U.S. patent application number 11/843455 was filed with the patent office on 2008-01-31 for meningeal-derived stem cells.
Invention is credited to Roy C. Ogle, Sunil Tholpady.
Application Number | 20080026462 11/843455 |
Document ID | / |
Family ID | 29736364 |
Filed Date | 2008-01-31 |
United States Patent
Application |
20080026462 |
Kind Code |
A1 |
Ogle; Roy C. ; et
al. |
January 31, 2008 |
MENINGEAL-DERIVED STEM CELLS
Abstract
Described herein are stem cells derived from the meninges;
specifically, the dura mater, pia mater or arachnoid mater. Methods
for isolating, differentiating and explanting these cells are
described, as well. In particular embodiments, the stem cells of
the present invention are differentiated into nerve cells, bone
cells, cartilage cells and Schwann cells. The stem cells of the
invention can be taken from a small biopsy, and rapidly expanded to
large populations of cells using specially defined media that
maintain their undifferentiated state. Use of the stem cells of the
present invention in biomedical applications is also described.
Inventors: |
Ogle; Roy C.; (Earlysville,
VA) ; Tholpady; Sunil; (Charlottesville, VA) |
Correspondence
Address: |
DAVIS WRIGHT TREMAINE LLP/Los Angeles
865 FIGUEROA STREET
SUITE 2400
LOS ANGELES
CA
90017-2566
US
|
Family ID: |
29736364 |
Appl. No.: |
11/843455 |
Filed: |
August 22, 2007 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10458102 |
Jun 10, 2003 |
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11843455 |
Aug 22, 2007 |
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60387793 |
Jun 11, 2002 |
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Current U.S.
Class: |
435/368 |
Current CPC
Class: |
C12N 2500/38 20130101;
C12N 2501/998 20130101; C12N 2501/135 20130101; C12N 2501/15
20130101; C12N 2500/25 20130101; C12N 2501/385 20130101; C12N
2501/39 20130101; C12N 5/0668 20130101; C12N 2501/33 20130101; C12N
2500/90 20130101; C12N 2501/70 20130101; C12N 2533/50 20130101;
C12N 2533/52 20130101; C12N 2500/44 20130101; C12N 2501/115
20130101; C12N 2500/46 20130101; C12N 5/0607 20130101; C12N 2501/11
20130101; C12N 2501/01 20130101; C12N 2533/54 20130101; C12N
2533/90 20130101 |
Class at
Publication: |
435/368 |
International
Class: |
C12N 5/06 20060101
C12N005/06 |
Goverment Interests
GOVERNMENT RIGHTS
[0002] This invention was made with United States Government
support under Grant No. SR01 DE010369 awarded by the National
Institutes of Health. The United States Government has certain
rights in the invention.
Claims
1. A stem cell line, comprising cells derived from a meningeal
tissue.
2. The stem cell line of claim 1, wherein said meningeal tissue is
selected from the group consisting of dura mater, pia mater,
arachnoid mater and combinations thereof.
3. The stem cell line of claim 1, wherein said meningeal tissue is
obtained by biopsy from a patient or aseptically from a fetus.
4. A composition comprising a substantially pure population of
meningeal-derived stem cells.
5. The composition of claim 4, wherein said population of
meningeal-derived stem cells includes greater than 80% of
totipotent or pluripotent meningeal-derived stem cells.
6. The composition of claim 4, wherein said population of
meningeal-derived stem cells includes greater than 90% of
totipotent or pluripotent meningeal-derived stem cells.
7. The composition of claim 4, wherein said population of
meningeal-derived stem cells includes greater than 99% of
totipotent or pluripotent meningeal-derived stem cells.
8-20. (canceled)
21. The composition of claim 4, further comprising a
pharmaceutically acceptable carrier.
22-32. (canceled)
33. The composition of claim 4, wherein the meningeal-derived stem
cells are obtained by a process, comprising: obtaining meningeal
tissue from a subject; washing said meningeal tissue in a
physiologic buffer to produce washed meningeal tissue; placing said
washed meningeal tissue in a solution including said physiologic
buffer and collagenase to produce dissociated meningeal tissue;
recovering said dissociated meningeal tissue; and plating said
dissociated meningeal tissue onto a culture substrate including a
growth medium to culture said meningeal-derived stem cells.
34. The composition of claim 33, wherein obtaining said meningeal
tissue is performed by a technique selected from biopsy from said
subject and aseptic removal from said subject.
35. The composition of claim 33, wherein said physiologic buffer is
selected from the group consisting of phosphate buffered saline
(PBS), Hanks balanced salt solution and combinations thereof.
36. The composition of claim 33, wherein placing said washed
meningeal tissue in said solution further includes maintaining said
washed meningeal tissue in said solution for about 5 to about 30
minutes at a temperature of about 37.degree. C.
37. The composition of claim 33, wherein recovering said
dissociated meningeal tissue further includes centrifuging said
meningeal tissue and washing a resulting pellet.
38. The composition of claim 33, wherein said culture substrate is
selected from the group consisting of a tissue culture plate
plastic, a laminin-covered substrate, a polyamino acid, fibronectin
and type I collagen.
39. The composition of claim 33, wherein said growth medium
includes Dulbecco's modified eagle medium (DMEM), about 10% fetal
bovine serum (FBS) and about 1% glutamine.
40. The composition of claim 4, wherein the meningeal-derived stem
cells are obtained by a process, comprising: obtaining meningeal
tissue from a subject; washing said meningeal tissue in a
physiologic buffer to produce washed meningeal tissue; placing said
washed meningeal tissue on a culture substrate including a growth
medium to culture said meningeal-derived stem cells.
41. The composition of claim 40, wherein obtaining said meningeal
tissue is performed by a technique selected from biopsy from said
subject and aseptic removal from said subject.
42. The composition of claim 40, wherein said physiologic buffer is
selected from the group consisting of phosphate buffered saline
(PBS), Hanks balanced salt solution and combinations thereof.
43. The composition of claim 40, wherein said culture substrate is
selected from the group consisting of a tissue culture plate
plastic, a laminin-covered substrate, a polyamino acid, fibronectin
and type I collagen.
44. The composition of claim 40, wherein said growth medium
includes Dulbecco's modified eagle medium (DMEM), about 10% fetal
bovine serum (FBS) and about 1% glutamine.
45. (canceled)
Description
RELATED APPLICATIONS
[0001] This application claims the benefit of priority under 35
U.S.C. .sctn. 119 of provisional U.S. application Ser. No.
60/387,793, filed Jun. 11, 2002, the contents of which are hereby
incorporated by reference.
FIELD OF THE INVENTION
[0003] The present invention is directed to stem cells and methods
of preparing populations of progenitor cells that differentiate
into a preselected cell type with high efficiency.
BACKGROUND OF THE INVENTION
[0004] The brain and spinal cord are enclosed and protected by the
meninges; tough and fibrous tissues comprising the dura mater and
pia mater. Developmentally, these tissues form partly from the
neural crest; a class of highly migratory and plastic cells that
also form several diverse tissue types, such as bone, cartilage,
muscle, gut, adrenal glands, etc. The dura mater has been shown to
regulate bone formation in the developing skull through tissue
interactions mediated by growth factors originating in the dura (L.
A. Opperman et al., "Tissue interactions with underlying dura mater
inhibit osseous obliteration of developing cranial sutures," Dev.
Dynamics, 198(4):312-322 (1993)). During skull regeneration in
humans and animals whose heads are still growing, the bone and
other connective tissues of the skull are formed from cellular
precursors in the dura (FIG. 1; D. B. Drake et al., "Calvarial
deformity regeneration following subtotal craniectomy for
craniosynostosis: a case report and theoretical implications," J.
Craniofacial Surg., 4(2):85-90 (1993)).
[0005] Moreover, there is extensive interest in developing methods
for using pluripotential stem cell populations for a wide variety
of potential therapeutic applications, including delivery of
therapeutic genes, correction of gene defects,
replacement/augmentation of existing dysfunctional cell populations
(e.g., dopaminergic neurons in Parkinsons Disease), and generation
of organs/tissues for surgical repair/replacement. However,
existing methods in the field have a number of major limitations
that relate to obtaining purified populations of the desired cell
types from pluripotent stem cells. By way of example, embryonic
stem cells pose interesting possibilities as several studies show
that these cells are pluripotent, however, the use of these cells
is mired in ethical and political considerations. It is therefore
likely that this technology will not be available for use in the
near future.
[0006] There is a need in the art for a stem cell population that
obviates the limitations of currently available stem cells; thereby
enabling further research in this field, and also the therapeutic,
clinical use of stem cells in various aspects of biomedicine. The
present invention is directed to such a novel stem cell population,
which is isolated from meningeal tissues. This stem cell population
has properties that provide significant advantages over the stem
cells currently available.
SUMMARY OF THE INVENTION
[0007] One aspect of the present invention is directed to stem
cells derived from the dura mater, the pia mater or the arachnoid
mater, as well as methodologies for isolation, differentiation and
explantation of these cells. Meningeal tissue may be explanted
(i.e., cells migrate out of adherent pieces of tissue) or
enzymatically dissociated to yield primitive mesenchymal cells. The
tissues may include those removed by biopsy from a patient or
tissues removed aseptically from a fetus. These cells exhibit
characteristics of "adult" stem cells or progenitors: robust
self-renewal and a high degree of developmental plasticity. The
stem cells may be readily propagated in culture (showing little
senescence after 20 passages), and are capable of differentiating
into various cell types. Thus, the meningeal-derived stem cells of
the present invention are multipotent.
[0008] The meningeal stem cells of the present invention can be
taken from a small biopsy, and rapidly expanded to large
populations of cells using a specially defined media that maintains
their undifferentiated state. Transformation to neural cells can be
accomplished rapidly (i.e., within several hours) and bone and
cartilage within two weeks, by adding factors that support and
maintain these cell phenotypes. Schwann cells, adipocytes and
fibroblasts may be rapidly produced, as well. In addition, the
number of cells that transform to a neural morphology is between
90% and 95%. Thus, these cells may have particular utility in
treating central nervous system (CNS) degenerative disorders and
spinal cord injuries. The rapid proliferative capacity and high
rate of neuronal differentiation makes these applications
well-suited for the stem cells, although numerous other
applications exist, as well.
[0009] In another aspect of the present invention, the use of stem
cells derived from the dura A, the pia mater or the arachnoid mater
in biomedical applications is described. For example, the stem
cells of the present invention may be used for tissue regeneration,
gene and drug delivery and cell replacement therapies. They may
find additional applications in research settings, as well as
alternate therapeutic modalities or clinical treatments.
BRIEF DESCRIPTION OF THE DRAWINGS
[0010] The file of this patent contains at least one drawing
executed in color. Copies of this patent with color drawings will
be provided by the Patent and Trademark Office upon request and
payment of the necessary fee.
[0011] FIG. 1 is executed in color, and illustrates a histological
identification of the meninges and cells within a newborn rat, in
human tissue and in human fetal tissue, in accordance with an
embodiment of the present invention. FIG. 1A depicts the dura mater
of a newborn rat; FIG. 1B depicts the dura cells of a newborn rat;
FIG. 1C depicts the dura cells (exposed to dexamethasone) of a
newborn rat; and FIG. 1D depicts the dura mater, pia mater and
meninges of a newborn rat. FIG. 1E depicts the frontal and parietal
bones of a human infant skull, and FIG. 1F depicts the dura mater
and osteogenic front of a human fetal skull.
[0012] FIG. 2 illustrates the neural differentiation of
meningeal-derived stem cells in accordance with an embodiment of
the present invention. FIG. 2A depicts meningeal cells on a tissue
culture plate; their flattened morphology is apparent. FIG. 2B
depicts meningeal cells exposed to steroid treatment; extensive
branching and dendritic morphologies are apparent. FIG. 2C depicts
meningeal cells exposed to antioxidant treatment; cells are mostly
bipolar-type neuronal morphologies.
[0013] FIG. 3 is executed in color, and illustrates osseous
differentiation of meningeal-derived stem cells in accordance with
an embodiment of the present invention. FIGS. 3A and 3B depict
untreated meningeal cells showing no staining for alkaline
phosphatase at low and high magnification, respectively. FIGS. 3C
and 3D depict meningeal cells plated onto MATRIGEL, showing intense
staining for alkaline phosphatase in cell condensations following
two weeks in culture.
[0014] FIG. 4 is executed in color, and illustrates chondrocytic
differentiation of meningeal-derived stem cells in accordance with
an embodiment of the present invention. Meningeal cells were
allowed to grow in micromass culture for four weeks in chondrocytic
differentiation media. Nodules were exposed to Alcian Blue, a dye
that specifically stains sulfated proteoglycans found in
cartilage.
[0015] FIG. 5 is executed in color, and illustrates differentiation
of meningeal-derived stem cells into Schwann cells in accordance
with an embodiment of the present invention. Cells were stained for
S-100. Cells treated with a final growth factor treatment step
stained more strongly (i.e., were more highly positive) for S-100
(FIG. 5A) as compared with cells that did not receive this final
treatment step (FIG. 5B).
DETAILED DESCRIPTION OF THE INVENTION
[0016] In describing and claiming the invention, the following
terminology will be used in accordance with the definitions set
forth below.
[0017] As used herein, "nucleic acid," "DNA," and similar terms
also include nucleic acid analogs, i.e., analogs having other than
a phosphodiester backbone. For example, the so-called "peptide
nucleic acids," which are known in the art and have peptide bonds
instead of phosphodiester bonds in the backbone, are considered
within the scope of the present invention.
[0018] As used herein a "gene" refers to the nucleic acid coding
sequence as well as the regulatory elements necessary for the DNA
sequence to be transcribed into messenger RNA (mRNA) and then
translated into a sequence of amino acids characteristic of a
specific polypeptide.
[0019] A "marker" is an atom or molecule that permits the specific
detection of a molecule comprising that marker in the presence of
similar molecules without such a marker. Markers include, for
example radioactive isotopes, antigenic determinants, nucleic acids
available for hybridization, chromophors, fluorophors,
chemiluminescent molecules, electrochemically detectable molecules,
molecules that provide for altered fluorescence-polarization or
altered light-scattering and molecules that allow for enhanced
survival of an cell or organism (i.e. a selectable marker). A
reporter gene is a gene that encodes for a marker.
[0020] As used herein, the term "purified" and like terms relate to
the isolation of a molecule or compound in a form that is
substantially free of contaminants normally associated with the
molecule or compound in a native or natural environment.
[0021] As used herein the term "pharmaceutically acceptable
carrier" encompasses any of the standard pharmaceutical carriers,
such as phosphate buffered saline (PBS), water and emulsions such
as an oil/water or water/oil emulsion, and various types of wetting
agents.
[0022] As used herein the term "totipotent" or "totipotential" and
like terms refers to cells that have the capability of developing
into a complete organism or differentiating into any cell type of
that organism.
[0023] As used herein the term "pluripotent" or "pluripotential"
and like terms refers to cells that cannot develop into a complete
organism, but retain developmental plasticity, and are capable of
differentiating into some of the cell types of that organism.
[0024] As used herein a "differentiated cell type" refers to a cell
that expresses gene products that are unique to that cell type. For
example, a nerve cell is a cell type that expresses specific
markers associated with smooth muscle cells, including .beta.-III
tubulin and neuron specific enolase (NSE).
[0025] The present invention is based on the inventors' surprising
discovery that stem cell populations may be derived from the
meninges. More specifically, the present invention is directed to
compositions comprising purified meningeal stem cells, and, more
particularly, stem cells isolated from the dura mater, pia mater or
arachnoid mater, as well as methodologies for the isolation,
differentiation and explantation of such stem cells. These stem
cells may be used in accordance with the present invention in a
wide variety of biomedical applications, including, but in no way
limited to, tissue regeneration, gene and drug delivery and cell
replacement therapies. The cells are unusual not only in their
anatomical location (the meninges have not heretofore been
identified as a source of stem cells), but also in their
behavior--the stem cells of the present invention are believed to
be the only stem cells that differentiate into osteoblasts without
dexamethasone treatment. In fact, the stem cells of the present
invention differentiate into neuronal cells in media containing
dexamethasone, while all other stem cells differentiate into
osteoblasts under similar treatment conditions.
[0026] The invention includes the generation of multiple cell types
from the multipotent cell or cells that reside in the meningeal
tissues surrounding and associated with the brain and spinal cord.
In particular, the invention is directed to the derivation of stem
cells from these tissues and the differentiation of these stem
cells into cell types beyond those that are normally associated
with the meninges. Although the procedures described herein produce
a total population of cells, individual, clonal cell lines may be
derived from the total population. Large quantities of cells may be
grown and harvested with the methods of the present invention for
applications in, for example, gene, drug and molecule screening and
delivery, tissue engineering, regeneration and replacement of
nerve, bone, cartilage, muscle, fat and other organs, and treatment
of spinal cord injury and CNS disorders such as Parkinson's
disease, Alzheimer's disease, dementia and multiple sclerosis.
Replacement or regeneration of tissue damaged through a variety of
physiologic and pathologic processes including aging, cancer,
trauma, infection, and congenital anomalies is an area of active
and intense investigation, and may also be within the scope of
conditions that may be addressed with the stem cells of the present
invention. In accordance with yet another embodiment of the present
invention, in vitro treatment includes insertion of a gene
construct for delivery on implantation of the cells of the present
invention.
[0027] In accordance with one embodiment of the present invention,
a composition comprising a substantially pure population of
totipotent or pluripotent cells is provided. The composition may
include a pharmaceutically acceptable carrier. In preferred
embodiments, the substantially pure population of cells comprises
greater than 80% of totipotent or pluripotent cells; more
preferably greater than 90% of totipotent or pluripotent cells; and
most preferably a purity of 99% or 100% of totipotent or
pluripotent cells. In one embodiment, a purified population of
meningeal-derived stem cells is provided, wherein greater than 60%
of the cells are induced to form nerve cells, bone cells, cartilage
cells, Schwann cells, adipocytes and fibroblasts by contacting the
cells with a nerve cell, bone cell, cartilage cell, Schwann cell,
adipocyte or fibroblast inducing agent, respectively.
[0028] The tissues for isolation of the stem cells may include
those removed by biopsy from patients or tissues removed
aseptically from fetuses by any of a host of methodologies that
will be readily understood and may be routinely performed by those
of skill in the art. By way of example, meningeal stem cells may be
prepared by obtaining a small, full thickness piece of tissue from
the meninges surrounding the brain or spinal cord. This piece of
tissue may include, e.g., an approximately 4 mm punch biopsy.
Alternatively, dural cells that adhere to calvarial fragments
(e.g., those removed as autologous grafting materials or for burr
holes during neurosurgery) may be suitable for use as a piece of
tissue in connection with the methods of the present invention.
[0029] In a further embodiment of the present invention, a method
is provided for isolating meningeal stem cells by enzymatic
digestion. The tissue may first be washed in a physiologic buffer
(e.g., PBS or Hanks balanced salt solution), and then placed in the
same solution containing collagenase (300 U) for a predetermined
length of time to dissociate the tissue (typically the tissue is
treated for about 5 to 30 minutes, and more preferably for about 15
minutes, at approximately 37.degree. C.). The resultant dissociated
tissue is then recovered, typically by centrifuging the tissue and
washing the resultant pellet. The pellet is then plated onto tissue
culture dishes containing a growth medium. Preferably, the growth
medium includes Dulbecco's Modified Eagle Medium (DMEM),
approximately 10% fetal bovine serum (FBS) and approximately 1%
glutamine. The tissue culture substrate can include, but is not
limited to, tissue culture plate plastic, polyamino acids,
fibronectin, type I collagen and various forms of laminin (e.g.,
pure mouse laminin-1 or MATRIGEL); all forms of laminin being
hereinafter included in the term "laminin." By way of example,
MATRIGEL (available from BD Biosciences Discovery Labware; Bedford,
Mass.; hereinafter "BD Biosciences") is over 90% laminin-1, with
the remaining portion including a mixture of type IV collagen,
perlecan and nidogen/entactin. This preparation is extracted from
the Engelbreth-Holm-Swarm (EHS) tumor of mice and is subjected to
multiple 45% ammonium sulfate precipitation to remove growth
factors. The use of laminin substrates is well known in the art,
and, by way of example, is described in L. A. Davis et al.,
"Embryonic heart mesenchymal cell migration on laminin," Dev.
Biol., 133:37-43 (1989); T. M. Sweeney et al., "Laminin potentiates
differentiation of PCC4azal embryonal carcinoma into neurons," J.
Cell Sci., 97:3-31 (1990); and T. M. Sweeney et al., "Repair of
critical size rat calvarial defects using extracellular matrix
gels," J. Neurosurg., 83(4):710-715 (1995).
[0030] In an alternate explanation isolation technique, the tissue
may be placed on the culture substrate with a minimal amount of
medium where it is allowed to adhere firmly; stem cells grow out of
the tissue onto the plate. As used herein, a "minimal amount" of
medium is a volume of medium sufficient to cover the tissue,
preventing drying, but not so much that the tissue will float or
become dislodged from the substrate before cells begin to emigrate.
Conversely, an "excessive amount" of medium is a volume of medium
in which the tissue floats and cells are unable to contact the
substrate; disadvantageous for explant outgrowth. Plastic or
laminin-covered culture substrates may be particularly advantageous
in this alternate isolation technique.
[0031] The stem cells that attach and grow in the culture dishes or
plates may be subcultured and expanded for several generations.
Cells may be passaged when they become 70-80% confluent, and are
not allowed to become completely confluent (although this does not
appear to alter differentiation capacity in the short term). No
changes were observed in the cells' doubling behavior or cell
characteristics over the longest culture period studied (i.e.,
sixty population doublings).
[0032] The self-renewal capacity that these cells demonstrate is
one of the characteristics common to all stem cells. Because the
cells possess a self-renewal capacity, it is not necessary
(although still possible and may be particularly useful if these
cells are studied as cell lines) to immortalize the cells using one
of the many transfection techniques commonly used in the art. These
techniques may be used to transfer genes of interest into the
meningeal stem cells. Self-maintenance is but one of several
characteristics that stem cells possess. Additionally, they have
the capacity to proliferate, to produce of a large number of
differentiated functional progeny, and to regenerate target tissue
after injury. Moreover, stem cells are generally flexible with
respect to the aforementioned functional capabilities (C. S. Potten
et al., "Stem cells: attributes, cycles, spirals, pitfalls and
uncertainties. Lessons for and from the crypt," Development--Supp.,
110(4):1001-20 (1990)).
[0033] While not wishing to be bound by any theory, it is believed
that the developmental origin of the meningeal cells as neural
crest derivatives confers multipotent differentiation capacity to
the cells. In accordance with various embodiments of the present
invention, the inventors have exploited this capacity of cells
derived from the dura mater to produce nerve, bone, cartilage and
Schwann cells, as well as adipocytes and fibroblasts. In alternate
embodiments of the present invention, meningeal-derived cells may
be turned into melanocytes and a variety of neural supporting cells
and muscle cell types.
[0034] There are several advantages to using meningeal-derived stem
cells in clinical biomedicine and research applications, as opposed
to other stem cell variants. The cells of the present invention are
a reservoir of developmental potential unique from any previously
described and will increase the armamentarium available for stem
cell-based therapies. A major advantage of this particular stem
cell is its capacity to differentiate into neural cells at a higher
rate and greater percentage than either bone marrow stem cells or
fat-derived stem cells (the other major adult stem cells known to
form neurons). As such, purer stem cell populations can be
implanted sooner into a damaged CNS than could be achieved with
other adult-derived stem cells. Additionally, purer populations are
believed to be advantageous because there is a larger biomass that
actively participates in restoration and regeneration.
[0035] Another advantage of the cells of the present invention is
that they can be derived from adult as well as fetal tissues. Thus,
there are fewer ethical or legal implications in their use than
with stem cells from embryonic and early fetal sources. Another
advantage relative to embryonic stem cells is that the cells of the
present invention can be derived from an individual, propagated and
differentiated in vitro, and delivered back to the same individual;
thereby avoiding rejection issues. These issues limit the use of
embryonic stem cells at present. The ability to transplant cells
without immunosuppressive drugs is also a major advantage, because
these drugs tend to impair wound healing and regenerative capacity.
Yet a further advantage of the cells of the present invention is
the decreased senescence observed in the cell line, which allows
for tremendous expansion--large masses of cells may be produced for
transplantation based on only a small biopsy.
EXAMPLES
[0036] The following Examples illustrate the differentiation of
meningeal-derived stem cells into distinct cell types in vitro.
Cells derived from both the covering of the brain and spinal cord
were isolated, cultured and exposed to conditions that caused
differentiation into cells with the morphology and specific gene
expression of neuroblasts, Schwann cells, osteoblasts,
chondrocytes, adipocytes and fibroblasts. The stem cells divide
rapidly, with population doubling times of 36 hours; nearly as fast
as the fastest primary human cell lines.
Example 1
Preparation of Nerve Cells
[0037] Cells from the meninges that are allowed to become 70%
confluent (FIG. 2A) are susceptible to differentiating into nerve
under two conditions: antioxidant treatment and steroid hormone
treatment. When cells are exposed to a neuronal pre-induction media
containing an antioxidant (DMEM, 20% FBS, 1 mM
.beta.-mercaptoethanol) for 24 hours, followed by treatment with
neuronal induction media also containing an antioxidant (DMEM, 5 mM
.beta.-mercaptoethanol) the cells differentiate into neural-like
cells within six hours (FIG. 2C). This can also be achieved with
other antioxidants (i.e., reducing agents), such as butylated
hydroxyanisole (BHA) (approximately 200 .mu.M), dithiothreitol
(DTT; i.e., Cleland's reagent), as well as dithioerythritol,
tributylphosphine, iodoacetamide, tris-phosphine HCl,
deoxythymidine-triphosphate trilithium salt,
diethylthiatricarbocyanine perchlorate, diethylthiatricarbocyanine
iodide and DECROLINE D (available from BASF Corporation; Mount
Olive, N.J.). These conditions are similar to those used to
differentiate bone marrow-derived stem cells and adipose-derived
stem cells (D. Woodbury et al., "Adult rat and human bone marrow
stromal cells differentiate into neurons," J. Neuroscience Res.,
61(4):364-70 (2000)). The cells that form have a bipolar morphology
and express nerve-specific markers (i.e., .beta.-III tubulin,
NSE).
[0038] The cells differentiate into what are morphologically
distinct subsets of neurons when exposed to small concentrations
(e.g., 100 .mu.M) of dexamethasone; a steroid hormone. A neuronal
induction media incorporating the same (DMEM, 10% FBS, 10 nM
dexamethasone) causes neural differentiation as with the
antioxidant media described above, but the cells cultured in this
steroid hormone media are highly dendritic and have complex
processes reminiscent of neural cells from the CNS (FIG. 2B). These
cells also express the specific neuronal marker gene .beta.-III
tubulin.
[0039] While not truly within the steroid class of compounds,
vitamin A and its derivatives (e.g., retinol, retinaldehyde and
retinoic acid) act through the steroid response elements and
elicited similar effects to dexamethasone, as did 1,25-dihydroxy
vitamin D.sub.3. These may therefore be used as substitutes for
dexamethasone. Other steroids that may be used in accordance with
this embodiment of the present invention include pregnenolone,
aldosterone, testosterone, estradiol and cortisol.
[0040] Neural cell differentiation may also be stimulated with
agents that stimulate increased intracellular cyclic AMP, including
dibuteryl cAMP (dbcAMP) or iso-butrymethylxanthine in the 0.5-10 mM
range.
Example 2
Preparation of Bone Cells
[0041] Dural cells were forced to adopt a bony phenotype by two
separate methods. The first included plating these cells on a
MATRIGEL or laminin substrate (100 .mu.g/cm.sup.2). Plated cells
expressed alkaline phosphatase (a differentiated bone marker)
within seven days, and adopted an osteocytic morphology (FIGS. 3C
& 3D). Untreated cells showed no staining for alkaline
phosphatase (FIGS. 3A & 3B).
[0042] The second method involved exposing the cells to organic and
inorganic phosphates. Inorganic phosphates included varying levels
(i.e., 3-6 mM) of sodium phosphate and potassium phosphate, and
organic phosphates included 10 mM .beta.-glycerol phosphate. In
addition, cells were exposed to 50 .mu.g/ml ascorbic acid. Cells
under these conditions also expressed alkaline phosphatase (data
not shown).
Example 3
Preparation of Cartilage
[0043] Cartilage development is fundamentally different from other
tissues in that complex three-dimensional interactions are required
to form nodules of cartilage in vitro. To accomplish this, a 10
.mu.L volume of a 1.times.10.sup.7 cells/mL suspension was plated
and allowed to attach to a tissue culture surface. This micromass
culture differentiated into cartilage within two weeks when placed
in media that contained 1.times.insulin-selenium-transferrin (ITS
diluted 100-fold; available under the tradename ITS+ PREMIX from BD
Biosciences) and 10 ng/ml transforming growth factor (TGF)-.beta.1.
The production of sulfated proteoglycan as demonstrated by Alcian
Blue (available from Sigma-Aldrich Co.; St. Louis, Mo.) staining is
indicative of chondrocytic differentiation (FIG. 4).
Example 4
Preparation of Schwann Cells
[0044] The dural stem cells have the capacity to differentiate into
nerve support cells or Schwann cells. In response to a multi-day,
multi-drug regimen, they became highly positive for S-100, a
Schwann cell marker. In addition, they assumed a neuronal phenotype
(FIG. 5). The treatments included serum withdrawal, basal medium
eagle (BME), retinoids and growth factors. Specifically, a first
treatment step included DMEM/1 mM BME, and was administered for one
day. A second treatment step included DMEM/10% FBS/70 ng/ml
retinoic acid, and was administered for three days. Finally, a
third treatment step included DMEM/10% FBS/5 .mu.M forskolin
(FSK)/200 ng/ml heregulin (HER)/10 ng/ml basic fibroblast growth
factor (bFGF)/5 ng/ml platelet-derived growth factor (PDGF), and
was administered for five days. Cells treated with this final
treatment step stained more strongly (FIG. 5A) for S-100 than those
that were not treated with this treatment step (FIG. 5B).
[0045] Schwann cell differentiation may also be stimulated with
agents that stimulate increased intracellular cyclic AMP, including
dibuteryl cAMP (dbcAMP) or iso-butrymethylxanthine in the 0.5-10 mM
range.
Example 5
Preparation of Adipocytes
[0046] Adipogenesis was induced in media consisting of basal media
(DMEM with 10% FBS), along with the following additives: 1 .mu.M
dexamethasone, 10 .mu.M insulin, 200 .mu.M indomethacin and 0.5 mM
isobutyl-methylxanthine (IBMX). Multiocula adipocytes positive for
peroxisome proliferator-activated receptor (PPAR)-gamma (data not
shown) began to appear between one and three weeks following
culture preparation.
Example 6
Preparation of Fibroblasts
[0047] The undifferentiated cells were grown in basal media (DMEM
with 10% FBS) along with 50 mM ascorbic acid. After loading a
native type I collagen gel with cells in basal media, a variety of
constructs were derived by manipulating: (1) the concentration of
collagen (from 1-10 mg/ml); (2) the cross-linking of the collagen
by gluteraldehyde treatment; and (3) the type and amount of
tensional force applied. By way of example, if the cells were given
two fixed points against which they contract, the structure formed
resembled a tendon (data not shown). If a sheet of cross-linked
collagen sponge was seeded with cells in basal media, the construct
resembled the fibroblasts of the dermis (data not shown).
[0048] While the description above refers to particular embodiments
of the present invention, it will be understood that many
modifications may be made without departing from the spirit
thereof. The accompanying claims are intended to cover such
modifications as would fall within the true scope and spirit of the
present invention. The presently disclosed embodiments are
therefore to be considered in all respects as illustrative and not
restrictive, the scope of the invention being indicated by the
appended claims, rather than the foregoing description, and all
changes which come within the meaning and range of equivalency of
the claims are therefore intended to be embraced therein.
* * * * *