Roles of Mesenchymal Cells in the Lung: From Lung Development to Chronic Obstructive Pulmonary Disease.

Mesenchymal cells are an essential cell type because of their role in tissue support, their multilineage differentiation capacities and their potential clinical applications. They play a crucial role during lung development by interacting with airway epithelium, and also during lung regeneration and remodeling after injury. However, much less is known about their function in lung disease. In this review, we discuss the origins of mesenchymal cells during lung development, their crosstalk with the epithelium, and their role in lung diseases, particularly in chronic obstructive pulmonary disease.

A model of the aged lung epithelium in idiopathic pulmonary fibrosis.

Idiopathic pulmonary fibrosis (IPF) is an age-related disorder that carries a universally poor prognosis and is thought to arise from repetitive micro injuries to the alveolar epithelium. To date, a major factor limiting our understanding of IPF is a deficiency of disease models, particularly in vitro models that can recapitulate the full complement of molecular attributes in the human condition. In this study, we aimed to develop a model that more closely resembles the aberrant IPF lung epithelium. By exposing mouse alveolar epithelial cells to repeated, low doses of bleomycin, instead of usual one-time exposures, we uncovered changes strikingly similar to those in the IPF lung epithelium. This included the acquisition of multiple phenotypic and functional characteristics of senescent cells and the adoption of previously described changes in mitochondrial homeostasis, including alterations in redox balance, energy production and activity of the mitochondrial unfolded protein response. We also uncovered dramatic changes in cellular metabolism and detected a profound loss of proteostasis, as characterized by the accumulation of cytoplasmic protein aggregates, dysregulated expression of chaperone proteins and decreased activity of the ubiquitin proteasome system. In summary, we describe an in vitro model that closely resembles the aberrant lung epithelium in IPF. We propose that this simple yet powerful tool could help uncover new biological mechanisms and assist in developing new pharmacological tools to treat the disease.

A Synthesis Concerning Conservation and Divergence of Cell Types across Epithelia.

Advances in single-cell RNA-seq (scRNA-seq) and computational analysis have enabled the systematic interrogation of the cellular composition of tissues. Combined with tools from developmental biology, cell biology, and genetics, these approaches are revealing fundamental aspects of tissue geometry and physiology, including the distribution, origins, and inferred functions of specialized cell types, and the dynamics of cellular turnover and differentiation. By comparing different tissues, such studies can delineate shared and specialized features of cell types and their lineage. Here, we compare two developmentally related murine epithelia, the airway and the small intestinal epithelia, which are both derived from the embryonic endodermal gut tube. We examine how airway and intestine generate and functionalize common archetypal cell types to fulfill similar shared physiologic functionalities. We point to cases in which similar cell types are repurposed to accommodate each tissue's unique physiologic role, and highlight tissue-specific cells whose specializations contribute to the distinct functional roles of each organ. We discuss how archetypal and unique cell types are incorporated within a cellular lineage, and how the regulation of the proportions of these cell types enables tissue-level organization to meet functional demands and maintain homeostasis.

Alveolus-like organoid from isolated tip epithelium of embryonic mouse lung.

Embryonic lungs were obtained from embryonic day 13.5 ICR mice. The lung-tip epithelium isolated using dispase treatment was embedded in low-growth factor Matrigel, cultured in DMEM/F12 medium containing 0.1% bovine serum albumin, supplemented with insulin, transferrin, and selenium (ITS), with or without fibroblast growth factor 7 (FGF7), and were observed for 14 days. With the addition of FGF7, the tip epithelium grew to form a cyst by culture day 7. Then, tubular tufts-like alveolus appeared around the cyst surface. Reverse transcription-polymerase chain reaction revealed that, with the addition of FGF7, the cultured lung explants expressed alveolar-type 1 cell markers, such as HopX and Aquaporin5, and type 2 cell markers, such as Lamp3 and Surfactant apoproteins (Sftp) C and D. Paraffin-embedded sections were stained with hematoxylin and eosin, and alveolar structures at culture day 14 were composed of squamous and cuboidal epithelial cells. Immunohistochemical studies showed that the squamous epithelial cells were positive for HopX, and the cuboidal epithelial cells were positive for pro-SftpC. Furthermore, transmission electron microscopic observation confirmed that the squamous epithelial cells were alveolar-type 1 cells and the cuboidal cells were type 2 cells, because they had many lamellar inclusion bodies. Embryonic lung-tip epithelium forms an alveolus-like organoid through the self organization with the aid of Matrigel, ITS, and FGF7. This method to make alveolus-like organoid in vitro is easy, reproducible, and economical. This method could have potential to solve many issues in alveolar epithelial cells in normal and pathological conditions.

Temporal differentiation of bovine airway epithelial cells grown at an air-liquid interface.

There is an urgent need to develop improved, physiologically-relevant in vitro models of airway epithelia with which to better understand the pathological processes associated with infection, allergies and toxicological insults of the respiratory tract of both humans and domesticated animals. In the present study, we have characterised the proliferation and differentiation of primary bovine bronchial epithelial cells (BBECs) grown at an air-liquid interface (ALI) at three-day intervals over a period of 42 days from the introduction of the ALI. The differentiated BBEC model was highly representative of the ex vivo epithelium from which the epithelial cells were derived; a columnar, pseudostratified epithelium that was highly reflective of native airway epithelium was formed which comprised ciliated, goblet and basal cells. The hallmark defences of the respiratory tract, namely barrier function and mucociliary clearance, were present, thus demonstrating that the model is an excellent mimic of bovine respiratory epithelium. The epithelium was fully differentiated by day 21 post-ALI and, crucially, remained healthy and stable for a further 21 days. Thus, the differentiated BBEC model has a three-week window which will allow wide-ranging and long-term experiments to be performed in the fields of infection, toxicology or general airway physiology.

Brushed nasal epithelial cells are a surrogate for bronchial epithelial CFTR studies.

Recent advances in the management of cystic fibrosis (CF) target underlying defects in the CF transmembrane conductance regulator (CFTR) protein, but efficacy analyses remain limited to specific genotype-based subgroups. Patient-derived model systems may therefore aid in expanding access to these drugs. Brushed human nasal epithelial cells (HNEs) are an attractive tissue source, but it remains unclear how faithfully they recapitulate human bronchial epithelial cell (HBE) CFTR activity. We examined this gap using paired, brushed HNE/HBE samples from pediatric CF subjects with a wide variety of CFTR mutations cultured at the air-liquid interface. Growth and structural characteristics for the two cell types were similar, including differentiation into mature respiratory epithelia. In electrophysiologic analysis, no correlation was identified between nasal and bronchial cultures in baseline resistance or epithelial sodium channel (ENaC) activity. Conversely, robust correlation was demonstrated between nasal and bronchial cultures in both stimulated and inhibited CFTR activity. There was close correlation in modulator-induced change in CFTR activity, and CFTR activity in both cell types correlated with in vivo sweat chloride measurements. These data confirm that brushed HNE cell cultures recapitulate the functional CFTR characteristics of HBEs with fidelity and are therefore an appropriate noninvasive HBE surrogate for individualized CFTR analysis.

Pulmonary IL-1ß expression in early life causes permanent changes in lung structure and function in adulthood.

Chorioamnionitis, mechanical ventilation, oxygen therapy, and postnatal infection promote inflammation in the newborn lung. The long-term consequences of pulmonary inflammation during infancy have not been well characterized. The aim of this study was to examine the impact of inflammation during the late saccular to alveolar stages of lung development on lung structure and function in adulthood. To induce IL-1ß expression in the pulmonary epithelium of mice with a tetracycline-inducible human IL-1ß transgene, doxycycline was administered via intraperitoneal injections to bitransgenic pups and their littermate controls on postnatal days (PN) 0, 0.5, and 1. Lung structure, inflammation, and airway reactivity were studied in adulthood. IL-1ß production in early life resulted in increased numbers of macrophages and neutrophils on PN21, but inflammation subsided by PN42. Permanent changes in alveolar structure, i.e., larger alveoli and thicker alveolar walls, were present from PN21 to PN84. Lack of alveolar septation thus persisted after IL-1ß production and inflammation had ceased. Early IL-1ß production caused goblet cell hyperplasia, enhanced calcium-activated chloride channel 3 (CLCA3) protein expression, and increased airway reactivity in response to methacholine on PN42. Lymphoid follicles were present adjacent to small airways in the lungs of adult bitransgenic mice, and levels of the B cell chemoattractant CXC-motif ligand (CXCL) 13 were elevated in the lungs of bitransgenic mice compared with controls. In conclusion, IL-1ß-induced pulmonary inflammation in early life causes a chronic lung disease in adulthood.

Functional characterization of a novel 3D model of the epithelial-mesenchymal trophic unit.

BACKGROUND/AIM: Epithelial-mesenchymal communication plays a key role in tissue homeostasis and abnormal signaling contributes to chronic airways disease such as COPD. Most in vitro models are limited in complexity and poorly represent this epithelial-mesenchymal trophic unit. We postulated that cellular outgrowth from bronchial tissue would enable development of a mucosal structure that recapitulates better in vivo tissue architecture. MATERIALS AND METHODS: Bronchial tissue was embedded in Matrigel and outgrowth cultures monitored using time-lapse microscopy, electrical resistance, light and electron microscopy. Cultures were challenged repetitively with cigarette smoke extract (CSE). RESULTS: The outgrowths formed as a multicellular sheet with motile cilia becoming evident as the Matrigel was remodeled to provide an air interface; cultures were viable for more than one year. Immunofluorescence and electron microscopy (EM) identified an upper layer of mucociliary epithelium and a lower layer of highly organized extracellular matrix (ECM) interspersed with fibroblastic cells separated by a basement membrane. EM analysis of the mucosal construct after repetitive exposure to CSE revealed epithelial damage, loss of cilia, and ECM remodeling, as occurs in vivo. CONCLUSIONS: We have developed a robust bronchial mucosal model. The structural changes observed following CSE exposure suggest the model should have utility for drug discovery and preclinical testing, especially those targeting airway remodeling.

The Effects of Passive Smoking on Laryngeal and Tracheal Mucosa in Male Wistar Rats During Growth: An Experimental Study.

Cigarettes contain toxic and carcinogenic substances. In this context, cigarette smoking, and similar activities, are associated with numerous pathologies, being considered a risk factor in up to 10% of the total number of deaths in adults. Recent evidence suggests that the exposure of children to smoking in the early days of their development causes many diseases. Using light microscopy, this study aims to analyze the possible histopathological effects of an experimental model of chronic inhalation of cigarette smoke (passive smoking) on the laryngeal and tracheal mucosa of young Wistar rats. A total of 24 young Wistar rats were studied for a period of 120 days. The animals were divided into two groups: passive smoking (n = 16) and control (n = 8). The level of exposure to cigarette smoke was evaluated from the urinary cotinine level. Although no cancerous lesions were identified, histopathological analysis in the laryngeal and tracheal mucosa of all the animals in the experimental group showed that the proportion of moderate and focal inflammation was higher in animals exposed to chronic inhalation of cigarette smoke (P = 0.041). Histopathologic analysis revealed moderate and focal inflammatory lesions in the region of the infraglottic mucosa in exposed animals, although without dysplastic or neoplastic lesions in the laryngeal and tracheal mucosa.

Gill remodelling and growth rate of striped catfish Pangasianodon hypophthalmus under impacts of hypoxia and temperature.

Gill morphometric and gill plasticity of the air-breathing striped catfish (Pangasianodon hypophthalmus) exposed to different temperatures (present day 27°C and future 33°C) and different air saturation levels (92% and 35%) during 6weeks were investigated using vertical sections to estimate the respiratory lamellae surface areas, harmonic mean barrier thicknesses, and gill component volumes. Gill respiratory surface area (SA) and harmonic mean water - blood barrier thicknesses (HM) of the fish were strongly affected by both environmental temperature and oxygen level. Thus initial values for 27°C normoxic fish (12.4±0.8g) were 211.8±21.6mm2g-1 and 1.67±0.12µm for SA and HM respectively. After 5weeks in same conditions or in the combinations of 33°C and/or PO2 of 55mmHg, this initial surface area scaled allometrically with size for the 33°C hypoxic group, whereas branchial SA was almost eliminated in the 27°C normoxic group, with other groups intermediate. In addition, elevated temperature had an astounding effect on growth with the 33°C group growing nearly 8-fold faster than the 27°C fish.

CYP21A2 expression is localized in the developing distal epithelium of the human perinatal lung and is compatible with in situ production and intracrine actions of active glucocorticoids.

Glucocorticoids play essential roles in lung development. We investigated for expression of CYP21A2 (21-hydroxylase) as well as for the presence of the corresponding protein and identification of CYP21A2-expressing cells in several human developing lungs. Expression of some related genes was also assessed. CYP21A2 and CYP17A1 (P450c17) mRNAs were found in all the 34 lung samples from 17 to 40 weeks' gestation at variable levels. No correlation was found according to sex but a correlation with age was detected for CYP17A1 only. In contrast, CYP11B1 (11ß-hydroxylase)- and CYP11B2 (aldosterone synthase)-mRNAs were not detected. Significant levels of the CYP21A2 protein were detected in all the analyzed samples, while only very low signals were detected for CYP17A1 protein. In situ hybridization revealed that CYP21A2 was almost exclusively expressed in the distal epithelium. It was reported that the lung distal epithelium of human fetuses also express 11ß-hydroxysteroid dehydrogenase type 2, which catalyzes cortisol inactivation into cortisone. Based on this information, intracrine glucocorticoid actions should take place from CYP21A2 products through the glucocorticoid receptor in the absence of cortisol. In contrast, mineralocorticoid receptor activation did not seem to depend on deoxycorticosterone produced from local activity of CYP21A2 because of the reported circulating amounts of aldosterone.

Update on Molecular Biology of Lung Development--Transcriptomics.

This article highlights some of the significant advances in our understanding of lung developmental biology made over the last few years, which challenge existing paradigms and are relevant to a fundamental understanding of this process. Additional comments address how these new insights may be informative for chronic lung diseases that occur, or initiate, in the neonatal period. This is not meant to be an exhaustive review of the molecular biology of lung development. For a more comprehensive, contemporary review of the cellular and molecular aspects of lung development, readers can refer to recent reviews by others.

Non-canonical WNT signalling in the lung.

The role of WNT signalling in metazoan organogenesis has been a topic of widespread interest. In the lung, while the role of canonical WNT signalling has been examined in some detail by multiple studies, the non-canonical WNT signalling has received limited attention. Reliable evidence shows that this important signalling mechanism constitutes a major regulatory pathway in lung development. In addition, accumulating evidence has also shown that the non-canonical WNT pathway is critical for maintaining lung homeostasis and that aberrant activation of this pathway may underlie several debilitating lung diseases. Functional analyses have further revealed that the non-canonical WNT pathway regulates multiple cellular activities in the lung that are dependent on the specific cellular context. In most cell types, non-canonical WNT signalling regulates canonical WNT activity, which is also critical for many aspects of lung biology. This review will summarize what is currently known about the role of non-canonical WNT signalling in lung development, homeostasis and pathogenesis of disease.

Ultrastructure of Developing Feline Nonciliated Bronchiolar Epithelial Cells.

The ultrastructure of the developing bronchiolar cell was studied in six age groups: prenatal (60 d post-conception); postnatal (1-, 7-, 14- and 21-day-old); and adult. Following intratracheal fixation, the lung tissue was processed for scanning and transmission electron microscopy. The lining of terminal bronchioles consists of cuboidal to columnar nonciliated bronchiolar cells (NBCs) and ciliated with or without microvilli. NBCs were recognized by indented centrally located nucleus. The apical surface extended beyond the surface of neighboring cells and was covered by minute microvilli, except in prenatal kittens. The NBCs of the adult were characterized by abundant mitochondria and glycogen inclusions. In prenatal kittens, the cytoplasm was filled with patches of alpha and beta form of glycogen. Postnatally, glycogen was reduced in quantity, became scattered throughout the cytoplasm and was predominantly of the beta form. Islands of cytoplasm, separated from the apical cytoplasm were observed in the lumen of adult bronchioles. This suggests an apocrine mode of secretion. The NBCs attain maturity by three weeks of age.

Developmental lung expression and transcriptional regulation of claudin-6 by TTF-1, Gata-6, and FoxA2.

BACKGROUND: Claudins are transmembrane proteins expressed in tight junctions that prevent paracellular transport of extracellular fluid and a variety of other substances. However, the expression profile of Claudin-6 (Cldn6) in the developing lung has not been characterized. METHODS AND RESULTS: Cldn6 expression was determined during important periods of lung organogenesis by microarray analysis, qPCR and immunofluorescence. Expression patterns were confirmed to peak at E12.5 and diminish as lung development progressed. Immunofluorescence revealed that Cldn6 was detected in cells that also express TTF-1 and FoxA2, two critical transcriptional regulators of pulmonary branching morphogenesis. Cldn6 was also observed in cells that express Sox2 and Sox9, factors that influence cell differentiation in the proximal and distal lung, respectively. In order to assess transcriptional control of Cldn6, 0.5, 1.0, and 2.0-kb of the proximal murine Cldn6 promoter was ligated into a luciferase reporter and co-transfected with expression vectors for TTF-1 or two of its important transcriptional co-regulators, FoxA2 and Gata-6. In almost every instance, TTF-1, FoxA2, and Gata-6 activated gene transcription in cell lines characteristic of proximal airway epithelium (Beas2B) and distal alveolar epithelium (A-549). CONCLUSIONS: These data revealed for the first time that Cldn6 might be an important tight junctional component expressed by pulmonary epithelium during lung organogenesis. Furthermore, Cldn6-mediated aspects of cell differentiation may describe mechanisms of lung perturbation coincident with impaired cell junctions and abnormal membrane permeability.

DA-Raf-dependent inhibition of the Ras-ERK signaling pathway in type 2 alveolar epithelial cells controls alveolar formation.

Alveolar formation is coupled to the spatiotemporally regulated differentiation of alveolar myofibroblasts (AMYFs), which contribute to the morphological changes of interalveolar walls. Although the Ras-ERK signaling pathway is one of the key regulators for alveolar formation in developing lungs, the intrinsic molecular and cellular mechanisms underlying its role remain largely unknown. By analyzing the Ras-ERK signaling pathway during postnatal development of lungs, we have identified a critical role of DA-Raf1 (DA-Raf)-a dominant-negative antagonist for the Ras-ERK signaling pathway-in alveolar formation. DA-Raf-deficient mice displayed alveolar dysgenesis as a result of the blockade of AMYF differentiation. DA-Raf is predominantly expressed in type 2 alveolar epithelial cells (AEC2s) in developing lungs, and DA-Raf-dependent MEK1/2 inhibition in AEC2s suppresses expression of tissue inhibitor of matalloprotienase 4 (TIMP4), which prevents a subsequent proteolytic cascade matrix metalloproteinase (MMP)14-MMP2. Furthermore, MMP14-MMP2 proteolytic cascade regulates AMYF differentiation and alveolar formation. Therefore, DA-Raf-dependent inhibition of the Ras-ERK signaling pathway in AEC2s is required for alveolar formation via triggering MMP2 activation followed by AMYF differentiation. These findings reveal a pivotal role of the Ras-ERK signaling pathway in the dynamic regulation of alveolar development.

Morphology of non-sensory epithelium during post-natal development of the rabbit vomeronasal organ.

The vomeronasal organ (VNO), because of its ability to detect pheromones, has an important role in many social and sexual behaviours in mammals. It also mediates defensive behaviours through detection of protein pheromone homologues. A detailed morphological description of the post-natal development of the 'non-sensory' epithelium (NSE) of the female rabbit is recorded. Histological techniques were used to study the NSE of the VNO in post-natal development of female rabbits. The study focused on the following post-natal ages: newborn, 1 week, 2 weeks and 1 month (five animals each) beside to two adult animals. The rabbit VNO was surrounded externally by bony capsule and internally by cartilaginous capsule. NSE was pseudostratified columnar partially ciliated epithelium without goblet cells. In addition to basal cells, NSE contained ciliated and three types of non-ciliated columnar cells (dark, pale and light). At birth, dark cells may have primary cilia. By 1 month, the cytoplasm became lighter with less free ribosomes. The pale cells had electron-lucent cytoplasm, which contained a few organelles. Mitotic figures were observed in basal and columnar cells, particularly during the first 2 weeks of post-natal development. Light columnar cells were common during the first week. Numerous leucocytes and a few nerve endings were detected intra-epithelial. Scanning electron microscope revealed a gradual increase in height of microvilli of non-ciliated cells. Ciliated cells had cilia and microvilli. Cells were arranged singly, in clumps or in a dense population of cells. The rabbit VNO-NSE had a unique morphological structure.

Lung progenitors from lambs can differentiate into specialized alveolar or bronchiolar epithelial cells.

BACKGROUND: Airways progenitors may be involved in embryogenesis and lung repair. The characterization of these important populations may enable development of new therapeutics to treat acute or chronic lung disease. In this study, we aimed to establish the presence of bronchioloalveolar progenitors in ovine lungs and to characterize their potential to differentiate into specialized cells. RESULTS: Lung cells were studied using immunohistochemistry on frozen sections of the lung. Immunocytochemistry and flow cytometry were conducted on ex-vivo derived pulmonary cells. The bronchioloalveolar progenitors were identified by their co-expression of CCSP, SP-C and CD34. A minor population of CD34(pos)/SP-C(pos)/CCSP(pos) cells (0.33% ± 0.31) was present ex vivo in cell suspensions from dissociated lungs. Using CD34 magnetic positive-cell sorting, undifferentiated SP-C(pos)/CCSP(pos) cells were purified (>80%) and maintained in culture. Using synthetic media and various extracellular matrices, SP-C(pos)/CCSP(pos) cells differentiated into either club cells (formerly named Clara cells) or alveolar epithelial type-II cells. Furthermore, these ex vivo and in vitro derived bronchioloalveolar progenitors expressed NANOG, OCT4 and BMI1, specifically described in progenitors or stem cells, and during lung development. CONCLUSIONS: We report for the first time in a large animal the existence of bronchioloalveolar progenitors with dual differentiation potential and the expression of specialized genes. These newly described cell population in sheep could be implicated in regeneration of the lung following lesions or in development of diseases such as cancers.

Chemosensory brush cells of the trachea. A stable population in a dynamic epithelium.

Tracheal brush cells (BCs) are specialized epithelial chemosensors that use the canonical taste transduction cascade to detect irritants. To test whether BCs are replaced at the same rate as other cells in the surrounding epithelium of adult mice, we used 5-bromo-2'-deoxyuridine (BrdU) to label dividing cells. Although scattered BrdU-labeled epithelial cells are present 5-20 days after BrdU, no BCs are labeled. These data indicate that BCs comprise a relatively static population. To determine how BCs are generated during development, we injected 5-day-old mice with BrdU and found labeled BCs and non-BC epithelial cells 5 days after BrdU. During the next 60 days, the percentage of labeled BCs increased, whereas the percentage of other labeled cell types decreased. These data suggest that BCs are generated from non-BC progenitor cells during postnatal tracheal growth. To test whether the adult epithelium retains the capacity to generate BCs, tracheal epithelial cells were recovered from adult mice and grown in an air-liquid interface (ALI) culture. After transition to differentiation conditions, BCs are detected, and comprise 1% of the total cell population by Day 14. BrdU added to cultures before the differentiation of BCs was chased into BCs, indicating that the increase in BC density is attributable to the proliferation of a non-BC progenitor. We conclude that: (1) BCs are normally a static population in adult mice; (2) BC progenitors proliferate and differentiate during neonatal development; and (3) BCs can be regenerated from a proliferative population resident in adult epithelium.

Long-term outcome of human cord blood-derived hematopoietic progenitor cells in murine lungs.

Intranasally delivered human cord blood-derived CD34+ hematopoietic progenitor cells have the capacity to engraft and undergo transdifferentiation to surfactant-containing alveolar epithelial type II cell-like cells in lungs of newborn mice. The aim of this study was to determine the long-term fate of such transplanted cells as well as their effects on alveolar development in neonatally injured lungs. Double transgenic CCSP+/FasL+ mice with inducible lung-specific FasL expression, targeted to induce respiratory epithelial apoptosis in the perinatal period, served as model of neonatal lung injury. Non-injured single transgenic CCSP+/FasL- littermates served as controls. Freshly isolated umbilical cord blood CD34+ cells (0.5 to 1.0×10(6)) were administered at postnatal day 5 by intranasal inoculation; sham controls received equal volume PBS. Engraftment, alveolar epithelial differentiation, lung growth, and alveolarization were evaluated one year after transplantation. Engrafted cord blood-derived cells, detected by human-specific FISH (fluorescent in situ hybridization) analysis, and cord blood-derived alveolar type II-like cells, detected by double immunofluorescence analysis, while sparse, were seen in all conditions and more frequent in double than single transgenic recipients. The total lung volume and volume of air-exchanging parenchyma, assessed by stereological volumetry, were significantly greater in CD34-treated double transgenic animals than in PBS-treated double transgenic controls. Alveolarization, assessed by histomorphometry, was equivalent in these groups. These results suggest that transdifferentiated alveolar epithelial cells, derived from cord blood CD34+ cells, can persist up to one year after intrapulmonary delivery. Cord blood-CD34+ cell administration appears to have growth-promoting effects in injured newborn lungs, without affecting alveolar development in this model.