U.S. patent application number 13/481318 was filed with the patent office on 2012-09-20 for mesenchymal stem cell and the method of use thereof.
This patent application is currently assigned to NC MEDICAL RESEARCH INC.. Invention is credited to Kazuo Hashi, Osamu Honmou, Teiji Uede.
Application Number | 20120237487 13/481318 |
Document ID | / |
Family ID | 26594623 |
Filed Date | 2012-09-20 |
United States Patent
Application |
20120237487 |
Kind Code |
A1 |
Honmou; Osamu ; et
al. |
September 20, 2012 |
MESENCHYMAL STEM CELL AND THE METHOD OF USE THEREOF
Abstract
Demyelinated axons were remyelinated in the demyelinated rat
model by collecting bone marrow cells from mouse bone marrow and
transplanting the mononuclear cell fraction separated from these
bone marrow cells.
Inventors: |
Honmou; Osamu; (Sapporo-shi,
JP) ; Hashi; Kazuo; (Sapporo-shi, JP) ; Uede;
Teiji; (Sapporo-shi, JP) |
Assignee: |
NC MEDICAL RESEARCH INC.
Tokyo
JP
|
Family ID: |
26594623 |
Appl. No.: |
13/481318 |
Filed: |
May 25, 2012 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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13040954 |
Mar 4, 2011 |
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13481318 |
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12076092 |
Mar 13, 2008 |
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13040954 |
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11189050 |
Jul 26, 2005 |
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12076092 |
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10330963 |
Dec 23, 2002 |
7098027 |
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11189050 |
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PCT/JP2001/005456 |
Jun 26, 2001 |
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10330963 |
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Current U.S.
Class: |
424/93.7 |
Current CPC
Class: |
A61P 25/18 20180101;
C12N 5/0664 20130101; C12N 5/0676 20130101; A61K 35/28 20130101;
A61P 25/28 20180101; C12N 5/0663 20130101; A61K 35/12 20130101;
A61K 9/0019 20130101; C12N 5/0665 20130101; A61P 25/08 20180101;
C12N 5/0668 20130101; C12N 2506/1353 20130101; A61P 9/10 20180101;
C12N 5/0622 20130101; A61K 2035/124 20130101; C12N 5/0662 20130101;
A61P 25/02 20180101; A61P 25/00 20180101; C12N 5/0667 20130101;
A61K 35/51 20130101; A61P 35/00 20180101; C12N 5/0666 20130101 |
Class at
Publication: |
424/93.7 |
International
Class: |
A61K 35/14 20060101
A61K035/14; A61K 35/28 20060101 A61K035/28; A61P 25/28 20060101
A61P025/28 |
Foreign Application Data
Date |
Code |
Application Number |
Jun 26, 2000 |
JP |
2000-190421 |
May 29, 2001 |
JP |
2001-160579 |
Claims
1. A method for treating neurodegenerative diseases, comprising:
injecting an effective amount of cells capable of differentiating
into neural cells or glia cells into a patient afflicted with a
neurodegenerative disease, wherein the cells are prepared by a
method comprising: diluting marrow cells or cord blood cells;
isolating a mononuclear cell fraction; and selecting cells having
surface markers SH2(+), SH3(+), SH4(+), CD29(+), CD44(+), CD14(-),
CD34(-), and CD45(-) from the mononuclear cell fraction, and
wherein the injected cells treat said patient's neurodegenerative
disease.
Description
CROSS-REFERENCE TO RELATED APPLICATION
[0001] This application is a Divisional of U.S. patent application
Ser. No. 13/040,954, filed Mar. 4, 2011, which is a Continuation of
application Ser. No. 12/076,092, filed Mar. 13, 2008, which is a
Continuation of application Ser. No. 11/189,050, filed Jul. 26,
2005, now abandoned, which is a Divisional of application Ser. No.
10/330,963, filed Dec. 23, 2002, now U.S. Pat. No. 7,098,027, which
is a Continuation-In-Part of PCT/JP01/05456, filed Jun. 26, 2001,
the contents of all of which are incorporated herein by reference
in their entirety.
TECHNICAL FIELD
[0002] The present invention relates to cells derived from bone
marrow cells, cord blood cells, or embryonic hepatic tissues that
can differentiate into neural cells, and cell fractions containing
such cells. It is expected that these cells and cell fractions can
be used to treat neurological diseases, particularly in autologous
transplantation therapy.
BACKGROUND ART
[0003] Transplantation of oligodendrocytes (i.e., oligodendroglia)
(Archer D. R., et al., 1994. Exp. Neurol. 125:268-77; Blakemore W.
F., Crang A. J., 1988. Dev. Neurosci. 10:1-11; Gumpel M., et al.
1987. Ann. New York Acad. Sci. 495:71-85) or myelin-forming cells,
such as Schwann cells (Blakemore W. F., 1977. Nature 266:68-9;
Blakemore W. F., Crang A. J., 1988. Dev. Neurosci. 10:1-11; Honmou
O. et al., 1996. J. Neurosci. 16:3199-208), or olfactory ensheating
cells (Franklin R. J. et al., 1996. Glia 17:217-24; Imaizumi T. et
al., 1998. J. Neurosci. 18(16):6176-6185; Kato T. et al., 2000.
Glia 30:209-218), can elicit remyelination in animal models and
recovery of electrophysiological function (Utzschneider D. A. et
al., 1994. Proc. Natl. Acad. Sci. USA. 91:53-7; Honmou O. et al.,
1996. J. Neurosci. 16:3199-208). It is possible to prepare such
cells from patients or other persons for cell therapy. However,
this method is considerably problematic because tissue material
must be collected from either the brain or nerves.
[0004] Neural progenitor cells or stem cells derived from brain
have the ability to self-renewal and differentiate into various
lineages of neurons and glia cells (Gage F. H. et al., 1995. Proc.
Natl. Acad. Sci. USA. 92:11879 83; Lois C., Alvarez-Buylla A.,
1993. Proc. Natl. Acad. Sci. USA. 90:2074-7; Morshead C. M. et al.,
1994. Neuron 13:1071-82; Reynolds B. A., Weiss S., 1992. Science
255:1707-10).
By transplantation into newborn mouse brain, human neural stem
cells collected from fetal tissues differentiate into neurons and
astrocytes (Chalmers-Redman R. M. et al., 1997. Neurosci.
76:1121-8; Moyer M. P. et al., 1997. Transplant. Proc. 29:2040-1;
Svendsen C. N. et al., 1997. Exp. Neurol. 148:135-46), and
myelinate the axons (Flax J. D. et al., 1998. Nat. Biotechnol.
16:1033-9). Remyelination and recovery of impulse conduction upon
transplantation of neural progenitor (stem) cells derived from
adult human brain into demyelinated rodent spinal cord have been
reported (Akiyama Y. et al., 2001. Exp. Neural.).
[0005] These studies have evoked great interest due to the
indicated possibility of the application of the above-mentioned
cells to regenerative strategy of neurological diseases (Akiyama Y.
et al., 2001. Exp. Neural.; Chalmers-Redman R. M. et al., 1997.
Neurosci. 76:1121-8; Moyer M. P. et al., 1997. Transplant. Proc.
29:2040-1; Svendsen C. N. et al., 1997. Exp. Neurol. 148:135-46;
Yandava B. D. et al., 1999. Proc. Natl. Acad. Sci. USA.
96:7029-34). However, in order to establish cell transplantation
therapy (including autologous transplantation) using these cells,
still problems, such as establishment of harvest method and
requirement of cell expansion using trophic factors, remain to be
solved.
[0006] According to the recent studies, neural stem cells were
revealed to be able to differentiate or transform into
hematopoietic cells in vivo, suggesting that neural progenitor
(stem)-cells are not restricted to the neural cell lineage
(Bjornson C. R. et al., 1999. Science 283:534-7). Furthermore, bone
marrow stromal cells (not mesenchymal stem cells in the bone
marrow) are reported to differentiate into astrocytes by the
injection into the lateral ventricles of neonatal mice (Kopen G. C.
et al., Proc. Natl. Acad. Sci. USA. 96:10711-6), and into neurons
in vitro when cultured under appropriate cell culture conditions
(Woodbury D. et al., 2000. J. Neurosci. Res. 61:364-70).
DISCLOSURE OF THE INVENTION
[0007] The present inventors have previously isolated and cultured
neural stem cells from adult human brain, and established some cell
lines. By studying their functions, the inventors newly discovered
that the neural stem cells have pluripotency and the ability to
self-renewal. Specifically, single-cell expansion of neural
progenitor (stem) cells obtained from adult human brain was
conducted to establish cell lines; the established cells were then
subjected to in vitro clonal analysis. The result showed that the
cell lines had pluripotency (namely, differentiation into neuron,
astroglia (or astrocyte), and oligodendroglia (i.e.,
oligodendrocyte)) and the ability to self-renewal (namely,
proliferation potency). Thus, these cells were confirmed to possess
the characteristics of neural stem cell.
[0008] Transplantation of these cells indeed resulted in very
favorable graft survival, migration, and differentiation in
cerebral ischemic model rats or injury model rats. Furthermore,
transplantation of the cells was found to result in functional
myelin sheath formation in spinal cord demyelination model rats.
Thus, such transplantation allows remyelination of the demyelinated
axon and restoration of the neural function in the rat spinal cord
demyelination model. Effectiveness of such transplantation therapy
using these cells was confirmed by histological,
electrophysiological, and behavior studies.
[0009] Judging from the above-described findings, transplantation
of cultured neural stem cells, which have been isolated from a
small amount of neural tissue collected from the cerebrum of a
patient, into the lesion of the brain or the spinal cord of the
patient seems to be a widely applicable in autotransplantation
therapy.
[0010] However, while not causing neurologic deficits, collecting
tissues containing neural stern cells from cerebrum is relatively
invasive. Thus, considering the need for establishing therapeutic
methods for various complicated diseases in the nervous system
today, it is crucial to establish a safer and simpler method for
autotransplantation therapy.
[0011] Thus, an objective of the present invention is to provide
cellular material that is useful in the treatment of neurological
diseases, and which can be prepared safely and readily. Another
objective of the present invention is to provide a method for
treating neurological diseases, preferably a method for
autotransplantation therapy, using the cellular material.
[0012] In view of the existing state as described above, to
establish donor cells the present inventors focused on the
technique of collecting bone marrow cells from bone marrow, a
simpler technique as compared to the collection of neural stem
cells and routinely used in today's medical practice. First, they
collected bone marrow cells from mouse bone marrow, isolated
mononuclear cell fraction, and then transplanted this fraction as
donor cells into spinal cord demyelination model rats.
Surprisingly, it was discovered that the demyelinated axon gets
remyelination by the treatment. Hence, the present inventors newly
revealed that the mononuclear cell fraction prepared from bone
marrow cells have the ability to differentiate into neural cells.
The present inventors also discovered that cell fractions
containing mesodermal stem cells, mesenchymal stem cell, stromal
cells, and AC133-positive cells, that were isolated from the
mononuclear cell fraction had the ability to differentiate into
neural cells. Besides bone marrow cells, these cell fractions can
also be prepared from cord blood cells. Furthermore, AC133-positive
cells can be prepared from embryonic hepatic tissues.
[0013] Thus, the present invention provides cell fractions
containing cells capable of differentiating into neural cells,
which are isolated from bone marrow cells, cord blood cells, and
embryonic hepatic tissues.
[0014] In another embodiment, such cell fractions contain
mesenchymal stem cells having the following character: SH2(+),
SH3(+), SH4(+), CD29(+), CD44(+), CD14(-), CD34(-), and
CD45(-).
[0015] In another embodiment, such cell fractions contain stromal
cells having the following characteristics: Lin(-), Sca-1(+),
CD10(+), CD11D(+), CD44(+), CD45(+), CD71(+), CD90(+), CD105(+),
CDW123(+), CD127(+), CD164(+), fibronectin (+), ALPH(+), and
collagenase-1(+).
[0016] In another embodiment, such cell fractions contain cells
having the character AC133(+).
[0017] In addition, the present invention provides cells capable of
differentiating into neural cells, which are contained in the
above-mentioned cell fraction.
[0018] Furthermore, the present invention provides compositions for
treating neurological disease, which contain the above-mentioned
mononuclear cell fractions or the above-mentioned cells. According
to a preferred embodiment of the present invention, the
neurological disease is selected from the group consisting of:
central and peripheral demyelinating diseases; central and
peripheral degenerative diseases; cerebral apoplexy (cerebral
infarction, cerebral hemorrhage, subarachnoid hemorrhage); brain
tumor; dysfunction of higher function of the brain (the term
"higher function of the brain" involves the cognitive function,
short and long memory, speech, etc.); psychiatric diseases;
dementia; infectious diseases; epilepsy; traumatic neurological
diseases; and infarction of spinal cord diseases.
[0019] Furthermore, the present invention provides therapeutic
methods for neurological diseases, which comprises transplanting of
the above-mentioned mononuclear cell fractions, cells, or
compositions. In preferred embodiments, the donor cells are derived
from a recipient.
[0020] The present invention provides mononuclear cell fractions
isolated from bone marrow cells, cord blood cells, or embryonic
hepatic tissues, wherein the fractions contain cells capable of
differentiating into neural cells. It is unclear whether the
differentiation of cells contained in the cell fractions provided
by the present invention into neural cells is caused by the
transformation of so-called hematopoietic cells into neural cells,
or, alternatively, by the differentiation of immature cells capable
of differentiating into neural cells that are comprised in bone
marrow cells, etc. However, the majority of the cells
differentiating into neural cells are assumed to be stem or
precursor cells, namely, cells having the self-renewal ability and
pluripotency. Alternatively, the cells differentiating into neural
cells may be stem or precursor cells which have differentiated to
some extent into endoderm or mesoderm.
[0021] Cells in a cell fraction of the present invention do not
have to be proliferated with any trophic factors (then again they
can proliferate in the presence of trophic factors). Thus, these
cells are simple and practical from the standpoint of the
development of autotransplantation technique for the diseases in
the neural, and are very beneficial in medical industry. In
general, a cell fraction of the present invention is derived from
vertebrate, preferably from mammal (for example, mouse, rat, human,
etc.).
[0022] A cell fraction of the present invention can be prepared by
subjecting bone marrow cells or cord blood cells collected from
vertebrate to density-gradient centrifugation at 2,000 rpm in a
solution for a sufficient time ensuring separation depending on
specific gravity, and then recovering the cell fraction with a
certain specific gravity within the range of 1.07 to 1.1 g/ml.
Herein, the phrase "a sufficient time ensuring separation depending
on specific gravity" refers to a time sufficient for the cells to
shift to a position in the solution according to their specific
gravity, which is typically about 10 to 30 minutes. The specific
gravity of the cell fraction to be recovered is within the range of
1.07 to 1.08 g/ml (for example, 1.077 g/ml). Solutions, such as
Ficoll solution and Percoll solution, can be used for the
density-gradient centrifugation, but is not limited thereto.
[0023] Specifically, first, bone marrow (5 to 10 .mu.l) collected
from a vertebrate is combined with a solution (2 ml L-15 plus 3 ml
Ficoll), and then centrifuged at 2,000 rpm for 15 minutes to
isolate a mononuclear cell fraction (approx. 1 ml). The mononuclear
cell fraction is combined with culture solution (2 ml NPBM) to wash
the cells, and then the cells are again centrifuged at 2,000 rpm
for 15 minutes. Then, the precipitated cells are recovered after
the removal of the supernatant. Besides femur, sources to obtain a
cell fraction of the present invention include sternum, and ilium
constituting the pelvis. Any other bone can serve as a source so
long as it is large enough. A cell fraction of the present
invention can also be prepared from bone marrows and cord blood
stored in bone marrow bank or cord blood bank.
[0024] Another embodiment of cell fractions of the present
invention includes a mononuclear cell fraction isolated and
purified from bone marrow cells or cord blood cells, which contains
mesodermal (mesenchymal) stem cells capable of differentiating into
neural cells. The term "mesodermal (mesenchymal) stem cell" refers
to cells that can copy (divide and proliferate) cells with the same
potential as themselves and that are capable of differentiating
into any type of cells constituting mesodermal (mesenchymal)
tissues. Mesodermal (mesenchymal) cells indicate cells constituting
tissues that are embryologically categorized to the mesoderms,
including blood cells. The mesodermal (mesenchymal) stem cell
includes, for example, cells characterized by SH2(+), SH3(+),
SH4(+), CD29(+), CD44(+), CD14(-), CD34(-), and CD45(-). A cell
fraction containing mesodermal (mesenchymal) stem cells can be
obtained, for example, by selecting cells having a cell surface
marker, such as SH2, as described above from the above-mentioned
cell fraction obtained by centrifuging bone marrow cells or cord
blood cells (the cell fraction according to claim 2).
[0025] Furthermore, a cell fraction containing mesodermal
(mesenchymal) stem cells capable of differentiating into neural
cells can be prepared by subjecting bone marrow cells or cord blood
cells collected from vertebrate to density-gradient centrifugation
at 900 G in a solution for a sufficient time ensuring separation
depending on specific gravity, and then recovering the cell
fraction with a certain specific gravity within the range of 1.07
to 1.1 g/ml. Herein, "a sufficient time ensuring separation
depending on specific gravity" refers to a time sufficient for the
cells to shift to a specific position corresponding to their
specific gravity in the solution for density-gradient
centrifugation, which is typically about 10 to 30 minutes. The
specific gravity of a cell fraction to be recovered varies
depending on the type of animal (for example, human, rat, and
mouse) from which the cells have been derived. A solution to be
used for density-gradient centrifugation includes Ficoll solution
and Percoll solution, but is not limited thereto.
[0026] Specifically, first, bone marrow (25 ml) or cord blood
collected from vertebrate is combined with an equal volume of PBS
solution, and then centrifuge at 900 G for 10 minutes. Precipitated
cells are mixed with PBS and then are recovered (cell
density=approx. 4.times.10.sup.7 cells/ml) to remove blood
components. Then, a 5-ml aliquot thereof is combined with Percoll
solution (1.073 g/ml), and centrifuged at 900 G for 30 minutes to
extract a mononuclear cell fraction. The extracted mononuclear cell
fraction is combined with a culture solution (DMEM, 10% FBS, 1%
antibiotic-antimycotic solution) to wash the cells, and is
centrifuged at 2,000 rpm for 15 minutes. Finally, the supernatant
is removed, precipitated cells are recovered and cultured at
37.degree. C. under 5% CO.sup.2 atmosphere.
[0027] Another embodiment of a cell fraction of the present
invention is a fraction of mononuclear cells isolated from bone
marrow cells or cord blood cells, which contains stromal cells
capable of differentiating into neural cells. Examples of stromal
cell include cells characterized by Lin(-), Sca-1(+), CD10(+),
CD11D(+), CD44(+), CD45(+), CD71(+), CD90(+), CD105(+), CDW123(+),
CD127(+), CD164(+), fibronectin (+), ALPH(+), and collagenase-1(+).
A cell fraction containing stromal cells can be prepared, for
example, by selecting cells having a cell surface marker, such as
Lin as described above, from the above-mentioned cell fraction
obtained by centrifuging bone marrow cells or cord blood cells (the
cell fraction according to claim 2).
[0028] Furthermore, such a cell fraction can be prepared by
subjecting bone marrow cells or cord blood cells collected from
vertebrate to density-gradient centrifugation at 800 G in a
solution for a sufficient time ensuring separation depending on
specific gravity, and then recovering the cell fraction with a
certain specific gravity within the range of 1.07 to 1.1 g/ml.
Herein, "a sufficient time ensuring separation depending on the
specific gravity" indicates a time sufficient for the cells to
shift to a specific position corresponding to their specific
gravity in the solution for density-gradient centrifugation, which
is typically about 10 to 30 minutes. The specific gravity of a cell
fraction to be recovered is preferably within the range of 1.07 to
1.08 g/ml (for example, 1.077 g/ml). A solution to be used for
density-gradient centrifugation includes Ficoll solution and
Percoll solution, but is not limited thereto.
[0029] Specifically, first, bone marrow or cord blood collected
from vertebrate is combined with an equal volume of a solution
(PBS, 2% BSA, 0.6% sodium citrate, and 1% penicillin-streptomycin).
A 5-ml aliquot thereof is combined with Ficoll+Paque solution
(1.077 g/ml) and centrifuged at 800 G for 20 minutes to obtain a
mononuclear cell fraction. The mononuclear cell fraction is
combined with a culture solution (Alfa MEM, 12.5% FBS, 12.5% horse
serum, 0.2% i-inositol, 20 mM folic acid, 0.1 mM 2-mercaptoethanol,
2 mM L-glutamine, 1 .mu.M hydrocortisone, 1% antibiotic-antimycotic
solution) to wash the cells, and then are centrifuged at 2,000 rpm
for 15 minutes. The supernatant is removed after centrifugation.
The precipitated cells are collected and then cultured at
37.degree. C. under 5% CO.sup.2 atmosphere.
[0030] Another embodiment of a cell fraction of the present
invention is a mononuclear cell fraction containing cells
characterized by AC133(+) capable of differentiating into neural
cells, which is isolated from bone marrow cells, cord blood cells,
or embryonic hepatic tissues. Such a cell fraction can be obtained,
for example, by selecting cells having a cell surface marker
including the above-mentioned AC133(+) from the cell fraction
obtained, as described above, by centrifuging bone marrow cells or
cord blood cells (the cell fraction according to claim 2).
[0031] Furthermore, the cell fraction can be obtained by subjecting
embryonic hepatic tissues collected from vertebrate to
density-gradient centrifugation at 2,000 rpm in a solution for a
sufficient time ensuring separation depending on specific gravity,
recovering a cell fraction within the range of a specific gravity
of 1.07 to 1.1 g/ml, and then recovering cells with the
characteristic of AC133(+) from the cell fraction. Herein, "a
sufficient time ensuring separation depending on specific gravity"
indicates a time sufficient for the cells to shift to a specific
position corresponding to their specific gravity in the solution
for density-gradient centrifugation, which is typically about 10 to
30 minutes. The solution to be used for density-gradient
centrifugation includes Ficoll solution and Percoll solution, but
is not limited thereto.
[0032] Specifically, first, liver tissue collected from vertebrate
is washed in L-15 solution, and then treated enzymatically in an
L-15 solution containing 0.01% DNaseI, 0.25% trypsin, and 0.1%
collagenase at 37.degree. C. for 30 minutes. Then, the tissue is
dispersed into single cells by pipetting. The single-dispersed
embryonic hepatic cells are centrifuged by the same procedure as
described for the preparation of the mononuclear cell fraction from
femur in Example 1(1). The cells thus obtained are washed, and then
AC133(+) cells are collected from the washed cells using an AC133
antibody. Thus, cells capable of differentiating into neural cells
can be prepared from embryonic hepatic tissues. The antibody-based
recovery of AC133(+) cells can be achieved using magnetic beads or
a cell sorter (FACS, etc.).
[0033] Transplantation of any of these cell fractions containing
mesodermal stem cells, mesenchymal stem cells, stromal cells, or
AC133-positive cells into demyelinated spinal cord can lead to
efficient remyelination of the demyelinated region. In particular,
the above-mentioned cell fraction containing mesenchymal stem cells
can engraft favorably and differentiate into neural cells such as
neurons or glia when transplanted into a stroke model or a cerebral
infarction model.
[0034] The present invention also provides cells capable of
differentiating into neural cells, which are contained in the
above-mentioned cell fraction. These cells include, for example,
neural stem cells, mesodermal stem cells, mesenchymal stem cells,
stromal cells, and AC133-positive cells which are contained in the
above-mentioned cell fraction, but are not limited thereto so long
as they can differentiate into neural cells.
[0035] The present invention also provides compositions for
treating neurological diseases, which comprise a cell fraction or
cells of the present invention. It is possible to transplant the
cell fractions or cells of the present invention without
modification. However, in order to improve the efficiency of
therapy, they may be transplanted as compositions combined with
various additives or introduced with genes. The preparation of
compositions of the present invention may comprise, for example,
(1) addition of a substance that improves the proliferation rate of
cells included in a cell fraction of the present invention or
enhances the differentiation of the cells into neural cells, or
introducing a gene having the same effect; (2) addition of a
substance that improves the viability of cells in a cell fraction
of the present invention in damaged neural tissues, or introducing
a gene having the same effect; (3) addition of a substance that
inhibits adverse effects of damaged neural tissue on the cells in a
cell fraction of the present invention, or introducing a gene
having the same effect; (4) addition of a substance that prolongs
the lifetime of donor cells, or introducing a gene having the same
effect; (5) addition of a substance that modulates the cell cycle,
or introducing a gene having the same effect; (6) addition of a
substance to suppress the immunoreaction or inflammation, or
introducing a gene having the same effect; (7) addition of a
substance that enhances the energy metabolism, or introducing a
gene having the same effect; (8) addition of a substance that
improves the migration of donor cells in host tissues, or
introducing a gene having the same effect; (9) addition of a
substance that improves blood flow (including inductions of
angiogenesis), or introducing a gene having the same effect; (10)
addition of a substance that cure the infectious diseases, or
introducing a gene having the same effect, or (11) addition of a
substance that cure the tumors, or introducing a gene having the
same effect, but is not limited thereto.
[0036] It is considered that the cells according to the present
invention are immobilized in the bone marrow by a distinct
mechanism involving a certain type of substance (proteins, etc.)
and do not normally move out into the peripheral blood. Therefore,
to make these cells enter the peripheral blood circulation,
conventionally, they are removed from the bone marrow, and then
administered intravenously. However, the studies conducted by the
present inventors gradually elucidated the mechanism of
immobilization of these cells in the bone marrow. The discovery
made by the present inventors showed that these cells, which had
been localized in the bone marrow, could be made to move out into
the peripheral blood by intravenous injection of active factors,
such as an antibody, a cytokine, chemicals, or a growth factor.
That is, therapeutic effect of bone marrow transplantation that is
similar to that of the aforementioned method can be expected from
intravenous injection of an active factor, such as an antibody, a
cytokine, chemicals, or a growth factor.
[0037] A cell fraction, cell, and composition of the present
invention can be used for treating neurological diseases. Target
neurological diseases for the therapy include, for example, central
and peripheral demyelinating diseases; central and peripheral
degenerative diseases; cerebral apoplexy (including cerebral
infarction, cerebral hemorrhage, and subarachnoid hemorrhage);
cerebral tumor; disorders of higher brain function including
dementia; psychiatric diseases; epilepsy, traumatic neurological
diseases (including head injury, cerebral contusion, and spinal
cord injury); infectious diseases; and infarction of spinal cord,
but are not limited thereto.
[0038] According to the present invention, cells derived from bone
marrow cells of a recipient can be transplanted as donor cells
(autotransplantation therapy). This has the following advantages:
(1) low risk of rejection for the transplantation; and (2) no
difficulty in using immunosuppressant. When autotransplantation
therapy is arduous, then cells derived from other person or
nonhuman animal may be used. Cells frozen for storage are also
usable. The donor cells may be derived from cord blood.
[0039] Bone marrow can be collected, for example, by anesthetizing
(by local or systemic anesthesia) an animal (including human) that
serves as a source, put a needle into the sternum or iliac of the
animal, and aspirating the bone marrow with a syringe. On the other
hand, it is an established technique to collect cord blood at birth
by putting needle directly into the umbilical cord, and aspirating
the blood from the umbilical cord using syringe, and to store the
blood.
[0040] Transplantation of cells into a patient can be performed,
for example, by first filling a syringe with cells to be
transplanted. Herein, the cells are suspended in an artificial
cerebrospinal fluid or physiological saline. Then, the damaged
neural tissue is exposed by surgery, and, with a needle, directly
injecting the cells into the lesion. Due to high migrating
potential of cells contained in a cell fraction of the present
invention, they can migrate in the neural tissues. Hence, cells can
be transplanted into a region adjacent to the lesion. Moreover,
injection of the cells into the cerebrospinal fluid is also
expected to be efficacious. In the case of the injection of the
cells into the cerebrospinal fluid, the cells can be injected into
a patient by typical lumbar puncture, without surgical operation
only under local anesthetization. Thus, the patient can be treated
in patient's sickroom (not in an operation room), which makes the
method preferable. Alternatively, intravenous injection (including
any systemic transplantations such as intravenous, intraarterial,
selective intraarterial administration) of the cells is also
expected to be effective. Thus, transplantation can be carried out
by a procedure based on typical blood transfusion, which is
advantageous in that the treatment can be performed in patient's
sickroom.
[0041] Furthermore, due to their high migrating potential, cells in
a cell fraction of the present invention can be used as a carrier
(vector) for genes. For example, the cells are expected to be
useful as a vector for gene therapy for various neurological
diseases such as brain tumor.
BRIEF DESCRIPTION OF THE DRAWINGS
[0042] FIG. 1 shows optical light micrographs of transections of
dorsal column of the spinal cord, which had been prepared at 1-mm
intervals. (A), normal axon; and (C), damaged demyelinated axon.
Patterns of dorsal column observed at higher magnification are
shown in (B) for normal axon and in (D) for demyelinated axon.
Scale bars: 250 .mu.m in (A) and (C); 10 .mu.m in (B) and (D).
[0043] FIG. 2 shows microphotographs demonstrating the
remyelination of rat spinal cord (A), after transplantation of
adult mouse bone marrow cells; and (C), after transplantation of
Schwann cells. Photomicrographs demonstrating remyelinated axon
observed at higher magnification are shown in (B), after
transplantation of bone marrow cells; and (D), after
transplantation of Schwann cells. Scale bars: 250 .mu.m in (A) and
(C); 10 .mu.m in (B) and (D).
[0044] FIG. 3 shows an electron micrograph of remyelinated axon
after the transplantation of bone marrow into the dorsal columns.
The tissue was treated with substrate X-Gal to detect transplanted
bone marrow cells containing the .beta.-galactosidase gene in the
damaged tissues (the reaction product is indicated with arrow).
When observed at higher magnification, basal lamina was found
around the fibers (wedge-shaped mark; scale bar 1 .mu.m). The
presence of large cytoplasmic and nuclear regions and basal lamina
in the transplanted cells indicates peripheral myelination.
BEST MODE FOR CARRYING OUT THE INVENTION
[0045] The present invention will be described in more detail below
with reference to examples based on specific experiments.
Example 1
Preparation of Bone Marrow Cells and Schwann Cells
[0046] (1) Bone Marrow Mononuclear Cells
[0047] Mouse bone marrow cells (10 .mu.l) were obtained from the
femur of adult LacZ (a structural gene of .beta.-galactosidase)
transgenic mice (The Jackson Laboratory, Maine, USA). The collected
sample was diluted in L-15 medium (2 ml) containing 3 ml Ficoll,
and centrifuged at 2,000 rpm for 15 minutes. Cells were collected
from the mononuclear cell fraction, and suspended in 2 ml
serum-free medium (NPMM: Neural Progenitor cell Maintenance
Medium). Following centrifugation (2,000 rpm, 15 min), the
supernatant was removed, and precipitated cells were collected and
re-suspended in NPMM.
[0048] (2) Schwann Cells
[0049] Primary Schwann cell cultures were established from the
sciatic nerve of neonatal mouse (P1-3) according to the method of
Honmou et al. (J. Neurosci., 16(10): 3199-3208, 1996).
Specifically, cells were released from sciatic nerve by enzymatic
and mechanical treatment. 8.times.10.sup.5 cells per plate were
plated onto 100-mm.sup.2 poly (L-lysine)-coated tissue culture
plates and the cells were cultured in Dulbecco's modified Eagle's
medium (DMEM) supplemented with 10% (vol/vol) fetal calf serum.
Example 2
Experimental Animal Preparation and Transplantation
[0050] (1) Preparation of Demyelinated Rat Model
[0051] Experiments were performed on 12 week old Wistar rats. A
localized demyelinated lesion was created in the dorsal columns
using X-ray irradiation and ethidium bromide injection (EB-X
treatment). Specifically, rats were anesthetized with ketamine (75
mg/kg) and xylazine (10 mg/kg) i.p., and a surface dose of 40 Grays
of X-ray was irradiated using Softex M-150 WZ radiotherapy machine
(100 kV, 1.15 mA, SSD 20 cm, dose rate 200 cGy/min) on the spinal
cord caudal to the tenth thoracic spine level (T-10) through a
2.times.4 cm opening in a lead shield (4 mm thick). Three days
after X-ray irradiation, rats were anesthetized as above, and
aseptic laminectomy of the eleventh thoracic spine (T-11) was
conducted. A demyelinating lesion was generated by the direct
injection of ethidium bromide (EB) into the dorsal columns via a
glass micropipette whose end was drawn. 0.5 saline containing 0.3
mg/ml EB was injected at the depths of 0.7 and 0.4 mm.
[0052] (2) Transplantation of Stem or Progenitor Cells that can
Differentiate or Transform into the Neural Lineages
[0053] 3 days after the EB injection, 1 .mu.l of the cell
suspension (1.times.10.sup.4 cells/.mu.l), which was obtained in
Example 1, was injected into the middle of the EB-X-induced lesion.
Transplanted rats were immunosuppressed with cyclosporin A (10
mg/kg/day).
Example 3
Histological Examination
[0054] Rats were deeply anesthetized by the administration of
sodium pentobarbital (60 mg/kg, i.p.), and perfused through the
heart cannula, first, with phosphate-buffer solution (PBS) and then
with a fixative containing 2% glutaraldehyde and 2%
paraformaldehyde in 0.14 M Sorensen's phosphate buffer, pH 7.4.
Following in situ fixation for 10 minutes, the spinal cord was
carefully excised, cut into 1 mm segments and kept in fresh
fixative. The tissue was washed several times in Sorensen's
phosphate buffer, post-fixed in 1% OsO.sub.4 for 2 hours at
25.degree. C., dehydrated by elevating the concentration of the
ethanol solution, passed through propylene oxide and embedded in
EPON. Then, the tissue was cut into sections (1 .mu.m),
counterstained with 0.5% methylene blue and 0.5% azure II in 0.5%
borax, and examined under light microscope (Zeiss: Axioskop FS).
Ultrathin sections were counterstained with uranyl and lead salts,
and examined with JEOL JEM1200EX electron microscope (JEOL, Ltd.,
Japan) at 60 kV.
[0055] A 50.times.50 .mu.m standardized region in the central core
of the dorsal columns in the spinal cords near the site wherein the
cells were initially injected was used for morphometric analysis.
The numbers of remyelinated axons and cell bodies associated with
the axons were counted within this region; and the density to
square millimeters was calculated. Furthermore, the diameters of
the axons and cell bodies, the number of cells with multi-lobular
nuclei, and cells showing myelination were examined in the same
standardized region. Measurements and counts were obtained from
five sections per rat, and five rats (n=5) were analyzed for each
experimental condition. All variances represent standard error
(.+-.SEM).
[0056] The dorsal column in the spinal cord mostly consists of
myelinated axons (FIG. 1A, B). The proliferation of endogenous
glial cells was inhibited by the irradiation of X-ray to the dorsal
columns of the lumbar spinal cord. Further, by the administration
of a nucleic acid chelator, ethidium bromide, glial cells such as
oligodendrocytes were found damaged and local demyelination
occurred. Such lesions generated according to this procedure are
characterized by almost complete loss of endogenous glial cells
(astrocytes and oligodendrocytes) and preservation of axons (FIG.
1C). Examination of the lesion with a light microscope under a
higher magnification revealed that congested areas consisting of
demyelinated axons are appositioned closely to one another
separated by areas wherein the debris of myelin exist and wherein
macrophages exist (FIG. 1D). The lesion occupied nearly the entire
dorso-ventral extent of the dorsal columns 5 to 7 mm
longitudinally. Almost none of the endogenous invasion of Schwann
cells or astrocytes occurs till the sixth to eighth week. With the
start of invasion, these glial cells begin to invade into the
lesion from peripheral borders. Thus, a demyelinated and glial-free
environment is present for at least 6 weeks in vivo.
[0057] Three weeks after transplantation of LacZ transgenic mouse
bone marrow cells (BM) into the central region of the lesion in
immunosuppressed and demyelinated rat models, extensive
remyelination of the demyelinated axons was observed (FIGS. 2A and
2B). Remyelination was observed across the entire coronal dimension
of the dorsal columns and throughout the antero-posterior extent of
the lesion. FIGS. 2C and 2D show the pattern of remyelination
observed after transplantation of allogeneic Schwann cells (SC)
into the EB-X lesion model. It is remarkable that large nuclear and
cytoplasmic regions were found around the remyelinated axons, a
characteristic of peripheral myelin, by both BM and SC
transplantations.
Example 4
Detection of .beta.-Galactosidase Reaction Products In Vivo
[0058] Three weeks after transplantation, .beta.-galactosidase
expressing myelin-forming cells were detected in vivo. Spinal cords
were collected and fixed in 0.5% glutaraldehyde in phosphate-buffer
for 1 h. Sections (100 .mu.m) were cut with a vibratome and
.beta.-galactosidase expressing myelin-forming cells were detected
by incubating the sections at 37.degree. C. overnight with X-Gal
(substrate which reacts with (.beta.-galactosidase to develop
color) at a final concentration of 1 mg/ml in X-Gal developer (35
mM K.sub.3Fe (CN).sub.6/35 mM K.sub.4Fe (CN).sub.63H.sub.2O/2 mM
MgCl.sub.2 in phosphate-buffered saline) to form blue color within
the cell. Sections were then fixed for an additional 3 h in 3.6%
(vol/vol) glutaraldehyde in phosphate-buffer (0.14 M), and were
examined with light microscope for the presence of blue reaction
product (.beta.-galactosidase reaction product). Prior to embedding
in EPON, the tissue was treated with 1% OsO.sub.4, dehydrated in a
graded series of ethanol, and soaked in propylene oxide for a short
period. Ultrathin sections were then examined under an electron
microscope without further treatment.
[0059] Under the electron microscope, most of the myelin-forming
cells derived from donor cells retained the basal membrane (FIG. 3:
wedge-shaped mark). Additionally, the myelin-forming cells had
relatively large nucleus and cytoplasm, which suggests the
formation of a peripheral nervous system-type myelin sheath.
[0060] It was confirmed that almost no endogenous remyelination by
oligodendrocytes or Schwann cells occurs for at least six weeks in
the lesion model used in the present experiment. Furthermore, the
donor cells that contained the reporter gene LacZ, i.e.,
X-Gal-positive cells, were observed to form myelin at the electron
microscopic level (FIG. 3: arrow).
[0061] Differentiation into neurons and glial cells could be
observed following the transplantation of bone marrow cells into
the EB-X lesions, but not by SC transplantation. Five percent of
lacZ-positive cells (transplanted bone marrow cells) in the EB-X
lesions showed NSE (Neuron Specific-Enolase)-immunoreactivity and
3.9% showed GFAP (Glial Fibrially Acidic Protein)-immunoreactivity,
indicating that some of the bone marrow cells can differentiate
into neurons or glial cells, respectively, in vivo.
[0062] Furthermore, employing antibodies, the present inventors
isolated mesenchymal stem cells with the characteristic of cell
markers SH2(+), SH3(+), CD29(+), CD44(+), CD14(-), CD34(-), and
CD45 (-) from the cell fraction obtained in Example 1 (1).
Furthermore, they discovered that transplantation of the cells into
the demyelinated regions of rat spinal cord results in more
efficient remyelination. It was also revealed that the cells
survived favorably and differentiated into neurons or neuronal
cells and glia cells when transplanted into cerebral infarction
model rats.
[0063] Further, the present inventors isolated stromal cells
characterized by the cell surface markers Lin (-), Sca-1(+),
CD10(+), CD11D(+), CD44(+), CD45(+), CD71(+), CD90(+), CD105(+),
CDW123(+), CD127(+), CD164(+), fibronectin (+), ALPH(+), and
collagenase-1(+) from the cell fraction obtained in Example 1 (1).
Transplantation of the cells into demyelinated regions of rat
spinal cord also resulted in efficient remyelination.
[0064] Further, the present inventors isolated cells characterized
by the cell surface marker AC133 (+) from the cell fraction
obtained in Example 1 (1). Transplantation of the cells into
demyelinated regions of rat spinal cord also resulted in efficient
remyelination.
[0065] In addition, the present inventors obtained a cell fraction
containing AC133-positive cells capable of differentiating into
neural cells from rat embryonic hepatic tissues by the following
procedure. Specifically, first, liver tissues collected from rat
fetuses were washed in L-15 solution, and then treated
enzymatically in L-15 solution containing 0.01% DNaseI, 0.25%
trypsin, and 0.1% collagenase at 37.degree. C. for 30 minutes.
Then, the tissue was dispersed into single cells by pipetting
several times. The single-dispersed embryonic hepatic tissues were
centrifuged as in Example 1 (1) (preparation of mononuclear cell
fraction from femur) to isolate a mononuclear cell fraction. The
obtained mononuclear cell fraction was washed, and then, AC133(+)
cells were recovered from the cell fraction using anti-AC133
antibody. The isolation of AC133-positive cells can be achieved
using magnetic beads or a cell sorter (FACS or the like).
Transplantation of the obtained AC133-positive cells into
demyelinated regions of rat spinal cord also resulted in efficient
remyelination.
INDUSTRIAL APPLICABILITY
[0066] As described above, the present invention provides fractions
of mononuclear cells isolated and purified by collecting bone
marrow-derived bone marrow cells, cord blood-derived cells, or
fetal liver-derived cells. Transplantation of such mononuclear cell
fractions into a demyelination model animal was confirmed to result
in remyelination of the demyelinated axon.
[0067] Cells for transplantation can be'relatively easily isolated
from a small quantity of bone marrow cell fluid aspirated from bone
marrow, and can be prepared for transplantation in several tens of
minutes after the cells are being collected. Thus, these cells can
serve as useful and regenerable cellular material for
autotransplantation in the treatment of demyelinating diseases.
[0068] This invention highlights the development of the
autotransplantation technique to treat demyelinating diseases in
the central nervous system. Furthermore, the use of the present
invention in transplantation and regeneration therapy for more
general and diffuse damage in the nervous system is envisaged. In
other words, this invention sheds light on autotransplantation
therapy against ischemic cerebral damage, traumatic cerebral
injury, cerebral degenerating diseases, and metabolic neurological
diseases in the central and peripheral nervous systems.
[0069] According to the present invention, cells in the
hematopoietic system (bone marrow or cord blood) are used as donor
cells. Thus, to treat neurological diseases, the cells may be
transplanted into the vessels instead of directly transplanting
them into neural tissues. Specifically, donor cells transplanted
into a vessel can migrate to the neural tissues and thereby
regenerate the neural tissues. Hence, the present invention is a
breakthrough in developing a therapeutic method for relatively
noninvasive transplantation.
[0070] Furthermore, the present invention adds significantly to
elucidate the mechanism underlying the differentiation of
non-neural cells such as hematopoietic cells and mesenchymal cells
into neural cells. When genes determining the differentiation are
identified and analyzed, use of such genes will allow efficient
transformation of a sufficient quantity of non-neural cells such as
hematopoietic cells and mesenchymal cells in a living body to
neural cells. Thus, the present invention is a breakthrough in the
field of "gene therapy" for inducing regeneration of neural
tissues.
* * * * *