U.S. patent application number 12/130656 was filed with the patent office on 2008-12-04 for isolation and growth of stem cells from hemangiomas.
This patent application is currently assigned to Nevada Cancer Institute. Invention is credited to Louis M. Fink, Yupo Ma, Milton Waner, David C. Ward.
Application Number | 20080299077 12/130656 |
Document ID | / |
Family ID | 40088484 |
Filed Date | 2008-12-04 |
United States Patent
Application |
20080299077 |
Kind Code |
A1 |
Ma; Yupo ; et al. |
December 4, 2008 |
ISOLATION AND GROWTH OF STEM CELLS FROM HEMANGIOMAS
Abstract
The present invention describes stem cells and progenitor cells
derived from hemangiomas, including testing of angiogenic
inhibitors using these cells. The invention as described is useful
in providing a process to culture and propagate hemangioma stem
cells and generate xenograft models to develop treatments for
infantile hemangiomas and other types of vascular lesions.
Inventors: |
Ma; Yupo; (Las Vegas,
NV) ; Fink; Louis M.; (Las Vegas, NV) ; Ward;
David C.; (Las Vegas, NV) ; Waner; Milton;
(New York, NY) |
Correspondence
Address: |
DLA PIPER LLP (US)
4365 EXECUTIVE DRIVE, SUITE 1100
SAN DIEGO
CA
92121-2133
US
|
Assignee: |
Nevada Cancer Institute
Las Vegas
NV
|
Family ID: |
40088484 |
Appl. No.: |
12/130656 |
Filed: |
May 30, 2008 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11809871 |
Jun 1, 2007 |
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12130656 |
|
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61050131 |
May 2, 2008 |
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Current U.S.
Class: |
424/85.2 ;
435/29; 435/325; 435/372; 435/378; 435/6.16; 435/7.1; 800/9 |
Current CPC
Class: |
C12N 15/8509 20130101;
C12Q 2600/136 20130101; A01K 67/0275 20130101; C12Q 1/6886
20130101; G01N 33/5011 20130101; A01K 2227/105 20130101; G01N
33/5073 20130101; A01K 2267/0331 20130101; A01K 2217/05 20130101;
A61P 35/00 20180101; C12N 2830/008 20130101; A01K 2267/0381
20130101; C07K 14/4705 20130101; C12N 2800/30 20130101 |
Class at
Publication: |
424/85.2 ;
435/325; 435/6; 435/29; 800/9; 435/372; 435/378; 435/7.1 |
International
Class: |
C12N 5/08 20060101
C12N005/08; C12N 5/06 20060101 C12N005/06; C12Q 1/68 20060101
C12Q001/68; C12Q 1/02 20060101 C12Q001/02; G01N 33/53 20060101
G01N033/53; A01K 67/02 20060101 A01K067/02; A61K 38/20 20060101
A61K038/20 |
Claims
1. A composition comprising an enriched population of stem cells,
wherein the stem cells form tumor spheres in vitro.
2. The composition of claim 1, wherein the stem cells are derived
from a human, heterologous vascular tumor.
3. The composition of claim 2, wherein the vascular tumor is a
hemangioma.
4. The composition of claim 1, wherein the stem cells express CD133
or SALL4, or a combination thereof.
5. The composition of claim 4, wherein the stem cells express
SALL4.
6. The composition of claim 1, wherein the stem cells grow in vitro
in the absence of serum or serum replacement.
7. The composition of claim 1, wherein the stem cells are capable
of differentiating into endothelial-like tissue when contacted with
VEGF.
8. A method of screening agents that modulate cell proliferation
comprising: a) contacting a progenitor cell or stem cell from
hemangioma tissue with a test agent; b) comparing a characteristic
property associated with the cells in the presence of the test
agent with characteristic property of the cells in the absence of
the test agent; and c) determining the affect of the test agent on
the property, wherein a test agent modulates cell proliferation
when a change in a property associated with the cell is
affected.
9. The method of claim 8, wherein the property is selected from the
group consisting of growth characteristics; tumor sphere formation;
karyotype; immunohistochemical profile; gene expression profile;
self-renewal capacity; and/or differentiation capacity.
10. The method of claim 9, wherein the growth characteristics are
determined by contacting the progenitor or stem cell contained
within a xenograft transplanted in a non-human animal.
11. The method of claim 8, wherein the immunohistochemical profile
comprises a tissue specific marker selected from the group
consisting of GLUT-1, merosin, Fc-gamma-RII, Lewis Y antigens,
.beta.4-integrin, CD133, and SALL4, or a combination thereof.
12. The method of claim 8, wherein the determining step comprises
generating global maps for SALL4 binding to promoter regions in the
presence and absence of the test agent.
13. The method of claim 8, further comprising determining the
effect of the test agent in the presence and absence of an
angiogenesis inhibitor.
14. The method of claim 8, wherein the test agent is an
angiogenesis inhibitor.
15. The method of claim 13 or 14, wherein the angiogenesis
inhibitor is selected from the group consisting of VEGFR-1, NRP-1;
angiopoietin 2, TSP-1, TSP-2, angiostatin, endostatin, vasostatin,
calreticulin, platelet factor 4, IMP, CDAI, Meth-1, Meth-2,
INF-.alpha., INF-.beta., INF-.gamma., CXCL10, IL-4, IL-12, IL-18,
prothrombin (kringle domain-2) antithrombin III fragment,
prolactin, VEGI, SPARC, osteopontin, maspin, canstatin,
proliferin-related protein, restin, bevacizumab,
carboxyamidotriazol, TNP-470, CM101, suramin, thrombospondin,
angiostatic steroids/heparin, cartilage-derived angiogenesis
inhibitory factor, matrix metalloproteinase inhibitors,
2-methoxyestradiol, Tecogalan, .alpha..sub.v.beta..sub.3
inhibitors, and linomide.
16. A non-human animal containing a xenograft transplant comprising
an hemangioma derived progenitor cell, stem cell, or differentiated
cell derived from human infantile hemangioma tissue, wherein the
stem cell or progenitor cell self renews in culture, and forms a
tumor sphere from a single cell in suspension.
17. The non-human animal of claim 16, wherein the differentiated
cell is an endothelial-like cell.
18. The non-human animal of claim 16, wherein the non-human animal
is murine.
19. A tumor sphere derived from an immortalized hemangioma cell
comprising a progenitor cell or stem cell from human infantile
hemangioma tissue.
20. The tumor sphere of claim 19, wherein the tumor sphere
comprises cells which express CD133 or SALL4, or a combination
thereof.
21. The tumor sphere of claim 20, wherein the cells express
SALL4.
22. The tumor sphere of claim 20, wherein the tumor sphere grows in
vitro in the absence of serum or serum replacement.
23. The tumor sphere of claim 20, wherein cells isolated from the
tumor sphere are capable of forming endothelial-like cells when
contacted with VEGF.
24. An immortalized hemangioma cell line comprising a progenitor
cell or stem cell from human infantile hemangioma tissue, wherein
the stem cell or progenitor cell self renews in culture, and forms
a tumor sphere from a single cell in suspension in vitro.
25. The immortalized hemangioma cell line of claim 24, wherein the
cell line is isolated from a human, heterologous vascular
tumor.
26. The immortalized hemangioma cell line of claim 25, wherein the
tumor sphere comprises cells which express CD133 or SALL4, or a
combination thereof.
27. The immortalized hemangioma cell line of claim 26, wherein the
cells express SALL4.
28. The immortalized hemangioma cell line of claim 27, wherein the
cells grow in vitro in the absence of serum or serum
replacement.
29. The immortalized hemangioma cell line of claim 24, wherein the
stem cell is capable of differentiating into endothelial-like
tissue when contacted with VEGF.
30. A method of producing stem cells comprising: a) isolating cells
from hemangioma tissue; b) culturing the cells of step (a) in serum
free media until tumor spheres are formed; c) isolating cells from
the tumor spheres; and d) culturing the isolated cells of step (c)
in serum free media.
31. The method of claim 30, wherein the stem cell expresses
SALL4.
32. The method of claim 30, wherein the stem cells are capable of
differentiating into endothelial-like tissue when contacted with
VEGF.
33. A method of treating hemangiomas comprising administering an
agent screened in claim 8.
34. The method of claim 33, further comprising co-administration of
one or more of the angiogenesis inhibitors selected from the group
consisting of VEGFR-1, NRP-1; angiopoietin 2, TSP-1, TSP-2,
angiostatin, endostatin, vasostatin, calreticulin, platelet factor
4, IMP, CDAI, Meth-1, Meth-2, INF-.alpha., INF-.beta., INF-.gamma.,
CXCL10, IL-4, IL-12, IL-18, prothrombin (kringle domain-2)
antithrombin III fragment, prolactin, VEGI, SPARC, osteopontin,
maspin, canstatin, proliferin-related protein, restin, bevacizumab,
carboxyamidotriazol, TNP-470, CM101, suramin, thrombospondin,
angiostatic steroids/heparin, cartilage-derived angiogenesis
inhibitory factor, matrix metalloproteinase inhibitors,
2-methoxyestradiol, Tecogalan, .alpha..sub.v.beta..sub.3
inhibitors, and linomide.
35. The method of claim 33, wherein the hemangioma is infantile
hemangioma.
36. A method of screening combinations of agents that modulate cell
proliferation comprising: a) contacting hemangioma derived stem
cells with one or more first agents that induce differentiation of
the stem cells; b) allowing the stem cells to differentiate; c)
contacting the differentiated cells with a second agent; and d)
determining the affect of the second agent on the differentiated
cells, wherein combining the first and second agents modulates
proliferation of the differentiated cell.
37. The method of claim 36, wherein the hemangioma derived stem
cells express CD133 or SALL4, or a combination thereof
38. The method of claim 36, wherein the first agent is selected
from the group consisting of retinoic acid, BMP, VEGF, GATA
factors, EGF, FGF-2, PDGF, HGF, laminin, nicotinamide, GM-CSF,
IL-3, collagen IV, dexamethasone, TGF-.beta., fibronectin,
erythropoietin, thrombopoietin, G-CSF, TNF-.alpha., and
insulin.
39. The method of claim 38, wherein the first agent is VEGF.
40. The method of claim 36, wherein differentiation is determined
by detecting the presence or absence of one or more cell-type
differentiation markers.
41. The method of claim 36, wherein the second agent is selected
from the group consisting of small molecules, nucleic acids,
proteins, polypeptides, peptides, and peptidomimetics.
42. The method of claim 41, wherein the second agent modulates the
expression of SALL4.
43. The method of claim 42, wherein the second agent is selected
from the group consisting of 5' azacytidine, 5'
aza-2-deoxycytidine, 1-B-D-arabinofuranosyl-5-azacytosine,
dihydroxy-5-azacytidine, MG132, PSI, lactacystin, expoxomicin, and
bortezomib.
44. The method of claim 42, wherein the second agent is an RNAi
which decreases the expression of SALL4.
45. The method of claim 36, wherein the second agent is selected
from the group consisting of VEGFR-1, NRP-1; angiopoietin 2, TSP-1,
TSP-2, angiostatin, endostatin, vasostatin, calreticulin, platelet
factor 4, IMP, CDAI, Meth-1, Meth-2, INF-.alpha., INF-.beta.,
INF-.gamma., CXCL10, IL-4, IL-12, IL-18, prothrombin (kringle
domain-2) antithrombin III fragment, prolactin, VEGI, SPARC,
osteopontin, maspin, canstatin, proliferin-related protein, restin,
bevacizumab, carboxyamidotriazol, TNP-470, CM101, suramin,
thrombospondin, angiostatic steroids/heparin, cartilage-derived
angiogenesis inhibitory factor, matrix metalloproteinase
inhibitors, 2-methoxyestradiol, Tecogalan,
.alpha..sub.v.beta..sub.3 inhibitors, and linomide.
46. The method of claim 36, wherein combining the first and second
agents inhibits proliferation of the differentiated cell.
47. A method of treating hemangiomas comprising administering a
combination of agents screened in claim 36.
Description
CROSS REFERENCE TO RELATED APPLICATION
[0001] This application claims priority under 35 U.S.C. .sctn.120
as a continuation-in-part of U.S. application Ser. No. 11/809,871,
filed Jun. 1, 2007, and claims benefit of priority under 35 U.S.C.
.sctn.119(e) to U.S. Provisional Application No. 61/050,131, filed
May 2, 2008, the disclosures, each of which, are incorporated by
reference in their entireties.
FIELD OF THE INVENTION
[0002] The present invention relates generally to stem cells, and
more specifically to progenitor cells and stem cells isolated from
human infantile hemangiomas, methods for culturing such cells, and
methods of treating disorders associated with hemangiomas.
BACKGROUND INFORMATION
[0003] Infantile hemangiomas, the most common tumors of infancy,
are vascular tumors characterized by rapid proliferation of
endothelial cells during the first few months of postnatal life
followed by slow spontaneous involution. Most disappear
spontaneously (immature hemangiomas), while others persist and
create cosmetic problems. Complications may follow overtreatment,
posttraumatic ulceration, or localized tissue hypertrophy from a
persistent angioma of the CNS, the face, or an extremity.
[0004] While many hemangiomas eventually "involute," the result is
not always cosmetically acceptable. Early intervention has been
shown to reduce the need for corrective surgery after "involution"
has occurred, or to, at least, minimize extensive corrective
surgeries in the future. Psycho-social scarring which occurs when a
child has been forced to live with a facial deformity until
"involution" has been completed can be avoided by early, aggressive
intervention, according to presently known treatment options.
[0005] Conventional treatment options for hemangiomas range from
surgical excision (followed, in cases of facial or neck
hemangiomas, with cosmetic surgery) to systemic corticosteroid
treatments, laser, and use of alpha-interferon. Recently,
cryosurgery and sclerotherapy, have been proposed additions to the
available treatment regimens for hemangiomas.
[0006] Each conventional treatment option carries potential side
effects. Clearly, surgery always presents risks, whether for
infection, unexpected patient reaction to anesthesia, and/or
unexpected aesthetic results.
[0007] While systemic corticosteroid treatment is suspected of
certain side effects, regardless of age, steroid treatment carries
decided risks if carried on beyond a child's first birthday.
Furthermore, hemangiomas do not warrant nor benefit from steroids
beyond the first birthday, in part, because proliferation of
hemangiomas tends to end by that point anyway. In any event, if
steroids are lowered too quickly or given intermittently, "rebound
growth" is possible, if not likely. Some investigators have
reported other side effects from steroid treatments. In one
investigation, children (29 percent) became more irritable,
depressed and/or napped less during treatment, although this
resolved as treatment was tapered and discontinued. Other
short-term side effects included gastric irritation, oral or
perineal yeast infection, recurrent otitis media, hypertension, and
myopathy.
[0008] As stated above, another treatment is sclerotherapy (i.e.,
injection of a chemical irritant into a vein to "harden" it). The
disadvantages of sclerotheraphy include the pain of injection,
swelling, and psychological strain associated therewith, as well as
the danger of necrosis if the sclerosis technique is flawed.
[0009] While certain treatments for hemangiomas are considered
typically effective, the psychological effects of hemangiomas alone
warrant continued pursuit of more effective treatment regimens for
hemangiomas, whether for use alone, or in concert with existing
treatment options. Also, because most patients receiving treatments
are infants or small children, patient tolerance for the treatment
options becomes of more paramount importance.
SUMMARY OF THE INVENTION
[0010] The present invention is based on the seminal discovery that
a stem cell population can be obtained from a hemangioma. Such
cells are useful as a tool for developing treatments of infantile
hemangiomas and other types of vascular lesions as well as for
identifying agents that are anti-angiogenic.
[0011] In one embodiment, a stem cell or progenitor cell derived
from hemangioma tissue is disclosed, where the stem cell or
progenitor cell self renews in culture. The stem cell or progenitor
typically forms a tumor sphere from a single cell in
suspension.
[0012] In one aspect, the tumor sphere is enriched for cells which
express CD133 or SALL4, or a combination thereof. In another
aspect, the cells can be cultured in vitro in the absence of serum
or serum replacement.
[0013] In a related aspect, the stem cell is capable of
differentiating into endothelial-like tissue when the cell is
contacted with vascular endothelial cell growth factor (VEGF).
[0014] In another embodiment, a method of screening agents that
modulate cell proliferation is disclosed. In one aspect, the method
includes contacting a progenitor cell or stem cell from hemangioma
tissue with a test agent, comparing a characteristic property of
the cells in the presence of the agent with the characteristic
property in the absence of the test agent, and determining the
effect of the test agent on the property. For example, a test agent
modulates cell proliferation when a change in a property associated
with the stem cell is affected (e.g., tumor sphere formation). In
another aspect, a property includes, but is not limited to, growth
characteristics; karyotype; immunohistochemical profile; gene
expression profile; self-renewal capacity; and/or differentiation
capacity.
[0015] In another aspect, the growth characteristics of a
progenitor or stem cell are determined by contacting the progenitor
or stem cell contained within a xenograft transplanted in a
non-human animal. In a further aspect, the immunohistochemical
profile is defined by one or more tissue specific markers, and
includes, but is not limited to, GLUT-1, merosin, Fc-gamma-RII,
Lewis Y antigens, .beta.4-integrin, CD133, or SALL4, or a
combination thereof.
[0016] In another aspect, the step of determining an effect of a
test agent on a stem or progenitor cell includes generating global
maps for SALL4 promoter binding in the presence and absence of the
agent.
[0017] In one aspect, the method includes determining the effect of
the test agent on a stem cell or progenitor cell in the presence
and absence of an angiogenesis inhibitor, where the inhibitor
includes VEGFR-1, NRP-1, angiopoietin 2, TSP-1, TSP-2, angiostatin,
endostatin, vasostatin, calreticulin, platelet factor 4, IMP, CDAI,
Meth-1, Meth-2, INF-.alpha., INF-.beta., INF-.gamma., CXCL10, IL-4,
IL-12, IL-18, prothrombin (kringle domain-2) antithrombin III
fragment, prolactin, VEGI, SPARC, osteopontin, maspin, canstatin,
proliferin-related protein, restin, bevacizumab,
carboxyamidotriazol, TNP-470, CM101, suramin, thrombospondin,
angiostatic steroids/heparin, cartilage-derived angiogenesis
inhibitory factor, matrix metalloproteinase inhibitors,
2-methoxyestradiol, Tecogalan, .alpha..sub.v.beta..sub.3
inhibitors, or linomide.
[0018] In another embodiment, a non-human animal containing a
xenograft transplant is disclosed, where the non-human animal
contains an immortalized hemangioma cell-derived progenitor cell,
stem cell, or differentiated cell from human infantile hemangioma
tissue, and where the stem cell or progenitor cell self renews in
culture, and which cell forms a tumor sphere from a single cell in
suspension. In a related aspect, the differentiated cell is an
endothelial-like cell. In a further related aspect, the non-human
animal is murine, e.g., a mouse.
[0019] In one embodiment, a tumor sphere is disclosed which is
derived from an hemangioma cell and includes a progenitor cell or
stem cell from human infantile hemangioma tissue. In a related
aspect, the tumor sphere comprises cells which express CD133 or
SALL4, or a combination thereof.
[0020] In another embodiment, a method of producing stem cells from
hemangiomas is disclosed including isolating cells from hemangioma
tissue, culturing the isolated cells from the tissue in serum free
media until tumor spheres are formed, isolating cells from the
tumor spheres, and culturing the isolated cells from the tumor
spheres in serum free media. The cells are typically characterized
as a single cell type via expression of SALL4 or CD133, or a
combination thereof.
[0021] In one embodiment, a method is provided for treating a
hemangioma including administering an agent identified in the
methods of screening herein. In a related aspect, the method
includes co-administering one or more of the angiogenesis
inhibitors including VEGFR-1, NRP-1; angiopoietin 2, TSP-1, TSP-2,
angiostatin, endostatin, vasostatin, calreticulin, platelet factor
4, IMP, CDAI, Meth-1, Meth-2, INF-.alpha., INF-.beta., INF-.gamma.,
CXCL10, IL-4, IL-12, IL-18, prothrombin (kringle domain-2)
antithrombin III fragment, prolactin, VEGI, SPARC, osteopontin,
maspin, canstatin, proliferin-related protein, restin, bevacizumab,
carboxyamidotriazol, TNP-470, CM101 suramin, thrombospondin,
angiostatic steroids/heparin, cartilage-derived angiogenesis
inhibitory factor, matrix metalloproteinase inhibitors,
2-methoxyestradiol, Tecogalan, .alpha..sub.v.beta..sub.3
inhibitors, or linomide.
[0022] In another embodiment, a method of screening combinations of
agents that modulate cell proliferation is disclosed including
contacting hemangioma derived stem cells with one or more first
agents that induce differentiation of a subpopulation of cells;
allowing the subpopulation of cells to differentiate; contacting
the differentiated cells with a second agent; and determining the
affect of the second agent on the differentiated cells, where
combining the first and second agents modulates proliferation of
the differentiated cell.
BRIEF DESCRIPTION OF THE DRAWINGS
[0023] FIG. 1 is a photomicrograph showing the initial formation of
a tumor sphere derived from primary culture. The tumor sphere is
beginning to form with fibroblast cells derived from the tissue
sample surrounding the forming sphere.
[0024] FIG. 2 is a photomicrograph showing the intermediate
formation of tumor spheres in culture.
[0025] FIG. 3 is a photomicrograph of a tumor sphere derived from
human hemangioma tissue under high power magnification.
[0026] FIG. 4 is a photomicrograph showing tumor spheres
dissociated into single cells.
[0027] FIG. 5 is a photomicrograph showing the tumor sphere formed
from a single cell colony.
[0028] FIG. 6 is a photomicrograph showing endothelial-like cells
differentiated from a single cell.
[0029] FIG. 7 shows immunohistochemical staining of tumor sphere
tissues with CD34, a marker for differentiated endothelial cells
and SALL4, a pluripotency marker associated with ES cells and
cancer stem cells.
[0030] FIG. 8 shows immunofluorescent staining of CD133-positive
and CD133-low hemangioma tumor spheres following induced
differentiation. A and B are infantile hemangioma tumor spheres
showing high expression of the progenitor cells in various organ
systems. C and D are tumor spheres differentiating toward the
endothelial lineage following treatment with VEGF. E shows
fibroblast cells derived form the same primary culture as the
hemangioma tumor spheres do not express CD133. F is a negative
control antibody demonstrating the specificity of
immunostaining.
DETAILED DESCRIPTION OF THE INVENTION
[0031] Before the present composition, methods, and methodologies
are described, it is to be understood that this invention is not
limited to particular compositions, methods, and experimental
conditions described, as such compositions, methods, and conditions
may vary. It is also to be understood that the terminology used
herein is for purposes of describing particular embodiments only,
and is not intended to be limiting, since the scope of the present
invention will be limited only in the appended claims.
[0032] As used in this specification and the appended claims, the
singular forms "a", "an", and "the" include plural references
unless the context clearly dictates otherwise. Thus, for example,
references to "a cell" includes one or more cells, and/or
compositions of the type described herein which will become
apparent to those persons skilled in the art upon reading this
disclosure and so forth.
[0033] Unless defined otherwise, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art to which this invention belongs. Any
methods and materials similar or equivalent to those described
herein can be used in the practice or testing of the invention, as
it will be understood that modifications and variations are
encompassed within the spirit and scope of the instant
disclosure.
[0034] As used herein "angioma," including grammatical variations
thereof, means a localized vascular lesion of the skin and
subcutaneous tissues, rarely of the CNS, that result from
hyperplasia of the blood or lymph vessels. For example, angiomas
include nevus flammeus hemangioma, infantile hemangioma, cavernous
hemangioma, spider angioma, and lymphangiomas.
[0035] There are three major classifications of congenital
hemangiomas: nevus flammeus (portwine stain); infantile hemangioma
(capillary hemangioma or "strawberry mark"); and cavernous
hemangioma.
[0036] Nevus flammeus hemangioma usually appears as a flat, pink,
red, or purplish lesion present at birth. These lesions represent
dilation of blood vessels commonly present in region of the back or
nape of the neck. Nevus flammeus of the trigeminal area may be a
component of the Sturge-Weber syndrome (leptomeningeal angiomatosis
with intracranial calcification). A nevus flammeus usually will not
fade, though splotchy small red macular lesions in the area above
the nose and on the eyelids may disappear in a few months.
[0037] Cavernous hemangioma usually appears as a raised red or
purplish lesion composed of large vascular spaces. The blood
vessels and frequently the lymphatics are often immature, in which
case the lesion may contain numerous arteriovenous shunts (i.e., a
direct connection between an artery and vein) and vascular
malformations. Cavernous hemangiomas rarely involute spontaneously.
Partial involution may follow ulceration, trauma, or
hemorrhage.
[0038] Infantile hemangioma usually appears as a raised bright red
lesion consisting of proliferations of endothelial cells. It
develops shortly after birth and tends to enlarge slowly during the
first several months of life. About 75 to 95% usually involute
spontaneously within 5 to 7 years; regression however is usually
incomplete, and at times a brownish pigmentation and scarring or
wrinkling of the skin remains.
[0039] Infantile hemangiomas arise from the over proliferation of
endothelial cells and present themselves as vascular appearing
tumors. These tumors are generally self involuting over time, but
can present complications if excessively large. Further, these
benign tumors arise shortly after birth with a proliferative phase
lasting up to one year. Following the proliferative phase is a
several year period of tumor regression. Usually this results in
complete regression of the vasculature. In rare cases, these tumors
can last well into middle age of adults. Surgical procedures to
remove the vasculature are often quite complicated due to location
and shear size of the tumor. However, removal of these disfiguring
tumors is highly desirable for much of the population. The
molecular nature of these tumors is unknown, yet may provide an
opportunity to cure a currently incurable disease.
[0040] Routine histopathology varies according to the stage of the
hemangioma. In early proliferation, hemangiomas are characterized
by nonencapsulated masses and dense cords of mitotically active,
plump endothelial cells in close association with pericytes. Few,
small caliber lumina are present. Special stains reveal
well-developed basement membranes around primitive vessels. Mast
cells are present in varying numbers in all stages. As the
hemangioma proliferates, the vascular lumina enlarge. An increase
of apoptotic endothelial cells and a decrease in plump, mitotically
active endothelial cells herald the involution phase. As involution
progresses, the endothelial cells continue to mature and assume a
flatter appearance. The vascular lumina continue to enlarge until
few, mature ectatic vessels remain. Much of the proliferating
endothelial cell mass is replaced with fibro-fatty tissue. Varying
degrees of epidermal atrophy, scar tissue, and loss of elastic
tissue can be seen in late involuting lesions.
[0041] While not being bound by theory, hemangiomas may
fundamentally be a stem cell disease. In the classical model for
tumors, phenotypically distinct cell populations all have
equivalent self renewal and proliferative capacity, where tumor
cell heterogeneity results from environmental factors as well as
ongoing mutations within tumor cells that give rise to diverse
populations of cells having similar tumorigenic potential.
Alternatively, tumors may contain a limited subset of cells that
share properties in common with stem cells in that they possess
indefinite capacity for self renewal and are capable of
differentiation into other cell types.
[0042] The present invention is based on the alternative model,
where hemangiomas may be caused by either over-proliferation of a
stem cell or progenitor cell population responsible for giving rise
to the vasculature of hemangiomas, or, alternatively, a loss in
regulation of differentiation of these cell populations. Following
this model, the involution described following the first years of
life corresponds to the depletion of these stem cell or progenitor
cell populations. It is likely that this depletion is from the
aging of the stem cell/progenitor cells resulting in apoptosis of
these cells. Conversely, these cell populations may also undergo a
terminal cell division resulting in the loss of self-renewal
capabilities and leading to tumor regression.
[0043] Currently, there is no cell line or mouse model available to
study infantile hemangiomas. These deficiencies render further
understanding of the cellular biology and genetic events involved
in infantile hemangiomas nearly impossible. In addition, testing
the existing angiogenic inhibitors and screening new drugs for
treatment of infantile hemangiomas has historically been a
difficult task due to the lack of a cell line or mouse model. The
ability to identify cell populations within hemangiomas, based on
structural features, lineage markers, growth characteristics,
expression profiles, and the like as described herein, allows the
skill artisan to distinguish cell populations within such angiomas
that possess indefinite self renewal capacity (i.e., stem cells)
from cells which are depleted of this activity.
[0044] The present invention discloses the identification and
isolation of a population of cells that possess indefinite self
renewal capacity generated from tumor spheres derived from
hemangioma tissue: i.e., hemangioma derived stem cells. In one
embodiment, a composition comprising an enriched population of stem
cells is disclosed, where the stem cells form tumor spheres in
vitro or in vivo. As used herein "stem cell" means a relatively
undifferentiated cell that retains the ability to divide to provide
cells that can be specialized and take the place of cells that die
or are lost. Further, "tumor sphere" means a globular cell cluster
that may be formed in suspension that contains progenitor/stem
cells, which cells retain self renewal capacity. Moreover, such
tumor spheres include clonally derived non-adherent colonies of
cells derived from a single tumor stem cell.
[0045] The cells described herein may be evaluated for
tissue-specific markers such as GLUT-1, merosin, Fc-gamma-RII, and
Lewis Y antigens. These markers may aid in differentiating
infantile hemangiomas (positive staining for all) from other
vascular neoplasms or malformations, such as the congenital
hemangiomas (e.g., rapidly involuting congenital hemangioma,
noninvoluting congenital hemangioma), kaposiform
hemangioendothelioma, tufted angioma, or pyogenic granuloma, none
of which stains positively for these antigens. These markers are
coexpressed by infantile hemangiomas and placental microvessels.
The present invention demonstrates that hemangioma tissues comprise
a population of stem cells which express SALL4 and/or CD133.
Accordingly, stem cells of the present invention may be further
described by the determining the percentage of cells which express
SALL4 and/or CD133.
[0046] SALL4 (and its isoforms SALL4A, SALL4B, and SALL4C), is a
human homolog to Drosophila spalt and belongs to a group of
C.sub.2H.sub.2 zinc finger transcription factors characterized by
multiple finger domains distributed over the entire protein. CD133
is a cell surface protein expressed in hematopoietic stem cells,
neuronal stem cells, glial stem cells, and endothelial progenitor
cells. Expression of CD133 can serve as a marker for primitive
cells.
[0047] In one aspect, the enriched population of hemangioma derived
stem cells may be defined as comprising a population of SALL4
and/or CD133 positive cells that is greater than the concentration
of SALL4 and/or CD133 positive cells in a non-concentrated
hemangioma tissue preparation obtained from a subject with an
angioma. As used herein "derived," including grammatical variations
thereof, means to be obtained from a parent source. For example,
stem cells of the present invention may be obtained from hemangioma
tissue biopsied from a subject with an angioma. In one embodiment,
the hemangioma derived stem cells may be obtained by isolating
cells from primary hemangioma tissue or a primary hemangioma tumor
sphere, culturing the cells in serum free media until tumor spheres
are formed, isolating cells from the tumor spheres, and culturing
the isolated cells in serum free media. In a related aspect, the
hemangioma derived stem cells are immortal. As used herein
"immortal," including grammatical variations thereof, means to
change a cell population with a finite life span to one possessing
a life span that may be maintained indefinitely under specific
conditions.
[0048] In another aspect, the enriched preparations of the
invention may be described as a function of the percentage of cells
having self renewal capacity. As used herein "self renewal
capacity" or "self renewal" means the ability of a cell to possess
the property of cell division which results in one or both daughter
cells (progeny) that have essentially the same ability to replicate
and generate differentiated cell lineages as the progenitor cell.
As used herein "progenitor cell" means a parental cell that gives
rise to a distinct cell lineage by a series of cell divisions.
Further, "differentiation capacity," as used herein, means the
ability of cell to give rise to a distinct cell lineage.
[0049] The enriched preparation of the invention may further be
described as a function of the percentage of SALL4 and/or CD133
positive cells that are capable of giving rise to a tumor sphere as
compared to a non-enriched population. Further, the stem/progenitor
cells of the present invention may be characterized by, for
example, tumor cell formation, growth characteristics (e.g.,
population doubling capability, doubling time, passages to
senescence), karyotype analysis (e.g., normal karyotype; maternal
or neonatal lineage), flow cytometry (e.g., FACS analysis),
immunohistochemistry and/or immunocytochemistry (e.g., for
detection of epitopes), gene expression profiling (e.g., gene chip
arrays; polymerase chain reaction (for example, reverse
transcriptase PCR, real time PCR, and conventional PCR)), protein
arrays, protein secretion (e.g., by Enzyme Linked ImmunoSorbent
Assay (ELISA)), and/or other methods known in the art. Moreover,
the stem/progenitor cells may be analyzed by limiting dilution
assay, primary sphere formation assay, and/or cell proliferation
assay.
[0050] Another key property of stem cells of the present invention
is their ability to differentiate into mature cells. In one
embodiment, hemangioma tumor sphere cells are capable of
differentiating into mature cells, when such tumor cells are
cultured in appropriate growth factors. Accordingly, the enriched
population of the present invention may also be described as a
function of the percentage of SALL4 and/or CD133 positive cells
which differentiate into mature cells. For example, cells from
hemangioma tumor spheres grown in VEGF differentiate into the
endothelial-like tissue (see Example 1). As used herein
"endothelial-like" means a cell which possesses morphology and/or
express markers that are normally characteristic of endothelial
cells. For example, morphologically, endothelial cells are
flattened cells of mesoblastic origin. Further, endothelial cells
express markers which include, but are not limited to, CD31, von
Willebrand factor, vascular endothelial growth factor receptor 1,
zonula occludens 1, and occludin.
[0051] The culture medium to support the growth and proliferation
of the cells of the present invention may be a chemically defined
serum-free medium that is supplemented with a source of mitogens
and survival factors to allow the growth of stem cells in
culture.
[0052] Conditions for culturing should be close to physiological
conditions. The pH of the culture medium should be close to
physiological pH, preferably between pH 6-8, more preferably
between about pH 7 to 7.8, with pH 7.4 being most preferred.
Physiological temperatures range between about 30.degree. C. to
40.degree. C. Cells are preferably cultured at temperatures between
about 32.degree. C. to about 38.degree. C., and more preferably
between about 35.degree. C. to about 37.degree. C. Similarly, cells
may be cultured in levels of O.sub.2 that are comparatively reduced
relative to O.sub.2 concentrations in air, such that the O.sub.2
concentration is comparable to physiological levels (1-6%), rather
than 20% O.sub.2 in air.
[0053] For example, primary cells may be cultured in KNOCKOUT.TM.
Dulbecco's Modified Eagle's Medium (DMEM), 10% KNOCKOUT.TM. Serum,
1.times. non-essential amino acids (NEAA), and 20 .mu.g/ml
basic-fibroblast growth factor (bFGF). Following growth of a
primary culture for approximately 1 to 2 day, 2 to 3 days, 3 days
to 1 week, tumor spheres will spontaneously form from the
homogenous mixture of primary culture. Spheres may be collected
using collagenase digestion, washed, and plated at an appropriate
density (e.g., 200 cells per milliliter) with DMEM-F12 nutrient
mix, 20 .mu.g/ml bFGF and NEAA on low-attachment plates. The tumor
spheres form and propagate using collagenase digestion, which
digests the spheres to single cells. These single cells may then be
used to form additional tumor spheres in vitro or grafts in vivo.
The stem cells of the present invention and stem cell progeny may
also be cryopreserved until they are needed by any method known in
the art. The cells can be suspended in an isotonic solution,
preferably a cell culture medium, containing a particular
cryopreservant. Such cryopreservants include dimethyl sulfoxide
(DMSO), glycerol and the like. These cryopreservants are used at a
concentration of 5-15%, preferably 8-10%. Cells are frozen
gradually to a temperature of -10.degree. C. to -150.degree. C.,
preferably -20.degree. C. to -100.degree. C., and more preferably
-150.degree. C. Further, the stem cells of the present invention
may be used to produce immortalized cell lines.
[0054] In one embodiment, due to the vascular origin of the benign
tumor, testing angiogenic inhibitors in vitro and in vivo may be
used to improve various treatment modalities. Additionally,
screening methods for identifying new drugs that target key
mechanisms in hemangioma propagation or specific genes with vital
roles in infantile hemangioma proliferation are also disclosed.
[0055] In one embodiment, a method of screening test agents that
modulate cell proliferation is disclosed including contacting a
stem cell from hemangioma tissue with an test agent, comparing a
characteristic property associated with the cells in the presence
of the agent with the characteristic property in the absence of the
test agent, and determining the affect of the test agent on the
property, where a test agent modulates (increases or decreases)
cell proliferation when a change in a property associated with the
stem cell is affected. In one aspect, the property includes growth
characteristics; tumor sphere formation; karyotype;
immunohistochemical profile; gene expression profile; self-renewal
capacity; and differentiation capacity. In one aspect, one or more
agents may induce apoptosis of the stem cell.
[0056] In another aspect, such agents may be beneficial in other
vascular legions arising from endothelial cells or similar
stem/progenitor cells. Further, the methods of the present
invention provide an avenue to culture and propagate hemangioma
stem cells, and generate grafts in non-human animals.
[0057] In one aspect, tumor spheres of the present invention may be
used as a xenograft. As used herein the term xenograft refers to a
tissue or organ, for example a tumor or a nondispersed piece of a
tumor, from a donor of one species, placed into a recipient of
another species.
[0058] Xenografts allow for in vivo proliferation of the tumor
sphere derived cells. The in vivo proliferation of the cells of the
present invention may be accomplished by injection of the cells
into non-human animals, preferably mammals, including murine
species.
[0059] In one embodiment, a non-human animal containing a xenograft
transplant is disclosed, where the non-human animal contains a
differentiated cell from human infantile hemangioma tissue, and
where the stem cell or progenitor cell self renews in culture. In a
related aspect, the differentiated cell is an endothelial-like
cell. As xenografts may be rejected by an intact immune system of
the recipient, in some embodiments the recipient is chosen or
manipulated to have an incompetent immune system. In one
embodiment, the recipient is a SCID mouse, Beige/SCID mouse,
NOD/SCID mice. The mouse is injected with varying numbers of cells
and observed for tumor formation. The injection method may be any
method known in the art, following the enrichment of the injected
population of cells.
[0060] Thus, the present invention provides an approach for the
treatment of infantile hemangiomas through analysis of an in vitro
and/or in vivo system. These systems allow for the development of
novel treatments for infantile hemangiomas and other types of
vascular lesions.
[0061] In one embodiment, a method of treating hemangiomas is
disclosed, including administering an agent identified by the
screening method above. In a related aspect, the method may further
include co-administration of one or more angiogenesis inhibitors
such as VEGFR-1, NRP-1; angiopoietin 2, TSP-1, TSP-2, angiostatin,
endostatin, vasostatin, calreticulin, platelet factor 4, IMP, CDAI,
Meth-1, Meth-2, INF-.alpha., INF-.beta., INF-.gamma., CXCL10, IL-4,
IL-12, IL-18, prothrombin (kringle domain-2) antithrombin III
fragment, prolactin, VEGI, SPARC, osteopontin, maspin, canstatin,
proliferin-related protein, restin, bevacizumab,
carboxyamidotriazol, TNP-470, CM101, suramin, thrombospondin,
angiostatic steroids/heparin, cartilage-derived angiogenesis
inhibitory factor, matrix metalloproteinase inhibitors,
2-methoxyestradiol, Tecogalan, .alpha..sub.v.beta..sub.3
inhibitors, and linomide.
[0062] The observation that only a sub-population of hemangioma
cells are stem cells, which cells maintain the ability to give rise
to cells by self renewal, suggests that involution occurs when
these cells lose this capacity. By screening agents which modulate
the self renewal/differentiation capacity of the cells of the
present invention, identification of therapeutic modalities which
only affect this sub-population may be identified. For example,
SALL4 expression maintains self-renewal properties in embryonic
stems cells (ESC), including that when SALL4 expression is
inhibited, self-renewal capacity may be modulated in these cells.
In one embodiment, a method of screening agents is disclosed
including contacting the cells of the present invention with an
agent which reduces the expression levels of SALL4. Agents include,
but are not limited to, known inhibitors of SALL4 expression such
as methylation inhibitors, including 5' azacytidine, 5'
aza-2-deoxycytidine, 1-B-D-arabinofuranosyl-5-azacytosine, or
dihydroxy-5-azacytidine, and proteosome inhibitors including MG132,
PSI, lactacystin, expoxomicin, or bortezomib.
[0063] The present invention also discloses that VEGF-induced
differentiated cells derived from hemangioma tumor spheres are
CD133 negative (Example 1), suggesting that differentiation of the
hemangioma derived stem cells results in loss of self renewal
capacity. As such, hemangiomas may be treated by "differential
sensitization," where the subpopulation of CD133 positive cells in
the tumor sphere are induced to differentiate by contacting such
cells with a first agent, including but not limited to a growth
factor, and subsequently contacting the differentiated cells with a
second agent that modulates a property associated with the
differentiated cell. In one embodiment, agents for use in
differential sensitization may be screened by contacting a
hemangioma tumor sphere with one or more agents that induce
differentiation of CD133 positive cells, subsequently allowing the
CD133 positive cells to differentiate, contacting the cells with a
second agent, and determining the affect of the second agent on the
differentiated cell, where the combination of the first and second
agent modulates proliferation of the differentiated cell (e.g.,
induces apoptosis). In one aspect, differentiation may be
determined by detecting the presence or absence of one or more
cell-type differentiation markers. In another aspect, the first
agent includes, but is not limited to, retinoic acid, BMP, VEGF,
GATA factors, EGF, FGF-2, PDGF, HGF, laminin, nicotinamide, GM-CSF,
IL-3, collagen IV, dexamethasone, TGF-.beta., fibronectin,
erythropoietin, thrombopoietin, G-CSF, TNF-.alpha., and insulin. In
a related aspect, the second agent includes, but is not limited to,
small molecules, nucleic acids, proteins, polypeptides, peptides,
and peptidomimetics. In one aspect, the second agent is an
angiogenesis inhibitor, and includes, but is not limited to,
VEGFR-1, NRP-1; angiopoietin 2, TSP-1, TSP-2, angiostatin,
endostatin, vasostatin, calreticulin, platelet factor 4, IMP, CDAI,
Meth-1, Meth-2, INF-.alpha., INF-.beta., INF-.gamma., CXCL10, IL-4,
IL-12, IL-18, prothrombin (kringle domain-2) antithrombin III
fragment, prolactin, VEGI, SPARC, osteopontin, maspin, canstatin,
proliferin-related protein, restin, bevacizumab,
carboxyamidotriazol, TNP-470, CM101, suramin, thrombospondin,
angiostatic steroids/heparin, cartilage-derived angiogenesis
inhibitory factor, matrix metalloproteinase inhibitors,
2-methoxyestradiol, Tecogalan, .alpha..sub.v.beta..sub.3
inhibitors, and linomide.
[0064] Combination therapy according to the present invention may
be performed alone or in conjunction with another therapy and may
be provided at home, the doctor's office, a clinic, a hospital's
outpatient department, or a hospital. The duration of the
combination therapy depends on the type of vascular disorder being
treated, the age and condition of the patient, the stage and type
of the patient's disease, and how the patient responds to the
treatment.
[0065] The compositions comprising agents of the present invention
may be administered orally, topically, parenterally, by inhalation
or spray or rectally in dosage unit formulations containing
conventional non-toxic pharmaceutically acceptable carriers,
adjuvants and vehicles.
[0066] The term "parenteral" as used herein includes percutaneous,
subcutaneous, peritumoral, intravascular (e.g., intravenous),
intramuscular, or intrathecal injection or infusion techniques and
the like. In addition, there is provided a pharmaceutical
formulation comprising an agent of the present invention and a
pharmaceutically acceptable carrier. Such combinations may be
present in association with one or more non-toxic pharmaceutically
acceptable carriers and/or diluents and/or adjuvants, and if
desired other active ingredients. The pharmaceutical compositions
may be in a form suitable for oral use, for example, as tablets,
troches, lozenges, aqueous or oily suspensions, dispersible powders
or granules, emulsion, hard or soft capsules, or syrups or
elixirs.
[0067] Pharmaceutical compositions of the invention may also be in
the form of oil-in-water emulsions. The oily phase may be a
vegetable oil or a mineral oil or mixtures of these. Suitable
emulsifying agents may be naturally-occurring gums, for example gum
acacia or gum tragacanth, naturally-occurring phosphatides, for
example soy bean, lecithin, and esters or partial esters derived
from fatty acids and hexitol, anhydrides, for example sorbitan
monooleate, and condensation products of the said partial esters
with ethylene oxide, for example polyoxyethylene sorbitan
monooleate. The emulsions may also contain sweetening and flavoring
agents.
[0068] Syrups and elixirs may be formulated with sweetening agents,
for example glycerol, propylene glycol, sorbitol, glucose or
sucrose. Such formulations may also contain a demulcent, a
preservative and flavoring and coloring agents. The pharmaceutical
compositions may be in the form of a sterile injectable aqueous or
oleaginous suspension. This suspension may be formulated according
to the known art using those suitable dispersing or wetting agents
and suspending agents that have been mentioned above. The sterile
injectable preparation may also be a sterile injectable solution or
suspension in a non-toxic parentally acceptable diluent or solvent,
for example as a solution in 1,3-butanediol. Among the acceptable
vehicles and solvents that may be employed are water, Ringer's
solution and isotonic sodium chloride solution. In addition,
sterile, fixed oils are conventionally employed as a solvent or
suspending medium. For this purpose any bland fixed oil may be
employed including synthetic mono- or diglycerides. In addition,
fatty acids such as oleic acid find use in the preparation of
injectables.
[0069] The combinations containing agents of the present invention
may also be administered in the form of suppositories, e.g., for
rectal administration of the drug. These compositions can be
prepared by mixing the drug with a suitable non-irritating
excipient that is solid at ordinary temperatures but liquid at the
rectal temperature and will therefore melt in the rectum to release
the drug. Such materials include cocoa butter and polyethylene
glycols.
[0070] For disorders of external tissues, e.g., mouth and skin, the
formulations are preferably applied as a topical gel, spray,
ointment or cream, or as a suppository, containing the active
ingredients in a total amount of, for example, 0.075 to 30% w/w,
preferably 0.2 to 20% w/w and most preferably 0.4 to 15% w/w. When
formulated in an ointment, the active ingredients may be employed
with either paraffinic or a water-miscible ointment base.
[0071] Alternatively, the active ingredients may be formulated in a
cream with an oil-in-water cream base. If desired, the aqueous
phase of the cream base may include, for example at least 30% w/w
of a polyhydric alcohol such as propylene glycol, butane-1,3-diol,
mannitol, sorbitol, glycerol, polyethylene glycol and mixtures
thereof. The topical formulation may desirably include a compound
which enhances absorption or penetration of the active ingredient
through the skin or other affected areas. Examples of such dermal
penetration enhancers include dimethylsulfoxide and related
analogs. The compounds of this invention can also be administered
by a transdermal device. Preferably topical administration will be
accomplished using a patch either of the reservoir and porous
membrane type or of a solid matrix variety. In either case, the
active agent is delivered continuously from the reservoir or
microcapsules through a membrane into the active agent permeable
adhesive, which is in contact with the skin or mucosa of the
recipient. If the active agent is absorbed through the skin, a
controlled and predetermined flow of the active agent is
administered to the recipient. In the case of microcapsules, the
encapsulating agent may also function as the membrane. The
transdermal patch may include the compound in a suitable solvent
system with an adhesive system, such as an acrylic emulsion, and a
polyester patch. The oily phase of the emulsions of this invention
may be constituted from known ingredients in a known manner. While
the phase may comprise merely an emulsifier, it may comprise a
mixture of at least one emulsifier with a fat or an oil or with
both a fat and an oil. Emulsifiers and emulsion stabilizers
suitable for use in the formulation of the present invention
include Tween 60, Span 80, cetostearyl alcohol, myristyl alcohol,
glyceryl monostearate, and sodium lauryl sulfate, among others. The
choice of suitable oils or fats for the formulation is based on
achieving the desired cosmetic properties, since the solubility of
the active compound in most oils likely to be used in
pharmaceutical emulsion formulations is very low. Thus, the cream
may be a non-greasy, non-staining and washable product with
suitable consistency to avoid leakage from tubes or other
containers. Straight or branched chain, mono- or dibasic alkyl
esters such as di-isoadipate, isocetyl stearate, propylene glycol
diester of coconut fatty acids, isopropyl myristate, decyl oleate,
isopropyl palmitate, butyl stearate, 2-ethylhexyl palmitate or a
blend of branched chain esters may be used. These may be used alone
or in combination depending on the properties required.
Alternatively, high melting point lipids such as white soft
paraffin and/or liquid paraffin or other mineral oils can be
used.
[0072] It will be understood that the specific dose level for any
particular subject will depend upon a variety of factors including
the activity of the specific compound employed, the age, body
weight, general health, sex, diet, time of administration, route of
administration, and rate of excretion, drug combination and the
severity of the particular disease undergoing therapy.
[0073] In the undifferentiated state, the stem cells of the present
invention divide and are therefore excellent targets for genetic
modification. The term "genetic modification" as used herein refers
to the stable or transient alteration of the genotype of a
precursor cell by intentional introduction of exogenous DNA. DNA
may be synthetic, or naturally derived, and may contain genes,
portions of genes, or other useful DNA sequences. The term "genetic
modification" as used herein is not meant to include naturally
occurring alterations such as that which occurs through natural
viral activity, natural genetic recombination, or the like. General
methods for the genetic modification of eukaryotic cells are known
in the art. See, Sambrook et al., Molecular Cloning: A Laboratory
Manual (Cold Spring Harbor Press, Cold Spring Harbor, N.Y., 1989);
Current Protocols in Molecular Biology, Ausubel et al., eds.,
(Wiley Interscience, New York, 1993)).
[0074] Many methods for introducing vectors into cells or tissues
are available and equally suitable for use with stem cells of the
present invention in vivo, in vitro, and ex vivo. Vectors may be
introduced into stem cells taken from the patient and clonally
propagated.
[0075] "Transformation," or "genetically modified" as defined
herein, describes a process by which exogenous DNA enters and
changes a recipient cell. Transformation may occur under natural or
artificial conditions according to various methods well known in
the art, and may rely on any known method for the insertion of
foreign nucleic acid sequences into a prokaryotic or eukaryotic
host cell. The method for transformation is selected based on the
type of host cell being transformed and may include, but is not
limited to, viral infection, electroporation, heat shock,
lipofection, and particle bombardment, and the like. The term
"transformed" cells includes stably transformed cells in which the
inserted DNA is capable of replication either as an autonomously
replicating plasmid or as part of the host chromosome, as well as
transiently transformed cells which express the inserted DNA or RNA
for limited periods of time.
[0076] Genetic modification of a cell may be accomplished using one
or more techniques well known in the gene therapy field. Mulligan R
C, Human Gene Therapy 5: 543-563 (1993). Viral transduction methods
may comprise the use of a recombinant DNA or an RNA virus
comprising a nucleic acid sequence that drives or inhibits
expression of a protein to infect a target cell. A suitable DNA
virus for use in the present invention includes but is not limited
to an adenovirus (Ad), adeno-associated virus (AAV), herpes virus,
vaccinia virus or a polio virus. A suitable RNA virus for use in
the present invention includes, but is not limited to, a retrovirus
or Sindbis virus. Several such DNA and RNA viruses exist that may
be suitable for use in the present invention.
[0077] Adenoviral vectors have proven especially useful for gene
transfer into eukaryotic cells (Graham F L & Prevec L, In
Vaccines: New Approaches to Immunological Problems, Ellis R V ed.,
363-390 (Butterworth-Heinemann, Boston, 1992).
[0078] "Non-viral" delivery include DNA-ligand complexes,
adenovirus-ligand-DNA complexes, direct injection of DNA,
CaPO.sub.4 precipitation, gene gun techniques, electroporation, and
lipofection. Mulligan R C, Science 260: 926-932 (1993). Any of
these methods are widely available to one skilled in the art and
would be suitable for use in the present invention. Other suitable
methods are available to one skilled in the art, and it is to be
understood that the present invention may be accomplished using any
of the available methods of transfection. Lipofection may be
accomplished by encapsulating an isolated DNA molecule within a
liposomal particle and contacting the liposomal particle with the
cell membrane of the target cell. Liposomes are self-assembling,
colloidal particles in which a lipid bilayer, composed of
amphiphilic molecules such as phosphatidyl serine or phosphatidyl
choline, encapsulates a portion of the surrounding media such that
the lipid bilayer surrounds a hydrophilic interior. Unilammellar or
multilammellar liposomes can be constructed such that the interior
contains a desired chemical, drug, or, as in the instant invention,
an isolated DNA molecule. Delivery by transfection, by liposome
injections, or by polycationic amino polymers may be achieved using
methods which are well known in the art (see, e.g., Goldman, C. K.
et al. Nature Biotechnology 15:462-466 (1997)).
[0079] Genetically modified stem cells of the present invention may
be subjected to tissue culture protocols known in the art (see,
U.S. Pat. Nos. 5,750,376 and 5,851,832, Spector et al., Cells: A
Laboratory Manual (Cold Spring Harbor Press, Cold Spring Harbor,
N.Y., 1998)). The stem cells may be genetically modified in culture
to promote differentiation, cell death, or immunogenicity. For
example, stem cells can be modified to enhance expression of
products that direct an immune response against the patient's
hemangioma. Alternatively, the stem cells may be subjected to
various proliferation protocols in vitro prior to genetic
modification. The protocol used depends upon the type of
genetically modified stem cell desired.
[0080] DNA or RNA may be isolated from the stem cells of the
present invention according to any of a number of methods well
known to those of skill in the art. For example, methods of
purification of nucleic acids are described in Tijssen, Laboratory
Techniques in Biochemistry and Molecular Biology Hybridization With
Nucleic Acid Probes, Part I. Theory and Nucleic Acid Preparation
(Elsevier, New York N.Y., 1993). "Sample" is used in its broadest
sense. A sample containing polynucleotides or polypeptides can be a
bodily fluid; an extract from a cell, chromosome, organelle, or
membrane isolated from a cell; genomic DNA, RNA, or cDNA in
solution or bound to a substrate; a cell; a tissue; a tissue print;
and the like.
[0081] Total RNA may be isolated using the TRIZOL reagent (Life
Technologies, Gaithersburg Md., USA), and mRNA is isolated using
oligo d(T) column chromatography or glass beads. Alternatively,
when target polynucleotides are derived from an mRNA, the target
polynucleotides may be a cDNA reverse transcribed from an mRNA, an
RNA transcribed from that cDNA, a DNA amplified from that cDNA, an
RNA transcribed from the amplified DNA, and the like. When the
target polynucleotide is derived from DNA, the target
polynucleotide may be DNA amplified from DNA or RNA reverse
transcribed from DNA.
[0082] Several technologies produce pools of restriction fragments
of limited complexity for electrophoretic analysis, such as methods
combining double restriction enzyme digestion with phasing primers
(see, e.g., European patent application EP 0 534 858 A1), or
methods selecting restriction fragments with sites closest to a
defined mRNA end (see, e.g., Prashar et al., Proc. Natl. Acad. Sci.
USA 93: 659-663 (1996)). Other methods statistically sample cDNA
pools, such as by sequencing sufficient bases (in each of multiple
cDNAs to identify each cDNA, or by sequencing short tags which are
generated at known positions relative to a defined mRNA end (see,
e.g., Velculescu, Science 270: 484-487 (1995)).
[0083] Methods of modifying RNA abundances and activities currently
fall within three classes, ribozymes, antisense species (PCT patent
application WO 88/09810), and RNA aptamers (Good et al., Gene
Therapy 4: 45-54 (1997)). Ribozymes are RNAs which are capable of
catalyzing RNA cleavage reactions. (PCT patent application WO
90/11364). Ribozyme methods involve exposing a cell to, inducing
expression in a cell, etc. of such small RNA ribozyme molecules.
(Grassi & Marini, Annals of Medicine 28: 499-510 (1996);
Gibson, Cancer and Metastasis Reviews 15: 287-299 (1996)).
[0084] The term "antisense," as used herein, refers to any
composition containing a nucleic acid sequence which is
complementary to the "sense" strand of a specific nucleic acid
sequence. Antisense molecules may be produced by any method
including synthesis or transcription. Once introduced into a cell,
the complementary nucleotides combine with natural sequences
produced by the cell to form duplexes and to block either
transcription or translation. The designation "negative" can refer
to the antisense strand, and the designation "positive" can refer
to the sense strand. Such antisense molecules include interfering
RNA (i.e., RNAi).
[0085] The term "RNAi" refers generally to a process in which a
double-stranded RNA molecule changes the expression of a nucleic
acid sequence with which the double-stranded or short hairpin RNA
molecule shares substantial or total homology. While not being
bound by theory, the mechanism of action may include, but is not
limited to, direct or indirect down regulation of the expression of
the SALL4 gene, decrease in SALL4 mRNA, and/or a decrease in SALL4
activity. The term "RNAi," including "short inhibitory RNA
(siRNA)," refer to RNA sequences that elicit RNA interference, and
which is transcribed from a vector. The terms "short hairpin RNA"
or "shRNA" refer to an RNA structure having a duplex region and a
loop region. This term should also be understood to specifically
include RNA molecules with stem-loop or panhandle secondary
structures. In some embodiments of the present invention, RNAis are
expressed initially as shRNAs.
[0086] RNAi is generally optimised by identical sequences between
the target and the RNAi. The RNA interference phenomenon can be
observed with less than 100% homology, but the complementary
regions must be sufficiently homologous to each other to form the
specific double stranded regions. The precise structural rules to
achieve a double-stranded region effective to result in RNA
interference have not been fully identified, but approximately 70%
identity is generally sufficient. Accordingly, in some embodiments
of the invention, the homology between the RNAi and SALL4 is at
least 70% nucleotide sequence identity, and may be at least 75%
nucleotide sequence identity. Homology includes, but is not limited
to, at least 80% nucleotide sequence identity, and is at least 85%
or even 90% nucleotide sequence identity. In one embodiment,
sequence homology between the target sequence and the sense strand
of the RNAi is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,
98%, 99% or 100% nucleotide sequence identity.
[0087] Another consideration is that base-pairing in RNA is subtly
different from DNA in that G will pair with U, although not as
strongly as it does with C, in RNA duplexes. Moreover, for RNAi
efficacy, it is more important that the antisense strand be
homologous to the target sequence. In some circumstances, it is
known that 17 out of 21 nucleotides is sufficient to initiate RNAi,
but in other circumstances, identity of 19 or 20 nucleotides out of
21 is required. While not being bound by theory, at a general
level, greater homology is required in the central part of a double
stranded region than at its ends. Some predetermined degree of lack
of perfect homology may be designed into a particular construct so
as to reduce its RNAi activity which would result in a partial
silencing or repression of the target gene's product, in
circumstances in which only a degree of silencing was sought. In
such a case, only one or two bases of the antisense sequence may be
changed. On the other hand, the sense strand is more tolerant of
mutations. While not being bound by theory, this may be due to the
antisense strand being the one that is catalytically active. Thus,
less identity between the sense strand and the transcript of a
region of a target gene will not necessarily reduce RNAi activity,
particularly where the antisense strand perfectly hybridizes with
that transcript. Mutations in the sense strand (such that it is not
identical to the transcript of the region of the target gene) may
be useful to assist sequencing of hairpin constructs and
potentially for other purposes, such as modulating dicer processing
of a hairpin transcript or other aspects of the RNAi pathway.
[0088] The terms "hybridizing" and "annealing" including
grammatical equivalents thereof, are used interchangeably in this
specification with respect to nucleotide sequences and refer to
nucleotide sequences that are capable of forming Watson-Crick base
pairs due to their complementarity. The person skilled in the art
would understand that non-Watson-Crick base-pairing is also
possible, especially in the context of RNA sequences. For example a
so-called "wobble pair" can form between guanosine and uracil
residues in RNA.
[0089] The RNA expression products lead to the generation of a
double-stranded RNA (dsRNA) complex for inducing RNA interference
and thus down-regulating or decreasing expression of a mammalian
gene. "dsRNA" refers to a ribonucleic acid complex comprising two
Watson-Crick base-paired complementary RNA strands. The dsRNA
complex comprises a first nucleotide sequence that hybridizes under
stringent conditions, including a wash step of 0.2.times.SSC at
65.degree. C., to a nucleotide sequence of at least one mammalian
gene and a second nucleotide sequence which is complementary to the
first nucleotide sequence. The first nucleotide sequence might be
linked to the second nucleotide sequence by a third nucleotide
sequence (e.g., an RNA loop) so that the first nucleotide sequence
and the second nucleotide sequence are part of the same RNA
molecule; alternatively, the first nucleotide sequence might be
part of one RNA molecule and the second nucleotide sequence might
be part of another RNA molecule. Thus, a dsRNA complex may be
formed by intramolecular hybridization or annealing or the ds RNA
complex is formed by intermolecular hybridization or annealing.
[0090] Oligonucleotides may be synthesized by standard methods
known in the art, e.g. by use of an automated DNA synthesizer (such
as are commercially available from Biosearch, Applied Biosystems,
etc.).
[0091] "Amplification," as used herein, relates to the production
of additional copies of a nucleic acid sequence. Amplification is
generally carried out using polymerase chain reaction (PCR)
technologies well known in the art. See, e.g., Dieffenbach C W
& Dveksler G S, PCR Primer, a Laboratory Manual 1-5 (Cold
Spring Harbor Press, Plainview, N.Y., 1995). Amplification can be
polymerase chain reaction (PCR), ligase chain reaction (LCR),
nucleic acid sequence-based amplification (NASBA), or T7 based RNA
amplification.
[0092] "Polypeptide" refers to an amino acid, amino acid sequence,
oligopeptide, peptide, or protein or portions thereof whether
naturally occurring or synthetic.
[0093] Methods for direct measurement of protein activity are well
known to those of skill in the art. Such methods include, e.g.,
methods which depend on having an antibody ligand for the protein,
such as Western blotting, see, e.g., Burnette, A. Anal. Biochem.
112: 195-203 (1981). Such methods also include enzymatic activity
assays, which are available for most well-studied protein drug
targets. Detection of proteins can be accomplished by
antibodies.
[0094] The term "antigenic determinant," as used herein, refers to
that fragment of a molecule (i.e., an epitope) that makes contact
with a particular antibody. When a protein or a fragment of a
protein is used to immunize a host animal, numerous regions of the
protein may induce the production of antibodies which bind
specifically to antigenic determinants (given regions or
three-dimensional structures on the protein). An antigenic
determinant may compete with the intact antigen (i.e., the
immunogen used to elicit the immune response) for binding to an
antibody.
[0095] Proteins isolated from an enriched population of stem cells
can be separated by two-dimensional gel electrophoresis systems.
Two-dimensional gel electrophoresis is well-known in the art and
typically involves iso-electric focusing along a first dimension
followed by SDS-PAGE electrophoresis along a second dimension. See,
e.g., Hames et al, Gel Electrophoresis of Proteins: A Practical
Approach (IRL Press, New York 1990); Lander, Science 274:536-539
(1996). The resulting electrophoretograms can be analyzed by
numerous techniques, including mass spectrometric techniques,
western blotting and immunoblot analysis using polyclonal and
monoclonal antibodies, and internal and N-terminal
micro-sequencing. Using these techniques, it is possible to
identify a substantial fraction of all the proteins produced under
given physiological conditions, including in cells (e.g., in
hemangioma derived stem cells) exposed to a drug, or in cells
modified by, e.g., deletion or over-expression of a specific
gene.
[0096] The purified stem cells of the present invention may be used
to make arrays or cDNA libraries using methods known in the art
(see, Sambrook et al., Molecular Cloning: A Laboratory Manual (Cold
Spring Harbor Press, Cold Spring Harbor, N.Y., 1989); Current
Protocols in Molecular Biology, Ausubel et al., eds., (Wiley
Interscience, New York, 1993)) to identify potential novel drug
targets.
[0097] Molecular biology comprises a wide variety of techniques for
the analysis of nucleic acids and proteins, many of which form the
basis of clinical diagnostic assays. These techniques include
nucleic acid hybridization analysis, restriction enzyme analysis,
genetic sequence analysis, and separation and purification of
nucleic acids and proteins (Sambrook et al., Molecular Cloning: A
Laboratory Manual, Second Ed. (Cold Spring Harbor Laboratory Press,
Cold Spring Harbor, N.Y., 1989). Many molecular biology techniques
involve carrying out numerous operations on a large number of
samples. For guidance to genomics and other molecular biological
methods useful in the invention, see Birren et al., Genome
Analysis: A Laboratory Manual Series, Volume 1, Analyzing DNA (Cold
Spring Harbor Press, Cold Spring Harbor, N.Y., 1997); Birren et
al., Genome Analysis: A Laboratory Manual Series, Volume 2,
Detecting Genes (Cold Spring Harbor Press, Cold Spring Harbor,
N.Y., 1998); Birren et al., Genome Analysis: A Laboratory Manual
Series, Volume 4, Mapping Genomes (Cold Spring Harbor Press, Cold
Spring Harbor, N.Y., 1999).
[0098] Nucleic acid hybridization analysis generally involves the
detection of a very small numbers of specific target nucleic acids
(DNA or RNA) with probes among a large amount of non-target nucleic
acids. Multiple sample nucleic acid hybridization analysis has been
conducted on a variety of filter and solid support formats. The
"dot blot" hybridization, involves the non-covalent attachment of
target DNAs to a filter, which are subsequently hybridized with a
labeled probes. The "dot blot" hybridization has been further
developed for multiple analysis (European Patent application EP 0
228 075) and for the detection of overlapping clones and the
construction of genomic maps (U.S. Pat. No. 5,219,726). Another
format, the so-called "sandwich" hybridization, involves attaching
oligonucleotide probes covalently to a solid support and using them
to capture and detect multiple nucleic acid targets (U.S. Pat. No.
4,751,177; PCT International patent application WO 90/01564).
Multiplex versions of these formats are called "reverse dot
blots."
[0099] Methods are known in the art for amplifying signal using
sensitive reporter groups (enzyme, fluorophores, radioisotopes,
etc.) and associated detection systems (fluorometers, luminometers,
photon counters, scintillation counters, etc.). These methods can
be combined with amplification methods, such as the polymerase
chain reaction (PCR) for the amplification of target nucleic acid
sequences. See, Innis et al., PCR Protocols: A Guide to Methods and
Applications, (Academic Press, 1990).
[0100] Analysis of the nucleic acids of the hemangioma derived stem
cells of the present invention may be carried out using multiple
sample nucleic acid hybridization methods on micro-formatted
multiplex or matrix devices (e.g., DNA chips or microarrays) (see,
Barinaga, Science 253: 1489 (1991); Bains, Bio/Technology 10:
757-758 (1992)). Guidance for the use of microarrays is provided by
Wang, E et al, Nature Biotechnology 18; 457-459 (2000); Diehn M et
al., Nature Genetics 25: 58-62 (2000).
[0101] Microarrays are known in the art and consist of a surface to
which probes that correspond in sequence to gene products (e.g.,
cDNAs, oligonucleotides, mRNAs, cRNAs, polypeptides, and fragments
thereof), can be specifically hybridized or bound at a known
position. In one embodiment, the microarray is an array (i.e., a
matrix) in which each position represents a discrete binding site
for a product encoded by a gene (e.g., a protein or RNA), and in
which binding sites are present for products of most or almost all
of the genes in the organism's genome.
[0102] The polynucleotides, polypeptides, or analogues are attached
to a solid support or substrate, which may be made from glass,
plastic (e.g., polypropylene, nylon), polyacrylamide,
nitrocellulose, or other materials. "Substrate" refers to any
suitable rigid or semi-rigid support to which polynucleotides or
polypeptides are bound and includes membranes, filters, chips,
slides, wafers, fibers, magnetic or nonmagnetic beads, gels,
capillaries or other tubing, plates, polymers, and microparticles
with a variety of surface forms including wells, trenches, pins,
channels and pores. The polynucleotides can be immobilized on a
substrate by any method known in the art. Preferably, the
substrates are optically transparent.
[0103] A variety of methods are currently available for making
arrays of biological macromolecules, such as arrays of nucleic acid
molecules or proteins. One method for making ordered arrays of DNA
on a porous membrane is a "dot blot" or "slot-blot" method. A more
efficient technique employed for making ordered arrays of fragments
uses an array of pins dipped into the wells, e.g., the 96 wells of
a microtiter plate, for transferring an array of samples to a
substrate, such as a porous membrane. An alternate method of
creating ordered arrays of nucleic acid sequences is described by
U.S. Pat. No. 5,143,854 (to Pirrung), and also by Fodor et al.,
Science 251:767-773 (1991). The method involves synthesizing
different nucleic acid sequences at different discrete regions of a
support. Khrapko et al., DNA Sequence 1:375-388 (1991) describes a
method of making an oligonucleotide matrix by spotting DNA onto a
thin layer of polyacrylamide, manually with a micropipette. U.S.
Pat. No. 5,807,522 (to Brown et al.) describes methods for
fabricating microarrays of biological samples by dispensing a known
volume of a reagent at each selected array position, by tapping a
capillary dispenser on the support under conditions effective to
draw a defined volume of liquid onto the support.
[0104] Spotters can use pin, ink-jet, and other technologies to
deposit samples onto the support material. Several of the more
common methods utilize metal pins, which can be either solid or
split. When the pins are dipped into wells that contain the
compounds of interest, each picks up a small amount of the
material. The pin is then brought into contact with the solid
support and a nanoliter volume is dispensed at the desired
location. In split pins (otherwise known as quills) a slot cut into
the head of the pin functions as a reservoir for the compound being
spotted. Quill pins are most often used with glass slides, while
solid pins are typically used for spotting membranes. Amersham
Pharmacia Biotech, GeneMachines, and other companies offer spotting
robots.
[0105] Ink-jet technology provides another method of spotting
microarrays. Adapted from the printer industry and redesigned for
use in biotechnological applications, this uses piezoelectric
crystal oscillators and an electrode guidance system to deposit the
compound in a precise location on the slide or membrane. Companies
such as Cartesian Technologies and ProtoGene Laboratories use this
technology.
[0106] A method for attaching the nucleic acids to a surface is by
printing on glass plates, as is described generally by PCT
publication WO 95/35505; DeRisi et al., Nature Genetics 14:457-460
(1996); Shalon et al., Genome Res. 6:639-645 (1996); and Schena et
al., Proc. Natl. Acad. Sci. USA 93: 10614-10619 (1995). Another
method for making microarrays is by making high-density
oligonucleotide arrays. Techniques are known for producing arrays
containing thousands of oligonucleotides complementary to defined
sequences, at defined locations on a surface using
photolithographic techniques for synthesis in situ (see, Fodor et
al., Science 251:767-773 (1991); Pease et al., Proc. Natl. Acad.
Sci. USA 91:5022-5026 (1994); Lockhart et al., Nature Biotech
14:1675 (1996); U.S. Pat. Nos. 5,578,832, 5,556,752; and
5,510,270).
[0107] U.S. Pat. No. 6,110,426 (to Shalon et al.) a method and
apparatus for fabricating microarrays of biological samples for
large scale screening assays, such as arrays of DNA samples to be
used in DNA hybridization assays for genetic research and
diagnostic applications. U.S. Pat. No. 6,221,674 (to Sluka et al.)
discloses a process is described for applying spatially defined
reagent areas to a solid phase which is characterized in that a
liquid containing an adsorptive binding reagent is contacted with
spatially defined areas of a solid phase which comprises an
essentially continuous metal or metal oxide surface for an adequate
time period to enable the formation of adsorptive bonds between the
binding reagent and the solid phase. A process is described in PCT
application WO 92/10092 which can be used to generate a plurality
of different structures on a glass support by means of
photoreactive compounds and irradiation using masks. A process is
described in U.S. Pat. No. 4,877,745 in which differently
functionalized spots can be applied to plastic supports by means of
ink-jet.
[0108] Among the vendors of microarrays and microarray technology
useage are Affymetrix, Inc. (USA), NimbleGen Systems, Inc.
(Madison, Wis., USA), and Incyte Genomics (USA) (producing
microarrays for core facilities in large industrial and academic
departments); Agilent Technologies (USA) and Graffinity
Pharmaceutical Design, GmbH (Germany) (which provide specific
services such as printing and fingerprinting arrays designed and
used by individual researchers); and CLONTECH Laboratories (Becton
Dickinson Bioscience) and BioRobotics, Ltd. (Great Britain) (which
provide the basic tools necessary for individual researchers to
carry out the entire process of producing microarrays, including
printing). See, Gwynne P & Heebner G, "DNA Chips and
Microarrays" Science (2001).
[0109] In contrast to plastic surfaces, metal and metal oxide
surfaces have the advantage that they can be coated with an exactly
defined matrix layer by self-assembly techniques. A self-assembled
monolayer (SAM) is formed for example when organic alkylthiols are
adsorbed onto a gold surface, the spontaneous organisation of such
a densely packed monolayer being based on strong specific
interactions between the support material and the adsorbent. Nuzzo
et al., J. Am. Chem. Soc. 105: 4481 (1983). In this manner it is
possible to apply an exactly defined monolayer of a binding matrix
to the surface of metals such as, e.g., gold or silver. Furthermore
the specific binding capability of self-assembled solid phases can
be further optimized by dilution of the specific solid phase
reactants as described in EP-A-0 515 615.
[0110] The coating of metal surfaces with microstructures based on
self-assembled monolayers is also known and can be used to attach
components isolated from solid tumor stem cells. Whitesides et al.,
Langmuir 10 (1994) 1498-1511 describe a process in which reagents
are stamped onto a noble metal surface by means of a special
microstructured silicone stamp. This enables microstructured
monolayers to be generated with zones that are spatially separated
from one another. Microstructures of self-assembled monolayers on
noble metal surfaces can be formed by irradiation through masks of
substrates whose whole area is covered with thiols and subsequent
washing. Hemminger et al., Langmuir 10: 626-628 (1994). Spatially
separate zones are also formed in this process which are all
identically functionalized. A further possibility of producing
reagent spots is firstly to apply gold spots to a support that are
already spatially separated from one another which are then
subsequently coated with reagents.
[0111] The binding of analytes to a functionalized solid phase
matrix according to the invention can for example be detected by
confocal scanner fluorescence microscopy or by plasmon resonance
spectroscopy. Ruthenhausler B et al., Nature, 332: 615-617
(1988).
[0112] U.S. Pat. No. 6,228,659 describes an apparatus for producing
a plurality of arrays of reagent regions is disclosed. A dispensing
assembly in the apparatus has a plurality of heads which are spaced
for depositing reagents at selected positions in different array
areas in a substrate.
[0113] Transcript arrays may be employed for analyzing the
transcriptional state in a cell, and especially for measuring the
transcriptional states of cells exposed to graded levels of a
therapy of interest such as graded levels of a drug of interest or
to graded levels of a disease state of interest. In one embodiment,
transcript arrays are produced by hybridizing detectably labeled
polynucleotides representing the mRNA transcripts present in a cell
(e.g., fluorescently labeled cDNA synthesized from total cell mRNA)
to a microarray. In alternative embodiments, the cDNA or RNA probe
can be synthesized in the absence of detectable label and may be
labeled subsequently, e.g., by incorporating biotinylated dNTPs or
rNTP, or some similar means (e.g., photo-cross-linking a psoralen
derivative of biotin to RNAs), followed by addition of labeled
streptavidin (e.g., phycoerythrin-conjugated streptavidin) or the
equivalent. The label for the probe may be selected from the group
consisting of biotin, fluorescent, radioactive, and enzymatic
labels. When fluorescently-labeled probes are used, many suitable
fluorophores are known, including fluorescein, lissamine,
phycoerytluin, rhodamine (Perkin Elmer Cetus), Cy2, Cy3, Cy3.5,
Cy5, Cy5.5, Cy7, Fluor X (Amersham) and others (see, e.g. Kricka,
Nonisotopic DNA Probe Techniques (Academic Press San Diego, Calif.,
1992). It will be appreciated that pairs of fluorophores are chosen
that have distinct emission spectra so that they can be easily
distinguished. In another embodiment, a label other than a
fluorescent label is used. For example, a radioactive label, or a
pair of radioactive labels with distinct emission spectra, may be
used (see Zhao et al., Gene 156:207 (1995); Pietu et al., Genome
Res. 6:492 (1996); see also, EXAMPLE 21). For example, .sup.32P may
be used.
[0114] These methods of attaching transcripts usually attach
specific polynucleotide sequences to very small specific areas of a
solid support, such as micro-wells of a DNA chip. These
hybridization formats are micro-scale versions of the conventional
"reverse dot blot" and "sandwich" hybridization systems. The
micro-formatted hybridization can be used to carry out "sequencing
by hybridization" (see, Barinaga, Science 253: 1489 (1991); Bains,
Bio/Technology 10: 757-758 (1992). Sequencing by hybridization
makes use of all possible n-nucleotide oligomers (n-mers) to
identify n-mers in an unknown DNA sample, which are subsequently
aligned by algorithm analysis to produce the DNA sequence (see,
U.S. Pat. No. 5,202,231; see also, United Kingdom patent
application GB 8810400 (1988); Southern et al., Genomics 13: 1008
(1992); Fodor et al., Nature 364: 555-556 (1993); Fodor et al.,
Science 251: 767-773 (1991); U.S. Pat. No. 5,143,854.
[0115] Probes can be synthesized, in whole or in part, on the
surface of a substrate using a chemical coupling procedure and a
piezoelectric printing apparatus, such as that described in PCT
publication WO 95/251116 (Baldeschweiler et al.). Alternatively,
the probe can be synthesized on a substrate surface using a
self-addressable electronic device that controls when reagents are
added (U.S. Pat. No. 5,605,662).
[0116] Furthermore, the probes do not have to be directly bound to
the substrate, but rather can be bound to the substrate through a
linker group. The linker groups are typically about 6 to 50 atoms
long to provide exposure to the attached polynucleotide probe.
Preferred linker groups include ethylene glycol oligomers,
diamines, diacids and the like. Reactive groups on the substrate
surface react with one of the terminal portions of the linker to
bind the linker to the substrate. The other terminal portion of the
linker is then functionalized for binding the polynucleotide
probe.
[0117] Devices and computer systems for forming and using arrays of
materials on a chip or substrate are known. For example, PCT
International patent applications WO 92/10588 and WO 95/11995, both
incorporated herein by reference, describe techniques for
sequencing or sequence checking nucleic acids and other materials.
Arrays for performing these operations can be formed in arrays
according to the methods of, for example, the pioneering techniques
disclosed in U.S. Pat. Nos. 5,445,934, 5,384,261 and 5,571,639.
Improved methods of forming high-density arrays of peptides,
polynucleotides, and other polymer sequences in a short period of
time have been devised using combinatorial solid phase synthesis.
Very Large Scale Immobilized Polymer Synthesis (VLSIPS) technology
has greatly advanced combinatorial solid phase polymer synthesis
and paved the way to clinical application of deoxyribonucleic acid
(DNA) array chips such as those sold under the name GENECHIP.TM..
Kozal et al., Nature Medicine 2: 753-759 (1996). VLSIPS technology
is disclosed in U.S. Pat. No. 5,143,854, PCT International patent
applications WO 90/15070), WO 92/10092, and WO 95/11995; and Fodor
et al., Science 251: 767-777 (1991).
[0118] Nucleic acid hybridization and wash conditions are chosen so
that the probe "specifically binds" or "specifically hybridizes" to
a specific array site, i.e., the probe hybridizes, duplexes or
binds to a sequence array site with a complementary nucleic acid
sequence but does not hybridize to a site with a non-complementary
nucleic acid sequence. As used herein, one polynucleotide sequence
is considered complementary to another when, if the shorter of the
polynucleotides is less than or equal to 25 bases, there are no
mismatches using standard base-pairing rules or, if the shorter of
the polynucleotides is longer than 25 bases, there is no more than
a 5% mismatch. Preferably, the polynucleotides are perfectly
complementary (no mismatches). It can easily be demonstrated that
specific hybridization conditions result in specific hybridization
by carrying out a hybridization assay including negative controls.
Optimal hybridization conditions will depend on the length (e.g.,
oligomer versus polynucleotide greater than 200 bases) and type
(e.g., RNA, DNA, PNA) of labeled probe and immobilized
polynucleotide or oligonucleotide. General parameters for specific
(i.e., stringent) hybridization conditions for nucleic acids are
described in Ausubel et al., Current Protocols in Molecular Biology
(Greene Publishing and Wiley-Interscience, New York 1987). Useful
hybridization conditions are also provided in, e.g., Tijessen,
Hybridization With Nucleic Acid Probes, (Elsevier Science
Publishers B.V., 1993) and Kricka, Nonisotopic DNA Probe
Techniques, (Academic Press, San Diego, Calif., 1992).
[0119] When cDNA complementary to the RNA of a cell is made and
hybridized to a microarray under suitable hybridization conditions,
the level of hybridization to the site in the array corresponding
to any particular gene will reflect the prevalence in the cell of
RNA transcribed from that gene. For example, when detectably
labeled (e.g., with a fluorophore) cDNA complementary to the total
cellular mRNA is hybridized to a microarray, the site on the array
corresponding to a gene (i.e., capable of specifically binding the
product of the gene) that is not transcribed in the cell will have
little or no signal (e.g., fluorescent signal), and a gene for
which the encoded mRNA is prevalent will have a relatively strong
signal.
[0120] U.S. Pat. No. 6,183,968 (to Bandman et al.) discloses
polynucleotide probes that can be used as hybridizable array
elements in a microarray, each of the polynucleotide probes having
at least a portion of a gene which encodes a protein associated
with cell proliferation.
[0121] When fluorescently labeled probes are used, the fluorescence
emissions at each site of a transcript array can be, preferably,
detected by scanning confocal laser microscopy. In one embodiment,
a separate scan, using the appropriate excitation line, is carried
out for each of the two fluorophores used. Alternatively, a laser
can be used that allows simultaneous specimen illumination at
wavelengths specific to the two fluorophores and emissions from the
two fluorophores can be analyzed simultaneously (see Shalon et al.,
Genome Research 6:639-645 (1996)). Signals are recorded and,
preferably, analyzed by computer, using commercially available
methods. The abundance sort program of the invention described in
U.S. Pat. No. 5,840,484 can be used to tabulate and sort by
frequency the mRNA transcripts corresponding to each gene
identified. Since some of the polynucleotide sequences are
identified solely based on expression levels, it is not essential
to know a priori the function of a particular gene in hemangioma
derived stem cells.
[0122] Transcript image comparisons can be obtained by methods well
known to those skilled in the art. Transcript levels and images can
be obtained and compared, for example, by a differential gene
expression assay based on a quantitative hybridization of arrayed
DNA clones (Nguyen et al. Genomics 29:207-216 (1995), based on the
serial analysis of gene expression (SAGE) technology (Velculescu et
al. Science 270:484-487 (1995)), based on the polymerase chain
reaction (Peng et al. Science 257:967-971 (1992), based on a
differential amplification protocol (U.S. Pat. No. 5,545,522), or
based on electronic analysis, such as comparative gene transcript
analysis (U.S. Pat. No. 5,840,484) or the GEMTOOLS gene expression
analysis program (Incyte Pharmaceuticals, Palo Alto, Calif., USA).
Preferably, comparisons between two or more transcript profiles are
performed electronically.
[0123] Measurement of the translational state may be performed
according to several methods. For example, whole genome monitoring
of protein (i.e., the "proteome,") can be carried out by
constructing a microarray in which binding sites comprise
immobilized, preferably monoclonal, antibodies specific to a
plurality of protein species encoded by the cell genome.
[0124] The microarrays describe above can be employed in several
applications including hemangioma diagnostics, prognostics and
treatment regimens, drug discovery and development, toxicological
and tumorgenicity studies, forensics, pharmacogenomics and the
like.
[0125] In one embodiment, the microarray is used to monitor the
progression of disease. Physicians can assess and catalog the
differences in gene expression between healthy and hemangioma
tissues by analyzing changes in patterns of gene expression
compared with hemangioma derived stem cells from the patient. The
stem cells of the present invention may also be used to monitor the
efficacy of treatment. For some treatments with known side effects,
the microarray is employed to "fine tune" the treatment regimen. A
dosage is established that causes a change in genetic expression
patterns indicative of successful treatment. Expression patterns
associated with undesirable side effects are avoided. This approach
may be more sensitive and rapid than waiting for the patient to
show inadequate improvement, or to manifest side effects, before
altering the course of treatment.
[0126] Alternatively, animal models which mimic infantile
hemangioma, rather than patients, may be used to characterize
expression profiles associated with a particular disease or
condition. This gene expression data may be useful in diagnosing
and monitoring the course of disease in a patient, in determining
gene targets for intervention, and in testing novel treatment
regimens.
[0127] Also, researchers can use the microarray to rapidly screen
large numbers of candidate drug molecules, looking for ones that
produce an expression profile similar to those of known therapeutic
drugs, with the expectation that molecules with the same expression
profile will likely have similar therapeutic effects. Thus, the
invention provides the means to determine the molecular mode of
action of a drug.
[0128] In various embodiments, the effects on the cell can be
determined by measuring gene expression, protein abundances,
protein activities, or a combination of such measurements. In
various embodiments, modifications to a putative target in the cell
can be made by modifications to the genes encoding the target,
modification to abundances of RNAs encoding the target,
modifications to abundances of target proteins, or modifications to
activities of the target proteins. The present invention provides
an improvement to these methods of drug discovery by providing the
hemangioma derived stem cells, for a more precise drug discovery
program.
[0129] An "expression profile" comprises measurement of a plurality
of cellular constituents that indicate aspects of the biological
state of a cell. Such measurements may include, e.g., RNA or
protein abundances or activity levels. Aspects of the biological
state of a cell of a subject, for example, the transcriptional
state, the translational state, or the activity state, are
measured. The collection of these measurements, optionally
graphically represented, is called the "diagnostic profile."
Aspects of the biological state of a cell which are similar to
those measured in the diagnostic profile, e.g., the transcriptional
state, are measured in an analogous subject or subjects in response
to a known correlated disease state or, if therapeutic efficacy is
being monitored, in response to a known, correlated effect of a
therapy. The collection of these measurements, optionally
graphically represented, is called herein the response profile."
The response profiles are interpolated to predict response profiles
for all levels of protein activity within the range of protein
activity measured. In cases where therapeutic efficacy is to be
monitored, the response profile may be correlated to a beneficial
effect, an adverse effect, such as a toxic effect, or to both
beneficial and adverse effects.
[0130] As is commonly appreciated, protein activities result from
protein abundances; protein abundances result from translation of
mRNA (balanced against protein degradation); and mRNA abundances
result from transcription of DNA (balanced against mRNA
degradation). Therefore, genetic level modifications to a cellular
DNA constituent alters transcribed mRNA abundances, translated
protein abundances, and ultimately protein activities. RNA level
modifications similarly alter RNA abundance and protein abundances
and activities. Protein level modifications alter protein
abundances and activities. Finally, protein activity modifications
are the most targeted modification methods. As is commonly
appreciated, it is ultimately protein activities (and the
activities of catalytically active RNAs) that cause cellular
transformations and effects. Also, most drugs act by altering
protein activities.
[0131] In one embodiment, cDNAs from two different cells (one being
the hemangioma derived stem cells of the invention) are hybridized
to the binding sites of the microarray. In the case of therapeutic
efficacy (e.g., in response to drugs) one cell is exposed to a
therapy and another cell of the same type is not exposed to the
therapy. In the case of disease states one cell exhibits a
particular level of disease state and another cell of the same type
does not exhibit the disease state (or the level thereof). The cDNA
derived from each of the two cell types are differently labeled so
that they can be distinguished. In one embodiment, for example,
cDNA from a cell treated with a drug (or exposed to a pathway
perturbation) is synthesized using a fluorescein-labeled dNTP, and
cDNA from a second cell, not drug-exposed, is synthesized using a
rhodamine-labeled dNTP. When the two cDNAs are mixed and hybridized
to the microarray, the relative intensity of signal from each cDNA
set is determined for each site on the array, and any relative
difference in abundance of a particular mRNA detected. The use of a
two-color fluorescence labeling and detection scheme to define
alterations in gene expression has been described, e.g., in Shena
et al., Science 270:467-470 (1995). An advantage of using cDNA
labeled with two different fluorophores is that a direct and
internally controlled comparison of the mRNA levels corresponding
to each arrayed gene in two cell states can be made, and variations
due to minor differences in experimental conditions (e.g.,
hybridization conditions) will not affect subsequent analyses.
[0132] Gene expression profiles of purified stem cells could give
clues for the molecular mechanisms of stem cell behavior. Terskikh
A V et al., Proc Natl Acad Sci USA 98(14): 7934-7939 (2001)
analyzed hematopoietic stem cells (HSC)-enriched cells by
comparison with normal tissue and mouse neurospheres (a population
greatly enriched for neural progenitor cells) by comparison with
terminally differentiated neural cells, using cDNA microarray
techniques and in situ hybridization, thus identifying potential
regulatory gene candidates. The invention provides an improved
method of drug discovery over the methods of Terskikh, in that the
use of the hemangioma derived stem cells of the invention can
provide a distinct set of drug targets when compared with a
patient's normal tissue (such as from the area of the hemangioma)
or compared with the other populations of cells obtained from the
hemangioma.
[0133] Several other methods for utilizing DNA chips are known,
including the methods described in U.S. Pat. Nos. 5,744,305;
5,733,729; 5,710,000; 5,631,734; 5,599,695; 5,593,839; 5,578,832;
5,556,752; 5,770,722; 5,770,456; 5,753,788; 5,688,648; 5,753,439;
5,744,306. U.S. Pat. No. 5,807,522 (to Brown et al.) discloses a
method to monitor early changes in a cell that correlate with
levels of a disease state or therapy and which precede detectable
changes in actual protein function or activity.
[0134] Moreover, microarrays of genomic DNA from hemangioma derived
stem cells can be probed for single nucleotide polymorphisms (SNP),
to localize the sites of genetic mutations that cause cells to
become tumorigenic. Guidance for such methods are available from
commercial vendors and may be found in general genetic method
books, such as those disclosed herein.
[0135] In some embodiments of the present invention, cells are
analyzed by chromatin immunoprecipitation (ChIP). A complete
protocol is provided by NimbleGen Systems Inc (Madison, Wis.). In
brief, cells are grown, cross-linked with formaldehyde and sheared
by sonication. In one aspect, anti-SALL4 antibody and rabbit serum
are used for ChIP. ChIP-purified DNA is blunt-ended, ligated to
linkers and subjected to low-cycle PCR amplification. Promoter
tiling arrays may be obtained from NimbleGen. The promoter array
may be designed as a single array containing 2.7 kb of each
promoter region. The promoter region is covered by 50-75 mer probes
at roughly 100 bp spacing dependent on the sequence composition of
the region. The arrays are hybridized, and the data may be
extracted according to NimbleGen standard procedures. Confirmation
of the predicted binding sites is performed using Quantitative
real-time PCR analysis of the amplicons that are applied to the
arrays.
[0136] A custom microarray may be obtained commercially (e.g., from
NimbleGgen Madison, Wis.) using maskless array synthesis. Each gene
is compared with all others using the BLAST program to remove
redundancies. Ten probe pairs for each target are selected from the
3' 1 kb of each target. Probes are spaced evenly over the length of
the target region (.ltoreq.1 kb), so that the exact spacing
depended on the length of the target sequence. Each probe may be
about 10, about 20, or about 25 nucleotides in length. In one
aspect, the probes are 24 nucleotides in length. For each perfect
match probe there is also a mismatch probe, which differs by one or
more nucleotides.
[0137] Labeled cDNA is hybridized to the oligonucleotide probes on
the microarray. After washing, arrays are stained with an affinity
tag (e.g., streptavidin-Cy3 conjugate, Amersham Biosciences,
Piscataway, N.J.), followed by washing and a blow dry step. Slides
may be scanned using a GenePix 4000B microarray scanner (Axon
Instruments, Union City, Calif., USA), and the feature intensities
extracted from the TIF files are calculated by the scanner software
using a proprietary application developed at NimbleGen (Madison,
Wis., USA). This application calculates mean signal intensities for
the pixels that define each feature (3.times.3 grid of pixels). The
intensities for each gene are calculated by taking the mean of the
intensities for the perfect match probes specific to each target
minus the mean of the intensity of the mismatch probes. Probes that
differ from the mean for the set by more than 3 SD are removed from
the set and the mean recalculated. Average differences
(recalculated mean) are used for subsequent analysis.
[0138] The hemangioma derived stem cells of the invention can be
used to raise anti-stem cell antibodies. In one embodiment, the
method involves obtaining an enriched population of hemangioma
derived stem cells or isolated hemangioma derived stem cells;
treating the population to prevent cell replication (for example,
by irradiation); and administering the treated cell to a human or
animal subject in an amount effective for inducing an immune
response to the stem cells. For guidance as to an effective dose of
cells to be injected or orally administered; see, U.S. Pat. Nos.
6,218,166, 6,207,147, and 6,156,305. In another embodiment, the
method involves obtaining an enriched population of hemangioma
derived stem cells or isolated hemangioma derived stem cells;
mixing the stem cells in an in vitro culture with immune effector
cells (according to immunological methods known in the art) from a
human subject or host animal in which the antibody is to be raised;
removing the immune effector cells from the culture; and
transplanting the immune effector cells into a host animal in a
dose that is effective to stimulate an immune response in the
animal.
[0139] The stem cells of the present invention may also be used to
prepare monoclonal antibodies. Monoclonal antibodies to hemangioma
derived stem cells may be prepared using any technique which
provides for the production of antibody molecules by continuous
cell lines in culture. These include, but are not limited to, the
hybridoma technique, the human B-cell hybridoma technique, and the
EBV-hybridoma technique (see, e.g., Kozbor, D. et al., J. Immunol.
Methods 81:31-42 (1985); Cote R J et al. Proc. Natl. Acad. Sci.
80:2026-2030 (1983); and Cole S P et al. Mol. Cell. Biol.
62:109-120 (1984)).
[0140] In addition, techniques developed for the production of
"chimeric antibodies," such as the splicing of mouse antibody genes
to human antibody genes to obtain a molecule with appropriate
antigen specificity and biological activity, may be used (see,
e.g., Morrison S L et al. Proc. Natl. Acad. Sci. 81:6851-6855
(1984); Neuberger M S et al. Nature 312:604-608 (1984); and Takeda
S et al. Nature 314:452-454 (1985)).
[0141] Various immunoassays may be used for screening to identify
antibodies having the desired specificity. Numerous protocols for
competitive binding or immunoradiometric assays using either
polyclonal or monoclonal antibodies with established specificities
are well known in the art.
[0142] The antibody can also be a humanized antibody. The term
"humanized antibody," as used herein, refers to antibody molecules
in which the amino acid sequence in the non-antigen binding regions
has been altered so that the antibody more closely resembles a
human antibody, and still retains its original binding ability.
Antibodies are humanized so that they are less immunogenic and
therefore persist longer when administered therapeutically to a
patient.
[0143] Human antibodies can be generated using the XENOMOUSE.TM.
technology from Abgenix (Fremont, Calif., USA), which enables the
generation and selection of high affinity, fully human antibody
product candidates to essentially any disease target appropriate
for antibody therapy. See, U.S. Pat. Nos. 6,235,883, 6,207,418,
6,162,963, 6,150,584, 6,130,364, 6,114,598, 6,091,001, 6,075,181,
5,998,209, 5,985,615, 5,939,598, and 5,916,771; Yang X et al.,
Crit. Rev Oncol Hemato 38(1): 17-23 (2001); Chadd H E & Charnow
S M. Curr Opin Biotechnol 12(2):188-94 (2001); Green L L, Journal
of Immunological Methods 231 11-23 (1999); Yang X-D et al., Cancer
Research 59(6): 1236-1243 (1999); and Jakobovits A, Advanced Drug
Delivery Reviews 31: 33-42 (1998). Antibodies with fully human
protein sequences are generated using genetically engineered
strains of mice in which mouse antibody gene expression is
suppressed and functionally replaced with human antibody gene
expression, while leaving intact the rest of the mouse immune
system.
[0144] Moreover, the generation of antibodies directed against
markers present in or on hemangioma derived stem cells of the
present invention may be used as a method of identifying targets
for drug development. The antibodies that are raised in an immune
response to the stem cells of the present invention may be used to
identify antigenic proteins on the hemangioma derived stem cells
using methods known in the art (Harlow, Using Antibodies: A
Laboratory Manual (Cold Spring Harbor Press, Cold Spring Harbor,
N.Y., 1999)) and can further be used to identify polynucleotides
coding for such proteins (Sambrook et al., Molecular Cloning: A
Laboratory Manual (Cold Spring Harbor Press, Cold Spring Harbor,
N.Y., 1989); Current Protocols in Molecular Biology, Ausubel et
al., eds., (Wiley Interscience, New York, 1993)). Once identified,
the proteins and polynucleotides can be compared with other
proteins and polynucleotides previously identified to be involved
in infantile hemangioma. In one embodiment, the XENOMOUSE.TM.
technology to produce fully human antibodies can be used to
generate antibodies directed against drug development targets (see,
Jeffrey Krasner, Boston Globe (Jul. 25, 2001) at F4). The present
invention provides an improvement to these antibody-based methods
of drug discovery by providing the hemangioma derived stem cells,
to which the immune response is raised, for a more precise drug
discovery program.
[0145] Alternatively, methods for raising an immune response can
take advantage of the "stem cell" qualities of the hemangioma
derived stem cell of the invention. Hemangioma derived stem cells,
hemangioma derived stem cell protein extracts, purified proteins
from hemangioma derived stem cells, or proteins derived from the
expression of cDNAs from hemangioma derived stem cells (see, above
for genetic modification of hemangioma derived stem cells) to
induce an immune response in an animal. The immune response can be
directed against hemangioma cells, as shown by standard
immunological methods. For example, the hemangioma derived stem
cells (enriched populations of isolated cells) or proteins can be
contacted with dendritic cells in culture, antigen presenting cells
in culture, or antigen presenting cells and T cells in culture.
Then antigen-stimulated cells are infused back into the
subject.
[0146] Alternatively, the hemangioma derived stem cells of the
invention can be genetically engineered to promote an immune
response against the stem cells. For example, hematopoietic stem
cells can be engineered to contain a T-cell receptor targeting a
tumor stem cell antigen. See, U.S. Pat. Nos. 5,914,108 and
5,928,638. Thus, T cell receptors that recognize antigens expressed
by tumor stem cells can be identified, then cloned into
hematopoietic stem cells. The engineered hematopoietic stem cells
may then be transplanted into a subject and allowed to engraft,
giving rise to large numbers of T cells that express receptors
recognizing the tumor stem cells. In a similar fashion, by
increasing the numbers of hemangioma derived stem cell-specific T
cells, an anti-hemangioma immune response may be potentiated.
[0147] Other means are also available for increasing the
anti-hemangioma immune response, including using the hemangioma
derived stem cells as the basis of a vaccine, using the hemangioma
derived stem cells to stimulate antigen presenting dendritic cells,
and using the hemangioma derived stem cells as an innoculum to
generate anti-hemangioma derived stem cell antibodies. Hemangioma
derived stem cells may be used as a vaccine by killing a subject's
hemangioma derived stem cells, such as by irradiation, and
re-administering the killed stem cells back into the subject in a
physiological/pharmaceutically and immunologically acceptable
carrier, for the purpose of generating an immune response against
the hemangioma derived stem cells. See, U.S. Pat. No. 4,960,716, in
which antibodies were raised to membrane vesicle preparations of
breast carcinoma cell cells; U.S. Pat. No. 4,584,268, in which
anti-human mammary epithelial antibody was produced from a membrane
fraction of delipidated human milk fat globules.
[0148] Dendritic cells from a subject may be cultured in vitro and
killed hemangioma derived stem cells from the same subject may be
added to the cultures to stimulate the dendritic cells. The
activated dendritic cells, presenting hemangioma derived stem cell
antigens, may then be re-administered to the subject to stimulate
the subject's anti-hemangioma response. Finally, hemangioma derived
stem cells can be administered to an animal such as a mouse, rat,
hamster, goat, sheep, rabbit, or donkey to generate antibodies
against the stem cells. Preferably, monoclonal anti-hemangioma
derived stem cell antibodies are made in mouse, rat, or hamster.
Monoclonal antibodies that are made in this way can then be
administered to subjects, or first humanized (as described above)
and then administered to subjects, to promote an immune response
against the hemangioma derived stem cells in the subject.
[0149] Furthermore, adenoviral vectors have proven especially
useful for gene transfer into eukaryotic cells for vaccine
development. Graham F L & Prevec L, In Vaccines: New Approaches
to Immunological Problems, Ellis R V ed., 363-390
(Butterworth-Heinemann, Boston, 1992).
[0150] The identification of biological pathways is an important
part of modem drug discovery process. Biological pathways in
hemangioma derived stem cells and other cell populations obtained
from hemangiomas, particularly pathways involved in drug actions,
i.e., pathways that originate at a drug target (e.g., proteins),
can be identified for use as shown by U.S. Pat. No. 5,965,352.
[0151] In one set of methods, drugs are screened to determine the
binding of test compounds to receptors, in which the binding
activates a cell's biological pathway to cause expression of
reporter polypeptides. Frequently the reporter polypeptides are
coded for on recombinant polypeptides, in which the coding
polynucleotide is in operable linkage with a promoter.
[0152] The terms "operably associated" or "operably linked," as
used herein, refer to functionally related polynucleotides. A
promoter is operably associated or operably linked with a coding
sequence if the promoter controls the translation of the encoded
polypeptide. While operably associated or operably linked nucleic
acid sequences can be contiguous and in the same reading frame,
certain genetic elements, e.g., repressor genes, are not
contiguously linked to the sequence encoding the polypeptide but
still bind to operator sequences that control expression of the
polypeptide.
[0153] The detectable signal can be fluorescence, absorbence, or
luminescence, depending on the reporter polypeptide, which can be,
for example, luciferase (firefly luciferase, Vibrio fisceri
luciferase, or Xenorhabdus luminescens luciferase), green
fluorescent protein, green fluorescent protein variant,
chloramphenicol acetyltransferase, .beta.-glucuronidase,
.beta.-galactosidase, neomycin phosphotransferase, guanine xanthine
phosphoribosyltransferase, thymidine kinase, .beta.-lactamase,
alkaline phosphatase, invertase, amylase (for yeast based assays)
human growth hormone (for activity based assays). The fluorescent
detectable signal can be fluorescence resonance energy transfer
(FRET), bioluminescence resonance energy transfer (BRET),
time-resolved fluorescence (TRF) or fluorescence polarization (FP).
Where appropriate, the detectable signal is detected by a machine
such as a fluorometer, luminometer, fluorescence microplate reader,
dual-monochromator microplate spectrofluorometer,
spectrophotometer, confocal microscope (laser scanner), or a
charge-coupled device (CCD). The detectable signal is determined by
comparing the amount of signal produced when the reporter
polypeptide is expressed in the hemangioma derived stem cell with
the signal produced when the reporter polypeptide is not expressed
in the hemangioma derived stem cell.
[0154] Another technique for drug screening provides for high
throughput screening of compounds (see, e.g., PCT application WO
84/03564.) In this method, large numbers of different small test
compounds are synthesized on a solid substrate, such as plastic
pins or some other surface. The test compounds are reacted with
hemangioma derived stem cells, or portions thereof, and washed.
Bound hemangioma stem cells are then detected by methods well known
in the art, using commercially available machinery and methods, for
example, the Automated Assay Optimization (AAO) software platforms
(Beckman, USA) that interface with liquid handlers to enable direct
statistical analysis that optimizes the assays; modular systems
from CRS Robotics Corp. (Burlington, Ontario), liquid handling
systems, readers, and incubators, from various companies using
POLARA.TM. (CRS), an open architecture laboratory automation
software for a Ultra High Throughput Screening System; 3P
(Plug&Play Peripherals) technology, which is designed to allow
the user to reconfigure the automation platform by plugging in new
instruments (ROBOCON, Vienna, Austria); the ALLEGRO.TM. system or
STACCATO.TM. workstation (Zymark), which enables a wide range of
discovery applications, including HTS, ultra HTS, and high-speed
plate preparation; MICROLAB Vector software (Hamilton Co., Reno,
Nev., USA) for laboratory automation programming and integration;
and others.
[0155] For any of these machines and methods, the assays measure a
response the target cells (hemangioma derived stem cells or
genetically modified hemangioma derived stem cells) that provides
detectable evidence that the test compound may be efficacious. The
detectable signal is compared to control cells and the detectable
signal identified by subtraction analysis. The relative abundance
of the differences between the "targeted" and "untargeted" aliquots
are simultaneously compared using a "subtraction" analysis
(differential analysis) technique such as differential display,
representational difference analysis (RDA), GEM-Gene Expression
Microarrays (U.S. Pat. No. 5,545,531), suppressive subtraction
hybridization (SSH) and direct sequencing (PCT patent application
WO 96/17957). The subtraction analysis can include the methods of
differential display, representational differential analysis (RDA),
suppressive subtraction hybridization (SSH), serial analysis of
gene expression (SAGE), gene expression microarray (GEM), nucleic
acid chip technology, or direct sequencing.
[0156] Hemangioma derived stem cells of the present invention are
particularly useful in the drug development process because
hemangioma stem cells provide a limited and enriched set of targets
for drug development. One of the most important steps in rational
drug design is the identification of a target, the molecule with
which the drug itself interacts.
[0157] Likewise, the genetically modified hemangioma derived stem
cell of the present invention is particularly useful in the drug
development. For example, the genetically modified stem cell can
contain polynucleotide with a promoter operably linked to the
polynucleotide encoding a reporter polypeptide. The reporter
polypeptide is expressed in the stem cell after a receptor of the
stem cell is activated by binding to a test compound or inactivated
by binding to a test compound. Such a detectable signal makes the
genetically modified hemangioma derived stem cell appropriate for
use in high throughput screening (HTS).
[0158] The detectable signal can be a result of a positive
selection or a negative selection. The positive selection includes
manipulations that test the ability of cells to survive under
specific culture conditions, ability to express a specific factor,
changes in cell structure, or differential gene expression.
[0159] The invention also provides a kit comprising packaging
material and a primary reagent contained within said packaging
material. The primary reagent is a hemangioma stem cell preparation
as described above. The packaging material includes a label that
indicates that the primary reagent can be used for identifying an
agent for reducing hemangiomas. Also, the invention provides a kit
for determining the activity level of a particular polynucleotide
or protein in a cell. Such kits contain arrays or microarrays
containing a solid phase, e.g., a surface, to which are bound,
either directly or indirectly, hemangioma derived stem cells
(enriched populations of or isolated), polynucleotides extracted
from such hemangioma derived stem cells, or proteins extracted from
such hemangioma derived stem cells. The kit may also contain probes
that are hybridized or bound to the hemangioma derived stem cell
components at a known location of the solid phase. These probes
consist of nucleic acids of known, different sequence, with each
nucleic acid being capable of hybridizing to an RNA species or to a
cDNA species derived therefrom. In particular, the probes contained
in the kits of this invention are nucleic acids capable of
hybridizing specifically to nucleic acid sequences derived from RNA
species which are known to increase or decrease in response to
perturbations correlated to the particular diseases or therapies to
be monitored by the kit. The probes contained in the kits of this
invention preferably substantially exclude nucleic acids which
hybridize to RNA species that are not increased or decreased in
response to perturbations correlated to the particular levels of
disease states or therapeutic effects to be determined by the
kit.
[0160] The following examples are intended to illustrate but not
limit the invention.
EXAMPLES
Example 1
Isolation of Tumor Spheres
[0161] Tissue samples were isolated from superficial raised patches
on the skin of male and female infants (less than 1 year), prior to
involution. Upon receipt, the tissue was minced and digested with
collagenase for 2 hours at 37.degree. C. Following digestion and
several wash steps, the tissue mixture was then filtered through a
100 .mu.m cell strainer and grown in culture using KNOCKOUT.TM.
Dulbecco's Modified Eagle's Medium (DMEM), 10% KNOCKOUT.TM. Serum,
1.times. non-essential amino acids (NEAA), and 20 .mu.g/ml
basic-fibroblast growth factor (bFGF). Following growth of this
primary culture for approximately three days, tumor spheres will
spontaneously form from the homogenous mixture of primary culture.
These spheres are collected using collagenase digestion, washed,
and plated at a density of 200 cells per milliliter with DMEM-F12
nutrient mix, 20 .mu.g/ml bFGF and NEAA on low-attachment plates.
The tumor spheres form and propagate using collagenase digestion,
which digest the spheres to single cells. These single cells then
form additional tumor spheres in vitro.
[0162] The tumor spheres derived from fresh human hemangioma
tissues grow for an indefinite period of time under serum-free
conditions (FIGS. 1-3). These tumor spheres bear similar key
properties of all normal stem cells and tumor stem cells, that is,
their unique ability to self-renew. One method to determine whether
tumor sphere cells have self-renewal capacity is to test their
capability for serial passages. The tumor spheres dissociated into
single cell suspensions continued to grow single cells in culture
for a week (FIG. 4). The single cell suspension was able to expand
and form new tumor spheres within one to two weeks (FIG. 5).
[0163] Another key property of stem cells is their ability to
differentiate into mature cells. To assess whether hemangioma tumor
sphere cells are able to differentiate into mature cells, tumor
cells were cultured in appropriate growth factors. Hemangioma tumor
spheres grown in DMEM/F12+50 .mu.g/ml VEGF+NEAA for 72 hours
differentiated into the endothelial-like tissue. Phase-contrast
microscopy analysis revealed changes in the cell morphology within
24 hours and endothelial-like tissues were formed (FIG. 6).
[0164] Although tumor sphere capacity for self renewal and
differentiation were demonstrated, demonstration of lineage
commitment to endothelial lineage by the tumor spheres was sought.
Immunohistochemical staining of the hemangioma tumor spheres
revealed that the cells were negative for CD34, and endothelial
marker. In contrast, these cells highly expressed the SALL4
protein, a marker for pluripotency expressed in embryonic stem
cells and cancer cells (FIG. 8).
[0165] To further demonstrate that the infantile hemangioma tumor
spheres are pluripotent, immunofluorescent staining was used to
demonstrate the expression of glycoprotein CD133. CD133 is a cell
surface protein expressed in hematopoietic stem cells, neuronal
stem cells, glial stem cells, and importantly, endothelial
progenitor cells. Expression of this protein serves as a marker for
primitive cells. As shown in FIG. 7, tumor spheres were
immuno-positive for the stem cell marker CD133 (FIGS. 8A and 8B),
while fibroblasts generated from the same primary tissues sample
were CD133 negative (FIG. 8E). After adding VEGF to the culture
medium, partially differentiated tumor spheres were only GFP
positive in undifferentiated cells with GFP-negative fibroblasts
surrounding the spheres. Expression of CD133 in the hemangioma
tumor spheres is consistent with their endothelial lineage.
[0166] Taken together, these data suggest that tumor spheres have
the stem cell properties of self-renewal and differentiation. Tumor
stem cells have been known to resist to chemotherapy and are likely
responsible for the initiation and progression of disease by
maintaining the production of new tumor cells. Therefore, the tumor
spheres derived from fresh infantile hemangioma tissues have many
potential uses.
Example 2
ChIP Assay
[0167] Isolated cells from tumor spheres (1.times.10.sup.6
cells/well in 6-well plates), are processed using a ChIP Assay Kit
(Upstate, Charlottesville, Va.) following the manufacture's
protocol. Briefly, cells are cross-linked by adding formaldehyde
(27 .mu.l of 37% formaldehyde/ml) and incubated for 10 min. Then,
chromatin is sonicated to an average size of approximately 500 bp
and immunoprecipitated with SALL4 antibodies, preimmune serum, or
anti-HA (hemagglutination) antibody. Antibodies for histone
modifications, histone H3 trimethy K4 and histone H3 dimethy K79,
may be purchased from Abeam (Cambridge, Mass.). Histone-DNA
crosslinks are reversed by heating at 65.degree. C. followed by
digestion with proteinase K (Invitrogen, Carlsbad, Calif.). DNA is
recovered by using a PCR purification kit (Qiagen, Valencia,
Calif.) and then used for PCR or QRT-PCR (quantitative real time
polymerase chain reaction).
[0168] Although the invention has been described with reference to
the above examples, it will be understood that modifications and
variations are encompassed within the spirit and scope of the
invention. Accordingly, the invention is limited only by the
following claims.
* * * * *