Identification of Novel Resident Pulmonary Stem Cells: Form and Function of the Lung Side Population
http://www.100md.com
《干细胞学杂志》
a Department of Medicine, Cardiovascular Pulmonary Research Section,
b Department of Medicine, Pulmonary Hypertension Center, and
c Cancer Center, Flow Cytometry Core, University of Colorado Health Sciences Center, Denver, Colorado, USA;
d Colorado State University Department of Microbiology, Immunology & Pathology, Ft. Collins, Colorado, USA
Key Words. Side population ? Lung side population stem cells ? Adult stem cells
Correspondence: Susan Majka, Ph.D., Department of Medicine, Cardiovascular Pulmonary Research Section, University of Colorado Health Sciences Center, 4200 East 9th Avenue, SOM 3811, mail stop B-133, Denver, Colorado 80262, USA. Telephone: 303-883-8786; Fax: 303-315-4871; e-mail: Susan.majka@uchsc.edu
ABSTRACT
Resident stem cell populations have been identified in a variety of adult tissues, including lung. These cells likely contribute to local tissue regeneration throughout life and as such represent a target population that may be studied and manipulated to functionally restore injured pulmonary tissue. The lung stem cell potential may be compromised in various diseases, such as pulmonary hypertension (PH), pulmonary fibrosis (PF), and emphysema. A delineation of the mechanisms by which stem cells fail to regenerate local tissue, whether due to inappropriate terminal differentiation or apoptosis, is vital to understanding the pathology of lung diseases to identify appropriate targets for therapy. Studying how the microenvironment under these circumstances influences stem cell differentiation may also aid in the development of stem cell–based regeneration strategies, i.e., autologous bone marrow (BM)–based therapies, stimulation of local precursors, and gene therapy.
Hematopoietic stem cells (HSCs) and multipotent adult progenitor cells (MAPCs) derived from BM are well-studied populations of pulmonary precursor cells and may be important for autologous therapies . However, the phenotypes and origin of true resident stem cells within the lung remain largely undefined. Resident lung stem cells may function to replace pulmonary tissue, including epithelium, mesenchyme, and vasculature. Tissue-specific stem cells are typically located at a specialized site, proximal to the cell type they will regenerate. The epithelial precursor cell niches in the lung are well studied and have been extensively reviewed (Table 1). In the lung, epithelial stem cells include the type II pneumocyte in the alveolus and the Clara cell in the bronchiole. Pulmonary epithelial precursors have also been identified in the tracheal submucosal gland ducts. The microenvironment, such as underlying mesenchyme, in these instances is believed to influence stem cell commitment to specific lineages . Recently, a more primitive putative adult stem cell population has been identified in the lung, the lung side population (SP) of cells, which seems to have both mesenchymal and epithelial potential (Fig. 1) . To date, no resident pulmonary vascular stem cell has been described.
Table 1. Potential pulmonary stem cells
Figure 1. Lung side population (SP) profile. (A): SP cells may be isolated from adult lung via Hoechst 33342 staining and fluorescence-activated cell sorting analysis. (B): SP cells are defined by the presence of epithelial and mesenchymal markers. The SP cells represent less than 1% of the total lung cells. The viability of cells after staining and sorting is typically greater than 85%. (C): Upon isolation, the cells appear round and bright, similar to bone marrow SP. Subpopulations of lung SP may represent epithelial precursors and in vitro begin to express (D) cytokeratin and (E) epidermal growth factor receptor. Magnification x 100.
STEM CELL CRITERIA
SP cells, regardless of tissue origin, are identified by their unique fluorescence-activated cell sorting (FACS) profile. When separated by a flow cytometer with a UV laser, SP cells are distinct from cells that take up the Hoechst 33342 dye. When stained with the vital DNA dye, Hoechst 33342, and excited by a UV (351- to 364-nm) laser, the SP cells exhibit a low blue (440- to 460-nm) and low red (>675-nm) fluorescent staining pattern. This pattern presents as an SP tailing off the main G0-G1 population (Fig. 1) and is created by an efflux of the Hoechst 33342 dye from the SP cells. This efflux is the result of the presence of a multidrug resistance-like (MDR) transporter in the SP cells (Figs. 1A, 1B). Strict adherence to the staining protocol as to timing, temperature, cell concentration, and Hoechst dye concentration is necessary to accurately identify the SP. It is helpful to include propidium iodide in the stain mixture as a dead cell discriminator. After staining, the sample must be maintained at 4°C to prevent additional efflux of the Hoechst 33342 dye. Additionally, the flow cytometer must be properly aligned for linear signal collection, and a high number of events (at least 100,000) must be collected to ensure statistical validity of the data.
SP cells are negative for all hematopoietic lineage (lin–) markers, and BM-derived SP cells reconstitute lethally irradiated mice in smaller numbers compared with the whole BM HSC compartment . These SP cells were initially identified in BM by Goodell and colleagues and functionally characterized in transplantation analyses as enriched primitive hematopoietic precursors, lacking all markers of differentiated blood lineages . Further functional analyses have shown this population as well as single-cell derivatives to have multipotent stem cell ability; they can engraft into cardiac myocytes/fibers, vascular endothelium, liver, and skeletal muscle . These studies along with novel technology to identify an HSC population using flow cytometry formed the basis for the identification of putative resident adult tissue-specific stem cell populations (Table 1) . SP cells have been identified in various tissues, such as skeletal muscle, liver, lung, brain, kidney, heart, intestine, mammary, and spleen, and in tumors associated with these organs .
RESIDENT LUNG SP CELLS
Transdifferentiation involves the conversion of a lineage-determined cell into another phenotypically distinct cell type. Although transdifferentiation of many cell types has been reported, single-cell or clonal analyses have not been performed to rule out the possibility that the population under investigation did not contain multiple cells types, or "uncommitted" cells, which then differentiate rather than "trans" differentiate. The most well-characterized lung epithelial is the alveolar type II cell, which gives rise to the alveolar epithelial type I cell, both of which maintain alveolar homeostasis . Danto et al. , using isolated type II cells, demonstrated that the commitment of type II cell to the type I lineage required continuous regulation via external signals and that, in the absence of these stimuli, the cell types were capable of reversible transdifferentiation between the two phenotypes . The evidence for transdifferentiation between these two epithelial cell types is the focus of extensive study; generally accepted functional criteria have been established and summarized by Fehrenbach . As far as the occurrence of additional transdifferentiation events in the lung, there are data suggesting that vascular smooth muscle cells and fibroblasts may be derived from pulmonary artery (PA) endothelial cells , alveolar type II cells may be derived from tracheal epithelium , and myofibroblasts may be derived from lipofibroblasts . Most recent reports of mature PA vascular endothelial cells transdifferentiating into smooth muscle suggest that such a process may contribute to both tissue regeneration and pathological vascular remodeling in response to injury. Shannon et al. isolated embryonic tracheal epithelium and demonstrated that in the absence of appropriate inductive mesenchymal signals, these cells had the potential to assume an alveolar type II cell phenotype with surfactant protein C expression; however, this ability is developmentally restricted. Boros et al. demonstrated the influence of increased oxygen tension on the transition from a fetal lipofibroblast to a more contractile myofibroblast. These studies suggest that nonconventional pathways of cell differentiation may contribute to pulmonary regeneration in response to injury. Thus, true transdifferentiation of a committed cell type has not been rigorously demonstrated. Although these studies provide a convincingly detailed characterization of both vascular and epithelial transdifferentiation, they require the evaluation of differentiation on a clonal level.
THE POTENTIAL FOR LUNG STEM CELLS TO CONTRIBUTE TO PULMONARY DISEASE
Recent evidence suggests the potential for the lung SP cells to be enriched for epithelial precursors as well as hematopoietic cells. The studies reviewed are in their early stages and provide a foundation for future in vitro and in vivo functional studies as well as the obvious necessity for the stem cell criteria to be used as a guideline. The mounting evidence as to the potential for BM-derived cells to participate and differentiate into pulmonary tissues during various disease processes has been previously summarized (Table 1). However, BM-derived precursors have not been shown to restore tissue function during disease processes and seem to offer little hope for the regeneration of functional tissue without manipulation. With more uniform isolation and rigorous characterization of the local lung-specific stem cell population, we will begin to understand how the local environment influences these changes. This knowledge will allow the manipulation of the local microenvironment to facilitate a more normal regenerative process via both the resident and BM-derived cells.
ACKNOWLEDGMENTS
Verfaillie C, Schwartz R, Reyes M et al. Unexpected potential of adult stem cells. Ann N Y Acad Sci 2003;996:231–234.
Gebb S, Shannon J. Tissue interactions mediate early events in pulmonary vasculogenesis. Dev Dyn 2000;217:159–169.
Shannon J, Nielsen L, Gebb S et al. Mesenchyme specifies epithelial differentiation in reciprocal recombinants of embryonic lung and trachea. Dev Dyn 1998;212:482–494.
Shannon J, Gebb S, Nielsen L. Induction of alveolar type II cell differentiation in embryonic tracheal epithelium in mesenchyme free culture. Development 1999;126:1675–1688.
Stenmark K, Gebb S. Lung Vascular Development 2003;28:133–137.
Asakura A, Rudnicki M. SP cells derived from diverse adult tissues are capable of in vitro hematopoietic differentiation. Exp Hematol 2001;30:1339–1345.
Giangreco A, Shen H, Reynolds S et al. Molecular phenotype of airway SP cells. Am J Physiol Lung Cell Mol Physiol 2004;286:L624–L630.
Summer R, Kotton D, Sun X et al. A. Origin and phenotype of lung sp cells. Am J Physiol Lung Cell Mol Physiol 2003;287:l477–l483.
Summer R, Kotton D, Sun X et al. SP cells and bcrp1 expression in lung. Am J Physiol Lung Cell Mol Physiol. 2004;285:97–104.
Anderson D, Gage F, Weissman I. Can stem cells cross lineage boundaries? Nat Med 2001;7:393–399.
Holden C, Vogel G. Plasticity: a time for reappraisal? Science 2002;296:2126–2128.
Lemischka I. The power of stem cells reconsidered. Proc Natl Acad Sci US A 1999;96:14193–14195.
Verfaillie C. Adult stem cells: assessing the case for pluripotency. Trends Cell Biol 2002;12:502–508.
Goodell MA, Brose K, Paradis G et al. Isolation and functional properties of murine hematopoietic stem cells that are replicating in vivo. J Exp Med 1996;183:1797–1806.
Goodell M, McKinney-Freeman S, Camargo F. Isolation and characterization of SP cells. In: Helgason CD, Miller CL, eds. Methods in Molecular Biology, Volume 290: Basic Cell Culture Protocols, 3rd ed. Totowa, NJ: Humana Press, 2004.
Jackson KJ, Majka SM, Wang H et al. Regeneration of ischemic cardiac muscle and vascular endothelium by adult stem cells. J Clin Invest 2001;107:1395–1402.
Camargo F, Green R, Capetenaki Y et al. Single HSC generate skeletal muscle through myeloid intermediates. Nat Med 2003;9:1520–1527.
Kotton D, Summer R, Fine A. Lung stem cells: new paradigms. Exp Hematol 2004;32:340–343.
Wulf G, Jackson K, Brenner M et al. Cells of the hepatic side population contribute to liver regeneration and can be replaced with BM stem cells. Haematologica 2003;88:368–378.
Bunting K. ABC transporters as phenotypic markers and functional regulators of stem cells. STEM CELLS 2002;20:11–20.
Zhou S, Schuetz J, Bunting K et al. The ABC transporter Bcrp1/ABCG2 is expressed in a wide variety of stem cells and is a molecular determinant of the side-population phenotype. Nat Med 2001;7:1028–1034.
Alvi A, Clayton H, Joshi C et al. Functional and molecular characterization of mammary side population cells. Breast Cancer Res 2002;5:R1–R8.
Hirschmann-Jax C, Foster A, Wulf G et al. A distinct SP of cells with a high drug efflux capacity in human tumor cells. Proc Natl Acad Sci U S A 2004;101:14228–14233.
Majka S, Jackson KJ et al. Distinct populations of adult stem cells in skeletal muscle differentially contribute to injury: induced vascular regeneration. J Clin Invest 2003;111:71–79.
Abe S, Boyer C, Liu X et al. Cells derived from the circulation contribute to the repair of lung injury. Am J Respir Crit Care Med 2004;170:1158–1163.
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b Department of Medicine, Pulmonary Hypertension Center, and
c Cancer Center, Flow Cytometry Core, University of Colorado Health Sciences Center, Denver, Colorado, USA;
d Colorado State University Department of Microbiology, Immunology & Pathology, Ft. Collins, Colorado, USA
Key Words. Side population ? Lung side population stem cells ? Adult stem cells
Correspondence: Susan Majka, Ph.D., Department of Medicine, Cardiovascular Pulmonary Research Section, University of Colorado Health Sciences Center, 4200 East 9th Avenue, SOM 3811, mail stop B-133, Denver, Colorado 80262, USA. Telephone: 303-883-8786; Fax: 303-315-4871; e-mail: Susan.majka@uchsc.edu
ABSTRACT
Resident stem cell populations have been identified in a variety of adult tissues, including lung. These cells likely contribute to local tissue regeneration throughout life and as such represent a target population that may be studied and manipulated to functionally restore injured pulmonary tissue. The lung stem cell potential may be compromised in various diseases, such as pulmonary hypertension (PH), pulmonary fibrosis (PF), and emphysema. A delineation of the mechanisms by which stem cells fail to regenerate local tissue, whether due to inappropriate terminal differentiation or apoptosis, is vital to understanding the pathology of lung diseases to identify appropriate targets for therapy. Studying how the microenvironment under these circumstances influences stem cell differentiation may also aid in the development of stem cell–based regeneration strategies, i.e., autologous bone marrow (BM)–based therapies, stimulation of local precursors, and gene therapy.
Hematopoietic stem cells (HSCs) and multipotent adult progenitor cells (MAPCs) derived from BM are well-studied populations of pulmonary precursor cells and may be important for autologous therapies . However, the phenotypes and origin of true resident stem cells within the lung remain largely undefined. Resident lung stem cells may function to replace pulmonary tissue, including epithelium, mesenchyme, and vasculature. Tissue-specific stem cells are typically located at a specialized site, proximal to the cell type they will regenerate. The epithelial precursor cell niches in the lung are well studied and have been extensively reviewed (Table 1). In the lung, epithelial stem cells include the type II pneumocyte in the alveolus and the Clara cell in the bronchiole. Pulmonary epithelial precursors have also been identified in the tracheal submucosal gland ducts. The microenvironment, such as underlying mesenchyme, in these instances is believed to influence stem cell commitment to specific lineages . Recently, a more primitive putative adult stem cell population has been identified in the lung, the lung side population (SP) of cells, which seems to have both mesenchymal and epithelial potential (Fig. 1) . To date, no resident pulmonary vascular stem cell has been described.
Table 1. Potential pulmonary stem cells
Figure 1. Lung side population (SP) profile. (A): SP cells may be isolated from adult lung via Hoechst 33342 staining and fluorescence-activated cell sorting analysis. (B): SP cells are defined by the presence of epithelial and mesenchymal markers. The SP cells represent less than 1% of the total lung cells. The viability of cells after staining and sorting is typically greater than 85%. (C): Upon isolation, the cells appear round and bright, similar to bone marrow SP. Subpopulations of lung SP may represent epithelial precursors and in vitro begin to express (D) cytokeratin and (E) epidermal growth factor receptor. Magnification x 100.
STEM CELL CRITERIA
SP cells, regardless of tissue origin, are identified by their unique fluorescence-activated cell sorting (FACS) profile. When separated by a flow cytometer with a UV laser, SP cells are distinct from cells that take up the Hoechst 33342 dye. When stained with the vital DNA dye, Hoechst 33342, and excited by a UV (351- to 364-nm) laser, the SP cells exhibit a low blue (440- to 460-nm) and low red (>675-nm) fluorescent staining pattern. This pattern presents as an SP tailing off the main G0-G1 population (Fig. 1) and is created by an efflux of the Hoechst 33342 dye from the SP cells. This efflux is the result of the presence of a multidrug resistance-like (MDR) transporter in the SP cells (Figs. 1A, 1B). Strict adherence to the staining protocol as to timing, temperature, cell concentration, and Hoechst dye concentration is necessary to accurately identify the SP. It is helpful to include propidium iodide in the stain mixture as a dead cell discriminator. After staining, the sample must be maintained at 4°C to prevent additional efflux of the Hoechst 33342 dye. Additionally, the flow cytometer must be properly aligned for linear signal collection, and a high number of events (at least 100,000) must be collected to ensure statistical validity of the data.
SP cells are negative for all hematopoietic lineage (lin–) markers, and BM-derived SP cells reconstitute lethally irradiated mice in smaller numbers compared with the whole BM HSC compartment . These SP cells were initially identified in BM by Goodell and colleagues and functionally characterized in transplantation analyses as enriched primitive hematopoietic precursors, lacking all markers of differentiated blood lineages . Further functional analyses have shown this population as well as single-cell derivatives to have multipotent stem cell ability; they can engraft into cardiac myocytes/fibers, vascular endothelium, liver, and skeletal muscle . These studies along with novel technology to identify an HSC population using flow cytometry formed the basis for the identification of putative resident adult tissue-specific stem cell populations (Table 1) . SP cells have been identified in various tissues, such as skeletal muscle, liver, lung, brain, kidney, heart, intestine, mammary, and spleen, and in tumors associated with these organs .
RESIDENT LUNG SP CELLS
Transdifferentiation involves the conversion of a lineage-determined cell into another phenotypically distinct cell type. Although transdifferentiation of many cell types has been reported, single-cell or clonal analyses have not been performed to rule out the possibility that the population under investigation did not contain multiple cells types, or "uncommitted" cells, which then differentiate rather than "trans" differentiate. The most well-characterized lung epithelial is the alveolar type II cell, which gives rise to the alveolar epithelial type I cell, both of which maintain alveolar homeostasis . Danto et al. , using isolated type II cells, demonstrated that the commitment of type II cell to the type I lineage required continuous regulation via external signals and that, in the absence of these stimuli, the cell types were capable of reversible transdifferentiation between the two phenotypes . The evidence for transdifferentiation between these two epithelial cell types is the focus of extensive study; generally accepted functional criteria have been established and summarized by Fehrenbach . As far as the occurrence of additional transdifferentiation events in the lung, there are data suggesting that vascular smooth muscle cells and fibroblasts may be derived from pulmonary artery (PA) endothelial cells , alveolar type II cells may be derived from tracheal epithelium , and myofibroblasts may be derived from lipofibroblasts . Most recent reports of mature PA vascular endothelial cells transdifferentiating into smooth muscle suggest that such a process may contribute to both tissue regeneration and pathological vascular remodeling in response to injury. Shannon et al. isolated embryonic tracheal epithelium and demonstrated that in the absence of appropriate inductive mesenchymal signals, these cells had the potential to assume an alveolar type II cell phenotype with surfactant protein C expression; however, this ability is developmentally restricted. Boros et al. demonstrated the influence of increased oxygen tension on the transition from a fetal lipofibroblast to a more contractile myofibroblast. These studies suggest that nonconventional pathways of cell differentiation may contribute to pulmonary regeneration in response to injury. Thus, true transdifferentiation of a committed cell type has not been rigorously demonstrated. Although these studies provide a convincingly detailed characterization of both vascular and epithelial transdifferentiation, they require the evaluation of differentiation on a clonal level.
THE POTENTIAL FOR LUNG STEM CELLS TO CONTRIBUTE TO PULMONARY DISEASE
Recent evidence suggests the potential for the lung SP cells to be enriched for epithelial precursors as well as hematopoietic cells. The studies reviewed are in their early stages and provide a foundation for future in vitro and in vivo functional studies as well as the obvious necessity for the stem cell criteria to be used as a guideline. The mounting evidence as to the potential for BM-derived cells to participate and differentiate into pulmonary tissues during various disease processes has been previously summarized (Table 1). However, BM-derived precursors have not been shown to restore tissue function during disease processes and seem to offer little hope for the regeneration of functional tissue without manipulation. With more uniform isolation and rigorous characterization of the local lung-specific stem cell population, we will begin to understand how the local environment influences these changes. This knowledge will allow the manipulation of the local microenvironment to facilitate a more normal regenerative process via both the resident and BM-derived cells.
ACKNOWLEDGMENTS
Verfaillie C, Schwartz R, Reyes M et al. Unexpected potential of adult stem cells. Ann N Y Acad Sci 2003;996:231–234.
Gebb S, Shannon J. Tissue interactions mediate early events in pulmonary vasculogenesis. Dev Dyn 2000;217:159–169.
Shannon J, Nielsen L, Gebb S et al. Mesenchyme specifies epithelial differentiation in reciprocal recombinants of embryonic lung and trachea. Dev Dyn 1998;212:482–494.
Shannon J, Gebb S, Nielsen L. Induction of alveolar type II cell differentiation in embryonic tracheal epithelium in mesenchyme free culture. Development 1999;126:1675–1688.
Stenmark K, Gebb S. Lung Vascular Development 2003;28:133–137.
Asakura A, Rudnicki M. SP cells derived from diverse adult tissues are capable of in vitro hematopoietic differentiation. Exp Hematol 2001;30:1339–1345.
Giangreco A, Shen H, Reynolds S et al. Molecular phenotype of airway SP cells. Am J Physiol Lung Cell Mol Physiol 2004;286:L624–L630.
Summer R, Kotton D, Sun X et al. A. Origin and phenotype of lung sp cells. Am J Physiol Lung Cell Mol Physiol 2003;287:l477–l483.
Summer R, Kotton D, Sun X et al. SP cells and bcrp1 expression in lung. Am J Physiol Lung Cell Mol Physiol. 2004;285:97–104.
Anderson D, Gage F, Weissman I. Can stem cells cross lineage boundaries? Nat Med 2001;7:393–399.
Holden C, Vogel G. Plasticity: a time for reappraisal? Science 2002;296:2126–2128.
Lemischka I. The power of stem cells reconsidered. Proc Natl Acad Sci US A 1999;96:14193–14195.
Verfaillie C. Adult stem cells: assessing the case for pluripotency. Trends Cell Biol 2002;12:502–508.
Goodell MA, Brose K, Paradis G et al. Isolation and functional properties of murine hematopoietic stem cells that are replicating in vivo. J Exp Med 1996;183:1797–1806.
Goodell M, McKinney-Freeman S, Camargo F. Isolation and characterization of SP cells. In: Helgason CD, Miller CL, eds. Methods in Molecular Biology, Volume 290: Basic Cell Culture Protocols, 3rd ed. Totowa, NJ: Humana Press, 2004.
Jackson KJ, Majka SM, Wang H et al. Regeneration of ischemic cardiac muscle and vascular endothelium by adult stem cells. J Clin Invest 2001;107:1395–1402.
Camargo F, Green R, Capetenaki Y et al. Single HSC generate skeletal muscle through myeloid intermediates. Nat Med 2003;9:1520–1527.
Kotton D, Summer R, Fine A. Lung stem cells: new paradigms. Exp Hematol 2004;32:340–343.
Wulf G, Jackson K, Brenner M et al. Cells of the hepatic side population contribute to liver regeneration and can be replaced with BM stem cells. Haematologica 2003;88:368–378.
Bunting K. ABC transporters as phenotypic markers and functional regulators of stem cells. STEM CELLS 2002;20:11–20.
Zhou S, Schuetz J, Bunting K et al. The ABC transporter Bcrp1/ABCG2 is expressed in a wide variety of stem cells and is a molecular determinant of the side-population phenotype. Nat Med 2001;7:1028–1034.
Alvi A, Clayton H, Joshi C et al. Functional and molecular characterization of mammary side population cells. Breast Cancer Res 2002;5:R1–R8.
Hirschmann-Jax C, Foster A, Wulf G et al. A distinct SP of cells with a high drug efflux capacity in human tumor cells. Proc Natl Acad Sci U S A 2004;101:14228–14233.
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