U.S. patent application number 15/837633 was filed with the patent office on 2018-04-12 for transplantation adjuvant in cell therapy using neural progenitor cells.
This patent application is currently assigned to Sumitomo Dainippon Pharma Co., Ltd.. The applicant listed for this patent is Sumitomo Dainippon Pharma Co., Ltd.. Invention is credited to Jun Takahashi.
Application Number | 20180098971 15/837633 |
Document ID | / |
Family ID | 51897974 |
Filed Date | 2018-04-12 |
United States Patent
Application |
20180098971 |
Kind Code |
A1 |
Takahashi; Jun |
April 12, 2018 |
Transplantation Adjuvant in Cell Therapy Using Neural Progenitor
Cells
Abstract
The present invention provides a transplantation adjuvant for
neural progenitor cells containing valproic acid and/or zonisamide
as an active ingredient.
Inventors: |
Takahashi; Jun; (Kyoto,
JP) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Sumitomo Dainippon Pharma Co., Ltd. |
Osaka |
|
JP |
|
|
Assignee: |
Sumitomo Dainippon Pharma Co.,
Ltd.
Osaka
JP
|
Family ID: |
51897974 |
Appl. No.: |
15/837633 |
Filed: |
December 11, 2017 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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14891601 |
Nov 16, 2015 |
9884045 |
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PCT/JP2013/080816 |
Nov 14, 2013 |
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15837633 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 31/423 20130101;
A61K 31/20 20130101; A61P 25/16 20180101; A61P 43/00 20180101; A61P
25/00 20180101; A61P 25/28 20180101; A61K 31/19 20130101; A61K
31/20 20130101; A61K 2300/00 20130101; A61K 31/423 20130101; A61K
2300/00 20130101 |
International
Class: |
A61K 31/423 20060101
A61K031/423; A61K 31/19 20060101 A61K031/19; A61K 31/20 20060101
A61K031/20 |
Foreign Application Data
Date |
Code |
Application Number |
May 16, 2013 |
JP |
2013-104329 |
Claims
1. (canceled)
2. The method according to claim 4, wherein the administering is
performed no sooner than two days before the transplanting.
3. The method according to claim 4, wherein the neural progenitor
cells are derived from iPS cells.
4. A method of treating a degenerative disease of dopaminergic
neurons in a subject in need thereof, comprising transplanting
neural progenitor cells in the subject, and administering valproic
acid to the subject before, after, or simultaneously with the
transplanting in an amount effective to increase a graft survival
rate of dopaminergic neurons induced from the neural progenitor
cells.
5. The method according to claim 4, wherein the degenerative
disease of dopaminergic neurons is Parkinson's disease.
6. The method according to claim 4, wherein the administering is
performed before the transplanting.
7. The method according to claim 4, wherein the administering is
performed after the transplanting.
8. The method according to claim 2, wherein the administering is
performed after the transplanting.
9. The method according to claim 4, wherein the subject is
human.
10. The method according to claim 4, wherein the method further
comprises administering the effective amount of zonisamide to the
subject.
11. The method according to claim 4, wherein the effective amount
is from 100 to 1200 mg per day of the valproic acid.
12. A method of improving retention rate of dopaminergic nerve
cells induced from neural progenitor cells after transplantation,
comprising transplanting neural progenitor cells in a subject, and
administering an effective amount of valproic acid to the subject
before, after, or simultaneously with the transplanting.
13. The method according to claim 12, wherein the administering is
performed no sooner than two days before the transplanting.
14. The method according to claim 12, wherein the neural progenitor
cells are derived from induced pluripotent stem (iPS) cells.
15. A method of treating a degenerative disease of dopaminergic
neurons, comprising the method of improving retention rate of
dopaminergic nerve cells induced from neural progenitor cells after
transplanting according to claim 12.
16. The method according to claim 15, wherein the degenerative
disease of dopaminergic neurons is Parkinson's disease.
17. The method according to claim 12, wherein the administering is
performed before the transplanting.
18. The method according to claim 12, wherein the administering is
performed after the transplanting.
19. The method according to claim 12, wherein the subject is
human.
20. The method according to claim 12, wherein the method further
comprises administering of an effective amount of zonisamide to the
subject.
21. The method according to claim 12, wherein the effective amount
is from 100 to 1200 mg per day of the valproic acid.
Description
TECHNICAL FIELD
[0001] The present invention relates to a transplantation adjuvant
in cell therapy using neural progenitor cells.
BACKGROUND ART
[0002] Parkinson's disease is a progressive neurodegenerative
disease, and is characterized by loss of nigrostriatal dopaminergic
nerves (dopaminergic neurons). It has been confirmed through
clinical studies made until now that a motor symptom of a
Parkinson's disease patient is improved by transplantation of fetal
midbrain cells. Based on this fact, cell replacement therapy is
presumed to be employed as a treatment method for Parkinson's
disease.
CITATION LIST
Non Patent Literature
[0003] Non Patent Literature 1: Hsieh, J. et al., Proc. Natl. Acad.
Sci. USA (2004) 101, 16659-16664
[0004] Non Patent Literature 2: Abematsu, M. et al., J Clin.
Invest., (2010) 120, 3255-3266
SUMMARY OF INVENTION
Problems to be Solved by the Invention
[0005] Pluripotent stem cells, particularly induced pluripotent
stem cells (iPS cells), have a possibility of supplying a large
amount of dopaminergic neurons. Therefore, pluripotent stem cells
are regarded as a novel donor cell source. According to the
findings obtained by the present inventors, however, neural
progenitor cells and dopaminergic neurons differentiated from stem
cells like iPS cells have an extremely low retention rate
(hereinafter sometimes referred to as "survival rate") after
transplantation into a brain.
[0006] Accordingly, an object of the present invention is to
provide a transplantation adjuvant for neural progenitor cells with
which a retention rate of dopaminergic neurons induced from
transplanted neural progenitor cells can be improved.
Means for Solving the Problems
[0007] The present inventors found that a retention rate of
dopaminergic neurons after transplantation is improved by
administering, to a subject, valproic acid or zonisamide, used as
an antiepileptic drug, as adjuvant in transplantation of neural
progenitor cells, resulting in accomplishing the present
invention.
[0008] It has been conventionally reported that valproic acid
differentiates hippocampal neural progenitor cells into neurons in
an in vitro system (Non Patent Literature 1), and that
differentiation into neurons is accelerated in a model mouse
suffering from spinal injury by administering valproic acid at the
same time as transplantation of neural stem cells (Non Patent
Literature 2). It has not been known, however, that valproic acid
improves a retention rate of differentiation induced dopaminergic
neurons after transplantation. Also with respect to zonisamide,
that is, an antiepileptic drug, there have been no findings about
improvement of a retention rate of neural progenitor cells and
dopaminergic neurons after transplantation.
[0009] Specifically, the present invention pertains to the
following:
[0010] [1] A transplantation adjuvant for neural progenitor cells
comprising valproic acid and/or zonisamide as an active
ingredient.
[0011] [2] The transplantation adjuvant according to [1] described
above, to be administered no sooner than two days before
transplanting the neural progenitor cells.
[0012] [3] The transplantation adjuvant according to [1] or [2]
described above, in which the neural progenitor cells are derived
from iPS cells.
[0013] [4] The transplantation adjuvant according to any one of [1]
to [3] described above, used for treating a degenerative disease of
dopaminergic neurons.
[0014] [5] The transplantation adjuvant according to [4] described
above, in which the degenerative disease of dopaminergic neurons is
Parkinson's disease.
[0015] [6] The transplantation adjuvant according to any one of [1]
to [5] described above, used for administering, to a human, 100 to
1200 mg per day of the valproic acid or 10 to 600 mg per day of the
zonisamide.
[0016] [7] The transplantation adjuvant according to any one of [1]
to [6] described above, to be administered, to a human, no sooner
than two days before transplanting the neural progenitor cells.
[0017] The present invention further pertains to the following:
[0018] [8] A method for improving a retention rate of dopaminergic
neurons induced from neural progenitor cells after transplantation,
including administering an effective amount of valproic acid and/or
zonisamide to a mammal into which the neural progenitor cells have
been transplanted.
[0019] [9] The method according to [8] described above, in which
the mammal is a human.
[0020] [10] The method according to [8] or [9] described above, in
which the effective amount of valproic acid and/or zonisamide is
administered no sooner than two days before transplanting the
neural progenitor cells.
[0021] [11] The method according to any one of [8] to [10]
described above, in which the neural progenitor cells are derived
from iPS cells.
[0022] [12] The method according to any one of [8] to [11]
described above, in which the mammal has a degenerative disease of
dopaminergic neurons.
[0023] [13] The method according to [12] described above, in which
the degenerative disease of dopaminergic neurons is Parkinson's
disease.
[0024] [14] The method according to any one of [8] to [13]
described above, in which the effective amount is 100 to 1200 mg
per day of the valproic acid or 10 to 600 mg per day of the
zonisamide.
[0025] [15] The method according to any one of [8] to [14]
described above, in which the effective amount of valproic acid
and/or zonisamide is administered to a human no sooner than two
days before transplanting the neural progenitor cells.
[0026] [16] Valproic acid and/or zonisamide for use in improving a
retention rate of dopaminergic neurons induced from neural
progenitor cells transplanted into a mammal
[0027] [17] The valproic acid and/or zonisamide according to [16]
described above, to be administered no sooner than two days before
transplanting the neural progenitor cells.
[0028] [18] The valproic acid and/or zonisamide according to [16]
or [17] described above, in which the neural progenitor cells are
derived from iPS cells.
[0029] [19] The valproic acid and/or zonisamide according to any
one of [16] to [18] described above, in which the mammal has a
degenerative disease of dopaminergic neurons.
[0030] [20] The valproic acid and/or zonisamide according to [19]
described above, in which the degenerative disease of dopaminergic
neurons is Parkinson's disease.
[0031] [21] The valproic acid and/or zonisamide according to any
one of [16] to [20] described above, used to be administered, to a
human, 100 to 1200 mg per day of the valproic acid or 10 to 600 mg
per day of the zonisamide.
[0032] [22] The valproic acid and/or zonisamide according to any
one of [16] to [21] described above, to be administered, to a
human, no sooner than two days before transplanting the neural
progenitor cells.
Effects of the Invention
[0033] According to the present invention, a transplantation
adjuvant for neural progenitor cells with which a retention rate of
dopaminergic neurons induced from transplanted neural progenitor
cells can be improved can be provided.
BRIEF DESCRIPTION OF DRAWINGS
[0034] FIG. 1 is a diagram illustrating a schedule for
differentiation induction of mouse iPS cells through neural
progenitor cells into midbrain dopaminergic neurons.
[0035] FIG. 2 illustrates RT-PCR results showing expression
time-course of respective marker genes caused by the
differentiation of mouse iPS cells through neural progenitor cells
into midbrain dopaminergic neurons.
[0036] FIG. 3 illustrates graphs, obtained in in vitro experiments,
of expression levels of respective marker molecules of neural
progenitor cells derived from mouse iPS cells.
[0037] FIG. 4 illustrates graphs, obtained in in vitro experiments,
of expression levels of respective marker molecules of midbrain
dopaminergic neurons derived from mouse iPS cells.
[0038] FIG. 5 illustrates a graph, obtained in in vitro
experiments, of an expression level of caspase 3 of midbrain
dopaminergic neurons derived from mouse iPS cells.
[0039] FIG. 6 illustrates graphs, obtained in in vivo experiments,
of expression levels of respective marker molecules in grafts
derived from mouse iPS cells.
[0040] FIG. 7 illustrates graphs, obtained in in vivo experiments,
of the volumes of grafts derived from mouse iPS cells and the
numbers of midbrain dopaminergic neurons present in the grafts.
[0041] FIG. 8 illustrates graphs, obtained in in vivo experiments,
of the numbers of midbrain dopaminergic neurons present in grafts
derived from human iPS cells.
EMBODIMENTS FOR CARRYING OUT THE INVENTION
[0042] Preferred embodiments of the present invention will now be
described in detail. It is noted, however, that the present
invention is not limited to the following embodiments.
[0043] A transplantation adjuvant for neural progenitor cells
(hereinafter sometimes simply referred to as the "transplantation
adjuvant") of the present embodiment contains, as an active
ingredient, valproic acid (chemical name: sodium
2-propylpentanoate) and/or zonisamide (chemical name:
1,2-benzisoxazole-3-methanesulfonamide) (hereinafter, valproic acid
and zonisamide are sometimes designated respectively as "VPA" and
"ZNS").
[0044] Here, a transplantation adjuvant means an agent for
assisting transplantation for attaining a desired effect of cell
transplantation by improving a retention rate of transplanted
cells, by leading transplanted cells into a desired cell type, by
preventing tumorigenesis of transplanted cells, or the like. A
transplantation adjuvant can be grasped as, for example, an agent
for improving survival rate, an agent for improving a graft
survival rate or an agent for improving a differentiation induction
for neurons of interest after transplantation. It can be determined
whether or not a retention rate of phenotypic neurons of interest
(midbrain dopaminergic neurons) after transplantation has been
improved depending on, for example, whether or not dopamine
producing cells remaining seven days to four weeks after the
transplantation or an increase rate of production or the like of
brain dopamine is statistically significant as compared with that
of a control, or whether or not the size of a graft is unchanged
with time. Here, since there is no problem even if a duration from
the transplantation to a test for making the aforementioned
determination is longer, the duration from the transplantation to
the test is not limited to "seven days to four weeks", but the
upper limit is not specified.
[0045] The transplantation adjuvant may contain, as an active
ingredient, valproic acid or zonisamide singly, or both valproic
acid and zonisamide. For example, valproic acid is available from
Sigma-Aldrich, and zonisamide is available from Sumitomo Dainippon
Pharma Co., Ltd.
[0046] The transplantation adjuvant may contain merely the active
ingredient of valproic acid and/or zonisamide alone, or may contain
both the active ingredient and another component. Examples of
another component include a pharmaceutically acceptable carrier, a
filler, a binder, a stabilizer, a buffer, a solubilizer, and an
isotonic agent. In addition, suitable another component can be
appropriately prepared in accordance with oral or parenteral
administration.
[0047] To "contain as an active ingredient" includes not only a
case where valproic acid or zonisamide is in a free form of an acid
or the like but also a case where the transplantation adjuvant
contains such a component in the form of a pharmaceutically
acceptable salt. An example of the pharmaceutically acceptable salt
includes sodium salt.
[0048] Although it is varied depending on various conditions
including the symptom, the age and the weight of a patient, in a
case of oral administration to a human, the transplantation
adjuvant can contain, as the active ingredient, 100 to 1200 mg or
400 to 1200 mg of valproic acid per daily dose. In the case of oral
administration to a human, the transplantation adjuvant can
contain, as the active ingredient, 10 to 600 mg or 25 to 200 mg of
zonisamide per daily dose.
[0049] The neural progenitor cells to which the transplantation
adjuvant is applied mean cells capable of differentiating into
neurons.
[0050] The neural progenitor cells may be cells isolated from a
brain tissue of a mammal such as a human. The neural progenitor
cells may be cells obtained through differentiation induction from
pluripotent stem cells such as embryonic stem cells (ES cells) and
iPS cells (which are respectively sometimes designated as ES
cell-derived cells and iPS-cell derived cells). An example of the
cells isolated from a brain tissue includes cells contained in a
midbrain tissue of an embryo described in, for example, Nature
Neuroscience, 2, 1137 (1999) or N. Engl. J. Med.; 334: 710-9
(2001). The neural progenitor cells may be dopamine-producing
progenitor cells.
[0051] If the neural progenitor cells are isolated from a brain
tissue of a mammal such as a human, they can be isolated by a known
method such as flow cytometry by using, as an index, a marker
molecule specifically expressed in the neural progenitor cells or
neurons, such as PSA-NCAM, CD24 or Corin.
[0052] If the neural progenitor cells are obtained through the
differentiation induction from stem cells such as ES cells or iPS
cells, any of known methods can be employed. Examples of a method
for differentiating the neural progenitor cells from iPS cells
include: (1) serum-free floating culture of embryoid bodies-like
aggregates (SFEB) (Watanabe K. et al., Nat. Neurosci. 8: 288-96,
2005), (2) a method for differentiating pluripotent stem cells
through culture on stromal cells (SDIA method) (Kawasaki H. et al.,
Neuron. 28: 31-40, 2000), (3) a method for culturing with an agent
added to Matrigel (Chambers S M. et al., Nat. Biotechnol. 27:
275-80, 2009), and (4) a method using a low molecular weight
compound (Morizane A. et al., J. Neurosci. Res. 89: 117-126, 2011).
As a method for isolating neural progenitor cells differentiated
from pluripotent stem cells, a method similar to one employed for
isolating the neural progenitor cells from a brain tissue of a
mammal such as a human may be employed.
[0053] Here, a pluripotent stem cell means a stem cell that has
pluripotency capable of differentiating into any of all cells
present in an organism, and in addition, has proliferation potency.
Examples of the pluripotent stem cell include, but are not
especially limited to, an embryonic stem (ES) cell, a cloned
embryo-derived embryonic stem (ntES) cell obtained by nuclear
transplantation, a spermatogonial stem cell (GS cell), an embryonic
germ cell (EG cell), an induced pluripotent stem (iPS) cell, and a
cultured fibroblast- or a bone marrow stem cell-derived pluripotent
stem cell (Muse cell). The pluripotent stem cell may be an ES cell,
an ntES cell or an iPS cell. In consideration of an ethical point,
the pluripotent stem cell may be an iPS cell.
[0054] An ES cell can be produced from a fertilized egg derived
from a mammal Examples of the mammal include a mouse, a rat, a
guinea pig, a hamster, a rabbit, a cat, a dog, a sheep, a pig, a
bovine, a horse, a goat, a monkey and a human. The mammal may be a
human.
[0055] Specifically, a blastocyst developed from a fertilized egg
is cultured together with feeder cells to increase an inner cell
mass. Thereafter, an operation to isolate cells derived from the
increased inner cell mass into single cells and to subculture the
resultant cells with the feeder cells is repeated, and thus, an ES
cell line can be obtained (Thomson J A. et al. (1998), Science.
282: 1145-1147 and H. Suemori et al. (2006), Biochem. Biophys. Res.
Commun., 345: 926-932).
[0056] An iPS cell can be produced from a somatic cell derived from
a mammal Examples of the mammal include a mouse, a rat, a guinea
pig, a hamster, a rabbit, a cat, a dog, a sheep, a pig, a bovine, a
horse, a goat, a monkey and a human. The mammal may be a human.
[0057] A specific example includes a cell that is obtained by
introducing a plurality of prescribed reprogramming factors into a
somatic cell such as a skin cell and has acquired multipotency.
Examples of the reprogramming factors include Oct3/4, Sox2, Sox1,
Sox3, Sox15, Sox17, Klf4, Klf2, c-Myc, N-Myc, L-Myc, Nanog, Lin28,
Fbx15, ERas, ECAT15-2, Tcl1, beta-catenin, Lin28b, Sal11, Sal14,
Esrrb, Nr5a2, Tbx3 and Glis1. One of these reprogramming factors
may be singly used or, a combination of these may be used. Examples
of the combination of the reprogramming factors include those
described in WO2007/069666, WO2008/118820, WO2009/007852,
WO2009/032194, WO2009/058413, WO2009/057831, WO2009/075119,
WO2009/079007, WO2009/091659, WO2009/101084, WO2009/101407,
WO2009/102983, WO2009/114949, WO2009/117439, WO2009/126250,
WO2009/126251, WO2009/126655, WO2009/157593, WO2010/009015,
WO2010/033906, WO2010/033920, WO2010/042800, WO2010/050626,
WO2010/056831, WO2010/068955, WO2010/098419, WO2010/102267,
WO2010/111409, WO2010/111422, WO2010/115050, WO2010/124290,
WO2010/147395, WO2010/147612, Huangfu D., et al. (2008), Nat.
Biotechnol., 26: 795-797, Shi Y, et al. (2008), Cell Stem Cell, 2:
525-528, Eminli S., et al. (2008), Stern Cells. 26:2467-2474,
Huangfu D., et al. (2008), Nat. Biotechnol. 26:1269-1275, Shi Y, et
al. (2008), Cell Stern Cell, 3, 568-574, Zhao Y, et al. (2008),
Cell Stern Cell, 3:475-479, Marson A., (2008), Cell Stern Cell, 3,
132-135, Feng B., et al. (2009), Nat. Cell Biol. 11:197-203, R. L.
Judson et al., (2009), Nat. Biotech., 27:459-461, Lyssiotis C A.,
et al. (2009), Proc. Natl. Acad. Sci. USA. 106:8912-8917, Kim J B.,
et al. (2009), Nature. 461:649-643, Ichida J K., et al. (2009),
Cell Stem Cell. 5:491-503, Heng J C., et al. (2010), Cell Stem
Cell. 6:167-74, Han J., et al. (2010), Nature. 463:1096-100, Mali
P., et al. (2010), Stem Cells. 28:713-720, Maekawa M., et al.
(2011), Nature. 474:225-9. The combination of the reprogramming
factors may be a combination of Oct3/4, Klf4 and Sox2.
[0058] Besides, iPS cells are available from specific institutions
(such as Kyoto University). For example, a 440A3 cell line, that
is, a mouse-derived iPS cell line obtained by introducing Oct3/4
gene, Klf4 gene and Sox2 gene, is available from Kyoto University.
Examples of a human-derived iPS cell line include 201B7, 409B2 and
1039A1.
[0059] Also when the neural progenitor cells are ES cell-derived
cells, similar effects can be attained.
[0060] The transplantation adjuvant can be used to be administered
to a mammal of interest before or after transplanting the neural
progenitor cells into the mammal or at the same time as the
transplantation. The transplantation adjuvant may be used to be
administered no sooner than two days before transplanting the
neural progenitor cells. If the transplantation adjuvant is
administered no sooner than two days before transplanting the
neural progenitor cells, the transplantation can be conducted in a
state where the active ingredient retains an effective blood
concentration. Here, "two days before transplanting the neural
progenitor cells" means two days before a day when the neural
progenitor cells are transplanted into the mammal of interest.
[0061] Examples of the mammal of interest include a mouse, a rat, a
guinea pig, a hamster, a rabbit, a cat, a dog, a sheep, a pig, a
bovine, a horse, a goat, a monkey and a human. The transplantation
adjuvant of the present embodiment may be used in a human.
[0062] Although it is varied depending on the purpose of
administration, the method of administration and the situation of
an administration subject (such as the sex, the age, the weight and
the condition of a disease), in a case of administration to a
human, for example, the transplantation adjuvant may be used so
that 100 to 1200 mg or 400 to 1200 mg of the valproic acid can be
administered per day. For example, the transplantation adjuvant may
be used so that 10 to 600 mg or 25 to 200 mg of the zonisamide can
be administered per day.
[0063] If the transplantation adjuvant is administered at the
above-described dose once a day, it may be used to be administered
to the mammal of interest at least once or more. If the
transplantation adjuvant is administered at the above-described
dose once a day, it may be used to be administered to the mammal of
interest 60 through 180 times or 90 through 120 times.
[0064] The route of the administration of the transplantation
adjuvant may be either oral administration or parenteral
administration, and may be oral administration. Examples of usually
employed dosage form include tablets, capsules, granules, fine
granules, powders, sublingual tablets, syrups and suspensions. The
transplantation adjuvant in a liquid form may be parenterally
administered as an injection. The above-described dosage forms can
be produced by mixing valproic acid and/or zonisamide as the active
ingredient with acceptable usual carrier, filler, binder,
stabilizer and the like. If the transplantation adjuvant is used as
an injection, an acceptable buffer, solubilizer, isotonic agent and
the like may be added thereto.
[0065] When the transplantation adjuvant is administered to the
mammal of interest, retention ratios of the neural progenitor cells
and dopaminergic neurons differentiated from the neural progenitor
cells after the transplantation are improved. Therefore, the
transplantation adjuvant may be also used for treating or
preventing a degenerative disease of dopaminergic neurons.
[0066] A degenerative disease of dopaminergic neurons refers to a
disease caused when the dopaminergic neurons are reduced, and the
examples include Parkinson's disease and dementia with Lewy
body.
[0067] The present invention has been specifically described with
reference to the embodiment so far, but the present invention is
not limited to the above-described embodiment. For example, the
transplantation adjuvant of the present invention may be added to
neural progenitor cells in vitro to prepare cells for
transplantation of dopaminergic neurons or the like, and
thereafter, the thus obtained cells for transplantation may be
transplanted into a brain region or the like. In this case, the
transplantation adjuvant is added in an amount sufficient for
differentiation into desired cells for transplantation, and after
retaining it for, for example, 48 to 192 hours, the resultant cells
are transplanted into a target brain region. After transplanting
the cells for transplantation into the target brain region, the
transplantation adjuvant of the present invention may be further
systemically administered to the transplanted mammal
Examples
Materials and Methods
Differentiation of Dopaminergic Neurons from Mouse iPS Cells
[0068] The 440A3 cells, that is, a mouse iPS cell line, were used
after 10 to 25 passages. The 440A3 cells produced by using a
plasmid vector having three genes of Oct3/4, Klf4 and Sox2 had a
green fluorescent protein (GFP) and a puromycin resistance gene
under control of Nanog enhancer and promotor. The GFP gene and the
puromycin resistance gene are activated merely when the 440A3 cells
are not differentiated. There is no report on integration of an
exogenous gene in the 440A3 cells.
[0069] The undifferentiated 440A3 cells were maintained in a DMEM
(Dulbecco's Modified Eagle Medium, manufactured by Wako Pure
Chemical Industries, Ltd.) together with mouse embryo fibroblasts
(MEF) (feeder cells) having been treated with mitomycin C. In this
manner, unintentionally differentiated 440A3 cells were removed.
The DMEM contained 1% fetal bovine serum (FBS), 5% knockout serum
replacement (KSR; manufactured by Invitrogen), 0.1 mM non-essential
amino acids, 1 mM sodium pyruvate, 0.1 mM 2-mercaptoethanol (2-ME;
manufactured by Invitrogen), 2000 U/ml leukemia inhibitory factor
(manufactured by Invitrogen), and 1.5 .mu.g/ml puromycin. In order
to differentiate and induce the iPS cells into neural cells,
serum-free floating culture of embryoid bodies-like aggregates
(SFEB) was employed. Specifically, aggregates of the 440A3 cells
were separated into individual cells by using 0.25% trypsin/1 mM
EDTA (ethylenediaminetetraacetic acid), and the resultant cells
were seeded in a 96-well low adhesion plate (product name:
Lipidure-Coat Plate A-US96, manufactured by NOF Corporation) at a
concentration (cell density) of 3000 cells/well. Thereafter,
re-aggregation of the 440A3 cells was induced in a differentiation
medium containing GMEM (Glasgow Minimum Essential Medium), 5% KSR,
0.1 mM non-essential amino acids, 1 mM sodium pyruvate and 0.1 mM
2-ME, and this day was defined as day 0 (FIG. 1). During this
differentiation process, various factors were added to the
differentiation medium for inducing midbrain dopaminergic
phenotypes as illustrated in FIG. 1. Specifically, from day 3 to
day 7 after starting the SFEB, 20 ng/ml of mouse FGF-8b
(manufactured by R & D Systems) was added, from day 4 to day 7
after starting the SFEB, 10 ng/ml of recombinant mouse sonic
hedgehog (C25II) N-terminus (manufactured by R & D Systems) was
added, and on and after day 7 after starting the SFEB, 1% N-2
supplement (manufactured by Gibco) and 200 nM ascorbic acid were
added. The KSR was removed from the differentiation medium on day 7
after starting the SFEB.
Fluorescence-Activated Cell Sorting (FACS)
[0070] On day 9 after starting the SFEB, the 440A3 cells were
washed with phosphate buffered saline (PBS(-)) twice. Thereafter,
the 440A3 cells were dissociated into single cells by five-minute
incubation performed at 37.degree. C. by using Accumax
(manufactured by Innovate Cell Technologies, product name). The
cells were collected with a FACS buffer. The FACS buffer was
constituted by PBS(-) containing 2% FBS, 20 mM D-glucose and 1%
penicillin/streptomycin (P/S, manufactured by Invitrogen). The
collected cells were mechanically dissociated into a single cell
suspension by a gentle pipetting operation. Next, the resultant
cells were incubated with a mouse anti-PSA-NCAM antibody (dilution
rate of 1:200, manufactured by Millipore) at 4.degree. C. for about
30 minutes. Thereafter, a washing operation using a centrifuge was
performed twice, and the resultant cells were further incubated for
30 minutes with a secondary antibody of a donkey anti-mouse IgG
(dilution rate of 1:400, manufactured by Invitrogen) labeled with
AlexaFluor 594. Dead cells and cell debris were excluded by using
7-aminoactinomycin-D (7-AAD, manufactured by BD Pharmigen) stain.
The remaining living cells were suspended again at a final
concentration (cell density) of 1.times.10.sup.7 cells/ml. Cell
sorting was conducted by using a FACSAria II cell sorter
(manufactured by Becton Dickinson And Company) equipped with a 488
nm argon laser, a 633 nm helium-neon laser, a 100 .mu.m nozzle and
a FACSDiva software program. A PSA-NCAM positive rate was
determined on the basis of a negative control not using a primary
antibody.
In vitro Treatment for Differentiation Inducing Neural Progenitor
Cells into Dopaminergic Neurons by using Test Compound
[0071] After the cell sorting, in order to induce the reaggregation
of the cells, a PSA-NCAM.sup.+ cell group was seeded in a DMEM/F12
medium (manufactured by Wako Pure Chemical Industries, Ltd.) in a
96-well plate at a concentration (cell density) of 20000
cells/well. The DMEM/F12 medium contained 1% N-2 supplement, 200 nM
ascorbic acid, 2% B27 supplement (manufactured by Invitrogen), 0.5
mM L-glutamine and 1% P/S. In order to prevent apoptosis, a ROCK
inhibitor, Y-27632 (manufactured by Wako Pure Chemical Industries,
Ltd.), was used at a concentration of 30 .mu.M during the cell
sorting process and the following overnight cultivation. On day 10
after starting the SFEB, any one of valproic acid (VPA)
(manufactured by Sigma-Aldrich), zonisamide (ZNS) sodium salt
(manufactured by Sumitomo Dainippon Pharma Co., Ltd.), 17.beta.
estradiol (E2) (manufactured by Sigma-Aldrich), a glial cell line
derived neurotrophic factor (GDNF) (manufactured by R & D
Systems) and PBS(-) was added to the medium for 4 days. Each of
VPA, ZNS and E2 was used at three different concentrations.
Specifically, the concentration of VPA was 0.01 mM, 0.1 mM or 1 mM,
the concentration of ZNS was 1 .mu.M, 10 .mu.M or 100 .mu.M, and
the concentration of E2 was 1 nM, 10 nM or 100 nM. GDNF was added
at a concentration of 20 mg/ml to be used as a positive control. In
order to neutralize the effect of VPA and E2, 2,5-dideoxyadenosine
(ddA, 100 .mu.M; manufactured by Santa Cruz Biotechnology, Inc.),
that is, an adenylate cyclase inhibitor, and ICI182780 (ICI, 2
.mu.M; manufactured by Wako Pure Chemical Industries, Ltd.), that
is, an estrogen receptor antagonist, were respectively added to the
media on day 10 after starting the SFEB.
Transplantation Experiment of Mouse iPS Cell-Derived Dopamine
Neural Progenitor Cells
[0072] Ten-week-old Sprague-Dawley rats (SD rats, available from
Shimizu Laboratory Supplies Co., Ltd.) were handled in accordance
with the Guidelines for Animal Experiments of Kyoto University.
Each SD rat was anesthetized, and donor cells were transplanted by
stereotaxic injection into striatums on both sides. Two cell
aggregates (containing 3.1.times.10.sup.5 cells on average)
obtained on day 9 after starting the SFEB were collected in 1 .mu.l
of PBS(-) so as to be used as the donor cells for the
transplantation in each tract. To the PBS(-), Y-27632 was added at
a final concentration of 30 .mu.M. Thereafter, intraperitoneal
injection of VPA (150 mg/kg/day), ZNS sodium salt (30 mg/kg/day),
E2 (80 .mu.M/kg/day) or a saline was conducted from two days before
transplanting the donor cells until a day of necropsy. For
immunosuppression, cyclosporin A (CsA, manufactured by Wako Pure
Chemical Industries, Ltd.) was administered to all the SD rats at a
daily dose of 10 mg/kg. Four weeks after the transplantation of the
donor cells, the brains of the SD rats were washed and fixed by
intracardially perfusing 4% paraformaldehyde under deep anesthesia.
On the day of necropsy, a blood sample was collected from each SD
rat one hour after the final injection of the test compound or CsA.
Such a sample was sent to SRL, Inc. (Tokyo, Japan) where the blood
concentration of the administered drug (test compound) was
measured.
Transplantation Experiment of Human iPS Cell-Derived Dopamine
Neural Progenitor Cells
[0073] Twelve-week-old SCID rats (produced by Institute of
Laboratory Animals, Graduate School of Medicine, Kyoto University)
were handled in accordance with the Guidelines for Animal
Experiments of Kyoto University. The SCID rats were anesthetized,
and donor cells were transplanted by stereotaxic injection into
striatums on both sides. Dopamine neural progenitor cells
(2.7.times.10.sup.5 cells on average) prepared from 1039A1 cell,
that is, a human iPS cell line, were collected in 2 .mu.l of PBS(-)
so as to be used as the donor cells for the transplantation in each
tract. To the PBS(-), Y-28632 was added at a final concentration of
30 .mu.M. Thereafter, intraperitoneal injection of VPA (150
mg/kg/day or 600 mg/kg/day, which are sometimes designated
respectively as a high dose and a low dose), ZNS sodium salt (30
mg/kg/day or 60 mg/kg/day, which are sometimes designated
respectively as a high dose and a low dose), or a saline was
conducted from two days before transplanting the donor cells until
a day of necropsy. Four weeks after the transplantation of the
donor cells, the brains of the SCID rats were washed and fixed by
intracardially perfusing 4% paraformaldehyde under deep anesthesia.
On the day of necropsy, a blood sample was collected from each SCID
rat one hour after the final injection of the test compound, and
the blood concentration of the drug (test compound) was
measured.
Reverse Transcriptase Polymerase Chain Reaction (RT-PCR)
[0074] Total RNA was extracted by using RNeasy Plus Mini Kit
(manufactured by Qiagen). The extracted total RNA was reverse
transcribed by using Super Script III First-Strand Synthesis System
(manufactured by Invitrogen). Each PCR was performed by using Hot
StartTaq DNA polymerase (manufactured by Qiagen). A reverse
transcriptase was not added so as to perform a control
amplification reaction for each primer. MEF was used as another
negative control. Gene sequences to be detected by the RT-PCR were
all known, and on the basis of the gene sequences, the primers were
designed and the molecular weights of amplified products were
estimated.
Immunofluorescence
[0075] In an in vitro experiment, a cell aggregate treated with any
of the above-described test compounds on day 14 after starting the
SFEB was fixed with 4% paraformaldehyde. Thereafter, the fixed cell
aggregate was frozen and sliced into thin sections each having a
thickness of 10 .mu.M by using a microtome for immunocytochemistry.
On the other hand, in an in vivo experiment (transplantation
experiment), the brain of a SD rat or a SCID rat was taken out
after the transplantation experiment to be fixed again with 4%
paraformaldehyde for 2 days. Thereafter, the fixed brain of the SD
rat or SCID rat was cryopreserved in 30% sucrose for 3 days,
frozen, and sliced into thin sections each having a thickness of 40
.mu.M for immunohistochemistry. The frozen sections of the sphere
(spherical cell mass) and the brain were subjected to a
permeabilization and blocking treatment in PBS(-) for 1 hour at
room temperature to be used as samples. The PBS(-) contained 0.3%
Triton-X and 2% donkey serum. Thereafter, each of the sections was
incubated overnight together with a primary antibody at 4.degree.
C. Primary antibodies used in this example are a rabbit
anti-tyrosine hydroxylase antibody (dilution rate of 1:400, TH;
manufactured by Millipore), a mouse anti-TH antibody (dilution rate
of 1:200, manufactured by Millipore), a sheep anti-TH antibody
(dilution rate of 1:400, manufactured by Millipore), a mouse
anti-Tub.beta.3 antibody (dilution rate of 1:1000, Tuj1;
manufactured by Covance Inc.), a rat anti-NURR1 antibody (dilution
rate of 1:1000, KAN Research Institute, Inc., Kobe, Japan), a
rabbit anti-Ki67 antibody (dilution rate of 1:1000, manufactured by
Novocastra: NCL-Ki67p), a rabbit anti-caspase 3 antibody (dilution
rate of 1:500, manufactured by Santa Cruz Biotechnology, Inc.), a
rat anti-M2M6 antibody (dilution rate of 1:50, manufactured by
Developmental Study Hybridoma Bank), a mouse anti-Nestin antibody
(dilution rate of 1:50; manufactured by Millipore), a rabbit
anti-Pitx3 antibody (dilution rate of 1:500; manufactured by
Chemicon International Inc.), a goat anti-HNF-3.beta. antibody
(dilution rate of 1:500, Foxa2; manufactured by Santa Cruz
Biotechnology, Inc.), a mouse anti-human Nuclei antibody (dilution
rate of 1:1000; manufactured by Millipore), and a mouse anti-NeuN
antibody (dilution rate of 1:500, manufactured by Chemicon
International Inc.). After washing with PBS (0.05% Tween-20) three
times, the resultant sample was incubated with an Alexa
Fluor-conjugated secondary antibody for 1 hour at room temperature.
After washing three more times, the sample was incubated with DAPI
for nuclear staining and mounted using Permaflow (Dako).
Immunoreactive cells were visualized with a confocal laser
microscope (Fluoview FV1000D; manufactured by Olympus Corporation).
In order to determine the percentage of positive cells for each
marker, the number of labelled cells was manually counted in at
least three independent experiments. The volume and the number of
Ki67.sup.+/Nestin.sup.+ cells in each graft were determined by
using BZ-II analysis software program (Keyence). In order to
estimate the number of immunoreactive cells in each graft, the
number of cells in every six grafts was manually counted, with
Abercrombie Correction (Abercrombie, 1946) applied.
Statistical Analysis
[0076] Statistical analysis was carried out by using GraphPad Prism
software program Ver. 5.0b (GraphPad Software). All quantitative
data was indicated in the form of mean.+-.SD (standard deviation),
and One-way ANOVA and Newman-Keuls post-hoc tests were used.
Differences were considered to be statistically significant for
P<0.05.
Results
Differentiation of Dopaminergic Neurons from Mouse iPS Cells
[0077] Dopaminergic neurons were differentiation induced from 440A3
cells, that is, a mouse iPS cell line, by employing the SFEB. The
440A3 cells proliferated continuously until day 14 after starting
the SFEB. In accordance with the differentiation induction,
expression of Nanog-GFP was gradually reduced, and the expression
was not substantially observed on day 9 after starting the SFEB.
RT-PCR photographs showing change with time of the expression of
respective gene markers are illustrated in FIG. 2. It was confirmed
that cell aggregates having pluripotency (Oct3/4.sup.+,
Nanog.sup.+) were differentiated into immature neural progenitor
cells (NPC) (Nestin.sup.+) on day 6 to 9 after starting the SFEB,
and thereafter differentiated into Tuj1.sup.+ neurons (FIG. 2). The
Tuj1.sup.+ neurons also expressed Lmx1a, Nurr1 and TH, that are,
dopaminergic neuron-specific markers.
[0078] The obtained cells also contained undifferentiated cells and
non-neural cells. Therefore, in order to obtain a highly
homogeneous population of NPC, PSA-NCAM.sup.+ cells were sorted by
using the FACS. PSA-NCAM is a cell adhesion molecule specifically
expressed on the surface of a neural cell. On day 9 after starting
the SFEB, about 60% of the cells were positive for PSA-NCAM
(PSA-NCAM.sup.+). The PSA-NCAM.sup.+ cells sorted by the FACS were
made to re-aggregate and allowed to mature for another 5 days. The
matured cells were subjected to immunocytochemistry. When a section
of the matured cell aggregate was immunofluorescence stained, most
of cells were Tuj1.sup.+ neurons, in which midbrain dopaminergic
neurons were present.
[0079] The dopaminergic neurons simultaneously expressed TH, NURR1,
FOXA2 and PITX3.
Influence of VPA and E2 on Differentiation into Dopaminergic
Neurons In Vitro
[0080] It was examined whether or not VPA, ZNS and E2 affect the
differentiation induction into dopaminergic neurons in vitro. From
day 10 to day 14 after starting the SFEB, re-aggregated
PSA-NCAM.sup.+ cells were cultured in the presence of VPA, ZNS or
E2. When the immunocytochemistry was performed on day 14 after
starting the SFEB, 90% or more of cells expressed Tuj1, that is, a
neuronal marker, in using any of the test compounds (FIG. 3(A)). In
control cells, 5.2.+-.1.1% of cells were TH.sup.+. On the contrary,
in cells cultured respectively in the presence of VPA (0.01 mM and
0.1 mM) and E2 (10 nM), ratios of TH.sup.+ cells were increased
about twice as large as that of the control (which ratios were
12.1.+-.1.5%, 11.7.+-.0.4% and 12.2.+-.2.3%, respectively) (FIG.
3(B)). In order to investigate whether or not such an effect of VPA
and E2 was caused via the cyclic AMP pathway or the estrogen
receptor, ddA, that is, an adenylate cyclase inhibitor, and ICI,
that is, an estrogen receptor antagonist, were respectively used.
When cells were cultured with 100 .mu.M of ddA and 2 .mu.M of ICI
respectively added to 0.1 mM of VPA and 10 nM of E2, the ratios of
increase in the TH.sup.+ cells were remarkably reduced (FIG. 3(C)).
On the other hand, even if ddA or ICI was singly added to cells,
the ratio of TH.sup.+ cells was not changed as compared with that
of the control.
[0081] Next, double-label immunocytochemistry was conducted for
marker molecules of midbrain dopaminergic neurons such as FOXA2,
NURR1, PITX3 and TH. In cells cultured with 0.1 mM of VPA added,
ratios of TH.sup.+ FOXA2.sup.+ cells and TH.sup.+ NURR1.sup.+ cells
were remarkably increased as compared with those of controls (which
ratios were respectively 1.00.+-.0.58% vs. 0.25.+-.0.22% and
1.00.+-.0.70% vs. 0.37.+-.0.32%, FIG. 4). Since a time period of
the differentiation induction was too short and the cells were
cultured without adding a cytokine such as GDNF, PITX3.sup.+ cells
were substantially not observed. These results suggested that the
differentiation into dopaminergic neurons and the acquisition of
midbrain-like dopaminergic neuron phenotype are accelerated by
culturing cells with VPA.
[0082] Next, with expression of caspase 3, that is, a marker of
apoptosis cells, used as an index, the influence of the
above-described test compounds on the survival rate of TH.sup.+
neurons in vitro was evaluated. In a control sphere, 18.0.+-.5.9%
of TH.sup.+ neurons expressed caspase 3. This result suggested that
one-fifth of dopaminergic neurons were undergoing apoptosis (FIG.
5). On the other hand, in cells cultured in the presence of VPA or
E2, a ratio of dopaminergic neurons undergoing apoptosis was low.
Among four groups, however, there was no significant
difference.
Influence of VPA on Differentiation of Transplanted NPC into
Neurons
[0083] Next, it was examined whether or not the systemic
administration of VPA, ZNS or E2 affects the survival rate and
differentiation of dopaminergic neurons in a graft. In this
transplantation experiment, on day 9 after starting the SFEB, a
cell population (3.1.times.10.sup.5 cells in two aggregates, in
PBS) not sorted by the FACS was transplanted into a striatum of a
SD rat. To the SD rat used for the transplantation, one of the
above-described agents and CsA, that is, an immunosuppressive
agent, were intraperitoneally administered every day from two days
before the transplantation until a day of sacrifice (for four weeks
after the transplantation). On the day of sacrifice, the blood
concentration of CsA was 3700.+-.898 ng/ml on average. The blood
concentrations of VPA, ZNS and E2 were respectively 158.5.+-.3.9
.mu.g/ml, 2.43.+-.0.13 .mu.g/ml and 1141.+-.926 pg/ml.
[0084] When the double-label immunohistochemistry was performed for
Nestin (a marker of NPC) and Ki67 (a marker of proliferating
cells), 15 to 20% of transplanted cells were Nestin.sup.+ cells,
but there was no significant difference among four groups (FIG.
6(A)). A ratio of Ki67.sup.+ cells in Nestin.sup.+ cells was very
low (<0.1%) in all grafts. It was suggested that the
Nestin.sup.+ cells were mostly quiescent or becoming post-mitotic
at this time point. On the other hand, when the
immunohistochemistry was performed for NeuN, that is, a marker of
mature neurons, in a SD rat administered with VPA, a ratio of
NeuN.sup.+ cells to the number of all the cells in a graft was
significantly increased as compared with that in a control SD rat
(which ratio was 77.9.+-.5.1% vs. 57.7.+-.9.4%, FIG. 6(B)). This
result suggested that VPA accelerates the differentiation of
transplanted NPC into neurons. The grafted cells were identified
using immunofluorescent staining for M2M6. The M2M6 is expressed
merely in grafted cells (mouse cells) and is not expressed in cells
of the SD rat, that is, a host. Four weeks after the
transplantation, grafts satisfactorily survived and no signs of
tumor formation were observed in all the groups. Regarding the
volume of graft, that of the SD rat administered with VPA was
smallest (4.33.+-.2.14 mm.sup.3), and that of the control SD rat
was largest (9.76.+-.3.19 mm.sup.3) There was, however, no
significant difference (FIG. 7(A)).
Improvement, attained by Administration of VPA or ZNS, of Retention
Rate of Midbrain Dopaminergic Neurons in Graft Containing Mouse iPS
Cell-derived Neural Progenitor Cells
[0085] The number of TH.sup.+ cells in a graft obtained four weeks
after the transplantation was compared among four groups. When the
double-label immunohistochemistry was performed, the number of
TH.sup.+ cells in a graft of a SD rat administered with VPA was
remarkably large as compared with that in a graft of a control SD
rat (which numbers were respectively 1396.+-.864 cells and
393.+-.311 cells, FIG. 7(B)).
[0086] In the graft of the control SD rat, merely a part of the
TH.sup.+ cells co-expressed FOXA2, that is, a midbrain marker
(24.7.+-.9.3%). On the contrary, in grafts of SD rats respectively
administered with VPA and ZNS, most of the TH.sup.+ cells were
FOXA2.sup.+ (the ratios of which were respectively 81.8.+-.33.6%
and 80.4 35 21.1%). It was revealed, through statistical analysis,
that the number of midbrain dopaminergic neurons (TH.sup.+
FOXA2.sup.+) in a graft of a SD rat administered with VPA or ZNS
was significantly increased as compared with that in a graft of a
control SD rat (the numbers of which were respectively 984.+-.770
cells, 835.+-.540 cells and 97.+-.76 cells, FIG. 7(C)). These
results suggested that the retention rate of dopaminergic neurons
differentiated from transplanted neural progenitor cells is
improved by systemic administration of VPA or ZNS. When ZNS was
administered, a ratio of the differentiation of the transplanted
NPC into neurons was almost equivalent to that of a control (FIG.
6(B)), and hence, it was suggested that there is a mutually
independent relationship between a retention rate attained after
transplantation and efficiency of differentiation of NPC into
neurons.
Survival Rate, attained by Administration of VPA or ZNS, of
Midbrain Dopaminergic Neurons in Graft containing Human iPS
Cell-derived Neural Progenitor Cells
[0087] The number of TH.sup.+ cells in a graft obtained four weeks
after transplantation was compared among five groups. When the
double-label immunohistochemistry was performed, the number of
TH.sup.+ cells in a graft of a SCID rat administered with ZNS at a
high dose was remarkably large as compared with that in a graft of
a control SCID rat (the numbers of which were respectively
6480.+-.2145 cells and 3026.+-.1349 cells, FIG. 8(A). A symbol "*"
used in the drawing indicates p<0.05). While a ratio of cells
co-expressing FOXA2, that is, a midbrain marker, in the TH.sup.+
cells was 76.3.+-.9.7% in the graft of the control SCID rat,
91.8.+-.6.2% of the TH.sup.+ cells were FOXA2.sup.+ in the graft of
the SCID rat administered with ZNS at a high dose. It was revealed,
through statistical analysis, that the number of midbrain
dopaminergic neurons (TH.sup.+ FOXA2.sup.+) in a graft of a SCID
rat administered with ZNS at a high dose is significantly increased
as compared with that in a graft of a control SCID rat (the numbers
of which were respectively 5889.+-.1821 cells and 2297.+-.1116
cells,
[0088] FIG. 8(B). Symbols "*" and "**" used in the drawing
respectively indicate p<0.05 and p<0.01.). These results
suggested that the retention rate of dopaminergic neurons
differentiated from transplanted neural progenitor cells is
improved by the systemic administration of ZNS.
INDUSTRIAL APPLICABILITY
[0089] A transplantation adjuvant of the present invention is
useful for improving, in transplantation of neural progenitor
cells, particularly iPS cell-derived neural progenitor cells, a
retention rate of dopaminergic neurons in a transplantation site of
a recipient's brain. Besides, if the transplantation adjuvant
contains valproic acid, the transplantation adjuvant is also useful
for accelerating differentiation of the neural progenitor cells
into dopaminergic neurons.
* * * * *