Viscoelastic Extracellular Matrix Enhances Epigenetic Remodeling and Cellular Plasticity.

Living tissue and extracellular matrices possess viscoelastic properties, but understanding how viscoelastic matrix regulates chromatin and the epigenome is limited. Here, we find that the regulation of the epigenetic state by the viscoelastic matrix is more pronounced on softer matrices. Cells on viscoelastic matrices exhibit larger nuclei, increased nuclear lamina ruffling, loosely organized chromatin, and faster chromatin dynamics, compared to those on elastic matrices. These changes are accompanied by a global increase in euchromatic marks and a local increase in chromatin accessibility at the cis -regulatory elements associated with neuronal and pluripotent genes. Consequently, viscoelastic matrices enhanced the efficiency of reprogramming fibroblasts into neurons and induced pluripotent stem cells, respectively. Together, our findings demonstrate the key roles of matrix viscoelasticity in the regulation of epigenetic state, and uncover a new mechanism of biophysical regulation of chromatin and cell reprogramming, with implications for the design of smart materials to engineer cell fate.

Substrate stiffness modulates the emergence and magnitude of senescence phenotypes.

Cellular senescence is a major driver of aging and disease. Here we show that substrate stiffness modulates the emergence and magnitude of senescence phenotypes post induction. Using a primary dermal fibroblast model of senescence, we show that decreased substrate stiffness accelerates cell-cycle arrest during senescence development and regulate expression of conventional protein-based biomarkers of senescence. We found that the expression of these senescence biomarkers, namely p21 WAF1/CIP1 ( CDKN1a ) and p16 INK4a ( CDKN2a ) are mechanosensitive and are in-part regulated by myosin contractility through focal adhesion kinase (FAK)-ROCK signaling. Interestingly, at the protein level senescence-induced dermal fibroblasts on soft substrates (0.5 kPa) do not express p21 WAF1/CIP1 and p16 INK4a at comparable levels to induced cells on stiff substrates (4GPa). However, cells do express CDKN1a, CDKN2a, and IL6 at the RNA level across both stiff and soft substrates. When cells were transferred from soft to stiff substrates, senescent cells recover an elevated expression expressing p21 WAF1/CIP1 and p16 INK4a at levels comparable to senescence cells on stiff substrates, pointing to a mechanosensitive regulation of the senescence phenotypes. Together, our results indicate that the induction of senescence programs depends critically on the mechanical environments of cells and that senescent cells actively respond and adapt to changing mechanical cues.

HIF2A gain-of-function mutation modulates the stiffness of smooth muscle cells and compromises vascular mechanics.

Heterozygous gain-of-function (GOF) mutations of hypoxia-inducible factor 2α (HIF2A), a key hypoxia-sensing regulator, are associated with erythrocytosis, thrombosis, and vascular complications that account for morbidity and mortality of patients. We demonstrated that the vascular pathology of HIF2A GOF mutations is independent of erythrocytosis. We generated HIF2A GOF-induced pluripotent stem cells (iPSCs) and differentiated them into endothelial cells (ECs) and smooth muscle cells (SMCs). Unexpectedly, HIF2A-SMCs, but not HIF2A-ECs, were phenotypically aberrant, more contractile, stiffer, and overexpressed endothelin 1 (EDN1), myosin heavy chain, elastin, and fibrillin. EDN1 inhibition and knockdown of EDN1-receptors both reduced HIF2-SMC stiffness. Hif2A GOF heterozygous mice displayed pulmonary hypertension, had SMCs with more disorganized stress fibers and higher stiffness in their pulmonary arterial smooth muscle cells, and had more deformable pulmonary arteries compared with wild-type mice. Our findings suggest that targeting these vascular aberrations could benefit patients with HIF2A GOF and conditions of augmented hypoxia signaling.

Biomaterials as vectors for the delivery of CRISPR-Cas9.

The emergence of the CRISPR-Cas9 gene editing system has brought much hope and excitement to the field of gene therapy and the larger scientific community. However, in order for CRISPR-based therapies to be translated to the clinical setting, there is an urgent need to develop optimized vectors for their delivery. The delivery vector is a crucial determinant of the therapeutic efficacy of gene editing and should be designed to accommodate various factors including the form of the payload, the physiological environment, and the potential immune responses. Recently, biomaterials have become an attractive option for the delivery of Cas9 due to their tunability, biocompatibility and increasing efficacy at drug delivery. Biomaterials offer a unique solution for creating tailored vectors to meet the demands of various applications that cannot be easily met by other delivery methods. In this review, we will discuss the various biomaterial systems that have been used to deliver Cas9 in its plasmid, mRNA and protein forms. In addition, the functions of these materials will be reviewed to understand their roles in Cas9 delivery. Finally, the immune challenges associated with Cas9 and the delivery materials will be discussed as an understanding of the immune responses along with the functions of biomaterials will ultimately guide the field in designing new delivery systems for the clinical applications of CRISPR-Cas9.

Enhanced elastin synthesis and maturation in human vascular smooth muscle tissue derived from induced-pluripotent stem cells.

Obtaining vascular smooth muscle tissue with mature, functional elastic fibers is a key obstacle in tissue-engineered blood vessels. Poor elastin secretion and organization leads to a loss of specialization in contractile smooth muscle cells, resulting in over proliferation and graft failure. In this study, human induced-pluripotent stem cells (hiPSCs) were differentiated into early smooth muscle cells, seeded onto a hybrid poly(ethylene glycol) dimethacrylate/poly (l-lactide) (PEGdma-PLA) scaffold and cultured in a bioreactor while exposed to pulsatile flow, towards maturation into contractile smooth muscle tissue. We evaluated the effects of pulsatile flow on cellular organization as well as elastin expression and assembly in the engineered tissue compared to a static control through immunohistochemistry, gene expression and functionality assays. We show that culturing under pulsatile flow resulted in organized and functional hiPSC derived smooth muscle tissue. Immunohistochemistry analysis revealed hiPSC-smooth muscle tissue with robust, well-organized cells and elastic fibers and the supporting microfibril proteins necessary for elastic fiber assembly. Through qRT-PCR analysis, we found significantly increased expression of elastin, fibronectin, and collagen I, indicating the synthesis of necessary extracellular matrix components. Functionality assays revealed that hiPSC-smooth muscle tissue cultured in the bioreactor had an increased calcium signaling and contraction in response to a cholinergic agonist, significantly higher mature elastin content and improved mechanical properties in comparison to the static control. The findings presented here detail an effective approach to engineering elastic human vascular smooth muscle tissue with the functionality necessary for tissue engineering and regenerative medicine applications. STATEMENT OF SIGNIFICANCE: Obtaining robust, mature elastic fibers is a key obstacle in tissue-engineered blood vessels. Human induced-pluripotent stem cells have become of interest due to their ability to supplement tissue engineered scaffolds. Their ability to differentiate into cells of vascular lineages with defined phenotypes serves as a potential solution to a major cause of graft failure in which phenotypic shifts in smooth muscle cells lead to over proliferation and occlusion of the graft. Herein, we have differentiated human induced-pluripotent stem cells in a pulsatile flow bioreactor, resulting in vascular smooth muscle tissue with robust elastic fibers and enhanced functionality. This study highlights an effective approach to engineering elastic functional vascular smooth muscle tissue for tissue engineering and regenerative medicine applications.

Fabrication of 3-dimensional multicellular microvascular structures.

Despite current advances in engineering blood vessels over 1 mm in diameter and the existing wealth of knowledge regarding capillary bed formation, studies for the development of microvasculature, the connecting bridge between them, have been extremely limited so far. Here, we evaluate the use of 3-dimensional (3D) microfibers fabricated by hydrogel electrospinning as templates for microvascular structure formation. We hypothesize that 3D microfibers improve extracellular matrix (ECM) deposition from vascular cells, enabling the formation of freestanding luminal multicellular microvasculature. Compared to 2-dimensional cultures, we demonstrate with confocal microscopy and RT-PCR that fibrin microfibers induce an increased ECM protein deposition by vascular cells, specifically endothelial colony-forming cells, pericytes, and vascular smooth muscle cells. These ECM proteins comprise different layers of the vascular wall including collagen types I, III, and IV, as well as elastin, fibronectin, and laminin. We further demonstrate the achievement of multicellular microvascular structures with an organized endothelium and a robust multicellular perivascular tunica media. This, along with the increased ECM deposition, allowed for the creation of self-supporting multilayered microvasculature with a distinct circular lumen following fibrin microfiber core removal. This approach presents an advancement toward the development of human microvasculature for basic and translational studies.

Comparative analysis of in vitro oxidative degradation of poly(carbonate urethanes) for biostability screening.

The resistance to oxidation and environmental stress cracking of poly(carbonate urethanes) (PCUs) has generated significant interest as potential replacements of poly(ether urethanes) in medical devices. Several in vitro models have been developed to screen segmented polyurethanes for oxidative stability. High concentrations of reactive oxygen intermediates produced by combining hydrogen peroxide and dissolved cobalt ions has frequently been used to predict long-term oxidative degradation with short-term testing. Alternatively, a 3% H2O2 concentration without metal ions is suggested within the ISO 10993-13 standard to simulate physiological degradation rates. A comparative analysis which evaluates the predictive capabilities of each test method has yet to be completed. To this end, we have utilized both systems to test three commercially available PCUs with low and high soft segment content: Bionate PCU and Bionate II PCUs, two materials with different soft segment chemistries, and CarboSil TSPCU, a thermoplastic silicone PCU. Bulk properties of all PCUs were retained with minor changes in molecular weight and tensile properties indicating surface oxidative degradation in the accelerated system after 36 days. Soft segment loss and surface damage were comparable to previous in vivo data. The 3% H2O2 method exhibited virtually no changes on the surface or in bulk properties after 12 months of treatment despite previous in vivo results. These results indicate the accelerated test method more effectively characterized the oxidative degradation profiles than the 3% H2O2 treatment system. The lack of bulk degradation in the 12-month study also supports the hydrolytic stability of these PCUs.