This study aimed to isolate, culture, and characterize duck primordial germ cells (PGCs) and to compare these cells with chicken PGCs. We first cultured Muscovy duck (Cairina moschata) circulating PGCs and gonadal PGCs (gPGCs) in the modified serum-containing medium used to amplify chicken PGCs. gPGCs were found to proliferate better in serum-free chemically defined medium than in serum-containing medium. Thereafter, gPGCs were similarly isolated from 2 other duck breeds, the Pekin duck (Anas platyrhynchos) and the hybrid mule duck (C. moschata × A. platyrhynchos), and amplified for a limited period of time in the chemically defined culture condition, but sufficiently to be characterized and transplanted. Cultured gPGCs of all 3 duck breeds were characterized by Periodic acid-Schiff staining, immunocytochemical staining, and expression analysis of germline-specific and pluripotency genes. Cultured duck gPGCs colonized the gonads after being genetically labeled and injected into recipient embryos. Taken together, these results demonstrate that duck PGCs retain their germline characteristics after being isolated, expanded in vitro, and genetically modified. Further studies are required to establish the optimal conditions for long-term culture of duck PGCs, which may involve supplementing the culture medium with other growth factors or compounds.

Scalable production of avian cell lines exhibits a valuable potential on therapeutic application by producing recombinant proteins and as the substrate for virus growth due to the special glycosylation occurs in avian species. Chicken primordial germ cells (cPGCs), a germinal pluripotent avian cell type, present the ability of self-renewal, an anchorage-independent cell growth and the ability to be genetically modified. This cell type could be an interesting bioreactor system for industrial purposes. This study sought to establish an expandable culture system with defined components for three-dimensional (3D) culture of cPGCs. cPGCs were cultured in medium supplemented with the functional polymer FP003. Viscoelasticity was low in this medium but cPGCs did not sediment in culture and efficiencies of space and nutrient utilization were thus enhanced and consequently their expansion was improved. The total number of cPGCs increased by 17-fold after 1 week of culture in 3D-FAot medium, an aseric defined medium containing FP003 polymer, FGF2 and Activin A as growth factors and Ovotransferrin as protein. Moreover, cPGC cell lines stably expressed the germline-specific reporter VASA:tdTOMATO, as well as other markers of cPGCs, for more than 1 month upon culture in 3D-FAot medium, indicating that the characteristics of these cells are maintained. In summary, this novel 3D culture system can be used to efficiently expand cPGCs in suspension without mechanical stirring, which is available for long-term culture and no loss of cellular properties was found. This system provides a platform for large-scale culture of cPGCs.

Background Cells, scaffolds, and factors are the triad of regenerative engineering; however, it is difficult to distinguish whether cells in the regenerative construct are from the seeded cells or host cells via the host blood supply. We performed a novel in vivo study to transplant enhanced green fluorescent pig mesenchymal stem cells (EGFP-pMSCs) into calvarial defect of DsRed pigs. The cell distribution and proportion were distinguished by the different fluorescent colors through the whole regenerative period. Method/Results Eight adult domestic Ds-Red pigs were treated with five modalities: empty defects without scaffold (group 1); defects filled only with scaffold (group 2); defects filled with osteoinduction medium-loaded scaffold (group 3); defects filled with 5 x 10 3 cells/scaffold (group 4); and defects filled with 5 x 10 4 cells/scaffold (group 5). The in vitro cell distribution, morphology, osteogenic differentiation, and fluorescence images of groups 4 and 5 were analyzed. Two animals were sacrificed at 1, 2, 3, and 4 weeks after transplantation. The in vivo fluorescence imaging and quantification data showed that EGFP-pMSCs were represented in the scaffolds in groups 4 and 5 throughout the whole regenerative period. A higher seeded cell density resulted in more sustained seeded cells in bone regeneration compared to a lower seeded cell density. Host cells were recruited by seeded cells if enough space was available in the scaffold. Host cells in groups 1 to 3 did not change from the 1st week to 4th week, which indicates that the scaffold without seeded cells cannot recruit host cells even when enough space is available for cell ingrowth. The histological and immunohistochemical data showed that more cells were involved in osteogenesis in scaffolds with seeded cells. Conclusion Our in vivo results showed that more seeded cells recruit more host cells and that both cell types participate in osteogenesis. These results suggest that scaffolds without seeded cells may not be effective in bone transplantation.

26 2 27 28 Keywords: mesenchymal stem cell, bone regeneration, enhanced green fluorescent, DsRed pig, 29 pig calvarial defect model 30 31 Abstract 32 Background: Cells, scaffolds, and factors are the triad of regenerative engineering;33 however, it is difficult to distinguish whether cells in the regenerative construct are from the 34 seeded cells or host cells via the host blood supply. We performed a novel in vivo study to 35 transplant enhanced green fluorescent pig mesenchymal stem cells (EGFP-pMSCs) into calvarial 36 defect of DsRed pigs. The cell distribution and proportion were distinguished by the different 37 fluorescent colors through the whole regenerative period. 38Method/Results: Eight adult domestic Ds-Red pigs were treated with five modalities:39 empty defects without scaffold (group 1); defects filled only with scaffold (group 2); defects 40 filled with osteoinduction medium-loaded scaffold (group 3); defects filled with 5 x 10 3 41 cells/scaffold (group 4); and defects filled with 5 x 10 4 cells/scaffold (group 5). The in vitro cell 42 distribution, morphology, osteogenic differentiation, and fluorescence images of groups 4 and 543 were analyzed. Two animals were sacrificed at 1, 2, 3, and 4 weeks after transplantation. The in 44 vivo fluorescence imaging and quantification data showed that EGFP-pMSCs were represented 45 in the scaffolds in groups 4 and 5 throughout the whole regenerative period. A higher seeded cell 46 density resulted in more sustained seeded cells in bone regeneration compared to a lower seeded 47 cell density. Host cells were recruited by seeded cells if enough space was available in the 48 scaffold. Host cells in groups 1 to 3 did not change from the 1st week to 4th week, which 3 49 indicates that the scaffold without seeded cells cannot recruit host cells even when enough space 50 is available for cell ingrowth. The histological and immunohistochemical data showed that more 51 cells were involved in osteogenesis in scaffolds with seeded cells. 52Conclusion: Our in vivo results showed that more seeded cells recruit more host cells and 53 that both cell types participate in osteogenesis. These results suggest that scaffolds without 54 seeded cells may not be effective in bone transplantation. 55 4 56 Introduction 57 Skeletal defects require surgery using bone grafts. Autografts are the gold standard for 58 bone grafting [1]; however, donor site morbidity and the limited amount of available donor tissue 59 restrict their application [2, 3]. Regenerative tissue engineering using cells, scaffolds, factors and 60 blood supply [4] has become an alternative method to treat skeletal bone defects. 61 Allografts may provide the same osteoconductive conduit for bony fusion as traditional 62 autografts and may have comparable biomechanical properties without amount restriction [5, 6]. 63 Although depleted of osteoprogenitor cells like mesenchymal stem cells (MSCs), the fusion rate 64 still reaches 73% to 100% in instrumented spinal fusion [7-16], making allograft a clinically 65 feasibl...

Despite the advances in coronary reperfusion in acute myocardial infarction (MI), post-MI heart failure is still a large burden of public health. Transmural infarction and extended fibrosis contribute largely to post-MI systolic dysfunction and heart failure, and thus cardioprotective strategies are crucial. Human amniotic membrane-derived mesenchymal stem cells (hAMSCs) have been shown with properties of immunomodulation, anti-inflammation, and low immunogenicity, which make them good candidates for cell therapies. In this study, a myocardial ischemia/reperfusion (I/R) model was established in rats, and hAMSCs were administered via the tail vein during coronary reperfusion. Compared to the control group, the rats receiving hAMSCs during the I/R procedure (hAMSC group) exhibited significantly better left ventricular ejection fractions after MI. Histological examinations of the hearts in hAMSC group showed minimal transmural infarction 4 weeks after the I/R procedure. Compared to the control group, hAMSC group had reduced size of cardiac fibrosis and less thinning of myocardial wall. In conclusion, intravenous hAMSCs limit transmural infarction, reduce fibrosis size, and improve left ventricular systolic function after MI in the animal model.

Chicken embryos are a powerful and widely used animal model in developmental biology studies. After the development of CRISPR technology, gene-edited chickens have been generated by transferring primordial germ cells (PGCs) after genetic modifications. However, the low inheritance caused by the competition between host germ cells and the transferred ones is the most common complication and largely reduces the production efficiency in this way. Here, we generated a gene-edited chicken, in which germ cells can be ablated in a drug-dependent manner, as recipients for gene-edited PGC transfer. We used the nitroreductase/metronidazole (NTR/Mtz) system for cell ablation, in which NTR produces cytotoxic alkylating agents from administered Mtz, causing cell apoptosis. The chicken Vasa homolog (CVH) gene locus is used to drive the expression of the NTR gene in a germ cell-specific manner. In addition, a fluorescent protein gene, mCherry, was also placed in the CVH locus to visualize the PGCs. We named this system germ cell-Specific AutonoMoUs RemovAl Induction (gSAMURAI). gSAMURAI chickens will be an ideal recipient to produce offspring derived from transplanted exogenous germ cells.

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