Characterization of mouse pulmonary mesenchymal stem cells isolated in vitro and the intervention effects on lung injury

Li WANG; Weijing ZHANG; Bing LIU; Xiusen LI

ABSTRACT

BACKGROUND:

There are several types of endogenons stem/progenitor cells in lung that develop from either endoderm or mesoderm precursors.To elucidate the characteristics of the intrapulmonary stem ceils wig help to understand their biological behavior in lung injury.

OBJECTIVE:

To isolate the mesenchymal stem cells (MSCs) from mouse lung tissues,and identify their morphology and growth characteristics,cell surface antigens,differentiation potential in vitro,stem cell properties,and to investigate the protective role in bleomycin challenged lung.DESIGN,TIME AND

SETTING:

The present randomized controlled in vivo animal experiment based on in vitro observation of cytology was performed at the Laboratory of Cell Biology,Institute of Basic Medical Science,the Academy of Military Medical Science between October 2005 and August 2007.MATERIALS Male(3-4 weeks) and female C57BL/6 (6-8 weeks) mice were used.Twenty female C57BL/6 mice were randomly and evenly divided into a pulmonary MSCs (PMSCs)-treated group and a myelosuppression group.

METHODS:

The lungs from male C57BL/6 mice were digested with collagenase Ⅱ,followed by centrifugation over a Ficoll step.The interface fraction was collected and cultured by adherent method.When the monolayer of adherent cells reached 70%-80%confluence,the adherent cells were detected and expanded in culture medium.The mice in the PMSCs-treated and myelosuppression groups were intraperitoneally administered busulfan to inhibit the immigration of bone marrow stem cells.In addition,pulmonary fibrosis injury was induced in these mice with bleomycin.The PMSCs-treated group was intravenously administered 5×105 PMSCs.At the same time,the myelosuppression group received 100 μ L of phosphate buffered saline.After 14 days,the lungs were taken to prepare paraffin-embedded section.MAIN OUTCOME

MEASURES:

Cell morphology,immunophenotype,specific markers of the induced cells,expression of gene peroxisome proliferator activated receptor γ,osteopontin,osteocalcin,prosurfactant protein C(SP-C),Oct-4 and Nanog,histological alteration of lung tissue sections.

RESULTS:

MSCs were successfully isolated from mouse lung tissue.The pulmonary MSCs(PMSCs) were fibroblast-like cells,and expanded rapidly in vitro for up to 40 passages.The phenotype of the PMSCs was Scu- 1+ ,CD44+ ,CD29+ ,CD105+ ,CD54+ ,CD34-,CD45-,CD11b,c-kif,and CD31.They did not express alveolar epithelial cell specific markers,such as SP-C,aquaporin-5,and clara cell secretory protein.It was noticeable that stem cell markers octamer-binding transcription factor 4 (Oct-4) and Nanog were expressed continuously in culture expanded cells.They could differentiate into adipocytes,osteoblasts and alveolar epithelial cells in vitro.Colony-forming assay demonstrated that the colony forming efficiency of the PMSCs was nearly 3%.The cells from single colony were capable of differentiating to osteocytes,adipocytes and alveolar epithelial cells.Intravenous administration of PMSCs could alleviate pulmonary injury and fibrosis induced by bleomycin,despite the bone marrow was intact or not.

CONCLUSION:

The PMSCs are able to extensively propagate in vitro,can efficiently switch to both mesenchymal and alveolar epithelial lineages in vitro,generate adipocytes,osteocytes,and the alveolar epithelial cells,and reduce pulmonary injury and fibrosis in bleomycin challenged lung in vivo,strongly suggesting their promising applications in cellular therapy against the lung injury.