Golnaz Karoubi

Traumatic injury, stenosis, and malignancy involving large segments of the airway are difficult to reconstruct and require novel solutions. Despite advances in surgical techniques, the reconstruction of long-segment tracheal defects remains a significant challenge. Several bioengineering approaches have been explored for tracheal regeneration in vitro and in vivo, using cells in combination with three dimentional (3D) biological or synthetic scaffolds. This paper reviews recent advances in developing bioengineered trachea and the technologies utilized toward generating transplantable tracheal grafts. Specifically, the review will focus on the recellularization of tissue-engineered grafts using natural or synthetic scaffolds, highlighting relevant cell types used to reconstitute tracheal epithelium and cartilage. The promise of newly explored paradigms, including the application of pluripotent stem cells, will be discussed with an overview of associated challenges and necessary steps for future translation. Overall, these advances provide a foundation for the development of clinically viable tracheal grafts, bringing engineered tracheal reconstruction closer to reality.

Recellularization of acellular lung scaffolds is a promising technique for the generation of bioengineered lungs. However, at the present time, pre-clinical evaluation of bioengineered lungs shows graft failure likely due to thrombus formation and bleeding from bronchi. This is believed to be due to the impairment of barrier function, incomplete vascular endothelial cell coverage, and limited endothelial cell function. Therefore, efficient recellularization of the vasculature region of the lung scaffold with functional endothelial cells is an important goal. Coating of decellularized tissues, prior to recellularization has shown improved vascular endothelial cell coverage and endothelial cell function. Here, we report the first demonstration of preconditioning decellularized lung vasculature with an anti‑CD31 antibody to enhance re-endothelialization and vascular function in bioengineered lungs. Presence of the anti-CD31 antibody (Ab) coating resulted in enhanced cell adhesion, viability, proliferation and expression of key endothelial cell genes including Claudin-1 (CLDN) and Endothelial Nitric Oxide Synthase (eNOS). Re-endothelialized scaffolds in the anti-CD31 Ab coated group had morphological reconstitution of the pulmonary vasculature and a higher percentage of endothelialized blood vessels. Markedly greater fractions of intravascular Dextran in the anti-CD31 Ab coated group (20.7 ± 2.5% vesus 51.7 ± 2.5%, p = 0.0001) demonstrated reduced vascular endothelium leakage. Importantly, scaffolds in the anti-CD31 Ab coated group showed reduced platelet aggregation, measured via staining and quantification for integrin αIIb, when perfused with whole-blood ex vivo (17.7 ± 3.8% vs. 6.7 ± 1.5%, p = 0.04); and in vivo following a 30 min-left orthotopic left lung transplant in the mouse (14.7 ± 2.5% vs. 4.3 ± 1.5% in the anti-CD31 group ( p = 0.004); suggesting reduced thrombus formation. Taken together, our results frame scaffold preconditioning with anti-CD31 Ab as a viable approach towards the generation of vascularized bioengineered lung constructs.

Human alveolar type 2 (AT2) cells play a crucial role in maintaining lung homeostasis and facilitating repair. Researchers are investigating AT2 cells in ex vivo platforms due to their essential roles in lung physiology, disease modeling, and drug testing. Herein, we present a comprehensive workflow for the isolation and culture of primary human AT2 cells, including an efficient enzyme-based lung tissue digestion protocol and automated purification of AT2 cells from both healthy and diseased lungs. By targeting the HTII-280 surface protein on AT2 cells, we employed an automated magnetic-activated cell sorting (MACS) separation strategy using the autoMACS® Pro Separator. AT2 cells from healthy and diseased lungs were grown in 3D culture and formed alveolospheres, elucidating that the isolation strategy did not affect cell functionality. This approach provides high AT2 cell yield and purity, offering scalability to generate sufficient quantities of cells for downstream applications, such as disease modeling and cell-based therapeutics.

Chronic respiratory diseases are a major global health concern. Lung epithelial dysfunction is a common underlying feature of many such conditions; hence, reconstructing the diseased epithelium with functional epithelial cells is a promising therapeutic approach. There are various endogenous stem cell and progenitor populations in the lung epithelium that can be utilized for transplantation. Additionally, pluripotent stem cells (PSCs) have emerged as a valuable source for generating therapeutic cells due to their capacity for indefinite self‐renewal and the availability of directed differentiation protocols to transform them into lung progenitors or mature lung epithelial cells. This review discusses the endogenous stem cell and progenitor populations of the lung epithelium, recent advances in developing directed differentiation protocols to generate these cells, and the application of both endogenous and PSC‐derived lung epithelial cells for disease modeling in vitro and as cell therapies in vivo. It provides valuable insights into the current progress of regenerative medicine within the respiratory field and highlights areas that require further research.

Background: Tracheal transplantation is indicated in cases where injury exceeds 50% of the organ in adults and 30% in children. However, transplantation is not yet considered a viable treatment option partly due to high morbidity and mortality associated with graft rejection. Recently, decellularization (decell) has been explored as a technique for creating bioengineered tracheal grafts. However, risk of post-operative stenosis increases due to the death of chondrocytes, which are critical to maintain the biochemical and mechanical integrity of tracheal cartilage. In this project, we propose a new de-epithelialization protocol that adequately removes epithelial, mucosal, and submucosal cells while maintaining a greater proportion of viable chondrocytes. Methods: The trachea of adult male outbred Yorkshire pigs were extracted, decontaminated, and decellularized according to the original and new protocols before incubation at 37 °C in DMEM for 10 days. Chondrocyte viability was quantified immediately following post-decellularization and on days 1, 4, 7, and 10. Histology was performed pre-decellularization, post-decellularization, and post-incubation. Results: The new protocol showed a significant (p < 0.05) increase in chondrocyte viability up to four days after de-ep when compared to the original protocol. We also found that the new protocol preserves ECM composition to a similar degree as the original protocol. When scaffolds created using the new protocol were re-epithelialized, cell growth curves were near identical to published data from the original protocol. Conclusion: While not without limitations, our new protocol may be used to engineer chimeric tracheal allografts without the need for cartilage regeneration.

Chemically directed differentiation of pluripotent stem cells (PSCs) into defined cell types is a potent strategy for creating regenerative tissue models and cell therapies. In vitro observations suggest that physical cues can augment directed differentiation. We recently demonstrated that confining human PSC-derived lung progenitor cells in a tube with a diameter that mimics those observed during lung development results in the alteration of cell differentiation towards SOX2⁻SOX9⁺ lung cells. Here we set out to assess the robustness of this geometric confinement effect with respect to different culture parameters in order to explore the corresponding changes in cell morphometry and determine the feasibility of using such an approach to enhance directed differentiation protocols. Culture of progenitor cells in polydimethylsiloxane (PDMS) tubes reliably induced self-organization into tube structures and was insensitive to a variety of extracellular matrix coatings. Cellular morphology and differentiation status were found to be sensitive to the diameter of tube cells that were cultured within but not to seeding density. These data suggest that geometric cues impose constraints on cells, homogenize cellular morphology, and influence fate status.

Here we align airway epithelial cells on hydrogel grooves and induce apical-basal polarization and cilia formation. Neither initial alignment, nor maintained alignment on the grooves induced polarized cilia alignment in the epithelial sheet.