Application of stem cells for cell therapy of respiratory diseases is a developing field. We have previously established several protocols for the differentiation of embryonic stem cells (ESC) into alveolar epithelial cells, which require a high degree of operator interference and result in a low yield of target cells. Herein, we have shown that, by provision of a medium conditioned using A549 cells and by integration of classic steps of ESC differentiation into a single step through encapsulation in hydrogels (three-dimensional) and culture in a rotary bioreactor, murine ESC (mESC) could be directed to differentiate into distal respiratory epithelial cells. Type I and II pneumocytes (with a yield of 50% for type II) and Clara cells were demonstrated by the expression of aquaporin 5, surfactant protein C, and Clara cell secretory protein, respectively. We identified target cells as early as day 5 of culture and stably maintained our differentiated cells in vitro for 100 days. Electron microscopy demonstrated microvilli and intracellular lamellar bodies (LB), and fluorescent staining confirmed the active process of exocytosis of these LB in differentiated type II cells. When these cells were decapsulated and cultured in static conditions in flask cultures (two-dimensional), they retained their characteristic type II phenotype and morphology. In conclusion, our protocol offers integrated bioprocessing, shorter time of differentiation, lower cost, no use of growth factors, high reproducibility, and high phenotypic and functional stability, as well as being amenable to automation and being scalable, which would move this field closer to future clinical applications.

Functional groups on a material surface affect the response of many cell types. As part of our strategy aimed at engineering lung tissue, we introduced functional groups into the surface of Poly(D,L-lactide) (PDLLA) films to improve its suitability for the culture of mature pulmonary epithelial cells (A549 line) using two different methods. The first method, aminolysis, can introduce primary amines into PDLLA films by transesterification using 1-15% of ethylenediamine in isopropanol. The second method, a branching modification, can generate amine-terminated or carboxylic acid-terminated tree-like branched architectures. All modified PDLLA surfaces exhibited lower water contact angles, i.e. are more hydrophilic than unmodified PDLLA. PDLLA treated with 15% ethylenediamine exhibited a rougher surface than the control, and PDLLA with branching modification had a droplet-like surface topography as visualized by atomic force microscopy (AFM). PDLLA treated with 15% ethylenediamine and branching modification with two and three generations enhanced the attachment of pulmonary epithelial cells measured using Hoechst dye. Immunostaining demonsatrated that amine-terminated branched architectures allowed for better focal adhesion point formation than the control 24 h after cell seeding. Furthermore, they also induced higher A549 cell populations and levels of activity after 4 days in culture measured using Hoechst dye and WST1 cell proliferation reagents, respectively. In contrast, carboxylic acid-terminated branching architectures were found to reduce the cell population size after 4 days. It was concluded that the concentration, type and distribution of surface functional groups can affect significantly the behavior of pulmonary epithelial cells growing on a PDLLA surface, and PDLLA film modified with. two or three generations of amine-terminated branched architectures is a suitable 2D scaffold for the culture of of pulmonary epithelial cells.

The fibroblast growth factors (FGFs) play an important role in the development of embryonic lung. In this study, we investigated the effects of mainly FGF 1, 2, and 10 at concentrations selected on the basis of data obtained from previous in vitro culture on the derivation of the pulmonary progenitors from murine embryonic stem cells cultured on gelatin or Matrigel-coated plates. For cells cultured on a gelatin-coated plate, high concentrations of FGF1 were found to enhance the expression of mRNAs for SPC and CC10, markers of distal airway epithelium, while high levels of FGF2 decreased the expression of RNAs for not only SPC, CC10 but also for the additional markers SPD and aquaporin 5. FGF10 at all tested concentrations was found to have no effect on the differentiation of pneumocytes when ESCs were grown on gelatin-coated plates. However, when differentiation was performed on Matrigel-coated plates, the addition of 60 ng/ml FGF10 enhanced the expression of pneumocyte markers, suggesting a synergic effect of FGF10 and extracellular matrix. In conclusion, growth factors were proven to be effective in the differentiation of pulmonary progenitors from mESCs. The need of signals from extracellular matrix proteins depends on the growth factors supplemented.

We have previously differentiated lung epithelium from human and murine embryonic stem cells (mESCs) and are now exploring the potential applications of these cells, including in the engineering of lung tissue constructs. In this study, we hypothesized that the differentiation and maintenance of lung epithelium derived from mESCs can be enhanced by extracellular matrix (ECM) proteins. Our established differentiation protocol was applied to mESCs grown on a range of ECMs: collagen I, laminin 332, fibronectin, Matrigel, and, as an experimental control, gelatin. The ECMs were coated onto tissue culture plastic (TCP) and poly-DL-lactic acid (PDLLA), a biodegradable polymer we have previously shown to support the growth of mature pneumocytes. Matrigel or Laminin-332 coating of either TCP or PDLLA film resulted in enhanced surfactant protein C gene expression in differentiating mESCs, a direct indication of the upregulation of lung epithelial differentiation. For each combination, changes in the contact angle and zeta potential of protein-coated TCP and PDLLA film confirmed protein adsorption. We conclude that the choice of the coating protein can greatly affect the differentiation of ESCs, and laminin-332-coated PDLLA provided an ECM-degradable scaffold combination that is suitable for engineering of lung tissue constructs.

The majority of methodologies for maintaining human embryonic stem cell (hESC) pluripotency require the use of human or animal feeder cell layers, the most common being murine embryonic fibroblasts. In this study, we applied a protocol aimed at maintaining hESCs in culture without exposure to animal cells or proteins. hESCs were encapsulated in 1.1% (w/v) calcium alginate hydrogels and grown in basic maintenance medium for a period of up to 260 days. Investigation of the cell aggregates formed within the hydrogels yielded no evidence of the formation of any of the three germ layers, although the hESCs retained their pluripotency and could differentiate when they were subsequently cultured in a conditioned environment. Immunohistochemistry and RT-PCR showed that the hESC aggregates expressed protein and gene markers characteristic of pluripotency including Oct-4, Nanog, SSEA-4, TRA-1-60 and TRA-1-81. At the ultrastructural level, the cells were arranged in closely packed clusters and showed no cytoplasmic organelles, suggesting an undifferentiated state. These data show that it is possible to maintain hESCs in an undifferentiated state, without passaging or embryoid body formation, and without animal contamination.

From time to time, an area of research erupts into the public arena and is promulgated as the new great white hope of medicine. The field of stem cells is one of the latest examples, with the media speculation on its potential applications being fueled and politicized by associated ethical issues concerning the use of embryonic stem cells and the occasional appearance of that ominous word cloning. For the basic scientist, stem cells offer new tools, for example in the investigation of developmental and pathogenetic pathways. For the clinician, the capacity of stem cells both to proliferate and to form different types of cells makes them, in theory at least, ideal for replacing or repairing damaged or diseased tissue. Sometimes the promise of medical research fulfils expectations, but too often it falls short of the hype. Stem/progenitor cell therapies are emerging in various medical disciplines, but their predicted status as a panacea has not yet been attained; the jury is still out. For the respiratory system, stem cell research has moved more slowly than that of many other organs, mainly because of the lung’s structural complexity, cellular heterogeneity, and the low turnover rate of its epithelia. However, major advances have been and continue to be made, including clarification of the events underlying embryonic lung development and the discovery of previously unknown regenerative pathways in the adult lung. Targeted activation of endogenous stem cell pools would exploit existing repair mechanisms and augment the body’s innate regenerative capability. Stem cells could form the basis of cell therapies and also offer the potential to create pulmonary tissue constructs ex vivo for implantation into the lung to repair more extensive damage. In addition, stem cells can be used to create in vitro models of lung development and disease for further investigation and manipulation. The 23rd Transatlantic Airway Conference was aimed at evaluating the progress made in our understanding of the stem cell biology of the lung and how far we may be from establishing effective therapies based on that understanding. The 2008 Conference took advantage of the unique nature of this series of meetings in bringing together not only physicians and research scientists at the center of the respiratory stem cell field but also a broad range of experts from related disciplines, including tissue engineering, bioengineering, and biophysics, to discuss stem and progenitor cell biology in relation to lung repair.

Embryonic stem cells (ESCs) derived from the preimplantation blastocyst are pluripotent and capable of indefinite expansion in vitro. As such, they present a cell source to derive a potentially inexhaustible supply of pulmonary cells and tissue. ESC-derived pulmonary epithelium could be used for in vitro cell or tissue models or, in the future, implanted into the damaged or diseased lung to effect repair. Efforts to date have largely focused on obtaining distal lung epithelial phenotypes from ESCs, notably alveolar epithelium. Several disparate methods have been developed to enhance differentiation of ESCs into pulmonary epithelial lineages; these are broadly based on recapitulating developmental signaling events, mimicking the physical environment, or forcibly reprogramming the ESC nucleus. Early findings of our preclinical experiments implanting differentiated ESCs into the injured lung are also described here. Future efforts will focus on maximizing ESC differentiation efficiency and yield of the target phenotype, as well as characterizing the function of derived cells in vivo and in vitro.

In this study we investigate the ability of Salmonella enterica serovar Typhi (S. Typhi) surface structures to influence invasion and adhesion in epithelial cell assay systems. In general, S. Typhi was found to be less adherent, invasive and cytotoxic than S. enterica serovar Typhimurium (S. Typhimurium). Culture conditions had little effect on adhesion of S. Typhi to cultured cells but had a marked influence on invasion. In contrast, bacterial growth conditions did not influence S. Typhi apical invasion of polarized cells. The levels of S. Typhi, but not S. Typhimurium, invasion were increased by application of bacteria to the basolateral surface of polarized cells. Expression of virulence (Vi) capsule by S. Typhi resulted in a modest reduction in adhesion, but profoundly reduced levels of invasion of non-polarized cells. However, Vi capsule expression had no affect on invasion of the apical or basolateral surfaces of polarized cells. Mutation of the staA, tcfA or pilS genes did not affect invasion or adhesion in either the presence or the absence of Vi capsule.

Embryonic stem (ES) cells have the potential to be a valuable cell source for use in regenerative medicine. So far, most efforts have been concentrated on the quality of ES cells and relatively little attention has been paid to the mechanisms that affect the quantity of ES cells in culture conditions, an aspect that gains importance when considering ES cells as a possible commercial commodity.

We have previously shown that embryonic stem cells (ESC) can be directed to differentiate into alveolar type II cells by provision of a serum-free medium designed for in vitro maintenance of mature alveolar epithelial cells (small airway growth medium: SAGM), although the target cell yield was low. SAGM comprises a basal serum-free medium (SABM) plus a series of defined supplements. In order to try increase the proportion of pneumocytes in differentiated cultures, we aimed in this study to determine the effects on murine ESC of each of the individual growth factors in SAGM. In accordance with our previous reports, expression of surfactant protein C (SPC) and its mRNA was used to monitor differentiation of type II pneumocytes. Surprisingly, we found that addition of each factor separately to SABM decreased the expression of SPC mRNA when compared with the effect of SABM alone. Thus, it seems that the observed enhancement by SAGM of pneumocyte differentiation from murine ESC can, in fact, be attributed to the provision of a serum-free environment.

The lungs are essential organs for life and, not surprisingly, respiratory diseases are a leading cause of death worldwide. The complex structure of the lung makes it a particularly difficult target for any tissue engineering strategy and the construction of lung tissue for human implantation remains a remote target. However, some progress is being made toward achieving targeted lung repair and the creation of a biohybrid device that can augment the function of damaged lung. Some of the most pivotal advances have been made in the area of stem cell biology, which has moved to the forefront of medical research in the past few years. Being the basis of natural pathways for tissue maintenance and repair, stem cells represent a key target for mediating repair in vivo. Targeted activation of endogenous stem cell pools can augment the body's innate regenerative capability. In cases where this approach is not sufficient, stem cells can provide an abundant, renewable source for the generation of pulmonary cells in vitro that can then be delivered to the lung, alone or on scaffolds, for the repair of even more extensive tissue damage. A further application of pulmonary cells grown in vitro from stem cells is the construction of in vitro screening systems for pharmaceutical or toxicological applications. This chapter describes the progress being made toward producing lung constructs.

Repair or regeneration of defective lung tissue would be of great clinical use. Potential cellular sources for the regeneration of lung tissue in vivo or lung tissue engineering in vitro include endogenous pulmonary stem cells, extrapulmonary circulating stem cells and embryonic stem cells. This review summarizes the recent research on each of these stem cell types and their potential for use in the treatment of lung injury and disease.

The capacity of embryonic stem cells for virtually unlimited self-renewal and differentiation capacity has opened up the prospect of widespread applications in biomedical research and regenerative medicine. For the latter, the cells provide hope that it will be possible to overcome the problems of donor tissue shortage and also, by making the cells immunocompatible with the recipient, implant rejection. Four years after the first derivation of human pluripotent cell lines from pre-implantation embryos, a great deal has been learnt about their biology and how differentiation can be encouraged towards particular cell lineages. However, considerable research is needed, not least into means to enrich and purify derivative cell lineages, before clinical trials can be considered. Copyright (C) 2002 John Wiley Sons, Ltd.

Current approaches have focused on deriving ESCs differentiation into chondrocytes from a cell source of spontaneously formed intact mesoderm in EB formation, resulting in limited yield. Our study aimed at upregulating chondrogenic differentiation of murine ESCs by enhancing mesoderm formation. Specifically, culture of mESCs with conditioned medium from a human hepatocarcinoma cell line resulted in a cell population with a gene expression pattern similar to that of primitive streak/nascent mesoderm, including up-regulation of brachyury, goosecoid, nodal, and cripto. From this cell population, reducing the embryoid body formation time resulted in enhancement of chondrogenic differentiation as evidenced by larger Alcian blue-stained cartilage nodules, higher production of sulfated glycosaminoglycan matrix, the presence of well-organised type II collagen and type II collagen, aggrecan and sox-9 gene expression. In conclusion, we present here a new approach to the generation of chondrocytes from mESCs that enhances yields and, thus, could have widespread applications in cartilage tissue engineering.

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. Three separate protocols are described here that utilize both murine and human ES cells. The distal lung epithelial phenotype is 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. Phenotypic analysis is demonstrated using immunocytochemistry and SP-C promoter-eGFP reporter gene expression in transgenic ES cells. These methods provide an increased efficiency of distal lung epithelial derivation from ES cells and, therefore, they 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.

Embryonic stem cells (ESCs) are being investigated increasingly for their potential as a cell source for tissue engineering. Antibiotics are regularly used in ESC culture media to control contamination, although they can be cytotoxic and interfere with protein synthesis. Our aim was to examine the effects of the frequently used antibiotics gentamicin and combined penicillin and streptomycin on ESC culture using differentiation of murine ESC into type II pneumocytes as a model. Antibiotics reduced the expression of the specific marker for type II pneumocytes, SPC mRNA, by up to 60%. We also identified an adverse effect on the growth rate of differentiating embryoid bodies, causing a significant ( p < 0.05) reduction of up to 40%, and an increase in population doubling time of up to 48%. No contamination was seen in any of the cultures. Our findings suggest that the routine use of antibiotics in ESC culture should be avoided as it may reduce the efficiency of the culture system.