Adult lung side population cells have mesenchymal stem cell potential
Introduction
Pulmonary hypertension, chronic obstructive pulmonary disease, interstitial pulmonary fibrosis and other adult lung conditions are a major cause of morbidity and mortality. Deaths as a result of these conditions have doubled in the last decade. Interestingly, while the lung exhibits tremendous regenerative capacity following tissue resection, regeneration and restoration of pulmonary function do not occur in many adult lung diseases. There is an increasing emphasis on the development of cell-based therapies to address these conditions, but the lung is a recalcitrant candidate for these strategies because of the diverse cell types and functions. We are focused on identifying and manipulating the endogenous lung stem cells.
Both the origins of stem cells for cell-based therapy and contributions of stem cells to pulmonary remodeling are currently under intense investigation. Specialized microenvironments, or niches, for resident multipotent mesenchymal stem cells (MSC) have been identified in many adult tissues [1., 2., 3.]. The normal differentiation processes of these stem cells may be disrupted by pathologic micro-environmental stimuli during disease, epigenetic changes or genetic mutation, which program their contribution to pathologic expansion at the expense of functional tissue regeneration.
Side population cells (SP) were first identified in bone marrow (BM) and are characterized based on their lateral location on a cytometric dot plot as a side arm off the main population [4]. SP cells demonstrate this unique profile when stained with the Hoechst 33342 vital dye, which fluoresces in red and blue when excited by ultraviolet (UV) laser. These cells decrease in fluorescence as the dye is pumped out of their cytoplasm via an ABCG2 transporter mechanism [5, 6]. Functional analyzes have defined the BM SP population as multipotent and enriched for hematopoietic stem cells (HSC), distinguished by uniform expression of CD45 [4, 7, 8]. In other adult tissues, CD45 expression varies and has been used to distinguish between hematopoietic SP and the non-hematopoietic (CD45neg) [9., 10., 11., 12.]. HSC ability is limited to the CD45pos SP fraction [8, 9].
SP cells have been reported to reside in many adult tissues other than BM, as well as tumors [9, 13]. Recent data suggest CD45neg SP cells may represent an enriched source of organ-specific pulmonary precursors with endothelial, epithelial and mesenchymal potential [2, 14, 15]. Endothelial precursors arise from the mesenchyme during development [16]. MSC have been isolated from tracheal aspirates from neonates in respiratory distress and from adult bronchoalveolar lavage [17., 18., 19., 20.]. The origin of MSC from these subjects and their significance to lung pathology is presently unknown.
We sought to characterize a source of lung MSC by isolating CD45neg lung SP single-cell clones, measuring their telomerase expression over time, and analyzing their chromosomal stability. We then assayed the mesenchymal differentiation potential of these cells over time using cultures at passage 3, as well as single-cell clones at passage 50. We hypothesized that these lung SP clones would exhibit stem cell characteristics and mesenchymal differentiation potential in vitro and thereby represent a potential source of MSC in the lung that may be used to study the fate of lung stem cells during pulmonary disease.
Section snippets
Isolation and immunophenotyping of lung SP cells
SP cells were isolated from 12-week-old murine adult C57Bl6 lungs using a 0.2% collagenase digest of lung tissue to obtain a single-cell suspension. Hoechst 33342 staining was performed to identify SP cells, as described previously using a DAKO MoFlo [4, 7, 9, 14, 21]. Isolated lung SP cells in these experiments were analyzed for expression of CD45 to separate the CD45 positive (hematopoietic) from negative (mesenchymal) subpopulations [9]. The gates were set using whole BM SP cells. Freshly
Isolation and characterization of hematopoietic and mesenchymal lung SP subpopulations
Co-staining of cells with markers indicative of stem cells, HSC, MSC and macrophages were compared with reported analyzes of BM HSC and MSC [2., 3., 4., 8, 10, 12., 13., 14., 15., 17, 18, 20, 25., 26., 27., 28.]. The results are summarized in Table 1. We selected for the SP fraction using lung tissue digested into a single-cell suspension stained with Hoechst 33342. The SP was analyzed based upon expression of CD45 and a second marker of interest to differentiate hematopoietic (CD45pos) from
Discussion
In these studies we sought to identify a resident lung stem cell population and were able to demonstrate that the CD45neg lung SP cells are a source of adult lung MSC. Fresh, cultured and clonal CD45neg lung SP cells express cell-surface determinants common to MSC. Using an in vitro cell culture system, we assayed the ability of high telomerase-expressing CD45neg lung SP cells and their single-cell clones to differentiate into mesenchymal lineage cells, specifically adipocytes, osteocytes and
Acknowledgements
We would like to thank Drs Lisa Johansen and Dwight Klemm for their review of the manuscript. This work was funded by grants to S. M. Majka from the American Heart Association (SDG-0335052N), the Cystic Fibrosis Foundation the UC-HSC Department of Medicine and the American Physiological Society Giles Filley Memorial Award. We acknowledge technical and scientific assistance from C. Childs in the University of Colorado Comprehensive Cancer Center Flow Cytometry Core and Cytogenetics Core (NIH 5
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