U.S. patent application number 15/100682 was filed with the patent office on 2016-10-13 for method for highly efficient conversion of human stem cells to lineage-specific neurons.
The applicant listed for this patent is THE JOHNS HOPKINS UNIVERSITY, KENNEDY KRIEGER INSTITUTE, INC.. Invention is credited to John Laterra, Mingyao Ying.
Application Number | 20160298080 15/100682 |
Document ID | / |
Family ID | 53274055 |
Filed Date | 2016-10-13 |
United States Patent
Application |
20160298080 |
Kind Code |
A1 |
Ying; Mingyao ; et
al. |
October 13, 2016 |
METHOD FOR HIGHLY EFFICIENT CONVERSION OF HUMAN STEM CELLS TO
LINEAGE-SPECIFIC NEURONS
Abstract
The present invention relates to the field of stem cells. More
specifically, the present invention provides methods and
compositions useful for the highly efficient conversion of human
stem cells to lineage-specific neurons. In a specific embodiment, a
method of inducing differentiation of human stem cells into
dopaminergic (DA) neurons comprises the steps of (a) transfecting
human stem cells with a lentiviral vector encoding Atoh1, wherein
the vector is Dox inducible; and (b) growing the transfected cells
in culture in the presence of Dox, Sonic Hedgehog (SHH) and FGF-8b
until DA neurons are induced.
Inventors: |
Ying; Mingyao; (Ellicott
City, MD) ; Laterra; John; (Baltimore, MD) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
THE JOHNS HOPKINS UNIVERSITY
KENNEDY KRIEGER INSTITUTE, INC. |
Baltimore
Baltimore |
MD
MD |
US
US |
|
|
Family ID: |
53274055 |
Appl. No.: |
15/100682 |
Filed: |
December 3, 2014 |
PCT Filed: |
December 3, 2014 |
PCT NO: |
PCT/US2014/068273 |
371 Date: |
June 1, 2016 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
61911238 |
Dec 3, 2013 |
|
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61P 25/00 20180101;
C12N 2501/119 20130101; C12N 2506/08 20130101; A61K 35/30 20130101;
C12N 2501/60 20130101; C12N 2501/115 20130101; C12N 2506/02
20130101; C12N 2740/15043 20130101; C12N 15/86 20130101; C12N
2533/52 20130101; C12N 5/0619 20130101; C12N 2501/01 20130101; C12N
2506/45 20130101; C12N 15/85 20130101; C12N 2501/15 20130101; C12N
2501/13 20130101; C12N 2500/38 20130101; C12N 2533/32 20130101;
C12N 2501/41 20130101; C12N 2510/00 20130101; C12N 2799/027
20130101 |
International
Class: |
C12N 5/0793 20060101
C12N005/0793; C12N 15/85 20060101 C12N015/85; C12N 15/86 20060101
C12N015/86; A61K 35/30 20060101 A61K035/30 |
Claims
1. A method for inducing differentiation of neuronal cells from
human stem cells comprising the steps of (a) transfecting human
stem cells with an expression vector encoding Atoh1; and (b)
growing the transfected cells in culture until the stem cells are
differentiated.
2. The method of claim 1, wherein the human stem cells are induced
pluripotent stem cells (iPSCs), embryonic stem cells (ESCs) or
neural stem cells (NSCs).
3. The method of claim 2, wherein the neural stem cells are fetal
or adult neural stem cells.
4. The method of claim 1, wherein the expression vector is a viral
vector.
5. The method of claim 4, wherein the viral vector is from a
lentivirus, adeno-associated virus, herpes simplex virus, Senai
virus or baculovirus.
6. The method of claim 5, wherein the viral vector is from a
lentivirus.
7. The method of claim 1, wherein the expression vector is
non-viral.
8. The method of claim 1, wherein the expression vector encoding
Atoh1 is inducible.
9. The method of claim 6, wherein the lentiviral expression vector
is doxycycline (Dox) inducible.
10. The method of claim 9, wherein the cells are grown in the
presence of a sufficient concentration of Dox in order to complete
differentiation into neuronal cells.
11. The method of claim 9, wherein step (b) further comprises the
steps outlined in Table 1.
12. The method of claim 1, wherein prior to step (b), the method
further comprises the step of transfecting the human stem cells
with an expression vector encoding NeuroD1.
13. The method of claim 12, wherein prior to step (b), the method
further comprises the step of transfecting the human stem cells
with an expression vector encoding Neurogenin 2.
14. The method of claim 1, wherein the expression vector also
encodes NeuroD1 and/or Neurogenin 2.
15. The method of claim 1, further comprising exposing the cells to
sufficient concentrations of additional growth factors.
16. A method of inducing differentiation of human stem cells into
dopaminergic (DA) neurons comprising the steps of: a. transfecting
human stem cells with a lentiviral vector encoding Atoh1, wherein
the vector is Dox inducible; and b. growing the transfected cells
in culture in the presence of Dox, Sonic Hedgehog (SHH) and FGF-8b
until DA neurons are induced.
17. The method of claim 16, wherein the human stem cells are
iPSCs.
18. The method of claim 16, wherein the human stem cells are
ESCs.
19. The method of claim 17, wherein step (b) further comprises the
steps outlined in Table 2.
20. The method of claim 17, wherein the human stem cells are
NSCs.
21. The method of claim 20, wherein step (b) further comprises the
steps outlined in Table 3.
22. The method of claim 16, wherein prior to step (b), the method
further comprises the step of transfecting the human stem cells
with an expression vector encoding NeuroD1.
23. The method of claim 22, wherein prior to step (b), the method
further comprises the step of transfecting the human stem cells
with an expression vector encoding Neurogenin 2.
24. The method of claim 16, wherein the lentiviral vector also
encodes NeuroD1 and/or Neurogenin 2.
25. A method for treating a patient suffering from a
neurodegenerative disease comprising the steps of: a. obtaining
stem cells from the patient; b. initiating differentiation of the
stem cells into a population of differentiated cells using the
methods of claim 16; c. analyzing the development of differentiated
neurons in culture; and d. transplanting the differentiated cells
into the patient's brain.
26. The method of claim 25, wherein the neurodegenerative disease
is Parkinson's disease and the differentiated cells are DA
neurons.
27. A population of neuronal cells prepared using the method of
claim 16.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims the benefit of U.S. Provisional
Application No. 61/911,238, filed Dec. 3, 2013, which is
incorporated herein by reference in its entirety.
FIELD OF THE INVENTION
[0002] The present invention relates to the field of stem cells.
More specifically, the present invention provides methods and
compositions useful for the highly efficient conversion of human
stem cells to lineage-specific neurons.
INCORPORATION-BY-REFERENCE OF MATERIAL SUBMITTED ELECTRONICALLY
[0003] This application contains a sequence listing. It has been
submitted electronically via EFS-Web as an ASCII text file entitled
"P12596-02_ST25.txt." The sequence listing is 21,808 bytes in size,
and was created on Dec. 3, 2014. It is hereby incorporated by
reference in its entirety.
BACKGROUND OF THE INVENTION
[0004] Optimized differentiation strategies are essential for
differentiating human stem cells (SCs) into lineage-specific
neuronal progenies in sufficient numbers and purity for
transplantation or disease modeling. Current strategies for
generating lineage-specific neurons from human pluripotent or
multipotent stem cells, including induced pluripotent cells
(iPSCs), embryonic stem cells (ESCs) and fetal neural stem cells
(NSCs), generally yields incomplete neuronal conversion and lineage
specification, which adversely affects in vivo engraftment and
function of these neurons and also leads to considerable safety
concerns regarding their potential for malignancy formation or
neural overgrowth. The present invention provides a novel and
optimized strategy for highly efficient differentiation of human
SCs to lineage-specific and functional neurons.
SUMMARY OF THE INVENTION
[0005] The present invention is based, at least in part, on the
development of methods for using transcription factors including
Atoh1, Neurogenin 2, and NeuroD1, to drive highly efficient
neuronal differentiation of human SCs into lineage-specific
neurons, such as dopaminergic (DA) neurons.
[0006] Cell replacement therapy using human stem cells (SCs) holds
great promise for treating neurological diseases in which neuronal
loss results in cognitive, extrapyramidal, and/or motor
dysfunction. Optimized differentiation strategies are essential to
differentiate human stem cells into lineage-specific neuronal
progenies in sufficient numbers and purity for transplantation or
disease modeling. As described herein, we have established a
strategy for differentiating human pluripotent or multipotent SCs
into lineage-specific and functional neurons. We found a defined
transcription factor that can induce the highly efficient
conversion of human SCs to lineage-specific neurons (e.g.,
dopaminergic (DA) neurons). This strategy can generate dopaminergic
neurons with >80% purity. The established strategy is novel and
highly applicable for disease modeling and cell replacement therapy
for several neurological disorders, including but not limited to
Parkinson's disease, spinal cord injury, amyotrophic lateral
sclerosis and hearing loss.
[0007] Current differentiation strategies for converting human SCs
to neurons act primarily on cell surface receptors or intracellular
signaling proteins to alter the level of multiple downstream
transcription factors and in turn to change the gene expression
profile and cell fate specification. These strategies lack
specificity and efficiency because they activate multiple signaling
cascades and transcription factors, only a fraction of which are
optimal and necessary to drive lineage-specific neuronal
differentiation. In certain embodiments, our differentiation
strategy uses a single transcription factor Atoh1 to induce highly
specific neuronal differentiation signaling, resulting in rapid and
highly efficient lineage-specific neuronal conversion. In other
embodiments, the strategy may include NeuroD1 and/or Neurogenin 2.
Our differentiation strategy saves the time for generating
lineage-specific neurons from human SCs by at least 50%. It also
generates lineage-specific neurons with >80% purity,
significantly higher than the purity (10-40%) normally achieved by
other methods.
[0008] Accordingly, in one aspect, the present invention provides
methods for differentiating stem cells into neuronal cells. In one
embodiment, a method for inducing differentiation of neuronal cells
from human stem cells comprises the steps of (a) transfecting human
stem cells with an expression vector encoding Atoh1; and (b)
growing the transfected cells in culture until the stem cells are
differentiated. In certain embodiments, the human stem cells are
induced pluripotent stem cells (iPSCs), embryonic stem cells (ESCs)
or neural stem cells (NSCs). In specific embodiments, the neural
stem cells are fetal or adult neural stem cells.
[0009] In particular embodiments, Atoh1 comprises SEQ ID NO:2. In
other embodiments, Atoh1 comprises an N-terminal flag tag and is
shown, for example, in SEQ ID NO:4.
[0010] In other embodiments, the expression vector is a viral
vector. In particular embodiments, the viral vector is from a
lentivirus, adeno-associated virus, herpes simplex virus, Senai
virus or baculovirus. In a specific embodiment, the viral vector is
from a lentivirus. In other embodiments, the expression vector is
non-viral. In a further embodiment, the expression vector encoding
Atoh1 is inducible. In a specific embodiment, the lentiviral
expression vector is doxycycline (Dox) inducible.
[0011] In a further embodiment, the cells are grown in the presence
of a sufficient concentration of Doxycycline (Dox) in order to
induce Atoh1 transgene expression and drive the differentiation of
human stem cells into neuronal cells. In a more specific
embodiment, step (b) further comprises the steps outlined in Table
1. In yet another embodiment, prior to step (b), the method further
comprises the step of transfecting the human stem cells with an
expression vector encoding NeuroD1. In a particular embodiment,
NeuroD1 comprises SEQ ID NO:8. In an alternative embodiment,
NeuroD1 comprises SEQ ID NO:9. In another embodiment, prior to step
(b), the method further comprises the step of transfecting the
human stem cells with an expression vector encoding Neurogenin 2.
In a particular embodiment, Neurogenin 2 comprises SEQ ID NO:6. In
alternative embodiments, the expression vector of step (a) also
encodes Neurogenin 2 and/or NeuroD1. The present invention further
contemplates the use of biologically active or functional fragments
of Atoh1, Neurogenin 2 and/or NeuroD1 in the present invention.
Furthermore, the methods of the present invention can further
comprise exposing the cells to sufficient concentrations of
additional growth factors.
[0012] In a specific embodiment, a method of inducing
differentiation of human stem cells into dopaminergic (DA) neurons
comprises the steps of (a) transfecting human stem cells with a
lentiviral vector encoding Atoh1, wherein the vector is Dox
inducible; and (b) growing the transfected cells in culture in the
presence of Dox, Sonic Hedgehog (SHH) and FGF-8b until DA neurons
are induced. In one embodiment, the human stem cells are iPSCs. In
another embodiment, the human stem cells are ESCs. In such
embodiments, step (b) further comprises the steps outlined in Table
2. In an alternative embodiment, the human stem cells are NSCs. In
such embodiment, step (b) further comprises the steps outlined in
Table 3. In yet another embodiment, prior to step (b), the method
further comprises the step of transfecting the human stem cells
with an expression vector encoding NeuroD1. In another embodiment,
prior to step (b), the method further comprises the step of
transfecting the human stem cells with an expression vector
encoding Neurogenin 2. In alternative embodiments, the lentiviral
vector of step (a) also encodes NeuroD1 and/or Neurogenin 2.
[0013] In another aspect, the present invention provides methods
for treating patients suffering from a neurodegenerative disease.
In one embodiment, a method for treating a patient suffering from a
neurodegenerative disease comprises the steps of (a) obtaining stem
cells from the patient; (b) initiating differentiation of the stem
cells into a population of differentiated cells using a method
described herein; (c) analyzing the development of differentiated
neurons in culture; and (d) transplanting the differentiated cells
into the patient's brain. In certain embodiments, the
neurodegenerative disease is Parkinson's disease and the
differentiated cells are DA neurons.
[0014] The present invention also provides a population of neuronal
cells prepared using any one of the methods described herein.
BRIEF DESCRIPTION OF THE FIGURES
[0015] FIG. 1. Atoh1 is induced during the differentiation of human
pluripotent stem cells into neurons. (A): Human induced pluripotent
stem cells (iPSCs) were differentiated into neurons following the
dual-SMAD inhibition protocol. By day 10 of differentiation, cells
expressed neural lineage markers (PAX6 and NESTIN). By day 40 of
differentiation, iPSC-derived neurons expressed neuronal markers
(TUJ1 and MAP2). Cell nuclei were counterstained with DAPI. Scale
bars=50 .mu.m. (B, C): Markers for pluripotent cells (NANOG),
neural (PAX6), and neuronal (Ngn2 and NEUROD1) lineages were
analyzed by quantitative real-time polymerase chain reaction
(qRT-PCR) during iPSC differentiation at days 0, 10, and 20. Atoh1
expression was analyzed by qRT-PCR (B) and Western blotting (C).
The data represent means.+-.SEM. *, p<0.01 compared with day 0.
Abbreviation: DAPI, 4',6-diamidino-2-phenylindole.
[0016] FIG. 2. Ectopic Atoh1 expression drives neuronal conversion
in induced pluripotent stem cells (iPSCs). (A): Diagram of the
lentiviral vector for Dox-inducible Atoh1 expression. (B): Dox
controls the on/off switch of Atoh1 expression. Human iPSCs were
infected with lentivirus harboring Dox-inducible Atoh1. Stable
Atoh1-iPSCs after puromycin selection were treated with or without
Dox for 48 hours and transferred to Dox-free medium. Whole cell
lysates collected on each indicated time point were subjected to
immunoblot using anti-FLAG antibody. (C): Atoh1-iPSCs were treated
with Dox (+Dox) for 3 days and changed to Dox-free medium (Dox
withdrawal) for 3 days. Cells were immunostained with FLAG
antibody. (D): Diagram of Atoh1-induced neuron differentiation
protocol. Atoh1 is induced by Dox from days 1 to 5. (E):
Immunostaining from cell cultures at differentiation day 6 shows
TUJ1 expression in Atoh1-induced cells but not in control cells.
(F): Bright-field microscope images show cell adhesion and neuronal
process formation in Atoh1-induced cells on differentiation day 10.
(G): Immunostaining shows the coexpression of TUJ1 and Synapsin in
Atoh1-induced neurons on differentiation day 36. (H, I): During a
5-day time period, an equal number of Atoh1-iPSCs received
different lengths of Dox treatment (from 1 to 5 days). After being
matured for 30 days, cells were immunostained against neuronal
marker TUJ1 and MAP2. The percentage of TUJ1+/MAP2+ cells over
DAPI+ cells and the total number of TUJ1+/MAP2+ cells were
quantified in 10 random-selected microscopic fields (p, p, 0.01
compared with cells that had 4- and 5-day Atoh1 induction). (J, K):
Atoh1-iPSCs were treated with Dox for 2 days and returned to
Dox-free medium for 3 days (J). The expression of Atoh1, NEUROD1,
and Ngn2 was measured by quantitative real-time polymerase chain
reaction in control, Atoh1 induction, and Atoh1 silencing samples
(*, p<01 compared with control). In (C) and (E-G), cell nuclei
were counterstained with DAPI. Scale bars=20 .mu.m. The data
represent means.+-.SEM. Abbreviations: Con, control; DAPI,
4',6-diamidino-2-phenylindole; Dox, doxycycline; FLAG-Atoh1,
FLAG-tagged Atoh1; IRES, internal ribosome entry site; Puro.sup.r,
puromycin selection marker; rtTA3, reverse tet-transactivator; TRE,
tet-inducible promoter; UBC, human ubiquitin C promoter.
[0017] FIG. 3. Neuron subtype specification in Atoh1-induced
neurons. (A): Atoh1-induced neurons derived from Atoh1-induced
pluripotent stem cells were allowed to mature in vitro, and cells
at differentiation day 36 were immunostained with antibodies
detecting dopaminergic (TH), GABAergic (GAD67), serotonergic
(serotonin), and glutamatergic (VGluT1) neuron subtypes. Cell
nuclei were counterstained with DAPI. Scale bars=50 .mu.m. (B):
Immunostained neurons from 10 random-selected microscopic fields
were counted to calculate the percentage of TH+, GAD67+,
serotonin+, and VGluT1+ cells over DAPI+ cells. The data represent
means.+-.SEM. Abbreviations: DAPI, 4',6-diamidino-2-phenylindole;
N.D., not detected; TH, tyrosine hydroxylase; VGluT, vesicular
glutamate transporter 1.
[0018] FIG. 4. Ectopic Atoh1 expression and cell extrinsic factors
induce dopaminergic (DA) neurons from PSCs. (A): Diagram of DA
neuron differentiation induced by Atoh1, SHH, and FGF8b. (B):
Atoh1-iPSCs were differentiated by Atoh1 induction alone or in
combination with SHH and FGF8b. The expression of DA lineage
markers was analyzed by quantitative real-time polymerase chain
reaction using cells at differentiation day 6. Control cells
followed the same differentiation protocol but did not receive Dox
treatment. Atoh1 induction in combination with SHH and FGF8b more
robustly induced DA lineage markers than Atoh1 alone or untreated
cells. The data represent means.+-.SEM. *, p<01 compared with
Atoh1 alone; .diamond-solid., p<01 compared with control. (C,
D): Atoh1-iPSCs were differentiated following the protocol shown in
(A). Atoh1-induced neurons at differentiation day 36 were
immunostained for neuronal marker (TUJ1) and DA neuron marker (TH).
Cell nuclei were counterstained with DAPI. Scale bars=20 .mu.m.
TH+/TUJ1+DA neurons derived from Atoh1-iPSCs and Atoh1-ESCs from 10
random-selected microscopic fields were counted to calculate the
percentage of TH+/TUJ1+ cells over DAPI+ cells. The data represent
means.+-.SEM. (E): Bright-field microscope image of Atoh1-induced
iPSC-derived DA neurons 7 days after being recovered from
cryopreservation. Scale bars=20 .mu.m. (F): Atoh1-iPSCs (lx
10.sup.6) were differentiated following the protocol shown in (A).
Cells were counted to calculate the number of NPCs (differentiation
day 7), DA neurons (differentiation day 14), and post-thaw DA
neurons (frozen at differentiation day 7 and cultured 7 days after
cryopreservation). Abbreviations: Atoh1+S/F, Atoh1 induction in
combination with SHH and FGF8b; DAPI,
4',6-diamidino-2-phenylindole; DAT, dopamine transporter; ESC,
embryonic stem cell; FGF, fibroblast growth factor; iPSC, induced
pluripotent stem cell; NPC, neuron precursor cell; SHH, Sonic
Hedgehog; TH, tyrosine hydroxylase.
[0019] FIG. 5. The expression of midbrain DA neuron markers and
dopamine release in Atoh1-induced neurons. (A-E): Atoh1-induced
pluripotent stem cells were differentiated following the protocol
shown in FIG. 4A. DA neurons at differentiation day 36 were
immunostained for midbrain DA neuron markers (FOXA2, NURR1, EN1,
TH, GIRK2, and DAT) and mature neuron marker (Synapsin). Cell
nuclei were counterstained with DAPI. The arrows and arrowhead in
(E) indicate DAT+ and DAT- neurons, respectively. Scale bars=20
.mu.m. (F, G): Representative HPLC chromatogram (F) and
quantification (G) of DA and its metabolites (DOPAC, 3-MT, and HVA)
released from Atoh1-induced DA neurons at differentiation day 36 in
response to KCl-evoked depolarization for 15 minutes. The data
represent means.+-.SEM (n=2). Abbreviations: DA, dopaminergic;
DAPI, 4',6-diamidino-2-phenylindole; DAT, dopamine transporter;
DOPAC, 3,4-dihydroxy-phenylacetic acid; GIRK2, G protein-regulated
inward-rectifier potassium channel 2; HVA, homovanillic acid; 3-MT,
3-methoxytyramine; TH, tyrosine hydroxylase.
[0020] FIG. 6. Electrophysiological properties of Atoh1-induced
dopaminergic (DA) neurons. (A): Differential interference contrast
image of a patched Atoh1-induced DA neuron. Scale bar=20 .mu.m. (B,
C): Atoh1-induced DA neurons derived from Atoh1-iPSCs showed
spontaneous spiking activity. This cell has a resting membrane
potential of 265 mV (B) (a zoomed view is shown in the right panel)
and an average spiking frequency of 4.8 Hz (C) (left panel). The
spontaneous spiking frequencies from 37 neurons were plotted in the
right panel of (C) with the means6 SEM marked inside. (D): Whole
cell current-clamp recording of action potentials evoked by 70 pA
current injection (top panel). Action potentials were suppressed by
sodium channel blocker (TTX) (middle panel). Action potentials
recovered after TTX withdrawal (bottom panel). (E): An
hyperpolarized injection of current (0.2 nA) evoked
hyperpolarization and rebound tonic spiking. A typical
hyperpolarization sag was observed in the upper panel, which was
dampened by ML252 (5 mM, a KCNQ2 inhibitor, lower panel). (F):
Voltage-clamp recording of Atoh1-induced neurons. Depolarized
sodium and potassium currents were evoked by elevation the membrane
potential to different levels (left panel). Both sodium and
potassium currents were attenuated by sodium and potassium channel
inhibitors (TTX [0.5 mM] and 4-AP [25 mM], respectively).
Abbreviations: ACSF, artificial cerebrospinal fluid; TTX,
tetrodotoxin.
[0021] FIG. 7. Neurotoxicity induced by 6-OHDA in Atoh1-induced
dopaminergic (DA) neurons. (A): Atoh1-inducedDAneurons derived from
Atoh1-iPSCs at differentiation day 36 were treated with 6-OHDA for
15 minutes. Bright-field microscope images show the morphological
signs of neuron death at 24 hours after treatment. Scale bar=100
.mu.m. (B): Cell death was quantified by LDH cytotoxicity assay.
The data represent means.+-.SEM (n=3). *, p<01 compared with
control. Abbreviations: LDH, lactate dehydrogenase; 6-OHDA,
6-hydroxydopamine.
[0022] FIG. 8. Neuronal maturation of Atoh1-induced neurons derived
from human ESCs. At differentiation day 36, Atoh1-induced neurons
derived from ESCs co-expressed the synaptic vesicle protein
Synapsin and neuronal marker TUJ1. Cell nuclei were counterstained
with DAPI. Bar: 20 .mu.m.
[0023] FIG. 9. Atoh1 induction for 2-5 days is sufficient for
neuronal conversion in iPSCs. During a 5-day time period, equal
number of Atoh1-iPSCs received different length of Dox treatment
(from 1 to 5 days). Brightfield microscope images show neurons at
differentiation day 36, which were also immunostained against
neuronal marker TUJ1 and MAP2. Cell nuclei were counterstained with
DAPI. Bar: 20 .mu.m.
[0024] FIG. 10. Atoh1-induced DA neurons from human ESCs at
differentiation day 36 were immunostained with neuronal marker
(TUJ1) and DA neuron marker (TH). Cell nuclei were counterstained
with DAPI. Bar: 20 .mu.m.
[0025] FIG. 11. Atoh1-induced DA neurons from human iPSCs at
differentiation day 36 were immunostained against GAD67, serotonin,
SOX2 and OCT4. Cell nuclei were counterstained with DAPI. Bar: 20
.mu.m.
[0026] FIG. 12. Atoh1 induces highly efficient DA neuron
differentiation in human ESCs and NSCs. (A) Atoh1-ESCs were
differentiated following the protocol as shown in FIG. 4A and Table
2. Atoh1-induced neuron cultures at Day 36 were immunostained with
antibody against neuronal lineage marker (TUJ1) and DA neuron
marker (TH). Cell nuclei were counterstained with DAPI. (Bar: 20
.mu.m). Cells from 10 random-selected microscopic fields were
counted to calculate the percentage of TH+/TUJ1+ cells over DAPI+
cells (left panel, Data represents Mean.+-.SEM). (B) Atoh1-NSCs
were differentiated following the protocol as shown in FIG. 4A and
Table 3. Atoh1-induced neuron cultures at Day 36 were immunostained
with antibody against neuronal lineage marker (TUJ1) and DA neuron
marker (TH). Cell nuclei were counterstained with DAPI. (Bar: 20
.mu.m). Cells from 10 random-selected microscopic fields were
counted to calculate the percentage of TH+/TUJ1+ cells over DAPI+
cells (left panel, Data represents Mean.+-.SEM).
[0027] FIG. 13. Synthetic mRNA encoding Atoh1 drives the
differentiation of human iPSCs into neurons. Ectopic Atoh1 (with
N-terminal FLAG tag) expression was detected in >90% of the
Atoh1-mRNA-transfected cells (FIG. 13A) but not in untransfected
control cells (FIG. 13B), as determined by FLAG immunofluorencence
staining After iPSCs received daily transfection of Atoh1 mRNA for
4 days, cells were replated (3.times.10.sup.5 cells per cm.sup.2)
on dishes pre-coated with poly-D-Lysine (1 .mu.g/ml) and laminin (1
.mu.g/ml). Neurons after being matured in vitro for 20 days
co-express the neuronal marker (TUJ1) and the mature neuron markers
(MAP2 and Synapsin) (FIGS. 13C and 13D). Overall, these results
demonstrate that Atoh1 can be induced in human SCs by delivering
synthetic Atoh1 mRNA, and Atoh1 mRNA delivery drives neuronal
conversion of human SCs.
[0028] FIG. 14. Neurogenin 2 can be induced by Atoh1 and also
drives the differentiation of human iPSCs into neurons. (A) Atoh1
induction in human iPSCs for 2 days activated known Atoh1 target
NeuroD1 and the proneural transcription factor Neurogenin 2 that
has not yet been defined as an Atoh1 target gene (FIG. 14A), as
determined by quantifying gene expression using quantitative
real-time PCR. (B) We further tested if the Atoh1 target gene
Neurogenin 2 can also drive lineage-specific neuronal conversion of
human iPSCs. We applied the same lentivirus-mediated gene delivery
system as that for Atoh1 expression (FIG. 2A) to achieve Dox
inducible expression of Neurogenin 2 in human SCs. By following the
differentiation strategy as shown in FIG. 14B, we successfully
generated TH+/TUJ1+DA neurons from human iPSCs by inducing
Neurogenin 2 in combination with two morphogens (sonic hedgehog
(SHH) and FGF-8b) (FIG. 14C). These results demonstrate that
Atoh1-induced gene targets (e.g. Neurogenin 2) can be used to drive
lineage-specific neuronal conversion of human SCs.
DETAILED DESCRIPTION OF THE INVENTION
[0029] It is understood that the present invention is not limited
to the particular methods and components, etc., described herein,
as these may vary. It is also to be understood that the terminology
used herein is used for the purpose of describing particular
embodiments only, and is not intended to limit the scope of the
present invention. It must be noted that as used herein and in the
appended claims, the singular forms "a," "an," and "the" include
the plural reference unless the context clearly dictates otherwise.
Thus, for example, a reference to a "protein" is a reference to one
or more proteins, and includes equivalents thereof known to those
skilled in the art and so forth.
[0030] Unless defined otherwise, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art to which this invention belongs. Specific
methods, devices, and materials are described, although any methods
and materials similar or equivalent to those described herein can
be used in the practice or testing of the present invention.
[0031] All publications cited herein are hereby incorporated by
reference including all journal articles, books, manuals, published
patent applications, and issued patents. In addition, the meaning
of certain terms and phrases employed in the specification,
examples, and appended claims are provided. The definitions are not
meant to be limiting in nature and serve to provide a clearer
understanding of certain aspects of the present invention.
[0032] Human pluripotent stem cells (PSCs), including embryonic
stem cells (ESCs) and induced pluripotent stem cells (iPSCs),
exhibit unique characteristics such as indefinite self-renewal
capacity and multi-lineage differentiation potential. Human PSCs,
especially patient-derived iPSCs, hold enormous promise for various
applications in regenerative medicine, including disease modeling,
drug development and cell replacement therapy. In order to fully
utilize these patient-specific iPSCs in regenerative medicine,
highly efficient differentiation strategies are required to drive
iPSCs into desired lineages and generate functional cell progenies,
such as various subtypes of neurons. Current protocols for
differentiating human PSCs into lineage-specific neurons (e.g.,
dopaminergic (DA) neurons) are based on embryoid body formation,
stromal feeder co-culture, selective survival conditions or
inhibitors of SMAD signaling. These PSC-derived neurons have
allowed scientists to study molecular mechanisms underlying various
neurological disorders, test potential drugs and optimize
strategies for cell replacement therapy. However, current neuron
differentiation protocols for PSCs involve months of stem cell
culture procedures and multiple reagents, which cause significant
variation especially for researchers who have limited practice in
PSC culture and desire functional neurons at high purity for
disease-in-a-dish models. A broad desire for a robust system to
generate human PSC-derived neurons motivated us to develop a highly
efficient strategy to generate lineage-specific functional neurons
using the proneural transcription factor Atoh1.
[0033] ATOH1 (the mammalian homolog of Drosophila Atonal) belongs
to the proneural transcription factors of the basic
helix-loop-helix (bHLH) family. Proneural transcription factors are
crucial in driving the acquisition of a generic neuronal fate and
regulating neuronal subtype specification during development. Atoh1
proteins form heterodimers with E proteins, and these heterodimers
function as transcriptional activators by binding E box motifs
(CANNTG) in the regulatory regions of their target genes. Atoh1 is
a key regulator of neurogenesis, governing the differentiation of
various neuronal lineages, including cerebellar granule neurons,
brainstem neurons, inner ear hair cells, and numerous components of
the proprioceptive and interoceptive systems, as well as some
nonneuronal cell types. Atoh1 can activate crucial neurogenic
transcription factors, such as NeuroD1, 2, 6 and Nhlh1, 2, to
initiate a neuronal differentiation program that later becomes
self-supporting and Atoh1-independent. Certain members of the
proneural transcription factor family, such as ASCL1, Ngn2 and
NeuroD1, have been successfully used to generate neurons from both
PSCs and somatic cells. However, the role of Atoh1 in the neuronal
differentiation and neuron subtype specification of human PSCs is
largely unknown, and, as a result, Atoh1-based strategies for the
neuronal conversion of human PSCs is still unavailable.
[0034] Here, we show that Atoh1 is induced during the neuronal
differentiation of human PSCs. By transiently inducing ectopic
Atoh1 expression, we are able to efficiently convert PSCs into
neurons. Atoh1 induction, in combination with two neural patterning
morphogens (Sonic Hedgehog (SHH) and fibroblast growth factor 8b
(FGF8b)), leads to rapid and highly efficient conversion of PSCs
into DA neurons that recapitulate key biochemical and
electrophysiological features of primary midbrain DA neurons. We
also demonstrate that Atoh1-induced DA neurons serve as a reliable
model for analyzing 6-OHDA-induced neurotoxicity in human midbrain
DA neurons. Since most symptoms of Parkinson's disease (PD) result
from the degeneration of midbrain DA neurons located in the
substantia nigra, Atoh1-induced DA neurons provide an in vitro
neuron model for mechanistic studies and drug testing for PD.
[0035] Here, we established a method for using Atoh1 to drive
highly efficient neuronal differentiation of human SCs into
lineage-specific neurons, such as DA neurons. Neuronal
differentiation strategies are widely applicable to various kinds
of human stem cells, including human embryonic stem cells, induced
pluripotent cells, fetal/adult neural stem cells and mesenchymal
stem cells. Thus, the present invention can be used in driving the
neuronal differentiation of these stem cells.
[0036] Here, we used lentivirus to deliver Atoh1 gene into human
stem cells. Atoh1 gene can also be delivered by other virus
vectors, including but not limited to adeno-associated virus,
herpes simplex virus, Sendai virus and baculovirus. Non-viral Atoh1
delivery is also feasible by using plasmid transfection, mRNA
transfection and recombinant cell-penetrating protein. Certain
chemical compounds can also be used to activate expression of
endogenous Atoh1. See U.S. Patent Publication No. 20090232780.
[0037] Here, we used Atoh1 in the differentiation of human stem
cells into dopaminergic neurons. Atoh1 is a key regulator of
neurogenesis, governing the differentiation of various neuronal
lineages (cerebellar granule and brainstem neurons, inner ear hair
cells, and numerous components of the proprioceptive and
interoceptive systems), as well as some nonneuronal cell types. We
anticipate that Atoh1 can be used to differentiate human stem cells
into different neuronal lineages, including but not limited to
motor neurons, cerebellar neurons, inner ear hair cells, and
numerous neuron lineages of the proprioceptive and interoceptive
systems.
[0038] Here we used Atoh1 in combination with sonic hedgehog and
FGF-8b as the morphogenes to drive the differentiation of human SCs
into DA neurons. Other reagents, such as puromorphamine and
CHIR99021, have also been successfully used to drive the DA neuron
differentiation of human SCs. Expression of specific genes,
including but not limited to FOXA2, LMX1A, PITX3 and NURR1, has
also been shown to differentiate human or mouse SCs into DA
neurons. We anticipate that these reagents and genes can be
incorporated into Atoh1-induced DA neuron differentiation
protocol.
[0039] Without further elaboration, it is believed that one skilled
in the art, using the preceding description, can utilize the
present invention to the fullest extent. The following examples are
illustrative only, and not limiting of the remainder of the
disclosure in any way whatsoever.
EXAMPLES
[0040] The following examples are put forth so as to provide those
of ordinary skill in the art with a complete disclosure and
description of how the compounds, compositions, articles, devices,
and/or methods described and claimed herein are made and evaluated,
and are intended to be purely illustrative and are not intended to
limit the scope of what the inventors regard as their invention.
Efforts have been made to ensure accuracy with respect to numbers
(e.g., amounts, temperature, etc.) but some errors and deviations
should be accounted for herein. Unless indicated otherwise, parts
are parts by weight, temperature is in degrees Celsius or is at
ambient temperature, and pressure is at or near atmospheric. There
are numerous variations and combinations of reaction conditions,
e.g., component concentrations, desired solvents, solvent mixtures,
temperatures, pressures and other reaction ranges and conditions
that can be used to optimize the product purity and yield obtained
from the described process. Only reasonable and routine
experimentation will be required to optimize such process
conditions.
Example 1
Proneural Transcription Factor Atoh1 Drives Highly Efficient
Differentiation of Human Pluripotent Stems Cells into Dopaminergic
Neurons
Materials and Methods
[0041] Cell Culture.
[0042] Human H1 ESC line was obtained from WiCell Research
Resources (WiCell, WI). Human iPSC line ND27760 (passage 25-30) was
derived from human skin fibroblasts from a PD patient with a SNCA
triplication that were obtained from the Coriell Cell Repositories.
Cell reprogramming was performed using non-integrating 4 factor
(SOX2/OCT4/KLF4/MYC) Sendai virus system (CytoTune-iPS
Reprogramming Kit, Life Technologies). The pluripotency of this
iPSC line has been characterized by immunocytochemistry for
pluripotent cell markers (NANOG, OCT4, TRA-1-60 and SSEA-3) and
embryoid body differentiation. Human ESCs and iPSCs were maintained
as feeder-free cultures in Essential 8 medium (Life Technologies)
or mTESR1 medium (Stemcell Technologies) in 5% CO.sub.2/95% air
condition at 37.degree. C., and were passaged using dispase (Life
Technologies). Karyotype analysis of G-banded metaphase chromosomes
was performed to confirm the chromosomal integrity of these ESCs
and iPSCs. All experiments involving human stem cells were
performed with the approval of the Johns Hopkins Medicine
Institutional Review Boards.
[0043] Lentiviral Transduction.
[0044] Human Atoh1 cDNA was constructed using high-fidelity PCR kit
(Roche) and cloned into pTRIPZ vector (Thermo Scientific) with AgeI
and MluI. The nucleic acid and amino acid sequences for Atoh1 are
shown in SEQ ID NOS:1-2, respectively. Trans-Lentiviral Packaging
System (Thermo Scientific) was used for lentivirus packaging. Cells
were infected by lentivirus at an MOI of 5 for 24 h with the
addition of TransDux Virus Infection solution (System Biosciences).
Stable cell lines were established by puromycin selection (0.5
.mu.g/ml). All recombinant DNA and lentivirus experiments were
performed following the National Institutes of Health
guidelines.
[0045] Cell Differentiation and Cryopreservation.
[0046] To measure Atoh1 expression during the neuronal conversion
of human PSCs, cells were differentiated following a dual-SMAD
inhibition protocol (Chambers et al., 27 NAT. BIOTECHNOL. 275-80
(2009). Noggin in this protocol was replaced by LDN193189 (100 nM,
Stemgent). For the Atoh1-induced neuron differentiation protocol,
cells were plated (8.times.10.sup.4 cells per cm.sup.2) on matrigel
(BD) in Essential 8 Medium (Life Technologies) with the ROCK
inhibitor (Y-27632, 10 .mu.M, Stemgent). Atoh1 was induced by
Doxycycline (0.5 .mu.g/ml, Sigma-Aldrich) in culture medium from
Day 1 to 5. From Day 1 to 3, cell culture medium was changed every
day and gradually shifted from Essential 6 Medium (Life
Technologies) to N2 Medium (DMEM/F12 medium with N2 supplement,
Life Technologies). Cells were cultured in N2 Medium until day 7,
dissociated using Accutase (Sigma-Aldrich) and replated
(3.times.10.sup.5 cells per cm.sup.2) on dishes pre-coated with
poly-D-Lysine (1 .mu.g/ml) and laminin (1 .mu.g/ml) using neuron
culture medium (Neurobasal Medium with B27 supplement, BDNF
(brain-derived neurotrophic factor, 20 ng/ml, PeproTech), GDNF
(Glial cell line-derived neurotrophic factor, 20 ng/ml, PeproTech),
TGF.beta.3 (transforming growth factor type .beta.3, 1 ng/ml,
R&D), ascorbic acid (0.2 mM, Sigma-Aldrich), dibutyryl cAMP
(0.5 mM, Sigma-Aldrich), and DAPT (10 .mu.M, Stemgent)). From Day 8
to Day 36, half of the cell culture medium was replenished every
3-4 days. For Atoh1-induced DA neuron differentiation protocol, the
protocol above was modified by adding SHH (SHH C25II, 100 ng/ml,
R&D) and FGF8b (100 ng/ml, PeproTech) from Day 1 to 5.
[0047] Atoh1-induced DA neuron precursors at differentiation Day 7
were dissociated using Accutase. 1.times.10.sup.6 cells were
cryopreserved in 1 ml freezing medium (40% Neurobasal Medium with
B27 supplement, 50% fetal bovine serum and 10% DMSO) using a
freezing container (Nalgene) in -80.degree. C. for 24 h and stored
in liquid nitrogen.
[0048] Atoh1-Mediated Differentiation of Human iPSCs into Mature
Neurons.
[0049] As shown in FIG. 2D, we established protocols for
differentiating Atoh1-iPSCs into neurons. The recipes for cell
culture media are listed in Table 1. Cells were plated
(4.times.10.sup.4 cells per cm.sup.2) on matrigel (BD) in Essential
8 Medium (Life Technologies) with ROCK inhibitor (Y-27632,
Stemgent). From Day 1 to Day 7, cell culture medium was changed
every day. From Day 8 to Day 36, half of the cell culture medium
was changed every 3-4 days. On differentiation day 6, Atoh1
dramatically increases the level of neuronal differentiation marker
(TUJ1) when compared with Dox-untreated control (FIG. 2E). On day
7, cells were dissociated using Accutase (Sigma-Aldrich) and
replated (3.times.10.sup.5 cells per cm.sup.2) on dishes pre-coated
with poly-D-Lysine (1 .mu.g/ml) and laminin (1 .mu.g/ml). After
cell passaging, cells differentiated by Atoh1 adhered and formed
neuronal processes (FIG. 2F, right panel). Cells without Atoh1
induction failed to attach and grow in neuron culture medium (FIG.
2F, left panel). Atoh1-induced neurons further matured in vitro,
and co-expressed neuronal marker (.beta.-tubulin III, TUJ1) and
synaptic resident protein (synapsin), suggesting the establishment
of synaptic terminals and neuronal maturation (FIG. 2G).
TABLE-US-00001 TABLE 1 Neuron differentiation media used in Atoh1-
induced protocol as shown in FIG. 2D Day Medium Additives 0 E8
Y-27632 1 E6 (75%) + N2 (25%) Doxycyline 2 E6 (50%) + N2 (50%)
Doxycyline 3 E6 (25%) + N2 (75%) Doxycyline 4 N2 Doxycyline 5 N2
Doxycyline 6 N2 (50%) + B27 (50%) 7 B27 8-36 B27 B/G/C/A/D E8:
Essential 8 Medium E6: Essential 6 Medium N2: DMEM/F12 medium with
N2 supplement (1:100) B27: neurobasal medium with B27 supplement
(1:50) Doxycyline (0.5 .mu.g/ml) Y-27632 (10 .mu.M) B/G/C/A/D:
BDNF(20 ng/ml)/GDNF(10 ng/ml)/Dibutyryl cAMP(0.5
mM)/AscorbicAcid(0.2 mM)/DAPT(10 .mu.M)
[0050] Quantitative Real-Time PCR (qRT-PCR).
[0051] Total RNA was extracted using the RNeasy Mini kit (Qiagen).
Reverse transcription was performed using MuLV reverse
transcriptase (Applied biosystems) and Oligo(dT) primers. qRT-PCR
was performed using SYBR Green PCR Master Mix (Applied Biosystems)
and IQ5 RT-PCR detection system (Bio-rad). All primer sequences are
listed in Table 1. Relative expression of each gene was normalized
to the 18S rRNA.
[0052] Western Blot.
[0053] Total cellular proteins were extracted with RIPA buffer
(Sigma-Aldrich) containing a protease and phosphatase inhibitor
cocktail (Calbiochem). SDS-PAGE was performed with 50 .mu.g total
cellular proteins per lane using 4-12% gradient Tris-glycine gels
(Lonza). Western blot was performed using Quantitative Western Blot
System (LI-COR Biosciences) following the manufacturer's
instructions. The primary antibodies were: mouse anti-FALG M2
(Sigma-Aldrich), rabbit anti-Atoh1 (Millipore) and mouse
anti-.beta.-actin (Sigma-Aldrich). Secondary antibodies were
labeled with IRDye infrared dyes and protein levels were quantified
with Odyssey Infrared Imaging System (LI-COR Biosciences).
[0054] Immunofluorescence and Cell Counting.
[0055] Differentiated cells were fixed in 4% PFA/1% sucrose in PBS
(pH 7.4) at room temperature, and blocked with 5% normal goat serum
and 0.2% Triton X-100. Primary antibodies (Table 2) were diluted in
5% normal goat serum and incubated with samples overnight at
4.degree. C. Cy3 and Alexa 488 labeled secondary antibodies were
applied for 2 hours. Samples were counterstained with DAPI and
mounted on glass slides using ProLong anti-fade kit (Life
Technologies).
[0056] The percentage of marker positive cells was determined in
samples derived from at least three independent experiments. In
Adobe Photoshop software, images from 10 randomly selected fields
were used for counting the number of DAPI-positive cells expressing
a specific marker.
[0057] Electrophysiological Recordings.
[0058] Voltage-clamp or current-clamp recordings were performed at
35.degree. C. in a chamber perfused with regular Artificial
Cerebrospinal Fluid (ACSF; in mM: NaCl, 124; KCl, 2.5; MgCl.sub.2,
1.3; CaCl.sub.2 2.5; NaH.sub.2PO.sub.4, 1; NaHCO.sub.3, 26.2;
glucose, 20; pH 7.4, equilibrated with 95% O.sub.2 and 5% CO.sub.2,
.about.310 mosm) which flowed at 4 ml/min. Patch electrodes were
pulled from borosilicate glass and had resistances of 2-4.0
M.OMEGA. when filled with an intracellular solution (in mM:
KMeSO.sub.4, 135; KCl, 5; HEPES, 5; EGTA free acid, 0.25; Mg-ATP,
2; GTP, 0.5; phosphocreatine-tris, 10; pH 7.3, .about.290
mosm).
[0059] Neurons were identified using a 10.times. objective mounted
on an upright microscope with transmitted light, and their neuronal
somata were then visualized through a 40.times. water immersion
objective using infrared differential interference contrast (DIC)
optics. The cell somatic recordings were made using an Axopatch
200B amplifier in combination with pClamp 9.0 software (Molecular
Devices). Neurons were voltage-clamped at -80 mV. R.sub.series and
R.sub.input were monitored using a 2.5 mV 100 ms depolarizing
voltage step in each recording sweep. Current traces were filtered
at 5 kHz, and digitized at 10 kHz using a Digidata 1322A interface,
and stored for off-line analysis. Leak and capacitative currents
were corrected by subtracting a scaled current elicited by a +2.5
mV step from the holding potential.
[0060] For current clamp recording, the same Axopatch 200B
amplifier was used; whole cell mode was achieved initially in the
voltage clamp configuration. Then, the recording was switched into
current clamp mode. The resting membrane potential was monitored
for more than five minutes. The experiment was discontinued if the
resting membrane potential became more positive than -40 mV. The
action potential was continually monitored for five minutes, and if
there was no threshold change, the reagent perfusion commenced. All
reagents were bought from Sigma (St Louis, Mo.) except TTX (abcam,
Cambridge, Mass.) and ML252 (Vanderbilt Center for Neuroscience
Drug Discovery).
[0061] High-Performance Liquid Chromatography (HPLC) Analysis.
[0062] On day 36 of differentiation, medium was replaced by HBSS
buffer with addition of 56 mM KCl (200 .mu.l per well in 24-well
plates) and incubated for 15 min at 37.degree. C. Medium was
collected and centrifuged (15,000 g for 15 min at 4.degree. C.) to
clear cell debris. Samples were immediately frozen in liquid
nitrogen and stored at -80.degree. C. For HPLC analysis, samples
were thawed and concentrated using a vacuum (Savant SDP 121P
ThermoSci) connected with refrigerated vapor trap (Savant RVT 5105
ThermoSci) and the freeze dried samples were resuspended in 10 mM
perchloric acid. Monoamines were analyzed by HPLC-ECD
(Electrochemical Detection) by dual channel coulchem III
electrochemical detector (Model 5300, ESA, Inc. Chelmsford, Mass.,
USA), and monoamines were separated by using a reverse phase C18
column (3 mm.times.150 mm C-18 RP-column, Acclaim Polar advantage
II, Thermo Scientific) with a flow rate of 0.600 mL/min. Monoamine
concentrations were quantified by comparison of the area under the
curve (AUC) to known standard dilutions.
[0063] 6-OHDA Treatment in DA Neurons and LDH Analysis.
[0064] Neuron culture medium was changed to neurobasal medium
before treatment. 6-OHDA was freshly prepared in vehicle solution
(ascorbic acid (0.15%) in H.sub.2O) and quickly added to the neuron
culture. Control cells were treated with vehicle solution alone.
After 15 min at 37.degree. C., the medium was removed and neurons
were gently washed twice with Neurobasal Medium. 200 .mu.l neuron
culture medium (Neurobasal Medium with B27 Supplement) was added to
each well and further incubated for 24 h. Cytotoxicity induced by
6-OHDA was measured using LDH Cytotoxicity Detection Kit (Roche)
following the manufacturer's protocol. The percentage cytotoxicity
was calculated using the following equation: Cytotoxicity
(%)=(Experiment Value-Low Control)/(High Control-Low
Control).times.100. (Low control: culture medium; High control:
total cell lysate).
[0065] Data Analysis and Statistics.
[0066] All results reported here represent at least three
independent replications. Statistical analysis was performed using
Prizm software (GraphPad). Post-hoc tests included the Students
t-test and the Tukey multiple comparison tests as appropriate. All
data are represented as mean value.+-.standard error of mean
(SEM).
[0067] For neurophysiological recordings, the recorded data were
first visualized with Clampfit 9.2, and exported to Matlab
(Mathworks, Natick, Mass.) for further analysis and plotting. The
recording traces are visualized with Igor 6.0 (WaveMetrics,
Portland, Oreg.). All group data are reported as mean.+-.STD except
otherwise stated.
Results
[0068] Atoh1 is Induced During the Neuronal Differentiation of
Human PSCs.
[0069] We followed a dual-SMAD inhibition protocol (Chambers et
al., 27 NAT. BIOTECHNOL. 275-80 (2009)) for differentiating human
iPSCs into neurons. Differentiated cells first expressed
neurectodermal marker (PAX6) and neural rosette marker (NESTIN) at
day 10 of differentiation (FIG. 1A, left). Mature neurons at day 40
of differentiation expressed neuronal marker .beta.-Tubulin III
(TUJ1) and MAP2 (FIG. 1A, right). We further examined the
expression of various markers at differentiation day 0, 10 and 20
by quantitative real-time PCR (qRT-PCR), which confirmed the
inhibition of pluripotency marker (NANOG) and the induction of
neural markers (PAX6, Ngn2 and NEUROD1) (FIG. 1B). Next, we
examined Atoh1 expression by qRT-PCR and found that ATOH1 was
induced at differentiation day 10 and 20 when compared to
undifferentiated cells (FIG. 1B). Western blotting also confirmed
the induction of Atoh1 protein at differentiation day 20 (FIG. 1C).
These data suggested that Atoh1 is involved in the neuronal
conversion of human PSCs, which warranted further study.
[0070] Ectopic Atoh1 Expression Induces Highly Efficient Neuronal
Conversion of Human PSCs.
[0071] To address whether Atoh1 induction is sufficient for the
neuronal differentiation of human PSCs, we established a
lentivirus-mediated gene delivery system to achieve Dox-inducible
Atoh1 expression in human PSCs. We constructed a Tet-On lentiviral
vector that harbors human Atoh1 transgene with N-terminal FLAG tag
(SEQ ID NO:3) (FIG. 2A). Human iPSCs and ESCs were infected with
Atoh1 lentivirus to establish stable cell lines (Atoh1-iPSC, and
Atoh1-ESC) after puromycin selection. Dox treatment for 48 h in
Atoh1-iPSCs induced Atoh1 expression as determined by
immunoblotting against FLAG tag, and transgenic Atoh1 expression
was turned off after Dox withdrawal (FIG. 2B). Immunostaining
against FLAG tag also confirmed Atoh1 induction after 3-day Dox
treatment and the silencing of Atoh1 transgene after Dox withdrawal
for 3 days (FIG. 2C).
[0072] Next, we induced ectopic Atoh1 expression in PSCs for
neuronal differentiation following a protocol outlined in FIG. 2D
(also see details in Materials and Methods). Atoh1-iPSCs were
maintained in a feeder-free culture system, and Atoh1 was induced
by Dox for 5 days to drive neuronal conversion. After Dox
withdrawal, neuronal precursors were passaged and allowed to
further mature in vitro. On differentiation day 6, Atoh1 induced
robust expression of the neuronal differentiation marker TUJ1,
which was not detected in Dox-untreated cells (FIG. 2E). On day 7,
cells were dissociated and replated on surfaces pre-coated for
neuron culture. Two days after cell passaging, Atoh1-induced cells
adhered and formed neuronal processes. In contrast, control cells
failed to attach or grow in neuron culture medium (FIG. 2F). After
further maturation in vitro for 30 days, Atoh1-induced neurons
co-expressed the synaptic vesicle protein Synapsin and neuronal
marker (TUJ1), demonstrating the establishment of synaptic
terminals and neuronal maturation (FIG. 2G). We also replicated
these results in Atoh1-ESC, in which Atoh1 also initiated the
neuronal differentiation process and generated mature neurons (FIG.
8).
[0073] To further optimize the Atoh1-mediated differentiation
strategy, we asked what is the minimum time of Atoh1 induction for
successful neuronal conversion. Equal numbers of Atoh1-iPSCs
received different durations of Dox treatment (1 to 5 days), after
which cells were replated and allowed to mature in vitro for
additional 30 days. Cells with Dox treatment for 1 day failed to
attach after replating. In contrast, Dox treatment for 2 to 5 days
successfully differentiated iPSCs into neurons expressing TUJ1 and
MAP2 (FIG. 9). By quantifying the number of TUJ1+/MAP2+ neurons, we
found that longer Atoh1 induction time did not increase the purity
of Atoh1-induced neurons (FIG. 2H), but did significantly increase
the yield of neurons, especially when comparing Atoh1 induction for
4-5 days to 2-day induction (FIG. 2I). To compare the level of
neurogenic signaling before and after silencing ectopic Atoh1
expression, we treated Atoh1-iPSCs with Dox for 2 days and then
withdrew Dox for 3 days (FIGS. 2J and 2K). As determined by
qRT-PCR, Atoh1 showed 49-fold up-regulation after Dox treatment and
decreased after Dox withdrawal. Two neurogenic transcription
factors (NEUROD1 and Ngn2) showed 6- and 5-fold induction,
respectively, in response to Atoh1 induction. After Dox withdrawal,
their expression did not decrease but increased further to 261- and
189-fold higher than control cells, respectively. These results
suggest that ectopic Atoh1 expression in PSCs initiates a
neurogenic program that becomes self-sustaining after the
withdrawal of ectopic Atoh1.
[0074] To determine the subtype specification of Atoh1-induced
neurons, we characterized neurons induced from Atoh1-iPSCs with
various neuron subtype markers (FIG. 3). By day 36 of
differentiation, .about.35% of Atoh1-induced neurons expressed
tyrosine hydroxylase (TH), the rate-limiting enzyme in DA synthesis
and a widely-used DA neuron marker. Fewer than 10% of total cells
expressed glutamate decarboxylase (GAD67) and serotonin, markers
for GABAergic and serotonergic neurons, respectively. Glutamatergic
neurons expressing Vesicular Glutamate Transporter 1 (VGluT1) were
not detected in Atoh1-induced neurons.
[0075] Atoh1-Mediated Differentiation of Human PSCs into DA
Neurons.
[0076] We found that ectopic Atoh1 expression preferentially drives
the differentiation of human PSCs to TH-expressing neurons,
suggesting a DA lineage specification. Two morphogens (SHH and
FGF-8b) for neural patterning have been widely used to drive DA
lineage specification during the neuronal conversion of human PSCs.
We combined Atoh1 induction with these two morphogens to
differentiate PSCs into DA neurons, following a protocol outlined
in FIG. 4A. Ectopic Atoh1 expression alone induced multiple DA
neuron markers, such as FOXA2, NURR1, LMX1A, OTX2, Ngn2, TH, DAT
and VMAT2, most of which were further upregulated significantly by
combining Atoh1 induction with SHH and FGF-8b (FIG. 4B). At day 36
of differentiation, Atoh1-induced neurons derived from both iPSCs
and ESCs co-expressed the neuronal marker (TUJ1) and the DA neuron
marker (TH) (FIG. 4C and FIG. 10). The Atoh1-mediated protocol
yielded DA neurons from human iPSCs and ESCs with 82.+-.8% and
84.+-.9% purity, respectively, as determined by the percentage of
TH+/TUJ1+ cells over DAPI+ cells (FIG. 4D).
[0077] In order to store Atoh1-induced DA neurons, Atoh1-induced DA
neuron precursor cells (NPCs) at differentiation day 7 were
cryopreserved, and these cells showed high viability and neuronal
morphology when being recovered from cryopreservation and cultured
for 7 days (FIG. 4E). From 1.times.10.sup.6 iPSCs, the
Atoh1-mediated protocol generated 4.7.times.10.sup.6 DA NPCs, and
yielded 2.7.times.10.sup.6 or 1.4.times.10.sup.6 DA neurons after
direct cell passaging or cryopreservation, respectively (FIG.
4F).
[0078] Next, we analyzed the expression of midbrain DA neuron
markers in Atoh1-induced neurons. By differentiation day 36, these
neurons expressed the midbrain DA neuron markers FOXA2, NURR1,
Engrailed 1 (EN1), TH, G-protein-regulated inward-rectifier
potassium channel 2 (GIRK2) and dopamine transporter (DAT) (FIGS.
5A, 5B, 5C, 5D and 5E), which are also expressed in midbrain DA
neurons located in substantia nigra pars compacta (SNpc). These
Atoh1-induced DA neurons show extensive TUJ1+ nerve fiber growth
and robust expression of synaptic vesicle protein Synapsin (FIG.
5E). GABAergic (GAD67+) or serotonergic (serotonin+) neurons were
not detected in Atoh1-induced neurons derived from iPSCs, and
undifferentiated iPSCs (SOX2+ or OCT4+) were also not detected
(FIG. 11).
[0079] Functional Characterization of Atoh1-Induced DA Neurons.
[0080] We asked if Atoh1-induced DA neurons exhibit key
physiological properties of mature midbrain DA neurons. Dopamine
release was quantified in Atoh1-induced DA neurons at
differentiation day 36. HPLC analysis demonstrated the release of
dopamine and its metabolites evoked by KCl depolarization (FIGS. 5F
and 5G).
[0081] It has been well established that midbrain DA neurons are
pacemaker neurons that discharge spontaneously at a rate between 1
and 10 Hz with an average rate of 4.5 Hz. To test whether
Atoh1-induced DA neurons display similar electrophysiological
properties to primary midbrain DA neurons, we performed patch-clamp
recording in Atoh1-induced DA neurons (n=57) derived from human
iPSCs at differentiation day 36-49 (FIG. 6A). In voltage-clamp
experiments, the series input resistance of these Atoh1-induced DA
neurons was 7.7.+-.3 M.OMEGA.; the input resistance was
295.8.+-.174.5 M.OMEGA., and the average resting membrane potential
was 75.3.+-.9.9 mV. 64.9% of these neurons showed spontaneous
spiking activity (FIG. 6B, n=37) with an amplitude of 66.1.+-.18.3
mV and a mean frequency of 6.2.+-.4.7 Hz (n=37; FIG. 6C). 26.3% of
these neurons discharged action potentials during current injection
either by depolarization or hyperpolarization (n=15), and only 8.7%
(n=5) of these neurons did not have typical action potential either
by positive or negative current injection.
[0082] We further investigated the maturation of intrinsic ion
channels in Atoh1-induced DA neurons. As shown in FIG. 6D, we first
injected current (70 pA) to a neuron to depolarize the membrane
potential. This induced a train of action potentials (FIG. 6D, up
panel), which were completely blocked by the sodium channel blocker
TTX (0.5 .mu.M, 5 minutes administration, FIG. 6D, middle panel).
This effect was reversed by TTX washout, after which the action
potentials recovered in 17 minutes (FIG. 6D, bottom panel).
[0083] Midbrain DA neurons have been found to also have KCNQ
potassium channels that contribute to their tonic spontaneous
activity, and during hyperpolarization these neurons display a
typical sag in voltage. Here, we injected negative current to
hyperpolarize the membrane of Atoh1-induced neurons (FIG. 6E). This
negative current injection produced hyperpolarization sag and
rebound action potentials that resemble the tonic spontaneous
spiking activity. Both events were blocked by ML252 (5 .mu.M, a
KCNQ2 inhibitor). We further investigated the voltage-sensitive
sodium and potassium currents in voltage-clamp mode by elevating
membrane potentials to different levels (FIG. 6F, left). After the
treatment of sodium and potassium channel inhibitors (TTX (0.5
.mu.M) and 4-AP (25 .mu.M), respectively), both sodium and
potassium currents are significantly attenuated (FIG. 6F,
right).
[0084] Atoh1-Induced DA Neurons are Sensitive to 6-OHDA
Treatment.
[0085] 6-hydroxydopamine (6-OHDA) is a neurotoxin widely used to
induce neurotoxicity both in vivo and in vitro to model DA neuron
loss in PD pathogenesis. In neuron cultures from the substantia
nigra of neonatal rat brains, 6-OHDA treatment at 40 .mu.M causes
selective DA neuron loss without affecting GABA neurons. Here, we
tested the response of Atoh1-induced DA neurons to 6-OHDA
treatment. 6-OHDA treatment (40 .mu.M and 100 .mu.M) for 15 minutes
caused morphological signs of neuron death, including cell
condensation and neurite fragmentation (FIG. 7A), and neuron death
was also confirmed by LDH Cytotoxicity assay (FIG. 7B). Thus,
Atoh1-induced DA neurons derived from human iPSCs are sensitive to
6-OHDA treatment at a concentration that selectively damages
primary DA neurons isolated from substantia nigra.
Discussion
[0086] Human iPSCs provide a unique cell resource for establishing
patient-specific disease models and for testing potential
therapies. Due to the limited resource of human neurons,
lineage-specific neurons derived from human iPSCs are the most
desirable cells for modeling various neurological disorders, such
as midbrain DA neurons for PD, striatal GABAergic neurons for
Huntington's disease, and cholinergic motor neurons for amyotrophic
lateral sclerosis. It is critical to develop highly efficient
protocols for neuronal conversion in PSCs, in order to translate
current iPSC-derived neuron models from small-scale laboratory
applications to large-scale personalized drug testing platforms.
Proneural transcription factors are core drivers of neurogenesis,
and multiple members in this family (e.g., ASCL1, Ngn2 and NeuroD1)
have been used to differentiate PSCs into neurons and more recently
transdifferentiate somatic cells into neurons. We now show that
Atoh1 is a highly efficient driver for neuronal conversion in PSCs,
and Atoh1 induction in combination with cell extrinsic factors
rapidly differentiates human PSC to functional DA neurons at high
purity.
[0087] Multiple proneural transcription factors, e.g., ASCL1, Ngn2,
and NeuroD1, are activated during the neuronal conversion of human
PSCs, which initiate and sustain a neurogenic transcriptional
network. We identified Atoh1 as a proneural transcription factor
that is also upregulated during this neuronal conversion process.
By using a Tet-On gene expression system to transiently induce
ectopic Atoh1 expression in PSCs, we found that Atoh1 induction
alone is sufficient for highly efficient neuronal conversion in
PSCs. We further determined that 2 days are the minimal amount of
time and that 4-5 days are ideal for transient Atoh1 induction in
order to achieve successful neuronal conversion in PSCs. Several
studies have suggested that Atoh1 and other proneural transcription
factors are able to activate a neurogenic transcription factor
network that over time becomes self-supporting. We also found that
the neurogenic transcription factor NeuroD1 and Ngn2 were induced
by ectopic Atoh1 and their expression was sustained after the
silencing of exogenous Atoh1. This demonstrates that Atoh1-induced
neuronal differentiation program in PSCs can become self-supporting
and independent of exogenous Atoh1. It is noteworthy to mention
that although exogenous Atoh1 was not detectable by western
blotting after Dox withdrawal, Atoh1 expression did not return to
baseline. This result is consistent with our previous result in
FIG. 1B showing that endogenous Atoh1 is upregulated during the
neuronal conversion of PSCs. The persistence of endogenous Atoh1
expression can be explained by the evidence that Atoh1 protein
binds to its own enhancer to establish an autoregulation loop for
maintaining its expression. Overall, our results support the
mechanism that transient Atoh1 expression in PSCs can activate a
cell-intrinsic program for neuronal commitment (a neuro-programming
process). This process might share similar features to somatic cell
reprogramming, where transient expression of reprogramming
transcription factors induces the remodeling of epigenetic markers
and drives cells into a self-sustaining pluripotent status. The
epigenetic dynamics during this neuro-programming process warrants
further studies, and a deep understanding of this process might
lead to more potent approaches for converting both PSCs and somatic
cells into neurons.
[0088] Proneural transcription factors have been shown to
coordinately control the acquisition of a generic neuronal fate and
the neuron subtype specification [9]. The functions of proneural
transcription factors during neural development are strongly
influenced by the spatial and temporal context including multiple
modifiers such as transcriptional cofactors and cell extrinsic
factors. Atoh1 has been found to drive the differentiation of
numerous neuronal populations (e.g., cerebellar granule neurons,
spinal cord neurons and inner ear hair cells), as well as diverse
nonneuronal cell types (e.g., Merkel cells and intestinal secretory
lineages), suggesting that the functions of Atoh1 depend on
specific developmental contexts. When ectopic Atoh1 was expressed
in the context of human PSCs, we detected a high percentage of
neurons expressing the DA marker TH, and multiple DA neuron markers
were induced in response to ectopic Atoh1 expression. Moreover, SHH
and FGF8b, two neural patterning morphogens for DA specification,
further promoted the expression of DA neuron markers and increased
the efficiency of Atoh1-induced DA neuron conversion. These results
suggest that Atoh1-induced neurons respond to extrinsic factors for
generating lineage-specific neurons. A recent report shows that, in
embryonic bodies derived from mouse ESCs, Atoh1 induction in
combination with extrinsic factors promotes the generation of
cerebellar granule neurons. Atoh1 induction has also been found to
induce inner ear hair cell-like cells from mouse ESC-derived
embryonic bodies. Overall, it is possible to derive different
neuron subtypes from human or mouse PSCs by controlling the
temporal induction of Atoh1 in various differentiation stages of
PSCs and combining Atoh1 induction with different cell intrinsic
and extrinsic factors (e.g., neuron-subtype-specific transcription
factors or morphogens). Other proneural transcription factors also
show this plasticity in specifying various neuron subtypes. For
example, ASCL1 has been used to generate glutamatergic/GABAergic,
DA and cholinergic neurons.
[0089] We established a highly efficient Atoh1-mediated approach
for generating lineage-specific functional neurons from human PSCs.
Inducible Atoh1 transgene was delivered using a single-vector
Tet-On lentivirus and is stable in PSCs after >15 passages with
puromycin selection (data not shown). Atoh1-induced DA neuron
cultures derived from human iPSCs and ESCs showed >80%
pan-neuronal purity and >80% DA subtype purity. This protocol
yields DA neurons from PSCs with a rate of return of >250% or
>100% after the cyropreservation of Atoh1-induced NPCs. The
cryopreservation of Atoh1-induced DA NPCs will enable us to
establish patient-specific DA neuron banks After in vitro
maturation, Atoh1-induced DA neurons expressed midbrain DA neuron
markers (such as GIRK2, NURR1, FOXA2 and DAT) and exhibited robust
synapse formation. The functional maturation of these DA neurons
was further confirmed by DA release and spontaneous spiking
activity. Overall, Atoh1-induced DA neurons derived from human
iPSCs recapitulate key features of primary midbrain DA neurons,
making this Atoh1-mediated approach particularly applicable for PD
modeling using patient-derived iPSCs. It has been reported that
primary DA neurons but not GABAergic neurons, from the substantia
nigra of neonatal rat brains, are sensitive to 6-OHDA treatment at
low concentration (40 .mu.M). We also demonstrated that
Atoh1-induced DA neurons showed similar 6-OHDA sensitivity to
primary midbrain DA neurons, supporting that Atoh1-induced DA
neurons can serve as a reliable neurotoxicity model for PD.
[0090] Due to the use of genome-integrating lentivirus for ectopic
Atoh1 expression, our Atoh1-induced DA neurons will not be optimal
for cell replacement therapy. However, we found that transient
Atoh1 expression for 3-5 days is sufficient for highly efficient
neuronal conversion in PSCs. Thus, non-integrating viruses (e.g.,
adenovirus and Sendai virus) should be suitable to overcome the
limitation due to using lentivirus. More recently, multiple
virus-free systems based on mRNA or protein delivery have been
established to generate transgene-free human iPSCs [42-44], and
these approaches can be applied to generate transplant-ready
Atoh1-induced DA neurons. It is also noteworthy that chemical
compounds that induce Atoh1 expression have been identified and
patented (Patent Publication Number: US20090232780A1), thus
providing another potential method for Atoh1 induction in PSCs that
warrants further testing.
Conclusion
[0091] Atoh1 is a potent driver for highly efficient neuronal
conversion in human PSCs. Atoh1 induction in combination with cell
extrinsic factors differentiates PSCs into functional DA neurons in
high purity. Atoh1-induced DA neurons derived from human iPSCs
recapitulate key features of primary midbrain DA neurons and
provide a useful cell model for studying the pathogenesis of both
familial PD and, more importantly, sporadic PD, and testing
potential PD therapies.
Example 2
Atoh1-Mediated Differentiation of Human ESCs and NSCs into DA
Neurons
[0092] Atoh1-ESCs were differentiated following the protocol as
shown in FIG. 4A and Table 2. Atoh1-induced neuron cultures at Day
36 co-expressed neuronal and DA neuron markers, .beta.-tubulin III
(TUJ1) and tyrosine hydroxylase (TH), respectively (FIG. 12A). The
Atoh1 induction protocol yielded DA neurons from human ESCs with
84.+-.9% purity as determined by the percentage of TUJ+/TH+ cells
(FIG. 12A). Atoh1-NSCs were differentiated following the protocol
as shown in FIG. 4A and Table 3. Atoh1-induced neuron cultures at
Day 36 co-expressed neuronal marker (.beta.-tubulin III, TUJ1) and
DA neuron marker (tyrosine hydroxylase, TH) (FIG. 12B). The Atoh1
induction protocol yielded DA neurons from human NSCs with 82.+-.8%
purity as determined by the percentage of TUJ+/TH+ cells (FIG.
12B).
TABLE-US-00002 TABLE 2 DA neuron differentiation media used in
Atoh1- induced protocol as shown in FIG. 4A Day Medium Additives 0
E8 Y-27632 1 E6 (75%) + N2 (25%) Doxycyline/SHH/FGF-8b 2 E6 (50%) +
N2 (50%) Doxycyline/SHH/FGF-8b 3 E6 (25%) + N2 (75%)
Doxycyline/SHH/FGF-8b 4 N2 Doxycyline/SHH/FGF-8b 5 N2
Doxycyline/SHH/FGF-8b 6 N2 (50%) + B27 (50%) 7 B27 8-36 B27
B/G/T/C/A/D E8: Essential 8 Medium E6: Essential 6 Medium N2:
DMEM/F12 medium with N2 supplement(1:100) B27: neurobasal medium
with B27 supplement(1:50) Doxycyline (0.5 .mu.g/ml); Y-27632 (10
.mu.M) SHH: Sonic Hedgehog (200 ng/ml); FGF-8b (100 ng/ml)
B/G/T/C/A/D: BDNF(20 ng/ml)/GDNF(10 ng/ml)/TGF-.beta.3(1
ng/ml)/Dibutyryl cAMP(0.5 mM)/AscorbicAcid(0.2 mM)/DAPT(10
.mu.M)
TABLE-US-00003 TABLE 3 DA neuron differentiation media used in
Atoh1-induced protocol as shown in FIG. 4A NSC Differentiation Day
Medium Additives 0 N2 bFGF 1 N2 Doxycyline/SHH/FGF-8b 2 N2
Doxycyline/SHH/FGF-8b 3 N2 Doxycyline/SHH/FGF-8b 4 N2
Doxycyline/SHH/FGF-8b 5 N2 Doxycyline/SHH/FGF-8b 6 N2 (50%) + B27
(50%) 7 B27 8-36 B27 B/G/T/C/A/D E8: Essential 8 Medium E6:
Essential 6 Medium N2: DMEM/F12 medium with N2 supplement(1:100)
B27: neurobasal medium with B27 supplement(1:50) bFGF(10 ng/ml);
SHH: Sonic Hedgehog (200 ng/ml); FGF-8b (100 ng/ml) B/G/T/C/A/D:
BDNF(20 ng/ml)/GDNF(10 ng/ml)/TGF-.beta.3(1 ng/ml)/Dibutyryl
cAMP(0.5 mM)/AscorbicAcid(0.2 mM)/DAPT(10 .mu.M)
Example 3
Atoh1-Mediated Differentiation of Human iPSCs into Dopaminergic
(DA) Neurons
[0093] As shown in FIG. 4A, we established protocols for
differentiating human iPSCs into DA neurons by Atoh1 and other
additives (sonic hedgehog and FGF-8b). The recipes for cell culture
media are listed in Table 2. Cells were plated (4.times.10.sup.4
cells per cm.sup.2) on matrigel (BD) in Essential 8 Medium (Life
Technologies) with ROCK inhibitor (Y-27632, Stemgent). From Day 1
to Day 7, cell culture medium was changed every day. From Day 8 to
Day 36, half of the cell culture medium was changed every 3-4 days.
At day 36 of differentiation, these neurons co-expressed neuronal
marker (.beta.-tubulin III, TUJ1) and DA neuron marker (tyrosine
hydroxylase, TH), the rate-limiting enzyme in the synthesis of
dopamine (FIG. 4C). The Atoh1 induction protocol yielded DA neurons
with 89.+-.6% purity as determined by the percentage of TUJ+/TH+
cells (FIG. 4D). Moreover, the Atoh1-induced DA neurons also
co-expressed other midbrain DA neuron markers, such as
G-protein-regulated inward-rectifier potassium channel 2 (GIRK2),
forkhead box protein A2 (FOXA2) and dopamine transporter (DAT)
(FIGS. 6A, 6B and 6C), which are also expressed in substantia nigra
pars compacta (SNPC) DA neurons. By day 60, these DA neurons show
extensive TUJ1+ nerve fiber growth and robustly express mature
neuron marker (synapsin) (FIG. 6C).
Example 4
Synthetic mRNA Encoding Atoh1 Drives the Differentiation of Human
iPSCs into Neurons
[0094] We further tested Atoh1 induction in iPSCs by using
synthetic mRNAs encoding Atoh1. A cDNA encoding Atoh1 protein with
an N-terminal FLAG tag was cloned into a DNA vector with T7
promoter for in vitro mRNA synthesis. The synthesis of Atoh1 mRNA
was performed by using the mMESSAGE mMACHINE.RTM. T7 ULTRA
Transcription Kit (Ambion). Synthetic Atoh1 mRNA was transfected
into human iPSCs cultured in the 24-well plate (0.25 .mu.g mRNA per
well) using a lipid-based mRNA transfection reagent. Ectopic Atoh1
expression was detected in >90% of the Atoh1-mRNA-transfected
cells (FIG. 13A) but not in untransfected control cells (FIG. 13B),
as determined by FLAG immunofluorencence staining After iPSCs
received daily transfection of Atoh1 mRNA for 4 days, cells were
replated (3.times.10.sup.5 cells per cm.sup.2) on dishes pre-coated
with poly-D-Lysine (1 .mu.g/ml) and laminin (1 .mu.g/ml). Neurons
after being matured in vitro for 20 days co-express the neuronal
marker (TUJ1) and the mature neuron markers (MAP2 and Synapsin)
(FIGS. 13C and 13D). Overall, these results demonstrate that Atoh1
can be induced in human SCs by delivering synthetic Atoh1 mRNA, and
Atoh1 mRNA delivery drives neuronal conversion of human SCs.
Example 5
Neurogenin 2 can be Induced by Atoh1 and Also Drives the
Differentiation of Human iPSCs into Neurons
[0095] Atoh1 can activate crucial neurogenic transcription factors,
such as NeuroD1, 2, 6 and Nhlh1, 2, to initiate a neuronal
differentiation program that later becomes self-supporting and
Atoh1-independent. Here, we found that Atoh1 induction in human
iPSCs for 2 days activated known Atoh1 target NeuroD1 and the
proneural transcription factor Neurogenin 2 that has not yet been
defined as an Atoh1 target gene (FIG. 14A), as determined by
quantifying gene expression using quantitative real-time PCR. We
further tested if the Atoh1 target gene Neurogenin 2 can also drive
lineage-specific neuronal conversion of human iPSCs. We applied the
same lentivirus-mediated gene delivery system as that for Atoh1
expression (FIG. 2A) to achieve Dox inducible expression of
Neurogenin 2 in human SCs. By following the differentiation
strategy as shown in FIG. 14B, we successfully generated
TH+/TUJ1+DA neurons from human iPSCs by inducing Neurogenin 2 in
combination with two morphogens (sonic hedgehog (SHH) and FGF-8b)
(FIG. 14C). These results demonstrate that Atoh1-induced gene
targets (e.g. Neurogenin 2) can be used to drive lineage-specific
neuronal conversion of human SCs.
Sequence CWU 1
1
911065DNAHomo sapiensmisc_feature(1)..(1065)Human Atoh1 nucleic
acid sequence 1atgtcccgcc tgctgcatgc agaagagtgg gctgaagtga
aggagttggg agaccaccat 60cgccagcccc agccgcatca tctcccgcaa ccgccgccgc
cgccgcagcc acctgcaact 120ttgcaggcga gagagcatcc cgtctacccg
cctgagctgt ccctcctgga cagcaccgac 180ccacgcgcct ggctggctcc
cactttgcag ggcatctgca cggcacgcgc cgcccagtat 240ttgctacatt
ccccggagct gggtgcctca gaggccgctg cgccccggga cgaggtggac
300ggccgggggg agctggtaag gaggagcagc ggcggtgcca gcagcagcaa
gagccccggg 360ccggtgaaag tgcgggaaca gctgtgcaag ctgaaaggcg
gggtggtggt agacgagctg 420ggctgcagcc gccaacgggc cccttccagc
aaacaggtga atggggtgca gaagcagaga 480cggctagcag ccaacgccag
ggagcggcgc aggatgcatg ggctgaacca cgccttcgac 540cagctgcgca
atgttatccc gtcgttcaac aacgacaaga agctgtccaa atatgagacc
600ctgcagatgg cccaaatcta catcaacgcc ttgtccgagc tgctacaaac
gcccagcgga 660ggggaacagc caccgccgcc tccagcctcc tgcaaaagcg
accaccacca ccttcgcacc 720gcggcctcct atgaaggggg cgcgggcaac
gcgaccgcag ctggggctca gcaggcttcc 780ggagggagcc agcggccgac
cccgcccggg agttgccgga ctcgcttctc agccccagct 840tctgcgggag
ggtactcggt gcagctggac gctctgcact tctcgacttt cgaggacagc
900gccctgacag cgatgatggc gcaaaagaat ttgtctcctt ctctccccgg
gagcatcttg 960cagccagtgc aggaggaaaa cagcaaaact tcgcctcggt
cccacagaag cgacggggaa 1020ttttcccccc attcccatta cagtgactcg
gatgaggcaa gttag 10652354PRTHomo sapiensMISC_FEATURE(1)..(354)Human
Atoh1 amino acid sequence 2Met Ser Arg Leu Leu His Ala Glu Glu Trp
Ala Glu Val Lys Glu Leu 1 5 10 15 Gly Asp His His Arg Gln Pro Gln
Pro His His Leu Pro Gln Pro Pro 20 25 30 Pro Pro Pro Gln Pro Pro
Ala Thr Leu Gln Ala Arg Glu His Pro Val 35 40 45 Tyr Pro Pro Glu
Leu Ser Leu Leu Asp Ser Thr Asp Pro Arg Ala Trp 50 55 60 Leu Ala
Pro Thr Leu Gln Gly Ile Cys Thr Ala Arg Ala Ala Gln Tyr 65 70 75 80
Leu Leu His Ser Pro Glu Leu Gly Ala Ser Glu Ala Ala Ala Pro Arg 85
90 95 Asp Glu Val Asp Gly Arg Gly Glu Leu Val Arg Arg Ser Ser Gly
Gly 100 105 110 Ala Ser Ser Ser Lys Ser Pro Gly Pro Val Lys Val Arg
Glu Gln Leu 115 120 125 Cys Lys Leu Lys Gly Gly Val Val Val Asp Glu
Leu Gly Cys Ser Arg 130 135 140 Gln Arg Ala Pro Ser Ser Lys Gln Val
Asn Gly Val Gln Lys Gln Arg 145 150 155 160 Arg Leu Ala Ala Asn Ala
Arg Glu Arg Arg Arg Met His Gly Leu Asn 165 170 175 His Ala Phe Asp
Gln Leu Arg Asn Val Ile Pro Ser Phe Asn Asn Asp 180 185 190 Lys Lys
Leu Ser Lys Tyr Glu Thr Leu Gln Met Ala Gln Ile Tyr Ile 195 200 205
Asn Ala Leu Ser Glu Leu Leu Gln Thr Pro Ser Gly Gly Glu Gln Pro 210
215 220 Pro Pro Pro Pro Ala Ser Cys Lys Ser Asp His His His Leu Arg
Thr 225 230 235 240 Ala Ala Ser Tyr Glu Gly Gly Ala Gly Asn Ala Thr
Ala Ala Gly Ala 245 250 255 Gln Gln Ala Ser Gly Gly Ser Gln Arg Pro
Thr Pro Pro Gly Ser Cys 260 265 270 Arg Thr Arg Phe Ser Ala Pro Ala
Ser Ala Gly Gly Tyr Ser Val Gln 275 280 285 Leu Asp Ala Leu His Phe
Ser Thr Phe Glu Asp Ser Ala Leu Thr Ala 290 295 300 Met Met Ala Gln
Lys Asn Leu Ser Pro Ser Leu Pro Gly Ser Ile Leu 305 310 315 320 Gln
Pro Val Gln Glu Glu Asn Ser Lys Thr Ser Pro Arg Ser His Arg 325 330
335 Ser Asp Gly Glu Phe Ser Pro His Ser His Tyr Ser Asp Ser Asp Glu
340 345 350 Ala Ser 31134DNAHomo
sapiensmisc_feature(1)..(1134)Human Atoh1 nucleic acid sequence
with N-terminal flag tag 3atggactaca aagaccatga cggtgattat
aaagatcatg atatcgatta caaggatgac 60gatgacaaga tgtcccgcct gctgcatgca
gaagagtggg ctgaagtgaa ggagttggga 120gaccaccatc gccagcccca
gccgcatcat ctcccgcaac cgccgccgcc gccgcagcca 180cctgcaactt
tgcaggcgag agagcatccc gtctacccgc ctgagctgtc cctcctggac
240agcaccgacc cacgcgcctg gctggctccc actttgcagg gcatctgcac
ggcacgcgcc 300gcccagtatt tgctacattc cccggagctg ggtgcctcag
aggccgctgc gccccgggac 360gaggtggacg gccgggggga gctggtaagg
aggagcagcg gcggtgccag cagcagcaag 420agccccgggc cggtgaaagt
gcgggaacag ctgtgcaagc tgaaaggcgg ggtggtggta 480gacgagctgg
gctgcagccg ccaacgggcc ccttccagca aacaggtgaa tggggtgcag
540aagcagagac ggctagcagc caacgccagg gagcggcgca ggatgcatgg
gctgaaccac 600gccttcgacc agctgcgcaa tgttatcccg tcgttcaaca
acgacaagaa gctgtccaaa 660tatgagaccc tgcagatggc ccaaatctac
atcaacgcct tgtccgagct gctacaaacg 720cccagcggag gggaacagcc
accgccgcct ccagcctcct gcaaaagcga ccaccaccac 780cttcgcaccg
cggcctccta tgaagggggc gcgggcaacg cgaccgcagc tggggctcag
840caggcttccg gagggagcca gcggccgacc ccgcccggga gttgccggac
tcgcttctca 900gccccagctt ctgcgggagg gtactcggtg cagctggacg
ctctgcactt ctcgactttc 960gaggacagcg ccctgacagc gatgatggcg
caaaagaatt tgtctccttc tctccccggg 1020agcatcttgc agccagtgca
ggaggaaaac agcaaaactt cgcctcggtc ccacagaagc 1080gacggggaat
tttcccccca ttcccattac agtgactcgg atgaggcaag ttag 11344377PRTHomo
sapiensMISC_FEATURE(1)..(377)Human Atoh1 amino acid sequence with
N-terminal flag tag 4Met Asp Tyr Lys Asp His Asp Gly Asp Tyr Lys
Asp His Asp Ile Asp 1 5 10 15 Tyr Lys Asp Asp Asp Asp Lys Met Ser
Arg Leu Leu His Ala Glu Glu 20 25 30 Trp Ala Glu Val Lys Glu Leu
Gly Asp His His Arg Gln Pro Gln Pro 35 40 45 His His Leu Pro Gln
Pro Pro Pro Pro Pro Gln Pro Pro Ala Thr Leu 50 55 60 Gln Ala Arg
Glu His Pro Val Tyr Pro Pro Glu Leu Ser Leu Leu Asp 65 70 75 80 Ser
Thr Asp Pro Arg Ala Trp Leu Ala Pro Thr Leu Gln Gly Ile Cys 85 90
95 Thr Ala Arg Ala Ala Gln Tyr Leu Leu His Ser Pro Glu Leu Gly Ala
100 105 110 Ser Glu Ala Ala Ala Pro Arg Asp Glu Val Asp Gly Arg Gly
Glu Leu 115 120 125 Val Arg Arg Ser Ser Gly Gly Ala Ser Ser Ser Lys
Ser Pro Gly Pro 130 135 140 Val Lys Val Arg Glu Gln Leu Cys Lys Leu
Lys Gly Gly Val Val Val 145 150 155 160 Asp Glu Leu Gly Cys Ser Arg
Gln Arg Ala Pro Ser Ser Lys Gln Val 165 170 175 Asn Gly Val Gln Lys
Gln Arg Arg Leu Ala Ala Asn Ala Arg Glu Arg 180 185 190 Arg Arg Met
His Gly Leu Asn His Ala Phe Asp Gln Leu Arg Asn Val 195 200 205 Ile
Pro Ser Phe Asn Asn Asp Lys Lys Leu Ser Lys Tyr Glu Thr Leu 210 215
220 Gln Met Ala Gln Ile Tyr Ile Asn Ala Leu Ser Glu Leu Leu Gln Thr
225 230 235 240 Pro Ser Gly Gly Glu Gln Pro Pro Pro Pro Pro Ala Ser
Cys Lys Ser 245 250 255 Asp His His His Leu Arg Thr Ala Ala Ser Tyr
Glu Gly Gly Ala Gly 260 265 270 Asn Ala Thr Ala Ala Gly Ala Gln Gln
Ala Ser Gly Gly Ser Gln Arg 275 280 285 Pro Thr Pro Pro Gly Ser Cys
Arg Thr Arg Phe Ser Ala Pro Ala Ser 290 295 300 Ala Gly Gly Tyr Ser
Val Gln Leu Asp Ala Leu His Phe Ser Thr Phe 305 310 315 320 Glu Asp
Ser Ala Leu Thr Ala Met Met Ala Gln Lys Asn Leu Ser Pro 325 330 335
Ser Leu Pro Gly Ser Ile Leu Gln Pro Val Gln Glu Glu Asn Ser Lys 340
345 350 Thr Ser Pro Arg Ser His Arg Ser Asp Gly Glu Phe Ser Pro His
Ser 355 360 365 His Tyr Ser Asp Ser Asp Glu Ala Ser 370 375
5819DNAHomo sapiensmisc_feature(1)..(819)Human Neurogenin2 nucleic
acid sequence 5atgttcgtca aatccgagac cttggagttg aaggaggaag
aggacgtgtt agtgctgctc 60ggatcggcct cccccgcctt ggcggccctg accccgctgt
catccagcgc cgacgaagaa 120gaggaggagg agccgggcgc gtcaggcggg
gcgcgtcggc agcgcggggc tgaggccggg 180cagggggcgc ggggcggcgt
ggctgcgggt gcggagggct gccggcccgc acggctgctg 240ggtctggtac
acgattgcaa acggcgccct tcccgggcgc gggccgtctc ccgaggcgcc
300aagacggccg agacggtgca gcgcatcaag aagacccgta gactgaaggc
caacaaccgc 360gagcgaaacc gcatgcacaa cctcaacgcg gcactggacg
cgctgcgcga ggtgctcccc 420acgttccccg aggacgccaa gctcaccaag
atcgagaccc tgcgcttcgc ccacaactac 480atctgggcac tcaccgagac
cctgcgcctg gcggatcact gcgggggcgg cggcgggggc 540ctgccggggg
cgctcttctc cgaggcagtg ttgctgagcc cgggaggagc cagcgccgcc
600ctgagcagca gcggagacag cccctcgccc gcctccacgt ggagttgcac
caacagcccc 660gcgccgtcct cctccgtgtc ctccaattcc acctccccct
acagctgcac tttatcgccc 720gccagcccgg ccgggtcaga catggactat
tggcagcccc cacctcccga caagcaccgc 780tatgcacctc acctccccat
agccagggat tgtatctag 8196272PRTHomo
sapiensMISC_FEATURE(1)..(272)Human Neurogenin2 amino acid sequence
6Met Phe Val Lys Ser Glu Thr Leu Glu Leu Lys Glu Glu Glu Asp Val 1
5 10 15 Leu Val Leu Leu Gly Ser Ala Ser Pro Ala Leu Ala Ala Leu Thr
Pro 20 25 30 Leu Ser Ser Ser Ala Asp Glu Glu Glu Glu Glu Glu Pro
Gly Ala Ser 35 40 45 Gly Gly Ala Arg Arg Gln Arg Gly Ala Glu Ala
Gly Gln Gly Ala Arg 50 55 60 Gly Gly Val Ala Ala Gly Ala Glu Gly
Cys Arg Pro Ala Arg Leu Leu 65 70 75 80 Gly Leu Val His Asp Cys Lys
Arg Arg Pro Ser Arg Ala Arg Ala Val 85 90 95 Ser Arg Gly Ala Lys
Thr Ala Glu Thr Val Gln Arg Ile Lys Lys Thr 100 105 110 Arg Arg Leu
Lys Ala Asn Asn Arg Glu Arg Asn Arg Met His Asn Leu 115 120 125 Asn
Ala Ala Leu Asp Ala Leu Arg Glu Val Leu Pro Thr Phe Pro Glu 130 135
140 Asp Ala Lys Leu Thr Lys Ile Glu Thr Leu Arg Phe Ala His Asn Tyr
145 150 155 160 Ile Trp Ala Leu Thr Glu Thr Leu Arg Leu Ala Asp His
Cys Gly Gly 165 170 175 Gly Gly Gly Gly Leu Pro Gly Ala Leu Phe Ser
Glu Ala Val Leu Leu 180 185 190 Ser Pro Gly Gly Ala Ser Ala Ala Leu
Ser Ser Ser Gly Asp Ser Pro 195 200 205 Ser Pro Ala Ser Thr Trp Ser
Cys Thr Asn Ser Pro Ala Pro Ser Ser 210 215 220 Ser Val Ser Ser Asn
Ser Thr Ser Pro Tyr Ser Cys Thr Leu Ser Pro 225 230 235 240 Ala Ser
Pro Ala Gly Ser Asp Met Asp Tyr Trp Gln Pro Pro Pro Pro 245 250 255
Asp Lys His Arg Tyr Ala Pro His Leu Pro Ile Ala Arg Asp Cys Ile 260
265 270 71071DNAHomo sapiensmisc_feature(1)..(1071)Human NeuroD1
nucleic acid sequence 7atgaccaaat cgtacagcga gagtgggctg atgggcgagc
ctcagcccca aggtcctcca 60agctggacag acgagtgtct cagttctcag gacgaggagc
acgaggcaga caagaaggag 120gacgacctcg aagccatgaa cgcagaggag
gactcactga ggaacggggg agaggaggag 180gacgaagatg aggacctgga
agaggaggaa gaagaggaag aggaggatga cgatcaaaag 240cccaagagac
gcggccccaa aaagaagaag atgactaagg ctcgcctgga gcgttttaaa
300ttgagacgca tgaaggctaa cgcccgggag cggaaccgca tgcacggact
gaacgcggcg 360ctagacaacc tgcgcaaggt ggtgccttgc tattctaaga
cgcagaagct gtccaaaatc 420gagactctgc gcttggccaa gaactacatc
tgggctctgt cggagatcct gcgctcaggc 480aaaagcccag acctggtctc
cttcgttcag acgctttgca agggcttatc ccaacccacc 540accaacctgg
ttgcgggctg cctgcaactc aatcctcgga cttttctgcc tgagcagaac
600caggacatgc ccccccacct gccgacggcc agcgcttcct tccctgtaca
cccctactcc 660taccagtcgc ctgggctgcc cagtccgcct tacggtacca
tggacagctc ccatgtcttc 720cacgttaagc ctccgccgca cgcctacagc
gcagcgctgg agcccttctt tgaaagccct 780ctgactgatt gcaccagccc
ttcctttgat ggacccctca gcccgccgct cagcatcaat 840ggcaacttct
ctttcaaaca cgaaccgtcc gccgagtttg agaaaaatta tgcctttacc
900atgcactatc ctgcagcgac actggcaggg gcccaaagcc acggatcaat
cttctcaggc 960accgctgccc ctcgctgcga gatccccata gacaatatta
tgtccttcga tagccattca 1020catcatgagc gagtcatgag tgcccagctc
aatgccatat ttcatgatta g 10718356PRTHomo
sapiensMISC_FEATURE(1)..(356)Human NeuroD1 amino acid sequence 8Met
Thr Lys Ser Tyr Ser Glu Ser Gly Leu Met Gly Glu Pro Gln Pro 1 5 10
15 Gln Gly Pro Pro Ser Trp Thr Asp Glu Cys Leu Ser Ser Gln Asp Glu
20 25 30 Glu His Glu Ala Asp Lys Lys Glu Asp Asp Leu Glu Ala Met
Asn Ala 35 40 45 Glu Glu Asp Ser Leu Arg Asn Gly Gly Glu Glu Glu
Asp Glu Asp Glu 50 55 60 Asp Leu Glu Glu Glu Glu Glu Glu Glu Glu
Glu Asp Asp Asp Gln Lys 65 70 75 80 Pro Lys Arg Arg Gly Pro Lys Lys
Lys Lys Met Thr Lys Ala Arg Leu 85 90 95 Glu Arg Phe Lys Leu Arg
Arg Met Lys Ala Asn Ala Arg Glu Arg Asn 100 105 110 Arg Met His Gly
Leu Asn Ala Ala Leu Asp Asn Leu Arg Lys Val Val 115 120 125 Pro Cys
Tyr Ser Lys Thr Gln Lys Leu Ser Lys Ile Glu Thr Leu Arg 130 135 140
Leu Ala Lys Asn Tyr Ile Trp Ala Leu Ser Glu Ile Leu Arg Ser Gly 145
150 155 160 Lys Ser Pro Asp Leu Val Ser Phe Val Gln Thr Leu Cys Lys
Gly Leu 165 170 175 Ser Gln Pro Thr Thr Asn Leu Val Ala Gly Cys Leu
Gln Leu Asn Pro 180 185 190 Arg Thr Phe Leu Pro Glu Gln Asn Gln Asp
Met Pro Pro His Leu Pro 195 200 205 Thr Ala Ser Ala Ser Phe Pro Val
His Pro Tyr Ser Tyr Gln Ser Pro 210 215 220 Gly Leu Pro Ser Pro Pro
Tyr Gly Thr Met Asp Ser Ser His Val Phe 225 230 235 240 His Val Lys
Pro Pro Pro His Ala Tyr Ser Ala Ala Leu Glu Pro Phe 245 250 255 Phe
Glu Ser Pro Leu Thr Asp Cys Thr Ser Pro Ser Phe Asp Gly Pro 260 265
270 Leu Ser Pro Pro Leu Ser Ile Asn Gly Asn Phe Ser Phe Lys His Glu
275 280 285 Pro Ser Ala Glu Phe Glu Lys Asn Tyr Ala Phe Thr Met His
Tyr Pro 290 295 300 Ala Ala Thr Leu Ala Gly Ala Gln Ser His Gly Ser
Ile Phe Ser Gly 305 310 315 320 Thr Ala Ala Pro Arg Cys Glu Ile Pro
Ile Asp Asn Ile Met Ser Phe 325 330 335 Asp Ser His Ser His His Glu
Arg Val Met Ser Ala Gln Leu Asn Ala 340 345 350 Ile Phe His Asp 355
9356PRTHomo sapiensMISC_FEATURE(1)..(356)Human NeuroD1 amino acid
sequence alt. 9Met Thr Lys Ser Tyr Ser Glu Ser Gly Leu Met Gly Glu
Pro Gln Pro 1 5 10 15 Gln Gly Pro Pro Ser Trp Thr Asp Glu Cys Leu
Ser Ser Gln Asp Glu 20 25 30 Glu His Glu Ala Asp Lys Lys Glu Asp
Asp Leu Glu Thr Met Asn Ala 35 40 45 Glu Glu Asp Ser Leu Arg Asn
Gly Gly Glu Glu Glu Asp Glu Asp Glu 50 55 60 Asp Leu Glu Glu Glu
Glu Glu Glu Glu Glu Glu Asp Asp Asp Gln Lys 65 70 75 80 Pro Lys Arg
Arg Gly Pro Lys Lys Lys Lys Met Thr Lys Ala Arg Leu 85 90 95 Glu
Arg Phe Lys Leu Arg Arg Met Lys Ala Asn Ala Arg Glu Arg Asn 100 105
110 Arg Met His Gly Leu Asn Ala Ala Leu Asp Asn Leu Arg Lys Val Val
115 120 125 Pro Cys Tyr Ser Lys Thr Gln Lys Leu Ser Lys Ile Glu Thr
Leu Arg 130 135 140 Leu Ala Lys Asn Tyr Ile Trp Ala Leu Ser Glu Ile
Leu Arg Ser Gly 145 150 155 160 Lys Ser Pro Asp Leu Val Ser Phe Val
Gln Thr Leu Cys Lys Gly Leu 165 170 175 Ser Gln Pro Thr Thr Asn Leu
Val Ala Gly Cys Leu Gln Leu Asn Pro 180 185 190 Arg Thr Phe Leu Pro
Glu Gln Asn Gln Asp Met Pro Pro His Leu
Pro 195 200 205 Thr Ala Ser Ala Ser Phe Pro Val His Pro Tyr Ser Tyr
Gln Ser Pro 210 215 220 Gly Leu Pro Ser Pro Pro Tyr Gly Thr Met Asp
Ser Ser His Val Phe 225 230 235 240 His Val Lys Pro Pro Pro His Ala
Tyr Ser Ala Ala Leu Glu Pro Phe 245 250 255 Phe Glu Ser Pro Leu Thr
Asp Cys Thr Ser Pro Ser Phe Asp Gly Pro 260 265 270 Leu Ser Pro Pro
Leu Ser Ile Asn Gly Asn Phe Ser Phe Lys His Glu 275 280 285 Pro Ser
Ala Glu Phe Glu Lys Asn Tyr Ala Phe Thr Met His Tyr Pro 290 295 300
Ala Ala Thr Leu Ala Gly Ala Gln Ser His Gly Ser Ile Phe Ser Gly 305
310 315 320 Thr Ala Ala Pro Arg Cys Glu Ile Pro Ile Asp Asn Ile Met
Ser Phe 325 330 335 Asp Ser His Ser His His Glu Arg Val Met Ser Ala
Gln Leu Asn Ala 340 345 350 Ile Phe His Asp 355
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