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Keywords:

  • Bioartificial lung;
  • lung transplantation;
  • perfusion decellularization;
  • regenerative medicine;
  • tissue engineering

Abstract

  1. Top of page
  2. Abstract
  3. Clinical Background
  4. Scientific Foundation for Bioartificial Lung Engineering
  5. Current Challenges in Bioartificial Lung Engineering
  6. Pathway to Translation via Intermediate Milestones
  7. Disclosure
  8. References

End-stage lung disease is a major health care challenge. Lung transplantation remains the definitive treatment, yet rejection and donor organ shortage limit its broader clinical impact. Engineering bioartificial lung grafts from patient-derived cells could theoretically lead to alternative treatment strategies. Although many challenges on the way to clinical application remain, important early milestones toward translation have been met. Key endodermal progenitors can be derived from patients and expanded in vitro. Advanced culture conditions facilitate the formation of three-dimensional functional tissues from lineage-committed cells. Bioartificial grafts that provide gas exchange have been generated and transplanted into animal models. Looking ahead, current challenges in bioartificial lung engineering include creation of ideal scaffold materials, differentiation and expansion of lung-specific cell populations and full maturation of engineered constructs to provide graft longevity after implantation in vivo. A multidisciplinary collaborative effort will not only bring us closer to the ultimate goal of engineering patient-derived lung grafts, but also generate a series of clinically valuable translational milestones such as airway grafts and disease models. This review summarizes achievements to date, current challenges and ongoing research in bioartificial lung engineering.


Abbreviations: 
BMP

bone morphogenic protein

CCSP

clara cell protein

COPD

chronic obstructive pulmonary disease

ECM

extracellular matrix

ESC

embryonic stem cell

FGF

fibroblast growth factor

SPC

surfactant-associated protein C

TTF-1

thyroid transcription factor 1

Clinical Background

  1. Top of page
  2. Abstract
  3. Clinical Background
  4. Scientific Foundation for Bioartificial Lung Engineering
  5. Current Challenges in Bioartificial Lung Engineering
  6. Pathway to Translation via Intermediate Milestones
  7. Disclosure
  8. References

End-stage lung disease is the fourth leading cause of death in the United States (1). Lung transplantation is the only definitive treatment, but the need for immunosuppression and donor organ shortage limit a larger clinical impact. Because less than 3000 suitable donor organs are transplanted per year, waitlist mortality exceeds 10% (2). Postoperatively, one out of three lung recipients will experience at least one episode of acute rejection in the first year, while only half of those surviving the first year will have preserved graft function beyond 5 years (3). Tissue engineering and regenerative medicine are novel disciplines attempting to develop alternative strategies to address end organ failure, donor organ shortage and chronic rejection. This review will summarize current progress in tissue engineering, pulmonary stem cell biology and whole organ engineering relevant to the development of a bioartificial lung.

Scientific Foundation for Bioartificial Lung Engineering

  1. Top of page
  2. Abstract
  3. Clinical Background
  4. Scientific Foundation for Bioartificial Lung Engineering
  5. Current Challenges in Bioartificial Lung Engineering
  6. Pathway to Translation via Intermediate Milestones
  7. Disclosure
  8. References

Engineering the lung's functional unit

Pulmonary tissue engineering is a multidisciplinary field aimed at generating constructs that can provide gas exchange to restore or replace lost lung function. The air–blood interface between epithelium and endothelium is the lung's functional unit. This interface must have a minimal diffusion length to facilitate efficient gas exchange between gas on the alveolar side and blood on the capillary side. Physiologic gas exchange further depends on hierarchical branching airways and vascular networks that both allow efficient perfusion and ventilation. One approach to engineering functional gas exchange units is engineering such networks. Synthetic scaffold materials such as polydimethylsiloxane can be micromilled into small networks to mimic the native lung's vasculature, providing physiologic shear stress and functional gas transfer across an acellular artificial membrane (4). Engineered to defined specifications, such constructs can be customized to match a patient's respiratory needs and pulmonary perfusion pressures via altering construct designs (4). Even specific cellular functions such as responses to nanoparticles, infection, cytokines and toxins can be mimicked in vitro when artificial scaffolds are seeded with cells (5). By reseeding constructs with a lung-specific heterogeneous cell population, mature organ functions such as surfactant production, mucociliary clearance and defense mechanisms can be introduced into engineered grafts. Although synthetic constructs can provide gas exchange for a limited period of time, longevity as a permanent implant may be challenging. Ideally, host cell engraftment and graft remodeling will mediate in vivo graft longevity and homeostasis. Aside from a potential clinical application as lung assist devices, engineered cellular constructs provide unique ex vivo lung models that allow high-throughput analyses of pathophysiologic responses to environmental and pharmacological molecules. With the advent of patient-specific pneumocytes derived from induced pluripotent stem (iPS) cells, such bioartificial lung constructs may allow rapid throughput drug screening and provide an isolated culture system to study cell fate along predefined vascular and airway architecture.

Deriving the ideal cell(s)

Although advances in micromilling and three-dimensional microfluidics computer modeling strive to generate artificial scaffolds capable of mimicking the native lung's microarchitecture, bioartificial lung constructs will require “recellularization” to successfully integrate into a host organism and maintain long-term in vivo function. To achieve tissue formation, appropriate stem or progenitor cell candidates must be identified, isolated and driven along a developmental path that reproduces native organ development and/or repair. Bioartificial lung engineering, therefore, requires a deep understanding of lung development and stem cell biology.

Pluripotent stem cells generated from adult cells represent one appealing cell source for clinical translation (6). Generation of patient-specific lung progenitors for rebuilding respiratory epithelium depends on signals that guide lung cell fate in organ development and repair. Lung and trachea derive from an outgrowth of the foregut endoderm and develop a hierarchical architecture (via branching morphogenesis) with corresponding supportive mesenchyme (vasculature and lymphatic structures). Early respiratory cell fate in the mid-foregut endoderm is demarcated by the expression of thyroid transcription factor 1 (TTF1, Nkx2.1). Although TTF1 expression is not exclusive to lung [also expressed in thyroid progenitors (7) and in the developing brain (8)], it is necessary for the development of mature lung epithelial cells. Beyond the cellular level, the lung develops in the context of other organs: developing cells respond to specific signal gradients. For instance, cardiac mesoderm plays an integral role in promoting respiratory progenitor cell instruction. High concentrations of fibroblast growth factor-2 (FGF2) result in high levels of lung-specific gene expression (TTF1, surfactant-associated protein C [SPC], clara cell protein [CCSP]) in the ventral endoderm whereras an absence of FGF2 induces an increased pancreatic-specific (Pdx1) gene expression (9,10). In addition to growth factors and spatial cues within the developing embryo, temporal patterns of specific signals further promote lung progenitor specification [e.g. transient BMP signaling induces proliferation of distal epithelial progenitor populations (11)]. As the number of spatial-temporal cues increases, the intricate interplay among these cues increases. A correct combination of transcriptional control, growth factors and spatial-temporal cues will be necessary to derive and differentiate patient specific cells to engineer bioartificial lungs.

Currently, the mechanisms and signals that delineate specified respiratory progenitor subpopulations are largely correlative. A few milestones, however, have been achieved in replicating development in vitro and thereby directly deriving lung progenitor cells from iPS/embryonic stem (ES) cells. SPC (12) and aquaporin 5 (13) expressing cells were first derived from ES cells using specific “small airway growth media” and cell-extract based reprogramming. Advanced culture conditions such as air–liquid interface culture induce the formation of tracheobronchial-like epithelia (including Clara cell-like phenotype) from ES cells (10,14). Coculture of ES cells with digested fetal lung mesenchyme induced pneumocyte differentiation and surfactant protein expression, highlighting the mesenchyme's role in lung development (15). Using more targeted stimulation via activin/nodal/Tgf beta signaling pathways, ES cell culture produced definitive endoderm progenitors (16). Further lung differentiation of such definitive endoderm via an intermediate “anteriorization” step recently produced a highly enriched (40%) TTF1 expressing population (17). In an alternative approach, a pure population of human type II alveolar cells was generated via antibiotic selection of transfected cell lines expressing SPC, an adult marker of type II alveolar cells (18). Such ES cell-derived pneumocytes engrafted in vivo and facilitated repair of bleomycin-injured airway epithelium, as quantified by improved lung function and decreased collagen deposition (19). Because the use of genetic alteration complicates clinical translation, current efforts now seek to use high-throughput chemical screening to identify key conditions that may generate, maintain and expand pure populations of lung progenitors derived from the anteriorized-definitive endoderm.

Although iPS/ES cells are fully pluripotent, resident lung epithelial progenitor cells (“adult stem cells”) are instrumental in everyday repair and renewal and represent an attractive alternative cell source for patient-specific bioartificial lung tissue engineering. Each section along the lung's hierarchical architecture has progenitor cell types that may self-renew in response to injury (10,20). The proximal tracheobronchial airways contain basal cells (21), Clara cells (not site specific and found throughout the whole lung epithelium) (22), neuroepithelial cells (23) and submucosal gland epithelial cells (24); distal bronchial airways contain nonCyp2f2 expressing (“variant”) Clara cells (25); and alveolar epithelium contains type II alveolar cells (26). In addition to lineage tracing confirmatory studies, current endeavors seek to understand the microenvironmental niches of these progenitor cells and their possible plasticity in response to injury. Once correctly identified and characterized, niche-specific progenitor cells and the signals that maintain cellular self-renewal may be utilized to regenerate functional epithelia.

Moving to the organ level

By applying the basic principles of tissue engineering and the lessons learned from lung developmental and stem cell biology, bioartificial lung engineering strives to regenerate the broad spectrum of specialized tissues of native lungs (e.g. conducting airways, vasculature, gas exchange tissue) in their three dimensional physiologic context. Currently, bioartificial lung engineering relies largely on the interaction of lung progenitor cells with their native niches to recapitulate tissue formation.

Constituting a portion of lung hierarchical niches and site-specific cues, extracellular matrix (ECM) proteins have been implicated in regulating lung development, repair and cell fate. In development, ECM proteins are necessary for directing correct structural remodeling. In repair, ECM proteins can signal both normal and pathological repair processes. Airway ECM, for instance, guides cytokeratin 5 (a marker for basal cells) and CXCR4 positive epithelial cells to reestablish tracheal pseudostratified columnar epithelium after epithelial injury (27). Conversely, intratracheal administration of collagen fragments induces accumulation of pulmonary neutrophils and fibrosis (28). ECM proteins alter lung cell fate via multiple direct pathways and regulates epithelial cell demographics by providing specific niches alongside other factors. For instance, ECM proteins facilitate Notch ligand postprocessing (29) and regulate the equilibrium between ciliated and mucin producing cell proliferation (30,31). Efforts to reconstitute proper physiologic lung matrix-cell “crosstalk” require scaffolds that mimic the native ECM with intact and site-specific three-dimensional signaling-cues.

In addition to creating scaffolds with site-specific microenviromental ECM cues to facilitate matrix-cell “crosstalk,” engineering proper lung physiology requires scaffolds that match the native lung's architectural complexity. A hierarchical vascular network is necessary to meet the metabolic needs of engrafted cells, facilitating the formation of three-dimensional tissue. Simultaneously, this network serves as the conduit for pulmonary blood flow and one compartment of the air–blood interface. A network of descending conducting airways is necessary for efficient ventilation and completion of the air–blood interface. Perfusion-decellularization of deceased donor lungs via anionic detergent perfusion through the native vasculature results in generation of acellular whole-lung ECM scaffolds. The unique characteristic of acellular native scaffolds is the whole organ preservation of multiple compartments (e.g. airway, vasculature, alveoli) in their physiologic proximal–distal relation (Figure 1; Refs. 32,33). Perfusion-decellularized native ECM scaffolds support the engraftment and growth of immature pneumocytes and endothelial cells (32–34). When cultured under biomimetic conditions, recellularized ECM scaffolds formed functional lung tissue with measurable gas exchange ex vivo and in vivo (32–34).

image

Figure 1. Bioartificial lung engineering on the basis of perfusion-decellularized matrix. Deceased donor lungs are cannulated via the pulmonary artery, vein and trachea. Cells and cellular debris are removed through perfusion with detergents and phosphate buffered saline via the pulmonary vasculature. Resulting acellular native lung ECM scaffolds are then mounted in a bioreactor and seeded with epithelial and endothelial cells via the trachea and pulmonary artery, respectively. After in vitro testing, the left lungs are removed, cannulated and transplanted in an orthotopic position after left pneumonectomy in rats. Grafts are perfused and ventilated via the recipient's vasculature and tracheobronchial tree.

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Current Challenges in Bioartificial Lung Engineering

  1. Top of page
  2. Abstract
  3. Clinical Background
  4. Scientific Foundation for Bioartificial Lung Engineering
  5. Current Challenges in Bioartificial Lung Engineering
  6. Pathway to Translation via Intermediate Milestones
  7. Disclosure
  8. References

Bioartificial lung engineering strives to recreate the broad spectrum of specialized tissues of native lungs (e.g. conducting airways, vasculature, gas exchange tissue) in their physiologic context by incorporating complex scaffolds, progenitor cells and multifaceted culturing conditions. Several approaches to bioartificial lung engineering provide promising progress toward clinical translation; however, significant hurdles remain (Figure 2).

image

Figure 2. Current approaches to bioartificial lung engineering. Seeding of endothelial and epithelial cells onto engineered (left panel) and native (right panel) matrix constructs achieves the formation of functional gas exchange tissue. Generation of a sustainable lung graft for long-term implantation will require a construct that approaches the cellular and architectural diversity of the native lung (middle panel). Engineered gas exchange devices lack the full hierarchy of the native lung, whereas lung bioengineering based upon native scaffolds provide this to a certain extent. Accurate epithelial differentiation along the proximal–distal spectrum has not yet been achieved with any of the current approaches. Derivation and subsequent seeding of the wide spectrum of appropriate cellular phenotypes at the appropriate sites will require new strategies for either approach.

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Challenges in scaffold materials and designs

Synthetic scaffolds are engineered using well-defined materials according to defined specifications and thus, allow generation of reproducible constructs. However, currently used synthetic scaffolds lack the entire spectrum of the native ECM protein composition. Furthermore, engineered constructs model single lung compartments and their function. Native ECM proteins are necessary for host derived defense and graft homeostasis. For instance, transmigration of leukocytes into air spaces is a physiologic response to airway injury and represents one subset of epithelial regulation of lung immunologic defense. Lung ECM proteoglycans direct neutrophil transmigration in the presence of bleomycin-induced epithelial injury (35). Thus, synthetic scaffolds devoid of native ECM proteins may provide gas exchange, but incompletely restore physiologic lung epithelial response to injury. Also, the architectural complexity of the lung is matched by a heterogeneous, but site-specific ECM protein composition along the proximal–distal airway: bronchoalveolar-specific variant Clara cells for instance coinhabit regions expressing specific isoforms of collagen IV unique to the bronchoalveolar junction (36). Therefore, native ECM proteins may guide physiologic lung functions in a site-specific manner and should be considered an essential scaffold component.

As more physiologic scaffold materials become available, scaffold architecture becomes the next hurdle. Synthetic scaffold designs used for lung-assist devices mimic the functional compartment (gas-exchange unit) with defined artificial materials that may provide ideal gas exchange. Besides generating increasingly compact vascular flow networks capable of physiologic blood flow and gas exchange, current efforts in artificial scaffold design are aimed at nanomilling other functional segments of the lung (conducting airway, bronchoalveolar junction, distal alveolar sacs) in one block, to recreate the lung's architectural hierarchy (Figure 2). Such constructs can be reseeded with correct site-specific populations of cells and corresponding ECM proteins. As an alternative to improved synthetic scaffold design, nanomilling technology and material selection, perfusion-decellularized native ECM scaffolds offer a blueprint to help guide creation of more sophisticated lung assist devices.

Challenges in cell differentiation

Understanding the continuum of cellular identity from stemness to differentiated lung tissue along the proximal–distal distribution is central to engineering bioartificial lungs. Cell origin and regenerative potential of region-specific lung cells must be accurately identified. The major distinct cell types (Figure 2) along the proximal–distal distribution are identified on the basis of protein expression: basal cells express P63/keratin-5; ciliated cells, FoxJ1; Clara cells, CCSP; type II pneumocytes, surfactant-protein, etc. Yet, these phenotypic classifications overlap and do not sufficiently define distinct subpopulations responsible for lung homeostasis. Although identification of lung epithelial “demographics” is the first step, recapitulating in vitro each distinct cell type's differentiation pathway is an even bigger challenge. Currently available techniques to guide differentiation of cell numbers required for human lung are extremely costly and labor-intensive, even with automated systems. Native lung ECM cues and three-dimensional biomimetic culture conditions may increase the efficiency of current protocols. ES cells for instance exhibit a growth preference for native decellularized lung ECM (in comparison to traditional culturing conditions) and also have a propensity to differentiate (or lose their stemness) in the native lung ECM when compared to traditional culture conditions (37).

Challenges at the organ level

Moving from single compartments to the whole organ level generates more compounded hurdles. Perfusion-decellularized native lung matrices may provide the complex hierarchical architecture of vasculature, conducting airways and alveoli, but also have major limitations. Perfusion-decellularized native matrices and synthetic scaffolds represent an incomplete “snap-shot” of a healthy lung's ECM, rather than that of a developing lung or a lung undergoing repair. Inflammatory cells and mesenchyme constantly remodel lung ECM in response to environmental stimuli. Priming of the ECM scaffold with growth factors and adhesion molecules before cell seeding may be necessary to induce tissue regeneration. A valid concern is that after incomplete recellularization, ECM proteins will be exposed and may initiate pathological reparative responses in vivo. Disrupting ECM composition with perfusion-decellularization may result in accelerated ECM scaffold degradation after recellularization or transplantation. When considering decellularization as a spectrum (and native matrix cues and degree of decellularization as inversely correlated factors), current protocols need to be optimized to best suit regenerative efforts. Selectively preserving (or substituting) key matrix proteins and cues found instrumental in guiding site-specific cellular plasticity will be essential.

Engineering bioartificial lungs at the whole organ level requires control of cell behavior in a three dimensional context. Current cell seeding strategies deliver a heterogeneous population of epithelial airway cells via gravity perfusion. Engrafted cells do not always accurately reconstitute the airway epithelial cellular demographics, as indicated by incorrect cell populations at different levels in recellularized grafts (Figure 2). Reseeding efforts to date have used immature pneumocytes, recapitulating fetal repair rather than true structure formation, thus bypassing the complex and highly dynamic earlier developmental stages. Despite these challenges, regenerated bioartificial whole lungs on the basis of current detergent-based perfusion-decellularization protocols contributed to gas exchange in vivo after experimental orthotopic transplantation for up to 1 week (34). Because of limited plasticity, immature pneumocytes failed to completely reconstitute epithelial architecture along a physiologic proximal–distal distribution in these experiments. As a result, graft longevity of these constructs in vivo is still limited by lack of functional mucociliary clearance.

Pathway to Translation via Intermediate Milestones

  1. Top of page
  2. Abstract
  3. Clinical Background
  4. Scientific Foundation for Bioartificial Lung Engineering
  5. Current Challenges in Bioartificial Lung Engineering
  6. Pathway to Translation via Intermediate Milestones
  7. Disclosure
  8. References

The concept of growing and successfully transplanting patient-specific bioartificial whole lungs seems fictional. A stepwise approach to this farfetched goal, however, will provide a better understanding of cell fate in lung development and repair, a series of clinically valuable translational milestones and may ultimately lead to success—an immunotolerated lung grown “on demand.” As a first milestone toward translation of regenerated grafts, clinical transplantation of regenerated large airways based upon decellularized human trachea has been performed successfully (38). Despite limited data on reconstituting proper tracheal architecture, early results suggest that regenerated constructs do serve as biologic prostheses and undergo remodeling and full integration in vivo. Taking this approach to the level of a whole lung with its entire architectural complexity, a segmental regenerative approach (large tracheobronchial, bronchioalveolar and alveolar airways) is warranted. Complete decellularization and recellularization may be the ultimate goal; however, until the technology has fully matured, repair of suboptimal donor organs using partial decellularization and repopulation with autologous progenitor cells may be an intermediate translational step and an opportunity to test the regenerative potential of candidate cell populations. Taking current technologies to clinical scale will require not only refined decellularization and scaffold preservation protocols, but also novel cell expansion, differentiation and reseeding techniques. A deeper understanding of lung progenitor cell hierarchy and cell-mesenchymal dynamics will enable not only phenotypic control in organ engineering, but also enable reparative cell therapy and modification of mesenchymal remodeling in vivo.

In summary, lung transplantation remains the definitive treatment for end stage lung disease. Tissue engineering and more recently organ bioengineering are novel disciplines striving to address donor organ shortage and the need for immunosuppression. The development of a transplantable bioartificial lung is far from reality. However, ongoing efforts will provide a forum for synergy among multiple disciplines. A stepwise approach to this ambitious goal will generate a series of clinically valuable milestones that will be more immediately applicable to the treatment of chronic lung disease and repair of donor lungs.

Disclosure

  1. Top of page
  2. Abstract
  3. Clinical Background
  4. Scientific Foundation for Bioartificial Lung Engineering
  5. Current Challenges in Bioartificial Lung Engineering
  6. Pathway to Translation via Intermediate Milestones
  7. Disclosure
  8. References

The authors of this manuscript have conflicts of interest to disclose as described by the American Journal of Transplantation. Dr. Ott is a member of the Board of Directors of Miromatrix Medical Inc. Dr. Ott is not a member of the management team at Miromatrix, does not participate in the daily activities of the Company, and recuses himself at the four annual Board meetings when any scientific or medical subjects are to be discussed that may conflict with his current or anticipated future research activities. He is compensated for his Board participation by the receipt of a small number of stock options and currently has no stock ownership in the Company.

References

  1. Top of page
  2. Abstract
  3. Clinical Background
  4. Scientific Foundation for Bioartificial Lung Engineering
  5. Current Challenges in Bioartificial Lung Engineering
  6. Pathway to Translation via Intermediate Milestones
  7. Disclosure
  8. References
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