A team of Japanese researchers has demonstrated that embryonic stem cells obtained from mice and humans can spontaneously organize themselves into cortical tissues when grown in a culture dish under special conditions.
Reporting in the journal Cell Stem Cell, the researchers show that the neurons generated form functioning short-range and long-range connections, and can be effectively integrated into existing neuronal circuits following transplantation into the brains of experimental animals.
Yoshiki Sasai, head of the Organogenesis and Neurogenesis Group at RIKEN's Center for Developmental Biology in Kobe, and his colleagues used a three-dimensional cell culture system which they had developed previously. This involves isolating tissue from mouse embryos and then treating it with enzymes which cause it to dissociate into a suspension of single cells. Within 2 days, the embryonic stem (ES) cells in the suspension spontaneously re-aggregate to form clumps and begin to differentiate when placed in a specially prepared culture medium.
Earlier work had shown that the ES cells aggregate into clumps of varying sizes and that in a typical experiment only 30-35% of the cells differentiate into mature neurons. In the new study, Sasai's group sought to improve the culture technique. By designing conditions in which the formation of the aggregates could be controlled tightly, they were able to coax the cultured ES cells to differentiate more efficiently.
Using their modified culture method, the researchers found that the dissociated ES cells aggregated far more quickly - they formed clumps within a matter of hours rather than days. The aggregates were of a uniform size and, remarkably, more than 95% of the cells within them formed a continuous sheet during the first 5 days in culture, which closely resembled the neuroepithelium that forms on the outer surface of the embryo during the earlier stages of neural development.
When cultered for several days longer, each of these sheets reformed into several round clusters called rosettes (right). The cells in the rosettes were organized into a distinctly layered structure which closely mimicked the developing neocortex. The cultured cells segregated themselves into four zones, with the thick inner layer (labelled purple) containing neural progenitors which divided asymmetrically to propagate themselves and to generate newborn neurons which then migrated outwards. Cells in each layer also expressed a specific set of genes; for example, the outermost layer contained neurons which synthesized Reelin, a protein that is normally expressed by the first cells to migrate into the developing cortex, and is thought to provide positional information for subsequent waves of neurons as they migrate out to and populate the developing cortex. Furthermore, the activation of these layer-specific genes occurred in the same temporal sequence that is seen during cortical development.
The researchers were also able to manipulate the regional identity of the neurons derived from the ES cell cultures. During development, the nervous system is patterned along its major axes by combinations of signalling molecules which activate and inhibit specific sets of genes, so that cells in a given location assume their proper identity. The researchers found that treating their cultures with each of a number of signalling molecules altered the gene expression patterns of the ES cells, so that they assumed the same fate as cells that are exposed to each signal during normal development.
The fate of differentiating neurons could also be influenced. During development of the cortex, the first cells to be generated form a transient outer of Cajal-Retzius cells, which are characterized by synthesis and secretion of the Reelin protein. Subsequently, the layers of the cortex are generated in an "inside-out" manner. The researchers found that forcing cells to exit the cell cyle (by blocking their division) at an early time point led them to differentiate into Cajal-Retzius cells, whereas doing so at a later point in time led them to differentiate into neurons that are located in deeper layers.
Cells cultured for 21 days showed patterns of spontaneous activity which differed from one neuron to the next in terms of frequency, duration and regularity. Although different patterns of activity were observed, each neuron examined increased its activity when the neurotransmitter glutamate was added to the culture; furthermore, all activity was effectively inhibited with application of tetrodotoxin, which blocks the sodium ion channels that are essential for generating nervous impulses. Surprisingly, the researchers also found that the neurons derived from their ES cell cultures exhibited calcium waves which synchronously activate neurons over distances of more than 1mm. This large-scale oscillatory activity is unique to neurons in the developing cortex, and is normally only seen in the first postnatal week.
Clumps of ES cells expressing green fluorescent protein were then injected into the lateral ventricles of forebrain slices dissected from embryonic or juvenile mice. Cells from the clumps were seen to invade the appropriate region of developing cortex in the slices and also to differentiate into neurons, extend processes and become effectively integrated into the circuitry. The same thing happened when the cells were transplanted into the frontal cortex of live newborn mice. Analysis of the animals' brains one month after transplantation revealed that axons from the transplanted cells had extended into deep layers of the cortex and to the other hemisphere of the brain through the corpus callosum. The axons had also formed bundles which projected further afield; they were observed in subcortical structures such as the striatum and thalamus; in the pons, the uppermost region of the brain stem; and in the pyramidal tract, which projects from the primary motor cortex down into the spinal cord, and which contains the axons of neurons which control voluntary movement.
Finally, the researchers found that their cell culture method was applicable to human ES cells. Just like ES cells isolated from mice, human cells spontaneously formed a neuroepithelial structure. But whereas the mouse ES cells formed several rosettes when cultured for about 1 week, the human cells instead formed one or a few mushroom-shaped structures. The cells in these structures exhibited the same properties as the mouse cells: they continued to differentiate to form a distinct layered structure. However, they remained viable for far longer - they could be maintained for up to 60 days, at which time they broke apart. By contrast, the rosettes formed by the mouse cells became disorganized after just 12 days in culture. The reason for this is unclear, but it may be because of a difference in the balance between the rate at which human and mouse ES cells renew themselves and generate differentiating neurons.
The unique method described in this study supports the formation of cerebral cortical tissue in the culture dish. The neurons in the tissue were generated in a spatially and temporally pattern that was tightly controlled, and this pattern evidently recapitulates the development of the cortex. This culture method could therefore provide a versatile system for researchers investigating the cellular and molecular mechanisms of cortical development.
This study further shows that ES cells can differentiate into neurons when transplanted into the mouse brain, and that those neurons can in turn function as viable progenitor cells for the projection neurons of the cerebral cortex. These findings therefore have potential future applications in regenerative medicine. The ability to manipulate the regional identity of neurons in this way could lead, for example, to new methods by which human ES cells are directed to differentiate into the typers of neurons which degenerate in Alzheimer's Disease, Parkinson's Disease or amyotrohoic lateral sclerosis.
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Eiraku, M. et al (2008). Self-Organized Formation of Polarized Cortical Tissues from ESCs and Its Active Manipulation by Extrinsic Signals. Cell Stem Cell 3: 519-532. DOI: 10.1016/j.stem.2008.09.002.
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