Acellular Myocardial Scaffolds and Slices Fabrication, and Method for Applying Mechanical and Electrical Simulation to Tissue Construct.

Cardiac tissue engineering/regeneration using decellularized myocardium has attracted great research attention due to its potential benefit to myocardial infarction (MI) treatment. Here, we described an optimal decellularization protocol to generate 3D porcine myocardial scaffolds with well-preserved cardiomyocyte lacunae, myocardial slices as a biomimetic cell culture and delivery platform, and a multi-stimulation bioreactor that is able to provide coordinated mechanical and electrical stimulations for facilitating cardiac construct development.

Iris stromal cell nuclei deform to more elongated shapes during pharmacologically-induced miosis and mydriasis.

Nuclear shape alteration in ocular tissues, which can be used as a metric for overall cell deformation, may also lead to changes in gene expression and protein synthesis that could affect the biomechanics of the tissue extracellular matrix. The biomechanics of iris tissue is of particular interest in the study of primary angle-closure glaucoma. As the first step towards understanding the mutual role of the biomechanics and deformation of the iris on the activity of its constituent stromal cells, we conducted an ex-vivo study in freshly excised porcine eyes. Iris deformation was achieved by activating the constituent smooth muscles of the iris. Pupillary responses were initiated by inducing miosis and mydriasis, and the irides were placed in a fixative, bisected, and sliced into thin sections in a nasal and temporal horizontal orientation. The tissue sections were stained with DAPI for nucleus, and z-stacks were acquired using confocal microscopy. Images were analyzed to determine the nuclear aspect ratio (NAR) using both three-dimensional (3D) reconstructions of the nuclear surfaces as well as projections of the same 3D reconstruction into flat two-dimensional (2D) shapes. We observed that regardless of the calculation method (i.e., one that employed 3D surface reconstructions versus one that employed 2D projected images) the NAR increased in both the miosis group and the mydriasis group. Three-dimensional quantifications showed that NAR increased from 2.52 ± 0.96 in control group to 2.80 ± 0.81 and 2.74 ± 0.94 in the mydriasis and miosis groups, respectively. Notwithstanding the relative convenience in calculating the NAR using the 2D projected images, the 3D reconstructions were found to generate more physiologically realistic values and, thus, can be used in the development of future computational models to study primary angle-closure glaucoma. Since the iris undergoes large deformations in response to ambient light, this study suggests that the iris stromal cells are subjected to a biomechanically active micro-environment during their in-vivo physiological function.

Preseeding of Mesenchymal Stem Cells Increases Integration of an iPSC-Derived CM Sheet into a Cardiac Matrix.

Cell sheet technology has demonstrated great promise in delivering a large amount of therapeutic cells for tissue repair, including in the myocardium. However, the lack of host integration remains one of the key challenges in using cell sheets for cardiac repair. Paracrine factors secreted by mesenchymal stem cells (MSCs) have been reported to facilitate tissue repair and regeneration in a variety of ways. It has been demonstrated that paracrine factors from MSCs could enhance scaffold recellularization and vascularization. In this study, we used an in vitro cardiac matrix mimic platform to examine the effects of hMSCs preseeding on the interactions between cell sheets and cardiac matrix. The fabricated human induced pluripotent stem cells-derived cardiomyocyte sheets were attached to a decellularized porcine myocardium slice with or without preseeding of hMSCs. The hMSCs preseeding significantly enhanced the interactions between cardiomyocyte sheets and cardiac matrix in terms of cell migration distance, cell distribution, and mature vascular and cardiomyocyte marker expressions in the matrix. Growth factor and matrix metalloproteinases array analysis suggested that hMSCs- induced vascularization and MMPs regulation are the two possible mechanisms that lead to the improved CMs and cardiac matrix interactions. Further examination of these two mechanisms will enable the development of new approaches to facilitate transplanted cells for tissue repair.

In Vivo Assessment of Decellularized Porcine Myocardial Slice as an Acellular Cardiac Patch.

Acellular cardiac patches made of various biomaterials have shown to improve heart function after myocardial infarction (MI). Extracellular matrix scaffold derived from a decellularized tissue has unique advantages to serve as an acellular cardiac patch due to its biomimetic nature. In this study, we examined the therapeutic outcomes of using a decellularized porcine myocardium slice (dPMS) as an acellular patch in a rat acute MI model. dPMSs with two different thicknesses (300 and 600 µm) were patched to the infarcted area of the rat myocardium, and their effects on cardiac function and host interactions were assessed. We found that the implanted dPMS firmly attached to host myocardium after implantation and prevented thinning of the left ventricular (LV) wall after an MI. A large number of host cells were identified to infiltrate into the implanted dPMS, and a significant number of vessel structures was observed in the dPMS and infarcted area. We detected a significantly higher density of M2 macrophages in the groups treated with dPMSs as compared to the MI group. Contraction of the LV wall and cardiac functional parameters (left ventricular ejection fraction and fractional shortening) was significantly improved in the treatment groups (300 and 600 µm dPMS) 4 weeks after surgery. Our results proved the therapeutic benefits of using dPMS as an acellular cardiac patch for the treatment of acute myocardial infarction.

A Thin Layer of Decellularized Porcine Myocardium for Cell Delivery.

Decellularized porcine myocardium has shown many benefits as a cell delivery scaffold for cardiac therapy. However, using full thickness decellularized myocardium as cardiac patch may lead to poor viability and inhomogeneous distribution of delivered cells, due to perfusion limitations. In this study, we explored the feasibility of decellularized porcine myocardial slice (dPMS) to construct a vascularized cardiac patch for cell delivery. Decellularized porcine myocardium was sliced into thin layers (thickness~300 µm). Adipose-derived Stem cells (ASCs) obtained from rat and pig were seeded on dPMS. The viability, infiltration, and differentiation of seeded ASCs were examined. The mechanical properties of dPMSs of various thickness and native myocardium were tested. We noticed dPMS supported attachment and growth of rat and pig ASCs. Both rat and pig ASCs showed high viability, similar patterns of proliferation and infiltration within dPMS. Rat ASCs showed expression of early-endothelial markers followed by mature-endothelial marker without any additional inducers on dPMS. Using rat myocardial infarction model, we delivered ASCs using dPMS patched to the infarcted myocardium. After 1 week, a higher number of transplanted cells were present in the infarcted area when cells were delivered using dPMS versus direct injection. Compared with MI group, increased vascular formation was also observed.

Recurring Amplification at 11q22.1-q22.2 Locus Plays an Important Role in Lymph Node Metastasis and Radioresistance in OSCC.

A key feature in the pathogenesis of OSCC is genetic instability, which results in altered expression of genes located in amplified/deleted chromosomal regions. In a previous study we have shown that the amplification of the 11q22.1-q22.2 region, encoding cIAP1 and cIAP2, is associated with lymph node metastasis and poor clinical outcome in OSCC. Here, we validate the aCGH results by nuc ish and detect a weak amplification at the 11q22.1-q22.2 locus in 37% of the 182 samples tested. We find positive correlation of 11q22.1-q22.2 amplification with lymph node metastasis, reduced survival, and increased cancer recurrence, and we observe that patients with 11q22.1-q22.2 amplification fail to respond to radiotherapy. We confirm the concurrent overexpression of cIAP1 and cIAP2 and observe differential subcellular localization of the two proteins in OSCC. To ascertain the roles of cIAP1/cIAP2 in lymph node metastasis and radioresistance, we use an in vitro pre-clinical model and confirm the role of cIAP1 in invasion and the role of cIAP2 in invasion and migration. Studies of other tumor types in which cIAP1 is overexpressed suggest that multi-regimen treatments including SMAC mimetics may be effective. Thus, the evaluation of 11q22.1-q22.2 amplifications in OSCC patients may help choose the most effective treatment.

Prevascularization of Decellularized Porcine Myocardial Slice for Cardiac Tissue Engineering.

Prevacularization strategies have been implemented in tissue engineering to generate microvasculature networks within a scaffold prior to implantation. Prevascularizing scaffolds will shorten the time of functional vascular perfusion with host upon implantation. In this study, we explored key variables affecting the interaction between decellularized porcine myocardium slices (dPMSs) and reseeded stem cells toward the fabrication of prevascularized cardiac tissue. Our results demonstrated that dPMS supports attachment of human mesenchymal stem cells (hMSCs) and rat adipose derived stem cells (rASCs) with high viability. We found that cell seeding efficiency and proliferation are dPMS thickness dependent. Compared to lateral cell seeding, bilateral cell seeding strategy significantly enhanced seeding efficiency, infiltration, and growth in 600 µm dPMS. dPMS induced endothelial differentiation and maturation of hMSCs and rASCs after 1 and 5 days culture, respectively. These results indicate the potential of dPMS as a powerful platform to develop prevascularized scaffolds and fabricate functional cardiac patches.

Current challenges in dedifferentiated fat cells research.

Dedifferentiated fat cells show great promises as a novel cell source for stem cell research. It has many advantages when used for cell-based therapeutics including abundance, pluripotency, and safety. However, there are many obstacles researchers need to overcome to make the next big move in DFAT cells research. In this review, we summarize the current main challenges in DFAT cells research including cell culture purity, phenotypic properties, and dedifferentiation mechanisms. The common methods to produce DFAT cells as well as the cell purity issue during DFAT cell production have been introduced. Current approaches to improve DFAT cell purity have been discussed. The phenotypic profile of DFAT cells have been listed and compared with other stem cells. Further studies on elucidating the underlying dedifferentiation mechanisms will dramatically advance DFAT cell research.

Tailoring material properties of cardiac matrix hydrogels to induce endothelial differentiation of human mesenchymal stem cells.

Cardiac matrix hydrogel has shown great promise as an injectable biomaterial due to the possession of cardiac-specific extracellular matrix composition. A cardiac matrix hydrogel facilitating neovascularization will further improve its therapeutic outcomes in cardiac repair. In this study, we explored the feasibility of tailoring material properties of cardiac matrix hydrogels using a natural compound, genipin, to promote endothelial differentiation of stem cells. Our results demonstrated that the genipin cross-linking could increase the mechanical properties of the cardiac matrix hydrogel to a stiffness range promoting endothelial differentiation of human mesenchymal stem cells (hMSCs). It also decreased the swelling ratio and prolonged degradation without altering gelation time. Human mesenchymal stem cells cultured on the genipin cross-linked cardiac matrix hydrogels showed great viability. After 1 day culture, hMSCs demonstrated down-regulation of early endothelial marker expression and up-regulation of mature endothelial marker expression. Especially for 1 mM genipin cross-linked cardiac matrix hydrogels, hMSCs showed particularly significant expression of mature endothelial cell marker vWF. These attractive results indicate the potential of using genipin cross-linked cardiac matrix hydrogels to promote rapid vascularization for cardiac infarction treatment through minimally invasive therapy.

Biocompatible flavone-based fluorogenic probes for quick wash-free mitochondrial imaging in living cells.

Mitochondria, vital organelles existing in almost all eukaryotic cells, play a crucial role in energy metabolism and apoptosis of aerobic organisms. In this work, we report two new flavone-based fluorescent probes, MC-Mito1 and MC-Mito2, for monitoring mitochondria in living cells. These two probes exhibit remarkably low toxicity, good cell permeability, and high specificity; these probes complement the existing library of mitochondrial imaging agents. The new dyes give nearly no background fluorescence, and their application does not require tedious postwashing after cell staining. The appreciable tolerance of MC-Mito2 encourages a broader range of biological applications for understanding the cell degeneration and apoptosis mechanism.