Radiation Induced and FACS-Sorted Senescent tdTOMp16+ Cells Upregulate Profibrotic Gene Expression in Mesenchymal Stem Cells (Stromal Cells).

PURPOSE/OBJECTIVE(S) The role of cellular senescence in radiation induced pulmonary fibrosis (RIPF) is unknown. Furthermore, clearance of senescent cells (SCs) could be an effective therapeutic strategy; however, the identification and targeted removal of only senescent cells by senolytic drugs is difficult. We isolated a pure population of radiation induced senescent and non-senescent cells using tdTOMp16+ mice and examined induction of fibrosis in target cells. MATERIALS/METHODS A stromal cell line was established from explanted tdTOMp16+ mouse bone marrow. The tdTOMp16+ stromal cell line was irradiated to each of several doses by Cesium Gamma Cell dose rate 300 cGy/min including 1.0 Gy, 2.5 Gy, 5 Gy, and 7.5 Gy under growth conditions of either 50% or 100% confluence. The number and percent of senescent cells, by p16 promoter linked to the red fluorochrome biomarker was quantitated. Irradiated red, irradiated non-red, and non-irradiated cells were sorted and cultured as single cells in 96-well plates, then monitored for cell division over four weeks. The expression of senescence markers, p16 and p21, was evaluated by qPCR, and SA-β-gal by fluorogenic substrate. To examine the effect of senescent cells on a target fibrosis-prone C57BL/6 line, sorted irradiated red, irradiated non-red, and non-irradiated cells were placed on the top level of transwells, and after 2 weeks induction of biomarkers of fibrosis including Ctgf, TGF-β, collagen 1a, and collagen 3 was measured in target cells in the lower level by qPCR. RESULTS After 5 Gy irradiation at 50% cell density, ∼9% of tdTOMp16+ cells became senescent (stably red in color) after 10 days, thus, defining the optimal conditions. Then, in bulk cultures, red cells were sorted by fluorescence automated cell sorter (FACS) to a pure population of red tdTOMp16+ cells. When cultured as single cells, none of 960 irradiated red cells divided after 2 weeks, 137 of these cells remained intact, and assume large and flat morphology. In contrast, 50 of 800 non-red irradiated cells divided, and 104 of 560 non-irradiated control cells divided. There was significant detection of senescent cell markers including: SA-β-gal, p16, and p21 in irradiated sorted red cells compared to irradiated non-red cells (P = 0.0096, P < 0.0001 and P = 0.0001 respectively based on scoring of 1000 cells in triplicate experiments for SA-β-gal and analyses from three independent experiments). Induction of biomarkers of fibrosis Ctgf, TGF-β, collagen 1a, and collagen 3 in C57BL/6 stromal target cells was significant by irradiated red, but not irradiated non-red or non-irradiated cells (P = 0.037, P = 0.14, P = 0.015, and P = 0.0259). CONCLUSION The data indicate that sorted irradiated red senescent tdTOMp16 stromal cells can be isolated as a pure non-dividing population and induce fibrotic markers in target cells in non-contact transwell cultures. This unique system can now be used to define the mechanism of senescent cell-induced radiation fibrosis, and the effects of senolytic drugs.