Transcript Jarman, Alvarez, & Freed

Neural Progenitor Cells as Replacement Therapy for Diseased and Aging Brains.
R.G. Jarman, E. Alveraz, C.R. Freed; Division of Clinical Pharmacology, Dept of Medicine and Neuroscience
Program. Univ CO Health Science Center. Denver, CO.
STRIATUM
Abstract
Neural progenitor cells, also known as neural stem cells and neurospheres, have been
shown to contain the ability to differentiate into any of the three classes of cells within
the central nervous system that includes neurons, astrocytes and oligodentrocytes. This
potential gives credence to the use of these cells as potential cell therapies for diseased
and aging brains. The presumed function of neural progenitor cells is to replace dying
cells throughout the course of natural life and perhaps, by a decrease in numbers or
functional capacity in later life, these cells may lose their replacement functions leading
to dementia and neurodegenerative diseases. Cell therapy with neural progenitors may
therefore help in relieving these types of ailment by simply allowing the cells access to
the diseased area of the brain and allowing them to differentiate into the appropriate cell
type as their natural function would dictate. Furthermore, the possibility that these cells
can be manipulated in to a specific type of neuron can become reality as been shown
with embryonic stem cells. By manipulating neural progenitor cells into a specific type
of neuron, many genetic and neurodegenerative diseases can potentially be slowed down
or disease progression may be stopped, by delivering genetically normal or mature
neurons that are lost during the progression of the disease.
To determine if neural progenitor cells have the ability to integrate and differentiate into
neurons and astrocytes we have transplanted two green fluorescent labeled neurospheres
into the striatum and hippocampus. The striatum is an involved in movement control
and is affected by Parkinson’s disease, Huntington’s disease and Glutaric Acidemia. The
hippocampus is involved in long-term memory and is associated with Alzheimer’s
disease and many cognitive and dementia abnormalities. Our goal for this initial study
was to determine if there are any differences in the neurosphere’s ability to integrate,
migrate and differentiate into neurons in the brains of old verse young rats. To
accomplish this goal 10 young and 10 old rats were transplanted with two neurosphers
that were infected with an adenovirus expressing green fluorescent protein into the
striatum and hippocampus. The neurospheres were allowed to differentiate for two
weeks at which time the animals were sacrificed. The brains were preserved and cut
into 30 micron frozen sections. The sections were observed for fluorescently labeled
cells (from the neurospheres) and their ability to integrate into the host nervous system
was determined by cell differentiation (morphology) and migration away from the
transplant tract. To determine what type of cells the neurosphere cells differentiated into
we will stain with a variety of neuronal and/or astrocyte specific markers and look for
co-expression with the fluorescent green cells that were transplanted.
This study will provide a foundation for the feasibility for further studies using these
cells in disease models. Furthermore, it may allow some insight on neural progenitor
cell characteristics in aged animals where we presume that the number of natural neural
progenitor cells are low and the aged brain is starving for repair.
Figure 5: Neurosphere cells can become
ASTROCYTES. Striatum and hippocampal
sections from rat brains one week following
transplant with neurospheres. To determine what
type of cells the neural progenitors differentiated
into, the sections were stained with an antibody to
the astrocyte specific Glial Fibillary Acidic Protein
(GFAP). Cells that have differentiated into
astrocytes are positive for GFP (green) and MAP2
(red) and are yellow.
Figure 2: One to two neurospheres labeled with green
fluorescent protein were transplanted into the striatum,
an area of the brain often affected by neurodegenerative
disorders including Parkinson’s disease. The bottom
panels show microscopic sections of brain with surviving
transplanted cells.
HIPPOCAMPUS
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A nearly infinite supply of brain stem cells can be obtained from one embryonic
or adult brain using specific growth factors.
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Stem cells can survive transplant into fetal and adult animal brains.
QUESTION
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Can brain stem cells repopulate the brain of old as well as young rats?
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METHODS
1) Neural stem cells were isolated from the midbrain of an
embryonic day 14 rat. The tissue was mechanically dissociated
and placed in an uncoated tissue culture plate in media contain
B27 supplements with 20ng/ml of epidermal growth factor
(EGF) and no serum.
Figure 3: One to two neurospheres were transplanted
into the hippocampus, an area of the brain controlling
memory which is abnormal in several dementias
including Alzheimer’s disease and Downs Syndrome. As
in figure 2, green transplanted cells survived and
developed into neurons and astrocytes.
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Figure 6: Counts of transplanted green fluorescent
protein labeled cells into the striatum and hippocampus
of old and young rats. Two weeks after transplanting
about 2000 cells labeled with GFP, animals were
sacrificed and brains were sectioned to determine the
number of GFP surviving cells. Counts show that neural
progenitor cells can survive and develop in the brains
regardless of age.
2) Two to four weeks after the initial plating, about 100 floating
spheres were infected with 1 x 108 transforming units of an
adenovirus expressing GFP.
3) One week following infection, one to two neurospheres
containing approximately 2000 cells were transplanted using
stereotaxic surgery into striatum and hippocampus of 24 month
and 6 week Fisher 344 rat.
CONCLUSIONS
4) Two weeks post-transplant the rats were perfused with
heparinized saline followed by 4% paraformaldehyde, brains
were then removed and cryopreserved.
Neural progenitor cells can be successfully
transplanted into aged animals at least as well
as into young animals.
5) The brains were cut into 40m sections using a cryostat.
Sections were observed for GFP expressing cells using a
fluorescence microscope.
Neural progenitor cells can differentiate into
neurons and astrocytes and can migrate away
from the transplant track.
6) Floating GFP positive sections were processed
immunohistochemically with antibodies specific for the neuronal
marker MAP2 or the astrocyte specific marker GFAP using the
appropriate secondary antibody conjugated with Texas Red.
These sections were analyzed using either confocal or standard
fluorescence microscopy.
Figure 4: Neurosphere cells can become
NEURONS. Transplanted green cells were
stained with an antibody to the neuron
specific microtubule associated protein
(MAP2). Cells that have differentiated into
neurons are positive for GFP (green) and
MAP2 (red) and are yellow.
Figure 1: A) Uninfected neurosphere with an approximate
diameter of 300 m. The estimated number of cells within a
sphere of this size is 1700 assuming a 25 m cell diameter.
B) Neurospheres 3 days after infection with an adenovirus
expressing GFP. C) An individual neurosphere induced to
differentiate and dissociate by attaching to a tissue culture
dish in the presence of 10% fetal bovine serum.