These results demonstrate that adult marrow cells can be recruited to airway epithelium and induced to express Cftr in mice otherwise lacking this protein. However, the number of observed chimeric epithelial cells is small and new strategies for enhancing airway epithelial remodeling by adult bone marrow-derived cells will be necessary for correction of defective CFTR-dependent chloride transport.

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.

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.

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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.

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. STEM CELLS TRANSLATIONAL MEDICINE 2014;3:194-205

We have previously demonstrated that hepatocyte proliferation induced by the mitogen 1,4-bis[2-(3,5-dichloropyridyloxy)] benzene (TCPOBOP) is independent of changes in cytokines, immediate early genes, and transcription factors that are considered to be necessary for regeneration of the liver after partial hepatectomy (PH) or necrosis. To further investigate the differences between mitogen-induced mouse hepatocyte proliferation and liver regeneration after PH, we have measured the expression of cyclin D1, cyclin D3, cyclin E, and cyclin A and of the cyclin-dependent kinases CDK2, CDK4, and CDK6. The involvement of the cyclin-dependent kinase inhibitors p21 and p27 and of the oncosuppressor gene p53 was also examined at different times after stimulation of hepatocyte proliferation. Results showed that a single administration of TCPOBOP caused a very rapid increase in the levels of cyclin D1, a G1 protein, when compared with two thirds PH (8 hours versus 30 hours). The early increase in cyclin D1 protein levels was associated with a faster onset of increased expression of S-phase-associated cyclin A (24 hours versus 36 hours with PH mice). Accordingly, measurement of bromodeoxyuridine (BrdU) incorporation revealed that, although approximately 8% of hepatocytes were BrdU-positive as early as 24 hours after TCPOBOP, no significant changes in BrdU incorporation were observed at the same time point after two thirds PH. The expression of other proteins involved in cell cycle control, such as cyclin-dependent kinases (CDK4, CDK2, CDK6), was also analyzed. Results showed that expression of CDK2 was induced much more rapidly in TCPOBOP-treated mice (2 hours) than in mice subjected to PH (36 hours). A different pattern of expression in the two models of hepatocyte proliferation, although less dramatic, was also observed for CDK4 and CDK6. Expression of the CDK inhibitors p21 and p27 and the oncosuppressor gene p53 variably increased after two thirds PH, whereas basically no change in protein levels was found in TCPOBOP-treated mice. The results demonstrate that profound differences in many cell cycle-regulatory proteins exist between direct hyperplasia and compensatory regeneration. Cyclin D1 induction is one of the earlier events in hepatocyte proliferation induced by the primary mitogen TCPOBOP and suggests that a direct effect of the mitogen on this cyclin may be responsible for the rapid onset of DNA synthesis observed in TCPOBOP-induced hyperplasia.