Cindi M. Morshead

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.

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.

Delayed Transplantation of Adult Neural Precursor Cells Promotes Remyelination and Functional Neurological Recovery after Spinal Cord Injury

Spinal cord injury (SCI) results in loss of oligodendrocytes demyelination of surviving axons and severe functional impairment. Spontaneous remyelination is limited. Thus, cell replacement therapy is an attractive approach for myelin repair. In this study, we transplanted adult brain-derived neural precursor cells (NPCs) isolated from yellow fluorescent protein-expressing transgenic mice into the injured spinal cord of adult rats at 2 and 8 weeks after injury, which represents the subacute and chronic phases of SCI. A combination of growth factors, the anti-inflammatory drug minocycline, and cyclosporine A immunosuppression was used to enhance the survival of transplanted adult NPCs. Our results show the presence of a substantial number of surviving NPCs in the injured spinal cord up to 10 weeks after transplantation at the subacute stage of SCI. In contrast, cell survival was poor after transplantation into chronic lesions. After subacute transplantation, grafted cells migrated >5 mm rostrally and caudally. The surviving NPCs integrated principally along white-matter tracts and displayed close contact with the host axons and glial cells. Approximately 50% of grafted cells formed either oligodendroglial precursor cells or mature oligodendrocytes. NPC-derived oligodendrocytes expressed myelin basic protein and ensheathed the axons. We also observed that injured rats receiving NPC transplants had improved functional recovery as assessed by the Basso, Beattie, and Bresnahan Locomotor Rating Scale and grid-walk and footprint analyses. Our data provide strong evidence in support of the feasibility of adult NPCs for cell-based remyelination after SCI.

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.

Hematopoietic competence is a rare property of neural stem cells that may depend on genetic and epigenetic alterations

The concept of stem-cell plasticity received strong support from a recent observation that extensively passaged, clonally derived neural stem cells could contribute to hematopoiesis. We investigated whether hematopoietic potential was a consistent or unusual feature of neural stem cells, and whether it depended on the extent of in vitro passaging before transplantation. Here we transplanted over 128 × 106 neurosphere cells into 128 host animals; however, we never observed contribution to hematopoiesis, irrespective of the number of passages and despite the use of an assay that could detect the contribution of a single blood stem cell to hematopoietic repopulation. Although extensively cultured neurosphere cells continued to generate neural progeny, marked changes in their growth properties occurred, including changes in growth-factor dependence, cell-cycle kinetics, cell adhesion and gene expression. Our results exclude hematopoietic competence as a consistent property of intravenously infused neural stem cells. However, the consistent changes that occurred during extended passaging are compatible with genetic or epigenetic alterations and suggest that rare transformation events may account for the neural-to-blood fate switch originally reported.

Activated Neural Stem Cells Contribute to Stroke-Induced Neurogenesis and Neuroblast Migration toward the Infarct Boundary in Adult Rats:

Stroke increases neurogenesis. The authors investigated whether neural stem cells or progenitor cells in the adult subventricular zone (SVZ) of rats contribute to stroke-induced increase in neurogenesis. After induction of stroke in rats, the numbers of cells immunoreactive to doublecortin, a marker for immature neurons, increased in the ipsilateral SVZ and striatum. Infusion of an antimitotic agent (cytosine-β-D-arabiofuranoside, Ara-C) onto the ipsilateral cortex eliminated more than 98% of actively proliferating cells in the SVZ and doublecortin-positive cells in the ipsilateral striatum. However, doublecortin-positive cells rapidly replenished after antimitotic agent depletion of actively proliferating cells. Depleting the numbers of actively proliferating cells in vivo had no effect on the numbers of neurospheres formed in vitro, yet the numbers of neurospheres derived from stroke rats significantly (P < 0.05) increased. Neurospheres derived from stroke rats self-renewed and differentiated into neurons and glia. In addition, doublecortin-positive cells generated in the SVZ migrated in a chainlike structure toward ischemic striatum. These findings indicate that in the adult stroke brain, increases in recruitment of neural stem cells contribute to stroke-induced neurogenesis, and that newly generated neurons migrate from the SVZ to the ischemic striatum.

Spatially controlled simultaneous patterning of multiple growth factors in three-dimensional hydrogels

Three-dimensional (3D) protein-patterned scaffolds provide a more biomimetic environment for cell culture than traditional two-dimensional surfaces, but simultaneous 3D protein patterning has proved difficult. We developed a method to spatially control the immobilization of different growth factors in distinct volumes in 3D hydrogels, and to specifically guide differentiation of stem/progenitor cells therein. Stem-cell differentiation factors sonic hedgehog (SHH) and ciliary neurotrophic factor (CNTF) were simultaneously immobilized using orthogonal physical binding pairs, barnase-barstar and streptavidin-biotin, respectively. Barnase and streptavidin were sequentially immobilized using two-photon chemistry for subsequent concurrent complexation with fusion proteins barstar-SHH and biotin-CNTF, resulting in bioactive 3D patterned hydrogels. The technique should be broadly applicable to the patterning of a wide range of proteins.

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

&NA; 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.

The ablation of glial fibrillary acidic protein-positive cells from the adult central nervous system results in the loss of forebrain neural stem cells but not retinal stem cells.

The adult mammalian forebrain subependyma contains neural stem cells (NSCs) capable of self‐renewal and multilineage differentiation. The in vivo identification of NSCs has not been definitively addressed using a loss of function approach. Using a transgenic mouse expressing herpes‐simplex virus thymidine kinase from the glial fibrillary acidic protein (GFAP) promotor, we have selectively killed dividing GFAP‐positive cells in the presence of ganciclovir (GCV) and shown a > 95% loss in the numbers of NSCs, as assayed by the formation of clonally derived neurospheres in vitro. This loss is seen following 3 days of GCV exposure in vivo or in vitro only and cannot be rescued by coculturing with pure astrocyte populations or control (green fluorescent protein‐expressing) subependymal cells. Exposure to GCV in vitro has no effect on adult retinal stem cells hence, we conclude that adult forebrain NSCs comprise a subpopulation of the GFAP‐positive cells within the subependyma.

Support for the immortal strand hypothesis neural stem cells partition DNA asymmetrically in vitro

The immortal strand hypothesis proposes that asymmetrically dividing stem cells (SCs) selectively segregate chromosomes that bear the oldest DNA templates. We investigated cosegregation in neural stem cells (NSCs). After exposure to the thymidine analogue 5-bromo-2-deoxyuridine (BrdU), which labels newly synthesized DNA, a subset of neural precursor cells were shown to retain BrdU signal. It was confirmed that some BrdU-retaining cells divided actively, and that these cells exhibited some characteristics of SCs. This asymmetric partitioning of DNA then was demonstrated during mitosis, and these results were further supported by real time imaging of SC clones, in which older and newly synthesized DNA templates were distributed asymmetrically after DNA synthesis. We demonstrate that NSCs are unique among precursor cells in the uneven partitioning of genetic material during cell divisions.