Lung-specific Cell Research Articles

Amniotic fluid-derived mesenchymal stem cells reduce inflammation and improve lung function following transplantation in a porcine model

BackgroundLung transplantation is hindered by low donor lung utilization rates. Infectious complications are reasons to decline donor grafts due to fear of post-transplant primary graft dysfunction. Mesenchymal stem cells are a promising therapy currently investigated in treating lung injury. Full-term amniotic fluid-derived lung-specific mesenchymal stem cell treatment may regenerate damaged lungs. These cells have previously demonstrated inflammatory mediation in other respiratory diseases, and we hypothesized that treatment would improve donor lung quality and post-operative outcomes. MethodsIn a transplantation model, donor pigs were stratified to either the treated or the non-treated group. Acute respiratory distress syndrome was induced in donor pigs and harvested lungs were placed on ex vivo lung perfusion before transplantation. Treatment consisted of three doses of 2x106 cells/kg: one during ex vivo lung perfusion and two after transplantation. Donors and recipients were assessed on clinically relevant parameters and recipients were followed for 3 days before evaluation for primary graft dysfunction (PGD). ResultsRepeated injection of the cell treatment showed reductions in inflammation seen through lowered immune cell counts, reduced histology signs of inflammation, and decreased cytokines in the plasma and bronchoalveolar lavage fluid. Treated recipients showed improved pulmonary function, including increased PaO2/FiO2 ratios and reduced incidence of PGD. ConclusionsRepeated injection of lung-specific cell treatment during EVLP and post-transplant was associated with improved function of previously damaged lungs. Cell treatment may be considered as a potential therapy to increase the number of lungs available for transplantation and the improvement of post-operative outcomes.

Unlocking the Future: Pluripotent Stem Cell-Based Lung Repair.

The human respiratory system is susceptible to a variety of diseases, ranging from chronic obstructive pulmonary disease (COPD) and pulmonary fibrosis to acute respiratory distress syndrome (ARDS). Today, lung diseases represent one of the major challenges to the health care sector and represent one of the leading causes of death worldwide. Current treatment options often focus on managing symptoms rather than addressing the underlying cause of the disease. The limitations of conventional therapies highlight the urgent clinical need for innovative solutions capable of repairing damaged lung tissue at a fundamental level. Pluripotent stem cell technologies have now reached clinical maturity and hold immense potential to revolutionize the landscape of lung repair and regenerative medicine. Meanwhile, human embryonic (HESCs) and human-induced pluripotent stem cells (hiPSCs) can be coaxed to differentiate into lung-specific cell types such as bronchial and alveolar epithelial cells, or pulmonary endothelial cells. This holds the promise of regenerating damaged lung tissue and restoring normal respiratory function. While methods for targeted genetic engineering of hPSCs and lung cell differentiation have substantially advanced, the required GMP-grade clinical-scale production technologies as well as the development of suitable preclinical animal models and cell application strategies are less advanced. This review provides an overview of current perspectives on PSC-based therapies for lung repair, explores key advances, and envisions future directions in this dynamic field.

PerfuPul-A Versatile Perfusable Platform to Assess Permeability and Barrier Function of Air Exposed Pulmonary Epithelia.

Complex in vitro models, especially those based on human cells and tissues, may successfully reduce or even replace animal models within pre-clinical development of orally inhaled drug products. Microfluidic lung-on-chips are regarded as especially promising models since they allow the culture of lung specific cell types under physiological stimuli including perfusion and air-liquid interface (ALI) conditions within a precisely controlled in vitro environment. Currently, though, such models are not available to a broad user community given their need for sophisticated microfabrication techniques. They further require systematic comparison to well-based filter supports, in analogy to traditional Transwells®. We here present a versatile perfusable platform that combines the advantages of well-based filter supports with the benefits of perfusion, to assess barrier permeability of and aerosol deposition on ALI cultured pulmonary epithelial cells. The platform as well as the required technical accessories can be reproduced via a detailed step-by-step protocol and implemented in typical bio-/pharmaceutical laboratories without specific expertise in microfabrication methods nor the need to buy costly specialized equipment. Calu-3 cells cultured under liquid covered conditions (LCC) inside the platform showed similar development of transepithelial electrical resistance (TEER) over a period of 14 days as cells cultured on a traditional Transwell®. By using a customized deposition chamber, fluorescein sodium was nebulized via a clinically relevant Aerogen® Solo nebulizer onto Calu-3 cells cultured under ALI conditions within the platform. This not only allowed to analyze the transport of fluorescein sodium after ALI deposition under perfusion, but also to compare it to transport under traditional static conditions.

Abstracts from The International Society for Aerosols in Medicine 23rd ISAM Congress Boise, Idaho May 22–26, 2021

Abstracts from The International Society for Aerosols in Medicine 23rd ISAM Congress Boise, Idaho May 22–26, 2021

Culture Methods and Choice of Cells Makes a Difference in Outcome of Tissue Engineering- a Perspective

This perspective reviews the effects of different tissue engineering techniques, for induced differentiation of human embryonic stem cells into pulmonary lineage specific cells, and validating cell based regenerative therapy to ameliorate pulmonary degeneration. The etiology of degeneration differs from disease to disease. Likewise, strategies to regenerate lost tissues in its heterogeneity, should also cater to the regeneration process. To customize the same, researchers ought to device strategies to repair, replace or regenerate, in the spatio- temporal format, healthy tissues in their heterogeneous multi-functional existence, such that when transplanted in vitro, they may find their niche and integrate seamlessly into the tissue system and manifest full-fledged functional competence in order to reverse the erstwhile effects of degeneration. Cell based lung repair or regeneration is unquestionably the most promising agenda of regenerative medicine. In one of our studies, human embryonic stem cell (hESC) line H7 was successfully differentiated into three non-ciliated alveolar epithelial cells. In another study, BJNhem19 and BJNhem20 stem cell lines were used to induce similar guided endodermal differentiation into pulmonary lineage cells. Both studies were done using similar protocols, where the hESCs were first plated on gamma irradiated mouse embryonic fibroblast (MEF) feeder cells and undifferentiated hESCs were taken through embroid body formation. They were then subjected to induction into lung specific cells, by defined growth factors in Small Airways Growth Medium (SAGM) and Bronchiolar Endothelial Growth Media (BEGM). The H7 cells were found to express, both intracellularly and on their surfaces, characteristic marker proteins. However, the BJNhem19 and BJNhem20 cells showed poor differential potential, and could not be successfully differentiated into lung- specific cells. This difference in differentiation potential may be due to several intrinsic limitations, like genetic heterogeneity, anomalous morphogen receptivity or dormant/ unresponsive cytoplasmic determinants. These observations indicate that the choice of cells may play an important factor in tissue engineering. This review addresses the different culture methods and conditions needed for guided differentiation of stem cells. This perspective also discusses culture conditions of mesenchymal stem cells from different origins and dendritic stem cells from bone marrow.

Culture Methods and Choice of Cells Makes a Difference in Outcome of Tissue Engineering - A Perspective

This perspective reviews the effects of different tissue engineering techniques, for induced differentiation of human embryonic stem cells into pulmonary lineage specific cells, and validating cell based regenerative therapy to ameliorate pulmonary degeneration. The etiology of degeneration differs from disease to disease. Likewise, strategies to regenerate lost tissues in its heterogeneity, should also cater to the regeneration process. To customize the same, researchers ought to device strategies to repair, replace or regenerate, in the spatio- temporal format, healthy tissues in their heterogeneous multi-functional existence, such that when transplanted in vitro, they may find their niche and integrate seamlessly into the tissue system and manifest full-fledged functional competence in order to reverse the erstwhile effects of degeneration. Cell based lung repair or regeneration is unquestionably the most promising agenda of regenerative medicine. In one of our studies, human embryonic stem cell line H7 was successfully differentiated into three non-ciliated alveolar epithelial cells. In another study, BJNhem19 and BJNhem20 stem cell lines were used to induce similar guided endodermal differentiation into pulmonary lineage cells. Both studies were done using similar protocols, where the hESCs were first plated on gamma irriadiated MEF feeder cells and undifferentiated hESCs were taken through embroid body formation. They were then subjected to induction into lung specific cells, by defined growth factors in Small Airways Growth Medium and Bronchiolar Endothelial Growth Media. The H7 cells were found to express, both intracellularly and on their surfaces, characteristic marker proteins. However, the BJNhem19 and BJNhem20 cells showed poor differential potential, and could not be successfully differentiated into lung- specific cells. This difference in differentiation potential may be due to several intrinsic limitations, like genetic heterogeneity, anomalous morphogen receptivity or dormant/ unresponsive cytoplasmic determinants. These observations indicate that the choice of cells may play an important factor in tissue engineering. This review addresses the different culture methods and conditions needed for guided differentiation of stem cells. This perspective also discusses culture conditions of mesenchymal stem cells from different origins and dendritic stem cells from bone marrow

Metabolic shift in lung alveolar cell mitochondria following acrolein exposure

Acrolein, an α,β unsaturated electrophile, is an environmental pollutant released in ambient air from diesel exhausts and cooking oils. This study examines the role of acrolein in altering mitochondrial function and metabolism in lung-specific cells. RLE-6TN, H441, and primary alveolar type II (pAT2) cells were exposed to acrolein for 4 h, and its effect on mitochondrial oxygen consumption rates was studied by XF Extracellular Flux analysis. Low-dose acrolein exposure decreased mitochondrial respiration in a dose-dependent manner because of alteration in the metabolism of glucose in all the three cell types. Acrolein inhibited glyceraldehyde-3-phosphate dehydrogenase (GAPDH) activity, leading to decreased substrate availability for mitochondrial respiration in RLE-6TN, H441, and pAT2 cells; the reduced GAPDH activity was compensated in pAT2 cells by an increase in the activity of glucose-6-phosphate dehydrogenase, the regulatory control of the pentose phosphate pathway. The decrease in pyruvate from glucose metabolism resulted in utilization of alternative sources to support mitochondrial energy production: palmitate-BSA complex increased mitochondrial respiration in RLE-6TN and pAT2 cells. The presence of palmitate in alveolar cells for surfactant biosynthesis may prove to be the alternative fuel source for mitochondrial respiration. Accordingly, a decrease in phosphatidylcholine levels and an increase in phospholipase A2 activity were found in the alveolar cells after acrolein exposure. These findings have implications for understanding the decrease in surfactant levels frequently observed in pathophysiological situations with altered lung function following exposure to environmental toxicants.

Bioartificial Lung Engineering

End-stage lung disease is a major health care challenge. Lung transplantation remains the definitive treatment, yet rejection and donor organ shortage limit its broader clinical impact. Engineering bioartificial lung grafts from patient-derived cells could theoretically lead to alternative treatment strategies. Although many challenges on the way to clinical application remain, important early milestones toward translation have been met. Key endodermal progenitors can be derived from patients and expanded in vitro. Advanced culture conditions facilitate the formation of three-dimensional functional tissues from lineage-committed cells. Bioartificial grafts that provide gas exchange have been generated and transplanted into animal models. Looking ahead, current challenges in bioartificial lung engineering include creation of ideal scaffold materials, differentiation and expansion of lung-specific cell populations and full maturation of engineered constructs to provide graft longevity after implantation in vivo. A multidisciplinary collaborative effort will not only bring us closer to the ultimate goal of engineering patient-derived lung grafts, but also generate a series of clinically valuable translational milestones such as airway grafts and disease models. This review summarizes achievements to date, current challenges and ongoing research in bioartificial lung engineering.

Engineering an artificial alveolar-capillary membrane: a novel continuously perfused model within microchannels

Introduction Pulmonary hypoplasia is a condition of the newborn that is characterized by underdeveloped lungs and poor outcome. One strategy in the treatment of patients with hypoplasia is to augment underdeveloped lungs using biocompatible artificial lung tissue. However, one central challenge in current pulmonary tissue engineering efforts remains the development of a stable bio-mimetic alveolar-capillary membrane. Accordingly, we have built a series of bio-mimetic microfluidic devices that specifically model the alveolar-capillary membrane. Current designs include a single-layer microchip that exposes alveolar and endothelial cell types to controlled fluidic stimuli. A more advanced multi-layered device allows for alveolar cells to be cultured at an air interface while allowing constant media nourishment and waste removal, thus better mimicking the physiologic milieu of the alveolar-capillary interface. Both devices possess the benefit of parallel testing. Material and methods Microdevices were fabricated using soft lithography in a biocompatible transparent polymeric material, polydimethyl siloxane, sealed covalently to glass. The multistage microdevice also integrated a suspended polyethylene terephthalate membrane connected via microfluidic channels to constant media and air access. Pulmonary endothelial (HMEC-1) and alveolar epithelial (A549) cell lines, along with fetal pulmonary cells (FPC) harvested from Swiss Webster mice at day 18 gestational age, were studied under multiple hydrodynamic shear conditions and liquid-to-cell ratio regimes. Cultures were examined for cell viability, function and proliferation to confluent monolayers. A549 cells cultured at an air-interface in a microdevice was also tested for their ability to maintain cell phenotype and function. Results The single-layer differential flow microdevice allowed for a systematic determination of the optimal growth conditions of various lung-specific cell types in a microfluidic environment. Our device showed a greater surfactant based decrease in surface tension of the alveolar hypophase in A549 cultures exposed to air as compared to submerged cultures. Conclusions We have successfully developed biomimetic microfluidic devices that specifically allow stable alveolar cell growth at the air-liquid interface. This work serves prerequisite towards an implantable artificial alveolar membrane.

2-DE analysis of breast cancer cell lines 1833 and 4175 with distinct metastatic organ-specific potentials: Comparison with parental cell line MDA-MB-231

Human MDA-MB-231 derived breast cancer cell lines 1833 and 4175 have different metastatic potentials in terms of their tissue tropisms and aggressiveness. Cell line 1833 is specifically metastatic to the bone. The highly aggressive cell line 4175 is specific to the lung. We performed 2-DE analysis of the cell lines. We found 16 significantly changed protein spots, 14 protein spots were identified. Expression of cathepsin D, triosephosphate isomerase, phosphoglycerate kinase 1, heme binding protein 1 and annexin 2 could be correlated with the in vitro aggressiveness of the respective cell lines. Interstitial collagenase and dimethylargininase 2 were exclusive to the cell line 1833 and might contribute to its bone specificity. Serpin B9, cathepsin B chain b, galectin 3 and HSP 27 were changed in the lung specific cell line 4175. The possible contribution of identified proteins to differences in metastatic behavior of the cell lines is discussed.

Inhibition of LPS-induced activation of alveolar macrophages by high concentrations of LPS-binding protein

Lipopolysaccharide (LPS)-binding protein regulates the effects of LPS on immunocompetent cells. By catalyzing the binding of LPS to membrane CD14, LPS-binding protein (LBP) potentiates both the inflammatory response and internalization of LPS. LBP-mediated transport of LPS into high density lipoprotein particles participates in LPS clearance. Elevated serum levels of LBP have been shown to elicit protective effects in vivo. Because the expression of LBP is upregulated in lung epithelial cells upon proinflammatory stimulation, we here investigated whether LBP modulates inflammatory responses by lung specific cells. The moderate elevation of LBP concentrations enhanced both LPS-induced signaling and LPS uptake by rat alveolar macrophages, whereas strongly elevated LBP levels inhibited both. In contrast, the lung epithelial cell line A549 responded to high concentrations of LBP by an enhanced LPS uptake which did not result in cellular activation, suggesting an anti-inflammatory function of these cells by clearing LPS.