The present invention relates to a method for inducing
differentiation of stem cells into midbrain-specific
dopaminergic neural precursor cells and for mass production
10 of stem cell-derived midbrain-specific dopaminergic neural
precursor cells.
The present invention was supported by a grant
(project number HI18C0096) from the Ministry of Health and
15 Welfare, Korea. The project is conducted in the project
name “Function- and efficacy-based development of
pluripotent stem cell-derived cell therapy product for
Parkinson’s disease” as the research named “Development of
frontier medical technique” by the managing company S.
20 Biomedics Co. Ltd. under the supervision of the Korea
health Industry Development Institute during April 30, 2018
to December 31, 2022.
This patent application claims priority to and the
benefit of Korean Patent Application No. 10-2019-0118370,
25 filed September 25, 2019, the entire contents of which are
2
incorporated herein by reference.
Description of the Prior Art
Stem cells refer to cells remaining in a pre5 differentiation phase and undergo differentiation into
specific cells upon exposure to specific differentiation
stimuli (environments). Unlike completely differentiated
cells that do not further differentiate, stem cells can
also show the proliferation (expansion) characteristic of
10 dividing in self-renewal to produce more of the same type
of the stem cells. In addition, stem cells, which
differentiate into specific cells in response to
differentiation stimuli, are characterized by the
differentiation plasticity that cell types to which stem
15 cells are differentiated depend on environments or stimuli
to which the stem cells are exposed.
Nowadays, extensive attention is paid to stem cells
for use as cell therapy products. Much research has also
been conducted into the use of stem cells as cell therapy
20 products for various neurological diseases caused by
neuronal injury. Among other diseases, cranial nerve
diseases are considered to be the most suitable target for
cell transplantation therapy because tissues in the brain
nervous system exhibits almost no immune rejection
25 responses, unlike the other tissues and thus are expected
3
to allow the long-term survival of cells transplanted from
the outside.
Meanwhile, a technique is required for effectively
differentiating stem cells into specific cells and
5 supplying specific cells in a desired time in order to
enhance the efficacy of stem cells as cell therapy
products.
However, thus far, techniques have not yet been
developed for differentiating stem cells into specific
10 cells (inter alia, dopaminergic neural cells) at such a
high efficiency as to allow clinical application and for
storing the cells at suitable stages.
SUMMARY OF THE INVENTION
15
Leading to the present disclosure, intensive and
thorough research into the induction of stem cells to
differentiate into midbrain-specific dopaminergic neural
precursor cells, conducted by the present inventors, with
20 the aim of developing cell therapy products for cranial
nerve diseases, resulted in conceiving a method capable of
mass production of midbrain-specific dopaminergic neural
precursor cells at so high efficiency as to allow clinical
application.
25 It is therefore a purpose of the present disclosure to
4
provide a method for inducing stem cells to differentiate
into dopaminergic neural precursors cells.
It is another purpose of the present disclosure to
provide a method for mass production of dopaminergic neural
5 precursor cells.
BRIEF DESCRIPTION OF THE DRAWINGS
The above and other aspects, features and advantages
10 of the present disclosure will be more apparent from the
following detailed description taken in conjunction with
the accompanying drawings, in which:
FIG. 1 is a schematic view illustrating a method for
inducing differentiation into dopaminergic neural precursor
15 cells according to an embodiment of the present disclosure;
FIGS. 2a and 2b are images showing an optimal duration
of stem cell culturing according to an embodiment of the
present disclosure (derived from embryonic stem cells);
FIG. 3 is an image showing an optimal duration of
20 embryoid body culturing according to an embodiment of the
present disclosure (derived from embryonic stem cells);
FIGS. 4a to 4c are images showing an optimal time of
treatment with SAG and CHIR99021 for embryoid body
culturing according to an embodiment of the present
25 disclosure (derived from embryonic stem cells);
5
FIGS. 5a and 5b are images showing whether or not the
step of forming embryoid bodies are necessary according to
an embodiment of the present disclosure (derived from
embryonic stem cells);
5 FIG. 6 shows images illustrating an optimal duration
of culturing for neural rosette generation (derived from
embryonic stem cells);
FIGS. 7a and 7b are images showing an optimal duration
of culturing for differentiation into dopaminergic neural
10 precursor cells according to an embodiment of the present
disclosure (derived from embryonic stem cells);
FIGS. 8a to 8d are graphs and images showing an
optimal duration of treatment with SAG and CHIR99021 for
differentiation into dopaminergic neural precursor cells
15 according to an embodiment of the present disclosure
(derived from embryonic stem cells);
FIGS. 9a to 9c are images showing optimal treatment
concentrations of SAG and CHIR99021 according to an
embodiment of the present disclosure (derived from
20 embryonic stem cells);
FIGS. 10a and 10b images showing an optimal treatment
concentration of ECM according to an embodiment of the
present disclosure (derived from embryonic stem cells);
FIGS. 11a and 11b are images showing the final
25 differentiation rate of dopaminergic neural precursor cells
6
according to an embodiment of the present disclosure (11a:
derived from embryonic stem cells, 11b: derived from
induced pluripotent stem cells);
FIG. 12 shows a graph, together with a table,
5 indicating the mass production of dopaminergic neural
precursor cells according to an embodiment of the present
disclosure (derived from embryonic stem cells); and
FIGS. 13a and 13b are plots showing in vivo efficacy
of dopaminergic neural precursor cells according to an
10 embodiment of the present disclosure (derived from
embryonic stem cells).
DETAILED DESCRIPTION OF THE EXEMPLARY EMBODIMENTS
15 In order to develop a cell therapy product for cranial
nerve disease, the present inventors have made effects to
conceive a method for inducing the differentiation of stem
cells into midbrain-specific dopaminergic neural precursor
cells. As a result, a method for mass production of
20 midbrain-specific dopaminergic neural precursor cells at so
high efficiency as to allow clinical application is
introduced.
In addition, the present inventors have established an
effective method capable of storing differentiation-induced
25 midbrain-specific dopaminergic neural precursor cells as a
7
working cell bank (WCB) at a suitable stage.
The present disclosure pertains to a method for
inducing stem cells to differentiate into dopaminergic
neural precursor cells and producing dopaminergic neural
5 precursor cells on a mass scale.
Below, a detailed description will be given of the
present disclosure.
According to an aspect thereof, the present disclosure
10 pertains to a method for inducing stem cells to
differentiate into dopaminergic neural precursor cells, the
method comprising the steps of:
a) culturing stem cells in a monolayer format;
b) forming and maintaining an embryoid body;
15 c) generating a neural rosette; and
d) differentiating the neural rosette into
dopaminergic neural precursor cells.
Hereinafter, a method for preparation of dopaminergic
20 neural cells will be described in detail.
Step a)
This step is a process in which undifferentiated stem
cells are stimulated by treatment with a BMP signaling
inhibitor and an activin/nodal signaling inhibitor. In
25 this process, the stem cells are differentiated into
8
ectodermal cells, especially neuroectodermal cells at
higher efficiency, compared to those that have not treated
with such materials.
The stem cells may be embryonic stem cells, induced
5 pluripotent stem cells (iPSCs), adult stem cells, somatic
cell nuclear transfer embryonic stem cells, or stem cells
generated by direct reprogramming.
This step may be conducted for 5-9 days or 8 days, but
without limitations thereto.
10 Differentiation within the range allows embryoid
bodies to be formed without collagenase. When departing
from the range, the differentiation does not guarantee the
generation of the desired cells, but may proceed to natural
differentiation or may give problems in the next step of
15 forming an embryoid body. Meanwhile, respective optimal
periods of time may be established for individual types of
the stem cells within the range because the working times
of the inhibitors differ from one stem cell type to
another.
20 In the step, a BMP signaling inhibitor and an
activin/nodal signaling inhibitor may be added daily from
1-3 days before the end of the step, but without
limitations thereto.
So long as it is known in the art, any BMP signaling
25 inhibitor may be available without limitations. Examples
9
of the BMP signaling inhibitor include dorsomorphin, Smad6,
Smad7, Noggin, Chordin, Gremlin, Sog (shortgastrulation),
Follistatin, DAN (differential screening = selected gene
aberrant in neuroblastoma), Cerberus, Dante, and/or PRDC
5 (Protein Related to DAN and Cerberus).
In the present disclosure, "dorsomorphin" is an
inhibitor against the BMP signaling pathway, acting to
inhibit BMP itself or repress the binding of BMP to a BMP
receptor.
10 Dorsomorphin is represented by the following Chemical
Formula 1:
[Chemical Formula 1]
In the step, the BMP signaling inhibitor may be used
15 at a concentration of 1.0 to 20.0 µM, at a concentration of
4.0 to 6.0 µM, or at the concentration of 5.0 µM, but
without limitations thereto.
A concentration departing from the range may cause
10
cell death. Meanwhile, respective optimal concentrations
may be established for individual types of the stem cells
within the range because the working concentrations of the
inhibitor differ from one stem cell type to another.
5 Selection may be made of various activin/nodal
signaling inhibitors known in the art, without limitations.
Particularly, the activin/nodal signaling inhibitor useful
in the present disclosure may be 4-(5-benzo[1,3]dioxol-5-
yl-4-pyridin-2-yl-1H-imidazol-2-yl)-benzimide, Smad6,
10 Smad7, and/or Follistatin.
In the present disclosure, "4-(5-benzo[1,3]dioxol-5-
yl-4-pyridin-2-yl-1H-imidazol-2-yl)-benzimide", known as
SB431542 in the art, inhibits the activin/nodal signaling
pathway by suppressing activin/nodal itself or preventing
15 activin/nodal from binding to the receptor thereof.
The 4-(5-Benzo[1,3]dioxol-5-yl-4-pyridin-2-yl-1Himidazol-2-yl)-benzimide is represented by the following
Chemical Formula 2:
[Chemical Formula 2]
20
11
In the present disclosure, the compound represented by
Chemical Formula 1 is used in combination with SB431542.
In the step, the activin/nodal signaling inhibitor may
be used at a concentration of 1.0 to 50.0 µM, at a
5 concentration of 4.0 to 6.0 µM, or at the concentration of
5.0 µM, but with no limitations thereto.
When departing from the range, a concentration of the
activin/nodal signaling inhibitor may cause cell death.
Meanwhile, respective optimal concentrations may be
10 established for individual types of the stem cells within
the range because the working concentrations of the
inhibitor differ from one stem cell type to another.
In the step, the cells may be cultured in a TeSR2 cell
culture medium. This is intended to use the cells for
15 clinical entry and cell therapy products. In addition to
the TeSR2 cell culture medium, any stem cell culture medium
that allows clinical entry may be selectively employed
without limitations.
20 Step b)
This step is a process in which an embryoid body is
formed and then stimulated by treatment with a sonic
hedgehog (SHH) signaling activator and a GSK-3 inhibitor
while being cultured. In this process, the embryoid body is
25 differentiated into dopaminergic neural precursor cells at
12
higher efficiency, compared to those that have not been
treated with such materials.
As used herein, the term “embryoid body” refers to a
three-dimensional aggregate of pluripotent stem cells of
5 which embryonic stem cells are representative. Pluripotent
stem cells within embryoid bodies can undergo
differentiation in the initial embryonic development stage
and cell specification along the three germ lineages of
endoderm, ectoderm, and mesoderm, which comprises all
10 somatic cell types.
This step may be conducted for 3-6 days, 4 days, 5
days, or 6 days, but without limitations thereto.
The formation and maintenance of the embryoid body
within the range can allow for the maximum differentiation
15 rate (yield) of dopaminergic neural precursor cells. When
the step is conducted beyond the range, the embryoid body
is badly affected and thus differentiates at a poor rate.
Meanwhile, respective optimal periods of time may be
established for individual types of the stem cells within
20 the range because the working periods of time differ from
one stem cell type to another.
In the step, the BMP signaling inhibitor and the
activin/nodal signaling inhibitor may be added daily from
the starting day of the step, whereby the efficiency of
25 differentiation into neuroectoderm can be further improved.
13
Moreover, in the step, an SHH signaling activator and
a GSK-3 inhibitor may be added daily from 2-6 days after
the starting of the step, but without limitations thereto.
Selection may be made of various SHH signaling
5 activators known in the art, without limitations. Examples
of the SHH signaling activator include smoothened agonist
(SAG), purmorphamine, halcinonide, fluticasone, clobetasol,
and/or fluocinonide.
As used herein, the term “smoothened agonist” (SAG)
10 refers to a small-molecule compound activating the sonic
hedgehog (SHH) signaling pathway. SHH plays a critical
role in the differentiation and distribution of
dopaminergic neurons of the ventral midbrain in the
neuroectodermal development stage.
15 In addition, as explained in the following Example
section, SAG acts to upregulate the expression of FOXA2,
which is one of the important markers for dopaminergic
neural precursor cells. FOXA2 (winged helix/forkhead box
A2) (HNF3beta) is a transcription factor which plays an
20 important role in the development of the central nervous
system (CNS) and has an influence on the expression of
various genes involved in midbrain-specific development and
on the formation of midbrain-specific dopaminergic neurons.
Meanwhile, the use of an SHH signaling activator alone
25 makes it impossible to differentiate stem cells into
14
midbrain-specific dopaminergic precursor cells because an
SHH signaling activator is involved, in conjunction with a
GSK-3 inhibitor, in the differentiation of midbrainspecific dopaminergic precursor cells at high yield, as
5 will described below.
SAG is represented by the following Chemical Formula
3:
[Chemical Formula 3]
10 In the step, the SHH signaling activator may be used
at a concentration of 0.1 to 5.0 µM, at a concentration of
0.6 to 5.0 µM, or at a concentration of 1.0 µM, but without
limitations thereto.
When the concentration exceeds the range, the
15 activator may induce differentiation into undesired cells.
Meanwhile, respective concentrations may be established for
individual types of the stem cells within the range because
the working concentration differs from one stem cell type
15
to another.
Selection may be made of various GSK-3 inhibitors
known in the art, without limitations. Particular examples
of the GSK-3 inhibitor include 6-[[2-[[4-(2,4-
5 dichlorophenyl)-5-(5-methyl-1H-imidazol-2-yl)-2-
pyrimidinyl]amino]ethyl]amino]-3-pyridinecarbonitrile
(CHIR99021), 3-(2,4-dichlorophenyl)-4-(1-methyl-1H-indol-3-
yl)-1H-pyrrole-2,5-dione (SB216763), N6-[2-[[4-(2,4-
dichlorophenyl)-5-(1H-imidazol-2-yl)-2-
10 pyrimidinyl]amino]ethyl]-3-nitro-2,6-pyridinediamine
(CHIR98014), TWS119, Tideglusib, 3-[(3-chloro-4-
hydroxyphenyl)amino]-4-(2-nitrophenyl)-1H-pyrrol-2,5-dione
(SB415286), (2'Z,3'E)-6-bromoindirubin-3'-oxime(BIO),
valproic acid, 5-iodo-7-β-D-ribofuranosyl-7H-pyrrolo[2,3-
15 d]pyrimidin-4-amine(Iodotubercidin), 1-azakenpaullone,
curcumin, olanzapine, and/or pyrimidine.
As used herein, “CHIR99021” is a small-molecule
compound activating the Wnt/beta-catenin signaling pathway
(GSK-3 inhibitor). The Wnt/beta-catenin signaling pathway
20 controls the ectoderm and neurogenesis stage and plays an
important role, together with SHH, in the differentiation
of midbrain dopaminergic neurons.
In addition, CHIR99021 acts to upregulate the
expression of LMX1A and En-1, which are important markers
25 for dopaminergic neural precursor cells, as illustrated in
16
the Example section, below.
When used alone, a GSK-3 inhibitor cannot
differentiate the embryoid bodies into dopaminergic neural
precursor cells because the GSK-3 inhibitor is involved, in
5 conjunction with an SHH activator, in the differentiation
of midbrain-specific dopaminergic precursor cells at high
yield. The use of the GSK-3 inhibitor alone may incite the
embryoid bodies to increasingly develop into hindbrain
cells. When continuously applied, the GSK-3 inhibitor is
10 involved in the proliferation of the cells, but may cause a
problem with safety upon transplantation.
CHIR99021 is represented by the following Chemical
Formula 4:
[Chemical Formula 4]
15
In this step, the GSK-3 inhibitor may be added at a
concentration of 0.1 to 5.0 µM, at a concentration of 1.6
to 5.0 µM, or at a concentration of 2.0 µM, but with no
17
limitations thereto.
When departing from the range, a concentration of the
GSK-3 inhibitor may cause the embryoid bodies to
differentiate into undesired cells. Meanwhile, respective
5 concentrations may be established for individual types of
the stem cells within the range because the working
concentration differs from one stem cell type to another.
In the step, the embryoid bodies may be cultured in
bFGF-free ES cell culture medium. However, so long as it
10 allows EB to be cultured therein, any culture medium may be
selectively used without limitations.
Step c)
This step is a process in which neural rosettes are
formed. In this process, selection can be made of the
15 cells that are fated to differentiate into neural cells
among the neuroectodermal cells.
As used herein, the term “neural rosette” refers to a
cluster of cells that are fated to develop into various
types of neural cells.
20 The step may be conducted for 3-6 days, 4 days, or 5
days, but without limitations thereto.
When the step is conducted for a period of time
departing from the range, neural rosettes are not
maintained to cease the differentiation or to cause the
25 differentiation to proceed in an undesired direction.
18
Meanwhile, respective optimal periods of time may be
established for individual types of the stem cells within
the range because the working periods of time differ from
one stem cell type to another.
5 In the step, an SHH signaling activator and a GSK-3
inhibitor may be added daily from the starting of the step.
In this process, the cells fated to differentiate into
dopaminergic neural precursor cells, other than general
neural rosettes that can differentiate into various types
10 of neural cells, can be obtained in a large amount.
In the step, the cells are cultured in a DMEM/F12 cell
culture medium. However, so long as it allows the
formation/maintenance of neural rosettes, any medium may be
selectively employed without limitations thereto.
15 The cell culture medium may further comprise N2
supplement CTS, human insulin, and bFGF.
Step d)
This step is a process in which neural rosettes are
differentiated into dopaminergic neural precursor cells.
20 In this process, selection is made of neural rosettes only,
with the exclusion of differentiated cells other than
neural cells, thereby reinforcing differentiation into
dopaminergic neural precursor cells.
As used herein, the term “neural cell” refers to a
25 cell that is a component of the nervous system and is
19
interchangeably used with neuron.
As used herein, the term “dopaminergic neural cell”
refers to a neural cell secreting the neurotransmitter
dopamine.
5 The term “precursor cell”, as used herein, refers to a
cell that can be divided just before expressing traits of
the cell that has undergone complete differentiation and is
interchangeably used with “progenitor” or “precursor”.
In the present disclosure, therefore, “dopaminergic
10 neural precursor cells” are cells that can be divided into
neural cells secreting dopamine after experiencing a
maturation stage since in vivo transplantation.
The step may be conducted for 8-10 days or 9 days, but
with no limitations thereto.
15 When the step is conducted for less than the lower
limit of the range, the differentiation rate of
dopaminergic precursor cells may decrease. A period of
time greater than the upper limit of the range may make
mass production impossible. Meanwhile, respective optimal
20 periods of time may be established for individual types of
the stem cells within the range because the working periods
of time differ from one stem cell type to another.
In the step, an SHH signaling activator and a GSK-3
inhibitor may be added daily from the starting of the step.
25 In this process, most (about 80% or more) of the cells can
20
differentiate into dopaminergic neural precursor cells with
the lapse of differentiation days.
The step is conducted by exchanging the medium with a
fresh medium every day and passaging the cells every three
5 days from the starting of the step, whereby the
dopaminergic neural precursor cells can be proliferated on
a large scale and maintained in the best state and the
differentiation rate can be improved.
In the step, the cells are cultured in a DMEM/F12 cell
10 culture medium. However, so long as it allows the
formation/maintenance of dopaminergic precursor cells, any
medium may be selectively employed without limitations
thereto.
The cell culture medium may further comprise N2
15 supplement CTS and B-27 supplement CTS.
The method may further comprise the following step:
e) proliferating the dopaminergic neural precursor
cells through passage.
Step e)
20 This step is a process in which the dopaminergic
neural precursor cells are produced in a large amount by
proliferation. This process can make stable cell supply
possible as well as increasing the differentiation rate of
dopaminergic neural precursor cells.
25 The differentiation rate to dopaminergic neural
21
precursor cells, induced by the method, may be 80% or
greater, but is not limited thereto.
The dopaminergic neural precursor cells induced by the
method may improve in the expression level of FOXA2, LMX1A,
5 and/or En1.
According to an embodiment of the present disclosure,
the dopaminergic neural precursor cells induced by the
method may alleviate symptoms of Parkinson’s disease.
The cell culture in each step of the method may
10 further comprise an extracellular matrix (ECM). This is
because undifferentiated stem cells and neural cells cannot
be attached to the culture dish by themselves, but can be
maintained and cultured with the aid of an extracellular
matrix or feeder cells.
15 The extracellular matrix may be, for example, laminin,
but is not limited thereto. In addition to laminin, other
extracellular matrices may be used alone or in combination.
Suitable extracellular matrices may differ from one type of
stem cells to another.
20 The extracellular matrix may be used at a
concentration of 3.5-5.5 µg/mL, 4.0 µg/mL, or 5.0 µg/mL,
but without limitations thereto.
When the concentration of the extracellular matrix
exceeds the range, differentiation into dopaminergic neural
25 precursor cells may be impossible or a problem with
22
adhesion may occur, causing problems in the production
process.
The neural precursor cells obtained by the method may
be used to treat neurodegenerative diseases, for example,
5 Alzheimer’s disease, Huntington’s disease, Parkinson’s
disease, and amyotrophic lateral sclerosis.
Another aspect of the present disclosure pertains to a
method for mass production of dopaminergic neural precursor
cells, the method comprising the following steps:
10 a) culturing stem cells;
b) forming and maintaining an embryoid body;
c) generating a neural rosette;
d) differentiating the neural rosette into
dopaminergic neural precursor cells; and
15 e) proliferating the dopaminergic neural precursor
cells through passage.
As for the method for mass production of dopaminergic
neural precursor cells, its descriptions in common with the
method for inducing differentiation into dopaminergic
20 neural precursor cells are omitted in order to avoid undue
redundancy leading to the complexity of this specification.
A better understanding of the present disclosure may
be obtained through the following examples which are set
25 forth to illustrate, but are not to be construed as
23
limiting the present disclosure.
Culturing of Human Embryonic Stem Cells (hESCs)
Undifferentiated hESCs (SNU32, the Korean Cell Line
5 Bank) to be differentiated into dopaminergic neural cells
were cultured in a CELLstart-coating dish containing a
TeSR2 (STEMCELL, SCR5860) medium.
In this regard, the undifferentiated stem cells were
cultured in a monolayer format. Under the principle of 7-
10 day culturing, cells were harvested using a scraper after
incubation with Versene (GIBCO, 15040-066) for 4 minutes in
a 37°C incubator when reaching a confluency of 90-95% and
then transferred into a 15-ml tube. The cells were
pipetted about 8-12 times with a 1000P pipette before
15 passage at 1:7 ratio (based on CELLstart-CTS coating dish),
and then maintained with the medium exchanged with a fresh
medium daily within 24 hours every day for 7 days.
Culturing of Induced Pluripotent Stem Cells (iPSCs)
20 Undifferentiated iPSCs (hFSiPS1, the National Stem
Cell Bank, Depository Authority: Division for Intractable
Disease at the Korean National Institute of Health) to be
differentiated into dopaminergic neural cells were cultured
in the same manner as the hESC culturing method.
25
24
Immunocytochemistry Assay
Cells were fixed for 10 min in a 4% paraformaldehyde
solution.
In order to smoothly penetrate into the cytoplasm,
5 each antibody was incubated with 0.1% Trition X-100 (in
PBS) for 15 min and then with 2% bovine serum albumin (BSA,
in PBS) for 1 hour at room temperature.
Subsequently, the primary antibodies (see Table 1,
below) were allowed to bind to the cells at 4°C. Secondary
10 antibodies suitable for the respective primary antibody
species (see Table 1, below) were used to confirm the
primary antibody-bound cells.
Finally, cell nuclei were imaged. In this regard, the
cells were incubated with 4’, 6-diamino-2-phenylindole
15 (DAPI) in PBS for 10 min to stain the nuclei which were
then imaged under a fluorescence microscope. Important
markers were identified and analyzed.
TABLE 1
Protein Species Manufacturer Cat. No. Dilution
LMX1A Goat Santa Cruz sc-54273 1:100
FOXA2(HNF3beta) Mouse Santa Cruz sc-374376 1:50
20
Gene Expression Assay (qRT-PCR)
Cells were harvested from which total RNA was then
25
isolated using the Easy-Spin®Total RNA extraction kit
(iNtRON Biotechnology). cDNA was synthesized from 1 µg of
the total RNA, using the PrimeScript™RT Master Mix (TAKARA
Bio Inc.). mRNA levels were quantitated by real time RT5 PCR using SYBR®Premix Ex Taq™(TAKARA Bio Inc.) and CFX96
Real-Time System(Bio-Rad). Primer sequences used in the
gene expression assay are given in Table 2, below.
TABLE 2
Gene Name Sequence (5'-3')
En-1 (Engrailed 1) F: CGT GGC TTA CTC CCC ATT TA
(SEQ ID NO: 1)
R: TCT CGC TGT CTC TCC CTC TC
(SEQ ID NO: 2)
GAPDH (Glyceraldehyde-3-
Phosphate Dehydrogenase)
F: CAA TGA CCC CTT CAT TGA CC
(SEQ ID NO: 3)
R: TTG ATT TTG GAG GGA TCT CG
(SEQ ID NO: 4)
10 EXAMPLE: Protocol for Differentiation into
Dopaminergic Neural Precursor Cell
After being stabilized through two passage rounds from
the time of thawing an MCB (Master Cell Bank), the hESCs or
iPSCs cultured above were subjected to the 3rd passage to
15 induce differentiation into dopaminergic neural precursor
cells on the passage culture dish.
Starting from the day (d0) on which the 3rd passage
culture dish was prepared, the cells were pre-treated with
26
5 µM dorsomorphin (hereinafter referred to as “DM”)
(Millipore, 171260) and 5 µM SB431542 (hereinafter referred
to as “SB”) (Sigma, S4317) in a hESC culture medium (TeSR2,
STEMCELL, SCR5860) for two days from differentiation day 6
5 (d6) to differentiation day 8 (d8) to increase the
feasibility of differentiation into neuroectoderms.
On differentiation day 8 (d8), the hESCs that were
being cultured in a monolayer format were subdivided into a
format of 1.5-mm grids by using 1-ml 26-G syringe and then
10 left for about 30 min in a 37°C incubator or incubated with
2 ml of collagenase (Animal Origin Free (CLSAFC),
Worthington, LS004138) for about 5 min in a 37°C incubator
to form a 1.5-mm square cell sheet which acts as a basis
for embryoid bodies, thereby forming embryoid bodies
15 (hereinafter referred to as “EM”). The EM thus induced was
cultured in a bFGF-free hESC culture medium (EB medium).
In this regard, while being incubated for 4 days until
differentiation day 12 (d12), the cells pretreated with 5
µM DM and 5 µM SB were further pretreated with the
20 patterning factors, 1.0 µM SAG (Millipore, 566661,
hereinafter referred to as “SAG”) and 2.0 µM CHIR99021
(Milteny, 130-106-539), to increase the occupancy degree of
midbrain dopaminergic neural precursor cells.
On differentiation day 12 (d12), a DMEM/F12 medium
25 supplemented with 20 µg/mL human insulin and 20 ng/mL bFGF
27
(mN2+b) was used to attach the EB formed in the previous
step to a Laminin-521-coating culture dish (pEB step),
followed by incubation with the patterning factors 1.0 µM
SAG (smoothened agonist) and 2.0 µM CHIR99021 for 5 days.
5 On differentiation day 17 (d17), neural rosettes
formed from the attached EB were separated by treating with
Accutase (Millipore, SCR003) for 2 min or using a method in
which a user processed a glass pipette and directly
separate the cells therewith. The neural rosettes were
10 then re-attached to a separate Laminin-521-coating culture
dish. For use in the re-attachment, DMEM/F12 medium
supplemented with N2 (N-2 supplement, CTS grade, GIBCO,
A1370701, hereinafter referred to as “N2”) and B-27(B-27
supplement xeno-free, CTS grade, GIBCO, A1486701,
15 hereinafter referred to as “B27”) (N2B27 medium), which is
used as a medium for culturing dopaminergic neural
precursor cells, was added with 1.0 µM SAG and 2.0 µM
CHIR99021. For re-attachment, the medium was further added
with 10 µM Y27632 and then used to aid the attachment of
20 the cells for one hour. After one hour, the medium was
exchanged with Y27632-free one. Until day 20 (d20), the
medium was exchanged daily with a fresh medium within 24
hours to continuously induce differentiation into
dopaminergic neural precursor cells. When treated with
25 Accutase, all of the cells, except for neural rosettes,
28
were separated and removed. Only the neural rosette
clusters were transferred to 15-ml tubes by using a scraper
and dissociated by pipetting up and down about 40 times
with a 200P pipette before re-attachment onto a Laminin5 521-coating culture dish.
On differentiation day 20 (d20), the dopaminergic
neural precursor cells were separated into single cells in
the presence of Accutase and re-attached at a density of
4.0x106 cells/35mm dish onto a Laminin-521-coating culture
10 dish containing an N2B27 medium supplemented with 1.0 µM
SAG and 2.0 µM CHIR99021. While the medium was exchanged
every day with a fresh medium, the cells were re-attached
at a density of 4.0x106 cells/35-mm dish every three days in
a Laminin-521-coating culture dish to proliferate the cells
15 in a large amount before preparing a working cell bank
(WCB) on day 26 (d26).
[Clinical entry] Dopaminergic neural precursor cells
in the WCB prepared on day 26 were dissociated into single
cells by using Accutase in the same manner as in the re20 attachment method. The dissociated single cells were
aliquoted into vials at a density of 3.0x106 cells/vial or
in a range guaranteed by a cryoprotectant.
[Preparation of transplant cells] The WCB was used 9
days after being thawed. For a 35-mm culture dish, 4.0x106
25 cells are needed. Thus, 2 vials (3.0x106 cells for each
29
vial) were pooled and living cells were counted with a
trypan blue solution. Among them, 4.0x106 cells were
attached to a 35-mm Laminin-521 coating culture dish
containing the N2B27 medium and cultured until
5 differentiation day 35 (d35), with the medium exchanged
every day with a fresh medium. Also, the differentiation
induction and proliferation was conducted by re-attachment
every three days until day 35 (d35).
A concrete protocol is illustrated in FIG. 1.
10
EXPERIMENTAL EXAMPLE 1: Step of Culturing Stem Cell
1-1. Optimal time of treatment with DM and SB431542
The same procedure as in the method according to the
present disclosure was conducted, with the exception of
15 treatment with DM and SB431542 from culturing day 8,
instead of culturing day 6.
As can be seen in FIG. 2a, treatment with DM and
SB431542 from culturing day 6 accounting for a stem cell
culturing step (the method of the present disclosure)
20 guaranteed better results in terms of neural rosette state
and yield, than treatment with DM and SB431542 from
culturing day 8 accounting for an embryoid body forming
step (conventional method).
This result suggests that pre-treatment with DM and
25 SB431542 from the undifferentiated cell stage prior to the
30
embryoid body formation can increase the feasibility of
differentiation into neuroectoderm upon embryoid body
formation as well as exceptionally improving the final rate
of differentiation into dopaminergic neural precursor
5 cells.
1-2. Optimal culturing duration
The same procedure as in the method according to the
present disclosure was conducted, with the exception that
10 following pretreatment with DM and SB431542 from culturing
day 7 to culturing day 9, grids for embryoid body formation
were established on culturing day 9, instead of the
establishment of grids for embryoid body formation on
culturing day 8.
15 As can be seen in FIG. 2b, when lines were drawn after
formation of embryoid bodies on culturing day 9, the cells
were floated on the medium because their adhesion became
very weak. The cells were detached from the bottom of the
culture dish before establishment of grids and were
20 difficult to form into 1.5-mm square cell sheets (the left
panel in FIG. 2b). In contrast, when grids for embryoid
body formation were established on culturing day 8 (the
method of the present disclosure), 1.5-mm cell sheets could
be normally made (the right panel in FIG. 2b).
25
31
EXPERIMENTAL EXAMPLE 2: Step of Forming and
Maintaining Embryoid Body
2-1. Optimal culturing duration
The same procedure as in the method according to the
5 present disclosure was conducted, with the exception that
EB formation was induced by treatment with DM and SB431542
until culturing day 13, instead of attachment of EB to the
culture dish on culturing day 12 (on day 4 of EM
formation/maintenance).
10 As shown in FIG. 3, embryoid bodies were, in the most
part, individually maintained well until day 4 (culturing
day 12), but since day 5 (culturing day 13), embryoid
bodies were attached to each other to form large aggregates
at high frequency.
15
2-2. Optimal time of treatment with SAG and CHIR99021
The same procedure as in the method according to the
present disclosure was conducted, with the exception of
treatment with SAG and CHIR99021 from culturing day 6 or 8,
20 instead of treatment with SAG and CHIR99021 from culturing
day 10.
As can be seen in FIGS. 4a to 4c, neural rosettes were
not normally formed and different morphologies of
differentiated cells were redundantly found after treatment
25 with SAG and CHIR99021 from differentiation day 6 (FIG.
32
4a). The neural rosettes that had been treated with SAG
and CHIR99021 from differentiation day 8 were
morphologically collapsed and showed a bad condition (FIG.
4b), compared to those treated with SAG and CHIR99021 from
5 day 10 (FIG. 4c).
2-3. Comparison of monolayer differentiation
Examination was made to show whether or not the
culturing/differentiation process could be simplified. In
10 this regard, differentiation into dopaminergic neural
precursor cells was carried out using the same protocol as
the method of the Example, with the exception of omitting
the embryoid body formation procedure (culturing days 8 to
12). Comparison between the conventional method and the
15 method of the present disclosure was made with respect to
the expression of FOXA2 and/or LMX1A on differentiation day
27.
As seen in FIGS. 5a and 5b, differentiation into
neuroectoderms in a monolayer format without embryoid body
20 formation (FIG. 5a) resulted in significantly low
expression levels of FOXA2 and/or LMX1A, compared to
differentiation with embryoid body formation (the method of
the present disclosure, FIG. 5b).
25 EXPERIMENTAL EXAMPLE 3: Step of Generating Neural
33
Rosette
3-1. Optimal differentiation duration
The same procedure as in the method of the present
disclosure was conducted, with the exception that neural
5 rosettes were generated until culturing day 18 (neural
rosette generation day 6), instead of re-attachment of
neural rosettes onto a separate culture dish on culturing
day 17(neural rosette generation day 5).
As shown in FIG. 6, neural rosettes were maintained
10 and proliferated in a good state until neural rosette
generation day 5 (the method of the present disclosure).
On day 6, however, the neural rosettes underwent a
whitening phenomenon starting from the outer part thereof
at high frequency. In addition, the central part of each
15 rosette turned black while the cells were morphologically
changed. That is, most of the cells were dead and
spontaneously detached off upon washing with PBS or upon
medium exchange. Therefore, there is a difficulty in
supplying neural rosettes.
20 Meanwhile, the formation of neural rosettes during the
proliferation of neural precursor cells is an initial check
point of determining whether differentiation into
neuroectoderms and midbrain dopaminergic neural cells was
successfully induced. The quantity of rosettes and the
25 duration of rosette maintenance are very important in
34
forming and quantitatively securing midbrain dopaminergic
neural precursor cells in future.
EXPERIMENTAL EXAMPLE 4: Step of Differentiation into
5 Dopaminergic Neural Precursor Cells
4-1. Optimal duration of differentiation
In order to establish the preparation time of WBC
which guarantees the highest rate of differentiation into
dopaminergic neural precursor cells, WCB was established on
10 culturing day 23, instead of culturing day 26. With
respect to the final rate of differentiation into
dopaminergic neural precursor cells, comparison between
this method and the method of the present disclosure was
made.
15 As can be seen in FIGS. 7a and 7b, a lower
differentiation rate was measured in the case where a WCB
was established and thawed on day 23 (FIG. 7a), compared to
the case where a WCB was established and thawed on day 26
(the method of the present disclosure, FIG. 7b).
20
4-2. Optimal duration of treatment with SAG and
CHIR99021
The cells were differentiated into dopaminergic neural
precursor cells in the same procedure as the method of the
25 present disclosure was conducted, with the exception of
35
treatment with SAG and CHIR99021 until culturing day 20 or
35, instead of treatment with SAG and CHIR99021 until
culturing day 26. With respect to En1 expression and cell
morphology, the method was compared to the method of the
5 present disclosure.
As shown in FIGS. 8a to 8d, the cells treated with SAG
and CHIR99021 until day 20 (FIG. 8a) were measured to more
rapidly decrease in En1 expression level with the
progression of differentiation, compared to the cells
10 treated with SAG and CHIR99021 until day 35 (FIG. 8b). In
addition, as can be seen in FIG. 8c, the cells treated with
SAG and CHIR99021 until day 35 started to take more mature
morphology in which differentiation was further proceeded
from dopaminergic neural precursor cells while not
15 decreasing in proliferative rate. In contrast, the cells
treated with SAG and CHIR99021 until day 26 (the method of
the present disclosure, FIG. 8d) was found to exhibit a
lower reduced expression level of En1 and take a precursor
cell morphology. Hence, selection was made of the method in
20 which the cells are treated with SAG and CHIR99021 until
day 26 (FIG. 8d).
EXPERIMENTAL EXAMPLE 5: Optimal Concentration of SAG
and CHIR99021
25 The cells were differentiated into dopaminergic neural
36
precursor cells in the same manner as the method of the
present invention, with the exception of treating the cells
with SAG and CHIR99021 at respective concentrations of 0.5
µM and 1.0 µM, or 1.0 µM and 1.5 µM, instead of 1.0 µM and
5 2.0 µM. With the expression of FOXA2 and/or LMX1A, this
method was compared to the method of the present
disclosure.
As seen in FIGS. 9a to 9c, higher expression levels of
FOXA2 and/or LMX1A were detected in the cells treated with
10 SAG and CHIR99021 at respective concentrations of 1.0 µM
and 2.0 µM (the method of the present disclosure, FIG. 9c),
compared to the cells treated with SAG and CHIR99021 at
respective concentrations of 0.5 µM and 1.0 µM (FIG. 9a) or
1.0 µM and 1.5 µM (FIG. 9b).
15
EXPERIMENTAL EXAMPLE 6: Optimal Concentration of ECM
With respect to CELLstart and various concentrations
(2-7 µg/mL) of ECM (Laminin 521), stem cells were cultured
using the same protocol as in the above Examples to conduct
20 a cell adhesion test.
The results are shown in FIGS. 10a and 10b. In
CELLstart, no problems happened until the steps of
culturing stem cells and maintaining neural rosettes.
However, the cells barely remained attached, but were
25 floated or grew in a mesh pattern in the step of
37
differentiating into dopaminergic precursor cells and
proliferating the same (FIG. 10a). At Laminin-521
concentrations of 2 µg/mL and 3 µg/mL, the cells exhibited
too poor adhesion to conduct the test. When Laminin-521
5 was used at concentrations of 6 µg/mL and 7 µg/mL, the
cells underwent spontaneous differentiation at high
frequency. In contrast, the Laminin-521 concentrations of
4 µg/mL and 5 µg/mL guaranteed relatively high adhesion,
with most preference for 5 µg/mL in terms of cell
10 morphology and count under the same time condition (FIG.
10b).
Based on the results, it was most reasonable to employ
CELLstart in the stem cell culturing step and Laminin-521
at the concentration of 5 µg/mL in all of the subsequent
15 steps except for the embryoid body step.
EXPERIMENTAL EXAMPLE 7: Mass Proliferation of
Dopaminergic Neural Precursor Cells (Increase in
Differentiation Rate)
20 As a rule, mass production in a GMP facility takes
separate protocols for MCB (Master Cell Bank) obtained by
proliferating and storing a large amount of
undifferentiated stem cells and for WCB (Working Cell Bank)
obtained by proliferating and storing a large amount of
25 dopaminergic neural precursor cells for transplantation.
38
Therefore, the time points of preparing WCB from MCB and
thawing WCB to proliferate dopaminergic precursor cells in
a large amount are very important in increasing the
differentiation rate while decreasing cell proliferation
5 rates to some degree to guarantee stability against
proliferation upon transplantation.
In this regard, stem cells were differentiated into
dopaminergic precursor cells by using the protocols of the
above Examples while monitoring expression levels of FOXA2
10 and/or LMX1A. As shown in FIGS. 11a and 11b, expression
levels of FOXA2 and/or LMX1A were measured to increasingly
increase from differentiation day 27 to day 36. Meanwhile,
when differentiation was conducted beyond day 35, the
differentiation rate was rather decreased and the cell
15 morphology increasingly escaped from the morphological
scope of the precursors (the same morphology as that upon
ceasing treatment with SAG and CHIR99021 on differentiation
day 35 in FIG. 8c). For this reason, the cells on
differentiation day 35 were determined to undergo the final
20 differentiation.
Accordingly, differentiation day 26 was set forth as
the time point of preparing WCB in the present disclosure
(because the same differentiation rate must be measured
between the method in which cells are not frozen, but are
25 differentiated in a continuous manner and the method in
39
which cells are frozen and thawed before differentiation,
based on the result of Experimental Example 4-1).
Selection was made of the cells on day 35 obtained by
thawing WCB on day 26 and proliferating the same for 9
5 days.
Therefore, as can be seen in FIG. 12, the protocol of
the present disclosure can produce about 130 billion
dopaminergic neural precursor cells from one vial (about
3x106 cells) of MCB (Master Cell Bank) through
10 differentiation.
EXPERIMENTAL EXAMPLE 8: In vivo Transplantation of
Cells Prepared Using the Inventive Protocol
8-1. Construction of 6-OHDA injured Parkinson’s
15 disease (PD)-model
Female Sprague-Dawley lineage rats (Orient Bio Inc.),
each weighing 200-250 g, were used as subjects. They were
anesthetized with a mixture of 30mg/kg Zoletil (Virbac) and
10 mg/kg Rompun (Bayer). According to the coordinates (AP -
20 0.40, ML -0.13, DV -0.70, TB -0.45), 3 µL of 30 mM 6-OHDA
was injected into the medial forebrain bundle of each rat
to create a hemi-parkinsonian model.
8-2. Behavioral Recovery of PD-model after
25 transplantation of cells prepared using inventive protocol
40
Stem cells were differentiated according to the
differentiation protocols of the above Examples. The
differentiated cells on differentiation day 35 (d35) were
suspended at the final concentration of 8.75x104 cell/µL in
5 PBS (CTS) to prepare a cell suspension for transplantation.
For a control, a group transplanted with PBS alone was
used. Four weeks after 6-OHDA injury in Experimental
Example 8-1, the animals were divided into groups and then
subjected to immunosuppression by intraperitoneally
10 injecting cyclosporine A (Chong Kun Dang Pharmaceutical
Corp) at a daily dose of 10 mg/kg thereto. The cell
suspension thus obtained was transplanted in an amount of 4
µL into each rat in a stereotactic manner according to the
coordinates (AP +0.08, ML -0.30, DV -0.40 and -0.50, TB -
15 0.24). Before transplantation, or 4, 8, 12, or 16 weeks
after transplantation, amphetamine (2.5 mg/kg, SigmaAldrich) was intraperitoneally injected, followed by
monitoring whether the rats rotated within 30 min following
injection. For comparison, the same experiment was carried
20 out with dopaminergic neural precursor cells derived from
H9 human embryonic stem cells (H9 hESCs, WiCell Inc., U. S.
A.).
Compared to the control, as shown in FIGS. 13a and
13b, the rats into which the cells differentiated by the
25 conventional method (the H9 embryonic stem cell-derived
41
dopaminergic neural precursor cells) were transplanted were
observed to improve in motor function significantly only 16
weeks after transplantation (FIG. 13a) whereas the rats
transplanted with the cells differentiated according to the
5 method (protocol) of the present disclosure were observed
to improve in motor function significantly all 8, 12, and
16 weeks after transplantation (FIG. 13b).
These results imply that the cells prepared according
to the method (protocol) of the present disclosure can
10 survive in vivo at higher efficiency and have a greater
effect of improving motor functions.
Industrial Applicability
The present invention relates to a method for inducing
15 differentiation of stem cells into midbrain-specific
dopaminergic neural precursor cells and for mass production
of stem cell-derived midbrain-specific dopaminergic neural
precursor cells.
20
As described hitherto, the present disclosure pertains
to a method for inducing the differentiation of stem cells
into dopaminergic neural precursor cells and a method for
mass production of dopaminergic neural precursor cells.
25 Having ability to effectively differentiate stem cells into
42
neural precursor cells, the methods of the present
disclosure can find advantageous applications in research
and development and commercialization associated therewith.

WE CLAIM

1. A method for inducing differentiation of stem cells
into dopaminergic neural precursor cells, the method
5 comprising the steps of:
a) culturing stem cells in a monolayer format;
b) forming and maintaining an embryoid body;
c) generating a neural rosette; and
d) differentiating the neural rosette into
10 dopaminergic neural precursor cells.
2. The method of claim 1, further comprising a step
of:
e) proliferating the dopaminergic neural precursor
15 cells through passage.
3. The method of claim 1, wherein the stem cells are
embryonic stem cells, induced pluripotent stem cells
(iPSCs), adult stem cells, somatic cell nuclear transfer
20 embryonic stem cells, or stem cells formed by direct
reprogramming.
4. The method of claim 1, wherein the stem cells are
cultured in a medium containing an extracellular matrix
25 (ECM).
44
5. The method of claim 1, wherein step a) is carried
out by daily adding a BMP signaling inhibitor and an
activin/nodal signaling inhibitor from 1-3 days before the
5 end of the step.
6. The method of claim 1, wherein step b) is carried
out by adding a BMP signaling inhibitor and an
activin/nodal signaling inhibitor daily from the starting
10 day of the step and adding an SHH (sonic hedgehog)
signaling activator and a GSK-3 inhibitor daily from 2-6
days after the starting day of the step.
7. The method of claim 1, wherein step c) is carried
15 out by adding an SHH signaling activator and a GSK-3
inhibitor daily from the starting day of the step.
8. The method of claim 1, wherein step d) is carried
out by adding an SHH signaling activator and a GSK-3
20 inhibitor daily from the starting day of the step.
9. The method of claim 1, wherein step d) is carried
out by exchanging the medium with a fresh medium every day
and passaging the cells every three days.
25
45
10. The method of claim 5 or 6, wherein the BMP
signaling inhibitor is dorsomorphin and the activin/nodal
signaling inhibitor is 4-(5-benzo[1,3]dioxol-5-yl-4-
pyridin-2-yl-1H-imidazol-2-yl)-benzimide (SB431542).
5
11. The method of any one of claims 6 to 8, wherein
the SHH signaling activator is SAG (smoothened agonist) and
the GSK-3 inhibitor is CHIR99021.
10 12. The method of claim 1, wherein the method has a
rate of differentiation into dopaminergic neural precursor
cells of 80 % or higher.
13. The method of claim 1, wherein dopaminergic neural
15 precursor cells have increased expression levels of FOXA2
and/or LMX1A.
14. The method of claim 1, wherein the dopaminergic
neural precursor cells alleviate symptoms of Parkinson’s
20 disease.
15. A method for mass production of dopaminergic
neural precursor cells, the method comprising the steps of:
a) culturing stem cells in a monolayer format;
25 b) forming and maintaining an embryoid body;
46
c) generating a neural rosette;
d) differentiating the neural rosette into
dopaminergic neural precursor cells; and
e) proliferating the dopaminergic neural precursor
5 cells through passage.