Humoral and contact interactions in astroglia/stem cell co-cultures

in the course of glia-induced neurogenesis


.Zs. Környei 1*,  V. Szlávik 1, B. Szabó 2, E. Gócza 3, A. Czirók 2, and E. Madarász 1


1 Institute of Experimental Medicine, Budapest, Hungary;  2 Department of Biological Physics, Eötvös University, Budapest, Hungary; 3 Agricultural Biotechnology Center, Gödöllő, Hungary



Astroglial cells support or restrict the migration and differentiation of neural stem cells depending on the developmental stage of the progenitors and the physiological state of the astrocytes. In the present study we show that astroglial cells instruct non-committed, immortalized neuroectodermal stem cells to adopt a neuronal fate, while they fail to induce neuronal differentiation of embryonic stem cells under similar culture conditions. Astrocytes induce neuron formation by neuroectodermal progenitors both through direct cell-to-cell contacts and via short-range acting humoral factors. Neuron formation takes place inside compact stem cell assemblies formed 30-60 hours after the onset of glial induction. Statistical analyses of time-lapse microscopic recordings show that direct contacts with astrocytes hinder the migration of neuroectodermal progenitors, while astroglia-derived humoral factors increase their motility. In non-contact co-cultures with astrocytes, altered adhesiveness prevents the separation of frequently colliding neural stem cells. In contact co-cultures with astrocytes, on the other hand, the restricted migration on glial surfaces keeps the cell-progenies together resulting in the formation of clonally proliferating stem cell aggregates. The data indicate that in vitro maintained parenchymal astrocytes i, secrete factors, which initiate neuronal differentiation of neuroectodermal stem cells and ii, provide a cellular microenvironment where stem cell/stem cell interactions can develop and the sorting out of the future neurons can proceed. In contrast to non-committed progenitors, postmitotic neuronal precursors leave the stem cell clusters, indicating that astroglial cells selectively support the migration of maturing neurons as well as the elongation of neurites.


Glia, 2005 Feb;49(3):430-44.


Key words: neural stem cell, astroglia, embryonic stem cell, differentiation, migration


Supplemental movies:


Non-induced NE-4C immortalized neuroectodermal stem cells on poly-L-lysin coated plastic surface: (16.4 MB), Movie1.mpeg (2.1 MB)

Field of view: 150um X 130um. Duration of recording: 48 hours. Number of frames: 289 (10 min/frame).

In monocultures of non-induced NE-4C cells the daughter cells depart from each other after each division. Colored trajectories show the migratory routes of consecutive generations of a representative NE-4C neuroectodermal stem cell. The cells were seeded onto poly-L-lysine coated plastic culture dish in serum free medium. The 48-hour long movie starts at 30 min. after attachment to the substrate.


Aggregate formation by an NE-4C neuroectodermal cell plated onto astroglial monolayer: (5.1 MB), Movie2.mpeg (2.5 MB)

Field of view: 70um X 80um. Duration of recording: 86 hours. Number of frames: 653 (10 min/frame).

On the surface of astrocytes, the progenies of individual NE-4C neural stem cells did not separate from each other in the course of repeated cell divisions. Synchronized divisions seem to result in a periodic loosening and compaction of the aggregate. The movie starts 30 min. after attachment to the substrate and lasts for 86 hours.


On the surface of astrocytes NE-4C neuroectodermal stem cells expand neurite-like processes: (0.6 MB), Movie3.mpeg (0.4 MB)

Field of view: 106um X 92um. Duration of recording: 2 hours and 40 minutes. Number of frames: 34 (5 min/frame).
Note the promptness (30±2 mm/h) of neurite extension.


Migration of NE-4C neural stem cell derived neuronal precursors on the surface of astrocytes: (19.5 MB), Movie4.mpeg (5.6 MB)

Field of view: 216um X 108um. Duration of recording: 60 hours. Number of frames: 364 (10 min/frame).
The movie starts 80 hours after seeding the cells. The birth (the terminal division) of a future neuron can be seen at the side of the aggregate, denoted by green circle. Note that the marked cells do not divide during the 60-hour period of the recording. The tracked cells were later identified as neuronal precursors by neuron-specific bIII-tubulin immunostaining.


Neurite elongation by an NE-4C derived neuron on the surface of astroglial cells: (7.4 MB), Movie5.mpeg (3.6 MB)

Field of view: 130um X 170um. Duration of recording: 17 hours. Number of frames: 364 (10 min/frame).
The recording starts at 120 hours after seeding and last for 17 hours. The tracked neurite was later shown to express neuron-specific bIII-tubulin.


Aggregate formation by NE-4C neuroectodermal cells in non-contact co-cultures with astrocytes: (16.4 MB), Movie6.mpeg (9.9 MB)

Field of view: 440um X 320um. Duration of recording: 48 hours. Number of frames: 579 (5 min/frame).

During the first 30 hours NE-4C cells display random collisions and separations. Sister cells separate from each other after each cell divison. From the 30th-36th  hours, however, altered adhesiveness prevents the separation of either randomly encountering or doubling cells. Astrocytes can be recognized by their flattened shape.



Other Movies:


Migration of an NE-4C neural stem cell derived neuronal precursor on the surface of astrocytes: (7,3 MB), Movie7.mpeg (2,7 MB)

Field of view: 160um X 200um. Duration of recording: 30 hours and 30 minutes. Number of frames: 178 (10 min/frame).


Development of a neurogenic NE-4C cell cluster on the surface of astrocytes: (17 MB), Movie8.mpeg (11,9 MB)

Field of view: 200um X 166um. Duration of recording: 100 hours. Number of frames: 2404 (5 min/frame).

Watch the extensive neurite-elongation at 70 hour.


Aggregation of NE-4C cells in non-contact co-cultures with astrocytes: (27.5 MB), Movie9.mpeg (14,3 MB)

Field of view: um 1200X 870um. Duration of recording: 72 hours. Number of frames: 864 (5 min/frame).




Related documents:


    Posters at 15th Biennal Meeting of International Society of Developmental Neuroscience, 2004, Edinburgh

*   Dynamics of astroglia-induced neurogenesis    pdf

*   Expression of neurogenic and region specific genes during in vitro induced neurogenesis of NE-4C neuroectodermal cells   pdf

*   Gap junction communication by NE-4C neuroectodermal stem cells   pdf

    Poster at IBRO International Workshop on Neuronal Circuits, 2004, Budapest

*   Astroglia – neural stem cell interaction   pdf

     Poster at Satellite Symposium to the Sixth IBRO World Congress of Neuroscience, 2003, Prague

*   Astroglia – stem cell interaction    pdf

     Poster at Faseb Summer Research Confererences / Retinoids, 2002, Tucson

*   Astroglial cells support the neuronal differentiation of immortalized neuroectodermal progenitor cells   pdf


Related sites:


*    Long-term automated live cell microscopy at Dept. of Biological Physics, Eotvos University, Budapest, Hungary
*    Laboratory of Neural Cell Biology at Institute of Experimental Medicine, Budapest, Hungary  

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