Isolation and Characterization of Novel Murine Epiphysis Derived Mesenchymal Stem Cells

BACKGROUND: While bone marrow (BM) is a rich source of mesenchymal stem cells (MSCs), previous studies have shown that MSCs derived from mouse BM (BMMSCs) were difficult to manipulate as compared to MSCs derived from other species. The objective of this study was to find an alternative murine MSCs source that could provide sufficient MSCs. METHODOLOGY/PRINCIPAL FINDINGS: In this study, we described a novel type of MSCs that migrates directly from the mouse epiphysis in culture. Epiphysis-derived MSCs (EMSCs) could be extensively expanded in plastic adherent culture, and they had a greater ability for clonogenic formation and cell proliferation than BMMSCs. Under specific induction conditions, EMSCs demonstrated multipotency through their ability to differentiate into adipocytes, osteocytes and chondrocytes. Immunophenotypic analysis demonstrated that EMSCs were positive for CD29, CD44, CD73, CD105, CD166, Sca-1 and SSEA-4, while negative for CD11b, CD31, CD34 and CD45. Notably, EMSCs did not express major histocompatibility complex class I (MHC I) or MHC II under our culture system. EMSCs also successfully suppressed the proliferation of splenocytes triggered by concanavalin A (Con A) or allogeneic splenocytes, and decreased the expression of IL-1, IL-6 and TNF-α in Con A-stimulated splenocytes suggesting their anti-inflammatory properties. Moreover, EMSCs enhanced fracture repair, ameliorated necrosis in ischemic skin flap, and improved blood perfusion in hindlimb ischemia in the in vivo experiments. CONCLUSIONS/SIGNIFICANCES: These results indicate that EMSCs, a new type of MSCs established by our simple isolation method, are a preferable alternative for mice MSCs due to their better growth and differentiation potentialities

Mixed Solid-State Fermentation of Okara and Copra Meal by Probiotics with Non-Starch Polysaccharide Enzymes and Its Effects on the Growth Performance and Ileal Microbiota in Broilers

With the global shortage of feed ingredients, the use of agricultural by-products has become an alternative to animal feed. Okara, a by-product of soymilk and tofu processing, is high in nutrients but contains non-starch polysaccharides (NSP) and has a high-water content, which are disadvantages in animal feed. Herein, we mixed okara and copra meal (CM) with probiotics (Lactobacillus species and Clostridium butyricum) and NSP enzymes (NSPases) for solid-state fermentation (SSF) to enhance okara feed value; the optimal parameters of fermented okara and CM (FOCM) and their effects on broiler growth performance and ileal microbiota were investigated. The result showed that FOCM in combination with NSPases and probiotics at 55% of the initial moisture content and 60 h fermentation time at 37 °C were able to degrade NSP and reduce sugar content. After fermentation, the total viable counts, lactic acid, and butyric acid contents in the FOCM were 8.6 log CFU/g, 3.7%, and 17.15%, respectively. During the fifth week of the feeding period and over the whole feeding period, broilers fed with 1.25% and 2.5% FOCM had a better feed conversion ratio (p p Escherichia-Shigella in the ileal content (p < 0.05). Collectively, supplementation of probiotics and enzymes during SSF was found to be effective in enhancing the nutritional value of FOCM. Moreover, dietary supplementation of FOCM improved the broiler feed conversion ratio, gut morphology, and ileal microbiota

Effect of different vitamin D3 metabolites on intestinal calcium homeostasis-related gene expression in broiler chickens

ABSTRACT The purpose of this study was to investigate the effects of vitamin D3 metabolites 1α-hydroxycholecalciferol (1α(OH)D3), 25-hydroxycholecalciferol (25(OH)2D3), and 1,25-dihydroxycholecalciferol (1,25(OH)2D3) on growth performance, bone quality, and intestinal calcium homeostasis-related gene expression in broiler chickens. One-day-old broilers were fed a basal diet and basal diet containing different vitamin D3 metabolites. The body weight, feed intake, and feed conversion ratio in control and experimental broilers were measured to assess the growth performance, mineral levels, and bone breaking strength. The duodenum was used to assess calcium homeostasis-related gene expressions by quantitative reverse transcription-PCR. No statistically significant difference was found in growth performance, mineral deposition, or bone breaking strength in broiler chickens after three weeks feeding with vitamin D3. However, supplementation of vitamin D3 metabolites tended to improve feed conversion rate, bone mineral deposition, and breaking strength in broiler chickens. The results demonstrated that vitamin D3 metabolites significantly upregulated calcium homeostasis-related genes, including calbindin, β-glucuronidase, TRPV6, and Na/Pi IIb cotransporter, mRNA levels after 12 h of feeding. The vitamin D3 metabolite 1,25(OH)2D3 was the most effective at regulating calcium homeostasis-associated gene expression after 6 h of feeding. Dietary vitamin D3 metabolites may alleviate the development of TD in broiler chickens and these effects probably occur through regulation of intestinal calcium homeostasis-related gene expression

Optimization of solid-state fermentation conditions of Bacillus licheniformis and its effects on Clostridium perfringens-induced necrotic enteritis in broilers

ABSTRACT In the present study, we examined the growth parameters of Bacillus licheniformis in solid-state fermentation (SSF) and evaluated the effects of Bacillus licheniformis-fermented products on Clostridium perfringens-challenged broilers. During four and six days of SSF, the highest viable biomass was observed at 5% glucose, 10% soybean meal, 3% yeast, and 50% initial moisture content. The Bacillus licheniformis SSF products were heat- and acid-resistant. Furthermore, the fermented products were able to inhibit the growth of Clostridium perfringens and Staphylococcus aureus in vitro. In feeding experiments, in a similar manner to the antibiotic treatment group, dietary supplementation of Bacillus licheniformis-fermented products significantly improved intestinal morphology and necrotic lesions under Clostridium perfringens challenge, accompanied by increased IFN-γ mRNA expression in the spleen and bursa of Fabricius. These results together suggest that Bacillus licheniformis-fermented products have potential for development as feed additives and use as possible substitutes for antibiotics to treat Clostridium perfringens in the poultry industry

Optimization of Emulsification Conditions on Ethanol Extract of Taiwanese Green Propolis Using Polysorbate and Its Immunomodulatory Effects in Broilers

Beeswax and resin are the main components of propolis, both of which are hydrophobic. The use of emulsifiers helps to improve the extraction of active propolis compounds and makes them more widely used. In this study, we investigated the optimal parameters for the emulsification of Taiwanese green propolis (TGP) using different polysorbates (polysorbate-20, polysorbate-60, and polysorbate-80) and evaluated the effects on the immunomodulatory response in broilers. The results showed that 4 mg/mL of TGP in combination with 2% polysorbate-60 at 60 &deg;C for 60 min significantly decreased the undissolved particle size of ethanol extract of TGP during the emulsification. The bioactive compounds of TGP, the propolins (C, D, F, G, and H), were also detected after emulsification. Supplementation of emulsified TGP (eTGP) in the drinking water of broilers before and after vaccination significantly enhanced the antibody titer response to infectious bronchitis virus at 28 days of age. In the lipopolysaccharide-challenged model, supplementation of eTGP in the drinking water of broilers decreased pro-inflammatory gene expression and increased anti-inflammatory gene expression. These results together suggested that the polysorbate-60 could effectively emulsify the ethanol extract of TGP. Moreover, eTGP could be used as a vaccine adjuvant and an immunomodulator to improve the immune response of broilers

The Effect of Feeding Restriction on the Microbiota and Metabolome Response in Late-Phase Laying Hens

This study investigated cecal bacterial community profile, cecal and serum metabolites, and its biosynthesis pathway in late-phase laying hens during 6 weeks feeding restriction (FR), using 16S rDNA as gene sequencing and non-targeted LC-MS/MS as metabolomics approach. We used three groups (ad libitum, FR20, and FR40). FR can reduce excessive fat in late-phase laying hens, while egg production rate is not affected, except for the FR40 group. In phylum level, FR20 had more population of Bacteriodetes and Firmicutes amongst groups. The same result is at genus level, FR20 were higher of the predominant genus (Bacteroides and Rikenellaceae_RC9_gut_group). Both of FR20 and FR40 reduced Proteobacteria as potential pathogenic bacteria. Non-targeted metabolomic analysis revealed that FR20 modified 20 metabolites in cecal and 10 metabolites in serum of laying hens, whereas 48 cecal metabolites and 31 serum metabolites has revealed in FR40. KEGG assay showed FR20 and FR40 upregulated lipid, carbohydrate, amino acid, nucleic acid pathway, and FR40 modified steroid metabolism in cecal analysis. In serum, only FR40 modified lipid, amino acid pathway, and carbohydrate biosynthesis were shown. This study showed that FR during late-phase laying hens altered the microbiome composition, modified metabolites profile and biosynthesis of the cecal as well as serum

Repair of bone fracture in mice after transplantation of EMSCs.

<p>(A) Fracture healing was assessed by X-ray after 14 days with or without EMSCs (at the fifth passage) transplantation. Arrowheads indicate the site of fracture. (B) The bone density of fracture site (dotted box in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036085#pone-0036085-g008" target="_blank">Figure 8A</a>) was quantified (n = 4). EMSCs transplantation significantly improved osteocalcification as compared to the control group. (C) The morphology of fracture sites were examined by H&E staining. Tissue disorganization was showed in the control group while the EMSCs transplanted group newly formed bone tissue (arrow) and capillaries (arrowhead). * represents <i>P</i><0.05. Scale bars represent 100 µm. Data are presented as mean ± s.d. and analyzed with Student's <i>t</i>-test. Control: SPONGOSTAN carried with complete medium-transplantation group; EMSC: SPONGOSTAN carried with EMSCs-transplantation group.</p

Establishment of EMSCs and BMMSCs.

<p>(A) Schematic protocol for the establishment of cultures of EMSCs and BMMSCs. (B) Phase-contrast micrograph of EMSCs in primary culture after seven days. Arrow heads indicate triangle, spindle-shaped, fibroblast-like EMSCs. Lymphohematopoietic cells are indicated by arrows. (C) Phase-contrast micrograph of BMMSCs in primary culture after seven days. Arrow heads indicate flat, fibroblast-like BMMSCs and arrows indicate lymphohematopoietic cells. (D) Phase contrast micrograph of EMSCs at the fifth passage showing that EMSCs maintained their shape during propagating. (E) Phase contrast micrograph of BMMSCs upon the fifth passage showing that BMMSCs became flatted with increasing passages. All the scale bars represent 100 µm.</p

Surface markers analysis of EMSCs after IFN-γ treatment and during increased serial passages.

<p>(A) MHC I and MHC II expression profiles for EMSCs (passage five) were analysed under 10 ng or (B) 100 ng IFN-γ treatment. EMSCs were positive for MHC I and MHC II after IFN-γ treatment. (C) EMSCs surface antigen profiles were detected via flow cytometric analysis during increasing passages. The markers of CD73, CD166, SSEA-4 and Sca-1 were decreased with propagating. Respective isotype control is indicated by the dotted line.</p

Transplanted EMSCs improved blood perfusion in ischemic limb.

<p>(A) Foot perfusion was evaluated by laser Doppler blood perfusion analysis at day 7 post ischemia. In color-coded images, red represents normal perfusion, while dark blue represents low or absent perfusion. (B) Quantification of the foot perfusion showed that EMSCs (at the fifth passage) transplantation significantly improved blood perfusion (n = 3). (C) H&E staining showed massive muscle degeneration in ischemic regions in the control group compared to the markedly reduced muscle degeneration in the EMSCs group. (D) Masson's Trichrome staining and (E) its quantification showed significantly larger fibrotic area in the control group than the EMSCs injected group. (F) Immunostaining of von Willebrand factor (vWF) of ischemic limb muscle and (G) its quantification showed significantly more vWF expressed cells (arrowhead) in the EMSCs injected group than the control group. Scale bars represent 100 µm. **represents <i>P</i><0.01. Data are presented as mean ± s.d. and analyzed with Student's <i>t</i>-test. Isch: ischemic limb; NI: non-ischemic limb; Control: complete medium injection-group; EMSC: EMSCs-injection group.</p