Ex vivo cartilage defect model for the evaluation of cartilage regeneration using mesenchymal stem cells

An ex vivo cartilage defect model for the evaluation of cartilage regeneration using mesenchymal stem cells (MSCs) was developed. Porcine chondrocytes and human MSCs were transplanted into cartilage defects created on the porcine osteochondral and chondral discs and cultivated for 3 weeks. Although the regeneration of cartilage-like tissues was observed after the transplantation of chondrocytes to defects on both of the osteochondral and chondral discs, the transplanted MSCs formed cartilage-like tissues only in the defect on the chondral disc, in which an in vivo cartilage-like structure was partly observed, and a degraded tissue was observed in the defect on the osteochondral disc. The effects of medium additives such as serum, transforming growth factor-β3 (TGF-β3), and fibroblast growth factor-2, and the transfection of TGF-β3 gene to MSCs could be clearly estimated using the cartilage defect model. In conclusion, a chondral disc with defects is useful for evaluating cartilage regeneration using MSCs.     

Lipopolysaccharide induces lipolysis and insulin resistance in adipose tissue from dairy cows

Intense and protracted adipose tissue (AT) fat mobilization increases the risk of metabolic and inflammatory periparturient diseases in dairy cows. This vulnerability increases when cows have endotoxemia—common during periparturient diseases such as mastitis, metritis, and pneumonia—but the mechanisms are unknown. Fat mobilization intensity is determined by the balance between lipolysis and lipogenesis. Around parturition, the rate of lipolysis surpasses that of lipogenesis, leading to enhanced free fatty acid release into the circulation. We hypothesized that exposure to endotoxin (ET) increases AT lipolysis by activation of classic and inflammatory lipolytic pathways and reduction of insulin sensitivity. In experiment 1, subcutaneous AT (SCAT) explants were collected from periparturient (n = 12) Holstein cows at 11 ± 3.6 d (mean ± SE) before calving, and 6 ± 1 d and 13 ± 1.4 d after parturition. Explants were treated with the endotoxin lipopolysaccharide (LPS; 20 µg/mL; basal = 0 µg/mL) for 3 h. The effect of LPS on lipolysis was assessed in the presence of the β-adrenergic agonist and promoter of lipolysis isoproterenol (ISO; 1 µM; LPS+ISO). In experiment 2, SCAT explants were harvested from 24 nonlactating, nongestating multiparous Holstein dairy cows and exposed to the same treatments as in experiment 1 for 3 and 7 h. The effect of LPS on the antilipolytic responses induced by insulin (INS = 1 µL/L, LPS+INS) was established during ISO stimulation [ISO+INS, LPS+ISO+INS]. The characterization of lipolysis included the quantification of glycerol release and the assessment of markers of lipase activity [adipose triglyceride lipase (ATGL), hormone-sensitive lipase (HSL), and phosphorylated HSL Ser563 (pHSL)], and insulin pathway activation (AKT, pAKT) using capillary electrophoresis. Inflammatory gene networks were evaluated by real-time quantitative PCR. In periparturient cows, LPS increased AT lipolysis by 67 ± 12% at 3 h across all time points compared with basal. In nonlactating cows, LPS was an effective lipolytic agent at 3 h and 7 h, increasing glycerol release by 115 ± 18% and 68.7 ± 16%, respectively, relative to basal. In experiment 2, LPS enhanced ATGL activity with minimal HSL activation at 3 h. In contrast, at 7 h, LPS increased HSL phosphorylation (i.e., HSL activity) by 123 ± 11%. The LPS-induced HSL lipolytic activity at 7 h coincided with the activation of the MEK/ERK inflammatory pathway. In experiment 2, INS reduced the lipolytic effect of ISO (ISO+INS: ?63 ± 18%) and LPS (LPS+INS: ?45.2 ± 18%) at 3 h. However, the antilipolytic effect of INS was lost in the presence of LPS at 7 h (LPS+INS: ?16.3 ± 16%) and LPS+ISO+INS at 3 and 7 h (?3.84 ± 23.6% and ?21.2 ± 14.6%). Accordingly, LPS reduced pAKT:AKT (0.11 ± 0.07) compared with basal (0.18 ± 0.05) at 7 h. Our results indicated that exposure to LPS activated the classic and inflammatory lipolytic pathways and reduced insulin sensitivity in SCAT. These data provide evidence that during endotoxemia, dairy cows may be more susceptible to lipolysis dysregulation and loss of adipocyte sensitivity to the antilipolytic action of insulin.     

Novel gene-modified-tissue engineering of cartilage using stable transforming growth factor-beta1-transfected mesenchymal stem cells grown on chitosan scaffolds

Rabbit bone marrow-derived mesenchymal stem cells (MSCs) were stably transfected with the TGF-beta1 gene in monolayer culture using Lipofectamine 2000. After transfection, the expression of cartilage-specific extracellular matrix was upregulated, whereas matrix metalloproteinases 1 and 3 (MMP 1 and 3) protein expressions and enzymatic activities were downregulated. Autologous MSCs modified with the TGF-beta1 gene were seeded into chitosan scaffolds to construct gene-modified cartilage, which was then implanted into the full-thickness articular cartilage defects of rabbits' knees. Twelve weeks after implantation, the defects were filled with regenerated hyaline-like cartilage tissue as confirmed by the positive immunohistochemical staining of collagen type II and intense toluidine blue staining of proteoglycan. Our findings suggest that the repair of cartilage defects can be enhanced by TGF-beta1 gene-modified-tissue engineering of cartilage on the basis of a strategy using MSCs, chitosan, and liposomal transfection.     

Effect of ascorbic acid on bone marrow-derived mesenchymal stem cell proliferation and differentiation

Mesenchymal stem cells (MSCs) derived from bone marrow are an important tool in tissue engineering and cell-based therapies because of their multipotent capacity. Majority of studies on MSCs have investigated the roles of growth factors, cytokines, and hormones. Antioxidants such as ascorbic acid can be used to expand MSCs while preserving their differentiation ability. Moreover, ascorbic acid can also stimulate MSC proliferation without reciprocal loss of phenotype and differentiation potency. In this study, we evaluated the effects of ascorbic acid on the proliferation, differentiation, extracellular matrix (ECM) secretion of MSCs. The MSCs were cultured in media containing various concentrations (0-500 microM) of L-ascorbate-2-phosphate (Asc-2-P) for 2 weeks, following which they were differentiated into adipocytes and osteoblasts. Ascorbic acid stimulated ECM secretion (collagen and glycosaminoglycan) and cell proliferation. Moreover, the phenotypes of the experimental groups as well as the differentiation potential of MSCs remained unchanged. The apparent absence of decreased cell density or morphologic change is consistent with the toxicity observed with 5-250 microM concentrations of Asc-2-P. The results demonstrate that MSC proliferation or differentiation depends on ascorbic acid concentration.     

Photoperiod affects gene expression of leptin and leptin receptors in adipose tissue from lactating dairy cows

Leptin is mainly secreted by adipocytes and is implicated in the regulation of metabolic status, feed intake, and body condition. Day length (DL) can affect leptin gene expression and secretion. The aim of the study was to evaluate the effect of DL on gene expression of leptin and leptin receptors in adipose tissue (AT). Four lactating and pregnant Holstein cows were housed in a climate-controlled chamber for 51 d. The first 30 d were used to adapt animals to the new housing conditions. During that period the DL adopted was 12 h light:12 h dark (12:12). The experimental period included 3 different and consecutive phases: 7 d of neutral DL (12:12); 7 d of long DL (18 h light:6 h dark); and 7 d of short DL (6 h light:18 h dark). Subcutaneous AT biopsies were performed at the end of each phase. Prolactin, growth hormone, cortisol, leptin, glucose, nonesterified fatty acids, β-OH-butyrate, and cholesterol were determined in plasma samples. Abundance of leptin mRNA, and Ob-Ra and Ob-Rb leptin receptor mRNA were determined in AT samples by ribonuclease protection assay. Day length did not affect feed intake or body condition score. Exposure to short DL significantly reduced milk yield (13.1 ± 2.2 vs. 15.8 ± 1.7 and 16.0 ± 2.0 kg/d for short vs. neutral and long DL, respectively). Plasma leptin, growth hormone, cortisol, nonesterified fatty acids, β-OH-butyrate, and glucose were not affected by DL; cholesterol was lowest under short DL (3.93 ± 0.38 vs. 4.36 ± 0.39 and 4.07 ± 0.38 mmol/L for short vs. neutral and long DL, respectively). Prolactin increased under long DL (134.82 ± 16.94 vs. 81.98 ± 20.25 and 96.16 ± 0.38 ng/mL for long vs. neutral and short DL, respectively). Gene expression of leptin and its receptors was affected by DL. Leptin mRNA increased under long DL (11.91 ± 0.84 vs. 7.82 ± 0.84 and 7.56 ± 0.84 pg of mRNA/μg of total RNA for long vs. neutral and short DL, respectively). Leptin receptors Ob-Ra and Ob-Rb mRNA were higher under long DL, whereas Ob-Ra and Ob-Rb mRNA were lower under short DL (Ob-Ra: 1.91 ± 0.41, 2.49 ± 0.41, and 0.65 ± 0.41 pg of mRNA/μg of total RNA for neutral, long, and short DL, respectively; Ob-Rb: 5.29 ± 0.79, 5.98 ± 0.68, and 2.02 ± 0.70 pg of mRNA/μg of total RNA for neutral, long, and short DL, respectively). Results of the present study appear to exclude an effect of feed intake and metabolic status on leptin gene expression. A prolactin-mediated effect of photoperiod on AT leptin modulation may be proposed in lactating dairy cows.     

Glucose-stimulated insulin secretion of various mesenchymal stem cells after insulin-producing cell differentiation

Mesenchymal stem cells (MSCs) are capable of crossing germinative layer borders and are obtainable in high numbers via in vitro cultures. Therefore, many researchers have searched for diverse sources of MSCs. Recently the generation of glucose-responsive insulin-producing cells (IPCs) from MSCs has shown immense potential for the treatment of type 1 diabetes mellitus (T1DM) due to a lack of pancreas donors. In this study, we compared the growth potency of four kinds of MSCs derived from bone marrow, Wharton's jelly, adipose tissue, and the periosteum. In addition, in vitro differentiation of these MSCs into IPCs was also investigated. After 2weeks of IPCs differentiation, we compared the expression of the insulin gene and protein using RT-qPCR and immunofluorescence staining. Only IPCs derived from periosteum-derived progenitor cells (PDPCs) showed a response to glucose concentration. Glucose stimulated insulin secretion was conclusive evidence of the potential functionality of IPCs. Therefore, PDPCs are a promising alternative stem cell source for IPCs differentiation.     

Combination of cytomegalovirus enhancer with human cellular promoters for gene-induced chondrogenesis of human bone marrow mesenchymal stem cells

High-expression plasmid vectors for human mesenchymal stem cells (MSCs) were constructed by combination of cytomegalovirus immediate-early enhancer with cellular promoters. MSCs transfected with the vector showed higher transgene production of a cytokine, which increased the differentiation level to chondrocytes.     

Generation of human induced pluripotent stem cells from oral mucosa

Induced pluripotent stem (iPS) cells are one of the most promising sources for cell therapy in regenerative medicine. Using a patient's own genetically identical and histocompatible cells is the ideal way to practice personalized regenerative medicine. For personalized iPS cell therapy, the prerequisites for cell source preparation are a simple and safe procedure, no aesthetic or functional damage, and quick wound healing. Oral mucosa fibroblasts (OFs) may have high potential to fulfill these requirements. In this study, biopsy was performed in a dental chair; no significant incisional damage was recognized and rapid wound healing (within a week) was observed. We generated human iPS cells from the isolated OFs via the retroviral gene transfer of OCT4, SOX2, c-MYC, and KLF4. Reprogrammed cells showed ES-like morphology and expressed undifferentiated markers such as OCT4, NANOG, SSEA4, TRA-1-60, and TRA-1-81. Subsequent in vitro and in vivo analyses confirmed the pluripotency of resultant iPS cells, which matched the criteria for iPS cells. In addition, we found that the endogenous expression levels of c-MYC and KLF4 in OFs were similar to those in dermal fibroblasts. Taken together, we propose that OFs could be a practical source for preparing iPS cells to achieve personalized regenerative medicine in the near future.