U.S. patent application number 13/363399 was filed with the patent office on 2012-05-24 for adipose tissue-derived stromal cells and methods of isolating and using the same.
Invention is credited to Keith L. March, Jalees Rehman.
Application Number | 20120129253 13/363399 |
Document ID | / |
Family ID | 28454666 |
Filed Date | 2012-05-24 |
United States Patent
Application |
20120129253 |
Kind Code |
A1 |
March; Keith L. ; et
al. |
May 24, 2012 |
ADIPOSE TISSUE-DERIVED STROMAL CELLS AND METHODS OF ISOLATING AND
USING THE SAME
Abstract
Adipose tissue-derived stromal cells and methods of isolating
and using the same. In at least one embodiment of isolated adipose
tissue-derived stromal cells of the present disclosure, the cells
are isolated by performing adipose tissue resection or suction on a
mammalian patient, dissecting tissue obtained from said tissue
resection or suction and dissociating said tissue into a cell
suspension, removing adipocytes from the cell suspension, culturing
the adipocyte-depleted cell suspension in EGM-2-MV media, and
isolating adipose tissue-derived stromal cells secreting vascular
endothelial growth factor (VEGF), hepatocyte growth factor (HGF),
and granulocyte-colony stimulating factor (G-CSF).
Inventors: |
March; Keith L.; (Carmel,
IN) ; Rehman; Jalees; (Zionsville, IN) |
Family ID: |
28454666 |
Appl. No.: |
13/363399 |
Filed: |
February 1, 2012 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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13279222 |
Oct 21, 2011 |
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13363399 |
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12569887 |
Sep 29, 2009 |
8067234 |
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13279222 |
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10508223 |
Jun 23, 2005 |
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PCT/US03/08582 |
Mar 19, 2003 |
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12569887 |
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60365498 |
Mar 19, 2002 |
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Current U.S.
Class: |
435/325 |
Current CPC
Class: |
A61P 9/00 20180101; C12N
2506/08 20130101; C12N 5/0619 20130101; C12N 2506/1384 20130101;
C12N 2506/13 20130101; C12N 2501/11 20130101; C12N 2501/115
20130101; C12N 2501/165 20130101; C12N 5/0653 20130101; A61K 35/12
20130101; C12N 5/0667 20130101; C12N 2501/105 20130101 |
Class at
Publication: |
435/325 |
International
Class: |
C12N 5/077 20100101
C12N005/077; C12N 5/10 20060101 C12N005/10 |
Claims
1. An isolated adipose tissue-derived stromal cell.
2. The isolated cell of claim 1, which expresses at least one
characteristic of transformed cell selected from the group
consisting of a cardiac cell, an endothelial cell, a smooth muscle
cell, a dopaminergic neuronal cell, and a hepatic cell.
3. The isolated cell of claim 1 which secretes at least one factor
selected from the group consisting of vascular endothelial growth
factor (VEGF), hepatocyte growth factor (HGF), granulocyte-colony
stimulating factor (G-CSF), and basic fibroblast growth factor
(bFGF).
4. The isolated cell of claim 3, cultured on matrigel.
5. The isolated cell of claim 4, cultured in DMEM and 10% fetal
bovine serum which express alpha-actin.
6. The isolated cell of claim 1, cultured in the presence of
5-azacytidine.
7. The isolated cell of claim 1, which secretes a factor selected
from the group consisting of pro-angiogenic factors, anti-apoptotic
factors, vasculoprotective factors, and cardioprotective
factors.
8. The isolated cell of claim 1, initially cultured in EGM-2-MV
media followed by subsequent culture in EBM-2 media, said cell
secreting vascular endothelial growth factor (VEGF), hepatocyte
growth factor (HGF), and granulocyte-macrophage colony stimulating
factor (GM-CSF) and granulocyte-colony stimulating factor
(G-CSF).
9. The isolated cell of claim 1, which expresses CD34 and does not
express VE-cadherin.
10. The isolated cell of claim 1, wherein mechanical separation is
performed on adipose tissue to dissociate the adipose tissue into a
cell suspension containing the isolated adipose tissue-derived
cell, the mechanical separation selected from the group consisting
of mechanical agitation, sonic energy, thermal energy, and a
combination of two or more of the same.
11. The isolated cell of claim 1, wherein chemical separation is
performed on adipose tissue to dissociate the adipose tissue into a
cell suspension containing the isolated adipose tissue-derived
cell, the chemical separation selected from the group consisting of
collagenase separation, dispase separation, trypsin separation, and
a combination of two or more of the same.
12. A defined cell population comprising the cell of claim 1.
13. The cell population of claim 12, wherein said cells comprise an
exogenous nucleic acid encoding a protein of interest.
14. The cell population of claim 13, wherein said exogenous nucleic
acid is selected from the group of nucleic acids encoding VEGF,
bFGF, IGF1, IGF2, HGF, cardiotrophin, myotrophin, nitric oxide
synthase 1, nitric oxide synthase 2, nitric oxide synthase 3, tumor
necrosis factor alpha, tumor necrosis factor beta, fibroblast
growth factor, pleotrophin, endothelin, and angiopoietin.
15. The isolated cell of claim 1, wherein the isolated cell is
obtained from a mammalian patient and is subsequently administered
back to the patient to treat the patient.
16. The isolated cell of claim 1, wherein the isolated cell is
initially harvested within a plurality of cells from a mammalian
patient, wherein the harvesting is selected from the group
consisting of liposuction and surgery.
17. Adipose tissue-derived stromal cells, isolated by: performing
adipose tissue resection or suction on a mammalian patient;
dissecting tissue obtained from said tissue resection or suction
and dissociating said tissue into a cell suspension; removing
adipocytes from the cell suspension; and isolating adipose
tissue-derived stromal cells from the cell suspension with
adipocytes removed.
18. The adipose tissue-derived stromal cells of claim 17, wherein
the step of removing adipocytes from the cell suspension further
comprises the step of exposing the cell suspension to red cell
lysis buffer.
19. The adipose tissue-derived stromal cells of claim 17, wherein
the adipose tissue-derived stromal cells are subsequently cultured
in EGM-2-MV media to effectively multiply the number of the
isolated adipose tissue-derived stromal cells.
20. The adipose tissue-derived stromal cells of claim 19, wherein
the adipose tissue-derived stromal cells secrete at least one
factor selected from the group consisting of a vascular endothelial
growth factor (VEGF), a hepatocyte growth factor (HGF), and a
granulocyte-colony stimulating factor (G-CSF).
21. Adipose tissue-derived stromal cells, isolated by: performing
adipose tissue resection or suction on a mammalian patient;
dissecting tissue obtained from said tissue resection or suction
and dissociating said tissue into a cell suspension; removing
adipocytes from the cell suspension; culturing the
adipocyte-depleted cell suspension in EGM-2-MV media; and isolating
adipose tissue-derived stromal cells secreting vascular endothelial
growth factor (VEGF), hepatocyte growth factor (HGF), and
granulocyte-colony stimulating factor (G-CSF).
22. The adipose tissue-derived stromal cells of claim 21, wherein
the isolated adipose tissue-derived stromal cells contain
pluripotent cells capable of differentiating into another cell
type.
Description
PRIORITY
[0001] The present application is related to, claims the priority
benefit of, and is a divisional application of, U.S. patent
application Ser. No. 13/279,222, filed Oct. 21, 2011, which is
related to, claims the priority benefit of, and is a divisional
application of, U.S. patent application Ser. No. 12/569,887, filed
Sep. 29, 2009 and issued as U.S. Pat. No. 8,067,234 on Nov. 29,
2011, which is related to, claims the priority benefit of, and is a
divisional application of, U.S. patent application Ser. No.
10/508,223, filed Jun. 23, 2005, which is related to, claims the
priority benefit of, and is a U.S. .sctn.371 National Stage
Application of, International Patent Application Serial No.
PCT/US2003/008582, filed Mar. 19, 2003, which is related to, and
claims the priority benefit of, U.S. patent application Ser. No.
60/365,498, filed Mar. 19, 2002. The contents of each of these
applications are hereby incorporated by reference in their entirety
into this disclosure.
BACKGROUND
[0002] The present disclosure relates to the fields of cardiology,
vascularization, and molecular biology. More specifically, methods
are provided for isolating stem cells from adipose tissue,
optionally inducing the stem cells to secrete endogenous or
exogenously provided growth factors, and re-administering such stem
cells to a patient for therapeutic benefit.
[0003] Several publications and patent documents are cited
throughout the specification in order to describe the state of the
art to which this disclosure pertains. Each of these references is
incorporated herein as though set forth in full.
[0004] Coronary artery disease (CAD) is a major cause of morbidity
and mortality, requiring bypass surgery or angioplasty in almost
1,000,000 patients/year in the USA. While some of these patients
form collateral vessels as alternative pathways for blood supply,
thus ameliorating or preventing ischemic myocardial damage, many do
not form the vascular networks to sufficiently compensate for the
loss of the original blood supply. Accordingly, many patients could
be helped by the development of compositions and methods which
would accelerate natural processes of post-natal collateral vessel
formation. Such approaches are broadly referred to as "therapeutic
angiogenesis", and encompass both angiogenesis (which strictly
speaking refers to capillary sprouting) and arteriogenesis (the
maturation and enlargement of existing vessels) (Isner J. M. and
Asahara T., J Clin Invest. (1999) 103:1231-1236; van Royen N. et
al., Cardiovasc Res. (2001) 49:543-553.)
[0005] An emerging therapeutic approach is the use of stem and
progenitor cell transplantation to improve angiogenesis.
Endothelial progenitor cells (EPCs) are cells present in bone
marrow or peripheral blood which co-express stem and progenitor
cell markers like CD34 or AC133, as well as endothelial markers
like VE-Cadherin and VEGF-Receptor-2 (KDR) (Rafii S., J Clin
Invest. (2000) 105:17-19). EPCs and hematopoietic stem cells (HSCs)
are thought to be derived from a common "hemangioblast" precursor
(Ribatti D. et al., J Hematother Stem Cell Res. (2000) 9:13-19;
Choi K., J Hematother Stem Cell Res. (2002) 11:91-101; Eichmann A.
et al., J Hematother Stem Cell Res. (2002) 11:207-214).
Interestingly, the cell surface marker CD34 is only found on either
hematopoietic stem/progenitor cells or endothelial cells (Rafii S.,
J Clin Invest. (2000) 105:17-19), which may be a reflection of the
common origin of these two cell lineages. In addition to the shared
"hemangioblastic" ancestry between HSCs and endothelial cells, HSCs
have also been suggested to trans-differentiate into either
endothelial progenitor cells or mature endothelial cells (Kang H.
J. et al., Br J Haematol. (2001) 113:962-969; Quirici N. et al., Br
J Haematol. (2001) 115:186-194; Gehling U. M. et al., Blood. (2000)
95:3106-3112). The recent discovery of circulating smooth muscle
progenitor cells, and the potential of HSCs to differentiate into
smooth muscle cells (Sata M. et al., Nat Med. (2002) 8:403-409;
Simper D. et al., Circulation (2002) 106:1199-1204) may suggest yet
another novel and intriguing link between the hematopoietic and
vascular cell lineages, now in the context of smooth muscle
cells.
[0006] Animal studies using hindlimb ischemia or myocardial
ischemia models in immune deficient rodents have demonstrated that
transplantation of about 10.sup.6 peripheral blood derived
[0007] EPCs (Kawamoto A. et al., Circulation (2001) 103:634-637;
Kalka C. et al., Proc Natl Acad Sci USA (2000) 97:3422-3427) can
result in increased angiogenesis. Remarkably, labeled peripheral
blood derived EPCs appear to home preferentially to ischemic areas
and incorporate into foci of neovascularization (Kawamoto A. et
al., Circulation (2001) 103:634-637; Kalka C. et al., Proc Natl
Acad Sci USA (2000) 97:3422-3427). In addition to the
above-mentioned studies on peripheral blood-derived cells, EPCs
derived from bone marrow, unpurified bone marrow mononuclear cells,
and HSCs have also been shown to enhance angiogenesis or show
endothelial differentiation in vivo in a variety of animal models
of ischemia (Kocher A. A. et al., Nat Med. (2001) 7:430-436;
Shintani S. et al., Circulation (2001) 103:897-903; Fuchs S. et
al., J Am Coll Cardiol (2001) 37:1726-1732; Kamihata H. et al.,
Circulation (2001) 104:1046-1052; Orlic D. et al., Proc Natl Acad
Sci USA (2001) 98:10344-10349; Jackson K. A. et al., J Clin Invest.
(2001) 107:1395-1402).
[0008] Transplantation of hematopoietic stem cells (HSCs) into
patients with myelodysplastic disorders or following myeloablative
radiochemotherapy is the most widespread application of stem cell
therapy. HSCs are characterized by surface markers like CD34, and
constitute less than 0.5-1% of the bone marrow (Gunsilius E. et
al., Biomed Pharmacother. (2001) 55:186-194), which is currently
the primary source of transplantable HSCs in the clinical setting.
The limited availability of HLA-compatible siblings (less than 30%)
(Tabbara I. A. et al., Arch Intern Med. (2002) 162:1558-1566) has
resulted in frequent use of non-HLA-compatible siblings as donors.
Although recently there has been some success in the reduction of
complications following allogeneic transplantation of HSCs, chronic
graft versus host disease and engraftment failure of allogeneic
cells remains a significant clinical problem (Tabbara I. A. et al.,
Arch Intern Med. (2002) 162:1558-1566). Autologous stem cell
transplantation circumvents these complications, however,
autologous cells from the bone marrow or peripheral blood may be
contaminated by malignant cells (Hahn U. and To L. B., In:
Schindhelm K., Nordon R. eds. Ex vivo Cell Therapy. San Diego,
Calif.: Academic Press; (1999) 99-126).
[0009] While the concept of using autologous peripheral blood
derived EPCs in patients seems attractive, based on animal studies,
one would need 12 liters of blood from a patient to isolate enough
cells to achieve a pro-angiogenic effect (Iwaguro H. et al.,
Circulation. (2002) 105:732-738). This amount of blood is not
readily available in a clinical setting. Human studies that have
used bone marrow cell transplantation in ischemic patients (Strauer
B. E. et al., Dtsch Med Wochenschr. (2001) 126:932-938;
Tateishi-Yuyama E. et al., Lancet. (2002) 360:427-435) suggest that
human angiogenic cell therapy requires at least cell numbers of
10.sup.7 to 10.sup.9, depending on the degree of stem cell purity
as well as the optimal delivery method.
[0010] The discovery of pluripotent cells in the adipose tissue
(Zuk P. A. et al., Tissue Engineering. (2001) 7:211-228) has
revealed a novel source of cells that may be used for autologous
cell therapy to regenerate tissue. The pluripotent cells reside in
the "stromal" or "non-adipocyte" fraction of the adipose tissue;
they were previously considered to be pre-adipocytes, i.e.
adipocyte progenitor cells, however recent data suggests a much
wider differentiation potential. Zuk et al. were able to establish
differentiation of such subcutaneous human adipose stromal cells
(ASCs) in vitro into adipocytes, chondrocytes and myocytes (Zuk P.
A. et al., Tissue Engineering. (2001) 7:211-228). These findings
were extended in a study by Erickson et al., which showed that
human ASCs could differentiate in vivo into chondrocytes (Erickson
et al., Biochem Biophys Res Commun. (2002) 290:763-769) following
transplantation into immune-deficient mice. More recently, it was
demonstrated that human ASCs were able to differentiate into
neuronal cells (Safford K. M. et al., Biochem Biophys Res Commun.
(2002) 294:371-379).
[0011] Given the many applications of stem cell therapy, a need
exists in the art for providing a more abundant and practical
source for such stem cells, and for enhancing the potential
therapeutic benefit and route of administration of these cells.
BRIEF SUMMARY
[0012] In accordance with the present disclosure, isolated, adipose
tissue derived stromal cells are provided. The cells of the present
disclosure may be induced to express at least one characteristic of
a variety of cell types including but not limited to cardiac cells,
endothelial cells, smooth muscle cells, dopaminergic neuronal
cells, hematopoietic cells, or hepatic cells. Further, the cells
may be induced to express a growth factor including but not limited
to vascular endothelial growth factor (VEGF), hepatocyte growth
factor (HGF), granulocyte-colony stimulating factor (G-CSF), and
basic fibroblast growth factor (bFGF), with or without subsequent
cell culture.
[0013] Another object of the present disclosure is to provide a
method of promoting angiogenesis, cardiomyogenesis, or regeneration
of hematopoietic cells in which the cells of the present disclosure
are delivered to a patient. The cells may be delivered by methods
which include but are not limited to retrograde coronary venous
infusion, retrograde delivery to other tissues, direct injection
into target tissue, intra-arterial or intra-coronary infusion via a
catheter, and systemic intravenous administration.
[0014] Another object of the disclosure is a method for isolating
adipose derived stem cells, and optionally culturing the cells in a
medium of interest. As a further option, these cells may be exposed
to a receptor ligand cocktail including but not limited to at least
one of VEGF, LIF, bFGF, IGF1, IGF2, HGF, cardiotrophin, myotrophin,
nitric oxide synthase 3, tumor necrosis factor alpha, tumor
necrosis factor beta, fibroblast growth factor, pleotrophin,
endothelin, and angiopoietin.
[0015] In yet a further embodiment of the present disclosure, the
adipose derived stromal cells may comprise an exogenous nucleic
acid encoding a protein of interest.
BRIEF DESCRIPTION OF THE DRAWINGS
[0016] FIGS. 1A-B show phase contrast micrographs of sub-confluent
(1A) and confluent (1B) cultures of human adipose stromal cells
grown in EGM2MV media, at 250.times. magnification.
[0017] FIG. 2 is a graph showing growth of human subcutaneous
adipose stromal cells.
[0018] FIG. 3 shows flow cytometric analysis of fresh human
subcutaneous adipose stromal cells.
[0019] FIG. 4 shows flow cytometric analysis of Sca-1 expression
(green) and the corresponding isotype control (pink.)
[0020] FIG. 5 shows flow cytometric analysis of CD-34 and
VE-cadherin expression on plated adipose stromal cells.
[0021] FIGS. 6A-B show human aortic endothelial cells (6A) and
human adipose stromal cells (6B) plated overnight on Matrigel.
[0022] FIG. 7 shows smooth-muscle alpha-actin staining on adipose
stromal cells.
[0023] FIG. 8 is a graph which shows secretion of growth factors
VEGF, HGF, and G-CSF, presented as mean.+-.standard error of mean
of pg/10.sup.6 cells.
[0024] FIG. 9 is a micrograph of GFP expression on porcine adipose
stromal cells. The fluorescent image is overlaid 10 on a phase
contrast image.
[0025] FIG. 10 shows flow cytometric analysis of murine adipose
stromal cells gated on CD45(-) cells. The R3 cells are an ASC
Sca-1(+)CD45(-)c-kit(-) population, while the R4 cells are Sca-1
(-)CD45 (-)c-kit(-).
[0026] FIGS. 11A-E show a series of micrographs (320.times.) taken
following 12 days of culture of Sca-1(+)CD45(-)c-kit(-) cells from
muscle tissue and adipose tissue, which shows adipocyte-like
differentiation (Oil-Red-0 stain, 11A and 11D) or neuron-like
differentiation (phase-contrast in 11B and 11E) when grown in the
respective differentiation media. Panel 11C shows staining for the
neuronal tau-protein (red) and nuclei staining (DAPI, blue) with
confocal microscopy.
[0027] FIGS. 12A-F are micrographs of porcine myocardium tissue,
showing the distribution of BrdU labeled cells (marked with arrows)
following administration by retrograde coronary venous delivery
(12A-D) and by direct injection (12E-F).
[0028] FIGS. 13A-B show laser Doppler imaging of the mouse
hindlimb, which demonstrates that the Rac -/- mouse (13A) has
persistent marked impairment of the ischemic leg (right leg)
perfusion, whereas the wild-type mouse (13B) shows significant
restoration of blood perfusion to the ischemic leg (right leg.)
High or normal blood perfusion is depicted in red, and low or
absent perfusion is shown in yellow and green.
[0029] FIG. 14 shows the ratio of perfusion to the ischemic leg to
that of the non-ischemic leg measured by laser Doppler imaging in
wild-type and Rac2 -/- mice.
[0030] FIGS. 15A-B show a representative depiction of limb necrosis
on day 10 in a media injected mouse (15A) and an adipose stromal
cell injected mouse (15B), which shows that adipose stromal cell
injection can attenuate ischemia-induced necrosis.
[0031] FIG. 16 is a graph showing that on day 10, adipose stromal
cell treated animals had a significantly reduced degree of limb
necrosis when compared to the control media injected mice.
DETAILED DESCRIPTION
[0032] Stem cell therapies are expected to someday provide a wide
range of treatment options for various diseases. However the
difficulties associated with obtaining stem cells in therapeutic
quantities has troubled the scientific community. As described
above, stem cells from blood and/or bone marrow cannot be isolated
in sufficient quantity for therapy without significant cell
expansion. Further, it is difficult to find an HLA matched donor,
and allogenic transplants pose a high risk of complications.
[0033] New methods of deriving stem cells from adipose tissue
provide an excellent solution to these challenges. The methods
disclosed herein will demonstrate that 10.sup.5 to 10.sup.6 cells
that are obtained from 5-10 grams of subcutaneous tissue can be
expanded 50-fold in approximately one week. Considering that the
simple outpatient procedure of liposuction can often yield 1 liter
of fat tissue even in non-obese patients, ASC cell numbers of
10.sup.8 to 10.sup.9 can be isolated from an individual with little
or no expansion.
[0034] Furthermore, the methods disclosed herein demonstrate ways
to induce these adipose stromal cells to secrete useful growth
factors which can promote new tissue growth. For example, ASC's can
be induced to secrete Vascular Endothelial Growth Factor (VEGF),
which is known to be an effective inducer of angiogenesis.
[0035] Methods provided herein combine efficient isolation of
therapeutic quantities of stem cells with inducing these stem cells
to secrete growth factors such as VEGF. These cells can then be
administered to a patient to promote vascularization and tissue
growth in areas of ischemic damage.
I. Definitions
[0036] The following definitions are provided to facilitate an
understanding of the present disclosure:
[0037] The term "autologous" implies identical nuclear genetic
identity between donor cells or tissue and those of the
recipient.
[0038] The term "adipose stromal cells" refers to the
"non-adipocyte" fraction of adipose tissue. The cells can be fresh,
or in culture. Adipose stromal cells contain pluripotent cells,
which have the ability to differentiate into cell types including
but not limited to adipocytes, cardiomyocytes, endothelial cells,
hematopoietic cells, hepatic cells, chondrocytes, osteoblasts,
neuronal cells, and myotubes. "Adipose stem cells" are cells within
the adipose stromal fraction which exhibit a stem cell phenotype,
such as CD45-/Sca-1+/c-kit- or CD45-/CD34+/c-kit-.
[0039] "Therapeutic quantities of stromal cells" or "therapeutic
quantities of stem cells" refers to the minimum amount of stem
cells which will produce a desired therapeutic effect. For example,
a therapeutic quantity of VEGF secreting stem cells is the quantity
which will produce therapeutically beneficial levels of
angiogenesis, when administered to a patient.
[0040] "Therapeutic angiogenesis" or "therapeutic vascular growth"
includes but is not limited to angiogenesis (such as capillary
sprouting) and arteriogenesis (such as the maturation and
enlargement of existing vessels.)
[0041] The term "regeneration" or "tissue regeneration" includes,
but is not limited to the growth, generation, or reconstruction of
new cells types or tissues from the ASCs of the instant disclosure.
These cells types or tissues include but are not limited to
endothelial cells, cardiomyocytes, hematopoietic cells, hepatic
cells, adipocytes, chondrocytes, osteoblasts, neuronal cells, and
myotubes. Cardiomyogenesis refers to cardiac tissue regeneration or
reconstruction. Tissue regeneration also includes bone marrow
repopulation using the ASCs of the present disclosure, which
express hematopoietic lineage cell marker(s).
[0042] "Growth factors" are molecules which promote tissue growth,
cellular proliferation, vascularization, and the like. These
include, but are not limited to Vascular Endothelial Growth Factors
A, B, C, D, and E (VEGF), Placental Growth Factor, Hepatocyte
Growth Factor (HGF), Granulocyte-Colony Stimulating Factor,
Granulocyte-macrophage Colony Stimulating Factor, Macrophage Colony
Stimulating Factor, Monocyte Chemotactic Factor, TGF family, FGF
family, pleiotrophin, endothelin, angiopoietins, and so forth.
[0043] "Multipotent" implies that a cell is capable, through its
progeny, of giving rise to several different cell types found in
the adult animal.
[0044] "Pluripotent" implies that a cell is capable, through its
progeny, of giving rise to all the cell types which comprise the
adult animal including the germ cells. Both embryonic stem and
embryonic germ cells are pluripotent cells under this
definition.
[0045] The term "transgenic" animal or cell refers to animals or
cells whose genome has been subject to technical intervention
including the addition, removal, or modification of genetic
information. The term "chimeric" also refers to an animal or cell
whose genome has modified.
[0046] The term "cultured" as used herein in reference to cells can
refer to one or more cells that are undergoing cell division or not
undergoing cell division in an in vitro environment. An in vitro
environment can be any medium known in the art that is suitable for
maintaining cells in vitro, such as suitable liquid media or agar,
for example. Specific examples of suitable in vitro environments
for cell cultures are described in Culture of Animal Cells: a
manual of basic techniques (3rd edition), 1994, R. I. Freshney
(ed.), Wiley-Liss, Inc.; Cells: a laboratory manual (vol. 1), 1998,
D. L. Spector, R. D. Goldman, L. A. Leinwand (eds.), Cold Spring
Harbor Laboratory Press; and Animal Cells: culture and media, 1994,
D. C. Darling, S. J. Morgan John Wiley and Sons, Ltd.
[0047] The term "cell line" as used herein can refer to cultured
cells that can be passaged at least one time without terminating.
The present disclosure relates to cell lines that can be passaged
at least 1, 2, 5, 10, 15, 20, 30, 40, 50, 60, 80, 100, and 200
times. Cell passaging is defined hereafter.
[0048] The term "suspension" as used herein can refer to cell
culture conditions in which cells are not attached to a solid
support. Cells proliferating in suspension can be stirred while
proliferating using apparatus well known to those skilled in the
art.
[0049] The term "monolayer" as used herein can refer to cells that
are attached to a solid support while proliferating in suitable
culture conditions. A small portion of cells proliferating in a
monolayer under suitable growth conditions may be attached to cells
in the monolayer but not to the solid support. Preferably less than
15% of these cells are not attached to the solid support, more
preferably less than 10% of these cells are not attached to the
solid support, and most preferably less than 5% of these cells are
not attached to the solid support.
[0050] The term "plated" or "plating" as used herein in reference
to cells can refer to establishing cell cultures in vitro. For
example, cells can be diluted in cell culture media and then added
to a cell culture plate, dish, or flask. Cell culture plates are
commonly known to a person of ordinary skill in the art. Cells may
be plated at a variety of concentrations and/or cell densities.
[0051] The term "cell plating" can also extend to the term "cell
passaging." Cells of the present disclosure can be passaged using
cell culture techniques well known to those skilled in the art. The
term "cell passaging" can refer to a technique that involves the
steps of (1) releasing cells from a solid support or substrate and
disassociation of these cells, and (2) diluting the cells in media
suitable for further cell proliferation. Cell passaging may also
refer to removing a portion of liquid medium containing cultured
cells and adding liquid medium to the original culture vessel to
dilute the cells and allow further cell proliferation. In addition,
cells may also be added to a new culture vessel which has been
supplemented with medium suitable for further cell
proliferation.
[0052] The term "proliferation" as used herein in reference to
cells can refer to a group of cells that can increase in number
over a period of time.
[0053] The term "permanent" or "immortalized" as used herein in
reference to cells can refer to cells that may undergo cell
division and double in cell numbers while cultured in an in vitro
environment a multiple number of times until the cells terminate. A
permanent cell line may double over 10 times before a significant
number of cells terminate in culture. Preferably, a permanent cell
line may double over 20 times or over 30 times before a significant
number of cells terminate in culture. More preferably, a permanent
cell line may double over 40 times or 50 times before a significant
number of cells terminate in culture. Most preferably, a permanent
cell line may double over 60 times before a significant number of
cells die in culture.
[0054] The term "precursor cell" or "precursor cells" as used
herein can refer to a cell or cells used to establish cultured
mammalian cells or a cultured mammalian cell line. A precursor cell
or cells may be isolated from nearly any cellular entity.
[0055] The term "reprogramming" or "reprogrammed" as used herein
can refer to materials and methods that can convert a cell into
another cell having at least one differing characteristic. Also,
such materials and methods may reprogram or convert a cell into
another cell type that is not typically expressed during the life
cycle of the former cell. For example, (1) a non-totipotent cell
can be reprogrammed into a totipotent cell; (2) a precursor cell
can be reprogrammed into a cell having a morphology of an EG cell;
and (3) a precursor cell can be reprogrammed into a totipotent
cell. An example of materials and methods for converting a
precursor cell into a totipotent cell having EG cell morphology is
described hereafter.
[0056] The term "isolated" as used herein can refer to a cell that
is mechanically separated from another group of cells. Examples of
a group of cells are a developing cell mass, a cell culture, a cell
line, and an animal.
[0057] The term "non-embryonic cell" as used herein can refer to a
cell that is not isolated from an embryo. Non-embryonic cells can
be differentiated or nondifferentiated. Non-embryonic cells can
refer to nearly any somatic cell, such as cells isolated from an ex
utero animal. These examples are not meant to be limiting.
[0058] The term "differentiated cell" as used herein can refer to a
precursor cell that has developed from an unspecialized phenotype
to a specialized phenotype. For example, embryonic cells can
differentiate into an epithelial cell lining the intestine.
Materials and methods of the present disclosure can reprogram
differentiated cells into totipotent cells. Differentiated cells
can be isolated from a fetus or a live born animal, for
example.
[0059] The term "undifferentiated cell" as used herein can refer to
a precursor cell that has an unspecialized phenotype and is capable
of differentiating. An example of an undifferentiated cell is a
stem cell.
[0060] The term "asynchronous population" as used herein can refer
to cells that are not arrested at any one stage of the cell cycle.
Many cells can progress through the cell cycle and do not arrest at
any one stage, while some cells can become arrested at one stage of
the cell cycle for a period of time. Some known stages of the cell
cycle are G1, S, G2, and M. An asynchronous population of cells is
not manipulated to synchronize into any one or predominantly into
any one of these phases. Cells can be arrested in the M stage of
the cell cycle, for example, by utilizing multiple techniques known
in the art, such as by colcemid exposure. Examples of methods for
arresting cells in one stage of a cell cycle are discussed in WO
97/07669, entitled "Quiescent Cell Populations for Nuclear
Transfer".
[0061] The terms "synchronous population" and "synchronizing" as
used herein can refer to a fraction of cells in a population that
are within a same stage of the cell cycle. Preferably, about 50% of
cells in a population of cells are arrested in one stage of the
cell cycle, more preferably about 70% of cells in a population of
cells are arrested in one stage of the cell cycle, and most
preferably about 90% of cells in a population of cells are arrested
in one stage of the cell cycle. Cell cycle stage can be
distinguished by relative cell size as well as by a variety of cell
markers well known to a person of ordinary skill in the art. For
example, cells can be distinguished by such markers by using flow
cytometry techniques well known to a person of ordinary skill in
the art. Alternatively, cells can be distinguished by size
utilizing techniques well known to a person of ordinary skill in
the art, such as by the utilization of a light microscope and a
micrometer, for example. In a preferred embodiment, cells are
synchronized by arresting them (i.e., cells are not dividing) in a
discreet stage of the cell cycle.
[0062] An "exogenous nucleic acid" as used herein refers to any
nucleic acid which is introduced into the ASCs of the present
disclosure, and encodes a protein of interest. Specific exogenous
nucleic acids encode proteins which include without limitation a
Vascular Endothelial Growth Factor A, B, C, D, and E (VEGF), bFGF,
IGF1, IGF2, Hepatocyte Growth Factor (HGF), Placental Growth
Factor, cardiotrophin, myotrophin, nitric oxide synthases 1, 2, or
3, Granulocyte-Colony Stimulating Factor, Granulocyte-macrophage
Colony Stimulating Factor, Macrophage Colony Stimulating Factor,
Monocyte Chemotatic Factor, TGF family, FGF family, pleiotrophin,
endothelin, angiopoietins, and genes promoting differentiation
along pre-determined pathways.
[0063] The term "modified nuclear DNA" as used herein can refer to
a nuclear deoxyribonucleic acid sequence of a cell of the present
disclosure that has been manipulated by one or more recombinant DNA
techniques. Examples of recombinant DNA techniques are well known
to a person of ordinary skill in the art, which can include (1)
inserting a DNA sequence from another organism (e.g., a human
organism) into target nuclear DNA, (2) deleting one or more DNA
sequences from target nuclear DNA, and (3) introducing one or more
base mutations (e.g., site-directed mutations) into target nuclear
DNA. Cells with modified nuclear DNA can be referred to as
"transgenic cells" or "chimeric cells" for the purposes of the
present disclosure. The phrase "modified nuclear DNA" may also
encompass "corrective nucleic acid sequence(s)" which replace a
mutated nucleic acid molecule with a nucleic acid encoding a
biologically active, phenotypically normal polypeptide. The
constructs utilized to generate modified nuclear DNA may optionally
comprise a reporter gene encoding a detectable product.
[0064] As used herein, the terms "reporter," "reporter system",
"reporter gene," or "reporter gene product" shall mean an operative
genetic system in which a nucleic acid comprises a gene that
encodes a product that when expressed produces a reporter signal
that is a readily measurable, e.g., by biological assay,
immunoassay, radioimmunoassay, or by calorimetric, fluorogenic,
chemiluminescent or other methods. The nucleic acid may be either
RNA or DNA, linear or circular, single or double stranded,
antisense or sense polarity, and is operatively linked to the
necessary control elements for the expression of the reporter gene
product. The required control elements will vary according to the
nature of the reporter system and whether the reporter gene is in
the form of DNA or RNA, but may include, but not be limited to,
such elements as promoters, enhancers, translational control
sequences, poly A addition signals, transcriptional termination
signals and the like.
[0065] "Selectable marker" as used herein refers to a molecule that
when expressed in cells renders those cells resistant to a
selection agent. Nucleic acids encoding selectable marker may also
comprise such elements as promoters, enhancers, translational
control sequences, poly A addition signals, transcriptional
termination signals and the like. Suitable selection agents include
antibiotic such as kanamycin, neomycin, and hygromycin.
[0066] Methods and tools for insertion, deletion, and mutation of
nuclear DNA of mammalian cells are well-known to a person of
ordinary skill in the art. See, Molecular Cloning, a Laboratory
Manual, 2nd Ed., 1989, Sambrook, Fritsch, and Maniatis, Cold Spring
Harbor Laboratory Press; U.S. Pat. No. 5,633,067, "Method of
Producing a Transgenic Bovine or Transgenic Bovine Embryo," DeBoer
et al., issued May 27, 1997; and U.S. Pat. No. 5,612,205,
"Homologous Recombination in Mammalian Cells," Kay et al., issued
Mar. 18, 1997. These methods include techniques for transfecting
cells with foreign DNA fragments and the proper design of the
foreign DNA fragments such that they effect insertion, deletion,
and/or mutation of the target DNA genome.
[0067] The adipose stromal cells defined herein can be altered to
harbor modified nuclear or cytoplasmic DNA.
[0068] Examples of methods for modifying a target DNA genome by
insertion, deletion, and/or mutation are retroviral or
adeno-associated viral insertion, artificial chromosome techniques,
gene insertion by electroporation, sonoporation, or chemical
methods, random insertion with tissue specific promoters,
homologous recombination, gene targeting, transposable elements,
and/or any other method for introducing foreign DNA. Other
modification techniques well known to a person of ordinary skill in
the art include deleting DNA sequences from a genome, and/or
altering nuclear DNA sequences. Examples of techniques for altering
nuclear DNA sequences are site-directed mutagenesis and polymerase
chain reaction procedures. Therefore, the present disclosure
relates in part to mammalian cells that are simultaneously
totipotent and transgenic. Such transgenic and totipotent cells can
serve as nearly unlimited sources of donor cells for production of
cloned transgenic animals.
[0069] The term "recombinant product" as used herein can refer to
the product produced from a DNA sequence that comprises at least a
portion of the modified nuclear DNA. This product can be a peptide,
a polypeptide, a protein, an enzyme, an antibody, an antibody
fragment, a polypeptide that binds to a regulatory element (a term
described hereafter), a structural protein, an RNA molecule, and/or
a ribozyme, for example. These products are well defined in the
art.
[0070] The term "promoters" or "promoter" as used herein can refer
to a DNA sequence that is located adjacent to a DNA sequence that
encodes a recombinant product. A promoter is preferably linked
operatively to an adjacent DNA sequence. A promoter typically
increases an amount of recombinant product expressed from a DNA
sequence as compared to an amount of the expressed recombinant
product when no promoter exists. A promoter from one organism can
be utilized to enhance recombinant product expression from a DNA
sequence that originates from another organism. In addition, one
promoter element can increase an amount of recombinant products
expressed for multiple DNA sequences attached in tandem. Hence, one
promoter element can enhance the expression of one or more
recombinant products. Multiple promoter elements are well-known to
persons of ordinary skill in the art.
[0071] The term "enhancers" or "enhancer" as used herein can refer
to a DNA sequence that is located adjacent to the DNA sequence that
encodes a recombinant product. Enhancer elements are typically
located upstream of a promoter element or can be located downstream
of a coding DNA sequence (e.g., a DNA sequence transcribed or
translated into a recombinant product or products). Hence, an
enhancer element can be located 100 base pairs, 200 base pairs, or
300 or more base pairs upstream or downstream of a DNA sequence
that encodes recombinant product. Enhancer elements can increase an
amount of recombinant product expressed from a DNA sequence above
increased expression afforded by a promoter element. Multiple
enhancer elements are readily available to persons of ordinary
skill in the art.
[0072] The term "nuclear transfer" as used herein can refer to
introducing a full complement of nuclear DNA from one cell to an
enucleated cell. Nuclear transfer methods are well known to a
person of ordinary skill in the art. See, e.g., Nagashima et al.,
1997, Mol. Reprod. Dev. 48: 339-343; Nagashima et al., 1992, J.
Reprod. Dev. 38: 73-78; Prather et al., 1989, Biol. Reprod. 41:
414-419; Prather et al., 1990, Exp. Zool. 255: 355-358; Saito et
al., 1992, Assis. Reprod. Tech. Andro. 259: 257-266; and Terlouw et
al., 1992, Theriogenology 37: 309. Nuclear transfer may be
accomplished by using oocytes that are not surrounded by a zona
pellucida.
[0073] The terms "transfected" and "transfection" as used herein
refer to methods of delivering exogenous DNA into a cell. These
methods involve a variety of techniques, such as treating cells
with high concentrations of salt, an electric field, liposomes,
polycationic micelles, or detergent, to render a host cell outer
membrane or wall permeable to nucleic acid molecules of interest.
These specified methods are not limiting and the present disclosure
relates to any transformation technique well known to a person of
ordinary skill in the art.
[0074] The term "antibiotic" as used herein can refer to any
molecule that decreases growth rates of a bacterium, yeast, fungi,
mold, or other contaminants in a cell culture. Antibiotics are
optional components of cell culture media. Examples of antibiotics
are well known in the art. See Sigma and DIFCO catalogs.
[0075] The term "feeder cells" as used herein can refer to cells
that are maintained in culture and are co-cultured with target
cells. Target cells can be precursor cells, adipose stromal cells,
cultured cells, and totipotent cells, for example. Feeder cells can
provide, for example, peptides, polypeptides, electrical signals,
organic molecules (e.g., steroids), nucleic acid molecules, growth
factors (e.g., bFGF), other factors (e.g., cytokines such as LIF
and steel factor), and metabolic nutrients to target cells. Certain
cells may not require feeder cells for healthy growth. Feeder cells
preferably grow in a mono-layer.
[0076] Feeder cells can be established from multiple cell types.
Examples of these cell types are fetal cells, mouse cells, and
oviductal cells. These examples are not meant to be limiting.
Tissue samples can be broken down to establish a feeder cell line
by methods well known in the art (e.g., by using a blender). Feeder
cells may originate from the same or different animal species as
precursor cells. Feeder cells can be established from fetal cells,
mammalian fetal cells, and murine fetal cells.
[0077] The term "receptor ligand cocktail" as used herein can refer
to a mixture of one or more receptor ligands. A receptor ligand can
refer to any molecule that binds to a receptor protein located on
the outside or the inside of a cell. Receptor ligands can be
selected from molecules of the cytokine family of ligands,
neurotrophin family of ligands, growth factor family of ligands,
and mitogen family of ligands, all of which are well known to a
person of ordinary skill in the art. Examples of receptor/ligand
pairs are: vascular endothelial growth factor receptor/vascular
endothelial growth factor, epidermal growth factor
receptor/epidermal growth factor, insulin receptor/insulin,
cAMP-dependent protein kinase/cAMP, growth hormone receptor/growth
hormone, and steroid receptor/steroid. It has been shown that
certain receptors exhibit cross-reactivity. For example,
heterologous receptors, such as insulin-like growth factor receptor
1 (IGFR1) and insulin-like growth factor receptor 2 (IGFR2) can
both bind IGF1. When a receptor ligand cocktail comprises a
stimulus, the receptor ligand cocktail can be introduced to a
precursor cell in a variety of manners known to a person of
ordinary skill in the art.
[0078] The term "cytokine" as used herein can refer to a large
family of receptor ligands well-known to a person of ordinary skill
in the art. The cytokine family of receptor ligands includes such
members as leukemia inhibitor factor (LIF); cardiotrophin 1 (CT-1);
ciliary neurotrophic factor (CNTF); stem cell factor (SCF), which
is also known as Steel factor; oncostatin M (OSM); and any member
of the interleukin (IL) family, including IL-6, IL-1, and IL-12.
The teachings of the present disclosure do not require the
mechanical addition of steel factor (also known as stem cell factor
in the art) for the conversion of precursor cells into totipotent
cells.
II. Methods of Isolating Autologous Stem Cells from Adipose
Tissues
[0079] In accordance with the present disclosure, it has been
discovered that therapeutic quantities of autologous stem cells may
be obtained from adipose tissue. Such stem cells can be obtained as
described herein.
[0080] For example, adipose tissue is obtained from an animal,
preferable a human, and most preferably from the patient who is the
intended recipient of the therapeutic stem cells. The tissue may be
obtained by numerous methods known in the art, including without
limitation liposuction, and surgery. Preferably, the adipose tissue
is obtained by liposuction, which is a simple, minimally invasive
procedure.
[0081] In an exemplary method, following collection, adipose tissue
is optionally washed, for example with saline (such as PBS) to
removed loose matter. Next, the tissue is digested with an enzyme,
(such as collagenase, dispase, or trypsin), and/or may also be
degraded by mechanical agitation, sonic energy, thermal energy, and
the like. The resultant product is then optionally filtered, and
then centrifuged to separate the stromal cells from the adipose
cells. Another round of washing and centrifugation may be performed
in order to further purify the cells. The cells may also be sorted
using flow cytometry or other cell sorting means, and further
isolated. Cells so obtained exhibit stem cell markers, eg.
CD34.
[0082] Other methods of isolating stem cells from adipose tissue
are known in the art, and are disclosed for example in Zuk P. A. et
al., Tissue Engineering (2001) 7:211-228).
III. Methods of Inducing Stem Cells to Differentiate and/or Express
Growth Factors
[0083] Stem cells derived from adipose tissue as set forth above
may be used in many therapeutic procedures. Transgenic stem cells
so obtained may also be optionally transfected at this point with a
"corrective nucleic acid sequence". The stem cells so obtained are
then passaged and exposed to a receptor ligand cocktail to induce
differentiation into the desired cell lineage as exemplified herein
below.
[0084] Tissues currently being developed from stem cells include,
but are not limited to: blood vessels (Kocher A. A. et al., Nature
Med. (2001) 7:430-436; Jackson K. A. et al., J. Clin. Invest.
(2001) 107:1395-1402), bone (Petite H. et al., Nature Biotech.
(2000) 18:959-963), cartilage (Johnstone B. et al., Clin. Orthop.
(1999) S156-S162), cornea (Tsai R. J. et al., N. Eng. J. Med.
(2000) 343:86-93), dentin (Gronthos S. et al., Proc. Natl. Acad.
Sci. USA (2000) 97:13625-13630), heart muscle (Klug M. G. et al.,
J. Clin. Invest. (1996) 98:216-224; review Boheler K. R. et al.,
Cir. Res. (2002) 91:189-201), liver (Lagasse E. et al., Nature Med.
(2000) 6:1229-1234), pancreas (Soria B. et al., Diabetes (2000)
49:1-6; Ramiya V. K. et al., Nature Med. (2000) 6:278-282), nervous
tissue (Bjorkland A., Novartis Found. Symp. (2000) 231:7-15; Lee S.
H. et al., Nature Biotechnology (2000) 18:675-679; Kim J. H. et
al., Nature (2002) 418:50-56), skeletal muscle (Gussoni E. et al.,
Nature (1999) 401:390-394), and skin (Pellegrini G. et al.,
Transplantation (1999) 68:868-879). Some of the tissues being
generated from stem cells are described in further detail
below.
Heart Muscle
[0085] The loss of cardiomyocytes from adult mammalian hearts is
irreversible and leads to diminished heart function. Methods have
been developed in which embryonic stem (ES) cells are employed as a
renewable source of donor cardiomyocytes for cardiac engraftment
(Klug, M. G. et al., J. Clin. Invest. (1996) 98:216-224). The ASC
cells of the present disclosure can be similarly
differentiated.
[0086] ES cells were first transfected by electroporation with a
plasmid expressing the neomycin resistance gene from an
.alpha.-cardiac myosin heavy chain promoter and expressing the
hygromycin resistance gene under the control of the
phosphoglycerate kinase (pGK) promoter. A proportion of cells
comprising an activatable .alpha.-cardiac myosin heavy chain
promoter will differentiate into cardiac cells. Transfected clones
were selected by growth in the presence of hygromycin (200
.mu.g/ml; Calbiochem-Novabiochem). Transfected ES cells were
maintained in the undifferentiated state by culturing in high
glucose DMEM containing 10% fetal bovine serum (FBS), 1%
nonessential amino acids, and 0.1 mM 2-mercaptoethanol. The medium
was supplemented to a final concentration of 100 U/ml with
conditioned medium containing recombinant LIF.
[0087] To induce differentiation, 2.times.10.sup.6 freshly
dissociated transfected ES cells were plated onto a 100-mm
bacterial Petri dish containing 10 ml of DMEM lacking supplemental
LIF. Regions of cardiogenesis were readily identified by the
presence of spontaneous contractile activity. For cardiomyocyte
selection, the differentiated cultures were grown for 8 days in the
presence of G418 (200 .mu.g/ml; GIBCO/BRL). Cultures of selected
ES-derived cardiomyocytes were digested with trypsin and the
resulting single cell preparation was washed three times with DMEM
and directly injected into the ventricular myocardium of adult
mice.
[0088] The culture obtained by this method after G418 selection is
more 99% pure for cardiomyocytes based on immunofluorescence for
myosin. The obtained cardiomyocytes contained well defined
myofibers and intercalated discs were observed to couple juxtaposed
cells consistent with the observation that adjacent cells exhibit
synchronous contractile activity. Importantly, the selected
cardiomyocytes were capable of forming stable intercardiac grafts
with the engrafted cells aligned and tightly juxtaposed with host
cardiomyocytes. As mentioned previously the adipose stromal cells
of the present disclosure can be similarly treated to generate
cardiomyocytes.
Endothelial Cells
[0089] Endothelial cells are critical to neo-vascularization, and
are known to secrete numerous growth factors which promote healing
and expansion. Administration of endothelial cells promotes
angiogenesis, resulting in increased vascularization and
development. Methods of inducing stem cells to differentiate to
endothelial cells are generally known in the art, and are disclosed
for example, in Balconi et al., Arterioscler Thromb Vasc Biol.
(2000) 20:1443-1451.
[0090] Stem cell lines were grown in the undifferentiated state
either on gelatin (0.1%)-coated Petri dishes (CJ7) or on a feeder
layer of STO murine fibroblasts.
[0091] To initiate cell differentiation cells were briefly
trypsinized and suspended in Iscove's modified Dulbecco's medium
with 15% FBS, 10 mg/mL insulin (Sigma), 100 U/mL penicillin, 100
mg/mL streptomycin, and 450 mmol/L monothioglycerol. A growth
factor cocktail was added to the culture medium to optimize
vascular differentiation and included: recombinant human VEGF
(Peprotech Inc) at 50 ng/mL; recombinant human erythropoietin
(Cilag AG), at 2 U/mL; human bFGF (Genzyme), at 100 ng/mL; and
murine interleukin 6 (Genzyme), at 10 ng/mL.
[0092] Cells were seeded in bacteriological Petri dishes
(1.5.times.10.sup.4 cells per 35-mm Petri dish) and cultured for 11
days, without further feeding, at 37.degree. C. in an incubator
with 5% CO.sub.2 in air and 95% relative humidity. The cells were
routinely examined for the presence of endothelium-like structures
by whole-mount preparation, by using rat mAb MEC 7.46 directly
against mouse PECAM as primary antibody and commercial rabbit
immunoglobulins to rat immunoglobulin (DAKO) as a secondary
antibody.
[0093] In some cases, endothelial cells were selected with the use
of sheep anti-mouse CD31mAb-coated magnetic beads (Dynabeads, Dynal
AS). In some experiments, endothelial cell lines could be obtained
without immuno selection. In those cases, after EB disaggregation,
the cells were immortalized by PmT. In previous studies, it was
observed that PmT specifically immortalizes endothelial cells and
not any other cell type.
[0094] A suitable culture medium for stem-cell derived endothelial
cells is DMEM with 20% FBS, supplemented with 2 mmol/L glutamine,
100 U/mL penicillin, 100 mg/mL streptomycin, 50 mg/mL endothelial
cell growth supplement (Sigma), and 100 mg/mL heparin (Sigma)
(Balconi et al., Arterioscler Thromb Vasc Biol. (2000)
20:1443-1451).
[0095] ASCs in accordance with the present disclosure can be
induced to form endothelial cells in a similar fashion.
Neuronal Cells
[0096] Parkinson's disease is caused by the loss of midbrain
neurons that synthesize the neurotransmitter dopamine. Delivery of
dopamine-synthesizing neurons to the midbrain should alleviate the
symptoms of the disease by restoring dopamine production. Stem
cells obtained using the methods of the present disclosure may be
differentiated into dopamine-synthesizing neurons utilizing the
exemplary protocols set forth below. (Lee, S. H. et al., Nature
Biotechnology, (2000) 18:675-679; Kim, J. H. et al., Nature (2002)
418:50-56).
[0097] In a murine model, mouse ES cells were first transfected by
electroporation with a plasmid expressing nuclear receptor
related-1 (Nurr1), a transcription factor that has a role in the
differentiation of midbrain precursors into dopamine neurons and a
plasmid encoding neomycin resistance. Transfected clones (Nurr1 ES
cells) were then subsequently isolated by culturing the cells in
G418. The Nurr1 ES cells were then expanded under cultures which
prevented differentiation (e.g., growth on gelatin-coated tissue
culture plates in the presence of 1,400 U/ml-I of leukemia
inhibitory factor (LIF; GIBCO/BRL, Grand Island, N.Y.) in ES cell
medium consisting of knockout Dulbecco's minimal essential medium
(GIBCO/BRL) supplemented with 15% FCS, 100 mM MEM nonessential
amino acids, 0.55 mM 2-mercaptoethanol, L-glutamine, and
antibiotics (all from GIBCO/BRL)). Selection of nestin-positive
cells, a marker of developmental neurons, was initiated by
replacing the ES cell medium by serum-free Dulbecco's modified
Eagle's medium (DMEM)/F12 (1:1) supplemented with insulin (5
.mu.g/ml), transferrin (50 .mu.g/ml), selenium chloride (30 nM),
and fibronectin (5 .mu.g/ml) (ITSFn) medium. After 6-10 days of
selection, expansion of nestin-positive cells was initiated.
Specifically, the cells were dissociated by 0.05% trypsin/0.04%
EDTA, and plated on tissue culture plastic or glass coverslips at a
concentration of 1.5-2.times.10.sup.5 cells/cm2 in N2 medium
modified (described in Johe K. et al., Genes Dev. (1996)
10:3129-3140), and supplemented with 1 .mu.g/ml of laminin and 10
ng/ml of bFGF (R&D Systems, Minneapolis, Minn.) in the presence
of murine N-terminal fragment of sonic hedgehog (SHH; 500 ng/ml)
and murine fibroblast growth factor (FGF) 8 isoform b (100 ng/ml;
both from R&D Systems). Before cell plating, dishes and
coverslips were precoated with polyornithine (15 mg/ml) and laminin
(1 .mu.g/ml, both from Becton Dickinson Labware, Bedford, Mass.).
Nestin-positive cells were again expanded for six days. The medium
was changed every two days. Differentiation was induced by removal
of basic FGF (bFGF). The differentiation medium consisted of N2
medium supplemented with laminin (1 mg/ml) in the presence of cAMP
(1 .mu.M) and ascorbic acid (200 .mu.M, both from Sigma St. Louis,
MC). The cells were incubated under differentiation conditions for
6-15 days.
[0098] 78% of Nurr1 ES cells were found to be induced into
dopamine-synthesizing, tyrosine hydroxylase (TH, 4 rate limiting
enzyme in the biosynthesis of dopamine) positive neurons by the
method set forth above. The resultant neurons were further
characterized to express a variety of midbrain- specific markers
such as Ptx3 and Engrailed 1 (En-1). The dopamine-synthesizing,
TH+cells were also grafted into a rodent model of Parkinson's
disease and were shown to extend axons, form functional synaptic
connections, perform electrophysiological functions expected of
neurons, innervate the striatum, and improve motor asymmetry.
[0099] The ASCs of the present disclosure may be induced to form
neuronal cells in a similar fashion.
Insulin-Producing Cells
[0100] An ideal treatment for diabetes is the restoration of
.beta.-cell function or mimicking the insulin secretory pattern of
these cells. Insulin-secreting cells derived from stem cells have
been generated by the following method and have been shown to be
capable of normalizing blood glucose levels in a diabetic mouse
model (Soria, B. 10 et al., Diabetes (2000) 49:1-6).
[0101] ES cells were transfected by electroporation with a plasmid
expressing .beta.-gal under the control of the human insulin
regulatory region and expressing the hygromycin resistance gene
under the control of the pGK promoter. Transfected clones were
selected by growth in the presence of hygromycin (200 pg/ml;
Calbiochem-Novabiochem). Transfected ES cells were maintained in
the undifferentiated state by culturing in high glucose Dulbecco's
modified Eagle's medium (DMEM) containing 10% fetal bovine serum
(FBS), 1% nonessential amino acids, 0.1 mM 2-mercaptoethanol, 1 mM
sodium pyruvate, 100 IU/ml penicillin, and 0.1 mg/ml streptomycin.
The medium was supplemented to a final concentration of 100 U/ml
with conditioned medium containing recombinant LIF.
[0102] To induce differentiation to an insulin-secreting cell line,
2.times.10.sup.6 hygromycin-resistant ES cells were plated onto a
100-mm bacterial Petri dish and cultured in DMEM lacking
supplemental LIF. For ES Ins/.beta.-gal selection, the
differentiated cultures were grown in the same medium in the
presence of 200 .mu.g/ml G418. For final differentiation and
maturation, the resulting clones were trypsinized and plated on a
100-mm bacterial Petri dish and grown for 14 days in DMEM
supplemented with 200 .mu.g/ml G418 and 10 mM nicotinamide (Sigma),
a form of Vitamin B3 that may preserve and improve beta cell
function. Finally, the resulting clusters were cultured for 5 days
in RPMI 1640 media supplemented with 10% FBS, 10 mM nicotinamide,
200 .mu.g/ml G418, 100 IU/ml penicillin, 0.1 mg/ml streptomycin,
and low glucose (5.6 mM).
[0103] For cell implantation, ES-derived insulin-secreting cells
were washed and resuspended in RPMI 1640 media supplemented with
10% FBS, 10 mM nicotinamide, 100 IU/ml penicillin, 0.1 mg/ml
streptomycin, and 5.6 mM glucose at 5.times.10.sup.6 cells/ml. The
mice to receive the implantation of ES-derived insulin-secreting
cells were male Swiss albino mice that had diabetic conditions
induced by a single intraperitoneal injection of streptozotocin
(STZ, Sigma) at 200 mg/kg body weight in citrate buffer.
1.times.10.sup.6 cells were injected into the spleen of mice under
anesthesia.
[0104] The ES-derived insulin-secreting cells produced from this
method produced a similar profile of insulin production in response
to increasing levels of glucose to that observed in mouse
pancreatic islets. Significantly, implantation of the ES-derived
insulin-secreting cells led to the correction of the hyperglycemia
within the diabetic mouse, minimized the weight loss experienced by
the mice injected with STZ, and lowered glucose levels after meal
challenges and glucose challenges better than untreated diabetic
mice and similar to control nondiabetic mice.
[0105] The ASCs of the present disclosure may be similarly
differentiated into insulin producing cells using similar
methods.
Hematopoietic Cells
[0106] Hematopoietic cells are used in numerous therapeutic
regimens such as replenishing bone marrow supply, or treating
hematological disorders, such as anemias, thalassemias, and so
forth. Stem cells can be differentiated into hematopoietic stem
cells by methods known in the art, and disclosed, for example, in
Howell et al., Experimental Hematology (2002) 30:915-924.
[0107] In Howell et al., CD45-Sca-1+c-kit- cells were incubated for
nine days in MEM+10% HS in the presence of 5 ng/mL mSCF, mFlt-3L,
and MGDF, which are cytokines. Cells were then expanded. Cells
expressed increased levels of c-kit.
[0108] The in vivo repopulating potential of these cells was then
assessed by transplanting 4.times.10.sup.4 cells into lethally
irradiated Boy J (CD45.1) recipient mice. Analysis of peripheral
blood after 4 months revealed that freshly isolated cells exhibited
7.9%.+-.2.9% chimerism in recipient mice. A mathematical derivation
of fold expansion of engraftment potential of cultured cells,
relative to fresh cells revealed that these cytokine cultured cells
have a 5 fold increase in relative hematopoietic repopulating
potential.
[0109] The ASCs of the present disclosure may be similarly
differentiated into hematopoietic cells, and used therapeutically
to treat hematopoietic disorders.
[0110] The recently described ability to genetically manipulate
human ES cells should allow for the rapid isolation of highly
uniform and singularly differentiated cells (Eiges R. et al.,
Current Biol. (2001) 11:514-518). A potential method to this end
would be to employ a similar method to that described above for
murine ES cells in which antibiotic resistance genes or selectable
marker genes are expressed under cell specific promoters.
Alternatively, cell-type specific transcription factors or any
other cell-type specific factors found to drive cell-type specific
differentiation can be expressed.
IV. Administration of Adipose Derived Stem Cells for Therapeutic
Benefit
[0111] The adipose derived stem cells of the present disclosure can
be used in a wide array of therapeutic procedures. These therapies
include administration of adipose stem cells, administration of
stem cells which secrete growth factors such as VEGF, and stem
cells which have been transfected to express a desired therapeutic
molecule.
[0112] These molecules may be administered by a variety of methods,
including retrograde coronary venous delivery, direct injection,
arterial infusion via catheter, systemic administration via IV, and
so forth.
[0113] Methods of delivery of molecules to the heart via retrograde
coronary venous delivery are generally known in the art and are
described for example in Boekstegers P. et al., Gene Ther (2000)
7:232-240, Herity N. A., Cathet. Cardiovasc. Intervent. (2000)
51:358-363, Suzuki et al., Circulation (2000) III-359-364, and Yock
et al., U.S. Pat. No. 6,346,098. The injection of cells via RCVD
can be at rates which achieve pressures of about 30-400 mm Hg, most
preferably about 50-150 mm Hg.
[0114] Retrograde coronary venous delivery is a suitable method for
delivery of other types of stem cells having therapeutic benefit.
Such stem cells include without limitation bone marrow derived stem
cells, peripheral blood mononuclear derived stem cells, and
skeletal tissue derived myocytes.
[0115] Methods of direct injection comprise primarily direct
infusion, ideally by hypodermic needle to the site where increased
vascularization is desired. For example, adipose stem cells of the
present disclosure may be administered via intramuscular injection,
to a tissue where angiogenesis is desired.
[0116] Methods of infusion via indwelling catheter generally
comprise placement of an indwelling catheter to a region where stem
cell therapy would be beneficial, and administration of the adipose
stem cells of the present disclosure through such a catheter.
[0117] The following non-limiting examples are provided to further
illustrate the present disclosure.
Example I
Isolation and Characterization of Stem Cells from Adipose
Tissue
Isolation of Stem Cells from Adipose Tissue
[0118] To isolate adipose stromal cells (ASCs,) subcutaneous
adipose tissue (5-15 grams) was obtained from obese patients
undergoing a gastroplasty procedure. The tissue was suspended in
PBS and dissected into smaller pieces using a scalpel. Collagenase
(Worthington Biochemical) was then added to the suspension and
placed in a shaker at 37.degree. C. for approximately 90 minutes.
The digested sample was filtered through a 750 micron Nitex filter
and a 50 micron filter, and rinsed with Dulbecco's Modified Eagle
Media (DMEM) with 10% Fetal Bovine Serum (FBS). The filtered
suspension was centrifuged at 200 g for 5 minutes and the top layer
consisting of adipocytes was discarded. After a second spin at 300
g for 5 minutes, any remaining adipocytes in the top layer were
again discarded. The cell pellet was re-suspended in red cell lysis
buffer and centrifuged at 300 g after 5-10 minutes of incubation at
3720 C. This cell pellet consisted of adipose stromal cells and was
re-suspended in the desired media. Adipose stromal cells (ASCs)
were plated on tissue culture flasks at densities ranging from 1000
to 10,000 cells per cm.sup.2. Non-adherent cells were discarded on
the following day. The majority of ASCs attach to the flask and
these adherent cells (FIG. 1) can be expanded in either DMEM media
with 10% FBS or in EGM2MV media (Clonetics), which contains the
growth factors VEGF, bFGF, EGF, IGF and 5% FBS. Cells cultured in
EGM2MV media, for example, can be expanded 50-fold in 8 days (FIG.
2) with their growth rate decreasing when they reach confluency.
ASCs continue to have a high proliferative activity when they are
passaged. ASCs were also isolated from porcine and murine adipose
tissue using similar isolation and culture protocols.
[0119] These experiments demonstrate that ASCs can be readily
isolated and rapidly expanded ex vivo from relatively small amounts
of adipose tissue, thus indicating that autologous ASCs may be used
to advantage in research and clinical protocols. Human
cardiovascular cell therapy studies suggest that 10.sup.8 to
10.sup.9 autologous cells may be required for clinical
applications.
[0120] Considering the fact that liposuction can yield up to 3
liters of subcutaneous fat tissue in a single outpatient procedure,
such cell numbers can be easily obtained from patients with little
or no expansion.
Expression of Stem Cell Markers on ASCs
[0121] To evaluate the expression of the stem cell marker CD-34,
adipose stromal cells were freshly isolated from human adipose
tissue and labeled with fluorescent antibodies against CD34 (BD
Biosciences). As CD34 is also expressed on mature endothelial cells
and endothelial progenitor cells, the isolated cells were
co-labeled with an antibody directed against human VE-cadherin (BD
Biosciences), a highly specific marker of endothelial cells (Rafii
S., J Clin Invest. (2000) 105:17-19). Data from a representative
experiment is shown in FIG. 3. The results indicate that the
majority of cells are CD34+/VE-cadherin- , thus suggesting that
most human adipose tissue derived stromal cells are non endothelial
CD34-positive stem or progenitor cells. The human adipose stromal
cell fraction also contains a small but distinct population of
CD34+/VE-cadherin+cells, which may represent either mature
endothelial cells or endothelial progenitor cells.
[0122] To determine whether the expression of stem cell markers
could also be found on adipose stromal cells in the mouse, murine
ASCs were isolated from mice using the same protocol that was used
for human ASC isolation. Murine ASCs were cultured, expanded in
DMEM/F12 media with 10% fetal bovine serum and passaged when
confluent. The passaged cells were finally detached with EDTA at
confluence and subsequently labeled with an antibody directed
against the specific murine stem cell marker Sca-1 (Stem Cell
Antigen-1). As shown in FIG. 4, greater than 70% of cultured murine
ASCs express the stem cell marker Sca-1. The high level of
expression of CD34 in human ASCs and of Sca-1 on murine ASCs
complements the data on ASC differentiation potential (Zuk P. A. et
al., Tissue Engineering (2001) 7:211-228; Erickson et al., Biochem
Biophys Res Commun. (2002) 290:763-769; Safford K. M. et al.,
Biochem Biophys Res Commun. (2002) 294:371-379) and provides
evidence that ASCs have stem cell characteristics.
Example II
Differentiation of Adipose Derived Stromal Cells
ASCs can Develop a Vascular Phenotype
[0123] A major pathway by which ASCs can enhance angiogenesis is by
differentiation of ASCs into a vascular cell phenotype. Since ASCs
are already known to differentiate into a number of cell types like
muscle, bone and neural cells, it was evaluated whether ASCs can
develop phenotypes that correspond to either vascular endothelial
cells or vascular smooth muscle cells. Plated adherent human ASCs
were evaluated by flow cytometry for the expression of the stem
cell marker CD34 and the endothelial marker VE-Cadherin, (FIG. 5).
While the majority of cells are still CD34+/VE-Cadherin- , there
has emerged a substantial proportion of cells that are
CD34+/VE-Cadherin+; this finding has been replicated consistently
with samples from several donors. This is in contrast to freshly
isolated ASCs, which express minimal VE-Cadherin (FIG. 3), prior to
plating. The appearance of a CD34+/VE-Cadherin+cell population
suggests that differentiation towards an endothelial phenotype may
have occurred. This experiment also demonstrates that the adherent
ASC population continues to express high levels of the stem cell
marker CD34 both in conjunction with the expression of VE-cadherin,
as well as in a VE-cadherin-negative population. In parallel
experiments (data not shown), it was found the human ASC population
began to express an additional endothelial marker, the VEGF
receptor-2, (KDR or flk-1) following passage, whereas they did not
express this marker at detectable levels when freshly isolated;
this finding supports the interpretation of direction towards an
endothelial lineage.
[0124] To examine whether ASCs can also manifest typical
endothelial behavior in vitro, their phenotype on Matrigel was
determined. Mature endothelial cells when plated overnight on
Matrigel (BD Biosciences) form tube- and cord-like structures,
reminiscent of a capillary network. Human subcutaneous ASCs that
had been cultured in EGM2MV media on Matrigel overnight were
plated, and the non-adherent cells were discarded the following
day. Human aortic endothelial cells were also plated as a positive
control in a separate Matrigel well (FIG. 6). The endothelial cells
formed the expected tube and cord-like structures. Interestingly,
ASCs also formed similar structures, further supporting the
potential of ASCs to develop an endothelial phenotype.
[0125] In parallel studies designed to evaluate whether ASCs could
also develop a smooth muscle phenotype, we stained ASCs cultured in
DMEM/10% FBS media for the smooth muscle-specific marker, smooth
muscle alpha-actin (FIG. 7). After culture in DMEM/10% FBS, roughly
40-50% of ASCs stain positive for alpha-actin, suggesting the
development of a smooth muscle or myofibroblast phenotype. ASCs
cultured in EGM2MV show essentially no cells staining positively
for SM-alpha actin.
[0126] Together, these data suggest that ASCs can develop
phenotypes of specific vascular cells, in a fashion that is
directly responsive to their growth factor and matrix environments,
suggesting a pliability of phenotype in these cells. These data
demonstrate that ASCs may be able to contribute to angiogenesis or
arteriogenesis directly by providing differentiated cells for
incorporation into nascent vascular structures.
Example III
Secretion of Growth Factors by Adipose Derived Stromal Cells
Secretion of Angiogenic Growth Factors by ASCs
[0127] Since stem and progenitor cells can secrete multiple growth
factors (Majka M. et al., Blood (2001) 97:3075-3085) and putative
therapeutic utilities could in part be related to this endocrine
function, the secretion of angiogenic growth factors by human
subcutaneous ASCs was evaluated. Human subcutaneous adipose stromal
cells were cultured in EGM-2-MV media to confluence, and then
switched into growth-factor free basal media (EBM-2, Clonetics) for
72 hours. Cell supernatants were collected and subsequently assayed
for angiogenic growth factors using ELISA and
Multi-Analyte-Profiling kits (R&D Systems) for Vascular
Endothelial Growth Factor (VEGF), Hepatocyte Growth Factor (HGF)
and Granulocyte-Colony Stimulating Factor (G-CSF). The cell number
was assessed at the end of the 72-hour period, and the secretion of
growth factors is expressed in pg/10.sup.6 ASCs. The cell
supernatants contained significant amounts of secreted VEGF, HGF
and G-CSF (FIG. 8). On the other hand, subcutaneous ASCs did not
secrete detectable levels of the arteriogenic growth factor basic
FGF. These findings indicate that in addition to the pluripotency
of ASCs, their endocrine or paracrine potential may have
significant therapeutic relevance; ASCs delivered to the heart in
the setting of coronary or peripheral arterial occlusive disease,
for example, may be able enhance angiogenesis not only by
differentiating into a vascular phenotype, but also by recruiting
resident mature vascular endothelial cells to integrate into the
nascent vascular network.
Example IV
Stromal Cells Can be Transfected with Mammalian Plasmids and Used
for Gene Therapy
Transfection of Adipose Derived Stromal Cells with
Green-Fluorescent Protein (GFP)
[0128] ASCs are suitable host cells for transfection by mammalian
expression plasmids, thus allowing for use of ASCs as cellular
"vectors" for gene therapy. Such transfected cells should
supplement the therapeutic effects of endogenously secreted growth
factors. Porcine ASCs were isolated from subcutaneous porcine
adipose tissue and cultured in EGM2MV media. Confluent ASC cultures
at passages 0 through 4 were suspended in PBS and electroporated
with green fluorescent protein (GFP) plasmid. Optimal conditions to
achieve high levels of transfection were an electroporation voltage
of 300V, capacitance of 9601 .mu.F and a cell concentration of
5.times.10.sup.4 to 1.5.times.10.sup.5 cells per .mu.g of DNA.
Electroporated cells were plated and GFP expression was assessed on
days 1-3 post transfection. Transfection efficiency as assessed by
either manual counting of GFP-positive cells (FIG. 9) or by flow
cytometric analysis of positive cells was routinely 50% or higher.
These data show that ASCs can be transfected with non-viral methods
and therefore be used as autologous cell vectors for gene therapy.
This application of ASCs will be particularly beneficial in the
therapeutic setting using plasmids, which encode exogenously
secreted factors, which complement the activity of endogenously
produced factors.
Example V
Further Characterization of Adipose Stromal Cells
Flow Cytometric Comparison of Murine ASCs and Muscle Derived Stem
Cells
[0129] Previous data indicated that the expression of Sca-1 on
murine ASCs, and therefore suggested similarity between the
subcutaneous ASC population and muscle derived
Sca-1(+)CD45(-)c-kit(-) stem cells. The phenotype of murine ASCs
was further defined by co-staining for the markers CD45 and c-kit.
As shown in FIG. 10, murine ASCs contain a significant
Sca-1(+)CD45(-)c-kit(-) cell population. These findings support and
extend the data showing the similarity of murine ASCs and the
muscle derived stem cells. This in turn provides further evidence
that ASCs may have a hematopoietic potential similar to what has
been shown for muscle-derived stem cells (Howell J. et al., Exp.
Hem. 30:915-924, 2002).
Example VI
Further Differentiation of Adipose Derived Stromal Cells
Differentiation of Sca-1(+)CD45(-)c-kit(-) Cells
[0130] To characterize the pluripotentiality of adipose
Sca-1(+)CD45(-)c-kit(-) cells in direct comparison to that of
muscle derived stem cells, Sca-1(+)CD45(-)c-kit(-) cells were
purified from both tissues by cell sorting, and cultured in either
neuronal or adipose differentiation media.
[0131] By day 12, muscle-derived Sca-1(+)CD45(-)c-kit(-) cells
differentiated into adipocyte-like (with IGF-1) or neuron-like
(with bFGF and PDGF) cells (FIGS. 11A and B). Neuronal
differentiation of these cells was confirmed by positive staining
for the tau-protein (FIG. 11C.) The similarly purified population
of adipose-derived Sca-1(+)CD45(-)c-kit(-) cells also responded to
these respective growth conditions by differentiating into
adipocyte-like (FIG. 11D) and neuron-like cells (FIG. 11E).
Staining for tau-protein for adipose-derived cells in neuronal
differentiation media was not yet available at the time. Control
experiments with Sca-1-negative cells did not demonstrate any
significant transdifferentiation, thus showing for the first time
that pluripotency of ASCs may be specifically observed in a
particular sorted population. These data suggest that
Sca-1(+)CD45(-)c-kit(-) cells within the uniquely accessible
adipose tissue compartment, have comparable differentiation
potential to similar cells derived from skeletal muscle, and thus
may represent a common stem cell in multiple tissues.
Example VII
Adipose Stromal Cells can be Delivered to the Myocardium Via
Retrograde Coronary Venous Delivery (RCVD) and Direct Injection
Delivery of ASCs to the Myocardium of Pigs Via RCVD
[0132] Juvenile farm pigs, 25-35 kg, were used. The anterior
interventricular vein (AIV) or posterior vein of left ventricle
(PVLV) was cannulated by a balloon-tipped catheter. The balloon was
inflated to prevent venous regurgitation and myocardial blush was
confirmed by 1.0-1.5 cc contrast injection.
[0133] Human adipose stromal cells, and porcine aortic endothelial
cells were delivered via retrograde coronary venous delivery
(RCVD.) Briefly, 5 ml of 2.times.10.sup.6 pre-labeled human adipose
stromal cells or porcine aortic endothelial cells were labeled with
either PKH26 or BrdU, and administered to pigs. Animals were
euthanized 1-1.5 hours post delivery.
[0134] The heart was then sliced transversely, perpendicular to the
apical-basal axis. Planimetry measurement was performed using NIH
Image. Cells in the area of myocardial blush were evaluated by
fluorescence microscopy or immunostaining for BrdU (See FIGS.
12A-D.)
[0135] Retrograde infusion was well tolerated in all animals
without adverse hemodynamic effects; the arterial pressure and
heart rate did not change significantly following delivery. The
retrograde infusion time was 5.6..+-.2.8 sec. Non-sustained
ventricular tachycardia was limited to the time of retrograde
infusion, consisting of 5 to 8 ectopic beats and ceasing
immediately following the injection.
[0136] As shown in FIGS. 12A-D, PKH26 and BrdU pre-labeled cells
were observed at the target myocardium tissue. From this example,
it is clear that multiple cell types can be delivered into the
myocardium using RCVD, and that RCVD results in widespread cell
distribution in the myocardial tissue.
[0137] Delivery of ASCs to the Myocardium of Pigs via Direct
Injection
[0138] Direct Injection of BrdU and PKH26 labeled cells was
performed using a tuberculine syringe. After the heart was exposed,
200 microliters of BrdU pre-labeled cell suspension containing
2.times.10.sup.5 cells was injected into the left ventricle free
wall, at nine discrete injection sites. See FIGS. 12E-F.
Alternatively, cells can be injected into the ventricular muscle
using needle devices which are introduced from the ventricular
lumen (injection into the endocardial surface), or into veins
and/or arteries coursing over the heart. These techniques for
intramuscular and intravascular injection are generally known in
the art.
[0139] The foregoing results demonstrate that delivery of stem
cells via RCVD gives rise to a wider distribution of cells in
target tissue than direct microinjection.
Example VIII
An In vivo Model for Assessment of Angiogenesis
In vivo Assessment of Angiogenesis Using a Mouse Hindlimb Ischemia
Model
[0140] An in vivo model of quantifying angiogenesis over time has
been established in a mouse model using hindlimb ischemia. This has
allowed identification of modulators of angiogenesis. In one study,
for example, the role of the GTP-binding signalling molecule Rac2
was evaluated. Wild-type mice (C57BL6) and Rac2-/- mice underwent
ligation of the femoral artery below the inguinal ligament.
[0141] Non-invasive laser doppler imaging (LDI) (Moor Instruments)
was performed on the ischemic and non-ischemic hindlimbs of all
mice on post-operative days 1, 4, 7, 10, 14 and 21, in order to
evaluate blood flux as a surrogate for perfusion. A representative
LDI image is shown in FIG. 13, where the Rac2-/- mouse has
significant impairment of blood flow to the ischemic leg (Panel
13A), manifested by a blue shift in the colors of the pseudocolored
pixels, while the wild-type mouse has recovered most of the
perfusion to the ischemic leg, reflected by a bilaterally symmetric
flux reflected in the pseudocolor image.
[0142] When the blood perfusion is measured over time and
quantitated as a ratio of blood perfusion in the ischemic to the
non-ischemic leg, it becomes apparent that the impairment in
recovery of limb perfusion in Rac2-/- mice when compared to
wild-type mice is first evident on post-surgery day 4 (FIG. 14).
This difference between the two groups is maintained through day
21. Statistical analysis using a between group repeated measures
ANOVA shows a highly significant between group difference. This
repeated in vivo assessment of angiogenesis allows one to not only
distinguish between global angiogenesis in a control and treatment
group, but also to observe time-dependent changes. This model
system can be used to quantify the effects of ASC transplantation
into ischemic mouse hindlimbs on time-dependent angiogenesis.
Intramuscular Injection of ASCs into Ischemic Hindlimbs Improves
Perfusion
[0143] Human ASCs were isolated and cultured in the presence of
endothelial growth media (EGM-2-MV, Clonetics) containing the
growth factors VEGF, bFGF, EGF and IGF. NOD/SCID immune deficient
mice (n=6, aged 8 months) underwent unilateral femoral artery
ligation and received intramuscular injection of either
4.times.10.sup.5 human ASCs per mouse (n=3) or media (n=3) into the
quadriceps, gastrocnemius and tibialis muscles of the ischemic
hindlimb on the subsequent day. Due to the advanced age of the mice
and the reduced immunologic competence, the endogenous angiogenesis
response of the mice was markedly blunted, thus resulting in some
degree of either toe or limb necrosis in all animals. However, by
day 10 of the study, mice receiving ASC injections had a remarkably
mitigated extent of limb necrosis (FIG. 15). This reduction in
necrosis was statistically significant (FIG. 16).
[0144] This study highlights the in vivo angiogenic potential of
the cells. An ongoing hindlimb ischemia experiment with younger
NOD/SCID mice has not resulted in any significant toe or limb
necrosis, thus allowing us to use Laser Doppler Imaging to detect
subtle perfusion differences between cell-treated and media-treated
animals.
[0145] The cells disclosed herein may optionally be administered by
retrograde coronary venous delivery, or other means set forth
above.
[0146] In conclusion, these preliminary data support the hypotheses
of our original proposal that ASCs have a phenotype and
pluripotentiality that is similar to that of muscle-derived stem
cells and that they can accordingly be used for cell therapies
targeted at enhancing angiogenesis and hematopoiesis. The key
advantage of identifying such cells in subcutaneous adipose tissue
is that they can be harvested by liposuction and therefore increase
the clinical feasibility of autologous cell therapy.
[0147] The previous examples and description set forth certain
embodiments of the present disclosure. It should be appreciated
that not all components or method steps of a complete
implementation of a practical system are necessarily illustrated or
described in detail. Rather, only those components or method steps
necessary for a thorough understanding of the present disclosure
have been illustrated and described in detail. Actual
implementations may utilize more steps or components or fewer steps
or components. Thus, while certain of the preferred embodiments of
the present disclosure have been described and specifically
exemplified above, it is not intended that the present disclosure
be limited to such embodiments. Various modifications may be made
thereto without departing from the scope and spirit of the present
disclosure, as set forth in the following claims.
* * * * *