Respiratory disease is the third leading cause of death in the industrialized world. Consequently, the trachea, lungs, and cardiopulmonary vasculature have been the focus of extensive investigations. Recent studies have provided new information about the mechanisms driving lung development and differentiation. However, there is still much to learn about the ability of the adult respiratory system to undergo repair and to replace cells lost in response to injury and disease. This review highlights the multiple stem/progenitor populations in different regions of the adult lung, the plasticity of their behavior in injury models, and molecular pathways that support homeostasis and repair.

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 hours, 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.

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.

Rationale Cardiac myocyte hypertrophy is the main compensatory response to chronic stress on the heart. p90 Ribosomal S6 Kinase (RSK) family members are effectors for extracellular signal-regulated kinases that induce myocyte growth. Although increased RSK activity has been observed in stressed myocytes, the functions of individual RSK family members have remained poorly defined, despite being potential therapeutic targets for cardiac disease. Objective To demonstrate that type 3 RSK (RSK3) is required for cardiac myocyte hypertrophy. Methods and Results RSK3 contains a unique N-terminal domain that is not conserved in other RSK family members. We show that this domain mediates the regulated binding of RSK3 to the muscle A-kinase anchoring protein (mAKAP) scaffold, defining a novel kinase anchoring event. Disruption of both RSK3 expression using RNA interference and RSK3 anchoring using a competing mAKAP peptide inhibited the hypertrophy of cultured myocytes. In vivo, RSK3 gene deletion in the mouse attenuated the concentric myocyte hypertrophy induced by pressure overload and catecholamine infusion. Conclusions Taken together, these data demonstrate that anchored RSK3 transduces signals that modulate pathologic myocyte growth. Targeting of signaling complexes that contain select kinase isoforms should provide an approach for the specific inhibition of cardiac myocyte hypertrophy and for the development of novel strategies for the prevention and treatment of heart failure.