Elsevier

Cytotherapy

Volume 10, Issue 2, 2008, Pages 140-151
Cytotherapy

Adult lung side population cells have mesenchymal stem cell potential

https://doi.org/10.1080/14653240801895296Get rights and content

Background

The development of stem cell therapy for pulmonary diseases remains a challenge. Many diverse cell types reside within the lung and a common stem cell has not yet been identified. A basic understanding of lung stem cell fate during disease may prove important for drug intervention as well as autologous therapies. Niches for resident mesenchymal stem cells (MSC) have been identified in many adult tissues and more recently in the lung. We present data to confirm the observation that non-hematopoietic CD45neg lung side population (SP) cells contain MSC, single cells capable of multilineage differentiation.

Methods

We carried these observations forward by analyzing the MSC potential of single-cell clones, as well as their chromosomal stability and telomerase activity.

Results

The expression of MSC markers was characterized in mouse CD45neg lung SP by flow cytometry on freshly isolated or cultured clonal populations. The karyotype of these cells was subsequently assayed by banding analysis, and telomerase activity was assessed using quantitative polymerase chain reaction. MSC differentiation potential was confirmed by the characteristic ability of single-cell clones to differentiate into cells of three mesenchymal lineages, chondrocytes, adipocytes and osteocytes. Differentiation was confirmed by histochemical analysis. All analyzed populations of CD45neg lung SP expressed mesenchymal markers (CD44, CD90, CD105, CD106, CD73 and Sca-I) and lacked hematopoietic markers (CD45, c-kit, CD11b, CD34 and CD14). The cultured and clonal CD45neg lung SP had normal chromosomal structures and expressed high levels of telomerase. After being expanded and cultured in differentiation medium, all populations of CD45neg lung SP demonstrated adipogenic, osteogenic and chrondrogenic potential. Adult CD45neg lung SP cells are a source of MSC.

Discussion

In defining this tissue-specific stem cell population in the lung, we are now better able to clarify a potential role for them in lung diseases.

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

References (44)

  • SummerR. et al.

    SP cells and bcrp1 expression in lung

    American Journal of Physiology Lung Cell and Molecular Physiology

    (2003)
  • TadjaliM. et al.

    Prospective isolation of murine HSC by expression of ABCG2/GFP allele

    Stem Cells

    (2006)
  • GoodellM.A. et al.

    Isolation and characterization of side population cells

    Met Mol Biol

    (2004)
  • McKinney-FreemanS. et al.

    Muscle-derived HSC are hematopoietic in origin

    PNAS

    (2002)
  • MajkaS.M. et al.

    Distinct populations of adult stem cells in skeletal muscle differentially contribute to injury-induced vascular regeneration

    Journal of Clinical Investigation

    (2003)
  • GiangrecoA. et al.

    Molecular phenotype of airway SP cells

    American Journal of Physiology Lung Cell Molecular Physiology

    (2004)
  • SummerR. et al.

    Origin and phenotype of lung SP cells

    American Journal of Physiology Lung Cell and Molecular Physiology

    (2003)
  • WuC. et al.

    Side population cells isolated from mesenchymal neoplasms have tumor initiating potential

    Cancer Res

    (2007)
  • IrwinD. et al.

    Hyperoxic induced changes in pulmonary lung side population of progenitor populations in response to hyperoxia

    Am J Physiol Lung Cell Mol Physiol

    (2007)
  • SummerR. et al.

    Isolation of an adult mouse lung mesenchymal progenitor cell population

    Am J Respir Cell Mol Biol

    (2007)
  • GebbS. et al.

    Tissue interactions mediate early events in pulmonary vasculogenesis

    Dev Dynam

    (2000)
  • GuptaN. et al.

    Intrapulmonary delivery of bone marrow-derived MSCs improves survival and attenuates endotoxin-induced acute lung injury in mice

    J Immunol

    (2007)
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