Non-invasive and real-time measurement of microvascular barrier in intact lungs.

Microvascular leak is a phenomenon witnessed in multiple disease states. In organ engineering, regaining a functional barrier is the most crucial step towards creating an implantable organ. All previous methods of measuring microvascular permeability were either invasive, lengthy, introduced exogenous macromolecules, or relied on extrapolations from cultured cells. We present here a system that enables real-time measurement of microvascular permeability in intact rat lungs. Our unique system design allows direct, non-invasive measurement of average alveolar and capillary pressures, tracks flow paths within the organ, and enables calculation of lumped internal resistances including microvascular barrier. We first describe the physiology of native and decellularized lungs and the inherent properties of the extracellular matrix as functions of perfusion rate. We next track changing internal resistances and flows in injured native rat lungs, resolving the onset of microvascular leak, quantifying changing vascular resistances, and identifying distinct phases of organ failure. Finally, we measure changes in permeability within engineered lungs seeded with microvascular endothelial cells, quantifying cellular effects on internal vascular and barrier resistances over time. This system marks considerable progress in bioreactor design for intact organs and may be used to monitor and garner physiological insights into native, decellularized, and engineered tissues.

Bioengineered lungs generated from human iPSCs-derived epithelial cells on native extracellular matrix.

The development of an alternative source for donor lungs would change the paradigm of lung transplantation. Recent studies have demonstrated the potential feasibility of using decellularized lungs as scaffolds for lung tissue regeneration and subsequent implantation. However, finding a reliable cell source and the ability to scale up for recellularization of the lung scaffold still remain significant challenges. To explore the possibility of regeneration of human lung tissue from stem cells in vitro, populations of lung progenitor cells were generated from human iPSCs. To explore the feasibility of producing engineered lungs from stem cells, we repopulated decellularized human lung and rat lungs with iPSC-derived epithelial progenitor cells. The iPSCs-derived epithelial progenitor cells lined the decellularized human lung and expressed most of the epithelial markers when were cultured in a lung bioreactor system. In decellularized rat lungs, these human-derived cells attach and proliferate in a manner similar to what was observed in the decellularized human lung. Our results suggest that repopulation of lung matrix with iPSC-derived lung epithelial cells may be a viable strategy for human lung regeneration and represents an important early step toward translation of this technology.

Controlled gas exchange in whole lung bioreactors.

In cellular, tissue-level or whole organ bioreactors, the level of dissolved oxygen is one of the most important factors requiring control. Hypoxic environments may lead to cellular apoptosis, while hyperoxic environments may lead to cellular damage or dedifferentiation, both resulting in loss of overall tissue function. This manuscript describes the creation, characterization and validation of a bioreactor system that can control oxygen delivery based on real-time metabolic demand of cultured whole lung tissue. A mathematical model describing and predicting gas exchange within the tunable bioreactor system is developed. In addition, the inherent gas exchange properties of the bioreactor and the inherent oxygen consumption rates of native rat lungs are determined, thereby providing a quantitative relationship between system parameters and levels of dissolved oxygen. Finally, the mathematical model is validated during whole lung culture under a range of system parameters. The system presented here provides a quantitative relationship between the concentration of dissolved oxygen, tissue oxygen consumption rates, and controllable system parameters that introduce gasses into the bioreactor. This relationship not only enables the maintenance of constant levels of dissolved oxygen throughout a culture period during which cells are replicating, but also provides noninvasive and real-time estimation of the metabolic and proliferative states of native or engineered lung tissue simply through dissolved oxygen measurements. Copyright © 2017 John Wiley & Sons, Ltd.

Efficient and Functional Endothelial Repopulation of Whole Lung Organ Scaffolds.

To date, efforts to generate engineered lung tissue capable of long-term function have been limited by incomplete barrier formation between air and blood and by thrombosis of the microvasculature upon exposure of blood to the collagens within the decellularized scaffold. Improved barrier function and resistance to thrombosis both depend upon the recapitulation of a confluent monolayer of functional endothelium throughout the pulmonary vasculature. This manuscript describes novel strategies to increase cell coverage of the vascular surface area, compared to previous reports in our lab and others, and reports robust production of multiple anticoagulant substances that will be key to long-term function in vivo once additional strides are made in improving barrier function. Rat lung microvascular endothelial cells were seeded into decellularized rat lungs by both the pulmonary artery and veins with the use of low-concentration cell suspensions, pulsatile, gravity-driven flow, and supraphysiological vascular pressures. Together, these strategies yielded 72.44 ± 10.52% endothelial cell nuclear coverage of the acellular matrix after 3-4 d of biomimetic bioreactor culture compared to that of the native rat lung. Immunofluorescence, Western blot, and PCR analysis of these lungs indicated robust expression of phenotypic markers such as CD31 and VE-Cadherin after time in culture. Endothelial-seeded lungs had CD31 gene expression of 0.074 ± 0.015 vs 0.021 ± 0.0023 for native lungs, p = 0.025, and VE-Cadherin gene expression of 0.93 ± 0.22 compared to that of the native lung at 0.13 ± 0.02, p = 0.023. Precursors to antithrombotic substances such as tissue plasminogen activator, prostacyclin synthase, and endothelial nitric oxide synthase were expressed at levels equal to or greater than those of the native lung. Engineered lungs reseeded with endothelial cells were implanted orthotopically and contained patent microvascular networks that had gas exchange function during mechanical ventilation on 100% O2 greater than that of decelluarized lungs. Taken together, these data suggest that these engineered constructs could be compatible with long-term function in vivo when utilized in future studies in tandem with improved barrier function.

Targeted proteomics effectively quantifies differences between native lung and detergent-decellularized lung extracellular matrices.

Extracellular matrix is a key component of many products in regenerative medicine. Multiple regenerative medicine products currently in the clinic are comprised of human or xenogeneic extracellular matrix. In addition, whole-organ regeneration exploits decellularized native organs as scaffolds for organotypic cell culture. However, precise understanding of the constituents of such extracellular matrix-based implants and scaffolds has sorely lagged behind their use. We present here an advanced protein extraction method using known quantities of proteotypic 13C-labeled peptides to quantify matrix proteins in native and decellularized lung tissues. Using quantitative proteomics that produce picomole-level measurements of a large number of matrix proteins, we show that a mild decellularization technique ("Triton/SDC") results in near-native retention of laminins, proteoglycans, and other basement membrane and ECM-associated proteins. Retention of these biologically important glycoproteins and proteoglycans is quantified to be up to 27-fold higher in gently-decellularized lung scaffolds compared to scaffolds generated using a previously published decellularization regimen. Cells seeded onto this new decellularized matrix also proliferate robustly, showing positive staining for proliferating cell nuclear antigen (PCNA). The high fidelity of the gently decellularized scaffold as compared to the original lung extracellular matrix represents an important step forward in the ultimate recapitulation of whole organs using tissue-engineering techniques. This method of ECM and scaffold protein analysis allows for better understanding, and ultimately quality control, of matrices that are used for tissue engineering and human implantation. These results should advance regenerative medicine in general, and whole organ regeneration in particular. STATEMENT OF SIGNIFICANCE: The extracellular matrix (ECM) in large part defines the biochemical and mechanical properties of tissues and organs; these inherent cues make acellular ECM scaffolds potent substrates for tissue regeneration. As such, they are increasingly prevalent in the clinic and the laboratory. However, the exact composition of these scaffolds has been difficult to ascertain. This paper uses targeted proteomics to definitively quantify 71 proteins present in acellular lung ECM scaffolds. We use this technique to compare two decellularization methods and demonstrate superior retention of ECM proteins important for cell adhesion, migration, proliferation, and differentiation in scaffolds treated with low-concentration detergent solutions. In the long term, the ability to acquire quantitative biochemical data about biological substrates will facilitate the rational design of engineered tissues and organs based on precise cell-matrix interactions.

Engineered Tissue-Stent Biocomposites as Tracheal Replacements.

Here we report the creation of a novel tracheal construct in the form of an engineered, acellular tissue-stent biocomposite trachea (TSBT). Allogeneic or xenogeneic smooth muscle cells are cultured on polyglycolic acid polymer-metal stent scaffold leading to the formation of a tissue comprising cells, their deposited collagenous matrix, and the stent material. Thorough decellularization then produces a final acellular tubular construct. Engineered TSBTs were tested as end-to-end tracheal replacements in 11 rats and 3 nonhuman primates. Over a period of 8 weeks, no instances of airway perforation, infection, stent migration, or erosion were observed. Histological analyses reveal that the patent implants remodel adaptively with native host cells, including formation of connective tissue in the tracheal wall and formation of a confluent, columnar epithelium in the graft lumen, although some instances of airway stenosis were observed. Overall, TSBTs resisted collapse and compression that often limit the function of other decellularized tracheal replacements, and additionally do not require any cells from the intended recipient. Such engineered TSBTs represent a model for future efforts in tracheal regeneration.

Comparative biology of decellularized lung matrix: Implications of species mismatch in regenerative medicine.

Lung engineering is a promising technology, relying on re-seeding of either human or xenographic decellularized matrices with patient-derived pulmonary cells. Little is known about the species-specificity of decellularization in various models of lung regeneration, or if species dependent cell-matrix interactions exist within these systems. Therefore decellularized scaffolds were produced from rat, pig, primate and human lungs, and assessed by measuring residual DNA, mechanical properties, and key matrix proteins (collagen, elastin, glycosaminoglycans). To study intrinsic matrix biologic cues, human endothelial cells were seeded onto acellular slices and analyzed for markers of cell health and inflammation. Despite similar levels of collagen after decellularization, human and primate lungs were stiffer, contained more elastin, and retained fewer glycosaminoglycans than pig or rat lung scaffolds. Human endothelial cells seeded onto human and primate lung tissue demonstrated less expression of vascular cell adhesion molecule and activation of nuclear factor-κB compared to those seeded onto rodent or porcine tissue. Adhesion of endothelial cells was markedly enhanced on human and primate tissues. Our work suggests that species-dependent biologic cues intrinsic to lung extracellular matrix could have profound effects on attempts at lung regeneration.

Analysis of AKAP7γ Dimerization.

A-kinase anchoring proteins (AKAPs) constitute a family of scaffolding proteins that contribute to spatiotemporal regulation of PKA-mediated phosphorylation events. In particular, AKAP7 is a family of alternatively spliced proteins that participates in cardiac calcium dynamics. Here, we demonstrate via pull-down from transfected cells and by direct protein-protein association that AKAP7γ self-associates. Self-association appears to be an isoform specific phenomenon, as AKAP7α did not associate with itself or with AKAP7γ. However, AKAP7γ did associate with AKAP7δ, suggesting the long isoforms of the AKAP can form heterodimers. Surface plasmon resonance found that the AKAP7γ self-association occurs via two high affinity binding sites with K D values in the low nanomolar range. Mapping of the binding sites by peptide array reveals that AKAP7γ interacts with itself through multiple regions. Photon counting histogram analysis (PCH) of AKAP7γ-EGFP expressed in HEK-293 cells confirmed that AKAP7γ-EGFP self-associates in a cellular context. Lastly, computational modeling of PKA dynamics within AKAP7γ complexes suggests that oligomerization may augment phosphorylation of scaffolded PKA substrates. In conclusion, our study reveals that AKAP7γ forms both homo- and heterodimers with the long isoforms of the AKAP and that this phenomenon could be an important step in mediating effective substrate phosphorylation in cellular microdomains.

Phosphorylation state-dependent interaction between AKAP7δ/γ and phospholamban increases phospholamban phosphorylation.

Changes in heart rate and contractility in response to sympathetic stimulation occur via activation of cAMP dependent protein kinase A (PKA), leading to phosphorylation of numerous substrates that alter Ca(2+) cycling. Phosphorylation of these substrates is coordinated by A-kinase anchoring proteins (AKAPs), which recruit PKA to specific substrates [1]. Phosphorylation of the PKA substrate phospholamban (PLB) is a critical determinant of Ca(2+) re-entry into the sarcoplasmic reticulum and is coordinated by AKAP7δ/γ [2,3]. Here, we further these findings by showing that phosphorylation of PLB requires interaction with AKAP7δ/γ and that this interaction occurs only when PLB is unphosphorylated. Additionally, we find that two mutants of PLB (R9C and Δ14), which are associated with dilated cardiomyopathy in humans, prevent association with AKAP7δ/γ and display reduced phosphorylation in vitro. This finding implicates the AKAP7δ/γ-PLB interaction in the pathology of the disease phenotype. Further exploration of the AKAP7δ/γ-PLB association demonstrated a phosphorylation state-dependence of the interaction. Computational modeling revealed that this mode of interaction allows for small amounts of AKAP and PKA (100-200nM) to regulate the phosphorylation of large quantities of PLB (50µM). Our results confirm that AKAP7γ/δ binding to PLB is important for phosphorylation of PLB, and describe a novel phosphorylation state-dependent binding mechanism that explains how phosphorylation of highly abundant PKA substrates can be regulated by AKAPs present at ~100-200 fold lower concentrations.

Arterial specification of endothelial cells derived from human induced pluripotent stem cells in a biomimetic flow bioreactor.

Endothelial cells (ECs) exist in different microenvironments in vivo, including under different levels of shear stress in arteries versus veins. Standard stem cell differentiation protocols to derive ECs and EC-subtypes from human induced pluripotent stem cells (hiPSCs) generally use growth factors or other soluble factors in an effort to specify cell fate. In this study, a biomimetic flow bioreactor was used to subject hiPSC-derived ECs (hiPSC-ECs) to shear stress to determine the impacts on phenotype and upregulation of markers associated with an anti-thrombotic, anti-inflammatory, arterial-like phenotype. The in vitro bioreactor system was able to efficiently mature hiPSC-ECs into arterial-like cells in 24 h, as demonstrated by qRT-PCR for arterial markers EphrinB2, CXCR4, Conexin40 and Notch1, as well protein-level expression of Notch1 intracellular domain (NICD). Furthermore, the exogenous addition of soluble factors was not able to fully recapitulate this phenotype that was imparted by shear stress exposure. The induction of these phenotypic changes was biomechanically mediated in the shear stress bioreactor. This biomimetic flow bioreactor is an effective means for the differentiation of hiPSC-ECs toward an arterial-like phenotype, and is amenable to scale-up for culturing large quantities of cells for tissue engineering applications.

Osteogenic performance of donor-matched human adipose and bone marrow mesenchymal cells under dynamic culture.

Adipose-derived mesenchymal cells (ACs) and bone marrow-derived mesenchymal cells (BMCs) have been widely used for bone regeneration and can be seeded on a variety of rigid scaffolds. However, to date, a direct comparison of mesenchymal cells (MC) harvested from different tissues from the same donor and cultured in identical osteogenic conditions has not been investigated. Indeed, it is unclear whether marrow-derived or fat-derived MC possess intrinsic differences in bone-forming capabilities, since within-patient comparisons have not been previously done. This study aims at comparing ACs and BMCs from three donors ranging in age from neonatal to adult. Matched cells from each donor were studied in three distinct bioreactor settings, to determine the best method to create a viable osseous engineered construct. Human ACs and BMCs were isolated from each donor, cultured, and seeded on decellularized porcine bone (DCB) constructs. The constructs were then subjected to either static or dynamic (stirring or perfusion) bioreactor culture conditions for 7-21 days. Afterward, the constructs were analyzed for cell adhesion and distribution and osteogenic differentiation. ACs demonstrated higher seeding efficiency than BMCs. However, static and dynamic culture significantly increased BMCs proliferation more than ACs. In all conditions, BMCs demonstrated stronger osteogenic activity as compared with ACs, through higher alkaline phosphatase activity and gene expression for various bony markers. Conversely, ACs expressed more collagen I, which is a nonspecific matrix molecule in most connective tissues. Overall, dynamic bioreactor culture conditions enhanced osteogenic gene expression in both ACs and BMCs. Scaffolds seeded with BMCs in dynamic stirring culture conditions exhibit the greatest osteogenic proliferation and function in vitro, proving that marrow-derived MC have superior bone-forming potential as compared with adipose-derived cells.

Spatiotemporal regulation of PKC via interactions with AKAP7 isoforms.

The regulation of kinases by scaffolding proteins greatly contributes to the fidelity of signal transduction. In the present study, we explored an interaction between the ubiquitous enzyme PKC (protein kinase C) and the scaffolding protein AKAP7 (A-kinase-anchoring protein 7). Using protein biochemistry and surface plasmon resonance approaches, we demonstrate that both AKAP7γ and AKAP7α are capable of high-affinity interactions with multiple isoenzymes of PKC. Furthermore, this interaction is achieved via multi-site binding on both proteins. FRET (fluorescence resonance energy transfer) analysis using a PKC activity reporter suggests that anchoring of the kinase within AKAP7 complexes enhances the phosphorylation of substrate proteins. Finally, we determined using FRAP (fluorescence recovery after photobleaching) and virtual modelling that AKAP7 restricts the mobility of PKC within cells by tethering it to subcellular compartments. Collectively, the results of the present study suggests that AKAP7 could play an integral role in dictating PKC localization and function in tissues where the two proteins are co-expressed.

Identification of AKAP79 as a protein phosphatase 1 catalytic binding protein.

The ubiquitously expressed and highly promiscuous protein phosphatase 1 (PP1) regulates many cellular processes. Targeting PP1 to specific locations within the cell allows for the regulation of PP1 by conferring substrate specificity. In the present study, we identified AKAP79 as a novel PP1 regulatory subunit. Immunoprecipitaiton of the AKAP from rat brain extract found that the PP1 catalytic subunit copurified with the anchoring protein. This is a direct interaction, demonstrated by pulldown experiments using purified proteins. Interestingly, the addition of AKAP79 to purified PP1 catalytic subunit decreased phosphatase activity with an IC(50) of 811 ± 0.56 nM of the anchoring protein. Analysis of AKAP79 identified a PP1 binding site that conformed to a consensus PP1 binding motif (FxxR/KxR/K) in the first 44 amino acids of the anchoring protein. This was confirmed when a peptide mimicking this region of AKAP79 was able to bind PP1 by both pulldown assay and surface plasmon resonance. However, PP1 was still able to bind to AKAP79 upon deletion of this region, suggesting additional sites of contact between the anchoring protein and the phosphatase. Importantly, this consensus PP1 binding motif was found not to be responsible for PP1 inhibition, but rather enhanced phosphatase activity, as deletion of this domain resulted in an increased inhibition of PP1 activity. Instead, a second interaction domain localized to residues 150-250 of AKAP79 was required for the inhibition of PP1. However, the inhibitory actions of AKAP79 on PP1 are substrate dependent, as the anchoring protein did not inhibit PP1 dephosphorylation of phospho-PSD-95, a substrate found in AKAP79 complexes in the brain. These combined observations suggest that AKAP79 acts as a PP1 regulatory subunit that can direct PP1 activity toward specific targets in the AKAP79 complex.