ABSTRACT
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 were used to assess their ability to differentiate into skeletal cell lineages. The colony forming ability, proliferation, alkaline phosphatase [ALP] activity, calcium content, and osteogenic gene expression were evaluated in both BMMSCs and BlCs cultures at days 7, 14, and 21
Results: qRT-PCR analysis revealed that the cells from both sources readily differentiated into mesodermal lineages. There was significantly higher colony forming ability in BM-MSCs compared to BlCs [P<0.05]. Alizarin red staining [ARS], calcium, and the ALP assay showed the same degree of mineral deposition in both BlCs and BM-MSCs. Gene expression levels of osteblastic markers indicated similar bone differentiation capacity for both BlCs and BM-MSCs at all time-points
Conclusion: characteristics of BlCs in vitro appear to be similar to BM-MSCs. Therefore, they could be considered as a substitute for BlCs for a regenerative approach with potential use in future clinical settings for regenerating human appendages
ABSTRACT
Objective: Dual inhibition of mitogen-activated protein kinase [MAPK] kinase [also known as MEK] and transforming growth factor beta [TGFbeta] 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 timepolymerase 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, Nanog, Sox2, Rex1, Dppa3, Tcf3, Utf1, Nodal, Dax1, Sall4 and beta-Catenin] and lower expression of early differentiation genes [Gata6, Lefty2 and Cdx2] in R2i condition compared to the serum condition. Moreover, the upstream region of Oct4 and Nanog showed a progressive increase in methylation levels in the upstream regions of the genes following in R2i or serum conditions, followed by a decrease of DNA methylation in ESCs obtained under R2i. However, the methylation level of ICM outgrowths in the serum condition was much higher than R2i, at levels that could have a repressive effect and therefore explain the absence of expression of these two genes in the serum condition
Conclusion: Our investigation revealed that generation of ESCs in the ground-state of pluripotency could be achieved by inhibiting the MEK and TGF-beta signaling pathways in the first 5 days of ESC derivation
ABSTRACT
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 reports 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 transient expression of green fluorescent protein in two genetically different hESC lines, Royan H5 [XX] and Royan H6 [XY], as well as human foreskin fibroblasts [hFF]. For long-term EGFP expression VASA and OLIG2 promoters [germ cell and motoneuron specific genes, respectively], were isolated and subsequently cloned into a pBluMAR5 plasmid backbone to drive EGFP expression. Flow cytometry analysis was performed two days after transfection to determine transient expression efficiency. Differentiation of drug resistant hESC colonies toward primordial germ cells [PGCs] was conducted to confirm stable integration of the transgene
Results: Transient and stable expression suggested a variable potential for different cell lines against transfection. Analysis of parameters that influenced gene transformation efficiency revealed that the vector concentrations from 20-60 Mug and the density of the subjected cells [5×105 and 1×106 cells] were not as effective as the genetic background and voltage rate. The present data indicated that in contrast to the circular form, the linearized vector generated more distinctive drug resistant colonies
Conclusion: Electroporation was an efficient tool for genetic engineering of hESCs compared to the chemical method. The genetic background of the subjected cell line for transfection seemed to be a fundamental factor in each gene delivery method. For each cell line, optimum voltage rate should be calculated as it has been shown to play a crucial role in cell death and rate of gene delivery
ABSTRACT
Recombinant vaccine technology is one of the most developed means in controlling infectious diseases. However, an effective vaccine against Shigella is still missing. To evaluate recombinant IpaC protein of Shigella as a vaccine candidate. In this study we cloned IpaC gene into an expression vector in prokaryotic system. The protein expression was evaluated by SDS-PAGE and Western-Blotting analysis. The recombinant protein was purified using Ni-NTA affinity chromatography. Guinea pigs were immunized with the recombinant protein and the level of immunogenicity was examined by ELISA and Western blotting of IpaC. Challenge test was done through the intraoculary injection of Shigella dysenteriae [6×108 CFU/eye] and after 48 hours was scored for keratoconjunctivitis. The results showed a remarkable level of immunogenicity in terms of antibody response and protection against keratoconjunctivitis in tested animals. The recombinant IpaC protein provided a protective system against Shigella dysenteriae type I during the challenge test. The results showed the potential of using recombinant IpaC in preparation of vaccine in perspective studies
ABSTRACT
For immunotherapy of human papillomavirus [HPV] -16-associated cervical cancers the E7 protein is considered a prime candidate. However it is a poor inducer of cytotoxic T-cell response, when being used as a singular antigen in protein vaccination. Hence, in this study we focused on the utilization of a vaccine delivery system for prevention or treatment of cervical cancer. In this experimental study, we designed and evaluated a novel fusion protein comprising HPV16 E7 antigen fused to Shiga toxin B-subunit [STxB] as both an antigen vector and an adjuvant. Then we designed two preventive and therapeutic tumor models to investigate the prevention and inhibition of TC-1 cell growth in female C57BL/6 mice, respectively. In each model, mice were immunized with the recombinant protein of E7-STxB or E7 without any adjuvant. We demonstrated that prophylactic immunization of E7-STxB protected mice against TC-1 cells. Also in the therapeutic model, E7-STxB inhibited TC-1 tumor growth inlungs. The results were significant when compared with the immunization of E7 singularly. We concluded that immunization with the E7-STxB protein without any adjuvant could generate anti-tumor effect in mice challenged with TC-1 cells. This research verifies the clinical applications and the future prospects of developing HPV16 E7 therapeutic vaccines fused to immunoadjuvants
Subject(s)
Animals, Laboratory , Papillomavirus E7 Proteins , Mice , Shiga Toxin , Shigella dysenteriae , Uterine Cervical Neoplasms , ImmunizationABSTRACT
Genus Shigella is one of the important members of the family Enterobacteriaceae. There are numerous antigens in Shigella carrying by a 220 kb plasmid. Among them, IpaD is the key virulence factor of S. flexneri. Apart from having effectors function that is essential for host cell invasion and intracellular survival, this protein also controls the secretion and translocation of other effector proteins into eukaryotic host cells. In the present study, we have cloned and expressed the ipaD in E. coli. The ipaD gene was amplified by PCR. Prokaryote expression vector pET-28a [+] - ipaD was constructed, and used to transform E. coli BL21DE3 plySs. The expression of recombinant protein induced by IPTG was examined by SDS-PAGE. Western blot were used to determine immunoreactivity of IpaD-His by a rabbit monoclonal antibodies against his-tag. SDS-PAGE demonstrated that the constructed prokaryotic expression efficiently produced IpaD at the 1 mmol/L of IPTG. IpaD protein was able to react with the rabbit monoclonal antibody against His-tag. IpaD is essential for Shigella spp invasion. N-terminal region is most significant functional fragment of IpaD. Purification of IpaD from the wild type of Shigella is difficult furthermore profound study on a specific domain on the N-terminal of IpaD by using the wild type of purified IpaD is not feasible