Generation of ips-like cells from COPD lung fibroblasts
RATIONALE:Induced pluripotent stem cells (iPSCs) promise to advance regenerative medicine in several distinct ways: as therapeutic agents themselves, as a source of differentiated cells, or, as in the current work, as a model system to develop therapy aimed at reprogramming abnormally functioning cells. In the current work we developed iPS-like cells from lung cells of COPD patients and controls. METHODS:Lentiviral particles for Oct3/4, Sox2, c-Myc and Klf4 were utilized to generate iPS-like cells from lung fibroblasts of three COPD and three non-COPD subjects. Cells were infected with lentiviral particles at MOI of 5 and transferred to six well plate containing feeder layer of mouse embryonic fibroblasts 48 hours after transfection. To enhance the generation of iPS-like rock inhibitor Y27632 was used after seven days of infection and 100ng b-fgf was used in the media for the last seven days of the reprogramming protocol. After enriching for colonies, iPS-like cells were moved to feeder independent plates using mTESR1® media and BD matrigel®. Alkaline phosphatase immunecytochemistry and real time PCR for undifferentiated markers Oct3/4 and Sox2 and were carried out to demonstrate the self-renewal and pluripotency capacity of the generated iPS-like cells. iPS-like cells were then used to prepare embryoid bodies (EBs) and the ability of these EBs to re-differentiate towards fibroblasts was assessed then iPS-like cells-derived fibroblasts from both groups were tested functionally by gel contraction and chemotaxis. RESULTS:iPS-like cells were successfully generated from both COPD and control adult lung fibroblasts. Morphology of iPS-like cells generated COPD and non-COPD lung fibroblasts was similar to the control iPSCs obtained from university of Wisconsin. Generated iPS-like cells expressed high levels of alkaline phosphatase and high levels of Oct3/4 and Sox2 were also detected by real time PCR. The iPS-like cells formed EBs in culture and differentiated into fibroblasts in three-dimension collagen gels. CONCLUSION: We demonstrated that fibroblasts cultured from the lungs of patients with COPD and controls can be induced to form iPS-like cells. The resulting "stem cells" can be used to induce differentiation and a fibroblast-like phenotype can be restored. This system should be able to help elucidate whether epigenetic alterations accumulate in structural cells in COPD.