Further defining lung SP cells: their origin and their heterogeneity, now if we only knew their fate!

Patricia J. Dubin and Jay K. Kolls

Department of Pediatrics, University of Pittsburgh, Pittsburgh, Pennsylvania 15213

TISSUE INJURY AND REPAIR are ongoing processes in the lung and result from acute and chronic exposure to environmental insults. There are a myriad of effectors of lung injury, including infectious agents, particulate and chemical pollutants, radiation, and host defense mechanisms gone awry. Many of these processes are ablative in nature and require repair mechanisms that regenerate mature lung tissue through cell proliferation and differentiation.

Fundamental to understanding mechanisms of repair are identifying and characterizing the cells that are potentially capable of repopulating the injured tissue. Currently, efforts are being made to identify 1) which cell(s) repopulates regions of injured lung; 2) what their source is (endogenous or resident cells vs. exogenous or recruited cells); and 3) whether they are pluripotent stem cells capable of self renewal or transient amplifying cells that are multipoint but more lineage committed. In the lung, multiple cell populations contribute to lung repair. Most, like the basal cells of the tracheal epithelium, alveolar type II cells, bone marrow-derived stem cells, and residential stem cells that potentially serve the vascular compartment appear to be anatomically localized (5, 8). Others, like the side population (SP) cells, have not yet been localized to a single lung compartment.

The molecular phenotype of hematopoietic stem cells (HSC) has been extensively characterized and is defined as a population of cells that are CD45+, Sca-1+, c-kit+, and Lin–. HSC are further characterized by their ability to rapidly efflux the DNA dye Hoechst 33342 (6, 7) via Brcp1 (ABCG2), an ATP-binding cassette transporter (11, 15). This phenotypic characteristic results in segregation of HSC within the SP, a subset of bone marrow cells that exhibit low blue and red fluorescence after being stained with Hoechst 33342. SP cells are highly enriched with cells capable of long-term reconstitution of the hematopoietic compartment following lethal irradiation and have been found in numerous species (6). More recently, in addition to first localizing these cells to bone marrow, investigators have found cells with similar efflux capacities in many tissues (4, 9) and the lung (4, 5a, 13). Despite similar markers, such as Sca-1, muscle SP cells (3) and lung SP cells (5a) are heterogeneous in CD45 expression compared with bone marrow SP cells.

SP cells are believed to be derived from the bone marrow and can be differentiated from committed tissue stem cells. In models of ablative radiation injury, CD45+ lung SP cells have demonstrated transient repopulation of the bone marrow and indicated that CD45+ lung SP cells are analogous to the short-term repopulating hematopoietic cell (1).

The report from Summer et al., one of the current articles in focus (Ref. 13a, see p. L477 in this issue), substantiates this conclusion by demonstrating that CD45+ lung SP cells lack detectable GATA-1, a transcription factor necessary for maintenance of HSC. The finding that the transcription factors GATA-2 and PU.1, involved in myeloid differentiation, were present in CD45+ lung SP cells suggests that these cells may serve as a specific myeloid progenitor pool within the lung. Previous analyses of CD45– lung SP cells have supported the conclusion that these cells are derived from various compartments within this organ. Immunohistochemical analysis of CD45– SP from the collagenase-digested lung demonstrated that 40% of these cells express the endothelial marker CD31, and RT-PCR analysis of gene expression detected the lung-related transcription factor HNF3b but not the epithelial transcription factor TTF1. RT-PCR analysis of gene expression in sorted CD45– SP cells from elastase-digested lung demonstrated that this method results in selection of cells with a molecular profile similar to the previously identified airway epithelial stem cell (5a, 10a). These cells, termed variant Clara cell secretory protein (CCSP) expressing cells, are CCSP+/CyP4502F2–. Vimentin was also detected in these preparations, but the alveolar epithelial type II cell marker surfactant protein C, the endothelial transcript platelet/endothelial cell adhesion molecule, and the fibroblast marker FSP-1 were not detected (5a).

In models of ablative radiation injury, CD45+ SP cells have been demonstrated to be sufficient for reconstitution of the bone marrow (1, 2). In addition, in one report, marked SP cells were shown to repopulate damaged lung (2) in irradiated mice, resulting in rare, but detectable, fibroblasts and alveolar and bronchial epithelial cells. These findings support the position that tissue SP cells are hematopoietically derived, pluripotent stem cells that may play an important role in tissue repair. Other studies have attempted to address whether SP cells that are localized to specific tissues maintain their pluripotency. Distinct populations of tissue-localized SP or Lin–, Sca-1+ cells that maintain hematopoietic activity have been identified in muscle (3, 12) and liver (14). Interestingly, these SP cells have not been identified in peripheral blood and are least frequently found in the bone marrow (0.79% of nucleated cells) compared with other tissues (0.96–15.1% of nucleated cells) (4).

The report by Summer et al. (13a) adds to our understanding of the lung SP and its function by phenotypically characterizing these cells. The authors have demonstrated that there are both CD45+ and CD45– lung SP cells and that both of these populations are contributed to by the bone marrow compartment, as demonstrated by whole bone marrow transplantation experiments. However, the data also suggest that SP cells have multiple origins since both the CD45+ and CD45– populations contain cells derived from the bone marrow and from untagged cells. Alternatively, the fraction of untagged cells could represent contamination of the transplanted population with a contaminating (CD45–) progenitor.

The results and conclusions of this study leave us with other questions for consideration. What prompts a change in CD45 expression and the concomitantly expressed differences in transcription factors? What are the functional implications for CD45– cells? Do they interact with other tissue-specific stem cells, such as tracheal basal cells or alveolar type II cells in repair? Is the lung SP cell of fetal origin and self regenerative or dependent on the bone marrow SP for repopulation? Is there a difference in SP cell contribution when considering repair in response to acute vs. chronic lung injury? Pursuing these avenues of study will lead to a more integrated understanding of the roles that the SP cell and the committed, tissue-specific stem cells play in lung repair.


    ACKNOWLEDGMENTS
 
The authors thank Dr. Susan D. Reynolds, Department of Environmental and Occupational Health, University of Pittsburgh for helpful discussion.


    FOOTNOTES
 

Address for reprint requests and other correspondence: J. K. Kolls, Children's Hospital/Univ. of Pittsburgh, 3705 Fifth Ave., Ste. 3765, Pittsburgh, PA 15213 (E-mail: jay.kolls{at}chp.edu)


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