Cell and Tissue Journal

Abstract Introduction: Gene therapy involves transferring genetic material into target cells to correct mutations or introduce new biological functions. Among delivery systems, lentiviral vectors are considered efficient and reliable tools due to their ability to integrate stably into the host genome and transduce both dividing and non-dividing cells. This property provides long-term gene expression, which is highly valuable for therapeutic and experimental applications. The PDX1 (Pancreatic and Duodenal Homeobox 1) gene plays a central role in pancreatic organogenesis and the regulation of insulin-producing beta cells. It acts as a transcription factor controlling genes critical for endocrine differentiation and insulin secretion. Chick embryos are a useful experimental model due to their accessibility, rapid development, and the responsiveness of their fibroblast and germ cells to gene transfer systems. These features make them suitable for studying gene delivery efficiency and expression stability.
Aim: The aim of this study was to construct and produce recombinant lentiviral vectors carrying the PDX1 gene in HEK293T-LentiX cells and evaluate their transfer efficiency in chick embryonic fibroblast and germ cells. This work was conducted to assess the potential of lentiviral systems for stable gene delivery in avian cells.
Materials and Methods: HEK293T-LentiX cells were selected as producer cells due to their high transfection efficiency and viral packaging capability. They were cultured in DMEM supplemented with 10% fetal bovine serum, penicillin, and streptomycin under standard incubation conditions (37°C, 5% CO₂). Lentiviral particles were generated using a three-plasmid packaging system, including a transfer vector containing the PDX1 gene and two helper plasmids. Transfection was carried out using the calcium phosphate method. After 48–72 hours, the viral-containing supernatant was collected, filtered, and concentrated. Viral titers were determined by evaluating GFP expression in target cells through fluorescence microscopy and flow cytometry. Chick embryonic fibroblast and primordial germ cells were isolated and infected with various viral concentrations in the presence of 8 µg/ml polybrene to enhance infection. After incubation, cells were examined for GFP signal as evidence of successful gene transfer.
Results: High-titer recombinant lentiviral particles were successfully produced in HEK293T cells. Fluorescence microscopy revealed strong GFP expression, confirming the presence of functional viral particles. Flow cytometry analysis provided quantitative confirmation of high viral titers. Following transduction, chick embryonic fibroblast and germ cells exhibited clear GFP expression, indicating efficient infection and gene transfer. The PDX1 gene was successfully delivered and expressed within target cells. Although transduction efficiency varied slightly between cell types, the overall results demonstrated that the lentiviral system provided stable and effective gene delivery to chick embryo-derived cells.
Discussion: The study confirmed that lentiviral vectors carrying the PDX1 gene could be efficiently produced and used to achieve stable gene transfer in chick embryonic cells. This system’s ability to integrate permanently into the host genome ensures consistent gene expression over time without repeated transfection. For functional genes like PDX1, this stability is crucial for maintaining insulin-related pathways and pancreatic cell differentiation. Chick embryos serve as an advantageous model because their cells are easily accessible, grow rapidly, and respond well to viral vectors. Such characteristics make them ideal for investigating genetic regulation during early development. Evaluation of viral titers using fluorescence microscopy and flow cytometry provided reliable data confirming efficient vector production. The integration of new tools such as CRISPR/Cas9 can further enhance lentiviral design precision, allowing targeted modification of specific genes. Combining these technologies may open promising avenues for studying metabolic disorders and for gene-based therapies.
Conclusion: Recombinant lentiviral vectors carrying the PDX1 gene were successfully generated and used to transduce chick embryonic fibroblast and germ cells. The system exhibited high production efficiency, stable gene expression, and suitability for in vitro studies. These findings demonstrate that lentiviral vectors represent a powerful and versatile platform for gene transfer and experimental modeling in avian systems. Moreover, coupling lentiviral vectors with genome editing technologies could expand future applications in regenerative medicine and genetic engineering.

Abstract Introduction: Titanium dioxide nanoparticles (TiO₂ NPs) are widely used in industry, medicine, food, and cosmetics. TiO₂ NPs are harmful to the environment and human health. Changes in the environment may especially affect the growing neurological system. Therefore, it is impossible to overlook their impact on the development of the embryo and reproductive success. A sensitive and widely used model for evaluating the teratogenic potential and developmental toxicity of different nanoparticles is the chicken embryo. At the outset of embryogenesis, the application of TiO₂ NPs enables the penetration into various tissues, such as brain precursor cells and structures. Furthermore, the organs are unable to remove the nanoparticles from the egg due to their isolation and enclosure from the mother and the environment. Consequently, the embryos are perpetually exposed to TiO₂ NPs during the 20-day embryogenesis period. In recent years, a growing number of studies have been conducted to examine the possible harmful effects of TiO₂ NPs due to worries about inadvertent exposure of NPs on humans.
Aim: In this study, the embryonic toxicity of various dosages of TiO₂ NPs was investigated in the chicken embryo's brain cortex.
Materials and Methods: In this study, 90 fertilized eggs were divided into five groups: control group (untreated group), four treatment groups that received 0 (sham), 12.5, 25, and 50 μg/mL of TiO₂ nanoparticles. The embryos' morphology, weight, and Crown-rump length (CRL) were assessed after 7, 9, and 13 days.   Tissue samples were collected from the cerebrum and cerebellum of the chick embryos. The specimens were immediately fixed in a 10% neutral buffered formalin solution for 48 hours. Subsequently, they were dehydrated through a series of ascending ethanol concentrations (70%, 80%, 90%, and 100%), with each step lasting two hours. The samples were then cleared in xylene for one hour, embedded in paraffin wax, and sectioned at a thickness of 5μm using a microtome. The sections were then mounted on glass slides.  Finally, all tissue sections were stained with hematoxylin and eosin, and the effects of TiO₂ NPs were examined on the histology, pathology, and histomorphometry of the chick embryo cerebrum and cerebellum tissues.
Results: The findings showed that TiO₂ NPs cause embryo death in all days at 25 and 50 μg/mL. In morphological studies, the weight and length of 13-day-old embryos treated with 50 μg/mL of TiO₂ NPs decreased. Counting of cells (neurons and glial cells) in the cerebral cortex of a 13-day-old chick embryo displayed a significant decrease in the experimental group of 50 μg/ml compared to the control and sham groups. The evaluations showed a decrease in the number of Purkinje cells of the cerebellum cortex in 13-day-old embryos treated with 50 μg/ml of TiO₂ NPs.   The group of 13-day-old embryos treated with 50 µg/ml had a considerable capillary hyperemia in the cerebellar cortex.
Conclusion: It was concluded that the in-ovo-administered TiO₂ NPs given immediately before incubation have adverse effects on the developing cerebellum and cerebrum. So that the increase of damage happened in older embryos, and the highest damage occurred on day 13 of incubation.

Abstract Aim: we aimed at presenting a simple and efficient method for isolating and characterizing the neural crest cells.
Material and Methods: The hen’s fertilized eggs were incubated for about 35h at 38°c and 55-60% humidity until the embryos reached to stages 10-12 according to Hamburger-Hamilton developmental stage table. Then the embryos were removed from the egg’s yolk and the neural tube was isolated and cultured for 24 h in a tissue culture dish to release neural crest cell. Then after, the neural tube was removed and allowed to NCC to expand for further 5 days. Finally, the cells were collected and subjected to PCR to study their gene expression profile.
Results: The neural tube released NCC and these cells proliferated in culture condition. They also expressed markers including Slug, Sox9 ,and Sox10 by the RT-PCR method.
Conclusion: The neural tube can release NCC in culture condition and these cells can proliferate in the presence of an appropriate medium.
 

Abstract Aim: The present study was designed to investigate the effects of chitosan/poly vinyl alcohol scaffold incorporated by NRBE (neonatal rat brain extract) on neuronal differentiation of P19 EC (embryonal carcinoma) stem cells.
Material and Methods: In order to induce neuronal phenotype, P19-derived embryonic carcinoma stem cells were cultured on a rat chitosan/poly (vinyl alcohol) scaffold with newborn rat brain extracts. To evaluate the survival potential, migration and differentiation of the three-dimensional culture cells, these cells were grafted to the developing central nervous system of the chick embryo. Finally, the transplanted cells were detected using specific staining and immunofluorescence methods.
Results: Cresyl-Violet specific staining confirmed the neuronal phenotype of the transplantation cells. Also, the expression of specific neural protein, synaptophysin, was shown using the immunofluorescence process.
Conclusion: The results of this study showed that P19 embryonic carcinoma stem cells can be used as a source of pluripotent cells in transplantation studies in order to investigate cellular and molecular aspects of developmental and cellular differentiation.
 

Abstract Aim: The somites are epithelial blocks of mesodermal cells differentiating to sclerotome and dermomyotome and notochord or axial mesoderm plays a major role in somitic cell differentiation to sclerotome. This study was aimed to evaluate the role of the notochord in sclerotomal differentiation of the somites in vitro. Material and Methods: In this experimental study, isolated notochords from chick embryo were encapsulated in alginate beads and co-cultured with somites for six days. In control group, the somites were cultured alone. Newly isolated and non-cultured somites were considered as somites on day 0. Eventually, morphology of cultured somitic cells was evaluated by inverted microscope and then RT-PCR method was used to analyze the sclerotomal and dermomyotomal gene expression profile. Results: In comparison with control group, the somitic cells co-cultured with notochord had highly differentiation potential and showed mesenchymal morphology with numerous slender processes. Gene expression profile of co-cultured somitic cells indicated upregulation of Pax1 and BMP4 and downregulation of MyoD, presenting sclerotomal cell characteristics. Conclusion: Embryonic notochord can induce in vitro sclerotomal differentiation in somites through upregulation of genes involving in this differentiation.