Lung Regeneration

Chapter 48

Lung Regeneration: The Bioengineering Approach Joan Nichols and Joaquin Cortiella Departments of Internal Medicine and Microbiology & Immunology, University of Texas Medical Branch at Galveston, Texas

Chapter Outline 48.1 Introduction 48.2 Scaffolds for Lung Engineering 48.2.1 Development of AC Natural Lung Scaffolds for Lung Engineering 48.2.2 Use of Nanotechnology to Design Scaffolds 48.3 Potential Cell Sources for Lung Engineering 48.3.1 Endogenous Cell Sources 48.3.2 Exogenous Cell Sources 48.3.3 Good Manufacturing Practice Considerations Related to Clinical Applications of Stem Cells 48.4 Engineering of Lung Tissue

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48.1 INTRODUCTION Lung transplantation is currently an appropriate treatment option for patients suffering from end-stage lung diseases when medical and surgical options have not been effective. Currently, lung transplantation is limited by the number of suitable donor organs available for use. The shortage of human lungs is exacerbated by the fact that the lung is easily compromised during the process of organ retrieval, and this results in rejection of the majority of procured organs [1,2]. Although ex vivo perfusion and reconditioning increase the number of lungs available for transplant, there are still too few organs available to meet the increasing need. One answer to the organ shortage problem is the development of patientspecific tissues or organs for transplantation. This has been a goal for many researchers working in biomedical engineering and regenerative medicine. Progress has been made in some areas of respiratory system organ engineering, such as for the trachea [3], but the field of lung tissue engineering has not progressed rapidly. This lack of progress is due to (i) the specialized needs of the lung in terms of strength and elasticity of the scaffold

Regenerative Medicine Applications in Organ Transplantation. © 2014 Elsevier Inc. All rights reserved.

48.4.1 Early In Vitro Production of Lung Tissue 48.4.2 In Vivo use of AC Lung Scaffolds 48.4.3 Role of AC Matrix in Lung Lineage Differentiation 48.4.4 Problems Associated with Use of AC Lung Scaffolds 48.4.5 The Development of AC Lung Scaffolds for Clinical Use 48.4.6 Vascularization of Engineered Lung 48.5 Conclusions References

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necessary to support engineered tissues, (ii) the lack of identification of an appropriate cell source for tissue generation, and (iii) the problems associated with development of methods which promote adequate vascularization of engineered tissues which is needed in order to support gas exchange.

48.2 SCAFFOLDS FOR LUNG ENGINEERING Although some attempts have been made to produce freefloating 3D matrix-independent, fluid-filled spheroids of airway epithelium [4], it is generally accepted that engineering lung tissue will require the development of a scaffold that meets the specialized needs of the lung. Of critical importance in selecting a scaffold for generation of lung tissue is the strength and elasticity of the material and the adsorption kinetics or capacity for cellular remodeling [5 7]. Since the primary physiological function to be replaced by engineered lung is gas exchange, an appropriate lung scaffold must allow gas exchange to occur unimpeded while providing adequate support to the

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cells and tissues. Lung scaffold design also needs to take into account the normal architecture and geometry of the lung as well as the need to support cell movement, transfer of nutrients into the tissues, and waste removal from tissues. For development of lung tissue, scaffolding must remain long enough to support tissue development without impeding the elasticity or altering the elastic recoil of the engineered tissue or any adjoining normal tissue [6,7]. Scaffold components that are not as elastic as normal lung have the potential to contribute to development of restrictive conditions similar to the disease process which occurs in patients with idiopathic pulmonary fibrosis or sarcoidosis [6,7]. In the case of obstructive diseases, scaffold materials that limit the size of the airway could potentially add to existing obstructive problems. Synthetic and natural polymers have been used as scaffolds to support efforts to engineer lung tissue. Synthetic polymers can be produced with a wide range of mechanical and chemical properties [8]. Degradable synthetic matrices that have been used to engineer lung tissue include polyglycolic acid (PGA) in the form of a felt sheet [9], PGA combined with pluronic F-127 [9], polylactic-co-glycolic acid (PLGA) [9] or poly-L-lactic-acid (PLLA) [9,10], poly-DL-lactic acid (PDLLA) [10], and polydimethylsiloxane [11]. Natural materials used in the past to engineer lung tissue include Matrigelt [12], Gelfoamt [13], EnglebrethHolms murine sarcoma basement membrane [14,15], and collagen [16 18]. Matrigelt (Becton-Dickinson) is a solubilized basement membrane preparation extracted from Engelbreth-Holm-Swarm (EHS) mouse sarcoma, a tumor rich in extracellular matrix (ECM) proteins including laminin, collagen IV, and heparin sulfate proteoglycans [19]. EHS and a variety of component matrix proteins found in EHS are commercially available. Collagen (generally type I) has been used as a scaffold for engineering a variety of tissues including lung, and a number of collagenbased scaffolds are available for clinical use. Gelfoamt (Pfizer) is a compressed sponge of porcine skin gelatin and was originally developed as a hemostatic device to arrest bleeding and promote clotting. There are limitations in the use of natural scaffold materials such as these due to their mechanical properties and variation in degradation rates. Scaffolds formed from these materials are also often very simple in terms of geometry, ECM composition, and lack the complexity as well as the elasticity or strength provided by normal lung ECM. Acellular (AC) natural scaffolds are composed of the ECM secreted by the resident cells of the tissue or organ from which they are produced. As a result, organ-specific scaffolds already possess the correct anatomical, chemical, and morphological structure of the natural tissue, and this has been shown to facilitate the constructive remodeling of many different organs in both preclinical animal

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studies and human clinical applications [20,21]. The composition and structure of AC scaffolds or natural organ ECM depends on the origin of the tissues and the physiologic functions provided by the tissues [20 23]. Apart from meeting biological requirements, the biophysical cues originating from ECM microstructure and mechanical properties have been shown to be a major influence on the growth and differentiation of cells and regulation of cell behavior [8,23 26]. It has been suggested that organ-specific ECM structure and mechanics may actually guide tissue patterning and stem cell differentiation [27,28]. It is also important to remember that changes in ECM during remodeling, particularly of collagen and elastin fibers forming natural scaffolds, may in fact interfere with normal respiratory mechanics [29 31].

48.2.1 Development of AC Natural Lung Scaffolds for Lung Engineering The structure of the lung is largely determined by the connective tissue network of the ECM and the organization of cells which generate the nonlinear mechanical properties which lead to the complex mechanical behavior of the lung which supports the process of breathing [31]. The ECM of normal lung parenchyma is composed primarily of collagen I, III, and elastin, and the principal function of these components is to form the mechanical scaffold that maintains the structure of the lung during the process of ventilation. Development of procedures for production of decellularized lung must allow for retention of key ECM components supporting lung functions while facilitating removal of cell debris and nucleic acids both of which could induce unwanted immune responses. Design of effective decellularization procedures are dictated by factors such as tissue density, tissue organization, or organ structure [22,23]. This is of particular importance for the lung since tissue density, and structure vary considerably among main stem bronchi, bronchioles, and distal lung. A variety of methods have been used to produce AC scaffolds from human tissues or organs [22,23], but these processes are not lung specific and do not take into account the delicate nature of lung structure. Techniques used for tissue and whole organ decellularization have been reviewed, including descriptions of solvents, detergents, physical agents, and enzymes [6,21 23]. Currently, there are only a few protocols for decellularization of lung [27,28,32 36], and these have been listed in Table 48.1. Protocols differ in the reagent used to facilitate cell removal, the physical conditions for decellularization, the duration of the process and, subsequently, in the ECM composition of the AC scaffold produced. Detergents used for the decellularization of lung include Triton X-100, sodium dodecyl sulfate (SDS),

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TABLE 48.1 Comparison of Current Decellularization (DC) Methods and Outcomes after Recellularization Protocol

Tissue Origin

Detergent Used

Condition For DC

Time ECM Remaining in AC for Lung Process

Cell Source

Cell Responses or Cell Products

Lwebuga- Human Mukasa JS lung [1986] [37]

Distilled water, then 0.1% Triton X, followed by 2% sodium deoxycholate

Immersion

48 1 h

Collagen I, III, IV, V; laminin and fibronectin but no fibronectin and collagen V in basement membrane

Rat AE II cells

Loss of lamellar bodies and change in morphology of AE cells

Cortiella J Rat lung [2010] [28]

1% SDS

Infusion followed by bioreactor immersion

3 5 weeks

Collagen I and elastin

mESC (C57BL6)

Prosurfactant protein C, surfactant protein A, CD31, cytokeratin-18 Clara cell secretory protein and TTF-1

Ott HC [2010] [38]

Rat lung

0.1% SDS

Infusion and immersion

120 min Collagen, proteoglycans, and elastic fibers

Rat fetal lung cells

Surfactant protein A, SPC, and TTF1 T1-α by FLCs

Petersen TH [2010] [39]

Rat lung

CHAPS

Infusion and immersion

180 min Collagen I, elastin, and laminin

Neonatal rat epithelium and microvascular lung endothelial cells

Prosurfactant protein C and pro-SPB Clara cell secretory protein, aquaporin-5

Price AP [2010] [40]

Mouse lung

0.1% Triton X 100 followed by 2% sodium deoxycholate

Infusion and immersion

48 1 h

Collagen I, elastin, glycosaminoglycans, and laminin

Murine fetal lung cells (E17)

Prosurfactant protein C Cytokeratin-18

Daly AB [2011] [41]

Mouse lung

0.1% Triton X 100 followed by 2% sodium deoxycholate

Infusion and immersion

3 days

Collagen I, collagen IV, laminin, fibronectin, but low levels of elastin and glycosaminoglycans

Mouse bone marrow MSCs

Transient expression of TTF-1 but no other lung lineage markers

Song JJ [2011] [42]

Rat lung

0.1% SDS

Perfusion

72 h

NA-Described in Ott HC [2010]

Rat fetal lung

TTF-1, pro-SPC, CC10, vimentin and ED-1 (CD68)

Bonvillan RW [2012] [43]

Rhesus macaque lung

0.1% Triton X 100 followed by 2% sodium deoxycholate

0.1% Triton X 100 followed by 2% sodium deoxycholate

3 days

GAGs are preserved. Collagen I, collagen IV, laminin, and some fibronectin

Rhesus macaque Cells adhered to Bone marrow scaffold MSCs and adipose tissue

mESC, murine embryonic stem cells; pro-SPC, prosurfactant protein C; pro-SPB, prosurfactant protein B; CC10, Clara cell 10 kD protein; TTF-1, thyroid transcription factor-1; AE cells, alveolar epithelial cells; SDS, sodium dodecyl sulfate.

sodium deoxycholate, and 3-[(3-cholamidopropyl) dimethylammonio]-1-propanesulfonate (CHAPS). Triton X-100 (C14H22O(C2H4O)n) is a nonionic surfactant which has a hydrophilic polyethylene oxide group and a hydrocarbon lipophilic or hydrophobic group. SDS is an organic compound with the formula CH3(CH2)11OSO3Na. It is an anionic surfactant used in both cleaning and hygiene products. Sodium deoxycholate (deoxycholic

acid) is a water-soluble, bile-acid, ionic detergent generally used in methods for protein isolation or as a component of many cell lysis buffers (e.g., RIPA buffer). CHAPS is a zwitterionic detergent used in the laboratory to solubilize biological macromolecules such as proteins or as a nondenaturing solvent in some procedures for protein purification. Comparisons of matrix composition after decellularization of rat or mouse lungs have

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shown in Figure 48.3A. In this decellularization system, catheters are attached to the trachea and pulmonary artery in order to allow fresh detergent to be flushed continually through the organ (Figure 48.3B) using peristaltic pumps (Figure 48.3C) to facilitate removal of cell debris and blood. Whole pig trachea-lungs or human trachea-lungs can be produced in this large perfusion system in hours to days depending on the detergent or detergent concentration used as well as the fluid flow rate (Figure 48.3D F). Computer programs can also be written, which control pump speed and lung expansion (Figure 48.4B). Often our research group has used a bronchoscope during the decellularization process to perform a gross examination of the airways or vascular system as shown in Figure 48.4C and D.

48.2.2 Use of Nanotechnology to Design Scaffolds

FIGURE 48.1 Perfusion system for decellularization of mouse or rat lung. Rat lungs were attached to a 16GX 3.5 in. spinal needle (Dyna Medical Corp, London, Ontario) and detergent was pumped through the trachea and continually removed from the decellularization chamber (a filtered 75 cm2 tissue culture flask) using peristalic pumps.

indicated that variations in the ECM composition may occur depending on the detergent used [37,39,44]. The basement membrane is composed of collagen type IV, laminin, and proteoglycans. The interstitial matrix of the lung is composed of fibrilar collagens types I, II, and VII as well as elastin, proteoglycans, and hyaluron. Use of CHAPS or Triton X-100 combined with sodium deoxycholate in the decellularization process allowed for retention of varying amounts of basement membrane components such as collagen IV, laminin, and proteoglycans, as well as components of the interstitial matrix collagen 1 and elastin. The use of SDS resulted in removal of most basement membrane components but left interstitial matrix components collagen I and elastin. Different physical systems used to facilitate decellularization include simple immersion in detergent, use of perfusion systems (Figure 48.1), or bioreactor chambers (Figure 48.2A C). Use of a bioreactor to facilitate removal of cell debris is shown in Figure 48.2. Whole rat trachealungs were placed in a bioreactor chamber (Synthecon, Houston, TX) containing SDS (Figure 48.2A) and allowed to rotate with perfusion of fresh detergent for a period of days to weeks depending on the concentration of the detergent and the flow rates used (Figure 48.2B D). Recently, more complex systems have been developed to produce large organ, AC pig or human trachea-lung such as the one

In the future it is possible that lung scaffolds with high levels of complexity may be fabricated using nanotechnology [38,40]. Current nanofabricating techniques allow the creation of nanometer-sized scaffolds formed from combinations of ECM proteins with surface qualities that facilitate cell attachment and influence cell fate determination. These methodologies could be applied to produce scaffolds that are made from strong, highly elastic material with architecture and pore size similar to that found in the normal alveolus. Nanoparticle design of scaffolds has been attempted using a variety of different approaches. One scaffold with the potential to meet these requirements features a novel inverted colloidal crystal (ICC) geometry formed from modified hydrogel [41,43,45]. Primary colloidal crystals can form hexagonally packed lattices of spheres, with a wide range of diameters from nanometers to micrometers. ICCs are similarly organized structures where the spheres are replaced with cavities leaving open interstitial spaces within which cells can grow and form tissues. When ICC cavities exceed the diameter of cells, they can be used as 3D cell scaffolds [43,45]. The open geometry of the ICC lattice, high porosity (74% of free space), and large surface area make ICC an attractive structure to support the development of lung alveolus formation [41,43,45]. One promising option for scaffold development may be to use a combination of electrospray and spinning referred to as electrospinning. In the production of electrospun materials, a high electric field is applied to a droplet of fluid creating an electrically charged jet of polymer solution or melt, which dries or solidifies to leave a polymer fiber, typically in the micro- or nanoscale. This process has been extensively reviewed by others [42,46]. These techniques can regulate surface topography down to the nanometer range and include

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FIGURE 48.2 Bioreactor system for decellularization of mouse or rat trachea-lung. (A) Fresh lung in bioreactor chamber containing 2% SDS at the beginning of the decellularization process. (B) Decellularized lung in 1% SDS at the end of the decellularization process. (C) Acellular rat lung scaffold after washing in DNAase and PBS with antibiotics and antimycotics. (D) Acellular whole trachea lung scaffold of rat. Protocol from [29].

nanoscale surface pattern fabrication, electrospinning, and self-assembly fabrication [42,46,47]. Electrospinning has been used to form 3D porous nonwoven mats or layers of biomaterials with geometries that support cell attachment, movement, and cell spreading. Electrospun meshes of collagen and elastin (1:1 ratio) have been produced from aqueous solution using this technology [48]. It is even possible to blend synthetic and natural polymers together to generate complex multicomponent scaffolds such as gelatin (degraded collagen) combined with either a conductive polymer, polyaniline, or with a mixture of PLA/ PGA. These mixed scaffolds supported cell attachment and, depending on the pore size, induced cells to display different morphologies [48].

48.3 POTENTIAL CELL SOURCES FOR LUNG ENGINEERING 48.3.1 Endogenous Cell Sources The lack of an adequate source of human cells for use in engineering lung remains a critical issue that continues to limit the development of functional engineered lung tissue

for transplantation. The generation of new lung tissue from lung progenitor cell populations is the most appealing option for a cell source since it would offer the possibility of autologous therapy or in situ therapy, which would minimize the risks of graft rejection and disease transmission. This is not the only option available, although, it is the least problematic one for the purpose of generation of an engineered lung for transplantation. The single most logical choice for a cell type to be used in the production of engineered lung tissue is the alveolar epithelial type II (AE II) cell of the distal lung. Regeneration of alveolar epithelia has been thought to be mediated by proliferation of existing AE II cells in the lung with their subsequent differentiation into alveolar epithelial type 1 (AE I) cell [49 55]. Adult AE II cells could be obtained from cadaveric human lungs not deemed suitable for transplantation. Currently, a variety of excellent procedures have been developed for the isolation of small numbers of AE II cells from human lung tissue [52 54]. Unfortunately, the current lack of isolation of sufficient numbers of endogenous lung progenitor cells or AE II cells as well as lack of proliferative capability of these cells in vitro make them an unsuitable choice for cell source for

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FIGURE 48.3 Large organ perfusion system for decellularization of pig lung. (A) Freshly isolated whole trachea-lung. (B) Picture of pig lung in the bioreactor chamber with cannula plaecd into pulmonary artery. (C) Image of chamber containing 2% SDS and cell debris on day 1 of the decellularization process. (D) Image of lung in chamber at end of decellularization process. (E) Lungs were expanded in the decellularization chamber to check for leaks in the pleura. (F) Acellular whole pig trachea-lung.

FIGURE 48.4 System for decellularization of human lung. (A) modified decellularization chamber showing the peristaltic pumps that were used to control flow into the trachea, pulmonary artery, and from the chamber into cell waste container. (B) Image of acellular lung scaffold produced from a pediatric lung. (C and D) Use of bronchoscope to examine the airways of the AC lung shown in panel B.

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Lung Regeneration: The Bioengineering Approach

generation of lung tissue at this time. It is possible that culture procedures may be developed in the future which facilitate isolation and proliferation of these cells. Defining endogenous stem or progenitor cell populations in animal or human lung has been difficult. This topic has been extensively reviewed [55 58]. Little is known regarding human endogenous lung stem and progenitor cells and their identity, organization, and regulation in the adult human lung. A number of excellent reviews on the topic exist [55 58], and although a number of putative stem or progenitor cell types have been identified in mice, many of these cell types have required that the lungs be damaged in order to identify cells involved in regeneration of epithelia [59 64]. Different epithelial cell types in distinct areas of the lung are sensitive to various agents and have been used in lung injury models to investigate stem cells in the adult lung [62 64]. The best approach for the identification of human lung stem or progenitor cells will require an approach based on isolation and clonal assay of cells similar to what has been done to identify mouse lung epithelial stem or progenitor cells [65]. There are few reports of lung-derived progenitor cells that have generated lung tissue in vitro or in vivo. One of the first reports regarding generation of lung tissue, by Nichols et al., described a mixed population of ovine ckit positive somatic lung progenitor cells (SLPCs) that were used to produce surfactant protein C (SPC) as well as CC10 expressing cells [8]. Later work by other groups using human c-kit 1 lung stem cells showed that these cells were capable of self-renewal and multipotency in vivo with formation of thyroid transcription factor 1 (TTF-1) and other markers of lung lineage as well as development of lung tissue in injured mouse lungs [66]. Most recently, a population of putative human E-Cad/ Lgr6þ stem cells isolated, and indefinitely expanded from human lungs, was shown to harbor both self-renewal capacity and the potency to differentiate in vitro and in vivo[67]. Single-cell injections of E-Cad/Lgr6þ cells in the kidney capsule of mice produce differentiated bronchioalveolar tissue, while retaining capacity for selfrenewal, as demonstrated by serial transplantations under the kidney capsule or in the lung. Further work though is needed to examine the regenerative capacity of these progenitor cells. As stem cell advances make patient-specific stem cells easier to produce, maintain, and differentiate into lung lineages, we may begin to understand what will be required to produce tissues worthy of future clinical application.

48.3.2 Exogenous Cell Sources Exogenous cells with the potential to be used to generate lung tissue include fetal lung-derived cells (FLC),

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embryonic stem cells (ESC), and induced pluripotent stem cells (iPSC). Cells derived from rat or mouse fetal lung tissue have been used consistently by a number of research groups to produce mature lung cell lineages [13,27,28,32,34], but similar work has not been done using human fetal lung cells. ESCs are pluripotent cells derived from the inner cell mass of the preimplantation embryos [68]. These cells can be maintained in culture in an undifferentiated state indefinitely, maintaining their proliferating capacity and have the unique potential to give rise to cells and tissues of all three embryonic germ layers [68]. ESCs have been shown to be able to differentiate into AE II cells as well as other lung lineage cells [32,69 75]. The first reports of derivation of a lung-specific cell from mouse ESCs showed production of a heterogeneous population of cells with low, differentiation efficiency [69 71]. Later studies where mouse ESCs were transferred in small airway growth medium (SAGM) showed a 20-fold increase in the expression of type II pneumocyte marker SPC [71]. The main disadvantages of directing ESC differentiation using a defined medium are the extensive assay time required and the low yields of target cells obtained [69 72]. One of the most promising studies using ESC circumvented the difficulties in generating a pure culture of ESC-derived AE II cells by generating stable transfected human embryonic stem cell (hESC) lines containing a single copy of an SPC-promoter-driven neomycin-resistant gene cassette [75]. In this study, production of AE II cells was accomplished without having to form embryotic bodies, and hESC lines were differentiated and cultured in the presence of neomycin to produce pure preparations of hESC-derived AE II cells with biological and phenotypic characteristics of AE II cells which demonstrated the ability to proliferate and differentiate into AE I cells [75]. iPSCs are derived by reprogramming adult cells into a primitive stem cell state. This process results in the creation of cells that are similar to ESCs in terms of their capability to differentiate into a variety of cell lineages, including endoderm that forms lung cell lineages [76]. Takahashi made the ground breaking discovery that four transcription factors (Klf4, Sox2, Oct4, and c-Myc), when introduced into mouse fibroblasts through retroviral transduction, led to the formation of cells with pluripotent properties [77]. Later, human pluripotent cells were generated by either the same set of transcriptional factors or another set of transcriptional factors that did not include the oncogenic transgene c-Myc [78]. There are few reports describing production of lung epithelial cells from iPSCs [78 80]. One report provides the most compelling argument for the use of a humanized version of a single lentiviral “stem cell cassette” vector to reprogram cells [79]. This single vector allowed for derivation of human

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iPSC containing a single excisable viral integration that on removal generates human iPSC free of integrated transgenes. This strategy was used to generate .100 lung disease-specific iPSC lines from individuals with a variety of diseases affecting the epithelial, endothelial, or interstitial compartments of the lung, including cystic fibrosis, α-1 antitrypsin deficiency-related emphysema, scleroderma, and sickle-cell disease. Use of this system was shown to robustly differentiate iPSC into definitive endoderm in vitro which is the developmental precursor tissue of lung epithelia [80]. The same techniques were used to generate lung and airway progenitor cells from mouse mesenchymal stromal cells (MSCs) and patientspecific cystic fibrosis iPSCs [80]. These disease-specific human lung progenitors formed respiratory epithelium when engrafted subcutaneously into immunodeficient mice [80]. Although the use of ESCs or fetal tissue as a source of progenitor cells has produced some promising results, tissues produced from these sources would have to be human leukocyte antigen (HLA) matched or engineered to express the graft recipient’s HLA repertoire in order to be used for human organ transplantation. This would require the development of ESC or fetal tissue banks and, if a perfect HLA or tissue match was not available, immunosuppressive treatment of recipients of the engineered tissues. Although there are many problems that still must be addressed for ESC or iPSC technologies, such as the low efficiency of differentiation to lung lineages or the tendency for tumors to evolve after transplantation, these cell types may offer the best solutions for a renewable cell source for use in the future in the engineering of lung tissue.

48.3.3 Good Manufacturing Practice Considerations Related to Clinical Applications of Stem Cells Production of engineered lung tissues from any cell source will require considerable cell expansion and manipulation in vitro prior to cell scaffold construct formation. Because of potential contamination or damage to tissues, related compliance requirements for “good manufacturing practice” (GMP) and “good tissue practice” will have to be followed for cell sources used in the production of engineered lung [81,82]. Mechanisms to confirm that cell differentiation only occurs along desired lineages will also be necessary. Evaluation of karyotypic stability of the cells used and the possibility of formation of genetic alterations due exclusively to the manipulation of cells in vitro will also need to be addressed. The same potential for self-renewal and plasticity that makes embryonic and iPSCs or fetal germ tissue attractive

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sources for production of engineered lung also raises concerns about their potential for tumorigenicity. Specific tumorigenic lung cancer stem cell populations [83] or transformed counterparts of lung-derived stem cells with the potential to give rise to carcinomas have been identified [84]. Regulatory frameworks may actually require the implantation of differentiated tissues rather than growth factor primed cell matrix constructs in order to limit problems associated with using stem or progenitor cells for the engineering of tissues. The risk that adult growth factor primed endogenous lung progenitor cells or adult AE II cells have the capacity to give rise to cancer or form tumors is fairly low since the transforming events necessary to promote tumorigenic properties in stem cells requires more than the normal effects of growth factor activation and priming. It is also important to note that there was no indication of tumor formation from implantation of in vitro amplified, growth factor primed SLPCs into immunodeficient mice by Nichols et al. although development of mature lung lineage cells was shown [8]. Further work is needed to define the tumorigenic capabilities of endogenous lung progenitor cell populations or other potential cell sources.

48.4 ENGINEERING OF LUNG TISSUE 48.4.1 Early In Vitro Production of Lung Tissue Early simple in vitro models of the lung, although very simplistic duplicated some rudimentary aspects of lung function. Information gained from simple one- or twocell-type model systems paved the way for the design and execution of later complex multicell engineered tissue equivalents and has been extensively reviewed by the authors [5,6]. The first report describing transplantation of cell scaffold constructs to generate lung tissue was done by Nichols et al. [8]. In this study ovine CD117 1 SLPCscaffold constructs were produced using a combination of PGA and/or PF-127. These constructs were implanted into both a large animal model (sheep) and a small animal model (nude mouse). In sheep, autologous cell scaffold constructs were implanted directly into the right upper lobe of the lung into a pocket created by a wedge resection. In nude mice, similar cell constructs were placed on the animals’ backs. Implanted constructs were well tolerated in these studies and lung tissue assembly was facilitated in vivo by the use of the PGA scaffold. In this same study, SLPC PGA constructs formed on felt sheets of PGA were implanted into the thoracic cavity of three adult sheep with attachment of the construct to the right main stem bronchus site following a full pneumonectomy (Figure 48.5A and B). When harvested after 3 months,

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FIGURE 48.5 Implantation of somatic progenitor cell/PGA construct after a pneumonectomy. In vivo tissueengineered ovine lung using SLPC/PGA construct. (A and B) Surgery showing implantation of construct at the right main stem bronchus site. Black arrow points to SLPC/PGA sheet being implanted at pneumonectomy site. (C) Engineered lung tissue after excision. (D) Section of engineered lung, produced 3 months after implantation, stained with hematoxylin and eosin (magnification 400X).

these implants were not shown to support development of lung epithelia but did form extremely well-vascularized fleshy tissue fragments (Figure 48.5C and D). Gelfoamt constructs (sponge with cells) delivered into the lung by injection of the constructs (sponge with fetal rat lungderived cells) into normal lung parenchyma has also been used to facilitate formation of lung tissue in vivo[13]. Most newly formed alveolar-like structures, though, were found close to the border between the sponge and the surrounding normal tissue with few found within the sponge itself. It is unclear why this occurred since the porosity of the Gelfoamt scaffold should have provided cells with an adequate microenvironment to support cell movement and tissue development. Mondrinos et al. developed cell scaffold constructs using fetal pulmonary cells (FPCs) grown on a Matrigelt scaffold [9 12]. To examine the contribution of donor-derived endothelial cells to tissue construct vascularization, FPC Matrigelt constructs were injected into the anterior abdominal cavity of adult graft recipients (C57Bl6 mice) [12]. Implantation of Matrigelt alone resulted in infiltration of host cells into the scaffold. In FPC Matrigelt-FGF2 constructs the tissues were shown to develop ductal epithelial structures with development of pro-SPC expressing epithelial cells and patent vasculature. Vascularization of the construct was enhanced by addition of FGF2, and even in FGF2 Matrigelt implantations alone there was significant increase in capillary density. One of the first descriptions using a preparation of human AC lung scaffold evaluated AE II cell adherence

and viability using strips of AC lung alveolar matrix seeded with rat AE II cells to examine the influence of ECM on cell attachment and morphology. The AC scaffolds described contained collagen I, II, IV, and V as well as laminin and fibronectin [33]. Rat AE II cells seeded on the strips of human AC lung scaffold took on some of the morphological characteristics of AE I cells such as loss of lamellar bodies and cytoplasmic flattening. From 1986 until 2010, there was no progress in the development of AC lung scaffolds for the engineering of lung tissue. This changed dramatically in 2010 when four research groups published their findings related to the production of engineered lung using whole trachea-lung mouse or rat AC scaffolds [27,28,32,34]. In these studies, whole AC trachea-lung scaffolds were seeded and cultured with mouse [34], rat fetal lung cells (FLCs) [28], neonatal rat [27], or murine embryonic stem cells (mESCs) [32]. All groups reported good cell attachment, survival of cells, and limited differentiation of cells into lung-specific cell phenotypes. All groups also reported that the recellularization of the scaffolds was not complete and that there were areas of the whole lung scaffolds that had not been adequately populated. Whole rat AC trachea-lung scaffold has also been shown to support attachment of murine bone marrow derived MSCs or C10 mouse lung epithelial cells following intratracheal inoculation [35,36]. Although the MSCs were cultured in small airway growth medium (SAGM), the MSCs predominately expressed genes consistent with a mesenchymal or osteoblast phenotype, and no airway genes or vascular genes were

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expressed. Similar results were found when AC nonhuman primate lungs produced using a perfusion decellularization system were populated with rhesus bone marrow derived MSCs and adipose tissue. [36].

48.4.2 In Vivo use of AC Lung Scaffolds Use of whole AC trachea-lungs to engineer tissue makes the process of transplantation into animal models or potential clinical applications easier to work with since the lungs (i) fit properly into the thoracic cavity, (ii) support functions of the lung similar to normal lung, and (iii) possess the appropriate anatomical structures (trachea or bronchus) that allow it to be sutured in place in order to facilitate orthotopic transplantation. Engineered lung produced on AC scaffolds has been transplanted into animal models although graft recipient survival was limited in these early studies [27,28,37]. For the first of these studies, orthotopic implantation of an engineered left lung derived from neonatal rat lung cells cultured on whole AC rat lung matrix for up to 8 days was done following a left thoracotomy [27]. When implanted into rats for short time intervals, 45 120 min, the engineered lungs were shown to participate in gas exchange although there was some bleeding into the airways (Figure 48.3C). A second group engineered lung using rat neonatal lung cells cultured on AC rat scaffold for 5 days [28]. The engineered lung was transplanted orthotopically following a leftsided pneumonectomy. In these initial experiments by this research group, graft function in vivo was limited to a few hours due to development of pulmonary edema in the engineered lung. Later studies by the same group used engineered lungs derived from AC rat scaffolds seeded with human umbilical vein endothelial cells (HUVECS) and rat FLCs cultured for 7 10 days [37]. For these experiments, engineered lungs were transplanted into recipient athymic rats following a pneumonectomy. Athymic rats receiving a pneumonectomy with no transplant, or rats receiving transplantation of cadaveric lungs from Sprague Dawley rats were used as controls. Cadaveric lungs were shown to have slightly higher compliance levels than engineered lungs on postoperative day 7. Oxygenation levels for both engineered and cadaveric recipients was higher than for pneumonectomized animals up to day 7 but gradually declined in rats receiving an engineered lung between 7 and 14 days. Compliance and gas exchange of the engineered lung in this study gradually declined after 7 days due to progressive graft consolidation and inflammation. Engineered lung grafts but not cadaveric grafts also induced the formation of a thick fibrous scar surrounding the graft, causing restriction of graft expansion. This scarring was potentially due to a residual natural killer (NK) cell response in the athymic recipient [37]. Despite the problems encountered

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following transplantation of these engineered tissues, this study provides hope that in the future we will be capable of engineering functional lung tissue capable of long term growth and survival in vivo.

48.4.3 Role of AC Matrix in Lung Lineage Differentiation Lung ECM plays a major role in support of biomechanics in the lung [85], but we are just beginning to understand the influence of lung ECM on the differentiation of stem or progenitor cells and of subsequent tissue formation. Studies to examine this role have been limited although the field of tensegrity or mechanosensing has long suggested that (i) regional variation of ECM remodeling that occurs during embryogenesis leads to local differentials in ECM structure and mechanics, (ii) changes in matrix compliance (e.g., increased stiffness when the thinned basement membrane is stretched) alter mechanical force balance across membrane receptors that mediate cellECM adhesion, and (iii) altering the level of forces that are transmitted to the internal cytoskeleton will produce cell distortion and change intracellular biochemistry, thereby switching cells between growth, differentiation, and apoptosis [26,85 88]. As mentioned earlier, there is extensive regional variation in ECM composition and stiffness as you progress from the trachea to the bronchi and bronchioles and then to distal lung. Changes in ECM structure and composition could influence cell adhesion and provide critical cues that orchestrate tissue formation and cell function [88]. There have been a few reports supporting a role for lung-specific scaffold or at least cell ECM interactions in influencing differentiation of stem or progenitor cells [10,32,86,87]. Early studies suggested that specific components of ECM materials could strongly influence the differentiation of ESC into pneumocytes [32]. One of the first reports regarding the influence of ECM on ESC differentiation into lung epithelial and endothelial lineages examined the efficiency of differentiation after allowing mESCs to attach to individual components of ECM. In these studies, ECM proteins collagen I, laminin, and fibronectin were shown to induce production of AE II cells from mESC cultured in 2D or 3D [10]. Production of surfactant proteins C and A and aquaporin-5 were enhanced by the presence of laminin. Similar results were found when culturing mESCs on whole AC rat lung scaffold [32,73]. Efficiency of differentiation was measured here by evaluation of CD31, cytokeratin-18, and pro-SPC expression and was shown to be increased by mESCs cultured on AC lung compared to commercially available matrices Gelfoamt, collagen I, or Matrigelt [32]. Production of organized lung tissue as well as significant production of surfactant proteins A and

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Lung Regeneration: The Bioengineering Approach

C were only seen for mESCs cultured on AC lung and not on any of the other matrices used. Other reports also support the concept that organ-specific stroma or ECM may even be required for proper site-specific differentiation and organization of lung tissues [87]. Comparison between liver- and lung-derived AC scaffolds indicated that liver derived scaffolds maintained the differentiation state of primary hepatocytes while lung-derived scaffolds allowed for both induction of lung lineage and maintenance of site-specific development of AE II cells [87].

48.4.4 Problems Associated with Use of AC Lung Scaffolds The clinical application and success of decellularized trachea suggests that AC scaffolds may retain adequate strength to support both physiologic and anatomic functions of the trachea [3,89]. This may or may not be true for the lung. There are some limitations that must be considered before use of decellularized natural AC scaffold for development of lung tissues for clinical applications. Problems associated with the use of AC or natural scaffold material include the possibility that natural materials may harbor bacteria or viruses if adequate steps have not been taken to ensure the cleanliness of the materials produced. Retention of unwanted materials could lead to induction of immunological reactions leading to inflammation and graft rejection. The process of decellularization can also lead to degradation of the ECM, resulting in depletion of collagen, glycosaminoglycan, and elastin which compromises the mechanical integrity of the scaffold and any tissues generated on it. We must consider the effects of the decellularization process on the mechanical integrity of the lung ECM prior to and after engineering of lung tissue. As we have mentioned previously, AC ECM may be weakened, damaged, or degraded during the decellularization process. Quantitative evaluation of the composition of lung scaffolds suggests that there are great variations in AC ECM related to specific detergents used [41,43]. We do not know if the alteration in ECM composition will be detrimental to the production of tissues for the purpose of transplantation. What we know is that increased ECM production leading to deposition of collagen types I and III in fibrotic lungs results in lower lung compliance during assessment of pulmonary function tests (PFTs). Compliance levels are an important indicator of lung elasticity and, in disease states, correlate well with collagen composition of the lung ECM. Some groups reported lower compliance values for AC compared to normal lung in vitro[27,28,34,37] and after transplantation [37]. Our understanding of the link between lung tissue structure and function is incomplete due to the complexity

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of the problem and may not be easily understood or evaluated properly without examination of the ensemble behavior of all constituents of the lung which includes cells and ECM [90,91]. Culturing of selected cells on the AC lung scaffold will result in immediate changes to the ECM. Modifications to and remodeling of the ECM begins the moment cells attach. We may need to carefully examine the modifications made by cells to the AC ECM in order to determine what components of the ECM are truly critical to development of functional engineered tissues. For example, although SDS treatment removed basement membrane components during the process of decellularization, mESCs began to produce laminin and collagen IV relatively quickly following attachment and replaced the missing ECM components [32]. A recent comparative analysis of whole decellularized mouse lungs produced using Triton X-100/sodium deoxycholate, SDS or CHAPs also showed that although there were differences in gelatinase activation and protein composition of the AC scaffold, binding and initial growth following intratracheal inoculation with MSCs or C10 cells were similar [39]. Although cells and cell debris have been removed from the scaffold along with the HLAs, which are responsible for graft rejection, this does not mean that the scaffolds are no longer immunogenic. The host immune response to transplanted tissues or organs is an important determinant of graft function and survival. Natural scaffolds may induce immunogenic responses and invoke immunological reactions leading to inflammation and thereby causing graft rejection. A hallmark of tissue injury is increased turnover of ECM proteins [92]. Failure to remove ECM degradation products from the site of tissue injury or tissue remodeling can result in the induction of inflammation in the host. Hyaluronan is an important component of the lung interstitium and functions to help maintain the structural integrity of the lung. This protein also plays a major role in cell signaling following lung injury. Fragments of hyaluronan have been shown to trigger toll-like receptor (TLR), TLR-2- and TLR-4-dependent inflammation activation pathways resulting in initiation of inflammatory responses [92]. The immune response to AC scaffolds has not been studied extensively, and we need to develop a better understanding of the host response to the AC scaffold alone [93]. Investigation of the immunomodulatory effects of natural ECM scaffolds has recently indicated that tissue source, decellularization method, and chemical cross-linking modifications of scaffolds can affect the presence of damage associated molecular patterns (DAMPS) within the biologic scaffold. Presence of these DAMPS has correlated with differences in cell proliferation, cell death, secretion of proinflammatory chemokines, and

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upregulation of TLR-4 proinflammatory molecules and may influence remodeling of the ECM and associated tissue [94].

48.4.5 The Development of AC Lung Scaffolds for Clinical Use Before discarded human lungs can be considered for use in clinical applications, there are some issues that must be addressed. Policies need to be developed related to organ procurement for development of AC scaffolds from discarded lungs. Production standards, processing and sterilization methods, evaluation of the product, and handling requirements prior to and after production of these scaffolds will have to be established. We will need to evaluate the influence of the manufacturing process on ECM composition and immunogenicity and determine what components of the AC scaffold ECM are critical for tissue engineering. Poorly manufactured lung scaffolds may also lead to an induction of immune responses in recipients, alter the process of tissue remodeling, and affect the functional outcome of the engineered lung. Natural materials may also harbor bacteria or viruses if adequate steps have not been taken to ensure the cleanliness of the materials produced. GMPs such as training and certification of personnel and design of clean room technologies in order to guarantee the safety and quality of each scaffold will also need to be developed. Sizing and long-term storage concerns need to be addressed as does the impact of potential complications related to the variability of each scaffold due to donor diversity. While xenografts are regulated as medical devices, most allograft tissue is classified as a Human Cell & Tissue/Product (HCT/P) by the FDA and not as a medical device. The FDA does not require a specific sterilization technique or Sterility Assurance Level (SAL) for allografts. Similar practices will have to be developed for the cell source to be used to engineer lung tissue for transplantation. 21 CFR Part 1271, became effective on April 4, 2001 for human tissues intended for transplantation that are regulated under section 361 of the PHS Act and 21 CFR Part 1270 [95]. This is a comprehensive plan for regulating human cells, tissues, or cellular and tissue based products that would include establishment of registration and product listing, donor suitability requirements, good tissue practice regulations, and other appropriate production requirements.

48.4.6 Vascularization of Engineered Lung Generation of a functional vasculature is essential to the clinical success of any engineered tissue construct and

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Lung

remains a key challenge for the generation of clinically applicable engineered lung as well as for the field of regenerative medicine. Oxygen and nutrients cannot be delivered to cells residing in the interior of large-volume scaffolds via diffusion alone. The topic of vascular engineering has been extensively reviewed elsewhere [96,97] and will not be covered in detail in this chapter. In brief, a variety of scaffold materials and scaffold-free systems have been used to generate engineered vessels, but complete biomimetic constructs have not yet been produced. The same problems which limit the field of lung engineering also plague the field of blood vessel engineering such as lack of design and construction of appropriate scaffolds and lack of identification of adequate cell sources. A few novel approaches that could be used to produce vascular networks for lung engineering are worth mentioning at this time. Most recently developed vascularizing approaches for biomaterials attempt to mimic cellular communication that occurs in tissues and organs through the use of multiple cell types and biochemically modified scaffolds [97]. These attempts to match natural biological and mechanical cues through the use of ECM components have inspired the creation of scaffolds that incorporate growth factors, integrin-binding peptides, vasculature-forming cells, and physical features that promote diffusion and transport [97]. One novel approach to engineering blood vessels used sandwiched, striped patterns of endothelial cell and fibroblast sheets to control the formation of vasculature in the tissue, and in doing so identified that cell-to-cell interactions were critical in the fabrication of a specific 3D tissue structure with specific endothelial cell orientation [98]. Advances in fabrication technology have enabled the construction of microfluidic networks in scaffolds. Microscale technologies such as photolithography, micromachining, and micromolding have been utilized to fabricate 2D microfluidic networks on various biomaterials. These techniques allow for design of microfluidic networks by combining an oxygen transport simulation with biomimetic principles governing biological vascular trees [99]. One such system formed using a lost mold shapeforming process based on microstereolithography was used in the fabrication of a porous scaffold containing a microfluidic vascular network which was shown to provide oxygen and support cell proliferation under static and perfusion conditions [99]. This approach established a practical basis for designing an effective microfluidic network in a cell-seeded scaffold. In vivo angiogenic sprouting occurs and generation of capillary beds occurs naturally and has been reproduced using 3D capillary beds in an in vitro microfluidic platform comprised of a biocompatible collagen I gel supported by a mechanical framework of alginate beads [100].The engineered vessels had patent lumens, formed

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Lung Regeneration: The Bioengineering Approach

robust B1.5 mm capillary networks, and supported the perfusion of 1 μm fluorescent beads through them. Similar procedures may be used to produce a functional alveolar-endothelial junction which is the first of many steps that will be necessary in the production of a fully vascularized engineered lung construct. In the future, further research into mechanisms for regulating the spatial aspects of vascularization will be essential in order to produce vascularized lung capable of surviving after transplantation.

[3]

[4]

[5] [6]

48.5 CONCLUSIONS While the studies outlined in this chapter have generated great enthusiasm about the potential for regeneration of lung tissue for therapeutic use, the reality is that engineering of all of the critical component parts of lung tissue which support the physiologic function of the lung has been limited. In order to produce engineered lung tissue in the future for regenerative therapeutics, we will have to (i) select the cell source with the highest potential for use in transplantation (HLA matched) which may require tissue typing and HLA matching of banked cells; (ii) select the best biocompatible and degradable scaffold suitable for lung development, that takes into consideration the type of lung disease to be treated (restrictive or obstructive); (iii) determine the best combinations of growth factors and culture conditions which promote cellular differentiation and increase the efficiency of differentiation of ESCs, FLCs, or autologous stem cells in a manner suitable for in vitro production of 3D tissue engineered culture; (iv) select the best conditions overall which promote 3D production of lung tissue. The progress toward development of individual components of engineered lung to date has been encouraging, particularly in the differentiation of ESCs and iPSCs into AE II cells. Although significant advances must be made before we will achieve engineered tissues worthy of clinical application we have progressed to the point of in vivo implantation of engineered tissues. We will need to develop a better understanding of factors promoting vascularization of engineered tissues before we can begin to engineer lung tissue capable of being used in clinical applications.

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PART | VII

Lung

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