Differentiation of canine adipose mesenchymal stem cells into insulin-producing cells: comparison of different culture medium compositions

The aim of this study was to differentiate canine adipose-derived mesenchymal stem cells (ADMSCs) into insulin-producing cells by using culture media with different compositions to determine the most efficient media. Stem cells isolated from the fat tissues close to the bitch uterus were distributed into 6 groups: (1) Dulbecco’s modified Eagle medium (DMEM)-high glucose (HG), β-mercaptoethanol, and nicotinamide; (2) DMEMHG, β-mercaptoethanol, nicotinamide, and exendin-4; (3) DMEMHG, β-mercaptoethanol, nicotinamide, exendin-4, B27, nonessential amino acids, and l-glutamine; (4) DMEMHG, β-mercaptoethanol, and nicotinamide (for the initial 8-d period), and DMEMHG, β-mercaptoethanol, nicotinamide, exendin-4, B27, nonessential amino acids, l-glutamine, and basic fibroblast growth factor (for the remaining 8-d period); (5) DMEMHG and fetal bovine serum; and (6) DMEM-low glucose and fetal bovine serum (standard control group). Adipose-derived mesenchymal stem cells from groups 1 to 5 gradually became round in shape and gathered in clusters.
These changes differed between the groups. In group 3, the cell clusters were apparently more in numbers and gathered as bigger aggregates. Dithizone staining showed that groups 3 and 4 were similar in terms of the mean area of each aggregate stained for insulin. However, only in group 4, the number of insulin aggregates and the total area of aggregates stained were significantly bigger than in the other groups. The mRNA expression of PDX1, BETA2, MafA, and Insulin were also confirmed in all the groups. We conclude that by manipulating the composition of the culture medium it is possible to induce canine ADMSCs into insulin-producing cells, and the 2-staged protocol that was used promoted the best differentiation.

Development of a 3D Tissue-Engineered Skeletal Muscle and Bone Co-culture System.

In vitro 3D tissue-engineered (TE) structures have been shown to better represent in vivo tissue morphology and biochemical pathways than monolayer culture, and are less ethically questionable than animal models. However, to create systems with even greater relevance, multiple integrated tissue systems should be recreated in vitro. In the present study, the effects and conditions most suitable for the co-culture of TE skeletal muscle and bone are investigated. High-glucose Dulbecco’s modified Eagle medium (HGDMEM) supplemented with 20% fetal bovine serum followed by HGDMEM with 2% horse serum is found to enable proliferation of both C2C12 muscle precursor cells and TE85 human osteosarcoma cells, fusion of C2C12s into myotubes, as well as an upregulation of RUNX2/CBFa1 in TE85s.
Myotube formation is also evident within indirect contact monolayer cultures. Finally, in 3D co-cultures, TE85 collagen/hydroxyapatite constructs have significantly greater expression of RUNX2/CBFa1 and osteocalcin/BGLAP in the presence of collagen-based C2C12 skeletal muscle constructs; however, fusion within these constructs appears reduced. This work demonstrates the first report of the simultaneous co-culture and differentiation of 3D TE skeletal muscle and bone, and represents a significant step toward a full in vitro 3D musculoskeletal junction model.

Reduced cystathionine-γ-lyase (CSE) expression is involved in high glucose induced MMP14 expression in adipocytes and adipose tissues.

In the present study, we investigate the effect of reduced cystathionine-γ-lyase (CSE) expression in high glucose induced metalloproteinases14 (MMP14) expression in adipocytes and visceral adipose tissues. Diabetic mice were prepared by injections of STZ and the expression of CSE, MMP14 in visceral adipose tissues were determined. Adipocytes were differentiated from 3T3-L1 cells and treated with high glucose (HG), H2S slow-releasing compound GYY4137 or transfected with CSE siRNA. Then the expression of CSE, MMP14 were determined by western blotting. CSE knockout mice were generated by crossing CSE+/- heterozygous mice and given intraperitoneally (i.p.) injections of GYY4137, and then the expression of CSE and MMP14 in visceral adipose tissues were determined by quantitative real-time PCR and western blotting.
The following results were obtained from the study. In adipose tissues of diabetic mice, the mRNA and protein expression of MMP14 increased while the mRNA and protein expression of CSE decreased. In 3T3-L1 adipocytes, both HG DMEM and CSE siRNA transfection increased the mRNA and protein of MMP14. The addition of GYY4137 inhibited HG-induced upregulation of MMP14 expression. In CSE knockout mice, the mRNA and protein expression of MMP14 in adipose tissues increased, which could be inhibited by i.p. injections of GYY4137. In conclusion, high glucose increased the expression of MMP14 in adipocytes and visceral adipose tissues through inhibiting the expression of CSE.

Probucol promotes high glucose-induced proliferation and inhibits apoptosis by reducing reactive oxygen species generation in Müller cells.

To explore the protective effect of probucol on human retinal Müller cells cultured in high glucose.Primary Müller cells from human retinas were cultured in complete DMEM. Third-generation Müller cells were identified using glutamine synthetase (GS) antibody and randomly divided into three groups: normoglycemia (NG, 5.5 mmol/L); hyperglycemia (HG, 30 mmol/L); and hyperglycemia (30 mmol/L) with probucol (10 μmol/L; HGPB). After a 24-h intervention, cell proliferation, apoptosis, and cellular reactive oxygen species (ROS) were measured with a CCK-8 kit, flow cytometry, and DCFH-DA probe, respectively. Kelch-like ECH-associated protein 1 (Keap1), NF-E2-related factor 2 (Nrf2), and glutamate cysteine ligase catalytic subunit (GCLC) protein expression were detected by immunofluorescence staining.
For NG, HG, and HGPB, optical density (OD) values for cell proliferation were 0.98 ± 0.23, 0.58 ± 0.11, and 0.73 ± 0.11; apoptotic rates were 2.79 ± 0.52%, 7.70 ± 0.44%, and 4.00 ± 0.95%; and intracellular ROS were 20.89 ± 5.14, 55.17 ± 14.07, and 26.28 ± 4.73, respectively. Compared to NG, OD was markedly decreased (P < 0.01), apoptosis was increased (P < 0.001), and intracellular ROS level was significantly higher than in HG (P < 0.01). Compared to HG, OD was markedly increased (P < 0.01), apoptosis was meaningfully decreased (P < 0.01), and intracellular ROS level was significantly lower than in HGPB (P < 0.01). GS, Keap1, Nrf2, and GCLC had positive expression. Probucol could inhibit intracellular ROS generation, promote proliferation, and decrease apoptosis of human retinal Müller cells cultured in high glucose. This might also be associated with Keap1/Nrf2/ARE oxidative stress signaling pathway activation.

High Glucose-reduced Apoptosis in Human Breast Cancer Cells Is Mediated by Activation of NF-κB.

Tumor cells rely on glycolysis for their energy supply with the production of lactate even in normoxia condition, which is named aerobic glycolysis or Warburg effect. Therefore, high glucose (HG) concentration provides a favorable condition for increasing proliferation, angiogenesis and decreasing apoptosis, but its molecular mechanisms are still unknown. The objective of this study is to investigate HG condition on tumor cells behavior including proliferation, apoptosis, and an angiogenesis mediator. In this study, MCF-7 derived from human breast adenocarcinoma, were cultured in DMEM with two different concentrations of glucose for 48 h (5.5 mM as normal glucose (NG) condition and 25 mM as HG condition).

DMEM (HG) With high-glucose, L-glutamine, and sodium pyruvate.

from Caisson Labs
DML10-1000ML | 1000 ml: 80.00 EUR

DMEM (HG) With high-glucose, L-glutamine, and sodium pyruvate.

from Caisson Labs
DML10-500ML | 500 ml: 71.00 EUR

DMEM (HG) With high-glucose, L-glutamine, and sodium pyruvate.

from Caisson Labs
DML10-6X1000ML | 6 x 1000 ml: 148.00 EUR

DMEM (HG) With high-glucose, L-glutamine, and sodium pyruvate.

from Caisson Labs
DML10-6X500ML | 6 x 500 ml: 101.00 EUR

DMEM (HG) With high-glucose, L-glutamine, and sodium pyruvate.

from Caisson Labs
DML26-500ML | 500 ml: 92.00 EUR

DMEM (HG) With high-glucose, L-glutamine, and sodium pyruvate.

from Caisson Labs
DML26-6X500ML | 6 x 500 ml: 217.00 EUR

DMEM (HG) With high-glucose, and L-glutamine. W/O sodium pyruvate.

from Caisson Labs
DML09-1000ML | 1000 ml: 80.00 EUR

DMEM (HG) With high-glucose, and L-glutamine. W/O sodium pyruvate.

from Caisson Labs
DML09-500ML | 500 ml: 71.00 EUR

DMEM (HG) With high-glucose, and L-glutamine. W/O sodium pyruvate.

from Caisson Labs
DML09-6X1000ML | 6 x 1000 ml: 148.00 EUR

DMEM (HG) With high-glucose, and L-glutamine. W/O sodium pyruvate.

from Caisson Labs
DML09-6X500ML | 6 x 500 ml: 101.00 EUR
We used Zingiber officinale extraction for the inhibition of NF-κB. Cell proliferation assay was done by direct counting, cell viability by MTT method, bcl-2 by Immunocytochemistry, apoptosis by Hoechst/PI double staining and vascular endothelium growth factor (VEGF) by ELISA.
Results showed that HG increased lactate production, significantly. HG increased cell proliferation, cell viability, VEGF secretion, and bcl-2 expression while it decreased apoptosis. However, when HG was combined with Zingiber officinale extraction, cell proliferation, cell viability, VEGF secretion and bcl-2 expression decreased and apoptosis increased significantly due to inhibition of NF-κB. Results revealed that HG increased cell proliferation, angiogenesis and decreased apoptosis due to activation of NF-κB pathway. Moreover, the probable mechanism of the activation of NF-κB in HG is increasing reactive oxygen species (ROS) in this condition that can activate NF-κB directly.

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