A. Trecartin1, R. Spurrier1, D. Warburton1, B. Driscoll1, M. Hiatt1, T. Grikscheit1 1Children’s Hospital Los Angeles,Pediatric Surgery,Los Angeles, CA, USA
Introduction: Lung disease is the fourth leading cause of death in the US and remains a significant cause of morbidity and mortality in pediatric and adult surgical patients. Lung stem cells and critical signaling from supporting mesenchymal cells are implicated in the pathogenesis of and recovery from lung injury. Bronchoalveolar stem cells (BASCs) appear to play a critical role in self-renewal and repair, differentiating into both club cells and type II alveolar epithelial cells in vitro. Definitive demonstration of BASC differentiation in-vivo has yet to be performed. A unique feature of BASCs is their expression of both surfactant protein C (SP-C) and the club cell marker CC10. To our knowledge there has been no previous successful development of tissue-engineered lung (TELu) containing these progenitors.
Methods: Lung organoid units (LOU) were prepared from actin-GFP murine lung tissue in a variation of a previously published protocol to generate OU from small intestine and were loaded onto poly-glycolic acid/poly-L lactic acid scaffolds. These scaffolds were then implanted subcutaneously into C57BL/6 hosts. Four weeks later the implants were harvested, preserved in paraffin, sectioned and prepared for immunofluorescence staining. A section of native actin-GFP mouse lung tissue was stained as a control. The slides were incubated with primary antibodies for T1α, SP-C, and CC10. After washing, the secondary antibodies labeled with Alexa Fluor® 488 for T1α’s primary antibody host, Alexa Fluor® 568 for SP-C, and Alexa Fluor® 680 for CC10 were placed. These were subsequently imaged in green, red, and far-red channels respectively.
Results: Both native lung and TELu show positive staining for T1α and SP-C indicating the presence of type I and II alveolar epithelial cells respectively. In some fields in TELu, clusters of multiple cells are identified with positive staining for both SP-C and CC10, indicating presence of bronchoalveolar stem/progenitor cells (BASCs).
Conclusion: TELu is a novel in-vivo model in which multiple lung epithelial subtypes are identified, including Type I and II alveolar epithelial cells and bronchoalveolar stem cells. BASCs are not identified in native lung in the larger clusters imaged in TELu, but usually are found at the bronchoalveolar junctions. Which may make this in-vivo model useful for better understanding the stem/progenitor cell functions that underpin regeneration of lung injury.