Stem Cells, Cell Therapies and Bioengineering in Lung Biology and Diseases 2023.

Repair and regeneration of a diseased lung using stem cells or bioengineered tissues is an exciting therapeutic approach for a variety of lung diseases and critical illnesses. Over the past decade increasing evidence from preclinical models suggests that cells, which are not normally resident in the lung can be utilized to modulate immune responses after injury, but there have been challenges in translating these promising findings to the clinic. In parallel, there has been a surge in bioengineering studies investigating the use of artificial and acellular lung matrices as scaffolds for three-dimensional lung or airway regeneration, with some recent attempts of transplantation in large animal models. The combination of these studies with those involving stem cells, induced pluripotent stem cell derivatives, and/or cell therapies is a promising and rapidly developing research area. These studies have been further paralleled by significant increases in our understanding of the molecular and cellular events by which endogenous lung stem and/or progenitor cells arise during lung development and participate in normal and pathologic remodeling after lung injury. For the 2023 Stem Cells, Cell Therapies, and Bioengineering in Lung Biology and Diseases Conference, scientific symposia were chosen to reflect the most cutting-edge advances in these fields. Sessions focused on the integration of "-omics" technologies with function, the influence of immune cells on regeneration, and the role of the extracellular matrix in regeneration. The necessity for basic science studies to enhance fundamental understanding of lung regeneration and to design innovative translational studies was reinforced throughout the conference.

3D Bioprinting Phototunable Hydrogels to Study Fibroblast Activation.

Phototunable hydrogels can transform spatially and temporally in response to light exposure. Incorporating these types of biomaterials in cell-culture platforms and dynamically triggering changes, such as increasing microenvironmental stiffness, enables researchers to model changes in the extracellular matrix (ECM) that occur during fibrotic disease progression. Herein, a method is presented for 3D bioprinting a phototunable hydrogel biomaterial capable of two sequential polymerization reactions within a gelatin support bath. The technique of Freeform Reversible Embedding of Suspended Hydrogels (FRESH) bioprinting was adapted by adjusting the pH of the support bath to facilitate a Michael addition reaction. First, the bioink containing poly(ethylene glycol)-alpha methacrylate (PEGαMA) was reacted off-stoichiometry with a cell-degradable crosslinker to form soft hydrogels. These soft hydrogels were later exposed to photoinitator and light to induce the homopolymerization of unreacted groups and stiffen the hydrogel. This protocol covers hydrogel synthesis, 3D bioprinting, photostiffening, and endpoint characterizations to assess fibroblast activation within 3D structures. The method presented here enables researchers to 3D bioprint a variety of materials that undergo pH-catalyzed polymerization reactions and could be implemented to engineer various models of tissue homeostasis, disease, and repair.

Human alveolar hydrogels promote morphological and transcriptional differentiation in iPSC-derived alveolar type 2 epithelial cells.

Alveolar type 2 epithelial cells (AT2s) derived from human induced pluripotent stem cells (iAT2s) have rapidly contributed to our understanding of AT2 function and disease. However, while iAT2s are primarily cultured in three-dimensional (3D) Matrigel, a matrix derived from cancerous mouse tissue, it is unclear how a physiologically relevant matrix will impact iAT2s phenotype. As extracellular matrix (ECM) is recognized as a vital component in directing cellular function and differentiation, we sought to derive hydrogels from decellularized human lung alveolar-enriched ECM (aECM) to provide an ex vivo model to characterize the role of physiologically relevant ECM on iAT2 phenotype. We demonstrate aECM hydrogels retain critical in situ ECM components, including structural and basement membrane proteins. While aECM hydrogels facilitate iAT2 proliferation and alveolosphere formation, a subset of iAT2s rapidly change morphology to thin and elongated ring-like cells. This morphological change correlates with upregulation of recently described iAT2-derived transitional cell state genetic markers. As such, we demonstrate a potentially underappreciated role of physiologically relevant aECM in iAT2 differentiation.

An Introduction to Engineering and Modeling the Lung.

Over the last decade, the field of lung biology has evolved considerably due to many advancements, including the advent of single-cell RNA (scRNA) sequencing, induced pluripotent stem cell (iPSC) reprogramming, and 3D cell and tissue culture. Despite rigorous research and tireless efforts, chronic pulmonary diseases remain the third leading cause of death globally, with transplantation being the only option for treating end-stage disease. This chapter will introduce the broader impacts of understanding lung biology in health and disease, provide an overview of lung physiology and pathophysiology, and summarize the key takeaways from each chapter describing engineering translational models of lung homeostasis and disease. This book is divided into broad topic areas containing chapters covering basic biology, engineering approaches, and clinical perspectives related to (1) the developing lung, (2) the large airways, (3) the mesenchyme and parenchyma, (4) the pulmonary vasculature, and (5) the interface between lungs and medical devices. Each section highlights the underlying premise that engineering strategies, when applied in collaboration with cell biologists and pulmonary physicians, will address critical challenges in pulmonary health care.

Decellularization and Recellularization Methods for Avian Lungs: An Alternative Approach for Use in Pulmonary Therapeutics.

The shortage of compatible allogeneic organs and an increase in the number of patients requiring long-term lung assist devices while waiting for lung transplantation have motivated scientists to explore alternatives to bioengineer new lungs, including through decellularization and recellularization processes. A novel approach for bioengineering an extracorporeal membrane oxygenator is based on the parenchymal structure of avian lungs which utilizes a cross-current unidirectional flow of air and blood rather than bidirectional airflow, and thus eliminates dead-space ventilation. This provides more efficient gas exchange than mammalian lungs. The novel approach utilized is to decellularize avian lungs and then to recellularize with patient-derived human lung epithelial and vascular endothelial cells with the goal of creating a fully functional structure that can be used as a gas-exchange device. Here, we present avian lung decellularization and recellularization methods for chicken and emu lungs, in order to study both small- and large-scale avian lung models. For decellularization, a detergent-based protocol is utilized, and different techniques are used to validate the de- and recellularization of those lungs, including microscopy, mass spectrometry, and immunohistochemical analyses. For recellularization, techniques for seeding different human lung cell types into the decellularized scaffolds are presented.