Technological advances in study of lung regenerative medicine: perspective from the 2019 Vermont lung stem cell conference

Regenerating complex organs, such as the lung, requires extensive knowledge about tissue homeostasis and the pathological molecular perturbations that occur as disease develops. Over the past decade, development of novel genomic, cellular and engineering technologies has increased our capacity to understand and even manipulate processes at a cellular and tissue level. The eighth edition of the “Stem Cells, Cell Therapies, and Bioengineering in Lung Biology and Diseases” workshop, held July 15–18, 2019, at the University of Vermont, Burlington, provided an interactive platform to discuss the application of cutting-edge technologies to rapidly evolving research areas of lung regeneration and repair. A technology that has rapidly gained ground since its introduction in 2006 is the use of induced pluripotent stem cells (iPSCs) in regenerative medicine. Through directed differentiation (i.e., the recapitulation of developmental milestones), the repertoire of iPSC-derived clinically relevant cells is expanding, with some already in clinical trials. In the lung field, impressive progress has been made over the past 10 years, and it is now possible to derive lung epithelial cells with distal (alveolar) and proximal (airway) attributes from human iPSCs [1,2]. The refinement of gene editing techniques has also allowed the correction of diseasecausing mutations in patient-specific iPSCs, such as the cystic fibrosis transmembrane regulator (CFTR) in cystic fibrosis, thereby providing a means to make functional and molecular comparisons between diseased and syngeneic iPSC-derived lung epithelium [3]. The application of computational methods to evaluate the similarity of iPSC-derived lung lineages to their in vivo counterparts [4] and the derivation of functional, engraftable lung stem cells with wide competency, such as airway basal stem cells [1], will enhance iPSC clinical relevance in lung regeneration.

Another prominent technology that is providing high-resolution characterization of cellular states in lung development, homeostasis and disease is single cell RNA sequencing (scRNAseq). In recent years, this technology has helped define rare and/or new lung cell populations such as the ionocytes [5,6], a CFTR-high population of airway epithelial cells, and has allowed deep phenotyping of human airways in smokers, as well as asthma and pulmonary fibrosis patients [7]. In parallel, analysis of time-series scRNAseq datasets has emerged as a powerful tool for defining transcriptional signatures of lung epithelial progenitors during human lung development [8] or reconstructing fate trajectories of iPSC-derived distal lung progenitors [9]. Moreover, scRNAseq data are being used to infer higher order relationships, such as intercellular signaling networks in the adult lung [10] or region-specific mesenchymal-epithelial interactions during foregut development [11]. Additionally, scRNAseq along with other “omics” technologies are an integral part of consortia efforts to characterize the processes of lung development (LungMAP), homeostasis and disease (Human Cell Atlas). Continuous improvement of dimensionality reduction and clustering methods along with introduction of intuitive, collaborative platforms for visualization and analysis of scRNAseq datasets will render scRNAseq a widely adopted tool for lung biologists and clinicians alike [12]. Nevertheless, scRNAseq findings must continue to be validated and expanded by a variety of other experimental approaches, such as loss-of-function studies, lineage tracing, protein-level confirmation, and spatial transcriptomics.

Resolving lung regeneration must be considered on multiple scales. Single cell omics technologies can provide a trove of information on cellular processes and states, yet it is emerging functions at the tissue and organ level that will determine the success of lung regenerative strategies. Generation of functional airway or lung tissue that can be transplanted would help fill unmet clinical needs for several lung conditions and diseases that have no other therapeutic options. Although there continues to be significant progress toward generation of functional lung and airway tissue in conjunction with the use of acellular lung scaffolds in preclinical models, it remains unclear whether an appropriate a scaffold source has been identified that can be scaled for the reproducible generation of tissue suitable for clinical transplantation [13]. One emerging method that could help address this gap is three-dimensional (3D) bioprinting, which provides a means to manufacture lung or airway tissue on demand for use in personalized medicine approach. Several groups have made progress in identifying potential bioinks, biomaterials and 3D-bioprinting techniques suitable for airways. However, there are still many critical studies that need to be performed including extensive in vivo testing to understand the potential for long-term tissue functioning.

Emerging technologies, such as scRNAseq, iPSCs and 3D bioprinting, will accelerate the path to successful cell therapies for respiratory diseases. These approaches may include stimulation of endogenous cellular regenerative pathways or engraftment of exogenous cells for regeneration of the injured or diseased lung. Moreover, the use of iPSCs to model monogenic or even complex lung diseases in vitro (disease-in-a-dish), combined with the power of 3D bioprinting to create complex structures with precise arrangement of multiple cell types will greatly advance the goal of in vitro lung organogenesis for studies of lung development and disease, high-throughput drug screening and bio-manufacturing. Ultimately, understanding of fundamental biological processes and the application of such knowledge to lung regenerative medicine is the key to the future development of safe and effective therapies.