Brain injury activates microglia that induce neural stem cell proliferation ex vivo and promote differentiation of neurosphere-derived cells into neurons and oligodendrocytes

Brain damage, such as ischemic stroke, enhances proliferation of neural stem/progenitor cells (NSPCs) in the subventricular zone (SVZ). To date, no reliable in vitro systems, which can be used to unravel the potential mechanisms underlying this lesion-induced effect, have been established. Here, we developed an ex vivo method to investigate how the proliferation of NSPCs changes over time after experimental stroke or excitotoxic striatal lesion in the adult rat brain by studying the effects of microglial cells derived from an injured brain on NSPCs. We isolated NSPCs from the SVZ of brains with lesions and analyzed their growth and differentiation when cultured as neurospheres. We found that NSPCs isolated from the brains 1-2 weeks following injury consistently generated more and larger neurospheres than those harvested from naive brains. We attributed these effects to the presence of microglial cells in NSPC cultures that originated from injured brains. We suggest that the effects are due to released factors because we observed increased proliferation of NSPCs isolated from non-injured brains when they were exposed to conditioned medium from cultures containing microglial cells derived from injured brains. Furthermore, we found that NSPCs derived from injured brains were more likely to differentiate into neurons and oligodendrocytes than astrocytes. Our ex vivo system reliably mimics what is observed in vivo following brain injury. It constitutes a powerful tool that could be used to identify factors that promote NSPC proliferation and differentiation in response to injury-induced activation of microglial cells, by using tools such as proteomics and gene array technology.     

A neural stem/precursor cell monolayer for neural tissue engineering

The purpose of this study was to prepare a monolayer of neural stem/precursor cells (NSPCs) for neural tissue engineering applications. Two components present in serum, fibronectin and epidermal growth factor (EGF) were added into DMEM/F12 medium (termed medium B) to examine the effect of the migration-, proliferation- and differentiation-promoting potential on the cultured NSPCs, isolated from embryonic rat cerebral cortex. Compared with the serum effect, medium B also permitted neurosphere attachment onto the substrate surface and cell migration out of neurospheres extensively, but enhanced more extensive cell division and slowed down NSPC differentiation to generate a confluent NSPC monolayer. It was found the medium B-treated NSPCs possessed the capability to form typical neurospheres or to undergo differentiation into neuron-related cell types on various biomaterial surfaces. Therefore, we proposed a two-stage process for wound healing or nerve conduit preparation. Extensive NSPC division and MAP2-positive neuron differentiation were manipulated in NSPCs cultured in the medium B followed by the neuronal differentiation-favorable medium. These results should be useful for controlling the proliferation and differentiation of NSPCs on various biomaterials and conduits in neuroscience research.     

Lack of a p21waf1/cip -dependent G1/S checkpoint in neural stem and progenitor cells after DNA damage in vivo

The cyclin-dependent kinase inhibitor p21(waf1/cip) mediates the p53-dependent G1/S checkpoint, which is generally considered to be a critical requirement to maintain genomic stability after DNA damage. We used staggered 5-ethynyl-2'deoxyuridine/5-bromo-2'-deoxyuridine double-labeling in vivo to investigate the cell cycle progression and the role of p21(waf1/cip) in the DNA damage response of neural stem and progenitor cells (NSPCs) after exposure of the developing mouse cortex to ionizing radiation. We observed a radiation-induced p21-dependent apoptotic response in migrating postmitotic cortical cells. However, neural stem and progenitor cells (NSPCs) did not initiate a p21(waf1/cip1) -dependent G1/S block and continued to enter S-phase at a similar rate to the non-irradiated controls. The G1/S checkpoint is not involved in the mechanisms underlying the faithful transmission of the NSPC genome and/or the elimination of critically damaged cells. These processes typically involve intra-S and G2/M checkpoints that are rapidly activated after irradiation. p21 is normally repressed in neural cells during brain development except at the G1 to G0 transition. Lack of activation of a G1/S checkpoint and apoptosis of postmitotic migrating cells after DNA damage appear to depend on the expression of p21 in neural cells, since substantial cell-to-cell variations are found in the irradiated cortex. This suggests that repression of p21 during brain development prevents the induction of the G1/S checkpoint after DNA damage.     

Extramedullary chitosan channels promote survival of transplanted neural stem and progenitor cells and create a tissue bridge after complete spinal cord transection

Transplantation of neural stem and progenitor cells (NSPCs) is a promising strategy for repair after spinal cord injury. However, the epicenter of the severely damaged spinal cord is a hostile environment that results in poor survival of the transplanted NSPCs. We examined implantation of extramedullary chitosan channels seeded with NSPCs derived from transgenic green fluorescent protein (GFP) rats after spinal cord transection (SCT). At 14 weeks, we assessed the survival, maturation, and functional results using NSPCs harvested from the brain (brain group) or spinal cord (SC group) and seeded into chitosan channels implanted between the cord stumps after complete SCT. Control SCT animals had empty chitosan channels or no channels implanted. Channels seeded with brain or spinal cord-derived NSPCs showed a tissue bridge, although the bridges were thicker in the brain group. Both cell types showed long-term survival, but the number of surviving cells in the brain group was approximately five times as great as in the SC group. In both the brain and SC groups at 14 weeks after transplantation, many host axons were present in the center of the bridge in association with the transplanted cells. At 14 weeks astrocytic and oligodendrocytic differentiation in the channels was 24.8% and 17.3%, respectively, in the brain group, and 31.8% and 9.7%, respectively, in the SC group. The channels caused minimal tissue reaction in the adjacent spinal cord. There was no improvement in locomotor function. Thus, implantation of chitosan channels seeded with NSPCs after SCT created a tissue bridge containing many surviving transplanted cells and host axons, although there was no functional improvement.     

Distinctive effects of arachidonic acid and docosahexaenoic acid on neural stem /progenitor cells

Arachidonic acid (ARA) and docosahexaenoic acid (DHA), which are the dominant polyunsaturated fatty acids in the brain, have crucial roles in brain development and function. Recent studies have shown that ARA and DHA promote postnatal neurogenesis. However, the direct effects of ARA on neural stem/progenitor cells (NSPCs) and the effects of ARA and DHA on NSPCs at the neurogenic and subsequent gliogenic stages are still unknown. Here, we analyzed the effects of ARA and DHA on neurogenesis, specifically maintenance and differentiation, using neurosphere assays. We confirmed that primary neurospheres are neurogenic NSPCs and that tertiary neurospheres are gliogenic NSPCs. Regarding the effects of ARA and DHA on neurogenic NSPCs, ARA and DHA increased the number of neurospheres, whereas neither ARA nor DHA had a detectable effect on NSPCs in the differentiation condition. In gliogenic NSPCs, DHA increased the number of neurospheres, whereas ARA had no such effect. In contrast, ARA increased the number of astrocytes, whereas DHA increased the number of neurons in the differentiation condition. These results suggest that ARA promotes the maintenance of neurogenic NSPCs and might induce the glial differentiation of gliogenic NSPCs and that DHA promotes the maintenance of both neurogenic and gliogenic NSPCs and might lead to the neuronal differentiation of gliogenic NSPCs.     

Novel subventricular zone early progenitor cell-specific adenovirus for in vivo therapy of central nervous system disorders reinforces brain stem cell heterogeneity

Neural stem/progenitor cells (NSPCs) have the potential to self-renew and to generate all neural lineages as well as to repopulate damaged areas in the brain. Our previous targeting strategies have indicated precursor cell heterogeneity between different brain regions that warrants the development of NSPC-specific delivery vehicles. Here, we demonstrate a target-specific adenoviral vector system for the in vivo manipulation of progenitor cells in the subventricular zone of the adult mouse brain. For this purpose, we identified a series of peptide ligands via phage display. The peptide with the highest affinity, SNQLPQQ, was expressed in conjunction with a bispecific adaptor molecule. To verify the targeting potential of the specific peptide, green fluorescent protein-expressing Ad vectors were coupled with the adaptor molecule and injected into the subventricular region of adult mice by stereotaxic surgery. An efficient and selective transduction of NSPCs in the SVZ was achieved, whereas hippocampal NSPCs were negative. Our results offer an expeditious and simple tool to produce retargeted viral vectors for a specific and direct in vivo manipulation of these progenitor cells. This powerful technique provides an opportunity to develop innovative strategies and express therapeutic genes in specific types of neural progenitor cells to allow success in treatment of brain disorders.     

Postnatal neurogenesis generates heterotopias, olfactory micronodules and cortical infiltration following single-cell Tsc1 deletion

Neurological symptoms in tuberous sclerosis complex (TSC) and associated brain lesions are thought to arise from abnormal embryonic neurogenesis due to inherited mutations in Tsc1 or Tsc2. Neurogenesis persists postnatally in the human subventricular zone (SVZ) where slow-growing tumors containing Tsc-mutant cells are generated in TSC patients. However, whether Tsc-mutant neurons from the postnatal SVZ contribute to brain lesions and abnormal circuit remodeling in forebrain structures remain unexplored. Here, we report the formation of olfactory lesions following conditional genetic Tsc1 deletion in the postnatal SVZ using transgenic mice or targeted single-cell electroporation. These lesions include migratory heterotopias and olfactory micronodules containing neurons with a hypertrophic dendritic tree. Most significantly, our data identify migrating glial and neuronal precursors that are re-routed and infiltrate forebrain structures (e.g. cortex) and become glia and neurons. These data show that Tsc1-mutant cells from the neonatal and juvenile SVZ generate brain lesions and structural abnormalities, which would not be visible using conventional non-invasive imaging. These findings also raise the hypothesis that micronodules and the persistent infiltration of cells to forebrain structures may contribute to network malfunction leading to progressive neuropsychiatric symptoms in TSC.     

Leptomeningeal-derived doublecortin-expressing cells in poststroke brain

Increasing evidence indicates that neural stem/progenitor cells (NSPCs) reside in many regions of the central nervous system (CNS), including the subventricular zone (SVZ) of the lateral ventricle, subgranular zone of the hippocampal dentate gyrus, cortex, striatum, and spinal cord. Using a murine model of cortical infarction, we recently demonstrated that the leptomeninges (pia mater), which cover the entire cortex, also exhibit NSPC activity in response to ischemia. Pial-ischemia-induced NSPCs expressed NSPC markers such as nestin, formed neurosphere-like cell clusters with self-renewal activity, and differentiated into neurons, astrocytes, and oligodendrocytes, although they were not identical to previously reported NSPCs, such as SVZ astrocytes, ependymal cells, oligodendrocyte precursor cells, and reactive astrocytes. In this study, we showed that leptomeningeal cells in the poststroke brain express the immature neuronal marker doublecortin as well as nestin. We also showed that these cells can migrate into the poststroke cortex. Thus, the leptomeninges may participate in CNS repair in response to brain injury.     

Cross-talk between neural stem cells and immune cells: the key to better brain repair?

Systemic or intracerebral delivery of neural stem and progenitor cells (NSPCs) and activation of endogenous NSPCs hold much promise as potential treatments for diseases in the human CNS. Recent studies have shed new light on the interaction between the NSPCs and cells belonging to the innate and adaptive arms of the immune system. According to these studies, the immune cells can be both beneficial and detrimental for cell genesis from grafted and endogenous NSPCs in the CNS, and the NSPCs exert their beneficial effects not only by cell replacement but also by immunomodulation and trophic support. The cross-talk between immune cells and NSPCs and their progeny seems to determine both the efficacy of endogenous regenerative responses and the mechanism of action as well as the fate and functional integration of grafted NSPCs. Better understanding of the dialog between NSPCs and innate and adaptive immune cells is crucial for further development of effective strategies for CNS repair.     

Polycystic kidney disease as a result of loss of the tuberous sclerosis 2 tumor suppressor gene during development

Somatic loss of function of the tuberous sclerosis 2 (TSC2) tumor suppressor gene leads to the development of benign and malignant lesions of the kidney, brain, uterus, spleen, and liver and germline loss of function of this tumor suppressor gene is embryonic lethal. In addition, the gene product of TSC2, tuberin, is necessary for normal function of the polycystic kidney disease 1 (PKD1) gene product, polycystin-1, which is required for normal cell-cell and cell-matrix interactions. We report here the development of severe polycystic kidney disease in three cases of young Eker rats carrying a germline inactivation of one allele of the Tsc2 gene. Extrarenal tumors were also noted in the spleen and uterus of these animals, which was remarkable given their young age and in the case of the spleen, diffuse involvement of the affected organ. A cell line (EKT2) was established from an affected kidney of one of these animals and used in conjunction with tissues from affected animals to elucidate the defect responsible for the development of these lesions. Affected cells were determined to have lost the wild-type Tsc2 allele while retaining two copies of chromosome 10 containing the mutant Tsc2 allele along with two normal copies of the Pkd1 gene. The genetic data, bilateral nature of the observed kidney disease, and extent of involvement of the spleen and kidney indicate that, in affected animals, loss of the wild-type Tsc2 allele occurred during embryogenesis, probably as a result of chromosome nondisjunction, with affected animals being mosaics for loss of Tsc2 gene function.     

Gallium nitride induces neuronal differentiation markers in neural stem/precursor cells derived from rat cerebral cortex

In the present study, gallium nitride (GaN) was used as a substrate to culture neural stem/precursor cells (NSPCs), isolated from embryonic rat cerebral cortex, to examine the effect of GaN on the behavior of NSPCs in the presence of basic fibroblast growth factor (bFGF) in serum-free medium. Morphological studies showed that neurospheres maintained their initial shape and formed many long and thick processes with the fasciculate feature on GaN. Immunocytochemical characterization showed that GaN could induce the differentiation of NSPCs into neurons and astrocytes. Compared to poly-d-lysine (PDL), the most common substrate used for culturing neurons, there was considerable expression of synapsin I for differentiated neurons on GaN, suggesting GaN could induce the differentiation of NSPCs towards the mature differentiated neurons. Western blot analysis showed that the suppression of glycogen synthase kinase-3β (GSK-3β) activity was one of the effects of GaN-promoted NSPC differentiation into neurons. Finally, compared to PDL, GaN could significantly improve cell survival to reduce cell death after long-term culture. These results suggest that GaN potentially has a combination of electric characteristics suitable for developing neuron and/or NSPC chip systems.     

The effects of peptide modified gellan gum and olfactory ensheathing glia cells on neural stem/progenitor cell fate

The regenerative capacity of injured adult central nervous system (CNS) tissue is very limited. Specifically, traumatic spinal cord injury (SCI) leads to permanent loss of motor and sensory functions below the site of injury, as well as other detrimental complications. A potential regenerative strategy is stem cell transplantation; however, cell survival is typically less than 1%. To improve cell survival, stem cells can be delivered in a biomaterial matrix that provides an environment conducive to survival after transplantation. One major challenge in this approach is to define the biomaterial and cell strategies in vitro. To this end, we investigated both peptide-modification of gellan gum and olfactory ensheathing glia (OEG) on neural stem/progenitor cell (NSPC) fate. To enhance cell adhesion, the gellan gum (GG) was modified using Diels-Alder click chemistry with a fibronectin-derived synthetic peptide (GRGDS). Amino acid analysis demonstrated that approximately 300 nmol of GRGDS was immobilized to each mg of GG. The GG-GRGDS had a profound effect on NSPC morphology and proliferation, distinct from that of NSPCs in GG alone, demonstrating the importance of GRGDS for cell-GG interaction. To further enhance NSPC survival and outgrowth, they were cultured with OEG. Here NSPCs interacted extensively with OEG, demonstrating significantly greater survival and proliferation relative to monocultures of NSPCs. These results suggest that this co-culture strategy of NSPCs with OEG may have therapeutic benefit for SCI repair.     

Ischemia-induced neural stem/progenitor cells in the pia mater following cortical infarction

Increasing evidence shows that neural stem/progenitor cells (NSPCs) can be activated in the nonconventional neurogenic zones such as the cortex following ischemic stroke. However, the precise origin, identity, and subtypes of the ischemia-induced NSPCs (iNSPCs), which can contribute to cortical neurogenesis, is currently still unclear. In our present study, using an adult mouse cortical infarction model, we found that the leptomeninges (pia mater), which is widely distributed within and closely associated with blood vessels as microvascular pericytes/perivascular cells throughout central nervous system (CNS), have NSPC activity in response to ischemia and can generate neurons. These observations indicate that microvascular pericytes residing near blood vessels that are distributed from the leptomeninges to the cortex are potential sources of iNSPCs for neurogenesis following cortical infarction. In addition, our results propose a novel concept that the leptomeninges, which cover the entire brain, have an important role in CNS restoration following brain injury such as stroke.     

Transplanted adult spinal cord-derived neural stem/progenitor cells promote early functional recovery after rat spinal cord injury

We examined the effect of spinal cord-derived neural stem/progenitor cells (NSPCs) after delayed transplantation into the injured adult rat spinal cord with or without earlier transplantation of bone marrow-derived mesenchymal stromal cells (BMSCs). Either BMSCs or culture medium were transplanted immediately after clip compression injury (27 g force), and then, 9 days after injury, NSPCs or culture medium were transplanted. Cell survival and differentiation, functional recovery, retrograde axonal tracing, and immunoelectron microscopy were assessed. A significant improvement in functional recovery based on three different measures was seen only in the group receiving NSPCs without BMSCs, and the improved recovery was evident within 1 week of transplantation. In this group, NSPCs differentiated mainly into oligodendrocytes and astrocytes, there was ensheathing of axons at the injury site by transplanted NSPCs, an increase in host oligodendrocytes, and a trend toward an increase in retrogradely labeled supraspinal nuclei. Transplantation of the BMSC scaffold resulted in a trend toward improved survival of the NSPCs, but there was no increase in function. Thus, transplantation of adult rat NSPCs produced significant early functional improvement after spinal cord injury, suggesting an early neuroprotective action associated with oligodendrocyte survival and axonal ensheathment by transplanted NSPCs.     

Therapeutic value of prenatal rapamycin treatment in a mouse brain model of tuberous sclerosis complex

Epileptic seizures, particularly infantile spasms, are often seen in infants with tuberous sclerosis complex (TSC) soon after birth. It is feared that there are long-term developmental and cognitive consequences from ongoing, frequent epilepsy. In addition, the hallmark brain pathology of TSC, cortical tubers and giant cells are fully developed at late gestational ages. These observations have led us to examine the benefit of prenatal rapamycin in a new fetal brain model of TSC. In this Tsc1(cc) Nes-cre(+) mouse model, recombination and loss of Tsc1 in neural progenitor cells leads to brain enlargement, hyperactivation of mTOR, and neonatal death on P0 due to reduced pup-maternal interaction. A single dose of prenatal rapamycin given to pregnant dams (1 mg/kg, subcutaneous) rescued the lethality of mutant mice. This one dose of prenatal rapamycin treatment reduced hyperactivation of the mTOR pathway in the mutant brain without causing apparent pregnancy loss. Continued postnatal rapamycin beginning at day 8 extended the survival of these mice to a median of 12 days with complete suppression of hyperactive mTOR. However, the rapamycin-treated mutants developed enlarged brains with an increased number of brain cells, displaying marked runting and developmental delay. These observations demonstrate the therapeutic benefit and limitations of prenatal rapamycin in a prenatal-onset brain model of TSC. Our data also suggest the possibility and limitations of this approach for TSC infants and mothers.     

A mouse model of tuberous sclerosis 1 showing background specific early post-natal mortality and metastatic renal cell carcinoma

Tuberous sclerosis complex (TSC) is an autosomal dominant disorder caused by mutations in either the TSC1 or the TSC2 genes and characterized by the development of benign hamartomatous growths in multiple organ systems. We have inactivated Tsc1 in the mouse germ line by gene targeting in ES cells and confirmed that the mutant allele (Tsc1-) has a recessive embryonic lethal phenotype. We found that a significant number (approximately 27%) of heterozygous (Tsc1+/-) mice on the C57BL/6 background died before weaning (P = 0.014) and show that these mice die in the post-natal period (P = 0.033), normally at 1-2 days, from unknown causes. Forty-four percent (7/16) of Tsc1+/- mice on a C3H background developed macroscopically visible renal lesions as early as 3-6 months, increasing to 95% (37/39) by 15-18 months. Renal lesions progressed from cysts through cystadenomas to solid carcinomas. Eighty percent (16/20) of Tsc1+/- mice on a Balb/c background exhibited solid renal cell carcinomas (RCC) by 15-18 months and in 41%, RCCs were > or = 5 mm, resulting in grossly deformed kidneys. Some RCCs had a sarcomatoid morphology of spindle cells in whorled patterns and metastasized to the lungs. We detected loss of the wild-type Tsc1 allele and elevated levels of p-mTOR and p-S6 in lesions from Tsc1+/- mice. This new murine model of hamartin deficiency exhibits a more severe phenotype than existing models.     

Controlled epi-cortical delivery of epidermal growth factor for the stimulation of endogenous neural stem cell proliferation in stroke-injured brain

One of the challenges in treating central nervous system (CNS) disorders with biomolecules is achieving local delivery while minimizing invasiveness. For the treatment of stroke, stimulation of endogenous neural stem/progenitor cells (NSPCs) by growth factors is a promising strategy for tissue regeneration. Epidermal growth factor (EGF) enhances proliferation of endogenous NSPCs in the subventricular zone (SVZ) when delivered directly to the ventricles of the brain; however, this strategy is highly invasive. We designed a biomaterials-based strategy to deliver molecules directly to the brain without tissue damage. EGF or poly(ethylene glycol)-modified EGF (PEG-EGF) was dispersed in a hyaluronan and methylcellulose (HAMC) hydrogel and placed epi-cortically on both uninjured and stroke-injured mouse brains. PEG-modification decreased the rate of EGF degradation by proteases, leading to a significant increase in protein accumulation at greater tissue depths than previously shown. Consequently, EGF and PEG-EGF increased NSPC proliferation in uninjured and stroke-injured brains; and in stroke-injured brains, PEG-EGF significantly increased NSPC stimulation. Our epi-cortical delivery system is a minimally-invasive method for local delivery to the brain, providing a new paradigm for local delivery to the brain.     

Pharmacological MRI of stem cell transplants in the 3-nitropropionic acid-damaged striatum

Blood oxygen level dependent (BOLD) pharmacological magnetic resonance imaging (phMRI) affords the non-invasive visualization of brain activity resulting from the administration of pharmacological compounds. Once the compound-responsive cells are lost, no change in activity is expected to occur. This principle therefore allows the assessment of neuronal loss or lack of signal transmission. These investigations can provide evidence of pathology in the absence of significant tissue loss and can be highly specific to determine which type of cell has been lost. Conversely, transplantation of cells replacing the lost neurons should restore normal signal transmission. We here demonstrate the application of phMRI to differentiate between rats with 3-nitroproprionic acid (3-NPA)-induced striatal lesions and 3-NPA-lesioned animals with neural stem cell transplants or controls. 3-NPA-induced lesions mainly involve striatal projection neurons that are responsive to dopamine agonists. The D2-agonist bromocriptine acts on these projection cells and loss of these through 3-NPA administration resulted in a significant decrease of locomotor activity and a substantial attenuation of the BOLD-response in the striatum. In contrast, lesioned animals that were grafted with neural stem cells exhibited an activity pattern akin to controls. Hence, grafting of neural stem cells exerts a functionally significant effect on striatal signal transmission that could underpin behavioral recovery.