SSR Marker Acquisition and Application from Transcriptome of Captive Chinese Forest Musk Deer (Moschus berezovskii).

Forest musk deer (Moschus berezovskii) is one of the most endangered medicinally important wild animals in the world. Forest musk deer farming is the main way of production of musk. However, the single provenance and lack of genetic information lead to reduced genetic diversity of forest musk deer. Therefore, more SSR markers need to be developed to identify forest musk deer germplasm. In this study, bone marrow derived mesenchymal cells were used to construct cDNA library for transcriptome sequencing. The datasets were de novo assembled and annotated. 9 polymorphic simple sequence repeat (SSR) markers were finally identified and used to detect population genetic diversity. 6.07 Gb clean data were generated using Illumina sequencing technology, and de novo assembled into 138,591 transcripts and 81,553 unigenes. 5,777 simple sequence repeats (SSRs) were identified, in which there were 578 repeating motif types, with mono-nucleotide and tri-nucleotides comprising 55.88% and 25.60%, respectively. 100 primer pairs were designed to validate amplification and polymorphism using DNA from fecal samples. 9 polymorphic SSRs were developed and used to detect population genetic diversity of 122 forest musk deer in 2 farms. The average number of alleles per locus varied from 4 to 15 (average = 8.3). The observed heterozygosity (HO) per locus ranged from 0.102 to 0.941, while the expected heterozygosity (HE) per locus was from 0.111 to 0.651. All loci deviated significantly from the Hardy-Weinberg equilibrium (p < 0.001). The polymorphism information content (PIC) of these loci varied from 0.108 to 0.619. 9 polymorphic SSR markers were developed in this research. These sites can be used for breeding planning and conservation of germplasm resources.

Embryonic Development of Parthenogenetic and Sexual Eggs in Lower Termites.

Worldwide, termites are one of few social insects. In this research, the stages of embryonic development in the parthenogenetic and sexual eggs of Reticulitermes aculabialis and R. flaviceps were observed and described. In R. flaviceps, the egg development of the FF and FM groups happened during the early phases of development, whereas in R. aculabialis, this appeared mainly during the late phase of development. The variance in the number of micropyles between the R. flaviceps FF colony type and the R. aculabialis FF colony type was statistically significant. Five stages of egg development were found in both types of R. aculabialis but only the sexual eggs of R. flaviceps. In R. flaviceps, 86% of the parthenogenetic eggs stopped growing during the blastoderm development, with the yolk cell assembling frequently in the center of the egg. According to the results of the single-cell transcriptome sequencing, we investigated the egg-to-larval expression level of genes (pka, map2k1, mapk1/3, hgk, mkp, and pax6) and indicated that the levels of essential gene expression in RaFF were considerably higher than in RfFF (p < 0.05). We also discovered that the oocyte cleavage rate in the FF colony type was considerably lower in R. flaviceps compared to R. aculabialis, which gave rise to a smaller number of mature oocytes in R. flaviceps. During ovulation in both species, oocytes underwent activation and one or two cleavage events, but the development of unfertilized eggs ceased in R. flaviceps. It was shown that termite oocyte and embryonic development were heavily influenced by genes with significant expressions. Results from the databases KEGG, COG, and GO unigenes revealed the control of numerous biological processes. This study is the first to complete a database of parthenogenetic and sexual eggs of R. flaviceps and R. aculabialis.

High-quality genome assembly and comparative genomic profiling of yellowhorn (Xanthoceras sorbifolia) revealed environmental adaptation footprints and seed oil contents variations.

Yellowhorn (Xanthoceras sorbifolia) is a species of deciduous tree that is native to Northern and Central China, including Loess Plateau. The yellowhorn tree is a hardy plant, tolerating a wide range of growing conditions, and is often grown for ornamental purposes in parks, gardens, and other landscaped areas. The seeds of yellowhorn are edible and contain rich oil and fatty acid contents, making it an ideal plant for oil production. However, the mechanism of its ability to adapt to extreme environments and the genetic basis of oil synthesis remains to be elucidated. In this study, we reported a high-quality and near gap-less yellowhorn genome assembly, containing the highest genome continuity with a contig N50 of 32.5 Mb. Comparative genomics analysis showed that 1,237 and 231 gene families under expansion and the yellowhorn-specific gene family NB-ARC were enriched in photosynthesis and root cap development, which may contribute to the environmental adaption and abiotic stress resistance of yellowhorn. A 3-ketoacyl-CoA thiolase (KAT) gene (Xso_LG02_00600) was identified under positive selection, which may be associated with variations of seed oil content among different yellowhorn cultivars. This study provided insights into environmental adaptation and seed oil content variations of yellowhorn to accelerate its genetic improvement.

Isolation and Identification of Bone Marrow Mesenchymal Stem Cells from Forest Musk Deer.

The forest musk deer (Moschus berezovskii) is an endangered animal that produces musk that is utilized for medical applications worldwide, and this species primarily lives in China. Animal-derived musk can be employed as an important ingredient in Chinese medicine. To investigate the properties of bone marrow mesenchymal stem cells (MSCs) obtained from the bone marrow of forest deer for future application, MSCs were isolated and cultivated in vitro. The properties and differentiation of these cells were assessed at the cellular and gene levels. The results show that 81,533 expressed genes were detected by RNA sequencing, and marker genes of MSCs were expressed in the cells. Karyotype analysis of the cells determined the karyotype to be normal, and marker proteins of MSCs were observed to be expressed in the cell membranes. Cells were differentiated into osteoblasts, adipocytes, and chondroblasts. The expression of genes related to osteoblasts, adipocytes, and chondroblasts was observed to be increased. The results of this study demonstrate that the properties of the cells isolated from bone marrow were in keeping with the characteristics of MSCs, providing a possible basis for future research.

Effects of small-molecule compounds on fibroblast properties in golden snub-nosed monkey (Rhinopithecus roxellana).

BACKGROUND: Golden snub-nosed monkey (Rhinopithecus roxellana) is an endangered primate species, whose molecular material for conservation purposes has not yet been maintained. Although small-molecule compounds (SMCs) have been reported to improve induced pluripotent stem cells (iPSCs), their efficiency in the interspecies-transferred nucleus is still unknown. METHODS: We thus used the fibroblasts from the golden snub-nosed monkey treated with SMC as donor cells, injected into the enucleated oocytes of goats, to test such efficiency. Gene expression profiles in the cell-constructed embryos with and without SMCs were compared by qPCR. RESULTS: The results show that cell morphology undergoes remarkable changes (volume is smaller than normal cells, and many black spots in the cytoplasm were found); pluripotent genes (Oct4, Sox2, and Nanog) significantly increased with SMC treatment. CONCLUSIONS: This study demonstrates that SMCs alter the properties of donor cells and promote the expression of pluripotent genes in hybrid embryos.

Effect of bFGF on fibroblasts derived from the golden snub-nosed monkey.

Golden snub-nosed monkeys are endangered animals in China, and their cells have been demonstrated to be important as genetic resources and in applications for advancing biological research. Moreover, in primary research, basic fibroblast growth factor (bFGF) is used to promote the proliferation of fibroblasts to create abundant cells for cryopreservation. To further investigate the effect of bFGF on the efficiency of preservation of fibroblasts obtained from an endangered species, a fibroblast cell line was isolated from a dead golden snub-nosed monkey. Cell viability and mitochondrial membrane potential were assessed using CCK8 and JC-1 assay kits. The karyotype was analyzed by chromosomal microarray analysis, while RNA sequencing and gene expression analyses were performed to assess molecular changes in response to bFGF. Flow cytometry was used to characterize changes in cell surface markers in response to bFGF treatment. The results showed that cells maintained typical fibroblast morphology, while cell viability and mitochondrial membrane potential were not significantly affected between three and eight passages (p > 0.05). We also observed that the addition of bFGF promoted fibroblast proliferation and increased mitochondrial membrane potential. In addition, the bFGF treatment did not alter the normal karyotype of cells, downregulating fibroblast-associated genes and upregulating those associated with cell regulation, including those of the WNT, PI3K and MAPK pathways. The addition of bFGF also increased CD29, CD90, CD105, CD34 and CD44 expression while decreasing that of CD14 and HLA-DR at the protein level. Taken together, these results demonstrate that bFGF may upregulate the WNT, PI3K and MAPK pathways to promote cell proliferation while also increasing the expression of genes and surface markers associated with mesenchymal and hematopoietic cell linages.

Physiological and transcriptomic analyses of yellow horn (Xanthoceras sorbifolia) provide important insights into salt and saline-alkali stress tolerance.

Yellow horn (Xanthoceras sorbifolia) is an oil-rich woody plant cultivated for bio-energy production in China. Soil saline-alkalization is a prominent agricultural-related environmental problem limiting plant growth and productivity. In this study, we performed comparative physiological and transcriptomic analyses to examine the mechanisms of X. sorbifolia seedling responding to salt and alkaline-salt stress. With the exception of chlorophyll content, physiological experiments revealed significant increases in all assessed indices in response to salt and saline-alkali treatments. Notably, compared with salt stress, we observed more pronounced changes in electrolyte leakage (EL) and malondialdehyde (MDA) levels in response to saline-alkali stress, which may contribute to the greater toxicity of saline-alkali soils. In total, 3,087 and 2,715 genes were differentially expressed in response to salt and saline-alkali treatments, respectively, among which carbon metabolism, biosynthesis of amino acids, starch and sucrose metabolism, and reactive oxygen species signaling networks were extensively enriched, and transcription factor families of bHLH, C2H2, bZIP, NAC, and ERF were transcriptionally activated. Moreover, relative to salt stress, saline-alkali stress activated more significant upregulation of genes related to H+ transport, indicating that regulation of intracellular pH may play an important role in coping with saline-alkali stress. These findings provide new insights for investigating the physiological changes and molecular mechanisms underlying the responses of X. sorbifolia to salt and saline-alkali stress.

Neuroprotective effects of the aerial parts of Polygala tenuifolia Willd extract on scopolamine-induced learning and memory impairments in mice.

Alzheimer's disease is a common neurodegenerative disease characterized by progressive cognitive dysfunction and behavioral impairment. Aerial parts of Polygala tenuifolia Willd (APT) is a traditional Chinese medicine used for the treatment of amnesia. The present study aimed to investigate the protective effects of APT on scopolamine-induced learning and memory impairments in mice. Scopolamine-induced mice were used to determine the effects of APT on learning and memory impairment. Mice were orally administered with APT (25, 50 and 100 mg/kg) and piracetam (750 mg/kg) for 14 days, and intraperitoneally injected with scopolamine (2 mg/kg) from days 8 to 14. Morris water maze and step-down tests were performed to evaluate learning and memory. Levels of acetylcholine (ACh), choline acetyltransferase (ChAT), acetylcholinesterase (AChE), interleukin (IL)-1ß, IL-10 and brain-derived neurotrophic factor (BDNF) in the hippocampus and frontal cortex were measured by ELISA. Superoxide dismutase (SOD), malondialdehyde (MDA) and glutathione (GSH) were measured via biochemical detection. The results demonstrated that APT ameliorated learning and memory impairment in scopolamine-induced mice. Correspondingly, APT significantly increased ACh and ChAT levels in the hippocampus and prefrontal cortex of scopolamine-induced mice. Additionally, treatment with APT significantly increased BDNF and IL-10 levels, and decreased IL-1ß and AChE levels in the same mice. Furthermore, APT significantly increased SOD activity and GSH content, and decreased MDA levels in brain tissue. These results indicated that APT may ameliorate learning and memory impairment by regulating cholinergic activity, promoting BDNF and inhibiting neuroinflammation and oxidative stress.

Acylation modification of konjac glucomannan and its adsorption of Fe (â ¢) ion.

A biodegradable adsorbent, modified konjac glucomannan (MKGM), was prepared by konjac glucomannan (KGM) acylated with phthalic anhydride catalyzed using concentrated sulfuric acid. The modified conditions such as reaction temperature, mass ratio of phthalic anhydride to KGM, catalyst dosage and reaction time were investigated, respectively. MKGM exhibited preferable adsorption performance for the removal of Fe (Ⅲ) ion. The adsorption behavior was discussed using the Langmuir and Freundlich isotherm models. The results showed that the Freundlich linear model was suitable for describing the adsorption process of Fe (Ⅲ). The maximum adsorption capacity of MKGM for Fe (Ⅲ) ion was 31.87 mg g-1 at 298 K. The kinetics studies suggested that adsorption process followed the pseudo-second-order model and the adsorption process was mainly controlled by both surface reactivity and intra-particle diffusion. Together with the evaluation of the thermodynamic parameters such as Gibbs free energy, enthalpy and entropy changes, the results indicated that the adsorption process of Fe (Ⅲ) was endothermic, feasible, and spontaneous in nature. Hence, as a bioadsorbent, the MKGM has a promising potential for the removal of Fe (Ⅲ) ion from aqueous solutions.

Integrated transcriptomic and metabolomic analyses of yellow horn (Xanthoceras sorbifolia) in response to cold stress.

Xanthoceras sorbifolia, a medicinal and oil-rich woody plant, has great potential for biodiesel production. However, little study explores the link between gene expression level and metabolite accumulation of X. sorbifolia in response to cold stress. Herein, we performed both transcriptomic and metabolomic analyses of X. sorbifolia seedlings to investigate the regulatory mechanism of resistance to low temperature (4 °C) based on physiological profile analyses. Cold stress resulted in a significant increase in the malondialdehyde content, electrolyte leakage and activity of antioxidant enzymes. A total of 1,527 common differentially expressed genes (DEGs) were identified, of which 895 were upregulated and 632 were downregulated. Annotation of DEGs revealed that amino acid metabolism, glycolysis/gluconeogenesis, starch and sucrose metabolism, galactose metabolism, fructose and mannose metabolism, and the citrate cycle (TCA) were strongly affected by cold stress. In addition, DEGs within the plant mitogen-activated protein kinase (MAPK) signaling pathway and TF families of ERF, WRKY, NAC, MYB, and bHLH were transcriptionally activated. Through metabolomic analysis, we found 51 significantly changed metabolites, particularly with the analysis of primary metabolites, such as sugars, amino acids, and organic acids. Moreover, there is an overlap between transcript and metabolite profiles. Association analysis between key genes and altered metabolites indicated that amino acid metabolism and sugar metabolism were enhanced. A large number of specific cold-responsive genes and metabolites highlight a comprehensive regulatory mechanism, which will contribute to a deeper understanding of the highly complex regulatory program under cold stress in X. sorbifolia.

High-fat-diet impaired mitochondrial function of cumulus cells but improved the efficiency of parthenogenetic embryonic quality in mice.

Global human health has been compromised by high-fat diets. This study aimed to investigate the relationship between a high-fat diet and parthenogenetic embryo quality. Mice fed a high-fat or a normal diet was used as treated or control groups, respectively. Estradiol (E2), total cholesterol (TC) and total triglyceride (TG) were detected by Enzyme-Linked ImmunoSorbent Assay (ELISA). Cumulus-oocyte complexes (COCs) were collected from the mice in the treated and control groups. The ultrastructure of COCs, the expression level of genes involved in mitochondrial and nuclear functions in cumulus cells and oocytes quality were evaluated with transmission electron microscopy, real-time quantitative polymerase chain reaction (RT-PCR) and artificial parthenogenesis, respectively. The results showed that the efficiency of parthenogenetic embryonic development in vitro was significantly higher in the treated group than in the control group (p < .05). The expression level of genes involved in mitochondrial function was lower in cumulus cells from the treated group than that from the control group (p < .05). The estradiol and cholesterol level in the serum and the expression level of P450 arom were higher in the treated group than the control group (p < .05). The reactive oxygen species (ROS) level was higher in culumus cells from the treated group than the control group, while the mitochondrial membrane potential was lower in cumulus cells from the treated group (p < .05). Accumulation of lipid droplets was only in cumulus but in oocyte, the results demonstrated that mitochondrial functions were impaired by a high-fat diet, but parthenogenetic embryonic development in vitro was improved, in controllable range of damage for the body.

Alterations of protein glycosylation in embryonic stem cells during adipogenesis.

The understanding of adipose tissue development is crucial for the treatment of obesity-related diseases. Adipogenesis has been extensively investigated at the gene and protein levels in recent years. However, the alterations in protein glycosylation during this process remains unknown, particularly that of parthenogenetic embryonic stem cells (pESCs), a type of ESCs with low immunogenicity and no ethical concerns regarding their use. Protein glycosylation markedly affects cell growth and development, cell-to-cell communication, tumour growth and metastasis. In the present study, the adipogenic potentials of J1 ESCs and pESCs were first compared and the results demonstrated that pESCs had lower adipogenic potential compared with J1 ESCs. Lectin microarray was then used to screen the alteration of protein glycosylation during adipogenesis. The results revealed that protein modification of GlcNAc and α-1-2-fucosylation increased, whereas α-1-6­fucosylation, α-2-6-sialylation and α-1-6-mannosylation decreased in J1 ESCs and pESCs during this process. In addition, α-1-3-mannosylation decreased only in pESCs. Lectin histochemistry and quantitative polymerase chain reaction of glycosyltransferase confirmed the results obtained by lectin microarray. Therefore, protein glycosylation of ESCs was significantly altered during adipogenesis, indicating that protein glycosylation analysis is not only helpful for studying the mechanism of adipogenesis, but may also be used as a marker to monitor adipogenic development.

Directing the Differentiation of Parthenogenetic Stem Cells into Tenocytes for Tissue-Engineered Tendon Regeneration.

Uniparental parthenogenesis yields pluripotent stem cells without the political and ethical concerns surrounding the use of embryonic stem cells (ESCs) for biomedical applications. In the current study, we hypothesized that parthenogenetic stem cells (pSCs) could be directed to differentiate into tenocytes and applied for tissue-engineered tendon. We showed that pSCs displayed fundamental properties similar to those of ESCs, including pluripotency, clonogenicity, and self-renewal capacity. pSCs spontaneously differentiated into parthenogenetic mesenchymal stem cells (pMSCs), which were positive for mesenchymal stem cell surface markers and possessed osteogenic, chondrogenic, and adipogenic potential. Then, mechanical stretch was applied to improve the tenogenic differentiation of pMSCs, as indicated by the expression of tenogenic-specific markers and an increasing COL1A1:3A1 ratio. The pSC-derived tenocytes could proliferate and secrete extracellular matrix on the surface of poly(lactic-co-glycolic) acid scaffolds. Finally, engineered tendon-like tissue was successfully generated after in vivo heterotopic implantation of a tenocyte-scaffold composite. In conclusion, our experiment introduced an effective and practical strategy for applying pSCs for tendon regeneration. Stem Cells Translational Medicine 2017;6:196-208.

Effects of Alcohol on Mitochondrial Functions of Cumulus Cells in Mice.

Alcohol is an important compound used in food, agriculture, and medicine. In this study, we investigated the effect of alcohol on oocyte quality in mice by exposing animals for different duration times during an estrous cycle. Cumulus-oocyte complexes were collected from mice after pregnant mare serum gonadotropin- and human chorionic gonadotropin-induced superovulation. Ovulation number, E2 level in serum, and parthenogenetic embryo development in vitro were evaluated. Mitochondrial gene expression, mitochondrial membrane potential, and reactive oxygen species (ROS) levels in the cumulus were also assessed. The results showed that acute exposure to alcohol did not affect ovulation time (p > 0.05). Blasocyst formation rate in vitro was significantly improved after 1 and 2 days of alcohol exposure (p < 0.01). Mitochondrial membrane potential was significantly increased after 1-4 days of alcohol exposure (p < 0.05), but it decreased after 5 days (p < 0.05). ROS levels remained relatively low after 2, 3, and 4 days of exposure (p < 0.05), and they significantly increased after 6 days (p < 0.05). In addition, alcohol altered the expression of mitochondrial and nuclear genes in the cumulus. Taken together, our data suggest that acute exposure to alcohol affects oocyte quality by influencing the function and gene expression in the cumulus. These results underscore potential implications for the development of human reproductive therapeutics.

Screening somatic cell nuclear transfer parameters for generation of transgenic cloned cattle with intragenomic integration of additional gene copies that encode bovine adipocyte-type fatty acid-binding protein (A-FABP).

Somatic cell nuclear transfer (SCNT) is frequently used to produce transgenic cloned livestock, but it is still associated with low success rates. To our knowledge, we are the first to report successful production of transgenic cattle that overexpress bovine adipocyte-type fatty acid binding proteins (A-FABPs) with the aid of SCNT. Intragenomic integration of additional A-FABP gene copies has been found to be positively correlated with the intramuscular fat content in different farm livestock species. First, we optimized the cloning parameters to produce bovine embryos integrated with A-FABP by SCNT, such as applied voltage field strength and pulse duration for electrofusion, morphology and size of donor cells, and number of donor cells passages. Then, bovine fibroblast cells from Qinchuan cattle were transfected with A-FABP and used as donor cells for SCNT. Hybrids of Simmental and Luxi local cattle were selected as the recipient females for A-FABP transgenic SCNT-derived embryos. The results showed that a field strength of 2.5 kV/cm with two 10-µs duration electrical pulses was ideal for electrofusion, and 4-6th generation circular smooth type donor cells with diameters of 15-25 µm were optimal for producing transgenic bovine embryos by SCNT, and resulted in higher fusion (80%), cleavage (73%), and blastocyst (27%) rates. In addition, we obtained two transgenic cloned calves that expressed additional bovine A-FABP gene copies, as detected by PCR-amplified cDNA sequencing. We proposed a set of optimal protocols to produce transgenic SCNT-derived cattle with intragenomic integration of ectopic A-FABP-inherited exon sequences.

Preclinical study of mouse pluripotent parthenogenetic embryonic stem cell derivatives for the construction of tissue-engineered skin equivalent.

BACKGROUND: Embryonic stem cell (ESC) derivatives hold great promise for the construction of tissue-engineered skin equivalents (TESE). However, harvesting of ESCs destroys viable embryos and may lead to political and ethical concerns over their application. In the current study, we directed mouse parthenogenetic embryonic stem cells (pESCs) to differentiate into fibroblasts, constructed TESE, and evaluated its function in vivo. METHODS: The stemness marker expression and the pluripotent differentiation ability of pESCs were tested. After embryoid body (EB) formation and adherence culture, mesenchymal stem cells (MSCs) were enriched and directed to differentiate into fibroblastic lineage. Characteristics of derived fibroblasts were assessed by quantitative real-time PCR and ELISA. Functional ability of the constructed TESE was tested by a mouse skin defects repair model. RESULTS: Mouse pESCs expressed stemness marker and could form teratoma containing three germ layers. MSCs could be enriched from outgrowths of EBs and directed to differentiate into fibroblastic lineage. These cells express a high level of growth factors including FGF, EGF, VEGF, TGF, PDGF, and IGF1, similar to those of ESC-derived fibroblasts and mouse fibroblasts. Seeded into collagen gels, the fibroblasts derived from pESCs could form TESE. Mouse skin defects could be successfully repaired 15 days after transplantation of TESE constructed by fibroblasts derived from pESCs. CONCLUSIONS: pESCs could be induced to differentiate into fibroblastic lineage, which could be applied to the construction of TESE and skin defect repair. Particularly, pESC derivatives avoid the limitations of political and ethical concerns, and provide a promising source for regenerative medicine.

A novel approach for the cryodesiccated preservation of tissue-engineered skin substitutes with trehalose.

Tissue-engineered skin (TES) holds great promise for wound healing in the clinic. However, optimized preservation methods remain an obstacle for its wide application. In this experimental work, we developed a novel approach to preserve TES in the desiccated state with trehalose. The uptake of trehalose by fibroblasts under various conditions, including the trehalose concentration, incubation temperature and time, was studied. The cell viability was investigated by the MTT assay and CFSE/PI staining after cryodesiccation and rehydration. TES was then prepared and incubated with trehalose, and the wound healing effect was investigated after desiccated preservation. The results showed that the optimized conditions for trehalose uptake by fibroblasts were incubation in 200 mM trehalose at 37 °C for 8 h. Cryodesiccated cells and TES maintained 37.55% and 28.31% viabilities of controls, respectively. Furthermore, cryodesiccated TES exhibited a similar wound healing effect to normal TES. This novel approach enabled the preservation and transportation of TES at ambient temperature with a prolonged shelf time, which provides great advantages for the application of TES.

Induction of matrix metalloproteinase-3 (MMP-3) expression in the microglia by lipopolysaccharide (LPS) via upregulation of glycoprotein nonmetastatic melanoma B (GPNMB) expression.

Substantial evidence suggests that inflammation is an important contributor to many neurodegenerative disorders. Activated microglial cells play an important role in releasing pro-inflammatory factors, including tumor necrosis factor-α (TNF-α) and interleukin-1ß (IL-1ß) for inducing inflammation. Recently, some reports have suggested that glycoprotein nonmetastatic melanoma B (GPNMB) is highly expressed in microglia after LPS treatment. However, the role of GPNMB in activated microglia is not clearly understood. In this study, we used RT-PCR and Western blotting to detect GPNMB and matrix metalloproteinase-3 (MMP-3) expressions in activated microglia. GPNMB small interfering RNA (siRNA) or MMP-3 inhibitor was applied on microglial BV2 cells, and ELISA was performed to measure the expressions of TNF-α and IL-1ß in BV2 cells. Levels of iNOS and NO in BV2 cells were also determined. We found that the levels of GPNMB and MMP-3 were significantly increased in BV2 cells after LPS treatment. Moreover, we found that GPNMB significantly upregulated the expression of MMP-3 in BV2 cells, and high expression of MMP-3 was dependent on the level of GPNMB. Inhibition of GPNMB or MMP-3 expression by GPNMB siRNA or MMP-3 inhibitor dramatically suppressed the expressions of TNF-α, IL-1ß, iNOS, and NO in activated microglia. All of these results suggest that GPNMB is involved in the inflammatory responses of microglia.

Improved establishment of embryonic stem (ES) cell lines from the Chinese Kunming mice by hybridization with 129 mice.

Chinese Kunming mice (Mus musculus Km), widely used as laboratory animals throughout China, remain very refractory for embryonic stem (ES) cell isolation. The present study was aimed to evaluate the effects of hybridization with 129/Sv mice, and culture media containing fetal bovine serum (FBS) or Knockout serum replacement (KSR) on ES cell isolation from Kunming mice. The results demonstrated that ES cells had been effectively isolated from the hybrid embryos of Kunming and 129/Sv mice using all three media containing 15% FBS, 15% KSR and their mixture of 14% KSR and 1% FBS, individually. These isolated ES cells had maintained in vitro undifferentiated for a long time, exhibiting all features specific for mouse ES cells. In addition, the rates of ES cell isolation in the medium containing 14% KSR and 1% FBS, was 46.67% and significantly higher than those in another two media containing only FBS or KSR (p < 0.05). Contrarily, no ES cell line had been established from Kunming mouse inbred embryos using the same protocols. These results suggested that ES cells with long-term self-renewal ability could be efficiently generated from hybrid embryos of Kunming and 129/Sv mice, and a small volume of FBS was necessary to isolate ES cells in the KSR medium when embryos and early ES cells cultured.

Directing parthenogenetic stem cells differentiate into adipocytes for engineering injectable adipose tissue.

The selection of appropriate seed cells is crucial for adipose tissue engineering. Here, we reported the stepwise induction of parthenogenetic embryonic stem cells (pESCs) to differentiate into adipogenic cells and its application in engineering injectable adipose tissue with Pluronic F-127. pESCs had pluripotent differentiation capacity and could form teratomas that include the three primary germ layers. Cells that migrated from the embryoid bodies (EBs) were selectively separated and expanded to obtain embryonic mesenchymal stem cells (eMSCs). The eMSCs exhibited similar cell surface marker expression profiles with bone morrow mesenchymal stem cells (BMSCs) and had multipotent differentiation capacity. Under the induction of dexamethasone, indomethacin, and insulin, eMSCs could differentiate into adipogenic cells with increased expression of adipose-specific genes and oil droplet depositions within the cytoplasm. To evaluate their suitability as seed cells for adipose tissue engineering, the CM-Dil labelled adipogenic cells derived from eMSCs were seeded into Pluronic F-127 hydrogel and injected subcutaneously into nude mice. Four weeks after injection, glistering and semitransparent constructs formed in the subcutaneous site. Histological observations demonstrated that new adipose tissue was successfully fabricated in the specimen by the labelled cells. The results of the current study indicated that pESCs have great potential in the fabrication of injectable adipose tissue.