Lung inflammatory environments differentially alter mesenchymal stromal cell behavior.

Mesenchymal stromal (stem) cells (MSCs) are increasingly demonstrated to ameliorate experimentally induced lung injuries through disease-specific anti-inflammatory actions, thus suggesting that different in vivo inflammatory environments can influence MSC actions. To determine the effects of different representative inflammatory lung conditions, human bone marrow-derived MSCs (hMSCs) were exposed to in vitro culture conditions from bronchoalveolar lavage fluid (BALF) samples obtained from patients with either the acute respiratory distress syndrome (ARDS) or with other lung diseases including acute respiratory exacerbations of cystic fibrosis (CF) (non-ARDS). hMSCs were subsequently assessed for time- and BALF concentration-dependent effects on mRNA expression of selected pro- and anti-inflammatory mediators, and for overall patterns of gene and mRNA expression. Both common and disease-specific patterns were observed in gene expression of different hMSC mediators, notably interleukin (IL)-6. Conditioned media obtained from non-ARDS BALF-exposed hMSCs was more effective in promoting an anti-inflammatory phenotype in monocytes than was conditioned media from ARDS BALF-exposed hMSCs. Neutralizing IL-6 in the conditioned media promoted generation of anti-inflammatory monocyte phenotype. This proof of concept study suggest that different lung inflammatory environments potentially can alter hMSC behaviors. Further identification of these interactions and the driving mechanisms may influence clinical use of MSCs for treating lung diseases.

Comparative Decellularization and Recellularization of Wild-Type and Alpha 1,3 Galactosyltransferase Knockout Pig Lungs: A Model for Ex Vivo Xenogeneic Lung Bioengineering and Transplantation.

BACKGROUND: A novel potential approach for lung transplantation could be to utilize xenogeneic decellularized pig lung scaffolds that are recellularized with human lung cells. However, pig tissues express several immunogenic proteins, notably galactosylated cell surface glycoproteins resulting from alpha 1,3 galactosyltransferase (α-gal) activity, that could conceivably prevent effective use. Use of lungs from α-gal knock out (α-gal KO) pigs presents a potential alternative and thus comparative de- and recellularization of wild-type and α-gal KO pig lungs was assessed. METHODS: Decellularized lungs were compared by histologic, immunohistochemical, and mass spectrometric techniques. Recellularization was assessed following compartmental inoculation of human lung bronchial epithelial cells, human lung fibroblasts, human bone marrow-derived mesenchymal stromal cells (all via airway inoculation), and human pulmonary vascular endothelial cells (CBF) (vascular inoculation). RESULTS: No obvious differences in histologic structure was observed but an approximate 25% difference in retention of residual proteins was determined between decellularized wild-type and α-gal KO pig lungs, including retention of α-galactosylated epitopes in acellular wild-type pig lungs. However, robust initial recellularization and subsequent growth and proliferation was observed for all cell types with no obvious differences between cells seeded into wild-type versus α-gal KO lungs. CONCLUSION: These proof of concept studies demonstrate that decellularized wild-type and α-gal KO pig lungs can be comparably decellularized and comparably support initial growth of human lung cells, despite some differences in retained proteins. α-Gal KO pig lungs are a suitable platform for further studies of xenogeneic lung regeneration.

CD11b+ and Sca-1+ Cells Exert the Main Beneficial Effects of Systemically Administered Bone Marrow-Derived Mononuclear Cells in a Murine Model of Mixed Th2/Th17 Allergic Airway Inflammation.

UNLABELLED: Systemic administration of bone marrow-derived mononuclear cells (BMDMCs) or bone marrow-derived mesenchymal stromal cells (MSCs) reduces inflammation and airway hyperresponsiveness (AHR) in a murine model of Th2-mediated eosinophilic allergic airway inflammation. However, since BMDMCs are a heterogeneous population that includes MSCs, it is unclear whether the MSCs alone are responsible for the BMDMC effects. To determine which BMDMC population(s) is responsible for ameliorating AHR and lung inflammation in a model of mixed Th2-eosinophilic and Th17-neutrophilic allergic airway inflammation, reminiscent of severe clinical asthma, BMDMCs obtained from normal C57Bl/6 mice were serially depleted of CD45, CD34, CD11b, CD3, CD19, CD31, or Sca-1 positive cells. The different resulting cell populations were then assessed for ability to reduce lung inflammation and AHR in mixed Th2/Th17 allergic airway inflammation induced by mucosal sensitization to and challenge with Aspergillus hyphal extract (AHE) in syngeneic C56Bl/6 mice. BMDMCs depleted of either CD11b-positive (CD11b+) or Sca-1-positive (Sca-1+) cells were unable to ameliorate AHR or lung inflammation in this model. Depletion of the other cell types did not diminish the ameliorating effects of BMDMC administration. In conclusion, in the current model of allergic inflammation, CD11b+ cells (monocytes, macrophages, dendritic cells) and Sca-1+ cells (MSCs) are responsible for the beneficial effects of BMDMCs. SIGNIFICANCE: This study shows that bone marrow-derived mononuclear cells (BMDMCs) are as effective as bone marrow-derived mesenchymal stromal cells (MSCs) in ameliorating experimental asthma. It also demonstrates that not only MSCs present in the pool of BMDMCs are responsible for BMDMCs' beneficial effects but also monocytes, which are the most important cell population to trigger these effects. All of this is in the setting of a clinically relevant model of severe allergic airways inflammation and thus provides further support for potential clinical use of cell therapy using MSCs, BMDMCs, and also adult cells such as monocytes in patients with severe asthma.

Residual Detergent Detection Method for Nondestructive Cytocompatibility Evaluation of Decellularized Whole Lung Scaffolds.

The development of reliable tissue engineering methods using decellularized cadaveric or donor lungs could potentially provide a new source of lung tissue. The vast majority of current lung decellularization protocols are detergent based and incompletely removed residual detergents may have a deleterious impact on subsequent scaffold recellularization. Detergent removal and quality control measures that rigorously and reliably confirm removal, ideally utilizing nondestructive methods, are thus critical for generating optimal acellular scaffolds suitable for potential clinical translation. Using a modified and optimized version of a methylene blue-based detergent assay, we developed a straightforward, noninvasive method for easily and reliably detecting two of the most commonly utilized anionic detergents, sodium deoxycholate (SDC) and sodium dodecyl sulfate (SDS), in lung decellularization effluents. In parallel studies, we sought to determine the threshold of detergent concentration that was cytotoxic using four different representative human cell types utilized in the study of lung recellularization: human bronchial epithelial cells, human pulmonary vascular endothelial cells (CBF12), human lung fibroblasts, and human mesenchymal stem cells. Notably, different cells have varying thresholds for either SDC or SDS-based detergent-induced cytotoxicity. These studies demonstrate the importance of reliably removing residual detergents and argue that multiple cell lines should be tested in cytocompatibility-based assessments of acellular scaffolds. The detergent detection assay presented here is a useful nondestructive tool for assessing detergent removal in potential decellularization schemes or for use as a potential endpoint in future clinical schemes, generating acellular lungs using anionic detergent-based decellularization protocols.

Systemic Administration of Human Bone Marrow-Derived Mesenchymal Stromal Cell Extracellular Vesicles Ameliorates Aspergillus Hyphal Extract-Induced Allergic Airway Inflammation in Immunocompetent Mice.

UNLABELLED: An increasing number of studies demonstrate that administration of either conditioned media (CM) or extracellular vesicles (EVs) released by mesenchymal stromal cells (MSCs) derived from bone marrow and other sources are as effective as the MSCs themselves in mitigating inflammation and injury. The goal of the current study was to determine whether xenogeneic administration of CM or EVs from human bone marrow-derived MSCs would be effective in a model of mixed Th2/Th17, neutrophilic-mediated allergic airway inflammation, reflective of severe refractory asthma, induced by repeated mucosal exposure to Aspergillus hyphal extract (AHE) in immunocompetent C57Bl/6 mice. Systemic administration of both CM and EVs isolated from human and murine MSCs, but not human lung fibroblasts, at the onset of antigen challenge in previously sensitized mice significantly ameliorated the AHE-provoked increases in airway hyperreactivity (AHR), lung inflammation, and the antigen-specific CD4 T-cell Th2 and Th17 phenotype. Notably, both CM and EVs from human MSCs (hMSCs) were generally more potent than those from mouse MSCs (mMSCs) in most of the outcome measures. The weak cross-linking agent 1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride was found to inhibit release of both soluble mediators and EVs, fully negating effects of systemically administered hMSCs but only partly inhibited the ameliorating effects of mMSCs. These results demonstrate potent xenogeneic effects of CM and EVs from hMSCs in an immunocompetent mouse model of allergic airway inflammation and they also show differences in mechanisms of action of hMSCs versus mMSCs to mitigate AHR and lung inflammation in this model. SIGNIFICANCE: There is a growing experience demonstrating benefit of mesenchymal stromal cell (MSC)-based cell therapies in preclinical models of asthma. In the current study, conditioned media (CM) and, in particular, the extracellular vesicle fraction obtained from the CM were as potent as the MSCs themselves in mitigating Th2/Th17-mediated allergic airway inflammation in a mouse model of severe refractory clinical asthma. Moreover, human MSC CM and extracellular vesicles were effective in this immunocompetent mouse model. These data add to a growing scientific basis for initiating clinical trials of MSCs or extracellular vesicles derived from MSCs in severe refractory asthma and provide further insight into the mechanisms by which the MSCs may ameliorate the asthma.

Recellularization of decellularized lung scaffolds is enhanced by dynamic suspension culture.

Strategies are needed to improve repopulation of decellularized lung scaffolds with stromal and functional epithelial cells. We demonstrate that decellularized mouse lungs recellularized in a dynamic low fluid shear suspension bioreactor, termed the rotating wall vessel (RWV), contained more cells with decreased apoptosis, increased proliferation and enhanced levels of total RNA compared to static recellularization conditions. These results were observed with two relevant mouse cell types: bone marrow-derived mesenchymal stromal (stem) cells (MSCs) and alveolar type II cells (C10). In addition, MSCs cultured in decellularized lungs under static but not bioreactor conditions formed multilayered aggregates. Gene expression and immunohistochemical analyses suggested differentiation of MSCs into collagen I-producing fibroblast-like cells in the bioreactor, indicating enhanced potential for remodeling of the decellularized scaffold matrix. In conclusion, dynamic suspension culture is promising for enhancing repopulation of decellularized lungs, and could contribute to remodeling the extracellular matrix of the scaffolds with subsequent effects on differentiation and functionality of inoculated cells.

Freshly thawed and continuously cultured human bone marrow-derived mesenchymal stromal cells comparably ameliorate allergic airways inflammation in immunocompetent mice.

UNLABELLED: Recent data suggest that freshly thawed previously frozen mesenchymal stromal cells (MSCs) may not have the same effectiveness or breadth of anti-inflammatory activities as do continuously cultured MSCs. This has significant implications for clinical use, in which many infusion schemes use frozen cells thawed at the bedside for administration. The available data, however, predominantly evaluate in vitro MSC properties, and so far there has been limited in vivo analysis. To further assess this issue, we compared freshly thawed (thawed) versus continuously cultured (fresh) human bone marrow-derived MSC (hMSC) administration in a mouse model of mixed Th2/Th17 allergic airway inflammation induced by Aspergillus hyphal extract (AHE) exposures in immunocompetent C57Bl/6 mice. Control cell populations included fresh versus thawed murine bone marrow-derived MSCs (mMSCs) and human lung fibroblasts (HLFs). Systemic administration of both thawed and fresh hMSCs and mMSCs, but not HLFs, at the onset of antigen challenge in previously sensitized mice significantly ameliorated the AHE-provoked increases in airway hyper-reactivity, lung inflammation, and antigen-specific CD4 T-cell Th2 and Th17 phenotype. Notably, there was no difference in effects of fresh versus thawed hMSCs or mMSCs on any outcome measured except for some variability in the effects on the bronchoalveolar lavage fluid composition. These results demonstrated potent xenogeneic effects of human MSCs in an immunocompetent mouse model of allergic airways inflammation and that thawed MSCs are as effective as fresh MSCs. The question of fresh versus thawed MSC effectiveness needs to be investigated carefully and may differ in different in vivo disease-specific models. SIGNIFICANCE: This study addressed whether freshly thawed mesenchymal stromal cells (MSCs) are as effective in in vivo settings as those that have been continuously cultured. It also provided further data demonstrating that xenogeneic use of MSCs in immunocompetent mice is as effective as murine MSCs. This information provides further support and direction for potential clinical use of MSCs in patients with severe asthma.

Comparative decellularization and recellularization of normal versus emphysematous human lungs.

Acellular whole human lung scaffolds represent a unique opportunity for ex vivo tissue engineering. However, it remains unclear whether lungs from individuals with chronic lung diseases such as chronic obstructive pulmonary disease (COPD) can be appropriately decellularized and recellularized. To assess this, cadaveric human lungs from normal (non-smoking) patients and from patients with COPD (smoking history) were decellularized and found by histochemical and immunohistochemical staining, electron microscopy, and mass spectrometry to retain characteristic histological architecture and extracellular matrix components (ECM) reflecting either normal or COPD, particularly emphysematous, origin. Inoculation of human bronchial epithelial cells, endothelial progenitor cells, bone marrow-derived mesenchymal stem cells, and lung fibroblasts via airway or vascular routes into small, excised segments of the decellularized lungs demonstrated that normal lung scaffolds robustly supported initial engraftment and growth of each cell type for up to one month. In contrast, despite initial binding, all cell types inoculated into decellularized emphysematous lungs did not survive beyond one week. However, cell attachment and proliferation on solubilized ECM homogenates of decellularized normal and emphysematous lungs coated onto tissue culture plates was comparable and not impaired, suggesting that the 3-dimensional decellularized emphysematous scaffolds may lack the necessary ECM architecture to support sustained cell growth.

Three-dimensional scaffolds of acellular human and porcine lungs for high throughput studies of lung disease and regeneration.

Acellular scaffolds from complex whole organs such as lung are being increasingly studied for ex vivo organ generation and for in vitro studies of cell-extracellular matrix interactions. We have established effective methods for efficient de and recellularization of large animal and human lungs including techniques which allow multiple small segments (∼ 1-3 cm(3)) to be excised that retain 3-dimensional lung structure. Coupled with the use of a synthetic pleural coating, cells can be selectively physiologically inoculated via preserved vascular and airway conduits. Inoculated segments can be further sliced for high throughput studies. Further, we demonstrate thermography as a powerful noninvasive technique for monitoring perfusion decellularization and for evaluating preservation of vascular and airway networks following human and porcine lung decellularization. Collectively, these techniques are a significant step forward as they allow high throughput in vitro studies from a single lung or lobe in a more biologically relevant, three-dimensional acellular scaffold.

Mesenchymal stromal cells mediate Aspergillus hyphal extract-induced allergic airway inflammation by inhibition of the Th17 signaling pathway.

Systemic administration of mesenchymal stromal cells (MSCs) suppresses airway inflammation and methacholine-induced airway hyper-responsiveness (AHR) in mouse models of T helper cell (Th) type 2-mediated eosinophilic allergic airway inflammation (AAI); however, the efficacy of MSCs in mouse models of severe Th17-mediated neutrophilic AAI has not yet been demonstrated. We assessed MSC effects in a mouse model of mixed Th2/Th17 AAI produced by mucosal exposure to Aspergillus fumigatus hyphal extract (AHE). Following sensitization produced by oropharyngeal AHE administration, systemic (tail vein) administration of syngeneic MSCs on the first day of challenge significantly reduced acute AHR predominantly through reduction of Th17-mediated airway inflammation. In parallel experiments, MSCs also mitigated AHR when administered during recurrent challenge 10 weeks after initial sensitization and challenge through reduction in systemic Th17-mediated inflammation. Investigation into potential mechanistic actions of MSCs in this model demonstrated that although T regulatory cells were increased in all AHE-treated mice, MSC administration did not alter T regulatory cell numbers in either the acute or recurrent model. Differential induction of interleukin-17a secretion was observed in ex vivo restimulation of mediastinal lymph node mixed-cell cytokine analyses. Although the mechanisms by which MSCs act to decrease inflammation and AHR in this model are not yet fully elucidated, decrease in Th17-mediated airway inflammation appears to play a significant role. These results provide a basis for further investigations of MSC administration as a potential therapeutic approach for severe refractory neutrophilic asthma.

Multipotent adult progenitor cells decrease cold ischemic injury in ex vivo perfused human lungs: an initial pilot and feasibility study.

BACKGROUND: Primary graft dysfunction (PGD) is a significant cause of early morbidity and mortality following lung transplantation. Improved organ preservation techniques will decrease ischemia-reperfusion injury (IRI) contributing to PGD. Adult bone marrow-derived adherent stem cells, including mesenchymal stromal (stem) cells (MSCs) and multipotent adult progenitor cells (MAPCs), have potent anti-inflammatory actions, and we thus postulated that intratracheal MAPC administration during donor lung processing would decrease IRI. The goal of the study was therefore to determine if intratracheal MAPC instillation would decrease lung injury and inflammation in an ex vivo human lung explant model of prolonged cold storage and subsequent reperfusion. METHODS: Four donor lungs not utilized for transplant underwent 8 h of cold storage (4°C). Following rewarming for approximately 30 min, non-HLA-matched allogeneic MAPCs (1 × 10(7) MAPCs/lung) were bronchoscopically instilled into the left lower lobe (LLL) and vehicle comparably instilled into the right lower lobe (RLL). The lungs were then perfused and mechanically ventilated for 4 h and subsequently assessed for histologic injury and for inflammatory markers in bronchoalveolar lavage fluid (BALF) and lung tissue. RESULTS: All LLLs consistently demonstrated a significant decrease in histologic and BALF inflammation compared to vehicle-treated RLLs. CONCLUSIONS: These initial pilot studies suggest that use of non-HLA-matched allogeneic MAPCs during donor lung processing can decrease markers of cold ischemia-induced lung injury.

Polymorphisms in the CD1d promoter that regulate CD1d gene expression are associated with impaired NKT cell development.

CD1d-restricted NKT cells comprise an innate-like T cell population that exerts significant influence over early events in the developing immune response. The frequency of NKT cells is highly variable in humans and in mice, but the basis for this variability remains unclear. In this study, we report a striking deficiency of type I NKT cells in the wild-derived inbred strains PWD/PhJ, SPRET/EiJ, and CAST/EiJ. Investigation of the underlying basis for the lack of type I NKT cells revealed that one strain, PWD/PhJ, exhibited a significant impairment in thymocyte and splenocyte CD1d gene and protein expression. Accordingly, both thymocytes and bone marrow-derived dendritic cells from PWD mice exhibited a significant impairment in the ability to present α-galactosylceramide to NKT cells. The impaired PWD CD1d gene expression was due to impaired CD1d promoter activity. Fine-mapping of the promoter activity revealed that two single nucleotide substitutions at positions -331 and -164 in the proximal promoter were each sufficient to account for the diminished PWD CD1d promoter activity. Examination of the strain distribution pattern of these polymorphisms revealed that, of 19 strains analyzed, only PWD and PWK mice possessed both CD1d promoter polymorphisms. A subsequent examination of the PWK strain revealed that it also exhibited impaired thymocyte CD1d expression and very low numbers of NKT cells. Taken together, these results provide new insight into the control of CD1d gene expression, and they have implications for the evolution of CD1d and type I NKT cells.

The effects of storage and sterilization on de-cellularized and re-cellularized whole lung.

Despite growing interest on the potential use of de-cellularized whole lungs as 3-dimensional scaffolds for ex vivo lung tissue generation, optimal processing including sterilization and storage conditions, are not well defined. Further, it is unclear whether lungs need to be obtained immediately or may be usable even if harvested several days post-mortem, a situation mimicking potential procurement of human lungs from autopsy. We therefore assessed effects of delayed necropsy, prolonged storage (3 and 6 months), and of two commonly utilized sterilization approaches: irradiation or final rinse with peracetic acid, on architecture and extracellular matrix (ECM) protein characteristics of de-cellularized mouse lungs. These different approaches resulted in significant differences in both histologic appearance and in retention of ECM and intracellular proteins as assessed by immunohistochemistry and mass spectrometry. Despite these differences, binding and proliferation of bone marrow-derived mesenchymal stromal cells (MSCs) over a one month period following intratracheal inoculation was similar between experimental conditions. In contrast, significant differences occurred with C10 mouse lung epithelial cells between the different conditions. Therefore, delayed necropsy, duration of scaffold storage, sterilization approach, and cell type used for re-cellularization may significantly impact the usefulness of this biological scaffold-based model of ex vivo lung tissue regeneration.

The effect of age and emphysematous and fibrotic injury on the re-cellularization of de-cellularized lungs.

Use of de-cellularized cadaveric lungs as 3-dimensional scaffolds for ex vivo lung tissue generation offers a new potential therapeutic approach for clinical lung transplantation. However, it is likely that some of the available cadaveric human lungs may be from older donors or from donors with previously existing structural lung diseases such as emphysema or pulmonary fibrosis. It is not known whether these lungs will be suitable for either de-cellularization or re-cellularization. To investigate this, we assessed the effects of advanced age, representative emphysematous and fibrotic injuries, and the combination of advanced age and emphysematous injury and found significant differences both in histologic appearance and in the retention of extracellular matrix (ECM) and other proteins, as assessed by immunohistochemistry and mass spectrometry, between the different conditions. However, despite these differences, binding, retention and growth of bone marrow-derived mesenchymal stromal cells (MSCs) over a 1-month period following intratracheal inoculation were similar between the different experimental conditions. In contrast, significant differences occurred in the growth of C10 mouse lung epithelial cells between the different conditions. Therefore, age, lung injury, and the cell type used for re-cellularization may significantly impact the usefulness of de-cellularized whole lungs for ex vivo lung tissue regeneration.

A nonhuman primate model of lung regeneration: detergent-mediated decellularization and initial in vitro recellularization with mesenchymal stem cells.

Currently, patients with end-stage lung disease are limited to lung transplantation as their only treatment option. Unfortunately, the lungs available for transplantation are few. Moreover, transplant recipients require life-long immune suppression to tolerate the transplanted lung. A promising alternative therapeutic strategy is decellularization of whole lungs, which permits the isolation of an intact scaffold comprised of innate extracellular matrix (ECM) that can theoretically be recellularized with autologous stem or progenitor cells to yield a functional lung. Nonhuman primates (NHP) provide a highly relevant preclinical model with which to assess the feasibility of recellularized lung scaffolds for human lung transplantation. Our laboratory has successfully accomplished lung decellularization and initial stem cell inoculation of the resulting ECM scaffold in an NHP model. Decellularization of normal adult rhesus macaque lungs as well as the biology of the resulting acellular matrix have been extensively characterized. Acellular NHP matrices retained the anatomical and ultrastructural properties of native lungs with minimal effect on the content, organization, and appearance of ECM components, including collagen types I and IV, laminin, fibronectin, and sulfated glycosaminoglycans (GAG), due to decellularization. Proteomics analysis showed enrichment of ECM proteins in total tissue extracts due to the removal of cells and cellular proteins by decellularization. Cellular DNA was effectively removed after decellularization (∼92% reduction), and the remaining nuclear material was found to be highly disorganized, very-low-molecular-weight fragments. Both bone marrow- and adipose-derived mesenchymal stem cells (MSC) attach to the decellularized lung matrix and can be maintained within this environment in vitro, suggesting that these cells may be promising candidates and useful tools for lung regeneration. Analysis of decellularized lung slice cultures to which MSC were seeded showed that the cells attached to the decellularized matrix, elongated, and proliferated in culture. Future investigations will focus on optimizing the recellularization of NHP lung scaffolds toward the goal of regenerating pulmonary tissue. Bringing this technology to eventual human clinical application will provide patients with an alternative therapeutic strategy as well as significantly reduce the demand for transplantable organs and patient wait-list time.

Initial binding and recellularization of decellularized mouse lung scaffolds with bone marrow-derived mesenchymal stromal cells.

Recellularization of whole decellularized lung scaffolds provides a novel approach for generating functional lung tissue ex vivo for subsequent clinical transplantation. To explore the potential utility of stem and progenitor cells in this model, we investigated recellularization of decellularized whole mouse lungs after intratracheal inoculation of bone marrow-derived mesenchymal stromal cells (MSCs). The decellularized lungs maintained structural features of native lungs, including intact vasculature, ability to undergo ventilation, and an extracellular matrix (ECM) scaffold consisting primarily of collagens I and IV, laminin, and fibronectin. However, even in the absence of intact cells or nuclei, a number of cell-associated (non-ECM) proteins were detected using mass spectroscopy, western blots, and immunohistochemistry. MSCs initially homed and engrafted to regions enriched in types I and IV collagen, laminin, and fibronectin, and subsequently proliferated and migrated toward regions enriched in types I and IV collagen and laminin but not provisional matrix (fibronectin). MSCs cultured for up to 1 month in either basal MSC medium or in a small airways growth media (SAGM) localized in both parenchymal and airway regions and demonstrated several different morphologies. However, while MSCs cultured in basal medium increased in number, MSCs cultured in SAGM decreased in number over 1 month. Under both media conditions, the MSCs predominantly expressed genes consistent with mesenchymal and osteoblast phenotype. Despite a transient expression of the lung precursor TTF-1, no other airway or alveolar genes or vascular genes were expressed. These studies highlight the power of whole decellularized lung scaffolds to study functional recellularization with MSCs and other cells.

Comparative assessment of detergent-based protocols for mouse lung de-cellularization and re-cellularization.

Several different detergent-based methods are currently being explored for de-cellularizing whole lungs for subsequent use as three-dimensional scaffolds for ex vivo lung tissue generation. However, it is not yet clear which of these methods may provide a scaffold that best supports re-cellularization and generation of functional lung tissue. Notably, the detergents used for de-cellularization activate matrix metalloproteinases that can potentially degrade extracellular matrix (ECM) proteins important for subsequent binding and growth of cells inoculated into the de-cellularized scaffolds. We assessed gelatinase activation and the histologic appearance, protein composition, and lung mechanics of the end product scaffolds produced with three different detergent-based de-cellularization methods utilizing either Triton-X 100/sodium deoxycholate (Triton/SDC), sodium dodecyl sulfate (SDS), or 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS). There were significant differences both in gelatinase activation and in the retention of ECM and other intracellular proteins, assessed by immunohistochemistry, mass spectrometry, and western blotting as well as in airways resistance and elastance of lungs de-cellularized with the different methods. However, despite these differences, binding and initial growth following intratracheal inoculation with either bone marrow-derived mesenchymal stromal cells or with C10 mouse lung epithelial cells was similar between lungs de-cellularized with each method. Therefore despite differences in the structural composition of the de-cellularized lungs, initial re-cellularization does not appear significantly different between the three de-cellularization approaches studied.

Slam haplotypes modulate the response to lipopolysaccharide in vivo through control of NKT cell number and function.

CD1d-restricted NKT cells make up an innate-like T cell subset that plays a role in amplifying the response of innate immune leukocytes to TLR ligands. The Slam locus contains genes that have been implicated in innate and adaptive immune responses. In this study, we demonstrate that divergent Slam locus haplotypes modulate the response of macrophages to the TLR4 ligand LPS through their control of NKT cell number and function. In response to LPS challenge in vivo, macrophage TNF production in Slam haplotype-2(+) 129S1/SvImJ and 129X1/SvJ mice was significantly impaired in comparison with macrophage TNF production in Slam haplotype-1(+) C57BL/6J mice. Although no cell-intrinsic differences in macrophage responses to LPS were observed between strains, 129 mice were found to be deficient in liver NKT cell number, in NKT cell cytokine production in response to the CD1d ligand alpha-galactosylceramide, and in NKT cell IFN-gamma production after LPS challenge in vivo. Using B6.129c1 congenic mice and adoptive transfer, we found that divergent Slam haplotypes controlled the response to LPS in vivo, as well as the diminished NKT cell number and function, and that these phenotypes were associated with differential expression of signaling lymphocytic activation molecule family receptors on NKT cells. These data suggest that the polymorphisms that distinguish two Slam haplotypes significantly modulate the innate immune response in vivo through their effect on NKT cells.