Nanotechnology has made a substantial impact on the introduction of nanomedicine. gene transfer demonstrated 17.2% of transfection effectiveness and a lot more than 80% of cell viability in stem cells. These ideals were greater than that of PEI. The manifestation of the shipped BMP2 gene in stem cells improved the osteogenic differentiation. These outcomes proven MK-8776 cost that chitosan-g-PEI can be with the capacity of applying in providing gene to stem cells and offering potential applications in stem cell-based gene therapy. and [14,15]. Nevertheless, this vector possesses high cytotoxicity with dose and molecular weight dependency [16] relatively. It hasn’t yet been found in human being research therefore. Lately, many pilot research had proven how the mix of chitosan and PEI can concurrently improve the transfection effectiveness and reduce the cytotoxicity [17-19]. This method could possibly be improved using the properties of targeted delivery [20-23] additional, prolonged blood flow [20], and stimuli-responsive [24] by particular structure modification. Nevertheless, many of these research were carried out in tumor cells such as HeLa [20,24-26], HepG2 [27], and A549 cells [28], or MK-8776 cost targeted for tumor treatment [21,29-31]. There are only a few studies MK-8776 cost left using it to deliver gene to somatic cell such as murine macrophage cells [22] and osteoarthritis [32]. In our previous study, the bioreducible low molecular weight PEI-conjugated chitosan (chitosan-g-PEI) was developed, characterized, and applied to deliver gene to osteoblast cells [33]. It was also simply tried in stem cells. However, there is a lack of a detailed study focused on the behavior of this vector in stem cells, which is very important in the regenerative medicine. Vectors usually show the cell type-dependent transfection properties because of the differences in cell cycle, cell division frequency, endocytic capacity, and metabolic activity [34]. Mesenchymal stem cells (MSCs) are usually more difficult to transfect [35]. In recent years, the investigation of MSCs and their clinical application have attracted extensive interests. Some nonviral vectors have demonstrated their efficiency in delivering BMP2 gene to MSC such as liposome and PEI [36,37]. So far, there are few nonviral vectors that have been applied in stem cells, leaving very limited choices for stem cell-based gene therapy. Therefore, chitosan-g-PEI should be expected to show its effect on stem cells. In this study, chitosan-g-PEI was evaluated on delivering BMP2 gene to bone marrow stem cells and compared with chitosan and PEI in terms of the transfection properties and the transgene function differentiation of BMSC into multilineage cells To assess the multilineage differentiation capacity, the obtained bone marrow stem cells (BMSC) underwent osteogenic, adipogenic, and chondrogenic induction by different culture media. For osteogenic differentiation, cells were cultured with osteogenic medium with -MEM supplemented with 10% FBS, 10?7?M dexamethasone (Sigma-Aldrich), 10?mM -glycerol phosphate (Sigma-Aldrich), and 50?mM ascorbate-2-phosphate (Sigma-Aldrich). After 3?weeks of differentiation, the mineralization was stained by Alizarin Red S staining. For adipogenic differentiation, cells were cultured with -MEM supplemented with 10% FBS, 10?6?M dexamethasone, 0.5?M isobutylmethylxanthine (IBMX, Sigma-Aldrich), and 10?ng/mL of insulin (Sigma-Aldrich) for 2?weeks. Lipid accumulation was identified by Oil Red O staining. For chondrogenic differentiation, cells (1??106) were seeded in polypropylene tubes with DMEM supplemented with 10?7?M dexamethasone, 1% insulin-transferrin-selenium (ITS, Sigma-Aldrich), 50?M ascorbate-2-phosphate, 1?mM sodium pyruvate (Sigma-Aldrich), 50?g/mL of proline (Sigma-Aldrich), and 20?ng/mL of TGF-3 (R&D Systems, Minneapolis, MN, USA). After 3?weeks in culture, the pellets MK-8776 cost were fixed in 10% buffered formalin for 48?h and embedded in paraffin. Then, 4?m thick MK-8776 cost sections were processed for toluidine blue staining (Sigma-Aldrich). Transfection efficiency and cytotoxicity The transfection efficiency was investigated by flow cytometry. Cells were seeded in 6-well plates at an initial density of 4??105 cell well?1 and allowed to reach 70% to PSEN1 80% confluence. Before transfection, cells were washed with.