Design and development of tissue engineered lung

Abstract

Before we can realize our long term goal of engineering lung tissue worthy of clinical applications, advances in the identification and utilization of cell sources, development of standardized procedures for differentiation of cells, production of matrix tailored to meet the needs of the lung and design of methods or techniques of applying the engineered tissues into the injured lung environment will need to occur. Design of better biomaterials with the capacity to guide stem cell behavior and facilitate lung lineage choice as well as seamlessly integrate with living lung tissue will be achieved through advances in the development of decellularized matrices and new understandings related to the influence of extracellular matrix on cell behavior and function. We have strong hopes that recent developments in the engineering of conducting airway from decellularized trachea will lead to similar breakthroughs in the engineering of distal lung components in the future.

Introduction

Tissue engineering for regenerative medicine involves the reconstruction of tissue equivalents with the capacity to replace physiologic functions of tissues or organs rendered nonfunctional due to disease or traumatic injury. Functionally, trachea and lung are formed from branching epithelial cell lined tubes that support the conduction of air to the numerous primary gas exchange units, the alveoli, where circulating blood is oxygenated. The alveoli form the air/blood or endothelial cell/epithelial cell interface that provides the critical functional aspect of the lung which allows for gas exchange. Cell types that provide the specific functions necessary in part of the lung are very different and the challenges in the development of engineered lung tissues for clinical use lie in the issues of complexity of lung and the number of different cell types that make up this organ. For the development of complex tissues such as the lung advances in the identification and utilization of cell sources, development of standardized procedures for differentiation of cells, production of matrix tailored to meet the needs of the lung and design of methods or techniques of applying the engineered tissues into the injured lung environment will need to occur before we can realize our long term goal of engineering lung tissue worthy of clinical applications.

Although there has not been a great deal of recent progress in the engineering of distal lung tissue for clinical applications there has been some progress in critical areas that impact the field of lung tissue engineering as a whole. Many advances have been made in the identification of both endogenous lung and exogenous stem cell populations with the potential to be used as a source for development of engineered tissues. A recent and striking development in the field is the recent use of acellular cadaveric trachea seeded with mesenchymal stem cells to engineer upper respiratory tract as a therapeutic strategy.1,2 This is a major step forward in the field of regenerative lung medicine and the engineered trachea provided the recipient with a functional conducting airway and immediately improved the patient's quality of life which has always been the goal of regenerative medicine as a whole.

Engineering of any tissue requires careful selection of a cell source which provides for the generation and maintenance of specific biological functions. Potential cell sources for the regeneration or generation of lung tissue include embryonic stem cells,3–6 endogenous pulmonary stem cells7–10 and extrapulmonary stem cells.11–15 The choice of stem cell populations used is critical for design of any stem cell based therapy or for engineering of functional lung replacement tissues. Over the past few years, progress been made toward defining embryonic lung epithelial progenitors.16,17 The first report showing derivation of lung-specific cell phenotypes from embryonic stem cells (ESC) used small airway growth medium (SAGM) to differentiate murine embryonic stem (mES) cells.3 This group later identified a defined medium for the differentiation of ES cells into alveolar epithelium and also showed mESC cultured in this defined medium could be induced to express the type II pneumocyte marker surfactant protein-C (SPC).6 Further work suggested that the effects of growth factors on ESC cell differentiation, such as epidermal growth factor (EGF), etinoic acid (RA) and triiodothyronine, cannot be predicted according to their role in lung development.18

ESC have also been induced to differentiate towards specific lung lineages using exposure to differentiated mature lung cell extracts.19,20 Media conditioned by culture of the A549 human lung adenocarcinoma cell line were effective in driving type II pneumocyte differentiation from both murine and human ESCs.21 Type II pneumocyte-like cells derived by this method were shown to have a well-differentiated ultrastructure containing lamellar bodies and apical microvilli, and also expressed SP-C. It is possible that transcription regulators from the cell extracts used on these studies were the critical element that contributed to the differentiation process making a thorough examination of the role of these factors in cell differentiation warranted in the future if we are to design culture or priming methods that consistently differentiate stem cells along lung specific lineages.

Based on observations that ES cell fate is affected by the microenvironment surrounding the cells, Van Vranken et al.22 examined the effects of pulmonary mesenchyme on the differentiation capacities of mES cells. Previous studies on the effect of mesenchyme type on pulmonary epithelium differentiation had shown that plasticity of this epithelium, depended on the type of mesenchyme it was cultured with.23 When murine embryoid bodies (EBs) were grown in direct or indirect cocultures with lung mesenchyme, there was evidence of formation of lung epithelial cells.22 ESCs have also been differentiated according to the activin A protocol which was used to produce terminally differentiated lung epithelial cells.6

The existence of region-specific stem or progenitor cells is generally accepted although endogenous lung stem cells are still ill defined in the lung, and the mechanisms that control their proliferation and differentiation are for the most part unknown. A great deal of progress has been made in the definition of both endogenous24–30 as well as exogenous lung cells.31,32 The possibility that lung disorders may one day be treated using endogenous lung stem cells, or with exogenously applied stem cells, is the focus of much research effort. In order to understand how mature lung cells are derived from stem cell precursors we must first understand the normal behavior of endogenous lung stem cells, and how they are influenced by their environment. The generation of new lung tissue from cells derived from endogenous lung progenitor cells is appealing, since it offers the possibility of autologous therapy or in situ therapy, which minimizes the risks of graft rejection and disease transmission. This is not the only option, although, it is the least problematic one for the purpose of transplantation and to date the only lung-derived progenitor cell that has been used to generate lung tissue in vitro as well as in vivo is a heterogeneous population of somatic lung progenitor cells (SLPCs) described by Cortiella et al.7 Other endogenous lung cells with potential to function in the future, for development of engineered lung tissues are the bronchio-alveolar duct junction cells which include the bronchio-alveolar stem cells (BASCs)8 and variant Clara cells.9 BASCs in mice are a rare Sca-1⁺ CD45⁻ CD31⁻ CD34⁺ stem cell population found at the bronchio-alveolar duct junction which co-express both the alveolar Type II cell marker pro-surfactant protein-C (pro SP-C) and the Clara cell marker, Clara cell protein 10 (CC10).8 Variant Clara cells express CC10 and resist injury by naphthalene treatment and have been shown to contribute to distal airway epithelial repair.9 There have also been numerous reports supporting the possibility of bone-marrow-derived cell engraftment in the lung and/or differentiation into lung lineages12,33 although these cell types have not yet been used to generate lung tissue in vitro or in vivo. A more systematic approach to identification and study of endogenous lung or respiratory tract region specific stem cells is required. Specifically, we need to know the various mechanisms by which progenitors can influence repair and tissue development and structure.

Recently a novel stem cell isolated from amniotic fluid and referred to as amniotic fluid stem cells (AFSC) has been described.34 Data suggest that these cells are multipotent, showing the ability to differentiate into cell types from each embryonic germ layer. Human amniotic fluid stem cells (hAFSC) were shown to integrate into murine lung and to differentiate into pulmonary lineages after injury. Using microinjection into cultured mouse embryonic lungs, hAFSC were seen to integrate into the epithelium and express the early human differentiation marker thyroid transcription factor 1 (TTF1). In adult nude mice following hyperoxia injury, tail vein-injected hAFSC were seen to localize in the distal lung and were seen to express TTF1 and the type II pneumocyte marker SP-C. Specific damage of Clara cells using naphthalene injury produced integration and differentiation of hAFSC at the bronchio-alveolar and bronchial sites with expression of Clara cell 10-kDa protein. These results illustrate the plasticity of hAFSC to respond in different ways to different types of lung damage by expressing specific alveolar versus bronchiolar epithelial cell lineage markers, depending on the type of injury to recipient lung.

One cell type that has recently received considerable attention is the Clara Cell Secretory Protein⁺ (CCSP), SP-C⁺ cell located in the terminal bronchioles.11 Data suggest that these cells can contribute to both bronchiolar and alveolar lineages and may be a stem cell for both components of the lung.

A critical but unanswered question regarding endogenous lung stem cells is whether these cells are a distinct cell population or whether multipotent embryonic progenitors persist through adulthood? Evidence does suggest that, apart from their ability to proliferate and replicate themselves, embryonic cells that form tissues during embryonic development are different from the adult cells that maintain and repair them after birth.17 The majority of the genes expressed in multipotent embryonic progenitors are not expressed by endogenous lung stem cells or in adult lung. Embryonic cells proliferate rapidly and undergo shape changes that mediate branching morphogenesis and patterning in terms of tissue development which lead eventually to organ formation.17,35 In contrast, endogenous lung stem cells which maintain homeostasis, have limited capability to proliferate and although they do alter their shape and develop into self assembled tissues, they do not necessarily possess the ability to form complex tissues or organs. There is no evidence at this time that a multipotent stem cell population exists in adult lung and each region of the adult organ—upper and lower trachea, bronchi, bronchioles, terminal bronchioles, alveoli—appears to be maintained by its own stem or progenitor cell population.26–28

Protocols to Differentiate Cells to Lung Lineages

A major challenge to the development of engineered lung lies in the lack of understanding the influence of priming treatments, addition of lung specific growth factors or consideration of environment in which the cells will reside after transplantation has on lung cell lineage choice. Cell-cell and cell-matrix interactions of this complex epithelium appear to play a large part of the regeneration mechanism of the pulmonary epithelium through paracrine, autocrine and endocrine pathways. The type of stimuli used to promote lung epithelial regeneration has also been found to have an important function in the determination of the particular cell type that contributes to the renewal of the lung epithelium. The potential for embryonic stem (ES) cells to differentiate into cells with a distal lung epithelial phenotype has been demonstrated using different in vitro culture methods.6,36,37 The distal lung epithelial phenotype has been induced through the use of embryonic distal lung mesenchyme in coculture systems with differentiating embryoid bodies or the use of soluble factors in defined media to maximize definitive endoderm formation and select and maintain the desired phenotype.38 These methods provide an increased efficiency of distal lung epithelial derivation from ES cells and therefore, provide the foundation for the development of a cell replacement product to treat chronic lung disease or a useful in vitro model for the study of lung disease and development. The main disadvantages of directing stem cell differentiation using defined mediums, coculture or conditioned medium are the extensive time required in order to differentiate the cells, the inability to adequately control differentiation and the low yields of preferred cell types that result from these methods. A network of transcription factors and signaling molecules are known to affect lung growth and therefore presumably progenitor cell proliferation and work in this area is critical for the development of better protocols and selection of growth factor combinations that consistently drive SC to differentiate along lung lineages.

Good Laboratory Practice or GLP Concerns

It is important to remember that use of any stem cell source to generate engineered lung tissues will require considerable cell expansion, manipulation and culture time in vitro prior to use and that evaluation of potential contamination damage to tissues required for compliance with requirements for “good manufacturing practice” and “good tissue practice” will have to be followed.39,40 The transplantation of live human cells into patients presents a great potential for new medical advances, in the past contemplated only in the in the realms of science fiction. However many scientific, ethical, political and regulatory issues must be addressed before these advances in stem cell based or tissue engineered therapies may be realized. On January 19, 2001, the US Food and Drug Administration published a registration and listing final rule that requires human cells, tissue and cellular and tissue-based product establishments to register with the agency and list their human cells, tissues, and cellular and tissue-based products. The final rule, 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 at www.fda.gov/CBER/tissue/tisreg.htm. This is a comprehensive plan for regulating human cells, tissues and cellular and tissue-based products that would include establishment registration and product listing, donor-suitability requirements, good tissue practice regulations and other requirements. Similar programs that deal with good practice have been instituted in most countries where stem cell based or tissue based treatments are being examined for clinical applications. Procedures to confirm that cell differentiation only occurs along desired lineages will also be a necessary component of these regulations. Compliance procedures will also eventually require evaluation of the karyotypic stability of the cells and assessment of the possibility of formation of genetic alterations due solely to the manipulation of cells in vitro. The same potential for self-renewal and plasticity that makes adult, fetal tissue derived or embryonic stem cells attractive cell sources for the eventual production of engineered tissues also raises concern about the possibility for tumorigenicity of the stem cell source. Regulatory frameworks may eventually decide to support implantation of differentiated tissues rather than growth factor primed cell-matrix constructs to reduce the risk of tumor formation. Even though human embryonic stem cells have been shown to generate cell types found in the lung, differentiation has not been consistently directed efficiently towards a single cell lineage. Although ESC are self renewing, the possibility of tumor formation exists41 which is an important consideration in the selection of clinically applicable cell sources. Endogenous lung stem cells pose less risk of becoming tumorogenic. The risk that growth factor-primed and activated adult or tissue specific stem cells have the capacity to give rise to cancer or can result in tumor formation is low. Reports identifying lung tissue specific tumorogenic lung cancer stem cell populations42 or transformed counterparts of lung-derived stem cells that have the potential to give rise to carcinomas8 have shown that the transforming events required to initiate tumorogenic properties in stem cells require more than the normal effects of growth factor activation and priming for the purposes of cell differentiation and induction of tissue formation. 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 severe combined immunodeficient mice by Cortiella et al.7

Matrix Design

The extracellular matrix (ECM) of any tissue serves to support the architecture and structure of the tissue but also plays a role in development, growth, physiology and response to injury.43 The proteins that comprise ECM include those that supply tensile strength of the tissue, support metabolic functions, promote cell migration and facilitate oxygenation and immune responsiveness. Materials that contain geometry supporting movement of nutrients into the tissues and waste removal away from tissues are essential. The behavior of a developing cell is determined by its location and geometry of the tissue and hence the matrix supporting that tissue.44 These are also expectations for any natural or synthetic matrix designed for three-dimensional (3D) development of tissue in order to replicate specific organ or tissue functions.45 Of critical importance in matrix selection for development of lung tissue are the biocompatibility, the elasticity and the adsorption kinetics of the material used.46 Biomaterials designed for use as matrix for regenerative medicine purposes fail to replicate the complexity of the ECM that is found in the lung. The nature of complex organs such as the lung may even require the development of hybrid scaffolds formed from more than one material to provide all of the above requirements. For development of lung tissue the scaffolding or matrix must also remain long enough to support cell growth and tissue development without impeding the elasticity or altering the elastic recoil of the engineered tissue or the surrounding normal lung tissue. In the lung ECM contributes to the overall mechanical properties of the lung which change after injury or disease.47,48 If a biomaterial designed for use in the lung is not as elastic as normal lung it could potentially contribute to development of a restrictive condition similar to what is caused by the restrictive scar tissue formation seen in idiopathic pulmonary fibrosis or sarcoidosis patients. Both synthetic and natural polymers have been studied for use in lung tissue engineering.46 Natural materials that have been used to grow lung tissue include collagen,49–52 Matrigel53,54 and Gelfoam.55 In vivo use of these natural scaffolds has been shown to support tissue growth although development of lung tissue using these materials has not been substantial.54,55

Matrix elasticity may even aid to direct stem cell lineage specification. Native mesenchymal stem cells were shown to specify cell lineage choice based on sensitivity to matrix-tissue elasticity.56 The critical role played by matrix material in lineage selection can be seen in studies by Coraux et al.4 who demonstrated the generation of Clara cells as well as a fully differentiated airway epithelium from mES cells. The differentiation pathways that were examined included direct culture of undifferentiated mES cells on various substrates, such as collagen type I, collagen type IV, collagen type VI and gelatine in both submerged cultures and air-liquid interface. Mouse ES cells grown on collagen type I show differentiation into Clara cells as early as day 8 in culture, with or without keratinocyte growth factor and RA. Ciliated cells were obtained after air-liquid interface culture of the type I collagen-induced mESC. When other substrates were used, Clara cells were obtained after day 15 in culture.4

ECM is not static and the composition and structure of the ECM are a function of the location within tissues and the physiologic requirements of a particular tissue.43 Biophysical cues originating from ECM microstructure and mechanical properties have been shown to be major signaling sources for the growth and differentiation of cells and to regulate fundamental cell behavior.57 Mechanical stretch alone has been shown to promote alveolar type II epithelial cell differentiation.58 It has been shown that there are changes in the distribution of ECM proteins in the lung according to the associated epithelial cell population.59,60 We know that changes in ECM structure and mechanics may actually guide tissue patterning.61–64 Understanding the critical roles that ECM physical and mechanical forces play in cell differentiation and tissue development must be taken into account in future design and fabrication of biomaterials that can adequately support lung tissue engineering.

When considering the engineering of complex tissues we need to remember that one of the best sites in the body to test implantation of relatively unvascularized engineered tissues will be the lung. For in vivo implantation techniques to be successful the metabolic and therefore oxygen needs of the implant need to be met. The pleural cavity is an oxygen rich environment and even without support of blood flow in the lung direct diffusion of oxygen65 or other gases66 occurs in the tissues. Although the lung environment has the capability to support tissue survival, oxygen diffusion through natural and synthetic matrices depends on the matrix ultrastructure and varies greatly between materials.67,68 Because of this it is possible that the best matrix to support engineering of lung tissue may be decellularized lung itself. There are however some limitations that we must consider before using a decellularized or natural extracellular matrix. We must consider the effects of the decellularization process on the matrix and the mechanical integrity of the extracellular matrix since it has been shown that structurally the matrix may be weakened or degraded during the decellularization process.69 The recent clinical use and success of decellularized trachea suggests that this may be the best choice of matrix to use to engineer replacement lung tissues in the future.