Circumferential Esophageal Reconstruction Using a Tissue-engineered Decellularized Tunica Vaginalis Graft in a Rabbit Model.

BACKGROUND: Pediatric surgeons have faced esophageal reconstruction challenges for decades owing to a variety of congenital and acquired conditions. This work aimed to introduce a reproducible and efficient approach for creating tissue-engineered esophageal tissue using bone marrow mesenchymal stem cells (BMSCs) cultured in preconditioned mediums seeded on a sheep decellularized tunica vaginalis (DTV) scaffold for partial reconstruction of a rabbit's esophagus. METHODS: DTV was performed using SDS and Triton X-100 solutions. The decellularized grafts were employed alone (DTV group) or after recellularization with BMSCs cultured for 10 days in preconditioned mediums (RTV group) for reconstructing a 3 cm segmental defect in the cervical esophagus of rabbits (n = 20) after the decellularization process was confirmed. Rabbits were observed for one month, after which they were euthanized, and the reconstructed esophagi were harvested for histological analysis. RESULTS: Six rabbits in the DTV group and eight rabbits in the RTV group survived until the end of the one-month study period. Despite histological examination demonstrating that both grafts completely repaired the esophageal defect, the RTV graft demonstrated a histological structure similar to that of the normal esophagus. The reconstructed esophagi in the RTV group revealed the arrangement of the different layers of the esophageal wall with the formation of newly formed blood vessels and Schwann-like cells. CONCLUSION: DTV xenograft is a novel scaffold that promotes cell adhesion and differentiation and might be effectively utilized for regenerating esophageal tissue, paving the way for future clinical trials in pediatric patients.

Tibial Nerve Repair in a Dog Model: Effect of Local and Systemic Administration of Erythropoietin.

PURPOSE: This study aimed to assess the effectiveness of erythropoietin (EPO) as a novel treatment for peripheral nerve injury after surgical repair of an induced tibial nerve injury in dogs. METHODS: Mongrel dogs (n = 27) were randomly divided into three equal groups. A complete tibial nerve injury was induced and repaired directly by stay sutures and the local application of 1 mL fibrin glue (control group). In the "systemic" group, 20,000 IU of EPO were given subcutaneously immediately after surgery and on the first and second days after surgery. In the "local" group, EPO was mixed with fibrin glue at 1,000 IU/mL. Lameness score, compound muscle action potential of the tibial nerve, and serum biochemical and histopathological examinations were performed to evaluate the treated dogs over the study period (12 weeks). RESULTS: EPO significantly improved the lameness score and compound muscle action potential in both the systemic and local groups. After 12 weeks, systemic and local groups showed earlier improvement in lameness, reaching scores of -1 and 0, respectively, in comparison with the control group, which did not reach a score of -1. The histological study revealed a normal architecture of the nerve bundles within connective tissue. The axons were aligned in a regular pattern, whereas the control group had disrupted and degenerated nerve axons with large gaps in between. CONCLUSIONS: EPO has an accelerating healing effect after tibial nerve surgical repair. Local EPO mimics systemic EPO treatment without systemic adverse effects. These findings indicated that EPO has a potential role in tibial nerve recovery and nerve regeneration. CLINICAL RELEVANCE: The findings of the present experimental study supported the beneficial effects of systemic and local EPO when combined with peripheral nerve surgical repair, potentially improving functional outcomes and enhancing faster recovery.

Calpain signaling: from biology to therapeutic opportunities in neurodegenerative disorders.

Neurodegenerative disorders represent a major and growing healthcare challenge globally. Among the numerous molecular pathways implicated in their pathogenesis, calpain signaling has emerged as a crucial player in neuronal dysfunction and cell death. Calpain is a family of calcium-dependent cysteine proteases that is involved in many biological processes, such as signal transduction, cytoskeleton remodeling, and protein turnover. Dysregulation of calpain activation and activity has been associated with several neurodegenerative diseases, including Alzheimer's, Parkinson's, and Huntington's diseases. Understanding the intricate structure of calpains is crucial for unraveling their roles in cellular physiology and their implications in pathology. In addition, the identification of diverse abnormalities in both humans and other animal models with deficiencies in calpain highlights the significant progress made in understanding calpain biology. In this comprehensive review, we delve into the recent roles attributed to calpains and provide an overview of the mechanisms that govern their activity during the progression of neurodegenerative diseases. The possibility of utilizing calpain inhibition as a potential therapeutic approach for treating neuronal dysfunctions in neurodegenerative disorders would be an area of interest in future calpain research.

Reconstruction of a partial esophageal defect using tunica vaginalis and buccal mucosa autograft: an experimental study in mongrel dogs.

In veterinary clinics, esophageal reconstruction is essential in many clinical situations. In this study, two autografts, the tunica vaginalis (TV) and the buccal mucosa (BM), were proposed to reconstruct a semi-circumferential cervical esophageal defect in dogs. This study aimed to verify whether these two grafts could successfully patch esophageal defects. Twelve male mongrel dogs were divided into two groups. Following cervical esophagoplasty, the defective area was patched with either a TV or a BM graft. Comprehensive clinical, serum biochemical, and histological analyses were performed to evaluate the two grafts. Throughout the study (120 days), the dogs survived the procedure well with minor complications. The lumen of the patched areas was covered with mucosa, with slight scar retraction. Compared with that of the natural esophagus, the average relative luminal diameter was not significantly decreased. Importantly, the measured cortisol and inflammatory marker levels returned to the preoperative levels after 14 days. Although histological examination revealed that both grafts repaired the esophageal defect with complete re-epithelialization, the BM graft showed a histological structure similar to that of the natural esophagus. Both grafts effectively repaired the esophageal defect with minor complications; therefore, both are recommended as promising low-cost clinical alternatives for cervical esophagoplasty in dogs.

Selective Calpain Inhibition Improves Functional and Histopathological Outcomes in a Canine Spinal Cord Injury Model.

Calpain activation has been implicated in various pathologies, including neurodegeneration. Thus, calpain inhibition could effectively prevent spinal cord injury (SCI) associated with neurodegeneration. In the current study, a dog SCI model was used to evaluate the therapeutic potential of a selective calpain inhibitor (PD150606) in combination with methylprednisolone sodium succinate (MPSS) as an anti-inflammatory drug. SCI was experimentally induced in sixteen mongrel dogs through an epidural balloon compression technique. The dogs were allocated randomly into four groups: control, MPSS, PD150606, and MPSS+PD150606. Clinical evaluation, serum biochemical, somatosensory evoked potentials, histopathological, and immunoblotting analyses were performed to assess treated dogs during the study. The current findings revealed that the combined administration of MPSS+PD150606 demonstrated considerably lower neuronal loss and microglial cell infiltration than the other groups, with a significant improvement in the locomotor score. The increased levels of inflammatory markers (GFAP and CD11) and calcium-binding proteins (Iba1 and S100) were significantly reduced in the combination group and to a lesser extent in MPSS or PD150606 treatment alone. Interestingly, the combined treatment effectively inhibited the calpain-induced cleavage of p35, limited cdk5 activation, and inhibited tau phosphorylation. These results suggest that early MPSS+PD150606 therapy after acute SCI may prevent subsequent neurodegeneration via calpain inhibition.

Enhancement of chronic wound healing with maltodextrin/ascorbic acid gel: a clinical evaluation of distal limb wounds in horses.

Delayed healing associated with distal limb wounds is highly challenging in equine clinical practice. This study aimed to evaluate healing rates between chronic non-granulating wounds of horse distal limbs that were treated with maltodextrin/ascorbic acid gel alone or in combination with povidone-iodine 1% solution and those treated with povidone-iodine 1% only throughout the study period (35 days) in clinical settings. The study was conducted on 18 adult horses (3-15 years old). Based on the treatment regimen utilized, the horses were divided into three groups (n = 6), with each group having a similar mean wound area. The percentages of wound contraction, epithelialization, and overall wound healing were determined weekly for each wound. By the end of the study, the total wound healing percentage was significantly increased between the study groups (p < 0.05). The use of maltodextrin/ascorbic acid gel resulted in considerable wound contraction, rapid epithelialization, and complication-free wound healing. Based on the findings of this study, maltodextrin/ascorbic acid gel, independently or in combination with a 1% povidone-iodine solution, might be applied as a safe and effective wound healing promoting agent in horses with chronic non-granulating wounds.

Augmentation cystoplasty in dogs: A comparative study of different tunica vaginalis grafts.

In veterinary practice, numerous urological disorders that cause bladder dysfunction necessitate augmentation cystoplasty (AC). The purpose of this study is to evaluate the dog tunica vaginalis allograft (DTVA), sheep tunica vaginalis xenograft (STVX) and sheep tunica vaginalis decellularized extracellular matrix (STVDEM) as graft materials for urinary bladder (UB) reconstruction following a 45±5% cystectomy model in dogs. In this study, 18 adult apparently healthy mongrel dogs of both sexes were divided into three groups (6 dogs each): the DTVA group, the STVX group, and the STVDEM group. The evaluation of the AC in different groups was carried out using clinical, hematological, serum biochemical, urine, ultrasonographic, retrograde positive cystogram, and histopathological analysis all over the study period of 12 weeks. The dogs in all groups survived the procedures, except three dogs died from both STVX and DTVA groups. The mean bladder capacity indicated that the DTVA and STVX groups had regained 82.22% and 68.62%, respectively, of their preoperative baseline capacity. Interestingly, the STVDEM group's bladder capacity increased to 113.70%. Although histological analysis revealed that the three grafts successfully rebuilt the bladder wall, the STVDEM demonstrated well-organized and well-differentiated epithelial and muscular tissues that resembled, but were not identical to, native UB tissues. As a result, STVDEM is proposed as an ideal and potential acellular graft for UB reconstruction in dogs, whereas DTVA and STVX could be employed in emergencies requiring UB reconstruction.

Exploring a Novel Fasudil-Phospholipid Complex Formulated as Liposomal Thermosensitive in situ Gel for Glaucoma.

PURPOSE: Fasudil hydrochloride (Fas), a rho-associated protein kinase inhibitor, proved to be promising for glaucoma management owing to its IOP lowering and antioxidant effects. However, its highly hydrophilic nature limits ocular permeation and bioavailability. Hence, the study objective was the development of Fas loaded vesicular system with high entrapment efficiency formulated as a thermosensitive gel for local administration aiming to enhance ocular retention and permeation and hence therapeutic efficacy. METHODS: Fasudil complex with phospholipid (Fas/PL) was prepared by solvent evaporation technique and characterized by Fourier transform infrared spectroscopy (FT-IR) and X-ray diffraction (XRD). Fas/PL was further formulated as liposomes by methanol injection method and characterized regarding colloidal properties, entrapment efficiency (EE%) and in vitro drug release. The prepared liposomes were incorporated into an optimized thermosensitive in situ gel (Fas/PL-LipoP407/HPMCgel) selected based on gelling time and temperature and rheological properties. The effect of incorporation into gel on the in vitro characteristics of liposomes was investigated. The in vitro mucoadhesive potential, ex vivo permeation, irritability and efficacy in a glaucoma rabbit model were also assessed. RESULTS: FT-IR and XRD suggested interactions between Fas and PL, proposing complexation. Fas/PL liposomal dispersions showed good colloidal properties (particle size: 132.5 ± 1.6 nm, zeta potential: -21.6 ± 0.9 and %EE 78.6 ± 0.3%) with sustained drug release. In situ thermosensitive gel (20% poloxamer 407 and 0.5% HPMC) showed optimum gelling properties. The selected gel formulation reduced burst release of the drug, enhanced mucoadhesive properties and prolonged corneal permeation ex vivo. HET-CAM test confirmed that the prepared formulations were non-irritant. In vivo pharmacodynamic study indicated improved bioavailability and significantly lower intraocular pressure (IOP) of Fas/PL-LipoP407/HPMC gel compared to drug solution and liposomal dispersion. CONCLUSION: The results present Fas/PL-LipoP407/HPMC gel as a potential platform for ophthalmic delivery of fasudil with improved pharmaceutical attributes and enhanced bioavailability and efficacy in glaucoma.

Tissue Harvesting Site Effect on the Canine Adipose Stromal Vascular Fraction Quantity and Quality.

Mesenchymal stem cells (MSCs) constitute a great promise for regenerative therapy, but these cells are difficultly recovered in large amounts. A potent alternative is the stromal vascular fraction (SVF), non-cultured MSCs, separated from adipose tissue (AT). We aim to evaluate AT harvesting site effect on the SVF cells' quantity and quality in dogs. Subcutaneous abdominal fat, falciform ligament and peri-ovarian fat were sampled. After SVF isolation, the trypan blue exclusion test and a hemocytometer were used to assess the cell viability and cellular yield. SVF cells were labeled for four surface antigenic markers, clusters of differentiation CD90, CD44, CD29, and CD45, and then examined by flow cytometry. Semi-quantitative RT-PCR was used to evaluate the gene expression of the former markers in addition to OCT-4 and CD34. SVF cells in the peri-ovarian AT recorded the highest viability% (99.63 ± 0.2%), as well as a significantly higher cellular yield (36.87 ± 19.6 × 106 viable cells/gm fat, p < 0.001) and a higher expression of adipose-derived mesenchymal stem cells AD-MSCs surface markers than that of other sites. SVF cells from the peri-ovarian site revealed a higher expression of MSC markers (CD90, CD44, and CD29) and OCT-4 compared to the other sites, with weak CD45 and CD34 expressions. The positive OCT-4 expression demonstrated the pluripotency of SVF cells isolated from different sites. To conclude, the harvesting site is a strong determinant of SVF cells' quantity and quality, and the peri-ovarian site could be the best AT sampling site in dogs.

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.

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.

NICD-mediated notch transduction regulates the different fate of chicken primordial germ cells and spermatogonial stem cells.

BACKGROUND: Notch signaling is mainly regulated by Notch1 during development of chicken germ stem cells; however, the molecular mechanisms that contribute to generation of these germ stem cells have not been thoroughly investigated. RESULTS: In our studies, Overexpression of the Notch1 NICD promoted development of the reproductive ridge, but inhibited the formation of seminiferous tubules. The formation efficiency of PGCs in the reproductive ridge following overexpression of NICD (7.5% ± 0.11) was significantly higher than that (4.9% ± 0.17, p < 0.05) following inhibition of NICD, While the formation efficiency of spermatogonial stem cells (SSCs) in the testes (12.7% ± 0.08) was significantly lower after NICD overexpression than that after inhibition of NICD (16.3% ± 0.16, p < 0.05). Using co-immunoprecipitation, we found that this anomaly stemmed from the reversal of dissociation of the Notch-regulated transcription factor CBF-1/RBP co-suppression complex during the differentiation of PGCs into SSCs. This dissociation of the CBF-1/RBP co-suppressing complex during the differentiation of ESCs into PGCs resulted in the release of HDAC1 and HDAC2 and the recruitment of mastermind-like 1 to form a coactive complex to promote the expression of the downstream transcription suppressor hairy/enhancer of split-1. Dynamic expression of transducin-like enhancer of split 3, TLE4, and C-terminal binding protein 2 during further differentiation of PGCs inhibited the dissociation of the CBF-1/RBP co-suppression complex and inhibited the expression of the downstream genes. CONCLUSIONS: In summary, Notch signaling plays diametrically opposing roles during normal development of chicken PGCs and SSCs, and these functions was determined by the expression of NICD, changes in the CBF-1/RBP complex composition, and histone modification.

Regulation of fibroblast growth factor 8 (FGF8) in chicken embryonic stem cells differentiation into spermatogonial stem cells.

Fibroblast growth factors (FGFs) are essential in regulating the formation of spermatogonial stem cells (SSCs). Here, we explored the effect of FGF8 on chicken SSCs formation by knockdown or overexpression of FGF8 in chicken embryonic stem cells (ESCs) both in vitro and in vivo. Our results showed that knockdown of FGF8 could facilitate the differentiation of ESCs into SSCs, overexpression of FGF8 could promote PGCs self-renewal, inhibit SSCs formation. This study further revealed the positive correlation between the expression level of FGF8 and MAPK/ERK signal. In the absence of FGF8, the expression of downstream genes such as FGFR2, GRB2, RAS, BRAF, RAF1, and MEK2 was not maintained, while overexpressing FGF8 enhances them. Thus, our study demonstrated that FGF8 can regulate germ cell fate by modulating the dynamic equilibrium between differentiation and self-renewal, which provides a new idea for the study of germ cell regulatory network.

Hsd3b2 associated in modulating steroid hormone synthesis pathway regulates the differentiation of chicken embryonic stem cells into spermatogonial stem cells.

Steroid hormones regulate differentiation of various types of cell during embryogenesis. Testosterone is one of the androgens that bind to receptors to regulate gene expression and promote spermatogenesis. Our results showed that testosterone, as a product of steroid hormones synthesis pathway, could facilitate the differentiation of embryonic stem cells (ESCs) into spermatogonial stem cells (SSCs). The analysis of the steroid hormones synthesis pathway demonstrated that 3beta-hydroxysteroid dehydrogenase2 (Hsd3b2) plays a major role in the synthesis of testosterone. In the absence of Hsd3b2, the expression of downstream genes such as Cyp1a1, Ugt1a1, and Hsd17b7 was not maintained. This reduction is probably due to the down-regulation of the steroid hormones synthesis pathway. Furthermore, qRT-PCR, immunofluorescence, and flow cytometry analysis confirmed that the steroid hormones synthesis pathway could facilitate the differentiation of ESCs. Altogether, these results lead to a model in which Hsd3b2 regulates ESCs differentiation via modulating the activity of steroid hormones synthesis pathway.

Regulation of Hedgehog Signaling in Chicken Embryonic Stem Cells Differentiation Into Male Germ Cells (Gallus).

The study aims to analyze the key signaling pathways in regulating the process of embryonic stem cells (ESCs) differentiation into spermatogonial stem cells (SSCs). Based on RNA Sequencing result, we further explored the specific regulating mechanisms of Hedgehog (HH) signaling in this process. HH signaling was found to be a crucial signaling pathway participating in the differentiation process of ESCs to SSCs. In Retinoic acid (RA) induced in vitro differentiation assay, the expression of two germ cell marker genes, integrin α6, and integrin ß1, was observed to significantly increase, while it decreased dramatically when IHH was knocked down. Fluorescence activated cell sorting analysis showed that the proportion of integrin α6+ and integrin ß1+ cells in the RA group was significantly higher than that in the RA + siRNA- Indian Hedgehog (IHH) group. In in vivo situations, siRNA-IHH could stably express in fertilized chicken embryos and significantly down-regulate the IHH expression. With real-time quantitative PCR and western blot, we identified that integrin α6 and integrin ß1 expression was significantly suppressed along with IHH inhibition in vivo. We concluded that Hedgehog signaling pathway positively regulates the differentiation of ESCs to male germ cells through signal transduction by IHH. J. Cell. Biochem. 118: 1379-1386, 2017. © 2016 Wiley Periodicals, Inc.

Basing RNA-seq explored the regulatory mechanism of the carbohydrate metabolism pathways during chicken male germ cell differentiation.

Our study aimed to explore the regulatory mechanism of the carbohydrate metabolism signaling pathways and related genes during the differentiation of chicken embryonic stem cells to male germ cells, providing the basis for improving the efficiency of the in vitro induction system. Cell sorting was used to obtain highly purified embryonic stem cells (ESCs), primitive germ cells (PGCs), and spermatogonial stem cells (SSCs). The total RNA was then extracted from each cell type. The transcriptions of ESCs, PGCs, and SSCs were sequenced by DNA microarray and mRNA sequencing (RNA-seq). The results were analyzed by gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway database. The key pathways and genes of carbohydrate metabolism were screened during the differentiation process of chicken male germ cell. We concluded that 419 differentially expressed genes enriched to 26 carbohydrate metabolism pathways during the differentiation process of ESCs to SSCs, all of the chondroitin sulfate (CS) signaling pathway was significant. We screened the key genes CHSY3, B3GAT1, CHPF, and B4GALT7 which was significantly expressed in CS pathway. Quantitative RT-PCR showed that the expression trend of these genes is consistent with DNA Microarray and RNA-seq results. Our study supports the opinion that CS pathway is significantly different during the differentiation of chicken male germ cell (P < 0.05) and that CHSY3, B3GAT1, CHPF, and B4GALT7 are key genes.

Regulatory mechanism of protein metabolic pathway during the differentiation process of chicken male germ cell.

We explored the regulatory mechanism of protein metabolism during the differentiation process of chicken male germ cells and provide a basis for improving the induction system of embryonic stem cell differentiation to male germ cells in vitro. We sequenced the transcriptome of embryonic stem cells, primordial germ cells, and spermatogonial stem cells with RNA sequencing (RNA-Seq), bioinformatics analysis methods, and detection of the key genes by quantitative reverse transcription PCR (qRT-PCR). Finally, we found 16 amino acid metabolic pathways enriched in the biological metabolism during the differentiation process of embryonic stem cells to primordial germ cells and 15 amino acid metabolic pathways enriched in the differentiation stage of primordial germ cells to spermatogonial stem cells. We found three pathways, arginine-proline metabolic pathway, tyrosine metabolic pathway, and tryptophan metabolic pathway, significantly enriched in the whole differentiation process of embryonic stem cells to spermatogonial stem cells. Moreover, for these three pathways, we screened key genes such as NOS2, ADC, FAH, and IDO. qRT-PCR results showed that the expression trend of these genes were the same to RNA-Seq. Our findings showed that the three pathways and these key genes play an important role in the differentiation process of embryonic stem cells to male germ cells. These results provide basic information for improving the induction system of embryonic stem cell differentiation to male germ cells in vitro.