Calcium influx differentially regulates migration velocity and directedness in response to electric field application.

Neural precursor cells (NPCs) respond to externally applied direct current electrical fields (DCEFs) by undergoing rapid and directed migration toward the cathode in a process known as galvanotaxis. It is unknown if the underlying mechanisms of galvanotactic migration is common to non-electrosensitive cells and if so, how NPCs and other galvanotactic cells sense and transduce electrical fields into cellular motility. In this study, we show that distinct aspects of NPC galvanotactic migration: motility (quantified through |velocity|) and directedness, are differentially regulated by calcium. We use low-Ca2+ culture conditions; an intracellular Ca2+ chelator; and voltage gated calcium channel (VGCC) inhibitors to specific channels expressed on NPCs, to demonstrate the role of Ca2+ influx in DCEF-induced NPC migration. Consistent with existing literature, we show Ca2+ is involved in F-actin polymerization that lengthens NPC membrane protrusions necessary for cellular motility. However, inhibiting Ca2+ results in reduced velocity but has no effect on DCEF-induced directedness. This dissociation between velocity and directedness reveal that these migration parameters can be independently regulated, thus suggesting a parallel process of sensing DCEFs by NPCs.

Injectable hydrogel promotes early survival of induced pluripotent stem cell-derived oligodendrocytes and attenuates longterm teratoma formation in a spinal cord injury model.

Transplantation of pluripotent stem cells and their differentiated progeny has the potential to preserve or regenerate functional pathways and improve function after central nervous system injury. However, their utility has been hampered by poor survival and the potential to form tumors. Peptide-modified biomaterials influence cell adhesion, survival and differentiation in vitro, but their effectiveness in vivo remains uncertain. We synthesized a peptide-modified, minimally invasive, injectable hydrogel comprised of hyaluronan and methylcellulose to enhance the survival and differentiation of human induced pluripotent stem cell-derived oligodendrocyte progenitor cells. Cells were transplanted subacutely after a moderate clip compression rat spinal cord injury. The hydrogel, modified with the RGD peptide and platelet-derived growth factor (PDGF-A), promoted early survival and integration of grafted cells. However, prolific teratoma formation was evident when cells were transplanted in media at longer survival times, indicating that either this cell line or the way in which it was cultured is unsuitable for human use. Interestingly, teratoma formation was attenuated when cells were transplanted in the hydrogel, where most cells differentiated to a glial phenotype. Thus, this hydrogel promoted cell survival and integration, and attenuated teratoma formation by promoting cell differentiation.

A new 'spin' on neural stem cells?

The existence of neural stem cells in the adult brain was essentially denied until the last decade. Within the past ten years, considerable progress has been made in examining the fundamental properties of neural stem cells. Most recently there has been much interest in the identification and precise location of the adult neural stem cells in vivo. Studies examining the localization of neural stem cells are controversial and suggest two distinct locations within the adult brain: the ependymal layer lining the ventricles, and the subependymal layer immediately adjacent to the ependyma.

A cell-survival factor (N-acetyl-L-cysteine) alters the in vivo fate of constitutively proliferating subependymal cells in the adult forebrain.

The adult mouse brain contains a population of constitutively proliferating subependymal cells that surround the lateral ventricle and are the direct progeny of the neural stem cell. Constitutively proliferating cells divide rapidly; 6 days after labeling, 60% of their progeny undergo cell death, 25% migrate to the olfactory bulbs, and 15% continue to proliferate within the subependyma. We have intraventricularly infused a cell survival factor N-acetyl-L-cysteine (NAC), which is known to have survival effects without concomitant proliferative effects on cells in vitro, and examined the resulting fate of cells spared from the normally occurring cell death. NAC infusion for 5 days results in a five-fold increase in the number of retrovirally labeled subependymal cells compared to saline-infused controls. The increase in the number of subependymal cells is directly proportional to the amount of time during which NAC is present and is not due to increased proliferation. While NAC is able to keep all the normally dying progeny alive, the cells spared from death remain confined to the subependyma lining the lateral ventricles and do not migrate to the olfactory bulbs (one normal fate of constitutively proliferating progeny) or into the surrounding brain parenchyma. When animals survive for an additional 6 days following NAC infusion, the number of retrovirally labeled subependymal cells returns to control values, indicating that the continued presence of NAC is necessary for cell survival. These data suggest that preventing cell death is not sufficient to keep all of the progeny of these cells in a proliferative mode.

Migrational analysis of the constitutively proliferating subependyma population in adult mouse forebrain.

Initial experiments to evaluate the in vivo fate(s) of constitutively proliferating subependymal cells determined that, following in vivo labeling of this population by infection with a retrovirus containing a beta-galactosidase reporter gene, there was a progressive and eventually complete loss of histochemically beta-galactosidase-positive cells within the lateral ventricle subependyma with increasing survival times of up to 28 days after retroviral infection. Subsequent experiments were designed to ascertain the potential contributions of: (i) the migration of subependymal cells away from the forebrain lateral ventricles; and (ii) the down-regulation of the retroviral reporter gene expression. Retroviral lineage tracing experiments demonstrate that a major in vivo fate for constitutively proliferating subependymal cells is their rostral migration away from the walls of the lateral ventricle to the olfactory bulb. Although down-regulation of retroviral reporter gene expression does not contribute to the loss of detection of beta-galactosidase-labeled cells from the lateral ventricle subependyma, it does result in an underestimation of the absolute number of retrovirally labeled cells in the olfactory bulb at longer survival times. Furthermore, a temporal decrease in the double labeling of beta-galactosidase-labeled cells with [3H]thymidine was observed, indicating that only a subpopulation of the migratory subependymal-derived cells continue to actively proliferate en route to the olfactory bulb. These two events may contribute to the lack of a significant increase in the total number of retrovirally labeled subependymal cells during rostral migration. Evidence from separately published studies suggests that cell death is also an important regulator of the size of the constitutively proliferating subependymal population. In summary, in vivo studies utilizing retroviral reporter gene labeling demonstrate that constitutively proliferating subependymal cells born in the lateral ventricle migrate rostrally to the olfactory bulb. Loss of proliferation potential and retroviral reporter gene down-regulation contribute to the lack of any significant increase in the total number of labeled cells recovered in the olfactory bulb.

Adult mammalian forebrain ependymal and subependymal cells demonstrate proliferative potential, but only subependymal cells have neural stem cell characteristics.

The adult derivatives of the embryonic forebrain germinal zones consist of two morphologically distinct cell layers surrounding the lateral ventricles: the ependyma and the subependyma. Cell cycle analyses have revealed that at least two proliferating populations exist in this region, one that is constitutively proliferating and one that is relatively quiescent and thought to include the endogenous adult neural stem cells. Earlier studies demonstrated that specific dissection of the region surrounding the lateral ventricles was necessary for the in vitro isolation of multipotent, self-renewing neural stem cells. However, in these studies, the ependymal layer was not physically separated from the subependymal layer to identify the specific adult laminar localization of the neural stem cells around the lateral ventricles. To determine which cellular compartment in the adult forebrain contained the neural stem cells, we isolated and cultured the ependyma separately from the subependyma and tested for the presence of neural stem cells using the in vitro neurosphere assay. We demonstrate that the ependymal cells can proliferate in vitro to form sphere-like structures. However, the ependymal cells generating spheres do not have the ability to self-renew (proliferate to form secondary spheres after dissociation) nor to produce neurons, but rather only seem to generate glial fibrillary acidic protein-positive ependymal cells when plated under differentiation conditions in culture. On the other hand, a subpopulation of subependymal cells do possess the self-renewing and multipotential characteristics of neural stem cells. Therefore, the adult forebrain neural stem cell resides within the subependymal compartment.

In vivo clonal analyses reveal the properties of endogenous neural stem cell proliferation in the adult mammalian forebrain.

The adult mammalian forebrain contains a population of multipotential neural stem cells in the subependyma of the lateral ventricles whose progeny are the constitutively proliferating cells, which divide actively throughout life. The adult mammalian brain is ideal for examining the kinetics of the stem cells due to their strict spatial localization and the limited and discrete type of progeny generated (constitutively proliferating cells). Clonal lineage analyses 6 days after retrovirus infection revealed that under baseline conditions 60% of the constitutively proliferating cells undergo cell death, 25% migrate to the olfactory bulb and 15% remain confined to the lateral ventricle subependyma (where they reside for approximately 15 days). Analysis of single cell clones 31 days after retroviral infection revealed that the stem cell divides asymmetrically to self-renew and give rise to constitutively proliferating cells. Following repopulation of the depleted subependyma the average clone size is 2.8 times larger than control, yet the absolute number of cells migrating to the olfactory bulb is maintained and the stem cell retains its asymmetric mode of division. The number of neural stem cells in the adult forebrain 33 days after repopulation of the subependyma was estimated using bromodeoxyuridine labeling of subepenydmal cells. There were calculated to be 1200-1300 cells between the rostral corpus callosum and rostral anterior commissure; these data support a lineage model similar to those based on stem cell behavior in other tissue types.

Transforming growth factor-alpha null and senescent mice show decreased neural progenitor cell proliferation in the forebrain subependyma.

The adult mammalian forebrain subependyma contains neural stem cells and their progeny, the constitutively proliferating progenitor cells. Using bromodeoxyuridine labeling to detect mitotically active cells, we demonstrate that the endogenous expression of transforming growth factor-alpha (TGFalpha) is necessary for the full proliferation of progenitor cells localized to the dorsolateral corner of the subependyma and the full production of the neuronal progenitors that migrate to the olfactory bulbs. Proliferation of these progenitor cells also is diminished with age (in 23- to 25-months-old compared with 2- to 4-months-old mice), likely because of a lengthening of the cell cycle. Senescence or the absence of endogenous TGFalpha does not affect the numbers of neural stem cells isolated in vitro in the presence of epidermal growth factor. These results suggest that endogenous TGFalpha and the effects of senescence may regulate the proliferation of progenitor cells in the adult subependyma, but that the number of neural stem cells is maintained throughout life.

Is there a neural stem cell in the mammalian forebrain?

Neural precursor cells have been of interest historically as the building blocks of the embryonic CNS and, most recently, as substrates for restorative neurological approaches. The majority of previous in vitro studies of the regulation of neural-cell proliferation by polypeptide growth factors, and in vivo studies of neural lineage, argue for the presence of precursors with limited proliferative or lineage potential in the mammalian CNS. This is in contrast to renewable tissues, such as the blood or immune system, skin epithelium and epithelium of the small intestinal crypts, which contain specialized, self-renewing cells known as stem cells. However, recent in vitro and in vivo studies from our and other laboratories lead us to conclude that neural stem cells, with self-renewal and multilineage potential, are present in the embryonic through to adult mammalian forebrain.

In vivo growth factor expansion of endogenous subependymal neural precursor cell populations in the adult mouse brain.

The lateral ventricle subependyma in the adult mammalian forebrain contains both neural stem and progenitor cells. This study describes the in situ modulation of these subependymal neural precursor populations after intraventricular administration of exogenous growth factors. In vivo infusion of epidermal growth factor (EGF) into adult mouse forebrain for 6 consecutive days resulted in a dramatic increase in the proliferation and total number of subependymal cells and induced their migration away from the lateral ventricle walls into adjacent parenchyma. Immediately after EGF infusion, immunohistochemical characterization of the EGF-expanded cell population demonstrated that >95% of these cells were EGF receptor- and nestin-positive, whereas only 0.9% and 0.2% labeled for astrocytic and neuronal markers, respectively. Seven weeks after EGF withdrawal, 25% of the cells induced to proliferate after 6d of EGF were still detectable; 28% of these cells had differentiated into new astrocytes and 3% into new neurons in the cortex, striatum, and septum. Newly generated oligodendrocytes were also observed. These in vivo results (1) confirm the existence of EGF-responsive subependymal neural precursor cells in the adult mouse forebrain and (2) suggest that EGF acts directly as a proliferation, survival, and migration factor for subependymal precursor cells to expand these populations and promote the movement of these cells into normal brain parenchyma. Thus, in situ modulation of endogenous forebrain precursor cells represents a novel model for studying neural development in the adult mammalian brain and may provide insights that will achieve adult replacement of neurons and glia lost to disease or trauma.

Neural stem cells in the adult mammalian forebrain: a relatively quiescent subpopulation of subependymal cells.

Dissection of the subependyma from the lateral ventricle of the adult mouse forebrain is necessary and sufficient for the in vitro formation of clonally derived spheres of cells that exhibit stem cell properties such as self-maintenance and the generation of a large number of progeny comprising the major cell types found in the central nervous system. Killing the constitutively proliferating cells of the subependyma in vivo has no effect on the number of stem cells isolated in vitro and induces a complete repopulation of the subependyma in vivo by relatively quiescent stem cells found within the subependyma. Depleting the relatively quiescent cell population within the subependyma in vivo results in a corresponding decrease in spheres formed in vitro and in the final number of constitutively proliferating cells in vivo, suggesting that a relatively quiescent subependymal cell is the in vivo source of neural stem cells.

Satellite cell proliferation in the adult rat trigeminal ganglion results from the release of a mitogenic protein from explanted sensory neurons.

Explant of trigeminal ganglia neurons in adult rats induces perineuronal glial proliferation of primarily satellite cells as opposed to Schwann cells. This proliferation begins at 15 h after explant culture and by 27 h there is a significant increase in glial proliferation as measured by scintillation counts of [3H]thymidine. Blocking protein synthesis between 0 and 3.5 h after explant culture (early) results in an enhanced proliferative response, while blocking protein synthesis between 3.5 and 7 h (late) causes a complete block of the proliferative response assessed at 27 h. Conditioned media experiments demonstrate that both the mitogenic and inhibitory signals are diffusible and heat labile. Finally, the addition of neurotrophic factors to rescue injured ganglionic neurons attenuates the proliferative glial response suggesting that injured neurons produce and release signals that induce glial proliferation.

Postmitotic death is the fate of constitutively proliferating cells in the subependymal layer of the adult mouse brain.

The early development of the mammalian forebrain involves the massive proliferation of the ventricular zone cells lining the lateral ventricles. A remnant of this highly proliferative region persists into adult life, where it is known as the subependymal layer. We examined the proliferation kinetics and fates of the mitotically active cells in the subependyma of the adult mouse. The medial edge, the lateral edge, and the dorsolateral corner of the subependymal layer of the rostral portion of the lateral ventricle each contained mitotically active cells, but the dorsolateral region had the highest percentage of bromodeoxyuridine (BrdU)-labeled cells per unit area. Repeated injections of BrdU over 14 hr revealed a proliferation curve for the dorsolateral population with a growth fraction of 33%, indicating that 33% of the cells in this subependymal region make up the proliferating population. The total cell cycle time in this population was approximately 12.7 hr, with an S-phase of 4.2 hr. To examine the fate of these proliferating cells, we injected low concentrations of a replication-deficient, recombinant retrovirus directly into the lateral ventricles of adult mice for uptake by mitotically active subependymal cells. Regardless of the survival time postinjection (10 hr, 1 d, 2 d, or 8 d), the number of retrovirally labeled cells per clone remained the same (1 or 2 cells/clone). This suggests that one of the progeny from each cell division dies. Moreover, the clones remained confined to the subependyma and labeled cells were not seen in the surrounding brain tissue. Thus, while 33% of the dorsolateral subependymal cells continue to proliferate in adult life, the fate of the postmitotic progeny is death.

Post-herpetic neuralgia: further post-mortem studies of cases with and without pain.

The pathological features associated with post-herpetic neuralgia require further study. We report here 5 cases, 3 with severe post-herpetic neuralgia (PHN) and 2 with no persistent pain. The findings of dorsal horn atrophy and cell, axon and myelin loss with fibrosis in the sensory ganglion were found only in patients with persistent pain. Marked loss of myelin and axons in the nerve and/or sensory root were found in cases with and without pain. Some evidence is presented for a more generalized subacute or chronic inflammatory process which may explain the clinical features of some patients. Further studies will be necessary to fully describe the morbid anatomy of this disorder.

Separate blood and brain origins of proliferating cells during gliosis in adult brains.

The response of the brain to injury involves the accumulation of a large number of proliferating cells at the site of damage. Neither the identity nor the origin of these cells is unequivocally established. We have investigated this proliferative response after unilateral kainic acid lesions in the striatum of adult mice by labeling with tritiated thymidine (3H-thy) or bromodeoxyuridine (Brdu) to identify cells passing through S-phase. Labeled cells were seen only ipsilaterally in coronal section and extended laterally from the subependymal zone lining the lateral ventricle, through the striatal kainic acid injection site and into the cortex. The maximum proliferative response, after a single pulse of 3H-thy administered 4 h before sacrifice, was seen 6 days post-lesion close to the injection site. The proliferating cells were not astrocytes, as neither 3H-thy- nor Brdu-labeled cells were double-labeled with antisera to glial fibrillary acidic protein after the lesion. Animals given 3H-thy on day 3 post-lesion and then sacrificed on days 4, 5 or 6 post-lesion showed cumulative increases in the number of proliferating cells at the injection site with no increases in the surrounding tissue. We hypothesized that this reflected the presence of 2 sources of labeled cells: (1) an exogenous population of blood cells coming in through the broken blood-brain barrier and accumulating at the injection site and (2) endogenous cells (microglia) which are normally quiescent in the adult but proliferate in response to injury. By irradiating adult mice (900 rads) we attempted to selectively remove the blood stem cell precursors which gave rise to the proposed exogenous source of cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Post-herpetic neuralgia: post-mortem analysis of a case.

The morphological and biochemical substrates of the severe pain in post-herpetic neuralgia (PHN) are unclear. This report is an autopsy study of a 67-year-old male with severe PHN during the last 5 years of his life over the right T7-8 dermatomes. The dorsal horn of the thoracic spinal cord of the affected side was atrophic from T4 to T8, with loss of both myelin and axons. Despite this, only the T8 ganglion was affected by fibrosis and cell loss and only the nerve roots at that level appeared affected. Markers of unmyelinated afferents (substance P), substantia gelatinosa neurons (opiate receptors), glial cells (glial fibrillary acidic protein), and descending spinal projections (dopamine-beta-hydroxylase and serotonin) were not different at affected versus non-affected spinal cord levels. The pain of PHN may result from the uninhibited activity of unmyelinated primary afferents as a result of the loss of myelinated afferent fibers and the possible presence of hypersensitive neurons in the dorsal horn.