U.S. patent application number 12/778938 was filed with the patent office on 2011-11-17 for integration-free human induced pluripotent stem cells from blood.
This patent application is currently assigned to iPierian, Inc.. Invention is credited to Stefan Irion.
Application Number | 20110281281 12/778938 |
Document ID | / |
Family ID | 44839577 |
Filed Date | 2011-11-17 |
United States Patent
Application |
20110281281 |
Kind Code |
A1 |
Irion; Stefan |
November 17, 2011 |
INTEGRATION-FREE HUMAN INDUCED PLURIPOTENT STEM CELLS FROM
BLOOD
Abstract
Provided herein are methods for generating human induced
pluripotent stem cells free from genomic integration of exogenous
transgenes by transfecting into nucleated blood cells one or more
DNA expression vectors (e.g., plasmid vectors) that do not contain
a mammalian origin of replication, and encode and permit expression
of one or more reprogramming factors (e.g., Oct4, Sox2, Klf4, and
c-Myc). Also provided herein are the integration-free human induced
pluripotent stem cells obtained by the methods described
herein.
Inventors: |
Irion; Stefan; (San
Francisco, CA) |
Assignee: |
iPierian, Inc.
South San Francisco
CA
|
Family ID: |
44839577 |
Appl. No.: |
12/778938 |
Filed: |
May 12, 2010 |
Current U.S.
Class: |
435/7.21 ;
435/366 |
Current CPC
Class: |
C12N 2501/603 20130101;
C12N 2501/125 20130101; C12N 2506/11 20130101; C12N 2510/00
20130101; C12N 2501/604 20130101; C12N 5/0696 20130101; C12N
2800/24 20130101; C12N 15/85 20130101; C12N 2501/602 20130101; C12N
2501/22 20130101; C12N 2501/2304 20130101; C12N 2501/606
20130101 |
Class at
Publication: |
435/7.21 ;
435/366 |
International
Class: |
G01N 33/567 20060101
G01N033/567; C12N 5/071 20100101 C12N005/071 |
Claims
1. A method for generating integration-free human induced
pluripotent stem cells, comprising transfecting human nucleated
blood cells with one or more plasmid DNA expression vectors
encoding reprogramming factors (a) Oct4, Sox2, Klf4, and c-Myc; (b)
Oct4, Sox2, and Klf4; (c) Oct4, Sox2, Klf4, c-Myc, and Nanog; or
(d) Oct 4, Sox2, Lin-28, and Nanog, expressing the encoded
reprogramming factors in the transfected nucleated blood cells, and
culturing the transfected nucleated blood cells under conditions
adapted to growth of human induced pluripotent stem cells,
identifying human induced pluripotent stem cell colonies,
identifying integration-free human induced pluripotent stem cells
that do not comprise exogenous DNA from the one or more plasmid DNA
nucleic acid expression vectors, wherein: (i) at least two of the
encoded reprogramming factors are linked by an intervening
self-cleaving peptide sequence; (ii) the transfected human
nucleated blood cells do not express an exogenous trans-acting
factor that binds to a replication origin of an extra-chromosomal
template or the one or more plasmid DNA expression vectors do not
comprise a mammalian origin of replication sequence; and (iii) the
transfection is performed only one time, or is performed only
within a single 24 hour period.
2. The method of claim 1, wherein the one or more plasmid DNA
expression vectors encode the reprogramming factors Oct4, Sox2,
Klf4, and c-Myc.
3. The method of claim 1, wherein the one or more plasmid DNA
expression vectors encode the reprogramming factors Oct4, Sox2,
Klf4, c-Myc, and Nanog.
4. (canceled)
5. (canceled)
6. The method of claim 1, wherein the transfection comprises
transfecting the nucleated blood cells with only one transfection
method.
7. (canceled)
8. The method of claim 1, wherein the one or more plasmid DNA
expression vectors further encode a reporter protein that is
expressed in the transfected nucleated blood cells.
9-11. (canceled)
Description
BACKGROUND OF THE INVENTION
[0001] The advent of cellular reprogramming technology to generate
human induced pluripotent stem (hiPS) cells combined with directed
differentiation in vitro has opened the door to vast opportunities
for more effective drug discovery and regenerative medicine.
Typically, an hiPS cell line is generated by transducing
fibroblasts expanded from a small skin biopsy with one or more
integrating retroviruses encoding reprogramming factors. The
resulting hiPS line contains one or more proviral integrations,
which may interfere with the use of the hiPS cell lines in several
applications including, for example, regenerative medicine
applications. Further, use of skin fibroblasts for generating hiPS
cells presents a number of disadvantages: isolation of a skin
biopsy is somewhat invasive for the patient; expansion of
fibroblasts from a skin biopsy prior to cellular reprogramming is
relatively slow and inefficient; and skin is often directly exposed
to environmental mutagens (e.g., UV irradiation) that may
compromise the genomic integrity of the fibroblasts and therefore
that of fibroblast-derived hiPS cell lines.
SUMMARY OF THE INVENTION
[0002] Described herein are human induced pluripotent stem (hiPS)
cells generated from nucleated blood cells and free from
genomically integrated exogenous nucleic acid sequences
("integration-free" hiPS cells), as well as methods for generating
such hiPS cells by using non-viral nucleic acid expression vectors
(e.g., plasmid expression vectors).
[0003] Accordingly, in one aspect provided herein is a method for
generating integration-free human induced pluripotent stem cells
that includes transfecting human nucleated blood cells with one or
more DNA expression vectors encoding reprogramming factors (a)
Oct4, Sox2, Klf4, and c-Myc; (b) Oct4, Sox2, and Klf4; (c) Oct4,
Sox2, Klf4, c-Myc, and Nanog; or (d) Oct 4, Sox2, Lin-28, and
Nanog, expressing the encoded reprogramming factors in the
transfected nucleated blood cells, and culturing the transfected
nucleated blood cells under conditions adapted to growth of human
induced pluripotent stem cells, and identifying integration-free
human induced pluripotent stem cells that do not comprise exogenous
DNA from the one or more nucleic acid expression vectors, wherein
the transfected human nucleated blood cells do not express an
exogenous trans-acting factor that binds to a replication origin of
an extra-chromosomal template. In some embodiments, the one or more
DNA expression vectors encode the reprogramming factors consisting
of Oct4, Sox2, Klf4, and c-Myc. In some embodiments, tIn other
embodiments, the one or more DNA expression vectors encode the
reprogramming factors Oct4, Sox2, Klf4, c-Myc, and Nanog. In some
embodiments, the one or more DNA expression vectors encode
reprogramming factors consisting of Oct4, Sox2, Klf4, c-Myc, and
Nanog. In other embodiments, the one or more DNA expression vectors
encode reprogramming factors consisting of Oct4, Sox2, and Klf4. In
some embodiments, the one or more DNA expression vectors do not
comprise a mammalian origin of replication.
[0004] In some embodiments, the transfection is performed within a
single 24 hour period. In other embodiments, the transfection is
performed only one time. In some embodiments, the transfection
comprises transfecting the nucleated blood cells with only one
transfection method.
[0005] In some embodiments, the human peripheral blood is from an
adult. In some embodiments, the transfection is done by
electroporation, nucleofection, or lipofection. In one embodiment,
the transfection is done by electroporation. In some embodiments,
the DNA expression vector is a plasmid vector. In some embodiments,
the one or more DNA expression vectors comprise two DNA expression
vectors.
[0006] In some embodiments, the method does not include introducing
or expressing a recombinase (e.g., Cre recombinase) or transposase
(e.g., PiggyBac transposase or Sleeping Beauty transposase) in hiPS
cells following the identification of hiPS cell colonies. In some
embodiments, the method does not include a DNA expression vector
excision step to generate the integration-free human induced
pluripotent stem cells.
[0007] In some embodiments, the nucleated blood cells are not
hematopoietic stem cells.
[0008] In some embodiments, the method includes transfecting the
nucleated blood cells with a single DNA expression vector. In some
embodiments, the one or more DNA expression vectors further encode
a reporter protein (e.g., a fluorescent protein, .beta.-lactamase,
or a luciferase) that is expressed in the transfected nucleated
blood cells. In some embodiments, where the one or more DNA
expression vectors further encode a reporter protein, the
identification step includes identifying human induced pluripotent
stem colonies that do not express the reporter protein.
[0009] In some embodiments, the one or more DNA expression vectors
encoding reprogramming factors do not encode Tert or SV40 Large
T-antigen.
[0010] In a related aspect provided herein is an integration-free
human iPS cell induced by transfecting nucleated blood cells from
human peripheral blood with one or more DNA expression vectors
encoding reprogramming factors (a) Oct4, Sox2, Klf4, and c-Myc; (b)
Oct4, Sox2, and Klf4; (c) Oct4, Sox2, Klf4, c-Myc, and Nanog; or
(d) Oct 4, Sox2, Lin-28, and Nanog, expressing the encoded
reprogramming factors in the transfected nucleated blood cells, and
culturing the transfected nucleated blood cells under conditions
adapted to growth of human induced pluripotent stem cells, and
identifying integration-free human induced pluripotent stem cells
that do not comprise exogenous DNA from the one or more nucleic
acid expression vectors, wherein the one or more DNA expression
vectors do not contain a functional mammalian origin of
replication. In some embodiments, the one or more DNA expression
vectors encode the reprogramming factors Oct4, Sox2, Klf4, and
c-Myc. In some embodiments, the just-mentioned integration-free
human induced pluripotent stem cell comprises a rearrangement in
the Vj immunoglobulin genomic locus (e.g., VJ or VDJ recombination
in the heavy and/or light chain locus) or a rearrangement of the
T-cell receptor genomic locus (e.g., VJ or VDJ recombination in the
alpha and/or beta chain locus). In some embodiments, the
just-mentioned integration-free human induced pluripotent stem cell
comprises a rearrangement in the VJ region of the immunoglobulin
genomic locus. In some embodiments, the just-mentioned
integration-free human induced pluripotent stem cell comprises
somatic hypermutation in the immunoglobulin genomic locus. In some
embodiments, the just-mentioned integration-free human induced
pluripotent stem cell comprises a genomic change commonly
associated with a B cell or T cell (e.g., junctional diversity,
somatic recombination, somatic hypermutation).
INCORPORATION BY REFERENCE
[0011] All publications and patent applications mentioned in this
specification are herein incorporated by reference to the same
extent as if each individual publication or patent application was
specifically and individually indicated to be incorporated by
reference.
BRIEF DESCRIPTION OF THE DRAWINGS
[0012] The novel features of the invention are set forth with
particularity in the appended claims. A better understanding of the
features and advantages of the present invention will be obtained
by reference to the following detailed description that sets forth
illustrative embodiments, in which the principles of the invention
are utilized, and the accompanying drawings of which:
[0013] FIG. 1 shows a pair of photomicrographs (at 4.times. and
10.times. magnification) of individual colonies from the
integration-free hiPS cell line 630.2, which was derived from adult
nucleated blood cells by a single transient transfection with a
plasmid encoding the reprogramming factors Oct4, Sox2, Klf4, and
c-Myc.
[0014] FIG. 2 shows quantitative flow sorting analysis for the
pluripotent stem cell surface markers SSEA-4 and TRA 1-60 from the
representative, integration-free hiPS cell line 630.6B (bottom
panels). The top two panels show negative controls for staining of
these cell surface markers. Staining for these two cell surface
markers showed that 95% or greater of the analyzed 630.6B hiPSC
line cells expressed SSEA-4 and TRA 1-60.
[0015] FIG. 3 is a bar graph depicting relative expression levels
of a panel of pluripotency marker genes (a, E-CADHERIN; b, DNMT3b;
d, GDF3; e, LIN28; f, NANOG; g, OCT4, I, SALL4; j, SOX2; k, TERT;
l, REX1) in the integration-free hiPS cell lines 630.6A, 630.6B,
71.67A, 71.67B, 71.89A compared to expression levels of these
pluripotency gene markers in hiPS cell lines derived using
retroviral delivery of transcription factors (IPRN18 and IPRN20),
and adult human fibroblasts. None of the pluripotency marker genes
are expressed in adult human Fibroblasts, but fibroblasts do
express similar levels of housekeeping genes (c, GAPDH; h, RPLPO).
Expression levels were normalized to the "housekeeping" gene GAPDH,
and are shown as levels relative to iPSC line IPRN18. The
expression values shown represent the mean value of duplicate
reactions.
[0016] FIG. 4 shows agarose gel electrophoresis analysis of RT-PCR
and genomic PCR for rtTA in hiPS cell lines 630.6B, 71.89, and
71.67 with separate primer pairs ("primer set 1" and "primer set
2," having one primer in common). The left panel shows PCR results
using primer set 1. Lane 1 shows a 100 bp ladder; lane 2 shows
amplification from an rtTA plasmid positive control with the
appropriate 609 bp amplicon band; lane 3 is a water-only negative
control PCR; lane 4 is from an hES (H7) cell line negative control
PCR; lanes 5, 7, and 9 show RT-PCR results for rtTA in hiPS cell
lines treated with Doxycyline (2 .mu.g/ml); lanes 6, 8, and 10 show
RT-PCR results for rtTA in the same hiPS cell lines, but in the
absence of Doxycycline treatment (note that rtTA expression was
under the control of a constitutive (CAG) promoter; lane 10 shows a
100 bp ladder; lanes 11-13 show the results of genomic PCR for rtTA
on the same hiPSC lanes. The right panel shows PCR results using
primer set 2. The right panel shows the same configuration of lanes
as the left panel, but results are from PCR reactions with a
different primer pair (primer set 2). All of the hiPS cell lines
tested negative for expression and genomic integration of the rtTA
transgene indicating that the rtTA vector had been lost over the
course of reprogramming nucleated blood cells.
[0017] FIG. 5 shows agarose gel electrophoresis analysis of RT-PCR
and genomic PCR for the Myc-Klf4-Oct4-Sox2 four reprogramming
factor transgene and the endogenous actin gene in hiPS cell lines
630.6B, 71.89, and 71.67. The left panel shows PCR results using
primer set 1. Lane 1 shows a 100 bp ladder; lane 2 shows
amplification from the PB-MKOS plasmid positive control with the
appropriate 616 bp amplicon band; lane 3 is a water-only negative
control PCR; lane 4 is from an hES (H7) cell line negative control
PCR; lanes 5, 7, and 9 show RT-PCR results for the expression of
the MKOS transgene in hiPS cell lines treated with Doxycyline (2
.mu.g/ml); lanes 6, 8, and 10 show RT-PCR results for rtTA in the
same hiPS cell lines, but in the absence of Doxycycline treatment;
lanes 11 is empty; lanes 12-14 show the results of genomic PCR for
the MKOS transgene on the same hiPSC lanes. The right panel shows
the results of RT-PCR analysis for expression of actin in the three
hiPSC lanes as a positive control for the quality of the RNA
samples and RT-PCR conditions. Lane 1 shows a 100 bp ladder; lane 2
is a water-only negative control PCR; lane 3 is from an hES (H7)
cell line positive control RT-PCR; lanes 4, 6, and 8 show RT-PCR
results for the expression of the endogenous actin gene in hiPS
cell lines treated with Doxycyline (2 .mu.g/ml); lanes 5, 7, and 9
show RT-PCR results for actin in the same hiPS cell lines, but in
the absence of Doxycycline treatment. All of the hiPS cell lines
tested negative for expression of the MKOS transgene. Genomic PCR
for the MKOS transgene (data not shown) likewise was negative for
the presence of the MKOS transgene indicating that the PB-MKOS
vector had been lost over the course of reprogramming nucleated
blood cells.
DETAILED DESCRIPTION OF THE INVENTION
I. Introduction
[0018] Described herein are integration-free human induced
pluripotent stem (hiPS) cells generated from nucleated blood cells
and methods for generating such hiPS cells. These methods and
compositions are based on the unexpected finding that human
nucleated blood cells are, amenable to cellular reprogramming by
transient transfection with virus-free nucleic acid expression
vectors (e.g., plasmid expression vectors) that (i) encode
reprogramming factors and permit their transient expression in
human cells, and (ii) do not encode an exogenous trans-acting
factor that binds to the replication origin to replicate an
extra-chromosomal template. In some embodiments, cellular
reprogramming of nucleated blood cells includes a single transient
transfection of one or more nucleic acid vectors encoding
reprogramming factors, or transfection of these one or more
expression vectors during only a single 24 hour period. Without
wishing to be bound by theory, it is believed that nucleated blood
cells are more amenable to reprogramming by transient transfection
because nucleated blood cells, in contrast to fibroblasts, have
very limited proliferative potential in vitro, which thereby limits
dilution/loss of transfected nucleic acid expression vectors in the
transfected population over time, and allows transient expression
of reprogramming factors at sufficiently high levels and for
sufficiently long times to effect induction of hiPS cells.
II. Definitions
[0019] "transient transfection," as used herein, refers to the
introduction of n exogenous nucleic acid into a mammalian cell by a
method that does not generally result in the integration of the
exogenous nucleic into the genome of the transiently transfected
mammalian cell. "mononucleated blood cells," as used herein, refers
to any of B cell lymphocytes, T cell lymphocytes, neutrophils,
eosinophils, basophils, monocytes, macrophages, dendritic cells,
and circulating hematopoietic stem cells. "mammalian origin of
replication," or "replication origin of an extra-chromosomal
template," as used herein, refers to a nucleic acid sequence that
permits episomal replication of a nucleic acid vector (e.g., a
plasmid) in mammalian cells. Examples of a mammalian origin of
replication include, but are not limited to, any of replication
origin of a lymphotrophic herpes virus or a gamma herpesvirus, an
adenovirus, SV40, a bovine papilloma virus, or a yeast,
specifically a replication origin of a lymphotrophic herpes virus
or a gamma herpesvirus corresponding to oriP of EBV. "reprogramming
factor," as used herein, refers to any gene product, though usually
a polypeptide, that alone or in combination with other
reprogramming factors or reprogramming agents reprograms a
postnatal somatic cell to become a pluripotent stem cell. "induced
pluripotent stem cell," as used herein, refers to a pluripotent
stem cell derived from a postnatal somatic cell by any combination
of forced expression of reprogramming factors alone or in
combination with one or more reprogramming agents. "nucleic acid
expression vector," as used herein, refers to a nucleic acid not
associated with viral proteins, competent to that encodes one or
more proteins to be expressed in a host mammalian cell. In some
cases, the nucleic acid expression vector is a DNA expression
vector, e.g., a plasmid, a minicircle, a PCR product expression
cassette, or a BAC. Such DNA expression vectors include a promoter,
a polyadenylation sequence, a kozak initiation sequence that allow
for transcription and translation of genes encoded by an expression
cassette included within the nucleic acid vector sequence. In other
cases, the nucleic acid expression vector is an RNA expression
vector, i.e., a cRNA that encodes one or more proteins for
expression in a host cell, and is competent for translation within
the mammalian host cell. "nucleated blood cell populations,"
"nucleated blood cells," or "NBCs," as used herein, refer to cells
including any of the following cell types present in peripheral
blood or cord blood: white blood cells, macrophages, monocytes,
dendritic cells, lymphocytes, T-cells, B-cells, NK cells,
granulocytes, basophils, eosinophils, neutrophils, and any
progenitor cell thereof. The term NBC, as used herein, also refers
to cells (e.g., macrophages, dendritic cells, etc.) derived from
cells isolated from the blood (e.g., monocytes). For example, the
term NBCs includes macrophages and/or dendritic cells obtained by
in vitro or in vivo maturation or differentiation of monocytes
present in, or purified from, peripheral blood obtained from a
subject. The term NBC, as used herein, also refers to both mature
and immature cells, e.g., mature or immature dendritic cells. A NBC
"subpopulation" refers to an NBC population that has been
specifically enriched for or depleted of any of the forgoing NBC
types.
III. Methods
Overview
[0020] The methods described herein are drawn to generation of
integration-free human iPS (hiPS) cells from nucleated blood cells
(NBCs). In some cases, NBCs are isolated directly from a blood
sample, transfected with one or more vectors for expression of one
or more reprogramming factors, cultured in a medium suitable for
human iPS cells (e.g., mTeSR medium), and then examined for the
development of colonies exhibiting human pluripotent stem cell
morphology and pluripotent gene expression patterns as described
herein. In other cases, a NBC fraction is cultured prior to prior
to reprogramming to enrich for or deplete particular cell types
within the NBC population as described herein. Of note, the
transfected human NBCs do not express an exogenous or endogenous
trans-acting factor that binds to a replication origin of an
extra-chromosomal template as is found in so-called "episomal
vectors." See, e.g., Yu et al (2009), Science, 324(5928):797-801.
Such trans-acting factors include, but are not limited, to EBNA-1
of the Epstein-Barr Virus, large T antigen of a mouse polyomavirus,
the large T antigen of a BK virus, E1 and E2 of a bovine papilloma
virus, and Epstein-Barr Nuclear Antigen-1 (EBNA-1), and sequence
variants of any of the foregoing that retain the ability to bind to
their respective replication origin element, also referred to
herein as a "mammalian origin of replication." Further, the methods
described herein do not require an excision step (e.g., by
expression of a recombinase or transpoase) to remove transfected
DNA expression vectors from the hiPS cells obtained by the
reprogramming methods described herein.
Isolation of NBCs
[0021] In some embodiments, a nucleated blood cell fraction is
obtained by the Ficoll-Hypaque method, as described in, e.g., Kanof
et al., (1993), Current Protocols in Immunology (J. E. Coligan, A.
M. Kruisbeek, D. H. Margulies, E. M. Shevack, and W. Strober,
eds.), ch. 7.1.1.-7.1.5, John Wiley & Sons, New York). In some
embodiments, the isolation of a nucleated blood cell fraction is
performed on a human blood sample (peripheral blood or cord blood)
volume of at least about 1 ml to about 50 ml, e.g., 3 ml, 7 ml, 8
ml, 10 ml, 12 ml, 15 ml, 20 ml, 25 ml, 30 ml, 35 ml, 37 ml, 40 ml,
42 ml, 45 ml, or another volume of peripheral blood or cord blood
from at least about 1 ml to about 50 ml. For the methods described
herein NBCs may be isolated from fresh whole blood samples; from
whole blood (or NBC-containing blood fraction) samples stored for
about two days to about five weeks in heparanized storage tubes; or
from cryopreserved whole blood (or NBC-containing fraction) samples
cryopreserved with DMSO in liquid nitrogen by standard methods
(see, e.g., Stevens et al (2007), Cancer Epidemiol Biomarkers Prev,
16(10):2160-2163.
[0022] In some cases, reprogramming is performed on NBC
subpopulations that are depleted of or enriched in certain cell
types. Specific cell populations can be depleted or enriched using
standard methods. For example, monocytes/macrophages can be
isolated by differential adherence on plastic. T cells, B cells,
macrophages, and granulocytes can be enriched or depleted, for
example, by positive and/or negative selection using antibodies to
cell type-specific surface markers. Examples of suitable cell
type-specific surface marker proteins for which antibodies commonly
used for selection or depletion of specific NBC subpopulations,
include, but are not limited to CD3 for T cells, CD19 for B cells,
CD14 for monocytes/macrophages and CD15 for granulocytes. Specific
cell populations can be enriched or depleted by incubating cells
with a specific primary monoclonal antibody (mAb), followed by
isolation of cells that bind the mAb by a number of well known
methods including fluorescence-activated cell sorting (FACS),
magnetic bead sorting, or magnetic-activated cell sorting
(MACST.TM.).
[0023] In some cases, NBCs are depleted of particular cell types by
negative selection, where the NBCs are incubated in the presence of
one or more antibodies against cell surface markers specific to the
undesired cell types. Afterwards, the labeled subpopulation of
cells is depleted (negative selection) from the NBC sample by any
of the above-mentioned methods, e.g., FACS. In some embodiments,
NBCs are depleted of B cells and T cells by incubating NBCs in the
presence of antibodies against CD3 (for T-cells) and CD19 (for B
cells) and selecting cells, e.g., by FACS, that are not labeled by
such antibodies.
[0024] In other cases, specific cell types, e.g., T-cells, are
isolated by incubating NBCs in the presence of antibodies against
cell surface markers specific to the desired cell types.
Afterwards, the labeled subpopulation of cells is selected
(positive selection) from the NBC sample by any of the
above-mentioned methods, e.g., FACS. In some embodiments,
monocytes/macrophages and/or granulocytes are selected by
incubating NBCs in the presence of CD14 and CD15 antibodies,
respectively, and then isolating the labeled cells by FACS or other
another suitable method as described herein or known in the
art.
[0025] In some cases NBCs are first subjected to a round of
negative selection to deplete one or more cell types (e.g., B-cells
and T-cells), and the remaining cell population is then subjected
to positive selection to isolate specific cell types
(monocytes/macrophages or granulocytes). In other embodiments,
positive selection is performed first on the NBCs, and then
followed by negative selection of the positively selected cell
population to deplete the positively selected cell population of
unwanted cell types.
[0026] In some embodiments, a NBC fraction is subjected to positive
selection with antibodies against CD14 and CD15, followed by FACS
or MACS for isolation of CD14.sup.+ or CD15.sup.+ cells. In other
embodiments, MACS is used to isolate CD14.sup.+ or CD15.sup.+
cells. In some embodiments, the isolated CD14+ cells are further
differentiated into macrophages or dendritic cells.
[0027] In other cases, a NBC fraction is subjected to negative
selection with antibodies against CD3 and CD19, followed by FACS or
MACS for isolation of CD3.sup.- and CD19.sup.- cells.
[0028] In some embodiments, PBNCs or a subpopulation of PBNCs are
cultured prior to reprogramming by the methods described herein. In
some cases, culture conditions are selected that favor selective
proliferation of some cell types from a starting population of
PBNCs. For example, where selective proliferation of cells of
myeloid lineage is preferred, culture of NBCs is carried out in the
presence of GM-CSF, G-CSF, IL-4, SCF alone or in combination.
Examples of suitable culture conditions can be found for
macrophages in U.S. Pat. No. 5,078,996, neutrophils/granulocytes in
U.S. patent publication No. 20030039661, or dendritic cells in U.S.
Pat. No. 5,851,756.
[0029] In some embodiments, the NBCs to be reprogrammed by the
methods described herein do not include hematopoietic stem
cells.
[0030] The NBCs can be derived from neonatal or postnatal blood
collected from a subject within the period from birth, including
cesarean birth, to death. For example, the tissue may be from a
subject who is >10 minutes old, >1 hour old, >1 day old,
>1 month old, >2 months old, >6 months old, >1 year
old, >2 years old, >5 years old, >10 years old, >15
years old, >18 years old, >25 years old, >35 years old,
>45 years old, >55 years old, >65 years old, >80 years
old, <80 years old, <70 years old, <60 years old, <50
years old, <40 years old, <30 years old, <20 years old or
<10 years old. The subject may be a neonatal infant. In some
cases, the subject is a child or an adult. In some examples, the
tissue is from a human of age 2, 5, 10 or 20 hours. In other
examples, the tissue is from a human of age 1 month, 2 months, 3
months, 4 months, 5 months, 6 months, 9 months or 12 months. In
some cases, the tissue is from a human of age 1 year, 2 years, 3
years, 4 years, 5 years, 18 years, 20 years, 21 years, 23 years, 24
years, 25 years, 28 years, 29 years, 31 years, 33 years, 34 years,
35 years, 37 years, 38 years, 40 years, 41 years, 42 years, 43
years, 44 years, 47 years, 51 years, 55 years, 61 years, 63 years,
65 years, 70 years, 77 years, or 85 years old.
[0031] The NBCs may be collected from subjects with a variety of
disease statuses. The cells can be collected from a subject who is
free of an adverse health condition. In other cases, the subject is
suffering from, or at high risk of suffering from, a disease or
disorder, e.g., a chronic health condition such as cardiovascular
disease, eye disease (e.g., macular degeneration), auditory
disease, (e.g., deafness), diabetes, obesity, cognitive impairment,
schizophrenia, depression, bipolar disorder, dementia,
neurodegenerative disease, Spinal Muscular Atrophy, Alzheimer's
Disease, Parkinson's Disease, Huntington's Disease, multiple
sclerosis, osteoporosis, liver disease, kidney disease, autoimmune
disease, arthritis, or a proliferative disorder (e.g., a cancer).
In other cases, the subject is suffering from, or at high risk of
suffering from, an acute health condition, e.g., stroke, spinal
cord injury, burn, or a wound. In certain cases, a subject provides
cells for his or her future use (e.g., an autologous therapy), or
for the use of another subject who may need treatment or therapy
(e.g., an allogeneic therapy). In some cases, the donor and the
recipient are immunohistologically compatible or HLA-matched.
Reprogramming NBCs by Introduction of Nucleic Acid Vectors
[0032] Where DNA expression vectors are used, such vectors include
a promoter competent to drive reprogramming factor gene
transcription in a plurality of cells to be reprogrammed within an
expression cassette encoding at least one reprogramming factor. In
some embodiments, the DNA expression vectors used in the
reprogramming methods described herein do not include loxP
transposition target sites for CRE recombinase, or a mammalian
origin of replication, e.g., the Epstein-Barr Virus oriP element
(Yates et al (1984), Proc. Natl. Acad. Sci. USA, 81:3806-3810.
Other examples of a mammalian origin of replication include, but
are not limited to, replication origin of a lymphotrophic herpes
virus or a gamma herpesvirus, an adenovirus, SV40, a bovine
papilloma virus, or a yeast, specifically a replication origin of a
lymphotrophic herpes virus or a gamma herpesvirus corresponding to
oriP of EBV. With reference to a replication origin that may serve
as a mammalian origin of replication, lymphotrophic herpes virus
may be Epstein Barr virus (EBV), Kaposi's sarcoma herpes virus
(KSHV), Herpes virus saimiri (HS), or Marek's disease virus (MDV).
Epstein Barr virus (EBV) and Kaposi's sarcoma herpes virus (KSHV)
are also examples of a gamma herpesvirus. DNA expression vectors
comprising a mammalian origin of replication are capable of being
stably replicated as extrachromosomal episomes even within cells
that actively proliferate, e.g., fibroblasts in the presence of a
sufficient level of serum. Examples of vectors comprising a
mammalian origin of replication are described in, e.g., U.S. patent
application Ser. No. 12/478,154. In some embodiments, the DNA
expression vectors suitable for the methods described herein do not
contain the mammalian origin of replication found in, e.g., any of
the following episomal vectors: pCEP4, pREP4, or pEBNA DEST. In
some embodiments, the DNA expression vectors suitable for the
methods described herein do not include a S/MAR (scaffold/matrix
attachment region) sequence. See, e.g., Piechaczek et al (1999),
Nucleic Acids Res, 27:426-428.
[0033] Examples of suitable promoters for driving mammalian cell
expression of the polypeptides described herein in include, but are
not limited to, constitutive promoters such as, CMV, CAG,
EF-1.alpha., HSV1-TK, SV40, EF-1.alpha., .beta. actin; PGK, and
inducible promoters, such as those containing TET-operator
elements. In certain embodiments, cell type-specific promoters are
used to drive expression of reprogramming factors in specific cell
types. Examples of suitable cell type-specific promoters useful for
the methods described herein include, but are not limited to, the
synthetic macrophage-specific promoter described in He et al
(2006), Human Gene Therapy, 17:949-959; the granulocyte and
macrophage-specific lysozyme M promoter (see, e.g., Faust et al
(2000), Blood, 96(2):719-726); and the myeloid-specific CD11b
promoter (see, e.g., Dziennis et al (1995), Blood, 85(2):319-329).
In some cases, an expression cassette encodes a polycistronic mRNA
(a "polycistronic expression cassette"), which, upon translation
gives rise to independent polypeptides comprising different amino
acid sequences or functionalities. In some embodiments, a
polycistronic expression cassette encodes a "polyprotein"
comprising multiple polypeptide sequences that are separated by
encoded by a picornavirus, e.g., a foot-and-mouth disease virus
(FMDV) viral 2A peptide sequence. The 2A peptide sequence acts
co-translationally, by preventing the formation of a normal peptide
bond between the conserved glycine and last proline, resulting in
ribosome skipping to the next codon, and the nascent peptide
cleaving between the Gly and Pro. After cleavage, the short 2A
peptide remains fused to the C-terminus of the `upstream` protein,
while the proline is added to the N-terminus of the `downstream`
protein. which during translation allow cleavage of the nascent
polypeptide sequence into separate polypeptides. See, e.g., Trichas
et al (2008), BMC Biol, 6:40. Two exemplary 2A nucleotide sequences
and their corresponding peptide sequences are shown below: [0034]
5'GGCAGTGGAGAGGGCAGAGGAAGTCTGCTAACATGCGGTGACGTCGAGGAGAAT
CCTGGCCCA3' (SEQ ID NO:1), which is translated into the peptide
sequence: [0035] GSGEGRGSLLTCGDVEENPGP (SEQ ID NO:2); or [0036]
5'GGTTCTGGCGTGAAACAGACTTTGAATTTTGACCTTCTCAAGTTGGCGGGA
GACGTGGAGTCCAACCCAGGGCCC3' (SEQ ID NO:3) [0037] which translates to
the sequence GSGVKQTNFDLLKLAGDVESNPGP (SEQ ID NO: 4)
[0038] In other embodiments, a polycistronic expression cassette
may incorporate one or more internal ribosomal entry site (IRES)
sequences between open reading frames incorporated into the
polycistronic expression cassette. IRES sequences and their use are
known in the art as exemplified in, e.g., Martinez-Sales, Curr Opin
Biotechnol, 10(5):458-464.
Reprogramming Factors
[0039] In various embodiments, nucleic acids encoding one or more
reprogramming factors useful in cellular reprogramming of human
NBCs into hiPS cells are used. Examples of suitable reprogramming
factor genes include, but are not limited to genes encoding a
polypeptide that comprises an amino acid sequence at least 80%
identical, e.g., at least 85%, 88%, 90%, 95%, 97%, or another
percent identical to the amino sequence of any of the following
human or mouse sequences: Oct 4 (GenBank Accession Nos.
NP.sub.--002692 and NP.sub.--038661.2, respectively), Sox2 (GenBank
Accession Nos. NP.sub.--003097.1 and AAH57574, respectively), Klf4
(GenBank Accession Nos. NP.sub.--004226.3 and NP.sub.--034767.2,
respectively), c-Myc (NP.sub.--002458.2 and NP.sub.--034979,
respectively), Nanog (AY230262.1 and NP.sub.--082292.1,
respectively), and Lin-28 (NP.sub.--078950.1 and NP.sub.--665832.1,
respectively). In some embodiments, the encoded reprogramming
factors may also include human or mouse activation-induced cytidine
deaminase (AID), (GenBank Accession Nos. (NP.sub.--065712.1 and
NP.sub.--033775.1, respectively). In some embodiments, the encoded
reprogramming factor amino acid sequences are from human. In other
embodiments, the encoded sequences are from mouse. In some
embodiments, a nucleic acid expression vector encodes the human
ortholog of any of Oct 4, Sox2, Klf4, c-Myc, Nanog, or Lin-28. In
other embodiments, a nucleic acid expression vector encodes the
mouse ortholog of any of Oct 4, Sox2, Klf4, c-Myc, Nanog, or
Lin-28. In some embodiments, an expression cassette is a
polycistronic expression cassette that encodes the amino acids
sequences of multiple reprogramming factors, the expression of
which is under the control of the same promoter. Such polycistronic
expression cassettes may include at least two, three, four, five,
or six reprogramming factors. In some cases, an expression cassette
includes the open reading frames for Oct 4 and Sox2. In other
cases, the expression cassette includes the open reading frames for
Oct 4, Sox2, and Klf4. In other embodiments, the expression
cassette includes the open reading frames for Oct 4, Sox2, Klf4,
and c-Myc. In further embodiments, the expression cassette includes
the open reading frames for Oct 4, Sox2, Nanog, and Lin-28. In some
embodiments, a polycistronic expression cassette for expression of
multiple reprogramming factors contains the sequence encoding the
2A peptide between the sequences encoding the various reprogramming
factors. A polycistronic expression cassette may contain the
sequences of reprogramming factors from 5' to 3' in any order. In
some cases, a polycistronic expression cassette comprises a nucleic
acid sequence encoding reprogramming factors in the order from 5'
to 3' c-Myc, K14, Oct4, Sox2 with intervening 2A sequences (lower
case) as shown in the following exemplary nucleotide and amino acid
sequences:
TABLE-US-00001 (SEQ ID NO: 5)
ATGCCCCTCAACGTGAACTTCACCAACAGGAACTATGACCTCGACTAC
GACTCCGTACAGCCCTATTTCATCTGCGACGAGGAAGAGAATTTCTAT
CACCAGCAACAGCAGAGCGAGCTGCAGCCGCCCGCGCCCAGTGAGGAT
ATCTGGAAGAAATTCGAGCTGCTTCCCACCCCGCCCCTGTCCCCGAGC
CGCCGCTCCGGGCTCTGCTCTCCATCCTATGTTGCGGTCGCTACGTCC
TTCTCCCCAAGGGAAGACGATGACGGCGGCGGTGGCAACTTCTCCACC
GCCGATCAGCTGGAGATGATGACCGAGTTACTTGGAGGAGACATGGTG
AACCAGAGCTTCATCTGCGATCCTGACGACGAGACCTTCATCAAGAAC
ATCATCATCCAGGACTGTATGTGGAGCGGTTTCTCAGCCGCTGCCAAG
CTGGTCTCGGAGAAGCTGGCCTCCTACCAGGCTGCGCGCAAAGACAGC
ACCAGCCTGAGCCCCGCCCGCGGGCACAGCGTCTGCTCCACCTCCAGC
CTGTACCTGCAGGACCTCACCGCCGCCGCGTCCGAGTGCATTGACCCC
TCAGTGGTCTTTCCCTACCCGCTCAACGACAGCAGCTCGCCCAAATCC
TGTACCTCGTCCGATTCCACGGCCTTCTCTCCTTCCTCGGACTCGCTG
CTGTCCTCCGAGTCCTCCCCACGGGCCAGCCCTGAGCCCCTAGTGCTG
CATGAGGAGACACCGCCCACCACCAGCAGCGACTCTGAAGAAGAGCAA
GAAGATGAGGAAGAAATTGATGTGGTGTCTGTGGAGAAGAGGCAAACC
CCTGCCAAGAGGTCGGAGTCGGGCTCATCTCCATCCCGAGGCCACAGC
AAACCTCCGCACAGCCCACTGGTCCTCAAGAGGTGCCACGTCTCCACT
CACCAGCACAACTACGCCGCACCCCCCTCCACAAGGAAGGACTATCCA
GCTGCCAAGAGGGCCAAGTTGGACAGTGGCAGGGTCCTGAAGCAGATC
AGCAACAACCGCAAGTGCTCCAGCCCCAGGTCCTCAGACACGGAGGAA
AACGACAAGAGGCGGACACACAACGTCTTGGAACGTCAGAGGAGGAAC
GAGCTGAAGCGCAGCTTTTTTGCCCTGCGTGACCAGATCCCTGAATTG
GAAAACAACGAAAAGGCCCCCAAGGTAGTGATCCTCAAAAAAGCCACC
GCCTACATCCTGTCCATTCAAGCAGACGAGCACAAGCTCACCTCTGAA
AAGGACTTATTGAGGAAACGACGAGAACAGTTGAAACACAAACTCGAA
CAGCTTCGAAACTCTGGTGCAggttctggcgtgaaacagactttgaat
tttgaccttctcaagttggcgggagacgtggagtccaacccagggccc
ATGGCTGTCAGCGACGCTCTGCTCCCGTCCTTCTCCACGTTCGCGTCC
GGCCCGGCGGGAAGGGAGAAGACACTGCGTCCAGCAGGTGCCCCGACT
AACCGTTGGCGTGAGGAACTCTCTCACATGAAGCGACTTCCCCCACTT
CCCGGCCGCCCCTACGACCTGGCGGCGACGGTGGCCACAGACCTGGAG
AGTGGCGGAGCTGGTGCAGCTTGCAGCAGTAACAACCCGGCCCTCCTA
GCCCGGAGGGAGACCGAGGAGTTCAACGACCTCCTGGACCTAGACTTT
ATCCTTTCCAACTCGCTAACCCACCAGGAATCGGTGGCCGCCACCGTG
ACCACCTCGGCGTCAGCTTCATCCTCGTCTTCCCCAGCGAGCAGCGGC
CCTGCCAGCGCGCCCTCCACCTGCAGCTTCAGCTATCCGATCCGGGCC
GGGGGTGACCCGGGCGTGGCTGCCAGCAACACAGGTGGAGGGCTCCTC
TACAGCCGAGAATCTGCGCCACCTCCCACGGCCCCCTTCAACCTGGCG
GACATCAATGACGTGAGCCCCTCGGGCGGCTTCGTGGCTGAGCTCCTG
CGGCCGGAGTTGGACCCAGTATACATTCCGCCACAGCAGCCTCAGCCG
CCAGGTGGCGGGCTGATGGGCAAGTTTGTGCTGAAGGCGTCTCTGACC
ACCCCTGGCAGCGAGTACAGCAGCCCTTCGGTCATCAGTGTTAGCAAA
GGAAGCCCAGACGGCAGCCACCCCGTGGTAGTGGCGCCCTACAGCGGT
GGCCCGCCGCGCATGTGCCCCAAGATTAAGCAAGAGGCGGTCCCGTCC
TGCACGGTCAGCCGGTCCCTAGAGGCCCATTTGAGCGCTGGACCCCAG
CTCAGCAACGGCCACCGGCCCAACACACACGACTTCCCCCTGGGGCGG
CAGCTCCCCACCAGGACTACCCCTACACTGAGTCCCGAGGAACTGCTG
AACAGCAGGGACTGTCACCCTGGCCTGCCTCTTCCCCCAGGATTCCAT
CCCCATCCGGGGCCCAACTACCCTCCTTTCCTGCCAGACCAGATGCAG
TCACAAGTCCCCTCTCTCCATTATCAAGAGCTCATGCCACCGGGTTCC
TGCCTGCCAGAGGAGCCCAAGCCAAAGAGGGGAAGAAGGTCGTGGCCC
CGGAAAAGAACAGCCACCCACACTTGTGACTATGCAGGCTGTGGCAAA
ACCTATACCAAGAGTTCTCATCTCAAGGCACACCTGCGAACTCACACA
GGCGAGAAACCTTACCACTGTGACTGGGACGGCTGTGGGTGGAAATTC
GCCCGCTCCGATGAACTGACCAGGCACTACCGCAAACACACAGGGCAC
CGGCCCTTTCAGTGCCAGAAGTGCGACAGGGCCTTTTCCAGGTCGGAC
CACCTTGCCTTACACATGAAGAGGCACTTTggctccggagagggcaga
ggaagtctgctaacatgcggtgacgtcgaggagaatcctggcccactc
gagATGGCTGGACACCTGGCTTCAGACTTCGCCTCCTCACCCCCACCA
GGTGGGGGTGATGGGTCAGCAGGGCTGGAGCCGGGCTGGGTGGATTCT
CGAACCTGGCTAAGCTTCCAAGGGCCTCCAGGTGGGCCTGGAATCGGA
CCAGGCTCAGAGGTATTGGGGATCTCCCCATGTCCGCCCGCATACGAG
TTCTGCGGAGGGATGGCATACTGTGGACCTCAGGTTGGACTGGGCCTA
GTCCCCCAAGTTGGCGTGGAGACTTTGCAGCCTGAGGGCCAGGCAGGA
GCACGAGTGGAAAGCAACTCAGAGGGAACCTCCTCTGAGCCCTGTGCC
GACCGCCCCAATGCCGTGAAGTTGGAGAAGGTGGAACCAACTCCCGAG
GAGTCCCAGGACATGAAAGCCCTGCAGAAGGAGCTAGAACAGTTTGCC
AAGCTGCTGAAGCAGAAGAGGATCACCTTGGGGTACACCCAGGCCGAC
GTGGGGCTCACCCTGGGCGTTCTCTTTGGAAAGGTGTTCAGCCAGACC
ACCATCTGTCGCTTCGAGGCCTTGCAGCTCAGCCTTAAGAACATGTGT
AAGCTGCGGCCCCTGCTGGAGAAGTGGGTGGAGGAAGCCGACAACAAT
GAGAACCTTCAGGAGATATGCAAATCGGAGACCCTGGTGCAGGCCCGG
AAGAGAAAGCGAACTAGCATTGAGAACCGTGTGAGGTGGAGTCTGGAG
ACCATGTTTCTGAAGTGCCCGAAGCCCTCCCACAGCAGATCACTCACA
TCGCCAATCAGCTTGGGCTAGAGAAGGATGTGGTTCGAGTATGGTTCT
GTAACCGGCGCCAGAAGGGCAAAAGATCAAGTATTGAGTATTCCCAAC
GAGAAGAGTATGAGGCTACAGGGACACCTTTCCCAGGGGGGGCTGTAT
CCTTTCCTCTGCCCCCAGGTCCCCACTTTGGCACCCCAGGCTATGGAA
GCCCCCACTTCACCACACTCTACTCAGTCCCTTTTCCTGAGGGCGAGG
CCTTTCCCTCTGTTCCCGTCACTGCTCTGGGCTCTCCCATGCATTCAA
ACgggtcgggtcaatgtactaactacgctttgttgaaactcgctggcg
atgttgaaagtaataaccccggtcctATGTATAACATGATGGAGACGG
AGCTGAAGCCGCCGGGCCCGCAGCAAGCTTCGGGGGGCGGCGGCGGAG
GAGGCAACGCCACGGCGGCGGCGACCGGCGGCAACCAGAAGAACAGCC
CGGACCGCGTCAAGAGGCCCATGAACGCCTTCATGGTATGGTCCCGGG
GGCAGCGGCGTAAGATGGCCCAGGAGAACCCCAAGATGCACAACTCGG
AGATCAGCAAGCGCCTGGGCGCGGAGTGGAAACTTTTGTCCGAGACCG
AGAAGCGGCCGTTCATCGACGAGGCCAAGCGGCTGCGCGCTCTGCACA
TGAAGGAGCACCCGGATTATAAATACCGGCCGCGGCGGAAAACCAAGA
CGCTCATGAAGAAGGATAAGTACACGCTTCCCGGAGGCTTGCTGGCCC
CCGGCGGGAACAGCATGGCGAGCGGGGTTGGGGTGGGCGCCGGCCTGG
GTGGCGGGCTGAACCAGCGCATGGACAGCTACGCGCACATGAACGGCT
GGAGCAACGGCAGCTACAGCATGATGCAGGAGCAGCTGGGCTACCCGC
AGCACCCGGGCCTCAACGCTCACGGCGCGGCACAGATGCAACCGATGC
ACCGCTACGTCGTCAGCGCCCTGCAGTACAACTCCATGACCAGCTCGC
AGACCTACATGAACGGCTCGCCCACCTACAGCATGTCCTACTCGCAGC
AGGGCACCCCCGGTATGGCGCTGGGCTCCATGGGCTCTGTGGTCAAGT
CCGAGGCCAGCTCCAGCCCCCCCGTGGTTACCTCTTCCTCCCACTCCA
GGGCGCCCTGCCAGGCCGGGGACCTCCGGGACATGATCAGCATGTACC
TCCCCGGCGCCGAGGTGCCGGAGCCCGCTGCGCCCAGTAGACTGCACA
TGGCCCAGCACTACCAGAGCGGCCCGGTGCCCGGCACGGCCAAATACG
GCACACTGCCCCTGTCGCACATGTGA,
which translates to the following amino acid sequence:
TABLE-US-00002 (SEQ ID NO: 6)
MPLNVNFTNRNYDLDYDSVQPYFICDEEENFYHQQQQSELQPPAPSED
IWKKFELLPTPPLSPSRRSGLCSPSYVAVATSFSPREDDDGGGGNFST
ADQLEMMTELLGGDMVNQSFICDPDDETFIKNIIIQDCMWSGFSAAAK
LVSEKLASYQAARKDSTSLSPARGHSVCSTSSLYLQDLTAAASECIDP
SVVFPYPLNDSSSPKSCTSSDSTAFSPSSDSLLSSESSPRASPEPLVL
HEETPPTTSSDSEEEQEDEEEIDVVSVEKRQTPAKRSESGSSPSRGHS
KPPHSPLVLKRCHVSTHQHNYAAPPSTRKDYPAAKRAKLDSGRVLKQI
SNNRKCSSPRSSDTEENDKRRTHNVLERQRRNELKRSFFALRDQIPEL
ENNEKAPKVVILKKATAYILSIQADEHKLTSEKDLLRKRREQLKHKLE
QLRNSGAGSGVKQTLNFDLLKLAGDVESNPGPMAVSDALLPSFSTFAS
GPAGREKTLRPAGAPTNRWREELSHMKRLPPLPGRPYDLAATVATDLE
SGGAGAACSSNNPALLARRETEEFNDLLDLDFILSNSLTHQESVAATV
TTSASASSSSSPASSGPASAPSTCSFSYPIRAGGDPGVAASNTGGGLL
YSRESAPPPTAPFNLADINDVSPSGGFVAELLRPELDPVYIPPQQPQP
PGGGLMGKFVLKASLTTPGSEYSSPSVISVSKGSPDGSHPVVVAPYSG
GPPRMCPKIKQEAVPSCTVSRSLEAHLSAGPQLSNGHRPNTHDFPLGR
QLPTRTTPTLSPEELLNSRDCHPGLPLPPGFHPHPGPNYPPFLPDQMQ
SQVPSLHYQELMPPGSCLPEEPKPKRGRRSWPRKRTATHTCDYAGCGK
TYTKSSHLKAHLRTHTGEKPYHCDWDGCGWKFARSDELTRHYRKHTGH
RPFQCQKCDRAFSRSDHLALHMKRHFGSGEGRGSLLTCGDVEENPGPL
EMAGHLASDFASSPPPGGGDGSAGLEPGWVDSRTWLSFQGPPGGPGIG
PGSEVLGISPCPPAYEFCGGMAYCGPQVGLGLVPQVGVETLQPEGQAG
ARVESNSEGTSSEPCADRPNAVKLEKVEPTPEESQDMKALQKELEQFA
KLLKQKRITLGYTQADVGLTLGVLFGKVFSQTTICRFEALQLSLKNMC
KLRPLLEKWVEEADNNENLQEICKSETLVQARKRKRTSIENRVRWSLE
TMFLKCPKPSLQQITHIANQLGLEKDVVRVWFCNRRQKGKRSSIEYSQ
REEYEATGTPFPGGAVSFPLPPGPHFGTPGYGSPHFTTLYSVPFPEGE
AFPSVPVTALGSPMHSNGSGQCTNYALLKLAGDVESNNPGPMYNMMET
ELKPPGPQQASGGGGGGGNATAAATGGNQKNSPDRVKRPMNAFMVWSR
GQRRKMAQENPKMHNSEISKRLGAEWKLLSETEKRPFIDEAKRLRALH
MKEHPDYKYRPRRKTKTLMKKDKYTLPGGLLAPGGNSMASGVGVGAGL
GGGLNQRMDSYAHMNGWSNGSYSMMQEQLGYPQHPGLNAHGAAQMQPM
HRYVVSALQYNSMTSSQTYMNGSPTYSMSYSQQGTPGMALGSMGSVVK
SEASSSPPVVTSSSHSRAPCQAGDLRDMISMYLPGAEVPEPAAPSRLH
MAQHYQSGPVPGTAKYGTLPLSHM
[0040] Various combinations of exogenous reprogramming factors can
be used to reprogram populations or subpopulations of NBCs. As
described herein, the exogenous reprogramming factors are delivered
to NBCs by introduction of one or more nucleic acid expression
vectors (DNA expression vectors or RNA expression vectors) encoding
the exogenous reprogramming factors. In some embodiments, the
exogenous reprogramming factors to be expressed include the four
factors Oct4, Sox2, Klf4, and c-Myc. In some embodiments the
exogenous reprogramming factors include Oct4, Sox2, Klf4, c-Myc,
and Nanog. In other embodiments, the exogenous reprogramming
factors include (i) the four reprogramming factors Oct4, Sox2, K14,
c-Myc, but without additional exogenous reprogramming factors, or
(ii) the five reprogramming factors Oct4, Sox2, Klf4, c-Myc, and
Nanog, but without additional exogenous reprogramming factors. In
other embodiments, the four exogenous reprogramming factors include
Oct4, Sox2, Nanog, and Lin-28, or Oct4, Sox2, Nanog, and Lin-28,
but without additional exogenous reprogramming factors.
[0041] In further embodiments, the exogenous reprogramming factors
include the three reprogramming factors Oct4, Sox2, and Klf4; or
include Oct4, Sox2, and K14, but without additional exogenous
reprogramming factors.
[0042] In some embodiments, the exogenous reprogramming factors do
not include Tert or SV40 Large T-antigen.
[0043] In some cases, expression of each exogenous reprogramming
factor is achieved by introducing a separate nucleic acid
expression vector (e.g., a DNA expression vector) encoding the
reprogramming factor into the NBCs to be reprogrammed. For example,
in some cases four DNA expression vectors are introduced into a NBC
host cell population or subpopulation to be reprogrammed, where
each plasmid expression vector encodes and drives expression of a
separate reprogramming factor in the host cells to be reprogrammed.
In some embodiments, the four DNA expression vectors (e.g.,
plasmids or minicircles) separately encode Oct4, Sox2, K14, Sox2,
and c-Myc. In other embodiments, the four DNA expression vectors
encode Oct4, Sox2, Lin-28, and Nanog. In some cases, three separate
DNA expression vectors encoding separately Oct4, Sox2, and Lin-28
are used in the reprogramming methods described herein.
[0044] In some cases, the reprogramming methods described utilize a
combination of nucleic acid expression vectors, some of which
encode and drive the expression of a single reprogramming factor
(e.g., Oct4), and the others encoding two, three, or four
reprogramming factors within a single, polycistronic expression
cassette, as described herein. In some embodiments, two nucleic
acid expression vectors are utilized, each encoding two
reprogramming factors linked to each other by a 2A peptide or
equivalent "autocleavage." In some embodiments, the two nucleic
acid expression vectors encode Oct4 and Sox2, and c-Myc and Klf4.
In some cases, the two nucleic acid expression vectors are DNA
expression vectors. In other cases, the two nucleic acid expression
vectors are RNA expression vectors.
[0045] In some cases, where DNA expression vectors encoding the
reprogramming factors are under the control of an inducible
promoter requiring an exogenous transactivator (e.g., the reverse
tetracycline transactivator or "rtTA"), a nucleic acid expression
vector for expression of the transactivator is introduced into the
NBCs in addition to the one or more nucleic acids encoding the
reprogramming factors. In some embodiments, the nucleic acid
expression vector encoding the transactivator is introduced into
the NBCs at same time as one or more nucleic acid expression
vectors encoding reprogramming factors. In other embodiments, the
nucleic acid expression vector encoding the transactivator is
introduced into the NBCs at a different time than the nucleic acid
expression vectors encoding the reprogramming factors. In one
embodiment, the reprogramming methods described herein include
introducing a DNA expression vector that includes a polycistronic
expression cassette for expression of Oct4, Sox2, Klf4, and Sox2
under the control of an inducible Tet-O promoter and a separate DNA
expression vector containing an expression cassette for rtTA under
the control of a constitutive promoter suitable for expression in
NBCs or NBC subpopulations.
[0046] In some embodiments, the nucleic acid expression vector
encoding one or more reprogramming factors further encode within
the same expression cassette one or more selection markers that
facilitates identification or selection of NBCs that have received
and express the reprogramming factors along with the selection
marker. Examples of marker genes include, but are not limited to,
genes encoding fluorescent proteins, e.g., Fluorescent Timer,
tandem-dimer (td)-Tomato mCherry, EGFP, DS-Red, monomeric Orange,
YFP, and CFP; genes encoding proteins conferring resistance to a
selection agent, e.g., the, Puro.sup.R, Puro.sup.R-.DELTA.TK,
Zeo.sup.R, Hygro.sup.R neo.sup.R gene, and the blasticidin
resistance gene. In some cases, the selection marker contains the
amino acid sequences of a fluorescent reporter and a selection
marker enzyme protein sequence fused to each other. Examples of
such fusion selection markers include, but are not limited to,
EGFP-Puro.sup.R, EGFP-Hygro.sup.R, Fluorescent Timer-Puro.sup.R,
and mCherry-Hygro.sup.R.
[0047] With respect to the nucleic acid and amino acid sequences
described herein, in some embodiments sequence variants (e.g.,
reprogramming factor sequence variants) may be utilized. In
general, polypeptide sequence variants, include functional variants
(as determined by appropriate assays) comprising an amino sequence
at least 75%, e.g., at least 80%, 85%, 90%, 95%, or any other
percent identical to those disclosed herein. With regard to the
polypeptide sequences described herein and variants thereof, the
structural and functional homology of two or polypeptides generally
includes determining the percent identity of their amino acid
sequences to each other. Sequence identity between two or more
amino acid sequences is determined by conventional methods. See,
for example, Altschul et al., (1997), Nucleic Acids Research,
25(17):3389-3402; and Henikoff and Henikoff (1982), Proc. Natl.
Acad. Sci. USA, 89:10915 (1992). Briefly, two amino acid sequences
are aligned to optimize the alignment scores using a gap opening
penalty of 10, a gap extension penalty of 1, and the "BLOSUM62"
scoring matrix of Henikoff and Henikoff (ibid.). The percent
identity is then calculated as: ([Total number of identical
matches]/[length of the longer sequence plus the number of gaps
introduced into the longer sequence in order to align the two
sequences])(100).
[0048] Those skilled in the art will appreciate that there are many
established algorithms available to align two amino acid sequences.
The "FASTA" similarity search algorithm of Pearson and Lipman is a
suitable protein alignment method for examining the level of
identity shared by an amino acid sequence disclosed herein and the
amino acid sequence of another peptide. The FASTA algorithm is
described by Pearson and Lipman (1988), Proc. Nat'l Acad. Sci. USA,
85:2444, and by Pearson (1990), Meth. Enzymol., 183:63. Briefly,
FASTA first characterizes sequence similarity by identifying
regions shared by the query sequence and a test sequence that have
either the highest density of identities (if the ktup variable is
1) or pairs of identities (if ktup=2), without considering
conservative amino acid substitutions, insertions, or deletions.
The ten regions with the highest density of identities are then
rescored by comparing the similarity of all paired amino acids
using an amino acid substitution matrix, and the ends of the
regions are "trimmed" to include only those residues that
contribute to the highest score. If there are several regions with
scores greater than the "cutoff" value (calculated by a
predetermined formula based upon the length of the sequence and the
ktup value), then the trimmed initial regions are examined to
determine whether the regions can be joined to form an approximate
alignment with gaps. Finally, the highest scoring regions of the
two amino acid sequences are aligned using a modification of the
Needleman-Wunsch-Sellers algorithm (Needleman and Wunsch (1970), J.
Mol. Biol., 48:444-453; Sellers (1974), SIAM J. Appl. Math.,
26:787), which allows for amino acid insertions and deletions.
Illustrative parameters for FASTA analysis are: ktup=1, gap opening
penalty=10, gap extension penalty=1, and substitution
matrix=BLOSUM62. These parameters can be introduced into a FASTA
program by modifying the scoring matrix file ("SMATRIX"), as
explained in Appendix 2 of Pearson (1990), Meth. Enzymol.,
183:63.
[0049] Suitable nucleic acids that may be utilized for the methods
described herein hybridize specifically under low, medium, or high
stringency conditions to a probe of at least 1000 nucleotides from
a nucleic acid encoding the amino acid sequence of any of SEQ ID
NOs:2, 4, 6, human Oct4, Sox2, Klf4, c-Myc, Nanog, or Lin-28. Low
stringency hybridization conditions include, e.g., hybridization
with a 1000 nucleotide probe of about 40% to about 70% GC content;
at 42.degree. C. in 2.times.SSC and 0.1% SDS. Medium stringency
hybridization conditions include, e.g., at 50.degree. C. in
0.5.times.SSC and 0.1% SDS. High stringency hybridization
conditions include, e.g., hybridization with the above-mentioned
probe at 65.degree. C. in 0.2.times.SSC and 0.1% SDS. Under these
conditions, as the hybridization temperature is elevated, a nucleic
acid with a higher homology can be obtained.
[0050] A number of considerations are useful to the skilled artisan
in determining if a particular amino acid sequence variant of the
polypeptides described herein is suitable for use in the methods
described herein. These considerations include, but are not limited
to: (1) known structure-function relationships such as a DNA
binding domain or a transactivation domain; (2) the presence of
amino acid sequence conservation among naturally occurring homologs
(e.g., in paralogs and orthologs) of the polypeptide, as revealed
by sequence alignment algorithms as described herein. Notably, a
number of bioinformatic algorithms are known in the art that
successfully predict the functional effect, i.e., "tolerance" of
particular amino substitutions in the amino acid sequence of a
protein on its function. Such algorithms include, e.g., pMUT, SIFT,
PolyPhen, and SNPs3D. For a review see, e.g., Ng and Henikoff
(2006), Ann Rev Genomics Hum Genet., 7:61-80. For example, pMUT
predicts with a high degree of accuracy (about 84% overall) whether
a particular amino acid substitution at a given sequence position
affects a protein's function based on sequence homology. See
Ferrer-Costa et al., (2005), Bioinformatics, 21(14):3176-3178;
Ferrer-Costa et al., (2004), Proteins, 57(4):811-819; and
Ferrer-Costa et al., (2002), J Mol Biol, 315:771-786. The PMUT
algorithm server is publicly available on the world wide web at:
//mmb2.pcb.ub.es:8080/PMut/. Thus, for any polypeptide amino acid
sequence, an "amino acid substitution matrix" can be generated that
provides the predicted neutrality or deleteriousness of any given
amino acid substitution on a given protein's function(s).
[0051] Non-naturally occurring sequence variants can be generated
by a number of known methods. Such methods include, but are not
limited to, "Gene Shuffling," as described in U.S. Pat. No.
6,521,453; "RNA mutagenesis," as described in Kopsidas et al.,
(2007), BMC Biotechnology, 7:18-29; and "error-prone PCR methods."
Error prone PCR methods can be divided into (a) methods that reduce
the fidelity of the polymerase by unbalancing nucleotides
concentrations and/or adding of chemical compounds such as
manganese chloride (see, e.g., Lin-Goerke et al., (1997),
Biotechniques, 23:409-412), (b) methods that employ nucleotide
analogs (see, e.g., U.S. Pat. No. 6,153,745), (c) methods that
utilize `mutagenic` polymerases (see, e.g., Cline, J. and Hogrefe,
H. H. (2000), Strategies(Stratagene Newsletter), 13:157-161 and (d)
combined methods (see, e.g., Xu et al., (1999), Biotechniques,
27:1102-1108. Other PCR-based mutagenesis methods include those,
e.g., described by Osuna et al., (2004), Nucleic Acids Res.,
32(17):e136 and Wong et al., (2004), Nucleic Acids Res., 10;
32(3):e26), and others known in the art.
Introduction of Nucleic Acid Expression Vectors into NBCs
[0052] In some embodiments of the reprogramming methods described
herein, any of the above-described nucleic acid expression vector
combinations are introduced into NBCs by transfection only within a
single period no greater than about 40 hours, e.g., no greater than
36 hours, 34 hours, 33 hours, 30 hours, 28 hours, 26 hours, 24
hours, 22 hours, 18 hours, 16 hours, or another period no greater
than about 40 hours. In some embodiments, transfection of the NBCs
with the one or more DNA expression vectors encoding the
reprogramming factors (e.g., Oct4, Sox2, Klf4, and c-Myc) are
introduced by transfection into the NBCs only once. In some
embodiments, where the one transfection is done by electroporation
(e.g., by nucleoporation) or any other method in the art that
rapidly introduces nucleic acids into cells (e.g., biolistics, or
laser-pulse-mediated transfection), the electroporation may include
multiple electroporation pulses within a period less than about 30
minutes, but this will be understood herein to be one transfection
only.
[0053] Nucleic acid expression vectors, e.g., DNA expression
vectors encoding reprogramming factors can be introduced into NBCs
or NBC subpopulations by a variety of methods known in the art.
Examples of high efficiency transfection efficiency methods include
capillary electroporation, as described in Kim et al (2008),
Biosensors and Bioelectronics, 23:1353-1360, and in PCT Patent
Application Publication No. WO2009129327, which is commercially
available under the trade name Neon.TM. (Invitrogen, Carlsbad,
Calif.); "nucleofection," as described in, e.g., Trompeter (2003),
J Immunol. Methods, 274(1-2):245-256, and in international patent
application publications WO2002086134, WO200200871, and
WO2002086129, transfection with lipid-based transfection reagents
such as Fugene.RTM. 6 and Fugene.RTM. HD (Roche), DOTAP, and
Lipofectamine.TM. LTX in combination with the PLUS (Invitrogen,
Carlsbad, Calif.), Dreamfect.TM. (OZ Biosciences, Marseille,
France), GeneJuice.TM. (Novagen, Madison, Wis.), polyethylenimine
(see, e.g., Lungwitz et al., (2005), Eur. J Pharm. Biopharm.,
60(2):247-266), and GeneJammer.TM. (Stratagene, La Jolla, Calif.),
and nanoparticle transfection reagents as described in, e.g., U.S.
patent application Ser. No. 11/195,066. Methods for preparation of
transfection-grade nucleic acid expression vectors and transfection
methods are well established. See, e.g., Sambrook and Russell
(2001), "Molecular Cloning: A Laboratory Manual," 3.sup.rd ed,
(CSHL Press); and Current Protocols in Molecular Biology, John
Wiley & Sons, N.Y. (2005), 9.1-9.14.
[0054] In some cases, in the transfection step for the
reprogramming methods described herein, a suitable ratio of nucleic
acid vector mass (in culture solution) to cells (e.g., NBCs) for
each nucleic acid expression vector to be introduced ranges from
about 0.1 .mu.g/10.sup.5 cells to about 3.0 .mu.g/10.sup.5 cells,
e.g., 0.20 .mu.g/10.sup.5 cells, 0.5 .mu.g/10.sup.5 cells, 0.75
.mu.g/10.sup.5 cells, 1.0 .mu.g/10.sup.5 cells, 1.5 .mu.g/10.sup.5
cells, 1.75 .mu.g/10.sup.5 cells, 2.0 .mu.g/10.sup.5 cells, 2.3
.mu.g/10.sup.5 cells, 2.7 .mu.g/10.sup.5 cells, or another vector
mass to cell ratio from about 0.1 .mu.g/10.sup.5 cells to about 3.0
.mu.g/10.sup.5 cells. In some embodiments, a suitable vector copy
number to cell ratio ranges from about 70,000 copies (in culture
solution)/cell to about 2.times.10.sup.6 copies (in culture
solution)/cell, e.g., 100,000 copies/cell, 200,000 copies/cell,
400,000 copies/cell, 650,000 copies/cell, 800,000 copies/cell,
1.0.times.10.sup.6 copies/cell, 1.2.times.10.sup.6 copies/cell,
1.5.times.10.sup.6 copies/cell, 1.75.times.10.sup.6 copies/cell, or
another vector copy number to cell ratio from about 70,000
copies/cell to about 2.0.times.10.sup.6 copies/cell.
[0055] Where, multiple nucleic acid vectors are to be introduced,
their mass ratios, or their copy number ratios may be varied from
about 1:10 to about 10:1, e.g., 1:8, 1:7, 1:4, 1:3, 1:1, 2:1, 3:1,
4:1, 6:1, 8:1, or another mass or copy number ratio from about 1:10
to about 10:1. For example, in some embodiments, where nucleic acid
vectors encoding Oct4, Sox2, Klf4, and c-Myc are introduced into
the NBCs to be reprogrammed, Oct 4 is introduced at a copy number
stoichiometric ratio of 3:1 relative to the one or more expression
cassettes encoding the other three reprogramming factors. In one
embodiment, the reprogramming method comprises introducing one
nucleic acid expression vector encoding Oct4, and another
containing a polycistronic expression cassette encoding c-Myc-,
Klf4, and Sox2. In some embodiments, the method includes the use of
one DNA expression vector containing a polycistronic vector
encoding, c-Myc, Klf4, Oct2, and Sox2 interconnected by an encoded
2A polypeptide, and under the control of a tet-inducible promoter,
or another inducible promoter known in the art, and another nucleic
acid expression vector encoding the cognate transactivator for the
inducible promoter, e.g., rtTA for a tet-inducibled promoter, and
allowing constitutive expression of the encoded transactivator,
where the nucleic acid expression vectors are provided at a mass
ratio of 1:2.
[0056] In some embodiments, the reprogramming methods include
introducing into NBCs or subpopulations of NBCs by electroporation
one or more DNA expression vectors encoding a combination of
reprogramming factors including any of the following: (a) Oct4,
Sox2, Klf4, and c-Myc; (b) Oct4, Sox2, and Klf4; (c) Oct4, Sox2,
Klf4, c-Myc, and Nanog; or (d) Oct 4, Sox2, Lin-28, and Nanog.
[0057] In one exemplary embodiment, the electroporation method is a
capillary electroporation method, as described in Kim et al (2008),
Biosens Bioelectron, 23(9):1353-1360, U.S. Patent Application
Publication No. 20070275454. In some embodiments, capillary
electroporation is performed on about 50,000 cells to about
1.times.10.sup.6 cells, e.g., 75,000 cells, 100,000 cells, 200,000
cells, 400,000 cells, 500,000 cells, 700,000 cells or another
number of cells from about 50,000 cells to about 1.times.10.sup.6
cells. Typically, the capillary electroporation is performed in a
volume of about 5 .mu.l to about 20 .mu.l of a suitable
transfection buffer containing the nucleic acid expression vectors
to be introduced, e.g., 10 .mu.l, 12 .mu.l, 15 .mu.l, or another
volume of vector-containing transfection buffer from about 5 .mu.l
to about 20 .mu.l. Capillary electroporation parameters that may be
optimized include, pulse voltage, pulse time, and number of pulses.
Pulse voltage may range from about 1500V to 3500V, e.g., about
1600V, 1700V, 1800V, 1850V, 1900V, 1950V, 2000V, 2100V, 2200V,
2500V, 2700V, 3000V, 3200V, or another voltage from about 1500V to
about 3500V. Pulse duration may range from about 10 milliseconds
(ms) to about 40 ms, e.g., about 15 ms, 20 ms, 25 ms, 27 ms, 30 ms,
32 ms, 35 ms, or another pulse duration from about 10 ms to about
40 ms. The number of pulses may be 1, 2, or 3 pulses. In some
embodiments, one or more nucleic acid expression vectors are
introduced into NBCs by capillary electroporation with 1 pulse at
1900V for 30 ms.
Culture of Cells for hiPS Cell Line Derivation Following
Transfection
[0058] After electroporation NBCs are transferred to 96-well ultra
low attachment (ULA) plates in a volume of about 200 .mu.l/well of
a medium suitable for culture of NBCs or subpopulations of NBCs
("hematopoietic culture medium"). After about 24 hours, transfected
NBCs are transferred to 24 well plates in a volume of the same
medium of about 0.5 ml to about 1.5 ml, e.g., about 0.7 ml, 0.8 ml,
1.0 ml, 1.2 ml, 1.3 ml, or another volume of medium from about 0.5
ml to about 1.5 ml. In some embodiments, culture of the transfected
NBCs is continued in hematopoietic culture medium within ULA tissue
culture ware (e.g., 96-well plates) for about two to about six
days, e.g., about three, four, five, or another culture period from
about two to about six days in hematopoietic culture medium. In
some embodiments, where expression of encoded reprogramming factors
is under inducible control of a transactivator (e.g., rtTA), the
inducing agent (e.g., doxycycline) is added after about one to
about three days following transfection to induce expression of the
reprogramming factors in the NBCs. Examples of suitable base media
include, but are not limited to, HSC GEM (Stemgenix; Amherst,
N.Y.)/Stemline (Sigma-Aldrich; St. Louis, Mo.) Hematopoietic Stem
Cell Expansion Medium, X-VIVO.TM. 15 (BioWhittaker; Walkersville,
Md.), HPGM (BioWhittaker; Walkersville, Md.), CellGro.RTM. SCGM
(Cellgenix, Gaithersburg, Md.), QBSF-60 (Quality Biological,
Gaithersburg, Md.), HemaPro.TM. (Celox, St. Paul, Minn.),
StemPro.RTM.-34 (Life Technologies; Grand Island, N.Y.) and
StemSpan H2000.TM./StemSpan SFEM (StemCell Technologies; Vancouver,
British Columbia) StemPro.RTM.-34 medium (Invitrogen). In some
embodiments, the foregoing media contain stem cell factor (SCF),
thrombopoietin (TPO), and granulocyte macrophage colony stimulating
factor (GM-CSF) at 100 ng/ml. In some embodiments, the suitable
medium is Sigma Stemline.RTM. Dendritic Cell Maturation Medium
(Sigma, Catalog #S3444) containing penicillin/streptomycin,
.beta.-mercaptoethanol, SCF at 100 ng/ml, granulocyte macrophage
colony stimulating factor (GM-CSF) at 20 ng/ml (growth factors from
R&D systems), and interleukin 4 (IL-4) at 20 ng/ml.
[0059] Following the period of hematopoietic culture, transfected
NBCs are transferred to 24-well Matrigel.TM.--(or other
extracellular matrix substrate) coated cell culture plates, in a
medium that is composed of about 50% medium suitable for
hematopoietic cell lineages and 50% of a medium suitable for human
embryonic stem (ES) or hiPS cell medium. Subsequently, a complete
hiPS medium change is then carried out about every two to about
four days. Suitable media for hiPS culture, particularly under
feeder cell-free conditions, for the methods described herein
include, but are not limited to, mTeSR.TM. (available, e.g., from
StemCell Technologies, Vancouver, Canada), See, e.g., Ludwig et al,
(2006), Nat Biotechnol., 24(2):185-187. In other cases, alternative
culture conditions for growth of hiPS cells are used, as described
for human ES cells in, e.g., Skottman et al., (2006), Reproduction,
132(5):691-698. Typically, culture medium suitable for maintenance
and passaging of hiPS cells includes fibroblast growth factor
(FGF-2) at a concentration of about 5 ng/ml to about 100 ng/ml. In
some cases, hiPS cells may be cultured under xeno-free conditions,
e.g., in "RegES" medium as described in Rajala et al (2010), PLoS
One, 5(4):e10246. In some embodiments, the transfected NBCs are
plated on mouse embryonic fibroblast (MEF) feeder cells in hES
culture medium.
[0060] In some cases after about 20 days to about 40 days of
maintaining transfected NBCs in hiPS cell medium (e.g., mTeSR.TM.),
e.g., about 21 days, 22 days, 24 days, 26 days, 30 days, 32 days,
34 days, 36 days, or another period from about 20 days to about 40
days, cultures are monitored for the presence of adherent colonies
of hiPSCs, which typically are made up of small cells having a high
nucleus to cytoplasm ratio. Individual colonies are then picked and
transferred individually to new wells for subcloning and
characterization.
[0061] After obtaining and characterizing hiPS cells, a vector
excision step, e.g., expression of a transposase or recombinase, is
not necessary to remove DNA expression vectors that had been
introduced during transfection of human NBCs
[0062] In some embodiments, transfected NBCs are cultured in the
presence of an inhibitor of the TGF-.beta. receptor pathway to
enhance the efficiency of reprogramming as described in
International Patent Application No. PCT/US 10/26451. Examples of
suitable TGF-.beta. receptor pathway inhibitors include, but are
not limited to, TGF-.beta. receptor pathway inhibitors having the
structure of any of Compounds I-IV:
##STR00001##
[0063] Suitable concentrations of the foregoing compounds range
from about 1.0 .mu.M to about 30 .mu.M, e.g., about 2 .mu.M, 5
.mu.M, 10 .mu.M, 12 .mu.M, 15 .mu.M, 20 .mu.M, 25 .mu.M, or another
concentration from about 1.0 .mu.M to about 30 .mu.M. In some
embodiments, transfected NBCs are cultured in the presence of the
TGF-.beta. receptor pathway inhibitor until putative hiPS cell
colonies are identified. In other cases, the transfected NBCs are
cultured in the presence of the TGF-.beta. receptor pathway
inhibitor for a more limited period of time following transfection,
e.g., about 2 days to 30 days, e.g., about 3 days, 4 days, 5 days,
7 days, 10 days, 12 days, 14 days, 16 days, 21 days, 24 days, or
another culture period from about 2 days to 30 days.
[0064] The production of integration-free hiPS cells by the
reprogramming methods described herein does not require the
expression of a recombinase or transposase in putative hiPS cells
to excise one or more DNA expression vectors encoding reprogramming
factors following identification and subcloning of putative hiPS
cell colonies. Examples of recombinases and transposases include,
but are not limited to, Cre-Recombinase, PiggyBac transposase, and
Sleeping Beauty transposase. In some embodiments, the methods
described herein do not include introducing or expressing a
recombinase or transposase in hiPS cells following the
identification of putative hiPS cell colonies as described herein.
Typically, the methods described herein do not include an excision
step to (e.g., by expression of a transposase or recombinase)
remove DNA expression vectors from hiPS cells to generate
integration-free hiPS cells.
[0065] The absence of integrated exogenous nucleic acids within
hiPSCs derived the methods described herein may be determined by
any of a number of standard methods known in the art, which
include, but are not limited to genomic PCR and Southern blot
hybridization to detect the presence of exogenous vector and/or
exogenous transgene sequences within the genome of NBC-derived
hiPSCs.
[0066] Analysis of hiPS Cells
[0067] Methods for identifying hiPS cells and hiPS cell colonies
are known in the art. For example, putative iPS cell colonies may
be tested for alkaline phosphatase (ALP) activity, and if positive,
may then be assayed for expression of a series of human embryonic
stem cell marker (ESCM) genes including, but not limited to, Nanog,
E-Cadherin, DNMT3b, TDGF1, Lin-28, Dnmt3b, Zfp42, FoxD3, GDF3,
CYP26A1, TERT, Oct 3/4, Sox2, Rex1, Sal14, and HPRT. See, e.g.,
Assou et al., (2007), Stem Cells, 25:961-973. Many methods for gene
expression analysis are known in the art. See, e.g., Lorkowski et
al., (2003), Analysing Gene Expression, A Handbook of Methods:
Possibilities and Pitfalls, Wiley-VCH. Examples of suitable nucleic
acid-based gene expression assays include, but are not limited to,
quantitative RT-PCR (qRT-PCR), microarray hybridization, dot
blotting, RNA blotting, RNAse protection, and SAGE.
[0068] In some embodiments, levels of ESCM gene mRNA expression
levels in putative iPS cells colonies are determined by qRT-PCR.
Putative iPS cell colonies are harvested, and total RNA is
extracted using the "Recoverall total nucleic acid isolation kit
for formaldehyde- or paraformaldehyde-fixed, paraffin-embedded
(FFPE) tissues" (manufactured by Ambion, Austin, Tex.). In some
instances, the colonies used for RNA extraction are fixed colonies,
e.g., colonies that have been tested for ALP activity. The colonies
can be used directly for RNA extraction, i.e., without prior
fixation. In an exemplary embodiment, after synthesizing cDNA from
the extracted RNA, the target gene is amplified using the
TaqMan.RTM. PreAmp mastermix (manufactured by Applied Biosystems,
Foster City, Calif.). Real-time quantitative PCR is performed using
an ABI Prism 7900HT using the following PCR primer sets (from
Applied Biosystems) for detecting mRNA of the above-mentioned ESCM
genes: Nanog, Hs02387400_g1, Dnmt3b, Hs00171876_ml, FoxD3,
Hs00255287_s1, Zfp42, Hs01938187_s1, TDGF1, Hs02339499_g1, TERT,
Hs00162669_ml, GDF3, Hs00220998_ml, CYP26A1, Hs00175627_ml, GAPDH,
Hs99999905_ml). Putative hiPS cell colonies may be assayed by an
immunocytochemistry method for expression of protein markers
including, but not limited to, SSEA-3, SSEA-4, TRA-1-60, TRA-1-81,
CD9, CD24, Thy-1, and Nanog. A wide range of immunocytochemistry
assays, e.g., fluorescence immunocytochemistry assays, are known as
described in, e.g., Harlow et al., (1988), Antibodies: A Laboratory
Manual 353-355, Cold Spring Harbor Laboratory, Cold Spring Harbor,
N.Y., and see also, The Handbook--A Guide to Fluorescent Probes and
Labeling Technologies (2004), Molecular Probes, Inc., Eugene, Oreg.
In some cases, immunofluorescence staining is followed by
quantitation of the number of cells immunopositive for one or more
of the above-mentioned ES-cell protein markers. Such quantitative
methods include, but are not limited to flow cytometry and image
cytometry.
[0069] It is generally believed that pluripotent stem cells have
the ability to form a teratoma, comprising ectodermal, mesodermal,
and endodermal tissues, when injected into an immunocompromised
animal. Induced cells or induced pluripotent stem cells (iPS) or ES
cell-like pluripotent stem cells may refer to cells having an in
vitro long-term self-renewal ability and the pluripotency of
differentiating into three germ layers, and said pluripotent stem
cells may form a teratoma when transplanted into a test animal such
as mouse.
[0070] The induced cells may be assessed for pluripotency in a
teratoma formation assay in an immunocompromised animal model. The
immunocompromised animal may be a rodent that is administered an
immunosuppressive agent, e.g., cyclosporin or FK-506. For example,
the immunocompromised animal model may be a SCID mouse. About
0.5.times.10.sup.6 cells to about 2.0.times.10.sup.6 cells e.g.,
0.6.times.10.sup.6 cells, 0.8.times.10.sup.6 cells,
1.0.times.10.sup.6 cells, 1.2.times.times.10.sup.6 cells,
1.5.times.times.10.sup.6 cells, 1.7.times.10.sup.6 cells, or other
number of induced cells from about 0.5.times.10.sup.6 cells to
about 2.0.times.10.sup.6 cells induced cells/mouse may be injected
into the medulla of a testis of a 7- to 8-week-old
immunocompromised animal. After about 6 to about 8 weeks, the
teratomas are excised after perfusing the animal with PBS followed
by 10% buffered formalin. The excised teratomas are then subjected
to immunohistological analysis. One method of distinguishing human
teratoma tissue from host (e.g., rodent) tissue includes
immunostaining for the human-specific nuclear marker HuNu.
Immunohistological analysis includes determining the presence of
ectodermal (e.g., neuroectodermal), mesodermal, and endodermal
tissues. Protein markers for ectodermal tissue include, but are not
limited to, nestin, GFAP, and integrin .beta.1. Protein markers for
mesodermal tissue include, but are not limited to, collagen II,
Brachyury, and osteocalcin. Protein markers for endodermal tissue
include, but are not limited to, alpha-fetoprotein (alpha.-FP) and
HNF3beta.
[0071] In some embodiments, the resulting integration free hiPSCs
have a rearrangement in the immunoglobulin genomic locus (e.g., VJ,
VDJ) or a rearrangement of the T-cell receptor genomic locus (e.g.,
VJ, VDJ). Such genomic rearrangements can be detected by a number
of techniques known in the art, e.g., genomic PCR or Southern blot
hybridization. In some embodiments, the resulting integration free
hiPSCs comprise one or more genomic changes commonly associated
with a B cell or T cell (e.g., junctional diversity, somatic
recombination, somatic hypermutations, etc.).
EXAMPLES
Example 1
Generation of Integration-Free hiPS Cells by Transient Transfection
of Nucleated Blood Cells with Reprogramming Factors Oct4, Sox2,
K14, and c-Myc
[0072] The original objective of this work was to evaluate the
ability of the piggyBac plasmid/transposon reprogramming system (as
described in Woltjen et al (2009), Nature, 458(7239) 766-770) to
reprogram PBMCs. This system, as reported in Woltejen et al (2009),
utilizes piggyBac transposase to initially drive genomic
integration of a transposon expression vector encoding four
reprogramming factors (Klf4, c-Myc, Oct4, and Sox2) and a separate
transposon vector encoding the reverse-tetracycline transactivator
(rtTA) to drive expression in trans of the four reprogramming
factors to induce reprogramming of somatic cells into iPS cells.
Subsequently, the same transposase is transiently expressed to
remove the integrated transposon(s) from the genome to obtain
integration-free hiPS cells, although to date this system has only
been demonstrated successfully in neonatal fibroblasts. The system
described by Woltjen includes three components that are used for
the reprogramming step:
(1) a transposon plasmid containing a Doxycycline-inducible
("tet-inducible") expression cassette encoding the open reading
frames of Klf4, c-Myc, Oct4, and Sox2 linked to each other by the
2A peptide sequence followed by an IRES element and
.beta.-galactosidase (transposon reprogramming vector), which
allows a polycistronic mRNA to be translated to yield the four
separate reprogramming factors and the .beta.-gal reporter protein;
(2) a transposon plasmid containing an expression cassette for
constitutive mammalian expression of the reverse-tetracycline
transactivator (rtTA vector), which, in the presence of doxycycline
drives expression of the four reprogramming factors from the above
transposon expression vector; and (3) a constitutive mammalian
expression plasmid for expression of the piggyBac transposase (PB
vector). Thus, it was expected that transfection of these three
plasmids in adult human NBCs followed by culture in the presence of
doxycycline would yield hiPS cells with an integrated reprogramming
vector. Surprisingly, it was found that while multiple hiPS cell
lines were obtained following transfection of these plasmids and
culture of human NBCs in the presence of doxycyline, none of the
obtained hiPSC lines contained an integrated reprogramming
vector
Isolation of NBCs
[0073] Whole blood was purchased from Zen-bio and shipped at either
ambient or 4.degree. C. overnight. Samples arrived on day 3 after
blood draw. Typically 10 ml per donor were requested. Samples were
then diluted 1:3 in Hanks buffered salt solution (HBSS) and layer
over a 15 ml Ficoll-Hypaque gradient. Samples were spun at
445.times.g for 45 minutes with no brakes at room temperature.
Mononuclear cell layer and granulocytes were then isolated. After
washing the cells in HBSS remaining red blood cells were lysed
using Tris Ammonium Chloride solution at 37.degree. C. for 5
minutes. Remaining cells were washed two more times and
counted.
[0074] In subsequent experiments cells were sorted after Ficoll
density gradients using the MoFlo cell sorter and sorted based on
the cell surface markers CD3, CD19, CD14 and CD15. Or such cells
were isolated from whole blood using the Miltenyi whole blood MACS
kit.
Delivery of Plasmid Vectors
[0075] Prior to electroporation, PBMCs (100,000 to 1,000,000) were
pelleted at 445.times.g and resuspended in 10 .mu.l of transfection
buffer (Neon.TM. transfection system, Invitrogen) containing 0.5
.mu.g PiggyBac transposase (PB) plasmid, 0.5 .mu.g reverse
tetracycline transactivator (rtTA) expression plasmid and 1 .mu.g
of plasmid containing a tet-responsive 2A peptide-linked 4 factor
reprogramming cassette with KlF4, c-Myc, Oct4, and Sox2 flanked by
piggyBac transposition elements (PB-MKOS plasmid). In one case,
however, the PB plasmid was omitted. Electroporation was executed
using the NEON system (Invitrogen) and conditions were 1900 V, 30
msec, single pulse. Immediately after electroporation cells were
transferred to 200 .mu.l of dendritic cell media (Stemline, Sigma),
containing Penicillin/Streptomycin, .beta.-mercaptoethanol (55
.mu.M), 100 ng/ml SCF, 20 ng/ml GM-CSF, 20 ng/ml IL-4 (all from
R&D Systems) in 96-well Ultra-Low Attachment (ULA) plates
(Corning).
Culture
[0076] After 24 hours, the cells were transferred to dendritic cell
media, containing Penicillin/Streptomycin, .beta.-mercaptoethanol
(55 .mu.M), 100 ng/ml SCF, 20 ng/ml GM-CSF, 20 ng/ml IL-4 and 2
.mu.g/ml Doxycycline in 24-well ULA plates (Corning).
[0077] After 4-5 days post-transfection, cells were transferred to
Matriger.TM. coated plates in the original media. Then 1 ml of
mTESR.TM. medium was added. Subsequently, the medium was changed
every 3-4 days by carefully removing the supernatant and replacing
it with fresh mTESR.TM.+2 .mu.g/ml Doxycycline.
Clonal Isolation and Expansion
[0078] Characteristic colonies appeared around day 30 of culture
and were isolated and expanded using standard technique.
Gene Expression Analysis
[0079] hiPS cell total RNA was isolated using Qiagen RNeasy kit
following manufacturer's instructions. RT-qPCR was performed using
ABI reagents and Fluidigm Biomark instrument. Taqman probes used
were Hs00170423_m1, Hs00171876_m1, Hs00220998_m1, Hs00702808_s1,
Hs02387400_g1, Hs00360675_m1, Hs00162669_m1, Hs00399279_m1,
Hs99999905_m1, Hs99999902_m1, Hs01053049_s1. The Oct4 probe was
custom designed. Data were analyzed using Fluidigm Gene Expression
and Spotfire software.
Results
[0080] NBCs were isolated using a Ficoll-Hypaque density gradient
and subsequently we introduced the following 3 plasmids into these
cells using the NEON system: pBASE, a plasmid encoding the piggyBAC
transposase; PB-rtTA, a plasmid encoding the reverse tetracycline
transactivator flanked by the piggyBAC terminal repeats and
PB-MKOS, a plasmid encoding the 4 transcription factors OCT4, KLF4,
SOX2 and CMYC (all of which were mouse orthologs), as well as an
IRES linked .beta.geo cassette conferring neomycin resistance and
lacZ enzymatic activity, again flanked by the piggyBAC terminal
repeats. In one experiment, as a negative control, the PB plasmid
was omitted for the electroporation. After a single pulse delivery
of these plasmids, the cells were allowed to recover at high
density for 24 hours. Typically cell survival was around 50%. The
next day we transferred all cells to a larger volume and added
Doxycycline to induce the expression of the transgenes. After 4
days in hematopoietic media adaptation of the transfected NBCs to
iPSC conditions was by transferring them onto Matrigel.TM. and
added an equal volume of mTESR.TM. iPSC media. After an additional
3-4 days all media was carefully removed and the supernatant was
harvested and centrifuged separately to recover loosely attached
and floating cells. Floating cells were then resuspended in mTESR
with Doxycycline and plated together with the adherent cells. This
procedure was routinely performed twice a week until we saw the
appearance of iPSC colonies at around day 30. Once colonies reached
a suitable size we individually isolated them and plated them on a
fresh plate of Matrigel in mTESR media. Out of 6 colonies from 3
different donors we were able to establish 6 iPSC lines (630.2,
630.6A, 630.6B, 71.67A, 71.67B, 71.89A) from 2 different donors in
two independent experiments. Interestingly one of these lines
(630.2 shown in FIG. 1) was derived from an experimental condition
in which the PiggyBac transposase was not included in the
transfection suggesting initially that either the other two
plasmids integrated into the genome without the aid of a
transposase or the plasmids remained episomal.
hiPS Cell Characterization
[0081] The iPSC lines were analyzed for the cell surface
pluripotency marker expression of TRA-1-60 and SSEA4 by FACS
analysis. Representative data are shown for hiPSC line 630.6B in
FIG. 2. The six hiPSC lines were then characterized for expression
of various pluripotency gene markers and compared to two hiPS cell
lines (IPRN18 and IPRN20), previously established by retroviral
transduction of the same four reprogramming factors, and
fibroblasts. The results of this analysis are shown for five of
these lines in FIG. 3, although the results were similar for hiPS
line not shown in FIG. 3 (630.2). The panel of pluripotency markers
included E-Cadherin ("a"), DNMT3b ("b"), GDF3 ("d"), Lin28 ("e"),
Nanog ("f"), Oct4 ("g"), Sal14 ("i"), Sox2 ("j"), Tert ("k"), Rex1
("l"); control "housekeeping genes" included GAPDH ("c") and RPLPO
("h"). The data were normalized to GAPDH expression and shown
relative to expression level values for iPSC line IPRN18. The bars
represent mean values of duplicate reactions. As shown, the
expression pattern for the pluripotency markers within the five
hiPS cell lines generated with plasmid vectors were quite similar
to that observed in the two control hiPS cell lines, and strikingly
different from the pluripotency marker expression pattern observed
in fibroblasts. These data confirmed the status of these six lines
as hiPS cell lines.
No Exogenous Sequences are Expressed or Detected in the NBC-Derived
hips Cell Lines Generated by Transfection
[0082] Since the tet-inducible expression cassette of the PB-MKOS
vector also encoded .beta.-galactosidase, it was possible, in
principle, to use tet-inducible .beta.-galactosidase expression as
a convenient marker for the presence/absence of the PB-MKOS and
rtTA transposon vectors in the hiPS cell lines before and after
genomic excision of these transposon vectors by transient
expression of PB transposase. Surprisingly, no .beta. galactosidase
activity could be detected following treatment of the hiPS cell
lines with Doxycycline (data not shown). This result suggested that
at least one of the transposon vectors had been lost in all of
these clonal lines, i.e., either the plasmid encoding the rtTA, the
plasmid encoding the MKOS reprogramming cassette, or both. To
confirm this we looked for: (A) the expression of the rtTA by
RT-PCR, and genomic integration of the rtTA transgene by genomic
PCR with two separate primer pairs in each experiment, and (B) the
expression of the MKOS transgene by RT-PCR, and genomic integration
of the MKOS transgene by genomic PCR with two separate primer pairs
in each experiment. As shown for the three lines (630.6B, 71.89 and
71.67) in FIG. 4, we could not detect the rtTA either by RT-PCR
(labeled "cDNA" in each panel) or PCR on genomic DNA (labeled
"gDNA" in each panel), although a control plasmid did amplify (lane
2 in left and right panels). Similarly, as shown in FIG. 5 for the
same three lines, we did not detect the expression of the MKOS
plasmid by RT-PCR (labeled "cDNA" in left panel) or genomic PCR
(labeled "gPCR" in left panel). On the other hand, abundant
expression of the housekeeping gene ACTB was observed (right
panel). We confirmed all of these results for the other three lines
(data not shown), and also confirmed in all six lines that an actin
amplicon could be detected by gPCR (data not shown). Based on these
data, it was concluded that none of the transposon vectors had
integrated into NBCs or the hiPS cell lines derived from the
starting NBCs.
[0083] A preliminary genomic PCR analysis for rearrangement for the
T-cell and B-cell receptor loci (data not shown) indicated that at
least two of the clones, 630.6B and 71.67B, were derived from cells
that had undergone T-cell receptor rearrangement, suggesting that
at least two of the hiPS cell lines were of T-cell origin.
[0084] Based on these data, it was concluded that transient
expression of the reprogramming factors in the transfected NBCs
from the reprogramming plasmid vector during the reprogramming
period was sufficient to induce hiPSCs without requiring genomic
integration of any of the transfected plasmids. It is likely that a
low level/absence of NBC proliferation in culture allows for
persistent extrachromosomal expression of reprogramming factors at
a high level and for a period sufficient to drive reprogramming
Conversely, it may be that cell proliferation is necessary for
transposon integration into the genome. While in the present
experiments, a two plasmid, inducible vector system was used, it is
believed that a one vector constitutive expression plasmid for
expression of the four reprogramming factors would work equally
well if not better. In summary, it is concluded that hiPSCs free of
transgene/vector integration can be readily generated by transient
transfection of one or more nucleic acid expression vectors for
expression of reprogramming factors.
[0085] While preferred embodiments of the present invention have
been shown and described herein, such embodiments are provided by
way of example only. Numerous variations, changes, and
substitutions are possible without departing from the invention. It
should be understood that various alternatives to the embodiments
of the invention described herein may be employed in practicing the
invention. It is intended that the following claims define the
scope of the invention and that methods and structures within the
scope of these claims and their equivalents be covered thereby.
Sequence CWU 1
1
7163DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 1ggcagtggag agggcagagg aagtctgcta
acatgcggtg acgtcgagga gaatcctggc 60cca 63221PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 2Gly
Ser Gly Glu Gly Arg Gly Ser Leu Leu Thr Cys Gly Asp Val Glu1 5 10
15Glu Asn Pro Gly Pro 20375DNAArtificial SequenceDescription of
Artificial Sequence Synthetic oligonucleotide 3ggttctggcg
tgaaacagac tttgaatttt gaccttctca agttggcggg agacgtggag 60tccaacccag
ggccc 75424PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 4Gly Ser Gly Val Lys Gln Thr Asn Phe Asp Leu Leu
Lys Leu Ala Gly1 5 10 15Asp Val Glu Ser Asn Pro Gly Pro
2054971DNAArtificial SequenceDescription of Artificial Sequence
Synthetic polynucleotide 5atgcccctca acgtgaactt caccaacagg
aactatgacc tcgactacga ctccgtacag 60ccctatttca tctgcgacga ggaagagaat
ttctatcacc agcaacagca gagcgagctg 120cagccgcccg cgcccagtga
ggatatctgg aagaaattcg agctgcttcc caccccgccc 180ctgtccccga
gccgccgctc cgggctctgc tctccatcct atgttgcggt cgctacgtcc
240ttctccccaa gggaagacga tgacggcggc ggtggcaact tctccaccgc
cgatcagctg 300gagatgatga ccgagttact tggaggagac atggtgaacc
agagcttcat ctgcgatcct 360gacgacgaga ccttcatcaa gaacatcatc
atccaggact gtatgtggag cggtttctca 420gccgctgcca agctggtctc
ggagaagctg gcctcctacc aggctgcgcg caaagacagc 480accagcctga
gccccgcccg cgggcacagc gtctgctcca cctccagcct gtacctgcag
540gacctcaccg ccgccgcgtc cgagtgcatt gacccctcag tggtctttcc
ctacccgctc 600aacgacagca gctcgcccaa atcctgtacc tcgtccgatt
ccacggcctt ctctccttcc 660tcggactcgc tgctgtcctc cgagtcctcc
ccacgggcca gccctgagcc cctagtgctg 720catgaggaga caccgcccac
caccagcagc gactctgaag aagagcaaga agatgaggaa 780gaaattgatg
tggtgtctgt ggagaagagg caaacccctg ccaagaggtc ggagtcgggc
840tcatctccat cccgaggcca cagcaaacct ccgcacagcc cactggtcct
caagaggtgc 900cacgtctcca ctcaccagca caactacgcc gcacccccct
ccacaaggaa ggactatcca 960gctgccaaga gggccaagtt ggacagtggc
agggtcctga agcagatcag caacaaccgc 1020aagtgctcca gccccaggtc
ctcagacacg gaggaaaacg acaagaggcg gacacacaac 1080gtcttggaac
gtcagaggag gaacgagctg aagcgcagct tttttgccct gcgtgaccag
1140atccctgaat tggaaaacaa cgaaaaggcc cccaaggtag tgatcctcaa
aaaagccacc 1200gcctacatcc tgtccattca agcagacgag cacaagctca
cctctgaaaa ggacttattg 1260aggaaacgac gagaacagtt gaaacacaaa
ctcgaacagc ttcgaaactc tggtgcaggt 1320tctggcgtga aacagacttt
gaattttgac cttctcaagt tggcgggaga cgtggagtcc 1380aacccagggc
ccatggctgt cagcgacgct ctgctcccgt ccttctccac gttcgcgtcc
1440ggcccggcgg gaagggagaa gacactgcgt ccagcaggtg ccccgactaa
ccgttggcgt 1500gaggaactct ctcacatgaa gcgacttccc ccacttcccg
gccgccccta cgacctggcg 1560gcgacggtgg ccacagacct ggagagtggc
ggagctggtg cagcttgcag cagtaacaac 1620ccggccctcc tagcccggag
ggagaccgag gagttcaacg acctcctgga cctagacttt 1680atcctttcca
actcgctaac ccaccaggaa tcggtggccg ccaccgtgac cacctcggcg
1740tcagcttcat cctcgtcttc cccagcgagc agcggccctg ccagcgcgcc
ctccacctgc 1800agcttcagct atccgatccg ggccgggggt gacccgggcg
tggctgccag caacacaggt 1860ggagggctcc tctacagccg agaatctgcg
ccacctccca cggccccctt caacctggcg 1920gacatcaatg acgtgagccc
ctcgggcggc ttcgtggctg agctcctgcg gccggagttg 1980gacccagtat
acattccgcc acagcagcct cagccgccag gtggcgggct gatgggcaag
2040tttgtgctga aggcgtctct gaccacccct ggcagcgagt acagcagccc
ttcggtcatc 2100agtgttagca aaggaagccc agacggcagc caccccgtgg
tagtggcgcc ctacagcggt 2160ggcccgccgc gcatgtgccc caagattaag
caagaggcgg tcccgtcctg cacggtcagc 2220cggtccctag aggcccattt
gagcgctgga ccccagctca gcaacggcca ccggcccaac 2280acacacgact
tccccctggg gcggcagctc cccaccagga ctacccctac actgagtccc
2340gaggaactgc tgaacagcag ggactgtcac cctggcctgc ctcttccccc
aggattccat 2400ccccatccgg ggcccaacta ccctcctttc ctgccagacc
agatgcagtc acaagtcccc 2460tctctccatt atcaagagct catgccaccg
ggttcctgcc tgccagagga gcccaagcca 2520aagaggggaa gaaggtcgtg
gccccggaaa agaacagcca cccacacttg tgactatgca 2580ggctgtggca
aaacctatac caagagttct catctcaagg cacacctgcg aactcacaca
2640ggcgagaaac cttaccactg tgactgggac ggctgtgggt ggaaattcgc
ccgctccgat 2700gaactgacca ggcactaccg caaacacaca gggcaccggc
cctttcagtg ccagaagtgc 2760gacagggcct tttccaggtc ggaccacctt
gccttacaca tgaagaggca ctttggctcc 2820ggagagggca gaggaagtct
gctaacatgc ggtgacgtcg aggagaatcc tggcccactc 2880gagatggctg
gacacctggc ttcagacttc gcctcctcac ccccaccagg tgggggtgat
2940gggtcagcag ggctggagcc gggctgggtg gattctcgaa cctggctaag
cttccaaggg 3000cctccaggtg ggcctggaat cggaccaggc tcagaggtat
tggggatctc cccatgtccg 3060cccgcatacg agttctgcgg agggatggca
tactgtggac ctcaggttgg actgggccta 3120gtcccccaag ttggcgtgga
gactttgcag cctgagggcc aggcaggagc acgagtggaa 3180agcaactcag
agggaacctc ctctgagccc tgtgccgacc gccccaatgc cgtgaagttg
3240gagaaggtgg aaccaactcc cgaggagtcc caggacatga aagccctgca
gaaggagcta 3300gaacagtttg ccaagctgct gaagcagaag aggatcacct
tggggtacac ccaggccgac 3360gtggggctca ccctgggcgt tctctttgga
aaggtgttca gccagaccac catctgtcgc 3420ttcgaggcct tgcagctcag
ccttaagaac atgtgtaagc tgcggcccct gctggagaag 3480tgggtggagg
aagccgacaa caatgagaac cttcaggaga tatgcaaatc ggagaccctg
3540gtgcaggccc ggaagagaaa gcgaactagc attgagaacc gtgtgaggtg
gagtctggag 3600accatgtttc tgaagtgccc gaagccctcc ctacagcaga
tcactcacat cgccaatcag 3660cttgggctag agaaggatgt ggttcgagta
tggttctgta accggcgcca gaagggcaaa 3720agatcaagta ttgagtattc
ccaacgagaa gagtatgagg ctacagggac acctttccca 3780gggggggctg
tatcctttcc tctgccccca ggtccccact ttggcacccc aggctatgga
3840agcccccact tcaccacact ctactcagtc ccttttcctg agggcgaggc
ctttccctct 3900gttcccgtca ctgctctggg ctctcccatg cattcaaacg
ggtcgggtca atgtactaac 3960tacgctttgt tgaaactcgc tggcgatgtt
gaaagtaata accccggtcc tatgtataac 4020atgatggaga cggagctgaa
gccgccgggc ccgcagcaag cttcgggggg cggcggcgga 4080ggaggcaacg
ccacggcggc ggcgaccggc ggcaaccaga agaacagccc ggaccgcgtc
4140aagaggccca tgaacgcctt catggtatgg tcccgggggc agcggcgtaa
gatggcccag 4200gagaacccca agatgcacaa ctcggagatc agcaagcgcc
tgggcgcgga gtggaaactt 4260ttgtccgaga ccgagaagcg gccgttcatc
gacgaggcca agcggctgcg cgctctgcac 4320atgaaggagc acccggatta
taaataccgg ccgcggcgga aaaccaagac gctcatgaag 4380aaggataagt
acacgcttcc cggaggcttg ctggcccccg gcgggaacag catggcgagc
4440ggggttgggg tgggcgccgg cctgggtggc gggctgaacc agcgcatgga
cagctacgcg 4500cacatgaacg gctggagcaa cggcagctac agcatgatgc
aggagcagct gggctacccg 4560cagcacccgg gcctcaacgc tcacggcgcg
gcacagatgc aaccgatgca ccgctacgtc 4620gtcagcgccc tgcagtacaa
ctccatgacc agctcgcaga cctacatgaa cggctcgccc 4680acctacagca
tgtcctactc gcagcagggc acccccggta tggcgctggg ctccatgggc
4740tctgtggtca agtccgaggc cagctccagc ccccccgtgg ttacctcttc
ctcccactcc 4800agggcgccct gccaggccgg ggacctccgg gacatgatca
gcatgtacct ccccggcgcc 4860gaggtgccgg agcccgctgc gcccagtaga
ctgcacatgg cccagcacta ccagagcggc 4920ccggtgcccg gcacggccaa
atacggcaca ctgcccctgt cgcacatgtg a 497161656PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
6Met Pro Leu Asn Val Asn Phe Thr Asn Arg Asn Tyr Asp Leu Asp Tyr1 5
10 15Asp Ser Val Gln Pro Tyr Phe Ile Cys Asp Glu Glu Glu Asn Phe
Tyr 20 25 30His Gln Gln Gln Gln Ser Glu Leu Gln Pro Pro Ala Pro Ser
Glu Asp 35 40 45Ile Trp Lys Lys Phe Glu Leu Leu Pro Thr Pro Pro Leu
Ser Pro Ser 50 55 60Arg Arg Ser Gly Leu Cys Ser Pro Ser Tyr Val Ala
Val Ala Thr Ser65 70 75 80Phe Ser Pro Arg Glu Asp Asp Asp Gly Gly
Gly Gly Asn Phe Ser Thr 85 90 95Ala Asp Gln Leu Glu Met Met Thr Glu
Leu Leu Gly Gly Asp Met Val 100 105 110Asn Gln Ser Phe Ile Cys Asp
Pro Asp Asp Glu Thr Phe Ile Lys Asn 115 120 125Ile Ile Ile Gln Asp
Cys Met Trp Ser Gly Phe Ser Ala Ala Ala Lys 130 135 140Leu Val Ser
Glu Lys Leu Ala Ser Tyr Gln Ala Ala Arg Lys Asp Ser145 150 155
160Thr Ser Leu Ser Pro Ala Arg Gly His Ser Val Cys Ser Thr Ser Ser
165 170 175Leu Tyr Leu Gln Asp Leu Thr Ala Ala Ala Ser Glu Cys Ile
Asp Pro 180 185 190Ser Val Val Phe Pro Tyr Pro Leu Asn Asp Ser Ser
Ser Pro Lys Ser 195 200 205Cys Thr Ser Ser Asp Ser Thr Ala Phe Ser
Pro Ser Ser Asp Ser Leu 210 215 220Leu Ser Ser Glu Ser Ser Pro Arg
Ala Ser Pro Glu Pro Leu Val Leu225 230 235 240His Glu Glu Thr Pro
Pro Thr Thr Ser Ser Asp Ser Glu Glu Glu Gln 245 250 255Glu Asp Glu
Glu Glu Ile Asp Val Val Ser Val Glu Lys Arg Gln Thr 260 265 270Pro
Ala Lys Arg Ser Glu Ser Gly Ser Ser Pro Ser Arg Gly His Ser 275 280
285Lys Pro Pro His Ser Pro Leu Val Leu Lys Arg Cys His Val Ser Thr
290 295 300His Gln His Asn Tyr Ala Ala Pro Pro Ser Thr Arg Lys Asp
Tyr Pro305 310 315 320Ala Ala Lys Arg Ala Lys Leu Asp Ser Gly Arg
Val Leu Lys Gln Ile 325 330 335Ser Asn Asn Arg Lys Cys Ser Ser Pro
Arg Ser Ser Asp Thr Glu Glu 340 345 350Asn Asp Lys Arg Arg Thr His
Asn Val Leu Glu Arg Gln Arg Arg Asn 355 360 365Glu Leu Lys Arg Ser
Phe Phe Ala Leu Arg Asp Gln Ile Pro Glu Leu 370 375 380Glu Asn Asn
Glu Lys Ala Pro Lys Val Val Ile Leu Lys Lys Ala Thr385 390 395
400Ala Tyr Ile Leu Ser Ile Gln Ala Asp Glu His Lys Leu Thr Ser Glu
405 410 415Lys Asp Leu Leu Arg Lys Arg Arg Glu Gln Leu Lys His Lys
Leu Glu 420 425 430Gln Leu Arg Asn Ser Gly Ala Gly Ser Gly Val Lys
Gln Thr Leu Asn 435 440 445Phe Asp Leu Leu Lys Leu Ala Gly Asp Val
Glu Ser Asn Pro Gly Pro 450 455 460Met Ala Val Ser Asp Ala Leu Leu
Pro Ser Phe Ser Thr Phe Ala Ser465 470 475 480Gly Pro Ala Gly Arg
Glu Lys Thr Leu Arg Pro Ala Gly Ala Pro Thr 485 490 495Asn Arg Trp
Arg Glu Glu Leu Ser His Met Lys Arg Leu Pro Pro Leu 500 505 510Pro
Gly Arg Pro Tyr Asp Leu Ala Ala Thr Val Ala Thr Asp Leu Glu 515 520
525Ser Gly Gly Ala Gly Ala Ala Cys Ser Ser Asn Asn Pro Ala Leu Leu
530 535 540Ala Arg Arg Glu Thr Glu Glu Phe Asn Asp Leu Leu Asp Leu
Asp Phe545 550 555 560Ile Leu Ser Asn Ser Leu Thr His Gln Glu Ser
Val Ala Ala Thr Val 565 570 575Thr Thr Ser Ala Ser Ala Ser Ser Ser
Ser Ser Pro Ala Ser Ser Gly 580 585 590Pro Ala Ser Ala Pro Ser Thr
Cys Ser Phe Ser Tyr Pro Ile Arg Ala 595 600 605Gly Gly Asp Pro Gly
Val Ala Ala Ser Asn Thr Gly Gly Gly Leu Leu 610 615 620Tyr Ser Arg
Glu Ser Ala Pro Pro Pro Thr Ala Pro Phe Asn Leu Ala625 630 635
640Asp Ile Asn Asp Val Ser Pro Ser Gly Gly Phe Val Ala Glu Leu Leu
645 650 655Arg Pro Glu Leu Asp Pro Val Tyr Ile Pro Pro Gln Gln Pro
Gln Pro 660 665 670Pro Gly Gly Gly Leu Met Gly Lys Phe Val Leu Lys
Ala Ser Leu Thr 675 680 685Thr Pro Gly Ser Glu Tyr Ser Ser Pro Ser
Val Ile Ser Val Ser Lys 690 695 700Gly Ser Pro Asp Gly Ser His Pro
Val Val Val Ala Pro Tyr Ser Gly705 710 715 720Gly Pro Pro Arg Met
Cys Pro Lys Ile Lys Gln Glu Ala Val Pro Ser 725 730 735Cys Thr Val
Ser Arg Ser Leu Glu Ala His Leu Ser Ala Gly Pro Gln 740 745 750Leu
Ser Asn Gly His Arg Pro Asn Thr His Asp Phe Pro Leu Gly Arg 755 760
765Gln Leu Pro Thr Arg Thr Thr Pro Thr Leu Ser Pro Glu Glu Leu Leu
770 775 780Asn Ser Arg Asp Cys His Pro Gly Leu Pro Leu Pro Pro Gly
Phe His785 790 795 800Pro His Pro Gly Pro Asn Tyr Pro Pro Phe Leu
Pro Asp Gln Met Gln 805 810 815Ser Gln Val Pro Ser Leu His Tyr Gln
Glu Leu Met Pro Pro Gly Ser 820 825 830Cys Leu Pro Glu Glu Pro Lys
Pro Lys Arg Gly Arg Arg Ser Trp Pro 835 840 845Arg Lys Arg Thr Ala
Thr His Thr Cys Asp Tyr Ala Gly Cys Gly Lys 850 855 860Thr Tyr Thr
Lys Ser Ser His Leu Lys Ala His Leu Arg Thr His Thr865 870 875
880Gly Glu Lys Pro Tyr His Cys Asp Trp Asp Gly Cys Gly Trp Lys Phe
885 890 895Ala Arg Ser Asp Glu Leu Thr Arg His Tyr Arg Lys His Thr
Gly His 900 905 910Arg Pro Phe Gln Cys Gln Lys Cys Asp Arg Ala Phe
Ser Arg Ser Asp 915 920 925His Leu Ala Leu His Met Lys Arg His Phe
Gly Ser Gly Glu Gly Arg 930 935 940Gly Ser Leu Leu Thr Cys Gly Asp
Val Glu Glu Asn Pro Gly Pro Leu945 950 955 960Glu Met Ala Gly His
Leu Ala Ser Asp Phe Ala Ser Ser Pro Pro Pro 965 970 975Gly Gly Gly
Asp Gly Ser Ala Gly Leu Glu Pro Gly Trp Val Asp Ser 980 985 990Arg
Thr Trp Leu Ser Phe Gln Gly Pro Pro Gly Gly Pro Gly Ile Gly 995
1000 1005Pro Gly Ser Glu Val Leu Gly Ile Ser Pro Cys Pro Pro Ala
Tyr 1010 1015 1020Glu Phe Cys Gly Gly Met Ala Tyr Cys Gly Pro Gln
Val Gly Leu 1025 1030 1035Gly Leu Val Pro Gln Val Gly Val Glu Thr
Leu Gln Pro Glu Gly 1040 1045 1050Gln Ala Gly Ala Arg Val Glu Ser
Asn Ser Glu Gly Thr Ser Ser 1055 1060 1065Glu Pro Cys Ala Asp Arg
Pro Asn Ala Val Lys Leu Glu Lys Val 1070 1075 1080Glu Pro Thr Pro
Glu Glu Ser Gln Asp Met Lys Ala Leu Gln Lys 1085 1090 1095Glu Leu
Glu Gln Phe Ala Lys Leu Leu Lys Gln Lys Arg Ile Thr 1100 1105
1110Leu Gly Tyr Thr Gln Ala Asp Val Gly Leu Thr Leu Gly Val Leu
1115 1120 1125Phe Gly Lys Val Phe Ser Gln Thr Thr Ile Cys Arg Phe
Glu Ala 1130 1135 1140Leu Gln Leu Ser Leu Lys Asn Met Cys Lys Leu
Arg Pro Leu Leu 1145 1150 1155Glu Lys Trp Val Glu Glu Ala Asp Asn
Asn Glu Asn Leu Gln Glu 1160 1165 1170Ile Cys Lys Ser Glu Thr Leu
Val Gln Ala Arg Lys Arg Lys Arg 1175 1180 1185Thr Ser Ile Glu Asn
Arg Val Arg Trp Ser Leu Glu Thr Met Phe 1190 1195 1200Leu Lys Cys
Pro Lys Pro Ser Leu Gln Gln Ile Thr His Ile Ala 1205 1210 1215Asn
Gln Leu Gly Leu Glu Lys Asp Val Val Arg Val Trp Phe Cys 1220 1225
1230Asn Arg Arg Gln Lys Gly Lys Arg Ser Ser Ile Glu Tyr Ser Gln
1235 1240 1245Arg Glu Glu Tyr Glu Ala Thr Gly Thr Pro Phe Pro Gly
Gly Ala 1250 1255 1260Val Ser Phe Pro Leu Pro Pro Gly Pro His Phe
Gly Thr Pro Gly 1265 1270 1275Tyr Gly Ser Pro His Phe Thr Thr Leu
Tyr Ser Val Pro Phe Pro 1280 1285 1290Glu Gly Glu Ala Phe Pro Ser
Val Pro Val Thr Ala Leu Gly Ser 1295 1300 1305Pro Met His Ser Asn
Gly Ser Gly Gln Cys Thr Asn Tyr Ala Leu 1310 1315 1320Leu Lys Leu
Ala Gly Asp Val Glu Ser Asn Asn Pro Gly Pro Met 1325 1330 1335Tyr
Asn Met Met Glu Thr Glu Leu Lys Pro Pro Gly Pro Gln Gln 1340 1345
1350Ala Ser Gly Gly Gly Gly Gly Gly Gly Asn Ala Thr Ala Ala Ala
1355 1360 1365Thr Gly Gly Asn Gln Lys Asn Ser Pro Asp Arg Val Lys
Arg Pro 1370 1375 1380Met Asn Ala Phe Met Val Trp Ser Arg Gly Gln
Arg Arg Lys Met 1385 1390 1395Ala Gln Glu Asn Pro Lys Met His Asn
Ser Glu Ile Ser Lys Arg 1400 1405 1410Leu Gly Ala Glu Trp Lys Leu
Leu Ser Glu Thr Glu Lys Arg Pro 1415 1420 1425Phe Ile Asp Glu Ala
Lys Arg Leu Arg Ala Leu His Met Lys Glu 1430 1435 1440His Pro Asp
Tyr Lys Tyr Arg Pro Arg Arg Lys Thr Lys Thr Leu 1445 1450 1455Met
Lys Lys Asp Lys Tyr Thr Leu Pro Gly Gly Leu Leu Ala Pro 1460 1465
1470Gly Gly Asn Ser Met Ala Ser Gly Val Gly Val Gly Ala Gly Leu
1475 1480
1485Gly Gly Gly Leu Asn Gln Arg Met Asp Ser Tyr Ala His Met Asn
1490 1495 1500Gly Trp Ser Asn Gly Ser Tyr Ser Met Met Gln Glu Gln
Leu Gly 1505 1510 1515Tyr Pro Gln His Pro Gly Leu Asn Ala His Gly
Ala Ala Gln Met 1520 1525 1530Gln Pro Met His Arg Tyr Val Val Ser
Ala Leu Gln Tyr Asn Ser 1535 1540 1545Met Thr Ser Ser Gln Thr Tyr
Met Asn Gly Ser Pro Thr Tyr Ser 1550 1555 1560Met Ser Tyr Ser Gln
Gln Gly Thr Pro Gly Met Ala Leu Gly Ser 1565 1570 1575Met Gly Ser
Val Val Lys Ser Glu Ala Ser Ser Ser Pro Pro Val 1580 1585 1590Val
Thr Ser Ser Ser His Ser Arg Ala Pro Cys Gln Ala Gly Asp 1595 1600
1605Leu Arg Asp Met Ile Ser Met Tyr Leu Pro Gly Ala Glu Val Pro
1610 1615 1620Glu Pro Ala Ala Pro Ser Arg Leu His Met Ala Gln His
Tyr Gln 1625 1630 1635Ser Gly Pro Val Pro Gly Thr Ala Lys Tyr Gly
Thr Leu Pro Leu 1640 1645 1650Ser His Met 165578PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 7Asp
Xaa Glu Xaa Asn Pro Gly Pro1 5
* * * * *