Three-Dimensional Imaging in Stem Cell-Based Researches.

Stem cells have an important role in regenerative therapies, developmental biology studies and drug screening. Basic and translational research in stem cell technology needs more detailed imaging techniques. The possibility of cell-based therapeutic strategies has been validated in the stem cell field over recent years, a more detailed characterization of the properties of stem cells is needed for connectomics of large assemblies and structural analyses of these cells. The aim of stem cell imaging is the characterization of differentiation state, cellular function, purity and cell location. Recent progress in stem cell imaging field has included ultrasound-based technique to study living stem cells and florescence microscopy-based technique to investigate stem cell three-dimensional (3D) structures. Here, we summarized the fundamental characteristics of stem cells via 3D imaging methods and also discussed the emerging literatures on 3D imaging in stem cell research and the applications of both classical 2D imaging techniques and 3D methods on stem cells biology.

Optogenetic control of neural differentiation in Opto-mGluR6 engineered retinal pigment epithelial cell line and mesenchymal stem cells.

In retinal degenerative disorders, when neural retinal cells are damaged, cell transplantation is one of the most promising therapeutic approaches. Optogenetic technology plays an essential role in the neural differentiation of stem cells via membrane depolarization. This study explored the efficacy of blue light stimulation in neuroretinal differentiation of Opto-mGluR6-engineered mouse retinal pigment epithelium (mRPE) and bone marrow mesenchymal stem cells (BMSCs). mRPE and BMSCs were selected for optogenetic study due to their capability to differentiate into retinal-specific neurons. BMSCs were isolated and phenotypically characterized by the expression of mesenchymal stem cell-specific markers, CD44 (99%) and CD105 (98.8%). mRPE culture identity was confirmed by expression of RPE-specific marker, RPE65, and epithelial cell marker, ZO-1. mRPE cells and BMSCs were transduced with AAV-MCS-IRES-EGFP-Opto-mGluR6 viral vector and stimulated for 5 days with blue light (470 nm). RNA and protein expression of Opto-mGluR6 were verified. Optogenetic stimulation-induced elevated intracellular Ca2+ levels in mRPE- and BMS-treated cells. Significant increase in cell growth rate and G1/S phase transition were detected in mRPE- and BMSCs-treated cultures. Pou4f1, Dlx2, Eomes, Barlh2, Neurod2, Neurod6, Rorb, Rxrg, Nr2f2, Ascl1, Hes5, and Sox8 were overexpressed in treated BMSCs and Barlh2, Rorb, and Sox8 were overexpressed in treated mRPE cells. Expression of Rho, Thy1, OPN1MW, Recoverin, and CRABP, as retinal-specific neuron markers, in mRPE and BMS cell cultures were demonstrated. Differentiation of ganglion, amacrine, photoreceptor cells, and bipolar and Muller precursors were determined in BMSCs-treated culture and were compared with mRPE. mRPE cells represented more abundant terminal Muller glial differentiation compared with BMSCs. Our results also demonstrated that optical stimulation increased the intracellular Ca2+ level and proliferation and differentiation of Opto-mGluR6-engineered BMSCs. It seems that optogenetic stimulation of mRPE- and BMSCs-engineered cells would be a potential therapeutic approach for retinal degenerative disorders.

Profilometry of an optical microfiber based on modal evolution.

The waist diameter of a tapered optical fiber (TOF) has been determined using the modal evolution during the tapering process of a single-mode optical fiber (SMF28) through the short-time Fourier transform (STFT) analysis. The STFT was utilized to calculate the cutoff moment of the different modes. By the knowledge of the cutoff diameter, the final diameter of the waist with accuracy better than 5 nm was measured. The TOF shape depends on the flame parameters, the material properties, and the stretching conditions. By calculating the TOF deformation rate of the TOF, the diameter of TOFs near the waist has been measured with an accuracy of 6.1%; moreover, the TOFs were fabricated with a non-uniform flame.

Optogenetic control of perforant pathway kindled seizures by photoinhibition of CA3 after insertion of Jaws into CA3 neuronal cells.

Medial temporal lobe epilepsy (MTLE) is among the most common and most drug-resistant types of epilepsies associated with remodeling of the trisynaptic circuit of the hippocampus. The cornu ammonis (CA)3 region, as the "pacemaker" of the circuit, and CA3 â†’ CA1 synapse (Schaffer collaterals) are potential targets for suppression of MTLE. We examined optogenetic manipulation of CA3 neurons in controlling the perforant pathway kindled seizures. One week after implantation of stimulating electrodes in perforant pathway, a recording electrode in CA1, and an optic fiber in CA3, rats underwent rapid kindling procedure. A lentivector with capability to move in retrograde monosynaptic direction and to insert the gene of red light sensitive opsin Jaws in neurons was injected into CA1 of the kindled rats. One week later, the kindled rats were stimulated at afterdischarge (AD) threshold under red light illumination to CA3; and duration of AD (ADD), generalized seizures (S5D), and total seizure behavior (SD) were recorded. Encoding Jaws in CA1, CA3, and entorhinal neuronal cells of the vector injected rats was verified by immunohistochemistry. More than 90% of CA1, CA3, and entorhinal neurons of the counted sections expressed Jaws. Red light (625 nm) illumination to CA3 of the kindled rats expressing Jaws entirely suppressed generalized seizures and significantly diminished ADD and SD. Encoding the light-sensitive chloride pump Jaws in the CA3, is an efficient optogenetic strategy to stop perforant pathway kindled seizures.

Multimode Fabry⁻Perot Interferometer Probe Based on Vernier Effect for Enhanced Temperature Sensing.

New miniaturized sensors for biological and medical applications must be adapted to the measuring environments and they should provide a high measurement resolution to sense small changes. The Vernier effect is an effective way of magnifying the sensitivity of a device, allowing for higher resolution sensing. We applied this concept to the development of a small-size optical fiber Fabry⁻Perot interferometer probe that presents more than 60-fold higher sensitivity to temperature than the normal Fabry⁻Perot interferometer without the Vernier effect. This enables the sensor to reach higher temperature resolutions. The silica Fabry⁻Perot interferometer is created by focused ion beam milling of the end of a tapered multimode fiber. Multiple Fabry⁻Perot interferometers with shifted frequencies are generated in the cavity due to the presence of multiple modes. The reflection spectrum shows two main components in the Fast Fourier transform that give rise to the Vernier effect. The superposition of these components presents an enhancement of sensitivity to temperature. The same effect is also obtained by monitoring the reflection spectrum node without any filtering. A temperature sensitivity of -654 pm/°C was obtained between 30 °C and 120 °C, with an experimental resolution of 0.14 °C. Stability measurements are also reported.

Measurement of milli-Newton axial force and temperature using a hybrid microsilica sphere Fabry-Perot sensor.

An optical fiber sensor based on the Hybrid Fabry-Perot for simultaneous measurement of milli-Newton axial force and temperature is proposed. This structure is composed of a single-mode optical fiber (SMF) integrated to a silica capillary tube (SCT) with polydimethylsiloxane (PDMS). A microsilica sphere cavity (MSSC) is fabricated at the tip of a capillary tube. Consequently, an air gap followed by a microsilica sphere forms two cascade cavities. To explain the transferring load from the SMF to the SCT through PDMS, a shearing mechanism is employed. The experimental results show that axial force and temperature sensitivities of the air gap cavity in the range of 0-3.43 mN and 30-65°C are 170 pm/mN and 24 pm/°C, respectively, while the MSSC did not show any force sensitivity due to its rigidity. However, its temperature sensitivity is 34 pm/°C. The different sensitivities enable us to implement simultaneous sensing of force and temperature.

Epidermal neural crest stem cell-derived glia enhance neurotrophic elements in an ex vivo model of spinal cord injury.

Growing evidence that cell-based therapies can improve recovery outcome in spinal cord injury (SCI) models substantiates their application for treatment of human with SCI. To address the effectiveness of these stem cells, potential candidates should be evaluated in proper SCI platform that allows direct real-time monitoring. In this study, the role of epidermal neural crest stem cells (EPI-NCSCs) was elucidated in an ex vivo model of SCI, and valproic acid (VPA) was administered to ameliorate the inhospitable context of injury for grafted EPI-NCSCs. Here the contusion was induced in organotypic spinal cord slice culture at day seven in vitro using a weight drop device and one hour post injury the GFP- expressing EPI-NCSCs were grafted followed by VPA administration. The evaluation of treated slices seven days after injury revealed that grafted stem cells survived on the injured slices and expressed GFAP, whereas they did not express any detectable levels of the neural progenitor marker doublecortin (DCX), which was expressed prior to transplantation. Immunoblotting data demonstrated that the expression of GFAP, BDNF, neurotrophin-3 (NT3), and Bcl2 increased significantly in stem cell treated slices. This study illustrated that the fate of transplanted stem cells has been directed to the glial lineage in the ex vivo context of injury and EPI-NCSCs may ameliorate the SCI condition through releasing neurotrophic factors directly and/or via inducing resident spinal cord cells.