Download what factors are involved in the stem cell differentiation process

CAN ENTERIC STEM CELLS BECOME CNS MEURONS AND GLIA?
Supervisor: Åsa Fex Svenningsen ([email protected]) IMM- Neurobiology, J.B. Winsløws vej 21,
5000 Odense C.
The over all aim of the greater project of which this exam work is a part, is to investigate
whether fetal neural crest derived stem/precursor cells (NCS) from the intestine (ENCS) can
differentiate into neurons and/or glia (oligodendrocytes and astrocytes) of the CNS. If our
results point in this direction we will continue to investigate adult intestinal stem cells that we
hope, in the future, can be used as a source for transplantation into the injured or diseased
CNS.
It is well known that stem cells exist in certain parts of the adult mammalian CNS, such as the
lateral ventricles and the hippocampus 1-3. Stem cells from the CNS have the ability to
differentiate into all types of CNS neurons and glia 4, but some CNS stem cells can also
become PNS cells 5-8. The process of fate determination, whether a stem cell will choose to
differentiate into a certain kind of neuron or glia, is not fully elucidated, although some
critical factors are known 7,9-11. Transdifferentiation, when CNS stem cells differentiate into
PNS committed cells or vice versa, is an even less known process and the signals involved are
also less known. Our group recently found that when fetal cortex stem cells (NSPCs)
transdifferentiate into PNS cells, soluble molecules secreted by cells in the immediate
surroundings drive the differentiation process 8.
We, and others, have suggested that some NCS also have the ability to transdifferentiate into
CNS cells 12,13. We have previously demonstrated that a small number of cells in dissociated
cultures of fetal rat and mouse dorsal root ganglia (DRG) develop spontaneously into
astrocytes and oligodendrocytes in culture 12. Cultured fetal dissociated DRG cells,
maintained as neurospheres, also have the ability to differentiate into CNS cells 13.
Several recent reports show that adult PNS contain stem cells 13-16. These cells (neural crest
stem cells, NCS) have been identified in DRGs 13,15,16, trigeminal ganglia 17 and intestine 14.
Interestingly, at least two groups have also reported that these cells can transdifferentiate into
CNS neurons and glia 13,16. The enteric part of the PNS, the ENS, has a great regenerative
capacity as well as plasticity, even in adult mammals 18,19. The ENS matures late in
development, and nerve cell proliferation also takes place late in development 20.
We have just developed a method to culture fetal enteric stem cells and would now like to
investigate their capacity to transdifferentiate into neurons and glia of the CNS.
Project description
Evaluation of the number of ENCS that can transdifferentiate into CNS committed cells. To
investigate this, a method previously described by us will be used 8:
The intestine of Embryonic day 14 actin EGFP mice will be dissociated and cultured into
neurosperes. At the same time cultures will be made of the fetal cerebellum from the wild
type embryos of the same litter. After 2 weeks the ENCS are dissociated and plated on top of
the fetal cerebellar cultures. ENCS are then cultured in close contact with the fetal CNS
(cerebellar) feeder cultures. After 10 days the cultures will be fixed and the cells will be
investigated using immunocytochemistry and fluoresce microscopy. The different cell species
and cell number will be investigated. The identification is made using antibodies specific for
neurons (MAP2, -tubulin) and glia (Astro1, GFAP for astrocytes and NG2, CNP and PLP
for oligodendrocytes) of the CNS and ENS (peripherin for neurons and GFAP for glia). The
ratio between transdifferentiated cells and the total number (seen with DAPI) will be
determined by manual counting as we have previously done using fluorescent conventional
microscopy 8,21. Other ENS stem cells will be differentiated using special differentiating
media and media conditioned by CNS cultures. These will be analyzed using qpcr and
primers specific for different CNS cells.
Methods
The project will involve dissection of embryonic mouse peripheral- and central nervous
system and primary cell culturing of ENCS cells as well as fetal primary cultures of the
cerebellum. To analyze the amount of the different cell species in the cell mixture of
differentiated ENCS and cerebellar cells, we will analyze digital pictures taken by our
fluorescent microscope. The results will be statistically analyzed. Other stem cells will be
differentiated using special differentiating media and media conditioned by CNS cultures.
These will be analyzed using qpcr.
References:
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