U.S. patent application number 15/156851 was filed with the patent office on 2016-12-08 for methods, kits, and compositions for stem cell self-renewal.
The applicant listed for this patent is STOWERS INSTITUTE FOR MEDICAL RESEARCH. Invention is credited to Justin C. Grindley, Linheng Li, John M. Perry.
Application Number | 20160355784 15/156851 |
Document ID | / |
Family ID | 39925972 |
Filed Date | 2016-12-08 |
United States Patent
Application |
20160355784 |
Kind Code |
A1 |
Perry; John M. ; et
al. |
December 8, 2016 |
Methods, Kits, and Compositions for Stem Cell Self-Renewal
Abstract
The present invention relates to methods and kits for expanding
a stem cell population. More particularly, the invention relates,
inter alia, to methods, kits, and compositions for expanding a stem
cell population, particularly a hematopoietic stem cell
population.
Inventors: |
Perry; John M.; (Olathe,
KS) ; Li; Linheng; (Leawood, KS) ; Grindley;
Justin C.; (Kansas City, MO) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
STOWERS INSTITUTE FOR MEDICAL RESEARCH |
Kansas City |
MO |
US |
|
|
Family ID: |
39925972 |
Appl. No.: |
15/156851 |
Filed: |
May 17, 2016 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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12802913 |
Jun 16, 2010 |
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15156851 |
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12589551 |
Oct 23, 2009 |
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12802913 |
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PCT/US2008/005230 |
Apr 23, 2008 |
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12589551 |
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60926065 |
Apr 23, 2007 |
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61066693 |
Feb 22, 2008 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61P 7/06 20180101; C12N
2501/70 20130101; A61P 3/00 20180101; C12N 5/0647 20130101; A61K
35/28 20130101; C12N 2502/11 20130101; A61P 35/02 20180101; C12N
2501/415 20130101; A61P 43/00 20180101; A61P 7/00 20180101; C12N
2501/40 20130101; A61P 35/00 20180101 |
International
Class: |
C12N 5/0789 20060101
C12N005/0789; A61K 35/28 20060101 A61K035/28 |
Claims
1. An ex vivo method for expanding the number of hematopoietic stem
cells (HSC) in a population of mononuclear cells (MNC) comprising
culturing the population of MNCs comprising at least one HSC in an
HSC expansion media for a period of time sufficient to expand the
number of HSCs in the MNC population, wherein the expanded HSCs are
functional with long term, multi-lineage, repopulating
potential.
2. The method according to claim 1, which provides HSCs that, upon
transplant into a recipient, exhibit greater than 5% donor
repopulation.
3. The method according to claim 1, which provides HSCs that, upon
transplant into a recipient, exhibit greater than 25% donor
repopulation.
4. The method according to claim 1, which provides HSCs that, upon
transplant into a recipient, exhibit greater than 45% donor
repopulation.
5. The method according to claim 1, which provides HSCs that, upon
transplant into a recipient, exhibit greater than 60% donor
repopulation.
6. The method according to claim 1, wherein the HSC expansion media
comprises a modulator of the Wnt pathway.
7. The method according to claim 6, wherein the modulator of the
Wnt pathway down-regulates GSK-3.beta..
8. The method according to claim 6, wherein the modulator of the
Wnt pathway is a reversible GSK-3.beta. inhibitor selected from the
group consisting of a small molecule, a biologic, an antisense RNA,
a small interfering RNA (siRNA), and combinations thereof.
9. The method according to claim 8, wherein the reversible
GSK-3.beta. inhibitor is a small molecule.
10. The method according to claim 8, wherein the reversible
GSK-3.beta. inhibitor is selected from the group consisting of
Hymenialdisine, Flavopiridol, Kenpaullone, Alsterpaullone,
Azakenpaullone, Indirubin-30-oxime, 6-Bromoindirubin-30-oxime
(BIO), 6-Bromoindirubin-30-acetoxime, Aloisine A, Aloisine B,
TDZD8, Compound 12, CHIR98014, CHIR99021 (CT99021), CT20026,
Compound 1, SU9516, ARA014418, Staurosporine, Compound 5a, Compound
29, Compound 46, GF109203x (bisindolylmaleimide I), Ro318220
(bisindolylmaleimide IX), SB216763, SB415286, I5, CGP60474,
Compound 8b, TWS119, Compound 1A, Compound 17, Lithium, Beryllium,
Zinc, small molecule GSK-3.beta. inhibitors (Vertex
Pharmaceuticals), NP-12 (Neuropharma), GSK-3.beta. inhibitors
(Amphora), GSK-3.beta. inhibitors (CrystalGenomics), SAR-502250
(Sanofi-Aventis), 3544 (Hoffmann-La Roche), GSK-3.beta. inhibitors
(Lundbeck), TDZD-8 (Cancer Center, University of Rochester),
pharmaceutically acceptable salts thereof, and combinations
thereof.
11. The method according to claim 10, wherein the GSK-3.beta.
inhibitor is CHIR99021.
12. The method according to claim 6, which provides HSCs that, upon
transplant into a recipient, exhibit greater than 60% donor
repopulation.
13. The method according to claim 1, wherein the HSC is obtained
from a mammalian tissue selected from the group consisting of cord
blood, peripheral blood, and bone marrow.
14. An expanded, substantially undifferentiated HSC population made
by the method according to claim 1.
15. An expanded, substantially undifferentiated HSC population made
by the method according to claim 6.
16. A kit for expanding, ex vivo, the number of hematopoietic stem
cells (HSC) in a population of mononuclear cells (MNC), the kit
comprising a GSK-3.beta. inhibitor, and instructions for the use of
the inhibitor, wherein, when used, the kit provides expanded HSCs
that are functional with long term, multi-lineage, repopulating
potential.
17. The kit according to claim 16, wherein the GSK-3.beta.
inhibitor is selected from the group consisting of Hymenialdisine,
Flavopiridol, Kenpaullone, Alsterpaullone, Azakenpaullone,
Indirubin-30-oxime, 6-Bromoindirubin-30-oxime (BIO),
6-Bromoindirubin-30-acetoxime, Aloisine A, Aloisine B, TDZD8,
Compound 12, CHIR98014, CHIR99021 (CT99021), CT20026, Compound 1,
SU9516, ARA014418, Staurosporine, Compound 5a, Compound 29,
Compound 46, GF109203x (bisindolylmaleimide I), Ro318220
(bisindolylmaleimide IX), SB216763, SB415286, I5, CGP60474,
Compound 8b, TWS119, Compound 1A, Compound 17, Lithium, Beryllium,
Zinc, small molecule GSK-3.beta. inhibitors (Vertex
Pharmaceuticals), NP-12 (Neuropharma), GSK-3.beta. inhibitors
(Amphora), GSK-3.beta. inhibitors (CrystalGenomics), SAR-502250
(Sanofi-Aventis), 3544 (Hoffmann-La Roche), GSK-3.beta. inhibitors
(Lundbeck), TDZD-8 (Cancer Center, University of Rochester),
pharmaceutically acceptable salts thereof, and combinations
thereof.
18. The kit according to claim 16, wherein the GSK-3.beta.
inhibitor is CHIR99021.
19. The kit according to claim 16, which provides HSCs that, upon
transplant into a recipient, exhibit greater than 60% donor
repopulation.
20. A media for carrying out ex vivo expansion of a stem cell in a
population of MNCs comprising a fluid media suitable for
maintaining viable stem cells and a GSK-3.beta. inhibitor present
in the media at a concentration sufficient to enable expansion of
the stem cell population while maintaining a long term,
multi-lineage, repopulating potential in the stem cells, wherein
the stem cells, when transplanted into a recipient, exhibit greater
than 5% donor repopulation.
21. An ex vivo method for expanding the number of cells capable of
supporting multi-lineage repopulation in a population of
mononuclear cells (MNC) comprising culturing the population of MNCs
comprising at least one hematopoietic stem cell (HSC) and at least
one hematopoietic progenitor cell in an HSC expansion media for a
period of time sufficient to expand the number of cells capable of
supporting multi-lineage repopulation in the MNC population.
22. The method according to claim 10, wherein the GSK-3.beta.
inhibitor is lithium, a pharmaceutically acceptable salt thereof,
or combinations thereof.
23. An expanded, substantially undifferentiated HSC population made
by the method according to claim 22.
24. The kit according to claim 16, wherein the GSK-3.beta.
inhibitor is lithium, a pharmaceutically acceptable salt thereof,
or combinations thereof.
25. The media according to claim 20, wherein the GSK-3.beta.
inhibitor is lithium, a pharmaceutically acceptable salt thereof,
or combinations thereof.
26. The method according to claim 21, wherein the HSC expansion
media comprises a reversible GSK-3.beta. inhibitor.
27. The method according to claim 26, wherein the reversible
GSK-3.beta. inhibitor is lithium, a pharmaceutically acceptable
salt thereof, or combinations thereof.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. patent
application Ser. No. 12/802,913, filed Jun. 16, 2010, which is a
continuation-in-part of U.S. patent application Ser. No.
12/589,551, filed Oct. 23, 2009, which is a continuation-in-part of
International Application Serial No. PCT/US2008/005230, filed Apr.
23, 2008. The International Application, PCT/US2008/005230, claims
benefit to U.S. Provisional Patent Application Ser. No. 60/926,065,
filed Apr. 23, 2007 and U.S. Provisional Patent Application Ser.
No. 61/066,693, filed Feb. 22, 2008. The entire contents of the
above-mentioned applications are hereby incorporated by reference
as if recited in full herein.
INCORPORATION BY REFERENCE OF SEQUENCE LISTING
[0002] This application contains references to amino acids and/or
nucleic acid sequences that have been filed concurrently herewith
as sequence listing text file "0310504con_Sequences", file size of
1.2 KB, created on May 17, 2016. The aforementioned sequence
listing is hereby incorporated by reference in its entirety
pursuant to 37 C.F.R. .sctn.1.52(e)(5).
FIELD OF THE INVENTION
[0003] The present invention relates to methods, kits, and
compositions for expanding a stem cell population, particularly an
hematopoietic stem cell population.
BACKGROUND OF THE INVENTION
[0004] Hematopoietic stem cells (HSCs) are clonogenic cells, which
possess the properties of both self-renewal (expansion) and
multilineage potential giving rise to all types of mature blood
cells. HSCs are responsible for hematopoiesis and undergo
proliferation and differentiation to produce mature blood cells of
various lineages while still maintaining their capacity for
self-renewal. The ability to self-renew maintains the HSC
population for the lifespan of an animal and also allows HSCs to
repopulate the bone marrow of lethally irradiated congenic
hosts.
[0005] Early HSC development displays a hierarchical arrangement,
starting from long-term (LT-) HSCs, which have extensive
self-renewal capability, followed by the expansion state, which
corresponds to short-term (ST-) HSCs (having limited self-renewal
ability) and proliferative multipotent progenitors (MPPs) (having
multipotent potential but no self-renewal capability). MPP is also
a stage of priming or preparation for differentiation. An MPP
differentiates and commits to become either a common lymphoid
progenitor (CLP), which gives rise to all the lymphoid lineages, or
a common myeloid progenitor (CMP), which produces all the myeloid
lineages. During this process, the more primitive population gives
rise to a less primitive population of cells, which is unable to
give rise to a more primitive population of cells. The intrinsic
genetic programs that control these processes including the
multipotential, self-renewal, and activation (or transient
amplification) of HSCs, and lineage commitment from MPP to CLP or
CMP, remain largely unknown.
[0006] To sustain constant generation of blood cells for the
lifetime of an individual, HSCs located in bone marrow niches
(Zhang, J. et al. Nature 425, 836-841, 2003; Calvi, L. M. et al.
Nature 425, 841-846, 2003; Kiel, M. J., et al. Cell 121, 1109-1121,
2005; Arai, F. et al. Cell 118, 149-161, 2004) must achieve a
balance between quiescence and activation so that immediate demands
for hematopoiesis are fulfilled, while long-term stem cell
maintenance is also assured. In adults, homeostasis between the
quiescent and activated states of stem cells is important to
protect HSCs from losing their potential for self-renewal and, at
the same time, support ongoing tissue regeneration (Li, L. and Xie,
T. Annu. Rev. Cell. Dev. Biol. 21, 605-631, 2005). Over-activation
and expansion of stem cells risks both eventual depletion of the
stem cell population and a predisposition to tumorigenesis.
Although some factors important for stem cell activation have been
identified (Heissig, B. et al. Cell 109, 625-637, 2002), the
molecular events governing the transition between quiescence and
activation are poorly understood.
[0007] Phosphatase and tensin homolog (PTEN) functions as a
negative regulator of the PI3K/Akt pathway, which plays crucial
roles in cell proliferation, survival, differentiation, and
migration (Stiles, B. et al. Dev. Biol. 273, 175-184, 2004). The
PTEN tumor suppressor is commonly mutated in tumors, including
those associated with lymphoid neoplasms, which feature deregulated
hematopoiesis (Mutter, G. L. Am. J. Pathol. 158, 1895-1898, 2001;
Suzuki, a. et al. Immunity 14, 523-534, 2001). PTEN-deficiency has
been associated with expansion of neural and embryonic stem cell
populations (Groszer, M. et al. Science 294, 2186-2189, 2001;
Kimura, T. et al. Development 130, 1691-1700, 2003). But, the role
of PTEN in stem cells and tumorigenesis and the recurrence of
tumors heretofore has been not understood.
[0008] PTEN functions as an antagonist of phosphatidyl inositol
3-kinase (PI3K) (Maehama T & Dixon J E. J Biol Chem.
273:13375-13378. 1998). The serine kinase Akt is downstream of the
PI3K signal (Cross D A, Alessi D R, Cohen P et al. Nature
378:785-789 1995). PTEN has been shown to inhibit Akt and thereby
inhibit the nuclear accumulation of .beta.-catenin (Persad S et al.
J Cell Biol. 153:1161-1174 2001).
[0009] Akt has a broad range of effects. Its major function is to
provide a survival signal and to block apoptosis, complementary to
its regulation of .beta.-catenin function. (Song, G. et al., J.
Cell. Mol. Med., 9(1): 59-71, 2005) Akt acts through a number of
proteins, including mammalian target of rapamycin (mTOR), the
Forkhead family of transcription factors (FoxO), BAD, caspase 9,
murine double minute 2 (Mdm2).
[0010] Akt can directly and indirectly activate the
serine/threonine kinase mTOR, which activates protein translation
through a signaling cascade. (LoPiccolo, J., et al., Anti-Cancer
Drugs, 18:861-874, 2007). Indirect activation occurs through
tuberous sclerosis complex-2 (TSC2), which, when in the
unphosphorylated state, forms a complex with tuberous sclerosis
complex-1 (TSC1, also known as hamartin). This complex promotes the
GTPase activity of Ras homolog enriched in brain (RHEB), which in
turn, acts to down-regulate mTOR activity. Upon phosphorylation by
Akt, however, the ability of the TSC1-TSC2 complex to promote
RHEB's GTPase activity is inhibited, and therefore, mTOR's activity
is promoted. (Cully, M. et al., Nat. Rev. Cancer, 6:184-192, 2006).
mTOR can also form a complex with Rictor, and this complex can
provide positive feedback on the Akt signaling cascade by
phosphorylating and activating Akt. (Sarbassov, D. D., et al.,
Science, 307: 1098-1101, 2005).
[0011] Akt also regulates cell survival through transcriptional
factors, including FoxO. Akt's phosphorylation of FoxO inhibits
FoxO, resulting in inhibition of transcription of several
proapoptotic genes, such as Fas-L, IGFBP1 and Bim. (Datta, S. R.,
et al., Cell, 91:231-241, 1997; Nicholson, K. M., et al., Cell
Signal, 14:381-395, 2002).
[0012] One of the down-stream targets of FoxO is p27 (Kip1), a
potent inhibitor of cyclin E/cdk2 complexes. (Wu, H. et al.,
Oncogene, 22: 3113-3122, 2003). FoxO factors induce expression of
p27, which can bind to cyclin E/cdk2 complexes and inhibit their
activity, resulting in a block in cellular proliferation.
(Burgering, B. M. T. & Medema, R. H., J. Leukocyte Biol.,
73:689-701, 2003). In addition, Akt itself can also directly
phosphorylate p27 on T157, resulting in the redistribution of p27
from the nucleus to the cytoplasm, away from cyclin E/cdk2
complexes. (Id.) Phosphorylation of p27 on T198 was critical for
the binding of p27 to 14-3-3 proteins, and through this pathway,
Akt may directly promote p27's degradation. (Fujita, N., et al., J.
Biol. Chem., 277(32): 28706-28713, 2002).
[0013] Another one of the targets of Akt in promoting cell survival
is BAD, a member of the Bcl-2 family of proteins. In the absence of
Akt phosphorylation, BAD forms a complex with Bcl-2 or Bcl-X on the
mitochondrial membrane and inhibits the anti-apoptotic potential of
Bcl-2 and Bcl-X. (Song, G. et al., J. Cell. Mol. Med., 9(1): 59-71,
2005) Akt phosphorylates BAD on Serine 136, thus releasing BAD from
the Bcl-2/Bcl-X complex. (Song, G. et al., J. Cell. Mol. Med.,
9(1): 59-71, 2005; Datta, S. R., et al., Genes Dev., 13:2905-2927,
1999). Therefore, Akt suppresses BAD-mediated apoptosis and
promotes cell survival.
[0014] Furthermore, by phosphorylation of pro-caspase-9 at Serine
196, Akt inhibits proteolytic processing of pro-caspase-9 to the
active form, caspase-9, which is an initiator and an effecter of
apoptosis (Cardone et al., 1998, Science, 282: 1318-1320, Donepudi,
M. & Grutter, M. G., Biophys. Chem., 145-152, 2002).
[0015] Additionally, Akt regulates cell survival via the Mdm2/p53
pathway. Akt can activate Mdm2 by direct phosphorylation, thereby
inducing the nuclear import of Mdm2 or the up-regulation of Mdm2's
ubiquitin ligase activity. (Mayo L. D., Donner D. B., 2001, Proc.
Natl., Acad. Sci. USA 98:11598-11603; Gottlieb T. M. et al,
Ocogene, 21: 1299-1303, 2002). Mdm2 negatively regulates the p53
protein, which may induce cell death in response to stresses (Oren
M., Cell Death Differ., 10:431-442, 2003), by targeting p53 for
ubiquitin-mediated proteolysis (Haupt, Y. et al., 1997, Nature 387:
296-299) or by binding to the transactivation domain of p53,
thereby inhibiting p53-mediated gene regulation. (Momand, J. et
al., Cell, 69: 1237-1245, 1992) One of the down-stream targets of
p53 is the p21 (CIP1/WAF1) gene. The p53 gene product binds to a
site located 2.4 kb upstream of the p21 coding sequence, and this
binding site confers p53-dependent transcriptional regulation.
(El-Deiry, W. S., et al., Cell, 75: 817-825, 1993) Thus,
down-regulation of p53 also down-regulates the transcription of
p21.
[0016] PTEN not only regulates p53 protein through antagonizing the
Akt-Mdm2 pathway, it can also directly regulate p53. First, PTEN
can enhance p53 transactivation in a phosphatase-independent manner
(Tang, Y. & Eng C., Cancer Research, 66: 736-742, 2006).
Second, PTEN forms a complex with p300 in the nucleus and plays a
role in maintenance of high p53 acetylation, which is the activated
form of p53. (Li A. et al., Molecular Cell, 23 (4): 575-587, 2006).
In turn, p53 may also activate the transcription of PTEN. (Cully,
M. et al., Nat. Rev. Cancer, 6:184-192, 2006).
[0017] Canonical signals in the Wnt pathway are involved in stem
cell proliferation. (Kim, L. & Kimmel, A. R. Current Drug
Targets 7:1411-1419, 2006). Glycogen synthase kinase 3 beta
(GSK-3.beta.) is a part of the Wnt signaling pathway, and its
primary substrate is .beta.-catenin. (Hagen, T et al., J. Biochem.
277(26):23330-23335). In the absence of canonical Wnt signaling,
GSK-3.beta. binds to .beta.-catenin and phosphorylates
.beta.-catenin, thereby targeting .beta.-catenin for ubiquitination
and followed by proteosome-mediated degradation, which is mediated
by Adenomatous Polyposis Coli (APC). (Id., Moon, R. T. et al.,
Science 296:1644-1646. 2002). Canonical Wnt signals induce the
release of .beta.-catenin from GSK-3.beta., thereby activating
.beta.-catenin. (Katoh, M & Katoh, M. Cancer Biol Ther.
5(9):1059-64, 2006). .beta.-catenin then localizes to the nucleus,
where it activates gene transcription. (Id.).
[0018] In view of the foregoing, it would be advantageous to
elucidate the interaction between Wnt and PTEN signaling pathways
and to provide new insights into molecular regulation of stem cell
proliferation and differentiation. It would also be advantageous to
use such insights to provide new methods, kits, and compositions
for expanding stem cells in vivo and ex vivo, which stem cells
would be of the kind and quantity sufficient to transplant into a
suitable recipient.
SUMMARY OF THE INVENTION
[0019] Thus, one embodiment of the invention is an ex vivo method
for expanding the number of hematopoietic stem cells (HSC) in a
population of mononuclear cells (MNC). This method comprises
culturing the population of MNCs comprising at least one HSC in an
HSC expansion media for a period of time sufficient to expand the
number of HSCs in the MNC population, wherein the expanded HSCs are
functional with long term, multi-lineage, repopulating
potential.
[0020] An additional embodiment of the invention is a kit for
expanding, ex vivo, the number of hematopoietic stem cells (HSC) in
a population of mononuclear cells (MNC). The kit comprises a
GSK-3.beta. inhibitor, and instructions for the use of the
inhibitor, wherein, when used, the kit provides expanded HSCs that
are functional with long term, multi-lineage, repopulating
potential.
[0021] A further embodiment of the invention is a media for
carrying out ex vivo expansion of a stem cell in a population of
MNCs. This media comprises a fluid media suitable for maintaining
viable stem cells and a GSK-3.beta. inhibitor present in the media
at a concentration sufficient to enable expansion of the stem cell
population while maintaining a long term, multi-lineage,
repopulating potential in the stem cells, wherein the stem cells,
when transplanted into a recipient, exhibit greater than 5% donor
repopulation.
[0022] Yet another embodiment of the invention is an ex vivo method
for expanding the number of cells capable of supporting
multi-lineage repopulation in a population of mononuclear cells
(MNC). This method comprises culturing the population of MNCs
comprising at least one hematopoietic stem cell (HSC) and at least
one hematopoietic progenitor cell in an HSC expansion media for a
period of time sufficient to expand the number of cells capable of
supporting multi-lineage repopulation in the MNC population.
[0023] Another embodiment of the invention is a method for
expanding a population of stem cells obtained from a tissue
selected from the group consisting of peripheral blood, cord blood,
and bone marrow. This method comprises modulating a PTEN pathway
and a Wnt pathway in the population of stem cells to expand the
number of stem cells.
[0024] Another embodiment of the invention is a method for ex vivo
expansion of a substantially undifferentiated stem cell population.
This method comprises modulating a PTEN pathway and a Wnt pathway
in the undifferentiated stem cell population to expand the number
of undifferentiated stem cells without significant differentiation
of the stem cell population.
[0025] Yet another embodiment of the invention is a method for ex
vivo expansion of an hematopoietic stem cell (HSC) population
obtained from a tissue selected from the group consisting of
peripheral blood, cord blood, and bone marrow. This method
comprises modulating a PTEN pathway and a Wnt pathway in the HSC
population to expand the HSC population to a sufficient quantity
while maintaining a multilineage differentiation potential in the
HSC population, which is sufficient for subsequent transplantation
into a patient in need thereof.
[0026] Another embodiment of the invention is an expanded,
substantially undifferentiated stem cell population made by a
method of the present invention. In a related embodiment, the
invention is an expanded HSC population made by a method of the
present invention.
[0027] An additional embodiment is a method for ex vivo expansion
of hematopoietic stem cells (HSCs) by at least 40-fold, the
expanded HSCs being competent to reconstitute an HSC lineage upon
transplantation into a mammalian patient in need thereof. This
method comprises culturing a population of HSCs in a suitable
culture medium comprising a PTEN inhibitor and a GSK-3.beta.
inhibitor.
[0028] A further embodiment of the invention is a kit for expanding
an hematopoietic stem cell (HSC) population for subsequent
transplantation into a patient in need thereof. The kit comprises a
PTEN inhibitor, a GSK-3.beta. inhibitor, and instructions for the
use of the inhibitors.
[0029] An additional embodiment is a media for carrying out ex vivo
expansion of a stem cell population. The media comprises a fluid
media suitable for maintaining viable stem cells and PTEN and
GSK-3.beta. inhibitors present in the media at concentrations
sufficient to enable expansion of the stem cell population while
maintaining a multilineage differentiation potential in the stem
cells.
[0030] A further embodiment is a method for administering an
hematopoietic stem cell (HSC) to a patient in need thereof. This
method comprises (a) culturing, in a suitable culture media, a
sample containing an HSC population in the presence of a modulator
of a molecule in the PTEN pathway and a modulator of a molecule in
the Wnt pathway for a period of time sufficient to expand the
number of HSCs in the sample to a number sufficient to transplant
into the patient; (b) removing from the culture the PTEN and Wnt
pathway modulators; and (c) administering the HSCs to the
patient.
[0031] A further embodiment of the invention is a method for
reconstituting bone marrow in a patient in need thereof. This
method comprises: (a) culturing, in a suitable culture media, a
sample containing an HSC population in the presence of a modulator
of a molecule in the PTEN pathway and a modulator of a molecule in
the Wnt pathway for a period of time sufficient to expand the
number of HSCs in the sample to a number sufficient to transplant
into the patient; (b) removing from the culture the PTEN and Wnt
pathway modulators; and (c) administering the HSCs to the
patient.
[0032] Another embodiment is a method for expanding a population of
hematopoietic stem cells (HSCs). This method comprises culturing a
population of HSCs under conditions sufficient to result in an
expansion of the HSC population by at least 40-fold, wherein the
expanded population of HSCs is suitable for transplantation into a
mammal in need thereof.
[0033] Yet another embodiment is a method for treating a patient in
need of a transplant selected from the group consisting of a bone
marrow transplant, a peripheral blood transplant, and an umbilical
cord blood transplant. This method comprises administering to the
patient a population of HSCs obtained by a method of the present
invention.
[0034] A further embodiment is a method for expanding a population
of hematopoietic stem cells (HSCs) comprising: (a) obtaining from a
mammal a tissue sample comprising an HSC population; (b) expanding,
in vitro, the HSC population from the sample, wherein: (i) the HSC
population expands by at least 40-fold; and (ii) the expanded HSC
population has the ability to reconstitute an hematopoietic lineage
for at least 4-weeks after transplantation into a recipient.
[0035] An additional embodiment is a method for reconstituting an
hematopoietic stem cell lineage in a recipient in need thereof.
This method comprises: (a) obtaining from a mammal a tissue sample
comprising an HSC population; (b) expanding, in vitro, the HSC
population from the sample, wherein: (i) the HSC population expands
by at least 40-fold; and (ii) the expanded HSC population has the
ability to reconstitute an hematopoietic lineage for at least
4-weeks after transplantation into a recipient in need thereof; and
(c) transplanting the expanded HSC population into a recipient in
need thereof.
[0036] A further embodiment of the invention is a method for
expanding a hematopoietic stem cell population in a mammal in need
of such expansion. This method comprises administering to the
mammal a therapeutically effective amount of a modulator of Wnt and
Akt for a period of time sufficient to expand the HSC population by
at least 40-fold with HSCs that possess the ability to reconstitute
an hematopoietic lineage in the mammal.
[0037] These and other aspects of the invention are further
disclosed in the detailed description and examples which
follow.
BRIEF DESCRIPTION OF THE DRAWINGS
[0038] The patent or application file contains at least one drawing
executed in color. Copies of this patent or patent application
publication with color drawing(s) will be provided by the Office
upon request and payment of the necessary fee.
[0039] The following drawings form part of the present
specification and are included to further demonstrate certain
aspects of the present invention. The invention may be better
understood by reference to one or more of these drawings in
combination with the detailed description of specific embodiments
presented herein.
[0040] FIG. 1A-FIG. 1K are a series of bar graphs and fluorescence
activated cell sorting ("FACS") analyses that collectively show
that loss of PTEN with constitutively active .beta.-catenin leads
to hematopoietic stem cell (HSC) expansion with loss of early
hematopoietic progenitors.
[0041] FIG. 1A is two bar graphs showing the absolute numbers (per
femur+tibia) of lineage negative, Sca-1.sup.+Kit.sup.+ (LSK) cells
in Scl-Cre negative control and Scl-Cre.sup.+ PTEN with
constitutively activated .beta.-catenin (Pten:Ctnnb1, also referred
to as Pten:.beta.-cat.sup.Act) double mutant and each single mutant
bone marrow (top) and spleen (bottom) as determined by FACS
analysis. (Harada, N., et al., Embo J, 18(21): 5931-42 1999.
Yilmaz, O. H., et al., Nature, 441:475-82 2006. Zhang, J., et al.,
Nature, 441(7092): 518-22 2006.) Mice are at 10 days post-induction
of Tamoxifen. Reduction of LSK cells in double mutant bone marrow
with expansion in the spleen is indicative of mobilization from
bone marrow to spleen. Scl-Cre is an HSC-specific Tamoxifen
inducible Cre-recombinase used to achieve conditional knockout of
LoxP flanked (floxed) Pten and Ctnnb1 alleles. (Gothert, J. R., et
al., Blood, 105(7): 2724-2732, 2005.)
[0042] FIG. 1B-FIG. 1C show representative results of FACS analysis
(pre-gated for lineage negative (Lin.sup.-) cells) of LSK cells
(right blue box) and myeloid progenitors (left blue box) in
control, single and double mutant bone marrow and spleen as
indicated. Mean frequencies are based on total cell number.+-.SD.
Cells were collected from mice at 6 weeks post-induction (wpi) of
Tamoxifen.
[0043] FIG. 1D shows the frequency of CD34.sup.- cells within the
LSK population in control, single and double mutants at 6 wpi.
[0044] FIG. 1E shows the absolute number of LSK CD34.sup.- cells in
control, single and double mutant bone marrow (tibia+femur) and
spleen at 6 wpi.
[0045] FIG. 1F and FIG. 1G are bar graphs showing the absolute
number of LSK cells per femur and tibia in control, Ctnnb1, Pten,
and Pten:Ctnnb1 double mutant bone marrow (FIG. 1F) and spleen
(FIG. 1G) at 6 weeks post-induction. While the percentage of LSKs
is increased in double mutants (see FIG. 1C), low cellularity of
bone marrow from double mutants yields only moderately increased
absolute numbers compared to control.
[0046] FIG. 1H and FIG. 1I are bar graphs and FACS analysis,
respectively, of percentage of LSK cells, which are Flk2.sup.-
(indicating long-term reconstituting (LT)-HSCs) in control, single
and double mutant bone marrow (as indicated) at 6 weeks
post-induction. Single mutants are not significantly different from
controls at this time point. Boxes in FIG. 1I indicate Flk2.sup.-
(LT HSC) cells.
[0047] FIG. 1J is a set of FACS analyses of CD45 in leukemic
Pten:Ctnnb1 mutant bone marrow. CD45 (high) blast crisis cells are
indicated (blue box, left panel). No blast cell population is
observed in control or Ctnnb1 mutants while a minor population has
been observed in 1 of 1 Pten single mutant mice at 6 weeks
post-induction (data not shown). The right panel shows LSK analysis
of leukemic Pten:Ctnnb1 mutant mouse bone marrow. Note the
conversion to blast cells (lower left) with only a remnant LSK
population (compare to FIG. 1C).
[0048] FIG. 1K is a bar graph showing early hematopoietic
progenitors defined by FACS analysis in control, single, and double
mutant bone marrow as indicated. Common myeloid progenitor (CMP);
granulocyte-monocyte progenitor (GMP); megakaryocyte-erythrocyte
progenitor (MEP); and common lymphoid progenitor (CLP).
[0049] FIG. 2A-FIG. 2J are a series of photographs, bar graphs, and
FACS analyses that collectively show that double mutant HSCs expand
dramatically in vitro and in vivo but fail to differentiate.
[0050] FIG. 2A is a series of photographs showing 100 LSK cells
isolated from control, active .beta.-catenin (Ctnnbl), Pten mutant,
and double mutant (Pten:Ctnnbl) mice after 10 days in culture
(original magnification 100.times.). Cell numbers are not
dramatically increased from 100 seeded LSKs in control while Ctnnb1
single mutant LSKs do not survive. In contrast, Pten single mutant
LSKs exhibit greater proliferation but appear more heterogeneous
indicating more significant differentiation. The greatest and most
homogeneous expansion occurs from Pten:Ctnnb1 double mutant
LSKs.
[0051] FIG. 2B is a set of photographs showing LSK cells from Pten
and Pten:Ctnnb1 mutants at 34 days culture (original magnification
200.times.). (Note: wild-type control cultures do not expand beyond
4 weeks; Ctnnbl mutant cultures do not survive beyond 10 days.)
Pten mutant HSC cultures appear more heterogeneous with significant
cell clumping and more irregular cell morphology. Also note the
spindle-shaped adherent cells (arrows) showing differentiation. In
contrast, double mutant HSC cultures exhibit consistent morphology.
Therefore, while Pten single mutant LSKs survive and expand, they
have undergone more significant differentiation than the much more
homogeneous Pten:Ctnnb1 double mutant LSKs.
[0052] FIG. 2C and FIG. 2D are bar graphs showing the results of an
expansion experiment. Pten and Pten:Ctnnb1 LSK seven week cultures
were counted and analyzed by FACS for maintenance of the LSK
phenotype (wild-type control and Ctnnb1 cultures did not survive
this long in vitro). Double mutant LSKs undergo>1,200 fold
expansion vs. 50 fold for Pten single mutant LSKs. LSK purity of
cultures is significantly greater in Pten:Ctnnb1 cultures
maintaining the LSK phenotype in about 85% of total live cells vs.
about 50% for Pten single mutant cultures.
[0053] FIG. 2E is a FACS analysis of a 7 week culture of
Pten:Ctnnb1 LSK cells (pre-gated on live, lineage negative cells)
along with isotype control. The boxed area indicates
Kit.sup.+Sca-1.sup.+ (LSK) cells.
[0054] FIG. 2F and FIG. 2G are FACS analyses showing a transplant
engraftment experiment. At 5 weeks culture (see FIG. 2B), Pten and
Pten:Ctnnb1 LSK cultures were re-sorted and 1000 LSK cells
(CD45.2.sup.+) from each were transplanted into lethally irradiated
(10Gy) CD45.1.sup.+ recipient mice along with 2.times.10.sup.5
congenic whole bone marrow competitor cells. Because wild-type
cells did not survive 5 weeks culture, 1000 fresh wild-type LSK
cells were also transplanted as a separate control group. At 4
weeks post-transplant, there was no detectable engraftment from
peripheral blood analysis of mice transplanted with either Pten or
Pten:Ctnnbl LSK cultures (data not shown). At 5 weeks
post-transplant, bone marrow from recipient mice was analyzed for
donor engraftment (CD45.2.sup.+ cells) and donor LSK cells
(CD45.2.sup.+ LSKs). FIG. 2F and FIG. 2G display representative
donor engraftment (left, boxed areas indicate CD45.2.sup.+ donor
cells) and donor LSK cell engraftment (right, boxed areas indicate
LSK cells) from bone marrow of mice transplanted with 1000 Fresh
LSK cells (FIG. 2F) or 1000 cultured Pten:Ctnnb1 LSK cells (FIG.
2G).
[0055] FIG. 2H-FIG. 2J are bar graphs showing the quantitative
analysis of donor (CD45.2.sup.+) cells (FIG. 2H), donor LSK cells
(FIG. 2I), and fold increase in donor LSKs (FIG. 2J) isolated from
bone marrow of recipient mice described in FIG. 2F and FIG. 2G at 5
weeks post-transplant.
[0056] FIG. 3A-FIG. 3K are schematics, photographs, bar graphs, and
FACS analyses demonstrating that ex vivo pharmacological
manipulation of the PTEN/Akt and Wnt/.beta.-catenin signaling
pathways cooperatively drive functional HSC expansion.
[0057] FIG. 3A is a schematic illustrating representative members
of the Wnt and PTEN pathways. Inhibition of GSK-3.beta. leads to
.beta.-catenin activation which blocks HSC differentiation.
Inhibition of PTEN leads to Akt activation which promotes survival.
Both pathways individually have been shown to promote HSC
proliferation.
[0058] FIG. 3B and FIG. 3C are photographs of HSCs. One hundred LSK
Flk2.sup.- cells were sorted from wild-type (C57BI/6) mice and
cultured in (1) media, (2) media+1 .mu.M CHIR99021 (GSK-3.beta.
inhibitor), (3) media+200 nM Dipotassium
Bis-peroxo(picolinato)oxovanadate (BpV(pic), a PTEN inhibitor), and
(4) media+1 .mu.M CHIR99021+200 nM BpV(pic). An alternative PTEN
inhibitor, Shikonin, was also utilized at 200 nM alone (5) or in
combination with 1 .mu.M CHIR99021 (6). Pictures are at 17 days
culture (FIG. 3B, original magnification 100.times.) and 23 days
(FIG. 3C, original magnification 40.times.). Compared to control,
both inhibitors applied individually yield greater expansion of LSK
cells indicating that GSK-3.beta. inhibition is not strictly
equivalent to constitutive activation of .beta.-catenin shown in
Ctnnb1 mutant LSKs while BpV(pic) yields similar results compared
to Pten mutant LSKs (see FIG. 2). Similar to double mutant LSKs
(FIG. 2), the greatest expansion is shown with both inhibitors
present (FIG. 3B/FIG. 3C, panel 4).
[0059] FIG. 3D is a series of photographs showing LSK Flk2.sup.-
cells at 28 days culture in the indicated media conditions
(original magnification 200.times.). Here, significant expansion
relative to control is observed with both inhibitors present
individually; however, significant differentiation/heterogeneity of
cell morphology is observed in both single inhibitor cultures,
including more variable cell size/morphology and/or differentiation
to adherent, spindle-shaped cells (middle panels). In contrast, and
quite surprisingly, expansion with homogeneity is achieved when
both inhibitors are present (last panel).
[0060] FIG. 3E is a FACS analysis of 28 day LSK Flk2.sup.- cells
cultured in media containing both inhibitors (200 nM BpV(pic) and 1
.mu.M CHIR99021). Cells were pre-gated on live, lineage negative
cells. The boxed area indicates Kit.sup.+Sca1.sup.+ (LSK) cells.
Greater than 90% of LSKs retain Flk2 negativity (data not shown).
The LSK Flk2.sup.- phenotype is maintained with high purity in
cultures containing both inhibitors.
[0061] FIG. 3F is a bar graph showing fold expansion of LSK
Flk2.sup.- cells after 28 days culture in the indicated conditions.
While both inhibitors added individually lead to significant
expansion compared to media without either inhibitor, the greatest
expansion (.about.270 fold) is observed when both inhibitors are
added together.
[0062] FIG. 3G and FIG. 3H are bar graphs showing the %
repopulation and % CD45.2.sup.+ cells from engrafted mice.
Twenty-eight day cultures (FIG. 3D-FIG. 3F) were re-sorted for LSK
Flk2.sup.- cells and 1000 LSK Flk2.sup.- cells (CD45.2.sup.+) from
each media condition were transplanted into lethally irradiated
(10Gy) CD45.1.sup.+ recipient mice along with 2.times.10.sup.5
congenic whole bone marrow competitor cells. At 4 weeks
post-transplant, peripheral blood was analyzed for donor (FIG. 3G)
and multi-lineage (FIG. 3H) engraftment. In FIG. 3G, each bar
represents an individual mouse; the horizontal-dashed line
represents the average "engraftment" of mice transplanted with
competitor cells only and thus the limit of detectability for true
engraftment. Long-term (4 month) engraftment has not been observed
from 28-day cultures (data not shown). 6 of 8 mice show >1%
engraftment when transplanted with LSK Flk2.sup.- cells cultured
with both inhibitors present compared to 4/8 with only CHIR99021
present, 0/10 with only BpV(pic) present, and 2/6with media only.
One percent or greater engraftment is a standard limit for
substantial engraftment. (Zhang, C. C., et al., Nat Med, 12(2):
240-5, 2006. Zhang, C. C. and H. F. Lodish, Blood, 105(11):
4314-20, 2005.) Thus, while use of both inhibitors together leads
to greatest expansion in LSKs (FIG. 3F), transplantation of
equivalent numbers of these cultured LSK Flk2.sup.- cells also
yields increased short-term engraftment/functionality when cultured
with both inhibitors compared to no or either single inhibitor
only.
[0063] FIG. 3I is a bar graph showing the fold expansion of LSK
Flk2.sup.- cells after 9 days culture in (1) media, (2) media+200
nM BpV(pic), (3) media+100 nM CHIR99021, and (4) media+200 nM
BpV(pic)+100 nM CHIR99021. Because long-term engraftment was not
observed from 28 day cultures (FIG. 3D-FIG. 3H and data not shown),
LSK Flk2.sup.- cells were cultured for only 9 days to test if both
expansion and long-term repopulation can be achieved. Similar
trends are observed here to the 28 day cultures (compare to FIG.
3F) although the extent of expansion is substantially reduced at
only 9 days compared to 28 days culture.
[0064] FIG. 3J is a FACS analysis of 9 day LSK Flk2.sup.- cells
cultured in media+200 nM BpV(pic)+100 nM CHIR99021. The boxed area
indicates Kit.sup.+Sca-1.sup.+ (LSK) cells. Cells were pre-gated on
live, lineage negative cells. Greater than 90% of LSKs retain Flk2
negativity (data not shown). Here, the levels of Sca-1 and Kit
appear normal compared to the Sca-1.sup.(high)Kit.sup.(high)
population shown from 28 day cultures (FIG. 3E).
[0065] FIG. 3K is a bar graph showing % repopulation of 10-day
cultured cells in mice. Ten day cultures were transplanted into
lethally irradiated (10Gy) CD45.1.sup.+ recipient mice along with
2.times.10.sup.5 congenic whole bone marrow competitor cells. The
total, non-adherent cell product after 10 days culture of 100
initial LSK Flk2.sup.- cells was transplanted per mouse. At 8 weeks
post-transplant, peripheral blood was analyzed for donor
engraftment. As in FIG. 3H, multi-lineage reconstitution was
observed from all mice exhibiting true engraftment (data not
shown). Each bar represents an individual mouse; the
horizontal-dashed line represents the average `engraftment` of mice
transplanted with competitor cells only and thus the limit of
detectability for true engraftment. Here, 3/7 mice transplanted
with LSK Flk2.sup.- cells cultured in the presence of both
inhibitors exhibited 1% or greater donor engraftment vs. no mice
reaching this threshold in the single or no inhibitor groups.
[0066] FIG. 4A-FIG. 4N show that ex vivo expansion of unsorted bone
marrow mononuclear cells enhances functional long-term
hematopoietic reconstitution relative to sorted, ex vivo expanded
HSCs.
[0067] FIG. 4A is a logarithmic plot of CD45.2 (donor) frequency of
total CD45.sup.+ cells in peripheral blood of transplant
recipients. Red line denotes limit of detectable engraftment as
determined by "engraftment" found in mice transplanted with
competitor cells only.
[0068] FIG. 4B is a linear plot of CD45.2 (donor) frequency of
total CD45.sup.+ cells in peripheral blood of transplant
recipients. Putative HSCs were identified by fluorescence activated
cell sorting (FACS) based upon cell-surface markers, including
lineage marker negative, Sca-1.sup.+, c-Kit.sup.+, Flk2.sup.-
(LSKF), sorted and cultured for 14 days. Bone marrow mononuclear
cells (MNCs) were also fractionated and the concentration of
LSKF.sup.- cells was determined. MNCs containing a known quantity
of LSKF.sup.- cells were cultured for 14 days. After 14 days, the
cellular product of these cultures was transplanted into
lethally-irradiated recipients at a dosage corresponding to an
original input into culture of 100 LSKF.sup.- cells per mouse for
sorted cultures and MNCs containing 5 LSKF.sup.- cells per mouse
for unsorted cultures. In addition, 100 freshly isolated, sorted
LSKF.sup.- cells per mouse and freshly isolated MNCs containing 5
LSKF.sup.- cells per mouse were transplanted into two additional
groups. 1.times.10.sup.5 competitor bone marrow cells congenic with
the hosts (CD45.1.sup.+) were included per mouse. At 4 weeks
post-transplant, peripheral blood was collected from each
transplant recipient, and donor vs. host derived hematopoietic
cells were determined by FACS analysis.
[0069] FIG. 4C shows the percentage of donor derived peripheral
blood cells (CD45.2.sup.+) contributing to the main hematopoietic
lineages (B lymphoid, T lymphoid, and myeloid cells) from
transplant recipients described in FIG. 4A and FIG. 4B at 4 weeks
post-transplantation.
[0070] FIG. 4D-FIG. 4F show repopulation data obtained from
peripheral blood samples from transplant recipients described in
FIG. 4A-FIG. 4C at 16 weeks post-transplant.
[0071] FIG. 4G-FIG. 4H show the results of a secondary
transplantation. At 16 weeks post-transplant, mice transplanted
with MNCs containing 5 LSKF.sup.- cells cultured for 14 days
described in FIG. 4A-FIG. 4F were sacrificed, and bone marrow was
isolated. A secondary transplantation was performed on new groups
of lethally irradiated mice by transplanting 1.times.10.sup.6 bone
marrow cells from the original transplant group per mouse. At 4
weeks post-transplant, peripheral blood was collected from each
transplant recipient and donor-derived repopulation was determined
as in FIG. 4A-FIG. 4B.
[0072] FIG. 4I shows the percentage of donor derived peripheral
blood cells (CD45.2.sup.+) contributing to the main hematopoietic
lineages from transplant recipients described in FIG. 4G-FIG. 4H at
4 weeks post-transplant.
[0073] FIG. 4J-FIG. 4L show repopulation data obtained from
peripheral blood samples from transplant recipients described in
FIG. 4G-FIG. 4I at 16 weeks post-transplant.
[0074] FIG. 4M-FIG. 4N show representative FACS plots of donor
(CD45.2) vs. host (CD45.1) cells obtained from peripheral blood
samples from recipients described in FIG. 4J-FIG. 4K.
[0075] FIG. 5A-FIG. 5F show that culturing with the small-molecule
inhibitor of GSK-3.beta., CHIR99021, enhances long-term engraftment
of ex vivo expanded HSCs.
[0076] FIG. 5A is a logarithmic plot of CD45.2 (donor) frequency of
total CD45.sup.+ cells in peripheral blood of transplant recipients
at 4 weeks post-transplant. FIG. 5B is a linear plot of the same.
Sorted LSKF.sup.- cells and MNCs with a known quantity of
LSKF.sup.- cells were cultured and transplanted as described in
FIG. 4A. Cultures contained media alone or media with 250 nM
CHIR99021 for each group.
[0077] FIG. 5C shows the percentage of donor derived peripheral
blood cells (CD45.2.sup.+) contributing to the main hematopoietic
lineages from transplant recipients described in FIG. 5A-FIG. 5B at
4 weeks post-transplant.
[0078] FIG. 5D-FIG. 5F show the repopulation data obtained from
peripheral blood samples from transplant recipients described in
FIG. 5A-FIG. 5C at 16 weeks post-transplant.
[0079] FIG. 6A-FIG. 6H show that the ex vivo expansion protocol
allows for elimination of bone marrow rescue cells and yields
engraftment equivalent to a one-hundred fold greater dosage of
freshly isolated cells.
[0080] FIG. 6A shows CD45.2 (donor) frequency of total CD45.sup.+
cells in peripheral blood of transplant recipients at 4 weeks
post-transplant. Mice transplanted with freshly isolated MNCs
containing 5 LSKF.sup.- cells (indicated by "X") do not survive
beyond 2-3 weeks post-transplant preventing measurement of
engraftment. For this experiment, MNCs with a known quantity of
putative HSCs were cultured with and without CHIR99021 for 14 days.
After 14 days, the cellular product of these cultures was
transplanted into lethally-irradiated recipients at a dosage
corresponding to an original input into culture of MNCs containing
5 LSKF.sup.- cells per mouse. Freshly isolated MNCs containing
either 5 or 500 LSKF- cells were also transplanted into 2
additional lethally irradiated groups of mice. No rescue/competitor
bone marrow cells were included.
[0081] FIG. 6B shows the percentage of donor derived peripheral
blood cells (CD45.2.sup.+) contributing to the main hematopoietic
lineages from transplant recipients described in FIG. 6A at 4 weeks
post-transplant.
[0082] FIG. 6C-FIG. 6D show repopulation data obtained from
peripheral blood samples from transplant recipients described in
FIG. 6A-FIG. 6B at 16 weeks post-transplant.
[0083] FIG. 6E shows the results of a secondary transplant. At 16
weeks post-transplant, mice transplanted with MNCs containing 5 or
500 LSKF.sup.- cells freshly isolated or cultured for 14 days
described in FIG. 6A-FIG. 6D were sacrificed and bone marrow
isolated. A secondary transplantation was performed on new groups
of lethally irradiated mice by transplanting 1.times.10.sup.6 bone
marrow cells from the original transplant group per mouse. At 4
weeks post-transplant, peripheral blood was collected from each
transplant recipient and donor-derived repopulation was determined.
Mice transplanted with freshly isolated MNCs containing 5
LSKF.sup.- cells (indicated by "X") do not survive beyond 2-3 weeks
post-transplant, thus preventing secondary transplantation.
[0084] FIG. 6F shows the percentage of donor derived peripheral
blood cells (CD45.2.sup.+) contributing to the main hematopoietic
lineages from transplant recipients described in FIG. 6E at 4 weeks
post-transplant.
[0085] FIG. 6G-FIG. 6H show repopulation data obtained from
peripheral blood samples from transplant recipients described in
FIG. 6E-FIG. 6F at 16 weeks post-transplant.
[0086] FIG. 7A-FIG. 7C show ex vivo expansion of human HSCs. In
FIG. 7A, bone marrow and mobilized peripheral blood was collected
from human patients. Putative HSCs (CD34.sup.+CD38.sup.- cells)
were identified by FACS analysis. Ex vivo expansion was performed
with and without CHIR99021. After 14 days culture, the cellular
product of these cultures was analyzed to determine the expansion
of CD34.sup.+CD38.sup.- cells. FIG. 7B-FIG. 7C are representative
FACS plots of CD34.sup.+CD38.sup.- cells prior to (FIG. 7B) and
following (FIG. 7C) ex vivo expansion.
[0087] FIG. 8A shows a .beta.-cat-pS552 immunoassaying of homed
GFP-HSCs. Detection of .beta.-cat-pS552.sup.+ (red) cells adjacent
or close to N-cadherin-LacZ.sup.+ (blue) osteoblasts (OB) which
have been identified with the HSC niche (Xie, Y. et al. Detection
of functional haematopoietic stem cell niche using real-time
imaging. Nature 457, 97-101 (2009); Zhang, J. et al. Identification
of the haematopoietic stem cell niche and control of the niche
size. Nature 425, 836-841 (2003)). "BM" indicates bone marrow. FIG.
8B-FIG. 8D show detection of dividing GFP.sup.+ HSCs (white
arrows). DAPI for nucleic staining, GFP for donor HSC, and red for
.beta.-cat-pS552 GFP signal was imposed on the merged image of
DAPI, DIC and red.
[0088] FIG. 9 shows the percent of Mac-1+ Gr1+ myeloid cells in
bone marrow and spleen at 8-9 weeks post-induction (wpi) in
control, single and double mutants as determined by FACS. Results
are graphed as mean.+-.SD.
[0089] FIG. 10A-FIG. 10F show that double mutant mice lose early
myeloid progenitors as mutant HSCs predominate. Data shown relate
to lethally irradiated recipient mice previously transplanted with
1,000 LSK Flk2.sup.- cells derived from control, single and double
mutant donors +200,000 congenic rescue bone marrow cells. FIG. 10A
shows FACS diagrams of LSK cells (right blue boxes) and myeloid
progenitors (left blue boxes) in control, single and double mutant
bone marrow (top panels) and spleen (bottom panels) as indicated.
As used herein, .beta.-cat.sup.Act is used interchangeably with
Ctnnb1, and Pten:.beta.-cat.sup.Act is used interchangeably with
Pten:Ctnnb1. Mice were at 9 or 10 wpi as indicated. Note the
LS.sup.LowK.sup.Mid population in double mutants at 9 wpi (red
arrows). FIG. 10B shows FACS analysis of early hematopoietic
progenitors in control, single and double mutant bone marrow at 9
wpi. FIG. 10C and FIG. 10D show the absolute number of bone marrow
(per tibia and femur) (FIG. 10C) or spleen (FIG. 10D) LSK cells and
early hematopoietic progenitors in control, single, and double
mutants at 9-10 wpi. Note the collapse of LSK and early progenitor
populations in double mutant bone marrow (red arrows) with
conversion to a dominant "blast" population (see also FIG. 12).
FIG. 10E shows percent donor engraftment at 9 wpi of
lethally-irradiated recipient mice previously transplanted with
1,000 LSK Flk2.sup.- cells derived from control, single and double
mutant donors +200,000 congenic rescue bone marrow cells. FIG. 10F
shows the EGFP-reporter expression of LSK Flk2.sup.- cells in
control, single and double mutants with the Z/EG transgenic
reporter construct at 9 wpi.
[0090] FIG. 11A-FIG. 11G shows Ctnnb1 (.beta.-cat.sup.Act) HSCs
undergo apoptosis whereas .beta.-catenin deletion prevents
PTEN-deficiency-induced HSC expansion but not myeloproliferative
disorder (MPD). To obtain the results shown in FIG. 11A, 1,000 LSK
Flk2.sup.- cells per well were sorted from bone marrow isolated
from uninduced control, Pten, Ctnnb1 (.beta.-cat.sup.Act) and
Pten:Ctnnb1 (Pten:.beta.-cat.sup.Act) mice. Within 12 hours of
sorting, OHT was added to the cultures for a final concentration of
1 .mu.M. Cultures depicted at 4 days post-in vitro induction. FIG.
11B shows control and Ctnnbl (.beta.-cat.sup.Act) cultures as
described in FIG. 11A at 48 hours post-in vitro induction. FIG. 11C
shows representative FACS plots distinguishing live (Sytox Green
negative) from dead (Sytox Green positive) cells. Cultures from
FIG. 11B were stained with Sytox Green and Annexin V according to
manufacturer's instructions (Vybrant Apoptosis Kit #9, Invitrogen)
and analyzed by FACS. Live cells were further gated for Annexin V
positive (apoptotic) cells. Numbers within gates represent the
average.+-.standard deviation from 3 independent experiments. FIG.
11D shows the absolute number of LSK cells and early progenitors in
spleen as determined by FACS analysis. Mice were transplanted with
control, .beta.-cat.sup.-/-, Pten, and Pten:.beta.-cat.sup.-/- mice
bone marrow as indicated; analysis is at 10 wpi. FIG. 11E-FIG. 11G
show the percent of Gr1.sup.+Mac-1.sup.+ cells (FIG. 11E), B-cells
(FIG. 11F), and T-cells (FIG. 11G) in bone marrow of mice described
in FIG. 11D as determined by FACS (see FIG. 20).
[0091] FIG. 12A-FIG. 12C show that Leukemia development and niche
disruption in double mutants. FIG. 12A shows a Kaplan-Meier
survival curve for control, single and double mutants (as indicated
in the figure legend) following tamoxifen induction (Scl-Cre system
unless otherwise specified). FIG. 12B shows H&E stained
sections of control and double mutant bone marrow at 9 wpi. White
arrow indicates grossly normal cellularity in trabecular bone area.
FIG. 12C shows FACS analysis of control, single and double mutant
bone marrow at 10 wpi demonstrating typical CD45 expression. Note
CD45.sup.High blast cells (blue box) only mainly appear in double
mutants. Blast cells from double mutants were further analyzed for
cell surface marker expression of the T-cell specific marker,
CD3.
[0092] FIG. 13A-FIG. 13C show that ex vivo expansion of HSCs is
enhanced by inhibition of GSK3.beta.. For the experimental results
shown in FIG. 13A, sorted LSK Flk2.sup.- cells and unsorted MNCs
containing a known quantity of LSK Flk2.sup.- cells (CD45.2.sup.+)
were cultured for 14 days in ST media with and without CHIR99021.
The cultured product of 100 sorted or 5 unsorted LSK Flk2.sup.-
cells per mouse were transplanted into lethally irradiated
recipients (CD45.1.sup.+). 5 freshly isolated, unsorted LSK
Flk2.sup.- cells per mouse were transplanted into a separate group.
1.times.10.sup.5 freshly isolated CD45.1.sup.+
competitor/radioprotective cells were also added per mouse.
Peripheral blood analysis of recipients at 16 weeks post-transplant
depicts % chimerism. FIG. 13B shows the percentage of donor-derived
peripheral blood cells (CD45.2.sup.+) contributing to the main
hematopoietic lineages (B lymphoid, T lymphoid, and myeloid cells)
from transplant recipients described in (FIG. 13A) at 16 weeks
post-transplantation. FIG. 13C shows representative FACS plots of
donor (CD45.2) vs. host (CD45.1) cells obtained from peripheral
blood samples at 16 weeks post-transplant from recipients described
in FIG. 13A.
[0093] FIG. 14 shows abundant .beta.-cat-pS552.sup.+ cells in
double mutant spleen. Spleen sections stained with .beta.-cat-p5552
antibody in control, single and double mutants at 3 dpi using
Mx1-Cre system. Original magnification 400.times. (upper panels)
and 1000.times. (lower panels).
[0094] FIG. 15 shows trichofolliculoma in double mutants using
Mx1-Cre mediated conditional knockout. Abdomen of Mx1-Cre+
Pten:Ctnnb1 (Pten:.beta.-cat.sup.Act) mutant (left panel, control
mouse at left). H&E stained section of hair follicle tumor
showing multiple, well-developed but densely packed hair follicles
in cross section (right panel).
[0095] FIG. 16A-FIG. 16B show vascular niche disruption by splenic
fibrosis in double mutants. FIG. 16A shows whole spleen isolated
from control, single and double mutants at 9 wpi. Three examples of
double mutant spleen exhibiting mild to severe fibrosis are shown.
Scale bar indicates 1 cm. FIG. 16B shows Masson's Trichrome stained
sections of control, single and double mutant spleens at 9 wpi. Red
arrows indicate examples of collagen fibers (light blue).
[0096] FIG. 17A-FIG. 17B show the number of different types of LSK
cells and early progenitors, as determined by FACS (see FIG. 10)
except that primary mutant mice were utilized here instead of
transplant recipients as in FIG. 10. Absolute number of bone marrow
(per tibia and femur) (FIG. 17A) or spleen (FIG. 17B) LSK cells and
early hematopoietic progenitors in control, single and double
mutants at 9-10 wpi. Note the collapse of LSK and early progenitor
populations in double mutant bone marrow (red arrows) with
conversion to a dominant "blast" population. Compare to FIG.
10.
[0097] FIG. 18 shows that Ctnnb1 (.beta.-cat.sup.Act) mutant HSCs
are not maintained in vivo. LSK Flk2.sup.- cells were sorted from
Scl-Cre negative control and Ctnnb1 (.beta.-cat.sup.Act) mutants at
2 and 16 wpi and genotyped for deletion of exon 3. Primers utilized
were: 5'-CGTGGACAATGGCTACTCAA-3' (forward) (SEQ ID NO: 1) and
5'-TGTCAGCTCAGGAATTGCAC-3' (reverse) (SEQ ID NO: 2) to yield
wild-type (911 bp) and .DELTA.Exon 3 alleles (683 bp). Note that
mice with the dominant .beta.-cat.sup.Act allele are all
heterozygous for this allele.
[0098] FIG. 19A-FIG. 19C show the functional reversibility of
myeloid differentiation blockage in double mutant HSCs. To obtain
the experimental results shown in FIG. 19A, LSK Flk2.sup.- cells
were sorted from uninduced control, Pten, and Pten:Ctnnb1
(Pten:.beta.-cat.sup.Act) mice into an HSC expansion media
containing 0.25 .mu.M 4-hydroxy-tamoxifen (OHT) and cultured for 3
days. Cultures were transduced with lentiviral vector control and
vector expressing shRNA targeting .beta.-catenin transcripts.
Colony forming unit (CFU) assays were performed on day 6. Images
depict typical colonies from control, Pten, and Pten:Ctnnb1
(Pten:.beta.-cat.sup.Act) cultures transduced with control vector
(left panels) and vector expressing shRNA targeting .beta.-catenin
(right panels). Scale bar indicates 0.5 mm. FIG. 19B shows the
quantification of colonies by type from FIG. 19A including early
erythoid progenitors (BFU-E, burst-forming unit-erythroid),
granulocyte-monocyte progenitors (CFU-GM, colony forming
unit-granulocyte/monocyte), and mixed early myeloid progenitors
(CFU-GEMM, granulocyte/erythroid/macrophage/megakaryocyte). Large
CFU (>0.5 mm diameter), which are further characterized in FIG.
19C and form only from double mutant cultures transduced with
control vector, are designated as primitive CFU. FIG. 19C shows
panels depicting representative plots of CD3 expression in control
and Pten:Ctnnb1 (Pten:.beta.-cat.sup.Act) cells transduced with
control vector and Pten:.beta.-cat.sup.Act cells transduced with
vector expressing shRNA targeting .beta.-catenin transcripts. CFU
were harvested, disaggregated into single-cell suspension and
subjected to FACS analysis for CD3 expression. Average percentage
of CD3.sup.+ cells from 3 experiments.+-.S.D. are shown.
[0099] FIG. 20A-FIG. 20G show hematopoietic lineage defects and
leukemogenesis in single vs. double mutants. As in FIG. 10, mice
here refer to transplant recipients of 1,000 LSK Flk2.sup.- cells
derived from control, single and double mutants as indicated along
with 2.times.10.sup.5 congenic rescue bone marrow cells. FIG. 20A
shows the percent of immature (B220.sup.Low, IgM.sup.+), mature
(B220.sup.High, IgM.sup.+) and Pre-Pro B (B220.sup.Low, IgM.sup.-)
cells in control, single and double mutant bone marrow at 8-9 wpi
as determined by FACS. FIG. 20B shows FACS diagrams illustrating
control and double mutant data on T-cell lineage quantified in FIG.
20C. FIG. 20C shows percent of CD3+, double and single positive T
cells in control, single and double mutant bone marrow at 8-9 wpi.
Note the logarithmic scale. FIG. 20D-FIG. 20E show Double Negative
(DN) populations in control, single and double mutant thymus at 8-9
wpi. Representative FACS plots of control (upper panel) and double
mutant (lower panel) thymus are shown in FIG. 20D. Note the
logarithmic scale in FIG. 20E. FIG. 20E-FIG. 20G show double and
single positive thymocyte populations from control, single and
double mutants. Representative FACS plots of control (left panel)
and double mutant (right panel) thymus (FIG. 20F). Results are
graphed as mean.+-.SD (FIG. 20G).
[0100] FIG. 21 shows that PI3K inhibition reverses ex vivo HSC
expansion and inhibits CHIR99021's ability to enhance this
expansion. Bone marrow MNCs were cultured for 10 days in an HSC
expansion media with and without 250 nM CHIR99021, along with the
indicated concentrations of PI3K inhibitor (NVP-BEZ235) (Maira, S.
M. et al. Identification and characterization of NVP-BEZ235, a new
orally available dual phosphatidylinositol 3-kinase/mammalian
target of rapamycin inhibitor with potent in vivo antitumor
activity. Molecular cancer therapeutics 7, 1851-1863 (2008)), and
then subjected to FACS analysis to determine expansion of LSK
Flk2.sup.- cells.
[0101] FIG. 22 shows that both CHIR99021 and lithium enhance
repopulation capacity of ex vivo expanded hematopoietic stem cells.
The experimental conditions of the results shown in FIG. 22 are as
follows. Bone marrow mononuclear cells (MNCs) were fractionated and
the concentration of lineage marker negative, Sca-1.sup.+,
c-Kit.sup.+, Flk2.sup.- (LSKF.sup.-) cells was determined. MNCs
containing a known quantity of LSKF.sup.- cells were cultured for
14 days in media containing SCF and Tpo (ST), ST media with 250 nM
CHIR99021 or ST media with 2 mM lithium chloride (LiCl) as
indicated. After 14 days, the cellular product of these cultures
was transplanted into lethally-irradiated recipients (10 Gray
units) at a dosage corresponding to an original input into culture
of MNCs containing 5 LSKF.sup.- cells per mouse for each group.
1.times.10.sup.5 competitor bone marrow cells congenic with the
hosts (CD45.1.sup.+) were included per mouse. At 4 weeks
post-transplant, peripheral blood was collected from each
transplant recipient, and donor vs. host derived hematopoietic
cells were determined by FACS analysis. Top panel shows a plot of
CD45.2 (donor) frequency of total CD45.sup.+ cells in peripheral
blood of transplant recipients from each group. Bottom panel shows
the percentage of donor derived peripheral blood cells
(CD45.2.sup.+) contributing to the main hematopoietic lineages (B
lymphoid, T lymphoid, and myeloid cells) from transplant recipients
as described above.
[0102] FIG. 23 shows that both CHIR99021 and lithium enhance
long-term repopulation capacity of ex vivo expanded hematopoietic
stem cells. The experimental conditions of the results shown in
FIG. 23 are as follows. Bone marrow mononuclear cells (MNCs) were
fractionated and the concentration of lineage marker negative,
Sca-1.sup.+, c-Kit.sup.+, Flk2.sup.- (LSKF.sup.-) cells was
determined. MNCs containing a known quantity of LSKF.sup.- cells
were cultured for 14 days in media containing SCF and Tpo (ST), ST
media with 250 nM CHIR99021 or ST media with 2 mM lithium chloride
(LiCI) as indicated. After 14 days, the cellular product of these
cultures was transplanted into lethally-irradiated recipients (10
Gray units) at a dosage corresponding to an original input into
culture of MNCs containing 5 LSKF.sup.- cells per mouse for each
group. 1.times.10.sup.5 competitor bone marrow cells congenic with
the hosts (CD45.1.sup.+) were included per mouse. At 16 weeks
post-transplant, peripheral blood was collected from each
transplant recipient, and donor vs. host derived hematopoietic
cells were determined by FACS analysis. The top panel of FIG. 23
shows a plot of CD45.2 (donor) frequency of total CD45.sup.+ cells
in peripheral blood of transplant recipients from each group. The
bottom panel shows the percentage of donor derived peripheral blood
cells (CD45.2.sup.+) contributing to the main hematopoietic
lineages (B lymphoid, T lymphoid, and myeloid cells) from
transplant recipients as described above.
[0103] FIG. 24 shows FACS diagrams with frequencies of early
progenitors in control and double mutants as indicated.
[0104] FIG. 25 shows the absolute number of LSK cells and early
hematopoietic progenitors in control and Pten mutant bone marrow
(tibia and femur) and spleen as determined by FACS analysis at 9-10
wpi.
[0105] FIG. 26A shows FACS diagrams (pre-gated Lin.sup.- cells)
demonstrating pre-culture and 14-day post-culture analysis of LSK
and early progenitor cells with and without addition of lithium.
Frequency of early myeloid (left, blue box) and LSK (right, blue
box) per total, live cells is shown .+-.SD. FIG. 26B shows the
quantification of total cell and LSK Flk2.sup.- cell expansion
after 14-day culture of MNC cells with and without 250 nM
CHIR99021.
[0106] FIG. 27A-FIG. 27C show ex vivo expansion of normal,
long-term repopulating HSC. The experimental conditions of the
results shown in FIG. 27A are as follows. MNCs containing a known
quantity of LSK Flk2.sup.- cells were cultured for 14 days with and
without CHIR99021. The cultured progeny of MNCs containing a
pre-culture dosage of 0.1, 0.3 or 1.0 LSK Flk2.sup.- cells were
transplanted into lethally irradiated recipients along with
1.times.10.sup.5 rescue/competitor cells (n=10 for each of 6
groups). Peripheral blood was analyzed at 16 weeks post-transplant
and competitive repopulating unit (CRU) frequency was determined
using L-Calc software (Stem Cell Technologies, Inc.) based on
Poisson statistics (Zhang, C. C. & Lodish, H. F. Murine
hematopoietic stem cells change their surface phenotype during ex
vivo expansion. Blood, Vol. 105, pages 4314-4320 (2005); Miller, C.
L. & Eaves, C. J. Expansion in vitro of adult murine
hematopoietic stem cells with transplantable lympho-myeloid
reconstituting ability. Proceedings of the National Academy of
Sciences of the United States of America, Vol. 94, pages
13648-13653 (1997)). FIG. 27B shows a serial transplantation assay.
The experimental conditions of the results shown in FIG. 27B are as
follows. Fresh MNCs containing 5 or 500 LSK Flk2.sup.- cells, or
the progeny of MNCs containing 5 LSK Flk2.sup.- cells cultured for
14 days with and without CHIR99021, were transplanted into lethally
irradiated recipients without rescue/competitor cells. At 16-17
weeks post-transplant, bone marrow isolated from 1.degree.
recipients was transplanted into 2.degree. recipients and, at 16-17
weeks post-secondary transplant, from 2.degree. into 3.degree.
recipients at a dosage of 1.times.10.sup.6 cells per mouse.
Peripheral blood was analyzed for percent donor repopulation at 16
weeks post-1.degree., 2.degree., and 3.degree. transplant (upper
panels) and percent mature donor-derived B, T and myeloid cells
(lower panels). FIG. 27C shows Kaplan-Meier survival curve for
3.degree. transplant recipients from FIG. 27B.
[0107] FIG. 28 shows the percent of Mac-1+ Gr1+ myeloid cells in
bone marrow and spleen in control, single and double mutants as
determined by FACS.
[0108] FIG. 29 shows long-term mobilization of LSK cells coupled
with increased myeloid differentiation in Pten single mutants.
Absolute number of LSK cells and early hematopoietic progenitors in
control and Pten mutant bone marrow (tibia and femur) (top panel)
and spleen (lower panel) were determined by FACS analysis at 15
wpi.
[0109] FIG. 30 shows that media without insulin was unable to
substantially expand HSCs. Bone marrow MNCs from C57BL/6 mice were
cultured for 10 days in ST media with or without insulin along with
the indicated concentrations of PI3K inhibitor (NVP-BEZ235) (Maira,
S. M. et al. Identification and characterization of NVP-BEZ235, a
new orally available dual phosphatidylinositol 3-kinase/mammalian
target of rapamycin inhibitor with potent in vivo antitumor
activity. Molecular cancer therapeutics 7, 1851-1863 (2008)) and
then analyzed by FACS to determine expansion of LSK Flk2.sup.-
cells.
DETAILED DESCRIPTION OF THE INVENTION
[0110] One embodiment of the invention is an ex vivo method for
expanding the number of hematopoietic stem cells (HSC) in a
population of mononuclear cells (MNC). This method comprises
culturing the population of MNCs comprising at least one HSC in an
HSC expansion media for a period of time sufficient to expand the
number of HSCs in the MNC population, wherein the expanded HSCs are
functional with long term, multi-lineage, repopulating
potential.
[0111] As used herein, "expand", "expanding" and like terms means
to increase the number of stem cells in the population relative to
the number of stem cells in the original population in vitro, in
vivo or ex vivo using any of the methods disclosed herein.
Preferably, the expansion is at least 40-fold compared to the
original number of stem cells in the population. More preferably,
the expansion is at least 80-fold, 100-fold, 150-fold, 200-fold,
250-fold, or 270-fold compared to the original number of stem
cells.
[0112] In the present invention, "stem cells" mean cells that
possess the ability to give rise to many different types of cells
and which have the ability to self-renew. Representative,
non-limiting examples of stem cells according to the present
invention include bronchioalveolar stem cells (BASCs), bulge
epithelial stem cells (bESCs), corneal epithelial stem cells
(CESCs), cardiac stem cells (CSCs), epidermal neural crest stem
cells (eNCSCs), embryonic stem cells (ESCs), endothelial progenitor
cells (EPCs), hepatic oval cells (HOCs), hematopoetic stem cells
(HSCs), keratinocyte stem cells (KSCs), mesenchymal stem cells
(MSCs), neuronal stem cells (NSCs), pancreatic stem cells (PSCs),
retinal stem cells (RSCs), and skin-derived precursors (SKPs).
[0113] Hematopoietic stem cells or HSCs, for example, have the
ability to self-renew (i.e., expand) and can give rise to all the
types of progenitor cells (such as, e.g., CMP, GMP, MEP and CLP)
and ultimately all the types of blood cells (such as e.g., red
blood cells, B lymphocytes, T lymphocytes, natural killer cells,
neutrophils, basophils, eosinophils, monocytes, macrophages, and
platelets) in the hematopoietic system.
[0114] As used herein, "mononuclear cells" or "MNC" mean blood
cells that have a one-lobed nucleus. MNCs include without
limitation monocytes, lymphocytes, plasma cells, macrophages, and
mast cells.
[0115] As used herein, "HSC expansion media" means any media
suitable for expanding the number of HSC population in a culture.
It includes without limitation, the particular media disclosed in
the Examples.
[0116] As used herein, cells with "long term, multi-lineage
repopulating potential" means cells that are capable of
repopulating many different types of blood cells in irradiated
recipients upon transplantation and/or cells that possess high
proliferative potential in vitro.
[0117] In one aspect of this embodiment, this method provides HSCs
that, upon transplant into a recipient, exhibit greater than 5%
donor repopulation, such as greater than 10%, 15%, 20%, 25%, 30%,
35%, 40%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, or 90% donor
repopulation. Preferably, the method provides HSCs that, upon
transplant into a recipient, exhibit greater than 25%, 35%, 45%, or
60% donor repopulation. Preferably, the recipient is a mammal, for
example, a primate, such as a human; or laboratory animals such as
mice, rats, dogs, and pigs. In the present invention, "recipient"
is used interchangeably with "patient."
[0118] In another aspect of this embodiment, the HSC expansion
media comprises a modulator of the Wnt pathway. Preferably, the
modulator of the Wnt pathway down-regulates GSK-3.beta.. As used
herein, "down-regulating" GSK-3.beta. means decreasing or
inhibiting the expression or the function of GSK-3.beta..
[0119] In the present invention, "a modulator of a Wnt Pathway" (or
"Wnt pathway modulator") is any agent that regulates the activity
of any member of the Wnt pathway, which results in, e.g., increased
.beta.-catenin expression in a stem cell, and/or increased
.beta.-catenin function in a stem cell, and/or increased
.beta.-catenin localization to a nucleus of a stem cell. A
modulator of the Wnt pathway may act upstream or downstream of Wnt.
Preferably, the modulator acts at GSK-3.beta.. Representative,
non-limiting examples of members of the Wnt pathway, include Wnt,
seven-transmembrane Frizzled (Fz), the single-pass, LDL
receptor-related proteins (LRP) 5/6, Axin, Dishevelled, glycogen
synthase kinase 3 beta (GSK-3.beta.), adenomatous polyposis coli
(APC), and .beta.-catenin. Inhibition of GSK-3.beta. leads to Akt
activation which promotes survival.
[0120] In a preferred embodiment, the modulator of the Wnt pathway
is a reversible GSK-3.beta. inhibitor selected from the group
consisting of a small molecule, a biologic, an antisense RNA, a
small interfering RNA (siRNA), and combinations thereof. As used
herein, "reversible" means that the effect of the down-regulation
is not permanent.
[0121] Preferably, the reversible GSK-3.beta. inhibitor is a small
molecule. Examples of reversible GSK-3.beta. inhibitors include
without limitation, Hymenialdisine, Flavopiridol, Kenpaullone,
Alsterpaullone, Azakenpaullone, Indirubin-30-oxime,
6-Bromoindirubin-30-oxime (BIO), 6-Bromoindirubin-30-acetoxime,
Aloisine A, Aloisine B, TDZD8, Compound 12, CHIR98014, CHIR99021
(CT99021), CT20026, Compound 1, SU9516, ARA014418, Staurosporine,
Compound 5a, Compound 29, Compound 46, GF109203x
(bisindolylmaleimide I), Ro318220 (bisindolylmaleimide IX),
SB216763, SB415286, I5, CGP60474, Compound 8b, TWS119, Compound 1A,
Compound 17, Lithium, Beryllium, Zinc, small molecule GSK-3.beta.
inhibitors (Vertex Pharmaceuticals), NP-12 (Neuropharma),
GSK-3.beta. inhibitors (Amphora), GSK-3.beta. inhibitors
(CrystalGenomics), SAR-502250 (Sanofi-Aventis), 3544 (Hoffmann-La
Roche), GSK-3.beta. inhibitors (Lundbeck), TDZD-8 (Cancer Center,
University of Rochester), pharmaceutically acceptable salts
thereof, or combinations thereof. Preferably, the GSK-3.beta.
inhibitor is CHIR99021, or lithium, a pharmaceutically acceptable
salt thereof, or combinations thereof.
[0122] In the present invention, the term "small molecule" includes
any chemical or other moiety, other than polypeptides and nucleic
acids, that can act to affect biological processes, particularly to
modulate members of the Wnt and PTEN pathways. Small molecules can
include any number of therapeutic agents presently known and used,
or that can be synthesized in a library of such molecules for the
purpose of screening for biological function(s). Small molecules
are distinguished from macromolecules by size. The small molecules
of the present invention usually have a molecular weight less than
about 5,000 daltons (Da), preferably less than about 2,500 Da, more
preferably less than 1,000 Da, most preferably less than about 500
Da.
[0123] Small molecules include without limitation organic
compounds, peptidomimetics and conjugates thereof. As used herein,
the term "organic compound" refers to any carbon-based compound
other than macromolecules such as nucleic acids and polypeptides.
In addition to carbon, organic compounds may contain calcium,
chlorine, fluorine, copper, hydrogen, iron, potassium, nitrogen,
oxygen, sulfur and other elements. An organic compound may be in an
aromatic or aliphatic form. Non-limiting examples of organic
compounds include acetones, alcohols, anilines, carbohydrates,
monosaccharides, oligosaccharides, polysaccharides, amino acids,
nucleosides, nucleotides, lipids, retinoids, steroids,
proteoglycans, ketones, aldehydes, saturated, unsaturated and
polyunsaturated fats, oils and waxes, alkenes, esters, ethers,
thiols, sulfides, cyclic compounds, heterocyclic compounds,
imidizoles, and phenols. An organic compound as used herein also
includes nitrated organic compounds and halogenated (e.g.,
chlorinated) organic compounds.
[0124] Preferred small molecules are relatively easier and less
expensively manufactured, formulated or otherwise prepared.
Preferred small molecules are stable under a variety of storage
conditions. Preferred small molecules may be placed in tight
association with macromolecules to form molecules that are
biologically active and that have improved pharmaceutical
properties. Improved pharmaceutical properties include changes in
circulation time, distribution, metabolism, modification,
excretion, secretion, elimination, and stability that are favorable
to the desired biological activity. Improved pharmaceutical
properties include changes in the toxicological and efficacy
characteristics of the chemical entity.
[0125] As used herein, the term "biologic" means products derived
from living sources as opposed to a chemical process. Non-limiting
examples of a "biologic" include proteins, conditioned media, and
partially purified products from tissues.
[0126] The terms "peptide," "polypeptide," and "protein" are used
interchangeably herein. In the present invention, these terms mean
a linked sequence of amino acids, which may be natural, synthetic,
or a modification or combination of natural and synthetic. The term
includes antibodies, antibody mimetics, domain antibodies,
lipocalins, targeted proteases, and polypeptide mimetics. The term
also includes vaccines containing a peptide or peptide fragment
intended to raise antibodies against the peptide or peptide
fragment.
[0127] "Antibody" as used herein includes an antibody of classes
IgG, IgM, IgA, IgD, or IgE, or fragments or derivatives thereof,
including Fab, F(ab')2, Fd, and single chain antibodies, diabodies,
bispecific antibodies, and bifunctional antibodies. The antibody
may be a monoclonal antibody, polyclonal antibody, affinity
purified antibody, or mixtures thereof, which exhibits sufficient
binding specificity to a desired epitope or a sequence derived
therefrom. The antibody may also be a chimeric antibody. The
antibody may be derivatized by the attachment of one or more
chemical, peptide, or polypeptide moieties known in the art. The
antibody may be conjugated with a chemical moiety. The antibody may
be a human or humanized antibody. These and other antibodies are
disclosed in U.S. Published Patent Application No. 20070065447.
[0128] Other antibody-like molecules are also within the scope of
the present invention. Such antibody-like molecules include, e.g.,
receptor traps (such as entanercept), antibody mimetics (such as
adnectins, fibronectin based "addressable" therapeutic binding
molecules from, e.g., Compound Therapeutics, Inc.), domain
antibodies (the smallest functional fragment of a naturally
occurring single-domain antibody (such as, e.g., nanobodies; see,
e.g., Cortez-Retamozo et al., Cancer Res. 2004 Apr. 15;
64(8):2853-7)).
[0129] Suitable antibody mimetics generally can be used as
surrogates for the antibodies and antibody fragments described
herein. Such antibody mimetics may be associated with advantageous
properties (e.g., they may be water soluble, resistant to
proteolysis, and/or be nonimmunogenic). For example, peptides
comprising a synthetic beta-loop structure that mimics the second
complementarity-determining region (CDR) of monoclonal antibodies
have been proposed and generated. See, e.g., Saragovi et al.,
Science. Aug. 16, 1991; 253(5021):792-5. Peptide antibody mimetics
also have been generated by use of peptide mapping to determine
"active" antigen recognition residues, molecular modeling, and a
molecular dynamics trajectory analysis, so as to design a peptide
mimic containing antigen contact residues from multiple CDRs. See,
e.g., Cassett et al., Biochem Biophys Res Commun. Jul. 18, 2003;
307(1):198-205. Additional discussion of related principles,
methods, etc., that may be applicable in the context of this
invention are provided in, e.g., Fassina, Immunomethods. October
1994; 5(2):121-9.
[0130] Targeted proteases are polypeptides which are capable of,
e.g., substrate-targeted inhibition of post-translational
modification such as disclosed in, e.g., U.S. Patent Application
Publication No. 20060275823.
[0131] In general, a polypeptide mimetic ("peptidomimetic") is a
molecule that mimics the biological activity of a polypeptide, but
that is not peptidic in chemical nature. While, in certain
embodiments, a peptidomimetic is a molecule that contains no
peptide bonds (that is, amide bonds between amino acids), the term
peptidomimetic may include molecules that are not completely
peptidic in character, such as pseudo-peptides, semi-peptides, and
peptoids.
[0132] "Antisense" molecules as used herein include antisense or
sense oligonucleotides comprising a single-stranded nucleic acid
sequence (either RNA or DNA) capable of binding to target mRNA
(sense) or DNA (antisense) sequences. The ability to derive an
antisense or a sense oligonucleotide, based upon a cDNA sequence
encoding a given protein is described in, for example, Stein and
Cohen, Cancer Res. 48:2659, (1988) and van der Krol et al.,
BioTechniques 6:958, (1988).
[0133] Antisense molecules can be modified or unmodified RNA, DNA,
or mixed polymer oligonucleotides. These molecules function by
specifically binding to matching sequences resulting in inhibition
of peptide synthesis (Wu-Pong, November 1994, BioPharm, 20-33)
either by steric blocking or by activating an RNase H enzyme.
Antisense molecules can also alter protein synthesis by interfering
with RNA processing or transport from the nucleus into the
cytoplasm (Mukhopadhyay & Roth, 1996, Crit. Rev. in Oncogenesis
7, 151-190). In addition, binding of single stranded DNA to RNA can
result in nuclease-mediated degradation of the heteroduplex
(Wu-Pong, supra). Backbone modified DNA chemistry, which have thus
far been shown to act as substrates for RNase H are
phosphorothioates, phosphorodithioates, borontrifluoridates, and
2'-arabino and 2'-fluoro arabino-containing oligonucleotides.
[0134] Antisense molecules may be introduced into a cell containing
the target nucleotide sequence by formation of a conjugate with a
ligand binding molecule, as described, e.g., in WO 91/04753.
Suitable ligand binding molecules include, but are not limited to,
cell surface receptors, growth factors, other cytokines, or other
ligands that bind to cell surface receptors. Preferably,
conjugation of the ligand binding molecule does not substantially
interfere with the ability of the ligand binding molecule to bind
to its corresponding molecule or receptor, or block entry of the
sense or antisense oligonucleotide or its conjugated version into
the cell. Alternatively, a sense or an antisense oligonucleotide
may be introduced into a cell containing the target nucleic acid
sequence by formation of an oligonucleotide-lipid complex, as
described, e.g., in WO 90/10448.
[0135] The term small interfering RNA ("siRNA") refers to small
inhibitory RNA duplexes that induce the RNA interference (RNAi)
pathway. (Elbashir, S. M. et al. Nature 411:494-498 (2001); Caplen,
N. J. et al. Proc. Natl. Acad. Sci. USA 98:9742-9747 (2001);
Harborth, J. et al. J Cell Sci. 114:4557-4565 (2001).) These
molecules can vary in length (generally 18-30 base pairs) and
contain varying degrees of complementarity to their target mRNA in
the antisense strand. Some, but not all, siRNA have unpaired
overhanging bases on the 5' or 3' end of the sense strand and/or
the antisense strand. The term "siRNA" includes duplexes of two
separate strands, as well as single strands that can form hairpin
structures comprising a duplex region. As used herein, siRNA
molecules are not limited to RNA molecules but further encompass
chemically modified nucleotides and non-nucleotides. siRNA
gene-targeting may be carried out by transient siRNA transfer into
cells, achieved by such classic methods as lipid-mediated
transfection (such as encapsulation in liposome, complexing with
cationic lipids, cholesterol, and/or condensing polymers,
electroporation, or microinjection). siRNA gene-targeting may also
be carried out by administration of siRNA conjugated with
antibodies or siRNA complexed with a fusion protein comprising a
cell-penetrating peptide conjugated to a double-stranded (ds)
RNA-binding domain (DRBD) that binds to the siRNA (see, e.g., U.S.
Patent Application Publication No. 2009/0093026).
[0136] In another preferred embodiment, the method comprises
culturing the population of MNCs comprising at least one HSC in any
of the HSC expansion media disclosed herein, and the method
provides HSCs that, upon transplant into a recipient, exhibit
greater than 5%, such as greater than 10%, 15%, 20%, 25%, 30%, 35%,
40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, or 90% donor
repopulation, preferably, greater than 60% donor repopulation.
[0137] In another aspect of this embodiment, the HSC is obtained
from a mammalian tissue selected from the group consisting of cord
blood, peripheral blood, and bone marrow.
[0138] A further aspect of this embodiment is an expanded,
substantially undifferentiated HSC population made by any of the
methods disclosed herein. Preferably, the substantially
undifferentiated HSC population is made using an HSC expansion
media comprising a modulator of the Wnt pathway. More preferably,
the substantially undifferentiated HSC population is made using an
HSC expansion media comprising lithium, a pharmaceutically
acceptable salt thereof, or combinations thereof.
[0139] A stem cell population is "substantially undifferentiated"
if a sufficient number of cells in that population retain the
ability to self-renew and can give rise to various differentiated
cell types when transplanted into a recipient, for example, in the
case of an HSC population, repopulating the HSC lineage when
transplanted. As used herein, "without significant differentiation"
means the expanded stem cell population has a sufficient number of
cells that maintain a multi-lineage differentiation potential that
the full scope of a target stem lineage may be regenerated upon
transplantation of the expanded stem cell population into a
recipient. Thus, e.g., in the case of an HSC population, the
expanded HSC population, when transplanted into a recipient, is
capable of regenerating the entire hematopoietic cell lineage.
[0140] An additional embodiment of the invention is a kit for
expanding, ex vivo, the number of hematopoietic stem cells (HSC) in
a population of mononuclear cells (MNC). The kit comprises a
GSK-3.beta. inhibitor, and instructions for the use of the
inhibitor, wherein, when used, the kit provides expanded HSCs that
are functional with long term, multi-lineage, repopulating
potential. In one aspect of this embodiment, the GSK-3.beta.
inhibitor is as disclosed herein. Preferably, the GSK-3.beta.
inhibitor is CHIR99021, or lithium, a pharmaceutically acceptable
salt thereof, or combinations thereof.
[0141] In another aspect of this embodiment, the kit provides HSCs
that, upon transplant into a recipient, exhibit greater than 5%,
such as greater than 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%,
55%, 60%, 65%, 70%, 75%, 80%, 85%, or 90% donor repopulation,
preferably, greater than 60% donor repopulation. The kit may be
packaged in any convenient manner and include additional reagents
and/or devices for carrying out its intended purpose.
[0142] A further embodiment of the invention is a media for
carrying out ex vivo expansion of a stem cell in a population of
MNCs. This media comprises a fluid media suitable for maintaining
viable stem cells and a GSK-3.beta. inhibitor present in the media
at a concentration sufficient to enable expansion of the stem cell
population while maintaining a long term, multi-lineage,
repopulating potential in the stem cells, wherein the stem cells,
when transplanted into a recipient, exhibit greater than 5% donor
repopulation, such as greater than 10%, 15%, 20%, 25%,
.sup.30%.sup., 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%,
85%, or 90% donor repopulation. Preferably, the GSK-3.beta.
inhibitor is lithium, a pharmaceutically acceptable salt thereof,
or combinations thereof.
[0143] Yet another embodiment of the invention is an ex vivo method
for expanding the number of cells capable of supporting
multi-lineage repopulation in a population of mononuclear cells
(MNC). This method comprises culturing the population of MNCs
comprising at least one hematopoietic stem cell (HSC) and at least
one hematopoietic progenitor cell in an HSC expansion media for a
period of time sufficient to expand the number of cells capable of
supporting multi-lineage repopulation in the MNC population.
[0144] As used herein, "cells capable of supporting multi-lineage
repopulation" means those cells that are capable of repopulating
many different types of blood cells in irradiated recipients upon
transplantation. Non-limiting examples of such cells include
HSCs.
[0145] As used herein, an "hematopoietic progenitor cell" means a
cell that has lost the capacity of self-renewal but is still able
to give rise to different types of blood cells. Non-limiting
examples of hematopoietic progenitor cells include CMP, GMP, MEP,
and CLP.
[0146] In one aspect of this embodiment, the HSC expansion media
comprises a reversible GSK-3.beta. inhibitor. Preferably, the
GSK-3.beta. inhibitor is lithium, a pharmaceutically acceptable
salt thereof, or combinations thereof.
[0147] Another embodiment of the invention is a method for
expanding a population of stem cells obtained from a tissue
selected from the group consisting of peripheral blood, cord blood,
and bone marrow. This method comprises modulating a PTEN pathway
and a Wnt pathway in the population of stem cells to expand the
number of stem cells.
[0148] In the present invention, "modulating", "modulation" and
like terms mean altering the signal transduction pathway, e.g., a
protein in the PTEN and/or Wnt pathways, including but not limited
to lowering or increasing the expression level of a protein,
altering the sequence of such a protein (by mutation,
pre-translational or post-translational modification or otherwise),
or inhibiting or activating such a protein (whether by binding,
phosphorylation, glycosylation, translocation or otherwise). Such
modulation may be achieved genetically or pharmacologically.
[0149] As used herein, "a modulator of a PTEN pathway" (or "PTEN
pathway modulator") is any agent that regulates the activity of any
member of the PTEN pathway, which results in, e.g., increased
.beta.-catenin expression in a stem cell, and/or increased
.beta.-catenin function in a stem cell, and/or increased
.beta.-catenin localization to a nucleus of a stem cell and/or
provides a survival signal complementary to .beta.-catenin. Thus, a
modulator of the PTEN pathway may act upstream or downstream of
PTEN; preferably the modulator acts at or downstream from PTEN.
Inhibition of PTEN leads to Akt activation which promotes survival
(FIG. 3A). Representative, non-limiting examples of members of the
PTEN pathway, include PTEN, phosphatidylinositol 3-kinase (PI3K),
the serine/threonine protein kinase Akt, and .beta.-catenin.
[0150] Representative non-limiting examples of PI3K modulators,
particularly PI3K activators, include pervanadate (Maude Tessier
and James R. Woodgett, J. Biol. Chem., 281(33):23978-23989 (2006)),
insulin (Hui, L., et al., Brain Research, 1052(1):1-9 (2005)),
insulin-like growth factor (Kenney, A. M., et al., Development,
131:217-228 (2004) and Datta, S. R., et al., Cell, 91:231-241
(1997)), platelet derived growth factor (Datta, S. R., et al., Cell
91:231-241 (1997)), carbachol (Cui, Q L, et al., Neurochem Int,
48:383-393 (2006)), nicotine (West, K. et al., J. Clinical
Investigation, 111:81-90 (2003)),
4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) (Id.),
adrenomedullin (AM) (Nikitenko, L L et al., British J. Cancer,
94:1-7 (2006)), lysophosphatidic acid, platelet activating factor,
macrophage simulating factor, and sphingosine-1-phosphate.
[0151] Representative non-limiting examples of Akt modulators,
particularly Akt activators, include Ro-31-8220 (Wen, H. et al.,
Cellular signaling, 15:37-45 (2003)); Nicotine (West, K. et al., J.
Clinical Investigation, 111:81-90 (2003)); carbachol (Cui Q L,
Fogle E & Almazan G Neurochem Int, 48:383-393 (2006));
4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) (West, K. et
al., J. Clinical Investigation, 111:81-90 (2003)); adrenomedullin
(AM) (Nikitenko, L L et al, British J. Cancer, 94:1-7 (2006));
lysophosphatidic acid; platelet activating factor, macrophage
simulating factor; sphingosine-1-phosphate; cAMP-elevating agents,
such as forskolin, chlorophenylthio-cAMP, prostaglandin-E1, and
8-bromo-cAMP (Song et al., J. Cell. Mol. Med., 9(1):59-71 (2005));
and growth factors, including insulin and insulin growth factor-1
(Datta, S. R., et al., Cell, 91:231-241 (1997)), and platelet
derived growth factor.
[0152] Additional preferred modulators of the present invention
include those that target mTOR, RHEB, FoxO, p27, BAD, caspase-9, or
p53. Representative non-limiting examples of such modulators
include mTOR modulators, particularly mTOR activators, such as
phosphatidic acid (PA) (see, e.g., WO/2006/027545; Foster, D. A.,
Cancer Res, 67(1):1-4 (2007); and Tee et al., J. Biol. Chem.
278:37288-96 (2003)); RHEB modulators, particularly RHEB-GTPase
inhibitors, such as RHEB antibodies (see, e.g., WO/2004/048536);
FoxO modulators, particularly FoxO inhibitors, such as FKH(DBD),
which is a truncated version of FKHRL1 (see, e.g., Gilley, J., et
al., J. Cell Biol. 162(4):613-622 (2003)); p27 modulators,
particularly p27 inhibitors, such as p27 antisense inhibitors and
triplex forming oligonucleotides, protein and peptide antagonists
(see, e.g., U.S. Pat. No. 5,958,769); BAD modulators, particularly
BAD inhibitors, such as 14-3-3 protein (see, e.g., S. Hsu et al.,
Molecular Endocrinology 11 (12):1858-1867 (1997)); caspase-9
modulators, particularly caspase-9 inhibitors, such as LB-84451 (LG
Life Sciences) and Z-LEHD-FMK Caspase Inhibitor (Thornberry, N. A.,
and Lazebnik, Y., Science 281:1312-1316 (1998)); and p53
modulators, particularly p53 inhibitors, such as Pifithrin-.alpha.
and its derivatives (see, e.g., Science, Komarov et al., 285
(5434): 1733-1737 (1999), Pietrancosta et al., Drug Dev Res
65:43-49 (2005)).
[0153] In one aspect of the present invention, modulating the PTEN
pathway comprises introducing a mutation into a population of stem
cells, which mutation results in modulation of a molecule in the
PTEN pathway. In the present invention, modulation of the PTEN
pathway also includes contacting the stem cells with a modulator of
a molecule in the PTEN pathway that leads to .beta.-catenin
activation. Representative, non-limiting examples of such
modulators include a small molecule, a biologic, an antisense RNA,
a small interfering RNA (siRNA), and combinations thereof. This
aspect of the invention further includes modulating the Wnt
pathway, which comprises introducing a mutation into a population
of stem cells that results in modulation of a molecule in the Wnt
pathway. In the present invention modulation of the Wnt pathway
also includes contacting the stem cells with a modulator of a
molecule in the Wnt pathway. Representative, non-limiting examples
of such a modulator include a small molecule, a biologic, an
antisense RNA, a small interfering RNA (siRNA), and combinations
thereof.
[0154] As used herein, "introducing a mutation" means any
conventional method for producing an alteration in the genetic
makeup of the stem cell population. Non-limiting examples for
introducing a mutation into a stem cell population include
mutagenesis via ultra-violet light irradiation, chemical
mutagenesis, targeted mutagenesis such as site directed mutagenesis
of a stem cell, and creation of a transgenic mouse.
[0155] In the present invention, the phrase "modulation of a
molecule in the PTEN pathway" means altering the function of a
member of the PTEN pathway, which altered function has an effect
similar to inhibiting or decreasing the function of PTEN.
Non-limiting examples of such "modulation" include constitutively
activating .beta.-catenin, constitutively activating Akt, or
loss-of-function or null alleles of PTEN. The phrase "modulation of
a molecule in the Wnt pathway" means blocking or decreasing the
function of a member of the Wnt pathway, which has an effect
similar to blocking or decreasing GSK-3.beta. function.
Non-limiting examples of such modulation include constitutively
activating .beta.-catenin and loss-of-function or null alleles of
GSK-3.beta..
[0156] "Modulators of a molecule in the PTEN pathway" are molecules
that cause, directly or indirectly, activation of .beta.-catenin.
Non-limiting examples of such molecules include those that activate
.beta.-catenin, activate Akt, activate PI3K, or inhibit PTEN.
"Modulators of a molecule in the Wnt pathway" are molecules that
directly or indirectly block or decrease the function of a member
of the Wnt pathway. Non-limiting examples of such molecules include
those that activate p-catenin or that inhibit GSK-3.beta., Axin, or
APC.
[0157] In another aspect of the present invention, modulating the
PTEN and Wnt pathways comprises contacting the stem cell population
with a small molecule inhibitor of the PTEN pathway and a small
molecule inhibitor of the Wnt pathway. Preferably, modulating the
PTEN and Wnt pathways comprises down-regulating PTEN and
GSK-3.beta., respectively. As used herein, "down-regulating" means
inhibiting or reducing the amount of or inhibiting or decreasing
the activity of PTEN and GSK-3.beta.. Such down-regulation may be
accomplished using, e.g. antisense RNA, siRNA, antibodies, or small
molecules.
[0158] Preferably, down-regulating PTEN and GSK-3.beta. comprises
contacting the stem cell population with: (a) a reversible PTEN
inhibitor selected from the group consisting of a small molecule, a
biologic, an antisense RNA, a small interfering RNA (siRNA), and
combinations thereof and (b) a reversible GSK-3.beta. inhibitor
selected from the group consisting of a small molecule, a biologic,
an antisense RNA, a small interfering RNA (siRNA), and combinations
thereof. In the present invention, genetic alteration of both the
PTEN and the Wnt pathways leads to an increased ability to
self-renew both in vitro as well as in vivo following long-term
culture but a failure to differentiate and thus a failure to
repopulate the hematopoietic system of transplant recipients. In
contrast, use of reversible down-regulators of both pathways, such
as, e.g., bpV(pic) and CHIR99021, allows for expansion of
functional HSCs, but (1) once the down-regulator is withdrawn,
cultured HSCs can differentiate unlike cultured HSCs from genetic
mutants, and (2) if such cultured HSC are transplanted, recipient
animals do not develop leukemia as genetic mutants do.
[0159] Preferably, both the reversible PTEN inhibitor and the
reversible GSK-3.beta. inhibitor are small molecules. In one
aspect, the reversible PTEN inhibitor is any molecule, such as a
small molecule, which is capable of inhibiting PTEN or a
down-stream member of the PTEN pathway, which inhibition leads to
.beta.-catenin activation. Preferably, the PTEN inhibitor is
selected from the group consisting of shikonin, a bisperoxovanadium
compound, SF-1751 (Semafore Pharmaceuticals), pharmaceutical salts
thereof, and combinations thereof. In this aspect, the
bisperoxovanadium compound is selected from the group consisting of
bpV(phen)2, bpV(pic), pharmaceutical salts thereof, and
combinations thereof.
[0160] In the present invention, the reversible GSK-3.beta.
inhibitor is any molecule that is capable of reversibly inhibiting
GSK-3.beta.. Preferably, such an inhibitor is selected from the
group consisting of Hymenialdisine, Flavopiridol, Kenpaullone,
Alsterpaullone, Azakenpaullone, Indirubin-30-oxime,
6-Bromoindirubin-30-oxime (BIO), 6-Bromoindirubin-30-acetoxime,
Aloisine A, Aloisine B, TDZD8, Compound 12, CHIR98014, CHIR99021
(CT99021), CT20026, Compound 1, SU9516, ARA014418, Staurosporine,
Compound 5a, Compound 29, Compound 46, GF109203x
(bisindolylmaleimide I), Ro318220 (bisindolylmaleimide IX),
SB216763, SB415286, I5, CGP60474, Compound 8b, TWS119, Compound 1A,
Compound 17, Lithium, Beryllium, Zinc, small molecule GSK-3.beta.
inhibitors (Vertex Pharmaceuticals), NP-12 (Neuropharma),
GSK-3.beta. inhibitors (Amphora), GSK-3.beta. inhibitors
(CrystalGenomics), SAR-502250 (Sanofi-Aventis), 3544 (Hoffmann-La
Roche), GSK-3.beta. inhibitors (Lundbeck), TDZD-8 (Cancer Center,
University of Rochester), pharmaceutically acceptable salts
thereof, and combinations thereof.
[0161] While the PTEN and GSK-3.beta. inhibitors may be contacted
with the stem cell population in any convenient manner that
achieves the desired level of stem cell expansion, it is preferred
that the inhibitors are co-administered. Moreover, multiple
GSK-3.beta. and PTEN inhibitors may be contacted with the stem
cells. Furthermore, the PTEN and GSK-3.beta. inhibitors may be
contacted/administered to the stem cells in concert with other
agents suitable for promoting stem cell self renewal. Preferably,
the PTEN inhibitor is bpV(pic) and the GSK-3.beta. inhibitor is
CHIR99021.
[0162] In an additional aspect of the present invention, the number
of stem cells is increased by a factor of at least 40-fold.
Preferably, the number of stem cells is increased by a factor of at
least 80-fold, such as at least 100-fold, including at least
150-fold, at least 200-fold, at least 250-fold, or at least
270-fold. Surprisingly and unexpectedly such levels of stem cell
expansion are achieved using the methods of the present
invention.
[0163] As noted above, the methods of the present invention may be
used to expand any population of stem cells. Representative,
non-limiting examples of stem cells are as set forth above.
Preferably, the stem cells that may be expanded according to the
methods of the present invention may selected from hematopoietic
stem cells (HSCs), endothelial progenitor cells (EPCs), mesenchymal
stem cells (MSCs), cardiac stem cells (CSCs), neuronal stem cells
(NSCs), and combinations thereof. More preferably, the stem cells
are HSCs.
[0164] Another embodiment of the invention is a method for ex vivo
expansion of a substantially undifferentiated stem cell population.
This method comprises modulating a PTEN pathway and a Wnt pathway
in the undifferentiated stem cell population to expand the number
of undifferentiated stem cells without significant differentiation
of the stem cell population.
[0165] A further embodiment of the invention is a method for ex
vivo expansion of an hematopoietic stem cell (HSC) population
obtained from a tissue selected from the group consisting of
peripheral blood, cord blood, and bone marrow. This method
comprises modulating a PTEN pathway and a Wnt pathway in the HSC
population to expand the HSC population to a sufficient quantity
while maintaining a multilineage differentiation potential in the
HSC population, which is sufficient for subsequent transplantation
into a patient in need thereof.
[0166] As used herein, "obtained" from a tissue means any
conventional method of harvesting or partitioning tissue from a
donor. For example, the tissue may obtained from a blood sample,
such as a peripheral or cord blood sample, or harvested from bone
marrow. Methods for obtaining such samples are well known to the
artisan. In the present invention, the samples may be fresh, i.e.,
obtained from the donor without freezing. Moreover, the samples may
be further manipulated to remove extraneous or unwanted components
prior to expansion. The samples may also be obtained from a
preserved stock. For example, in the case of peripheral or cord
blood, the samples may be withdrawn from a cryogenically or
otherwise preserved bank of such blood. Such samples may be
obtained from any suitable donor. Preferably, the donor is a
mammal, for example, a primate, such as a human; or laboratory
animals such as mice, rats, dogs, and pigs. Furthermore, the sample
may be obtained from an autologous or allogeneic donor or source.
Preferably, the sample is obtained from an autologous source.
[0167] In this method, "maintaining a multilineage differentiation
potential" means that the expanded HSC population has the ability,
when transplanted into a patient in need of such a transplant, to
regenerate all the types of progenitor cells e.g., CMP, GMP, MEP,
and CLP, and ultimately all the types of blood cells including,
e.g., red blood cells, B lymphocytes, T lymphocytes, natural killer
cells, neutrophils, basophils, eosinophils, monocytes, macrophages,
and platelets in the hematopoietic system.
[0168] In the present invention, that quantity of expanded HSCs,
which is "sufficient for subsequent transplantation" generally
corresponds to that number of HSCs, which would result in greater
than about 1% engraftment after transplantation. This is one
accepted measure of a successful transplant. In the present
invention, any conventional method may be used to determine the %
engraftment, including the one set forth in the Examples. Such a
measure may be carried out with or without competitor cells,
typically and preferably, without competitor cells. (Zhang, C. C.,
et al., Nat Med, 12(2): 240-5, 2006. Zhang, C. C. and H. F. Lodish,
Blood, 105(11): 4314-20, 2005).
[0169] In the above described ex vivo expansion methods, modulating
the PTEN and Wnt pathways may be achieved as previously set forth.
Modulating the PTEN and Wnt pathways may include contacting the
stem cell population with a small molecule inhibitor of the PTEN
pathway and a small molecule inhibitor of the Wnt pathway.
Modulating the PTEN and Wnt pathways may include down-regulating
PTEN and GSK-3.beta., respectively. Preferably, down-regulating the
PTEN and Wnt pathways comprises contacting the stem cell population
with a reversible PTEN inhibitor and a reversible GSK-3.beta.
inhibitor as previously described. Preferably, both the reversible
PTEN inhibitor and the reversible GSK-3.beta. inhibitor are small
molecules.
[0170] The reversible PTEN inhibitor may be selected from the group
consisting of shikonin, a bisperoxovanadium compound, SF-1751
(Semafore Pharmaceuticals), pharmaceutical salts thereof, and
combinations thereof. Preferably, the bisperoxovanadium compound is
selected from the group consisting of bpV(phen)2, bpV(pic),
pharmaceutical salts thereof, and combinations thereof.
[0171] The reversible GSK-3.beta. inhibitor may be selected from
the group consisting of Hymenialdisine, Flavopiridol, Kenpaullone,
Alsterpaullone, Azakenpaullone, Indirubin-30-oxime,
6-Bromoindirubin-30-oxime (B10), 6-Bromoindirubin-30-acetoxime,
Aloisine A, Aloisine B, TDZD8, Compound 12, CHIR98014, CHIR99021
(CT99021), CT20026, Compound 1, SU9516, ARA014418, Staurosporine,
Compound 5a, Compound 29, Compound 46, GF109203x
(bisindolylmaleimide I), Ro318220 (bisindolylmaleimide IX),
SB216763, SB415286, I5, CGP60474, Compound 8b, TWS119, Compound 1A,
Compound 17, Lithium, Beryllium, Zinc, small molecule GSK-3.beta.
inhibitors (Vertex Pharmaceuticals), NP-12 (Neuropharma),
GSK-3.beta. inhibitors (Amphora), GSK-3.beta. inhibitors
(CrystalGenomics), SAR-502250 (Sanofi-Aventis), 3544 (Hoffmann-La
Roche), GSK-3.beta. inhibitors (Lundbeck), TDZD-8 (Cancer Center,
University of Rochester), pharmaceutically acceptable salts
thereof, and combinations thereof.
[0172] In these ex vivo expansion methods, preferably, the PTEN
inhibitor is bpV(pic), and the GSK-3.beta. inhibitor is CHIR99021.
In these methods, it is preferred that the stem cell is selected
from HSCs, endothelial progenitor cells, (EPCs), mesenchymal stem
cells (MSCs), cardiac stem cells (CSCs), neuronal stem cells
(NSCs), and combinations thereof. Preferably the stem cell is an
HSC. In these methods, the HSC is obtained from a mammalian, e.g.,
primate or human, tissue selected from the group consisting of cord
blood, peripheral blood, and bone marrow.
[0173] In another aspect of the method for ex vivo expansion of an
hematopoietic stem cell (HSC) population, the expansion of the
number of stem cells is by at least 40-fold, such as e.g., by at
least 80-fold, including at least 100-fold, at least 150-fold, at
least 200-fold, at least 250-fold, or at least 270-fold.
[0174] Yet another embodiment of the present invention is an
expanded, substantially undifferentiated stem cell population made
by a method of the present invention, such as, e.g., the method for
ex vivo expansion of a substantially undifferentiated stem cell
population or the method for ex vivo expansion of an hematopoietic
stem cell (HSC) population.
[0175] An additional embodiment of the present invention is a
method for ex vivo expansion of hematopoietic stem cells (HSCs) by
at least 40-fold, wherein the expanded HSCs, are competent to
reconstitute an HSC lineage upon transplantation into a mammalian
patient in need thereof. This method comprises culturing a
population of HSCs in a suitable culture medium comprising a PTEN
inhibitor and a GSK-3.beta. inhibitor.
[0176] In this aspect of the invention, "competent to reconstitute
an HSC lineage" means that the expanded HSCs, when transplanted
into a suitable mammalian patient, result in greater than 1%
engraftment in the recipient, which engrafted cells are able to
differentiate into the cell lineages necessary to have a normal
functioning hematopoietic system. In this method, a "suitable
culture medium", "fluid media" and "media" which are used
interchangeably herein, mean physiologically balanced salt
solutions that can maintain a stem cell population for a required
period of time, which solution may be supplemented with the PTEN
and GSK-3.beta. modulator/inhibitors of the present invention. Such
base culture media are well known in the arts. A non-limiting
example of a suitable base culture medium for HSCs is StemSpan
Media (Stem Cell Technologies; Cat. No. 09600), which is
supplemented with 10 ug/ml Heparin, 0.5.times.
Penicillin/Streptomycin, 10 ng/ml recombinant mouse (rm) Stem Cell
Factor, and 20 ng/ml rm-Thrombopoietin.
[0177] Typically, the culture media also includes from about 100 to
about 1000 nM of the PTEN inhibitor. The culture media may further
include from about 50 nM to about 500 nM of the GSK-3.beta.
inhibitor. In the present invention, when a range is recited, any
value within that range, including the endpoints, is contemplated.
Preferably, the culture media includes both the PTEN and the
GSK-3.beta. inhibitors at the concentrations indicated. For
example, the media may contain as the PTEN inhibitor, bpV(pic), and
as the GSK-3.beta. inhibitor, CHIR99021.
[0178] In one aspect of this embodiment, the HSCs are obtained from
a mammalian tissue, preferably primate or human tissue, which is
selected from cord blood, peripheral blood, and bone marrow. In
this embodiment, the number of HSCs is expanded by a factor of at
least 80-fold, such as at least 100-fold, including at least
150-fold, at least 200-fold, at least 250-fold, or at least
270-fold.
[0179] Yet another embodiment of the present invention is a kit for
expanding an hematopoietic stem cell (HSC) population for
subsequent transplantation into a patient in need thereof. The kit
comprises a PTEN inhibitor and a GSK-3.beta. inhibitor as described
above and instructions for the use of the inhibitors. Preferably,
in the kit, the PTEN inhibitor is bpV(pic) and the
GSK-3.beta..quadrature. inhibitor is CHIR99021. The kit and the
components therein may be packaged in any suitable manner for
distribution and/or storage.
[0180] A further embodiment of the present invention is a media for
carrying out ex vivo expansion of a stem cell population. The media
comprises a fluid media suitable for maintaining viable stem cells
and PTEN and GSK-3.beta. inhibitors present in the media at
concentrations sufficient to enable expansion of the stem cell
population while maintaining a multilineage differentiation
potential in the stem cells.
[0181] In this embodiment, a "concentration sufficient to enable
expansion" means the minimum concentration of the PTEN and
GSK-3.beta. inhibitors, which are sufficient to achieve the desired
level of stem cell renewal, e.g., expansion sufficient for
successful engraftment.
[0182] In one aspect of this embodiment, expansion of the number of
stem cells is by a factor selected from the group consisting of at
least 40-fold, at least 80-fold, at least 100-fold, at least
150-fold, at least 200-fold, at least 250-fold, or at least
270-fold.
[0183] A further embodiment of the present invention is a method
for administering an hematopoietic stem cell (HSC) to a patient in
need thereof. The method comprises (a) culturing, in a suitable
culture media, a sample containing an HSC population in the
presence of a modulator of a molecule in the PTEN pathway and a
modulator of a molecule in the Wnt pathway for a period of time
sufficient to expand the number of HSCs in the sample to a number
sufficient to transplant into the patient; (b) removing from the
culture the PTEN and Wnt pathway modulators; and (c) administering
the HSCs to the patient. In this embodiment, the culture media,
sample, and PTEN and GSK-3.beta. modulators are previously
described.
[0184] An additional embodiment of the present invention is a
method for reconstituting bone marrow in a patient in need thereof.
The method comprises culturing, in a suitable culture media, a
sample containing an HSC population in the presence of a modulator
of a molecule in the PTEN pathway and a modulator of a molecule in
the Wnt pathway for a period of time sufficient to expand the
number of HSCs in the sample to a number that is sufficient to
transplant into the patient. Next, the PTEN and Wnt pathway
modulators are removed from the culture. Then, the expanded HSCs
are administered to the patient in any conventional manner.
[0185] In this method, "reconstituting bone marrow" means
restoration of all or a portion of the bone marrow in a patient
suffering from a disease in which normal bone marrow function has
been compromised. Non-limiting examples of such diseases include
aplastic anemia, myelodysplastic syndromes (MDS), paroxysmal
nocturnal hemoglobinuria (PNH), and blood cancers, such as
leukemia. Thus, as used herein, "reconstituted" means that the
transplanted HSCs are able to successfully engraft in the host and
differentiate into all the cell lineages typically found in or
derived from bone marrow.
[0186] In this method, "a period of time sufficient to expand the
number of HSCs" means the minimum amount of time to expand the HSCs
in culture to a point where there is a sufficient number of HSCs
for one or more transplantations. Typically, such a period of time
may be at least about 10 days in culture. Under certain
circumstances, it may be desirable to expand the stem cell, e.g.,
HSC, population beyond what is required for a single
transplantation. For example, it may be desirable to expand the
stem cell, e.g., HSC, population to a number sufficient for
multiple transplantations, such as e.g., from about 2 to about 100
transplantations. In these circumstances, the excess cells may be
preserved for later use by any conventional method, such as e.g.,
by cryo-preservation.
[0187] As indicated previously, "a number sufficient to transplant"
means the minimum number of stem cells, e.g., HSCs, necessary to
achieve greater than 1% engraftment in a recipient. "Administering
the HSCs to the patient" means conventional methods for delivering
HSCs to the patient, including but not limited to, delivering the
HSCs surgically and/or intravenously. In this embodiment, the
tissue the HSCs are obtained from, and the PTEN and GSK-3.beta.
inhibitors are as previously described.
[0188] An additional embodiment of the present invention is a
method for expanding a population of hematopoietic stem cells
(HSCs). This method comprises culturing a population of HSCs under
conditions sufficient to result in an expansion of the HSC
population by at least 40-fold, wherein the expanded population of
HSCs is suitable for transplantation into a mammal in need thereof.
In this embodiment the "conditions sufficient to result in an
expansion of the HSC population" are those conditions that can
result in expansion of HSCs in culture by, e.g., at least 40-fold,
such as, e.g., by at least 80-fold, at least 150-fold, at least
200-fold, at least 250-fold, or at least 270-fold. "Suitable for
transplantation into a mammal" means that the number and quality of
HSCs is sufficient to support greater than 1% engraftment in a
mammalian recipient, such as, e.g., a primate recipient, including
an human recipient, in need thereof.
[0189] Yet another embodiment of the present invention is a method
for treating a patient in need of a bone marrow transplant, a
peripheral blood transplant, or a cord blood transplant comprising
administering to the patient a population of HSCs obtained by a
method disclosed herein, particularly the methods for expanding a
population of hematopoietic stem cells (HSCs).
[0190] A further embodiment of the present invention is a method
for expanding a population of hematopoietic stem cells (HSCs). The
method comprises (a) obtaining from a mammal a tissue sample
comprising an HSC population; (b) expanding, in vitro, the HSC
population from the sample, wherein (i) the HSC population expands
by at least 40-fold; and (ii) the expanded HSC population has the
ability to reconstitute an hematopoietic lineage for at least
4-weeks, such as for example at least 8-weeks, after
transplantation into a recipient. In this embodiment the "ability
to reconstitute an hematopoietic lineage" means that the expanded
HSC population when transplanted into a recipient will result in
greater than 1% engraftment of HSC in a recipient. In one aspect of
this embodiment, the HSC population expands by at least 80-fold,
such as e.g., at least 100-fold, including at least 150-fold, at
least 200-fold, at least 250-fold, or at least 270-fold. In another
aspect of this embodiment, the mammal is a primate, including a
human. Preferably, the human requires a peripheral blood
transplant, a cord blood transplant, or a bone marrow transplant.
In a further aspect, the tissue sample is obtained from a tissue
selected from the group consisting of cord blood, peripheral blood,
and bone marrow.
[0191] An additional embodiment of the present invention is a
method for reconstituting an hematopoietic stem cell lineage in a
recipient in need thereof. The method comprises(a) obtaining from a
mammal a tissue sample comprising an HSC population; (b) expanding,
in vitro, the HSC population from the sample, wherein: (i) the HSC
population expands by at least 40-fold, such as for example, by at
least 80-fold, including at least 100-fold, at least 150-fold, at
least 200-fold, at least 250-fold, or at least 270-fold, and (ii)
the expanded HSC population has the ability to reconstitute an
hematopoietic lineage for at least 4-weeks, for example, at least
8-weeks, after transplantation into a recipient in need thereof;
and (c) transplanting the expanded HSC population into a recipient
such as a mammal, including a primate or human, in need
thereof.
[0192] In this embodiment, "reconstituting an hematopoietic stem
cell lineage" means that the expanded HSCs, when transplanted into
a recipient result in greater than 1% engraftment of hematopoietic
cells, which are able to differentiate into the normal
hematopoietic lineages. In this embodiment, the human recipient
requires a peripheral blood transplant, a cord blood transplant or
a bone marrow transplant. Thus, in a further aspect, the tissue
sample is obtained from a tissue selected from the group consisting
of cord blood, peripheral blood, and bone marrow. The sample may be
obtained from an autologous or allogeneic source. Preferably, the
sample is obtained from an autologous source.
[0193] In the present invention, it is preferred that the expanded
HSC population comprises HSCs that have a phenotype selected from
the group consisting of CD34.sup.- or
CD34.sup.+/CD38.sup.-/low/Thy-1.sup.+/CD90.sup.+/Kit.sup.-/lo/Lin.sup.-/C-
D133.sup.+VEGFR2.sup.+, which are markers for the most primitive
and long-term undifferentiated human HSCs;
CD150.sup.+/CD48.sup.-/CD244.sup.-, which is a marker for human
HSCs and their progenitors; and/or
CD150.sup.-/CD48.sup.-/CD244.sup.+ and
CD150.sup.-/CD48.sup.+/CD244.sup.+, which are markers for
non-self-renewing multipotent hematopoietic progenitors, and
combinations thereof. (See, e.g., Mimeault, M., et al., Stem Cells:
A Revolution in Therapeutics--Recent Advances in Stem Cell Biology
and Their Therapeutic Applications in Regenerative Medicine and
Cancer Therapies. Clin Pharmacol Ther., 82(3):252-64 (2007)).
[0194] The exact proportions of HSCs having these markers in the
population is not critical, so long as the expanded HSC population
as a whole is sufficient to result in at least 1% engraftment in a
recipient.
[0195] In another embodiment, the invention is a method for
expanding a hematopoietic stem cell population in a mammal in need
of such expansion. This method comprises administering to the
mammal a therapeutically effective amount of a modulator of Wnt and
Akt for a period of time sufficient to expand the HSC population by
at least 40-fold with HSCs that possess the ability to reconstitute
an hematopoietic lineage in the mammal.
[0196] In this method, the respective modulators of Wnt and Akt may
be any molecule, such as a small molecule, a biologic, an antisense
RNA, a siRNA, or combinations thereof, which acts directly or
indirectly to activate .beta.-catenin. Preferably, the Wnt
modulator is selected from a Wnt polypeptide, QS11 (Zhang, Q. et
al., PNAS, 104(18):7444-8 (2007)),
2-amino-4-[3,4-(methylenedioxy)benzyl-amino]-6-(3-methoxyphenyl)pyrimidin-
e (Liu, J. et al., Angew Chem Int Ed Engl. 44(13):1987-90 (2005)),
deoxycholic acid (R. Pai et al., Mol Biol Cell. 15(5):2156-63
(2004)), and combinations thereof. Preferably, the modulator of Akt
is selected from the group consisting of Ro-31-8220 (Wen, H. et
al., Cellular signaling, 15:37-45 (2003)); Nicotine (West, K. et
al., J. Clinical Investigation, 111:81-90 (2003)); carbachol (Cui Q
L, Fogle E & Almazan G Neurochem Int, 48:383-393 (2006));
4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) (West, K. et
al., J. Clinical Investigation, 111:81-90 (2003)); adrenomedullin
(AM) (Nikitenko, L L et al, British J. Cancer, 94:1-7 (2006));
lysophosphatidic acid; platelet activating factor, macrophage
simulating factor; sphingosine-1-phosphate; cAMP-elevating agents,
such as forskolin, chlorophenylthio-cAMP, prostaglandin-E1, and
8-bromo-cAMP (Song et al., J. Cell. Mol. Med., 9(1):59-71 (2005));
and growth factors, including insulin and insulin growth factor-1
(Datta, S. R., et al., Cell, 91:231-241 (1997)), platelet derived
growth factor, and combinations thereof.
[0197] In this method, the Wnt and Akt modulators may be
administered using any regimen that effectively expands the HSC
population by at least 40-fold with HSCs that possess the ability
to reconstitute an hematopoietic lineage in the mammal. Preferably,
the Wnt and Akt modulators are co-administered.
[0198] In the present invention, a "therapeutically effective
amount" is an amount sufficient to effect beneficial or desired
results. In terms of treatment of a mammal, a "therapeutically
effective amount" of a modulator is an amount sufficient to treat,
manage, palliate, ameliorate, or stabilize a condition, such as a
bone marrow disease, in the mammal. A therapeutically effective
amount can be administered in one or more doses.
[0199] The therapeutically effective amount is generally determined
by a physician on a case-by-case basis and is within the skill of
one in the art. Several factors are typically taken into account
when determining an appropriate dosage. These factors include age,
sex and weight of the patient, the condition being treated, the
severity of the condition and the form of the drug being
administered.
[0200] Effective dosage forms, modes of administration, and dosage
amounts may be determined empirically, and making such
determinations is within the skill of the art. It is understood by
those skilled in the art that the dosage amount will vary with the
route of administration, the rate of excretion, the duration of the
treatment, the identity of any other drugs being administered, the
age, size, and species of animal, and like factors well known in
the arts of medicine and veterinary medicine. In general, a
suitable dose of a modulator according to the invention will be
that amount of the modulator, which is the lowest dose effective to
produce the desired effect. The effective dose of a modulator maybe
administered as two, three, four, five, six or more sub-doses,
administered separately at appropriate intervals throughout the
day.
[0201] A modulator, particularly a Wnt or Akt modulator of the
present invention, may be administered in any desired and effective
manner: as pharmaceutical compositions for oral ingestion, or for
parenteral or other administration in any appropriate manner such
as intraperitoneal, subcutaneous, topical, intradermal, inhalation,
intrapulmonary, rectal, vaginal, sublingual, intramuscular,
intravenous, intraarterial, intrathecal, or intralymphatic.
Further, a modulator, particularly a Wnt or Akt modulator, of the
present invention may be administered in conjunction with other
treatments. A modulator, particularly a Wnt or Akt modulator, of
the present invention maybe encapsulated or otherwise protected
against gastric or other secretions, if desired.
[0202] While it is possible for a modulator, particularly a Wnt or
Akt modulator, of the invention to be administered alone, it is
preferable to administer the modulator as a pharmaceutical
formulation (composition). Such pharmaceutical formulations
typically comprise one or more modulators as an active ingredient
in admixture with one or more pharmaceutically-acceptable carriers
and, optionally, one or more other compounds, drugs, ingredients
and/or materials. Regardless of the route of administration
selected, the modulator, particularly a Wnt or Akt modulator, of
the present invention is formulated into
pharmaceutically-acceptable dosage forms by conventional methods
known to those of skill in the art. See, e.g., Remington's
Pharmaceutical Sciences (Mack Publishing Co., Easton, Pa.).
[0203] Pharmaceutically acceptable carriers are well known in the
art (see, e.g., Remington's Pharmaceutical Sciences (Mack
Publishing Co., Easton, Pa.) and The National Formulary (American
Pharmaceutical Association, Washington, D.C.)) and include sugars
(e.g., lactose, sucrose, mannitol, and sorbitol), starches,
cellulose preparations, calcium phosphates (e.g., dicalcium
phosphate, tricalcium phosphate and calcium hydrogen phosphate),
sodium citrate, water, aqueous solutions (e.g., saline, sodium
chloride injection, Ringer's injection, dextrose injection,
dextrose and sodium chloride injection, lactated Ringer's
injection), alcohols (e.g., ethyl alcohol, propyl alcohol, and
benzyl alcohol), polyols (e.g., glycerol, propylene glycol, and
polyethylene glycol), organic esters (e.g., ethyl oleate and
tryglycerides), biodegradable polymers (e.g.,
polylactide-polyglycolide, poly(orthoesters), and
poly(anhydrides)), elastomeric matrices, liposomes, microspheres,
oils (e.g., corn, germ, olive, castor, sesame, cottonseed, and
groundnut), cocoa butter, waxes (e.g., suppository waxes),
paraffins, silicones, talc, silicylate, etc. Each pharmaceutically
acceptable carrier used in a pharmaceutical composition comprising
a modulator of the invention must be "acceptable" in the sense of
being compatible with the other ingredients of the formulation and
not injurious to the subject. Carriers suitable for a selected
dosage form and intended route of administration are well known in
the art, and acceptable carriers for a chosen dosage form and
method of administration can be determined using ordinary skill in
the art.
[0204] Pharmaceutical compositions comprising a modulator of the
invention may, optionally, contain additional ingredients and/or
materials commonly used in pharmaceutical compositions. These
ingredients and materials are well known in the art and include (1)
fillers or extenders, such as starches, lactose, sucrose, glucose,
mannitol, and silicic acid; (2) binders, such as
carboxymethylcellulose, alginates, gelatin, polyvinyl pyrrolidone,
hydroxypropylmethyl cellulose, sucrose and acacia; (3) humectants,
such as glycerol; (4) disintegrating agents, such as agar-agar,
calcium carbonate, potato or tapioca starch, alginic acid, certain
silicates, sodium starch glycolate, cross-linked sodium
carboxymethyl cellulose and sodium carbonate; (5) solution
retarding agents, such as paraffin; (6) absorption accelerators,
such as quaternary ammonium compounds; (7) wetting agents, such as
cetyl alcohol and glycerol monosterate; (8) absorbents, such as
kaolin and bentonite clay; (9) lubricants, such as talc, calcium
stearate, magnesium stearate, solid polyethylene glycols, and
sodium lauryl sulfate; (10) suspending agents, such as ethoxylated
isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters,
microcrystalline cellulose, aluminum metahydroxide, bentonite,
agar-agar and tragacanth; (11) buffering agents; (12) excipients,
such as lactose, milk sugars, polyethylene glycols, animal and
vegetable fats, oils, waxes, paraffins, cocoa butter, starches,
tragacanth, cellulose derivatives, polyethylene glycol, silicones,
bentonites, silicic acid, talc, salicylate, zinc oxide, aluminum
hydroxide, calcium silicates, and polyamide powder; (13) inert
diluents, such as water or other solvents; (14) preservatives; (15)
surface-active agents; (16) dispersing agents; (17) control-release
or absorption-delaying agents, such as hydroxypropylmethyl
cellulose, other polymer matrices, biodegradable polymers,
liposomes, microspheres, aluminum monosterate, gelatin, and waxes;
(18) opacifying agents; (19) adjuvants; (20) wetting agents; (21)
emulsifying and suspending agents; (22), solubilizing agents and
emulsifiers, such as ethyl alcohol, isopropyl alcohol, ethyl
carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate,
propylene glycol, 1,3-butylene glycol, oils (in particular,
cottonseed, groundnut, corn, germ, olive, castor and sesame oils),
glycerol, tetrahydrofuryl alcohol, polyethylene glycols and fatty
acid esters of sorbitan; (23) propellants, such as
chlorofluorohydrocarbons and volatile unsubstituted hydrocarbons,
such as butane and propane; (24) antioxidants; (25) agents which
render the formulation isotonic with the blood of the intended
recipient, such as sugars and sodium chloride; (26) thickening
agents; (27) coating materials, such as lecithin; and (28)
sweetening, flavoring, coloring, perfuming and preservative agents.
Each such ingredient or material must be "acceptable" in the sense
of being compatible with the other ingredients of the formulation
and not injurious to the subject. Ingredients and materials
suitable for a selected dosage form and intended route of
administration are well known in the art, and acceptable
ingredients and materials for a chosen dosage form and method of
administration may be determined using ordinary skill in the
art.
[0205] Pharmaceutical compositions suitable for oral administration
may be in the form of capsules, cachets, pills, tablets, powders,
granules, a solution or a suspension in an aqueous or non-aqueous
liquid, an oil-in-water or water-in-oil liquid emulsion, an elixir
or syrup, a pastille, a bolus, an electuary or a paste. These
formulations may be prepared by methods known in the art, e.g., by
means of conventional pan-coating, mixing, granulation or
lyophilization processes.
[0206] Solid dosage forms for oral administration (capsules,
tablets, pills, dragees, powders, granules and the like) may be
prepared by mixing the active ingredient(s) with one or more
pharmaceutically-acceptable carriers and, optionally, one or more
fillers, extenders, binders, humectants, disintegrating agents,
solution retarding agents, absorption accelerators, wetting agents,
absorbents, lubricants, and/or coloring agents. Solid compositions
of a similar type maybe employed as fillers in soft and hard-filled
gelatin capsules using a suitable excipient. A tablet may be made
by compression or molding, optionally with one or more accessory
ingredients. Compressed tablets may be prepared using a suitable
binder, lubricant, inert diluent, preservative, disintegrant,
surface-active or dispersing agent. Molded tablets may be made by
molding in a suitable machine. The tablets, and other solid dosage
forms, such as dragees, capsules, pills and granules, may
optionally be scored or prepared with coatings and shells, such as
enteric coatings and other coatings well known in the
pharmaceutical-formulating art. They may also be formulated so as
to provide slow or controlled release of the active ingredient
therein. They may be sterilized by, for example, filtration through
a bacteria-retaining filter. These compositions may also optionally
contain opacifying agents and may be of a composition such that
they release the active ingredient only, or preferentially, in a
certain portion of the gastrointestinal tract, optionally, in a
delayed manner. The active ingredient can also be in
microencapsulated form.
[0207] Liquid dosage forms for oral administration include
pharmaceutically-acceptable emulsions, microemulsions, solutions,
suspensions, syrups and elixirs. The liquid dosage forms may
contain suitable inert diluents commonly used in the art. Besides
inert diluents, the oral compositions may also include adjuvants,
such as wetting agents, emulsifying and suspending agents,
sweetening, flavoring, coloring, perfuming and preservative agents.
Suspensions may contain suspending agents.
[0208] Pharmaceutical compositions for rectal or vaginal
administration may be presented as a suppository, which maybe
prepared by mixing one or more active ingredient(s) with one or
more suitable nonirritating carriers which are solid at room
temperature, but liquid at body temperature and, therefore, will
melt in the rectum or vaginal cavity and release the active
compound. Pharmaceutical compositions which are suitable for
vaginal administration also include pessaries, tampons, creams,
gels, pastes, foams or spray formulations containing such
pharmaceutically-acceptable carriers as are known in the art to be
appropriate.
[0209] Dosage forms for the topical or transdermal administration
include powders, sprays, ointments, pastes, creams, lotions, gels,
solutions, patches, drops and inhalants. The active compound may be
mixed under sterile conditions with a suitable
pharmaceutically-acceptable carrier. The ointments, pastes, creams
and gels may contain excipients. Powders and sprays may contain
excipients and propellants.
[0210] Pharmaceutical compositions suitable for parenteral
administrations comprise one or more modulator in combination with
one or more pharmaceutically-acceptable sterile isotonic aqueous or
non-aqueous solutions, dispersions, suspensions or emulsions, or
sterile powders which may be reconstituted into sterile injectable
solutions or dispersions just prior to use, which may contain
suitable antioxidants, buffers, solutes which render the
formulation isotonic with the blood of the intended recipient, or
suspending or thickening agents. Proper fluidity can be maintained,
for example, by the use of coating materials, by the maintenance of
the required particle size in the case of dispersions, and by the
use of surfactants. These compositions may also contain suitable
adjuvants, such as wetting agents, emulsifying agents and
dispersing agents. It may also be desirable to include isotonic
agents. In addition, prolonged absorption of the injectable
pharmaceutical form may be brought about by the inclusion of agents
which delay absorption.
[0211] In some cases, in order to prolong the effect of a drug
containing a modulator of the present invention, it is desirable to
slow its absorption from subcutaneous or intramuscular injection.
This may be accomplished by the use of a liquid suspension of
crystalline or amorphous material having poor water solubility.
[0212] The rate of absorption of the drug then depends upon its
rate of dissolution which, in turn, may depend upon crystal size
and crystalline form. Alternatively, delayed absorption of a
parenterally-administered drug may be accomplished by dissolving or
suspending the drug in an oil vehicle. Injectable depot forms may
be made by forming microencapsule matrices of the active ingredient
in biodegradable polymers. Depending on the ratio of the active
ingredient to polymer, and the nature of the particular polymer
employed, the rate of active ingredient release can be controlled.
Depot injectable formulations are also prepared by entrapping the
drug in liposomes or microemulsions which are compatible with body
tissue. The injectable materials can be sterilized for example, by
filtration through a bacterial-retaining filter.
[0213] The formulations may be presented in unit-dose or multi-dose
sealed containers, for example, ampules and vials, and may be
stored in a lyophilized condition requiring only the addition of
the sterile liquid carrier, for example water for injection,
immediately prior to use. Extemporaneous injection solutions and
suspensions may be prepared from sterile powders, granules and
tablets of the type described above.
[0214] The following examples are provided to further illustrate
the methods and compositions of the present invention. These
examples are illustrative only and are not intended to limit the
scope of the invention in any way.
EXAMPLES
Example 1
Loss of PTEN with Constitutively Active .beta.-Catenin Leads to HSC
Expansion with Loss of Early Hematopoietic Progenitors
Animals
[0215] All mice used in this study were housed in the animal
facility at Stowers Institute for Medical Research (SIMR) and
handled according to Institute and NIH guidelines. All procedures
were approved by the IACUC of SIMR. Pten/constitutively active
.beta.-catenin double mutant mice were induced by intra-peritoneal
injection of Tamoxifen (Sigma, Cat. No. T5648) everyday for 5 days
using 5 mg on day 1 and 2 mg on days 2-5 each dissolved in 0.1 ml
of corn oil (Sigma, Cat. No. C8267) (complete dissolution was
achieved by 42.degree. C. water bath sonication for about 5
minutes). Mx-1 Cre induction was achieved by 250 .mu.g injection of
polyl:C every other day utilizing 1 dose (for the Mx-1 Cre
Pten:.beta.-cat.sup.Act model) or 5 doses (for the Mx1-Cre
Pten:.beta.-cat.sup.-/- transplant model). Scl-Cre, Pten,
.beta.-cat.sup.Act, and .beta.-cat.sup.-/- mice were obtained from
Joachim Goethert (University of Duisburg-Essen, Germany), Hong Wu
(UCLA, Los Angeles, Calif.), Makoto Taketo (Kyoto University,
Japan) and the Jackson Laboratory (Bar Harbor, Me.),
respectively.
Histology
[0216] Paraffin sections of spleen, tumors or decalcified femurs
were stained with H&E or Masson's Trichrome as indicated.
Immunofluorescent Assays
[0217] GFP.sup.+ HSCs were sorted and transplanted as previously
reported (Xie, Y. et al. Detection of functional haematopoietic
stem cell niche using real-time imaging. Nature 457, 97-101
(2009)). Femurs and tibias were fixed in 4% PFA or
Zn.sup.2+-Formalin and processed for paraffin and frozen sections,
respectively. For immunofluorescent staining, after antigen
retrieval using EZ Retriever Microwave (BioGenex, San Ramon,
Calif.) for 10 minutes at 95.degree. C. in citrate buffer,
non-specific antibody binding was blocked by incubating slides with
1.times. Universal Block (BioGenex, #HK085-5k) at room temperature
for 1 hour. p.beta.-cat-5552 was stained as previously reported
(He, X. C. et al. PTEN-deficient intestinal stem cells initiate
intestinal polyposis. Nat Genet 39, 189-198 (2007)).
Induction Of PTEN/Constitutively Active .beta.-Catenin Double
Mutant Mice
[0218] The inventors have previously demonstrated that PTEN
deficiency leads to excessive intestinal stem cell (ISC)
proliferation resulting in intestinal polyposis, a pre-cancerous
neoplasia (He, X. C. et al. PTEN-deficient intestinal stem cells
initiate intestinal polyposis. Nat Genet 39, 189-198 (2007)). Akt
has been shown to phosphorylate .beta.-catenin at serine residue
552 (S552), with the resulting phosphorylated form of
.beta.-catenin being nuclear localized in ISCs. An antibody
highly-specific for .beta.-catenin phosphorylated at S552
(.beta.-cat-p5552) reveals that cells with nuclear (activated)
.beta.-cat-pS552 initiate ISC expansion, resulting in polyposis in
PTEN-deficient mice. Staining with .beta.-cat-pS552 antibody shows
simultaneous activation of the two pathways. Considering its role
in ISCs, it was hypothesized that .beta.-cat-pS552 antibody may
also recognize activated HSCs. To investigate this, purified HSCs
which express green fluorescent protein (GFP.sup.+ HSCs) was
transplanted into irradiated and non-irradiated mice. The
recipients were sacrificed, and their bone sections were stained
with anti-.beta.-cat-pS552 antibody. With irradiation, a condition
previously shown to result in rapid HSC expansion (Xie, Y. et al.
Detection of functional haematopoietic stem cell niche using
real-time imaging. Nature 457, 97-101 (2009)), purified
GFP.sup.+-HSCs were observed to be adjacent to their endosteal
niche with 5 of 40 GFP.sup.+ HSCs costaining as
.beta.-cat-pS552.sup.+ cells, some of which were in the process of
active division (FIG. 8A-FIG. 8D). However, without irradiation, a
condition where HSCs do not expand (Id.), 0 of 15 GFP.sup.+ HSCs
were found to be .beta.-cat-pS552.sup.+ (data not shown).
.beta.-cat-pS552 antibody were also used to visualize
Wnt/.beta.-catenin and PTEN/Akt signaling pathway interaction in
control, single, and double mutant spleen. At 3 days post-induction
(dpi), control and single mutant spleens showed only rare and
lightly stained cells, while double mutants exhibited more intense
and abundant .beta.-cat-pS552 staining (FIG. 14). These results
support the importance of activated Akt and .beta.-catenin
interaction in normal but proliferating HSCs and show that this
pathway interaction is enhanced in double mutants compared to
single mutants or control.
[0219] The consequences of combining both conditional Pten deletion
with constitutive activation of .beta.-catenin (.beta.-cat.sup.Act)
(Ctnnb1.sup.tm1Mmt) (Harada, N. et al. Intestinal polyposis in mice
with a dominant stable mutation of the beta-catenin gene. The EMBO
journal 18, 5931-5942 (1999)) was studied using Mx1-Cre. This
interferon-inducible system results in tissue non-specific knockout
of LoxP-flanked (floxed) alleles and has been used in previous
studies focusing on either Pten or .beta.-cat.sup.Act single
mutants (Kirstetter, P., Anderson, K., Porse, B. T., Jacobsen, S.
E. & Nerlov, C. Activation of the canonical Wnt pathway leads
to loss of hematopoietic stem cell repopulation and multilineage
differentiation block. Nat Immunol 7, 1048-1056 (2006); Scheller,
M. et al. Hematopoietic stem cell and multilineage defects
generated by constitutive [beta]-catenin activation. Nature
Immunology 7, 1037-1047 (2006); Yilmaz, O. H. et al. Pten
dependence distinguishes haematopoietic stem cells from
leukaemia-initiating cells. Nature 441, 475-482 (2006); Zhang, J.
et al. PTEN maintains haematopoietic stem cells and acts in lineage
choice and leukaemia prevention. Nature 441, 518-522 (2006)).
Mx1-Cre.sup.+ Pten:.beta.-cat.sup.Act double mutants exhibit severe
tumors consisting of well-formed but densely packed hair follicles
which rapidly cover the body (FIG. 15), suggesting a stem cell
proliferation defect (Gray, H. R. & Helwig, E. B.
Trichofolliculoma. Arch Dermatol. 86, 99-105 (1962)).
[0220] Because this severe tumor phenotype made it impossible to
complete long-term studies of the hematopoietic system in these
double mutants using Mx1-Cre, Pten and .beta.-cat.sup.Act
(Ctnnb1.sup.fl/fl) single and double mutants were crossed with the
tamoxifen-inducible HSC-SCL-Cre-ER.sup.T strain (hereafter referred
to as Scl-Cre), which allowed for studying the effects in the
hematopoietic system, in particular those effects which initiate
primarily from HSCs (Gothert, J. R., et al., In vivo fate-tracing
studies using the Scl stem cell enhancer: embryonic hematopoietic
stem cells significantly contribute to adult hematopoiesis." Blood,
2005. 105(7): p. 2724-2732). The cross is set forth in more detail
below.
[0221] Mice with homozygous floxed (fl) alleles of Pten
(Pten.sup.fl/fl) were bred with Ctnnb1.sup.fl/fl mice in which exon
3 of the mouse .beta.-catenin gene (where all phosphorylation
target serine/threonine residues are located) was sandwiched by two
loxP sequences. (Harada, N., et al., Embo J, 18(21): 5931-42 1999.
Yilmaz, O. H., et al., Nature, 441:475-82 2006. Zhang, J., et al.,
Nature, 441(7092): 518-22 2006.) Double heterozygous mice from this
cross were then crossed to generate Pten.sup.fl/fl Ctnnb1.sup.fl/+
mice (since Ctnnb1 is a gain-of-function allele, only heterozygous
mice for Ctnnb1 are necessary). Concurrently, Pten.sup.fl/fl mice
were bred with Scl-Cre.sup.+ transgenic mice to generate
Scl-Cre.sup.+ Pten.sup.fl/+ mice. These were then crossed to
generate Scl-Cre.sup.+ Pten.sup.fl/fl mice ("Pten"). Finally,
Pten.sup.fl/fl Ctnnb1.sup.fl/+ mice were bred with Scl-Cre.sup.+
Pten.sup.fl/fl mice to generate Scl-Cre.sup.+ Pten.sup.fl/fl
Ctnnb1.sup.fl/+ mice ("Pten:Ctnnb1"). Scl-Cre mice were also bred
with Ctnnb1.sup.fl/fl mice to generate the single mutant
Scl-Cre.sup.+ Ctnnb1.sup.fl/+ mice ("Ctnnb1"). Mice lacking Scl-Cre
("Scl-Cre negative" or "Control") were used as controls.
HSC Analysis
[0222] For phenotype analysis, hematopoietic cells were harvested
from bone marrow (femur and tibia), spleen, peripheral blood, and
thymus. Red blood cell lysis was performed using hemolysis buffer
(0.16M ammonium chloride, Sigma Cat. No. A9434). Cells were stained
for lineage markers using CD3, CD4, CD8, B220, IgM, Mac-1, Gr1, and
Ter119 antibodies along with Kit, and Sca-1 for LSK analysis or
these markers along with IL-7R.alpha., CD34 and CD16/32 for
progenitor analysis (Akashi, K., et al., A clonogenic common
myeloid progenitor that gives rise to all myeloid lineages. Nature
2000. 404(6774): p. 193-7). Flk2 was added as indicated for LT-HSC
analysis.
[0223] Unless otherwise indicated, all antibodies were obtained
from eBiosciences (San Diego, Calif.) as indicated below:
Fluorescein isothiocyanate (FITC) conjugated CD3 antibody (Cat. No.
11-0452-85), FITC conjugated CD4 antibody (Cat. No. 11-0042-85),
FITC conjugated CD8 antibody (Cat. No. 11-0081-85), FITC conjugated
B220 antibody (Cat. No. 11-0452-85), FITC conjugated Ter119
antibody (Cat. No. 11-5921-85), FITC conjugated Mac-1 antibody
(Cat. No. 11-0112-85), FITC conjugated Gr1 antibody (Cat. No.
11-5931-85), FITC conjugated IgM antibody (Cat. No. 11-5790-85),
Phycoerythrin (PE) conjugated Sca-1 antibody (Cat. No. 12-5981-83),
Allophycocyanin (APC) conjugated Kit antibody (Cat. No.
17-1171-83), Biotin conjugated CD135 (Flk-2) antibody (Cat. No.
13-1351-85), PE-Cy5 conjugated CD127 (IL-7R.alpha.) antibody (Cat.
No. 15-1271-83), PE-Cy7 conjugated CD16/32 (Fc.gamma.RII/III)
antibody (Cat. No. 25-0161-82), Biotin conjugated CD34 antibody
(Cat. No. 13-0341-85), Streptavidin conjugated PE-Cy7 antibody
(Cat. No. 25-4317-82), Streptavidin conjugated APC-Cy7 antibody
(Cat. No. 10-4317-82), APC conjugated Gr1 antibody (Cat. No.
17-5931-82), APC conjugated B220 antibody (Cat. No. 17-0452-83), PE
conjugated Mac-1 antibody (Cat. No. 12-0112-83), and PE conjugated
CD3 antibody (Cat. No. 12-0031-85).
[0224] Antibody stained cells were sorted by FACS using a MoFlo
(Dako, Ft. Collins, Colo.) flow cytometer and/or a CyAn ADP (Dako,
Ft. Collins, Colo.), and analyzed for lineage negative,
Sca-1.sup.+Kit.sup.+ (LSK) cells in Scl-Cre negative control and
Scl-Cre.sup.+ PTEN with constitutively activated .beta.-catenin
(Pten:Ctnnb1; also referred to as Pten:.beta.-cat.sup.Act) double
mutant bone marrow and spleen. Data analysis was performed using
FlowJo software (Ashland, Oreg.).
[0225] In order to study the consequences of Pten deletion combined
with .beta.-catenin activation on stem cells in vivo, HSCs and
early progenitors were analyzed from single and double mutants bred
onto the Scl-Cre line. At 10 days post-induction (dpi), Pten:Ctnnb1
(hereafter mutants are Scl-Cre.sup.+ unless otherwise specified as
Mx1-Cre.sup.+) LSK cells were slightly reduced in bone marrow but
significantly increased in spleen (p<0.001), suggesting a
mobilization of HSCs. Similar to previous reports (Yilmaz, O. H. et
al. Pten dependence distinguishes haematopoietic stem cells from
leukaemia-initiating cells. Nature 441, 475-482 (2006); Zhang, J.
et al. PTEN maintains haematopoietic stem cells and acts in lineage
choice and leukaemia prevention. Nature 441, 518-522 (2006)), LSK
cells in Pten mutants were also significantly increased in spleen
(p<0.05), though this expansion was not as great as in double
mutant spleen (FIG. 1A). At 4 weeks post-induction (wpi), early
myeloid progenitors including common myeloid,
megakaryocyte-erythroid, and granulocyte-monocyte progenitors
(CMPs, MEPs, and GMPs, respectively) (Akashi, K., Traver, D.,
Miyamoto, T. & Weissman, I. L. A clonogenic common myeloid
progenitor that gives rise to all myeloid lineages. Nature 404,
193-197 (2000)) were significantly reduced in Pten:Ctnnb1 bone
marrow. No other dramatic differences were observed between
control, single, and double mutant bone marrow and spleen (data not
shown).
[0226] By 6 wpi, the frequency of LSK cells (lineage negative,
Sca-1.sup.+, Kit.sup.+), which are highly enriched in HSCs,
significantly increased in Pten:Ctnnb1 bone marrow, although the
absolute number of LSK cells was not significantly increased in
bone marrow due to low cellularity (FIG. 1B and FIG. 1F; cells were
pre-gated on live, lineage negative cells.). Strikingly, LSK cells
in spleen, which increased only modestly in Pten single mutants,
were dramatically increased in double mutants (FIG. 1C and FIG.
1G). However, the frequency of progenitor cells (lineage negative,
Sca-1.sup.-, Kit.sup.+ cells) decreased in both bone marrow and
spleen in double mutants, indicating a failure to differentiate
(FIG. 1B-FIG. 1C).
[0227] Because LSK cells are heterogeneous, the LSK population was
further examined based on Flk2 and CD34 expression, the negative
fractions of which further define long-term reconstituting HSCs
(Himburg, H. A. et al., "Pleiotrophin regulates the expansion and
regeneration of hematopoietic stem cells" Nat Med Vol. 16, pages
475-482 (2010); Sato, et al., "Maintenance of pluripotency in human
and mouse embryonic stem cells through activation of Wnt signaling
by a pharmacological GSK-3-specific inhibitor" Nat Med Vol. 10,
55-63 (2004)). LSKs may be subdivided based on Flk2 expression,
which allowed for further enrichment long-term reconstituting (LT)
HSCs (Flk2.sup.-) from short-term reconstituting (ST) HSCs
(Flk2.sup.+) (Christensen, J. L. & Weissman, I. L. Flk-2 is a
marker in hematopoietic stem cell differentiation: a simple method
to isolate long-term stem cells. Proceedings of the National
Academy of Sciences of the United States of America 98, 14541-14546
(2001)). Double mutants exhibited a higher frequency of both
Flk2.sup.- and CD34.sup.- cells within the LSK population than
control or either single mutant (FIG. 1D, FIG. 1H, and FIG. 1I).
The absolute number of LSK CD34.sup.- cells was also significantly
increased in both bone marrow and spleen of double mutants compared
to both control and single mutants (FIG. 1E).
[0228] Despite the substantial increase in phenotypic HSCs, further
characterization of early myeloid progenitors showed that both
frequency and absolute numbers of common myeloid,
megakaryocyte-erythroid, and granulocyte-monocyte progenitors
(CMPs, MEPs, and GMPs, respectively) were significantly reduced in
double mutants (FIG. 1B-FIG. 1C) (Akashi, K., Traver, D., Miyamoto,
T. & Weissman, I. L. A clonogenic common myeloid progenitor
that gives rise to all myeloid lineages. Nature 404, 193-197
(2000)). However, the frequency and absolute numbers of common
lymphoid progenitors (CLP) was similar to control (FIG. 1K and FIG.
24) (Nakorn et al., "Myeloerythroid-restricted progenitors are
sufficient to confer radioprotection and provide the majority of
day 8 CFU-S." J. clinical investigation Vol. 109, pages 1579-1585
(2002); Datta et al. "Akt phosphorylation of BAD couples survival
signals to the cell-intrinsic death machinery." Cell Vol. 91, pages
231-241 (1997)). These data demonstrate that phenotypic HSCs are
substantially expanded in double mutants compared to control and
single mutants. Furthermore, this expansion is coupled with reduced
differentiation of myeloid progenitors but without significant
corresponding increases in early lymphoid differentiation. In
contrast, Pten single mutants exhibit excessive myeloid
differentiation, progressing to MPD (FIG. 9).
[0229] By 6 wpi, about 50% of double mutants began to develop
leukemia with substantial blast cell (CD45.sup.High) populations
(FIG. 1J and data not shown) (Borowitz, M. J., Guenther, K. L.,
Shults, K. E., Stelzer, G. T. Immunophenotyping of acute leukemia
by flow cytometric analysis. Am. J. Clin. Pathol. 100, 534-540
(1993)). As shown in FIG. 1J, CD45 (high) blast crisis cells are
indicated in the blue box of the left panel. LSK analysis of
leukemic Pten:Ctnnb1 mutant mouse bone marrow was also performed
(FIG. 1J, right panel). Note the conversion to blast cells (lower
left) with only a remnant LSK population (compare to FIG. 1C).
These leukemic mice were excluded from the analyses presented in
the rest of FIG. 1 and FIG. 24 because their LSK population was
reduced when blast cells increased and out-competed other cells,
which was accompanied by stromal/niche disruption (see below). By
10-11 wpi, all double mutants had to be euthanized due to severe
leukemia comprised of early T-lymphoid precursors. In comparison,
no blast cell population is observed in control or Ctnnb1 single
mutants while a minor one was observed in 1 of 8 Pten single mutant
mice at 6 weeks post-induction (data not shown). The long-term
phenotype of double as well as single mutants is detailed in FIG.
10, FIG. 12, FIG. 16, FIG. 17, FIG. 20, and FIG. 28. Overall,
double mutants ultimately exhibited reductions in most major
hematopoietic lineages except for early T-cell precursors, which
predominated. Interestingly, double mutants at 9 wpi frequently
exhibited a unique population of lineage negative, Sca-1.sup.Low,
Kit.sup.Low or LSK-like cells prior to the complete dominance of
leukemic blast cells. Preliminary evidence indicates that this
population is particularly enhanced in leukemia-initiating or
cancer stem cell activity (FIG. 10A and data not shown).
[0230] Together, this data demonstrates the phenotypic effect of
the genetic loss of PTEN coupled with constitutive activation of
.beta.-catenin in HSCs. While loss of PTEN alone results in a
slight but significant expansion in splenic HSCs due to
mobilization from the bone marrow, double mutant HSCs exhibit the
greatest mobilization at 10 days post-induction. By six weeks
post-induction, only double mutant splenic HSCs are dramatically
increased while single mutants are not significantly different from
controls. In addition, this dramatic increase in HSCs is not
accompanied by an increase in early hematopoietic progenitors;
rather these early progenitors are all reduced with the exception
of CLPs which are not significantly different from control. HSCs
accumulate dramatically in the spleen of double, but not single,
mutants by proliferation with reduced differentiation. Thus,
surprisingly and unexpectedly, loss of PTEN coupled with the
constitutive activation of .beta.-catenin drives stem cell
self-renewal while neither pathway individually is capable of
driving long-term self-renewal.
Example 2
In Vitro Culture of Control and Mutant LSK Cells
Cell Culture
[0231] LSK or LSK Flk2.sup.- cells were sorted into 96-well
U-bottom tissue culture plates at 100 cells/well with 200 .mu.l
media/well. Cells were incubated at 37.degree. C., 5% O.sub.2, 5%
CO.sub.2 (balance N.sub.2) for the indicated number of days.
One-half total volume of media (see Table 1, below for the base
media) was carefully pipetted from the top and replaced with fresh
media every other day.
TABLE-US-00001 TABLE 1 Base Media Components Source StemSpan Media:
(Iscove's-modified Stem Cell Technologies; Dulbecco's medium (IMDM)
supplemented Cat. No. 09600 with 1% bovine serum albumin, 10 .mu.g
ml.sup.-1 recombinant human insulin, 200 .mu.g ml.sup.-1
iron-saturated transferrin, 0.1 mM 2-mercaptoethanol and 2 mM
glutamine.) 10 .mu.g/ml Heparin Sigma, Cat. No. H-3149 0.5X
Penicillin/Streptomycin Sigma, Cat. No. P4333 10 ng/ml recombinant
mouse (rm) Biovision, Cat. No. Stem Cell Factor 4328-10 20 ng/ml
rm-Thrombopoietin Cell Sciences, Inc, Cat. No. CRT401B
Double Mutant HSCs Expand Dramatically In Vitro and In Vivo but
Fail to Differentiate.
[0232] For the following experiments, the base media from Table 1
was further supplemented with 20 ng/ml rm-IGF-2 (R&D Systems,
Cat. No. 792-MG) and 10 ng/ml recombinant human FGF-1 (Affinity
BioReagents, Cat. No. ORP16010).
[0233] The ability of HSCs isolated from Mx1-Cre.sup.+ single and
double mutants to expand in vitro was examined. Lineage negative,
Sca-1.sup.+, Kit.sup.+ (LSK) cells (a population highly enriched in
HSCs) were sorted from wild-type (control), single, and double
mutant Mx1-Cre.sup.+ bone marrow and cultured in defined media
based on a previous report regarding ex vivo HSC expansion (Zhang,
C. C. & Lodish, H. F. Murine hematopoietic stem cells change
their surface phenotype during ex vivo expansion. Blood 105,
4314-4320 (2005)). After 10 days culture, control LSK cells had
undergone a modest expansion; however, Ctnnb1 LSK cells did not
survive, suggesting they had undergone apoptosis. In contrast, Pten
LSK cultures expanded to a greater degree than control, while the
best expansion was observed from double mutant cultures (FIG. 2A).
Pten and Pten:Ctnnb1 cultures continued to expand up to 5 weeks in
vitro (FIG. 2B); however, control cultures began to decline after 4
weeks (data not shown). Unlike control, both Pten and Pten:Ctnnbl
cultures remained robust after 5 weeks, but Pten:Ctnnb1 cultures
contained far more cells and their appearance was more homogenous
than Pten cultures (FIG. 2B). At 7 weeks, a portion of the
remaining Pten and Pten:Ctnnb1 cultures was re-analyzed by
fluorescence-activated cell scanning (FACS) analysis to determine
how many cells had maintained their LSK phenotype (FIG. 2E). Due to
long-term culture, these cells expressed unusually high levels of
Kit and Sca-1. While LSK cells from Pten cultures had expanded
50-fold, Pten:Ctnnb1 cultures expanded more than 1,200-fold (FIG.
2C). In addition, the purity of LSK cells (% of total cells
maintaining the LSK phenotype) was significantly higher in
Pten:Ctnnb1 cultures compared to Pten only (84% vs. 52%,
respectively, FIG. 2D).
Example 3
Transplantation Analysis of Pten and Pten:Ctnnb1 LSK Cells after 5
Weeks of Culture
[0234] For the following experiments, cells were cultured in the
same manner as described in Example 2. As in Example 2, the base
media of Table 1 was supplemented with 20 ng/ml rm-IGF-2 (R&D
Systems, 792-MG) and 10 ng/ml recombinant human FGF-1 (Affinity
BioReagents, ORP16010).
[0235] While Pten and especially Pten:Ctnnb1 cultures exhibited
significant expansion in LSK cells, whether these cells were
functional in vivo was determined.
[0236] At 5 weeks culture, Pten and Pten:Ctnnb1 LSK cultures were
re-sorted and 1000 LSK cells (CD45.2.sup.+) from each were
transplanted into lethally irradiated (10Gy) CD45.1.sup.+ recipient
mice along with 2.times.10.sup.5 congenic whole bone marrow
competitor cells. Because wild-type cells did not survive 5 weeks
culture, 1000 fresh wild-type LSK cells were also transplanted as a
separate control group. Peripheral blood analysis at 4 weeks
post-transplantation revealed robust repopulation in mice
transplanted with fresh/uncultured control cells as expected;
however, mice transplanted with either Pten or Pten:Ctnnb1 cultured
cells did not exhibit repopulation (data not shown). At 5 weeks
post-transplant, bone marrow from recipient mice was analyzed for
donor engraftment (CD45.2.sup.+ cells) and donor LSK cells
(CD45.2.sup.+ LSKs).
[0237] To determine whether LSK or other donor-derived (CD45.2+)
cells remained in the bone marrow of mice transplanted with
cultured cells, bone marrow was analyzed for donor (CD45.2+) and
LSK cells. While the control group exhibited robust repopulation of
CD45.2+ bone marrow cells, few CD45.2+ cells were maintained as LSK
cells as expected (FIG. 2C-FIG. 2D and FIG. 2F). In contrast,
recipients transplanted with in vitro expanded Pten or Pten:Ctnnb1
mutant LSK cells exhibited few donor-derived total bone marrow
cells (FIG. 2G-FIG. 2H). However, a large portion of Pten:Ctnnb1
donor-derived cells were maintained as LSK cells in recipients,
whereas those from Pten only cultures were similar in number to
control (FIG. 2G-FIG. 2I). In order to determine whether ex vivo
expanded donor cells had further expanded in vivo following
transplantation, the total number of donor LSK cells in total bone
marrow per mouse were estimated (Smith, L. H. & Clayton, M. L.
Distribution of injected 59Fe in mice. Exp. Hematol. 20, 82-86
(1970)). As shown in FIG. 2J, the expansion of total donor-derived
LSK cells in transplant recipients was modest and similar between
control and Pten (8.6.+-.1.4 and 13.+-.6.3, respectively), but
significantly greater in recipients transplanted with cultured
Pten:Ctnnb1 LSK cells (43.+-.3.4).
[0238] Collectively, these data demonstrate that double mutant HSCs
can be cultured longer and with far greater expansion than either
single mutant or control HSCs. However, permanent genetic
alteration of both pathways leads to an increased ability to
self-renew both in vitro as well as in vivo following long-term
culture but a failure to differentiate and thus repopulate the
hematopoietic system of transplant recipients. This further
demonstrates the ability of the PTEN and .beta.-catenin signaling
pathways to cooperatively drive stem cell expansion by
proliferation without differentiation.
Example 4
Transplanted Double Mutant LSK Flk2.sup.- Cells are Maintained as
Phenotypic HSCs In Vivo
[0239] Initially, primary (non-transplanted) animals were used for
phenotypic analysis. These mice eventually exhibited severe
non-hematopoietic defects, including reduction of the marrow cavity
and splenic fibrosis resulting in disruption of splenic niches
(FIG. 16). Consequently, LT-HSC transplantations were used to
verify Scl-Cre specificity (FIG. 10). Comparing these transplant
groups with the initial data from primary mutants revealed an
essentially identical phenotypic manifestation of defects between
transplant and non-transplant groups, demonstrating that
non-hematopoietic effects are due to interaction between the
hematopoietic system and stroma rather than from defects arising
from the stroma (FIG. 17 and data not shown).
[0240] The health of double mutants typically declined by 9 wpi
(see below). LSK cells and early progenitors from control, single,
and double mutant bone marrow and spleen at 9-10 wpi were analyzed
by FACS (FIG. 10A-FIG. 10B). Absolute number of LSK cells were
reduced in bone marrow and spleen of Ctnnbl single mutants but
increased in the spleens of Pten single mutants (FIG. 10A and FIG.
10C-FIG. 10D). CMPs and MEPs were increased in Pten bone marrow and
spleen (FIG. 10B-FIG. 10D). In contrast, LSK cells and all early
progenitors including CMPs, MEPs, GMPs and CLPs (Akashi, K.,
Traver, D., Miyamoto, T. & Weissman, I. L. A clonogenic common
myeloid progenitor that gives rise to all myeloid lineages. Nature
404, 193-197 (2000); Kondo, M., Weissman, I. L. & Akashi, K.
Identification of clonogenic common lymphoid progenitors in mouse
bone marrow. Cell 91, 661-672 (1997)) were severely depleted in
Pten:Ctnnb1 bone marrow. Interestingly, when leukemic cells
predominated but health had not yet severely declined, Pten:Ctnnb1
mutants exhibited a distinct population of Lin- Sca-1.sup.Low
Kit.sup.Mid (LS.sup.LowK.sup.Mid) cells at 9 wpi (FIG. 10A, panels
IV and IX). At 10 wpi when health had severely declined, this
population was typically absent and only leukemic blast cells
remained (FIG. 10A, panels V and X).
[0241] CD45.1 (recipient) and CD45.2 (donor) markers were used to
measure engraftment levels in recipients at 9-10 wpi. 1,000 sorted
LSK Flk2.sup.- cells from control, single, and double mutant mice
(CD45.2.sup.+) were transplanted along with 2.times.10.sup.5
competitor (CD45.1.sup.+) bone marrow cells into
lethally-irradiated CD45.1.sup.+ recipients. As expected, robust
engraftment was observed in recipients of 1,000 control LSK
Flk2-cells (77.+-.6%) (FIG. 10E). Pten mutants exhibited somewhat
higher average engraftment of 88.+-.4%. The highest and most
consistent engraftment of 97.+-.1.5% was exhibited in Pten:Ctnnb1
mice. In contrast, average engraftment was only 32.+-.36% in Ctnnb1
mutants, with half the recipients exhibiting little to no
engraftment. The relatively poor and variable engraftment observed
in the Ctnnb1 transplant group may be due to a minor portion of
HSCs that escaped knockout of the floxed Ctnnb1 allele. Indeed,
previous reports have shown that phenotypically defined HSCs in
Ctnnb1 mutants are no longer functional (Kirstetter, P., Anderson,
K., Porse, B. T., Jacobsen, S. E. & Nerlov, C. Activation of
the canonical Wnt pathway leads to loss of hematopoietic stem cell
repopulation and multilineage differentiation block. Nat Immunol 7,
1048-1056 (2006); Scheller, M. et al. Hematopoietic stem cell and
multilineage defects generated by constitutive [beta]-catenin
activation. Nature Immunology 7, 1037-1047 (2006)). In order to
test this, LSK Flk2- cells from Scl-Cre negative (control) as well
as Ctnnb1 mutants at 2 and 16 wpi were sorted and genotyped for
presence of the knockout allele. At 2 wpi, the mutant Ctnnb1 allele
was present; however, by 16 wpi no cells containing mutant Ctnnb1
allele remained, demonstrating that Ctnnb1 mutant HSCs are not
maintained long-term (FIG. 18). In contrast, Pten:Ctnnb1 HSCs were
highly dominant and almost wholly out-competed all HSCs found
within the competitor bone marrow cells.
[0242] To verify this, the Z/EG reporter system was included to
determine which cells had undergone Cre-mediated excision of their
floxed alleles (Novak, A., Guo, C., Yang, W., Nagy, A. & Lobe,
C. G. Z/EG, a double reporter mouse line that expresses enhanced
green fluorescent protein upon Cre-mediated excision. Genesis 28,
147-155 (2000)). The Z/EG reporter system activates expression of
enhanced green fluorescent protein (EGFP) upon Cre-mediated
excision.
[0243] Given the impaired myeloid differentiation and accumulation
of phenotypic HSCs in double mutants, whether mutant LSK Flk2.sup.-
cells were maintained in recipient mice was tested. LSK Flk2.sup.-
cells was gated in each recipient group, and GFP expression in this
subpopulation was observed. As expected, mice transplanted with
Scl-Cre negative (control)-Z/EG donor LSK Flk2- cells exhibited no
EGFP.sup.+ LSK Flk2.sup.- cells (0.8.+-.0.8%) (FIG. 10F).
Similarly, recipients of Ctnnb1-Z/EG LSK Flk2.sup.- cells also had
very few EGFP+ LSK Flk2- (1.6.+-.0.9%) cells, further demonstrating
that essentially only those LSK Flk2- cells escaping knockout
induction remained. LSK Flk2.sup.- cells from Pten-Z/EG transplant
recipients exhibited a minor GFP.sup.+ population (8.6.+-.2.1%),
demonstrating that some mutant LSK Flk2.sup.- cells remained even
after 9-10 wpi, although most had differentiated or had been
otherwise lost. In contrast, LSK Flk2- cells from Pten:Ctnnb1-Z/EG
transplant recipients were 90.0.+-.4.0% EGFP+. These data
demonstrate that, while Ctnnb1 mutant phenotypic HSCs are not
maintained and Pten mutant phenotypic HSCs differentiate, double
mutant phenotypic HSCs are maintained in vivo.
Example 5
Differentiation Block and Dominant Phenotype of Pten:Ctnnb1 Mutant
HSCs
Colony Forming Unit (CFU) Assays
[0244] CFU Assays were performed according to manufacturer's
instructions using complete methylcellulose media with Epo (Cat.
No. M3434, Stem Cell Technologies, Inc., Vancouver, Canada).
Lentiviral Production/HSC Transduction
[0245] To knockdown mouse .beta.-catenin in HSCs and their progeny,
lentiviruses in which mouse .beta.-catenin short-hairpin RNAs
(shRNAs) and an IRES GFP label are driven by a MSCV (murine stem
cell virus) LTR promoter were generated. Lentiviral constructs were
produced by directionally cloning DNA oligonucleotides
corresponding to two Ctnnb1 shRNAs (sequences HP_224742 and
HP_240000; SEQ ID NOs: 3-4, RNAicodex, Cold Spring, N.Y.) into the
Gateway.RTM. entry vector pEN-LmiRc3 (Invitrogen), then recombining
with the destination vector pDSL-hpIG (Zhu, X. et al. A versatile
approach to multiple gene RNA interference using microRNA-based
short hairpin RNAs. BMC molecular biology 8, 98 (2007)). The
control viral construct drives a luciferase shRNA (Id.) from the
same vector backbone. Virus was produced in 293T cells by
co-transfection of the virus plasmid with packaging plasmids
(pRC-CMV-Rall, HDM-Tat16, HDM-HGPM2 and HDM-VSVG, a gift from Dr.
Jeffrey M. Rosen, Baylor College of Medicine), and was purified by
poly-ethylene-glycol (PEG) precipitation (0.45 .mu.m-filtered
supernatant was precipitated with 10% PEG-8000, 1.5% fetal bovine
serum for 72 hours at 4.degree. C., then pelleted at 1,500 g for 10
minutes), followed by ultracentrifugation through a sucrose cushion
(al Yacoub, N., Romanowska, M., Haritonova, N. & Foerster, J.
Optimized production and concentration of lentiviral vectors
containing large inserts. The journal of gene medicine 9, 579-584
(2007)). Titres were established using 293T cells due to the
limited numbers of HSCs available. Viral transduction of HSCs was
performed overnight in ST media with 8 .mu.M final polybrene at a
multiplicity of infection of 20-50 relative to the initial cell HSC
number (500 cells).
Experimental Results
[0246] Although putative HSCs can be highly enriched by cell
surface marker phenotype, bona fide HSCs are functionally defined.
When genetic mutation compromises function, formal proof that a
putative HSC population represents true HSCs can be precluded. This
is the case for mutants with constitutively active .beta.-catenin
because differentiation is blocked. Whether LSK Flk2.sup.- cells
isolated from double mutants could recover multilineage
differentiation capacity if .beta.-catenin transcripts were
degraded by RNA interference (RNAi) were determined. LSK Flk2.sup.-
cells from uninduced control, Pten, and Pten:Ctnnb1 mice were
sorted and induced knockout in vitro with 4-hydroxy-tamoxifen (OHT)
added for 3 days in culture. At day 3, HSC cultures were transduced
using lentiviral vectors targeting .beta.-catenin transcripts by
RNAi as set forth below.
[0247] At day 6, myeoloid-specific colony forming unit (CFU) assays
were performed on these HSC cultures. While knockdown of
.beta.-catenin in control and Pten HSC cultures did not
significantly affect colony formation, knockdown of .beta.-catenin
in double mutant cultures resulted in reversal from a novel CFU
phenotype to a CFU phenotype similar to Pten single mutants (FIG.
19A-FIG. 19B). Specifically, double mutant HSC cultures transduced
with control vector formed large CFU (>0.5 mm) which were not
produced in control or Pten cultures. Interestingly, these
primitive CFU were mostly CD3.sup.+ (T lymphoid) cells not found in
control colonies or Pten colonies (FIG. 19C). These novel CFUs are
apparently the result of myeloid differentiation blockage and
leukemic transformation in double mutants. In contrast, double
mutant cultures transduced with short-hairpin (sh) RNA targeting
.beta.-catenin produced only small CFU morphologically and
quantitatively similar to Pten CFU. These smaller colonies further
contained only minor proportions of CD3.sup.+ cells. In addition,
the number of colonies was shifted toward a predominance of
granulocyte/monocyte progenitors (CFU-GM) similar to Pten single
mutants (FIG. 19A-FIG. 19C). These data demonstrate that the
differentiation blockage exhibited by double mutant LSK Flk2.sup.-
cells is functionally reversible via knockdown of .beta.-catenin,
supporting the idea that the phenotypically defined HSCs expanding
in double mutants are, indeed, bona fide, though functionally
compromised, HSCs.
Example 6
Pten Single Mutants Exhibit Increased Myeloid Differentiation
[0248] Though not nearly as large as the increase observed in
double mutants, Pten single mutants exhibited increased frequency
and absolute number of LSK CD34.sup.- cells in spleen at 6 wpi
(FIG. 1C and FIG. 1E). At 9-10 wpi, early myeloid progenitors were
substantially increased in bone marrow and especially spleen of
Pten single mutants (FIG. 25), as were more mature myeloid cells
(FIG. 28). Indeed, the trend of moderate but variable LSK cell
expansion in spleen, coupled with increased myeloid
differentiation, was sustained for at least 15 wpi (FIG. 29).
[0249] In order to more comprehensively study the role of
.beta.-catenin interaction with the PTEN/Akt signaling pathway,
mice with floxed null alleles of .beta.-catenin (Ctnnb1.sup.tm2Kem)
(Cobas, M. et al. Beta-catenin is dispensable for hematopoiesis and
lymphopoiesis. The Journal of experimental medicine 199, 221-229
(2004)) were obtained and crossed to Mx1-Cre and Mx1-Cre Pten
mutants, allowing for the combination of conditional deletion of
.beta.-catenin (.beta.-cat.sup.-/-), Pten, and
Pten:.beta.-cat.sup.-/-. As with the Pten:Ctnnb1 compound mutant,
primary animals were studied; however, this was difficult to pursue
because .beta.-cat.sup.-/- mice typically had to be sacrificed by
15 dpi, while Pten:.beta.-cat.sup.-/- double mutants rarely
maintained adequate health beyond 7 dpi (data not shown).
[0250] In order to study long-term and hematopoietic-specific
defects in single and double knockout mutants, whole bone marrow
transplantations were performed. Bone marrow from control
(Cre.sup.-), Mx1-Cre.sup.+ single and double mutant donors into
lethally irradiated Ptprc recipients using 1.times.10.sup.6
cells/recipient prior to induction. At 10 wpi of transplant
recipients, 5 mice from each group were sacrificed, and LSK cells
as well as early progenitors were analyzed by FACS. Unlike
Pten:.beta.-cat.sup.Act double mutants, none of the
Pten:.beta.-cat.sup.-/- double mutants exhibited signs of leukemia
by 10 wpi (data not shown). Consistent with previous reports,
.beta.-cat.sup.-/- single mutants did not exhibit any defects in
absolute numbers of LSK or early progenitors (Cobas, M. et al.
Beta-catenin is dispensable for hematopoiesis and lymphopoiesis.
The Journal of experimental medicine 199, 221-229 (2004)); however
Pten single mutants exhibited an expansion of LSK cells as well as
CMPs and MEPs in the spleen (FIG. 11D). Interestingly,
Pten:.beta.-cat.sup.-/- double knockout transplant recipients did
not exhibit an expansion of LSK cells in the spleen, while CMPs and
MEPs were increased compared to control but less than Pten only
mutants (FIG. 11D). In contrast, analysis of more mature
hematopoietic lineages revealed similar increases in Mac-1.sup.+
Gr1.sup.+ cells between Pten single and Pten:.beta.-cat.sup.-/-
double knockouts, indicative of MPD, while lymphoid lineages were
similarly reduced in both Pten and Pten:.beta.-cat.sup.-/-
transplant recipients (FIG. 11 E-FIG. 11G). These results
demonstrate that loss of .beta.-catenin rescues the LSK cell
expansion observed in Pten mutant spleen and partially rescues the
early myeloid progenitor cell expansion, although MPD development
still occurs. Relative to the number of LSK cells, Pten:
.beta.-cat.sup.-/- mutants expanded early myeloid progenitors as
well as or greater than Pten single mutants (FIG. 11D). Thus, loss
of .beta.-catenin appears to primarily rescue HSC-specific effects,
with the downstream events that lead to MPD being separable,
.beta.-catenin-independent phenomena. These data also further
confirm that the Wnt/.beta.-catenin and PTEN/Akt pathways
cooperatively interact in driving HSC expansion.
Example 7
.beta.-cat.sup.Act Single Mutant HSCs Rapidly Undergo Apoptosis In
Vitro
[0251] .beta.-cat.sup.Act single mutants exhibited a reduced
frequency of LSK CD34.sup.- cells in bone marrow at 6 wpi (FIG.
1B); however, long-term defects in .beta.-cat.sup.Act single
mutants was not observed. Indeed, although efficient knockout in
sorted .beta.-cat.sup.Act HSCs at 2 wpi was detected, at 16 wpi
HSCs with the mutant .beta.-cat.sup.Act allele were not present in
induced .beta.-cat.sup.Act mice (FIG. 18). Similarly, the
transplantation experiments demonstrated that .beta.-cat.sup.Act
single mutant HSCs were not maintained in vivo (FIG. 10F).
[0252] Because the exact time point when .beta.-cat.sup.Act HSCs
were lost in vivo is not known, whether .beta.-cat.sup.Act HSCs
undergo apoptosis was tested. LSK Flk2.sup.- cells were isolated
from uninduced mice, genetic deletion was induced in vitro, and the
resulting cultures were then visually monitored. These experiments
revealed that, by 4 days post-induction, no .beta.-cat.sup.Act LSK
Flk2.sup.- cells survived; whereas control, Pten, and particularly
double mutant LSK Flk2.sup.- cells survived and expanded (FIG.
11A). At 48 hours post-induction, although some .beta.-cat.sup.Act
LSK Flk2.sup.- cells remained, their numbers were reduced relative
to control (FIG. 11B). Whether these cells were undergoing
apoptosis was tested by Annexin V staining. Unlike control, the
majority of .beta.-cat.sup.Act LSK Flk2.sup.- cells at 48 hours
post-induction were either undergoing apoptosis or already dead,
demonstrating that constitutive activation of .beta.-catenin in LSK
Flk2.sup.- cells in vitro results in rapid apoptosis (FIG. 11C).
These data demonstrate that while most Pten mutant HSCs
differentiate, .beta.-cat.sup.Act mutant HSCs undergo rapid
apoptosis in vitro, exhibit functional failure in vivo, and are not
maintained in recipients. In contrast, double mutant LT-HCSs were
phenotypically maintained 9-10 wpi, becoming the dominant HSC
population in transplant recipients. These data demonstrate that
.beta.-cat.sup.Act mutant HSCs have a survival defect and that this
survival defect can be rescued by additional loss of Pten.
Example 8
Unlike Single Mutants, Double Mutants Rapidly and Consistently
Develop Leukemia
[0253] Control animals (Scl-Cre negative littermates) remained
healthy as expected, and Ctnnb1 mutants also remained healthy
through at least 20 wpi. In contrast, about 30% of Pten single
mutants had to be sacrificed by 20 wpi, but the majority survived
through at least 28 wpi. Pten:Ctnnb1 double mutants exhibited a far
more rapid decline in health than Pten single mutants. Double
mutants typically survived until at least 8 wpi when a minority had
to be sacrificed due to poor condition (FIG. 12A). By 11 wpi,
however, all double mutants had to be sacrificed. Histological
examination of Pten:Ctnnb1 bone marrow at 9-10 wpi revealed that
the bone shaft (diaphysis) became substantially filled with bone,
while trabecular bone regions (metaphysis), reported to be enriched
in sites containing the HSC niche (Xie, Y. et al. Detection of
functional haematopoietic stem cell niche using real-time imaging.
Nature 457, 97-101 (2009); Arai, F. et al. Tie2/angiopoietin-1
signaling regulates hematopoietic stem cell quiescence in the bone
marrow niche. Cell 118, 149-161 (2004); Calvi, L. M. et al.
Osteoblastic cells regulate the haematopoietic stem cell niche.
Nature 425, 841-846 (2003); Zhang, J. et al. Identification of the
haematopoietic stem cell niche and control of the niche size.
Nature 425, 836-841 (2003)), were largely hypo-cellular with areas
that appeared grossly normal (FIG. 11B). In contrast, no obvious
defects were apparent in either single mutant bone marrow (data not
shown). Splenomegaly in Pten:Ctnnb1 mutants at 9-10 wpi was
observed, with the spleen exhibiting severe hypo-cellularity and
fibrosis (FIG. 16). In contrast, gross appearance of single mutant
spleen was normal at 9-10 wpi. Furthermore, the hypo-cellularity
and fibrosis observed in double mutant spleen was present even in
wild-type mice transplanted with LSK Flk2- cells from Pten:Ctnnb1
donors. The stromal abnormalities observed were most likely a
consequence of loss of negative inhibition from hematopoietic cells
to normal stroma rather than of defects originating in the
stroma.
[0254] Suspecting that acute leukemia/lymphoma caused the rapid
decline in health of Pten:Ctnnb1 mutants between 8-11 wpi, FACS
analysis was used to examine the abundance of CD45.sup.High
primitive blast cells in control, single, and double mutants
(Borowitz, M. J., Guenther, K. L., Shults, K. E., Stelzer, G. T.
Immunophenotyping of acute leukemia by flow cytometric analysis.
Am. J. Clin. Pathol. 100, 534-540 (1993)). As shown in FIG. 5C,
Pten:Ctnnb1 mutants exhibited a conversion to predominantly
leukemic blast cells in the bone marrow by 9-10 wpi. This was
observed in all double mutants examined (n>20). In contrast,
control and Ctnnb1 mice never exhibited a significant blast
population. Typically, Pten mutants were also similar to control
regarding bone marrow CD45 expression level at 9-10 wpi, although
2/16 exhibited a minor blast population (data not shown). These
data demonstrate that all Pten:Ctnnb1 mutants develop a severe
acute leukemia by 9-10 wpi while single mutants do not. Lineage
marker analysis further characterized the leukemic cells to express
the T-cell specific marker CD3, revealing the leukemia to be T-cell
acute lymphocytic leukemia or T-ALL (FIG. 5C).
[0255] To further investigate hematopoietic lineage defects in
Pten:Ctnnb1 mutants and to characterize the type of leukemia, the
major hematopoietic lineages was examined in bone marrow at 8-9 wpi
(FIG. 20A-FIG. 20G). Most prominently, CD3.sup.+ cells in double
mutants did not express more differentiated T-cell markers, CD4 or
CD8. Overall, more than 75% of total bone marrow cells in double
mutants were CD3.sup.+ but CD4 and CD8 negative, compared with less
than 5% in control (FIG. 20B-FIG. 20C). To further define the
origin and nature of the T-ALL observed in double mutants, T-cell
development in thymus was also examined. Double negative (DN) early
T-cell precursors lack CD3, 4 and 8 expression, and their stage of
maturation can be distinguished by CD25 and/or CD44 expression.
While less than 5% of thymocytes were within the DN subset in
control and single mutants, the majority of thymocytes were within
this subset in double mutants (FIG. 20D-FIG. 20E). Although both
single mutants were similar to control, Pten:Ctnnb1 mice exhibited
a large increase in DN CD25- CD44- cells (FIG. 20D-FIG. 20E). Also,
while the majority of thymocytes were double positive precursors in
control and single mutants as expected, this population was
essentially absent from Pten:Ctnnbl mice (FIG. 20E-FIG. 20G). These
data demonstrate that the T-ALL observed in double mutants involves
expansion of an early thymic progenitor, resulting in the
accumulation of immature T-lineage precursors.
[0256] Self-renewal has been proposed to require the concurrence of
three events, proliferation while preventing apoptosis and blocking
differentiation (Zhang, J. & Li, L. BMP signaling and stem cell
regulation. Dev Biol 284, 1-11 (2005)). However, the imposition of
differentiation on proliferating stem cells--or apoptosis for stem
cells that fail to properly differentiate--is critical to HSC
homeostasis and cancer prevention. By studying the individual and
combined effects of PTEN and .beta.-cat mutants, the inventors have
discovered that HSC self-renewal is cooperatively controlled by the
PTEN/Akt and Wnt/.beta.-catenin pathways acting in a manner
consistent with this tripartite view of self-renewal. Switching
from a non-tissue specific method of gene disruption to generating
HSC-specific conditional mutants using the Scl-Cre system allowed
for the study of defects arising primarily from HSCs and for the
long-term, controlled study of double mutants. With this more
refined model, it was found that Pten deletion results in
relatively moderate HSC proliferation but sustained phenotypic HSC
expansion. However, this is coupled with increased myeloid
differentiation. Pten deletion also results in Akt activation, a
potent cell survival factor which prevents apoptosis (Datta, S. R.
et al. Akt phosphorylation of BAD couples survival signals to the
cell-intrinsic death machinery. Cell 91, 231-241 (1997); Salmena,
L., Carracedo, A. & Pandolfi, P. P. Tenets of PTEN Tumor
Suppression. Cell 133, 403-414 (2008)). In contrast,
Wnt/.beta.-catenin signaling blocks differentiation, but additional
signals are needed for HSC expansion. Similarly, all .beta.-catenin
and most Pten single mutants fail to develop leukemia, which
requires aberrant self-renewal. However, these experiments
demonstrates that only in cooperation can Wnt/.beta.-catenin and
PTEN/Akt signaling drive self-renewal and expansion without
extensive differentiation. Although permanent mutation in both
these pathways ultimately leads to T-ALL, transient,
pharmacological manipulation allows for the expansion of functional
HSCs. Thus, at the stem cell level, the interaction between these
two pathways coordinates the necessary components of self-renewal,
with each pathway making unique as well as joint contributions to
HSC expansion.
[0257] These findings that Pten:Ctnnbl double mutants expand HSCs
to a greater degree than single mutants and that compound loss of
both .beta.-catenin and Pten rescues Pten-deficiency-induced HSC
expansion demonstrate that the effects of Pten loss on HSCs are
partially mediated through .beta.-catenin. Rapamycin treatment has
been reported to prevent the formation of leukemia-initiating cells
in Pten mutants and to restore normal HSC function, indicating that
mammalian target of rapamycin (mTor) is also an important mediator
of the effects of Pten-deficiency (Yilmaz, O. H. et al. Pten
dependence distinguishes haematopoietic stem cells from
leukaemia-initiating cells. Nature 441, 475-482 (2006)).
[0258] A recent study using VE-cadherin-Cre mediated deletion of
Pten has demonstrated that leukemic stem cells are highly enriched
in a relatively rare population of Kit.sup.Mid CD3+ Lin- cells,
which appear to be driven by increased .beta.-catenin activation
(Guo, W. et al. Multi-genetic events collaboratively contribute to
Pten-null leukaemia stem-cell formation. Nature 453, 529-533
(2008)). Thus, excessive self-renewal driven by Wnt/.beta.-catenin
and PTEN/Akt interaction may be important in cancer stem cell
development as well as normal HSC self-renewal. Defining the
origins and characteristics of cancer stem cells is critical if
they are to be detected, if their formation is to be prevented, or
if they are to be selectively eliminated. In the instant
application, the HSC-like LS.sup.LowK.sup.Mid population frequently
observed in double mutants prior to being out-competed by leukemic
blast cells (FIG. 10A) may be of particular interest. As a
primitive population these could be cancer stem cells or they could
be the ultimate source of a more mature population of CD3+ cancer
stem cells, possibilities that require further testing.
Example 9
Ex Vivo Pharmacological Manipulation of the PTEN/Akt and
Wnt/.beta.-Catenin Signaling Pathways Cooperatively Drive
Functional HSC Expansion
[0259] In double mutants, permanent genetic alteration leads to
enhanced self-renewal, while differentiation is blocked except
toward early T-cell commitment, ultimately resulting in T-ALL. The
conversion of essentially all bone marrow cells to competitive
leukemic blast cells along with the niche disruption prevents
sustained HSC expansion in double mutants. However, reversible,
pharmacological manipulation of the PTEN/Akt and/or
Wnt/.beta.-catenin pathways may allow for the transient enhancement
of self-renewal in vitro with the capacity to function as normal
HSCs following removal of these agents and in vivo
transplantation.
[0260] This concept was tested by utilizing a small molecule
inhibitor of GSK3.beta. (CHIR99021) (Ring, D. B. et al. Selective
glycogen synthase kinase 3 inhibitors potentiate insulin activation
of glucose transport and utilization in vitro and in vivo. Diabetes
52, 588-595 (2003); Schmid, A. C., Byrne, R. D., Vilar, R. &
Woscholski, R. Bisperoxovanadium compounds are potent PTEN
inhibitors. FEBS Lett 566, 35-38 (2004)). GSK3.beta. inhibits
.beta.-catenin by targeting .beta.-catenin for proteosomal
degradation and acts in the Wnt/.beta.-catenin pathway. Indeed,
CHIR99021, which is the most specific and potent small molecule
inhibitor of the Wnt/.beta.-catenin pathway reported (Ring, D. B.
et al. Selective glycogen synthase kinase 3 inhibitors potentiate
insulin activation of glucose transport and utilization in vitro
and in vivo. Diabetes 52, 588-595 (2003)), has been shown to
promote embryonic stem (ES) cell self-renewal and expansion (Ying,
Q.-L. et al. The ground state of embryonic stem cell self-renewal.
Nature 453, 519-523 (2008)).
[0261] The inventors have developed a defined culture system
utilizing only two cytokines, stem cell factor (SCF) and
thrombopoietin (Tpo) (ST media), which have been shown previously
to support HSC expansion in vitro (Zhang, C. C. & Lodish, H. F.
Murine hematopoietic stem cells change their surface phenotype
during ex vivo expansion. Blood 105, 4314-4320 (2005)). While
addition of CHIR99021 increased the expansion of LSK Flk2.sup.-
cells, addition of a small molecule inhibitor of PI3K (NVP-BEZ235)
(Maira, S. M. et al. Identification and characterization of
NVP-BEZ235, a new orally available dual phosphatidylinositol
3-kinase/mammalian target of rapamycin inhibitor with potent in
vivo antitumor activity. Molecular cancer therapeutics 7, 1851-1863
(2008)) decreased the ability of LSK Flk2.sup.- cells to expand in
a dose-dependent manner (FIG. 21). Also, the ability of CHIR99021
to enhance expansion was negated by PI3K inhibition (FIG. 21).
Together, these data demonstrate that stimulation of PI3K signaling
is required for substantial HSC expansion in our culture system.
Indeed, a recent study reports that the growth factor pleiotrophin
expands HSCs, in part, by activating PI3K signaling (Himburg, H. A.
et al. "Pleiotrophin regulates the expansion and regeneration of
hematopoietic stem cells" Nat Med Vol 16, pages 475-482
(2010)).
[0262] One hundred LSK Flk2.sup.- cells were sorted from wild-type
(C57BI/6) mice and cultured in (1) media, (2) media+1 .mu.M
CHIR99021 (a GSK-3.beta. inhibitor, a gift from Dr. Sheng Ding),
(3) media+200 nM Dipotassium Bis-peroxo(picolinato)oxovanadate
(BpV(pic), a PTEN inhibitor, available from Calbiochem, Cat. No.
203705), (4) media+1 .mu.M CHIR99021+200 nM BpV(pic), (5) media+200
nM Shikonin (also a PTEN inhibitor, available from Calbiochem, Cat.
No. 565850), and (6) media+200 nM Shikonin+1 .mu.M CHIR99021. (FIG.
3B-FIG. 3C). Cells were cultured as described above. Cells were
examined at 17 days of culture (FIG. 3B, original magnification
100.times.) and 23 days (FIG. 3C, original magnification
40.times.). Compared to control, both inhibitors applied
individually exhibited greater expansion of LSK cells indicating
that GSK-3.beta. inhibition is not strictly equivalent to
constitutive activation of .beta.-catenin shown in Ctnnb1 mutant
LSKs, while BpV(pic) exhibited similar results compared to Pten
mutant LSKs (see FIG. 2). Similar to double mutant LSKs (FIG. 2),
the greatest expansion occurred with both inhibitors present (FIG.
3B/FIG. 3C panel 4).
[0263] LSK Flk2.sup.- cells at 28 days culture in the indicated
media conditions were examined (FIG. 3D, original magnification
200.times.). Here, significant expansion relative to control was
observed with both inhibitors present individually; however,
significant differentiation/heterogeneity of cell morphology was
observed in both cases, including more variable cell
size/morphology and/or differentiation to adherent, spindle-shaped
cells (middle panels). In contrast, expansion with homogeneity was
achieved when both inhibitors were present (last panel).
[0264] FACS analysis of 28 day LSK Flk2.sup.- cells cultured in
media+BpV(pic)+CHIR99021 (FIG. 3E) was performed. Cells were
pre-gated on live, lineage negative cells. Greater than 90% of LSKs
retained Flk2 negativity (data not shown). Thus, the LSK Flk2.sup.-
phenotype was maintained with high purity in cultures containing
both inhibitors.
[0265] Fold expansion of LSK Flk2.sup.- cells after 28 days culture
in (1) media, (2) media+BpV(pic), (3) media+CHIR99021, and (4)
media+CHIR99021+BpV(pic) were analyzed. While each inhibitor added
individually led to significant expansion compared to media without
either inhibitor, the greatest expansion (.about.270 fold) was
observed when both inhibitors were added together.
Example 10
Transplantation Analysis of Cultured Sorted LSK Cells after Ex Vivo
Pharmacological Manipulation
Cell Harvest and Repopulation
[0266] Cells were harvested from the wells prior to transplantation
by pipetting up and down several times before transferring to a
fresh tube. Residual was then collected by adding more media and
repeating. Cells were washed in DMEM (Invitrogen, Cat. No. 31053)
without phenol red and added to the appropriate number of whole
bone marrow rescue cells from a congenic donor (for 200,000 rescue
cells+1,000 re-sorted LSK Flk2.sup.- cells (FIG. 3F-FIG. 3H) or the
non-adherent product of 10 days culture of 100 LSK Flk2.sup.-
cultured cells (FIG. 3I-FIG. 3K) per mouse as indicated). Cells
were injected into lethally irradiated (10 Grays, single dose)
Ptprc (CD45.1.sup.+) recipient mice through the tail vein using an
insulin syringe.
[0267] Repopulation was measured at 4 weeks post-transplant by
collection of periperal blood, red blood cell lysis, and staining
of CD45.1 (recipient) compared to CD45.2 (donor) engraftment using
antibodies purchased from eBiosciences (FITC conjugated CD45.2
(Cat. No. 11-0454-85) and PE-Cy5 conjugated CD45.1 (Cat. No.
15-0453-82)). Mice transplanted with rescue/competitor cells only
were used as a control to determine the limits of repopulation
detection. Multi-lineage reconstitution was determined by CD3, B220
(for lymphoid) and Gr1, Mac-1 (for myeloid), as described
above.
Transplantation Analysis of 28 Day Cultures
[0268] Cells cultured for 28 days in (1) media, (2) media+BpV(pic),
(3) media+CHIR99021 and (4) media+CHIR99021 (1 .mu.M)+BpV(pic) (200
nM) were re-sorted for LSK Flk2.sup.- cells. One thousand LSK
Flk2.sup.- cells (CD45.2.sup.+) from each media condition were
transplanted into lethally irradiated (10Gy) CD45.1.sup.+ recipient
mice along with 2.times.10.sup.5 congenic whole bone marrow
competitor cells. At 4 weeks post-transplant, peripheral blood was
analyzed for donor (FIG. 3G) and multi-lineage (FIG. 3H)
engraftment. In FIG. 3G, each bar represents an individual mouse.
The horizontal-dashed line represents the average `engraftment` of
mice transplanted with competitor cells only and, thus, the limit
of detectability for true engraftment. Long-term (4 month)
engraftment has not been observed from 28-day cultures (data not
shown). Six of 8 mice show >1% engraftment when transplanted
with LSK Flk2.sup.- cells cultured with both inhibitors present
compared to 4/8 with only CHIR99021 present, 0/10 with only
BpV(pic) present, and 2/6 with media only. One percent or greater
engraftment is a standard limit for substantial engraftment.
(Zhang, C. C., et al., Nat Med, 12(2): 240-5, 2006. Zhang, C. C.
and H. F. Lodish, Blood, 105(11): 4314-20, 2005). Thus, while both
inhibitors together leads to greatest expansion in LSKs (FIG. 2F),
transplantation of equivalent numbers of these cultured LSK
Flk2.sup.- cells also leads to increased short-term
engraftment/functionality when cultured with both inhibitors
compared to no or either single inhibitor only.
[0269] While all mice with genetic alterations resulting in
constitutively active .beta.-catenin and loss of PTEN will develop
leukemia and must be sacrificed due to poor health within 8-10
weeks post-mutation induction (FIG. 1I and data not shown), no mice
transplanted with LSK Flk2.sup.- cells cultured in either inhibitor
singly or in combination has shown any sign of leukogenesis up to
16 weeks post-transplantation. All such mice appeared healthy
unlike 8-10 weeks post-induction genetically double mutant mice,
exhibiting no loss of body weight, anemia, loss of appetite,
lethargy, hunched posture, etc. Thus, the effects of the inhibition
of both pathways using, e.g., BpV(pic) and CHIR99021, is
reversible.
Transplantation Analysis of 10 Day Cultures
[0270] Cells cultured for 9 days in (1) media, (2) media+BpV(pic)
(200 nM), (3) media+CHIR99021 (100 nM), and (4) media+CHIR99021
(100 nM)+BpV(pic) (200 nM) were re-sorted for LSK Flk2.sup.- cells,
and fold expansion of LSK Flk2.sup.- cells after 9 days culture in
the indicated conditions was determined (FIG. 31). Because
long-term engraftment was not observed from 28 day cultures (FIG.
3D-FIG. 3H and data not shown), LSK Flk2.sup.- cells were cultured
for only 9 days to test if both expansion and long-term
repopulation could be achieved. Similar trends were observed here
when compare to the 28 day cultures (compare to FIG. 9F) although
the extent of expansion was substantially reduced at only 9 days
versus 28 days culture.
[0271] FACS analysis was performed on 9 day LSK Flk2.sup.- cells
cultured in media+BpV(pic) (200 nM)+CHIR99021 (100 nM) (FIG. 3J).
Cells were pre-gated on live, lineage negative cells. Greater than
90% of LSKs retain Flk2 negativity (data not shown). Here, the
levels of Sca-1 and Kit appear normal compared to the
Sca-1.sup.(high)Kit.sup.(high) population shown from 28 day
cultures (FIG. 1E).
[0272] Ten day cultures were transplanted into lethally irradiated
(10Gy) CD45.1.sup.+ recipient mice along with 2.times.10.sup.5
congenic whole bone marrow competitor cells. The total,
non-adherent cell product after 10 days culture of 100 initial LSK
Flk-2 cells was transplanted per mouse. At 8 weeks post-transplant,
peripheral blood was analyzed for donor (FIG. 3G) and multi-lineage
(FIG. 3H) engraftment. As shown, multi-lineage reconstitution was
observed from all mice exhibiting true engraftment (data not
shown). In FIG. 3G, each bar represents an individual mouse; the
horizontal-dashed line represents the average `engraftment` of mice
transplanted with competitor cells only and thus the limit of
detectability for true engraftment. Here, 3/7 mice transplanted
with LSK Flk2.sup.- cells cultured in the presence of both
inhibitors exhibited 1% or greater donor engraftment compared to no
mice reaching this threshold in the single or no inhibitor
groups.
[0273] Collectively, these data demonstrate that the PTEN/Akt and
Wnt/.beta.-catenin signaling pathways can be manipulated
pharmacologically to drive HSC expansion. Functional, short-term
HSCs show highest reconstitution ability when cultured in the
presence of both inhibitors. Substantial longer-term reconstitution
(8 weeks) occurs only when HSCs are cultured in the presence of
both inhibitors but not when cultured with either single inhibitor
or in the absence of either inhibitor. Thus, the pharmacological
manipulation of both pathways simultaneously results in the
greatest expansion of functional HSCs. This effect is reversible
because recipient animals did not develop leukemia as genetic
mutants did (FIG. 1) and cultured HSCs were able to differentiate
unlike cultured HSCs from genetic mutants (FIG. 2).
Example 11
Transplantation Analysis of HSCs in a Population of Bone Marrow
Mononuclear Cells
Materials and Methods
[0274] For the experiments set forth in this Example, a particular
HSC expansion media was used. This HSC expansion media consists of
the following ingredients: (1) StemSpan Media (Stem Cell
Technologies; Cat. No. 09600) (StemSpan Media consists of
Iscove's-modified Dulbecco's medium (IMDM) supplemented with 1%
bovine serum albumin, 10 .mu.g/ml recombinant human insulin, 200
.mu.g/ml iron-saturated transferrin, 0.1 mM 2-mercaptoethanol and 2
mM glutamine.); (2) 10 ug/ml Heparin (Sigma; Cat. No. H-3149); (3)
0.5.times. Penicillin/Streptomycin (Sigma; Cat. No. P4333); (4) 10
ng/ml recombinant mouse (rm) or recombinant human (rh) Stem Cell
Factor (SCF) (Biovision; Cat. No. 4328-10 or 4327-10,
respectively); (5) 20 ng/ml rm or rh Thrombopoietin (Tpo) (Cell
Sciences, Inc; Cat. No. CRT401B or CRT400B, respectively).
CHIR99021 (250 nM) (Stemgent, Inc; Cat. No. 04-0004) may be added
to this HSC expansion media as indicated.
[0275] Note that the optimal base media for expanding phenotypic
HSCs (StemSpan SFEM, Stem Cell Technologies, Inc.) contained a high
concentration of insulin, a major stimulator of the PI3K/Akt
pathway. Because the same media without insulin was unable to
substantially expand HSCs, expansion of phenotypic HSCs is
dependent on insulin in our culture system (FIG. 30). Similarly,
SCF, a typical cytokine utilized for ex vivo HSC expansion, also
activates the PI3K pathway. It was found that Tpo or SCF alone
could not expand HSCs in culture, both Tpo and SCF were necessary
for substantial expansion (data not shown).
[0276] Cells were cultured in 96-well U-bottom tissue culture
plates (Becton, Dickinson and Company; Cat. No. 353077).
[0277] Antibodies used are listed below and as set forth in Example
1. The following antibodies were obtained from eBiosciences: FITC
conjugated CD45.2 (Cat. No. 11-0454-85), PE-Cy5 conjugated CD45.1
(Cat. No. 15-0453-82), PE conjugated CD34 (Cat. No. 12-0349-73),
and APC conjugated CD38 (Cat. No. 17-0389-73).
[0278] Cell counts were obtained using a Quanta cell
counter/cytometer (Beckman-Coulter). Cell sorting and analysis were
performed using a MoFlo (Dako, Ft. Collins, Colo.) flow cytometer
and/or a CyAn ADP (Dako, Ft. Collins, Colo.). Frequency of LSK
Flk.sup.- cells was determined by analyzing >3.times.10.sup.5
cells per sample independently in triplicate.
[0279] Bone marrow cells were harvested from C57BI/6 (CD45.2) mice
and made into a single cell suspension by gently drawing through a
22 g needle several times. Mobilized peripheral blood or bone
marrow from human patients was harvested at the University of
Kansas Medical Center (Kansas City, Mo. USA). Because red blood
cell (RBC) lysis was determined to severely inhibit functional HSC
expansion, cells were not exposed to any RBC lysis procedure.
Mononuclear cells were isolated from mouse bone marrow using
Histopaque 1077 (Sigma; Cat. No. 10771) and human blood or bone
marrow using Ficoll-Paque PLUS (Stem Cell Technologies; Cat. No.
07917) according to the manufacturers' instructions. Cells were
washed and resuspended in HSC expansion media. Cells were counted
and a fraction of mononuclear cells (MNCs) were stained for lineage
markers using CD3, CD4, CD8, B220, IgM, Mac-1, Gr1, and Ter119
antibodies along with Kit, Sca-1, and Flk2 for mouse HSC analysis
or CD34 and CD38 for human HSC analysis. 1.times.10.sup.6 cells/0.1
ml were stained at 4.degree. C. for 30 minutes using 0.05 .mu.g of
antibody for each lineage marker and 0.2 .mu.g for remaining
antibodies. Cells were washed twice in staining buffer (1.times.
Phosphate buffered saline (PBS) (Mediatech, Inc, Cat. No.
20-031-CV)+2% fetal bovine serum (FBS) (Gibco-BRL, Cat. No.
16140-071)). Frequency of putative HSCs (lineage negative,
Sca-1.sup.+, Kit.sup.+, Flk-1.sup.- for mouse or CD34.sup.+,
CD38.sup.- cells for human) was determined by analyzing
>3.times.10.sup.5 cells per sample independently in triplicate.
MNCs were then plated at 100 putative HSCs (along with
2.5-5.0.times.10.sup.4 MNCs depending on frequency of putative HSCs
in the particular sample--typically 0.2-0.4%) in 200 .mu.l of HSC
expansion media per well in a 96-well U bottom plate (Becton,
Dickinson and Company; Cat. No. 353077). MNCs were also plated at
50 LSK Flk2.sup.- cells (along with 1.7-5.0.times.10.sup.4 MNC
cells depending on frequency of putative HSC in the particular
sample--typically 0.1-0.3%) in 200 .mu.l of HSC expansion media per
well in a 96-well U-bottom plate.
[0280] Cells were incubated at 37.degree. C. with 5% CO.sub.2 and
5% O.sub.2 (balance N.sub.2) for 14 days. Cultures were checked
daily and cell pellets accumulating at the bottom of each well
which exceeded 2 mm in diameter were split into new wells at a 1:1
ratio (splitting involved resuspension of the culture cell pellet
by gently pipetting up and down 5-7 times and removing 1/2 of the
volume of the original well and placing it into a fresh well. That
volume of fresh media was then replaced in each "old" and "new"
well). It is critical for optimal HSC expansion that cell pellets
are maintained at a density of 1-2 mm in size. Splitting is
typically required at day 1 and every 2-3 days thereafter. In
parallel, putative HSCs were sorted into 96-well U bottom plates at
100 putative HSCs per well. Sorted putative HSCs were handled
equivalently to unsorted cultures. After 14 days culture, the total
culture product was harvested by pipetting up and down 10 times and
combining into a test tube. Cells were washed and resuspended in
DMEM (Invitrogen; Cat. No. 31053) in a volume equivalent to 5
original input putative HSCs per 100 .mu.l for unsorted HSC
cultures (for example, a well containing MNCs with 100 putative
HSCs along with its descendant wells resulting from splitting would
be resuspended in 2,000 .mu.l) or 100 original input putative HSCs
per 100 .mu.l for sorted HSC cultures.
[0281] For competitive repopulation assays, 1.times.10.sup.5 bone
marrow cells congenic with the host (CD45.1.sup.+) were included
per mouse. 100 .mu.l of cultured cells or cells freshly isolated
and quantified in the same manner were transplanted into lethally
irradiated (10 Grays, single dose) Protein tyrosine phosphatase,
receptor type, C (Ptprc or CD45.1) recipient mice through the tail
vein using an insulin syringe (29 gauge). Mice were placed on
Batril.RTM. water (Bayer Healthcare, LLC, Shawnee Mission, KS) 3
days prior to irradiation which continued for 2 weeks
post-irradiation. Repopulation was measured every 4 weeks
post-transplant by collection of periperal blood, red blood cell
lysis and staining of CD45.1 (recipient) vs. CD45.2 (donor)
engraftment. Mice transplanted with rescue/competitor cells only
were used as a control to determine the limits of detectable
repopulation (typically 0.2%). Multi-lineage reconstitution was
determined by CD3, B220 (for T and B lymphoid, respectively) and
Gr1, Mac-1 (for myeloid) gating on donor (CD45.2.sup.+) cells. For
secondary transplantation, the original, primary transplant
recipients were sacrificed and bone marrow was harvested from the
femur, made into a single-cell suspension, and strained through a
70 .mu.M cell strainer (BD Biosciences; Cat. No. 21008-952). Bone
marrow cells were counted and transplanted as above at a dosage of
1.times.10.sup.6 per mouse.
Experimental Results
[0282] The effect of GSK3.beta. inhibition (using lithium or the
small molecule inhibitor CHIR99021) in the HSC culture system was
tested. It was found that increasing concentrations of GSK3.beta.
inhibitor resulted in increasing proportions of LSK cells relative
to early myeloid progenitors. Specifically, without GSK3.beta.
inhibition, the frequency of early myeloid progenitors was more
than twice as high as that of LSK cells. However, addition of
GSK3.beta. inhibitor decreased the frequency of myeloid progenitors
but increased the frequency of LSK cells, yielding equivalent
frequencies of early myeloid and LSK cells (FIG. 26A). Although the
pre-culture frequency of LSK cells was only 0.14.+-.0.08% in bone
marrow MNCs, this increased to 9.5.+-.0.9% after 14 day culture in
ST media and further increased to 15.1.+-.1.1% with GSK3.beta.
inhibitor addition. Although the total number of cells after
culture was not increased, the total number of LSK Flk2.sup.- cells
increased 78-fold on average which further increased to 93-fold
with GSK3.beta. inhibitor addition (FIG. 26B). Interestingly, these
data partially mimic the genetic mutant animal models disclosed
above.
[0283] To determine whether the culture method expands not only
phenotypic but also functional HSCs, in vivo transplantation assays
were performed. The results show that ex vivo expansion of unsorted
bone marrow mononuclear cells enhances functional long-term
hematopoietic reconstitution in vivo relative to sorted, ex vivo
expanded HSCs (FIG. 4). These data demonstrate that the culture
methodology set forth above results in substantial expansion of
functional HSCs with long-term, multi-lineage repopulating
potential. The presence of non-stem cells is critical to this
expansion, demonstrating that the typical practice of purifying
specific putative HSC populations is not ideal for the ex vivo
expansion of HSCs. Indeed, the cultured product of MNCs containing
only 5 putative HSCs exhibits increased repopulation potential
compared to 100 sorted putative HSCs which are either freshly
isolated or also cultured. Secondary transplant experiments further
demonstrate that functional, long-term repopulating HSCs have been
expanded with all recipients exhibiting >25% donor repopulation
with the average being >60%. In contrast, an equivalent sample
of unexpanded MNCs yields long-term (16+ weeks) donor repopulation
of <1% in all recipients, with recipients being at or below
levels of detectable engraftment. This culture expansion protocol
meets rigorous functional tests, including the ability to yield
high levels of repopulation even in the presence of 10.sup.5 fresh,
uncompromised competitor cells and in serial transplantation
experiments, conditions that are generally more rigorous than those
encountered clinically.
[0284] The results further showed that culture with the
small-molecule inhibitor of GSK-3.beta., CHIR99021, enhances
long-term engraftment of ex vivo expanded HSCs (FIG. 5 and FIG.
13). While 100 sorted, putative HSCs cultured without CHIR99021
yield average repopulation of 1.1%, culturing with CHIR99021 yields
average repopulation of 12.3%. Similarly, unsorted MNC cultures in
the absence and presence of CHIR99021 yields average repopulation
of 37.4 and 64.8%, respectively. These data demonstrate that ex
vivo expansion in the presence of a small molecule inhibitor of
GSK-3.beta., CHIR99021, substantially increases the level of
long-term, multi-lineage engraftment.
[0285] Similarly, culture with another small-molecule inhibitor of
GSK-3.beta., lithium, also enhanced long-term engraftment of ex
vivo expanded HSCs, as shown in FIG. 22 and FIG. 23.
[0286] To quantify the number of functional HSC resulting from the
culture system, limiting-dilution, competitive repopulating unit
(CRU) assays were performed (Delaney, C. et al. Notch-mediated
expansion of human cord blood progenitor cells capable of rapid
myeloid reconstitution. Nat Med Vol. 16, pages 232-236 (2010); Zhu,
X. et al. A versatile approach to multiple gene RNA interference
using microRNA-based short hairpin RNAs. BMC molecular biology Vol.
8, page 98 (2007)). Poisson statistical analysis of n=60 total
recipient mice showed that, based on pre-culture number of LSK
Flk2.sup.- cells, the cultured progeny contained a frequency of 1/2
CRU (95% confidence interval: 1/1 to 1/5) for ST media conditions,
but when CHIR99021 was added, the frequency was 1/0.4 (95%
confidence interval: 1/0.2 to 1/0.8) (FIG. 27A). Thus, addition of
CHIR99021 increases the CRU frequency by an average of 5-fold (
2/0.4).
[0287] More mature, radioprotective cells are necessary for
short-term survival of recipients transplanted with purified HSCs,
and early myeloid progenitors have been found to confer this
radioprotective effect (Na Nakorn, T., Traver, D., Weissman, I. L.
& Akashi, K. Myeloerythroid-restricted progenitors are
sufficient to confer radioprotection and provide the majority of
day 8 CFU-S. The Journal of clinical investigation 109, 1579-1585
(2002)). Considering the high frequency of progenitor cells present
in the culture system (FIG. 26A), the inventors hypothesized that
the culture system might allow for the transplantation of only ex
vivo expanded cells without the necessity of any fresh
competitor/radioprotective bone marrow cells. Eliminating the need
for fresh, whole bone marrow cells would make the culture system
more relevant to and useful in a potential clinical
setting--indeed, short-term repopulation is equally critical to
survival. To test this, after 14 days culture in ST media with and
without CHIR99021, only the cultured product of MNCs containing 5
LSK Flk2.sup.- cells were transplanted into lethally irradiated
recipients. For comparison, fresh, uncultured MNCs containing 5 LSK
Flk2.sup.- cells per mouse were also transplanted. In addition,
because the average 14-day expansion of LSK Flk2.sup.- cells in the
unsorted cultures was approximately 100-fold (FIG. 26B), fresh,
uncultured MNCs containing 500 LSK Flk2.sup.- cells (100.times.
uncultured cells) were also transplanted into lethally irradiated
recipients for comparison. No rescue/competitor cells were added in
these groups. While recipients of uncultured MNCs containing 5 LSK
Flk2.sup.- cells had to be sacrificed due to bone marrow failure
between 2-3 weeks post-irradiation, mice transplanted with only
cultured cells containing 5 LSK Flk2.sup.- cells or fresh MNCs
containing 500 LSK Flk2.sup.- cells recovered. In these surviving
groups, all primary recipients exhibited robust donor engraftment
(>90%) with no significant difference between groups (FIG.
27B).
[0288] To determine if CHIR99021 was affecting the long-term
potential of HSCs, the primary recipients were euthanized, and
serial bone marrow transplantation into secondary,
lethally-irradiated recipients was performed. At 16 weeks
post-secondary transplant, donor reconstitution was 67.3.+-.20.6%
for ex vivo expansion in ST media and 90.6.+-.4.8% in ST media with
CHIR99021 (FIG. 6G and FIG. 6H). Notably, there was no significant
difference in long-term, multi-lineage donor reconstitution between
mice receiving ex vivo expanded HSCs in the presence of CHIR99021
and mice receiving a 100-fold greater dosage of fresh LSK
Flk2.sup.- cells (90.6.+-.4.8% vs. 90.1.+-.3.1, respectively;
p=0.88). Thus, at 16 weeks, secondary transplant recipients of
cells cultured in ST media exhibited reduced average repopulation
compared to secondary recipients of cells cultured in ST+CHIR99021
or 100.times. uncultured cells.
[0289] Bone marrow cells from these secondary recipients were then
transplanted into tertiary recipients. At 16 weeks, average
repopulation of tertiary recipients of cells cultured in ST media
was again further reduced relative to recipients of cells cultured
with CHIR99021 or 100.times. uncultured cells. Notably, tertiary
recipients of cells cultured with CHIR99021 or 100.times.
uncultured cells exhibited equivalent levels of repopulation with
no statistical difference between the two groups (P=0.9) (FIG.
27B).
[0290] While tertiary recipients of cells cultured without
CHIR99021 all succumbed to bone marrow failure by 6 months
post-transplantation, recipients of cells cultured with CHIR99021
exhibited survival rates similar to tertiary recipients of
100.times. uncultured cells (FIG. 27C). Importantly, even those
mice euthanized due to poor health did not exhibit signs of
leukemia but rather succumbed to bone marrow failure, exhibiting
low overall blood cell counts (data not shown). Thus, unlike the
results of permanent genetic manipulation in vivo, the transient ex
vivo manipulation of the PI3K/Akt and/or Wnt/.beta.-catenin
pathways does not result in leukemic transformation. These data
demonstrate that functional LT-HSCs can be expanded ex vivo to a
significantly greater degree when they are not fractionated from
more mature cells. Furthermore, by manipulating the
Wnt/.beta.-catenin pathways, inhibition of GSK3.beta., e.g. with
CHIR99021 or with lithium, during ex vivo HSC expansion
substantially enhances long-term donor reconstitution.
[0291] These data demonstrate that the ex vivo expansion protocol
allows for transplantation of only the cultured product of MNCs
containing 5 putative HSCs, resulting in long-term survival of
recipients. No fresh, rescue bone marrow cells are required. In
contrast, transplantation of fresh, unexpanded MNCs containing 5
putative HSCs does not allow any of the recipients to survive
beyond 2-3 weeks, the typical survival time of mice receiving
lethal irradiation without transplantation (Na Nakorn, T., Traver,
D., Weissman, I. L. & Akashi, K. Myeloerythroid-restricted
progenitors are sufficient to confer radioprotection and provide
the majority of day 8 CFU-S. The Journal of clinical investigation,
Vol. 109, 1579-1585 (2002)). Thus, in addition to the expansion of
long-term repopulating HSCs, short-term radioprotective cells are
also expanded utilizing the ex vivo expansion protocol. With the
inclusion of CHIR99021 during ex vivo expansion, the level of
repopulation of recipients of ex vivo expanded MNCs containing 5
putative HSCs is equivalent to fresh, unexpanded MNCs containing
500 putative HSCs at 16 weeks post-secondary transplantation. This
data demonstrates that the ex vivo expansion protocol allows for
long-term repopulation equivalent to a 100-fold greater dose of
fresh, unexpanded cells.
[0292] The data obtained from experiments involving ex vivo
expansion of human HSCs (FIG. 7) indicate that the culture
methodology developed in the mouse system should translate into the
human system, allowing for substantial expansion of HSCs in
culture. This should allow for currently limited sources of HSCs,
such as umbilical cord blood, which is widely available but is low
in cell number, to be utilized with greater efficacy.
[0293] HSCs are known to be able to undergo considerable expansion
in vivo and are the most extensively studied stem cell system. It
is somewhat paradoxical, therefore, that they remain difficult to
expand in culture, although progress has been recently achieved,
particularly by activation of the Notch pathway (Himburg, H. A. et
al. Pleiotrophin regulates the expansion and regeneration of
hematopoietic stem cells. Nat Med 16, 475-482 (2010); Antonchuk et
al., HOXB4-induced expansion of adult hematopoietic stem cells ex
vivo. Cell 109, 39-45 (2002); Butler, J. M. et al. Endothelial
cells are essential for the self-renewal and repopulation of
Notch-dependent hematopoietic stem cells. Cell stem cell 6, 251-264
(2010); Delaney, C. et al. Notch-mediated expansion of human cord
blood progenitor cells capable of rapid myeloid reconstitution. Nat
Med 16, 232-236 (2010)). Typically, only modest expansion being
consistently achieved, while more significant expansion is usually
coupled with substantial differentiation (North, T. E. et al.
Prostaglandin E2 regulates vertebrate haematopoietic stem cell
homeostasis. Nature 447, 1007-1011 (2007).; Kobayashi, M., Laver,
J. H., Kato, T., Miyazaki, H. & Ogawa, M. Thrombopoietin
supports proliferation of human primitive hematopoietic cells in
synergy with steel factor and/or interleukin-3. Blood 88, 429-436
(1996); Antonchuk, J., Sauvageau, G. & Humphries, R. K.
HOXB4-induced expansion of adult hematopoietic stem cells ex vivo.
Cell 109, 39-45 (2002); Varnum-Finney, B. et al. Pluripotent,
cytokine-dependent, hematopoietic stem cells are immortalized by
constitutive Notch1 signaling. Nat Med 6, 1278-1281 (2000)). In
addition, ex vivo expansion protocols often use genetically
modified cells or feeder layers which may present difficulties in
translating to a clinical setting.
[0294] Driving self-renewal appears to require activation of
certain proto-oncogenes along with simultaneous inhibition of
certain tumor suppressors, a combination that limits regenerative
capacity and makes substantial expansion difficult without risking
oncogenesis or stem cell exhaustion (Reya, T. et al. A role for Wnt
signaling in self-renewal of haematopoietic stem cells. Nature 423,
409-414 (2003); Yilmaz, O. H. et al. Pten dependence distinguishes
haematopoietic stem cells from leukaemia-initiating cells. Nature
441, 475-482 (2006); Zhang, J. et al. PTEN maintains haematopoietic
stem cells and acts in lineage choice and leukaemia prevention.
Nature 441, 518-522 (2006); Varnum-Finney, B. et al. Pluripotent,
cytokine-dependent, hematopoietic stem cells are immortalized by
constitutive Notch1 signaling. Nat Med 6, 1278-1281 (2000);
Matsuoka, S. et al. Fbxw7 acts as a critical fail-safe against
premature loss of hematopoietic stem cells and development of
T-ALL. Genes Dev 22, 986-991 (2008); Park, I. K. et al. Bmi-1 is
required for maintenance of adult self-renewing haematopoietic stem
cells. Nature 423, 302-305 (2003); Perry, J. M. & Li, L.
Self-renewal versus transformation: Fbxw7 deletion leads to stem
cell activation and leukemogenesis. Genes Dev. 22, 1107-1109
(2008)). Simultaneous manipulation of proto-oncogene and tumor
suppressor activity can achieve substantial stem cell expansion in
vitro; however, it is critical to balance this transient expansion
with return to conditions that mimic the in vivo situation where
relative quiescence is recovered and tumor suppressors are
reactivated.
[0295] Although sorting specific populations enriched in HSCs has
been the typical methodology utilized for culturing HSCs, the
inventors found ex vivo HSC expansion to be best supported when
cultured in the presence of more mature cells. It was observed that
sorted LSK Flk2.sup.- cells typically declined in number within the
first 48 hours in culture. Indeed, when LSK Flk2.sup.- cells were
sorted, substantial HSC expansion was achieved only after some
differentiation had occurred, yielding HSCs in an environment
surrounded by mature cells, a situation similar to that encountered
by HSCs in vivo. It may be that HSCs negatively inhibit
self-renewal and even survival of other HSCs in close proximity,
helping to maintain stem cells as a rare population in vivo.
Culturing unsorted HSCs combined with other technical procedures
results in robust functional HSC expansion. While the ex vivo HSC
expansion protocol yielded robust long-term reconstitution, even in
competitive repopulation assays, competitor or rescue bone marrow
cells were not necessary following ex vivo expansion, demonstrating
that radioprotective cells also expanded in the culture system.
Utilizing a small molecule inhibitor of GSK3.beta., the ex vivo HSC
expansion protocol allowed for expansion of LT-HSCs which performed
equivalently to a 100-fold greater dosage of uncultured cells even
in long-term, serial transplant recipients. Beyond this expansion,
there is a distinct practical advantage with the disclosed culture
system. By employing a serum-free with relatively low
concentrations of only two cytokines, but without the necessity of
feeder layers, cell sorting, or the use of fresh bone marrow for
radioprotection, this culture system may have clinical value if
developed for humans.
[0296] In summary, this process for the ex vivo expansion of
hematopoietic stem cells utilizes defined culture media
supplemented with low concentrations of only two specific cytokines
and does not require complicated schemes such as cell sorting or
contaminating cellular feeder layers. Therefore, it allows for
fast, simple and relatively inexpensive expansion of functional
HSCs. In addition, HSC transplantation following myeloablative
therapy requires the transplantation of radioprotective cells,
typically whole bone marrow cells, for short-term survival prior to
the establishment of long-term hematopoiesis by HSCs (Paquette, R.
& Dorshkind, K. Optimizing hematopoietic recovery following
bone marrow transplantation. The Journal of clinical investigation
109, 1527-1528 (2002)). This HSC expansion protocol also expands
these radioprotective cells, allowing for the transplantation of
the cultured product alone. When cultured with the small molecule
CHIR99021, an inhibitor of GSK-3.beta., this ex vivo expansion
protocol allows for long-term repopulation equivalent to a 100-fold
greater dose of fresh, unexpanded cells.
Example 12
Culturing of HSC in Media Containing Hiologics
[0297] Anti-GSK-3.beta. and anti-PTEN antibodies may be made in
accordance with procedures known in the art (or purchased, e.g.,
from Sigma, ExactAntigene, and Biocom pare).
[0298] One hundred LSK Flk2.sup.- cells are sorted from wild-type
(C57BI/6) mice and are cultured in (1) media, (2) media+an
GSK-3.beta. antibody, (3) media+an anti-PTEN antibody, and (4)
media+anti-GSK-3.beta. and anti-PTEN antibodies. Cells are cultured
as described above. Cells are examined at 9 days, 17 days and 23
days of culture. The greatest expansion of HSCs is expected to
occur when both antibodies are present.
Example 13
Culturing of HSC in Media Containing siRNA or RNAi
[0299] PTEN siRNA and GSK-3b siRNA may be made in accordance with
procedures known in the art. (See, e.g., Mise-Omata S et al.
Biochem Biophys Res
[0300] Commun. 328(4):1034-42 2005, or may be purchased from
Biocompare).
[0301] One hundred LSK Flk2.sup.- cells are sorted from wild-type
(C57BI6) mice and are cultured in (1) media, (2) media+GSK-3.beta.
siRNA, (3) media+PTEN siRNA, and (4) media+GSK-3.beta. siRNA and
PTEN siRNA. Cells are cultured as described above. Cells are
examined at 9 days, 17 days and 23 days of culture. The greatest
expansion of HSC is expected to occur when both siRNAs are
present.
[0302] All documents cited in this application are hereby
incorporated by reference as if recited in full herein.
[0303] Although illustrative embodiments of the present invention
have been described herein, it should be understood that the
invention is not limited to those described, and that various other
changes or modifications may be made by one skilled in the art
without departing from the scope or spirit of the invention.
Sequence CWU 1
1
4120DNAArtificialSynthetic Construct 1cgtggacaat ggctactcaa
20220DNAArtificialSynthetic Construct 2tgtcagctca ggaattgcac
20397DNAArtificialSynthetic Construct; HP_224742 3tgctgttgac
agtgagcgac cagtgtgggt gaatacttta tagtgaagcc acagatgtat 60aaagtattca
cccacactgg ctgcctactg cctcgga 97497DNAArtificialSynthetic
Construct; HP_240000 4tgctgttgac agtgagcgcg gaccaggtgg tagttaataa
tagtgaagcc acagatgtat 60tattaactac cacctggtcc ttgcctactg cctcgga
97
* * * * *