U.S. patent application number 13/857449 was filed with the patent office on 2013-12-12 for pluripotent cells from rat and other species.
This patent application is currently assigned to The University Court of The University of Edinburgh. The applicant listed for this patent is Austin Gerard Smith, Qi-Long Ying. Invention is credited to Austin Gerard Smith, Qi-Long Ying.
Application Number | 20130333058 13/857449 |
Document ID | / |
Family ID | 37809760 |
Filed Date | 2013-12-12 |
United States Patent
Application |
20130333058 |
Kind Code |
A1 |
Ying; Qi-Long ; et
al. |
December 12, 2013 |
Pluripotent Cells From Rat and Other Species
Abstract
Pluripotent cells are derived and maintained in a self-renewing
state in serum-free culture medium comprising a MEK inhibitor, a
GSK3 inhibitor and an antagonist of an FGF receptor.
Inventors: |
Ying; Qi-Long; (Los Angeles,
CA) ; Smith; Austin Gerard; (Cambridge, GB) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Ying; Qi-Long
Smith; Austin Gerard |
Los Angeles
Cambridge |
CA |
US
GB |
|
|
Assignee: |
The University Court of The
University of Edinburgh
Edinburgh
GB
|
Family ID: |
37809760 |
Appl. No.: |
13/857449 |
Filed: |
April 5, 2013 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
11832317 |
Aug 1, 2007 |
8431395 |
|
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13857449 |
|
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Current U.S.
Class: |
800/9 ;
800/14 |
Current CPC
Class: |
C12N 2501/115 20130101;
C12N 2501/16 20130101; A01K 67/0275 20130101; A01K 2217/00
20130101; A01K 2227/105 20130101; C12N 2501/70 20130101; A61K
49/0008 20130101; A01K 2227/103 20130101; A01K 2227/108 20130101;
C12N 5/0606 20130101; A01K 2227/101 20130101; C12N 2501/235
20130101 |
Class at
Publication: |
800/9 ;
800/14 |
International
Class: |
A61K 49/00 20060101
A61K049/00 |
Foreign Application Data
Date |
Code |
Application Number |
Aug 1, 2006 |
GB |
0615327.4 |
Jan 10, 2007 |
GB |
0700479.9 |
Claims
1-160. (canceled)
161. A genetically engineered rat obtained by a method comprising
genetically modifying a rat pluripotent cell expressing two or more
of Nanog, Oct4, FGF4, and Sox-2; and introducing the pluripotent
cell into a rat embryo to produce a genetically modified rat,
wherein the rat pluripotent cell contributes to the germline of the
genetically modified rat.
162. The genetically engineered rat of claim 161, wherein the rat
pluripotent cell expresses Nanog, Oct4, and Sox-2.
163. The genetically engineered rat of claim 161, wherein the rat
pluripotent cell further expresses alkaline phosphatase.
164. The genetically engineered rat of claim 161, wherein the rat
pluripotent cell expresses Rex1, Stella, FGF4, and Sox-2.
165. The genetically engineered rat of claim 161, wherein the rat
pluripotent cell does not express FGF5.
166. The genetically engineered rat of claim 161, wherein the rat
pluripotent cell is capable of forming a teratoma or
teratocarcinoma in which differentiated cells from all three germ
layers are present.
167. The genetically engineered rat of claim 161, wherein the rat
pluripotent cell is capable of growth and/or proliferation as a
single cell in culture.
168. The genetically engineered rat of claim 161, wherein the
pluripotent rat cell is derived from a blastocyst by a method
comprising: culturing the blastocyst in the presence of a MEK
inhibitor and a GSK3 inhibitor, to obtain an inner cell mass;
isolating and dissociating the primary outgrowths of the inner cell
mass; isolating a cell or cells from the dissociated primary
outgrowths of the inner cell mass; and culturing the isolated cell
or cells in the presence of a MEK inhibitor, a GSK3 inhibitor, and
an antagonist of an FGF receptor.
169. The genetically engineered rat of claim 168, wherein the
pluripotent rat cell expresses one or more of Nanog, Oct4, FGF4,
Sox-2, and alkaline phosphatase.
170. A genetically engineered rat obtained as the progeny of the
genetically engineered rat of claim 161.
171. The genetically engineered rat of claim 161, wherein the
genetically engineered rat is homozygous null for a gene of
interest.
172. The genetically engineered rat of claim 161, wherein the
genetically modifying comprises replacing a gene in the rat with a
corresponding human gene.
173. The genetically engineered rat of claim 161, wherein the
genetically modifying comprises: knocking out one or more genes of
interest; or targeted insertion of a gene of interest.
174. The genetically engineered rat of claim 161 for use in drug
discovery and/or testing.
175. The genetically engineered rat of claim 161 for use as a model
for human or animal disease.
176. The genetically engineered rat of claim 175 for use in
assessments of behavioural recovery or cognitive repair.
Description
[0001] This application claims the benefit under 35 U.S.C.
.sctn.119 of Great Britain Application No. 0615327.4, filed Aug. 1,
2006, and Great Britain Application No. 0700479.9, filed Jan. 10,
2007, all of which are incorporated herein by reference.
[0002] The present invention relates to maintenance of a self
renewing phenotype in pluripotent stem cells. The methods and
compositions provided are suitable for culturing and isolating
pluripotent stem cells such as embryonic stem (ES) cells,
especially mammalian, including rat, mouse, bovine, ovine, porcine
and human, stem cells. In particular this invention relates to
self-renewing cultures of rat, mouse and human pluripotent cells
and to methods and compositions therefor.
[0003] The establishment and maintenance of in vitro pluripotent
stem cell cultures in the presence of medium containing serum and
Leukaemia Inhibitory Factor (LIF) is well known (Smith et al.
(1988) Nature 336: 688-90). Such methods have been used to maintain
pluripotent embryonic stem (ES) cells from "permissive" strains of
mice over many passages. Maintenance and self renewal of
pluripotent stem cell cultures is further supported where the stem
cells are cultured in the presence of feeder cells or extracts
thereof, usually mouse fibroblast cells. Under such conditions it
is possible to maintain human ES cells in a pluripotent state over
many passages in culture.
[0004] In many cases ES cells can only be maintained, or are best
maintained, using medium that contains serum or serum extract, and
hence is undefined, or using cell culture conditions that require
the presence of other cells, such as the fibroblast feeder cells
used to maintain human ES cells. But any undefined component,
whether in the medium or produced by e.g. the feeder cells,
potentially interferes with or hinders research into ES cell
propagation and differentiation. This prevents development of good
manufacturing practices for therapeutic and other applications of
ES cells and their progeny. Some defined ES cell media are known
but alternative and preferably improved defined media are
needed.
[0005] In prior applications by the applicants, WO-A-03/095628 and
a later as yet unpublished application, culturing pluripotent stem
cells, such as ES cells, in serum-free media comprising (1)
agonists of gp130 (e.g. LIF) and (2) agonists of the TGF-.beta.
superfamily (e.g. BMP4) or Id signalling pathways is used to
promote self renewal of the stem cells for multiple passages. In
the presence of gp130 signalling, an agonist of the TGF-.beta.
superfamily or the Id signalling pathway surprisingly provided a
self renewal stimulus rather than a pro-differentiation signal.
Nevertheless, ever improved efficiencies in maintaining pluripotent
cells in a self renewing state and media for transferring
pluripotent cells away from feeder cells or away from
feeder-conditioned medium is desired.
[0006] Sato N, et al, Nat. Med. 2004, January 10(1) pp 55-63
describe the effects of a Glycogen Synthase Kinase 3 (GSK3)
inhibitor, 6-bromoindirubin-3'-oxime, on mouse and human ES cells
in serum containing medium. These effects, however, were observed
only over a very short time frame, too short for firm conclusions
to be drawn, and the influence of unknown factors in the undefined
media used in that study may be significant. The inventors of the
present invention have tried but failed to repeat the results, and
have in fact found effects opposite to those described.
[0007] For preparation of ES cell culture media it is desired to
provide individual media components in as pure a form as possible.
However, most media components are cytokines the purity of which is
compromised by the need to manufacture them in cellular systems and
then remove potential contaminants from the production broth.
Another problem with some cytokines is that they have a narrow
range of concentration over which they are effective and non-toxic.
Media components which have a broader range and/or are less toxic
at higher concentrations would be highly useful. Cytokines can also
have limited stability in storage, and more stable media components
are sought.
[0008] An object of the invention is to overcome or at least
ameliorate problems in the art, preferably to provide alternative,
more preferably improved, methods of culturing and culture media
suitable for pluripotent stem cells, which are capable of
supporting self-renewal of said stem cells for many passages. A
further object of the invention is to provide an alternative
culturing system that permits maintenance of a pluripotent stem
cell culture in vitro until differentiation of the cells can be
induced in a controlled manner. A still further object of the
invention is to provide methods and compositions that enhance the
derivation and isolation of pluripotent stem cells and facilitate
their derivation and isolation from organisms refractory to ES cell
isolation or from which pluripotent stem cells have not yet been
isolated. A further, related, object of the invention is to provide
pluripotent stem cells from such organisms.
BRIEF DESCRIPTION OF THE DRAWINGS
[0009] FIGS. 1A-1B show mouse ES cells derived and maintained
according to the invention and shows high efficiency of chimera
contribution by these ES cells.
[0010] FIG. 2 shows passage 4 mouse ES cells grown in accordance
with the invention.
[0011] FIG. 3 shows that mouse ES cells grown in accordance with
the invention, are Oct4 positive.
[0012] FIG. 4 shows a colony of rat ES cells isolated in accordance
with the invention.
[0013] FIG. 5 shows that rat ES cells isolated in accordance with
the invention express Nanog.
[0014] FIG. 6 shows that rat ES cells isolated in accordance with
the invention express Oct4.
[0015] FIG. 7 shows that rat ES cells isolated in accordance with
the invention express Cdx-2.
[0016] FIG. 8 shows that rat ES cells isolated in accordance with
the invention are alkaline phosphatase positive.
[0017] FIG. 9 shows RT-PCR expression data in four rat ES cell
lines isolated in accordance with the invention.
[0018] FIG. 10 shows sections of mature differentiated tissue in a
teratoma generated from rat ES cells isolated in accordance with
the invention.
[0019] FIGS. 11A-D show rat ICM outgrowths and primary colonies
retain expression of Oct4 and Nanog in 3i. Figure A shows
immunofluorescence stainings of ICMs for Oct4 and Cdx2 after 3 days
in culture in the presence of serum and LIF, or in 3i. Figure aaB
shows quantification of Oct4 and Cdx2 immunopositive cells in
cultured rat ICMs. FIG. 11C shows Oct4 and Nanog immunostaining of
ICMs after 4 days culture in 3i. FIG. 11D shows primary colony in
3i 4 days after disaggregation of ICM outgrowth.
[0020] FIGS. 12A-12E shows the morphology, clonal expansion, and
chromosome complement of rat embryo derived cells propagated in 3i.
FIG. 12A shows representative colony of cells passaged on 3i on
feeders. FIG. 12B shows cells passaged without feeders on
fibronectin in 3i. FIG. 12C shows immunofluorescent staining of 3i
colonies for Oct4 and Nanog. FIG. 12D shows RT-PCR analysis of
marker expression in rat embryo derived 3i cells compared with
E14Tg2a mouse ES cells (mES), rat ExS cells (rExS) and rat ExS
cells expressing mouse Oct and mouse Nanog (rExS+mO/mN) transgenes.
FIG. 12E shows metaphase chromosomes from 3i rat ES cells (line
C).
[0021] FIGS. 13A-13C show the differentiation of rat 3i cells in
vitro. FIG. 13A shows phase contrast and immunofluorescence images
of cells following exposure to serum plus LIF on feeders. FIG. 13B
shows differentiated cells after 8 days culture on gelatine in
serum-free medium without 3i. FIG. 13C shows characterization of
the X-chromosome status in XX rat ES cells. Immunofluorescence for
H3K27me3 (green) in undifferentiated and 6 days differentiated XX
rat 3i cells and in XX rat neural stem (NS) cells. Undifferentiated
cells show diffuse staining (lack of inactive X) while
differentiated cells and somatic stem cells exhibit the presence of
a H3K27me3 nuclear body (inactive X). Cells were counterstained
with Dapi (blue).
[0022] FIGS. 14A-14D show the formation of teratomas and
contribution to chimaeras. FIG. 14A shows histological sections of
teratomas. Keratinised epithelium (line C), striated muscle (line
B), gut epithelium (line C). FIG. 14B shows images of intact embryo
and head to tail transverse sections (i-x) of E10.5 chimaera
generated using GFP expressing line D 3i cells. GFP fluorescence in
green, DAPI nuclear staining in blue. FIG. 14C shows coat colour
chimaeras derived from cells of line C. Albino snouts denote the
recipient Fischer strain (hooded albino), body pigmentation the
introduced DA strain cells. FIG. 14D shows microsatellite analyses
of genomic DNA in adult coat colour chimaeras. Polymorphic
microsatellite regions D1Rat122 and D3Rat17 were amplified by PCR
from genomic DNA of tail, ear and blood and analysed by agarose gel
and fluorescent detection methods.
[0023] FIGS. 15A-F show the in vitro undifferentiated and
differentiated states of a representative ovine ES cell line (7a,
passage 5 or greater).
[0024] FIG. 16 shows RT-PCR data for a representative ovine ES cell
line in the undifferentiated state (7a, passage 5).
[0025] FIG. 17 shows a comparison of the percentage of bovine
embryos that formed embryonal outgrowths and were taken to passage
1 among embryos cultured with media 1-4. % outgrowths:
.chi..sup.2.sub.3=12.49, p=0.0059; % passaged
.chi..sup.2.sub.3=9.96, p=0.0189. a There is a positive interaction
between the observation (i.e. % outgrowths, % passaged) and the
treatment. b There is a negative association between the
observation and the treatment.
[0026] FIG. 18 shows a comparison of the percentage of embryos that
formed embryonic stem cell lines by Passage 3 (P.sub.3) or Passage
6 (P.sub.6) among embryos cultured with media 1-4. P.sub.3:
.chi..sup.2.sub.3=12.02, p=0.0073; P.sub.6:
.chi..sup.2.sub.3=13.81, p=0.0032. a There is a positive
association between the observation (i.e. % cell lines at P.sub.3
or P.sub.6) and the treatment. b There is a negative association
between the observation and the treatment.
[0027] FIG. 19 shows a comparison of colony expansion at Passage 0
of bovine embryonic stem cell colonies from embryos cultured with
media 1-4.
[0028] FIG. 20 shows a comparison of colony expansion during the
first passage (i.e. P.sub.23+1) of an established bovine ES cell
line, BES-4, cultured with media 1-4.
[0029] FIGS. 21A-21H show a comparison of the morphology of bovine
embryonic stern cell colonies cultured with (a, b) control medium,
(c, d) medium 2, (e, f) medium 3 of (g, h) medium 4 for (a, c, e,
g) 37 days or (b, d, f, h) 57 days. Cultures were at passages 4, 5
or 6.
[0030] FIGS. 22A-22D show a comparison of the morphology of bovine
embryonic stem cell colonies from an established bovine ES cell
line, BES-1, cultured with (a) control medium, (b) medium 2, (c)
medium 3 or (d) medium 4 for 10 days. Cultures were at P.sub.19+1.
Scale bar=2 mm.
[0031] FIGS. 23A-23D show a comparison of the morphology of bovine
embryonic stem cells from colonies isolated with (a) control
medium, (b) medium 2, (c) medium 3 or (d) medium 4. Cultures were
at P.sub.5 or P.sub.7.
[0032] FIGS. 24A-24D show a comparison of the morphology of bovine
embryonic stem cells from an established bovine ES cell line,
BES-1, cultured with (a) control medium, (b) medium 2, (c) medium 3
or (d) medium 4. Cultures were at P.sub.18+1.
[0033] FIG. 25 shows a comparison of gene expression profiles of
pluripotency during culture for ES cells from ES cell lines
isolated in media 1-4. Control (medium 1) line at P.sub.1, P.sub.3,
P.sub.7; medium 2 line at P.sub.1, P.sub.3, P.sub.4; medium 3 line
at P.sub.2, P.sub.3, P.sub.6; medium 4 line at P.sub.1, P.sub.3,
P.sub.6. Gene order for all gel figures, from Left to Right:
.beta.-actin, Oct4, Rex1, Sox2, SSEA1, Alkaline Phosphatase and
Nanog.
[0034] FIGS. 26A-26B show a comparison of the gene expression
profiles of pluripotency for bovine ES cells from ES cell lines
isolated in media 1-4, demonstrating some of the variation at (a)
Passage 1 and (b) later passages, where control examples are from
P.sub.4 and P.sub.7, medium 2 examples are from P.sub.4 and
P.sub.6, medium 3 examples are from P.sub.5 and P.sub.6 and medium
4 examples are from P.sub.4 and P.sub.6. Gene order for all gel
figures, from Left to Right: .beta.-actin, Oct4, Rex1, Sox2, SSEA1,
Alkaline Phosphatase and Nanog.
[0035] FIG. 27 shows a comparison of gene expression profiles of
pluripotency for bovine ES cells from an established ES cell line,
BES-4, cultured with media 1-4 for 1, 4 or 5 passages. Gene order
for all gel figures, from Left to Right: .beta.-actin, Oct4, Rex1,
Sox2, SSEA1, Alkaline Phosphatase and Nanog.
[0036] FIGS. 28A-28D show a comparison of the responses of explants
from an established bovine ES cell line, when placed in conditions
that promote in vitro differentiation. (a) Embryoid body formed
from explants cultured with medium 2. (b) Embryoid body formed from
explants cultured with medium 3. (c) and (d) Cells from explants
cultured with medium 4 had attached to the culture vessel and begun
to differentiate.
[0037] According to a first aspect of the present invention,
inhibition of all of GSK3 and MEK and a FGF receptor in a
pluripotent cell is used to promote self-renewal of the cell.
[0038] In accordance with the present invention, pluripotent stem
cells, such as ES cells, are cultured in medium, preferably
serum-free, comprising a MEK inhibitor, a GSK3 inhibitor and an
antagonist of a FGF receptor (e.g. a small molecule GSK3 inhibitor
and a small molecule MEK inhibitor and a small molecule FGFR
antagonist). Self renewal of the stem cells for multiple passages
is thereby promoted. Hence, inhibition of GSK3, MEK and FGF
receptor signalling in the pluripotent cells provides a self
renewal stimulus.
[0039] The invention has a number of applications. A combination of
GSK3 and MEK, MEK and FGFR or GSK3, MEK and FGFR inhibition can be
used to grow pluripotent cells, especially ES cells, and, where
they have been derived or grown on feeders, to adapt pluripotent
cells, especially ES cells, to grow without feeder cells or a layer
of feeder cells, often referred to as feeders or feeder cells. A
method of expanding stem cells in culture comprises culturing the
cells in the presence of a GSK3 inhibitor and a MEK inhibitor, in
the presence of a MEK inhibitor and an antagonist of an FGF
receptor, or preferably in the presence of a GSK3 inhibitor, a MEK
inhibitor and an antagonist of a FGF receptor. Culture medium can
be prepared containing one or more GSK3 inhibitors and MEK
inhibitors, one or more MEK inhibitors and FGFR antagonists and,
optionally, one or more MEK inhibitors, GSK3 inhibitors and FGFR
antagonists. ES cells can be derived using GSK3 inhibitors and MEK
inhibitors, using MEK inhibitors and FGFR antagonists, or using
GSK3 inhibitors, MEK inhibitors and FGFR antagonists, including ES
cells from organisms from which ES cells have not hitherto been
isolated.
DETAILED DESCRIPTION OF THE INVENTION
[0040] Reference to pluripotent cells includes but is not limited
to reference to embryonic stem (ES) cells. Characteristic
properties of pluripotent cells, including ES cells, include the
expression of multiple genes associated with the pluripotent stage
of development, the ability to differentiate into cells
representative of any and all tissue types present in the source
animal, the ability to contribute to chimeras and, particularly,
the ability to contribute to the germ line of chimeras. For example
true pluripotent cells, such as ES cells, would be expected to
express many, if not all, of the pluripotency-associated genes
Nanog, Oct4, FGF4, Sox-2 and alkaline phosphatase. In particular,
expression of Nanog, Oct4 and Sox-2 is widely regarded as providing
a definitive initial indication that a cell is an ES cell. Germ
line transmission in chimeras and the ability to generate teratomas
or teratocarcinomas comprising differentiated cells from all three
primary germ layers (i.e. endoderm, mesoderm and ectoderm) are also
widely regarded as definitive indications of a cell being an ES
cell.
[0041] Reference to GSK3 inhibition refers to inhibition of one or
more GSK3 enzymes. Thus a GSK3 inhibitor can inhibit one member,
several members or all members of the family of GSK3 enzymes. The
family of GSK3 enzymes is well-known and includes but is not
limited to GSK3-.alpha. and GSK3-.beta.. A number of variants have
been described (see e.g. Schaffer et al.; Gene 2003; 302(1-2):
73-81). In specific embodiments GSK3-.beta. is inhibited.
GSK3-.alpha. inhibitors are also suitable, and in general
inhibitors for use in the invention inhibit both. A wide range of
GSK3 inhibitors are known, by way of example, the inhibitors CHIR
98014, CHIR 99021, AR-AO144-18, TDZD-8, SB216763 and SB415286.
Other inhibitors are known and useful in the invention. In
addition, the structure of the active site of GSK3-.beta. has been
characterised and key residues that interact with specific and
non-specific inhibitors have been identified (Bertrand et al.; J
Mol. Biol. 2003; 333(2): 393-407). This structural characterisation
allows additional GSK inhibitors to be readily identified.
[0042] The inhibitors of certain embodiments are specific for
GSK3-.beta. and GSK3-.alpha., substantially do not inhibit erk2 and
substantially do not inhibit cdc2. Preferably the inhibitors have
at least 100 fold, more preferably at least 200 fold, very
preferably at least 400 fold selectivity for human GSK3 over mouse
erk2 and/or human cdc2, measured as ratio of IC.sub.50 values;
here, reference to GSK3 IC.sub.50 values refers to the mean values
for human GSK3-.beta. and GSK3-.alpha.. Good results have been
obtained with CHIR 99021 and CHIR 98014, which are both specific
for GSK3. Examples of GSK3 inhibitors are described in Bennett C,
et al, J. Biol. Chem., vol. 277, no. 34, Aug. 23, 2002, pp
30998-31004 and in Ring D B, et al, Diabetes, vol. 52, March 2003,
pp 588-595. Suitable concentrations for use of CHIR 99021 are in
the range 0.01 to 100, preferably 0.1 to 20, more preferably 0.3 to
10 micromolar.
[0043] GSK3 inhibition can also be conveniently achieved using RNA
mediated interference (RNAi). Typically, a double-stranded RNA
molecule complementary to all or part of a GSK3 gene is introduced
into pluripotent cells, thus promoting specific degradation of
GSK3-encoding mRNA molecules. This post-transcriptional mechanism
results in reduced or abolished expression of the targeted GSK3
gene. Suitable techniques and protocols for achieving GSK3
inhibition using RNAi are known.
[0044] Reference to a MEK inhibitor herein refers to MEK inhibitors
in general. Thus, reference to a MEK inhibitor refers to any
inhibitor a member of the MEK family of protein kinases, including
MEK1, MEK2 and MEK3. Reference is also made to MEK1, MEK2 and MEK3
inhibitors. A MEK inhibitor can inhibit one member, several members
or all members of the family of MEK kinases. Examples of suitable
MEK inhibitors, already known in the art, include but are not
limited to the MEK1 inhibitors PD184352 and PD98059, inhibitors of
MEK1 and MEK2 U0126 and SL327, and those discussed in Davies et al
(2000) (Davies S P, Reddy H, Caivano M, Cohen P. Specificity and
mechanism of action of some commonly used protein kinase
inhibitors. Biochem J. 351, 95-105). In particular, PD184352 has
been found to have a high degree of specificity and potency when
compared to other known MEK inhibitors. Other MEK inhibitors and
classes of MEK inhibitors are described in Zhang et al. (2000)
Bioorganic & Medicinal Chemistry Letters; 10:2825-2828. Anthrax
lethal factor has also been found to exhibit an MAPKK inhibitory
profile similar to that of PD098059 (Duesbery et al, 1999). Further
suitable MEK inhibitors include PD032509, U0126, SL327 and
CI-1055.
[0045] Inhibition of MEK kinases can also be conveniently achieved
using RNA-mediated interference (RNAi). Typically, a
double-stranded RNA molecule complementary to all or part of a MEK
gene is introduced into pluripotent cells, thus promoting specific
degradation of MEK-encoding mRNA molecules. This
post-transcriptional mechanism results in reduced or abolished
expression of the targeted MEK gene. Suitable techniques and
protocols for achieving MEK inhibition using RNAi are known.
[0046] Other approaches for achieving MEK inhibition include using
a compound that inhibits or reduces activity of a component of the
ras/MAPK cascade. For example, the compound inhibits one or more
mitogen activated protein kinases, for example ERK1 and ERK2.
Alternatively, the compound inhibits SHP-2, for example by
inhibiting binding of the enzyme to gp130, having a similar effect.
In a further embodiment, the inhibitor inhibits MEK.
[0047] Inhibition of MEK may alternatively be achieved by
downregulation of a component of the ras/MAPK cascade. MKP-3 is an
example of a MAP kinase phosphatase and a known downregulator of
the ERKs, and has been introduced into an ES cell by way of a
transgene.
[0048] A number of assays for identifying kinase inhibitors,
including GSK3 inhibitors and MEK inhibitors, are known. For
example, Davies et al (2000) describe kinase assays in which a
kinase is incubated in the presence of a peptide substrate and
radiolabelled ATP. Phosphorylation of the substrate by the kinase
results in incorporation of the label into the substrate. Aliquots
of each reaction are immobilised on phosphocellulose paper and
washed in phosphoric acid to remove free ATP. The activity of the
substrate following incubation is then measured and provides an
indication of kinase activity. The relative kinase activity in the
presence and absence of candidate kinase inhibitors can be readily
determined using such an assay. Downey et al. (1996) J Biol Chem.;
271(35): 21005-21011 also describes assays for kinase activity
which can be used to identify kinase inhibitors.
[0049] Reference to an antagonist of fibroblast growth factor (FGF)
receptor (FGFR) refers to a polypeptide or small molecule or other
antagonist of a FGF receptor, typically inhibiting FGFR1 and/or
FGFR2. Thus, a FGF receptor antagonist can be an antagonist of one,
several or all members of the FGF receptor family, including but
not limited to FGFR1, FGFR2, FGFR3 and FGFR4. Members of the FGF
receptor family typically comprise three immunoglobulin-like
domains and present a region of acidic amino acids (the acidic box)
which can participate in the binding of a member of the FGF family
to a FGF receptor. In some cases, molecules comprising only two
immunoglobulin-like domains can also function as FGF receptors. A
number of FGFR antagonists are known, including but not limited to
SU5402 and PD173074. Suitable concentrations of SU5402 are in the
micromolar range, such as from 0.1-20 .mu.M, preferably 0.5-10
.mu.M, especially in the range 1-5 .mu.M. We have found that
PD173074 can substitute for SU5402 and is fully effective at about
100-fold lower concentrations, consistent with its higher affinity
for the FGF receptor. Thus, suitable concentrations for PD173074
are in the range 1-200 nM, preferably from 5-100 nM, especially in
the range 10-50 nM. It is also known to inhibit FGR receptor
signalling by transgene expression of a dominant negative mutant
FGF receptor. In embodiments of the invention, however, it is
preferred to use a small molecule antagonist and not a transgenic
based antagonism.
[0050] Suitable assays for identifying antagonists of FGF receptors
are known. For example, a cell line in which signalling via a FGF
receptor activates expression of a reporter gene can be used to
assess the activity of a potential antagonist.
[0051] It has advantageously been found that the use of a MEK
inhibitor in combination with a GSK3 inhibitor and an antagonist of
the FGF receptor improves the propagation of ES cells.
[0052] In preferred embodiments between around 0.1 .mu.M and around
25 .mu.M MEK inhibitor are used. Further preferably, between around
0.1 .mu.M and around 5 .mu.M MEK inhibitor are used, more
preferably from 0.2 .mu.M to 2 .mu.M.
[0053] Particularly preferred media according to the invention
comprise 0.8 .mu.M PD184352, 3 .mu.M CHIR99021 and/or 3 .mu.M
SU5402. A particularly preferred medium comprises 0.8 .mu.M
PD184352, 3 .mu.M CHIR99021 and 3 .mu.M SU5402, preferably in N2B27
medium. The concentration of SU5402 can be optimized to suit
different pluripotent cell lines, typically in the range 1-5 .mu.M
(e.g. 2 .mu.M).
[0054] In examples below, we have cultured mouse ES cells in the
presence of a GSK3 inhibitor together with a MEK inhibitor and, in
a specific example, an antagonist of the FGF receptor to promote
self renewal. In other specific examples, a method of promoting
self-renewal of mouse pluripotent cells in culture comprises
inhibiting GSK3 and MEK or inhibiting GSK3, MEK and an FGF
receptor.
[0055] Optionally, activating gp130 downstream signalling can also
be employed to further enhance the promotion of self renewal by
inhibiting GSK3 and MEK. Molecules that activate gp130 downstream
signalling are sometimes referred to as gp130 activators or gp130
agonists. Activation of one or more gp130 downstream signalling
pathways can be achieved by use of a cytokine acting through gp130,
for example a cytokine or other agonist of the LIF receptor.
Cytokines capable of acting through gp130, and thus of activating
gp130 signal transduction, include but are not limited to LIF,
ciliary neurotrophic factor (CNTF), cardiotrophin, oncostatin M,
IL-6 plus sIL-6 receptor, hyper IL-6 and IL-11. Suitable cytokines
include mimetics, fusion proteins or chimaeras that can bind to
and/or activate signalling though gp130. The role of cytokines
acting through gp130 in the presence of serum is well established,
but the capacity of those cytokines to sustain undifferentiated
cells in the absence of serum is limited.
[0056] An advantage of the invention is that in the presence of a
GSK3 inhibitor, a MEK inhibitor and, optionally, an antagonist of
the FGF receptor, pluripotent cells can be grown in defined medium.
A particular advantage associated with using the combination of a
GSK3 inhibitor, a MEK inhibitor and an antagonist of the FGF
receptor is that it is not necessary for the medium to contain
other growth factors, such as insulin, N2B27, or a gp130 agonist
(e.g. LIF). The present invention therefore enables alternative
and/or improved culture of ES cells in medium that is free of
serum, serum extract, feeder cells and feeder cell extract.
[0057] Purported embryonic stem cells have been reported from a
number of mammalian sources including mouse (Bradley et al (1984)
Nature 309: 255-56), American mink (Mol Reprod Dev (1992) December;
33(4):418-31), pig and sheep (3 Reprod Fertil Suppl (1991);
43:255-60), hamster (Dev Biol (1988) May; 127(1):224-7) and cow
(Roux Arch Dev Biol (1992); 201: 134-141). It is noted, however,
that in some instances multipotent or pluripotent cells have been
designated "ES cells" or "ES-like cells" in the absence of complete
characterization. ES cell status has sometimes been assigned based
on cell morphology, the observation of some spontaneous
differentiation in culture, the formation of cystic embryoid
bodies, or the expression of a gene associated with the pluripotent
stage of development. However, more rigorous characterization of
purported ES cells is desirable in order to establish whether the
cells are indeed ES cells. This is confirmed in U.S. Pat. No.
6,271,436, which indicates that many previous attempts to derive ES
cells, e.g. porcine ES cells, have failed to demonstrate
conclusively that the reported cells were pluripotent ES cells.
[0058] Characteristic properties of ES cells include the expression
of multiple genes associated with the pluripotent stage of
development, the ability to differentiate into cells representative
of any and all tissue types present in the source animal, the
ability to contribute to chimeras and, particularly, the ability to
contribute to the germ line of chimeras. For example true ES cells
would be expected to express many, if not all, of the
pluripotency-associated genes Nanog, Oct4, FGF4, Sox-2 and alkaline
phosphatase. In particular, expression of Nanog, Oct4 and Sox-2 is
widely regarded as providing a definitive initial indication that a
cell is an ES cell. Germ line transmission in chimeras and the
ability to generate teratomas or teratocarcinomas comprising
differentiated cells from all three primary germ layers (i.e.
endoderm, mesoderm and ectoderm) are also widely regarded as
definitive indications of a cell being an ES cell.
[0059] Specific examples herein use mouse and human ES cells and
also rat cells from primary outgrowths. It will be appreciated that
the methods and compositions of the present invention are suitable
for adaptation to culturing of other mammalian or avian pluripotent
cell cultures, including primate, especially human, rodent,
especially mouse and rat, bovine, ovine, and porcine pluripotent
stem cells, especially ES cells. In other embodiments, the methods
and compositions of the invention can be used to culture
pluripotent cells from primary outgrowths of bovine, ovine and
porcine embryos.
[0060] The methods and compositions of the present invention are
particularly suitable for culturing pluripotent cells, including ES
cells, that express one or more, preferably any two or more, of
Nanog, Oct4, FGF4, Sox-2 and alkaline phosphatase. In preferred
embodiments, the pluripotent cells express Nanog, Oct4, and Sox-2.
Typically the pluripotent cells are morphologically
undifferentiated in culture and can be maintained in culture for a
prolonged period, typically in excess of at least 10, 20 or 50
passages, without significant differentiation or loss of viability
or loss of pluripotent cell characteristics. It is envisaged that
pluripotent cells isolated or cultured in the media or using the
methods of the invention will be capable of being maintained in
culture for about two weeks or longer. For example, the cells may
be capable of being maintained in culture for four, six, eight,
ten, twelve or more weeks and still substantially retain their
original characteristics, including the specific characteristics of
pluripotent cells described herein. By way of example, rat
pluripotent cells isolated using the procedures described herein
have been subjected to continuous culture for six months with no
deterioration in growth rate and no significant
differentiation.
[0061] Such characteristics include the ability to contribute to a
chimera, including contributing to the germ line of the chimera.
For example mouse ES cells can contribute to a chimera, for example
when injected into a mouse blastocyst. Similarly, rat pluripotent
and ES cells, provided for the first time herein, can contribute to
a chimera in which all cells of the chimera are rat cells. ES cells
derived from other mammals, e.g. as described herein, are similarly
able to contribute to a chimera. The ES cells will also be capable
of forming a teratoma or teratocarcinoma, for example following
injection of ES cells into immunodeficient mice. Typically the
teratoma or teratocarcinoma will comprise differentiated cells from
all three germ layers, although in some cases cells from only one
or two of the germ layers may be observed.
[0062] A second aspect of the invention provides a method of
culture of pluripotent cells, especially ES cells, so as to promote
self renewal, comprising maintaining the cells in medium
containing:-- [0063] (1) an inhibitor of GSK3; [0064] (2) an
inhibitor of MEK; and, optionally, [0065] (3) an antagonist of an
FGF receptor.
[0066] Methods of the invention can be used generally for growing
pluripotent cells, including growing ES cells in medium which is
free of serum and free of serum extract, which cells have
previously been passaged in the presence of serum or serum extract.
Preferably, such methods are also carried out in the absence of
feeder cells and/or feeder cell extracts. For example, culture of
ES cells can be carried out comprising the steps of:-- [0067]
maintaining the ES cells in a pluripotent state in culture,
optionally on feeders; [0068] passaging the ES cells at least once;
[0069] withdrawing the serum or the serum extract from the medium
and withdrawing the feeders (if present), so that the medium is
free of feeders, serum and serum extract; and [0070] subsequently
maintaining ES cells in a pluripotent state in the presence of an
inhibitor of GSK3, a MEK inhibitor and, optionally, an FGFR
antagonist.
[0071] The present invention also provides a method of obtaining a
transfected population of ES cells, comprising:-- [0072]
transfecting ES cells with a construct encoding a selectable
marker; [0073] plating the ES cells; [0074] culturing the ES cells
in the presence of a MEK inhibitor, a GSK3 inhibitor and,
optionally, an FGFR antagonist; and [0075] selecting for cells that
express the selectable marker.
[0076] Further optionally, the cells are cultured in the presence
of a MEK inhibitor, a GSK3 inhibitor and an activator of a gp130
downstream signalling pathway.
[0077] The selectable marker may encode antibiotic resistance, a
cell surface marker or another selectable marker as described e.g.
in EP-A-0695351, and preferably comprises a nucleotide sequence
encoding the selectable marker operatively linked to a promoter
which preferentially expresses the selectable marker in desired
cells.
[0078] In a further embodiment, the present invention provides a
method of culture of pluripotent, especially ES, cells, comprising
the steps of transferring an individual cell to a culture vessel,
such as an individual well on a plate, and culturing the cell in
the presence of a GSK3 inhibitor, a MEK inhibitor and, optionally,
an FGFR antagonist, so as to obtain a clonal population of
pluripotent, especially ES, cells, all of which are progeny of a
single cell. Optionally, the cells may also be cultured in the
presence of an activator of gp130 downstream signalling
pathways.
[0079] Once a stable, homogenous culture of ES cells is obtained,
the culture conditions can be altered to direct differentiation of
the cells into one or more cell types selected from ectodermal,
mesodermal or endodermal cell fates. Addition of, or withdrawal of
cytokines and signalling factors, can enable the derivation of
specific differentiated cell populations at high efficiency.
Differentiation of an ES cell towards a non-neuroectodermal fate
may be achieved by maintaining the ES cell in the presence of a
cytokine acting through gp130, a MEK inhibitor and a GSK3 inhibitor
and then withdrawing the cytokine whilst maintaining the GSK3
inhibitor and MEK inhibitor and/or adding a further signalling
molecule capable of directing differentiation. Alternatively, the
cells may be maintained in the presence of a MEK inhibitor and a
GSK3 inhibitor and then differentiation directed by withdrawing one
or both of the inhibitors and/or adding a signalling molecule
capable of directing differentiation. The methods described above
all optionally include the step of obtaining and/or isolating a
differentiated cell which is the product of the process.
[0080] Further aspects of the invention provide for cell culture
media. One medium is for self-renewal of pluripotent, especially
ES, cells, the medium comprising an inhibitor of GSK3, an inhibitor
of MEK and an FGFR antagonist. The medium may also optionally
comprise an activator of a gp130 downstream signalling pathway.
Another medium of the invention is a stem cell culture medium,
comprising an inhibitor of GSK3, a MEK inhibitor and an FGFR
antagonist. All media preferably further comprises basal medium.
Further, all media is preferably free of an agonist of gp130, hence
is preferably free of LIF.
[0081] The invention provides medium that is free of serum and
serum extract. One such medium comprises:-- [0082] basal medium;
[0083] a MEK inhibitor; [0084] a GSK3 inhibitor; and [0085] an
iron-transporter; wherein the medium is optionally free of serum
and serum extract.
[0086] The medium preferably also comprises an FGFR antagonist. The
medium may also optionally comprise an activator of a gp130
downstream signalling pathway.
[0087] Preferred medium for pluripotent stem cells, especially rat
or mouse cells, may be free of serum and of gp130 agonists and
comprises a MEK inhibitor, a GSK3 inhibitor, and an antagonist of
an FGF receptor. Substitutions of media components can be made as
described herein.
[0088] Basal medium is medium that supplies essential sources of
carbon and/or vitamins and/or minerals for the cells. The basal
medium is generally free of protein and incapable on its own of
supporting self-renewal of cells. The iron transporter provides a
source of iron or provides the ability to take up iron from the
culture medium. Suitable iron transporters include transferrin and
apotransferrin. It is preferred that the medium further comprises
one or more of insulin or insulin-like growth factor and albumin
(preferably recombinant) or albumin substitute, and is free of
feeder cells and feeder cell extract. The medium may also comprise
an inhibitor of apoptosis or any other component that promotes the
maintenance of pluripotent cells in culture. For example, the
medium can comprise a ROCK inhibitor, e.g. Y-27632 (Watanabe et
al., Nature Biotechnology (2007) 25(6): 681-686).
[0089] A particular medium of the invention comprises MEK
inhibitor, GSK3 inhibitor, insulin, albumin and transferrin, with
or without additional basal medium. In this medium, LIF can be
optionally included and can be substituted by other activators of
gp130 signalling, though preferred medium comprises the gp130
receptor binding cytokine, LIF, suitable concentrations of which
are generally between 10 U/ml and 1000 U/ml, more preferably
between 50 U/ml and 500 U/ml, even more preferably in the region of
100 U/ml. The GSK3 and MEK inhibitors are preferably as described
herein in more detail.
[0090] The invention further provides a method of deriving a
pluripotent cell from a blastocyst, comprising:-- [0091] (1)
obtaining a blastocyst; [0092] (2) culturing the blastocyst in the
presence of a MEK inhibitor, a GSK3 inhibitor and, optionally, an
antagonist of an FGF receptor, to obtain an inner cell mass; [0093]
(3) dissociating the inner cell mass; [0094] (4) isolating a cell
or cells from the dissociated inner cell mass; and [0095] (5)
culturing the isolated cell or cells in the presence of a MEK
inhibitor, a GSK3 inhibitor and an antagonist of an FGF
receptor.
[0096] Optionally, the isolated cell or cells are cultured in the
presence of a MEK inhibitor, a GSK3 inhibitor and an activator of
gp130 downstream signalling. An antagonist of an FGF receptor may
also be present.
[0097] Preferably, the method comprises culturing the blastocyst in
LIF, more preferably for a period of from 2 to 4 days. The isolated
cell or cells are preferably cultured in serum free medium.
Typically, the cells are replated as clumps. The blastocyst is also
preferably cultured in serum free medium, optionally in the absence
of an agonist of the BMP receptor.
[0098] It is further preferred, according to the invention, that
culture of cells is carried out in an adherent culture, which may
be promoted by the inclusion of a cell adhesion protein on culture
substrate. It is also preferred to culture pluripotent cells
according to the invention in monolayer culture, though it is
optional for cells to be grown in suspension culture or as pre-cell
aggregates; cells can also be grown on beads or on other suitable
scaffolds such as membranes or other 3-dimensional structures.
[0099] Surprisingly, it has been found that the compositions and
methods of the present invention are particularly suitable for the
derivation of pluripotent and ES cells from rats. Although some
reports claim that rat pluripotent cells have been produced
(Vassilieva et al. (2000) Exp Cell Res 258(2): pp 361-373; Schultze
et al. (2006) Methods Mol Biol 329: pp 45-58), the evidence
presented is far from conclusive. Schultze et al. acknowledge that
most attempts to derive and maintain rat ES cell lines have failed
and that researchers have been forced to abandon this line of
research. Similarly, Vassilieva et al. observe that previous
attempts to generate rat ES cells have failed as the purported ES
cells could only be maintained in culture for a short time or the
experiments were not repeatable. Moreover, cell lines described in
the art have been demonstrated not to be pluripotent ES cells. The
purported designation "ES cell" has been assigned based on the
observation of limited differentiation in culture, the formation of
embryoid bodies or the expression of an incomplete set of
pluripotency-associated markers, often only one gene. However, such
indications do not permit unambiguous identification of ES cells,
and may merely indicate that multipotent, not pluripotent, cells
have been isolated. Many key indicators of pluripotency have not
been demonstrated, and there remains considerable doubt as to
whether rat ES cells have in fact been isolated. In particular,
there have been no reported rat cell lines for which (i) the
ability to form teratomas or teratocarcinomas comprising
differentiated tissues from all three germ layers, (ii) the ability
to colonise the germ line of a chimera or (iii) the coordinated
expression of multiple pluripotency-associated genes, especially
coordinated expression of Nanog, Oct4 and Sox-2, have been
demonstrated.
[0100] In a comparative example, set out in detail below, a report
of obtaining purported rat ES cells has been reproduced without
success.
[0101] However, the methods of the invention have been shown to be
suitable for the derivation of rat pluripotent and ES cells, which
have been isolated from dissociated primary outgrowths of the inner
cell mass of rat blastocysts. Thus, the present invention provides
a method of deriving a pluripotent rat cell from a blastocyst,
comprising:-- [0102] (1) obtaining a blastocyst; [0103] (2)
culturing the blastocyst in the presence of a MEK inhibitor and a
GSK3 inhibitor, to obtain an inner cell mass; [0104] (3) isolating
and dissociating the primary outgrowths of the inner cell mass;
[0105] (4) isolating a cell or cells from the dissociated primary
outgrowths of the inner cell mass; and [0106] (5) culturing the
isolated cell or cells in the presence of a MEK inhibitor, a GSK3
inhibitor and, optionally, an antagonist of an FGF receptor.
[0107] Typically, the rat blastocyst will be cultured for a period
of from 2 to 4 days, e.g. 3 days. The blastocyst may also be
cultured in the presence of a MEK inhibitor, a GSK inhibitor and an
antagonist of an FGF receptor. The isolated rat pluripotent cells
typically have the properties of pluripotent and ES cells described
herein.
[0108] In some embodiments, the method is modified such that the
inner cell mass is isolated from the blastocyst by removal of the
trophectoderm, and the inner cell mass is cultured in the presence
of a MEK inhibitor, a GSK3 inhibitor and an antagonist of an FGF
receptor
[0109] The methods described herein are also suitable for deriving
pluripotent cells from other animals. For example, the steps
described herein have been performed on bovine and ovine
blastocysts, thus allowing the derivation of pluripotent cells from
bovine and ovine primary outgrowths. The methods described herein
have also been used to derive human pluripotent cells from human
blastocysts. The methods can also be performed on porcine
blastocysts to derive pluripotent cells from porcine primary
outgrowths.
[0110] The methods described herein have also been used to derive
pluripotent cells from non-permissive strains of mice. Thus, the
present invention also provides murine pluripotent cells from
strains of mouse that have hitherto been considered to be
non-permissive to the derivation of ES cells.
[0111] A further aspect of the invention provides a rat pluripotent
cell obtainable by a method of the invention. The invention also
provides a pluripotent rat cell expressing two or more of Nanog,
Oct4, FGF4 and Sox-2. Preferably the pluripotent rat cell expresses
Nanog, Oct4 and Sox-2. The pluripotent rat cell may also express
alkaline phosphatase and may possess any of the characteristics of
pluripotent and ES cells described herein, such as the ability to
colonise the germ line of a chimera and the ability to form a
teratoma or teratocarcinoma comprising differentiated tissues
derived from endoderm, mesoderm and ectoderm.
[0112] Accordingly, the present invention provides a pluripotent
rat cell expressing Rex1, Stella (Dppa3), FGF4 and Sox-2. The
pluripotent rat cells of the invention may also express one or more
additional markers of the pluripotent state and or markers
associated with the inner cells mass, including Err.beta., Pecam 1,
Tbx3, and Gbx2. The pluripotent rat cells do not express FGF5. In
addition, the rat pluripotent cells of the invention do not
express, or do not express significant levels of, genes associated
with the epiblast and early germ layers such as Otx2, Eomes, Foxa2,
brachyury, Gata6, Sox17 and Cer1. Analysis of rat pluripotent cells
of the invention has failed to detect expression of the hypoblast
and definitive endoderm markers Gata6 and Sox17, the mesoderm
markers brachyury and Flk1, and the neurectoderm markers Pax6 and
nestin. These characteristics, and other characteristics of the rat
pluripotent cells of the invention, will also be exhibited by other
mammalian pluripotent cells of the invention.
[0113] Thus, the pluripotent cells of the present invention can be
distinguished from the stem cells (referred to as EpiSCs) recently
derived from the post-implantation egg cylinder stage epiblast
(Tesar P. J. et al. Nature (2007) 448: 196-9; Brons I. G. M. et al.
Nature (2007) 448: 191-5) which show little or no ability to
reincorporate into the preimplantation embryo and contribute to
chimaeras. In contrast to the pluripotent cells of the present
invention, EpiSCs cannot be readily propagated after dissociation
into single cells, do not express markers of ES cells or early
epiblast, and require maintenance in FGF2 plus activin, under which
conditions the pluripotent cells of the invention are induced to
differentiate.
[0114] The pluripotent cells of the invention, including rat,
ovine, bovine and porcine pluripotent cells, are morphologically
undifferentiated in culture. The pluripotent cells of the invention
are capable of being maintained in culture for about 2 weeks or
longer. Preferably the pluripotent cells are capable of being
maintained in culture for, four, six, eight, ten or twelve weeks or
longer, more preferably for about three, six, nine or twelve months
or longer. After being maintained in culture, the progeny of the
pluripotent cell retain one or more, preferably all, of the
characteristics of the original pluripotent cell.
[0115] The pluripotent cells of the invention are capable of
contributing to a chimaera. In particular they are capable of
contributing to a chimaera in which all cells of the chimaera are
cells of the same species as the pluripotent cell. For example, if
a pluripotent rat cell is used to produce a chimaera, all of the
cells of the chimaera are rat cells. The pluripotent cells of the
invention are preferably capable of contributing to the germ line
of a chimaera.
[0116] The pluripotent cells of the invention are capable of
forming a teratomas or teratocarcinoma in which differentiated
cells from all three germ layers, i.e. endoderm, mesoderm and
ectoderm, are present.
[0117] It is also preferred that the pluripotent cells of the
invention have a normal karyotype, i.e. the karyotype of the
pluripotent cells corresponds to the normal karyotype of cells from
the same species and/or strain.
[0118] Typically, the pluripotent cells of the invention, including
rat, ovine, bovine and porcine pluripotent cells, exhibit one or
more, preferably two, three, four or five, and most preferably all
of the following characteristics: [0119] a) the ability to form a
chimaera; [0120] b) the capability of growth and/or proliferation
as a single cell in culture; [0121] c) expression of Rex1, Stella,
FGF4 and Sox-2; [0122] d) they are induced to differentiate or fail
to grow in the presence of activin and/or FGF; [0123] e) they are
not induced to differentiate or die by activin receptor blockade;
and [0124] f) growth and/or proliferation of the pluripotent cell
is supported by the presence of a MEK inhibitor, a GSK3 inhibitor
and, optionally, an antagonist of an FGF receptor.
[0125] Without wishing to be bound by any particular theory, it is
believed that MEK inhibition is particularly important for
maintenance of the pluripotent phenotype. Thus, the invention also
provides pluripotent stem cells and populations thereof maintained
in the presence of a MEK inhibitor. Preferably, the pluripotent
cells and populations are maintained in the presence of a MEK
inhibitor and one or more additional factors suitable for
maintaining the pluripotent state, including but not limited to a
GSK3 inhibitor, an antagonist of an FGF receptor and a cytokine
acting through gp130.
[0126] The invention also provides a population of pluripotent rat
cells as described herein. The populations of rat pluripotent cells
obtained by particular methods of the invention and thus provided
by the invention have been found to be highly homogenous,
containing at least 85%, preferably at least 90% more preferably at
least 95% pluripotent cells, assessed according to the
characteristics mentioned above. In specific embodiments of the
invention cultures of rat pluripotent cells of 98% purity and more
have been obtained. For example, the invention provides a
population of pluripotent rat cells in which at least 95% of the
cells retain the characteristics of pluripotent cells. Preferably
at least 95% of the cells express Nanog and/or Oct4.
[0127] In another aspect, the invention provides a culture of
pluripotent rat cells, comprising pluripotent rat cells and a
culture medium comprising a MEK inhibitor. Preferably the medium
further comprises a GSK3 inhibitor and/or an antagonist of an FGF
receptor. Optionally, the medium contains one or more additional
components suitable for supporting the growth of pluripotent cells,
for example one or more of the components described herein.
[0128] The invention also provides pluripotent cells derived from
other animals described herein, e.g. bovine, ovine and porcine
pluripotent cells, obtainable by a method of the invention. Such
pluripotent cells, according to the invention, will possess one or
more of the characteristics of pluripotent and ES cells described
herein.
[0129] Accordingly, the invention provides a pluripotent mammalian
cell other than a pluripotent mouse cell expressing two or more of
Nanog, Oct4, FGF4 and Sox-2. Preferably the pluripotent cell
expresses Nanog, Oct4 and Sox-2. More preferably, the pluripotent
cell further expresses alkaline phosphatase. In preferred
embodiments, the pluripotent cell expresses Rex1, Stella, FGF4 and
Sox-2. Typically, the pluripotent cell does not express FGF5. The
pluripotent cells of the invention may also express one or more
additional markers of the pluripotent state and or markers
associated with the inner cells mass, including Err.beta., Pecam 1,
Tbx3, and Gbx2. In addition, the pluripotent cells of the invention
do not express, or do not express significant levels of, genes
associated with the epiblast and early germ layers such as Otx2,
Eomes, Foxa2, brachyury, Gata6, Sox17 and Cer1.
[0130] The invention also provides populations of pluripotent cells
as described herein, including populations of bovine, ovine and
porcine pluripotent cells. The populations of pluripotent cells
obtained by particular methods of the invention and thus provided
by the invention preferably contain at least 85%, preferably at
least 90% more preferably at least 95% pluripotent cells, assessed
according to the characteristics mentioned above. In some specific
embodiments, the invention provides populations containing 98%
pluripotent cells and more. For example, the invention provides a
population of pluripotent cells in which at least 95% of the cells
retain the characteristics of pluripotent cells. Preferably at
least 95% of the cells express Nanog and/or Oct4.
[0131] It has been found that populations of ES cells cultured in
the media described herein, particularly media comprising a MEK
inhibitor, a GSK3 inhibitor and an antagonist of an FGF receptor,
display significantly greater homogeneity than ES cells maintained
in conventional media, e.g. when assayed by immunostaining for
pluripotency associated markers such as Nanog or Oct4. The
maintenance of pluripotency using the media described herein
provides a particular advantage in that highly homogeneous
populations of pluripotent cells can be maintained for extended
periods in culture without significant cell differentiation and
without the need for regular selection for pluripotent cells and
against differentiated cell types.
[0132] In another aspect, the invention provides a culture of
pluripotent cells, comprising pluripotent cells and a culture
medium comprising a MEK inhibitor. Preferably the medium further
comprises a GSK3 inhibitor and/or an antagonist of an FGF receptor.
Optionally, the medium contains one or more additional components
suitable for supporting the growth of pluripotent cells, for
example one or more of the components described herein.
[0133] Culture medium used in the examples of the invention
preferably also comprises serum albumin. This can be used in
purified or preferably recombinant form, and if in a recombinant
form this has the advantage of absence of potential contaminating
factors, cytokines etc. The culture medium does not need to contain
serum albumin and this component can be omitted or replaced by
another bulk protein or by a synthetic polymer (polyvinyl alcohol)
as described by Wiles et al.
[0134] A particularly preferred medium of the invention is one that
is fully defined. This medium does not contain any components which
are undefined, that is to say components whose content is unknown
or which may contain undefined or varying factors that are
unspecified. An advantage of using a fully defined medium is that
efficient and consistent protocols for culture and subsequent
manipulation of pluripotent cells can be derived. Further, it is
found that maintenance of cells in a pluripotent state is
achievable with higher efficiency and greater predictability and
that when differentiation is induced in cells cultured using a
defined medium the response to the differentiation signal is more
homogenous than when undefined medium is used.
[0135] Methods of the invention also include a method of obtaining
a differentiated cell comprising culturing a pluripotent cell as
described and allowing or causing the cell to differentiate,
wherein the cell contains a selectable marker which is capable of
differential expression in the desired differentiated cell compared
with other cell types, including pluripotent stem cells, whereby
differential expression of the selectable marker results in
preferential isolation and/or survival and/or division of the
desired differentiated cells. The selectable marker may be
expressed in the desired differentiated cells but not expressed in
other cell types, or the level of expression may differ between
desired differentiated cells and other cell types, thereby allowing
selection for expression of the selectable marker. The
differentiated cell can be a tissue stem or progenitor cell,
including a multipotential or a unipotential stem or progenitor
cell, and may be a terminally differentiated cell. The invention
also provides differentiated cells obtainable by the method and
populations of such differentiated cells.
[0136] Examples of differentiated cells that can be obtained
according to the invention include gonadal stem cells, somatic
stem/progenitor cells, haematopoietic stem cells, epidermal stem
cells and neuronal stem cells. In one embodiment, the
differentiated cell is a neural cell. It will be apparent that
other differentiated cell types and populations of differentiated
cells can be obtained according to this method.
[0137] Generally also, the invention extends to a cell obtained by
following any of the methods of the invention described herein.
Cells of the invention can be used in assays for drug discovery.
Cells of the invention may also be used for cell therapy, and thus
a method of the invention comprises using a combination of
inhibition of MEK and inhibition of GSK3 and, optionally,
antagonism of FGF signalling to derive and/or maintain pluripotent
cells, deriving cells for cell therapy therefrom and using those
cells in cell therapy. Optionally, the combination is used in the
absence of an activator of gp130 downstream signalling.
[0138] The invention further provides additional methods for
deriving rat, ovine, bovine, and porcine pluripotent cells and
pluripotent cells from strains of mouse considered to be
non-permissive to the derivation of ES cells.
[0139] Accordingly, the invention provides a method of deriving a
pluripotent rat cell from a blastocyst, comprising:-- [0140] (1)
obtaining a blastocyst; [0141] (2) culturing the blastocyst in the
presence of a MEK inhibitor and a GSK3 inhibitor, to obtain an
inner cell mass; [0142] (3) isolating and dissociating the primary
outgrowths of the inner cell mass; [0143] (4) isolating a cell or
cells from the dissociated primary outgrowths of the inner cell
mass; and [0144] (5) culturing the isolated cell or cells in the
presence of a MEK inhibitor, a GSK3 inhibitor and an antagonist of
an FGF receptor.
[0145] In a related aspect, the invention provides a method of
deriving a pluripotent mammalian cell from a blastocyst,
comprising:-- [0146] (1) obtaining a blastocyst; [0147] (2)
culturing the blastocyst to obtain an inner cell mass; [0148] (3)
dissociating the inner cell mass; [0149] (4) isolating a cell or
cells from the dissociated inner cell mass; and [0150] (5)
culturing the isolated cell or cells in the presence of a MEK
inhibitor, a GSK3 inhibitor and an antagonist of an FGF
receptor.
[0151] Optionally, the method comprises culturing the blastocyst in
LIF. In preferred embodiments, the blastocyst is cultured in the
presence of a MEK inhibitor, a GSK3 inhibitor and, optionally, an
antagonist of an FGF receptor.
[0152] In specific embodiments, the blastocyst is a rat blastocyst,
a bovine blastocyst, an ovine blastocyst, a porcine blastocyst or a
murine blastocyst from a mouse strain that is non-permissive for
the derivation of ES cells.
[0153] In some embodiments the cell or cells of step (4) are
isolated from dissociated primary outgrowths of the inner cell
mass.
[0154] The methods of deriving pluripotent cells provided by the
invention preferably provide a pluripotent cell that expresses one
or more of Nanog, Oct4, FGF4, Sox-2 and alkaline phosphatase, more
preferably any two or more of Nanog, Oct4, FGF4, Sox-2 and alkaline
phosphatase. In a preferred embodiment, the pluripotent cell
expresses Nanog, Oct4 and Sox-2 and preferably the pluripotent cell
further expresses, alkaline phosphatase. In a particularly
preferred embodiment, the pluripotent cell expresses Rex1, Stella,
FGF4 and Sox-2. It is also preferred that the pluripotent cell does
not express FGF5.
[0155] The methods of the invention permit the provision of a
pluripotent cell that is morphologically undifferentiated in
culture. Typically, the pluripotent cell is capable of being
maintained for extended periods in culture, as described herein.
For example, the pluripotent cell is capable of being maintained in
culture for about two weeks or longer. Preferably, the pluripotent
cell is capable of being maintained in culture for about 6 months
or longer. Typically, the progeny of the pluripotent mammalian cell
retain the characteristics of the original pluripotent mammalian
cell after being maintained in culture.
[0156] The methods of the invention provide pluripotent cells that
are capable of contributing to a chimera. In particular, they are
capable of contributing to a chimaera in which all cells of the
chimera are cells of the same species as the pluripotent mammalian
cell. Preferably, the pluripotent cells are capable of contributing
to the germ line of a chimera.
[0157] The pluripotent cells produced by the methods of the
invention are additionally capable of forming a teratoma or
teratocarcinoma in which differentiated cells from all three germ
layers are present. The pluripotent cells are capable of growth
and/or proliferation as a single cell in culture, are induced to
differentiate or fail to grow in the presence of activin and/or FGF
and are not induced to differentiate by activin receptor blockade.
Growth and/or proliferation of the pluripotent mammalian cells is
supported by the presence of a MEK inhibitor, a GSK3 inhibitor and,
optionally, an antagonist of an FGF receptor.
[0158] In further aspects of the invention, there are provided a
rat pluripotent cell, an ovine pluripotent cell, a bovine
pluripotent cell, a porcine pluripotent cell and a murine
pluripotent cell from a non-permissive strain of mouse obtainable
by the methods of the invention. The invention also provides
populations of pluripotent cells obtainable by the methods of the
invention.
[0159] In preferred embodiments of all aspects of the invention the
pluripotent cell or cells are ES cells.
[0160] A further aspect of the invention provides a method of
obtaining a genetically engineered rat comprising genetically
modifying a rat pluripotent cell and introducing the pluripotent
cell into a rat embryo to produce a genetically modified rat.
[0161] A still further aspect of the invention provides a method of
obtaining a genetically engineered non-human mammal other than a
mouse comprising genetically modifying a mammalian pluripotent cell
and introducing the pluripotent cell into a non-human mammalian
embryo to produce a genetically modified mammal.
[0162] The genetically engineered mammal may, for example, be any
non-human mammal including a rat, cow, sheep or pig. In a preferred
embodiment the genetically engineered mammal is homozygous null for
a gene of interest. In another embodiment, the genetically
modifying comprises replacing a gene in the rat or non-human mammal
with a corresponding human gene. In further embodiments, the
genetically modifying comprises knocking out one or more genes of
interest or targeted insertion of a gene of interest. Such genetic
manipulation can be carried out using techniques readily available
to the person of skill in the art.
[0163] The invention also provides a genetically engineered rat and
genetically engineered non-human mammal other than a mouse
obtainable by the methods of the invention. Such genetically
engineered mammals can be used in several applications, for example
in assessing the physiological role of one or more genes of
interest. Another use of a genetically engineered rat or a
genetically engineered non-human mammal of the invention is in drug
discovery and/or testing. This can, in some embodiments, include
toxicity studies, e.g. to evaluate potential side effect of a
potential drug. In further embodiments, genetically engineered rats
or genetically engineered non-human mammals of the invention are
used as models for human or animal disease.
[0164] The use of genetically modified rats of the invention may be
particularly advantageous as the rat is the preferred model
organism in many areas of biomedical research. For example,
assessments of behavioural recovery and cognitive repair are
considerably more sophisticated in rats than in mice.
[0165] A number of advantages of the invention are described above
or apparent. Cell culture components may be identified which are
relatively non-toxic and cell permeable. The MEK inhibitors, GSK3
inhibitors and FGFR antagonists used in specific embodiments of the
invention can be purified easily, especially compared to, say,
purification of protein cytokines. Recombinant proteins can be
expensive to make and the small molecule medium components may be
more cheaply produced and more stable in storage, with a wider
effective concentration range. In addition, the use of MEK
inhibitors, GSK3 inhibitors and/or FGFR antagonists permits the
derivation and maintenance of previously unobtainable mammalian
pluripotent cell types.
[0166] Specific embodiments set out below used a combination of
CHIR 99021, PD184352 and SU5402 in a serum-free, fully defined
medium and gave improved self renewal of mouse ES cells with very
little differentiation. It is occasionally reported when culturing
ES cells in the presence of BMP that there is some neurogenesis.
This was not seen in the examples of the invention.
[0167] The invention is now further described in specific
examples.
[0168] In the examples the term 2i medium or 2i is used to indicate
medium comprising a MEK inhibitor and an antagonist of an FGF
receptor. The term 3i medium or 3i is used to indicate medium
comprising a MEK inhibitor, a GSK3 inhibitor and an antagonist of
an FGF receptor.
EXAMPLES
Example 1
[0169] Mouse ES cells were grown in medium containing CHIR99021,
PD184352 and SU5402, prepared as follows: --
Concentrations of the Three Inhibitors/Antagonist:
TABLE-US-00001 [0170] Final Concentration Initial when added to
Compound concentration Dilutions media CHIR99021 10 mM Aliquot
stock in 20 .mu.l aliquots. 3 .mu.M Store at -20 Initial 1:10
dilution with N2B27 This >1 yr media = 1 mM. concentration Store
at 4.degree. C. Add diluted stock to was used for all media at
1:333 to make 3 .mu.M final. cell lines PD184352 10 mM Aliquot
stock in 10 .mu.l aliquots. 0.8 .mu.M Store at -20 Initial 1:100
dilution in N2B27 = Some cell lines >1 yr 1 ml of 100 .mu.M,
store at 4.degree. C. Add to were grown in media at 1:125 for 0.8
.mu.M final. concentrations varying in the range = 0.5-1 .mu.M
SU5402 5 mM Initial 1:10 dilution = 0.5 mM in 2 .mu.M Store at -20
N2B27. Some cell lines >1 yr Add to media at 1:250 for final may
need to be concentration of 2 .mu.M optimised, range = 1-5
.mu.M
Media:
Preparation of DMEM/F12--N2 Medium
[0171] To 100 ml of DMEM/F12 (Gibco 42400-010) add 1 ml of N2
100.times. stock solution. The final concentration of each
component of N2 in the DMEM/F12 medium is:
TABLE-US-00002 Insulin 25 .mu.g/ml Putrescine 16 .mu.g/ml
Transferrin 100 .mu.g/ml Sodium Selenite 30 nM Progesterone 6 ng/ml
BSA 50 .mu.g/ml
Preparation of Neurobasal/B27:
[0172] To 100 ml Neurobasal medium (Gibco 21103-049) add 2 ml of
B27 (Gibco 17504-044) and 1-2M L-glutamine (TC stores 1:100)
Preparation of N2 B27 medium:
[0173] Mix DMEM/F12-N2 medium with Neurobasal/B27 medium at the
ration of 1:1. The media was used to dilute all compounds and grow
the cells.
[0174] The medium was used for derivation and maintenance of ES
cells from 129 strain mice, and also for derivation of ES cells
from the non-peimissive mouse strain CBA and partially permissive
strain C57BL/6. The medium was used for maintenance of cells in
primary outgrowths of rat embryos, indicating that pluripotent rat
cells are maintained by the medium in culture.
[0175] Thus, ES cells are maintained in a combination of a GSK3
inhibitor, a MEK inhibitor and an antagonist of an FGF receptor and
the invention also provides culture methods and media therefor.
Example 2
[0176] Rat embryos were collected at 4.5 dpc (E4.5), the zonas
removed in acid Tyrodes, and immunosurgery performed to remove the
trophectoderm. The inner cell masses were cultured in 4-well plates
on feeder layers of gamma-irradiated DIA-M cells (described in
Buehr et al. 2003) in the following medium (3I medium):
[0177] 3 .mu.M Chiron 99021 (a GSK inhibitor)
[0178] 0.8 .mu.M PD184352 (a MEK inhibitor)
[0179] 2 .mu.M SU5402 (an FGF receptor antagonist)
[0180] in N2B27.
Penicillin and streptomycin may be added if necessary.
[0181] If desired, primary cultures of mouse embryonic fibroblasts
(MEFs) can be used as feeder cells rather than DIA-M cells.
[0182] These cultures were maintained for 3 days. After that time
the small outgrowths were disaggregated manually, and small clumps
of cells were moved on to fresh DIA-M feeders in the same
medium.
[0183] These cultures were maintained for two weeks. The medium was
changed every 2-3 days and the cultures were inspected regularly.
If small, clear colonies appeared, these were transferred further
in similar conditions, and disaggregated if large enough.
[0184] After 2 weeks of culture, colonies of undifferentiated cells
were seen in some cultures. Some of these colonies adhered to the
feeders but more commonly they rounded up and lifted off the
substrate. Cultures were passaged by disaggregating colonies,
either with trypsin-EDTA or manually (by drawing a colony into a
narrow pipette and expelling the cell clumps into a fresh well).
Cultures were maintained on DIA-M feeders and in 3I medium at all
times.
[0185] Cells derived and cultured in this way were morphologically
undifferentiated (FIG. 4) and express the pluripotency-associated
genes Nanog (FIGS. 5 and 9) and Oct4 (FIGS. 6 and 9) as well as
FGF4, Sox-2 (FIG. 9) and alkaline phosphatise (FIG. 8). The rat
cells also expressed the trophectoderm marker Cdx2 (FIGS. 7 and
9).
REFERENCES
[0186] Buehr, M., Nichols, J., Stenhouse, F., Mountford, P.,
Greenhalgh, C. J., Kantachuvesiri, S., Brooker, G., Mullins, J. M.,
Smith, A. G. (2003). Rapid loss of oct-4 and pluripotency in
cultured rodent blastocysts and derivative cell lines. Biol Reprod
68: 222-229.
Example 3
[0187] Cells from the rat 148C ES cell line, which was isolated as
described in Example 2, were used to generate a teratoma using the
standard methodology. In brief, rat ES cells were injected under
the kidney capsule of immunodeficient SCID mice. FIG. 10 shows
sections of a teratoma generated in one such experiment, in which
the animal developed a large tumour and was sacrificed 33 days
after the injection.
[0188] Mature differentiated tissues can clearly be seen in the
sections, notably epidermis, striated muscle and gut epithelium.
These tissues are derived, respectively, from ectoderm, mesoderm
and endoderm. Thus, differentiated cells derived from each of the
three primary germ layers are present, indicating that rat 148C ES
cells are pluripotent.
Example 4
Derivation of Pluripotent Stem Cells from Rat Blastocysts
[0189] Although embryonic stem (ES) cells have been derived from
inbred mice since 1981.sup.1,2, they have not been authenticated
for other species. Repeated failure to establish ES cells from
other rodents, plus evidence that primate embryo-derived cells
differ in key properties from mouse ES cells.sup.3, calls into
question the relationship between cultivated stem cells and
pluripotent cells of the embryo.sup.4-7. Here we culture rat
embryos in conditions designed to shield the pluripotent state of
early epiblast from inductive differentiation signals. This results
in reproducible establishment of cultures of cells that express the
pluripotency markers Oct4.sup.8 and Nanog.sup.9. These cells are
morphologically undifferentiated and show stable long term
expansion. They share molecular features with mouse ES cells and
can be induced to differentiate in vitro and form
multidifferentiated teratomas in immunocompromised adult mice.
Phenotypically and functionally they are distinct from the recently
described egg-cylinder derived EpiSCs.sup.3,10. Most importantly,
upon injection into rat blastocysts they can give extensive
contributions to chimaeras. We conclude that pluripotent cells with
characteristics of ES cells can be isolated from rat embryos. We
suggest that ES cell derivation may be a general facility from
naive epiblast cells placed in non-inductive culture.
[0190] ES cells arise from pluripotent mouse epiblast cells in the
synthetic context of tissue culture.sup.4,6. It is unclear whether
ES cells themselves are a product of this artificial environment or
represent a specific phase of ontogeny that is captured in
cultures.sup.11. Empirical experience is that ES cells can
reproducibly be derived from certain inbred mouse strains using
fibroblast feeders and/or the cytokine leukaemia inhibitory factor
(LIF) in combination with selected batches of foetal calf serum or
the growth factor bone morphogenetic protein.sup.4,12. However, the
same conditions do not yield ES cells from all mouse strains and
not at all from the rat.sup.13,14. We.sup.7 and others.sup.15,16
have reported the derivation of cell lines from rat embryos that
have superficial morphological similarities to ES cells but do not
express meaningful levels of the key transcription factor
determinants of ES cell identity, Oct4 and Nanog.sup.17, and are
not capable of definitive germlayer differentiation in vitro, in
tumours or in chimaeras. In our hands such cells give rise only to
extraembryonic trophectoblast and hypoblast lineages and we refer
to them as extraembryonic stem (ExS) cells.sup.7. It is possible
that repeated failures to derive true ES cells from rat embryos
reflect some fundamental difference between the pluripotent
epiblast of mouse and rat embryos.sup.18. Alternatively the culture
construction may be inappropriate for maintaining pluripotency,
except on particular modified genetic backgrounds produced by
extensive inbreeding of laboratory mice.
[0191] We have formulated a combination of three small molecule
inhibitors (3i) to eliminate inductive stimuli for differentiation
in mouse ES cell culture (Ying et al, submitted). 3i enables
efficient maintenance of pluripotency both in established cell
lines and during de novo derivation from mouse embryos. To
determine whether this principle was restricted to mice or may be
more broadly applicable, we examined the effect of 3i on rat embryo
cells. Retained expression of the transcriptional determinant Oct4
can be used as a surrogate assay for presence of undifferentiated
cells in primary outgrowths.sup.7. Oct4 is rapidly extinguished in
ICMs cultured in serum containing medium.sup.6,7 or in serum-free
medium. Inner cell masses (ICMs) were isolated from E4.5 rat
blastocysts by immunosurgery and plated on feeders to facilitate
attachment, and also as a source of leukaemia inhibitory factor
(LIF).sup.7. Cultures were fixed after 3 or 4 days and analysed by
immunostaining (FIG. 11a-c). Concurrent with the previously
described loss of Oct4, we observed up-regulation of Cdx2, the
trophectoderm determinant and antagonist of Oct4.sup.19. These
changes were not prevented by feeders or soluble LIF, or by culture
in serum-free medium without inhibitors. In contrast, in the
presence of 3i, we found that Oct4 protein is maintained by the
majority of cells in the outgrowth and expression of Cdx2 was
suppressed. Furthermore, a second critical marker of pluripotent
status, Nanog.sup.9, which is down-regulated in similar fashion to
Oct4 in cultured ICMs, was maintained in serum-free medium
supplemented with 3i. We dissociated ICMs cultured in 3i medium for
3 days into small clumps, and replated them in the same conditions.
Cells remained viable and appeared to proliferate, forming colonies
of morphologically undifferentiated cells that continued to express
Oct4 and Nanog 4 days later (FIG. 1d).
[0192] Following on from these indications that 3i sustains the
uncommitted state in primary cultures, we investigated longer term
effects. A total of 35 ICMs from rat embryos of two different
strains, DA and Fischer 344, were plated on feeders in 3i. After 3
days, 11 of the ICMs had attached and proliferated such that they
could be mechanically broken up into small clumps and replated in
fresh wells. From four of these replated ICMs, small colonies of
undifferentiated morphology developed. After 10 days each of these
colonies was transferred intact into a new well. Four days later
all had increased appreciably in size. The colonies were then
dissociated manually and replaced. Further undifferentiated
colonies emerged in all cases. We found that these cultures could
then be passaged repeatedly, resulting in the establishment of four
cell lines, two derived from the Fischer embryos and two from DA
embryos. All 4 lines were similar in morphology and growth
characteristics. They proliferate as three dimensional aggregates
of tightly packed cells (FIG. 12a), typical of ES cells cultured in
3i (Ying et al., submitted). Individual cells have the high nucleus
to cytoplasm ratio and prominent nucleoli characteristic of ES
cells. They are apolar and lack processes, cytoarchitectural
features, or other signs of specialization. Routinely cells were
passaged every 3-4 days when colonies reached an intermediate size
because they have a tendency to detach from the feeder layer if
they become too large. We found that undifferentiated cells were
difficult to maintain on gelatin-coated plastic in 3i. Adhesion was
poor without feeders and cells tended to aggregate and detach from
the substrate. However, on fibronectin-coated dishes attachment was
improved and adequate for propagation of undifferentiated cells
(FIG. 12b), indicating that the major contribution of feeders is to
support cell adhesion rather than provide a specific self-renewal
signal. The colonies can readily be dissociated manually by
pipetting or enzymatically using Accutase. After dissociation,
single cells isolated by micropipette can readily give rise to
undifferentiated colonies that can be serially passaged (FIG. 12b).
Cells can be cryopreserved and recovered by conventional
procedures. Cultures have been expanded continuously for 6 months
with no deterioration in growth rate and little or no overt
differentiation.
[0193] To assess the potential identity of these rat cells we
examined expression of key markers of pluripotency and lineage
commitment. By immunofluorescence the cells show nuclear expression
of Oct-4 and Nanog (FIG. 12c). RT-PCR analysis confirmed expression
of these genes along with Rex-1, Err.beta., Sox2, Stella and the
Oct4/Sox2 target Fgf4 (FIG. 12d). Transcripts for the hypoblast and
definitive endoderm markers Gata6 and Sox17, the mesoderm markers
brachyury and Flk1, or the neuroectoderm markers Pax6 and nestin
were not detected. Primers for amplification of Nanog were designed
against sequences specific for the rat gene. These primers
consistently yielded products of anticipated size from rat cells
but not from mouse ES cells. Conversely, mouse specific primers did
not yield any product from the rat cells. The rat identity of the
cells was confirmed by preparation of metaphase spreads (FIG. 12e).
All 4 lines contained the metacentric and acrocentric chromosomes
characteristic of the rat, and distinct from mouse which has only
telocentric chromosomes.
[0194] In two repeat experiments a total of 41 further ICMs were
cultured in 3i. Of these, 13 attached and formed cell masses. These
were disaggregated after three days, and primary undifferentiated
colonies subsequently appeared in 6 wells. All 6 of these cultures
yielded continuously expandable undifferentiated cell lines. These
cultures are indistinguishable morphologically from the original 4
lines and all express both Oct4 and Nanog. Subsequently we have
derived a further 4 lines from 18 disaggregated ICMs. We conclude
that the derivation procedure is reproducible and robust.
[0195] We investigated the effect of altering the culture
conditions on maintenance of the undifferentiated state. We found
that the LIF-producing DIA-M feeders used in the derivations.sup.7,
could be replaced by standard mouse embryo fibroblasts (MEFs) with
no evident compromise. LIF is not included in culture medium,
consistent with the sufficiency of 3i for mouse ES cells. Rat cells
transferred from 3i to medium supplemented with serum and LIF or
BMP and LIF differentiated into various morphologies and lost
expression of Oct4 and Nanog (FIG. 13a). These observations
indicate that the 3i rat cells have reduced responsiveness to
gp130/Stat3 signalling compared with mouse ES cells.sup.20,21. This
may be a significant contributory factor to previous failures to
derive rat ES cells.
[0196] Cells cultured without inhibitors differentiated into
various morphologies (FIG. 13b). Culture in Fgfr inhibitor and
inhibitor of Mek activation (2i) was adequate to maintain the
undifferentiated population without the GSK3 inhibitor, as also
seen with mouse ES cells. These findings are consistent with the
hypothesis that blockade of FGF/Erk signaling is the critical
requirement to sustain the naive pluripotent state (Ying et al.,
submitted).
[0197] A hallmark of undifferentiated female ES cells and of naive
epiblast is that both X chromosomes are active and upon
differentiation one copy is inactivated.sup.22-24. We stained XX 3i
rat cells with an antibody against trimethylated histone 3 lysine
27 (H3K27), an epigenetic silencing mark that distinguishes the
inactive X chromosome in interphase nuclei.sup.25. Cells maintained
in 3i showed only diffuse immunoreactivity whereas cells
differentiated after exposure to serum for 6 days exhibited a
prominent nuclear body diagnostic of the inactive X (FIG. 13c).
These data indicate that in undifferentiated XX 3i rat cells the
two X chromosomes are active and that one X chromosome is
inactivated during differentiation. Therefore 3i rat cells exhibit
the appropriate epigenetic status of early epiblast and ES
cells.
[0198] When cultured in suspension in serum-free medium without 3i
cells died. However if serum or Matrigel were added, the cells
aggregated into embryoid body-like structures (FIG. 13d). RT-PCR
analysis of these structures revealed down-regulation of Oct4 and
Nanog, and appearance of endodermal and mesodermal markers (FIG.
13e). To characterize further the differentiation capacity of 3i
rat cells we injected cells of both DA and Fischer derived lines
under the kidney capsule of 11 immunocompromised SCID mice. After
30-55 days, animals were sacrificed. Seven exhibited a macroscopic
tissue mass at the site of the injection varying in size from less
than a gram to over 4 grams. Growths were obtained from both lines
tested. Histological analysis of two specimens, one from each line,
revealed classical features of a teratoma, with multiple
differentiated cell types and structures including striated muscle,
bone, cartilage, keratinised epithelia, secretory epithelia of the
gut, and many others (FIG. 14a). We conclude that 3i rat cell lines
are capable of producing teratomas and are competent for mature
multilineage differentiation.
[0199] The defining feature of authentic ES cells is their capacity
to colonise host embryos and contribute differentiated progeny to
all three germlayers of chimaeric animals. Following lipofection
and selection in puromycin we derived a pool of cells that express
green fluorescent protein (GFP) constitutively from integrated
plasmid. These cells were injected into blastocysts to track the
contribution to developing embryos. Two out of eight embryos
harvested at E10.5 showed widespread contribution of GFP labelled
cells (FIG. 14b). Examination of cryosections confirmed
incorporation into tissues of all three germ layers.
[0200] We also injected unmodified 3i rat cells into blastocysts to
determine their potential to contribute to post-natal chimaeras.
The genetically determined coat colour distinctions between agouti
DA and albino Fischer provide a convenient indicator of chimaerism
when cells of one strain are introduced into embryos of the other.
Out of 15 pups generated from transferred embryos, 2 animals
exhibited coat colouring diagnostic of the presence of introduced
DA cells in Fischer hosts (FIG. 14b). These animals are heavily
pigmented indicating a contribution of DA cells. The albino host
contribution is apparent only on the head, a consequence of the
presence of the hooded allele in the Fischers. This pattern is
characteristic of rat chimaeras between albino hooded and fully
pigmented strains.sup.26. To confirm and quantitate the chimaeric
nature of these animals we undertook microsatellite analysis of
tail biopsies. This revealed that cells from both contributing
genotypes were present in proportions of 25:75 and 75:25
(DA:Fischer), respectively, in the two animals (FIG. 14d). These
chimaeras have developed into healthy fertile adults. Both are
male, however, and therefore will not support development of
gametes from the introduced cells, retrospectively identified as
XX.
[0201] Recently the derivation of cell lines from post-implantation
egg cylinder stage epiblast has been reported in mice and
rats.sup.3,10. These so called EpiSCs have multilineage
differentiation potential in vitro but exhibit a range of
phenotypic, molecular and developmental differences from true ES
cells. Most notably they show little or no ability to be
reincorporated into the pre-implantation embryo and contribute to
chimaeras. In contrast the 3i rat cells do contribute to chimaeras.
They also express Rex1, Stella (Dppa3) and alkaline phosphatase,
markers of ES cells and early epiblast that are absent in EpiSCs.
Furthermore 3i rat cells can readily be propagated after
dissociation into single cells whereas maintained cell-cell contact
appears critical for EpiSCs. We tested whether the 3i rat cells
could be maintained in the specific growth conditions, FGF2 plus
activin, that are required to maintain EpiSCs. We found that they
behaved like mouse ES cells and differentiated when 3i was replaced
with activin plus FGF2, whether on feeders or fibronectin-coated
dishes. In addition, the activin receptor inhibitor SB431452, which
induces differentiation of EpiSCs.sup.10 but not of mouse ES cells,
did not impede propagation of undifferentiated 3i rat cells. A
final difference between 3i cells and EpiSCs is that the latter are
derived from post-implantation embryos rather than blastocysts. We
also tested whether blastocyst-derived rat ExS cells.sup.7 could be
maintained in 3i and found that they do not survive. Therefore 3i
rat cells are distinct from other cell lines established from
cultured rat embryos.
[0202] Collectively these findings demonstrate that use of 3i
enables the derivation of rat cell lines with cardinal features of
ES cells: long-term self-renewal; pluripotency; and capacity for
incorporation into the developing embryo. They also express the key
molecular markers of the ES cell state, Nanog, Oct4, Sox2 and Rex1.
The availability of rat ES cells opens the door to application of
gene targeting and related genome engineering technologies in the
species of choice for many areas of biomedical research.
Availability of 3i rat cells immediately creates opportunities to
use more advanced in vivo models for testing the capacity of in
vitro stem cell differentiation to produce cells that can integrate
and function in adult tissues. For example, evaluation of cognitive
repair is considerably more sophisticated in rats than in mice.
[0203] The efficient preservation of pluripotency in cultured rat
embryo cells using the neutralizing 3i culture system suggests that
derivation of ES cells may indeed represent the capture of resident
embryo cells rather than a tissue culture creation. Isolation of ES
cells therefore may not entail extensive transcriptional or
epigenetic reprogramming.sup.5,6,27. Rather ES cells may represent
a simple continuation by default of early epiblast proliferation
when inductive signals are eliminated (Ying et al, submitted). This
raises the possibility that culture formulations based on the 3i
principle may be sufficient for derivation of ES cells from other
mammals, including livestock species.
Methods
[0204] Details of RT-PCR and microsatellite analyses, teratoma
generation, and antibodies are provided in the supplementary
information.
[0205] Immunosurgery.
[0206] Zonae were removed from 41/2 dpc rat blastocysts with acid
Tyrodes, and the blastocysts incubated at 37.degree. in 20%
anti-rat whole serum (Sigma) for 3 hours. Blastocysts were washed
and incubated for 20 minutes in rat serum as a source of
complement, and the lysed trophectoderm removed by pipetting
[0207] Culture Procedure.
[0208] Feeder cells were prepared from gamma-irradiated mouse
fibroblasts.sup.7 plated on gelatin in 4-well plates at a density
of 1.5.times.10.sup.4 cells per well. Isolated ICMs and passaged
cell lines were plated on the feeders in N2B27 medium.sup.28
containing Fgf receptor inhibitor SU5402, 2 .mu.M, inhibitor of
Mek1/2 activation PD184352, 0.8 .mu.M, and the GSK3 inhibitor
CHIR99021, 3 .mu.M (3i; Ying et al., submitted). The 3i cell lines
were routinely passaged by aspirating the colonies into fine
pipettes and transferring the resultant disaggregated cells and
small clumps to fresh plates. For embryoid body formation, samples
of approximately 200 dissaggregated cells were transferred into 10
.mu.l hanging drops. These were cultured for 2 days and cell
aggregates collected and transferred into bacteriological dishes
for a further 5 days.
[0209] Transfection Protocol.
[0210] Colonies were dissociated with Accutase (Sigma), pelleted in
GMEM containing 10% FCS then resuspended in serum-free 3i culture
medium and plated in a final volume of 400 .mu.l. A mixture of 0.25
.mu.g ScaI-linearised pPyCAGgfpIP plasmid.sup.29 DNA, 0.25 .mu.l
PLUS reagent and 0.625 .mu.l Lipofectamine LTX (Invitrogen
Corporation) was added and cultures incubated overnight. The
reagents were removed after 18 hours and replaced with fresh
serum-free 3i culture medium. Stable transfectants were selected in
0.5 .mu.g/ml puromycin applied 48 hours after transfection.
Polyclonal lines were obtained by pooling several resistant
colonies maintained continuously in puromycin.
[0211] Chimaera Generation.
[0212] 4.5 dpc rat embryos were collected by noon on the day of
injection, and cultured for a further 2-3 hours in KSOM medium to
allow maximum cavitation. Cell lines were disaggregated in Accutase
and 10-12 cells injected into the blastocyst cavities of recipient
embryos. Injected embryos were transferred into the uteri of 3.5
dpc pregnant rats. Due to unavailability of vasectomised male rats
we were obliged to use naturally mated recipients for this study,
which does reduce the frequency of implantation of transferred
embryos.
[0213] Vibratome Sectioning.
[0214] Fluorescent embryos were embedded in a 1:1 mixture
containing 20% gelatin and 20% albumin in PBS then fixed overnight
at 4.degree. C. in 4% paraformaldehyde. The gelatin blocks were
then embedded in a `setting solution` with 10% glutaraldehyde
overnight at 4.degree. C. Transverse sections were cut in a Series
1000 Vibratome at 100 .mu.m and then treated with PBS/0.1% Tween20
for 10 minutes prior to mounting under coverslips in Vectashield
(Vector Laboratories) containing DAPI nuclear fluorescent stain.
Sections were examined by confocal microscopy within 24 hours.
[0215] Microsatellite genotyping. Fluorescent-tagged oligos were
used to amplify the rat microsatellite regions D1Rat122 (forward
oligo, 6-FAM-CTGCTCCACCTGCCTGTATT, reverse oligo,
TCCCTTTGCAATAGACAATGG) and D3Rat17 (forward oligo
VIC-TCATTTTCCTTCCTCTCTCTCA, reverse oligo AAGACAAAATGCTGGAGGGA)
from genomic DNA of tail, ear and blood. PCR reactions were
amplified on a PTC-200 thermocycler (MJ Research) using GoTaq Flexi
DNA Polymerase (Promega Corporation, #M8305) under the following
conditions; 95.degree. C. 2 minutes, followed by 35 cycles of
94.degree. C. 30 seconds, 56.degree. C. or 62.degree. C. 30 seconds
and 72.degree. C. 30 seconds, with a final extension at 72.degree.
C. for 5 minutes. The annealing temperature was 56.degree. C. and
62.degree. C. for D1Rat122 and D3Rat17 respectively. The PCR
products were diluted 1 in 100 in sterile, distilled water. l.mu.l
of this was diluted in 9 .mu.l Hi-Di Formamide (Applied Biosystems,
#4311320) containing LIZ-500 internal size standard (Applied
Biosystems, #4322682). The resulting products were detected on an
ABI capillary 3730 DNA analyzer and visualised on a 4% agarose gel.
The D1Rat122 alleles are 230 bp and 255 bp for DA and Fischer rat
strains respectively. The D3Rat17 alleles are 140 bp and 178 bp for
DA and Fischer strains respectively.
[0216] Teratoma Generation.
[0217] Approximately 200-400 cells were injected under kidney
capsules of SCID mice (BALB/c JHan Hsd-Prkdc scid.). Tumours were
collected at various times, and their weight determined by weighing
tumour and kidney, and subtracting the weight of the contralateral
uninjected kidney. Tumours were embedded in paraffin wax,
sectioned, and stained with Masson's trichrome
[0218] Antibodies.
[0219] Plates upon which cells were growing were fixed in 4% PFA in
PBS, permeabilised with PBST (0.3% Triton in PBS) and blocked in 1%
BSA and 10% goat serum in PBST for 2 hours at room temperature. The
primary antibodies were anti-oct-4 C10 (Santa Cruz Biotechnology,
1:200) anti-nanog (Abcam, 1:200) and anti-cdx2 (Abcam, 1:80), which
were left on the plates overnight at 4.degree. F. The secondaries
were Alexa 488 IgG2b goat anti-mouse (for oct-4) Alexa 568 goat
anti-rabbit IgG (for nanog) and Alexa 568 IgG1, goat anti mouse
(for cdx2), all at a concentration of 1:1000, left on the plates
for 2 hours at RT. Plates were counterstained with DAPI. Omission
of the primary antibodies resulted in no fluorescence (nanog, cdx2)
or a faint overall background (oct-4). For X chromosome
immunostaining, cells were plated onto microscope slides
(SuperFrost Plus, VWR international) and then incubated for
approximately 3 hours. Cells were then fixed in 4% paraformaldehyde
for 15 min at room temperature and permeabilized for 10 min in 0.5%
Triton X. Antiserum against H3K27me3 (generous gift from Thomas
Jenuwein) was used at 1:500.
[0220] RT-PCR.
[0221] RNA was purified from around 50 colonies using RNeasy Mini
Kit (Qiagen, #74104). cDNA was subsequently generated by Oligo-dT
priming using SuperScript First-Strand Synthesis System (Invitrogen
Corporation, #11904-018). Typically one tenth of cDNA was PCR
amplified on a PTC-200 thermocycler (MJ Research) using GoTaq Flexi
DNA Polymerase (Promega Corporation, #M8305) under the following
conditions; 94.degree. C. 2 minutes, followed by 30 cycles of
94.degree. C. 20 seconds, 50.degree. C. 20 seconds and 72.degree.
C. 1 minute, with a final extension at 72.degree. C. for 5 minutes.
The primer sequences used and the product sizes are listed
below.
TABLE-US-00003 Product Rat mRNA Rat primer sequence size
.beta.-Actin For - CACTGGCATTGTGATGGACT 427 bp Rev -
ACGGATGTCAACGTCACACT Brachyury For - AACTGCGAGTGGGTCTGGAAG 451 bp
Rev - TGGGTCTCGGGAAAGCAGTG Cdx2 For - CCGAATACCACGCACACCATC 394 bp
Rev - CTTTCCTTGGCTCTGCGGTTC Eras For - CGAGCGGTGTGGGTAAAAGTG 501 bp
Rev - GGTGTCGGGTCTTCTTGCTTG Err.beta. For - TGTGCGGGGACATTGCTTCTG
436 bp Rev - TCCCGATCTGCCAAGTCACAG FGF4 For - CGGGGTGTGGTGAGCATCTTC
202 bp Rev - CCTTCTTGGTCCGCCCGTTC GATA-6 For -
TCATCACGACGGCTTGGACTG 467 bp Rev - GCCAGAGCACACCAAGAATCC Kdr For -
ATACACCTGCACAGCGTACAG 271 bp Rev - TCCCGCATCTCTTTCACTCAC Nanog For
- GCCCTGAGAAGAAAGAAGAG 356 bp Rev - CTGACTGCCCCATACTGGAA Nestin For
- AGAGAAGCGCTGGAACAGAG 234 bp Rev - AGGTGTCTGCAACCGAGAGT Oct-4 For
- GGGATGGCATACTGTGGAC 412 bp Rev - CTTCCTCCACCCACTTCTC Pax6 For -
GAGACTGGCTCCATCAGACC 212 bp Rev - CTAGCCAGGTTGCGAAGAAC Rex-1 For -
TTCTTGCCAGGTTCTGGAAGC 277 bp Rev - TTTCCCACACTCTGCACACAC Sox2 For -
GGCGGCAACCAGAAGAACAG 414 bp Rev - GTTGCTCCAGCCGTTCATGTG Sox17 For -
AGGAGAGGTGGTGGCGAGTAG 268 bp Rev - GTTGGGATGGTCCTGCATGTG Stella For
- TCCTACAACCAGAAACACTAG 304 bp Rev - GTGCAGAGACATCTGAATGG Mouse
Product mRNA Mouse primer sequence size Nanog For -
ATGAAGTGCAAGCGGTGGCAGAAA 464 bp Rev - CCTGGTGGAGTCACAGAGTAGTTC
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Comparative Example 1
[0251] EP1726640, which names Teratani and Ochiya as inventors,
purports to describe the provision of a rat embryonic stem cell.
However, in the following experiments, the present applicant has
shown that the culture conditions described by Teratani and Ochiya
do not support the survival or proliferation of isolated rat ICMs
and hence these conditions are not suitable for the derivation or
maintenance of rat ES cells.
I. Culture of Established Rat ES Cells (Derived in 3i Conditions)
According to the Teratani and Ochiya Protocol
[0252] A. Medium was made up as specified in the Teratani and
Ochiya protocol (that is: rLIF, KSR and other components as
specified in EP1726640, in the concentrations specified, in
DMEM)
[0253] B. Feeder layers were prepared from gamma-irradiated MEFs
(primary cultures of mouse embryonic fibroblasts, passage 3)
[0254] C. Colonies from established rat ES cell lines (derived
according to the procedures described in Example 4-lines oct-4 1
(passage 12), DA 2 (passage .about.10) C (passage 22) and D
(passage 20) were disaggregated and transferred to wells of a
4-well plate, in medium as in A and on feeders as in B. This was
labelled plate 253. Cultures prepared from the same lines were
maintained in parallel in 3i conditions
[0255] D. Results [0256] Day 2. Lines oct-4 1 and DA 2: poor:
rounded cells appearing on surface of colonies. Lines C and D:
still look undifferentiated [0257] Day 6. All poor. Much cell
death, much differentiation as cobble-like cells on bottom of the
dish, also as detaching rounded cells. Cells do not appear to have
proliferated. Medium changed [0258] Day 8. All cells differentiated
or dead. Cultures consist of large lumps of opaque brown material,
with many cells outgrowing on the bottom of the wells [0259]
Control cultures (in 3i) appear undifferentiated, and have
proliferated extensively
[0260] C. Conclusions. All cells cultured in the conditions
specified in the Teratani and Ochiya protocol died or
differentiated within 8 days: this protocol, therefore, is
incapable of maintaining rat ES cells in a viable, undifferentiated
and proliferative state.
II. Attempted Derivation of Rat ES Cell Lines in the Conditions
Specified in the Teratani and Ochiya Protocol
[0261] A. Medium and feeder layers were as described in IA and B,
above
[0262] B. Rat blastocysts (4.5 dpc) were collected and subjected to
immunosurgery.
[0263] C. 7 isolated inner cell masses (ICMs) were transferred to
the medium specified in Teratani and Ochiya (rLIF, KSR, etc.)
(plate 258 B)
[0264] D. As controls, 6 isolated ICMs were transferred to 3i
medium (plate 258 A)
[0265] E. Several outgrowths could be seen in the control well (A)
but none in the well with the Teratani and Ochiya conditions (B).
The cultures were fixed.
[0266] F. After DAPI staining, ICM outgrowths were identified in
the 3i control group. Cells in all had proliferated extensively
[0267] G. 1/7 outgrowths could be identified in the Teratani/Ochiya
group. It was extremely small and identifiable as an outgrowth only
after DAPI staining.
[0268] H. Conclusions. The Teratani and Ochiya protocol does not
support the survival or proliferation of isolated rat ICMs. There
is no evidence to support the assertion that rat ES cells can be
derived by this method.
Example 5
Derivation and Characterization of Ovine Pluripotent Cells
[0269] The following example describes the provision of ovine
embryonic stem (ES) cells that can not be obtained by conventional
methods. The ovine ES cells have been established and produced by
performing a procedure comprising the following steps (A)-(D),
using a novel, serum-free culture system:
(A) dissociating an inner cell mass formed by the culture of ovine
blastocysts, remaining in a state of cell aggregation, (B)
culturing primary embryonic stem cells resulting from the culture
of the dissociated inner cell mass until it can be transferred or
passaged, (C) dissociating the primary embryonic stem cells, which
have become capable of being transferred or passaged, remaining in
a state of cell aggregation, followed by passaging and culturing
the same, and (D) further transferring or passaging and culturing
the cells to establish an embryonic stem cell line.
[0270] The methods for ovine ES cell establishment, the method for
characterizing the established ovine ES cells and the method for
subculturing ovine ES cells are as follows;
1. Ovine.
[0271] The methods are suitable for deriving ES cells from any
strain of ovine. For example, ES cells can be derived from ovine
strains such as Merino strain and the like.
2. Feeder Cells.
[0272] It is preferable to use feeder cells for the establishment
and the subsequent culture and transfer of ovine ES cells. Feeder
cells may be ones derived from any species, and are preferably
normal fibroblasts rather than but not to the exclusion of
established and/or immortalised lines of feeder cells (e.x., DIAMs
(C3H10T1); Rathjen P D, Toth S, Willis A, Heath J K, Smith A G.
Differentiation inhibiting activity is produced in
matrix-associated and diffusible forms that are generated by
alternative promoter usage. Cell 1990: 62:1105-1114), with or
without genetic-based selection markers. Specifically, normal mouse
embryonic fibroblasts can be mentioned, but not to the exclusion of
new born or adult fibroblasts. More specifically, primary cultured
cells of normal mouse embryonic fibroblasts between the 12th and
16th days of pregnancy can be used. As the normal fibroblasts, for
example, normal fibroblasts of 129sv fetal mouse at the 12.5th day
are exemplified. The feeder cells can be prepared by a conventional
method or commercially available products (mouse fetal fibroblasts;
ATCC) can also be utilized. It is preferable to use the feeder
cells inactivated by the treatment with mitomycin C,
gamma-irradiation and the like. It may also be preferable to
pre-coat the intended culture vessel with a matrix such as, but not
exclusively, gelatin, fibronectin, laminin and the like.
3. Culture Medium.
[0273] For the production, establishment and culture of the ovine
ES cells, a substantially serum-free culture medium was used. The
specific compositions of the culture media used are detailed
below;
a) Culture Medium for Ovine ES Cell Establishment.
[0274] A culture medium used in the steps from blastocysts to inner
cell mass formation is referred to as "culture medium for ovine ES
cell establishment". An example of the composition is as described
in Dattena M, et al (Dattena M, Mara L, Bin T A A, Cappai P.
Lambing rate using vitrified blastocysts is improved by culture
with BSA and hyaluronan. Mol Reprod Dev. 2007 January;
74(1):42-7).
b) Culture Medium for Ovine ES Cells.
[0275] A culture medium used in the culture after inner cell mass
formation (including culture of established ovine ES cells) is
referred to as "culture medium for ovine ES cells" or hereafter
"N2B27+3i". A specific example of composition; 100 mL Dulbecco's
modified Eagle medium/F12 with 5 mM L-alanyl-glutamine or 2 mM
L-glutamine (Invitrogen/Gibco) (Table II), 1 mL of 100.times.N2
supplement (Table II; 25 .mu.g/mL insulin, 16 .mu.g/mL putrescine,
100 .mu.g/mL transferrin, 30 nM sodium selenite, 6 ng/mL
progesterone, 50 .mu.g/mL bovine or human serum albumin, final
concentrations) added to 100 mL (1:1 v/v) Neurobasal medium
(Invitrogen/Gibco) (Table II), 2 mL 50.times. B27 supplement (Table
II; 2 mg/L L-alanine, 3.7 mg/L L-glutamate, 441 mg/L 1-glutamine,
7.76 mg/L L-proline, 0.10 mg/L biotin, 0.34 mg/L vitamin B12, 0.02
mg/L corticosterone, 0.0063 mg/L progesterone, 0.1 mg/L vitamin A,
0.1 mg/L retinol acetate, 4 mg/L insulin, 0.002 mg/L
triiodo-L-thryonine, 25 mg/L sodium pyruvate, 0.047 mg/L lipoic
acid, 1 mg/L vitamin E, 1 mg/mL D,L-.alpha.-tocopherol acetate, 2.5
mg/L catalase, 1 mg/L reduced glutathione, 2.5 mg/L superoxide
dismutase, 2 mg/L L-carnitine, 1 mg/L ethanolamine, 15 mg/L
D(+)-galactose, 2600 mg/L HEPES, 16.1 mg/L putrescine, 0.016 mg/L
sodium selenite, 0.194 mg/L zinc sulphate, 1 mg/L linoleic acid, 1
mg/L linolenic acid, 2500 mg/L bovine or human serum albumin, 5
mg/L transferring, final concentrations), 3 .mu.M CHIR99021, 0.8
.mu.M PD184352, 2 .mu.M SU5402 [Antibiotic-Antimicrotics solution
(optional)].
4. Culture Conditions.
[0276] The temperature of ovine ES cell culture in the production,
establishment and culture of ovine ES cells was within the range of
35.degree. C.-37.5.degree. C., and is preferably 37.degree. C. The
culture was carried out in a humidified 5% CO.sub.2 incubator used
for typical cell culture.
5. Method for Ovine ES Cell Establishment.
[0277] A specific example of the method for ovine ES cell
establishment is described as follows;
a) Oocyte (Embryo in Blastocyst Stage) Collection.
[0278] An ovine for oocyte collection was selected. Oocyte
collection was carried out by a conventional method described in
Dattena M, et al (Dattena M, Mara L, Bin T A A, Cappai P. Lambing
rate using vitrified blastocysts is improved by culture with BSA
and hyaluronan. Mol Reprod Dev. 2007 January; 74(1):42-7), although
it will be appreciated that other conventional methods of oocyte
collection can also be used. Specifically, ovines were naturally
crossed, the female ovine for oocyte sampling was sacrificed to
excise a uterus after vaginal plug detection. This uterus was
perfused with a suitable medium to recover fertilized oocytes
(embryos). The development or maturation proceeded from fertilized
oocytes (embryos) through moluras to blastocysts (embryos in
blastocyst stage) and was confirmed by microscopic observations
that the development has proceeded to blastocyst stage. Preferably,
the development proceeds up to the late blastocyst stage and where
the inner cell masses are present and visible.
b) Formation and Isolation of Inner Cell Masses.
[0279] The blastocysts obtained in the aforementioned 5a) were
confirmed microscopically, and zona pellucidas were removed using,
for example, Acidic Tyrode (pH 2.5), hyaluronidase, pronase. Then,
feeder cells treated with mitomycin C were seeded onto 0.1%
gelatine/PBS-coated culture dishes, 5-10 zona pellucida-removed
ovine blastocysts were transferred to each of the dishes, and the
culture was started using the aforementioned "N2B27+3i" culture
medium for ovine ES cell establishment.
[0280] Between the 1st and 8th days of the culture, zona
pellucida-removed ovine blastocysts (late stage) adhered to the
feeder cells. On 5-10 days after adhesion, the inner cell mass from
the blastocysts was mechanically dissociated using a tapered glass
Pasteur pipette or the like. The inner cell mass fragments or
aggregates of 5-20 cells were transferred to a like culture
dishe(s) aforementioned containing the "N2B27+3i" culture medium
for ovine ES cell establishment. At the time, the inner cell mass
was not dissociated with a protease such as trypsin-EDTA and the
like, but mechanically dissociated as described above.
c) Establishment of Ovine ES Cells.
[0281] In the 0.1% gelatin/PBS-coated culture dishes wherein the
feeder cells were seeded, inner cell masses dissociated as
described above were cultured in the "N2B27+3i" culture medium for
ovine ES cells. Primary ES cell colonies appeared between the 4th
and 10th days of the culture. The appearance of the primary ES cell
colony was confirmed by microscopic observations and the ES cells
observed are referred to as "primary ES cells". By continuing the
culture for about 5-10 days thereafter, the primary ES cell colony
became capable of being further transferred or passaged. The "state
capable of being passaged" used herein means a state wherein the
number of cells constituting the primary ES cell colony formed has
reached approximately 200-600. While microscopically confirming
that it has such morphology, the ES cell colony was separated using
a tapered glass Pasteur pipette. This separated ES cell colony was
transferred to a sterile intermediate culture vessel containing a
culture medium for ovine ES cells, and dissociated enzymatically to
single cells with a protease such as trypsin-EDTA, preferably in
the presence of an apoptosis inhibitor (e.x., Y-27632; Watanabe K
et al. A ROCK inhibitor permits survival of dissociated human
embryonic stem cells. Nature May 27 (2007)), or mechanically into
cell aggregates consisting of about 5-20 cells. The dissociated ES
cell colony was subjected to a primary culture (cells at passage 1)
in a culture medium for ovine ES cells in a 0.1% gelatin/PBS-coated
culture dish wherein the feeder cells were seeded. An ES cell
colony appeared after about 2-4 days and became in a state capable
of being passaged about 5-10 days after the beginning of the
culture. The passage of the cells thereafter was performed as
above.
6) Presentation of Ovine ES Cells.
[0282] The ovine ES cell is expected to be characterised some or
all of the following properties (a)-(m), many of which have been
experimentally confirmed:
(a) the ES cell colony presenting as compact, near homogenous, (b)
expressing Oct3/4 gene, Sox2 gene, Stella gene, Rex1 gene, FGF4
gene and Nanog gene, (c) positive for alkaline phosphatase
activity, (d) having an embryoid body forming ability, (e)
differentially expressing SSEA (Stage-Specific Embryonic
Antigen)-1, -3 and -4, (f) expressing TRA (Tumour Rejection
Antigen)-1-60, -1-81, (g) having the same number of chromosomes as
does a normal ovine cell, (h) capable of being subcultured and
remaining in the undifferentiated state, (i) having in vitro
pluripotency state, (j) having a potential to differentiate to
cells of three embryonic germ lineages, (k) having in vivo teratoma
formation ability, (l) having an ability to produce a chimaeric
ovine and, (m) ability to be ovine germ-line capable.
[0283] The present invention provides an ovine ES cell retaining
all the properties of an ES cell shown in the aforementioned
(a)-(m) for the first time. The aforementioned attributes (a)-(m)
can be analyzed by the following methods that the established ovine
ES cell retains the properties as an ES cell, that is the
properties as an ES cell maintaining an undifferentiated state.
6.1) Morphology.
[0284] The ovine ES cells maintained a compact, near homogenous
colony as visualised by microscopy. (FIGS. 15A-D)
6.2) Expression of Gene Markers for Undifferentiated State.
[0285] The presence or absence of expression of representative
undifferentiated state gene markers such as Oct3/4, Nanog, Stella,
FGF4, Sox2 and Rex-1 are characteristic of ES cells. Ovine ES cells
were analysed by reverse transcriptase polymerase chain reaction
(RT-PCR) for the expression of the undifferentiated state gene
markers (Table I). The housekeeping gene, .beta.-Actin, was also
used to assess the integrity of the cDNA. Commercial kits were used
to extract mRNA from the samples and perform the synthesis of cDNA
by RT-PCR (TRIzol reagent and SuperScript III First Strand
Synthesis System for RT-PCR, Invitrogen) according to the
manufacturer's instructions. Briefly, TRIzol extracted mRNA was
converted to cDNA by SuperScript III reverse transcription in 20
.mu.L RT reaction volume containing 4 .mu.L SxFirst Strand Buffer,
1 .mu.L DTT (0.1M), 1 .mu.L dNTP's (10 mM), 1 .mu.L random primers
(10 .mu.M), 1 .mu.L RNA inhibitor, 1 .mu.L Superscript III and 7.5
.mu.L H.sub.2O and 3 .mu.L mRNA sample. A corresponding RT
negative, without Superscript III, was also prepared to determine
genomic contamination. The reaction was then processed in a Thermal
Cycler (Perkin Elmer) with the following cycle parameters:
50.degree. C. for 60 mins and 70.degree. C. for 15 mins. The cDNA
was then used for PCR with primers of the undifferentiated state
gene markers (Table I). A standard 25 .mu.L PCR reaction volume was
used containing 2.54, 10.times.PCR buffer, 1.5 .mu.L MgCl.sub.2 (25
mM), 0.5 .mu.L dNTPs (10 mM), 1 .mu.L Forward Primer (10 .mu.M), 1
.mu.L Reverse Primer (10 .mu.M), 0.2 .mu.L recombinant Taq DNA
Polymerase, 17.3 .mu.L H.sub.2O and 1 .mu.L RT sample. A reaction
sample was then cycled using the following parameters: 94.degree.
C. for 5 mins, 35 cycles of (94.degree. C. for 45s, 55.degree. C.
for 45 sec and 72.degree. C. for 45 sec), and 72.degree. C. for 5
mins. Expression of Oct3/4, Nanog and Sox2 was visualised after a
small volume of the final reaction aforementioned was
electrophoresed on a 2.5% agarose gel, stained with ethidium
bromide and photographed under UV illumination (FIG. 16).
6.3) Alkaline Phosphatase Activity.
[0286] Undifferentiated ovine ES cell were observed to expresses an
alkaline phosphatase in large amounts. The expression of the
alkaline phosphatase can be easily determined using various
commercially available alkaline phosphatase detection kits. In the
present example the ALP tissue staining kit (Sigma) was used
according to the manufacturer's instructions. (FIG. 15B).
6.4) Embryoid Body Forming Ability.
[0287] Embryoid body formation by culturing ES cell and/or
aggregates using a non-coated culture dish under conditions without
feeder cells. The embryoid body formation was confirmed by
microscopic observations of the appearance of regular or irregular
spheroid-like body formed by cell aggregation after ovine ES cells
were cultured using a culture medium for ovine ES cells without 3
.mu.M CHIR99021, 0.8 .mu.M PD184352, 2 .mu.M SU5402 (`3i`) in a
non-coated culture dish for about 2 days to 14 days. (FIGS. 15E and
15F).
6.5) Expression of Nuclear or Cell Surface Antigens.
[0288] One of the characteristics used in identifying the
undifferentiated state of stem cells, including ES cell, is the
detection of nuclear Oct3/4 and Nanog, and cell surface antigens
(SSEA (stage-specific embryonic antigen)-1, -3, -4; TRA (Tumour
Rejection Antigen)-1-60, -1-81) whose expression amounts change
specifically as a consequence upon their differentiation. The
expression of the cell surface markers was evaluated by an
immunostaining using ES Cell Characterization Kits (Millipore)
according to the manufacturer' instructions (data not shown).
6.5) Chromosome Number.
[0289] Normal, expected chromosome number for ovine diploid cells
can be confirmed by analyzing the chromosome number by G-banding
method (e.x., Sumner, A. T., Cancer Genet Cytogenet. 6: 59-87
(1982)) that the established ovine ES cell is a normal ES cell
maintaining the chromosome number (2n=54) of ovine from which it
originates.
6.6) Maintenance of Undifferentiated State.
[0290] The established ES cell can be subcultured with maintenance
of undifferentiated state, characteristically that it can be
subcultured until at least 30 passages. The maintenance of
undifferentiated state can be confirmed by performing a subculture
according to the subculture method of the ovine ES cell (the
aforementioned 3), and determining the aforementioned (6)
presentation of ovine ES cells.
6.7) Pluripotency.
[0291] ES cell spontaneously differentiates into various cells
through an embryoid body by culturing it under conditions without
feeder cells. This property of ES cell can be observed by forming
an embryoid body by the method described in the aforementioned
6.4), then transferring the embryoid body to a gelatin-coated
culture dish and culturing for about 7 days to 14 days. The
appearance of neuron-like cell, adipose-like cell or epiderm-like
cell or the like can be confirmed by the characteristic morphology
of the each cell.
6.8) In Vitro Differentiation by Culture in the Presence of
Serum.
[0292] Moreover, the ES cell has a property that it differentiates
by culture in the presence of 20% serum. Preferably, the ovine ES
cell differentiates by culture in the presence of 2-20% serum. The
differentiation of ovine ES cell in vitro can be confirmed by the
disappearance of alkaline phosphatase activity, or disappearance of
expression of ES cell undifferentiated state marker genes such as
Oct3/4, Nanog and the like (aforementioned 6 (a)-(f)).
6.9) Differentiation Potential to Cells of Three Embryonic Germ
Lineages.
[0293] ES cells have a potential of differentiating to cells of
three embryonic germ (endoderm, mesoderm, ectoderm) lineages. This
property of ES cells can be confirmed by extracting RNA from an
embryoid body (aforementioned 6.4), and analyzing the expression of
each marker gene for ectodermal cell (e.g., neuron), mesodermal
cell (e.g., cardiomyocyte) and endodermal cell (e.g., hepatocyte)
by RT-PCR.
6.10) Ability of Teratoma Formation.
[0294] The transplantation of ES cells to a homologous or innate
immunodeficient heterologous animal can lead to teratoma formation.
The teratoma is the designation of a mixed tumor wherein various
tissues derived from three embryonic germs, endoderm, mesoderm and
ectoderm, are randomly present in a tumor. The formation of the
teratoma can be confirmed by transplanting ovine ES cells to the
subdermic and the like of a homologous animal or a heterologous
animal with a primary immune deficiency, and macroscopically
observing the presence of a bulbous growth after several months. It
can be confirmed that the teratoma formed has three embryonic germ
structures by sectioning the excised teratoma, staining with
hematoxylin/eosin and observing the morphology of the tissues and
cells microscopically.
6.11) Chimaeric Ovine-Producing Ability.
[0295] A chimaeric ovine can be produced by introducing ES cells
into a homologous or heterologous ovine. The production of the
chimaeric ovine can be carried out by, for example, the following
method. To facilitate confirmation of the production of the
chimaeric ovine, a marker gene (e.g., GFP, X-gal, luciferase, etc.)
may be previously introduced into the ovine ES cell. Specifically,
a recombinant ovine ES cell incorporating a vector containing such
marker gene in the ES cell chromosome is established by
incorporating the aforementioned vector into the ovine ES cell
chromosome by an electroporation method and the like, followed by
selection in a culture medium supplemented with a drug. The
recombinant ovine ES cells are, for example, transplanted into a
blastocoele of a ovine blastocyst or into morula stage or 16-cell
stage embryo by a microscopic manipulation and developed with an
inner cell mass or as a part of an inner cell mass (microinjection
method: Gordon J. W. et al., Proc. Natl. Acad. Sci. USA., 77:
7380-7384 (1980)). Alternatively, zona pellucidas are removed from
two 8-cell embryos and the embryos are co-cultured with the
aforementioned recombinant ovine ES cells to form an aggregate.
When the resulting aggregate is cultivated, one blastocyst is
obtained (cell aggregate method: Dvorak P. et al., Int. J. Dev.
Biol., 39: 645-652 (1995)). The embryo (egg for transplantation)
obtained above is transplanted in the uterus of a pseudopregnant
female ovine prepared by natural crossbreeding with a male ovine
after vasoligation treatment and developed, whereby a chimaeric
ovine can be produced.
[0296] That the obtained chimaeric ovine has cells and tissues
derived from an ES cell, or an established rat ES cell has a
chimaeric ovine-producing ability can be confirmed by, for example,
genomic PCR using genomic DNA extracted from various tissues of
chimaeric ovine as a template and marker gene (marker gene
introduced into ES cell)-specific primers. Moreover,
differentiation of ES cell to the cell of each tissue lineage can
be confirmed, for example, by sectioning each tissue of a chimaeric
ovine, and detecting the presence of a marker gene expression
product (marker protein) based on the properties of the marker
protein used.
TABLE-US-00004 TABLE I Ovine Primer sequences. Expected Product
Gene Forward Primer (5'-3') Reverse Primer (5'-3') size (base
pairs) .beta.-Actin ggcacccagcacaatgaaga cgactgctgtcaccttcaccg 340
Oct4 agtgagaggcaacctggagag gacagacaccoaquaaagac 331 Sox2
catccacagcaaatgacagc tttctgcaaagctcctaccg 251 NANOG
agccccagagtgaaaccactgtc gtgttctcacagacccagctg 164
TABLE-US-00005 TABLE II Type DMEMF12 Neurobasal N2 B27
Concentration 1x 1x 1x 1x Constituent concentration mg/L mg/L mg/L
mg/L INORGANIC SALTS, METALS CaCl.sub.2 (anhyd) 116.6 200.0
CuSO.sub.4.cndot.5H.sub.2O 0.0013 Fe(NO.sub.3).cndot.9H.sub.2O
(Iron III) 0.05 0.1 FeSO.sub.4.cndot.7H.sub.2O 0.417 KCl 311.8 400
MgCl.sub.2 (anhydrous) 28.64 77.3 MgSO.sub.4 (anhydrous) 48.84 NaCl
6995.5 3000 NaHCO.sub.3 2438 2200 NaH.sub.2PO.sub.4.cndot.H.sub.2O
62.5 125 Na.sub.2HPO.sub.4.cndot.H.sub.2O 71.02
ZnSO.sub.4.cndot.7H.sub.2O 0.432 OTHER COMPONENTS D-(+)-Galactose
15 D-(+)-Glucose (Dextrose) 3151 4500 Phenol Red 8.1 8.1 HEPES 2600
2600 Hypoxanthine.cndot.Na 2.39 Linolenic acid 1 Linoleic acid
0.042 1 Lipoic acid 0.105 0.047 Sodium Putrescine.cndot.2HCl 0.081
16.11 16.11 Sodium Selenite (Na.sub.2SeO.sub.3.cndot.XH.sub.2O)
0.0052 0.016 Sodium Pyruvate 55 25 AMINO ACIDS L-Alanine 4.45 2
L-alanyl-L-glutamine 542 L-Arginine.cndot.HCl 147.5
L-Asparagine.cndot.H.sub.2O 7.5 L-Aspartic acid 6.65
L-Cysteine.cndot.H.sub.2O 17.56 L-Cystine.cndot.2HCl 31.29
L-Glutamic acid 7.35 3.7 L-Glutamine 441 L-Glycine 18.75
L-Histidine.cndot.HCl.cndot.H.sub.2O 31.48 L-Isoleucine 54.47
L-Leucine 59.05 L-Lysine.cndot.HCl 91.25 L-Methionine 17.24
L-Phenylalanine 35.48 L-Proline 17.25 7.76 L-Serine 26.25
L-Threonine 53.45 L-Tryptophan 9.02
L-Tyrosine.cndot.2Na.cndot.2H.sub.2O 55.79 L-Valine 52.85 VITAMINS
D,L-a-tocopherol (Vit E) 1 D,L-a-tocopherol acetate (Vit E) 1
Biotin (Vit B27/H) 0.0035 0.1 L-Carnitine.cndot.HCl (Vit Bt) 2 D-Ca
panthenate (Vit B5) 2.24 4 Folic acid (Vit M) 2.65 4 (myo)
i-Inositol (Vit Bh) 12.6 7.2 Niacinamide (Vit B3) 2.02 4
Pyridoxine.cndot.HCl (Vit B6) 2.031 4 all trans Retinol 0.1 Retinyl
acetate (Retinol, Vit A) 0.1 Riboflavine (Vit B2) 0.219 0.4
Thiamine.cndot.HCl (Vit B1) 2.17 4 Thymidine (deoxy ribonucleoside)
0.365 Vitamin B12 (Cobalamin) 0.68 0.34 0.34 PROTEINS Albumin
(Bovine or Human serum) 50 2500 Cortisone 0.02 Insulin (Full Chain)
5, 25 4 Transferrin 100 5 Progesterone 0.0063 0.0063 T3
(Triiodothyronine) 0.002 OTHER Catalase 2.5 Choline chloride 8.98 4
Ethanolamine.cndot.HCl 1 L-Glutathione (reduced) 1 Superoxide
dismutase (SOD) 2.5 pH 7.0-7.4 ND ND ND Osmolarity (mOsm/kg
H.sub.2O) 290-330 ND ND ND
Example 6
Derivation and Characterisation of Bovine Pluripotent Cells
Aim
[0297] To assess the efficacy of 3i media to isolate bovine ES
cells and to maintain bovine ES cell lines.
Materials and Methods
Experimental Design
[0298] To assess ES cells isolation, Day 7 or 8 blastocysts that
were produced in vitro (n=105) were randomly allocated to each of
four treatments: [0299] 1. Control (N2B27) [0300] 2. 3i medium
(with hLIF) [0301] 3. 3i medium (without hLIF) [0302] 4.
Millipore-Chemicon ESGRO Complete
[0303] When the putative ES cells expanded they were passaged, and
at this time samples were collected to characterize gene
expression. After 30 days of culture, ES cell cultures and
treatments were evaluated and treatments that did not yield ES cell
lines were discontinued. ES cell lines were cultured for a further
60 days, for a total of 90 days. The proportion of blastocysts in
each treatment that formed embryonal outgrowths, and were cultured
to Passage 1, Passage 3 and Passage 6 was used to compare the
treatments. Morphology and expression of pluripotency genes (i.e.
oct4, rex1, sox2, ssea1, alkaline phosphatase, nanog) were also
used to compare bovine ES cells in different treatments. All
primers generate bands in cultured bovine ES cells except Nanog,
which to date has only generated bands in blastocysts.
[0304] To assess maintenance of established bovine ES cell lines,
established lines (n=3) were passaged to the same four treatments
used in the ES cell isolation experiments, i.e. media 1-4 as
described above. These established cell lines were cultured for up
to 6 passages (-60 days). Morphology and expression of pluripotency
genes (i.e. oct4, rex1, nanog, ssea1) were used to compare
established bovine ES cell lines cultured in different
treatments.
In Vitro Production of Embryos
[0305] Oocytes were collected one day a week to generate
blastocysts in vitro for 4 consecutive weeks.
Cumulus-oocyte-complexes (COCs) were aspirated from ovaries
obtained from an abattoir, and bovine blastocysts were produced in
vitro using previously described methods (1, 2). Briefly, COCs
aspirated from ovaries were cultured for 24 hours in TCM-199
supplemented with .beta.-estradiol, luteinising hormone and fetal
bovine serum to trigger oocyte maturation. COCs with expanded
cumulus were presumed to contain mature oocytes, and were
co-cultured for 24 hours with bovine spermatozoa in IVF-TALP
medium, containing heparin, penicillamine, epinephrine and
hypotaurine. Putative zygotes were then cultured for 6-7 days in
SOF medium (3) supplemented with essential and non-essential amino
acids, tri-sodium citrate dehydrate, myo-inositol and bovine serum
albumin. Gametes were cultured at 39.degree. C. in a humidified gas
environment of 5% CO.sub.2 in air, and embryos were cultured at
39.degree. C. in a humidified gas environment of 5% CO.sub.2/5%
O.sub.2/90% N.sub.2. From the total set of blastocysts, some
blastocysts (n=15) were processed for characterization (positive
control for RT-PCR) and the rest (n=105) were used for ES CELLS
isolation.
ES Cell Isolation and Culture
[0306] Zonae pellucidae were mechanically removed from the
blastocysts and the blastocyst was pressed onto a feeder layer of
mouse embryonic fibroblasts (MEFs) that had been inactivated by
mitomycin C treatment. Different batches of MEF feeders were used
during the study, but each batch was used for all treatments. The
pressed blastocysts on MEFs were cultured with a complex medium
(.alpha.-MEM) supplemented with serum, non-essential amino acids
and growth factors (human LIF, (FGF, EGF) at 39.degree. C. in a
humidified gas environment of 5% CO.sub.2 in air. After 7-9 days
the embryos formed embryonal outgrowths containing putative bovine
ES CELLSs, which were mechanically cut from the primary culture
(Passage 0) and pressed to fresh feeder layers (Passage 1). When
the putative ES CELLSs had expanded (range: 6-26 days), they were
passaged to fresh feeder layers. At the time of passage, samples of
putative ES CELLSs were placed in lysis buffer (500 .mu.l Lysis
Buffer: 458.5 .mu.l DEPC Water, 25 .mu.l DTT (0.1 M), 4 .mu.l
IPEGAL and 12.5 .mu.l RNA inhibitor) and stored at -80.degree. C.
for subsequent analysis of pluripotency gene transcripts. Culture
media were changed every 2-3 days during culture. The nomenclature
used to refer to the passage number of the cell lines isolated in
this study was P.sub.x, where x=the number of passages in this
study.
[0307] Established bovine ES cell lines (BES-1: P.sub.19,183 days;
BES-3: P.sub.18,206 days; BES-4: P.sub.23,210 days) were passaged
to each of the four treatments. These established lines were
cultured as described above. The nomenclature used to refer to the
passage number of the established cell lines after they had been
allocated to this study was P.sub.x+y, where x=passage number
before this study and y=the number of passages in this study.
RT-PCR Analysis of Gene Expression
[0308] Blastocysts (positive control) and putative bovine ES cells
from recently isolated or established bovine ES cell lines were
analysed by reverse transcriptase polymerase chain reaction
(RT-PCR) for the expression of the pluripotency genes oct4, rex1,
sox2, ssea1, alkaline phosphatase (AP) and nanog. The housekeeping
gene, .beta.-Actin, was used to assess the quality of cDNA. A
commercial kit was used to extract mRNA from the samples
(Dynabeads.RTM. mRNA DIRECT.TM. Micro Kit, Invitrogen). The
standard protocol recommended by the manufacturer was used to
extract the mRNA. Briefly, mRNA was converted to cDNA by Reverse
Transcription. A 20 .mu.l RT system was used: 4 .mu.l 5*First
Strand Buffer, 1 .mu.l DTT (0.1 M), 1 .mu.l dNTP Mixture (10 mM), 1
.mu.l random primers (10 .mu.M), 1 .mu.l RNA inhibitor, 1 .mu.l
Superscript III and 7.5 .mu.l H.sub.2O and 3 .mu.l sample. A
corresponding RT negative, without Superscript III, was prepared to
check for genomic contamination. All reagents were purchased from
Invitrogen. The prepared solution was processed in a Mycycler
Thermal Cycler (Bio-rad) with the following program: 50.degree. C.
for 60 mins and 70.degree. C. for 15 mins. cDNA quality was checked
by PCR with .beta.-Actin primers. Proved cDNA was then used for PCR
with primers of the pluripotency markers. A standard 25 .mu.l PCR
system was used: 2.5 .mu.l 10* PCR buffer, 1.5 .mu.l MgCl.sub.2 (25
mM), 0.5 .mu.l dNTP Mixture (10 mM), 1 .mu.l primer Forward (10
.mu.M), 1 .mu.l primer Reverse (10 .mu.M), 0.2 .mu.l Taq DNA
Polymerase, recombinant, 17.3 .mu.l H.sub.2O and 1 .mu.l sample.
All reagents were purchased from Fisher Biotec. The prepared
solution was processed in a Mycycler Thermal Cycler (Bio-rad) with
the following program: 94.degree. C. for 5 mins, 35 cycles of
(94.degree. C. for 45 s, 55.degree. C. for 45 s and 72.degree. C.
for 45 s), and 72.degree. C. for 5 mins.
[0309] The PCR products for OCT4 and Rex1 have been confirmed by
sequencing. The PCR products for SSEA1, Sox2, Alkaline Phosphatase
and Nanog have not yet been sequenced, but the size of the product
is the same as that of the target genes.
[0310] The primers used for this study were as follows:
TABLE-US-00006 Markers Primer F Primer R Size .beta.-Actin
ggcacccagcacaat cgactgctgtcaccttc 340 gaaga accg Oct4
agtgagaggcaacct gacagacaccgagggaa 331 ggagag agac Rex1
gcagaatgtgggaaa gactgaataaacttctt 220 gcct gc SSEA1 gagtcaatacgtgac
ctgaagtagccgcgata 330 cgtggac gacag SOX2 catccacagcaaatg
tttctgcaaagctccta 251 acagc ccg NANOG tgtgctcaatgacag
tagaagcctgggtattc 210 atttcag tgc AP ggacccaggaaacca
gaatgagggagtgagca 378 aagtc aacc
Differentiation In Vitro
[0311] Explants excised from bovine ES cell colonies were placed in
conditions to promote random differentiation (i.e. low-adhesion
culture vessel, no feeder layer, no LIF, no growth factors), and
cultured at 39.degree. C. in a humidified gas environment of 5%
CO.sub.2 in air. Culture medium was changed every 3-4 days during
culture.
Data Analysis
[0312] Continuous data (days in culture) are reported as arithmetic
mean.+-.standard error of the mean (S.E.M.). These data were
limited and therefore not analysed statistically. Category data
were analysed by a chi square analysis of contingency tables (4,
5). If the analysis returned a significant difference in the data
set (i.e. p.ltoreq.0.05), standardized residuals and percentage
deviations were used to determine which observed chi square
frequencies differed most from the expected frequencies, based on
the null hypothesis (5). These differences are reported as positive
or negative associations between the observation and the
treatment.
Results
Growth in Culture
Isolation and Passage of Bovine ES Cells
[0313] The proportion of embryos that formed embryonal outgrowths
varied significantly between treatments (.chi..sup.2.sub.3=12.49,
p=0.0059), and 80% or more of the embryos cultured with media 2-4
formed outgrowths (FIG. 17). There was a negative association
between the % outgrowths and Control group, so that fewer embryos
formed outgrowths when cultured with Control medium. The proportion
of embryonal outgrowths that were passaged varied significantly
between treatments (.chi..sup.2.sub.3=9.96, p=0.0189), with 65% or
more of the blastocysts cultured with SCS1 or SCS2 being passaged
(FIG. 17). There was a positive association between % Passaged and
medium 2 (3i plus hLIF), and a negative association between %
Passaged and Control group. The proportion of bovine ES cell lines
at Passage 3 (.chi..sup.2.sub.3=12.02, p=0.0073) and Passage 6
(.chi..sup.2.sub.3=13.81, p=0.0032) varied significantly between
treatments (FIG. 18). More than 60% of blastocysts cultured with
media 2 and 3 (3i with or without hLIF) were passaged to Passage 3,
and there was a negative association between the % Cell lines at P3
and Control group. More than 40% of blastocysts cultured with
medium 2 or 3 were passaged to Passage 6, and there was a positive
association between the % Cell lines at P6 and medium 2 and 3
groups and a negative association between the % Cell lines at P6
and medium 4 and Control groups. During isolation at Passage 0, ES
cell colonies expanded at a similar rate when cultured with any of
the four media (FIG. 3). After 50 days of culture, all cell lines
isolated with medium 2 (52.8.+-.0.56 days) or medium 3
(54.0.+-.1.13 days) had progressed to P6 or later, while some cell
lines isolated with Control (n=2) or medium 4 (n=3) had not yet
been passaged to Passage 6 (i.e. still in Passage 5).
Culture of Established Bovine ES Cell Lines
[0314] Growth of one of the established bovine ES cell lines,
BES-1, was retarded by MEF feeders that were not completely
inactivated (see Results: General Morphology in Culture). This cell
line was only cultured in the treatments for three passages and the
data were not analysed, but explants from BES-1 were tested for
their potential to differentiate in vitro after culture in the
treatments (see below). The other two established bovine ES cell
lines, BES-3 and BES-4, were cultured for 8 passages in each of the
four media. There were no apparent differences among treatments or
between cell lines in explant survival, number of passages
supported, or the number of days to complete 8 passages (Table 3).
During culture with each of media 1-4, ES cell colonies from
established bovine ES cell lines expanded at a similar rate (FIG.
20).
TABLE-US-00007 TABLE 3 Comparison of explant survival, number of
completed passages and days in culture to end of Passage 8 of
established bovine ES cell lines, BES-3 and BES-4, cultured with
media 1-4. BES-3 BES-4 Medium 1 2 3 4 1 2 3 4 Explant 36/37 23/23
12/12 13/13 25/27 31/31 31/31 18/18 Survival Completed 8 8 8 8 8 8
8 8 Passages Days in 74 80 80 80 81 76 76 76 Culture to Complete 8
Passages
General Morphology in Culture
[0315] There were no obvious differences in colony morphology
between newly isolated bovine ES cells and those that formed from
established cell lines. Bovine ES cell colonies cultured with
Control medium were round to oval in shape, with an undulate border
(FIG. 21a, 21b, 22a). The colonies were mostly flat (i.e. two
dimensional), with few vertical undulations. Putative stem cells
were mostly concentrated toward the centre of the colony, and the
outer region of the colony contained no apparent ES cells. In some
regions, fluid accumulated between the ES cells and the feeder,
lifting the ES cell layer to form dome and spherical structures. ES
cell colonies cultured with medium 2 (3i plus hLIF) (FIG. 21c, 21d,
22b) or medium 3 (3i without hLIF) (FIG. 21e, 21f, 22c) were round
to oval in shape, with a very distinct, undulate border. These
colonies were more three dimensional in form than those cultured
with Control medium, having a dense centre with many vertical
undulations radiating from the centre. Fluid had accumulated under
some colonies so that they were convex. Colonies had dense, raised
linear structures radiating from the centre. Putative ES cells were
concentrated at the base of these linear structures where they
elevated from the colony. Less dense regions of putative ES cells
were located away from these structures, mostly toward the centre
of the colony and some were located near the colony edge.
Spherical, fluid-filled structures, resembling embryoid bodies, had
formed near the raised linear structures after more than 10 days
culture. ES cell colonies cultured with medium 4 were irregular in
shape with an undulate, distinct border, similar to colonies
cultured with Control medium (FIG. 21g, 21h, 22d). The colonies
were three dimensional, with dense regions of cells forming a
fibrous, undulating network throughout the colony that had no
regular pattern. In some regions, fluid accumulated between the ES
cells and the feeder, forming dome and spherical structures. Cells
in the colony were diffuse and patchy, but the centre of the colony
was more dense and raised. Individual cells were quite apparent and
the putative ES cells were in patches throughout the colony, but
not apparent near the colony edge. Although the different
treatments triggered differences in colony morphology, they did not
appear to promote differences in morphology of putative ES cells
(FIGS. 23 and 24).
[0316] Some batches of MEF feeders cultured with Control medium had
a typical morphology in that they formed a uniform layer on the
culture vessel that remained relatively unchanged throughout the
passage. When cultured with any of media 2-4, the cells of the
feeder began to round up and detach from the feeder layer by Day 3
of the passage, but this response was more acute with medium 4.
Other batches of MEF feeders did not behave in a typical manner
when cultured with Control medium, in that cloudy regions appeared
throughout the layer and the cells seemed to continue to grow.
These MEF feeders peeled away from the culture vessel by about Day
12 of the passage. When cultured with medium 2 or 3, these batches
of MEFs looked very healthy and had a morphology that was typical
of feeders cultured with Control medium. When these MEF batches
were cultured with medium 4, the cells of the feeder rounded up and
detached from the feeder layer by Day 6-8 of the passage.
Appearance and Behaviour During Passage
[0317] Sheets of bovine ES cells (explants) were reasonably easy to
excise mechanically using 29G needles from colonies cultured with
Control medium. There was little movement of the entire bovine ES
cell colony during mechanical passage. When explants were excised,
the ES cell colonies appeared to be a single layer of cells that
grew over the top of the MEF feeder layer. Explants from ES cell
colonies cultured with medium 2 or 3 were more difficult to excise
because the ES cell colonies appeared to be more dense, spongy and
pliable. Much of the colony moved when needles were used to cut
through the sheet of cells. Explants cultured with medium 2 or 3
were generally more easy to attach to fresh MEF feeder plates at
passage, than were explants from colonies cultured with Control
medium. ES cell colonies cultured with medium 4 moved significantly
during passage, similar to colonies cultured with medium 2 or
medium 3. The putative bovine ES cells near the centre of colonies
cultured in medium 4 medium were quite friable at passage and
readily dispersed to smaller clumps making it difficult to excise
explants for passage. Medium 4 explants were quite refractile in
appearance.
Expression of Pluripotency Genes
[0318] Oct4 was frequently and commonly expressed in bovine ES
cells isolated in any of the four media, and other genes were never
expressed without oct4 (FIGS. 25 and 26). Rex1 and ssea1 were also
frequently expressed in bovine ES cells isolated in any of the four
media, and were almost always expressed concurrently. Occasionally,
rex1 was expressed without ssea1 (FIGS. 25 and 26: Control and
medium 3: not shown), but ssea1 was never expressed without rex1.
Sox2 and AP were less frequently expressed and always concurrently
with oct4, rex1 and ssea1. Sox2 and AP were expressed in bovine ES
cells isolated in any of the four media, but their expression was
more common in earlier passages and in bovine ES cells isolated in
3i media (medium 2 or 3). Nanog was very rarely expressed and, on
both occasions (FIG. 26: Control & medium 3), it was expressed
concurrently with oct4, rex1 and ssea1. The only population of
cells analysed that expressed all six pluripotency genes was bovine
ES cells at Passage 1 isolated in medium 3 (3i without hLIF) (FIG.
26).
[0319] The gene expression of bovine ES cells from established ES
cell lines cultured with each of the four media differed in two
aspects (FIG. 27). Firstly, rex1 and ssea1 were expressed in the
absence of oct4, and secondly, the most commonly expressed genes
were rex1 and ssea1, rather than oct4. More genes were expressed in
established cell lines after a number of passages cultured with
media 2-4, including oct4 when it had not been present at earlier
passages.
Ability to Differentiate In Vitro
[0320] Within 3 days explants of one of the established lines
(BES-1) excised from colonies cultured with medium 2 or 3 had
formed fluid-filled, spherical structures and cell aggregates
typical of embryoid bodies (EBs), when placed into conditions to
promote in vitro differentiation (FIG. 28a, 28b). These EBs were
similar in form to those formed by explants excised from colonies
cultured with Control medium. One explant in medium 2 had attached
to the dish. Within 3 days, 2 explants excised from colonies
cultured with medium 4 had attached to the dish. Individual cells
had attached to the culture vessel and had begun to differentiate,
in that they had numerous, long processes extending from a main
cell body (FIG. 28c, 28d). One explant had formed an aggregate of
cells with attached vesicles. The EB cultures from BES-1 became
contaminated with bacteria after 10 days culture and were
discarded. Explants from colonies isolated in this study also
formed EBs and attached colonies when placed into conditions to
promote in vitro differentiation.
Discussion
[0321] Different media induced varied colony morphology, cell
interactions and attachment to the feeder layer, as demonstrated by
microscopic observation and behaviour at passage. During mechanical
passage bovine ES cell colonies cultured with Control medium moved
very little, suggesting that the bovine ES cells were attached to
the feeder layer or to the culture vessel. Unlike human or mouse ES
cell colonies, bovine ES cell colonies grow over the top of the
feeder layer. The dense, spongy, pliable form of bovine ES cell
colonies cultured with 3i media indicates these colonies may have
been multilayered. During mechanical passage bovine ES cell
colonies cultured with media 2-4 moved significantly, suggesting
there was little connection to the MEFs or culture vessel. This is
not surprising, since these three media had a detrimental effect on
MEF feeder cells, causing them to round up and detach from the
feeder. Individual cells in putative bovine ES cell regions were
quite apparent in colonies cultured with medium 4, possibly
indicating there were less connections between cells. While colony
morphology varied greatly among media, differences in cell
morphology were less apparent. Colony morphology and behaviour at
passage was similar for newly isolated bovine ES cells and those
that formed form established cell lines.
[0322] The 3i media (media 2 and 3) were more efficient for the
isolation of bovine ES cells since more blastocysts formed
embryonal outgrowths and were taken to Passage 1. These two media
were also superior for bovine ES cell expansion and maintenance,
since there were more cell lines at Passage 3 and Passage 6. All
cell lines in 3i media (media 2 and 3) were at Passage 6 or later,
while some cell lines cultured with medium 4 and Control medium
remained viable at Passage 5 at the end of this study. This
suggests bovine ES cell growth and development was faster in 3i
media. In this study, Control medium was least efficient for the
isolation and subsequent maintenance of bovine ES cells, since
fewer embryos formed outgrowths and were passaged, and there were
fewer cell lines at Passage 3 and Passage 6 (i.e. all parameters
analysed).
[0323] There was a problem with some MEF feeder layers used in this
study, in that they continued to grow and were probably not
completely inactivated by the mitomycin C treatment. This may have
selectively disadvantaged the control group as media 2-4 appeared
to be detrimental to the MEF feeders and slowed the growth of any
non-inactivated feeder cells, whereas Control medium had no effect
on growth of the feeders. However, the same batches of MEF feeders
were used in all four treatment groups in this study, so
comparisons between treatments are valid. Differences in growth and
development of established bovine ES cell lines were not apparent
among the four media.
[0324] OCT4, Rex1 and SSEA1 are strong, robust pluripotency markers
in bovine ES cells, and this was true for recently isolated ES
cells and for established bovine ES cell lines. Oct4, Rex1 and
ssea1 are usually expressed in bovine ES cells and, in this study,
other pluripotency genes were never expressed in their absence. In
newly isolated bovine ES cells the expression of rex1 and ssea1 was
dependent on the expression of oct4, and the expression of ssea1
was dependent on the expression of rex1. The expression of these
three genes was independent of treatment group. The expression of
other pluripotency genes was irregular, sporadic and related to
culture media, and culture with 3i media induced expression of
other pluripotency genes. In established bovine ES cell lines, oct4
was often not expressed in early passages in this study, yet rex1
and ssea1 were expressed. Interestingly, oct4 was expressed in
established ES cell lines after culture in media 2-4, even though
it had not been detected in earlier passages. This suggests that
the media induced expression of other pluripotency genes, which is
similar to newly isolated ES cells. The OCT4 primers used in this
study produce one clear band in blastocysts and in vivo embryos,
but two bands in in vitro isolated ES cells. The smaller band has
been sequenced and confirmed as OCT4, and the larger band is not
genomic DNA, since the negative controls do not show the presence
of this band. Our in vitro culture system may induce a pseudogene
or isoform of oct4 to be expressed and to confirm this, the second
band is being sequenced.
[0325] There were no significant differences in bovine ES cells
cultured with medium 2 or 3 (3i medium), which varied only by the
presence of hLIF in medium 2. This indicates that hLIF had little
additional effect on the bovine ES cells.
Conclusions
[0326] Of the four media tested, the two 3i media were most
efficient for the isolation of bovine ES cells. Growth and
development of bovine ES cells and expression of pluripotency genes
were superior in these two media. In addition, bovine ES cells
isolated in 3i media showed potential to differentiate in vitro.
Such pronounced differences in growth and development were not
apparent with the established bovine ES cell lines. However more
pluripotency genes were expressed after culture in 3i media, so
these media appear to enhance the pluripotency component of bovine
ES cell lines.
REFERENCES
[0327] 1. Lonergan P, O'Kearney-Flynn M, Boland, M. P. (1999)
Effect of protein supplementation and presence of an antioxidant on
the development of bovine zygotes in synthetic oviduct fluid medium
under high or low oxygen tension. Theriogenology 51: 1565-1576
[0328] 2. Holm P, Booth P J, Schmidt M H, Greve T, Callesen H
(1999) High bovine blastocyst development in a static in vitro
production system using SOFaa medium supplemented with sodium
citrate and myo-inositol with or without serum-proteins.
Theriogenology 52: 683-700 [0329] 3. Tervit H R, Whittingham, DG,
Rowson L E A (1972) Successful culture in vitro of sheep and cattle
ova. Journal of Reproduction and Fertility 30: 493-497 [0330] 4.
Zar J H (1999) Biostatistical Analysis. IE 4.sup.th edn, Prentice
Hall, Upper Saddle River [0331] 5. Fowler J, Cohen L, Jarvis P
(1998) Practical Statistics for Field Biology. 2.sup.nd Edn, John
Wiley and Sons, New York
Sequence CWU 1
1
60120DNAArtificialsynthetic oligonucleotide 1ctgctccacc tgcctgtatt
20221DNAArtificialsynthetic oligonucleotide 2tccctttgca atagacaatg
g 21322DNAArtificialsynthetic oligonucleotide 3tcattttcct
tcctctctct ca 22420DNAArtificialsynthetic oligonucleotide
4aagacaaaat gctggaggga 20520DNAArtificialsynthetic oligonucleotide
5cactggcatt gtgatggact 20620DNAArtificialsynthetic oligonucleotide
6acggatgtca acgtcacact 20721DNAArtificialsynthetic oligonucleotide
7aactgcgagt gggtctggaa g 21820DNAArtificialsynthetic
oligonucleotide 8tgggtctcgg gaaagcagtg 20921DNAArtificialsynthetic
oligonucleotide 9ccgaatacca cgcacaccat c
211021DNAArtificialsynthetic oligonucleotide 10ctttccttgg
ctctgcggtt c 211121DNAArtificialsynthetic oligonucleotide
11cgagcggtgt gggtaaaagt g 211221DNAArtificialsynthetic
oligonucleotide 12ggtgtcgggt cttcttgctt g
211321DNAArtificialsynthetic oligonucleotide 13tgtgcgggga
cattgcttct g 211421DNAArtificialsynthetic oligonucleotide
14tcccgatctg ccaagtcaca g 211521DNAArtificialsynthetic
oligonucleotide 15cggggtgtgg tgagcatctt c
211620DNAArtificialsynthetic oligonucleotide 16ccttcttggt
ccgcccgttc 201721DNAArtificialsynthetic oligonucleotide
17tcatcacgac ggcttggact g 211821DNAArtificialsynthetic
oligonucleotide 18gccagagcac accaagaatc c
211921DNAArtificialsynthetic oligonucleotide 19atacacctgc
acagcgtaca g 212021DNAArtificialsynthetic oligonucleotide
20tcccgcatct ctttcactca c 212120DNAArtificialsynthetic
oligonucleotide 21gccctgagaa gaaagaagag
202220DNAArtificialsynthetic oligonucleotide 22ctgactgccc
catactggaa 202320DNAArtificialsynthetic oligonucleotide
23agagaagcgc tggaacagag 202420DNAArtificialsynthetic
oligonucleotide 24aggtgtctgc aaccgagagt
202519DNAArtificialsynthetic oligonucleotide 25gggatggcat actgtggac
192619DNAArtificialsynthetic oligonucleotide 26cttcctccac ccacttctc
192720DNAArtificialsynthetic oligonucleotide 27gagactggct
ccatcagacc 202820DNAArtificialsynthetic oligonucleotide
28ctagccaggt tgcgaagaac 202921DNAArtificialsynthetic
oligonucleotide 29ttcttgccag gttctggaag c
213021DNAArtificialsynthetic oligonucleotide 30tttcccacac
tctgcacaca c 213120DNAArtificialsynthetic oligonucleotide
31ggcggcaacc agaagaacag 203221DNAArtificialsynthetic
oligonucleotide 32gttgctccag ccgttcatgt g
213321DNAArtificialsynthetic oligonucleotide 33aggagaggtg
gtggcgagta g 213421DNAArtificialsynthetic oligonucleotide
34gttgggatgg tcctgcatgt g 213521DNAArtificialsynthetic
oligonucleotide 35tcctacaacc agaaacacta g
213620DNAArtificialsynthetic oligonucleotide 36gtgcagagac
atctgaatgg 203724DNAArtificialsynthetic oligonucleotide
37atgaagtgca agcggtggca gaaa 243824DNAArtificialsynthetic
oligonucleotide 38cctggtggag tcacagagta gttc
243920DNAArtificialsynthetic oligonucleotide 39ggcacccagc
acaatgaaga 204021DNAArtificialsynthetic oligonucleotide
40cgactgctgt caccttcacc g 214121DNAArtificialsynthetic
oligonucleotide 41agtgagaggc aacctggaga g
214221DNAArtificialsynthetic oligonucleotide 42gacagacacc
gagggaaaga c 214320DNAArtificialsynthetic oligonucleotide
43catccacagc aaatgacagc 204420DNAArtificialsynthetic
oligonucleotide 44tttctgcaaa gctcctaccg
204523DNAArtificialsynthetic oligonucleotide 45agccccagag
tgaaaccact gtc 234621DNAArtificialsynthetic oligonucleotide
46gtgttctcac agacccagct g 214720DNAArtificialsynthetic
oligonucleotide 47ggcacccagc acaatgaaga
204821DNAArtificialsynthetic oligonucleotide 48cgactgctgt
caccttcacc g 214921DNAArtificialsynthetic oligonucleotide
49agtgagaggc aacctggaga g 215021DNAArtificialsynthetic
oligonucleotide 50gacagacacc gagggaaaga c
215119DNAArtificialsynthetic oligonucleotide 51gcagaatgtg ggaaagcct
195219DNAArtificialsynthetic oligonucleotide 52gactgaataa acttcttgc
195322DNAArtificialsynthetic oligonucleotide 53gagtcaatac
gtgaccgtgg ac 225422DNAArtificialsynthetic oligonucleotide
54ctgaagtagc cgcgatagac ag 225520DNAArtificialsynthetic
oligonucleotide 55catccacagc aaatgacagc
205620DNAArtificialsynthetic oligonucleotide 56tttctgcaaa
gctcctaccg 205722DNAArtificialsynthetic oligonucleotide
57tgtgctcaat gacagatttc ag 225820DNAArtificialsynthetic
oligonucleotide 58tagaagcctg ggtattctgc
205920DNAArtificialsynthetic oligonucleotide 59ggacccagga
aaccaaagtc 206021DNAArtificialsynthetic oligonucleotide
60gaatgaggga gtgagcaaac c 21
* * * * *