Note: Descriptions are shown in the official language in which they were submitted.
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STEM CELLS BEARING AN FGF RECEPTOR
ON THE CELL SURFACE
pCf/US99/19730
FIELD OF INVENTION
The present invention is directed to a new
phenotype of stem cells which contain a fibroblast growth
factor receptor (FGFR) on the cell surface thereof and
further have a phenotype indicative of a primitive state.
The present invention is further directed to subpopulations
l0 thereof having a phenotype indicative of endothelial or
stromal cells.
BACKGROUND OF THE INVENTION
The ability of tissues and organs to develop,
remodel, regenerate, and repair depends on the existence of
stem cells (also known as progenitor cells) that, upon
division, form more differentiated progeny. Stem cells have
been found in the epidermis, the intestinal epithelium, and
the hematopoietic system. There is mostly indirect evidence
of stem cells in mesenchymal tissues. In vivo and in vitro
2o studies have provided evidence of osteogenic precursor cells
in bone marrow and other stromal cell preparations. However,
the identity of cells in these tissues and their relationship
to cells with classical stem cell characteristics have yet to
be established.
Endothelial cells are part of the normal bone
marrow stroma. Long-term cultures of human bone marrow
contain a complex mix of stromal cells including adipocytes,
.fibroblasts, endothelial cells, macrophages, and smooth
muscle cells. Endothelial cells and hematopoietic cells are
3o thought to be derived from the common progenitor cells,
hemangioblasts.
Cell surface molecules on various types of cells,
and particularly hematopoietic cells, are given a cluster of
differentiation (CD) designation in which each CD molecule
designation describes a surface molecule (marker)
identifiable by a cluster of monoclonal antibodies that
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display the same cellular reactivity. CD designations are
assigned at regularly held international workshops on human
leukocyte differentiation antigens. For example, the CD19
marker is specific to B cells, and the CD33 marker is
specific to myeloid cells. At the present time, it is not
known how many of the markers associated with differentiated
cells are also present on stem cells. One marker which has
been indicated as being present on stem cells is CD34.
However, this marker is also found on a significant. number of
lineage-committed progenitors. Other markers which are known
or thought to be present primarily on stem cells, i.e.,
"primitive" markers, include AC133 (Yin et al, 1997; Buhring
et al, 1999), Thy-1 (hurray et al, 1995) and c-kit (D'Arena
et al, 1998).
It is known that a small number of circulating
CD34+ hematopoietic stem cells are present in peripheral
blood. As the major source of CD34' hematopoietic stem cells
in the adult is the bone marrow, the purpose of this small,
circulating CD34+ cell population is unknown. One explanation
2o is that the bone marrow is "leaky", and the stem cells
escape, circulate and return to the marrow. A second
possibility is that the function of these circulating stem
cells is to seed sites, such as the liver and the spleen,
which can function as additional sites of hematopoiesis in a
crisis.
The human CD34+ hematopoietic population isolated
from bone marrow, cord blood, and peripheral blood is a
heterogeneous population that contains hematopoietic stem
cells. Recent evidence indicates that circulating CD34+ cells
3o also contain endothelial stem cells, which may also circulate
(Asahara et al, 1997; Nieda et al, 1997; Shi et al, 1998; Lin
et al, 1998). Asahara et al (1997) have shown that CD34+
cells isolated from the peripheral blood can be incorporated
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into the endothelium of ischaemic blood vessels of recipient
animals. Purified umbilical cord blood CD34' cells also give
rise to von Willebrand factor-expressing endothelial cells in
vitro, providing additional evidence for a circulating
progenitor endothelial cell (Nieda et al, 1997). In
addition, bone marrow derived CD34+ cells also contain a
transplantable stromal stem cell (Prockop, 1997; Pereira et
al, 1998).
Recently, convincing evidence has been presented
(Goan et al, 1997) that human CD34' progenitor cells from
peripheral blood or cord blood that were transplanted into
NOD/SCID immunodeficient mice gave rise to human stromal
cells. The human stromal cells expressed the endothelial
cell-specific vascular endothelial growth factor (VEGF)
receptor-2 (KDR) and von Willebrand factor, indicating that
they were of endothelial origin. There is also recent
evidence that infusion of whole bone marrow cells into
recipient mice results in fibroblasts of donor origin in a
number of non-hematopoietic tissues (Prockop, 1997; Pereira,
1998), indicating that stromal progenitor cells reside in the
bone marrow. As CD34 has been shown to be expressed by bone
marrow stromal precursor cells (Simmons et al, 1991), it is
possible that these stromal progenitors reside in the bone
marrow within the CD34+ progenitor population.
The CD34+ progenitor population is, therefore, a
heterogeneous fraction that may include precursor cells of
the hematopoietic, endothelial, and stromal/fibroblast
lineages. In addition, pluripotent mesenchymal stem cells
capable of differentiating into cells of the osteogenic,
chondrogenic, tendonogenic, adipogenic and myogenic lineages
have been shown to reside within the bone marrow
microenvironment (Majumdar et al, 1998). There is recent
literature indicating that circulating endothelial
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progenitor/stem cells exist, and that stromal stem cells in
marrow serve as a source for continual renewal of cells in a
number of non-hematopoietic tissues. A common embryological
precursor that gives rise to both hematopoietic and
endothelial cells has recently been identified (Suda et al,
1997; Choi et al, 1998; Caprioli et al, 1998).
Recent evidence has also shown that embryonic stem
(ES) cells can give rise to endothelial cells (Hirashima et
al, 1999).
1o Fibroblast growth factors (FGFs) can synergize with
other factors to stimulate hematopoietic progenitor cell
proliferation (Wilson et al, 1991; Quito et al, 1996;
Allouche, 1995; Yuen et al, 1998, U.S. Patent 5,612,211; U.S.
Patent 5,817,773). It has also been shown that basic FGF
(FGF-2) acts to antagonize cytokines that induce
differentiation (Burger et al, 1994). In addition, low
amounts of FGF-2, on the order of 10-100 pg/ml, induce a more
primitive phenotype in human K562 leukemic cells.
It would be very useful to be able to isolate stem
2o cells which are progenitors of endothelial and/or stromal
cells. The more primitive the stem cell, the more useful it
is in bone marrow transplantation. Furthermore, endothelial
stem cells and stromal stem cells, or a stem cell which is a
progenitor of both, would find many utilities in repairing
damaged vasculature and in treating other conditions where
endothelial or stromal cells need to be replenished.
SUi~IARY OF THE INVENTION
It is an object of the present invention to
overcome the deficiencies in the prior art.
It is another object of the present invention to
isolate a new phenotype of stem cells.
It is a further object of the present invention to
identify and isolate stem cells for use in stem cell
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transplantation.
It is yet another object of the present invention
to identify and isolate endothelial and/or stromal stem
cells.
A small population of cells having a "primitive
phenotype", such as CD34+ or CD34-lin-, has been isolated
which express cell surface receptors for fibroblast growth
factor (FGF). The population of cells bearing FGF receptors
(FGFR) are designated as FGFR+. The FGFR+ primitive phenotype
l0 cell population has several unique properties:
(1) The CD34'FGFR' cells are predominantly present
in the region of the fluorescence-activated cell sorter
profile having low forward scatter (FSC) and low side scatter
(SSC). Thus, the majority of the cells of the population are
very small and of low granularity. These small cells are
located in the FSC/SSC region of the fluorescence-activated
cell sorter profile that is normally not analyzed, as this
area contains many of the dead and apoptotic cells.
Interestingly, this region has recently been shown to be the
2o site of a mesenchymal stem cell population (Zohar et al,
1997). The CD34-lin-FGFR+ cells have FSC/SSC properties that
are similar to those of the CD34+FGFR' cells. This CD34-lin-
FGFR+ population also includes significant numbers of cells
with higher FSC properties.
(2) The CD34'FGFR+ cells are deeply dormant, which
is characteristic of a stem cell population. They do not
proliferate in culture until 30-60 days after isolation.
The FGFR+ primitive phenotype cell population is a
unique stem cell population that is a precursor cell for
3o endothelium and/or hematopoiesis and/or stroma. The FGFR+
primitive phenotype cell population, obtained either from
general circulation, the bone marrow, cord blood or embryonic
cells, is capable of forming endothelial, blood and stromal
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cells, depending on the need at the time.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 is a bar graph summarizing the fourteen
experiments of Example 1 and showing the percent of CD34+FGFR+
cells which express the indicated third antigens.
Figures 2A-2F are flow cytometry plots for
Experiment 7 of Example 1. Figure 2A is a dot plot of FSC
versus SSC of all events with region R1 drawn to eliminate
the area containing most of the cell debris and doublets.
1o Figure 2B is a histogram showing the intensity of staining
with the dye 7-aminoactinomycin D (7-AAD). The region R2 is
drawn to delineate live cells. Figure 2C is a dot plot of
SSC versus CD34 gated on R1 AND R2. The region R3 is drawn to
delineate CD34+ cells. Figure 2D is a dot plot of FSC versus
SSC gated on R1 AND R2 AND R3, thus showing the FSC/SSC
characteristics of live CD34' cells in R1. Figure 2E is a dot
plot of CD34 versus FGFR gated on R1 AND R2. The region R4
is drawn to delineate CD34+FGFR+. Figure 2F is a dot plot of
FSC versus SSC gated on R1 AND R2 AND R4, thereby showing the
characteristics of live CD34+FGFR+ cells in R1.
Figure 3 is a bar graph showing the percent of live
FGFR+ cells co-expressing the indicated antigens.
Figure 4 is a bar graph showing the growth of
various cell populations in the presence or absence of FGF-2
or a combination of FGF-2 plus VEGF.
DETAILED DESCRIPTION OF TI3E INVENTION
Stem cells, when transplanted, can restore the
production of hematopoietic, endothelial and stromal cells to
a patient who has lost such production due to, for example,
radiation therapy. By isolating FGFR+ primitive phenotype
cells, preferably CD34'FGFR+ or CD34-lin-FGFR~, from other
cells in the body, it is possible to obtain relatively pure
stem cells, preferably separate from contaminating cells and
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other substances, so that the stem cells can be safely
transplanted into a patient in need thereof.
The unique isolated cells of the present invention
are separated from other cells by virtue of their CD34+ or
CD34-lin- state and possession of fibroblast growth factor
receptors. The cells can be isolated by conventional
techniques for separating cells, such as those described in
Civin, U.S. Patents 4,714,680, 4,965,204, 5,035,994, and
5,130,144, Tsukamoto et al 5,750,397, and Loken et al, U.S.
l0 Patent 5,137,809, all of which are hereby incorporated by
reference in their entirety. Thus, for example, a CD34-
specific monoclonal antibody or an FGFR-specific antibody can
be immobilized, such as on a column or on magnetic beads.
The entire cell population may then be passed through the
column or added to the magnetic beads. Those which remain
attached to the column or are attached to the magnetic beads,
which may then be separated magnetically, are those cells
which contain a marker which is recognized by the antibody
used. Thus, if the anti-CD34 antibody is used, then the
2o resulting population will be greatly enriched in CD34+ cells.
If the antibody used is FGFR, then the resulting population
will be greatly enriched in FGFR+ cells. That population may
then be enriched in the other marker by repeating the steps
using a solid phase having attached thereto an antibody to
the other marker.
Another way to sort CD34iFGFR+ cells is by means of
flow cytometry, most preferably by means of a fluorescence-
activated cell sorter (FACS~, such as those manufactured by
Becton-Dickinson under the names FACScan or FACSCalibur. By
means of this technique, the cells having a CD34 marker
thereon are tagged with a particular fluorescent dye by means
of an anti-CD34 antibody which has been conjugated to such a
dye. Similarly, the FGFR marker of the cells are tagged with
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a different fluorescent dye by means of an anti-FGFR antibody
which is conjugated to the other dye. When the stained cells
are placed on the instrument, a stream of cells is directed
through an argon laser beam that excites the fluorochrome to
emit light. This emitted light is detected by a photo-
multiplier tube (PMT) specific for the emission wavelength of
the fluorochome by virtue of a set of optical filters. The
signal detected by the PMT is amplified in its own channel
and displayed by a computer in a variety of different
l0 forms-e. g., a histogram, dot display, or contour display.
Thus, fluorescent cells which emit at one wavelength, express
a molecule that is reactive with the specific fluorochrome-
labeled reagent, whereas non-fluorescent cells or fluorescent
cells which emit at a different wavelength do not express
this molecule but may express the molecule which is reactive
with the fluorochrome-labeled reagent which fluoresces at the
other wavelength. The flow cytometer is also semi-
quantitative in that it displays the amount of fluorescence
(fluorescence intensity) expressed by the cell. This
correlates, in a relative sense, to the number of the
molecules expressed by the cell.
Flow cytometers are also equipped to measure non-
fluorescent parameters, such as cell volume or light
scattered by the cell as it passes through the laser beam.
Cell volume is usually a direct measurement. The light
scatter PMTS detect light scattered by the cell either in a
forward angle (forward scatter: FSC) or at a right angle
(side scatter; SSC). FSC is usually an index of size,
whereas SSC is an index of cellular complexity, although both
parameters can be influenced by other factors.
Preferably, the flow cytometer is equipped with
more than one PMT emission detector. The additional PMTs may
detect other emission wavelengths, allowing simultaneous
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detection of more than one fluorochrome, each in individual
separate channels. Computers allow the analysis of each
channel or the correlation of each parameter with another.
Fluorochromes which are typically used with FRCS machines
include fluorescein isothiocyanate (FITC), which has an
emission peak at 525 nm (green), R-phycoerythrin (PE) , which
has an emission peak at 575 nm (orange-red), propidium iodide
(PI), which has an emission peak at 620 nm (red), 7-
aminoactinomycin D (7-AAD), which has an emission peak at 660
nm (red), R-phycoerythrin Cy5 (RPE-Cy5), which has an
emission peak at 670 nm (red), and allophycocyanin (APC),
which has an emission peak at 655-750 nm (deep red).
These and other types of FRCS machines may have
the additional capability to physically separate the various
fractions by deflecting the cells of different properties
into different containers.
Any other method for isolating the CD34+FGFR;
population of a starting material, such as bone marrow,
peripheral blood or cord blood, may also be used in
2o accordance with the present invention. The various
subpopulations of the present invention may be isolated in
similar manners.
The isolated cell population of this invention can
be used in therapeutic methods, such as stem cell
transplantation, as well as other therapeutic methods as
described below, as well as others that are readily apparent
to those skilled in the art. For example, the isolated cell
populations can be administered directly by intravenous route
to a mammalian patient requiring a bone marrow transplant in
an amount sufficient to reconstitute the patient's
hematopoietic and immune system. Precise, effective
quantities can be readily determined by those skilled in the
art and will depend, of course, upon the exact condition
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being treated by the therapy. In many applications, however,
an amount containing approximately the same number of stem
cells found in one-half to one liter of aspirated marrow
should be adequate.
Thus, a suspension of human cells from marrow or
blood comprising cells which are positive both for CD34 and
for fibroblast growth factor receptors, preferably
substantially free of cells that are not positive for both
CD34 and fibroblast growth factor receptors, can restore the
production of hematopoietic cells to a human lacking
production of these cells. A suspension of these isolated
cells is administered to a patient in need thereof in an
effective amount to restore production of hematopoietic/
endothelial/stromal cells.
The patients in need of this product are those with
a specific requirement for hematopoietic, endothelial or
stromal cells. For example, patients with vascular injury,
persons with genetic defects in their hematopoietic, stromal
or endothelial cells, such as collagen deficiency, adenosine
deaminase deficiency, or clotting factor deficiency. It is
expected that the circulating stem cells will selectively
home to sites of hematopoietic, endothelial or stromal cell
damage/deficiency.
Other "primitive" phenotype indicators besides
CD34+ are also known. These include AC133+ (Buhring et al,
1994; Yin et al, 1997), Thy-1 (hurray et al, 1995) and c-kit+
(Buhring et al, 1994; D'Arena et al, 1998). Another
population of primitive cells is CD34'lin-. The present
inventors have discovered that the CD34+FGFR' population of
the preferred embodiment of the present invention contains
significant amounts of the AC133 marker (approximately 64~),
the Thy-1 marker (approximately 52~) or the c-kit marker
(approximately 57~). Many of these cells have more than one
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primitive marker.
Furthermore, certain markers are known to be
endothelial markers. These include VE-Cadherin (also known
as CD144) (Vittet et al, 1996), TIE-1 (also known as TIE)
(Suda et al, 1997), TEK (also known as TIE-2) (Suda et al,
1997; Hamaguchi et al, 1999) and CD31 (also known as PECAM)
(Watt et al, 1993). Significant quantities of each of these
markers were also found on the CD34+FGFR+ population. About
865 of this population co-expresses VE-Cadherin, about 70~
1o co-expresses CD31; about 47~ co-expresses TEK; and about 33~
co-expresses TIE-1. This data indicates that the CD34~FGFR+
population includes a primitive population of cells which are
precursors of endothelial cells. The subpopulation with
these endothelial markers can be isolated and are also part
of the present invention. Certain additional markers are
known to be stromal cell markers, such as STRO-1 (Gronthos et
al, 1994). A subpopulation of the CD34+FGFR+ cells of the
present invention which is also STRO-1+ is a primitive
population of cells which are precursors of stromal cells.
The subpopulation with these stromal markers can be isolated
and are also part of the present invention.
The results as to co-expression of additional
markers were obtained from fluorescence-activated cell sorter
(FRCS) analysis using specific antibodies to cell surface
antigens. The antibodies were labeled with three or four
different fluorochromes. The results show that 100 of live
FGFR+Thy-1' cells co-express VE-Cadherin, 97~ of live
FGFR+AC133+ cells co-express VE-Cadherin, 91~ of live
FGFR+AC133+ cells co-express Thy-1, and 67~ of live FGFR+TEK+
3o cells co-express Thy-1. Cell sorter systems using additional
fluorochromes will be able to allow the separation of those
cells in the CD34+FGFR+ population which also express two or
more of the various other primitive or endothelial markers
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discussed above. Similarly, other markers, such as the
vascular endothelial growth factor-receptor (VEGF-R) (also
known as KDR), which is a marker for endothelial cells, can
also be included in such analyses. It is predicted that a
subpopulation of CD34+FGFR+Thy-1+VEGF-R' cells exist, which
subpopulation can be identified and isolated by such systems
by one of ordinary skill in the art without undue
experimentation. This subpopulation represents a progenitor
population capable of developing into either or both of the
hematopoietic and endothelial lineages.
The isolated CD34'FGFR+ cells grow exceedingly
slowly in culture with a long lag of 4-6 weeks. The cells
grow in an FGF-dependent manner, as shown in Example 3 and
Table 5. A long dormant period is associated with a stem
cell phenotype, indicating that these cells have growth
characteristics compatible with stem cells.
While the CD39+FGFR' cells can be isolated in
substantial purity, i.e., in a substantially homogeneous
population, by the methods discussed above, such as, for
2o example, by means of the FACS apparatus, it is not always
necessary that the CD34+FGFR+ stem cell population of the
present invention be present in substantial purity. For
example, for most purposes, it is sufficient if the
population of cells contains greater than 90$ of human stem
cells characterized as CD39+ and FGFR+ or FGFR+ with another
indication of primitive phenotype. Other aspects of the
present invention include subpopulations of the FGFR+
primitive phenotype population which are substantially
homogeneous for other markers. This includes the
3o subpopulation of the FGFR' primitive phenotype human stem
cells which are also positive for any one or more of the
endothelial markers, any one or more additional primitive
markers and/or any one or more of stromal cell markers, which
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subpopulation is substantially homogeneous or is a
composition wherein greater than 90~ of said cells are FGFR+
primitive phenotype and. positive for one or more of those
additional markers. Thus, the subpopulation may be a
substantially homogeneous population or a composition in
which greater than 90~ of the cells therein are CD34+FGFR+ (or
CD34'lin'FGFR+) and TIE-1+. The subpopulation may also be a
substantially homogeneous population or a composition in
which greater than 90~ of the cells therein are CD34+FGFR+ (or
CD34'lin'FGFR+) and CD31' and/or TEK+ and/or VEGFR+ and/or VE-
Cadherin' and/or positive for any other endothelial marker.
Similarly, the subpopulation may be a substantially
homogeneous population or a composition in which greater than
90$ of the cells therein are CD39+FGFR+ (or CD39'lin'FGFR+) and
positive for one or more of the other primitive markers, such
as AC133, Thy-1 and c-kit. The subpopulation may also be a
substantially homogeneous population or a composition in
which greater than 90~ of the cells therein are CD34+FGFR+ (or
CD34'lin'FGFR+) and also positive for one or more of the other
primitive markers and further positive for one or more of the
endothelial markers. The subpopulation may also be a
substantially homogeneous population or a composition in
which greater than 90$ of the cells therein are CD34+FGFR' (or
CD34'lin'FGFR~) and also positive for one or more of stromal
markers, such as CD34+FGFR' (or CD34'lin'FGFR') and STRO-1+.
Such subpopulations are also contemplated by the present
invention.
With some of the utilities of the present
invention, such as, for example, bone marrow transplantation,
the stem cell population may be a substantially smaller
percent of the total cell count being administered. The
remaining cells may be filler cells, which may be cells
incapable of replicating. Alternatively, the remaining cells
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may be any of the other types of cells from which the cells
of the present invention are originally separated. Thus, for
example, the present invention also comprehends populations
containing at least 20~ of any of the phenotypes of the
present invention, i . a . , CD34+FGFR+, CD34-lin-FGFR',
CD34+FGFR+Thy-1', CD34+FGFR+TIE-1', CD34+FGFR+CD31',
CD34+FGFR+VEGF-R+, CD34+FGFR+Thy-1+VEGF-R', etc. Such a low
purity subpopulation still defines over the prior art and yet
maintains many of the advantages of the present invention for
to many of its proposed utilities. Compositions having greater
than 30~, 40~, 50~, 60~, 70$ or 80~s of cells of any of the
phenotypes of the present inventions are also considered to
be part of the present invention.
Another way of defining the cellular compositions
of the present invention is as a suspension of human cells,
comprising pluripotent stem cells or endothelial stem cells
which are substantially free of mature lymphoid and myeloid
cells. Cells substantially all of which are of the FGFR+
primitive phenotype are substantially free of mature lymphoid
and myeloid cells.
Although the present invention has thus far been
primarily described with respect to the preferred embodiment
of CD34+FGFR+ cells, it should be understood that FGFR+ cells
having other indications of primitive phenotype are also
contemplated in accordance with the present invention. For
example, CD34- cells which are also negative for lineage
markers (lin-) may be even more primitive than CD34+ cells
(see Zanjani et al, 1999). Thus, the CD34-lin- phenotype is
also considered to be an indication of primitiveness in
3o accordance with the present invention. Additionally, when
embryonic stem cells are used as the source of cells from
which the population of the present invention is to be
separated, all such cells, by definition, have a primitive
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phenotype. Thus, FGFR+ cells separated from an embryonic stem
cell source will inherently be FGFR+ cells with a primitive
phenotype. Furthermore, other primitive markers, such as
AC133, Thy-1 and c-kit, may also be used as markers for
primitiveness. Thus, in its broadest aspect, the present
invention relates to phenotypes which are FGFR', as well as
positive for any phenotype indicating primitive state cells,
including, but not limited to, CD34+, CD34-lin-, being
embryonic stem cells, AC133+,.Thy-1' and c-kit+. The present
1o invention further relates to subpopulations thereof as
described above.
The pluripotent FGFR+ primitive phenotype stem
cells, or pluripotent stem cells of any of the other
phenotypes of the present invention, have considerable
commercial use, including one or more of the following:
(1) For bone marrow transplantation.
(2) To target delivery of anti-tumor agents.
Endothelial stem cells of the present invention can be used
to target the delivery of angiostatic agents and anti-tumor
2o agents to the rapidly proliferating vascular bed associated
with tumors. Endothelial cells are long-lived, and the stem
cells can be used as vectors to deliver angiostatic/antitumor
agents to the rapidly expanding vascular bed associated with
tumors without affecting the stable endothelium of
established blood vessels.
(3) To coat valves and devices used in surgical
procedures. The endothelial stem cells of the present
invention can be used to coat valves and implant devices,
eliminating many of the clotting problems currently
3o associated with these devices. The endothelial stem cells
can be cultured from specific individuals so that valves,
implant devices, etc., may be coated with autologous
endothelial cells. Panels of HLA-matched endothelial stem
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cells can also be produced for these purposes.
(4) As vectors for genetic engineering.
Genetically engineered stem cells "homing" to the
endothelium, bone marrow, or connective tissue stroma are
long-lived and can secrete proteins, such as adenosine
deaminase or clotting factors, as well as other proteins,
such as t-PA, which promote thrombolysis. A wild-type gene
can be incorporated into the endothelial stem cells, either
by homologous or random recombination. With allogeneic
to endothelial stem cells, normal cells lacking the genetic
defect can be used therapeutically. Other indications for
gene therapy are introduction of drug resistance genes to
enable normal stem cells to have an advantage and be subject
to selective pressure, e.g., the multiple drug resistance
gene. Disease other than those associated with endothelial
cells may also be treated, where the disease is related to
the lack of a particular excreted product, such as a hormone,
enzyme, interferon, factor, or the like. By employing an
appropriate regulatory initiation region, inducible
2o production of the deficient protein is achieved so that
production of the protein parallels natural production, even
though production is in a different cell type from the cell
type that normally produces such protein. A ribozyme,
antisense or other message can be inserted to inhibit
particular gene products or susceptibility to disease.
(5) To repair sites of vascular injury.
Engineered endothelial stem cells secreting such factors as
tissue plasminogen activator, designed to help prevent
restenosis after balloon angioplasty, can be infused.
Asahara et al (1997) have demonstrated that endothelial stem
cells selectively home to sites of vascular damage.
(6) To isolate new molecules that are important in
stem and tumor cell biology.
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(7) To form endothelium, stroma and blood cells,
depending on the need at the time.
The stem cells of the present invention can be
expanded in number by long-term in vitro culture with minimal
differentiation until needed. However, the stem cells can
produce blood cells when treated with the appropriate
hematopoietic growth and differentiation factors, or form
tubular network structures characteristic of endothelial
cells upon treatment with the appropriate agents, or form
fibroblast-like stromal cells upon treatment with the
appropriate agents. Additionally, the cultured stem cells of
the present invention can mature into functionally competent
blood cells in vivo, capable of mediating antigen-specific
immune responses, repopulating lympho-hematopoietic organs,
and prolonging survival of animals with a destroyed
hematopoietic system.
Although the phenotype of the particular stem cells
isolated according to the present invention is new, one
skilled in the art could readily, without undue
2o experimentation, be able to use the stem cells of the present
invention in the capacities described above, as well as in
other capacities. Illustrations of such use are found in
Wagner et al, U.S. Patent 5,744,347.
The following materials and methods are applicable
to all of the following examples.
Materials and Methods
Source of Human Cells
Fresh or frozen cells from bone marrow, cytokine
mobilized peripheral blood or cord blood were obtained from
3o donors after informed consent.
Bone marrow or cord blood samples were diluted
with 4 volumes RPMI 1640 medium containing 10~ fetal calf
serum (FCS). Mononuclear cells were separated on a
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Histopaque~-1077 density gradient (Sigma Diagnostics, St.
Louis, MO) and washed x 2 with PBS-citrate (PBS containing
13.6 mmol/L sodium citrate, 1 mmol/L adenosine and 2 mmol/L
theophylline).
Leukapheresis samples were not normally subjected
to Histopaque~-1077 density gradient centrifugation but were
washed X 2 with PBS-citrate before proceeding to the
filtration step.
PBS-citrate washed cells (from all sources) were
filtered through a 40 ~m nylon cell strainer (Falcon 2340,
Becton-Dickinson, New Jersey). Cells were resuspended in 2
ml PBS-citrate and overlaid on a 3 ml PBS-citrate/10~ bovine
serum albumin (BSA) cushion and then centrifuged for 10
minutes at 200 g at room temperature, to remove platelets
(Thoma et al, 1994). This step was repeated once or twice.
If necessary DNase was used to digest DNA from
cell debris.
Imiaunomaanetic Set~aration
Samples were enriched for CD34+ cells by one of the
2o following methods:
(a) magnetic activated cell sorting (MACS)
columns, using anti-CD34 antibodies coated
onto uniform, supermagnetic, polystyrene
beads (Miltenyi Biotec, Auburn, CA).
Separation was carried out on a MiniMACS
device according to manufacturer's
recommendation (Mitenyi Biotec, Auburn, CA).
(b) magnetic separation using the Dynal CD34
Progenitor Cell Selection System (Dynal A.S.,
Oslo, Norway), which also uses anti-CD34
antibodies immobilized onto microbeads.
In both techniques magnetically labeled cells are separated
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from other cells by means of a magnetic field.
Antibodies and Reagents
The following antibodies were used for
immunofluorescent staining: CD39-FITC, CD34-PE, CD34-RPE-
CYS, c-kit-PE, CD38-FITC, Mouse IgG-FITC, Mouse IgG-PE,
Mouse IgG-RPE-CyS, Mouse IgG2a, streptavidin-PE and goat-
anti-mouse-RPE were purchased from Dako (Dako A/S, Glostrup,
Denmark). Thy-1-FITC and Thy-1-PE were obtained from
Immunotech (Immunotech, France). CD31-FITC, Mouse IgG
Biotin , CD34-APC and Mouse IgG APC were obtained from
Caltag (Caltag Laboratories, Burlingame, CA). HLA-DR-FITC,
Mouse IgG and Goat IgG were purchased from Sigma (Sigma~,
St. Louis MO) and CD34-APC, CD31-PE, Mouse IgG-APC from
Becton-Dickinson (Becton-Dickinson, San Jose, CA). AC133-PE
was obtained from Miltenyi (Miltenyi Biotec, Auburn, CA).
VE-Cadherin-FITC was a kind gift from Dr. W. A. Muller,
Cornell University, New York. TIE 1-FITC, TIE 1-PE,
biotinylated TIE 1 and biotinylated TEK were generous gifts
from Dr. T. Suda, Kumamoto University, Kumamoto, Japan.
FGF-R1 antibody was obtained from Dr. W. L. McKeehan, Texas
A and M University, Houston, TX or commercially, from QED
Bioscience Inc.(San Diego, CA). Anti-FGF-R1-FITC was either
purchased from QED or prepared in our laboratory. Anti-FGF-
R1-APC was produced, purified and conjugated to
allophycocyanin (APC) in our own laboratory. Conjugation of
the antibody to APC was performed using a PhycolinkTM
conjugation kit, PJ25C , purchased from Prozyme (Prozyme,
San Leandro, CA) .
Cell Staining and Flow Cytometry
3o MACS-selected or Dynal-selected CD34+ cells were
resuspended in PBS/0.1~ BSA/0.01$ NaN3/Aprotinin 20~,g/ml
(PBS/BSA/N3/Aprotinin). Fc receptors and non specific
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WO 00/12683 PCT/US99/19730
binding of immunoglobulins to cell surfaces were blocked
with human IgG and either mouse or goat IgG where
appropriate. Cells were incubated with appropriate
antibodies for 30 minutes on ice. After washing x 2 with
PBS/BSA/N3/Aprotinin, cells were analyzed on a FACSCalibur
flow cytometer (Becton-Dickinson, San Jose, CA), equipped
with an argon laser to excite FITC, PE and RPE-CY5
fluorochromes and a helium-neon diode, with time delay
adjusted according to manufacturer's recommendations, for
to excitation of allophycocyanine (APC). 30,000 to 150,000
CD34+ selected cells were analyzed using CellQuest software
(Becton-Dickinson, San Jose, CA). The dye, 7-
aminoactinomycin D (7-AAD) (Sigma, St. Louis, MO), was used
in some experiments to identify dead cells. This was done
to ensure that the CD34'FGFR' population was a viable
population.
Cell Sortiac
To isolate and study the growth characteristics of
the CD34'"FGFR+ population, CD34+ selected cells were obtained
as previously described. These cells were incubated with
antibodies to CD34 and FGF-R1 (as described above) and
sorted on a Coulter Epics Elite (Beckman Coulter Inc.,
Fullerton, CA) into CD34+FGFR' and CD34+ FGFR- populations.
Sorted cells were incubated in a variety of media, including
RPMI 1640, aMEM, DMEM and long term culture medium (LTCM)
(Myelocult H5100 from StemCell Technologies Inc., Vancouver,
Canada) containing 12.5 horse serum and 12.5 FCS in the
presence or absence of growth factors such as FGF-2 and
VEGF. Growth was assessed by determining the number of
cells present at various time points.
EXAMPLE 1: CD34+FGFR+ Co-Expression of Other Antigens
CD34-enriched cells were purified from cytokine
mobilized peripheral blood (PB), bone marrow (BM), or cord
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blood (CB), using magnetic separation techniques (Dynal or
MiniMacs). Fluorescent-labeled antibodies were used to assay
for CD34+FGFR+ cells and a percentage of CD34+FGFR+ was
determined for each experiment. The results of the fourteen
experiments which were run are shown in Table 1. For the
fourteen experiments, a mean of 4.4$ (~ 2.3~) of CD34+ cells
expressed FGFR.
The presence of a third antigen on the CD34'FGFR;
cells was also ascertained using fluorescent antibodies. The
1o mean percentage of CD34'FGFR+ cells expressing a particular
third antigen is shown in Table 1 and is shown graphically in
Figure 1. The error bars indicate the standard deviation
(SD) and the numbers show the number of experiments assaying
a particular antigen.
The experiments were run by submitting the CD34+ or
CD34'lin- enriched cells to the Becton-Dickinson FACSCalibur
machine using the CellQuest System software for three- and
four-color analysis. The techniques were similar to those
described in "Flow Cytometry Analysis Using the Becton-
Dickinson FACScan", Current Protocols in Immunoloctv, unit
5.4, pages 5.4.1-5.4.19 (Supplement 16, 1995).
- 21-
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WO 00/12683 PCT/US99/19730
It can be seen that in Experiment 7 results were
obtained for third antigens from each of the panel of nine
different antigens. The results of Experiment 7 are
tabulated in Table 2. Furthermore, flow cytometry plots for
Experiment 7 are shown in Figures 2A-2F. Figure 2A is a dot
plot of forward scatter (FSC) versus side scatter (SSC) of
all events. FSC gives an indication of cell size, i.e., low
FSC equals small size. SSC gives an indication of cell
granularity or complexity, i.e., low SSC equals low
granularity. A region R1 is drawn to delineate cells of low-
to-high FSC and low-to-medium SSC. This eliminates the area
containing most of the cell debris and doublets.
Figure 2B is a histogram showing the intensity of
staining with the dye 7-AAD (Philpott et al, 1996). 7-AAD is
a large molecule which is not readily taken up by cells with
intact cell membranes and which stains dead cells intensely.
The histogram is gated on R1, and the region R2 is drawn to
delineate live cells.
Figure 2C is a dot plot of SSC versus CD34 gated on
R1 AND R2. The gates are set using Boolean logic in which
the convention used for R1 AND R2 is R1*R2, and in this
instance the gate delineates live cells in R1. The region R3
is drawn to delineate CD34+ cells in this plot. FITC was used
to label the CD34+ cells.
Figure 2D is a dot plot of FSC versus SSC gated on
R1 AND R2 AND R3 (R1*R2*R3), i.e., this plot uses
"backgating" to show the FSC/SSC characteristics of live CD34+
cells in R1.
Figure 2E is a dot plot of CD34 versus FGFR gated
on R1 AND R2, i.e., gated on live cells in R1. The region R4
is drawn to delineate CD34'FGFR+ cells.
Figure 2F is a dot plot of FSC versus SSC gated on
R1 AND R2 AND R4 (Rl*R2*R4), i.e., this plot uses
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"backgating" to show the characteristics of live CD34+FGFR+
cells in RI. It is noted that the majority of live CD34'FGFR'
cells have low FSC and low SSC, i.e., they are small cells
with low granularity. The position of these cells on the
scatter dot plot is somewhat unusual, as it appears in the
region which is conventionally ignored.
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Table 3 shows the results of some of the samples
from Experiment 8. In these samples, CD39 staining was not
included in order that other combinations of antigens could
be assayed. Figure 3 shows these results graphically. One
can see that all live FGFR+Thy-1+ cells co-express VE-Cadherin
as do 97$ of live FGFR+AC133+ cells. Since 99$ of CD34+FGFR+
cells in Experiment 8 were found to be VE-Cadherin+, one can
extrapolate from this experiment that the phenotype of the
CD34~FGFR' population is CD34+FGFR+VE-Cadherin+Thy-1+AC133' and
that approximately two-thirds are TEK+.
Table 3
Experiment 8: Analysis of Live FGFR+ Cells from
Cytokine Mobilized Peripheral Blood
Number Number $ of LIVE
Sampleof of FGFR'
# LIVE FGFR' Cells population
Cells of
Each
Phenotype
LIVE FGFR'VE-CADHERIN'609 96
1 635 LIVE FGFR'THY-1' S39 84 la
2 LIVE FGFR'VE-CADHERIN'THY-1'535 89 lb
0
LIVE FGER'VE-CADHERIN'550 93
2 594 LIVE FGFR'AC133' 316 53 2a
LIVE FGFR'VE-CADHERIN'AC133'307 52 2b
LIVE FGFR'THY-1' 270 76
2 3 356 LIVE FGFR'AC133' 198 56 3a
5
LIVE FGFR'THY'AC133' 181 51 3b
LIVE FGFR'THY-1' 941 70
4 627 LIVE FGFR'TEK' 399 69 4a
LIVE FGFR'THY-1'TEK' 269 93 4b
Table 3 shows that in this experiment:
All live FGFR'THY-1' cells co-express VE-Cadherin (la, lb)
About 97$ of live FGFR'AC133' cells co-express VE-Cadherin
(2a, 2b)
At least 91$ of live FGFR'AC133' cells co-express THY-1 (3a,
3b)
At least 67~k of live FGFR'TEK' cells co-express THY-1 (4a, 9b)
ExamQle 2 - CD34-lin-FGFR+ Co-Expression of Other Antiaens
The following experiment was conducted using the
techniques described for Example 1. Lineage depletion to
obtain lin- cells was conducted as described in Bhatia et al,
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1997, and Bhatia et al, 1998, using Dynal beads and magnetic
separation. The results obtained are summarized in Table 4.
It can be seen that 77% of CD34-lin'FGFR+ cells co-express
CD31, 40% of CD34'lin'FGFR+ cells co-express c-kit, 47$ of
CD34-lin'FGFR+ cells co-express VE-Cadherin, and 18% of CD39'
lin-FGFR+ cells co-express TIE-1.
TABLE 4
Expt CD34 % Cell
of Number
CD34i
or
CD34-LIN'FGFR'
Cells
Expressing
Third
Antigen
{a) T 96% ofCD34'LIN-FGFR+cellsare CD31? 572
- 77% ofCD34-LIN-FGFR'cellsare CD31* 211
Y 97% ofCD34'LIN-FGFR'cellsare c-kit' 1500
- 40% ofCD34'LIN'FGFRicellsare c-kit' 107
(b) t 75% ofCD34+LIN-FGFR'cellsare VE-Cadherin' 417
- 47% ofCD39'LIN-FGFR'cellsare VE-Cadherin' 570
42% ofCD34'LIN-FGFR+cellsare TIE 1~ 237
- 13% ofCD34-LIN-FGFR'cellsare TIE 1' 162
(c) T 37% ofCD34+LIN'FGFR+cellsare TIE 1' 1133
- 23% ofCD34-LIN-FGFR'cellsare TIE ly ~ 62~
Example 3: Growth Characteristics
Cytokine mobilized peripheral blood cells were
enriched for CD34+ cells using magnetic separation. The cells
were stained with fluorescent-labeled antibodies to CD34 and
FGFR and sorted on a fluorescence-activated cell sorter
(FACS) into FGFR' and FGFR' populations. Cells were seeded at
1000 cells per well into collagen/gelatin coated wells
containing long-term culture medium (LTCM) with either (a) no
additions, (b) 10 ng/ml FGF-2 or (c) 10 ng/ml FGF-2 plus 10
ng/ml VEGF, and incubated at 37'C in a humidified incubator.
After 4-6 weeks, the number of cells per well were counted.
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The results are shown in Table 5 and shown graphically in
Figure 4. It can be seen that the FGFR+ population grows in
an FGF dependent manner and that the addition of VEGF
together with FGF-2 further increases the growth of the
cells.
Table 5
Cell Growth
(cells/well)
ADDITIONS
Expt Population None FGF-2~ FGF-2 + VEGF
(10 ng/ml) (both at 10 ng/ml)
(i) CD39'FGFR" 1734 11978 ND
CD34"FGFR' 1244 2500 ND
(ii) CD34" 1617 1457 2311
CD34'FGFR' 3227 8057 17140
CD34'FGFR- 1993 2098 3093
All references cited herein, including journal
articles or abstracts, published or unpublished U.S. or
foreign patent applications, issued U.S. or foreign patents,
or any other references are entirely incorporated by
reference herein, including ali data, tables, figures and
text present in the cited references. Additionally, the
entire contents of the references cited within the references
cited herein are also incorporated by reference in their
entirety.
Reference to known method steps, conventional
method steps, known methods or conventional methods is not in
any way an admission that any aspect, description or
embodiment of the present invention is disclosed, taught or
suggested in the relevant art.
The foregoing description of the specific
embodiments will so fully reveal the general nature of the
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invention that others can, by applying current knowledge,
readily modify and/or adapt for various applications such
specific embodiments without undue experimentation and
without departing from the generic concept, and, therefore,
such adaptations and modifications should and are intended to
be comprehended within the meaning and range of equivalents
of the disclosed embodiments. It is to be understood that
the phraseology or terminology employed herein is for the
purpose of description and not of limitation. The means,
materials, and steps for carrying out various disclosed
functions may take a variety of alternative forms without
departing from the invention. Thus the expressions "means
to..." and "means for...", or any method step language, as
may be found in the specification above and/or in the claims
below, followed by a functional statement, are intended to
define and cover whatever structural, physical, chemical or
electrical element or structure, or whatever method step,
which may now or in the future exist which carries out the
recited function, whether or not precisely equivalent to the
embodiment or embodiments disclosed in the specification
above, i.e., other means or steps for carrying out the same
function can be used; and it is intended that such
expressions be given their broadest interpretation.
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REFERENCES
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(1995) .
Asahara et al, "Isolation of putative progenitor endothelial
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Bhatia et al, "Purification of primitive human hematopoietic
cells capable of repopulating immune-deficient mice",
Proc. Natl. Acad Sci IUSA."Z 94:5320-5325 (1997).
Bhatia et al, "A newly descovered class of human
hematopoietic cells with SCID-repopulating activity",
Nat. Med. 4(9):1038-1045 (1998).
Buhring et al, "Expression of novel surface antigens on early
hematopoietic cells", Ann. NY Acad. Sci 872:25-38
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Burger et al, "bFGF antagonizes TGF-~i-mediated erythroid
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Caprioli et al, "Blood-borne seeding by hematopoietic and
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Choi et al, "A common precursor for hematopoietic and
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D'Arena et al, "Thy-1 (Cdw90) and c-kit receptor (CD117)
expression on CD34+ hematopoietic progenitor cells: a
five dimensional flow cytometric study", Haematolocrica
83:587-592 (1998) .
de Wynter et al, "Comparison of purity and enrichment of CD34+
cells from bone marrow, umbilical cord and peripheral
blood (primed for apheresis) using five separation
systems", Stem Cells 13:524-532 (1995).
Goan et al, "A bone marrow stroma-forming potential of human
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