Stroke is a neurodegenerative disorder and the leading cause of disability in adult humans. Treatments to support efficient recovery in stroke patients are lacking. Several studies have demonstrated the ability of grafted neural stem cells (NSCs) to partly improve impaired neurological functions in stroke-subjected animals. Recently, we reported that NSCs from human fetal striatum and cortex exhibit region-specific differentiation in vitro, but survive, migrate and form neurons to a similar extent after intrastriatal transplantation in newborn rats. Here, we have transplanted the same cells into the stroke-damaged striatum of adult rats. The two types of NSCs exhibited a similar robust survival (30%) at 1 month after transplantation, and migrated throughout the damaged striatum. Striatal NSCs migrated farther and occupied a larger volume of striatum. In the transplantation core, cells were undifferentiated and expressed nestin and, to a lesser extent, also GFAP, betaIII-tubulin, DCX and calretinin, markers of immature neural lineage. Immunocytochemistry using markers of proliferation (p-H3 and Ki67) revealed a very low content of proliferating cells (<1%) in the grafts. Human cells outside the transplantation core differentiated, exhibited mature neuronal morphology and expressed mature neuronal markers such as HuD, calbindin and parvalbumin. Interestingly, striatal NSCs generated a greater number of parvalbumin+ and calbindin+ neurons. Virtually none of the grafted cells differentiated into astrocytes or oligodendrocytes. Based on these data, human fetal striatum- and cortex-derived NSCs could be considered potentially safe and viable for transplantation, with strong neurogenic potential, for further exploration in animal models of stroke.

Neural stem cells (NSCs) derived from human fetal striatum and transplanted as neurospheres survive in stroke-damaged striatum, migrate from the implantation site, and differentiate into mature neurons. Here, we investigated how various steps of neurogenesis are affected by intrastriatal transplantation of human NSCs at different time points after stroke and with different numbers of cells in each implant. Rats were subjected to middle cerebral artery occlusion and then received intrastriatal transplants of NSCs. Transplantation shortly after stroke (48 hours) resulted in better cell survival than did transplantation 6 weeks after stroke, but the delayed transplantation did not influence the magnitude of migration, neuronal differentiation, and cell proliferation in the grafts. Transplanting greater numbers of grafted NSCs did not result in a greater number of surviving cells or increased neuronal differentiation. A substantial number of activated microglia was observed at 48 hours after the insult in the injured striatum, but reached maximum levels 1 to 6 weeks after stroke. Our findings show that the best survival of grafted human NSCs in stroke-damaged brain requires optimum numbers of cells to be transplanted in the early poststroke phase, before the inflammatory response is established. These findings, therefore, have direct clinical implications.

BackgroundMagnetic resonance imaging (MRI) is a promising tool for monitoring stem cell-based therapy. Conventionally, cells loaded with ironoxide nanoparticles appear hypointense on MR images. However, the contrast generated by ironoxide labeled cells is neither specific due to ambiguous background nor quantitative. A strategy to overcome these drawbacks is 19F MRI of cells labeled with perfluorocarbons. We show here for the first time that human neural stem cells (NSCs), a promising candidate for clinical translation of stem cell-based therapy of the brain, can be labeled with 19F as well as detected and quantified in vitro and after brain implantation.Methodology/Principal FindingsHuman NSCs were labeled with perfluoropolyether (PFPE). Labeling efficacy was assessed with 19F MR spectroscopy, influence of the label on cell phenotypes studied by immunocytochemistry. For in vitro MRI, NSCs were suspended in gelatin at varying densities. For in vivo experiments, labeled NSCs were implanted into the striatum of mice. A decrease of cell viability was observed directly after incubation with PFPE, which re-normalized after 7 days in culture of the replated cells. No label-related changes in the numbers of Ki67, nestin, GFAP, or βIII-tubulin+ cells were detected, both in vitro and on histological sections. We found that 1,000 NSCs were needed to accumulate in one image voxel to generate significant signal-to-noise ratio in vitro. A detection limit of ∼10,000 cells was found in vivo. The location and density of human cells (hunu+) on histological sections correlated well with observations in the 19F MR images.Conclusion/SignificanceOur results show that NSCs can be efficiently labeled with 19F with little effects on viability or proliferation and differentiation capacity. We show for the first time that 19F MRI can be utilized for tracking human NSCs in brain implantation studies, which ultimately aim for restoring loss of function after acute and neurodegenerative disorders.

Human fetal brain is a potential source of neural stem cells (NSCs) for cell replacement therapy in neurodegenerative diseases. We explored whether NSCs isolated from cortex and striatum of human fetuses, aged 6-9 weeks post-conception, maintain their regional identity and differentiate into specific neuron types in culture and after intrastriatal transplantation in neonatal rats. We observed no differences between cortex- and striatum-derived NSCs expanded as neurospheres in proliferative capacity, growth rate, secondary sphere formation, and expression of neural markers. After 4 weeks of differentiation in vitro, cortical and striatal NSCs gave rise to similar numbers of GABAergic and VMAT2- and parvalbumin-containing neurons. However, whereas cortical NSCs produced higher number of glutamatergic and tyrosine hydroxylase- and calretinin-positive neurons, several-fold more neurons expressing the striatal projection neuron marker, DARPP-32, were observed in cultures of striatal NSCs. Human cortical and striatal NSCs survived and migrated equally well after transplantation. The two NSC types also generated similar numbers of mature NeuN-positive neurons, which were several-fold higher at 4 months as compared to at 1 month after grafting. At 4 months, the grafts contained cells with morphologic characteristics of neurons, astrocytes, and oligodendrocytes. Many of neurons were expressing parvalbumin. Our data show that NSCs derived from human fetal cortex and striatum exhibit region-specific differentiation in vitro, and survive, migrate, and form mature neurons to the same extent after intrastriatal transplantation in newborn rats.

Mitochondrial uptake of calcium in excitotoxicity is associated with subsequent increase in reactive oxygen species (ROS) generation and delayed cellular calcium deregulation in ischemic and neurodegenerative insults. The mechanisms linking mitochondrial calcium uptake and ROS production remain unknown but activation of the mitochondrial permeability transition (mPT) may be one such mechanism. In the present study, calcium increased ROS generation in isolated rodent brain and human liver mitochondria undergoing mPT despite an associated loss of membrane potential, NADH and respiration. Unspecific permeabilization of the inner mitochondrial membrane by alamethicin likewise increased ROS independently of calcium, and the ROS increase was further potentiated if NAD(H) was added to the system. Importantly, calcium per se did not induce a ROS increase unless mPT was triggered. Twenty-one cyclosporin A analogs were evaluated for inhibition of calcium-induced ROS and their efficacy clearly paralleled their potency of inhibiting mPT-mediated mitochondrial swelling. We conclude that while intact respiring mitochondria possess powerful antioxidant capability, mPT induces a dysregulated oxidative state with loss of GSH-and NADPH-dependent ROS detoxification. We propose that mPT is a significant cause of pathological ROS generation in excitotoxic cell death.

Over the past few decades, a variety of different reagents for stem cell maintenance and differentiation have been commercialized. These reagents share a common goal in facilitating the manufacture of products suitable for cell therapy while reducing the amount of non-defined components. Lessons from developmental biology have identified signalling molecules that can guide the differentiation process in vitro, but less attention has been paid to the extracellular matrix used. With the introduction of more biologically relevant and defined matrices, that better mimic specific cell niches, researchers now have powerful resources to fine-tune their in vitro differentiation systems, which may allow the manufacture of therapeutically relevant cell types. In this review article, we revisit the basics of the extracellular matrix, and explore the important role of the cell–matrix interaction. We focus on laminin proteins because they help to maintain pluripotency and drive cell fate specification.This article is part of the theme issue ‘Designer human tissue: coming to a lab near you’.

During stroke, the reduction of blood flow leads to undersupply of oxygen and nutrients and, finally, to cell death, but also to upregulation of pro-angiogenic molecules and vascular remodeling. However, the temporal profile of vascular changes after stroke is still poorly understood. Here, we optimized steady-state contrast-enhanced magnetic resonance imaging (SSCE MRI) and followed the dynamic changes in vascular architecture for up to 4 weeks after transient middle cerebral artery occlusion (MCAO) in rats. Using MRI diffusion measurements and the changes of transversal relaxation rates ΔR2 and ΔR2* after injection of a superparamagnetic contrast agent, SSCE MRI provided several hemodynamic parameters: relative cerebral blood volume (rCBV), rCBV in small vessels, microvascular density, and relative vessel size. Six rats underwent SSCE MRI before MCAO and at 7, 14, 21 and 28 days after surgery. 5-Bromo-2'deoxyuridine (BrdU) was injected between days 2 and 7 to label proliferating cells during this time. SSCE MRI depicted a decrease in microvessel density and an increase in vessel size in the ischemic striatum after stroke. A persistently decreased MRI vessel density was confirmed with histology at 28 days. BrdU + endothelial cells were found in regions close to the infarct indicating endothelial cell proliferation during the first week after MCAO; however, late-stage angiogenesis, as would be reflected by increased vessel density, was not detected. The optimized SSCE MRI protocol was used to follow spatio-temporal changes of important vessel characteristics, which may contribute to a better understanding of the role of angiogenesis at different stages after stroke.

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