U.S. patent application number 10/175277 was filed with the patent office on 2002-12-12 for derivation of pluripotential embryonic cell lines from domestic animals.
This patent application is currently assigned to Babraham Institute. Invention is credited to Evans, Martin John, Moor, Robert Michael, Notaranni, Elena.
Application Number | 20020187549 10/175277 |
Document ID | / |
Family ID | 26294421 |
Filed Date | 2002-12-12 |
United States Patent
Application |
20020187549 |
Kind Code |
A1 |
Evans, Martin John ; et
al. |
December 12, 2002 |
Derivation of pluripotential embryonic cell lines from domestic
animals
Abstract
A method of selecting and growing pluripotential embryonic stem
cells isolated from an ungulate species blastocysts of embryos that
develop by way of an embryonic disc is disclosed. The method
comprises growing blastocysts in tissue culture growth medium which
includes both heat-inactivated new born calf serum and
heat-inactivated fetal calf serum; disaggregating the blastocysts
either after spontaneous hatching or after mechanical removal of
the zone pellucida; growing stem cell colonies from the
disaggregated cells in issue culture growth medium; selecting stem
cell colonies by morphological characteristics; and growing the
selected stem cells in tissue culture growth medium. The cells are
round cells, tightly packed with large nuclei in relation to
cytoplasm, and fairly prominent nucleoli. They grow in tightly
adherent coloedes and as the colonies get larger the cells tend to
flatten out in the center of the colony. The outer, less flattened
cells of a larger colony or all the cells of a smaller colony
without central flattening are readily disaggregated by
trypsinization into small spherical cells which have a bright phase
contrast appearance, and if observed after a short time of
incubation at 37 C. they show lobular pseudopodia
Inventors: |
Evans, Martin John;
(Cambridge, GB) ; Moor, Robert Michael;
(Cambridge, GB) ; Notaranni, Elena; (Cambridge,
GB) |
Correspondence
Address: |
FOLEY AND LARDNER
SUITE 500
3000 K STREET NW
WASHINGTON
DC
20007
US
|
Assignee: |
Babraham Institute
|
Family ID: |
26294421 |
Appl. No.: |
10/175277 |
Filed: |
June 20, 2002 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10175277 |
Jun 20, 2002 |
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07669403 |
Apr 23, 1991 |
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6436701 |
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07669403 |
Apr 23, 1991 |
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PCT/GB89/01103 |
Sep 21, 1989 |
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Current U.S.
Class: |
435/325 ;
800/14 |
Current CPC
Class: |
C12N 5/0603 20130101;
C12N 5/0606 20130101 |
Class at
Publication: |
435/325 ;
800/14 |
International
Class: |
C12N 005/06; A01K
067/027 |
Foreign Application Data
Date |
Code |
Application Number |
Aug 9, 1989 |
GB |
89 18203.4 |
Sep 21, 1988 |
GB |
88 22158.5 |
Claims
1. Pluripotential embryonic stem cells isolated from in vitro
treatment of non-human and non-rodent blastocysts.
2. Stem cells according to claim 1 from ungulate species.
3. Stem cells according to claim 1 from bovine species.
4. Stem cells according to claim 1 from porcine species.
5. A blastocyst to which has been introduced one or more stem cells
according to any of claims 1 to 4.
6. An embryonic cell to which has been introduced by nuclear
transfer a nucleus of a stem cell according to any of claims 1 to
4.
7. A chimeric animal which is the progeny of a blastocyst according
to claim 5 or an embryonic cell according to claim 6.
8. A method of obtaining pluripotential embryonic stem cells
according to any of claims 1 to 4, comprising growing blastocysts
in tissue culture growth medium which includes both new born calf
serum and foetal calf serum (both sera having been heat inactivated
before use); causing disaggregation of the blastocysts either after
spontaneous hatching or after mechanical removal of the zona
pellucida; growing the disaggregated cells in tissue culture growth
medium; selecting stem cell colonies by morphological
characteristics; and growing the selected stem cells in tissue
culture growth medium; wherein the morphologically selected cells
grow in distinctive flat polarised epithelial colonies which tend
to spread to form monolayers and the cells are round, have
relatively large clear nuclei, have prominent nucleoli and
relatively little cytoplasm.
9. A method according to claim 8, further including passaging the
selected stem cells by trypsinisation onto fresh tissue culture
growth medium at intervals to prevent differentiation of the cells
and to maintain a cell line in culture.
10. A method which comprises introducing into a blastocyst one or
rare stem cells made according to the method of claim 8.
11. A method which comprises introducing by nuclear transfer into
an embryonic cell a nucleus of a stem cell made according to the
method of claim 8.
12. A method which comprises introduction to the uterus of a
pseudo-pregnant foster mother animal, a viable embryo obtained
using a technique involving any of the methods of claim 8 to 11, so
as to produce progeny in the form of a chimeric animal.
Description
Mammalian Genetics
[0001] Traditional genetics depended upon mutations or pre-existing
genetic polymorphisms which were discovered in a species. The only
experimental approach to widen the scope of genetic variants
available for study was mutagenesis followed by specific screening
or fortuitous recovery of relevant alleles. Animal breeding depends
upon selection from suitable variation either in or introduced into
the stock. The major tool for genetic analysis was breeding
segregation studies and direct phenotypic analysis.
[0002] Notwithstanding the great theoretical and practical interest
in mammalian genetics the conventional genetic analysis of
experimental mammals, because of their relatively small litter
sizes and long life cycles, has been handicapped vis-a-vis other
experimental animal systems. Mammalian genetics has, however,
benefitted from the study of human genetics where although there is
no experimental breeding, detailed observation of a very large
population has allowed investigation both of polymorphisms and of
very rare mutations, and statistical methods for analysis of data
available from human pedigrees of genetic segregation have become
highly refined.
[0003] Added to this, methods for non-meiotic genetic
analysis--somatic cell genetics and more recently direct
molecular-biological analysis, have been and indeed are being very
effectively applied as well as being combined with pedigree
analysis so that mammalian genetic maps and knowledge of gene
sequence data are advancing rapidly.
[0004] This is, however, an observational analysis. Our
understanding of genetic function and also, in experimental
animals, the practical application of our genetic knowledge
requires the ability deliberately to modify the genome, preferably
in a manner not entirely reliant upon screening the accidents of
nature. Thus the concept of a reverse mammalian genetics emerges
where the effect of specific genetic modification may be studied in
the context of the intact organism.
[0005] Genes of interest are now not only being identified from the
results of the intensifying mammalian genetic analysis but
importantly also through their molecular biology and by cross
homology to those of other species. In a great number of cases
there are genes which have been identified in mice through their
molecular biology, by analogy with those of other species (e.g.
human disease syndromes or Drosophila genetics) or from the
biochemistry of their protein products but for which there is no
lack-of-function allele and thus no rigorous genetic test of
function. Neither are these mutations of protein structure or of
genetic control. These can only be provided by creating such
alleles. In the field of practical application to domestic farm
animals, potential alteration of normal physiology which may be
desirable, deletion or modification of function of controlling
genes may be just as Important as overexpression of others. The
technology to undertake such targetted gene deletion or
modification has now reached feasibility as exemplified by creation
of null alleles at the hypoxanthine phosphorbosyl transferase
gene--HPRT--locus [1] [2]. The development of methods which will
allow targetting and screening for deletion of function or specific
Codification of any gene whose sequence is known is now well
underway and this is clearly a realistic proposal. It will become
an available routine technique for mouse cells in the next year or
so and with the development of domestic animal embryonic stem cells
will be immediately applicable to these species and may well become
the transgenic route of choice.
Mammalian Transgenesis
[0006] Transgenic animals possess an alteration in their DNA which
has been stably incorporated into the genome as a result of
intentional experimental intervention. Typically this results from
the additional exogenous foreign DNA or novel DNA constructs. With
the advent of specific gene targetting we should not necessarily
exclude from the definition of transgenesis specific modification
of endogenous gene sequences by direct experimental
manipulation.
[0007] A fully experimental approach to mammalian genetics is very
rapidly becoming a reality through the use both of conventional
zygote injection transgenics and of embryonic stem cells. The
latter approach allows extensive in vitro genetic manipulation,
selection and screening prior to whole animal reconstruction. Thus
both an experimental molecular genetics and the ability to design
genetic changes in animals are available. For practical purposes
the mouse has been the species of choice for such studies but it is
important to be able to extend the methods developed in the mouse
to larger domestic farm animal species with the intention of their
practical application. The new experimental mammalian genetics
allows for testing of genetic modifications in vivo and designing
genetic modification of a target species. One of the most important
prospects is the construction of experimental animal models of
disease for pharmaceutical testing and developments. The other is
for specific modification of domestic farm animals to create more
desirable qualities for food production, disease resistance, and
biopharmaceutical protein production.
Methods for Transgenesis
[0008] Although DNA micro-injection is the most commonly used
method of generating transgenic animals, alternatives include
embryo infection using recombinent retroviral vectors incorporating
the transgene and also the use of pluripotential embryonic stem
(ES) cells.
[0009] In order to introduce genetic alterations into a mammal it
is necessary to transform genetically a cell the progeny of which
can give rise to all or to the desired part of the intact organism.
Zygote micro-injection [4] achieves transgenesis by transformation
of the embryo's genome at the single cell or very early cleavage
stage. As the germ line in mammals is segregated from somatic
progenitor lineages at the early primitive-streak stage
transformation of embryonic cells before this stage for instance by
retroviral vector infection of cleavage embryos [5] may provide
animals which are transgenic both somatically and in the germ line.
Genetic transformation of cells after this stage will lead to
either germ line or somatic genetic mosaicism. Where stem cells may
be isolated from the organism these may be transformed and used to
re-colonise their target tissue and the use of such techniques is
exemplified by haematopoetic stem cell manipulation e.g. [7]. This
type of approach to somatic transgenesis is likely to be the only
ethical route for human gene therapy and could well prove
particularly useful for genetic modification of domestic livestock
and when the stem cells may be maintained in tissue culture prior
to their use to reconstitute their target tissue there is the
advantage that selection for the desired transformants may preceed
reconstitution. See for example Edwards' use of transient culture
of mammary epithelial stem cells [8]. Cells isolated from an embryo
before segregation of the germ line are able to provide a genetic
vehicle for germ line transgenesis. Whereas embryonic stem cells
have been isolated from mice [9] [10] and cells which seem likely
to have such properties from hamster, [11] it is by no means
apparent that cells of a similar type may be necessarily isolated
from other non-rodent embryos. Moreover it is unlikely that the
methods as described for mouse and utilised for hamster will be
directly applicable to other embryos. Indeed the reported failure
(notwithstanding the optimistic title) of some competent
researchers in the field [12] to isolate sheep embryonic stem cells
by a method based upon that used for mouse embryonic stem cells
indicates this. Others have isolated cells but failed to maintain
lines or demonstrate their pluripotentiality [15]. Past failures
may have been due to the expectation that the cells would be
fast-growing and resemble those of the mouse. It was indeed
reported that malignant transformation was necessary in order to
overcome the inherent quiescence of the embryonic disc [6].
[0010] There have also been numerous attempts which have been
orally reported at various scientific meetings which have been
unsuccessful. When the mouse embryonic stem cells were first
isolated virtually every expected property was predicted and the
embryonic stage at which they might be found was clearly identified
[13]. None of this background is available for putative ungulate
embryonic stem cells.
[0011] We have discovered that the methods which have been
established and described for the isolation of embryonic stem cells
from mouse embryos and successfully applied to hamster embryos are
NOT applicable to ungulate embryos as exemplified by bovine and
porcine embryos. In particular the most important step in embryonic
stem-cell isolation--identification and isolation of the stem cells
from other cell types is quite differently based as is the
necessary tissue-culture handling of the cells.
Early Mammalian Development: Theoretical Consideration for the
Isolation of Stem Cells from Ungulates
[0012] In mammals, the pattern of embryonic development from
fertilisation to implantation is broadly similar between species:
fertilisation of the oocyte occurs in the oviduct, and the zygote
is transported to the uterus whilst undergoing a series of mitotic
divisions. At each division cell size decreases, and so the volume
of the embryo remains constant. A blastocyst is formed at a certain
stage, when a cavity appears within the embryo. At this time the
cells have differentiated into two types, the trophoblast and the
inner cell mass, destined to become the fetal portion of the
placenta and the fetus, respectively. However, by the time of
implantation great differences are observed between species. In
particular in the mouse, implantation into the uterine epithelium
is an invasive and rapid process (14). In ungulates, in particular
in cattle, sheep and pigs, implantation occurs only after a
considerable delay during which the trophectoderm proliferates
rapidly and the inner cell mass forms a quiescent embryonic disc.
In these cases implantation involves loose association between the
fetal cells and the maternal cotyledons (14). Thus early
development at the time when stem cells may be isolated is very
significantly different in many species including ungulates from
the mouse.
[0013] Procedures for the isolation of murine embryonic stem cell
lines are now well established. Success in the isolation of
embryonic stem cells from the mouse depended on the recognition of
the need for careful timing, so that ICM cells are committed to the
ICM lineage and yet are free from the influence of differentiated
derivatives [9]. Although, by extrapolation, it can be argued that
stem cells may be isolated similarly from the ungulates and other
species, we anticipated that problems would arise in that exactly
analogous stages do not exist in the embryos of mice and ungulates
owing to difference in their development. We inferred that a
different strategy will be required for the ungulates as, for
example, the rate of development is much slower and the early
embryonic ectoderm is present in a discoid arrangement and not as a
solid mass as in the 5 day mouse embryo. These considerations led
us to predict that the embryonic cells of the pre-implanting
embryo, owing to the obligatory period of metabolic quiescence of
the embryonic disc, would not be culturable with facility in vitro;
and that stem cells, if isolated, would not necessarily resemble
mouse embryonic stem cells in morphology or growth
characteristics.
[0014] In these situations recognition of the stem cell types would
be difficult. Nevertheless, we are able to define those conditions
which are sufficient for the isolation of stem cells from
ungulates, and for preventing cell differentiation sufficiently for
cell lines to be established. It is likely that the cell type
required and the means of its isolation may prove more general than
that of embryonic stem cell isolation in the mouse as many other
mammalian embryos--for example primate embryos--develop through an
embryonic disc more similar to the structure found in ungulates
than the epiblast and egg cylinder seen in the mouse and some other
rodents.
Method of Isolation and Culture of Embryonic Stem Cells from
Ungulate Embryos
[0015] 1. Tissue Culture Medium
[0016] Dulbecco's modified DMEM culture medium supplimented with 5
to 10 percent of both foetal and new born calf serum, non-essential
amino acids to the Eagles formula and 0.1 Millimolar
2-mercaptoethanol is used. Particular attention to selection of the
sera is essential and unlike most sera used for mouse embryonic
stem cell derivation, it was found that heat-inactivation of the
serum at 56 degrees centigrade for 30 minutes was necessary.
[0017] 2. Explantation of the Embryo
[0018] Both species of embryo behave differently in culture from
the mouse. In the case of the bovine embryos the procedure is as
follows:
[0019] Either fresh 6-day embryos flushed from in vivo or
preferably embryos which have been grown in vitro from in vitro
fertilised in vitro matured oocytes (supplied by Animal
Biotechnology Cambridge Ltd.) at 6-7 days of
development--unhatched, fully expanded blastocysts--are grown from
1 or two extra days in tissue culture medium. They either hatch
spontaneously or are freed from their zona pellucida mechanically
and allowed to explant upon the bottom of a petri dish containing
an STO fibroblast feeder layer as previously described [9]. Unlike
the case with a mouse embryo the inner-cell-mass derived cells do
not form a central egg cylinder. Their derivatives which ray be
isolated as the precursors of the embryonic stem cells are found on
the periphery of the explant. These are isolated by careful
trypsinisation using trypsin/EGTA/polyvinyl alcohol (0.25%:0.1
mM:10 ug/ml respectively) and replated as below.
[0020] For porcine embryos the procedure is: Embryos from the stage
of hatching (6.5 days) to trophoblast expansion (11 days) are
either explanted intact in which case most of the trophoblast layer
dies, or preferably dissected to isolate the embryonic disc before
this is explanted onto a fibroblast feeder layer typically
inactivated STO fibroblasts. Primary outgrowths may be recognised
to be the precursors of the embryonic stem cells and these are
disaggregated and passaged. These primary stem cell outgrowths are
different from the established cell lines and appear as more
translucent and flatter tightly-packed epithelial colonies.
[0021] 3. Feeder Cells and Growth Factors
[0022] It is sufficient to use STO fibroblasts as feeder cells for
either species. It is not necessary to use exogenous growth factors
or conditioned media.
[0023] 4. Recognition and Isolation of the Stem Cells
[0024] Unlike those of the mouse and hamster, the ungulate
embryonic stem cells do not form multilayered colonies but grow in
distinctive flat polarised epithelial colonies which eventually
spread to form monolayers. The cells are larger than those of the
mouse, have large clear nuclei, several prominent nucleoli and
relatively little cytoplasm. The cell size is found to vary from
isolation to isolation and with growth conditions but the general
morphology and appearance is distinctive.
[0025] Other non-epithelial cell types may be observed and isolated
but these do not have the differentiative properties described.
[0026] 5. Maintenance of Cells in Culture
[0027] Cells are passaged 1 in 4-5 by trypsinisation onto fresh
feeder cells at 3-4 day intervals or just before they attain
confluency. Failure to passage prior to confluency results in the
onset of spontaneous differentiation which if allowed to continue
leads to loss of the cell line. Undifferentiated colonies may be
able to be recovered after partial differentiation of the culture.
The cells will grow without feeders but their ability to form
embryoid bodies becomes compromised.
[0028] 6. Verification of the Stem Cells
[0029] These cells differentiate readily in culture either
spontaneously or in response to morphogens such as dimethyl
sulphoxide or retinoic acid. The main differentiated derivatives
are fibroblast, nerve endoderm and muscle which are representative
of the three germ layers and verifies the pluripotentiality of the
cells. Moreover these cells will form tumours when transplanted
beneath the kidney capsule of an irradiated nude mouse, these
tumours being teratocarcinomas showing a variety of differentiated
cell types (FIGS. 7a and 7b).
[0030] On aggregation in vitro embryoid bodies are formed. These
are distinctly different from those of the mouse by virtue of the
fact that they show a polarisation akin to that seen in those
formed by human teratocarcinoma cells. Explanation of these
embryoid bodies into a tissue culture dish results in rapid and
extensive differentiation.
[0031] All these observations demonstrate that the cultures
described are indeed embryonic stem cells.
[0032] The present invention can provide stem cells, and a general
method as exemplified above for isolation of embryonic stem cells
from all embryos in which development is via an embryonic disc in
particular ungulate embryos (such as porcine embryos and bovine
embryos). The invention can provide for the derivation of such
cells from embryos carrying a particular genetic background or
specific mutations. For example derivation of such cells from
high-pedigree agricultural stock. It can also provide a method for
preparation and use of such cells for differentiation and
developmental studies in vitro, together with a method of use of
such cells as a source of any other differentiated cell in vitro or
in vivo. Furthermore, the invention can provide for the use of such
cells to repopulate an embryo of the same species thus giving rise
to a chimaeric animal, particularly a chimaeric animal in which
some or all of the germ cells are derived from the tissue-culture
cells; for example a chimaeric animal in which some or all of the
germ cells are derived from the tissue-culture cells where the
embryonic stem cells have been genetically modified or selected for
genetic modification in culture. Stem cells according to the
invention can be cultured either transiently or maintained as a
cell line to provide nuclei for nuclear transfer into enucleated
oocytes or other embryonic cells, e.g. using cells with specific
genetic properties either by virtue of their provenance from
specific embryos or otherwise by specific genetic modification.
[0033] The invention can allow development of embryos from cells
which have received a necleus from an embryonic stem cell cultured
in vitro. It can allow the use of stem cells genetically
transformed in such a way as to introduce a novel protein
production in a specific part (e.g. the mammary gland, the liver)
of a subsequently derived chimaeric animal or the offspring of such
a chimaeric animal, or a subsequently derived nuclear-cloned animal
or the offspring of such an animal to provide nuclei for neclear
transfer into enucleated oocytes or other embryonic cells.
[0034] Stem cells of the invention may be used in techniques of
genetic transformation and may be used in the creation of embryos
to produce a genetically transformed living animal by embryo
transfer.
[0035] The scope of this invention extends to cover not only the
stem cells per se, but to embryos and genetically transformed
animals derived therefrom. The invention also covers essentially
non-biological methods for production of stem cells, embryos and
animals.
[0036] According to one aspect of the invention there is provided a
bovine stem cell line, and a method for its production.
[0037] This aspect of the invention also provides a cell culture
system comprising bovine stem cells.
[0038] Bovine stem cells are conveniently produced by growing
bovine blastocysts (fertilised in vivo or in vitro) in suitable
tissue culture growth medium. One preferred medium consists of a
mixture of 75 parts of Dulbecco's Modified Eagles Medium (DMEM) to
25 parts of Ham's F12M Medium, supplemented with non essential
amino acids (Eagle's) (about 1% by volume), beta mercapto ethanol
10.sup.-4M, 10% new born calf serum and 10% foetal calf serum, both
sera having been heat inactivated by treatment at 56.degree. C. for
30 minutes before use.
[0039] After hatching the blastocysts are desirably treated to
cause disaggregation. This is preferably effected by treating the
blastocysts about 1 day after hatching by soaking for about 10
minutes in trypsin (0.25% Difco trypsin 1 in 250) supplemented with
10.sup.-4 molar EGTA (ethylene glycol tetracetic acid) and 10 ug/ml
polyvinyl alcohol, followed by physical treatment to cause
disaggregation, e.g. by sucking and blowing through a small
pipette.
[0040] Hence in a preferred aspect the present invention provides a
method of obtaining bovine stem cells, involving the steps of
growing bovine blastocysts in tissue culture growth medium, and
soaking the blastocysts about 1 day after hatching for about 10
minutes in trypsin supplemented with EGTA and polyvinyl alcohol to
cause disaggregation of the blastocysts.
[0041] After disaggregation, the disrupted blastocyst cells may be
replated and grown in a further supply of the growth medium
together with inactivated STO cells, resulting in the growing up of
colonies of cells of at least 2 different types.
[0042] After a suitable incubation line colonies of stem cells can
be selected by morphology, as described below. The cells are grown
up in the growth medium plus inactivated STO cells, with the cells
passaged about once a week.
[0043] After the 4th or 5th passage then cells can be subjected to
further treatment of different types.
[0044] For example, the cells can be frozen if required for
storage.
[0045] Alternatively the fresh cells can be introduced to a host
blastocyst e.g. using conventional micromanipulation techniques.
Typically between 1 and 15 cells are introduced to a host
blastocyst. The blastocyst can then be introduced to the uterus of
a pseudopregnant foster mother where it may develop into a
chimaeric animal.
[0046] Prior to introduction to a host blastocyst, the cells can if
desired by manipulated in culture by known techniques, e.g. by DNA
transformation, targetted mutation by homologous recombination, or
infection with retroviral vectors, so enabling modification of the
resulting animals and enabling production of an animal having
desired characteristics.
[0047] In a further aspect the present invention provides a method
of obtaining bovine stem cells, comprising growing bovine
blastocysts in tissue culture growth medium; causing disaggregation
of the blastocysts after hatching; growing the disaggregated cells
in tissue culture growth medium; selecting stem cell colonies by
morphological characteristics; and growing the selected stem cells
in tissue culture growth medium.
[0048] The invention also provides a blastocyst to which has been
introduced one or more bovine stem cells of the invention, and the
chimaeric progeny of such a blastocyst.
[0049] The invention will be further described, by way of
illustration, in the following example and with reference to the
accompanying figures.
[0050] FIG. 1 is a photograph of colonies of stem cells growing in
cell culture; and
[0051] FIG. 2 is a photograph of a monolayer of bovine stem
cells.
EXAMPLE
[0052] Bovine embryos are treated by the following procedure
[0053] The embryos may be fertilised in vivo, in which case they
are obtained by flushing from a cow by known techniques at 5-6 days
embryonic development. However, it is preferred to use embryos
fertilised in vitro and the following work was carried out using
fresh expanded bovine blastocysts derived by in vitro fertilisation
and obtained from Animal Biotechnology Cambridge Limited,
Cambridge, England. Similar material may also be obtained from
other commercial sources. The blastocysts may also be obtained in
frozen form, but it is preferred to use fresh blastocysts.
[0054] The blastocysts are grown on a culture dish, in tissue
culture growth medium on a feeder layer of mytomycin inactivated
STO cells. The medium used consists of a mixture of 75 parts of
Dulbecco's Modified Eagles medium (DMEM) to 25 parts of Ham's F12M
Medium, supplemented with non essential amino acis (Eagle's) (about
1% by volume), beta mercapto ethanol to 10.sup.-4 molar, 50
units/ml penicillin G, 10% new born calf serum and 10% fetal calf
serum, both sera having been heat inactivated at 56.degree. C. for
30 minutes before use. The blastocysts are incubated at 37.degree.
C. in a carbon dioxide gas humidified incubator in an atmosphere of
about 5% carbon dioxide in air.
[0055] The incubating blostocysts are periodically examined using a
microscope, say twice daily, until hatching is observed. The timing
of this varies with different embryos, but will typically be after
about 2 days.
[0056] The hatched blastocysts are then treated in one of two
alternative ways.
[0057] 1) In a first approach, about 1 day after hatching the
blastocysts are treated by being soaked for about 10 minutes in
trypsin (0.25% Difco trypsin 1 in 250) supplemented with 10.sup.-4
molar EGTA (ethylene glycol tetracetic acid) and 10 ug/ml polyvinyl
alcohol. This mixture acts to disaggregate the cells of the
blastocysts while maintaining cell viability.
[0058] Using a small pipette, with a tip about 50 to 100 microns in
diameter the cells are then physically disaggregated by sucking and
blowing, causing the cells to fall into clusters, breaking down the
blastocyst structure.
[0059] The disrupted blastocyst is immediately subjected to further
treatment, described below.
[0060] 2) In an alternative approach the hatched blastocysts are
left in the tissue culture medium and allowed to develop further.
After about 3-4 days development it is observed that the inner cell
mass of the blastocyst is found in clusters of rounded cells
located around the edge of the explant. At this stage the trypsin
mixture used in 1) above is added to the culture dish and left for
about 10 minutes. It is found that the cell clusters at the edge of
the explant loosen more easily than others, and these cells are
picked off using a small pipette with a tip about 20 to 30 microns
in diameter, and transferred for further treatment.
[0061] After treatment by method 1) or 2) the disrupted blastocyst
cells are replated into a tissue culture dish surface treated with
a gelatin solution. A further supply of the growth medium described
above is added, together with about 10.sup.-6 inactivated STO
cells, and the dish placed in an incubator at 37.degree. C.
[0062] The cells are periodically examined by microscope, e.g.
daily. Colonies of cells of at least 2 different types are observed
growing up.
[0063] After a suitable incubation time colonies believed to be
stem cells are selected by morphology: stem cells have the
following features:
[0064] a) They are round cells, tightly packed with large nuclei in
relation to cytoplasm, and fairly prominent nucleoli.
[0065] b) They grow in tightly adherent colonies. As the colonies
get larger the cells tend to flatten out in the centre of the
colony, with the colony having an outer rim of cells of the form
described in a).
[0066] c) On trypsinisation of such a colony using the trypsin
mixture described in 1) above it may be seen that the outer, less
flattened cells of a larger colony or all the cells of a smaller
colony without central flattening are radily disaggregated into
small spherical cells which have a bright phase contrast
appearance, and if observed after a short time of incubation at
37.degree. C. show lobular pseudopodia.
[0067] Such colonies are illustrated in FIG. 1.
[0068] Suitable colonies, believed to be of stem cells, are
selected and transferred to another dish, to which is added a
further supply of the growth medium together with inactivated STO
cells. The dish is placed is an incubator at 37.degree. C. to allow
colonies of the cells to grow up.
[0069] The cells are passaged about once a week, i.e. subjected to
the trypsin treatment as described in 1) above and replated onto a
new dish. This is to stop differentiation.
[0070] If the cells are to be stored they are forzen after the 4th
or 5th passage.
[0071] Otherwise, cells at this stage can be introduced to another,
host, bovine blastocyst: typically between 1 and 15 cells are
introduced to a blastocyst using conventional micromanipulation
techniques. The blastocyst can then be introduced to the uterus of
the pseudopregnant foster mother in known manner, or maintained in
an artificial environment, in the uterus develop into a chimaeric
animal containing DNA from both the host blastocyst and the
introduced cells. Chimaerism can be detected in known ways, e.g. by
use of genetic markers, or possibly simply by visual
inspection.
[0072] Prior to introduction to a host blastocyst, the cells can be
manipulated in culture by known techniques, e.g. by DNA
transformation or infection with retroviral vectors, so enabling
modification of the resulting animals and enabling production of an
animal having desired characteristics.
[0073] According to another aspect of the invention there is
provided a porcine stem cell line, and a method for its production.
This aspect of the invention also provides a cell culture system
comprising porcine stem cells.
[0074] This aspect of the invention will now be further described,
by way of illustration in the following example and with reference
to the accompanying figures in which are:
[0075] FIG. 3. Appearance of primary colony resulting from
attachment of inner cell mass from 8 d blastocyst. The flattened,
translucent colonies are arrowed:
[0076] Morphologies of colonies resulting from disaggregated
primary outgrowths of inner cell masses. FIG. 4a; colony of cells
producing large, trophoblast-like cells, which are visible at the
perimeter. This culture was derived from a 7 d blastocyst. FIG. 4b;
and 4c; colonies of stem-like cells, which are epithelial, adherent
and have large nuclei and prominent nucleoli. The colony shown in
FIG. 4b derived from a 7 d blastocyst, and that in 4c from an 8 d
blasocyst.
[0077] FIG. 5. Established porcine cell line showing morphological
differentiation. FIG. 5a. Nest of undifferentiated cells. FIG. 5b.
Confluent monolayer of cells. FIG. 5c. Confluent monolayer showing
morphological differentiation into neuron-like cells.
[0078] FIG. 6a. Aggregates formed by porcine cell line following
culture for 7 d on a non-adhesive substratum.
[0079] FIGS. 6b to 6d. Outgrowths of cells from aggregates which
were permitted to reattach to a substratum. Several differentiated
cell types are visible; 6 b epithelial, 6 c muscle and fibroblastic
and 6 d nerve-like.
[0080] Hatched blastocysts were recovered by retrograde uterine
flushing from Large British White gilts at 7-9 days post oestrus.
The animals were naturally mated and not superovulated. Either
intact blastocysts or the inner cell masses manually dissected from
them were explanted onto STO fibroblast mitotically-inactivated
feeder cells in a manner similar to that described by Evans &
Kaufman (1981) [9] for mouse blastocysts. The medium used was
Dulbecco's modified Eagle's medium supplemented with 10% new born
calf serum and 5% to 10% fetal calf serum, and 0.1 millimolar
2-mercaptoethanol, and neither conditioned medium nor exogenous
growth factors were added.
[0081] In order to stimulate organised differentiation, cells were
disaggregated by trypsinisation and then seeded onto culture dishes
which had been coated with a layer of 0.5% agarose as described by
Magrane (1982) [3] for stimulation of formations of embryoid bodies
by human teratocarcinoma cell cultures. To enhance cell
differentiation both mercaptoethanol and fetal calf serum were
omitted from the medium.
Establishment of Cultures
[0082] We have by these methods of cultures been able routinely to
establish cultures from explanted porcine embryos. Although the
success rate is variable it is often high with as many as 6
successful cultures being derived from 8 explanted blastocysts in
one expert.
[0083] When blastocysts or embryonic discs are brought into
culture, they attach within one day. The primary outgrowths consist
of colonies of large flat, highly-translucent epithelial cells
(FIG. 3). These are clearly of very different appearance and
culture morphology to murine EK cells. Portions of trophectoderm
dissected from blastocysts between the ages of 7-10 days and
cultured in the sane way were unable to form colonies or
outgrowths.
[0084] The primary outgrowths were dissaggregated 7-14 days after
explantation and passaged to fresh feeder layers. Pregressively
growing colonies were formed which grew as a monolayer with very
distinct colony boundaries. The cells are epithelioid with large
clear nuclei containing 2-4 prominent nucleoli, and relatively
sparse cytoplasm. Some differences in the appearance of these cells
have been noticed between different isolates which is principally
related to cell size.
[0085] FIG. 4 shows small colonies of large, undifferentiated cells
which continuously produce cells with morphological characteristics
of trophoblast giant cells. Such cultures have been maintained for
4 months in continuous culture. Cultures of this type have only
been observed to arise from 7 day embryos.
[0086] FIG. 4b shows a cell type which is more stable and able to
grow in larger colonies. These cells are the most common isolate.
Several cell lines of the type shown in FIG. 4b have been derived
from both 7 and 9 day embryos. One cell line has been maintained in
continuous culture for more than one year with passaging 1 in 4
every 5-7 days without change of cell phenotype. It therefore
appears to be immortal. differentiation of these cells occurs
spontaneously when the cells are permitted to reach high density
(FIG. 5). Overtly differentiated cells fail to reattach on passage
leading to regeneration of undifferentiated cultures.
[0087] FIG. 4c shows a colony of smaller cells which are a more
rarely isolated form.
[0088] All of these cell types grow more slowly than and differ in
appearance to mouse embryonic stem cells. They show spontaneous
differentiation in culture mainly into trophoblast-like cells or
endoderm-like cells. Other differentiated (mainly fibroblastoid)
cell types are also formed.
Differentiation into Embryoid Bodies
[0089] As a test of differentiation potential (Martin & Evans
1975) [16] cells from the cell line which had been maintained for
12 months were induced to form aggregates by seeding onto a
non-adhesive substratum. After several days an outer smooth layer
of cuboidal epithelial cells appeared at one end of the aggregates
and at the other pole there were more loosely attached cells of a
more ragged appearance. Extensive differentiation occurred when the
embryoid bodies were permitted to attach to the substratum, by
replating onto tissue-culture dishes (FIG. 6b). Cells migrated and
multiplied to form dense cultures, with several types visible,
including epithelium, endoderm, muscle and neural cells. These
differentiated cells are representative derivatives of all three
embryonic germ layers, and suggest that the stem-cell-like culture
represents a primary ectodermal lineage of the pre-somite
embryo.
Discussion
[0090] The cell lines isolated have an appearance considerably
different although slightly reminiscent of murine embryonic stem
cells. In appearance and form of growth they are more similar to
sane cell lines derived from human testicular teratocarcinomas. The
form of development of their aggregates when maintained in
suspension is very similar to that of a human teratocarcinoma cell
line Hutt KEB (as described in Magrane 1982) [3]. We tentatively
conclude that these differentiating structures are indeed
homologous to the murine embryoid bodies and this conclusion is
strengthened by the observations reported here of a more extensive
in vitro differentiation following their re-explanation onto a
tissued-culture surface. One of us (MJE) has previously speculated
that the assymetric form of the Hutt KEB embryoid bodies reflects
the development of the human embryo via an embryonic disc in
contrast to the mouse egg cylinder. It is interesting to note here
that in another species where early development is via an embryonic
disc with a clearly epithelioid embryonic epiblast, the isolated
cells grow more as a monolayer than in the piling colonies typical
of mouse EC and EK cells and differentiation of their embryoid
bodies is clearly assymetric. This distinctly different behaviour
from that of mouse EK cells may be a general feature of those
non-rodent embryos where embryonic development is via an embryonic
disc.
[0091] We conclude that pluripotent embryonic lineages may be
derived from the pig and can be maintained in culture.
[0092] These are very different both in appearance, growth
characteristics and behaviour to those previously described in the
mouse. The clear similarity between porcine cell lines and those
seen from bovine embryos strongly supports the suggestion that this
type of embryonic cell lineage is the form of cell line which will
be obtained from mammalian species in general developing via an
embryonic disc, examples of such species being e.g. ungulates. We
are currently evaluating the potential of our embryonic cells, to
determine (a) their relationship to normal embryonic cells, and to
determine their distinctive features compared to the murine EK
lineages, and (b) whether there exist restricted potency stem cell
populations which may be transitory in nature, and (c) the origin
and nature of those cells which are capable of expressing
pluripotency under certain conditions. It is notable that the rate
of proliferation of undifferentiated, stem-like cells in explants
of porcine embryos is slower compared with those derived from
marine embryos. This may reflect another important difference in
pre-implantational development in these species, that is the period
of quiescence of the inner cell mass in ungulates up to the time of
gastrulation.
[0093] As these new cell lines may be considered homologous to
murine EK cells they have the potential as a vector for genetic
manipulation by their incorporation into a normal fertile pig via
embryo chimaerism leading to their contribution to the germ cell
line.
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