Transplantation of human embryonic stem cell-derived alveolar epithelial type II cells abrogates acute lung injury in mice.

Respiratory diseases are a major cause of mortality and morbidity worldwide. Current treatments offer no prospect of cure or disease reversal. Transplantation of pulmonary progenitor cells derived from human embryonic stem cells (hESCs) may provide a novel approach to regenerate endogenous lung cells destroyed by injury and disease. Here, we examine the therapeutic potential of alveolar type II epithelial cells derived from hESCs (hES-ATIICs) in a mouse model of acute lung injury. When transplanted into lungs of mice subjected to bleomycin (BLM)-induced acute lung injury, hES-ATIICs behaved as normal primary ATIICs, differentiating into cells expressing phenotypic markers of alveolar type I epithelial cells. Without experiencing tumorigenic side effects, lung injury was abrogated in mice transplanted with hES-ATIICs, demonstrated by recovery of body weight and arterial blood oxygen saturation, decreased collagen deposition, and increased survival. Therefore, transplantation of hES-ATIICs shows promise as an effective therapeutic to treat acute lung injury.

A pure population of lung alveolar epithelial type II cells derived from human embryonic stem cells.

Alveolar epithelial type II (ATII) cells are small, cuboidal cells that constitute approximately 60% of the pulmonary alveolar epithelium. These cells are crucial for repair of the injured alveolus by differentiating into alveolar epithelial type I cells. ATII cells derived from human ES (hES) cells are a promising source of cells that could be used therapeutically to treat distal lung diseases. We have developed a reliable transfection and culture procedure, which facilitates, via genetic selection, the differentiation of hES cells into an essentially pure (>99%) population of ATII cells (hES-ATII). Purity, as well as biological features and morphological characteristics of normal ATII cells, was demonstrated for the hES-ATII cells, including lamellar body formation, expression of surfactant proteins A, B, and C, alpha-1-antitrypsin, and the cystic fibrosis transmembrane conductance receptor, as well as the synthesis and secretion of complement proteins C3 and C5. Collectively, these data document the successful generation of a pure population of ATII cells derived from hES cells, providing a practical source of ATII cells to explore in disease models their potential in the regeneration and repair of the injured alveolus and in the therapeutic treatment of genetic diseases affecting the lung.

myogenin: Human candidate gene and knockout mouse effect

The myogenin gene encodes an evolutionarily conserved basic helix-loop-helix transcription factor that regulates the expression of skeletal muscle-specific genes and its homozygous deletion results in mice who die of respiratory failure at birth. The histology of skeletal muscle in the myogenin null mice is reminiscent of that found in some severe congenital myopathy patients, many of whom also die of respiratory complications and provides the rationale that an aberrant human myogenin (myf4) coding region could be associated with some congenital myopathy conditions.^ With PCR, we found similarly sized amplimers for the three exons of the myogenin gene in 37 patient and 40 control samples. In contrast to the GeneBank sequence for human myogenin, we report several differences in flanking and coding regions plus an additional 659 and 498 bps in the first and second introns, respectively, in all patients and controls. We also find a novel (CA)-dinucleotide repeat in the second intron. No causative mutations were detected in the myogenin coding regions of genomic DNA from patients with severe congenital myopathy.^ Severe congenital myopathies in humans are often associated with respiratory complications and pulmonary hypoplasia. We have employed the myogenin null mouse, which lacks normal development of skeletal muscle fibers as a genetically defined severe congenital myopathy mouse model to evaluate the effect of absent fetal breathing movement on pulmonary development.^ Significant differences are observed at embryonic days E14, E17 and E20 of lung:body weight, total DNA and histologically, suggesting that the myogenin null lungs are hypoplastic. RT-PCR, in-situ immunofluorescence and EM reveal pneumocyte type II differentiation in both null and wild lungs as early as E14. However, at E14, myogenin null lungs have decreased BrdU incorporation while E17 through term, augmented cell death is detected in the myogenin null lungs, not seen in wild littermates. Absent mechanical forces appear to impair normal growth, but not maturation, of the developing lungs in myogenin null mouse.^ These investigations provide the basis for delineating the DNA sequence of the myogenin gene and and highlight the importance of skeletal muscle development in utero for normal lung organogenesis. My observation of no mutations within the coding regions of the human myogenin gene in DNA from patients with severe congenital myopathy do not support any association with this condition. ^

Mechanism of silica -induced apoptosis in human alveolar macrophages

The mechanisms involved in the development of pulmonary silicosis have not been well defined, however most current evidence implicates a central role for alveolar macrophages in this process. We propose that the fibrotic potential of a particulate depends upon its ability to cause apoptosis in alveolar macrophage (AM). The overall goal of this study was to determine the mechanism of silica-induced apoptosis of AM. Human AM were treated with fibrogenic, poorly fibrogenic and nonfibrogenic model particulates, such as, silica, amorphous silica and titanium dioxide, respectively (equal surface area). Treatment with silica resulted in apoptosis in human AM as observed by morphology, DNA fragmentation and Cell Death ELISA assays. In contrast, amorphous silica and titanium dioxide demonstrated no significant apoptotic potential. To elucidate the possible mechanism by which silica causes apoptosis, we investigated the role of the scavenger receptor (SR) in silica-induced apoptosis. Cells were pretreated with and without SR ligand binding inhibitors, polyinosinic acid (Poly I), fucoidan and high density lipoprotein (HDL), prior to silica treatment. Pretreatment with Poly I and fucoidan resulted in significant inhibition of silica-induced apoptosis suggesting that silica-induced AM apoptosis is mediated via the SR. Further, we examined the involvement of interleukin converting enzyme (ICE) family of proteases in silica-mediated apoptosis. Silica activated ICE, Ich-1L, cpp32 beta and cleavage of PARP. Taken together, these results suggested that (1) fibrogenic particulates, such as, silica caused apoptosis of alveolar macrophages, (2) this apoptotic potential of fibrogenic particulates may be a critical factor in initiating an inflammatory response resulting in fibrosis, (3) silica-induced apoptosis of alveolar macrophages may be due to the interaction of silica particulates with the SR, and (4) silica-induced apoptosis involves the activation of the ICE family of proteases. An understanding of the molecular events involved in fibrogenic particulate-induced apoptosis may provide a useful insight into the mechanism involved in particulate-induced fibrosis. ^