U.S. patent application number 14/077776 was filed with the patent office on 2014-10-16 for ex vivo expansion of human hematopoietic stem cells.
This patent application is currently assigned to Whitehead Institute for Biomedical Research. The applicant listed for this patent is Whitehead Institute for Biomedical Research. Invention is credited to Harvey Lodish, ChengCheng Zhang.
Application Number | 20140308747 14/077776 |
Document ID | / |
Family ID | 39790348 |
Filed Date | 2014-10-16 |
United States Patent
Application |
20140308747 |
Kind Code |
A1 |
Zhang; ChengCheng ; et
al. |
October 16, 2014 |
Ex Vivo Expansion of Human Hematopoietic Stem Cells
Abstract
Methods and kits for expanding the number of hematopoietic stem
cells are provided. The methods comprise incubating cells in medium
comprising isolated IGFBP-2 and an angiopoietin-like protein
(Angptl). Expanded HSCs are provided as well as culture media and
kits for the expansion of human HSCs in a defined medium. Methods
of administering expanded human HSCs to and individual are provided
as well as methods of treating an individual by administering
certain growth factors and cytokines.
Inventors: |
Zhang; ChengCheng;
(Richardson, TX) ; Lodish; Harvey; (Brookline,
MA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Whitehead Institute for Biomedical Research |
Cambridge |
MA |
US |
|
|
Assignee: |
Whitehead Institute for Biomedical
Research
Cambridge
MA
|
Family ID: |
39790348 |
Appl. No.: |
14/077776 |
Filed: |
November 12, 2013 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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12598770 |
Dec 14, 2009 |
8609411 |
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PCT/US08/62365 |
May 2, 2008 |
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14077776 |
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60927668 |
May 4, 2007 |
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61014006 |
Dec 14, 2007 |
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Current U.S.
Class: |
435/406 |
Current CPC
Class: |
A61P 7/00 20180101; C12N
2501/17 20130101; C12N 2501/91 20130101; A61P 19/08 20180101; C12N
2500/90 20130101; C12N 2501/113 20130101; C12N 2501/125 20130101;
C12N 2501/105 20130101; A61P 43/00 20180101; A61P 5/00 20180101;
C12N 2501/145 20130101; C12N 5/0647 20130101; A61P 7/06
20180101 |
Class at
Publication: |
435/406 |
International
Class: |
C12N 5/0789 20060101
C12N005/0789 |
Goverment Interests
GOVERNMENT FUNDING
[0002] This technology was made with support from the United States
government under grant numbers RO1 DK 067356-01, 1 K01 CA
120099-01, and 075/P-IRFT, awarded by the National Institute of
Health, and the United States government has certain rights in the
technology.
Claims
1-25. (canceled)
26. A method of promoting the expansion of hematopoietic stem cells
in culture, comprising culturing cells in a culture medium
comprising angiopoietin 2 under conditions sufficient for expansion
of the cells, wherein at least one of the cells is a hematopoietic
stem cell.
27. The method of claim 26, wherein the angiopoietin 2 is a human
angiopoietin 2.
28. The method of claim 26, wherein the cells are cultured for ten
days.
29. The method of claim 26, wherein the culture medium is serum
free medium.
30. The method of claim 29, wherein the culture medium further
comprises at least one additional factor selected from the group
consisting of insulin growth factor (IGF), fibroblast growth factor
(FGF), thrombopoietin (TPO), and stem cell factor (SCF).
31. The method of claim 26, wherein the cells are CD45+, Sca-1+
bone marrow cells.
32. The method of claim 26, wherein the cells are side population
cells.
33. A culture medium suitable for expanding hematopoietic stem
cells in vitro comprising a serum free medium and angiopoietin
2.
34. The culture medium of claim 33, wherein the angiopoietin 2 is
human angiopoietin 2.
35. The culture medium of claim 33, wherein the culture medium
further comprises at least one additional factor selected from the
group consisting of insulin growth factor (IGF), fibroblast growth
factor (FGF), thrombopoietin (TPO), and stem cell factor (SCF).
36. The culture medium of claim 33, comprising at least two
additional factors selected from the group consisting of insulin
growth factor (IGF), fibroblast growth factor (FGF), thrombopoietin
(TPO), and stem cell factor (SCF).
37. The culture medium of claim 35, wherein the IGF is IGF-2.
38. The culture medium of claim 35, wherein the FGF is FGF-1.
39. The culture medium of claim 35, further comprising IGF-2,
FGF-1, SCF and TPO.
40. The method of claim 30, further comprising IGF-2, FGF-1, SCF
and TPO.
41. The method of claim 26, wherein the angiopoietin 2 has the
amino acid sequence of SEQ ID NO: 1 or an amino acid sequence
having at least 60% sequence identity thereto.
42. The culture medium of claim 33, wherein the angiopoietin 2 has
the amino acid sequence of SEQ ID NO: 1 or an amino acid sequence
having at least 60% sequence identity thereto.
43. A kit for in vitro expansion of hematopoietic stem cells
comprising a) angiopoietin 2, and b) instructions for expanding
hematopoietic stem cells in vitro.
44. The kit of claim 43, wherein the angiopoietin 2 is human
angiopoietin 2.
45. The kit of claim 43, wherein the angiopoietin 2 has the amino
acid sequence of SEQ ID NO: 1 or an amino acid sequence having at
least 60% sequence identity thereto.
Description
RELATED APPLICATIONS
[0001] This application is a continuation of U.S. application Ser.
No. 12/598,770 filed Nov. 4, 2009, which is a national stage
application of International Application No. PCT/US2008/062365
filed May 2, 2008, which claims the benefit of and priority to U.S.
Provisional Application No. 60/927,668 entitled "Ex Vivo Expansion
of Human Hematopoietic Stem Cells" filed May 4, 2007, now expired
and U.S. Provisional Application No. 61/014,006 entitled "Method
for Expansion and Analysis of Cultured Hematopoietic Stem Cells"
filed Dec. 14, 2007, now expired, the contents of all of which
applications are herein incorporated by reference in their
entirety.
FIELD
[0003] This disclosure relates to methods and compositions for the
in vitro and/or ex vivo propagation of human hematopoietic stem
cells.
BACKGROUND OF THE TECHNOLOGY
[0004] The hematopoietic stem cell (HSC), through proliferation and
differentiation, gives rise to all lymphoid, myeloid, and erythroid
cells. Pluripotent HSCs are thus the basis of bone marrow
transplantation and are considered attractive target cells for
hematopoietic gene therapy for many clinical conditions. However,
these important clinical applications have been severely hampered
by the low numbers of HSCs that can be obtained from an animal, as
well as difficulties in culturing HSCs in vitro and expanding HSCs
for subsequent administration to a patient.
[0005] Culture and propagation of stem cells, such as hematopoietic
stem cells, typically requires supplementation with unknown factors
that allow the stem cells to survive and multiply in number. The
unknown factors can be supplied by co-culturing the stem cells with
feeder cells which secrete an undefined panel of factors, or can be
supplied by adding undefined serum products to the growth medium.
Such supplemented medium contains many unknown factors and
therefore is not chemically defined.
[0006] The presence of unknown factors is problematic when the stem
cells are being prepared for in vivo use, especially in humans. In
many instances, the unknown factors are from non-human sources
(such as bovine serum products). The non-human components may cause
an immune reaction in the recipient, or the undefined components
could include undetected pathogenic agents such as prions or
viruses that would be detrimental to the recipient of the stem
cells. There is a need for methods and compositions that allow the
in vitro and/or ex vivo propagation of human HSCs in a chemically
defined medium, while maintaining the pluripotency of the
propagated cells.
SUMMARY
[0007] Provided herein is a defined culture medium for expanding
human hematopoietic stem cells. The defined medium includes certain
growth factors that synergize with each other to stimulate
expansion of human HSCs. Surprisingly, as demonstrated herein, a
factor produced by non-transfected 293T cells promotes the in vitro
expansion of human HSCs. That factor is shown herein to be
insulin-like growth factor binding protein 2 (IGFBP-2). The finding
that IGFBP-2 promotes the expansion of human hematopoietic stem
cells is unexpected in light of the inhibitory effects that
exogenous IGFBP-2 has on cell proliferation in different
IGF-dependent cell culture systems. (Hoeflich, et al., Canc. Res.
61:8601-8619 (2001)).
[0008] As demonstrated herein, IGFBP-2, in combination with one or
more Angptl proteins promotes the expansion of human HSCs in a
defined culture medium. In some embodiments, human HSCs are
expanded in a defined medium by 250 fold or more.
[0009] In some embodiments, the method expanding human HSCs
comprises incubating human cells in a defined culture medium. The
defined culture medium can comprise IGFBP-2 and an
angiopoietin-like protein (Angptl). In some embodiments, the
defined culture medium can comprise IGFBP-2, Angptl5, fibroblast
growth factor 1 (FGF-1), thrombopoietin (TPO), and stem cell factor
(SCF). In some embodiments, the method comprises incubating human
cells for five days in a defined culture medium.
[0010] In some embodiments, the human cells are primary human
cells. In some embodiments, the human cells include at least one
cell that is capable of differentiating into one or more blood cell
types. In some embodiments, the human cells include at least one
hematopoietic stem cell.
[0011] In some embodiments, the human cells have been selected for
cells that express a surface marker selected from the group
consisting of CD133 and CD34 prior to being incubated.
[0012] Methods of administering hematopoietic stem cells to an
individual are also provided. In some embodiments, the method
comprises obtaining cells from the individual or a donor. In some
embodiments, at least one of the cells is capable of
differentiating into one or more blood cell types. The cells are
expanded in vitro as provided herein. In some embodiments, the
cells are incubated in a defined culture medium comprising an
IGFBP-2 and a growth factor selected from the group consisting of
angiopoietin 2 or an Angptl. The incubated cells are then
administered into the individual.
[0013] Methods of treating a patient comprising administering an
IGFBP-2 and an Angptl to the individual are provided.
[0014] Hematopoietic stem cells that have been expanded in vitro as
described herein are also provided.
[0015] Culture media and kits for expanding human hematopoietic
stem cells in vitro are also provided. In some embodiments, the kit
comprises a defined medium suitable for culturing hematopoietic
stem cells, an isolated IGFBP-2, and another growth factor selected
from the group consisting of angiopoietin 2 or an Angptl. The
growth factors can be supplied as separate components, a cocktail,
or can be supplied already in combination with HSC growth
medium.
[0016] In addition, methods are provided for expanding stem cells
in culture, including hematopoietic stem cells, by culturing a
population of cells that contains stem cells in a culture medium
which contains an effective amount of an angiopoietin, such as
angiopoietin 2, under conditions sufficient for expansion of the
cells. Isolated hematopoietic stem cells are also provided wherein
the isolated hematopoietic cells specifically bind an angiopoietin.
Culture media and kits for expanding hematopoietic stem cells in
vitro are also provided. The culture media and kits comprise an
angiopoietin, such as angiopoietin 2 and instructions for expanding
hematopoietic stem cells in vitro.
[0017] As a result of the methods and compositions provided herein,
human HSCs can be expanded in a defined medium while maintaining
pluripotency. The cells can be expanded in vitro for research use,
or can be expanded in vitro for subsequent administration to an
individual (also referred to herein as ex vivo expansion).
[0018] The various embodiments described herein can be
complimentary and can be combined or used together in a manner
understood by the skilled person in view of the teachings contained
herein.
BRIEF DESCRIPTION OF FIGURES
[0019] FIG. 1A shows total cell number versus days in culture of
total human cord blood cells in the presence of Angptl5 (squares)
or Angptl3 (diamonds).
[0020] FIG. 1B shows the amount of human chimerism in the bone
marrow of NOD/SCID mice transplanted with 1.times.10.sup.6
uncultured human mononuclear cord blood cells (col. 1), or the
progeny of 1.times.10.sup.6 initial human cord blood cells cultured
in serum free STIF plus Angptl5 (col. 2) or Angptl3 (col. 3). Each
symbol represents the engraftment of a single transplanted mouse
assayed at two months post-transplant (n=5-12). (* Significantly
different from lane 1 value. Student's t-test, p<0.001.)
[0021] FIG. 2A is a bar graph showing percent repopulation using
murine HSCs after culturing in serum-free IMDM supplemented with 10
ng/ml SCF, 20 ng/ml TPO, 20 ng/ml IGF-2, and 10 ng/ml FGF-1, bar 1;
freshly collected conditioned medium from 293T cells, bar 2; or in
the same conditioned medium after freeze/thaw, bar 3.
[0022] FIG. 2B, top panel shows a representative FACS analysis of
the repopulation of myeloid and lymphoid lineages in mice that
received cultured cells from conditions represented by bar 2 of
FIG. 2A, at 5 months post-transplant and a bar graph; the bottom
panel shows a summary of the percent repopulation data from six
mice that received cultured cells from conditions represented by
bar 2 of FIG. 2A for T-lymphoid (bar 1), B-lymphoid (bar 2), and
myeloid (bar 3) cells.
[0023] FIG. 3 shows a Western blot of IGFBP-2 (lane 1), serum-free
3T3 conditioned medium (lane 2), and serum-free 293T conditioned
medium (lane 3) separated on an SDS PAGE gel and probed with
anti-IGFBP-2 antibody.
[0024] FIG. 4A shows percent repopulation 1 month (left panel) and
4 months (right panel) after engraftment of mice with murine HSCs
after culturing in STIF medium plus Angptl3 (col. 1 and 4), STIF
medium plus Angptl3 and IGFBP-2 (col. 2 and 5), STIF medium plus
Angptl3 and Timp-1 (col. 3 and 6).
[0025] FIG. 4B shows percent repopulation 1 month (left panel) and
4 months (right panel) after engraftment of mice with murine HSCs
after culturing in STF medium plus Angptl3 (col. 1 and 4), STF
medium plus IGFBP-2 (col. 2 and 5), STF medium plus Angptl3 and
IGFBP-2 (col. 3 and 6).
[0026] FIG. 4C shows limiting dilution analysis of the repopulating
ability of adult BM SP CD45.sup.+Sca-1.sup.+ cells before culture
(left) and after culture for 21 days in conditioned STF medium
containing 100 ng/ml of purified Angptl3 and 500 ng/ml IGFBP-2
(right).
[0027] FIG. 5A shows cell number over time in days of human HSCs
cultured in STF medium containing Angptl 5 (squares), or cultured
in STF medium (diamonds).
[0028] FIG. 5B shows % repopulation by 8000 fresh cells (col. 1),
15000 fresh cells (col. 2), 8000 cells cultured in STF medium (col.
3), or 8000 cells cultured in STF medium containing Angptl5 and
IGFBP-2 (col. 4).
[0029] FIG. 5C shows FACS analysis of human hematopoietic
engraftment at 2 months in a representative mouse that was
transplanted with uncultured (fresh) or cultured human cord blood
CD133.sup.+ cells.
[0030] FIG. 5D shows the summary of multilineage engraftment data
from mice transplanted with uncultured cells (left panel) and cells
cultured in STF medium containing Angptl5 and IGFBP-2 (right panel)
showing % repopulation with myeloid (CD15/66b+, cols. 1, 4),
B-lymphoid (CD34-CD19/20.sup.+, cols. 2, 5), and primitive
(CD34.sup.+, cols. 3, 6) human cells.
[0031] FIG. 5E shows % repopulation of secondary recipients with
total hematopoietic (CD45/71.sup.+, col. 1), myeloid
(CD15/66b.sup.+, col. 2), B-lymphoid (CD34-CD19/20.sup.+, col. 3),
and primitive (CD34.sup.+, col. 5) human cells, transplanted with
bone marrow from the primary mice transplanted with cultured in STF
medium containing Angptl5 and IGFBP-2 (lane 4 of FIG. 5B) and
transplanted into sublethally irradiated secondary recipients.
[0032] FIG. 6A shows total cell number over time of
2.times.10.sup.5 human cord blood CD133.sup.+ cells in STF medium
containing Angptl5 and IGFBP-2 cultured in low levels of O.sub.2
(diamonds) and normal levels of O.sub.2 (squares).
[0033] FIG. 6B shows the number of CD34.sup.+ primitive cells over
time for human HSCs cultured in STF medium containing Angptl5 and
IGFBP-2 cultured in low levels of O.sub.2 (diamonds) and normal
levels of O.sub.2 (squares).
[0034] FIG. 6C shows limiting dilution analysis of the repopulating
ability of cells before culture.
[0035] FIG. 6D shows limiting dilution analysis of the repopulating
ability of cells after culture for 10 days in STF medium containing
500 ng/ml of Angptl5 and 100 ng/ml IGFBP-2 in low levels of O.sub.2
(squares) and normal levels of O.sub.2 (diamonds).
[0036] FIG. 6E shows multilineage engraftment in NOD/SCID
recipients transplanted with 20,000 uncultured CD133.sup.+ cells
(left panel, n=8) or cultured progeny from 5,000 initial CD
133.sup.+ cells at normal O.sub.2 (right panel, n=10).
[0037] FIG. 7 is a bar graph showing percent repopulation 4 months
after transplant with 20 CD45.2 bone marrow SP Sca-1.sup.+
CD45.sup.+ cells cultured in STIF medium with angiopoietin 2
together with 1.times.10.sup.5 CD45.1 bone marrow cells into CD45.1
recipients (n=4-5).
[0038] FIG. 8 shows SEQ ID NOs. 3-6, amino acid sequences for
exemplary angiopoietin-like proteins.
[0039] FIG. 9 shows SEQ ID NO. 1, the amino acid sequence for an
exemplary angiopoietin 2 protein and SEQ ID NO. 2, the amino acid
sequence for an exemplary IGFBP-2 protein.
DETAILED DESCRIPTION
[0040] Methods for propagating and/or expanding hematopoietic stem
cells (HSCs) are provided, as well as human HSCs produced by the
methods. In some embodiments, the human HSCs are expanded in a
defined medium. As a result of the methods provided herein, ex vivo
expanded human HSCs are available that are free from unknown
factors or contaminants typically present in cultured cells.
Culture Medium
[0041] As described herein, suitable cells are incubated in a
defined (also referred to herein as chemically defined) medium.
Defined or chemically defined medium refers to a nutritive medium
for culturing cells where every component and quantity thereof
present in the medium is known. In some embodiments, the medium is
a liquid. In other embodiments, the medium can be a solid such as a
tablet or a powder or semisolid material such as a gel. In still
other embodiments, the medium can be a liquid that includes a solid
structure such as a mesh, porous bead(s), and the like. The defined
medium can comprise a base mixture of components, such as
Dulbecco's MEM, IMDM, X-Vivo 15 (Cambrex), RPMI-1640 and StemSpan
(Stem Cell Technologies). The base mixture can be supplemented with
known quantities of other components such as heparin, serum
albumin, insulin, transferrin, and the like, or combinations
thereof. In some embodiments, the medium is supplemented with 10
.mu.g/ml heparin. The added components can be derived, for example,
from any suitable animal source, including, human, bovine, and
murine sources. For example, StemSpan comprises IMDM supplemented
with bovine serum albumin, human insulin, and human transferrin.
The added components and growth factors can be isolated from a
biological source (such as tissue, serum, or conditioned medium) or
can be recombinantly produced. Suitable hosts for producing
recombinant growth factors or other components include, for
example, bacteria, yeast, or cell culture. The cell culture can be,
for example, insect cell culture, or mammalian cell culture. The
growth factor can be glycosylated. In some embodiments, the growth
factor is glycosylated in the same or substantially the same manner
as the naturally occurring growth factor. The growth factors or
other added components described herein can be from any suitable
animal, including, for example, mouse, non-human primate, and
human.
[0042] An "isolated" or "purified" component or growth factor is
substantially free of other materials with which it is associated
with when produced (e.g., as produced by the biological source or
by recombinant methods such as expression in transfected cells). In
some embodiments, isolated means less than 0.1%, less than 0.01%,
or less than 0.001% of the other materials with which the component
or growth factor is associated with when produced is present. In
another embodiment, the defined medium is serum free.
[0043] The exemplary sequences of growth factors having the GenBank
Accession Nos. provided herein are hereby incorporated by
reference.
IGFBP-2
[0044] In some embodiments, the defined medium includes isolated
insulin-like growth factor binding protein 2 (IGFBP-2). IGF Binding
Proteins (IGFBPs) are a family of circulating proteins that bind
IGF-1 and IGF-2 with an affinity equal or greater than that of the
IGF receptors. IGFBP-2 is also known to have inhibitory effects on
cell proliferation in different IGF-dependent cell culture systems.
(Hoeflich, et al., Canc. Res. 61:8601-8619 (2001)). Surprisingly,
as demonstrated herein, IGFBP-2 has a positive effect on the in
vitro expansion of human HSCs.
[0045] An exemplary IGFBP-2 protein sequence is provided, for
example, in GenBank as Accession Number AAA36048 (human
insulin-like growth factor binding protein 2; SEQ ID NO: 2, FIG.
9). In addition to IGFBP-2, the skilled artisan will further
appreciate that suitable IGFBP-2 includes those proteins and/or
polypeptides that have changes in the naturally occurring amino
acid sequence wherein the altered sequence retains at least some
functional ability of native IGFBP-2. Suitable alterations include
changes to or elimination of non-essential amino acid residues as
well as conservative amino acid changes (e.g., replacing an amino
acid residue with an amino acid residue having a similar side
chain).
[0046] Suitable IGFBP-2 shares at least 60% sequence identity with
SEQ ID NO. 9. In other embodiments, suitable IGFBP-2 shares at
least 70% or at least 80% or at least 90%, or at least 95%, or at
least 96, 97, 98, or 99% sequence identity with SEQ ID NO. 9 or a
biologically active portion thereof.
[0047] Suitable analogs of IGFBP-2 include fragments retaining the
desired activity and related molecules. Molecules capable of
binding the corresponding receptor of IGFBP-2 and initiating one or
more biological actions associated with binding to the IGFBP-2
receptor are also within the scope of the technology (e.g.,
methods, HCSs, media, and kits) provided herein.
Angiopoietin-Like Proteins
[0048] The one or more angiopoietin-like protein (Angptl) can be
any member of a family of secreted glycosylated proteins that are
similar in structure to angiopoietins (Oike et al., Int. J.
Hematol. 80:21-8 (2004)). Angptl proteins contain an N-terminal
coiled-coil domain and a C-terminal fibrinogen-like domain. Unlike
angiopoietins, Angptl proteins do not bind to the tyrosine kinase
receptor Tie2. Angptl proteins include Angptl 1, 2, 3, 4, 5, 6, and
7. Angptl proteins also include microfibrillar-associated
glycoprotein 4 (Mfap4), and analogs and equivalents thereof.
Angptl2 has been described by Kim, I. et al. J Biol Chem 274,
26523-8 (1999)). In addition, Angptl proteins are available
commercially (R&D Systems, Abnova Corp). In one embodiment, the
Angptl is Angptl 3. In another embodiment, the Angptl is Angptl
5.
[0049] Exemplary Angptl proteins are provided, for example in
GenBank as Accession Number AAH12368 (human Angptl 1: SEQ ID NO 3;
human Angptl2 precursor; SEQ ID NO: 4) Accession Number AAH58287
(human Angptl3 precursor; SEQ ID NO: 5) Accession Number AAH23647
(human Angptl4; SEQ ID NO: 6) and Accession Number AAH49170 (human
Angptl5; SEQ ID NO: 7). SEQ ID NOs: 3 through 7 are shown in FIG.
8. Other suitable Angptl proteins share at least 60% sequence
identity with any one of SEQ ID NOs: 3 to 7. In other embodiments,
suitable Angptl proteins share at least 70% or at least 80% or at
least 90%, or at least 95%, or at least 96, 97, 98, or 99% sequence
identity with an exemplary Angptl sequence such as SEQ ID NOs: 3,
4, 5, 6, or 7, or biologically active portions thereof. An
exemplary sequence for Angptl7 is found in GenBank Accession No.
AAH01881. An exemplary sequence for Mfap4 is found in GenBank
Accession No. NP.sub.--002395.
[0050] In addition to sequences provided above for Angptls, the
skilled artisan will further appreciate that suitable Angptls
include those proteins and/or polypeptides that have changes in the
naturally occurring amino acid sequence wherein the altered
sequence retains at least some functional ability of the native
Angptl. Suitable alterations include changes to or elimination of
non-essential amino acid residues as well as conservative amino
acid changes (e.g., replacing an amino acid residue with an amino
acid residue having a similar side chain).
[0051] Suitable analogs of Angptls include fragments retaining the
desired activity and related molecules. For example, a suitable
analog of an Angptl is a fragment of the angiopoietin-like protein
containing the coiled coil domain. For example, the coiled coil
domain of an angiopoietin-like protein. Another analog is the
fibrinogen-like domain. Fragments of Angptls such as the
coiled-coil domain and the fibrinogen-like domain may be easier to
express and to purify compared to full-length protein. Molecules
capable of binding the corresponding receptor of the Angptl and
initiating one or more biological actions associated with binding
to the Angptl receptor are also within the scope of the technology
provided herein.
Angiopoietin 2
[0052] In some embodiments, the defined medium includes
angiopoietin 2. An exemplary angiopoietin 2 protein sequence is
provided, for example, in GenBank as Accession Number
NP.sub.--001138 (human angiopoietin 2; SEQ ID NO: 1, FIG. 9). In
addition, the skilled artisan will further appreciate that suitable
angiopoietin 2 includes proteins and/or polypeptides that have
changes in the naturally occurring amino acid sequence wherein the
altered sequence retains at lease some functional ability of native
angiopoietin 2. Suitable alterations include changes to or
elimination of non-essential amino acid residues as well as
conservative amino acid changes (e.g., replacing an amino acid
residue with an amino acid residue having a similar side
chain).
[0053] Suitable angiopoietin 2 shares at least 60% sequence
identity with SEQ ID NO. 1. In other embodiments, suitable
angiopoietin 2 shares at least 70% or at least 80% or at least 90%,
or at least 95%, or at least 96, 97, 98, or 99% sequence identity
with SEQ ID NO. 1 or a biologically active portion thereof.
[0054] Suitable analogs of angiopoietin 2 include fragments
retaining the desired activity and related molecules. Molecules
capable of binding the corresponding receptor of angiopoietin 2 and
initiating one or more biological actions associated with binding
to the angiopoietin 2 receptor are also within the scope of the
technology provided herein.
Other Growth Factors
[0055] In addition to IGFBP-2, angiopoietin 2, or one or more
Angptls, other growth factors or cytokines useful to promote
expansion of hematopoietic stem cells in methods of the technology
can include one or more of: fibroblast growth factor (FGF), insulin
growth factor, thrombopoietin (TPO), and stem cell factor (SCF).
Accordingly, in another embodiment, the media includes at least two
of FGF, IGF, TPO and SCF or analogs and equivalents thereof.
Equivalents thereof include molecules having similar biological
activity to these factors (i.e. FGF, TPO, IGF and SCF) as wild-type
or recombinantly produced cytokines. Analogs include fragments
retaining the desired activity and related molecules. For example,
TPO is a ligand of the mp1 receptor, thus molecules capable of
binding the mp1 receptor and initiating one or more biological
actions associated with TPO binding to mp1 are also within the
scope of the technology. An example of a TPO mimetic is found in
Cwirla et. al., Science 276:1696 (1997).
[0056] Cytokines and growth factors are commercially available from
several vendors such as, for example, Amgen (Thousand Oaks,
Calif.), R & D Systems (Minneapolis, Minn.) and Immunex
(Seattle, Wash.).
[0057] As indicated above, the concentrations of cytokines or
growth factors range from about 0.1 ng/mL to about 1.0 .mu.g/mL. In
another embodiment, from about 1 ng/mL to about 500 ng/mL of the
factor is used. In another embodiment, from about 10 ng/ml to 100
ng/ml of the factor is used. Other useful concentrations of the
growth factors can be readily determined by one of ordinary skill
in the art using the teachings contained herein.
[0058] In another embodiment, FGF-1, TPO, and SCF are also included
in the medium. In another embodiment the SCF is present at 10
ng/ml, TPO at 20 ng/ml, and FGF-1 at 10 ng/ml. In another
embodiment, IGF-2, FGF-1, TPO, and SCF are also included in the
medium. Other useful concentrations of the growth factors or
cytokines can be readily determined by one of ordinary skill in the
art using the teachings contained herein.
[0059] As described herein, identity or homology to a given amino
acid sequence can be determined as the percentage of identity
between two sequences. The homology can be determined using methods
known in the art, such as by means of computer programs such as GAP
provided in the GCG program package (Program Manual for the
Wisconsin Package, Version 8, August 1994, Genetics Computer Group,
575 Science Drive, Madison, Wis., USA 53711) (Needleman, S. B. and
Wunsch, C. D., (1970), Journal of Molecular Biology, 48, p.
443-453).
Ex Vivo Cultures of Hematopoietic Stem Cells
Cells
[0060] The cell or cells to be cultured can include any cell that
is capable of differentiating into one or more blood cell types.
Exemplary blood cell types include phagocytic immune cells (e.g.,
granulocytes), monocytes (e.g., macrophage precursor cells),
macrophages, eosiniphils, erythrocytes, platelet forming cells
(e.g., megakaryocytes), T lymphocytes, B lymphocytes, and natural
killer (NK) cells. Suitable cells include primary cells obtained
from an individual or donor. Suitable cells can also be capable of
self renewal, that is, capable of propagating or increasing in
number and remaining at the same developmental stage as the parent
cell.
[0061] Suitable cells can be isolated, for example, from any known
source of hematopoietic stem cells, including, but not limited to,
bone marrow, peripheral blood, mobilized peripheral blood (MPB),
fetal liver, and umbilical cord blood. Umbilical cord blood is
discussed, for example, in Issaragrishi et al., N. Engl. J. Med.
332:367-369 (1995). Bone marrow cells can be obtained from a source
of bone marrow, including but not limited to, ilium (e.g., from the
hip bone via the iliac crest), tibia, femora, vertebrate, or other
bone cavities. Other sources of stem cells include, but are not
limited to, ES cells, embryonic yolk sac, fetal liver, and fetal
spleen. Methods for obtaining cells from an individual or donor are
well known in the art.
[0062] For isolation of bone marrow, an appropriate solution can be
used to flush the bone, including, but not limited to, salt
solution, optionally supplemented with fetal calf serum (FCS) or
other naturally occurring factors, in conjunction with an
acceptable buffer. In an embodiment, the buffer is at low
concentration, generally from about 5 to about 25 mM. Convenient
buffers include, but are not limited to, HEPES, phosphate buffers
and lactate buffers. Bone marrow can also be aspirated from the
bone in accordance with conventional techniques.
[0063] Suitable cells and the hematopoietic cells of the technology
can be derived from any animal, where hematopoietic stem cells are
present. Suitable animals include human, non-human primate, cow,
horse, dog, cat, mouse and the like. In an embodiment, the cells
are human cells, in still another embodiment, the cells are murine
cells.
[0064] Animal models for long-term engrafting potential of
candidate human hematopoietic stem cell populations include the
non-obese diabetic/severe combined immunodeficiency mouse
(NOD/SCID) model, the SCID-hu bone model (Kyoizumi et al. (1992)
Blood 79:1704; Murray et al. (1995) Blood 85(2) 368-378) and the in
utero sheep model (Zanjani et al. (1992) J. Clin. Invest. 89:1179).
For a review of animal models of human hematopoiesis, see Srour et
al. (1992) J. Hematother. 1:143-153 and the references cited
therein. An in vitro model for stem cells is the long-term
culture-initiating cell (LTCIC) assay, based on a limiting dilution
analysis of the number of clonogenic cells produced in a stromal
co-culture after 5 to 8 weeks (Sutherland et al. (1990) Proc. Nat'l
Acad. Sci. 87:3584-3588). The LTCIC assay has been shown to
correlate with another commonly used stem cell assay, the
cobblestone area forming cell (CAFC) assay, and with long-term
engrafting potential in vivo (Breems et al. (1994) Leukemia
8:1095).
[0065] As used herein, expansion or propagation includes any
increase in cell number. Expansion includes, for example, an
increase in the number of hematopoietic stem cells over the number
of HSCs present in the cell population used to initiate the
culture. The methods provided herein provide for the increased
survival of existing cells, such as hematopoietic stem cells. The
term survival refers to the ability to continue to remain alive or
function.
[0066] The methods provided herein can be used to stimulate the
expansion of any stem cells which expand in the presence of
angiopoietin 2, and/or an angiopoietin-like protein and/or IGFBPs,
including other types of adult stem cells such as endothelial
progenitor cells (Shi, Q. et al. (1998), Blood. 92, 362-367), bone
marrow stromal stem cells (Owen, M. (1988), J. Cell Science Supp.
10, 63-76), mesenchymal stem cells (Pittenger, M. F. and Marshak,
D. R. (2001), Marshak, D. R., Gardner, D. K., and Gottlieb, D. eds.
Cold Spring Harbor, N.Y.: Cold Spring Harbor Laboratory Press
349-374), and skeletal muscle stem cells (Gussoni, E., et al.
(1999), Nature. 401, 390-394), embryonic stem cells, as well as
others. In another embodiment, the stem cells are endothelial
progenitor cells, which are believed to share the same
precursor--hemangioblasts--as HSCs.
[0067] Subpopulations of cells can also be used in the methods
provided herein. For example, a purified "side population" (SP)
cells obtained from bone marrow or other sources can be used. Other
enriched populations of HSCs can also be used. Methods for
isolating enriched populations of HSCs are known to those in the
art, e.g. methods for obtaining SP cells are described in Goodell
et al., J. Exp. Med. 183, 1797-806 (Apr. 1, 1996).
[0068] In addition, a subpopulation of cells enriched for stem
cells can be used in the methods described herein. Separation of
stem cells from a cell population can be performed by any number of
methods, including cell sorting, (e.g., fluorescence activated cell
sorting) magnetic beads, and packed columns. The methods typically
rely on the presence of certain cell surface markers characteristic
of stem cells and/or the absence of certain cell surface markers
characteristic of differentiated cells. The methods can also rely
on functional assays to measure the engraftment or differentiation
potential of the population of cells. Such markers and functional
assays are known in the art.
[0069] An example of a enriched for stem cells is a population of
cells selected for the CD34.sup.+Thy.sup.-1.sup.+LIN.sup.-
phenotype as described in U.S. Pat. No. 5,061,620. A population of
this phenotype typically has an average CAFC frequency of
approximately 1/20 (Murray et al. (1995) supra; Lansdorp et al., J.
Exp. Med. 177:1331 (1993)). Methods for isolating highly enriched
populations of hematopoietic stem cells are further provided in
U.S. Pat. No. 5,681,559.
[0070] As described herein, hematopoietic stem cells have the
ability to differentiate into any of several types of blood cells,
including red blood cells, white blood cells, including lymphoid
cells and myeloid cells. As described herein, HSCs include
hematopoietic cells having long-term engrafting potential in vivo.
Long term engrafting potential (e.g., long term hematopoietic stem
cells) can be determined using animal models or in vitro
models.
[0071] The cells can be enriched for stem cells or immature cells,
e.g. in a blood cell lineage, prior to culturing according to the
methods provided herein. Cells populations highly enriched in stem
cells and methods for obtaining them are described in WO 95/05843;
WO 95/03693 and WO 95/08105. In a some embodiments, the one or more
cells comprise a population of cells that is substantially enriched
in hematopoietic stem cells. In other embodiments, the cells
cultured according to the methods provided herein are substantially
free of stromal cells.
[0072] In some embodiments, the cells used in the methods provided
herein are selected or enriched for the presence of or absence of
particular markers on the surface of the cell. For example, in some
embodiments, the cells are selected for the presence of stem cell
markers particular for the animal source of the primary tissue. In
other embodiments, the cells are selected for the absence of
lineage specific markers. In some embodiments, the cells are
selected from the presence of particular markers and the absence of
other markers. Methods for isolating cells that have particular
markers or that do not have particular markers are well known to
those skilled in the art.
[0073] Regarding lineage specific markers, the absence or low
expression of lineage specific markers can be identified by the
lack of binding of antibodies specific to the lineage specific
markers. The cells or source of cells for use in the methods
provided herein can be subjected to negative selection techniques
to remove those cells that express lineage specific markers and
retain those cells which are lineage negative ("Lin.sup.-").
Lin.sup.- generally refers to cells which lack markers such as
those associated with T cells (such as CD2, 3, 4 and 8), B cells
(such as B220, CD48, CD10, 19 and 20), myeloid cells (such as
Mac-1, Gr-1, CD14, 15, 16 and 33), natural killer ("NK") cells
(such as CD244, CD2, 16 and 56), RBC (such as Ter119, and
glycophorin A), megakaryocytes (CD41), mast cells, eosinophils or
basophils. Methods of negative selection are known in the art.
Lineage specific markers also include CD38, HLA-DR and CD71.
[0074] Various techniques can be employed to separate the cells by
initially removing cells of dedicated lineage or having a
particular phenotype. Procedures for separation can include, but
are not limited to, physical separation, magnetic separation (using
antibody-coated magnetic beads), affinity chromatography, cytotoxic
agents joined to a monoclonal antibody or used in conjunction with
a monoclonal antibody, including, but not limited to, complement
and cytotoxins, and "panning" with antibody attached to a solid
matrix, e.g., plate, elutriation or any other convenient technique.
Techniques providing accurate and rapid separation include, but are
not limited to, flow cytometry (e.g., fluorescence activated cell
sorting) and cytospin.
[0075] The use of physical separation techniques include, but are
not limited to, those based on differences in physical (density
gradient centrifugation and counter-flow centrifugal elutriation),
cell surface (lectin and antibody affinity), and vital staining
properties (mitochondria-binding dye rho123 and DNA-binding dye
Hoechst 33342). These procedures are well known to those of skill
in this art.
[0076] The cells obtained either with or without enrichment for
hematopoietic stems cells as described above can be used
immediately or frozen at liquid nitrogen temperatures and stored.
The frozen cells can be thawed and used in the methods described
herein.
Cell Culture
[0077] In some embodiments of expanding HSC cells, the cells,
obtained, for example from a primary tissue source or from a
suitable animal, are incubated in a suitable medium. Suitable
conditions comprise incubating at 33.degree. to 39.degree. C., and
preferably around 37.degree. C. HSCs can be cultured in an oxygen
concentration of 1 to 10%. In some embodiments, the HSCs are
cultured under hypoxic conditions. In some embodiments, the cells
are incubated under normoxic conditions Normoxic conditions can be,
for example, 5% CO.sub.2 and oxygen at 15% or more. In some
embodiments normoxic conditions are 21% O.sub.2. Hypoxic conditions
can be, for example, 5% CO.sub.2 and 5% O.sub.2.
[0078] Media can be replaced throughout the culture period. In
another embodiment, half of the medium is replaced twice per week
with fresh media. The cells can be cultured from 3 to 30 days. In
another embodiment, the population of cells including HSCs is
cultured for at least four weeks. In another embodiment, the
population of cells including HSCs is cultured for up to two weeks.
In another embodiment, the population of cells including HSCs is
cultured for 7 to 14 days. In another embodiment, the population of
cells including HSCs is cultured for 10 days.
[0079] HSCs can be propagated by culturing or incubating one or
more cells in an expansion container and in a volume of a suitable
medium. The cells can be cultured such that the culture well
contains about 1-100 cells per well. Where the population of cells
is bone marrow, the cells can be cultured at a density of about
1.times.10.sup.2 cells to about 1.times.10.sup.7 cells/mL of
medium. In another embodiment, the cells can be cultured at a
density of about 1.times.10.sup.5 cells to about 1.times.10.sup.6
cells/mL of medium. In another embodiment, the population of cells
comprises Side Population (SP) bone marrow cells. The SP bone
marrow cells can be cultured at lower density, for example from
about 1.times.10.sup.2 to 5.times.10.sup.3 cells/ml. In a separate
aspect, the population of cells can be derived from mobilized
peripheral blood. The mobilized peripheral blood cells can be
cultured at a density of about 20,000 cells/mL to about 50,000
cells/mL; in another embodiment, the mobilized peripheral blood
cells is cultured at a density of about 50,000 cells/mL.
[0080] Any suitable expansion container, flask, or appropriate tube
such as a 12, 24 or 96 well plate, 12.5 cm.sup.2 T flask or
gas-permeable bag can be used in the methods provided herein. Such
culture containers are commercially available from Falcon, Corning
or Costar. As used herein, "expansion container" also is intended
to include any chamber or container for expanding cells whether or
not free standing or incorporated into an expansion apparatus.
[0081] Hematopoietic stem cells that have been expanded in vitro as
described herein are also provided. It is understood that the
descendants of stem cells grown in culture may not be completely
identical (either morphologically, genetically, or phenotypically)
to the parent cell. However, as provided herein, the descendants of
the stem cells possess at least some ability to differentiate into
one or more blood cell types as described, supra. Functional
characteristics, such as the ability to develop into one or more
blood cell types can be measured, for example, using methods and
lineage markers as described herein.
Uses for Ex Vivo Expanded Hematopoietic Stem Cells
[0082] The expanded cultured hematopoietic stem cells of the
technology can be used for a variety of applications, including
transplantation, drug discovery, gene cloning, gene delivery, and,
gene expression.
Transplantation
[0083] The hematopoietic stem cells provided herein can be
administered to a subject or an individual. In some embodiments,
the hematopoietic stem cells produced by the methods provided
herein are used in cell-based therapies, such a bone marrow
transplantation. Suitable subject or individuals include any animal
as described above. The subject or individual can be any animal
suitable for studying hematopoiesis or cell-based therapies in
vivo. vertebrate. The subject or individual can be any animal in
need of cell-based therapy. In some embodiments, the individual is
a mammal. Mammals include, but are not limited to, humans,
non-human primates, mice, cows, horses, dogs, cats and the like. In
a preferred embodiment, the mammal is a human.
[0084] The transplanted stem cells can be autologous (derived from
the individual being treated), allogenic (derived from a donor of
the same species), or obtained from a histocompatibly matched
donor. In some embodiments, the transplanted stem cells can be
xenogenic (derived from a animal of a different species from the
recipient). Human autologous and allogeneic bone marrow
transplantations are currently used as therapies for diseases such
as leukemia, lymphoma, and other life-threatening diseases.
[0085] With respect to administering the expanded cells provided
herein to a patient, an effective amount of expanded cells may
range from as few as several hundred or fewer to as many as several
million or more. It will be appreciated that the number of expanded
cells to be administered will vary depending on the specifics of
the disorder to be treated, including but not limited to size or
total volume to be treated, as well as the needs and condition of
the recipient, among other factors familiar to the medical
professional. In some embodiments, between 10.sup.3 and 10.sup.10
cells per 100 kg person are administered or transplanted into the
subject or individual. Methods of administering or transplanting
are well known in the art and include, for example, infusion.
Expanded cells provided herein can be administered, for example, by
intravenous infusion.
[0086] In some embodiments, a single administration of cells is
provided. In other embodiments, multiple administrations are used.
Multiple administrations can be provided over periodic time periods
such as an initial treatment regime of 3 to 7 consecutive days, and
then repeated at other times.
[0087] The expanded cells can be used for reconstituting the full
range of hematopoietic cells in an individual following therapies
such as, but not limited to, radiation treatment and chemotherapy.
Such therapies destroy hematopoietic cells either intentionally or
as a side-effect of bone marrow transplantation or the treatment of
lymphomas, leukemias and other neoplastic conditions, e.g., breast
cancer.
[0088] Expanded cells provided herein are also useful as a source
of cells for specific hematopoietic lineages. The maturation,
proliferation and differentiation of expanded hematopoietic cells
into one or more selected lineages may be effected through
culturing the cells with appropriate factors including, but not
limited to, erythropoietin (EPO), colony stimulating factors, e.g.,
GM-CSF, G-CSF, or M-CSF, SCF, Flt-3 ligand, interleukins, e.g.,
IL-1, -2, -3, -4, -5, -6, -7, -8, -13, etc., or with stromal cells
or other cells which secrete factors responsible for stem cell
regeneration, commitment, and differentiation.
Drug Discovery
[0089] Hematopoietic stem cells provided by the methods described
herein are useful for drug discovery. For example, culture
conditions or growth factors that promote or inhibit such
biological responses of stem cells can be identified by exposing
the cells to the conditions or factors to be tested. In this way
one may also identify, for example, receptors for these factors or
agents that interfere with the biological activity of the
factor.
[0090] The hematopoietic stem cells produced by the methods
provided herein can be used in assays for differentiating stem
cells into various hematopoietic lineages. These assays may be
readily adapted in order to identify substances such as factors
which, for example, promote or inhibit stem cell self-regeneration,
commitment, or differentiation.
Gene Cloning Strategies
[0091] The hematopoietic cells provided herein can be used to
identify and clone genes whose expression is associated with
proliferation, commitment, differentiation, and maturation of stem
cells or other hematopoietic cells, e.g., by subtractive
hybridization or by expression cloning using monoclonal antibodies
specific for target antigens associated with these biological
events or characteristic of a hematopoietic cell type.
Gene Delivery and Expression
[0092] Hematopoietic stem cells are also important targets for gene
delivery and expression in a subject. Accordingly the hematopoietic
cells provided herein can be genetically altered prior to
reintroducing the cells into an individual. For example a gene
whose expression is expected to have a therapeutic effect on the
individual can be introduced into one more of the hematopoietic
cells provided herein. The cells can be genetically altered before
or after being cultured and/or expanded as described herein.
Methods for introducing genes into the cultured cells are well
known in the art.
[0093] In some aspects of the technology, individuals can be
treated by supplementing, augmenting and/or replacing defective
and/or damaged cells with cells that express a therapeutic gene.
The cells may be derived from cells of a normal matched donor or
stem cells from the individual to be treated (i.e., autologous). By
introducing normal genes in expressible form, individuals suffering
from such a deficiency can be provided the means to compensate for
genetic defects and eliminate, alleviate or reduce some or all of
the symptoms.
[0094] Expression vectors may be introduced into and expressed in
autologous or allogeneic expanded hematopoietic cells, or the
genome of cells may be modified by homologous or non-homologous
recombination by methods known in the art. In this way, one may
correct genetic defects in an individual or provide genetic
capabilities naturally lacking in stem cells. For example, diseases
including, but not limited to, .beta.-thalassemia, sickle cell
anemia, adenosine deaminase deficiency, recombinase deficiency, and
recombinase regulatory gene deficiency may be corrected in this
fashion. Diseases not associated with hematopoietic cells may also
be treated, e.g., diseases related to the lack of secreted proteins
including, but not limited to hormones, enzymes, and growth
factors. Inducible expression of a gene of interest under the
control of an appropriate regulatory initiation region will allow
production (and secretion) of the protein in a fashion similar to
that in the cell which normally produces the protein in nature.
Transduction of Hematopoietic Stem Cell Cultures
[0095] The hematopoietic stem cells provided herein can be
genetically modified. The introduction of the gene into the
hematopoietic stem cell can be by standard techniques, e.g.
infection, transfection, transduction or transformation. The HSC
cells can be transduced with a therapeutic gene. For example, the
transduction can be via a viral vector such as a retroviral vector
(e.g. as described in for example, WO 94/29438, WO 97/21824 and WO
97/21825) or a pox viral vector. When transduction is ex vivo, the
transduced cells are subsequently administered to the recipient.
Thus, the technology provided herein encompasses treatment of
diseases amenable to gene transfer into HSCs, by administering the
gene ex vivo or in vivo by the methods disclosed herein. For
example, diseases including, but not limited to,
.beta.-thalassemia, sickle cell anemia, adenosine deaminase
deficiency, recombinase deficiency, recombinase regulatory gene
deficiency, etc. can be corrected by introduction of a therapeutic
gene. Other indications of gene therapy are introduction of drug
resistance genes to enable normal stem cells to have an advantage
and be subject to selective pressure during chemotherapy. Suitable
drug resistance genes include, but are not limited to, the gene
encoding the multidrug resistance (MDR) protein.
[0096] Examples of modes of gene transfer include e.g., naked DNA,
CaPO.sub.4 precipitation, DEAE dextran, electroporation, protoplast
fusion, lipofection, cell microinjection, and viral vectors,
adjuvant-assisted DNA, gene gun, catheters, etc. In another
embodiment, a viral vector is used.
[0097] One or more polynucleotide of interest can be inserted into
a vector using methods well known in the art. For example, insert
and vector DNA can be contacted, under suitable conditions, with a
restriction enzyme to create complementary ends on each molecule
that can pair with each other and be joined together with a ligase.
Alternatively, synthetic nucleic acid linkers can be ligated to the
termini of restricted polynucleotide. These synthetic linkers
contain nucleic acid sequences that correspond to a particular
restriction site in the vector DNA. Additionally, an
oligonucleotide containing a termination codon and an appropriate
restriction site can be ligated for insertion into a vector
containing, for example, some or all of the following: a selectable
marker gene, such as the neomycin gene for selection of stable or
transient transfectants in mammalian cells; enhancer/promoter
sequences from the immediate early gene of human CMV for high
levels of transcription; transcription termination and RNA
processing signals from SV40 for mRNA stability; SV40 polyoma
origins of replication and ColE1 for proper episomal replication;
versatile multiple cloning sites; and T7 and SP6 RNA promoters for
in vitro transcription of sense and antisense RNA. Other means are
well known and available in the art.
[0098] Modification of hematopoietic stem cells can comprise the
use of an expression cassette created for either constitutive or
inducible expression of the introduced transgene. Such an
expression cassette can include regulatory elements such as a
promoter, an initiation codon, a stop codon, and a polyadenylation
signal. Suitable elements that are operable in the stem cells or in
cells that arise from the stem cells after infusion into an
individual can be used. Moreover, it is necessary that these
elements be operably linked to the nucleotide sequence that encodes
the protein such that the nucleotide sequence can be expressed in
the stem cells and thus the protein can be produced. Initiation
codons and stop codons are generally considered to be part of a
nucleotide sequence that encodes the protein.
[0099] Examples of promoters that may be used to cause expression
of the introduced sequence in specific cell types include Granzyme
A for expression in T-cells and NK cells, the CD34 promoter for
expression in stem and progenitor cells, the CD8 promoter for
expression in cytotoxic T-cells, and the CD11b promoter for
expression in myeloid cells. In addition, regulatable promoters can
be used. Regulatable promoters such as inducible promoters are
available commercially.
[0100] The exogenous genetic material that includes the transgene
operably linked to the regulatory elements may remain present in
the cell as a functioning cytoplasmic molecule, a functioning
episomal molecule or it may integrate into the cell's chromosomal
DNA. Exogenous genetic material may be introduced into cells where
it remains as separate genetic material in the form of a plasmid.
Alternatively, linear DNA, which can integrate into the chromosome,
may be introduced into the cell. When introducing DNA into the
cell, reagents, which promote DNA integration into chromosomes, may
be added. DNA sequences, which are useful to promote integration,
may also be included in the DNA molecule. Alternatively, RNA may be
introduced into the cell.
[0101] Selectable markers can be used to monitor uptake of the
desired gene into the hematopoietic stem cells of the technology.
These marker genes can be under the control of any promoter or an
inducible promoter. These are well known in the art and include
genes that change the sensitivity of a cell to a stimulus such as a
nutrient, an antibiotic, etc. Genes include those for neo, puro,
and tk, multiple drug resistance (MDR), etc. Other genes express
proteins that can readily be screened for such as green fluorescent
protein (GFP), blue fluorescent protein (BFP), luciferase, and
LacZ.
[0102] As used herein, therapeutic gene can be an entire gene or
only the functionally active fragment of the gene capable of
compensating for the deficiency in the patient that arises from the
defective endogenous gene. Therapeutic gene also encompasses
antisense oligonucleotides or genes useful for antisense
suppression and ribozymes for ribozyme-mediated therapy.
Therapeutic genes that encode dominant inhibitory oligonucleotides
and peptides as well as genes that encode regulatory proteins and
oligonucleotides also are encompassed by this technology.
Generally, gene therapy will involve the transfer of a single
therapeutic gene although more than one gene may be necessary for
the treatment of particular diseases. The therapeutic gene can be a
normal, e.g., wild-type, copy of the defective gene or a functional
homolog. In a separate embodiment, the therapeutic gene is a
dominant inhibiting mutant of the wild-type. More than one gene can
be administered per vector or alternatively, more than one gene can
be delivered using several compatible vectors. Depending on the
genetic defect, the therapeutic gene can include the regulatory and
untranslated sequences. For gene therapy in human patients, the
therapeutic gene will generally be of human origin although genes
from other closely related species that exhibit high homology and
biologically identical or equivalent function in humans may be
used, if the gene product does not induce an adverse immune
reaction in the recipient. For example, a primate insulin gene
whose gene product is capable of converting glucose to glycogen in
humans would be considered a functional equivalent of the human
gene. The therapeutic gene suitable for use in treatment will vary
with the disease. For example, a suitable therapeutic gene for
treating sickle cell anemia is a normal copy of the globin gene. A
suitable therapeutic gene for treating SCID is the normal ADA
gene.
EXAMPLES
Results
Culture of Total Human Cord Blood Cells in the Presence of Angptl5
or Angptl3 Stimulates Ex Vivo Expansion of Human HSCs.
[0103] Total human cord blood cells were cultured in STIF medium
containing Angptl3 or Angptl5. (Conklin, D. et al. Genomics 62,
477-82 (1999); Zeng, L. et al. J Hum Genet. 48, 159-62 (2003)).
[0104] 2.5.times.10.sup.7 total cord blood cells were seeded at a
density of 1.times.10.sup.6 cells/ml serum-free STIF medium
containing 100 ng/ml Angptl3 or Angptl5, and total cell numbers
were counted at indicated time. After 23 days of culture, the
number of total cells in the presence of Angptl3 increased about 10
fold to 2.6.+-.0.3.times.10.sup.8 (FIG. 1A. diamonds), and the
cells cultured with Angptl5 increased to 2.2.+-.0.3.times.10.sup.8
(squares). The cultured cells contained mostly suspension cells
with a minor adherent subpopulation.
[0105] NOD/SCID repopulation assays were conducted to test whether
ex vivo-expanded cells were capable of engraftment. FIG. 1B shows
the amount of human chimerism in the bone marrow of NOD/SCID mice
transplanted with 1.times.10.sup.6 uncultured human mononuclear
cord blood cells, or the cultured progeny of 1.times.10.sup.6
initial human cord blood cells. Each symbol represents the
engraftment of a single transplanted mouse assayed at two months
post-transplant (n=5-12). (* Significantly different from lane 1
value. Student's t-test, p<0.001.) Thus, 1.times.10.sup.6 or
3.times.10.sup.6 uncultured cells, or the cultured progenies of
1.times.10.sup.6 initial cells were injected into sublethally
irradiated NOD/SCID recipients. When 9.2.times.10.sup.6 cells,
cultured with Angptl5 for 19 days (which is the progeny of
1.times.10.sup.6 initially plated total cord blood cells), were
transplanted, an average human hematopoietic chimerism of 8.8% was
observed 2 months after transplantation (FIG. 1B, lane 2). This is
much greater than the 0.6% engraftment shown by the equivalent
1.times.10.sup.6 uncultured cells (FIG. 1B, lane 1; p<0.001,
student's t-test). The cells cultured in the presence of Angptl3
for 23 days also engrafted recipients, with an average chimerism of
7.1% (FIG. 1B, lane 3).
Non-Transfected 293T Cells Stimulates Ex Vivo Expansion of HSCs
[0106] Surprisingly, as demonstrated herein, serum-free conditioned
medium collected from non-transfected 293T cells stimulates ex vivo
expansion of HSCs
[0107] Twenty freshly isolated CD45.2 bone marrow SP Sca-1+ CD45+
cells were cultured for 10 days in serum-free IMDM supplemented
with 10 ng/ml SCF, 20 ng/ml TPO, 20 ng/ml IGF-2, and 10 ng/ml FGF-1
(STIF medium; FIG. 2A, bar 1), in freshly collected serum-free
conditioned STIF medium from 293T cells (bar 2), or in the same
conditioned medium after freeze/thaw (bar 3). The cultured cells
were co-transplanted with 1.times.10.sup.5 CD45.1 total bone marrow
cells into CD45.1 recipients (n=5-6).
[0108] FIG. 2B shows the multilineage contribution of cultured
cells at conditions represented by bar 2 of FIG. 2A at 5 months
post-transplant (n=6). Data shown in the top panel are
representative FACS plots of peripheral blood mononuclear cells
from one mouse at 5 months post-transplant (from bar 2 of FIG. 1A).
Percentages of cells in each quadrant are listed. The summary of
percent repopulation data from mice in bar 2 of FIG. 1A for
T-lymphoid, B-lymphoid, and myeloid cells is plotted in the bottom
panel.
[0109] Serum-free 293T conditioned medium was analyzed by mass
spectrometry in order to identify potential candidate proteins that
stimulated ex vivo expansion of HSCs. Peptides from several
proteins were identified. A partial list of peptides identified in
the mass spectrometry analysis of the fraction of serum-free IMDM
based conditioned medium of 293T cells that contained proteins
smaller than 70 kD is shown in Table I. Proteins found in common
with the control serum-free IMDM sample are not shown.
TABLE-US-00001 TABLE I Accession No. P18965 PO1033 POCOP6 P35555
P36955 Gene IGFBP-2 Timp-1 NPS Fibrillin-1 PEDF # peptides 37 20 12
13 15 Total 46 22 24 22 22 peptides % of 8.45% 5.32% 15.01% 5.70%
2.99 coverage M.W. 35114 23156 10096 26129 46313
[0110] As demonstrated herein, IGFBP-2 is expressed in serum-free
293T conditioned medium. FIG. 3 shows western blot analysis of
purified human IGFBP-2 (positive control; lane 1), serum-free 3T3
conditioned medium (negative control; lane 2), and serum-free 293T
conditioned medium (lane 3) detected by anti-human IGFBP-2
polyclonal antibody.
Purified IGFBP-2 Stimulates Ex Vivo Expansion of HSCs.
[0111] As demonstrated herein, IGFBP-2 stimulates ex vivo expansion
of HSCs. Twenty CD45.2 bone marrow SP Sca-1.sup.+CD45.sup.+ cells
were cultured for 5 days in STIF medium with 100 ng/ml Angptl3
(bars 1 and 4); in the same medium with 500 ng/ml IGFBP-2 (bars 2
and 5); and in the same medium with 200 ng/ml Timp-1 (bars 3 and 6)
(see FIG. 4A). The cells were then cotransplanted with
1.times.10.sup.5 CD45.1 total bone marrow cells into CD45.1
recipients (n=5). Engraftments at 1 month or 4 months
post-transplant are shown in FIG. 4A. (* Significantly different
from bars 4 and 6 values. Student's t-test, p<0.05.)
[0112] Twenty CD45.2 bone marrow SP Sca-1.sup.+ CD45.sup.+ cells
were cultured for 10 days in serum-free medium with 10 ng/ml SCF,
20 ng/ml TPO, 10 ng/ml FGF-1 (STF medium), and 100 ng/ml Angptl3
(bars 1 and 4); in STF medium with 500 ng/ml IGFBP-2 (bars 2 and
5); and in STF medium with 500 ng/ml IGFBP-2 and 100 ng/ml Angptl3
(bars 3 and 6) (see FIG. 4B). The cells were then cotransplanted
with 1.times.10.sup.5 CD45.1 total bone marrow cells into CD45.1
recipients (n=6-7). Engraftments at 1 month or 4 months
post-transplant are shown in FIG. 4B. (* and ** Significantly
different from bar 4 or bar 5 value respectively. Student's t-test,
p<0.05.)
[0113] FIG. 4C shows limiting dilution analysis of the repopulating
ability of adult BM SP CD45.sup.+Sca-1.sup.+ cells before culture
(left) and after culture for 21 days in serum-free conditioned STF
medium containing 100 ng/ml of purified Angptl3 and 500 ng/ml
IGFBP-2 (right). Irradiated CD45.1 congenic mice were injected with
1.times.10.sup.5 CD45.1 BM competitor cells and 1, 5, 25, or 100
freshly isolated SP CD45.sup.+Sca-1.sup.+ cells (left; n=24) or the
cultured progenies of 0.2, 1, 4, or 10 SP CD45+Sca-1+ cells (right;
n=26). 100 Freshly isolated SP Sca-1+CD45+ cells and the cultured
progeny of 4 or 10 input cells repopulated all recipients and these
data points are not plotted. Plotted is the percentage of recipient
mice containing less than 1% CD45.2 populations in nucleated
peripheral blood cells 4 months after transplant versus the number
of cells injected.
Culture of Human Cord Blood CD133.sup.+ Cells in the Presence of
Angptl5 and IGFBP-2 Stimulates Ex Vivo Expansion of HSCs by Over
250 Fold.
[0114] It was surprisingly found that IGFBP-2 can replace IGF-2 in
supporting the ex vivo expansion of mouse HSCs. In addition, in the
presence of STF medium, IGFBP-2 promoted the ex vivo expansion
human cord blood HSCs CD133.sup.+ cells by over 250 fold.
[0115] Culture of 1.times.10.sup.5 human cord blood CD133.sup.+
cells was initiated in serum-free STF medium, or in serum-free STF
medium supplemented with 500 ng/ml Angptl5 and 500 ng/ml IGFBP-2
and cultured in a low O.sub.2 environment (5% O.sub.2). Total cell
numbers were counted. As shown in FIG. 5A, the number of total
cells increased greater than 200 fold after 11 days of culture
either in serum-free STF medium or serum-free STF medium containing
Angptl5 and IGFBP-2.
IGFBP-2 and Angptl5 Promote Engraftment and Chimerism of Human Cord
Blood Cells in a Mouse Model
[0116] The amount of human chimerism in the bone marrow of NOD/SCID
mice transplanted with 8,000 or 15,000 uncultured (fresh) human
cord blood CD 133.sup.+ cells, or the progeny from 8,000 initial
CD133.sup.+ cells cultured in serum-free STF medium with or without
Angptl5 and IGFBP-2 for 11 days was measured. 8,000 uncultured
CD133.sup.+ cells were capable of engraftment in 1 out of 7
recipients 2 months post-transplant, and the average chimerism was
0.2% (FIG. 5B, lane 1). 15,000 uncultured CD133.sup.+ cells showed
an increased but still modest engraftment; there was positive
engraftment of 4 of 8 mice (average chimerism 2.0% of total cells;
FIG. 5B, lane 2). In striking contrast, 2.1.times.10.sup.6 cells
after culture with serum free STF medium with Angptl5 and IGFBP-2,
that is, the progeny of 8,000 initial cells after 11 days of
culture, engrafted all recipient mice, and showed significantly
increased chimerism relative to 8,000 or 15,000 uncultured sells
(average 39.5%) (FIG. 5B, lane 4; p<0.05, student's t-test). The
cultured progeny of the same number of initial cells (8,000)
cultured in STF medium without Angptl5 and IGFBP-2 (now
1.6.times.10.sup.6 cells) only exhibited a poor engraftment,
similar to that of their uncultured counterparts (FIG. 5B, lane 3).
Thus, Angptl5 and IGFBP-2 support the ex vivo expansion of human
SCID-repopulating cells (SRCs) in a defined medium. Each symbol
represents the engraftment of a single transplanted mouse assayed
at two months post-transplant (n=7-8). (* Significantly different
from lanes 1-3 values. Student's t-test, p<0.05.)
[0117] FIG. 5C shows human hematopoietic engraftment at 2 months in
a representative mouse that was transplanted with uncultured
(fresh) or cultured human cord blood CD133.sup.+ cells.
Representative FACS plots of bone marrow cells from one mouse at
the condition represented by lane 1 of (FIG. 5B) (Fresh), or at the
condition represented by lane 4 of (FIG. 5B) (Cultured), at 2
months post-transplant. Percentages of cells in each quadrant are
listed. Transplant with cells cultured in serum-free STF medium
containing Angptl5 and IGFBP-2 (lane 4 of FIG. 5B) displayed a much
higher engraftment of total hematopoietic (CD45/71.sup.+), myeloid
(CD15/66b.sup.+), B-lymphoid (CD34.sup.-CD19/20.sup.+), and
primitive (CD34.sup.+) human cells than the mouse transplanted with
uncultured cells (lane 1 of FIG. 5B).
[0118] The summary of multi-lineage engraftment of mice
transplanted with uncultured cells (FIG. 5B lane 2) and cells
cultured in STF medium containing Angptl5 and IGFBP-2 (FIG. 5B lane
4) is shown in FIG. 5D. The progeny of 8,000 cells, after culture,
repopulated myeloid and lymphoid lineages 2 months post-transplant,
demonstrating the expansion of human stem cell activity. Some mice
transplanted with uncultured cells had zero percent donor
repopulation and these data points are not plotted. (* Values are
significantly different from the values of the uncultured cells.
Student's t-test, p<0.05.) FIG. 5D shows human hematopoietic
engraftment at 2 months in a representative mouse that was
transplanted with uncultured or cultured human cord blood
CD133.sup.+ cells. The progeny of 8,000 cells, after culture,
repopulated myeloid and lymphoid lineages 2 months post-transplant,
demonstrating the expansion of human stem cell activity.
Expansion of Human HSCs in Culture
[0119] To measure the self-renewal potential of SCID-repopulating
cells (SRCs), bone marrow was collected from the primary mice
transplanted with uncultured cells (lane 2 of FIG. 5B) or cells
cultured in STF medium containing Angptl5 and IGFBP-2 (lane 4 of
FIG. 5B) and transplanted them into sublethally irradiated
secondary recipients. Bone marrow aspirate from one hind leg from a
primary recipient was used to transplant two secondary recipients.
Multilineage engraftment in secondary NOD/SCID recipients was
assayed at 5-8 weeks post-transplant (n=12 mice transplanted).
While uncultured cells could not engraft secondary recipients (not
shown), the cultured cells, again, showed positive engraftment
after secondary transplantation (FIG. 5E). These data indicate a
net expansion of human HSCs during the initial culture period. Two
additional independent experiments demonstrated that human HSCs
were dramatically expanded in culture using the methods described
herein.
Limiting Dilution Analysis of Human Cord Blood CD133.sup.+ Cells
Transplanted into NOD/SCID Mice after Culture at Normal or Low
Oxygen Levels.
[0120] The culture system provided herein dramatically expands
human SRCs as demonstrated by several additional independent
experiments. In one representative experiment, 2.times.10.sup.5
human cord blood CD133.sup.+ cells were cultured in STF medium
containing 500 ng/ml Angptl5 and 100 ng/ml IGFBP-2 under normal or
low O.sub.2 conditions. The numbers of total cells (FIG. 6A) and
CD34.sup.+ cells (FIG. 6B) were counted. After 10 days of culture,
the number of total cells in these two conditions did not differ
significantly and both increased greater than 200 fold (FIG. 6A).
Nevertheless, a higher number of CD34.sup.+ primitive cells was
observed after 5 days of culture at normal O.sub.2 versus low
O.sup.2 (FIG. 6B).
[0121] Limiting dilution assays were performed to quantitate the
SRC frequencies before (FIG. 6C) and after (FIG. 6D) culture. As
demonstrated in FIGS. 6B and 6C, after a 10-day culture, the number
of SRCs cultured in serum-free STF medium containing Angptl5 and
IGFBP-2 at low or normal O.sub.2 increased by 8- or 20-fold,
respectively. Plotted is the percentage of recipient mice
containing less than 1% human hematopoietic populations in
recipient mouse bone marrow 6-8 weeks after transplant versus the
number of input or input-equivalent cells injected. The progeny of
10,000 input cells cultured at normal or low O.sub.2 repopulated
all recipients and these data points (zero percent negative mice)
are not plotted. The frequency of repopulating cells (CRU) for this
particular sample of uncultured CD133.sup.+ cells is 1 per 64,075
cells (95% confidence interval for mean: 1/23,919 to 1/171,643,
n=25). That is, as calculated from Poisson statistics, injection of
on average of 64,075 uncultured human CD133.sup.+ cells is
sufficient to repopulate 63% (=1-1/e) of transplanted mice. When
cells were cultured in STF medium containing Angptl5 and IGFBP-2 at
low O.sub.2, the CRU frequency was 1/7,814 input equivalent cells
(95% confidence interval for mean: 1/3,432 to 1/17,791, n=26),
.about.8 fold greater than that of the uncultured cells.
Strikingly, when the cells were cultured at normal O.sub.2, the CRU
frequency increased to 1/3,209 input equivalent cells (95%
confidence interval for mean: 1/1,889 to 1/5,453, n=27). This
indicates that the total number of functional SRCs increased
.about.20 fold.
[0122] FIG. 6E shows multilineage engraftment in NOD/SCID
recipients transplanted with 20,000 uncultured CD133.sup.+ cells
(left panel, n=8) or cultured progeny from 5,000 initial
CD133.sup.+ cells at normal O.sub.2 (right panel, n=10). Some mice
transplanted with uncultured cells had zero percent donor
repopulation and these data points are not plotted. (total
hematopoietic (cols. 1, and 5, CD45/71+), myeloid (cols. 2 and 6,
CD15/66b+), B- lymphoid (cols. 3 and 7, CD34-CD19/20+), and
primitive (cols. 4 and 8 CD34+) lineages are shown. (* Value is
significantly different from the value of the uncultured cells.
Student's t-test, p<0.05.) As demonstrated in FIG. 6E, these
cultured cells had much greater levels of multi-lineage engraftment
than uncultured cells.
[0123] It has been suggested that hypoxia improves expansion of
human SRCs. (Danet, et al., J Clin Invest 112, 126-35 (2003)). As
demonstrated herein, profound expansion of SRCs was observed in a
hypoxia condition. Unexpectedly, the SRC expansion was even greater
under normoxic pressure.
[0124] Human cord blood CD34.sup.+ cells cultured in the presence
of Angptl5 and IGFBP-2 also achieved expansion of SRCs (data not
shown).
[0125] The use of Angptls and IGFBP-2 for the ex vivo expansion of
human HSCs allows increased clinical use of cord blood for bone
marrow transplantation because human HSCs can now be expanded ex
vivo in a defined medium. The technology provided herein will be
useful for the development of novel strategies of cell and gene
therapies that utilize HSCs.
Angiopoietin 2
Competitive Reconstitution Analysis
[0126] Twenty CD45.2 donor cells were cultured for 10 d in
serum-free conditioned STIF medium or in the same medium with 500
ng/ml purified human angiopoietin 1, human angiopoietin 2, mouse
angiopoietin 3, or human angiopoietin 4. The cells cotransplanted
with 1.times.10.sup.5 freshly isolated CD45.1 competitor bone
marrow cells, into recipient mice. The mixture injected
intravenously via the retro-orbital route into each of a group of
6-9 week old CD45.1 mice previously irradiated with a total dose of
10 Gy. Reconstitution 4 months post transplant was measured. To
measure reconstitution of transplanted mice, peripheral blood was
collected at the indicated times post-transplant and the presence
of CD45.1.sup.+ and CD45.2.sup.+ cells in lymphoid and myeloid
compartments were measured as described (Zhang, C. C. and Lodish,
H. F. Blood 105, 4314-20 (2005)). Briefly, peripheral blood cells
were collected by retro-orbital bleeding, followed by lysis of red
blood cells and staining with anti-CD45.2-FITC, and anti-CD45.1-PE,
or anti-Thy1.2-PE (for T-lymphoid lineage), anti-B220-PE (for
B-lymphoid lineage), anti-Mac-1-PE, anti-Gr-1-PE (cells costaining
with anti-Mac-1 and anti-Gr-1 were deemed the myeloid lineage), or
anti-Ter119-PE (for erythroid lineage) monoclonal antibodies (BD
Pharmingen). As shown in the FIG. 7, angiopoietin 2 stimulates ex
vivo expansion of HSCs.
Methods
[0127] Mice.
[0128] C57 BL/6 CD45.2 and CD45.1 mice were purchased from the
Jackson Laboratory or the National Cancer Institute. NOD/SCID
(NOD.CB17-Prkdcscid/J) mice were purchased from the Jackson
Laboratory and were maintained at the Whitehead Institute animal
facility. All animal experiments were performed with the approval
of M.I.T. Committee on Animal Care.
[0129] Culture Medium.
[0130] Serum-free STIF medium is StemSpan serum-free medium
(StemCell Technologies) supplemented with 10 .mu.g/ml heparin
(Sigma), 10 ng/ml mouse SCF, 20 ng/ml mouse TPO, 20 ng/ml mouse
IGF-2 (all from R&D Systems), and 10 ng/ml human FGF-1
(Invitrogen). Serum-free STF medium is the same medium without
IGF-2. Indicated amounts of purified Angptl3 (a gift from R&D
Systems), Angptl5 (Abnova, Taiwan), or IGFBP-2 (R&D Systems)
were added. Conditioned medium was collected from confluent 293T or
3T3 cells after overnight culture.
[0131] Mouse HSC Culture.
[0132] Twenty BM SP Sca-1.sup.+ CD45.sup.+ cells isolated from 8-10
week old C57BL/6 CD45.2 mice were plated in one well of a U-bottom
96-well plate (3799; Corning) with 160 .mu.l of indicated medium.
Cells were cultured at 37.degree. C. in 5% CO.sub.2 and normal
O.sub.2. For the purpose of competitive transplantation, cells were
pooled from at least 6 culture wells and mixed with competitors
before the indicated numbers of cells were transplanted into each
mouse.
[0133] Human Cell Culture.
[0134] Human total cord blood mononuclear cells were purchased from
Cambrex. Cells were plated at 1.times.10.sup.6 cells/ml of STIF
medium, with 100 ng/ml Angptl3 or Angptl5. Medium volume was
increased by adding fresh medium at day 5, 8, 12, 15, and 18 to
maintain cell densities at 5.times.10.sup.5-1.5.times.10.sup.6
cells/ml. Cells were cultured at 37.degree. C. in 5% CO.sub.2 and
normal O.sub.2. Human cyropreserved cord blood CD133.sup.+ cells
used in the experiments of FIGS. 5 and 6 were purchased from
Cambrex and StemCell Technologies Inc. Cells were plated at
1.times.10.sup.4 cells/well in one well of a U-bottom 96-well plate
(3799; Corning) with 200 .mu.l of the indicated medium for 2 days.
At day 3, cells were pooled from individual wells and transferred
to 6-well plates at 5.times.10.sup.4 cells/ml. Fresh medium was
added at days 4 and 7 to keep the cell density at 2.times.10.sup.5
cells/ml (day 4) or 7.times.10.sup.5/ml (day 7). Cells were
cultured at 37.degree. C. in 5% CO.sub.2, and normal O.sub.2 or 5%
O.sub.2 (low O.sub.2) levels.
[0135] NOD/SCID Transplant.
[0136] Uncultured or cultured progeny of human total cord blood
mononuclear cells or CD133.sup.+ cells at indicated days were
collected and injected intravenously via the retro-orbital route
into sub-lethally irradiated (350 rad) NOD/SCID mice. Six to eight
weeks or at indicated time after transplantation, bone marrow
nucleated cells from transplanted animals were analyzed by flow
cytometry for the presence of human cells. For secondary
transplantations, bone marrow aspirates from one hind leg of a
primary recipient were used to transplant two secondary recipients,
as described. (Hogan, et al., Proc Natl Acad Sci USA 99, 413-8
(2002)). Calculation of CRUs in limiting dilution experiments was
conducted using L-Calc software (StemCell Technologies). (Zhang, et
al.,. Proc Natl Acad Sci USA 103, 2184-9 (2006)). Mice were
considered to be positive for human HSC engraftment when at least
1% (for primary transplantation) or 0.1% (for secondary
transplantation) CD45/71.sup.+ human cells were detected among the
mouse bone marrow cells.
[0137] Flow Cytometry.
[0138] Donor bone marrow cells were isolated from 8-10 week old
C57BL/6 CD45.2 mice. SP Sca-1.sup.+ CD45.sup.+ cells were isolated
as described Zhang, C. C. et al., Nat Med 12, 240-5 (2006). For
analyzing repopulation of mouse HSCs, peripheral blood cells of
recipient CD45.1 mice were collected by retro-orbital bleeding,
followed by lysis of red blood cells and staining with
anti-CD45.2-FITC, and anti-CD45.1-PE, or anti-Thy1.2-PE (for
T-lymphoid lineage), anti-B220-PE (for B-lymphoid lineage),
anti-Mac-1-PE, anti-Gr-1-PE (cells costaining with anti-Mac-1 and
anti-Gr-1 were deemed the myeloid lineage), or anti-Ter119-PE (for
erythroid lineage) monoclonal antibodies (BD Pharmingen). The
"Percent repopulation" shown in all Figures except FIG. 1B was
based on the staining results of anti-CD45.2-FITC and
anti-CD45.1-PE. In all cases FACS analysis of the above listed
lineages was also performed to confirm multilineage
reconstitution.
[0139] For analyzing human hematopoietic engraftment in NOD/SCID
mice, a published protocol was followed. (Cashman, et al., J Exp
Med 196, 1141-9 (2002)). Briefly, bone marrow cells from recipient
NOD/SCID mice were stained with anti-human CD45-PE, CD71-PE,
CD15-FITC, and CD66b-FITC to quantify the total human hematopoietic
(CD45/71.sup.+) cell population as well as the subset of
exclusively granulopoietic (CD 15/66b.sup.+) cells within this
population. Cells were stained with anti-human CD34-FITC and
anti-human CD19-PE and CD20-PE to quantify human progenitor
(CD34.sup.+) and B-lineage (CD34.sup.-CD19/20.sup.+) populations.
In the experiment of FIG. 1, only total human hematopoietic
(CD45/71.sup.+) engraftment was measured. Anti-human CD34-FITC was
used to quantitate CD34.sup.+ cells in culture. All anti-human
antibodies were purchased from Becton Dickinson.
[0140] FACS Sorting.
[0141] Donor bone marrow cells were isolated from 8-10 week old
C57BL/6 CD45.2 mice. To sort SP Sca-1.sup.+CD45.sup.+ cells, adult
mouse bone marrow SP cells (stained as previously described (Zhang,
C. C. and Lodish, H. F., Blood 103, 2513-21 (2004); Zhang, C. C.
and Lodish, H. F., Blood 105, 4314-20 (2005)) were further stained
with anti-Sca-1-PE and anti-CD45-FITC followed by cell sorting on a
MoFlo.RTM. sorter.
[0142] Competitive Reconstitution Analysis.
[0143] The indicated numbers of mouse CD45.2 donor cells were mixed
with 1.times.10.sup.5 freshly isolated CD45.1 competitor bone
marrow cells, and the mixture injected intravenously via the
retro-orbital route into each of a group of 6-9 week old CD45.1
mice previously irradiated with a total dose of 10 Gy. To measure
reconstitution of transplanted mice, peripheral blood was collected
at the indicated times post-transplant and the presence of
CD45.1.sup.+ and CD45.2.sup.+ cells in lymphoid and myeloid
compartments were measured as described (Zhang, C. C. and Lodish,
H. F. Blood 103, 2513-21 (2004), Zhang, C. C. and Lodish, H. F.,
Blood (2005)). Calculation of CRUs in limiting dilution experiments
was conducted using L-Calc software (StemCell Technologies) (Zhang,
et al., Proc Natl Acad Sci USA 103, 2184-9 (2006)).
[0144] Western Blots.
[0145] Purified proteins or crude proteins in conditioned medium
were analyzed by electrophoresis on 4-12% NuPage Bis-Tris
polyacrylamide gels (Invitrogen), and proteins were electroblotted
onto nitrocellulose membranes. The membranes were probed with
anti-human IGFBP-2 polyclonal antibody (AF674, R&D Systems) at
0.1 .mu.g/ml, followed with the horseradish peroxidase-conjugated
donkey-anti-goat antibody and detected by a chemiluminescence kit
(Millipore).
[0146] While the technology has been particularly shown and
described with reference to specific embodiments, it should be
understood by those skilled in the art that various changes in form
and detail may be made therein without departing from the spirit
and scope of the technology as defined by the appended claims.
Sequence CWU 1
1
71496PRTHomo sapiens 1Met Trp Gln Ile Val Phe Phe Thr Leu Ser Cys
Asp Leu Val Leu Ala 1 5 10 15 Ala Ala Tyr Asn Asn Phe Arg Lys Ser
Met Asp Ser Ile Gly Lys Lys 20 25 30 Gln Tyr Gln Val Gln His Gly
Ser Cys Ser Tyr Thr Phe Leu Leu Pro 35 40 45 Glu Met Asp Asn Cys
Arg Ser Ser Ser Ser Pro Tyr Val Ser Asn Ala 50 55 60 Val Gln Arg
Asp Ala Pro Leu Glu Tyr Asp Asp Ser Val Gln Arg Leu 65 70 75 80Gln
Val Leu Glu Asn Ile Met Glu Asn Asn Thr Gln Trp Leu Met Lys 85 90
95 Leu Glu Asn Tyr Ile Gln Asp Asn Met Lys Lys Glu Met Val Glu Ile
100 105 110 Gln Gln Asn Ala Val Gln Asn Gln Thr Ala Val Met Ile Glu
Ile Gly 115 120 125 Thr Asn Leu Leu Asn Gln Thr Ala Glu Gln Thr Arg
Lys Leu Thr Asp 130 135 140 Val Glu Ala Gln Val Leu Asn Gln Thr Thr
Arg Leu Glu Leu Gln Leu 145 150 155 160Leu Glu His Ser Leu Ser Thr
Asn Lys Leu Glu Lys Gln Ile Leu Asp 165 170 175 Gln Thr Ser Glu Ile
Asn Lys Leu Gln Asp Lys Asn Ser Phe Leu Glu 180 185 190 Lys Lys Val
Leu Ala Met Glu Asp Lys His Ile Ile Gln Leu Gln Ser 195 200 205 Ile
Lys Glu Glu Lys Asp Gln Leu Gln Val Leu Val Ser Lys Gln Asn 210 215
220 Ser Ile Ile Glu Glu Leu Glu Lys Lys Ile Val Thr Ala Thr Val Asn
225 230 235 240Asn Ser Val Leu Gln Lys Gln Gln His Asp Leu Met Glu
Thr Val Asn 245 250 255 Asn Leu Leu Thr Met Met Ser Thr Ser Asn Ser
Ala Lys Asp Pro Thr 260 265 270 Val Ala Lys Glu Glu Gln Ile Ser Phe
Arg Asp Cys Ala Glu Val Phe 275 280 285 Lys Ser Gly His Thr Thr Asn
Gly Ile Tyr Thr Leu Thr Phe Pro Asn 290 295 300 Ser Thr Glu Glu Ile
Lys Ala Tyr Cys Asp Met Glu Ala Gly Gly Gly 305 310 315 320Gly Trp
Thr Ile Ile Gln Arg Arg Glu Asp Gly Ser Val Asp Phe Gln 325 330 335
Arg Thr Trp Lys Glu Tyr Lys Val Gly Phe Gly Asn Pro Ser Gly Glu 340
345 350 Tyr Trp Leu Gly Asn Glu Phe Val Ser Gln Leu Thr Asn Gln Gln
Arg 355 360 365 Tyr Val Leu Lys Ile His Leu Lys Asp Trp Glu Gly Asn
Glu Ala Tyr 370 375 380 Ser Leu Tyr Glu His Phe Tyr Leu Ser Ser Glu
Glu Leu Asn Tyr Arg 385 390 395 400Ile His Leu Lys Gly Leu Thr Gly
Thr Ala Gly Lys Ile Ser Ser Ile 405 410 415 Ser Gln Pro Gly Asn Asp
Phe Ser Thr Lys Asp Gly Asp Asn Asp Lys 420 425 430 Cys Ile Cys Lys
Cys Ser Gln Met Leu Thr Gly Gly Trp Trp Phe Asp 435 440 445 Ala Cys
Gly Pro Ser Asn Leu Asn Gly Met Tyr Tyr Pro Gln Arg Gln 450 455 460
Asn Thr Asn Lys Phe Asn Gly Ile Lys Trp Tyr Tyr Trp Lys Gly Ser 465
470 475 480Gly Tyr Ser Leu Lys Ala Thr Thr Met Met Ile Arg Pro Ala
Asp Phe 485 490 495 2328PRTHomo sapiens 2Met Leu Pro Arg Val Gly
Cys Pro Ala Leu Pro Leu Pro Pro Pro Pro 1 5 10 15 Leu Leu Pro Leu
Leu Pro Leu Leu Leu Leu Leu Leu Gly Ala Ser Gly 20 25 30 Gly Gly
Gly Gly Ala Arg Ala Glu Val Leu Phe Arg Cys Pro Pro Cys 35 40 45
Thr Pro Glu Arg Leu Ala Ala Cys Gly Pro Pro Arg Val Ala Pro Pro 50
55 60 Ala Ala Val Ala Ala Val Ala Gly Gly Ala Arg Met Pro Cys Ala
Glu 65 70 75 80Leu Val Arg Glu Pro Gly Cys Gly Cys Cys Ser Val Cys
Ala Arg Leu 85 90 95 Glu Gly Glu Ala Cys Gly Val Tyr Thr Pro Arg
Cys Gly Gln Gly Leu 100 105 110 Arg Cys Tyr Pro His Pro Gly Ser Glu
Leu Pro Leu Gln Ala Leu Val 115 120 125 Met Gly Glu Gly Thr Cys Glu
Lys Arg Arg Asp Ala Glu Tyr Gly Ala 130 135 140 Ser Pro Glu Gln Val
Ala Asp Asn Gly Asp Asp His Ser Glu Gly Gly 145 150 155 160Leu Val
Glu Asn His Val Asp Ser Thr Met Asn Met Leu Gly Gly Gly 165 170 175
Gly Ser Ala Gly Arg Lys Pro Leu Lys Ser Gly Met Lys Glu Leu Ala 180
185 190 Val Phe Arg Glu Lys Val Thr Glu Gln His Arg Gln Met Gly Lys
Gly 195 200 205 Gly Lys His His Leu Gly Leu Glu Glu Pro Lys Lys Leu
Arg Pro Pro 210 215 220 Pro Ala Arg Thr Pro Cys Gln Gln Glu Leu Asp
Gln Val Leu Glu Arg 225 230 235 240Ile Ser Thr Met Arg Leu Pro Asp
Glu Arg Gly Pro Leu Glu His Leu 245 250 255 Tyr Ser Leu His Ile Pro
Asn Cys Asp Lys His Gly Leu Tyr Asn Leu 260 265 270 Lys Gln Cys Lys
Met Ser Leu Asn Gly Gln Arg Gly Glu Cys Trp Cys 275 280 285 Val Asn
Pro Asn Thr Gly Lys Leu Ile Gln Gly Ala Pro Thr Ile Arg 290 295 300
Gly Asp Pro Glu Cys His Leu Phe Tyr Asn Glu Gln Gln Glu Ala Arg 305
310 315 320Gly Val Asp Thr Gln Arg Met Gln 325 3491PRTHomo sapiens
3Met Lys Thr Phe Thr Trp Thr Leu Gly Val Leu Phe Phe Leu Leu Val 1
5 10 15 Asp Thr Gly His Cys Arg Gly Gly Gln Phe Lys Ile Lys Lys Ile
Asn 20 25 30 Gln Arg Arg Tyr Pro Arg Ala Thr Asp Gly Lys Glu Glu
Ala Lys Lys 35 40 45 Cys Ala Tyr Thr Phe Leu Val Pro Glu Gln Arg
Ile Thr Gly Pro Ile 50 55 60 Cys Val Asn Thr Lys Gly Gln Asp Ala
Ser Thr Ile Lys Asp Met Ile 65 70 75 80Thr Arg Met Asp Leu Glu Asn
Leu Lys Asp Val Leu Ser Arg Gln Lys 85 90 95 Arg Glu Ile Asp Val
Leu Gln Leu Val Val Asp Val Asp Gly Asn Ile 100 105 110 Val Asn Glu
Val Lys Leu Leu Arg Lys Glu Ser Arg Asn Met Asn Ser 115 120 125 Arg
Val Thr Gln Leu Tyr Met Gln Leu Leu His Glu Ile Ile Arg Lys 130 135
140 Arg Asp Asn Ser Leu Glu Leu Ser Gln Leu Glu Asn Lys Ile Leu Asn
145 150 155 160Val Thr Thr Glu Met Leu Lys Met Ala Thr Arg Tyr Arg
Glu Leu Glu 165 170 175 Val Lys Tyr Ala Ser Leu Thr Asp Leu Val Asn
Asn Gln Ser Val Met 180 185 190 Ile Thr Leu Leu Glu Glu Gln Cys Leu
Arg Ile Phe Ser Arg Gln Asp 195 200 205 Thr His Val Ser Pro Pro Leu
Val Gln Val Val Pro Gln His Ile Pro 210 215 220 Asn Ser Gln Gln Tyr
Thr Pro Gly Leu Leu Gly Gly Asn Glu Ile Gln 225 230 235 240Arg Asp
Pro Gly Tyr Pro Arg Asp Leu Met Pro Pro Pro Asp Leu Ala 245 250 255
Thr Ser Pro Thr Lys Ser Pro Phe Lys Ile Pro Pro Val Thr Phe Ile 260
265 270 Asn Glu Gly Pro Phe Lys Asp Cys Gln Gln Ala Lys Glu Ala Gly
His 275 280 285 Ser Val Ser Gly Ile Tyr Met Ile Lys Pro Glu Asn Ser
Asn Gly Pro 290 295 300 Met Gln Leu Trp Cys Glu Asn Ser Leu Asp Pro
Gly Gly Trp Thr Val 305 310 315 320Ile Gln Lys Arg Thr Asp Gly Ser
Val Asn Phe Phe Arg Asn Trp Glu 325 330 335 Asn Tyr Lys Lys Gly Phe
Gly Asn Ile Asp Gly Glu Tyr Trp Leu Gly 340 345 350 Leu Glu Asn Ile
Tyr Met Leu Ser Asn Gln Asp Asn Tyr Lys Leu Leu 355 360 365 Ile Glu
Leu Glu Asp Trp Ser Asp Lys Lys Val Tyr Ala Glu Tyr Ser 370 375 380
Ser Phe Arg Leu Glu Pro Glu Ser Glu Phe Tyr Arg Leu Arg Leu Gly 385
390 395 400Thr Tyr Gln Gly Asn Ala Gly Asp Ser Met Met Trp His Asn
Gly Lys 405 410 415 Gln Phe Thr Thr Leu Asp Arg Asp Lys Asp Met Tyr
Ala Gly Asn Cys 420 425 430 Ala His Phe His Lys Gly Gly Trp Trp Tyr
Asn Ala Cys Ala His Ser 435 440 445 Asn Leu Asn Gly Val Trp Tyr Arg
Gly Gly His Tyr Arg Ser Lys His 450 455 460 Gln Asp Gly Ile Phe Trp
Ala Glu Tyr Arg Gly Gly Ser Tyr Ser Leu 465 470 475 480Arg Ala Val
Gln Met Met Ile Lys Pro Ile Asp 485 490 4493PRTHomo sapiens 4Met
Arg Pro Leu Cys Val Thr Cys Trp Trp Leu Gly Leu Leu Ala Ala 1 5 10
15 Met Gly Ala Val Ala Gly Gln Glu Asp Gly Phe Glu Gly Thr Glu Glu
20 25 30 Gly Ser Pro Arg Glu Phe Ile Tyr Leu Asn Arg Tyr Lys Arg
Ala Gly 35 40 45 Glu Ser Gln Asp Lys Cys Thr Tyr Thr Phe Ile Val
Pro Gln Gln Arg 50 55 60 Val Thr Gly Ala Ile Cys Val Asn Ser Lys
Glu Pro Glu Val Leu Leu 65 70 75 80Glu Asn Arg Val His Lys Gln Glu
Leu Glu Leu Leu Asn Asn Glu Leu 85 90 95 Leu Lys Gln Lys Arg Gln
Ile Glu Thr Leu Gln Gln Leu Val Glu Val 100 105 110 Asp Gly Gly Ile
Val Ser Glu Val Lys Leu Leu Arg Lys Glu Ser Arg 115 120 125 Asn Met
Asn Ser Arg Val Thr Gln Leu Tyr Met Gln Leu Leu His Glu 130 135 140
Ile Ile Arg Lys Arg Asp Asn Ala Leu Glu Leu Ser Gln Leu Glu Asn 145
150 155 160Arg Ile Leu Asn Gln Thr Ala Asp Met Leu Gln Leu Ala Ser
Lys Tyr 165 170 175 Lys Asp Leu Glu His Lys Tyr Gln His Leu Ala Thr
Leu Ala His Asn 180 185 190 Gln Ser Glu Ile Ile Ala Gln Leu Glu Glu
His Cys Gln Arg Val Pro 195 200 205 Ser Ala Arg Pro Val Pro Gln Pro
Pro Pro Ala Ala Pro Pro Arg Val 210 215 220 Tyr Gln Pro Pro Thr Tyr
Asn Arg Ile Ile Asn Gln Ile Ser Thr Asn 225 230 235 240Glu Ile Gln
Ser Asp Gln Asn Leu Lys Val Leu Pro Pro Pro Leu Pro 245 250 255 Thr
Met Pro Thr Leu Thr Ser Leu Pro Ser Ser Thr Asp Lys Pro Ser 260 265
270 Gly Pro Trp Arg Asp Cys Leu Gln Ala Leu Glu Asp Gly His Asp Thr
275 280 285 Ser Ser Ile Tyr Leu Val Lys Pro Glu Asn Thr Asn Arg Leu
Met Gln 290 295 300 Val Trp Cys Asp Gln Arg His Asp Pro Gly Gly Trp
Thr Val Ile Gln 305 310 315 320Arg Arg Leu Asp Gly Ser Val Asn Phe
Phe Arg Asn Trp Glu Thr Tyr 325 330 335 Lys Gln Gly Phe Gly Asn Ile
Asp Gly Glu Tyr Trp Leu Gly Leu Glu 340 345 350 Asn Ile Tyr Trp Leu
Thr Asn Gln Gly Asn Tyr Lys Leu Leu Val Thr 355 360 365 Met Glu Asp
Trp Ser Gly Arg Lys Val Phe Ala Glu Tyr Ala Ser Phe 370 375 380 Arg
Leu Glu Pro Glu Ser Glu Tyr Tyr Lys Leu Arg Leu Gly Arg Tyr 385 390
395 400His Gly Asn Ala Gly Asp Ser Phe Thr Trp His Asn Gly Lys Gln
Phe 405 410 415 Thr Thr Leu Asp Arg Asp His Asp Val Tyr Thr Gly Asn
Cys Ala His 420 425 430 Tyr Gln Lys Gly Gly Trp Trp Tyr Asn Ala Cys
Ala His Ser Asn Leu 435 440 445 Asn Gly Val Trp Tyr Arg Gly Gly His
Tyr Arg Ser Arg Tyr Gln Asp 450 455 460 Gly Val Tyr Trp Ala Glu Phe
Arg Gly Gly Ser Tyr Ser Leu Lys Lys 465 470 475 480Val Val Met Met
Ile Arg Pro Asn Pro Asn Thr Phe His 485 490 5460PRTHomo sapiens
5Met Phe Thr Ile Lys Leu Leu Leu Phe Ile Val Pro Leu Val Ile Ser 1
5 10 15 Ser Arg Ile Asp Gln Asp Asn Ser Ser Phe Asp Ser Leu Ser Pro
Glu 20 25 30 Pro Lys Ser Arg Phe Ala Met Leu Asp Asp Val Lys Ile
Leu Ala Asn 35 40 45 Gly Leu Leu Gln Leu Gly His Gly Leu Lys Asp
Phe Val His Lys Thr 50 55 60 Lys Gly Gln Ile Asn Asp Ile Phe Gln
Lys Leu Asn Ile Phe Asp Gln 65 70 75 80Ser Phe Tyr Asp Leu Ser Leu
Gln Thr Ser Glu Ile Lys Glu Glu Glu 85 90 95 Lys Glu Leu Arg Arg
Thr Thr Tyr Lys Leu Gln Val Lys Asn Glu Glu 100 105 110 Val Lys Asn
Met Ser Leu Glu Leu Asn Ser Lys Leu Glu Ser Leu Leu 115 120 125 Glu
Glu Lys Ile Leu Leu Gln Gln Lys Val Lys Tyr Leu Glu Glu Gln 130 135
140 Leu Thr Asn Leu Ile Gln Asn Gln Pro Glu Thr Pro Glu His Pro Glu
145 150 155 160Val Thr Ser Leu Lys Thr Phe Val Glu Lys Gln Asp Asn
Ser Ile Lys 165 170 175 Asp Leu Leu Gln Thr Val Glu Asp Gln Tyr Lys
Gln Leu Asn Gln Gln 180 185 190 His Ser Gln Ile Lys Glu Ile Glu Asn
Gln Leu Arg Arg Thr Ser Ile 195 200 205 Gln Glu Pro Thr Glu Ile Ser
Leu Ser Ser Lys Pro Arg Ala Pro Arg 210 215 220 Thr Thr Pro Phe Leu
Gln Leu Asn Glu Ile Arg Asn Val Lys His Asp 225 230 235 240Gly Ile
Pro Ala Glu Cys Thr Thr Ile Tyr Asn Arg Gly Glu His Thr 245 250 255
Ser Gly Met Tyr Ala Ile Arg Pro Ser Asn Ser Gln Val Phe His Val 260
265 270 Tyr Cys Asp Val Ile Ser Gly Ser Pro Trp Thr Leu Ile Gln His
Arg 275 280 285 Ile Asp Gly Ser Gln Asn Phe Asn Glu Thr Trp Glu Asn
Tyr Lys Tyr 290 295 300 Gly Phe Gly Arg Leu Asp Gly Glu Phe Trp Leu
Gly Leu Glu Lys Ile 305 310 315 320Tyr Ser Ile Val Lys Gln Ser Asn
Tyr Val Leu Arg Ile Glu Leu Glu 325 330 335 Asp Trp Lys Asp Asn Lys
His Tyr Ile Glu Tyr Ser Phe Tyr Leu Gly 340 345 350 Asn His Glu Thr
Asn Tyr Thr Leu His Leu Val Ala Ile Thr Gly Asn 355 360 365 Val Pro
Asn Ala Ile Pro Glu Asn Lys Asp Leu Val Phe Ser Thr Trp 370 375 380
Asp His Lys Ala Lys Gly His Phe Asn Cys Pro Glu Gly Tyr Ser Gly 385
390 395 400Gly Trp Trp Trp His Asp Glu Cys Gly Glu Asn Asn Leu Asn
Gly Lys 405 410 415 Tyr Asn Lys Pro Arg Ala Lys Ser Lys Pro Glu Arg
Arg Arg Gly Leu 420 425 430 Ser Trp Lys Ser Gln Asn Gly Arg Leu Tyr
Ser Ile Lys Ser Thr Lys 435 440 445 Met Leu Ile His Pro Thr Asp Ser
Glu Ser Phe Glu 450 455 4606406PRTHomo sapiens 6Met Ser Gly Ala Pro
Thr Ala Gly Ala Ala Leu Met Leu Cys Ala Ala 1 5 10
15 Thr Ala Val Leu Leu Ser Ala Gln Gly Gly Pro Val Gln Ser Lys Ser
20 25 30 Pro Arg Phe Ala Ser Trp Asp Glu Met Asn Val Leu Ala His
Gly Leu 35 40 45 Leu Gln Leu Gly Gln Gly Leu Arg Glu His Ala Glu
Arg Thr Arg Ser 50 55 60 Gln Leu Ser Ala Leu Glu Arg Arg Leu Ser
Ala Cys Gly Ser Ala Cys 65 70 75 80Gln Gly Thr Glu Gly Ser Thr Asp
Leu Pro Leu Ala Pro Glu Ser Arg 85 90 95 Val Asp Pro Glu Val Leu
His Ser Leu Gln Thr Gln Leu Lys Ala Gln 100 105 110 Asn Ser Arg Ile
Gln Gln Leu Phe His Lys Val Ala Gln Gln Gln Arg 115 120 125 His Leu
Glu Lys Gln His Leu Arg Ile Gln His Leu Gln Ser Gln Phe 130 135 140
Gly Leu Leu Asp His Lys His Leu Asp His Glu Val Ala Lys Pro Ala 145
150 155 160Arg Arg Lys Arg Leu Pro Glu Met Ala Gln Pro Val Asp Pro
Ala His 165 170 175 Asn Val Ser Arg Leu His Arg Leu Pro Arg Asp Cys
Gln Glu Leu Phe 180 185 190 Gln Val Gly Glu Arg Gln Ser Gly Leu Phe
Glu Ile Gln Pro Gln Gly 195 200 205 Ser Pro Pro Phe Leu Val Asn Cys
Lys Met Thr Ser Asp Gly Gly Trp 210 215 220 Thr Val Ile Gln Arg Arg
His Asp Gly Ser Val Asp Phe Asn Arg Pro 225 230 235 240Trp Glu Ala
Tyr Lys Ala Gly Phe Gly Asp Pro His Gly Glu Phe Trp 245 250 255 Leu
Gly Leu Glu Lys Val His Ser Ile Thr Gly Asp Arg Asn Ser Arg 260 265
270 Leu Ala Val Gln Leu Arg Asp Trp Asp Gly Asn Ala Glu Leu Leu Gln
275 280 285 Phe Ser Val His Leu Gly Gly Glu Asp Thr Ala Tyr Ser Leu
Gln Leu 290 295 300 Thr Ala Pro Val Ala Gly Gln Leu Gly Ala Thr Thr
Val Pro Pro Ser 305 310 315 320Gly Leu Ser Val Pro Phe Ser Thr Trp
Asp Gln Asp His Asp Leu Arg 325 330 335 Arg Asp Lys Asn Cys Ala Lys
Ser Leu Ser Gly Gly Trp Trp Phe Gly 340 345 350 Thr Cys Ser His Ser
Asn Leu Asn Gly Gln Tyr Phe Arg Ser Ile Pro 355 360 365 Gln Gln Arg
Gln Lys Leu Lys Lys Gly Ile Phe Trp Lys Thr Trp Arg 370 375 380 Gly
Arg Tyr Tyr Pro Leu Gln Ala Thr Thr Met Leu Ile Gln Pro Met 385 390
395 400Ala Ala Glu Ala Ala Ser 405 7388PRTHomo sapiens 7Met Met Ser
Pro Ser Gln Ala Ser Leu Leu Phe Leu Asn Val Cys Ile 1 5 10 15 Phe
Ile Cys Gly Glu Ala Val Gln Gly Asn Cys Val His His Ser Thr 20 25
30 Asp Ser Ser Val Val Asn Ile Val Glu Asp Gly Ser Asn Ala Lys Asp
35 40 45 Glu Ser Lys Ser Asn Asp Thr Val Cys Lys Glu Asp Cys Glu
Glu Ser 50 55 60 Cys Asp Val Lys Thr Lys Ile Thr Arg Glu Glu Lys
His Phe Met Cys 65 70 75 80Arg Asn Leu Gln Asn Ser Ile Val Ser Tyr
Thr Arg Ser Thr Lys Lys 85 90 95 Leu Leu Arg Asn Met Met Asp Glu
Gln Gln Ala Ser Leu Asp Tyr Leu 100 105 110 Ser Asn Gln Val Asn Glu
Leu Met Asn Arg Val Leu Leu Leu Thr Thr 115 120 125 Glu Val Phe Arg
Lys Gln Leu Asp Pro Phe Pro His Arg Pro Val Gln 130 135 140 Ser His
Gly Leu Asp Cys Thr Asp Ile Lys Asp Thr Ile Gly Ser Val 145 150 155
160Thr Lys Thr Pro Ser Gly Leu Tyr Ile Ile His Pro Glu Gly Ser Ser
165 170 175 Tyr Pro Phe Glu Val Met Cys Asp Met Asp Tyr Arg Gly Gly
Gly Trp 180 185 190 Thr Val Ile Gln Lys Arg Ile Asp Gly Ile Ile Asp
Phe Gln Arg Leu 195 200 205 Trp Cys Asp Tyr Leu Asp Gly Phe Gly Asp
Leu Leu Gly Glu Phe Trp 210 215 220 Leu Gly Leu Lys Lys Ile Phe Tyr
Ile Val Asn Gln Lys Asn Thr Ser 225 230 235 240Phe Met Leu Tyr Val
Ala Leu Glu Ser Glu Asp Asp Thr Leu Ala Tyr 245 250 255 Ala Ser Tyr
Asp Asn Phe Trp Leu Glu Asp Glu Thr Arg Phe Phe Lys 260 265 270 Met
His Leu Gly Arg Tyr Ser Gly Asn Ala Gly Asp Ala Phe Arg Gly 275 280
285 Leu Lys Lys Glu Asp Asn Gln Asn Ala Met Pro Phe Ser Thr Ser Asp
290 295 300 Val Asp Asn Asp Gly Cys Arg Pro Ala Cys Leu Val Asn Gly
Gln Ser 305 310 315 320Val Lys Ser Cys Ser His Leu His Asn Lys Thr
Gly Trp Trp Phe Asn 325 330 335 Glu Cys Gly Leu Ala Asn Leu Asn Gly
Ile His His Phe Ser Gly Lys 340 345 350 Leu Leu Ala Thr Gly Ile Gln
Trp Gly Thr Trp Thr Lys Asn Asn Ser 355 360 365 Pro Val Lys Ile Lys
Ser Val Ser Met Lys Ile Arg Arg Met Tyr Asn 370 375 380 Pro Tyr Phe
Lys 385
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