U.S. patent application number 10/520271 was filed with the patent office on 2005-11-10 for methods of implating mesenchymal stem cells for tissue repair and formation.
Invention is credited to Aslan, Hadi, Gazit, Dan, Gazit, Zulma.
Application Number | 20050249731 10/520271 |
Document ID | / |
Family ID | 30115956 |
Filed Date | 2005-11-10 |
United States Patent
Application |
20050249731 |
Kind Code |
A1 |
Aslan, Hadi ; et
al. |
November 10, 2005 |
Methods of implating mesenchymal stem cells for tissue repair and
formation
Abstract
The invention relates to methods of isolating and implanting
mesenchymal stem cells for tissue repair or formation, without
prior culture expansion of the mesenchymal stem cells. In
particular, the invention relates to methods of isolating
mesenchymal stem cells from bone marrow, for repairing or inducing
formation of bone, without prior culture expansion of the
mesenchymal stem cells. The invention further relates to an
isolated, non-culurally expanded human adult mesenchymal stem cell
population.
Inventors: |
Aslan, Hadi; (Magar, IL)
; Gazit, Dan; (Jerusalem, IL) ; Gazit, Zulma;
(Jerusalem, IL) |
Correspondence
Address: |
Martin Moynihan
Anthony Castorina
2001 Jefferson Davis Highway
Suite 207
Arlington
VA
22202
US
|
Family ID: |
30115956 |
Appl. No.: |
10/520271 |
Filed: |
January 14, 2005 |
PCT Filed: |
July 15, 2003 |
PCT NO: |
PCT/IL03/00587 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
60396010 |
Jul 16, 2002 |
|
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|
Current U.S.
Class: |
424/144.1 ;
424/93.7; 435/366 |
Current CPC
Class: |
C07K 16/2884 20130101;
A61K 35/28 20130101; A61P 19/08 20180101; C12N 5/0663 20130101;
A61P 43/00 20180101; A61K 2035/124 20130101; C07K 16/289 20130101;
C07K 16/2896 20130101; C07K 16/2842 20130101 |
Class at
Publication: |
424/144.1 ;
424/093.7; 435/366 |
International
Class: |
A61K 045/00; A61K
039/395; C12N 005/08 |
Claims
1. A method of obtaining an isolated, non-culture expanded
mesenchymal stem cell, comprising the following steps: Contacting a
human cell population with an antibody that binds to a surface
molecule expressed on a mesenchymal stem cell within said human
cell population, so as to form a cell-antibody-complex; Recovering
said mesenchymal stem cell; Maintaining said recovered mesenchymal
stem cell under conditions preventing significant cellular
expansion; thereby obtaining a non-culture expanded mesenchymal
stem cell.
2. The method of claim 1, wherein said human cell population
comprises unfractionated bone marrow, unfractionated human blood,
unfractionated human dermis, unfractionated human periosteum,
unfractionated muscle or unfractionated human fat.
3. The method of claim 1, wherein the recovered mesenchymal stem
cell is capable of further differentiating into a differentiated
cell of mesenchymal tissue lineage.
4. The method of claim 3, wherein said mesenchymal tissue lineage
is bone, cartilage, fat, tendon, ligament, muscle or marrow
stroma.
5. The method of claim 3, wherein said mesenchymal tissue lineage
is kidney tissue, liver tissue, spleen tissue or neuronal
tissue.
6. The method of claim 1, wherein said antibody interacts with at
least one human CD105 antigen.
7. The method of claim 1, wherein said antibody interacts with at
least one human CD29 or CD44 antigen.
8. The method of claim 1, wherein said antibody is supported on a
column, plastic, array or magnetic bead.
9. The method of claim 1, wherein said mesenchyal stem cell is
further genetically engineered to express a protein of
interest.
10. The method of claim 9, wherein said protein of interest is a
macromolecule necessary for cell growth, morphogenesis,
differentiation, or tissue building and combinations thereof.
11. The method of claim 9, wherein said macromolecule necessary for
cell growth, morphogenesis, differentiation, and/or tissue building
and combinations thereof is a bone morphogenic protein, a bone
morphogenic-like protein, an epidermal growth factor, a fibroblast
growth factor, a platelet derived growth factor, an insulin like
growth factor, a transforming growth factor, a vascular endothelial
growth factor, Ang-1, PIGF and combinations thereof.
12. Use of an isolated, non-culture expanded mesenchymal stem cell
in the preparation of a medicament for administration to a subject,
wherein the non-culture expanded mesenchymal stem cell is obtained
via the method of claim 1.
13. Use of an isolated, non-culture expanded mesenchymal stem cell
in the preparation of a medicament for stimulating or enhancing
tissue repair in a subject, wherein the non-culture expanded
mesenchymal stem cell is obtained via the method of claim 1.
14. Use of an isolated, non-culture expanded mesenchymal stem cell
in the preparation of a medicament for stimulating or enhancing
tissue formation in a subject, wherein the non-culture expanded
mesenchymal stem cell is obtained via the method of caim 1.
15. Use of an isolated, non-culture expanded mesenchymal stem cell
in the preparation of a medicament for maintaining or increasing
bone volume, bone quality, or bone strength in a subject, wherein
the non-culture expanded mesenchymal stem cell is obtained via the
method of claim 1.
16. Isolated, non-culture expanded human adult mesenchymal stem
cells.
17. The mesenchymal stem cells of claim 16, wherein said
mesenchymal stem cells express CD105, CD29 and/or CD44 cell surface
antigens.
18. The mesenchymal stem cells of claim 16, wherein at least 50% of
said mesenchymal stem cells expressing CD105, express CD29 and/or
CD44 cell surface antigens.
19. The mesenchymal stem cells of claim 16, wherein less than 25%
of said mesenchymal stem cells expressing CD105, express CD45,
CD14, CD34 and/or CD31 cell surface antigens.
20. The mesenchymal stem cells of claim 16, wherein said
mesenchymal stem cells are capable of further differentiation to
cells of mesenchymal tissue lineage.
21. The mesenchymal stem cells of claim 20, wherein said
mesenchymal tissue lineage is bone, cartilage, fat, tendon,
ligament, muscle or marrow stroma.
22. The mesenchymal stem cells of claim 16, wherein said
mesenchymal stem cells are engineered to express at least one
protein of interest.
23. The mesenchymal stem cells of claim 22, wherein said protein of
interest is a macromolecule necessary for cell growth,
morphogenesis, differentiation, tissue building or combinations
thereof.
24. The mesenchymal stem cells of claim 23, wherein said
macromolecule necessary for cell growth, morphogenesis,
differentiation, and/or tissue building is a bone morphogenic
protein, a bone morphogenic-like protein, an epidermal growth
factor, a fibroblast growth factor, a platelet derived growth
factor, an insulin like growth factor, a transforming growth
factor, a vascular endothelial growth factor, Ang-1, PIGF or
combinations thereof.
Description
FIELD OF THE INVENTION
[0001] The invention relates to methods of isolating and
transplanting mesenchymal stem cells for tissue repair or
formation, without the step of culturally expanding the mesenchymal
stem cells. In another embodiment, the invention relates to methods
of isolating and transplanting mesenchymal stem cells from bone
marrow, for repairing or inducing formation of bone, without the
step of culturally expanding the mesenchymal stem cells.
BACKGROUND OF THE INVENTION
[0002] Mesenchymal stem cells (MSCs) are considered one of the most
promising tools for cell and cell-mediated gene therapy in skeletal
repair. MSCs were shown to have the potential to differentiate into
several lineages including bone (Haynesworth et al., 1992),
cartilage (Mackay et al., 1998, Yoo et al., 1998), fat (Pittenger
et al., 1996), tendon (Young et al., 1998), muscle, and stroma
(Reviewed by Caplan et al, 2001). The main source known of MSCs in
adult humans is the bone marrow compartment that contains several
cell types including cells of the hematopoietic lineage,
endothelial cells, and mesenchymal stem cells, which are part of
the marrow stromal system (Pittenger et al, 1999).
[0003] Several protocols were recently established in order to
enable regeneration and filling of large bone defects, utilizing
human MSCs expanded in culture as both the cells differentiating
into osteogenic cells, and as the vehicles delivering the
therapeutic gene product such as BMP-2 (Turgeman et al., 2001). It
was recently shown that in combination with BMP-2, hMSCs are able
to heal full-thickness non-union bone defects (Turgeman et al.,
2001), and in recent studies it was shown that human MSCs can be
transduced by retroviral vectors and maintain stable expression of
the therapeutic gene after in vivo transplantation (Lee et al.,
2001). Within these studies, the MSCs were isolated from the bone
marrow, expanded in culture, in some cases genetically engineered,
and transplanted in-vivo. The culture expansion stage is extremely
costly, time consuming, and in many cases, the cells may lose their
multipotentiality and fail to achieve the desired goal. In
contrast, very few studies described the use of non-cultured
freshly isolated human MSCs. Horwitz et al., (1999) showed that
human MSCs present in unprocessed bone marrow allografts engraft
and may provide stem cell reservoir for osteoblast differentiation
and renewal. The isolation of hMSCs-enriched population requires
efficient and reproductive isolation method. Few methods were
described for the isolation of MSCs, including enhancement of the
plastic adherence property of the cells by using selected lots of
fetal calf serum (Kadiyala et al., 1997, Pittenger et al., 1999),
immunomagnetic isolation based on the presence of the STRO-1
surface molecule (Gronthos and Simmons, 1995). Both methods are
very hard to be reproduced by other labs and no studies were done
to show the differentiation potential of cells before culture
expansion. Majumdar et al. (2000) showed that cells from human BM
aspirates were isolated by the anti-CD105 (endoglin) antibodies,
differentiated to chondrogenic cells after culture expansion, and
showed immunophenotype distinctive to MSCs, suggesting that these
CD105+ cells contain the osteogenic MSCs population.
SUMMARY OF THE INVENTION
[0004] The invention relates to methods of isolating non-culture
expanded mesenchymal stem cells for use in the preparation of
medicaments for tissue repair or formation.
[0005] A method of obtaining an isolated, non-culture expanded
mesenchymal stem cell, comprising the following steps: contacting a
human cell population with an antibody that binds to a surface
molecule expressed on a mesenchymal stem cell within the human cell
population, so as to form a cell-antibody-complex; recovering the
mesenchymal stem cell; maintaining the recovered mesenchymal stem
cell under conditions preventing significant cellular expansion;
thereby obtaining a non-culture expanded mesenchymal stem cell. In
another embodiment, the sample comprising a human cell population
is contacted with enrichment growth medium so as to obtain a mixed
sample prior to forming the cell-antibody-complex, as described
hereinabove.
[0006] In another embodiment, there is provided a method of use of
an isolated, non-culture expanded mesenchymal stem cell for the
preparation of a medicament for administration to a subject,
wherein the non-culture expanded mesenchymal stem cell is isolated
via obtaining a sample comprising a human cell population;
contacting the sample with an antibody that binds to a surface
molecule expressed on a mesenchymal stem cell so as to form a
cell-antibody-complex; recovering the mesenchymal stem cell,
thereby obtaining a non-culture expanded mesenchymal stem cell. In
another embodiment, the sample comprising a human cell population
is contacted with enrichment growth medium so as to obtain a mixed
sample prior to forming the cell-antibody-complex, as described
hereinabove.
[0007] In another embodiment, there is provided a method of use of
an isolated, non-culture expanded mesenchymal stem cell in the
preparation of a medicament for stimulating or enhancing tissue
repair in a subject, wherein the non-culture expanded mesenchymal
stem cell is isolated via obtaining a sample comprising a human
cell population; contacting the sample with an antibody that binds
to a surface molecule expressed on a mesenchymal stem cell so as to
form a cell-antibody-complex; recovering the mesenchymal stem cell,
thereby obtaining a non-culture expanded mesenchymal stem cell. In
another embodiment, the sample comprising a human cell population
is contacted with enrichment growth medium so as to obtain a mixed
sample prior to forming the cell-antibody-complex, as described
hereinabove.
[0008] In another embodiment, there is provided a method of use of
an isolated, non-culture expanded mesenchymal stem cell in the
preparation of a medicament for stimulating or enhancing tissue
formation in a subject, wherein the non-culture expanded
mesenchymal stem cell is is isolated via obtaining a sample
comprising a human cell population; contacting the sample with an
antibody that binds to a surface molecule expressed on a
mesenchymal stem cell so as to form a cell-antibody-complex;
recovering the mesenchymal stem cell, thereby obtaining a
non-culture expanded mesenchymal stem cell. In another embodiment,
the sample comprising a human cell population is contacted with
enrichment growth medium so as to obtain a mixed sample prior to
forming the cell-antibody-complex, as described hereinabove.
[0009] In another embodiment, there is provided a method of use of
an isolated, non-culture expanded mesenchymal stem cell in the
preparation of a medicament for maintaining or increasing bone
volume, bone quality, or bone strength in a subject, wherein the
non-culture expanded mesenchymal stem cell is isolated via
obtaining a sample comprising a human cell population; contacting
the sample with an antibody that binds to a surface molecule
expressed on a mesenchymal stem cell so as to form a
cell-antibody-complex; recovering the mesenchymal stem cell,
thereby obtaining a non-culture expanded mesenchymal stem cell. In
another embodiment, the sample comprising a human cell population
is contacted with enrichment growth medium so as to obtain a mixed
sample prior to forming the cell-antibody-complex, as described
hereinabove.
[0010] In another embodiment, this invention provides isolated,
non-culture expanded human adult mesenchymal stem cells. In one
embodiment, the isolated non-culture expanded human adult
mesenchymal stem cells express CD105, CD29 and/or CD44 cell surface
antigens.
BRIEF DESCRIPTION OF THE DRAWINGS
[0011] FIG. 1 shows the immunophenotype of bone marrow-derived
mononuclear cells. Mononuclear cells (MNCs) were obtained from
fresh human BM by separation on a density-gradient. Aliquots of
1.times.10.sup.6 cells were staied with the indicated mouse
anti-human antibodies. The percent (%) of cells in the quadrants
were determined.
[0012] FIG. 2 shows in vitro differentiation of expanded hMSCs
isolated with CD105 microbeads to osteogenic lineage. A: Alkaline
phosphatase activity in cell lysates was assayed at 1, 2, and 3
weeks after the addition of osteogenic supplement. Activity was
assessed as the release of p-nitrophenyl per minute normalized to
total cell protein (in micrograms). B: Calcium deposition measured
in cell lysates, assessed as the formation of
Calcium-Cresolphthalein Complexon complex, and expressed as optical
density (OD) at 575 nm, which is directly proportional to the
calcium concentration in the sample. Note significant increase
(*P<0.05) in the cells cultured with osteogenic supplement
(+Suppl.) compared to the cells cultured without osteogenic
supplement (-Suppl.). The bars represent the mean (.+-.SEM) ALP
(A), or calcium deposition (B), from three individual wells.
[0013] FIG. 3 demonstrates morphology and flow cytometry analysis
of culture-expanded CD105+ cells. The cells were isolated from bone
marrow using microbeads coupled-antibody against CD105, plated in
media and maintained in culture as indicated. Photomicrograph of
10-day cultured CD105+ cells (A, X 40), and whole BM-MNCs (B, X
40). C: Expression of surface molecules on culture expanded
(passage 3-5) CD105+ cells.
[0014] FIG. 4 demonstrates the in vivo osteogenic potential of
fresh BM-derived CD105+ cells. A 5-mm-diameter circular defect was
created in the radius of 6-8-week-old male CD-1 nude mice.
Non-cultured, fresh BM-derived CD105+ cells (isolated by the CD105
microbeads) were mounted on collagen sponges and transplanted in
the defect site. Twenty days post transplantation, mice were
sacrificed, calvariae dissected from other soft tissues, analyzed
by x-ray, and histologic analysis for evidence of new bone
formation. A, B: X-rays of the calvariae specimens transplanted
with BM-CD105+ (A), and BM-CD105- cells (B), note radio-opaque
region in the defect transplanted with BM-CD105+ cells (A, doubled
arrows). C, D, E, F: Micrograph of calvariae specimens transplanted
with BM-CD105+ cells (C, D), and BM-CD105- cells (E, F). Note newly
formed bone on the margins of the defect in specimen transplanted
with BM-CD105+ cells (arrows).
[0015] FIG. 5 demonstrates engraftment and differentiation of
non-cultured hMSCs transplanted at an ectopic site. Non-cultured
hMSCs, isolated by the RosetteSep.TM. were labeled with the
fluorescent cell tracer, DiI and implanted with rhBMP. Two weeks
following transplantation, implants were harvested, fixed and
embedded in OCT. A&B: H&E staining of sections of these
implants revealed newly formed cartilage and bone (arrowheads),
mainly at the periphery of the implant (original magnification: A:
10 X, B: 40 X). DiI-labeled cells with chondrocyte (D1, arrow) and
osteoblast (D2, doubled arrow) morphology were evident, as
well.
DETAILED DESCRIPTION OF THE INVENTION
[0016] The invention relates to methods of isolating and implanting
mesenchymal stem cells for tissue repair or formation, without
culture expansion of the mesenchymal stem cells. In one embodiment,
the invention relates to methods of isolating mesenchymal stem
cells from bone marrow, for repairing or inducing formation of a
bone without prior culture expansion of the mesenchymal stem
cells.
[0017] The tissue is, in one embodiment, a cartilage, or, in
another embodiment, a bone or in another embodiment, a ligament
tissue. In another embodiment, the tissue is any mammalian tissue
in need repair, such as neuronal tissue, striated muscle, cardiac
muscle, spleen, liver or kidney, wherein mesenchymal stem cells
play a role in such repair.
[0018] Mesenchymal Stem Cells (MSCs) are multi-potent cells that
can replicate yet maintain their status as undifferentiated cells
while possessing the potential to differentiate into specific
mesenchymal tissues lineages, including bone, cartilage, fat,
tendon, muscle and bone marrow stroma. As used herein, term "MSC"
or "mesenchymal stem cells" of the invention comprises, without
limitation, embryonic MSC, adult MSC or cord blood stem cells.
[0019] This invention provides, in one embodiment, a simple,
efficient, and fast platform, for using isolated mesenchymal cells,
without culture expansion of the cells prior to their incorporation
into a medicament for use in tissue repair or, in another
embodiment, for use in tissue formation, or, in another embodiment,
for use in bone repair.
[0020] The invention provides, in another embodiment, for the use
of isolated, non-culture expanded MSC cells in the preparation of a
medicament, for administration at any site of interest.
[0021] The term "use of isolated, non-culture expanded MSC cells in
the preparation of a medicament" refers hereinabove to use of the
isolated MSC cells immediately after their isolation or 0-12 hours
following their isolation, while avoiding the step of cellular
expansion in culture, i.e. cells are kept under conditions
significantly preventing their proliferation in culture, prior to
their use. By the terms "significantly preventing their
proliferation in culture" and "maintaining recovered mesenchymal
stem cell under conditions preventing significant cellular
expansion", it is to be understood that cellular proliferation is
drastically reduced, as a reflection of the culture conditions, the
length of time present in culture, or the environmental conditions
under which the recovered mesenchymal stem cells are kept.
[0022] In one embodiment, cells are cultured for short period of
time, which does not exceed 24 hours. In one embodiment, cells are
cultured between 1 and 24 hours. In another embodiment, cells are
cultured between 1 and 5, or in another embodiment, between 5 and
10, or in another embodiment, between 10 and 15, or in another
embodiment, 15 and 20, or in another embodiment, between 20 and 24
hours prior to their use for any method or application of this
invention. It is to be understood that any period of time that is
less than 24 hours is to be considered as part of this invention,
as long as the cells do not undergo expansion while in culture.
[0023] In one embodiment, the cells are engineered to express a
protein of interest prior to culture. In another embodiment,
engineered cells are directly utilized for the methods and/or
applications listed herein.
[0024] MSCs isolated via the methods disclosed herein, are not
culture expanded, hence do not come in contact with plastics or
polymers used in long-term cell culture procedures, they are not
damaged by stress conditions or infection, which are common
occurrences for cells subjected prolonged culture periods.
[0025] In one embodiment, MSCs isolated via the methods of this
invention, which are to be utilized for the applications listed
herein, are utilized immediately following isolation. In another
embodiment, cells are transiently cultured (ie less than 24 hours,
as discussed hereinabove), prior to their use. In another
embodiment, cells may be frozen following isolation, and stored for
any length of time that does not radically compromise cell
function, pluripotency or viability. Frozen cells may then be
thawed and used for methods and applications listed herein.
[0026] The term "mesenchymal stem cell" or "MSC" is used
interchangeably for cells which are not terminally differentiated,
which can divide without limit, to yield cells that are either stem
cells, or which, irreversibly differentiate to yield a new type of
cell. It is important to note that mesenchymal stem cells and
progenitors of the invention can be isolated from different source
tissues. In one embodiment, the source tissue is skin, or, in
another embodiment, bone marrow, or, in another embodiment, muscle,
or, in another embodiment, fat, or, in another embodiment, liver.
In addition, any cell type with stem cell properties or
demonstrating differentiation plasticity, for example, without
limitation, cells from the source of bone marrow, muscle, spleen or
any other tissue. Bone marrow cells may be obtained from iliac
crest, femora, tibiae, spine, rib or other medullary spaces. Other
sources of human mesenchymal stem cells include, without
limitation, embryonic yolk sac, placenta, fat, umbilical cord,
fetal and adolescent skin, muscle tissue and blood.
[0027] In one embodiment, there is provided a method of obtaining
an isolated, non-culture expanded mesenchymal stem cell, wherein
the cell is obtained by positive selection, via the use of an
antibody, which binds to epitope on the cell surface. The method
comprises the following steps: obtaining a sample comprising a
human cell population; contacting the sample with an antibody that
binds to a surface molecule so as to form a cell-antibody-complex;
recovering the mesenchymal stem cell; thereby obtaining an
isolated, non-culture expanded. The antibody may recognize CD105,
as is shown in Example 1 or, may also be other antibodies, which
bind to the mesenchymal stem cell surface molecule such as CD44 and
CD29. The antibodies may be supported on a column, plastic, array
or magnetic bead.
[0028] Upon forming an antigen-antibody complex, the
antigen-antibody complexes are separated from the other
subpopulations which are not bound to the antibody by a column, for
example. Where the antibody is supported by a magnetic bead, a
magnetic column can be used.
[0029] The step of recovering the cells from the antibodies is
performed by washes with suitable buffers, known to one skilled in
the art. In another embodiment, there is provided a method of
obtaining an isolated, non-culture expanded mesenchymal stem cell
by negative selection comprising the following steps: obtaining a
sample comprising a human cell population; administering to the
sample enrichment growth medium so as to obtain a mixed sample;
separating the mixed sample so as to obtain a mesenchymal stem
cell; recovering the mesenchymal stem cell; thereby obtaining an
isolated, non-culture expanded mesenchymal stem cell from a sample.
The enrichment growth medium can be, for example without
limitation, RosetteSep.TM. Mesenchymal Enrichment Cocktail
(StemCell Technologies BC, Canada). The step of separating the
mixed sample so as to obtain a mesenchymal stem cell is known in
the art and is performed, for example without being limited, by the
use of a centrifuge. In another embodiment the cells can be
negative selected by the use of antibodies, which are directed to
the surface of other subpopulation, which exist in the sample. For
example, as is shown in U.S. Pat. No. 5,965,436, a bone marrow
sample may be subjected to negative selection for removal of
megakaryocytes from other hematopoietic cells via antibody sorting
and mesenchymal cell subpopulations are than obtained. It is to be
understood that any method employing mesenchymal stem cell
selection, without culture expansion, whether via positive or
negative selection, is considered as part of this invention.
[0030] In another embodiment, the cell can be separated according
to U.S. Pat. No. 6,043,066 by utilizing electric fields to
selectively inactivate and render non-viable particular
subpopulations of cells in a suspension, while not adversely
affecting other desired subpopulations. According to the methods,
the cells can be selected on the basis of intrinsic or induced
differences in a characteristic electroporation threshold; which
can depend, for example, on a difference in cell size and/or
critical dielectric membrane breakdown voltage.
[0031] The presence of MSCs in isolated cells may be verified by
specific cell surface markers which are identified with unique
monoclonal antibodies, for example, see U.S. Pat. No.
5,486,359.
[0032] Example 1 demonstrated that when cells were positively
selected and isolated with CD105 antibodies, as well as by negative
selection, the CD105+ cells were found to stain positive for the
CD105 (endoglin), CD29 (Beta 1 Integrin) and CD44 (Hyaluronate)
surface markers. The isolated cells were found to stain negatively
for hematopoietic markers CD14 (macrophage marker), and CD45
(leukocyte common antigen). These results demonstrated that cells
isolated according to the methods presented herein were MSCs. In
addition, as shown assays measuring calcium deposition and ALP
activity, in cells cultured in the presence of ascorbate,
beta-glycerophosphate, and dexamethasone, demonstrated CD105+ MSC
differentiation to osteoblasts, thus, these isolated MSCs express
surface antigens characteristic of mesenchymal stem cells alone,
can regenerate in culture without differentiating, and can
differentiate into specific mesenchymal lineages when either
induced in vitro (Example 2) or in vivo, when placed at the site of
damaged tissue (Example 3).
[0033] Although human MSCs are normally present in bone marrow in
minute amounts, which greatly decrease with age (from about
1/10,000 cells in a relatively young patient to as few as
1/2,000,000 in an elderly patient), this invention provides a
process for obtaining isolated cells for administration to a
subject without prior culture expansion.
[0034] New bone formation was evident when non-cultured CD105+
cells were transplanted in nude mice that underwent critical-sized
non-healing skull defects (Example 3). These results demonstrated
that non-culture expanded bone marrow derived CD105+ cells are
osteogenic cells that can be used for bone repair. In addition, as
multi-potent cells, non-culture expanded MSCs can be utilized in
other application such as cell therapy for tissue repair,
hematopietic reconstitution and in any kind of procedure utilized
in regenerative medicine.
[0035] In another embodiment, there is provided a method of use of
an isolated, non-culture expanded mesenchymal stem cell in the
preparation of a medicament for administration to a subject,
wherein the non-culture expanded mesenchymal stem cell is obtained
via the methods described herein.
[0036] The medicament is formulated for use topically,
systemically, or locally as an injectable and/or transplant and/or
device, usually by adding necessary buffers. The cells when
suspended in appropriate buffers are referred to
compositions/cultures of non-culture expanded cells. When
formulated for administration, the non-culture expanded cells used
in this invention are, of course, in a pyrogen-free,
physiologically acceptable form. Further, the non-culture expanded
cells, may be injected in a viscous form for delivery to the site
of tissue damage. Topical administration may be suitable for wound
healing and tissue repair. In one embodiment, therapeutically
useful agents may also optionally be included in the cell
composition as described above, or, in other embodiments, may
alternatively or additionally, be administered simultaneously or
sequentially with the composition in the methods of the
invention.
[0037] In another embodiment, the compositions of the present
invention may be used in conjunction with presently available
treatments for tendon/ligament injuries, such as suture (e.g.,
vicryl sutures or surgical gut sutures, Ethicon Inc., Somerville,
N.J.) or tendon/ligament allograft or autograft, in order to
enhance or accelerate the healing potential of the suture or
graft.
[0038] The choice of a carrier material is based on
biocompatibility, biodegradability, mechanical properties, cosmetic
appearance and interface properties. The particular application of
the compositions/cultures of non-culture expanded cells will define
the appropriate formulation. In another embodiment the non-culture
expanded cells can be mixed with a matrix. Potential matrices for
compositions/cultures of non-culture expanded cells may be
biodegradable and chemically defined. Further matrices are
comprised of pure proteins or extracellular matrix components.
Other potential matrices are non-biodegradable and chemically
defined. Preferred matrices include collagen-based materials,
including sponges, is such as Helistat.RTM. (Integra LifeSciences,
Plainsboro, N.J.), or collagen in an injectable form, as well as
sequestering agents, which may be biodegradable, for example,
hyalouronic acid-derived. Biodegradable materials, such as
cellulose films, or surgical meshes, may also serve as matrices.
Such materials could be sutured into an injury site, or wrapped
around the tendon/ligament. Another preferred class of carriers are
polymeric matrices, where the cell of the invention can be mixed
with polymers of poly lactic acid, poly glycolic acid and
copolymers of lactic acid and glycolic acid. These matrices may be
in the form of a sponge, or in the form of porous particles, and
may also include a sequestering agent. Suitable polymer matrices
are described, for example, in WO93/00050.
[0039] In another embodiment, there is provided a method of use of
an isolated, non-culture expanded mesenchymal stem cell in the
preparation of a medicament for stimulating or enhancing tissue
repair in a subject, wherein the non-culture expanded mesenchymal
stem cell is obtained via the methods described herein. In one
embodiment, the mesenchymal stem cells are cultured for short
periods of time (less than 24 hours). In another embodiment, the
cells are transfected or transduced prior to their administration
to the subject. In another embodiment, the method is utilized for
repairing damage to connective tissue, or in another embodiment non
connective tissue.
[0040] In another embodiment, there is provided a method of use of
an isolated, non-culture expanded mesenchymal stem cell in the
preparation of a medicament for stimulating or enhancing tissue
formation in a subject, wherein the non-culture expanded
mesenchymal stem cell is obtained via the methods described
herein.
[0041] The term tissue includes, in one embodiment, a connective
tissue and in another embodiment a non-connective tissue.
[0042] The term "connective tissue" refers hereinabove to bone,
cartilage, ligament, skin, fat, tendon, muscle, meniscus and
interval disc tissue of a mammalian tissue and stroma.
[0043] In another embodiment, the compositions/cultures of
non-culture expanded cells of the invention may comprise, other
therapeutically useful agents such as, and without being limited
to, cytokines, chemokines, leukemia inhibitory factor
(LIF/HILDA/DIA), migration inhibition factor, MP52, growth factors
including epidermal growth factor (EGF), fibroblast growth factor
(FGF), platelet derived growth factor (PDGF), transforming growth
factors (TGF-alpha and TGF-beta), and fibroblast growth factor-4
(FGF-4), parathyroid hormone (PTH), insulin-like growth factors
(IGF-I and IGF-II), or combinations thereof. Portions of these
agents may also be used in compositions of the present invention.
Such a composition may be useful for treating defects of the
embryonic joint where tendon, ligaments, and bone form
simultaneously at contiguous anatomical locations, and may be
useful for regenerating tissue at the site of tendon attachment to
bone. It is contemplated that the compositions of the invention may
also be used in wound healing, such as skin healing and related
tissue repair. The types of wounds include, but are not limited to
burns, incisions and ulcers.
[0044] In another embodiment at least one other agent can be added
such as agent that promotes hematopoiesis, such as, for example a
cytokine, which participates in hematopiesis. Some non-limiting
examples are: CSF-1, G-CSF, GM-CSF, interleukins, interferons, or
combinations thereof.
[0045] In another embodiment, an agent that promotes the delivery
of systemic proteins such as Factor IX, VIII, Growth hormone etc.
may be provided to the subject following the incorporation of
engrafted mesenchymal stem cells into bone marrow following
transplantation.
[0046] As used herein, the term "inducing formation" refers to a
use in tissue renewal or regeneration so as to ameliorate
conditions of tissue, degeneration, depletion or damage such as
might be caused by aging, genetic or infectious disease, accident
or any other cause, in humans, livestock, domestic animals or any
other animal species. In another embodiment the tissue formation is
required for tissue development in livestock, domestic animals or
any other animal species in order to achieve increased growth for
commercial or any other purpose. In another embodiment the tissue
formation is required in plastic surgeries, such as, and without
being limited to, facial reconstruction in order to obtain a
stabilized shape.
[0047] As used herein, the term "enhancing tissue repair" or
"repairing" refers to healing and/or regeneration of tissue
injuries, tears, deformities or defects, and prophylactic use in
preventing tissue damage.
[0048] In another embodiment, the invention provides a method of
use of an isolated, non-culture expanded mesenchymal stem cell in
the preparation of a medicament for treating and preventing
osteoporosis, which results from a decrease in estrogen, which may
be caused by menopause or ovariectomy in women. Use of isolated,
non-culture expanded mesenchymal stem cell in the preparation of a
medicament for prevention of accelerated bone resorption and
inhibition of a decrease of bone volume, bone quality and bone
strength is also provided by the invention. In accordance with the
methods of the invention, a pharmaceutical composition containing
an extract from inflamed tissue as an effective component may be
used to maintain or increase bone volume, bone quality, and bone
strength. Trabecular connectivity and trabecular unconnectivity may
be maintained at healthy levels with the pharmaceutical
compositions of the present invention. Osteoporosis and its
symptoms such as decreased bone volume, bone quality, and bone
strength, decreased trabecular connectivity, and increased
trabecular unconnectivity may be treated or prevented by
administration of a pharmaceutically effective amount of the
extract to a patient in need thereof.
[0049] Thus, there is provided a method for maintaining or
increasing bone volume, bone quality, or bone strength in a
subject, comprising the use of an isolated, non-culture expanded
mesenchymal stem cell in the preparation of a medicament for
maintaining or increasing bone volume, bone quality, or bone
strength in a subject, wherein the non-culture expanded mesenchymal
stem cell is obtained via the methods described herein.
[0050] It is to be understood that the production of any
composition or medicament that utilizes non-culture expanded
mesenchymal stem cells, is to be considered as part of this
invention.
[0051] The compositions/medicaments/cultures of this invention may
be utilized in the following applications: (1) for regenerating
mesenchymal tissues which have been damaged through acute injury,
abnormal genetic expression or acquired disease; (2) repairing
damaged mesenchymal tissue by removal of small aliquots of bone
marrow, isolation of their mesenchymal stem cells and treatment of
damaged tissue with non cultured MSCs combined with a biocompatible
carrier suitable for delivering MSCs to the damaged tissues
site(s); (3) for producing various mesenchymal tissues; (4) for
detecting and evaluating growth factors relevant to MSC
self-regeneration and differentiation into committed mesenchymal
lineages; (5) for detecting and evaluating inhibitory factors which
modulate MSC commitment and differentiation into specific
mesenchymal lineages; (6) for developing mesenchymal cell lineages
and assaying for factors associated with mesenchymal tissue
development. MSCs have pivotal role in the bone marrow environment
and have the ability to support hematopoiesis as indicated by many
crucial cytokines and growth factors that they constitutively
express and secrete (Majumdar et al., 2000); (7) for stimulation of
skeletal development in livestock, domestic animals or any other
animal species in order to achieve increased growth for commercial
or any other purpose; and (8) for treatment of neoplasia or
hyperplasia of bone or cartilage or any other tissue, in humans,
livestock, domestic animals or any other animal species
[0052] For these reasons, it's obvious that MSCs are very important
for hematopoietic reconstitution. The non-cultured MSCs can be used
in combination with hematopoietic transplants.
[0053] In another embodiment, the cells can be genetically
engineered to express a protein of interest prior to the
application to the subject in need. The protein of interest is any
macromolecule, which is necessary for cell growth, morphogenesis,
differentiation, tissue building or combinations thereof. These
are, for example, a bone morphogenic protein, a bone
morphogenic-like protein, an epidermal growth factor, a fibroblast
growth factor, a platelet derived growth factor, an insulin like
growth factor, a transforming growth factor, a vascular endothelial
growth factor, cytokines related to hematopoiesis, factors for
systemic delivery as such as GH, factor VIII, factor IX or
combinations thereof.
[0054] The term "cells engineered to express a protein of interest"
is defined hereinabove as a cell or to a tissue which had been
modified via molecular biologic techniques, for example via
recombinant DNA technology, to express any macromolecule which is
necessary for cell growth, morphogenesis, differentiation, tissue
building or combinations thereof. In another embodiment, cells are
thus modified in order to produce an increased amount of any
macromolecule, which is necessary for cell growth, morphogenesis,
differentiation, tissue building or combinations thereof. The term
"increased amount" refers hereinabove to at least 10 times more
than an amount normally produced.
[0055] The step of `genetically engineered a cell to express a
protein of interest` is performed by the transfection or
transduction of the cell with a nucleic acid encoding the protein
of interest.
[0056] The term "transfection" or "transfected cells" refer to
cells in which DNA is integrated into the genome by a method of
transfection, i.e. by the use of plasmids or liposomes.
[0057] The term "transduction" or "transduced cells" refers to
viral DNA transfer for example, by phage or retroviruses. The
nucleic acid, which encodes the protein of interest, can be
introduced by a vector molecule, as well, and represents an
additional embodiment of this invention.
[0058] The vector molecule can be any molecule capable of being
delivered and maintained, within the target cell, or tissue such
that the gene encoding the product of interest can be stably
expressed. In one embodiment, the vector utilized in the present
invention is a viral or retroviral vector or a non-viral DNA
plasmid. According to one aspect, the method includes introducing
the gene encoding the product into the cell of the mammalian tissue
for a therapeutic or prophylactic use. -The viral vectors, used in
the methods of the present invention, can be selected from the
group consisting of (a) a retroviral vector, such as MFG or pLJ;
(b) an adeno-associated virus; (c) an adenovirus; and (d) a herpes
virus, including but not limited to herpes simplex 1 or herpes
simples 2 or (e) lentivirus. Alternatively, a non-viral vector,
such as a DNA plasmid vector, can be used. Any DNA plasmid vector
known to one of ordinary skill in the art capable of stable
maintenance, within the targeted cell, or tissue upon delivery,
regardless of the method of delivery utilized is within the scope
of the present invention. Non-viral means for introducing the gene
encoding for the product into the target cell are also within the
scope of the present invention. Such non-viral means can be
selected from the group consisting of (a) at least one liposome,
(b) Ca.sub.3 (PO.sub.4).sub.2, (c) electroporation, (d)
DEAE-dextran, and (e) injection of naked DNA.
[0059] The term "nucleic acid" refers to polynucleotides or to
ologonucleotides such as deoxyribonucleic acid (DNA), and, where
appropriate, ribonucleic acid (RNA) or mimetics thereof. The term
should also be understood to include, as equivalents, analogs of
either RNA or DNA made from nucleotide analogs, and, as applicable
to the embodiment being described, single (sense or antisense) and
double-stranded polynucleotides. This term includes
oligonucleotides composed of naturally occurring nucleobases,
sugars and covalent internucleoside (backbone) linkages as well as
oligonucleotides having non-naturally-occurring portions which
function similarly. Such modified or substituted oligonucleotides
are often preferred over native forms because of desirable
properties such as, enhanced cellular uptake, enhanced affinity for
nucleic acid target and increased stability in the presence of
nucleases.
[0060] The dosage of the treatment, which is the amount of the
cells that are administered in order to obtain a therapeutic
effect, is affected by various factors which modify the action of
the non-cultured cells' composition, e.g., amount of tendon or
ligament tissue desired to be formed, the site of tendon or
ligament damage, the condition of the damaged tendon or ligament,
the size of a wound, type of damaged tissue, the patient's age,
sex, and diet, the severity of any infection, time of
administration and other clinical factors. The dosage may vary with
the type of matrix used in the reconstitution and the types of
additional proteins in the composition. The addition of other known
growth factors, such as IGF-I (insulin like growth factor I), to
the final composition, may also affect the dosage. Progress can be
monitored by periodic assessment of tendon/ligament-like tissue
formation, or tendon or ligament growth and/or repair. The progress
can be monitored by methods known in the art, for example, X-rays
(CT), ultra-sound, MRI, arthroscopy and histomorphometric
determinations.
[0061] In another embodiment, this invention provides non-culture
expanded human adult mesenchymal stem cells. The cells are isolated
via the methods disclosed herein, and may be utilized in any of the
above-mentioned applications.
[0062] In one embodiment, the mesenchymal stem cells will express
CD105, CD29 and/or CD44 cell surface antigens. In another
embodiment, at least 50%, or in another embodiment, at least 55%,
or, in another embodiment, at least 60%, or, in another embodiment,
at least 65%, or, in another embodiment, at least 70%, or, in
another embodiment, at least 75%, or, in another embodiment, at
least 80%, or, in another embodiment, at least 85%, or, in another
embodiment, at least 90%, or, in another embodiment, at least 95%,
or, in another embodiment, from about 95-100%, of the mesenchymal
stem cells expressing CD105, will express CD29 and/or CD44 cell
surface antigens.
[0063] In another embodiment, less than 25% of the mesenchymal stem
cells expressing CD105 will express CD45, CD14, CD34 and/or CD31
cell surface antigens. In another embodiment, less than 20%, or, in
another embodiment, less than 15%, or, in another embodiment, less
than 10%, or, in another embodiment, less than 7%, or, in another
embodiment, less than 5%, or, in another embodiment, between 0 and
5% of the mesenchymal stem cells expressing CD105, will express
CD45, CD14, CD34 and/or CD31 cell surface antigens.
[0064] It is to be understood that the mesenchymal stem cells are
capable of further differentiation to cells of mesenchymal tissue
lineage. As discussed hereinabove, the mesenchymal tissue lineage
may comprise bone, cartilage, fat, tendon, ligament, muscle or
marrow stroma.
[0065] In another embodiment, the cells of the invention may be
engineered to express at least one protein of interest, where, the
protein of interest may comprise a macromolecule necessary for cell
growth, morphogenesis, differentiation, tissue building or
combinations thereof, each representing a separate embodiment of
the invention.
[0066] In another embodiment, the macromolecule necessary for cell
growth, morphogenesis, differentiation, and/or tissue building is a
bone morphogenic protein, a bone morphogenic-like protein, a
cytokine, a chernokine, a hormone, an epidermal growth factor, a
fibroblast growth factor, a platelet derived growth factor, an
insulin like growth factor, a transforming growth factor, a
vascular endothelial growth factor, Ang-1, PIGF or combinations
thereof.
EXAMPLES
Experimental Procedures
[0067] Isolation of hMSCs-Enriched Cell Populations:
[0068] 1) Immunomaznetic Isolation of BM-CD105+ Cells:
[0069] Human Bone Marrow (BM) was recovered from heparinized
trabecular bone samples obtained from patients undergoing
corrective orthopaedic surgery (approved by the Helsinki Committee
Board of the Hadassah Medical Center, Jerusalem, Israel). The
BM-containing trabecular bone sample was flushed with PBS
(Biological Industries, Kibbutz Beit Haemek, Israel). To isolate
mono-nuclear cells, whole BM was layered over lymphocyte separation
medium (LSM, ICN-Cappel Inc., Aurora, Ohio, USA), and centrifuged
at 900 g for 30 minutes, room temperature, without a break.
[0070] Mononuclear cells were washed once with PBS and twice with
magnetic-activated cell sorting (MACS) buffer (PBS with 0.5% BSA, 2
mM EDTA, PH7.2), counted, and resuspended in MACS buffer at a
concentration of 10.sup.7 cells per 80 ul, transferred to 1.5 ml
test tube, to which directly conjugated mouse anti-human CD105
antibody-microbeads (Miltenyi Biotec, Germany) were added and
placed on rotator for 45-50 min at 4.degree. C. in the dark. The
cells were washed with PBS, resuspended in MACS buffer and
separated on a magnetic column MS+ according to the manufacturer's
recommendation. Cells that passed through the column were
considered CD105- cells. To recover the CD105+ cells, the column
was removed from the magnet and the cells were flushed out with
MACS buffer. The CD105-, and CD105+ cells were then recovered by
centrifugation for future use.
[0071] 2) BM-hMSCs Enrichment:
[0072] Human BM cell aliquots, flushed from trabecular bone samples
with complete growth medium, were mixed with RosetteSep.TM.
Mesenchymal Enrichment Cocktail (StemCell Technologies, BC, Canada)
at 50 ul cocktail/1 ml BM, and incubated at room temperature for 20
minutes. The BM-enrichment cocktail mixture was then diluted with 2
volumes of PBS containing 2% FCS and 1 mM EDTA, layered over LSM
and centrifuged at 900 g for 30 minutes, room temperature, without
brake.
[0073] Mononuclear cells collected from the interface were
considered as mesenchymal stem cells-enriched populations, and were
washed with PBS and counted for further use.
[0074] Cell Culture:
[0075] Either CD105+ or MSCs-enriched populations were resuspended
with DMEM supplemented with 2 mM L-glutamine, 100 units/ml
penicillin, 100 units/ml streptomycin, and 10% FCS (Biological
Industries, Kibbutz Beit Haemek, Israel) and plated in tissue
culture dishes at a density of 10,000-15,000 cells/cm.sup.2 growth
area, at 37.degree. C. in 5% CO.sub.2 in air. Medium was changed
after 72 h and then after 3-4 days. At day 14-16, cells were
detached by incubation with 0.25% trypsin-EDTA for 5-10 min. The
cells were then re-plated at a density of 5000-6000 cells/cm.sup.2
for expansion. The cells were subcultured as mentioned when reached
90% confluency, and were used for assays or stored in 85% complete
medium (DMEM+10% FCS), 5% BSA and 10% DMSO (Sigma, St. Louis, Mo.,
USA) in liquid nitrogen for future use.
[0076] Flow Cytomeotry
[0077] Aliquots (0.5-1.5.times.10.sup.6 cells) of fresh human bone
marrow mononuclear cells or culture-expanded hMSCs were used for
the analysis of cell surface molecules. Cells were washed with PBS,
resuspended with FACS buffer consisting of 2% BSA and 0.1% sodium
azide (Sigma, St. Louis, Mo., USA) in PBS, and stained with the
fluorochrome-conjugated mouse anti-human CD 105 (Serotec), CD44,
CD29, CD14, CD34, CD45 monoclonal antibodies (DAKO) according to
the manufacturer's recommendation, using the mouse monoclonal
isotype antibodies (IgG1, IgG2) to determine non-specific
fluorescence. Cells were then washed with PBS and resuspended with
0.5 ml FACS buffer and analysed for the expression of the human
indicated antigens by FACscan (Becton-Deckinson), and the
Cell-Quest software for data collection and analysis.
[0078] In-Vitro Differentiation Assays.
[0079] In order to induce the osteogenic differentiation of hMSCs
in vitro, bone marrow derived hMSCs isolated either by the CD105
microbeads or the RosetteSep.TM. enrichment cocktail were plated at
a density of 3000 cells/cm.sup.2 in DMEM+10% FCS containing
osteogenic supplement consisting of: 0.05 mM ascorbic
acid-2-phosphate, 10 mM beta-glycerophosphate, and 0.1 M
dexamethasone (Sigma, St. Louis, Mo., USA). Cells grown with
DMEM+10% FCS without osteogenic supplement were used as the
negative control. At 1, 2, and 3 weeks after addition of
supplement, cells were lysed with alkaline buffer solution (Sigma,
St. Louis, Mo., USA), containing 0.5% Triton X100, and 10 mM MgCl2
(for alkaline phosphatase, ALP assay), or 0.5 N HCl solution (for
calcium deposition assay). For ALP assay, lysates were incubated
with assay buffer containing 0.75 M 2-amino-2-methyl-1-propranolol
pH 10.3 for 10 min at 37C with p-nitrophenylphosphate as a
substrate. For calcium depositions assay, cell lysates were
incubated at 4 C, for 24 hrs with gentle shaking. Samples were then
assayed for calcium content using the Calcium kit (Sigma, St.
Louis, Mo., USA). Protein content was mesaured using the BCA
protein assay kit (Pierce, Rockford, Ill.).
[0080] BM-hMSCs Transplantation at an Ectopic Site:
[0081] BM-hMSCs isolated via either CD105 microbeads or
RosetteSep.TM. cocktail were immediately labeled with Vybrant DiI
cell-labeling solution (Molecular Probes). Labeling was performed
by re-suspending cells in serum-free DMEM at a concentration of
1.times.10.sup.6 cells/ml, mixed with 15 .mu.l per ml Vybrant DiI
solution, and incubated for 30 minutes at 37.degree. C. and 5%
CO.sub.2 on shaker, in the dark. DiI-labeled non-cultured cells
(1-1.5.times.10.sup.6/implant) were mixed with 5 .mu.g recombinant
human BMP-2 (rhBMP2), mounted on a collagen sponge (Duragen) and
transplanted subcutaneously into 6-8 week old NOD/SCID mice.
Implants were harvested 2 and 4 weeks post-transplantation.
[0082] Transplantations into Skull Defects:
[0083] For the skull-defect assay, we used male CD-1 nude mice. The
mice were anesthetized with ketamine-xylazine mixture, the scalp
was dissected to the skull and a 5-mm-diameter full-thickness
circular skull defect. This defect, a nonhealing critical-sized
defect (Krebsbach et al., 1998), was created at the apex of the
skull with use of a dental burr, with minimal penetration of the
dura. The mice were divided into three groups, and a collagen
sponge (Duragen) cut to form-fit the defect was used as a matrix.
The scalp was closed with use of nylon suture. At 3 weeks, mice
were sacrificed and the skull specimens were dissected free from
the soft tissue and fixed in 4% formalin, decalcified by decal
rapid solution, paraffm embedded, and 5-7 micron sections were
prepared and stained by H&E for the evaluation of new bone
formation.
Experimental Results
Example 1
[0084] Isolation of hMSCs and Flow Cytometry:
[0085] In order to characterize the CD105+ cell population in human
bone marrow (BM), human BM-derived mononuclear cells were analyzed
for co-expression of CD105 antigen and other hematopoietic markers.
Marker FACS analysis of freshly separated human BM mononuclear
cells demonstrated that CD105 was co-expressed with other
hematopoietic markers at low levels, except for CD45, which was
co-expressed at higher percentages on CD105 cells (about 40%, FIG.
1C). More importantly, however, was the minimal expression of CD14
(a monocyte marker) on CD105+ cells, indicating that macrophages,
the hematopoietic cell population from bone marrow with the
greatest adherence to plastic, did not comprise the CD105+ cell
population isolated (FIG. 1E).
[0086] CD105+ cells were isolated from human BM mononuclear cells
via CD105-microbeads, then plated for expansion. Colonies of
fibroblastic-like cells appeared within 7-10 days following initial
plating, and were a morphologically homogenous population, whereas
whole BM mononuclear cells (without separation via CD105
microbeads) were a mixed population of cells, consisting of
fibroblast- and macrophage-like cells (FIGS. 2A and 2B). Marker
FACS analysis of the culture-expanded hMSCs (passages 3-6) isolated
by this method demonstrated cells positive for CD105, CD29 and CD44
and negative for CD14 and CD45 (FIG. 2C).
Example 2
[0087] In vitro osteogenic differentiation:
[0088] In order to determine the in vitro osteogenic potential of
CD105+ cells, CD105+ cells were cultured following expansion
(passages 3-5) under conditions inducing differentiation of
mesenchymal stem cells to cells of osteogenic lineage (Pittenger et
al., 1999). Significant increase in alkaline phosphatase activity
was evident in induced hMSCs (with supplement), as compared to
non-induced hMSCs (without supplement), during the 3 weeks of
differentiation (FIG. 3A). Continual, significant increase in
calcium deposition (expressed as OD at 575 nm) was observed during
the differentiation period (1, 2, and 3 weeks, FIG. 3B).
Example 3
[0089] Non-Culture Expanded, CD105+ hMSCs Form Cartilage and Bone
in vivo:
[0090] Freshly isolated, non-culture expanded hMSCs isolated via
CD105 microbead separation were transplanted into skull-defects
induced in CD-1 nude mice, in order to determine their in vivo
osteogenic potential. New bone formation was observed in sections
processed and stained with H&E, whereas no evidence of newly
formed bone was observed in similarly injured mice transplanted
with collagen inserts containing CD105- cells (FIG. 4) or collagen
inserts alone (data not shown).
[0091] Osteogenic differentiation of non-culture expanded hMSCs in
vivo was also demonstrated via implanting the cells into NOD/SCID
mice. Non-culture expanded hMSCs (1-1.5.times.10.sup.6 cells)
labeled with DiI were mixed with rhBMP2, loaded onto collagen
sponges (Duragen) serving as scaffolding and transplanted under the
skin of NOD/SCID mice. Cartilage and bone formation was detected in
harvested implants, two weeks post-transplantation (FIGS. 5A and
B). Non-culture expanded hMSCs were identified by confocal
microscopy in newly formed cartilage and bone tissue. DiI-labeled
chondrocytes (FIG. 5C) and osteoblasts (FIG. 5D) were detected
within the implant.
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