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

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.

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

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

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

Immunohistochemical assessment of galectin-3 during pre-implantation in mouse endometrium

Background: galectin-3 [Gal-3], a beta-galactoside-binding lectin, is a multifunctional lectin that involves in a number of critical biological processes

Objective: the purpose of this study was to investigate the expression pattern of Gal-3 in mouse endometrium during estrus phase of estrous cycle and pre-implantation

Materials and Methods: in this experimental study 42 NMRI female mice were divided in seven different groups. Ovulation in NMRI female mice was stimulated by injecting hMG and hCG. Estrus phase was considered as stimulated and un-stimulated groups. The other groups of mice were mated, and the day of vaginal plug formation was considered as the day 1 of pregnancy. The mice of all groups were sacrificed on different days of pre-implantation period and their uterine horns were fixed and avid in- biotin complex method of immunohistochemistry [IHC] was applied

Results: in estrus group, Gal-3 immunoreactivity in luminal epithelium was strong, in stromal cells very strong, in glandular epithelium very weak and endothelial cells very strong. No identifiable difference was observed in un-stimulated and stimulated estrus phase. In test groups, days 1-2, insignificant difference of Gal-3 expression was observed. On day 3, luminal epithelium and stromal cells showed significant decrease in comparison to estrus and day 1 [p=0.001]. On the 4[th] and 5[th] days, luminal epithelium and stromal cells showed significant decrease in comparison to estrus phase and days 1-3 [p=0.0001]

Conclusion: the data suggested that successful implantation is probably associated with the downregulation of Gal-3 in the mouse endometrium at the beginning of pregnancy

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

Focal adhesion kinase [FAK] involvement in human endometrial remodeling during the menstrual cycle

Endometrial remodeling occurs during each menstrual cycle in women. Reports have shown that, in a variety of cell types, processes such as proliferation, signaling complex formation and extra cellular matrix remodeling require a cytoplasmic tyrosine kinase, focal adhesion kinase [FAK]. The present study has focused on the expression pattern of FAK in human endometrium during the menstrual cycle. The purpose of this study was to ascertain the probable function of FAK in menstrual cycle changes and the role of FAK in tissue repair and tissue remodeling in vivo. Formalin-fixed paraffin-embedded endometrial samples were obtained from 400 pre-menopausal, non-pregnant women, who underwent hysterectomy and biopsy for benign diseases. Forty six samples with no tissue abnormalities were studied and ABC staining method of immuno-histochemistry methods was applied. Positive staining of FAK by different cell types of human endometrium was scaled and compared with each other by using histologic score method. All different cell types of endometrium showed various patterns of FAK expression in different stages of menstruation. FAK in glandular and luminal epithelial cells is up-regulated during the early proliferative [EP] to mid-secretory [MS] phases. FAK in stromal cells is up-regulated during the EP, early and MS phases in comparison to the late secretory [LS] phase. FAK expression in endothelial cells is up-regulated during the EP and MS phases in comparison to LS phase. This study showed that endometrial FAK expression is a phase-dependent manner during the menstrual cycle. It appears that up-regulation of FAK during the proliferative phases is responsible for endometrial regeneration and high expression of FAK in the EP and MS phases may associate with the implantation. Down-regulation of FAK during the LS phase may facilitate apoptosis in human endometrium. It seems that FAK as a key kinase plays a critical role in endometrial remodeling that it may regulate by steroid hormones

expression of signal regulatory protein-alpha in normal and osteoarthritic human articular cartilage and its involvement in chondrocyte mechano-transduction response

Signal regulatory proteins [SIRP] belong to immunoglobulin super family [IgSF] and relate to integrin signaling cascades. It has been shown that SIRP alpha is expressed in a variety of cells including myeloid cells and neurons. In the present study the expression of this IgSF member in articular chondrocytes was investigated. Using a panel of anti-SIRPa antibodies, immunohistochemistry, Western-blotting and electrophysiology methods, expression of SIRPa and its role in chondrocyte mechano-transduction were assessed. No identifiable positive signal was obtained by using immunohistochemistry methods on frozen and paraffin sections. SIRP alpha is expressed by both normal and osteoarthritis cultured chondrocytes. The electrophysiological response of chondrocytes in the presence of SE7C2 mAb was significantly inhibited whereas; SE5A5 did not show any modification in this response. It seems likely that SIRP alpha could be associated with other proteins such as integrins, CD47 and ion channels, which contribute to the electrophysiological response of human articular chondrocytes. In any case, this study has provided a specific functional role for SIRP alpha in chondrocyte mechano-transduction

Evaluation of CD98 expression in normal and osteoarthritic human articular chondrocytes

Recent studies have provided evidence that integrins play roles in recognition of mechanical stimuli and its translation into a cellular response. Integrin signaling may be regulated by a number of mechanisms including accessory proteins such as CD98 [4F2 antigen]. To determine CD98 expression by human articular chondrocytes and its involvement in human articular mechanotransduction. CD98 expression was assessed by immunostaining of cryostat sections of snap frozen articular cartilage and in cultured cells by western blotting. Chondrocytes enzymatically isolated from macroscopically normal and osteoarthritic [OA] articular cartilage were grown in short term, primary monolayer culture and used in a resting state or following mechanical stimulation at 0.33Hz. Human articular chondrocytes express CD98 and immunoreactivity revealed a similar heterogeneous pattern of CD98 in both normal and osteoarthritic [OA] human articular cartilage. No role of CD98 was detected by electrophysiological study. It appears that CD98 is expressed in a similar pattern in both normal and osteoarthritic [OA] cartilage. Although we detected no role for CD98 in chondrocyte mechanotransduction, it may be involved in other biological functions in chondrocyte intracellular signaling events

Differential immunohistochemical expression pattern of galectin-3 in normal and osteoarthritic human articular cartilage

Previous studies have shown that Galectin-3, a member of lectin family, is expressed in developing cartilage in mouse embryo and also in growth plate of long bones. In the present work, the expression pattern of Galectin-3 in normal and various grades of osteoarthritic [OA] human articular cartilage has been studied. Using immunohistochemistry and standard western blotting, the in vivo and in vitro expression pattern of Galectin-3 in normal and OA human articular cartilage were assessed. Immunohistochemical studies showed a similar pattern of Galectin-3 expression in normal and mild OA but severe OA showed different strong expression in all zones of human articular cartilage. Increased expression pattern of Galectin-3 in advanced stages of OA may occur as a result of the imbalance of chondrocyte homeostasis that occurs in OA cartilage and provides a condition to modify normal chondrocyte to an OA chondrocyte

Isolation and differentiation of mouse embryonic stem cells

Recently, embryonic stem [ES] cells have become very important resources in basic medical researches. These cells can differetiate into derivatives of all primary germ layers. In order to isolate embryonic stem cells in vitro, the blastocyst were cultured and the morphological aspects, population doubling time, alkalin phosphatse and differentiation properties of the cells were investigated. The balstocysts from NMRI mice were cultured for 3 days up to time that inner cell mass [ICM] reach to the outgrowth stage. The cells were disaggregated and trypsinized every 3 days until the appearance of the colonies of ES cells. The colony positive cells were fixed and stained for alkaline phosphatase. The ES cells were cultured in suspension state for 5 days, at the same time Leukaemia Inhibitory Factor [LIF] was removed from media to form embryoid bodies[EBs]. The EBs were cultured for 8 - 20 days on collagen coated dish to induce the spontaneouse differentiation. During the 6-9 days after the disaggregation of ICM in the expansion stage, the colony of ES cells appeared as a flat monolayer mass with strike boundaries and nondistinguish cytoplasm including a few nuclei. In colony formation stage, the morphology changed from flat monolayer to round multilayer with strike define boundaries. Undifferentiated cells were seen as intensely small cells attached together compactly with high nucleus/cytoplasm [N/C] ratio. The cells of colonies tend to differetiate by separation from each other and became larger and diffused on substrate by attaching to dish. The positive alkaline phosphatase cells were seen in typical morphology of ES colonies. The EBs cells were seen in culture after 5 days in suspension and began to spontaneously differentiate into various types of cells such as nerve and hematopoitic lineages.Despite strike morphology of ES colonies, it is difficult to distinguish the differentiated from undifferentiated cell colonies in the colony formation stage. New ES cells are capable to give rise into EBs and are susceptible of spontaneously differentiation in various type of cells