Platforms to test the temporospatial capabilities of carrier systems in delivering growth factors to benefit vascular bioengineering.

In this study we produced a set of in vitro culture platforms to model vascular cell responses to growth factors and factor delivery vehicles. Two of the systems (whole vessel and whole lung vascular development) were supported by microfluidic systems facilitating media circulation and waste removal. We assessed vascular endothelial growth factor (VEGF) delivery by Pluronic F-127 hydrogel, 30 nm pore-sized microparticles (MPs), 60 nm pore-sized MP or a 50/50 mixture of 30 and 60 nm pore-sized MP. VEGF was delivered to porcine acellular lung vascular scaffolds (2.5 cm2 square pieces or whole 3D segments of acellular blood vessels) as well as whole acellular lung scaffolds. Scaffold-cell attachment was examined as was vascular tissue formation. We showed that a 50/50 mixture of 30 and 60 nm pore-sized silicon wafer MPs allowed for long-term release of VEGF within the scaffold vasculature and supported vascular endothelial tissue development during in vitro culture.

Production and utilization of acellular lung scaffolds in tissue engineering.

Pulmonary disease is a worldwide public health problem that reduces the quality of life and increases the need for hospital admissions as well as the risk for premature death for those affected. For many patients, lung transplantation is the only chance for survival. Unfortunately, there is a significant shortage of lungs for transplantation and since the lung is the most likely organ to be damaged during procurement many lungs deemed unacceptable for transplantation are simply discarded. Rather than discarding these lungs they can be used to produce three-dimensional acellular (AC) natural lung scaffolds for the generation of engineered lung tissue. AC scaffolds are lungs whose original cells have been destroyed by exposure to detergents and physical methods of removing cells and cell debris. This creates a lung scaffold from the skeleton of the lungs themselves. The scaffolds are then used to support adult, stem or progenitor cells which can be grown into functional lung tissue. Recent studies show that engineered lung tissues are capable of surviving after in vivo transplantation and support limited gas exchange. In the future engineered lung tissue has the potential to be used in clinical applications to replace lung functions lost following injury or disease. This manuscript discusses recent advances in development and use of AC scaffolds to support engineering of lung tissues.

Production and transplantation of bioengineered lung into a large-animal model.

The inability to produce perfusable microvasculature networks capable of supporting tissue survival and of withstanding physiological pressures without leakage is a fundamental problem facing the field of tissue engineering. Microvasculature is critically important for production of bioengineered lung (BEL), which requires systemic circulation to support tissue survival and coordination of circulatory and respiratory systems to ensure proper gas exchange. To advance our understanding of vascularization after bioengineered organ transplantation, we produced and transplanted BEL without creation of a pulmonary artery anastomosis in a porcine model. A single pneumonectomy, performed 1 month before BEL implantation, provided the source of autologous cells used to bioengineer the organ on an acellular lung scaffold. During 30 days of bioreactor culture, we facilitated systemic vessel development using growth factor-loaded microparticles. We evaluated recipient survival, autograft (BEL) vascular and parenchymal tissue development, graft rejection, and microbiome reestablishment in autografted animals 10 hours, 2 weeks, 1 month, and 2 months after transplant. BEL became well vascularized as early as 2 weeks after transplant, and formation of alveolar tissue was observed in all animals (n = 4). There was no indication of transplant rejection. BEL continued to develop after transplant and did not require addition of exogenous growth factors to drive cell proliferation or lung and vascular tissue development. The sterile BEL was seeded and colonized by the bacterial community of the native lung.

Giving new life to old lungs: methods to produce and assess whole human paediatric bioengineered lungs.

We report, for the first time, the development of an organ culture system and protocols to support recellularization of whole acellular (AC) human paediatric lung scaffolds. The protocol for paediatric lung recellularization was developed using human transformed or immortalized cell lines and single human AC lung scaffolds. Using these surrogate cell populations, we identified cell number requirements, cell type and order of cell installations, flow rates and bioreactor management methods necessary for bioengineering whole lungs. Following the development of appropriate cell installation protocols, paediatric AC scaffolds were recellularized using primary lung alveolar epithelial cells (AECs), vascular cells and tracheal/bronchial cells isolated from discarded human adult lungs. Bioengineered paediatric lungs were shown to contain well-developed vascular, respiratory epithelial and lung tissue, with evidence of alveolar-capillary junction formation. Types I and II AECs were found thoughout the paediatric lungs. Furthermore, surfactant protein-C and -D and collagen I were produced in the bioengineered lungs, which resulted in normal lung compliance measurements. Although this is a first step in the process of developing tissues for transplantation, this study demonstrates the feasibility of producing bioengineered lungs for clinical use. Copyright © 2016 John Wiley & Sons, Ltd.

Tissue-engineered lung: an in vivo and in vitro comparison of polyglycolic acid and pluronic F-127 hydrogel/somatic lung progenitor cell constructs to support tissue growth.

In this study, we describe the isolation and characterization of a population of adult-derived or somatic lung progenitor cells (SLPC) from adult mammalian lung tissue and the promotion of alveolar tissue growth by these cells (both in vitro and in vivo) after seeding onto synthetic polymer scaffolds. After extended in vitro culture, differentiating cells expressed Clara cell 10kDa protein, surfactant protein-C, and cytokeratin but did not form organized structures. When cells were combined with synthetic scaffolds, polyglycolic acid (PGA) or Pluronic F-127 (PF-127), and maintained in vitro or implanted in vivo, they expressed lung-specific markers for Clara cells, pneumocytes, and respiratory epithelium and organized into identifiable pulmonary structures (including those similar to alveoli and terminal bronchi), with evidence of smooth muscle development. Although PGA has been shown to be an excellent polymer for culture of specific cell types in vitro, in vivo culture in an immunocompetent host induced a foreign body response that altered the integrity of the developing lung tissue. Use of PF-127/cell constructs resulted in the development of tissue with less inflammatory reaction. These data suggest that the therapeutic use of engineered tissues requires both the use of specific cell phenotypes, as well as the careful selection of synthetic polymers, to facilitate the assembly of functional tissue.

A composite tissue-engineered trachea using sheep nasal chondrocyte and epithelial cells.

This study evaluates the feasibility of producing a composite engineered tracheal equivalent composed of cylindrical cartilaginous structures with lumens lined with nasal epithelial cells. Chondrocytes and epithelial cells isolated from sheep nasal septum were cultured in Ham's F12 media. After 2 wk, chondrocyte suspensions were seeded onto a matrix of polyglycolic acid. Cell-polymer constructs were wrapped around silicon tubes and cultured in vitro for 1 wk, followed by implanting into subcutaneous pockets on the backs of nude mice. After 6 wk, epithelial cells were suspended in a hydrogel and injected into the embedded cartilaginous cylinders following removal of the silicon tube. Implants were harvested 4 wk later and analyzed. The morphology of implants resembles that of native sheep trachea. H&E staining shows the presence of mature cartilage and formation of a pseudo-stratified columnar epithelium, with a distinct interface between tissue-engineered cartilage and epithelium. Safranin-O staining shows that tissue-engineered cartilage is organized into lobules with round, angular lacunae, each containing a single chondrocyte. Proteoglycan and hydroxyproline contents are similar to native cartilage. This study demonstrates the feasibility of recreating the cartilage and epithelial portion of the trachea using tissue harvested in a single procedure. This has the potential to facilitate an autologous repair of segmental tracheal defects.

Autologous tissue-engineered trachea with sheep nasal chondrocytes.

OBJECTIVE: This study was designed to evaluate the ability of autologous tissue-engineered trachea shaped in a helix to form the structural component of a functional tracheal replacement. METHODS: Nasal septum were harvested from six 2-month-old sheep. Chondrocytes and fibroblasts were isolated from tissue and cultured in media for 2 weeks. Both types of cells were seeded onto separate nonwoven meshes of polyglycolic acid. The chondrocyte-seeded mesh was wound around a 20-mm-diameter x 50-mm-long helical template and then covered with the fibroblast-seeded mesh. In 2 separate studies the implants were placed either in a subcutaneous pocket in the nude rat (rat tissue-engineered trachea) or in the neck of a sheep (sheep tissue-engineered trachea). Rat tissue-engineered tracheas were harvested after 8 weeks and analyzed by means of histology and biochemistry. Sheep tissue-engineered tracheas were harvested from the neck at 8 weeks and anastomosed into a 5-cm defect in the sheep trachea. RESULTS: Sheep receiving tissue-engineered trachea grafts survived for 2 to 7 days after implantation. Gross morphology and tissue morphology were similar to that of native tracheas. Hematoxylin-and-eosin staining of rat tissue-engineered tracheas and sheep tissue-engineered tracheas revealed the presence of mature cartilage surrounded by connective tissue. Safranin-O staining showed that rat tissue-engineered tracheas and sheep tissue-engineered tracheas had similar morphologies to native tracheal cartilage. Collagen, proteoglycan, and cell contents were similar to those seen in native tracheal tissue in rat tissue-engineered tracheas. Collagen and cell contents of sheep tissue-engineered tracheas were elevated compared with that of normal tracheas, whereas proteoglycan content was less than that found in normal tracheas. CONCLUSIONS: This study demonstrated the feasibility of recreating the cartilage and fibrous portion of the trachea with autologous tissue harvested from single procedure. This approach might provide a benefit to individuals needing tracheal resection.

Comparison of tracheal and nasal chondrocytes for tissue engineering of the trachea.

BACKGROUND: This study was undertaken to evaluate the feasibility of creating engineered tracheal equivalents grown in the shape of cylindrical cartilaginous structures using sheep nasal cartilage-derived chondrocytes. We also tested sheep tracheal and nasal septum for cell yield and quality of the engineered cartilage each produced. METHODS: Nasal septum and tracheal tissue were harvested from sheep. Chondrocytes from each were separately isolated from the tissues and suspended in culture media. Tracheal and nasal chondrocytes were seeded onto separate polyglycolic acid matrices. Cell-polymer constructs were cultured for 1 week and then wrapped around a 7-mm diameter x 30-mm length silicon tube and implanted subcutaneously on the back of nude mice for 8 weeks (each, n = 6). Both of the tissue-engineered tracheas (TET) were harvested and analyzed for histological, biochemical, and biomechanical properties. These values were compared with native sheep trachea. RESULTS: The morphology and histology of both tracheal-chondrocyte TET and nasal-chondrocyte TET closely resembled that of native sheep trachea. Safranin-O staining showed that tissue-engineered cartilage was organized into lobules with round, angular lacunae, each containing a single chondrocyte. Chondrocytes from the trachea or nasal septum produced tissue with similar mechanical properties and had similar glycosaminoglycan and hydroxyproline content. CONCLUSIONS: This study demonstrates that the property of TET using nasal chondrocytes is similar to that obtained using tracheal chondrocytes. This has the potential benefit of facilitating an autologous approach for repair of segmental tracheal defects using an easily obtained chondrocyte population.

Effects of the decellularization method on the local stiffness of acellular lungs.

Lung bioengineering, a novel approach to obtain organs potentially available for transplantation, is based on decellularizing donor lungs and seeding natural scaffolds with stem cells. Various physicochemical protocols have been used to decellularize lungs, and their performance has been evaluated in terms of efficient decellularization and matrix preservation. No data are available, however, on the effect of different decellularization procedures on the local stiffness of the acellular lung. This information is important since stem cells directly sense the rigidity of the local site they are engrafting to during recellularization, and it has been shown that substrate stiffness modulates cell fate into different phenotypes. The aim of this study was to assess the effects of the decellularization procedure on the inhomogeneous local stiffness of the acellular lung on five different sites: alveolar septa, alveolar junctions, pleura, and vessels' tunica intima and tunica adventitia. Local matrix stiffness was measured by computing Young's modulus with atomic force microscopy after decellularizing the lungs of 36 healthy rats (Sprague-Dawley, male, 250-300 g) with four different protocols with/without perfusion through the lung circulatory system and using two different detergents (sodium dodecyl sulfate [SDS] and 3-[(3-cholamidopropyl) dimethylammonio]-1-propanesulfonate [CHAPS]). The local stiffness of the acellular lung matrix significantly depended on the site within the matrix (p<0.001), ranging from ∼ 15 kPa at the alveolar septum to ∼ 60 kPa at the tunica intima. Acellular lung stiffness (p=0.003) depended significantly, albeit modestly, on the decellularization process. Whereas perfusion did not induce any significant differences in stiffness, the use of CHAPS resulted in a ∼ 35% reduction compared with SDS, the influence of the detergent being more important in the tunica intima. In conclusion, lung matrix stiffness is considerably inhomogeneous, and conventional decellularization procedures do not result in substantially different local stiffness in the acellular lung.

Modeling the lung: Design and development of tissue engineered macro- and micro-physiologic lung models for research use.

Respiratory tract specific cell populations, or tissue engineered in vitro grown human lung, have the potential to be used as research tools to mimic physiology, toxicology, pathology, as well as infectious diseases responses of cells or tissues. Studies related to respiratory tract pathogenesis or drug toxicity testing in the past made use of basic systems where single cell populations were exposed to test agents followed by evaluations of simple cellular responses. Although these simple single-cell-type systems provided good basic information related to cellular responses, much more can be learned from cells grown in fabricated microenvironments which mimic in vivo conditions in specialized microfabricated chambers or by human tissue engineered three-dimensional (3D) models which allow for more natural interactions between cells. Recent advances in microengineering technology, microfluidics, and tissue engineering have provided a new approach to the development of 2D and 3D cell culture models which enable production of more robust human in vitro respiratory tract models. Complex models containing multiple cell phenotypes also provide a more reasonable approximation of what occurs in vivo without the confounding elements in the dynamic in vivo environment. The goal of engineering good 3D human models is the formation of physiologically functional respiratory tissue surrogates which can be used as pathogenesis models or in the case of 2D screening systems for drug therapy evaluation as well as human toxicity testing. We hope that this manuscript will serve as a guide for development of future respiratory tract model systems as well as a review of conventional models.

Isotopic study of L-Arginine kinetics in the lung during pseudomonas sepsis in an ovine model.

UNLABELLED: The objective of the study is to investigate how L-Arginine pulmonary metabolism is altered in response Pseudomonas aeruginosa (P. aeruginosa) induced septic conditions using an ovine model. METHODS: Seven female sheep were infused with a primed-constant infusion of L-[(15)N2-guanidino, 5, 5, (2)H2] L-Arginine for 28 hs. After the initial 4 hs of the L-Arginine infusion, a continuous infusion of live Pseudomonas aeruginosa bacteria started for 24 hs. A NO synthase (NOS) inhibitor, N(G)-Methyl-L-arginine (L-NMA), infusion was added during the last 4 hs of the bacterial infusion. Blood samples were taken at specific time points for isotopic enrichment during control, septic and NOS blocking phases of the study. RESULTS: We observed that the level of total delivery of L-Arginine to the lungs was significantly decreased in septic phase after 24 hours of pseudomonas infusion. In contrast, the fractional uptake and metabolism of L-Arginine by the lungs was doubled during septic phase relative to the control phase (MARG-basal = 100% vs. MARG-septic = 220 ± 56%, P < 0.05). NO production in the lungs was also significantly increased. Infusion of L-NMA markedly blunted this elevated NO production and attenuated the total arginine metabolized in the septic lungs (MARG-septic = 220 ± 56% vs. MARG-NO blocking = -25 ± 20%; P < 0.05). We demonstrated sepsis induced by P. aeruginosa infusion caused an increase in the fractional uptake and metabolic rate of arginine in the lungs. Furthermore, our data suggests that arginine was mainly consumed via arginine - NO pathway, which might be responsible for this enhanced arginine metabolic activity in the septic lungs.

Novel in vitro respiratory models to study lung development, physiology, pathology and toxicology.

Detailed studies of lung pathology in patients during the course of development of acute lung injury or respiratory distress are limited, and in the past information related to lung-specific responses has been derived from the study of lungs from patients who died at autopsy or from animal models. Development of good in vitro human tissue models would help to bridge the gap in our current knowledge of lung responses and provide a better understanding of lung development, physiology and pathology. In vitro models of simple one-cell or two-cell culture systems as well as complex multicellular lung analogs that reproduce defined components of specific human lung responses have already been realized. A benefit of current in vitro lung models is that hypotheses generated from review of data from human or animal disease studies can be tested directly in engineered human tissue models. Results of studies done using simple in vitro lung systems or more complex three-dimensional models have already been used to examine cell-based responses, physiologic functions, pathologic changes and even drug toxicity or drug responses. In the future we will create models with specific genetic profiles to test the importance of single gene products or pathways of significance. Recent development of microfluidics-based models that support high-throughput screening will allow early-stage toxicity testing in human systems and faster development of new and innovative medical products. Model design in the future will also allow for evaluation of multiple organ systems at once, providing a more holistic or whole-body approach to understanding human physiology and responses.

Neurogenic and neuro-protective potential of a novel subpopulation of peripheral blood-derived CD133+ ABCG2+CXCR4+ mesenchymal stem cells: development of autologous cell-based therapeutics for traumatic brain injury.

INTRODUCTION: Nervous system injuries comprise a diverse group of disorders that include traumatic brain injury (TBI). The potential of mesenchymal stem cells (MSCs) to differentiate into neural cell types has aroused hope for the possible development of autologous therapies for central nervous system injury. METHODS: In this study we isolated and characterized a human peripheral blood derived (HPBD) MSC population which we examined for neural lineage potential and ability to migrate in vitro and in vivo. HPBD CD133+, ATP-binding cassette sub-family G member 2 (ABCG2)+, C-X-C chemokine receptor type 4 (CXCR4)+ MSCs were differentiated after priming with ß-mercaptoethanol (ß-ME) combined with trans-retinoic acid (RA) and culture in neural basal media containing basic fibroblast growth factor (FGF2) and epidermal growth factor (EGF) or co-culture with neuronal cell lines. Differentiation efficiencies in vitro were determined using flow cytometry or fluorescent microscopy of cytospins made of FACS sorted positive cells after staining for markers of immature or mature neuronal lineages. RA-primed CD133+ABCG2+CXCR4+ human MSCs were transplanted into the lateral ventricle of male Sprague-Dawley rats, 24 hours after sham or traumatic brain injury (TBI). All animals were evaluated for spatial memory performance using the Morris Water Maze (MWM) Test. Histological examination of sham or TBI brains was done to evaluate MSC survival, migration and differentiation into neural lineages. We also examined induction of apoptosis at the injury site and production of MSC neuroprotective factors. RESULTS: CD133+ABCG2+CXCR4+ MSCs consistently expressed markers of neural lineage induction and were positive for nestin, microtubule associated protein-1ß (MAP-1ß), tyrosine hydroxylase (TH), neuron specific nuclear protein (NEUN) or type III beta-tubulin (Tuj1). Animals in the primed MSC treatment group exhibited MWM latency results similar to the uninjured (sham) group with both groups showing improvements in latency. Histological examination of brains of these animals showed that in uninjured animals the majority of MSCs were found in the lateral ventricle, the site of transplantation, while in TBI rats MSCs were consistently found in locations near the injury site. We found that levels of apoptosis were less in MSC treated rats and that MSCs could be shown to produce neurotropic factors as early as 2 days following transplantation of cells. In TBI rats, at 1 and 3 months post transplantation cells were generated which expressed markers of neural lineages including immature as well as mature neurons. CONCLUSIONS: These results suggest that PBD CD133+ABCG2+CXCR4+ MSCs have the potential for development as an autologous treatment for TBI and neurodegenerative disorders and that MSC derived cell products produced immediately after transplantation may aid in reducing the immediate cognitive defects of TBI.

Isotopic study of L-Arginine kinetics in the lung during pseudomonas sepsis in an ovine model.

UNLABELLED: The objective of the study is to investigate how L-Arginine pulmonary metabolism is altered in response Pseudomonas aeruginosa (P. aeruginosa) induced septic conditions using an ovine model. METHODS: Seven female sheep were infused with a primed-constant infusion of L-[(15)N2-guanidino, 5, 5, (2)H2] L-Arginine for 28 hs. After the initial 4 hs of the L-Arginine infusion, a continuous infusion of live Pseudomonas aeruginosa bacteria started for 24 hs. A NO synthase (NOS) inhibitor, N(G)-Methyl-L-arginine (L-NMA), infusion was added during the last 4 hs of the bacterial infusion. Blood samples were taken at specific time points for isotopic enrichment during control, septic and NOS blocking phases of the study. RESULTS: We observed that the level of total delivery of L-Arginine to the lungs was significantly decreased in septic phase after 24 hours of pseudomonas infusion. In contrast, the fractional uptake and metabolism of L-Arginine by the lungs was doubled during septic phase relative to the control phase (MARG-basal = 100% vs. MARG-septic = 220 ± 56%, P < 0.05). NO production in the lungs was also significantly increased. Infusion of L-NMA markedly blunted this elevated NO production and attenuated the total arginine metabolized in the septic lungs (Mnitrate-septic = 43.6 ± 5.7 vs. Mnitrate-septic + L-NMA = 13.4 ± 5.1 umol/kg/min; p < 0.05). We demonstrated sepsis induced by P. aeruginosa infusion caused an increase in the fractional uptake and metabolic rate of arginine in the lungs. Furthermore, our data suggests that arginine was mainly consumed via arginine - NO pathway, which might be responsible for this enhanced arginine metabolic activity in the septic lungs.

Production and assessment of decellularized pig and human lung scaffolds.

The authors have previously shown that acellular (AC) trachea-lung scaffolds can (1) be produced from natural rat lungs, (2) retain critical components of the extracellular matrix (ECM) such as collagen-1 and elastin, and (3) be used to produce lung tissue after recellularization with murine embryonic stem cells. The aim of this study was to produce large (porcine or human) AC lung scaffolds to determine the feasibility of producing scaffolds with potential clinical applicability. We report here the first attempt to produce AC pig or human trachea-lung scaffold. Using a combination of freezing and sodium dodecyl sulfate washes, pig trachea-lungs and human trachea-lungs were decellularized. Once decellularization was complete we evaluated the structural integrity of the AC lung scaffolds using bronchoscopy, multiphoton microscopy (MPM), assessment of the ECM utilizing immunocytochemistry and evaluation of mechanics through the use of pulmonary function tests (PFTs). Immunocytochemistry indicated that there was loss of collagen type IV and laminin in the AC lung scaffold, but retention of collagen-1, elastin, and fibronectin in some regions. MPM scoring was also used to examine the AC lung scaffold ECM structure and to evaluate the amount of collagen I in normal and AC lung. MPM was used to examine the physical arrangement of collagen-1 and elastin in the pleura, distal lung, lung borders, and trachea or bronchi. MPM and bronchoscopy of trachea and lung tissues showed that no cells or cell debris remained in the AC scaffolds. PFT measurements of the trachea-lungs showed no relevant differences in peak pressure, dynamic or static compliance, and a nonrestricted flow pattern in AC compared to normal lungs. Although there were changes in content of collagen I and elastin this did not affect the mechanics of lung function as evidenced by normal PFT values. When repopulated with a variety of stem or adult cells including human adult primary alveolar epithelial type II cells both pig and human AC scaffolds supported cell attachment and cell viability. Examination of scaffolds produced using a variety of detergents indicated that detergent choice influenced human immune response in terms of T cell activation and chemokine production.

In vitro human bone marrow analog: clinical potential.

Bone marrow is the primary site of hematopoiesis in adult humans. Bone marrow can be cultured in vitro but few simple culture systems fully support hematopoiesis beyond a few months. Human bone marrow analogs are long-term in vitro cultures of marrow stromal and hematopoietic stem cells that can be used to produce cells and products normally harvested from human donors. Bone marrow analog systems should exhibit confluence of the stromal cell populations, persistence of hematopoietic progenitor cells, presence of active regions of hematopoiesis and capacity to produce mature cell types for extended periods of time. Although we are still years away from realizing clinical application of products formed by artificial bone marrow analogs, the process of transitioning this research tool from bench to bedside should be fairly straightforward. The most obvious application of artificial marrow would be for production of autologous hematopoietic CD34(+) stem cells as a stem cell therapy for individuals experiencing bone marrow failure due to disease or injury. Another logical application is for 'blood farming', a process for large-scale in vitro production of red blood cells, white blood cells or platelets, for transfusion or treatment. Other possibilities include production of nonhematopoietic stem cells such as osteogenic stromal cells, osteoblasts and rare pluripotent stem cells. Bone marrow analogs also have great potential as ex vivo human test systems and could play a critical role in drug discovery, drug development and toxicity testing in the future.

Influence of acellular natural lung matrix on murine embryonic stem cell differentiation and tissue formation.

We report here the first attempt to produce and use whole acellular (AC) lung as a matrix to support development of engineered lung tissue from murine embryonic stem cells (mESCs). We compared the influence of AC lung, Gelfoam, Matrigel, and a collagen I hydrogel matrix on the mESC attachment, differentiation, and subsequent formation of complex tissue. We found that AC lung allowed for better retention of cells with more differentiation of mESCs into epithelial and endothelial lineages. In constructs produced on whole AC lung, we saw indications of organization of differentiating ESC into three-dimensional structures reminiscent of complex tissues. We also saw expression of thyroid transcription factor-1, an immature lung epithelial cell marker; pro-surfactant protein C, a type II pneumocyte marker; PECAM-1/CD31, an endothelial cell marker; cytokeratin 18; alpha-actin, a smooth muscle marker; CD140a or platelet-derived growth factor receptor-alpha; and Clara cell protein 10. There was also evidence of site-specific differentiation in the trachea with the formation of sheets of cytokeratin-positive cells and Clara cell protein 10-expressing Clara cells. Our findings support the utility of AC lung as a matrix for engineering lung tissue and highlight the critical role played by matrix or scaffold-associated cues in guiding ESC differentiation toward lung-specific lineages.

Design and development of tissue engineered lung: Progress and challenges.

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.

In vitro analog of human bone marrow from 3D scaffolds with biomimetic inverted colloidal crystal geometry.

In vitro replicas of bone marrow can potentially provide a continuous source of blood cells for transplantation and serve as a laboratory model to examine human immune system dysfunctions and drug toxicology. Here we report the development of an in vitro artificial bone marrow based on a 3D scaffold with inverted colloidal crystal (ICC) geometry mimicking the structural topology of actual bone marrow matrix. To facilitate adhesion of cells, scaffolds were coated with a layer of transparent nanocomposite. After seeding with hematopoietic stem cells (HSCs), ICC scaffolds were capable of supporting expansion of CD34+ HSCs with B-lymphocyte differentiation. Three-dimensional organization was shown to be critical for production of B cells and antigen-specific antibodies. Functionality of bone marrow constructs was confirmed by implantation of matrices containing human CD34+ cells onto the backs of severe combined immunodeficiency (SCID) mice with subsequent generation of human immune cells.

Engineering of a complex organ: progress toward development of a tissue-engineered lung.

Although there has been slow progress in the engineering of the lung, recent advances in the use of stem or progenitor cells leading to the reliable production of component parts of the lung show promise for the future development of engineered lung tissue. Progress toward the goal of developing an engineered lung will only be accomplished through the parallel development of effective and functional tissue-engineered components that include both upper and lower respiratory tract as well as scaffold material suitable for use in the lung. The knowledge acquired from developing each individual component of lung will, over time, be integrated to allow for the development of larger complex organ structures. To accomplish the goal of developing engineered lung for regenerative medicine, many advances will be required in scaffold design and production, including improved biocompatibility, improved elasticity, and better control of scaffold ultrastructure and porosity. Development of new materials designed to meet the anatomic and physiologic needs of the lung must occur before we can begin to realize the goal of engineering functional lung tissue. Better understanding of factors promoting cell adhesion, migration, differentiation, and vascularization of grafts and lung regeneration as a whole is also needed. Advances in the development of mathematical models to examine the conditions that promote lung morphogenesis and tissue growth for computational investigations of tissue development will also be necessary if we are to realistically evaluate the production of lung tissue strictly from the engineering perspective. It is obvious that engineering of lung tissue will require a multidisciplinary approach if we are to eventually succeed in our attempts to produce tissues worthy of clinical application in the future.