Isolation And Characterization Of Lung Resident Mesenchymal Stem Cells Capable Of Differentiating Into Alveolar Epithelial Type Ⅱ Cells

Background:At present, more and more studies have shown that the LR-MSCs are closely related to the occurrence and development of the pulmonary fibrosis and other lung diseases. And also studies have found that, these cells can differentiate into skin cells or fibroblasts in the pathological environment, and then participate in the repair process of lung injury and pulmonary fibrosis progress. However, there is no effective method to separate and purify the LR-MSCs in vitro, and there is no specific inducer to build the differentiation system of the LR-MSCs. In view of this, we first isolated、 purified and identified the LR-MSCs with the optimized method of flow sorting; then an in vitro model was established by co-culture of the LR-MSCs and alveolar type II epithelial cells(ATII cells) with the trans-well technology. It will lay the foundation for investigating the specific molecular mechanisms of the LR-MSCs differentiation.Aims: 1. Isolate, purify and identify LR-MSCs in vitro;2. Establish the air-liquid interface co-culture system of LR-MSCs and ATII cell to induce the LR-MSCs to differentiate into ATII cells.Methods:1. The lung stem cells single-cell suspension was prepared each time from lungs of at least5C57BL/6(4-6wk old) using dispease enzyme and collagenase. We first precultured the digested lung cells, and then the Sca-1+CD45-CD31-cell population was sorted by FACS Aria I. Then the sorted cells were cultured in the low glucose DMEM medium containing15%FBS.2. We investigated the surface marker expression, gene expression and DNA profile between Sca-1+CD45’CD31’cell population and BM-MSCs.3. In the air-liquid interface co-culture model, LR-MSCs were mixed with the purchased ATII cells a ratio of1:1and cultured in the upper compartment of the culture well. After7days and14days co-culture, the expressions of epithelial markers on LR-MSCs were detected by immunofluorescence.Results:1. The Sca-1+CD45’CD31"cell population was successfully sorted by FACS. And the LR-MSCs were demonstrated as a homogeneous fibroblast-like and spindle-shaped morphology.2. Similar to bone marrow derived mesenchymal stem cells (BM-MSCs), these cells expressed Sca-1, CD29, CD90, CD44and CD106, but not CD31or CD45;shared the same gene expression file with the BM-MSCs; had a similar DNA profile and could be serially passaged with all the properties maintained. So, we identified these cells as LR-MSCs.3. In the indirect co-culture system, the ATII cells could induce LR-MSCs differentiation into epithelial cells. A subset of LR-MSCs presented an epithelia-like cell shape change from a long shuttle-type shift to polygonal, round shape. Some LR-MSCs expressed epithelial-specific proteins CK18, CK19, Occludin, SP-C.Conclusions:In this experiment, the LR-MSCs were isolated and identified successfully; in the indirect trans-well system, LR-MSCs were co-cultured with the ATII cells, and the ATII cells can induce LR-MSCs differentiation into epithelial ATII cell. This approach may hold promise in using LR-MSC for treatment in lung disease.