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.

Characterization of a three-dimensional organotypic co-culture skin model for epidermal differentiation of rat adipose-derived stem cells

Objective: the organotypic co-culture is a well-known technique to examine cellular interactions and their roles in stem cell proliferation and differentiation. This study aims to evaluate the effects of dermal fibroblasts [DFs] on epidermal differentiation of adipose-derived stem cells [ASCs] using a three-dimensional [3D] organotypic co-culture technique

Materials and Methods: in this experimental research study, rat DFs and ASCs were isolated and cultured separately on electrospun polycaprolactone [PCL] matrices. The PCL matrices seeded by ASCs were superimposed on to the matrices seeded by DFs in order to create a 3D organotypic co-culture. In the control groups, PCL matrices seeded by ASCs were placed on matrices devoid of DFs. After 10 days, we assessed the expressions of keratinocyte-related genes by real-time reverse transcriptase-polymerase chain reaction [RT-PCR] and expression of pan-cytokeratin protein by immunofluorescence in the differentiated keratinocyte-like cells from co-culture and control groups. Keratinocyte-like cell morphologies were also observed by scanning electron microscopy [SEM]

Results: the early, intermediate, and terminal differentiation keratinocyte markers-Cytokeratin14, Filaggrin, and Involucrin significantly expressed in the co-culture groups compared to the control ones [P<0.05]. We observed pan-cytokeratin in keratinocyte-like cells of both groups by immunofluorescence. SEM observation of the co-culture groups showed that the differentiated keratinocyte-like cells developed a polygonal cobblestone shape, considered characteristic of keratinocytes

Conclusion: the 3D organotypic co-culture bilayered construct that consisted of DFs and ASCs was an effective technique for epidermal differentiation of ASCs. This co-culture might be useful for epidermal differentiation of stem cells for future applications in skin regeneration

The influence of substrate topography and biomaterial substance on skin wound healing / 대한해부학회지

Tissue engineering is a new field of which the main purpose is to regenerate and repair the damaged tissues. Scaffolds serve as three dimensional matrices for neo-organogenesis and their substance can be biologic or synthetic. Natural polymers have good interactions with the cells and synthetic biomaterials are also highly useful in biomedical application because of their biocompatible properties. In addition to scaffold substance, surface properties of biomaterials have an important role in tissue engineering. In this study, we examined whether substrate substance is important for wound healing or its surface topography. Therefore, we fabricated two matrices, electrospun polycaprolactone (PCL) nanofibers and collagen/chitosan film, and implanted them to the same rat models. After 2 weeks, the sizes of healing wounds were measured and their cellular structures were evaluated by histochemistry and mmunohistochemistry. Histological staining showed a good level of cellularization and epidermis-dermis formation in PCL implant while no determinable epithelium was observed after 2 weeks in collagen-chitosan graft. Immunohistochemical study demonstrated the highly expressed pancytokeratin in PCL graft while its expression was weak in underdeveloped epidermis of collagen-chitosan implantation. In conclusion, this study suggested that PCL nanofibers with high surface area had a more ideal property than natural collagen-chitosan film, therefore the structure and topography of a matrix seemed to be more important in wound healing than its material substance.

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.

In Vitro Toxic Effects of Zinc Oxide Nanoparticles on Rat Adipose Tissue-Derived Mesenchymal Stem Cells

Objective: Zinc oxide nanoparticles [ZnO-NPs] are increasingly used in sunscreens, biosensors, food additives, pigments, manufacture of rubber products, and electronic materials. There are several studies about the effects of NPs on dermal fibroblast or keratinocytes, but very little attention has been directed towards adipose-derived mesenchymal stem cells [ASCs]. A previous study has revealed that ZnO-NPs restricted the migration capability of ASCs. However, the potential toxicity of these NPs on ASCs is not well understood. This study intends to evaluate the effects of ZnO-NPs on subcutaneous ASCs

Materials and Methods: In this experimental study, In order to assess toxicity, we exposed rat ASCs to ZnO-NPs at concentrations of 10, 50, and 100 Mug/ml for 48 hours. Toxicity was evaluated by cell morphology changes, cell viability assay, as well as apoptosis and necrosis detection

Results: ZnO-NPs concentration dependently reduced the survival rates of ASCs as revealed by the trypan blue exclusion and 3-[4,5-Dimethylthiazol-2-yl]-2,5-diphenyltetrazolium-bromide [MTT] tests. ZnO-NPs, at concentrations of 10 and 50 Mug/ml, induced a significant increase in apoptotic indices as shown by the annexin V test. The concentration of 10 Mug/ml of ZnO-NPs was more toxic

Conclusion: Lower concentrations of ZnO-NPs have toxic and apoptotic effects on subcutaneous ASCs. We recommend that ZnO-NPs be used with caution if there is a dermatological problem

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.

Healing potential of mesenchymal stem cells cultured on a collagen-based scaffold for skin regeneration

Wound healing of burned skin remains a major goal in public health. Previous reports showed that the bone marrow stem cells were potent in keratinization and vascularization of full thickness skin wounds. In this study, mesenchymal stem cells were derived from rat adipose tissues and characterized by flowcytometry. Staining methods were used to evaluate their differentiation ability. A collagen-chitosan scaffold was prepared by freeze-drying method and crosslinked by carbodiimide-based crosslinker. The results of immunecytochemistry and PCR experiments confirmed the adipose-derived stem cells [ASC] in differentiation to the keratinocytes under the treatment of keratinocyte growth factor. The isolated ASC were seeded on the scaffolds and implanted at the prepared wounds. The scaffolds without cells were considered as a control and implanted on the other side of the rat. Histopathological analyses confirmed the formation of new tissue on the scaffold-cell side after 14 days with the formation of dermis and epidermis. These results indicated the capacity of ASC in differentiation to keratinocytes and also wound healing in vivo