U.S. patent application number 10/952096 was filed with the patent office on 2005-07-07 for cultivation of primate embryonic stem cells.
Invention is credited to Levenstein, Mark, Thomson, James A..
Application Number | 20050148070 10/952096 |
Document ID | / |
Family ID | 24079156 |
Filed Date | 2005-07-07 |
United States Patent
Application |
20050148070 |
Kind Code |
A1 |
Thomson, James A. ; et
al. |
July 7, 2005 |
Cultivation of primate embryonic stem cells
Abstract
The invention relates to methods for culturing human embryonic
stem cells by culturing the stem cells in an environment
essentially free of mammalian fetal serum and in a stem cell
culture medium including amino acids, vitamins, salts, minerals,
transferring, insulin, albumin, and a fibroblast growth factor that
is supplied from a source other than just a feeder layer the
medium. Also disclosed are compositions capable of supporting the
culture and proliferation of human embryonic stem cells without the
need for feeder cells or for exposure of the medium to feeder
cells.
Inventors: |
Thomson, James A.; (Madison,
WI) ; Levenstein, Mark; (Madison, WI) |
Correspondence
Address: |
QUARLES & BRADY LLP
FIRSTAR PLAZA, ONE SOUTH PINCKNEY STREET
P.O. BOX 2113 SUITE 600
MADISON
WI
53701-2113
US
|
Family ID: |
24079156 |
Appl. No.: |
10/952096 |
Filed: |
September 28, 2004 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
10952096 |
Sep 28, 2004 |
|
|
|
09522030 |
Mar 9, 2000 |
|
|
|
Current U.S.
Class: |
435/366 ;
435/404 |
Current CPC
Class: |
C12N 5/0606 20130101;
C12N 2501/115 20130101; C12N 2500/90 20130101 |
Class at
Publication: |
435/366 ;
435/404 |
International
Class: |
C12N 005/08 |
Claims
We claim:
1. A method of culturing human embryonic stem cells, comprising:
culturing the stem cells in a culture essentially free of mammalian
fetal serum and in a stem cell culture medium including amino
acids, vitamins, salts, minerals, transferrin or a transferrin
substitute, insulin or an insulin substitute, albumin, and a
fibroblast growth factor that is supplied from a source other than
just a feeder layer and is present in a concentration of at least
about 100 ng/ml, the medium capable of supporting the culture and
proliferation of human embryonic stem cells without the need for
feeder cells or for exposure of the medium to feeder cells.
2. The method of claim 1, wherein the culture is essentially free
of any animal serum.
3. A method of culturing human embryonic stem cells in defined
media without serum and without fibroblast feeder cells, the method
comprising: culturing the stem cells in a culture medium containing
albumin, amino acids, vitamins, minerals, at least one transferrin
or transferrin substitute, at least one insulin or insulin
substitute, the culture medium essentially free of mammalian fetal
serum and containing at least about 100 ng/ml of a fibroblast
growth factor capable of activating a fibroblast growth factor
signaling receptor, wherein the growth factor is supplied from a
source other than just a fibroblast feeder layer, the medium
supported the proliferation of stem cells in an undifferentiated
state without feeder cells or conditioned medium.
4. The method of claim 3, wherein said culturing step includes the
embryonic stem cells proliferating in culture for over one month
while maintaining the potential of the stem cells to differentiate
into derivatives of endoderm, mesoderm, and ectoderm tissues, and
while maintaining the karyotype of the stem cells.
5. A culture of human embryonic stem cells comprising: human
embryonic stem cells; and a stem cell medium comprising containing
albumin, amino acids, vitamins, minerals, at least one transferrin
or transferrin substitute, at least one insulin or insulin
substitute, the culture medium essentially free of mammalian fetal
serum and containing at least about 100 ng/ml of a fibroblast
growth factor capable of activating a fibroblast growth factor
signaling receptor, the medium capable of culturing stem cells in
the absence of serum and in the absence of feeder cells and also in
the absence of medium exposed to feeder cells, wherein the culture
is capable of maintaining the stem cells in an undifferentiated
state indefinitely.
Description
CROSS REFERENCES TO RELATED APPLICATIONS
[0001] This application is a continuation-in-part application of
U.S. patent application Ser. No.
[0002] 09/522,030 filed Mar. 9, 2000.
STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH
[0003] To be determined.
BACKGROUND OF THE INVENTION
[0004] The present invention relates to methods for culturing
primate embryonic stem cell cultures and culture media useful
therewith.
[0005] Primate (e.g. monkey and human) pluripotent embryonic stem
cells have been derived from preimplantation embryos. See for
example, U.S. Pat. No. 5,843,780 and J. Thomson et al., 282 Science
1145-1147 (1998). The disclosure of these publications and of all
other publications referred to herein are incorporated by reference
as if fully set forth herein. Notwithstanding prolonged culture,
these cells stably maintain a developmental potential to form
advanced derivatives of all three embryonic germ layers.
[0006] Primate (particularly human) ES cell lines have widespread
utility in connection with human developmental biology, drug
discovery, drug testing, and transplantation medicine. For example,
current knowledge of the post-implantation human embryo is largely
based on a limited number of static histological sections. Because
of ethical considerations the underlying mechanisms that control
the developmental decisions of the early human embryo remain
essentially unexplored.
[0007] Although the mouse is the mainstay of experimental mammalian
developmental biology, and although many of the fundamental
mechanisms that control development are conserved between mice and
humans, there are significant differences between early mouse and
human development. Primate/human ES cells should therefore provide
important new insights into their differentiation and function.
[0008] Differentiated derivatives of primate ES cells could be used
to identify gene targets for new drugs, used to test toxicity or
teratogenicity of new compounds, and used for transplantation to
replace cell populations in disease. Potential conditions that
might be treated by the transplantation of ES cell-derived cells
include Parkinson's disease, cardiac infarcts, juvenile-onset
diabetes mellitus, and leukemia. See e.g. J. Rossant et al. 17
Nature Biotechnology 23-4 (1999) and J. Gearhart, 282 Science
1061-2 (1998).
[0009] Long term proliferative capacity, developmental potential
after prolonged culture, and karyotypic stability are key features
with respect to the utility of primate embryonic stem cell
cultures. Cultures of such cells (especially on fibroblast feeder
layers) have typically been supplemented with animal serum
(especially fetal bovine serum) to permit the desired proliferation
during such culturing.
[0010] For example, in U.S. Pat. Nos. 5,453,357, 5,670,372 and
5,690,296 various culture conditions were described, including some
using a type of basic fibroblast growth factor together with animal
serum. Unfortunately, serum tends to have variable properties from
batch to batch, thus affecting culture characteristics.
[0011] In WO 98/30679 there was a discussion of providing a
serum-free supplement in replacement for animal serum to support
the growth of certain embryonic stem cells in culture. The serum
replacement included albumins or albumin substitutes, one or more
amino acids, one or more vitamins, one or more transferrins or
transferrin substitutes, one or more antioxidants, one or more
insulins or insulin substitutes, one or more collagen precursors,
and one or more trace elements. It was noted that this replacement
could be further supplemented with leukemia inhibitory factor,
steel factor, or ciliary neurotrophic factor. Unfortunately, in the
context of primate embryonic stem cell cultures (especially those
grown on fibroblast feeder layers), these culture media did not
prove satisfactory.
[0012] In the context of nutrient serum culture media (e.g. fetal
bovine serum), WO 99/20741 discusses the benefit of use of various
growth factors such as bFGF in culturing primate stem cells.
However, culture media without nutrient serum are not
described.
[0013] In U.S. Pat. No. 5,405,772 growth media for hematopoietic
cells and bone marrow stromal cells are described. There is a
suggestion to use fibroblast growth factor in a serum-deprived
media for this purpose. However, conditions for growth of primate
embryonic stem cells are not described.
[0014] The first human embryonic stem cell cultures were grown
using a layer of fibroblast feeder cells, which has the property of
enabling the human embryonic stem cells to be proliferated while
remaining undifferentiated. Later, it was discovered that it is
sufficient to expose the culture medium to feeder cells, to create
what is called conditioned medium, which had the same property as
using feeder cells directly. Without the use of either feeder cells
or conditioned medium, human embryonic stem cells in culture could
not be maintained in an undifferentiated state. Since the use of
feeder cells, or even the exposure of the medium to feeder cells,
risks contamination of the culture with unwanted material, avoiding
the use of feeder cells and conditioned medium is desirable. Medium
which has not been exposed to feeder cells is referred to here as
unconditioned medium.
[0015] It can therefore be seen that a need still exists for
techniques to stably culture primate embryonic stem cells without
the requirement for use of animal serum.
BRIEF SUMMARY OF THE INVENTION
[0016] In one aspect the invention provides a method of culturing
primate embryonic stem cells. One cultures the stem cells in a
culture essentially free of mammalian fetal serum (preferably also
essentially free of any animal serum) and in the presence of
fibroblast growth factor that is supplied from a source other than
just a fibroblast feeder layer. In a preferred form, the fibroblast
feeder layer, previously required to sustain a stem cell culture,
is rendered unnecessary by the addition of sufficient fibroblast
growth factor.
[0017] Fibroblast growth factors are essential molecules for
mammalian development. There are currently more then twenty known
fibroblast growth factor ligands and five signaling fibroblast
growth factor receptors therefor (and their spliced variants). See
generally D. Ornitz et al., 25 J. Biol. Chem. 15292-7 (1996); U.S.
Pat. No. 5,453,357. Slight variations in these factors are expected
to exist between species, and thus the term fibroblast growth
factor is not species limited. However, we prefer to use human
fibroblast growth factors, more preferably human basic fibroblast
growth factor produced from a recombinant gene. This compound is
readily available in quantity from Gibco BRL-Life Technologies and
others.
[0018] It should be noted that for purposes of this patent the
culture may still be essentially free of the specified serum even
though a discrete component (e.g. bovine serum albumin) has been
isolated from serum and then is exogenously supplied. The point is
that when serum itself is added the variability concerns arise.
However, when one or more well defined purified component(s) of
such serum is added, they do not.
[0019] Preferably the primate embryonic stem cells that are
cultured using this method are human embryonic stem cells that are
true ES cell lines in that they: (i) are capable of indefinite
proliferation in vitro in an undifferentiated state; (ii) are
capable of differentiation to derivatives of all three embryonic
germ layers (endoderm, mesoderm, and ectoderm) even after prolonged
culture; and (iii) maintain a normal karyotype throughout prolonged
culture. They are therefore referred to as being pluripotent.
[0020] The culturing permits the embryonic stem cells to stably
proliferate in culture for over one month (preferably over six
months; even more preferably over twelve months) while maintaining
the potential of the stem cells to differentiate into derivatives
of endoderm, mesoderm, and ectoderm tissues, and while maintaining
the karyotype of the stem cells.
[0021] In another aspect the invention provides another method of
culturing primate embryonic stem cells. One cultures the stem cells
in a culture essentially free of mammalian fetal serum (preferably
also essentially free of any animal serum) and in the presence of a
growth factor capable of activating a fibroblast growth factor
signaling receptor, wherein the growth factor is supplied from a
source other than just a fibroblast feeder layer. While the growth
factor is preferably a fibroblast growth factor, it might also be
other materials such as certain synthetic small peptides (e.g.
produced by recombinant DNA variants or mutants) designed to
activate fibroblast growth factor receptors. See generally T.
Yamaguchi et al., 152 Dev. Biol. 75-88 (1992)(signaling
receptors).
[0022] In yet another aspect the invention provides a culture
system for culturing primate embryonic stem cells. It has a human
basic fibroblast growth factor supplied by other than just the
fibroblast feeder layer. The culture system is essentially free of
animal serum.
[0023] Yet another aspect of the invention provides cell lines
(preferably cloned cell lines) derived using the above method.
"Derived" is used in its broadest sense to cover directly or
indirectly derived lines.
[0024] Variability in results due to differences in batches of
animal serum is thereby avoided. Further, it has been discovered
that avoiding use of animal serum while using fibroblast growth
factor can increase the efficiency of cloning.
[0025] It is therefore an advantage of the present invention to
provide culture conditions for primate embryonic stem cell lines
where the conditions are less variable and permit more efficient
cloning. Other advantages of the present invention will become
apparent after study of the specification and claims.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0026] In some of the following experiments one of the inventors
here used the methods and culture systems of the invention to
culture human ES cell lines without adding serum to the culture
medium. Two clonally derived human ES cell lines proliferated for
over eight months after clonal derivation and maintained the
ability to differentiate to advanced derivatives of all three
embryonic germ layers.
[0027] In another of the experiments set forth below, it has now
been demonstrated that the addition of relatively large amounts of
a human fibroblast growth factor (FGF) aids in the culture and
growth of human embryonic stem cells, even in the absence of both
serum and feeder cells. This permits the culture of stem cells that
have never been exposed either to animal cells or to media in which
animal cells have been cultured.
[0028] Techniques for the initial derivation, culture, and
characterization of the human ES cell line H9 were described in J.
Thomson et al., 282 Science 1145-1147 (1998). The experiments
described below were conducted with this and other cells lines, but
the processes and results are independent of the particular ES
cells lines.
[0029] It is described here that the addition of FGF aids in the
cultivation and cloning of human ES cells. This phenomenon occurs
because of the action of FGF in interacting with FGF receptors in
the human ES cells. It is not particularly critical which of the
many known FGF variants are used in the culture. Here basic FGF, or
bFGF, also known as FGF2, is commonly used, but that is only
because bFGF is the cheapest and most readily commercially
available member of the FGF family of factors. More then twenty
different FGF family members have been identified referred to as
FGF-1 through FGF-27. While the concentration of FGF here is given
in amounts of bFGF, it should be understood that this is intended
to quantify the amount of stimulation of the FGF receptors and that
the concentration of FGF may have to adjusted, upward or downward,
for other members of the FGF family. For bFGF, the preferred
concentration of FGF in the ES cell medium is in the range of about
0.1 to about 1000 ng/ml, with concentrations in excess of about 100
ng/ml being sufficient to avoid the need for both serum and feeder
cells.
[0030] Human ES cell cultures in the defined human ES cell media
described below in the examples can be cultivated indefinitely in
the complete absence of fibroblast feeder cells and without
conditioned media. The human ES cells retain all of the
characteristics of human ES cells including characteristic
morphology (small and compact with indistinct cell membranes),
proliferation and the ability to differentiate into many, if not
all, the cell types in the human body. The human ES cells will also
retain the characteristic that they can form all three primordial
cell layers when injected into immuno-compromised mice. In
particular, the ES cells retain the ability to differentiate into
ectoderm, mesoderm and endoderm. The ES cells still exhibit markers
indicative of ES cell status, such as expression of the nuclear
transcription factor Oct4, which is associated with pluripotency.
Throughout the process and at its end, the human ES cells retain
normal karyotypes.
EXAMPLES
[0031] In the first experiments described here human ES cells were
plated on irradiated (35 gray gamma irradiation) mouse embryonic
fibroblasts. Culture medium for the present work consisted of 80%
"KnockOut" Dulbeco's modified Eagle's medium (DMEM) (Gibco BRL,
Rockville, Md.), 1 mM L-Glutamine, 0.1 mM .beta.-mercaptoethanol,
and 1% nonessential amino acids stock (Gibco BRL, Rockville, Md.),
supplemented with either 20% fetal bovine serum (HyClone, Logan,
Utah) or 20% KnockOut SR, a serum-free replacement originally
optimized for mouse ES cells (Gibco BRL, Rockville, Md.). The
components of KnockOut SR are those described for serum
replacements in WO 98/30679.
[0032] In alternative experiments medium was supplemented with
either serum or the aforesaid serum replacer KnockOut SR, and
either with or without human recombinant basic fibroblast growth
factor (bFGF, 4 ng/ml). The preferred concentration range of bFGF
in the culture was between 0.1 ng/ml to 500 ng/ml.
[0033] To determine cloning efficiency under varying culture
conditions, H-9 cultures were dissociated to single cells for 7
minutes with 0.05% trypsin/0.25% EDTA, washed by centrifugation,
and plated on mitotically inactivated mouse embryonic fibroblasts
(10.sup.5 ES cells per well of a 6-well plate). To confirm growth
from single cells for the derivation of clonal ES cell lines,
individual cells were selected by direct observation under a
stereomicroscope and transferred by micropipette to individual
wells of a 96 well plate containing mouse embryonic fibroblasts
feeders with medium containing 20% serum replacer and 4 ng/ml
bFGF.
[0034] Clones were expanded by routine passage every 5-7 days with
1 mg/ml collagenase type IV (Gibco BRL, Rockville, Md.). Six months
after derivation, H9 cells exhibited a normal XX karyotype by
standard G-banding techniques (20 chromosomal spreads analyzed).
However, seven months after derivation, in a single karyotype
preparation, 16/20 chromosomal spreads exhibited a normal XX
karyotype, but 4/20 spreads demonstrated random abnormalities,
including one with a translocation to chromosome 13 short arm, one
with an inverted chromosome 20, one with a translocation to the
number 4 short arm, and one with multiple fragmentation.
Subsequently, at 8, 10, and 12.75 months after derivation, H9 cells
exhibited normal karyotypes in all 20 chromosomal spreads
examined.
[0035] We observed that the cloning efficiency of human ES cells in
previously described culture conditions that included animal serum
was poor (regardless of the presence or absence of bFGF). We also
observed that in the absence of animal serum the cloning efficiency
increased, and increased even more with bFGF. It has now been
established that the addition of FGF facilitated the cultivation of
human ES cells in general and is of particular help in facilitating
the cloning of human ES cultures.
[0036] The data expressed below is the total number of colonies
resulting from 10.sup.5 individualized ES cells plated, .+-.
standard error of the mean (percent colony cloning efficiency).
With 20% fetal serum and no bFGF there was a result of 240.+-.28.
With 20% serum and bFGF the result was about the same, 260.+-.12.
In the absence of the serum (presence of 20% serum replacer) the
result with no bFGF was 633.+-.43 and the result with bFGF was
826.+-.61. Thus, serum adversely affected cloning efficiency, and
the presence of the bFGF in the absence of serum had an added
synergistic benefit insofar as cloning efficiency.
[0037] The long term culture of human ES cells in the presence of
serum does not require the addition of exogenously supplied bFGF,
and (as noted above) the addition of bFGF to serum-containing
medium does not significantly increase human ES cell cloning
efficiency. However, in serum-free medium, bFGF increased the
initial cloning efficiency of human ES cells.
[0038] Further, it has been discovered that supplying exogenous
bFGF is very important for continued undifferentiated proliferation
of primate embryonic stem cells in the absence of animal serum. In
serum-free medium lacking exogenous bFGF, human ES cells uniformly
differentiated by two weeks of culture. Addition of other factors
such as LIF (in the absence of bFGF) did not prevent the
differentiation.
[0039] The results perceived are particularly applicable to clonal
lines. In this regard, clones for expansion were selected by
placing cells individually into wells of a 96 well plate under
direct microscopic observation. Of 192 H-9 cells plated into wells
of 96 well plates, two clones were successfully expanded (H-9.1 and
H-9.2). Both of these clones were subsequently cultured
continuously in media supplemented with serum replacer and
bFGF.
[0040] H9.1 and H9.2 cells both maintained a normal XX karyotype
even after more than 8 months of continuous culture after cloning.
The H-9.1 and H-9.2 clones maintained the potential to form
derivatives of all three embryonic germ layers even after long term
culture in serum-free medium. After 6 months of culture, H9.1 and
H9.2 clones were confirmed to have normal karyotypes and were then
injected into SCID-beige mice.
[0041] Both H9.1 and H9.2 cells formed teratomas that contained
derivatives of all three embryonic germ layers including gut
epithelium (endoderm) embryonic kidney, striated muscle, smooth
muscle, bone, cartilage (mesoderm), and neural tissue (ectoderm).
The range of differentiation observed within the teratomas of the
high passage H9.1 and H9.2 cells was comparable to that observed in
teratomas formed by low passage parental H9 cells.
[0042] It should be appreciated from the description above that
while animal serum is supportive of growth it is a complex mixture
that can contain compounds both beneficial and detrimental to human
ES cell culture. Moreover, different serum batches vary widely in
their ability to support vigorous undifferentiated proliferation of
human ES cells. Replacing serum with a clearly defined component
reduces the variability of results associated with this serum batch
variation, and should allow more carefully defined differentiation
studies.
[0043] Further, the lower cloning efficiency in medium containing
serum suggests the presence of compounds in conventionally used
serum that are detrimental to stem cell survival, particularly when
the cells are dispersed to single cells. Avoiding the use of these
compounds is therefore highly desired.
[0044] The present invention has been described above with respect
to its preferred embodiments. Other forms of this concept are also
intended to be within the scope of the claims. For example, while
recombinantly produced human basic fibroblast growth factor was
used in the above experiments, naturally isolated fibroblast growth
factor should also be suitable. Further, these techniques should
also prove suitable for use on monkey and other primate cell
cultures.
[0045] Thus, the claims should be looked to in order to judge the
full scope of the invention.
[0046] Additional investigations later were directed to the culture
of ES cells lines in higher concentrations of FGF but in the
absence of both serum and feeder cells. Three different medium
formulations have been used in this work, and those medium
formulations are referred to here as UM100, BM+ and DHEM. The
nomenclature UM100 refers to unconditioned medium to which has been
added 100 ng/ml of bFGF. The UM100 medium does contain the Gibco
Knockout Serum Replacer product but does not include or require the
use of fibroblast feeder cells of any kind. The BM+ medium is basal
medium (DMEM/F12) plus additives, described below, that also
permits the culture of cells without feeder cells, but this medium
omits the serum replacer product. Lastly, the name DHEM refers to a
defined human embryonic stem cell medium. This medium, also
described below, is sufficient for the culture, cloning and
indefinite proliferation of human ES cells while being composed
entirely of inorganic constituents and only human proteins, as
opposed to the BM+ medium which contains bovine albumin.
[0047] Culture of human ES cells lines H1 and H9 in
UM100/BM+/DHEM
[0048] UM100 media was prepared as follows: unconditioned media
(UM) consisted of 80% (v/v) DMEM/F12 (Gibco/Invitrogen) and 20%
(v/v) Knockout-Serum Replacer (Gibco/Invitrogen) supplemented with
1 mM glutamine (Gibco/Invitrogen), 0.1 mM .beta.-mercaptoethanol
(Sigma--St. Louis, Mo.), and 1% nonessential amino acid stock
(Gibco/Invitrogen). To complete the media 100 ng/ml bFGF was added
and the medium was filtered through a 0.22 uM nylon filter
(Nalgene).
[0049] BM+ medium was prepared as follows: 16.5 mg/ml BSA (Sigma),
196 .mu.g/ml Insulin (Sigma), 108 .mu.g/ml Transferrin (Sigma), 100
ng/ml bFGF, 1 mM glutamine (Gibco/Invitrogen), 0.1 mM
.beta.-mercaptoethanol (Sigma), and 1% nonessential amino acid
stock (Gibco/Invitrogen) were combined in DMEM/F12
(Gibco/Invitrogen) and the osmolality was adjusted to 340 mOsm with
SM NaCl. The medium was then filtered through a 0.22 uM nylon
filter (Nalgene).
[0050] DHEM media was prepared as follows: 16.5 mg/ml HSA (Sigma),
196 .mu.g/ml Insulin (Sigma), 108 .mu.g/ml Transferrin (Sigma), 100
ng/ml bFGF, 1 mM glutamine (Gibco/Invitrogen), 0.1 mM
.beta.-mercaptoethanol (Sigma), 1% nonessential amino acid stock
(Gibco/Invitrogen), vitamin supplements (Sigma), trace minerals
(Cell-gro.RTM.), and 0.014 mg/L to 0.07 mg/L selenium (Sigma), were
combined in DMEM/F12 (Gibco/Invitrogen) and the osmolarity was
adjusted to 340 mOsm with SM NaCl. It is noted that the vitamin
supplements in the media may include thiamine (6.6 g/L), reduced
glutathione (2 mg/L) and ascorbic acid PO.sub.4. Also, the trace
minerals used in the media are a combination of Trace Elements B
(Cell-gro.RTM., Cat #: MT 99-175-Cl and C (Cell-gro.RTM., Cat #: MT
99-176-Cl); each of which is sold as a 1,000.times.solution. It is
well known in the art that Trace Elements B and C contain the same
composition as Cleveland's Trace Element I and II, respectively.
(See Cleveland, W. L., Wood, I. Erlanger, B. F., J Imm. Methods 56:
221-234, 1983.) The medium was then filtered through a 0.22 uM
nylon filter (Nalgene). Finally, sterile, defined lipids
(Gibco/Invitrogen) were added to complete the medium.
[0051] H1 or H9 human embryonic stem cells previously growing on
MEF (mouse embryonic fibroblast) feeder cells were mechanically
passaged with dispase (1 mg/ml) and plated onto Matrigel (Becton
Dickinson, Bedford, Mass.). Appropriate medium was changed daily
until cell density was determined to be adequate for cell passage.
Cells were then passaged with dispase as described and maintained
on Matrigel (Becton Dickinson).
[0052] Growth Rates
[0053] To determine the growth rate of human ES cells in the
various media, cells were plated at a density of about
2.times.10.sup.5 cells/well in a 6-well tissue culture dish
(Nalgene). On days 3, 5, and 7 duplicate wells were treated with
trypsin/EDTA (Gibco/Invitrogen), individualized and cell numbers
were counted. On day 7 an additional well was treated with dispase,
counted, and used to re-seed a new plate at a cell density of about
2.times.10.sup.5 cells/well. Growth rates were collected for 3
consecutive passages. Growth rate experiments show that
UM100-cultured human ES cells grow as robustly as CM-cultured human
ES cells.
[0054] Attachment Dynamics
[0055] To determine the attachment rate of human ES cells in the
various media cells were plated at a density of 2.times.10.sup.5
cells/well in a 6-well tissue culture dish (Nalgene). At time
points ranging from 30 minutes to 48 hours unattached cells were
washed away and attached cells were removed with trypsin/EDTA
(Gibco/Invitrogen) and counted. These experiments were performed to
examine if the UM100 growth rate data was due to a combination of
better cell attachment and slower growth as opposed to equivalent
growth rates for UM100 and CM. We found that attachment percentages
were equivalent for both media at all time points tested. Thus,
they grow at the same rate.
[0056] FACS Analysis of Human ES Cells
[0057] Human ES cells were removed from a 6-well tissue culture
plate (Nalgene) with trypsin/EDTA (Gibco/Invitrogen) +2% chick
serum (ICN Biomedicals, Inc., Aurora, Ohio)for 10 min. at
37.degree. C. The cells were diluted in an equal volume of FACS
Buffer (PBS+2% FBS+0.1% Sodium Azide) and filtered through an 80
.mu.M cell strainer (Nalgene). Pellets were collected for 5 min. at
1000 RPM and resuspended in 1 ml 0.5% paraformaldehyde. Human ES
cells were fixed for 10 min. at 37.degree. C. and the pellets were
collected as described. The ES cells were resuspended in 2 ml FACS
Buffer and total cell number was counted with a hemacytometer.
Cells were pelleted as described and permeablized for 30 min. on
ice in 90% methanol. Human ES cells were pelleted as described and
1.times.10.sup.5 cells were diluted into 1 ml of FACS Buffer+0.1%
Triton X-100 (Sigma) in a FACS tube (Becton Dickinson). hESC were
pelleted as described and resuspended in 50 .mu.l of primary
antibody diluted in FACS Buffer+0.1% Triton X-100 (Sigma). Samples
of appropriate control antibodies were applied in parallel. HESC
were incubated overnight at 4.degree. C. Supernatants were poured
off and cells were incubated in the dark for 30 min. at room
temperature in 50 .mu.l of secondary antibody (Molecular
Probes/Invitrogen). FACS analysis was performed in a Facscalibur
(Becton Dickinson) cell sorter with CellQuest Software (Becton
Dickinson). This method for performing FACS analysis allows one to
detect cell surface markers, to thus show that you have ES cells.
The result observed was that human ES cells cultured in UM100 were
90% positive for Oct-4 as a population. This is comparible to
CM-cultured ES cells and confirms that the cells are an ES cell
population.
[0058] Results
[0059] Cells of human ES cell line H1 have now been cultivated in
the UM100 medium for over 14 passages (112 days) while retaining
the morphology and characteristics of human ES cells. H1 cells were
cultivated in the BM+ medium for over 6 passages (70 days) while
retaining the morphology and characteristics of human ES cells. H9
cells have been cultivated in DHEM medium for over 5 passages (67
days). Currently, H9 and H7 human ES cells are also being
cultivated in UM100 medium. Subsequent testing of the BM+ and
UM100-cultured cells established normal karyotypes. This was
demonstrated by FACS analysis discussed above.
[0060] Industrial Applicability
[0061] The present invention provides methods for culturing primate
embryonic stem cells, and culture media for use therewith.
* * * * *