Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
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EMBRYONIC AND ADULT CNSSTEM CELLS
BACKGROUND OF THE lNV~:N'l'ION
1. FIELD OF THE lNv~NlIoN
The present invention relates to a technology
where stem cells from embryonic and adult brain
are isolated, propagated, and differentiated
efficiently in culture to generate large numbers
of nerve cells. This technology, for the first
time, enables one to generate large numbers of
many different kinds of neurons found in a normal
brain and provides a new foundation for gene
therapy, cell therapy, novel growth factor
screening, and drug screening for nervous system
disorders.
2. DESCRIPTION OF THE RELATED ART
The brain is composed of highly diverse nerve
cell types making specific interconnections and,
once destroyed, the nerve cells (neurons) do not
regenerate. In addition, the brain is protected
by a blood-brain barrier that effectively blocks
the flow of large molecules into the brain,
rendering peripheral injection of potential growth
factor drugs ineffective. Thus, a major challenge
currently facing the biotechnology industry is to
find an efficient mechanism for delivering
SUBSTITUTE SHEET (RULE 26)
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potential gene therapy products directly into the
brain in order to treat nervous system disorders.
Moreover, for a degenerative disease like
Parkinson's, the most comprehensive approach to
regain a lost neural function may be to replace
the damaged cells with healthy cells, rather than
just a single gene product. Thus, current and
future success of gene therapy and cell therapy
depends upon development of suitable cells that
lo can (1) carry a healthy copy of a disease gene
(i.e., a normal gene), (2) be transplanted into
the brain, and (3) be integrated into the host's
neural network. This development ideally requires
cells of neuronal origin that (1) proliferate in
culture to a large number, (2) are Ame~Ahle to
various methods of gene transfer, and (3)
integrate and behave as the cells of a normal
brain. However, there have been no such cells for
therapeutic purposes since neurons do not divide
and therefore cannot be propagated in culture.
As alternatives, various transformed cells of
neural and non-neural origins such as glias,
fibroblasts, and even muscle cells, which can be
proliferated in culture, have been used as
possible vehicles for delivering a gene of
interest into brain cells. However, such cells do
not and cannot be expected to provide neuronal
functions. Another alternative approach has been
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to force a neural cell of unknown origin to divide
in culture by genetically modifying some of its
properties, while still retaining some of its
ability to become and function as a neuron.
Although some "immortalized" cells can display
certain features of a neuron, it is unclear
whether these altered cells are truly a viable
alternative for clinical purposes.
A developing fetal brain contains all of the
lo cells germinal to the cells of an adult brain as
well as all of the programs necessary to
orchestrate them toward the final network of
neurons. At early stages of development, the
nervous system is populated by germinal cells from
which all other cells, mainly neurons, astrocytes,
and oligodendrocytes, derive during subsequent
stages of development. Clearly, such germinal
cells that are precursors of the normal brain
development would be ideal for all gene-based and
cell-based therapies if these germinal cells could
be isolated, propagated, and differentiated into
mature cell types.
The usefulness of the isolated primary cells
for both basic research and for therapeutic
application depends upon the extent to which the
isolated cells resemble those in the brain. Just
how many different kinds of precursor cells there
are in the developing brain is unknown. However,
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several distinct cell types may exist:
a precursor to neuron only ("committed
neuronal progenitor'~ or "neuroblast"),
a precursor to oligodendrocyte only
("oligodendroblast"),
a precursor to astrocyte only (~astroblast~ ?
a bipotential precursor that can become either
neuron or oligodendrocyte, neuron or astrocyte,
and oligodendrocyte or astrocyte, and
a multipotential precursor that maintains the
capacity to differentiate into any one of the
three cell types.
Fate mapping analysis and transplantation
studies in vivo have shown that different neuronal
1~ types and non-neuronal cells can be derived from
the same precursor cells'~5. In vi tro analyses
have also suggested that multipotential cells.are
present in the developing brain67. Lineage
analysis alone, however, does not directly
identify the multipotential cells; nor does it
define the mechanisms that drive them to different
fates. Precursor cells from the central nervous
system (CNS) have been expanded in vi tro and
differentiation into neurons and glia has been
observed8~12 and, as detailed below, markedly
different cell types have been obtained even when
the culture conditions used were seemingly the
same.
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Because of the current lack of understanding
of histogenesis during brain development, many
investigators have used various terms loosely to
describe the cells that they have studied, e.g.,
neuronal progenitor, neural precursor,
neuroepithelial precursor, multipotential stem
cell, etc. Thus, the nature of the cells so far
described in the literature and culture conditions
for obtaining them can only be compared to each
other by their reported differentiation capacity.
The entire subject of the isolation,
characterization, and use of stem cells from the
CNS has recently been reviewed 333438.
In summary, conditions have not been found to
date, despite many reports, to successfully
identify, propagate, and differentiate
multipotential stem cells. A useful compilation
of studies reporting culture of CNS precursor
cells is found in Table 3, p. 172, of a recent
review34 and further extended below.
Vicario-Abejon, C., ~ohe, K., Hazel, T.,
Collazo, D. & McKaY, R., Functions of basic
fibroblast qrowth factor and neurotroPhins in the
differentiation of hiPPocamPal neurons, Neuron 15,
105-114 (1995) 12.
Cells expanded by Vicario-Abej-on et al. are
significantly different from those described in
the present invention although the starting tissue
(embryonic hippocampus), the mitogen (basic
fibroblast growth factor, bFGF), and the basal
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medium (N2) are similar in both reports. Almost
all of the cells expanded by Vicario-Abejon et al.
failed to differentiate into any cell types but
died in the absence of bFGF (as stated in the
paper, pg. 106). This is also reflected in Fig. 3
of the paper where the number of MAP2 positive
neurons is exceedingly low (50-100 cells out of an
initial cell number of approximately 80,000 per
well; i.e., far less than 1% in all reported
conditions). Thus, difference~ in culture
conditions, subtle as they may be, can yield cells
with significantly different properties and this
is, in fact, consistent with the main observation
of the present invention that the extracellular
environment can shift the developmental properties
of the C~S stem cells.
Vicario-Abejon et al. used the following
culture conditions which differ from the those
described in the present invention:
1. Used enzymatic dissociation, 0.1-0.25%
trypsin + 0.4% DNAse I for the initial tissue
dissociation a~ well as subsequent passaging. In
the present invention, enzymatic dissociation
effectively causes proteolyses of FGF receptors
and causes cells to become unresponsive to bFGF
and leads to differentiation.
2. Used 10~ fetal bovine serum to stop the
trypsin activity and to prime the cells from 4
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hours to overnight before switching to serum free
medium. In the present invention, serum even at
less than 1% concentration shifts stem cells to
astrocytic fate.
3. Cells were seeded at much higher density
of 45,000 cells per cm2 and then grown to
confluence before passaging by trypsin and serum.
In the present invention, high cell density
inhibits proliferation and causes spontaneous
differentiation even in the presence of bFGF.
4. bFGF was given only intermittently every
2-3 days, and at 5 ng/ml, less than the optimal
concentration disclosed in the present invention.
This condition leads to partial differentiation of
cells and subsequent heterogeneity of cell types
in culture.
5. Basal medium consisting of "N2" components
consisted of 5 ng/ml insulin, less than the
optimal concentration disclosed in the present
invention.
Ray, ~., Peterson, D., Schinstine, M. & Gaqe,
F., Proli_eration, differentiation, and lonq-term
culture o_ Primary hiPpocampal neurons, Proc.
Natl . Acad. Sci . USA 90, 3602-3606 (1993)'~.
This study used culture conditions that are
very similar to those described by Vicario-Abejon
et al.--bFGF as the primary mitogen, serum-free
medium, and E16 hippocampus. However, it reports
isolation and expansion of a precursor population
(neuroblasts) quite different from the cells of
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Vicario-Abejon et al. (undefined) as well as the
multipotential stem cells described in the present
invention. The reported cells had the following
properties which markedly contrast from those of
CNS stem cells:
1. The expanded cells under the reported
condition are mitotic neurons with antigenic
expressions of neurofilament, nestin,
neuron-specific enolase, galactocerebroside, and
MAP2 (Table I, p. 3604). The expanding CNS stem
cells reported in the present invention express
nestin, only, are negative for the above antigens,
and are, therefore, a molecularly distinct
population of cells from those described by Ray et
al.
2. Ultrastructural analysis of the expanded
cells in culture "demonstrated their histotypic
neuronal morphology". The expanding CNS stem
cells exhibit entirely different, non-neuronal
morphology.
3. The mitotic "neurons" had a doubling time
of 4 days and could be passaged and grown as
continuous cell lines. The CNS stem cells double
at every 20-24 hours and exhibit a characteristic
regression of mitotic and differentiative capacity
over time so that they cannot be maintained as
stable cell lines indefinitely.
4. The culture system by Ray et al. generates
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"nearly pure neuronal cell cultures". The culture
system in the present invention generates
multipotential stem cells that can differentiate
into all three major cell types of the brain,
i.e., neurons, oligodendrocytes, and astrocytes.
Ray et al. used the following culture
conditions which differ from those of the present
invention.
1. Embryonic hippocampi were mechanically
triturated without the use of an enzyme; however,
cells were plated approximately lOO,oO0 cells per
cm2, optimal for neuronal survival, but almost 10
times higher cell density than optimal for
expansion of CNS stem cells.
2. bFGF was given at 20 ng/ml,
intermittently, at every 3-4 days.
3. Basal "N2" medium contained 5 ~g/ml
insulin, less than optimal. Medium change was
also prolonged at every 3-4 days.
4. Cells were passaged by using trypsin.
In conclusion, even seemingly small
differences in culture conditions can result in
isolation of vastly different cell types.
R Y, J. and Gaqe, F.H., Spinal cord
neuro~lasts proliferate in response to basic
fibro~last qrowth factor, J. Neurosci . 14,
3548-3564 (1994) 39.
Ray and Gage report isolation and propagation
of cells "that have already committed to a
neuronal pathway are and expressing neuronal
g
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phenotypes (neuroblasts)" from spinal cord using
bFGF. Again, although the primary mitogen is
bFGF, their culture conditions are different and
obtained cells markedly different from CNS stem
cells.
1. E14-E16 spinal cord was used, a much later
stage of development than optimal for stem cells.
2. The tissue was dissociated enzymatically
by papain and DNase.
3. Initial plating was done in 10% fetal
bovine serum.
4. There was a preliminary enrichment for a
non-adherent cell population.
5. There was intermittent medium change and
bFGF supplement, every 3-4 days.
Gaqe, F.H., Coates, P.W., Palmer, T.D., Kuhn,
H.G., Fisher, L.J., Suhonen, JØ, Peterson, D.A.,
Suhr, S.T. & RaY, J., Survival and differentiation
of adult neuronal proqenitor cells transPlanted to
the adult brain, Proc. Natl. Acad. Sci. USA 92,
11879-11883 (1995) 35.
Gage et al. report isolation, propagation, and
transplantation of cells from adult hiPpocampus.
These mixtures of cells were maintained in culture
for one year through multiple passages. 80~ of
them exhibit rather unusual properties such as
co-expressing glial and neuronal antigens while
remaining mitotic. These properties are not
exhibited by stem cells isolated from the adult
striatal subventricular zone.
Again, using bFGF as a primary mitogen, the
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authors derived markedly different cells than CNS
stem cells reported in the present invention.
Gritti, A. et al., Multipotertial stem cells
from the adult mouse brain proli_erate and
self-renew in response to basic _ibroblast qrowth
factor, J. Neurosci . 16, 1091-1100 tl996) 40.
These authors report isolation and propagation
of multipotential stem cells from the
subventricular zone of adult brain by using bFGF.
A significant difference in culture conditions
used by Gritti et al. is that the cells are
propagated as aqqreqated spheres without
attachment to plate surface. Culture conditions
by Gritti et al. require this aggregation of cells
into spheres, using either bFGF or epidermal
growth factor (EGF), as an essential step for
propagating multipotential cells. This
aggregation step alone essentially distinguishes
the reported culture system from that of the
present invention. The aggregation promotes
undefined cell-cell interactions and results in
uncontrollable differentiation/fate-shifts and
overall in much less expansion and
differentiation. Furthermore, this culture system
and the result obtained by Gritti et al. are
limited to adult brain where extremely small
number of cells were obtained (105 cells per brain)
and have not been extended to various regions of
embryonic brain.
The procedure in the present invention permits
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propagation of stem cells throughout the
developing CNS as well as the striatum of the
adult brain. It also uses adherent culture and
actively avoids cell-cell contact and high cell
density. As a result, it permits much more
efficient expansion of the cells in an
undifferentiated multipotential state and much
more precise and efficient control over
differentiation of the expanded cells.
ReYnolds, B. & Weiss, S., Generation of
neurons and astrocytes from isolated cells of the
adult mammalian central nervous sYstem~ Science
255, 1707-1710 ~1992)l5.
ReYnolds, B., Tetzlaff, W. & Weiss, S., A
multipotent EGF-responsive striatal embrYonic
proqenitor cell Produces neurons and astrocytes,
J. Neurosci. 12, 4565-45~4 (1992~9.
Ve-covi, A.L., Reyno_ds, B.A., Fraser, D.D.,
and We ss, S., bFGF requ_ates the proliferative
fate o~ unipotent (neuronal) and biPotent
(neuronal/ astroqlial) EGF-qenerated CNS
proqenitor cells, Neuron 11, 95~-966 (1993) 4~ .
These three studies describe the original
sphere cultures of neural precursor cells from
adult and embryonic brain using EGF (epidermal
growth factor). The expanded cells differentiate
into neurons and astrocytes, but not into
oligodendrocytes, and thus are thought to be a
bipotential population, rather than
multipotential. Another distinguishing property
of the cells is that they respond only to EGF and
not to bFGF in particular, whereas CNS stem cells
respond similarly to both EGF and bFGF. Again,
the sphere culture conditions are not comparable
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to those employed in the present invention because
they require cell aggregation in which many
additional undefined interactions are expected to
occur.
Ahmed, S., ~eynolds, B.A., and Weiss, S., BDNF
enhances the di_ferentiation but not the survival
of ~NS stem cel_-derived neuronal precursors, J.
Neurosci . 15, 5765-5778 (1995) 42 .
This paper reports the effects of
brain-derived growth factor (BDNF) on sphere
cultures of embryonic neural precursor cells
propagated with EGF. There is no further
enhancement of the culture system per se.
Svendsen, C.N., Fawcett, J.W., Bentlaqe, C. &
Dunnett, S.B., Increased survival of rat
EGF-qenerated CNS precursor cells usinq B27
supplemented medium, Exp . Brain Res . 102, 407-414
(1995) 36.
This study utilizes the sphere culture with
EGF as described above to test a commercially
available medium supplement called "B27". The
study simply reports that use of B27 enhances cell
survival (not neuronal survival) in a mixed
culture containing neurons, astrocytes, and
oligodendrocytes.
KilPatrick, T.J. and Bartlett, P.F., Cloninq
and qrowth of multipotentia_ neural Precursors:
requirements for Proliferat_on and
differentiation, Neuron 10, 255-265 (1993) 43.
The authors report existence of multipotential
precursor cells in E10 mouse telencephalon by
culturing single cells from the brain in bFGF plus
serum. The results were based on 700 cells
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expanded clonally for 10 days, some of which, when
differentiated in the presence of bFGF, serum, and
astrocyte conditioned medium, could give rise to
neurons. There was no mass expansion of the
cells.
;Cilpatrick, T.J. and Bartlett, P.F., Cloned
mult_potential precursors from the mouse cerebrum
requzre FGF-2, whereas qlial restricted precursors
are stimulated with either FGF-2 or EGF, J.
Neurosci. 15, 3653-3661 (1995)44.
The authors utilize the clonal culture system
reported in the above-described reference43 to test
mitogenic efficacy of bFGF and EGF on cortical
cells from E10 and E17 embryos. Again, the
culture condition applies strictly to microculture
in serum containing medium to demonstrate
existence of different precursor cells in
developing brain. There is no mass expansion,
long-term culture, or systematic differentiation
protocol.
Baetqe, E.E., Neural stem cells for CNS
transplantation, Ann. N.Y. Acad. Sci . 695, 285
tl993)45.
This is a brief review paper summarizing
various studies directed to isolating precursor
cells and their derivatives in culture. It is
somewhat outdated and most of the-relevant
original studies cited have been discussed above.
Bartlett, P.F. et al., Requlation of neural
Precursor differentiation in the embrYonic and
adult forebrain, Clin. ExP. Pharm. Physiol. 22,
559-562 (1995) 46.
This is also a brief review paper summarizing
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mostly previous works from the authors' laboratory
in regard to their microculture studies where
differentiation potentials of certain clones of
precursors are tested in the presence of acidic
FGF (aFGF), bFGF, serum, and/or astrocyte
conditioned medium.
In addition, Sabate et al. 32 reported the
culturing of a human neural progenitor with
undefined differentiation capacity. Davis and
Temple6 demonstrated the existence of
multipotential stem cells in cortex by
co-culturing with epithelial cells for short term
(less than 100 cells altogether).
However, cell differentiation could not be
controlled in any of the reported studies which
precluded analysis of their lineage relations and
the mechanisms regulating fate choice.
The present invention provides a method for
efficiently propagating the undifferentiated
germinal cells, i.e., stem cells of the central
nervous system (CNS), in culture and defines
conditions to effectively turn the
undifferentiated cells into mature cell types.
These undifferentiated cells or "CNS stem cells~
display the multipotential capacity to
differentiate into all three major cell types of a
mature brain -- neurons, astrocytes, and
oligodendrocytes. Moreover, the same culture
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conditions enable isolation, expansion, and
differentiation of equivalent multipotential cells
from the adult brain.
Since the initial disclosure, additional
reports have appeared. Most recent research on
and use of CNS stem cells and neural progenitors
have been further reviewed47~52. In addition,
Reynolds and Weiss53 reported that embryonic
striatal progenitors generated as spheres using
EGF were able to differentiate into all three cell
types including oligodendrocytes, astrocytes, and
neurons. The frequency of EGF-responsi~e cells
was limited to only 1% of the initial primary
culture. Subcloning to establish self-renewal was
questionable since up to 500 cells/well were used
to generate the secondary "clones".
Differentiation of the cells was induced by
incorporating 1% serum in the medium. However, no
data demonstrating all three cell types were
presented from single-cell derived clones.
Weiss et al. 54 reported that multipotential
CNS stem cells could be isolated from adult spinal
cord and third and fourth ventricles by using a
combination of EGF and bFGF but not with either
alone.
Svendsen et al.55 reported that neural
precursor cells isolated from striatum and
mesencephalon of 16 day old rat embryos (E16),
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when grafted into lesioned adult rat brains,
failed to differentiate into neurons. They also
reported that EGF-generated mesencephalon cells
but not striatal cells differentiated into
tyrosine hydroxylase (TH)-positive neurons, albeit
in very low number (0.002~). There were no
characterization of cells in vitro to ensure that
the primary culture used contained no post-mitotic
neurons carrying over from the tissue, especially
given that the result could only be obtained with
E16 tissue when most TH cells are already born.
Schinstine and Iacovitti56 reported that some
of the astrocytes derived from EGF-generated
neural precursor cells expressed neuronal antigens
such as tau and MAP2. Qian et al. 57 reported that
different concentrations of bFGF proliferate stem-
like cells of E10 mouse cortex with varying
differentiation potentials ranging from only
neuronal to multipotential.
Palmer et al.65 reported that multipotential
CNS stem cells could be isolated from adult rat
hippocampus. 84% of the cells they expanded,
however, co-expressed MAP2c and 04, immature
neuronal and oligodendroglial markers. Only 0.2%
were MAP2ab positive and less than 0.01% were
positive for other neuronal markers such as tau
and neurofilament 200. Such properties are quite
different from the properties described in the
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Examples in the present application.
Fin:Ley et al. 66 reported that the mouse
embryonic carcinoma cells line, P19, can form
neuronal polarity and be eletrophysiologically
active when induced by retinoic acid and serum.
Strubing et al. 67 reported that embryonic stem
cells grown in serum-containing medium could
differentiate into electrophysiologically active
neurons in vitro. Okabe et al. 68 also reported
differentiation of some of embryonic stem cells
into neurons in vitro.
Gritti et al. 40 reported that multipotential
stem cells could be isolated from adult mouse
subependyme by EGF and bFGF, which when
differentiated, could be eletrophysiologically
active and express GABA-, gluatamate-, and ChAT-
immunoreactivities, but not others. The frequency
of such neurons, however, was not documented and
thus it is difficult to ascertain how efficient
neuronal maturation was. Moreover, these neuronal
phenotypes derived from dividing stem cells were
not directly demonstrated by BrdU labeling. This
is particularly relevant since aggregate cultures
are extremely prone to be contaminated by primary
neurons from the tissue, which carry over for
several passages. Weiss et al.49, in fact, stated
that only GABA-positive cells could be obtained
from their cultures. Most of the GABA-positive
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cells may ~e oligodendrocytes.
Feldman et al. 69 reported electrophysiological
studies of EGF-generated rat neural precursors.
They found that most, if not all, electro-
physiologically active cells are in fact non-
neuronal, and that glial cells do contain voltage-
sensitive Na channels that evoke action potential-
like conductances.
Results such as these illustrate that
identifying CNS stem cells, defining conditions
that stably maintain CNS stem cell properties for
long-term, and controlling their differentiation
into mature cell types are neither obvious nor
predictable to those skilled in this art.
SUMMARY OF THE INVENTION
The present invention discloses an in vi tro
culture of stem cells of the central nervous
system oE a mammal, a method for the in vi tro
culture of the stem cells, and a method for the
differentiation of the stem cells.
In the in vi tro culture of the stem cells of
the central nervous system of a mammal, the stem
cells maintain the multipotential capacity to
differentiate into neurons, astrocytes, and
ollgodendrocytes. The stem cells can be derived
from central nervous system tissue from a human,
fetus or adult. Furthermore, the central nervous
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system tissue may be hippocampus, cerebral cortex,
striatum, septum, diencephalon, mesencephalon,
hindbrain, or spinal cord.
Furthermore, the stem cells can differentiate
to mature neurons exhibiting axon-dendrite
polarity, synaptic terminals, and localization of
proteins involved in synaptogenesis and synaptic
activity including neurotransmitter receptors,
transporters, and processing enzymes. In
addition, the stem cells retain their capacity to
generate subtypes of neurons having molecular
differences among the subtypes.
In the method for the in vitro culture of the
stem cells, where the stem cells maintain the
multipotential capacity to differentiate into
neurons, astrocytes, and oligodendrocytes, cells
from the central nervous system are:
a) dissociated by mechanical trituration;
b) plated at the optimal initial density of
1 x 106 cells (from hippocampus and septum) or 1.5
x lo6 cells (from other CNS regions) per 10 cm
plate precoated with poly-ornithine and
fibronectin;
c) cultured in the complete-absence of
serum;
d) supplied daily with a growth factor
selected from the group consisting of
i) basic fibroblast growth factor
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(bFGF) at a concentration of at least 10 ng/ml,
ii) EGF at a concentration of at least
10 ng/ml,
iii) TGF-alpha at a concentration of at
least 10 ng/ml, and
iv) acidic FGF (aFGF) at a
concentration of at least 10 ng/ml plus 1 ~g/ml
heparln;
e) replaced 100~ of culture medium every two
days with fresh medium;
f) passaged at every 4 days after plating by
treating the cultured cells with saline solution
and scraping the cells from the plate; and
g) replated passaged cells at 0.5 x 106 cells
per 10 cm plate precoated with poly-ornithine and
fibronectin.
The method is applicable with stems cells
derived from central nervous system tissue from a
human, fetus or adult. Again, the central nervous
system tissue may be hippocampus, cerebral cortex,
striatum, septum, diencephalon, mesencephalon,
hindbrain, or spinal cord.
In the method for the differentiation of an
in vitro culture of stem cells of the central
nervous system of a mammal, where the stem cells
maintain the multipotential capacity to
differentiate into neurons, astrocytes, and
oligodendrocytes, cells from the central nervous
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system are:
a) dissociated by mechanical trituration;
b) plated at the optimal initial density of
1 X 106 cells (from hippocampus and septum) or 1.5
x 1o6 cells (from other CNS regions) per 10 cm
plate precoated with poly-ornithine and
fibronectin;
c) cultured in the complete absence of
serum;
d) supplied daily with a growth factor
selected from the group conslsting of
i) basic fibroblast growth factor
(bFGF) at a concentration of at least 10 ng/ml,
ii) EGF at a concentration of at least
~5 10 ng/ml,
iii) TGF-alpha at a concentration of at
least 10 ng/ml, and
iv) acidic FGF (aFGF) at a
concentration of at least 10 ng/ml plus 1 ~g/ml
~0 heparin;
e) replaced 100% of culture medium every two
days with fresh medium;
f) passaged at every 4 days after plating by
treating the cultured cells with saline solution
~5 and scraping the cells from the plate;
g) replated passaged cells at 0.5 x 106 cells
per 10 cm plate precoated with poly-ornithine and
fibronectin; and
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h) removed the growth factor either by
rinsing the cells with saline solution or by
treating with trypsin and subsequently trypsin
inhibitor, and then continued to culture the cells
in the serum-free medium without the growth
factor.
Furthermore, differentiation may be
specifically directed by adding a second growth
factor to the cultured cells either before or
after removing the first growth factor from the
cultured cells. The second or added growth factor
may be platelet-derived growth factor (PDGF),
ciliary neurotropic factor (CNTF), leukemia
inhi~itory factor (LIF), or thyroid hormone,
iodothyronine (T3).
The present invention also discloses an in
vitro culture of region-specific, terminally.
differentiated, mature neurons derived from
cultures of mammalian multipotential CNS stem
cells and an in vitro culture method for
generation of the differentiated neurons.
In the method for in vi tro generation of
region-specific, terminally dlfferentiated, mature
neurons from cultures of mammalian multipotential
CNS stem cells, multipotential CNS stem cells from
a specific region are cultured in a chemically
defined serum-free culture medium containing a
growth factor; the medium is replaced with growth
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factor-free medium; the stem cells are harvested
by trypsinization; plated at a density of between
100,000 to 250,000 cells per square centimeter;
and cultured in a glutamic acid-free chemically
defined serum-free culture medium. The specific
region of the CNS from which the multipotential
stems cells are derived are selected from the
group consisting of cortex, olfactory tubercle,
retina, septum, lateral ganglionic eminence,
medial ganglionic eminence, amygdala, hippocampus,
thalamus, hypothalamus, ventral and dorsal
mesencephalon, brain stem, cerebellum, and spinal
cord.
In addition, the chemically defined
serum-free culture medium may be selected from N2
(DMEM/F12, glucose, glutamine, sodium bicarbonate,
25 ~g/ml insulin, 100 ~g/ml human apotransferrin,
25 nM progesterone, 100 ~M putrescine, 30 nM
sodium selenite, pH 7.28) or N2-modified media.
The growth factor may be selected from the group
consisting of bFGF, EGF, TGF-alpha and aFGF.
Furthermore, the glutamic acid-free
chemically defined serum-free culture medium may
be supplemented with between 10-100 ng/ml of
brain-derlved neurotropic factor. The method is
applicable to multipotential CNS stem cells
derived from central nervous system tissue from
any ~AmmAl, including rat and human.
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The present invention also discloses in vi tro
cultures of region-speclfic, terminally
differentiated, mature neurons derived from
cultures of mammalian multipotential CNS stem
cells from a specific region of the CNS. The
specific region from which the multipotential
stems cells are derived are selected from the
group consisting of cortex, olfactory tubercle,
retina, septum, lateral ganglionic eminence,
medial ganglionic eminence, amygdala, hippocampus,
thalamus, hypothalamus, ventral and dorsal
mesencephalon, brain stem, cerebellum, and spinal
cord. Likewise, the in vi tro culture of region-
specific differentiated neurons may be derived
from any mammalian multipotential CNS stem cell,
including rat and human.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure lA shows the controlled
differentiation of CNS stem cells at high density.
Rapidly dividing nestin-positive precursor cells
were labelled with BrdU during the last 24 hours
of proliferation. Differentiation was then
initiated by withdrawal of bFGF (day 0) and
continued for up to 6 days. At indicated times,
cells were fixed and stained for BrdU and neuronal
antigens. Ratios of cells double-stained for BrdU
and each neuronal antigen to total BrdU positive
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(BrdU+) cells are shown. Up to 50~ of BrdU+ cells
expressed neuronal antigens and their expression
was time-dependent. MAP2 positive (MAP2+), filled
circle; TuJ1 positive (TuJ1+), grey diamond;
neurofilament L positive (neurofilament L+), open
square; neurofilament M positive (neurofilament
M+), filled triangle.
Figure lB shows proportions of MAP2+ neurons
(-), GalC+ oligodendrocytes (+), and GFAP+
astrocytes (o) in differentiated clones. Clones
of various sizes ranging from 39 cells to 2800
cells were differentiated for 6 days and analyzed
for two cell types per clone by double
immunohistochemistry. A partial list is given in
Table I and immunostaining shown in Fig. 3. The
number of neurons increased with increasing clone
size, constituting 50~ of the clone.
Figure lC shows a comparison of the mitogenic
efficacies of epidermal growth factor (EGF) and
basic fibroblast growth factor (bFGF). Cells
(initial density of 1 x 104 per plate) acutely
dissociated from E16 hippocampus (HI), E14 cortex
(CTX) and striatum (ST), and adult subependymal
layer (Adult) were expanded with either EGF (20
ng/ml) or bFGF (10 ng/ml). Colonies arising after
10 days of expansion were stained for nestin, an
intermediate filament protein characteristic for
CNS precursor cells1314. Relative number of
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colonies averaged from at least 2 experiments for
each region are shown (bFGF = 1). Twenty- to
fifty-fold more nestin+ colonies per plate were
present when embryonic cells were grown in bFGF
(dotted bar) than in EGF (striped bar). At high
densities (1 x lo6 and 2.5 x 106), bFGF condition
gave 10-fold higher BrdU+/nestin+ cells than EGF.
Both growth factors were equally mitogenic for the
adult cells. When EGF- and bFGF-expanded clones
were differentiated, neurons and oligodendrocytes
were found in similar quantities; however,
ÉGF-expanded clones gave rise to significantly
higher number of GFAP+ astrocytes.
Figures 2A-D show a typical clone of CNS stem
cells. Cells were marked by a circle on the plate
within 24 hours of plating before the first
mitosis and then expanded up to 10 days (Fig. 2A).
Higher magnification view of another clone before
differentiation, immunostained with anti-nestin
antibody, is shown in Figure 2B. Note the
homogeneous radial morphology of the
nestin-positive cells consistent with the
nestin-positive morphology in neuroepithelium in
vivo. Figure 2C shows a sister clone at low
magnification, which has been differentiated for 6
days and immunostained with a neuron-specific
antibody, TuJl. Note the widespread and
non-localized presence of TuJ1-positive neurons
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across the entire clone. A higher magnification
view of the same cells is shown in Figure 2D. The
TuJ1-positive cells assume typical neuronal
morphology. Heterogeneous morphologies in the
non-neuronal TuJ1-negative cells are apparent.
Figures 3A-J show examples of representative
clones of embryonic hippocampal cells (3A, 3C, 3E,
3G, 3I) and adult subependymal cells (3B, 3D, 3F,
3H, 3J) double-stained with combinations of
lo antibodies to reveal different cell types within
indlvidual clones: anti-MAP2, neuronal;
anti-GalC, oligodendrocytic; anti-GFAP,
astrocytic. The two immunoreactions were
developed sequentially and distinguished by using
two distinct chromogens via alkaline phosphatase
reaction (blue, indicated by arrows) versus horse
radish peroxidase reaction (red, indicated by
arrow heads).
The cells in Figures 3A and 3B were
double-stained with anti-MAP2 (neuronal, arrows)
and anti-GFAP (astrocytic, arrow heads) and show
that bFGF-expanded clones derived from embryonic
or adult brain differentiate into both neurons and
astrocytes. (Oligodendrocytes ar~ unstained in
this staining.)
The cells in Figures 3C and 3D were
double-stained with anti-GalC (oligodendrocytic,
arrows) and anti-GFAP (astrocytic, arrow heads)
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and show that bFGF-expanded clones derived from
embryonic or adult brain differentiate into both
oligodendrocytes and astrocytes. (Neurons are
unstained in this staining.)
Figures 3E and 3F show clones differentiated
in the presence of platelet-derived growth factor
tPDGF). The cells were double stained with
anti-MAP2 (neuronal, arrows) and anti-GFAP
(astrocytic). Most cells were MAP2+ and only a
few were GFAP+.
Figures 3G and 3H show clones differentiated
in the presence of ciliary neurotrophic factor
(CNTF). The cells were double-stained with
anti-MAP2 (neuronal) and anti-GFAP (astrocytic,
arrow heads). All cells were intensely GFAP+.
Figures 3I and 3J show clones differentiated
in the presence of thyroid hormone, tri-
iodothyronine (T3). The cells were double-stained
with anti-GalC (oligodendrocytic, arrows) and
anti-GFAP (astrocytic, arrow heads). GFAP+ and,
particularly, GalC+ cells increased. MAP2+ cells
decreased (Table IV).
Figure 4 shows the differentiation of human
CNS stem cells into neuron in high density
culture. bFGF-expanded CNS cells at high density
were differentiated by withdrawal of bFGF (~WD").
The number of neurons expressing tau protein was
determined by immunocytochemistry in culture
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during the expansion phase ("Before WD") versus
after differentiation ("After WD"). The dramatic
increase in postmitotic neurons only after the
withdrawal of bFGF indicates that they were
generated from the dividing stem cells.
Figures 5A-F show human stem cells stained
with human-specific anti-tau antiserum (Chemicon)
which identify neurons. Proliferating human CNS
stem cells in high density culture do not express
tau protein, a neuronal marker (Figure 5A). After
6 days of differentiation, however, many cells
with typical neuronal morphology express high
level of tau protein (Figure 5B). In order to
further demonstrate that these neurons have indeed
derived from dividing stem cells, the stem cells
were labeled with 10 ~M bromodeoxyuridine (BrdU),
an indicator of mitosis, for 24 hours just prior
to the bFGF withdrawal. They were then
differentiated for 6 days, double stained with
human specific anti-tau antiserum (FITC, green)
and anti-BrdU antibody (Rhodamine, red). Figure
5C shows a high magnification view of subsequent
tau-positive neurons as seen through FITC
fluorescence. Figure 5D shows the same field of
view as in Figure 5D but seen through rhodamine
fluorescence to reveal BrdU-positive nuclei. Most
tau-positive neurons are also positive for BrdU,
demonstrating that they were derived from mitotic
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stem cells before the bFGF withdrawal.
In order to further demonstrate multi-
potentiality of human CNS stem cells, they were
cultured at clonal density as described for rodent
cells. Figure 5E shows a typical clone at low
magnification, which has been expanded from a
single cell for 20 days, subsequently
differentiated for 12 days, and immunostained with
the neuron specific, anti-MAP2 antibody. Neurons
are abundant in the clone. Figure 5F shows a
higher magni~ication view of the clone in Figure
5E to indicate that the MAP2-positive cell are of
typical neuronal morphology.
Figures 6A-D demonstrate directed
differentiation of human CNS stem cells. Human
CNS stem cells after 16 days of expansion were
grown clonally for an additional 20 days and then
differentiated in the presence or absence of
single factors, PDGF ~10 ng/ml), CNTF (10 ng/ml),
or T3 (3 ng/ml). Figure 6A shows a untreated
control clone, with approximately 50%
MAP2-positive neurons (arrows) and 2-10%
GFAP-positive astrocytes (arrow heads). Figure 6B
shows a PDGF-treated clone, where 75% of cells are
MAP2-positive neurons (arrows) and 2-10% GFAP-
positive astrocytes (arrow heads). Figure 6C
shows a CNTF-treated clone, where B5% are
GFAP-positive astrocytes (arrow heads) and only 9%
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MAP2-positive neurons (arrows). Figure 6D shows a
T3-treated clone with increased number of 04-
and/or GalC-positive oligodendrocytes (arrows) and
of GFAP -positive astrocytes (arrow heads).
Figures 7A-7I show that a large number of
mature neurons with correct axon-dendritic
polarity and synaptic activity can be obtained
routinely from long-term expanded CNS stem cells.
Hippocampal stem cells from E16 rat embryos were
expanded in culture for 16 days and through 4
passages Just before the last passage, rapidly
dividing cells were labeled with 10 ~M BrdU for
overnight, passaged by using trypsin, and plated
onto chamber slides. Cells were maintained for 21
days to allow constitutive differentiation and
maturation of neurons. Subsequently, the extent
of maturation and neuronal subtypes generated were
analyzed by immunocytochemistry.
Fig. 7A: Neurons stained with TuJl antibody
viewed at low magnification (100x) to illustrate
that production of neurons is efficient.
Fig. 7B: Typical morphology of neurons
revealed by TuJl antibodies (400x).
Fig. 7C: Typical morphology~of neurons
revealed by MAP2 antibodies (400x).
Fig. 7D: Neurons stained with synapsin
antibody. Only mature neurons containing synaptic
vesicles are stained.
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Fig. 7E: BrdU staining. All cells in the
culture, neurons and glia, are labeled with BrdU.
Fig. 7F: Synapsin and BrdV double staining.
Mature synapsin-positive cells are also
BrdU-positive, demonstrating that they are derived
from mitotic stem cells in culture.
Fig. 7G: Punctate anti-synapsin antibody
staining marks the presynaptic axon terminals
specifically in large mature neurons.
Fig. 7H: The synapsin-positive structures are
closely apposed to dendritic processes revealed by
MAP2 antibody staining.
Fig. 7I: MAP2 and synapsin proteins are
closely associated but not co-localized,
suggesting presynaptic-postsynaptic interaction.
Figs. 8A-I show that stem cell-derived neurons
express various neurotransmitter receptors and
transporters expected to be involved in synaptic
transmission as detected by RT-PCR. Long-term
expanded stem cells derived from E16 rodent cortex
were differentiated for 14 days and harvested to
prepare RNA. Undifferentiated stem cells were
also prepared in order to compare differentiation-
specific induction. RNA from a whole brain of
adult rat was used as a positive control. Primers
specific for NMDA (N-methyl-D-aspartate) and AMPA
(a-amino-3-hydroxy-5-methyl-4-isoxazole
proprionic acid) families of glutamate receptor
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subtypes as well as for various GABA transporters
were used. Note especially the specific induction
of NMDA R1, AMPA R1 and AMPA R2 receptors in
differentiated cells.
Figures 9A-F show examples of typical neurons
derived from rat embryonic hippocampal stem cells
which had been expanded in vitro for 16 days
(approximately 16 cell divisions through 4
passages) and differentiated for 21 days total.
Mitotic CNS stem cells were pulse-labeled with
bromodeoxyuridine (BrdU) for the last 24 hours
prior to differentiation. Resulting neurons were
triple-immunostained with antibodies against BrdU
(Fig. 9A), MAP2ab (Fig. 9B), and synapsin (Fig.
9C). The composite view of the triple stained
cell is shown in Flg. 9D. The BrdU-labeling
demonstrates that the differentiated neuron
derived from a mitotic precursor in the culture
and that it is a terminally differentiated neuron
since it retained the mitotic label during the
prolonged differentiation phase. MAP2ab is a
well-established neuron-specific protein present
only in mature neurons and localized mostly in
dendrites. Synapsin is a well-est-ablished
synaptic vesicle protein and thus localizes
synaptic terminals in axons. The triple-labeled
neurons as shown in Fig. 9D established that long-
term expanded mitotic CNS stem cells terminally
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differentiate into mature neurons with proper
subcellular polarization containing distinct
dendritic (post-synaptic) and axonal (presynaptic)
structures expected of fully functional neurons.
Other synaptic vesicle proteins also localize in
the same pattern of punctate axon terminals
apposed to soma and dendrites. Fig. 9E and Fig.
9F show another hippocampal CNS stem cell derived
mature neuron double-stained for synaptophysin and
MAP2ab, respectively.
Figure 10 shows a field of neurons from
hippocampal CNS stem cells viewed by transmission
electron microscopy. The abundant presence of
synapses containing synaptic vesicles and post-
synaptic densities are evident.
Figures 11 A-D show intracellular electro-
physiological recordings from single neurons
obtained from rat E15.5 septal CNS stem cells.
Consistent with the morphology, these recordings
show that the CNS stem cell-derived neuronal
networks are also electrophysiologically active.
Thus, when individual cells were stimulated with
electrode, they conducted action potentials (Fig.
llA), demonstrated presence of var-ious
voltage-sensitive ion channels (Fig. llB), and
evoked excitatory and inhibitory postsynaptic
potentials in response to bath application of the
excitatory neurotransmitter, glutamate (Fig. 11 C
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and D). These examples establish beyond doubt
that CNS stem cells give rise to terminally
differentiated, electrophysiologically functional,
neuronal networks.
Diverse neuronal phenotypes seen in vivo are
obtained from the CNS stem cell cultures.
Examples of some of these neuronal phenotypes are
shown in Figures 12-18 and Table VII.
Figs. 12 A-B show the expression of dopamine
receptors D1 and D2 from CNS stem cells isolated
from E15 . 5 lateral and medial ganglionic eminence.
Total RNAs were isolated from respective CNS stem
cell cultures differentiated for varying periods
( 0-20 days). Shown is the electrophoresis pattern
of the DNA amplified by RT-PCR ( reverse
transcription-polymerase chain reaction). Results
from five independent culture preparations, run in
parallel in a single gel, are shown. The numbers
above the lanes indicate the days of
differentiation. D1 = dopamine receptor, D1; D2 =
dopamine receptor, D2.
Figures 13 A-D show cholinergic neurons from
septal CNS stem cells. CNS stem cells derived
from E16 septum were differentiated for 18-21
days. The cholinergic neurons were assessed by
acetylcholine esterase histochemistry (not shown),
by immunost~;n;ng for acetylcholine transferase
(Fig. 1 3A ) and for acetylcholine transporter (Fig.
RECTIFIED S~'EE~ ~RULF 91~
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13C). In each case, CNS stem cells were incubated
with the mitotic label, BrdU (10 ~M), for 24 hours
just before switching to the differentiation
condition ~Fig. 13B and D).
Figures 14A-F show neuropeptide-containing
neurons obtained from rat 15.5 lateral ganglionic
eminence (striatum) CNS stem cells. They are a
neuropeptide Y-positive (Fig. 14A), BrdU-positive
(Fig. 14B) neuron, a met-enkephalin-positive (Fig.
14C), BrdU-positive (Fig. 14D) neuron, and a leu-
enkephalin-positive (Fig. 14E), BrdU-positive
(Fig. 14F) neuron.
Figures 15A-F show typical morphologies of
several subtypes of neurons derived from CNS stem
cell of rat E12.5 ventral mesencephalon. Figure
15A and B show a TH-positive and BrdU-positive
neuron (Fig. 15A-TH staining; Fig. 15B-BrdU
staining). Figure 15 C and D show another TH-
positive and MAP2ab-positive neuron (Fig. 15C-TH
staining; Fig. 15D-MAP2ab staining). Figure 15E
shows neurons stained with anti-GABA antibody.
Figure 15F shows neurons stained by acetylcholine
esterase histochemistry.
Figures 16A-D show examples of neurons from
spinal cord stem cells. Figure 16A shows an
acetylcholine esterase-positive neuron derived
from rat E13.5 spinal cord CNS stem cells, which
is also BrdU-positive (Fig. 16B). Cholinergic
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neurons are shown by acetylcholine transferase
staining (Fig. 16 C), which are also BrdU-positive
~Fig. 16D).
Figures 17A and B show GABAergic neurons
derived from rat E15.5 hippocampal CNS stem cells,
which have been double-stained for glutamic acid
decarboxylase (Fig. 17A) and GABA (Fig. 17B).
Figure 17C and D show a hippocampal calretinin-
positive (Fig. 17C), MAP2ab-positive (Fig. 17D)
neuron.
Figures 18A-F show neurons derived from rat
E13.5 thalamus and hypothalamus CNS stem cells.
Figure 18A shows thalamic neurons stained for tau;
Figure 18B shows the same field of view stained
for BrdU. Figure 18C shows a hypothalamic neuron
stained for tau; Figure 18D shows the same field
of view stained for BrdU. Figure 18E and F show
synapsin-positive neurons from thalamus and
hypothalamus CNS stem cells, respectively.
DETAILED DESCRIPTION OF THE INVENTION
In this application, conditions are defined
which permit mass expansion up to 109 fold in
culture and controlled differentia~tion of
multipotential CNS stem cells from the embryonic
and adult brain of mammals. In both cases, clones
derived from single cells differentiate into
neurons, astrocytes, and oligodendrocytes.
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Addition of single factors can dramatically shift
the proportion of cell types within a clone.
The procedure for isolating, propagating, and
differentiating the CNS stem cells are given in
detail below. The procedure contains four
essential steps that must be followed in concert
for successful isolation and differentiation of
the CNS stem cells. The four essential steps are
as follows:
(1) The initial dissociation of cells from
tissue is done by mechanical trituration and not
by enzymatic digestion. With adult tissue, it is
necessary to first enzymatically digest the tissue
and then dissociate the cells from the tissue by
mechanical trituration.
Trituration means gentle agitation of cell
aggregates caused by fluid movement occurring
during repetitive pipetting action by which
individual cells become loose and dissociated from
neighboring cells. Trituration is done in a
saline solution free of divalent cations whose
absence aids break-up of interactions among
cell-adhesion proteins on cell surface. Rapidly
dividing stem cells in the ventri~ular zone are
only weakly adherent and simply removing the
divalent cations from the medium and gentle
agitation by pipetting are sufficient to
dissociate the tissue into mostly single cells.
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The cells are then cultured in the complete
absence of serum. Even a brief exposure to serum
deleteriously affects the differentiation capacity
of the stem cells so that they are no longer able
to differentiate into neurons and
oligodendrocytes. Precoating the plates with
poly-L-ornithine and fibronectin facilita~es the
adhesion of the cells to the plates.
(2) The CNS stem cells display an innate
property to differentiate spontaneously, which
reflects a regulatory mechanism controlling cell
cycle depending upon the free concentration of
growth factor, the mitogen. In order to suppress
the differentiation of the stem cells into other
cell types and to maintain homogeneity, the growth
factor must be supplied daily at a concentration
of 10 ng/ml or higher. The growth factor can be
selected from (1) basic fibroblast growth factor
(bFGF), (2) EGF, (3) TGF-alpha, or (4) acidic FGF
(aFGF). If acidic fibroblast growth factor is
selected, heparin at a concentration of 1 ~g/ml
must also be supplied.
(3) Even a continuous supply of bFGF or
other selected growth factor is insufficient to
inhibit differentiation if the culture is allowed
to reach a critical density of greater than
approximately 50%. This is most likely because of
yet undefined endogenous factor(s) secreted by the
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dividing cells themselves which antagonize the
action of bFGF. Thus, in order to remove such
factors from the culture and to reduce cell-cell
interaction as much as possible, the cells must be
passaged frequently at every 4 days after plating,
and replating should be done at low density of
approximately 0.5 x 106 per 10 cm plate, i.e., in
the range of 1 x 102 to 1 x 106 cells per 10 cm
plate, precoated with poly-ornithine and
fibronectin.
(4) Passaging the cells by trypsin results
in proteolytic removal of a bFGF receptor
component and disables the mitogenic effect of
bFGF. The turn-over rate of the receptor is
sufficiently slow during which period the cells
fail to recognize the mitogen and activate the
differentiation pathway.
In order to circumvent this process, the
cells are treated with Hank's buffered saline
solution (HBSS) to remove divalent cations in the
culture which disrupts the ionic interactions
between the cadherins and the integrins on the
cell surface and extracellular matrix proteins on
the culture plate, causing the cells to round up.
At this point the stem cells can be scraped from
the plate with a scraper without damaging the
cells.
Other cells in culture maintain tightly bound
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to the plate and scraping eliminates them, thus
allowing effective selection of rapidly dividing
undifferentiated stem cells.
Differentiation of the CNS stem cells is
achieved by simply removing the mitogen, bFGF or
other selected growth factor, from the medium.
Specification of the cell types, i.e., neurons,
oligodendrocytes, and astrocytes, occurs
constitutively. In order for the effective
controlled differentiation, the cells must be in a
homogeneous state which can be achieved by
following steps 1-4, above.
These procedures yield a culture system for
obtaining a homogeneous population of the CNS stem
cells that can be differentiated into neurons,
oligodendrocytes, and astrocytes with control and
efficiency. The highlights of the features of
this system are:
(1) production of a large number of the CNS
stem cells with the potential to form many
different neuronal subtypes, oligodendrocytes, and
astrocytes that can be transplanted into a brain;
(2) controlled differentiation in vitro
under serum-free conditions which allows the
search for novel growth factors and cytokines;
(3) rapidly dividing cells accessible to
genetic manipulation for introduction of foreign
genes;
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(4) generation of mature neurons in vitro
suitable for genetic and pharmacological
screening; and
(5) direct derivation of intermediate
precursor cells from the stem cells for enrichment
of a single population of cells.
The isolation of the CNS stem cells in the
above-described manner further permits directed
differentiation of the cells by treating them with
specific growth factors. One practical
significance of this directed differentiation to
biotechnology is that a single cell type can be
enriched in vitro. Thus, a novel application of
previously discovered growth factors PDGF3'
(platelet-derived growth factor), CNTF (ciliary
neurotrophic factor), and T3 (thyroid hormone,
tri-iodothyronine) would be to direct the CNS stem
cells to generate neurons, astrocytes, and
oligodendrocytes, respectively.
Another practical significance, especially
for PDGF, is that PDGF-induced neurons appear to
be actually neuronal progenitors that can further
proliferate and expand in culture by PDGF. These
cells differentiate only to neurons or to neurons
and oligodendrocytes and differ from the stem
cells. Isolation of neuronal progenitors from
mammalian CNS by PDGF has not been described
prevlously .
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EXAMPLES
1. Isolation of CNS Stem Cells from
Embryonic Rat Brain
Rat embryonic hippocampus (gestation day 16;
day of conception is day 1, Taconic Farm) were
dissected in Hank's buffered saline solution
(HBSS) and dissociated by brief mechanical
trituration in HBSS. The cells were collected by
centrifugation and resuspended in a serum-free
medium containing DMEM/F12, glucose, glutamine,
sodium bicarbonate, 25 ~g/ml insulin, 100 ~g/ml
human apotransferrin, 25 nM progesterone, 100 ~M
putrescine, 30 nM sodium selenite, pH 7.2~, plus 10
ng/ml recombinant human basic fibroblast growth
factor'2 (bFGF; R&D Inc.).
1 x 106 cells were plated per 10 cm plastic
tissue culture plate precoated with 15 ~g/ml
poly-L-ornithine and 1 ~g/ml bovine plasma
fibronectin (Gibco). bFGF was added daily and
media change was every 2 days. Cells were
passaged at 50% confluence (4 days after initial
plating) by briefly incubating them in HBSS and
scraping with a cell scraper.
Cells with multipotential capacity were found
throughout the developing neuroepithelium. Under
identical culture conditions, similar cells could
be prepared from other regions of the developing
CNS including cerebral cortex, striatum, septum,
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diencephalon, mesencephalon, hindbrain, and spinal
cord. From E14 cortex and striatum and E16
hippocampus, approximately 70% of acutely
dissociated cells responded to bFGF within 2 days
of plating by undergoing mitosis.
2. Propaqation of CNS Stem Cells from
Embryonic Rat Brain
a) Mass Expansion
Hippocampal cells isolated from embryonic rat
brains were expanded by daily addition of basic
fibroblast ~rowth factor (bFGF) in serum-free
medium. Continuous supply of bFGF was important
to repress differentiation and to maintain a
homogeneous population of rapidly dividing cells
expressing nestin, an intermediate filament
protein characteristic for CNS precursor cellsl3l4.
Less than 1~ of the cells expressed the astroglial
marker GFAP or the oligodendroglial markers, 04
and GalC.
The cells were passaged 4 days after plating
during which time cell number increased rapidly
with an average cell doubling time of
approximately 24 hours. Passaged cells were
replated at 0.5 x 106cells per 10 cm plate and
were allowed to propagate further. Cells could be
passaged up to five times in this manner for a
total of 20 days in vitro during which time a
yield of 220 cells could be ideally expected.
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After this time period, the mitotic rate of the
cells declined rapidly and the cells gradually
lost their multipotential capacity, exhibiting
glial characteristics and unable to differentiate
into neurons.
Large numbers of cells from cortex, striatum,
and septum isolated from embryos of 12-18 days of
gestation could also be expanded in mass culture
in the same manner. The time course of the
expansion was similar to that of hippocampal
cells. Continuous expansion was again limited by
the constitutive loss of multipotentiality after
about 20 days of cell division. Thus, this
regression appears to be a characteristic property
of CNS stem cells.
b) Clonal expansion
No simple antigenic marker is available which
uniquely identifies multipotential stem cells from
other precursors in vitro. Identity of a
precursor population can only be ascertained by
the cell's differentiation capacity. The
conditions defined for mass culture in this
application also permitted clonal expansion where
cells were plated at extremely low- cell density so
that single cells were well isolated.
Differentiation capacity of the cells
expanded in mass culture was assessed at each
passage by plating 200 cells per lO cm plate and
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cultured under conditions as described above.
Within 24 hours of plating, well isolated single
cells were marked with a 3 mm ring (Nikon) on the
bottom of the plate. Initial viability of the
marked single cells was 5-10~ and each plate
typically yielded 10-20 marked clones. Only a
single cell resided in each circle. The
subsequent population of cells within each circle
are progeny of that single cell. Clones were
expanded for up to 10 days (500-2000 cells).
Average double time was approximately 24 hours.
3. Differentiation and AnalYsis of CNS Stem
Cells from Embryonic Rat Brain
Developmental potential of expanded cells was
tested by directly differentiating the cells.
Withdrawal of bFGF initiated differentiation
within 24 hours. To initiate differentiation of
high density cells, rapidly dividing cells, which
had been in culture for 12 days and passaged three
times, were incubated for the last 24 hours with
10 ~M BrdU (bromodeoxyuridine) prior to passaging.
80-85~ of the cells incorporated BrdU. The cells
were harvested either by scraping or by using
trypsin followed by soybean trypsin inhibitor in
the serum-free medium. They were plated in
duplicate at 40,000 cells/cm2 into multi-well
chamber slides (LabTek) precoated with
poly-L-ornithine and fibronectin, and cultured in
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the serum-free medium without bFGF. At indicated
times, the cells were fixed and stained with
various antibodies according to standard
procedure.
Immunopositive cells were counted under 400x
magnification. At least five fields with a total
cell number greater than 1,000 per sample were
counted. Results shown (Fig. lA) are cell counts
averaged from two experiments. Antibody reagents
used were: anti-nestin antiserum; monoclonal
anti-MAP2 (clone HM-2, Sigma) and anti-tau
antiserum (Sigma), monoclonal anti-neurofilament L
and M (clones NR4 and NN18, Boehringer-Manheim),
anti-beta tubulin type III (TuJl), monoclonal
anti-GFAP (ICN), A2B5 (ATCC), 04, and
anti-galactocerebroside (GalC).
Over a 6 day period, there was a progressive
increase in the number of cells expressing several
well established neuron-specific antigens,
including MAP2a, b and c, beta tubulin type 3
(TuJ1), tau, and neurofilaments L, M, and H (Fig.
lA). Up to 50% of the cells expressed the
neuronal antigens and exhibited complex neuronal
morphology. The remaining cells expressed GFAP,
GalC/04, or nestin. While neurons and glia have
been observed previously in expanded culture,
these examples are the first to establish that the
differentiation of proliferating precursor cells
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can be initiated at a precise time point and that
multiple cell types arise rapidly. These
conditions permit large scale lineage analysis in
vitro.
To determine if the precursor population
contains separate committed progenitors that
independently give rise to neurons and glia,
rapidly dividing cells were plated at clonal
density (200 cells per 10 cm plate) and
well-isolated single cells were marked with 3 mm
diameter circles. 5-10% of the marked single
cells survived and proliferated with a doubling
time of 24 hours to generate clones. After
various periods of expansion (clone sizes ranging
from 24 to 21~ cells), differentiation of clones
was initiated by washing the plates once with HBSS
and culturing in the same medium but in the
absence of bFGF (Fig. 2).
For subcloning (data shown in Table II), the
clonal plates were washed and briefly left in HBSS
until the cells rounded up. Clones of 500-2000
cells were picked in 50 ~l volume with an
adjustable pipetter while viewing through a
microscope. Each clone was replated in a 10 cm
plate and single cells were marked and cultured as
before.
Cell types within clones were analyzed during
the first six days of differentiation by
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double-staining with combinations of cell-type
specific antibodies that react with mutually
exclusive cells:
neurons = MAP2+, tau+, TuJ1+, neurofilament
L+, or neurofilament M+;
astrocytes = GFAP+; and
oligodendrocytes = 04+ or GalC+.
Double staining was done sequentially using a
commercial kit (Zymed) according to the
manufacturer instructions. For oligodendrocyte
staining, cells fixed with 4% paraformaldehyde
were stained first for the cell-surface antigens
04 or GalC without permeabilization. The first
antibody was developed with alkaline phosphatase
reaction (blue) and the second with peroxidase
reaction (red) (Zymed).
As in high density culture, by six days, 50%
of the cells in a clone expressed neuronal
antigens lncluding MAP2, tau, and beta tubulin
type III (Table I, Fig. 3A and C). In Table I,
clones of hippocampal precursor cells were
expanded, differentiated, and analyzed as
described above. A partial list of typical clones
are presented above. Total number~of cells (clone
size) and cells stained positive for cell-type
specific antigens are shown. Their relative
proportion is given in percentage in parenthesis.
A total of 48 clones was quantified from 4
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different passages in 6 separate experiments.
Total Average indicates the average composition of
each cell types in the 48 clones.
Neurofllament expression was delayed under
these conditions. On average, 8~ of the cells in
a clone were GalC+ and had typical o}igodendrocyte
morphology. An additional 8% expressed GFAP and
displayed a characteri9tic astrocytic morphology.
The rem~ining cell~ were unstained by any of the
antibodies specific for differentiated cell types
but reacted with A2~5 and/or anti-nestln
antibodies. A maximum of 20% of the celis died
during differentiation. Identical results were
obtained whether clones were obtained from acutely
dissociated cells with no prior passage or from
cel}s after 4 passages ~26 days ln ~itro).
Cells with multipotential capacity were found
throughout the developing neuroepithelium. Under
identical culture conditions, similar cells could
be prepared from other regions of the developing
CNS including cerebral cortex, striatum, septum,
diencephalon, mesencephalon, hin~?hrain, and spinal
cord. When clonally ~xr~n~e~, almost all of the
clones contained multiple cell types defined by
both morphology and antigen expression with
neurons constituting 50% of the clone.
Proliferating clones of the multipotential
cells contained uniform morphology and patterns of
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antlgen expression. Yet, the separation of
neuronal and non-neuronal morphologies occurred
rapidly within 24 hours and only after the mitogen
withdrawal. The early neurons were evenly
distributed throughout the clone without obvious
polarity or localization, suggesting the absence
of committed neuronal progenitors during clonal
expansion. Moreover, the number of neurons
increased linearly with increasing clone size and
reproducibly constituted 50% of the clone (Fig.
lB).
In order to further determine if expanding
clones consisted of proliferating committed
progenitors, clones were picked and replated
again. 10-15% of the cells gave rise to second
generation clones. Again, all of the subclones
contained neurons, astrocytes, oligodendrocytes,
and unstained cells (Table II). More
specifically, cell type composition of subclones
obtained from three independent clones, HI6, HI8,
HI19, are shown in Table II. A total of 84
subclones was quantified from 13 independent
parental clones in two separate experiments. Only
a partial list is presented. Total- Average
indicates the average composition of each cell
type from the 84 subclones. No subclone consisted
of only one cell type. These data indicate that
the multipotential precursors undergo symmetric
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diviQions to generate daughter cell~ with
multipotential capacity.
4. Isolationl ProPaqation, Differentiation,
and AnalYsis o~ CNS Stem Cells from Adult Rat
Brain
The subependymal layer of adult rat brain
contains mitotic nestin positive cells that could
be expanded in aggregate culture in the presence
of epidermal growth factor (EGF) but not bFGFls.
Some of the cells ~n aggregates showed neuronal
and astrocytic properties. To more fully define
their developmental capacity, the mitotic
population ~1% of 1 x 105 cells/brain) lining the
}ateral ventricle of adult rat striatum was
expanded in the presence of bFG~ and compared to
the embryonic precursors. Forebrain slices from
250 g adult rat brains (10-20 per experiment) were
prepared and the subependymal region of strlatum
lining the lateral ventricles were cut out under
microscope in oxygenated HBSS. The cells were
dissociated by incubating minced tissues at room
temperature for 10 minutes with trypsin (1 mg/ml),
hyaluronidase ~0.7 mg/ml), and kynurenic acid (0.2
mg/ml) ln oxygenated HBSS. They were washed once
in HBSS with 0.7 mg~ml ovomucoid and 0.2 mg/ml
kynurenlc acid, resuspended, and mechanically
triturated in the same solu~ion. Dissociated
cells were recovered by centrifugation and
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cultured in the serum-free medium plus bFGF ~10
ng/ml) as described for the embryonic cells.
The morphology and growth characteristics of
the nestin-positive adult cells were similar to
those of embryonic cells. Following bFGF
withdrawal, marked clones differentiated into
multiple cell types expressing MAP2, TuJ1, GFAP,
and GalC (Fig. 3B and D). Strikingly, the same
high proportion of neurons were found in
differentiated clones of adult cells as in the
embryonic clones (Table III). More specifically,
Table III shows the cell type composition of
differentiated clones derived from adult
subependymal cells. 23 clones from three
independent experiments were quantified.
5. Isolation, Propaqation, Differentiation,
and Analysis of CNS Stem Cells from EmbrYonic and
Adult Rat Brains
Acutely dissociated cells from various
regions of embryonic brain were cultured in the
presence of either EGF (20 ng/ml) or bFGF (10
ng/ml) under identical conditions as described
above. Acutely dissociated adult cells were
prepared as described above and cultured under
identical condition as the embryonic cells. The
possible effect of initial cell density on the
mitogenic response was tested by varying the
initial cell density from 1 x 104 to 2.5 x 106 per
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plate. At low density, efficiency of colony
formation was measured; at high density,
BrdU+/nestin+ mitotic cells per field were
counted. EGF- and bFGF-expanded colonies were
also differentiated by withdrawing the mitogens
and cell types analyzed as described above.
Under the culture conditions of these
examples, EGF was an equally effective mitogen as
bFGF for adult cells (Fig. lC) and, when clones
were differentiated, they gave rise to all three
cell types. EGF-expanded embryonic clones, with
and without passage, also differentiated into all
three cell types. Unlike the adult cells,
however, EGF was at least 10-fold less effective
than bFGF as a mitogen for the embryonic cells
from several different regions, regardless of
initial cell density (Fig. lC). Thus, with the
exception of the proliferative effects of EGF,
these data reveal that the multipotential cells
from embryonic and adult CNS are remarkably
similar. TGF~ (10 ng/ml) was also a mitogen for
the multipotential cells and was indistinguishable
from EGF, while aFGF (10 ng/ml) in the presence of
heparin (1 ~g/ml) mimicked the ef~ects of bFGF.
6. Directed Differentiation of CNS Stem
Cells from Embryonic and Adult Rat Brain
The clonal analysis suggests that the
~0 multipotential precursors are not committed prior
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to mitogen withdrawal and thus extracellular
signals may regulate cell type determination. We
tested whether the proportion of the cell types
generated within a clone could be influenced by
growth factors and cytokines either during
proliferation or differentiation.
Influence of growth factors on cell type
specification was tested by adding them to the
culture two days before the withdrawal of bFGF and
during the 6 days of differentiation. Factors
were added daily and medium was changed every 2
days. At the end of the 6 days, the clones were
analyzed for cell type composition by
double-staining as described above. Final
concentrations of the factors were 10 ng/ml
PDGF-AA, -AB, or -BB, 10 ng/ml CNTF, and 3 ng/ml
T3.
In embryonic clones, the proportion of
neurons increased significantly in the presence of
PDGF (10 ng/ml, -AA, -~3, or -BB) during the
differentiation. Up to 80~ of the cells were
neuronal with MAP2, tau, TuJ1, or NF-M expression,
and fewer cells expressed 04, GalC, and GFAP (Fig.
3E and F, Table IV). More specifi~ally, Table IV
shows the average clonal composition of each cell
type obtained when clones were differentiated for
6 days either in the absence (Untreated) or
presence of different factors. Clonal plates were
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prepared from cells after 0-3 passages. Clone
size ranged from 17 to 5336 cells. Differentiated
cell types were analyzed as described above.
The cells expressing the neuronal antigens
showed a less mature morphology under these
conditions. When treated with ciliary
neurotrophic factor (CNTF), clones gave rise
almost exclusively to astrocytes (Fig. 3G and H,
Table IV). Remarkably, less than 1% of the cells
were MAP2-positive in this condition. The
CNTF-treated cells were intensely GFAP-positive
and all showed a flat, astrocytic morphology. LIF
showed identical effects as CNTF.
Thyroid hormone, tri-iodothyronine (T3),
influenced the differentiation of the
multipotential precursors toward a mixed glial
fate (Fig. 3I and J, Table IV). Astrocytes and
oligodendrocytes were both increased 3-fold and
there was a marked decrease in the proportion of
neurons. As in the untreated clones, GalC- and
04-positive cells showed characteristic
oligodendrocyte morphologies. The clones were of
similar size in all the experiments and numerical
analysis of dead cells showed that. selective cell
death cannot account for the changes in the
proportion of cell types. Similar results were
obtained with multipotential stem cells from
embryonic cortex and striatum. Furthermore, the
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multipotential cells derived from subepe~dymal
layer of the adult brain showed quantitatively
similar differentiation responses to PDGF, CNTF,
and T3 (Fig. 3, Table IV). This emphasizes the
general nature of these pathways.
Other factors that were tested during this
study and showed no significant instructive effect
on cell type determination were: NGF, NT-3, BDNF,
TGFbl, ILlb, IL2-11, G-CSF, M-CSF, GM-CSF,
oncostatin M, stem cell factor, erythropoietin,
interferon gamma, 9-cis and all-trans retinoic
acid, retinyl acetate, dexamethasone, and
corticosterone.
7. Isolation, ExPansion, Differentiation,
and AnalYsis of CNS Stem Cells from Human Fetal
Brain
Tissues from various regions of human fetal
brains were obtained from fetuses of 45 to 114
days of ~estation periods. The tissues were
dissociated in HBSS by mechanical trituration as
described above. Cells were collected by
centrifugation, resuspended, plated at 1 x 106
cells per 10 cm plate, and expanded in the
serum-free medium plus 10 ng/ml bFGF under
conditions identical to those described for rodent
fetal CNS stem cells above.
Approximately 25-50% of the human cells
depending upon the age and the region had CNS stem
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cell morphology and responded to bFGF by rapid
cell division. Human CNS stem cells were expanded
in culture for up to 36 days. Average doubling
time was approximately 48 hours, which contrasts
with the rodent counterpart with 24 hour doubling
time. Upon withdrawal of bFGF, the
differentiation of human fetal CNS stem cells
occurred rapidly and multiple cell types arose.
In high density culture, the cells were
differentiated for up to 13 days and subsequent
cell types present were analyzed by immunocyto-
chemistry as described for rodent cell culture.
Before differentiation by bFGF withdrawal, few
tau-positive neurons were present in the culture
(Fig. 4). In contrast, after the bFGF withdrawal,
up to 40% of the bFGF-expanded human fetal brain
cells in mass culture were neurons immunoreactive
with human-specific anti-tau antiserum. A
majority of the tau-positive neurons in culture
could be labeled with BrdU (bromodeoxyuridine),
the indicator of mitosis, within 24 hours prior to
the bFGF withdrawal (Fig. 5C and D). This result
demonstrates that the culture conditions defined
for rodent CNS stem cells applies~e~ually well for
efficient expansion and differentiation of human
CNS stem cells to generate large numbers of
neurons in culture.
In order to further analyze the multi-
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potentiality of the human fetal CNS stem cells in
mass culture, dividing cells were plated at clonal
density (100-200 cells per 10 cm plate) and
further expanded for 20 days (clone size = 2l~).
Subsequently, clones were differentiated by
withdrawing bFGF and analyzed for cell types
immunoreactive for neuron-, astrocyte-, or
oligodendrocyte-specific antibodies. Almost all
of clonally expanded human fetal cells
differentiated to give rise to all three cell
types--neurons, astrocytes, and oligdendrocytes
(Fig. 5E and F). As with rodent stem cells,
MAP2-positive neurons comprised approximately 50%
of the clone (Table V). The remaining cells were
of large elongated glial morphology.
Approximately 10% of the cells expressed mature
astrocytic antigen, GFAP, and about 2% expressed
oligodendrocytic antigens 04 or galactocerebroside
~GalC) (Table V). This clonal analysis thus
demonstrates that the culture system described
here permits efficient isolation, mass-expansion,
and differentiation of multipotential stem cells
from human fetal CNS.
8. Directed Differentiation of CNS Stem
Cells from Human Fetal Brain
In addition to the mulitpotentiality and
self-renewing properties of CNS stem cells, the
capacity to differentiate into one cell type in
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response to an extracellular slgnal is the key
defining property of rodent CNS stem cells as
demonstrated above. The three extracellular
factors, PDGF, CNTF, and T3, also directed the
differentiation of the human CNS cell clones in an
identical manner (Table V; Fiq. 6A-D). Thus, in
the presence of PDGF, MAP2-positive neuronal cells
increased to 71% of a clone, significantly higher
than the 46% in the untreated control culture. In
contrast, in the presence of CNTF, MAP2-posi~ive
cells decreased and GFAP-positive astrocytes
increased dramatically to 85% of the ciones. T3
increased 04- or GalC-positive oligodendro~lial
cells as well as GFAP-positive astroqlial cells,
while MAP2-positive neurons decreased (Table V).
These results demonstrate the similarities
quantitatively between the human and the rodent
CNS stem cells and the universal applicability of
the precent culture system for efficient expansion
and differentiation of ~m~l ian CNS stem cells.
9. Maturation, SYna~toqenesis. and Diversity
of Stem Cell-Derived Neurons In ~i ~ro
Multipotentiality of CNS stem cells and their
directed differentiation by defined extracellular
signals unequivocally establish that neurons
derive from stem cells directly. Thus, the origin
of neuronal diversity seen in mature brain starts
from CNS stem cells. Can the CNS stem cells
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expanded in culture for long-term retain the
ability to mature to form axonal-dendritic
polarity, to interact with other cells and form
synap~es? In order to investigate the extent to
which CNS stem cell-derived neurons can mature in
vitro under serum-free condition, stem cells
derived from embryonic rat hippocampus were
allowed to differentiate for up to 21 days at high
density.
Subsequently, neurons were stained with
various antibodies recognizing either axon- or
dendrite- specific proteins. Synapsin,
synaptophysin, synaptobrevin, and syntaxin are
proteins found in synaptic vesicles of mature
neurons at axon terminals and are involved in
exocytosis of neurotransmitters. All four
proteins were highly co-localized in the stem
cell-derived neurons, in punctate pattern, most
likely delineating the axon terminals. The
processes bearing the synaptic vesicle proteins
were thin, highly elaborate, traveled long
distance, and decorated the perimeter of
neighboring neurons (Fig. 7G). They contained
axon-specific proteins such as tau and
neurofilament and were devoid of dendrite specific
proteins such as MAP2a and MAP2b (Fig. 7H and 7I).
These results indicate that, similar to
neurons generated in vivo, the stem cell-derived
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neurons display proper axon-dendrite polarity and
exhibit synaptic activity. Stem cell-derived
neurons also expressed major neurotransmitter
receptors, transporters, and processing enzymes
S important for neurotransmitter functions. These
included members of glutamate receptors, GABA
receptors, and dopamine receptors (Fig. 8).
Furthermore, the stem cells retain their capacity
to generate subtypes of neurons having molecular
differences among the subtypes.
10. In Vitro Generation of All Neuronal
SubtyPes Found in the Mature Brain bY
Differentiatinq CNS Stem Cells
Understanding the molecular programs that
govern the organization of complex neuronal
diversity in the mammalian adult brain is a major
goal of developmental neurobiology. Most of the
structural domains in the adult brain and
subpopulations of postmitotic neurons comprising
them are generated during embryonic development.
The developmental properties of the immediate
precursor cells that give rise to specific
neurons, however, are largely unknown. Also
unclear are the precise stage of differentiation
and the general molecular principle by which
neurons ac~uire their neurotransmitter phenotypes.
One emerging line of evidences is that from
early stages of development, neural tube and brain
CA 022~9484 1998-12-31
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vesicles are patterned by spatial and temporal
expression of a number of nuclear and secreted
proteins5'359. This evidence is consistent with the
hypothesis that the early neuroepithelium is
composed of predetermined precursor cells and that
mature cortical organization, for example, derives
from a predetermined early "proto-cortex. "60
The idea of predetermined neuroepithelium,
however, is at odds with other observations from
in vivo fate mapping studies and transplantation
studies56l~63. One main conclusion from these
experiments is that certain precursor
population(s) are multipotential and/or widely
plastic in respect to the neuronal versus glial
lineages as well as neuronal phenotypes such as
neurotransmitter phenotypes and laminar or
regional destination. In order to reconcile these
two sets of seemingly contradicting observations,
several major issues must be addressed. What is
the differentiation capacity of the precursor cell
that directly gives rise to terminally
differentiated neurons? What information, if any,
does that precursor cell contain in respect to
specific phenotype of the neurons?~
To answer these questions, we have
successfully isolated from early rat
neuroepithelium multipotential precursor cells,
CNS stem cells, and examined quantitatively their
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differentiation capacity in vitrofi4 (see also
Examples 1-3, 5 and 6). Under constitutive
conditions with no exogenous influence, CNS stem
cell clones differentiated into all three major
cell types-- neurons, astrocytes, and
oligodendrocytes. In the presence of single
extracellular factors, however, their fate choice
could be directed toward single cell types.
Moreover, such multipotential stem cells were by
far the majority of expandable populations in
culture suggesting that they are abundant in the
neuroepithelium. These properties are also shared
by CNS stem cells from human fetal brain. Thus,
these are the defining properties of mammalian CNS
stem cells, which constitute the majority of
embryonic CNS and are the direct precursors to
neurons of the adult brain.
What, then, is the developmental capacity of
multipotential CNS stem cells in respect to
neuronal phenotypes? In this Example, we examined
the extent of information embedded in the isolated
CNS stem cells to guide terminal differentiation
and maturation of neurons and for generation of
specific subpopulations of neurons. We found that
although CNS stem cells are widely distributed in
large numbers throughout the neuroepithelium and
are equally multipotential in respect to the three
major cell types, CNS stem cells derived from a
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distinct region give rise to neuronal phenotypes
appropriate for that region only. We conclude
that the information specifying region-specific
neuronal phenotypes is present in the
multipotential stem cell state, that this
information is stably inherited through many cell
divisions in vitro, and that, when differentiated
under constitutive conditions in the absence of
external influence, CNS stem cells are non-
equivalent and each gives rise to only restrlctedsets of neurons appropriate for the region from
where the CNS stem cells originated.
In Examples, 1-3, 5 and 6, we limited the
differentiation of CNS stem cell clones only to
the earliest time point of maturation at which all
three cellular phenotypes, i.e., neurons,
astrocytes and oligodendrocytes, could be sampled
without encountering significant cell death.
Hence, neuronal differentiation was limited only
to early stages of differentiation. We decided to
examine to what extent CNS stem cell-derived
neuron~ could differentiate in vitro under
constitutive conditions, that is, in serum-free,
defined minimal medium in the abse-nce of exogenous
factors.
Neuronal differentiation encompasses many
distinct phases of cellular maturation. One of
the earliest characteristics of a functional
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neuron to be expected is the polarization of a
neuron into distinct compartments, i.e., soma,
dendrite and axon. We examined various defined
culture conditions to promote differentiation of
clones of multipotential CNS stem cells into
polarized neurons. Under clonal conditions, we
found that neuronal survival is too limited to
permit systematic characterization of late
phenotypes of neuronal differentiation.
Interestingly, addition of various commercially
available neurotrophic factors including NGF and
FGF families could not overcome this barrier.
However, simply increasing the cell density
of differentiating CNS stem cells was sufficient
for effective neuronal survival, and polarized
neurons with mature morphology could be
reproducibly obtained after 14-21 days in N2
medium in the absence of glutamate and any other
exogenous factors. Although not essential,
occasional supplementation of the culture with
brain-derived neurotrophic factor (BDNF) further
facilitated long-term neuronal survival generally
and was, therefore, used in this Example.
Specifically, CNS stem cells-were isolated
and expanded under defined conditions as
previously described above in Examples 1-3, 5 and
6. Different neurons were derived by isolating
CNS stem cells from different regions of the
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central nervous system and from different stages
of the CNS development. Differentiation
conditions for obtaining all neuronal phenotypes
were identical and different neurons derived only
from allowing expression of inherent information
already embedded in the expanded CNS stem cells.
More specifically, at the last mitotic cycle,
differentiation was overtly triggered by
withdrawal of mitogen, e.g., bFGF, by replacing
the growth medium with mitogen-free medium. At
the same time, or some days later without any
differential consequence, the cells were harvested
by trypsinization and centrifugation according to
conventional procedures. Trypsin was inactivated
by adding trypsin inhibitor. The resulting cell
pellet was resuspended in the same N2 growth
medium without bFGF or any other factor and plated
at high cell density, optimally at 125,000 cells
per square centimeter, onto tissue culture plates
precoated with poly-L-ornithine (15 ~g/ml) and
fibronectin (1 ~g/ml) or laminin (1 ~g/ml). Two
to four days later, the N2 medium was replaced by
N2 medium without glutamic acid. The high cell
density was necessary for efficient neuronal
differentiation and the absence of glutamic acid
was necessary to permit long-term survival of
mature neurons.
Neurons were maintained for long periods (up
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to 30 days) under these conditions with the medium
changed every 3-4 days. Supplementing the medium
with 20 ng/ml of recombinant human BDNF further
facilitated neuronal survival and maturation.
After 12-30 days of differentiation, cells were
fixed with 4% paraformaldehyde and neuronal
phenotypes were identified by immunocytochemistry
against marker proteins.
In order to directly demonstrate that the
lo mature neurons and various subtypes of neurons in
culture were directly produced from the mitotic
CNS stem cells, CNS stem cells from various
embryonic brain regions (see below for examples)
which had been expanded in vitro for long-term
(approximately 16 days and 16 cell divisions
through 4 passages) were overtly differentiated
for 21 days total as described above. Mitotic CNS
stem cells were pulse-labeled with bromodeoxy-
uridine (BrdU) for the last 24 to 48 hours prior
to differentiation. From all regions, up to 86%
of MAP2ab-positive neurons were also positive for
BrdU. By 24 hour-BrdU labeling, approximately
50%-75% of the neurons immunopositive for the
antigens specific for different neuronal subtypes
were also positive for BrdU. Prior to the overt
differentiation step, no neurons expressing MAP2ab
or any other subtype-specific antigens were
observed in any of the CNS stem cell cultures.
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Thus, consistent with the previous examples
(Examples 1-8), all of the neurons and neuronal
subtypes reported below were produced exclusively
from long-term expanded, mitotic, CNS stem cells.
Neurons thus obtained contained distinct
localization of dendritic proteins such as MAP2ab
from axonal proteins such as tau, neurofilaments,
and several synaptic vesicle proteins. Shown in
Fig. 9 are typical neurons derived from rat
embryonic hippocampal stem cells. Mature neurons
were triple-immunostained with antibodies against
BrdU (Fig. 9A), MAP2ab (Fig. 9B), and synapsin
(Fig. 9C). Combined staining is shown in Fig. 9D.
Figures 9E and F show another typical example
of hippocampal stem cell-derived neurons
double-stained for synaptophysin (Fig. 9E), a
synaptic vesicle protein labeling axon terminals,
and MAP2ab (Fig. 9F) labeling dendritic process.
These immunostaining results demonstrate
polarization of neurons into axons and dendrites
and significantly suggest numerous synaptic
junctions. Further examination of these
morphologies by electron microscopy confirmed the
abundant presence of synapses containing synaptic
vesicles and synaptic densities (Fig. 10).
These neuronal networks were also functional
electrophysiologically. They conducted action
potentials (Fig. llA), contained various voltage-
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sensitive ion channels (Fig. llB), and transmitted
excitatory and inhibitory postsynaptic potentials
when evoked by bath application of the excitatory
neurotran~mitter, glutamate (Fig. llC and D).
Thus, these results unequivocally demonstrate that
all of the information necessary and sufficient to
form mature neurons and synaptogenesis from the
mitotic state is self-contained and stable within
the long-term expanded CNS stem cells.
Long-term expanded CNS stem cells derived
from several different regions of the neuro-
epithelium gave rise to distinct subpopulations of
neurons. Thus, CNS stem cells were isolated from
several dlfferent regions of rat embryonic CNS at
times known to be at the beginning or in the midst
of neurogenesis-- embryonic gestation day 15.5 (E
15.5) cortex (CTX), septum (SEP), lateral
ganglionic eminence (LGE), medial ganglionic
eminence (MGE), hippocampus, E13.5 thalamus,
hypothalamu~, E12.5 ventral and dorsal
mesencephalon, and E11.5-E13.5 spinal cords. From
each of these regions, almost homogeneous cultures
of CNS stem cells could be expanded for long term
(typically for 16 days with average doubling time
of 24 hours) according to the culture conditions
described previously64.
General properties of the expanding CNS stem
cells such as morphology, mitotic rate, and
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differentiation characteristics were
indistinguishable among different regions
including hippocampus which has been described
above in detail64. In clonal analysis, each of
these regions contain many CNS stem cell clones
with multipotential capacity to differentiate into
neurons, astrocytes, and oligodendrocytes in
relative proportions identical to the previously
detailed hippocampal stem cell clones64.
The compelling question then is whether there
is only one kind of stem cell that constitutes the
entire neuroepithelium and that regional
specification and neuronal diversity occur at
subsequent stages of development or whether the
CNS stem cells at the multipotential stage contain
the information for reglonally distinct neuronal
phenotypes.
Lateral and medial ganglionic eminence are
two closely adjacent structures that develop in
parallel into striatum and globus pallidus in the
adult brain. Dopamine receptors, D1 and D2 are
expressed in striatum, but only D2 is present in
pallidus. The expression of D1 and D2 receptors
from CNS stem cells isolated from-E16 lateral and
medial ganglionic eminence was examined by RT-PCR
~Fig. 12). Prior to differentiation, CNS stem
cells from either region expressed no dopamine
receptors. After 9 days of differentiation, D1
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and D2 receptors were expressed in LGE-derived
stem cells, but only D2 receptor was expressed in
cells from MGE. This differential pattern was
stable throughout the differentiation course up to
21 days examined (Fig. 12).
Cholinergic neurons of septum have been
critically attributed to the onset of Alzheimer's
disease. These neurons appear during E15-E18 in
rats. CNS stem cells derived from E16 septum were
differentiated for 18-21 days under the defined
conditions as described above and the presence of
cholinergic neurons were assessed by acetylcholine
esterase histochemistry and by immunostaining for
acetylcholine transferase and for vesicular
acetylcholine transporter. Figure 13A shows a
septal CNS stem cell-derived cholinergic neuron
immuno-stained for acetylcholine transferase.
Figure 13B shows the same field of view as Fig.
13A stained for the mitotic label BrdU. Figure
13C and D show another example of a cholinergic
neuron double-stained for vesicular acetylcholine
transporter and BrdU, respectively.
Table VII summarizes the number of MAP2ab-
positive neurons per square centimeter and the
proportions of different neuronal phenotypes
relative to the total MAP2ab-positive neurons
derived from CNS stem cells of several different
regions and ages. Approximately 4-5% of the MAP2
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positive neurons were cholinergic. In contrast,
hippocampal and cortical CNS stem cells gave rise
to no cholinergic neurons.
About 0.4% of neurons derived from LGE and
MGE CNS stem cells also expressed vesicular
acetylcholine transporter, a specific marker of
cholinergic neurons (Table VII). Approximately
2.8~ to 10.7% of LGE and MGE-derived neurons
contained several different neuropeptides such as
neuropeptide Y, met-enkephalin, and leu-enkephalin
(Fig. 14; Table VII). Figures 14A, C, and D show
typical LGE CNS stem cell-derived neurons stained
for neuropeptide Y, met-enkephalin, and leu-
enkephalin, respectively. Figures 14B, D, and F
show the immunostaining for BrdU of the same
fields as in Figs. 14A, C, and E, respectively.
When CNS stem cells expanded from E12.5
ventral mesencephalon were differentiated,
approximately 2.6 + 0.3% of MAP2 positive neurons
expressed tyrosine hydroxylase, the key enzyme for
dopamine synthesis and a well-established marker
of dopaminergic neurons (Table VII). Figure 15A
shows a typical CNS stem cell derived TH-positive
neuron and Figure 15B shows the corresponding BrdU
staining of the same field. All TH-positive cells
are neurons as shown by double-staining for TH and
MAP2ab (Figs. 15C and D, respectively). Most of
the remaining neurons were positive for the marker
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of GABAergic neurons, glutamic acid decarboxylase
(GAD) as well as for GABA itself (Fig. 15E) and/or
for acetylcholine esterase (Fig. 15F; Table VII)
which is known to be expressed in monoaminergic
neurons in this area.
CNS stem cells derived from dorsal
mesencephalon, in contrast, generated no
TH-positive neurons (Table VII). Almost all
neurons of this area (100.9 + 9.1%) expressed
acetylcholine esterase (Table VII). They are most
likely monoaminergic neurons, consistent with the
in vivo pattern. Significantly, no TH-positive
neurons arose from CNS stem cells derived from
cortex, septum, hippocampus, striatum, and spinal
cord (Table VII). Thus, in parallel with the
known in vivo expression pattern, generation of
T~-positive neurons were unique to ventral
mesencephalon CNS stem cells in vitro.
CNS stem cells from E13.5 cervical and
thoracic spinal cords were expanded and
differentiated. 1.2 + 0.1% of MAP2 positive
neurons were cholinergic containing vesicular
acetylcholine transporter (Table VII).
Cholinergic neurons also expressing acetylcholine
transferase and BrdU-positive are shown in Figures
16C and D, respectively. 39.3 + 2.5% of the
neurons expressed acetylcholine esterase (Table
VII), most of which are expected to be
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monoaminergic. A typical acetylcholine esterase-
positive and BrdU-positive neuron is shown in
Figure 16A and B, respectively.
Neurons derived from E15.5 hippocampal and
cortical CNS stem cells did not express tyrosine
hydroxylase, acetylcholine esterase, acetylcholine
transferase, and vesicular acetylcholine
transporter (Table VII). This is appropriate for
known absence of these markers in hippocampus in
vivo. About 30~ of MAP2ab-positive neurons were
GABAergic, indicated by expression of GAD and
GABA. Figures 17A and B show typical examples of
GAD- and GABA-positive staining, respectively,
which completely overlap. A typical hippocampal
calretinin- and MAP2ab-positive neuron is shown in
Figures 17C and D, respectively.
Mature neurons can be also be derived with
equal efficiency from E13.5 thalamus and
hypothalamus. These neurons contain exceptionally
long axonal processes. A typical thalamic neuron
stained for the axonal protein, tau, and BrdU is
shown in Figures 18A and B, respectively. A
typical hypothalamic neuron stained for tau and
BrdU is shown in Figures 18C and ~, respectively.
Synapsin staining of thalamic and hypothalamic
neurons is shown in Figures 18E and F,
respectively.
The examples given above have been selected
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based upon only well-established in vivo
populations available in the literature and also
upon the well-defined markers commercially
available. A summary of the proportions of
s different neuronal phenotypes from various
regional CNS stem cells are shown in Table VII.
These examples are only a part of the neuronal
diversity present in the CNS stem cell-derived
cultures.
In summary, these results conclusively
demonstrate that distinct subpopulations of
neurons are generated in culture from expanded CNS
stem cells and that the types of neurons generated
are restricted in a region-specific manner
approximately corresponding to in vivo patterns of
expression. The information which specifies
neuronal phenotype is therefore embedded in the
multipotential stem cell state. Moreover, this
specifying information is heritably stable through
many cell divisions and enacted during the
subsequent differentiation process. These results
directly demonstrate that the mammalian
neuroepithelium is indeed divided in a mosaic
pattern of different kinds of multipotential CNS
stem cells with heritable, restricted information
which specify neuronal phenotypes in the absence
of any other interactions. Thus, all neuronal
subtypes found in the mature mammalian brain can
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be generated in vitro by differentiating
appropriate CNS stem cells.
These neurons and the CNS stem cells capable
of differentiating into such neurons provide the
key element for gene therapy, cell therapy, and
identification of novel therapeutic molecules
(proteins, peptides, DNA, oligonucleotides,
synthetic and natural organic compounds) directed
to nervous system disorders.
SIGNIFICANCE OF CNS STEM CELL TECHNOLOGY
sehavior of the stem cells in vi tro provides
important insights on the CNS development.
Efficient proliferation and controlled
differentiation of the precursor cells permitted a
quantitative analysis of their developmental
capacity. They display properties expected of
stem cells: rapid proliferation, multi-
potentiality, and self-regeneration. Moreover,
the cells from adult brain were quantitatively
equivalent to the embryonic cells, indicating that
stem cells persist in the adult.
The multipotential cells could be efficiently
isolated from many regions of the developing CNS,
indicating that they are abundant throughout the
neuroepithelium. This contrasts with the
widely-held notion that stem cells are rare.
Differentiation of the stem cells can be
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effectively directed by extracellular factors that
are known to be present during CNS developmentl6~23.
This suggests that different extracellular factors
can act on a single class of stem cells to
generate different cell types. A similar
instructive mechanism has also been observed in
vitro with stem cells isolated from the peripheral
nervous system24.
Multiple cell types appear rapidly when the
stem cells are differentiated in vitro. In
contrast, neurons, astrocytes, and oligo-
dendrocytes appear at distinct times in vivo.
Clearly, additional mechanisms must regulate the
fate choice in vivo. Temporal and spatial
expression of extracellular factors and their
receptors may be a part of the mechanism.
Another mechanism of fate choice regulation
in vivo may involve intermediate stages of
differentiation. Identification of the
bipotential oligodendrocyte precursor cell, 0-2A,
from postnatal optic nerve directly demonstrated
that restricted progenitors are produced during
development2526. The stem cells are distinct from
the 0-2A cells. Their origins, properties, and
developmental capacities differ. Given that the
stem cells differentiate into oligodendrocytes,
the differentiation pathway may involve an
obligatory intermediate stage, a committed
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progenitor state like the 0-2A cell. The similar
responses of both cells to T3 and CNTF272B may
reflect this common step.
There is also evidence from lineage analysis
in vivo and in vitro for the presence of other
lineage restrictions, including bipotential
neuronal and oligodendrocyte precursors and
committed neuronal progenitors672930. These may
also arise from differentiating stem cells. The
clonal assay described here will permit the
relative contributions of lineage commitment
versus instructive and selective factors on these
intermediate cells to be quantitatively defined.
In summary, the present application reveals
that:
(1) most regions of the fetal brain and
spinal cord can be made to multiply in culture
under completely defined culture conditions to
yield up to a 1,000,000,000-fold increase in cell
number;
(2) the homogeneous stem cell culture can be
triggered to differentiate under precisely
controlled conditions where up to 50% of the cells
differentiate into neurons while the remaining
cells become astrocytes and oligodendrocytes;
(3) many different kinds of neurons are
generated in culture;
(4) growth factors have been identified that
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effectively direct the stem cells to differentiate
into a single cell type, i.e., neuron, astrocyte,
or oligodendrocyte; and
(5) equivalent stem cells have been isolated
and expanded from adult subependymal layer by
using a similar procedure.
These enumerated results provide the
following advantages over the current state of the
art. First, this CNS stem cell technology permits
large scale culture of homogeneous stem cells in
an undifferentiated state. The longer that the
cells can be maintained in the stem cell state,
the higher the yield of neurons that can be
derived from the culture, thereby enabling more
efficient gene transfer and large scale selection
of those cells carrying the gene of interest.
Second, this culture system permits
controlled differentiation of the stem cells where
50~ of the expanded cells now turn into neurons.
This efficient differentiation, combined with
efficient proliferation, routinely yields more
than 100 million neurons from the neocortex of one
rat fetal brain in a two-week period.
Third, the differentiation of~the stem cells
into neurons, astrocytes, and oligodendrocytes
occurs constitutively where all three cell types
continue to mature in culture, most likely due to
nurturing interactions with each other, as during
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CA 022~9484 1998-12-31
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normal brain development. Many different types of
neurons arise, which respond to many growth
factors and contain neurotransmitters and their
receptors. Thus, a significant portion of the
brain development can be recapitulated in a
manipulable environment, thereby hlghlighting the
potential to extract and test novel neurotropic
factors normally secreted by these cells.
Finally, these results permit the
establishment of conditions by which dividing
immature neurons can be derived directly from the
stem cells and expanded further to allow large
scale isolation of specific kinds of neurons in
culture.
POTENTIAL COMMERCIAL APPLICATIONS
The stem cell technology of the present
invention can be developed for direct application
to many different aspects of therapy and drug
discovery for nervous system disorders. Outlined
below are four examples for potential commercial
applications, i.e., gene therapy for Parkinson's
disease, cell therapy, search for novel growth
factors, and assays for drug screening.
The CNS stem cells more than meet the
technical criteria as vehicles for gene therapies
and cell therapies in general. The stem cells can
be expanded rapidly under precisely controlled,
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reproducible conditions. Furthermore, these cells
are readily accessible to all standard gene
transfer protocols such as ~ia retroviruses,
adenoviruses, liposomes, and calcium phosphate
treatment, as well as subsequent selection and
expansion protocols. The expanded stem cells
efficiently differentiate into neurons en masse.
In addition, it should be emphasized that two
additional properties of the stem cells make them
unique as the fundamental basis of therapeutic
development directed at the human nervous system.
First, once stem cells are triggered to
differentiate into mature cell types, all of the
molecular interactions are in place within the
culture system to generate, to mature, and to
survive a variety of different cell types and
neuronal subtypes. These interactions
recapitulate a significant portion of the natural
brain development process. Therefore, the stem
cells, as vehicles of gene therapy and cell
therapy, refurnish not only a single potential
gene or factor to be delivered but also the whole
infrastructure for nerve regeneration.
Second, the stem cells in culture are
expanded from the multipotential germinal
precursors of the normal brain development.
Hence, these stem cells retain the capacity to
become not only three different cell types but
CA 022~9484 1998-12-31
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also many different types of neurons depending
upon the environmental cues to which they are
exposed. This broad plasticity, which is the
inherent property of the stem cells, distinctly
suggests that, once transplanted, the cells may
retain the capacity to conform to many different
host brain regions and to differentiate into
neurons specific for that particular host region.
These intrinsic properties of the primary stem
cells are far different from the existing
tumorigenic cell lines where some neuronal
differentiation can be induced under artificial
conditions. Therefore, with these unique
properties, the expandable human CNS stem cells
contain significant commercial potential by
themselves with little further development.
1. Gene TheraPY for Parkinson's Disease
Parkinson's Disease results mainly from
degeneration of dopamine releasing neurons in the
substantia nigra of the brain and the resulting
depletion of dopamine neurotransmitter in the
striatum. The cause of this degeneration is
unknown but the motor degeneratio~ symptoms of the
disease can be alleviated by peripherally
administering the dopamine precursor, L-dopa, at
the early onset of the disease. As the disea~e
continues to worsen, L-dopa is no longer effective
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and currently no further treatment is available.
One promising treatment being developed is to
transplant dopamine-rich substantia nigra neurons
from fetal brain into the striatum of the brain of
the patient. Results obtained from various
clinical centers look extremely optimistic.
However, it is estimated that up to 10 fetal
brains are needed in order to obtain enough cells
for one transplant operation. This requirement
renders unfeasible the wide application of the
transplantation of primary neurons as a
therapeutic reality. This is exactly the type of
problem solved by the CNS stem cell technology of
the present application, whereby a small number of
cells can be expanded in culture up to a
1,000,000,000 fold.
It is now widely recognized that
transplantation of dopamine producing cells is the
most promising therapy of severe Parkinson's
Disease and that a stable cell population or cell
line genetically engineered to produce dopamine is
essential to effective therapy. Tyrosine
hydroxylase (TH) is the key enzyme for dopamine
synthesis. Human CNS stem cells derived from
fetal basal ganglia can be produced which express
the tyrosine hydroxylase (T~) gene. These cells
can be expanded, differentiated, and transplanted
into the patient's striatum. Since the cells are
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originally derived from the primordial striatum,
they would have the best chance of integrating
into this region of the brain. Production of such
cells and their successful transplantation into
animal models will result in the most promising
application of gene therapy to date.
2. Cell TherapY
In most neurological diseases, unlike
Parkinson's Disease, the underlying cause of
symptoms cannot be attributed to a single factor.
This condition renders the therapeutic approach of
introducing a single gene by gene therapy
ineffective. Rather, replacement of the host
neuronal complex by healthy cells will be
required. Since CNS stem cells are the natural
germinal cells of the developing brain with the
capacity to become the cells of the mature brain,
the stem cells from the spinal cord and different
regions of the brain may be used directly to
repopulate degenerated nerves in various
neuropathies.
Several specific stem cell lines that over
express various growth factors that are currently
in clinical trials are being developed. This
application combines the unique plasticity of the
stem cells and growth factor-mediated gene therapy
to provide not only the benefit of the targeted
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delivery of the protein but also more broad
neuronal regeneration in specific areas.
Primary examples of growth factors currently
in clinical trials or under full development by
various companies are listed below in Table VI31.
So far, tests of growth factors have been limited
to direct peripheral injection of large doses,
which brings significant risks of side effects
since most growth factors affect many different
populations of neurons and non-neural tissues and
with a short half-life. These problems can be
overcome by generating from the CNS stem cells
several cell populations or cell lines stably
expressing these growth factors and demonstrating
their capacity to differentiate into neurons and
to secrete the growth factors in specific
peripheral and central regions.
3. Search for Novel Growth Factors
One of the central principles of modern
neurobiology is that each of the major projection
neurons, if not all neurons, requires specific
signals (trophic factors) to reach their target
cells and survive. Neuropathies in many diseases
may be caused by or involve lack of such growth
factors. These growth factors represent the next
generation of preventive and therapeutic drugs for
nervous system disorders, and hence the enormous
capitalization invested in the search and
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development of novel growth factors by the
biotechnology industry.
Implicit in the observation that a variety of
mature neurons can be produced from CNS stem cell
culture is that various growth factors are
secreted by the differentiating cells for
determination of cell types, maturation, and
continued support for their survival and that the
cells contain the necessary receptor machinery to
respond to those growth factors and probably
others. Most of the growth factors known so far
in the nervous system were discovered by their
effects on peripheral nerves and they most likely
represent a very minor fraction of existing growth
factors in the brain.
Search for growth factors from the brain has
been difficult mainly becauqe particular neuro~al
cell types are difficult to isolate from the brain
and maintain in defined culture conditions.
Differentiation of the stem cells into neurons
overcomes this problem and opens new assays to
screen potential growth factors.
4. Assays for Druq Screenin~
As more and more neurotransmitter receptors
and signal transducing proteins are being
identified from the brain, it is becoming clear
that the dogma of one neurotransmitter activating
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one receptor is an oversimplification. Most
receptor complexes in neurons are composed of
protein subunits encoded by several genes and each
gene synthesizes many different variations of the
protein. These variations result in a wide range
of possible receptor combinations, and not a
single receptor, that can interact with a
neurotransmitter. Consequently, a range of signal
output may be produced by a single
neurotransmitter action. The specific signal
effected by a neurotransmitter on a neuron, then,
depends on which receptor complex is produced by
the cell. Thus, cellular diversity must parallel
the molecular diversity and constitute a major
structural element underlying the complexity of
brain function.
Drug discovery by traditional pharmacology
had been performed without the knowledge of such
complexity using whole brain homogenate and
animals, and mostly produced analogs of
neurotransmitters with broad actions and side
effects. The next generation of pharmaceutical
drugs aimed to modify specific brain functions may
be obtained by screening potential chemicals
against neurons displaying a specific profile of
neurotransmitters, receptors complexes, and ion
channel~.
CNS stem cells expanded and differentiated
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into neurons in culture express several
neurotransmitters and receptor complexes. Many
cell lines derived from stem cells and neuronal
progenitors of different regions of the brain can
be developed which, when differentiated into
mature neurons, would display a unique profile of
neurotransmitter receptor complexes. Such
neuronal cell lines will be valuable tools for
designing and screening potential drugs.
In summary, the CNS stem cell technology of
this application offers broad and significant
potentials for treating nervous system disorders.
The following scientific articles have been
cited throughout this application.
1. Turner, D.L. & Cepko, C.L., Nature 328,
131-136 (1987).
2. Gray, G., Glover, J., Majors, J. & Sanes,
J., Proc. Natl. Acad. Sci. USA 85, 7356-7360
(1988).
3. Wetts, R. & Fraser, S., Science 239,
1142-1145 (1988).
4. McConnell, S., Curr. Opin. Neurobiol. 2,
23-27 (1992).
5. Walsh, C. & Cepko, C.L.,-Nature 362,
632-635 (1993).
6. Davis, A.A. & Temple, S., Nature 372,
263-266 (1994).
7. Williams, B. & Price, J., Neuron 14,
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1181-1188 (1995).
8. Cattaneo, E. & McKay, R.D.G., Nature 347,
762-765 (1990).
9. Reynolds, B., Tetzlaff, W. & Weiss, S.,
J. Neurosci. 12, 4565-4574 (1992).
10. Ray, J., Peterson, D., Schinstine, M. &
Gage, F., Proc. Natl. Acad. Sci. USA 90, 3602-3606
(1993).
11. Ghosh, A. & Greenberg, M., Neuron 15,
89-103 (1995).
12. Vicario-Abejon, C., Johe, K., Hazel, T.,
Collazo, D. & McKay, R., Neuron 15, 105-114
(1995)-
13. Frederiksen, K. & McKay, R.D.G., J.
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14. Lendahl, U., Zimmermann, L.B. & McKay,
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While the invention has been described in
connection with what is presently considered to be
the most practical and preferred embodiments, it
is to be understood that the invention is not
limited to the disclosed embodiments, but on the
contrary i5 intended to cover various
modifications and equivalent arrangements included
within the spirit and scope of the appended
claims.
Thus, it is to be understood that variations
in the present invention can be made without
departing from the novel aspects of this invention
as defined in the claims. All patents and
scientific articles cited herein are hereby
incorporated by reference in their entirety and
relied upon.
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TABLE I:
CELL TYPE COMPOSITION OF DIFFERENTIATED CLONES
Clones of Embryonic Hippocampal Precursor Cells
Passaqe Clone Size MAP2+ (~) GalC+ (~) GFAP+ (%)
1 319 145 (45) 41 (13)
1 451 245 ~54) 0 (0)
1 1237 634 (51) 9 (l)
1 2197 956 (44) 42 (2)
1 27791617 (58) 336 (12)
4 71 10 (14)5 (7)
4 139 14 (10)4 (3)
4 296 21 (7)139 (47)
4 341 54 (16)38 (ll)
4 420 39 (9)25 (6)
4 600 35 (6)60 (10)
4 662 66 (10)62 (9)
4 141 42 (30) 4 (3)
4 427 220 (52) 15 (4)
4 610 306 (50) 29 (5)
Total Average:48.6+1.6% 8.4+1.0%7.8+2.3%
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TABLE II:
CELL TYPE COMPOSITION OF DIFFERENTIATED CLONES
Subclones from Clones o~ Embryonic Hippocampal
Precursor Cells
Subclone Clone Size MAP2+ (%) GalC+ (%) GFAP+(~)
HI6.1 337 22 (7) 99 (29)
HI6.2 338 13 (4) 157 (46)
HI6.3 537 132 (25) 48 (9)
HI6.4 565 98 (17) 28 (5)
HI6.5 831 96 (12) 107 (13)
HI6.6 886 158 (18) 134 (15)
HI6.7 893 135 (15) 66 (7)
HI6.8 950 154 (16) 53 (6)
HI6.9 951 112 (12) 120 (13)
HI6.10 970 105 (11) 95 (10)
HI19.1 84 11 (13) 0 (0)
HI19.2 211 45 (21) 0 (0)
HI19.3 363 61 (17) 18 (5)
HI19.4 697 172 (25) 5 (1)
HI19.5 861 135 (16) 57 (7)
HI19.61469 401 (27) 123 (8)
HI19.71841 486 (26) 179 (10)
HI8.1 88 4 (5) 0 (0)
HI8.2 104 3 (3) 0 (0)
HI8.3 193 16 (8) 28 (15)
HI8.4 237 14 (6) 39 (16)
HI8.5 384 65 (17) 119 (31)
HI8.6 402 26 (6)75 (19)
HI8.7 554 49 (9)45 (8)
HI8.8 571 23 (4)49 (9)
HI8.9 662 41 (6)118 (18)
HI8.10 669 46 (7)46 (7)
HI8.11 827 57 (7)18 (2)
HI8.12 836 92 (11) 97 (12)
HI8.131084 104 (10) 53 (5)
HI8.141268 124 (10) 163 (13)
HI8.151284 75 (6) 193 (15)
Total Average: 20.1+1.4%8.9+1.1%10.0+0.7
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TABLE III:
CELL TYPE COMPOSITION OF ~IFFERENTIATED CLONES
Clones of Adult Subependymal Cells
Passaqe Clone Size MAP2+ (%) GalC+ (%) GFAP+ (%)
1 73 6 (8) 37 (51)
1 159 56 (35) 42 (26)
1 173 57 (33) 26 (15)
1 185 71 (38) 32 (17)
1 230 97 (42) 39 (17)
1 273139 (51) 56 (21)
1 387117 (30) 45 (12)
1 554237 (43) 84 (15)
1 675280 (41) 74 (11)
1 847399 (47) 155 (18)
1 496 23 (5)92 (19)
1 526 7 (1)115 (22)
1 644 19 (3)26 (4)
1 713 22 (3)179 (25)
1 1112 56 (5)235 (21)
0 278153 (55) 6 (2)
0 305145 (48) 19 (6)
1 411156 (38) 68 (17)
0 513242 (47) 3 (1)
0 532246 (46) 26 (5)
0 538283 (53) 10 (2)
0 584277 (47) 32 (5)
0 1012498 (49) 5 (0)
Total Average:41.7+2.6% 4.2+1.2% 19.6+2.7%
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TABLE IV: EFFECT OF EXTRACELLULAR FACTORS ON CELL
TYPE DETERMINATION
~Antibody) Untreated +PDGF +CNTF +T3
(~) (%) (%) (~)
A. Embryonic
Neuron (MAP2)45.9 81.0 ~~9 11.5
Neuron (TuJ1)9.9 72.4 N.D. N.D.
Neuron (NF-M)1.0 53.0 N.D. N.D.
Oligodendrocyte (GalC) 7.42.8 4.5 21.2
Astrocyte (GFAP) 6.3 2.097.3 20.7
B. Adult
Neuron (MAP2)36.8 73.911.8 35.2
-Neuron (TuJ1)47.9 72.4N.D. N.D.
Oligodendrocyte (GalC)4.8 N.D. N.D. 47.4
Astrocyte (GFAP) 20.3 2.2 72.9 32.4
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TA~LE V: DIRECTED DIFFERENTIATION OF bFGF-EXPANDED
HUMAN CNS STEM CELLS
(Antibody) Untreated +PDGF+CNTF +T3
~%~ (%) (%) (%)
Neuron
(MAP2+) 45.9+2.3 71.4+1.99.4+1.616.9+2.4
Oligodendrocytes
(04+/Galc+) 2.6+0.8 0.8+0.30.9+0.1 25.3+2.8
Astrocytes
(GFAP+)10.5+1.8 7.1+1.2 85.2+1.9 37.1+3.4
Dead 5.9+0.7 3.5+0.4 2.0+0.5 8.2+0.6
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TABLE VI: NEUROTROPIC FACTORS AND DISEASES
Neurotropic factor: Disease
Nerve growth factor: Alzheimer's Disease
Diabetic neuropathy
Taxol neuropathy
Compressive neuropathy
AIDS-related neuropathy
Brain-derived
growth factor: Amyotrophic lateral sclerosis
Neurotrophin 3: Large f iber neuropathy
Insulin-like
growth factor: Amyotrophic lateral sclerosis
Vincristine neuropathy
Taxol neuropathy
Ciliary neurotrophic
factor: Amyotrophic lateral sclerosis
Glia-derived
neurotrophic
factor: Parkinson's Disease
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~ I o ~
o U~
O O ~ ~
~ ~1 +1 Z :Z Z
1~ _
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~ O ~ ~, O
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ll N
C Z Z ~ 2 Z
''' dP r~ ~
n In ,~
Ei ~ ~ a a ~
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d~ ~O O ,~
O ~~ tD O ~ ~1
Z --+1 +1 +1 ~ ~D
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TABLE VII LEGEND:
'Numbers for different neuronal phenotypes
are given as the percentage of MAP2ab-
positive neurons per square centimeter for
each region. MAP2, microtubule associated
protein a and b; TH, tyrosine hydroxylase;
AchE, acetylcholine esterase; VAT,
vesicular acetylcholine transporter; ChAT,
choline acetyl transferase; GAD, glutamic
acid decarboxylase; NPY, neuropeptide Y;
L-Enk, leu-enkephalin; M-Enk, met-
enkephalin.
2Regions from where CNS stem cells were
derived. SEPT, E15.5 septum; LGE, E15.5
lateral ganglionic eminence; MGE, E15.5
medial ganglionic eminence; HI, E15.5
hippocampus; VM, E12.5 ventral
mesencephalon; DM, E12.5 dorsal
mesencephalon; SPC, E13.5 spinal cord.
3Shown are the average number of MAP2ab-
positve cells per square centimeter.
Initial number of cells plated for all
regions was 125,000 cells per square
centimeter. + standard mean error.
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