Although p53 and Ras have different molecular pathways, together they control key cellular procedures (38)

Although p53 and Ras have different molecular pathways, together they control key cellular procedures (38). with liposomes carrying exogenous genes, have excellent mobility, are capable of undergoing multilineage differentiation (2) and have low immunogenicity (3). These properties make BM-MSCs effective carrier cells for biological treatments of tumors. Using stem cells as carriers to target drug delivery to malignant tumors alone may reduce the adverse reactions caused by systemic drug distribution (4). Furthermore, using genetically altered BM-MSCs as tumor target gene therapy vectors may enhance anti-tumor effects, providing a novel method for tumor therapy (5,6). The stem cell niche is the microenvironment in which stem cells exist. The stem cell niche allows conversation between stem cells to regulate their function and fate, and it is a critical factor in stem cell homeostasis. The stem cell niche is able to tightly regulate stem cell self-renewal and proliferation by signal molecules (7). It has been reported that BM-MSCs undergoing long-term culture may undergo spontaneous changes in terms of their biological characteristics, and may even undergo malignant transformation (8C10). These results suggest that alterations to the cell microenvironment may affect the differentiation and proliferation of stem cells; however, the molecular mechanisms responsible for these alterations have not been fully elucidated. It has not yet been reported whether changes to BM-MSC biological characteristics in the lung microenvironment are caused by cytokines, signaling molecules or cellular interactions. To identify the risk of BM-MSCs undergoing malignant transformation when being used for biological therapies in the tumor microenvironment, the present study utilized a Transwell chamber to co-culture BM-MSCs and lung cancer A549 cells to simulate a tumor microenvironment. From this, it was possible to investigate whether BM-MSCs are able to spontaneously undergo changes in proliferation, migration and differentiation in the tumor microenvironment and whether it was possible to maintain BM-MSC genetic stability in these specific culture conditions. The results of the current study may provide an experimental basis for the clinical application of stem EPHA2 cell therapy. Materials and methods Cells and cell culture BM-MSCs (Cyagen Biosciences, Inc., Santa Clara, CA, USA) and human lung cancer A549 cells (stored in the Provincial-Level Key Laboratory for Molecular Medicine of Major Diseases and The Prevention and Treatment with Traditional Chinese Medicine Research in Gansu Colleges and Universities, Lanzhou, China) were cultured in complete medium, consisting of Dulbecco’s altered Eagle’s medium/F12 supplemented with 10% fetal bovine serum (Hyclone; GE Healthcare Life Sciences, Logan, UT, USA). The culture medium was replenished every 2C3 days. Cell aggregates were typically formed after 24 h incubation in a humidified chamber at 37C (5% CO2). Cell aggregates were grown in suspension for 3C5 days before they began to attach to the bottom of the culture bottle. When the cells covered 80C90% of NGD-4715 the bottom of the bottle, they were digested with 0.25% trypsin to perform a co-culture experiment. Establishment of co-culture system A non-contact co-culture system of NGD-4715 BM-MSCs and lung cancer A549 cells was established using a Transwell suspension culture chamber with polyethylene terephthalate film combined with a 6-pore plate (Corning 3450; Corning, Inc., NGD-4715 Corning, NY, USA). The BM-MSC and A549 groups were groups in which BM-MSC cells and A549 cells were cultured respectively, in impartial wells NGD-4715 of a 6-well plate. The co-BM-MSC group, including BM-MSCs and A549 cells, co-cultured in the transwell system (BM-MSCs in the upper chamber and A549 cells in the lower chamber). The number of cells seeded per chamber for each group is usually 5104 cells. Cells were cultured in 6-well plates (Corning 3450) made up of the aforementioned complete medium at 37C (5% CO2 incubator). Culture medium was replenished every 48 h and cell growth state was observed under an inverted microscope. On day 7 of culture, cell culture was terminated and single cell suspensions were prepared for detection. Analysis of cell morphology, cell cycle and cell viability The aforementioned cells were observed every 24 h during culture periods to detect changes in cell morphology using an inverted microscope. The partial harvested cell suspensions were fixed at 4C in 70% ethanol overnight. Propidium iodide (PI) and RNase A were subsequently added (final concentration 50 g/ml; Beckman Coulter, Inc., Brea, CA, USA) and incubated at 37C for 30 min. Following staining, the cells were washed once.