U.S. patent application number 13/750772 was filed with the patent office on 2013-08-01 for methods and compositions for modulating angiogenesis and vasculogenesis.
This patent application is currently assigned to SanBio, Inc.. The applicant listed for this patent is SanBio, Inc.. Invention is credited to Casey Case, Monique Dao, Ciara Tate.
Application Number | 20130195817 13/750772 |
Document ID | / |
Family ID | 47710337 |
Filed Date | 2013-08-01 |
United States Patent
Application |
20130195817 |
Kind Code |
A1 |
Dao; Monique ; et
al. |
August 1, 2013 |
METHODS AND COMPOSITIONS FOR MODULATING ANGIOGENESIS AND
VASCULOGENESIS
Abstract
Disclosed herein are methods and compositions for stimulating
angiogenesis, using cells descended from marrow adherent stromal
cells that have been transfected with sequences encoding a Notch
intracellular domain. Applications of these methods and
compositions include treatment of ischemic disorders such as
stroke.
Inventors: |
Dao; Monique; (Huntington
Beach, CA) ; Tate; Ciara; (Montpellier, FR) ;
Case; Casey; (San Mateo, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
SanBio, Inc.; |
Mountain View |
CA |
US |
|
|
Assignee: |
SanBio, Inc.
Mountain View
CA
|
Family ID: |
47710337 |
Appl. No.: |
13/750772 |
Filed: |
January 25, 2013 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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61591486 |
Jan 27, 2012 |
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61637740 |
Apr 24, 2012 |
|
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61709619 |
Oct 4, 2012 |
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Current U.S.
Class: |
424/93.21 |
Current CPC
Class: |
A61K 35/34 20130101;
A61K 38/1866 20130101; C12N 2533/90 20130101; C12N 2510/02
20130101; C12N 5/0662 20130101; C12N 2502/28 20130101; C12N 2501/42
20130101; C12N 5/0668 20130101; A61K 38/1866 20130101; A61K 48/00
20130101; A61K 35/28 20130101; A61P 9/00 20180101; A61K 45/06
20130101; A61P 25/00 20180101; C12N 5/0697 20130101; C12N 2502/1347
20130101; A61K 35/28 20130101; A61P 9/10 20180101; C12N 5/0663
20130101; A61K 2300/00 20130101; A61K 35/44 20130101; A61L 27/3834
20130101; A61K 2300/00 20130101 |
Class at
Publication: |
424/93.21 |
International
Class: |
A61K 35/28 20060101
A61K035/28; A61K 45/06 20060101 A61K045/06; C12N 5/071 20060101
C12N005/071; A61K 38/18 20060101 A61K038/18 |
Claims
1. A method for the repair of ischemic damage in a subject, the
method comprising administering to the subject a population of
SB623 cells; wherein the SB623 cells are obtained by: (a) providing
a culture of mesenchymal stem cells, (b) contacting the cell
culture of step (a) with a polynucleotide comprising sequences
encoding a Notch intracellular domain (NICD) wherein said
polynucleotide does not encode a full-length Notch protein, (c)
selecting cells that comprise the polynucleotide of step (b), and
(d) further culturing the selected cells of step (c) in the absence
of selection.
2. The method of claim 1, wherein the repair of ischemic damage is
selected from the group consisting of augmentation of angiogenesis,
prevention of endothelial cell death, enhancement of endothelial
cell survival, stimulation of endothelial cell proliferation,
enhancement of blood vessel branching, and provision of one or more
angiogenic factors.
3. The method of claim 2, wherein the angiogenic factor is selected
from the group consisting of one or more of angiogenin,
angiopoietin-2, epidermal growth factor, basic fibroblast growth
factor, leptin, platelet-derived growth factor-BB, and placental
growth factor.
4. The method of claim 1, wherein the ischemic damage occurs in the
central nervous system of the subject.
5. The method of claim 4, wherein the ischemic damage occurs in the
brain of the subject.
6. A composition for use in the treatment of ischemic damage in a
subject, the composition comprising: (1) a population of SB623
cells; wherein the SB623 cells are obtained by: (a) providing a
culture of mesenchymal stem cells, (b) contacting the cell culture
of step (a) with a polynucleotide comprising sequences encoding a
Notch intracellular domain (NICD) wherein said polynucleotide does
not encode a full-length Notch protein, (c) selecting cells that
comprise the polynucleotide of step (b), and (d) further culturing
the selected cells of step (c) in the absence of selection; and (2)
a pro-angiogenic agent.
7. The composition of claim 6, wherein the pro-angiogenic agent is
a polypeptide.
8. The composition of claim 6, wherein the pro-angiogenic agent is
a nucleic acid.
9. The composition of claim 7, wherein the polypeptide is a
transcription factor that activates expression of a pro-angiogenic
protein.
10. The composition of claim 9, wherein the pro-angiogenic protein
is vascular endothelial growth factor (VEGF).
11. The composition of claim 10, wherein the transcription factor
is a non-naturally-occurring zinc finger protein that activates
transcription of the VEGF gene.
12. A method for the repair of ischemic damage in a subject, the
method comprising administering to the subject an effective amount
of the composition of claim 6.
13. The method of claim 12, wherein the repair of ischemic damage
is selected from the group consisting of augmentation of
angiogenesis, prevention of endothelial cell death, enhancement of
endothelial cell survival, stimulation of endothelial cell
proliferation, enhancement of blood vessel branching, and provision
of one or more angiogenic factors.
14. The method of claim 13, wherein the angiogenic factor is
selected from the group consisting of one or more of angiogenin,
angiopoietin-2, epidermal growth factor, basic fibroblast growth
factor, leptin, platelet-derived growth factor-BB, and placental
growth factor.
15. The method of claim 12, wherein the ischemic damage occurs in
the central nervous system of the subject.
16. The method of claim 15, wherein the ischemic damage occurs in
the brain of the subject.
17. The method of claim 16, wherein the ischemic damage results
from stroke.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application claims the benefit of U.S. Provisional
Patent Application No. 61/591,486 filed on Jan. 27, 2012, U.S.
Provisional Patent Application No. 61/637,740 filed on Apr. 24,
2012, and U.S. Provisional Patent Application No. 61/709,619 filed
on Oct. 4, 2012; the specifications and drawings of which are
incorporated herein by reference in their entireties for all
purposes.
STATEMENT REGARDING FEDERAL SUPPORT
[0002] Not applicable.
FIELD
[0003] The present disclosure is in the fields of angiogenesis and
vasculogenesis; e.g., for the treatment of ischemic events such as
stroke. It is also in the field of stem cells and cells derived
from stem cells by genetic manipulation.
BACKGROUND
[0004] In stable stroke, reinstating vascular flow is imperative
for restoring nutrient supply in the brain. To repair vascular
damage after prolonged ischemia, at least two sequential steps are
needed. The first step is angiogenic sprouting of endothelial cells
(ECs); this process entails the initial proliferation of
endothelial cells and remodeling of the surrounding extracellular
matrix. VEGF-mediated proliferation of ECs and matrix
metalloproteinases are among the major components of angiogenic
sprouting. The second step is vessel stabilization; a process that
relies on recruitment of vascular smooth muscle cells to encase the
young vessels. Monocytes and pericytes are also involved in vessel
stabilization, producing the appropriate arteriogenic factors and
extracellular matrix proteins. In the absence of vessel
stabilization by smooth muscle cells and pericytes, regression of
nascent vasculature can occur.
[0005] Marrow stromal cells (MSCs, also known as mesenchymal stem
cells)) have been shown to promote revascularization after cerebral
artery occlusion and traumatic brain in jury. Omori et al. (2008)
Brain Res. 1236:30-38; Onda et al. (2008) J. Cereb. Blood Flow
Metab. 28:329-340; Pavlichenko et al. (2008) Brain Res.
1233:203-213; Xiong et al. (2009) Brain Res. 1263:183-191. SB623
cells are a derivative of marrow stromal cells, obtained by
transfecting marrow stromal cells with a vector containing
sequences encoding a Notch intracellular domain (NICD). See, for
example, U.S. Pat. No. 7,682,825 and Dezawa et al. (2004) J. Clin.
Investig. 13:1701-1710. SB623 cells elicit functional improvement
in experimental stroke models. See, for example, U.S. Pat. No.
8,092,792 and Yasuhara et al. (2009) Stem Cells and Development
18:1501-1514. Although the secretome of SB623 cells is comparable
to that of the parental MSCs; different levels of specific trophic
factors have been observed to be secreted by MSCs, as compared to
SB623 cells. See, for example, Tate et al. (2010) Cell
Transplantation 19:973-984; U.S. Patent Application Publication No.
2010/0266554. Moreover, many of the factors whose expression levels
differ between MSCs and SB623 cells have been reported to be
involved in vascular regeneration.
[0006] Because stroke is a leading cause of adult disability in the
United States, and is the second leading cause of death worldwide,
there remains a need for treatments to restore blood supply to, and
promote reperfusion of, regions of stroke-induced ischemic damage
in the brain.
SUMMARY
[0007] The present inventors have discovered that descendents of
mesenchymal stem cells that have been transfected with sequences
encoding a Notch intracellular domain (i.e., SB623 cells) have the
surprising property of being able to synthesize and secrete factors
that promote angiogenesis. Because angiogenesis, i.e., the
formation of new blood vessels, is a critical part of the
endogenous repair process in brain injury and disease, this
discovery provides new methods of treatment for vascular disorders
such as stroke.
[0008] Accordingly, the present disclosure provides, inter
alia:
[0009] 1. A method for augmenting angiogenesis in a subject, the
method comprising administering to the subject a population of
SB623 cells; wherein the SB623 cells are obtained by (a) providing
a culture of mesenchymal stem cells, (b) contacting the cell
culture of step (a) with a polynucleotide comprising sequences
encoding a Notch intracellular domain (NICD) wherein said
polynucleotide does not encode a full-length Notch protein, (c)
selecting cells that comprise the polynucleotide of step (b), and
(d) further culturing the selected cells of step (c) in the absence
of selection.
[0010] 2. The method of embodiment 1, wherein the augmentation of
angiogenesis occurs in the central nervous system.
[0011] 3. The method of embodiment 2, wherein the augmentation of
angiogenesis occurs in the brain.
[0012] 4. A method for repairing ischemic damage in a subject, the
method comprising administering to the subject a population of
SB623 cells; wherein the SB623 cells are obtained by (a) providing
a culture of mesenchymal stem cells, (b) contacting the cell
culture of step (a) with a polynucleotide comprising sequences
encoding a Notch intracellular domain (NICD) wherein said
polynucleotide does not encode a full-length Notch protein, (c)
selecting cells that comprise the polynucleotide of step (b), and
(d) further culturing the selected cells of step (c) in the absence
of selection.
[0013] 5. The method of embodiment 4, wherein the ischemic damage
occurs in the central nervous system.
[0014] 6. The method of embodiment 5, wherein the ischemic damage
occurs in the brain.
[0015] 7. The method of embodiment 6, wherein the ischemic damage
results from stroke.
[0016] 8. A method for enhancing survival of endothelial cells, the
method comprising contacting the endothelial cells with a
population of SB623 cells; wherein the SB623 cells are obtained by
(a) providing a culture of mesenchymal stem cells, (b) contacting
the cell culture of step (a) with a polynucleotide comprising
sequences encoding a Notch intracellular domain (NICD) wherein said
polynucleotide does not encode a full-length Notch protein, (c)
selecting cells that comprise the polynucleotide of step (b), and
(d) further culturing the selected cells of step (c) in the absence
of selection.
[0017] 9. The method of embodiment 8, wherein the method prevents
the death of endothelial cells.
[0018] 10. The method of either of embodiments 8 or 9, wherein the
endothelial cells are in a subject.
[0019] 11. The method of embodiment 10, wherein the endothelial
cells are in the central nervous system of the subject.
[0020] 12. The method of embodiment 11, wherein the endothelial
cells are in the brain of the subject.
[0021] 13. A method for stimulating proliferation of endothelial
cells, the method comprising contacting the endothelial cells with
a population of SB623 cells; wherein the SB623 cells are obtained
by (a) providing a culture of mesenchymal stem cells, (b)
contacting the cell culture of step (a) with a polynucleotide
comprising sequences encoding a Notch intracellular domain (NICD)
wherein said polynucleotide does not encode a full-length Notch
protein, (c) selecting cells that comprise the polynucleotide of
step (b), and (d) further culturing the selected cells of step (c)
in the absence of selection.
[0022] 14. The method of embodiment 13, wherein the endothelial
cells are in a subject.
[0023] 15. The method of embodiment 14, wherein the endothelial
cells are in the central nervous system of the subject.
[0024] 16. The method of embodiment 15, wherein the endothelial
cells are in the brain of the subject.
[0025] 17. A method for enhancing the branching of blood vessels,
the method comprising contacting the vessels with a population of
SB623 cells; wherein the SB623 cells are obtained by (a) providing
a culture of mesenchymal stem cells, (b) contacting the cell
culture of step (a) with a polynucleotide comprising sequences
encoding a Notch intracellular domain (NICD) wherein said
polynucleotide does not encode a full-length Notch protein, (c)
selecting cells that comprise the polynucleotide of step (b), and
(d) further culturing the selected cells of step (c) in the absence
of selection.
[0026] 18. The method of embodiment 17, wherein the blood vessels
are in a subject
[0027] 19. The method of embodiment 18, wherein the blood vessels
are in the central nervous system of the subject.
[0028] 20. The method of embodiment 19, wherein the blood vessels
are in the brain of the subject.
[0029] 21. A method for augmenting angiogenesis in a subject, the
method comprising administering to the subject (1) a population of
SB623 cells; wherein the SB623 cells are obtained by (a) providing
a culture of mesenchymal stem cells, (b) contacting the cell
culture of step (a) with a polynucleotide comprising sequences
encoding a Notch intracellular domain (NICD) wherein said
polynucleotide does not encode a full-length Notch protein, (c)
selecting cells that comprise the polynucleotide of step (b), and
(d) further culturing the selected cells of step (c) in the absence
of selection; and (2) a pro-angiogenic agent.
[0030] 22. The method of embodiment 21, wherein the augmentation of
angiogenesis occurs in the central nervous system.
[0031] 23. The method of embodiment 22, wherein the augmentation of
angiogenesis occurs in the brain.
[0032] 24. The method of embodiment 21, wherein the pro-angiogenic
agent is a nucleic acid.
[0033] 25. The method of embodiment 21, wherein the pro-angiogenic
agent is a polypeptide.
[0034] 26. The method of embodiment 25, wherein the polypeptide is
a transcription factor that activates expression of a
pro-angiogenic protein.
[0035] 27. The method of embodiment 26, wherein the pro-angiogenic
protein is vascular endothelial growth factor (VEGF).
[0036] 28. The method of embodiment 27, wherein the transcription
factor is a non-naturally-occurring zinc finger protein that
activates transcription of the VEGF gene.
[0037] 29. A method for repairing ischemic damage in a subject, the
method comprising administering to the subject (1) a population of
SB623 cells; wherein the SB623 cells are obtained by (a) providing
a culture of mesenchymal stem cells, (b) contacting the cell
culture of step (a) with a polynucleotide comprising sequences
encoding a Notch intracellular domain (NICD) wherein said
polynucleotide does not encode a full-length Notch protein, (c)
selecting cells that comprise the polynucleotide of step (b), and
(d) further culturing the selected cells of step (c) in the absence
of selection; and (2) a pro-angiogenic agent.
[0038] 30. The method of embodiment 29, wherein the ischemic damage
occurs in the central nervous system.
[0039] 31. The method of embodiment 30, wherein the ischemic damage
occurs in the brain.
[0040] 32. The method of embodiment 31, wherein the ischemic damage
results from stroke.
[0041] 33. The method of embodiment 29, wherein the pro-angiogenic
agent is a nucleic acid.
[0042] 34. The method of embodiment 29, wherein the pro-angiogenic
agent is a polypeptide.
[0043] 35. The method of embodiment 34, wherein the polypeptide is
a transcription factor that activates expression of a
pro-angiogenic protein.
[0044] 36. The method of embodiment 35, wherein the pro-angiogenic
protein is vascular endothelial growth factor (VEGF).
[0045] 37. The method of embodiment 36, wherein the transcription
factor is a non-naturally-occurring zinc finger protein that
activates transcription of the VEGF gene.
[0046] 38. A method for treating stroke in a subject, the method
comprising administering to the subject (1) a population of SB623
cells; wherein the SB623 cells are obtained by (a) providing a
culture of mesenchymal stem cells, (b) contacting the cell culture
of step (a) with a polynucleotide comprising sequences encoding a
Notch intracellular domain (NICD) wherein said polynucleotide does
not encode a full-length Notch protein, (c) selecting cells that
comprise the polynucleotide of step (b), and (d) further culturing
the selected cells of step (c) in the absence of selection; and (2)
a pro-angiogenic agent.
[0047] 39. The method of embodiment 38, wherein the pro-angiogenic
agent is a nucleic acid.
[0048] 40. The method of embodiment 38, wherein the pro-angiogenic
agent is a polypeptide.
[0049] 41. The method of embodiment 40, wherein the polypeptide is
a transcription factor that activates expression of a
pro-angiogenic protein.
[0050] 42. The method of embodiment 41, wherein the pro-angiogenic
protein is vascular endothelial growth factor (VEGF).
[0051] 43. The method of embodiment 42, wherein the transcription
factor is a non-naturally-occurring zinc finger protein that
activates transcription of the VEGF gene.
[0052] 44. The method of any of embodiments 8, 9, or 13, further
comprising administering a pro-angiogenic agent along with the
SB623 cells.
[0053] 45. The method of embodiment 44, wherein the endothelial
cells are in a subject.
[0054] 46. The method of embodiment 45, wherein the endothelial
cells are in the central nervous system of the subject.
[0055] 47. The method of embodiment 46, wherein the endothelial
cells are in the brain of the subject.
[0056] 48. The method of embodiment 44, wherein the pro-angiogenic
agent is a nucleic acid.
[0057] 49. The method of embodiment 44, wherein the pro-angiogenic
agent is a polypeptide.
[0058] 50. The method of embodiment 49, wherein the polypeptide is
a transcription factor that activates expression of a
pro-angiogenic protein.
[0059] 51. The method of embodiment 50, wherein the pro-angiogenic
protein is vascular endothelial growth factor (VEGF).
[0060] 52. The method of embodiment 51, wherein the transcription
factor is a non-naturally-occurring zinc finger protein that
activates transcription of the VEGF gene.
[0061] 53. The method of embodiment 17, further comprising
administering a pro-angiogenic agent along with the SB623
cells.
[0062] 54. The method of embodiment 53, wherein the blood vessels
are in a subject.
[0063] 55. The method of embodiment 54, wherein the blood vessels
are in the central nervous system of the subject.
[0064] 56. The method of embodiment 55, wherein the blood vessels
are in the brain of the subject.
[0065] 57. The method of embodiment 53, wherein the pro-angiogenic
agent is a nucleic acid.
[0066] 58. The method of embodiment 53, wherein the pro-angiogenic
agent is a polypeptide.
[0067] 59. The method of embodiment 58, wherein the polypeptide is
a transcription factor that activates expression of a
pro-angiogenic protein.
[0068] 60. The method of embodiment 59, wherein the pro-angiogenic
protein is vascular endothelial growth factor (VEGF).
[0069] 61. The method of embodiment 60, wherein the transcription
factor is a non-naturally-occurring zinc finger protein that
activates transcription of the VEGF gene.
[0070] 62. A method for providing an angiogenic factor to a
subject, wherein the method comprises administering to the subject
a population of SB623 cells; wherein the SB623 cells are obtained
by (a) providing a culture of mesenchymal stem cells, (b)
contacting the cell culture of step (a) with a polynucleotide
comprising sequences encoding a Notch intracellular domain (NICD)
wherein said polynucleotide does not encode a full-length Notch
protein, (c) selecting cells that comprise the polynucleotide of
step (b), and (d) further culturing the selected cells of step (c)
in the absence of selection.
[0071] 63. The method of embodiment 62, wherein the subject is
suffering from an ischemic disorder.
[0072] 64. The method of embodiment 63, wherein the subject is
suffering from a disease or disorder of the central nervous
system.
[0073] 65. The method of embodiment 62, wherein the trophic factor
is selected from the group consisting of one or more of angiogenin,
angiopoietin-2, epidermal growth factor, basic fibroblast growth
factor, heparin-binding epithelial growth factor-like growth
factor, hepatocyte growth factor, leptin, platelet-derived growth
factor-BB, placental growth factor and vascular endothelial growth
factor.
[0074] 66. The method of embodiment 65, wherein the trophic factor
is vascular endothelial growth factor.
[0075] 67. A method for providing vascular endothelial growth
factor to a subject, wherein the method comprises administering to
the subject a population of SB623 cells; wherein the SB623 cells
are obtained by (a) providing a culture of mesenchymal stem cells,
(b) contacting the cell culture of step (a) with a polynucleotide
comprising sequences encoding a Notch intracellular domain (NICD)
wherein said polynucleotide does not encode a full-length Notch
protein, (c) selecting cells that comprise the polynucleotide of
step (b), and (d) further culturing the selected cells of step (c)
in the absence of selection.
[0076] 68. The method of embodiment 67, wherein the subject is
suffering from an ischemic disorder.
[0077] 69. The method of embodiment 68, wherein the subject is
suffering from a disease or disorder of the central nervous
system.
BRIEF DESCRIPTION OF THE DRAWINGS
[0078] FIG. 1 shows measurements of the fraction of cells permeable
to propidium iodide in cultures of HUVECs that have been starved
for serum and growth factors. Left-most bar shows results obtained
from control serum/growth factor-starved HUVECs; center bar shows
results for serum/growth factor-starved HUVECs cultured for seven
days in the presence of conditioned medium from MSCs, and the
right-most bar shows results for serum/growth factor-starved HUVECs
cultured for seven days in the presence of conditioned medium from
SB623 cells. Values shown are mean.+-.SD for three separate donors
of MSCs and SB623 cells; * indicates p<0.05 compared to control
group.
[0079] FIG. 2 shows measurement of the fraction of cells expressing
Bcl-2 in cultures of HUVECs that have been starved for serum and
growth factors. Left-most bar shows results obtained from control
serum/growth factor-starved HUVECs; center bar shows results for
serum/growth factor-starved HUVECs cultured for seven days in the
presence of conditioned medium from MSCs, and the right-most bar
shows results for serum/growth factor-starved HUVECs cultured for
seven days in the presence of conditioned medium from SB623 cells.
Results were obtained by measuring fluorescence of cells stained
with a fluorescein-conjugated anti-Bcl-2 antibody and subtracting
fluorescence of cells exposed to fluorescein-conjugated IgG. Values
shown are mean.+-.SD for three separate donors of MSCs and SB623
cells; * indicates p<0.05 compared to control group.
[0080] FIG. 3 shows measurement of the fraction of cells expressing
Ki67 in cultures of HUVECs that have been starved for serum and
growth factors. Left-most bar shows results obtained from control
serum/growth factor-starved HUVECs; center bar shows results for
serum/growth factor-starved HUVECs cultured for seven days in the
presence of conditioned medium from MSCs, and the right-most bar
shows results for serum/growth factor-starved HUVECs cultured for
seven days in the presence of conditioned medium from SB623 cells.
Results were obtained by measuring fluorescence of cells stained
with a fluorescein-conjugated anti-Ki67 antibody and subtracting
fluorescence of cells exposed to fluorescein-conjugated IgG. Values
shown are mean.+-.SD for three separate donors of MSCs and SB623
cells; * indicates p<0.05 compared to control group.
[0081] FIG. 4 shows phase-contrast photomicrographs of HUVECs
following culture for 16 hours in conditioned media from MSCs or
SB623 cells. The left-most photograph shows cells cultured in
conditioned medium from MSCs; the center photograph shows cells
cultured in conditioned medium from SB623 cells; and the right-most
photograph shows cells cultured in commercial culture medium
without added conditioned medium.
[0082] FIG. 5 shows measurement of the effect of conditioned medium
on tube formation by HUVECs. Left-most bar shows results obtained
from control serum/growth factor-starved HUVECs; center bar shows
results for serum/growth factor-starved HUVECs cultured for 16
hours in the presence of conditioned medium from MSCs, and the
right-most bar shows results for serum/growth factor-starved HUVECs
cultured for 16 hours in the presence of conditioned medium from
SB623 cells. Values shown are mean.+-.SEM for three separate donors
of MSCs and SB623 cells.
[0083] FIGS. 6A-6C show photographs of aortic rings after culture
for 10 days in unconditioned medium (A), MSC conditioned medium
(B), or SB623 cell conditioned medium (C).
[0084] FIGS. 7A and 7B show measurements of vessel sprouting and
branching in an aortic ring assay. FIG. 7A shows counts of new
vessels and of branchpoints in the new vessels. For each of the
three pairs of bars, the left bar shows measurements of new vessel
formation and the right bar shows measurements of vessel branching.
The left-most pair of bars ("Control") shows results obtained from
control aortic rings; the center pair of bars ("MSC CM") shows
results obtained from aortic rings cultured for 10 days in MSC
conditioned medium; and the right-most pair of bars ("SB623 CM")
shows results obtained from aortic rings cultured for 10 days in
SB623 cell conditioned medium. FIG. 7B show ratios of branchpoints
to new vessels for control aortic rings (left bar), rings cultured
10 days in MSC conditioned medium (center bar) and rings cultured
10 days in SB623 cell conditioned medium (right bar).
[0085] Values shown are Mean.+-.SEM for 7 donor pairs. "*"
indicates p<0.05 compared to control group.
[0086] FIG. 8 shows levels of four different trophic factors in
conditioned medium from MSCs (light bars) and SB623 cells (dark
bars). Protein levels are expressed as picograms per ml of
conditioned medium per 10.sup.6 cells. Conditioned media from MSCs
(and SB623 cells derived therefrom) from four different human
donors were tested, as indicated in the figure. Levels of
angiogenin, angiopoietin-2, heparin-binding epidermal growth
factor-like growth factor (HB-EGF), and placental growth factor
(PIGF) are shown.
[0087] FIG. 9 shows levels of ten different cytokines in
conditioned medium from MSCs and SB623 cells. Cells for production
of conditioned medium were obtained from four different donors (D1,
D2, D3 and D4), as indicated in the figure. This figure highlights
the vast amounts of VEGF produced by MSCs and SB623 cells, compared
to the levels of the other trophic factors tested. Abbreviations
are given in the legend to Table 1 (Example 6).
[0088] FIGS. 10A and 10B show the effects of a VEGF receptor
inhibitor on improvements in HUVEC viability promoted by SB623
cell-conditioned medium. FIG. 10A shows the fraction of cells
permeable to propidium iodide in cultures of HUVECs that had been
starved for serum and growth factors. Left-most bar shows results
obtained from control serum/growth factor-starved HUVECs; center
bar shows results for serum/growth factor-starved HUVECs cultured
for five days in the presence of conditioned medium from SB623
cells, and the right-most bar shows results for serum/growth
factor-starved HUVECs cultured for five days in the presence of
conditioned medium from SB623 cells and 50 nM SU5416. Results were
averaged from two donors; "*" indicates p<0.05 with respect to
control cultures; "#" indicates p<0.05 with respect to cultures
exposed to SB623 cell conditioned medium and SU5416.
[0089] FIG. 10B shows measurement of the fraction of cells
expressing Bcl-2 in a culture of HUVECs that had been starved for
serum and growth factors. Left-most bar shows results obtained from
control serum/growth factor-starved HUVECs; center bar shows
results for serum/growth factor-starved HUVECs cultured for five
days in the presence of conditioned medium from SB623 cells, and
the right-most bar shows results for serum/growth factor-starved
HUVECs cultured for five days in the presence of conditioned medium
from SB623 cells and 50 nM SU5416. Results, averaged from duplicate
donors, were obtained by measuring fluorescence of cells stained
with a fluorescein-conjugated anti-Bcl-2 antibody and subtracting
fluorescence of cells exposed to fluorescein-conjugated IgG.
[0090] FIG. 11 shows measurement of the fraction of cells
expressing Ki67 in HUVEC cultures exposed to SB623 cell conditioned
medium in the presence and absence of the VEGFR2 inhibitor SU5416,
and by control cells cultured in the absence of CM. The left-most
(clear) bar shows results obtained from control serum/growth
factor-starved HUVECs; the center (black) bar shows results for
serum/growth factor-starved HUVECs cultured in the presence of
conditioned medium from SB623 cells, and the right-most (gray) bar
shows results for serum/growth factor-starved HUVECs cultured in
the presence of conditioned medium from SB623 cells and 50 nM
SU5416. Values shown are mean.+-.SEM for two separate donors of
SB623 cells. "*" indicates p<0.05 with respect to the negative
control cultures (no conditioned medium); "#" indicates p<0.05
with respect to SU5416-treated cultures.
[0091] FIG. 12 shows the effects of a VEGF receptor inhibitor on
the enhancement of tube formation by HUVECs promoted by MSC- and
SB623 cell-conditioned media. The top row shows cells cultured in
the absence of the inhibitor. The left-most panel of the top row
("neg") shows a phase-contrast photomicrograph of control HUVECs
following culture for 16 hours in Opti-MEM Medium. The second panel
from the left ("+10 ng VEGF") shows a phase-contrast
photomicrograph of HUVECs following culture for 16 hours in
Opti-MEM Medium to which 10 ng/ml VEGF was added. The third panel
from the left ("+MSC-CM") shows a phase-contrast photomicrograph of
HUVECs following culture for 16 hours in MSC-conditioned medium.
The rightmost panel ("+SB623-CM") shows a phase-contrast
photomicrograph of HUVECs following culture for 16 hours in SB623
cell-conditioned medium. Panels in the bottom row show
photomicrographs of HUVECs under the same conditions as in the top
row but with the addition of 50 nM SU5416.
[0092] FIG. 13 shows quantitation of tube formation by HUVECs
exposed to SB623 cell conditioned medium in the presence and
absence of the VEGFR2 inhibitor SU5416, and by control cells
cultured in the absence of CM.
[0093] For each time point, the left-most (clear) bar shows results
obtained from control serum/growth factor-starved HUVECs; the
center (black) bar shows results for serum/growth factor-starved
HUVECs cultured in the presence of conditioned medium from SB623
cells, and the right-most (gray) bar shows results for serum/growth
factor-starved HUVECs cultured in the presence of conditioned
medium from SB623 cells and 50 nM SU5416. Values shown are
mean.+-.SEM for three separate donors of SB623 cells. "*" indicates
p<0.05 with respect to the negative control cultures (no
conditioned medium); "#" indicates p<0.05 with respect to
SU5416-treated cultures.
[0094] FIG. 14 shows the effects of a VEGF receptor inhibitor on
enhancement of vessel outgrowth and branching promoted by SB623
cell-conditioned medium in an aortic ring assay. In the upper row,
the left panel shows a photomicrograph of an aortic ring after
culture for 10 days on a RGF-basement gel in OptiMEM medium
("Negative control"). The center panel shows a photomicrograph of
an aortic ring after culture for 10 days in SB623 cell conditioned
medium ("+SB623-CM"). The right panel shows a photomicrograph of an
aortic ring after culture for 10 days in SB623 cell conditioned
medium containing 50 nM SU5416 ("+SB623-CM+SU5416"). Enlargements
of certain regions of each photomicrograph are shown in the lower
row.
DETAILED DESCRIPTION
[0095] Disclosed herein are new methods and compositions for
modulation of angiogenesis. In particular, factors secreted by
SB623 cells (cells descended from MSCs that have been transfected
with a vector containing sequences encoding a Notch intracellular
domain) promote survival and proliferation of endothelial cells in
vitro under serum- and growth factor-deprived conditions, and
stimulate vascular tube formation by human umbilical vein
endothelial cells. In addition, conditioned medium from SB623 cells
promoted endothelial sprouting and branching in a rodent aortic
ring assay.
[0096] Practice of the present disclosure employs, unless otherwise
indicated, standard methods and conventional techniques in the
fields of cell biology, toxicology, molecular biology,
biochemistry, cell culture, immunology, oncology, recombinant DNA
and related fields as are within the skill of the art. Such
techniques are described in the literature and thereby available to
those of skill in the art. See, for example, Alberts, B. et al.,
"Molecular Biology of the Cell," 5.sup.th edition, Garland Science,
New York, N.Y., 2008; Voet, D. et al. "Fundamentals of
Biochemistry: Life at the Molecular Level," 3.sup.rd edition, John
Wiley & Sons, Hoboken, N.J., 2008; Sambrook, J. et al.,
"Molecular Cloning: A Laboratory Manual," 3.sup.rd edition, Cold
Spring Harbor Laboratory Press, 2001; Ausubel, F. et al., "Current
Protocols in Molecular Biology," John Wiley & Sons, New York,
1987 and periodic updates; Freshney, R. I., "Culture of Animal
Cells: A Manual of Basic Technique," 4.sup.th edition, John Wiley
& Sons, Somerset, N.J., 2000; and the series "Methods in
Enzymology," Academic Press, San Diego, Calif.
[0097] For the purposes of the present disclosure, "angiogenesis"
refers to the formation of new vasculature (e.g., blood vessels;
e.g., veins, arteries, venules, arterioles, capillaries).
Angiogenesis can occur by sprouting of new vessels from an existing
vessel, and/or by branching of a vessel. Angiogenesis also includes
the attendant processes of matrix remodeling and cell recruitment
(e.g., recruitment of smooth muscle cells, monocytes and/or
pericytes).
[0098] "MSCs" refer to adherent, non-hematopoietic stem cells
obtained from bone marrow. These cells are variously known as
mesenchymal stem cells, mesenchymal stromal cells, marrow adherent
stromal cells, marrow adherent stem cells and bone marrow stromal
cells.
[0099] Stroke
[0100] "Stroke" is the name given to conditions resulting from
impairment of blood flow in the brain. Such cerebrovascular
impairment can result, for example, from intracranial hemorrhage,
or from reduction or blockage of blood flow in the brain (i.e.,
cerebral ischemia). Ischemic blockages can result from thrombosis
(i.e., formation of a clot in situ in a cranial vessel or a vessel
supplying the brain) or from a cerebral embolism (i.e., migration
of a clot to a site in the brain). The damage resulting from
ischemic or hemorrhagic stroke usually results in impairment of
neurological function. Additional information relating to different
types of stroke, and their characteristics, is found in co-owned
U.S. Pat. No. 8,092,792; the disclosure of which is incorporated by
reference in its entirety herein for the purpose of describing
different types of stroke and their characteristics.
[0101] Mesenchymal Stem Cells (MSCs)
[0102] The present disclosure provides methods for promoting
angiogenesis by transplanting SB623 cells to a site of ischemic
injury in a subject. SB623 cells are obtained from marrow adherent
stromal cells, also known as mesenchymal stem cells (MSCs), by
expressing the intracellular domain of the Notch protein in the
MSCs. MSCs are obtained by selecting adherent cells (i.e., cells
that adhere to tissue culture plastic) from bone marrow.
[0103] Exemplary disclosures of MSCs are provided in U.S. patent
application publication No. 2003/0003090; Prockop (1997) Science
276:71-74 and Jiang (2002) Nature 418:41-49. Methods for the
isolation and purification of MSCs can be found, for example, in
U.S. Pat. No. 5,486,359; Pittenger et al. (1999) Science
284:143-147 and Dezawa et al. (2001) Eur. J. Neurosci.
14:1771-1776. Human MSCs are commercially available (e.g.,
BioWhittaker, Walkersville, Md.) or can be obtained from donors by,
e.g., bone marrow aspiration, followed by selection for adherent
bone marrow cells. See, e.g., WO 2005/100552.
[0104] MSCs can also be isolated from umbilical cord blood. See,
for example, Campagnoli et al. (2001) Blood 98:2396-2402; Erices et
al. (2000) Br. J. Haematol. 109:235-242 and Hou et al. (2003) Int.
J. Hematol. 78:256-261.
[0105] Notch Intracellular Domain
[0106] The Notch protein is a transmembrane receptor, found in all
metazoans, that influences cell differentiation through
intracellular signaling. Contact of the Notch extracellular domain
with a Notch ligand (e.g., Delta, Serrate, Jagged) results in two
proteolytic cleavages of the Notch protein, the second of which is
catalyzed by a .gamma.-secretase and releases the Notch
intracellular domain (NICD) into the cytoplasm. In the mouse Notch
protein, this cleavage occurs between amino acids gly1743 and
val1744. The NICD translocates to the nucleus, where it acts as a
transcription factor, recruiting additional transcriptional
regulatory proteins (e.g., MAM, histone acetylases) to relieve
transcriptional repression of various target genes (e.g., Hes
1).
[0107] Additional details and information regarding Notch signaling
are found, for example in Artavanis-Tsakonas et al. (1995) Science
268:225-232; Mumm and Kopan (2000) Develop. Biol. 228:151-165 and
Ehebauer et al. (2006) Sci. STKE 2006 (364), cm7. [DOI:
10.1126/stke.3642006 cm7].
[0108] Cell Culture and Transfection
[0109] Standard methods for cell culture are known in the art. See,
for example, R. I. Freshney "Culture of Animal Cells: A Manual of
Basic Technique," Fifth Edition, Wiley, New York, 2005.
[0110] Methods for introduction of exogenous DNA into cells (i.e.,
transfection) are also well-known in the art. See, for example,
Sambrook et al. "Molecular Cloning: A Laboratory Manual," Third
Edition, Cold Spring Harbor Laboratory Press, 2001; Ausubel et al.,
"Current Protocols in Molecular Biology," John Wiley & Sons,
New York, 1987 and periodic updates.
[0111] SB623 Cells
[0112] In one embodiment for the preparation of SB623 cells, a
culture of MSCs is contacted with a polynucleotide comprising
sequences encoding a Notch intracellular domain (NICD); e.g., by
transfection; followed by enrichment of transfected cells by drug
selection and further culture. See, for example, U.S. Pat. No.
7,682,825 (Mar. 23, 2010); U.S. Patent Application Publication No.
2010/0266554 (Oct. 21, 2010); and WO 2009/023251 (Feb. 19, 2009);
all of which disclosures are incorporated by reference, in their
entireties, for the purposes of describing isolation of mesenchymal
stem cells and conversion of mesenchymal stem cells to SB623 cells
(denoted "neural precursor cells" and "neural regenerating cells"
in those documents).
[0113] In these methods, any polynucleotide encoding a Notch
intracellular domain (e.g., vector) can be used, and any method for
the selection and enrichment of transfected cells can be used. For
example, in certain embodiments, MSCs are transfected with a vector
containing sequences encoding a Notch intracellular domain and also
containing sequences encoding a drug resistance marker (e.g.
resistance to G418). In additional embodiments, two vectors, one
containing sequences encoding a Notch intracellular domain and the
other containing sequences encoding a drug resistance marker, are
used for transfection of MSCs. In these embodiments, selection is
achieved, after transfection of a cell culture with the vector or
vectors, by adding a selective agent (e.g., G418) to the cell
culture in an amount sufficient to kill cells that do not comprise
the vector but spare cells that do. Absence of selection entails
removal of said selective agent or reduction of its concentration
to a level that does not kill cells that do not comprise the
vector. Following selection (e.g., for seven days) the selective
agent is removed and the cells are further cultured (e.g., for two
passages).
[0114] Preparation of SB623 cells thus involves transient
expression of an exogenous Notch intracellular domain in a MSC. To
this end, MSCs can be transfected with a vector comprising
sequences encoding a Notch intracellular domain wherein said
sequences do not encode a full-length Notch protein. All such
sequences are well known and readily available to those of skill in
the art. For example, Del Amo et al. (1993) Genomics 15:259-264
present the complete amino acid sequences of the mouse Notch
protein; while Mumm and Kopan (2000) Devel. Biol. 228:151-165
provide the amino acid sequence, from mouse Notch protein,
surrounding the so-called S3 cleavage site which releases the
intracellular domain. Taken together, these references provide the
skilled artisan with each and every peptide containing a Notch
intracellular domain that is not the full-length Notch protein;
thereby also providing the skilled artisan with every
polynucleotide comprising sequences encoding a Notch intracellular
domain that does not encode a full-length Notch protein. The
foregoing documents (Del Amo and Mumm) are incorporated by
reference in their entireties for the purpose of disclosing the
amino acid sequence of the full-length Notch protein and the amino
acid sequence of the Notch intracellular domain, respectively.
[0115] Similar information is available for Notch proteins and
nucleic acids from additional species, including rat, Xenopus,
Drosophila and human. See, for example, Weinmaster et al. (1991)
Development 113:199-205; Schroeter et al. (1998) Nature
393:382-386; NCBI Reference Sequence No. NM.sub.--017167 (and
references cited therein); SwissProt P46531 (and references cited
therein); SwissProt Q01705 (and references cited therein); and
GenBank CAB40733 (and references cited therein). The foregoing
references are incorporated by reference in their entireties for
the purpose of disclosing the amino acid sequence of the
full-length Notch protein and the amino acid sequence of the Notch
intracellular domain in a number of different species.
[0116] In additional embodiments, SB623 cells are prepared by
introducing, into MSCs, a nucleic acid comprising sequences
encoding a Notch intracellular domain such that the MSCs do not
express exogenous Notch extracellular domain. Such can be
accomplished, for example, by transfecting MSCs with a vector
comprising sequences encoding a Notch intracellular domain wherein
said sequences do not encode a full-length Notch protein.
[0117] Additional details on the preparation of SB623 cells, and
methods for making cells with properties similar to those of SB623
cells which can be used in the methods disclosed herein, are found
in U.S. Pat. No. 7,682,825; and U.S. Patent Application Publication
Nos. 2010/0266554 (Oct. 21, 2010) and 2011/0229442 (Sep. 22, 2011);
the disclosures of which are incorporated by reference herein for
the purposes of providing additional details on, and alternative
methods for the preparation of, SB623 cells, and for providing
methods for making cells with properties similar to those of SB623
cells. See also Dezawa et al. (2004) J. Clin. Invest.
113:1701-1710.
[0118] Uses
[0119] As disclosed herein, the inventors have discovered that
descendants of mesenchymal stem cells in which a Notch
intracellular domain has been transiently expressed (i.e., SB623
cells) have angiogenic activity; and that said cells synthesize and
secrete angiogenic factors. Accordingly, transplantation of SB623
cells is useful for treatment of disorders in which a therapeutic
benefit can be achieved by increasing angiogenesis in a subject.
Such disorders include, but are not limited to, cerebral ischemia
(e.g., stroke), cardiac ischemia (e.g., ischemic heart disease),
ischemia of the bowel (e.g., ischemic colitis, mesenteric
ischemia), ischemia of the limb, cutaneous ischemia, ocular
ischemic syndrome (e.g., retinal ischemia) and cerebral palsy.
[0120] Thus, SB623 cells as described herein can be used in a
number of methods related to stimulation of angiogenesis. These
include, but are not limited to, treatment of any of the disorders
mentioned in the previous paragraph, augmentation of angiogenesis,
repair of ischemic damage, preventing death of endothelial cells,
enhancing survival of endothelial cells, stimulating proliferation
of endothelial cells, and/or enhancing the branching of blood
vessels,
[0121] Such methods can be performed in vitro or in a subject. The
subject can be a mammal, preferably a human. Stimulation of
angiogenesis by SB623 cells, and the attendant effects of such
stimulation as disclosed herein, can occur, for example, in the
central nervous system (e.g., in the brain).
[0122] Transplantation of SB623 cells can also be used in methods
for providing one or more angiogenic trophic factors to a subject.
Such factors include, but are not limited to, angiogenin,
angiopoietin-2, epidermal growth factor, basic fibroblast growth
factor, heparin-binding epithelial growth factor-like growth
factor, hepatocyte growth factor, leptin, platelet-derived growth
factor-BB, placental growth factor and vascular endothelial growth
factor.
[0123] In additional embodiments, SB623 cells can be used in
combination with a second pro-angiogenic agent, in combination
therapies for increasing angiogenesis in a subject. Said
combination therapies can be used for all of the purposes set forth
above. The second pro-angiogenic agent can be, e.g., a small
molecule drug, a nucleic acid or a polypeptide (e.g., antibody,
transcription factor). Exemplary nucleic acids are triplex-forming
nucleic acids, ribozymes and siRNAs that activate expression of
angiogenic proteins and/or block expression of anti-angiogenic
proteins. Exemplary antibodies are those that bind to and/or
inhibit the activity of angiogenic proteins (or other angiogenic
agents). Exemplary transcription factors are those that inhibit
transcription of a gene encoding one or more anti-angiogenic
protein(s), as well as those that activate the transcription of one
or more pro-angiogenic protein(s). Anti-angiogenic and
pro-angiogenic proteins are known in the art. Exemplary
anti-angiogenic proteins include pigment epithelium derived factor
(PEDF) and placental growth factor (PlGF). Exemplary pro-angiogenic
proteins include vascular endothelial growth factor (VEGF)
angiopoietin, and hepatocyte growth factor (HGF).
[0124] In certain embodiments, transcription factors as disclosed
above are non-naturally-occurring (engineered) transcription
factors. An example of such a non-naturally-occurring transcription
factor is a non-naturally-occurring zinc finger protein that has
been engineered to bind to a DNA sequence in cellular chromatin
that regulates transcription of a target gene (e.g., a VEGF gene).
Said engineered zinc finger transcription factors comprise, in
addition to an engineered zinc finger DNA-binding domain, a
transcriptional regulatory domain (e.g., a transcriptional
activation domain or a transcriptional repression domain), as are
known in the art.
[0125] Methods for engineering zinc finger DNA binding domains, to
bind to a DNA sequence of choice, are well-known in the art. See,
for example, Beerli et al. (2002) Nature Biotechnol. 20:135-141;
Pabo et al. (2001) Ann. Rev. Biochem. 70:313-340; Isalan et al.
(2001) Nature Biotechnol. 19:656-660; Segal et al. (2001) Curr.
Opin. Biotechnol. 12:632-637; Choo et al. (2000) Curr. Opin.
Struct. Biol. 10:411-416. Zinc finger binding domain are engineered
to have a novel binding specificity, compared to a
naturally-occurring zinc finger protein. Engineering methods
include, but are not limited to, rational design and various types
of empirical selection methods. Rational design includes, for
example, using databases comprising triplet (or quadruplet)
nucleotide sequences and individual zinc finger amino acid
sequences, in which each triplet or quadruplet nucleotide sequence
is associated with one or more amino acid sequences of zinc fingers
which bind the particular triplet or quadruplet sequence. See, for
example, U.S. Pat. Nos. 6,140,081; 6,453,242; 6,534,261; 6,610,512;
6,746,838; 6,866,997; 7,067,617; U.S. Patent Application
Publication Nos. 2002/0165356; 2004/0197892; 2007/0154989;
2007/0213269; and International Patent Application Publication Nos.
WO 98/53059 and WO 2003/016496.
[0126] Exemplary selection methods, including phage display,
interaction trap, hybrid selection and two-hybrid systems, are
disclosed in U.S. Pat. Nos. 5,789,538; 5,925,523; 6,007,988;
6,013,453; 6,140,466; 6,200,759; 6,242,568; 6,410,248; 6,733,970;
6,790,941; 7,029,847 and 7,297,491; as well as U.S. Patent
Application Publication Nos. 2007/0009948 and 2007/0009962; WO
98/37186; WO 01/60970 and GB 2,338,237.
[0127] Enhancement of binding specificity for zinc finger binding
domains has been described, for example, in U.S. Pat. No. 6,794,136
(Sep. 21, 2004). Additional aspects of zinc finger engineering,
with respect to inter-finger linker sequences, are disclosed in
U.S. Pat. No. 6,479,626 and U.S. Patent Application Publication No.
2003/0119023. See also Moore et al. (2001a) Proc. Natl. Acad. Sci.
USA 98:1432-1436; Moore et al. (2001b) Proc. Natl. Acad. Sci. USA
98:1437-1441 and WO 01/53480.
[0128] Transcriptional activation and repression domain are known
in the art. See, e.g., Science 269:630 (1995). Exemplary
transcriptional activation domains include p65, VP16 and VP64.
Exemplary transcriptional repression domains include KRAB, KAP-1,
MAD, FKHR, ERD and SID. Functional domains from nuclear hormone
receptors can act as either activators or repressors, depending
upon the presence of a ligand. See also U.S. Pat. No.
7,985,887.
[0129] Formulations, Kits and Routes of Administration
[0130] Therapeutic compositions comprising SB623 cells as disclosed
herein are also provided. Such compositions typically comprise the
SB623 cells and a pharmaceutically acceptable carrier.
Supplementary active compounds can also be incorporated into SB623
cell compositions (see below).
[0131] The therapeutic compositions disclosed herein are useful
for, inter alia, stimulating angiogenesis after occurrence of a
stroke or other ischemic injury in a subject. Accordingly, a
"therapeutically effective amount" of a composition comprising
SB623 cells can be any amount that stimulates angiogenesis. For
example, dosage amounts can vary from about 100; 500; 1,000; 2,500;
5,000; 10,000; 20,000; 50,000; 100,000; 500,000; 1,000,000;
5,000,000 to 10,000,000 cells or more (or any integral value
therebetween); with a frequency of administration of, e.g., once
per day, twice per week, once per week, twice per month, once per
month, depending upon, e.g., body weight, route of administration,
severity of disease, etc.
[0132] Various pharmaceutical compositions and techniques for their
preparation and use are known to those of skill in the art in light
of the present disclosure. For a detailed listing of suitable
pharmacological compositions and techniques for their
administration one may refer to texts such as Remington's
Pharmaceutical Sciences, 17th ed. 1985; Brunton et al., "Goodman
and Gilman's The Pharmacological Basis of Therapeutics,"
McGraw-Hill, 2005; University of the Sciences in Philadelphia
(eds.), "Remington: The Science and Practice of Pharmacy,"
Lippincott Williams & Wilkins, 2005; and University of the
Sciences in Philadelphia (eds.), "Remington: The Principles of
Pharmacy Practice," Lippincott Williams & Wilkins, 2008.
[0133] The cells described herein can be suspended in a
physiologically compatible carrier for transplantation. As used
herein, the term "physiologically compatible carrier" refers to a
carrier that is compatible with the SB623 cells and with any other
ingredients of the formulation, and is not deleterious to the
recipient thereof. Those of skill in the art are familiar with
physiologically compatible carriers. Examples of suitable carriers
include cell culture medium (e.g., Eagle's minimal essential
medium), phosphate buffered saline, Hank's balanced salt
solution+/-glucose (HBSS), and multiple electrolyte solutions such
as Plasma-Lyte.TM. A (Baxter).
[0134] The volume of a SB623 cell suspension administered to a
patient will vary depending on the site of implantation, treatment
goal and number of cells in solution. Typically the amount of cells
administered to a patient will be a therapeutically effective
amount. As used herein, a "therapeutically effective amount" or
"effective amount" refers to the number of transplanted cells which
are required to effect treatment of the particular disorder; i.e.,
to produce a reduction in the amount and/or severity of the
symptoms associated with that disorder. For example, in the case of
stroke, transplantation of a therapeutically effective amount of
SB623 cells results in new vessel growth, vessel sprouting and
vessel branching, e.g., in an area that has been damaged by
ischemia. Therapeutically effective amounts vary with the type and
extent of ischemic damage, and can also vary depending on the
overall condition of the subject.
[0135] The disclosed therapeutic compositions can also include
pharmaceutically acceptable materials, compositions or vehicles,
such as a liquid or solid filler, diluent, excipient, solvent or
encapsulating material, i.e., carriers. These carriers can, for
example, stabilize the SB623 cells and/or facilitate the survival
of the SB623 cells in the body. Each carrier should be "acceptable"
in the sense of being compatible with the other ingredients of the
formulation and not injurious to the subject. Some examples of
materials which can serve as pharmaceutically-acceptable carriers
include: sugars, such as lactose, glucose and sucrose; starches,
such as corn starch and potato starch; cellulose and its
derivatives, such as sodium carboxymethyl cellulose, ethyl
cellulose and cellulose acetate; powdered tragacanth; malt;
gelatin; talc; excipients, such as cocoa butter and suppository
waxes; oils, such as peanut oil, cottonseed oil, safflower oil,
sesame oil, olive oil, corn oil and soybean oil; glycols, such as
propylene glycol; polyols, such as glycerin, sorbitol, mannitol and
polyethylene glycol; esters, such as ethyl oleate and ethyl
laurate; agar; buffering agents, such as magnesium hydroxide and
aluminum hydroxide; alginic acid; pyrogen-free water; isotonic
saline; Ringer's solution; ethyl alcohol; phosphate buffer
solutions; and other non-toxic compatible substances employed in
pharmaceutical formulations. Wetting agents, emulsifiers and
lubricants, such as sodium lauryl sulfate and magnesium stearate,
as well as coloring agents, release agents, coating agents,
sweetening, flavoring and perfuming agents, preservatives and
antioxidants can also be present in the compositions.
[0136] Exemplary formulations include, but are not limited to,
those suitable for parenteral administration, e.g., intrapulmonary,
intravenous, intra-arterial, intra-ocular, intra-cranial,
sub-meningial, or subcutaneous administration, including
formulations encapsulated in micelles, liposomes or drug-release
capsules (active agents incorporated within a biocompatible coating
designed for slow-release); ingestible formulations; formulations
for topical use, such as eye drops, creams, ointments and gels; and
other formulations such as inhalants, aerosols and sprays. The
dosage of the compositions of the disclosure will vary according to
the extent and severity of the need for treatment, the activity of
the administered composition, the general health of the subject,
and other considerations well known to the skilled artisan.
[0137] In additional embodiments, the compositions described herein
are delivered locally to a site of ischemic damage. Localized
delivery allows for the delivery of the composition
non-systemically, thereby reducing the body burden of the
composition as compared to systemic delivery. Such local delivery
can be achieved, for example, by intra-cranial injection, or
through the use of various medically implanted devices including,
but not limited to, stents and catheters, or can be achieved by
inhalation, phlebotomy, or surgery. Methods for coating,
implanting, embedding, and otherwise attaching desired agents to
medical devices such as stents and catheters are established in the
art and contemplated herein.
[0138] Another aspect of the present disclosure relates to kits for
carrying out the administration of SB623 cells, optionally in
combination with another therapeutic agent, to a subject. In one
embodiment, a kit comprises a composition of SB623 cells,
formulated in a pharmaceutical carrier, optionally containing,
e.g., a pro-angiogenic agent (see below), formulated as
appropriate, in one or more separate pharmaceutical
preparations.
[0139] Combination Therapies
[0140] In certain embodiments, SB623 cell compositions can be used
in combination with other compositions comprising substances that
stimulate angiogenesis ("pro-angiogenic agents"), e.g., for
treatment of stroke. The compositions can be administered
sequentially in any order or concurrently. Accordingly, therapeutic
compositions as disclosed herein can contain both SB623 cells and a
pro-angiogenic agent. In additional embodiments, separate
therapeutic compositions, one comprising SB623 cells and the other
comprising a pro-angiogenic agent, can be administered to the
subject, either separately or together.
[0141] In certain embodiments, a pro-angiogenic agent is a protein
(e.g., fibroblast growth factor, platelet-derived growth factor,
transforming growth factor alpha, hepatocyte growth factor,
vascular endothelial growth factor, sonic hedgehog, MAGP-2, HIF-1,
PR-39, RTEF-1, c-Myc, TFII, Egr-1, ETS-1) or a nucleic acid
encoding such a protein. See, for example, Vincent et al. (2007)
Gene Therapy 14:781-789. In other embodiments, a pro-angiogenic
agent can be a small RNA molecule (e.g., siRNA, shRNA, microRNA) or
a ribozyme that targets a nucleic acid encoding an inhibitor of
angiogenesis. In additional embodiments, a pro-angiogenic agent can
be a triplex-forming nucleic acid that binds to DNA sequences
regulating the expression of a protein that inhibits angiogenesis,
such as to block transcription of the gene encoding the
protein.
[0142] In additional embodiments, a pro-angiogenic agent is a
transcription factor that activates expression of a pro-angiogenic
molecule (e.g., protein). Naturally-occurring transcription factors
(such as, for example, HIF-1alpha) that regulate the expression of
pro-angiogenic proteins, are known. In addition, synthetic
transcriptional regulatory proteins can be constructed by genetic
engineering. For example, methods for the design of zinc finger
DNA-binding domains that bind to a sequence of interest, and
methods for the fusion of such zinc finger DNA-binding domains to
transcriptional activation and repression domains, have been
described. See, for example, U.S. Pat. Nos. 6,534,261; 6,607,882;
6,785,613; 6,794,136; 6,824,978; 6,933,113; 6,979,539; 7,013,219;
7,177,766; 7,220,719; and 7,788,044. These methods can be used to
synthesize non-naturally-occurring proteins that activate
transcription of any gene encoding a pro-angiogenic protein. In
addition, synthetic zinc finger transcriptional activators of the
vascular endothelial growth factor (VEGF) gene have been described.
See, e.g., U.S. Pat. Nos. 7,026,462; 7,067,317; 7,560,440;
7,605,140; and 8,071,564. Accordingly, a non-naturally-occurring
(i.e., synthetic) zinc finger protein that activates transcription
of the VEGF gene can be used, in combination with SB623 cells, for
augmenting angiogenesis, e.g., in the treatment of stroke.
[0143] In additional embodiments, a natural or synthetic
transcriptional regulatory protein (e.g., a synthetic zinc finger
transcriptional regulatory protein) that inhibits transcription of
an anti-angiogenic molecule can be used as a pro-angiogenic
agent.
EXAMPLES
Example 1
Conditioned Medium
[0144] MSCs and SB623 cells were obtained and/or prepared as
described. See, for example, U.S. Pat. No. 7,682,825 (Mar. 23,
2010) and U.S. Patent Application Publications Nos. 2010/0266554
(Oct. 21, 2010), 2010/0310529 (Dec. 9, 2010), 2011/0229442 (Sep.
22, 2011), and 2011/0306137 (Dec. 15, 2011); the disclosures of
which are incorporated by reference in their entireties for the
purposes of describing the preparation of SB623 cells (variously
referred to as "neural precursor cells" and "neural regenerating
cells" in those documents). Cells were cultured in growth medium,
which contained alph.alpha.-MEM (Mediatech, Herndon, Va.)
supplemented with 10% fetal bovine serum (FBS, Hyclone, Logan,
Utah), 2 mM L-glutamine and 1% penicillin/streptomycin (both from
Invitrogen, Carlsbad, Calif.). MSCs and SB623 cells typically
expressed CD29, CD90 and CD105; and did not express CD31, CD34, or
CD45, as determined by flow cytometry.
[0145] For use in the experiments described herein, frozen MSCs and
SB623 cells from the same human donor were thawed, re-plated in
growth medium, and allowed to recover for approximately one week.
To obtain conditioned medium, cells were grown to approximately 90%
confluence (15,000 cells/cm.sup.2), the plates were rinsed once
with phosphate buffered saline (PBS) and the medium was then
replaced with OptiMEM.RTM. medium (Invitrogen, Carlsbad, Calif.),
maintaining the same cell density. Conditioned medium was collected
72 hours later. Frozen samples of conditioned medium were slowly
warmed to 37.degree. C. prior to use.
Example 2
Effect of SB623 Cell-Secreted Factors on HUVEC Survival
[0146] Cerebral ischemia can result in loss of nutrient supply to
the affected area. To determine if soluble factors from SB623 cells
and MSCs have restorative effects on nutrient-deprived endothelial
cells, human umbilical vein endothelial cells (HUVECs) were
cultured in medium depleted of serum and growth factors for 24
hours, then exposed to conditioned medium (CM) from MSCs or SB623
cells. Control cultures remained in serum- and growth
factor-depleted medium without addition of CM. Viability and
proliferative capacity of the HUVECs were then assessed.
[0147] For these experiments, human umbilical vein endothelial
cells were passed twice, then 7.5.times.10.sup.5 cells were plated
in EBM-2/ECGS medium (Endothelial Basal Medium-2/Endothelial Cell
Growth Supplements; Lonza, Walkersville, Md.) on T-75 flasks coated
with 0.1% gelatin and cultured for 24 hours. The HUVEC monolayers
were rinsed twice with warm PBS and incubated with 12 ml of fresh
EBM-2 medium overnight at 37.degree. C., 5% CO.sub.2. Effects of CM
were then assessed by withdrawing 6 ml of medium from each flask,
and replacing it with 6 ml fresh OptiMEM (control), 6 ml MSC
conditioned medium, or 6 ml SB623 cell conditioned medium
(conditioned media prepared as described in Example 1). After 7
days, non-adherent and adherent cells were collected, centrifuged
at 1400 rpm for 5 min, and divided into three fractions for
subsequent staining analyses (PI, Bcl-2 and Ki67).
[0148] To quantify cell death, cells were stained with propidium
iodide (PI), since dead cells are permeable to PI. Cells were
stained with 5 ug/ml PI for 30 min at room temperature, and flow
cytometry acquisition and analysis were conducted using the FL-2
logarithmic channel of a BD FACSCalibur CellQuest program (BD
Biosciences, San Jose, Calif.). For this assay, 3 different human
donor pairs were tested. The results are shown in FIG. 1. In
control HUVEC cultures maintained in nutrient-deprived medium for 7
days, more than 70% of the cells were positive for propidium iodide
staining. Addition of either SB623- or MSC-conditioned medium
significantly reduced the percentage of propidium iodide positive
cells (p<0.05).
[0149] These results indicate that both MSC conditioned medium and
SB623 cell conditioned medium significantly reduced death of
endothelial cells (i.e., reduced the number of propidium
iodide-positive HUVECs) resulting from serum and growth factor
starvation.
[0150] Bcl-2 is an anti-apoptotic protein originally identified as
being overexpressed in certain B-cell lymphomas. Accordingly, the
fraction of cells expressing the Bcl-2 protein was measured in
serum/growth factor-starved HUVECs as an indicator of their
apoptotic potential. For Bcl-2 measurement, cells were fixed in 4%
paraformaldehyde and permeabilized with 0.1% Triton-X100 for one
hour. Following permeabilization, cells were stained for one hour,
on ice, with fluorescein-conjugated anti-Bcl-2 antibody, then
samples were washed, acquired, and analyzed on the FL-1 channel of
a BD FACSCalibur. Cells exposed to fluorescein-conjugated IgG were
used as a negative control. For these assays, 3 different human
donor pairs were tested.
[0151] The results, shown in FIG. 2, indicate that presence of
either MSC conditioned medium or SB623 cell conditioned medium
significantly increased the fraction of Bcl-2-positive cells in
cultures of serum-starved endothelial cells.
[0152] The fact that conditioned medium from MSCs or from SB623
cells decreased the number of dead (PI-positive) cells and
increased of the number of cells expressing the anti-apoptotic
Bcl-2 protein shows that both MSCs and SB623 cells secrete factors
that enhance endothelial cell survival.
Example 3
Effect of SB623 Cell-Secreted Factors on HUVEC Proliferation
[0153] Ki67 is a protein present in cells exiting from the GO
(quiescent) phase of the cell cycle; therefore Ki67 levels can be
used as a measure of cell proliferation. The fraction of cells
expressing Ki67 protein was measured in HUVECs that had been
starved for serum and growth factors, then cultured with
conditioned medium from either MSCs or SB623 cells.
[0154] For Ki67 measurement, HUVECs were cultured and exposed to CM
as described in Example 2. Cells were fixed in 4% paraformaldehyde
and permeabilized with 0.1% Triton-X100 for one hour. Following
permeabilization, cells were stained for one hour on ice with
fluorescein-conjugated anti-KI67 antibody, then samples were
washed, acquired, and analyzed on the FL-1 channel of a BD
FACSCalibur. Cells exposed to fluorescein-conjugated IgG were used
as a negative control. For these assays, 3 different human donor
pairs were tested.
[0155] FIG. 3 shows that culture of starved HUVECs in the presence
of conditioned medium from either MSCs or SB623 cells resulted in
an increased fraction of cells expressing Ki67, compared to control
HUVECs not exposed to conditioned medium. The fact that conditioned
medium from MSCs or from SB623 cells increased the number of cells
expressing the proliferation-associated Ki67 protein shows that
both MSCs and SB623 cells secrete factors that enhance endothelial
cell proliferation.
[0156] The results presented in this and the previous example
revealed significant increases in survival and proliferation of
HUVECs when these endothelial cells were cultured for 7 days with
MSC- or SB623 cell-conditioned medium, compared to culture in
unconditioned medium (p<0.05).
Example 4
Effect of SB623 Cell-Secreted Factors on Tube Formation by
Endothelial Cells
[0157] A HUVEC tube formation assay was used to test the ability of
MSCs and SB623 cells to elaborate factors that stimulate vessel
formation. See, for example, E J Smith & CA Staton, "Tubule
formation assays," in Angiogenesis Assays--A Critical Appraisal of
Current Techniques, (Staton, Lewis & Bicknell, eds.). John
Wiley & Sons, Ltd., West Sussex, UK, pp. 65-87, 2006; and
Goodwin (2007) Microvasc. Res. 74:172-183.
[0158] HUVECs were passed five times in EBM-2/ECGS medium, then
transferred to alph.alpha.-MEM/0.5% FBS/2 mM glutamine/pen-strep,
at a density of 1.times.10.sup.5 cells/ml. After 24 hours, HUVECs
were harvested using 0.25% trypsin-EDTA, rinsed, and resuspended in
.alpha.-MEM/2 mM glutamine/pen-strep at a density of
1.times.10.sup.5 cells/ml. A mixture of 75 ul of HUVECs plus 75 ul
of either MSC- or SB623-conditioned medium (Example 1), or 75 ul
OptiMEM medium as a negative control, was added to each well of a
96-well plate that had been pre-treated by adding 50 ul of Reduced
Growth Factor (RGF)-basement gel (Invitrogen, Carslsbad, Calif.)
per well and incubating the plates at 37.degree. C. for 45 minutes.
For this assay, MSCs were obtained from 3 different human donors,
and a portion of the MSCs from each donor were converted to SB623
cells.
[0159] After 16 hours, the cultures were examined by phase contrast
microscopy and photographed. The number of complete tubes (formed
by contiguous cells) was quantified by an experimenter blinded to
the group. A photograph showing results from one of the three
donors is shown in FIG. 4. The results of assays using MSCs and
SB623 cells from all three donors, summarized in FIG. 5, indicate
that tube formation is strongly enhanced by conditioned medium from
either MSCs or SB623 cells. Thus, MSCs and SB623 cells secrete
factors that promote vasculogenesis.
Example 5
Effect of SB623 Cell-Secreted Factors on Vessel Outgrowth and
Branching
[0160] Restoration of vasculature after ischemic injury requires
that surviving endothelial cells receive signals that prompt their
migration and invasion. Such signals may arise from vascular smooth
muscle cells, monocytes, and/or macrophages, among others. To test
for secretion of factors involved in vessel sprouting and
branching, the aortic ring assay was used. See, for example,
Nicosia & Ottinetti (1990) Lab. Invest. 63:115-122 and Nicosia
(2009) J. Cell. Mol. Med. 13:4113-4136.
[0161] For preparation of aortic rings, adult Sprague-Dawley rats
were euthanized prior to dissection. After clamping off its two
ends, the aorta was removed and placed in ice-cold
.alpha.-MEM/pen-strep medium prior to removal of the external
adipose layer. Adipose-free aorta was rinsed twice with ice-cold
EBM-2/pen-strep medium before being sectioned into rings of 1.0 mm
thickness. The aortic rings were then transferred to plates
containing EBM-2/pen-strep medium and incubated at 37.degree. C.,
5% CO.sub.2 for 6 days, with the medium replaced with fresh
EBM-2/pen-strep medium on day 3, to deplete any endogenous rat
angiogenic factors. At that point, the medium was replaced with
alpha-MEM/pen-strep medium and culture was continued for 24
hours.
[0162] On day 0 of the aortic ring assay (seven days after
beginning of culture), 50 .mu.l of reduced growth factor (RGF)
basement gel was deposited per well of a 24-well plate. An
individual aortic ring was placed in the middle of each gel-coated
well and overlaid with an additional 25 .mu.l of RGF-basement gel.
After allowing 30 minutes at 37.degree. C./5% CO.sub.2 for
solidification of the gel, 500 .mu.l of .alpha.-MEM/2 mM
glutamine/pen-strep was added to each well and incubation was
continued for an additional 30 minutes. Then, 500 ul of either MSC-
or SB623-derived conditioned medium (Example 1) was added. As a
negative control, 500 .mu.l of OptiMEM medium was used in place of
conditioned medium.
[0163] To assess the angiogenic activity of MSC- and SB623-derived
factors, phase contrast photographs were taken on Day 10, and
results were quantified by an experimenter blinded to the group, by
counting vessel outgrowth and branching. Growth of new vessels was
quantitated by measuring the number of vessels growing out from the
ring; and vessel branching was quantitated by measuring the number
of branchpoints present in vessels growing out from the aortic
ring. For this assay, 7 different human donor pairs were
tested.
[0164] Representative results from Day 10 samples are shown in FIG.
6, and results from seven sets of 10-day cultures are summarized
and quantitated in FIG. 7. FIG. 7A shows that conditioned medium
from both MSCs and SB623 cells stimulated an increase in the number
of newly-sprouted vessels and in the degree of branching, compared
to control aortic rings. Moreover, significant increases in vessel
branching were observed in rings cultured in SB623 cell-conditioned
medium (FIG. 6C, FIG. 7A), compared with either rings cultured in
MSC-conditioned medium (FIG. 6B, FIG. 7A) or rings cultured in
unconditioned medium (FIG. 6A, FIG. 7A). These results indicate
that MSCs and SB623 cells secrete factors that enhance vessel
sprouting and vessel branching. In particular, SB623 cells secrete
factors that greatly enhance vessel branching (see FIG. 7B).
[0165] The data presented in the foregoing examples indicate that
SB623 cell-secreted soluble factors promote several aspects of
angiogenesis, which contribute to recovery in the injured
brain.
Example 6
Statistics
[0166] For each experiment (which included 3-4 wells/group), a mean
value was obtained for: (1) the treatment condition for each cell
type (either MSC- or SB623 cell-derived conditioned medium; one
value per human donor tested) and (2) the untreated group (one
value for each round of testing). For statistical comparison
(SigmaStat, SystatSoftware, Chicago, Ill.) each of these values
were used and comparisons were made using one way ANOVA between the
following groups (1) Control (unconditioned medium; n=3), (2)
MSC-conditioned medium (n=3-5); and (3) SB623 cell-conditioned
medium (n=3-5). Additional pair-wise comparisons were made using
Tukey's test. An alpha value of 0.05 was used to determine whether
the means were significantly different.
Example 7
Identification of Angiogenic Factors Secreted by MSCs and SB623
Cells
[0167] The levels of certain cytokines and trophic factors in
conditioned medium from MSCs and SB623 cells were measured. To
obtain conditioned medium, MSCs or SB623 cells were cultured in
growth medium to .about.90% confluence (15,000 cells/cm.sup.2), at
which point medium was removed, the cells were rinsed in PBS, and
Opti-MEM.RTM. medium (Invitrogen, Carlsbad, Calif.) was added to
give a concentration of .about.150,000 cells/ml. The conditioned
medium was collected 72 hours later and assayed using a
Quantibody.RTM.Human Angiogenesis Array 1 (RayBiotech, Norcross,
Ga.) according to the manufacturer's instructions. For each source
of MSCs, a portion of the cells were cultured directly as MSCs and
a portion were converted to SB623 cells. Thus, a culture of MSCs
from a particular donor and a culture of SB623 cells made from
those MSCs, are referred to as a matched "donor pair." In this
experiment, four donor pairs were assayed. Results, expressed as
protein concentration, were normalized to the number of cells
present in the culture when the conditioned medium was collected.
FIG. 8 shows results, by donor, for angiogenin, ANG-2, HB-EGF and
P1GF. FIG. 9 shows results for these four factors, and six others,
also by donor, and highlights the large amounts of VEGF produced by
MSCs and SB623 cells.
[0168] Table 1 shows protein levels averaged among the four donor
pairs for the ten factors tested. Although levels of trophic
factors secreted were variable among the different donors (as
shown, for example, in FIGS. 8 and 9), levels of four of the
factors (angiogenin, angiopoietin-2, HB-EGF and P1GF) were
consistently different between MSCs and SB623 cells. Angiogenin,
ANG-2 and HB-EGF were more highly expressed by SB623 cells, while
higher concentrations of P1GF were produced by MSCs.
TABLE-US-00001 TABLE 1 Levels of Angiogenic trophic factors in
conditioned medium from MSCs and SB623 Cells MSCs SB623 Cells
FACTOR AVG SD AVG SD Angiogenin 741 178 985 271 ANG-2 540 252 641
275 EGF -- n/a -- n/a bFGF 53 7 40 17 HB-EGF 205 162 282 228 HGF
123 55 143 75 Leptin 397 226 437 213 PDGF-BB 18 22 16 18 PIGF 300
178 171 85 VEGF 30,503 9229 38,119 8692 Abbreviations are as
follows. ANG-2: angiopoietin-2; EGF: epidermal growth factor; bFGF:
basic fibroblast growth factor/fibroblast growth factor 2; HB-EGF:
heparin-binding epidermal growth factor-like growth factor; HGF:
hepatocyte growth factor; PDGF-BB: platelet-derived growth
factor-BB; PIGF: placental growth factor; VEGF: vascular
endothelial growth factor. Numbers refer to cytokine levels
expressed as pg/ml/10.sup.6 cells. "AVG" refers to the average
value from 4 sources of MSCs and 4 sources of SB623 cells from
which conditioned medium was obtained; "SD" refers to standard
deviation. "--" indicates that levels, if any, were below the limit
of detection in the assay; "n/a" indicates "not applicable"
Example 8
Effect of an Inhibitor of VEGF Signaling on HUVEC Viability and
Proliferation
[0169] In light of the large amounts of VEGF secreted by both MSCs
and SB623 cells, the contribution of VEGF to the pro-angiogenic
activities of MSC- and SB623-conditioned media was tested using an
inhibitor of VEGF signaling. SU5416 (VEGFR2 kinase inhibitor III,
EMD Millipore, Billerica, Mass.) blocks downstream signaling by
VEGF receptor 2 (Flk-1) and, to a lesser extent, by VEGF receptor 1
(Flt-1) and other receptor tyrosine kinases, thereby inhibiting
angiogenesis.
[0170] HUVEC viability assays (propidium iodide uptake and Bcl-2
expression) were conducted as described in Example 2 on two batches
of SB623 cell-conditioned medium, in the presence and absence of 50
nM SU5416; except that cells were cultured for five days, instead
of seven, before assay. The inhibitor was added to cultures 30
minutes before addition of CM. Since higher concentrations of
SU5416 can inhibit receptor tyrosine kinases other than VEGFR2,
this SU5416 concentration was chosen so that VEGFR2 signaling (but
not signaling by, e.g., PDGF receptor, EGF receptors, or Flt3) was
inhibited. The results, shown in FIGS. 10A and 10B, indicate that
more cells take up PI (FIG. 10A) and fewer cells express the
anti-apoptotic Bcl-2 protein (FIG. 10B) when HUVECs are cultured in
SB623 conditioned medium and SU5416, than when they are cultured in
SB623 cell-conditioned medium alone. Thus, inhibition of VEGF
receptor activity partially reduces the positive effect of SB623
cell-conditioned medium on viability of HUVECs, pointing to a role
of the VEGF protein in these effects.
[0171] The effect of the VEGF receptor inhibitor on stimulation of
HUVEC proliferation by SB623 cell factors was also assessed. Assays
for expression of Ki67 were conducted as described in Example 3,
except that 50 nM SU5416 was added to cultures 30 minutes before
addition of conditioned medium, and cells were cultured for five
days, instead of seven, before assay. The results, shown in FIG. 11
and averaged from two donors, indicate that the enhancement of
HUVEC proliferation observed in the presence of conditioned medium
from SB623 cells was partially reversed by inhibition of
VEGFR2.
[0172] These results point to a role for VEGF, in addition to other
SB623 cell-derived factors, in the pro-survival and
pro-proliferative activity of MSC and SB623 cell conditioned
media.
Example 9
Effect of an Inhibitor of VEGF Signaling on Tube Formation by Human
Umbilical Vein Endothelial Cells (HUVECs)
[0173] HUVEC tube formation assays with conditioned medium from
MSCs and SB623 cells were conducted as described in Example 4, in
the presence and absence of the VEGF2 receptor inhibitor SU5416.
Cells cultured in the absence of conditioned medium were used as
negative controls; and cells cultured in the presence of VEGF (10
ng/ml were used as positive controls. The results, shown in FIG.
12, indicate that VEGF, MSC-conditioned medium and SB623
cell-conditioned medium all promote tube formation; while the
VEGFR2 inhibitor SU5416 reduces the stimulation of tube formation
by all of these agents.
[0174] Quantitation of tube formation was conducted, as described
in Example 4, for HUVECs exposed to SB623 cell conditioned medium
in the presence and absence of SU5416, at 16 and 40 hours after
plating. The results, shown in FIG. 13, indicate that, at both time
points, inhibition of VEGFR2 completely reversed the positive
effect of CM on tube formation.
Example 10
Effect of an Inhibitor of VEGF Signaling on Vessel Outgrowth and
Branching in an Aortic Ring Assay
[0175] Aortic ring angiogenesis assays were conducted as described
in Example 5 on one batch of SB623 cell-conditioned medium, in the
presence and absence of 50 nM SU5416. The inhibitor was added to
cultures 30 minutes before addition of CM and rings were assayed
after 10 days of culture. The results indicate that the vessel
outgrowth and branching resulting from culture of aortic rings in
SB623 cell-conditioned medium (FIG. 14, compare left and center
panels) was reduced in the presence of the VEGF receptor inhibitor
SU5416 (FIG. 14, compare center and right panels). These results
provide further evidence for the role of VEGF in the pro-angiogenic
activities of SB623 cell-conditioned medium.
[0176] The results obtained using a VEGF receptor inhibitor
(described above), while confirming the importance of VEGF to these
processes (particularly tube formation, vessel outgrowth and vessel
branching) do not rule out the participation of additional factors
(other than VEGF) in the pro-angiogenic activities of MSC- and
SB623 cell-conditioned media.
* * * * *