Alveolar Type II Cells or Mesenchymal Stem Cells: Comparison of Two Different Cell Therapies for the Treatment of Acute Lung Injury in Rats.

The use of cell therapies has recently increased for the treatment of pulmonary diseases. Mesenchymal stem/stromal cells (MSCs) and alveolar type II cells (ATII) are the main cell-based therapies used for the treatment of acute respiratory distress syndrome (ARDS). Many pre-clinical studies have shown that both therapies generate positive outcomes; however, the differences in the efficiency of MSCs or ATII for reducing lung damage remains to be studied. We compared the potential of both cell therapies, administering them using the same route and dose and equal time points in a sustained acute lung injury (ALI) model. We found that the MSCs and ATII cells have similar therapeutic effects when we tested them in a hydrochloric acid and lipopolysaccharide (HCl-LPS) two-hit ALI model. Both therapies were able to reduce proinflammatory cytokines, decrease neutrophil infiltration, reduce permeability, and moderate hemorrhage and interstitial edema. Although MSCs and ATII cells have been described as targeting different cellular and molecular mechanisms, our data indicates that both cell therapies are successful for the treatment of ALI, with similar beneficial results. Understanding direct cell crosstalk and the factors released from each cell will open the door to more accurate drugs being able to target specific pathways and offer new curative options for ARDS.

Efferocytosis of apoptotic alveolar epithelial cells is sufficient to initiate lung fibrosis.

Type II alveolar epithelial cell (AEC) apoptosis is a prominent feature of fibrotic lung diseases and animal models of pulmonary fibrosis. While there is growing recognition of the importance of AEC injury and apoptosis as a causal factor in fibrosis, the underlying mechanisms that link these processes remain unknown. We have previously shown that targeting the type II alveolar epithelium for injury by repetitively administering diphtheria toxin to transgenic mice expressing the diphtheria toxin receptor off of the surfactant protein C promoter (SPC-DTR) develop lung fibrosis, confirming that AEC injury is sufficient to cause fibrosis. In the present study, we find that SPC-DTR mice develop increased activation of caspase 3/7 after initiation of diphtheria toxin treatment consistent with apoptosis within AECs. We also find evidence of efferocytosis, the uptake of apoptotic cells, by alveolar macrophages in this model. To determine the importance of efferocytosis in lung fibrosis, we treated cultured alveolar macrophages with apoptotic type II AECs and found that the uptake induced pro-fibrotic gene expression. We also found that the repetitive intrapulmonary administration of apoptotic type II AEC or MLE-12 cells induces lung fibrosis. Finally, mice lacking a key efferocytosis receptor, CD36, developed attenuated fibrosis in response to apoptotic MLE-12 cells. Collectively, these studies support a novel mechanism linking AEC apoptosis with macrophage pro-fibrotic activation via efferocytosis and reveal previously unrecognized therapeutic targets.

Intratracheal instillation of alveolar type II cells enhances recovery from acute lung injury in rats.

BACKGROUND: Acute lung injury (ALI) and acute respiratory distress syndrome (ARDS) are characterized by excess production of inflammatory factors. Alveolar type II (ATII) cells help repair damaged lung tissue, rapidly proliferating and differentiating into alveolar type I cells after epithelial cell injury. In ALI, the lack of viable ATII favors progression to more severe lung injury. ATII cells regulate the immune response by synthesizing surfactant and other anti-inflammatory proteins and lipids. Cross-talk between ATII and other cells such as macrophages may also be part of the ATII function. The aim of this study was to test the anti-inflammatory and reparative effects of ATII cells in an experimental model of ALI. METHODS: In this study ATII cells (2.5 × 106 cells/animal) were intratracheally instilled in rats with HCl and lipopolysaccharide (LPS)-induced ALI and in healthy animals to check for side effects. The specific effect of ATII cells was compared with fibroblast transplantation. RESULTS: ATII cell transplantation promoted recovery of lung function, decrease mortality and lung inflammation of the animals with ALI. The primary mechanisms for benefit were paracrine effects of prostaglandin E2 (PGE2) and surfactant protein A (SPA) released from ATII cells that modulate alveolar macrophages to an anti-inflammatory phenotype. To our knowledge, these data are the first to provide evidence that ATII cells secrete PGE2 and SPA, reducing pro-inflammatory macrophage activation and ALI. CONCLUSION: ATII cells and their secreted molecules have shown an ability to resolve ALI, thereby highlighting a potential novel therapeutic target.

Safety and Tolerability of Alveolar Type II Cell Transplantation in Idiopathic Pulmonary Fibrosis.

BACKGROUND: Idiopathic pulmonary fibrosis (IPF) is a progressive and fatal lung disease with limited response to currently available therapies. Alveolar type II (ATII) cells act as progenitor cells in the adult lung, contributing to alveolar repair during pulmonary injury. However, in IPF, ATII cells die and are replaced by fibroblasts and myofibroblasts. In previous preclinical studies, we demonstrated that ATII-cell intratracheal transplantation was able to reduce pulmonary fibrosis. The main objective of this study was to investigate the safety and tolerability of ATII-cell intratracheal transplantation in patients with IPF. METHODS: We enrolled 16 patients with moderate and progressive IPF who underwent ATII-cell intratracheal transplantation through fiberoptic bronchoscopy. We evaluated the safety and tolerability of ATII-cell transplantation by assessing the emergent adverse side effects that appeared within 12 months. Moreover, pulmonary function, respiratory symptoms, and disease extent during 12 months of follow-up were evaluated. RESULTS: No significant adverse events were associated with the ATII-cell intratracheal transplantation. After 12 months of follow-up, there was no deterioration in pulmonary function, respiratory symptoms, or disease extent. CONCLUSIONS: Our results support the hypothesis that ATII-cell intratracheal transplantation is safe and well tolerated in patients with IPF. This study opens the door to designing a clinical trial to elucidate the potential beneficial effects of ATII-cell therapy in IPF.

Neonatal Type II Alveolar Epithelial Cell Transplant Facilitates Lung Reparation in Piglets With Acute Lung Injury and Extracorporeal Life Support.

OBJECTIVES: Type II alveolar epithelial cells have potential for lung growth and reparation. Extracorporeal membrane oxygenation is used as life support for lung impairment resulting from acute respiratory distress syndrome. We hypothesized that intratracheal transplantation of isogeneic primary type II alveolar epithelial cells in combination with extracorporeal membrane oxygenation may facilitate lung reparation for acute lung injury (ALI). DESIGN: A randomized, controlled experiment. SETTING: An animal laboratory in a university pediatric center. SUBJECTS: Twenty-eight 4- to 6-week young piglets, weighing 7-8 kg. INTERVENTIONS: Type II alveolar epithelial cells from neonatal male piglet lungs were isolated, purified, cultured, and labeled with chemical stain PKH26. After 3-6 hours of induction of ALI by IV endotoxin and mechanical ventilation (MV), young female piglets were allocated to five groups (n = 5): ALI-MV, ALI treated with MV; ALI-EC, ALI treated with both MV and venovenous extracorporeal membrane oxygenation; ALI-EC-T, ALI-EC protocol plus intratracheal type II alveolar epithelial cell transplant; CON-MV, healthy animals treated with MV; and CON-EC-T, healthy animals treated with venovenous extracorporeal membrane oxygenation. After 24 hours, animals were weaned from treatment for recovery in the ensuing 14 days, with their lungs assessed for injury and reparation. MEASUREMENTS AND MAIN RESULTS: Lung injury for animals in ALI-MV was moderate to severe, whereas much milder injuries in ALI-EC-T and ALI-EC were found. More PKH26-labeled type II alveolar epithelial cells were detected by fluorescence in the lungs of ALI-EC-T than in CON-EC-T as further verified by the expression of messenger RNA of sex-determining region of Y chromosome. Electromicroscopically intact type II alveolar epithelial cells and prominent lattice-like tubular myelin were also found in ALI-EC-T and CON-MV but not in ALI-EC. The hydroxyproline level in lung tissue was significantly lower in ALI-EC-T than in ALI-EC and ALI-MV, with most of the lung histopathologic and pathobiologic manifestations in favor of ALI-EC-T. CONCLUSIONS: The preliminary data suggested that type II alveolar epithelial cell transplant facilitated lung reparation for ALI in this model.

Differentiation of mouse induced pluripotent stem cells into alveolar epithelial cells in vitro for use in vivo.

Alveolar epithelial cells (AECs) differentiated from induced pluripotent stem cells (iPSCs) represent new opportunities in lung tissue engineering and cell therapy. In this study, we modified a two-step protocol for embryonic stem cells that resulted in a yield of ∼9% surfactant protein C (SPC)(+) alveolar epithelial type II (AEC II) cells from mouse iPSCs in a 12-day period. The differentiated iPSCs showed morphological characteristics similar to those of AEC II cells. When differentiated iPSCs were seeded and cultured in a decellularized mouse lung scaffold, the cells reformed an alveolar structure and expressed SPC or T1α protein (markers of AEC II or AEC I cells, respectively). Finally, the differentiated iPSCs were instilled intratracheally into a bleomycin-induced mouse acute lung injury model. The transplanted cells integrated into the lung alveolar structure and expressed SPC and T1α. Significantly reduced lung inflammation and decreased collagen deposition were observed following differentiated iPSC transplantation. In conclusion, we report a simple and rapid protocol for in vitro differentiation of mouse iPSCs into AECs. Differentiated iPSCs show potential for regenerating three-dimensional alveolar lung structure and can be used to abrogate lung injury.

Activation of canonical wnt pathway promotes differentiation of mouse bone marrow-derived MSCs into type II alveolar epithelial cells, confers resistance to oxidative stress, and promotes their migration to injured lung tissue in vitro.

The differentiation of mesenchymal stem cells (MSCs) into type II alveolar epithelial (AT II) cells in vivo and in vitro, is critical for reepithelization and recovery in acute lung injury (ALI), but the mechanisms responsible for differentiation are unclear. In the present study, we investigated the role of the canonical wnt pathway in the differentiation of mouse bone marrow-derived MSCs (mMSCs) into AT II cells. Using a modified co-culture system with murine lung epithelial-12 (MLE-12) cells and small airway growth media (SAGM) to efficiently drive mMSCs differentiation, we found that GSK 3ß and ß-catenin in the canonical wnt pathway were up-regulated during differentiation. The levels of surfactant protein (SP) C, SPB, and SPD, the specific markers of AT II cells, correspondingly increased in mMSCs when Wnt3a or LiCl was added to the co-culture system to activate wnt/ß-catenin signaling. The expression of these factors was depressed to some extent by inhibiting the pathway with the addition of DKK 1. The differentiation rate of mMSCs also depends on their abilities to accumulate and survive in inflammatory tissue. Our results suggested that the activation of wnt/ß-catenin signaling promoted mMSCs migration towards ALI mouse-derived lung tissue in a Transwell assay, and ameliorated the cell death and the reduction of Bcl-2/Bax induced by H(2) O(2), which simultaneously caused reduced GSK 3ß and ß-catenin in mMSCs. These data supports a potential mechanism for the differentiation of mMSCs into AT II cells involving canonical wnt pathway activation, which may be significant to their application in ALI.

Transplantation of alveolar type II cells stimulates lung regeneration during compensatory lung growth in adult rats.

OBJECTIVE: It is controversial whether lung regeneration contributes to compensatory lung growth after pulmonary resection in mature individuals. The objectives of this study were to clarify the molecular mechanisms that regulate the process of compensatory lung growth and investigate the influence of transplantation of lung cells enriched in alveolar type II cells on compensatory lung growth. METHODS: Serial changes of morphology and gene expression were examined in the remnant right lung after pneumonectomy in adult male Wistar rats. One day after surgery, animals received endotracheal transplants of rat lung cells enriched in alveolar type II cells at a dose of 2.5 × 10(6) cells. Serial morphologic changes were examined in comparison with pneumonectomy alone. Engraftment of lung cells was validated with a sex-mismatch model. RESULTS: The alveolar density with mean linear intercept was always lower in pneumonectomized rats than in sham surgical controls for 6 months after surgery. Microarray analysis revealed that multiple genes related to proliferation (but not specific alveolar development) were initially up-regulated and then returned to normal after 1 month. In the pneumonectomized rats with transplantation, the alveolar density was equivalent to that in the sham controls. Engraftment of the transplanted cells from male donors in the alveoli of female recipients was proven by detection of Y-chromosome positive cells and quantified by real-time polymerase chain reaction for the Sry gene. This occurred in pneumonectomized rats but not in sham controls. CONCLUSIONS: We postulate that lung cell transplantation stimulates lung regeneration in the remnant lung after pneumonectomy in mature rats.