U.S. patent application number 16/279991 was filed with the patent office on 2019-09-19 for wnt pathway stimulation in reprogramming somatic cells with nuclear reprogramming factors.
The applicant listed for this patent is Whitehead Institute for Biomedical Research. Invention is credited to Brett Chevalier, Ruth Foreman, Rudolf Jaenisch, Alexander Marson, Richard A. Young.
Application Number | 20190284537 16/279991 |
Document ID | / |
Family ID | 40429192 |
Filed Date | 2019-09-19 |
United States Patent
Application |
20190284537 |
Kind Code |
A1 |
Chevalier; Brett ; et
al. |
September 19, 2019 |
WNT PATHWAY STIMULATION IN REPROGRAMMING SOMATIC CELLS WITH NUCLEAR
REPROGRAMMING FACTORS
Abstract
The invention provides compositions and methods of use in
reprogramming somatic cells. Compositions and methods of the
invention are of use, e.g., for generating or modulating (e.g.,
enhancing) generation of induced pluripotent stem cells by
reprogramming somatic cells. The reprogrammed somatic cells are
useful for a number of purposes, including treating or preventing a
medical condition in an individual. The invention further provides
methods for identifying an agent that reprograms somatic cells to a
pluripotent state and/or enhances the speed and/or efficiency of
reprogramming. Certain of the compositions and methods relate to
modulating the Wnt pathway.
Inventors: |
Chevalier; Brett; (Malden,
MA) ; Marson; Alexander; (Cambridge, MA) ;
Young; Richard A.; (Boston, MA) ; Foreman; Ruth;
(Somerville, MA) ; Jaenisch; Rudolf; (Brookline,
MA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Whitehead Institute for Biomedical Research |
Cambridge |
MA |
US |
|
|
Family ID: |
40429192 |
Appl. No.: |
16/279991 |
Filed: |
February 19, 2019 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15429114 |
Feb 9, 2017 |
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16279991 |
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14822653 |
Aug 10, 2015 |
9593311 |
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15429114 |
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12675681 |
Aug 24, 2010 |
9102919 |
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PCT/US2008/010249 |
Aug 29, 2008 |
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14822653 |
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61188190 |
Aug 6, 2008 |
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60967028 |
Aug 31, 2007 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61P 35/02 20180101;
A61P 25/28 20180101; A61K 35/545 20130101; A61P 25/14 20180101;
A61P 25/16 20180101; A61P 17/02 20180101; A61P 31/18 20180101; C12N
5/0696 20130101; A61P 5/00 20180101; C12N 2501/604 20130101; A61P
35/00 20180101; C12N 2502/45 20130101; C12N 2510/00 20130101; A61P
43/00 20180101; A61P 37/02 20180101; C12N 2501/602 20130101; C12N
2501/415 20130101; A61P 7/06 20180101; A61P 21/02 20180101; A61P
25/00 20180101; C12N 2502/99 20130101; C12N 2501/603 20130101; A61K
38/00 20130101; C12N 5/0696 20130101 |
International
Class: |
C12N 5/074 20060101
C12N005/074; A61K 35/545 20060101 A61K035/545 |
Goverment Interests
[0002] The invention described herein was supported, in whole or in
pert, by grants 5-ROI-HDO45022, 5-R37-CA084198, and 5-ROI-CA067869
to RJ and by NIH grant HG002668 from the National Institutes of
Health. The Untied States government has certain rights in the
invention.
Claims
1.-65. (canceled)
66. A method of identifying or screening an agent useful for
reprogramming a mammalian somatic cell comprising: (i) identifying
an agent that modulates an activity of a Wnt pathway or an agent
that substitutes for a reprogramming factor in reprogramming
mammalian somatic cells to a pluripotent state; and (ii) testing
the effect of the agent on reprogramming a mammalian somatic
cell.
67. The method of claim 66, wherein the method in step (i)
comprises identifying an agent that modulates an activity of a Wnt
pathway.
68. The method of claim 67, wherein the method further comprises in
step (i): (a) providing a medium containing the agent; (b)
culturing a population of mammalian somatic cells in the medium;
and (c) measuring the activity of the Wnt pathway, thereby
determining if the agent modulates the activity of the Wnt
pathway.
69. The method of claim 67, further comprises in step (ii)
determining, after a period of time, whether cells having one or
more characteristics of pluripotent cells are present after
maintaining the mammalian somatic cells and their progeny in
culture for the time period.
70. The method of claim 66, wherein the mammalian somatic cells are
genetically modified or transiently transfected to express Oct4,
Sox2, and Klf.
71. The method of claim 66, wherein the method in step (i)
comprises identifying an agent that substitutes for a reprogramming
factor in reprogramming mammalian somatic cells to a pluripotent
state.
72. The method of claim 71, wherein the method in step (i)
comprises: (a) introducing into the mammalian somatic cell one or
more reprogramming factors, wherein the reprogramming factor to be
substituted is not introduced into the cell; (b) contacting a
population of mammalian somatic cells with a Wnt pathway activator
selected from the group consisting of: an exogenous, soluble,
biologically active Wnt protein that binds to a Wnt receptor and
activates the Wnt pathway, and a small molecule GSK-3 inhibitor
that activates the Wnt pathway; (c) contacting the mammalian
somatic cells with a candidate agent; (d) maintaining the mammalian
somatic cells in a cell culture system for a suitable period of
time; and (e) determining whether cells having one or more
characteristics of pluripotent cells are present in said culture
system, wherein the agent that substitutes for the reprogramming
factor in reprogramming mammalian somatic cells is identified if
cells having one or more characteristics of pluripotent cells are
present at levels greater than would be expected had the mammalian
somatic cells not been contacted with the candidate agent.
73. The method of claim 72, wherein the method is for identifying
an agent that substitutes for Klf in reprogramming mammalian
somatic cells to a pluripotent state, and wherein the method
comprises in step (a) introducing into the mammalian somatic cell
reprogramming factors Sox2 and Oct4, but not Klf.
74. The method of claim 72, wherein the method is for identifying
an agent that substitutes for Sox2 in reprogramming mammalian
somatic cells to a pluripotent state, and wherein the method
comprises in step (a) introducing into the mammalian somatic cell
reprogramming factors Klf and Oct4, but not Sox.
75. The method of claim 72, wherein the method is for identifying
an agent that substitutes for Oct4 in reprogramming mammalian
somatic cells to a pluripotent state, and wherein the method
comprises in step (a) introducing into the mammalian somatic cell
reprogramming factors Sox2 and Klf, but not Oct4.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims the benefit of priority to U.S.
Provisional Patent Application 60/967,028, filed Aug. 31, 2007, and
U.S. Provisional Patent Application 61/188,190, filed Aug. 6, 2008,
both of which are incorporated herein by reference.
BACKGROUND OF THE INVENTION
[0003] Stem cells are cells that are capable of self-renewal and of
giving rise to more differentiated cells. Embryonic stem (ES) cells
can differentiate into the multiple specialized cell types that
collectively comprise the body. In addition to being of immense
scientific interest, the property of pluripotency gives human ES
cells great clinical promise for applications m regenerative
medicine such as cell/tissue replacement therapies for disease.
[0004] Several different methods are currently used to obtain ES
cells. In one method, an ES cell line is derived from the inner
cell mass of a normal embryo in the blastocyst stage (See U.S. Pat.
Nos. 3,843,780 and 6,200,806, Thompson, J. A. et al. Science,
282:1143-7, 1998). A second method for creating pluripotent ES
cells utilizes somatic cell nuclear transfer (SCNT). In this
technique, the nucleus is removed from a normal egg, thus removing
the genetic material. The nucleus of a donor diploid somatic cell
is introduced directly into the enucleated oocyte, e.g., by
micromanipulation, or the donor diploid somatic cell is placed next
to the enucleated egg and the two cells are fused. The resulting
cell has the potential to develop into an early embryo from which
the portion containing the stem cell producing inner cell mess can
be obtained. In a third method, the nucleus of a human cell is
transplanted into an enucleated animal oocyte of a species
different from the donor cell. See, e.g., U.S. Pat. Pub. No.
20010012513. The resultant chimeric cells are used for the
production of pluripotent ES cells, in particular human-like
pluripotent ES cells. Disadvantages of this technique are that
these chimeric cells may contain unknown viruses and retain the
mitochondria of the animal species.
[0005] The traditional ES cell isolation methods suffer from
several limitations when applied to generating human ES cells.
These include ethical controversies associated with the source of
the cells as well as technical challenges. A significant limitation
to the productive utilization of ES cells for clinical applications
is the difficulty associated with generating ES cells that are
genetically matched to individual patients. There exists a
significant need for alternative methods of generating pluripotent
cells.
SUMMARY OP THE INVENTION
[0006] The present invention provides compositions and methods for
reprogramming somatic cells to a less differentiated state. In
certain embodiments the compositions and methods permit
reprogramming of somatic cells to pluripotent, embryonic stem
cell-like cells ("ES-like cells").
[0007] In one aspect, the invention provides a method of
reprogramming a somatic mammalian cell comprising culturing the
cell in the presence of an extracellular signaling molecule so that
the cell becomes reprogrammed.
[0008] In one aspect the invention provides a method of
reprogramming a somatic mammalian cell comprising culturing the
cell in Wnt conditioned cell culture medium so that the cell
becomes reprogrammed. In certain embodiments the method comprises
culturing the somatic cell so that the cell is induced to become
pluripotent. In certain embodiments the Wnt conditioned cell
culture medium comprises Wnt3a conditioned medium (Wnt3a-CM).
[0009] In another aspect the invention provider a method of
reprogramming a somatic mammalian cell comprising contacting the
cell with an agent that increases the activity of a Wnt pathway so
that the cell is induced to become pluripotent. In some embodiments
the agent is a soluble, biologically active Wnt protein, e.g., a
Wnt3a protein. In some embodiments the agent is selected from the
group consisting of: (i) small molecules that mimic the effect of
Wnt3a conditioned medium or soluble, bio logically active Wnt
proteins, e.g., by interacting with cell receptors) for Wnt; (ii)
agents that modulate the interaction between .beta.-catenin and a
member of the TCF/LEF family and/or modulate the expression or
activity of a member of the TCF/LEF family, (iii) agents that
inhibit expression or activity of an endogenous inhibitor of the
Wnt pathway.
[0010] The invention provides somatic cells reprogrammed using the
inventive methods.
[0011] Cell culture media containing a Wnt3a activator and an
additional reprogramming agent capable of substituting for
engineered expression of Oct4, Klf4, and/or Sox2 (or any
combination thereof) are additional aspects of this invention.
Further aspects of the invention are (I) a composition comprising:
(i) a cell that has been modified to increase its expression of
Oct4, Klf4, and/or Sox2, or any subset of these; and (ii) a Wnt
pathway modulator, e.g., a Wnt pathway activator; (2) a composition
comprising: (i) a cell that has been modified to increase its
expression or intracellular level of one or more reprogramming
factors, wherein the reprogramming factor(s) is/are optionally
selected from Oct4, Klf4, and/or Sox2, or any subset of these; and
(ii) Wnt conditioned medium; (3) a composition comprising: (i) a
cell that has been modified to increase its expression or
intracellular level of one or more reprogramming factors, wherein
the reprogramming factor(s) is/are optionally selected from Oct4,
Nanog, Lin28 and/or Sox2, or any subset of these; and (ii) a Wnt
pathway activator; and (4) a composition comprising: (i) a cell
that has been modified to increase its expression or intracellular
level of one or more reprogramming factors, wherein the
reprogramming factor(s) is/are optionally selected from Oct4,
Nanog, Lin28 and/or Sox2, or any subset of these; and (ii) Wnt
conditioned medium.
[0012] The invention also provides methods for identifying an agent
that reprograms somatic cells to a less differentiated state and/or
contributes to such reprogramming in combination with one or more
other agents. In certain of the methods, somatic cells are
contacted with an agent that increases Wnt pathway activity and a
candidate agent. Cells are assessed for pluripotency
characteristics. The presence of it least a subset of pluripotency
characteristics indicates that the agent as capable of
reprogramming somatic cells to a less-differentiated state. The
agents identified by the present invention can then by used to
reprogram somatic cells by contacting somatic cells with the
agents.
[0013] The present invention further provides methods for treating
a condition in an individual in need of treatment for a condition.
In certain embodiments, somatic cells are obtained from the
individual and reprogrammed by the methods of the invention. The
reprogrammed cells may be expanded in culture. Pluripotent
reprogrammed cells (which refers to the original reprogrammed cells
and/or their progeny that retain the property of pluripotency) are
maintained under conditions suitable for the cells to develop into
cells of a desired cell type or cell lineage. In some embodiments,
the cells are differentiated m vitro using protocols, such as those
known in the art. The reprogrammed cells of a desired cell type are
introduced into the individual to treat the condition. In certain
embodiments, the somatic cells obtained from the individual contain
a mutation in one or more genes. In these instances, in certain
embodiments the somatic cells obtained from the individual are
first treated to repair or compensate for the defect, e.g., by
introducing one or more wild type copies of the gene(s) into the
cells such that the resulting cells express the wild type version
of the gene. The cells are then introduced into the individual.
[0014] In certain embodiments, the somatic cells obtained from the
individual are engineered to express one or more genes following
their removal from the individual. The cells may be engineered by
introducing a gene or expression cassette comprising a gone into
the cells. The introduced gene may be one that is useful for
purposes of identifying, selecting, and/or generating a
reprogrammed cell. In certain embodiments the introduced gene(s)
contribute to initiating and/or maintaining the reprogrammed state,
in certain embodiments the expression produces) of the introduced
gene(s) contribute to producing the reprogrammed state but are
dispensable for maintaining the reprogrammed state.
[0015] In certain other embodiments, methods of the invention can
be used to treat individuals in need of a functional organ. In the
methods, somatic cells are obtained from an individual in need of a
functional organ, and reprogrammed by the methods of the invention
to produce reprogrammed somatic cells. Such reprogrammed somatic
cells are then cultured under conditions suitable for development
of the reprogrammed somatic cells into e desired organ, which is
then introduced into the individual.
[0016] In Anther summary, the invention provides a method of
reprogramming a somatic mammalian cell comprising contacting the
somatic mammalian cell with an agent that modulates a Wnt pathway
so that the somatic mammalian cell becomes reprogrammed, in certain
embodiments of the invention the method comprises reprogramming the
somatic mammalian cell to a pluripotent state. In certain aspects,
the invention provides improvements in methods of generating
induced pluripotent stem (iPS) cells. For example, in certain
aspects the invention enhances reprogramming somatic cells to
pluripotency that have not been engineered to express c-Myc. In
certain aspects, the inventive methods facilitate generating
homogenous ES-like colonies. In some embodiments, the inventive
methods enhance formation of homogenous, ES-like colonies without
imposing a selection step that requires genetic modification of the
initial somatic cells.
[0017] In certain embodiments of the invention, the method
comprises culturing the cell in Wnt-conditioned medium. In certain
embodiments, the method comprises culturing the cell in
Wnt3a-conditioned medium. In certain embodiments, the cell is a
human cell. In certain embodiments the cell is a mouse cell. In
certain embodiments, the cell is a non-human primate cell. In
certain embodiments, the somatic mammalian cell is a terminally
differentiated cell. In certain embodiments the cell is a
fibroblast or immune system cell (e.g., B or T cell). In certain
embodiments, the somatic mammalian cell is not a terminally
differentiated cell. For example, the somatic mammalian cell may be
a precursor cell, e.g., a neural precursor or hematopoietic
precursor cell. In certain embodiments, the method is practiced in
vitro. In certain embodiments, contacting the cell comprises
culturing the cell in culture medium containing the agent. In
certain embodiments, contacting comprises culturing the cell in
culture medium comprising the agent for at leant 10 days. In
certain embodiments, contacting comprises culturing the cell in
culture medium comprising the agent for at least 12 or at least 15
days or at least 20 days. In certain embodiments, the somatic cell
is genetically modified to contain a nucleic add sequence encoding
a selectable marker, operably linked to a promoter for an
endogenous pluripotency gene thereby allowing selection of cells
that have been reprogrammed to pluripotency while in other
embodiments the somatic cell is not genetically modified to contain
a nucleic acid sequence encoding a selectable marker operably
linked to a promoter for an endogenous pluripotency gene thereby
allowing selection of cells that have been reprogrammed to
pluripotency. In certain embodiments, the somatic cell is modified
to express or contain at least one reprogramming factor at levels
greater than normally present in somatic cells of that type. In
some embodiments, the reprogramming factor is Oct4. In some
embodiments, the reprogramming factor is Sox2. In some embodiments
the reprogramming footer is Klf4. In some embodiments the
reprogramming factor is Nanog. In some embodiments the
reprogramming factor is Lin28. In some embodiments the
reprogramming factor(s) are Oct4 and Sox2, In some embodiments the
reprogramming factor(s) are Oct4, Sox2, and Klf4. In certain
embodiments, the somatic cell is not genetically modified to
express c-Myc at levels greater than normally present in somatic
cells of that cell type. In certain embodiments, the cell is also
contacted with a second agent that modulates the Wnt pathway. In
certain embodiments, the somatic cell is cultured in medium
containing exogenous soluble, biologically active Wnt protein. In
certain embodiments, the Wnt protein is Wnt3a protein. In certain
embodiments, the method fort her comprises confirming that the
reprogrammed cell is pluripotent. In certain embodiments, the
method is practiced on a population of cells and the method further
comprises separating cells that are reprogrammed to a pluripotent
state from cells that are not reprogrammed to a pluripotent state.
In certain embodiments, the method further comprises administering
the reprogrammed cell to a subject. In certain embodiments, the
method further comprises differentiating the cell to a desired cell
type in vitro after reprogramming the cell. In certain embodiments,
the method further comprises administering the differentiated cell
to a subject.
[0018] The invention also provides a method of treating an
individual in need thereof comprising: (a) obtaining somatic cells
from the individual; (b) reprogramming at least some of the somatic
cells by a method comprising contacting the somatic mammalian cells
with an agent that modulates the Wnt pathway (e.g., a Wnt pathway
activator); and (c) administering at least some of the reprogrammed
cells to the individual, optionally after differentiating the cells
into one or more desired cell types. In some embodiments, the
individual is a human. In some embodiments, the method is practiced
on a population of cells and farther comprises separating cells
that are reprogrammed to a pi on potent state from cells that are
not reprogrammed to a pluripotent state. In some embodiments, the
method farther comprises differentiating the cell m vitro and,
optionally, administering the differentiated cell to an individual
in need of treatment for a condition for which cell therapy is of
use. For example, cells may be differentiated along a desired cell
lineage such as a neural lineage, a muscle lineage, etc.
[0019] The invention farther provides composition comprising (i) a
somatic mammalian cell that has been modified or treated so that it
expresses or contains at least one reprogramming factor at levels
greater than would be the case without such modification or
treatment; and (ii) an agent dial increases activity of a Wnt
pathway and contributes to reprogramming the somatic cell to a
pluripotent state. In certain embodiments, the agent is Wnt3a
protein. In certain embodiments, the agent is a small molecule.
[0020] The invention farther provides a method of identifying an
agent useful for modulating the reprogramming of mammalian somatic
cells to a pluripotent state comprising: (a) culturing a population
of mammalian somatic cells in medium containing an agent that
modulates activity of a Wnt pathway and a candidate agent; and (b)
determining; after a suitable period of time, whether cells having
one or more characteristics of ES cells are present after
maintaining the cells and their progeny in culture for a suitable
lime period, wherein the candidate agent is identified as being
useful for modulating the reprogramming of mammalian somatic cells
to a pluripotent state. If cells having one or more characteristics
of ES cells are present at levels different than would be expected
had the medium not contained the candidate agent.
[0021] In certain embodiments, the characteristics are selected
from: colony morphology, expression of an endogenous gene expressed
selectively by ES cells, expression of a detectable marker operably
linked to expression control sequences of a gene expressed
selectively by ES cells, ability to differentiate into cells having
characteristics of endoderm, mesoderm, and ectoderm when injected
into immunocompromised mice, and ability to participate in
formation of chimeras that survive to term. In certain embodiments,
the cells have been modified to express at least one reprogramming
factor. In certain embodiments, the medium is Wnt-conditioned
medium.
[0022] In certain embodiments, the medium is Wnt3a-conditioned
medium, in certain embodiments, the agent that modulates activity
of a Wnt pathway is Wnt3a protein, in certain embodiments, the
agent that modulates activity of a Wnt pathway is a small molecule.
In certain embodiments, the candidate agent is a small molecule. In
certain embodiments, the method comprises identifying an agent
useful for enhancing the reprogramming of mammalian somatic cells,
wherein the candidate agent is identified as being useful for
enhancing the reprogramming of mammalian somatic cells to a
pluripotent state if cells having one or more characteristics of ES
cells are present at levels greater than would be expected had the
medium not contained the candidate agent. In certain embodiments,
step (b) comprises determining whether cell colonies having one or
more characteristics of ES cell colonies are present after
maintaining the cells and their progeny in culture for a suitable
time period, wherein the candidate agent is identified as being
useful for modulating the reprogramming of mammalian somatic cells
to a pluripotent state if cell colonies having one or more
characteristics of ES cell colonies are present at levels different
than would be expected had the medium not contained the candidate
agent. In certain embodiments, the cells express at least one
reprogramming factor.
[0023] The invention also provides a method of identifying an agent
useful for reprogramming mammalian somatic cells to a pluripotent
state comprising: (a) contacting a population of mammalian somatic
cells with an agent that increases Wnt pathway activity and a
candidate agent; (b) maintaining the cells in a cell culture system
for a suitable period of time; and (c) determining whether cells
having one or more characteristics of ES cells are present in said
culture system, wherein the agent is identified as being useful for
reprogramming mammalian somatic cells to an ES-like state if cells
having one or more characteristics of ES cells are present at
levels greater than would be expected had the cells not been
contacted with the candidate agent.
[0024] In certain embodiments of the invention, the characteristics
are selected from: colony morphology, expression of an endogenous
gene expressed selectively by ES cells, expression of a detectable
marker operably linked to expression control sequences of a gene
expressed selectively by ES cells, ability to differentiate into
cells having characteristics of endoderm, mesoderm, and ectoderm
when injected into immunocompromised mice, and ability to
participate in formation of chimeras that survive to term.
[0025] In certain embodiments, the agent that increases Wnt pathway
activity is Wnt3a protein. In certain embodiments, the candidate
agent is a small molecule. In certain embodiments, the cells
express at least one reprogramming factor. In certain embodiments,
step (b) comprises determining whether cell colonies having one or
more characteristics of ES cell colonies are present after
maintaining the cells and their progeny in culture for a suitable
time period, wherein the candidate agent is identified as being
useful for modulating the reprogramming of mammalian somatic cells
to a pluripotent state if cell colonies having one or more
characteristics of ES cell colonies are present at levels different
than would be expected had the medium not contained the candidate
agent.
[0026] The invention also provides a method of reprogramming a
somatic mammalian cell comprising culturing the cell in the
presence of an extracellular signaling molecule so that the cell
becomes reprogrammed. In certain embodiments, said extracellular
signaling molecule is a molecule whose binding to an extracellular
domain of a cellular receptor initiates or modifies a signal
transduction pathway within the cell. In certain embodiments, the
signal transduction pathway is the Wnt pathway.
[0027] The invention also provides a method of identifying a Wnt
pathway modulator useful formulating the reprogramming of mammalian
somatic cells to a pluripotent state comprising: (a) culturing a
population of mammalian somatic cells in medium containing the Wnt
pathway modulator, (b) determining, after a suitable period of
time, whether cells having one or more characteristics of ES cells
are present after maintaining the cells and their progeny in
culture for a suitable time period, wherein the Wnt pathway
modulator is identified as being useful for modulating the
reprogramming of mammalian somatic cells to a pluripotent state if
cells having one or more characteristics of ES cells ere present et
levels different than would be expected had the medium not
contained the Wnt pathway modulator.
[0028] In certain embodiments, the method comprises (i) testing at
least 10 Wnt pathway modulators; and (ii) identifying one or more
of the Wnt pathway modulators as having significantly greater
effect on reprogramming speed or efficiency than at least 50% of
the other Wnt pathway modulators tested. In certain embodiments,
the method comprises testing at least 20, at least 50, or at least
100 Wnt pathway modulators. In some embodiments, the method
comprises identifying one or more of the Wnt pathway modulators as
having significantly greater effect on reprogramming speed or
efficiency than at least 75%, or at least 90% of the other Wnt
pathway modulators tested. In certain embodiments, the Wnt pathway
modulators tested are small molecules. In certain embodiments, the
Wnt pathway modulators tested are structurally related. For
example, they may be members of a set of compounds, e.g., a
combinatorial compound library, synthesized based on a common core
structure or they may be derivatives obtained by modifying a core
structure or lead compound such as by making substitutions or
additions at one or more positions. In certain embodiments, the Wnt
pathway modulator is identified as being useful for increasing the
speed or efficiency of reprogramming cells to an ES-tike state if,
after a suitable time period, cells having one or more
characteristics of ES cells ere present in numbers greater than
would be expected had the medium not contained the Wnt pathway
modulator. In certain embodiments, the Wnt pathway modulator is
identified as being useful for increasing the speed or efficiency
of reprogramming cells to a pluripotent state if, after a suitable
time period, cell colonies having one or more characteristics of ES
cell colonies, are present in numbers greater than would be
expected had the medium not contained the Wnt pathway modulator.
For example, the methods may result in an increased percentage of
colonies having features of ES cell colonies and/or the colonies
may be more homogenous than would be the case in the absence of the
Wnt pathway modulator.
[0029] The invention further provides a cell culture composition
comprising: (a) cell culture medium containing a Wnt pathway
modulator; and (b) a plurality of mammalian somatic cells, wherein
(i) the cells are genetically modified or transiently transfected
to express one or more reprogramming factors; (ii) the cells are
genetically modified to contain a nucleic acid sequence encoding a
selectable marker, operably linked to a promoter for an endogenous
pluripotency gene, thereby allowing select km of cells have been
reprogrammed to pluripotency, or (iii) the cell culture medium
contains one or more small molecules, nucleic acids, or
polypeptides that substitute for a reprogramming Actor other than
c-Myc.
[0030] In certain embodiments, the cell culture medium comprises
Wnt-3a CM. In certain embodiments, the medium contains a small
molecule that modulates the Wnt pathway.
[0031] In certain embodiments, the one or more reprogramming Adore
are selected from: Oct4, Nanog, Sox2, Lin2l, and Klf4. The
invention further provides a composition comprising: an iPS cell
and an agent that modulates, e.g., activates, the Wnt pathway. In
certain embodiments the agent that activates the Wnt pathway is
Wnt3a protein. In certain embodiments the agent that activates the
Wnt pathway is a small molecule.
[0032] In certain embodiments, the invention provides use of an
agent that modulates a Wnt pathway in the manufacture of a
medicament for reprogramming a somatic mammalian cell.
[0033] It is contemplated that all embodiments described herein are
applicable to the various aspects of the invention. It is also
contemplated that the various embodiments of the invention and
elements thereof can be combined with one or more other such
embodiments and/or elements whenever appropriate.
BRIEF DESCRIPTION OF THE DRAWINGS
[0034] FIG. 1. Wnt3a promotes epigenetic reprogramming, a.
Schematic representation of the experimental time-line. MEFs were
infected with DOX-inducible lentivirus, split into cultures with
and without Wnt3-CM treatment, and then induced with DOX (day 0).
G418 selection was initialed at fixed time points after induction
and Wnt3a-CM treatment was maintained for 7 days of selection. DOX
and G418 were maintained until resistant colonies were assessed, b.
G418-resistant colony counts from MEFs overexpressing
Oct4/Sox2/Klf4/c-Myc in standard ES cell media or with Wnt3a-CM
treatment, c. Phase images of G418 resistant colonies formed with
and without Wnt3a-CM treatment, d. G418-resistant colony counts
from MEFs infected with different combination of reprogramming
fedora in the presence and absence of Wnt3a-CM. G418 resistant
colonies emerged without c-Myc transduction in the presence of
Wnt3a-CM. e. Phase image of Myc[-] G418 resistant colony formed
with Wnt3a-CM treatment. In this experiment, no colonies were
observed for Myc[-] cells in the absence of Wnt3a-CM. f.
G418-resistant colony counts from MEFs over-expressing
Oct4/Sox2/Klf4 (Myc[-]) or Oct4/Sox2/Klf4/c-Myc (Myc[+]) in the
presence (red bars) and absence (gray bars) of Wnt3a-CM. G418
selection was initiated on day 5 or day 10 post-induction as
indicated and colonies (in a 32-cm.sup.2 area) were assessed on day
20 .mu.g. Scatter plots comparing GFP intensity to
autofluorescence, using flow cytometry, in Oct4-GFP cells on day 20
post-induction of Oct4/Sox2/Klf4, reveal a GFP expressing
population of cells (indicated with an arrow) only with Wnt3a-CM
treatment, h. Phase image of GFP expressing Myc[-] cells derived
with Wnt3a-CM treatment and without any genetic selection.
[0035] FIG. 2. Induction of Pluripotency in Wnt Stimulated cells,
a-d. Immunostaining reveals induction of pluripotency markers,
Nanog (a-b) and SSEA-1(c-d) in Wnt3a-CM treated Myc[-] cells, e-g.
Wnt3a-CM treated Myc[-] tines formed teratomas when injected into
SOD mice subcutaneously. Teratomas from Oct4/Sox2/Klf4/Wnt3aCM iPS
tines showed evidence of differentiated cells of three germ layers
similar to teratomas formed from V6.5 mES injections. Arrows
indicated neural tissue in (e), cartilage in (f), and endodermal
cells in (g), h. Oct4/Sox2/Klf4/Wnt3aCM iPS lines derived without
selection gave rise to chimeric mice (as shown on the left) with
agouti coat color and pigmented eyes (in contrast to wild type
Balb/c mouse, right) providing evidence of contribution so somatic
cells. Coat color of offspring confirmed that the
Oct4/Sox2/Klf1/Wnt3aCM iPS line generated here is
germline-competent (data not shown).
[0036] FIG. 3. Wnt/.beta.-catenin stimulation enhances iPS colony
formation in absence of c-Myc retrovirus, a. Counts are shown for
G418 resistant colonies in Oct4/Sox2/Klf4 over-expressing MEFs
cultured in ES cell media, MEF conditioned media, Wnt3a
over-expressing conditioned media, and Wnt3a over-expressing
conditioned media with ICG001 (4 .mu.M). Selection was initiated on
day 15 post-induction, and colonies were assessed on day 28.
Wnt3a-CM treatment was maintained until day 22. Mean number of
counts from triplicate experiments is displayed with error bars
indicating S.D. b. Counts are shown for G418 resistant colonies (in
a 32-cm.sup.2 area) in Oct4/Sox2/Klf4/c-Myc over-expressing MEFs
cultured in ES cell media, Wnt3a over-expressing conditioned media,
and Wnt3a over-expressing conditioned media with ICG-001 (4 .mu.M).
Selection was initiated on day 10 poet-induction, Wnt3a-CM was
maintained until day 17, and colonies were assessed on day 20. c.
Wnt stimulation promotes the formation of iPS cells in the absence
of c-Myc transduction. This could be due to: i) direct regulation
by the Wnt pathway of key endogenous pluripotency factors, such as
Oct4, Sox2 and Nanog as suggested by genomic studies in ES cells
(Cole et al., 2008), ii) Wnt pathway-induced activation of
endogenous Myc (He et al., 1998; Cole et al., 2008), or other cell
proliferation genes, accelerating the sequential process of forming
iPS colonies.
[0037] FIG. 4. (a) Timeline of initial experiments showing ability
of Wnt3a conditioned medium to promote generation of iPS cells.
Expression of the pluripotency-inducing factors was induced on day
2. Expression of GFP and colony formation were assessed as
indicated (b). Wnt3a promotes iPS cell formation in cells
over-expressing Oct4, Sox2, Klf4 and c-Myc; FIG. 4C Wnt3a promotes
iPS cell formation in cells over-expressing Oct4, Sox2, Klf4
without engineered expression of c-Myc; (c) Wnt3a promotes iPS cell
formation in cells over-expressing Oct4, Sox2, Klf4 without
engineered expression of c-Myc.
[0038] FIG. 5. Structure of ICG-001.
DETAILED DESCRIPTION OF THE INVENTION
Introduction and Definitions
[0039] The present invention relates to compositions and methods
for reprogramming somatic cells, e.g., for reprogramming somatic
cells to pluripotency in vitro. The invention provides methods for
reprogramming somatic cells to a less differentiated state. The
resulting cells are referred to herein as "reprogrammed somatic
cells" ("RSC") herein, or in some embodiments as induced
pluripotent stem (iPS) cells if reprogrammed to a pluripotent
state. The term "somatic cell" refers to any cell other then a gem
cell, a cell present in or obtained from e pre-implantation embryo,
or a cell resulting from proliferation of such a cell in vitro. In
some embodiments the somatic cell is a "non-embryonic somatic
cell", by which is meant a somatic cell that is not present in or
obtained (torn an embryo and does not result from proliferation of
such a cell in vitro, to some embodiments the somatic cell is an
"adult somatic cell", by which is meant a cell that is present in
or obtained from an organism other than an embryo or a fetus or
results from proliferation of such a cell in vitro. Unless
otherwise indicated the methods for reprogramming cell to a less
differentiated state are performed in vitro, i.e., they are
practiced using isolated somatic cells maintained in culture.
[0040] The invention encompasses the recognition that naturally
occurring signaling molecules that modulate the expression of
endogenous ES cell transcription factors are promising candidates
for soluble agents that enhance reprogramming. The invention also
encompasses the recognition that modulating foe biological pathways
with which such naturally occurring signaling molecules internet is
of use to enhance (e.g., increase speed and/or efficiency of)
reprogramming. The invention also encompasses the recognition that
agents (whether naturally occurring or synthetic, e.g., small
molecules) that modulate the biological pathways with which such
naturally occurring signaling molecules interact, are promising
candidates for soluble agents that enhance reprogramming.
[0041] As described in more detail below, certain embodiments of
the invention are based at least in pert on the recognition that
modulating, e.g., activating, the Wnt pathway is of use in
reprogramming somatic cells. Certain of the methods comprising
increasing activity of the Wnt pathway in somatic cells such that
at least some of the cell become reprogrammed, e.g., to a
pluripotent state. Certain of the methods comprise culturing
somatic cells in Wnt conditioned medium such that at least some of
the cells become reprogrammed, e.g., to a pluripotent state.
[0042] Reprogramming, as used herein, refers to a process that
alters or reverses the differentiation state of a somatic cell. The
cell can be either partially or terminally differentiated prior to
reprogramming. Reprogramming encompasses complete reversion of the
differentiation state of a somatic cell to a pluripotent state. As
known in the art, a "pluripotent" cell has the ability to
differentiate into or give rise to cells derived from all three
embryonic germ layers (endoderm, mesoderm and ectoderm) and
typically has the potential to divide in vitro for a long period of
time, e.g., greater than one year or more than 30 passages. ES
cells are an example of pluripotent cells. Reprogramming also
encompasses partial reversion of the differentiation state of a
somatic cell to a multipotent state. A "multipotent" cell is a cell
that is able to differentiate into some hot not all of the cells
derived from all three germ layers. Thus, a multipotent cell is a
partially differentiated cell. Adult stem cells are multipotent
cells. Adult stem cells include, for example, hematopoietic stem
cells and neural stem cells. Reprogramming also encompasses partial
reversion of the differentiation state of a somatic cell to a state
that renders the cell more susceptible to complete reprogramming to
a pluripotent state when subjected to additional manipulations such
as those described herein. Such contacting may result in expression
of particular genes by the cells, which expression contributes so
reprogramming. In certain embodiments of the invention,
reprogramming of a somatic cell causes the somatic cell to assume a
pluripotent, ES-like state. The resulting cells are referred to
herein as reprogrammed pluripotent somatic cells or induced
pluripotent stem (iPS) cells.
[0043] Reprogramming involves alteration, e.g., reversal, of at
least some of the heritable patterns of nucleic acid modification
(e.g., methylation), chromatin condensation, epigenetic changes,
genomic imprinting, etc., that occur during cellular
differentiation as a zygote develops into an adult Reprogramming is
distinct from simply maintaining the existing undifferentiated
state of a cell that is already pluripotent or maintaining the
existing less than fully differentiated state of a cell that is
already a multipotent cell (e.g., a hematopoietic stem cell).
Reprogramming is also distinct (tom promoting the self-renewal or
proliferation of cells that are already pluripotent or multipotent,
although the compositions and methods of the invention may also be
of use for such purposes. Certain of the compositions and methods
of the present invention contribute to establishing the pluripotent
state. The methods may be practiced on cells that fully
differentiated and/or restricted to giving rise only to cells of
that particular type, rather than on cells that are already
multipotent or pluripotent.
[0044] Somatic cells are treated many of a variety of ways to cause
reprogramming according to the methods of the present invention.
The treatment can comprise contacting the cells with one or more
agent(s) that contribute to reprogramming ("reprogramming agent").
Such contacting may be performed by maintaining the cell in culture
medium comprising the agent(s). In some embodiments the somatic
cells are genetically engineered. The somatic cell may be
genetically engineered to express one or more reprogramming agents
as described further below.
[0045] In the methods of the present invention somatic cells may,
in general, be cultured under standard conditions of temperature,
pH, and other environmental conditions, e.g., as adherent cells in
tissue culture plates at 37.degree. C. in an atmosphere containing
5-10% CO.sub.2. The cells and/or the culture medium are
appropriately modified to achieve reprogramming as described
herein, to certain embodiments, the somatic cells are cultured on
or in the presence of a material that mimics one or more features
of the extracellular matrix or comprises one or more extracellular
matrix or basement membrane components. In some embodiments
Matrigel.TM. is used. Other materials include proteins or mixtures
thereof such as gelatin, collagen, fibronectin, etc. In certain
embodiments of the invention the somatic cells are cultured in the
presence of a feeder layer of cells. Such cells may, for example,
be of murine or human origin. They may be irradiated, chemically
inactivated by treatment with a chemical inactivator such as
mitomycin c, or otherwise treated to inhibit their proliferation if
desired. In other embodiments the somatic cells are cultured
without feeder cells.
[0046] Generating pluripotent or multipotent cells by somatic cell
reprogramming using the methods of the present invention has a
number of advantages. First, the methods of the present invention
allow one to generate autologous pluripotent cells, which are cells
specific to and genetically matched with an individual. The cells
are derived from somatic cells obtained from the individual. In
general, autologous cells are less likely than non-autologous cells
to be subject to immunological rejection. Second, the methods of
the present invention allow the artisan to generate pluripotent
cells without using embryos, oocytes, and/or nuclear transfer
technology. Applicants' results demonstrate that (i) somatic cells
can be reprogrammed to an ES-tike state without the need to
engineer the cells to express an oncogene such as c-Myc; and (ii)
reprogramming of somatic cells can at least in part be effected by
means other than engineering the cells to express reprogramming
factors, i.e., by contacting the cells with a reprogramming agent
other than a nucleic acid or viral vector capable of being taken up
and causing a stable genetic modification to the cells. In
particular, the invention encompasses the recognition that
extracellular signaling molecules, e.g., molecules that when
present extracellularly bind to cell surface receptors and activate
intracellular signal transduction cascades, are of use to reprogram
somatic cells. The invention Anther encompasses the recognition
that activation of such signaling pathways by means other than the
application of extracellular signaling molecules is also of use to
reprogram somatic cells. In addition, the methods of the present
invention enhanced the formation of colonies of BS-like cells that
were detectable based on morphological criteria, without the need
to employ a selectable marker. The present disclosure thus reflects
several fundamentally important advances in the area of in vitro
somatic cell reprogramming technology. While certain aspects of the
invention are exemplified herein using Wnt pathway signaling, the
methods of the invention encompass activation of other signaling
pathways for purposes of reprogramming somatic cells.
[0047] Definitions of certain terms useful for understanding
aspects of the invention are presented below:
[0048] "Agent" as used herein means any compound or substance such
as, but not limited to, a small molecule, nucleic acid,
polypeptide, peptide, drug, ion, etc.
[0049] A "cell culture medium" (also referred to herein as a
"culture medium" or "medium") is a medium for culturing edit
containing nutrients that maintain cell viability and support
proliferation. The cell culture medium may contain any of the
following in an appropriate combination: salt(s), buffer(s) amino
acids, glucose or other sugar(s), antibiotics, serum or serum
replacement, and other components such as peptide growth factors,
etc. Cell culture media ordinarily used for particular cell types
are known to those skilled in the art. Some son-limiting examples
are provided herein.
[0050] "Cell line" refers to a population of largely or
substantially identical cells that has typically been derived from
a single ancestor cell or from a defined and/or substantially
identical population of ancestor cells. The cell line may have been
or may be capable of being maintained in culture for an extended
period (e.g., months, years, for an unlimited period of time). It
may have undergone a spontaneous or induced process of
transformation conferring an unlimited culture lifespan on the
cells. Cell lines include all those cell Knee recognized in the art
as such. It will be appreciated that cells acquire mutations and
possibly epigenetic charges over time such that at least some
properties of individual cells of a cell line may differ with
respect to each other.
[0051] The term "exogenous" refers to a substance present in a cell
or organism other than its native source. For example, the terms
"exogenous nucleic acid" or "exogenous protein" refer to a nucleic
acid or protein that has been introduced by a process involving the
hand of man into s biological system such as a cell or organism in
which it is not normally found or In which it is found in lower
amounts. A substance will be considered exogenous if it is
introduced into a cell or an ancestor of the cell that inherits the
substance. In contrast, the term "endogenous" refers to a substance
feat is native to the biological system.
[0052] "Expression" refers to the cellular processes involved in
producing RNA and proteins and as appropriate, secreting proteins,
including where applicable, but not limited to, for example,
transcription, translation, folding, modification and processing.
"Expression products" include RNA transcribed from a gene and
polypeptides obtained by translation of mRNA transcribed from a
gene.
[0053] A "genetically modified" or "engineered" cell as used herein
refers to a cell into which an exogenous nucleic acid has been
introduced by a process involving the hand of man (or a descendant
of such a cell feat has inherited at least a portion of the nucleic
acid). The nucleic acid may for example contain a sequence that is
exogenous to the cell, it may contain native sequences (i.e.,
sequences naturally found in the cells) but in a non-naturally
occurring arrangement (e.g., a coding region linked to a promoter
from a different gene), or altered versions of native sequences,
etc. The process of transferring the nucleic into the cell can be
achieved by any suitable technique. Suitable techniques include
calcium phosphate or lipid-mediated transfection, electroporation,
and transduction or infection using a viral vector. In some
embodiments the polynucleotide or a portion thereof is integrated
into the genome of the cell. The nucleic acid may have subsequently
been removed or excised from the genome, provided that such removal
or excision results in a detectable alteration in the cell relative
to an unmodified but otherwise equivalent cell.
[0054] "Identity" refers to the extent to which the sequence of two
or more nucleic acis or polypeptides is the same. The percent
identity between a sequence of interest and a second sequence over
a window of evaluation, e.g., over the length of the sequence of
interest, may be computed by aligning the sequences, determining
the number of residues (nucleotides or amino acids) within the
window of evaluation that are opposite an identical residue
allowing the introduction of gaps to maximize identity, dividing by
the total number of residues of the sequence of interest or the
second sequence (whichever is greater) dial fill within the window,
and multiplying by 100. When computing the number of identical
residues needed to achieve a particular percent identity, fractions
are to be rounded to the nearest whole number. Percent identity can
be calculated with the use of a variety of computer programs known
in the art. For example, computer programs such as BLAST2, BLASTN,
BLASTP, Gapped BLAST, etc., generate alignments and provide percent
identity between sequences of interest. The algorithm of Karlin and
Altschul (Karlin and Altschul, Proc. Natl. Acad. Sci. USA
87:22264-2268, 1990) modified as in Karlin and Altschul, Proc.
Natl. Acad. Sci. USA 90:5873-5877, 1993 is incorporated into the
NBLAST and XBLAST programs of Altschul et al. (Altschul, et al., J.
Mol. Biol. 215:403-410, 1990). To obtain gapped alignments for
comparison proposes, Gapped BLAST is utilized as described in
Altschul et al. (Altschul, et al. Nucleic Acids Res. 25: 3389-3402,
1997). When utilizing BLAST and Gapped BLAST programs; the default
parameters of the respective programs may be used. A PAM250 or
BLOSUM62 matrix may be used. Software for performing BLAST analyses
is publicly available through the National Center for Biotechnology
Information (NCBI). See the Web site having URL
www.ncbi.nlm.nih.gov for these programs. In a specific embodiment,
percent identity is calculated using BLAST2 with default parameters
as provided by the NCBI.
[0055] "Isolated" or "partially purified" as wed herein refers, in
the case of a nucleic acid or polypeptide, to a nucleic acid or
polypeptide separated from at least one other component (e.g.,
nucleic acid or polypeptide) that it present with the nucleic acid
or polypeptide as found in its natural source and/or that would be
present with the nucleic acid or polypeptide when expressed by a
cell, or secreted in the case of secreted polypeptides. A
chemically synthesized nucleic acid or polypeptide or one
synthesized using in vitro transcription/translation is considered
"isolated". An "isolated cell" is a cell that has been removed from
an organism in which it was originally found or a descendant of
such a cell. Optionally the cell has been cultured hi vitro, e.g.,
in the presence of other cells. Optionally the cell is later
introduced into a second organism or reintroduced into the organism
from which it (or the cell from which it is descended) was
isolated.
[0056] The term "gene whose function is associated with
pluripotency", as used herein, refers to a gene whose expression
under normal conditions (e.g., in the absence of genetic
engineering or other manipulation designed to alter gene
expression) occurs in and is typically restricted to pluripotent
stem cells, and is crucial for their functional identity as such.
It will be appreciated that the polypeptide encoded by a gene
functionally associated with pluripotency may be present as a
maternal factor in the oocyte. The gene may be expressed by at
least some cells of the embryo, e.g., throughout at least a portion
of the preimplantation period and/or in germ cell precursors of the
adult.
[0057] "Modulate" is used consistently with its use in the art,
i.e., meaning to cause or facilitate a qualitative or quantitative
change, alteration, or modification in a process, pathway, or
phenomenon of interest. Without limitation, such change may be an
increase, decrease, or change in relative strength or activity of
different components or branches of the process, pathway, or
phenomenon. A "modulator" is an agent that causes or facilitates a
qualitative or quantitative change, alteration, or modification in
a process, pathway, or phenomenon of interest.
[0058] The term "pluripotency factor" is used refer to the
expression product of a gene whose function is associated with
pluripotency, e.g., a polypeptide encoded by the gene. In some
embodiments the pluripotency factor is one that is normally
substantially not expressed in somatic cell types that constitute
the body of an adult animal (with the exception of germ cells or
precursors thereof). For example, the pluripotency Actor may be one
whose average level in ES edit is at least 50-fold or 100-fold
greater then its avenge level in those terminally differentiated
cell types present in the body of an adult mammal. In some
embodiments, the pluripotency factor is one that is essential to
maintain the viability or pluripotent state of ES cells in vivo
and/or ES cells derived using conventional methods. Thus if the
gene encoding the factor is knocked out or inhibited (i.e., its
expression is eliminated or substantially reduced), the ES cells
are not formed, die or, in some embodiments, differentiate. In some
embodiments, inhibiting expression of a gene whose function is
associated with pluripotency in an ES cell (resulting in, e.g., a
reduction in the average steady state level of RNA transcript
and/or protein encoded by the gene by at least 50%, 60%, 70%, 80%,
90%, 95%, or more) insults in a cell that is viable but no longer
pluripotent. In some embodiments the gene is characterized in that
its expression in an ES cell decreases (resulting in, e.g., a
reduction m the average steady state level of RNA transcript and/or
protein encoded by the gene by at least 50%, 60%, 70%, 80%, 90%,
95%, or more) when the cell differentiates into a terminally
differentiated cell.
[0059] A "pluripotency inducing gene", as used herein, refers to a
gene whose expression, contributes to reprogramming somatic cells
to a pluripotent state. "Pluripotency inducing factor" refers to an
expression product of a pluripotency inducing gene. A pluripotency
inducing factor may, but need not be, a pluripotency factor.
Expression of an exogenously introduced pluripotency inducing
factor may be transient, i.e., it may be needed during at least a
portion of the reprogramming process in order to induce
pluripotency and/or establish a stable pluripotent stele but
afterwards not required to maintain pluripotency. For example, the
factor may induce expression of endogenous genes whose function is
associated with pluripotency. These genes may then maintain the
reprogrammed cells in a pluripotent state.
[0060] "Polynucleotide" is used herein interchangeably with
"nucleic acid" to indicate a polymer of nucleosides. Typically a
polynucleotide of this invention is composed of nucleosides that
are naturally found in DNA or RNA (e.g., adenosine, thymidine,
guanosine, cytidine, uridine, deoxyadenosine, deoxythymidine,
deoxyguanosine, and deoxycytidine) joined by phosphodiester bonds.
However the term encompasses molecules comprising nucleosides or
nucleoside analogs containing chemically or biologically modified
bases, modified backbones, etc., whether or not found in naturally
occurring nucleic acids, and such molecules may be preferred for
certain applications. Where this application refers to a
polynucleotide it is understood that both DNA, RNA, and in each
case both single- and double-stranded forms (and complements of
each single-stranded molecule) are provided. "Polynucleotide
sequence" as used herein can refer to the polynucleotide material
itself and/or to the sequence information (i.e. the succession of
letters used as abbreviations for bases) that biochemically
characterizes a specific nucleic acid. A polynucleotide sequence
presented herein is presented in a 5' to 3' direction unless
otherwise indicated.
[0061] "Polypeptide" refers to a polymer of amino acids. The terms
"protein" and "polypeptide" are used interchangeably heroin. A
peptide is a relatively short polypeptide, typically between about
2 and 60 amino acids in length. Polypeptides used herein typically
contain amino acids such as the 20 L-amino acids that are most
commonly found in proteins. However, other amino acids and/or amino
acid analogs known in the art can be used. One or more of the amino
acids in a polypeptide may be modified, for example, by the
addition of a chemical entity such as a carbohydrate group, a
phosphate group, a fatty acid group, a linker for conjugation,
functionalization, etc. A polypeptide that has a nonpolypeptide
moiety covalently or noncovalently associated therewith is still
considered a "polypeptide". Exemplary modifications include
glycosylation and palmitoylation. Polypeptides may be purified from
natural sources, produced using recombinant DMA technology,
synthesized through chemical means such as conventional solid phase
peptide synthesis, etc. The term "polypeptide sequence" or "amino
acid sequence" as used herein can refer to the polypeptide material
itself and/or to the sequence information (i.e., the succession of
letters or three letter codes used as abbreviations for amino add
names) that biochemically characterizes a polypeptide. A
polypeptide sequence presented herein is presented in an N-terminal
to C-terminal direction unless otherwise indicated.
[0062] "Polypeptide variant" refers to any polypeptide differing
from a naturally occurring polypeptide by amino acid insertion(s),
deletion(s), and/or substitution(s), Variants may be naturally
occurring or created using, e g, recombinant DNA techniques or
chemical synthesis. In some embodiments amino acid "substitutions"
are the result of replacing one amino acid with another amino acid
having similar structural and/or chemical properties, i.e.,
conservative amino acid replacements. "Conservative" amino acid
substitutions may be made on the basis of similarity in any of a
variety or properties such as side chain size, polarity, charge,
solubility, hydrophobicity, hydrophilicity, and/or amphipathicity
of the residues involved. For example, the non-polar (hydrophobic)
amino acids include alanine, leucine, isoleucine, valine, glycine,
proline, phenylalanine, tryptophan and methionine. The polar
(hydrophilic), neutral amino acids include serine, threonine,
cysteine, tyrosine, asparagine, and glutamine. The positively
charged (basic) amino acids include arginine, lysine and histidine.
The negatively charged (acidic) amino acids include aspartic acid
and glutamic acid. Insertions or deletions may range in size from
about 1 to 20 amino acids, e.g., 1 to 10 amino acids. In some
instances larger domains may be removed without substantially
affecting function. In certain embodiments of the invention the
sequence of a variant can be obtained by malting no more than a
total of 5, 10, 15, or 20 amino acid additions, deletions, or
substitutions to the sequence of a naturally occurring enzyme. In
some embodiments not more than 1%, 5%, 10%, or 20% of the amino
acids in a polypeptide are insertions, deletions, or substitutions
relative to the original polypeptide. Guidance in determining which
amino acid residues may be replaced, added, or deleted without
eliminating or substantially reducing activities of interest, may
be obtained by comparing the sequence of the particular polypeptide
with that of homologous polypeptides (e.g., from other organisms)
and minimizing the number of amino acid sequence changes made in
regions of high homology (conserved regions) or by replacing amino
acids with those found in homologous sequences since amino acid
residues that are conserved among various species are more likely
to be important for activity than amino acids that are not
conserved.
[0063] "Purified" or "substantially purified" as used herein denote
that the indicated nucleic acid or polypeptide is present in the
substantial absence of other biological macromolecules, e.g.,
polynucleotides, proteins, and the like. In one embodiment, the
polynucleotide or polypeptide is purified such that it constitutes
at least 90% by weight, e.g., at least 9556 by weight, e.g., at
least 99% by weight, of the polynucleotide(s) or polypeptide(s)
present (but water, buffers, ions, and other small molecules,
especially molecules having a molecular weight of leas than 1000
daltons, can be present).
[0064] "RNA interference" is used herein consistently with its
meaning in the art to refer to a phenomenon whereby double-stranded
RNA (dsRNA) triggers the sequence-specific degradation or
translational repression of a corresponding mRNA having
complementarity to a strand of the dsRNA. It will be appreciated
that the complementarity between the strand of the dsRNA and the
mRNA need not be 100% but need only be sufficient to mediate
inhibition of gene expression (also referred toss "silencing"
or"knockdown"). For example, the degree of complementarity is such
that the strand can either (i) guide cleavage of the mRNA in the
RNA-induced silencing complex (RISC); or (ii) cause translational
repression of the mRNA. In certain embodiments the double-stranded
portion of the RNA is less than about 30 nucleotides in length,
e.g., between 17 and 29 nucleotides in length. In mammalian cells,
RNAi may be achieved by introducing an appropriate double-stranded
nucleic acid into the cells or expressing a nucleic acid in cells
that is then processed intracellularly to yield dsRNA therein.
Nucleic acids capable of mediating RNAi are referred to herein as
"RNAi agents". Exemplary nucleic acids capable of mediating RNAi
area short hairpin RNA (shRNA), a short interfering RNA (siRNA),
and a microRNA precursor. These terms are well known and are used
herein consistently with their meaning in the art. siRNAs typically
comprise two separate nucleic acid strands that are hybridized to
each other to form a duplex. They can be synthesized. In vitro,
e.g., using standard nucleic acid synthesis techniques. They can
comprise a wide variety of modified nucleosides, nucleoside analogs
and can comprise chemically or biologically modified bases,
modified backbones, etc. Any modification recognized in the art as
being useful for RNAi can be used. Some modifications result in
increased stability, cell uptake, potency, etc. In certain
embodiments the siRNA comprises a duplex about 19 nucleotides in
length and one or two 3' overhangs of 1-5 nucleotides in length,
which may be composed of deoxyribonucleotides. shRNA comprise
single nucleic acid strand that contains two complementary portions
separated by a predominantly non-selfcomplementary region. The
complementary portions hybridize to form a duplex structure and the
non-selfcomplementary region forms a loop connecting the 3' end of
one strand of the duplex and the 5' end of the other strand. shRNAs
undergo intracellular processing to generate siRNAs.
[0065] MicroRNAs (miRNAs) are small, non-coding, single-stranded
RNAs of about 21-25 nucleotides (in mammalian systems) that inhibit
gene expression in a sequence-specific manner. They are generated
intracellularly from precursors having a characteristic secondary
structure comprised of a short hairpin (about 70 nucleotides in
length) containing a duplex that often includes one or more regions
of imperfect complementarity. Naturally occurring miRNAs are only
partially complementary to their target mRNA and typically act via
transitional repression. RNAi agents modelled on endogenous
microRNA precursors are of use in the invention. In some
embodiments, a sequence encoding the stem portion of a stem-loop
structure or encoding a complete stem-loop can be inserted into a
nucleic acid comprising at least a portico of an endogenous
microRNA primary transcript, e.g., in place of the sequence that
encodes the endogenous microRNA or minimum (.about.70 nucleotide)
microRNA hairpin.
[0066] "Reprogramming factor" refers to a gene, RNA, or protein
that promotes or contributes to cell reprogramming, e.g., in vitro.
In aspects of the invention relating to reprogramming factor(s),
the invention provides embodiments in which the reprogramming
factor(s) are of interest for reprogramming somatic cells to
pluripotency in vitro. Examples of reprogramming factors of
interest for reprogramming somatic cells to pluripotency in vitro
are Oct4, Nanog, Sox2, Lin28, Klf4, c-Myc, and any gene/protein
that can substitute for one or more of these in a method of
reprogramming somatic cells in vitro. "Reprogramming to a
pluripotent state in vitro", or "reprogramming to pluripotency in
vitro", is used herein to refer to in vitro reprogramming methods
that do not require and typically do not include nuclear or
cytoplasmic transfer or cell fusion, e.g., with oocytes, embryos,
gam cells, or pluripotent cells. Any embodiment or claim of the
invention may specifically exclude compositions or methods relating
to or involving nuclear or cytoplasmic transfer or cell fusion,
e.g., with oocytes, embryos, germ cells, or pluripotent cells.
[0067] "Selectable marker" refers to a gene, RNA, or protein that
when expressed, confers upon cells s selectable phenotype, such as
resistance to a cytotoxic or cytostatic agent (e.g., antibiotic
resistance), nutritional prototrophy, or expression of a particular
protein that can be used as a basis to distinguish cells that
express the protein from cells that do not. Proteins whose
expression can be readily detected such as a fluorescent or
luminescent protein or an enzyme that acts on a substrate to
produce a colored, fluorescent, or luminescent substance
("detectable markers") constitute a subset of selectable markets.
The presence of a selectable marker linked to expression control
elements native to a gene that is normally expressed selectively or
exclusively in pluripotent cells makes it possible to identify and
select somatic cells that have been reprogrammed to a pluripotent
state. A variety of selectable marker genes can be used, such as
neomycin resistance gene (neo), puromycin resistance gene (pure),
guanine phosphoribosyl transferase (gpt), dihydrofolate reductase
(DHPR), adenosine deaminase (ada), puromycin-N-acetyltransferase
(PAC), hygromycin resistance gene (hyg), multidrug resistance gene
(mdr), thymidine kinase (TK), hypoxanthine-guanine
phosphoribosyltransferase (HPRT), and hisD gene. Detectable markers
include green fluorescent protein (GFP) blue, sapphire, yellow,
red, orange, and cyan fluorescent proteins and variants of any of
these. Luminescent proteins such as luciferase (e.g., firefly or
Renilla luciferase) are also of use. As will be evident to one of
skill in the art, the term "selectable marker" as used herein can
refer to a gene or to an expression product of the gene, e.g., an
encoded protein.
[0068] In some embodiments the selectable marker confers a
proliferation and/or survival advantage on cells that express it
relative to cells that do not express it or that express it at
significantly lower levels. Such proliferation and/or survival
advantage typically occurs when the cells are maintained under
certain conditions, i.e., "selective conditions". To ensure an
effective selection, a population of cells can be maintained for a
under conditions and for a sufficient period of time such that
cells that do not express the marker do not proliferate and/or do
not survive and are eliminated from the population or their number
is reduced to only a very small friction of the population. The
process of selecting cells that express a marker that confers a
proliferation and/or survival advantage by maintaining a population
of cells under selective conditions so as to largely or completely
eliminate cells that do not express the marker is referred to
hereto as "positive selection", and the marker is said to be
"useful for positive selection". Negative selection and marker
useful for negative selection are also of interest m certain of the
methods described herein. Expression of such markers confers a
proliferation and/or survival disadvantage on cells that express
the marker relative to cell that do not express the marker or
express it at significantly lower levels (or, considered another
way, cells that do not express the marker have a proliferation
and/or survival advantage relative to cells that express the
marker). Cells that express the marker can therefore be largely or
completely eliminated from a population of cells when maintained in
selective conditions for a sufficient period of time.
[0069] The terms "treat", "treating", "treatment", etc., as applied
to an isolated cell, include subjecting the cell to any kind of
process or condition or performing any kind of manipulation or
procedure on the cell. As applied to a subject, the terms refer to
providing medical or surgical attention, care, or management to an
individual. The individual is usually ill or injured, or at
increased risk of becoming ill relative to an average member of the
population and in need of such attention, care, or management.
[0070] The term "Wnt", or "Wnt protein" as used herein refers to a
polypeptide having a naturally occurring amino acid sequence of a
Wnt protein or a fragment, variant, or derivative thereof that at
least in part retains the ability of the naturally occurring
protein to bind to Wnt receptors) and activate Wnt signaling. In
addition to naturally-occurring allelic variants of the Wnt
sequences that may exist in the population, it will be appreciated
that, as is the case for virtually all proteins, a variety of
changes can be introduced into the sequences listed under the
accession numbers in Table 1 (referred to as "wild type" sequences)
without substantially altering the functional (biological) activity
of the polypeptides. Such variants are included within the scope of
the term "Wnt", "Wnt protein", etc.
[0071] The variant could be, e.g., a polypeptide at least 80%, 85%,
90%, 95%, 98%, or 99% identical to frill length Wnt. The variant
could be a fragment of fully length Wnt. The variant could be a
naturally occurring splice variant. The variant could be a
polypeptide at least 80%, 83%, 90%, 95%, 98%, or 99% identical to a
fragment of Writ, wherein the fragment is at least 50%, 60%, 70%,
80%, 85%, 90%, 95%, 98%, or 99% as long as the full length wild
type polypeptide or a domain thereof having an activity of interest
such as the ability to bind to a Wnt receptor. In some embodiments
the domain is at least 100, 200, 300, or 400 amino acids in length,
beginning at any amino acid position in the sequence and extending
toward the C-terminus. Variations known in the art to eliminate or
substantially reduce the activity of the Wnt protein are preferably
avoided. In some embodiments, the variant lacks an N- and/or
C-terminal portion of the fell length polypeptide, e.g., up to 10,
20, or 50 amino acids from either terminus is lacking. In some
embodiments the polypeptide has the sequence of a mature Wnt
polypeptide, by which is meant a Wnt polypeptide that has had one
or more portions such as a signal peptide removed during normal
intracellular proteolytic processing (e.g., during co-translational
or post-translational processing). In some embodiments wherein the
Wnt protein is produced other than by purifying it from cells that
naturally express it, the protein is a chimeric polypeptide, by
which is meant that it contains portions from two or more different
species, lit some embodiments wherein the Wnt protein is produced
other than by purifying it from cells that naturally express it,
the protein is a Wnt derivative, by Wnt is meant that the protein
comprises additional sequences not related to Wnt so long as those
sequences do not substantially reduce the biological activity of
the protein.
[0072] One of skill in the art will be aware of, or will readily be
able to ascertain, whether a particular Wnt variant, fragment, or
derivative is functional using assays known in the art. For
example, the ability of a variant of a Wnt polypeptide to bind to a
Wnt receptor can be assessed using standard protein binding assays.
Convenient assays include measuring the ability to activate
transcription of a reporter construct containing a TCP binding site
operably linked to a nucleic acid sequence encoding a delectable
marker such as luciferase. One assay involves determining whether
the Wnt variant induces phosphorylation of .beta.-catenin.
Phosphorylation status can be determined using any suitable method,
e.g., immunoblotting. Other assays involve testing fee variant or
fragment for known biological activities of Wnt. See, e.g., Barker,
N. and Clevers, H., Nat Rev Drag Discov. 5(12):997-1014, 2006,
which describes assays suitable for identifying agents that
modulate Wnt pathway activity. Such essay may readily be adapted to
identity or confirm activity of agents that activate Wnt pathway
activity. In certain embodiments of the invention a functioned
variant or fragment has at least 50%, 60%, 70%, 80%, 90%, 95% or
more of the activity of the full length wild type polypeptide.
[0073] "Wnt pathway activity" or "Wnt signaling" refers to the
series of biochemical events that ensues following binding of a
stimulatory ligand (e.g., a Wnt protein) to a receptor for a Wnt
family member, ultimately leading to changes in gene transcription
and, if in vivo, often leading to a characteristic biological
effect in an organism.
[0074] Reprogramming Somatic Cells by Activating the Wnt
Pathway
[0075] The present invention provides the recognition that
activating the Wnt pathway is of use to reprogram somatic cells.
The invention provides tire additional recognition that activating
the Wnt pathway increases the efficiency of reprogramming of
somatic cells, e.g., when such cells are subjected to a treatment
that would result in reprogramming of at least some cells.
"Increase the efficiency of reprogramming" means to cause an
increase in the percentage of cells that undergo reprogramming when
a population of cells is subjected to a reprogramming treatment,
typically resulting in a greater number of individual colonies of
reprogrammed cells after a given time period. In some embodiments
of the invention, activating the Wnt pathway according to the
invention increases the number of reprogrammed cells and/or the
number of colonies of reprogrammed cells and/or the percentage of
cells that undergo reprogramming. The invention further provides
the recognition that activating the Wnt pathway enables
reprogramming of somatic cells that have not been genetically
modified to increase their expression of an oncogene such as c-Myc.
The invention thus provides ways to substitute for engineered
expression of c-Myc in any method of reprogramming somatic cells
that would otherwise involve engineering cells to express c-Myc. In
some embodiments of the invention, activating the Wnt pathway is
sufficient to allow reprogramming under conditions in which
reprogramming would not otherwise occur.
[0076] The invention provides methods for generating reprogrammed
somatic edit comprising modulating, e.g., increasing, activity of
the Wnt pathway. The invention further provides compositions of use
in the methods. In one aspect, the invention provides a method of
reprogramming a somatic cell comprising modulating, e.g.,
increasing Wnt pathway activity in the cell. The invention farther
provides improved methods for reprogramming of somatic cells, the
method comprising subjecting somatic cells to a treatment that may
reprogram at least some of the cells, wherein the improvement
comprises increasing the activity of a Wnt pathway in said cells.
The treatment may be any treatment known in the art to be of use to
reprogram somatic cells or considered to be of potential use far
this purpose. In certain embodiments of the invention Wet pathway
activity is increased using activators of the Wnt pathway such as
small molecules, soluble Wnt proteins, or agents that mediate RNA
interference and thereby inhibit endogenous inhibitors of the Wnt
pathway. In certain embodiments somatic cells to be reprogrammed
are cultured in Wnt conditioned medium. In any of the embodiments
of the invention, unless otherwise indicated or evident from the
context, "reprogramming" can refer to reprogramming to a
pluripotent state.
[0077] Wnts are a family of secreted proteins important for a wide
array of developmental and physiological processes (Mikels, A J and
Nusse, R., Oncogene, 25: 7461-7468, 2006). Wnts are related to one
another in sequence and strongly conserved in structure and
function across multiple species. Thus a Wnt protein displaying
activity in one species may be used in other species to activate
the Wnt pathway in such species and may be expected to display
similar activity. Wnt family members include Wnt1, Wnt2, Wnt2b
(also called Wnt13), Wnt3, Wnt3a, Wnt4, Wnt5a, Wnt5b, Wnt6, Wnt7a,
Wnt7b, Wnt7c, Wnt8, Wnt8a, Wnt8b, Wnt5c, Wnt10a, Wnt10b, Wnt11,
Wnt14, Wnt15, or Wnt16. Sequences of Wnt genes and proteins are
known in the art. One of skill in the art can readily find the Gene
ID, accession numbers, and sequence information for Wnt family
members and other genes and proteins of interest herein in publicly
available databases (see Table 1 for examples).
TABLE-US-00001 TABLE 1 Wnt pathway proteins, effectors, and
regulators Gene Gene ID Accession numbers (mRNA/protein) Wnt3a
(mouse) 22416 NM_009522/NP_033548 Wnt3a (human) 89780
NM_033131/NP_149122 .beta.-catenin (mouse) 12387
NM_007614/NP_031640 .beta.-catenin (human) 1499
NM_001098209/NP_001091679 NM_001098210/NP_001091680
NM_001904/NP_001895 GSK3.alpha. (mouse) 606496
NM_001031667/NP_001026837 GSK3.alpha. (human) 2931
NM_019884/NP_063937 GSK3.beta. (mouse) 56637 NM_019827/NP_062801
GSK3.beta. (human) 605004 NM_002093/NP_002084 Sox2 (mouse) 20674
NM_011443/NP_035573 Sox2 (human) 6657 NM_003106/NP_003097 KIf4
(mouse) 16600 NM_010637/NP_034767 KIf4 (human) 9314
NM_004235/NP_004226 Oct4 (mouse) 18999 NM_013633/NP_038661 Oct4
(human) 5460 NM_203289/NP_976034 Oct4 (human) 5460
NM_002701/NP_002692 Nanog (mouse) 71950 NM_028016.2/NP_082292.1
Nanog (human) 79923 NM_024865/NP_079141 Lin28 (mouse) 83557
NM_145833/NP_665832 Lin28 (human) 79727 NM_024674/NP_078950
[0078] Wnt signaling is initiated by interaction of Wnt proteins
with a variety of receptors, including members of the Frizzled (Fz)
family of transmembrane receptors and members of the
low-density-lipoprotein receptor-related protein (LRP) family
(e.g., LRP5/LRP6). The extracellular Wnt signal stimulates
intracellular signal transduction cascades including the canonical
pathway, which regulates gene expression in the nucleus (reviewed
by Logan C Y and Nusse, R. Annu, Rev. Cell Dev. Biol., 20:781-810,
2004) and several non-canonical pathways (reviewed by Kohn, A D and
Moon, R T, Cell Calcium, 38:439-446, 2005). Briefly, Wnt signaling
via the canonical pathway leads to stabilization and nuclear
localization of .beta.-catenin, which assembles with members of the
T-cell factor/lymphoid enhancer factor (TCF/LEF) family of
transcription Actors to form complexes that generally activate
transcription. In the absence of Wnt signaling .beta.-catenin is
instead targeted for degradation by the .beta.-catenin destruction
complex, and TCF/LEFs form complexes that generally repress
transcription. In the absence of Wnt signaling, kinases such as
glycogen synthase kinase-3 (GSK3) and casein kinase 1 (CK1)
phosphorylate .beta.-catenin, which as a consequence is ubiquinated
and targeted for destruction by the proteasome. Activation of the
Wnt pathway thus results in diminished phosphorylation of
.beta.-catenin, thereby leading to its stabilization. Several
endogenous proteins have been identified as inhibitors of Wnt
signaling, including Dickkopf (Dkk), breakpoint cluster region
protein (Bcr), proteins comprising a WIF (Wnt inhibitory Actor)
domain etc.
[0079] In certain embodiments of the invention the reprogramming
methods comprise contacting a cell with an agent (that modulates,
e.g., increases, the activity of a Wnt pathway. In some
embodiments, increasing the Wnt pathway induces the cell to become
pluripotent and possess features characteristic of ES cells. The
methods are thus of use to generate pluripotent, ES-like cells (iPS
cells). In certain embodiments of the invention a treatment that
causes increased activity of a Wnt pathway is one that results in
increased intracellular levels of .beta.-catenin. In certain
embodiments of the invention, a treatment that causes increased
activity of a Wnt pathway is one that results in increased nuclear
translocation of .beta.-catenin. In certain embodiments of the
invention, a treatment that causes increased activity of a Wnt
pathway is one capable of causing changes. In gene expression
characteristic of cells exposed to a source of biologically active
Wnt protein. In some embodiments of the invention, reprogramming is
modulated using e Wnt pathway inhibitor.
[0080] A considerable advance towards the goal of reprogramming
somatic cells to a pluripotent state in vitro was achieved when it
was shown that cell lines with some of the properties of ES cells
could be produced by introducing genes encoding four transcription
factors associated with pluripotency, i.e., Oct3/4, Sox2, c-Myc and
Klf4, into mouse skin fibroblasts via retroviral infection, and
then selecting cells that expressed a marker of pluripotency,
Fbx15, in response to these factors (Takahashi, K. & Yamanaka,
S. Cell 126, 663-676, 2006). However, the resulting cells differed
from ES cells in their gene expression and DNA methylation patterns
and when injected into normal mouse blastocysts did not result in
live chimeras (animals carrying cells throughout their bodies from
both the original blastocyst and from the introduced cells).
Subsequent work improved on these results by performing more
rigorous select ion, resulting in derivation of stable reprogrammed
cell lines that, based on reported transcriptional, imprinting
(expression of elides predetermined by die parent from which they
originated) and chromatin-modification profiles, appeared
essentially identical to ES cells (Okita, K., et al., 448, 313-317,
2007; Wernig, M. et al. Nature 448, 318-324, 2007; Maherali, N. et
al. Cell Stem Cell 1, 33-70, 2007). Somatic cells that have been
reprogrammed to a pluripotent state in vitro using these methods or
other methods (e.g., involving application of small molecules) are
referred to herein consistently with usage in the art as "induced
plan potent stem" (iPS) cells. Subsequently, it was shown that
human somatic cells can also be reprogrammed to pluripotency using
these factors. Furthermore, it was demonstrated that the
combination of Oct4, Nanog, Sox2, and Lin28 was also able to
reprogram somatic cells to a pluripotent state in vitro (Yu J,
Science, 318(5858): 1917-20, 2007). However, generation of these
cells also Involved engineering the cells to express multiple
transcription factors and employed retroviral transduction.
[0081] Applicants have now shown that an increased number of
colonies comprised of ES-like cells developed when somatic cells
genetically engineered to express Oct4, Sox2, Klf4, and c-Myc were
cultured with Wnt3a conditioned medium than when the cells were
cultured in medium conditioned by control cells or in standard cell
culture medium conventionally used for the propagation of ES cells.
Applicants further showed that colonies comprised of ES-like cells
developed when somatic cells engineered to express Oct4, Sox2, and
Klf4 but not modified to express c-Myc were cultured in Wnt3a
conditioned medium, whereas colonies of ES-like cells did not form
within the 20 day time period shown in FIG. 1 when such cells were
cultured in unconditioned medium or medium conditioned by control
cells. In both cases, the colonies displayed morphological features
characteristic of ES cell colonies and expression of a detectable
marker indicative of Oct4 expression. By all criteria tested, the
cells appear to be pluripotent, ES-like cells (iPS cells).
Furthermore, culturing the somatic cells in Wnt3a conditioned
medium appeared to select for reprogrammed cells. The colonies
formed in the presence of Wnt3a conditioned medium appeared more
homogenous than those obtained in the absence of Wnt3a conditioned
medium. The methods are thus of use to facilitate identification of
reprogrammed cells, and optionally to facilitate separation of such
cells from cells that have not become reprogrammed, without the
need for chemical selection relying on so introduced genetic
element such as a gene whose expression product confers drug
resistance or fluorescence. The methods are thus of use to generate
reprogrammed cells that do not carry genetic modifications for
purposes of selection or detection of the reprogrammed cells.
Furthermore, the methods are of use to increase the average
percentage of reprogrammed cells in a colony comprising
reprogrammed cells relative to the average percentage of cells that
would be reprogrammed in the absence of an agent that increases Wnt
pathway activity.
[0082] Applicants and others have noticed that some iPS-like cells
can form without infecting the cells with c-Myc virus. However,
this is a low-efficiency event and could be at least in part a
result of insertional mutagenesis wherein a viral integration event
directly activates c-Myc or c-Myc target gene(s). In Applicants'
experiments, at very toe time points, some colonies were seen on
the plates dun were overexpressing Klf4, Sox2 and Oct4 (without
introducing c-Myc virus), even without Wnt conditioned medium.
Wnt-conditioned medium significantly reduced the time required and
increased the efficiency of the reprogramming process. One aspect
of the invention is that the faster timing of reprogramming
achieved using the methods of the invention will facilitate the use
of transient means of overexpression of pluripotency Inducing
factors for iPS formation (for example, transient transfection)
and/or reprogramming by treating somatic cells with reprogramming
agents such as proteins, small molecules, etc., instead of viral
infection. In addition, Applicants propose that increased
efficiency of iPS formation using the methods of the invention
could be of particular use in reprogramming human cells, either
with or without Myc overexpression.
[0083] Without limitation, the methods are thus of use to Increase
the speed of reprogramming somatic cells to iPS cells. Thus, the
invention provides a method of increasing the speed of
reprogramming somatic cells, comprising culturing a population of
somatic mammalian cells in Wnt conditioned cell culture medium so
that at least acme of the cells are induced to become ES-like cells
within a shorter period of time than would be the case in the
absence of Wnt conditioned medium. The invention also provides a
method of increasing the speed of reprogramming somatic cells
comprising activating the Wnt pathway in a cultured population of
somatic cells so that at least some of the cells are induced to
become ES-like cells within a shorter period of time than would be
the case if the Wnt pathway was not activated. The invention also
provides a method of increasing the speed of reprogramming somatic
cells comprising culturing a population of somatic mammalian cells
in the presence of an agent that increases Wnt pathway activity so
that at least some of the cells are induced to become ES-like cells
within a shorter period of time than would be the case in the
absence of said agent. In some embodiments of the invention, the
period of time is 7 days, while in other embodiments the period of
time is 10, 15, or 20 days. In some embodiments of the invention,
the cells are treated (e.g., genetically engineered) so that they
express Sox2, Klf4, Oct4, and c-Myc at levels greater than would be
the case in the absence of such treatment. In some embodiments of
the invention, the cells are treated so that they overexpress Sox2,
Klf4, and Oct4 at levels greater than would be the case in the
absence of such treatment, but are not genetically engineered to
overexpress c-Myc. One method of treatment is infecting the cells
with viruses (e.g., retrovirus, lentivirus) or transfecting the
cells with viral vectors (e.g., retroviral, lentiviral) that
contain the sequences of the factors operably linked to suitable
expression control elements to drive expression in the cells
following infection or transfection and, optionally integration
into the genome as known in the art. Further details regarding the
compositions and methods of the invention are provided below.
[0084] The invention provides a method of reprogramming a somatic
cell, comprising culturing the cell in Wnt conditioned cell culture
medium so that the cell becomes reprogrammed. In some embodiments,
culturing the cell in Wnt conditioned cell culture medium induces
the cell to become pluripotent and possess features characteristic
of ES cells. The methods are thus of use to generate pluripotent,
ES-like cells (iPS cells). In some embodiments, the Wnt conditioned
cell culture medium comprises Wnt3a conditioned medium.
[0085] The term "conditioned medium" refers to cell culture medium
that has previously been used for culturing cells. A conditioned
medium is characterized in that it contains soluble substances,
e.g., signaling molecules, growth factors, hormones etc., which are
produced by cells during their cultivation and released into the
medium. As used herein, "Wnt conditioned medium" refers to
conditioned medium that has been previously used for culturing
cells that produce and secrete Wnt. The medium may be father
described by reference to a particular Wnt protein produced by the
cells. For example, "Wnt3a conditioned medium" refers to
conditioned medium that has been previously used for culturing
cells that produce Wnt3a. The cells may also produce other Writs in
addition to the particular Wnt specifically referred to. Any
embodiment of the invention employing Wnt conditioned medium may
employ Wnt3a conditioned medium unless otherwise indicated.
[0086] It will be appreciated that certain Wnts have similar
biological activities to Wnt3a and/or are closely related in
sequence to Wnt3a. Conditioned media prepared using cells that
produce such Wnts are used in certain embodiments of the
invention.
[0087] Conditioned medium may be prepared by methods known in the
art. Such methods typically comprise culturing a first population
of cells in a cell culture medium, and then harvesting the medium
(typically without harvesting the cells). The harvested medium may
be filtered to remove cell debris, etc. The conditioned medium
(containing components secreted into the medium by the cells) may
then be used to support the growth of a second population of cells.
The cells are cultured in the medium for sufficient time to allow
adequate concentration of released factors such as Wnt (and/or
consumption of media components) to produce a medium that supports
the reprogramming of somatic cells. In some embodiments, medium is
conditioned by culturing for 24 h at 37.degree. C. However, longer
or shorter periods can be used such as between 24 and 72 hours. The
cells can be used to condition multiple batches medium over
additional culture periods, for as tong as the cells retain their
ability to condition the medium in an adequate fashion for the
desired purpose.
[0088] The medium in which the cells are cultured to produce
conditioned medium may be conventional cell culture medium capable
of maintaining viability of the cells. In some embodiments, the
medium ii chemically defined. In some embodiments, the medium is
similar or identical in composition to medium conventionally used
to culture embryonic stem cells of the same species as the somatic
cells to be reprogrammed using the conditioned medium. The base
medium used for conditioning can have any of a number of different
compositions, depending in part on the types of cells used. The
medium must be able to support culture of the cell line used for
the conditioning of the medium. In some embodiments, medium also
supports culture of somatic cells prior to their being reprogrammed
and, optionally, somatic cells that have been reprogrammed.
However, the conditioned medium can be supplemented with other
components, combined with other medium, etc., after conditioning so
as to tender it suitable for culturing somatic cells and
reprogrammed somatic cells.
[0089] Suitable base media can be made from the following
components: Dulbecco's modified Eagle's medium (DMEM), Invitrogen
Cat. No. 11965-092; Knockout Dulbecco's modified Eagle's medium (KO
DMEM), Invitrogen Cat. No. 10829-018; Ham's F12/50% DMEM basal
medium; 200 mM L-glutamate, Invitrogen Cat No. 15039-027;
non-essential amino acid solution, Invitrogen Cat. No. 11140-050;
beta-mercaptoethanol; human recombinant basic fibroblast growth
factor (bFGF). Exemplary serum-containing ES medium is made with
80% DMEM (typically KO DMEM), 20% defined fetal bovine serum (FBS)
not heat inactivated, 1% non-essential amino acids, 1 mM L
glutamine, and 0.1 mM .beta.-mercaptoethanol. The medium is
filtered and stored at 4.degree. C. for no longer than 2 weeks.
Scrum-free ES medium may be prepared with 80% KO DMEM, 20% scrum
replacement, 1% non-essential amino acids, 1 mM L-glutamine, and
0.1 mM .beta.-mercaptoethanol and a serum replacement such as
Invitrogen Cat No. 10828-028. The medium is filtered and stored at
4.degree. C. Before combining with the cells used for conditioning,
human bFGF can be added to a final concentration of 4 ng/mL. Stem
Pro.RTM. hESC SFM (Invitrogen Cat. No. A1000701), a fully defined,
serum- and feeder-free medium (SFM) specialty formulated for the
growth and expansion of human embryonic stem cells, is of use.
[0090] The cells used to prepare the conditioned medium may
naturally produce Wnt. In some embodiments the cells used to
prepare the medium are genetically engineered to increase their
expression of Wnt, e.g., by transfecting them with a cDNA encoding
Wnt, wherein the Wnt coding sequence is operably linked to
expression control sequences active in the cells. See, e.g., Cai,
L, et al., Cell Res. 17:62-72, 2007. In some embodiments, the cells
produce and secrete Wnt into their medium resulting in medium
having a concentration of between 100 ng/ml and 1000 ng/ml Wnt
protein. In some embodiments, the cells produce and secrete Wnt
into their medium resulting in medium having a concentration of
between 200 ng/ml and 500 ng/ml Wnt protein. Cells that overexpress
Wnt could also be used as feeder cells for purposes of
reprogramming somatic cells.
[0091] Conditioned medium may be combined with unconditioned medium
prior to use. For brevity, the resulting medium is still referral
to as conditioned medium if it comprises at least 5% conditioned
medium by volume. In some embodiments the amount (by volume) of
conditioned medium is at least 1096, e.g., at least 10%, 20%, 30%,
40%, 50%, 60%, 70%, 80%, 90% or more conditioned medium. In some
embodiments, the amount of conditioned medium is between about 50%
and 75% by volume. The unconditioned medium may be standard cell
culture medium. In some embodiments the unconditioned medium is
medium conventionally used for propagating ES cells of the same
species as the somatic cells to be reprogrammed.
[0092] The conditioned medium may be used immediately after being
harvested from the cells used to produce it or may be stored (e.g.,
at about 4.degree. C. or frozen) prior to use. The median may be
stored under conditions and for a time period consistent with
maintaining the ability of the conditioned medium to support
reprogramming in the methods of the invention. Without limitation,
such conditions and time may be consistent with maintaining at
least 20% of the original biological activity of secreted Wnt
present in the medium, which may be assessed using methods
mentioned above. The conditioned medium may be concentrated or
otherwise processed, e.g., using standard methods, provided such
concentration or processing is consistent with maintaining the
ability of the concentrate to support reprogramming when added to
unconditioned medium. Without limitation, such concentration or
processing may be consistent with maintaining at least 20% of the
original biological activity of secreted Wnt present in the medium.
As noted in the Examples, Applicants' results suggest dial normal
fibroblasts (not engineered to overexpress Wnt) may secrete
factors, perhaps including Wnt3a, that promote reprogramming,
raising the possibility that somatic cells undergoing reprogramming
in vitro, e.g., cells in culture that have been treated with
retrovirus or otherwise engineered to express Oct4, Sox2, Klf4, and
optionally c-Myc, may secrete such factors and thus contribute to
their own reprogramming. In certain embodiments of the present
invention, Wnt-conditioned medium has a greater concentration of
Wnt protein and/or Wnt pathway activating activity than would be
the case when unmodified somatic cells, e.g., fibroblasts,
undergoing reprogramming are cultured in medium known in the art to
be useful for culturing somatic cells undergoing reprogramming. In
some embodiments, such concentration and/or Wnt pathway activating
ability may be at least 1.5, 2, 5, 10, 20, or more times as great
as present in medium in which control fibroblasts are cultured as
described in Example 5.
[0093] Certain methods of the invention involve contacting a
somatic cell in vitro with one or mote defined agent(s) that
modulate, e.g., increase, Wnt pathway activity. The cells may be
maintained in standard cell culture medium known in the art. The
agent(s) may be added to the medium prior to using it to culture
the cells or during cell culture. The term "defined agent" in this
context means that the structure, sapience, or identity of the
agent that modulates, e.g., increases, Wnt pathway activity is
known and/or the agent is chemically synthesized and/or the agent
is (prior to addition to the medium) isolated or at least partially
purified. For example, the agent may not be an uncharacterized or
unidentified component of conditioned medium, cell or tissue lysate
or extract, cell cytoplasm or nuclear material, etc.
[0094] A variety of agents may be used to increase Wnt pathway
activity. Such agents are referred to herein as "Wnt pathway
activators" or "Wnt agonists". The Wnt pathway activator may act
directly by interacting with a Wnt receptor or indirectly by
interacting with one or more intracellular components of the Wnt
signaling pathway such as .beta.-catenin, a kinase or phosphatase
that acts on .beta.-catenin, a transcription (actor that assembles
with .beta.-catenin, etc. The activator may increase expression of
Wnt or a Wnt pathway component such as .beta.-catenin. In certain
embodiments the Wnt pathway activator increases activity of the Wnt
pathway to levels sufficient to enhance reprogramming of somatic
cells. In certain embodiments of the invention the Wnt pathway
activator inhibits degradation of .beta.-catenin, thereby enhancing
reprogramming of somatic cells. In certain embodiments of the
invention, it is of interest to inhibit the Wnt pathway in somatic
cells or in reprogrammed somatic cells. For example, Wnt pathway
inhibitors can be used to characterize or explore the mechanism by
which reprogramming occurs and/or to identify reprogramming agents
(e.g., agents that do not act via the Wnt pathway). Furthermore, m
certain embodiments of the invention, Wnt pathway inhibitors (e.g.,
small molecules, siRNA, proteins, etc.) may be of use to facilitate
differentiation of reprogrammed, pluripotent cells to a desired
cell type, e.g., in in vitro differentiation protocols.
[0095] In certain embodiments of the invention, the Wnt pathway
activator or inhibitor is s protein or smell molecule that binds to
a Wnt receptor. For example, the Wnt pathway activator can be a
soluble, biologically active Wnt protein.
[0096] In some embodiments the concentration of Wnt protein added
to the medium is between 10 and 10,000 ng/ml, e.g., between 100 and
5,000 ng/ml, e.g., between 1,000 and 2,500 ng/ml or between 2,500
and 5,000 ng/ml, or between 5,000 and 10,000 ng/ml.
[0097] As noted above certain Wnts have similar biological
activities to Wnt3a and/or are closely related in sequence to
Wnt3a. Such Wnts and/or agents that mimic the activity of such Wnts
are used in certain embodiments of the invention.
[0098] The Wnt protein may be isolated from naturally occurring
sources (e.g., mammalian cells that naturally produce the protein),
produced in eukaryotic or prokaryotic cells using recombinant
expression technology, or chemically synthesized. Soluble,
biologically active Wnt proteins may be prepared in purified form
using methods known in the art. See, e.g., U.S. Pat. Pub. No.
20040248803 and Willert, K, et al., Nature, 423:448-52, 2003. In
certain embodiments the soluble, biologically active Wnt protein is
Wnt3& In certain embodiments the Wnt protein is co- or
post-translationally modified as occurs when the Wnt protein is
produced in a host cell that naturally expresses the Wnt protein.
In other embodiments the Wnt protein is not co- or
post-translationally modified as in nature, in certain embodiments
the soluble, biologically active Wnt protein is modified with a
lipid moiety such as palmitate. The lipid moiety may be attached to
a conserved cysteine. For example, in certain embodiments the Wnt
protein is palmitoylated on a conserved cysteine as known in the
art. In certain embodiments the Wnt protein is glycosylated as
occurs when the Wnt protein is produced in a mammalian host cell
that naturally expresses the Wnt protein. In other embodiments the
Wnt protein is not glycosylated as found in nature. Recombinant
mouse Wnt3a is commercially available (e.g., from Millipore cat.
no. GF145 or R&D Systems cat no. 1324-WN-002).
[0099] In certain embodiments of the invention the Wnt pathway
activator is an agent that increases the level of .beta.-catenin,
promotes its nuclear localization, or otherwise activates
.beta.-catenin signaling.
[0100] In certain embodiments of the invention the Wnt pathway
activator is a small molecule, by which is meant an organic
compound having multiple carbon-carbon bonds and a molecular weight
of less than 1500 daltons. Typically such compounds comprise one or
more functional groups that mediate structural interactions with
proteins, e.g., hydrogen bonding, and typically include at least an
amine, carbonyl, hydroxyl or carboxyl group, and in some
embodiments at least two of the functional chemical groups. The
small molecule agents may comprise cyclic carbon or heterocyclic
structures and/or aromatic or polyaromatic structures substituted
with one or more chemical functional groups and/or heteroatoms.
[0101] In certain embodiments of the invention the Wnt pathway
activator is an agent that inhibits glycogen synthase kinase 3
(GSK3). These agents effectively "turn on" the Wnt pathway without
the need for extracellular Wm. GSK3 is a serine/threonine kinase,
originally identified as a regulator of glucose metabolism
(reviewed in Frame and Cohen, Biochem J 359:1-16, 2001; see also
Cohen, Biochem Soc Trans 7:159-80, 1979; Embi et al., Bur J Biochem
107:519-27, 1980). "GSK3" as used herein refers to either or both
isoforms of GSK3(GSK3.alpha. and GSK3.beta.). Inhibitors that
inhibit either or both of these isoforms are of use. In certain
embodiments the GSK3 inhibitor specifically inhibits GSK3 and does
not substantially inhibit the majority of other mammalian kinases.
In some embodiments the GSK3 inhibitor does not substantially
inhibit at least 10 diverse mammalian kinases. In some embodiments
the GSK3 inhibitor specifically inhibits both GSK3.beta. and
GSK3.alpha.. In some embodiments the GSK3 inhibitor specifically
inhibits GSK3.beta. but not GSK3.beta.. For example, the IC50 for
GSK3.alpha. may be at least 10-fold as great as for GSK3.beta.. In
some embodiments the GSK3 inhibitor specifically inhibits GSK3a but
not GSK3.alpha.. For example, the IC50 for GSK3.beta. may be at
least 10-fold as great as for GSK3.alpha.. In certain embodiments
the IC50 of the GSK3 inhibitor for GSK3 is at least 10-fold lower
than its IC50 for the majority of other mammalian kinases. In
certain embodiments the IC50 of the GSK3 inhibitor for GSK3 is less
than 10 .mu.M. In certain embodiments the IC50 of the GSK3
inhibitor for GSK3 is less than 1 .mu.M. It will be understood that
the GSK3 inhibitor should be capable of entering cells in
sufficient quantities under the conditions used so as to inhibit
GSK3 therein. In some embodiments the concentration of GSK3
inhibitor for used is at least equal to the IC50 of the compound as
measured in vitro. In some embodiments the concentration of GSK3
inhibitor used is no more than 100 times the IC50 of the compound
as measured in vitro. In some embodiments the concentration used
ranges between 0.5 and 50-fold the IC50 of the agent as measured in
vitro.
[0102] Many potent and selective small molecule inhibitors of GSK3
have now been identified (Wagman A S, Johnson K W, Bussiere D E,
Curr Pharm Des., 10(10): 1105-37, 2004). Exemplary GSK3 inhibitors
of use include the following: (1) BIO:
(2'Z,3'E)-6-Bromoindirubin-3'-oxime, 6-bromoindirubin-3-oxime (BIO)
is a potent, reversible and ATP-competitive GSK-3 inhibitor
(Polychronopoulos, P. et al. J. Med. Chem. 47, 935-946, 2004). (2)
AR-A014418: N-(4-Methoxybenzyl)-N'-(5-nitro-1,3-thiazol-2-yl)urea.
AR-A014418, inhibits GSK3 (IC50=104 nM), in an ATP-competitive
manner (Ki=38 nM). AR-A014418 does not significantly inhibit cdk2
or cdk5 (IC50>100 .mu.M) or 26 other kinases, demonstrating high
specificity for GSK3 (Bhat, R., et al., J. Biol. Chem. 278,
45937-45945, 2003). (3) SB 216763:
3-(2,4-Dichlorophenyl)-4-(1-methyl-1H-indol-3-yl)-1H-pyrrole-2,5-dione,
See, e.g., Smith, D. G., et al, Bioorg. Med. Chem. Lett. 11,
635-639, (2001) and Cross, D. A., et al, J. Neurochem. 77, 94-102,
(2001), (4) SB 415286:
3-[(3-Chloro-4-hydroxyphenyl)amino]-4-(2-nitrophenyl)-1H)-pyrrol--
2,5-dione. SB 415286 is described in Smith, D. G., et si, Bioorg.
Med. Chem. Lett. 11, 635-639, 2001 and Coughlan, M. P., et al,
Chem. Biol. 10, 793-803, 2000, TDZD-8:
4-Benzyl-2-methyl-2,4-thiadiazolidine-3,5-dione. This compound is a
selective inhibitor of GSK-3, a thiadiazolidinone derivative, a
non-ATP competitive inhibitor of GSK-3.beta. (IC50=2 .mu.M). It
does not inhibit Cdk-1/cyclin B, CK-II, PKA or PKC at >100
.mu.M. It has been proposed to bind to the kinase site of GSK-30.
(Martinez et al., J. Med. Chem. 45, 1292-1299, 2002); CHIR-911 and
CHIR-837 (also retorted to as CT-99021 and CT-98023 respectively).
Chiron Corporation (Emeryville, Calif.) and related compounds are
of use. Lithium chloride, sodium valproate, and GSK3 inhibitor II
(Calbiochem) are other GSK3 inhibitors of use. Additional GSK3
inhibitors are described in U.S. Pat. Nos. 6,057,117 and 6,608,063;
U.S. patent application publications 20040092535, 20040209878,
20050054663. Other GSK3 inhibitors of use are described in
WO/2003/049739, which discloses PYRIMIDINE COMPOUNDS USEFUL AS
GSK-3 INHIBITORS; WO/2002/085909, which discloses 9-DEAZAGUANINE
DERIVATIVES AS INHIBITORS OF GSK-3, WO/2003/011287, which discloses
PYRAZOLON DERIVATIVES AS INHIBITORS OF GSK-3, WO/2005/039485,
and/or WO/2006/091737.
[0103] In certain embodiments of the invention the Wnt pathway
activator is a casein kinase 1 (CK1) inhibitor. Examples include
D4476, IC261, and CKI-7 (see, e.g., Rena, G., et al. EMBO reports
5(1), 60-65, 2004). Compounds that inhibit CK1 and GSK3 are
disclosed in U.S. Pat. No. 7,098,204.
[0104] In certain embodiments of the invention the Wnt pathway
activator is an activator of a phosphatase that naturally
dephosphorylates .beta.-catenin at one or more of the sites
phosphorylated by GSK3 or CK1.
[0105] The CREB binding protein (CBP) and the closely related
protein p300 can assemble with .beta.-catenin and act as
.beta.-catenin binding transcriptional co-activators. For example,
to generate a transcriptionally active complex, .beta.-catenin
recruits the transcriptional coactivators. CREB-binding protein
(CBP) or its closely related homolog p300 (Hecht et al., EMBO J.
19:1839-50 (2000); Takemaru et al., J. Cell Biol. 149:249-54
(2000)) well other component of the basel transcription machinery.
Other .beta.-catenin co-activators include TBP, BRG1, BCL9/PYG,
etc. The invention encompasses directly or indirectly modulating
the interactions between .beta.-catenin and any one or more of
these co-activators so as to enhance the reprogramming of somatic
cells. For example, the invention encompasses altering the relative
participation of .beta.-catenin in any one or mere of these
complexes relative to its participation in one or more other
complexes. Agent such as small molecules may be used to selectively
disrupt interaction of .beta.-catenin with a particular
co-activator, thereby potentially reducing transcription that would
inhibit reprogramming or favor differentiation. Selective
disruption may shift the balance towards interaction with a
different co-activator to form a complex that enhances
reprogramming. The agent may act directly on the complex or
indirectly, e.g., by causing post-translational modification such
as phosphorylation of .beta.-catenin or a co-activator. In one
embodiment, the agent is a compound described in U.S. Patent Pub.
No. 20070128669 or an analog or derivative thereof, or an agent
having the same mechanism of action. .beta.-catenin interacting
protein (also known as ICAT or CTNNBIP1) binds .beta.-catenin and
inhibits interaction between .beta.-catenin and TCP family members
(Gottardi, et al., Am J Physiol Cell Physiol. 286(4):C747-56,
2004). The encoded protein is a negative regulator of the Wnt
signaling pathway. The invention encompasses inhibiting ICAT (which
tom includes any transcript variants or family members that inhibit
the interaction of .beta.-catenin and TCP) in order to activate the
Wnt pathway. In certain embodiments of the invention, the agent
that activates a Wnt pathway does so by inhibiting expression or
activity of an endogenous inhibitor or negative regulator of the
Wnt pathway. In some embodiments, the agent inhibits expression by
RNA interference (RNAi). In some embodiments, the agent inhibits
expression or activity of GSK3, ICAT, CK1, or CTNNBIP1.
[0106] In some embodiments an inhibitor of use in the present
invention is an RNAi agent One of skill in the art will be able to
identify an appropriate RNAi agent to inhibit expression of a gene
of interest. See, e.g., Yu, J-Y., et at, Molecular Therapy, 7(2):
228-236, 2003. The RNAi agent may inhibit expression sufficiently
to reduce the average steady state level of the RNA transcribed
from the gene (e.g., mRNA) or Its encoded protein by, e.g., by at
least 50%, 60%, 70%, 80%, 90%, 95%, or mono). The RNAi agent may
contain a sequence between 17-29 nucleotides long, e.g., 19-23
nucleotides long that is 100% complementary to the mRNA or contains
up to 1, 2, 3, 4, or 5 nucleotides, or up to about 10-30%
nucleotide, that do not participate in Watson-Crick base pairs when
aligned with the mRNA to achieve the maximum number of
complementary base pain. The RNAi agent may contain a duplex
between 17-29 nucleotide long in which all nucleotides participate
in Watson-Crick base pairs or in which up to about 10-30% of the
nucleotides do not participate in a Watson-Crick base pair. One of
skill in the art will be aware of which sequence characteristics
are often associated with superior siRNA functionality and
algorithms and rules by which such siRNAs can be designed (see,
e.g., Jagla, B., et al, RNA, 11(6):864-72, 2005). The methods of
the invention can, but need not, employ siRNAs having such
characteristics. In some embodiments, the sequence of either or
both strands of the RNAi agent is/are chosen to avoid silencing
non-target genes, e.g., the strand(s) may have less than 70% 90%,
or 90% complementarity to any mRNA other than the target mRNA. In
some embodiments, multiple different sequences are used. Table 1
lists the Gene IDs of the human and mouse genes encoding GSK3 and
the nucleic acid (mRNA) and protein sequence accession numbers.
RNAi agents capable of silencing mammalian genes are commercially
available (e.g., from suppliers such as Qiagen, Dharmacon,
Invitrogen, etc.). If multiple isoforms exist, one can design si RN
As or shRNAs targeted against a region present in all of the
isoforms expressed in a given cell of interest.
[0107] Methods for silencing genes by transfecting cells with siRNA
or constructs encoding shRNA are known in the art. To express an
RNAi agent in somatic cells, a nucleic acid construct comprising a
sequence that encodes the RNAi agent, operably linked to suitable
expression control elements, e.g., a promoter, can be introduced
into the cells as known in the art. For purposes of the present
invention a nucleic acid construct that comprises s sequence that
encodes an RNA or polypeptide of interest, the sequence being
operably linked to expression control elements such as a promoter
that direct transcription in a cell of interest, is referred to as
an "expression cassette", The promoter am be an RNA polymerase I,
II, or III promoter functional in somatic mammalian cells. In
certain embodiments, expression of the RNAi agent is conditional.
In some embodiments, expression is regulated by placing the
sequence that encodes the RNAi agent under control of a regulatable
(e.g., inducible or repressible) promoter.
[0108] Constitutively active versions of proteins such as
.beta.-catenin or other components of the Wnt signalling pathway
are also of use. N-terminal truncation or deletion of the potential
GSK-3 phosphorylation site in the N-terminal region or a missense
mutation of the serine or threonine residues therein results in the
accumulation of truncated or normal sized .beta.-catenin and then
in activation of .beta.-catenin-mediated signal (do La Costa PNAS,
95(15): 8847-8851, 1998). Dominant negative versions of endogenous
proteins that inhibit Wnt signalling are also of use. In some
embodiments, somatic cells are engineered to express these
proteins. In some embodiments, the protein is added to the culture
medium.
[0109] In some embodiments, cells are treated to enhance uptake of
a Wnt pathway activator that acts intracellularly. For example, the
cell membrane may be partially permeabilized. In some embodiments,
a Wnt pathway activator is modified to comprise an amino acid
sequence that enhances cellular uptake of molecules by cells (also
referred to as a "protein transduction domain"). Such
uptake-enhancing amino acid sequences are found, e.g., in HIV-1 TAT
protein, the herpes simplex virus 1 (HSV-1) DNA-binding protein
VP22, the Drosophila Antennapedia (Antp) transcription Actor, etc.
Artificial sequences are also of use. See, e.g., Fischer et al,
Bioconjugate Chem., Vol. 12, No. 6, 2001 and U.S. Pat. No.
6,835,810.
[0110] Without limitation, the invention contemplates use in the
methods of the present invention of any of the compositions and
approaches disclosed in U.S. Patent Pub. No. 20060147435 as being
useful for promoting Wnt/.beta.-catenin signaling.
[0111] In some embodiments of the invention, somatic cells are
treated so that they express a Wnt protein at levels greater than
would be the case without such treatment. In some embodiments,
somatic cells are genetically engineered to stably or transiently
express a Wnt protein at levels greater titan would be the case
without such treatment. In acme embodiments of the invention
somatic cells are treated so that they express a Wnt pathway
component such as .beta.-catenin or a TCF/LEF at levels greater
then would be the case without such treatment. In some embodiments
of the invention, somatic cells are genetically engineered to
stably or transiently express a Wnt pathway component such as
.beta.-catenin or a TCF/LEF at levels greater than would be the
case without such treatment.
[0112] Methods of the invention may include treating the cells with
multiple reprogramming agents either concurrently (i.e., during
time periods that overlap at least in part) or sequentially and/or
repeating the stops of treating the cells with an agent. The agent
used in the repeating treatment may be the same as, or different
from, the one used during the first treatment. The cells may be
contacted with a reprogramming agent for varying periods of time.
In some embodiments, the cells are contacted with the agent for a
period of time between 1 hour and 60 days, e.g., between 10 and 30
days, e.g., for about 15-20 days. Reprogramming agents may be added
each time the cell culture medium is replaced. The reprogramming
agents) may be removed prior to performing a selection to enrich
for pluripotent cells or assessing the cells for pluripotency
characteristics.
[0113] Reprogramming agents or candidate reprogramming agents of
interest include a variety of compounds. Exemplary compounds
include agents that inhibit histone deacetylation, e.g., histone
deacetylase (HDAC) inhibitors and agents that inhibit DMA
methylation, e.g., DNA methyltransferase inhibitors. Without
wishing to be bound by theory, DNA demethylation can regulate gene
expression by "opening" the chromatin structure detectable as
increased nuclease sensitivity. This remodeling of chromatin
structure allows transcription factors to bind to the promoter
regions, assembly of the transcription complex, and gene
expression.
[0114] The major classes of HDAC inhibitors include (a) Small chain
fatty acids (e.g., valproic acid); (b) hydroxamate small molecule
inhibitors (e.g., SAHA and PXD101); (c) Non-hydroxamate small
molecule inhibitors, e.g., MS-275; and (d) Cyclic peptides; e.g.,
depsipeptide (ace, e.g., Carey N and La Thangue N B, Curr Opin
Pharmacol.; 6(4):369-75, 2006). Examples of histone deacetylase
inhibitors include Trichostatin A:
[R-(E,E)]-7-[4-(Dimethylamino)phenyl]-N-hydroxy-4,6-dimethyl-7-oxo-2,4-he-
ptadienamide, which inhibits histone deacetylase at nanomolar
concentrations; resultant histone hyper-acetylation leads to
chromatin relaxation rod modulation of gene expression. (Yoshida,
M., et al., Bioessays 17, 423-430, 1995; Minucci, S., et al., Proc.
Natl. Acid. Sci. USA 94, 11295-11300, 1997; Brehm, A., et al.,
1998; Medina, V., et al., Induction of caspase-3 protease activity
and apoptosis by butyrate and trichostatin A (inhibitors of histone
deacetylase): dependence on protein synthesis and synergy with a
mitochondrial/cytochrome c-dependent pathway. Cancer Res. 57,
3097-3707, 1997; Kim, M. S., et al., Inhibition of histone
deacetylase increases cytotoxicity to anti cancer drugs targeting
DNA. Cancer Res. 63, 7291-7300, 2003); Apicidin:
Cyclo[(2S)-2-amino-8-oxodecanoyl-1-methoxy-L-tryptophyl-L-isoleucyl-(2R)--
2-piperidinecarbonyl] (Kwon, S. H., et al. J. Biol. Chem. 18, 2073,
2002; Han, J. W., et al. Cancer Res. 60, 6068, 2000; Colletti, S.
L., et al. Bioorg. Med. Chem. 11, 107, 2001; Kim J. S., et al.
Biochem. Biophys. Res. Commun. 281, 866, 2001).
[0115] A variety of DNA methylation inhibitors are known in the art
and are of use in the invention. Sec, e.g., Lyko, F. and Brown, R.,
JNCI Journal of the National Cancer Institute, 97(20):1498-t 506,
2005. Inhibitors of DNA methylation include nucleoside DNA
methyltransferase inhibitors such as decitabine
(2'-deoxy-5-azacytidine), 5-azadeoxycytidine, and zebularine,
non-nucleoside inhibitors such as the polyphenol
(-)-epigallocatechin-3-gallate (EGCG) and the small molecule RG108
(2-(1,3-dioxo-1,3-dihydro-2H-isoindol-2-yl)-3-(1H-indol-3-yl)propan-
oic acid), compounds described in WO2005085196 and phthalimides,
succinimides and related compounds as described in WO2007007034.
Three additional classes of compounds art: (1) 4-Aminobenzoic acid
derivatives, such as the antiarrhythmic drug procainamide and the
local anesthetic procaine; (2) the psammaplins, which also inhibits
histone deacetylase (Pina, I. C., J Org Chem., 68(10):3866-73,
2003); and (3) oligonucleotides, including siRNAs, shRNAi, and
specific antisense oligonucleotides, such as MG98. DNA methylation
inhibitors may act by a variety of different mechanisms. The
nucleoside inhibitors are metabolized by cellular pathways before
being incorporated into DNA. Alter incorporation, they function as
suicide substrates for DNMT enzymes. The nonnucleoside inhibitors
procaine, epigallocatechin-3-gallate (EGCG), and RG108 have been
proposed to inhibit DNA methyhransferases by masking DNMT target
sequences (i.e., procaine) or by blocking the active site of the
enzyme (i.e., EQCG and RG108). In some embodiments of the
invention, combinations of DNA methylation inhibitors are used. In
some embodiments, the concentrations are selected to minimize toxic
effects on cells. In some embodiments agents that incorporate into
DNA (or whose metabolic products incorporate into DNA) are not
used.
[0116] DNA methyltransferase (DNMT1,3a, and/or 3b) and/or one or
more HDAC family members can alternatively or additionally be
inhibited using RNAi agents.
[0117] The invention encompasses use of Wnt-conditioned medium,
soluble Wnt or small molecules that modulate the Wnt signaling
pathway in combination with other transient cues, e.g., small
molecules, that can replace Oct4, Sox2, Klf4, Nanog, and/or Lin28
retroviruses in reprogramming somatic cells to pluripotency. The
invention provides a composition comprising a Wnt pathway modulator
and at least one compound selected from the group consisting of:
HDAC inhibitors and DNA methylation inhibitors. The invention
provides a composition comprising a Wnt pathway modulator, at least
one HDAC inhibitor, and at least one DNA methylation inhibitor. The
invention provides cell culture medium containing any of the above
combinations of agents. In certain embodiments, the HDAC inhibitor
is any HDAC inhibitor mentioned above. In certain embodiments, the
DNA methylation inhibitor is any HDAC inhibitor mentioned above. In
certain embodiments, the Wnt pathway modulator activates the Wnt
pathway. In certain embodiments, the cell culture medium comprises
Wnt-conditioned medium, e.g., Wnt3a-CM, as the source of Wnt
pathway modulator. In certain embodiments, the Wnt pathway
modulator is a small molecule. In certain embodiments, the
composition comprises somatic cells. In certain embodiments, the
somatic cells are engineered to express at least one of the
transcription factors Oct4, Nanog, Sox2, Klf4, and Lin28.
[0118] Somatic Cells and Reprogrammed Somatic Cells
[0119] Somatic cells of use the invention may be primary cells
(non-immortalized cells), such as those freshly isolated from an
animal, or may be derived from a cell line capable or prolonged
proliferation in culture (e.g., for longer than 3 months) or
indefinite proliferation (immortalised cells). Adult somatic cells
may be obtained from individuals, e.g., human subjects, and
cultured according to standard cell culture protocols available to
those of ordinary skill w the art. The cells may be maintained in
cell culture following their isolation from a subject. In certain
embodiments, the cells are passaged once or more following their
isolation from the individual (e.g., between 2-5, 5-10, 10-20,
20-50, 50-100 times, or mote) prior to their use in a method of the
invention. They may be frozen and subsequently thawed prior to use.
In some embodiments, the cells will have been passaged no more than
1, 2, 5, 10, 20, or 50 times following their isolation from the
individual prior to their use in a method of the invention.
[0120] Somatic cells of use in the present invention include
mammalian cells, such as, for example, human cells, non-human
primate cells, or mouse cells. They may be obtained by well-known
methods from various organs, e.g., skin, lung, pancreas, liver,
stomach, intestine, heart, reproductive organs, Madder, kidney,
urethra and other urinary organs, etc., generally from any organ or
tissue containing live somatic cells. Mammalian somatic cells
useful in various embodiments of the present invention include, for
example, fibroblasts, adult stem cells, sertoli cells, granulosa
cells, neurons, pancreatic islet cells, epidermal cells, epithelial
cells, endothelial cells, hepatocytes, hair follicle cells,
keratinocytes, hematopoietic cells, melanocytes, chondrocytes,
lymphocytes (B and T lymphocytes), erythrocytes, macrophages,
monocytes, mononuclear cells, cardiac muscle cells, skeletal muscle
cells, etc., generally any living somatic cells.
[0121] Somatic cells may be treated so as to cause them to express
or contain one or mote reprogramming factor, pluripotency factor,
and/or pluripotency inducing factor, at levels greeter then would
be the case in the absence of such treatment. For example, somatic
cells may be genetically engineered to express one or more genes
encoding one or more such factor(s) and/or may be treated with
agent(s) that increase expression of one or more endogenous genes
encoding such factors and/or stabilize such factor(s). The agent
could be, for example, a small molecule, a nucleic acid, a
polypeptide, etc. In some embodiments, factors such as pluripotency
factors are introduced into somatic cells, e.g., by microinjection
or by contacting the cells with the factors under conditions in
which the factors are taken up by the cells. In some embodiments,
the factors are modified to incorporate a protein transduction
domain. In some embodiments, the cells are permeabilized or
otherwise treated to increase their uptake of the factors,
Exemplary factors are discussed below.
[0122] The transcription factor Oct4 (also called Pou5fl, Oct-3,
Oct3/4) is en example of a pluripotency factor. Oct4 has been shown
to be required for establishing and maintaining the
undifferentiated phenotype of ES cells and plays a major role in
determining early events in embryogenesis and cellular
differentiation (Nichols et al., 1998, Cell 95:379-391; Niwa et
al., 2000, Nature Genet 24:372-376). Oct4 expression is
down-regulated as stem cells differentiate into more specialized
cells. Nanog is another example of a pluripotency factor. Nanog is
a homeobox-containing transcription factor with an essential
function in maintaining the pluripotent cells of toe inner cell
mass and in the derivation of ES cells from these. Furthermore,
overexpression of Nanog is capable of maintaining toe pluripotency
and self-renewing characteristics of ESCs under what normally would
be differentiation-inducing culture conditions. (See Chambers et
al., 2003, Cell 113: 643-655; Mitsui et al., Cell. 2003,
113(5):631-42) Sox2, another pluripotency factor, is an HMO
domain-containing transcription factor known to be essential for
normal pluripotent cell development and maintenance (Avilion, A.,
et al., Genes Dev. 17, 126-140, 2003). Klf4 is a Kruppel-type zinc
finger transcription (actor initially identified as a Klf family
member expressed in the gut (Shields, J. M, et al., J. Biol. Chem.
271:20009-20017, 1996). Overexpression of Klf4 in mouse ES cells
was found to prevent differentiation in embryoid bodies formed in
suspension culture, suggesting that Klf4 contributes to ES self
renewal (Li, Y., et al., Blood 105:635-637, 2005). Sox2 is a member
of the family of SOX (sex determining region Y-box) transcription
factors and is important for maintaining ES cell self-renewal.
c-Myc is a transcription factor that plays a myriad of roles in
normal development and physiology as well as being an oncogene
whose dysregulated expression or mutation is implicated in various
types of cancer (reviewed in Pelengaris S, Khan M., Arch Biochem
Biophys. 416(2):129-36, 2003; Cole M D, Nikiforov M A, Curr Top
Microbiol Immunol., 302:33-50, 2006). In some embodiments, such
factors are selected from: Oct4, Sox2, Klf4, and combinations
thereof. In some embodiments, a different, functionally overlapping
Klf family member such as Klf2 is substituted for Klf4. In some
embodiments, the factors include at least Oct4. In some
embodiments, the factors include at least Oct4 and a Klf family
member, e.g., Klf2. Lin28 is a developmentally regulated RNA
binding protein. In some embodiments, somatic cells are treated so
that they express or contain one or more reprogramming factors
selected from: Oct4, Sox2, Klf4, Nanog, Lin28, and combinations
thereof. CCAAT/enhancer-binding-protein-alpha (C/EBPalpha) is
another protein that promotes reprogramming at least in certain
cell types, e.g., lymphoid cells such as B-lineage cells, is
considered a reprogramming factor for such cell types, and b of use
in certain embodiments of the invention, e.g., in combination with
one or more of the pluripotency genes and/or Wnt pathway modulators
described herein.
[0123] Other genes of interest are involved in chromatin remodeling
and/or are have been shown to be important for maintaining
pluripotency of ES cells. Optionally the gene is one that is
downregulated as the cells differentiate and/or is not expressed in
adult somatic cells. Other genes of interest encode microRNA
precursors that have been associated with multipotency or
pluripotency and/or dial are naturally expressed in multipotent or
pluripotent cells. Other genes of interest include encode RNAi
agents that Inhibit genes that are targets, of endogenous microRNAs
that are naturally expressed in multipotent or pluripotent
cells.
[0124] In one embodiment, the exogenously introduced gene may be
expressed from a chromosomal locus other than the chromosomal locus
of an endogenous gene whose function is associated with
pluripotency. Such a chromosomal locus may be a locus with open
chromatin structure, and contain gene(s) whose expression is not
required in somatic cells, e.g., the chromosomal locus contains
gene(s) whose disruption will not cause cells to die. Exemplary
chromosomal loci include, for example, the mouse ROSA 26 locus and
type II collagen (Col2a1) locus (See Zambrowicz et al., 1997).
[0125] Methods for expressing genes in cells are known in the art
Generally, a sequence encoding a polypeptide or functional RNA such
as an RNAi agent is operably linked to appropriate regulatory
sequences. The term regulatory sequence includes promoters,
enhancers and other expression control elements. Exemplary
regulatory sequences are described in Goeddel; Gets Expression
Technology: Methods in Enzymology, Academic Press, San Diego,
Calif. (1990). For instance, any of a wide variety of expression
control sequences that control the expression of a DNA sequence
when operatively linked to it may be used in these vectors to
express cDNAs.
[0126] The exogenously introduced gene may be expressed from an
inducible or repressible regulatory sequence such that its
expression can be regulated. The term "inducible regulatory
sequence", as used herein, refers to a regulatory sequence that, in
the absence of an inducer (such as a chemical and/or biological
agent) or combination of inducers, does not direct expression, or
directs low levels of expression of an operably linked nucleic acid
sequence such as a cDNA, and, in response to an inducer, its
ability to direct expression is enhanced. Exemplary inducible
promoters include, for example, promoters that respond to heavy
metals (CRC Boca Raton, Fla. (1991), 167-220; Brinster et al.
Nature (1982), 296, 39-42), to thermal shocks, to hormones (Lee et
al. P.N.A.S. USA (1988), 85, 1204-1208; (1981), 294, 228-232; Klock
et al. Nature (1987), 329, 734-736; Israel and Kaufman, Nucleic
Adds Res. (1989), 17, 2589-2604), promoters that respond to
chemical agents, such as glucose, lactose, galactose or antibiotic.
A "repressible regulatory sequence" is one that directs expression
of an operably linked nucleic acid sequence in the absence of a
specific agent or combination of agents that inhibits
expression.
[0127] A tetracycline-inducible promoter is an example of an
inducible promoter that responds to an antibiotic. See Gossen, M.
and Bujard, H., Annu Rev Genet. Vol. 36: 153-173 2002 and
references therein. The tetracycline-inducible promoter comprises a
minimal promoter linked operably to one or more tetracycline
operator(s). The presence of tetracycline or one of its analogues
leads to the binding of a transcription activator to the
tetracycline operator sequences, which activates the minimal
promoter and hence the transcription of the associated cDNA.
Tetracycline analog includes any compound that displays structural
similarity with tetracycline and is capable of activating a
tetracycline-inducible promoter. Exemplary tetracycline analogs
include, for example, doxycycline, chlorotetracycline and
anhydrotetracycline.
[0128] In some embodiments of the invention, expression of an
introduced gene, e.g., a gene encoding a reprogramming factor or
RNAi agent is transient. Transient expression can be achieved by
transient transfection or by expression from a regulatable
promoter. In some embodiments, expression can be regulated by, or
is dependent on, expression of a site-specific recombinase.
Recombinase systems include the Cre-Lox and Flp-Frt systems, among
others (Gossen, M. and Bujard, H., 2002). In some embodiments, a
recombinase is used to him on expression by removing a stopper
sequence that would otherwise separate the coding sequence from
expression control sequences. In some embodiments, a recombinase is
used to excise at least a portion of a gene after pluripotency has
been induced. In some embodiments, the recombinase is expressed
transiently, e.g., it becomes undetectable after about 1-2 days,
2-7 days, 1-2 weeks, etc. In some embodiments the recombinase is
introduced from external sources. Optionally the recombinase in
these embodiments a protein transduction domain.
[0129] Reprogrammed somatic cells may be assessed for one or mote
pluripotency characteristic(s). The presence of pluripotency
characteristic(s) indicates that the somatic cells have been
reprogrammed to a pluripotent state. The lean "pluripotency
characteristics", as used herein, refers to characteristics
associated with and indicative of pluripotency, including, for
example, the ability to differentiate into cells derived from all
three embryonic germ layers all types and a gene expression pattern
distinct for a pluripotent cell, including expression of
pluripotency factors and expression of other ES cell markers.
[0130] To assess potentially reprogrammed somatic cells for
pluripotency characteristics, one may analyze such cells for
particular growth characteristics and ES cell-like morphology.
Cells may be injected subcutaneously into immunocompromised SC ID
mice to determine whether they induce teratomas (a standard assay
for ES cells). ES-like cells can be differentiated into embryoid
bodies (another ES specific feature). Moreover, ES-like cells can
be differentiated in vitro by adding certain growth factors known
to drive differentiation into specific cell types. Self-renewing
opacity, marked by induction of telomerase activity, is another
pluripotency characteristic that can be monitored. One may carry
out functional assays of the reprogrammed somatic cells by
introducing thorn into blastocysts and determining whether the
cells are capable of giving rise to all cell types. See Hogan et
al., 2003. If the reprogrammed cells are capable of forming a few
cell types of the body, they are multipotent; if the reprogrammed
cells are capable of forming all cell types of the body including
germ cells, they are pluripotent.
[0131] One may also examine the expression of an individual
pluripotency lector in the (reprogrammed somatic cells to assess
their pluripotency characteristics. Additionally or alternately,
one may assess the expression of other ES cell markets.
Stage-specific embryonic I 5 antigens-1, -3, and -4 (SSEA-1,
SSEA-3, SSEA-4) are glycoproteins specifically expressed in early
embryonic development and are markers for ES cells (Solter and
Knowles, 1978, Proc. Natl. Acad. Sci. USA 75:5565-5569; Kannagi et
al., 1983, EMBO J 2:2355-2361). Elevated expression of the enzyme
alkaline phosphatase (AP) is another marker associated with
undifferentiated embryonic stem cells (Wobus et al. 1984, Exp. Cell
152:212-219; Pease et al., 1990, Dev. Biol. 141:322-352).
Additional ES cell markers are described in Ginis, I., et al., Dev.
Biol., 269:369-380, 2004 and in The International Stem Cell
Initiative, Adewumi O, et al., Nat Biotech not., 25(7):803-16, 2007
and references therein. For example, TRA-1-60, TRA-1-81, GCTM2 and
GCT343, and the protein antigens CD9, ThyI (also known as CD90),
class I HLA, NANOG, TDGF1, DNMT3B, GABRB3 and GDF3, REX-1, TERT,
UTF-1, TRF-1, TRF-2, connexin43, connexin45, Foxd3, FGFR-4, ABCG-2,
and Glut-1 are of use.
[0132] One may perform expression profiling of the reprogrammed
somatic cells to assess their pluripotency characteristics.
Pluripotent cells, such as embryonic stem cells, and multipotent
cells, such as adult stem cells, are known to have a distinct
pattern of global gene expression. See, for example, Ramalho-Samos
et al., Science 298:597-600, 2002; Ivanova et al., Science
298:601-604, 2002; Boyer, L A, et al. Nature 441, 349, 2006, and
Bernstein, B E, et al., Cell 125 (2), 315, 2006. One may assess DNA
methylation, gene expression, and/or epigenetic state of cellular
DNA, and/or developmental potential of the cells, e.g., as
described in Wernig, M., et al., Nature, 448:318-24, 2007. Celia
that are able to form teratomas containing cells having
characteristics of endoderm, mesoderm, and ectoderm when injected
into SCID mice and/or possess ability to participate (following
injection into murine blastocysts) in formation of chimeras that
survive to term are considered pluripotent Another method of use to
assess pluripotency is determining whether the cells have
reactivated a silent X chromosome.
[0133] Somatic cells may be reprogrammed to gain either a complete
set of the pluripotency characteristics. Alternatively, somatic
cells may be reprogrammed to gain only a subset of the pluripotency
characteristics.
[0134] Certain methods of the invention include a step of selecting
cells that express a marker that is expressed by multi potent or
pluripotent cells. The marker may be sped Really expressed in such
cells. Standard cell separation methods, e.g., flow cytometry,
affinity separation, etc may be used. Alternately or additionally,
one could select cells that do not express markers characteristic
of somatic cells from which the potentially reprogrammed cells were
derived and which are not expressed in ES cells generated using
conventional methods. Other methods of separating cells may utilize
differences in average cell size or density that may exist between
in impotent cells and somatic cells. For example, cells can be
filtered through materials having pores that will allow only
certain cells to pass through.
[0135] In some embodiments, the somatic cells contain a nucleic
acid comprising regulatory sequences of a gene encoding a
pluripotency factor operably linked to a selectable or detectable
marker (e.g., GPP or neo). The nucleic acid sequence encoding the
marker may be integrated at the endogenous locus of the gene
encoding the pluripotency factor (e.g., Oct4) or the construct may
comprise regulatory sequences operably linked to the marker.
Expression of the marker may be used to select, identify, and/or
quantify reprogrammed cells.
[0136] Any of the methods of the invention that relate to
generating a reprogrammed somatic cell may include a step of
obtaining a somatic cell or obtaining a population of somatic cells
from an individual in need of cell therapy. Reprogrammed somatic
cells are generated, selected, or identified from among the
obtained cells or cells descended from the obtained cells.
Optionally the cell(s) are expended in culture prior to generating,
selecting, or identifying reprogrammed somatic cell(s) genetically
matched to the donor.
[0137] In some embodiments colonies are subcloned and/or passaged
once or more in order to obtain a population of cells enriched for
ES-like cells. The enriched population may contain at least 95%,
96%, 97%, 98%, 99% or more, e.g., 100% ES-like cells. The invention
provides cell Knee of somatic cells that have been stably and
heritably reprogrammed to an ES-like state.
[0138] In some embodiments, the methods are practiced using somatic
cells that are not genetically engineered for purposes of
identifying or selecting reprogrammed cells. The resulting
reprogrammed somatic cells do not contain exogenous genetic
material that has been introduced into said cells (or ancestors of
said cells) by the hand of man, e.g., for purposes of identifying
or selecting reprogrammed cells. In some embodiments, the somatic
cells and reprogrammed somatic cells derived therefrom do contain
exogenous genetic material in their genome, but such genetic
material is introduced for purposes of correcting a genetic defect
in such cells or enabling such cells to synthesize a desired
protein for therapeutic purposes and is not used to identify or
select reprogrammed cells.
[0139] In some embodiments, the methods employ morphological
criteria to identify reprogrammed somatic cells from among a
population of somatic cells that are not reprogrammed. In some
embodiments, the methods employ morphological criteria to identify
somatic cells that have been reprogrammed to an ES-like state from
among a population of cells that are not reprogrammed or are only
pertly reprogrammed to an ES-like state. "Morphological criteria"
is used in a broad sense to refer to any visually detectable
feature or characteristic of the cells or colonies. Morphological
criteria include, e.g., the shape of the colonies, the sharpness of
colony boundaries, the density, small size, and rounded shape of
the cells relative to non-reprogrammed cells, etc. FIG. 1 shows
colonies of cells displaying morphological criteria indicative of
cells that have been reprogrammed to an ES-like state. Note the
dense colonies composed of small, rounded cells, and the sharp
colony boundaries. The invention encompasses identifying and,
optionally, isolating colonies (or cells from colonies) wherein the
colonies display one or more such characteristics. The reprogrammed
somatic cells may be identified as colonies growing in a first cell
culture dish (which term refers to any vessel, plate, dish,
receptacle, container, etc., in which living cells can be
maintained in vitro) and the colonies, or portions thereof,
transferred to a second cell culture dish, thereby isolating
reprogrammed somatic cells. Hie cells may then be further
expended.
Methods of Screening for an Agent that Reprograms or Contributes to
Reprogramming Somatic Cells
[0140] The present invention also provides methods for identifying
an agent that, alone or in combination with one or more other
agents, reprograms somatic cells to a less differentiated state.
The invention further provides agents identified according to the
methods, in one embodiment, the methods comprise contacting somatic
cells with a Wnt pathway activator and a candidate agent and
determining whether the presence of the candidate agent results in
enhanced reprogramming (e.g., increased reprogramming speed and/or
efficiency) relative to that which would occur if cells had not
been contacted with the candidate agent. In some embodiments, the
Wnt activator and candidate agent are present together in the cell
culture medium while in other embodiments the Wnt activator and the
candidate agent are not present together (e.g., the cells are
exposed to the agents sequentially). The cells may be maintained in
culture liar, e.g., at least 3 days, at least 5 days, up to 10
days, up to 15 days, up to 30 days, etc., during which time they
are contacted with the Wnt activator and the candidate agent for
all or pert of the time. In some embodiments, the agent is
identified as an agent that reprograms cells if there are at least
2, 5, or 10 times as many reprogrammed cells or colonies comprising
predominantly reprogrammed cells after said time period than if the
cells have not been contacted with the agent.
[0141] A candidate agent can be any molecule or supramolecular
complex, e.g. a polypeptide, peptide (which is used herein to refer
to a polypeptide containing 60 amino acids or less), small organic
or inorganic molecule (i.e., molecules having a molecular weight
less than 1,500 Da, 1000 Da, or 500 Da), polysaccharide,
polynucleotide, etc. which is to be tested for ability to reprogram
cells. In some embodiments, the candidate agents are organic
molecules, particularly small organic molecules, comprising
functional groups that mediate structural interactions with
proteins, e.g., hydrogen bonding, and typically include at least an
amine, carbonyl, hydroxyl or carboxyl group, and in some
embodiments at least two of the functional chemical groups. The
candidate agents may comprise cyclic carbon or heterocyclic
structures and/or aromatic or polyaromatic structures substituted
with one or more chemical functional groups and/or heteroatoms.
[0142] Candidate agents are obtained from a wide variety of
sources, as will be appreciated by those in the art, including
libraries of synthetic or natural compounds. In some embodiments,
candidate agents are synthetic compounds. Numerous techniques are
available for the random and directed synthesis of a wide variety
of organic compounds and biomolecules. In some embodiments, the
candidate modulators are provided as mixtures of natural compounds
in the form of bacterial, fungal, plant and animal extracts,
fermentation broths, conditioned media, etc., that are available or
readily produced.
[0143] In some embodiments, a library of compounds is screened. A
library is typically a collection of compounds that can be
presented or displayed such that the compounds can be identified in
a screening assay. In some embodiments, compounds in the library
are housed in individual wells (e.g., of microtiter plates),
vessels, tubes, etc., to facilitate convenient transfer to
individual wells or vessels for contacting cells, performing
cell-free assays, etc. The library may be composed of molecules
having common structural features which differ in the number or
type of group attached to the main structure or may be completely
random. Libraries include but are not limited to, for example,
phage display libraries, peptide libraries, polysome libraries,
aptamer libraries, synthetic small molecule libraries, natural
compound libraries, and chemical libraries. Methods for preparing
libraries of molecules are well known in the art and many libraries
are available from commercial or non-commercial sources. Libraries
of interest include synthetic organic combinatorial libraries.
Libraries, such as, synthetic small molecule libraries and chemical
libraries can comprise a structurally diverse collection of
chemical molecules. Small molecules include organic molecules often
having multiple carbon-carbon bonds. The libraries can comprise
cyclic carbon or heterocyclic structure and/or aromatic or
polyaromatic structures substituted with one or more functional
groups. In some embodiments, the small molecule has between 5 and
50 carbon atoms, e.g., between 7 and 30 carbons. In some
embodiments, the compounds are macrocyclic. Libraries of interest
also include peptide libraries, randomized oligonucleotide
libraries, and the like. Libraries can be synthesized of peptoids
and non-peptide synthetic moieties. Such libraries can further be
synthesised which contain non-peptide synthetic moieties which are
less subject to enzymatic degradation compared so their naturally
occurring counterparts, Small molecule combinatorial libraries may
also be generated. A combinatorial library of small organic
compounds may comprise a collection of closely related analogs dial
differ from each other in one or more points of diversity and are
synthesized by organic techniques using multi-step processes.
Combinatorial libraries can include a vast number of small organic
compounds. A "compound array" as used herein is a collection of
compounds identifiable by their spatial addresses in Cartesian
coordinates and arranged such that each compound has a common
molecular core and one or more variable structural diversity
elements. The compounds in such a compound array are produced in
parallel in separate reaction vessels, with each compound
identified and tracked by its spatial address. Examples of parallel
synthesis mixtures and parallel synthesis methods are provided in
U.S. Pat. No. 5,712,171. In some embodiments, mixtures containing
two or mote compounds, extracts or other preparations obtained from
natural sources (which may comprise dozens of compounds or more),
and/or inorganic compounds, eta., are screened.
[0144] In one embodiment, the methods of the invention are used to
screen "approved drugs". An "approved drug" is any compound (which
term includes biological molecules such as proteins and nucleic
acids) which has been approved for use in humans by the FDA or a
similar government agency in another country, for any purpose. This
can be a particularly useful class of compounds to screen because
it represents a set of compounds which are believed to be safe and,
at least in the case of FDA approved dregs, therapeutic for at
least one purpose. Thus, there is a high likelihood that these
drugs will at least be safe for other purposes.
[0145] Representative examples of libraries that could be screened
include DIVERSet.TM., available from ChemBridge Corporation, 16981
Via Tazon, San Diego, Calif. 92127. DIVERSet contains between
10,000 and 50,000 drug-like, hand-synthesized small molecules. The
compounds are pre-selected to form a "universal" library that
covers the maximum pharmacophore diversity with the minimum number
of compounds and is suitable for either high throughput or lower
throughput screening. For description of additional libraries, see,
for example, Tan, et al., Am. Chem Soc. 120, 8565-8566, 1998; Floyd
C D, Leblanc C, Whittaker M, Prog Med Chem 36:91-168, 1999.
Numerous libraries are commercially available. e.g., from
AnalytiCon USA Inc., P.O. Box 5926, Kingwood, Tex. 77325;
3-Dimensional Pharmaceuticals, Inc., 665 Stockton Drive, Suite 104,
Extern, Pa. 19341-1151; Tripos, Inc., 1699 Hanky Rd., St Louis,
Mo., 63144-2913, etc. For example, libraries based on quinic acid
and shikimic acid, hydroxyproline, santonine, dianhydro-D-glucitol,
hydroxypipecolinic acid, andrographolide, piperazine-2-carboxylic
acid based library, cytosine, etc., are commercially available.
[0146] In some embodiments, the candidate agents are cDNAs from a
cDNA expression library prepared from cells, e.g., pluripotent
cells. Such cells may be embryonic stem cells, oocytes,
blastomeres, teratocarcinomas, embryonic germ cells, inner cell
mess cells, etc.
[0147] It will be appreciated that the candidate reprogramming
agent to be tested is typically one that is not present in standard
culture medium, or if present is present in lower amounts than when
used in the present invention.
[0148] It will also be appreciated that a useful reprogramming
agent or other form of reprogramming treatment need not be capable
of reprogramming all types of somatic cells and need not be capable
of reprogramming all somatic cells of a given cell type. Without
limitation, a candidate agent that results in a population that is
enriched for reprogrammed cells by a factor of 2, 5, 10, 50, 100 or
more (i.e., the fraction of reprogrammed cells in the population is
2, 5, 10, 50, or 100 times more than present in a starting
population of cells heated in the same way but without being
contacted with the candidate agent) is of use.
[0149] In some embodiments of the invention, the inventive
screening method is used to identify an agent or combination of
agents that substitutes for Klf4 in reprogramming cells to an
ES-tike state. The method may be practiced using somatic cells
engineered to express Sox2 and Oct4 and contacted with a Wnt
pathway activator. In some embodiments, the method is used to
identity an agent that substitutes for Sox2 in reprogramming cells
to an BS-like state. The method may be practiced using somatic
cells engineered to express Klf4 and Oct4 and contacted with a Wnt
pathway activator. In some embodiments, the method is used to
identify an agent that substitutes for Oct4 in reprogramming cells
to an ES-like state. The method may be practiced using somatic
cells engineered to express Sox2 and Klf 4 and contacted with a Wnt
pathway activator. It is contemplated that engineered expression of
Klf4, Sox2, Oct4, and c-Myc is replaced by treating somatic cells
with a combination of small molecules and/or polypeptides or other
agents that do not involve modification of the genome. In some
embodiments, the methods are practiced using human cells. In some
embodiments, the methods are practiced using mouse cells. In some
embodiments, the methods are practiced using non-human primate
cells.
[0150] The invention encompasses testing Wnt pathway modulators,
e.g., libraries of small molecules known or suspected to modulate
the Wnt pathway, to identity those that are effective in enhancing
reprogramming and/or have superior ability to enhance reprogramming
somatic cells to pluripotency, e.g., relative to other compounds
tested. In some embodiments, at least 10, at least 20, at least 50,
at least 100, or at least 1,000 small molecules, e.g., structurally
related molecules, at least some of which are known or believed to
modulate Wnt pathway activity, are tested. In some embodiments, a
Wnt inhibitor is used to confirm that a compound that enhances
reprogramming and is suspected of doing so by modulating Wnt
pathway activity docs in feet act via the Wnt pathway. For example,
if the Wnt pathway inhibitor blocks the effect of a test compound
on reprogramming, it may be concluded feat tire test compound acts
via the Wnt pathway.
[0151] The methods and compositions of the present invention
relating to Wnt pathway modulation may be applied to or used in
combination with various other methods and compositions useful for
somatic cell reprogramming and/or for identifying reprogramming
agents for use in somatic cell reprogramming. Such combined methods
and compositions are aspects of the invention. For example, some
embodiments of the invention employ cell types (e.g., neural stem
cells or progenitor cells) that naturally express one or more
reprogramming factors at levels higher than such factor(s) are
expressed in many other cell types (see, e.g., Eminli, et al.,
Reprogramming of Neural Progenitor Cells into iPS Cells in the
Absence of Exogenous Sox2 Expression, Stem Cells. 2008 Jut 17, epub
ahead of print).
[0152] The methods and compositions may be used together with
methods and compositions disclosed in PCT/US2008/004516, which is
incorporated heroin by reference:
[0153] Genetically homogeneous `secondary` somatic cells that carry
reprogramming fedora as defined doxycycline (dox)-inducible
transgenes have been derived Wenrig, et al., A novel drug-inducible
transgenic system for direct reprogramming of multiple somatic cell
types. Nature Biotechnology; published online 1 Jul. 2008;
doi:10.1038/nbt1483). These cells were produced by infecting
fibroblasts with dox-inducible lentiviruses, reprogramming by dox
addition, selecting induced pluripotent stem cells and producing
chimeric mice. Cells derived from these chimeras reprogram upon dox
exposure without the need for viral infection with efficiencies 25-
to 50-fold greater than those observed using direct infection and
drug selection for pluripotency marker reactivation. In some
embodiments of the invention, such secondary somatic cells are used
in embodiments of the present invention and/or secondary somatic
cells are generated without use of c-Myc virus by employing Wnt
pathway stimulation as described herein. The instant invention
contemplates use of Wnt pathway modulation in compositions and
methods relating to secondary somatic cells.
[0154] In some embodiments of the invention, the somatic cells
contain a nucleic acid sequence encoding a selectable marker,
operably linked to a promoter of an endogenous pluripotency gene,
e.g., Oct4 or Nanog. The sequence encoding the marker may be
integrated into the genome at the endogenous locus. The selectable
marker may be, e.g., a readily detectable protein such as a
fluorescent protein, e.g., GPP or a derivative thereof. Expression
of the marker is indicative of reprogramming and can thus be used
to identify or select reprogrammed cells, quantify reprogramming
efficiency, and/or to identify, characterize, or use agents that
enhance reprogramming and/or are being tested for their ability to
enhance reprogramming.
[0155] Reprogrammed Somatic Cells and Uses Thereof
[0156] The present invention provides reprogrammed somatic cells
(RSCs), including induced pluripotent stem cells (iPS cells),
produced by the methods of the invention. Those cells have numerous
applications in medicine, agriculture, and other areas of interest,
some of which are described here.
[0157] The invention provides methods for the treatment or
prevention of s condition in a mammal. In one embodiment, the
methods involve obtaining somatic cells from the individual,
reprogramming the somatic cells so obtained by methods of the
present invention to obtain RSCs, e.g., iPS cells. The RSCs are
then cultured under conditions suitable for their development into
cells of a desired cell type. The developed cells of the desired
cell type are introduced into the individual to treat the
condition. In an alternative embodiment, the methods start with
obtaining somatic cells from the individual, reprogramming the
somatic cells so obtained by methods of the present invention. The
RPCs are then cultured under conditions suitable for development of
the RPCs into a desired organ, which is harvested and introduced
into the individual to treat the condition. The condition may be
any condition in which cell or organ function is abnormal and/or
reduced below normal levels. Thus, the invention encompasses
obtaining somatic cells from an individual in need of cell therapy,
reprogramming the cells by a process that comprises activating a
Wnt pathway and/or culturing the cells in Wnt conditioned medium,
optionally differentiating reprogrammed somatic cells them to
generate cells of one or more desired cell types, and introducing
the cells into the individual. An individual m need of cell therapy
may suffer from any condition, wherein the condition or one or more
symptoms of the condition can be alleviated by administering cells
to the donor and/or in which the progression of the condition can
be slowed by administering cells to the individual. The method may
include a step of identifying or selecting reprogrammed somatic
cells and separating them from cells that are not reprogrammed.
[0158] The RSCs in certain embodiments of the present invention are
ES-like cells, also referred to as iPS cells, and thus may be
induced to differentiate to obtain the desired cell types according
to known methods to differentiate ES cells, for example, the iPS
cells may be induced to differentiate into hematopoietic stem
cells, muscle cells, cardiac muscle cells, liver cells, pancreatic
cells, cartilage cells, epithelial cells, urinary tract cells,
nervous system cells (e.g., neurons) etc., by culturing such cells
in differentiation medium and under conditions which provide for
edit differentiation. Medium sod methods which result in the
differentiation of embryonic stem cells obtained using traditional
methods are known in the art, as are suitable culturing conditions.
Such methods and culture conditions may be applied to the iPS cells
obtained according to the present invention. See, e.g., Trounson,
A., The production and directed differentiation of human embryonic
stem cells, Endocr Rev. 27(2):206-19, 2006 and references therein,
all of which are incorporated by reference, for some examples. See
also Yao, S., et al, Long-term self-renewal and directed
differentiation of human embryonic stem cells in chemically defined
conditions, Proc Natl Acad. Sci USA, 103(18): 6907-6912, 2006 and
references therein, all of which ere incorporated by reference.
[0159] Thus, using known methods and culture medium, one skilled in
the sit may culture the reprogrammed pluripotent cells to obtain
desired differentiated cell types, e.g., neural cells, muscle
cells, hematopoietic cells, etc. The subject cells may be used to
obtain any desired differentiated cell type. Such differentiated
human cells afford a multitude of therapeutic opportunities. For
example, human hematopoietic stem cells derived from cells
reprogrammed according to the present invention may be used in
medical treatments requiring bone marrow transplantation. Such
procedures are used to treat many diseases, e.g., late stage
cancers and malignancies such as leukemia. Such cells are also of
use to treat anemia, diseases that compromise the immune system
such as AIDS, etc. The methods of the present invention can also be
used to treat, prevent, or stabilise a neurological disease such as
Alzheimer's disease, Parkinson's disease, Huntington's disease, or
ALS, lysosomal storage diseases, multiple sclerosis, or a spinal
cord injury. For example, somatic cells may be obtained from the
individual in need of treatment, and reprogrammed to gain
pluripotency, and cultured to derive neurectoderm cells that may be
used to replace or assist the normal function of diseased or
damaged tissue.
[0160] Reprogrammed cells that produce a growth factor or hormone
such as insulin, etc., may be administered to a mammal for the
treatment or prevention of endocrine disorders. Reprogrammed
epithelial cells may be administered to repair damage to the lining
of a body cavity or organ, such as a lung, gut, exocrine gland, or
urogenital tract. It is also contemplated that reprogrammed cells
may be administered to a mammal to treat damage or deficiency of
cells in an organ such as the bladder, brain, esophagus, fallopian
tube, heart, intestines, gallbladder, kidney, liver, lung, ovaries,
pancreas, prostate, spinal cord, spleen, stomach, testes, thymus,
thyroid, trachea, ureter, urethra, or uterus.
[0161] The present invention has the potential to provide an
essentially unlimited supply of genetically matched cells suitable
for transplantation. Such a supply would address the significant
problem associated with current transplantation methods, i.e.,
rejection of the transplanted tissue which may occur because of
host versus graft or graft versus host rejection. RSCs may also be
combined with a matrix to form a tissue or organ in vitro or in
vivo that may be used to repair or replace a tissue or organ in a
recipient mammal. For example, RSCs may be cultured in vitro in the
presence of a matrix to produce a tissue or organ of the
urogenital, cardiovascular, or musculoskeletal system.
Alternatively, a mixture of the cells and a matrix may be
administered to a mammal for the formation of the desired tissue in
vivo. The RSCs produced according to the invention may be used to
produce genetically engineered or transgenic differentiated cells,
e.g., by introducing a desired gene or genes, or removing all or
part of an endogenous gene or genes of RSCs produced according to
the invention, and allowing such cells to differentiate into the
desired cell type. One method for achieving such modification is by
homologous recombination, which technique can be used to insert,
delete or modify a gene or genes at a specific site or sites in the
genome.
[0162] This methodology can be used to replace defective genes or
to introduce genes which result in the expression of
therapeutically beneficial proteins such as growth factors,
hormones, lymphokines, cytokines, enzymes, etc. For example, the
gene encoding brain derived growth factor may be introduced into
human embryonic or stem-like cells, the cells differentiated into
neural cells and the cells transplanted into a Parkinson's patient
to retard the loss of neural cells during such disease. Using known
methods to introduced desired genes/mutations into ES cells, RSCs
may be genetically engineered, and the resulting engineered cells
differentiated into desired cell types, e.g., hematopoietic cells,
neural cells, pancreatic cells, cartilage cells, etc. Genes which
may be introduced into the RSCs include, for example, epidermal
growth factor, basic fibroblast growth factor, glial derived
neurotrophic growth factor, insulin-like growth factor (I and II),
neurotrophin3, neurotrophin-4/5, ciliary neurotrophic factor,
AFT-1, cytokine genes (interleukins, interferons, colony
stimulating factors, tumor necrosis factors (alpha and beta),
etc.), genes encoding therapeutic enzymes, collagen, human serum
albumin, etc.
[0163] Negative selection systems known in the art can be used for
eliminating therapeutic cells from a patient if desired. For
example, cells transfected with the thymidine kinase (TK) gene will
lead to the production of embryonic (e.g., ES-like) cells
containing the TK gene. Differentiation of these cells will lead to
the isolation of therapeutic cells of interest which also express
the TK gene. Such cells may be selectively eliminated at any time
from a patient upon gancyclovir administration. Such a negative
selection system is described in U.S. Pat. No. 5,698,446. In other
embodiments the cells are engineered to contain a gene that encodes
a toxic product whose expression is under control of an inducible
promoter. Administration of the inducer causes production of the
toxic product, leading to death of the cells. Thus any of the
somatic cells of the invention may comprise a suicide gene,
optionally contained in an expression cassette, which may be
integrated into the genome. The suicide gene is one whose
expression would be lethal to cells. Examples include genes
encoding diphtheria toxin, cholera toxin, ricin, etc. The suicide
gene may be under control of expression control elements that do
not direct expression under normal circumstances in the absence of
a specific inducing agent or stimulus. However, expression can be
induced under appropriate conditions, e.g. (i) by administering an
appropriate inducing agent to a cell or organism or (ii) if a
particular gene (e.g., an oncogene, a gene involved in the cell
division cycle, or a gene indicative of dedifferentiation or loss
of differentiation) is expressed in the cells, or (iii) if
expression of a gene such as a cell cycle control gene or a gene
indicative of differentiation is lost. See, e.g., U.S. Pat. No.
6,761,884. In some embodiments the gene is only expressed following
a recombination event mediated by a site-specific recombinase. Such
an event may bring the coifing sequence into operable association
with expression control elements such as a promoter. Expression of
the suicide gene may be induced if it is desired to eliminate cells
(or their progeny) from the body of a subject after the cells (or
their ancestors) have been administered to a subject. For example,
if a reprogrammed somatic cell gives rise to a tumor, the tumor can
be eliminated by inducing expression of the suicide gene. In some
embodiments tumor formation is inhibited because the cells are
automatically eliminated upon dedifferentiation or lose of proper
cell cycle control.
[0164] Examples of diseases, disorders, or conditions that may be
treated or prevented include neurological, endocrine, structural,
skeletal, vascular, urinary, digestive, integumentary, blood,
immune, auto-immune, inflammatory, endocrine, kidney, bladder,
cardiovascular, cancer, circulatory, digestive, hematopoietic, and
muscular diseases, disorders, and conditions. In addition,
reprogrammed cells may be used for reconstructive application, such
as for repairing or replacing tissues or organs. In some
embodiments, it may be advantageous to include growth factors and
proteins or other agents that promote angiogenesis. Alternatively,
the formation of tissues can be effected totally in vitro, with
appropriate culture media and conditions, growth factors, and
biodegradable polymer matrices.
[0165] With respect to the therapeutic methods of the invention the
administration of RSCs to a mammal is not limited to a particular
mode of administration, dosage, or frequency of dosing; the present
invention contemplates all modes of administration, including
intramuscular, intravenous, intraarticular, intralesional,
subcutaneous, or any other route sufficient to provide a dose
adequate to prevent or treat a disease. The RSCs may be
administered to the mammal in a single dose or multiple doses. When
multiple doses are administered, the doses may be separated from
one another by, for example, one week, one month, one year, or ten
years. One or more growth factors, hormones, interleukins,
cytokines, or other cells may also be administered before, during,
or after administration of the cells to further bias them towards a
particular cell type.
[0166] The RSCs of the present invention may be used as an in vitro
model of differentiation, in particular for the study of genes
which are involved in the regulation of early development.
Differentiated cell tissues and organs generated using the
reprogrammed cells may be used to study effects of drugs and/or
identify potentially useful pharmaceutical agents.
[0167] Further Applications of Somatic Cell Reprogramming Methods
and Reprogrammed Cells
[0168] The reprogramming methods disclosed herein may be used to
generate RSCs, e.g., iPS cells, for a variety of animal species.
The RSCs generated can be useful to produce desired animals.
Animals include, for example, avians and mammals as well as any
animal that is an endangered species. Exemplary birds include
domesticated birds (e.g., quail, chickens, ducks, geese, turkeys,
and guinea hens). Exemplary mammals include murine, caprine, ovine,
bovine, porcine, canine, feline and non-human primate. Of these,
preferred members include domesticated animals, including, for
examples, cattle, pigs, horses, cows, rabbits, guinea pigs, sheep,
and goats.
[0169] Methods for Gene Identification
[0170] The invention provides methods for identifying a gene whose
expression inhibits generation of reprogrammed cells. One method
comprises: (i) activating the Wnt pathway in somatic cells; (ii)
reducing expression of a candidate gene by RNAi; (iii) determining
whether reducing expression of the candidate gene results in
Increased efficiency of reprogramming and, if so, identifying the
candidate gene as one whose expression inhibits reprogramming of
somatic cells. One method comprises: (i) culturing somatic cells in
Wnt conditioned medium; (ii) reducing expression of a candidate
gene by RNAi; (iii) determining whether reducing expression of the
candidate gene results in increased efficiency of reprogramming
and, if so, identifying the candidate gene as one whose expression
inhibits reprogramming of somatic cells. Optionally the somatic
cells are engineered to express at least one gene selected from:
Oct4, Sox2, Nanog, Lin28, and Klf4. Optionally the cells are
contacted with Wnt pathway modulator. Libraries of shRNA or siRNA
of use m the method are commercially available. The identified gene
is a target for inhibition in order to enhance cellular
reprogramming. Agents that inhibit the gene (either RNAi agents or
other agents such as small molecules) are of use to reprogram
somatic cells, e.g., in conjunction with a Wnt activator.
EXEMPLIFICATION
[0171] The invention now being generally described, it will be more
readily understood by reference to the following example, which are
included merely for purposes of illustration of certain aspects and
embodiments of the present invention, and are not intended to limit
the invention.
[0172] Materials and Methods for Example 1
[0173] Cell Culture, Viral Infections, Induction of Gene
Expression.
[0174] Cells wore cultured in 15% FBS, DMEM-KO, Penn/Step,
Glutamine, Nonessential amino acids, .beta.-ME, and LIF. Mouse
embryo fibroblasts (MEFs) with an Oct4-IRES-eGFP construct
(Meissner, A., et al., Nature Biotechnology, Direct reprogramming
of genetically unmodified fibroblasts into pluripotent stem cells.
Published online: 27 Aug. 2007|dot:10.1038/nbt1335) inserted into
the endogenous Oct4 locus were infected with lentiviral vectors
driving fee doxycycline-inducible expression of Oct4, Sox2, Klf4
and c-Myc or only Oct4, Sox2, and Klf4. The vectors were based on
the FUGW lentiviral vector backbone (Lois C, et al., Science 2002;
295:868-872), modified to include a tet-inducible promoter. Two
days following infliction cells were split and induced with
doxycycline in the presence or absence of Wnt3a conditioned media
(used in a 1:1 dilution with normal ES media with 2.times.LIF).
These cells were monitored for GFP expression by flow cytometry at
day 13 and again at day 20. In parallel, MEFs with
doxycycline-inducible Oct4 expressed from the collagen locus and
Oct4-IRES-(neo resistance) inserted into the endogenous Oct4 locus
were infected wife lentiviruses driving the overexpression of
either Sox2, Klf4 and c-Myc or Sox2 and Klf4. Again, two days
following infection cells were split and induced with doxycycline
in the presence or absence of Wnt3a conditioned media. Separate
plates of these cells were selected wife G418 at day 7 and day 13
respectively. Following at least one week of G418 selection,
resistant colonies were examined and counted.
[0175] Conditioned Medium.
[0176] Wnt3a conditioned media (CM) was collected from mouse L
cells feat had been transfected wife Wnt3a cDNA (Shibamoto et al.
1998). These cells are available through ATCC (CRL-2647) along with
the untransfected parent cell line (CRL-2648) to use for control
conditioned medium. Wnt3a transfected cells secrete Wnt, reaching
levels up to 400 ng/mL of the Wnt3a protein in their growth media.
The basal medium consisted of DMEM, 15% FBS, Penn/Strep, Glutamine
and nonessential amino acids, prepared according the protocol of
Single et al. (Single, et al., Biochem Biophys Res Common.,
345(2):789-95, 2006). The media collected from the secreting
fibroblasts was filtered and diluted 1:1 wife regular ES cell media
(15% FBS, DMEM-KO, Penn/Step, Glutamine, Nonessential amino acids,
.beta.-ME, and LIF). This media was then used to treat ES cells.
The Wnt3a conditioned media has been shown by Applicants and others
to activate the Wnt signaling pathway in ES cells, as demonstrated
by immunoblots examining beta-catenin phosphorylation.
Example 1: Generation of ES-Like Cells Using Wnt3a Conditioned
Media
[0177] We hypothesized that stimulation of the stimulation of the
Wnt pathway using soluble factors could modulate the efficiency of
inducing pluripotency in somatic cells. This Example describes
initial experiments undertaken to determine the effect of Wnt
pathway stimulation on reprogramming. Cells containing an
Oct4-IRES-eGFP or Oct4-IRES-neo construct were infected with lend
viral vectors encoding either three or four factors as described
above. Expression of the pluripotency factors was induced on day 2.
In some experiments, cells were cultured in Wnt3a conditioned media
or unconditioned media as shown in FIG. 4A (top) from days 2-13.
GFP expression was analyzed by FACS on day 13 and 20. In other
experiments, cells were cultured in Wnt3a conditioned media or
unconditioned media as shown in FIG. 4A (bottom) from days 2-13 or
2-20. G418 selection was imposed on day 7 or 13. Surviving colonies
were counted on day 20.
[0178] Results showed that Wnt3a conditioned media increases the
rate of iPS formation in fibroblasts transduced with the four
reprogramming transcription factors. As shown in FIG. 4B, Wnt3a
promotes iPS cell formation in cells over-exposing Oct4, Sox2, Klf4
and c-Myc. Selectable cells overexpressing Oct4, Sox2, Klf4 and
c-Myc formed robust G418-resistant colonies earlier in the presence
of Wnt3a conditioned media than in the absence of this media. When
selected at day 7, only small colonies formed in the absence of
Wnt, none of which could be propagated in culture. Colonies formed
in the presence of Wnt conditioned media at this point were larger
and could be passaged as clones. When selection was started at day
13, colonies were observed in the absence of Wnt3a conditioned
medium that could be propagated. Although there wore fewer colonies
at this time point in the presence of Wnt conditioned media than in
the absence of Wnt conditioned media, the colonies that did form
were large, relatively homogenous in appearance and again could be
maintained in culture. This result suggests that Wnt3a conditioned
medium not only increased the rate of reprogramming hut also
selected for colonies of reprogrammed cells.
[0179] Wnt3a conditioned media also allows iPS cells to be formed
without addition of the oncogenic transcription footer c-Myc.
Whereas no iPS cells were formed in our initial experiment when
fibroblasts were transduced with Oct4, Sox2 and Klf4, we did
observe iPS colonies with these three factors when cells were grown
in Wnt3a conditioned media. These colonies appear to be true iPS
cells based on morphology and activation of the endogenous Oct4
locus, an event normally restricted to pluripotent cells. As shown
in FIG. 4C, in the presence of Wnt3a conditioned media, robust
neo-resistant colonies were observed in Oct4, Sox2, Klf4
overexpressing cells selected at both day 7 and day 13. In the
absence of Wnt conditioned media, no cells not infected with c-Myc
virus were found to be neo-resistant at either time point Without
selection, Oct4-IRES-eGFP cells infected with Sox2 and Klf4 lend
virus were found to express GFP (indicative of activation of the
endogenous Oct4 locus) by day 20 only in the presence of Wnt
conditioned media.
[0180] Discussion
[0181] The findings described above are significant for at least
two major reasons. First, there is great interest m creating iPS
cells that do not have viral integrations of the oncogenic c-Myc
transcription factor. Chimaeric mice with iPS cells made with Myc
show high rates of cancer associated with somatic reactivation of
the c-Myc virus. Even in vitro we note that iPS cell lines
generated with the c-Myc virus contain a mixed population with some
cells appearing morphologically much like ES cells and others
growing more like transformed, cancerous cells. Our results
obtained thus for indicate that iPS lines created without c-Myc in
Wnt3a conditioned media appear to be more homogeneously ES-like in
their morphology. Second, Wnt3a conditioned media appears to exert
a selective effect favoring the formation of large, homogenous
colonies. Use of Wnt3a conditioned medium or Wnt3a pathway
activators could thus be used as an alternative selection process
rather than imposing a selection step that requires genetic
modification of the initial somatic cells. Use of Wnt3a conditioned
medium or Wnt3a pathway activators during reprogramming would liras
provide a valuable improvement to any method of reprogramming
somatic cells currently known in the art or developed in the
future.
[0182] Materials and Methods for Examples 2-8
[0183] Cell Culture.
[0184] V6.5 (C57BL/6-129) murine ES cells and iPS cells were grown
under typical ES conditions on irradiated mouse embryonic
fibroblasts (MEFs). Transgenic MEFs used in the infections with
DOX-inducible lend viruses (T. Brambrink, R. Foreman, Cell Stem
Cell 2, 151-159 (2008)) were harvested at 13.5 dpc and selected on
2 .mu.g/ml puromycin from embryos after blastocyst injection of
Oct4-IRES-GFPneo/Oct4-inducible ES cells (M. Wernig, A. Meissner,
Nature 448, 318-324 (2007)) or harvested from F1 matings between
R26-M2rtTA mice (C. Beard, K. Hochedlinger, Genesis 44, 23-28
(2006)) and Oct4-GFP mice (A. Meissner, M. Wernig, Nat Biotechnol
25, 1177-1181 (2007). Wnt3a conditioned media and control
conditioned media was generated according to standard protocols
(ATCC) (K. Willert, J. D. Brown, Nature 423, 448-452 (2003),
described also above) and used in a 1:1 ratio with standard ES cell
medium). Wnt inhibitor ICG-001 was dissolved in DMSO to a stock
concentration of 0.1M. The final, working concentration of the Wnt
inhibitor was 4 uM.
[0185] Viral Transduction.
[0186] Tetracycline inducible lentiviral constructs expressing the
cDNAs for Oct4, Klf4, Sox2 and c-Myc were used as previously
described (Brambrink, supra). Virus was prepared by transfecting
HBK293T cells with a mixture of viral plasmid and packaging
constructs expressing the viral packaging functions and the VSV-G
protein (Eugene, Roche). Medium was replaced 24 hours after
transfection and viral supernatants were collected at 48 hours and
72 hours. After filtration, supernatants were pooled and
2.5.times.10.sup.5 MEFs were incubated with viral supernatants and
fresh media at a ratio of 1:1 for 24 hours. Infected cells wens
then split at ratios from 1:5 to 1:12 onto gelatin-coated 10 cm
dishes. One day following the split, ES media was supplemented with
2 ug/ml DOX and, in the appropriate dishes, conditioned media
and/or chemical inhibitor.
[0187] Immunostaining and Antibodies Cells were stained as
described previously (Wernig, supra). Antibodies against Nanog
(Bethyl) and SSEA1 (RAD systems, Minneapolis, Minn.) were used
according to supplier recommendations.
[0188] Teratoma Formation
[0189] Teratoma formation was assayed as previously described.
Briefly, cells were trypsinized and 5.times.10.sup.5 cells were
injected subcutaneously into SCID mice. After 14-21 days, teratomas
were dissected, fixed in 10% phosphate-buffered formalin overnight
and subsequently embedded in paraffin wax using a Tissue-Tek VIP
embedding machine (Miles Scientific, Naperville, Ill.) and a Thermo
Shandon Histocenter 2 (Thermo Fisher Scientific, Waltham, Mass.).
Sections were cut at a thickness of 2 .mu.m using a Leice RM2065
(Leica, Wetzlar, Germany) and stained with hematoxylin and eosin
(K. Hochedlinger, Y. Yamada, Cell 121, 465-477 (2005).
[0190] Blastocyst Injection.
[0191] Injections of iPS cells into Balb/c host blastocysts were
carried out as previously described (Beard, supra).
Example 2: Further Experiments Relating to Generation of
ES-Like
[0192] To further define the effect of Wnt3a on reprogramming, we
infected MEFs that harbor a doxycycline (DOX)-inducible Oct4 cDNA
(Hochedlinger, 2005) with DOX-inducible lentiviral vectors encoding
Sox2, Klf4, and c-Myc (Brambrink, et al., 2008). These cells also
contained a G418 resistance cassette in the endogenous Oct4 locus
allowing for drug selection of iPS cells (Meissner et al.,
2007).
[0193] Four-factor expression was induced by addition of DOX in
cells cultured in the presence or absence of Wnt3a conditioned
medium (Wnt3a-CM), G418 selection was initiated after 5 days and
the number of drug resistant colonies was determined 24 days after
induction (FIG. 1a). FIG. 1b shows that the total number of drug
resistant colonies was increased more that 7 fold when the cells
were cultured in Wnt3a-CM. We also noted that the drug resistant
colonies were larger and more ES-cell like by morphology when
cultured in Wnt3a-CM than in BS cell medium (FIG. 1c). Furthermore,
the colonies that appeared in Wnt3a-CM with G418 selection
initiated on Day 5 could be further propagated, in contrast to the
small colonies derived in standard ES cell medium.
[0194] Given that Wnt3a-CM had a positive effect on reprogramming
in concert with the four transcription Sectors, we next examined if
Wnt3a-CM could substitute for any of the nuclear factors, to
parallel experiments, fibroblasts were transduced with subsets of
the transcription footers and observed in the presence and absence
of Wnt3a-CM (FIGS. 1d and 1e). No resistant colonies formed in the
absence of Oct4 or Klf4 infection. One colony was observed in the
absence of Sox2 retrovirus, but this colony could not be not be
further propagated in ES cell culture conditions. In contrast, in
the presence of Wnt3a-CM, multiple robust G418-resistant colonies
formed in the absence of c-Myc in cells over-expressing Oct4, Sox2
and Klf4 (FIGS. 1d and 1e). Similar to colonies from MEFs
transduced with all four Actors, these iPS lines could be
propagated in standard ES cell media without further selection and
retained ES cell morphology, in replicate experiments,
G418-resistant colonies were formed occasionally with no c-Myc
transduction in the absence Wnt3a-CM. However, consistent with
published reports (8,9), these colonies were rare. In the
following, iPS cells generated with only three factors and without
c-Myc will be designated as Myc.sup.[-] iPS cells.
[0195] To more closely examine the effects of Wnt3a-CM treatment on
the reprogramming process, Oct4/Sox2/Klf4 and Oct4/Sox2/Klf4/c-Myc
over-expressing MEFs were cultured with and without and Wnt3a-CM,
and G418 selection was initiated at different times after DOX
addition. FIG. If shows that when three factor over-expressing
cells were cultured in Wnt3a-CM medium about 3 fold more
Myc.sup.[-] iPS colonies appeared when G418 was added at day 5 and
about 20 fold more colonies when G418 was added at day 10 after
induction as compared to cultivation in ES cell medium (FIG. 1f,
left panel) Wnt3a-CM medium also increased the number of drug
resistant colonies after induction of all four factors, though the
fold increase was less pronounced than in three factor induced
cells (FIG. 1f, right panel). These results indicate that Wnt3a-CM
increased the number of drug resistant colonies in both three
factor and four factor induced cells, with the most pronounced
effect on three factor over-expressing cells with selection applied
at the later time point.
Example 3: Generation of Myc.sup.[-] iPS Clones without Genetic
Selection
[0196] Recently, iPS cells have been generated without c-Myc
retrovirus (Myc[-]), but in the absence of exogenous c-Myc the
efficiency and kinetics of reprogramming are significantly reduced
(Nakagawa et al., 2008; Wernig et al., 2008). We tested whether
Wnt3a-CM would also aid in the generation of iPS cells in the
absence of selection for Oct4 reactivation. For this, cells with
GFP driven by the endogenous Oct4 promoter were utilized (Meissner,
et al., 2008). Oct4/Sox2/Klf4 infected cells with and without
Wnt3a-CM treatment were analyzed for GFP expression by flow
cytometry at days 10, 15 and 20 after DOX induction. No GFP
positive cells were present with or without Wnt3a-CM treatment on
day 10 or day 15. By day 20 a small population of GFP expressing
cells was detected in cells cultured in Wnt3a-CM but not in
standard ES cell medium (FIG. 1g). The Wnt3a-CM exposed cultures
formed GFP expressing colonies with morphology typical for ES or
iPS cells (FIG. 1b). However, unlike four footer transduced cells,
which usually form a highly heterogeneous population of cells when
propagated without selection, the Oct4/Sox2/Klf4/Wnt3a-CM colonies
appeared homogenously ES-like similar to previously reported
Myc.sup.[-] iPS clones (Nakagawa, et al., 2006.
Example 4: Developmental Potential of Myc.sup.[-] iPS Cells Derived
with Wnt3a-CM
[0197] Several assays were performed to characterize the
developmental potential of Myc.sup.[-] iPS cells derived with
Wnt3a-CM treatment. Immunocytochemistry confirmed the expression of
markers of pluripotency, including the nuclear factor Nanog (FIGS.
2a and 2b), and the surface glycoprotein SSEA1 (FIGS. 2c and 2d).
Functional assays confirmed dial, like ES cells, these iPS cells
were pluripotent. When injected into SCID mice subcutaneously, the
Myc.sup.[-] iPS cells gave rise to teratomas with histological
evidence of cells differentiating into all three germ layers (FIGS.
2e, 2f and 2g). More importantly, Myc.sup.[-] iPS cells derived
with Wnt3a-CM treatment contributed to the formation of
differentiated tissues in chimeric mice (FIG. 2h). These results
indicate that Wnt3a-CM treated Myc.sup.[-] clones are pluripotent
cells that are morphologically and functionally indistinguishable
from ES cells.
Example 5: Effect of Small Molecule Wnt Pathway Inhibitor on
Generation of IPS Myc.sup.[-] and IPS Cells in the Presence of
Wnt3a-CM
[0198] To quantify the effects of Wnt3a-CM, triplicate experiments
were performed on Oct4/Sox2/Klf4-inducible, G418 selectable MEFs
(FIG. 3a). G418 was added to the cultures et 15 days after
infection to select for cells that had reactivated the Oct4 locus.
When scored on day 28 alter infection, only a few Myc[-] G418
resistant colonies (between 0-3 colonies forming on each ten
centimetre plate) were detected in standard ES cell culture
conditions. In contrast, .about.20 fold more drug resistant
colonies formed when G418 selection was initiated on
Wnt3a-CM-treated cells, consistent with the conclusion that
activation of the Wnt pathway enhances reprogramming. It should be
noted that conditioned medium from control fibroblasts lacking
Wnt3a over-expression also caused a moderate increase in the number
of G418-resistant colonies relative to standard ES medium,
suggesting that normal fibroblasts may secrete factors, perhaps
including Wnt3a, that promote reprogramming.
[0199] To independently assess the effect of Wnt3a on
reprogramming, we cultured cells in the presence of ICG-001 (Teo et
al., 2005; McMillan and Kahn, 2005; see FIG. 5), an inhibitor of
the Wnt/.beta.-catenin pathway. FIG. 3a (right columns) shows that
4 .mu.M ICG-001 strongly inhibited the effect of Wnt3a-CM on
Myc.sup.[-] iPS formation. The effects of Wnt3a-CM and ICG-001 were
also examined in MEFs over-expressing all four reprogramming
factors, including c-Myc (FIG. 3b). High numbers of G418 resistant
colonies were observed in both standard ES cell media and Wnt3a-CM
in four factor reprogrammed cells, with only a subtle increase in
the number of colonies with Wnt3aCM. In contrast to the dramatic
effect of ICG-001 on Myc.sup.[-] cells, at the same dose, the
compound had only a subtle effect on the number of G418 colonies in
c-Myc transduced cells, and a relatively high number of resistant
colonies were observed under these conditions. At higher doses of
ICG-001, iPS colony numbers were further reduced, but even at 25
.mu.M multiple Oct4/Sox2/Klf4/c-Myc iPS colonies were observed
(data not shown). These results are consistent with the notion that
Wnt3a can, at least in part, replace the role of c-Myc in
reprogramming.
[0200] The Wnt signaling pathway has been shown to connect directly
to the core transcriptional regulatory circuitry of ES cells,
suggesting a mechanism by which this pathway could directly promote
the induction of the pluripotency in the absence of c-Myc
transduction (FIG. 3c). The Wnt signaling pathway has been shown to
connect directly to the cote transcriptional regulatory circuitry
of ES cells, suggesting a mechanism by which this pathway could
directly promote the induction of the pluripotency in the absence
of c-Myc transduction (FIG. 2c). In ES cells, Tcf3 occupies and
regulates the promoters of Oct4, Sox2 and Nanog (Cole et al., 2008;
Tam et al., 2008; Yi et al., 2008). In MEFs, these endogenous
pluripotency transcription factors are silenced. During
reprogramming, as exogenous Oct4, Sox2 and Klf4 contribute to the
reactivation of the endogenous pluripotency factors (Jaenisch and
Young, 2008), Wnt signaling could directly potentiate the effect of
these transcription factors, as it does in ES cells (Cole et al.,
2008). Additionally or alternately, Wnt could serve to activate
endogenous c-Myc directly, thereby substituting for exogenous
c-Myc. Indeed, c-Myc is a well-established target of the Wnt
pathway in colorectal cancer cells (He et al., 1098). In ES cells,
Tcf3 occupies the c-Myc promoter, and Wnt5a positively contributes
to expression of the gene (Cole et al., 2008), The fact that
enforced over-expression of c-Myc counteracts the negative effect
of the Wnt inhibitor ICG-001 on the reprogramming process suggests
that Wnt stimulation could be acting upstream of the endogenous
Myc. Wnt-induced effects on cell proliferation, mediated by c-Myc
or other endogenous proliferation factors, could help to accelerate
the sequence of events that lead to the generation of Myc[-] iPS
colonies.
[0201] A major goal of current research is to identify transient
cues that can reprogram somatic cells, eliminating the need for
retroviruses. The studies described here establish that Wnt
stimulation can be used to enhance the efficiency of reprogramming
in combination with nuclear Actors, Oct4, Sox2 and Klf4. By
enhancing the efficiency of reprogramming in the absence of c-Myc
retrovirus, soluble Wnt or small molecules that modulate the Wnt
signaling pathway will likely prove useful in combination with
other transient cues that can replace the remaining
retroviruses.
Example 6: Identification of Additional Reprogramming Agents
[0202] Example 3 is modified in that the medium farther contains,
in addition to Wnt3a-CM, a candidate reprogramming agent to be
tested for its potential to enhance or inhibit reprogramming. In
some embodiments the cells are infected so that they express only 2
of the following 3 reprogramming factors: Oct4, Klf4, and Sox2.
Agents that enhance generating of reprogrammed cells (e.g.,
increase speed or efficiency of reprogramming) are identified. The
process is repeated to identify agents capable of substituting for
engineered expression of Oct4, Klf4, and/or Sox2 in reprogramming
somatic cells.
Example 7: Identification of Additional Reprogramming Agents
[0203] Example 3 is modified in that the Wnt3a-CM medium farther
contains a candidate reprogramming agent. In some embodiments, the
cells are infected so that they express only 1 or 2 of the
following reprogramming factors: Oct4, Lin28, Sox2, and Nanog
(e.g., Oct4 only, Oct-4 and Sox2). Agents that enhance generating
of reprogrammed cells are identified. The process is repeated to
identify agents capable of substituting for engineered expression
of Oct4, Lin28, Sox2, and/or Nanog in reprogramming somatic
cells.
Example 8: Use of Small Molecule Wnt Pathway Modulator in
Reprogramming
[0204] Example 3 is repeated except that instead of using Wnt3a-CM,
ES cell medium containing a small molecule Wnt pathway activator is
used.
REFERENCES
[0205] Brambrink, T., Foreman, R., Welstead, G. G., Lengner, C. J.,
Wernig, M., Suh, H., and Jaenisch, R. (2008). Sequential expression
of pluripotency markers during direct reprogramming of mouse
somatic cells. Cell Stem Cell 2, 151-159. [0206] Cai. L, Ye, Z.,
Zhou, B. Y., Mali, P., Zhou, C., and Cheng, L. (2007). Promoting
human embryonic stem cell renewal or differentiation by modulating
Wnt signal and culture conditions. Cell Res 17, 62-72. [0207] Cole,
M. F., Johnstone, S. E., Newman, J. J., Kagey, M. H., and Young, R.
A. (2008). Tcf3 is an integral component of the core regulatory
circuitry of embryonic stem cells. Genes and Development 15;
22(6):746-55 (2008). [0208] Hanna, J., Wernig, M., Markoulaki, S.,
Sun, C. W., Meissner, A., Cassady, J. P., Beard, C., Brambrink, T.,
Wu, L. C., Townes, T. M., and Jaenisch. R. (2007). Treatment of
sickle cell anemia mouse model with iPS cells generated from
autologous skin. Science 318, 1920-1923. [0209] He, T. C., Sparks,
A. B., Rago, C., Hermeking, H., Zawel, L., da Costa, L. T., Morin,
P. J., Vogelstein, B., and Kinder, K. W. (1998). Identification of
c-MYC as a target of the APC pathway. Science 281, 1509-1512.
[0210] Hochedlinger, K., Yamada, Y., (2005) Cell. 121, 465-477.
[0211] Jaenisch, R., and Young, R. A. (2008). Stem cells, the
molecular circuitry of pluripotency and nuclear reprogramming. Cell
132, 567-582. [0212] Kim, J., Chu, J., Shen, X., Wang, J., and
Orkin, S. H. (2008). An extended transcriptional network for
pluripotency of embryonic atom cells. Cell 132, 1049-1061. [0213]
Knoepfler, P. S. (2008). Why Myc? An unexpected ingredient in the
stem cell cocktail. Cell Stem Cell 2, 18-21. [0214] McMillan, M.,
and Kahn, M. (2005). Investigating Wnt signaling: a chemogenomic
safari. Drug Discov Today 10, 1467-1474. [0215] Meissner, A.,
Wernig, M., and Jaenisch, R. (2007). Direct reprogramming of
genetically unmodified fibroblasts into pluripotent stem cells. Nat
Biotechnol 25, 1177-1181. [0216] Nakagawa, M., Koyanagi, M.,
Tanabe, K., Takahashi, K., Ichisaka, T., Aoi, T., Okita, K.,
Mochiduki, Y., Takizawa, N., and Yamanaka, S. (2008). Generation of
induced pluripotent stem cells without Myc from mouse and human
fibroblasts. Nat Biotechnol 26, 101-106. [0217] Ogawa, K.,
Nishinakamura, R., Iwamatsu, Y., Shimosato, D., and Niwa, H.
(2006). Synergistic action of Wnt and LlF in maintaining
pluripotency of mouse ES cells. Biochem Biophys Res Common 343,
159-166. [0218] Okita, K., Ichisaka, T., and Yamanaka, S. (2007).
Generation of germline-competent induced pluripotent stem cells.
Nature 448, 313-317. [0219] Reya, T., and Clevens, H. (2005). Wnt
signalling in stem cells and cancer. Nature 434, 843-850. [0220]
Sato, N., Meijer, L., Skaltsounis, L, Greengard, P., and Brivanlou,
A. H. (2004). Maintenance of pluripotency in human and mouse
embryonic stem cells through activation of Wnt signaling by a
pharmacological GSK-3-specific inhibitor. Nat Med 10, 55-63. [0221]
Single, D. K., Schneider, D. J., LeWinter, M. M., and Sobel, B. E.
(2006), wnt3a but not wnt11 supports self-renewal of embryonic stem
cells. Biochem Biophys Res Community, 780-795. [0222] Stadtfeld,
M., Maherali, N., Breault, D. T., and Hochedlinger, K. (2008).
Defining molecular cornerstones during fibroblast to iPS cell
reprogramming in mouse. Cell Stem Cell in press. [0223] Takahashi,
K., Tanabe, K., Ohnuki, M., Narita, M., Ichisaka, T., Tomoda, K.,
and Yamanaka, S. (2007). Induction of pluripotent stem cells from
adult human fibroblasts by defined factors. Cell 131, 861-871
[0224] Takahashi, K., and Yamanaka, S. (2006). Induction of
pluripotent stem cells from mouse embryonic and adult fibroblast
cultures by defined factors. Cell 126, 663-676. [0225] Tam, W. L,
Lim, C. Y., Han, J., Zhang, J., Ang. Y. S., Ng, H. H., Yang, H.,
and Lim, B. (2008). Tcf3 Regulates Embryonic Stem Cell Pluripotency
and Self-Renewal by the Transcriptional Control of Multiple Lineage
Pathways. Stem Cells. [0226] Teo, J. L, Ma, H., Nguyen, C., Lam,
C., and Kahn. M. (2005). Specific inhibition of CBP/beta-catenin
interaction rescues defects in neuronal differentiation caused by
apresenilin-1 mutation. Proc Natl Acad. Sci USA 102, 12171-12176.
[0227] Wernig, M., Meissner, A., Cassady, J. P., and Jaenisch, R.
(2008), c-Myc is dispensable for direct reprogramming of mouse
fibroblasts. Cell Stem Cell 2, 10-12. [0228] Willert, K., Brown, J.
D., Danenberg, E., Duncan, A. W., Weissman, I. L., Reya, T., Yales,
J. R., 3rd, and Nusse, R. (2003). Wnt proteins are lipid-modified
and can act astern cell growth factors. Nature 423, 448-452. [0229]
Yi, F., Pereira, L., and Merrill, B. J. (2008). Tcf3 Functions as a
Steady State Limiter of Transcriptional Programs of Mouse Embryonic
Stem Cell Self-Renewal Stem Cells. [0230] Yu, J., Vodyanik, M. A.,
Smuga-Otto, K., Antosiewicz-Bourget, J., Frane, J. L., Tian, S.,
Nie, J., Jonadottir, G. A., Ruotti, V., Stewart, R., et al. (2007).
Induced pluripotent stem cell lines derived from human somatic
cells. Science 318, 1917-1920.
[0231] The practice of the present invention will employ, unless
otherwise indicated, conventional techniques of mouse genetics,
developmental biology, cell biology, cell culture, molecular
biology, transgenic biology, microbiology, recombinant DNA, and
immunology, which are within the skill of the art. Such techniques
are described in the literature. See, for example, Current
Protocols in Cell Biology, ed. by Bonifacino, Dasso,
Lippincott-Schwartz, Hartford, and Yamada, John Wiley and Sons,
Inc., New York. 1999; Manipulating the Mouse Embryos, A Laboratory
Manual, 3.sup.rd Ed., by Hogan et al., Cold Spring Contain
Laboratory Press, Cold Spring Contain, New York, 2003; Gene
Targeting: A Practical Approach, IRL Press at Oxford University
Press, Oxford, 1993; and Gene Targeting Protocols, Human Press,
Totowa, N.J., 2000. All patents, patent applications and references
cited herein are incorporated in their entirety by reference.
[0232] One skilled in the art readily appreciates that the present
invention is well adapted to carry out the objects and oblate the
ends and advantages mentioned, as well as those inherent therein.
The methods, systems and kits are representative of certain
embodiments, are exemplary, and are not intended as limitations on
the scope of the invention. Modifications therein and other uses
will occur to those skilled in the art. These modifications are
encompassed within the spirit of the invention and are defined by
the scope of the claims. It will be readily apparent to a person
skilled in the art that varying substitutions and modifications may
be made to the invention disclosed herein without departing from
the scope and spirit of the invention.
[0233] The articles "a" and "an" as used herein in the
specification and in the claims, unless clearly indicated to the
contrary, should be understood to Include the plural referents.
Claims or descriptions that include "or" between one or more
members of a group are considered satisfied if one, more than one,
or all of the group members are present in, employed in, or
otherwise relevant to a given product or process unless indicated
to the contrary or otherwise evident from the context. The
invention includes embodiments in which exactly one member of the
group is present in, employed in, or otherwise relevant to a given
product or process. The invention also includes embodiments in
which more then one, or all of the group members are present in,
employed in, or otherwise relevant to a given product or process.
Furthermore, it is to be understood that the invention encompasses
all variations, combinations, and permutations m which one or more
limitations, elements, clauses, descriptive terms, etc., from one
or mote of the listed claims is introduced into another claim
dependent on the same base claim (or, as relevant, any other claim)
unless otherwise indicated or unless it would be evident to one of
ordinary skill in the art that a contradiction or inconsistency
would arise. Where elements are presented as lists, e.g., in
Markush group or similar format, it is to be understood that each
subgroup of the elements is also disclosed, and any element(s) can
be removed from the group. It should it be understood that, in
general, where the invention, or aspects of the invention, is/are
referred to as comprising particular elements, features, etc.,
certain embodiments of the invention or aspects of the invention
consist, or consist essentially of, such elements, features, etc.
For purposes of simplicity those embodiments have not in every case
been specifically set forth herein. It should also be understood
that any embodiment of the invention can be explicitly excluded
from the claims, regardless of whether the specific exclusion is
recited in the specification. For example, any Wnt modulator, e.g.,
any Wnt pathway activating agent, any somatic cell type, any
reprogramming agent, etc., may be excluded.
[0234] Where ranges are given herein, the invention includes
embodiments in which the endpoints are included, embodiments in
which both endpoints are excluded, and embodiments in which one
endpoint is included and the other is excluded. It should be
assumed that both endpoints are included unless indicated
otherwise. Furthermore, it is to be understood that unless
otherwise indicated or otherwise evident from the context and
understanding of one of ordinary skill in the art, values feat are
expressed as ranges can assume any specific value or subrange
within the stated ranges in different embodiments of the invention,
to the tenth of the unit of the lower limit of the range, unless
the context clearly dictates otherwise, it is also understood that
where a series of numerical values is stated herein, the invention
includes embodiments that relate analogously to any intervening
value or range defined by any two values in the aeries, and that
the lowest value may be taken as a minimum and the greatest value
may be taken as a maximum. Numerical values, as used herein,
include values expressed as percentages. For any embodiment of the
invention in which a numerical value is prefaced by "about" or
"approximately", the invention includes an embodiment in which the
exact value is netted. For any embodiment of the invention in which
a numerical value is not prefaced by "about" or "approximately",
the invention includes an embodiment in which the value is prefaced
by "about" or "approximately". "Approximately" or "about" is
intended to encompass numbers that fall within a range of .+-.10%
of a number, in some embodiments within .+-.5% of a number, in some
embodiments within .+-.1%, in some embodiments within .+-.0.5% of a
number, in some embodiments within .+-.0.1% of a number unless
otherwise stated or otherwise evident from the context (except
where such number would impermissibly exceed 100% of a possible
value).
[0235] Certain claims are presented in dependent form for the sake
of convenience, but Applicant reserves the right to rewrite any
dependent claim in independent form to include the limitations of
the independent claim and any other claim(s) on which such claim
depends, and such rewritten claim is to be considered equivalent in
all respects to the dependent claim in whatever form it is in
(either amended or unamended) prior to being rewritten in
independent format. It should also be understood that, unless
clearly indicated to the contrary, in any methods claimed herein
that include more than one act, the order of the acts of the method
is not necessarily limited to the older. In which the acts of the
method are recited, but the invention includes embodiments in which
the order is so limited.
* * * * *
References