Side population (SP) cells are a rare subset of cells found in various tissues that are highly enriched for stem cell activity. SP cells can be isolated by dual-wavelength flow cytometry because of their capacity to efflux Hoechst dye, a process mediated by the ATP-binding cassette transporter breast cancer resistance protein (Bcrp) 1. By performing flow cytometry of enzymedigested mouse lung stained with Hoechst dye, we found that SP cells comprise 0.03–0.07% of total lung cells and are evenly distributed in proximal and distal lung regions. By RT-PCR, we found that lung SP cells express hepatocyte nuclear factor-3β, but not thyroid transcription factor-1. Surface marker analysis revealed lung SP cells to be stem cell antigen 1 positive, Bcrp1 positive, lineage marker negative, and heterogeneous at the CD45 locus. As expected, we did not detect lung SP cells in Bcrp1-deficient animals. We, therefore, employed nonisotopic in situ hybridization and immunostaining for Bcrp1 as a strategy to localize these cells in vivo. Expression was observed in distinct lung cell types: bronchial and vascular smooth muscle cells and round cells within the distal air space. We confirmed the expression of Bcrp1 in primary bronchial smooth muscle cell cultures (BSMC) and in lavaged distal airway cells, but neither possessed the capacity to efflux Hoechst dye. In BSMC, Bcrp1 was localized to an intracellular compartment, suggesting that the molecular site of Bcrp1 expression regulates SP phenotype.
Contained within the adult lung are differentiated mesenchymal cell types (cartilage, smooth muscle, and myofibrobasts) that provide structural support for airways and vessels. Alterations in the number and phenotype of these cells figure prominently in the pathogenesis of a variety of lung diseases. While these cells are thought to arise locally, progenitors have yet to be purified. In previous work, we developed a method for isolating progenitors from lung tissue: this technique takes advantage of the unique ability of cell populations enriched for somatic stem and progenitor activity to efflux the vital dye Hoechst 33342, a feature that permits isolation by flow cytometry-based procedures. Using this method, we determined that a rare population of mesenchymal progenitors resides within the CD45- CD31- Hoechst low fraction of the adult murine lung. Similar to other mesenchymal progenitors, these cells express Sca-1, CD106, and CD44; can be serially passaged; and can differentiate to smooth muscle, cartilage, bone, and fat. Overall, these findings demonstrate that a phenotypically distinct mesenchymal progenitor resides within the adult murine lung, and provide a scheme for their isolation and study.
activation and an emphysema-like phenotype in adiponectin-deficient mice.
An ongoing controversy is the role of marrow cells in populating the alveolar epithelium. In this study, we employed flow cytometry and histologic techniques to evaluate this process. Donor bone marrow was harvested from transgenic mice expressing the LacZ or eGFP gene ubiquitously, or under the control of the human surfactant protein (SP)-C promoter, and transplanted into lethally irradiated, neonatal mice. In recipients transplanted with marrow that express eGFP or lacZ ubiquitously, light microscopy revealed cells whose morphology and location were compatible with a type II cell phenotype. Consistent with this, fluorescent microscopy suggested colocalization of eGFP and pro-SP-C proteins in single cells. In mice transplanted with SP-C-eGFP marrow, engraftment was not detectable by histology or flow cytometry. We therefore used deconvolution microscopy to reanalyze histologic sections that were thought to show marrow-derived type II cells. We found that all putative marrow-derived pneumocytes resulted from the overlapping fluorescent signals of an endogenous pro-SP-Cϩ type II cell and a donor-derived eGFPϩ cell. Taken together, our observations underscore the technical difficulties associated with evaluating engraftment in lung, and argue against a contributory role for marrow cells in populating the alveolar epithelium.
Side population (SP) cells are a select cell population identified by a capacity to efflux Hoechst dye that are highly enriched for stem/progenitor cell activity. In this study, we found that SP cells comprised of CD45(+) and CD45(-) subtypes are present in the embryonic lung (E-SP) at levels varying with gestational age. Long-term in vivo competitive blood reconstitution studies demonstrated that hematopoeitic stem cell capacity resided within the CD45(+) E-SP cell subset. Immunophenotyping of CD45(-) E-SP cells determined that this population consists of two subtypes: CD31(-) and CD31(+). Limited gene expression profiling indicated that CD45(-)/CD31(-) E-SP cells have features of smooth muscle precursors, and give rise to smooth muscle in culture. On the other hand, CD45(-)/CD31(+) E-SP cells express genes characteristic of endothelium, but by themselves do not grow or differentiate in culture. Co-culture of CD45(-)/CD31(+) and CD45(-)/CD31(-) E-SP cells, however, resulted in the formation of complex tubular networks that express markers of endothelium. Together, these findings illustrate that embryonic lung SP cells are heterogeneous, composed of hematopoeitic and nonhematopoeitic progenitors, and may play a key role in the formation of the lung vasculature.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.