ABSTRACT
Background: Expansion and differentiation of stem cells relies on the soluble materials as well as the physical conditions of their microenvironment. Several methods have been studied in attempt to enhance the growth and differentiation rates of different adult stem cells extracted from different sources
Objectives: The purpose was to improve the three-dimensional [3D] culture condition of the semi-permeable polymeric beads for encapsulation of the human adipose-derived mesenchymal stem cells [hADSCs] by modifying the ratio of the alginate-gelatin composition
Materials and Methods: Following isolation and characterization of hADSCs by flow cytometry and their functional differentiation, encapsulation in the alginate and alginate/gelatin compositions were performed. Moreover, the stability, swelling, size frequency, growth kinetics, and cytotoxicity of the beads were measured to meet proper condition in the designed experimental and control culture conditions. Finally, the growth rates of the cells in different experimental groups and control were measured and analyzed statistically
Results: Viability decreased in 2 and 3 percent alginate once compared to 1% alginate in beads [p
Conclusions: These findings suggested for the first time that the composite of alginate/gelatin beads with the ratio of 50:50 might provide a suitable culture condition for the encapsulation and in vitro expansion of the hADSCs
ABSTRACT
Piwil2, a member of Ago/Piwi gene family containing Piwi and PAZ domains, has been shown to be ectopically expressed in different cancer cells, especially its remarkable expression in cancer stem cells [CSCs], and is also known to be essential for germ line stem cell self-renewal in various organisms. The hypothesis that CSC may hold the key to the central problem of clinical oncology and tumor relapse leads to more anticancer treatment studies. Due to emerging controversies and extreme difficulties in studying of CSC, like the cells using in vivo models, more attempts have expended to establish different in vitro models. However, the progress was slow owing to the problems associated with establishing proper CSC cultures in vitro. To overcome these difficulties, we prompted to establish a novel stable cell line over-expressing Piwil2 to develop a potential proper in vitro CSC model. In this experimental study, mouse embryonic fibroblasts [MEFs] were isolated and electroporated with a construct containing Piwil2 cDNA under the control of the cytomegalovirus promoter [CMV]. Stable transfectants were selected, and the established MEF-Piwil2 cell line was characterized and designated as CSC-like cells using molecular markers. Functional assays, including proliferation, migration, and invasion assays were performed using characterized CSC like cells in serum-free medium. Additionally, MEF-Piwil2 cell density and viability were measured by direct and indirect methods in normoxic and hypoxic conditions. The results of reverse transcriptase-polymerase chain reaction [RT-PCR], western blot, and immunocytochemistry revealed an overexpression for Piwil2 in the transfected Piwil2 cells both in the RNA and protein levels. Furthermore, analysis of the kinetic and stoichiometric parameters demonstrated that the specific growth rate and the yield of lactate per glucose were significantly higher in the MEF-Piwil2 group compared to the MEF cells [ANOVA, p<0.05]. Also, analysis of functional assays including migration and invasion assays demonstrated a significantly higher number of migrated and invaded cells in the MEF-Piwil2 compared to that of the MEF cells [ANOVA, p<0.05]. The MEF-Piwil2 cells tolerated hypoxia mimetic conditions [CoCl[2]] with more than 95% viability. According to the molecular and functional studies, it has been realized that Piwil2 plays a key role[s] in tumor initiation, progression and metastasis. Therefore, Piwil2 can be used not only as a common biomarker for tumor, but also as a target for the development of new anticancer drug. Finally, the main outcome of our study was the establishment of a novel CSC-like in vitro model which is expected to be utilized in understanding the complex roles played by CSC in tumor maintenance, metastasis, therapy resistance or cancer relapse