological processes. Emerging evidence suggests a direct link between miRNAs and development of several diseases including type 2 diabetes [T2D]. In this study, we aimed to investigate the effect of predicted miRNA and target genes on insulin resistance
Materials and Methods: this experimental study was conducted on the C2C12 cell line. Using bioinformatics tools miRNA-135 and two respective target genes-insulin receptor [Insr] and vesicle associated membrane protein 2 [Vamp2]- were selected as potential factors involved in insulin resistance process. Levels of glucose uptake miRNA expression and respective gene targets were determined after cell transfaction by miR-135
Results: it was determined that Insr gene expression was significantly down-regulated in miR-135 transfected C2C12 cell line [P
Conclusion: our data indicated no change on the Vamp2 expression level after miRNA transfection, while expression level of Insr was reduced and miR-135 expression was contrarily increased leading to poor stimulation of glucose uptake through insulin, and development of insulin resistance phenotype in C2C12 cell line
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Objective: In the present study we investigated the effect of a dynamic culture in a shake flask bioreactor [SFB] on the proliferation and differentiation to osteoblasts for human mesenchymal stem cells [hMSCs] cultured on multilayered electrospun PCL-nHA scaffolds
Methods: First, we prepared PCL-nHA scaffolds by electrospinning. After culturing the hMSCs on the scaffolds in a static state, the seeded scaffolds were divided into two groups [static and SFB culture] and incubated up to 21 days. We assessed biocompatibility and cell differentiation by the MTT, calcium, and alkaline phosphatase [ALP] assays on days 7, 14, and 21
Results: The MTT assay evaluated hMSCs proliferation rate on the scaffold layers. There was greater cell proliferation [optical density values] on the layers in the bioreactor [OD=2.18] compared to the static state condition [OD=1.68] on day 21. In order to study osteogenic differentiation, we determined the amount of calcium deposition and ALP activity. We observed a 1.6-fold greater level of calcium deposition for the dynamic culture compared to the static culture, which showed increased cell differentiation within the bioreactor on day 21. The ALP results showed that during 14 days, ALP activity within the bioreactor was 1.55-fold higher than the static culture
Conclusion: The SFB culture displayed a higher proliferation and differentiation of stem cells on PCL-nHA multilayered scaffolds compared to the static state condition
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Background: Monolayer electrospun scaffolds have already been used in bone tissue engineering due to their high surface-tovolume ratio, interconnectivity, similarity to natural bone extracellular matrix [ECM], and simple production
Objectives: The aim of this study was to evaluate the dynamic culture effect on osteogenic differentiation and mineralizationi into a compact cellular multilayer nHA-PCL electrospun construct. The dynamic culture was compared with static culture
Materials and Methods: The calcium content, alkaline phosphatase [ALP] activity and cell viability were investigated on days 3 and 7
Results: When the dynamic culture compared to static culture, the mineralization and ALP activity were increased in dynamic culture. After 7 days, calcium contents were 41.24 and 20.44 micro g.[cm[3]][-1], and also normalized ALP activity were 0.32 and 0.19 U.mg[-1] in dynamic and static culture, respectively. Despite decreasing the cell viability until day 7, the scanning electron microscopy [SEM] results showed that, due to higher mineralization, a larger area of the construct was covered with calcium deposition in dynamic culture
Conclusions: The dynamic flow could improve ALP activity and mineralization into the compact cellular multilayer construct cultured in the perfusion bioreactor after 7 days. Fluid flow of media helped to facilitate the nutrients transportation into the construct and created uniform cellular construct with high mineralization. This construct can be applied for bone tissue engineering
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Objective: Prostate cancer is the fifth most common cancer. In 2012, it was the second leading cause of cancer death for men worldwide. The PI3K/AKT pathway plays an essential role in pathogenesis of prostate cancer; the key role of this pathway in cancer progression makes it an attractive target for prostate cancer therapy. MicroRNAs [miRNAs] that regulate gene expression have a special ability to simultaneously control multiple genes and pathways which make them candidates for therapeutics. This study aims to determine miRNAs which target the PI3K/AKT pathway and evaluate them in prostate cancer cell lines
Methods: In order to determine an effective miRNA for the PI3K/AKT pathway, we assessed six genes from this pathway which have been proposed as drug targets in ten different prediction algorithms. Next, the candidate miRNAs were analyzed in expression profile and pathway analysis databases. Expression of candidate miRNAs in control and prostate cancer cell lines were subsequently evaluated
Results: According to bioinformatics, the miR-29 family could target the most genes from this list. Other bioinformatic estimates confirmed these results. The miR-29 family showed significant downregulation in prostate cancer cell lines LNCAP, PC3 and DU-145 compared to control samples
Conclusion: These results propose the possibility of using the miR-29 family to inhibit the PI3K/AKT pathway in prostate cancer
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Objective: More similarity to in vivo may help to increase the proliferation and differentiation of cells at in vitro culture. The present study has investigated the effect of a dynamic culture medium and nano hydroxyapatite [nHA] presence on proliferation and differentiation of mesenchymal stem cells [MSCs] to bone cells using electrospun polycaprolactone [PCL] scaffolds
Methods: We prepared PCL and PCL-nHA scaffolds by electrospinning. After static culturing of the scaffolds with MSCs, the scaffolds, were divided into two groups of static and dynamic cultures. The dynamic culture scaffolds were placed on a shaker. Cell proliferation and differentiation at days 3, 7 and 14 were investigated by MTT, and the calcium and alkaline phosphatase assays
Results: The obtained results from the MTT assay on day 14 showed an increase of 1.1 times in cell proliferation in the dynamic culture compared to the static culture. During this period, the calcium content produced by cells in the dynamic culture at day 14 were 1.23 times higher for PCL scaffolds and 1.46 times higher for PCL-nHA scaffolds compared to the static culture. Alkaline phosphatase levels for the dynamic state PCL scaffold were 1.24 times more and for PCL-nHA scaffolds were 1.28 times more compared to the static culture at day 14
Conclusion: The obtained results from dynamic culture, showed higher proliferation and differentiation of stem cells to bone for both PCL and PCL-nHA scaffolds compared to the static culture. The amount of cell proliferation and differentiation in the scaffolds that contained nHA was more than scaffolds that did not have nHA
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Objective: The host immune response against minor donor blood groups may be considered a significant problem in certain groups of patients that undergo transfusions such as those who require repeat transfusions [thalassemia]. A proposed solution is to coat the surface antigens on red blood cells [RBCs] by covalent binding of methoxy polyethylene glycol [mPEG]. This study aims to determine the storage time of PEGylated cells before injection and its effective time during in vivo conditions
Methods: We used mPEG activated by succinimidyl valerate [SVA] to PEGlayte the cells. The stability of the created coating during in vitro conditions was investigated by three methods: counting the numbers of free cells, flow cytometry and qualitative investigation. The appropriate concentration of mPEG for rabbit RBC PEGylation was determined by electron microscopy. The effective time of PEGylated rabbit RBCs was determined with flow cytometric analysis after the injection. In addition, we investigated the serum biochemical properties at 24 hours after the injection
Results: The appropriate concentration of 15 mg/mL for rabbit RBC PEGylation was determined. At 48 hours after injection, 83% of the cells that were alive in the host circulatory system kept their polymeric coating
Conclusion: We determined that 18 days was an appropriate storage time for PEGylated RBCs under in vitro conditions. The effective time of 14 days was determined for PEGylated RBCs by tracking the cells in vivo. An investigation of the serum biochemical properties of rabbits at 24 hours after the injection showed that the RBC coating significantly inhibited stimulation of the host immune system and cell destruction
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Lentiviral vectors [LVs] are useful vehicle for genetransfer to dividing and non-dividing cells and genetic manipulations. However, the use of lentiviruses in studies requires an accurate titration technique.Quantitative real-time PCR [qPCR] is a sensitive technique for the indication and quantitation of retrovirals particles. In this study, we used the qPCR for lentiviral vector titeration. The puromycin resistance gene as templates for an SYBR green-based real-time qPCR method and detect lentiviral copy number integrated lentiviral DNA. Consequently, this studyshowed that theusing ofantibioticresistance genesviral particles titration maybeefficient with highly accuracy
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Skeletal muscle is a highly differentiated tissue with very specific functions which has low potential of regeneration. Skeletal muscle injuries especially in athletes almost have lead to muscular dysfunctions and healing may be prolonged for several years. Therefore, working on skeletal muscle differentiation remained an importance in biomedical researches. Adipose derived stem cells [ADSCs] are novel source of mesenchymal stem cells which are an excellent alternative for satellite cells in in-vitro skeletal muscle differentiation. Differentiation potential of ADSCs on both tissue culture plate [TCP] and also on Poly l-lactide acid [PLLA] electrospun fibrous nano-scaffold which now is widely used at tissue engineering investigations has studied in this research. Scanning electron microscopy [SEM] and Tensile test were performed for evaluating scaffold properties. Hydrocortisone has considered a critical factors for skeletal muscle differentiation while, the recommended concentrations of it for inducing myogenesis in stem cells is yet discussing. Statistical analysis of our results from colorimetric MTT assay for various concentrations of hydrocortisone showed that the concentration of 10[-7] mol/L is the optimum dose for myogenic differentiation of murine ADSCs which was used on both TCP and PLLA scaffolds and skeletal myosin fiber formations was confirmed with immunocytochemistry. DAPI staining proved myocytes nuclei and syncytium formations. Our results also showed that ADSCs and PLLA nano-scaffolds are the suitable biomaterials for engineering skeletal muscle tissue