Production, purification and titration of a lentivirus-based vector for gene delivery purposes

Viral vectors are valuable tools to deliver genetic materials into cells. Vectors derived from human immunodeficiency virus type 1 are being widely used for gene delivery, mainly because they are able to transduce both dividing and non-dividing cells which leads to stable and long term gene expression. In addition, these types of vectors are safe, with low toxicity, high stability and cell type specificity. Therefore, this work was aimed to produce lentivirus-based vector using a three-plasmid system. To produce this system, the eGFP marker gene was cloned into the plasmid pWPXLd. Subsequently, this vector plasmid, along with packaging plasmids, psPAX2 and envelope plasmid, pMD2.G, was co-transfected into packaging cell line (293T) using calcium phosphate method. 48 h post transfection, the constructed viral vector was harvested, purified and concentrated and stored at −80 °C for next experiments. The titration of the vector was carried out, using ELISA, flowcytometry, and fluorescent microscopy. Finally, transduction of HEK-293T, CHO, HepG2, MCF-7, MEFs and Jurkat cell lines was carried out with indicated cell numbers and multiplicities of infections of the vector in the presence of polybrene. Using this system, high titer lentivirus at titers of up to 2 × 108 transducing units/ml (TU/ml) was successfully generated and its transduction efficacy was improved by seven to over 20-fold in various cell types. We demonstrate the applicability of this vector for the efficient transduction of dividing and non-dividing cells, including HEK-293T, CHO, HepG2, MCF-7, MEFs and Jurkat cell line. Transduction efficiency yielded titers of (6.3 ± 1.2) 105 TU/ml. Furthermore, lentivirus transferred transgene was expressed at high level in the target cells and expression was followed until 90 days after transduction. Thus, the vector generated in this work, might be able to deliver the transgene into a wide range of mammalian cells. © 2014, Springer Science+Business Media Dordrecht

Cytogentic analysis of human dermal fibroblasts (HDFs) in early and late passages using both karyotyping and comet assay techniques

Human dermal fibroblasts (HDFs) are a potential source of somatic cells for genetic manipulation and tissue engineering. Confirmation of cytogenetic stability of these cells is an essential step for cell nuclear transfer and generation of a suitable and functional induced pluripotent stem cells line. HDF cells were isolated and cultured from human foreskin samples. Cytogenetic stability of these cells was evaluated in early (3-4) and late (10-15) passages using karyotype test and alkaline comet assay techniques. HDF cells in early and late passages showed normal karyotype but by comet assay abnormality and DNA damages in late passages of HDFs were observed. Also, the parameters of alkaline comet assay in early passages of HDFs compared with late passages and positive control groups more significantly were different (p < 0.05). These findings indicate that single-strand breaks or DNA damage after many passages may have occurred in HDF cells. Our results demonstrate that only early passages of HDF cells maintain cytogenetic stability and are good candidates for gene reprogramming. In conclusion, karyotype testing alone can not be used for detection of all signs of cytogenetic abnormality and DNA damages of cells. So, for precise evaluation of DNA damage and cytogenetic instability of fibroblast cells comet assay and karyotype techniques could complement each other

Effects of Feeder Layers, Culture Media, Conditional Media, Growth Factors, and Passages Number on Stem Cell Optimization

Stem cells are undifferentiated and self-renewal cells which could be obtained from the body or artificially derived from an adult somatic cell by forced expression of specific genes. In recent years stem cells are widely used in laboratory for tissue engineering and therapeutic applications. There are different factors and conditions that affect the stem cell culture such as feeder layers, atmosphere, kind of medium, growth factors, passages number, and conditional media with animal or human sources. Optimization of stem cell culture for medical approaches and regenerative medicine is important. Therefore, in the present study, the effect of these factors and agents on optimization of stem cell culture has been discussed. This review study showed that optimization of feeder layer, atmosphere, and using supplemented media with essential growth factors could help in maintaining the stem cells in undifferentiated state in vitro. The present study indicated that optimization of stem cell culture depends on the kind of each cell type and using stem cells in low passage number could decrease chromosomal abnormalities and DNA damages. For inhibiting the stem cell contamination by feeder cell lines, culture of these cells on feeder free systems like Matrigel matrix in conditioned media supplemented with essential growth factors is useful. Also, for eradicating immune system responses and reducing the risk of animal pathogen transfer, culture of stem cells on human feeder in optimized media is suitable for therapeutic approaches and regenerative medicine

Effects of Phenanthrene and Pyrene on Cytogenetic Stability of Human Dermal Fibroblasts Using Alkaline Comet Assay Technique

In the present study, the influence of phenanthrene and pyrene on cytogenetic stability of human dermal fibroblasts using alkaline comet assay was evaluated. The cells were isolated from foreskin samples obtained from healthy male infants and in low passages (P 1–3) were exposed to various concentrations (0.0900, 0.0625, 0.0320, and 0.0066 ppm) of phenanthrene and pyrene. Then, alkaline comet assay was performed to investigate the influence of these compounds on DNA damage and cytogenetic stability of human dermal fibroblasts. In the present work H2O2 treatment was used as a positive control of comet assay to create DNA damages. The analysis of alkaline comet assay parameters by CaspLab software showed the tail DNA% in high concentration (0.0900 ppm) of phenanthrene and pyrene in the exposed cells got increased as compared to normal cells, while head DNA% decreased. Also, similar to positive control (H2O2), DNA damage with long tail comet was observed in high concentration of these compounds as compared to normal cells. The comparison of comet assay parameters specially head DNA% and tail DNA% between each concentrations of phenanthrene and pyrene with other groups (healthy cells and H2O2 treatment) by ANOVA and post hoc Tukey test showed that these parameters were more significantly different (p < 0.05). These results indicated that phenanthrene and pyrene even in low dosage are dangerous and can increase the DNA damage and affect on cytogenetic stability of dermal fibroblasts. These findings suggested that phenanthrene and pyrene could increase the related diseases and risk of skin cancer in exposed individuals

Effects of lentiviral vectors on DNA damage of human dermal fibroblasts (HDFs)

In present study we evaluated the DNA damages and cytogenetic stability of transducted and non-transducted human dermal fibroblasts (HDFs) by enhanced green fluorescent protein (eGFP) lentiviral vector using karyotyping, comet assay, and molecular techniques. HDFs were isolated from human foreskin samples and eGFP-expressing lentiviral vector were transfected into HEK-293T cells to produce lentiviruses. Then, HDFs at passage 2 were transducted with concentrated eGFP lentivirus and transducted HDFs were detected by fluorescent microscope. The expression levels of cell cycle genes include two subunits of anaphase promoting complex (APC) in transducted and nontransducted HDFs were measured by quantitative real-time PCR and finally, karyotype test and comet assay was performed to evaluate the DNA damages and cytogenetic stability in both groups. The results of karyotype analysis were not showed any abnormalities in karyotype of transducted HDFs by eGFP in compared to normal cells. The mean values of alkaline comet assay parameters on non-transducted (normal cells), eGFP-transducted group and positive control (H2O2 treatment) were calculated by CaspLab software. The comparison of mean difference of comet assay parameters include tail length, comet length, tail moment, and Olive tail moment by T test between eGFP-transducted HDFs and other groups (positive control and non-transducted HDFs) were statistically significant (p≤0.05). The alkaline comet assay on HDFs in eGFP-transducted group was showed small tail and indicated slight genetic damage compared with non-transducted group. Furthermore, the analysis of real-time PCR on expression of APC2 and APC7 genes in non-transducted HDFs compared with eGFP-transducted HDFs were not significant (p≤0.05). These findings indicated that integration of lentiviral vectors in first passage of transducted HDFs could not disturb the DNA structure and create chromosome instability. So in genetic engineering and gene transformation these vectors in first passages are useful

A study of cytogenetic stability of induced pluripotent stem cells using karyotyping and comet assay techniques

Background & Aims: Induced pluripotent stem cells (iPSCs) have the capability to undergo unlimited selfrenewal and differentiation into all cell types in the body. These cells are artificially derived from a nonpluripotent cell, typically human dermal fibroblasts (HDFs). The study of cytogenetic stability of these cells, in order to use iPS cells and apply studies in therapeutic applications, is essential. Methods: In the present experimental study, HDFs were isolated and cultured from human foreskin samples. The cytogenetic stability of these cells was evaluated in early passages (1-3) of HDFs using karyotype test and alkaline comet assay technique. The HDF cells treatment with hydrogen peroxide (H2O2) was used as a positive control for alkaline comet assay. The iPS cells with low passage (4-7) derived from reprogrammed HDFs were cultured on mouse embryonic fibroblast (MEF) feeder layer and cytogenetic stability of these cells were evaluated in early passages using karyotype test and alkaline comet assay technique. Results: The iPS cells in early passages (4-7) had normal karyotype (46, XY) and DNA damage and comet were not observed in these cells. In addition, HDF cells showed normal karyotype in early passages (1-3), but using comet assay, abnormality and DNA damages were observed in positive control (HDFs treated with H2O2). The comparison of alkaline comet assay parameters of iPS and HDF cells with positive control group showed statistically significant differences (P < 0.05). Conclusion: Since the comet assay is a sensitive technique for finding DNA damage, it is best if cytogenetic stability of these cells were evaluated before performing functional experiments on iPS cells. Therefore, for the precise evaluation of DNA damage and cytogenetic stability of iPS cells, the two techniques could complement each other. © 2015, Kerman University of Medical Sciences. All rights reserved

Comparison between the cultures of human induced pluripotent stem cells (hiPSCs) on feeder- and serum-free system (Matrigel matrix), MEF and HDF feeder cell lines

Human induced pluripotent stem cells (hiPSCs) are a type of pluripotent stem cells artificially derived from an adult somatic cell (typically human fibroblast) by forced expression of specific genes. In recent years, different feeders like inactivated mouse embryonic fibroblasts (MEFs), human dermal fibroblasts (HDFs), and feeder free system have commonly been used for supporting the culture of stem cells in undifferentiated state. In the present work, the culture of hiPSCs and their characterizations on BD Matrigel (feeder- and serum-free system), MEF and HDF feeders using cell culture methods and molecular techniques were evaluated and compared. The isolated HDFs from foreskin samples were reprogrammed to hiPSCs using gene delivery system. Then, the pluripotency ability of hiPSCs cultured on each layer was determined by teratoma formation and immunohistochemical staining. After EBs generation the expression level of three germ layers genes were evaluated by Q-real-time PCR. Also, the cytogenetic stability of hiPSCs cultured on each condition was analyzed by karyotyping and comet assay. Then, the presence of pluripotency antigens were confirmed by Immunocytochemistry (ICC) test and alkaline phosphatase staining. This study were showed culturing of hiPSCs on BD Matrigel, MEF and HDF feeders had normal morphology and could maintain in undifferentiated state for prolonged expansion. The hiPSCs cultured in each system had normal karyotype without any chromosomal abnormalities and the DNA lesions were not observed by comet assay. Moreover, upregulation in three germ layers genes in cultured hiPSCs on each layer (same to ESCs) compare to normal HDFs were observed (p<0.05). The findings of the present work were showed in stem cells culturing especially hiPSCs both MEF and HDF feeders as well as feeder free system like Matrigel are proper despite benefits and disadvantages. Although, MEFs is suitable for supporting of stem cell culturing but it can animal pathogens transferring and inducing immune response. Furthermore, HDFs have homologous source with hiPSCs and can be used as feeder instead of MEF but in therapeutic approaches the cells contamination is a problem. So, this study were suggested feeder free culturing of hiPSCs on Matrigel in supplemented media (without using MEF conditioned medium) resolves these problems and could prepare easy applications of hiPSCs in therapeutic approaches of regenerative medicine such as stem-cell therapy and somatic cell nuclear in further researches

Changes of AML 1 and P53 tumor suppressor gene expression in patients de novo acute myeloid leukemia

P53 and AML1are two important tumor suppressor genes in regulation of hematopoiesis with a critical role in keeping balance between proliferation and differentiation. Alternations in the expression of these genes can be resulted in malignancy. The present study investigated the expression levels of P53 and AML1 genes in 82 de novo AML patient specimens against normal control group using Real-Time-PCR. The results presented in this study revealed that AML1 gene expression was significantly higher and P53 gene expression levels was significantly lower in patients with AML in comparison with the normal control group (P = 0.016 and P = 0.002). Furthermore, the correlation between P53 and AML1 was significant and positive (P= 0.037 and r= 0.231). The lower levels of P53 expression were expected and in line with the normal role of this gene as a tumor suppressor gene, however AML1 over expression was in contrast with of its well-known role in myeloid maturation. However, This findings suggest that despite the current established role this genes in myeloid cell differentiation, oncogenic form of AML1 (AML1a) has possibly increased and high expression of this isoform may act as an inhibitor for other normal AML1 isoforms and P53 as well