U.S. patent application number 14/244554 was filed with the patent office on 2014-10-30 for neurodegenerative diseases and methods of modeling.
This patent application is currently assigned to President and Fellows of Harvard College. The applicant listed for this patent is President and Fellows of Harvard College. Invention is credited to Francesco Paolo Di Giorgio, Kevin C. Eggan.
Application Number | 20140322237 14/244554 |
Document ID | / |
Family ID | 41608753 |
Filed Date | 2014-10-30 |
United States Patent
Application |
20140322237 |
Kind Code |
A1 |
Eggan; Kevin C. ; et
al. |
October 30, 2014 |
NEURODEGENERATIVE DISEASES AND METHODS OF MODELING
Abstract
Disclosed are embryonic stem cells and motor neurons derived
from mice carrying transgenic alleles of the normal or mutant human
SOD1 gene. Also disclosed are in vitro systems employing such SOD1
transgenic motor neurons for the study of neural degenerative
disease.
Inventors: |
Eggan; Kevin C.; (Boston,
MA) ; Di Giorgio; Francesco Paolo; (Cambridge,
MA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
President and Fellows of Harvard College |
Cambridge |
MA |
US |
|
|
Assignee: |
President and Fellows of Harvard
College
Cambridge
MA
|
Family ID: |
41608753 |
Appl. No.: |
14/244554 |
Filed: |
April 3, 2014 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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13750452 |
Jan 25, 2013 |
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14244554 |
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12386337 |
Apr 15, 2009 |
8470594 |
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13750452 |
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61200293 |
Nov 26, 2008 |
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61124229 |
Apr 15, 2008 |
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Current U.S.
Class: |
424/172.1 ;
424/93.6; 424/94.1; 435/29; 435/375; 435/5; 435/7.1; 514/44R;
514/9.7 |
Current CPC
Class: |
A61K 38/22 20130101;
G01N 2800/28 20130101; A61K 39/395 20130101; A61K 31/7088 20130101;
C12N 5/10 20130101; A61K 38/43 20130101; G01N 2333/90283 20130101;
G01N 33/5058 20130101; A61K 35/76 20130101 |
Class at
Publication: |
424/172.1 ;
435/29; 435/5; 435/7.1; 435/375; 514/9.7; 514/44.R; 424/94.1;
424/93.6 |
International
Class: |
G01N 33/50 20060101
G01N033/50; A61K 35/76 20060101 A61K035/76; A61K 31/7088 20060101
A61K031/7088; A61K 38/43 20060101 A61K038/43; A61K 39/395 20060101
A61K039/395; A61K 38/22 20060101 A61K038/22 |
Goverment Interests
GOVERNMENT SUPPORT
[0002] This invention was made with Government support under RO1
HD046732-01A1 awarded by the National Institute of Health. The
Government has certain rights in the invention.
Claims
1-14. (canceled)
15. A method of identifying an agent that affects the survival of a
mutant motor neuron comprising: providing a mutant motor neuron
comprising a SOD1 mutant allele; providing a test agent; contacting
the mutant motor neuron with the test agent; and determining the
effect of the test agent on survival of the mutant motor neuron by
comparing the survival of the mutant motor neuron to the survival
of a control motor neuron lacking the SOD1 mutant allele, which
control motor neuron is contacted with the test agent for a period
of time and under conditions identical to that of the SOD1 mutant
motor neuron.
16. The method of claim 15, wherein the SOD1 mutant motor neuron
comprises a mutation associated with a neurodegenerative
disease.
17. The method of claim 15, wherein the control motor neuron is
wild type.
18. The method of claim 15, wherein the test agent comprises an
agent selected from the group consisting of: a cell, a small
molecule, a hormone, a vitamin, a nucleic acid molecule, an enzyme,
an antibody, an amino acid, a virus, and combinations thereof.
19. The method of claim 15, further comprising providing a glial
cell, which glial cell negatively affects survival of the mutant
motor neuron.
20. The method of claim 19, wherein the glial cell comprises a SOD1
mutant allele.
21. The method of claim 20, wherein the SOD1 mutant allele of the
glial cells is transgenic.
22. The method of claim 20, wherein the SOD1 mutant allele of the
glial cell comprises a mutation associated with amyotrophic lateral
sclerosis.
23. The method of claim 22, wherein the SOD1 mutant allele of the
glial cell comprises a glycine to alanine substitution at amino
acid position 93.
24-75. (canceled)
76. A method of promoting the survival of a motor neuron, the
method comprising contacting the motor neuron with an agent that
inhibits the expression or activity of a gene or a product of a
gene in Table 2.
77. The method of claim 76, wherein the gene in Table 2 is a gene
involved in inflammation.
78. The method of claim 76, wherein the agent inhibits the
expression or activity of a prostaglandin D receptor.
79. The method of claim 76, wherein the motor neuron is contacted
with the agent in vitro.
80. The method of claim 76, wherein the motor neuron is contacted
with the agent in vivo.
81. The method of claim 78, wherein the agent is selected from the
group consisting of a cell, a small molecule, a hormone, a vitamin,
a nucleic acid molecule, an enzyme, an antibody, an amino acid, a
virus, and combinations thereof.
82. The method of claim 78, wherein the agent is a small
molecule.
83. The method of claim 78, wherein the agent is MK-0542.
84. A method of mediating motor neuron microglial toxicity, the
method comprising contacting the motor neuron with an agent that
reduces the expression or activity of a prostaglandin D2 (PGD2)
receptor and thereby mediate motor neuron microglial toxicity.
85. The method of claim 84, wherein the motor neuron is contacted
with the agent in vitro.
86. The method of claim 84, wherein the motor neuron is contacted
with the agent in vivo.
87. The method of claim 84, wherein the agent is selected from the
group consisting of a cell, a small molecule, a hormone, a vitamin,
a nucleic acid molecule, an enzyme, an antibody, an amino acid, a
virus, and combinations thereof.
88. The method of claim 84, wherein the agent is a small
molecule.
89. The method of claim 84, wherein the agent is MK-0542.
Description
RELATED APPLICATIONS
[0001] This application is copending with, shares at least one
common inventor with, and claims the benefit of U.S. Provisional
Application No. 61/124,229, filed Apr. 15, 2008, and U.S.
Provisional Application No. 61/200,293, filed Nov. 26, 2008. The
entire contents of the prior applications are hereby incorporated
by reference.
[0003] The patent or application file contains at least one drawing
executed in color. Copies of this patent or patent application
publication with color drawing(s) will be provided by the Office
upon request and payment of the necessary fee.
BACKGROUND
[0004] Amyotrophic Lateral Sclerosis ("ALS"), also known as Lou
Gehrig's disease, is a progressive neurodegenerative disease
characterized by the loss of upper and lower motor neurons,
culminating in muscle wasting and death from respiratory failure
(Boillee, S., Vande Velde, C. & Cleveland, D. W. ALS: a disease
of motor neurons and their nonneuronal neighbors. Neuron 52, 39-59,
2006). The majority of ALS cases are apparently sporadic, with 90%
of patients presenting disease symptoms without a family history of
ALS. The remaining 10% of ALS patients are diagnosed with familial
ALS (Boillee et al., 1996; Brown, R. H., Jr. Amyotrophic lateral
sclerosis. Insights from genetics. Arch Neurol 54, 1246-50, 1997;
Cole, N. & Siddique, T. Genetic disorders of motor neurons.
Semin Neurol 19, 407-18, 1999). Approximately 25% of the familial
cases of ALS are caused by dominant mutations in the gene encoding
super oxide dismutase (SOD1) (Rosen, D. R. et al. Mutations in
Cu/Zn superoxide dismutase gene are associated with familial
amyotrophic lateral sclerosis. Nature 362, 59-62, 1993).
Identification of pathogenic alleles of SOD1 has led to the
production of transgenic mouse and rat models for the study of ALS
(Gurney, M. E. et al. Motor neuron degeneration in mice that
express a human Cu,Zn superoxide dismutase mutation. Science 264,
1772-5, 1994; Nagai, M. et al. Rats expressing human cytosolic
copper-zinc superoxide dismutase transgenes with amyotrophic
lateral sclerosis: associated mutations develop motor neuron
disease. J Neurosci 21, 9246-54, 2001; Bruijn, L. I. et al.
ALS-linked SOD1 mutant G85R mediates damage to astrocytes and
promotes rapidly progressive disease with SOD1-containing
inclusions. Neuron 18, 327-38, 1997; Wong, P. C. et al. An adverse
property of a familial ALS-linked SOD1 mutation causes motor neuron
disease characterized by vacuolar degeneration of mitochondria.
Neuron 14, 1105-16, 1995). Overproduction of pathogenic human SOD1
protein in mice and rats leads to late onset, progressive
neurodegenerative disease (Gurney et al., 1994; Bruijn et al.,
1997; Wong et al., 1995). Studies of the SOD1 animal models have
led to the identification and study of intrinsic pathogenic
characteristics of ALS motor neurons including the formation of
protein aggregates, cytoskeletal abnormalities, proteosome
dysfunction and increased sensitivity to cell death signals
(Boillee et al., 2006; Bruijn, L. I., Miller, T. M. &
Cleveland, D. W. Unraveling the mechanisms involved in motor neuron
degeneration in ALS. Annu Rev Neurosci 27, 723-49, 2004).
[0005] Studies of chimeric mice suggest that non-cell autonomous
processes contribute to motor neuron death in ALS (Clement, A. M.
et al. Wild-type nonneuronal cells extend survival of SOD1 mutant
motor neurons in ALS mice. Science 302, 113-7, 2003). In animals
bearing both wild-type cells and cells harboring the SOD1G93A
transgene, wild-type neurons surrounded by transgenic non-neuronal
cells acquired cellular phenotypes characteristic of ALS (Clement
et al., 2003). Conversely, transgenic neurons associated with
wild-type non-neuronal cells were increasingly spared. However,
these animal studies did not identify which cells were involved in
the pathological interactions with motor neurons due to the complex
cellular milieu of both the spinal chord and the muscle.
Conditional mutagenesis experiments in which the SOD1 transgene was
specifically removed from motor neurons and microglial cells led to
an increase in animal lifespan, again suggesting the SOD1 protein
can have both cell autonomous and non-cell autonomous affects in
the disease (Boillee, S. et al. Onset and progression in inherited
ALS determined by motor neurons and microglia. Science 312,
1389-92, 2006). However, these experiments could not address the
direct effect of cellular interactions with motor neurons in the
disease because of the use of death as an endpoint.
BRIEF SUMMARY OF THE INVENTION
[0006] In certain embodiments, the present invention provides an
embryonic stem cell comprising a mutation in a gene involved in
motor neuron development and/or maintenance. In certain
embodiments, the present invention provides a motor neuron
generated by differentiating such an embryonic stem cell under
conditions wherein the embryonic stem cell adopts a motor neuron
cell fate. In certain embodiments, the present invention provides
an embryonic stem cell and/or a motor neuron comprising a mutation
in the SOD1 gene. For example, an embryonic stem cell and/or motor
neuron of the present invention may comprise a SOD1 mutation
wherein a glycine is substituted for the wild type alanine at
position 93 of the SOD1 amino acid sequence (referred to herein as
a "SOD1G93A" mutation or allele). In certain embodiments, such a
mutation in a SOD1 gene is associated with a neurodegenerative
disease.
[0007] In certain embodiments, an embryonic stem (ES) cell is
derived from a mouse bearing a transgene comprising a SOD1 allele,
such as without limitation, a SOD1G93A allele. In certain
embodiments, such a mouse bears a transgene comprising a human
SOD1G93A allele. Such a transgenic mouse is known to recapitulate
many pathologies of the human ALS disease. In certain embodiments,
an embryonic stem (ES) cell is a human ES cell bearing a transgene
comprising a SOD1 allele, such as without limitation, a SOD1G93A
allele. In certain embodiments, transgenic ES cells are
differentiated into motor neurons in large numbers (e.g., such as
by one or more methods described in Wichterle, H., Lieberam, I.,
Porter, J. A. & Jessell, T. M. Directed differentiation of
embryonic stem cells into motor neurons. Cell 110, 385-97, 2002)
and co-cultured either with ES-derived cells that arise during the
differentiation process and/or with other cells that contribute to
the survival, maintenance and/or differentiation of such transgenic
ES cells. For example, transgenic ES cells may be differentiated
into motor neurons in the presence of primary mouse and/or human
glial cells. In certain embodiments, such primary mouse and/or
human glial cells comprise a wild-type genotype. In certain
embodiments, such primary mouse and/or human glial cells comprise a
non-wild-type genotype. For example, such glial cells may comprise
a mutant SOD1 allele, e.g., a SOD1G93A allele. Such a mutant SOD1
allele may be provided as a transgene.
[0008] In certain embodiments, motor neurons of such cultures
display one or more abnormalities typical of a phenotype observed
in a particular disease. In certain embodiments, motor neurons of
such cultures display one or more abnormalities typical of a
phenotype observed in a neurodegenerative disease. For example,
such motor neurons may display abnormalities typical of those seen
in the motor neurons of ALS patients and/or ALS transgenic
animals.
[0009] In certain embodiments, the present invention provides novel
in vitro model systems to study ALS and/or other neurodegenerative
diseases, in which the factors directly influencing motor neuron
development, differentiation and/or survival can be investigated.
Certain of such systems are based on the differentiation of
embryonic stem (ES) cells derived from mice comprising a mutant
SOD1 allele. Certain of such systems are based on the
differentiation of human embryonic stem (ES) cells comprising a
mutant SOD1 allele. An exemplary mutant SOD1 allele that can be
used in accordance with methods and compositions of the present
invention is the SOD1G93A mutation, although systems of the present
invention are not limited to this mutation.
[0010] In certain embodiments, in vitro model systems of the
present invention are used to screen for a test agent that affects
the development, differentiation and/or survival of motor neurons.
In certain embodiments, such in vitro model systems are used to
screen for a test agent that affects the survival of wild type
motor neurons. In certain embodiments, such in vitro model systems
are used to screen for a test agent that affects the development,
differentiation and/or survival of motor neurons that comprise one
or more mutations. For example, such mutant motor neurons may
comprise a mutation associated with a neurodegenerative disease,
such as for example, ALS. In certain embodiments, such mutant motor
neurons comprise a mutation in the SOD1 gene, e.g. a SOD1G93A
mutation. In certain embodiments, the invention provides methods of
identifying an agent that affects the survival of a mutant motor
neuron. For example, certain of such methods comprise providing a
mutant motor neuron comprising a SOD1 mutant allele, providing a
test agent, contacting the mutant motor neuron with the test agent,
and determining the effect of the test agent on survival of the
mutant motor neuron by comparing the survival of the mutant motor
neuron to the survival of a control motor neuron lacking the SOD1
mutant allele, which control motor neuron is contacted with the
test agent for a period of time and under conditions identical to
that of the SOD1 mutant motor neuron. In certain embodiments, a
test agent is a cell, a small molecule, a hormone, a vitamin, a
nucleic acid molecule, an enzyme, an antibody, an amino acid,
and/or a virus. In certain embodiments, the test agent is an agent
that reduces the expression or activity of a gene or a product of a
gene in Table 2 (e.g., a product of a gene in Table 2 which is
involved in inflammation, an immune response, transcription,
signaling, or a metabolic pathway). In certain embodiments, the
test agent is an agent that reduces the expression or activity of a
prostaglandin D receptor.
[0011] In certain embodiments, the invention provides methods of
identifying a factor that has a non-cell autonomous effect on the
survival of a motor neuron. For example, certain of such methods
comprise providing a motor neuron, identifying a first cell, which
first cell negatively affects survival of the motor neuron,
identifying a second cell, which second cell does not negatively
affect survival of the motor neuron, isolating a factor from the
either the first or second cell, wherein the factor is either: i) a
factor from the first cell that contributes to the negative effect
on survival of the motor neuron; or ii) a factor from the second
cell that contributes to survival of the motor neuron. In certain
embodiments, the first cell, second cell or both is a glial cell.
In certain embodiments, the first cell, second cell or both
comprises a mutation that is associated with amyloid lateral
sclerosis, e.g. a SOD1 mutation such as without limitation a
SOD1G93A allele.
[0012] In certain embodiments, the invention provides methods of
identifying a factor that has a non-cell autonomous effect on the
survival of a motor neuron. For example, certain of such methods
comprise providing a motor neuron, culturing the motor neuron in
the presence of a test cell such that survival of the motor neuron
is negatively affected as compared to survival of a motor neuron
cultured in the presence of a control cell, and identifying a
factor present in the test cell, which factor contributes to the
negative effect on survival of the motor neuron. In certain
embodiments, the invention provides methods of identifying a factor
that has a non-cell autonomous effect on the survival of a motor
neuron, which methods comprise providing a motor neuron, culturing
the motor neuron in the presence of a test cell such that survival
of the motor neuron is negatively positively affected as compared
to survival of a motor neuron cultured in the presence of a control
cell, and identifying a factor that is absent in the test cell,
which factor contributes to the positive effect on survival of the
motor neuron.
[0013] In certain embodiments, the invention provides methods of
identifying a test agent that modulates the non-cell autonomous
effect of a test cell on the survival of a motor neuron. For
example, certain of such methods comprise providing a motor neuron,
culturing the motor neuron in the presence of a (i) test cell such
that survival of the motor neuron is negatively affected as
compared to survival of a motor neuron cultured in the presence of
a control cell, and (ii) a test agent, wherein a change in the
survival of the motor neuron in the presence of the test agent as
compared to the survival of the motor neuron in the absence of the
test agent indicates that the test agent modulates the non-cell
autonomous effect of a test cell. In certain embodiments, the test
agent is an agent that reduces the expression or activity of a gene
or a product of a gene in Table 2 (e.g., a product of a gene in
Table 2 which is involved in inflammation, an immune response,
transcription, signaling, or a metabolic pathway). In certain
embodiments, the test agent is an agent that reduces the expression
or activity of a prostaglandin D receptor.
[0014] In certain embodiments, the invention provides methods of
identifying a factor that has a cell autonomous effect on the
survival of a motor neuron. For example, certain of such methods
comprise providing a mutant motor neuron comprising a first SOD1
mutation, providing a control motor neuron lacking the first SOD1
mutation, culturing the mutant motor neuron, determining the effect
of the first SOD1 mutation on survival of the mutant motor neuron
by comparing the survival of the mutant motor neuron to the
survival of the control motor neuron, which control motor neuron is
cultured for a period of time and under conditions identical to
that of the mutant motor neuron, and isolating a factor from the
either the mutant motor neuron or the control motor neuron, wherein
the factor is either: i) a factor from the mutant motor neuron that
contributes to the negative effect on survival of the motor neuron;
or ii) a factor from the control motor neuron that contributes to
survival of the motor neuron.
[0015] In certain embodiments, in vitro model systems of the
present invention are used to screen for a factor that has a
non-cell autonomous effect on the development, differentiation
and/or survival of a motor neuron, e.g. a SOD1 mutant motor neuron
such as a SOD1G93A mutant motor neuron. In certain embodiments,
such a factor comprises a factor originating from another motor
neuron. In certain embodiments, such a factor comprises a factor
originating from another cell that is not a motor neuron. In
certain embodiments, such a factor originates from a glial cell.
Such a factor may have a negative effect on the development,
differentiation and/or survival of a motor neuron. Alternatively,
such a factor may contribute to the development, differentiation
and/or survival of a motor neuron such that its absence negatively
affects survival of the motor neuron.
[0016] In vitro model systems of the present invention are useful
for the identification of factors, agents, etc. that both
positively and negatively affect motor neuron survival. In certain
embodiments, one or more factors, agents, etc. that affect motor
neuron survival are identified by using SOD1 mutant glial cells,
e.g. glial cells comprising a SOD1G93A mutation. In certain
embodiments, in vitro model systems of the present invention
utilize human motor neurons and/or other cell types derived from
human ES cells. Such in vitro model systems may be advantageously
employed for the human physiological validation of findings from
animal models, such as, without limitation, animal models that
recapitulate neurodegenerative diseases such as ALS and/or other
neurodegenerative diseases. Additionally or alternatively, such in
vitro model systems may be advantageously employed to identify
novel factors, agents, etc. that affect human motor neuron
development and/or contribute to a disease state, such as, without
limitation, a neurodegenerative disease, e.g. ALS, in the absence
of an animal model. Additionally or alternatively, such in vitro
model systems may be advantageously employed to illuminate the
target, efficacy, toxicity, mode of action, etc. of factors,
agents, etc. that affect human motor neuron development and/or
contribute to a disease state, such as, without limitation, a
neurodegenerative disease, e.g. ALS.
BRIEF DESCRIPTION OF THE DRAWING
[0017] FIG. 1 shows one embodiment of derivation of Hb9GFP; SOD1
mouse ES cell lines. PCRs (FIG. 1A) for human SOD1 and Il2, and
(FIG. 1B) for GFP in Hb9::GFP, Hb9::GFP; SOD1 and Hb9GFP; SOD1G93A
ES cell lines. (FIG. 1C) Expression of human SOD1 protein in
Hb9GFP, Hb9GFP; SOD1 and Hb9GFP; SOD1G93A ES cell lines. (FIG. 1D)
E10.5 chimera generated using the Hb9GFP ES cell line.
[0018] FIG. 2 shows one embodiment of differentiation of the SOD1
G93A mouse ES cell lines into motor neurons. (FIG. 2A) GFP
expression of the ES cell lines during different phases of the
differentiation into motor neurons. Expression of neuronal markers
(FIG. 2B) Tuj1, (FIG. 2C) Hb9, (FIG. 2D) Isl1, (FIG. 2E) Choline
Acetyl-Transferase (ChAT) in motor neurons derived from HB9GFP;
SOD1G93A ES cell lines at 9 and 14 days after the beginning of
differentiation process. (FIG. 2F) Presence of GFAP positive cells
surrounding a GFP motor neuron after 28 days. The white arrows
indicate the nuclei of motor neurons immunoreactive for hb9 or Isl1
proteins.
[0019] FIG. 3 shows the effect of genetic background on motor
neuron survival. (FIG. 3A) GFP positive motor neurons derived from
SOD1G93A and Hb9GFP 61 days after differentiation. Number of GFP
positive cells derived from (FIG. 3B) Hb9GFP and (FIG. 3C) SOD1G93A
ES cell lines present 15, 30, 45 and 60 days after dissociation of
EBs plated at two different concentrations (8.times.10.sup.5 and
4.times.10.sup.5 per well). (FIG. 3D) Number of GFP positive motor
neurons derived from Hb9GFP and SOD1G93A 15 and 30 days after EB
dissociation plated at the concentration of 8.times.10.sup.5 (D)
and 4.times.10.sup.5 (F) per well. (FIGS. 3E, 3G). Same experiments
in (FIGS. 3D, 3F) analyzed as percent of GFP positive motor neurons
derived from Hb9GFP and SOD1G93A cell lines present at day 15,
which still remain at 30 days.
[0020] FIG. 4 shows intracellular aggregation of SOD1 protein in
cultured motor neurons. Expression of human SOD1 protein in motor
neurons derived from (FIG. 4A) SOD1G93A and (FIG. 4B) SOD1 cell
lines at day 7, 14 and 21 after EBs dissociation. (FIG. 4C)
Co-expression of ubiquitin and SOD1 protein in cells derived from
the SOD1G93A cell line, 28 days after differentiation. (FIG. 4D)
Percentage of GFP-Positive motor neurons with SOD1 inclusions
present after 21 days in culture.
[0021] FIG. 5 shows that glial cell genotype directly affects motor
neuron survival in culture. (FIG. 5A) GFP-positive motor neurons at
5, 7, 14, 21 and 28 days in culture after EB dissociation. (FIG.
5B) Graph shows percentage of Hb9GFP positive cells over time in
all the conditions studied. Experiments were made in triplicate and
results were normalized to the number of cells found at 7 days in
vitro.
[0022] FIG. 6 shows the percentage of differentiating EB cells that
express GFP. FACS analysis of cells dissociated from EBs after 5
days of treatment with retinoic acid and shh. (FIG. 6A) Non
transgenic cell line, (FIG. 6B) Hb9GFP, (FIG. 6C) Hb9GFP; SOD1,
(FIG. 6D) Hb9GFP; SOD1G93A. The dot plots are representative of one
experiment, but the percentages are the average of three different
experiments. Calcein blue was used to assay the viability of cells
during sorting.
[0023] FIG. 7 shows expression of neuronal marker Isl1 in cells
derived from SOD1G93A mouse ES cell lines. Expression of neuronal
marker Isl1 in GFP positive motor neurons (white arrows) and
non-neuronal cells (white stars) 14 days after differentiation.
[0024] FIG. 8 shows quantitative and qualitative analysis of SOD1
protein inclusions in ES cell derived motor neurons. (FIG. 8A)
Average area of SOD1 inclusions in SOD1 and SOD1G93A derived motor
neurons. (FIG. 8B) Average length of SOD1 inclusions in SOD1 and
SOD1G93A derived motor neurons. (FIG. 8C) Integrated Optical
Density of SOD1 inclusions in SOD1 and SOD1G93A derived motor
neurons. (FIG. 8D) Representative SOD1G93A ES cell derived motor
neurons, 21 days after EB dissociation, displaying intracellular
inclusion of the SOD1 protein. (FIG. 8E) Distribution of inclusion
bodies per cell in SOD1 and SOD1G93A derived motor neurons. Results
are graphed as mean+/-S.E.M.
[0025] FIG. 9 shows activation of Caspase 3 and cytoplasmic
localization of Cytochrome C in SOD1G93A motor neurons 14 days
after EB dissociation. (FIG. 9A) A SOD1G93A derived motor neuron is
immunoreactive with an antibody specific to the activated form of
caspase 3 (white arrow). Other cell types are also immunoreactive
for the staining (white stars). (FIG. 9B) The same motor neuron at
a larger magnification is clearly visible in fragmented nuclei
(DAPI) that is also immunoreactive for the antibody for Caspase 3.
(FIG. 9C). The cytoplasm of a subset of motor neurons in culture
also stained broadly with an antibody specific to Cytochrome C,
suggesting that cell death pathways are activated.
[0026] FIG. 10 shows characterization of primary glial monolayers
derived from SOD1 and SOD1 G93A mice. (FIG. 10A)
Immuno-fluorescence Analysis of GFAP and S100 expression in SOD1
and SOD1G93A glial monolayers at 7 and 14 days. (FIGS. 10B, 10C)
Summary of immuno-fluorescent analysis of glia markers GFAP, 5100,
RC2, Vimentin, CD 11 b, CNPase for both wt glia (FIG. 10B) and
SOD1G93A glia (FIG. 10C) at different time points.
[0027] FIG. 11 shows one embodiment of differentiation of human ES
cells into motor neurons. (FIG. 11A) Diagram outlining the protocol
used to differentiate human ES cells into motor neurons:
Undifferentiated human ES cell colonies are dissociated in
collagenase, and grown as EBs for the first 14 days in EB media,
then are induced to a rostrocaudal identity with retinoic acid (RA)
and Shh for another 14 days. Finally, EBs are matured in the
presence of GDNF for 14 more days. At this point the EBs can either
be plated whole or dissociated with papain and then plated. (FIG.
11B) Immunohistochemistry was performed to detect neuronal markers
PAX 6, NKX 6.1, ISL1/2, and HB9 in EB sections at 14, 28, and 42
days after collagenase treatment of undifferentiated human ES
cells. (FIG. 11C) Percentage of cells immuno-reactive for HB9 after
treatment with or without RA and Shh. (FIG. 11D) Percentage of
cells immuno-reactive for HB9 after 42 days of differentiation in
different HuES cell lines.
[0028] FIG. 12 shows characterization of the Hb9::GFP human ES cell
line. (FIG. 12A) DNA construct used for the electroporation of
human ES cells. (FIG. 12B) GFP expression during different stages
of the differentiation from human ES cells into motor neurons.
Expression of neuronal markers (FIG. 12C) HB9, (FIG. 12D) PAX6,
(FIG. 12E) ISL1/2, (FIG. 12F) Choline Acetyl-Transferase (ChAT) in
motor neurons derived from Hb9::GFP human ES cells.
[0029] FIG. 13 shows the effect of glial cells over expressing
SOD1G93A on human ES cell-derived motor neurons. (FIG. 13A)
Experimental design: embryonic stem cells were differentiated into
motor neurons, and an equal number of cells were plated on two
different glial monolayers; one derived from mice over-expressing
SOD1G93A, and the other derived from non-transgenic mice (WT).
Motor neurons were counted after 10 and 20 days in co-culture.
(FIG. 13B) Number of HB9 positive cells 10 days after plating on
SOD1G93A or non-transgenic (WT) glia. (FIG. 13C) Number of HB9
positive cells 20 days after plating on SOD1G93A or non-transgenic
(WT) glia. (FIG. 13D) Images of HB9/Tuj1 positive cells 20 days
after plating on SOD1G93A glia or non-transgenic (WT) glia. (FIG.
13E) Number of Hb9::GFP cells 20 days after plating on SOD1G93A
glia or non-transgenic (WT) glia or glia over-expressing the wild
type form of human SOD1 (SOD1WT). Images of Hb9::GFP cells in the
three different co-culture conditions (FIGS. 13F-13H).
[0030] FIG. 14 shows the specificity of the toxic effect of glia
overexpressing SOD1G93A on motor neurons. (FIG. 14A) Experimental
design: embryonic stem cells were differentiated into motor
neurons, and an equal number of cells were plated on two different
glial monolayers; one derived from mice over-expressing the
mutation SOD1G93A, and the other derived from non-transgenic mice
(WT). Human ES cell derived interneurons were counted after 20 days
in co-culture using two different markers, CHX10 and LHX2. (FIG.
14B) Number of LHX2 positive cells 20 days after plating on
SOD1G93A glia or non-transgenic (WT) glia. (FIG. 14C) Number of
CHX10 positive cells 20 days after plating on SOD1 G93A or
non-transgenic (WT) glia. (FIG. 14D) Image of LHX2/Tuj1 positive
cells 20 days after plating on SOD1G93A glia or non-transgenic (WT)
glia. (FIG. 14E) Image of CHX10/Tuj1 positive cells 20 days after
plating on SOD1G93A glia or non-transgenic (WT) glia. (FIG. 14F)
Experimental design: embryonic stem cells were differentiated into
motor neurons and same number of cells was plated on two different
MEF monolayers; one derived from mice over-expressing the mutation
SOD1G93A, and the other derived from non-transgenic mice (WT).
Motor neurons were counted after 20 days to compare the two
conditions. (FIG. 14G) Image of HB9/Tuj1 positive cells 20 days
after plating on SOD1G93A or non transgenic (WT) MEF. (FIG. 14H)
Number of HB9 positive cells 20 days after plating on SOD1 G93A or
non-transgenic (WT) MEF.
[0031] FIG. 15 shows neuronal marker expression at different time
points during one embodiment of differentiation from human ES cells
toward the motor neuron fate. (FIG. 15A) Percent of sectioned EBs
(n=20) staining positive for PAX6, NKX6.1, ISL1/2, or HB9 at day 0,
day 14, day 28, and day 42 of differentiation. (FIG. 15B) Percent
of cells per sectioned EB (n=3) staining positive for PAX6, NKX6.1,
ISL1/2, or HB9 at day 0, day 14, day 28, and day 42 of
differentiation.
[0032] FIG. 16 shows the effect of glia on human ES cell derived
neurons. (FIG. 16A) Expression of the glial marker glial fibrillary
acidic protein (GFAP) and the neuronal marker Tuj1 in mouse-derived
glia cells. (FIGS. 16B-16C) Different density and morphology of
human neurons 1 day after plating on a glial layer or on
laminin.
[0033] FIG. 17 shows analysis of one embodiment of human ES cell
derived motor neurons. Expression of neuronal markers (FIG. 17A)
ISL1/2, (FIG. 17B) HB9, (FIG. 17C) Choline Acetyl-Transferase
(ChAT) in motor neurons derived from human ES cells.
[0034] FIG. 18 shows characterization of a Hb9::GFP human ES cell
line. (FIGS. 18A-18C) Expression of HB9 protein in Hb9::GFP
positive cells. (FIG. 18D) Number of Hb9::GFP cells that are
immunoreactive to Hb9 antibody (Hb9+Hb9::GFP), or not
immunoreactive to Hb9 antibody (Hb9::GFP only). Expression of
neuronal markers (FIG. 18E) NKX2.2, (FIG. 18F) NKX6.1, (FIG. 18G)
LHX2, (FIG. 18H) CHX10, in motor neurons derived from Hb9::GFP
human ES cells.
[0035] FIG. 19 shows one embodiment of overexpression of human
SOD1G93A that results in protein aggregation in mouse ES cell
derived motor neurons, but not glia. (FIG. 19A) Human SOD1
expression in mouse motor neurons derived from Hb9::GFP mouse ES
cells over expressing SOD1G93A, after 21 days in culture. (FIG.
19B) Human SOD1 expression in glia derived from mice
over-expressing the mutation SOD1G93A, after 3 months in
culture.
[0036] FIG. 20A is a Venn Diagram presenting the overlap among
transcripts selectively over expressed in SOD1G93A glia and in SOD1
WT glia with respect to WT glia.
[0037] FIG. 20B is a table listing a subset of genes over expressed
in SOD1 G93A glia but not in SOD1 WT glia or WT glia.
[0038] FIG. 20C is a graph showing the percentage of Hb9::GFP cells
remaining on non-transgenic (WT) glia after 20 days of treatment
with GMFb, Rantes, Cxcl 7, Mcp 2, Shh or PGD2 compared to the
untreated condition (Ctrl) (n=3).
[0039] FIG. 20D shows images of Hb9::GFP positive cells after 20
days of treatment with prostaglandin D2 (PGD2) or without treatment
(Ctrl) on WT Glia.
[0040] FIG. 20E is a graph showing the percentage of Hb9::GFP cells
remaining on WT glia or SOD1G93A glia after 20 days of treatment
with the inhibitor of Prostaglandin D2 receptor (MK 0524)
(n=3).
[0041] FIG. 20F shows images of Hb9::GFP positive cells after 20
days of treatment with an inhibitor of Prostaglandin D2 receptor
(MK 0524) or without treatment (Ctrl) on SOD1G93A glia.
DESCRIPTION OF CERTAIN EMBODIMENTS OF THE INVENTION
[0042] In certain embodiments, the present invention provides
compositions and methods for detailed mechanistic studies of the
interactions between cells such as, without limitation, motor
neurons and other cells such as, without limitation, glia. In
certain embodiments, a motor neuron of the present invention
comprises a mutant motor neuron comprising an allele associates
with a neurodegenerative disease, such as, without limitation, ALS.
In certain embodiments, a mutant motor neuron comprises a SOD1
mutant allele associated with ALS. For example, a mutant motor
neuron may comprise a SOD1G93A allele. In certain embodiments,
compositions and methods of the present invention provide an assay
for diffusible factor(s), agent(s), etc. toxic to motor neurons. In
certain embodiments, the present invention provides a high
throughput cell based assay for small molecules that promote
survival of mutant SOD1 motor neurons. The present disclosure
validates the use of ES cells carrying disease-causing genes to
study disease mechanisms.
[0043] Certain embodiments of the present invention are discussed
in detail below. Those of ordinary skill in the art will
understand, however, that various modifications to these
embodiments are within the scope of the appended claims. It is the
claims and equivalents thereof that define the scope of the present
invention, which is not and should not be limited to or by this
description of certain embodiments.
DEFINITIONS
[0044] "Agent", "Test agent": The terms "agent" and "test agent" as
used herein refer to a compound or other entity that is tested to
determine whether it has an effect on the differentiation,
development and/or survival of a cell. As non-limiting examples, a
test agent may comprise a cell, a small molecule, a hormone, a
vitamin, a nucleic acid molecule, an enzyme, an amino acid, and/or
a virus. Those of ordinary skill in the art will be aware of other
test agents that may be tested for their effect(s) on
differentiation, development and/or survival of a cell. In certain
embodiments, a differentiating cell is subjected to a test agent
before, during and/or after differentiation to determine its
effect(s) on differentiation, development and/or survival of a
cell. For example, an embryonic stem cell undergoing
differentiation into a cell type of interest may be subjected to a
test agent before, during and/or after differentiation. In certain
embodiments, an embryonic stem cell undergoing differentiation into
a motor neuron is subjected to a test agent before, during and/or
after differentiation. In certain embodiments, a test agent that is
identified as having one or more effects on the differentiation,
development and/or survival of a cell is used in the treatment,
prevention and/or cure of a disease of interest.
[0045] "Embryonic stem cell", "ES cell": The terms "embryonic stem
cell" and "ES cell" as used herein refer to an undifferentiated
stem cell that is derived from the inner cell mass of a blastocyst
embryo and is pluripotent, thus possessing the capability of
developing into any organ or tissue type or, at least potentially,
into a complete embryo. Embryonic stem cells appear to be capable
of proliferating indefinitely, and of differentiating into all of
the specialized cell types of a mammal, including the three
embryonic germ layers (endoderm, mesoderm, and ectoderm), and all
somatic cell lineages and the germ line. As non-limiting examples,
embryonic stem cells have been shown to be capable of being induced
to differentiate into cardiomyocytes (Paquin et al., Proc. Nat.
Acad. Sci., 99:9550-9555, 2002), hematopoietic cells (Weiss et al.,
Hematol. Oncol. Clin. N. Amer., 11(6):1185-98, 1997; also U.S. Pat.
No. 6,280,718), insulin-secreting beta cells (Assady et al.,
Diabetes, 50(8):1691-1697, 2001), and neural progenitors capable of
differentiating into astrocytes, oligodendrocytes, and mature
neurons (Reubinoff et al., Nature Biotechnology, 19:1134-1140,
2001; also U.S. Pat. No. 5,851,832). One of ordinary skill in the
art will be aware of other cell types that have been derived from
embryonic stem cells.
[0046] "SOD1": As will be clear from context, the term "SOD1" as
used herein refers to either the gene encoding superoxide dismutase
1 or the enzyme encoded by this gene. The SOD1 gene or gene product
is known by other names in the art including, but not limited to,
ALS1, Cu/Zn superoxide dismutase, indophenoloxidase A, IPOA, and
SODC_HUMAN. Those of ordinary skill in the art will be aware of
other synonymous names that refer to the SOD1 gene or gene product.
The SOD1 enzyme neutralizes supercharged oxygen molecules (called
superoxide radicals), which can damage cells if their levels are
not controlled. The human SOD1 gene maps to cytogenetic location
21q22.1. Certain mutations in SOD1 are associated with ALS in
humans including, but not limited to, Ala4Val, Gly37Arg and
Gly93Ala, and more than one hundred others. Those of ordinary skill
in the art will be aware of these and other human mutations
associated with ALS. Certain compositions and methods of the
present invention comprise or employ cells comprising a SOD1
mutation.
[0047] "Stem-cell producing condition": The term "stem-cell
producing condition" as used herein refers to a condition or set of
conditions that permits and/or drives a cell to become a stem cell.
In certain embodiments, an embryonic cell is permitted and/or
driven to become an embryonic stem cell by subjecting such an
embryonic cell to a stem-cell producing condition. For example, an
embryonic blastomere may permitted and/or driven to become an
embryonic stem cell by isolating the embryonic blastomere from the
inner cell mass of a blastocyst and culturing the embryonic
blastomere under stem-cell producing conditions, such that at least
one blastomere proliferates into a pluripotent embryonic stem cell.
In certain embodiments, a transgenic embryonic stem cell is
generated by producing a transgenic cell according to one or more
methods of the present invention, allowing the transgenic cell to
develop into a transgenic blastocyst comprising a plurality of
transgenic blastomeres, isolating one or more transgenic
blastomeres from the inner cell mass of the transgenic blastocyst,
and culturing the isolated transgenic blastomere(s) under stem-cell
producing conditions such that at least one transgenic blastomere
develops into a pluripotent transgenic embryonic stem cell.
Embryonic Stem Cells and their Generation
[0048] Stem cells typically share two important characteristics
that distinguish them from other types of cells. First, they are
unspecialized cells that are capable of maintaining their
unspecialized state and of renewing themselves for long periods
through cell division. Second, under appropriate conditions, they
can be induced to differentiate into cells with specialized
functions. Several types of stem cells have been identified
including adult stem cells, umbilical cord stem cells, and
embryonic stem cells.
[0049] Embryonic stem cells may be characterized by any of several
criteria, which criteria will be known by those of ordinary skill
in the art. For example, embryonic stem cells are typically capable
of continuous indefinite replication in vitro. Continued
proliferation for a long period of time (e.g., 6 months, one year
or longer) of culture is a sufficient evidence for immortality, as
primary cell cultures without this property fail to continuously
divide for such a length of time (Freshney, Culture of animal
cells. New York: Wiley-Liss, 1994). In certain embodiments,
embryonic stem will continue to proliferate in vitro under
appropriate culture conditions for longer than one year, and
maintain the developmental potential to contribute all three
embryonic germ layers throughout this time. Such developmental
potential can be demonstrated by the injection of embryonic stem
cells that have been cultured for a prolonged period (over a year)
into SCID mice and then histologically examining the resulting
tumors. However, length of time in culture is not the sole criteria
that may be used to identify an embryonic stem cell, and even
though cells have grown in culture for less than 6 months, such
cells may nevertheless be embryonic stem cells.
[0050] Additionally or alternatively, embryonic stem cells may be
identified by the expression of certain markers, including but not
limited to cell surface markers. As will be understood by those of
ordinary skill in the art, embryonic stem cells from different
species will exhibit species-specific markers on their cell
surfaces. For example, Thomson (U.S. Pat. Nos. 5,843,780 and
6,200,806, each of which is incorporated herein in its entirety by
reference) discloses certain cell surface markers that may be used
to identify embryonic stem cells derived from primates.
Furthermore, Stage Specific Embryonic Antigens (SSEAs) are
monoclonal antibodies that recognize defined carbohydrate epitopes
and may also be used to identify embryonic stem cells. Embryonic
stem cells derived from different species exhibit different
patterns of SSEAs. For example, undifferentiated primate ES cells
(including human ES cells) express SSEA-3 and SSEA-4, but not
SSEA-1. Conversely, undifferentiated mouse ES cells express SSEA-1,
but not SSEA-3 or SSEA-4. Additionally or alternatively, markers
that are not exhibited on the surface of a cell may be used to
identify an embryonic stem cell. For example, the homeodomain
transcription factor Oct 4 (also termed Oct-3 or Oct3/4) is
frequently used as a marker for totipotent embryonic stem cells.
Those of ordinary skill in the art will be aware of cell surface
and other markers that are useful in identifying embryonic stem
cells, including markers diagnostic of a given species, that can be
used to identify an embryonic stem cell from that species.
[0051] Additionally or alternatively, embryonic stem cells may be
identified by the capacity to develop into all of the specialized
cell types of a mammal, including the three embryonic germ layers
(endoderm, mesoderm, and ectoderm), and all somatic cell lineages
and the germ line. Additionally and/or alternatively, embryonic
stem cells may be identified by the capacity to participate in
normal development when transplanted into a preimplantation embryo
to generate a chimeric embryo.
[0052] Cultured cells that have proliferated in cell culture for a
long period of time (e.g., six or more months) without
differentiating, are pluripotent, and appear genetically normal are
typically considered to be embryonic stem cells. In certain
embodiments, an embryonic stem cell of the present invention
comprises a human embryonic stem cell. In certain embodiments, an
embryonic stem cell of the present invention comprises a non-human
embryonic stem cell. For example, a non-human embryonic stem cell
of the present invention may include, but is not limited to, a
mouse, rat, pig, sheep, goat, and/or a primate stem cell. Those of
ordinary skill in the art will be aware of other non-human stem
cells that may be used in accordance with the present
invention.
[0053] The capacity of embryonic stem cells (ES) to self renew in
culture, while retaining their pluripotent potential, provides the
opportunity to produce virtually unlimited numbers of
differentiated cell types to replenish those lost as a consequence
of disease (Evans, M. J. & Kaufman, M. H. Establishment in
culture of pluripotential cells from mouse embryos. Nature 292,
154-6, 1981; Martin, G. R. Isolation of a pluripotent cell line
from early mouse embryos cultured in medium conditioned by
teratocarcinoma stem cells. Proc Natl Acad Sci USA 78, 7634-8,
1981). An alternative, but equally important potential of ES cells
is to provide insights into disease mechanisms (Lerou, P. H. &
Daley, G. Q. Therapeutic potential of embryonic stem cells. Blood
Rev 19, 321-31, 2005; Ben-Nun, I. F. & Benvenisty, N. Human
embryonic stem cells as a cellular model for human disorders. Mol
Cell Endocrinol 252, 154-9, 2006). ES cells carrying the genes
responsible for a particular disease can be induced to
differentiate into the cell types affected in that disease. Studies
of the differentiated cells in culture could provide important
information regarding the molecular and cellular nature of events
leading to pathology.
[0054] In certain embodiments, this approach is used to develop an
in vitro model of Amyotrophic Lateral Sclerosis ("ALS"). As
described more fully below in the Examples section of the present
application, embryonic stem cell lines were derived from normal
mice, and from mice that over-express either the wild-type human
SOD1 transgene or the mutant SOD1G93A transgene, the latter of
which is responsible for one type of familial ALS (see Example
section). Using the methods established by Wichterle et al (2002)
the three ES cell lines were differentiated into motor neurons in
culture. The wild-type SOD1 and the mutant SOD1G93A motor neurons
produce high levels of the corresponding human SOD1 proteins, and
they both display properties that characterize bone fide motor
neurons. These motor neurons could be maintained in long-term
culture, providing the opportunity to detect differences between
the mutant SOD1G93A ES cell-derived motor neurons and those derived
from control cell lines.
[0055] In certain embodiments, embryonic stem cells are generated
by culturing cells from the inner cell mass in a culture dish that
is coated with a feeder layer comprising mouse embryonic skin cells
that have been treated so they will not divide. Such a feeder layer
gives the inner cell mass cells a sticky surface to which they can
attach and also releases nutrients into the culture medium. In
certain embodiments, cells from the inner cell mass are cultured in
a culture dish that is not coated with a feeder layer. Such
embodiments provide certain advantages including reduction of the
risk that viruses or other macromolecules in the mouse cells may be
transmitted to the cultured cells.
[0056] In certain embodiments, embryonic stem cells are generated
by subjecting cells to stem-cell producing conditions. Stem-cell
producing conditions are known to those of ordinary skill in the
art and can often vary between species. For example, leukemia
inhibitory factor (LIF) is necessary and sufficient to prevent
differentiation of mouse embryonic stem cells and to allow them to
grow in an undifferentiated state indefinitely. Conversely, for
primate embryonic stem cells, at least one group has reported that
growth on a fibroblast feeder layer is required to prevent them
from differentiating (see e.g., U.S. Pat. Nos. 5,843,780 and
6,200,806, incorporated herein by reference in their entirety). One
of ordinary skill in the art will be aware of appropriate stem-cell
producing conditions including, but not limited to, culture media
and/or culturing conditions that permit and/or drive a cell of a
given species to become a stem cell.
[0057] In certain embodiments, embryonic stem cells of the present
invention are generated by any of a variety of methods disclosed in
U.S. Provisional Patent Application No. 60/926,525, filed Apr. 26,
2007, which is incorporated herein by reference in its entirety.
For example, in certain embodiments, an embryonic stem cell is
generated by transferring nuclear-derived genetic material from a
donor cell to a recipient cell to generate a transgenic cell, after
which the transgenic cell is allowed to develop into a blastocyst
and a blastomere cell from the inner cell mass is isolated and/or
cultured (and optionally passaged for several generations) under
stem-cell producing conditions, resulting in generation of an
embryonic stem cell syngenic with the nuclear-derived genetic
material removed from the donor cell used to generate the
transgenic cell.
[0058] In certain embodiments, embryonic stem cells of the present
invention are generated such that the generated embryonic stem
cells comprise a mutation associated with a disease of interest.
For example, embryonic stem cells may be generated which contain a
mutation associated with a neurodegenerative disease. Exemplary
neurodegenerative diseases include, but are not limited to: ALS,
Parkinson's disease, and Alzheimer's disease. Those of ordinary
skill in the art will be aware of other neurodegenerative diseases
of interest, as well as mutations associated with such
diseases.
[0059] In certain embodiments, an embryonic stem cell is generated
that comprises a mutation associated with ALS. For example, an
embryonic stem cell may be generated that comprises a mutation in
the SOD1 gene, e.g., Ala4Val, Gly37Arg and/or Gly93Ala. In certain
embodiments, an embryonic stem cell is generated that comprises a
SOD1G93A allele. In certain embodiments, an embryonic stem cell is
generated that comprises a human SOD1G93A allele.
[0060] In certain embodiments, an embryonic stem cell comprises a
mutation in a gene associated with neurodegenerative disease, which
gene is present as a transgene. For example, an endogenous gene
associated with a neurodegenerative disease may be deleted or
otherwise inactivated in such an embryonic stem cell by any of a
variety of techniques known to those skilled in the art, and a
transgene comprising a mutant copy of the endogenous gene may be
introduced into the embryonic stem cell by any of a variety of
techniques known to those skilled in the art. In certain
embodiments, such a transgene is integrated into the genome of the
embryonic stem cell. In certain embodiments, such a transgene is
not integrated into the genome of the embryonic stem cell.
[0061] Once embryonic stem cell lines are established, batches of
such embryonic stem cell lines can be frozen and for future
culturing and/or experimentation.
Differentiation into Motor Neurons
[0062] In certain embodiments of the present invention, an
embryonic stem cell is subjected to conditions that result in the
embryonic stem cell differentiating into a motor neuron. For
example, embryonic stem cells may be dissociated into a single-cell
suspension, allowed to spontaneously aggregate into embryoid bodies
over a first period of time (e.g. 48 hours, although such a period
of time may be increased or decreased depending on other conditions
to which the embryonic stem cells are subjected), and then treated
with a suitable differentiation factor or factors for a second
period of time such that the embryonic stem cells differentiate
into motor neurons. By way of example, such differentiation factors
may include retinoic acid (RAc) and soluble sonic hedgehog (Shh),
which may be administered for, e.g., 5 days. Other differentiation
factor(s) and condition(s) will be known to those of ordinary skill
in the art.
[0063] In certain embodiments, a motor neuron differentiated from
an embryonic stem cell comprises a mutation in a gene associated
with neurodegenerative disease. As non-limiting examples, such a
neurodegenerative disease may include ALS, Parkinson's disease,
Alzheimer's disease or any number of other neurodegenerative
diseases known to those of skill in the art. A variety of genes are
known to be associated with neurodegenerative diseases. As one
non-limiting example, mutations in the SOD1 gene are known to be
associated with the neurodegenerative disease ALS. For example, in
humans, Gly92Ala, Ala4Val and Gly37Arg mutations are associated
with the onset and progression of ALS. Those of ordinary skill in
the art will be aware of other SOD1 mutations associated with ALS.
In certain embodiments, compositions and method of the present
invention comprise or employ human motor neurons comprising a
SOD1G93A mutation such as Gly92Ala, Ala4Val and/or Gly37Arg.
[0064] In mice, the dominant SOD1G93A mutation is associated with
ALS-like phenotype. Thus, in certain embodiments, the present
invention comprises mouse motor neurons comprising a SOD1G93A
mutation. In certain embodiments, the present invention comprises
human motor neurons comprising a SOD1G93A mutation.
[0065] A number of changes characteristic of neurodegeneration in
ALS were observed in mouse mutant SOD1G93A motor neurons between 14
and 28 days (for additional detail, see Examples section below).
First, the SOD1G93A protein changed its intracellular localization,
forming inclusions that increased in size and density. Second, the
levels of ubiquitin increased. Third, some motor neurons expressed
activated caspase-3 and displayed cytoplasmic staining with
cytochrome c antibodies. Finally, a significant difference in
survival was observed between mutant SOD1G93A motor neurons and the
controls. Thus, many of the late onset pathologies observed in both
human ALS and SOD1G93A mice are recapitulated in this in vitro
model, including the loss of motor neurons, which is ultimate cause
of symptoms in patients.
[0066] In certain embodiments, methods of the present invention
comprise using human and/or non-human SOD1 mutant motor neurons to
screen for test agents that affect motor neuron differentiation,
development and/or survival. In certain embodiments, methods of the
present invention comprise using such SOD1 mutant motor neurons to
identify a factor that has a non-cell autonomous effect on the
differentiation, development and/or survival of a motor neuron.
[0067] In certain embodiments, a motor neuron differentiated from
an embryonic stem cell comprises a mutation in a gene associated
with neurodegenerative disease, which gene is present as a
transgene. For example, an endogenous gene associated with a
neurodegenerative disease may be deleted or otherwise inactivated
in an embryonic stem cell from which such a motor neuron is derived
by any of a variety of techniques known to those skilled in the
art, and a transgene comprising a mutant copy of the endogenous
gene may be introduced into the embryonic stem cell by any of a
variety of techniques known to those skilled in the art. In certain
embodiments, such a transgene is integrated into the genome of the
differentiated motor neuron. In certain embodiments, such a
transgene is not integrated into the genome of the differentiated
motor neuron.
Conditions Affecting Motor Neuron Differentiation, Development
and/or Survival
[0068] The present invention encompasses the recognition that
proper differentiation, development and/or survival of a cell can
be influenced by its environment. For example, non-cell autonomous
processes can contribute to the differentiation, development and/or
survival of a cell. In certain embodiments, the present invention
provides novel system and compositions for studying such non-cell
autonomous processes and for identifying factors that mediate such
non-cell autonomous processes.
[0069] In certain embodiments, methods and compositions of the
present invention are used to study non-cell autonomous processes
that contribute to the proper differentiation, development and/or
survival of a motor neuron. For example, both autonomous defects in
motor neurons and toxic non-cell autonomous interactions with other
cell types in the spinal cord have been implicated in ALS pathology
(Bruijn et al., 2004; Clement et al., 2003; Boillee et al., 1995;
Beers, D. R. et al. Wild-type microglia extend survival in PU.1
knockout mice with familial amyotrophic lateral sclerosis. Proc
Natl Acad Sci USA 103, 16021-6, 2006). Methods and compositions of
the present invention are well suited to the identification and
study of factors that mediate non-cell autonomous effects of other
cell types on motor neurons, leading to ALS.
[0070] Several studies have suggested that cells within the spinal
cord may have pathological, non-cell autonomous affects on motor
neurons or on the rate of disease progression (Clement et al.,
2003; Boillee et al., 2006). However, these studies were of limited
utility since they were not able to resolve the identity of cell
types that caused these affects and/or were not able to determine
whether they acted directly to affect motor neuron survival. The
present invention encompasses the discovery and recognition that
cultures of ES cell derived motor neurons contain other cell types,
including astroglia, and that these ES cell derived cells have a
non-cell autonomous affect on motor neuron survival in vitro (see
Examples section below). The effects of co-culturing motor neurons
with primary glia from SOD1G93A mice and mice expressing the
wild-type SOD1 protein were systematically examined. It was
discovered that mutant SOD1G93A glia reduced the survival of both
wild type and mutant motor neurons. However, the effect was
significantly greater on mutant SOD1G93A motor neurons. Therefore,
the presently described studies show for the first time that an ALS
genotype in glial cells directly and negatively affects the
survival of motor neurons and they confirm that there is a cell
autonomous component to motor neuron degeneration.
[0071] Consistent with the present disclosure, Nagai et al. have
shown that primary astroctye cultures expressing ALS-associated
mutant SOD1 proteins contain diffusible factor(s) that are toxic to
both primary and ES cell-derived motor neurons (Makiko Nagai, D. B.
R., Tetsuya Nagata, Alcmene Chalazonitis, Thomas M. Jessell, Hynek
Wichterle, Serge Przedborski. Astrocytes expressing ALS-associated
SOD1 mutants release factors selectively toxic to spinal motor
neurons. Nature Neuroscience, 2007). In Nagai et al.'s study, motor
neurons were the only cell types affected by these mutant glial
cells and only SOD1G93A glial cells, not muscle cells or
fibroblasts, adversely affected motor neuron survival. Although in
Nagai et al's study, mutant primary neurons exhibited morphometric
alterations, their survival up to 14 days in culture was
indistinguishable from that of their wild-type counterparts. In the
presently described studies, differences in survival between
wild-type SOD1 and mutant SOD1G93A ES cell-derived motor neurons
were observed at 14 and 28 days in culture. The differences between
the two studies may originate in the source (embryo or ES
cell-derived) or number of the motor neurons used and the timeframe
of the investigations.
[0072] In certain embodiments, the present invention provides
methods for identifying and studying non-cell autonomous factors
produced by glial and/or other cells, which factors influence the
differentiation, development and/or survival of motor neurons. For
example, motor neurons may be cultured in the present of mutant
glial cells, and the survival of such motor neurons may be compared
to the survival of motor neurons cultured in the presence of wild
type glial cells. A difference in survival of motor neurons
indicates that a mutation present in such a glial cell is important
in mediating proper survival of motor neurons. In certain
embodiments, such a mutation in a glial cell results in an
alteration in the quantity and/or quality of a protein encoded by a
gene in which the mutation is located, which protein may be a
factor that contributes to proper survival of motor neurons. In
certain embodiments, a mutation in a glial cell results in an
alteration in the quantity and/or quality of a protein that is not
encoded by gene in which the mutation is located. For example, a
mutation may alter the quantity and/or quality of a produced
transcription factor, which transcription factor contributes to the
proper regulation and/or expression of a second protein, which
second protein may be a factor that mediates proper survival of
motor neurons. In certain embodiments, a mutation in a glial cell
results in an alteration in the quantity and/or quality of a factor
that contributes to proper survival of motor neurons, which factor
is not a protein (e.g. a small molecule, a lipid, a hormone, etc.).
Those of ordinary skill in the art will be aware of a variety of
other ways in which a mutation in a particular gene may affect a
factor that contributes to the proper survival of motor
neurons.
[0073] In certain embodiments, embryonic stem cells are induced to
differentiate into motor neurons in the presence of glial cells.
Such embodiments are useful in the study of normal motor neuron
differentiation, development and/or survival, and can be expected
to provide useful insights into possible causes, treatments and/or
cures of various neurodegenerative diseases.
[0074] In certain embodiments, embryonic stem cells are induced to
differentiate into motor neurons in the presence of mutant glial
cells, and differentiation, development and/or survival of such
motor neurons may be compared to differentiation, development
and/or survival of motor neurons cultured in the presence of wild
type glial cells. A difference in differentiation, development
and/or survival of the motor neurons indicates that a mutation
present in the glial cell contributes to proper differentiation,
development and/or survival of motor neurons. In certain
embodiments, a mutation in a glial cell results in an alteration in
the quantity and/or quality of a protein encoded by gene in which
the mutation is located, which protein is a factor that contributes
to proper differentiation, development and/or survival of motor
neurons. In certain embodiments, a mutation in a glial cell results
in an alteration in the quantity and/or quality of a protein that
is not encoded by gene in which the mutation is located. For
example, a mutation may alter the quantity and/or quality of a
produced transcription factor, which transcription contributes to
the proper regulation and/or expression of a second protein, which
second protein is a factor that mediates proper differentiation,
development and/or survival of motor neurons. In certain
embodiments, a mutation in a glial cell results in an alteration in
the quantity and/or quality of a factor that is important for
proper differentiation, development and/or survival of motor
neurons, which factor is not a protein (e.g. a small molecule, a
lipid, a hormone, etc.). Those of ordinary skill in the art will be
aware of a variety of other ways in which a mutation in a
particular gene may affect a factor that is important in the proper
survival of motor neurons.
[0075] In certain embodiments, mutant glial cells to be used in
accordance with the present invention to identify and/or study
factors that contribute to proper differentiation, development
and/or survival of motor neurons comprise a mutation in a gene
associated with a neurodegenerative disease. As but a few
non-limiting examples, such a neurodegenerative disease may include
ALS, Parkinson's disease, Alzheimer's disease or any number of
other neurodegenerative diseases known to those of skill in the
art. A variety of genes are known to be associated with
neurodegenerative diseases. As one non-limiting example, mutations
in the SOD1 gene are known to be associated with the
neurodegenerative disease ALS. Thus, in certain embodiments, mutant
glial cells to be used in accordance with the present invention to
identify and/or study factors that contribute to proper
differentiation, development and/or survival of motor neurons
comprise a mutation in the SOD1 gene. In humans, Gly92Ala, Ala4Val
and Gly37Arg mutations are associated with the onset and
progression of ALS. Thus, in certain embodiments, mutant glial
cells to be used in accordance with the present invention to
identify and/or study factors that contribute to proper
differentiation, development and/or survival of motor neurons
comprise a SOD1G93A mutation such as Gly92Ala, Ala4Val and/or
Gly37Arg. Those of ordinary skill in the art will be aware of a
variety of other SOD1 mutant alleles associated with ALS, which
mutant alleles can be advantageously used in accordance with one or
more of the embodiments described herein.
[0076] In certain embodiments, the present invention provides
methods for identifying and/or studying non-cell autonomous factors
produced by non-glial cells, which factors influence the
differentiation, development and/or survival of motor neurons.
Non-limiting examples of such non-glial cells that can influence
differentiation, development and/or survival of motor neurons
include microglial cells, oligodendrocytes, astrocytes, other
neuronal cell types in spinal cords (e.g. interneurons) and/or
other cells that are in contact with neurons (e.g. muscle cells).
Those of ordinary skill in the art will be aware of a variety of
other non-glial cells that can influence the differentiation,
development and/or survival of motor neurons.
[0077] In certain embodiments, methods of the present invention
comprise identifying a factor that has a non-cell autonomous effect
on survival of a motor neuron. In certain embodiments, such methods
comprise providing a motor neuron, identifying a first glial cell,
which first glial cell negatively affects survival of the motor
neuron, identifying a second glial cell, which second glial cell
does not negatively affect survival of the motor neuron, isolating
a factor from the either the first or second glial cell, wherein
the factor is either: i) a factor from the first glial cell that
contributes to the negative effect on survival of the motor neuron;
or ii) a factor from the second glial cell that contributes to
survival of the motor neuron.
[0078] Factors that influence differentiation, development and/or
survival can be identified by any of a variety of methods known to
those of ordinary skill in the art. In certain embodiments, such a
factor is directly identified after determining that a given wild
type or mutant cell contributes to proper differentiation,
development and/or survival of a cell of interest such as, e.g., a
motor neuron. Non-limiting examples of such methods include
fractionation, mass spectrometry, protein chip analysis (e.g., if
such a factor comprises a protein), chromatography, etc. Those of
ordinary skill in the art will be aware of and will be able to
employ suitable techniques for directly identifying such a
factor.
[0079] In certain embodiments, such a factor is identified
indirectly. For example, a factor may comprise a protein encoded by
a gene. In such embodiments, a gene that encodes such a factor may
be identified by any of a variety of techniques such as, for
example, differential display, gene chip analysis, RT-PCR, direct
sequencing, etc. Those of ordinary skill in the art will be aware
of and will be able to employ suitable techniques for identifying
such a factor indirectly.
[0080] In certain embodiments, a combination of two or more factors
may together contribute to differentiation, development and/or
survival of a cell of interest such as, for example, a motor
neuron. Methods and compositions of the present invention may be
advantageously used to identify such a combination of factors.
Disease Modeling and Drug Screening
[0081] In certain embodiments, the present invention offers great
potential for developing better models for the study of human
disease and/or better methods of treatment. In certain embodiments,
methods of the present invention employ embryonic stem cells and/or
differentiated cells that comprise alterations (e.g., deletions,
rearrangements, duplications, substitutions, etc.) in genes
associated with a particular disease. In certain embodiments, a
disease of interest is a neurodegenerative disease. Exemplary
neurodegenerative diseases that can be studied using compositions
and methods of the present invention include, but are not limited
to, ALS, Parkinson's disease, and Alzheimer's disease. Those
skilled in the art will be aware of a number of other
neurodegenerative diseases that can be studied using compositions
and methods the present invention.
[0082] In certain embodiments, a disease of interest is modeled
and/or studied by inducing an embryonic stem cell line (that has,
for example, been generated by any of the variety of methods of the
present invention to contain one or more alterations in one or more
genes associated with a disease of interest) to differentiate by
culturing such a cell line under appropriate differentiation
conditions. For example, an embryonic stem cell line may be
generated that contains one or more alterations in one or more
genes associated with a neurological degenerative disease, e.g.
ALS, or any other neurodegenerative disease of interest. Such an
embryonic cell line may be induced to differentiate into motor
neurons by subjecting it to appropriate differentiation conditions.
Those of ordinary skill in the art will be aware of appropriate
differentiation conditions. By observing differentiation,
development and/or survival of such a motor neuron and comparing it
to differentiation, development and/or survival of a motor neuron
derived from an embryonic stem cell line that does not contain the
genetic alteration(s) associated with the neurodegenerative disease
of interest, the practitioner can achieve a better understanding of
the genetic basis of disease progression and pathogenesis.
[0083] Additionally or alternatively, an embryonic stem cell line
that is generated to contain one or more alterations in one or more
genes associated with a disease of interest may be used to screen
for agents (e.g., a cell, a small molecule, a hormone, a vitamin, a
nucleic acid molecule, an enzyme, an antibody, an amino acid, a
virus, etc.) that can be used, for example, in the treatment,
prevention and/or cure of that disease. For example, such an
embryonic stem cell line may be induced to differentiate into a
cell type associated with the disease of interest by placing it
under appropriate differentiation conditions. Before, during and/or
after differentiation, such a cell may be subjected to a test agent
in order to determine whether that agent has an effect on
differentiation, development and/or survival of the cell. In
certain embodiments, an embryonic stem cell comprising a mutation
associated with a neurodegenerative disease is induced to
differentiate into a motor neuron, which motor neuron is subjected
to an agent before, during and/or after differentiation.
[0084] In certain embodiments, compositions and methods of the
present invention are useful in studying and/or modeling diseases
that to date, have not been amenable to such study and/or modeling.
For instance, in many cases, by the time a patient is diagnosed
with a particular disease, the early events of disease progression
and pathogenesis have already occurred, making it difficult or
impossible to determine and track the molecular, cellular, or other
changes that occur during the course of the disease. Using
inventive methods and compositions disclosed herein, researchers
will now be able to determine and study such molecular, cellular,
or other changes, leading to a better understanding of disease
progression and pointing the way to more effective treatments.
[0085] In certain embodiments, methods of the present invention
comprise identifying an agent that affects the survival of a SOD1
mutant motor neuron. In certain embodiments, such methods comprise
providing a SOD1 mutant motor neuron, providing a test agent,
contacting the SOD1 mutant motor neuron with the test agent, and
determining the effect of the test agent on survival of the SOD1
mutant motor neuron by comparing the survival of the SOD1 mutant
motor neuron to the survival of a control motor neuron lacking the
SOD1 mutant allele, which control motor neuron is contacted with
the test agent for a period of time and under conditions identical
to that of the SOD1 mutant motor neuron. In certain embodiments, a
SOD1 motor neuron used in such methods is derived from an embryonic
stem cell. In certain embodiments, a SOD1 motor neuron used in such
methods comprises a SOD1 mutation associated with a
neurodegenerative disease of interest, e.g. ALS.
[0086] In certain embodiments, compositions and methods of the
present invention are used to study and/or model diseases and/or to
screen for agents that can be used in the treatment, prevention
and/or cure of diseases, which compositions or methods comprise or
make use of human embryonic stem cell lines and/or differentiated
cells derived from such human embryonic stem cell lines.
[0087] In certain embodiments, compositions and methods of the
present invention are used to study and/or model diseases and/or to
screen for agents that can be used in the treatment, prevention
and/or cure of diseases, which compositions or methods comprise or
make use of non-human embryonic stem cell lines and/or
differentiated cells derived from such non-human embryonic stem
cell lines. Use of non-human stem cell lines and/or differentiated
cells derived from them is advantageous when ethical and/or
practical limitations prevent the use of human stem cell lines.
Non-limiting examples of non-human stem cell lines (and/or
differentiated cells derived from them) that may be used in
accordance with the present invention to study and/or model
diseases and/or to screen for agents that can be used in the
treatment, prevention and/or cure of diseases include mouse, rat,
and primate stem cell lines. Those of ordinary skill in the art
will be aware of a variety of other non-human stem cell lines that
will be useful, and those of ordinary skill in the art will be able
to generate such stem cell lines by employing one or more methods
of the present invention.
[0088] Compositions and methods of the present invention may be
used to study and/or model of any of a variety of diseases or
conditions. Non-limiting examples of such diseases or conditions
include childhood congenital malformations, sickle cell anemia,
neurological diseases such as amyotrophic lateral sclerosis (also
known as Lou Gehrig's disease), Parkinson's disease, Alzheimer's
disease or any of a variety of other neurological diseases, Down
syndrome (a condition that arises in patients with trisomy for
chromosome 21 resulting in dysregulated signaling through the
NFAT/calcineurin pathway), etc. One of ordinary skill in the art
will be aware of a variety of other disease conditions that may be
modeled and/or studied by generating embryonic stem cells according
to one or more methods of the present invention.
[0089] In certain embodiments, compositions and methods of the
present invention comprise or make use of human embryonic stem cell
lines and/or differentiated cells derived from a patient suffering
from and/or predicted to suffer from a disease of interest.
Patient-specific, immune-matched human embryonic stem cells have
the potential to be of great biomedical importance for studies of
disease and development. For example, certain patients may respond
better to a given therapy or drug regimen than other patients.
Additionally or alternatively, certain patients may experience
fewer and/or less severe side effects after being administered a
given therapy or drug regimen than other patients. By utilizing
embryonic stem cells that contain the genetic complement of a
patient suffering from and/or predicted to suffer from a disease of
interest, and permitting such cells to differentiate into a cell
type associated with that disease, it will be possible to better
predict which therapy or drug regimen will be most beneficial
and/or result in the least detrimental side effects.
[0090] Furthermore, patient-specific human embryonic stem cells
have the potential to be of great biomedical importance for the
discovery and/or development of patient-specific agents that can be
used to prevent and/or treat a disease of interest. By utilizing
embryonic stem cells containing the genetic complement of a patient
suffering from a disease of interest, permitting such cells to
differentiate into a cell type associated with that disease, and
subjecting such cells to one or more test agents before, during
and/or after differentiation, discovery and/or development of an
agent that will be most beneficial and/or result in the least
detrimental side effects for that particular patient will be
facilitated. Those of ordinary skill in the art will be able to
apply methods and compositions of the present invention to the
discovery and/or development of an agent specific for a patient
and/or disease of interest.
[0091] In certain embodiments, a disease of interest is modeled
and/or studied by inducing an embryonic stem cell line to
differentiate into a cell type of interest by culturing such a cell
line under appropriate differentiation conditions, wherein the
embryonic stem cell line is differentiated in the presence of one
or more different cell types that contribute to proper
differentiation, development and/or survival of the cell type of
interest. For example, an embryonic stem cell line containing one
or more alterations in one or more genes associated with a
neurological degenerative disease, e.g. ALS or any other
neurodegenerative disease of interest, may be induced to
differentiate into a motor neuron in the presence of one or more
glial cells. By observing the differentiation, development and/or
survival of such a motor neuron and comparing it to the
differentiation, development and/or survival of a motor neuron
derived from an embryonic stem cell line that does not contain the
genetic alteration(s) associated with the neurodegenerative disease
of interest, the practitioner can achieve a better understanding of
the genetic basis of disease progression and pathogenesis.
[0092] In certain embodiments, an embryonic stem cell line
containing a wild type genome is induced to differentiate into a
cell type of interest in the presence of one or more different cell
types that contribute to proper differentiation, development and/or
survival of the cell type of interest. By observing the
differentiation, development and/or survival of such a cell type of
interest and comparing it to the differentiation, development
and/or survival of a cell type of interest in the absence of such
different cell types, the practitioner can achieve a better
understanding of the genetic basis of normal differentiation,
development and/or survival a cell type of interest.
[0093] In certain embodiments, a cell type of interest is induced
to differentiate in the presence of one or more different cell
types that contribute to proper differentiation, development and/or
survival of the cell type of interest, wherein the one or more
different cell types comprise one or more mutations that affect
differentiation, development and/or survival of the cell type of
interest. By observing the differentiation, development and/or
survival of the cell type of interest in such an environment and
comparing it to the differentiation, development and/or survival of
a cell type of interest differentiated in the presence of wild type
different cell types, the practitioner can achieve a better
understanding of non-cell autonomous factors and processes that
contribute to proper differentiation, development and/or survival
of the cell type of interest.
[0094] In certain embodiments, the present invention provides
systems and methods for identifying agents (e.g., a cell, a small
molecule, a hormone, a vitamin, a nucleic acid molecule, an enzyme,
an antibody, an amino acid, a virus, etc.) that can be used, for
example, in the treatment, prevention and/or cure of a disease of
interest, wherein the differentiation, development and/or survival
of a cell type of interest implicated in the onset and/or
progression of the disease of interest is influenced by one or more
different cell types. For example, such an embryonic stem cell line
may be induced to differentiate into a cell type associated with
the disease of interest by placing it under appropriate
differentiation conditions in the presence of one or more different
cell types that contribute to proper differentiation, development
and/or survival of the cell type of interest. Before, during and/or
after differentiation, such a cell may be subjected to a test agent
in order to determine whether that agent has an effect on
differentiation, development and/or survival of the cell.
[0095] In certain embodiments, an embryonic stem cell comprising a
mutation associated with a neurodegenerative disease is induced to
differentiate into a motor neuron in the presence of glial cells,
which motor neuron is subjected to an agent before, during and/or
after differentiation. In certain embodiments, such glial cells are
wild type. In certain embodiments, such glial cells comprise one or
more mutations that alter the proper differentiation, development
and/or survival of the motor neuron. For example, such glial cells
may comprise a mutation that induces the motor neuron to display a
phenotype characteristic of a disease of interest, such as for
example, a neurodegenerative disease including, without limitation,
ALS. In certain embodiments, such glial cells comprise a mutation
in the SOD1 gene, for example a SOD1G93A mutation. Thus, in certain
embodiments, the present invention provides systems and methods for
identifying agent(s) that prevent, ameliorate, or reverse the
adverse effects of such SOD1G93A mutant glial cells on the proper
differentiation, development and/or survival of a motor neuron,
including both wild type and mutant motor neurons. In certain
embodiments, such identified agents are used to prevent, treat
and/or cure ALS.
[0096] As described in the Examples herein, it has been discovered
that certain genes are overexpressed in glia having a mutation in
the SOD1 gene. Moreover, it has been discovered that agents that
target such genes or expression products of such genes can promote
survival of motor neurons (e.g., motor neurons produced and/or
cultured according to a method described herein). Accordingly, in
some embodiments, the present invention provides methods of
identifying a test agent that modulates survival of a motor neuron,
wherein the test agent targets a gene or product of a gene
overexpressed in SOD1 mutant glia. In some embodiments, a test
agent targets (e.g., inhibits expression or activity of) a gene or
product of a gene in Table 2 (e.g., a gene or product of a gene
selected from serine (or cysteine) preptidase inhibitor, Glade A,
member 1b (Serpina1b); protein tyrosine phosphatase, non-receptor
type 7 (Ptpn7); poly (ADP-ribose) polymerase family, member 12
(Zc3hdc1); prostaglandin D2 receptor (Ptgdr); glia maturation
factor, beta (Gmfb); ATP-binding cassette, sub-family A (ABC1),
member 5 (Abca5); developing brain homeobox 2 (Dbx2); RAB6B, member
RAS oncogene family (Rab6b); cut-like 1 (Cut11); adenosine
deaminase (Ada); receptor coactivator 6 interacting protein
(Ncoa6ip); interferon-induced protein 35 (i1i35); RAB, member of
RAS oncogene family-like 2A (Rab12a); STEAP family member 4
(A1481214); cytoglobin (cygb); Duffy blood group, chemokine
receptor (Dfy); chondrolectin (Chod1); neurexin 1 (NRXN1); defensin
beta 11 (Defb11); RUN and SH3 domain containing 2 (RUSC2);
matrilin-4 (matn4); X-linked lymphocyte-regulated 3A (Xlr3a); C-C
motif chemokine 8 (ccl8); T-cell immunoglobulin and mucin domain
containing 4 (Timd4); odd-skipped related 2 (osr2); RIKEN cDNA
9130213B05 gene (9130213B05Rik); reversion-inducing-cysteine-rich
protein with kazal motifs (Reck); olfactory receptor 116 (Olfr116);
protogenin homolog (Prtg; A230098A12Rik); sonic hedgehog (Shh);
formyl peptide receptor 1 (Fpr1); pro-platelet basic protein
(chemokine (C-X-C motif) ligand 7; CXCL7); DnaJ (Hsp40) homolog,
subfamily B, member 3 (DNAJB3); defensin beta 10 (Deft 10);
apolipoprotein A-II (Apoa2); collagen, type I, alpha 2 (Col1a2);
islet cell autoantigen 1-like (Ical1; 1700030B17Rik); ATPase, class
II, type 9A (Atp9a); chemokine (C-C motif) ligand 5 (CCL5); solute
carrier family 39 (zinc transporter), member 14 (Sc139A14); serum
amyloid A 3 (Saa3); RIKEN cDNA 3632451O06 gene (3632451O06Rik);
attractin like 1 (Atrnl1); Alstrom syndrome 1 (Alms 1); NK2
homeobox 2 (Nkx2-2); kallikrein related-peptidase 8 (Klk8; Prss19);
histone cluster 1, H4k (Hist1h4k); EPH receptor B2 (Ephb2);
synaptotagmin XII (Syt12); forkhead box Q1 (Foxq1); splicing
factor, arginine/serine-rich 16 (Sfrs16); LanC lantibiotic
synthetase component C-like 1 (Lancl1); and MARCKS-like 1 (M1p)).
In some embodiments, a test inhibits expression or activity of a
gene or gene product in Table 2 which is involved in inflammation.
In some embodiments, a test agent inhibits expression or activity
of a prostaglandin D receptor.
[0097] In certain embodiments, a test agent that inhibits
expression or activity of a gene or product of a gene in Table 2
includes a small molecule, an antibody, a hormone, a vitamin, a
nucleic acid molecule, an enzyme, an amino acid, and/or a
virus.
[0098] In certain embodiments, a test agent is a nucleic acid
molecule, e.g., a nucleic acid molecule that mediates RNA
interference. RNA interference refers to sequence-specific
inhibition of gene expression and/or reduction in target RNA levels
mediated by an at least partly double-stranded RNA, which RNA
comprises a portion that is substantially complementary to a target
RNA. Typically, at least part of the substantially complementary
portion is within the double stranded region of the RNA. In some
embodiments, RNAi can occur via selective intracellular degradation
of RNA. In some embodiments, RNAi can occur by translational
repression. In some embodiments, RNAi agents mediate inhibition of
gene expression by causing degradation of target transcripts. In
some embodiments, RNAi agents mediate inhibition of gene expression
by inhibiting translation of target transcripts. Generally, an RNAi
agent includes a portion that is substantially complementary to a
target RNA. In some embodiments, RNAi agents are at least partly
double-stranded. In some embodiments, RNAi agents are
single-stranded. In some embodiments, exemplary RNAi agents can
include small interfering RNA (siRNA), short hairpin RNA (shRNA),
and/or microRNA (miRNA). In some embodiments, an agent that
mediates RNAi includes a blunt-ended (i.e., without overhangs)
dsRNA that can act as a Dicer substrate. For example, such an RNAi
agent may comprise a blunt-ended dsRNA which is >25 base pairs
length. RNAi mechanisms and the structure of various RNA molecules
known to mediate RNAi, e.g. siRNA, shRNA, miRNA and their
precursors, are described, e.g., in Dykxhhorn et al., 2003, Nat.
Rev. Mol. Cell. Biol., 4:457; Hannon and Rossi, 2004, Nature,
431:3761; and Meister and Tuschl, 2004, Nature, 431:343; all of
which are incorporated herein by reference.
[0099] In some embodiments, a nucleic acid that mediates RNAi
includes a 19 nucleotide double-stranded portion, comprising a
guide strand and an antisense strand. Each strand has a 2 nt 3'
overhang. Typically the guide strand of the siRNA is perfectly
complementary to its target gene and mRNA transcript over at least
17-19 contiguous nucleotides, and typically the two strands of the
siRNA are perfectly complementary to each other over the duplex
portion. However, as will be appreciated by one of ordinary skill
in the art, perfect complementarity is not required. Instead, one
or more mismatches in the duplex formed by the guide strand and the
target mRNA is often tolerated, particularly at certain positions,
without reducing the silencing activity below useful levels. For
example, there may be 1, 2, 3, or even more mismatches between the
target mRNA and the guide strand (disregarding the overhangs).
[0100] Molecules having the appropriate structure and degree of
complementarity to a target gene will exhibit a range of different
silencing efficiencies. A variety of additional design criteria
have been developed to assist in the selection of effective siRNA
sequences. Numerous software programs that can be used to choose
siRNA sequences that are predicted to be particularly effective to
silence a target gene of choice are available (see, e.g., Yuan et
al., 2004, Nuc. Acid. Res., 32:W130; and Santoyo et al., 2005,
Bioinformatics, 21:1376; both of which are incorporated herein by
reference).
[0101] As will be appreciated by one of ordinary skill in the art,
RNAi may be effectively mediated by RNA molecules having a variety
of structures that differ in one or more respects from that
described above. For example, the length of the duplex can be
varied (e.g., from about 17-29 nucleotides); the overhangs need not
be present and, if present, their length and the identity of the
nucleotides in the overhangs can vary (though most commonly
symmetric dTdT overhangs are employed in synthetic siRNAs).
[0102] In certain embodiments, the present invention provides
methods of modulating survival of a motor neuron by contacting a
motor neuron (in vitro or in vivo) with an agent that reduces the
expression or activity of a prostaglandin D receptor. Agents that
reduce expression or activity (e.g., antagonize) prostaglandin D
receptors include MK 0524
((3R)-4-(4-chlorobenzyl)-7-fluoro-5-(methylsulfonyl)-1,2,3,4-tetrahydrocy-
clopenta[b]indol-3-yl]-acetic acid; Sturino et al., J. Med. Chem.
50(4):794-806, 2007) and analogs thereof, and compounds disclosed
in Mitsumori et al., Curr. Pharm. Des., 10(28):3533-8, 2004;
Beaulieu et al., Bioorg Med Chem Lett. 18(8):2696-700, 2008; Torisu
et al., Eur J Med Chem. 40(5):505-19, 2005; U.S. Pat. Pub. Nos.
20010051624, 20030055077, 20040180934, 20070244131, 20070265278,
20070265291, 20080194600, and U.S. Pat. No. 7,153,852.
Human Stem Cells and Cells Derived from Them
[0103] Although mouse genetics has provided a sophisticated
understanding of the cellular and molecular mechanisms that
contribute to familial ALS, it cannot inform us as to the actual
relevance of its findings to human patients. In fact, due to the
fundamental differences between human and mouse physiology, many
observations made in mouse disease models have not translated well
to human experimental systems or to the clinic. For example,
diabetes has been "cured" many times over in the NOD mouse model of
disease. However, few of the observations and experimental
therapies developed in this mouse model have proven relevant to the
human disease (Shoda, L. K. et al. A comprehensive review of
interventions in the NOD mouse and implications for translation.
Immunity. 23, 115-26, 2005). Similarly, mutations in the RB gene
that lead to Retinoblastoma in human patients cause an independent
range of tumors in mice carrying the same genetic lesion (Goodrich,
D. W. Lee, W.H. Molecular characterization of the retinoblastoma
susceptibility gene. Biochim Biophys Acta. 1155, 43-61, 1993;
Williams B. O. et al. Extensive contribution of Rb-deficient cells
to adult chimeric mice with limited histopathological consequences.
EMBO J., 13, 4251-9, 1994). As a result many therapeutics developed
in animal, or based on drug-targets discovered in animal models,
fail in clinical trials (Shoda et al., 2005; Gawarylewski, A. The
trouble with animal models. The Scientist. 21 (7), 45-51, 2007;
Rubin, L. L. Stem cells and drug discovery: the beginning of a new
era? Cell. 132, 549-52, 2008). The cost of these failures is
substantial (Gawarylewski et al., 2007; Rubin, 2008).
[0104] Considerable time, effort and expense would be saved if
fundamental observations made in animal models could be routinely
validated in the relevant human cell types. A potential solution is
to use human embryonic stem cells as a renewable source of these
cells for the study of disease and for drug target validation. The
discoveries described herein have demonstrated the usefulness of
motor neurons derived from human ES cells in validating findings
from mouse models of ALS (see e.g., Examples 7 and 8 below). It has
been found that glia cells over-expressing the SOD1G93A mutation
negatively affect the viability of human ES cell derived motor
neurons in a time dependent manner. Such non-cell autonomous effect
of glia is specific for motor neurons, as it does not seems
interfere with the survival of human interneurons.
[0105] In certain embodiments, in vitro model systems of the
present invention utilize human motor neurons derived from human ES
cells. In certain embodiments, a human motor neuron differentiated
from a human embryonic stem cell comprises a mutation in a gene
associated with neurodegenerative disease. As non-limiting
examples, such a neurodegenerative disease may include ALS,
Parkinson's disease, Alzheimer's disease or any number of other
neurodegenerative diseases known to those of skill in the art. A
variety of genes are known to be associated with neurodegenerative
diseases. As one non-limiting example, mutations in the SOD1 gene
are known to be associated with the neurodegenerative disease ALS.
For example, in humans, Gly92Ala, Ala4Val and Gly37Arg mutations
are associated with the onset and progression of ALS. Those of
ordinary skill in the art will be aware of other SOD1 mutations
associated with ALS. In certain embodiments, compositions and
method of the present invention comprise or employ human motor
neurons comprising a SOD1G93A mutation such as Gly92Ala, Ala4Val
and/or Gly37Arg.
[0106] In certain embodiments, in vitro model systems of the
present invention comprising human motor neurons may be
advantageously employed for the human physiological validation of
findings from animal models, such as, without limitation, animal
models (e.g., mouse) that recapitulate in whole or in part
neurodegenerative diseases such as ALS and/or other
neurodegenerative diseases.
[0107] In certain embodiments, in vitro model systems of the
present invention comprising human motor neurons may be
advantageously employed to identify novel factors, agents, etc.
that affect motor neuron development and/or contribute to a disease
state, such as, without limitation, a neurodegenerative disease,
e.g. ALS, in the absence of an animal model. In certain
embodiments, in vitro model systems of the present invention
comprising human motor neurons may be advantageously employed to
illuminate the target, efficacy, toxicity, mode of action, etc. of
factors, agents, etc. that affect motor neuron development and/or
contribute to a disease state, such as, without limitation, a
neurodegenerative disease, e.g. ALS.
[0108] In certain embodiments, methods of the present invention
employ human mutant motor neurons that comprise a mutation in a
gene associated with a neurodegenerative disease, for example ALS.
One non-liming example of a gene associated with the
neurodegenerative disease ALS is SOD1. A variety of SOD1 mutant
alleles are known to be associated with ALS, including without
limitation, SOD1G93A. In certain embodiments, methods of the
present invention utilize human motor neurons comprising a SOD1
mutant allele (e.g., SOD1G93A) to screen for test agents that
affect motor neuron differentiation, development and/or survival.
In certain embodiments, methods of the present invention comprise
using such SOD1 mutant motor neurons to identify a factor that has
a non-cell autonomous effect on the differentiation, development
and/or survival of a motor neuron.
[0109] In certain embodiments, methods of the present invention
employ human mutant cells that are not motor neurons, which mutant
cells comprise a mutation in a gene associated with a
neurodegenerative disease, for example ALS. As but one non-limiting
example, certain methods the present invention employ glial cells
comprising a mutation in a gene associated with a neurodegenerative
disease such as ALS. In certain embodiments, methods the present
invention employ glial cells comprising a mutation in a SOD1 gene,
such as, without limitation, SOD1G93A.
[0110] Those skilled in the art will be aware of other gene
mutations associated with neurodegenerative diseases, and will be
able to use methods and compositions described herein to validate
results of animal models, to identify novel factors, agents, etc.
that affect motor neuron development and/or contribute to a disease
state, and/or to illuminate the target, efficacy, toxicity, mode of
action, etc. of factors, agents, etc. that affect motor neuron
development and/or contribute to a disease state.
Non-Human Applications
[0111] Embryonic stem cells of the present invention and/or cells
derived from them can be advantageously used in the study and/or
modeling of human diseases, although one of ordinary skill in the
art will understand that the present disclosure is not limited to
human applications. Thus, for example, non-human embryonic stem
cell lines may be used in the study and/or modeling of diseases
associated with pets (e.g., cats, dogs, rodents, etc.) as well as
commercially important domestic animals (e.g., cows, sheep, pigs,
etc.). Additionally or alternatively, non-human embryonic stem cell
lines may be used to screen for agents that can be used in the
prevention and/or treatment of diseases associated with pets and/or
commercially important domestic animals.
EXAMPLES
Example 1
Derivation of ES Cell Lines from the SOD1G93A ALS Mouse Model
[0112] Embryonic stem cell lines were derived by crossing
hemizygous mice carrying either the pathogenic (mutant) SOD1G93A
transgene or the non-pathogenic (wild-type) SOD1 transgene (Gurney
et al., 1994) with hemizygous mice carrying a transgenic reporter
gene in which green fluorescent protein (GFP) expression is
controlled by promoter elements from the Hb9 gene (Hb9::GFP)
(Wichterle et al., 2002). The Hb9 gene encodes a homeodomain
transcription factor that is expressed in postmitotic motor neurons
(Arber, S. et al. Requirement for the homeobox gene Hb9 in the
consolidation of motor neuron identity. Neuron 23, 659-74, 1999;
Thaler, J. et al. Active suppression of interneuron programs within
developing motor neurons revealed by analysis of homeodomain factor
HB9. Neuron 23, 675-87, 1999). This Hb9::GFP transgene provides a
marker for the differentiation of ES cells into motor neurons
(Wichterle et al., 2002). Blastocyst stage embryos were retrieved
from the progeny of these crosses and used to derive ES cell lines
that were genotyped using the polymerase chain reaction (PCR) (FIG.
1A,B). These analyses identified ES cell lines that carried only
the Hb9::GFP transgene (Hb9GFP), lines that carried both Hb9::GFP
and the wild-type SOD1 transgene (SOD1), and a line that carried
both Hb9::GFP and the mutant SOD1G93A transgene (SOD1G93A).
[0113] To determine whether these ES cells recapitulate the proper
expression pattern of the Hb9::GFP reporter transgene, we assessed
GFP fluorescence in the undifferentiated ES cells and in chimeras
created by injecting these cells into non-transgenic blastocysts.
Each of the undifferentiated cell lines lacked obvious GFP
expression (For example FIG. 2A). However, in El 0.5 chimeras
created with these cells, highly specific GFP expression was
observed in the developing eye, hindbrain and spinal chord where
Hb9 is known to be expressed (FIG. 1D) (Wichterle et al., 2002;
Thaler et al., 1999). Immunostaining with antibodies that
preferentially recognize the human SOD1 protein confirmed the PCR
genotyping of the cell lines and showed that both the SOD1 and
SOD1G93A transgenes are expressed in the undifferentiated ES cells
(FIG. 1C).
Example 2
Production and Characterization of Motor Neurons by In Vitro
Differentiation of SOD1G93A ES Cells
[0114] To determine whether pathogenic properties associated with
ALS can be recapitulated in vitro, we generated motor neurons by
differentiating the transgenic ES cell lines as previously
described (Wichterle et al., 2002). Briefly, ES cells were
dissociated into a single cell suspension, allowed to spontaneously
aggregate into embryoid bodies (EBs) over 48 hours and then treated
with retinoic acid (RA) and soluble Sonic Hedgehog (Shh)
protein.sup.1 (Wichterle et al., 2002) for 5 days.
[0115] We found that the SOD1G93A genotype does not interfere with
the initial specification or differentiation of motor neurons, as
no significant qualitative or quantitative differences were
observed in the differentiation of the three cell lines. GFP
expression in EBs derived from the different cell lines, including
SOD1 G93A, first appeared 5 days after treatment with Shh and RA
(FIG. 2A). Two days later, when EBs were dissociated with papain
and plated, GFP positive cells with an obvious neuronal morphology
could be observed (FIG. 2A). We used fluorescence activated cell
sorting (FACS) to determine the percentage of differentiating ES
cells that expressed GFP and found no statistically significant
differences between the three cell lines (Hb9GFP 33%+/-4%, SOD1
33%+/-9%, SOD1G93A26%+/-6%) (see FIG. 6).
[0116] To confirm that the EB cells expressing GFP differentiated
into bone fide motor neurons, we dissociated the EBs and performed
immunostaining with antibodies that recognize proteins known to be
expressed in motor neurons (FIG. 2 B,C,D,E). As was previously
observed with normal ES cell lines (Wichterle et al., 2002), we
found that GFP positive cells derived from the SOD1G93A cell line
expressed a neuronal form of tubulin (Tuj1, FIG. 2B), the
transcription factors Hb9 and Isl1/2 (FIG. 2 C,D; FIG. 7) and the
enzymatic machinery required to generate acetylcholine (Chat, FIG.
2E).
Example 3
The SOD1G93a Genotype Affects the Survival of Motor Neurons in
Culture
[0117] ALS is a late onset, progressive neurodegenerative disease,
and mice carrying the human SOD1G93A transgene develop symptoms as
a consequence of motor neuron loss after several weeks. Therefore,
it seemed possible that motor neurons derived from ES cells might
display neurodegenerative properties only after they have been
maintained in culture for a prolonged length of time. To determine
the period of time that ES cell derived motor neurons can survive
in culture, we dissociated day 7 Hb9GFP and SOD1G93A EBs and plated
the resulting mixture of GFP positive and negative cells at two
different densities in the presence of neurotrophic factors
(Wichterle et al., 2002) (FIG. 3). We observed that the number of
GFP positive cells decreased precipitously during the first two
weeks, and then continued to decrease over the following weeks.
However, GFP positive cells could still be detected in both HB9GFP
and SOD1G93A derived cultures 54 days after plating (FIG. 3A).
Although GFP positive cells were present in both cultures at later
time points, the number of cells expressing GFP decreased more
rapidly in the SOD1G93A (FIG. 3C) cultures than in Hb9GFP control
cultures (FIG. 3B).
[0118] To confirm the affect of the SOD1G93A genotype on the number
of GFP positive cells, we differentiated both the SOD1G93A and
Hb9GFP ES cells into motor neurons, plated equal numbers of cells
at two different concentrations (8.times.10.sup.5 (n=3) and
4.times.10.sup.5 (n=3) EB cells per well) and counted the number of
GFP positive neurons in the cultures at 2 and 4 weeks (FIG.
3D,E,F,G). Under both plating conditions, significantly fewer GFP
positive cells were observed in the SOD1G93A cultures at both 2 and
4 weeks (FIG. 3D,E,F,G).
Example 4
Histopathological Hallmarks of ALS can be Observed in ES
Cell-Derived Motor Neurons
[0119] Hb9GFP and SOD1G93A ES cells differentiate into motor
neurons at a similar efficiency, but the cultures show differences
in the number of GFP positive cells over time. Thus, a pathological
process may underlie the preferential loss of GFP positive cells in
the SOD1G93A cultures. To investigate these processes and to
determine whether they mirror events that occur during the
progression of ALS, we examined motor neurons in culture for the
presence of histopathological hallmarks of the disease. Motor
neurons in ALS patients and transgenic mice carrying the SOD1G93A
allele accumulate protein inclusions that are recognized by
antibodies specific to the SOD1 protein (Boillee et al., 2006;
Bruijn et al., 19970. We therefore determined whether aggregation
of the mutant SOD1 protein accompanies the loss of GFP positive
motor neurons in SOD1G93A cultures by staining with antibodies
specific to the human SOD1 protein at 7, 14 and 21 days following
EB dissociation (FIG. 4A). At 7 and 14 days following dissociation
both the wild-type SOD1 protein and the mutant SOD1G93A protein
were localized broadly and evenly in the cytoplasm of GFP positive
motor neurons (FIG. 4B,A). However, at 14 days punctate structures
staining brightly with the SOD1 antibody could be observed in a
small proportion of motor neurons expressing the SOD1 G93A protein,
(FIG. 4A).
[0120] When cultures were examined 21 days after dissociation, a
shift in protein localization was observed in the SOD1G93A motor
neurons (FIG. 4A,D). In 43/54 (79.75.+-.7.75%) of the motor neurons
selected at random for analysis by GFP expression, the SOD1 G93A
protein localized to inclusions in the perinuclear space, in the
cell body and also in the neural processes (FIG. 4A,D supplemental
FIG. 3). When control motor neurons expressing wild-type SOD1 were
examined 21 days after differentiation (FIG. 4B,D), we observed
inclusions in a smaller proportion of cells 23/64 (35.93.+-.0.43%).
The inclusions in motor neurons expressing the SOD1G93A allele were
also significantly larger in area (FIG. 8A), significantly longer
(FIG. 8B) and displayed a higher optical density, suggesting that
they contained more SOD1 protein at a higher concentration (FIG.
8C).
[0121] The levels of ubquitinated proteins are significantly
elevated in motor neurons of ALS patients and SOD1G93A transgenic
animals during neural degeneration (Bruijn et al., 1997; Ince, P.
G., et al., Amyotrophic lateral sclerosis associated with genetic
abnormalities in the gene encoding Cu/Zn superoxide dismutase:
molecular pathology of five new cases, and comparison with previous
reports and 73 sporadic cases of ALS. J Neuropathol Exp Neurol 57,
895-904, 1998; Wang, J. et al. Copper-binding-site-null SOD1 causes
ALS in transgenic mice: aggregates of non-native SOD1 delineate a
common feature. Hum Mol Genet 12, 2753-64, 2003; Watanabe, M. et
al. Histological evidence of protein aggregation in mutant SOD1
transgenic mice and in amyotrophic lateral sclerosis neural
tissues. Neurobiol Dis 8, 933-41, 2001). Examination of the ES
cell-derived motor neurons revealed an increase in staining with
anti-ubiquitin antibodies, relative to other cells in the culture.
This staining often colocalized with the SOD1 protein inclusions
(FIG. 4C).
[0122] The death of motor neurons in ALS patients and transgenic
mice carrying mutant SOD1 genes occurs through activation of
programmed cell death pathways. Apoptosis in these cells has been
proposed to be mediated through the release of cytochrome c and the
activation of caspase-3 (Pasinelli, P., Houseweart, M. K., Brown,
R. H., Jr. & Cleveland, D. W. Caspase-1 and -3 are sequentially
activated in motor neuron death in Cu,Zn superoxide
dismutase-mediated familial amyotrophic lateral sclerosis. Proc
Natl Acad Sci USA 97, 13901-6, 2000; Raoul, C. et al. Motoneuron
death triggered by a specific pathway downstream of Fas.
potentiation by ALS-linked SOD1 mutations. Neuron 35, 1067-83,
2002). Examination of the SOD1G93A motor neurons 14 days after
dissociation of EBs, revealed that some neurons expressed activated
caspase-3 and contained diffuse cytoplasmic staining with
cytochrome c specific antibodies (FIG. 9A,B,C). Thus, the SOD1G93A
motor neurons appear to initiate cell death pathways in vitro that
are similar to those activated in vivo during the course of disease
(Pasinelli et al., 2000).
Example 5
SOD1G93A Glial Cells Adversely Affect Survival of ES Cell Derived
Motor Neurons
[0123] Both autonomous defects in motor neurons and toxic non-cell
autonomous interactions with other cell types in the spinal cord
have been implicated in ALS pathology (Bruijn et al., 2004; Clement
et al., 2003; Boillee et al., 2006). Only a subset of cells in EBs
became motor neurons under our differentiation conditions. We
therefore considered the possibility that cells within the EBs
might also develop into other cell types that normally associate
with neurons in the spinal cord. These additional cells might, as
suggested by chimeric experiments, contribute non-cell autonomously
to the loss of neurons in SOD1G93A cultures. Glial cells are
closely associated with motor neurons, and both are derived from a
common progenitor in vivo (Zhou, Q. & Anderson, D. J. The bHLH
transcription factors OLIG2 and OLIG1 couple neuronal and glial
subtype specification. Cell 109, 61-73, 2002). We therefore
addressed the possibility that glial cells are present in our
cultures. To determine if these cells, were also produced by our
differentiation protocol, we stained SOD1G93A cultures with
antibodies specific to the glial fibrillary acidic protein (GFAP)
(Bignami, A. & Dahl, D. Astrocyte-specific protein and
neuroglial differentiation. An immunofluorescence study with
antibodies to the glial fibrillary acidic protein. J Comp Neurol
153, 27-38, 1974). Indeed, GFAP positive cells were found in close
association with GFP positive motor neurons (FIG. 2F). We found
that approximately 30% of the cells at 28 days were GFAP positive
(Hb9GFP: 31%, SOD1: 32%, SOD1G93A: 36%). Thus, it seemed possible
that the SOD1G93A glial cells in these cultures might be adversely
affecting motor neuron survival.
[0124] To examine this possibility, we differentiated the three ES
cell lines (Hb9GFP, SOD1 and SOD1 G93A) into motor neurons and
plated them on established monolayers of primary glia isolated from
the cortex of neonatal mice with differing SOD1 genotypes
(wild-type SOD1, and the mutant SOD1G93A) (Banker, G. & Goslin,
K. Culturing nerve cells, xii, 666, 11 of plates (MIT Press,
Cambridge, Mass., 1998). During the first seven days after plating
on either glial monolayer, motor neurons of all genotypes increased
in size and took on a more mature morphology (FIG. 5A). However, by
14 days after plating there was a 50% decrease in the number of
wild-type SOD1 derived motor neurons in co-cultures with SOD1G93A
glia compared to the same preparation of neurons plated on
wild-type SOD1 glia. Similarly, we did not see a significant
reduction in the number of Hb9GFP motor neurons when plated on SOD1
glia, but did see a reduction of 30% if the same neurons were
co-cultured with SOD1G93A glia (FIG. 5B). These data suggest that
wild-type SOD1 glia provide a permissive environment for motor
neuron growth and differentiation, comparable to other
well-established in vitro systems for motor neuron culture (Ullian,
E. M., Harris, B. T., Wu, A., Chan, J. R. & Barres, B. A.
Schwann cells and astrocytes induce synapse formation by spinal
motor neurons in culture. Mol Cell Neurosci 25, 241-51, 2004;
Allen, N. J. & Barres, B. A. Signaling between glia and
neurons: focus on synaptic plasticity. Curr Opin Neurobiol 15,
542-8, 2005; Ullian, E. M., Christopherson, K. S. & Barres, B.
A. Role for glia in synaptogenesis. Glia 47, 209-16, 2004). In
contrast, when we co-cultured wild type motor neurons (SOD1 or
Hb9GFP) with glia from SOD1G93A mice, we observed a marked
reduction in survival (FIG. 5 A,B).
[0125] We next investigated the affect of the presence of the
mutant SOD1 transgene in motor neurons in the co-culture system.
When mutant SOD1G93A motor neurons were plated on wild-type SOD1
glial cells, there was a 27% decrease in the number of neurons
between 7 and 14 days. The loss of motor neurons increased to 75%
when the mutant SOD1G93A motor neurons were cultured with mutant
SOD1G93A glia (FIG. 5 A,B).
[0126] Together, our results show that the SOD1G93A genotype in
glial cells has a negative effect on motor neuron survival
regardless of the motor neuron genotype. However, a greater
negative effect is observed with the SOD1G93A motor neurons. Thus,
glial cells have a non-cell autonomous effect on motor neuron
survival and mutant motor neurons are more sensitive to the
effect.
[0127] To determine whether the differing influences of the
wild-type SOD1 and the mutant SOD1 G93A glial cell cultures could
be explained by the presence of different proportions of distinct
glial cell types, we characterized the two populations by
immunostaining with established glial markers. We did not observe a
significant difference in the wild-type SOD1 and the mutant
SOD1G93A glial populations over time (FIG. 10).
Example 6
Methods
[0128] Examples 1 through 5 were performed using the following
methods:
[0129] Derivation of Mouse Embryonic Stem Cells.
[0130] ES cell lines were derived from crosses between mice
transgenic for Hb9:GFP (Jackson lab, Stock Number:005029) and mice
transgenic for SOD1.sup.G93A (Jackson lab, Stock Number:004435) or
SOD1.sup.WT (Jackson lab, Stock Number:002297). Transgenic Hb9:GFP
females were injected IP with 7.5 units of pregnant mares' serum
(Calbiochem) followed 46-50 h later with 7.5 units of human
chorionic gonadotropin (HCG) (Calbiochem). After administration of
HCG, females were mated with either SOD1 G93A or SOD1 transgenic
males. Females were scarified three days later and blastocysts were
flushed from the uterine horn with mES cell media (Knockout-DMEM
(GIBCO), 15% Hyclone Fetal Bovine Serum (Hyclone), 10.000 unit
Penicillin and 1 mg/ml Streptomicin (GIBCO), 2 mM Glutamine
(GIBCO), 100 mM non-essential aminoacid (GIBCO), 55 mM
beta-mercapto-ethanol (GIBCO), 1,000 units/ml leukocyte inhibiting
factor (Chemicon)). Blastocysts were then plated individually into
one 10 mm-well of a tissue culture dish containing a feeder layer
of mitotically inactivated mouse embryonic fibroblast, in the
presence of mES cell media supplemented with the MEK kinase
inhibitor PD98059 (cell signaling, inc). 48 hours after plating
embryos, one half volume of fresh media was added to each culture
well. Starting three days after plating, the culture media was
changed.
[0131] Four to five days after plating, ICM-derived outgrowth were
observed, dislodged from the rest of the cells with a pasteur
pipette, washed once in a drop of PBS and then incubated for 10
minutes in a drop of 0.25% trypsin at 37 C. The ES cells clumps
were then gently dissociated with a Pasteur pipette filled with
mouse ES cell media and transferred onto a fresh layer of
fibroblasts in a 10 mm tissue culture well. For routine culture,
the mouse ES cells are generally split 1:6 with a solution of 0.25%
trypsin (GIBCO) every 2-3 days.
[0132] Generation of Chimeric Embryos.
[0133] Chimeric embryos were generated as previously described
(Hogan, B. Manipulating the mouse embryo: a laboratory manual,
xvii, 497 p. (Cold Spring Harbor Laboratory Press, Plainview, N.Y.,
1994). Blastocysts were collected from the uterus of non-transgenic
pregnant females mice 3.5 days postcoitum. The ES cells were
injected (about 10 for each blastocyst) using a microinjection
pipette with a diameter of 12-15 .mu.m applying a brief pulse of
the Piezo (Primetech, Ibaraki, Japan) on one side of the blastocyst
and pushing the needle through the zona and trophectoderm layer
into the blastocoel cavity. Ten injected blastocysts were
transferred to each uterine horn of 2.5 days postcoitum
pseudopregnant Swiss females that had mated with vasectomized
males. Recipient mothers were sacrificed at 10.5 days postcoitum,
and embryos were quickly removed from the uterus and placed in a
dish with cold PBS for whole mount analysis of GFP
fluorescence.
[0134] Differentiation of mES Cells into Motor Neurons.
[0135] Mouse ES cells were differentiated into motor neurons
according to methods previously described (Boillee et al., 2006).
The ES cells were grown at 70-80% confluence in a 10 cm plate
(Falcon) in mouse ES cells media. To form EB's, cells were washed
once with a PBS solution to eliminate the traces of media and then
incubated with 1 ml of 0.25% trypsin (GIBCO) for 5-10 minutes at
room temperature. After this the cells were resuspended in 10 ml of
DM1 media (DMEM-F12 (GIBCO), 10% Knockout serum (GIBCO), pen strep,
glutamine (GIBCO) and 2-mercaptoethanol (GIBCO)), they were counted
and plated at a concentration of 200.000/ml in Petri dishes
(Falcon). After 2 days the embryoid bodies were split from 1 dish
into 4 new Petri dishes containing DM1 medium supplemented with RA
(100 nM .mu.M; stock: 1 mM in DMSO, Sigma) and sonic hedgehog (300
nM, R&D Systems). The media is changed after 3-4 days. On day 7
the embryoid bodies were dissociated in single cells suspension.
The EBs were collected in a 15 ml falcon tube, centrifuged at 1000
rpm for 5 min, washed once with PBS and incubated in Earle's
Balanced Salt Solution with 20 units of papain and 1000 units of
Deoxyribonuclease I (Worthington Biochemical Corporation) for 30-60
minutes at 37.degree. C. The mixture was then triturated with a 10
ml pipette and centrifuged for 5 minutes at 1000 RPM. The resulting
cell pellet was washed once with PBS and finally resuspended in
supplemented F12 media (F12 medium (GIBCO) with 5% horse serum
(GIBCO), B-27 supplement (GIBCO), N2 supplement (GIBCO)) with
neurotrophic factors (GDNF, CNTF, and BDNF (10 ng/ml, R&D
Systems)). The cells were then counted and plated on
Poly-D-Lysine/Laminin CultureSlides (BD biosciences) or on a layer
of primary glia cells. For the motor neurons survival experiments,
GFP positive cells with visible axons and dendrites were counted at
different time points after plating (7, 14, 21, 28 days).
[0136] Polymerase Chain Reaction.
[0137] All PCR reactions were performed using an MJ Research
Thermal Cycler, and TaKaRa Ex Taq HS (Takara) enzymes. For the SOD1
genotyping of the newly derived ES cell lines and transgenic mice
the forward primer: CAT CAG CCC TAA TCC ATC TGA (SEQ ID NO:1) and
reverse primer: CGC GAC TAA CAA TCA AAG TGA (SEQ ID NO:2) amplified
a 236 bp fragment in the 4.sup.th exons of the gene. As an internal
control a set of primers that amplified a 324 bp fragment of the
IL-2 gene (forward: CTA GGC CAC AGA ATT GAA AGA TCT (SEQ ID NO:3),
reverse: CAT CAG CCC TAA TCC ATC TGA (SEQ ID NO:4)) were used. The
annealing temperature for this reaction was 60.degree. C. for 35
cycles. For GFP a set of primers (forward: AAG TTC ATC TGC AAC ACC
(SEQ ID NO:5), reverse: TCC TTG AAG AAG ATG GTG CG (SEQ ID NO:6))
that amplified a fragment of 173 bp of the gene were used, with an
annealing temperature of 60.degree. C. for 35 cycles.
[0138] FACS.
[0139] For FACS analysis a BD biosciences LSRII flow cytometer was
used. The embryoid bodies were dissociated with papain and
resuspended in cold PBS with 2% FBS, Calcein blue (Invitrogen) was
used to assay the cell viability. The cells were then analyzed
using a non transgenic mouse embryonic stem cell lines as a
negative control. The FACS Diva software package (BD Biosciences)
was used for data analysis
[0140] Glia Cultures.
[0141] Glia monolayers were obtained from P2 mice born from matings
between transgenic SODG93A. Tissue was isolated in Calcium and
Magnesium Free-Hanks's BSS (HBSS). Under a dissecting microscope
the cortex was isolated and carefully striped of the meninges. The
tissue was split in small pieces then transferred to a 50 ml
centrifuge tube in a final volume of 12 ml of HBSS. Tissue
digestion was performed using trypsin--EDTA (GIBCO BRLno.25200) and
1% DNAse (Sigma no. DN-25) at 37.degree. C. for 15 min, swirling
the mixture and periodically. We collected the dissociated tissue
and triturated using a fire polish Pasteur glass pipette and
filtered the combined supernatant through a 72 gm nylon mesh (NITEX
100% polyamide Nylon Fiber TETKO Inc.) to remove any undissociated
tissue. The filtered material was centrifuged at 1000 rpm for 5 min
to pellet the cells, resuspend in 2 ml of Glia medium (Minimum
Essential Medium with Earl's salts, GIBCO BRL no. 11095-080, 20%
Glucose, Penicillin-streptomycin, GIBCO BRL no. 15145-014, and 10%
Horse Serum GIBCO BRL no. 26050-070) and cell number was counted.
The yield from one brain was generally enough to plate one T75
flask (Falcon no. 3084). Once monolayers were confluent (generally
in 10 to 14 days) cells were replated on 24 or 12 well multiwell
dishes over poly-D-Lysine (0.5 mg/ml for 30 min RT) coated glass
cover slips.
[0142] Immunocytochemistry Analysis.
[0143] The cells were fixed with 4% paraformaldehyde-PBS, blocked
and permeabilized with BSA (1%)-Triton X100 (0.1%). After
incubating overnight with the following antibodies: mouse
monoclonal anti-Tuj1 (Covance), Islet 1 Islet2, RC2 (Developmental
Studies Hybridoma Bank University of Iowa, IA, USA) anti-sod1
(SIGMA), S100 (Chemicon), CNPase (Abeam) and rabbit anti-HB9 (Tom
Jessell, Columbia University), GFAP (Chemicon) anti-ubiquitin
(DAKO); goat anti-Vimentin (Chemicon); rat anti-CD 11 b (Abeam)
(see Table 1)
Table 1 describes primary antibodies used. Antiserum (host
species), working dilution and source.
TABLE-US-00001 Antiserum Dilution Source Tuj 1(anti-Mouse) 1/1000
Covance Islet 1(anti-Mouse) 1/1 Hybridoma Bank ChAT (anti-Goat)
1/100 Chemicon Hb9 (anti-Rabbit) 1/1000 Jessel Lab GFAP
(anti-Rabbit) 1/1000 Chemicon S100 (anti-Mouse) 1/100 Chemicon RC2
(anti-Mouse) 1/1 Hybridoma Bank Vimentin (anti-goat) 1/100 Chemicon
CD 11b (anti-Rat) 1/100 Abcam hSOD1 (anti-Mouse) 1/200 Sigma
Caspase 3(anti-Rabbit) 1/2000 BD Pharmingen Ubiquitin (anti-Rabbit)
1/200 DAKO Cytochrome C (anti-Mouse) 1/200 Abcam
[0144] The cells were then incubated with Donkey anti-rabbit
conjugated to Cy3 (1:100; 2 h) and Donkey anti mouse conjugated to
Cy5 second antibodies (1:100; 2 h; Jackson ImmunoResearch (West
Grove, Pa., USA.). After mounting the samples in Vectashield
(Vector Labs, Burlingame, Calif., USA), confocal or epi-fluorescent
microscopy was performed using Olympus FV 1000, 40.times. and
60.times. oil immersion objective 1.45 NA or fluorescent microscope
Olympus IX70. Image acquisition was performed using FLUOVIEW
software 4.0 for relative fluorescence analysis, all settings such
as exposure time, magnification and gain were maintained constant
for all samples. Offline analysis of relative intensities for all
the samples was done using Metamorph 4.5 (Downingtown, Pa., USA).
Only cells having morphological features of neurons (i.e., phase
bright soma and several neurites) were considered for subsequent
analysis.
[0145] Neuronal Density.
[0146] HB9-GFP positive motor neurons were counted for condition
studied (Zeiss microscope, 40.times. 1.3 NA, oil immersion
objective). The density of neurons, normalize as a percent of
initial number counted at 7 DIV or 14 DIV was established for all
conditions studied. These experiments were carried out three times
as independent experiments.
[0147] Data Analysis.
[0148] The data was obtained from control and SOD1 G93A motor
neurons in parallel conditions (sister plates) to reduce
dispersion. Statistical analysis were performed using Student's
t-Test or ANOVA and are expressed as arithmetic mean.+-.S.E.M.;
t-test values of * P<0.05, ** P<0.01, *** P<0.005 were
considered statistically significant. Each set of data presented
was performed in sister cultures to reduce variability. Similar
significances were found expressing the data in cumulative
distributions plots. Therefore, we chose to present the data as
mean.+-.E.M. to simplify its presentation. The kinetic analysis was
done using MiniAnalysis 5.0 (Synaptosoft).
Example 7
Human ES cell lines are sensitive to SOD1G93A glia
[0149] To generate a large supply of motor neurons from human ES
cells for the study of ALS we adapted a recently reported method
for the production of these cells within embryoid bodies (EBs)
(Singh Roy, N. et al. Enhancer-specified GFP-based FACS
purification of human spinal motor neurons from embryonic stem
cells. Exp Neurol. December; 196(2):224-34, 2005) (FIG. 11a).
Undifferentiated, self-renewing HuES 3 ES cells (Cowan, C. A. et
al. Derivation of embryonic stem-cell lines from human blastocysts.
N Engl J Med March 25; (13):1353-6, 2004) were dissociated into
small clumps using collagenase treatment and then allowed to
spontaneously differentiate in suspension for 14 days (FIG. 11a,
b). Staining of the resulting EBs with the neuronal progenitor
marker PAX6 (FIG. 11b) demonstrated that a substantial percentage
(29%+/-16%, FIG. 15a, b) contained cells differentiating down the
neuronal lineage. To direct these progenitors towards an anterior
and ventral motor neuron identity, we cultured the EBs another 14
days in the presence of retinoic acid (RA) and a small molecule
agonist of the sonic hedgehog (SHH) pathway. Under the influence of
these morphogens the population of PAX6 positive progenitors
expanded (45%+/-15%; FIG. 1b; FIG. 15a, b) and expression of the
ventral progenitor markers NKX6.1 and ISL1/2 was induced (FIG. 11b;
FIG. 15a,b). To promote motor neuron differentiation and survival,
we then transferred these 28 day-old EBs to media containing
neurotrophic factors for a final 14 days. After 42 days of
differentiation, the number of progenitors expressing PAX6 and
NKX6.1 had begun to decline (FIG. 15a, b), while the number of
cells expressing ISl1/2 continued to increase (FIG. 11b; FIG. 15a,
b). In addition, expression of the HB9 transcription factor, which
is expressed in maturing post-mitotic motor neurons, was detected
in 8% of all cells (FIG. 11b; FIG. 15a, b). Furthermore, when
plated on laminin, these EBs elaborated impressive neuronal
processes (FIG. 11).
[0150] To further characterize the putative motor neurons contained
within these EBs, the 42 day-old EBs were dissociated with papain
and the resulting cells plated directly onto glial mono layers
prepared from the cortex of neonatal mice (FIG. 16). We found, as
had been previously reported with neurons derived from mouse ES
cells (Song, H. et al. Astroglia induce neurogenesis from adult
neural stem cells. Nature. May 2; 417(6884):39-44, 2002) that
culturing human ES cell derived neurons on a glial monolayer
promoted their survival (FIG. 16b, c). Co-staining of cells with
antibodies specific to a neuronal form of tubulin (Tuj1) and the
transcription factors Hb9 and Isl1/2 (FIG. 17a, b), as well as
co-staining for Hb9 and choline acetyl transferase (Chat) (FIG.
17c) confirmed that many neurons isolated from these EBs were
differentiating towards a motor neuron identity.
[0151] To ensure that the appearance of motor neurons within these
EBs was dependent on the influences of RA and SHH, we repeated our
differentiation scheme in the absence of one or both of these
morphogens and counted the number of HB9 positive cells (FIG. 11c).
When SHH or RA activity were removed individually, the frequency of
cells expressing HB9 fell to 0.7% (+/-0.2) and 1.1% (+/-0.5%)
respectively. If both signaling molecules were omitted, less than
0.2% of the dissociated cells expressed HB9 (0.17%+/-0.07%). We
further confirmed the robustness of our approach for generating
motor neurons by differentiating six independent human ES cells
lines and then quantifying the number of HB9 positive cells within
the resulting EBs (FIG. 11d). We found that HuES1, HuES3, HuES5 and
HuES9 ES cell lines all differentiated with a similar efficiency
(HuES 1: 7.1%+/-1.8%; HuES 3: 8.5%+/-0.5%; HuES 5: 4.7%+/-0.8%;
HuES 9: 7.7%+/-1.5%), while HuES 12 cells differentiated at a lower
efficiency (2.8%+/-1.3%) and HuES 13 cells at a higher efficiency
13.9% (+/-3.8%). These results are consistent with a recent report
that suggests independent human ES cell lines can have varying
abilities to differentiate into certain cell types (Osafune, K. et
al. Marked differences in differentiation propensity among human
embryonic stem cell lines. Nat Biotechnol. March; 26(3):313-5,
2008).
[0152] In order to identify living motor neurons in cultures of
differentiating human ES cells, we generated a stable transgenic
human ES cell line in which sequences coding for the green
fluorescent protein (GFP) were under the control of the murine Hb9
promoter (Wichterle et al., 2002) (FIG. 12). To validate that this
transgenic cell line accurately reported HB9 expression, we
differentiated the cells, plated them on glial monolayers and
co-stained with antibodies specific to GFP and HB9. HB9 expression
was observed in 95% of GFP positive cells (FIG. 12c; FIG. 18a-d).
We next investigated whether these GFP positive cells expressed
other markers that would be consistent with a maturing motor neuron
identity (FIG. 12; FIG. 18). We observed considerable overlap
between GFP and expression of NKX6.1 (FIG. 180 but no co-expression
with NKX2.2 (FIG. 18e), confirming that GFP positive cells had
acquired the correct dorsal-ventral identity (Jessell TM. Neuronal
specification in the spinal cord: inductive signals and
transcriptional codes. Nat Rev Genet. October; 1(1):20-9, 2000).
Additionally these cells expressed ISL1/2 (FIG. 12e) and ChAT (FIG.
12f) but no longer expressed the progenitor marker PAX6 (FIG. 12d).
Antibody co-staining experiments also demonstrated that GFP
positive cells did not co-express makers found in other neuronal
subtypes such as the interneuron markers CHX10 (FIG. 18h) and LHX2
(FIG. 18g).
[0153] The results that we have described thus far confirm that it
is possible to reproducibly generate a large supply of human motor
neurons from embryonic stem cells. We next sought to use these
human neurons to ask whether they, like their mouse counterparts,
are sensitive to the non-cell autonomous effect of glial cells
overexpressing a mutant SOD1 gene product. To this end, we
dissociated 42 day-old EBs and plated the resulting cells on
primary glial monolayers derived from either SOD1G93A transgenic or
control mice (FIG. 13a). After 10 days a significant difference
(p<0.05) in the number of HB9 positive motor neurons was seen
between the two culture conditions (FIG. 13b) is already
appreciable. In cultures containing SOD1 G93A glia less than half
as many motor neurons remained (131+/-53) as in cultures containing
non-transgenic control glia (269+/-44) (FIG. 13b). The deficit in
motor neuron survival in co-cultures with SOD1G93A glia became even
more pronounced after 20 days (FIG. 13c, d). We next sought to
confirm that the toxic effect of glia we observed in our initial
experiments was due to the action of the mutant SOD1 protein rather
than mere SOD1 protein over-expression. Motor neuron preparations
were generated from the Hb9::GFP human ES cell line and co-cultured
for 20 days with non-transgenic glia or glia which either
over-expressed the wild-type human SOD1 protein or the mutant
SOD1G93A protein (FIG. 13e-h). There was no discernable difference
between the number of GFP positive motor neurons present in culture
with the non-transgenic Glia (304+/-60; FIG. 13e, h) or with glia
over-expressing the wild type SOD1 protein (328+/-30; FIG. 13e, g).
In contrast, there was a highly significant reduction (p<0.01)
in the number GFP positive motor neurons (127+/-16; FIG. 13e, f)
present in culture with the SOD1G93A Glia, confirming that the
non-cell autonomous effect of glia was mediated through the mutant
SOD1 protein.
[0154] In both patients and mice carrying mutant alleles of the
SOD1 gene, intracellular aggregation of the SOD 1 protein is often
documented and has been associated with motor neuron death (Boillee
et al., 2006). We therefore wondered whether the toxic effect of
glial cells expressing the mutant SOD1 protein that we observed was
a downstream consequence of protein aggregation. To address this,
we separately cultured primary mouse glia and mouse ES cell derived
motor neurons carrying the same SOD1G93A transgene and stained the
cultures with antibodies specific for the human SOD1 protein. After
21 days in culture, the SOD1 protein in mouse motor neurons was
observed to aggregate into cytoplasmic and perinuclear inclusions
(FIG. 19a) (Di Giorgio, F. P., Carrasco, M. A., Siao, M. C.,
Maniatis, T. & Eggan, K. Non-cell autonomous effect of glia on
motor neurons in an embryonic stem cell-based ALS model. Nat.
Neurosci. 10, 608-614, 2007). In contrast, even after more than 90
days in culture, the SOD1 protein was found to be broadly and
diffusely localized in the cytoplasm of all glial cells (FIG. 19b),
suggesting that the mutant protein is mediating its effect in these
cells through a mechanism independent of protein aggregation.
[0155] ALS leads to the specific degeneration of motor neurons.
Therefore, if the toxic effect of glial cells that we have observed
is relevant to ALS then we might expect that other spinal cord
neuronal types such as interneurons would not be sensitive to it.
During our characterization of human ES cell derived motor neurons
we noted that additional neurons expressing the transcription
factors CHX10 and LHX2, indicative of V2 and D1 interneuron
differentiation, were also produced (FIG. 18g, h). To test whether
these neuronal types were affected by co-culture with mutant glia,
we dissociated 42 day-old EBs, plated equal numbers of cells on
either SOD1G93A glia or non-transgenic glia (FIG. 14a-e) and after
20 days of culture stained for Tuj1 and either LHX2 (FIG. 4d) or
CHX10 (FIG. 14e). We found that neurons expressing either of these
interneuron markers were unaffected by culture with mutant glia
(FIG. 14b, c), in striking contrast to the sensitivity of motor
neurons to this culture environment.
[0156] To determine if the toxic effect of mutant glial cells was
the consequence of a specific activity within this cell type rather
then a general property of any cell over expressing the SOD1G93A
mutation, we plated motor neuron preparations on mouse embryonic
fibroblasts (MEFs) prepared from SOD1G93A and non-transgenic
sibling embryos (FIG. 14f-h). After 20 days of co-culture we did
not observe a significant difference between the number of HhB9,
Ttuj1 double positive motor neurons present on SOD1G93A MEFs
(204+/-28) or non-transgenic MEFs (197+/-23) (FIG. 14g, h),
consistent with the hypothesis that astrocytes are specifically
responsible for the non-cell autonomous effect we observed (Di
Giorgio et al., 2007; Nagai et al., 2007).
Example 8
Methods
[0157] Example 7 was performed using the following methods:
[0158] Growth of Human Embryonic Stem Cells.
[0159] The HuES cell lines were obtained from Doug Melton and
cultured as described by Cowan et al. (Cowan et al., 2004). The
hESCs were maintained on a feeder layer of inactivated mouse
embryonic fibroblasts (GlobalStem) in human ES cell media (KO-DMEM
(Gibco), 10% KO Serum Replacement, 10,000 units Penicillin and 1
mg/ml Streptomicin (GIBCO), 2 mM Glutamine (GIBCO), 100 .mu.M
non-essential amino acids (GIBCO), 55 .mu.M beta-mercapto-ethanol
(GIBCO), 10% Plasmanate (Bayer), 10 ng/mL bFGF2 (GIBCO)). The cells
were cultured at 37.degree. C. and 5% CO.sub.2. Media was replaced
daily for the duration of hESC expansion and the cells in these
conditions were passaged every 5-7 days using a solution with 0.05
trypsin (GIBCO).
[0160] Differentiation of Human Embryonic Stem Cells into Motor
Neurons.
[0161] For differentiation into motor neurons, the cells were
allowed to reach 80-90% confluency, washed once with PBS, and then
incubated for 15 minutes at 37.degree. C. in a solution of 1 g/L
Collagenase IV (GIBCO) in DMEM-F12 (GIBCO).
[0162] Using a cell scraper, the ES cell colonies were scraped and
washed off the plate, centrifuged for 5 minutes at 1000 RPM and
resuspended in human ES cell media without bFGF2 or plasmanate in
low attachment 6-well plates.
[0163] After 24 hours, the cells had aggregated to form embryoid
bodies (EBs), and the media was changed to remove debris by
centrifuging the EBs and resuspending in fresh in human ES cell
media without bFGF2 or plasmanate in low attachment 6-well plates.
EBs were cultured as such for 13 more days, with half of the media
changed every two days, and a complete media change every week.
After 14 days, the EBs were induced toward a caudal and ventral
identity using retinoic acid (1 .mu.M, Sigma) and an agonist of the
Shh signaling pathway (1 .mu.M) in N2 media: 1:1
DMEM:F-12+Glutamate (Gibco), 10,000 units Penicillin and 1 mg/mL
Streptomicin (Gibco), 1% N2 Supplement (Gibco), 0.2 mM ascorbic
acid (Sigma-Aldrich), 0.16% D-(+)-Glucose (Sigma-Aldrich), BDNF (10
ng/ml, R&D Systems), for another 14 days. The EBs were then
matured for a final 14 days in N2 media with GNDF (10 ng/mL,
R&D Systems). After 42 days of differentiation, the EBs were
dissociated. To dissociate the EBs, they were centrifuged at 1000
rpm for 5 min in a 15 ml falcon tube, and then washed once with PBS
to eliminate residual media. The EBs were then incubated for 60
minutes at 37.degree. C. in Earle's Balanced Salt Solution with 20
units of papain and 1000 units of Deoxyribonuclease I (Worthington
Biochemical Corporation). EBs were triturated using a 2 mL
serological pipette every 15-20 minutes during this incubation.
When an almost single cell suspension was achieved, the cells were
centrifuged for 5 minutes at 1000 RPM. The resulting cell pellet
was washed once with PBS and then resuspended in N2 media with
neurotrophic factors (GDNF, and BDNF (10 ng/ml, R&D Systems)).
These cells were then counted and plated on Poly-D-Lysine/Laminin
CultureSlides (BD biosciences) or on a layer of primary glial
cells. Depending on the experiment, motor neurons or intemeurons
were counted 10 or 20 days after plating.
[0164] Generation of the HuES 3 Hb9::GFP Cell Line.
[0165] To generate the Hb9::GFP HuES 3 cell line, HuES 3 cells were
electroporated with a plasmid containing a neomycin resistance
cassette and the coding sequence of Green Fluorescent Protein under
transcriptional control of a 9 kb murine Hb9 promoter restriction
fragment. The plasmid was a kind gift of Hynek Witcherle (Columbia
University) and was a modification of the construct described in
Witcherle et al. (2002). The elctroporation was performed as
described in Zwaka P T. et al. (Zwaka T P, Thomson J A. Homologous
recombination in human embryonic stem cells. Nat Biotechnol. March;
21(3):319-21, 2003).
[0166] Undifferentiated HUES 3 cells were grown as described below.
Once the cells reached 80-90% confluency, they were dissociated in
trypsin and counted. Approximately 1.0.times.10.sup.7 were
resuspended in 0.7 mL of human ES cell media and mixed with 0.1 mL
of the same media containing 30 .mu.g of linearized vector. This
mix of cells and DNA was then transferred to a 0.4 cm cuvette and
exposed to a pulse of 320 V, 200 .mu.F at room temperature. After
10 minutes at room temperature the cells were plated on a 10 cm
dish of MEF, and 48 hours after electroporation the cells were
switched to media containing G418 (50 .mu.g/mL, GIBCO). Selection
media was changed daily for 14 days, after which we picked and
expanded 24 resistant human ES cell colonies. In order to assay GFP
expression, we differentiated six of these resistant clones into
motor neurons and immunostained for GFP and HB9 co-expression. Two
of these clones gave rise GFP positive cells that elongate green
axons, however only one clone was validated by immunoreactivity to
the Hb9 antibody and used in subsequent experiments.
[0167] Immunocytochemistry Analysis.
[0168] Cells were fixed with 4% para-formaldehyde for 30 minutes at
room temperature. After fixation, the cells were washed 3 times
with PBS for 10 minutes and then treated for 1 hour in a blocking
solution (PBS (Cellgro), donkey serum (10%, Jackson
Immunoresearch)) plus Triton X (0A %, Sigma) for permeabilization.
After blocking, the cells were incubated overnight at 4.degree. C.
with primary antibodies: mouse anti-beta tubulin III (Covance);
rabbit anti-beta tubulin III (SIGMA); Pax6, Nkx6.1, Nkx2.2, Isl 1,
Hb9 (DSHB); Chx10, Lhx2 (Santa Cruz Technologies); ChAT (Chemicon);
rabbit anti-GFP conjugated Alexa fluor 488 (Molecular Probes); in
the blocking solution. After the overnight incubation the cells
were washed 3 times in PBS for 10 minutes. Localization of antigens
was visualized by incubating for 1 hour at room temperature using
the respective secondary antibodies (Alexa fluor 594 or 488;
Molecular Probes). Finally, the samples were washed again in PBS 3
times and mounted using a solution with or without DAPI. Images
were taken using a fluorescent Olympus 1X70 microscope.
[0169] Primary Glial Cultures.
[0170] P1-P3 mouse pups transgenic for SOD1G93A, SOD1WT or
non-transgenic pups were sacrificed by using an approved method of
euthanasia. Under a dissection microscope, the parenquima were
isolated and the meninges were carefully stripped away with fine
forceps. The tissue was then dissected into small pieces and
transferred to a solution containing 12 ml of HBSS, 1.5 ml of
trypsin (GIBCO) and 1% DNAse (Sigma) and incubated at 37.degree. C.
for 15 min, swirling the mixture periodically. The supernatant
containing the dissociated cells was then collected and 3 ml of
serum was added to inhibit trypsin activity.
[0171] The cells were then centrifuged at 1000 rpm for 5 min,
resuspended in Glia medium: (Minimun Essential Medium with Earl's
salts (GIBCO), D-(+)-Glucose 20% (Sigma), Penicillin-streptomycin
(GIBCO), 10% Horse Serum (GIBCO)) and plated at the concentration
of 80,000 cells per mL in T75 flasks (Falcon). After the glia
reached confluency, they were replated onto Poly-D-Lysine/Laminin
CultureSlides (BD biosciences).
[0172] Data Analysis.
[0173] Statistical analysis was performed using Student's t-Test
and are expressed as arithmetic mean.+-.S.D.; t-test values of *
P<0.05, ** P<0.01, were considered statistically
significant.
Example 9
Identification of Candidate Genes Involved in SOD1G93a Glial
Toxicity
[0174] To better understand how the expression of a mutant gene
that causes ALS can perturb the normal phenotype of astrocytes, and
to identify genes that may have a role in their toxic effect on
motor neurons, we used oligonucleotide arrays to compare the global
gene expression profiles of glia overexpressing the mutant SOD1G93A
protein with two different sets of controls: non-transgenic glia
and glia overexpressing the wild type form of the human SOD1
protein.
[0175] We identified 135 genes whose expression was significantly
(P<0.001) increased more than 2 fold in SOD1G93A glia when
compared to non-transgenic glia. Of these 135 genes, 53 genes were
exclusively up-regulated in the mutant glia, and not in glia
over-expressing the wt SOD1 protein (FIG. 20A). We found that 13 of
these 53 genes (24%) have previously been identified to have a role
either in inflammatory or immune processes. Genes overexpressed
more than 2 fold (P<0.001) in SOD1G93A glia with respect to WT
glia and SOD1WT glia are listed in Table 2. We narrowed our
analysis to a subset of these genes deemed to be of particular
interest because of their known role as pro-inflammatory factors
and their substantially increased expression in mutant glia (FIG.
20B). The prostaglandin D2 (PGD2) receptor was up-regulated more
than 14 fold in SOD1G93A glia compared to the control sample. Three
different cytokines were also shown to be over expressed in mutant
glia: Mcp2, Cxcl7, and Rantes. Also found to be highly (>13
fold) up-regulated in these microarrays, was the gene encoding
glial maturation factor beta (GMFb), which has been shown to induce
a pro-inflammatory state in astrocytes (Zaheer et al., J.
Neurochem. 101:364-376, 2007). Finally, we found that the
expression of SHH and the SHH responsive genes NKX2.2 and DBX2 was
modestly increased in the mutant glia, suggesting that this
signaling pathway might be activated in response to the actions of
the mutant SOD1 protein.
[0176] Microarray Analysis.
[0177] Glia were derived from P1-P3 mouse pups as described above.
Once the cells reached confluence, total RNA was isolated using
Trizol (Invitrogen) from three different biological replicates for
each type of glia. RNA was amplified by one round of T7
transcription using the Illumina TotalPrep RNA Amplification Kit.
Illumina Bead Array Reader. Analysis was done using the Illumina
Bead Studio Program.
[0178] Data Analysis.
[0179] Statistical analysis was performed using Student's t-Test
and are expressed as arithmetic mean.+-.S.D.; t-test values of *
P<0.05, ** P<0.01, were considered statistically
significant.
TABLE-US-00002 TABLE 2 Genes overexpressed in SOD1G93A mutant glia
SYMBOL SEARCH_KEY Fold diff. G93A vs N.T Gene function Serpina1b
NM_009244.2 17.64285714 protease Ptpn7 scl0320139.8_83 15.5
signaling/immuno response Zc3hdc1 NM_172893.1 14.61111111 unknown
Ptgdr NM_008962.2 14.125 inflammation Gmfb NM_022023.1 13.07692308
inflammation Abca5 NM_147219.1 11.45454545 signaling Dbx2
scl0223843.1_155 11.35714286 transcription factor Rab6b
scl0270192.9_201 6.363157895 signaling Cutl1 scl013047.4_12 5.4
transcription factor Ada NM_007398.2 4.962962963 metabolic Ncoa6ip
NM_054089.2 4.654545455 unknown Ifi35 scl40880.4.1_8 4.369047619
inflammation Rabl2a NM_026817.1 4.081081081 unknown Al481214
scl0004149.1_262 3.84 unknown Cygb NM_030206.1 3.833333333
transport Dfy NM_010045.1 3.305084746 inflammation Chodl
scl48930.7.1_272 3.186440678 structural Nrxn1 scl0001711.1_8
3.178723404 signaling Defb11 NM_139221.1 3.164556962 immuno
response Rusc2 scl25518.12_3 3.086787565 unknown Nrxn1
scl0001711.1_8 3.023584906 signaling Matn4 NM_013592.2 3.006147541
structural Xlr3a NM_011726.1 2.837696335 unknown Ccl8 NM_021443.1
2.776623377 inflammation Timd4 scl41638.9.1_29 2.760869565 immuno
response Osr2 scl47995.5.83_129 2.738461538 transcription factor
9130213B05Rik scl27589.4_81 2.6 unknown Reck scl053614.23_117
2.594262295 signaling Olfr116 NM_146632.1 2.574712644 unknown
A230098A12Rik scl36723.20_445 2.505050505 unknown Shh
scl28000.7.1_29 2.475247525 signaling Fpr1 scl50268.2.1_13
2.447058824 inflammation Cxcl7 NM_023785.1 2.43324937 inflammation
Dnajb3 scl16502.1.45_71 2.429906542 structural Defb10 NM_139225.1
2.348387097 immuno response Apoa2 NM_013474.1 2.335526316 metabolic
Col1a2 scl012843.30_10 2.326599327 structural 1700030B17Rik
scl16712.12_256 2.319796954 unknown Atp9a scl18294.24.1_12
2.317307692 metabolic Ccl5 NM_013653.1 2.299539171 inflammation
Slc39a14 scl00213053.1_19 2.288590604 transporter Saa3
scl31343.5.1_35 2.25437788 inflammation 3632451O06Rik
scl45626.8_445 2.203812317 unknown Atrnl1 scl0226255.12_147
2.14556962 unknown Alms1 scl29818.18.1_0 2.137614679 unknown Nkx2-2
scl18553.4.1_4 2.112612613 transcription factor Prss19 NM_008940.1
2.076923077 signaling Hist1h4k NM_178211.1 2.038550501 unknown
Ephb2 scl0013844.2_257 2.017094017 receptor Syt12 NM_134164.2
2.016666667 signaling Foxq1 NM_008239.3 2.016666667 transcription
factor Sfrs16 scl31678.22.1_32 2.01518785 unknown Lancl1
NM_021295.1 2.010822511 immuno response Mlp scl0017357.2_67
2.010471204 signaling
Example 10
Human ES Cell Derived Motor Neurons can be Used to Identify
Neurotoxic Factors
[0180] In order to investigate the possible involvement of
candidate factors and signaling pathways in the glial mediated
neurotoxicity we have observed, we tested the effect of these
candidate gene products, or molecules that activate them, on motor
neuron survival in co-cultures with wild type glial cells.
Non-transgenic glia were individually pretreated for 1 day with
either one of the three cytokines MCP2, Cxcl7, or Rantes; with
GMFb; an agonist of SHH pathway; or with PGD2. Glia were pretreated
for 1 day with either MCP2 (100 ng/ml; Peprotech), Cxcl7 (100
ng/ml; Peprotech), Rantes (100 ng/ml; Peprotech), GMFb (250 ng/ml;
Peprotech), an agonist of Shh pathway (1 .mu.M), PGD2 (10 .mu.M;
Chayman Chemical) or MK 0524 (10 .mu.M; Chayman Chemical). After
the pretreatment for 24 hours, a cellular preparation containing
Hb9::GFP human ES cell-derived motor neurons, dissociated from EBs,
was added to the glia at the concentration of 30,000 cells/well.
Replicate cultures were individually maintained for 20 days in the
presence of each of the 6 factors, fixed, and the numbers of GFP
positive motor neurons quantified.
[0181] We found that treatment with GMFb did not significantly
affect the number of human ES cell derived motor neurons compared
to the control condition (95%+/-9%). Likewise, the presence of any
one of the three cytokines (Rantes, Cxcl7 and Mcp2), or the SHH
agonist, did not seem to negatively affect the number of GFP
positive motor neurons (respectively 108%+/-20%; 102%+/-12%;
103%+/-8%; 97%+/-12%) (FIG. 20C). However, when the cells were
treated for 20 days with PGD2, we found a dramatic decrease in the
number of motor neurons compared to the control condition
(19%+/-2%; p<0.01) (FIGS. 20C and 20D), suggesting that
prostaglandin D2 signaling contributes to motor neuron toxicity in
this system.
Example 11
Inhibition of the Prostaglandin D2 Receptor Rescues Motor Neuron
Loss
[0182] To determine if there was a direct relationship between the
toxic effect of prostaglandin signaling on motor neurons and the
SOD1G93A glial mediated neurotoxicity, we tested whether a specific
antagonist of the prostaglandin D2 receptor, MK 0524 (Sturino et
al., J. Med. Chem., 50:794-806, 2007), could counteract or
ameliorate the toxic effect of mutant glia on motor neurons.
SOD1G93A glia and wt glia were pretreated for 1 day with the
prostaglandin D2 receptor inhibitor, human motor neurons were
added, and cultures were maintained for 20 days both in the
presence and absence of the drug. We found that the presence of MK
0524 did not affect motor neuron numbers when they were co-cultured
with wildtype glia. (100%+/8%, FIG. 20E). However, when human motor
neurons plated on SOD1G93A glia were treated with the inhibitor,
there was a statistically significant (p<0.05) increase in the
number of GFP positive neurons (32%, relative to untreated neurons
plated on the same glia) (FIGS. 20E and 20F). These experiments
suggest that inhibitors of PGD2 signaling do not generally act to
promote motor neuron survival and instead act to specifically
counteract the toxic effects of glial cells carrying the ALS
mutation.
[0183] The foregoing description is to be understood as being
representative only and is not intended to be limiting. Alternative
methods and materials for implementing the invention and also
additional applications will be apparent to one of skill in the
art, and are intended to be included within the accompanying claims
Sequence CWU 1
1
6121DNAArtificialsynthetic primer 1catcagccct aatccatctg a
21221DNAArtificialsynthetic primer 2cgcgactaac aatcaaagtg a
21324DNAArtificialsynthetic primer 3ctaggccaca gaattgaaag atct
24421DNAArtificialsynthetic primer 4catcagccct aatccatctg a
21518DNAArtificialsynthetic primer 5aagttcatct gcaacacc
18620DNAArtificialsynthetic primer 6tccttgaaga agatggtgcg 20
* * * * *