Homing and Engraftment of Bone Marrow Derived Cells in Irradiated Mouse Lungs.

Abstract 4600

To determine the degree to which murine bone marrow migrates to and can repopulate damaged lung, we studied lung stem cell expression of the Clara Cell Specific Protein (CCSP). Several different models have been used to deplete the lung stem cells. We used CCtk mice which expresses the HSV-tk under control of the CCSP promoter. Subcutaneous administration of gancicylovir (50 mg/ml) over a 7 day period using Alzet osmotic pumps results in the reduction of CCSP RNA and CCSP positive cells. To determine if a reduced dose of gancicylovir reduced CCSP cells to a nonfatal degree and opened a stem cell niche for homing of hematopoietic cells, CCtk mice had osmotic pumps placed subcutaneously containing a lower dose of 25 mg/ml of gancicylovir. The lungs were explanted at serial times up to 7 days after pump placement. RNA was extracted and real time polymerase chain reaction (RT-PCR) using primers specific for CCSP was used to quantitate CCSP expression. CCtk mice treated with gancicylovir showed a 70% reduction of lung CCSP expression. CCtk mice implanted with osmotic pumps containing 25 mg/ml gangcicylovir and injected intravenously with bone marrow from sex-mismatched GFP+ transgenic FVB male mouse 2 days later were followed for thirty days. The mice were sacrificed, lungs removed, single cell suspensions prepared, stained with a PE labeled anti-CD45 monoclonal antibody and analyzed by flow cytometry for GFP positive, CD45 negative cells. No GFP+, CCSP+ cells cells were detected. Injection of FVB mice with naphthalene (which kills differentiated but not stem CCSP+ cells) at 200 mg/kg intraperitoneally resulted in an 87% decrease in CCSP expression. When injected with bone marrow cells from GFP+ transgenic mice at serial times to 7 days and sacrificed at day 30, again no GFP+ CCSP+ cells were identified. In a third attempt to remove additional CCSP cells and prepare sites for homing of marrow cells, FVB mice were treated with naphthalene (200 mg/kg) followed by 20 Gy irradiation to the lungs 48 hr later, then injected with GFP+ bone marrow, 24 hr later. The mice were sacrificed 30 days later, and examined for GFP+, CD45- cells. Low numbers of GFP+ CCSP+ cells were detected. The data indicates a low baseline level of hematopoietic stem cell potential to home and differentiate to CCSP positive lung cells.

Supported by NIH-2R01CA119927-08A1.