Supplementary MaterialsSupplementary Components: Supplementary Number 1: identification of cell differentiation in lung differentiation platform. to fibrosis as demonstrated with the elevated levels of markers for epithelial-mesenchymal transition and myofibroblast (and studies. Improved gene expressions of connective cells growth element and but also for lung restoration . However, the effects of ionizing radiation on these CD45?CD54+CD157+ LSCs have not been investigated. Here, we demonstrated that these LSCs are more sensitive to radiation damage than their differentiated alveolar cells. In addition, using the fibrosis PCR array and immunostaining analyses, we showed that these irradiated LSCs underwent AECII and myofibroblast differentiation after irradiation and were involved in the fibrogenic response. Nintedanib, a tyrosine kinase inhibitor, L-Ascorbyl 6-palmitate is currently used to reduce the pace of decrease in lung function in individuals with idiopathic pulmonary fibrosis . A single published study implied that nintedanib offers antifibrotic activity after partial lung irradiation in mouse models; however, this cannot be monitored from the computed tomography imaging . Cells restoration and airway redesigning involving the differentiation of LSCs are essential to the maintenance of lung homeostasis. The characterization of the radiation response of LSCs and their differentiated alveolar cells used in the present study is a critical approach to better define and understand the pathophysiology of fibrosis. Moreover, using cultured L-Ascorbyl 6-palmitate stem cells and differentiated cells of the lung may provide an easy-to-follow and less time-consuming platform for drug testing and pave the way for tissue executive and stem cell therapy in the radiation research. 2. Materials and Methods 2.1. Mice and Irradiation CD-1 (ICR) mice were purchased from BioLasco (Taiwan). Rays was shipped utilizing a 6 MV X-ray linear accelerator in rays and Proton Therapy Middle, Chang Gung Memorial Hospital, Linkou, Taiwan. For experiments, cells (denseness: 2.5 104 cells/cm2) were exposed to 2, 4, or 8?Gy. For experiments, neonatal CD-1 mice were treated with or without 8 or 15?Gy whole-body irradiation. 2.2. Cell Tradition Main lung L-Ascorbyl 6-palmitate stem cell (LSC) tradition was performed as previously explained . Chuk LSCs were isolated from neonatal CD-1 mice by FACS sorting using phycoerythrin- (PE-) conjugated anti-CD157 (BioLegend, CA, USA), fluorescein isothiocyanate- (FITC-) conjugated anti-CD54 (BD Biosciences, CA, USA), and allophycocyanin- (APC-) conjugated anti-CD45 (eBioscience, CA, USA) antibodies. Isolated CD45?CD54+CD157+ cells or irradiated cells were taken care of in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% FBS, 1% insulinCtransferrinCselenium (ITS), and 1?ng/ml epidermal growth factors (EGF) (all from Thermo Fisher Scientific, CA, USA) through several passages inside a collagen I-coated plate. To conduct differentiation studies, the attached LSCs were incubated in MCDB-201 medium (Sigma-Aldrich, MO, USA) supplemented with 1% FBS, 1% ITS, and 10?ng/ml EGF for 7 or 14 days to induce AECII or AECI cells. To determine the fibrogenic effect of transforming growth element beta (TGF-(5?ng/ml) or L-Ascorbyl 6-palmitate CTGF (50?ng/ml) for 3 days. 2.3. Immunofluorescence Staining and Quantification Briefly, irradiated cells were washed, fixed in 4% paraformaldehyde/phosphate-buffered saline (PBS), and then clogged with 3% bovine serum albumin (BSA) in PBS for 30?min. Cells were incubated with main antibodies at 4C over night. The following antibodies were used: anti-CD157 (BD Pharmingen, CA, USA); antiprosurfactant protein C (SP-C) (Millipore, CA, USA); antipodoplanin, also known as T1 alpha (T1lung cell differentiation experiments. LSCs were isolated from neonatal ICR mice and then sequentially differentiated into alveolar cells by tradition with MCDB-201 medium. LSCs, AECII, and AECI cells were examined through immunostaining with anti-CD157, anti-pro-SP-C, and anti-T1antibodies. Level bars, 100?= 3, ? 0.05) relative to initial cell number. (c) Immunostaining of 100) or AECI cells ( 75) relative to the initial sample. (e) The cell morphology of irradiated lung cells at day 3 postirradiation. Scale bars, 100?(AECI marker). A decreased level of CD157 and increased levels of SP-C and T1were observed in LSC samples treated with 8?Gy.