U.S. patent application number 10/153972 was filed with the patent office on 2003-01-02 for directed in vitro differentiation of marrow stromal cells into neural cell progenitors.
Invention is credited to Deng, Weiwen, Prockop, Darwin J..
Application Number | 20030003090 10/153972 |
Document ID | / |
Family ID | 26851036 |
Filed Date | 2003-01-02 |
United States Patent
Application |
20030003090 |
Kind Code |
A1 |
Prockop, Darwin J. ; et
al. |
January 2, 2003 |
Directed in vitro differentiation of marrow stromal cells into
neural cell progenitors
Abstract
The invention relates to methods for inducing marrow stromal
cells to differentiate into neural cells by way of increasing
intracellular levels of cyclic AMP. The invention also encompasses
methods of producing a neural cell by causing a marrow stromal cell
to differentiate into a neural cell by increasing intracellular
levels of cyclic AMP. Methods for treating a human patient in need
of neural cells are also disclosed, as well as methods for treating
a human patient having a disease, condition, or disorder of the
central nervous system.
Inventors: |
Prockop, Darwin J.; (New
Orleans, LA) ; Deng, Weiwen; (Metairie, LA) |
Correspondence
Address: |
MORGAN, LEWIS & BOCKIUS LLP
1701 MARKET STREET
PHILADELPHIA
PA
19103-2921
US
|
Family ID: |
26851036 |
Appl. No.: |
10/153972 |
Filed: |
May 23, 2002 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
60294281 |
May 30, 2001 |
|
|
|
Current U.S.
Class: |
424/93.21 ;
435/368 |
Current CPC
Class: |
C12N 2506/1353 20130101;
C12N 5/0618 20130101; A61K 35/12 20130101; C12N 2501/01
20130101 |
Class at
Publication: |
424/93.21 ;
435/368 |
International
Class: |
A61K 048/00; C12N
005/08 |
Goverment Interests
[0002] The invention was made in part using funds obtained from the
U.S. Government (National Institutes of Health Grant Nos. AR47161
and AR42210) and the U.S. government may have certain rights in the
invention.
Claims
We claim:
1. A method of producing a neural cell, said method comprising
contacting an isolated marrow stromal cell with a cyclic
AMP-stimulating compound, thereby increasing the intracellular
level of cyclic AMP in said marrow stromal cell and producing a
neural cell.
2. The method of claim 1, wherein said cyclic AMP-stimulating
compound is selected from the group consisting of epinephrine,
isoproterenol, forskolin, isobutylmethylxanthine (IBMX), dibutyryl
cyclic AMP (dbcAMP), and a combination of IBMX and dbcAMP.
3. The method of claim 2, wherein said cyclic AMP-stimulating
compound is a combination of IBMX and dbcAMP.
4. The method of claim 3, wherein said IBMX is present in a
concentration of from about 0.01 millimolar to about 5.0
millimolar, and wherein said dbcAMP is present in a concentration
of from about 0.1 millimolar to about 10.0 millimolar.
5. The method of claim 4, wherein said IBMX is present at 0.5
millimolar and further wherein said dbcAMP is present at 1
millimolar.
6. The neural cell of claim 1, wherein said cell is human.
7. A method of treating a human patient having a disease, disorder,
or condition of the central nervous system, said method comprising
administering to a patient neural cells produced by the method of
claim 1, thereby treating the disease, disorder, or condition.
8. The method of claim 7, wherein said neural cells are transfected
with an isolated nucleic acid encoding a therapeutic protein,
wherein when said protein is expressed in said cells said protein
serves to effect treatment of said disease, disorder, or
condition.
9. The neural cell of claim 8, wherein said cell is human.
10. The method of claim 7, wherein said disease, disorder, or
condition of the central nervous system is selected from the group
consisting of Alzheimer's disease, Parkinson's disease,
Huntington's disease, amyotrophic lateral sclerosis, a tumor, a
trauma, elderly dementia, Tay-Sach's disease, Sandhoff s disease,
Hurler's syndrome, Krabbe's disease, birth-induced traumatic
central nervous system injury, epilepsy, multiple sclerosis,
trauma, tumor, stroke, and spinal cord injury.
11. A method of treating a human patient in need of neural cells,
said method comprising obtaining marrow stromal cells from a human
donor, producing neural cells by the method of claim 1, and
transplanting said neural cells into said human patient in need of
said neural cells, thereby treating said human patient in need of
neural cells.
12. A method of inducing differentiation of an isolated marrow
stromal cell into a neural cell, said method comprising contacting
said marrow stromal cell with a cyclic AMP stimulating
compound.
13. The method of claim 12, wherein said compound is selected from
the group consisting of epinephrine, isoproterenol, forskolin,
IBMX, dbcAMP, and a combination of IBMX and dbcAMP.
14. The method claim 13, wherein said compound is a combination of
IBMX and dbcAMP.
15. The method of claim 14, wherein said IBMX is present in a
concentration of from about 0.01 millimolar to about 5.0 millimolar
and wherein said dbcAMP is present in a concentration of from about
0.01 millimolar to about 10.0 millimolar.
16. The method of claim 15, wherein said IBMX is present at a
concentration of 0.5 millimolar and wherein said dbcAMP is present
at a concentration of 1 millimolar.
17. The neural cell of claim 9, wherein said cell is human.
18. A method of treating a human patient having a disease,
disorder, or condition of the central nervous system, said method
comprising administering to a patient neural cells produced by the
method of claim 12.
19. The method of claim 18, wherein said neural cells are
transfected with an isolated nucleic acid encoding a therapeutic
protein, wherein when said protein is expressed in said cells said
protein serves to effect treatment of said disease, disorder, or
condition.
20. The neural cell of claim 19, wherein said cell is human.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application claims priority under 35 U.S.C.
.sctn.119(e) to U.S. Provisional Application No. 60/294,281, filed
May 30, 2001.
BACKGROUND OF THE INVENTION
[0003] Human marrow stromal cells (hMSCs) are multipotential adult
stem cells that contribute to the regeneration of tissues such as
bone, cartilage, fat, and muscle (1997, Friedenstein, et al., Exp.
Hematol. 4(5):267-274; 1997, Prockop, D J, Science,
276(5309):71-74; 1999, Pittenger, et al., Science,
284(5411):143-147; 1998, Ferrari et al., Science,
279(5356):1528-1530).
[0004] The recent discovery of stem cell populations in the central
nervous system (CNS) has generated intense interest, since the
brain has long been regarded as incapable of regeneration (Reynolds
and Weiss, 1992, Science 255:1707-1710; Richards et al., 1992,
Proc. Natl. Acad. Sci. USA 89:8591-8595; Morshead et al., 1994,
Neuron 13:1071-1082). Neural stem cells (NSCs) are capable of
undergoing expansion and differentiating into neurons, astrocytes
and oligodendrocytes in vitro (Reynolds and Weiss, 1992, Science
255:1707-1710; Johansson et al., 1999, Cell 96:25-34; Gage et al.,
1995, Annu. Rev. Neurosci. 18:159-192; Vescovi et al., 1993, Neuron
11:951-966). NSCs back transplanted into the adult rodent brain
survive and differentiate into neurons and glia, raising the
possibility of therapeutic potential (Lundberg et al., 1997, Exp.
Neurol. 145:342-360; Lundberg et al., 1996, Brain Res. 737:295-300;
Renfranz et al., 1991, Cell 66:713-729; Flax et al., 1998, Nature
Biotech. 16:1033-1039; Gage et al., 1995, Proc. Natl. Acad. Sci.
USA 92:11879-11883; Svendsen et al., 1997, Exp. Neurol.
148:135-146). However, the inaccessibility of NSC sources deep in
the brain severely limits clinical utility. The recent report
demonstrating that NSCs can generate hematopoietic cells in vivo
suggests that hematopoietic stem cell populations may be less
restricted than previously thought (Bjornson, 1999, Science
283:534-537).
[0005] Recent data suggest that MSCs can also be induced to
differentiate into neural cells in vivo. It has been found that
hMSCs integrate and migrate along the known pathway for the
migration of neural stem cells after being infused into rat brain
(Azizi, et al., 1998, PNAS, 95(7):3908-3913). Other data
demonstrate that mouse MSCs (mMSCs) labeled with BrdU migrate to
both forebrain and cerebellum without disruption of normal brain
structure when injected into the lateral ventricle of a neonatal
mouse (Kopen, et al., 1999, PNAS, 96(19):10711-10716). Some of the
mMSCs differentiated into cells that had astrocyte morphology and
expressed the astrocyte-specific protein glial fibrillary acid
protein (GFAP). Further, some of the mMSCs appeared in the
olfactory bulb and the internal granular layer of the cerebellum,
both of which are neuron-rich regions. Finally, the Kopen study
also demonstrated that some BrdU-labeled mMSCs found in the
reticular formation of the brain stem were positive for
neurofilament.
[0006] Other investigations report conditions under which MSCs may
be differentiated in culture into neural-like cells. Woodbury et
al. demonstrate that cells may be differentiated either by serum
withdrawal and exposure to beta-mercaptoethanol (BME), or by
treatment of the MSCs with butylated hydroxytoluene (BHT) and
dimethylsulfoxide (DMSO) (Woodbury et al., 2000, J. Neurosci. Res.,
61(4):364-370). Others report that MSCs may be differentiated into
neural-like cells by treatment with epidermal growth factor (EGF)
followed by brain derived growth factor (BDGF), or by co-culture
with a suspension of rat or mouse midbrain cells (Sanchez-Ramos et
al., 2000, Exp. Neurol., 164(2):247-256).
[0007] However, until the present invention, a need has existed to
elucidate the early steps of neural differentiation so that, cells
at different early stages of differentiation may be identical and
used in therapy. The present invention fulfills this need.
SUMMARY OF THE INVENTION
[0008] The present invention encompasses a method of producing a
neural cell. The method comprises increasing the intracellular
level of cyclic AMP in an isolated marrow stromal cell, thereby
producing a neural cell. In a preferred aspect, the neural cell
produced by the method is human.
[0009] In another aspect, the method comprises increasing the
intracellular level of cyclic AMP by treatment of the marrow
stromal cell with isobutylmethylxanthine (IBMX) and dibutyryl
cyclic AMP (dbcAMP).
[0010] The invention also includes a method of treating a human
patient having a disease, disorder, or condition of the central
nervous system. The method comprises administering to a patient
neural cells produced by the method of increasing intracellular
levels of cyclic AMP in an isolated marrow stromal cell.
[0011] In a preferred embodiment, the neural cells used to practice
this method are transfected with an isolated nucleic acid encoding
a therapeutic protein, wherein when the protein is expressed in the
neural cells the protein serves to effect treatment of the disease,
disorder, or condition. Preferably, the transfected neural cell is
human.
[0012] In one aspect, the disease, disorder, or condition of the
central nervous system is selected from the group consisting of
Alzheimer's disease, Parkinson's disease, Huntington's disease,
amyotrophic lateral sclerosis, a tumor, a trauma, elderly dementia,
Tay-Sach's disease, Sandhoff's disease, Hurler's syndrome, Krabbe's
disease, birth-induced traumatic central nervous system injury,
epilepsy, multiple sclerosis, trauma, tumor, stroke, and spinal
cord injury.
[0013] The invention also encompasses a method of treating a human
patient in need of neural cells. The method comprises obtaining
marrow stromal cells from a human donor, producing neural cells by
the increasing intracellular levels of cyclic AMP in the marrow
stromal cells, and transplanting the neural cells into the human
patient in need of the neural cells, thereby treating the human
patient in need of neural cells.
[0014] A method of inducing differentiation of an isolated marrow
stromal cell into a neural cell comprising contacting the marrow
stromal cell with a compound which increases the intracellular
levels of cyclic AMP is also contemplated by the invention.
Preferably, the compound is a combination of IBMX and dbcAMP, and
more preferably, the neural cell so differentiated is human.
[0015] A method of treating a patient having a disease, disorder,
or condition of the central nervous system is also contemplated by
the invention. The method comprises administering to a patient
neural cells differentiated by the method of contacting an isolated
marrow stromal cell with a compound which increases the
intracellular levels of cyclic AMP.
[0016] In one aspect, the neural cells so differentiated are
transfected with an isolated nucleic acid encoding a therapeutic
protein, wherein when the protein is expressed in the neural cells
the protein serves to effect treatment of the disease, disorder, or
condition. Preferably, the differentiated neural cell is human.
[0017] In another aspect, the disease, disorder, or condition of
the central nervous system is selected from the group consisting of
Alzheimer's disease, Parkinson's disease, Huntington's disease,
amyotrophic lateral sclerosis, a tumor, a trauma, elderly dementia,
Tay-Sach's disease, Sandhoff's disease, Hurler's syndrome, Krabbe's
disease, birth-induced traumatic central nervous system injury,
epilepsy, multiple sclerosis, trauma, tumor, stroke, and spinal
cord injury.
BRIEF DESCRIPTION OF THE DRAWINGS
[0018] The foregoing summary, as well as the following detailed
description of the invention, will be better understood when read
in conjunction with the appended drawings. For the purpose of
illustrating the invention, there are shown in the drawings
embodiment(s) which are presently preferred. However, it should be
understood that the invention is not limited to the precise
arrangements and instrumentalities shown. In the drawings:
[0019] FIG. 1, comprising FIGS. 1A-1D, is a quartet of images
depicting induction of neural morphology in hMSCs. FIG. 1A is an
image depicting untreated hMSCs. FIGS. 1B, 1C, and 1D depict hMSCs
treated with 0.5 millimolar isobutylmethylxanthine (IBMX) and 1
millimolar dibutyryl cyclic AMP (dbcAMP) for 6 days. Arrows
represent differentiated neuron-like cells while undifferentiated
hMSCs are indicated by an arrow head. 200.times.Magnification.
[0020] FIG. 2 is a graph representing cellular proliferation and
neural differentiation in hMSCs treated with 0.5 millimolar IBMX
and 1 millimolar dbcAMP. The data points represent mean +/-
standard deviation of results of experiments performed in
triplicate. Cells were scored as hMSCs with neural morphology by
the presence of a refractile cell body, multipolar processes, and
growth-cone-like structures.
[0021] FIG. 3 is an image depicting a Western blot assay of
expression of vimentin, neuron-specific enolase (NSE), MAP1B, TuJ,
NF-M, MAP-2, tau, S-100, GFAP, MBP, and alpha-tubulin in treated
and untreated hMSCs. Lane C shows expression of the listed neural
markers in control, human brain extract. Lane 1 shows expression in
untreated hMSCs, and Lane 2 shows expression in hMSCs treated with
0.5 millimolar IBMX and 1 millimolar dbcAMP for 6 days.
DETAILED DESCRIPTION OF THE INVENTION
[0022] The invention herein described demonstrates another way to
differentiate MSCs to cells with many characteristics of early
neurons and glia. The method of differentiation involves increasing
levels of intracellular cyclic AMP by treating the MSCs with
dibutyryl cyclic AMP (dbcAMP) and isobutylmethylxanthine
(IBMX).
[0023] The invention comprises and utilizes the discovery that
increasing cyclic AMP levels in marrow stromal cells mediates
differentiation of the cells into neural cells expressing a variety
of early-expressed neuron-specific markers (e.g. neuron-specific
enolase (NSE), vimentin, MAP1B, and the like). The cells exhibit
other neuron-like phenotypic characteristics such as, but not
limited to, spherical and refractile cell bodies exhibiting typical
neural perikaryal appearance, cell bodies extending long processes
terminating in growth cones and filopodia typical of neurons. Thus,
the methods disclosed herein induce marrow stromal cell
differentiation into immature neural cells. Such methods are
crucial in the development of cell-based therapeutics for treatment
of central nervous system (CNS) disorders, diseases or conditions.
Indeed, prior to the present invention, the lack of clarity with
respect to neural differentiation has severely impeded the
development of CNS therapeutics.
[0024] The invention includes a method of inducing an isolated
marrow stromal cell to differentiate into an isolated neural cell.
Embodiments of the method of the invention are described in the
Examples section herein. Generally, bone marrow cells are isolated
from a donor, stromal cells are obtained therefrom, and the stromal
cells are subsequently cultured in vitro on culture plates using
standard cell culture techniques, e.g., as described in the
materials and methods section of the Examples. Preferably, the
donor is a human, however, the invention is intended to encompass a
mammalian donor and should not be limited to the specific donors
disclosed herein.
[0025] To induce the immature neural phenotype, the cells are
treated with an effective amount of a compound capable of
increasing cyclic AMP levels in marrow stromal cells. This compound
is introduced into the cell culture for a period of time. The
length of time may vary according to the precise method being
contemplated and should not be construed as limiting the invention
in any way. After treatment, the cells are prepared for Western
blot assay to determine expression patterns of various neural
markers. Neural morphology is evident within about two days, see
FIG. 1 for example, and the morphology becomes more evident
steadily over time.
[0026] In one embodiment of the invention, a combination of
isobutylmethylxanthine (IBMX) and dibutyryl cyclic AMP (dbcAMP) is
used to increase the levels of intracellular cyclic AMP. This
particularly preferred embodiment is more fully discussed in the
Examples section herein disclosed. However, the invention is not
limited to the compounds disclosed herein and should be construed
to include all compounds capable of increasing intracellular cyclic
AMP levels in a marrow stromal cell. Such compounds include, but
are not limited to epinephrine, isoproterenol, and forskolin.
[0027] Powder forms of IBMX and dbcAMP, prepared in water, may be
administered to isolated marrow stromal cells in culture in
combination in a range of 0.01 millimolar to 5.0 millimolar IBMX
and 0.1 millimolar to 10.0 millimolar dbcAMP. Preferably, the range
is 0.2 millimolar to 1.0 millimolar IBMX and 0.2 millimolar to 2.0
millimolar dbcAMP. Most preferably, a combination of IBMX at a
concentration of 0.5 millimolar and dbcAMP at a concentration of 1
millimolar is administered to the isolated marrow stromal cells in
culture. The IBMX/dbcAMP solution is preferably prepared in water.
It is also preferred that IBMX and dbcAMP be used as a combined
solution.
[0028] Administration of a cAMP-stimulating compound is preferably
delivered to from about thirty cells to about one million cells per
fifty-eight square centimeter culture dish. More preferably, the
range of cells is from about ten thousand to about one million
cells per dish, and most preferable, the cAMP-stimulating compound
is administered to about one million cells per fifty-eight square
centimeter dish.
[0029] Treatment of the cells with a cAMP-stimulating compound can
last from 0.5 to 60 days. Preferably, the treatment will extend
from 5 to 10 days, and more preferably, the cells are treated for 6
days. The number of days of treatment of the marrow stromal cells
with a cAMP-stimulating compound is dependent upon the development
of neural morphology.
[0030] Neural identity can be confirmed by Western blot assay for
detection of early neural markers. Examples of such early markers
are neuron-specific enolase (NSE), MAP1B, and TuJ. Progressive
differentiation of the marrow stromal cell to the neural cell
corresponds with an increase in each of these markers, indicating
that neural cells are produced. Other procedures may also be
employed to determine neural identity.
[0031] It is apparent from the data disclosed herein that it is
possible to differentiate isolated marrow stromal cell into neural
cells in vitro. Neural cells so differentiated are useful in
treating patients afflicted with any of a wide variety of central
nervous system diseases, disorders, or conditions.
[0032] The invention also includes a method for producing an
isolated neural cell from isolated marrow stromal cells. The method
comprises differentiating an isolated marrow stromal cell in the
same general manner as recited above, thereby producing an isolated
neural cell.
[0033] The isolated neural cell recited in both of the methods
above may be transfected with an isolated nucleic acid encoding a
therapeutic protein. The therapeutic protein, when expressed, will
treat a patient having a disease, disorder, or condition of the
central nervous system.
[0034] A wide plethora of therapeutic proteins are well known in
the art and are set forth in, for example, WO 96/30031 and WO
99/43286. Such examples include, but are not limited to, cytokines,
chemokines, neurotrophins, other trophic proteins, growth factors,
antibodies, and glioma toxic protein. When the transfected neural
cells encoding such proteins are administered to a patient, the
neural cells will therapeutically influence cells, which are
already present in the central nervous system. For example,
transfected neural cells which are introduced into the central
nervous system may be used to repair any central nervous system
damage, and/or to combat tumors of the central nervous system.
[0035] International patent applications WO 96/30031 and WO
99/43286 also describe use of MSCs in therapies for a wide variety
of CNS diseases, disorders, or conditions, which include, but are
not limited to, genetic diseases of the CNS (e.g., Tay-Sach's,
Sandhoff's disease, Hurler's syndrome, Krabbe's disease),
birth-induced traumatic CNS injury, adult CNS diseases, disorders
or conditions (e.g., Parkinson's, Alzheimer's, and Huntington's
diseases, elderly dementia, epilepsy, amyotropic lateral sclerosis,
multiple sclerosis, trauma, tumors, stroke, and the like) and
degenerative diseases and traumatic injury of the spinal cord.
[0036] Among neonates and children, transfected neural cells may be
used for treatment of a number of genetic diseases of the central
nervous system, including, but not limited to, Tay-Sachs disease
and the related Sandhoff's disease, Hurler's syndrome and related
mucopolysaccharidoses and Krabbe's disease. To varying extents,
these diseases also produce lesions in the spinal cord and
peripheral nerves and they also have non-neurological effects.
While the non-neurological effects of these diseases may be
treatable by bone marrow transplantation, the central nervous
system effects do not improve despite bone marrow transplantation.
The method of the present invention is useful to address the
central nervous system effects of these types of diseases. In
addition, in neonates and children, head trauma during birth or
following birth is treatable by introducing these neural cells
directly into the central nervous system of the children. Central
nervous system tumor formation in children is also treatable using
the methods of the present invention.
[0037] Adult diseases of the central nervous system are also
treatable by administering isolated neural cells to the adult. Such
adult diseases include but are not limited to, Parkinson's disease,
Alzheimer's disease, spinal cord injury, stroke, trauma, tumors,
degenerative diseases of the spinal cord such as amyotropic lateral
sclerosis, Huntington's disease and epilepsy. Treatment of multiple
sclerosis is also contemplated.
[0038] Treatment of spinal cord injuries is also possible using the
method of the present invention. Prior art methods of treating
spinal cord injuries involve using fibroblast cells to deliver
neurotrophins to the site of spinal cord lesions in animals. The
neurotrophins, delivered in this manner, reduce the lesion or
otherwise treat the injury. However, fibroblasts produce large
amounts of collagen, causing fibrosis at the site of the lesion,
thus negating the therapeutic effects of the treatment. Delivery of
neurotrophins to spinal cord lesions using transfected neural cells
is advantageous over prior art methods because neural cells do not
produce large amounts of collagen and therefore should not cause
fibrosis.
[0039] The isolated neural cell recited in both of the methods
above may also be transfected with an isolated nucleic acid
encoding a regulatory protein. The regulatory protein, when
expressed, will regulate the expression of a protein involved in a
disease, disorder, or condition of the central nervous system,
thereby controlling the disease state. The regulatory protein may
be, for example, neural growth factor, brain derived growth factor,
epidermal growth factor, fibroblast growth factor, glial derived
growth factor, and stem cell factor.
[0040] The invention further includes a method of treating a human
patient having a disease, disorder, or condition of the central
nervous system by administering the differentiated neural cells of
the invention to the central nervous system of the patient. Methods
of treating a human patient using MSCs are described in WO 96/30031
and WO 99/43286, which are incorporated by reference as if set
forth in their entirety herein. Methods of administering
differentiated neural cells to a patient are identical to those
used for MSCs as described in WO 96/30031 and WO 99/43286. The
methods encompass introduction of an isolated nucleic acid encoding
a therapeutic protein into differentiated neural cells and also
encompassing differentiated neural cells themselves in cell-based
therapeutics where a patient is in need of the administration of
such cells. The differentiated neural cells are preferably
administered to a human, and further, the neural cells are
preferably administered to the central nervous system of the human.
In some instances, the differentiated neural cells are administered
to the corpus striatum portion of the human brain. The precise site
of administration of the neural cells will depend on any number of
factors, including but not limited to, the site of the lesion to be
treated, the type of disease being treated, the age of the human
and the severity of the disease, and the like. Determination of the
site of administration is well within the skill of the artisan
versed in the administration of cells to mammals.
[0041] The mode of administration of the differentiated neural
cells to the central nervous system of the human may vary depending
on several factors including but not limited to, the type of
disease being treated, the age of the human, whether the neural
cells have isolated DNA introduced therein, and the like.
Generally, cells are introduced into the brain of a mammal by first
creating a hole in the cranium through which the cells are passed
into the brain tissue. Cells may be introduced by direct injection,
by using a shunt, or by any other means used in the art for the
introduction of compounds into the central nervous system.
Intravenous administration may also be used to introduce the cells
into a patient.
[0042] Definitions
[0043] The articles "a" and "an" are used herein to refer to one or
to more than one (i.e. to at least one) of the grammatical object
of the article. By way of example, "an element" means one element
or more than one element.
[0044] As used herein, "central nervous system" should be construed
to include brain and/or the spinal cord of a mammal. The term may
also include the eye and optic nerve in some instances.
[0045] As used herein, "stromal cells", "isolated marrow stromal
cells", and "MSCs" are used interchangeably and are meant to refer
to the small fraction of cells in bone marrow which can serve as
stem cell-like precursors of osteocytes, chondrocytes, monocytes,
and adipocytes and which are isolated from bone marrow by their
ability adhere to plastic dishes. Marrow stromal cells may be
derived from any animal. In some embodiments, stromal cells are
derived from primates, preferably humans.
[0046] As used herein, the term "therapeutic protein" is meant to
refer to a protein which can compensate for the protein encoded by
a defective gene and/or insufficient gene expression that is
causally linked to the disease or symptoms of the disease, disorder
or condition characterized by a gene defect. The presence of the
protein alleviates, reduces, prevents, or causes to be alleviated,
reduced or prevented, the causes and/or symptoms that characterize
the disease, disorder, or condition. A therapeutic protein is also
meant to refer to a protein which down-regulates a hyperexpressed
gene, upregulates a hypoexpressed gene, speeds a repair process,
increases replication or differentiation of exogenous or endogenous
stem cells, or causes synthesis of a compound or compounds that
improve the function or survival of neural cells.
[0047] As used herein, a "disease, disorder or condition" which can
be treated with a therapeutic protein is meant to refer to a
disease, disorder or condition that can be treated or prevented by
the presence of a protein which alleviates, reduces, prevents or
causes to be alleviated, reduced or prevented, the causes and/or
symptoms that characterize the disease, disorder or condition.
Diseases, disorders and conditions which can be treated with a
therapeutic protein include diseases, disorders and conditions
characterized by a gene defect as well as those which are not
characterized by a gene defect but which nonetheless can be treated
or prevented by the presence of a protein which alleviates,
reduces, prevents or causes to be alleviated, reduced or prevented,
the causes and/or symptoms that characterize the disease, disorder
or condition.
[0048] The term "isolated nucleic acid" should be construed to
refer to a nucleic acid sequence, or segment, or fragment which has
been purified from the sequences which flank it in a naturally
occurring state, e.g., a DNA fragment which has been removed from
the sequences which are normally adjacent to the fragment e.g., the
sequences adjacent to the fragment in a genome in which it
naturally occurs. The term also applies to nucleic acids which have
been substantially purified from other components which naturally
accompany the nucleic acid, e.g., RNA or DNA or proteins which
naturally accompany it in the cell.
[0049] As used herein, "transfected cells" is meant to refer to
cells to which a gene construct has been provided using any
technology used to introduce nucleic acid molecules into cells such
as, but not limited to, classical transfection (calcium phosphate
or DEAE dextran mediated transfection), electroporation,
microinjection, liposome-mediated transfer, chemical-mediated
transfer, ligand mediated transfer or recombinant viral vector
transfer.
[0050] The term "differentiation" as used herein, should be
construed to mean the induction of a differentiated phenotype in an
undifferentiated cell by coculturing the undifferentiated cell in
the presence of a substantially homogeneous population of
differentiated cells, in the presence of products of differentiated
cells or in the presence of an inducer of cell differentiation.
[0051] The term "neural cell" as used herein should be construed to
mean an MSC differentiated such that it expresses at least one of
the following neural markers: neuron-specific enolase (NSE), TuJ,
vimentin, and MAP1B.
[0052] The term "neuron" as used herein should be construed to mean
a nerve cell capable of receiving and conducting electrical
impulses from the central nervous system. A nerve cell or "neuron"
typically comprises a cell body, an axon, axon terminals, and
dendrites.
[0053] As used herein, the term "cAMP-stimulating compound" is
meant to refer to those compounds which increase intracellular
cyclic AMP levels in a cell. Examples of such cAMP-stimulating
compounds include, but are not limited to epinephrine,
isoproterenol, forskolin, IBMX and dbcAMP.
EXAMPLES
[0054] The invention is further described in detail by reference to
the following experimental examples. These examples are provided
for purposes of illustration only, and are not intended to be
limiting unless otherwise specified. Thus, the invention should in
no way be construed as being limited to the following examples, but
rather, should be construed to encompass any and all variations
which become evident as a result of the teaching provided
herein.
[0055] The materials and methods are now discussed.
[0056] Isolation and culture of hMSCs
[0057] Twenty milliliters of bone marrow aspirate were taken from
the iliac crest of normal donors ranging in age from 19 to 49 years
old. Isolation and culture of hMSCs were carried out as previously
described by DiGirolamo et al. (1999, Br. J. Haematol.,
107(2):275-281). Briefly, aspirate was diluted 1:1 with Hanks'
balanced salt solution (HBSS; Gibco-BRL, St. Louis, Mo.) and
layered over 10 milliliters of Ficoll (Ficoll-Paque; Pharmacia).
After centrifugation at 2,500 g for 30 minutes, the mononuclear
cell layer was recovered from the gradient interface and washed
with HBSS. The mononuclear cells were centrifuged at 1,500 g for 15
minutes and resuspended in complete culture medium (alpha-MEM;
Gibco-BRL) containing 20% fetal bovine serum (FBS; lot-selected for
rapid growth of hMSCs, Atlanta Biologicals), 100 units per
milliliter of penicillin, 100 micrograms per milliliter of
streptomycin; and 2 millimolar L-glutamine (Gibco-BRL). All of the
cells were plated in 25 milliliters of medium in a 150 centimeters
squared culture dish (Falcon) and incubated at 37 degrees Celsius
with 5 percent humidified carbon dioxide. After 24 hours,
non-adherent cells were discarded, and adherent cells were
thoroughly washed twice with phosphate-buffered saline (PBS). Fresh
complete culture medium was added or replaced every 3 or 4 days.
The cells were grown to approximately 70 to 90 percent confluency
over about 14 days. The cells (from passage 0) were harvested with
0.25 percent trypsin and 1 millimolar EDTA for 5 minutes at 37
degrees Celsius, replated in 75 centimeters squared flasks (Falcon)
at 5,000 cells per centimeter squared, and again grown to near
confluency. The cells (from passage 1) were harvested with the same
concentrations of trypsin and EDTA, suspended at approximately one
million to two million cells per milliliter in 5 percent DMSO and
30 percent FBS, and frozen as one milliliter aliquots in liquid
nitrogen. To expand a culture, a frozen stock of hMSCs was thawed,
plated at 5,000 cells per centimeter squared, and grown to
approximately 70 to 90 percent confluency over about 3 to 7 days.
The cells (from passage 2) were harvested with the same
concentrations of trypsin and EDTA and diluted 1:3 per passage for
further expansion.
[0058] Neural differentiation protocol
[0059] Powder forms of Isobutylmethylxanthine (IBMX) were
dissolution Dimethyl Sulfoxide (DMSO). Powder forms of Dibutyryl
Cyclic AMP (dbcAMP) were dissolved in dH.sub.2O. A final
concentration of 0.5 millimolar IBMX (Sigma)/1 millimolar dbcAMP
(Sigma) was achieved and this solution was added to 10 milliliters
of complete culture medium containing one million hMSCs (passage 2)
in a fifty-eight square centimeter tissue culture dish (Falcon).
IBMX/dbcAMP and complete culture medium were replaced at three days
and the incubation continued through six days.
[0060] Western blot analysis
[0061] Cells were rinsed with cold phosphate buffered saline (PBS)
twice and drained. Whole cell lysates were prepared by adding 0.5
milliliters of detergent-based cell lysis buffer (1 percent (w/w)
NP-40, 0.5 percent (w/v) sodium deoxycholate, 0.1 percent (w/v)
sodium dodecylsulfate (SDS), prepared in PBS) plus leupeptin (final
concentration at 0.1 milligrams per milliliter, freshly prepared
and added; Sigma), and scraping the cells into a centrifuge tube.
The cells were further lysed by flushing them 3 times through a 1
milliliter capacity syringe with a 21 gauge needle, and then
phenylmethylsulfonyl fluoride (PMSF; final concentration of 574
micromolar prepared in isopropanol, Sigma) was added to the cell
suspension. The sample was incubated on ice for 45 minutes,
centrifuged at 15,000 g for 30 minutes at 4 degrees Celsius, and
the supernatant was collected. Protein content was assayed
calorimetrically (Micro Protein Kit, Sigma). Five micrograms of the
cell lysate were loaded onto a 4 to 10 percent or 4 to 20 percent
polyacrylamide gradient gel. After electrophoresis, the protein was
transferred by electroelution onto a nitrocellulose membrane.
Immunodetection of each of the neuron markers shown in Table 1 was
performed with the following primary antibodies: rabbit anti-NSE
(ICN Biomedicals, 1:10,000 dilution), mouse anti-vimentin (DAKO,
1:1,000 dilution), rabbit anti-MAP1B (1:10,000 dilution; 1994,
Black et al., J. Neurosci. 14:857-870), mouse anti-TuJ-1 (BabCo,
1:2,000 dilution), mouse anti-alpha-tubulin (Sigma, 1:4,000
dilution), rabbit anti-neurofilament M (NF-M, Chemicon
International, Inc., 1:1,000 dilution), mouse anti-MAP2 (2a+2b,
Pharmingen, 1:1,000 dilution), mouse anti-tau (Tau-2, Pharmingen,
1:500 dilution), mouse anti-S-100 (Neomarkers, 1:500 dilution),
mouse anti-human GFAP (DAKO, 1:500 dilution), and mouse anti-myelin
basic protein (MBP, Chemicon International, 1:1,000 dilution).
[0062] The secondary antibody was horseradish peroxidase conjugated
to either goat anti-rabbit IgG or anti-mouse IgG. The membranes
were processed using enhanced chemiluminescence (ECL Western
blotting detection reagents, Amersham Pharmacia Biotech). About 0.5
micrograms of human brain extract (Clontech) was used as a
control.
[0063] The results of the experimental examples are now
discussed.
[0064] Induction of Neural Morphology on hMSCs
[0065] hMSCs (FIG. 1A) were induced to differentiate in culture by
incubation with 0.5 millimolar IBMX/1 millimolar dbcAMP. Typical
neural cells were identified as early as two days later (FIGS. 1B,
1C, and 1D). After 6 days, neural cells accounted for about 25% of
the total population (FIG. 2). The cells had morphological features
typical of neurons such as refractile cell bodies and long
branching processes with growth cone-like terminal structures that
frequently made contact with undifferentiated hMSCs. There was a
reduced rate of cellular proliferation, but there was no obvious
evidence of cell death. However, after IBMX/dbcAMP was withdrawn
from the complete culture medium of the hMSCs that were treated for
6 days, all neural cells died within several days. The remaining
cells stopped dividing and showed senescence morphology. The data
suggested that the differentiation was not reversible.
[0066] Biochemical Analysis of Cell Phenotype
[0067] Using the Western blot assay, it was determined that the
untreated hMSCs expressed several markers characteristic of neural
cells such as MAP1B, NSE, TuJ-1 and vimentin (FIG. 3). Using
alpha-tubulin as a control, it was demonstrated that the expression
levels of both NSE and vimentin were increased after incubation
with 0.5 millimolar IBMX and 1 millimolar dbcAMP. The increase in
NSE and vimentin mRNAs coincided with the appearance of neural
cells in the cultures. However, there was no change in the
expression level of either MAP1B or TuJ-1 (FIG. 3). Since NSE,
MAP1B, and TuJ-1 are early neuron-characteristic markers, and
vimentin is an early marker for glia, the data suggested that hMSCs
differentiated in vitro into some early progenitors of either
neurons or glia. Expression of NF-M, MAP2, tau, S-100, GFAP, and
MBP, all markers of mature neurons (FIG. 3), was not detected in
either untreated or IBMX/dbcAMP-treated hMSCs.
[0068] IBMX is a phosphodiesterase inhibitor and dbcAMP is a cAMP
analogue. Both agents are known to elevate intracellular cAMP
levels. Moore et al. (1996, Mol.& Chem. Neuropathol.
29(2-3):107-126) found that IBMX or dbcAMP can greatly increase the
extension of processes in a medulloblastoma cell line, MCD-1. The
formation of long processes induced by IBMX was associated with a
decrease in cell proliferation as evidenced by a reduction in
numbers of cells incorporating 5-bromo-2-deoxyuridine (BrdU). Bang
et al. (1994, PNAS, 91(12):5330-5334) found that elevation of cAMP
through addition of dbcAMP and IBMX induced a neuronal morphology
in human prostate carcinoma cells. The changes also included
increased expression of NSE, terminal differentiation, G.sub.1
synchronization, growth arrest, and loss of clonogenicity. Cox et
al. (1999, Cancer Res., 59(15):3821-3830) also found agents that
can elevate intracellular cAMP such as epinephrine, isoproterenol,
forskolin, IBMX, and dbcAMP can induce prostate tumor cells to
assume many of the characteristics of neuroendocrine cells. The
cells reverted to their original phenotype when the agents were
removed. With C6 glioma cells, both Sharma et al. (1987, J.
Neurosci. Res., 17(2):135-141) and Ghosh et al. (1997, Cell Biol.
Int., 21(9):551-557) found that dbcAMP induced neural
differentiation.
[0069] In the experiments presented here, MSCs were cultured under
conditions that increase intracellular cAMP, and it was determined
that a fraction of the cells in the cultures developed some of the
phenotypic features of neural cells. The results were similar but
not identical to the observations recently reported by Woodbury et
al. and Sanchez-Ramos et al. using different culture conditions
(Table 1). Similar morphological changes were seen with all three
experimental conditions, but the number of neural-like cells varied
widely. Our results were similar to those of Woodbury et al. in
that we saw an increased expression of NSE, but no expression of
GFAP. In contrast, Sanchez-Ramos et al. observed expression of GFAP
both before and after differentiation under their conditions.
Expression of either NF-M or tau that Woodbury et al. observed
after differentiation was not detected. Under the present
conditions, there was increased expression of vimentin, as is seen
in differentiation of glia. MAP1B and TuJ-1, two markers for early
neurons, were expressed at about the same levels before and after
differentiation. MAP-2, a marker for mature neurons, was negative.
S-100 and MBP, markers for mature astrocytes and oligodendroglia,
were also negative. Therefore, the results suggest that the cells
differentiated into early neural progenitors under conditions that
increase intracellular cAMP but not into mature cells of any
specific lineage. Differentiation of the cells into mature neural
cells will probably require a combination of the conditions tested
to date and functional assays such as the membrane potentials of
putative neurons.
1 TABLE 1 SANCHEZ- RAMOS WOODBURY EGF/BDNF BME or and RA INVENTION
Conditions DMSO/BHA or co-culture DbcAMP Species Rat/Human
Mouse/Human and IBMX Percent Cells Greater than 50 0.2 to 5 Human
with Neural Cyto- Cyto- 25 Morphology chem Western chem Western
Cytochem Western NSE +/+++ +/+++ +/+++ NF-M 0/+++ 0/0 Tau 0/+++ 0/0
Neu-N 0/++ +/++ ++/++ Nestin 0/+ to 0 /++ ++/0 GFAP 0/0 /++ ++/++
0/0 TrkA 0/+++ Vimentin +/++ MAP1B ++/++ TuJ-1 ++/++ MAP-2 0/0 0/0
S-100 0/0 MBP 0/0 Fibronectin +++/+ Comparisons of observations on
differentiation of MSCs. Observations are presented as 0 to +++
scores before/after differentiation. The scores are approximations
based on data presented in different formats by Woodbury et al. and
Sanchez-Ramos et al. Parallel assays by immunocytochemistry
(Cytochem) and Western blots were not performed in many of the
experiments.
[0070] The morphological changes of the hMSCs coincide with an
increase in NSE and vimentin expression. However, several markers
for mature neurons and glia are not expressed, indicating that the
present invention may be useful in studying the early steps of
neural cell differentiation. The results also indicate that these
early progenitor neural cells have a potential therapeutic use in
treating diseases, conditions and disorders of the central nervous
system.
[0071] The disclosure of every patent, patent application, and
publication cited herein is hereby incorporated herein by reference
in its entirety.
[0072] While this invention has been disclosed with reference to
specific embodiments, it is apparent that other embodiments and
variations of this invention can be devised by others skilled in
the art without departing from the true spirit and scope of the
invention. The appended claims include all such embodiments and
equivalent variations.
* * * * *