The intimal and medial linings of the pulmonary artery consist largely of vascular endothelial cells (VECs) and vascular smooth muscle cells (VSMCs), respectively. The migration of these cell types to a potential tissue-engineered pulmonary valve (TEPV) implant process is therefore of interest in understanding the valve remodeling process. Visualization and cell tracking by MRI, which employs hypointense contrast achievable through the use of superparamagnetic iron oxide (SPIO) microparticles to label cells, provides a method in which this can be studied. We investigated the SPIO labeling efficiency of human VECs and VSMCs, and used two- and three-dimensional gradient echo sequences to track the migration of these cells in agar gel constructs. Protamine sulfate (4.5 µg/mL) was used to enhance SPIO uptake and was found to have no influence on cell viability or proliferation. MRI experiments were initially performed using a 9.4-T scanner. The results demonstrated that the spatial positions of hypointense spots were relatively unchanged over 12 days. Subsequent MR experiments performed at 7 T demonstrated that three-dimensional imaging provided the best spatial resolution to assess cell fate. R(2)* maps were bright in SPIO cell-encapsulated gels in comparison with unlabeled counterparts. Signal voids were ruled out as hypointense regions owing to the smooth exponential decay of T(2)* in these voxels. As a next step, we intend to use the SPIO cell labeling and MR protocols established in this study to assess whether hemodynamic stresses will alter the vascular cell migratory patterns. These studies will shed light on the mechanisms of vascular remodeling after TEPV implantation.

Successful approaches to tissue engineering smooth muscle tissues utilize biodegradable scaffolds seeded with autologous cells. One common problem in using biological scaffolds specifically is the difficulty of inducing cellular penetration and controlling de novo extracellular matrix deposition=remodeling in vitro. Our hypothesis was that small intestinal submucosa (SIS) exposed to specific mechanical stimulation regimes would modulate the synthesis of de novo collagen and elastin by bladder smooth muscle cells (BSMC) within the SIS matrix. We further hypothesized that the cytokines vascular endothelial growth factor (VEGF) and fibroblast growth factor-2 (FGF-2), two key growth factors involved in epithelial mesenchymal signaling, will promote the cellular penetration into SIS necessary for mechanical stimulation. BSMC were seeded at 0.5Â10 6 cells=cm 2 onto the luminal side of SIS specimens. VEGF (10 ng=mL) and FGF-2 (5 ng=mL) were added to each insert in the media every other day for up to 7 days in static culture. Following static culture, specimens were stretched stripbiaxially under 15% peak strain at either 0.5 or 0.1 Hz for an additional 7 days. Following the culture period, specimens were assayed histologically and biochemically for cellular penetration, proliferation, elastin, collagen, and protease activity. Histological analyses demonstrated that in standard culture media, BSMC remained on the surface of the SIS while both FGF-2 and VEGF profoundly promoted ingrowth of the BSMC into the SIS. The penetration of the cells in response to these cytokines was confirmed using a Transwell assay. Following cellular penetration, BSMC produced significant amounts of elastic fibers under cyclic mechanical stretching at 0.1 Hz under 15% stretch, as evidenced by colorimetric assay and histology using a Verhoeff-Van Gieson stain. Protease activity was assessed in the media and found to be statistically increased in static culture following FGF-2 treatment. These findings demonstrate, for the first time, the capability of BSMC to produce histologically apparent elastin fibers in vitro. Moreover, our results suggest that a strategy involving growth factors and controlled mechanical stimulation may be used to engineer functional, elastin-rich tissue replacements using decellularized biologically derived scaffolds.

Mesenchymal stem cells are currently tested as a promising tool for the treatment of a wide range of human diseases. Enhanced therapeutic potential of spheroids formed from these cells has been proved in numerous studies, however, the fundamental basics of this effect are still being discussed. In this work, we showed that endometrial mesenchymal stem/stromal cells (eMSCs) assembled in spheroids possess a higher therapeutic efficacy compared to cells grown in monolayer in the treatment of the defects that are non-specific for eMSC tissue origin -skin wounds. With the purpose to elucidate the possible causes of superior spheroid potency, we compared the tolerance of eMSC cultivated in spheres and monolayer to the stress insults. Using genetically encoded hydrogen peroxide biosensor HyPer, we showed that three-dimensional configuration (3D) helped to shield the inner cell layers of spheroid from the external H 2 O 2 -induced oxidative stress. However, the viability of oxidatively damaged eMSCs in spheroids appeared to be much lower than that of monolayer cells. An extensive analysis, which included administration of heat shock and irradiation stress, revealed that cells in spheroids damaged by stress factors activate the apoptosis program, while in monolayer cells stress-induced premature senescence is developed. We found that basal down-regulation of anti-apoptotic and autophagy-related genes provides the possible molecular basis of the high commitment of eMSCs cultured in 3D to apoptosis. We conclude that predisposition to apoptosis provides the programmed elimination of damaged cells and contributes to the transplant safety of spheroids. In addition, to investigate the role of paracrine secretion in the wound healing potency of spheroids, we exploited the in vitro wound model (scratch assay) and found that culture medium conditioned by eMSC spheroids accelerates the migration of adherent cells. We showed that 3D eMSCs upregulate transcriptional activator, hypoxia-inducible factor (HIF)-1, and secret ten-fold more HIF-1-inducible pro-angiogenic factor VEGF

Application of genetically encoded biosensors of redox-active compounds promotes the elaboration of new methods for investigation of intracellular redox activities. Previously, we have developed a method to measure quantitatively the intracellular concentration of hydrogen peroxide (H 2 O 2 ) in living cells using genetically encoded biosensor HyPer. In the present study, we refined the method and applied it for comparing the antioxidant system potency in human cells of different phenotypes by measuring the gradient between the extracellular and cytoplasmic H 2 O 2 concentrations under conditions of H 2 O 2 -induced external oxidative stress. The measurements were performed using cancer cell lines (K-562 and HeLa), as well as normal human cells – all expressing HyPer in the cell cytoplasm. As normal cells, we used three isogenic lines of different phenotypes – mesenchymal stem/stromal cells (MSCs), induced pluripotent stem cells (iPSCs) derived from MSCs by reprogramming, and differentiated iPSC progenies with the phenotype resembling precursory MSCs. When exposing cells to exogenous H 2 O 2 , we showed that at low oxidative loads (<50 μM of H 2 O 2 ) the gradient depended on extracellular H 2 O 2 concentration. At high loads (>50 μM of H 2 O 2 ), which caused the exhaustion of thioredoxin activity in the cell cytoplasm, the gradient stabilized, pointing out that it is the functional status of the thioredoxin-depended enzymatic system that drives the dependence of the H 2 O 2 gradient on the oxidative load in human cells. At high H 2 O 2 concentrations, the cytoplasmic H 2 O 2 level in cancer cells was found to be several hundred times lower than the extracellular one. At the same time, in normal cells, extracellular-to-intracellular gradient amounted to thousands of times. Upon reprogramming, the potency of cellular antioxidant defense increased. In contrast, differentiation of iPSCs did not result in the changes in antioxidant system activity in the cell cytoplasm, assuming that intensification of the H 2 O 2 -detoxification processes is inherent to a period of early human development.

The study of proliferation regulation in human pluripotent stem cells is crucial to gain insights into understanding the physiology of these cells. However, redox regulation of the pluripotent cell cycle remains largely unexplored. Here, using human embryonic stem cells (hESCs) as well as human induced pluripotent stem cells (hiPSCs), we demonstrate that the level of reactive oxygen species (ROS) in pluripotent cells oscillates in accordance with the cell cycle progression with the peak occurring at transition from S to G2/M phase of the cycle. A decrease of this level by antioxidants leads to hindered S-phase initiation and progression but does not affect the early-G1-phase or mitosis. Cells exposed to antioxidants in the early-G1-phase accumulate the phosphorylated retinoblastoma protein and overcome the restriction point but are unable to accumulate the main regulators of the S phase—CYCLIN A and GEMININ. Based on the previous findings that CYCLIN A stability is affected by redox homeostasis disturbances in somatic cells, we compared the responses to antioxidant treatments in hESCs and in their differentiated fibroblast-like progeny cells (difESCs). In difESCs, similar to hESCs, a decrease in ROS level results in the disruption of S-phase initiation accompanied by a deficiency of the CYCLIN A level. Moreover, in antioxidant-treated cells, we revealed the accumulation of DNA breaks, which was accompanied by activation of the apoptosis program in pluripotent cells. Thus, we conclude that maintaining the physiological ROS level is essential for promotion of proliferation and accurate DNA synthesis in pluripotent cells and their differentiated descendants.