To overcome loss of stem-like properties and spontaneous differentiation those hinder the expansion and application of human mesenchymal stem cells (hMSCs), we have clonally isolated permanent and stable human MSC lines by ectopic overexpression of primary cell cultures of hMSCs with HPV 16 E6E7 and human telomerase reverse transcriptase (hTERT) genes. These cells were found to have a differentiation potential far beyond the ordinary hMSCs. They expressed trophoectoderm and germline specific markers upon differentiation with BMP4 and retinoic acid, respectively. Furthermore, they displayed higher osteogenic and neural differentiation efficiency than primary hMSCs or hMSCs expressed HPV16 E6E7 alone with a decrease in methylation level as proven by a global CpG island methylation profile analysis. Notably, the demethylated CpG islands were highly associated with development and differentiation associated genes. Principal component analysis further pointed out the expression profile of the cells converged toward embryonic stem cells. These data demonstrate these cells not only are a useful tool for the studies of cell differentiation both for the mesenchymal and neurogenic lineages, but also provide a valuable source of cells for cell therapy studies in animal models of skeletal and neurological disorders.
Fibroblast heterogeneity in human periodontal tissues was characterized by cloning and immuno-histochemical techniques. Cell suspensions from primary cultures gingival (GF) and PDL fibroblasts (PF) were cloned. The relative intensity of double-labeled immunofluorescence, using specific antibodies to the extracellular matrix (ECM) molecules collagen type I (CI), type III (CIII), and fibronectin (Fn), was measured by photometry. Most clones derived from either GF or PF showed positive intracellular staining for both CI and Fn, and CIII and Fn. However, there were variations in fluorescence intensity for CI and Fn, ranging from relatively weak to strongly positive. The fluorescence for CI and CIII was relatively weak in most isolated GF clones in contrast to their PF clones. These observations coupled with studies of growth and cellular morphology in individual clones suggest that: 1) GF and PF contain functionally heterogeneous subpopulations; and 2) the synthesis and expression of extracellular matrix molecules of GF may be essentially different from that of PF.
In the present work, RGDS (Arg-Gly-Asp-Ser) was immobilized on PLLA scaffolds with plasma treatment. The amount of immobilization, determined by HPLC, was confirmed to be in the effective order. Results from the culture of rat osteosarcoma (ROS), osteoblastic-like cells, demonstrate that the immobilization of RGDS could effectively enhance the attachment of ROS cells on PLLA and increase the cell density in PLLA scaffolds. In addition, experiments of in vitro mineralization indicate that there were more cells and mineralization focci in the RGDS-immobilized scaffolds, suggesting a tendency to form bone-like tissues, compared with the unmodified PLLA scaffold. On the other hand, the PLLA scaffolds immobilized with RGES (Arg-Gly-Glu-Ser) were much less effective in promotion of ROS attachment, suggesting that the enhancement on cell attachment was mainly due to the recognition of RGDS by the adhesion receptors on the cell membrane. The results presented in this work demonstrate that RGDS could be successfully immobilized on PLLA scaffolds with plasma treatment and such modification can make PLLA scaffolds more suitable for culture of osteoblast-like cells and for generation of bone-like tissues.
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