Characteristics of melanocyte spheroids formed through different biomaterial-induced processes.

BACKGROUND/PURPOSE: Cell behaviors in three-dimensional spheroids are known to be different from those in monolayer cultures; however, very few studies have compared the characteristics of cell spheroids formed through different biomaterial-induced processes. This study investigated the mechanism of melanocyte spheroid formation by using membranes composed of two hydrophilic polymer-based biomaterials, namely chitosan and polyvinyl alcohol (PVA). Our findings revealed that different spheroid-forming processes occurred on the two biomaterials. METHODS: Human melanocytes were provided by the cell bank of the Department of Dermatology, National Taiwan University Hospital. The cell viability was determined through the MTT (3-(4,5-dimethylthiazol-2-yl)-diphenyl tetrazolium bromide; Sigma) colorimetric assay. The cell living rate was determined using the trypan blue exclusion test. The amount of fibronectin adsorbed was quantified through Western blot analysis. Statistical significance was calculated using one-way analysis of variance (ANOVA) followed by Duncan's test, and p values <0.05 or <0.01 was considered significant. RESULTS: In the study, the melanocytes attached to, migrated on, and aggregated on the chitosan surface and then formed spheroids. By contrast, on the PVA surface, the melanocytes directly aggregated to form three-dimensional spheres in suspension. The proliferative ability and survival rate of the melanocytes were considerably higher on the chitosan membranes than on the PVA membranes. CONCLUSION: We concluded that only cell-cell interactions dominated in melanocytes seeded on the PVA membrane, whereas cell-cell and cell-substrate interactions occurred on the chitosan membranes and further enhanced cellular functions. The chitosan-induced spheroids could probably overcome the diffusion and assimilation of trophic factors.

Epithelial cell adhesion molecule (EpCAM) complex proteins promote transcription factor-mediated pluripotency reprogramming.

Epithelial cell adhesion molecule (EpCAM) is a transmembrane glycoprotein that is highly expressed in embryonic stem cells (ESCs) and its role in maintenance of pluripotency has been suggested previously. In epithelial cancer cells, activation of the EpCAM surface-to-nucleus signaling transduction pathway involves a number of membrane proteins. However, their role in somatic cell reprogramming is still unknown. Here we demonstrate that EpCAM and its associated protein, Cldn7, play a critical role in reprogramming. Quantitative RT-PCR analysis of Oct4, Sox2, Klf4, and c-Myc (OSKM) infected mouse embryonic fibroblasts (MEFs) indicated that EpCAM and Cldn7 were up-regulated during reprogramming. Analysis of numbers of alkaline phosphatase- and Nanog-positive clones, and the expression level of pluripotency-related genes demonstrated that inhibition of either EpCAM or Cldn7 expression resulted in impairment in reprogramming efficiency, whereas overexpression of EpCAM, EpCAM plus Cldn7, or EpCAM intercellular domain (EpICD) significantly enhanced reprogramming efficiency in MEFs. Furthermore, overexpression of EpCAM or EpICD significantly repressed the expression of p53 and p21 in the reprogramming MEFs, and both EpCAM and EpICD activated the promoter activity of Oct4. These observations suggest that EpCAM signaling may enhance reprogramming through up-regulation of Oct4 and possible suppression of the p53-p21 pathway. In vitro and in vivo characterization indicated that the EpCAM-reprogrammed iPSCs exhibited similar molecular and functional features to the mouse ESCs. In summary, our studies provide additional insight into the molecular mechanisms of reprogramming and suggest a more effective means of induced pluripotent stem cell generation.

Study on the effects of nylon-chitosan-blended membranes on the spheroid-forming activity of human melanocytes.

Though reported limitedly in tissue engineering, modification of cellular functions can be achieved by culturing them into multicellular spheroids. We have shown melanocytes form spheroids on chitosan surface. However, how biomaterials promote spheroid formation has never been systemically investigated. In this work, nylon, which inhibits melanocyte spheroid formation, and chitosan, which promotes melanocyte spheroid formation, are used to prepare nylon/chitosan-blended membranes. Membranes composed of pure nylon, pure chitosan and various ratios of nylon and chitosan are employed to examine their effects on spheroid formation. Melanocytes show better adhesion to nylon membranes than that to chitosan membranes. In blended membranes, as more nylon is incorporated, cell adhesion increases and the trend for spheroid formation decreases. Melanocytes can only form spheroids on membranes with poorer cell adhesion. Examining the surface of the blended membranes shows phase separation of nylon and chitosan. As nylon content increases, the nylon phase on the membrane surface increases and thereby enhances cell adhesion. The opposite trend for cell adhesion and spheroid formation substantiates our hypothesis of spheroid formation on biomaterials: a balance between cell-substrate interaction and cell-cell interaction. The decrease in cell-substrate interaction tilts the balance to a state more favorable for spheroid formation. Our work can serve as a model to investigate the relative strengths of cell-cell and cell-substrate interactions and also pave way to design blended membranes with desired physical properties while preserving the spheroid-forming activity.

Enhanced cell survival of melanocyte spheroids in serum starvation condition.

Autologous melanocyte transplantation for vitiligo treatment by use of melanocyte suspension has drawbacks including cell damage in cell preparation and transportation, difficult manipulation and low engraftment rate in acral vitiligious lesions. We have proposed the concept of cellular patch as an alternative solution. In the development of melanocyte patches, we have shown that chitosan membrane supports the growth and phenotype expression of melanocytes. Surprisingly, melanocytes spontaneously grow into three-dimensional spheroids on chitosan-coated surface. In this work, we demonstrate that, compared with monolayered melanocytes, melanocyte spheroids show a better survival in growth factor and serum-deprived condition. Survival of melanocytes is further ameliorated when a greater portion of melanocytes is precultured into spheroidal morphology. Melanocyte spheroids disintegrate and the cells return to a physiological dendritic morphology after they are reinoculated on collagen I-coated surface. Our results show that melanocytes are morphologically transformable depending on the substratum used and spheroidal melanocytes have a superior survival to that of monolayered dendritic melanocytes in stringent conditions. Preculturing melanocytes into spheroids can provide melanocytes a survival advantage. Chitosan-based melanocyte patch can be a promising method to enhance the engraftment rate and facilitate the cell preparation and transplantation procedures in melanocyte transplantation for vitiligo treatment.