Vladimer Darsalia

Persistent production of neurons from adult brain stem cells during recovery after stroke.

Neural stem cells in the subventricular zone of adult rodents produce new striatal neurons that may replace those that have died after stroke; however, the neurogenic response has been considered acute and transient, yielding only small numbers of neurons. In contrast, we show herein that striatal neuroblasts are generated without decline at least for 4 months after stroke in adult rats. Neuroblasts formed early or late after stroke either differentiate into mature neurons, which survive for several months, or die through caspase‐mediated apoptosis. The directed migration of the new neurons toward the ischemic damage is regulated by stromal cell‐derived factor‐1α and its receptor CXCR4. These results show that endogenous neural stem cells continuously supply the injured adult brain with new neurons, which suggests novel self‐repair strategies to improve recovery after stroke.

Forebrain ependymal cells are Notch-dependent and generate neuroblasts and astrocytes after stroke

Neurons are continuously generated from stem cells in discrete regions in the adult mammalian brain. We found that ependymal cells lining the lateral ventricles were quiescent and did not contribute to adult neurogenesis under normal conditions in mice but instead gave rise to neuroblasts and astrocytes in response to stroke. Ependymal cell quiescence was actively maintained by canonical Notch signaling. Inhibition of this pathway in uninjured animals allowed ependymal cells to enter the cell cycle and produce olfactory bulb neurons, whereas forced Notch signaling was sufficient to block the ependymal cell response to stroke. Ependymal cells were depleted by stroke and failed to self-renew sufficiently to maintain their own population. Thus, although ependymal cells act as primary cells in the neural lineage to produce neurons and glial cells after stroke, they do not fulfill defining criteria for stem cells under these conditions and instead serve as a reservoir that is recruited by injury.

Long-term accumulation of microglia with proneurogenic phenotype concomitant with persistent neurogenesis in adult subventricular zone after stroke.

Neural stem cells (NSCs) in the adult rat subventricular zone (SVZ) generate new striatal neurons during several months after ischemic stroke. Whether the microglial response associated with ischemic injury extends into SVZ and influences neuroblast production is unknown. Here, we demonstrate increased numbers of activated microglia in ipsilateral SVZ concomitant with neuroblast migration into the striatum at 2, 6, and 16 weeks, with maximum at 6 weeks, following 2 h middle cerebral artery occlusion in rats. In the peri‐infarct striatum, numbers of activated microglia peaked already at 2 weeks and declined thereafter. Microglia in SVZ were resident or originated from bone marrow, with maximum proliferation during the first 2 weeks postinsult. In SVZ, microglia exhibited ramified or intermediate morphology, signifying a downregulated inflammatory profile, whereas amoeboid or round phagocytic microglia were frequent in the peri‐infarct striatum. Numbers of microglia expressing markers of antigen‐presenting cells (MHC‐II, CD86) increased in SVZ but very few lymphocytes were detected. Using quantitative PCR, strong short‐ and long‐term increase (at 1 and 6 weeks postinfarct) of insulin‐like growth factor‐1 (IGF‐1) gene expression was detected in SVZ tissue. Elevated numbers of IGF‐1‐expressing microglia were found in SVZ at 2, 6, and 16 weeks after stroke. At 16 weeks, 5% of microglia but no other cells in SVZ expressed the IGF‐1 protein, which mitigates apoptosis and promotes proliferation and differentiation of NSCs. The long‐term accumulation of microglia with proneurogenic phenotype in the SVZ implies a supportive role of these cells for the continuous neurogenesis after stroke.

Stroke-Induced Neurogenesis in Aged Brain

Background and Purpose— Stroke induced by middle cerebral artery occlusion (MCAO) triggers increased neurogenesis in the damaged striatum and nondamaged hippocampus of young adult rodents. We explored whether stroke influences neurogenesis similarly in the aged brain. Methods— Young adult (3 months) and old (15 months) rats were subjected to 1 hour of MCAO, and new cells were labeled by intraperitoneal injection of 5-bromo-2′-deoxyuridine 5′-monophosphate (BrdU), a marker for dividing cells, for 2 weeks thereafter. Animals were euthanized at 7 weeks after the insult, and neurogenesis was assessed immunocytochemically with antibodies against BrdU and neuronal markers with epifluorescence or confocal microscopy. Results— Young and old rats exhibited the same increased numbers of new striatal neurons after stroke, despite basal cell proliferation in the subventricular zone being reduced in the aged brain. In contrast, both the number of stroke-generated granule cells and basal neurogenesis in the dentate subgranular zone were lower in old compared with young animals. Also, the ability of newly formed cells to differentiate into neurons was impaired in the aged dentate gyrus. Conclusions— Basal neurogenesis is impaired in the subgranular and subventricular zones of aged animals, but both regions react to stroke with increased formation of new neurons. The magnitude of striatal neurogenesis after stroke is similar in young and old animals, indicating that this potential mechanism for self-repair also operates in the aged brain.

Survival, migration and neuronal differentiation of human fetal striatal and cortical neural stem cells grafted in stroke-damaged rat striatum

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, βIII‐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.

Cell Number and Timing of Transplantation Determine Survival of Human Neural Stem Cell Grafts in Stroke-Damaged Rat Brain

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.

Human fetal cortical and striatal neural stem cells generate region-specific neurons in vitro and differentiate extensively to neurons after intrastriatal transplantation in neonatal rats.

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.

Suppression of stroke-induced progenitor proliferation in adult subventricular zone by tumor necrosis factor receptor 1.

Stroke induced by middle cerebral artery occlusion leads to transiently increased progenitor proliferation in the subventricular zone (SVZ) and long-lasting striatal neurogenesis in adult rodents. Tumor necrosis factor-α (TNF-α) is upregulated in stroke-damaged brain. Whether TNF-α and its receptors influence SVZ progenitor proliferation after stroke is unclear. Here we show that the increased proliferation 1 week after stroke occurred concomitantly with elevated microglia numbers and TNF-α and TNF receptor-1 (TNF-R1) gene expression in the SVZ of wild-type mice. TNF receptor-1 was expressed on sorted SVZ progenitor cells from nestin-green fluorescent protein reporter mice. In animals lacking TNF-R1, stroke-induced SVZ cell proliferation and neuroblast formation were enhanced. In contrast, deletion of TNF-R1 did not alter basal or status epilepticus-stimulated cell proliferation in SVZ. Addition of TNF-α reduced the size and numbers of SVZ neurospheres through a TNF-R1-dependent mechanism without affecting cell survival. Our results provide the first evidence that TNF-R1 is a negative regulator of stroke-induced SVZ progenitor proliferation. Blockade of TNF-R1 signaling might be a novel strategy to promote the proliferative response in SVZ after stroke.

Anterograde delivery of brain-derived neurotrophic factor to striatum via nigral transduction of recombinant adeno-associated virus increases neuronal death but promotes neurogenic response following stroke.

To explore the role of brain‐derived neurotrophic factor for survival and generation of striatal neurons after stroke, recombinant adeno‐associated viral vectors carrying brain‐derived neurotrophic factor or green fluorescent protein genes were injected into right rat substantia nigra 4–5 weeks prior to 30 min ipsilateral of middle cerebral artery occlusion. The brain‐derived neurotrophic factor–recombinant adeno‐associated viral transduction markedly increased the production of brain‐derived neurotrophic factor protein by nigral cells. Brain‐derived neurotrophic factor was transported anterogradely to the striatum and released in biologically active form, as revealed by the hypertrophic response of striatal neuropeptide Y‐positive interneurons. Animals transduced with brain‐derived neurotrophic factor‐recombinant adeno‐associated virus also exhibited abnormalities in body posture and movements, including tilted body to the right, choreiform movements of left forelimb and head, and spontaneous, so‐called ‘barrel’ rotation along their long axis. The continuous delivery of brain‐derived neurotrophic factor had no effect on the survival of striatal projection neurons after stroke, but exaggerated the loss of cholinergic, and parvalbumin‐ and neuropeptide Y‐positive, γ‐aminobutyric acid‐ergic interneurons. The high brain‐derived neurotrophic factor levels in the animals subjected to stroke also gave rise to an increased number of striatal cells expressing doublecortin, a marker for migrating neuroblasts, and cells double‐labelled with the mitotic marker, 5‐bromo‐2′‐deoxyuridine‐5′monophosphate, and early neuronal (Hu) or striatal neuronal (Meis2) markers. Our findings indicate that long‐term anterograde delivery of high levels of brain‐derived neurotrophic factor increases the vulnerability of striatal interneurons to stroke‐induced damage. Concomitantly, brain‐derived neurotrophic factor potentiates the stroke‐induced neurogenic response, at least at early stages.

A simple method for large-scale generation of dopamine neurons from human embryonic stem cells

Dopamine (DA) neurons derived from human embryonic stem cells (hESCs) are potentially valuable in drug screening and as a possible source of donor tissue for transplantation in Parkinsons disease. However, existing culture protocols that promote the differentiation of DA neurons from hESCs are complex, involving multiple steps and having unreliable results between cultures. Here we report a simple and highly reproducible culture protocol that induces expandable DA neuron progenitors from hESCs in attached cultures. We found that the hESC‐derived neuronal progenitors retain their full capacity to generate DA neurons after repeated passaging in the presence of basic fibroblast growth factor (bFGF) and medium conditioned with PA6 stromal cells. Using immunocytochemistry and RT‐PCR, we found that the differentiated DA neurons exhibit a midbrain phenotype and express, e.g., Aldh1a, Ptx3, Nurr1, and Lmx1a. Using HPLC, we monitored their production of DA. We then demonstrated that the expanded progenitors are possible to cryopreserve without loosing the dopaminergic phenotype. With our protocol, we obtained large and homogeneous populations of dopaminergic progenitors and neurons. We conclude that our protocol can be used to generate human DA neurons suitable for the study of disease mechanisms, toxicology, drug screening, and intracerebral transplantation.