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.

BACKGROUND

A nearly infinite supply of brain stem cells can be obtained from one embryonic or adult brain using specific growth factors.

Stem cells can survive transplant into fetal and adult animal brains.

QUESTION

Can brain stem cells repopulate the brain of old as well as young rats?

AbstractNeural 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.

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.

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.

4) Two weeks post-transplant the rats were perfused with heparinized saline followed by 4% paraformaldehyde, brains were then removed and cryopreserved.

5) The brains were cut into 40m sections using a cryostat. Sections were observed for GFP expressing cells using a fluorescence microscope.

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 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.

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.

STRIATUM

HIPPOCAMPUS

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.

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 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 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.

Transplanted Cell Survival

Mean

GF

P P

osit

ive C

ells

0

50

100

150

200

250

300

350

400

Young Old

STRIATUM n=10

HIPPOCAMPUS n=9

CONCLUSIONS

Neural progenitor cells can be successfully transplanted into aged animals at least as well as into young animals.

Neural progenitor cells can differentiate into neurons and astrocytes and can migrate away from the transplant track.