Fluorescent probes as a tool for cell population tracking in spontaneously active neural networks derived from human pluripotent stem cells.

Applications such as 3D cultures and tissue modelling require cell tracking with non-invasive methods. In this work, the suitability of two fluorescent probes, CellTracker, CT, and long chain carbocyanine dye, DiD, was investigated for long-term culturing of labeled human pluripotent stem cell-derived neural cells. We found that these dyes did not affect the cell viability. However, proliferation was decreased in DiD labeled cell population. With both dyes the labeling was stable up to 4 weeks. CT and DiD labeled cells could be co-cultured and, importantly, these mixed populations had their normal ability to form spontaneous electrical network activity. In conclusion, human neural cells can be successfully labeled with these two fluorescent probes without significantly affecting the cell characteristics. These labeled cells could be utilized further in e.g. building controlled neuronal networks for neurotoxicity screening platforms, combining cells with biomaterials for 3D studies, and graft development.

Impedance spectroscopy in monitoring the maturation of stem cell-derived retinal pigment epithelium.

The development and differentiation of stem cell-derived impermeable retinal pigment epithelium (RPE) with tight junctions (TJs) is a gradual process that is, at confluence, controlled by cell-to-cell contact. The objective of this study was to evaluate the use of electric impedance spectroscopy (EIS) to follow the maturation and development of barrier function in human embryonic stem cell-derived RPE (hESC-RPE). Barrier function was assessed using EIS, permeability measurements, and microscopic inspection in intact cells and following calcium sequestration with ethylene glycol tetraacetic acid (EGTA). The results showed that the cultures with the most mature morphology had the highest impedance and the lowest permeability values. The EIS of samples of high integrity fitted well to the equivalent model of a single RC circuit, whereas the semicircular shape of the Nyquist plots was distorted for samples of lower integrity. EGTA treatment resulted in lower impedance values and changes in the shapes of plots. Our results show that EIS-as a measure of overall maturity and integrity of the epithelium-is useful when evaluating the maturity of cell cultures. It is highly warranted in future transplantation therapies and in in vitro cell culture models in drug development.

Markers of pluripotency and differentiation in human neural precursor cells derived from embryonic stem cells and CNS tissue.

Cell transplantation therapies for central nervous system (CNS) deficits such as spinal cord injury (SCI) have been shown to be effective in several animal models. One cell type that has been transplanted is neural precursor cells (NPCs), for which there are several possible sources. We have studied NPCs derived from human embryonic stem cells (hESCs) and human fetal CNS tissue (hfNPCs), cultured as neurospheres, and the expression of pluripotency and neural genes during neural induction and in vitro differentiation. mRNA for the pluripotency markers Nanog, Oct-4, Gdf3, and DNMT3b were downregulated during neural differentiation of hESCs. mRNA for these markers was found in nonpluripotent hfNPC at higher levels compared to hESC-NPCs. However, Oct-4 protein was found in hESC-NPCs after 8 weeks of culture, but not in hfNPCs. Similarly, SSEA-4 and CD326 were only found in hESC-NPCs. NPCs from both sources differentiated as expected to cells with typical features of neurons and astrocytes. The expressions of neuronal markers in hESC-NPCs were affected by the composition of cell culture medium, while this did not affect hfNPCs. Transplantation of hESC-NPC or hfNPC neurospheres into immunodeficient mouse testis or subcutaneous tissue did not result in tumor formation. In contrast, typical teratomas appeared in all animals after transplantation of hESC-NPCs to injured or noninjured spinal cords of immunodeficient rats. Our data show that transplantation to the subcutaneous tissue or the testes of immunodeficient mice is not a reliable method for evaluation of the tumor risk of remaining pluripotent cells in grafts.

Caspase 6 expression in the rat hippocampus during epileptogenesis and epilepsy.

The molecular basis of neuronal circuit reorganization during epileptogenesis is poorly understood. Such data are, however, critical for the search of new targets for the prevention of epileptogenesis. Here, we extended our previous studies on caspases in epileptogenesis by investigating the expression and activity of caspase 6 at different phases of the epileptic process in rats. Epileptogenesis was triggered by kainate-induced status epilepticus (SE) under video-electroencephalography-monitoring. Caspase 6 activity was measured fluorometrically in the hippocampus 8 h, 24 h, 48 h, 1 week, and 4 weeks after SE. Caspase 6 expression was examined using Western blot and immunohistochemistry. Our data demonstrated that the SE-induced increase in the expression of cleaved caspase 6 and its intraneuronal localization were dependent on the time delay from SE induction. Double-labeling with a neuronal marker, NeuN, indicated that within the first 48 h, caspase 6 immunoreactivity was present both in the hippocampal pyramidal cells and hilar neurons, some of which were also terminal transferase dUTP-end labeling-positive. This was coincident with a transient 18-fold increase in caspase 6 enzymatic activity. At the 1-week and 4-week time points, elevated caspase 6 immunoreactivity was detected in the dendritic processes and neuropil. These findings indicate that caspase 6 expression remains elevated long after the occurrence of acute cell death during epileptogenesis and epilepsy. Further, caspase 6 protein is not exclusively located in the somata of neurons, but also in dendrites. These data suggest that caspase 6 has functions other than execution of programmed cell death in epileptogenic hippocampus.

Administration of caspase 3 inhibitor during and after status epilepticus in rat: effect on neuronal damage and epileptogenesis.

Symptomatic temporal lobe epilepsy typically develops in three phases: brain damage --> epileptogenesis --> spontaneous seizures (epilepsy). The challenge is to prevent epileptogenesis after injury. We hypothesized that alleviation of damage by caspase inhibitors will reduce epileptogenesis or at least have disease-modifying effects (less severe epilepsy, milder cognitive decline). Epileptogenesis was triggered by amygdala stimulation-induced status epilepticus (SE) in rats and spontaneous seizures were monitored with video-electroencephalography (EEG). First, we tested the neuroprotective effect of a 1-week treatment with caspase 1, 3 or 9 inhibitors (3 micro g/d/i.c.v., started 3 h after the beginning of SE). The least damage to the hippocampus was observed in animals treated with the caspase 3 inhibitor (z-DEVD-fmk) which reduced the enzyme activity to 6% of that in the vehicle group. Thus, z-DEVD-fmk was chosen for long-term studies, in which the treatment regime remained the same except the dose was doubled (6 micro g/d/i.c.v.). Video-EEG monitoring was performed for 3 to 4 weeks, starting either 8 or 14 weeks after SE. One group of animals was tested in water-maze and fear-conditioning tests, and all animals were perfused for histological analysis. Treatment with the caspase 3 inhibitor neither prevented the development of epilepsy, nor had any disease-modifying effects. Mossy fibre sprouting, however, was reduced. The present data indicate that administration of z-DEVD-fmk monotherapy was not antiepileptogenic despite its short-term neuroprotective effects. These findings challenge the idea that prevention of cell death is the primary target for the development of antiepileptogenic compounds.