U.S. patent application number 10/622206 was filed with the patent office on 2004-03-11 for method for production of neuroblasts.
Invention is credited to Gage, Fred H., Ray, Jasodhara.
Application Number | 20040048373 10/622206 |
Document ID | / |
Family ID | 26669174 |
Filed Date | 2004-03-11 |
United States Patent
Application |
20040048373 |
Kind Code |
A1 |
Gage, Fred H. ; et
al. |
March 11, 2004 |
Method for production of neuroblasts
Abstract
A method for producing a neuroblast and a cellular composition
comprising an enriched population of neuroblast cells is provided.
Also disclosed are methods for identifying compositions which
affect neuroblasts and for treating a subject with a neuronal
disorder, and a culture system for the production and maintenance
of neuroblasts.
Inventors: |
Gage, Fred H.; (La Jolla,
CA) ; Ray, Jasodhara; (San Diego, CA) |
Correspondence
Address: |
Lisa A. Haile, J.D., Ph.D.
GRAY CARY WARE & FREIDENRICH LLP
Suite 1100
4365 Executive Drive
San Diego
CA
92121-2133
US
|
Family ID: |
26669174 |
Appl. No.: |
10/622206 |
Filed: |
July 18, 2003 |
Related U.S. Patent Documents
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Application
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Filing Date |
Patent Number |
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10622206 |
Jul 18, 2003 |
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09915229 |
Jul 24, 2001 |
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6599695 |
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09915229 |
Jul 24, 2001 |
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08884427 |
Jun 27, 1997 |
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6265175 |
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08884427 |
Jun 27, 1997 |
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08445075 |
May 19, 1995 |
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08445075 |
May 19, 1995 |
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08147843 |
Nov 3, 1993 |
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5766948 |
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08147843 |
Nov 3, 1993 |
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08001543 |
Jan 6, 1993 |
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Current U.S.
Class: |
435/368 |
Current CPC
Class: |
C12N 2533/32 20130101;
A61K 48/00 20130101; C12N 2500/25 20130101; A61P 25/28 20180101;
C12N 2500/90 20130101; C12N 2501/115 20130101; C12N 2510/04
20130101; C12N 2501/13 20130101; C12N 5/0618 20130101; C12N
2501/235 20130101; C12N 5/0623 20130101; A61P 25/16 20180101; C12M
23/20 20130101; C12N 2501/11 20130101; A61P 25/08 20180101; C12N
5/0619 20130101; A61K 35/12 20130101; C12N 2502/08 20130101; C12N
2501/392 20130101; A61P 25/14 20180101 |
Class at
Publication: |
435/368 |
International
Class: |
C12N 005/08 |
Claims
1. A method of producing a neuroblast in vitro, the method
comprising culturing a neuronal cell in a vessel in a serum-free
basal media supplemented with at least one trophic factor wherein a
surface in the vessel allows attachment of the neuronal cell.
2. The method of claim 1, wherein the neuronal cell is derived from
neural tissue selected from the group consisting of hippocampus,
cerebellum, spinal cord, cortex, striatum, basal forebrain, ventral
mesencephalon, and locus ceruleus.
3. The method of claim 1, wherein the trophic factor is selected
from the group consisting of nerve growth factor, brain derived
neurotrophic factor, neurotrophin, fibroblast growth factor,
platelet derived growth factor, epidermal growth factor, insulin
growth factor, and transforming growth factor.
4. The method of claim 3, wherein the fibroblast growth factor is
basic fibroblast growth factor.
5. The method of claim 3, wherein the neurotrophin is
neurotrophin-3.
6. The method of claim 1, wherein the surface in the vessel is
treated with a polybasic amino acid to allow attachment of the
neuronal cell.
7. The method of claim 6, wherein the polybasic amino acid is
polyornithine.
8. The method of claim 1, wherein the surface in the vessel is
treated with an extracellular matrix molecule to allow attachment
of the neuronal cell.
9. The method of claim 8, wherein the extracellular matrix molecule
is selected from the group consisting of laminin, collagen and
fibronectin.
10. The method of claim 1, wherein the neuronal cell is cultured in
serum-containing media prior to culture in serum-free media.
11. A method for identifying a composition which affects a
neuroblast which comprises: (a) incubating components comprising
the composition and the neuroblast, wherein the incubating is
carried out under conditions sufficient to allow the components to
interact; and (b) measuring the effect on the neuroblast caused by
the composition.
12. The method of claim 11, wherein the effect is inhibition of the
neuroblast.
13. The method of claim 11, wherein the effect is stimulation of
the neuroblast.
14. The method of claim 11, wherein the neuroblast is derived from
neural tissue selected from the group consisting of hippocampus,
cerebellum, spinal cord, cortex, striatum, basal forebrain, ventral
mesencephalon, and locus ceruleus.
15. The method of claim 11, wherein the neuroblast is
immortalized.
16. The method of claim 15, wherein the neuroblast is immortalized
by the introduction to the neuroblast of at least one oncogene.
17. The method of claim 15, wherein the oncogene is selected from
the group consisting of v-myc, SV40 large T antigen and adenovirus
E1A.
18. The method of claim 11, wherein the neuroblast further
comprises at least one exogenous gene.
19. The method of claim 18, wherein the exogenous gene encodes a
receptor.
20. The method of claim 19, wherein the receptor is selected from
the group consisting of receptors which bind adrenaline,
noradrenaline, glutamate, serotonin, dopamine, GABA, and
acetylcholine.
21. A culture system useful for the production and maintenance of a
neuroblast comprising: (a) a serum-free basal media containing at
least one trophic factor; and (b) a vessel, wherein a surface in
the vessel allows attachment of the neuroblast.
22. The culture system of claim 21, wherein the neuroblast is
derived from neural issue selected from the group consisting of
hippocampus, cerebellum, spinal cord, cortex, striatum, basal
forebrain, ventral mesencephalon, and locus ceruleus.
23. The culture system of claim 21, wherein the trophic factor is
basic fibroblast growth factor.
24. The culture system of claim 21, wherein the trophic factor is
present at a concentration of from about 1 ng/ml to about 100
ng/ml.
25. The culture system of claim 21, wherein the trophic factor is
present at a concentration of from about 5 ng/ml to about 70
ng/ml.
26. The culture system of claim 21, wherein the trophic factor is
present at a concentration from about 15 ng/ml to about 60
ng/ml.
27. The culture system of claim 21, wherein the glucose is present
at a concentration from about 0.01% to about 1.5%.
28. The culture system of claim 21, wherein the glucose is present
at a concentration from about 0.1% to about 0.6%.
29. The culture system of claim 21, wherein the surface in the
vessel is treated with a polybasic amino acid.
30. The culture system of claim 29, wherein the polybasic amino
acid is polyomithine.
31. The culture system of claim 21, wherein the surface in the
vessel is treated with an extracellular matrix molecule.
32. The culture system of claim 31, wherein the extracellular
matrix molecule is selected from the group consisting of laminin,
collagen, and fibronectin.
33. A method of treating a subject with a neuronal cell disorder
comprising administering to the subject a therapeutically effective
amount of neuroblast.
34. The method of claim 33, wherein the neuroblast contains an
exogenous gene.
35. The method of claim 34, wherein the exogenous gene encodes an
oncogene.
36. The method of claim 35, wherein the oncogene is selected from
the group consisting of v-myc, SV40 large T antigen and adenovirus
E1A.
37. The method of claim 34, wherein the exogenous gene encodes a
receptor.
38. The method of claim 37, wherein the receptor is selected from
the group consisting of receptors which bind adrenaline,
noradrenaline, glutamate, serotonin, dopamine, GABA, and
acetylcholine receptor.
39. The method of claim 34, wherein the exogenous gene encodes a
ligand.
40. The method of claim 39, wherein the ligand is selected from the
group consisting of adrenaline, noradrenaline, glutamate, dopamine,
acetylcholine, gamma-aminobutyric acid, and serotonin.
41. The method of claim 33, wherein the neuronal disorder is
selected from the group consisting of Alzheimer's disease,
Parkinson's disease, Huntington's disease, stroke, and spinal cord
damage.
42. A cellular composition comprising an enriched population of
neuroblast cells.
43. The composition of claim 42, wherein the neuroblast is derived
from neural tissue selected from the group consisting of
hippocampus, cerebellum, spinal cord, cortex, striatum, basal
forebrain, ventral mesencephalon, and locus ceruleus.
44. The composition of claim 42, wherein the neuroblast is
immortalized.
45. The composition of claim 44, wherein immortalization is
achieved by the introduction to the cell of at least one
ancogene.
46. The composition of claim 45, wherein the oncogene is selected
from the group consisting of v-myc, SV40 large T antigen and
adenovirus E1A.
47. The composition of claim 42, wherein the neuroblast further
comprises at least one exogenous gene.
48. The composition of claim 47, wherein the, exogenous gene
encodes a receptor.
49. The composition of claim 48, wherein the receptor is selected
from the group consisting of receptors which bind adrenaline,
noradrenaline, glutamate, serotonin, dopamine, GABA, and
acetylcholine.
50. The composition of claim 47, wherein the exogenous gene encodes
a ligand.
51. The composition of claim 50, wherein the ligand is selected
from the group consisting of adrenaline, noradrenaline, glutamate,
dopamine, acetylcholine, gamma-aminobutyric acid, and serotonin.
Description
[0001] This application is a continuation-in-art of application
Ser. No. 08/001,543 filed on Jan. 6, 1993.
BACKGROUND OF THE INVENTION
[0002] 1. Field of the Invention
[0003] This invention relates generally to cell populations derived
from neurons, which are denoted neuroblasts, methods for the
production and long-term in vitro culture of these cell
populations, and the use of neuroblasts in the treatment of various
neuronal disorders as well as the identification of compositions
which affect neuroblasts.
[0004] 2. Description of Related Art
[0005] Only a few neuronal cell types have been reported to divide
in the adult brain and adult neurons do not survive well in vitro.
To date, even with all of the recent advances in neurobiology,
genetics, immunology and molecular biology, no reliable procedure
exists to establish cell lines from the central nervous system
(CNS) and neuronal tissues in the absence of immortalization. The
generation of clonal cell lines from different regions of the brain
is important and will greatly facilitate the discovery of new
neurotrophic factors and their receptors, and enhance the
understanding of their function.
[0006] The central nervous system contains two major classes of
cells known as neurons and glial cells. Glial cells include
astrocytes, oligodendrocytes and microglia. There are hundreds of
different types of neurons and many different neurotraphic factors
which influence their growth and differentiation. Depending on the
type of neuron and the region of the brain in which the neuron
resides, a different neurotrophic factor or specific combination of
factors affect the survival, proliferation and differentiation of
the neuron. Each type of neuron responds to different combinations
of neurotransmitters, neurotrophic factors, and other molecules in
its environment
[0007] To date, neuropharmacological studies in the CNS have been
delayed by the lack of cell systems needed to investigate
potentially useful neuroactive compounds. In live animals, the
complexity of the brain makes it difficult to effectively measure
which cellular receptors are being targeted by these compounds.
Additionally, the expense involved in live animal research and the
current controversies stemming from animal rights movements have
made in vivo animal studies less acceptable for initial research.
Primary cells from neuronal issue are often used for CNS studies,
however, long-term culture of primary neurons has not been
achieved. Also, only a few attempts to achieve not only long term
culture, but actual proliferation of neuronal cells have been
reported. In fact, the, proliferation of neuronal cells has proven
so elusive that it has become ingrained in the scientific community
that neuronal cells do not proliferate in vitro. As a consequence,
fresh dissections must be performed for each study in order to
obtain the necessary neuronal cell types, resulting in costly
research with increased variability in the experimental
results.
[0008] While some neuronal tumorogenic cells exist they are few in
number and are not well characterized. In general, these tumor cell
lines do not mimic the biology of the primary neurons from which
they were originally established and, as a result, are not suitable
for drug discovery screening programs. An vitro primary cultures
that would be more phenotypically representative of primary cells
and that could generate continuous cultures of specific neuronal
cell lines capable of proliferation would be invaluable for
neurobiological studies and CNS drug discovery efforts, as well as
therapy.
[0009] It has become increasingly apparent that more defined
conditions and further refinements in culture methodology are
necessary to produce neuronal cell lines which would enhance the
yield of information from in vitro studies of the nervous system.
Recognition of cell type and developmental stage-specific
requirements for maintaining neural cells in culture as well as the
development of a broader range of culture conditions are required.
However, in order to achieve these goals it is critical to develop
optimal culture methods which mimic in vivo conditions which are
devoid of the biological fluids used in conventional culture
techniques.
[0010] Recently, several researchers have isolated and immortalized
progenitor cells from various regions of the brain and different
stages of development. Olfactory and cerebellum cells have been
immortalized using the viral myc (v-myc) ancogene to generate cell
lines with neuronal and glial phenotypes (Ryder, et al., J.
Neurobiology, 21:356, 1990). Similar studies by Snyder, et al.
(Cell, 68:33, 1992) resulted in multipotent neuronal cell lines
which were engrafted into the rat cerebellum- to form neurons and
glial cells. In other studies, murine neuroepithelial cells were
immortalized with a retrovirus vector containing c-myc and were
cultured with growth factors to form differentiated cell types
similar to astrocytes and neurons. (Barlett, et al.,
Proc.Natl.Acad.Sci.USA, 85:3255,1988).
[0011] Epidermal growth factor (EGF) has been used to induce the in
vitro proliferation of a small number of cells isolated from the
striatum of the adult mouse brain (Reynolds and Weiss, Science,
255:1707 1992). Clusters of these cells had antigenic properties of
neuroepithelial stem cells and under appropriate conditions, these
cells could be induced to differentiate into astrocytes and neurons
with phenotypes characteristic of the adult striatum in vivo
However, it should be noted that these differentiated neurons were
not cultured for lengthy periods of time nor was there any evidence
that these cells could be frozen and then thawed and
recultured.
[0012] Cattaneo and McKay (Nature, 347:762, 1990) performed
experiments using rat striatum to determine the effect of nerve
growth factor (NGF) on proliferation of neuronal precursor cells.
The cells were dissected from rat embryonic striaturn and exposed
to both NGF and basic fibroblast growth factor (bFGF, also known as
FGF2). These cells were cultured only nine days in vitro, at which
time they had differentiated into neurons as determined by assay
with neuron-specific markers.
[0013] Neuronal precursor cells from the cerebral hemispheres of 13
day old rat embryos have been cultured for up to 8 days in the
presence of bFGF at 5 ng/ml (Gensberger, et at., FEBS Lett 217:1,
19874. At this concentration, bFGF stimulated only short-term
proliferation Proliferation and differentiation of primary neurons
from both fetal and adult striatum in response to a combination of
NGF and bFGF or only EGF have also been reported (Catteneo, et al.,
supra; Reynolds and Weiss, supra).
[0014] In view of the foregoing, there is a need for a long-term in
vitro culture system which would allow large scale production and
maintenance of a neuronal cell population which will proliferate
and can be passaged and subcultured over time. Such homogenous in
vitro neuronal cultures will prove invaluable in studying cell
populations, the interactions between these cells and the effects
of various neuroactive compositions on these cells.
SUMMARY OF THE INVENTION
[0015] Recognizing the importance of a system for producing and
maintaining neuronal cells in vitro, the inventors developed a
method and a culture system for producing continuous fetal and
adult neuronal cell lines. The development of primary neuronal
cultures maintained as cell lines, known as neuroblasts, using
neurotrophic factors in the absence of oncogenic immortalization,
now permits investigation of fundamental questions regarding the
biochemical and cellular properties of these cells and the dynamics
of interaction between their cellular and chemical environment
[0016] The neuroblasts of the invention can advantageously be used
to stably incorporate genetic sequences encoding various receptors,
ligands and neurotransmitters, for example, for use in the
treatment of subjects with neuronal disorders and for identifying
compositions which interact with these molecules.
BRIEF DESCRIPTION OF THE DRAWINGS
[0017] FIG. 1 shows BrdU staining and NeuroTag.TM. binding of
primary neurons in culture A. Primary neurons were labeled with
BrdU for 1 day; and B. for 4 days C. The neuronal nature of primary
cells was determined by binding with tetanus toxin (NeuroTagr.TM.).
Cell bodies and processes of all cells in culture were stained.
Calibration bar=20 .mu.m.
[0018] FIG. 2 illustrates photomicrographs showing the
morphological changes that occur during the culture and passaging
of primary neurons. A. Primary cell culture after 4 days of plating
in N2+bFGF. B. Primary cells 4 days in culture after passage
(passage 3). Cells were larger and interconnected by processes that
also increased in size. Small proliferating cells were visible in
the culture. C. Cells passaged (passage 3) and kept in culture for
.about.14 days in the presence of bFGF. Negative magnification
33.times..
[0019] FIG. 3 shows transmission electron micrographs of primary
neurons in culture. A. A pyramidal-shaped primary hippocampal
neuron showing both the soma and processes, including a major
apical process (arrow) and a finer caliber process (arrowhead).
Bar=10 .mu.m. B. Enlarged view of the neuronal soma shown in panel
A. Bar=1 .mu.m. C. A portion of the major apical process of the
neuron shown in panel A. Bar=1 .mu.m. D. Contact between two
neuritic processes. Bar=0.1 .mu.m.
[0020] FIG. 4 shows scanning electron micrographs of primary
neurons in culture. A. Overview of primary hippocampal neurons in
culture including well-differentiated pyramidal somata (arrow) with
large processes containing multiple levels of branching and
less-differentiated, rounded neurons with large, extended processes
(arrowheads). Bar=50 .mu.m B. A major apical dendrite emerging from
a well-differentiated pyramidal neuron showing a smooth, regular
caliber process just proximal to the first (major) bifurcation with
several smaller processes, possibly axons emerging from it. The
PORN/laminin coating the vessel surface can be seen as a porous
carpeting which is absent in some patches. Bar=2 m C. A
well-differentiated neuron (in the middle of the field) possessing
a large pyramidal soma (compare to FIG. 3A) and a large apical
dendrite (arrowheads) contacted by a number of processes from other
neurons. Other less-differentiated neurons which are fixed in the
process of dividing were also present (arrows). Bar=20 .mu.m. D.
Enlarged view of the dividing neuron in the upper field of view in
panel C. Bar=10 .mu.m.
DETAILED DESCRIPTION OF THE INVENTION
[0021] The present invention provides an in vitro method for
producing an isolated neuronal cell population. These cells, termed
neuroblasts can be produced by utilizing methodology which
comprises culturing a neuronal cell in a serum-free media
supplemented with at least one trophic factor using a vessel which
allows attachment of the cell. This method allows the generation of
continuous, neuronal cell cultures from different regions of the
brain, from both fetal and adult tissue, which are capable of
proliferation.
[0022] The invention also provides a method of identifying
compositions which affect a neuroblast, such as by inhibiting or
stimulating the neuroblast proliferation. A culture system useful
for the production and maintenance of a neuroblast comprising a
serum-free basal media containing at least one trophic factor and a
vessel which allows attachment of the neuroblast is also provided.
An enriched population of neuroblast cells produced by the method
of the invention is also provided and can be further utilized for
the treatment of a subject with a neuronal cell disorder or
alternatively, to screen compositions which affect the
neuroblast.
[0023] As used herein, the term "neuroblast" refers to a non-glial
cell of neuronal lineage which has been perpetualized. Neuronal
"perpetualization" refers to the procedure whereby a non-glial cell
of the neuronal lineage is treated with growth factors such that it
is capable of indefinite maintenance, growth and proliferation in
vitro. Typically, a primary culture, one in which the tissue is
removed from an animal, is placed in a culture vessel in
appropriate fluid medium, and has a finite lifetime. In contrast,
continuous cell lines proliferate and thus can be subcultured,
i.e., passaged repeatedly into new culture vessels. Continuous cell
lines can also be stored for long periods of time in a frozen state
in the vapor phase of liquid nitrogen when a cryopreservatve is
present, e.g., 10% dimethylsulfoxide or glycerol. The neuroblast of
the invention can be maintained in long-term culture as a cell line
closely resembling primary cultures, but without resort to
oncogenic immortalization. Rather, "perpetualization" establishes a
continuous culture from a primary neuronal cell by utilizing a
specific growth factor or combination of growth factors. This
perpetualization technique is novel in that no gene transfer or
genetic manipulation is required and, as a consequence, the cells
more closely resemble primary cultures.
[0024] There are hundreds of different types of neurons, each with
distinct properties. Each type of neuron produces and responds to
different combinations of neurotransmitters and neurotrophic
factors. Neurons do not divide in the adult brain, nor do they
generally survive long in vitro. The method of the invention
provides for the isolation and growth of perpetualized neurons, or
neuroblasts, in vitro; from virtually any region of the brain and
spinal cord. Either embryonic or adult neurons can be utilized for
the development of neuroblast cell lines. The neuronal cell of the
invention, which is utilized for production of a neuroblast, can be
derived from any fetal or adult neural tissue, including tissue
from the hippocampus, cerebellum, spinal cord, cortex (e.g., motor
or somatosensory cortex), striatum, basal forebrain (cholenergic
neurons), ventral mesencephalon (cells of the substantia nigra),
and the locus ceruleus (neuroadrenaline cells of the central
nervous system).
[0025] The liquid media for production of a neuroblast of the
invention is supplemented with at least one trophic factor to
support the growth and proliferation of a neuroblast Trophic
factors are molecules which are involved in the development and
survival of neurons. They are often synthesized in the brain, have
specific receptors, and influence the survival and function of a
subset of neurons. Examples of such factors include nerve growth
factor (NGF), brain-derived neurotrophic factor (BDNF),
neurotrophin-3, -4, and -5 (NTF-3, -4, -5), ciliary neurotrophic
factor (CNTF), basic fibroblast growth factor (bFGF), acidic
fibroblast growth factor (aFGF), platelet derived growth factor
(PDGF), epidermal growth factor (EGF), insulin-like growth factor-I
and -II (IGF-I, -II), transforming growth factor (TGF) and
lymphocyte infiltrating factor/cholinergic differentiating factor
(LIF/CDF). The specificity and selectivity of a trophic factor are
determined by its receptor. Preferably, the trophic factor utilized
in the invention is a neurotrophic factor. Preferably, the
neurotrophic factor added to the basal media for production of a
neuroblast according to the method of the invention is bFGF. The
neurotrophic factor which allows growth and proliferation of the
neuroblast in vitro will depend on the tissue origin of the
neuroblast. However, for most neuronal cells, bFGF will be the
preferred neurotrophic factor.
[0026] The vessel utilized for production of a neuroblast must
provide a surface which allows attachment of the neuronal cell.
Such vessels are also preferred once the isolated neuroblast
culture has been produced. The surface used to enhance attachment
of the neuronal cell can be the actual inner layer of the vessel or
more indirectly, the surface of a supplemental insert or membrane
which resides within the vessel. Attachment may be accomplished by
any means which allows the cell to grow as a monolayer on a vessel.
Attachment enhancing surfaces can be produced directly, such as by
advantageous selecting of appropriate plastic polymers for the
vessel or, indirectly, as by treating the surface in the vessel by
a secondary chemical treatment Therefore, "attachment" refers to
the ability of a cell to adhere to a surface in a tissue culture
vessel, wherein the attachment promoting surface is in direct
contact with neuronal cells, which otherwise would grow in a
three-dimensional cellular aggregate in suspension. Attachment, or
adherence, of a neuronal cell to the vessel surface allows it to be
perpetualized.
[0027] In addition to interactions with soluble factors, most cells
in vivo, including neuronal cells, are in contact with an
extracellular matrix, a complex arrangement of interactive protein
and polysaccharide molecules which are secreted locally and
assemble into an intricate network in the spaces between cells.
Therefore, the addition of an extracellular matrix protein to the
surface of the culture vessel forms an insoluble matrix which
allows neuronal cells in culture to adhere in a manner which
closely corresponds to the in vivo extracellular matrix The
neuroblast of the invention can be preferably produced by coating
the surface of a vessel, such as a tissue culture dish or flask,
with a polybasic amino acid composition to allow initial
attachment. Such compositions are well known in the art and include
polyornithine and polylysine. Most preferably, the polybasic amino
acid of the invention is polyornithine. Additionally, the surface
of the vessel may be coated with a known extracellular matrix
protein composition to enhance the neuroblast's ability to grow and
form processes on the substrate. Such compositions include laminin,
collagen and fibronectin. Other extracellular matrix proteins that
can be used in conjunction with a polybasic amino acid will be
apparent to one of skill in the art. Additionally, for the
production of adult neuroblasts, it is preferable to initially
culture the cells in the presence of serum.
[0028] The neuroblast of the invention is useful as a screening
tool for neuropharmacological compounds which affect a biological
function of the neuroblast. Thus, in another embodiment, the
invention provides a method for identifying a composition which
affects a neuroblast comprising incubating the components, which
include the composition to be tested and the neuroblast, under
conditions sufficient to allow the components to interact, then
subsequently measuring the effect the composition on the neuroblast
The observed effect on the neuroblast may be either inhibitory or
stimulatory. For example, a neuroactive compound which mimics a
neurotransmitter or binds to a receptor and exhibits either an
antagonistic or agonist effect, thereby inhibiting or stimulating a
biological response in the neuroblast, can be identified using the
method of the invention. The occurrence of a biological response
can be monitored using standard techniques known to those skilled
in the art. For example, inhibition or stimulation of a biological
response may be identified by the level of expression of certain
genes in the neuroblast. Such genes may include early response
genes such as fos, myc or jun (Greenberg, M. and Ziff, E. Nature,
311:433, 1984; eds. Burck, et al., in Oncogenes, 1988,
Springer-Verlag, New York.). Other genes, including those which
encode cell surface markers can also be used as indicators of the
effects neuropharmacological compounds on the neuroblasts of the
invention Methods for measurement of such effects include Northern
blot analysis of RNA (transcription), SDS-PAGE analysis of protein
(translation), [C.sup.3H]-thymidine uptake (DNA synthesis) and
antibody reactivity (both intracellular and extracellular). Other
commonly used methods will be apparent to those of skill in the
art.]
[0029] Neuroactive drugs which act similarly to those already known
to affect neuronal cells can thus be identified. For example, new
drugs that alleviate anxiety, analogously to Valium, which augment
or stimulate the action of the important inhibitory transmitter
gamma-aminobutyric acid (GABA), can be identified. Antidepressants,
such as Prozac, enhance the action of serotonin, an indoleamine
with a wide variety of functions. Other drugs can be readily
identified using the neuroblasts according to the method of the
invention. Other examples include psychoactive compounds. For
example, cocaine facilitates the action of dopamine, whereas
certain antipsychotics antagonize or inhibit this catecholamine.
Another example is nicotine which activates the acetylcholine
receptors which are distributed throughout the cerebral cortex.
Therefore, by using neuroblasts derived from neuronal cells from
the appropriate regions of the brain, drugs and trophic factors
which bind various receptors and would produce similar effects on
neuronal cells can be identified using the method of the
invention.
[0030] As described above, perpetualization of a neuronal cell can
be, accomplished without the use of oncogenic intervention.
However, if desired the neuroblast of the invention may be
immortalized to maintain the cell at a defined developmental stage.
The present techniques for immortalization typically involve the
transfection of an oncogene to the cell, therefore, immortalization
of a neuroblast can be achieved by introduction of at least one
oncogene to the neuroblast. Transfection of the oncogene can be
accomplished by several conventional methods well known to those
skilled in the art, including using recombinant retroviruses,
chemical, or physical methods. Recombinant retrovirus transfer is
the preferred method of the invention for immortalization of
neuroblasts.
[0031] The host neuroblast can be immortalized with a particular
onco'gene by such methods of transfection as calcium phosphate
co-precipitation, conventional mechanical procedures such as
microinjection, insertion of a plasmid encased in liposomes, or by
use of viral vectors. For example, one method is to use a
eukaryotic viral vector, such as simian virus 40 (SV40) or bovine
papilloma virus, to transiently infect or transform the neuroblast
(Eukaryotic Viral Vectors, Cold Spring Harbor Laboratory, Gluzman
ed., 1982).
[0032] Various viral vectors which can be utilized for
immortalization as taught herein include adenovirus, herpes virus,
vaccinia, and preferably, an RNA virus such as a retrovirus.
Preferably, the retroviral vector is a derivative of a murine or
avian retrovirus. Examples of retroviral vectors in which a single
foreign gene can be inserted include, but are not limited to:
Moloney murine leukemia virus (MoMuLV), Harvey murine sarcoma virus
(HaMuSV), murine mammary tumor virus (MuMTV), and Rous Sarcoma
Virus (RSV). A number of additional retroviral vectors can
incorporate multiple genes. All of these vectors can transfer or
incorporate a gene for a selectable marker so that transduced cells
can be identified and generated.
[0033] Since recombinant retroviruses are defective, they require
assistance in order to produce infectious vector particles. This
assistance can be provided, for example, by using helper cell lines
that contain plasmids encoding all of the structural genes of the
retrovirus (gag, env, and pol genes) under the control of
regulatory sequences within the long terminal repeat (LTR). These
plasmids are missing a nucleotide sequence which enables the
packaging mechanism to recognize an RNA transcript for
encapsidation. Helper cell lines which have deletions of the
packaging signal include, but are not limited to .psi.2, PA317,
PA12, CRIP and CRE, for example. These cell lines produce empty
virions, since no genome is packaged. If a retroviral vector is
introduced into such cells in which the packaging signal is intact,
but the structural genes are replaced by other genes of interest,
the vector can be packaged and vector virion produced. The vector
virions produced by this method can then be used to infect a tissue
cell line, such as NIH 3T3 cells, to produce large quantities of
chimeric retroviral virions.
[0034] Alternatively, NIH 3T3 or other issue culture cells can be
directly transfected with plasmids encoding the retroviral
structural genes gag, pol and env, by conventional calcium
phosphate or lipofection transfection. These cells are then
transfected with the vector plasmid containing the genes of
interest. The resulting cells release the retroviral vector into
the culture medium.
[0035] Herpes virus-based vectors may also be used to transfer
genes into a neuroblast. Since herpes viruses are capable of
establishing a latent infection and an apparently non-pathogenic
relationship with some neural cells, such vector based on HSV-1,
for example, may be used. Similarity, it should be possible to take
advantage other human and animal viruses that infect cells of the
CNS efficiently, such as rabies virus, measles, and other
paramyxoviruses and even the human immunodeficiency retrovirus
(HIV), to develop useful delivery and expression vectors.
[0036] When a recombinant retrovirus is engineered to contain an
immortalizing oncogene, the oncogene can be any one of those known
to immortalize. For example, such commonly used immortalizing genes
include genes of the myc family (both c-myc and v-myc) (Bartlett,
et al., Proc.Natl.Acad.Sci.USA 85:3255, 1988), adenovirus genes
(E1a 12s and E1a 13s) (Ruley, et al., Nature 304:602, 1983), the
polyoma large T antigen and SV40 large T antigen (Frederiksen, et
al., Neuron 1:439, 1988). Preferably, the oncogene used to
immortalize the neuroblast of the invention is v-myc. Other genes,
for example other nuclear oncogenes, that immortalize a cell but
may require a second gene for complete transformation, will be
known to those of skill in the art.
[0037] The same transfection methods described above for
immortalization of a neuroblast can be utilized to transfer other
exogenous genes to the neuroblast of the invention. An "exogenous
gene" refers to genetic material from outside the neuroblast which
is introduced into the neuroblast. An example of a desirable
exogenous gene which would be useful for the method of identifying
neuropharmacological compounds is a gene for a receptor molecule.
For example, such neuronal receptors include the receptor which
binds dopamine, GABA, adrenaline, noradrenaline, serotonin,
glutamate, acetylcholine and various other neuropeptides. Transfer
and expression of a particular receptor in a neuroblast of specific
neural origin, would allow identification of neuroactive drugs and
trophic factors which may be useful for the treatment of diseases
involving that neuroblast cell type and that receptor. For example,
a neuroactive compound which mimics a neurotransmitter and binds to
a receptor and exhibits either an antagonistic or agonist effect,
thereby inhibiting or stimulating a response in the neuroblast, can
be identified using the method of the invention.
[0038] In another embodiment, the invention provides a culture
system useful for the production and maintenance of a neuroblast
comprising a serum-free basal media containing at least one trophic
factor and a vessel having a surface which allows attachment of the
neuroblast. The culture system can be utilized to produce a
neuroblast from any tissue of neural origin as described above. The
"serum-free basal media" of the invention refers to a solution
which allows the production and maintenance of a neuroblast. The
basal media is preferably a commonly used liquid tissue culture
media, however, it is free of serum and supplemented with various
defined components which allow the neuroblast to proliferate. Basal
media useful in the culture system of the invention is any tissue
culture media well known in the art, such as Dulbecco's minimal
essential media, which contains appropriate amino acids, vitamins,
inorganic salts, a buffering agent, and an energy source. Purified
molecules, which include hormones, growth factors, transport
proteins, trace elements, vitamins, and substratum-modifying
factors are added to the basal media to replace biological fluids.
For example, progesterone, sodium selenite, putrescine, insulin and
transferrin are typically added to the basal media to enhance
neuroblast growth and proliferation. For the culture system of the
invention, only two of the defined supplements are necessary to
sustain growth of neurons alone (transferrin and insulin), whereas
the combination of the five supplements above have a highly
synergistic growth-stimulating effect Deletion of any single
supplement results in markedly diminished growth of the neuroblast.
An example of a preferred prototype medium which contains these
elements is. N2 medium (Bottenstein and Sato, et al.,
Proc.Natl.Acad.Sci.USA, 76:514, 1979). The optimal concentration of
the supplements are as follows: 5 .mu.g/ml insulin, 100 .mu.g/ml
transferrin, 20 nM progesterone, 100 .mu.M putrescine, and 30 nM
selenium (as Na.sub.2SeO.sub.3).
[0039] The basal media of the culture system further contains at
least one trophic factor for the production and maintenance of a
neuroblast. Most preferably, neurotrophic factors are utilized and
specifically bFGF. bFGF is utilized in the basal media at a
concentration from about 1 ng/ml to about 100 ng/ml, more
specifically from about, 5 ng/ml to about 70 ng/ml, and most
preferably from about 15 ng/ml to about 60 ng/ml. Neural cultures
are generally maintained at pH 72-7.6. A higher requirement for
glucose is also necessary for neural as opposed to non-neural
cells. Therefore, the basal media of the invention contains a
concentration of from about 0.01% to about 1.0% glucose and
preferably from about 0.1% to about 0.6% glucose.
[0040] The invention also provides a cellular composition
comprising an enriched population of neuroblast cells. The
composition preferably contains a majority of or at least about 90%
neuroblasts. The neuroblast cells are derived from any CNS neural
tissue such as from any region of the brain, as described above, or
from the spinal cord. The neuroblast may be further immortalized
with an oncogene, or it may contain an exogenous gene encoding a
receptor or a ligand for a receptor.
[0041] The present invention also provides a method of treating a
subject with a neuronal cell disorder which comprises administering
to the subject a therapeutically effective amount of the neuroblast
of the invention. "Therapeutically effective" as used herein,
refers to that amount of neuroblast that is of sufficient quantity
to ameliorate the cause of the neuronal disorder. "Ameliorate"
refers to a lessening of the detrimental effect of the neuronal
disorder in the patient receiving the therapy. The subject of the
invention is preferably a human, however, it can be envisioned that
any animal with a neuronal disorder can be treated with the
neuroblast of the invention. Preferably, the neuroblast is derived
from neuronal tissue of the same species as the species of the
subject receiving therapy.
[0042] The method of treating a subject with a neuronal disorder
entails intracerebral grafting of neuroblasts to the region of the
CNS having the disorder. Where necessary, the neuroblast can be
genetically engineered to contain an exogenous gene. The disorder
may be from either disease or trauma (injury). Neuroblast
transplantation, or "grafting" involves transplantation of cells
into the central nervous system or into the ventricular cavities or
subdurally onto the surface of a host brain. Such methods for
grafting will be known to those skilled in the art and are
described in Neural Grafting in the Mammalian CNS, Bjorklund and
Stenevi, eds., (1985), incorporated by reference herein. Procedures
include intraparenchymal transplantation, (i.e., within the host
brain) achieved by injection or deposition of tissue within the
host brain so as to be apposed to the brain parenchyma at the time
of transplantation.
[0043] Administration of the neuroblasts of the invention into
selected regions of the recipient subject's brain may be made by
drilling a hole and piercing the dura to permit the needle of a
microsyringe to be inserted. The neuroblasts can alternatively be
injected intrathecally into the spinal cord region. The neuroblast
preparation of the invention permits grafting of neuroblasts to any
predetermined site in the brain or spinal cord, and allows multiple
grafting simultaneously in several different sites using the same
cell suspension and permits mixtures of cells from different
anatomical regions. The present invention provides a method for
transplanting various neural tissues, by providing previously
unavailable proliferating neuroblasts and a culture system for
production of these neuroblas's in order to grow a sufficient
number of cells for in vitro gene transfer followed by in vivo
implantation.
[0044] The neuroblast used for treatment of a neuronal disorder may
optionally contain an exogenous gene, for example, an oncogene, a
gene which encodes a receptor, or a gene which encodes a ligand.
Such receptors include receptors which respond to dopamine, GABA,
adrenaline, noradrenaline, serotonin, glutamate, acetylcholine and
other neuropeptides, as described above. Examples of ligands which
may provide a therapeutic effect in a neuronal disorder include
dopamine, adrenaline, noradrenaline, acetylcholine,
gamma-aminobutyric acid and serotonin. The diffusion and uptake of
a required ligand after secretion by a donor neuroblast would be
beneficial in a disorder where the subject's neural cell is
defective in the production of such a gene product. A neuroblast
genetically modified to secrete a neurotrophic factor, such as
nerve growth factor, (NGF), might be used to prevent degeneration
of cholinergic neurons that might otherwise die without treatment.
Alternatively, neuroblasts to be grafted into a subject with a
disorder of the basal ganglia, such as Parkinson's disease, can be
modified to contain an exogenous gene encoding L-DOPA, the
precursor to dopamine. Parkinson's disease is characterized by a
loss of dopamine neurons in the substantia-nigra of the midbrain,
which have the basal ganglia as their major target organ.
Alternatively, neuroblasts derived from substantia-nigra neuronal
cells which produce dopamine could be introduced into a Parkinson's
patient brain to provide cells which "naturally" produce
dopamine.
[0045] Other neuronal disorders that can be treated similarly by
the method of the invention include Alzheimer's disease,
Huntington's disease, neuronal damage due to stroke, and damage in
the spinal cord. Alzheimer's disease is characterized by
degeneration of the cholinergic neurons of the basal forebrain. The
neurotransmitter for these neurons is acetylcholine, which is
necessary for their survival. Engraftment of cholinergic
neuroblasts, or neuroblasts containing an exogenous gene for a
factor which would promote survival of these neurons can be
accomplished by the method of the invention, as described.
Following a stroke, there is selective loss of cells in the CA1 of
the hippocampus as well as cortical cell loss which may underlie
cognitive function and memory loss in these patients. Once
identified, molecules responsible for CA1 cell death can be
inhibited by the methods of this invention. For example, antisense
sequences, or a gene encoding an antagonist can be transferred to a
neuroblast and implanted into the hippocampal region of the
brain.
[0046] The method of treating a subject with a neuronal disorder
also contemplates the grafting of neuroblasts in combination with
other therapeutic procedures useful in the treatment of disorders
of the CNS. For example, the neuroblasts can be co-administered
with agents such as growth factors, gangliosides, antibiotics,
neurotransmitters, neurohormones, toxins, neurite promoting
molecules and antimetabolites and precursors of these molecules
such as the precursor of dopamine, L-DOPA.
[0047] The following examples are intended to illustrate but not
limit the invention. While they are typical of those that might be
used, other procedures known to those skilled in the art may
alternatively be used.
EXAMPLES
[0048] The following examples show neuronal proliferation of
hippocampal, spinal cord, substania nigra, basal forebrain, and
other neuronal tissue cells from fetal rats cultured over 5 months
with bFGF. In addition, adult hippocampus was cultured with bFGF in
a defined media for more than 7 months. The examples also provide
methodology for the generation, differentiation and long term
culture of numerous cell types from fetal and adult neuronal tissue
and describe the morphological, immunocytochemical, ultrastructural
and molecular characteristics of proliferating non-neuronal and
neuronal cell types in the adult bFGF treated cultures.
[0049] Proliferating cells that incorporated bromodeoxyuridine were
immunopositive for neuron-specific enolase. Cells with polarized
morphologies typical of well-differentiated neurons were
immunopositive for the high molecular weight subunit of
neurofilament protein (NFh), characteristic of mature neurons, the
middle and low subunits of neurofilament protein and
microtubule-associated protein 2 (MAP-2). Cells from adult
mammalian hippocampus were capable of proliferation as well as
long-term neurogenesis and neuronal differentiation in vitro. These
cells may be a source of replacement cells in neuronal grafting.
Further, the induction of proliferation and differentiation of
these cells in vivo would be useful for replacement or augmentation
of neuronal loss or degeneration.
Example 1
Materials and Methods
[0050] Materials: DMEM:F12 medium, N2 supplement and laminin were
obtained from Gibco/BRL (Bethesda, Mo.); polyomithine (PORN) was
obtained from Sigma (St Louis, Mo.). Recombinant bFGF was from
Syntex/Synergen Consortium (Boulder, Colo.). Bovine bFGF was
purchased from R&D, Minneapolis, Minn. NeuroTag.TM. green was
obtained from Boehringer Mannheim, Indianapolis, Ind. Cell
proliferation kit containing bromodeoxyuridine (BrdU), anti-BrdU
antibody and streptavidin/Texas Red was purchased from Amersham.
Arlington Heights, Ind. The antibodies used to determine the
phenotypes of cells in culture were obtained from the following
sources and used at the indicated dilutions: polyclonal rabbit
antifleurofilament 200 (NF) (1:500; Chemicon International,
Temecula, Calif.), monoclonal anti-neuron specific enolase (NSE)
(1:50; DAKO, Carpenteria, Calif.), monoclonal anti-glia fibrillary
acidic protein (GFAP) (1:500-1:10,000; Amersham, Arlington Heights,
Ill.), monoclonal anti-vimentin (1:800; Boehringer Mannheim,
Indianapolis, Ind.), monoclonal anti-OX-42 (1:5000; Serotec,
Indianapolis, Ind.), polyclonal anti-galactocerebroside (Gal C)
(1:5000; Advanced Immunochemical Services, Long Beach, Calif.),
monoclonal anti-microtubule associated protein (MAP 2) (1:500;
Sigma Immunochemicals, St. Louis, Mo.), polyclonal anti-fibronectin
(1:2000; Telios, La Jolla, Calif.). Polyclonal nestin antibody
(1:15,000) was from Dr. R. McKay, MIT, Cambridge, Mass., and
monoclonal high affinity bFGF receptor antibody (1:20) was from Dr.
A. Baird, Whittier Institute, La Jolla, Calif. Polyclonal anti-GFAP
(1:2000) was from Dr. L F. Eng, Stanford University, Palo Alto,
Calif.
[0051] Cell Culture. The brains of Fisher 344 rats (E16) were
dissected, the meninges were removed and the hippocampi were
isolated. Hippocampi were transferred to a 15 ml tissue culture
tube and the volume was adjusted to 1-2 ml with phosphate-buffered
saline (pH 7.4) supplemented with 0.6% glucose (PBS-G). Hippocampi
were mechanically dissociated by trituration with a pasteur pipet
(.about.20.times.) followed by trituration with a pasteur pipet
fire-polished to significantly reduce the pipet bore
(.about.20.times.). The cell suspension was pelleted by
centrifugation at 1000 rpm for 5 minutes at room temperature. Cells
were taken up in .about.20 ml N2 medium (1:1 mixture of DMEM:F12
containing 20 nM progesterone, 30,nM sodium selenite, 100 .mu.M
putrescine, 3.9 mM glutamine, 5 .mu.M/ml insulin, 100 .mu.g/ml
transferrin) and the cell number was quantified with a
hemocytometer. Tissue culture plates were coated with polyomithine
(PORN; 10 .mu.g/ml) followed by laminin (10 .mu.g/ml).
Approximately 0.5-10.times.10.sup.6 cells/well were plated on
PORN/laminin-coated 6 well plates in N2 medium containing 20 ng/ml
bFGF (N2+bFGF) and cultured at 37.degree. C. in 5% CO.sub.2. Medium
was changed every 3-4 days with fresh N2+bFGF. For passaging, cells
were trypsinized (ATV trypsin, Irvine Scientific, Santa Ana,
Calif.) and then taken up in N2+bFGF. Cells were pelleted by
centrifugation and supernatant containing trypsin was removed.
Cells were resuspended in 10 ml N2.div.bFGF and plated. Cells could
be frozen in liquid nitrogen in N2+bFGF+10% dimethylsulfoxide
(DMSO). For culturing, cells were thawed quickly at 37.degree. C.,
added to 10 ml N2+bFGF, centrifuged to remove DMSO, resuspended in
fresh N2+bFGF and plated as described before.
[0052] BrdU Incorporation Experiments. Primary neurons (passage 5)
were grown for 3 days, whereupon the media was changed. On the
following day cells were incubated with BrdU for either 1 day or 4
days. Cells were fixed, washed and then treated with a monoclonal
antibody against BrdU for 1 hour After washing, cells were reacted
with biotinylated anti-mouse antibody (Vector Laboratories,
Burlingame, Calif.) followed by streptavidin/Texas Red complex.
Stained cultures were examined with a BioRad MRC600 confocal
scanning laser microscope equipped with a krypton-argon laser using
the YHS filter set (568 EX, 585 LP). Confocal fluorescent and
Nomarski transmitted collected images were transferred to an Apple
Macintosh Quadra 700, merged using Adobe Photoshop 2.01, and
printed out on a GCC film recorder.
[0053] Neurotag.TM. Binding. Primary neurons (passage 5) grown in
culture for 6 days were incubated with 10 .mu.g/ml recombinant
tetanus toxin C fragment conjugated to fluorescein isothiocyanate
(NeuroTag.TM.) in N2+bFGF and bovine serum albumin (0.1 mg/ml) for
2 hours. After washing the cells were examined in a BioRad confocal
microscope as described for BrdU stained cells except using the BHS
filter (488 ED, 515 LP).
[0054] Immunohistochemistry. Cells were passaged (passage 3; 4 days
in culture after plating), grown in a 24-well plate, fled in 4%
paraformaldehyde in PBS, and then permeabilized with 0.25% Triton
X-100 in Tris buffered saline. Cells were incubated overnight at
4.degree. C. with polyclonal or monoclonal antibodies in the
presence of 1% normal horse serum (for monoclonal antibody) or 10%
normal goat serum (for polyclonal antibody). After washing, cells
were incubated with biotin conjugated goat anti-rabbit IgG or horse
anti-mouse IgG antibodies (Vector Laboratories, Burlingame, Calif.)
for 1 hour at room temperature, followed by incubation for 1 hour
at room temperature with a pre-formed mixture of
avidin-biotinylated horseradish peroxidase complex (Vectastain
Elite ABC kit). The reaction products were visualized with
diaminobenzidine (DAB) histochemistry.
[0055] Transmission Electron Microscopy (TEM). Cultures (passage 3;
four days after plating) grown on LabTek.TM. permanox slides (Ted
Pella, Inc., Redding, Calif.) were fixed in 2% glutaraldehyde in
100 mM PO.sub.4 at 37.degree. C. for 2 hours and then rinsed and
postfixed in 1% aqueous OSO.sub.4 for 1 hour at room temperature.
Cultures were then dehydrated in a graded ethanol series,
infiltrated with Araldite resin and polymerized in situ. The glass
slide was separated from the polymerized resin from which blocks of
cultured cells were cut and glued to resin blanks. Sections were
cut parallel to the culture substrate at a thickness of 70 nm.
Sections collected on 300 mesh copper grids were stained with
uranyl acetate and lead citrate and examined with a Phillips CM10
transmission electron microscope at 80 kV.
[0056] Scanning Electronic Microscopy (SEM). Cultures (passage 2;
four days after plating) grown on LabTek.TM. glass slides were
prepared as for TEM up through ethanolic dehydration. The plastic
chambers were then removed, leaving the sealing gasket in place,
and the slide was placed into a Pelco critical point dryer.
Following drying, the slide was coated with gold-palladium to a
thickness of 300 .ANG. in a Technics sputter coater. The cells were
examined in a Cambridge Stereoscan 360 scanning electron microscope
at 10 kV.
[0057] Gene Transfer into Neurons. Approximately 1.times.10.sup.6
producer cells were plated on PORN/laminin coated wells in a 6 well
plate and grown overnight at 37.degree. C. in 5% CO.sub.2. Virus
from producer cells was collected after overnight incubation in
DMEM (Dulbecco's minimum essential medium) containing 5% fetal calf
serum (FCS) or 5% bovine calf serum (BCS). Virus containing media
was filtered through 0.45 .mu.m filters and then mixed with
polybrene (8 .mu.g/ml) and bFGF (20 ng/ml). Media was removed from
neuronal cultures and virus containing media was added to neuronal
cultures and incubated overnight at 37.degree. C. in 5% CO.sub.2.
After this infection period, the media was removed and replaced
with N2 media containing 20 ng/ml bFGF. When the expression vector
contained the neomycin resistant gene, the infected cells were
selected in the presence of G418 (400 .mu.g/ml). Cells were
passaged and maintained as described above.
[0058] The expression vectors and producer cells used were as
follows:
[0059] 1. Avian v-myc gene was expressed from MLV-LTR promoter and
bacterial neomycin resistant gene was expressed from thymidine
kinase (TK) promoter (Ryder, et al., J. Neurobiol., 21:356375,
1989; Kaplan, et al., J. Virol, 61:1731-1.734, 1987; and Land, et
al., Mol. Cell. Biol., 6:1917-1925, 1986). A producer line was
generated from .psi.2 cells. These cells grew in DMEM containing
10%. FCS and 400 .mu.g/ml G418. The day before the infection, the
medium was changed with fresh DMEM containing 5% FCC.
[0060] 2. Bacterial .beta.-galactosidase gene was expressed from
the MLV-LTR promoter. This expression vector contains a part of the
gag gene and produces very high titer virus. There is no neomycin
resistant gene in this vector. This vector was from Dr. Richard
Mulligan, MIT, Cambridge, Mass.
[0061] A promoter line was generated from CRIP cells. These cells
grow in DMEM containing 10% BOB. The day before the infection, the
medium was changed with DMEM containing 5% BCS.
Example 2
Growth of Neurons In Vitro
[0062] The chemically defined medium, N2 (Bottenstein and Sato,
Proc. Natl. Acad. Sci. USA, 76: 514-517, 1980; Bottenstein, J. E,
In: Cell culture in the neurosciences, J. E Bottenstein and G. H.
Sato, Eds., Plenum Press, New York, N.Y., pp 3-43, 1985; di Porizo,
et al., Nature, 288:370-373, 1980), has been used to reproducibly
generate short-term virtually pure neuronal cultures (Bottenstein,
et al., Exp. Cell Res., 125:183-190, 1980; Barnes and Sato, Anal.
Biochem., 102:255-302, 1980) This medium does not support the
survival or proliferation of non-neuronal cells and it is possible
to obtain >95% pure neuronal culture. In defined medium, primary
cultures of hippocampal neurons die within 7 days but can be
maintained for 24 weeks in the presence of hippocampal explants, a
feeder layer of astrocytes, in astrocyte-conditioned medium
(Banker, G. A., Science, 209:809-610, 1980) or in the presence of
bFGF (Walicke and Baird, Proc. Natl. Acad. Sci. USA, 83:3012-3016,
1986; Walicke; P. A.; J. Neurosci., 8:2618-2627, 1988; Walicke, et
al., In: Prog. Brain Res., vol. 78, D. m Gash and J. R. Sladek,
Eds. (Elsevier Science Publishers B.V.), pp 333-338, 1988).
However, cells continued to die slowly and few cells remained after
1 month (Walicke, P., et al., Proc. Natl. Acad. Sci. USA,
83:3012-3016, 1986).
[0063] bFGF at 20 ng/ml, a concentration of about 100 fold higher
concentration than that used before to study the survival and
elongation of axons (Walicke, P. et al., Proc. Natl. Acad. Sci.
USA, 83:3012-3016, 1986; Walicke, P. A., J. Neurosci., 8:2618-2627,
1988), showed dramatic proliferative effects on hippocampal cells.
This proliferative property of bFGF was used to promote continued
proliferation of primary hippocampal cells to form a long-term
culture. Cells cultured in 20 ng/ml bFGF began proliferating by 2
days, with a doubling time of 4 days. Primary cells became contact
inhibited for growth and reached a plateau after day 7, although
growth continued within aggregates (FIG. 2C).
[0064] To test whether division was occurring in all cells or only
in a subpopulation, cultures were incubated with BrdU for 1 or 4
days and the labeled nuclei were visualized by indirect
immunofluorescence using an anti-BrdU-antibody (FIGS. 1A, B).
[0065] FIG. 1 shows BrdU staining and NeuroTag.TM. binding of
primary neurons in culture. Primary neurons were labeled with BrdU
for 1 day (A) and for 4 days (B). Only a few cells were stained on
day 1, but by day 4 all cells were stained, indicating that all
cells in the culture were proliferating. The neuronal nature of
primary cells was determined by binding with tetanus toxin
(NeuroTag.TM.) (C). Cell bodies and processes of all cells in
culture were stained. Calibration bar=20 .mu.m. After day 1, the
nuclei of only a small fraction of cells were immunostained (FIG.
1A) but almost the entire cell population was immunostained after 4
days of incubation with BrdU (FIG. 1B).
[0066] To establish long-term cultures, cells were trypsinized and
passaged. The passaged cells (up to 6 passages tested) grew as well
as the original culture did Cells were frozen in liquid nitrogen,
thawed and cultured again. When cells at different passage numbers
were thawed and re-cultured, they grew equally well regardless of
the passage number. Freeze-thawed cells showed the same morphology
as the cells kept continuously in culture.
[0067] Other cells derived from neuronal tissue have also been
studied for their ability to grow and be maintained in N2 media in
the presence of bFGF. Table 1 shows the optimum concentrations of
bFGF for culture of the various cell lines.
1TABLE 1 REGION OF CNS CONCENTRATION OF bFGF (np/ml)* Hippocampus
20 Septum 100 Striatum 20 Cortex 20 Locus Coeruleus 50 Ventral
Mesencephalon 50 Cerebellum 20 Spinal Cord 20 *Optimum
concentration of bFGF used for culture
Example 3
Characterization of Cells
[0068] Several independent criteria were used to show tat the cells
in the cultures were indeed neurons. These included their
morphological characteristics during growth, expression of neuronal
markers and ultrastructural analysis by transmission and scanning
electron microscopy.
[0069] Cell morphology in culture was similar to that described for
short-term cultures of neurons (Banker and Cowan, Brain Res.,
126:397425, 1977; Banker and Cowan, J. Comp. Neurol., 187:469-494,
1979) (FIGS. 2A, B, C). FIG. 2 illustrates photomicrographs showing
the morphological changes that occur during the culture and
passaging of primary neurons. A shows primary cell culture after 4
days of plating in N2.div.bFGF contained numerous proliferating and
process-bearing cells. B shows' primary cells 4 days in culture
after passage (passage 3). Cells were larger and interconnected by
processes that also increased in size. Small proliferating cells
were visible in the culture. C shows cells passaged (passage 3) and
kept in culture for .about.14 days in the presence of bFGF formed
aggregates and were interconnected by an extensive network of
processes forming a lattice-type pattern (Negative magnification
33.times.).
[0070] Cells were immunostained for several different antigenic
markers. Cells were stained with anti-NF (200 KD) antibody (D);
with anti-NSE antibody (E) or with anti-GFAP antibody (F). Although
all cells stained with anti NF or anti-NSE antibodies, no cell
staining was observed with anti-GFAP antibody (Negative
magnification 33.times. (D,E); 66.times. (F)).
[0071] Calls began to proliferate by day 2 and newborn cells were
small and bipolar in shape. Short processes roughly equal in length
to cell bodies started to emerge from parent cells. Over the next
2-3 days, 1 or 2 of the processes started to grow rapidly and
contacted the neighboring cells (FIG. 2A). By day 7, both the cell
bodies and the processes had increased in size and an extensive
interconnecting network of processes had formed. This morphological
progression resembled hippocampal pyramidal neuronal morphologies
previously described in vitro (Banker and Cowan, Brain Res.,
126:397-425, 1977; Banker and Cowan, J. Comp. Neurol., 187:469-494,
1979). When cells growing in culture for 1-2 weeks were passaged,
more of these cells had processes than did the-cells newly cultured
from the brain (FIG. 2B). It is possible that many of these
processes survived passaging, albeit partially amputated. Cells
passaged and kept in culture for 14 days in the presence of bFGF
formed aggregates and were interconnected by an extensive network
of processes forming a lattice-type pattern (FIG. 2C). Few cells
divided in open areas; most cell division occurred in the
aggregates.
[0072] The cultures were characterized by immunostaining or
different antigenic markers (FIGS. 2D, E, F; Table 1). All cell
somata and their processes immunostained strongly with an antibody
against NF protein which is specifically expressed by neurons (FIG.
2D). Similarly, anti-NSE antibody stained all cells in our culture
(FIG. 2E; Table 1). The neuronal nature of the cells proliferating
in response to bFGF was further demonstrated by the binding of
tetanus toxin, a specific marker for neurons (Neale, et al., Soc.
Neurosci. Abst, 14:547, 1988). NeuroTag.TM. green stained cell
bodies and processes of all cells in the culture (FIG. 1C),
indicating that the cells were neurons and that no or very few
non-neuronal cells were present in the cultures. The large optical
depth of field with the objective used (10.times.) fails to
demonstrate the localization of NeuroTag.TM. signal as membrane
bound.
[0073] The cultures were tested by immunostaining for the presence
of non-neuronal cells (Table 2). Lack of immunostaining with
antibodies against GFAP indicated the absence of astrocytes (FIG.
2F). In a control experiment anti-GFAP antibody (Amersham), at the
same concentration (1:10,000) immunostained rat C6 and 9L and human
U373 glioma cells. The absence of oligodendrocytes and fibroblasts
in our cultures was demonstrated by the lack of staining for Gal C,
vimentin or fibronectin (Table 2). As a control, rat C6, 9L and
human U373 glioma cells were stained with vimentin (1:800) at the
same concentration as used for neuronal cultures. The results of
immunostaining for other antigenic markers are shown in Table 2:
these data support the conclusion that the cultures consist of
neurons uncontaminated by non-neuronal cells.
2TABLE 2 PROPERTIES OF PRIMARY HIPPOCAMPAL NEURONS - ANTIGENIC
MARKERS FOR NEURONS AND NON-NEURONAL CELLS CULTURING
CHARACTERISTICS Substrate Dependency Yes Basic FGF Dependency Yes
Freeze-Thaw Viability Yes ANTIGENIC MARKERS CELL SPECIFICITY
Neurofilament (NF) Neurons ++.sup.a GFAP Glia -.sup.b Nestin Stem
cells ++ Vimentin Glia precursors/fibroblasts - NSE Neurons +.sup.c
OX-42 Microglia/macrophages - Galactocerebroside Oligodendrocytes -
MAP2 Dendrites + Basic FGF receptor Neurons/glia + Fibronectin
Fibroblasts - ++.sup.a cells were labeled strongly -.sup.b cells
were not labeled +.sup.c cells were labeled weakly
Example 4
Analysis of Perpetualized Neurons In Vitro
[0074] Analysis of primary neurons in culture at the
ultrastructural level demonstrated the histotypic neuronal
morphology of these cells (FIGS. 3 and 4), in agreement with
previous ultrastructural studies (Bartlett and Banker, J.
Neurosci., 4:19440-19453, 1984. Rothman and Cowan, J. Comp.
Neurol., 195:141-155, 1981; Peacock, et al., Brain Res,
169:231-246, 1979). FIG. 3 shows transmission electron micrographs
of primary neurons in culture. A shows a pyramidal-shaped primary
hippocampal neuron showing both the soma and processes, including a
major apical process (arrow) and a finer caliber process
(arrowhead). Bar=10 .mu.m. B shows an enlarged view of the neuronal
soma shown in panel A. Bar=1 .mu.m. C shows a portion of the major
apical process of the neuron shown in panel A. This process is
dominated by microtubules and polysomal ribosomes identifying it as
a primary dendrite. Bar=1 .mu.m. D shows contact between two
neuritic processes. Bar=0.1 .mu.m.
[0075] The well-differentiated neurons exhibited a histotypic
pyramidal morphology, including a primary, apical dendrite with
multiple ramifications, finer caliber axons, and characteristic
nuclear morphology (FIGS. 3 and 4). A TEM micrograph of a
pyramidal-shaped primary hippocampal neuron is shown in FIG. 3A.
The level of this section encompasses both the soma and processes,
including a major apical process (arrow) and a finer caliber
process emerging from the basal aspect of the soma (arrowhead).
Other processes from adjacent neurons are also seen. The soma of
the neuron has a euchromatic nucleus with a peripheral rim of
heterochromatin and a somewhat reticulated nucleolus (FIG. 38).
Mitochondria and microtubules are present in the perikaryal
cytoplasm, which is dominated by rosettes of polysomal ribosomes. A
portion of the major apical process of the neuron is dominated by
microtubules and polysomal ribosomes identifying it as a primary
dendrite (FIG. 3C) Contact between 2 neuritic processes is shown in
FIG. 3D. The larger process containing a mitochondrion,
microtubules and vesicles is being contacted by a swollen,
bouton-ike structure arising from a finer caliber process. The
junction between such processes is typically vague and immature at
this age in culture. Although the membranes at the site of contact
appear to be uniformly parallel, there is little indication of
further assembly of synaptic structures. The contents of the
bouton-like process ending are unclear, appearing to be an
accumulation of vesicles, with a possible coated vesicle near the
site of contact.
[0076] FIG. 4 shows scanning electron micrographs of primary
neurons in culture. A shows an overview of primary hippocampal
neurons' in culture including well-differentiated pyramidal somata
(arrow) with large processes containing multiple levels of
branching and less-differentiated, rounded neurons with large,
extended processes (arrowheads). Bar=50 .mu.m. B shows a major
apical dendrite emerging from a well-differentiated pyramidal
neuron showing a smooth, regular caliber process just proximal to
the first (major) bifurcation with several smaller processes,
possibly axons emerging from it. The PORN/laminin coating the
vessel surface can be seen as a porous carpeting which is absent in
some patches. Bar=2 .mu.m. C shows a well-differentiated neuron (in
the middle of the field) possessing a large pyramidal soma (compare
to FIG. 3A) and a large apical dendrite (arrowheads) contacted by a
number of processes from other neurons. Other less-differentiated
neurons which are fixed in the process of dividing were also
present (arrows). Bar 20 .mu.m. D shows an enlarged view of the
dividing neuron in the upper field of view in panel C. The membrane
connecting the two daughter cell components is clearly continuous,
although cytokinesis is apparently underway. Note the process
extension from this less-differentiated neuron, indicating some
degree of differentiation during mitosis. Bar=10 .mu.m.
[0077] Scanning EM of primary hippocampal neurons in culture showed
the diversity of morphologies present, with some
well-differentiated pyramidal somata (FIG. 4A; arrow) extending
large processes which show multiple levels of branching and some
less-differentiated, rounded neurons. Even these rounded neurons
possess large, extended processes (FIG. 4A; arrowheads). Closer
examination of the major dendritic processes arising from the well
differentiated neurons shows large caliber processes with acute
bifurcations (FIG. 48). A number of small caliber, axon-like
processes are seen emerging from these major apical dendrites (FIG.
4B). Well-differentiated neurons typically possess a large
pyramidal soma (FIG. 4C compare to FIG. 3A). When
less-differentiated neurons are examined, many of these are found
to have been fixed in the process of dividing (FIG. 4C; arrows). A
closer view of the dividing neuron shows that, although cytokinesis
is apparently underway, the membrane connecting the two daughter
cell components is clearly continuous. The daughter cell component
to the right is extending a fine caliber, possibly axonal, process
into the foreground. Extending from this component into the upper
right of the field is another thicker, dendrite-like process which
undergoes several levels of branching.
[0078] In contrast to the previous, ultrastructural reports (Banker
and Cowen, Brain Res., 126.397-425, 1977; Banker and Cowen, J.
Comp. Neurol., 187:469-494, 1979; 29 Rothman, et al., J. Comp.
Neurol., 195:141-155, 1981), the perpetualized neurons had fine
caliber axonal processes which emerged from the soma in a
histotypic manner in addition to the dendritic origin (FIGS. 3A and
4D). These somatic axonal extensions may be the result of the high
levels of trophic support. Less-differentiated neurons typically
had rounder somata with fewer, less elaborate processes. Even
rounded neurons, differentiated adequately to extend processes,
appeared capable of proliferating (FIG. 4D). Neuronal processes and
somata have been identified based on both the ultrastructural
surface morphology and organelle content, which clearly
demonstrates that both the well-differentiated and proliferating,
less-differentiated cells are neurons.
Example 5
[0079] Effects of Different Growth Factors on Cell Culturing
[0080] Tissues were dissected from the specific areas of the
central nervous system (CNS) and dissociated as described in
EXAMPLE 1. After centrifugation, cells were resuspended in N2
medium and cells were counted. Approximately 0.5-1.0.times.10.sup.6
cells were plated an PORN/laminin coated 24 well plates in N2
medium containing different growth factors at different
concentrations, depending on the specific region of the CNS. Cells
were cultured at 37.degree. C. in 5% CO.sub.2. Cells were examined,
and if necessary, counted in 5 separate areas in a well at day 1,
4, and 7 to determine the growth rates in the presence of various
growth factors (TABLE 3).
[0081] In some experiments, no proliferation of cells was observed
in the presence of certain growth factors. In some cases there was
massive cell death, although a small population of cells survived
up to day 4. These surviving cells did not look healthy, however,
addition of bFGF at 20-100 ng/ml (depending on the origin of the
tissue), in N2 medium rescued these surviving cells as evidenced by
this proliferation (see a, TABLE 3).
3TABLE 3 EFFECTS OF DIFFERENT GROWTH FACTORS ON PROLIFERATION OF
CNS NEURONS Region Growth Factor Concentration Effect Hippocampus
bFGF 20 ng/ml ++ NGF.sup.a 20 ng/ml - EGF 20 ng/ml + BDNF 20 ng/ml
- NT3 ND* + Septum bFGF 100 ng/ml ++ NGF.sup.a 100 ng/ml -
EGF.sup.a 100 ng/ml - BDNF.sup.a 100 ng/ml - NT3.sup.a ND* - Locus
Ceruleus bFGF 50 ng/ml ++ NT3 ND* - Ventral bFGF 50 ng/ml ++
Mecencephalon BDNF 50 ng/ml - EGF 50 ng/ml - Cerebellum bFGF 20
ng/ml ++ EGF 20 ng/ml + NGF 20 ng/ml - BDNF 50 ng/ml - NT3 50 ng/ml
- Spinal Cord bFGF 20 ng/ml ++ NT3 20 ng/ml + *conditioned medium
from genetically modified fibroblasts expressing NT3 was used;
ND--not determined ++ high proliferation - no proliferation +
moderate proliferation .sup.acells could be rescued and
proliferated by bFGF
Example 6
Preparation of Adult Neuronal Cultures
[0082] Hippocampi of normal adult Fisher rats were dissociated and
grown in serum-free culture containing bFGF as described in Example
1. Briefly, hippocampi were dissected from normal adult (>3 mo)
rat brains. Most of the choroid plexus, ependymal lining and
subependymal zone was removed. Cells were dissociated mechanically
and enzymatically using methods described previously (Ray, et al.,
1993, supra) with the following modifications: After enzymatic
dissociation in a papain-protease-DNase (PPD) solution (Hank's
balanced salt solution supplemented with 4 mM MgSO.sub.4 and 0.01%
papain, 0.1% neutral protease and 0.01% DNasel), cells were
centrifuged at 1000 g for 3 min, resuspended and triturated in 1 ml
of DMEM:F12 (1:1) high glucose medium (Irvine Scientific)+10% fetal
bovine serum (10% FBS) (Sigma). Cells were plated onto uncoated
plastic T-75 culture flasks (Costar) at 1.times.10.sup.6 viable
cells per flask in 10% FBS medium overnight. Lower cell densities
were used with smaller culture flasks or Lab-Tek slide chambers
(Nunc). Cells were occasionally plated onto cultureware previously
coated with polyomithine/laminin as described in Example 1. The
medium was removed the next morning and replaced with, serum-free
medium: DMEM:F12.div.N2 (GIBCO) at 1 ml/100 ml medium (N2),+bFGF
(recombinant human bFGF, Syntex/Synergen Consortium; (Ray, et al.,
supra) at 20 ng/ml. Flasks were incubated 1-3 weeks, when half of
the medium was removed and replaced with the same volume of fresh
N2+bFGr. Partial medium exchange was made 1-2.times. weekly or as
needed. Cultures were examined and photographed using phase
contrast microscopy (Nikon Diaphot).
[0083] In a number of experiments cells were harvested and
transferred directly to new flasks or Lab-Tek slide chambers where
they attached immediately and started proliferating, or
occasionally passaged using trypsinization with ATV trypsin (Irvine
Scientific), followed by washing, centrifugation and re-plating in
N2+bFGF.
[0084] Primary cultures of neurons from adult rat hippocampi were
replicated more than 15 times. To determine whether 10% FBS or N2
medium could account for the observed effects, some cultures were
grown in 10% FBS or N2. Only cultures with bFGF developed large
numbers of neurons. Some dissections were made of the CA1, CA3 and
dentate gyrus regions. Neurons were generated from all three
regions. Cultures are described in three overlapping temporal
stages: early, middle and late.
[0085] Early cultures (1-21 days) were characterized by cell
attachment to the substrate, cell proliferation and expression of
mature neuronal features. After clearing cell debris in the medium,
single cells that were phase-bright and round and doublet cells,
suggestive of cell division, were observed at two days in vitro
(d.i.v.). Numerous phase-bright cell bodies displayed processes
tipped with growth cones. Cells of neuronal morphologies, i.e.,
phase-bright multipolar cell body with thin branching processes,
were observed as early as 5 d.i.v. Processes developing complex
branching patterns and evidence of incomplete cytokinesis or
potential synapse formation between presumptive sibling neurons
were observed as early as 8 d.i.v. (Nikon phase contrast-2
microscope/negative magnification 33.times.46.times.).
[0086] For SEM, cultures were fixed in 2% glutaraldehyde in 0.1 M
PBS, osmicated in 1% aqueous osmium tetroxide, dehydrated in a
graded ethanol series, critical point dried with liquid carbon
dioxide, attached to stubs with silver paste, sputter coated to 300
.ANG. with gold/palladium and examined and photographed in a
Cambridge Scanning Electron Microscope (Stereoscan 360).
[0087] Examination of the three-dimensional morphology of
early/intermediate stage cultures using scanning electron
microscopy (SEM) revealed numerous cells of both neuronal and
epithelioid phenotypes. Lacy neural networks were observed as with
phase microscopy. Cells that appeared to be dividing were also
observed. Higher magnification revealed that the processes between
cells and cell aggregates interpreted at the light microscope level
as a single process were frequently 2 or more fasciculated
processes.
[0088] Intermediate cultures (approximately 14-60 days) were
characterized by increasing numbers of cells, the presence of
neural networks, the development of mature neurons and initial cell
aggregate formation. Rudimentary networks of fine processes
connecting small cell clusters were observed as early as 14 d.i.v.
Networks of cells displaying neuronal morphologies became more
extensive and complex. Cells in the cultures were heterogeneous
although individual patches of neural networks displayed a uniform
morphological phenotype. Individual cells away from clusters or
networks also developed well differentiated morphological features
characteristic of mature neurons, with large phase-bright
multipolar-cell bodies and long thin processes that branched
repeatedly. Processes of these cells often measured nearly 1000
.mu.m, and large indented nuclei and prominent nucleoli could be
seen in different focal planes. Some neurons displayed small
thorn-like projections indistinguishable from dendritic spines on
processes.
[0089] Late cultures (approximately 2 to 7 months) were
characterized by increasing numbers of cells to confluence,
increasing cell aggregates connected by processes and a background
of individual cells. When substrate space was available, cells with
multiple thin processes characteristic of earlier stages continued
to be observed. Call aggregates were connected by cable-like
neurites. Large numbers of cell aggregates developed and the entire
substrate became covered with cell aggregates and individual cells
that appeared to have migrated from the cell aggregates. While many
background cells displayed features typical of neurons, some cells
expressed features typical of astrocytic glia
Example 7
Gene Expression in Cultured Neuronal Cells
[0090] The presence of NFh and GFAP was further confirmed by
reverse transcriptase-polymerase chain reaction (RT-PCR) with RNA
obtained from cells harvested after different times in culture.
[0091] RNA was extracted using the guanidinium cesium chloride
(CsCl) method (Current Protocols in Molecular Biology, Vol. 1,
Wiley Interscience, NY, F.M. Ausubel, et al., eds, 1988). The
pellets were solubilized in 1 ml solution D (4.0 M guinidine
thiocyanate, 25 mM Na citrate, 0.5% sarcosyl and DEPC treated
H.sub.2O) after thawing, triturated gently and the cell lysate was
transferred to CsCl previously poured into centrifuge tubes. The
level of the CsCl was marked, and the tubes were weighed and
balanced. The tubes were centrifuged in a Beckman Ultracentrifuge
overnight at 40,000 rpm at 20.degree. C. The next morning, solution
D was removed, and the interface washed with solution D. The CsCl
solution was carefully poured off, and the RNA pellet was rinsed
with 70% EtOH (made with DEPC water). After the pellet was dry it
was solubilized in DEPC-H.sub.2O and the remainder was stored in
EtOH at -70.degree. C.
[0092] A RT-PCR method was used to obtain cDNA's (Ausubel, et al.,
1988, supra). The reaction tube contained 4 .mu.l RNA (10-100 ng),
8 .mu.l (sufficient DEPC-H.sub.2O to bring the volume up to 20
.mu.l), 2 .mu.l 10.times.PCR buffer, 2 .mu.l 10 mM d NTP's, 1 .mu.l
random hexamers, 3 .mu.l 24 mM MgCl, 0.125 .mu.l AMV-RT and 0.5
.mu.l RNasin. A drop of Nujol mineral oil was added to each tube
and the reaction was run in a Perkin Elmer Thermal Cycler:
42.degree. C.--75 min; 95.degree. C.--10 min; and held at 4.degree.
C.
[0093] A PCR method was used to further amplify the specific
desired cDNAs from the cDNAs obtained above. Each reaction tube
contained 5 .mu.l cDNA, 9.5 .mu.l PCR buffer, 7.25 .mu.l
MgCl.sub.2, 0.2 or 0.3 .mu.l .sup.32P-dCTP, 1.5 .mu.l 10 mM dNTP's,
0.5 Taq polymerase, 6.mu., 1 primers (2 .mu.l [1 .mu.l (F (Forward
rx):5')+1 .mu.l R (reverse:3') each of RPL 27, NFh, GFAP, NGF or
bFGF) and sufficient H.sub.2O to bring the volume to 100 .mu.l. The
reacton was run in a Perkin Elmer Thermal Cycler: 94.degree. C.--10
min and held at 4.degree. C.
[0094] AmpliTaq DNA polymerase was from Perkin-Elmer, AMV reverse
transcriptase, random oligonucleotide hexamer primers and
recombinant RNasin ribonuclease inhibitor were from Promega,
specific primers were made to order. dNTP's were from New England
Nuclear. The primers were as follows:
[0095] NFh
4 Forward (F) primer: 5'-GAGGAGATAACTGAGTACCG-3' Reverse (A)
primer: 5'-CCAAAGCCAATCCGACACTC-3'
[0096] GFAP
5 F primer: 5'-ACCTCGGCACCCTGAGGCAG-3' R primer:
5'-CCAGCGAGTCAACCTTCCTC-3'
[0097] Gel electrophoresis of cDNA-samples obtained from PCR
amplification was done on a 6% non-denaturing polyacrylamide gel.
Some samples and their corresponding digests were run on agarose
gels using ethidium bromide to bind and illuminate the DNA under UV
light A 123 bp molecular ladder was run in a lane beside the
samples. Electrophoresis was done for varying periods of time, and
the resulting gels were dried for 1 hr on a gel drier.
Autoradiographic films of dried acrlyamide gels were developed for
periods, of time ranging from several hours to 10 days.
[0098] Relative levels of mRNA were analyzed quantitatively using
densitometry over cDNA bands identified as NFh, GFAP, NGF and bFGF
from Northern blots of cultures grown 36 to 117 days. A diverging
pattern of mRNA expression was apparent. Expression of message for
NFh was relatively low. At about 2 months, the relative levels
switched and expression of mRNA for GFAP increased over time then
dropped dramatically at about 4 months in culture, while expression
of NFh fell over time, then rose slightly after 4 months in
culture.
[0099] Digests of NFh were performed on samples remaining from
earlier reactions using a cocktail consisting of 40 .mu.l sample, 5
.mu.l React #6 buffer (50 mM Tris, pH 7.4, 6 mm MgCl.sub.2, 50 mM
KCl, 50 mM NaCl) and 5 .mu.l Pvu II restriction enzyme, and reacted
for 1 hr at 37.degree. C. The products were run along with a 123 bp
molecular ladder on a 6% acrylamide gel. The gel was dried, exposed
on film for varying periods of time, and the resulting
autoradiograms were examined for bands at the predicted molecular
weight levels. mRNA for both NFh and GFAP was present in all
cultures at the times examined.
Example 8
Immunocytochemistry
[0100] To determine whether cells expressed antigens typical of
neural tissue, cultures were processed for immunocytochemistry.
Cells were fixed for 30 min in 4% paraformaldehyde at room
temperature or 37.degree. C., incubated with 0.6% H.sub.2O.sub.2 in
TBS followed by incubation in blocking solution. Me cultures were
incubated with primary antibody at appropriate dilutions overnight
at 4.degree. C. The next day cells were rinsed with diluent and
incubated in secondary antibody for 1 hr at room temperature,
rinsed with TBS and incubated in ABC solution (equal amounts of
avidin and biotin) for 1 hr at room temperature. They were rinsed
with TBS and incubated with DAB-NiCl for variable reaction times,
rinsed with TBS, dried overnight, dehydrated through graded series
of alcohol and mounted in histoclear. Antibodies were from the
following sources and used at the dilutions indicated. Monoclonal
antibodies: high molecular weight subunit of neurofilament protein
(NF-H 200 kD; 1:24); middle molecular weight subunit of
neurofilament protein (NF-M 160: 1:10); glial filament acidic
protein (GFAP; 1:100) and synaptophysin (1:10) (Boehringer
Mannheim); calbindin (Cal-b; 1:200) and microtubule associated
protein 2 (MAP2; 1:500) (Sigma); neuron-specific enolase (NSE;
1:200) (DAKO). Polyclonal antibodies: NF-H 200 (1:250); NF-M 150
(1:500); NF-L 68 (1:125); GFAP (1:1000) and gamma amino butyric
acid (GABA; 1:200) (Chemicon); NSE (1:800) (Polysciences);
galactocerebrocide (Gal-C; 1:5000). (Advanced Immuno Chem.); bFGF
(1:1000); (Whittier Institute, La Jolla, Calif.). Normal horse and
goat serum, biotinylated goat anti-rabbit IgG, horse anti-mouse IgG
and ABC Vectastain Elite kit were from Vector Laboratories
(Burlingame, Calif.). There was no detectable staining when primary
antibody was omitted and replaced with non-immune serum.
[0101] Neuro-specific enolase (NSE)-positive cells were observed in
early cultures. By 170 d.i.v., a majority of the cells were
immunoreactive for high molecular weight neurofilament protein
(NFh, 200 kD) that is characteristic for adult neurons, as well as
the middle and low molecular weight of NF. Most NF-positive cells
also had morphological properties of neurons. A small subpopulation
of cells (less than 10%) was immunopositive for calbindin, which is
specific for granule cells. Cells with neuronal phenotypes were
also immunopositive for MAP2 with reaction product localized to the
cytoplasm of cell bodies and proximal processes. Cells with
astroglial morphology stained for GFAP Less than 1% of the cells
strained for GABA. Many cells were immunopositive for bFGF and a
few small round cells were immunopositive for
galactocerebroside.
Example 9
Characterization of Neuronal Cell Growth In Vitro
[0102] To determine whether cells were proliferating and, if so,
assess the nature of such cell types, cultures were incubated in
bromodeoxyuridine (BrdU) for 36 hours and then dual labeled for
immunofluorescence with BrdU and neuron-specific enolase (NSE) or
glial filament acidic protein (GFAP).
[0103] For BrdU incorporation, cells were cultured in glass Lab-Tek
slide chambers for 11 days. The medium was replaced with fresh
N2+bFGF containing bromodeoxyuridine (BrdU) labeling reagent (1
.mu.l/ml medium; Amersham) and the cultures were incubated an
additional 1 or 4 days for a total of 12 or 15 days. Cells were
fixed in 4% paraformaldehyde in phosphate-buffered saline (PBS) for
30 min, washed with PBS, blocked with 10% normal donkey serum
(Jackson ImmunoResearch Labs) in PBS, and reacted with monoclonal
antibody to BrdU (BrdU; undiluted; Amersham) followed by donkey
anti-mouse IgG coupled to Cy-5 (Jackson ImmunoResearch Labs). Some
cultures were dual labeled with polyclonal antibody against neuron
specific enolase (NSE; 1:800) or antibody against glial fibriliary
acidic protein (GFAP; 1:1000). Secondary antibody for the
polyclonals was donkey ant-rabbit IgG conjugated to fluorescein
isothiocyanate (FITC; Jackson ImmunoResearch Labs). Slides were
mounted in Slow-Fade mounting reagent (Molecular Probes). Cells
were visualized using a BioRad MRC 600 Confocal Scanning Laser
Microscope. Images were collected and transferred to an Apple
Macintosh Quadra 700, merged using Adobe Photoshop 101 and printed
out on a GCC film recorder. Confocal scanning microscopy revealed
cells immunoreactive for NSE and BrdU, as well as BrdU and GFAP
positive cells showing that cells expressing neuronal and glial
cell markers dividing in these cultures.
[0104] To determine if cell numbers in culture were increasing,
cells were counted over a 2 week period in 10 random fields.
Thirty-seven percent of cells originally attached had survived by
the second day in culture. Within 5 days, cell numbers had risen to
slightly above their original level, and by the end of seven days,
there were nearly twice as many cells. By the end of 2 weeks, there
were almost five times as many cells as on the first day in
culture.
[0105] The most important result of this study is the demonstration
of neuronal proliferation from normal adult hippocampus when
cultured with bFGF. Neurons survive and proliferate abundantly for
long periods of time, more than 200 d.i.v. to date; this is the
first such demonstration.
[0106] It has been reported that initiation of cell division of
isolated adult brain (striatal) cells in culture requires epidermal
growth factor (EGF), but not bFGF at 20 ng/ml, and a non-adhesive
substrate (Reynolds & Weiss, Science, 755:1707, 1992). In
contrast, the present data supports that: 1) bFGF at 20 ng/ml acts
as a strong mitogen and as a survival factor in adult as well as
fetal hippocampal cultures; 2) proliferation occurs in
substrate-bound cells and aggregates, i.e., cells not in
suspension, and 3) many, if not most, of the cells that attach are
bFGF-responsive.
[0107] Limited neuronal division as been reported over short times
in other culture systems of adult brain (Reynolds, supra; Richards,
et al. Proc. Natl. Acad. Sci, USA, 89:8591, 1992). In the present
study proliferation was confirmed by BrdU incorporation and the
finding that cell numbers increased almost 500% over a 2 week
period. Although it has been reported that less that 1% of adult
striatal cells initially plated proliferate (Reynolds, et al.
supra), in the cultures described herein nearly 40% of cells which
initially attached survived to the second day in culture,
suggesting that many cells are present in adult hippocampus that
have the capacity to proliferate.
[0108] Evidence from several independent experiments supports the
idea that most cells in these cultures not only are neurons, but
they are mature neurons which express morphological, biochemical
and molecular features characteristic of adult neurons. While glia
are also generated, glia were a minority phenotype in most
cultures. Similar findings have been reported for fetal rat
hippocampus neurons cultured with bFGF (Ray, et al., 1993,
supra).
[0109] The source of the proliferating neurons for the adult brain
remains to be determined. The cells could be mature functioning
neurons that were saved and induced to proliferated by high
concentration of bFGF; the cells could be stem cells of suspected
proliferation zones; or the cells could be partially committed
neurons (neuroblasts) that have become quiescent due to a reduction
in high levels of bFGF present only in the embryonic brain and/or
because of contact inhibition. While it is unlikely that all
neurons that were observed and generated could be accounted for the
mature neurons saved following plating, work is in progress to
determine whether mature differentiated neurons are capable of in
vitro survival and proliferation through dedifferentiation. It is
possible that the subventricular zone (SVZ) could be the source of
these cells, since SVZ of mammalian forebrain has been shown to be
the source of these cells that differentiate into neurons and glia
in adult mice (Clois, et al., Proc. Nat'l Acad. Sci. U.S.A.,
79:2074, 1993). However, it is not likely that the SVZ could have
served as the main source of proliferating cells in the cultures of
the invention, since the ependymal lining/SVZ, along with choroid
plexus, was stripped away. These neurons could be derived from a
small population of embryonic stem cells that survives in the adult
brain in a dormant, non-proliferative state, as has been suggested
exists in adult mouse striatum (Reynolds, et al., supra).
Alternatively, these neurons could be neuronal precursor cells
existing in adult mammalian brain that require discrete epigenetic
signals for their proliferation and differentiation as has been
speculated for adult mouse brain (Richards, et al., supra).
[0110] In addition to stem cells of the SVZ, there is a large
population of neuroblast in the normal adult mammalian hippocampus
that can be induced to generate large numbers of neurons over long
periods of time under appropriate in vitro conditions. It is
possible that this could also be true in vivo, a concept that has
profound implications for basic and clinical neuroscience. This
could mean that normal hippocampus and, by extension, normal CNS
has a reservoir of cells that can be activated under appropriate
conditions to replicate large numbers of neurons. Thus, neuroblasts
could be present not only in cultures of fetal CNS, but also in
cultures of adult CNS and in the adult CNS in situ.
Example 10
Long-Term Culture of Neurons from Adult Hippocampus
[0111] Brains of adult Fisher rats (>3 months old) were
dissected, the menengies removed, and the hippocampai dissected
out. The tissues were transferred to a 15 ml tissue culture tube
and washed three times with 5 ml Dulbecco's phosphate buffered
saline (D-PBS). After the last wash, the tissue was pelleted by
centrifugation at 1000 g for 3 min and the wash solution removed
The tissue was suspended in 5 ml papain-neutral protease-DNase
(PPD) solution and incubated at 37.degree. C. for 20-30 min with
occasional shaking. The solution was made in Hank's balanced salt
solution supplemented with 12.4 mM MgSO.sub.4 containing 0.01%
papain, 0.1% neutral protease and 0.01% DNase I (London, R. M. and
Robbins, R. J., Method. Enzymol., 124:412-424, 1986).
[0112] Hippocampai were mechanically dissociated by tituration with
a medium bore pasteur pipet (about 20 times). Cells were pelleted
by centrifugation at 1000 g for 3 min. The cells were resuspended
in 1 ml DMEM:F12 (1:1) medium containing 10% fetal bovine serum,
3.9 mM glutamine (complete medium). Cell clumps were mechanically
dissociated by tituration with medium to fine bore pasteur pipets
(about 20 times with each). Cells were washed with 5-10 ml complete
medium twice by centrifugation. Cells were taken up in 1 ml
complete medium, dissociated by tituration and counted in a
hemocytometer. Cells were plated at a density of 1.times.10.sup.6
cells/T75 flasks (Coaster) and incubated at 37.degree. C. in 5%
CO.sub.2/95% air incubator. After incubation for 18-24 hours, the
medium was changed with N2 medium (1:1 mixture of DMEM/F-12
containing 20 nM progesterone, 30 nM sodium selenite, 100 .mu.M
putrescine, 3.9 mM glutamine, insulin (5 .mu.g/ml) and transferrin
(100 .mu.g/ml)] containing 20 ng/ml FGF-2 (bFGF). To date cells
have been cultured for at least 7 months and have been cultured
from 15 different independent dissections.
[0113] The neuronal nature of cells were determined by examination
of morphology at light and scanning microscope levels.
Immunocytochemical analysis showed that these cells expressed
neuron-specific enolase, neurofilament medium and high molecular
weight proteins, MAP-2, and calbindin (only a small population).
Some cells in these cultures also stained for GFAP indicating the
presence of astrocytes in these cultures. The proliferation of
adult neuronal cells in cultures was determined by
bromodeoxyuridine (BrdU) incorporation. The nuclei of cells
expressing neuron-specific enolase were immunostained with an
antibody against BrdU indicating cell proliferation in culture.
Example 11
In Vivo Survival of Perpetual Hippocampal Neurons After Grafting in
the Adult Brain
[0114] Embryonic hippocampal neurons were cultured in N2 medium
containing 20 ng/ml bFGF. Cells were passaged and allowed to grow
until 7080% confluent. The medium was replaced with fresh medium
(N2+bFGF) containing .sup.3H-thymidine (1 .mu.Ci/ml; specific
activity: 25 Ci/mmol) and allowed to grow for 3.5 days. Cells were
harvested from flasks by trypsinization and washed with D-PBS 3
times by centrifugation. Cells were resuspended in 2 mls of D-PBS
containing 20 ng/ml bFGF, dissociated by tituration and counted in
a hemocytometer. After centrifugation to remove the supernatant,
cells were resuspended at a concentration of 60,000 cells/.mu.l.
One microliter of cell suspension was injected in the hippocampus
of adult Fisher rats (>3 months old). Animals were perfused with
4% paraformaldehyde, and the brains removed. Brain sections were
treated with antibodies with calbindin, GFAP and NF-H proteins. The
sections were dipped in emulsion and developed after 6 weeks.
Number of cells with at least 12 grains on them were counted in
every 12 sections for each animal (3 animal total). At three weeks,
an average of 17% cells implanted in the brain survived (Table
4).
6TABLE 4 SURVIVAL OF PERPETUAL HIPPOCAMPAL NEURONS IN ADULT RAT
HIPPOCAMPUS AVERAGE # CELLS ANIMAL WITH GRAINS/SECTION % CELLS
SURVIVING 1 222 15 2 233 19 3 244 17
[0115] Although the invention has been described with reference to
the presently preferred embodiment, it should be understood that
various modifications can be made without departing from the spirit
of the invention.
Sequence CWU 1
1
4 1 20 DNA Artificial Sequence Forward primer for PCR 1 gaggagataa
ctgagtaccg 20 2 20 DNA Artificial Sequence Reverse primer for PCR 2
ccaaagccaa tccgacactc 20 3 20 DNA Artificial Sequence Forward
primer for PCR 3 acctcggcac cctgaggcag 20 4 20 DNA Artificial
Sequence Reverse primer for PCR 4 ccagcgactc aaccttcctc 20
* * * * *