U.S. patent application number 12/610071 was filed with the patent office on 2010-02-25 for compositions and methods for isolation, propagation, and differentiation of human stem cells and uses thereof.
This patent application is currently assigned to LEVESQUE BIOSCIENCES, INC.. Invention is credited to Michel Levesque, Toomas Neuman.
Application Number | 20100047911 12/610071 |
Document ID | / |
Family ID | 26977548 |
Filed Date | 2010-02-25 |
United States Patent
Application |
20100047911 |
Kind Code |
A1 |
Neuman; Toomas ; et
al. |
February 25, 2010 |
COMPOSITIONS AND METHODS FOR ISOLATION, PROPAGATION, AND
DIFFERENTIATION OF HUMAN STEM CELLS AND USES THEREOF
Abstract
The invention is directed to the field of human stem cells and
includes methods and compositions for isolating, propagating, and
differentiating human stem cells. The invention provides
therapeutic uses of the methods and compositions, including
autologous transplantation of treated cells into humans for
treatment of Parkinson's and other neuronal disorders.
Inventors: |
Neuman; Toomas; (Santa
Monica, CA) ; Levesque; Michel; (Beverly Hills,
CA) |
Correspondence
Address: |
KNOBBE MARTENS OLSON & BEAR LLP
2040 MAIN STREET, FOURTEENTH FLOOR
IRVINE
CA
92614
US
|
Assignee: |
LEVESQUE BIOSCIENCES, INC.
Beverly Hills
CA
|
Family ID: |
26977548 |
Appl. No.: |
12/610071 |
Filed: |
October 30, 2009 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10216677 |
Aug 8, 2002 |
7632680 |
|
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12610071 |
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60310727 |
Aug 8, 2001 |
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60312714 |
Aug 16, 2001 |
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Current U.S.
Class: |
435/377 ;
435/404 |
Current CPC
Class: |
A61K 35/12 20130101;
C12N 2501/385 20130101; C12N 5/0619 20130101; C12N 2501/40
20130101; C12N 2501/119 20130101; C12N 2501/115 20130101; C12N
2501/01 20130101; C12N 2501/13 20130101; C12N 2501/23 20130101 |
Class at
Publication: |
435/377 ;
435/404 |
International
Class: |
C12N 5/0797 20100101
C12N005/0797; C12N 5/00 20060101 C12N005/00 |
Claims
1-30. (canceled)
31. A cell culture medium for differentiating central nervous
system (CNS) stem cells comprising RA, dBcAMP, FGF8, and GDNF.
32. The cell culture medium of claim 30 comprising 10.sup.-6 M RA,
1 mM dBcAMP, 20 ng/mi FGF8 and 20 ng/ml GDNF.
33. A method of causing central nervous system stem cells to
differentiate in vitro comprising culturing CNS stem cells in the
cell culture medium of claim 30.
34. The cell culture medium of claim 31 comprising about 10.sup.-8
to about 10.sup.-4 M RA, about 0.01 to about 3 mM dBcAMP, about
0.02 to about 200 ng/ml FGF8, and about 0.02 to about 200 ng/ml
GDNF.
Description
RELATED APPLICATIONS
[0001] The present application claims priority under 35 U.S.C.
.sctn.119(e) to U.S. Provisional Application Nos. 60/310,727 filed
Aug. 8, 2001 and 60/312,714 filed Aug. 16, 2001.
FIELD OF THE INVENTION
[0002] The invention is directed to the field of human stem cells
and includes methods and compositions for isolating, propagating,
and differentiating human stem cells. The invention includes
therapeutic uses of the methods and compositions, including
autologous transplantation of treated cells into humans for
treatment of Parkinson's and other diseases.
[0003] BACKGROUND OF THE INVENTION
[0004] Fetal tissue has been transplanted into human patients to
treat Parkinsonism and other neurodegenerative diseases and is a
promising treatment for many other conditions, including
neurotrauma and injuries to the spinal cord. Fetal cells are very
useful because they are multipotent, meaning that they have the
potential to turn into many different kinds of specialized cells,
for example a neuron (a brain cell) or a liver cell. A multipotent
cell is sometimes called a stem cell. A cell that changes from a
multipotent state into a specialized cell is said to differentiate
into a differentiated (i.e., specialized) cell. Once a cell
differentiates into a specialized cell it does not naturally return
to a multipotent state. Thus, any cell that is not yet fully
committed to a particular specialized cell type is referred to
hereinafter as a "stem cell."
[0005] Transplantation of fetal tissue has had limited success. One
technical limitation is that implanted fetal cells do not
necessarily differentiate into the desired cell type. For example,
a fetal cell put into the brain of a Parkinson's patient does not
necessarily become a type of neuron that benefits the patient, such
as a dopaminergic neuron. In addition, significant moral, ethical,
and technological issues make a non-fetal source of cells
desirable.
[0006] Scientists have learned that stem cells exist in mammals at
all stages of development, including the adult stage. Adult stem
cells are more specialized than fetal stem cells but have the
natural potential to become one of a wide variety of cell types.
The stem cell types are commonly named according to the tissue
where they reside: for example, bone marrow stem cells, epidermal
(skin) stem cells, or central nervous system stem cells. Many
hospitals routinely capture bone marrow stem cells from patients
undergoing chemotherapy. The cells are preserved outside of the
body during treatment and subsequently implanted following
treatment.
[0007] A substantial body of literature describes therapies based
on introducing cells into patients. These therapies include
treatments of Alzheimer's disease and Parkinson's disease. Such
therapies are described, for example, in "Survival and
differentiation of adult neuronal progenitor cells transplanted to
the adult brain" by F H. Gage et al., Proceedings of the National
Academy of Science U.S.A. 92:11879-83 (1995); "Site-specific
migration and neuronal differentiation of human neural progenitor
cells after transplantation in the adult rat brain" by R. A.
Fricker et al., Journal of Neuroscience, 19:5990-6005 (1999); and
"Self-repair in the brain" by A. Bjorklund and O. Lindvall, Nature
405:892-3,895 (2000).
Parkinson's Disease and Intracerebral Transplantation
[0008] Parkinson's disease is a neurodegenerative disease
characterized by profound loss of dopaminergic neurons in the
substantia nigra. The loss of dopaminergic neurons in the
substantia nigra results in the degeneration of the nigrostriatal
dopamine system that regulates motor function. This, in turn, leads
to motor dysfunction, consisting of poverty and slowness of
voluntary movements, tremor, stooped posture, rigidity, and gait
disturbance. There has been no cure for Parkinson's disease.
[0009] Modern knowledge of the pathogenesis of Parkinson's disease
indicates that successful functional restoration can be achieved by
replacing the lost dopamine in the damaged area of the brain. This
understanding inspired attempts to replace dopamine by grafting
dopamine producing cells, for example, fetal tissue rich in
dopaminergic neurons, cells from the adrenal medulla, or
dopaminergic neurons from another species, such as pigs, to the
degenerated striatum. The ability of intracerebral grafts to induce
behavioral recovery in brain-damaged recipients rests on the
multitude of trophic, neurohumoral, and synaptic mechanisms that
may allow the implanted tissue to promote host brain function and
repair. To what extent intracerebral implants can be functionally
integrated with the host brain, particularly in man, is still
poorly understood and remains a topic for further clinical
investigation. The chances for extensive integration may be
greatest for very small neural grafts or cell suspensions.
Re-innervation of these small, solid grafts seems to be a function
of the rapid availability of a rich vascular bed or contact with
cerebral spinal fluid. Evidence suggests that the grafts may be
revascularized very quickly, perhaps within hours, especially as
cell suspensions (Leigh et al., 1994). The results of these types
of cell replacement therapies are encouraging, but heterogeneity of
transplanted cells, risks for immunological rejection and other
problems related to the transplantable material have raised
numerous concerns about cell-based therapies.
Fetal Dopaminergic Neurons
[0010] Clinical data clearly demonstrate that fetal mesencephalic
dopamine neurons obtained from a human fetus can survive and
function in the brains of patients with Parkinson's disease.
Unfortunately, functional recovery after transplantation has been
only partial, and both the reproducibility and efficacy of the
procedure must be significantly improved. Nevertheless, early
publications on transplantation of fetal dopaminergic neurons have
demonstrated success. In a study from Great Britain an initial 12
patients had fetal tissue from a single donor placed
stereotactically into the caudate (Hitchcock et al., 1989). The
patients showed improvement within one week and levodopa dosage
(the more traditional therapy) was reduced by 29% within the first
three months and by 24% within the first six months. Follow-up on
nine patients demonstrated a 29% improvement in the Webster rating
scale at three months and a 42% improvement at six months. Other
early reports indicated similar improvement (Freed et al., 1989).
Since the late 1980's, when human fetal tissue transplants began,
it has been estimated that over 500 patients with Parkinson's
disease have received fetal implants. The results in two series of
experiments in the United States have been reported. Freed et al.
have made observations in seven patients followed from 12 to 46
months after mesencephalic fetal transplants (Freed et al., 1992).
Two of these patients had unilateral implants into the caudate and
putamen and five had bilateral implants into the putamen only.
Long-term moderate improvement was reported, and the Sinemet dosage
was substantially reduced. The improvement was related to the
presence or absence of immunosuppressant drugs. In another series
of experiments the improvement appeared to be more mild (Spencer et
al., 1992). These less impressive results may be related to the
cryopreservation of the transplanted fetal tissue and the older age
of the tissue. A major conclusion from these results is that
implantation of fetal dopamine-rich mesencephalic tissue can lead
to a therapeutically valuable, sustained improvement in motor
function in patients with idiopathic Parkinson's disease (see Lang
and Lozano, 1998).
[0011] The main limitations of current fetal cell-transplantation
procedures are the practical, ethical and safety issues related to
the use of fetal tissue. The large number of fetal dopaminergic
neurons that are needed to obtain therapeutic effects in patients
restricts the applications of transplantation procedures to highly
specialized medical centers. Current transplantation techniques
result in survival of 5-20% of the transplanted neurons.
Consequently, cells from 3 to 5 fetuses yield only 100,000-150,000
surviving dopaminergic neurons (Lindvall, 1997). Animal experiments
have demonstrated that inhibition of cell death by caspase
inhibitors, free radical scavengers, and neurotrophic factors may
increase dopamine neuron survival 2 to 3 fold (Sinclair et al.,
1996, Zawada et al., 1998, Schierle et al., 1999). Application of
these additions to human clinical protocols may increase the cell
suvival and reduce the number of fetal cells rquired for efficient
therapeutic effect. The main focus of current research is
developing techniques to improve survival and growth of
transplanted dopaminergic neurons.
Autologous Adrenal Medulla Grafts
[0012] Backlund and his group in Stockholm, Sweden started human
transplants based on experimentation by collaborators in Lund,
Sweden. In their experiments, cell suspensions were
stereotactically placed into the caudate. Although their results
were not spectacular, probably because they implanted relatively
pure suspensions of neurons without associated glial cells, their
experiments opened up the field to other investigators.
Subsequently, Dr. Ignacio Madrazo and Dr. Drucker-Colin described a
series of 54 patients with Parkinson's disease who showed marked
improvement in their disease some months after they had received a
transplant of autologous adrenal medulla to the caudate nucleus of
their brain (Madrazo et al., 1987). Their success seems stem from
changing their protocol so that they implant very small pieces of
adrenal gland (and, more recently, fetal grafts that have open
access to cerebral spinal fluid so that graft viability is
maintained until neovascularization). Following Madrazo's results,
Allen et al., at Vanderbilt University and Jiao et al. in Beijing,
China, reported on multiple patients with severe Parkinson's
disease who had improved after undergoing a technique very similar
to Madrazo's.
CNS Stem Cell Propagations Differentiation and Transplantation
[0013] An alternative approach to treating a neurodegenerative
disease such as Parkinson's disease is to take tissue from a
patient or donor, isolate the central nervous stem cells from the
tissue, cause the stem cells to differentiate into the desired type
of neurons, and implant the neurons into the appropriate region in
the patient's brain. This approach is referred to as autologous
transplantation because the cells are taken from a patient and
implanted into the same patient. The same process could be
applicable to many neuronal diseases and disorders in addition to
Parkinson's disease. For example, the process could be used to
treat spinal cord damage.
[0014] Following removal of an appropriate tissue sample, the
process would involve three steps: isolation, propagation, and
differentiation. In the isolation step, stem cells are preferably
separated from all the other cells in a tissue sample.
Alternatively, the tissue may be placed in a chemical environment
that preferentially facilitates the growth of stem cells. In the
propagation step the stem cells are kept alive and preferably
encouraged to multiply, for example from a few cells into tens of
thousands of cells. In the differentiation step, the cells are
preferably caused to develop into the type of cell that is suitable
for the application. For example, in the case of a Parkinson's
patient, at least a portion of the stem cells are preferably caused
to develop into neurons. Following differentiation, the
differentiated cells may be implanted into the patient. In the case
of a Parkinson's patient, the differentiated cells would preferably
be implanted in the patient's brain. For the treatment of spinal
cord injury, the differentiated cells would be placed in the spinal
cord at or near the site of injury.
[0015] Although such treatments have been contemplated, there
continues to be a need for actual compositions and methods for
isolation, propagation, and differentiation of stem cells for
treating nervous system pathologies.
SUMMARY OF THE INVENTION
[0016] In one aspect, the invention provides compositions and
methods for the isolation, propagation, and differentiation of
cells for treating pathologies. The cells that are propogated
include stem cells. In another aspect, the invention provides
treatments for neurotrauma and neurodegenerative diseases,
including Parkinson's disease. These compositions, methods and
treatments may be used in a wide variety of autologous and
homologous cell therapy applications, including, without
limitation, replacement of lost and defective cells and delivery of
therapeutic products. Other applications include drug design and
drug testing.
[0017] According to certain embodiments, the present invention
provides methods for treating neurodegenerative disorders,
including by not limited to, Alzheimer's disease, Parkinson's
disease, Huntington's chorea, amyotrophic lateral sclerosis and
other motor neuropathies. According to certain other embodiments
the present invention provides methods for treating patients with
nervous system damage, including, but not limited to, central
nervous system ("CNS") trauma (e.g., spinal cord injury) and
strokes.
[0018] In one embodiment, a method is provided for treating a
patient suffering from a central nervous system disorder. Central
nervous system stem cells are obtained and differentiated by
culturing in vitro in differentiation medium. The differentiation
medium preferably comprises FGF8 and GDNF. Following
differentiation the cells are transplanted into the central nervous
system of the patient.
[0019] The central nervous system neurons may be obtained from the
patient or from an unrelated donor. In one embodiment the CNS stem
cells are obtained by surgicaly removing a sample of central
nervous system tissue from the patient, preferably a sample of
cortex from the patient's frontal lobe.
[0020] In one embodiment the differentiation medium additionally
comprises one or more compounds selected from the group consisting
of all-trans retinoic acid (RA) and dibutyryl cyclic AMP (dBcAMP).
In a particular embodiment the differentiation medium comprises
10.sup.-6 RA, 1 mM dBcAMP, 20 ng/ml FGF8 and 20 ng/ml GDNF.
[0021] In another embodiment, the CNS stem cells are proliferated
in vitro prior to differentiation. The CNS stem cells are
preferably proliferated by culturing them in proliferation medium
comprising bFGF, LIF and EGF. In a particular embodiment the
proliferation medium comprises 20 ng/ml bFGF, 20 ng/ml LIF and 20
ng/ml EGF.
[0022] In one embodiment the stem cells are differentiated for
three days prior to transplantation.
[0023] In another embodiment the patient is suffering from
Parkinson's disease. In this case, the differentiated cells are
preferably transplanted into the caudate nucleus in the patient's
brain. In a further embodiment the patient is suffering from an
acute spinal cord injury.
[0024] In a further aspect, the invention provides a method of
treating a patient suffering from Parkinson's disease by obtaining
CNS stem cells from the patient, proliferating the stem cells by
culturing the cells in a first medium comprising bFGF, LIF and EGF,
differentiating the cells by culturing the cells in a second medium
comprising RA, dBcAMP, GDNF and FGF8, and transplanting the
differentiated cells into the patient's brain. In one embodiment
the cells are differentiated for about three days prior to
transplantation. Preferably the cells are transplanted into the
caudate nucleus and/or putamen in the patient's brain. The patient
preferably receives from about 0.5 million to about 80 million
cells, more preferably about 0.5 million to about 10 million cells,
and still more preferably 4 million to about 8 million cells.
[0025] The CNS stem cells are preferably obtained from the cortex
of the patient during craniotomy.
[0026] In one embodiment the first medium comprises about 0.2 to
about 200 ng/ml each of bFGF, LIF and EGF, more preferably about 20
ng/ml of each.
[0027] In another embodiment the second medium preferably comprises
about about 10.sup.-8 to about 10.sup.-4 M RA, about 0.01 to about
3 mM dBcAMP, about 0.02 to about 200 ng/ml FGF8, and about 0.02 to
about 200 ng/ml GDNF, more preferably about 10.sup.-6 M RA, about 1
mM dBcAMP, about 20 ng/ml FGF8 and about 20 ng/ml GDNF.
[0028] A cell culture medium for stimulating the proliferation of
CNS stem cells is provided that comprises basic fibroblast growth
factor (bFGF), leukemia inhibitor factor (LIF), and epidermal
growth factor (EGF), preferably in an amount that is sufficient to
support proliferation of stem cells. The stem cells are preferably
central nervous system stem cells, more preferably human central
nervous system stem cells. In one embodiment the preferred
concentration of bFGF, LIF, and EGF is each from about 0.2 to about
200 ng per ml of the medium. More preferably the concentration of
bFGF, LIF and EGF is each within the range of about 2 to about 200
ng/ml, and yet more preferably about 5 to about 50 ng per ml. Even
more preferably the medium comprises about 20 ng/ml each of bFGF,
LIF and EGF. In one embodiment the medium additionally comprises
B27 (GIBCO) supplement.
[0029] A method of stimulating the proliferation of stem cells,
preferably CNS stem cells, is also provided, comprising culturing
the stem cells in cell culture medium comprising bFGF, LIF and
EGF.
[0030] A cell culture medium for differentiating stem cells,
preferably CNS stem cells, is also provided. The medium preferably
comprises an amount of Fibroblast Growth Factor Eight (FGF-8) and
Glial Cell Line- Derived Neurotrophic Factor (GDNF) that supports
the differentiation of at least a portion of a treated population
of stem cells into a neuronal phenotype. In one embodiment the
medium preferably comprises about 0.02 to about 200 ng FGF-8 and
about 0.02 to about 200 ng GDNF per ml of medium in which the stem
cells are cultured. In a further embodiment, the composition
additionally comprises all-trans retinoic acid (RA) and/or
dibutyryl cyclic AMP (dBcAMP). In a particular embodiment RA is
preferably present in the medium at a concentration of about
10.sup.-8 to about 10.sup.-4 M. In another embodiment dBcAMP is
preferably present in the medium at a concentration of about 0.01
to about 3 mM. More preferably the culture medium comprises about
10.sup.-6 RA, about 1 mM dBcAMP, about 20 ng/ml FGF8 and about 20
ng/ml GDNF.
[0031] A method is also provided for stimulating the
differentiation of stem cells, preferably CNS stem cells,
comprising culturing the stem cells in vitro in culture medium
comprising RA, dBcAMP, FGF8 and GDNF.
[0032] In one aspect, the invention includes methods of treating
brain cells, including stem cells, taken from a patient suffering
from a neurodegenerative disease. In one embodiment the patient is
human. The methods are preferably performed on stem cells that have
been removed from a patient and are being cultured in vitro in a
medium, but prior to the reimplantation of the cells into the
patient's central nervous system. In one embodiment, stem cells
that have been removed from a patient are cultured in a composition
that stimulates stem cell propagation. The composition preferably
comprises basic fibroblast growth factor (bFGF), leukemia inhibitor
factor (LIF), and epidermal growth factor (EGF). In one embodiment
the preferable concentration of bFGF, LIF, and EGF is about 0.2 to
about 200 ng each per ml of cell culture medium, more preferably
about 2 to about 200 ng per ml and even more preferably about 5 to
about 50 ng per ml.
[0033] In another embodiment the stem cells are cultured in a
composition that stimulates stem cell differentiation. The
composition preferably comprises an amount of Fibroblast Growth
Factor Eight (FGF-8) and Glial Cell Line- Derived Neurotrophic
Factor (GDNF) that supports the differentiation of at least a
portion of a treated population of stem cells into a neuronal
phenotype. In one embodiment the composition preferably comprises
about 0.02 to about 2000 ng FGF-8 and about 0.02 to about 2000 ng
GDNF per ml of medium in which the stem cells are cultured. In a
further embodiment, the composition preferably additionally
comprises all-trans retinoic acid (RA) and/or dibutyryl cyclic AMP
(dBcAMP). In a particular embodiment RA is preferably present in
the medium at a concentration of about 10.sup.-8 to 10.sup.-4 M. In
another embodiment dBcAMP is preferably present in the medium at a
concentration of about 0.01 to about 3 mM.
[0034] In yet another aspect, the invention includes methods for
treating a patient suffering from a neurodegenerative disease by
culturing stem cells taken from the patient in one or more
compositions that support the proliferation and/or differentiation
of the stem cells. The cells are then transplanted back into the
patient in a location chosen to maximize the therapeutic benefit.
In one aspect a patient suffering from Parkinson's disease is
treated. Stem cells are obtained, preferably central nervous system
stem cells, and at least a portion of the cells are caused to
differentiate into dopaminergic neurons, The differentiated cells
are implanted into the brain of a patient. In another embodiment, a
patient suffering from spinal cord trauma is treated. In this
embodiment, stem cells are obtained and at least a portion of the
stem cells are caused to differentiate into GABAergic cells. The
differentiated cells are implanted into the spinal cord of the
patient. In another embodiment, neurons are implanted into the
central nervous system of a patient at or near a pattern-generation
portion of the central nervous system. Preferably, stem cells are
obtained from the patient to be treated. Alternatively, they may be
obtained from a related or unrelated donor. Compositions described
above may be used to cause the stem cell to propagate and
differentiate into GABAergic and/or dopaminergic neurons.
[0035] In a further aspect, the invention includes methods for
causing stem cells to differentiate into one or more of a variety
of neuronal cell types, including cholinergic, dopaminergic, and
glutamatergic neurons.
[0036] Differentiation of the stem cells in vivo is not required in
the methods of the present invention. However, in some embodiments
further differentiation may occur in vivo. The methods of the
invention allow control of the numbers and types of cells while the
cells are in vitro. In some embodiments the methods of the
invention are used with stem cells taken from a patient into whom
the cells will be transplanted back following in vitro propagation
and/or differentiation (autograft). In other embodiments the stem
cells for use in the methods are obtained from a person other than
the patient to be treated, as in a homologous graft. Or the
techniques could be used for treating cells derived from an animal
source and the resulting cells could be transplanted into humans
(xenograft).
[0037] The invention is described in terms of certain embodiments
set forth herein; these embodiments are not meant to limit the
scope or spirit of the invention. Other variations of the
embodiments described herein will be apparent to those skilled in
these arts.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT
[0038] The treatment of human neurodegenerative diseases and
neurotrauma by cell replacement or transplantation requires a
reliable source of neuronal cells. CNS stem cells are preferable
because of the ability to propagate and differentiate the cells in
vitro. IN addition, the use of CNS stem cells overcomes many of the
technical and ethical problems associated with the use of fetal
tissue.
[0039] Methods for isolating CNS stem cells, propagating and
differentiating them in culture and using them to treat neuronal
disorders are described herein.
[0040] It has been demonstrated that the entire ventricular
neuroaxis including spinal cord of the adult mammalian central
nervous system (CNS) contains multipotent stem cells ("CNS stem
cells" or "neuronal stem cells"; Morshead and van der Kooy, 1992,
Reynolds and Weiss, 1992, Lois and Alvarez-Buylla, 1993, 1994,
Morshead et al., 1994, Weiss et al., 1996a, b). The isolation of
putative stem cells form the embryonic (Davis and Temple, 1994) and
adult rodent CNS (Reynolds and Weiss, 1992, Lois and Alvarez
Buylla, 1993, Gritti, et al., 1996, Suhonen et al., 1996) has been
accomplished by means of growth factor stimulation.
[0041] Multipotent stem cells are localized in the proliferative
zones of the developing and adult nervous system. However, in
rodents, CNS stem cells reside in a variety of locations in the
nervous system (Palmer et al., 1999) including areas where there is
no detectable neurogenesis, such as in the optic nerve and spinal
cord. Fetal stem cells have been isolated form several areas
including spinal cord (Vescovi et al., 1999, Carpenter, et al.,
1999), while adult stem cells have been isolated from hippocampus,
subventricular zone, cortex and neocortex (Kukekov et al., 1999;
unpublished data). Neuronal stem cells are localized, for example,
in the ependymal zone of the brain and spinal cord. Continuous
neurogenesis also occurs in the dentate gyrus of the hippocampus
(Altman and Das, 1965, Bayer et al., 1982, Cameron et al., 1993).
Palmer et al (1995) demonstrated that basic fibroblast growth
factor supports proliferation of hippocampus neuronal stem cells in
vitro.
[0042] Regional and temporal differences in the expression of
regulatory genes in the proliferative compartment suggest the
presence of different stem cell populations. Differences in fetal
and adult CNS stem cell populations have been identified by
characterizing the developmental potential of neurospheres derived
from naturally aborted fetal and adult human brain. Analysis of the
expression patterns of regulatory genes known to be important for
neuronal differentiation (maturing) shows that fetal and adult
human CNS stem cell isolates display similar proliferation
kinetics, differentiate into the three major cell types of the
nervous system, and express similar sets of regulatory genes (Palm
et al., 2000). However, each of the individual CNS stem cell
isolates could be distinguished by its specific gene expression and
developmental potential.
[0043] Nervous system stem cells have been isolated from human
embryonic and adult brain (Svendsen et al., 1997, Chalmers-Redman
et al., 1997, Vescovi et al., 1999, Kukekov et al., 1999). These
cells survive transplantation into the adult rodent brain, and
differentiate into neurons and glia with no subsequent tumor
formation (Svendsen et al., 1997, Vescovi et al., 1998, Fricker, et
al., 1999). Further, neuronal stem cells have an extended
self-renewal capacity and possess the potential to give rise to all
three major brain cell types. Human CNS stem cell lines display
remarkable functional stability, as their growth characteristics,
dependence of growth factors, and potential for neuronal
differentiation remain unchanged over extensive subculturing
(Vescovi et al., 1999, Fricker et al., 1999, Carpenter et al.,
1999, our unpublished data). In addition, the expression of late
neuronal antigens such as neurofilament proteins and the
acquisition of distinct neuronal electrophysiological properties
show that even the progeny of long-term cultured stem cells can
accomplish full neuronal maturation. As a result, virtually
unlimited numbers of neuronal cells could be generated under
standardized conditions. Several laboratories have cultured human
stem cells in vitro without detectable changes in growth
characteristics and differentiation potential for prolonged
periods, up to several years (Capenter et al., 1999; Vescovi et
al., 1999; unpublished data).
[0044] Stem cells also provide the unprecedented opportunity to
control some critical parameters in clinical transplantation.
Neuronal stem cells differentiate spontaneously into neurons,
astrocytes, and oligodendrocytes after plating onto substrates
which stimulate adhesion and differentiation, for example
poly-L-ornithine or laminin. In addition, these multipotent CNS
stem cells proliferate and expand in response to epidermal growth
factor ("EGF") and basic fibroblast growth factor ("bFGF") and
differentiate into neurons, astrocytes and oligodendrocytes
(Reynolds and Weiss, 1992, Morshead et al., 1994, Weiss et al.,
1996b ). Human CNS stem cells continue to proliferate in vitro in
the presence of bFGF and EGF (Svendsen et al., 1997; Kukekov et al.
1999) or LIF (Carpenteretal. 1999; Fricker et al., 1999).
[0045] In view of the advantages described above, multipotent CNS
stem cells and their derivatives represent an ideal cell population
for cell based therapies. However, in order for stem cells to form
useful connections with host neurons following transplantation, the
stem cells must differentiate into a useful neuronal type for the
particular situation. Thus the phenotype (neurotransmitter profile,
neurotransmitter and other membrane receptors, second messenger
systems) of new neurons generated from the differentiation of stem
cells preferably corresponds to the phenotype of neurons at the
site of injury. Cell morphology and expression of neuronal cell
type specific antigens may be analyzed to evaluate neuronal
differentiation. For example, antibodies to nestin may be used as
markers of stem cell phenotypes. Expression of neurofilament L and
M, neuronal specific enolase, and GAP-43 may be used as general
neuronal markers.
[0046] Progress in transplantation experiments suggests that CNS
stem cells can be successfully used as a neuronal replacement after
CNS injury. In the ideal situation the transplanted stem cells will
differentiate into the types of neurons which will make correct
connections (synapses) and serve as a substrate for descending and
ascending long fiber tract neurons. Unfortunately, stem cells
differentiate mostly into astrocytes after transplantation.
Additionally, after transplantation, neuronal stem cells
differentiate into neurons in the areas of the nervous system where
neurogenesis occurs in the adults, such as dentate gyrus of the
hippocampus and olfactory bulb, but not in other regions. These
data indicate that signals that are necessary for neuronal
differentiation are missing in most parts of the adult nervous
system, including the spinal cord. Thus, in order to successfully
use neuronal stem cells in transplantation therapeutically it will
be important to initiate neuronal differentiation of stem cells
before transplantation. Methods are provided herein for propagating
stem cells in vitro, causing stem cells to differentiate into
particular types of neurons and methods for treating patients
suffering from CNS injury or disease or disorder by transplantation
of differentiated stem cells.
[0047] The present invention is based, in part, on the experimental
finding that adult human brain contains neuronal stem cells that
can be isolated, propagated in vitro and differentiated into
dopamine secreting neurons. The dopamine producing neurons survive
transplantation into animal and human striatum and are
therapeutically beneficial in the treatment of Parkinson's disease.
By manipulating the environment during differentiation, stem cells
can be caused to differentiate into other neuronal types, such as
GABAergic neurons.
[0048] In one aspect the invention provides methods of treating
cell samples that include stem cells, preferably taken from patient
suffering from a neurodegenerative disease. The methods are
preferably performed while the cells are cultured in vitro in a
medium and prior to the reimplantation of the cells into the
patient's central nervous system for therapy of the
neurodegenerative disease. The methods may include the step of
culturing the stem cells in a composition comprising an amount of
basic fibroblast growth factor (bFGF), leukemia inhibitor factor
(LIF), and epidermal growth factor (EGF) that is sufficient to
support proliferation of the stem cells in vitro. In addition,
prior to transplantation the stem cells are preferably cultured in
a medium comprising an amount of Fibroblast Growth Factor Eight
(FGF-8) and Glia Derived Neurotrophic Factor (GDNF) that is
sufficient to support differentiation of at least a portion of a
group of the stem cells into a neuronal phenotype. The medium
preferably further includes RA and dBcAMP. In a preferred
embodiment the methods are used to produce dopaminergic cells that
are implanted into the brain of a patient suffering from
Parkinson's disease. In another embodiment the methods are used to
make GABAergic neurons that are implanted into the brain or spinal
cord of a patient. The preferred implantation site is the pattern
generator.
[0049] In one embodiment of the present invention, human CNS cells
that include neuronal stem cells are differentiated in vitro,
resulting in the production of dopaminergic neuronal cells that can
be used to replace lost dopaminergic neurons in the brain of a
patient suffering from Parkinson's disease. Stem cells are
preferably isolated from the patient's own brain (autologous
transplantation). Alternatively, stem cells may be isolated from a
related donor, an unrelated donor of the same species, or a donor
from a different species. Stem cells are preferably initially
propagated in vitro in the presence of growth factors. Prior to
transplantation, cells are transformed into dopamine producing
cells, as well as other cells.
[0050] A purified culture of treated cells may be prepared for
administration to a patient suffering from Parkinson's disease. The
preparations preferably contain approximately 0.5 to 80 million
cells and more preferably contain 0.5 to 20 million cells, and
still more preferably 4-10 million cells. A preparation preferably
contains dopamine secreting neurons representing about 5 to about
30% of the neuronal cells in the preparation. Methods are included
for screening the cells for differentiation (cell type and
activity), presence of adventitious agents, purity, sterility,
mycoplasma, and endotoxins. Testing for differentiation is
preferably performed about four months after initiation of stem
cell culture.
A. Definitions
[0051] The terms "CNS stem cell" and "neuronal stem cell" refer to
multipotent cells obtained from the central nervous system that can
be caused to differentiate into cells that posses one or more
biological activities of a neuronal cell type.
[0052] As used herein, "treatment" is an approach for obtaining
beneficial or desired clinical results. For purposes of this
invention, beneficial or desired clinical results include, but are
not limited to, alleviation of symptoms, diminishment of extent of
disease, stabilized (i.e., not worsening) state of disease, delay
or slowing of disease progression, amelioration or palliation of
the disease state, and remission (whether partial or total),
whether detectable or undetectable. "Treatment" can also mean
prolonging survival as compared to expected survival if not
receiving treatment. "Treatment" is an intervention performed with
the intention of preventing the development of a disorder or
altering the pathology of a disorder. Accordingly, "treatment"
refers to both therapeutic treatment and prophylactic or
preventative measures. Specifically, the treatment may directly
prevent, slow down or otherwise decrease the pathology of cellular
degeneration or damage, such as the pathology of nerve cells, or
may render the cells, e.g. neurons, more susceptible to treatment
by therapeutic agents. In a preferred embodiment, the treatment
reduces or slows down the decline and/or restores function.
[0053] The term "patient" refers to those in need of treatment,
including those already with a disorder as well as those in which a
disorder is to be prevented.
[0054] The "pathology" of a (chronic) neurodegenerative disease or
acute nervous system injury includes all phenomena that affect the
well being of the patient including, without limitation, neuronal
dysfunction, degeneration, injury and/or death.
[0055] The terms "CNS disease" and "CNS disorder" are used in the
broadest sense and include any condition that is associated with
the central nervous system and would benefit from treatment of the
present invention. This includes chronic and acute disorders or
diseases including those pathological conditions which predispose
the mammal to the disorder in question. A preferred disorder to be
treated in accordance with the present invention is Parkinson's
disease. The terms include, without limitation, neurodegenerative
disorders as well as trauma or acute injury.
[0056] The terms "neurodegenerative disease" and "neurodegenerative
disorder" are used in the broadest sense to include all disorders
the pathology of which involves neuronal degeneration and/or
dysfunction, including various conditions involving spinal muscular
atrophy or paralysis; and other human neurodegenerative diseases.
Neurodegenerative diseases include, without limitation, Alzheimer's
disease and Parkinson's disease. In a preferred embodiment, the
neurodegenerative disease that is treated by the methods disclosed
herein is Parkinson's disease.
[0057] "Mammal" for purpose of treatment refers to any animal
classified as a mammal, including humans, domestic and farm
animals, and zoo, sport or pet animals, such as dogs, horses,
sheep, cats, cows, etc. Preferably, the mammal is human.
[0058] "Dopaminergic neuron," is used broadly herein and refers to
any cell that contains the neurotransmitter dopamine. Similarly,
"GABAergic neuron" as used herein, refers to any cells that contain
the neurotransmitter GABA. Expression of enzymes involved in the
synthesis or degradation of the different neurotransmitters is
preferably analyzed to determine specificity of neuronal
differentiation. For example, dopaminergic neurons may be
identified by positive staining for tyrosine hydroxylase (TH) or
dopa decarboxylase (DDC).
Propagation of Cells In Vitro
[0059] Stem cells for use in the methods disclosed herein are not
limited in any way and may be obtained from any source. Preferably
the stem cells are neuronal stem cells that are obtained from a
mammalian source. For example, they may be obtained from the
central nervous system of an adult, juvenile or fetal mammal,
preferably a human. Alternatively, the stem cells may be obtained
from an available in vitro culture of neuronal stem cells.
Regardless of the technique used, both stem cells and fully
differentiated cells are obtained.
[0060] In one embodiment a tissue sample is obtained from the
central nervous system of a mammal. The sample may be obtained by
any method known in the art, such as by biopsy or by a surgical
procedure. Such methods for obtaining tissue samples are well known
in the art. The sample is preferably obtained from a part of the
central nervous system that is known to comprise stem cells.
[0061] In a preferred embodiment, a small tissue sample is obtained
from a donor during a craniotomy procedure. The sample may be, for
example, a sample of cortex. Preferably, the donor is the patient
that will receive an autologous transplant of differentiated
cells.
[0062] In a particular embodiment, a stereotactic biopsy of the
temporal periventricular ependyma following sterotactic MRI of the
brain is performed under local anesthesia. The selected temporal
region is preferably of the non-dominant hemisphere. The
periventricular ependyma is preferably reached from an orthogonal
approach to the anterior temporal lobe
[0063] Optionally, the tissue sample is cut into small pieces,
preferably from about 0.1 to about 10 mm.sup.3, more preferably
from about 0.5 to about 1 mm.sup.3.
[0064] The tissue sample is treated to dissociate individual cells
by any method known in the art. In one embodiment the tissue sample
is transferred into trypsin solution that preferably comprises
about 0.02 mg trypsin/ml in Verseen (Gibco). The tissue is
incubated at 37.degree. C. for approximately 10-20 minutes, after
which trypsin inhibitor is added.
[0065] The sample is triturated mechanically, such as with a
Pasteur pipette and the cell suspension is centrifuged, the pellet
washed with culture media and the cells are plated.
[0066] Preferably the cells are plated at a density of about 5,000
to about 10,000 viable cells/ml culture media in tissue culture
dishes, such as 6 well Nunc dishes.
[0067] The cells are cultured in culture media that comprises
growth factors that stimulate proliferation. The culture media
comprises one or more growth factors selected from the group
consisting of basic fibroblast growth factor (bFGF), leukemia
inhibitor factor (LIF), and epidermal growth factor (EGF).
Preferably the culture medium comprises amounts of bFGF, LIF and
EGF that are sufficient to support proliferation of the stem cells.
More preferably the culture media comprises bFGF, LIF, and EGF at a
concentration of about 0.2 to about 200 ng each per ml of the
medium, even more preferably at a concentration of from about 2 to
about 200 ng each per ml of the medium; and yet more preferably at
a concentration of from about 5 to about 50 ng each per ml of the
medium.
[0068] In one embodiment the medium comprises DMEM/F-12, 20 ng/ml
bFGF and 20 ng/ml LIF. In another embodiment the medium
additionally comprises 20 ng/ml EGF. In a further embodiment the
medium comprises B27 supplement (Gibco).
[0069] The cells are preferably grown as neurospheres. They may be
propagated in vitro in the culture medium for any length of time.
Preferably the media is changed about every third day, and
neurospheres are dissociated by mechanical trituration every 10 to
15 days.
[0070] Optionally the cells may be passaged at high density (about
10.sup.4 to about 5.times.10.sup.4 cells/mm.sup.2) every 10 to 15
days.
[0071] When desired, the cells may be used in transplantation
therapy. Preferably, however, the cells are first differentiated,
as described below.
Differentiation of CNS Stem Cells into Neurons In Vitro
[0072] One of the greatest advantages of using stem cells for
transplantation is that cell differentiation can be initiated when
desired and fine-tuned by modifying the growth conditions. A very
small portion of human stem cells naturally differentiate into
dopaminergic neurons (tyrosine hydroxylase (TH)--positive) upon
withdrawal of growth factors and initiation of differentiation.
Svendson (1997) reported that less than 0.01% of total cells become
TH positive in vitro. Treatment of stem cells with a combination of
IL-1b, IL-11, and GDNF induces formation of more neurons in cell
culture (Carpenter et al., 1999).
[0073] Development and survival of substantia nigra dopaminergic
neurons depends on the expression of orphan nuclear hormone
receptor Nurr1 in these cells and over-expression of Nurr1 in stem
cells stimulates dopaminergic differentiation when these cells are
exposed to glial condition media (Wagner et al., 1999). Also other
laboratories have reported effect of glial conditioned media on
differentiation of dopaminergic neurons (Daadi and Weiss, 1999).
Analysis of development of mesencephalic neurons during
embryogenesis has revealed that sonic hedgehog (SHH) and FGF8 are
essential for development of mesencephalic dopaminergic neurons (Ye
et al., 1998). Unfortunately, treatment of stem cells with SHH and
FGF8 does not increase the number of TH positive cells in CNS stem
cell cultures. There are reports (Studer et al., 1998) that
expansion of precursor cells in the presence of bFGF results in
significant differentiation of dopaminergic neurons. Large clusters
of differentiated neurons contained 18% dopaminergic neurons.
[0074] Treatment of human CNS stem cells growing as neurospheres
with all-trans retinoic acid, dibutyryl cAMP, FGF8, and GDNF was
found to cause differentiation of neuronal stem cells into
dopaminergic neurons. Without treatment, less than 1% of
differentiated cells were TH positive while after treatment about
5-30% of differentiated cells were TH positive.
[0075] Neuronal stem cells are caused to differentiate by culturing
the cells in a differentiation media that comprises one or more
factors that stimulates differentiation. Cells are preferably
plated on poly-ornithine or laminin coated tissue culture plates
and contacted with the differentiation media. Cells are preferably
dissociated into smaller aggregates of about 50 to about 200 cells
prior to plating by mechanical trituration. Approximately
2.times.106 cells are plated, for example, on 100 mm tissue culture
plates.
[0076] Preferably the media comprises one or more factors selected
from the group consisting of fibroblast growth factor 8 (FGF8),
glia derived neurotrophic factor (GDNF); all-trans retinoic acid
(RA) and diburyry cyclic AMP (dBcAMP). The cells are preferably
cultured in the differentiation media for approximately 1 to 6
days, more preferably for about 3 days, prior to being used for
transplantation, as described below.
[0077] In one embodiment the neuronal stem cells are cultured in
media comprising an amount of Fibroblast Growth Factor 8 (FGF-8)
and/or Glia Derived Neurotrophic Factor (GDNF) that is sufficient
to support differentiation of at least a portion of a group of the
stem cells into a neuronal phenotype. In one embodiment the
preferred concentration of FGF-8 and GDNF in the medium is about
0.02 to about 200 ng each per ml of medium.
[0078] In another embodiment the neuronal stem cells are incubated
in media that comprises all-trans retinoic acid (RA) and dibutyryl
cyclic AMP (dBcAMP). The preferred concentration of RA in the
medium is about 10.sup.-8 to about 10.sup.-4 M, more preferably
about 10.sup.-6 M to about 10.sup.-4 M, and the preferred
concentration of dBcAMP in the medium is about 0.01 to about 3 mM,
more preferably about 1 mM.
[0079] In a further embodiment, the neuronal stem cells are
incubated in media that comprises RA, dBcAMP, FGF8 and GDNF.
Preferably, the concentration of RA is about 10-6 M, the
concentration of dBcAMP is about 1 mM, the concentration of FGF8 is
about 20 ng/ml and the concentration of GDNF is about 20 ng/ml.
[0080] The media is preferably F12/DMEM serum free medium
supplemented with B27 growth supplement (Gibco) as well as the
desired differentiation factors as described above.
[0081] Following differentiation, cells are preferably collected by
mild trypsinization (about 0.01% trypsin in Verseen), washed twice
with D-PBS and resuspended in a small volume of D-PBS. In a typical
experiment, differentiation for three days yields about 0.7 to
about 2 million dopaminergic cells. However, a much wider range of
dopaminergic cells can be obtained depending on the volume and
source of the original cells.
[0082] For the treatment of Parkinson's disease, a culture having
5% to 30% of the neurons as dopaminergic neurons is preferably used
for transplantation.
Transplantation of Human CNS Stem Cells
[0083] Following proliferation and differentiation, neuronal stem
cells are transplanted into a patient. Preferably the patient will
receive from about 0.5 million to about 80 million cells, more
preferably about 0.5 million to about 10 million cells, and still
more preferably 4 million to about 8 million cells. In a preferred
embodiment, this corresponds to about 25,000 to about 24,000,000
dopaminergic neurons, more preferably about 25,000 to about
3,000,000 dopaminergic neurons, and still more preferably 200,000
to about 2,400,00 dopaminergic neurons. Prior to transplantation,
cells are preferably washed at least 3 times with PBS to reduce the
level of growth factors from the differentiation media.
[0084] In the preferred embodiment, neuronal stem cells are
investigated to determine the proportion of the type of neuron of
interest prior to transplantation.
[0085] The patients will have the cells implanted following
standard surgical procedures. The cells may be implanted anywhere
in the central nervous system. The exact location will depend upon
the type of neuronal disorder from which the patient is suffering,
as well as the patients particular pathology. One of skill in the
art can determine the location of the transplant and optimize the
transplantation procedure.
[0086] In one embodiment a patient suffering from Parkinson's
disease is transplanted with cells differentiated as described
above. The patient will have the surgery performed under local
anesthesia in an operating room. The differentiated cells are
implanted in the caudate nucleus and putamen, preferably
bilaterally using stereotactic techniques. Preferably the
implantation is MRI-guided.
[0087] In a preferred embodiment the stereotactic frame is fixed to
the patients skull following administration of local anesthesia.
The caudate nucleus and putamen are visualized with MRI.
Thereafter, ten passes with very thin sterotactic needles are made
about 4 mm apart in the caudate and putamen. Four trajectories for
needle tracks in the caudate and six tracks in the putamen are
calculated to avoid the posterior limb of the internal capsule. The
entry points for the caudate and putamen are preferably at two
different sites on the surface.
[0088] Within each track, a single cell suspension of 50 .mu.l,
averaging about 2 million cells, is slowly delivered, preferably
over the course of about 10 minutes.
[0089] In transplantation experiments, human stem cell progeny
showed an engraftment efficiency comparable to that of fetal tissue
in rat brain (Vescovi et al., 1999, Fricker et al., 1999, our
unpublished data). Transplantation of untreated CNS stem cells into
6-OH lesioned rat striatum revealed that a small number of
dopaminergic neurons develops (Svendsen et al., 1997). They
transplanted stem cells, giving less than 0.01% TH positive neurons
in vitro, into lesioned brain and observed significant
physiological effect in 2 animals using rotation test. The brains
of these two animals contained TH positive neurons in the striatum.
These experiments suggest that human stem cells have the potential
to differentiate into dopaminergic neurons inside the striatum. We
also could argue that transplantation of undifferentiated stem
cells into Parkinson's disease patients would result in development
of dopaminergic neurons and reduction of symptoms. However,
transplantation of committed or differentiated dopaminergic neurons
will likely have significantly better results.
Articles of Manufacture
[0090] In another aspect the invention contemplates an article of
manufacture containing materials useful for the collection,
propagation, differentiation and transplantation of human CNS stem
cells. The article of manufacture comprises one or more containers
and a label or package insert on or associated with the container.
Suitable containers include, for example, boxes, bottles, vials,
syringes etc. The containers may be formed from a variety of
materials such as glass and plastic. The containers may hold one or
more compounds selected from the group consisting of bFGF, LIF,
EGF, FGF8, GDNF, RA and dBcAMP. The containers may also hold other
components necessary for the isolation, propagation,
differentiation and transplantation of neuronal stem cells. The
label or package insert indicates how to use the one or more
compounds to cause the stem cells to proliferate and differentiate
in vitro into the desired type of neuronal cell. Optionally, the
label may also indicate how to use the components of the article of
manufacture to obtain stem cells and/or to transplant
differentiated cells into a patient.
[0091] The foregoing written specification is considered to be
sufficient to enable one skilled in the art to practice the
invention. Various modifications of the invention, in addition to
those shown and described herein, will become apparent to those
skilled in the art from the foregoing description and fall within
the scope of the appended claims.
[0092] It is understood that the application of the teachings of
the present invention to a specific problem or situation will be
within the capabilities of one having ordinary skill in the art in
light of the teachings contained herein.
[0093] Further details of the invention are illustrated by the
following non-limiting Examples. The disclosures of all citations
in the specification are expressly incorporated herein by
reference.
EXAMPLE 1
Isolation and Propagation of Adult Multipotent CNS Stem Cells
[0094] Neuronal stem cells were isolated from the brains of 14
adult human patients. Two isolates were obtained from prefrontal
cortex, twelve isolates from hippocampus and two isolates from the
subventricular zone. A detailed analysis of isolated cells was
undertaken for three isolates and compared to fetal stem cells that
were isolated from 21 week fetal brain.
[0095] CNS stem cells were isolated from the adult hippocampus of
three patients with intractable epilepsy and from the 21 week old
fetal brain, as described below. Specimens were obtained in
accordance with IRB protocols obtained through the Cedars Sinai
Medical Center, Los Angeles, Calif. (IRB protocol #2475 and
2565).
[0096] Isolated stem cells from the adult CNS were cultured as
described below and proliferated in vitro as neurospheres
expressing nestin and musashi-1 mRNAs, markers that are
characteristic of neural stem cells (Lendahl et al., 1990,
Sakakibara et al. 1996). Growth characteristics of hippocampal and
cortical adult CNS stem cells were similar, with a doubling time of
approximately 4 days for both types of neurons. Cells were grown in
the presence of basic FGF, EGF and LIF (see below), which are
growth factors for neuronal stem cells. On average, variability in
growth rates was less than 15%.
[0097] To examine developmental potential of adult CNS stem cells,
the expression of the cell-type specific markers .beta.
III-tubulin, glial fibrillary acidic protein (GFAP), and
galactocerebroside (GalC) were analyzed upon differentiation of
fetal and adult neurospheres, as described below. The
differentiated cells comprised 20-50% astrocytes (GFAP+), 5-30%
neurons (.beta.-III-tubulin+) and 1-5% oligodendrocytes (GalC+).
Significant variances in the developmental potential of stem cell
clones from fetal and adult CNS were not detected. However, the
ratio of neurons to astrocytes and oligodendrocytes varied notably
from one stem cell isolate to another (Table 1). The percentage of
neurons, astrocytes and oligodendrocytes was evaluated. Data
presented as mean %.+-.SEM.
TABLE-US-00001 TABLE 1 Quantitation of differentiation of
individual fetal (F1, F2) and adult (AD1, AD2, AD3) CNS stem cell
isolates into neurons, astrocytes and oligodendrocytes. The
percentage of neurons, astrocytes and oligodendrocytes was
evaluated. Data presented as mean % .+-. SEM. Stem Cell Isolates F1
F2 AD1 AD2 AD3 Neurons (.beta.III-tubulin) 5 .+-. 2 23 .+-. 5 11
.+-. 2 30 .+-. 6 19 .+-. 5 Astrocytes (GFAP) 50 .+-. 9 29 .+-. 5 46
.+-. 7 49 .+-. 8 20 .+-. 7 Oligodendrocytes (GalC) 5 .+-. 2 1 .+-.
1 1 .+-. 1 3 .+-. 2 1 .+-. 1
Isolation of CNS Stem Cells
[0098] Resected human brain tissue was placed into ice-cold
DMEM/F-12 (GIBCO) containing penicillin-streptomycin, for further
dissection. The tissue was cut into small pieces and trypsinized
(0.02 mg/ml trypsin in Verseen (GIBCO)) at 37.degree. C. for 5
minutes. After adding trypsin inhibitor mixture (Clonetics), the
tissue was mechanically triturated. Cell suspension was centrifuged
at 400 rpm for 5 minutes, the pellet was washed once with
DMEM/F-12. Cells were plated at density of 5000-10,000 viable
cells/ml in media composed of DMEM/F-12, B27 supplement (GIBCO) and
growth factors bGFG (20 ng/ml; Peprotec), EGF (20 ng/ml; Peprotec),
LIF (20 ng/ml, Peprotec) and penicillin/streptomycin (GIBCO). Stem
cells were grown as neurospheres and the media was changed every
three days. The neurospheres were dissociated by mechanical
trituration every 12-15 days. All further analyses were performed
on stem cell isolates with comparable passage number. In cultures
which were subsequently used for transplantation into the striatum
of Parkinson's patients, antibiotics were omitted 30 days before
transplantation.
Counting Cells
[0099] Every five days neurospheres were dissociated using trypsin
and single cells were counted using hemocytometer after trypan blue
straining to identify living cells.
Differentiation of Cells and Immunostaining
[0100] Differentiation of neurospheres was initiated by plating
cells onto laminin coated tissue culture plates in growth media
containing all-trans retinoic acid (RA; 10.sup.-6 M) and dibutyryl
cyclic AMP (dBcAMP; 1 mM). Cells were fixed with 4%
paraformaldehyde in PBS seven days after plating and immunostained.
Antibody against type III .beta.-tubulin (1:500, Sigma) was used to
detect neurons, anti-GFAP (1:500, DAKO) to detect astrocytes and
anti-GalC (1:25, Rosche) to detect oligodendrocytes. The secondary
antibodies that were used were goat anti-mouse FITC (1:200, Sigma)
and goat anti-rabbit rhodamine (1:200, Boehringer). Immunostained
cells were counted in five separate, randomly chosen fields in each
culture using 20.times. objective. Total cell number was counted
using DAPI (Molecular Probes) stained nuclei. The numbers were
summed and percentages were calculated.
Conclusion
[0101] It was found that adult human CNS contains multipotent stem
cells that continue to proliferate in vitro in the presence of
appropriate growth factors (bFGF, LIF and EGF) and can be caused to
differentiate into neurons, astrocytes and oligodendrocytes. The
culture conditions described above make it possible to propagate
adult human CNS stem cells in vitro for use in cell replacement
therapies, as described herein.
EXAMPLE 2
Dopaminergic Differentiation of Stem Cells in Vitro
[0102] A low ratio of dopaminergic neurons in the progeny of CNS
stem cells was observed. As a result, the ability to stimulate
differentiation of CNS stem cells into dopaminergic neurons by
manipulating the growth conditions was investigated. It has been
shown that several growth factors and transcriptional regulators
are involved in determining dopaminergic phenotype of mesencephalic
neurons. A large number of different growth factors and agents that
are known to stimulate neuronal differentiation were tested.
[0103] Treatment of dissociated neurospheres with 10.sup.-6 M
all-trans retinoic acid (RA), 1 mM dibutyryl cyclic AMP (dBcAMP),
FGF8 (20 ng/ml), and GDNF (20 ng/ml) results in development of high
number dopamine producing cells, as measured by counting tyrosine
hydroxylase (TH) and dopa decarboxylase (DDC) positive cells (Table
2). Analyses of dopamine production and secretion using HPLC
analysis also clearly indicated that cell cultures that contain the
highest number of TH and DDC positive cells also synthesize and
secrete dopamine. Dopamine secretion in these experiments was
induced by elevated levels of KCl.
[0104] Reverse-phase HPLC analysis demonstrated the neuronal
synthesis and secretion of dopamine. Dopamine concentration in
culture media of differentiated cells 10 days after initiation of
differentiation varied from 0 to 100.+-.45 pg/ml in different stem
cell isolates. Stimulation of dopamine secretion by exposing
dopamine "positive" cultures to 50 mM KCl for 30 minutes lead to an
approximately three-fold increase in dopamine levels in the culture
media (345.+-.74 pg/ml, n=3). Levels of dopamine synthesis and
secretion correlated well with the ratios of TH and DDC positive
cells in culture (Table 2). Thus, cultures with higher number of TH
and DDC positive cells also contained more dopamine and secreted
more dopamine in response to KCl depolarization. Both the
immunostaining and dopamine secretion demonstrate the presence of
functional dopaminergic neurons in the treated cultures.
[0105] After treatment of cells with RA, dBcAMP, FGF8 and GDNF
10-40% of cells have differentiated into neurons as detected by
neurofilament L and beta tubulin III staining. Up to 30% of these
neurons express TH and DDC and are considered to be dopamine
synthesizing cells. A large variation in dopaminergic
differentiation between different stem cell isolates suggests that
dopaminergic differentiation preferably be tested during the early
phases of culturing of stem cells for transplantation.
TABLE-US-00002 TABLE 2 Dopaminergic differentiation of adult human
CNS stem cells after treatment with 10.sup.-4 M all-trans retinoic
acid (RA), 1 mM dibutyryl cyclic AMP (dBcAMP), FGF8 (20 ng/ml), and
GDNF (20 ng/ml). Dopamine % Dopamine Secretion Cells % Neurons %
TH+ Cells DDC+ Cells pg/ml pg/ml H1 35 .+-. 6 13 .+-. 3 12 .+-. 4
100 .+-. 40 350 .+-. 82 H2 11 .+-. 3 0.5 .+-. 0.02 0.5 .+-. 0.01 0
0 H3 24 .+-. 4 27 .+-. 3 22 .+-. 6 120 .+-. 39 370 .+-. 75 H4 14
.+-. 3 20 .+-. 4 18 .+-. 5 75 .+-. 31 210 .+-. 65 H5 25 .+-. 7 1
.+-. 0.1 1 .+-. 0.1 0 0 C1 39 .+-. 7 29 .+-. 7 32 .+-. 9 110 .+-.
28 340 .+-. 62
[0106] In addition to dopaminergic neurons, differentiated cultures
contained astrocytes, oligodendrocytes and several other types of
neurons. Also, in many cases a population of cells was identified
that do not express any of the differentiation markers, indicating
that these cells are not neurons, astrocytes or oligodendrocytes
but likely remain in an undifferentiated resting stage. The
development of GABAergic, cholinergic, glycinergic and
glutamatergic neurons was also analyzed in differentiated cultures.
Table 3 summarizes the results of differentiation of stem
cells.
TABLE-US-00003 TABLE 3 Development of astrocytes, oligodendrocytes,
GABAergic, cholinergic, glycinergic and glutamatergic neurons from
adult human CNS stem cells after treatment with 10.sup.-6 M
all-trans retinoic acid (RA), 1 mM dibutyryl cyclic AMP (dBcAMP),
FGF8 (20 ng/ml), and GDNF (20 mg/ml). % % % Clone % neurons GFAP+ %
GalC % GABA % CHAT Glycine glutamate H1 35 .+-. 6 60 .+-. 3 2 .+-.
1 60 .+-. 8 1 .+-. 1 0 10 .+-. 3 H2 11 .+-. 3 55 .+-. 6 1 .+-. 1 90
.+-. 6 0 0 0 H3 25 .+-. 4 70 .+-. 7 5 .+-. 3 56 .+-. 8 2 .+-. 1 7
.+-. 3 2 .+-. 1 H4 14 .+-. 3 75 .+-. 5 1 .+-. 1 80 .+-. 7 1 .+-. 1
1 .+-. 1 1 .+-. 1 H5 25 .+-. 7 55 .+-. 7 5 .+-. 2 95 .+-. 7 0 0 0
C1 97 .+-. 7 60 .+-. 5 1 .+-. 1 50 .+-. 7 3 .+-. 3 5 .+-. 3 5 .+-.
2
Methods:
[0107] Human CNS stem cells were grown in F12/DMEM serum free media
(GIBCO) supplemented with B27 growth supplement (GIBCO), 20 ng/ml
each of human recombinant bFGF, EGF and LIF (all from Pepro Tech,
Inc.). CNS stem cells were grown as neurospheres in 25 cm.sup.2 or
75 cm.sup.2 Falcon tissue culture flasks. The culture media was
changed every second day and neurospheres were dissociated by
mechanical trituration every 12-15 days.
[0108] To initiate differentiation, cells were plated onto
poly-ornithine coated tissue culture dishes in F12/DMEM serum free
medium (GIBCO) supplemented with B27 growth supplement (GIBCO),
10.sup.-6 M all-trans retinoic acid, 1 mM dibutyryl cyclic AMP,
FGF8 (20 ng/ml), and GDNF (20 ng/ml) and cultured 10 days. Before
plating cells were dissociated into smaller aggregates (50-200
cells) by mechanical trituration in growth media. Differentiation
was evaluated by immunological staining using antibodies against
known cell type markers. Cell cultures were fixed for 20 min at
room temperature with 4% paraformaldehyde in PBS, washed 3 times in
PBS, pH 7.4, permeabilized using a 10 min incubation with 0.1%
TritonX-100, and washed again with PBS. Cultures were then
incubated in 3% normal goat serum in PBS with 0.1% Tween 20 for at
least 1 h at room temperature. Blocking was followed by incubation
with primary antibodies in 1% goat serum+0.1% Tween 20 for at lest
2 hours at room temperature. Antibody against type III
.beta.-tubulin (1:100, Chemicon) was used to detect neurons and
antibody against tyrosine hydroxylase (TH) and dopa decarboxylase
(DDC) to detect dopaminergic cells. Antibodies against gamma amino
acid decarboxylase (GAD) were used to identify GABAergic neurons
(1:1000, Chemicon), anti L-glutamate to detect glutamatergic
neurons (1:50, Signature Immunologics), antiglycine to detect
glycinergic neurons (1:100, Signature Immunologics) and anticholine
acetyl transferase (CHAT) to detect cholinergic neurons (1:100,
Chemicon). The cultures were washed in PBS at least 3 times and
incubated with secondary antibodies diluted in 1% goat serum with
0.2% Tween 20 for 1 hour at room temperature in the dark. The
secondary antibodies were goat anti-mouse FITC (1:200, Sigma) and
goat anti-mouse rhodamine (1:200, Boehringer).
HPLC Analysis
[0109] 1 ml of growth media from growing cultures or cultures that
had been stimulated to induce dopamine secretion (by addition of 55
mM KCl for 30 minutes) was collected. Dopamine was immediately
stabilized by adding to the culture media 881 .mu.1 of 85%
orthophosphoric acid and 4.4 mg of metabisulfite. Samples were sent
to an HPLC facility where analyses were performed. Dopamine was
extracted from samples using the aluminum extraction method and
analyzed with a reverse-phase C18 column in a MD-TM mobile phase
(Esa Inc.) Results were validated by co-elution with dopamine
standards.
Conclusion
[0110] Treatment of adult human CNS stem cells propagated in the
presence of bFGF, EGF and LIF with all-trans RA, dibutyryl cAMP,
FGF8 and GDNF results in differentiation of neurons that synthesize
and secrete dopamine. The ratio of differentiated dopaminergic
neurons varies in different stem cell isolates from 0 to 30% of the
total number of neurons. Additionally, astrocytes, oligodendrocytes
and several other types of neurons, including GABAergic,
cholinergic, glycinergic and glutamatergic neurons, develop in
treated cultures.
EXAMPLE 3
Transplantation of Stem Cell Derived Dopaminergic Cells into
Striatum
[0111] A major problem related to the transplantation of fetal
dopaminergic cells, and other cell types, is the low survival rate
of transplanted cells. Current transplantation techniques result in
suvival of 5-20% of the transplanted neurons. Consequently, cells
obtained from 3 to 5 fetuses yield 100,000-150,000 surviving
dopaminergic neurons (Lindvall 1997). Using stem cell derived
dopaminergic neurons, it is possible to increase the number of
transplanted cells as well as to optimize the timing of
transplantation.
[0112] Transplantation of untreated human CNS stem cells into rat
striatum resulted in development and survival of only human
astrocytes while transplantation into neurogenic regions such as
hippocampus and subventricular zone resulted in development of
different neuronal types (unpublished results; Fricker et al.,
1999, Vescovi et al., 1999). These results clearly indicate that
transplantation of untreated human CNS stem cells into rat striatum
results in development of astrocytes and not neurons.
[0113] Human CNS stem cell derived dopaminergic neurons were
transplanted into rat striatum and analyzed for both survival of
neurons and survival of tyrosine hydroxylase (TH) positive cells in
particular. Table 4 summarizes the results relating to survival of
TH positive cells in rat striatum at 3 weeks and 2 months after
transplantation.
[0114] It was observed that fully differentiated cells (10 days) do
not survive transplantation. An optimal time for transplantation
was found to be 2-4 days after initiation of differentiation. Based
on these results, 3 day differentiated cells were used in the
transplantation experiments described below.
[0115] Human CNS stem cells were treated with 10.sup.-6 M all-trans
retinoic acid (RA), 1 mM dibutyryl cyclic AMP (dBcAMP), FGF8 (20
ng/ml), and GDNF (20 ng/ml) for 3 days and transplanted into rat
striatum. Three different human CNS stem cell isolates H3, H4 and
C1 were used in these experiments. Results are shown in Table
4.
TABLE-US-00004 TABLE 4 Survival of human CNS stem cell derived
neurons and TH positive cells three weeks and 2 months after
transplantation into rat striatum. Number of Human % of TH+ Clone
Cells Counted % of Neurons Human Cells 3 weeks H3 3000 .+-. 50 28
.+-. 8 8 .+-. 4 H4 3000 .+-. 50 20 .+-. 5 3 .+-. 1 C1 3000 .+-. 100
50 .+-. 10 15 .+-. 6 2 months H3 3000 .+-. 50 30 .+-. 7 15 .+-. 6
H4 3000 .+-. 50 15 .+-. 3 1 .+-. 1 C1 3000 .+-. 50 50 .+-. 9 25
.+-. 7
[0116] Human anti-nucleus antibodies were used to identify
migration of transplanted human cells in rat striatum. More than
80% of transplanted cells had migrated less than 0.5 mm from the
transplant site at both 3 weeks and 2 months after transplantation.
However, single human cells were detected as far as 5 mm from the
transplantation site.
[0117] Proliferation of transplanted human cells in rat brain was
studied using bromdeoxyuridine (BrdU) labeling. BrdU was
intraperitoneally injected into transplatned rats 18 days and 55
days after transplantation (n=3 animals). Animals were euthanized
three days after BrdU injection at 21 days and 2 months after
transplantation, respectively. Immunohistochemical analysis of BrdU
incorporation in brain sections did not reveal any BrdU positive
human cells in the transplanted brains. Based on these data, 3
weeks and 2 months after transplantation human CNS stem cells do
not proliferate in rat brain.
Methods:
[0118] Human CNS stem cells were grown in F12/DMEM serum free media
(GIBCO) supplemented with B27 growth supplement (GIBCO), 20 ng/ml
of human recombinant bFGF, LIF and EGF (all from Pepro Tech, Inc.).
CNS stem cells were grown as neurospheres in 25 cm.sup.2 or 75
cm.sup.2 Falcon tissue culture flasks. The media was changed every
second day and neurospheres were dissociated by mechanical
trituration every 12-15 days.
[0119] For stimulating differentiation, cells were plated onto
poly-ornithine coated tissue culture dishes in F12/DMEM serum free
medium (GIBCO) supplemented with B27 growth supplement (GIBCO),
10.sup.-6 M all-trans retinoic acid, 1 mM dibutyryl cyclic AMP,
FGF8 (20 ng/ml), and GDNF (20 ng/ml) and cultured 3 days. Before
plating cells were dissociated into smaller aggregates (50-200
cells) by mechanical trituration in growth media.
[0120] Prior to transplantation, cells were differentiated for 3
days as described above, then collected by mild trypsinization
(0.01% trypsin in Verseen, 5 minutes at room temperature), washed
twice with F12/DMEM medium, resuspended in Dulbecco-modified
phosphate-balanced salt solution (D-PBS, GIBCO), washed twice with
D-PBS and resuspended in small volume of D-PBS.
[0121] Stereotactic surgery was performed under deep ketamin
xylazine anesthesia. The immune system of the rats receiving
transplants was suppressed by intramuscular injection of
cyclosporin every second day. Rats received 3 .mu.l of cell
suspension from a 5 .mu.l Hamilton syringe. The cell suspension
contained approximately 100,000 cells and was delivered bilaterally
in the striatum according to the following coordinates from bregma:
Anterior=.+-.0.6, Lateral=2.8 and Ventral -4.8, -4.2. The tooth bar
was set at -2.3, and ventral coordinates were taken from dura.
Brains were analyzed after 3 weeks (n=4) and 2 months (n=4). Rats
were euthanized in a CO.sub.2 chamber. Brains were removed and the
striatum was dissected and placed in 4% paraformaldehyde overnight.
Coronal and sagittal sections were cut on a cryo microtome at a
thickness of 15 .mu.m.
Immunohistochemistry
[0122] Sections were incubated with primary antibodies following
blocking with 3% BSA and 0.02% Tween 20 for 16 hours at 4.degree.
C. All primary antibodies were diluted in PBS containing 3% BSA and
0.02% Tween 20. Antibodies used in this study were anti-human
nucleus antibodies (1:50 Chemicon), anti-type iii .beta.-tubulin
antibodies (1:100 Chemicon) and anti-tyrosine hydroxylase
antibodies (1:100, Sigma). For double staining, sections were
incubated simultaneously with two primary and secondary antibodies.
The secondary antibodies were goat anti-mouse FITC (1:200, Sigma)
and goat anti-rabbit rhodamine (1:200, Boehringer).
EXAMPLE 5
Transplantation of Stem Cell Derived Neurons into Spinal Cord
[0123] As discussed above, a major problem related to the
transplants of stem cells or stem cell-derived neural cells is the
low survival rate of transplanted cells. In addition,
transplantation of untreated human CNS stem cells into spinal cord
was found to result in development of astrocytes and not neurons.
Thus, transplantation of differentiated CNS stem cells into spinal
cord was investigated.
[0124] Differentiated human CNS stem cell-derived neurons were
transplanted into rat spinal cord and analyzed survival of neurons
and other cell types. Table 5 summarizes the results of survival of
neurons in rat spinal cord 3 weeks and 2 months after
transplantation. Fully differentiated cells (10 days) did not
survive transplantation. Survival was found to be highest when
cells were transplanted 2-4 days after initiation of
differentiation. As a result, 3 day differentiated cells in the
transplantation experiments.
[0125] Human CNS stem cells were treated with 10.sup.-6 M all-trans
retinoic acid (RA), 1 mM dibutyryl cyclic AMP (dBcAMP), FGF8 (20
ng/ml), and GDNF (20 ng/ml) for 3 days and transplanted into rat
striatum. Three different human CNS stem cell isolates H3, H4, and
C1 were used in these experiments. Results are shown in Table
5.
TABLE-US-00005 TABLE 5 Survival of human CNS stem cell-derived
neurons three weeks and 2 months after transplantation into rat
spinal cord. Clone Number of Human Cells Counted % of Neurons 3
weeks H3 3000 .+-. 50 29 .+-. 8 H4 3000 .+-. 100 22 .+-. 5 C1 3000
.+-. 100 51 .+-. 10 2 months H3 3000 .+-. 100 31 .+-. 7 H4 3000
.+-. 50 15 .+-. 3 C1 3000 .+-. 50 49 .+-. 9
[0126] Human anti-nucleus antibodies were used to identify
migration of transplanted human cells in rat spinal cord. More than
80% of transplanted cells were found to migrate less than 0.5 mm
from the transplant site both 3 weeks and 2 months after
transplantation. However, single human cells were detected as far
as 5 mm from the transplantation site.
[0127] Proliferation of transplanted human cells in rat spinal cord
was analyzed using bromdeoxyuridine (BrdU) labeling. BrdU was
intraperitoneally injected into transplanted rats 18 days and 55
days after transplantation (n=3 animals). Animals were euthanized
three days after BrdU injection, 21 days and 2 months after
transplantation, respectively. Immunohistochemical analyses of BrdU
incorporation in spinal cord sections did not reveal any BrdU
positive human cells indicating that 3 weeks and 2 months after
transplantation human CNS stem cells do not proliferate in rat
spinal cord.
[0128] Further, we analyzed survival of specific cell types after
differentiation of human CNS stem cell clone C1 cells into
GABAergic, cholinergic, glutamateric and glycinergic neurons.
[0129] To analyze survival and maintenance of cholinergic and
GABAergic neurons, differentiated neurons were transplanted into
rat spinal cord. Animals were sacrificed at 30 days and 3 months.
Double labeling immunohistochemical staining was used to identify
specific human neuronal types in the rat spinal cord. Labeled cells
were counted and efficiency of transplantation calculated. Table 6
summarizes the results for survival of GABAergic and cholinergic
neurons in rat spinal cord 3 weeks and 2 months after
transplantation.
TABLE-US-00006 TABLE 6 Survival of GAGAergic and cholinergic
neurons developed from the adult human CNS stem cells after
transplantation into rat spinal cord. Number of Human GABAergic/
Time Cells Counted % of Neurons Cholinergic 3 weeks GABAergic 3000
.+-. 50 28 .+-. 8 333 .+-. 3/<1 Cholinergic 3000 .+-. 50 20 .+-.
5 0/49 .+-. 5 2 months GABAergic 3000 .+-. 50 30 .+-. 7 25 .+-.
4/<1 Cholinergic 3000 .+-. 50 15 .+-. 3 0/38 .+-. 4
[0130] The data clearly demonstrate that a high percentage of
GABAergic and cholinergic neurons survive 3 weeks and 2 months
after surgery when transplanted 3 days after initiation of
differentiation in vitro.
EXAMPLE 6
[0131] A patient suffering from a spinal cord injury is treated by
transplantation of differentiated CNS stem cells. CNS stem cells
are collected and cultured in vitro. The cells are treated to
stimulate proliferation, followed by treatment to induce
differentiation into one or more specific cell types.
Differentiated cells are then transplanted into the injured area of
the spinal cord.
Preparation of Transplanted Cells
[0132] Autologous neuronal stem cells isolated from the patients
own central nervous system are used in the transplantation process.
Alternatively, embryonic or adult CNS stem from other sources, such
as a donor or from existing cell lines, such as those maintained at
Cedars-Sinai Medical Center, are used.
Autologous Adult Stem Cells
[0133] Small fragment of brain tissue (ependymal lining) are
obtained during brain biopsy procedure by standard methods.
[0134] Surgically removed human brain tissue is placed into
ice-cold DMEM/F-12 containing penicillin-streptomycin, for further
dissection. The tissue is cut into small pieces (0.5-1 mm.sup.3)
and transferred into trypsin solution (0.02 mg/ml in Verseen
(GIBCO) and incubated at 37.degree. C. for 10-20 minutes. After
incubation, trypsin inhibitor mixture (Clonetics) is added and the
tissue is triturated mechanically with a Pasteur pipette. The cell
suspension is centrifuged at 400 rpm for 5 minutes, the pellet is
washed once with DMEM/F-12 and cells are plated at a density of
5000-10,000 viable cells/ml in 6-well Nunc tissue-culture dishes in
media. The media comprises MDME/F-12 (1:1) with Hepes buffer,
glucose, sodium bicarbonate, and glutamine and is supplemented with
B27 supplement (GIBCO), basic fibroblast growth factor (bFGF) (20
ng/ml), leukemia inhibitory factor (LIF) (20 ng/ml) and optionally
epidermal growth factor EGF (20 ng/ml).
Cell Culture
[0135] Similarly, adult or embryonic CNS stem cells from donors are
grown in F12/DMEM serum free medium (GIBCO) supplemented with B27
growth supplement (GIBCO), 20 ng/ml of human recombinant LIF, bFGF
(both from Pepro Tech, Inc.) and optionally EGF (20 ng/ml). Cells
are grown as a neurospheres. The media is changed every third day
and neurospheres are dissociated by mechanical trituration after
every 10-12 days.
Established Cell Lines From Our Tissue Bank
[0136] Embryonic and adult CNS stem cells are also available from
cell banks. These CNS progenitor cells are also grown in F12/DMEM
serum free medium (GIBCO) supplemented with B27 growth supplement
(GIBCO), 20 ng/ml of human recombinant LIF, bFGF and optionally
EGF.
[0137] To generate bulk cultures for transplantation, CNS stem
cells are be passaged at high density (10.sup.4-5.times.10.sup.4
cells /cm.sup.2) every 10-12 days.
Induction of Stem Cells to Differentiate into Different Types of
Excitatory and Inhibitory Neurons
[0138] CNS stem cells are plated onto poly-ornithine coated tissue
culture dishes in F12/DMEM serum free medium (GIBCO) supplemented
with B27 growth supplement (GIBCO) and 10.sup.-6 M all-trans
retinoic acid. The media may optionally also comprise one or more
additional components selected from the group consisting of dBcAMP
(1 mM), FGF8 (20 ng/ml) and GDNF (20 ng/ml). Cells are cultured for
3 days, after which individual treatments may be done to
differentiate the cells into cholinergic, GABAergic, glutamatergic,
and/or glycinergic neurons, as discussed below. Before plating
cells are dissociated into smaller aggregates (50-200 cells) by
mechanical trituration in growth media.
[0139] Cholinergic differentiation--cells are cultured in media
containing IL-6 (20ng/ml), NT-3 (200ng/ml), and antisense
thiomodified oligonucleotides (5 .mu.M) blocking expression of
helix-loop-helix transcriptional regulators Id1 and Id2.
[0140] GABAergic differentiation--cells are cultured in media
containing brain derived neurotrophic factor (BDNF, 20 ng/ml), and
antisense thiomodified oligonucleotides (5 .mu.M) blocking
expression of HE1 negative regulator of neurogenic genes.
[0141] Glutamatergic differentiation--cells are cultured in media
containing 1 mM dibutyryl cyclic AMP and BDNF (20ng/ml).
[0142] Glycinergic differentiation--cells are cultured in media
containing 1 mM dibutyryl cyclic AMP and neurotrophin 3 (NT-3, 20
ng/ml).
[0143] Differentiation is evaluated by immunological staining using
antibodies against choline acetyl transferase (cholinergic
neurons), glutamic acid decarboxylase (GABAergic), glutamate
(glutamatergic), and glycine (glycinergic).
Collection of Stem Cells for Transplantation
[0144] Cells are differentiated for 3 days, then collected by mild
trypsinization (0.01% trypsin in Verseen, 5 minute at room
temperature), washed twice with F12/DMEM medium, resuspended in
Dulbecco modified phosphate balanced salt solution (GIBCO) and
transplanted into the patient's spinal cord.
EXAMPLE 7
Treatment of Mammals Suffering Striatal Damage by Transplantation
of Differentiated Neuronal Stem Cells
[0145] Differentiated human neural stem cells were transplanted
into control and 6-OHDA lesioned rat striatum. Rotation response to
amphetamine was analyzed.
[0146] Three independent cell isolates of stem cells, H3, H4 and C1
were used. The three isolates have similar characteristics, but
differ in their potential to develop into dopaminergic neurons.
Clones H3 and C1 achieve a high level of dopaminergic
differentiation and survival after transplantation while clone H4
does not differentiate well into dopamine producing cells.
Behavioral recovery was observed in animals transplanted with cells
from clones H3 and C1, but no recovery was seen in H4 transplanted
animals.
[0147] Table 7 summarizes the results of behavioral recovery of
6-OHDA lesioned animals. Sixty days after transplantation rotation
scores had improved in 5 animals transplanted with cell clones H3
and C1. compared to pre-transplantation scores, rotation scores for
these five animals were reduced 78% on average (from 50 to 95).
Animals transplanted with clone H4 and control animals showed no
behavioral improvement. Immunohistochemical analyses using
antibodies against TH showed presence of TH-positive cells in the
striatum of transplanted animals. The number of TH-positive cells
was significantly higher in animals transplanted with clones H3 and
C1 compared to H4. Immunohistochemical data support the finding
that behavioral recovery is related to the presence of dopamine
producing cells.
[0148] Human CNS stem cells were treated with 10-6 M all-trans
retinoic acid (RA), 1 mM dibutryryl cyclic AMP (dBcAMP), FGF8 (20
ng/ml) and GDNF (20 ng/ml) for 3 days and transplanted into
lesioned rat brain. Three different human CNS stem cell isolates
H3, H4 and C1 were used. Results are in Table 7.
TABLE-US-00007 TABLE 7 Behavioral recovery of 6-OHDA lesioned
animals after transplantation of stem cell-derived dopamine
producing cells. change in rotational behavior Clone animal 20 days
40 days 60 days H3 1 -40 -45 -50 2 -10 -12 -12 3 -60 -80 -95 4 -20
-60 -90 C1 1 -5 -5 -10 2 -50 -65 -75 3 -45 -70 -80 4 0 -5 -5 H4 1
-5 -5 -10 2 0 +5 +20 3 -5 -5 -5 4 0 0 +20 Control 1 -5 -10 -10 2 0
+20 +40 3 +20 +45 +60 4 +5 +25 +40
[0149] Animals were closely evaluated every day for abnormal
clinical signs. Each animal was examined once in the morning and
once in the afternoon for visual signs of motor dysfunction or
other adverse signs (i.e., ataxia, paralysis, tremors, seizures,
hypoactivity, etc.).
[0150] After final testing animals were sacrificed and analyzed for
the survival, migration and differentiation of transplanted cells
using immunohistochemical techniques. Brain sections were analyzed
for general histopathology using hematoxyline/eosin staining to
detect inflammation, tumor formation, morphological defects,
etc.
[0151] The number of surviving cells was analyzed using anti-human
ribonucleoprotein antibody. The differentiation of transplanted
cells into different cell types is analyzed using double labeling
immunohistochemistry. Analyses are done at different distances from
the site of transplantation (1, 3, 5, 7, 10, 15, 20 mm from the
transplant) to evaluate migration of transplanted cells.
Methods
[0152] Cells
[0153] Human CNS stem cells were grown in F12/DMEM serum free media
(GIBCO) supplemented with B27 growth supplement (GIBCO), 20ng/ml of
human recombinant bGFG, LIF and EGF (all from Pepro Tech, Inc.).
CNS stem cells were grown as neurospheres in 25 cm.sup.2 Falcon
tissue culture flasks. The media was changed every second day and
spheres were dissociated by mechanical trituration after every
12-15 days.
[0154] For differentiation, cells were plated onto poly-ornithine
coated tissue culture dishes in F12/DMEM serum free medium (GIBCO)
supplemented with B27 growth supplement (GIBCO), 10.sup.-6 M
all-trans retinoic acid, 1 mM dibutyryl cyclic AMP, FGF9 (20
ng/ml), and GDNF (20 ng/ml) and cultured 3 days. Prior to plating,
cells were dissociated into smaller aggregates (50-200 cells) by
mechanical trituration in growth media.
[0155] Prior to transplantation, cells were differentiated for 3
days, then collected by mild trypsinization (0.01% trypsin in
Verseen, 5 minutes at room temperature), washed twice with F12/DMEM
medium, resuspended in Dulbecco-modified phosphate-balanced salt
solution (D-PBS, GIBCO), washed twice with D-PBS and resuspended in
a small volume of D-PBS. 100,000 ocells were collected for each
transplantation.
[0156] Lesion and Transplantation
[0157] Stereotactic surgery was performed under deep ketamine
xylazine anesthesia. The immune system was suppressed by
intramuscular injection of cyclosporin every second day. Animals
were lesioned by unilateral injection of 6-hydroxydopamine (6-OHDA)
at two sites along the medial forebrain bundle. Each injection
contained 16 .mu.g of 6-OHDA-HBr in 3 .mu.l of ascorbic acid (0.2
mg/ml) in 0.9% saline. Injected animals were analyzed for
amphetamine-induced rotational behavior 2 and 3 weeks before
transplantation. 5 mg of d-amphetamine sulfate per kg of body
weight was injected i.p. Only animals with pre-transplantation
scores of over 8 rotations per minute were included in the study.
Unilaterally lesioned animals received 3 .mu.l of cell suspension
in the striatum according to the following coordinates from bregma:
Anterior=+0.9, Lateral=2.8 and Ventral -4.8, -4.2. The tooth bar
was set at -2.3, and ventral coordinates are taken from dura.
100,000 cells were transplanted using a 5 .mu.l Hamilton syringe.
Animals were analyzed for amphetamine-induced rotational behavior
20, 40 and 60 days after transplantation.
Immunohistochemistry
[0158] Animals were sacrificed 60 days after transplantation,
brains sectioned and immunostained. Sections were incubated after
blocking with 3% BSA and 0.02% Tween 20 for 16 hours at 4.degree.
C. All primary antibodies were diluted in PBS containing 3% BSA and
0.02% Tween 20. Antibodies that were used in this study were
anti-human nucleus antibodies (1:50, Chemicon) to detect human
cells, antitype III .beta.-tubulin antibodies (1:100, Chemicon) to
detect neurons, anti tyrosine hydroxylase (1:100 Sigma) and dopa
decarboxylase (1:200, Chemicon) antibodies to detect dopaminergic
cells, anti gamma amino acid decarboxylase (GAD) antibodies to
identify GABAergic neurons (1:1000, Chemicon), anti L-glutamate
antibodies to detect glutamatergic neurons (1:50, Signature
Immunologics), anti-glycine antibodies to detect glycinergic
neurons (1:100, Signature Immunologics), anticholine acetyl
transferase (CHAT) to detect cholinergic neurons (1:100 Chemicon)
and anti GFAP antibodies (1:500, DAKO) to detect astrocytes. For
double staining sections were incubated simultaneously with two
primary and secondary antibodies. The second antibodies were goat
anti-mouse FITC (1:200, Sigma) and goat anti-rabbit rhodamine
(1:200, Boehringer).
EXAMPLE 8
Tumorigenicity of Stem Cells
[0159] Stem cell were tested for their ability to form tumors
following transplantation. Adult human neuronal stem cells were
grown in F12/DMEM serum free medium (GIBCO) supplemented with B27
growth supplement (GIBCO), 20 ng/ml of human recombinant bFGF, EGF
and LIF (all from Pepro Tech, Inc.). Stem cells were grown as
neurospheres, media changed every second day and spheres
dissociated by trituration every 12 to 15 days. 200,000 cells in
growth media were stereotactically injected into anesthetized rat
(n=14) and nude mouse (n=8) hippocampus and striatum. Animals were
euthanized 5 months later adn analyzed for tumor formation. Rats
were euthanized using a CO.sub.2 chamber. Brains were removed and
placed in 4% paraformaldehyde overnight. Serial sagittal sections
were cut on a cryomicrotome at a thicness of 15 .mu.M and every
tenth section was stained with hematoxylin eosin for histological
examination. Macroscopic and histological examination did not
reveal any detectable neoplasm in the body or brain of transplanted
animals.
EXAMPLE 9
Treatment of Patients with Parkinson's Disease
[0160] Cells are extracted from the central nervous system of a
Parkinson's patient using standard procedures and propagated in a
first mixture of growth factors. A preferred first mixture
comprises F12/DMEM serum free medium (e.g., GIBCO) supplemented
with B27 growth supplement (e.g., GIBCO), 20 ng/ml each of human
recombinant bFGF, LIF and EGF (PEPROTECH, INC.). CNS stem cells are
grown as neurospheres, preferably in 25 cm.sup.2 or 75 cm.sup.2
FLACON tissue culture flasks. The media is changed about every
second day and spheres are dissociated by mechanical trituration
when necessary, preferably every 12-15 days.
[0161] When sufficient quantities of cells are available for
transplantation, cells are caused to differentiate. Cells are
plated onto poly-ornithine coated tissue culture dishes in F12/DMEM
serum free medium (e.g., GIBCO) supplemented with a second
composition. The second composition preferably comprises B27 growth
supplement (GIBCO), 10.sup.-6 M all-trans retinoic acid, 1 mM
dibutyryl cyclic AMP, FGF8 (20 ng/ml), and GDNF (20 ng/ml). Cells
are preferably cultured about 3 days prior to transplantation.
Before plating, cells are dissociated into smaller aggregates
(about 50-200 cells) by mechanical trituration in growth
medium.
[0162] Prior to transplantation, cells are differentiated for three
days, then collected by mild trypsinization (0.01% trypsin in
VERSION, 5 minutes at room temperature), washed twice in F12/DMEM
medium, resuspended in Dulbecco-modified phosphate-balanced salt
solution (D-PBS, GIBCO), washed twice with D-PBS and resuspended in
a small volume of D-PBS. About 100,000 to about 300,000 cells are
collected and used for one transplantation. The cells are
reimplanted into the Parkinson's patient according to standard
methods.
EXAMPLE 10
Treatment of a Patient with Parkinson's Disease
[0163] Cells were extracted from a small piece of cortex from the
frontal lobe of a Parkinson's patient in 1999. The sample was
recovered during a craniotomy procedure. Cells were propagated in
F12/DMEM serum free medium (e.g., GIBCO) supplemented with B27
growth supplement (e.g., GIBCO), 20 ng/ml human recombinant bFGF,
20 ng/ml LIF and 20 ng/ml EGF (PEPROTECH, INC.). CNS stem cells
were grown as neurospheres in 25 cm.sup.2 or 75 cm.sup.2 FLACON
tissue culture flasks. The media was changed every second day and
spheres were dissociated by mechanical trituration every 12-15
days. The cells were proliferated until they numbered about six
million neural stem cells and/or neurons.
[0164] To induce differentiation, cells were plated onto
poly-ornithine coated tissue culture dishes in F12/DMEM serum free
medium (GIBCO) supplemented with B27 growth supplement (GIBCO),
10.sup.-6 M all-trans retinoic acid (RA), 1 mM dibutyryl cyclic AMP
(dBcAMP), FGF8 (20 ng/ml), and GDNF (20 ng/ml) and cultured 3 days.
Before plating, cells were dissociated into smaller aggregates
(about 50-200 cells) by mechanical trituration in growth medium.
Prior to transplantation, cells were collected mild trypsinization
(0.01% trypsin in VERSENE, 5 minutes at room temperature), washed
twice in F12/DMEM medium, resuspended in Dulbecco-modified
phosphate-balanced salt solution (D-PBS, GIBCO), washed twice with
D-PBS and resuspended in a small volume of D-PBS.
[0165] Cells were implanted into the Parkinson's patient's left
putamen in March 1999. The results of the treatment were determined
to be successful. At three-month post-operatively, clinical motor
scores improved by 37% and the oral dopaminergic intake decreased
by 60%. F-DOPA PET studies showed a 55.1% increase in dopamine
uptake in the left putamen. The patient continued to exhibit
significant improvements in both clinical motor scores and
increased dopamine uptake at three years post-implantation.
[0166] The procedures for removing and reimplanting the cells are
known to persons skilled in the art of brain surgery.
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