The only cells in an organism that could do any other sort of cell until 2006 (except sperm or egg) were known as embryonic stem cells, ESC

Insulin is an important component of stem cell cultures; however, its role in the proliferation of avian primordial germ cells (PGCs) is unknown. The proliferation of PGCs in cultures varies and the growth factors and signaling pathways necessary to induce the proliferation of PGCs in chickens are unknown. Therefore, we conducted the present study to investigate the effect of insulin on the survival and proliferation of PGCs. In this study, we observed that under this culture system, PGCs proliferate in the presence of insulin, but do not proliferate in the absence of insulin. Furthermore, in insulin-lacking media, the expression of pluripotency genes, including LIN28, NANOG, POUV, and SOX2, was markedly decreased. Similarly, the expression of cell adhesion proteins ZO-1, Occludin, and JAM-A was significantly reduced. Elevated levels of ROS, GSSG, and MDA reduced the redox capacity of the cells and induced apoptosis. Subsequent transcriptome analyses revealed that insulin is one of the key factors in the proliferation of chicken PGCs through the regulation of downstream genes by PI3K/AKT, ECM–receptor interaction, Wnt, and P53 signaling, and that these downstream genes may be important for PGCs’ proliferation and survival.

Background: The importance of embryonic stem cells (ESCs) in chickens is undeniable, as they can be applied across various fields, including animal modeling, developmental biology, cell fate research, drug screening, toxicity testing, and gene function studies. However, a widely applicable culture system for chicken ESCs has yet to be developed. Objectives: This study aimed to investigate the effects of different culture systems on the derivation and maintenance of chicken ESCs, with a focus on optimizing the selected culture conditions. Methods: To achieve this, we tested the effectiveness of various species-specific ESC media in the derivation and culture of chicken PGCs, while incorporating different small molecule compounds to optimize the process. The pluripotency and differentiation potential of the resulting ESC-like cells were also evaluated. Results: The combination of PD0325901, SB431542, and LIF (R2i+LIF system) was found to be effective in generating chicken ESC-like clones. Further experiments showed that enhancing the R2i+LIF system with cytokines such as SCF and FGF2 significantly extended the culture period and increased the passage number of chicken ESC-like cells. These ESC-like cells were characterized through positive alkaline phosphatase staining and the expression of pluripotency markers POUV, NANOG, and SOX2. Additionally, differentiation assays confirmed their ability to form the three germ layers. Conclusions: The newly developed culture system provides suitable conditions for the short-term culture of chicken ESCs. However, further optimization is required to establish a system that can sustain long-term maintenance.

Aromatase, a crucial enzyme for estrogen synthesis, plays a vital role in gender determination and differentiation. This study aimed to establish an inducible knockout model of the chicken CYP19A1 gene, which encodes aromatase, to support gender control in chickens. We selected the most efficient sgRNA target site and constructed an inducible knockout model based on the Tet-on system. The knockout efficiency reached 80% with 20 μg/mL DOX induction in vitro. The encapsulation of the plasmid with PEI and injection into eggs achieved a knockout efficiency of 45% in ovo. qRT-PCR analysis revealed a significant downregulation of female-related genes (CYP19A1, FOXL2, ESR1) and upregulation of male-related genes (DMRT1, SOX9, AMH) in female chicken embryos after induction. Western blotting showed decreased protein expression of CYP19A1 and FOXL2, and increased SOX9 expression in female embryos post-DOX induction. Elisa detection further confirmed lower estradiol levels in the gonads of induced female embryos compared to normal and non-induced females. These findings demonstrate the successful establishment of an inducible knockout system for the CYP19A1 gene in chickens, providing theoretical and technical support for the creation of new breeding materials for gender control.

The utilization of chicken embryonic-derived pluripotent stem cell (PSC) lines is crucial in various fields, including growth and development, vaccine and protein production, and germplasm resource protection. However, the research foundation for chicken PSCs is relatively weak, and there are still challenges in establishing a stable and efficient PSC culture system. Therefore, this study aims to investigate the effects of the FGF2/ERK and WNT/β-catenin signaling pathways, as well as different feeder layers, on the derivation and maintenance of chicken embryonic-derived PSCs. The results of this study demonstrate that the use of STO cells as feeder layers, along with the addition of FGF2, IWR-1, and XAV-939 (FIX), allows for the efficient derivation of chicken PSC-like cells. Under the FIX culture conditions, chicken PSCs express key pluripotency genes, such as POUV, SOX2, and NANOG, as well as specific proteins SSEA-1, C-KIT, and SOX2, indicating their pluripotent nature. Additionally, the embryoid body experiment confirms that these PSC-like cells can differentiate into cells of three germ layers in vitro, highlighting their potential for multilineage differentiation. Furthermore, this study reveals that chicken Eyal–Giladi and Kochav stage X blastodermal cells express genes related to the primed state of PSCs, and the FIX culture system established in this research maintains the expression of these genes in vitro. These findings contribute significantly to the understanding and optimization of chicken PSC culture conditions and provide a foundation for further exploration of the biomedical research and biotechnological applications of chicken PSCs.

Background: Chicken Primordial Germ Cells (PGCs) are one of the few germ cells that can be cultured for a long time in vitro, but challenges remain such as low culture efficiency and unclear roles of nutrient factors and signaling pathways. Method: In this study, protein kinase B (AKT) pathway activator insulin-like growth factor 1 (IGF-1) was screened for its ability to promote cell proliferation by transcriptome results using various inhibitors of pathway activation. The effects of IGF-1 on PGCs were evaluated through EdU assays, qRT-PCR, flow cytometry, and migration experiments. Results: This study systematically examined the effects of insulin and IGF-1 on the proliferation, cell cycle, ferroptosis, migration capacity, and establishment efficiency of PGCs. The findings demonstrated that IGF-1 exhibited comparable effects to insulin and could effectively replace insulin in PGC culture systems. Conclusions: The research results are expected to provide a solid theoretical basis for optimizing the chicken PGC cultivation system and promoting its practical application.