The constitutively active pSMAD2/3 relatively improves the proliferation of chicken primordial germ cells.

In many multicellular organisms, mature gametes originate from primordial germ cells (PGCs). Improvements in the culture of PGCs are important not only for developmental biology research, but also for preserving endangered species, and for genome editing and transgenic animal technologies. SMAD2/3 appear to be powerful regulators of gene expression; however, their potential positive impact on the regulation of PGC proliferation has not been taken into consideration. Here, the effect of TGF-ß signaling as the upstream activator of SMAD2/3 transcription factors was evaluated on chicken PGCs' proliferation. For this, chicken PGCs at stages 26-28 Hamburger-Hamilton were obtained from the embryonic gonadal regions and cultured on different feeders or feeder-free substrates. The results showed that TGF-ß signaling agonists (IDE1 and Activin-A) improved PGC proliferation to some extent while treatment with SB431542, the antagonist of TGF-ß, disrupted PGCs' proliferation. However, the transfection of PGCs with constitutively active SMAD2/3 (SMAD2/3CA) resulted in improved PGC proliferation for more than 5 weeks. The results confirmed the interactions between overexpressed SMAD2/3CA and pluripotency-associated genes NANOG, OCT4, and SOX2. According to the results, the application of SMAD2/3CA could represent a step toward achieving an efficient expansion of avian PGCs.

Hydrogel-mediated delivery of microRNA-92a inhibitor polyplex nanoparticles induces localized angiogenesis.

Localized stimulation of angiogenesis is an attractive strategy to improve the repair of ischemic or injured tissues. Several microRNAs (miRNAs) such as miRNA-92a (miR-92a) have been reported to negatively regulate angiogenesis in ischemic disease. To exploit the clinical potential of miR-92a inhibitors, safe and efficient delivery needs to be established. Here, we used deoxycholic acid-modified polyethylenimine polymeric conjugates (PEI-DA) to deliver a locked nucleic acid (LNA)-based miR-92a inhibitor (LNA-92a) in vitro and in vivo. The positively charged PEI-DA conjugates condense the negatively charged inhibitors into nano-sized polyplexes (135 ± 7.2 nm) with a positive net charge (34.2 ± 10.6 mV). Similar to the 25 kDa-branched PEI (bPEI25) and Lipofectamine RNAiMAX, human umbilical vein endothelial cells (HUVECs) significantly internalized PEI-DA/LNA-92a polyplexes without any obvious cytotoxicity. Down-regulation of miR-92a following the polyplex-mediated delivery of LNA-92a led to a substantial increase in the integrin subunit alpha 5 (ITGA5), the sirtuin-1 (SIRT1) and Krüppel-like factors (KLF) KLF2/4 expression, formation of capillary-like structures by HUVECs, and migration rate of HUVECs in vitro. Furthermore, PEI-DA/LNA-92a resulted in significantly enhanced capillary density in a chicken chorioallantoic membrane (CAM) model. Localized angiogenesis was substantially induced in the subcutaneous tissues of mice by sustained release of PEI-DA/LNA-92a polyplexes from an in situ forming, biodegradable hydrogel based on clickable poly(ethylene glycol) (PEG) macromers. Our results indicate that PEI-DA conjugates efficiently deliver LNA-92a to improve angiogenesis. Localized delivery of RNA interference (RNAi)-based therapeutics via hydrogel-laden PEI-DA polyplex nanoparticles appears to be a safe and effective approach for different therapeutic targets.

Towards maturation of human otic hair cell-like cells in pluripotent stem cell-derived organoid transplants.

Human otic organoids generated from pluripotent stem cells (PSCs) provide a promising platform for modeling, drug testing, and cell-based therapies of inner ear diseases. However, providing the appropriate niche that resembles inner ear development and its vasculature to generate otic organoids is less conspicuous. Here, we devised a strategy to enhance maturation of otic progenitor cells toward human hair cell-like cells (HCLCs) by assembling three-dimensional (3D) otic organoids that contain human PSC-derived otic cells, endothelial cells, and mesenchymal stem cells (MSCs). Heterotopic implantation of otic organoids, designated as grafted otic organoids (GOs), in ex ovo chick embryo chorioallantoic membrane (CAM) stimulated maturation of the HCLCs. Functional analysis revealed the presence of voltage-gated potassium currents without detectable sodium currents in these cells in the GOs. Our results demonstrated that implantation of 3D heterotypic cell mixtures of otic organoids improved maturation of human HCLCs. This GO-derived HCLCs could be an attractive source for drug discovery and other biomedical applications.

In vitro improvement of quail primordial germ cell expansion through activation of TGF-beta signaling pathway.

Avian primordial germ cells (PGCs) have valuable potentials to cell-based approaches for transgenic bird production. In this regard, improvement of avian PGC expansion in vitro is necessary. Among experimental avian species, quail is a good model for transgenic technology, especially due to its short generation time. In the present study, we have examined the proliferative effects of transforming growth factor ß (TGF-ß) on the quail PGCs. After isolation of quail PGCs from blood (Hamburger-Hamilton [HH stages 13-15]) and gonads (HH stages 28-30), these cells were cultured on quail embryonic fibroblasts (QEF). Our results indicated th at cultured gonadal-derived PGCs proliferated 400 times in comparison to 100 times for blood PGCs over 40-50 days. Upon in vitro exposure to TGF-ß inducers by Activin or the inducer of definitive endoderm 1 (IDE1) small molecule, the number of gonad PGCs significantly increased to 26% and 64%, respectively. In contrast, inhibition of the TGF-ß signaling pathway by SB431542 resulted in a significant reduction in the numbers of PGCs (P < 0.001). Moreover, Phosphorylation of SMAD2/3 in the IDE1 group was higher compared to the Activin-treated ones. We confirmed the PGC identification with periodic acid-Schiff (PAS) staining, anti-SSEA1, ß-catenin, ß-integrin, and Nanog immunofluorescence staining. Exogenously IDE1 treated-PGCs migrated toward the embryonic gonads after transplantation into the heart of the recipient embryo at HH stages 13-15. Our results suggested that the application of IDE1 small molecule into the culture of quail PGCs represented a step toward achieving efficient expansion of the avian PGCs.

Scientometric analysis and perspective of IgY technology study.

Egg yolk immunoglobulin (IgY) is a class of antibody that is produced in birds against pathogens. Therefore, hyperimmunization of birds can produce a specific antibody in the egg against target antigen for a wide range of applications in diagnostic, prophylactic or treatment in human and veterinary medicine which is known today as IgY technology. Until now, the number of articles, patents and clinical studies on IgY technology has increased significantly. Hence, there is a fact that scientometric studies are needed to gain a deeper understanding of the research for the commercialization of IgY technology. Until now, no scientometric research has been directed toward IgY technology. In view of this, we conducted scientometric analysis in the WoS database. A total of 1,029 IgY-related papers were obtained including 981 journal articles and 48 reviews. The visualization of this literature showed an increasing trend in the number of IgY-related publications over the 4 decades, especially after 2008 to 2021. China, the United States, Canada, Japan, and Germany had the largest number of publications, with 220, 148, 91, 76, and 72, respectively. Among all the research institutions, Dalian University of Technology, Alberta University and Northwest Agriculture and Forestry University published the most of the articles, respectively. Among authors, Dr. Xiaoying Zhang had the highest number of publications with 21. The top most cited publications were from Dr. da Silva with 38 citations. Keywords co-occurrence network analysis showed that the correlation between different keywords is large, especially IgY, antibodies and immunoglobulin which is consistent with the rapid increase in the number of publications. Finally, through this data analysis, we hope that our result could help IgY technology to more maturity toward industrialization and commercialization.

Trends in industrialization and commercialization of IgY technology.

IgY technology refers to the strategic production process involved in generating avian immunoglobulin (IgY) against target antigens in a much more cost-effective manner with broad applications in the fields of diagnostics, prophylaxis, and therapeutics for both human and veterinary medicine. Over the past decade, promising progress in this research area has been evident from the steep increase in the number of registered manufacturing companies involved in the production of IgY products, the number of patents, and the notable number of clinical trials underway. Hence, it is crucial to conduct a prospective analysis of the commercialization and marketing potential of IgY-based commercial products for large-scale applications. This review revealed that the number of IgY patent applications increased steeply after 2010, with the highest of 77 patents filed in 2021. In addition, 73 industries are reportedly involved in marketing IgY products, out of which 27 were promoting biotherapeutics for human and veterinary medicine and 46 were in the diagnostic field. IgY antibodies are being used as primary and secondary antibodies, with approximately 3729 and 846 products, respectively. Biotherapeutic product consumption has notably increased as a food supplement and as a topical application in human and veterinary medicine, which are under different clinical phases of development to reach the market with around 80 and 56 products, respectively. In contrast, the number of IgY products as parenteral administrations and licensed drugs is not well developed given the lack of technical standards established for IgY registration and industrialization, as well as the restriction of the nature of polyclonal antibodies. However, recent ongoing research on functional IgY fragments indicates a promising area for IgY applications in the near future. Therefore, retrospective analysis with speculations is mandatory for IgY technology maturation toward industrialization and commercialization.

Chicken Interspecies Chimerism Unveils Human Pluripotency.

Human pluripotent stem cells (hPSCs) are commonly kept in a primed state but also able to acquire a more immature naive state under specific conditions in vitro. Acquisition of naive state changes several properties of hPSCs and might affect their contribution to embryonic development in vivo. However, the lack of an appropriate animal test system has made it difficult to assess potential differences for chimera formation between naive and primed hPSCs. Here, we report that the developing chicken embryo is a permissive host for hPSCs, allowing analysis of the pluripotency potential of hPSCs. Transplantation of naive-like and primed hPSCs at matched developmental stages resulted in robust chimerism. Importantly, the ability of naive-like but not of primed hPSCs to form chimera was substantially reduced when injected at non-matched developmental stages. We propose that contribution to chick embryogenesis is an informative and versatile test to identify different pluripotent states of hPSCs.

Tissue-Specific Microparticles Improve Organoid Microenvironment for Efficient Maturation of Pluripotent Stem-Cell-Derived Hepatocytes.

Liver organoids (LOs) are receiving considerable attention for their potential use in drug screening, disease modeling, and transplantable constructs. Hepatocytes, as the key component of LOs, are isolated from the liver or differentiated from pluripotent stem cells (PSCs). PSC-derived hepatocytes are preferable because of their availability and scalability. However, efficient maturation of the PSC-derived hepatocytes towards functional units in LOs remains a challenging subject. The incorporation of cell-sized microparticles (MPs) derived from liver extracellular matrix (ECM), could provide an enriched tissue-specific microenvironment for further maturation of hepatocytes inside the LOs. In the present study, the MPs were fabricated by chemical cross-linking of a water-in-oil dispersion of digested decellularized sheep liver. These MPs were mixed with human PSC-derived hepatic endoderm, human umbilical vein endothelial cells, and mesenchymal stromal cells to produce homogenous bioengineered LOs (BLOs). This approach led to the improvement of hepatocyte-like cells in terms of gene expression and function, CYP activities, albumin secretion, and metabolism of xenobiotics. The intraperitoneal transplantation of BLOs in an acute liver injury mouse model led to an enhancement in survival rate. Furthermore, efficient hepatic maturation was demonstrated after ex ovo transplantation. In conclusion, the incorporation of cell-sized tissue-specific MPs in BLOs improved the maturation of human PSC-derived hepatocyte-like cells compared to LOs. This approach provides a versatile strategy to produce functional organoids from different tissues and offers a novel tool for biomedical applications.

A Cell-Free SDKP-Conjugated Self-Assembling Peptide Hydrogel Sufficient for Improvement of Myocardial Infarction.

Biomaterials in conjunction with stem cell therapy have recently attracted attention as a new therapeutic approach for myocardial infarction (MI), with the aim to solve the delivery challenges that exist with transplanted cells. Self-assembling peptide (SAP) hydrogels comprise a promising class of synthetic biomaterials with cardiac-compatible properties such as mild gelation, injectability, rehealing ability, and potential for sequence modification. Herein, we developed an SAP hydrogel composed of a self-assembling gel-forming core sequence (RADA) modified with SDKP motif with pro-angiogenic and anti-fibrotic activity to be used as a cardioprotective scaffold. The RADA-SDKP hydrogel was intramyocardially injected into the infarct border zone of a rat model of MI induced by left anterior descending artery (LAD) ligation as a cell-free or a cell-delivering scaffold for bone marrow mesenchymal stem cells (BM-MSCs). The left ventricular ejection fraction (LVEF) was markedly improved after transplantation of either free hydrogel or cell-laden hydrogel. This cardiac functional repair coincided very well with substantially lower fibrotic tissue formation, expanded microvasculature, and lower inflammatory response in the infarct area. Interestingly, BM-MSCs alone or in combination with hydrogel could not surpass the cardiac repair effects of the SDKP-modified SAP hydrogel. Taken together, we suggest that the RADA-SDKP hydrogel can be a promising cell-free construct that has the capability for functional restoration in the instances of acute myocardial infarction (AMI) that might minimize the safety concerns of cardiac cell therapy and facilitate clinical extrapolation.