ABSTRACT

Neural stem cells reside in defined areas of the adult mammalian brain, including the dentate gyrus of the hippocampus. Rat neural stem/progenitor cells (NSPCs) isolated from this region retain their multipotency in vitro and in vivo after grafting into the adult brain. Recent studies have shown that endogenous or grafted NSPCs are activated after an injury and migrate toward lesioned areas. In these areas, reactive astrocytes are present and secrete numerous molecules and growth factors; however, it is not currently known whether reactive astrocytes can influence the lineage selection of NSPCs. We investigated whether reactive astrocytes could affect the differentiation, proliferation, and survival of adult NSPCs by modelling astrogliosis in vitro, using mechanical lesion of primary astrocytes. Initially, it was found that conditioned medium from lesioned astrocytes induced astrocytic differentiation of NSPCs without affecting neuronal or oligodendrocytic differentiation. In addition, NSPCs in coculture with lesioned astrocytes also displayed increased astrocytic differentiation and some of these NSPC-derived astrocytes participated in glial scar formation in vitro. When proliferation and survival of NSPCs were analyzed, no differential effects were observed between lesioned and nonlesioned astrocytes. To investigate the molecular mechanisms of the astrocyte-inducing activity, the expression of two potent inducers of astroglial differentiation, ciliary neurotrophic factor and leukemia inhibitory factor, was analyzed by Western blot and shown to be up-regulated in conditioned medium from lesioned astrocytes. These results demonstrate that lesioned astrocytes can induce astroglial differentiation of NSPCs and provide a mechanism for astroglial differentiation of these cells following brain injury.

SummaryThis paper describes a system for in vitro cell migration analysis. Adult neural stem/progenitor cells are studied using time-lapse bright-field microscopy and thereafter stained immunohistochemically to find and distinguish undifferentiated glial progenitor cells and cells having differentiated into type-1 or type-2 astrocytes. The cells are automatically segmented and tracked through the time-lapse sequence. An extension to the Chan-Vese Level Set segmentation algorithm, including two new terms for specialized growing and pruning, made it possible to resolve clustered cells, and reduced the tracking error by 65%. We used a custom-built manual correction module to form a ground truth used as a reference for tracked cells that could be identified from the fluorescence staining. On average, the tracks were correct 95% of the time, using our new segmentation. The tracking, or association of segmented cells, was performed using a 2-state Hidden Markov Model describing the random behaviour of the cells. By re-estimating the motion model to conform with the segmented data we managed to reduce the number of tracking parameters to essentially only one. Upon characterization of the cell migration by the HMM state occupation function, it was found that glial progenitor cells were moving randomly 2/3 of the time, while the type-2 astrocytes showed a directed movement 2/3 of the time. This finding indicates possibilities for celltype specific identification and cell sorting of live cells based on specific movement patterns in individual cell populations, which would have valuable applications in neurobiological research.

Stroke is one of the leading causes of chronic disability and death in the Western world. Today, no treatment can repair the cellular loss associated with an ischemic lesion. However, the discovery and dynamic regulation of neural stem/progenitor cells in the adult mammalian brain has resulted in exciting possibilities for future therapeutic interventions. Endogenous or grafted neural stem/progenitor cells are activated following an ischemic insult. These cells undergo directed migration towards infarcted areas, and differentiate in response to the insult. Unfortunately, the results of this regenerative effort are limited compared to the amount of tissue loss. This could be due to low survival of the recruited cells, but could also be explained by insufficient activation or dysfunctional lineage selection. Whether the lineage selection of neural stem/progenitor cells is altered following a lesion in the brain, what signals that are responsible for their activation or whether these cells can participate in post-lesion regeneration, astrogliosis or neuroprotection have yet to become clear. A greater understanding of these processes is necessary for finding ways to improve the endogenous regenerative capacity. We found that reactive astrocytes, a prominent part of the post-ischemic environment, induced astroglial differentiation of adult neural stem/progenitor cells in vitro. Moreover, astrocytes derived from these cells were shown to participate in glial scar formation in vitro. After studying gene expression in the peri-infarct region following focal ischemia, the expression of several genes was induced. We chose to focus our attention on one of these genes and its product, thyrotropin-releasing hormone (TRH). Immunoreactivity for TRH was found in several areas in both lesioned and intact brain regions, including in microglia present in the areas surrounding the lesion. Furthermore, TRH receptors were expressed on cultured neural stem/progenitor cells and TRH potently induced the proliferation of these cells. TRH is an interesting target for stroke treatment, but it also has many central effects in the brain and systemic administration may prove problematic. An interesting protocol for local delivery of TRH would be by grafting stem/progenitor cells, genetically engineered to secrete the peptide. In order to create a foundation for neuroprotective gene therapy, we developed efficient methods for non-viral transfection of neural stem/progenitor cells. Since neural stem/progenitor cells migrate towards the ischemic area we wanted to investigate whether these cells secreted factors that could protect neurons against excitotoxicity, the main inducer of cell death following a stroke. Mass spectrometric analysis of factors secreted from cultured neural stem/progenitor cells led to the identification of a novel neuroprotective peptide, which we termed pentinin. This peptide potently reduced excitotoxicity in both mature and immature neurons in an ex vivo hippocampal slice model. The results presented in this the...

J. Neurochem. (2009) 109, 858–866. Abstract Although the potential of adult neural stem cells to repair damage via cell replacement has been widely reported, the ability of endogenous stem cells to positively modulate damage is less well studied. We investigated whether medium conditioned by adult hippocampal stem/progenitor cells altered the extent of excitotoxic cell death in hippocampal slice cultures. Conditioned medium significantly reduced cell death following 24 h of exposure to 10 μM NMDA. Neuroprotection was greater in the dentate gyrus, a region neighboring the subgranular zone where stem/progenitor cells reside compared with pyramidal cells of the cornis ammonis. Using mass spectrometric analysis of the conditioned medium, we identified a pentameric peptide fragment that corresponded to residues 26–30 of the insulin B chain which we termed ‘pentinin’. The peptide is a putative breakdown product of insulin, a constituent of the culture medium, and may be produced by insulin‐degrading enzyme, an enzyme expressed by the stem/progenitor cells. In the presence of 100 pM of synthetic pentinin, the number of mature and immature neurons killed by NMDA‐induced toxicity was significantly reduced in the dentate gyrus. These data suggest that progenitors in the subgranular zone may convert exogenous insulin into a peptide capable of protecting neighboring neurons from excitotoxic injury.