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Updated: Apr 6, 2026
Technique for Isolation and Culture of Rat Jaw Bone Marrow Mesenchymal Stem Cells
Published on: May 31, 2024
Interactions between rat alveolar epithelial cells and bone marrow-derived mesenchymal stem cells: an in vitro
Hiroyuki Ito1, Tokujiro Uchida, Koshi Makita
1Department of Anesthesiology, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo, 113-8519, Japan, hiro-ito.mane@tmd.ac.jp.
Background:
Bone marrow-derived mesenchymal stem cells (BMSCs) reduced the severity of acute lung injury after transplantation in multiple experimental studies, and several paracrine soluble factors secreted by the cells likely contribute to their therapeutic effect. The direct interactions between BMSCs and alveolar epithelial cells (AECs) may be an important part of their beneficial effects. Therefore, we assessed the interactions between BMSCs and AECs using a co-culture model of these two cell types from rats.
Methods:
BMSCs and AECs were co-cultured using a Transwell system under the following conditions: (1) separated co-culture-AECs seeded on the insert and BMSCs in the base of the well; and (2) mixed co-culture-AECs on top of the monolayer of BMSCs on the culture insert and no cells in the base of the well. After 21 days of culture, the cells on the membrane of the culture insert were fixed and stained with antibodies against the receptor for advanced glycation end-products (RAGE), surfactant protein D (SP-D), and zona occludens protein-1, and then analyzed by confocal microscopy.
Results:
In the separated co-culture condition, the phenotype of the AECs was maintained for 21 days, and cluster formation of SP-D-positive cells was induced in the AEC monolayer. We also found cluster formations of phospholipid-positive cells covered with RAGE-positive epithelial cells. In the mixed co-culture condition, the BMSCs induced alveolar-like structures covered with an epithelial cell layer. To determine the effect of keratinocyte growth factor (KGF) on this three-dimensional structure formation, we treated the mixed co-cultures with siRNA for KGF. While KGF siRNA treatment induced a significant reduction in surfactant protein transcript expression, formation of the alveolar-like structure was unaffected. We also assessed whether Gap26, a functional inhibitor of connexin-43, could mitigate the effect of the BMSCs on the AECs. However, even at 300 μM, Gap26 did not inhibit formation of the alveolar-like structure.
Conclusions:
BMSCs release soluble factors that help maintain and sustain the AEC phenotype for 21 days, and direct interaction between these two cell types can induce a cyst-like, three-dimensional structure covered with AECs.
