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Mesenchymal Stem Cell Differentiation to Lung Alveolar Epithelial Cells: A Potential Mechanism for Repair After Ischemia-Reperfusion Injury
R. Castanos1, Y. Jin1, J. O. Chan1, L. M. Backhus1, R. M. Bremner*2, V. A. Starnes*1, M. A. Smith1. 1University of Southern California, Los Angeles, CA, 2St. Joseph's Hospital and Medical Center, Phoenix, AZ,
BACKGROUND: Mesenchymal stem cells (MSC) have the capacity to differentiate into a variety of tissue lineages. Here, we sought to determine whether MSC have the capacity to differentiate into alveolar epithelial cells as a possible mechanism for repair of lung injury secondary to ischemia-reperfusion after lung transplantation.
METHODS: MSC previously isolated from the rat circulation using an aortic pouch entrapment model were cultured and analyzed for the capacity to differentiate into fat, bone, and cartilage cells. The MSC were then transfected with a retroviral vector to express enhanced green fluorescent protein (eGFP). The eGFP transfected MSC (MSC-eGFP) were then co-cultured with a known rat alveolar epithelial cell line, RLE-6TN, for 10 days. Subsequently, these co-cultured MSC-eGFP (ccMSC-eGFP) were separated using fluorescent activated cell sorting and placed back in tissue culture. The ccMSC-eGFP, along with MSC-eGFP and RLE-6TN cells, were then analyzed for alveolar epithelial cell protein expression including thyroid transcription factor-1 (TTF-1), the alveolar type I cell marker RTI-40, and the alveolar type II cell marker surfactant protein C (SPC) using immunohistochemistry, western blot and reverse transcriptase polymerase chain reaction (RT-PCR) techniques.
RESULTS: After eGFP transfection, the MSC maintained the capacity to differentiate into fat, cartilage, and bone cells, as demonstrated by light microscopy and immunohistochemistry. RLE-6TN, the known rat alveolar epithelial cell line, demonstrated expression of TTF-1 by immunohistochemistry, western blot, and RT-PCR; SPC by immunohistochemistry and RT-PCR; and RTI-40 by RT-PCR. The ccMSC-eGFP but not MSC-eGFP cultured alone demonstrated alveolar epithelial cell morphology by light microscopy. ccMSC-eGFP but not MSC-eGFP cultured alone demonstrated TTF-1 expression by immunohistochemistry, western blot, and RT-PCR. ccMSC-eGFP demonstrated substantially greater expression of SPC by immunohistochemistry and RT-PCR, as well as RTI-40 by RT-PCR, compared to MSC-eGFP cultured alone.
CONCLUSIONS: These data suggest that circulating MSCs have the capacity and may be induced to differentiate into alveolar epithelial cells by being placed in proximity with known alveolar epithelial cells. Circulating MSC may serve as a mechanism for repair and repopulation of alveolar epithelial cells after lung injury.
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