Role of PI3K/AKT signaling pathway involved in self-renewing and maintaining biological properties of chicken primordial germ cells.

Avian primordial germ cells (PGCs) are important culture cells for the production of transgenic chickens and preservation of the genetic resources of endangered species; however, culturing these cells in vitro proves challenging. Although the proliferation of chicken PGCs is dependent on insulin, the underlying molecular mechanisms remain unclear. In the present study, we explored the expression of the PI3K/AKT signaling pathway in PGCs, investigated its effects on PGC self-renewal and biological properties, and identified the underlying mechanisms. Our findings indicated that although supplementation with the PI3K/AKT activator IGF-1 failed to promote proliferation under the assessed culture conditions, the PI3K/AKT inhibitor LY294002 resulted in retarded cell proliferation and reduced expression of germ cell-related markers. We further demonstrated that inhibition of PI3K/AKT regulates the cell cycle and promotes apoptosis in PGCs by activating the expression of BAX and inhibiting that of Bcl-2. These findings indicated that the PI3K/AKT pathway is required for cell renewal, apoptosis, and maintenance of the reproductive potential in chicken PGCs. This study aimed to provide a theoretical basis for the optimization and improvement of a culture system for chicken PGCs and provide insights into the self-renewal of vertebrate PGCs as well as potential evolutionary changes in this unique cell population.

Autophagy activation alleviates the LPS-induced inflammatory response in endometrial epithelial cells in dairy cows.

PROBLEM: Endometritis is a common disease that affects dairy cow reproduction. Autophagy plays a vital role in cellular homeostasis and modulates inflammation by regulating interactions with innate immune signaling pathways. However, little is known about the regulatory relationship between autophagy and inflammation in bovine endometrial epithelial cells (BEECs). Thus, we aimed to determine the role of autophagy in the inflammatory response in BEECs. METHODS OF STUDY: In the present study, the expression levels of proinflammatory cytokines were measured by quantitative real-time polymerase chain reaction. Changes in the nuclear factor-κB (NF-κB) pathway and autophagy were determined using immunoblotting and immunocytochemistry. The induction of autophagosome formation was visualized by transmission electron microscopy. RESULTS: Our results demonstrated that autophagy activation was inhibited in LPS-treated BEECs, while activation of the NF-κB pathway and the mRNA expression of IL-6, IL-8, and TNF-α were increased. Furthermore, blocking autophagy with the inhibitor chloroquine increased NF-κB signaling pathway activation and proinflammatory factor expression in LPS-treated BEECs. Conversely, activation of autophagy with the agonist rapamycin inhibited the NF-κB signaling pathway and downregulated proinflammatory factors. CONCLUSIONS: These data indicated that LPS-induced inflammation was related to the inhibition of autophagy in BEECs. Thus, the activation of autophagy may represent a novel therapeutic strategy for eliminating inflammation in BEECs.

The biogenesis, identification, and functionality of circWWP2 in lipopolysaccharide stimulated macrophages.

CircRNA, a non-coding RNA, is an ideal biomarker and a suitable potential therapeutic target for various disease due to its high stability, species conservation and cell/tissue specificity. Our previous study has found a circular RNA WWP2 (circWWP2) was significantly decreased in chicken macrophages during bacterial infection. However, the function of circWWP2 in chicken macrophages remains unclear. In this study, it was demonstrated that circWWP2 was a stable circular RNA created by back-splicing of exons 2 to 4 of WWP2 via PCR amplification, Sanger sequencing, RNase R exonuclease digestion, and RT-qPCR. Moreover, bioinformatics analysis showed circWWP2 could interact with 13 miRNAs and target 3,264 genes, which were significantly enriched in lysosomes, IgA-producing intestinal immune networks for IgA production, and Notch signaling pathway. Furthermore, CCK8 and RT-qPCR indicated that overexpression of circWWP2 could promote lipopolysaccharide (LPS)-induced cellular injury by decreasing cell viability and increasing the expression levels of pro-inflammatory cytokines and pro-apoptosis genes, and NO production. CircWWP2 may exert a potential target for the treatment of bacterial infection. Further experiments are necessary to validate the specific mechanism that circWWP2 regulates LPS induced cellular immune responses.

Clonally derived chicken primordial germ cell lines maintain biological characteristics and proliferative potential in long-term culture.

Chicken primordial germ cells (PGCs) are important cells with significant implications in preserving genetic resources, chicken breeding and production, and basic research on genetics and development. Currently, chicken PGCs can be cultured long-term in vitro to produce single-cell clones. However, systematic exploration of the cellular characteristics of these single-cell clonal lines has yet to be conducted. In this study, single-cell clonal lines were established from male and female PGCs of Rugao Yellow Chicken and Shouguang Black Chicken, respectively, using a micropipette-based method for single-cell isolation and culture. Analysis of glycogen granule staining, mRNA expression of pluripotency marker genes (POUV, SOX2, NANOG), germ cell marker genes (DAZL, CVH), and SSEA-1, EMA-1, SOX2, C-KIT, and CVH protein expression showed positive results, indicating that PGCs maintain normal cellular properties after single-cell cloning. Furthermore, tests on proliferation ability and gene expression levels in PGC single-cell clonal lines showed high expression of the pluripotency-related genes and TERT compared to control PGCs, and PGC single-cell clonal lines demonstrated higher proliferation ability. Finally, green fluorescent protein (GFP)-PGC single-cell clonal lines were established, and it was found that these single-cell clonal lines could still migrate into the gonads of recipients, suggesting their potential for germ-line transmission. This study systematically validated the normal cellular characteristics of PGC single-cell clonal lines, indicating that they could be applied in genetic modification research on chickens.

Effects of Insulin on Proliferation, Apoptosis, and Ferroptosis in Primordial Germ Cells via PI3K-AKT-mTOR Signaling Pathway.

Primordial germ cells (PGCs) are essential for the genetic modification, resource conservation, and recovery of endangered breeds in chickens and need to remain viable and proliferative in vitro. Therefore, there is an urgent need to elucidate the functions of the influencing factors and their regulatory mechanisms. In this study, PGCs collected from Rugao yellow chicken embryonic eggs at Day 5.5 were cultured in media containing 0, 5, 10, 20, 50, and 100 µg/mL insulin. The results showed that insulin regulates cell proliferation in PGCs in a dose-dependent way, with an optimal dose of 10 µg/mL. Insulin mediates the mRNA expression of cell cycle-, apoptosis-, and ferroptosis-related genes. Insulin at 50 µg/mL and 100 µg/mL slowed down the proliferation with elevated ion content and GSH/oxidized glutathione (GSSG) in PGCs compared to 10 µg/mL. In addition, insulin activates the PI3K/AKT/mTOR pathway dose dependently. Collectively, this study demonstrates that insulin reduces apoptosis and ferroptosis and enhances cell proliferation in a dose-dependent manner via the PI3K-AKT-mTOR signaling pathway in PGCs, providing a new addition to the theory of the regulatory role of the growth and proliferation of PGC in vitro cultures.

The First Crested Duck Genome Reveals Clues to Genetic Compensation and Crest Cushion Formation.

The Chinese crested (CC) duck is a unique indigenous waterfowl breed, which has a crest cushion that affects its survival rate. Therefore, the CC duck is an ideal model to investigate the genetic compensation response to maintain genetic stability. In the present study, we first generated a chromosome-level genome of CC ducks. Comparative genomics revealed that genes related to tissue repair, immune function, and tumors were under strong positive selection, indicating that these adaptive changes might enhance cancer resistance and immune response to maintain the genetic stability of CC ducks. We also assembled a Chinese spot-billed (Csp-b) duck genome, and detected the structural variations (SVs) in the genome assemblies of three ducks (i.e., CC duck, Csp-b duck, and Peking duck). Functional analysis revealed that several SVs were related to the immune system of CC ducks, further strongly suggesting that genetic compensation in the anti-tumor and immune systems supports the survival of CC ducks. Moreover, we confirmed that the CC duck originated from the mallard ducks. Finally, we revealed the physiological and genetic basis of crest traits and identified a causative mutation in TAS2R40 that leads to crest formation. Overall, the findings of this study provide new insights into the role of genetic compensation in adaptive evolution.

Isolation of Swine Bone Marrow Lin-/CD45-/CD133 + Cells and Cardio-protective Effects of its Exosomes.

BACKGROUND: The identification in murine bone marrow (BM) of CD133 + /Lin-/CD45- cells, possessing several features of pluripotent stem cells, encouraged us to investigate if similar population of cells could be also isolated from the swine BM. Heart failure is the terminal stage of many cardiovascular diseases, and its key pathological basis is cardiac fibrosis (CF). Research showed that stem cell derived exosomes may play a critical role in cardiac fibrosis. The effect of exosomes (Exos) on CF has remained unclear. OBJECTIVE: To establish an isolation and amplification method of CD133 + /Lin-/CD45- cells from newbron swine BM in vitro, explore an highly efficient method to enrich swine bone marrow derived CD133 + /Lin-/CD45- cells and probe into their biological characteristics further. Furher more, to extract exosomes from it and explore its effect on CF. METHODS: The mononuclear cells isolated from swine bone marrow by red blood cell (RBC) lysing buffer were coated by adding FcR blocking solution and coupled with CD133 antibody immunomagnetic beads, obtaining CD133 + cell group via Magnetic Activated Cell Sorting (MACS). In steps, the CD133 + /Lin-/CD45- cells were collected by fluorescence-activated cell sorting (FACS) labeled with CD133, Lin and CD45 antibodies, which were cultured and amplified in vitro. The biological features of CD133 + /Lin-/CD45- cells were studied in different aspects, including morphological trait observed with inverted microscope, ultrastructural characteristics observed under transmission electron microscope, expression of pluripotent markersidentified by immunofluorescent staining and Alkaline phosphatase staining. The Exos were extracted using a sequential centrifugation approach and its effects on CF were analyzed in Angiotensin II (Ang-II) induced-cardiac fibrosis in vivo. Rats in each group were treated for 4 weeks, and 2D echocardiography was adopted to evaluate the heart function. The degree of cardiac fibrosis was assessed by Hematoxylin-Eosin (HE) and Masson's trichrome staining. RESULTS: The CD133 + /Lin-/CD45- cells accounted for about 0.2%-0.5% of the total mononuclear cells isolated from swine bone marrow. The combination of MACS and FACS to extract CD133 + /Lin-/CD45- cells could improved efficiency and reduced cell apoptosis. The CD133 + /Lin-/CD45- cells featured typical traits of pluripotent stem cells, the nucleus is large, mainly composed of euchromatin, with less cytoplasm and larger nucleoplasmic ratio, which expressed pluripotent markers (SSEA-1, Oct-4, Nanog and Sox-2) and alkaline phosphatase staining was positive.Animal experiment indicated that the cardiac injury related indexes (BNP、cTnI、CK-MB and TNF-α), the expression of key gene Smad3 and the degree of cardiac fibrosis in Exo treatment group were significantly reduced compared with the control group. 4 weeks after the treatment, cardiac ejection fraction (EF) value in the model group showed a remarkable decrease, indicating the induction of HF model. While Exo elevated the EF values, demonstrating cardio-protective effects. CONCLUSION: The CD133 + /Lin-/CD45- cells derived from swine bone marrow were successfully isolated and amplified, laying a good foundation for further research on this promising therapeutic cell. The Exos may be a promising potential treatment strategy for CF.

Role and function of the Hintw in early sex differentiation in chicken (Gallus gallus) embryo.

Mono-Sex culturing is an important methodology for intensive livestock and poultry production. Here, Hintw was identified as a potential key gene in sex-determination process in chickens via RNA-seq. Then we developed an effective method to interfere or overexpress Hintw in chicken embryos through the intravascular injection. QRT-PCR, ELISA and H&E staining were used to detect the effects of Hintw on gonadal development of chicken embryos. Results showed that Hintw exhibited a female-biased expression pattern in the early stage of PGCs (primordial germ cells) in embryonic gonads. The qRT-PCR analysis showed that Foxl2, Cyp19a1 in females were upregulated under the overexpression of Hintw, while Sox9 and Dmrt1 were downregulated Hintw. Overexpression of Hintw can promote the development of gonadal cortex, while interference with Hintw show the opposite result. Additionally, we found that overexpression of the Hintw in male chicken embryos could inhibit androgen levels and increase estrogen levels. On the other hand, interfering with Hintw in female chicken embryos decreased estrogen levels and increased androgen levels. In conclusion, this work sets the basis for the understanding of the molecular regulatory network for the sex-determination process in chicken embryos as well as providing the theoretical basis for mono-sex culturing of poultry.

Bisphenol A regulates apolipoprotein A1 expression through estrogen receptors and DNA methlylation and leads to cholesterol disorder in rare minnow testis.

Bisphenol A (BPA) is a well-known plasticizer that widely distributed in the aquatic environment. BPA has many adverse effects on reproduction. However, few studies have investigated the mechanism of BPA affecting reproduction from the perspective of lipid metabolism. Apolipoprotein A1 (ApoA1) is the major component of high-density lipoprotein (HDL), and plays critical roles in reverse cholesterol transport (RCT). In this study, in order to investigate the effect and molecular mechanism of BPA on testicular ApoA1 and the role of ApoA1 in BPA induced abnormal spermatogenesis, adult male rare minnow Gobiocypris rarus were exposed to 15 µg/L of BPA for 1, 3 and 5 weeks. Results showed that BPA could significantly affect testicular ApoA1 mRNA and protein levels, testicular cholesterol levels, plasmatic sex hormone levels and the integrity of sperm head membrane. The main mechanism of BPA regulating ApoA1 expression is to alter Esr recruitment and CpG sites DNA methylation in ApoA1 promoter. The induced ApoA1 up-regulated high density lipoprotein cholesterol levels and enhanced RCT, and finally decreased the testicular free cholesterol levels. This is likely a key mechanism by which BPA induces sex hormone disorder and sperm head membrane damage. The present study reveals the mechanism by which BPA interferes with spermatogenesis from the perspective of cholesterol transport.

Promotion of Differentiating Bone Marrow Mesenchymal Stromal Cells (BMSCs) into Cardiomyocytes via HCN2 and HCN4 Cotransfection.

AIM: Investigation of the influences HCN2 and HCN4 has on bone marrow mesenchymal stromal cells (BMSCs) on cardiomyocyte differentiation. METHODS: Miniature adult pigs were used for bone marrow extraction and isolation of BMSCs. The identification of these BMSCs was done by using flow cytometry for the detection of expressed surface antigens CD45, CD11B, CD44, and CD90. Using HCN2 and HCN4 genes cotransfected into BMSCs as group HCN2+HCN4 while myocardial induction solution was used to induced BMSC differentiation in the BMSC induction group. Myocardial marker proteins α-actin and cTnT were detected by immunofluorescence staining, while α-actin, cTnT, and Desmin myocardial marker proteins expressed were detected by Western blot. The whole-cell patch-clamp technique was used to identify and detect cellular HCN2 channels, HCN4 channel current activation curve, and the inhibitory effect of CsCl on heterologous expression currents. RESULTS: Flow cytometry results showed that CD45 and CD11B were expressed negatively while CD90 and CD44 were positive. Post HCN2 and HCN4 gene transfection, immunofluorescence staining, and Western blot showed significantly increased HCN2, HCN4, α-actin, and cTnT expressed in group HCN2+HCN4 were, which could be compared to the expression levels in the BMSC-induced group. The HCN2+HCN4 group was able to document cell membrane channel ion currents that were similar to If properties. CONCLUSION: HCN2 and HCN4 overexpression can considerably enhance the MSC ability to differentiate into cardiomyocytes in vitro and restore the ionic current.

H3K9me2 regulates early transcription factors to promote mesenchymal stem­cell differentiation into cardiomyocytes.

Studies have shown that histone H3 at lysine 9 (H3K9me2) is an important epigenetic modifier of embryonic development, cell reprogramming and cell differentiation, but its specific role in cardiomyocyte formation remains to be elucidated. The present study established a model of 5­Azacytidine­induced differentiation of rat bone mesenchymal stem cells (MSCs) into cardiomyocytes and, on this basis, investigated the dimethylation of H3K9me2 and its effect on cardiomyocyte formation by knockdown of H3K9me2 methylase, euchromatic histone­lysine N­methyltransferase 2 (G9a) and H3K9me2 lysine demethylase 3A (KDM3A). The results demonstrated that, in comparison with the normal induction process, the knockdown of G9a could significantly reduce the H3K9me2 level of the MSCs in the induced model. Reverse transcription­quantitative (RT­q) PCR demonstrated that the expression of cardiac troponin T(cTnT) was significantly increased. In addition, flow cytometry demonstrated that the proportion of cTnT­positive cells was significantly increased on day 21. With the knockdown of KDM3A, the opposite occurred. In order to explore the specific way of H3K9me2 regulating cardiomyocyte formation, western blotting and RT­qPCR were used to detect the expression of key transcription factors including GATA binding protein 4 (GATA­4), NK2 Homeobox 5 (Nkx2.5) and myocyte enhancer factor 2c (MEF2c) during cardiomyocyte formation. The decrease of H3K9me2 increased the expression of transcription factors GATA­4, Nkx2.5 and MEF2c in the early stage of myocardial development while the increase of H3K9me2 inhibited the expression of those transcription factors. Accordingly, it was concluded that H3K9me2 is a negative regulator of cardiomyocyte formation and can participate in cardiomyocyte formation by activating or inhibiting key transcription factors of cardiomyocytes, which will lay the foundation for the optimized induction efficiency of cardiomyocytes in in vitro and clinical applications.

Production of viable chicken by allogeneic transplantation of primordial germ cells induced from somatic cells.

The allogeneic transplantation of primordial germ cells (PGCs) derived from somatic cells overcomes the limitation of avian cloning. Here, we transdifferentiate chicken embryo fibroblasts (CEFs) from black feathered Langshan chickens to PGCs and transplant them into White Plymouth Rock chicken embryos to produce viable offspring with characteristics inherited from the donor. We express Oct4/Sox2/Nanog/Lin28A (OSNL) to reprogram CEFs to induced pluripotent stem cells (iPSCs), which are further induced to differentiate into PGCs by BMP4/BMP8b/EGF. DNA demethylation, histone acetylation and glycolytic activation elevate the iPSC induction efficiency, while histone acetylation and glycolytic inhibition facilitate PGCs formation. The induced PGCs (iPGCs) are transplanted into the recipients, which are self-crossed to produce 189/509 somatic cells derived chicken with the donor's characteristics. Microsatellite analysis and genome sequencing confirm the inheritance of genetic information from the donor. Thus, we demonstrate the feasibility of avian cloning from somatic cells.

5-Azacytidine-Induced Cardiomyocyte Differentiation of Very Small Embryonic-Like Stem Cells.

The use of stem cells in generating cell-based pacemaker therapies for bradyarrhythmia is currently being considered. Due to the propensity of stem cells to form tumors, as well as ethical issues surrounding their use, the seed cells used in cardiac biological pacemakers have limitations. Very small embryonic-like stem cells (VSELs) are a unique and rare adult stem cell population, which have the same structural, genetic, biochemical, and functional characteristics as embryonic stem cells without the ethical controversy. In this study, we investigated the ability of rat bone marrow- (BM-) derived VSELs to differentiate in vitro into cardiomyocytes by 5-Azacytidine (5-AzaC) treatment. The morphology of VSELs treated with 10 µM 5-AzaC increased in volume and gradually changed to cardiomyocyte-like morphology without massive cell death. Additionally, mRNA expression of the cardiomyocyte markers cardiac troponin-T (cTnT) and α-sarcomeric actin (α-actin) was significantly upregulated after 5-AzaC treatment. Conversely, stem cell markers such as Nanog, Oct-4, and Sox2 were continuously downregulated posttreatment. On day 14 post-5-AzaC treatment, the positive expression rates of cTnT and α-actin were 18.41 ± 1.51% and 19.43 ± 0.51%, respectively. Taken together, our results showed that rat BM-VSELs have the ability to differentiate into cardiomyocytes in vitro. These findings suggest that VSELs would be useful as seed cells in exploring the mechanism of biological pacemaker activity.

C1EIP Functions as an Activator of ENO1 to Promote Chicken PGCs Formation via Inhibition of the Notch Signaling Pathway.

The production of germ cells, especially primordial germ cells (PGCs), is important for avian stem cells and reproduction biology. However, key factors involved in the regulation of PGCs remain unknown. Here, we report a PGC-related marker gene: C1EIP (Chromosome 1 Expression in PGCs), whose activation and expression are regulated by the transcription factor STAT3 (signal transducer and activator of transcription 3), histone acetylation, and promoter methylation. C1EIP regulates PGCs formation by mediating the expression of PGC-associated genes, such as CVH (Chicken Vasa Homologous) and CKIT (Chicken KIT proto-oncogene). C1EIP knockdown during embryonic development reduces PGC generation efficiency both in vitro and in ovo. Conversely, C1EIP overexpression increases the formation efficiency of PGCs. C1EIP encodes a cytoplasmic protein that interacts with ENO1 (Enolase 1) in the cytoplasm, inhibits the Notch signaling pathway, and positively regulates PGC generation. Collectively, our findings demonstrate C1EIP as a novel gene involved in PGC formation, which regulates genes involved in embryonic stem cell differentiation through interaction with ENO1 and subsequent inhibition of the Notch signaling pathway by the impression of Myc (MYC proto-oncogene).

P53 and H3K4me2 activate N6-methylated LncPGCAT-1 to regulate primordial germ cell formation via MAPK signaling.

Long noncoding RNAs (lncRNAs) participate in the formation of primordial germ cells (PGCs); however, the identity of the key lncRNAs and the molecular mechanisms responsible for the formation of PGCs remain unknown. Here, we identify a key candidate lncRNA (lncRNA PGC transcript-1, LncPGCAT-1) via RNA sequencing of embryonic stem cells, PGCs, and Spermatogonial stem cells (SSCs). Functional experiments confirmed that LncPGCAT-1 positively regulated the formation of PGCs by elevating the expression of Cvh and C-kit while downregulating the pluripotency(Nanog) in vitro and in vivo; PAS staining of genital ridges in vivo also showed that interference with LncPGCAT-1 can significantly reduce the number of PGCs in genital ridges, while overexpression of LncPGCAT-1 had the opposite result. The result of luciferase reporter assay combined with CHIP-qPCR showed that the expression of LncPGCAT-1 was promoted by the transcription factor P53 and high levels of H3K4me2. Mechanistically, the luciferase reporter assay confirmed that mitogen-activated protein kinase 1 (MAPK1) was the target gene of LncPGCAT-1 and gga-mir-1591. In the ceRNA system, high levels of N6 methylation of LncPGCAT-1 enhanced the adsorption capacity of LncPGCAT-1 for gga-mir-1591. Adsorption of gga-mir-1591 activated the MAPK1/ERK signaling cascade by relieving the gga-mir-1591-dependent inhibition of MAPK1 expression. Moreover, LncPGCAT-1 interacted with interleukin enhancer binding factor 3 (ILF3) to regulate the ubiquitination of P53 and phosphorylation of JNK. Interaction with ILF3 resulted in positive self-feedback regulation of LncPGCAT-1 and activation of JNK signaling, ultimately promoting PGC formation. Altogether, the study expands our knowledge of the function and molecular mechanisms of lncRNAs in PGC development.

Comparison between curcumin and all-trans retinoic acid in the osteogenic differentiation of mouse bone marrow mesenchymal stem cells.

The use of bone marrow mesenchymal stem cells (BMSCs) has great potential in cell therapy, particularly in the orthopedic field. BMSCs represent a valuable renewable cell source that have been successfully utilized to treat damaged skeletal tissue and bone defects. BMSCs can be induced to differentiate into osteogenic lineages via the addition of inducers to the growth medium. The present study examined the effects of all-trans retinoic acid (ATRA) and curcumin on the osteogenic differentiation of mouse BMSCs. Morphological changes, the expression levels of the bone-associated gene markers bone morphogenetic protein 2, runt-related transcription factor and osterix during differentiation, an in vitro mineralization assay, and changes in osteocalcin expression revealed that curcumin supplementation promoted the osteogenic differentiation of BMSCs. By contrast, the application of ATRA increased osteogenic differentiation during the early stages, but during the later stages, it decreased the mineralization of differentiated cells. In addition, to the best of our knowledge, the present study is the first to examine the effect of curcumin on the osteogenic potency of mouse embryonic fibroblasts (MEFs) after reprogramming with human lim mineralization protein (hLMP-3), which is a positive osteogenic regulator. The results revealed that curcumin-supplemented culture medium increased hLMP-3 osteogenic potency compared with that of MEFs cultured in the non-supplemented medium. The present results demonstrate that enrichment of the osteogenic culture medium with curcumin, a natural osteogenic inducer, increased the osteogenic differentiation capacity of BMSCs as well as that of MEFs reprogrammed with hLMP-3.

Functional characterization of the Sox2, c-Myc, and Oct4 promoters.

To better understand the mechanisms in transcriptional regulation, we analyzed the promoters of the reprogramming key genes Sox2, c-Myc, and Oct4. Here, we cloned different 5' deletions of the goat Sox2, c-Myc, and Oct4 promoters, and evaluated their functions by green fluorescent protein reporter system and dual-luciferase reporter system. Site-directed mugagenesis and epigenetic modifiers were used to explore the influence of transcription binding sites and epigenetic status on the promoters. The results suggested that the basal promoters were located in the - 109 to 49, - 147 to 1, and - 96 to 30 bp regions of the Sox2, c-Myc, and Oct4 promoters. The transcription factors that identified to influence the Sox2, c-Myc, and Oct4 promoter activities were Elf-1 and activating protein 2 (AP-2), C/EBP and Sp1, and Mzf1 and Sp1, respectively. The epigenetic alternation of the Sox2, c-Myc, and Oct4 promoters by 5-aza-2'-deoxycytidine or/and trichostatin A significantly increased the promoter activities. In conclusion, the result determined the core promoter areas of the Sox2, c-Myc, and Oct4 genes, and identified the transcription factors that influence their promoter activities. We also verified that the Sox2, c-Myc, and Oct4 promoters were hypermethylated and hypoacetylated.

Direct conversion of mouse embryonic fibroblast to osteoblast cells using hLMP-3 with Yamanaka factors.

Large bone defects and bone loss after fractures remain significant challenges for orthopedic surgeons. Our study aims to find an available, applicable and biological treatment for bone regeneration overcoming the limitations in ESC/iPSC technology. We directly reprogrammed the mouse embryonic fibroblast (MEF) into osteoblast cells using different combinations of Yamanaka factors with human lim mineralization protein-3 (hLMP-3). LMP is an intracellular LIM-domain protein acting as an effective positive regulator of the osteoblast differentiation. After transduction, cells were cultured in osteogenic medium, and then examined for osteoblast formation. The expression of osteogenic markers (BMP2, Runx2 and Osterix) during reprogramming and in vitro mineralization assay revealed that the best reprogramming cocktail was (c-Myc - Oct4) with hLMP-3. In addition, both immunofluorescent staining and western blot analysis confirmed that osteocalcin (OCN) expression increased in the cells treated with the c-Myc/Oct4/hLMP3 cocktail than using hLMP-3 alone. Furthermore, this reprogramming cocktail showed efficient healing in an induced femoral bone defect in rat animal model one month after transplantation. In the present study, we reported for the first time the effect of combining Yamanaka factors with hLMP-3 to induce osteoblast cells from MEF both in vitro and in vivo.

The establishment of clonally derived chicken embryonic fibroblast cell line (CSC) with high transfection efficiency and ability as a feeder cell.

This study established a single cloned chicken embryonic fibroblast (CEF) cell line. It solves the main problem of the instability of a cultured primary cell and its impact on the experiment. In this study, CEF pass through this crisis and formed a continuous cell line after subculture. We isolated single postcrisis CEF by a mouth pipette under a convert microscope then established a single cloned cell line named CSC-1-5 which passaged continuously from 96-well plates to 60 mm culture plates. CSC has a normal chicken diploid karyotype, no tumorigenicity, and a high G2/M phase cell ratio. We found that Fugene could mediate the transfection of CSCs efficiently; it was significantly improved compared with the primary cells. It could also promote the proliferation of chicken embryonic stem cell as a feeder layer.

DNA Methylation and Regulatory Elements during Chicken Germline Stem Cell Differentiation.

The production of germ cells in vitro would open important new avenues for stem biology and human medicine, but the mechanisms of germ cell differentiation are not well understood. The chicken, as a great model for embryology and development, was used in this study to help us explore its regulatory mechanisms. In this study, we reported a comprehensive genome-wide DNA methylation landscape in chicken germ cells, and transcriptomic dynamics was also presented. By uncovering DNA methylation patterns on individual genes, some genes accurately modulated by DNA methylation were found to be associated with cancers and virus infection, e.g., AKT1 and CTNNB1. Chicken-unique markers were also discovered for identifying male germ cells. Importantly, integrated epigenetic mechanisms were explored during male germ cell differentiation, which provides deep insight into the epigenetic processes associated with male germ cell differentiation and possibly improves treatment options to male infertility in animals and humans.