Regenerating the lung: harnessing the untapped potential of AT2 cells

Runny noses, body aches, and chills – dreaded signs that the flu season is upon us. The cough, however, remains one of the most painful flu symptoms and is a clear indication that the influenza virus has successfully invaded the lungs. Though the lungs provide critical protection from environmental insults, they are also highly susceptible to injury caused by influenza. Fortunately, the lungs have a remarkable ability to regenerate in response to infection and disease. Damage to the lung often results in loss of alveolar type 1 (AT1) cells, which make up the majority of the lung epithelial surface and mediate oxygen and carbon dioxide gas exchange. Regeneration of AT1 cells is therefore critical for restoring pulmonary function following injury. To address this regenerative need, alveolar type 2 (AT2) cells repopulate the lung epithelial surface with new AT1 cells. Under homeostatic conditions, AT2 cells secrete surfactant to prevent lung collapse; however, following damage to the lung, AT2 cells function as facultative stem cells to regenerate the AT1 cell compartment. This regenerative response is often plagued by the formation of dysplastic tissue, which hinders lung recovery. New tools and therapies are therefore needed to promote lung regeneration and limit dysplasia in response to injury.

To help improve lung regeneration, Aaron Weiner, a third year DSRB graduate student in Andrew Vaughan’s lab, is turning towards the use of organoids – “mini” organs that can be grown in a dish and that recapitulate the native tissue in both structure and function. Transplanting organoids into damaged tissue facilitates tissue regeneration in animal models of disease and injury. For example, intestinal organoids can be successfully transplanted into damaged colonic tissue to aid in crypt regeneration. However, within the lung, developing transplantable AT2 organoids has been limited by current culturing methods, which require co-culturing the organoids with mesenchymal cells to provide critical niche-derived factors that support AT2 cell growth. AT2 organoids are not conducive for transplantation, as introducing ectopic mesenchymal cells into an injured lung may worsen tissue damage and contribute to fibrosis. Developing mesenchyme-free culture conditions is therefore necessary if AT2 organoids are to be used to facilitate lung regeneration.

Weiner’s recent publication in Regenerative Medicine describes his work identifying mesenchyme-free culturing methods that promote AT2 organoid growth and transplantation. First, Weiner and colleagues isolated fresh AT2 cells from the lungs of mice and grew them into organoids using twelve different mesenchyme-free culture conditions. By measuring organoid diameter and AT2 cell-specific gene expression, the authors quantitatively determined the specific cocktail of culture components that produce the healthiest AT2 organoids. Interestingly, they found that the use of growth factors and developmental signaling pathway modulators facilitates organoid growth and renders AT2 organoids independent of mesenchymal co-culture.

Results from Aaron Weiner's recent Regenerative Medicine publication demonstrate that primary AT2 cells can successfully engraft into injured lungs. Figure provided by Weiner and edited by CAMB newsletter staff.

Weiner and colleagues next asked whether AT2 cells grown ex vivo are functionally competent to repair injured lung. He isolated AT2 cells from the mouse lung, grew them as organoids using the newly developed culture conditions, and then transplanted them back into mice with influenza-injured lungs. Weiner observed successful engraftment of the transplanted AT2 organoids into the injured lung and found that subsets of engrafted AT2 cells retain their cell identity. These results demonstrate that AT2 cells can be successfully grown into organoids without using mesenchymal co-culture and can be subsequently transplanted into injured lungs.

Surprisingly, Weiner found that some engrafted AT2 cells adopt a dysplastic fate in the recipient lung. Weiner initially considered this finding a setback, as the ultimate goal of AT2 cell transplantation is to promote lung regeneration while limiting dysplasia. To interrogate this puzzling finding further, the authors transplanted freshly isolated primary AT2 cells into injured lungs to determine if cells that have never been cultured maintain their correct cell lineage following engraftment. Indeed, primary AT2 cells transplanted into influenza-injured lungs produced robust engraftments, maintained their AT2 cell fate, and even differentiated into AT1 cells, thereby contributing to regeneration of the injured lung epithelium. Encouragingly, primary AT2 cells did not produce dysplastic cells upon transplantation. To extend these findings even further, Weiner collaborated closely with the Morrisey, Worthen, and Shen labs at Penn to test primary AT2 cell engraftment in other lung injury modes, such as acid- and chemotherapeutic-induced injury. Again, in all injury conditions, primary AT2 cells engrafted into the lung without contributing to the formation of dysplastic cells.

In a final set of experiments, Weiner performed a pulse oximetry assay to measure blood-oxygen saturation, a metric of overall lung function and a method to see if primary AT2 transplants provide physiological benefit to injured recipients. Weiner showed that transplantation of primary AT2 cells into influenza-injured lungs not only improves pulmonary function but tended to allow for quicker recovery than control mock-transplanted lungs. This critical experiment demonstrated that transplantation of primary AT2 cells into injured lungs improves regeneration and function, opening up further areas of investigation for therapies to treat lung injury.

The results from this study provide important insight into AT2 cell biology and lung regeneration. Weiner and colleagues show that AT2 cells can be successfully grown into organoids without the support of mesenchymal cells. Even more, these culture conditions offer an attractive ex vivo model that can be used to further study AT2 cells. The authors also demonstrate that primary AT2 cells can engraft into injured lungs, maintain their cell fate, and aid in the regeneration and restoration of lung function. When reflecting on these findings, Weiner notes that the “most exciting thing for [him] was pushing the limits of how “stem-like” AT2 cells really are and figuring out if they could really regenerate the lung.” He explains that his current work highlights the robustness of AT2 cells as progenitors and facultative stem cells. Even after being removed from their native environment and undergoing transplantation into an injured lung, these cells maintain their stem cell ability and can robustly regenerate AT1 cells.

Weiner acknowledges that there is much more exciting work to be done to understand AT2 cell function. When asked about his future plans, Weiner eagerly describes his interest in understanding the precise mechanisms that enable AT2 cells to regenerate AT1 cells in the injured lung. As a starting point, Weiner cites recent findings that suggest AT2 cells dedifferentiate to a more pluripotent-like state prior to differentiating into AT1 cells. In the future, Weiner would like to follow up on these results to help identify the mechanisms that enable AT2 cell dedifferentiation.

The findings reported by Weiner and colleagues ultimately enhance our understanding of the regenerative capabilities of AT2 cells and have great implications for healing patients suffering from lung injury. Weiner hopes that through future investigations of AT2 cells and their ability to regenerate the lung, “we can use more specific, better defined subsets of cells to help heal ourselves.”

Reference: Weiner, A.I. et al. (2019). Mesenchyme- free expansion and transplantation of adult alveolar progenitor cells: steps towards cell-based regenerative therapies. NPJRegenerativeMedicine. 4: 17 (eCollection).

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