U.S. patent application number 14/308048 was filed with the patent office on 2016-05-19 for methods and compositions for producing germ cells from bone marrow derived germline stem cells.
The applicant listed for this patent is The General Hospital Corporation. Invention is credited to Joshua Johnson, Jonathan L. Tilly.
Application Number | 20160137978 14/308048 |
Document ID | / |
Family ID | 35428927 |
Filed Date | 2016-05-19 |
United States Patent
Application |
20160137978 |
Kind Code |
A1 |
Tilly; Jonathan L. ; et
al. |
May 19, 2016 |
METHODS AND COMPOSITIONS FOR PRODUCING GERM CELLS FROM BONE MARROW
DERIVED GERMLINE STEM CELLS
Abstract
The present invention relates to the use of bone marrow derived
germline stem cells and their progenitors, methods of isolation
thereof, and methods of use thereof.
Inventors: |
Tilly; Jonathan L.;
(Windham, NH) ; Johnson; Joshua; (New Haven,
CT) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
The General Hospital Corporation |
Boston |
MA |
US |
|
|
Family ID: |
35428927 |
Appl. No.: |
14/308048 |
Filed: |
June 18, 2014 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
11131153 |
May 17, 2005 |
|
|
|
14308048 |
|
|
|
|
60572222 |
May 17, 2004 |
|
|
|
60574187 |
May 24, 2004 |
|
|
|
60586641 |
Jul 9, 2004 |
|
|
|
Current U.S.
Class: |
424/93.7 ;
435/366; 435/377 |
Current CPC
Class: |
A61P 15/12 20180101;
C12N 5/0634 20130101; A61P 15/08 20180101; C12N 5/0611 20130101;
C12N 2506/11 20130101; C12N 5/0609 20130101; A61P 35/00 20180101;
A61K 35/14 20130101; A61K 2035/124 20130101; C12P 21/06 20130101;
A61P 15/18 20180101; A61P 15/16 20180101; A61K 35/28 20130101; A61B
17/43 20130101 |
International
Class: |
C12N 5/0735 20060101
C12N005/0735; A61K 35/28 20060101 A61K035/28 |
Goverment Interests
STATEMENT OF POTENTIAL GOVERNMENT INTEREST
[0002] The United States government has certain rights in this
invention by virtue of grant numbers R01-AG12279 and R01-AG24999
from the National Institute on Aging of the National Institutes of
Health.
Claims
1. An isolated bone marrow cell that is mitotically competent, has
an XX karyotype and expresses Vasa, Oct-4, Dazl, Stella, Fragilis
and optionally, Nobox, c-Kit and Sca-1.
2. The isolated cell of claim 1, wherein the cell can produce
oocytes after a duration of at least 1 week, 1 to about 2 weeks,
about 2 to about 3 weeks, about 3 to about 4 weeks or more than
about 5 weeks post transplantation into a host.
3. The isolated cell of claim 1, wherein the cell can produce
oocytes after a duration of less than 1 week post transplantation
into a host.
4. The isolated cell of claim 1, wherein the cell can produce
oocytes after a duration of less than about 24 to about 48 hours
post transplantation into a host.
5. The isolated cell of claim 2, wherein the cell is a bone marrow
derived female germline stem cell.
6. The isolated cell of claim 3, wherein the cell is a bone marrow
derived female germline stem cell progenitor.
7. The isolated cell of claim 1, wherein the cell is a mammalian
cell.
8. The isolated cell of claim 1, wherein the cell is a human
cell.
9. The isolated cell of claim 1, wherein the cell is a
non-embryonic cell.
10. The isolated cell of claim 1, wherein the cell expresses
Nobox.
11. The isolated cell of claim 1, wherein the cell expresses
c-Kit.
12. The isolated cell of claim 1, wherein the cell expresses
Sca-1.
13. A method of in vitro fertilization of a female subject, said
method comprising the steps of: a) producing an oocyte by culturing
the isolated cell of claim 1 in the presence of an agent that
differentiates the cell into an oocyte; b) fertilizing the oocyte
in vitro to form a zygote; and c) implanting the zygote into the
uterus of a female subject.
14. A method of oocyte production, comprising culturing the
isolated cell of claim 1 in the presence of an agent that
differentiates the cell into an oocyte, thereby producing an
oocyte.
15. The method of claim 14, wherein the agent is selected from the
group consisting of a transforming growth factor, bone morphogenic
protein, Wnt family protein, kit-ligand, leukemia inhibitory
factor, meiosis-activating sterol, modulator of Id protein function
and modulator of Snail/Slug transcription factor function.
16. A pharmaceutical composition comprising a purified population
of cells that are mitotically competent, have an XX karyotype and
express Vasa, Oct-4, Dazl, Stella, Fragilis and optionally, Nobox,
c-Kit and Sca-1 and a pharmaceutically acceptable carrier.
17. The pharmaceutical composition of claim 16, wherein the cells
are purified from the bone marrow.
18. The pharmaceutical composition of claim 16, wherein the cells
are mammalian cells.
19. The pharmaceutical composition of claim 16, wherein the cells
are human cells.
20. The pharmaceutical composition of claim 16, wherein the
purified population of cells is about 50 to about 55%, about 55 to
about 60%, about 65 to about 70%, about 70 to about 75%, about 75
to about 80%, about 80 to about 85%, about 85 to about 90%, about
90 to about 95% or about 95 to about 100% of the cells in the
composition.
21-69. (canceled)
Description
RELATED APPLICATIONS/PATENTS & INCORPORATION BY REFERENCE
[0001] This application is a continuation of U.S. application Ser.
No. 11/131,153, filed on May 17, 2005, which claims the benefit of
U.S. Provisional Application Ser. No. 60/572,222, filed on May 17,
2004, U.S. Provisional Application Ser. No. 60/574,187, filed on
May 24, 2004, and U.S. Provisional Application Ser. No. 60/586,641,
filed on Jul. 9, 2004, the contents each of which are incorporated
herein in their entireties by reference.
[0003] Each of the applications and patents cited in this text, as
well as each document or reference cited in each of the
applications and patents (including during the prosecution of each
issued patent; "application cited documents"), and each of the PCT
and foreign applications or patents corresponding to and/or
claiming priority from any of these applications and patents, and
each of the documents cited or referenced in each of the
application cited documents, are hereby expressly incorporated
herein by reference, and may be employed in the practice of the
invention. More generally, documents or references are cited in
this text, either in a Reference List before the claims, or in the
text itself; and, each of these documents or references ("herein
cited references"), as well as each document or reference cited in
each of the herein cited references (including any manufacturer's
specifications, instructions, etc.), is hereby expressly
incorporated herein by reference.
BACKGROUND OF THE INVENTION
[0004] A basic doctrine of reproductive biology, which states that
mammalian females lose the capacity for germ-cell renewal during
fetal life, has only recently been successfully challenged by
Johnson et al., (2004) Nature 428: 145. Johnson et al. are the
first to conclusively demonstrate that juvenile and adult mouse
ovaries possess mitotically active germ cells that, based on rates
of oocyte degeneration and clearance, sustain oocyte and follicle
production in the postnatal mammalian ovary. However, it remains
unclear whether the precursors of germ cells are confined
exclusively to the ovaries or whether extra-ovarian sites in the
body, contain precursors having the ability to form germ cells.
[0005] Previously, Green and Bernstein (1970) Int. J. Radiat. Biol.
Vol. 17 (1): 87, had attempted to show that cells not derived from
reproductive organs can repopulate the testicular germinal
epithelium in a series of bone marrow-inoculation experiments. In
these experiments, a male test rat, which was sterilized by
whole-body irradiation, received injections of bone marrow from a
donor rat, which was sterilized by testes-specific irradiation.
These experiments failed to provide evidence that germinal
epithelium of the test rat could be repopulated to reinitiate
spermatogenesis upon injection of bone marrow cells obtained from
the donor rat. Therefore, it was not believed that bone marrow
derived cells could successfully repopulate the germline of the
mammalian gonads.
SUMMARY OF THE INVENTION
[0006] It has now been shown that bone marrow derived germline stem
cells can repopulate the germline of reproductive organs, and thus
restore gonadal function. Methods of the invention relate to the
use of bone marrow derived germline stem cells and their
progenitors to, among other things, replenish or expand germ cell
reserves of the testes and ovary, to enhance or restore fertility,
and in females, to ameliorate symptoms and consequences of
menopause.
[0007] In one aspect, the present invention provides compositions
comprising bone marrow derived female germline stem cells.
[0008] In one embodiment, the present invention provides
compositions comprising bone marrow derived female germline stem
cells, wherein the cells are mitotically competent and express Oct
4, Vasa, Dazl, Stella, Fragilis, and optionally Nobox, c-Kit and
Sca-1. Consistent with their mitotically competent phenotype, bone
marrow derived female germline stem cells of the invention do not
express growth/differentiation factor-9 ("GDF-9"), zona pellucida
proteins (e.g., zona pellucida protein-3, "ZP3"), histone
deacetylase-6 ("HDAC6") and synaptonemal complex protein-3
("SCP3"). Upon transplantation into a host, bone marrow derived
female germline stem cells of the invention can produce oocytes
after a duration of at least 1 week, more preferably 1 to about 2
weeks, about 2 to about 3 weeks, about 3 to about 4 weeks or more
than about 5 weeks post transplantation.
[0009] In another aspect, the present invention provides
compositions comprising progenitor cells derived from bone marrow
derived female germline stem cells. In one embodiment, the present
invention provides compositions comprising bone marrow derived
female germline stem cell progenitors, wherein the cells express
Oct 4, Vasa, Dazl, Stella, Fragilis, and optionally Nobox, c-Kit
and Sca-1 and wherein the cells do not express GDF-9, zona
pellucida proteins, HDAC6 and SCP3. Upon transplantation into a
host, bone marrow derived female germline stem cell progenitors of
the invention can produce oocytes after a duration of less than 1
week, preferably about 24 to about 48 hours post
transplantation.
[0010] In another embodiment, the present invention provides an
isolated bone marrow cell, wherein the cell is mitotically
competent and expresses Oct 4, Vasa, Dazl, Stella, Fragilis, and
optionally Nobox, c-Kit and Sca-1. Preferably, the cell is a bone
marrow derived female germline stem cell, or its progenitor cell,
having an XX karyotype. Preferably, the bone marrow derived female
germline stem cells, or their progenitor cells, are non-embryonic,
mammalian, and even more preferably, human.
[0011] In another embodiment, the present invention provides
purified populations of bone marrow derived female germline stem
cells and/or their progenitor cells. In specific embodiments, the
purified population of cells is about 50 to about 55%, about 55 to
about 60%, about 65 to about 70%, about 70 to about 75%, about 75
to about 80%, about 80 to about 85%, about 85 to about 90%, about
90 to about 95% or about 95 to about 100% of the cells in the
composition.
[0012] In yet another embodiment, the present invention provides
pharmaceutical compositions comprising bone marrow derived female
germline stem cells, and/or their progenitor cells, and a
pharmaceutically acceptable carrier. The pharmaceutical
compositions can comprise purified populations of bone marrow
derived female germline stem cells and/or their progenitor
cells.
[0013] Compositions comprising bone marrow derived female germline
stem cells of the invention can be provided by direct
administration to ovarian tissue, or indirect administration, for
example, to the circulatory system of a subject (e.g., to the
extra-ovarian circulation).
[0014] In yet another aspect, the invention provides methods for
manipulating bone marrow derived germline stem cells, or their
progenitor cells, in vivo, ex vivo or in vitro as described herein
below.
[0015] In one embodiment, the invention provides a method for
expanding bone marrow derived female germline stem cells, or their
progenitor cells, in vivo, ex vivo or in vitro, comprising
contacting bone marrow derived female germline stem cells, or their
progenitor cells, with an agent that increases the amount of bone
marrow derived female germline stem cells, or their progenitor
cells, by promoting proliferation or survival thereof, thereby
expanding the bone marrow derived female germline stem cells, or
their progenitor cells. In a preferred embodiment, the agent
includes, but is not limited to, a hormone or growth factor (e.g.,
insulin-like growth factor ("IGF"), transforming growth factor
("TGF"), bone morphogenic protein ("BMP"), Wnt protein, or
fibroblast growth factor ("FGF")), a cell-signaling molecule (e.g.,
sphingosine-1-phosphate ("S1P"), or retinoic acid ("RA")), or a
pharmacological or pharmaceutical compound (e.g., an inhibitor of
glycogen synthase kinase-3 ("GSK-3"), an inhibitor of apoptosis
such as a Bax inhibitor or a caspase inhibitor, an inhibitor of
nitric oxide production, or an inhibitor of HDAC activity).
[0016] In another embodiment, the invention provides a method for
identifying an agent that promotes proliferation or survival of a
bone marrow derived female germline stem cell, or its progenitor
cell, comprising contacting the bone marrow derived female germline
stem cells, or their progenitor cells, with a test agent; and
detecting an increase in the number of bone marrow derived female
germline stem cells, or their progenitor cells, thereby identifying
an agent that promotes proliferation or survival of a bone marrow
derived female germline stem cell, or its progenitor cell.
[0017] In yet another embodiment, the invention provides a method
for using the female germline stem cells, or their progenitor
cells, to characterize pharmacogenetic cellular responses to
biologic or pharmacologic agents, comprising isolating bone marrow
derived female germline stem cells, or their progenitor cells, from
a population of subjects, expanding said cells in culture to
establish a plurality of cell cultures, optionally differentiating
said cells into a desired lineage, contacting the cell cultures
with one or more biologic or pharmacologic agents, identifying one
or more cellular responses to the one or more biologic or
pharmacologic agents, and comparing the cellular responses of the
cell cultures from different subjects.
[0018] In yet another embodiment, the invention provides a method
for oocyte production, comprising culturing a bone marrow derived
female germline stem cell, or its progenitor cell, in the presence
of an agent that differentiates a bone marrow derived female
germline stem cell, or its progenitor cell, into an oocyte, thereby
producing an oocyte. In a preferred embodiment, the agent includes,
but is not limited to, a hormone or growth factor (e.g., a TGF, BMP
or Wnt family protein, kit-ligand ("SCF") or leukemia inhibitory
factor ("LIF")), a signaling molecule (e.g., meiosis-activating
sterol, "FF-MAS"), or a pharmacologic or pharmaceutical agent
(e.g., a modulator of Id protein function or Snail/Slug
transcription factor function).
[0019] In yet another embodiment, the invention provides a method
for in vitro fertilization of a female subject, said method
comprising the steps of: [0020] a) producing an oocyte by culturing
a bone marrow derived female germline stem cell, or its progenitor,
in the presence of an oocyte differentiation agent; [0021] b)
fertilizing the oocyte in vitro to form a zygote; and [0022] c)
implanting the zygote into the uterus of a female subject.
[0023] In yet another embodiment, the invention provides a method
for in vitro fertilization of a female subject, said method
comprising the steps of: [0024] a) producing an oocyte by
contacting a bone marrow derived female germline stem cell, or its
progenitor cell, with an agent that differentiates said cell(s)
into an oocyte; [0025] b) fertilizing the oocyte in vitro to form a
zygote; and [0026] c) implanting the zygote into the uterus of a
female subject.
[0027] In yet another embodiment, the invention provides a method
for identifying an agent that induces differentiation of a bone
marrow derived female germline stem cell, or its progenitor cell,
into an oocyte comprising contacting bone marrow derived female
germline stem cells, or their progenitor cells, with a test agent;
and detecting an increase in the number of oocytes, thereby
identifying an agent that induces differentiation of a bone marrow
derived female germline stem cell, or its progenitor.
[0028] In yet another embodiment, the present invention provides a
method for oocyte production, comprising providing a bone marrow
derived female germline stem cell, or its progenitor cell, to a
tissue, preferably the ovary, wherein the cell engrafts into the
tissue and differentiates into an oocyte, thereby producing an
oocyte.
[0029] In yet another embodiment, the present invention provides a
method for inducing folliculogenesis, comprising providing a bone
marrow derived female germline stem cell, or its progenitor cell,
to a tissue, preferably the ovary, wherein the cell engrafts into
the tissue and differentiates into an oocyte within a follicle,
thereby inducing folliculogenesis.
[0030] In yet another embodiment, the present invention provides a
method for treating in Fertility in a female subject in need
thereof comprising administering a therapeutically effective amount
of a composition comprising bone marrow derived female germline
stem cells, or their progenitor cells, to the subject, wherein the
cells engraft into a tissue, preferably ovarian tissue, and
differentiate into oocytes, thereby treating infertility. Except
where expressly stated herein, the female subject in need of
fertility treatment is not a subject who has undergone prior
chemotherapy or radiotherapy.
[0031] In yet another embodiment, the present invention provides a
method for restoring fertility to a female subject having undergone
chemotherapy or radiotherapy (or both treatments) and who desires
restored fertility, comprising administering a therapeutically
effective amount of bone marrow derived female germline stem cells,
or their progenitor cells, to the subject, wherein the cells
engraft into a tissue, preferably ovarian tissue, and differentiate
into oocytes, thereby restoring fertility in the subject.
Preferably, the bone marrow derived female germline stem cells
comprise a purified sub-population of cells obtained from the bone
marrow. Chemotherapeutic drugs include, but are not limited to,
busulfan, cyclophosphamide, 5-FU, vinblastine, actinomycin D,
etoposide, cisplatin, methotrexate, doxorubicin, among others.
Radiotherapy includes, but is not limited to, ionizing radiation,
ultraviolet radiation, X-rays, and the like.
[0032] In yet another embodiment, the present invention provides a
method for protecting fertility in a female subject undergoing or
expected to undergo chemotherapy or radiotherapy (or both
treatments), comprising providing an agent that protects against
reproductive injury prior to or concurrently with chemotherapy or
radiotherapy (or both treatments) and providing a bone marrow
derived female germline stem cell, or its progenitor cell, to the
subject, wherein the cell engrafts into a tissue, preferably
ovarian tissue, and differentiates into an oocyte, thereby
protecting fertility in the subject. The protective agent can be
S1P, a Bax antagonist, or any agent that increases SDF-1
activity.
[0033] In yet another embodiment, the present invention provides a
method for repairing damaged ovarian tissue, comprising providing a
therapeutically effective amount of a composition comprising bone
marrow derived female germline stem cells, or their progenitor
cells, to the tissue, wherein the cells engraft into the tissue and
differentiate into oocytes, thereby repairing the damaged tissue.
Damage can be caused, for example, by exposure to cytotoxic
factors, hormone deprivation, growth factor deprivation, cytokine
deprivation, cell receptor antibodies, and the like. Except where
expressly stated herein, the damage is not caused by prior
chemotherapy or radiotherapy. Damage can also be caused be diseases
that affect ovarian function, including, but not limited to cancer,
polycystic ovary disease, genetic disorders, immune disorders,
metabolic disorders, and the like.
[0034] In yet another embodiment, the present invention provides a
method for restoring ovarian function in a female subject having
undergone chemotherapy or radiotherapy (or both treatments) and who
desires restored ovarian function, comprising administering a
therapeutically effective amount of bone marrow derived female
germline stem cells, or their progenitor cells, to an ovary of the
subject, wherein the cells engraft into the ovary and differentiate
into oocytes within the ovary, thereby restoring ovarian function
in the subject.
[0035] In yet another embodiment, the present invention provides a
method for restoring ovarian function in a menopausal female
subject, comprising administering a therapeutically effective
amount of a composition comprising bone marrow derived female
germline stem cells, or their progenitor cells, to the subject,
wherein the cells engraft into the ovary and differentiate into
oocytes, thereby restoring ovarian function. The menopausal female
subject can be in a stage of either peri- or post-menopause, with
said menopause caused by either normal (e.g., aging) or
pathological (e.g., surgery, disease, ovarian damage)
processes.
[0036] Restoration of ovarian function can relieve adverse symptoms
and complications associated with menopausal disorders, including,
but not limited to, somatic disorders such as osteoporosis,
cardiovascular disease, somatic sexual dysfunction, hot flashes,
vaginal drying, sleep disorders, depression, irritability, loss of
libido, hormone imbalances, and the like, as well as cognitive
disorders, such as loss of memory; emotional disorders, depression,
and the like.
[0037] Methods of the present invention can be used in the
production of other reproductive cell types. Accordingly, in yet
another aspect, the present invention provides compositions
comprising bone marrow derived male germline stem cells, wherein
the bone marrow derived male germline stem cells are mitotically
competent and express Vasa and Dazl. Bone marrow derived male
germline stem cells of the invention carry an XY karyotype, whereas
bone marrow derived female germline stem cells of the invention
carry an XX karyotype. Preferably, the bone marrow derived male
germline stem cells are non-embryonic, mammalian, and even more
preferably, human.
[0038] In one embodiment, the invention provides an isolated bone
marrow cell that is mitotically competent, has an XY karyotype and
expresses Vasa and Dazl.
[0039] In another embodiment, the present invention provides a
method for restoring or enhancing spermatogenesis, comprising
providing a bone marrow derived male germline stem cell, or its
progenitor cell, to the testes of a male subject, wherein the cell
engrafts into the seminiferous epithelium and differentiates into a
sperm cell, thereby restoring or enhancing spermatogenesis.
[0040] In yet another embodiment, the present invention provides a
method for restoring fertility to a male subject having undergone
chemotherapy or radiotherapy (or both) and who desires restored
fertility, comprising administering a therapeutically effective
amount of bone marrow derived male germline stem cells, or their
progenitor cells, to the subject, wherein the cells engraft into
the seminiferous epithelium and differentiate into sperm cells,
thereby restoring fertility.
[0041] In yet another embodiment, the invention provides a method
for reducing the amount of bone marrow derived germline stem cells,
or their progenitor cells, in vivo, ex vivo or in vitro, comprising
contacting bone marrow derived germline stem cells, or their
progenitor cells, with an agent that reduces cell proliferation,
thereby reducing the amount of bone marrow derived germline stem
cells, or their progenitor cells. In a preferred embodiment, the
agent includes, but is not limited to, a hormone or growth factor
(e.g., TGF-.beta.), a peptide antagonist of mitogenic hormones or
growth factors (e.g., the BMP antagonists, Protein Related to DAN
and Cerberus ("PRDC") and Gremlin), or a pharmacological or
pharmaceutical compound (e.g., a cell cycle inhibitor, or an
inhibitor of growth factor signaling).
[0042] In yet another embodiment, the invention provides a method
for reducing the amount of bone marrow derived germline stem cells,
or their progenitor cells, in vivo, ex vivo or in vitro, comprising
contacting bone marrow derived germline stem cells, or their
progenitor cells, with an agent that inhibits cell survival or
promotes cell death, thereby reducing the amount of bone marrow
derived germline stem cells, or their progenitor cells. In a
preferred embodiment, the agent the that inhibits cell survival
includes, but is not limited to, a hormone, growth factor or
cytokine (e.g., a pro-apoptotic tumor necrosis factor ("TNF") super
family member such as TNF-.alpha., Fas-ligand ("FasL") and TRAIL),
an antagonist of pro-survival Bcl-2 family member function, a
signaling molecule (e.g., a ceramide), or a pharmacological or
pharmaceutical compound (e.g., an inhibitor of growth factor
signaling). In a preferred embodiment, the agent the that promotes
cell death includes, but is not limited to, a pro-apoptotic tumor
necrosis factor superfamily member (e.g., TNF-.alpha., FasL and
TRAIL), agonist of pro-apoptotic Bcl-2 family member function and
ceramide.
[0043] In yet another embodiment, the invention provides a method
for identifying an agent that reduces proliferation or survival, or
promotes cell death, of a bone marrow derived germline stem cell,
or its progenitor cell, comprising contacting bone marrow derived
germline stem cells, or their progenitor cells, with a test agent;
and detecting a decrease in the number of bone marrow derived
germline stem cells, or their progenitor cells, thereby identifying
an agent that reduces proliferation or survival, or promotes cell
death, of a female germline stem cell, or its progenitor cell.
[0044] In yet another embodiment, the present invention provides a
method for contraception in a male or female subject comprising
contacting bone marrow derived germline stem cells, or their
progenitor cells, of the subject with an agent that decreases the
proliferation, function or survival of bone marrow derived germline
stem cells, or their progenitor cells, or the ability of said cells
to produce new oocytes or sperm cells or other somatic cell types
required for fertility, thereby providing contraception to the
subject.
[0045] In yet another aspect, the present invention provides kits
for use in employing various agents of the invention.
[0046] In one embodiment, the present invention provides a kit for
expanding a bone marrow derived female germline stem cell, or its
progenitor cell, in vivo, ex vivo or in vitro, comprising an agent
that promotes cell proliferation or survival of the bone marrow
derived female germline stem cell, or its progenitor cell, and
instructions for using the agent to promote cell proliferation or
survival of the bone marrow derived female germline stem cell, or
its progenitor, thereby expanding a female germline stem cell, or
its progenitor cell in accordance with the methods of the
invention.
[0047] In another embodiment, the present invention provides a kit
for oocyte production, comprising an agent that differentiates a
bone marrow derived female germline stem cell, or its progenitor
cell, into an oocyte and instructions for using the agent to
differentiate a bone marrow derived female germline stem cell, or
its progenitor cell, into an oocyte in accordance with the methods
of the invention.
[0048] In yet another embodiment, the present invention provides a
kit for oocyte production, comprising an agent that increases the
amount of bone marrow derived female germline stem cells, or their
progenitor cells, by promoting proliferation or survival thereof,
and instructions for using the agent to increase the amount of bone
marrow derived female germline stem cells or their progenitor
cells, thereby producing oocytes in accordance with the methods of
the invention.
[0049] In yet another embodiment, the present invention provides a
kit for oocyte production comprising an agent that differentiates
bone marrow derived female germline stem cells, or their progenitor
cells, into oocytes and instructions for using the agent to
differentiate the bone marrow derived female germline stem cells,
or their progenitor cells, into oocytes, thereby producing oocytes
in accordance with the methods of the invention.
[0050] In yet another embodiment, the present invention provides a
kit for treating infertility in a female subject in need thereof
comprising an agent that increases the amount of bone marrow
derived female germline stem cells, or their progenitor cells, by
promoting proliferation or survival thereof and instructions for
using the agent to increase the amount of bone marrow derived
female germline stem cells or their progenitor cells, thereby
treating infertility in the subject in accordance with the methods
of the invention.
[0051] In yet another embodiment, the present invention provides a
kit for treating infertility in a female subject in need thereof
comprising an agent that differentiates bone marrow derived female
germline stem cells, or their progenitor cells, into oocytes, and
instructions for using the agent to differentiate bone marrow
derived female germline stem cells, or their progenitor cells, into
oocytes, thereby treating infertility in the subject in accordance
with the methods of the invention.
[0052] In yet another embodiment, the present invention provides a
kit for protecting fertility in a female subject undergoing or
expected to undergo chemotherapy or radiotherapy (or both
treatments), comprising an agent that that protects bone marrow
derived female germline stem cells, or their progenitor cells,
against reproductive injury and instructions for using the agent to
protect bone marrow derived female germline stem cells, or their
progenitor cells, against reproductive injury thereby protecting
fertility in the female subject in accordance with the methods of
the invention.
[0053] In yet another embodiment, the present invention provides a
kit for restoring ovarian function in a post-menopausal female
subject comprising an agent that increases the amount of bone
marrow derived female germline stem cells, or their progenitor
cells, by promoting proliferation or survival thereof and
instructions for using the agent to increase the amount of bone
marrow derived female germline stem cells or their progenitor
cells, thereby restoring ovarian function in the subject in
accordance with the methods of the invention.
[0054] In yet another embodiment, the present invention provides a
kit for restoring ovarian function in a post-menopausal female
subject comprising an agent that differentiates bone marrow derived
female germline stem cells, or their progenitor cells, into
oocytes, and instructions for using the agent to differentiate bone
marrow derived female germline stem cells, or their progenitor
cells, into oocytes, thereby restoring ovarian function in the
subject in accordance with the methods of the invention.
[0055] In another embodiment, the present invention provides a kit
for reducing the amount of bone marrow derived germline stem cells,
or their progenitor cells, in vivo, ex vivo or in vitro, comprising
an agent that inhibits cell survival or promotes cell death and
instructions for using the agent to inhibit cell survival or
promote cell death of the bone marrow derived germline stem cells,
or their progenitor cells, thereby the reducing the amount of bone
marrow derived germline stem cells, or their progenitor cells, in
accordance with the methods of the invention.
[0056] In yet another embodiment, the present invention provides a
kit for contraception in a male or female subject comprising an
agent that decreases the proliferation, function or survival of
bone marrow derived germline stem cells, or their progenitor cells,
or the ability of said cells to produce new oocytes or other
somatic cell types required for fertility and instructions for
using the agent to decrease the proliferation, function or survival
of bone marrow derived germline stem cells, or their progenitor
cells, or the ability of said cells to produce new oocytes or sperm
cells or other somatic cell types required for fertility, thereby
providing contraception to the subject in accordance with the
methods of the invention.
BRIEF DESCRIPTION OF THE DRAWINGS
[0057] The patent or application file contains at least one drawing
executed in color. Copies of this patent or patent application
publication with color drawings will be provided by the Office upon
request and payment of the necessary fee.
[0058] FIGS. 1A-1E depict several views of an analysis of germ
cells/progenitors in adult ovaries, in which: FIG. 1A shows
immunohistochemical analysis of SSEA1 expression (red, with nuclei
highlighted by propidium iodide in blue) in adult mouse ovaries;
FIG. 1B shows a higher magnification of the stage-specific
embryonic antigen 1+(SSEA1) cells shown in FIG. 1A; FIG. 1C shows
immunohistochemical analysis of SSEA1 expression (red, with nuclei
highlighted by propidium iodide in blue) in ovaries from different
mice; FIG. 1D shows single SSEA1+ cell in an adult ovary, including
cell surface expression of the antigen; FIG. 1E shows the gene
expression profile of isolated and residual cell fractions prepared
from adult mouse ovaries following SSEA1 antibody-based magnetic
bead sorting; and FIG. 1E shows the ribosomal gene, L7, amplified
as an internal loading control. No product was observed in any mock
reverse-transcribed (Mock) ovarian RNA samples.
[0059] FIGS. 2A-2F depict several views indicating that bone marrow
contains germ cells, in which: FIG. 2A shows germline marker
expression in bone marrow (BM) of adult wild-type female mice with
analysis of adult mouse ovary RNA and mock, mock
reverse-transcribed RNA samples, using the L7, "house-keeping"
gene; FIGS. 2B and 2C show analysis of MVH immunoreactivity (red,
with nuclei highlighted by propidium iodide in blue; scale bar=5
mm) in bone marrow of adult wild-type female mice; FIG. 2D also
show analysis of MVH immunoreactivity mouse ovary in parallel as a
positive control for the immunostaining shown in FIGS. 2B and 2C,
demonstrating a restricted expression of MVH (red) to germ cells
(oocytes); FIG. 2E shows real-time PCR analysis of Mvh levels in
bone marrow or peripheral blood of adult female mice during the
indicated stages of the estrous cycle, in which the data shown
represent the combined results from an analysis of 3-4 mice per
group, with mean levels at estrus set as the reference point for
comparisons to other stages of the cycle following normalization
against beta-actin for sample loading, and wherein for mice in
estrus, Mvh expression in bone marrow was detected during linear
amplification in only 1 of the 3 samples analyzed; and FIG. 2F
shows the number of non-atretic primordial oocyte-containing
follicles in adult female mice at the indicated stages of the
estrous cycle (mean.+-.SEM, n=4 mice per group).
[0060] FIGS. 3A-3B depict several views showing the properties of
bone marrow-derived germ cells, in which FIG. 3A shows quantitative
analysis of Mvh levels in crude (Total) and lineage-depleted (lin-)
bone marrow samples without or with further fractionation by FACS
based on cell-surface expression of Sca-1 or c-Kit in which: all
the remaining lin- cells not represented in the Sca-1-/c-Kit+ cell
fraction were pooled and analyzed together; and the data shown
represent the combined results from an analysis of 3 adult female
mice, with mean Mvh levels in the crude bone marrow sample set as
the reference point for comparisons following normalization against
beta-actin for sample loading; and FIG. 3B shows germline marker
expression in adherent bone marrow-derived cells following a total
of three serial passages (P3) over a six-week period in-vitro for
BM (freshly isolated bone marrow), and mock, mock
reverse-transcribed RNA samples using beta-actin as the
"house-keeping" gene.
[0061] FIG. 4 presents results indicating that bone marrow
transplantation (BMT) reverses chemotherapy-induced ovarian
failure. The number of non-atretic immature follicles present in
the ovaries of wild-type female mice 60 days after treatment with
busulfan and cyclophosphamide on day 42 postpartum without or with
BMT 1 or 7 days later (mean.+-.S.E.; n=5 mice per group, with 4 of
the 5 mice exposed to chemotherapy without subsequent BMT
completely lacking immature oocytes).
[0062] FIGS. 5A-5E present results indicating that BMT sustains
both short and long-term oocyte production in adult wild-type
female mice sterilized by chemotherapy, in which: FIG. 5A shows
representative ovarian histology in adult female mice 2 months
after treatment with vehicle and no BMT (control) (corpora lutea
denoted by asterisks); FIG. 5B shows combination chemotherapy
without BMT; FIG. 5C shows combination chemotherapy with BMT
performed 7 days later (corpora lutea denoted by asterisks); and
FIGS. 5D and 5E show ovarian histology of adult wild-type female
mice 11.5 months after combination chemotherapy (cyclophosphamide
and busulfan) followed by BMT on day 42 postpartum, with follicles
at various stages of maturational development highlighted
(insets).
[0063] FIGS. 6A-6F depict several views of the histology of various
samples of mouse ovaries, in which: FIG. 6A shows the histology of
postpartum day 4 wild-type ovaries; FIG. 6B shows the histology of
Atm-null ovaries; FIG. 6C shows a magnification of the histology
shown in FIG. 6A; FIG. 6D shows a magnification of the histology
shown in FIG. 6B; FIG. 6E shows the representative histology of
wild-type ovaries from adult mice; and FIG. 6F shows the
representative histology of Atm-null ovaries from adult mice.
[0064] FIG. 7 depicts expression of germline marker genes in Atm
deficient mouse ovaries by RT-PCR analysis of Oct4, Mvh (Vasa),
Dazl and Stella expression in ovaries of adult Atm-null (-/-)
female mice. The ribosomal gene, L7, was amplified as an internal
loading control; no product was observed in mock
reverse-transcribed (Mock) ovarian RNA samples.
[0065] FIGS. 8A and 8B depict views of ovaries in a chemotherapy
conditioned mouse that received exogenous, wild-type bone marrow,
in which: FIG. 8A shows ovaries in a chemotherapy conditioned
(bisulfan, cyclophosphamide) wild-type mouse that received
exogenous, wild-type bone marrow; and FIG. 8B shows ovaries in a
chemotherapy conditioned (bisulfan, cyclophosphamide) Atm-null
mouse that received exogenous, wild-type bone marrow. Both the
wild-type mouse and the Atm-null mouse that received exogenous,
wild-type bone marrow after sterilizing doses of chemotherapy
exhibited normal oocytes within normal appearing follicles.
[0066] FIG. 9 depicts analysis of germline markers in bone marrow
of humans. Expression of DAZL and STELLA in bone marrow collected
from 4 human female donors between 24-36 years of age. As a
negative control, germline markers were not detected in two
different adult human uterine (Ut) endometrial samples analyzed in
parallel. Glyceraldehyde 3 phosphate dehydrogenase (GAPDH), a house
keeping gene, amplified as an internal loading control.
[0067] Mock, mock reverse-transcribed RNA samples.
DETAILED DESCRIPTION OF THE INVENTION
Definitions
[0068] "Bone marrow derived germline stem cells" are any
multipotent cells obtained from bone marrow that include a
population of female or male germline stem cells.
[0069] "Expansion" refers to the propagation of a cell or cells
without terminal differentiation. "Isolation phenotype" refers to
the structural and functional characteristics of the bone marrow
derived germline stem cells upon isolation. "Expansion phenotype"
refers to the structural and functional characteristics of the bone
marrow derived germline stem cells during expansion. The expansion
phenotype can be identical to the isolation phenotype, or
alternatively, the expansion phenotype can be more differentiated
than the isolation phenotype.
[0070] "Differentiation" refers to the developmental process of
lineage commitment. A "lineage" refers to a pathway of cellular
development, in which precursor or "progenitor" cells undergo
progressive physiological changes to become a specified cell type
having a characteristic function (e.g., nerve cell, muscle cell or
endothelial cell). Differentiation occurs in stages, whereby cells
gradually become more specified until they reach full maturity,
which is also referred to as "terminal differentiation." A
"terminally differentiated cell" is a cell that has committed to a
specific lineage, and has reached the end stage of differentiation
(i.e., a cell that has fully matured). Oocytes are an example of a
terminally differentiated cell type.
[0071] The term "isolated" as used herein refers to a bone marrow
derived germline stem cell or its progenitor cell, in a
non-naturally occurring state (e.g., isolated from the body or a
biological sample, such as bone marrow, from the body).
[0072] "Progenitor cells" as used herein are germ lineage cells
that are 1) derived from germline stem cells of the invention as
the progeny thereof which contain a set of common marker genes; 2)
are in an early stage of differentiation; and 3) retain mitotic
capacity.
[0073] "Progeny" as used herein are all cells derived from bone
marrow derived germline stem cells of the invention, including
progenitor cells, differentiated cells, and terminally
differentiated cells.
[0074] "Derived from" as used herein refers to the process of
obtaining a daughter cell.
[0075] "Engraft" refers to the process of cellular contact and
incorporation into an existing tissue of interest (e.g., ovary) in
vivo.
[0076] "Agents" refer to cellular (e.g., biologic) and
pharmaceutical factors, preferably growth factors, cytokines,
hormones or small molecules, or to genetically-encoded products
that modulate cell function (e.g., induce lineage commitment,
increase expansion, inhibit or promote cell growth and survival).
For example, "expansion agents" are agents that increase
proliferation and/or survival of bone marrow derived germline stem
cells. "Differentiation agents" are agents that induce bone marrow
derived germline stem cells to differentiate into committed cell
lineages, such as oocytes and sperm cells.
[0077] A "follicle" refers to an ovarian structure consisting of a
single oocyte surrounded by somatic (granulosa without or with
theca-interstitial) cells. Somatic cells of the gonad enclose
individual oocytes to form follicles. Each fully formed follicle is
enveloped in a complete basement membrane. Although some of these
newly formed follicles start to grow almost immediately, most of
them remain in the resting stage until they either degenerate or
some signal(s) activate(s) them to enter the growth phase. For
reviews on ovarian structure, function and physiology, see Gougeon,
A., (1996) Endocr Rev. 17:121-55; Anderson, L. D., and Hirshfield,
A. N. (1992) Md Med J. 41: 614-20; and Hirshfield, A. N. (1991) Int
Rev Cytol. 124: 43-101.
[0078] A "sperm cell" refers to a male germ cell, in either a
pre-meiotic (i.e., mitotically competent) or post-meiotic state of
development, including a fully mature spermatozoan.
"Spermatogenesis" is the developmental process by which a sperm
cell is formed.
[0079] "Mitotically competent" refers to a cell that is capable of
mitosis, the process by which a cell divides and produces two
daughter cells from a single parent cell.
[0080] A "non-embryonic" cell refers to a cell that is obtained
from a post-natal source (e.g., infant, child or adult tissue).
[0081] A "subject" is a vertebrate, preferably a mammal, more
preferably a primate and still more preferably a human. Mammals
include, but are not limited to, primates, humans, farm animals,
sport animals, and pets.
[0082] The term "obtaining" as in "obtaining the agent" is intended
to include purchasing, synthesizing or otherwise acquiring the
agent (or indicated substance or material).
[0083] The terms "comprises", "comprising", are intended to have
the broad meaning ascribed to them in U.S. Patent Law and can mean
"includes", "including" and the like.
EMBODIMENTS OF THE INVENTION
I. Bone Marrow Derived Germline Stem Cells
[0084] Methods of the invention relate to the use of bone marrow
derived germline stem cells, or progenitors of bone marrow derived
germline stem cells, to restore or increase germ cell production.
Methods of the invention can be used to, among other things,
enhance or restore fertility, and in females, to ameliorate
symptoms and consequences of menopause.
[0085] Without wanting to be bound by theory, it is understood that
one or more mechanisms can be involved with the ability of bone
marrow derived germline stem cells to repopulate reproductive
organs. Female germline stem cells have been detected in the bone
marrow, which may therefore serve as a reservoir for stem cells
having the capacity to repopulate and/or expand the germ cell
supply of reproductive organs. Male germline stem cells also exist
in the bone marrow of male subjects. Other sub-populations of cells
in the bone marrow, such as hematopoietic stem cells, may likewise
have the ability to repopulate and/or expand the germ cell supply
of reproductive organs (Herzog, E. L., et al., (2004) Blood
102(10): 3483), for example, through de-differentiation into a
multipotent progenitor cell (see U.S. Pat. No. 6,090,625) which in
turn migrates through peripheral blood to the reproductive tract,
engrafts into an organ (e.g., ovary or testes) as a germline stem
cell or a progenitor of a germline stem cell and differentiates
into an oocyte (ovary) or sperm (testis).
[0086] As described herein, germline stem cells have been detected
in the bone marrow of male and female subjects. Bone marrow derived
female germline stem cells express markers including Oct 4, Vasa,
Dazi, Stella, Fragilis, and optionally Nobox, c-Kit and Sca-1. Bone
marrow derived female germline stem cells are mitotically competent
(i.e., capable of mitosis) and accordingly, do not express GDF-9,
zona pellucida proteins (e.g., ZP3), HDAC6 or SCP3.
[0087] The present invention also provides bone marrow derived
female germline stem cell progenitors. Bone marrow derived female
germline stem cell progenitors of the invention can circulate
throughout the body and most preferably can be localized in bone
marrow, peripheral blood and ovary. Progenitor cells of the
invention express Oct 4, Vasa, Dazl, Stella, Fragilis, and
optionally Nobox, c-Kit and Sca-1 but do not express GDF-9, zona
pellucida proteins (e.g., ZP3), HDAC6 or SCP3.
[0088] Bone marrow derived female germline stem cells and their
progenitor cells have functional distinctions. Upon transplantation
into a host, bone marrow derived female germline stem cells of the
invention can produce oocytes after a duration of at least 1 week,
more preferably 1 to about 2 weeks, about 2 to about 3 weeks, about
3 to about 4 weeks or more than about 5 weeks post transplantation.
Bone marrow derived female germline stem cell progenitors have the
capacity to generate oocytes more rapidly than bone marrow derived
female germline stem cells. Upon transplantation into a host, bone
marrow derived female germline stem cell progenitors of the
invention can produce oocytes after a duration of less than 1 week,
preferably about 24 to about 48 hours post transplantation.
[0089] Oct-4 is a gene expressed in bone marrow derived female
germline stem cells and their progenitor cells. The Oct-4 gene
encodes a transcription factor that is involved in the
establishment of the mammalian germline and plays a significant
role in early germ cell specification (reviewed in Scholer (1991),
Trends Genet. 7(10): 323-329). In the developing mammalian embryo,
Oct-4 is downregulated during the differentiation of the epiblast,
eventually becoming confined to the germ cell lineage. In the
germline, Oct-4 expression is regulated separately from epiblast
expression. Expression of Oct-4 is a phenotypic marker of
totipotency (Yeom et al. (1996), Development 122: 881-888).
[0090] Stella is a gene expressed in bone marrow derived female
germline stem cells and their progenitor cells. Stella is a novel
gene specifically expressed in primordial germ cells and their
descendants, including oocytes (Bortvin et al. (2004) BMC
Developmental Biology 4(2):1-5). Stella encodes a protein with a
SAP-like domain and a splicing factor motif-like structure. Embryos
deficient in Stella expression are compromised in preimplantation
development and rarely reach the blastocyst stage. Thus, Stella is
a maternal factor implicated in early embryogenesis.
[0091] Dazl is a gene expressed in bone marrow derived female
germline stem cells and their progenitor cells. The autosomal gene
Dazi is a member of a family of genes that contain a consensus RNA
binding domain and are expressed in germ cells. Loss of expression
of an intact Dazl protein in mice is associated with failure of
germ cells to complete meiotic prophase. Specifically, in female
mice null for Dazl, loss of germ cells occurs during fetal life at
a time coincident with progression of germ cells through meiotic
prophase. In male mice null for Dazl, germ cells were unable to
progress beyond the leptotene stage of meiotic prophase I. Thus, in
the absence of Dazl, progression through meiotic prophase is
interrupted (Saunders et al. (2003), Reproduction,
126:589-597).
[0092] Vasa is a gene expressed in bone marrow derived female
germline stem cells and their progenitor cells. Vasa is a component
of the germplasm that encodes a DEAD-family ATP-dependent RNA
helicase (Liang et al. (1994) Development, 120:1201-1211; Lasko et
al. (1988) Nature, 335:611-167). The molecular function of Vasa is
directed to binding target mRNAs involved in germ cell
establishment (e.g., Oskar and Nanos), oogenesis, (e.g., Gruken),
and translation onset (Gavis et al. (1996) Development, 110:
521-528). Vasa is required for pole cell formation and is
exclusively restricted to the germ cell lineage throughout the
development. Thus, Vasa is a molecular marker for the germ cell
lineage in most animal species (Toshiaki et al. (2001) Cell
Structure and Function 26:131-136). Because Vasa has been
associated with inhibition of cell migration, expression of Vasa in
progenitor cells of the invention may be differentially regulated,
depending on the migratory state of the progenitor. For example,
while in the bone marrow, the progenitor may express Vasa, and
while migrating to the reproductive tract, the progenitor may down
regulate expression.
[0093] Fragilis is a gene expressed in bone marrow derived female
germline stem cells and their progenitor cells. Fragilis is a
putative interferon-inducible gene that codes for a transmembrane
protein associated with the acquisition of germ cell competence by
epiblast cells (Saitou, M. et al. (2002) Nature 418:293-300).
Extraembryonic ectoderm is able to induce fragilis expression in
epiblast tissue. Fragilis is expressed in proximal epiblast at a
region in which primordial germ cell (PGC)-competent cells reside
according to clonal analysis (Lawson, K A et al. (1994) In Wiley,
Chichester (Ciba Foundation Symposium 182): 68-91). As these
proximal cells move to the posterior proximal region during
gastrulation, fragilis expression increases within a community of
cells at the base of the incipient allantoic bud. Cells with the
highest expression of fragilis initiate the germ
cell-characteristic expression of TNAP and stella/PGC-7 (Ginsburg,
M. et al. (1990) Development 110:521-528; Sato, M. et al. (2002)
Mech Dev 113:91-94) and show repression of Hox genes.
[0094] Nobox is a gene that is optionally expressed in bone marrow
derived female germline stem cells and their progenitor cells Nobox
(Newborn Ovary Homeobox) is a gene active in ovaries and testes
that regulates the transition of a primordial germ cell into a
primary follicle. Female mice lacking the Nobox gene lose all of
their follicles by 6 weeks of life and are essentially menopausal
by that time; males have normal testes but are 30% less fertile
(Rajkovic, A. et al. (2004) Science 305 (5687): 1157-1159). Nobox
appears to govern the activity of genes crucial to the development
of follicles, which hold the immature eggs cells or oocytes. These
follicles are supposed to thicken as the mouse develops; without
Nobox, the follicles do not develop, and the oocytes
deteriorate.
[0095] c-Kit is a gene that is optionally expressed in bone marrow
derived female germline stem cells and their progenitor cells.
c-Kit is a proto-oncogene that encodes a transmembrane protein
tyrosine kinase receptor that is structurally similar to the
receptors for colony-stimulating factor-1 (CSF-1) and platelet
derived growth factor. c-Kit has been found to play a pivotal role
in the normal growth and differentiation of embryonic melanoblasts.
c-kit, and its ligand have been demonstrated to be essential to the
processes of germ cell migration, proliferation and survival in the
rodent. The expression of c-kit mRNA and protein is germ cell
specific in human fetal gonads and are consistent with an important
role for the c-kit/kit ligand signalling system in germ cell
proliferation and survival in the developing human gonad (Robinson,
L. L., et al. (2001) Mol Hum Reprod 7(9):845-52).
[0096] Sca-1 is a gene that is optionally expressed in bone marrow
derived female germline stem cells and their progenitor cells.
Sca-1 (stem cell antigen 1, Ly-6A/E) is an 18 kDa
phosphatidylinositol-anchored protein and member of the Ly-6
antigen family. Sca-1 has been used in the isolation of
hematopoietic stem cells (purification to homogeneity) from mouse
bone marrow (Van de Rijn, M. et al. (1989) Proc. Natl. Acad. Sci.
USA 86:4634; Spangrude, G. I. et al. (1988) Science 241:58).
Sca-1.sup.+ HSCs can be found in the adult bone marrow, fetal liver
and mobilized peripheral blood and spleen within the adult animal
(Morrison, S. J. et al. (1997) Proc. Natl. Acad. Sci. USA 94:1908).
Additionally, Sca-1 may be involved in regulating both B and T cell
activation (Codias, E. K. et al., (1990) J. Immunol. 145:1407).
[0097] Bone marrow derived female germline stem cells and their
progenitor cells do not express GDF-9, a gene expressed in cells
that have already started to differentiate into oocytes. Growth
differentiation factor-9 (GDF-9) is a member of the transforming
growth factor-.beta. superfamily, expressed specifically in
ovaries. CDF-9 mRNA can be found in neonatal and adult oocytes from
the primary one-layer follicle stage until after ovulation (Dong,
J. et al (1996) Nature 383: 531-5). Analysis of GDF-9 deficient
mice reveals that only primordial and primary one-layer follicles
can be formed, but a block beyond the primary one-layer follicle
stage in follicular development occurs, resulting in complete
infertility.
[0098] Bone marrow derived female germline stem cells and their
progenitor cells do not express ZP1, ZP2, and ZP3, which are gene
products that comprise the zona pellucida of the oocyte. Their
expression is regulated by a basic helix-loop-helix (bHLH)
transcription factor, FIG.alpha.. Mice null in FIG.alpha. do not
express the Zp genes and do not form primordial follicles (Soyal,
S. M., et al (2000) Development 127: 4645-4654). Individual
knockouts of the ZP genes result in abnormal or absent zonae
pellucidae and decreased fertility (Zp1; Rankin T, et al (1999)
Development. 126: 3847-55) or sterility (Zp2, Rankin T L, et al.
(2001) Development 128: 1119-26; ZP3, Rankin T et al (1996)
Development 122: 2903-10). The ZP protein products are
glycosylated, and subsequently secreted to form an extracellular
matrix, which is important for in vivo fertilization and
pre-implantation development. Expression of the ZP proteins is
precisely regulated and restricted to a two-week growth phase of
oogenesis. Zp mRNA transcripts are not expressed in resting
oocytes, however once the oocytes begin to grow, all three Zp
transcripts begin to accumulate.
[0099] Bone marrow derived female germline stem cells and their
progenitor cells do not express HDAC6. HDACs, or histone
deacetylases are involved in ovarian follicle development. HDAC6 in
particular can be detected in resting germinal vesicle-stage
(primordial) oocytes (Verdel, A., et al. (2003) Zygote 11: 323-8;
FIG. 16). HDAC6 is a class II histone deacetylase and has been
implicated as a microtubule-associated deactylase (Hubbert, C. et
al, (2002) Nature 417: 455-8). HDACs are the target of inhibitors
including, but not limited to, trichostatin A and trapoxin, both of
which are microbial metabolites that induce cell differentiation,
cell cycle arrest, and reversal of the transformed cell
morphology.
[0100] Bone marrow derived female germline stem cells and their
progenitor cells do not express SCP3, consistent with observations
that they are pre-meiotic stem cells (i.e., diploid). The
synaptonemal complex protein SCP3 is part of the lateral element of
the synaptonemal complex, a meiosis-specific protein structure
essential for synapsis of homologous chromosomes. The synaptonemal
complex promotes pairing and segregation of homologous chromosomes,
influences the number and relative distribution of crossovers, and
converts crossovers into chiasmata. SCP3 is meiosis-specific and
can form multi-stranded, cross-striated fibers, forming an ordered,
fibrous core in the lateral element (Yuan, L. et al, (1998) J.
Cell. Biol. 142: 331-339). The absence of SCP3 in mice can lead to
female germ cell aneuploidy and embryo death, possibly due to a
defect in structural integrity of meiotic chromosomes (Yuan, L. et
al, (2002) Science 296: 1115-8).
[0101] Bone marrow derived female germline stem cells and their
progenitor cells can be isolated by standard means known in the art
for the separation of stem cells from the marrow (e.g., cell
sorting). Preferably, the isolation protocol includes generation of
a kit.sup.+/lin.sup.- fraction that is depleted of hematopoietic
cells. Additional selection means based on the unique profile of
gene expression (e.g., Vasa, Oct-4, Dazl, Stella, Fragilis) can be
employed to further purify populations of cells comprising bone
marrow derived female germline stem cells and their progenitor
cells. Compositions comprising bone marrow derived female germline
stem cells and their progenitor cells can be isolated and
subsequently purified to an extent where they become substantially
free of the biological sample from which they were obtained (e.g.
bone marrow).
[0102] Bone marrow derived female germline stem cell progenitors
can be obtained from female germline stem cells by, for example,
expansion in culture. Thus, the progenitor cells can be cells
having an "expansion phenotype."
II. Administration
[0103] Compositions comprising bone marrow derived germline stem
cells or their progenitor cells can be provided directly to the
reproductive organ of interest (e.g., ovary or testes).
Alternatively, compositions comprising bone marrow derived germline
stem cells or their progenitors can be provided indirectly to the
reproductive organ of interest, for example, by administration into
the circulatory system (e.g., to the extra-ovarian circulation).
Following transplantation or implantation, the cells can engraft
and differentiate into germ cells (e.g., oocytes or sperm cells).
"Engraft" refers to the process of cellular contact and
incorporation into an existing tissue of interest (e.g., ovary) in
vivo. Expansion and differentiation agents can be provided prior
to, during or after administration to increase production of germ
cells in vivo.
[0104] Compositions of the invention include pharmaceutical
compositions comprising bone marrow derived germline stem cells or
their progenitor cells and a pharmaceutically acceptable carrier.
Administration can be autologous or heterologous. For example, bone
marrow derived germline stem cells, or their progenitor cells, can
be obtained from one subject, and administered to the same subject
or a different, compatible subject.
[0105] Bone marrow derived germline stem cells of the invention or
their progeny (e.g., progenitors, differentiated progeny and
terminally differentiated progeny) can be administered via
localized injection, including catheter administration, systemic
injection, localized injection, intravenous injection, intrauterine
injection or parenteral administration. When administering a
therapeutic composition of the present invention (e.g., a
pharmaceutical composition), it will generally be formulated in a
unit dosage injectable form (solution, suspension, emulsion).
[0106] Compositions of the invention can be conveniently provided
as sterile liquid preparations, e.g., isotonic aqueous solutions,
suspensions, emulsions, dispersions, or viscous compositions, which
may be buffered to a selected pH. Liquid preparations are normally
easier to prepare than gels, other viscous compositions, and solid
compositions. Additionally, liquid compositions are somewhat more
convenient to administer, especially by injection. Viscous
compositions, on the other hand, can be formulated within the
appropriate viscosity range to provide longer contact periods with
specific tissues. Liquid or viscous compositions can comprise
carriers, which can be a solvent or dispersing medium containing,
for example, water, saline, phosphate buffered saline, polyol (for
example, glycerol, propylene glycol, liquid polyethylene glycol,
and the like) and suitable mixtures thereof.
[0107] Sterile injectable solutions can be prepared by
incorporating the cells utilized in practicing the present
invention in the required amount of the appropriate solvent with
various amounts of the other ingredients, as desired. Such
compositions may be in admixture with a suitable carrier, diluent,
or excipient such as sterile water, physiological saline, glucose,
dextrose, or the like. The compositions can also be lyophilized.
The compositions can contain auxiliary substances such as wetting,
dispersing, or emulsifying agents (e.g., methylcellulose), pH
buffering agents, gelling or viscosity enhancing additives,
preservatives, flavoring agents, colors, and the like, depending
upon the route of administration and the preparation desired.
Standard texts, such as "REMINGTON'S PHARMACEUTICAL SCIENCE", 17th
edition, 1985, incorporated herein by reference, may be consulted
to prepare suitable preparations, without undue
experimentation.
[0108] Various additives which enhance the stability and sterility
of the compositions, including antimicrobial preservatives,
antioxidants, chelating agents, and buffers, can be added.
Prevention of the action of microorganisms can be ensured by
various antibacterial and antifungal agents, for example, parabens,
chlorobutanol, phenol, sorbic acid, and the like. Prolonged
absorption of the injectable pharmaceutical form can be brought
about by the use of agents delaying absorption, for example,
aluminum monostearate and gelatin. According to the present
invention, however, any vehicle, diluent, or additive used would
have to be compatible with the bone marrow derived germline stem
cells or their progenitors.
[0109] The compositions can be isotonic, i.e., they can have the
same osmotic pressure as blood and lacrimal fluid. The desired
isotonicity of the compositions of this invention may be
accomplished using sodium chloride, or other pharmaceutically
acceptable agents such as dextrose, boric acid, sodium tartrate,
propylene glycol or other inorganic or organic solutes. Sodium
chloride is preferred particularly for buffers containing sodium
ions.
[0110] Viscosity of the compositions, if desired, can be maintained
at the selected level using a pharmaceutically acceptable
thickening agent. Methylcellulose is preferred because it is
readily and economically available and is easy to work with. Other
suitable thickening agents include, for example, xanthan gum,
carboxymethyl cellulose, hydroxypropyl cellulose, carbomer, and the
like. The preferred concentration of the thickener will depend upon
the agent selected. The important point is to use an amount that
will achieve the selected viscosity. Obviously, the choice of
suitable carriers and other additives will depend on the exact
route of administration and the nature of the particular dosage
form, e.g., liquid dosage form (e.g., whether the composition is to
be formulated into a solution, a suspension, gel or another liquid
form, such as a time release form or liquid-filled form).
[0111] A method to potentially increase cell survival when
introducing the cells into a subject in need thereof is to
incorporate bone marrow derived germline stem cells or their
progeny (e.g., in vivo, ex vivo or in vitro derived) of interest
into a biopolymer or synthetic polymer. Depending on the subject's
condition, the site of injection might prove inhospitable for cell
seeding and growth because of scarring or other impediments.
Examples of biopolymer include, but are not limited to, cells mixed
with fibronectin, fibrin, fibrinogen, thrombin, collagen, and
proteoglycans. This could be constructed with or without included
expansion or differentiation factors. Additionally, these could be
in suspension, but residence time at sites subjected to flow would
be nominal. Another alternative is a three-dimensional gel with
cells entrapped within the interstices of the cell biopolymer
admixture. Again, expansion or differentiation factors could be
included with the cells. These could be deployed by injection via
various routes described herein.
[0112] Those skilled in the art will recognize that the components
of the compositions should be selected to be chemically inert and
will not affect the viability or efficacy of the bone marrow
derived germline stem cells or their progenitors as described in
the present invention. This will present no problem to those
skilled in chemical and pharmaceutical principles, or problems can
be readily avoided by reference to standard texts or by simple
experiments (not involving undue experimentation), from this
disclosure and the documents cited herein.
[0113] One consideration concerning the therapeutic use of bone
marrow derived germline stem cells and their progeny is the
quantity of cells necessary to achieve an optimal effect. In
current human studies of autologous mononuclear bone marrow cells,
empirical doses ranging from 1 to 4.times.10.sup.7 cells have been
used with encouraging results. However, different scenarios may
require optimization of the amount of cells injected into a tissue
of interest. Thus, the quantity of cells to be administered will
vary for the subject being treated. In a preferred embodiment,
between 10.sup.4 to 10.sup.8, more preferably 10.sup.5 to 10.sup.7,
and still more preferably, 3.times.10.sup.7 stem cells of the
invention can be administered to a human subject.
[0114] Less cells can be administered directly to the ovary or
testes. Preferably, between 10.sup.2 to 10.sup.6, more preferably
10.sup.3 to 10.sup.5, and still more preferably, 10.sup.4 bone
marrow derived germline stem cells can be administered to a human
subject. However, the precise determination of what would be
considered an effective dose may be based on factors individual to
each patient, including their size, age, sex, weight, and condition
of the particular patient. As few as 100-1000 cells can be
administered for certain desired applications among selected
patients. Therefore, dosages can be readily ascertained by those
skilled in the art from this disclosure and the knowledge in the
art.
[0115] Bone marrow derived germline stem cells of the invention can
comprise a purified population of germline stem cells or their
progenitors. Those skilled in the art can readily determine the
percentage of cells in a population using various well-known
methods, such as fluorescence activated cell sorting (FACS).
Preferable ranges of purity in populations comprising female
germline stem cells or their progenitors are about 50 to about 55%,
about 55 to about 60%, and about 65 to about 70%. More preferably
the purity is about 70 to about 75%, about 75 to about 80%, about
80 to about 85%; and still more preferably the purity is about 85
to about 90%, about 90 to about 95%, and about 95 to about 100%.
Purity of female germline stem cells or their progenitors can be
determined according to the cell surface marker profile within a
population. Dosages can be readily adjusted by those skilled in the
art (e.g., a decrease in purity may require an increase in
dosage).
[0116] The skilled artisan can readily determine the amount of
cells and optional additives, vehicles, and/or carrier in
compositions and to be administered in methods of the invention.
Typically, any additives (in addition to the active stem cell(s)
and/or agent(s)) are present in an amount of 0.001 to 50% (weight)
solution in phosphate buffered saline, and the active ingredient is
present in the order of micrograms to milligrams, such as about
0.0001 to about 5 wt %, preferably about 0.0001 to about 1 wt %,
still more preferably about 0.0001 to about 0.05 wt % or about
0.001 to about 20 wt %, preferably about 0.01 to about 10 wt %, and
still more preferably about 0.05 to about 5 wt %. Of course, for
any composition to be administered to an animal or human, and for
any particular method of administration, it is preferred to
determine therefore: toxicity, such as by determining the lethal
dose (LD) and LD.sub.50 in a suitable animal model e.g., rodent
such as mouse; and, the dosage of the composition(s), concentration
of components therein and timing of administering the
composition(s), which elicit a suitable response. Such
determinations do not require undue experimentation from the
knowledge of the skilled artisan, this disclosure and the documents
cited herein. And, the time for sequential administrations can be
ascertained without undue experimentation.
III. Oocyte Production
[0117] In one embodiment, the present invention provides a method
for oocyte production, comprising providing a bone marrow derived
female germline stem cell, or its progenitor, to a female subject,
and more preferably to the ovary of said subject, wherein the cell
engrafts into the ovary and differentiates into an oocyte.
[0118] Preferably, the engrafted cells undergo folliculogenesis,
wherein the cells differentiate into an oocyte and become enclosed
within a follicle. Preferably, the engrafted cells differentiate
into an oocyte within a follicle of the ovary. Folliculogenesis is
a process in which an ovarian structure consisting of a single
oocyte is surrounded by somatic (granulosa without or with
theca-interstitial) cells. Somatic cells of the gonad enclose
individual oocytes to form follicles. Each fully formed follicle is
enveloped in a complete basement membrane. Although some of these
newly formed follicles start to grow almost immediately, most of
them remain in the resting stage until they either degenerate or
some signal(s) activate(s) them to enter the growth phase. A method
of the invention can induce folliculogenesis by providing a bone
marrow derived female germline stem cell, or its progenitor, to a
tissue (e.g., ovarian tissue) by any one of several routes of
administration. The bone marrow derived female germline stem cell,
or its progenitor, can engraft into the tissue and differentiate
into an oocyte within a follicle.
[0119] The number of bone marrow derived female germline stem
cells, or their progenitor cells can be increased by increasing the
survival or proliferation of existing bone marrow derived female
germline stem cells, or their progenitor cells.
[0120] Agents (e.g., expansion agents) which increase proliferation
or survival of bone marrow derived female germline stem cells, or
their progenitor cells include, but are not limited to, a hormone
or growth factor (e.g., a IGF, TGF, BMP, Wnt protein or FGF), a
cell-signaling molecule (e.g., S1P or RA), or a pharmacological or
pharmaceutical compound (e.g., an inhibitor of GSK-3, an inhibitor
of apoptosis such as a Bax inhibitor or caspase inhibitor, an
inhibitor of nitric oxide production, or an inhibitor of HDAC
activity).
[0121] Agents comprising growth factors are known in the art to
increase proliferation or survival of stem cells. For example, U.S.
Pat. Nos. 5,750,376 and 5,851,832 describe methods for the in vitro
culture and proliferation of neural stem cells using TGF. An active
role in the expansion and proliferation of stem cells has also been
described for BMPs (Zhu, G. et al, (1999) Dev. Biol. 215: 118-29
and Kawase, E. et al, (2001) Development 131: 1365) and Wnt
proteins (Pazianos, G. et al, (2003) Biotechniques 35: 1240 and
Constantinescu, S. (2003) J. Cell Mol. Med. 7: 103). U.S. Pat. Nos.
5,453,357 and 5,851,832 describe proliferative stem cell culture
systems that utilize FGFs. The contents of each of these references
are specifically incorporated herein by reference for their
description of expansion agents known in the art.
[0122] Agents comprising cell-signaling molecules are also known in
the art to increase proliferation or survival of stem cells. For
example, Sphingosine-1-phosphate is known to induce proliferation
of neural progenitor cells (Harada, J. et al, (2004) J. Neurochem.
88: 1026). U.S. Patent Application No. 20030113913 describes the
use of retinoic acid in stem cell self renewal in culture. The
contents of each of these references are specifically incorporated
herein by reference for their description of expansion agents known
in the art.
[0123] Agents comprising pharmacological or pharmaceutical
compounds are also known in the art to increase proliferation or
survival of stem cells. For example, inhibitors of glycogen
synthase kinase maintain pluripotency of embryonic stem cells
through activation of Wnt signaling (Sato, N. et al, (2004) Nat.
Med. 10: 55-63). Inhibitors of apoptosis (Wang, Y. et al, (2004)
Mol. Cell. Endocrinol. 218: 165), inhibitors of nitric oxide/nitric
oxide synthase (Matarredona, E. R. et al, (2004) Brain Res. 995:
274) and inhibitors of histone deacetylases (Lee, J. H. et al,
(2004) Genesis 38: 32-8) are also known to increase proliferation
and/or pluripotency. For example, the peptide humanin is an
inhibitor of Bax function that suppresses apoptosis (Guo, B. et al,
(2003) Nature 423: 456-461). The contents of each of these
references are specifically incorporated herein by reference for
their description of expansion agents known in the art.
[0124] Oocyte production can be further increased by contacting
bone marrow derived female germline stem cells, or their progenitor
cells, with an agent that differentiates bone marrow derived female
germline stem cells or their progenitor cells into oocytes (e.g.,
differentiation agents). Such differentiation agents include, but
are not limited to, a hormone or growth factor (e.g., TGF, BMP, Wnt
protein, SCF or LIF), a signaling molecule (e.g.,
meiosis-activating sterol, "FF-MAS"), or a pharmacologic or
pharmaceutical agent (e.g., a modulator of Id protein function or
Snail/Slug transcription factor function).
[0125] Agents comprising growth factors are known in the art to
induce differentiation of stem cells. For example, TGF-.beta. can
induce differentiation of hematopoietic stem cells (Ruscetti, F. W.
et al, (2001) Int. J. Hematol. 74: 18). U.S. Patent Application No.
2002142457 describes methods for differentiation of cardiomyocytes
using BMPs. Pera et al describe human embryonic stem cell
differentiation using BMP-2 (Pera, M. F. et al, (2004) J. Cell Sci.
117: 1269). U.S. Patent Application No. 20040014210 and U.S. Pat.
No. 6,485,972 describe methods of using Wnt proteins to induce
differentiation. U.S. Pat. No. 6,586,243 describes differentiation
of dendritic cells in the presence of SCF. U.S. Pat. No. 6,395,546
describes methods for generating dopaminergic neurons in vitro from
embryonic and adult central nervous system cells using LIF. The
contents of each of these references are specifically incorporated
herein by reference for their description of differentiation agents
known in the art.
[0126] Agents comprising signaling molecules are also known to
induce differentiation of oocytes. FF-Mas is known to promote
oocyte maturation (Marin Bivens, C. L. et al, (2004) BOR papers in
press). The contents of each of these references are specifically
incorporated herein by reference for their description of
differentiation agents known in the art.
[0127] Agents comprising pharmacological or pharmaceutical
compounds are also known in the art to induce differentiation of
stem cells. For example, modulators of Id are involved in
hematopoietic differentiation (Nogueria, M. M. et al, (2000) 276:
803) and Modulators of Snail/Slug are known to induce stem cell
differentiation (Le Douarin, N. M. et al, (1994) Curr. Opin. Genet.
Dev. 4: 685-695; Plescia, C. et al, (2001) Differentiation 68:
254). The contents of each of these references are specifically
incorporated herein by reference for their description of
differentiation agents known in the art.
[0128] The present invention also provides methods for reducing
bone marrow derived female germline stem cells, or their progenitor
cells, in vivo, ex vivo or in vitro, comprising contacting bone
marrow derived female germline stem cells or their progenitor cells
with an agent that reduces cell proliferation, inhibits cell
survival or promotes cell death. Unwanted proliferation of the
cells of the invention can give rise to cancerous and pre-cancerous
phenotypes (e.g., germ cell tumors, ovarian cancer). Such methods
can be used to control unwanted proliferation (e.g., cancer) or for
contraceptive measures by reducing the numbers of germline stem
cells, and optionally their progenitors or oocytes.
[0129] Agents that reduce cell proliferation include, but are not
limited to, a hormone or growth factor (e.g., TGF-.beta.), a
peptide antagonist of mitogenic hormones or growth factors (e.g.,
the BMP antagonists, PRDC and Gremlin), or a pharmacological or
pharmaceutical compound (e.g., a cell cycle inhibitor, or an
inhibitor of growth factor signaling).
[0130] Agents that inhibit cell survival include, but are not
limited to, a hormone, growth factor or cytokine (e.g., a
pro-apoptotic TNF super family member such as TNF-.alpha., FasL and
TRAIL), an antagonist of pro-survival Bcl-2 family member function,
a signaling molecule (e.g., a ceramide), or a pharmacological or
pharmaceutical compound (e.g., an inhibitor of growth factor
signaling). Pro-survival Bcl-2 family members include Bcl-2, Bcl-xl
(Cory, S. and Adams, J. M. (2000) Nat Rev Cancer 2(9):647-656;
Lutz, R. J. (2000) Cell Survival Apoptosis 28:51-56), Bcl-W
(Gibson, L., et al. (1996) Oncogene 13, 665-675; Cory, S. and
Adams, J. M. (2000) Nat Rev Cancer 2(9):647-656), Mel-1 (Kozopas,
K. M., et al. (1993) Proc. Natl. Acad. Sci. U.S.A. 90:3516-3520;
Reynolds, J. E., et al. (1994) Cancer Res. 54:6348-6352; Cory, S.
and Adams, J. M. (2000) Nat Rev Cancer 2(9):647-656) and A1 (Cory,
S. and Adams, J. M. (2000) Nat Rev Cancer 2(9):647-656; Gonzales,
J., et al. (2003) Blood 101(7):2679-2685; Reed, J. C. (1997) Nature
387:773-776).
[0131] Agents that promote cell death include, but are not limited
to, a pro-apoptotic tumor necrosis factor superfamily member (e.g.,
TNF-.alpha., FasL and TRAIL), agonist of pro-apoptotic Bcl-2 family
member function and ceramide. Pro-apoptotic Bcl-2 family members
include Bax (Oltvai, Z N, et al. (1993): Cell 74: 609-619), Bak
(Chittenden, T, et al. (1995) Nature 374:733-736), Bid (Luo, X., et
al. (1998) Cell 94:481-490), Hrk (Inohara, N. et al. (1997) EMBO J
16(7):1686-1694), Bod (Hsu, et al. (1998) Mol Endocrinol.
12(9):1432-1440), Bim (O'Connor, L., et al. (1998) EMBO J.
17(2):385-395), Noxa (Oda, E., et al. (2000) Science 288,
1053-1058; Yakovlev, A. G., et al. (2004) J Biol Chem
279(27):28367-28374), puma (Nakano, K. and Vousden, K. H. (2001)
Mol Cell 7(3):683-694), Bok (Yakovlev, A. G., et al. (2004) J Biol
Chem 279(27):28367-28374; Hsu, S Y, et al. (1997) Proc Natl Acad
Sci USA. 94(23):12401-6) and Bcl-xs (Boise, L. H., et al. (1993)
Cell 74:597-608).
[0132] Several agents are known in the art to inhibit cell
proliferation or survival or promote cell death, including PRDC
(Sudo et al, (2004) J. Biol. Chem., advanced publication), TNF
(Wong, G. et al, (2004) Exp. Neurol. 187: 171), FasL (Sakata, S. et
al, (2003) Cell Death Differ. 10: 676) and TRAIL (Pitti, R M, et
al. (1996) J Biol Chem 271: 12687-12690; Wiley, S R, et al. (1995)
Immunity 3: 673-682). Ceramide mediates the action of tumor
necrosis factor on primitive human hematopoietic cells
(Maguer-Satta, V. et al, (2000) Blood 96: 4118-23).
Agonist/antagonist of Bcl-2 family members, such as Bcl-2, Bcl-XL,
Bcl-W, Mel-1, A1, Bax, Bak, Bid, Hrk, Bod, Bim, Noxa, Puma, Bok and
Bcl-xs, are known to inhibit stem cell survival (Lindsten, T. et
al, (2003) J. Neurosci. 23: 11112-9). Agents comprising
pharmacological or pharmaceutical compounds are also known in the
art to inhibit cell survival. For example, inhibitors of growth
factor signaling, such as QSulf1, a heparan sulfate
6-O-endosulfatase that inhibits fibroblast growth factor signaling,
can inhibit stem cell survival (Wang, S. et al, (2004) Proc. Natl.
Acad. Sci. USA 101: 4833). The contents of each of these references
are specifically incorporated herein by reference for their
description of agents known in the art to inhibit cell
survival.
[0133] Agents can be provided directly to the reproductive organ of
interest. Alternatively, agents can be provided indirectly to the
reproductive organ of interest, for example, by administration into
the circulatory system.
[0134] Agents can be administered to subjects in need thereof by a
variety of administration routes. Methods of administration,
generally speaking, may be practiced using any mode of
administration that is medically acceptable, meaning any mode that
produces effective levels of the active compounds without causing
clinically unacceptable adverse effects. Such modes of
administration include oral, rectal, topical, intraocular, buccal,
intravaginal, intracisternal, intracerebroventricular,
intratracheal, nasal, transdermal, within/on implants, e.g., fibers
such as collagen, osmotic pumps, or grafts comprising appropriately
transformed cells, etc., or parenteral routes. The term
"parenteral" includes subcutaneous, intravenous, intramuscular,
intraperitoneal, intragonadal or infusion. Intravenous or
intramuscular routes are not particularly suitable for long-term
therapy and prophylaxis. A particular method of administration
involves coating, embedding or derivatizing fibers, such as
collagen fibers, protein polymers, etc. with therapeutic proteins.
Other useful approaches are described in Otto, D. et al., J.
Neurosci. Res. 22: 83 and in Otto, D. and Unsicker, K. J. Neurosci.
10: 1912.
[0135] In vitro and ex vivo applications can involve culture of the
bone marrow derived germline stem cells or their progenitors with
the selected agent to achieve the desired result. Cultures of cells
(from the same individual and from different individuals) can be
treated with differentiation agents of interest to stimulate, for
example, the production of oocytes or sperm cells, which can then
be used for a variety of therapeutic applications (e.g., in vitro
fertilization).
[0136] Differentiated cells derived from cultures of the invention
can be implanted into a host. The transplantation can be
autologous, such that the donor of the stem cells from which organ
or organ units are derived is the recipient of the engineered
tissue. The transplantation can be heterologous, such that the
donor of the stem cells from which organ or organ units are derived
is not that of the recipient of the engineered-tissue. Once
transferred into a host, the differentiated cells the function and
architecture of the native host tissue.
[0137] Bone marrow derived germline stem cells and the progeny
thereof can be cultured, treated with agents and/or administered in
the presence of polymer scaffolds. Polymer scaffolds are designed
to optimize gas, nutrient, and waste exchange by diffusion. Polymer
scaffolds can comprise, for example, a porous, non-woven array of
fibers. The polymer scaffold can be shaped to maximize surface
area, to allow adequate diffusion of nutrients and growth factors
to the cells. Taking these parameters into consideration, one of
skill in the art could configure a polymer scaffold having
sufficient surface area for the cells to be nourished by diffusion
until new blood vessels interdigitate the implanted
engineered-tissue using methods known in the art, Polymer scaffolds
can comprise a fibriliar structure. The fibers can be round,
scalloped, flattened, star-shaped, solitary or entwined with other
fibers. Branching fibers can be used, increasing surface area
proportionately to volume.
[0138] Unless otherwise specified, the term "polymer" includes
polymers and monomers that can be polymerized or adhered to form an
integral unit. The polymer can be non-biodegradable or
biodegradable, typically via hydrolysis or enzymatic cleavage. The
term "biodegradable" refers to materials that are bioresorbable
and/or degrade and/or break down by mechanical degradation upon
interaction with a physiological environment into components that
are metabolizable or excretable, over a period of time from minutes
to three years, preferably less than one year, while maintaining
the requisite structural integrity. As used in reference to
polymers, the term "degrade" refers to cleavage of the polymer
chain, such that the molecular weight stays approximately constant
at the oligomer level and particles of polymer remain following
degradation.
[0139] Materials suitable for polymer scaffold fabrication include
polylactic acid (PLA), poly-L-lactic acid (PLLA), poly-D-lactic
acid (PDLA), polyglycolide, polyglycolic acid (PGA),
polylactide-co-glycolide (PLGA), polydioxanone, polygluconate,
polylactic acid-polyethylene oxide copolymers, modified cellulose,
collagen, polyhydroxybutyrate, polyhydroxpriopionic acid,
polyphosphoester, poly(alpha-hydroxy acid), polycaprolactone,
polycarbonates, polyamides, polyanhydrides, polyamino acids,
polyorthoesters, polyacetals, polycyanoacrylates, degradable
urethanes, aliphatic polyester polyacrylates, polymethacrylate,
acyl substituted cellulose acetates, non-degradable polyurethanes,
polystyrenes, polyvinyl chloride, polyvinyl flouride, polyvinyl
imidazole, chlorosulphonated polyolifins, polyethylene oxide,
polyvinyl alcohol, Teflon.RTM., nylon silicon, and shape memory
materials, such as poly(styrene-block-butadiene), polynorbornene,
hydrogels, metallic alloys, and oligo(.epsilon.-caprolactone)diol
as switching segmentloligo(p-dioxyanone)diol as physical crosslink.
Other suitable polymers can be obtained by reference to The Polymer
Handbook, 3rd edition (Wiley, N. Y., 1989).
[0140] Factors, including but not limited to nutrients, growth
factors, inducers of differentiation or de-differentiation,
products of secretion, immunomodulators, inhibitors of
inflammation, regression factors, hormones, or other biologically
active compounds can be incorporated into or can be provided in
conjunction with the polymer scaffold.
[0141] Agents of the invention may be supplied along with
additional reagents in a kit. The kits can include instructions for
the treatment regime or assay, reagents, equipment (test tubes,
reaction vessels, needles, syringes, etc.) and standards for
calibrating or conducting the treatment or assay. The instructions
provided in a kit according to the invention may be directed to
suitable operational parameters in the form of a label or a
separate insert. Optionally, the kit may further comprise a
standard or control information so that the test sample can be
compared with the control information standard to determine if
whether a consistent result is achieved.
IV. Spermatogenesis
[0142] Methods of the present invention can be used in the
production of other reproductive cell types. Accordingly, in one
embodiment, the present invention provides a method for restoring
or enhancing spermatogenesis, comprising providing a bone marrow
derived male germline stem cell, or its progenitor, to the testes
of a male subject, wherein the cell engrafts into the seminiferous
epithelium and differentiates into a sperm cell. Administration of
a bone marrow derived male germline stem cell, or its progenitor,
to the testes is preferably carried out by testicular injection.
Direct injection into the testes advantageously circumvents the
blood barrier, and provides cells to suitable locations, such as
the seminiferous epithelium.
[0143] Spermatogenesis can be further increased by contacting
compositions comprising bone marrow derived male germline stem
cells, or their progenitor cells, with an agent that differentiates
bone marrow male germline derived stem cells or their progenitor
cells into sperm cells (e.g., differentiation agents). Such
differentiation agents can be, but are not limited to, those
described herein.
[0144] Spermatogenesis, or the formation of spermatocytes from
spermatogonia, can be regulated by numerous factors. Regulators of
apoptosis, including Bax, Bcl.sub.XL family members, and caspase
family members, can modulate spermatogenesis and affect male
fertility (Said, T. M., et al. (2004) Hum. Reprod. Update 10:
39-51; Yan, W. et al, (2003) Mol. Endocrinol. 17: 1868). Caspases
have been implicated in the pathogenesis of multiple andrological
pathologies, such as, inter alia, impaired spermatogenesis,
decreased sperm motility, and increased levels of sperm DNA
fragmentation. Caspase inhibitors, such as survivin and FLIP, can
be used to regulate apoptotic events during spermatogenesis
(Weikert S., (2004) Int. J. Androl. 27: 161; Giampietri, C. et al,
(2003) Cell Death Differ. 10: 175). Similarly, Bax inhibitors such
as humanin, are also implicated in spermatogenic apoptosis (Guo, B.
et al., (2003) Nature 423: 456).
[0145] Growth factors, such as fibroblast growth factor-4 (Hirai,
K. et al, (2004) Exp. Cell Res. 294: 77) can also influence
spermatogenesis. FGF-4 can play a critical role as a survival
factor for germ cells by protecting them from apoptosis. Upon FGF-4
stimulation in Sertoli cells, lactate production was induced, which
is indispensable for germ cell survival. FGF-4 stimulation can also
reduce DNA fragmentation in Sertoli cells.
[0146] Bone morphogenetic protein (BMP) signaling pathways have
also been implicated in maintenance of germ line stem cells in
Drosophila (Kawase, E. et al, (2004) Development 131: 1365-75;
Pellegrini, M. et al, (2003) J. Cell Sci. 116: 3363). BMP4
stimulation of cultured spermatogonia can induce Smad-mediated
proliferation, as well as differentiation through the c-kit gene.
Additionally, BMP signals from somatic cells were shown to be
essential for maintaining germline stem cells through repression of
the bam expression, indicating that Bmp signals from the somatic
cells maintain germline stem cells at least in part, by repressing
bam expression in the testis.
[0147] Transforming growth factor (TGF) can also repress barn
expression in testis. Maintenance and proliferation of germ line
stem cells and their progeny depends upon the ability of these
cells to transduce the activity of a somatically expressed
TGF-.beta. ligand, known in Drosophila as the BMP5/8 ortholog Glass
Bottom Boat (Shivdasani, A. A. and Ingham, P. W. (2003) Curr. Biol.
13: 2065). TGF-3 signaling represses the expression of barn, which
is necessary and sufficient for germ cell differentiation, thereby
maintaining germ line stem cells and spermatogonia in their
proliferative state.
[0148] Sphingosine-1-phosphate (S1P) is also known to affect the
survival and proliferation of germ line stem cells and
spermatogonia. In a study where irradiated testicular tissue was
treated with S1P, the numbers of primary spermatocytes and
spermatogonia were higher than untreated tissues, indicating that
S1P treatment can protect germ line stem cells against cell death
induced by radiation (Otala, M. et al, (2004) Biol Reprod. March;
70(3):759-67).
[0149] Glial-derived neurotrophic factor was found to markedly
amplify germline stem cells in murine testis (Kubota, H. et al,
(2004) Biol. Reprod. 71(3):722-31). Transplantation analysis
demonstrated not only germline stem cells enrichment, but also
differentiation from stem cells into sperm (Yornogida, K. et al,
(2003) Biol. Reprod. 69: 1303).
[0150] The present invention also provides methods for reducing
bone marrow derived male germline stem cells, or their progenitor
cells, in vivo, ex vivo or in vitro, comprising contacting bone
marrow derived male germline stein cells or their progenitor cells
with an agent that reduces cell proliferation, inhibits cell
survival or promotes cell death. Unwanted proliferation of the
cells of the invention can give rise to cancerous and pre-cancerous
phenotypes (e.g., germ cell tumors, testicular cancer). Such
methods can be used to control unwanted proliferation (e.g.,
cancer) or for contraceptive measures by reducing the numbers of
germline stem cells, and optionally their progenitors or sperm
cells.
[0151] Agents that reduce cell proliferation include, but are not
limited to, a hormone or growth factor (e.g., TGF-.beta.), a
peptide antagonist of mitogenic hormones or growth factors (e.g.,
the BMP antagonists, PRDC and Gremlin), or a pharmacological or
pharmaceutical compound (e.g., a cell cycle inhibitor, or an
inhibitor of growth factor signaling).
[0152] Agents that inhibit cell survival include, but are not
limited to, a hormone, growth factor or cytokine (e.g., a
pro-apoptotic TNF super family member such as TNF-.alpha., FasL and
TRAIL), an antagonist of pro-survival Bcl-2 family member function,
a signaling molecule (e.g., a ceramide), or a pharmacological or
pharmaceutical compound (e.g., an inhibitor of growth factor
signaling).
[0153] Agents that promote cell death include, but are not limited
to, a pro-apoptotic tumor necrosis factor superfamily member (e.g.,
TNF-.alpha., FasL and TRAIL), agonist of pro-apoptotic Bcl-2 family
member function and ceramide.
IV. Screening Assays
[0154] The invention provides methods for identifying modulators,
i.e., candidate or test compounds or agents (e.g., proteins,
peptides, peptidomimetics, peptoids, small molecules or other
drugs) which modulate bone marrow derived germline stem cells or
the progenitors thereof. Agents thus identified can be used to
modulate, for example, proliferation, survival and differentiation
of a bone marrow derived germline stem cell or its progenitor e.g.,
in a therapeutic protocol.
[0155] The test agents of the present invention can be obtained
singly or using any of the numerous approaches in combinatorial
library methods known in the art, including: biological libraries;
peptide libraries (libraries of molecules having the
functionalities of peptides, but with a novel, non-peptide backbone
which are resistant to enzymatic degradation but which nevertheless
remain bioactive; see, e.g., Zuckermaun, R. N. (1994) et al., J.
Med. Chem. 37:2678-85): spatially addressable parallel solid phase
or solution phase libraries; synthetic library methods requiring
deconvolution; the `one-bead one-compound` library method; and
synthetic library methods using affinity chromatography selection.
The biological library and peptoid library approaches are limited
to peptide libraries, while the other four approaches are
applicable to peptide, non-peptide oligomer or small molecule
libraries of compounds (Lam (1997) Anticancer Drug Des.
12:145).
[0156] Examples of methods for the synthesis of molecular libraries
can be found in the art, for example in: DeWitt et al. (1993) Proc.
Natl. Acad. Sci. U.S.A. 90:6909; Erb et al. (1994) Proc. Natl.
Acad. Sci. USA 91:11422; Zuckermann et al. (1994) J. Med. Chem.
37:2678; Cho et al. (1993) Science 261:1303; Carrell et al. (1994)
Angew. Chem. Int. Ed. Engl. 33:2059; Carell et al (1994) Angew.
Chem. Int. Ed. Engl. 33:2061; and Gallop et al. (1994) J. Med.
Chem. 37:1233.
[0157] Libraries of compounds may be presented in solution (e.g.,
Houghten (1992), Biotechniques 13:412-421), or on beads (Lam
(1991), Nature 354:82-84), chips (Fodor (1993) Nature 364:555-556),
bacteria (Ladner, U.S. Pat. No. 5,223,409), spores (Ladner U.S.
Pat. No. 5,223,409), plasmids (Cull et al. (1992) Proc Natl Acad
Sci USA 89:1865-1869) or on phage (Scott and Smith (1990) Science
249:386-390; Devlin (1990) Science 249:404-406; Cwirla et al.
(1990) Proc. Natl. Acad. Sci. 87:6378-6382; Felici (1991) J. Mol.
Biol. 222:301-310; Ladner supra.).
[0158] Chemical compounds to be used as test agents (i.e.,
potential inhibitor, antagonist, agonist) can be obtained from
commercial sources or can be synthesized from readily available
starting materials using standard synthetic techniques and
methodologies known to those of ordinary skill in the art.
Synthetic chemistry transformations and protecting group
methodologies (protection and deprotection) useful in synthesizing
the compounds identified by the methods described herein are known
in the art and include, for example, those such as described in R.
Larock (1989) Comprehensive Organic Transformations, VCH
Publishers; T. W. Greene and P. G. M. Wuts, Protective Groups in
Organic Synthesis, 2nd ed., John Wiley and Sons (1991); L. Fieser
and M. Fieser, Fieser and Fieser's Reagents for Organic Synthesis,
John Wiley and Sons (1994); and L. Paquette, ed., Encyclopedia of
Reagents for Organic Synthesis, John Wiley and Sons (1995), and
subsequent editions thereof.
[0159] In one aspect the compounds are organic small molecules,
that is, compounds having molecular weight less than 1,000 amu,
alternatively between 350-750 amu. In other aspects, the compounds
are: (i) those that are non-peptidic; (ii) those having between 1
and 5, inclusive, heterocyclyl, or heteroaryl ring groups, which
may bear further substituents; (iii) those in their respective
pharmaceutically acceptable salt forms; or (iv) those that are
peptidic.
[0160] The term "heterocyclyl" refers to a nonaromatic 3-8 membered
monocyclic, 8-12 membered bicyclic, or 11-14 membered tricyclic
ring system having 1-3 heteroatoms if monocyclic, 1-6 heteroatoms
if bicyclic, or 1-9 heteroatoms if tricyclic, said heteroatoms
selected from O, N, or S (e.g., carbon atoms and 1-3, 1-6, or 1-9
heteroatoms of N, O, or S if monocyclic, bicyclic, or tricyclic,
respectively), wherein 0, 1, 2 or 3 atoms of each ring can be
substituted by a substituent.
[0161] The term "heteroaryl" refers to an aromatic 5-8 membered
monocyclic, 8-12 membered bicyclic, or 11-14 membered tricyclic
ring system having 1-3 heteroatoms if monocyclic, 1-6 heteroatoms
if bicyclic, or 1-9 heteroatoms if tricyclic, said heteroatoms
selected from O, N, or S (e.g., carbon atoms and 1-3, 1-6, or 1-9
heteroatoms of N, O, or S if monocyclic, bicyclic, or tricyclic,
respectively), wherein 0, 1, 2, 3, or 4 atoms of each ring can be
substituted by a substituent.
[0162] The term "substituents" refers to a group "substituted" on
an alkyl, cycloalkyl, aryl, heterocyclyl, or heteroaryl group at
any atom of that group. Suitable substituents include, without
limitation, alkyl, alkenyl, alkynyl, alkoxy, halo, hydroxy, cyano,
nitro, amino, SO.sub.3H, perfluoroalkyl, perfluoroalkoxy,
methylenedioxy, ethylenedioxy, carboxyl, oxo, thioxo, imino (alkyl,
aryl, aralkyl), S(O).sub.nalkyl (where n is 0-2), S(O).sub.n aryl
(where n is 0-2), S(O).sub.n heteroaryl (where n is 0-2),
S(O).sub.n heterocyclyl (where n is 0-2), amine (mono-, di-, alkyl,
cycloalkyl, aralkyl, heteroaralkyl, and combinations thereof),
ester (alkyl, aralkyl, heteroaralkyl), amide (mono-, di-, alkyl,
aralkyl, heteroaralkyl, and combinations thereof), sulfonamide
(mono-, di-, alkyl, aralkyl, heteroaralkyl, and combinations
thereof), unsubstituted aryl, unsubstituted heteroaryl,
unsubstituted heterocyclyl, and unsubstituted cycloalkyl. In one
aspect, the substituents on a group are independently any one
single, or any subset of the aforementioned substituents.
[0163] Combinations of substituents and variables in compounds
envisioned by this invention are only those that result in the
formation of stable compounds. The term "stable", as used herein,
refers to compounds which possess stability sufficient to allow
manufacture and which maintains the integrity of the compound for a
sufficient period of time to be useful for the purposes detailed
herein (e.g., transport, storage, assaying, therapeutic
administration to a subject).
[0164] The compounds described herein can contain one or more
asymmetric centers and thus occur as racemates and racemic
mixtures, single enantiomers, individual diastereomers and
diastereomeric mixtures. All such isomeric forms of these compounds
are expressly included in the present invention. The compounds
described herein can also be represented in multiple tautomeric
forms, all of which are included herein. The compounds can also
occur in cis- or trans- or E- or Z-double bond isomeric forms. All
such isomeric forms of such compounds are expressly included in the
present invention.
[0165] Test agents of the invention can also be peptides (e.g.,
growth factors, cytokines, receptor ligants).
[0166] Screening methods of the invention can involve the
identification of an agent that increases the proliferation or
survival of bone marrow derived germline stem cells or the
progenitors thereof. Such methods will typically involve contacting
bone marrow derived stem or progenitor cells with a test agent in
culture and quantitating the number of new bone marrow derived stem
or progenitor cells produced as a result. Comparison to an
untreated control can be concurrently assessed. Where an increase
in the number of stem or progenitor cells is detected relative to
the control, the test agent is determined to have the desired
activity.
[0167] In practicing the methods of the invention, it may be
desirable to employ a purified population of bone marrow derived
germline stem cells or the progenitors thereof. A purified
population of bone marrow derived germline stem cells or the
progenitors thereof can have about 50-55%, 55-60%, 60-65% and
65-70% purity. More preferably the purity is about 70-75%, 75-80%,
80-85%; and still more preferably the purity is about 85-90%,
90-95%, and 95-100%.
[0168] Increased amounts of bone marrow derived female germline
stem cells or the progenitors thereof can also be detected by an
increase in gene expression of genetic markers including an Oct-4,
Dazl, Stella Vasa, Fragilis, Nobox and c-Kit. The level of
expression can be measured in a number of ways, including, but not
limited to: measuring the mRNA encoded by the genetic markers;
measuring the amount of protein encoded by the genetic markers; or
measuring the activity of the protein encoded by the genetic
markers.
[0169] The level of mRNA corresponding to a genetic marker can be
determined both by in situ and by in vitro formats. The isolated
mRNA can be used in hybridization or amplification assays that
include, but are not limited to, Southern or Northern analyses,
polymerase chain reaction analyses and probe arrays. One diagnostic
method for the detection of mRNA levels involves contacting the
isolated mRNA with a nucleic acid molecule (probe) that can
hybridize to the mRNA encoded by the gene being detected. The
nucleic acid probe is sufficient to specifically hybridize under
stringent conditions to mRNA or genomic DNA. The probe can be
disposed on an address of an array, e.g., an array described below.
Other suitable probes for use in the diagnostic assays are
described herein.
[0170] In one format, mRNA (or cDNA) is immobilized on a surface
and contacted with the probes, for example by running the isolated
mRNA on an agarose gel and transferring the mRNA from the gel to a
membrane, such as nitrocellulose. In an alternative format, the
probes are immobilized on a surface and the mRNA (or cDNA) is
contacted with the probes, for example, in a two-dimensional gene
chip array described below. A skilled artisan can adapt known mRNA
detection methods for use in detecting the level of mRNA encoded by
the genetic markers described herein.
[0171] The level of mRNA in a sample can be evaluated with nucleic
acid amplification, e.g., by rtPCR (Mullis (1987) U.S. Pat. No.
4,683,202), ligase chain reaction (Barany (1991) Proc. Natl. Acad.
Sci. USA 88:189-193), self sustained sequence replication (Guatelli
et al. (1990) Proc. Natl. Acad. Sci. USA 87:1874-1878),
transcriptional amplification system (Kwoh et al. (1989) Proc.
Natl. Acad. Sci. USA 86:1173-1177), Q-Beta Replicase (Lizardi et
al. (1988) Bio/Technology 6:1197), rolling circle replication
(Lizardi et al., U.S. Pat. No. 5,854,033) or any other nucleic acid
amplification method, followed by the detection of the amplified
molecules using techniques known in the art. As used herein,
amplification primers are defined as being a pair of nucleic acid
molecules that can anneal to 5' or 3' regions of a gene (plus and
minus strands, respectively, or vice-versa) and contain a short
region in between. In general, amplification primers are from about
10 to 30 nucleotides in length and flank a region from about 50 to
200 nucleotides in length. Under appropriate conditions and with
appropriate reagents, such primers permit the amplification of a
nucleic acid molecule comprising the nucleotide sequence flanked by
the primers.
[0172] For in situ methods, a cell or tissue sample can be
prepared/processed and immobilized on a support, typically a glass
slide, and then contacted with a probe that can hybridize to mRNA
that encodes the genetic marker being analyzed.
[0173] Screening methods of the invention can involve the
identification of an agent that differentiates bone marrow derived
germline stem cells, or their progenitor cells, into oocytes or
sperm cells. Such methods will typically involve contacting the
bone marrow derived stem or progenitor cells with a test agent in
culture and quantitating the number of new oocytes or sperm cells
produced as a result. Comparison to an untreated control can be
concurrently assessed. Where an increase in the number of oocytes
is detected relative to the control, the test agent is determined
to have the desired activity. The test agent can also be assayed
using a biological sample (e.g., ovarian tissue); subsequent
testing using a population of stem or progenitor cells may be
conducted to distinguish the functional activity of the agent
(e.g., differentiation rather then increase in proliferation or
survival) where the result is ambiguous.
[0174] Increased amounts of oocytes be detected by a decrease in
gene expression of bone marrow derived female germline stem or
progenitor genetic markers including Oct-4, Dazl, Stella Vasa,
Fragilis, Nobox or c-Kit or an increase in oocyte markers, such as
HDAC6, GDF9 and ZP3.
VI. Methods of Treatment
[0175] Bone marrow derived germline stem cells of the invention or
their progenitors can be used in a variety of therapeutic
applications (e.g., sperm/oocyte generation for in vivo restoration
or ex vivo procedures including in vitro fertilization).
Accordingly, methods of the invention relate to, among other
things, the use of bone marrow derived germline stem cells, or
their progenitor cells, to provide germ cells in the treatment of
reproductive disorders.
[0176] Compositions comprising bone marrow derived germline stem
cells or their progenitor cells can be provided directly to the
reproductive organ of interest (e.g., ovary or testes).
Alternatively, compositions comprising bone marrow derived germline
stem cells or their progenitor cells can be provided indirectly to
the reproductive organ of interest, for example, by administration
into the circulatory system (e.g., to extra-ovarian
circulation).
[0177] Thus, the present invention provides methods for treating
infertility in a female subject comprising providing a bone marrow
derived female germline stem cell, or its progenitor, to a female
subject in need thereof, wherein the cell engrafts into a tissue
(preferably ovarian tissue) and differentiates into an oocyte,
which can later be provided for fertilization following ovulation
in the subject. Alternatively, the engrafted oocyte can be
harvested from the subject and provided for in vitro fertilization
or somatic cell nuclear transfer. Except where expressly stated
herein, the female subject in need of fertility treatment is not a
subject who has undergone chemotherapy or radiotherapy.
[0178] The present invention also provides methods for treating
infertility comprising administering an agent that increases the
amount of bone marrow derived female germline stem cells, or their
progenitor cells, by increasing the proliferation or survival of
the bone marrow derived female germline stem cells or their
progenitor cells, thereby enhancing oocyte production. Agents can
be provided directly to the reproductive organ of interest.
Alternatively, agents can be provided indirectly to the
reproductive organ of interest, for example, by administration into
the circulatory system.
[0179] The present invention also provides methods for repairing
damaged ovarian tissue, comprising providing a bone marrow derived
female germline stem cell, or its progenitor cell, to the ovarian
tissue, wherein the cell engrafts into the ovarian tissue and
differentiates into an oocyte. Except where expressly stated
herein, the ovarian tissue was not damaged by chemotherapy or
radiotherapy. Damage can be caused, for example, by exposure to
cytotoxic factors, hormone deprivation, growth factor deprivation,
cytokine deprivation, cell receptor antibodies, and the like.
[0180] Where damage may be caused by an anticipated course of
chemotherapy and/or radiotherapy, administration of an agent that
protects against reproductive injury prior to or concurrently with
chemotherapy and/or radiotherapy can protect fertility and enhance
the restoration methods described herein. The protective agent
includes, but is not limited to, S1P, Bax, or any agent that
increases SDF-1 activity (i.e., SDF-1 mediated migration and homing
of stem cells). For a description of the use of S1P in protecting
reproductive systems, see U.S. application Ser. No. 10/217,259,
filed on Aug. 12, 2002 and published as 20030157086 on Aug. 21,
2003, the contents of which are herein incorporated by
reference.
[0181] The present invention also provides methods for restoring
ovarian function in a menopausal female subject, comprising
providing a bone marrow derived female germline stem cell, or its
progenitor, to the subject, wherein the cell engrafts into the
ovary and differentiates into an oocyte. The menopausal female
subject can be in a stage of either peri- or post-menopause, with
said menopause caused by either normal (e.g., aging) or
pathological (e.g., surgery, disease, ovarian damage)
processes.
[0182] Ovarian function in a post-menopausal female can also be
restored by administering an agent that increases the amount of
bone marrow derived female germline stem cells or their progenitors
(e.g., by increasing the number or life span of bone marrow derived
female germline stem cells, as well as by increasing the
differentiation of bone marrow derived female germline stem cells
into oocytes).
[0183] Restoration of ovarian function can relieve adverse symptoms
and complications associated with menopause, including, but not
limited to, somatic disorders such as osteoporosis, cardiovascular
disease, somatic sexual dysfunction, hot flashes, vaginal drying,
sleep disorders, depression, irritability, loss of libido, hormone
imbalances, and the like, as well as cognitive disorders, such as
loss of memory; emotional disorders, depression, and the like.
[0184] Bone marrow derived germline stem cells of the invention,
their progenitors or their progeny, can be administered as
previously described, and obtained by all methods known in the art.
Bone marrow derived germline stem cells of the invention can be
autologous (obtained from the subject) or heterologous (e.g.,
obtained from a donor). Heterologous cells can be provided together
with immunosuppressive therapies known in the art to prevent immune
rejection of the cells.
[0185] Bone marrow derived germline stem cells of the present
invention can be isolated by bone marrow aspiration. For increased
yield from female donors, it may be desirable to coordinate
isolation with appropriate stages of the female reproductive cycle
that exhibit higher levels of female germline stem cells in the
bone marrow, as described in Example 1. U.S. Pat. No. 4,481,946,
incorporated herein expressly by reference, describes a bone marrow
aspiration method and apparatus, wherein efficient recovery of bone
marrow from a donor can be achieved by inserting a pair of
aspiration needles at the intended site of removal. Through
connection with a pair of syringes, the pressure can be regulated
to selectively remove bone marrow and sinusoidal blood through one
of the aspiration needles, while positively forcing an intravenous
solution through the other of the aspiration needles to replace the
bone marrow removed from the site. The bone marrow and sinusoidal
blood can be drawn into a chamber for mixing with another
intravenous solution and thereafter forced into a collection bag.
The heterogeneous cell population can be further purified by
identification of cell-surface markers to obtain the bone marrow
derived germline stem cell compositions for administration into the
reproductive organ of interest.
[0186] U.S. Pat. No. 4,486,188 describes methods of bone marrow
aspiration and an apparatus in which a series of lines are directed
from a chamber section to a source of intravenous solution, an
aspiration needle, a second source of intravenous solution and a
suitable separating or collection source. The chamber section is
capable of simultaneously applying negative pressure to the
solution lines leading from the intravenous solution sources in
order to prime the lines and to purge them of any air. The solution
lines are then closed and a positive pressure applied to redirect
the intravenous solution into the donor while negative pressure is
applied to withdraw the bone marrow material into a chamber for
admixture with the intravenous solution, following which a positive
pressure is applied to transfer the mixture of the intravenous
solution and bone marrow material into the separating or collection
source.
[0187] According to methods of the invention, bone marrow can be
harvested during the lifetime of the subject, but a pre-menopausal
harvest is recommended. Furthermore, harvest prior to illness
(e.g., cancer) is desirable, and harvest prior to treatment by
cytotoxic means (e.g., radiation or chemotherapy) will improve
yield and is therefore also desirable.
[0188] U.S. Pat. No. 5,806,529 describes a method for bone marrow
transplantation from an HLA-nonmatched donor to a patient which
comprises conditioning the patient under a suitable regimen
followed by transplant of a very large dose of stem cells (at least
about 3-fold greater than the conventional doses used in T
cell-depleted bone marrow transplantation). The patient is
conditioned under lethal or supralethal conditions for the
treatment of malignant or non-malignant diseases, or under
sublethal conditions for the treatment of non-malignant diseases.
The transplant may consist of T cell-depleted bone marrow stem
cells and T cell-depleted stem cell-enriched peripheral blood cells
from the HLA-nonmatched donor. Preferably a relative of the
patient, which donor was previously treated with a drug, e.g. a
cytokine such as granulocyte colony-stimulating factor (G-CSF).
[0189] Where radiation or chemotherapy is conducted prior to
administration, transplantation of bone marrow derived germline
stem cells of the invention, their progenitors or their progeny
should optimally be provided within about one month of the
cessation of therapy. However, transplantation at later points
after treatment has ceased can be done with derivable clinical
outcomes.
[0190] As described herein, germline stem cells have been detected
in the bone marrow. Therefore, bone marrow derived germline stem
cells, and their progenitor cells, that can be used in the methods
of the invention can comprise a purified sub-population of cells
including, but not limited to male and female germline stem
cells.
[0191] Purified bone marrow derived germline stem cells, and their
progenitor cells, can be obtained by standard methods known in the
art, including cell sorting by FACs. Isolated bone marrow can be
sorted using flow cytometers known in the art (e.g., a BD
Biosciences FACScalibur cytometer) based on cell surface expression
of Sca-1 (van de Rijn et al., (1989) Proc. Natl. Acad. Sci. USA 86,
4634-4638) and/or c-Kit (Okada et al., (1991) Blood 78, 1706-1712);
(Okada et al., (1992) Blood 80, 3044-3050) following an initial
immunomagnetic bead column-based fractionation step to obtain
lineage-depleted (lin.sup.-) cells (Spangrude et al., (1988)
Science 241, 58-62); (Spangrude and Scollay, (1990) Exp. Hematol.
18, 920-926), as described (Shen et al., (2001) J. Immunol. 166,
5027-5033); (Calvi et al., (2003) Nature 425, 841-846).
[0192] For serial passage-based enrichment of bone marrow derived
germline stem cells, and their progenitor cells, in-vitro
(Meirelles and Nardi, (2003) Br. J. Haematol. 123, 702-711);
(Tropel et al., (2004) Exp. Cell Res. 295, 395-406), isolated bone
marrow can be plated on plastic in Dulbecco's modified Eagle's
medium (Fisher Scientific, Pittsburgh, Pa.) with 10% fetal bovine
serum (Hyclone, Logan, Utah), penicillin, streptomycin, L-glutamine
and amphotericin-B. About forty-eight hours after the initial
plating, the supernatants containing non-adherent cells can be
removed and replaced with fresh culture medium after gentle
washing. The cultures can then be maintained and passed once
confluence is reached (e.g., for a total of about three times over
the span of about 6 weeks) at which time the cultures can be
terminated to collect adherent cells for analysis.
[0193] Prior to administration, bone marrow derived germline stem
cells, their progenitors or their progeny, described herein can
optionally be genetically modified, in vitro, in vivo or ex vivo,
by introducing heterologous DNA or RNA or protein into the cell by
a variety of recombinant methods known to those of skill in the
art. These methods are generally grouped into four major
categories: (1) viral transfer, including the use of DNA or RNA
viral vectors, such as retroviruses (including lentiviruses),
Simian virus 40 (SV40), adenovirus, Sindbis virus, and bovine
papillomavirus, for example; (2) chemical transfer, including
calcium phosphate transfection and DEAE dextran transfection
methods; (3) membrane fusion transfer, using DNA-loaded membranous
vesicles such as liposomes, red blood cell ghosts, and protoplasts,
for example; and (4) physical transfer techniques, such as
microinjection, electroporation, or direct "naked" DNA
transfer.
[0194] The bone marrow derived germline stem cells of the
invention, their progenitors or their progeny, can be genetically
altered by insertion of pre-selected isolated DNA, by substitution
of a segment of the cellular genome with pre-selected isolated DNA,
or by deletion of or inactivation of at least a portion of the
cellular genome of the cell. Deletion or inactivation of at least a
portion of the cellular genome can be accomplished by a variety of
means, including but not limited to genetic recombination, by
antisense technology (which can include the use of peptide nucleic
acids, or PNAs), or by ribozyme technology, for example. The
altered genome may contain the genetic sequence of a selectable or
screenable marker gene that is expressed so that the cell with
altered genome, or its progeny, can be differentiated from cells
having an unaltered genome. For example, the marker may be a green,
red, yellow fluorescent protein, Pi-galactosidase, the neomycin
resistance gene, dihydrofolate reductase (DHFR), or hygromycin, but
are not limited to these examples.
[0195] In some cases, the underlying defect of a pathological state
is a mutation in DNA encoding a protein such as a metabolic
protein. Preferably, the polypeptide encoded by the heterologous
DNA lacks a mutation associated with a pathological state. In other
cases, a pathological state is associated with a decrease in
expression of a protein. A genetically altered bone marrow derived
germline stem cell, or its progeny, may contain DNA encoding such a
protein under the control of a promoter that directs strong
expression of the recombinant protein. Alternatively, the cell may
express a gene that can be regulated by an inducible promoter or
other control mechanism where conditions necessitate highly
controlled regulation or timing of the expression of a protein,
enzyme, or other cell product. Such stem cells, when transplanted
into a subject suffering from abnormally low expression of the
protein, produce high levels of the protein to confer a therapeutic
benefit. For example, the bone marrow derived germline stem cell of
the invention, its progenitor or its progeny, can contain
heterologous DNA encoding genes to be expressed, for example, in
gene therapy. Bone marrow derived germline stem cells of the
invention, their progenitors or their progeny, can contain
heterologous DNA encoding Atm, the gene responsible for the human
disease Ataxia-telangiectasia in which fertility is disrupted.
Providing Atm via bone marrow derived female germline stem cells,
their progenitors or their progeny, can further relieve defects in
ovarian function. DNA encoding a gene product that alters the
functional properties of bone marrow derived germline stem cells in
the absence of any disease state is also envisioned. For example,
delivery of a gene that inhibits apoptosis, or that prevents
differentiation would be beneficial.
[0196] Insertion of one or more pre-selected DNA sequences can be
accomplished by homologous recombination or by viral integration
into the host cell genome. The desired gene sequence can also be
incorporated into the cell, particularly into its nucleus, using a
plasmid expression vector and a nuclear localization sequence.
Methods for directing polynucleotides to the nucleus have been
described in the art. The genetic material can be introduced using
promoters that will allow for the gene of interest to be positively
or negatively induced using certain chemicals/drugs, to be
eliminated following administration of a given drug/chemical, or
can be tagged to allow induction by chemicals (including but not
limited to the tamoxifen responsive mutated estrogen receptor)
expression in specific cell compartments (including but not limited
to the cell membrane).
[0197] Calcium phosphate transfection can be used to introduce
plasmid DNA containing a target gene or polynucleotide into
isolated or cultured bone marrow derived germline stem cells or
their progenitors and is a standard method of DNA transfer to those
of skill in the art. DEAE-dextran transfection, which is also known
to those of skill in the art, may be preferred over calcium
phosphate transfection where transient transfection is desired, as
it is often more efficient. Since the cells of the present
invention are isolated cells, microinjection can be particularly
effective for transferring genetic material into the cells. This
method is advantageous because it provides delivery of the desired
genetic material directly to the nucleus, avoiding both cytoplasmic
and lysosomal degradation of the injected polynucleotide. This
technique has been used effectively to accomplish bone marrow
derived modification in transgenic animals. Cells of the present
invention can also be genetically modified using
electroporation.
[0198] Liposomal delivery of DNA or RNA to genetically modify the
cells can be performed using cationic liposomes, which form a
stable complex with the polynucleotide. For stabilization of the
liposome complex, dioleoyl phosphatidylethanolamine (DOPE) or
dioleoyl phosphatidylcholine (DOPQ) can be added. Commercially
available reagents for liposomal transfer include Lipofectin (Life
Technologies). Lipofectin, for example, is a mixture of the
cationic lipid N-[1-(2,3-dioleyloxy)propyl]-N--N--N-trimethyl
ammonia chloride and DOPE. Liposomes can carry larger pieces of
DNA, can generally protect the polynucleotide from degradation, and
can be targeted to specific cells or tissues. Cationic
lipid-mediated gene transfer efficiency can be enhanced by
incorporating purified viral or cellular envelope components, such
as the purified G glycoprotein of the vesicular stomatitis virus
envelope (VSV-G). Gene transfer techniques which have been shown
effective for delivery of DNA into primary and established
mammalian cell lines using lipopolyamine-coated DNA can be used to
introduce target DNA into the bone marrow derived germline stem
cells described herein.
[0199] Naked plasmid DNA can be injected directly into a tissue
mass formed of differentiated cells from the isolated bone marrow
derived germline stem cells or their progenitors. This technique
has been shown to be effective in transferring plasmid DNA to
skeletal muscle tissue, where expression in mouse skeletal muscle
has been observed for more than 19 months following a single
intramuscular injection. More rapidly dividing cells take up naked
plasmid DNA more efficiently. Therefore, it is advantageous to
stimulate cell division prior to treatment with plasmid DNA.
Microprojectile gene transfer can also be used to transfer genes
into stem cells either in vitro or in vivo. The basic procedure for
microprojectile gene transfer was described by J. Wolff in Gene
Therapeutics (1994), page 195. Similarly, microparticle injection
techniques have been described previously, and methods are known to
those of skill in the art. Signal peptides can be also attached to
plasmid DNA to direct the DNA to the nucleus for more efficient
expression.
[0200] Viral vectors are used to genetically alter bone marrow
derived germline stem cells of the present invention and their
progeny. Viral vectors are used, as are the physical methods
previously described, to deliver one or more target genes,
polynucleotides, antisense molecules, or ribozyme sequences, for
example, into the cells. Viral vectors and methods for using them
to deliver DNA to cells are well known to those of skill in the
art. Examples of viral vectors that can be used to genetically
alter the cells of the present invention include, but are not
limited to, adenoviral vectors, adeno-associated viral vectors,
retroviral vectors (including lentiviral vectors), alphaviral
vectors (e. g., Sindbis vectors), and herpes virus vectors.
[0201] Peptide or protein transfection is another method that can
be used to genetically alter bone marrow derived germline stem
cells of the invention and their progeny. Peptides including, but
not limited to, Pep-1 (commercially available as Chariot.TM.) and
MPG, can quickly and efficiently transport biologically active
proteins, peptides, antibodies, and nucleic acids directly into
cells, with an efficiency of about 60% to about 95% (Morris, M. C.
et al, (2001) Nat. Biotech. 19: 1173-1176). Without wishing to be
bound by theory, the peptide forms a non-covalent bond with the
macromolecule of interest (i.e., protein, nucleic acid). The
binding reaction stabilizes the protein and protects it from
degradation. Upon delivery into the cell of interest, such as stem
cells of the invention, the peptide-macromolecule complex
dissociates, leaving the macromolecule biologically active and free
to proceed to its target organelle. Delivery can occur in the
presence of absence of serum. Uptake and delivery can occur at
4.degree. C., which eliminates endosomal processing of incoming
macromolecules. Movement of macromolecules through the endosomal
pathway can modify the macromolecule upon uptake. Peptides such as
Pep-1, by directly delivering a protein, antibody, or peptide of
interest, bypass the transcription-translation process.
[0202] Methods of the invention can provide oocyte reserves for use
in ex vivo procedures, such as somatic cell nuclear transfer.
Employing recombinant techniques prior to nuclear transfer will
allow for the design of customized oocytes and ultimately produce
embryos from which embryonic stem cells can be derived. In
addition, genetic manipulation of donor DNA prior to nuclear
transfer will result in embryos that possess the desired
modification or genetic trait.
[0203] Methods of somatic cell nuclear transfer are well known in
the art. See U.S. application Ser. No. 10/494,074, filed on Mar.
24, 2004 and published as 20050064586; Wilmut et al. (1997) Nature,
385, 810-813; Wakayama, et al. (1998) Nature 394: 369-374; and
Teruhiko et al., (1999) PNAS 96:14984-14989. Nuclear
transplantation involves the transplantation of donor cells or cell
nuclei into enucleated oocytes. Enucleation of the oocyte can be
performed in a number of manners well known to those of ordinary
skill in the art. Insertion of the donor cell or nucleus into the
enucleated oocyte to form a reconstituted cell is usually by
microinjection of a donor cell under the zona pellucida prior to
fusion. Fusion may be induced by application of a DC electrical
pulse across the contact/fusion plane (electrofusion), by exposure
of the cells to fusion-promoting chemicals, such as polyethylene
glycol, or by way of an inactivated virus, such as the Sendai
virus. A reconstituted cell is typically activated by electrical
and/or non-electrical means before, during, and/or after fusion of
the nuclear donor and recipient oocyte. Activation methods include
electric pulses, chemically induced shock, penetration by sperm,
increasing levels of divalent cations in the oocyte, and reducing
phosphorylation of cellular proteins (as by way of kinase
inhibitors) in the oocyte. The activated reconstituted cells, or
embryos, are typically cultured in medium well known to those of
ordinary skill in the art and then transferred to the womb of an
animal.
[0204] Methods for the generation of embryonic stem cells from
embryos are also well known in the art. See Evans, et al. (1981)
Nature, 29:154-156; Martin, et al. (1981) PNAS, 78:7634-7638;
Smith, et al. (1987) Development Biology, 121:1-9; Notarianni, et
al. (1991) J. Reprod. Fert., Suppl. 43:255-260; Chen R L, et al.
(1997) Biology of Reproduction, 57 (4):756-764; Wianny, et al.
(1999) Theriogenology, 52 (2):195-212; Stekelenburg-Hamers, et al.
(1995) Mol. Reprod. 40:444-454; Thomson, et al. (1995) PNAS, 92
(17):7844-8 and Thomson (1998) Science, 282 (6):1145-1147.
Accordingly, embryos produced from oocytes of the invention can be
genetically modified, either through manipulation of the oocyte in
vitro prior to fertilization or manipulation of donor DNA prior to
nuclear transfer into the enucleated oocyte, to produce embryos
having a desired genetic trait.
VII. In Vitro Fertilization
[0205] Oocytes produced from bone marrow derived female germline
stem cells of the invention, or their progenitor cells, as
described herein can also be used for methods of in vitro
fertilization. Accordingly, the invention provides methods for in
vitro fertilization of a female subject, comprising the steps of:
[0206] a) producing an oocyte by culturing a bone marrow derived
female germline stem cell, or its progenitor, in the presence of an
oocyte differentiation agent; [0207] b) fertilizing the oocyte in
vitro to form a zygote; and [0208] c) implanting the zygote into
the uterus of a female subject.
[0209] Methods of in vitro fertilization are well known in the art,
and are now rapidly becoming commonplace. Couples are generally
first evaluated to diagnose their particular infertility
problem(s). These may range from unexplained infertility of both
partners to severe problems of the female (e.g., endometriosis
resulting in nonpatent oviducts with irregular menstrual cycles or
polycystic ovarian disease) or the male (e.g., low sperm count with
morphological abnormalities, or an inability to ejaculate normally
as with spinal cord lesions, retrograde ejaculation, or reversed
vasectomy). The results of these evaluations also determine the
specific procedure to be performed for each couple.
[0210] Procedures often begin with the administration of a drug to
down-regulate the hypothalamic/pituitary system (LHRH agonist).
This process decreases serum concentrations of the gonadotropins,
and developing ovarian follicles degenerate, thereby providing a
set of new follicles at earlier stages of development. This permits
more precise control of the maturation of these new follicles by
administration of exogenous gonadotropins in the absence of
influences by the hypothalamic pituitary axis. The progress of
maturation and the number of growing follicles (usually four to ten
stimulated per ovary) are monitored by daily observations using
ultrasound and serum estradiol determinations. When the follicles
attain preovulatory size (18-21 mm) and estradiol concentrations
continue to rise linearly, the ovulatory response is initiated by
exogenous administration of human chorionic gonadotropins
(hCG).
[0211] Oocytes can be obtained from bone marrow derived female
germline stem cells, or their progenitor cells, as previously
described herein. Bone marrow derived female germline stem cells,
or the progenitor cells, can be cultured in the presence of an
oocyte differentiation agent which induces differentiation into
oocytes. The differentiation agent can be supplied exogenously
(e.g., added to the culture medium) or from endogenous sources
during co-culture with allogenic or heterogenic ovarian tissue.
Bone marrow derived female germline stem cells, or their
progenitors, can also be cultured in a tissue-engineered structure
wherein the differentiation agent is either exogenously or
endogenously supplied and oocytes are obtained.
[0212] Individual oocytes can be evaluated morphologically and
transferred to a petri dish containing culture media and
heat-inactivated serum. A semen sample is provided by the male
partner and processed using a "swim up" procedure, whereby the most
active, motile sperm will be obtained for insemination. If the
female's oviducts are present, a procedure called GIFT (gamete
intrafallopian transfer) can be performed at this time. By this
approach, oocyte-cumulus complexes surrounded by sperm are placed
directly into the oviducts by laproscopy. This procedure best
simulates the normal sequences of events and permits fertilization
to occur within the oviducts. Not surprisingly, GIFT has the
highest success rate with 22% of the 3,750 patients undergoing ova
retrieval in 1990 having a live delivery. An alternative procedure
ZIFT (zygote intrafallopian transfer) permits the selection of in
vitro fertilized zygotes to be transferred to oviducts the day
following ova retrieval. Extra zygotes can be cryopreserved at this
time for future transfer or for donation to couples without female
gametes. Most patients having more serious infertility problems,
however, will require an additional one to two days incubation in
0.5 culture so that preembryos in the early cleavage states can be
selected for transfer to the uterus. This IVF-UT (in vitro
fertilization uterine transfer) procedure entails the transcervical
transfer of several 2-6 cell (day 2) or 8-16 (day 3) preembryos to
the fundus of the uterus (4-5 preembryos provides optimal
success).
[0213] Procedures for in vitro fertilization are also described in
U.S. Pat. Nos. 6,610,543 6,585,982, 6,544,166, 6,352,997,
6,281,013, 6,196,965, 6,130,086, 6,110,741, 6,040,340, 6,011,015,
6,010,448, 5,961,444, 5,882,928, 5,827,174, 5,760,024, 5,744,366,
5,635,366, 5,691,194, 5,627,066, 5,563,059, 5,541,081, 5,538,948,
5,532,155, 5,512,476, 5,360,389, 5,296,375, 5,160,312, 5,147,315,
5,084,004, 4,902,286, 4,865,589, 4,846,785, 4,845,077, 4,832,681,
4,790,814, 4,725,579, 4,701,161, 4,654,025, 4,642,094, 4,589,402,
4,339,434, 4,326,505, 4,193,392, 4,062,942, and 3,854,470, the
contents of which are specifically incorporated by reference for
their description of these procedures.
[0214] The following examples are put forth for illustrative
purposes only and are not intended to limit the scope of what the
inventors regard as their invention.
EXAMPLES
Example 1
Extra-Ovarian Female Germline Progenitor Cell Reservoirs
[0215] The restricted pattern of SSEA1 expression in the adult
mouse ovary (FIG. 1a, b) suggested that the number of germline stem
cells/progenitors thereof is relatively small. However, this would
be incongruous with recent studies indicating that germline stem
cells must offset an extremely robust rate of oocyte death for the
gonads to remain functional throughout reproductive life (Johnson,
J. et al (2004) Nature 428, 145-150) as well as with the ability of
adult mouse ovaries to rapidly generate hundreds of new primordial
oocyte-containing follicles. For details, see U.S. application Ser.
No. ______, filed on May 17, 2005 as Attorney Docket No.
51588-62054, the contents of which are herein incorporated by
reference. Accordingly, the possibility that a larger germline stem
cell reservoir exists somewhere outside of the ovaries was
considered. The first clue in this regard was provided by the
location of SSEA1.sup.+ cells in the medullary region of the ovary,
which is the principal entry and exit point for major blood vessels
that supply the female gonads. SSEA1.sup.+ cells may represent
germline stem cells/progenitors thereof en-route to, rather than
resident in, the ovary.
[0216] During embryogenesis, primordial germ cells (PGCs) and
hematopoietic stem cells (TSCs) are known to originate from the
same region--the proximail epiblast (Lawson and Hage (1994), Ciba
Found. Symp. 182, 68-84, 84-91. Early HSCs then colonize the
aorta-gonad-mesonepric region of the developing embryo prior to
migration into the fetal liver (Medvinsky and Dzierzak, (1996) Cell
86, 897-906), at roughly the equivalent time that PGCs enter the
same region of the embryo to colonize the fetal gonads (McLaren,
(2003) Dev. Bio. 262, 1-15); Molyneaux and Wylie, (2004) Int. J.
Dev. Biol. 48, 537-544). In postnatal life, the hematopoietic
system is maintained by stem cells that eventually home to and
reside in the bone marrow (Morrison et al., (1995) Annu. Rev. Cell
Dev. Biol. 11, 35-71); Attar and Scadden, (2004) Leukemia 18,
1760-1768). This information, along with the reported ability of
PGCs to generate primitive HSCs in-vitro (Rich, (1995) Blood 86,
463-472) and the increasing number of studies demonstrating the
multi-lineage potential of adult bone marrow-derived cells (Herzog
et al., (2003) Blood 102, 3483-3493); (Grove et al., (2004) Stem
Cells 22, 487-500); (Heike and Nakahata, (2004) Int. J. Hematol.
79, 7-14), prompted an investigation as to whether a molecular
signature consistent with the presence of germ cells could be
identified in adult female bone marrow.
[0217] For PCR analysis, total RNA was extracted from each sample
of bone marrow isolated from adult female mice and 1 .mu.g was
reverse transcribed (Superscript II RT; Invitrogen) using oligo-dT
primers. Amplification via 28-35 cycles of PCR was then performed
using Taq polymerase and Buffer-D (Epicentre) with primer sets
specific for each gene. For each sample, RNA encoded by the
ribosomal gene L7 (mouse studies) was amplified and used as a
loading control (`house-keeping` gene). All PCR products were
isolated, subcloned and sequenced for confirmation.
[0218] For immunohistochemical detection, ovaries, testes and bones
(femurs) were fixed in 4% neutral-buffered paraformaldehyde, and
bones were then decalcified for 72 hr in formic acid-EDTA. The
tissues were subsequently sectioned for immunohistochemical
analysis using antibodies specific for MVH (T. Noce; Fujiwara et
al., (1994) Cell Struct. Funct. 26, 131-136), HDAC6 (2162; Cell
Signaling Technology, Beverly, Mass.), NOBOX (A. Rajkovic; Suzumori
et al., (2002) Science 305, 1157-1159), or GDF-9 (AF739; R&D
Systems, Minneapolis, Minn.) after high temperature antigen
unmasking, as recommended by each supplier. The sections were
mounted with propidium iodide (Vectashield; Vector Laboratories,
Burlingame, Calif.) or TO-PRO-3 iodide (Molecular Probes, Eugene,
Oreg.) to visualize nuclei, and images were captured using a Zeiss
LSM 5 Pascal Confocal Microscope.
[0219] These experiments confirmed expression of Oct4, which in
adult mice is restricted to the germ lineage (Scholer et al.,
(1989) EMBO J. 8, 2543-2550); (Yoshimizu et al., (1999) Dev. Growth
Differ. 41, 675-684), as well as Mvh, Dazl, Stella and a fifth
germline marker gene termed Fragilis (Saitou et al., (2002) Nature
418, 293-300), in bone marrow isolated from adult female mice
(FIGS. 2A-2D). In addition, expression of the female germ
cell-specific homeobox gene, Nobox (Suzumori et al., (2002) Mech.
Dev. 111, 137-141), which is critical for directing expression of
Oct4 and Gdf9 in primordial oocytes as well as for folliculogenesis
(Rajkovic et al., 2004) Science 305, 1157-1159), was also detected
in bone marrow of adult females (FIG. 2A).
[0220] In light of these results, several public microarray
databases were searched to provide independent confirmation of the
findings that multiple germline markers are expressed in mouse and
human bone marrow. For example, expression of Mvh, Dazl, Stella and
Fragilis have been identified in mouse bone marrow (Benson et al.,
(2004) Nucleic Acids Res. 32 Database Issue, D23-D26); (Su et al.,
(2004) Proc. Natl. Acad. Sci. USA 101, 6062-6067), and expression
of STELLA has been demonstrated in human bone marrow (GenBank
Accession CV414052 from Dias Neto et al., 2000). Given the large
number of studies documenting the germline-restricted nature of
Vasa gene expression throughout the animal kingdom (Roussell and
Bennett, (1993) Proc. Natl. Acad. Sci. USA 90, 9300-9304);
(Fujiwara et al., (1994) Proc. Natl. Acad. Sci. USA 91,
12258-12262); (Komiya et al., (1994) Dev. Biol. 162, 354-363);
(Rongo et al., (1997) Cold Spring Harb. Symp. Quant. Biol. 62,
1-11); (Ikenishi,) (1998) Growth Differ. 40, 1-10); (Braat et al.,
(1999) RNA. Dev. Dyn. 216, 153-167); (Castrillon et al., (2000)
Proc. Natl. Acad. Sci. USA 97, 9585-9590); (Noce et al., (2001)
Cell Struct. Funct. 26, 131-136); (Dearden et al., (2003) Dev.
Genes Evol. 212, 599-603); (Fabioux et al., (2004) Biochem.
Biophys. Res. Commun. 320, 592-598) Mvh was selected as a
representative endpoint to next quantitatively assess potential
changes in the levels of germline marker expression in bone marrow
during the female reproductive cycle. Using real-time PCR with
standardization against the levels of .beta.-actin mRNA in each
sample, marked estrous cycle-related changes in Mvh expression in
bone marrow of adult female mice were uncovered, with a 9.52-fold
difference noted between estrus (nadir) and metestrus (peak) (FIG.
2E). A parallel evaluation of ovarian germ cell dynamics in the
same animals revealed a striking positive correlation between the
estrous cycle-related changes in bone marrow Mvh expression and
primordial follicle numbers, with ovaries at metestrus containing
over 800 more primordial follicles than ovaries at estrus (FIG.
2F). In light of these findings, the levels of Mvh in bone marrow
of adult females in metestrus were compared to levels present in
adult ovaries, which contain thousands of Mvh-expressing oocytes
(Fujiwara et al., 1994; Noce et al., 2001; see also FIG. 2D), or in
bone marrow of adult male mice. These experiments demonstrated that
Mvh transcript levels in bone marrow of adult females at metestrus
were 1.6% of those detected in adult ovaries (Table 1).
TABLE-US-00001 TABLE 1 Quantitative analysis of Mvh expression in
adult mice. Tissue Analyzed Fold Difference in Mvh Levels Female
Bone Marrow - Estrus (1.0) Female Bone Marrow - Metestrus 9.52
Ovary 598.29 Male Bone Marrow 1.72
Levels of Mvh expression in bone marrow of adult female mice at
estrus were used as a reference point for comparisons, and all data
were normalized against the levels of f-actin mRNA in each sample
prior to analysis.
[0221] Interestingly, Mvh expression was also detected in bone
marrow of adult male mice, with a level of expression slightly less
than 20% of that detected in bone marrow of adult females at
metestrus (Table 1). In addition, male bone marrow was also
positive for Dazl expression, whereas Stella expression was below
detectable limits.
[0222] Using established bone marrow fractionation protocols, bone
marrow samples were sorted based on cell surface stem cell markers
and quantitatively analyzed the resultant cell fractions for Mvh
levels. Briefly, bone marrow was isolated from adult female mice
and sorted using a BD Biosciences FACScalibur cytometer based on
cell surface expression of Sca-1 (van de Rijn et al., (1989) Proc.
Natl. Acad. Sci. USA 86, 4634-4638) and/or c-Kit (Okada et al.,
(1991) Blood 78, 1706-1712); (Okada et al., (1992) Blood 80,
3044-3050) following an initial immunomagnetic bead column-based
fractionation step to obtain lineage-depleted (lin.sup.-) cells
(Spangrude et al., (1988) Science 241, 58-62); (Spangrude and
Scollay, (1990) Exp. Hematol. 18, 920-926), as described (Shen et
al., (2001) J. Immunol. 166, 5027-5033); (Calvi et al., (2003)
Nature 425, 84.1-846). For serial passage-based enrichment of bone
marrow-derived stein cells in-vitro (Meirelles and Nardi, (2003)
Br. J. Haematol. 123, 702-711); (Tropel et al., (2004) Exp. Cell
Res. 295, 395-406), bone marrow isolated from adult female mice was
plated on plastic in Dulbecco's modified Eagle's medium (Fisher
Scientific, Pittsburgh, Pa.) with 10% fetal bovine serum (Hyclone,
Logan, Utah), penicillin, streptomycin, L-glutamine and
amphotericin-B. Forty-eight hr after the initial plating, the
supernatants containing non-adherent cells were removed and
replaced with fresh culture medium after gentle washing. The
cultures were then maintained and passed once confluence was
reached for a total of three times over the span of 6 weeks, at
which time the cultures were terminated to collect adherent cells
for analysis.
[0223] After removal of differentiated cells committed to
hematolymphoid lineages by negative selection, Mvh expression was
retained in the lineage-depleted (lin.sup.-) cell fraction with
levels comparable to those observed in crude bone marrow (FIG. 3A).
Subsequent separation of the lin.sup.- cells based on cell surface
expression of Sca-1 (van de Rijn et al., 1989) or c-Kit (Okada et
al., 1991) further revealed that the majority of Mvh-expressing
cells were negative for expression of Sca-I but positive for c-Kit
(Sca-1.sup.-/c-Kit.sup.+) (FIG. 3A). Moreover, expression of the
other germline markers co-segregated with Mvh in the
Sca-1.sup.-/c-Kit.sup.+ cell fraction. In parallel experiments,
in-vitro culture of adult female bone marrow-derived cells on
plastic under conditions shown previously to permit the progressive
enrichment of stem cells from bone marrow (Meirelles and Nardi,
(2003) Br. J. Haematol. 123, 702-711); (Tropel et al., (2004) Exp.
Cell Res. 295, 395-406), demonstrated that all of the germline
markers present in freshly isolated bone marrow samples were
expressed by the adherent cell fraction and remained so following
multiple serial passages over a 6-week period (FIG. 3B).
Example 2
Bone Marrow Transplantation Reverses Pathological Ovarian
Failure
[0224] To assess the functional capacity of bone marrow-derived
germ cells to produce new oocytes, bone marrow was isolated from
adult wild-type female mice and transplanted using standard
procedures into recipient adult females sterilized by treatment
with a combination of cyclophosphamide and busulphan to destroy the
existing pre- and post-meiotic germ cell pools prior to BMT.
[0225] Bone marrow was harvested from adult (6-10 weeks of age)
wild-type C57BL/6 female mice on the day of transplantation, and
2-5.times.10.sup.7 cells were injected intravenously via the tail
vein into recipients using standard procedures. To prepare
recipients, female mice received 0.5 mg anti-CD4 antibody (GK1.5)
(Dialynas, D. P. et al. (1984) J. Immunol. 131, 2445-2451) and 1 mg
anti-CD8 antibody (2.43) (Sarmiento, M. et al. (1980). Immunol.
125, 2665-2672) one week prior to a second injection of each
antibody along with 120 mg kg.sup.-1 cyclophosphamide (Cytoxan;
Bristol-Meyers Squibb) and 12 mg kg.sup.-1 busulphan (Sigma) at 6
weeks of age. Mice were conditioned before BMT with
cyclophosphamide and busulphan, the latter of which selectively
removes the contribution of germline stem cells to adult gonadal
function in both male and female mice. Bone marrow transplantation
was performed 1 or 7 days later. Animals were then euthanized for
collection and analysis of ovaries at the indicated times following
BMT.
[0226] Two months later, very few, if any, immature oocytes or
follicles were detected in the ovaries of those females given
cyclophosphamide and busulphan alone (FIG. 4). However, ovaries of
mice receiving BMT after combination chemotherapy possessed
hundreds of oocyte-containing follicles at all stages of
development, including the resting primordial stage that is most
susceptible to the cytotoxic actions of these drugs (FIGS. 4 and
5C).
[0227] Histological evaluations further substantiated that the
chemotherapy regimen essentially destroyed the ovaries--which,
after treatment, were composed of little more than stromal and
interstitial cells with a random cystic follicle or old corpus
luteum occasionally observed (FIG. 5B). By comparison, ovaries of
mice receiving BMT, even when the transplants were given a week
after inflicting the damage to the tissue, possessed a spectrum of
maturing follicles as well as corpora lutea indicative of a
resumption of normal ovulatory cycles (FIG. 5C). Furthermore,
oocytes and follicles were found in ovaries of
chemotherapy-sterilized females more than 11 months after the
initial transplantation (FIGS. 5D-SE), indicating that bone
marrow-derived germ cells are capable of sustaining long-term
oocyte production.
Example 3
Bone Marrow Transplantation Rescues Oocyte Production in Atm
Mutants
[0228] Atm.sup.-/- (homozygous null) mice, created by targeted
inactivation of the Atm gene, display many of the hallmarks of the
Ataxia-telangiectasia syndrome in humans, including growth
retardation, defects in T lymphocyte maturation and infertility
(Bagley et al. (2004) Blood 12: 1). Atm-deficient male and female
mice have been shown to be infertile due to the complete loss of
the production of mature gametes, i.e., spermatozoa and oocytes
(Barlow, C. et al. (1996) Cell 86: 159). These gametogenesis
defects in mutant mice lacking Atm result from apoptosis and
degeneration of the developing gametes that exhibit aberrant early
stages of meiosis, detected as early as the leptotene stage
(Barlow, C. et al. (1998) Development 125: 4007). Ovaries from
Atm-deficient females were shown to be completely barren of oocytes
and follicles by at least 11 days of age postpartum (Barlow, C. et
al. (1998) Development 125: 4007).
[0229] To first confirm and extend these findings, the ovaries of
wild-type mice were compared with ovaries from Atm gene-deficient
mice. Representative histology of postpartum day 4 wild-type (FIG.
6A, magnified in C) and Atm-null (B, D) ovaries is shown in FIG. 6.
Representative histology of adult wild-type (E) and Atm-null (F)
ovaries from adult mice is also shown in FIG. 6. In keeping with
past reports, ovaries from Atm-null animals, irrespective of
postnatal age, are barren of oocytes. However, in light of the
recent detection of pre-meiotic germline stem cells in the
postnatal mouse ovary (Johnson et al., (2004) Nature 428: 145), it
was possible that pre-meiotic germline stem cells were present and
capable of self-renewal, but ongoing oocyte production was
impossible due to failed meiotic entry in the absence of Atm.
[0230] The expression of germline lineage markers in the
Atm-deficient ovary versus wild-type controls was performed by
reverse-transcription followed by PCR (RT-PCR) and representative
data (n=3) are shown in FIG. 7. As predicted, the pluripotency
marker Oct-4 (Brehm et al., (1998) APMIS 106: 114) and the germline
markers Dazl (McNeilly et al., (2000) Endocrinology 141:4284);
(Nishi et al., (1999) Mol Hum Reprod 5: 495); Stella (Bortvin et
al., (2004) BMC Dev Biol 23: 2 and the mouse Vasa homologue, Mvh
(Fujiwara, Y. et al. (1994) Proc. Natl. Acad. Sci. USA 91,
12258-12262) are all expressed in the Atm-deficient ovary at
postnatal day 71. Semi-quantitative comparison of the relative
levels of these genes by examination of the loading control L7
shows that, as expected, these genes are expressed at much lower
levels than in wild-type ovaries containing oocytes. The
contralateral ovary in each animal used for RT-PCR analysis was
prepared for histology, and the sampling and examination of
histological sections from Atm-null mice did not reveal any oocytes
or structures resembling follicles, as expected. Thus,
Atm-deficiency results in a pool of germline stem cells that
persist into adult life (day 71) but these cells cannot, as
reported, produce viable oocytes due to the meiotic defect that
results in gamete death when Atm is absent.
[0231] Whether extra-ovarian cells have the ability to form germ
cells was further investigated. Due to its phenotype, the Atm-null
mouse was selected for evaluation as these animals are genetically
incapable of producing oocytes. If oocytes were detected in the
ovaries of Atm-null mice that received the transplants, they must
be derived from the tissue transplanted (i.e., bone marrow) based
on the nature of the Arm defect in the host animal. Bone marrow
transplantation as performed as described above.
[0232] Although the mutant females are genetically incapable of
generating oocytes from early germ cells, Atm-null female mice were
nonetheless conditioned with cyclophosphamide and busulfan (see
above) to eliminate the possibility of host germ cell contribution
to oocyte production following BMT. In contrast to the complete
absence of oocytes in non-transplanted Atm mutants, both the
wild-type mouse and the Atm-null mouse that received exogenous,
wild-type bone marrow exhibited normal oocytes within normal
appearing follicles (FIG. 8). Oocyte containing follicles were
found in transplanted Atm-null females for at least 11 months after
the initial BMT.
[0233] These results indicate that transplanted wild-type bone
marrow contains female germline stem cells which can then go on to
successfully differentiate into oocytes via meiosis since these
transplanted cells contain functional Atm.
[0234] From a clinical perspective, the finding that BMT rescues
oocyte production in female mice that were Atm-null or sterilized
by chemotherapy is of considerable interest. Accordingly, women
treated for cancer or other ovarian damage can respond similarly so
long as human bone marrow contains female germline stem cells. Bone
marrow samples were collected from human female donors between the
ages of 24-36. Expression of female germline markers Dazl and
Stella was detected, whereas a parallel analysis of adult human
uterine endometrium showed no expression of these genes, indicating
that human bone marrow contains female germline stem cells (FIG.
9).
REFERENCES
[0235] Allen, E. (1923). Ovogenesis during sexual maturity. Am. J.
Anat. 31, 439-470. [0236] Attar, E. C., and Scadden, D. T. (2004).
Regulation of hematopoietic stem cell growth. Leukemia 18,
1760-1768. [0237] Barlow, C., Hirotsune, S., Paylor, R., Liyanage,
M., Eclkhaus, M., Collins, F., Shiloh, Y., Crawley, J. N., Ried,
T., Tagle, D., and Wynshaw-Boris, A. (1996). Atm-deficient mice: a
paradigm of ataxia telangiectasia. Cell 86, 159-171. [0238] Barlow.
C., Liyanage, M., Moens, P. B., Tarsounas, M., Nagashirna, K.,
Brown, K., Rottinghaus, S., Jackson, S. P., Tagle, D., Ried, T.,
and Wynshaw-Boris, A. (1998). Atm deficiency results in severe
meiotic disruption as early as leptonema of prophase I. Development
125, 4007-4017. [0239] Benson, D. A., Karsch-Mizrachi, I., Lipman,
D. J., Ostell, J., and Wheeler, D. L. (2004). GenBank: update.
Nucleic Acids Res. 32 Database issue, D23-D26.
[0240] Bonadonna, G., and Valagussa, P. (1985). Adjuvant systemic
therapy for resectable breast cancer. J. Clin. Oncol. 3, 259-275.
[0241] Borum, K. Oogenesis in the mouse. (1961). A study of meiotic
prophase. Exp. Cell Res. 24, 495-507. [0242] Braat, A. K.,
Zandbergen, T., van de Water, S., Goos, H. J., and Zivkovic, D.
(1999). Charatcerization of zebrafish primordial germ cells:
morphology and early distribution of vasa RNA. Dev. Dyn. 216,
153-167. [0243] Brinster, C. J., Ryu, B. Y., Avarbock, M. R.,
Karagenc, L., Brinster, R. L., and Orwig, K. E. (2003). Restoration
of fertility by germ cell transplantation requires effective
recipient preparation. Biol. Reprod. 69, 412-420. [0244] Brinster,
R. L. (2002). Germline stem cell transplantation and transgenesis.
Science 296, 2174-2176. [0245] Bucci, L. R., and Meistrich, M. L.
(1987). Effects of busulfan on murine spermatogenesis:
cytotoxicity, sterility, sperm abnormalities, and dominant lethal
mutations. Mutat. Res. 176, 259-268. [0246] Calvi, L. M., Adams, G.
B., Weibrecht, K. W., Weber, J. M., Olson, D. P., Knicht, M. C.,
Martin, R. P., Schipani, E., Divietti, P., Bringhurst, F. R.,
Milner, L. A., Kronenberg, H. M., and Scadden, D. T. (2003).
Osteoblastic cells regulate the haematopoietic stem cell niche.
Nature 425, 841-846. [0247] Canning, J., Takai, Y., and Tilly, J.
L. (2003). Evidence for genetic modifiers of ovarian follicular
endowment and development from studies of five inbred mouse
strains. Endocrinology 144, 9-12. [0248] Capela, A., and Temple, S.
(2002). LeX/ssea-1 is expressed by adult mouse CNS stem cells,
identifying them as nonependymal. Neuron 35, 865-875. [0249]
Castrillon, D. H., Quade, B. J., Wang, T. Y., Quigley, C., and
Crum, C. P. (2000). The human VASA gene is specifically expressed
in the germ cell lineage. Proc. Natl. Acad. Sci. USA 97, 9585-9590.
[0250] Cohen, P., and Pollard, J. W. (2001). Regulation of meiotic
recombination and prophase I progression in mammals. BioEssays 23,
996-1009. [0251] Cooke, H. J., Lee, M., Kerr, S., and Ruggiu, M.
(1996). A murine homologue of the human DAZ gene is autosomal and
expressed only in male and female gonads. Hum. Mol. Genet. 5,
513-516. [0252] Cooper R. L., Goldman, J., and Vandenbergh, J. G.
(1993). Monitoring of estrous cyclicity in the laboratory rodent by
vaginal lavage. In Methods in Reproductive Toxicology, R. E. Chapin
and J. J. Heindel, eds. (Orlando, Fla.: Academic Press), pp. 45-56.
[0253] Dearden, P., Grbic, M., and Donly, C. (2003). Vasa
expression and germ-cell specification in the spider mite
Tetranychus urticae. Dev. Genes Evol. 212, 599-603. [0254] Deng,
W., and Lin, H. (2001). Asymmetric germ cell division and oocyte
determination during Drosophila oogenesis. Int. Rev. Cytol. 203,
93-138. [0255] Dialynas, D. P., Quan, Z. S., Wall, K. A., Pierres,
A., Quintans, J., Loken, M. R., Pierres, M., and Fitch, F. W.
(1984). Characterization of the murine T cell surface molecule
designated L3T4, identified by monoclonal antibody GK1.5:
similarity of L3T4 to the human Leu 3/T4 molecule. J. Immunol. 131,
2445-2451. [0256] Dias Neto, E., Correa, R. G., Verjovski-Almeida,
S., Briones, M. R., Nagai, M. A., da Silva, W. Jr., Zago, M. A.,
Bordin, S., Costa, F. F., Goldman, G. H., Carvalho, A. F.,
Matsukuma, A., Baia, G. S., Simpson, D. H., Brunstein, A., de
Oliveira, P. S., Bucher, P., Jongeneel, C. V., O'Hare, M. J.,
Soares, F., Brentani, R. R., Reis, L. F., de Souza, S. J., and
Simpson, A. J. (2000). Shotgun sequencing of the human
transcriptome with ORF expressed sequence tags. Proc. Natl. Acad.
Sci. USA 97, 3491-3496. [0257] Di Giacomo, M., Barchi, M., Baudet,
F., Edelman, W., Keeney, S., and Jasin, M. (2005). Distinct
DNA-damage-dependent and -independent responses drive the loss of
oocytes in recombination-defective mouse mutants. Proc. Natl. Acad.
Sci. USA 102, 737-742. [0258] Dong, J., Albertini, D. F.,
Nishimori, K., Kumar, T. R., Lu, N., and Matzuk, M. M. (1996).
Growth differentiation factor-9 is required during early ovarian
folliculogenesis. Nature 383, 531-535. [0259] Erickson, G. F., and
Shimasaki, S. (2000). The role of the oocyte in folliculogenesis.
Trends Endocrinol. Metab. 11, 193-198. [0260] Fabioux, C., Huvet,
A., Lelong, C., Robert, R., Pouvereau, S., Daniel, J. Y., Minguant,
C., Le Pennec, M. (2004). Oyster vasa-like gene as a marker of the
germline cell development in Crassostrea gigas. Biochem. Biophys.
Res. Commun. 320, 592-598.
[0261] Faddy, M. J., Gosden, R. G., Gougeon, A., Richardson, S. J.,
and Nelson, J. F. (1992). Accelerated disappearance of ovarian
follicles in mid-life: implications for forecasting menopause. Hum.
Reprod. 7, 1342-1346. [0262] Fox, M., Damjanov, I.,
Martinez-Hernandez, A., Knowles, B. B., and Solter, D. (1981).
Immunohistochemical localization of the early embryonic antigen
(SSEA-1) in post-implantation mouse embryos and fetal and adult
tissues. Dev. Biol. 83, 391-398. [0263] Franchi, L. L., Mandl, A.
M., and Zuckerman, S. (1962). The development of the ovary and the
process of oogenesis. In The Ovary, S. Zuckerman, ed. (New York,
N.Y.: Academic Press), pp. 1-88. [0264] Fujiwara, Y., Komiya, T.,
Kawabata, H., Sato, M., Fujimoto, H., Furusawa, M., and Noce, T.
(1994). Isolation of a DEAD-family protein gene that encodes a
murine homolog of Drosophila vasa and its specific expression in
germ cell lineage. Proc. Natl. Acad. Sci. USA 91, 12258-12262.
[0265] Geijsen, N., Horoschak, M., Kim, K., Gribnau, J., Eggan, K.,
and Daley, G. Q. (2004). Derivation of embryonic germ cells and
male gametes from embryonic stem cells. Nature 427, 148-154. [0266]
Generoso, W. M., Stout, S. K. & Huff, S. W. (1971). Effects of
alkylating agents on reproductive capacity of adult female mice.
Mutat. Res. 13, 171-184. [0267] Gilboa, L., and Lehmann, R. (2004).
Repression of primordial germ cell differentiation parallels germ
line stem cell maintenance. Curr. Biol. 14, 981-986. [0268] Gosden,
R. G. (1996). The vocabulary of the egg. Nature 383, 485-486.
[0269] Gosden, R. G. (2004). Germline stem cells in the postnatal
ovary: is the ovary more like a testis? Hum. Reprod. Update 10,
193-195. [0270] Gosden, R. G., Laing, S. C., Felicio, L. S.,
Nelson, J. F., and Finch, C. E. (1983). Imminent oocyte exhaustion
and reduced follicular recruitment mark the transition to
acyclicity in aging C57BL/6J mice. Biol. Reprod. 28, 255-260.
[0271] Green, E. L., and Bernstein, S. E. (1970). Do cells outside
the testes participate in repopulating the germinal epithelium
after irradiation? Negative results. Int. J. Radiat. Biol. Relat.
Stud. Phys. Chem. Med. 17, 87-92. [0272] Grove, J. E., Bruscia, E.,
and Krause, D. S. (2004). Plasticity of bone marrow-derived stem
cells. Stem Cells 22, 487-500. [0273] Hadjantonakis, A. K.,
Gertsenstein, M., Ikawa, M., Okabe, M., and Nagy, A. (1998).
Generating green fluorescent mice by germline transmission of green
fluorescent ES cells. Mech. Dev. 76, 79-90. [0274] Heike, T., and
Nakahata, T. (2004). Stem cell plasticity in the hematopoietic
system. Int. J. Hematol. 79, 7-14. [0275] Hershlag, A., and
Schuster, M. W. (2004). Return of fertility after autologous stem
cell transplantation. Fertil. Steril. 77, 419-421. [0276] Herzog,
E. L., Chai, L., and Krause, D. S. (2003). Plasticity of
marrow-derived stem cells. Blood 102, 3483-3493. [0277] Hirshfield,
A. N. (1991). Development of follicles in the mammalian ovary. Int.
Rev. Cytol. 124, 43-101. [0278] Ikenishi, K. (1998). Germ plasm in
Caenorhabditis elegans, Drosophila and Xenopus. Dev. Growth Differ.
40, 1-10. [0279] Johnson, J., Canning, J., Kaneko, T., Pru, J. K.,
and Tilly, J. L. (2004). Germline stem cells and follicular renewal
in the postnatal mammalian ovary. Nature 428, 145-150. [0280]
Kanatsu-Shinohara, M., Inoue, K., Lee, J., Yoshimoto, M., Ogonuki,
N., Miki, H., Baba, S., Kato, T., Kazuki, Y., Toyokuni, S.,
Toyoshima, M., Niwa, O., Oshimura, M., Heike, T., Nakahata, T.,
Ishino, F., Ogura, A., and Shinohara, T. (2004). Generation of
pluripotent stem cells from neonatal mouse testis. Cell 119,
1001-1012. [0281] Komiya, T., Itoh, K., Ikenishi, K., and Furusawa,
M. (1994). Isolation and characterization of a novel gene of the
DEAD box protein family which is specifically expressed in germ
cells of Xenopus laevis. Dev. Biol. 162, 354-363. [0282] Lawson, K.
A., and Hage, W. J. (1994). Clonal analysis of the origin of
primordial germ cells in the mouse. Ciba Found. Symp. 182, 68-84,
84-91. [0283] Lin, H. (2002). The stem-cell niche theory: lessons
from flies. Nat. Rev. Genet. 3, 931-940. [0284] Marani, E., van
Oers, J. W., Tetteroo, P. A., Poelmann, R. E., van der Veeken, J.,
and Deenen, M. G. (1986). Stage specific embryonic carbohydrate
surface antigens of primordial germ cells in mouse embryos: FAL
(S.S.E.A.-1) and globoside (S.S.E.A.-3). Acta Morphol. Neerl.
Scand. 24, 103-110. [0285] Matzuk, M. M., Burns, K. H., Viveiros,
M. M., and Eppig, J. J. (2002). Intercellular communication in the
mammalian ovary: oocytes carry the conversation. Science 296,
2178-2180. [0286] McGrath, S. A., Esquela, A. F., and Lee, S. J.
(1995). Oocyte-specific expression of growth/differentiation
factor-9. Mol. Endocrinol. 9, 131-136. [0287] McLaren, A. (1984).
Meiosis and differentiation of mouse germ cells. Symp. Soc. Exp.
Biol. 38, 7-23. [0288] McLaren, A. (2003). Primordial germ cells in
the mouse. Dev. Biol. 262, 1-15. [0289] Medvinsky, A., and
Dzierzak, E. (1996). Definitive hematopoiesis is autonomously
initiated by the AGM region. Cell 86, 897-906. [0290] Meirelles, L.
da S., and Nardi, N. B. (2003). Murine marrow-derived mesenchymal
stem cell: isolation, in vitro expansion, and characterization. Br.
J. Haematol. 123, 702-711. [0291] Milhem, M., Mahmud, N., Lavelle,
D., Araki, H., DeSimone, J., Saunthararajah, Y., and Hoffman, R.
(2004). Modification of hematopoietic stem cell fate by 5aza 2'
deoxycytidine and trichostatin A. Blood 103, 4102-4110. [0292]
Mintz, B., and Russell, E. S. (1957). Gene-induced embryological
modification of primordial germ cells in the mouse. J. Exp. Zool.
134, 207-230. [0293] Molyneaux, K., and Wylie, C. (2004).
Primordial germ cell migration. Int. J. Dev. Biol. 48, 537-544.
[0294] Morita, Y., Perez, G. I., Paris, F., Miranda, S., Ehleiter,
D., Haimovitz-Friedman, A., Fuks, Z., Xie, Z., Reed, J. C.,
Schuchman, E. H., Kolesnick, R. N., and Tilly, J. L. (2000). Oocyte
apoptosis is suppressed by disruption of the acid sphingomyelinase
gene or by sphingosine-1-phosphate therapy. Nat. Med. 6, 1109-1114.
[0295] Morrison, S. J., Uchida, N., Weissman, I. L. (1995). The
biology of hematopoietic stem cells. Annu. Rev. Cell Dev. Biol. 11,
35-71. [0296] Noce, T., Okamoto-Ito, S., and Tsunekawa, N. (2001).
Vasa homolog genes in mammalian germ cell development. Cell Struct.
Funct. 26, 131-136. [0297] Okada, S., Nakauchi, H., Nagayoshi, K.,
Nishikawa, S., Nishikawa, S., Miura, Y., and Suda. T. (1991).
Enrichment and characterization of murine hematopoietic stem cells
that express c-kit molecule. Blood 78, 1706-1712. [0298] Okada, S.,
Nakauchi, H., Nagayoshi, K., Nishikawa, S., Miura, Y., and Suda, T.
(1992). In vive and in vitro stem cell function of c-kit- and
Sca-1-positive murine hematopoietic cells. Blood 80, 3044-3050.
[0299] Perez, G. I., Knudson, C. M., Leykin, L., Korsmeyer, S. J.
& Tilly, J. L. (1997). Apoptosis-associated signaling pathways
are required for chemotherapy-mediated female germ cell
destruction. Nat. Med. 3, 1228-1232. [0300] Perez, G. I., Robles,
R., Knudson, C. M., Flaws, J. A., Korsmeyer, S. J., and Tilly, J.
L. (1999). Prolongation of ovarian lifespan into advanced
chronological age by Bax-deficiency. Nat. Genet. 21, 200-203 [0301]
Peters, H. (1969). The development of the mouse ovary from birth to
maturity. Acta Endocrinol. 62, 98-116. [0302] Peters, H. (1970).
Migration of gonocytes into the mammalian gonad and their
differentiation. Phil. Trans. Roy. Soc. Lond. B. 259, 91-101.
[0303] Philpott, C. C., Ringuette, M. J., and Dean, J. (1987).
Oocyte-specific expression and developmental regulation of ZP3, the
sperm receptor of the mouse zona pellucida. Dev. Biol. 121,
568-575. [0304] Pfaffl, M. W. (2001). A new mathematical model for
relative quantification in real-time RT-PCR. Nucleic Acids Res. 29,
e45. [0305] Rajkovic, A., Pangas, S. A., Ballow, D., Suzumori, N.,
and Matzuk, M. M. (2004). NOBOX deficiency disrupts early
folliculogenesis and oocyte-specific gene expression. Science 305,
1157-1159. [0306] Rich, I. N. (1995). Primordial germ cells are
capable of producing cells of the hematopoietic system in vitro.
Blood 86, 463-472. [0307] Richardson, S. J., Senikas, V., and
Nelson, J. F. (1987). Follicular depletion during the menopausal
transition: evidence for accelerated loss and ultimate exhaustion.
J. Clin. Endocrinol. Metab. 65, 1231-1237. [0308] Rongo, C.,
Broihier, H. T., Moore, L., Van Doren, M., Forbes, A., and Lehmann,
R. (1997). Germ plasm assembly and germ cell migration in
Drosophila. Cold Spring Harb. Symp. Quant. Biol. 62, 1-11. [0309]
Roussell, D. L., and Bennett, K. L. (1993). glh-1, a germ-line
putative RNA helicase from Caenorhabditis, has four zinc fingers.
Proc. Natl. Acad. Sci. USA 90, 9300-9304. [0310] Ryu, B. Y., Orwig,
K. E., Avarbock, M. R., and Brinster, R. L. (2003). Stem cell and
niche development in the postnatal rat testis. Dev. Biol. 263,
253-263. [0311] Saitou, M., Barton, S. C., and Surani, M. A.
(2002). A molecular programme for the specification of germ cell
fate in mice. Nature 418, 293-300.
[0312] Salooja, N., Chatterjee, R., McMillan, A. K., Kelsey, S. M.,
Newland, A. C., Milligan, D. W., Franklin, I. M., Hutchinson, R.
M., Linch, D. C., and Goldstone, A. H. (1994). Successful
pregnancies in women following single autotransplant for acute
myeloid leukemia with a chemotherapy ablation protocol. Bone Marrow
Transplant. 13, 431-435. [0313] Salooja, N., Szydlo, R. M., Socie,
G., Rio, B., Chatterjee, R., Ljungman, P., Van Lint, M. T., Powles,
R., Jackson, G., Hinterberger-Fischer, M., Kolb, H. J., and
Apperley, J. F; Late Effects Working Party of the European Group
for Blood and Marrow Transplantation. (2001). Pregnancy outcomes
after peripheral blood or bone marrow transplantation: a
retrospective study. Lancet 358, 271-276. [0314] Salustri, A.,
Fulop, C., Camaioni, A., and Hascall, V. C. (2004).
Oocyte-granulosa cell interactions. In The Ovary, 2nd Edition, P.
C. K. Leung and E. Y. Adashi, eds. (San Diego: Elsevier Academic
Press), pp. 131-143. [0315] Samuelsson, A., Fuchs, T., Simonsson,
B., and Bjorkholm, M. (1993). Successful pregnancy in a 28-year-old
patient autografted for acute lymphoblastic leukemia following
myeloablative treatment including total body irradiation. Bone
Marrow Transplant. 12, 659-660. [0316] Sanders, J. E., Hawley, J.,
Levy, W., Gooley, T., Buckner, C. D., Deeg, H. J., Doney, K.,
Storb, R., Sullivan, K., Witherspoon, R., and Appelbaum, F. R.
(1996). Pregnancies following high-dose cyclophosphamide with or
without high-dose busulfan or total-body irradiation and bone
marrow transplantation. Blood 87, 3045-3052. [0317] Sarmiento, M.,
Glasebrook, A. L., and Fitch, F. W. (1980). IgG or IgM monoclonal
antibodies reactive with different determinants on the molecular
complex bearing Lyt2 antigen block T cell-mediated cytolysis in the
absence of complement. J. Immunol. 125, 2665-2672. [0318] Scholer,
H. R., Hatzopoulos, A. K., Balling, R., Suzuki, N., and Gruss, P.
(1989). A family of octamer-specific proteins present during mouse
embryogenesis: evidence for germline-specific expression of an Oct
factor. EMBO J. 8, 2543-2550. [0319] Sette, C., Dolci, S., Geremia,
R., and Rossi, P. (2000). The role of stem cell factor and of
alternative c-kit gene products in the establishment, maintenance
and function of germ cells. Int. J. Dev. Biol. 44, 599-608. [0320]
Shen, H., Cheng, T., Olszak, I., Garcia-Zepeda, E., Lu, Z.,
Herrmann, S., Falon, R., Luster, A. D., and Scadden, D. T. (2001).
CXCR-4 desensitization is associated with tissue localization of
hematopoietic progenitor cells. J. Immunol. 166, 5027-5033. [0321]
Shiromiza, K., Thorgeirsson, S. S., and Mattison, D. R. (1984).
Effect of cyclophosphamide on ocyte and follicle number in
Sprague-Dawley rats, C57BL/6N and DBA/2N mice. Pediatr. Pharmacol.
4, 213-221. [0322] Soyal, S. M., Amleh, A., and Dean. J. (2000).
FIG.quadrature., a germ cell-specific transcription factor required
for ovarian follicle formation. Development 127, 4645-4654. [0323]
Spangrude, G. J., and Scollay, R. (1990). A simplified method for
enrichment of mouse hematopoietic stem cells. Exp. Hematol. 18,
920-926. [0324] Spangrude, G. J., Heimfeld, S., and Weissman, I. L.
(1988). Purification and characterization of mouse hematopoietic
stem cells. Science 241, 58-62. [0325] Spradling, A. C. (1993).
Developmental genetics of oogenesis. In The Development of
Drosophila melanogaster, Volume I, M. Bate and A. Martinez Arias,
eds. (Cold Spring Harbor, N.Y.: Cold Spring Harbor Press), pp.
1-70. [0326] Spradling, A. H., Drummond-Barbosa, D., and Kai, T.
(2001). Stem cells find their niche. Nature 414, 98-104. [0327] Su,
A. I., Cooke, M. P., Ching, K. A., Hakak, Y., Walker, J. R.,
Wiltshire, T., Orth, A. P., Vega, R. G., Sapinoso, L. M., Moqrich,
A., Patapoutian, A., Hampton, G. M., Schultz, P. G., and Hogenesch,
J. B. (2004). A gene atlas of the mouse and human protein-encoding
transcriptomes. Proc. Natl. Acad. Sci. USA 101, 6062-6067. [0328]
Suzumori, N., Yan, C., Matzuk, M. M., and Rajkovic, A. (2002).
Nobox is a homeobox-encoding gene preferentially expressed in
primordial and growing oocytes. Mech. Dev. 111, 137-141. [0329]
Szabo, P. E., Hubner, K., Schbler, H., and Mann, J. R. (2002).
Allele-specific expression of imprinted genes in mouse migratory
primordial germ cells. Mech. Dev. 115, 157-160. [0330] te Velde, E.
R., and Pearson, P. L. (2002). The variability of female
reproductive ageing. Hum. Reprod. Update 8, 141-154. [0331] Telfer,
E. E. (2004). Germline stem cells in the postnatal mammalian ovary:
a phenomenon of prosimian primates and mice? Reprod. Biol.
Endocrinol. 2, 24. [0332] Tilly, J. L. (2001). Commuting the death
sentence: how oocytes strive to survive. Nat. Rev. Mol. Cell Biol.
2, 838-848. [0333] Tilly, J. L. (2003). Ovarian follicle
counts--not as simple as 1, 2, 3. Reprod. Biol. Endocrinol. 1, 11.
[0334] Tropel, P., Noel, D., Platet, N., Legrand, P., Benabid,
A.-L., and Berger, F. (2004). Isolation and characterisation of
mesenchymal stem cells from adult mouse bone marrow. Exp. Cell Res.
295, 395-406. [0335] Tsuda, M., Sasaoka, Y., Kiso, M., Abe, K.,
Haraguchi, S., Kobayashi, S., and Saga, Y. (2003). Conserved roles
of nanos proteins in germ cell development. Science 301, 1239-1241.
[0336] Van de Rijn, M., Heimfeld, S., Spangrude, G. J., and
Weissman, I. L. (1989). Mouse hematopoietic stem-cell antigen Sca-1
is a member of the Ly-6 antigen family. Proc. Natl. Acad. Sci. USA
86, 4634-4638. [0337] van den Hurk, R., and Zhao, J. (2005).
Formation of mammalian oocytes and their growth, differentiation
and maturation within ovarian follicles. Theriogenology 63,
1717-1751. [0338] Williams, D. E., de Vries, P., Namen, A. E.,
Widmer, M. B., and Lyman, S. D. (1992). The Steel factor. Dev.
Biol. 151, 368-376. [0339] Wegnum, A. W., Eaves, A. C., and Thomas,
T. E. (2003). Identification and isolation of hematopoietic stem
cells. Arch. Med. Res. 34, 461-475. [0340] Xu, Y., Aslley, T.,
Brainerd, E. E., Bronson, R. T., Meyn, M. S., and Baltimore, D.
(1996). Targeted disruption of ATM leads to growth retardation,
chromosomal fragmentation during meiosis, immune defects, and
thymic lymphoma. Genes Dev. 10, 2411-2422. [0341] Yeom, Y. I.,
Fuhrmann, G., Ovitt, C. E., Brehm, A., Ohbo, K., Gross, M., Hubner,
K., and Scholer, H. R. (1996). Germline regulatory element of Oct-4
specific for the totipotent cycle of embryonal cells. Development
122, 881-894. [0342] Yoshimizu, T., Sugiyama, N., De Felice, M.,
Yeom, Y. I., Ohbo, K., Masuko, K., Obinata, M., Abe, K., Scholer,
H. R., and Matsui, Y. (1999). Germline-specific expression of the
Oct-4/green fluorescent protein (GFP) transgene in mice. Dev.
Growth Differ. 41, 675-684. [0343] Yuan, L., Liu, J. G., Hoja, M.
R., Wilbertz, J., Nordqvist, K., and Hoog, C. (2002). Female germ
cell aneuploidy and embryo death in mice lacking the
meiosis-specific protein SCP3. Science 296, 1115-1118. [0344] Zhu,
C. H., and Xie, T. (2003). Clonal expansion of ovarian germline
stem cells during niche formation in Drosophila. Development 130,
2579-258. [0345] Zuckerman, S. (1951). The number of oocytes in the
mature ovary. Recent Prog. Horm. Res. 6, 63-108. [0346] Zuckerman,
S., and Baker, T. G. (1977). The development of the ovary and the
process of oogenesis. In The Ovary, S. Zuckerman and B. J. Weir,
eds. (New York, N.Y.: Academic Press), pp. 41-67.
* * * * *