U.S. patent application number 11/837999 was filed with the patent office on 2008-01-31 for blood vessel formation from endothelial colony forming cells.
This patent application is currently assigned to Indiana University Research and Technology Corporation. Invention is credited to David A. Ingram, Daniel Prater, Mervin C. Yoder.
Application Number | 20080025956 11/837999 |
Document ID | / |
Family ID | 34865406 |
Filed Date | 2008-01-31 |
United States Patent
Application |
20080025956 |
Kind Code |
A1 |
Yoder; Mervin C. ; et
al. |
January 31, 2008 |
BLOOD VESSEL FORMATION FROM ENDOTHELIAL COLONY FORMING CELLS
Abstract
Methods and compositions to form fully functional blood vessels
in vivo using endothelial colony forming cells (ECFCs) are
disclosed. Culturing ECFCs in a support material results in
association of ECFCs in vitro and formation of blood vessels in
vivo upon implantation. Direct administration of cultured ECFCs
form blood vessels in vivo. Formation of blood vessels is useful in
treating a variety of medical conditions including ischemia and
hypoxia.
Inventors: |
Yoder; Mervin C.;
(Indianapolis, IN) ; Ingram; David A.;
(Indianapolis, IN) ; Prater; Daniel; (Fishers,
IN) |
Correspondence
Address: |
BARNES & THORNBURG LLP
11 SOUTH MERIDIAN
INDIANAPOLIS
IN
46204
US
|
Assignee: |
Indiana University Research and
Technology Corporation
Indianapolis
IN
46206
|
Family ID: |
34865406 |
Appl. No.: |
11/837999 |
Filed: |
August 13, 2007 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11055182 |
Feb 9, 2005 |
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11837999 |
Aug 13, 2007 |
|
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60822166 |
Aug 11, 2006 |
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60543114 |
Feb 9, 2004 |
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60542949 |
Feb 9, 2004 |
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60573052 |
May 21, 2004 |
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60637095 |
Dec 17, 2004 |
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Current U.S.
Class: |
424/93.7 ;
435/325; 435/402 |
Current CPC
Class: |
G01N 33/5005 20130101;
A61P 43/00 20180101; C12N 5/0692 20130101; A61P 9/00 20180101; C12N
2501/145 20130101; C12N 2501/125 20130101; C12N 2501/26 20130101;
G01N 2333/70596 20130101; G01N 33/56966 20130101; C12N 5/0068
20130101; C12N 2502/28 20130101; G01N 2333/70589 20130101; C12N
2533/54 20130101; C12N 2533/90 20130101; A61K 2035/124 20130101;
C12N 2501/22 20130101; C12N 5/0647 20130101 |
Class at
Publication: |
424/093.7 ;
435/325; 435/402 |
International
Class: |
A61K 35/12 20060101
A61K035/12; A61P 9/00 20060101 A61P009/00; C12N 5/02 20060101
C12N005/02; C12N 5/08 20060101 C12N005/08 |
Claims
1. A method for forming a functional vasculature in vivo in a host,
the method comprising: (a) culturing a plurality of isolated high
proliferative-potential endothelial colony forming cells (HPP-ECFC)
in a support material; (b) implanting the support material
comprising the endothelial colony forming cells to a target site;
and (c) forming the functional vasculature in vivo in the target
site in the host.
2. The method of claim 1, wherein the support material is an
artificial matrix.
3. The method of claim 2, wherein the matrix is a gel comprising
one or more components selected from the group consisting of
collagen, fibronectin, gelatin, laminin and any extracellular
matrix constituent.
4. The method of claim 1, wherein the ECFCs are circulating
ECFCs.
5. The method of claim 1, wherein the target site comprises an
ischemic injury.
6. The method of claim 1, wherein the target site is selected from
the group consisting of heart, lungs, kidney, liver, and
pancreas.
7. The method of claim 1, wherein the host is a human.
8. The method of claim 1, wherein the target site requires vessel
formation.
9. The method of claim 1, wherein the endothelial colony forming
cells comprise a density of about 1-2 million cells per milliliter
volume of implantation support material.
10. The method of claim 1, wherein the endothelial colony forming
cells are cultured for about 12-48 hours prior to implantation.
11. The method of claim 1, wherein the support material is
viscous.
12. The method of claim 1, wherein the support material provides
three-dimensional cell growth.
13. The method of claim 1, wherein the support material comprising
the cells is administered directly to the target site.
14. A method for forming functional blood vessels in vivo in a
host, the method comprising: (a) culturing cord blood endothelial
colony forming cells (ECFCs) in vitro; (b) administering the
cultured cells to a target site; and (c) forming the vasculature in
vivo in the host.
15. An implantable scaffold for treating an ischemic injury in a
host, the scaffold comprising high proliferative-potential
endothelial colony forming cells (HPP-ECFC), wherein the
endothelial colony forming cells associate to form blood vessels in
vivo.
16. The scaffold of claim 15 comprising a growth factor.
17. An engineered tissue comprising a biologically compatible
support material and a plurality of cultured high
proliferative-potential endothelial colony forming cells (HPP-ECFC)
that initiate vessel formation.
18. A composition comprising an effective amount of high
proliferative-potential endothelial colony forming cells (HPP-ECFC)
culture on a biocompatible gel material.
19. The composition of claim 19 comprises one or more components of
an extra cellular matrix.
20. The composition of claim 19, wherein the ECFCs are cultured on
a growth medium for endothelial cells.
Description
[0001] This application claim priority to U.S. Ser. No. 60/822,166
filed Aug. 11, 2006 and is a continuation-in-part of U.S. Ser. No.
11/055,182 filed Feb. 9, 2005, which claims priority to U.S. Ser.
No. 60/543,114 filed Feb. 9, 2004, U.S. Ser. No. 60/542,949 filed
Feb. 9, 2004, U.S. Ser. No. 60/573,052 filed May 21, 2004 and U.S.
Ser. No. 60/637,095 filed Dec. 17, 2004.
BACKGROUND
[0002] Cord blood circulating endothelial colony forming cells are
used to form functional blood vessels in vivo. Methods reproducibly
generate human vessels from the cord blood circulating colony
forming cells upon implantation into a desired target site.
[0003] During embryogenesis, blood vessels are formed de novo by
the patterned assembly of angioblasts in a process termed
vasculogenesis. Once an intact vascular system has been
established, the development of new blood vessels occurs via the
sprouting of endothelial cells from postcapillary venules or the
maturation and de novo growth of collateral conduits from larger
diameter arteries. These two mechanisms of new blood vessel
formation are termed angiogenesis and arteriogenesis, respectively.
A population of human circulating CD34+ cells that could
differentiate ex vivo into cells with endothelial cell-like
characteristics have been termed "endothelial progenitor cells"
(EPCs) and may also contribute to new vessel formation. Efforts are
focused on defining the role of EPCs in the repair of damaged
vascular endothelium or in tumor angiogenesis and on translating
these experimental observations into human clinical trials for
repair of vascular injury and/or ischemic tissue or as a novel
strategy for anticancer therapy.
[0004] New vessel formation occurs via vasculogenesis,
angiogenesis, or arteriogenesis. Blood post-natal vasculogenesis
has been purported to be an important mechanism for angiogenesis
via marrow derived circulating endothelial progenitor cells
(EPCs).
[0005] Angiogenesis (neoangiogenesis) is the process of new vessel
formation from pre-existing vessels; this is the process reported
to give rise to new vessels in adult subjects. Recent studies
indicate that marrow-derived EPCs may play minimal or no role in
neovascularization of tumors, vessel repair, or normal vessel
growth and development. These conflicting reports have raised
questions about the function of EPCs in vascular homeostasis and
repair. The controversies surrounding these fundamental questions
may in part originate from the heterogenous phenotypic definitions
of EPCs and a lack of functional clonogenic assays to isolate and
accurately describe the proliferative potential of EPCs. However,
there is no uniform definition of an EPC, which makes
interpretation of these studies problematic and prohibits
reproduction of cell types suitable for clinical use. Although a
hallmark of stem and progenitor cells (e.g. hematopoietic,
intestinal, neuronal) is their ability to proliferate and give rise
to functional progeny, EPCs are primarily defined by the expression
of selected cell surface antigens. Sole dependence on cell surface
expression of molecules can be problematic because the expression
may vary with the physiologic state of the cell. A hallmark of many
stem or progenitor cells in various tissues is their ability to
give rise to numerous differentiated progeny to provide sufficient
cells for tissue homeostasis.
[0006] Models for stem cell differentiation leading to endothelial
and hematopoietic cells are of interest because of the clinical
value of stem cells and their progeny. Methods previously used do
not guarantee that single endothelial cells have been isolated and
characterized to identify the progenitors. Endothelial cell
proliferation in vivo in normal, mature arterial, venous, and
capillary vessels in most mammals is reported to be extremely low,
if not nonexistent.
[0007] Both hematopoietic stem and progenitor cells (HSC/Ps) are
enriched in umbilical cord compared to adult peripheral blood. Cord
blood is currently used as an alternative resource of hematopoietic
stem cells for transplantation of patients with a variety of
hematological disorders and malignancies.
[0008] Thousands of patients require a hematopoietic stem cell
(HSC) transplant each year. Nearly 2/3 of the patients are unable
to find a human leukocyte antigen (HLA) compatible match for the
transplant. This is particularly true for many ethnic populations
and under-represented minorities. Only 1/3 of Caucasian patients
find suitable matched sibling grafts--the most compatible source
with the least graft versus host disease (GVHD) complications.
[0009] Human umbilical cord blood is known to be an alternative
source of HSCs for clinical transplantation. Whether or not the
donor cord blood is a full major histocompatible match to the
recipient or is mismatched, cord blood cells engraft and repopulate
conditioned hosts as a treatment for a variety of congenital or
acquired hematologic disorders. Even if the cord blood graft is
mismatched with the recipient by two or more loci, the incidence
and severity of GVHD is significantly less than that observed for
transplantation of a similarly mismatched adult marrow or mobilized
peripheral blood graft.
[0010] Limitations to a more widespread use of cord blood for
transplant include the fact that only a limited number of HSC and
progenitor cells are present in a graft. Because most patients do
not have a matched sibling donor, most cord blood grafts are
transplanted into mismatched recipients. Multiple studies report
that the dose of cord blood cells in a graft may be a factor for
patient survival when the graft comes from an unrelated donor.
Transplant related mortality is reported as 20% in recipients that
obtained a cord blood graft with more than 1.7.times.10.sup.5 CD34+
cells/kg versus 75% in those receiving fewer CD34+ cells in the
graft. Finding a method to effectively expand cord blood HSC ex
vivo to increase the number of cells in a graft, would be a major
advance for clinical transplantation and would have a significant
commercial market.
SUMMARY
[0011] Method and compositions using endothelial colony forming
cells to provide vasculature are disclosed. For example, methods to
suspend human cord blood circulating endothelial colony forming
cells in collagen-fibronectin gels to form functional blood vessels
in vivo are disclosed. In this embodiment, for example, the
suspended cells self-associate in about 12-24 hours in an incubator
and are implanted in vivo. The implanted cells autonomously form
vascular structures that connect to nearby blood vessels in the
host and carry blood cells, thus indicating that the newly formed
vessels are functional.
[0012] Endothelial colony forming cells (ECFCs) participate in
vascularization. Cultured ECFCs participate in endothelial network
formation (capillary formation) and transplanted ECFCs are
incorporated into sites of neovascularization in vivo. For example,
transplanted human ECFCs formed capillaries. Transplantation of
ECFCs functionally augment neovascularization in response to
ischemia, e.g., hindlimb ischemia.
[0013] Cord blood colony forming cells are used to form vessels in
human subjects with diminished vessel forming ability and critical
limb ischemia.
[0014] Single-cell colony assays were developed to describe novel
hierarchy among mammalian endothelial progenitor cells (EPCs)
isolated from peripheral blood and umbilical cord and from
endothelial cells isolated from umbilical or adult blood vessels. A
distinct population of progenitor cells from human, bovine, porcine
and rat biological samples was identified based on clonogenic and
proliferative potential.
[0015] Endothelial progenitor cells (EPCs) were isolated from adult
peripheral and umbilical cord blood and expanded exponentially ex
vivo. In contrast, human umbilical vein endothelial cells (HUVECs)
or human aortic endothelial cells (HAECs) derived from vessel walls
are widely considered to be differentiated, mature endothelial
cells (ECs) and are utilized as "controls" for EPC studies.
However, similar to adult and cord blood derived EPCs, HUVECs and
HAECs derived from vessel walls can be passaged for at least 40
population doublings in vitro. Diversity of EPCs exists in human
vessels and provides a conceptual framework for determining both
the origin and function of EPCs in maintaining vessel integrity.
EPCs are therefore readily obtained for clinical use e.g. grafts,
either from peripheral blood or from biopsies of human vessels.
[0016] A method for forming a functional vasculature in vivo in a
host, the method includes the steps of:
[0017] (a) culturing a plurality of isolated high
proliferative-potential endothelial colony forming cells (HPP-ECFC)
in a support material;
[0018] (b) implanting the support material comprising the
endothelial colony forming cells to a target site; and
[0019] (c) forming the functional vasculature in vivo in the target
site in the host.
[0020] A support material may be an artificial matrix and the
matrix may be a gel that includes one or more components selected
from the group consisting of collagen, fibronectin, gelatin,
laminin and any extracellular matrix constituent.
[0021] In an embodiment, the ECFCs are circulating ECFCs. For
example, target site comprises an ischemic injury and may be
selected from the group consisting of heart, lungs, kidney, liver,
and pancreas. Methods and compositions disclosed herein are
applicable in humans. A suitable target site for example may
require new vessel formation.
[0022] Endothelial colony forming cells include a density of about
1-2 million cells per milliliter volume of implantation support
material. Other suitable ranges include 1-10 or 10-100 million
cells per milliliter volume of implantation support material.
Cultured ECFCs without any implantation material or matrix material
may also directly administered or delivered to a target site.
Concentration of ECFC may range from 1-10 or from 0.1-10 million
cells/ml.
[0023] In an embodiment, the endothelial colony forming cells are
cultured for about 12-48 hours prior to implantation. Other
suitable incubation periods include for example, 6 hours, 10 hours,
15 hours, 20 hours, 36 hours, 42 hours, 48 hours, 3 days and 1
week. Suitable incubation temperatures include for example,
30-37.degree. C., 24.degree. C.
[0024] In an aspect, the support material is viscous and the
support material provides three-dimensional cell growth. In an
aspect, the support material that includes the cells is
administered directly to the target site.
[0025] A method for forming functional blood vessels in vivo in a
host, the method includes the steps of:
[0026] (a) culturing cord blood endothelial colony forming cells
(ECFCs) in vitro;
[0027] (b) administering the cultured cells to a target site;
and
[0028] (c) forming the vasculature in vivo in the host.
[0029] A method of augmenting blood supply at a target site, the
method includes the steps of providing a matrix seeded with high
proliferative-potential endothelial colony forming cells (HPP-ECFC)
and inducing formation of a functional vasculature to increase
blood supply at the target site.
[0030] A method of repairing an injury or preventing an injury to
an endothelial surface, the method includes the steps of culturing
high proliferative-potential endothelial colony forming cells
(HPP-ECFC) and providing cultured endothelial colony forming cells
at the endothelial surface to repair or prevent the injury to
endothelial surface.
[0031] An implantable scaffold for treating an ischemic injury in a
host, the scaffold includes high proliferative-potential
endothelial colony forming cells (HPP-ECFC), wherein the
endothelial colony forming cells associate to form blood vessels in
vivo. The scaffold may also include a growth factor.
[0032] An engineered tissue includes a biologically compatible
support material and a plurality of cultured high
proliferative-potential endothelial colony forming cells (HPP-ECFC)
that initiate vessel formation.
[0033] A tissue repair device includes an effective amount of high
proliferative-potential endothelial colony forming cells (HPP-ECFC)
culture on a biocompatible gel material. The tissue repair device
includes one or more components of an extra cellular matrix. The
tissue repair device includes endothelial colony forming cells that
are cultured on a growth medium for endothelial cells.
BRIEF DESCRIPTION OF THE DRAWINGS
[0034] FIG. 1 shows a scheme for transplantation of cord blood
ECFC. Cells (1-2.times.10.sup.6) derived from colonies of
circulating endothelial colony forming cells (ECFCs) established
from human cord blood were implanted in 1 mL of type 1 collagen and
fibronectin solution that hardens upon incubation at 37 degrees C.
The gels are incubated in 24 well plates overnight. The ECFC
containing gels are recovered from the plates and implanted
subcutaneously in anaesthetized NOD/SCID mice. At 2-4 weeks later,
the gels are imaged using a two photon microscope for visualization
of blood flow in an anaesthetized animal. After the imaging the
gels are recovered, fixed, and stained with monoclonal antibodies
(anti-human CD31 or anti-mouse CD31) to identify the human
vessels.
[0035] FIG. 2 shows (a) Human CD31 staining of collagen/FN gel
implants; (b) is a higher magnification of (a). Antibody specific
for human CD31 was used for the identification. Arrows indicate
human cells expressing CD31. These cells are lining the surface of
vessels and thus, are endothelial cells. The small round
non-staining mouse red blood cells inside the vessels are
shown.
[0036] FIG. 3 shows murine CD31 staining of collagen/FN gel
implants. Arrows point to the mouse endothelial cells expressing
CD31 in the subcutaneous tissues outside of the gels. No staining
of vessels or endothelial cells inside of the gels indicates that
the human cells are not stained by this antibody.
DETAILED DESCRIPTION
[0037] Human endothelial colony forming cells (ECFC), implanted
subcutaneously in immunodeficient mice spontaneously form vessels
that function to carry murine blood cells. A revised model for
considering the cellular events involved in new vessel formation
emphasizes the interacting role of macrophages and ECFC.
[0038] In an embodiment, methods of producing endothelial cell
tubules or blood vessels in vivo include the steps of preparing a
solution comprising collagen and fibronectin; suspending
endothelial cells in the solution; warming the suspension so that
the collagen gels to produce a three-dimensional gel; polymerizing
the collagen within the solution to form a three-dimensional gel;
and implanting the three-dimensional gel produced into an
animal.
[0039] In an embodiment, methods and compositions are disclosed
wherein the ECFCs associate to form structures characteristic of
mature microvessels in vivo. The ECFCs form vasculature that are
perfused by blood.
[0040] In an embodiment, methods and composition for promoting
vascularization/revascularization in an animal include the steps of
preparing a solution of a compatible biological polymer material
and suspending ECFCs in the solution, wherein the suspended ECFCs
associate in vitro; and directly injecting the solution into an
animal of choice, including humans to form functional blood vessels
in vivo.
[0041] Endothelial cell progenitor named a high proliferative
potential-endothelial colony forming cell (HPP-ECFC) displays high
proliferative potential (up to 100 population doublings compared to
20-30 doublings in adult blood EPC. HPP-ECFC were not only isolated
from cord blood but from umbilical and adult blood vessels.
HPP-ECFC cells can be replated at a single cell level and the
majority of cells proliferate with regeneration of at least
secondary HPP-ECFCs. Further, monolayers of cord blood endothelial
cells derived from HPP-ECFCs demonstrate a 2.5-fold decrease in
population doubling times (PDT) and at least a 2-fold increase in
cumulative population doubling levels (CPDL) compared to adult
LPP-ECFCs (during the same time of culture ex vivo). In contrast to
other populations of endothelial progenitor cells isolated from
cord blood utilizing different methodologies, cord blood HPP-ECFC
progeny uniformly express endothelial cell antigens and not
hematopoietic specific cell antigens. Thus, HPP-ECFC are enriched
in human umbilical cord blood and were not found in adult
peripheral blood. HPP-ECFC were also found in endothelial cells
derived from mammalian blood vessels e.g. umbilical vein and human
aortic vessels. These HPP-ECFCs appear in cultures of freshly
plated cord blood mononuclear cells within 10 days, whereas adult
blood LPP-ECFCs rarely appear before 14 days after plating.
[0042] Cell cultures derived from HPP-ECFC are homogenous and
display markers uniformly. These cells exhibit homogenous
proliferative and clonogenic potential. Because some of these cell
cultures are derived from a single HPP-ECFC, the resulting cells
are homogenous and are not mixtures of cells.
[0043] Further, cord blood colonies consistently appeared larger
compared to adult colonies. There were distinct differences in the
size, frequency and time of appearance between adult and cord blood
endothelial cell colonies. These observations show that cord blood
EPCs are composed of HPP-ECFC, LLP-ECFC, and clusters, whereas
adult blood EPCs are composed of LPP-ECFCs and clusters.
[0044] The complete hierarchy of HPP-ECFC, LPP-ECFC, clusters and
mature endothelial cells can be isolated from any blood vessel in a
living mammalian donor using the techniques described herein.
[0045] HPP-ECFCs give rise to all subsequent stages of endothelial
progenitors in addition to replating into secondary HPP-ECFCs. Low
proliferative potential-endothelial colony forming cells (LPP-ECFC)
arising from single cells form colonies, which contain greater than
50 cells, but do not form at least secondary LPP-ECFC colonies upon
replating. They do give rise to endothelial cell clusters (less
than 50 cells). Finally, endothelial cell clusters can arise from a
single cell but contain less that 50 cells, and do not replate into
colonies or clusters.
[0046] Hematopoietic stem and progenitor cells are enriched in
umbilical cord compared to adult peripheral blood. One intriguing
observation was that EPCs were also enriched in umbilical cord
blood compared to adult peripheral blood. Further, cord blood
derived EPCs contain high levels of telomerase activity, which may
account for the observation that these cells can be expanded for at
least 100 population doublings without obvious signs of cell
senescence. At the single cell level, some cord blood EPC-derived
cells can be expanded 10.sup.7 to 10.sup.12 fold.
[0047] Percent of dividing single cells giving rise to a colony
with the number of cells in the quantitative ranges shown (HPP,
LPP, clusters) (e) Representative photomicrographs (50.times.
magnification) of the different endothelial cell clusters (<50
cells), LPP (about 51-2000 cells), and HPP (about 2000-to
>10,000 cells) derived from a single cord blood or adult
EPC-derived endothelial cell. Results are representative of 4 other
independent experiments utilizing cells from different donors.
Scale bar in photomicrographs represents 100 .mu.m. LPP-ECFC
(51-2000 cells) and HPP-ECFC (2000>10,000 cells) ranges are
approximations.
[0048] The average level of telomerase activity in the adult
samples was 4.+-.4% and of the cord blood samples 34.+-.10% of the
telomerase activity of the HeLa cells. Comparison of telomerase
activity of early and late passage adult and cord blood EPC-derived
endothelial cells. PD indicates the cumulative population doubling
level of the cells tested. P indicates telomerase activity in HeLa
cells, which were used as a positive control. N indicates a
negative control. Three other experiments utilizing early and late
passage cord blood and adult EPC-derived endothelial cells from
three different donors showed similar results.
[0049] The HPP-ECFC are small cells (nuclear diameter 8-10 microns)
with minimal cytoplasmic spreading (diameters vary from 12-22
microns) with nuclear to cytoplasmic ratio >0.8. LPP-ECFC are
more heterogenous in size but are larger than HPP-ECFC. LPP-ECFC
nuclei vary in size from 10.5-12.5 microns and have more
cytoplasmic spreading (varying from 25-60 microns) with a ratio
>0.4 but <0.5. Endothelial clusters are nearly mature
endothelial cells with nuclei that vary from 13.0-16.5 microns and
have cytoplasmic diameters that vary from 65-80 microns and nuclear
to cytoplasmic ratios of >0.2 but <0.3. Mature differentiated
endothelial cells are large very well spread cells with nuclear
diameters that range from 17.0-22.0 microns and cytoplasmic
diameters from 85-105 microns and nuclear to cytoplasmic ratios
similar to endothelial clusters. Therefore, HPP-ECFC are very
distinctly smaller than any of the other EPC and quite smaller than
the mature endothelial cells
[0050] In an aspect, isolated endothelial colony forming cells have
the following characteristics:
[0051] (a) express cell surface antigens that are characteristic of
endothelial cells, such as CD31, CD105, CD146, and CD144;
[0052] (b) do not express cells surface antigens that are
characteristic of hematopoietic cells, such as CD45 and CD14;
[0053] (c) ingest acetylated LDL; and
[0054] (d) form capillary- like tubes in Matrigel.TM.
(extracellular matrix proteins).
[0055] The isolated cells classified as HPP-ECFC also
[0056] (a) replate into at least secondary colonies of at least
2000 cells when plated from a single cell;
[0057] (b) exhibit high proliferation;
[0058] (c) proliferate from a single cell; and
[0059] (d) express high levels of telomerase, at least 34% of that
expressed by HeLa cells. HPP-ECFC also display a high nuclear to
cytoplasmic ratio that is >0.8, cell diameters <22 microns,
and at least 10.sup.7 progeny derive from a single cell.
[0060] A method of isolating endothelial colony cells includes
steps of:
[0061] (a) culturing cells from a biological sample on supports
coated with extracellular matrix proteins;
[0062] (b) selecting cells that adhere to the supports and form
replatable colonies; and
[0063] (c) selecting single cells from the colonies.
[0064] The biological sample may be mammalian cord blood, or blood
vessel. Human, bovine, porcine and rat sources are suitable. A
single cell assay for types of endothelial cells includes the steps
of:
[0065] (a) cell sorting of biological samples using a specific
sorting method;
[0066] (b) culturing the single sorted cells on extracellular
matrix protein under defined conditions; and
[0067] (c) enumerating specific colony sizes, morphology, and
proliferative potential to determine the type of endothelial cell
e.g. HPP-ECFC.
[0068] A method of enriching for HPP-ECFC includes the steps
of:
[0069] (a) cell sorting of biological samples using a specific
sorting method; and
[0070] (b) culturing of single sorted cells on extracellular matrix
proteins under defined conditions.
[0071] A method for expanding hematopoietic stem cells ex vivo,
include the steps of:
[0072] (a) culturing HPP-ECFC cells on collagen coated solid
supports; and
[0073] (b) expanding hematopoietic stem cells (HSC) by co-culturing
with HPP-ECFC cells wherein the HPP-ECFCs are derived from human
cord blood cells and the HSC cells are derived from human bone
marrow.
[0074] A method for improving the percentage of hematopoietic stem
cells in a graft in a mammal, includes the step of:
[0075] (a) co-culturing human bone marrow cells with cord blood
HPP-ECFC to form a product; and
[0076] (b) transplanting a suitable amount of the product into the
mammal wherein the cells are CD45+ cells derived from human bone
marrow, and the mammal is a NOD-SCID mouse.
[0077] Cord blood high proliferative potential--endothelial colony
forming cells (HPP-ECFCs) in co-culture with autologous or
unrelated cord blood, mobilized adult peripheral blood, or
marrow-derived HSC expands the number of HSC cells and results in
an increase in HSC and an increase in HSC repopulating activity
leading to higher levels of engraftment in a recipient subject.
[0078] Use of a feeder layer of cells derived from high
proliferative potential-endothelial colony forming cells(HPP-ECFCs)
from human umbilical cord blood, stimulates growth and survival of
repopulating hematopoietic stem and progenitor cells. Stimulation
of growth and survival was determined by increased numbers of
progenitor cells in in vitro culture and increased levels of human
cell engraftment in the NOD/SCID immunodeficient mouse transplant
system.
[0079] As used herein, the terms "encapsulated" and "embedded"
refer to being entrapped and/or surrounded by a matrix or a support
material. In the case of ECFCs, encapsulated or embedded cells may
be entirely or partially surrounded by matrix material; they may be
able to move through, proliferate in and remodel the matrix.
[0080] "Matrix" as used herein, refers to the surrounding substance
within which ECFCs associate or is contained. Artificial matrix
refers to a matrix that is not naturally found.
[0081] "Three-dimensional cell culture" or "3-D cell culture" as
used herein, refers to cell cultures wherein cell expansion can
occur in any direction.
[0082] "Tissue cell culture" as used herein refers to an
aggregation of cells and intercellular matter performing one or
more functions in an organism. Examples of tissues include, but are
not limited to, epithelium, connective tissues (e.g., bone, blood,
cartilage), muscle tissue and nerve tissue.
[0083] "Two-dimensional cell culture" or "2-D cell culture" as used
herein, refers to conventional monolayer cell culture. Generally,
every cell in a 2-D culture directly contacts the substratum and
the cultures, therefore, generally expand horizontally as they
proliferate.
[0084] "Vascularization" as used herein, refers to the formation of
new blood vessels. This includes formation of additional blood
vessels from existing blood vessels. "Blood vessel" also includes
capillary-like structures that are fully functional to support the
transport of blood.
[0085] "Transplantation" as used herein, generally refers to the
process by which a body part, organ, tissue or cell is transferred
from one organism to another organism or transferred to an organism
from an artificial source such as an organ or tissue harvested from
cell or tissue culture systems. Grafts may include any tissue or
body part or cells that are transferred to a desired site in
vivo.
[0086] Support material as used herein refers to any biologically
compatible substance that can support the association of ECFCs to
form blood vessels. Suitable support material includes for example,
biologically compatible polymer material selected from the group
consisting of collagen, elastin, fibrinogen, fibrin, fibronectin,
gelatin, laminin, vitronectin, hyaluronan, heparan sulfate, agar,
agarose, alginate, chitosan and combinations thereof Suitable
support material can be selected from the group consisting of
collagen-fibronectin, collagen-gelatin, collagen-agarose,
collagen-chitosan, collagen-chitosan-agarose,
collagen-chitosan-gelatin, collagen-vitronectin-agarose,
collagen-vitronectin-gelatin, collagen-vitronectin-chitosaan
collagen-fibronectin-agarose, collagen-fibronectin-gelatin
collagen- fibronectin-chitosan, collagen-laminin-agarose,
collagen-laminin-gelatin, collagen-laminin-chitosan.
[0087] The support material or the implant material may further
incorporate an additional agent selected from the group consisting
of excipients, growth factors, vitamins, minerals, ions, gases,
crosslinking agents, active agents, carriers and combinations
thereof
[0088] ECFCs are also implanted or `seeded` into an artificial
structure capable of supporting three-dimensional tissue formation.
These structures, typically called scaffolds, are useful, both ex
vivo as well as in vivo, to recapitulating the in vivo milieu and
allowing cells to influence their own microenvironments. Scaffolds
usually serve at least one of the following purposes: allow cell
attachment and migration; deliver and retain cells and biochemical
factors; enable diffusion of vital cell nutrients and expressed
products; exert certain mechanical and biological influences to
modify the behavior of the cell phase. To achieve the goal of
tissue reconstruction, scaffolds may meet some specific
requirements. A high porosity and an adequate pore size are
necessary to facilitate cell seeding and diffusion throughout the
whole structure of both cells and nutrients. Biodegradability is a
factor since scaffolds may be absorbed by the surrounding tissues
without the necessity of a surgical removal. The rate at which
degradation occurs may coincide with the rate of tissue formation:
this means that while cells are fabricating their own natural
matrix structure around themselves, the scaffold is able to provide
structural integrity within the body and eventually it will break
down leaving the neotissue, newly formed tissue which will take
over the mechanical load. Injectability is also useful for clinical
uses.
[0089] Many different materials (natural and synthetic,
biodegradable and permanent) are useful. Biomaterials are
engineered to have suitable properties and functional
customization: injectability, synthetic manufacture,
biocompatibility, non-immunogenicity, transparency, nano-scale
fibers (nanomaterial), low concentration, resorption rates.
[0090] Scaffolds or implantable material may also be constructed
from natural materials:
[0091] in particular different derivatives of the extracellular
matrix are suitable for their ability to support cell growth.
Proteic materials, such as collagen or fibrin, and polysaccharidic
materials, like chitosan or glycosaminoglycans (GAGs) are
suitable.
[0092] As used herein, "consisting essentially of" is intended to
mean that the composition includes components that are primarily
responsible for blood vessel formation, e.g., ECFCs and any other
component that does not materially affect the formation of blood
vessels in vivo.
[0093] The content and teaching of co-pending applications U.S.
Ser. No. 60/822,166 filed Aug. 11, 2006, U.S. Ser. No. 11/055,182
filed Feb. 9, 2005, U.S. Ser. No. 60/543,114 filed Feb. 9, 2004,
U.S. Ser. No. 60/542,949 filed Feb. 9, 2004, U.S. Ser. No.
60/573,052 filed May 21, 2004 and U.S. Ser. No. 60/637,095 filed
Dec. 17, 2004 are hereby expressly incorporated by reference as
they relate to the isolation and characterization of endothelial
colony forming cells. Vessel formation in fibronectin gel is for
example, described in U.S. Publication No. 20040072342, the
disclosure of which is hereby incorporated by reference. Some
examples of engineering tissues can be found in U.S. Publication
Nos. 20050031598, and 20040009589, the disclosures of which are
incorporated by reference.
EXAMPLES
[0094] The following examples are illustrative embodiments and are
not intended to limit the scope of the disclosure.
Example 1
Characterization of Endothelial Cell Colonies (EPCs) Isolated from
Human Umbilical Cord Blood
[0095] Cells were expanded in culture while maintaining an
endothelial cell phenotype.
[0096] In contrast to previously described endothelial progenitor
cells isolated from cord blood, the present disclosure relates that
cells isolated from human umbilical cord blood, i.e. cord blood
HPP-ECFCs and progeny, can be cultured for at least 100 population
doublings and expanded exponentially even when beginning with a
single cell. Further, these cells do not express the hematopoietic
cell specific surface antigens, CD45 and most also do not express
CD14, and do not form hematopoietic cell colonies in methycellulose
assays. In addition, the HPP-ECFC progeny rapidly form vessels in
Matrigel.TM. (extracellular matrix proteins), upregulate VCAM-1 in
response to either IL-1 or TNF-.alpha. stimulation, and express
endothelial cell specific antigens, which confirms their
endothelial cell identity. These cells were designated high
proliferative potential-endothelial colony forming cells
(HPP-ECFCs).
[0097] Cord blood HPP-ECFCs demonstrate greater replicative
kinetics compared to adult blood (which are composed of LPP-ECFC,
clusters, and mature endothelial cells). While endothelial
progenitors are reported to express AC133, CD34, and Flk1, HPP-ECFC
progeny and EOC progeny display similar frequencies of cells
expressing AC133, CD34, and Flk1 antigens and, therefore, these
cell surface markers do not permit discrimination of cells with
differing proliferative potentials.
[0098] Endothelial outgrowth cells appear two to four weeks after
culture of MNCs isolated from adult peripheral blood and are
characterized by their exponential growth in vitro (however, EOC
display lower levels of telomerase, do not replete into secondary
LPP-ECFC, and reach replicative senescence long before TPP-ECFC).
In contrast, HPP-ECFC generated from human umbilical cord blood
MNCs, emerged five to ten days after culture of cord blood MNCs in
complete EGM-2 media on tissue culture plates coated with type I
collagen. Discrete adherent cell colonies appeared and displayed
the "cobblestone" morphology of endothelial cells. The morphology
and appearance of the colonies was similar to, but distinct from,
that previously described for adult peripheral blood derived EOC
colonies and clearly distinct from adherent circulating endothelial
cells or macrophages. The HPP-ECFC colonies are large and are
composed of a mixture of small round, long thin, and large
flattened round cells whereas the EOC are nearly homogenously
composed of long thin cells. The colony-derived cells were
subcultured and expanded cells derived from these colonies were
used for immunophenotyping, functional testing and measurement of
growth kinetics. After initial passage, the cells formed monolayers
of spindle shaped cells with "cobblestone" morphology.
Immunophenotyping revealed that the cells uniformly expressed the
endothelial cell surface antigens, CD31, CD141, CD105, CD146,
CD144, vWF, and flk-1. The cells did not express the hematopoietic
cell surface specific antigens, CD45 and CD14, confirming that the
monolayers were not contaminated with hematopoietic cells.
[0099] Confirming that the monolayers derived from the adherent
colonies were endothelial cells, the cells ingested acetylated-low
density lipoprotein (Ac-LDL) or (Dil-AC-LDL). These cellular
functions are characteristic of endothelial cells. Cells
subcultured from the adherent colonies uniformly incorporated
AC-LDL, formed vessels in Matrigel.TM. (extracellular matrix
proteins) after seeding varying numbers of cells, and upregulated
VCAM-1 in response to both rhTNF-.alpha. or rhIL-1 stimulation.
[0100] The growth kinetics of cord blood HPP-ECFC and progeny were
measured as a function of time. Strikingly, cord blood HPP-ECFC and
progeny could be exponentially expanded in culture for at least 100
population doublings without signs of senescence, and the number of
cells increased 10.sup.20 fold over a period of 100 days in
culture. A representative growth curve of cord blood endothelial
cells, illustrates the proliferative potential of these cells in
vitro. Thus, based on immunophenotyping, functional testing, and an
analysis of growth kinetics, it was shown that colonies of
endothelial cells (designated HPP-ECFCs) can be uniquely cultured
from cord blood MNCs and passaged into confluent monolayers of
exponentially expandable endothelial cells.
Example 2
HPP-ECFC Colonies are Present in Human Umbilical Cord Blood but Not
in Adult Peripheral Blood
[0101] 50-100 milliliters of peripheral blood was collected from
healthy adult donors or from umbilical cords of normal term infants
and isolated MNCs. Cells were seeded into tissue culture plates
coated with extracellular mature molecules in complete EGM-2 media
and observed for colony formation over the next one to six weeks.
The number of colonies per equivalent volume of blood was increased
15 fold in cord blood compared to adult peripheral blood (Table I).
Similar differences in colony formation were also observed when
equivalent numbers of cord and adult MNCs were plated. Although
adult EOC coloniestypically formed between 2-4 weeks after
initiation of culture, cord blood HPP-ECFC colonies appeared within
5-10 days. Finally, immunophenotyping of the cells isolated from
endothelial colonies from both cord blood and adult peripheral
blood by flow cytometry revealed that the colony-cells uniformly
expressed the endothelial cell surface antigens, CD31, CD105,
CD146, CD144, vWF, and flk-1 and not the hematopoietic cell surface
antigens CD45 and CD14, confirming their endothelial cell identity.
Immunophenotyping of the adult EOCs was consistent with previously
published studies. Thus, endothelial colony forming cell HPP-ECFC
are present in cord blood and represent a different cell type
compared to adult peripheral blood EOCs (which represent
LPP-ECFCs).
Example 3
The Proliferative Rate of Cord Blood HPP-ECFCs is Greater than
Adult Blood EOCs
[0102] Given the differences in the frequency and the time of
appearance of colony formation of cells from cord blood compared to
adult peripheral blood, a question was whether there were
differences in the proliferative kinetics of adult and cord blood
cells. Early passage monolayers of HPP-ECFC were established from
cord blood and EOC established from adult peripheral blood, and
cells were cultured in complete EGM-2 media on type I collagen
coated plates. Input cell numbers were counted for determination of
population doubling times (PDT) and cumulative population doubling
levels (CPDL) in long-term cultures, which were measurements used
to quantitate and to compare the proliferative kinetics of cord
blood and adult blood derived cells. Cells were cultured for at
least 10 passages to accurately quantitate the PDT and CPDL.
Results testing multiple cell lines from different donors, showed
that there was a 2.5 fold decrease in the PDT of cord blood
HPP-ECFCs compared to adult EOC controls. Further, consistent with
a decrease in PDT, culture of cord blood cells demonstrated a
significant increase in CPDLs compared to serial passage of adult
EOCs. Thus, although both cord and adult cells can be expanded in
culture, the proliferative potential of cord blood HPP-ECFC and
progeny is greater compared to adult EOC. Cord blood derived
HPP-ECFC also demonstrate greater proliferative potential at the
single cell level compared to adult blood EOCs.
[0103] The proliferative and clonogenic capacity of individual cord
blood HPP-ECFC-derived endothelial cells or adult EOCs at the
single cell level was determined. A novel experimental method was
designed to quantitate the proliferative and clonogenic capacity of
single cord blood HPP-ECFC-derived endothelial cells and adult
EOCs.
[0104] Early passage cord blood HPP-ECFC-derived endothelial cells
or adult EOC progeny were initially transduced with a retrovirus
encoding a green fluorescent protein (GFP) and selected for
expression of GFP. Transduction efficiency of both cord and adult
endothelial cells was greater that 95%. Following selection, one
GFP expressing HPP-ECFC-derived endothelial cell or adult
EOC-derived endothelial cell was plated by fluorescent cytometry
sorting (using a sorting nozzle with a diameter .gtoreq.100 microns
and a sheath flow pressure of .ltoreq.9 pounds per square inch)
into one well of a 96 well tissue culture plate coated with type I
collagen and filled with 200 .mu.l of EGM-2 media. Immediately
following placement, individual wells were examined to ensure that
only one endothelial cell had been placed into each well.
Endothelial cells were then cultured for 14 days, and one half of
the media was changed every 4 days with fresh EGM-2 media. At the
end of 14 days, the number of GFP expressing endothelial cells was
counted.
[0105] The number of single cells undergoing at least one cell
division was significantly greater for cord blood HPP-ECFC- derived
endothelial cells compared to adult EOC-derived endothelial cells.
In scoring the number of cells in each well at the end of 14 days,
it was clear that single cord blood HPP-ECFC-derived endothelial
cells divided more and produced larger colonies compared to adult
EOC-derived endothelial cells. Because of differences in the
capacity of single cord blood HPP-ECFC-derived endothelial cells to
divide and form colonies compared to adult EOC-derived endothelial
cells, the number of cells in each well, which demonstrated at
least one cell division, were counted. Although, most of the single
adult EOC-derived endothelial cells (which had divided), produced
clusters of between 2 and 50 cells, some did give rise to secondary
colonies of up to 500 cells, but only a single colony of >2000
cells arose from any of the single sorted adult EOC-derived
endothelial cells. However, greater than 60% of the cord blood
HPP-ECFC-derived endothelial cells (which had divided), formed well
circumscribed secondary colonies consisting of at least 2000 cells,
and numerous single sorted cells gave rise to colonies composed of
>10,000 cells (to the inventors' knowledge, no adult EOC-derived
endothelial cells ever produced such a colony).
[0106] Secondary cell colonies derived from either single adult
EOC-derived or cord blood HPP-ECFC-derived endothelial cells were
serially replated to determine if these cells could form more
colonies. Secondary colonies derived from single adult EOC-derived
endothelial cells never gave rise to tertiary colonies after
replating in 24 well or 6 well type I collagen coated tissue
culture plates in multiple independent experiments. Single cells
plated remained quiescent and did not proliferate. However, most of
the secondary colonies derived from single cord blood HPP-ECFC-
derived endothelial cells, which produced greater than 2000 cells,
could be replated under the same experimental conditions to form
tertiary endothelial colonies. Single primary cord blood
HPP-ECFC-derived endothelial cells can produce secondary colonies,
which can be subsequently serially passaged to produce from
10.sup.7-10.sup.12 endothelial cells.
[0107] Given the similarities of this unique and newly identified
population of cord blood derived endothelial colony forming cells
to the hematopoietic high proliferative potential-colony forming
cells (HPP-CFC; the most primitive multipotent hematopoietic
progenitor that can be cultured in an in vitro clonogenic assay)
these cells are named "high proliferative potential-endothelial
colony forming cells (HPP-ECFC)". In summary, these cells are
different from the previously described adult EOCs in the following
ways: (1) HPP-ECFCs have higher proliferative kinetics when
cultured under the same experimental conditions as adult EOCs, (2)
HPP-ECFCs appear at earlier timepoints in culture from plated cord
blood MNCs compared to adult EOCs derived from plated adult
peripheral MNCs, (3), HPP-ECFCs have higher clonogenic potential at
the single cell level compared to adult EOCs. (4) HPP-ECFCs can be
serially replated to form at least secondary HPP-ECFC colonies
while/whereas adult EOCs do not display this potential, and
HPP-ECFC display high levels of telomerase.
Example 4
Growth Kinetics of EPC-Derived Cord Blood and Adult Endothelial
Cells
[0108] Progenitor cells of different lineages are defined and
discriminated by their clonogenic and proliferative potential.
Because of the differences in cord blood and adult EPC colony
formation, the proliferative kinetics of EPC-derived cord blood and
adult endothelial cells were compared. Initially cells derived from
cord blood and adult endothelial cell colonies were plated at
limiting cell dilutions to test whether the cells would form
secondary colonies and grow to confluence. Interestingly, the cell
progeny derived from both adult and cord blood EPC colonies formed
secondary cell colonies of various sizes before growing to
confluence. However, colonies derived from cord blood EPC-derived
cell progeny were consistently larger and contained smaller cells
compared to adult colonies.
[0109] Cell monolayers were serially passaged to determine the
proliferative potential of EPC-derived cord blood and adult
endothelial cells. Remarkably, cord blood EPC-derived cells could
be expanded for at least 100 population doublings without obvious
signs of senescence. In contrast, adult EPC-derived cells could be
passaged for only 20-30 population doublings. To quantitate and
compare the proliferative kinetics of cord blood and adult
EPC-derived cells, the population doubling times (PDT) and
cumulative population doubling levels (CPDL) were calculated during
a defined time in culture (60 days). There was a 2.5 fold decrease
in the PDT and a 1.5 fold increase in the CPDLs of cord blood
EPC-derived cells compared to adult EPC-derived cells. The PDT and
CPDL of adult EPCs was similar to two recent reports, which tested
the proliferative kinetics of EPC-derived cells isolated from
healthy adult donors.
[0110] The proliferation of cord blood and adult EPC-derived cells
in response to either rhVEGF or rhbFGF stimulation, which are two
endothelial cell mitogens were compared. Cord blood and adult
EPC-derived cells were serum starved and then cultured in the
presence or absence of either rhVEGF or rhbFGF. Cells were cultured
for 16 hours, and pulsed with tritiated thymidine before harvest to
measure DNA synthesis. Cord blood EPC-derived cells displayed
greater DNA synthesis in response to either rhVEGF or rhbFGF
stimulation compared to adult EPC-derived cells. Collectively,
these results demonstrate that the proliferative rate and
expandability of cord blood EPC-derived cells is greater than adult
EPC-derived cells in both short and long term assays. Further, cord
blood and adult EPC-derived endothelial cells form distinct cell
colonies of various sizes and morphology when plated at limiting
dilution.
Example 5
Quantitation of the Clonogenic and Proliferative Potential of
Single Cord Blood and Adult Endothelial Cells Derived from EPC
Colonies
[0111] Cord blood and adult EPC colonies yield cells with different
proliferative and clonogenic potential. However, a rigorous test
for the clonogenic potential of a progenitor cell is to determine
whether a single cell will divide and form a colony in the absence
of other cells. Therefore, an assay was developed to quantitate the
proliferative and clonogenic potential of single cord blood and
adult endothelial cells derived from EPC colonies.
[0112] Cord blood and adult endothelial cells derived from the
initial EPC colonies were transduced with a retrovirus encoding
EGFP and selected for EGFP expression. Following selection, one
EGFP expressing endothelial cell was plated by FACS into one well
of a 96 well tissue culture plate coated with type I collagen and
filled with complete EGM-2 media. Endothelial cells were cultured,
and the number of EGFP-expressing endothelial cells was counted at
the end of 14 days as disclosed herein.
[0113] The percentage of single cells undergoing at least one cell
division was increased five fold for cord blood endothelial cells
compared to adult cells. Further, the average number of cell
progeny derived from a single cord blood endothelial cell was 100
fold greater compared to the number of cells derived from an
individual adult cell. Greater than 80% of the single adult
endothelial cells which divided gave rise to small colonies or
clusters of cells ranging in number from 2-50 cells. However, some
single adult endothelial cells did form colonies containing greater
than 500 cells. In contrast, at least 60% of the single plated cord
blood endothelial cells which divided formed well-circumscribed
colonies containing between 2,000 and 10,000 cells in the 14 day
culture period. The single cell studies demonstrate that there are
different types of cord and adult EPCs, which can be discriminated
by their proliferative and clonogenic potential, and that EPCs
display a hierarchy of proliferative potentials similar to the
hematopoietic progenitor cell hierarchy.
Example 6
The Cell Progeny of Single Cord Blood Endothelial Cells can be
Serially Replated and Expanded Exponentially in Long-Term
Cultures
[0114] In the hematopoietic cell system, the most proliferative
progenitor cell type is termed the high proliferative
potential-colony forming cell (HPP-ECFC). The HPP-ECFC is defined
by its ability to form large cell colonies, which yield individual
cells that have the potential to form at least secondary colonies
upon serial replating. The clonal progeny derived from a single
plated cord blood or adult EPC-derived cell were trypsinized,
replated and cultured into 24-well tissue culture plates for 7
days. After plating the clonal progeny of over 1000 single adult
EPC-derived cells into 24 well plates, only one secondary colony
was detected in the wells after 14 days of culture. In contrast,
approximately one half (205 of 421) of the clonal progeny of single
plated cord blood EPC-derived cells formed secondary colonies or
rapidly grew to confluence in 24 well plates. Since secondary
colonies were not detected in those wells that had rapidly grown to
confluence in 5 days, a limiting dilution analysis was performed on
the confluent monolayer. At least nine percent of the single cells
plated from this monolayer formed an endothelial cell colony,
containing greater than 100 cells. This result verifies that
individual cells derived from cord blood EPCs are capable of
forming secondary colonies.
[0115] The long-term proliferative potential of the cells derived
from a single plated cord blood EPC-derived endothelial cell was
tested. Secondary colonies or confluent cell monolayers derived
from single cord blood endothelial cells were serially passaged
into progressively larger tissue culture plates. The cell progeny
of 11 single endothelial cells, originally derived from three
different cord blood donors were tested. Single cord blood
endothelial cells yielded at least 10.sup.7 cells in long-term
culture. The average CPDL of the eleven single cord blood
endothelial cells tested was 30.8. Thus, a population of high
proliferative EPCs in cord blood, which form secondary and tertiary
colonies.
Example 7
EPC-Derived Cord Blood Endothelial Cells Contain High Levels of
Telomerase Activity
[0116] Endothelial cells derived from cord blood EPCs were serially
passaged beyond Hayflick's limit for at least 100 population
doublings. The only other reported primary endothelial cells with
similar growth kinetics are those genetically engineered to
overexpress telomerase. Thus, telomerase activity was measured in
cord blood and adult EPC-derived cells as a potential molecular
explanation for the differences in their growth kinetics. Both
early and late passage cord blood EPC-derived progeny display
significantly elevated levels of telomerase activity compared to
adult EPC-derived cells, reminiscent of the previously described
primary endothelial cells lines, which overexpress telomerase.
Thus, consistent with extensive proliferative potential, cord blood
EPC-derived cells retain high levels of telomerase activity
(34.+-.10% of the telomerase activity of an equal number of HeLa
cells) with serial passage in culture.
Example 8
Expansion of Repopulating Stem and Progenitor Cells Ex Vivo
[0117] Cord blood high proliferative potential--endothelial colony
forming cell (HPP-ECFC) in co-culture with autologous or unrelated
cord blood, mobilized adult peripheral blood, or marrow-derived
HSC, expands the number of HSC cells and results in an increase in
HSC and an increase HSC repopulating activity leading to higher
levels of engraftment in a recipient subject.
[0118] Co-culture of HPP-ECFC from cord blood with human HSC
increases hematopoietic progenitor cell numbers and enhances
engraftment of human hematopoietic cells in NOD/SCID mice, an assay
for in vivo measure of human HSC function.
[0119] Human cord blood HPP-ECFC-derived endothelial cells
co-cultured with human cord blood or mobilized adult peripheral
blood CD34.sup.+CD38.sup.- cells (enriched in HSC activity) for up
to 7 days (with added cytokines) results in an enhancement in human
CD45.sup.- cell engraftment in sublethally irradiated NOD/SCID mice
by >100 fold.
[0120] A method of collection, isolation, and expansion of the
HPP-ECFC and the particular method for co-culturing the HPP-ECFC
with human stem cells are novel. HPP-ECFC can be collected from any
cord blood sample, expanded, frozen, and stored. These cells can
then be thawed, expanded, and used in co-culture to expand human
cord blood, marrow-derived, or mobilized adult peripheral blood
stem and progenitor cell samples. The expanded product can then be
used for transplantation purposes (after regulatory agency
approval).
Example 9
Vessel Formation Using ECFCs
[0121] This example demonstrates the vessel forming capability of
human cord blood derived endothelial colony forming cells (ECFCs)
that are established in cell culture. A general scheme is
illustrated in FIG. 1 and the identification of vessel formation in
FIGS. 2-3. The ECFCs are isolated using the methods disclosed
herein and are then suspended in collagen and/or fibronectin gels
for vessel formation. In one aspect, 10.sup.6 ECFCs/ml of gel
material are used. The ECFCs in the gels are cultured for about
12-24 hours in an incubator. The ECFCs in the gels initiate vessel
formation by initiating association among the cells. The gel
implants with cultured ECFCs are then implanted at a desired site.
Vessel formation is analyzed by blood flow analysis (e.g., confocal
imaging or immuno histochemistry, IHC), if necessary. Further
staining by CD31 markers are also performed to verify the presence
of blood vessel formation. FIGS. 2-3 show human CD31 staining of
collagen/FN gel implants. The vessel formation is indicated by
arrows. The amount of cells and/or the nature of the gel material
used may vary depending on the treatment site and the nature of the
therapeutic process. For example, ECFCs in the range of about
10.sup.5-10.sup.7/ml of gel, or 10.sup.4-10.sup.8/ml of gel are
suitable for vessel formation in vivo.
[0122] In an embodiment, the ECFCs are cultured and are directly
introduced to a target site or in the circulatory system without
the presence of any support material, e.g., matrix material. The
ECFCs may adhere to the target site and associate to form blood
vessels or otherwise repair the endothelial lining. In an
embodiment, the associated ECFCs are introduced to a target site by
direct administration of cultured ECFCs.
Example 10
Clinical Therapy Using Cultured ECFCs to Enhance Blood Vessel
Formation
[0123] This example demonstrates the use of cultured ECFCs for a
variety of clinical applications. Clinical indications for ECFC
therapy to enhance blood vessel formation to alleviate ischemia and
hypoxia to tissues would include individuals with peripheral
arterial disease. This disorder strikes middle-aged and elderly
individuals and patients that smoke or suffer from diabetes are
more susceptible. Diminished arterial blood flow, particularly in
the lower extremities, can lead to pain with walking and eventual
inability to walk, tissue necrosis, and need for amputation.
[0124] Use of ECFC via direct administration into an ischemic site
would form new vessels in the affected areas, improve blood flow,
and salvage the extremities. In diabetic patients with poor wound
healing and diminished blood flow, ECFC administration into the
sites enhance the overall blood flow and improve the healing
process. Patients requiring skin and/or bone tissue grafts
following major trauma or repair of tissue defects following cancer
and chemotherapy benefit by improved graft perfusion and faster
tissue repair. Another population likely to benefit from ECFC
therapy include patients with myocardial ischemia. Administration
of cultured ECFCs, for example, in a gel, into the ischemic region
enhances recovery of perfusion and salvage cardiomyocytes with
overall improved long term outcomes.
[0125] Any clinically significant condition that can be alleviated
by increased blood supply through formation new blood vessels or
through repairing of existing blood vessels are treated with the
ECFCs described herein.
Materials and Methods
Adult Peripheral and Umbilical Cord Blood Samples
[0126] Fresh blood samples (50-100 ml) were collected by
venipuncture and anticoagulated in citrate phosphate dextrose
solution from healthy human volunteers (males and females between
the ages of 22 and 50). Human umbilical cord blood samples (20-70
ml) from healthy newborns (38-40 weeks gestational age, males and
females) were collected in sterile syringes containing citrate
phosphate dextrose solution as the anticoagulant. Written informed
consent was obtained from all mothers before labor and delivery.
The Institutional Review Board at the Indiana University School of
Medicine approved all protocols.
Buffy Coat Cell Preparation
[0127] Human mononuclear cells (MNCs) were obtained from either
adult peripheral or umbilical cord blood. Briefly, 20-100 ml of
fresh blood was diluted one to one with Hanks Balanced Salt
Solution (HBSS) (Invitrogen, Grand Island, N.Y.) and overlayed onto
an equivalent volume of Ficoll-Paque (Amersham Biosciences) a
ficoll density gradient material. Cells were centrifuged for 30
minutes at room temperature at 1800 rpms (740.times.g). MNCs were
isolated and washed three times with EBM-2 medium (Cambrex,
Walkersville, Md.) supplemented with 10-20% fetal bovine serum
(Hyclone, Logan, Utah), 2% penicillin/streptomyocin (Invitrogen)
and 0.25 .mu.g/ml of amphotericin B (Invitrogen) (complete EGM-2
medium).
Culture and Quantitative Analysis of Endothelial Outgrowth
Cells
[0128] Buffy coat MNCs were initially re-suspended in 12 ml of
EGM-2 medium (Cambrex) supplemented with 10% fetal bovine serum, 2%
penicillin/streptomyocin and 0.25 .mu.g/ml of amphotericin B
(complete EGM-2 medium). Four milliliters of cells were then seeded
onto three separate wells of a six well tissue culture plate (BD
Biosciences, Bedford Mass.) previously coated with extra cellular
matrix proteins e.g. type I rat tail collagen (BD Biosciences)
vitronectin, fibronectin, collagen type 10, polylysine. The plate
was incubated at 37.degree. C., 5% CO.sub.2 in a humidified
incubator. After 24 hours of culture, the non-adherent cells and
debris were carefully aspirated, and the remaining adherent cells
were washed one time with 2 ml of EGM-2 medium. After washing, 4 ml
of EGM-2 medium was added to each well. EGM-2 medium was changed
daily until day 7 of culture and then every other day until the
first passage.
[0129] Colonies of cells initially appeared between 5 days and 22
days of culture and were identified as well circumscribed
monolayers of cobblestone appearing cells. Colonies were enumerated
by visual inspection using an inverted microscope at 40.times.
magnification.
[0130] For passaging, cells were removed from the original collagen
coated tissue culture plates using 0.05% trypsin-0.53 mM EDTA
(Invitrogen), resuspended in 10 ml of EGM-2 media and plated onto
75 cm.sup.2 tissue culture flasks coated with type I rat tail
collagen. Monolayers of endothelial cells were subsequently
passaged after becoming 90-100% confluent.
Culture of HUVECs and HAECs
[0131] Two approaches were used to directly isolate the endothelial
cells from arterial or venous vessels. In the first approach, a 20
G blunt end needle was inserted into one end of an incised vessel
and the vascular contents (plasma with blood cells) were flushed
out the opposite end using sterile saline. Vascular clamps were
then applied to isolate each end of the vessel (3-5 cm in length).
A solution of 0.1% collagenase in Hanks balanced salt solution
(HBSS) was injected through the vessel wall via a 23 G needle, and
the vessel segments were incubated for 5 min at 37.degree. C. The
vascular clamp from one end of the vessel was then removed and the
endothelial cells were expelled via infusion of a cell dissociation
buffer (Gibco) (injected through the distal end of the vessel
opposite the "open" end of the vessel). The vessel segments were
infused with a minimum of 10 mL of cell dissociation buffer. The
suspended cells were centrifuged at 350.times.g and washed in EBM-2
media with 10% FBS, counted, and viability checked using Trypan
blue exclusion.
[0132] The second approach is best suited for large diameter
vessels (>1 cm). The vessel was incised along the entire length
and opened with the endothelial lumen exposed. Any remaining blood
cells and plasma were washed away with HBSS. The endothelium was
removed by firm scraping with a rubber policeman in a single
end-to-end motion. The cells adhering to the rubber policemen were
washed free by swirling the policemen in a solution of EBM-2 with
10% FBS in a 6 cm tissue culture well (precoated with extracellular
matrix proteins). Cells were cultured with visual examination each
day. Colonies of endothelium emerge in 3-10 days. The adherent
endothelial colonies were removed by trypsin-EDTA and transferred
to T 25 flasks that were coated with extracellular matrix proteins.
Cryopreserved human umbilical vein endothelial cells (HUVECs) and
human aortic endothelial cells (HAECs) were obtained from Cambrex
at passage three. Cells were seeded in 75 cm.sup.2 tissue culture
flasks precoated with type I rat tail collagen in complete EGM-2
medium for passage.
Growth Kinetics and Estimate of Replicative Capacity of EPCs.
[0133] At the time of first passage cells were enumerated by a
trypan blue exclusion assay (Sigma, St. Louis, Mo.). Monolayers of
cells were then grown to 90% confluence and passaged. At each
passage, cells were enumerated for calculation of a growth kinetic
curve, population doubling times (PDTs), and cumulative population
doubling levels (CPDLs).
[0134] The number of population doublings (PDs) occurring between
passages was calculated according to the equation: PD=log.sub.2
(C.sub.H/C.sub.S) where C.sub.H is the number of viable cells at
harvest and C.sub.S is the number of cells seeded. The sum of all
previous PDs determined the CPDL at each passage. The PDT was
derived using the time interval between cell seeding and harvest
divided by the number of PDs for that passage. Matrigel.TM.
(extracellular matrix proteins) assays and uptake of acetylated-low
density lipoprotein (Ac-LDL or Dil-Ac-LDL)
[0135] Matrigel.TM. (extracellular matrix proteins) assays were
performed. Briefly, early passage (2-3) HPP-ECFC-derived or
EPC-derived endothelial cells were seeded onto 96 well tissue
culture plates previously coated with 30 .mu.l of Matrigel.TM.
(extracellular matrix proteins) (BD Biosciences) at a cell density
of 5000-20,000 cells per well. Cells were observed every two hours
for capillary-like tube formation.
[0136] To assess the ability of attached HPP-ECFC and progeny or
EPC and progeny to incorporate Ac-LDL or Dil-Ac-LDL), 10 .mu.g/ml
of Ac-LDL (Biomedical Technologies Inc., Stoughton, Mass.) was
added to the media of cells cultured in a 6 well type I rat tail
collagen coated tissue culture plate. Cells were incubated for 30
minutes or 4 hours at 37.degree. C. and then washed three times
with phosphate buffered saline (PBS) stained with 1.5 .mu.g/ml of
DAPI (Sigma)and examined for uptake of Ac-LDL or Dil-Ac-LDL by
using a fluorescent microscope.
Immunophenotyping of Endothelial Cells by Fluorescence
Cytometry
[0137] Early passage (1-2) or (3-4) HPP-ECFC and progeny or EPC and
progeny (5.times.10.sup.5) were incubated at 4.degree. C. for 30-60
minutes with varying concentrations of the primary or isotype
control antibody as outlined below in 100 .mu.l of PBS and 2% FBS.
Cells were washed three times with PBS containing 2% FBS and
analyzed by fluorescence activated cell sorting (FACS.COPYRGT.)
(Becton Dickinson, San Diego, Calif.). Directly conjugated primary
murine monoclonal antibodies against human CD31 conjugated to
fluorescein isothiocyanate (FITC) (BD Pharmingen, San Diego,
Calif.) were used at a 1:20 dilution, human CD34 conjugated to
allophycocyanin (APC) (BD Pharmingen) at a 1:25 dilution, human
CD14 conjugated to FITC (BD Pharmingen) at a 1:10 dilution, human
CD45 conjugated to FITC (BD Pharmingen) at a 1:10 dilution, human
CD117 conjugated to APC (BD Pharmingen) at a 1:100 dilution, human
CD146 conjugated to phycoerythrin (PE) (BD Pharmingen) at a 1:10
dilution, human AC133 conjugated to PE (Miltenyi Biotec, Auburn,
Calif.) at a 1:5 dilution, human CD141 conjugated to FITC (Cymbus
Biotechnology, Chandlers Ford, UK) at a 1:10 dilution, human CD105
(BD Pharmingen) conjugated to Alexa Fluor 647 (Alexa Fluor 647
monoclonal antibody labeling kit, Molecular Probes, Eugene, Oreg.)
at a 1:100 dilution, and human CD144 conjugated to Alexa Fluor 647
at a 1:100 dilution.
[0138] To test for cell surface expression of vascular cell
adhesion molecule (VCAM-1) after activation by a cell agonist,
serum starved endothelial cells were stimulated with either 10
ng/ml of recombinant human interleukin one (IL-1) (Peprotech, Rocky
Hill, N.J.) or 10 ng/ml of recombinant human tumor necrosis
factor-alpha (TNF-.alpha.) (Peprotech) for 4 hours at 37.degree. C.
Following stimulation, cell surface expression of VCAM-1 was tested
utilizing a primary antibody against human VCAM-1 conjugated to
FITC (BD Pharmingen) at a 1:20 dilution. For all isotype controls
for immunopherotyping and UCAM-1 expression, the following
antibodies were used: mouse IgG.sub.2a, .kappa., conjugated to FITC
(BD Pharmingen), mouse IgG.sub.1, .kappa. conjugated to FITC (BD
Pharmingen), mouse IgG.sub.1, .kappa. conjugated to PE (BD
Pharmingen), and mouse IgG.sub.1, .kappa. conjugated to APC (BD
Pharmingen).
[0139] For detection of cell surface expression of von Willebrand
factor (vWF) and flk-1, cells were fixed in acetone for 10 minutes
at room temperature, washed two times with PBS, and blocked and
permeabilized for 30 minutes with PBS, 3% nonfat dry milk, and 0.1%
Triton X-100 (Sigma). We used 2 .mu.g/ml of a primary antibody
directed against human vWF (Dako, Carpenteria, Calif.) and a
biotinylated primary antibody directed against human flk-1 (Sigma)
at a 1:20 dilution. The secondary antibody used for vWF was a goat
anti-rabbit antibody conjugated to FITC (BD Pharmingen) at a 1:100
dilution and the secondary antibody used for flk-1 was streptavidin
conjugated to APC (BD Pharmingen) at a 1:100 dilution. For the
isotype control for vWF, we used rabbit Ig primary antibody (Dako)
at a 1:100 dilution with anti-rabbit Ig secondary antibody
conjugated to FITC (BD Pharmingen) at a 1:100 dilution. For the
isotype control for flk-1, we used a biotinylated mouse IgG.sub.1,
.kappa. (BD Pharmingen) primary antibody at a 1:100 dilution with a
streptavidin APC secondary antibody (BD Pharmingen) at a 1:100
dilution.
Telomerase Activity Assay
[0140] For detection of telomerase activity, the telomeric repeat
amplification protocol (TRAP) was employed in the form of a
TRAP-eze telomerase detection kit (Oncor, Gaithersburg, Md.).
Briefly, 1000 cultured HPP-ECFC or EPC colonies were absorbed onto
filter papers and lysed in TRAP assay buffer. The lysed material
was subjected to PCR amplification and the PCR products (6-bp
incremental ladder) were electrophoresed on a non-denaturing
polyacrylamide gel and visualized by DNA staining or radiolabeled
with .sup.32P. PCR products were loaded as neat or 1/10 or 1/100
dilutions and the level of intensity of staining compared to the
HELA cell line (1000 cells) positive control.
Thymidine Incorporation Assays
[0141] Endothelial colony-derived endothelial cells were deprived
of growth factors and cultured in EBM-2 media supplemented with 5%
FBS for 24 hours. Next, 3.times.10.sup.4 cells were plated in each
well of 6-well tissue culture dishes pre-coated with type I
collagen and cultured for 16 hours in EBM-2 media supplemented with
1% FBS. Cells were then cultured in EBM-2 without serum for an
additional eight hours to ensure quiescence. Cells were stimulated
in EBM-2 media supplemented with 10% FBS with 25 ng/ml of
recombinant human vascular endothelial growth factor (rhVEGF)
(Peprotech), 25 ng/ml of recombinant human basic fibroblast growth
factor (rhbFGF) (Peprotech) or no growth factors, as indicated, in
a 37.degree. C., 5% CO.sub.2, humidified incubator. Some cells were
cultured in EBM-2 media without growth factors or FBS. Cells were
cultured for 16 hours, and 1 .mu.Ci of tritiated thymidine (Perkin
Elmer Life Sciences Products, Boston, Mass.) was added 5 hours
prior to the harvest. Cells were lysed with 0.1 N sodium hydroxide
for one hour. Lysates were collected into 5 ml of liquid scintilant
(Fisher Scientific, St. Louis, Mo.) and .beta. emission was
measured. Assays were performed in triplicate.
Generation of GALV-Pseudotyped MFG-EGFP
[0142] The MFG-EGFP retrovirus vector expresses the enhanced green
fluorescent protein (EGFP) under the control of the Moloney murine
leukemia virus long terminal repeat (LTR) and has been previously
described by Pollok et al. (2001). For generation of the
GALV-pseudotyped vector, supernatant from an amphotrophic MFG-EGFP
clone was used to infect the PG13 packaging line (American Type
Culture Collection (ATCC), Manassas, Va.), and infected cells were
isolated by single cell cloning. Individual clones were screened
for titer by infecting 5.times.10.sup.5 human erythroleukemic cells
(HEL) (ATCC) and determining the percent EGFP expression 48 hours
after end-point dilution of supernatant. MFG-EGFP clone 5 has a
titer of 0.5-1.times.10.sup.6 infectious units/ml and was used for
experiments.
Retroviral Transduction of Endothelial Cells
[0143] Early passage (1-2) endothelial colony-derived endothelial
cells were transduced with equivalent starting titers of MGF-EGFP
supernatant. Six well non-tissue culture plates were coated with 5
.mu.g/cm.sup.2 fibronectin CH-296 (Takara Shuzo, Otsu, Japan) for 2
hours at room temperature or overnight at 4.degree. C. Plates were
washed one time with PBS, and endothelial cells were plated at
5.times.10.sup.4 cells/cm.sup.2 for transduction. Cells were
infected with retrovirus supernatant diluted 1:1 with complete
EGM-2 for 4 hours on 2 consecutive days with a change of complete
EGM-2 media for overnight incubation. After the second round of
infection, cells were harvested, counted and analyzed for EGFP
expression by fluorescence cytometry.
Single Cell Assays
[0144] Early passage (1-4) endothelial colony-derived endothelial
cells, transduced with the MFG-EGFP retrovirus, were sorted by
fluorescence cytometry for EGFP expression. A FACS Vantage Sorter
(Becton Dickenson) was used (sort nozzle .gtoreq.100 microns at a
sheath pressure of .ltoreq.9 pounds per square inch) to place one
single endothelial cell expressing EGFP into each well of a 96 well
flat bottom tissue culture plate pre-coated with type I collagen
containing 200 .mu.l of complete EGM-2 media. Individual wells were
examined under a fluorescence microscope at 50.times. magnification
to ensure that only one cell had been placed into each well. Cells
were cultured at 37.degree. C., 5% CO.sub.2 in a humidified
incubator. Media was changed every four days by removing 100 .mu.l
and replacing it with 100 .mu.l of fresh complete EGM-2 media. At
day 14, each well was examined for the growth of endothelial cells
from the single plated cell. To quantitate the frequency of
dividing single endothelial cells, the number of wells, which had 2
or more endothelial cells with a fluorescent microscope at
100.times. magnification were counted. To enumerate the number of
cells per well, the cells were counted by visual inspection with a
fluorescent microscope at 100.times. magnification (less than 50
cells per well), or the cells were trypsinized and counted them
with a hemacytometer utilizing a trypan blue exclusion assay (more
than 50 cells per well).
[0145] The long term proliferative and replating potential of
endothelial cells derived from a single cell was determined. At day
14 after initiation of culture, individual wells containing greater
than 50 cells were trypsinized, collected in 500 .mu.l of complete
EGM-2 media and subcultured to a 24 well tissue culture dish coated
with type I collagen. Four days after subculturing the cells, the
media was aspirated and replaced with 500 .mu.l of fresh complete
EGM-2 media. On day 7, wells were examined for colony growth or
cell confluence by visual inspection with a fluorescent microscope
at 50.times. magnification. Cells were then trypsinized, counted,
and subcultured in a 6 well tissue culture plate precoated with
type I collagen. Following 7 days of culture in a six well plate,
10-12 wells, which contained confluent cell monolayers, for
long-term cultures were selected under the conditions disclosed
herein. For each sample, PDT and CPDL were calculated.
[0146] CD Markers: CD14 (lipopolysaccharide receptor), CD31
(platelet endothelial cell adhesion molecule), CD34 (sialomucin),
CD45 (common leukocyte antigen), CD105 (endoglin), CD117 (c-Kit
receptor), CD133 (prominin 1), CD141 (thrombomodulin), CD144
(vascular endothelial cadherin), CD146 (endothelial associated
antigen, S-endo-1), flk-1 (fetal liver kinase-1, receptor for
vascular endothelial growth factor 2).
Confocal Imaging of EPC
[0147] Passage 3-5 EPC were grown in a T 75 flask for four days
using EBM-2 media with 10% added FBS. When cells reached
confluence, media was aspirated, 5 ml of sterile PBS was added to
the flask, and then aspirated, trypsin--EDTA was added and the
flask was incubated for 5 min at 37.degree. C. To quench the
trypsin, 5 mL of EBM-2 media with 10% FBS was added and the
released EPC were centrifuged at 350.times.G for 10 min. The
pelleted cells were washed with PBS and then resuspended in EBM-2
media with 10% FBS.
[0148] Glass chamber slides (4 chamber configuration; Corning) were
coated with extracellular matrix proteins (e.g. collagen type 1 or
4, fibronectin, or vitronectin) over night at 4.degree. C. and then
washed with sterile PBS in the morning. The PBS was aspirated and
cells in EBM-2 media with 10% FBS were added at 50 cells per
chamber and incubated at 37.degree. C. in 5% CO.sub.2 for 7
days.
[0149] EPC containing slides were washed twice with PBS and cells
were fixed in acetone for 10 min at room temperature, washed twice
with PBS, and blocked and permeabilized for 30 minutes with PBS, 3%
nonfat dry milk, and 0.1% Triton X100. To highlight the plasma
membrane of the cells, a primary antibody to CD146 conjugated to
phycoerythrin (PE) was added (1 .mu.g/mL) to the fixed cells along
with 1.5 mg/mL DAPI for nuclear staining. After a 30 minute
incubation, cells were washed twice in PBS and examined for
fluorescence using a Zeiss 510 confocal microscope. An ultraviolet
laser (351/364 nm excitation) and a helium-neon laser (543 nm
excitation) were used to excite the DAPI and PE-labeled cells
through a 40.times. water objective with the zoom kept on
0.7.times. magnification. Images were captured in a single plane
and displayed as monochromatic images for presentation. NIH Image
software was used to quantify the nuclear and cytoplasmic diameters
of cells from various EPC colony types. TABLE-US-00001 TABLE 1
Enumeration of the number and time of appearance of endothelial
progenitor cell colonies isolated from adult peripheral and
umbilical cord blood mononuclear cells. Adult Peripheral Blood
Umbilical Cord Blood Number of Day of Number of Day of Colonies/
First Colonies/ First Donor 20 ml Blood Colony Donor 20 ml Blood
Colony 1 0.50 15 1 8.18 7 2 0.83 17 2 5.45 6 3 0.35 13 3 13.97 7 4
0.83 21 4 4.92 7 5 0.58 13 5 9.79 7 6 0.17 22 6 1.14 8 7 0.00 -- 7
4.09 10 8 0.60 14 8 9.55 6 9 1.00 18 9 11.33 6 10 0.40 17 10 0.67 6
11 0.60 14 11 16.00 5 12 0.60 17 12 6.00 7 13 0.00 -- 13 16.00 6 14
0.33 12 15 1.33 11 16 0.67 13 17 0.00 -- 18 1.00 16
Co-Culture of HPP-ECFC and CD34+ Cells Expands NOD-SCID
Repopulating Cells.
[0150] Human CD34+ bone marrow cells, which have previously been
shown to harbor marrow repopulating cells in NOD/SCID mice (SRCs)
were isolated. Typically 0.5-1.0.times.10.sup.6 human marrow CD34+
cells are injected into NOD/SCID mice in order to achieve a level
of human CD45+ chimerism of 5-50%. Initially only 9.times.10.sup.3
CD34+ cells were injected into NOD-SCID mice as a control on the
day of harvest from human bone marrow. 9.times.10.sup.3 CD34+ cells
were cultured in the presence of SCF, G-CSF, TPO, and Flt-3 for
seven days. These are the growth factors currently used to
maximally expand HSCs ex vivo. 9.times.10.sup.3 CD34+ cells were
co-cultured with monolayers of cord blood HPP-ECFC derived progeny
in the absence of growth factors for seven days. Following seven
days of culture, the cultured CD34+ cells were injected into
NOD-SCID mice and the peripheral blood of transplanted mice was
tested for the presence of human cells four weeks after
transplantation. Co-culture of CD34+ cells with growth factors for
7 days increased the percentage of human cells detected in NOD-SCID
mice 8 weeks after transplantation 10 fold compared to CD34+ cells
injected shortly after isolation from human bone marrow. Despite
injecting a very limited number of cells compared to prior studies,
co-culture of CD34+ cells with cord blood HPP-ECFC-derived cells
increased the percentage of human cells detected in NOD-SCID mice 8
weeks after transplantation 260 fold. Both human myeloid and
lymphoid lineages were detected eight weeks after transplantation
indicating that multilineage reconstitution of the hematopoietic
system was achieved with CD34+cells co-cultured with cord blood
HPP-ECFC.
[0151] Starting with 2 T75 flasks of confluent monolayers of
HPP-ECFC-derived cells, cells were first washed with Hanks balanced
salt solution without calcium or magnesium (HBBS), then 1.5 mL of
Trypsin EDTA (Gibco) was added to each flask for 1 minute. Next 8.5
mL of endothelial basal medium 2 (EBM2) (Cambrex) with 10% fetal
bovine serum (FBS) (Hyclone), was added and suspended cells were
collected and counted via Trypan blue exclusion on a
hemacytometer.
[0152] HPP-ECFC- derived cells were plated at 3.times.10.sup.5
cells/well onto collagen 1 precoated 6 well tissue culture plates
(BD Biosciences). Cells were cultured with endothelial growth
medium 2 (EGM2) (Cambrex) supplemented with 10% FBS and cultured
overnight. The following morning the confluent cell monolayers were
washed with EBM2+10% FBS twice and then co-cultured with 9,000
CD34.sup.+ CD38.sup.dim Lin.sup.- (CD4, 8, 11b, 14, 24, 31, 33, and
glycophorin A) adult human bone marrow-derived cells collected by
fluorescence activated cell sorting resuspended in 4 mL of EBM2+10%
FBS+human megakaryocyte growth derived factor (MGDF) (100 ng/mL),
granulocyte colony stimulating factor (G-CSF) (100 ng/mL), and stem
cell factor (SCF) (100 ng/mL), and flt-3 ligand (100 ng/mL). Cells
were cultured in 37.degree. C. 5% CO2 humidified incubator for 7
days without disturbance. In some cultures the CD34.sup.+ cells
were co-cultured with the HPP-ECFC in EBM2+10% FBS and no added
growth factors.
[0153] A 5 mL pipette was used to aspirate the nonadherent cells
and media (4 mL) at the end of the 7 day co-culture. Wells were
washed once with 2 mL phosphate buffered saline (PBS) and the PBS
with nonadherent cells added to the original aspirate. To the same
wells, 1 mL of cell dissociation buffer (Gibco) was added for 4
minutes at room temperature, and then the cell dissociation buffer
and loosened cells were titrated in the well gently before
aspiration and adding to the original aspirate. Finally, the
HPP-ECFC monolayers were washed one final time with 2 mL of PBS and
this solution with scant cells was added to the original aspirate.
The final volume of media and cells was 9 mL.
[0154] The cell suspension was centrifuged at 1500 rpm
(514.times.g) at room temperature for 10 minutes. The solution was
removed and the cell pellet was dislodged mechanically then
resuspended in 1/2-1 mL of EBM2+10% FBS. Cells were counted in
Trypan blue on a hemacytometer. Recovered cells were plated in
progenitor assays or injected intravenously into NOD/SCID mice.
[0155] The method outlined above may be modified to provide a graft
for a human transplant. In this instance, the HPP-ECFC progeny is
plated in T75 flasks or in a perfusion chamber system to permit
large numbers of CD34+ hematopoietic stem cells (autologous or
allergenic human cord blood-, mobilized peripheral blood-, or
marrow-derived) to be expanded in the presence of the cord blood
HPP-ECFC. Systems are used that will permit the donor CD34+ cells
to be cultured with the HPP-ECFC progeny without the cells directly
touching and, thus, the donor CD34+ cells can be expanded,
recovered, and transplanted into the human patient without the
donor cells being "contaminated" with the cord blood HPP-ECFC
progeny.
Cellularized Gel for In Vivo Implants
[0156] The gel mixture is made following standard protocols. Cells
are apportioned to tubes. The gel mixture is added to cells. The
cellularized gel mixture is apportioned to 12 well plates to warm
the mixture (e.g., 37 C). The cell culture media is added to
solidified gel for overnight culture. The cellularized gel is
transferred to a desired site.
[0157] The gel mixture is kept cold to prevent gelation.
Volume Calculations
[0158] Since the gel material is highly viscous, the exact volume
intended is not recovered (some material always sticks to the
tubes), so if 4 gel implants are needed, a total volume of 2 ml gel
is used. This is added to wells of a 12 well plate. For
implantation, 1/2 of the well contents are used for a single
implant. If 2 ml of gel (final) are needed, then 1.2 times the
final amount is prepared to enable accurately retrieval of a
viscous mixture from the tent tube (thus for 2 ml.times.1.2=2.4 mL.
Additional gel material can also be prepared because some times
bubbles hinder recovery of gel material from the tubes.
Calculation to Add All Reagents Necessary for Making 2 mL
Cellularized Gel (for 4 Implants In Vivo).
[0159] EBM-2 (Cambrex, no supplements) w/10% serum and 1%
antibiotics are needed. HEPES at a concentration of 1M (Cambrex
17-737E) is obtained. Sodium bicarbonate at 1.5 mg/ml (Sigma) is
obtained. Rat Tail type 1 collagen at 3.88 mg/ml (Becton Dickinson)
is obtained. Fibronectin (100 .mu.g/ml; Chemicon, plasma
fibronectin, FCO10-10 mg) is obtained. Fetal bovine serum (Hyclone
SH30070.03) is obtained.
[0160] The pH is adjusted to 7.4 with IN NaOH at 10 .mu./ml
collagen gel mixture. EBM-2-0.302 mL; FBS-0.440 mL; HEPES 0.110 mL;
NaHCO.sub.3 0.088 mL; Collagen 1.700 mL; and fibronectin 0.440 mL
are mixed. The total volume is 3.08 mL of gel constituents. All the
constituents are kept on ice to prevent unwanted gelation.
[0161] The cells are divided and transferred to tubes. Generally
between 1-2 million cells/mL gel are used. 1.2 times the final cell
number needed is resuspended in (example if 1 million/mL wanted add
1.2 million) in 330 .mu.l GM-2.
[0162] Cell volume occupies about 0.030 mL so the total cells in
EGM-2 equals 0.0360 mL. The gel mixture (as generated above) is
added to cells in the tubes. 840 .mu.l of the gel mix is added per
tube, with 0.036 mL of cells in EGM-2 (volume=7.2 mL). The
cellularized gel mixture is transferred to a plate to warm in an
incubator.
[0163] 1 mL of cellularized gel mixture is recovered from the 1.2
mL in the tube and is added to one well of a 12-well plate. The
cellularized gel is placed in 37 degree C. incubator for about
15-30 minutes to harden. The gel becomes opaque and white in
appearance. The hardened gel is not clear. Growth media is added to
solidified gel. 1 mL of EGM-2 is added per gel, by gently
overlaying media onto gel. This assures adequate hydration during
the overnight incubation.
[0164] Implantation
[0165] In a laminar flow hood, 7-12 week old NOD/SCID mice are
anesthetized with 1/5% lsoflurane, and Nair is applied spot-wise
bilaterally to the abdomen for--2 minutes. Nair is wiped away
w/gauze, the skin cleaned with sterile PBS, then the sites of
incision are saturated with povidine solution and allowed to dry.
The skin is pinched away from the abdominal muscles and through a
small incision, a pouch is formed by dissecting the skin and
subcutaneous tissues from the muscle layer (procedure is repeated
on other side of animal). One-half of each gel is then inserted
into each pouch. The gel is positioned without tearing or
mutilating the gel, and the skin is closed with suture. Mice are
monitored daily for infection, change in diet, weight, and the
like. Harvesting occurs 21-28 days post-implantation.
* * * * *