Search Results (26)

Small extracellular vesicles (sEVs) derived from mesenchymal stem cells have emerged as promising therapeutic agents in regenerative dermatology. This study evaluated the safety and efficacy of Bio-Pulsed avian mesenchymal stem cell-derived sEVs (AMSC-sEVs), topically applied for hair follicle stimulation and skin rejuvenation. Two prospective, single-arm clinical trials were conducted: one involving 30 participants using a hair ampoule over 60 days, and the other involving 30 participants applying a facial essence for 28 days. Objective measurements demonstrated significant improvements in the anagen/telogen hair ratio, reduced shedding, increased collagen density, and reduced wrinkle depth and pigmentation. Small RNA sequencing and qPCR profiling confirmed that Bio-Pulsed AMSC-sEVs were enriched with regenerative microRNAs, such as miR-21-5p and miR-199a-5p, associated with anti-inflammatory and anti-aging effects. No adverse events were reported. These findings suggest that Bio-Pulsed AMSC-sEVs may offer a safe, non-invasive, and cell-free approach to enhance skin and hair regeneration in human subjects. Full article

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. Full article

Background: Histone deacetylase 4 (HDAC4) is a member of the class II histone deacetylase family, whose members play a crucial role in various biological processes. An in-depth investigation of the transcriptional characteristics of chicken HDAC4 can provide fundamental insights into its function. Methods: We examined HDAC4 expression in chicken embryonic stem cells (ESC) and spermatogonial stem cells (SSC) and cloned a 444 bp fragment from upstream of the chicken HDAC4 transcription start site. Subsequently, we constructed pEGFP-HDAC4 and a series of 5′-deletion luciferase reporter constructs, which we transfected into DF-1 cells to measure their transcriptional activity. The regulatory mechanisms of chicken HDAC4 expression were investigated by performing trichostatin A (TSA) treatment, deleting putative transcription factor binding sites, and altering transcription factor expression levels. Results: HDAC4 exhibited higher expression in SSC than in ESC. We confirmed that the upstream region from −295 bp to 0 bp is the core transcriptional region of HDAC4. TSA effectively inhibited HDAC4 transcription, and bioinformatics analysis indicated that the chicken core HDAC4 promoter sequence exhibits high homology with those of other avian species. The myelocytomatosis viral oncogene homolog (MYC) and hypoxia-inducible factor 1 α (HIF1A) transcription factors were predicted to bind to this core region. Treatment with TSA for 24 h resulted in the upregulation of MYC and HIF1A, which repressed HDAC4 transcription. Conclusions: Our results provide a basis for subsequent investigations into the regulation of HDAC4 expression and biological function. Full article

Background: The Jun proto-oncogene (JUN), also referred to as C-JUN, is an integral component of the JNK signaling pathway, which is crucial for the formation and differentiation of spermatogonial stem cells (SSCs). Investigations into the transcriptional regulation of chicken JUN can offer a molecular foundation for elucidating its mechanistic role in SSCs. Methods: In this study, we successfully cloned a 2000 bp upstream sequence of the JUN transcription start site and constructed a series of pGL3 recombinant vectors containing JUN promoters of varying lengths. Results: We verified the promoter activity of the 2000 bp upstream sequence by assessing the fluorescence intensity of DF-1 and identified the promoter activities of different regions via dual-luciferase assays. The transcription of JUN and its promoter region spanning −700 to 0 bp was modulated by an activator of the JNK signaling pathway. Bioinformatics analysis revealed that this −700 to 0 bp region was highly conserved among avian species and predicted the presence of binding sites for Wilms tumor 1 (WT1) and CCAAT/enhancer binding protein alpha (CEBPA). The JNK signaling pathway activator was found to upregulate the expression of these transcription factors in DF-1 cells. Through the deletion of binding sites and the overexpression of WT1 and CEBPA, we demonstrated that WT1 inhibited the transcription of JUN, while CEBPA promoted it. Conclusions: In conclusion, the −700 to 0 bp region is the key region of the JUN promoter, with WT1 inhibiting JUN transcription. The results of the study not only provide ideas for exploring the regulatory mechanism of JUN in chicken SSCs, but also lay an important foundation for the study of avian SSCs. Full article

Background/Objectives: Induced pluripotent stem cells (iPSCs) reprogrammed from somatic cells into cells with most of the ESC (embryonic stem cell) characteristics show promise toward solving ethical problems currently facing stem cell research and eventually yield clinical grade pluripotent stem cells for therapies and regenerative medicine. In recent years, an increasing body of research suggests that the chemical induction of pluripotency (CIP) method can yield iPSCs in vitro, yet its application in avian species remains unreported. Methods: Herein, we successfully obtained stably growing chicken embryonic fibroblasts (CEFs) using the tissue block adherence method and employed 12 small-molecule compounds to induce chicken iPSC formation. Results: The final optimized iPSC induction system was bFGF (10 ng/mL), CHIR99021 (3 μM), RepSox (5 μM), DZNep (0.05 μM), BrdU (10 μM), BMP4 (10 ng/mL), vitamin C (50 μg/mL), EPZ-5676 (5 μM), and VPA (0.1 mM). Optimization of the induction system revealed that the highest number of clones was induced with 8 × 10⁴ cells per well and at 1.5 times the original concentration. Upon characterization, these clones exhibited iPSC characteristics, leading to the development of a stable compound combination for iPSC generation in chickens. Concurrently, employing a deletion strategy to investigate the functionality of small-molecule compounds during induction, we identified CHIR99021 and BrdU as critical factors for inducing chicken iPSC formation. Conclusions: In conclusion, this study provides a reference method for utilizing small-molecule combinations in avian species to reprogram cells and establish a network of cell fate determination mechanisms. Full article

Avian primordial germ cells (PGCs) are essential in avian transgenic research, germplasm conservation, and disease resistance breeding. However, cultured PGCs are prone to fragmentation and apoptosis, regulated at transcriptional and translational levels, with N6-methyladenosine (m6A) being the most common mRNA modification. Resveratrol (RSV) is known for its antioxidant and anti-apoptotic properties, but its effects on PGCs and the underlying mechanisms are not well understood. This study shows that RSV supplementation in cultured PGCs improves cell morphology, significantly enhances total antioxidant capacity (p < 0.01), reduces malondialdehyde levels (p < 0.05), increases anti-apoptotic BCL2 expression, and decreases Caspase-9 expression (p < 0.05). Additionally, RSV upregulates the expression of m6A reader proteins YTHDF1 and YTHDF3 (p < 0.05). m6A methylation sequencing revealed changes in mRNA m6A levels after RSV treatment, identifying 6245 methylation sites, with 1223 unique to the control group and 798 unique to the RSV group. Combined analysis of m6A peaks and mRNA expression identified 65 mRNAs with significantly altered methylation and expression levels. Sixteen candidate genes were selected, and four were randomly chosen for RT-qPCR validation, showing results consistent with the transcriptome data. Notably, FAM129A and SFRP1 are closely related to apoptosis, indicating potential research value. Overall, our study reveals the protective effects and potential mechanisms of RSV on chicken PGCs, providing new insight into its use as a supplement in reproductive stem cell culture. Full article

Induced pluripotent stem cells (iPSCs) were first generated by Yamanaka in 2006, revolutionizing research by overcoming limitations imposed by the use of embryonic stem cells. In terms of the conservation of endangered species, iPSC technology presents itself as a viable alternative for the manipulation of target genetics without compromising specimens. Although iPSCs have been successfully generated for various species, their application in nonmammalian species, particularly avian species, requires further in-depth investigation to cover the diversity of wild species at risk and their different protocol requirements. This study aims to provide an overview of the workflow for iPSC induction, comparing well-established protocols in humans and mice with the limited information available for avian species. Here, we discuss the somatic cell sources to be reprogrammed, genetic factors, delivery methods, enhancers, a brief history of achievements in avian iPSC derivation, the main approaches for iPSC characterization, and the future perspectives and challenges for the field. By examining the current protocols and state-of-the-art techniques employed in iPSC generation, we seek to contribute to the development of efficient and species-specific iPSC methodologies for at-risk avian species. The advancement of iPSC technology holds great promise for achieving in vitro germline competency and, consequently, addressing reproductive challenges in endangered species, providing valuable tools for basic research, bird genetic preservation and rescue, and the establishment of cryobanks for future conservation efforts. Full article

Aves ranks among the top two classes for the highest number of endangered and extinct species in the kingdom Animalia. Notably, the IUCN Red List classified the green peafowl as endangered. This highlights promising strategies using genetics and reproductive technologies for avian wildlife conservation. These platforms provide the capacity to predict population trends and enable the practical breeding of such species. The conservation of endangered avian species is facilitated through the application of genomic data storage and analysis. Storing the sequence is a form of biobanking. An analysis of sequence can identify genetically distinct individuals for breeding. Here, we reviewed avian genomics and stem cell approaches which not only offer hope for saving endangered species, such as the green peafowl but also for other birds threatened with extinction. Full article

The potential of viruses as appropriate vectors for the development of new therapeutic strategies, as well as for the design of molecular (DNA, RNA, and/or protein) vaccines via substitution of nucleotide sequences, has been proven. Among the most appropriate DNA and/or RNA fragments, members belonging to families Parvoviridae (particularly adeno-associated virus, AAV) and Poxviridae have frequently been suggested for this purpose. In previous studies, the vaccine avipoxvirus strains FK (fowl) and Dessau (pigeon) have been proven able to infect mammalian cells (as well as avian cells), and to replicate productively in a small number of them; thus, we may be able to adapt them using incubation, and in these conditions. Additionally, we have previously proved, based on AAV recombinant DNA vectors, that it is possible to transfer appropriate genes of interest via mouse embryonic stem cells (mESCs). In the current study, we develop methods for the application of the same vaccine avipoxviral strains, based on the AAV DNA genome recombinant constructs, to be used for gene transfer in cells, for the transfer of DNA and/or RNA fragments (for the suppression of unwanted viral and/or cellular genes), and for the production of molecular (DNA, RNA, and/or protein) anti-cancer and anti-viral vaccines. To this end, sub-populations of embryonic mammalian cells infected with the two forms of both vaccine avipoxviral strains were frozen in the presence of cryo-protector dimethylsulfoxide (DMSO), subsequently thawed, and re-incubated. In most cases, the titers of the intra-cellular forms of the two strains were higher than those of their extra-cellular forms. These data were explained by the probable existence of the intra-cellular forms as different sub-forms, including those integrated in the cellular genome proviruses at a given stage of the cellular infection, and suggest the possibility of transferring nucleotide (DNA and/or RNA) fragments between cellular and viral genomes; this is due to the influence of activated fusion processes on DMSO, as well as drastic temperature variations. Full article

Among amniotic skin appendages, avian feathers and mammalian hairs protect their stem cells in specialized niches, located in the collar bulge and hair bulge, respectively. In chickens and alligators, label retaining cells (LRCs), which are putative stem cells, are distributed in the hinge regions of both avian scutate scales and reptilian overlapping scales. These LRCs take part in scale regeneration. However, it is unknown whether other types of scales, for example, symmetrically shaped reticulate scales, have a similar way of preserving their stem cells. In particular, the foot sole represents a special interface between animal feet and external environments, with heavy mechanical loading. This is different from scutate-scale-covered metatarsal feet that function as protection. Avian reticulate scales on foot soles display specialized characteristics in development. They do not have a placode stage and lack β-keratin expression. Here, we explore the molecular and cellular characteristics of avian reticulate scales. RNAscope analysis reveals different molecular profiles during surface and hinge determination compared with scutate scales. Furthermore, reticulate scales express Keratin 15 (K15) sporadically in both surface- and hinge-region basal layer cells, and LRCs are not localized. Upon wounding, the reticulate scale region undergoes repair but does not regenerate. Our results suggest that successful skin appendage regeneration requires localized stem cell niches to guide regeneration. Full article

The proto-oncogene myc has been intensively studied primarily in vertebrate cell culture systems. Myc transcription factors control fundamental cellular processes such as cell proliferation, cell cycle control and stem cell maintenance. Myc interacts with the Max protein and Myc/Max heterodimers regulate thousands of target genes. The genome of the freshwater polyp Hydra encodes four myc genes (myc1-4). Previous structural and biochemical characterization showed that the Hydra Myc1 and Myc2 proteins share high similarities with vertebrate c-Myc, and their expression patterns suggested a function in adult stem cell maintenance. In contrast, an additional Hydra Myc protein termed Myc3 is highly divergent, lacking the common N-terminal domain and all conserved Myc-boxes. Single cell transcriptome analysis revealed that the myc3 gene is expressed in a distinct population of interstitial precursor cells committed to nerve- and gland-cell differentiation, where the Myc3 protein may counteract the stemness actions of Myc1 and Myc2 and thereby allow the implementation of a differentiation program. In vitro DNA binding studies showed that Myc3 dimerizes with Hydra Max, and this dimer efficiently binds to DNA containing the canonical Myc consensus motif (E-box). In vivo cell transformation assays in avian fibroblast cultures further revealed an unexpected high potential for oncogenic transformation in the conserved Myc3 C-terminus, as compared to Hydra Myc2 or Myc1. Structure modeling of the Myc3 protein predicted conserved amino acid residues in its bHLH-LZ domain engaged in Myc3/Max dimerization. Mutating these amino acid residues in the human c-Myc (MYC) sequence resulted in a significant decrease in its cell transformation potential. We discuss our findings in the context of oncogenic transformation and cell differentiation, both relevant for human cancer, where Myc represents a major driver. Full article

Avian models are valuable for studies of development and reproduction and have important implications for food production. Rapid advances in genome-editing technologies have enabled the establishment of avian species as unique agricultural, industrial, disease-resistant, and pharmaceutical models. The direct introduction of genome-editing tools, such as the clustered regularly interspaced short palindromic repeats (CRISPR) system, into early embryos has been achieved in various animal taxa. However, in birds, the introduction of the CRISPR system into primordial germ cells (PGCs), a germline-competent stem cell, is considered a much more reliable approach for the development of genome-edited models. After genome editing, PGCs are transplanted into the embryo to establish germline chimera, which are crossed to produce genome-edited birds. In addition, various methods, including delivery by liposomal and viral vectors, have been employed for gene editing in vivo. Genome-edited birds have wide applications in bio-pharmaceutical production and as models for disease resistance and biological research. In conclusion, the application of the CRISPR system to avian PGCs is an efficient approach for the production of genome-edited birds and transgenic avian models. Full article

With a history of more than 100 years of different applications in various scientific fields, the chicken chorioallantoic membrane (CAM) assay has proven itself to be an exceptional scientific model that meets the requirements of the replacement, reduction, and refinement principle (3R principle). As one of three extraembryonic avian membranes, the CAM is responsible for fetal respiration, metabolism, and protection. The model provides a unique constellation of immunological, vascular, and extracellular properties while being affordable and reliable at the same time. It can be utilized for research purposes in cancer biology, angiogenesis, virology, and toxicology and has recently been used for biochemistry, pharmaceutical research, and stem cell biology. Stem cells and, in particular, mesenchymal stem cells derived from adipose tissue (ADSCs) are emerging subjects for novel therapeutic strategies in the fields of tissue regeneration and personalized medicine. Because of their easy accessibility, differentiation profile, immunomodulatory properties, and cytokine repertoire, ADSCs have already been established for different preclinical applications in the files mentioned above. In this review, we aim to highlight and identify some of the cross-sections for the potential utilization of the CAM model for ADSC studies with a focus on wound healing and tissue engineering, as well as oncological research, e.g., sarcomas. Hereby, the focus lies on the combination of existing evidence and experience of such intersections with a potential utilization of the CAM model for further research on ADSCs. Full article

Fifteen years after their discovery, telocytes (TCs) are yet perceived as a new stromal cell type. Their presence was initially documented peri-digestively, and gradually throughout the interstitia of many (non-)cavitary mammalian, human, and avian organs, including skin. Each time, TCs proved to be involved in diverse spatial relations with elements of interstitial (ultra)structure (blood vessels, nerves, immune cells, etc.). To date, transmission electron microscopy (TEM) remained the single main microscopic technique able to correctly and certainly attest TCs by their well-acknowledged (ultra)structure. In skin, dermal TCs reiterate almost all (ultra)structural features ascribed to TCs in other locations, with apparent direct implications in skin physiology and/or pathology. TCs’ uneven distribution within skin, mainly located in stem cell niches, suggests involvement in either skin homeostasis or dermatological pathologies. On the other hand, different skin diseases involve different patterns of disruption of TCs’ structure and ultrastructure. TCs’ cellular cooperation with other interstitial elements, their immunological profile, and their changes during remission of diseases suggest their role(s) in tissue regeneration/repair processes. Thus, expanding the knowledge on dermal TCs could offer new insights into the natural skin capacity of self-repairing. Moreover, it would become attractive to consider that augmenting dermal TCs’ presence/density could become an attractive therapeutic alternative for treating various skin defects. Full article

Mesenchymal stem cell (MSC)-derived extracellular vesicles (exosomes) possess regeneration, cell proliferation, wound healing, and anti-senescence capabilities. The functions of exosomes can be modified by preconditioning MSCs through treatment with bio-pulsed reagents (Polygonum multiflorum Thunb extract). However, the beneficial effects of bio-pulsed small extracellular vesicles (sEVs) on the skin or hair remain unknown. This study investigated the in vitro mechanistic basis through which bio-pulsed sEVs enhance the bioactivity of the skin fibroblasts and hair follicle cells. Avian-derived MSCs (AMSCs) were isolated, characterized, and bio-pulsed to produce AMSC-sEVs, which were isolated, lyophilized, characterized, and analyzed. The effects of bio-pulsed AMSC-sEVs on cell proliferation, wound healing, and gene expression associated with skin and hair bioactivity were examined using human skin fibroblasts (HSFs) and follicle dermal papilla cells (HFDPCs). Bio-pulsed treatment significantly enhanced sEVs production by possibly upregulating RAB27A expression in AMSCs. Bio-pulsed AMSC-sEVs contained more exosomal proteins and RNAs than the control. Bio-pulsed AMSC-sEVs significantly augmented cell proliferation, wound healing, and gene expression in HSFs and HFDPCs. The present study investigated the role of bio-pulsed AMSC-sEVs in the bioactivity of the skin fibroblasts and hair follicle cells as mediators to offer potential health benefits for skin and hair. Full article