N-acetyl-cysteine blunts 6-hydroxydopamine- and L-buthionine-sulfoximine-induced apoptosis in human mesenchymal stromal cells.

Parkinson disease (PD) is characterized by the loss of dopaminergic (DAergic) neurons linked to environmental toxicants that cause oxidative stress (OS). The aim of this investigation was to establish the molecular response of human mesenchymal stroma cells (MSCs) depleted of glutathione (GSH) by the specific inhibitor L-buthionine-sulfoximine (BSO) to 6-hydroxydopamine (6-OHDA) and/or N-acetylcysteine (NAC) co-treatment. We found that treatment with BSO (10 mM) plus 6-OHDA (200 µM) induced apoptosis in MSCs through an oxidative stress (OS) mechanism involving H2O2, reflected by the detection of dichlorofluorescein-positive (DCF+) cells and oxidation of DJ-1 Cys106-SH into DJ-1 Cys106-SO3; an almost complete reduction in glutathione peroxidase 1 (GPX1) expression; activation of the transcription factor c-JUN, the pro-apoptotic protein BAX and BH-3-only protein PUMA; loss of mitochondrial membrane potential (∆Ψm); activation of the protease caspase-3 (CASP3) and apoptosis-inducing factor (AIF); chromatin condensation; and DNA fragmentation. Strikingly, co-treatment of MSCs with NAC (5 mM) and BSO + 6-OHDA significantly reduced the expression of OS and cell death markers but were unable to restore the expression of GPX1 compared to the expression in untreated or treated cells with NAC only. These findings highlighted the importance of the maintenance of the GSH-dependent (e.g., GPX1, GSH synthesis) and -independent (e.g., ROS scavenger molecules and thiol reducing activity) antioxidant systems (e.g., NAC) in the protection of MSCs from detrimental stress stimuli, thereby increasing the survival of stromal cells.

PARKIN overexpression in human mesenchymal stromal cells from Wharton's jelly suppresses 6-hydroxydopamine-induced apoptosis: Potential therapeutic strategy in Parkinson's disease.

BACKGROUND AIMS: Stem cell transplantation is an excellent option for regenerative or replacement therapy. However, deleterious microenvironmental and endogenous factors (e.g., oxidative stress) compromise ongoing graft survival and longevity. Therefore, (transient or stable) genetically modified cells may be reasonably thought to resist oxidative stress-induced damage. Genetic engineering of mesenchymal stromal cells (MSCs) obtained from Wharton's jelly tissue may offer some therapeutic potential. PARKIN is a multifunctional ubiquitin ligase able to protect dopaminergic cells against stress-related signaling. We, therefore, evaluated the effect of the neurotoxicant 6-hydroxydopamine (6-OHDA) on regulated cell death signaling in MSCs and investigated whether overexpression of PARKIN in MSCs was capable of modulating the effect of 6-OHDA. METHODS: We transiently transfected Wharton's jelly-derived MSCs with an mCherry-PARKIN vector using the Lipofectamine LTX method. Naïve MSCs and MSCs overexpressing PARKIN were exposed to increasing concentrations of 6-OHDA. We used light and fluorescence microscopy, flow cytometry, immunocytochemistry staining, in-cell Western and Western blot analysis. RESULTS: After 12-24 h of 6-OHDA exposure, we detected dichlorofluorescein (DCF)-positive cells (80%) indicative of reactive oxygen species (H2O2) production, reduced cell viability (40-50%), decreased mitochondrial membrane potential (ΔΨm, ~35-45%), DNA fragmentation (18-30%), and G1-arrested cell cycle in the MSCs. 6-OHDA exposure increased the expression of the transcription factor c-JUN, increased the expression of the mitochondria maintenance Phosphatase and tensin homologue-induced putative kinase 1 (PINK1) protein and increased the expression of pro-apoptotic PUMA, caspase-3 and apoptosis-inducing factor (AIF). 6-OHDA exposure also significantly augmented the oxidation of the oxidative stress sensor, DJ-1. Overexpression of PARKIN in MSCs not only significantly reduced the expression of cell death and oxidative stress markers but also significantly reduced DCF-positive cells (~50% reduction). DISCUSSION: 6-OHDA induced apoptosis in MSCs via generation of H2O2, activation of c-JUN and PUMA, mitochondrial depolarization and nuclei fragmentation. Our findings suggest that PARKIN protects MSCs against 6-OHDA toxicity by partly interacting with H2O2, reducing the expression of c-JUN, PUMA, AIF and caspase-3, and maintaining the mitochondrial ΔΨm.

Fast transdifferentiation of human Wharton's jelly mesenchymal stem cells into neurospheres and nerve-like cells.

BACKGROUND: The human mesenchymal stem cells derived from Wharton's jelly tissue (hWJ-MSCs) represent a tool for cell-based therapies and regenerative medicine. hWJ-MSCs form neurospheres (NSs) within 3-7 days. No data is available to establish the neuro-phenotypic markers and time of formation of nerve-like (NLCs) and glial cells from NSs derived from hWJ-MSCs. NEW METHOD: hWJ-MSCs were incubated with Fast-N-Spheres medium for 24 and 72h. The new formed NSs were in turn incubated with forskolin in neurogenic NeuroForsk medium for 1-7days. RESULTS: hWJ-MSCs cultured with Fast-N-Spheres medium trans-differentiated into NSs in just 24h compared to 72h for hWJ-MSCs cultured with classic growth factor medium. The NSs generated from the Fast-N-Spheres medium expressed reduced levels SOX2, OCT4 and NANOG, as markers of pluripotency compared to undifferentiated hWJ-MSCs. The formed NSs exposed to NeuroForsk medium differentiated into NLCs in 4days as evidenced by high levels of protein expression of the neuronal markers, and no expression of the glial marker GFAP. COMPARISON WITH EXISTING METHOD(S): Currently, the formation and harvest of NSs is expensive and time consuming. Published protocols require 3-7days to form NSs from whole human umbilical cord MSCs. We report for the first time, to our knowledge, the differentiation of NSs-derived from hWJ-MSCs into NLCs. CONCLUSIONS: The fastest method to obtain NSs and NLCs from hWJ-MSCs takes only five days using the two-step incubation media Fast-N-Spheres and NeuroForsk.

Integration of Multi-Plane Tissue Doppler and B-Mode Echocardiographic Images for Left Ventricular Motion Estimation.

Although modern ultrasound acquisition systems allow recording of 3D echocardiographic images, tracking anatomical structures from them is still challenging. In addition, since these images are typically created from information obtained across several cardiac cycles, it is not yet possible to acquire high-quality 3D images from patients presenting varying heart rhythms. In this paper, we propose a method to estimate the motion field from multi-plane echocardiographic images of the left ventricle, which are acquired simultaneously during a single cardiac cycle. The method integrates tri-plane B-mode and tissue Doppler images acquired at different rotation angles around the long axis of the left ventricle. It uses a diffeomorphic continuous spatio-temporal transformation model with a spherical data representation for a better interpolation in the circumferential direction. This framework allows exploiting the spatial relation among the acquired planes. In addition, higher temporal resolution of the transformation in the beam direction is achieved by uncoupling the estimation of the different components of the velocity field. The method was validated using a realistic synthetic dataset including healthy and ischemic cases, obtaining errors of 0.14 ± 0.09 mm for displacements, 0.96 ± 1.03% for longitudinal strain and 3.94 ± 4.38% for radial strain estimation. In addition, the method was also demonstrated on a healthy volunteer and two patients with ischemia.

Improved myocardial motion estimation combining tissue Doppler and B-mode echocardiographic images.

We propose a technique for myocardial motion estimation based on image registration using both B-mode echocardiographic images and tissue Doppler sequences acquired interleaved. The velocity field is modeled continuously using B-splines and the spatiotemporal transform is constrained to be diffeomorphic. Images before scan conversion are used to improve the accuracy of the estimation. The similarity measure includes a model of the speckle pattern distribution of B-mode images. It also penalizes the disagreement between tissue Doppler velocities and the estimated velocity field. Registration accuracy is evaluated and compared to other alternatives using a realistic synthetic dataset, obtaining mean displacement errors of about 1 mm. Finally, the method is demonstrated on data acquired from six volunteers, both at rest and during exercise. Robustness is tested against low image quality and fast heart rates during exercise. Results show that our method provides a robust motion estimate in these situations.

3D strain assessment in ultrasound (Straus): a synthetic comparison of five tracking methodologies.

This paper evaluates five 3D ultrasound tracking algorithms regarding their ability to quantify abnormal deformation in timing or amplitude. A synthetic database of B-mode image sequences modeling healthy, ischemic and dyssynchrony cases was generated for that purpose. This database is made publicly available to the community. It combines recent advances in electromechanical and ultrasound modeling. For modeling heart mechanics, the Bestel-Clement-Sorine electromechanical model was applied to a realistic geometry. For ultrasound modeling, we applied a fast simulation technique to produce realistic images on a set of scatterers moving according to the electromechanical simulation result. Tracking and strain accuracies were computed and compared for all evaluated algorithms. For tracking, all methods were estimating myocardial displacements with an error below 1 mm on the ischemic sequences. The introduction of a dilated geometry was found to have a significant impact on accuracy. Regarding strain, all methods were able to recover timing differences between segments, as well as low strain values. On all cases, radial strain was found to have a low accuracy in comparison to longitudinal and circumferential components.