Impact of Cell Density on Differentiation Efficiency of Rat Adipose-derived Stem Cells into Schwann-like Cells

BACKGROUND AND OBJECTIVES: Schwann-like (SC-like) cells induced from adipose-derived stem cells (ASCs) may be one of the ideal alternative cell sources for obtaining Schwann cells (SCs). They can be used for treating peripheral nerve injuries. Co-culture with SCs or exposure to glial growth factors are commonly used for differentiation of ASCs to SC-like cells. However, the effect of initial cell density as an inductive factor on the differentiation potential of ASCs into the SC-like cells has not been yet investigated. METHODS AND RESULTS: ASCs were harvested from rat and characterized. The cells were seeded into the culture flasks at three different initial cell densities i.e. 2×10³, 4×10³ and 8×10³ cells/cm² an overnight and differentiated toward SC-like cells using glial growth factors. After two weeks, the differentiation rate of ASCs to SC-like cells at different densities was assessed by immunofluorescence, fluorescence-activated cell sorting analysis and real time RT-PCR. Expression of the typical SCs markers, S-100 proteins and glial fibrillary acidic protein (GFAP) protein, was observed in all cell densities groups although the number of S100-positive and GFAP-positive cells, and the expression of p75(NTR) mRNA, another SC marker, were significantly higher at the density of 8×10³ cells/cm² when compared with the other cell densities groups (p<0.001). CONCLUSIONS: The results suggest that the higher differentiation rate of ASCs to SC-like cells can be obtained at initial cell density of 8×10³ cells/cm², possibly via increased cell-cell interaction and cell density-dependent influence of glial growth factors.

Human sperm quality and metal toxicants: protective effects of some flavonoids on male reproductive function

Background: Metals can cause male infertility through affection of spermatogenesis and sperm quality. Strong evidences confirm that male infertility in metal-exposed humans is mediated via various mechanisms such as production of reactive oxygen species [ROS]. Flavonoids have antioxidant and metal chelating properties which make them suitable candidates for neutralizing adverse effects of metals on semen quality. In the current study, we have evaluated the effects of five types of flavonoids [rutin, naringin, kaempferol, quercetin, and catechin] on recovery of sperm motility and prevention of membrane oxidative damage from aluminum chloride [AlCl3], cadmium chloride [CdCl2], and lead chloride [PbCl4]

Materials and Methods: In this experimental study, motility and lipid peroxidation of metalexposed sperm was investigated in the presence of different concentrations of five kinds of flavonoids. Malondialdehyde [MDA] production was assessed as a lipid peroxidation marker

Results: Aluminum chloride [AlCl3], cadmium chloride [CdCl2], and lead chloride [PbCl4] diminished sperm motility. Treatment of metal-exposed sperm with rutin, naringin, and kaempferol attenuated the negative effects of the metals on sperm motility. Quercetin and catechin decreased the motility of metal-exposed sperm

Conclusion: Based on the MDA production results, only AlCl3 significantly induced lipid peroxidation. Treatment with rutin, naringin, and kaempferol significantly decreased MDA production

Differentiation of adipose-derived stem cells into Schwann-like cells: fetal bovine serum or human serum? / 대한해부학회지

Access to autologous Schwann cells is limited due to lack of donor site and its difficult isolation and culture. Therefore, one of the possible ways to obtain to Schwann cells is to differentiate mesenchymal stem cells into glial pathway using various materials and protocols. The aim of this study was to compare the effects of fetal bovine serum and human serum on Schwann cell differentiation of adipose-derived stem cells to choose the best serum for use in future research. For this purpose, after isolation of human adipose-derived stem cells, it was characterized and differentiated into Schwann cell lineage using two protocols which one of them contained fetal bovine serum and the other human serum. At the end, morphological evaluation declared an increased detachment of cells in response to human serum. On the other side, immunocytochemistry showed that there was a significant increase in the number of cells expressing glial fibrillary acidic proteins and S100 in fetal bovine serum-treated group when compared to human serum-treated one (P<0.05). It was concluded that fetal bovine serum was more effective than allogeneic human serum in Schwann cell differentiation of adipose-derived stem cells.

Expression of surface markers and myogenic potential of rat bone marrow- and adipose-derived stem cells: a comparative study / 대한해부학회지

In recent years, examination and comparison of the biological characteristics of bone marrow- and adipose-derived mesenchymal stem cells (MSCs) from various perspectives have come into the focus of stem cell research, as these cells should be well characterized in order to utilize them in future cellular therapies. Therefore, in the present study, surface protein markers and the skeletal myogenic differentiation potential of rat bone marrow- and adipose-derived MSCs were examined. The expression of CD44, CD45, CD73, and CD90 on bone marrow- and adipose-derived MSCs was characterized using flow cytometry. Subsequently, the stem cells were differentiated into myogenic lineages, and the expression of the skeletal myogenic markers MyoD1, Myog, and Myh2 was studied in cells using real time polymerase chain reaction and immunofluorescence. Our results reveal that the pattern of CD marker expression differs between these 2 types of MSCs to some extent, whereas no significant difference was observed with respect to their myogenic differentiation potential. Therefore, we concluded that despite the differences observed in the biological features of these 2 types of MSCs, their myogenic potential appears to be similar, and that adipose-derived stem cells may be useful in skeletal muscle tissue engineering, due to their easy isolation and capacity for rapid expansion in a short time span.

Morphological changes of bovine nasal chondrocytes induced by interleukin-1 alpha

A study of the histological events under interleukin-1 alpha [IL-1alpha] induction of bovine nasal cartilage [BNC] could result in useful data to better understand the mechanisms involved in tissue breakdown in joint diseases. The aim of this study was to investigate the effects of IL-1alpha on chondrocyte phenotype and extracellular matrix [ECM] changes in BNC explants. In this experimental study, samples were divided into two groups. Group I [control group] BNC explants were cultured only in Dulbecco's modified Eagle's medium [DMEM]. In group II, BNC explants were treated with IL-1alpha [10 ng/ml] for 28 days. Then, samples were harvested on culture days 3, 7, 14, 21 and 28 and chondrocyte morphology and ECM alterations were assessed by invert microscopy and histology by hematoxylin and eosin [H and E] and Alcian blue. Cell viability was evaluated by the lactate dehydrogenase [LDH] assay test. Data were analyzed by the t test and p<0.05 was considered significant. IL-1alpha induced significant morphological changes in cartilage. In the presence of IL-1alpha, most chondrocytes transformed into a fibroblast-like morphology with a granular black point appearance. An increase in the cell: matrix ratio was observed and there were decreased numbers of chondrocytes.IL-1alpha induced breakdown of ECM. We observed partial degradation of ECM between days 7-14 and complete degradation occurred between days 21-28 of culture. The LDH levels increased. IL-1alpha induced morphological changes in chondrocytes and increased destruction of cartilage ECM. There was a parallel correlation between proteoglycan degradation and changes in chondrocyte morpholgy

Protective effects of interleukin-4 on tissue destruction and morphological changes of bovine nasal chondrocytes in vitro

Previous studies have shown that some cytokines have protective effects on cartilage in joint diseases. In the current study, effects of IL-4 against morphological changes and tissue degradation induced by IL-1 Alpha on bovine nasal cartilage [BNC] explants were investigated. Fresh BNC samples were prepared from a slaughterhouse under sterile conditions. BNC explants culture was treated with both IL-l Alpha [10 ng/ml] and IL-4 [50 ng/ml] at the same time for 28 days. The morphological characteristics of explants were assessed by using histology techniques and invert microscopy. Matrix metalloproteinase-1 [MMP-1] production was assessed within different days by using Western blotting. IL-l Alpha induced prominent cartilage morphology degradation. The pro and active form of MMP-1 band substantially increased at day 21 of culture. In the presence of both IL-l Alpha and IL-4, chondrocytes preserved their ordinary normal phenotype with intact extracellular matrix. In addition, a significant reduction in pro-MMP-1and inhibition of active MMP-1 was seen. In conclusion, IL-4 could be regarded as a potential candidate in cartilage protecting against the degradation changes of IL-l Alpha. It seems that the preservation effect of IL-4 is associated with significant reduction of MMP-1

Design and fabrication of anatomical bioreactor systems containing alginate scaffolds for cartilage tissue engineering

The aim of the present study was to develop a tissue-engineering approach through alginate gel molding to mimic cartilage tissue in a three-dimensional culture system. The perfusion biomimetic bioreactor was designed to mimic natural joint. The shear stresses exerting on the bioreactor chamber were calculated by Computational Fluid Dynamic [CFD]. Several alginate/bovine chondrocyte constructs were prepared, and were cultured in the bioreactor. Histochemical and immunohistochemical staining methods for the presence of glycosaminoglycan[GAG], overall matrix production and type II collagen protein were performed, respectively. The dynamic mechanical device applied a linear mechanical displacement of 2 mm to 10 mm. The CFD modeling indicated peak velocity and maximum wall shear stress were 1.706x10-3 m/s and 0.02407 dyne/cm2, respectively. Histochemical and immunohistochemical analysis revealed evidence of cartilage-like tissue with lacunas similar to those of natural cartilage and the production of sulfated GAG of matrix by the chon-drons, metachromatic territorial matrix-surrounded cells and accumulation of type II collagen around the cells. The present study indicated that when chondrocytes were seeded in alginate hydrogel and cultured in biomimetic cell culture system, cells survived well and secreted newly synthesized matrix led to improvement of chondrogenesis

Combination effects of prednisolone and interleukin-4 protect bovine nasal cartilage explants from interleukin-1alpha induced degradation

Current treatments for joint diseases are moderately successful, but unfortunately are associated with significant side effects. This study was undertaken to investigate the combination effects of IL-4 and prednisolone on tissue characteristics and production of matrix metalloproteinase-1[MMP-1] in IL-lalpha-treated bovine nasal cartilage [BNC] explants. BNC explants were cultured in DMEM with IL-lalpha [10 ng/ml], IL-4 [50 ng/ml] and prednisolone [1 or 1,000 nM] at the same time for 28 days. At days 3, 7, 14, 21and 28, the media were collected and replaced with fresh media, and the removed media were stored at -20[degree sign] C. The alterations of tissue characteristics were assessed by using histology techniques. Western-blot method was used to determine the effects of IL-4 and prednisolone combination on MMP-1 production. The cell viability was evaluated by using lactate dehydrogenase assay test. In the presence of IL-lalpha alone, most chondrocytes were transformed into fibroblast-like morphology with pyknotic nuclei at day 28. In addition, a clear band of MMP-1 and extracellular matrix [ECM] degradation were observed. In combination of IL-4 and prednisolone, chondrocytes preserved their ordinary normal features. MMP-1 band formation was completely inhibited and ECM absolutely showed normal characteristics. IL-4 and prednisolone did not show cytotoxicity effects on BNC explant culture. This combination can strongly preserve cartilage from degradation features and the data possibly suggest that the combination of IL-4 and prednisolone could be a candidate for alternative therapy in joint diseases

Degradation of extracellular matrix molecules in interleukin-la treated bovine nasal cartilage

This work aimed to show and compare the degradation time of some of cartilage extracellular matrix components using an in vitro model for cartilage degradation induced by interleukin-la. It is known that elucidation of molecular events under Interleukin-la induction of bovine nasal cartilage could obtain useful data to understand more about involving mechanisms for tissue breakdown in joint disease. The cartilage was taken from an adult bovine in a local slaughterhouse. After removing the whole perichondrium, the equal 2 mm diameter pieces of bovine nasal cartilage were punched out and cultured in Dulbecco's modified Eagle's medium DMEM with or without 10 ng/ml Interleukin-la for 24 days. Each 3 days, the media were removed and exchanged by fresh media, and the removed media were stored in -20°C. Sodium dodecyl sulphate/polyacrylamid-gel electrophoresis SDS-PAGE and Western-blot methods were used for analyzing the samples. The first fragment of fibromodulin [FM] was seen at day 6 and further fragments were appeared at day 18. Cartilage oligomeric matrix protein [COMP] releasing was as a successw pattern during culture period and the first fragment was found at day 6. Collagen IX fragments were seen at day 9 and in a progressive pattern until the end of the study. This study shows that FM and COMP could be considered as the suitable candidates for studying the mechanisms that participate in the cartilage degradations