Bone Marrow Derived Lung Epithelial Cells Are Derived Predominantly From Nonhematopoietic Cells.

Abstract 2615

Since 1998, there have been many discoveries that bone marrow derived cells (BMDC) possess the ability to cross lineage barriers and differentiate into mature, non-hematopoietic cells of multiple tissues. In recent years, several studies demonstrated engraftment of BMDC as epithelial cells of the lung. One of the main unanswered questions is which population(s) of BMDC are responsible for this engraftment. Previous investigations reported a lack of marrow derived lung epithelial cells (MDLE) after transplantation of hematopoietic stem cells (Thy1^loLSK), while others found MDLE in the lungs of mice undergoing whole bone marrow transplantation. We therefore hypothesized that primitive, non-hematopoietic stem cells residing in the BM are the source of MDLE, and tested directly whether nonhematopoietic bone marrow derived cells have the capacity to engraft as epithelial cells of the lung. To test this hypothesis, we used Vav-cre transgenic mice, in which Cre recombinase is expressed uniquely within cells of the hematopoietic lineage, to determine which BM cell populations can engraft as surfactant protein C (SPC) expressing type 2 pneumocytes in surfactant-protein-C-null (SPC-KO) mice. Live hematopoietic and non-hematopoietic BM cells were separated by FACS-sorting based on expression of YFP in cells from ROSA-YFP/Vav-Cre mice that express YFP only in cells committed to the hematopoiesis. YFP positive (hematopoietic) or YFP negative (non-hematopoietic) cells were transplanted into irradiated SPC-KO mice. For transplant recipients of YFP negative cells, 1 million whole bone marrow cells from a recipient type SPC-KO mouse were cotransplanted to provide hematopoietic rescue following irradiation. MDLE were assessed by detection of alveolar type 2 cells expressing wild type SPC, which could only be derived from the donor cells in SPC-KO recipients. Methods of detection included confocal microscopic analysis of sorted CD45 negative lung cells and tissue sections stained for SPC and epithelial specific cytokeratin or TTF1 by immunofluorescence. In addition, isolated lung cells were stained in suspension and analyzed for donor derived type 2 cells by Imagestream technology for expression of SPC and cytokeratin. SPC positive type 2 cells were found in the lungs of 4 out of 6 mice receiving YFP^negative, non-hematopoietic BM cells, with 1 out of 16,000 total nucleated cells expressing SPC on lung tissue sections. In contrast, donor derived SPC positive type 2 pneumocytes were exceedingly rare in mice receiving YFP^positive, hematopoietic cells and were found in just 1 out of 9 mice, with only 1 out of 144,000 total nucleated cells being positive for SPC on tissue sections. These findings were confirmed by highly sensitive RT-PCR for wild type SPC mRNA. We conclude that the non-hematopoietic fraction of murine BM contains cells capable of engrafting as epithelial cells of the lung. Our data support the hypothesis of a primitive, pluripotent stem cell population residing in adult bone marrow.