Chabiotech Co., Ltd.

During embryogenesis, specific proteins expressed in cells have key roles in the formation of differentiated cells and tissues. Delivery of specific proteins into specific cells, both in vitro and in vivo, has proved to be exceedingly difficult. In this study, we developed a safe and efficient protein delivery system using encapsulation of proteins into biodegradable poly-(lactic-co-glycolic acid) (PLGA) nanoparticles (NPs). The PLGA NPs were used to deliver proteins into human mesenchymal stem cells (hMSCs). Fluorescent markers loaded into the PLGA NPs were used to verify the internalization of NPs into hMSCs using FACS analysis and confocal microscopy. With these methods, we demonstrated that the encapsulated model proteins are readily delivered into hMSCs, released from the NP vehicles, and, finally, moved into the cytosols. Using chondrogenesis-related proteins such as aggrecan and cartilage oligomeric matrix protein (COMP), chondrogenic differentiation of hMSCs treated with aggrecan and COMP encapsulated PLGA NPs was clearly observed and caused to differentiate into chondrocytes.

Mesenchymal stem cells (MSCs) have unique immunologic properties that may someday prove useful in cell-based therapy for various degenerative diseases. Its potential is limited, however, by several factors, including the rarity of these cells and difficulty in isolating them. To evaluate their potential as new sources for cell therapy, we isolated MSCs from human fetal tissue (hfMSC) derived from spontaneous abortus (8∼10 weeks) then studied their cell cycle and cell surface marker expression using a fluorescence-activated cell sorter (FACS), as well as the expression of differentiation markers using real-time polymerase chain reaction (RT-PCR). The hfMSCs were able to undergo PCR up to 20 times without displaying significant changes in morphology or expression of various stemness markers (Nanog and human telomerase reverse transcriptase [hAFP]), including germ layer markers (hNF68, alpha-cardiac actin, and hAFP). Also, teratomas were not seen in mice with severe combined immunodeficiency syndrome (SCID) that received a transplantation of hfMSCs with hTERT activity. The FACS analysis revealed that the majority of hfMSCs express mesenchymal markers CD13, CD44, CD71, CD90, CD105, CD253a, and HLA-ABC, but did not express CD31, CD34, CD38, CD45, and HLA-DR. Interestingly, hfMSCs derived from the cell membrane during early passages were negative for both HLA-ABC and HLA-DR, although HLA-ABC expression was detected during later passages (>20 passages). We found that hfMSCs could be differentiated into an osteogenic lineage; this was indicated by modulation of osteoblast markers specific for mRNA. We conclude that hfMSCs could be used as a new source of cells to treat patients with osteogenic diseases, as well as to understand the mechanisms of immunosuppression by MSCs.

The establishment of new technology for genetic modification in human embryonic stem (ES) cell lines has raised great hopes for achieving new ground in basic and clinical research. Recently, lentiviral vector technology has been shown to be highly effective and therefore could emerge as a popular tool for human ES cell genetic modification. The objectives of this study were to evaluate the efficiency of promoters in lentiviral gene delivery systems in mammalian ES cells, including mouse, monkey, and human, and to construct efficient and optimized conditions for lentivirus-mediated transfection systems. Mammalian ES cells were transfected with self-inactivating (SIN) human immunodeficiency virus type-1 (HIV-1)-based lentiviral vectors containing the human polypeptide chain elongation factor-1alpha (EF-1alpha) promoter or cytomegalovirus (CMV) promoter and analyzed by fluorescence-activated cell sorting (FACS) analysis for the expression of the enhanced green fluorescent protein (eGFP) reporter gene. The efficiency of the EF-1alpha promoter was higher than that of the CMV promoter in all ES cells tested. The EF-1alpha promoter efficiently drove gene expression (14.74%) compared with CMV promoter (3.69%) in human ES cells. We generated a stable eGFP+ human ES cell line (CHA3-EGFP human ES cells) that continuously expressed high levels of EGFP ( approximately 95%) from the EF-1alpha promoter and was maintained for up to 60 weeks with undifferentiated proliferation. The established CHA3-EGFP human ES cell lines were characterized as being negative for nondifferentiation markers and teratoma formation. These results imply that genetic modification by lentiviral vectors with specific promoters in ES cells constitute a powerful tool for guided differentiation as well as gene therapy.