U.S. patent application number 16/917008 was filed with the patent office on 2020-10-22 for generation of induced pluripotent stem cells from normal human mammary epithelial cells.
This patent application is currently assigned to CEDARS-SINAI MEDICAL CENTER. The applicant listed for this patent is CEDARS-SINAI MEDICAL CENTER. Invention is credited to Xiaojiang Cui, Loren A. Ornelas, Dhruv Sareen.
Application Number | 20200332316 16/917008 |
Document ID | / |
Family ID | 1000004929352 |
Filed Date | 2020-10-22 |
United States Patent
Application |
20200332316 |
Kind Code |
A1 |
Cui; Xiaojiang ; et
al. |
October 22, 2020 |
GENERATION OF INDUCED PLURIPOTENT STEM CELLS FROM NORMAL HUMAN
MAMMARY EPITHELIAL CELLS
Abstract
Described herein are reprogramming techniques allowing for
production of mammary-derived iPSCs ("m-iPSCs"). The m-iPSCs
described herein exhibit all the hallmarks of stem cell identity
including round cluster, bright colony morphology, clonal
expansion, and pluripotent marker expression (alkaline phosphatase
expression, Oct-4, nanog, etc.) Further refined techniques allow
for generation of m-iPSCs under essentially defined conditions.
Inventors: |
Cui; Xiaojiang; (Arcadia,
CA) ; Sareen; Dhruv; (Porter Ranch, CA) ;
Ornelas; Loren A.; (Los Angeles, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
CEDARS-SINAI MEDICAL CENTER |
Los Angeles |
CA |
US |
|
|
Assignee: |
CEDARS-SINAI MEDICAL CENTER
Los Angeles
CA
|
Family ID: |
1000004929352 |
Appl. No.: |
16/917008 |
Filed: |
June 30, 2020 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
14904641 |
Jan 12, 2016 |
10738323 |
|
|
PCT/US2014/046405 |
Jul 11, 2014 |
|
|
|
16917008 |
|
|
|
|
61845590 |
Jul 12, 2013 |
|
|
|
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12N 5/0696 20130101;
C12N 2501/603 20130101; C12N 15/85 20130101; C12N 2501/602
20130101; C12N 2506/09 20130101; C12N 2501/604 20130101; C12N
2501/415 20130101; C12N 2501/608 20130101; C12N 2501/727 20130101;
C12N 2501/115 20130101; C12N 15/8509 20130101; C12N 2510/00
20130101; C12N 2501/235 20130101 |
International
Class: |
C12N 15/85 20060101
C12N015/85; C12N 5/074 20060101 C12N005/074 |
Claims
1-22. (canceled)
23. A method of generating human epithelial cell-derived induced
pluripotent stem cells (ep-iPSCs), comprising: providing a quantity
of human epithelial cells; transfecting the human epithelial cells
with vectors encoding Oct4, Sox2, Nanog, Kruppel-like Factor 4
(KLF4), L-Myc, Lin28, SV40 Large T Antigen (SV40LT) and p53 shRNA;
plating the cells on a culture vessel coated with a substrate; and
culturing the cells in an induction media, wherein the
transfection, plating and the culturing generates colonies of human
epithelial-derived induced pluripotent stem cells (ep-iPSCs),
wherein the induction media comprises HA-100, CHIR99021, PD0325901,
and A83-01, wherein the plating of the transfected cells on the
culture vessel coated with the substrate further comprises
culturing in norm oxygen conditions, and wherein the plated mammary
cells express the transfected Oct4, Sox2, Nanog, KLF4, L-Myc,
Lin28, SV40LT and p53 shRNA.
24. The method of claim 23, wherein the epithelial cells are from
primary cells.
25. The method of claim 23, wherein the epithelial cells are from a
tumor.
26. The method of claim 23, wherein the epithelial cells are from a
cell line.
27. The method of claim 23, wherein the at least one vector
comprises an episomal vector.
28. The method of claim 27, wherein the episomal vector comprises
an oriP/EBNA1 vector.
29. The method of claim 23, wherein transfecting epithelial cells
comprises nucleofection or lipofection.
30. The method of claim 23, wherein plating the cells on a culture
vessel coated with a substrate further comprises culturing in norm
oxygen conditions.
31. The method of claim 23, wherein the substrate comprises
Matrigel.
32. The method of claim 23, wherein culturing the cells in an
induction media is for a period of 10-31 days.
33. A composition of ep-iPSCs produced by the method of claim
23.
34. The composition of claim 33, wherein the ep-iPSCs are capable
of serial passaging as a cell line.
35. A multi-potent cell produced by culturing the composition of
claim 34 in the presence of a differentiation agent.
36. A method of reprogramming a human epithelial cell, comprising:
providing a quantity of human epithelial cells; expanding the human
mammary cells to about 90% confluence; culturing the human
epithelial cells with vectors encoding Oct4, Sox2, Nanog,
Kruppel-like Factor 4 (KLF4), L-Myc, Lin28, SV40 Large T Antigen
(SV40LT) and p53 shRNA; plating the cells on a culture vessel
coated with a substrate; and culturing the cells in an induction
media, wherein the expanding, the transfecting, the plating and the
culturing reprograms the human mammary cells to a less
differentiated state, wherein the induction media comprises HA-100,
CHIR99021, PD0325901, and A83-01, wherein the plating of the
transfected cells on the culture vessel coated with the substrate
further comprises culturing in norm oxygen conditions, and wherein
the plated mammary cells express the transfected Oct4, Sox2, Nanog,
KLF4, L-Myc, Lin28, SV40LT and p53 shRNA.
37. The method of claim 36, wherein the epithelial cells are from
primary cells.
38. The method of claim 36, wherein the epithelial cells are from a
tumor.
39. The method of claim 36, wherein the epithelial cells are from a
cell line.
Description
FIELD OF THE INVENTION
[0001] Described herein are compositions and methods related to
pluripotent stem cells derived from mammary tissue. Such
compositions and methods find application in regenerative
medicine.
BACKGROUND
[0002] There is growing evidence that in many cancers, tumors are
initiated maintained by rare populations of dysregulated cells with
stem cell-like properties, collectively known as cancer stem cells
("CSCs"). These CSCs possess the hallmark stem cell properties of
self-renewal and multipotent differentiation capacity. It is also
these same properties of CSCs that promote the pronounced effects
of CSCs in cancer disease generation and progression, through
initiation of tumor formation, chemoresistance, bulk generation of
heterogenous tumor cells, and malignancy. Despite this increasing
evidence for the critical role of CSCs in cancer development, the
cellular origins of CSCs remains highly obscured and positive
identification of mammary CSCs ("maCSCs") in the specific context
of breast cancer remains elusive. The picture is even more
difficult to ascertain, given that the mere existence of human
mammary stem cells ("maSCs") is controversial. This lack of
understanding creates highly divergent possibilities of maCSCs
arising from endogenous stem cells altered through genetic
mutation, or from dedifferentiation of adult somatic cells. Which
mechanism accounts for breast cancer pathogenesis remains a totally
unanswered question.
[0003] Clearly, establishing the existence and identifying the
biological characteristics of human maSCs and their progeny would
also be a helpful first step in advancing identification of mammary
CSCs ("maCSCs") in breast cancer. An improving understanding of
breast cancer as subtypes possessing genetic signatures similar to
a cell-of-origin, identifying both maSCs and maCSCs would shed
light on crucial questions related to mechanisms of stem-cell
origin or adult dedifferentiation. Ultimately, identifying the
relevant pathological actors and mechanisms of cancer
parthenogenesis would allow distinguishing between maSCs, normal
mammary gland tissue cells, bulk tumor cells and maCSCs, thereby
allowing development of targeted therapeutic approaches for cancer
treatment. Present efforts to hone in on maSC or maCSC populations
are severely hampered by the fact that primary normal and tumor
mammary epithelial cells can only be cultured for short periods of
time before they cease proliferating and undergo senescence. Thus,
there is a great need in the art for platforms allowing for
generation of materials relevant to mammary development and breast
cancer formation.
[0004] Described herein are induced pluripotent stem cell ("iPSC")
related reprogramming techniques allowing for production of
mammary-derived iPSCs ("m-iPSCs"). Importantly, the m-iPSCs
described herein exhibit all the hallmarks of stem cell identity
including round cluster, bright colony morphology, clonal
expansion, and pluripotent marker expression (alkaline phosphatase
expression, Oct-4, nanog, etc.) Further refined techniques allow
for generation of m-iPSCs under essentially defined conditions.
SUMMARY OF THE INVENTION
[0005] Described herein is a method of generating human
mammary-derived induced pluripotent stem cells (m-iPSCs), including
providing a quantity of human mammary cells, transfecting the human
mammary cells with at least one vector encoding at least one
reprogramming factor, plating the cells on a culture vessel coated
with a substrate, and culturing the cells in an induction media,
wherein the transfection, plating and culturing generates colonies
of human mammary-derived induced pluripotent stem cells
(m-iPSCs).
[0006] In other embodiments, the mammary cells are from primary
cells. In other embodiments, the mammary cells are from a tumor. In
other embodiments, the mammary cells are from a cell line. In other
embodiments, the at least one vector encodes at least one
reprogramming factor selected from the following group: Oct4, Sox2,
Nanog, Kruppel-like Factor 4 (KLF4), L-Myc, Lin28, SV40 Large T
Antigen (SV40LT) and p53 shRNA. In other embodiments, the at least
one vector is an episomal vector. In other embodiments, the
episomal vector is an oriP/EBNA1 vector. In other embodiments,
transfecting mammary cell includes nucleofection or lipofection. In
other embodiments, plating the cells on a culture vessel coated
with a substrate further includes culturing in norm oxygen
conditions. In other embodiments, the substrate is Matrigel. In
other embodiments, the induction media includes one or more of the
following compounds: HA-100, CHIR99021, PD0325901, and A83-01. In
other embodiments, culturing the cells in an induction media is for
a period of 10-31 days.
[0007] Also described herein is composition of m-iPSCs produced by
the method including providing a quantity of human mammary cells,
transfecting the human mammary cells with at least one vector
encoding at least one reprogramming factor, plating the cells on a
culture vessel coated with a substrate, and culturing the cells in
an induction media, wherein the transfection, plating and culturing
generates colonies of human mammary-derived induced pluripotent
stem cells (m-iPSCs). In other embodiments, the m-iPSCs are capable
of serial passaging as a cell line. In other embodiments, a
multi-potent cell is produced by culturing the composition of
m-iPSCs capable of serial passaging as a cell line in the presence
of a differentiation agent.
[0008] Further described herein is a method of reprogramming a
human mammary cell, including providing a quantity of human mammary
cells, culturing the human mammary cells in the presence of at
least one vector and/or at least one reprogramming agent, plating
the cells on a culture vessel coated with a substrate, and
culturing the cells in an induction media, wherein the
transfection, plating and culturing reprograms the human cell to a
less differentiated state. In other embodiments, the mammary cells
are from primary cells. In other embodiments, the mammary cells are
from a tumor. In other embodiments, the mammary cells are from a
cell line. In other embodiments, the at least one vector encodes at
least one reprogramming factor including Oct4, Sox2, Nanog,
Kruppel-like Factor 4 (KLF4), L-Myc, Lin28, SV40 Large T Antigen
(SV40LT) and p53 shRNA. In other embodiments, the at least one
vector encodes at least one reprogramming factor includes a
microRNA. In other embodiments, the at least one reprogramming
agent is a small molecule including HA-100, PD0325901, SB431542,
CHIR99021, A83-01 and/or Y-27632.
BRIEF DESCRIPTION OF THE DRAWINGS
[0009] FIG. 1. Reprogramming of mammary cells into m-iPSC colonies.
As shown, human mammary epithelial cells from primary cultures
retain individual cell, or small aggregate morphology after
nucleofection (Day 1). Clusters of cells begin to form (Day 4) with
elongated fibroblast-like morphology appearing (Day 6). These cells
begin to possess a distinct stem cell colony-like formation cluster
morphology (Day 10), with bright, compact colonies being visible
thereafter (Day 13). Cells can be further cultured with retention
of stem-like morphology (Day 21).
[0010] FIG. 2. Reprogrammed m-iPSC colonies express pluripotent
markers. As shown in phase contrast, m-iPSCs possess the
characteristic bright, compact colony morphology, with Alkaline
Phosphatase (AP)-FITC staining clearly displaying expression of
high levels of AP. Overlay of fluorescent and phase contrast image
is shown, showing a high number of efficiently reprogrammed m-iPSC
cell within colonies.
[0011] FIG. 3. Picking m-iPSCs colonies. Individual colonies of
reprogrammed m-iPSCs were isolated and expanded. Various examples
are shown, including at lower and high magnification. In such
examples, colonies possess the bright, round morphology of stem
cells, along with a high cystoplasm-to-nucleus ratio.
[0012] FIG. 4. Clonal expansion of m-iPSCs colonies. Additional
example of identifying cells for clonal expansion are shown. As
reprogramming is not 100% efficient, individual cells were
identified based on morphology, and clonally expanded as
described.
[0013] FIG. 5. Pluripotent marker expression of m-iPSCs cells. Cell
line CS01i-MECn1 (Clone 1) could successfully be maintained and
passaged in culture without loss of pluripotent marker expression.
In this example, colonies of plated m-iPSCs expressed pluripotent
markers, Oct4, Nanog, SSEA, Tra-1-60, and AP.
[0014] FIG. 6. Pluripotent marker expression of m-iPSCs cells. Cell
line CS01i-MECn4 (Clone 4) could successfully be maintained and
passaged in culture without loss of pluripotent marker expression.
In this example, colonies of plated m-iPSCs expressed pluripotent
markers, Oct4, Nanog, SSEA, Tra-1-60, and AP.
[0015] FIG. 7. Pluripotent marker expression of m-iPSCs cells. Cell
line CS01i-MECn6 (Clone 6) could successfully be maintained and
passaged in culture without loss of pluripotent marker expression.
In this example, colonies of plated m-iPSCs expressed pluripotent
markers, Nanog, Tra-1-60, and AP.
DETAILED DESCRIPTION
[0016] All references cited herein are incorporated by reference in
their entirety as though fully set forth. Unless defined otherwise,
technical and scientific terms used herein have the same meaning as
commonly understood by one of ordinary skill in the art to which
this invention belongs. Allen et al., Remington: The Science and
Practice of Pharmacy 22.sup.nd ed., Pharmaceutical Press (Sep. 15,
2012); Hornyak et al., Introduction to Nanoscience and
Nanotechnology, CRC Press (2008); Singleton and Sainsbury,
Dictionary of Microbiology and Molecular Biology 3rd ed., revised
ed., J. Wiley & Sons (New York, N.Y. 2006); Smith, March's
Advanced Organic Chemistry Reactions, Mechanisms and Structure
7.sup.th ed., J. Wiley & Sons (New York, N.Y. 2013); Singleton,
Dictionary of DNA and Genome Technology 3rd ed., Wiley-Blackwell
(Nov. 28, 2012); and Green and Sambrook, Molecular Cloning: A
Laboratory Manual 4th ed., Cold Spring Harbor Laboratory Press
(Cold Spring Harbor, N.Y. 2012), provide one skilled in the art
with a general guide to many of the terms used in the present
application. For references on how to prepare antibodies, see
Greenfield, Antibodies A Laboratory Manual 2nd ed., Cold Spring
Harbor Press (Cold Spring Harbor N.Y., 2013); Kohler and Milstein,
Derivation of specific antibody-producing tissue culture and tumor
lines by cell fusion, Eur. J. Immunol. 1976 July, 6(7):511-9; Queen
and Selick, Humanized immunoglobulins, U.S. Pat. No. 5,585,089
(1996 December); and Riechmann et al., Reshaping human antibodies
for therapy, Nature 1988 Mar. 24, 332(6162):323-7.
[0017] One skilled in the art will recognize many methods and
materials similar or equivalent to those described herein, which
could be used in the practice of the present invention. Indeed, the
present invention is in no way limited to the methods and materials
described. For purposes of the present invention, the following
terms are defined below.
[0018] As used in the description herein and throughout the claims
that follow, the meaning of "a," "an," and "the" includes plural
reference unless the context clearly dictates otherwise. Also, as
used in the description herein, the meaning of "in" includes "in"
and "on" unless the context clearly dictates otherwise.
[0019] As described, a chief limitation in studies relating to
isolation and identification of mammary stem cells ("maSCs") and
mammary cancer stem cells ("maCSCs") is a lack of source material,
given that primary mammary cell cultures rapidly reach senescence.
Reprogramming techniques related to induced pluripotent stem cells
("iPSCs") allows a new avenue of approach by allowing for
generation of a renewal source material of mammary cells for study.
Reprogramming techniques related to generation iPSCs have been
established a variety of cell types ranging from fibroblasts to
neurons. Nevertheless, despite this apparent extensibility across
different cells, there have not been reports of successful
techniques applicable to mammary cells. What is known is that the
INK4/ARF locus, which is active in primary mammary cells, is a
barrier to reprogramming. The INK4/ARF genetic locus encodes for
several factors, including p15INK4B (also known as CDKN2B),
p16INK4A (also known as CDKN2A) and ARF, which positively regulate
the p53 and retinoblastoma tumour suppressor pathways to inhibit
cell proliferation and promote cellular senescence. In this regard,
it may be understood that not all somatic cell sources for
reprogramming are created equal, and mammary cells may possess
barriers preventing their successful reprogramming and
dedifferentiation. In any case, generation of iPSCs, regardless of
tissue source would necessarily possess the hallmark properties of
self-renewal, and pluripotency as demonstrated by expansion
capacity, pluripotent marker expression (e.g., alkaline
phosphatase, Oct-4, nanog, Sox-2, etc.) and multi-lineage
tumorigenesis in immunocompromised animals (e.g., SCID mouse)
establishing differentiation into various progeny.
[0020] Generally, different approaches for reprogramming somatic
cells can include integrative, or integration-defective viral
delivery, episomal delivery, direct RNA delivery, direct protein
delivery, chemical induction and combinations of these features. As
described further herein, the adoption of episomal vectors allows
for generation of iPSCs substantially free of the vectors used in
their production, as episomal or similar vectors do not encode
sufficient viral genome sufficient to give rise to infection or a
replication-competent virus. At the same time, these vectors do
possess a limited degree of self-replication capacity in the
beginning somatic host cells. This self-replication capacity
provides a degree of persistent expression understood to be
beneficial in allowing the dedifferentiation process to initiate
take hold in a target host cell.
[0021] One example of a plasmid vector satisfying these criteria
includes the Epstein Barr oriP/Nuclear Antigen-1 ("EBNA1")
combination, which is capable of limited self-replication and known
to function in mammalian cells. As containing two elements from
Epstein-Barr virus, oriP and EBNA1, binding of the EBNA1 protein to
the virus replicon region oriP maintains a relatively long-term
episomal presence of plasmids in mammalian cells. This particular
feature of the oriP/EBNA1 vector makes it ideal for generation of
integration-free iPSCs.
[0022] More specifically, persistent expression of reprogramming
factor encoded in an oriP/EBNA1 vector occurs across multiple cell
divisional cycles. Sufficiently high levels of reprogramming
factors across several cell divisions allows for successful
reprogramming even after only one infection. While sustained
expression of reprogramming factors is understood to be beneficial
during initial programming stages, otherwise unlimited constitutive
expression would hamper subsequent stages of the reprogramming
process. For example, unabated expression of reprogramming factors
would interfere with subsequent growth, development, and fate
specification of the host cells.
[0023] At the same time, a further benefit is the eventual removal
of the reprogramming factor transgenes, as a small portion of
episomes is lost per cell cycle. This is due to the asymmetric
replication capacity of the host cell genome and episomal
self-replication and it is estimated that approximately 0.5% of
vector is lost per generation. Gradual depletion of plasmids during
each cell division is inevitable following propagation leading to a
population of integration-free iPSCs. The persistent, yet eventual
abrogation of reprogramming factor expression on oriP/EBNA1 is
highly coincident with the needs for different stages of the
reprogramming process and eliminates the need for further
manipulation steps for excision of the reprogramming factors, as
has been attempted through use of transposons and excisable
polycistronic lentiviral vector elements. Although oriP/EBNA1 has
been applied by others in reprogramming studies, the reported
efficiencies are extremely low (as few as 3 to 6 colonies per
million cells nucleofected), which may be due, in-part, to reliance
on large plasmids encoding multiple reprogramming factors (e.g.,
more than 12 kb), negatively impacting transfection efficiency.
[0024] In addition to these choices in vector designs, the specific
combinations of reprogramming factors implemented in the literature
have varied. As mentioned, reprogramming factors that have been
used include pluripotency-related genes Oct-4, Sox-2, Lin-28,
Nanog, Sa114, Fbx-15 and Utf-1. These factors are traditionally
understood to be expressed early during development and are
involved in the maintenance of the pluripotent potential of a
subset of cells that will constituting the inner cell mass of the
pre-implantation embryo and post-implantation embryo proper. Their
ectopic expression of is believed to allow the establishment of an
embryonic-like transcriptional cascade that initates and propagates
an otherwise dormant endogenous core pluripotency program within a
host cell. Certain other reprogramming determinants, such as Tert,
Klf-4, c-Myc, SV40 Large T Antigen ("SV40LT") and short hairpin
RNAs targeting p53 ("shRNA-p53") have been applied. There
determinants may not be potency-determining factors in and of
themselves, but have been reported to provide advantages in
reprogramming. For example, TERT and SV40LT are understood to
enhance cell proliferation to promote survival during
reprogramming, while others such as short hairpin targeting of p53
inhibit or eliminate reprogramming barriers, such as senescence and
apoptosis mechanisms. In each case, an increase in both the speed
and efficiency of reprogramming is observed. In addition, microRNAs
("miRNAs") are also known to influence pluripotency and
reprogramming, and some miRNAs from the miR-290 cluster have been
applied in reprogramming studies. For example, the introduction of
miR-291-3p, miR-294 or miR-295 into fibroblasts, along with
pluripotency-related genes, have also been reported to increase
reprogramming efficiency.
[0025] While various vectors and reprogramming factors in the art
appear to present multiple ingredients capable of establishing
reprogramming in cells, a high degree of complexity occurs when
taking into account the stoichiometric expression levels necessary
for successful reprogramming to take hold. For example, somatic
cell reprogramming efficiency is reportedly fourfold higher when
OCT-4 and SOX2 are encoded in a single transcript on a single
vector in a 1:1 ratio, in contrast to delivering the two factors on
separate vectors. The latter case results in a less controlled
uptake ratio of the two factors, providing a negative impact on
reprogramming efficiency. One approach towards addressing these
obstacles is the use of polycistronic vectors, such as inclusion of
an internal ribosome entry site ("IRES"), provided upstream of
transgene(s) that is distal from the transcriptional promoter. This
organization allows one or more transgenes to be provided in a
single reprogramming vector, and various inducible or constitutive
promoters can be combined together as an expression cassette to
impart a more granular level of transcriptional control for the
plurality of transgenes. These more specific levels of control can
benefit the reprogramming process considerably, and separate
expression cassettes on a vector can be designed accordingly as
under the control of separate promoters.
[0026] Although there are advantages to providing such factors via
a single, or small number of vectors, upper size limitations on
eventual vector size do exist, which can stymie attempts to promote
their delivery in a host target cell. For example, early reports on
the use of polycistronic vectors were notable for extremely poor
efficiency of reprogramming, sometimes occurring in less than 1% of
cells, more typically less than 0.1%. These obstacles are due,
in-part, to certain target host cells possessing poor tolerance for
large constructs (e.g., fibroblasts), or inefficient processing of
IRES sites by the host cells. Moreover, positioning of a factor in
a vector expression cassette affects both its stoichiometric and
temporal expression, providing an additional variable impacting
reprogramming efficiency. Thus, some improved techniques can rely
on multiple vectors each encoding one or more reprogramming factors
in various expression cassettes. Under these designs, alteration of
the amount of a particular vector for delivery provides a coarse,
but relatively straightforward route for adjusting expression
levels in a target cell.
[0027] In some instances, there may be further benefits in altering
the chemical and/or atmospheric conditions under which
reprogramming will take place. For example, as the pre-implantation
embryo is not vascularized and hypoxic (similar to bone marrow
stem-cell niches) reprogramming under hypoxic conditions of 5%
O.sub.2, instead of the atmospheric 21% O.sub.2, may further
provide an opportunity to increase the reprogramming efficiency.
Similarly, chemical induction techniques have been used in
combination with reprogramming, particularly histone deacetylase
(HDAC) inhibitor molecule, valproic acid (VPA), which has been
found wide use in different reprogramming studies. At the same
time, other small molecules such as MAPK kinase (MEK)-ERK ("MEK")
inhibitor PD0325901, transforming growth factor beta ("TGF-.beta.")
type I receptor ALK4, ALK5 and ALK7 inhibitor SB431542 and the
glycogen synthase kinase-3 ("GSK3") inhibitor CHIR99021 have been
applied for activation of differentiation-inducing pathways (e.g.
BMP signaling), coupled with the modulation of other pathways (e.g.
inhibition of the MAPK kinase (MEK)-ERK pathway) in order to
sustain self-renewal. Other small molecules, such as Rho-associated
coiled-coil-containing protein kinase ("ROCK") inhibitors, such as
Y-27632 and thiazovivin ("Tzv") have been applied in order to
promote survival and reduce vulnerability of pSCs to cell death,
particularly upon single-cell dissociation. Finally, in some
instances, techniques such as nucleofection allow for enhanced
transfer directly into the cell nucleus and the cytoplasm, without
relying on cell division for the transfer of DNA into the
nucleus.
[0028] Further, in some instances, various sub-combinations of
reprogramming factors, chemical and/or atmospheric conditions
described herein may be deployed to reprogram, but not
dedifferentiate somatic mammary cells into a fully pluripotent
state. For example, while it is understood that generation of
m-iPSCs in some instances may possess useful properties for
recapitulation of maSC and maCSC candidate phenotypes,
reprogramming resulting in incomplete, partial, or aberrant
reprogramming not resulting in acquisition of pluripotency may
further prove to be useful in reprogramming somatic mammary cells
directly into maSCs or maCSC candidate phenotypes. Such approaches
may be described as conditional reprogramming, transformation, or
other terms understood in the art. But in any case, such an
approach avoids the need for recapitulation, and may be regarded as
a means for direct conversion of mammary somatic cells into
possible maSC and maCSC candidates.
[0029] Following successful reprogramming, clonal selection allows
for generation of pluripotent stem cell lines. Ideally, such cells
possess requisite morphology (i.e., compact colony, high nucleus to
cytoplasm ratio and prominent nucleolus), self-renewal capacity for
unlimited propagation in culture (i.e., immortal), and with the
capability to differentiate into all three germ layers (e.g.,
endoderm, mesoderm and ectoderm). Further techniques to
characterize the pluripotency of a given population of cells
include injection into an immunocompromised animal, such as a
severe combined immunodeficient ("SCID") mouse, for formation of
teratomas containing cells or tissues characteristic of all three
germ layers.
[0030] In addition to the choice of delivery vectors, reprogramming
factor combinations, and conditions for reprogramming, further
variations must consider the nature of the host target cell for
reprogramming. As described, a wide variety of cells have served as
sources for reprogramming including fibroblasts, stomach and liver
cell cultures, human keratinocytes, adipose cells, and frozen human
monocyte. There appears to be a wide and robust potential for
dedifferentiation across many tissues sources. Nevertheless, it is
widely understood that depending on the donor cell type,
reprogramming is achieved with different efficiencies and kinetics.
For example, although fibroblasts remain the most popular donor
cell type for reprogramming studies, other types of cells such as
human primary keratinocytes transduced with Oct-4, Sox-2, Klf-4 and
c-Myc have been reported to reprogram 100 times more efficiently
and two-fold faster. Additionally, some other cell types, such as
cord blood cells, may only require a subset of reprogramming
factors, such as Oct-4 and Sox-2 for dedifferentiation to take
hold, while neural progenitor cells may only require Oct-4. Without
being bound to any particular theory, it is believed that
differences in reprogramming efficiency and/or reprogramming factor
requirements of specific host cells result from high endogenous
levels of certain reprogramming factors and/or intrinsic epigenetic
states that are more amenable to reprogramming.
[0031] Importantly, it is generally understood that tissue-specific
iPSC lines also possess subtle differences resulting from the
specific cell-of-origin. These properties are colloquially referred
to "parental memory". This aspect of tissue-specific cells is
believed to arise, in-part, from differences in epigenetic
methylation status, divergent telomeric lengths, which can manifest
themselves in lineage preference, or other structural and
functional alterations upon differentiation. Extending these
techniques, disease-specific iPSCs can also be generated from adult
cells that harbor genetic mutations or other alterations, thereby
providing a useful model of cellular development for understanding
disease initiation and progression. Together, iPSC technology
provides an alternative approach for identification of mammary
gland development by creation of mammary-derived iPSCs. These cells
can then be recapitulated into maSCs, maCSCs, and various cellular
intermediates generated during mammary gland development or as a
model for breast cancer disease.
[0032] It is notable that few, if any, studies report iPSC
generation from mammary cells, indicating possible difficulty in
deriving iPSCs from this tissue type. Even within the single tissue
type of mammary cells, it is noted that primary mammary cell
cultures may pose specific challenges or properties that are
different from cells in an established mammary cell lines. For
example, it is known that widely studied breast cell lines such as,
non-tumor initiating MCF10 contain depleted INK4/ARF locus. MCF10
cells are therefore more amenable to reprogramming compared to
primary cultures, considering the barrier function of this
particular genetic locus as involved in p53-related senescence.
Similarly, other cell lines such as MCF7 are more susceptible for
neoplastic transformation than primary mammary cell cultures or
MCF10. This adds a dimension wherein MCF7 may be highly compatible
with iPSC generation (based on extand replicative and
differentiation capacity) and subsequent differentiation capacity
mirroring maCSC generation based on other translocations and/or
parental memory in that particular cell line. It may be considered
that primary mammary cell cultures represent a more accurate
representation of normal mammary cell function and development, as
lacking technical artifacts persistent in established mammary cell
lines. Choice of iPSC generation from primary mammary cell cultures
or specific mammary cell lines may rest on the eventual application
of interest, although for the reasons described above, such primary
mammary cell culture may prove to be the most difficult cells to
reprogram and dedifferentiate.
[0033] Described herein is a composition including a culture of
mammary-derived iPSCs ("m-iPSCs"). In certain embodiments, the
m-iPSCs are derived from a somatic cell via reprogramming. In
certain embodiments, the human mammary cell is from a primary
culture of cells, a biopsy sample isolated from a human subject,
such as normal healthy tissue and/or solid tumor tissue. In certain
embodiments, the human mammary cell is a basal/myoepithelial or
luminal cell. In other embodiments, the human mammary cell is from
a cell line, such as MCF7 or MCF10.
[0034] In different embodiments, reprogramming includes
applications of reprogramming factors, including one or more of
following: Oct4, Sox2, Klf4, c-Myc, Lin28, SV40-LT, p53 short
hairpin RNA ("shRNA"), and nanog. In different embodiments, the
reprogramming factors are encoded by a vector. In different
embodiments, the vector can be, for example, a non-integrating
episomal vector, plasmid, retrovirus (integrating and
non-integrating) and/or other genetic elements known to one of
ordinary skill. In different embodiments, the vector encodes one or
more reprogramming factors, and combinations of vectors can be used
together to deliver one or more of Oct4, Sox2, Klf4, c-Myc, Lin28,
SV40-LT, p53 shRNA and nanog. For example, oriP/EBNA1 is an
episomal vector that can encode a vector combination of multiple
reprogramming factors, such as pCXLE-hUL, pCXLE-hSK,
pCXLE-hOCT3/4-shp53-F, and pEP4 EO2S T2K.
[0035] In various embodiments, one can reprogram human mammary
epithelial cells ("HMECs") via plasmid nucleofection of
combinations of oriP/EBNA1 based vectors pCXLE-hUL, pCXLE-hSK,
pCXLE-hOCT3/4-shp53-F, and pEP4 EO2S T2K plasmid vectors. In some
reprogramming methods, sub-combinations of these vectors are used.
In various embodiments, 0.1 .mu.g to 0.5 .mu.g, 0.5 .mu.g to 1
.mu.g, 1 .mu.g to 2.5 .mu.g, 2.5 .mu.g to 5 .mu.g, or 5 .mu.g or
more of each plasmid is used. 1.5 .mu.g per plasmid After
nucleofection, cells can be on a substrate coated dish, such as
Matrigel, and fed with Mammary Epithelium Basal/myoepithelial
Medium ("MEBM"). In certain embodiments, norm-oxygen conditions
(e.g., 5% O.sub.2) during reprogramming may aid efficiency of the
reprogramming. Other examples of norm-oxygen conditions includes
less than 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2% O.sub.2 or less.
Cells in MEBM are cultured for 48h and gradually changed to
Reprogramming Medium ("RM") consisting of DMEM/F12, 1% Glutamax, 1%
NEAA, 1% N2, 2% B27, 1% antibiotic-antifungal, 0.1 mM
beta-mercaptoethanol, 100 ng/mL basic fibroblast growth factor
(bFGF), and 1000 units/mL human Leukemia Inhibitory Factor (hLIF).
An alternative RM formulation includes 64 mg/L L-Ascorbic Acid,
19.4 mg/L insulin, 100 .mu.g/L FGF, 10.7 mg/L transferrin, 14
.mu.g/L sodium, selenite, and 543 mg/L NaHCO.sub.3 with volume up
to 1 L of DMEM/F12 media. It is understood that such components can
be at variable concentrations depending on the desired application,
such as less than 1 mg/L, 1-10 mg/L, 10 mg/L to 50 mg/L, 50 mg/L to
100 mg/L, 100 mg/L to 250 mg/L, 250 mg/L or more of a media
component. In other embodiments, small molecules can be added to RM
to enhance reprogramming efficiency. Such small molecules include
components of a modified "3i" medium, composed of: 1) HA-100 (10
.mu.M) glycogen synthase kinase 3.beta. inhibitor of the
Wnt/.beta.-catenin signaling pathway (CHIR99021, 3 .mu.M) MEK
pathway inhibitor (PD 0325901, 0.5 .mu.M) Selective inhibitor of
TGF-.beta. type I receptor ALK5 kinase, type I activin/nodal
receptor ALK4 and type I nodal receptor ALK7 (A 83-01, 0.5 .mu.M).
Other possible small molecules include histone deacetylase (HDAC)
inhibitor molecule, valproic acid (VPA), MAPK kinase (MEK)-ERK
("MEK") inhibitors, transforming growth factor beta ("TGF-.beta.")
type I receptor ALK4, ALK5 and ALK7 inhibitor SB431542, glycogen
synthase kinase-3 ("GSK3") inhibitors, Rho-associated
coiled-coil-containing protein kinase ("ROCK") inhibitors, such as
Y-27632 and thiazovivin ("Tzv") In various embodiments,
concentrations of these small molecules can range from 0.1 .mu.M to
0.25 .mu.M, 0.25 .mu.M to 0.5 .mu.M, 0.5 .mu.M to 1 .mu.M, 1 .mu.M
to 5 .mu.M, 5 .mu.M-10 .mu.M, 10 .mu.M to 15 .mu.M, 15 .mu.M to 20
.mu.M, or 20 .mu.M or more. Finally, colonies with ES/iPSC-like
morphology would appear at days 25, 26, 27, 28, 29, 30, or 31
post-nucleofection. In different embodiments for which partial,
conditional reprogramming is of interest, other types of
reprogrammed cells can appear at different time periods, such as
before 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 day
post-nucleofection.
[0036] In various embodiments, HMECs are cultured in MEBM in a T-75
flask until cells reached approximately 90% confluence.
Reprogramming of the HMECs was achieved by plasmid nucleofection,
performed using oriP/EBNA1 based pCXLE-hUL, pCXLE-hSK,
pCXLE-hOCT3/4-shp53-F, and pEP4 EO2S T2K plasmid vectors and Amaxa
Human Dermal Fibroblast Nucleofector Kit. HMECs (1.times.10.sup.6
cells per nucleofection) were harvested and centrifuged at 200 g
for 5 minutes. The cell pellet was re-suspended carefully in
Nucleofector Solution (VPD-1001, Lonza) and combined with episomal
plasmids (1.5 .mu.g per plasmid) expressing, Oct4, Sox 2, Klf4,
c-Myc, Lin28, SV40LT and p53 shRNA. The cell/DNA suspension was
transferred into the Nucleofector.RTM. and the E-010 program
applied. Immediately after nucleofection, cells were plated on BD
Matrigel coated dishes and fed with MEBM. All cultures were be
maintained under norm-oxygen conditions (5% O.sub.2) during
reprogramming, which further enhances the efficiency of iPS cell
generation. The media was kept on for 48h and gradually changed to
RM consisting of DMEM/F12, 1% Glutamax, 1% NEAA, 1% N2, 2% B27, 1%
antibiotic-antifungal, 0.1 mM beta-mercaptoethanol, 100 ng/mL basic
fibroblast growth factor (bFGF), and 1000 units/mL human Leukemia
Inhibitory Factor (hLIF). In other embodiments, small molecules can
be added to RM to enhance reprogramming efficiency. An alternative
RM formulation includes 64 mg/L L-Ascorbic Acid, 19.4 mg/L insulin,
100 .mu.g/L FGF, 10.7 mg/L transferrin, 14 .mu.g/L sodium,
selenite, and 543 mg/L NaHCO.sub.3. In addition, small molecules
were supplemented in the RM to enhance reprogramming efficiency.
The small molecules used were, 1) HA-100 (10 .mu.M), 2) glycogen
synthase kinase 3.beta. inhibitor of the Wnt/.beta.-catenin
signaling pathway (CHIR99021, 3 .mu.M), 3) MEK pathway inhibitor
(PD 0325901, 0.5 .mu.M). Selective inhibitor of TGF-.beta. type I
receptor ALK5 kinase, type I activin/nodal receptor ALK4 and type I
nodal receptor ALK7 (A 83-01, 0.5 .mu.M). Fresh RM was added daily
to the conditioned media. This was repeated daily for the next 4
days. On the 7th day post nucleofection, all medium was aspirated
from the wells and cells were fed with RM. Media was changed every
3rd day to fresh RM for the next 13 days (day 20 post
nucleofection).
[0037] In other embodiments, the reprogramming factors are
delivered by techniques known in the art, such as nuclefection,
transfection, transduction, electrofusion, electroporation,
microinjection, cell fusion, among others. In other embodiments,
the reprogramming factors are provided in a cellular extract of a
pluripotent stem cell. In various embodiments, the m-iPSCs are
capable of differentiating into mammary stem cells ("maSCs")
candidates. In other embodiments, the m-iPSCs are capable of
differentiating into mammary cancer stem cells ("maCSCs")
candidates. In various embodiments, the m-iPSCs possess features of
pluripotent stem cells. Some exemplary features of pluripotent stem
cells including differentiation into cells of all three germ layers
(ectoderm, endoderm, mesoderm), either in vitro or in vivo when
injected into an immunodeficient animal, expression of pluripotency
markers such as Oct4, Sox2, nanog, TRA-1-60, TRA-1-81, SSEA4, high
levels of alkaline phosphatase ("AP") expression, indefinite
propagation in culture, among other features recognized and
appreciated by one of ordinary skill.
[0038] Establishment of m-iPSC cell lines provides an in vitro
model that could be used for recapitulating the formation of
candidate maSCs. In alternative embodiments, reprogramming allows
for direct conversion into candidate maSCs. A key challenge is that
relatively little is known about the ontology of maSCs during
embryonic development. Milk lines are the first visible embryonic
mammary gland structures arising from migration of cells from the
embryonic ectoderm. Therefore, various ligands or other factors
influencing ectoderm fate-specification, such as manipulation of
Notch, Wnt, bone morphogenetic protein ("BMP"), and fibroblast
growth factor ("FGF") signaling pathways can be applied to promote
ectodermal cell lineage development from m-iPSCs, these
differentiated cells providing a population for which to generate
populations of maSC candidates. For example, overexpression of BMP
antagonist, Noggin, is known to lead to defects in ectodermal organ
development, Notch pathway signaling prevents differentiation into
alternative fates by promoting ectodermal development, and FGFs are
essential regulators of the specification of the ectodermal stem
cells from the ventral skin to form mammary placodes during
embryogenesis.
[0039] Studies using growth factor cocktail combinations can be
supplemented by heterotopic tissue recombination assays in order to
replicate aspects of the complex tempo-spatial events that occur
during embryogenesis. For example, m-iPSCs can be cultured in the
presence of mammary mesenchyme and/or mammary epithelium in order
to promote generation of candidate maSCs. Under any of these
approaches, generation of candidate maSC candidate populations is
followed by further characterization of stem-cell like properties.
As described, several different types of possible maSC populations
and mammary progenitor cells in the human breast can possess
varying degrees of stem cell-like properties. This is due, in-part,
to the functional complexity of the breast in mammalian
development. The breast is unlike most veterbrate organs that are
structured during embryongenesis and maintain their basic form
throughout an organisms life. Instead, mammary gland development
includes distinct chains of events related to embryogenesis (bud
development, generation of mammary fat pad, luminal formation),
puberty (formation of ductal network and terminal end buds), and
pregnancy (expansion of ductal tree and differentiation into
lobular alveoli with secretory epithelium). The primitive cell
types in the breast provide the expansion and differentiation
capacity behind these events, thereby accounting for the existence
of multiple maSC populations and progenitor cells. A key obstacle
presented by the existence of this myriad of cells is their
relatively similar marker expression profile. For example, it has
been suggested that luminal progenitor cells are
Lin.sup.-CD29.sup.loCD49f.sup.+CD61.sup.+Kit.sup.+, whereas
alveolar epithelial cells are suggested to be
Lin.sup.-CD29.sup.loCD49f.sup.+CD61.sup.-, and myoepithelial cells
are suggested to be
Lin.sup.-CD29.sup.hiCD49f.sup.hiCD24.sup.+CD61.sup.-. This small
divergence in marker expression amongst different cells provides
limited analytical resolution.
[0040] Similarly, generation of candidate maCSCs from m-iPSCs, or
direct conversion of mammary somatic cells into candidates maCSCs,
can be achieved by exploiting the oncgogenic properties of CSCs.
For example, application of anticancer chemotherapeutic compounds
such as Taxol or Actinomycin D can be applied as a selection factor
to select for chemoresistant maCSCs. Alternatively, culturing of
m-iPSCs in the presence of carcinoma cultured media has been
reported to induced CSC-like phenotype in mouse maSCs, and a
similar approach can be adapted for human m-iPSCs. Further, given
that CSCs are widely understood to result from dysregulation of
stem cell-related pathways, perturbation of m-iPSCs to cause
transformation into maCSCs can be explored. An alternative approach
can rely on overexpression of pluripotency markers such as Oct4,
Sox 2, and nanog, or relying on a cell source including high
potential for transformation, such as MCF7 cell line. Candidate
maCSCs can be characterized for biochemical and functional
properties using the following described techniques. For example,
certain CSC markers, such as CD133.sup.+ and ALDH1.sup.+ have been
identified as common among CSCs from several different cancer
diseases, and such markers can provide an initial screen for
generation of mammary-specific CSCs. In addition, increasing
understanding of breast cancer has demonstrated via genetic
signatures that certain subtypes as possess features in common with
different cells-of-origin. These same genetic signatures for
subtypes such as claudin-low or nomal-breast-like can then be used
as a mechanism to screen various maCSCs generated by the described
method, thereby establishing a link between the generated maCSCs
and features of not only breast cancer, but specific breast cancer
subtypes.
[0041] Further functional studies for confirming maCSC identity can
include exposure to anticancer Taxol or Actinomycin D as a measure
of chemoresistance, or ionizing radiation. Moreover, a variety of
tumor sphere formation or invasion assays are well-known in the
art, and such methods can be applied in establishing the functional
properties of the generated maCSCs and subsequent roles in breast
cancer development.
[0042] Also described herein is a method of producing m-iPSCs, or a
method of reprogramming somatic mammary cells. In certain
embodiments, the method includes providing a quantity of human
mammary cells, reprogramming the mammary cells using one or more
vectors, each vector encoding one or more reprogramming factors. In
some embodiments, the method includes further culturing the
reprogrammed mammary cells to produce an m-iPSC cell. In other
embodiments, methods allows for direct conversion into a candidate
maSC or candidate maCSC. In certain embodiments, the human mammary
cell is from a primary culture of cells, a biopsy sample isolated
from a human subject, such as normal healthy tissue and/or solid
tumor tissue. In certain embodiments, the human mammary cell is a
basal/myoepithelial or luminal cell. In other embodiments, the
human mammary cell is from a cell line, such as MCF7 or MCF10. In
different embodiment, the one or more vectors include use of
oriP/EBNA1 based vector. In other embodiments, reprogramming the
mammary cells using one or more vectors, each vector encoding one
or more reprogramming factors can include examples such as
pCXLE-hUL, pCXLE-hSK, pCXLE-hOCT3/4-shp53-F, and pEP4 EO2S T2K. In
different embodiments, reprogramming the mammary cells using one or
more vectors includes use of nucleofection. In certain embodiments,
mammary cells are treated with sodium butyrate to improve m-iPSC
yield. In a different embodiment, further culturing the
reprogrammed mammary cells to produce a m-iPSC cell includes
culturing on a tissue culture vessel coated with a substrate, such
as extracellular matrix (ECM) or Matrigel coated dishes. In other
embodiments, the reprogramming and/or further culturing is under
norm-oxygen conditions, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,
12, 13, 14, 15, 16, 17, 18, 19, or 20% O.sub.2. In another
embodiment, reprogramming and/or further culturing includes use of
an induction media. In certain embodiments, the induction media
includes a one or more of the following: an inhibitor of MYLK, PKA,
and PKC pathways, an inhibitor the Wnt/.beta.-catenin signaling
pathways, such as an inhibitor of glycogen synthase kinase 3.beta.,
an inhibitor of MEK pathway, and an inhibitor of TGF-.beta.
pathways, such as type I receptor ALK5 kinase, type I activin/nodal
receptor ALK4 and type I nodal receptor ALK7. For example, an
exemplary combination of inhibitors in an induction media can
include HA-100 (MYLK, PKA, and PKC pathways inhibitor), CHIR99021
(GSK3 inhibitor), PD0325901 (MEK inhibitor) and/or A83-01
(TGF-.beta. inhibitor). In different embodiments, further culturing
with induction media is for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12
days or more after nucleofection. In different embodiments, further
culturing the reprogrammed mammary cells is for a total culture
period of 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,
23, 24, 25, 26, 27, 28, 29, 30, 30 days or more.
[0043] In various embodiments, one can reprogram human mammary
epithelial cells ("HMECs") via plasmid nucleofection of
combinations of oriP/EBNA1 based vectors pCXLE-hUL, pCXLE-hSK,
pCXLE-hOCT3/4-shp53-F, and pEP4 EO2S T2K plasmid vectors. In some
reprogramming methods, sub-combinations of these vectors are used.
In various embodiments, 0.1 .mu.g to 0.5 .mu.g, 0.5 .mu.g to 1
.mu.m, 1 .mu.g to 2.5 .mu.g, 2.5 .mu.g to 5 .mu.g, or 5 .mu.g or
more of each plasmid is used. 1.5 .mu.g per plasmid After
nucleofection, cells can be on a substrate coated dish, such as
Matrigel, and fed with Mammary Epithelium Basal/myoepithelial
Medium ("MEBM"). In certain embodiments, norm-oxygen conditions
(e.g., 5% O.sub.2) during reprogramming may aid efficiency of the
reprogramming. Other examples of norm-oxygen conditions includes
less than 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2% O.sub.2 or less.
Cells in MEBM are cultured for 48h and gradually changed to
Reprogramming Medium ("RM") consisting of DMEM/F12, 1% Glutamax, 1%
NEAA, 1% N2, 2% B27, 1% antibiotic-antifungal, 0.1 mM
beta-mercaptoethanol, 100 ng/mL basic fibroblast growth factor
(bFGF), and 1000 units/mL human Leukemia Inhibitory Factor (hLIF).
An alternative RM formulation includes 64 mg/L L-Ascorbic Acid,
19.4 mg/L insulin, 100 .mu.g/L FGF, 10.7 mg/L transferrin, 14
.mu.g/L sodium, selenite, and 543 mg/L NaHCO.sub.3 with volume up
to 1 L of DMEM/F12 media. It is understood that such components can
be at variable concentrations depending on the desired application,
such as less than 1 mg/L, 1-10 mg/L, 10 mg/L to 50 mg/L, 50 mg/L to
100 mg/L, 100 mg/L to 250 mg/L, 250 mg/L or more of a media
component. In other embodiments, small molecules can be added to RM
to enhance reprogramming efficiency. Such small molecules include
components of a modified "3i" medium, composed of: 1) HA-100 (10
.mu.M), 2) glycogen synthase kinase 3.beta. inhibitor of the
Wnt/.beta.-catenin signaling pathway (CHIR99021, 3 .mu.M), 3) MEK
pathway inhibitor (PD 0325901, 0.5 .mu.M), 4) Selective inhibitor
of TGF-.beta. type I receptor ALK5 kinase, type I activin/nodal
receptor ALK4 and type I nodal receptor ALK7 (A 83-01, 0.5 .mu.M).
Other possible small molecules include histone deacetylase (HDAC)
inhibitor molecule, valproic acid (VPA), MAPK kinase (MEK)-ERK
("MEK") inhibitors, transforming growth factor beta ("TGF-.beta.")
type I receptor ALK4, ALK5 and ALK7 inhibitor SB431542, glycogen
synthase kinase-3 ("GSK3") inhibitors, Rho-associated
coiled-coil-containing protein kinase ("ROCK") inhibitors, such as
Y-27632 and thiazovivin ("Tzv") In various embodiments,
concentrations of these small molecules can range from 0.1 .mu.M to
0.25 .mu.M, 0.25 .mu.M to 0.5 .mu.M, 0.5 .mu.M to 1 .mu.M, 1 .mu.M
to 5 .mu.M, 5 .mu.M-10 .mu.M, 10 .mu.M to 15 .mu.M, 15 .mu.M to 20
.mu.M, or 20 .mu.M or more. Finally, colonies with ES/iPSC-like
morphology would appear at days 25, 26, 27, 28, 29, 30, or 31
post-nucleofection. In different embodiments for which partial,
conditional reprogramming is of interest, other types of
reprogrammed cells can appear at different time periods, such as
before 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 day
post-nucleofection.
[0044] In various embodiments, HMECs are cultured in MEBM in a T-75
flask until cells reached approximately 90% confluence.
Reprogramming of the HMECs was achieved by plasmid nucleofection,
performed using oriP/EBNA1 based pCXLE-hUL, pCXLE-hSK,
pCXLE-hOCT3/4-shp53-F, and pEP4 EO2S T2K plasmid vectors and Amaxa
Human Dermal Fibroblast Nucleofector Kit. HMECs (1.times.10.sup.6
cells per nucleofection) were harvested and centrifuged at 200 g
for 5 minutes. The cell pellet was re-suspended carefully in
Nucleofector Solution (VPD-1001, Lonza) and combined with episomal
plasmids (1.5 .mu.g per plasmid) expressing, Oct4, Sox 2, Klf4,
c-Myc, Lin28, SV40LT and p53 shRNA. The cell/DNA suspension was
transferred into the Nucleofector.RTM. and the E-010 program
applied. Immediately after nucleofection, cells were plated on BD
Matrigel coated dishes and fed with MEBM. All cultures were be
maintained under norm-oxygen conditions (5% O.sub.2) during
reprogramming, which further enhances the efficiency of iPS cell
generation. The media was kept on for 48h and gradually changed to
RM consisting of DMEM/F12, 1% Glutamax, 1% NEAA, 1% N2, 2% B27, 1%
antibiotic-antifungal, 0.1 mM beta-mercaptoethanol, 100 ng/mL basic
fibroblast growth factor (bFGF), and 1000 units/mL human Leukemia
Inhibitory Factor (hLIF). In other embodiments, small molecules can
be added to RM to enhance reprogramming efficiency. An alternative
RM formulation includes 64 mg/L L-Ascorbic Acid, 19.4 mg/L insulin,
100 .mu.g/L FGF, 10.7 mg/L transferrin, 14 .mu.g/L sodium,
selenite, and 543 mg/L NaHCO.sub.3. In addition, small molecules
were supplemented in the RM to enhance reprogramming efficiency.
The small molecules used were, 1) HA-100 (10 .mu.M), 2) glycogen
synthase kinase 3.beta. inhibitor of the Wnt/.beta.-catenin
signaling pathway (CHIR99021, 3 .mu.M), 3) MEK pathway inhibitor
(PD 0325901, 0.5 .mu.M), 4) Selective inhibitor of TGF-.beta. type
I receptor ALK5 kinase, type I activin/nodal receptor ALK4 and type
I nodal receptor ALK7 (A 83-01, 0.5 .mu.M). Fresh RM was added
daily to the conditioned media. This was repeated daily for the
next 4 days. On the 7th day post nucleofection, all medium was
aspirated from the wells and cells were fed with RM. Media was
changed every 3rd day to fresh RM for the next 13 days (day 20 post
nucleofection).
[0045] Further described herein is a composition including a
culture of mammary stem cell candidates ("maSCs"). In various
embodiments, the candidate maSCs originate from a luminal or
basal/myoepithelial compartment, or both. In other embodiments, the
culture of maSCs were generated from a primary culture of cells, a
biopsy sample isolated from a human subject, such as normal healthy
tissue and/or solid tumor tissue. In certain embodiments, the human
mammary cell is a basal/myoepithelial or luminal cell. In other
embodiments, the human mammary cell is from a cell line, such as
MCF7 or MCF10. In various embodiments, the maSCs express the
following panel of markers: CD1d, CD10, CD24, CD29, CD49, CD61,
CD133, epithelial cell adhesion molecule (EpCAM), Lin, Muc-1,
Thy-1. In other embodiments, the maSCs express Oct4, Sox2, nanog,
TRA-1-60, TRA-1-81, SSEA4. In different embodiments, the maSCs can
be differentiated luminal progenitors, luminal cells,
basal/myoepithelial progenitors, basal/myoepithelial cells,
myoepithelial progenitors, myoepithelial cells. In various
embodiments, the maSCs are capable of forming a mammary gland from
a single cell. In other embodiments, the maSCs are capable of
repopulating a compartment of a mammary gland, such as
basal/myoepithelial or luminal compartments of a host subject.
[0046] Also described herein a method of generating a population of
candidate maSCs, including providing a quantity of human m-iPSCs,
and inducing the formation of candidate maSCs. In different
embodiments, inducing the formation of candidate maSCs include use
of ligands or other factors influencing ectoderm
fate-specification. This includes, for example, ligands or other
factors related to Notch, Wnt, bone morphogenetic protein ("BMP"),
and fibroblast growth factor ("FGF") signaling pathways. In another
embodiment, inducing the formation of maSCs include co-culture with
a cell layer. This include, for example, culturing m-iPSCs in the
presence of mammary mesenchyme and/or mammary epithelium.
[0047] Further described herein is a composition including a
culture of mammary cancer stem cells ("maCSCs") candidates. In
various embodiments, the candidate maCSCs originate from a luminal
or basal/myoepithelial compartment, or both. In other embodiments,
the culture of maCSCs were generated from a primary culture of
cells, a biopsy sample isolated from a human subject, such as
normal healthy tissue and/or solid tumor tissue. In certain
embodiments, the human mammary cell is a basal/myoepithelial or
luminal cell. In certain embodiments, the human mammary cell is a
basal/myoepithelial or luminal cell. In other embodiments, the
human mammary cell is from a cell line, such as MCF7 or MCF10. In
different embodiments, maCSCs may express one or more markers such
as one or more of following: ALDH1A (also known as ALDH1A1), CD24,
CD44, CD45, CD90, CD105, CD117, CD133, CD166, EpCAM, ESA, ABCBS,
ABCG2, SCA, Snail, Slug and SOX2. For example, a mCSC may express
CD44.sup.+/CD24.sub.low or CD73.sup.+CD90.sup.+. In other
embodiments, mCSCs possess nuclear localization of developmental
pathway related molecules, thereby demonstrating activation of
proteins such as Gli1 (hedgehog signaling), Notch1 (Notch
signaling), and/or .beta.-Catenin (Wnt signaling). In other
embodiments, maCSCs possess activation of TGF-.beta.-related
pathways, such as activation of SMAD signaling proteins, such as
Smad1, Smad3 and Smad5. In other embodiments, maCSCs are capable of
forming tumors composed of multilineage cells in vivo. This can
include formation of tumor tissues when injected in an
immunosuppressed, or immunodeficient mouse. In other embodiments,
the maCSCs possess a molecular signature similar to subsets of
breast cancer, includingluminal A, luminal B, luminal C,
molecular-apocrine, basal/myoepithelial, or normal-breast-like
cancer. In other embodiments, maCSCs possess a molecular signature
similar to subsets of breast cancer, such as ER.sup.+Her2.sup.+,
ER.sup.+Her2.sup.-, ER.sup.-Her2.sup.+, and ER.sup.-Her2.sup.-. In
other embodiments, the CSCs are resistant to an anticancer drug
such as Taxol, Fulvestant. and Actinomycin D. In other embodiments,
the maCSCs are resistant to ionizing radiation.
[0048] Also described herein a method of generating a population of
candidate maCSCs, including providing a quantity of human m-iPSCs,
and inducing the formation of maCSCs. In other embodiments, the
method includes inducing the formation of maSCs. In other
embodiments, inducing the formation of maCSCs include application
of anticancer chemotherapeutic compounds such as Taxol or
Actinomycin as a selection factor. In other embodiments, inducing
the formation of maCSCs includes exposure to ionizing or carcinoma
cultured media. In other embodiments, inducing the formation of
maCSCs includes application of reprogramming factors, such as Oct4,
Sox 2, and nanog.
Example 1
Generation of Human Mammary Epithelial Cell-Derived iPSCs Using
Episomal Plasmids
[0049] Generally, the inventors adapted reported iPSCs
reprogramming techniques to produce mammary-derived iPSCs
("m-iPSCs"). Certain modifications include, for example,
nucleofection of a specific 5 plasmid combination each encoding one
or more specific reprogramming factors, and modified "3i"
pluripotency media to establish the reprogrammed m-iPSC
cultures.
[0050] Briefly, reprogramming of the HMECs was achieved by plasmid
nucleofection of oriP/EBNA1 based pCXLE-hUL, pCXLE-hSK,
pCXLE-hOCT3/4-shp53-F, and pEP4 EO2S T2K plasmid vectors. Unlike
viral transduction, these genes do not integrate and are instead
expressed episomally (extrachromosomal) in a transient fashion.
After nucleofection, cells were plated on BD Matrigel coated
dishes, fed with Mammary Epithelium Basal/myoepithelial Medium
("MEBM"), and maintained under norm-oxygen conditions (5% O.sub.2)
during reprogramming. Subsequently, small molecules were
supplemented in the Reprogramming Medium ("RM") to enhance
reprogramming efficiency, including modified "3i" medium, composed
of: 1) HA-100 (10 .mu.M), 2) glycogen synthase kinase 3.beta.
inhibitor of the Wnt/.beta.-catenin signaling pathway (CHIR99021, 3
.mu.M), 3) MEK pathway inhibitor (PD 0325901, 0.5 .mu.M), 4)
Selective inhibitor of TGF-.beta. type I receptor ALK5 kinase, type
I activin/nodal receptor ALK4 and type I nodal receptor ALK7 (A
83-01, 0.5 .mu.M). Finally, colonies with ES/iPSC-like morphology
would appear at day 25-31 post-nucleofection. Exemplary differences
in cell morphology undergoing reprogramming are shown in FIG. 1,
colonies of m-iPSCs expressing alkaline phosphatase are shown in
FIG. 2.
Example 2
Nucleofection Using Non-Integrating Episomal Vectors
[0051] Human mammary epithelial cells (HMECs, obtained from ATCC)
were cultured in MEBM in a T-75 flask until cells reached
approximately 90% confluence. Reprogramming of the HMECs was
achieved by plasmid nucleofection, performed using oriP/EBNA1 based
pCXLE-hUL, pCXLE-hSK, pCXLE-hOCT3/4-shp53-F, and pEP4 EO2S T2K
plasmid vectors (Addgene). Amaxa Human Dermal Fibroblast
Nucleofector Kit was utilized to make the virus-free iPSC lines.
This method has a significant advantage over viral transduction,
because genes do not integrate and are instead expressed episomally
(extrachromosomal) in a transient fashion. Briefly, HMECs
(1.times.10.sup.6 cells per nucleofection) were harvested and
centrifuged at 200 g for 5 minutes. The cell pellet was
re-suspended carefully in Nucleofector Solution (VPD-1001, Lonza)
and combined with episomal plasmids (1.5 .mu.g per plasmid)
expressing, Oct4, Sox 2, Klf4, c-Myc, Lin28, SV40LT and p53 shRNA.
The cell/DNA suspension was transferred into the Nucleofector.RTM.
and the E-010 program applied.
Example 3
Induction of Stem Cell Pluripotency
[0052] Immediately after nucleofection, cells were plated on BD
Matrigel coated dishes and fed with MEBM. All cultures were be
maintained under norm-oxygen conditions (5% O.sub.2) during
reprogramming, which further enhances the efficiency of iPS cell
generation. The media was kept on for 48h and gradually changed to
reprogramming media consisting of DMEM/F12, 1% Glutamax, 1% NEAA,
1% N2, 2% B27, 1% antibiotic-antifungal, 0.1 mM
beta-mercaptoethanol, 100 ng/mL basic fibroblast growth factor
(bFGF), and 1000 units/mL human Leukemia Inhibitory Factor (hLIF).
In addition, small molecules were supplemented in the RM to enhance
reprogramming efficiency. The small molecules used were, 1) HA-100
(10 .mu.M), 2) glycogen synthase kinase 3.beta. inhibitor of the
Wnt/.beta.-catenin signaling pathway (CHIR99021, 3 .mu.M), 3) MEK
pathway inhibitor (PD 0325901, 0.5 .mu.M), 4) Selective inhibitor
of TGF-.beta. type I receptor ALK5 kinase, type I activin/nodal
receptor ALK4 and type I nodal receptor ALK7 (A 83-01, 0.5 .mu.M).
Fresh RM was added daily to the conditioned media. This was
repeated daily for the next 4 days. On the 7th day post
nucleofection, all medium was aspirated from the wells and cells
were fed with RM. Media was changed every 3rd day to fresh RM for
the next 13 days (day 20 post nucleofection).
Example 4
Generation of iPSC Colonies
[0053] Colonies with ES/iPSC-like morphology appeared at day 25-31
post-nucleofection. Subsequently, colonies with the best morphology
were picked on day 31 and transferred to BD Matrigel.TM. Matrix for
feeder-independent maintenance of hiPSCs in chemically-defined
mTeSR.RTM.1 medium. Examples of hMEC-iPSC colonies are shown in
FIG. 3. hMEC-iPSC colonies display bright morphology, high
cytoplasm to nucleus ratio as shown in FIG. 4.
[0054] Six independent iPS cell clones were picked, further
expanded and cryopreserved. Three colonies are shown and are
designated CS01i-MECn1 (Clone 1), CS01i-MECn4 (Clone 4), and
CS01i-MECn6 (Clone 6) are shown in FIGS. 5, 6, and 7, respectively.
Each cell line expressed alkaline phosphatase, Oct4, nanog, SSEA4
and/or TRA-1-60 markers as shown.
Example 5
Generation of iPSC Colonies Under Essentially Defined
Conditions
[0055] Extending the above studies, the Inventors have derived
suitable media conditions which are essentially defined as shown in
Table 1. Application of the above media composition during
reprogramming achieves the same results, with reduced growth factor
complexity.
TABLE-US-00001 TABLE 1 Essentially Defined Media Conditions
suggested amount concentration needed to Stock for 1 L make 100 ml
DMEM/F12 1 L 100 ml L-Ascorbic 5 G 64 mg/L 6.4 mg Acid Insulin 10
mg/ml 19.4 mg/L 194 .mu.l FGF 10 ug/ml 100 .mu.g/L 100 .mu.l
Transferrin 50 mg 10.7 mg/L 1.07 mg Sodium selenite 10 G 14 .mu.g/L
0.0014 mg NaHCO3 500 G 543 mg/L 54.3 mg
[0056] The various methods and techniques described above provide a
number of ways to carry out the invention. Of course, it is to be
understood that not necessarily all objectives or advantages
described may be achieved in accordance with any particular
embodiment described herein. Thus, for example, those skilled in
the art will recognize that the methods can be performed in a
manner that achieves or optimizes one advantage or group of
advantages as taught herein without necessarily achieving other
objectives or advantages as may be taught or suggested herein. A
variety of advantageous and disadvantageous alternatives are
mentioned herein. It is to be understood that some preferred
embodiments specifically include one, another, or several
advantageous features, while others specifically exclude one,
another, or several disadvantageous features, while still others
specifically mitigate a present disadvantageous feature by
inclusion of one, another, or several advantageous features.
[0057] Furthermore, the skilled artisan will recognize the
applicability of various features from different embodiments.
Similarly, the various elements, features and steps discussed
above, as well as other known equivalents for each such element,
feature or step, can be mixed and matched by one of ordinary skill
in this art to perform methods in accordance with principles
described herein. Among the various elements, features, and steps
some will be specifically included and others specifically excluded
in diverse embodiments.
[0058] Although the invention has been disclosed in the context of
certain embodiments and examples, it will be understood by those
skilled in the art that the embodiments of the invention extend
beyond the specifically disclosed embodiments to other alternative
embodiments and/or uses and modifications and equivalents
thereof.
[0059] Many variations and alternative elements have been disclosed
in embodiments of the present invention. Still further variations
and alternate elements will be apparent to one of skill in the art.
Among these variations, without limitation, are sources of
mammary-derived stem cells, method of detecting biomarkers,
prognostic and/or diagnostic panels that include mammary-derived
stem cells and their differentiated progeny, and the particular use
of the products created through the teachings of the invention.
Various embodiments of the invention can specifically include or
exclude any of these variations or elements.
[0060] In some embodiments, the numbers expressing quantities of
ingredients, properties such as concentration, reaction conditions,
and so forth, used to describe and claim certain embodiments of the
invention are to be understood as being modified in some instances
by the term "about." Accordingly, in some embodiments, the
numerical parameters set forth in the written description and
attached claims are approximations that can vary depending upon the
desired properties sought to be obtained by a particular
embodiment. In some embodiments, the numerical parameters should be
construed in light of the number of reported significant digits and
by applying ordinary rounding techniques. Notwithstanding that the
numerical ranges and parameters setting forth the broad scope of
some embodiments of the invention are approximations, the numerical
values set forth in the specific examples are reported as precisely
as practicable. The numerical values presented in some embodiments
of the invention may contain certain errors necessarily resulting
from the standard deviation found in their respective testing
measurements.
[0061] In some embodiments, the terms "a" and "an" and "the" and
similar references used in the context of describing a particular
embodiment of the invention (especially in the context of certain
of the following claims) can be construed to cover both the
singular and the plural. The recitation of ranges of values herein
is merely intended to serve as a shorthand method of referring
individually to each separate value falling within the range.
Unless otherwise indicated herein, each individual value is
incorporated into the specification as if it were individually
recited herein. All methods described herein can be performed in
any suitable order unless otherwise indicated herein or otherwise
clearly contradicted by context. The use of any and all examples,
or exemplary language (e.g. "such as") provided with respect to
certain embodiments herein is intended merely to better illuminate
the invention and does not pose a limitation on the scope of the
invention otherwise claimed. No language in the specification
should be construed as indicating any non-claimed element essential
to the practice of the invention.
[0062] Groupings of alternative elements or embodiments of the
invention disclosed herein are not to be construed as limitations.
Each group member can be referred to and claimed individually or in
any combination with other members of the group or other elements
found herein. One or more members of a group can be included in, or
deleted from, a group for reasons of convenience and/or
patentability. When any such inclusion or deletion occurs, the
specification is herein deemed to contain the group as modified
thus fulfilling the written description of all Markush groups used
in the appended claims.
[0063] Preferred embodiments of this invention are described
herein, including the best mode known to the inventors for carrying
out the invention. Variations on those preferred embodiments will
become apparent to those of ordinary skill in the art upon reading
the foregoing description. It is contemplated that skilled artisans
can employ such variations as appropriate, and the invention can be
practiced otherwise than specifically described herein.
Accordingly, many embodiments of this invention include all
modifications and equivalents of the subject matter recited in the
claims appended hereto as permitted by applicable law. Moreover,
any combination of the above-described elements in all possible
variations thereof is encompassed by the invention unless otherwise
indicated herein or otherwise clearly contradicted by context.
[0064] Furthermore, numerous references have been made to patents
and printed publications throughout this specification. Each of the
above cited references and printed publications are herein
individually incorporated by reference in their entirety.
[0065] In closing, it is to be understood that the embodiments of
the invention disclosed herein are illustrative of the principles
of the present invention. Other modifications that can be employed
can be within the scope of the invention. Thus, by way of example,
but not of limitation, alternative configurations of the present
invention can be utilized in accordance with the teachings herein.
Accordingly, embodiments of the present invention are not limited
to that precisely as shown and described.
* * * * *