Generation and characterization of giant panda induced pluripotent stem cells.

The giant panda (Ailuropoda melanoleuca) stands as a flagship and umbrella species, symbolizing global biodiversity. While traditional assisted reproductive technology faces constraints in safeguarding the genetic diversity of giant pandas, induced pluripotent stem cells (iPSCs) known for their capacity to differentiate into diverse cells types, including germ cells, present a transformative potential for conservation of endangered animals. In this study, primary fibroblast cells were isolated from the giant panda, and giant panda iPSCs (GPiPSCs) were generated using a non-integrating episomal vector reprogramming method. Characterization of these GPiPSCs revealed their state of primed pluripotency and demonstrated their potential for differentiation. Furthermore, we innovatively formulated a species-specific chemically defined FACL medium and unraveled the intricate signaling pathway networks responsible for maintaining the pluripotency and fostering cell proliferation of GPiPSCs. This study provides key insights into rare species iPSCs, offering materials for panda characteristics research and laying the groundwork for in vitro giant panda gamete generation, potentially aiding endangered species conservation.

Intestinal acetic acid regulates the synthesis of sex pheromones in captive giant pandas.

As a typical solitary animal, adult giant pandas rely on chemical signals (sex pheromones) to transmit reproductive information during oestrous. Although researchers have confirmed that the gut microbiota is related to the emission and reception of sex pheromones, there is no clear correlation between the gut microbes and the synthesis of sex pheromone of giant pandas, that is, which gut microbes and microbial metabolites are participate in the synthesis of giant panda's sex pheromone. As a mirror of gut microbiota, fecal microbiota can reflect the composition of gut microbiota and its interaction with host to some extent. The purpose of this study is to explore how the gut microbes affect the synthesis of sex pheromones in captive giant pandas by combining analysis of the fecal microbiome and metabolomics. The results of correlation and microbial function analysis show that intestinal microorganisms such as Veillonellaceae and Lactobacillilaceae are associated with the synthesis of short chain fatty acid (acetic acid) and volatile ester metabolites, such as 1-butanol, 3-methyl, acetate, acetic acid, hexyl ester and 3-hexen-1-ol, acetate, (Z). In summary, based on this study, we believe that volatile metabolites such as fecal acetate participate in the process of mate preference of captive giant pandas and affect their expression of natural mating behavior. The possible mechanism is that the gut microbes can promote the synthesis of key chemical signaling substances in perianal glands through mediated intermediate fecal metabolites, thus affecting the normal information exchange between giant pandas individuals. The results of this study have greatly enriched our understanding of gut microbes regulating the synthesis of sex pheromones in giant pandas.

Characteristics of mesenchymal stem cells and their exosomes derived from giant panda (Ailuropoda melanoleuca) endometrium.

Conservation of genetic resources is an important way to protect endangered species. At present, mesenchymal stem cells (MSCs) have been isolated from the bone marrow and umbilical cords of giant pandas. However, the types and quantities of preserved cell resources were rare and limited, and none of MSCs was derived from female reproductive organs. Here, we first isolated MSCs from the endometrium of giant panda. These cells showed fibroblast morphology and expressed Sox2, Klf4, Thy1, CD73, CD105, CD44, CD49f, and CD105. Endometrium mesenchymal stem cells (eMSCs) of giant panda could induce differentiation into three germ layers in vitro. RNA-seq analysis showed that 833 genes were upregulated and 716 genes were downregulated in eMSCs compared with skin fibroblast cells. The results of GO and the KEGG analysis of differentially expressed genes (DEGs) were mainly focused on transporter activity, signal transducer activity, pathways regulating pluripotency of stem cells, MAPK signaling pathway, and PI3K-Akt signaling pathway. The genes PLCG2, FRK, JAK3, LYN, PIK3CB, JAK2, CBLB, and MET were identified as hub genes by PPI network analysis. In addition, the exosomes of eMSCs were also isolated and identified. The average diameter of exosomes was 74.26 ± 13.75 nm and highly expressed TSG101 and CD9 but did not express CALNEXIN. A total of 277 miRNAs were detected in the exosomes; the highest expression of miRNA was the has-miR-21-5p. A total of 14461 target genes of the whole miRNAs were predicted and proceeded with functional analysis. In conclusion, we successfully isolated and characterized the giant panda eMSCs and their exosomes, and analyzed their functions through bioinformatics techniques. It not only enriched the conservation types of giant panda cell resources and promoted the protection of genetic diversity, but also laid a foundation for the application of eMSCs and exosomes in the disease treatment of giant pandas.

Possible Effects of Early Maternal Separation on the Gut Microbiota of Captive Adult Giant Pandas.

Maternal deprivation (MD) in early life induces dysbiosis in the host gut microbiota, which is a key determinant of abnormal behavior in stress model individuals. Compared with the early parenting environment of the wild, captive giant pandas face frequent and premature maternal separation. Will this lead to imbalance in intestinal flora and stress in captive giant pandas? The purpose of this research is to evaluate the possible adverse effects of the traditional parenting mode on the gut microbiota of captive giant pandas. The results showed that the frequent and premature maternal separation at early stages of the young did not change α and ß diversity indices of the gut microbes, but it increased the relative abundance of s_Clostridium_tetani and s_Clostridium_sp_MSJ_8 (significantly positively correlated with the metabolism of propionic acid) and also the concentrations of fecal metabolites that are related to stress (N-acetyl-l-aspartic acid and corticosterone) in the intestinal tract of giant pandas in adulthood. Thereby, the function of protein digestion and absorption in the intestines of captive giant pandas was decreased, and the metabolism of short-chain fatty acids was disturbed. In conclusion, the parenting experience of early maternal separation could adversely affect the stress caused by the unfavorable parenting environment in the early life of captive giant pandas related to the gut microbiota of the captive giant pandas in adulthood.

Comparison of transcriptome profiles of mesenchymal stem cells derived from umbilical cord and bone marrow of giant panda (Ailuropoda melanoleuca).

Mesenchymal stem cells (MSCs) have pluripotent differentiation ability and play an important role in human clinical cell therapy. While, the research on MSCs in endangered wild animals is extremely rare. In our previous studies, the bone marrow mesenchymal stem cells (bmMSCs) and umbilical cord mesenchymal stem cells (ucMSCs) of giant panda (Ailuropoda melanoleuca) were successfully isolated. We aimed to characterize the differences in gene expression profiles between these two types of MSCs using RNA sequencing (RNA-Seq) and to determine which potential pathways are involved in functional regulation. In total, 1079 significantly differentially expressed genes (DEGs) were identified, of which 478 genes were upregulated and 601 genes were downregulated. The significantly enriched Gene Ontology (GO) terms mainly contained cell adhesion, biological adhesion, intracellular signal transduction, molecular function regulator, Ras protein signal transduction, small GTPase mediated signal transduction, and regulation of Rho protein signal transduction. The most enrichment pathways of DEGs enriched in Kyoto Encyclopedia of Genes Genomes (KEGG) were PI3K-AKT signaling pathway, Rap1 signaling pathway, MAPK signaling pathway, Hippo signaling pathway, Wnt signaling pathway, cGMP-PKG signaling pathway and Signaling pathways regulating pluripotency of stem cells. In addition, quantitative real time polymerase chain reaction (qRT-PCR) showed that the AKT3, CDK2, MAPK3, mTOR, PI3K and PTK2 genes associated with PI3K-AKT pathway were highly expressed (P < 0.01), and Caspase-3 was low expressed (P < 0.05) in ucMSCs group when compared with bmMSCs. After treatment with the PI3K inhibitor LY294002, genes AKT3, CDK2, MAPK3, mTOR, and PTK2 were significantly decreased in ucMSCs (P < 0.01), and Caspase-3 was significantly up regulated (P < 0.001). In conclusion, we for the first time compared and analyzed the transcriptome profiles of giant panda ucMSCs and bmMSCs, and found the PI3K-AKT pathway was highly activated and might be a key signaling pathway in the ucMSCs regulation. This study will be beneficial for the research on MSCs proliferation regulation and differentiation of giant pandas in the future, and lay the foundation for MSCs application and clinical therapy for endangered wild animals.

Combined urine metabolomics and 16S rDNA sequencing analyses reveals physiological mechanism underlying decline in natural mating behavior of captive giant pandas.

The decline in natural mating behavior is the primary reason underlying in the poor population growth of captive giant pandas. However, the influencing factors and underlying mechanisms remain unclear to data. It is speculated that the decline in natural mating behavior could be related to the psychological stress caused by captivity, which restricts their free choice of mates. In order to test this hypothesis, we performed urinary metabolomics analysis using Ultra-High-Performance Liquid Chromatography-Mass Spectrometry (UHPLC/-MS) combined with 16S rDNA sequencing for exploring the physiological mechanism underlying the decline in the natural mating behavior of captive giant panda. The results demonstrated that the decline in mating ability could be related to abnormalities in arginine biosynthesis and neurotransmitter synthesis. Additionally, the relative abundance of bacteria from the Firmicutes, Proteobacteria, and Actinobacteria phyla and the Acinetobacter, Weissella, and Pseudomonas genus was significantly reduced in the group with low natural mating behavior. These findings imply that the inhibition of arginine synthesis induced by environmental changes could be related to the poor libido and failure of mate selection in captive giant pandas during the breeding period. The results also demonstrate the relationship between the altered urinary microbes and metabolites related to arginine and neurotransmitter synthesis. These findings may aid in understanding the mechanism underlying environment-induced mate selection in captive giant pandas and propose a novel strategy for determining the sexual desire of giant pandas based on urinary microbes. The method would be of great significance in improving the natural reproductive success rate of captive giant pandas.

Single-cell RNA-sequencing analysis and characterisation of testicular cells in giant panda (Ailuropoda melanoleuca).

CONTEXT: The giant panda (Ailuropoda melanoleuca ) is a rare and endangered species to be preserved in China. The giant panda has a low reproductive capacity, and due to the scarcity of samples, studies on testes from giant panda are very limited, with little knowledge about the process of spermatogenesis in this species. AIMS: To establish the gene expression profiles in cells from the testis of a giant panda. METHODS: The 10×Genomics single-cell RNA-sequencing platform was applied to cells from the testis of an adult giant panda. KEY RESULTS: We identified eight testicular cell types including six somatic and two germ cell types from our single-cell RNA-sequencing datasets. We also identified the differentially expressed genes (DEGs) in each cell type, and performed functional enrichment analysis for the identified testicular cell types. Furthermore, by immunohistochemistry we explored the protein localisation patterns of several marker genes in testes from giant panda. CONCLUSIONS: Our study has for the first time established the gene expression profiles in cells from the testis of a giant panda. IMPLICATIONS: Our data provide a reference catalogue for spermatogenesis and testicular cells in the giant panda, laying the foundation for future breeding and preservation of this endangered species.

Isolation and characterization mesenchymal stem cells from red panda (Ailurus fulgens styani) endometrium.

Endometrial mesenchymal stem cells (eMSCs) are undifferentiated endometrial cells with self-renewal, multidirectional differentiation and high proliferation potential. Nowadays, eMSCs have been found in a few species, but it has never been reported in endangered wild animals, especially the red panda. In this study, we successfully isolated and characterized the eMSCs derived from red panda. Red panda eMSCs were fibroblast-like, had a strong proliferative potential and a stable chromosome number. Pluripotency genes including Klf4, Sox2 and Thy1 were highly expressed in eMSCs. Besides, cultured eMSCs were positive for MSC markers CD44, CD49f and CD105 and negative for endothelial cell marker CD31 and haematopoietic cell marker CD34. Moreover, no reference RNA-seq was used to analyse the eMSCs transcriptional expression profile and key pathways. Compared with skin fibroblast cell group, 9104 differentially expressed genes (DEGs) were identified, among which are 5034 genes upregulated, 4070 genes downregulated and the top 20 enrichment pathways of DEGs in Gene Ontology (GO) and the Kyoto Encyclopedia of Genes Genomes (KEGG) mainly associated with G-protein coupled receptor signalling pathway, carbohydrate derivative binding, nucleoside binding, ribosome biogenesis, cell cycle, DNA replication, Ras signalling pathway and purine metabolism. Among the DEGs, some representative genes about promoting MSCs differentiation and proliferation were upregulated and promoting fibroblasts proliferation were downregulated in eMSCs group. Red panda eMSCs also had multiple differentiation ability and could differentiate into adipocytes, chondrocytes and hepatocytes. In conclusion, we, for the first time, isolated and characterized the red panda eMSCs with ability of multiplication and multilineage differentiation in vitro. The new multipotential stem cell could be beneficial not only for the germ plasm resources conservation of red panda, but also for basic or pre-clinical studies in the future.

Contrasting Vaginal Bacterial Communities Between Estrus and Non-estrus of Giant Pandas (Ailuropoda melanoleuca).

Bacterial infection and imbalance of bacterial community in the genitourinary system of giant panda could affect the reproductive health. In severe cases, it can also lead to abortion. In this study, 13 of vaginal secretions in the estrue (E) group and seven of vaginal secretions in the non-estrue (NE) group were used to study the composition and diversity of vaginal bacterial communities between estrus and non-estrus by 16S rRNA gene sequencing analysis. The results showed that the vaginal microbiome in giant pandas shared the same top five abundant species between estrus and non-estrus at the phylum level. However, the vaginal microbiome changed significantly during estrus at the genus level. In top 10 genera, the abundance of Escherichia, Streptococcus, and Bacteroides in the E group was significantly higher than that in the NE group (p<0.05); Azomonas, Porphyromonas, Prevotella, Campylobacter, and Peptoniphilus in the NE group was significantly higher than that in the E group (p<0.05). The richness and diversity of vaginal microbiome in giant panda on estrus were significantly lower than those on non-estrus (p<0.05). It is noteworthy that the abundance of Streptococcus, Escherichia, and Bacteroides of vagina in giant pandas maintained low abundance in the daily. Whereas, they increased significantly during estrus period, which may play an important role in female giant pandas during estrus period. It was hypothesized that hormones may be responsible for the changes in the vaginal microbiome of giant pandas between estrus and no-estrus stages.

Isolation and characterization of mesenchymal stem cells from umbilical cord of giant panda.

Umbilical cord-derived mesenchymal stem cells (UC-MSCs) constitute a class of cells with significant self-renewal and multilineage differentiation properties and have great potential for therapeutic applications and the genetic conservation of endangered animals. In this study, we successfully isolated and cultured UC-MSCs from the blood vessels of giant panda umbilical cord (UC). The cells were arranged in a vortex or cluster pattern and exhibited a normal karyotype, showing the morphological characteristics of fibroblasts. In addition, we found that basic fibroblast growth factor (bFGF) and epidermal growth factor (EGF) promoted cell proliferation, whereas stem cell factor (SCF) did not promote cell proliferation. Cultured UC-MSCs were negative for CD34 (hematopoietic stem cell marker) and CD31 (endothelial cell marker), but positive for MSC markers (CD44, CD49f, CD105, and CD73) and stem cell markers (KLF4, SOX2, and THY1). Similar to other MSCs, giant panda UC-MSCs have multiple differentiation ability and can differentiate into adipocytes, osteoblasts and chondrocytes. Giant panda UC-MSCs are new resources for basic research as cell models following their differentiation into different cell types and for future clinical treatments of giant panda diseases.

Characteristics of Mesenchymal Stem Cells Isolated from the Bone Marrow of Red Pandas.

Mesenchymal stem cells (MSC) have strong therapeutic potential due to their capacity for self-renewal and multilineage differentiation. MSCs can also be useful in preserving the current genetic diversity of endangered wildlife. To date, MSCs from various species have been studied, but only a few species of endangered wild animals have been reported. Adult bone marrow (BM) is a rich source of mesenchymal stem cells. The aim of this study was to isolate and characterize MSCs derived from the BM of red pandas. Red panda BM-MSCs isolated from five individuals were fibroblast-like cells, similar to other species. Cultured BM-MSCs with normal karyotype were negative for the hematopoietic line marker CD34 and the endothelial cell marker CD31 but were positive for MSC markers, including CD44, CD105 and CD90. RT-PCR and western blot analysis showed self-renewal and pluripotency genes, including Oct4, Sox2 and Klf4, were also expressed in red panda BM-MSCs. Finally, red panda BM-MSCs had the potential for differentiation into osteogenic, adipogenic and neuron-like cells by using a combination of previously reported protocols for other species. We have therefore demonstrated that cells harvested from red panda bone marrow are capable of extensive in vitro multiplication and multilineage differentiation, which is an essential step toward their use in the preservation of red pandas biological diversity and future studies on MSC applications in endangered species.

Effects of extender type on the quality of post-thaw giant panda (Ailuropoda melanoleuca) semen.

Sperm cryopreservation is an essential approach for assisted reproduction and genetic resources conservation in captive giant pandas. Cryopreservation, however, leads to a significant decrease in sperm quality and, consequently, a low fertilization rate. Therefore, it is mandatory to disclose more suitable and efficient freezing strategies for sperm cryopreservation. In the present study, we compared for the first time the performance of two commercial freeze extender (INRA96 versus TEST) freezing methods on post-thawed semen quality. Semen cryopreserved with the INRA96 showed better total motility (73.00 ± 4.84% vs 57.56 ± 3.60%, P < 0.001), membrane integrity (60.92 ± 2.27% vs 40.53 ± 2.97%, P < 0.001) and acrosome integrity (90.39 ± 2.74% vs 84.26 ± 4.27%, P < 0.05) than stored with TEST. There was no significant difference in DNA integrity after thawing between the two extenders (95.69 ± 3.60% vs 94.26 ± 4.84%). In conclusion, the INRA96 method showed to be better for giant panda sperm cryopreservation and should therefore be recommended for use in order to increase success of artificial insemination.

Mesenchymal to Epithelial Transition Mediated by CDH1 Promotes Spontaneous Reprogramming of Male Germline Stem Cells to Pluripotency.

Cultured spermatogonial stem cells (GSCs) can spontaneously form pluripotent cells in certain culture conditions. However, GSC reprogramming is a rare event that is largely unexplained. We show GSCs have high expression of mesenchymal to epithelial transition (MET) suppressors resulting in a developmental barrier inhibiting GSC reprogramming. Either increasing OCT4 or repressing transforming growth factor ß (TGF-ß) signaling promotes GSC reprogramming by upregulating CDH1 and boosting MET. Reducing ZEB1 also enhances GSC reprogramming through its direct effect on CDH1. RNA sequencing shows that rare GSCs, identified as CDH1+ after trypsin digestion, are epithelial-like cells. CDH1+ GSCs exhibit enhanced reprogramming and become more prevalent during the course of reprogramming. Our results provide a mechanistic explanation for the spontaneous emergence of pluripotent cells from GSC cultures; namely, rare GSCs upregulate CDH1 and initiate MET, processes normally kept in check by ZEB1 and TGF-ß signaling, thereby ensuring germ cells are protected from aberrant acquisition of pluripotency.

Ectopic POU5F1 in the male germ lineage disrupts differentiation and spermatogenesis in mice.

Expression levels of the pluripotency determinant, POU5F1, are tightly regulated to ensure appropriate differentiation during early embryogenesis. POU5F1 is also present in the spermatogonial stem cell/progenitor cell population in mice and it is downregulated as spermatogenesis progresses. To test if POU5F1 downregulation is required for SSCs to differentiate, we produced transgenic mice that ubiquitously express POU5F1 in Cre-expressing lineages. Using a Vasa-Cre driver to produce ectopic POU5F1 in all postnatal germ cells, we found that POU5F1 downregulation was necessary for spermatogonial expansion during the first wave of spermatogenesis and for the production of differentiated spermatogonia capable of undergoing meiosis. In contrast, undifferentiated spermatogonia were maintained throughout adulthood, consistent with a normal presence of POU5F1 in these cells. The results suggest that POU5F1 downregulation in differentiating spermatogonia is a necessary step for the progression of spermatogenesis. Further, the creation of a transgenic mouse model for conditional ectopic expression of POU5F1 may be a useful resource for studies of POU5F1 in other cell lineages, during tumorogenesis and cell fate reprogramming.

Histone lysine methylation exhibits a distinct distribution during spermatogenesis in pigs.

Spermatogenesis is a continual process throughout the adult life of a male, which is governed by unique transcriptional regulation and massive alterations of chromatin. Histone modification was one of the underlying epigenetic mechanisms during spermatogenesis. It has been shown that methylation of histone lysine exhibits a distinct distribution in mice during spermatogenesis and some histone lysine methylation is essential for male fertility. However, the dynamic change of methylated histone in porcine testis tissue was largely unknown. Here, we studied the dynamic modulation of three types of methylation (monomethylation, dimethylation, and trimethylation) of H3K4, H3K27, and H4K20 during spermatogenesis in pigs. The results showed that H3K4me2/3, H3K27me3, and H4K20me1/2/3 were extensively localized in adult pig testis. Interestingly, we found that undifferentiated spermatogonia contained strongly H4K20me2 and H4K20me3, but little H4K20me1, whereas the differentiated spermatogonia possessed H4K20me1 and H4K20me2 and little H4K20me3. The findings of this study help for the understanding of epigenetic modifications during spermatogenesis in pigs and provide information for further studies.

THY1 is a surface marker of porcine gonocytes.

Gonocytes are important for the study of spermatogenesis. Identification and isolation of gonocytes has been reported in rodents but not in pigs due to a lack of molecular markers for gonocytes. The objective of this study was to identify THY1 expression in porcine testicular tissue and subsequently utilise THY1 as a marker to isolate and enrich porcine gonocytes from testes of newborn piglets. Immunohistochemical analysis showed that THY1 was expressed in gonocytes. Double-immunofluorescent analysis of THY1 and ZBTB16 indicated that THY1 and ZBTB16 were partially co-localised in gonocytes. Double-immunofluorescent analysis of both THY1 and GATA4 suggested that THY1(+) cells were not Sertoli cells. Magnetic-activated cell sorting of THY1(+) cells yielded a cell population with an enrichment of UCHL1(+) gonocytes 3.4-fold of that of the unsorted testicular cell population. Western blot and quantitative reverse transcription-polymerase chain reaction analyses confirmed that the selected THY1(+) fraction had a higher expression of UCHL1 than the unsorted cells. In conclusion, the study demonstrated that THY1 is a surface marker of gonocytes in testes of pre-pubertal boars and could be utilised to identify and isolate porcine gonocytes. The findings will also facilitate culture and manipulation of male germline stem cells.

In vitro propagation of male germline stem cells from piglets.

PURPOSE: To study the effects of serum and growth factors on propagation of porcine male germline stem cells (MGSCs) in vitro and develop a culture system for these stem cells. METHODS: Fresh testicular cells from neonatal piglets were obtained by mechanical dissociation and collagenase-trypsin digestion. After differential plating, non-adhering cells were cultured in media supplemented with different concentrations of serum (0, 1 %, 2 %, 5 %, 10 %). After 10 days of primary culture, the cells were maintained in media supplemented with different concentrations of growth factors (basic fibroblast growth factor and epidermal growth factor at 1, 5, 10 ng/ml). The number of MGSC-derived colonies with different sizes was determined in each treatment to assess the effects of serum concentrations and growth factors. RESULTS: The number of MGSC-derived colonies was significantly higher in the presence of 1 % rather than 10 % fetal bovine serum (FBS). Basic fibroblast growth factor (bFGF) at 1, 5 ng/ml and epidermal growth factor (EGF) at 5, 10 ng/ml significantly promoted colony formation. Immunocytochemistry, reverse transcriptase-polymerase chain reaction (RT-PCR) and xenotransplantation assays demonstrated the presence of functional stem cells in cultured cell population. CONCLUSIONS: In vitro propagation of porcine MGSCs could be maintained in the presence of 1 % FBS and supplementation of growth factors for 1 month.