Evaluation of porcine intestinal organoids as an in vitro model for mammalian orthoreovirus 3 infection.

BACKGROUND: Mammalian orthoreovirus type 3 (MRV3), which is responsible for gastroenteritis in many mammalian species including pigs, has been isolated from piglets with severe diarrhea. However, the use of pig-derived cells as an infection model for swine-MRV3 has rarely been studied. OBJECTIVES: This study aims to establish porcine intestinal organoids (PIOs) and examine their susceptibility as an in vitro model for intestinal MRV3 infection. METHODS: PIOs were isolated and established from the jejunum of a miniature pig. Established PIOs were characterized using polymerase chain reaction (PCR) and immunofluorescence assays (IFAs) to confirm the expression of small intestine-specific genes and proteins, such as Lgr5, LYZI, Mucin-2, ChgA, and Villin. The monolayered PIOs and three-dimensional (3D) PIOs, obtained through their distribution to expose the apical surface, were infected with MRV3 for 2 h, washed with Dulbecco's phosphate-buffered saline, and observed. Viral infection was confirmed using PCR and IFA. We performed quantitative real-time reverse transcription-PCR to assess changes in viral copy numbers and gene expressions linked to intestinal epithelial genes and antiviral activity. RESULTS: The established PIOs have molecular characteristics of intestinal organoids. Infected PIOs showed delayed proliferation with disruption of structures. In addition, infection with MRV3 altered the gene expression linked to intestinal epithelial cells and antiviral activity, and these effects were observed in both 2D and 3D models. Furthermore, viral copy numbers in the supernatant of both models increased in a time-dependent manner. CONCLUSIONS: We suggest that PIOs can be an in vitro model to study the infection mechanism of MRV3 in detail, facilitating pharmaceutical development.

Immunomodulatory effects of canine mesenchymal stem cells in an experimental atopic dermatitis model.

Mesenchymal stem cells (MSCs) have the potential to differentiate into multi-lineage cells, suggesting their future applicability in regenerative medicine and biotechnology. The immunomodulatory properties of MSCs make them a promising replacement therapy in various fields of animal research including in canine atopic dermatitis (AD), a skin disease with 10-15% prevalence. We investigated the immunomodulatory effects of MSCs in an experimental canine AD model induced by Dermatophagoides farinae extract ointment. Canine adipose tissue-derived MSCs (cAT-MSCs) were differentiated into mesodermal cell lineages at the third passage. Alterations in immunomodulatory factors in control, AD, and MSC-treated AD groups were evaluated using flow cytometric analysis, enzyme-linked immunosorbent assay, and quantitative reverse transcription PCR. In the MSC-treated AD group, the number of eosinophils decreased, and the number of regulatory T cells (Tregs) increased compared to those in the AD group. In addition, the immunoglobulin E (IgE) and prostaglandin E2 levels were reduced in the MSC-treated AD group compared to those in the AD group. Furthermore, the filaggrin, vascular endothelial growth factor, and interleukin-5 gene expression levels were relatively higher in the MSC-treated AD group than in the AD group, however, not significantly. cAT-MSCs exerted immunomodulatory effects in an AD canine model via a rebalancing of type-1 and -2 T helper cells that correlated with increased levels of Tregs, IgE, and various cytokines.

Novel method to repair articular cartilage by direct reprograming of prechondrogenic mesenchymal stem cells.

Age-related cartilage loss is worsened by the limited regenerative capacity of chondrocytes. The role of cell-based therapies using mesenchymal stem cells is gaining interest. Adipose tissue-derived mesenchymal stem cells (ADSCs) are an attractive source to generate the optimal number of chondrocytes required to repair a cartilage defect and regenerate hyaline articular cartilage. Here, we report an outstanding technique to prepare chondrocytes for cartilage repair using canine ADSCs. We hypothesized that external electrical fields promote prechondrogenic condensation without requiring genetic modifications or exogenous factors. We analyzed the effect of electrical stimulation (ES) on the differentiation of ADSC micromass into chondrocytes. Highly compact structures were formed within 3 days of ES of canine ADSC micromass. The expression of type I collagen gene was abolished in these cells compared with that in control micromass cultures and monolayer cultures. We further found that ES enhanced the production of proteoglycan, a highly produced extracellular matrix component in chondrocytes. Additionally, single-cell RNA sequencing analysis showed that canine ADSC micromass undergoing ES developed a prechondrogenic cell aggregation, suggesting their metabolic conversion, biogenesis, and calcium ion change. Collectively, our findings demonstrate the capacity of ES to drive the chondrogenesis of ADSCs in the absence of exogenous factors and confirm its commercial potential as a budget-friendly therapy for the repair of cartilage defects.

Canine Natural Killer Cell-Derived Exosomes Exhibit Antitumor Activity in a Mouse Model of Canine Mammary Tumor.

Natural killer (NK) cells are key immune cells engaged in fighting infection and malignant transformation. In this study, we found that canine NK cell-derived exosomes (NK-exosomes) separated from activated cytotoxic NK cell supernatants express specific markers including CD63, CD81, Alix, HSP70, TSG101, Perforin 1, and Granzyme B. We examined the antitumor effects of NK-exosomes in an experimental murine mammary tumor model using REM134 canine mammary carcinoma cell line. We observed changes in tumor size, tumor initiation, progression, and recurrence-related markers in the control, tumor group, and NK-exosome-treated tumor group. We found that the tumor size in the NK-exosome-treated tumor group decreased compared with that of the tumor group in the REM134-driven tumorigenic mouse model. We observed significant changes including the expression of tumorigenesis-related markers, such as B cell-specific Moloney murine leukemia virus insertion site 1 (Bmi-1), vascular endothelial growth factor (VEGF), matrix metallopeptidase-3 (MMP-3), interleukin-1ß (IL-1ß), IL-6, tumor necrosis factor-α (TNF-α), multidrug resistance protein (MDR), tumor suppressor protein p53 (p53), proliferating cell nuclear antigen (PCNA), and the apoptotic markers, B cell lymphoma-2 associated X (Bax) and B cell lymphoma-extra large (Bcl-xL) belonging to the Bcl-2 family, in the tumor group compared with those in the control group. The expression of CD133, a potent cancer stem cell marker, was significantly higher than that of the control. By contrast, the NK-exosome-treated tumor group exhibited a significant reduction in Bmi-1, MMP-3, IL-1ß, IL-6, TNF-α, Bax, Bcl-xL, and PCNA expression compared with that in the tumor group. Furthermore, the expression of CD133, which mediates tumorigenesis, was significantly decreased in the NK-exosome-treated tumor group compared with that in the tumor group. These findings indicate that canine NK-exosomes represent a promising therapeutic tool against canine solid tumors, including mammary carcinoma.

Bovine tongue epithelium-derived cells: A new source of bovine mesenchymal stem cells.

Mesenchymal stem cells (MSCs) possess the ability to differentiate into multiple cell lineages, and thus, confer great potential for use in regenerative medicine and biotechnology. In the present study, we attempted to isolate and characterize bovine tongue tissue epithelium-derived MSCs (boT-MSCs) and investigate the culture conditions required for long-term culturing of boT-MSCs. boT-MSCs were successfully isolated by the collagenase digestion method and their proliferative capacity was maintained for up to 20 or more passages. We observed a significant increase in the proliferation of boT-MSCs during the 20 consecutive passages under low-glucose Dulbecco's modified Eagle's medium culture condition among the three culture conditions. These boT-MSCs presented pluripotency markers (octamer-binding transcription factor 3/4 (Oct3/4) and sex determining region Y-box2 (Sox2)) and cell surface markers, which included CD13, CD29, CD44, CD73, CD90, CD105, CD166, and major histocompatibility complex (MHC) class I (MHC-I) but not CD11b, CD14, CD31, CD34, CD45, CD80, CD86, CD106, CD117, and MHC-II at third passage. Moreover, these boT-MSCs could differentiate into mesodermal (adipocyte, osteocyte, and chondrocyte) cell lineages. Thus, the present study suggests that the tongue of bovines could be used as a source of bovine MSCs.

Effects of three-dimensional spheroid culture on equine mesenchymal stem cell plasticity.

Mesenchymal stem cells (MSCs) are useful candidates for tissue engineering and cell therapy fields. We optimize culture conditions of equine adipose tissue-derived MSCs (eAD-MSCs) for treatment of horse fractures. To investigate enhancing properties of three-dimensional (3D) culture system in eAD-MSCs, we performed various sized spheroid formation and determined changes in gene expression levels to obtain different sized spheroid for cell therapy. eAD-MSCs were successfully isolated from horse tailhead. Using hanging drop method, spheroid formation was generated for three days. Quantitative real-time PCR was performed to analyze gene expression. As results, expression levels of pluripotent markers were increased depending on spheroid size and the production of PGE2 was increased in spheroid formation compared to that in monolayer. Ki-67 showed a remarkable increase in the spheroid formed with 2.0 × 105 cells/drop as compared to that in the monolayer. Expression levels of angiogenesis-inducing factors such as VEGF, IL-6, IL-8, and IL-18 were significantly increased in spheroid formation compared to those in the monolayer. Expression levels of bone morphogenesis-inducing factors such as Cox-2 and TGF-ß1 were also significantly increased in spheroid formation compared to those in the monolayer. Expression levels of osteocyte-specific markers such as RUNX2, osteocalcin, and differentiation potential were also significantly increased in spheroid formation compared to those in the monolayer. Therefore, spheroid formation of eAD-MSCs through the hanging drop method can increases the expression of angiogenesis-inducing and bone morphogenesis-inducing factors under optimal culture conditions.

Effect of donor age on the proliferation and multipotency of canine adipose-derived mesenchymal stem cells.

Research into adipose tissue-derived mesenchymal stem cells (AD-MSCs) has demonstrated the feasibility of their use in clinical applications due to their ease of isolation and abundance in adipose tissue. We isolated AD-MSCs from young and old dogs, and the cells were subjected to sequential sub-passaging from passage 1 (P1) to P7. Canine AD-MSCs (cAD-MSCs) were examined for proliferation kinetics, expression of molecules associated with self-renewal, expression of cell surface markers, and differentiation potentials at P3. Cumulative population doubling level was significantly higher in cAD-MSCs of young donors than in those of old donors. In addition, expressions of CD73, CD80, Oct3/4, Nanog, cell survival genes and differentiation potentials were significantly higher in young donors than in old donors. The present study suggests that donor age should be considered when developing cell-based therapies for clinical application of cAD-MSCs.

Extensive characterization of feline intra-abdominal adipose-derived mesenchymal stem cells.

Mesenchymal stem cells (MSCs) isolated from various tissues have been well characterized for therapeutic application to clinical diseases. However, in contrast to MSCs from other animal species, the characteristics of feline MSCs have not been fully documented. In this study, we conducted extensive characterization of feline adipose tissue-derived MSCs (fAD-MSCs). Study fAD-MSCs were individually isolated from the intra-abdominal adipose tissues of six felines. The expression levels of cell surface markers and pluripotent markers were evaluated. Next, proliferation capacity was analyzed by performing cumulative population doubling level (CPDL) and doubling time (DT) calculation assays. Differentiation potentials of fAD-MSCs into mesodermal cell lineages were analyzed by examining specific staining and molecular markers. All fAD-MSCs positively expressed cell surface markers such as CD29, CD44, CD90, CD105, CD166, and MHC-I, while CD14, CD34, CD45, and CD73 were negatively expressed. The CPDL of the fAD-MSCs was maintained until passage 5 to 6 (P5 to P6), whereas DT increased after P3 to P4. Also, stem cell-specific pluripotent markers (Oct3/4, Nanog, and SSEA-4) were detected. Importantly, all fAD-MSCs demonstrated mesodermal differentiation capacity. These results suggest that fully characterized fAD-MSCs could be beneficial when considering the use of these cells in feline disease research.

Chondrogenic potential and anti-senescence effect of hypoxia on canine adipose mesenchymal stem cells.

Mesenchymal stem cells (MSCs) have the ability to differentiate into multi-lineage cells, which confers great promise for use in regenerative medicine. In this study, canine adipose MSCs (cAD-MSCs) were isolated from canine adipose tissue. These cells clearly represented stemness (Oct4, Sox2, and Nanog) and differentiation potential into the mesoderm (adipocytes, chondrocytes, and osteoblasts) at early passages. The aim of this study was to evaluate the effects of hypoxia on the differentiation potential into mesoderm, and the expression of anti-apoptotic genes associated with cell survival for the optimal culturing of MSCs. We observed that the proliferation of the cAD-MSCs meaningfully increased when cultured under hypoxic condition than in normoxic condition, during 7 consecutive passages. Also, we found that hypoxia strongly expressed anti-senescence related genes such as HDAC1 (histone deacetylase 1), DNMT1 (DNA (cytosine-5)-methyltransferase 1), Bcl-2 (inhibitor of apoptosis), TERT (telomerase reverse transcriptase), LDHA (lactate dehydrogenase A), SLC2A1 (glucose transporter), and DKC1 (telomere holoenzyme complex) and differentiation potential of cAD-MSCs into chondrocytes, than seen under the normoxic culture conditions. We also examined the multipotency of hypoxic conditioned MSCs using quantitative real-time RT-PCR. We found that the expression levels of stemness genes such as Oct-4, Nanog, and Sox-2 were increased in hypoxic condition when compared to the normoxic condition. Collectively, these results suggest that hypoxic conditions have the ability to induce proliferation of MSCs and augment their chondrogenic potential. This study suggests that cell proliferation of cAD-MSC under hypoxia could be beneficial, when considering these cells for cell therapies of canine bone diseases.

Comparative studies on proliferation, molecular markers and differentiation potential of mesenchymal stem cells from various tissues (adipose, bone marrow, ear skin, abdominal skin, and lung) and maintenance of multipotency during serial passages in miniature pig.

Mesenchymal stem cells (MSCs) have the ability to differentiate into multi-lineage cells, which confers great promise for use in regenerative medicine. In this study, MSCs were isolated from adipose tissue, bone marrow, ear skin, lung, and abdominal skin of miniature pigs (mpMSCs), and the optimal medium (DMEM/F12-Glutamax) was selected for the culturing of mpMSCs. As a result, proliferation of the mpMSCs derived from all tissues was steadily increased when cultured with DMEM/F12-Glutamax during 14 consecutive passages. The cells harbored MSC surface markers (CD34-, CD45-, CD29+, CD44+, CD90+, and CD105+), whose levels of expression differed among the tissue sources and declined over sub-passaging. In addition, the expression of stemness markers (Oct4, Sox2, and Nanog) and differentiation into mesoderm (adipocytes, chondrocytes, and osteoblasts) were clearly represented at early passage; however, expression of stemness markers decreased, and differentiation potential was lost over sequential sub-passaging, which should be considered in the selection of mpMSC for MSC-based application.

Gene cloning and enzymatic properties of hyperthermostable beta-glycosidase from Thermus thermophilus HJ6.

A microorganism (strain HJ6) producing extracellular beta-glycosidase was isolated from a hot springs located in Arima-cho, Hyogo, Japan. The cells were long-rods (2-4 microm) about 0.4 microm in diameter, and formed yellow-colored colonies, like most other strains of the genus Thermus. The pH and temperature for optimal growth were 6.5 and 80 degrees C. Thus, the HJ6 strain displayed a higher optimal temperature than other described Thermus sp. The gene encoding beta-glycosidase (TtbetaGly) was cloned, sequenced, and comprised of 1296 nucleotides encoding a protein (431 amino acids) with a predicted molecular mass of 48.7 kDa. TtbetaGly was expressed in Escherichia coli cells, and the recombinant protein was purified to homogeneity. The optimal temperature and pH for beta-glycosidase activity were found to be 90 degrees C and 8.5, respectively. The half-life of heat inactivation was about 30 min at 95 degrees C indicating that TtbetaGly had higher thermostability than beta-glycosidases from other Thermus sp. The results of the kinetics experiment indicated that beta-D-fucoside and beta-D-glucoside were better substrates of TtbetaGly than beta-D-galactoside. The catalytic efficiency (k(cat)/K(m)) of TtbetaGly at 80 degrees C increased 70-fold to that at 40 degrees C, indicating that this enzyme was activated at high temperatures. Thin layer chromatography showed that the enzyme had transglycosylation activity at high temperature and that various transfer products were formed in the reaction with lactose or cellobiose.