U.S. patent application number 14/791479 was filed with the patent office on 2015-10-29 for suspension culture of human embryonic stem cells.
The applicant listed for this patent is Asterias Biotherapeutics, Inc.. Invention is credited to Yan Li, Ramkumar Mandalam, Isabelle Nadeau-Demers.
Application Number | 20150307838 14/791479 |
Document ID | / |
Family ID | 37595746 |
Filed Date | 2015-10-29 |
United States Patent
Application |
20150307838 |
Kind Code |
A1 |
Mandalam; Ramkumar ; et
al. |
October 29, 2015 |
Suspension Culture of Human Embryonic Stem Cells
Abstract
This disclosure provides an improved system for culturing human
embryonic stem cells. The cells are cultured in suspension so as to
maximize the production capacity of the culture environment. The
new culture system of this invention allows for bulk proliferation
of hES cells in a more cost-effective manner, which facilitates
commercial production of important products for use in human
therapy.
Inventors: |
Mandalam; Ramkumar; (Union
City, CA) ; Li; Yan; (Tallahassee, FL) ;
Nadeau-Demers; Isabelle; (San Francisco, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Asterias Biotherapeutics, Inc. |
Menlo Park |
CA |
US |
|
|
Family ID: |
37595746 |
Appl. No.: |
14/791479 |
Filed: |
July 6, 2015 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
11917993 |
Mar 4, 2008 |
9074181 |
|
|
PCT/US2006/023976 |
Mar 4, 2008 |
|
|
|
14791479 |
|
|
|
|
60693266 |
Jun 22, 2005 |
|
|
|
Current U.S.
Class: |
435/366 ;
435/405 |
Current CPC
Class: |
C12N 2501/26 20130101;
C12N 5/0606 20130101; C12N 2501/115 20130101; C12N 2501/15
20130101; C12N 2501/125 20130101 |
International
Class: |
C12N 5/0735 20060101
C12N005/0735 |
Claims
1. A culture of human embryonic stem (hES) cells in suspension,
wherein the hES cells are substantially undifferentiated.
2. The culture of claim 1, wherein the cells are cultured in a
medium containing fibroblast growth factor at a concentration of at
least about 40 ng/mL.
3. The culture of claim 2, wherein the medium also contains
transforming growth factor beta (TGF.beta.), stem cell factor
(SCF), or Flt3 ligand (Flt3L).
4. The culture of any preceding claim, wherein the cells are
cultured in a medium containing one or more soluble or suspended
extracellular matrix components.
5. The culture of claim 4, wherein the extracellular matrix
component(s) include human laminin and/or human fibronectin.
6. The culture of any preceding claim, wherein the cells are
cultured in suspension with solid microparticle.
7. The culture of claim 7, wherein the microparticle are coated
with one or more soluble or suspended extracellular matrix
components.
8. A method for culturing hES cells, comprising: a) suspending the
cells in a nutrient medium; b) maintaining the cells in suspension
while culturing; c) changing the medium periodically; d) optionally
splitting the culture from time to time so as to reduce cell
density; and finally e) harvesting cells from the culture.
9. The method of claim 8, wherein the cells are cultured in a
medium containing fibroblast growth factor at a concentration of at
least about 40 ng/mL.
10. The method of claim 9, wherein the medium also contains
transforming growth factor beta (TGF.beta.), stem cell factor
(SCF), or Flt3 ligand (Flt3L).
11. The method of any of claims 8-10, wherein the cells are
cultured in a medium containing one or more soluble or suspended
extracellular matrix components.
12. The method of claim 11, wherein the extracellular matrix
component(s) include human laminin and/or human fibronectin.
13. The method of any of claims 8-12, wherein the cells are
cultured in suspension with solid microparticle.
14. The method of claim 13, wherein the microparticle are coated
with one or more soluble or suspended extracellular matrix
components.
15. The method of any of claims 8-14, wherein the cells are
cultured in suspension for at least two months.
16. The method of any of claims 8-15, wherein the cells undergo at
least a 3-fold expansion while in suspension culture.
17. The method of any of claims 8-16, further comprising plating
the harvested cells back onto a solid surface and continuing to
culture the cells so as to maintain a substantially
undifferentiated cell population.
18. The method of any of claims 8-17, further comprising
differentiating the harvested cells.
19. A system or kit for culturing hES cells in suspension,
comprising a nutrient medium that contains at least 40 ng/mL
fibroblast growth factor.
20. The system or kit of claim 19, also comprising an extracellular
matrix component or microparticle for adding to the medium.
Description
OTHER PATENT APPLICATIONS
[0001] This application claims priority to U.S. Ser. No.
60/693,266, filed Jun. 22, 2005.
BACKGROUND
[0002] Regenerative medicine is benefiting from recent advances
relating to the isolation, culture, and use of various types of
progenitor cells. This disclosure provides further improvements for
the commercial development of human pluripotent stem cells and
their derivatives.
[0003] Embryonic stem cells have two very special properties:
First, unlike other normal mammalian cell types, they can be
propagated in culture almost indefinitely, providing a virtually
unlimited supply. Second, they can be used to generate a variety of
tissue types of interest as a source of replacement cells and
tissues for use in tissue therapy, or for use in the screening of
pharmaceutical agents.
[0004] Thomson et al. (U.S. Pat. No. 5,843,780; Proc. Natl. Acad.
Sci. USA 92:7844, 1995) were the first to successfully isolate and
propagate pluripotent stem cells from primates. They subsequently
derived human embryonic stem (hES) cell lines from human
blastocysts (Science 282:114, 1998). Gearhart and coworkers derived
human embryonic germ (hEG) cell lines from fetal gonadal tissue
(Shamblott et al., Proc. Natl. Acad. Sci. USA 95:13726, 1998; and
U.S. Pat. No. 6,090,622). Both hES and hEG cells have the
long-sought characteristics of pluripotent stem cells: they can be
cultured extensively without differentiating, they have a normal
karyotype, and they are capable of producing a number of important
cell types.
[0005] A significant challenge to the use of pluripotent stem cells
for therapy is that they are traditionally cultured on a layer of
feeder cells to prevent differentiation (U.S. Pat. No. 5,843,780;
U.S. Pat. No. 6,090,622). According to Thomson et al. (Science
282:114, 1998), hPS cells cultured without feeders soon die, or
differentiate into a heterogeneous population of committed cells.
Leukemia inhibitory factor (LIF) inhibits differentiation of mouse
ES cells, but it does not replace the role of feeder cells in
preventing differentiation of human ES cells.
[0006] U.S. Pat. No. 6,800,480 (Geron Corp.) is entitled Methods
and materials for the growth of primate-derived primordial stem
cells. International Patent Publication WO 01/51616 (Geron Corp.)
is entitled Techniques for growth and differentiation of human
pluripotent stem cells. An article by Xu et al. (Nature
Biotechnology 19:971, 2001) is entitled Feeder-free growth of
undifferentiated human embryonic stem cells. An article by
Lebkowski et al. (Cancer J. 7 Suppl. 2:S83, 2001) is entitled Human
embryonic stem cells: culture, differentiation, and genetic
modification for regenerative medicine applications. International
Patent Publication WO 03/020920 is entitled Culture System for
Rapid Expansion of Human Embryonic Stem Cells. An article by Li et
al. (Biotechnology and Bioengineering, Published Online: 21 Jun.
2005) is entitled Expansion of human embryonic stem cells. These
publications report exemplary culture reagents and techniques for
propagating embryonic stem cells in an undifferentiated state, and
their use in preparing cells for human therapy.
[0007] The information provided in the section below further
advances the science of hES cell culture that will facilitate
growing and manipulating undifferentiated pluripotent stem cells,
and help realize the full commercial potential of embryonic cell
therapy.
SUMMARY OF THE INVENTION
[0008] This disclosure provides an improved system for culturing
and proliferating primate pluripotent stem (hES) cells. The
suspension culture system of this invention enables the user to
produce high-quality embryonic stem cells in a rapid and
volume-efficient mode, for use in therapy and drug discovery.
[0009] One aspect of this invention is a culture of human embryonic
stem (hES) cells in suspension, wherein the hES cells are
substantially undifferentiated. The culture may contain one or more
of the following: fibroblast growth factor at a high concentration,
other medium additives such as TGF.beta., stem cell factor (SCF),
or Flt3 ligand (Flt3L), one or more soluble or suspended
extracellular matrix components such as laminin and/or fibronectin,
or solid microparticles of various kinds, optionally porous in
nature or coated with extracellular matrix.
[0010] Another aspect of the invention is a method for culturing
hES cells, comprising: suspending the cells in a nutrient medium;
maintaining the cells in suspension while culturing in a system
such as described above; changing the medium periodically;
optionally splitting the culture from time to time so as to reduce
cell density; and finally harvesting cells from the culture.
[0011] Another aspect of this invention is a system or kit for
culturing hES cells in suspension, comprising one or more of the
components already referred to, or described below.
[0012] These and other aspects of the invention will be apparent
from the description that follows.
DRAWINGS
[0013] FIG. 1 shows colonies of hES cells after six passages on a
solid surface in unconditioned medium supplemented with growth
factors. (A) mEF conditioned ES medium (control)+bFGF (8 ng/mL);
(B) X-VIVO.TM. 10+bFGF (40 ng/mL); (C) X-VIVO.TM. 10+bFGF (40
ng/mL)+stem cell factor (SCF, Steel factor) (15 ng/mL); (D)
X-VIVO.TM. 10+bFGF (40 ng/mL)+Flt3 ligand (75 ng/mL); (E)
QBSF.TM.-60+bFGF (40 ng/mL). All three base media (ES medium,
X-VIVO.TM. 10, and QBSF.TM.-60) can be used to expand hES cells in
feeder-free culture. In this illustration, the cells growing in
combination shown in (C) expanded 8.2-fold per passage, whereas
those in conditioned medium expanded 2.2-fold.
[0014] FIG. 2 shows the gene expression profile of hTERT and
Oct3/4, measured by real time RT-PCR, as described in Example
1.
[0015] FIG. 3 demonstrates that cells cultured in unconditioned
medium retain their pluripotency. hES cells passaged 7 times in mEF
conditioned medium, or unconditioned X-VIVO.TM. 10 medium
containing bFGF and SCF. The cells were then differentiated into
embryoid bodies, plated, and analyzed by immunocytochemistry for
phenotypic markers representing each of the three germ layers. The
cells stain for .alpha.-fetoprotein (representing endoderm); muscle
actin (representing mesoderm), and .beta.-tubulin III (representing
ectoderm).
[0016] FIG. 4 shows the cell count of hES cells grown in suspension
culture in spinner flasks (Example 3). After the culture became
established, the cells continued to thrive at the same density
(Upper Panel). When passaged back to standard surface culture they
reverted to typical morphology of the undifferentiated phenotype:
namely, distinct colonies of cells having the classic hES cell
morphology (Lower Panel).
[0017] FIG. 5 is taken from an experiment in which the cells grown
in suspension (FIG. 4) were differentiated into embryoid bodies,
plated, and then analyzed by immunocytochemistry for specific cell
types (Top Row). Cells maintained throughout by standard surface
culture are also shown (Bottom Row). The cells cultured in
suspension maintained full pluripotency, demonstrating the
effectiveness of the suspension culture system in maintaining the
important properties of undifferentiated hES cells.
[0018] FIG. 6 shows the cell count of another suspension culture in
spinner flasks.
[0019] FIG. 7 shows cells from a different hES cell line maintained
in suspension culture on a shaker device. After four weeks, the
cells were plated back onto a solid surface, and showed classic
undifferentiated hES cell morphology, as shown here. A culture was
continued in this fashion for over three months, showing
substantial proliferation of the cells in suspension.
DETAILED DESCRIPTION
[0020] Previous technology for growing primate pluripotent stem
(hES) cells has involved culturing on a solid surface: either
fibroblast feeder cells (U.S. Pat. No. 6,200,806), or extracellular
matrix (U.S. Pat. No. 6,800,480). The feeder-free technology can be
optimized to allow for rapid expansion WO 03/020920, substantially
reducing the cost of hES cell production for commercial
purposes.
[0021] The information disclosed below provides a new system that
further advances the art of hES cell culture. Specifically, the
production capacity of the culture is no longer constrained by the
two dimensional size of the culture surface, and makes fuller use
of the three-dimensions of the entire culture vessel. Growth
conditions have been identified that permit hES cells to be
cultured in suspension for over three months (Example 4). hES cells
cultured in suspension maintain phenotypic characteristics of
undifferentiated cells, and maintain the full potential to
differentiate into tissue types representing any of the three germ
layers (Example 3).
[0022] The ability to culture hES cells in a three-dimensional
space should make the bulking up of hES cells an even more
cost-effective process, and provides further opportunities to
optimize the production capacity and growth rate of hES cell
cultures. The use of suspension cultures also facilitates the
adaptation of hES cell culture methods to a closed system, where
cells and media are introduced and recovered from the system in a
sterile manner, but the system can otherwise be handled in a less
scrupulous environment.
[0023] Further advantages of the invention will be understood from
the sections that follow.
DEFINITIONS
[0024] Prototype "primate Pluripotent Stem cells" (pPS cells) are
pluripotent cells derived from pre-embryonic, embryonic, or fetal
tissue at any time after fertilization, and have the characteristic
of being capable under the right conditions of producing progeny of
several different cell types. pPS cells are capable of producing
progeny that are derivatives of each of the three germ layers:
endoderm, mesoderm, and ectoderm, according to a standard
art-accepted test, such as the ability to form a teratoma in a
suitable host, or the ability to differentiate into cells having
markers for tissue types of all three germ layers in culture.
[0025] Included in the definition of pPS cells are embryonic cells
of various types, exemplified by hES cells, defined below;
embryonic stem cells from other primates, such as Rhesus or
marmoset stem cells (Thomson et al., Proc. Natl. Acad. Sci. USA
92:7844, 1995; Developmental Biology 38:133, 1998); and human
embryonic germ (hEG) cells (Shamblott et al., Proc. Natl. Acad.
Sci. USA 95:13726, 1998). Other types of pluripotent cells are also
included in the term. Any cells of primate origin that are capable
of producing progeny that are derivatives of all three germinal
layers are included, regardless of whether they were derived from
embryonic tissue, fetal tissue, or other sources. It is beneficial
to use pPS cells that are karyotypically normal and not derived
from a malignant source.
[0026] Prototype "human Embryonic Stem cells" (hES cells) are
described by Thomson et al. (Science 282:1145, 1998; U.S. Pat. No.
6,200,806). The scope of the term covers pluripotent stem cells
that are derived from a human embryo at the blastocyst stage, or
before substantial differentiation of the cells into the three germ
layers. Those skilled in the art will appreciate that except where
explicitly required otherwise, the term includes primary tissue and
established lines that bear phenotypic characteristics of hES
cells, and derivatives of such lines that still have the capacity
of producing progeny of each of the three germ layers.
[0027] hES cell cultures are described as "undifferentiated" when a
substantial proportion of stem cells and their derivatives in the
population display morphological characteristics of
undifferentiated cells, clearly distinguishing them from
differentiated cells of embryo or adult origin. Undifferentiated
hES cells are easily recognized by those skilled in the art, and
typically appear in the two dimensions of a microscopic view with
high nuclear/cytoplasmic ratios and prominent nucleoli. It is
understood that colonies of undifferentiated cells within the
population will often be surrounded by neighboring cells that are
differentiated. Nevertheless, the undifferentiated colonies persist
when the population is cultured or passaged under appropriate
conditions, and individual undifferentiated cells constitute a
substantial proportion of the cell population. Cultures that are
substantially undifferentiated contain at least 20%
undifferentiated hES cells on an ongoing basis, and may contain at
least 40%, 60%, or 80% in order of increasing preference (in terms
percentage of cells with the same genotype that are
undifferentiated).
[0028] Whenever a culture or cell population is referred to in this
disclosure as proliferating "without differentiation", what is
meant is that after proliferation, the composition is substantially
undifferentiated according to the preceding definition. Populations
that proliferate through at least four passages (.about.20
doublings) without differentiation will contain substantially the
same proportion of undifferentiated cells (or possibly a higher
proportion of undifferentiated cells) when evaluated at the same
degree of confluence as the originating culture.
[0029] A "nutrient medium" is a medium for culturing cells
containing nutrients that promote proliferation. The nutrient
medium typically contains isotonic saline, buffer, a protein source
(in the form of one or more added proteins or amino acids), and
potentially other exogenously added nutrients and growth
factors.
[0030] A "conditioned medium" is prepared by culturing a first
population of cells in a medium, and then harvesting the medium.
The conditioned medium (along with anything secreted into the
medium by the cells) may then be used to support the growth of a
second population of cells. Where a particular ingredient or factor
is described as having been added to the medium, what is meant is
that the factor (or a cell or particle engineered to secrete the
factor) has been mixed into the medium by deliberate
manipulation.
[0031] A "fresh medium" is a medium that has not been purposely
conditioned by culturing with a different cell type before being
used with the cell type it is ultimately designed to support.
Otherwise, no limitations are intended as to its manner of
preparation, storage, or use. It is added fresh (by exchange or
infusion) into the ultimate culture, where it may be consumed or
otherwise processed by the cell types that are present.
General Techniques
[0032] General methods in molecular genetics and genetic
engineering are described in the current editions of Molecular
Cloning: A Laboratory Manual, (Sambrook et al., Cold Spring
Harbor); Gene Transfer Vectors for Mammalian Cells (Miller &
Calos eds.); and Current Protocols in Molecular Biology (F. M.
Ausubel et al. eds., Wiley & Sons). Cell biology, protein
chemistry, and antibody techniques can be found in Current
Protocols in Protein Science (J. E. Colligan et al. eds., Wiley
& Sons); Current Protocols in Cell Biology (J. S. Bonifacino et
al., Wiley & Sons) and Current Protocols in Immunology (J. E.
Colligan et al. eds., Wiley & Sons.). Reagents, cloning
vectors, and kits for genetic manipulation referred to in this
disclosure are available from commercial vendors such as BioRad,
Stratagene, Invitrogen, ClonTech, and Sigma-Aldrich Co.
[0033] Cell culture methods are described generally in the current
edition of Culture of Animal Cells: A Manual of Basic Technique (R.
I. Freshney ed., Wiley & Sons); General Techniques of Cell
Culture (M. A. Harrison & I. F. Rae, Cambridge Univ. Press),
and Embryonic Stem Cells: Methods and Protocols (K. Turksen ed.,
Humana Press). Other references of interest include Culture Is Our
Business (M. McLuhan, Ballantine Books, 1970); and Understanding
Media (M. McLuhan, Signet, 1970). Tissue culture supplies and
reagents are available from commercial vendors such as Gibco/BRL,
Nalgene-Nunc International, Sigma Chemical Co., and ICN
Biomedicals.
Sources of Stem Cells
[0034] Embryonic stem cells can be isolated from blastocysts of
members of the primate species (U.S. Pat. No. 5,843,780; Thomson et
al., Proc. Natl. Acad. Sci. USA 92:7844, 1995). Human embryonic
stem (hES) cells can be prepared from human blastocyst cells using
primary mouse fibroblast feeder cells, according to the techniques
described by Thomson et al. (U.S. Pat. No. 6,200,806; Science
282:1145, 1998; Curr. Top. Dev. Biol. 38:133 ff., 1998) and
Reubinoff et al, Nature Biotech. 18:399, 2000. hES cell lines can
also be derived on human feeders (U.S. Pat. No. 6,642,048), or in
conditions entirely free of feeder cells (US 2002/0081724 A1).
Equivalent cell types to hES cells include their pluripotent
derivatives, such as primitive ectoderm-like (EPL) cells, as
outlined in WO 01/51610 (Bresagen).
[0035] The illustrations provided in the Example section ensue from
work done with hES cells. However, except where otherwise required,
the invention can be practiced using other cells that meet the
definition of primate pluripotent stem cells.
[0036] By no means does the practice of this invention require that
a human blastocyst be disaggregated in order to produce the hES or
embryonic stem cells for practice of this invention. hES cells can
be obtained from established lines obtainable from public
depositories (for example, the WiCell Research institute, Madison
Wis. U.S.A., or the American Type Culture Collection, Manassas Va.,
U.S.A.). Human Embryonic Germ (hEG) cells can be prepared from
primordial germ cells as described in Shamblott et al., Proc. Natl.
Acad. Sci. U.S.A. 95:13726, 1998 and U.S. Pat. No. 6,090,622. U.S.
Patent Publication 2003/0113910 A1 reports pluripotent stem cells
derived without the use of embryos or fetal tissue. It may also be
possible to reprogram other progenitor cells into hES cells by
using a factor that induces the pluripotent phenotype (Chambers et
al., Cell 113:643, 2003; Mitsui et al., Cell 113:631, 2003). Under
appropriate conditions, any cell with appropriate proliferative and
differentiation capacities can be used for the derivation of
differentiated tissues for use according to this invention.
Propagation of hES Cells
[0037] Initially, most scientists in the field preferred to culture
hES cells a layer of feeder cells to prevent differentiation, as
originally described by Thomson (U.S. Pat. No. 5,843,780; U.S. Pat.
No. 6,090,622).
[0038] Early on, scientists at Geron discovered culture systems in
which the elements contributed by the feeder cells to promote
proliferation of the undifferentiated phenotype could be provided
in another form. U.S. Pat. No. 6,800,480, WO 01/51616 (Geron
Corp.), and Xu et al. (Nature Biotechnology 19:971, 2001) describe
a feeder-free culture environment that supports proliferation
without differentiation.
[0039] One aspect of the feeder-free culture method is to support
the hES cells by culturing on an extracellular matrix. The matrix
can be deposited by preculturing and lysing a matrix-forming cell
line (U.S. Pat. No. 6,800,480), such as the STO mouse fibroblast
line (ATCC Accession No. CRL-1503), or human placental fibroblasts.
The matrix can also be coated directly into the culture vessel with
isolated matrix components. Matrigel.RTM. is a soluble preparation
from Engelbreth-Holm-Swarm tumor cells that gels at room
temperature to form a reconstituted basement membrane. Other
suitable extracellular matrix components may include laminin,
fibronectin, proteoglycan, vitronectin, entactin, heparan sulfate,
and so on, alone or in various combinations. The matrix components
may be human, and/or produced by recombinant expression. Substrates
that can be tested using the experimental procedures described
herein include not only other extracellular matrix components, but
also polyamines, hydrogels, and other commercially available
coatings.
[0040] Another aspect of the feeder-free culture system is the
nutrient medium. The medium will generally contain the usual
components to enhance cell survival, including isotonic buffer
(i.e., a buffer that is isotonic when adjusted to working
concentration), essential minerals, and either serum or a serum
replacement of some kind.
[0041] A direct way to introduce hES supportive factors is to
precondition the medium with primary mouse embryonic fibroblasts
(mEF) which can be prepared as described in U.S. Pat. No. 6,200,806
or WO 01/51616. Also suitable as feeder cells are telomerized cell
lines, and human cell lines obtained from differentiating hES cells
(U.S. Pat. No. 6,642,048) or other primitive cell types. hES cell
medium can be conditioned by culturing the feeder cells (typically
irradiated or otherwise inactivated). Medium conditioned by
culturing for 1-2 hours at 37.degree. C. contains a concentration
of factors that support hES cell culture for about 1-2 days.
However, the conditioning period can be adjusted upwards or
downwards, determining empirically what constitutes an adequate
period.
[0042] As an alternative to conditioned medium, hES cells can be
grown in fresh (non-conditioned) medium containing added factors
that invoke the appropriate signal transduction pathways in
undifferentiated cells. A suitable base medium for use without
conditioning can be identified empirically. The medium typically
contains a neutral buffer (such as phosphate and/or high
concentration bicarbonate) in isotonic solution; a protein nutrient
(e.g., serum such as FBS, serum replacement, albumin, or essential
and non-essential amino acids such as glutamine). It also typically
contains lipids (fatty acids, cholesterol, an HDL or LDL extract of
serum) and other ingredients found in most stock media of this kind
(such as insulin or transferrin, nucleosides or nucleotides,
pyruvate, a sugar source such as glucose, selenium in any ionized
form or salt, a glucocorticoid such as hydrocortisone and/or a
reducing agent such as .beta.-mercaptoethanol).
[0043] Many suitable commercially available base media have been
developed for culturing proliferative cell types like hematopoietic
cells. Exemplary are X-VIVO.TM. 10 expansion medium (Biowhittaker)
and QBSF.TM.-60 (Quality Biological Inc.) (Example 1). See also WO
98/30679 (Life Technologies Inc.) and U.S. Pat. No. 5,405,772
(Amgen). The X-VIVO.TM. 10 formulation contains pharmaceutical
grade human albumin, recombinant human insulin and pasteurized
human transferrin. Exogenous growth factors, artificial stimulators
of cellular proliferation or undefined supplements are not included
in the X-VIVO.TM. 10 medium. They are also devoid of any
protein-kinase C stimulators. QBSF.TM.-60 is a serum-free
formulation that contains recombinant or pasteurized human
proteins. Other potential alternatives are Ex-Cell VPRO.TM. medium
made by JRH Biosciences, and HyQ CDM4.TM. made by Hyclone.
[0044] The base medium is supplemented with additives that promote
proliferation of the undifferentiated phenotype while inhibiting
differentiation. Fibroblast growth factor at high concentration is
especially effective to promote hES cell proliferation without
differentiation. Exemplary are basic FGF (FGF-2), and FGF-4, but
other members of the family can also be used. Equivalent forms are
species homologs, artificial analogs, antibodies to the respective
FGF receptor, and other receptor activating molecules. It has been
determined from gene expression analysis that undifferentiated hES
cells express receptors for acidic FGF (FGF-1). At a high
concentration, FGF alone is sufficient to promote growth of hES
cells in an undifferentiated state (Examples 1 and 2).
Concentrations of FGF effective for promoting undifferentiated hES
cell growth on their own usually have a lower bound of about 20,
30, or 40 ng/mL, with a practical upper bound of about 200, 500, or
1000 ng/mL. Concentrations of at least 60, 80, or 100 ng/mL bFGF
are both reliable and cost effective. Equivalent concentrations of
other forms and analogs of FGF can be determined empirically by
weaning cultures from bFGF into the proposed substitute, and
monitoring the culture for differentiation according to the marker
system described below.
Culturing hES Cells in Suspension
[0045] It has now been discovered that hES cells can be grown in
suspension culture, rather than letting the cells grow on a solid
substrate.
[0046] hES cells expanded by another culture method (or obtained
from a primary source) are inoculated into a vessel adapted to keep
the cells in suspension. The vessel walls are typically inert or
resistant to adherence of undifferentiated hES cells. There is also
a means for preventing the cells from settling out, such as a
stirring mechanism like a magnetically or mechanically driven stir
bar or paddle, a shaking mechanism (typically attached to the
vessel by the outside), or an inverting mechanism (i.e., a device
that rotates the vessel so as to change the direction of gravity
upon the cells).
[0047] Vessels suitable for suspension culture for process
development include the usual range of commercially available
spinner or shaker flasks. Fermentors suitable for commercial
production are Celligen Plus (New Brunswick Scientific Co.) and the
Stirred-Tank Reactor.TM. (Applikon Inc.). These bioreactors can be
continuously perfused with medium or used in a fed-batch mode, and
come in various sizes.
[0048] Nutrient medium that helps maintain the undifferentiated
phenotype and supports growth is replaced as needed (for example,
by letting the cells settle out, replacing the medium, and then
resuspending the cells). Growth is monitored, and the culture is
split when required to make room for further growth. After a
suitable culture period, the cells are harvested and used for their
intended purpose.
[0049] Media and other components designed for growing hES cells in
the absence of feeders on a solid surface may also work in
suspension cultures. Either conditioned or fresh media can be used
(Example 4). However, the dynamics of suspension culture provide
the user with a further opportunity to optimize the various
components of the culture system. Without intending to be limited
by theory, it is a hypothesis of this invention that suspension
culture will be enhanced if the hES cells are permitted to form
small undifferentiated clusters (the three-dimensional equivalent
of an undifferentiated colony on a solid surface), possibly
surrounded by cells partly differentiated into stromal cells--or if
the hES cells are dispersed, but shielded from the dynamic fluid
forces that otherwise might cause differentiation.
[0050] Optimization of the suspension culture system can be
accomplished by empirical testing. Undifferentiated cells from a
previous surface or suspension culture are passaged to the test
condition, and cultured for a week or more. The cells can be
examined periodically for characteristics of hES cells, for
example, using the marker system described in the next section, and
illustrated in Example 1. The cells can also be passaged back to a
well-established culture system, and evaluated for classic
morphological features of undifferentiated cells (Example 3). If
the hES cells are intended ultimately for differentiation into a
particular tissue type, then the ultimate test may not be the
marker profile of the undifferentiated culture, but the ability of
the cells to differentiate as required. The pluripotency of hES
suspension cultures can be confirmed by sampling the cells, and
either producing teratomas in SCID mice, or by staining EB-derived
cells for markers representing all three germ layers (Example 3).
The user can thereby optimize the system to achieve a high growth
rate while retaining the full pluripotency of the cells (or at
least the ability of the cells to differentiate into the intended
tissue of interest).
[0051] Aspects of the culture system that may benefit from further
optimization include the nutrient medium. Alternative base media
and alternative FGF additives are listed in the previous section.
It may also be advantageous to use one or more additional
additives, such as the following: [0052] stem cell factor (SCF,
Steel factor), other ligands or antibodies that dimerize c-kit, and
other activators of the same signal transduction pathway [0053]
ligands for other tyrosine kinase related receptors, such as the
receptor for platelet-derived growth factor (PDGF), macrophage
colony-stimulating factor, Flt-3 ligand and vascular endothelial
growth factor (VEGF) [0054] factors that elevate cyclic AMP levels,
such as forskolin [0055] factors that induce gp130, such as LIF or
Oncostatin-M; [0056] hematopoietic growth factors, such as
thrombopoietin (TPO) [0057] transforming growth factors, such as
TGF.beta.1 [0058] other growth factors, such as epidermal growth
factor (EGF) [0059] neurotrophins, such as CNTF
[0060] With a view to protecting the cells from adhering to each
other, adhering to the vessel wall, or forming clusters that are
too big, it may be beneficial to include an anti-clumping agent,
such as those sold by Invitrogen (Cat #0010057AE).
[0061] While the cells have some capacity to form their own
extracellular matrix to a limited extent, it may also be beneficial
to include one or more extracellular matrix components either
dissolved or suspended in the medium. A suitable working range to
keep laminin in suspension is about 10 to 33 .mu.g/mL. Other
candidate matrix components for suspension cultures include some of
those listed earlier, particularly fibronectin, proteoglycan,
vitronectin, and artificial equivalents thereof. The extracellular
matrix may assist the cells in forming small aggregates of an
appropriate size.
[0062] Alternatively or in addition, the suspension culture may
contain particulate carriers that create surfaces within the
suspension, but still provide the benefits of culturing the cells
in a three-dimensional space. The cells are cultured and passaged
in the same way, except that the particles are retained in the
vessel during medium exchange, and more particles are added when
the cells are split.
[0063] One type of microcarrier is solid spherical or
semi-spherical particles made from glass, plastic, dextran having a
positive charge to augment cell attachment (Cytodex), and so on.
Another type is disk-shaped culture plastic, such as the Fibra-cel
Disks.TM. sold by New Brunswick Scientific Co, Inc. A gram of these
disks provide a surface area of 1200 cm.sup.2. Solid carriers are
optionally coated with an hES cell friendly extracellular matrix,
such as laminin, so that the attached cells have the same
microenvironment as cells plated onto a solid surface.
[0064] Another type of microcarrier is macroporous particles of
various pore sizes that permit the cells to reside in the interior
as well as the outside, to potentially enhance the protective
effect. In order to recover the hES cells with minimal disruption,
it is beneficial to use particles made of a material such as
agarose that can easily be dissolved or dispersed by gentle
mechanical or enzymatic action, thereby releasing the cells for
harvest or further culture.
Characteristics of Undifferentiated hES Cells
[0065] Human ES cells cultured according to this invention have the
characteristic morphological features of undifferentiated stem
cells. In the two dimensions of a standard microscopic image, hES
cells have high nuclear/cytoplasmic ratios in the plane of the
image, prominent nucleoli, and compact colony formation with poorly
discernable cell junctions. Cell lines can be karyotyped using a
standard G-banding technique (available at many clinical
diagnostics labs that provides routine karyotyping services, such
as the Cytogenetics Lab at Oakland Calif.) and compared to
published human karyotypes. It is desirable to obtain cells that
have a "normal karyotype", which means that the cells are euploid,
wherein all human chromosomes are present and are not noticeably
altered.
[0066] hES cells can be characterized by expressed cell markers
detectable by antibody (flow cytometry or immunocytochemistry) or
by reverse transcriptase PCR. hES cells typically have
antibody-detectable SSEA-4, Tra-1-60, and Tra-1-81, but little
SSEA-1, and have alkaline phosphatase activity. Panels of suitable
markers detectable at the mRNA level are listed in application US
2003/0224411 A1 (Geron Corp.) Exemplary are Cripto,
gastrin-releasing peptide (GRP) receptor, podocalyxin-like protein
(PODXL), human telomerase reverse transcriptase (hTERT), and the
POU transcription factor Oct 3/4.
[0067] As already described, an important feature of propagated hES
cells is a potential to differentiate into cells of all three germ
layers: endoderm, mesoderm, and ectoderm. Pluripotency of hES cells
can be confirmed by forming teratomas in SCID mice, and examining
them for representative tissues of all three germ layers.
Alternatively, pluripotency can be determined by allowing hES cells
to differentiate non-specifically (for example, by forming embryoid
bodies), and then determining the cell types represented in the
culture by immunocytochemistry (Example 3). Potential of hES cells
to differentiate into particular cell lines can be determined
according to procedures described in the next section.
Uses of Propagated hES Cells
[0068] This invention provides a method by which large numbers of
pluripotent cells can be produced on a commercial scale. The cells
are useful for a number of research and commercial purposes in the
undifferentiated form, or can be directed to differentiate into a
particular cell type.
[0069] Undifferentiated hES cells can be used to screen for factors
(such as small molecule drugs, peptides, polynucleotides, and the
like) or conditions (such as culture conditions or manipulation)
that affect the characteristics of hES cells in culture. hES
cultures can also be used for the testing of pharmaceutical
compounds in drug research. Assessment of the activity of candidate
pharmaceutical compounds generally involves combining the
differentiated cells of this invention with the candidate compound,
determining any resulting change, and then correlating the effect
of the compound with the observed change. Cytotoxicity or metabolic
effects can be determined by cell viability, morphology, the
expression or release of certain markers, receptors or enzymes, DNA
synthesis or repair, and so on.
[0070] hES cells cultured according to this invention can be used
to make differentiated cells of various commercially and
therapeutically important tissue types.
Liver Cells
[0071] Hepatocytes can be differentiated from hES cells using an
inhibitor of histone deacetylase, as described in U.S. Pat. No.
6,458,589 and PCT publication WO 01/81549 (Geron Corporation).
Undifferentiated hES cells are cultured in the presence of an
inhibitor of histone deacetylase.
[0072] Staged protocols for differentiating hES cells into
hepatocytes are described, in US 2005/0037493 A1 (Geron Corp.).
Cells are cultured with several combinations of differentiation and
maturation agents in sequence, causing the hES cells to
differentiate first into early endoderm or hepatocyte precursors,
and then to mature hepatocyte-like cells. Briefly, differentiation
into endoderm-like cells can be initiated using either butyrate,
DMSO or fetal bovine serum, optionally in combination with
fibroblast growth factors. Differentiation can then continue using
a commercially available hepatocyte culture medium, including
factors such as hepatocyte growth factor (HGF), epidermal growth
factor (EGF), and/or bone morphogenic protein (e.g., BMP-2, 4, or
7) in various combinations. Final maturation may be enhanced by the
presence of agents such as dexamethazone or Oncostatin M. Cells are
obtained that express asialoglycoprotein, glycogen storage,
cytochrome p450 enzyme expression; glucose-6-phosphatase activity,
and morphological features of hepatocytes.
Nerve Cells
[0073] Neural cells can be generated from hES cells according to
the method described in U.S. Pat. No. 6,833,269; Carpenter et al.,
Exp Neurol. 2001; 172(2):383-97; and WO 03/000868 (Geron
Corporation). Undifferentiated hES cells or embryoid body cells are
cultured in a medium containing one or more neurotrophins and one
or more mitogens, generating a cell population in which at least
.about.60% of the cells express A2B5, polysialylated NCAM, or
Nestin and which is capable of at least 20 doublings in culture.
Exemplary mitogens are EGF, basic FGF, PDGF, and IGF-1. Exemplary
neurotrophins are NT-3 and BDNF. The use of TGF-.beta. Superfamily
Antagonists, or a combination of cAMP and ascorbic acid, can be
used to increase the proportion of neuronal cells that are positive
for tyrosine hydroxylase, a characteristic of dopaminergic neurons.
The proliferating cells can then be caused to undergo terminal
differentiation by culturing with neurotrophins in the absence of
mitogen.
[0074] Oligodendrocytes can be generated from hES cells by
culturing them as cell aggregates, suspended in a medium containing
a mitogen such as FGF, and oligodendrocyte differentiation factors
such as triiodothyronine, selenium, and retinoic acid. The cells
are then plated onto a solid surface, the retinoic acid is
withdrawn, and the population is expanded. Terminal differentiation
can be effected by plating on poly-L-lysine, and removing all
growth factors. Populations can be obtained in which over 80% of
the cells are positive for oligodendrocyte markers NG2
proteoglycan, A2B5, and PDGFR.alpha., and negative for the neuronal
marker NeuN. See PCT publication WO 04/007696 and Keirstead et al.,
J Neurosci. 2005; 25(19):4694-705.
Heart Cells
[0075] Cardiomyocytes or cardiomyocyte precursors can be generated
from hES cells according to the method provided in WO 03/006950.
The cells are cultured in suspension with fetal calf serum or serum
replacement, and optionally a cardiotrophic factor that affects
DNA-methylation, such as 5-azacytidine. Alternatively,
cardiomyocyte clusters can be differentiated directly on a solid
substrate using a combination of Activin A and Bone Morphogenic
Protein 4: Spontaneously contracting cells can then be separated
from other cells in the population, by density centrifugation.
[0076] Further process steps can include culturing the cells so as
to form clusters known as Cardiac Bodies.TM. removing single cells,
and then dispersing and reforming the Cardiac Bodies.TM. in
successive iterations. Populations are obtained with a high
proportion of cells staining positive for cTnI, cTnT,
cardiac-specific myosin heavy chain (MHC), and the transcription
factor Nkx2.5. See WO 03/006950, Xu et al., Circ Res. 2002;
91(6):501-8; and PCT/US2005/009081 (Geron Corporation).
Other Cell Types
[0077] Islet cells can be differentiated from hES cells by
initiating differentiation of hES cells by culturing in a medium
containing a combination of several factors selected from Activin
A, a histone deacetylase inhibitor (such as butyrate), a mitogen
(such as bFGF); and a TGF-.beta. Superfamily antagonist (such as
noggin). The cells can then be matured by culturing with
nicotinamide, yielding a cell population in which at least 5% of
the cells express Pdx1, insulin, glucagon, somatostatin, and
pancreatic polypeptide. See WO 03/050249 (Geron Corp.).
[0078] Hematopoietic cells can be made by coculturing hES cells
with murine bone marrow cells or yolk sac endothelial cells was
used to generate cells with hematopoietic markers (U.S. Pat. No.
6,280,718). Hematopoietic cells can also be made by culturing hES
cells with hematogenic cytokines and a bone morphogenic protein, as
described in US 2003/0153082 A1 and WO 03/050251 (Roberts
Institute).
[0079] Mesenchymal progenitors can be generated from hES cells
according to the method described in WO 03/004605. The hES-derived
mesenchymal cells can then be further differentiated into
osteoblast lineage cells in a medium containing an osteogenic
factor, such as bone morphogenic protein (particularly BMP-4), a
ligand for a human TGF-.beta. receptor, or a ligand for a human
vitamin D receptor (WO 03/004605; Sotile et al., Cloning Stem Cells
2003; 5(2):149-55). Chondrocytes or their progenitors can be
generated by culturing hES cells in microaggregates with effective
combinations of differentiation factors listed in WO 03/050250
(Geron Corp.).
[0080] Other differentiation methods known in the art or
subsequently developed can be applied to hES cells cultured
according to this invention. hES derived cells can be used for drug
screening, preparing pharmaceutical compositions, research, and
many other worthwhile purposes.
Commercial Distribution
[0081] Components of the culture system of this invention may be
offered for sale, sold, or otherwise distributed from the place of
manufacture for use by another entity for any purpose. Components
may also be sold or distributed together in various useful
combinations, such as two or more of the following: [0082] media
suitable for culturing hES cells in suspension factors [0083]
extracellular matrix components or thickeners present in or to be
added to the medium [0084] microcarriers present in or to be added
to the medium [0085] vessels adapted for suspension culture [0086]
the hES cells themselves, either growing in the culture system, or
stored in another form, but intended for use in the culture
system
[0087] The products and product combinations are packaged in
suitable containers, optionally in kit form, and may be accompanied
by written information on the use of the materials according to
this invention--such as maintaining or expanding hES cells. The
information may be written in any language on any communication
medium accessible and understandable by the intended user. It may
take the form of a label on the container or the kit, or a product
insert packaged with the container and distributed together.
Equivalent forms are descriptions, instructions, or explanations
written in hard copy or in electronic form available to the user or
the intended user as reference or resource material associated with
the commercially distributed product. [0088] The examples that
follow are illustrations not meant to limit the claimed
invention
EXAMPLES
Example 1
Growing Pluripotent Stem Cells in Rapid Expansion Medium
[0089] A line of hES cells was obtained that had originally been
grown on mouse embryonic fibroblast feeder cells, and then expanded
for 20 passages in a feeder-free environment comprising
Matrigel.RTM. extracellular matrix and conditioned medium,
according to U.S. Pat. No. 6,800,480; as elaborated in Xu et al.,
Stem Cells 2005; 23(3):315-23.
[0090] The hES cells were next weaned onto X-VIVO.TM.10 expansion
medium from Biowhittaker; or QBSF.TM.-60 from Quality Biological
Inc. For use in these experiments, the X-VIVO.TM. 10 medium was
supplemented with the usual goodies: namely, 2 mM L-glutamine, 1%
non-essential amino acids (Gibco), 0.1 mM .beta.-mercaptoethanol,
and 8 ng/mL bFGF. The medium was further supplemented with 8 ng/mL
or 40 ng/mL of bFGF (Gibco); 40 ng/mL of bFGF and 15 ng/mL of SCF
(R & D System); or 40 ng/mL of bFGF and 75 ng/mL of Flt3 ligand
(R & D System). QBSF.TM.-60 medium was supplemented with 0.1 mM
.beta.-mercaptoethanol, 1% non-essential amino acids (Gibco) and 40
ng/mL of bFGF. hES cells cultured in mEF conditioned medium were
used as control in these experiments.
[0091] The hES cells were first passaged onto Matrigel.RTM. coated
plates using collagenase IV, and cultured for 2 days with
conditioned medium. On day 2, the conditioned medium was replaced
with 80% unconditioned ES medium plus 20% expansion medium. Cells
were fed fresh daily and passaged weekly. The proportion of
expansion medium was increased by 20% approximately every 2 days
until the cells were completely weaned, and then grown until they
had been passaged 6 more times.
[0092] FIG. 1 shows colonies of hES cell at the end of 6 passages
(sufficient for full adaptation) in the following media: (A) mEF
conditioned medium+bFGF (8 ng/mL); (B) X-VIVO.TM. 10+bFGF (40
ng/mL); (C) X-VIVO.TM. 10+bFGF (40 ng/mL)+stem cell factor (SCF,
Steel factor) (15 ng/mL); (D) X-VIVO.TM. 10+bFGF (40 ng/mL)+Flt3
ligand (75 ng/mL); (E) QBSF.TM.-60+bFGF (40 ng/mL).
[0093] The following table shows the average total cell expansion
per passage, for undifferentiated hES cells cultured for 4 passages
in mEF conditioned medium, or for 7 passages in X-VIVO.TM. 10 or
QBSF.TM.-60.
TABLE-US-00001 TABLE 1 Growth Rates for ES Cell Cultures Average
Cell Expansion Medium per Passage mEF conditioned medium 2.2 fold
X-VIVO .TM. 10 + bFGF (40 ng/mL) 6.0 fold X-VIVO .TM. 10 + bFGF (40
ng/mL) + 8.2 fold SCF (15 ng/mL) X-VIVO .TM. 10 + bFGF (40 ng/mL) +
5.0 fold Flt3 ligand (75 ng/mL) QBSF .TM.-60 + bFGF (40 ng/mL) 6.4
fold
The average expansion of cells per passage in X-VIVO.TM. 10 and
QBSF.TM.-60 was greater than the cells cultured in mEF conditioned
medium culture. The cells in mEF conditioned medium were passaged
on average every 7 days, while the cells in X-VIVO.TM. 10 and
QBSF.TM.-60 were passaged on average every 5 days. Thus, the rate
of expansion in unconditioned X-VIVO.TM. 10 or QBSF.TM.-60 was
.about.3.2 to 5.2 times faster than in mEF conditioned ES
medium.
[0094] FIG. 2 shows the gene expression profile of hTERT and
Oct3/4. The RNA was isolated from the cells using High Pure RNA
Isolation Kit (Roche Diagnostics) and evaluated by Taqman.TM. assay
(real time RT-PCR). The gene expression in each of the test
condition is plotted relative to expression in the control culture.
Taking into consideration the instrument error and assay
variability, differences in expression between the test and control
samples are only significant if greater than 2-fold. The analysis
shows expression of hTERT and Oct-3/4 decreases somewhat upon
adaptation to unconditioned X-VIVO.TM. 10 or QBSF.TM.-60 medium
(first four bars in each set), but returns to standard levels when
the cells are passaged back into mEF conditioned medium (last three
bars in each set).
[0095] To confirm that cells cultured in unconditioned medium
retain their pluripotency, embryoid bodies were formed and analyzed
by immunocytochemistry for phenotypic markers representing each of
the three germ layers. After passage 7 in expansion medium, the
cells were dissociated into small clumps using 200 U/mL collagenase
IV at 37.degree. C. for 10 min, placed in suspension culture in
differentiation medium (DMEM+10% FBS) for 4 days, then transferred
onto poly-L-ornithine hydrobromide coated plates for a further 10
days. They were fixed in 4% paraformaldehyde, permeabilized, and
labeled by immunocytochemistry.
[0096] FIG. 3 shows the results. hES cells passaged 7 times in
unconditioned X-VIVO.TM. 10 medium stained for .alpha.-fetoprotein
(representing endoderm); muscle actin (representing mesoderm), and
.beta.-tubulin III (representing ectoderm).
[0097] Thus, hES cells can be expanded in fresh (non-conditioned)
media in a feeder-free environment at a rapid rate suitable for
commercial production--as much as 2- to 5-fold faster or more
compared with growth in conditioned medium or on feeder cells. The
cells retain the morphology of undifferentiated hES cells, and can
be differentiated into derivative cells representing all three germ
layers.
Example 2
Culture of hES Cells in a Defined System Free of Animal-Based
Products
[0098] hES cells cultured in MEF-CM on Matrigel.RTM. were passaged
to a fresh (non-conditioned) serum free medium: X-VIVO.TM. 10
supplemented with Glutamine, non-essential amino acids and
.beta.-mercaptoethanol, plus 80 ng/mL human basic FGF on
Matrigel.RTM., and then adapted to surfaces coated with human
laminin. Alternatively, cryopreserved cells were directly thawed
into the same medium containing 80 ng/mL hbFGF. The cells were
passaged every 5-6 days using Collagenase IV.
[0099] Cultures grown under these conditions were similar or better
than cultures on Matrigel.RTM.. (A) morphology for cells grown in
mEF conditioned medium; (B) morphology fin defined medium on
laminin; (C) Surface marker SSEA-4 expression in mEF-CM (H1p62) or
defined medium (H1p34+28); (D) Expression of surface marker
Tra-1-60 in mEF-CM or defined medium. Culture performance in the
defined medium on laminin was superior: very large ES cell colonies
were observed, with colonies representing about 80% of the culture.
Levels of marker expression were as follows:
TABLE-US-00002 TABLE 2 Marker Expression in Defined Culture
Conditions Culture Surface marker expression Relative gene
expression Passage no. medium SSEA-4 Tra-1-60 hTERT Oct3/4 Cripto
Experiment 1: H1p41 Conditioned 79% 93% 1.00 1.00 1.00 H1p31 + 10
Defined 92% 87% 2.85 .+-. 0.58* 0.74 .+-. 0.04 1.82 .+-. 0.62
Experiment 2: H1p44 Conditioned 81% 91% 1.00 1.00 1.00 H1p34 + 11
Defined 77% 84% 1.11 .+-. 0.38 0.57 .+-. 0.24 0.76 .+-. 0.39
Experiment 3: H1p47 Conditioned 78% 92% 1.00 1.00 1.00 H1p35 + 12
Defined 80% 86% 2.00 .+-. 0.15 0.86 .+-. 0.20 3.12 .+-. 0.91 *mean
.+-. SD for 3 RT-PCR determinations
[0100] Expression of other markers characteristic of
undifferentiated hES cells was also comparable: Measured by
real-time PCR, the levels of hTERT and Cripto were the same or
greater in defined medium compared with mEF-CM, while the
expression of Oct 3/4 was lower by about 28% (average of three
experiments). TRAP analysis showed that the cells retained
telomerase enzyme activity.
[0101] Cells grown in completely defined culture system at p34+11
(75 days) were harvested and used to make teratomas in SCID mice to
evaluate pluripotency. The teratomas showed evidence for pigmented
epithelium (endoderm); renal tissue (endoderm); mesenchymal tissue
(mesoderm); and neural tubes (ectoderm). This confirms that the
cells retained their pluripotency.
Example 3
Culturing hES Cells in Suspension
[0102] With a view to increasing the yield of hES cells per culture
volume, the cells were cultured in suspension, and then evaluated
for morphology and their ability to form differentiated cells
representative of all three germ layers.
[0103] H9 hES cells grown on Matrigel.RTM. were harvested from
6-well plates and seeded into a spinner flask under the following
condition [0104] Vessel: 100 mL spinner flask [0105] Inoculation
(seeding) density: 3.6.times.10.sup.5 cells/mL; [0106] Medium
volume: 50 mL per spinner flask [0107] Medium used: mEF conditioned
medium containing bFGF (8 ng/mL) [0108] Agitation rate: 20 rpm
(Bellco carrier magnetic stirrer) [0109] Atmosphere: 37.degree. C.
CO2 incubator [0110] Medium exchange: Every other day (exchanged by
letting the cells settle down and replacing the supernatant) H9 hES
cells were maintained in the spinner flask under these conditions
for 6 days.
[0111] FIG. 4 (upper panel) shows the results. Following an initial
decline during which the culture was established, the cell number
began to rise from day 2 through day 6.
[0112] At this point, the cells were plated back into a 6-well
plate coated with Matrigel.RTM. to determine whether they still had
the phenotype of undifferentiated cells. The culture continued
using mEF-CM medium containing bFGF (8 ng/mL).
[0113] FIG. 4 (lower panel) shows the results. After a single
passage, the cells grew and exhibited morphology of
undifferentiated cells.
[0114] Pluripotency of the cells was evaluated by forming embryoid
bodies. The cells were harvested from confluent culture using
Collagenase IV, and transferred to a low attachment 6-well plate in
DMEM+20% FBS.
[0115] EBs were formed and maintained for four days. The EBs were
then replated onto polyornithine-coated chamber slides. After a
further 11 days, the EB outgrowths were stained for
.alpha.-fetoprotein (endoderm), muscle actin (mesoderm) and
.beta.-tubulin with neuron morphology (ectoderm).
[0116] FIG. 5 shows the results. The top row shows cells
differentiated from the hES cells maintained in suspension culture,
and then plated back onto laminin under standard conditions. The
lower row shows cells differentiated from the same hES cell line
that were maintained throughout as plated cells. As shown in these
images, hES cells maintained in suspension maintained their full
capacity to differentiate into derivatives of all three-germ
layers.
[0117] In another experiment, H9 hES cells were cultured under the
following conditions: [0118] Vessel: 100 mL spinner flask [0119]
Inoculation (seeding) density: 3.5.times.10.sup.5 cells/mL [0120]
Medium volume: 50 mL per spinner flask [0121] Medium used:
mEF-CM+bFGF (8 ng/mL) [0122] Agitation rate: 20 rpm (as before)
[0123] Medium exchange: Once every three days
[0124] FIG. 6 shows the results. Once the culture was established,
the cells were maintained happily for the full 12 day period.
Example 4
Long-Term Suspension Culture
[0125] This experiment was done with another hES cell line. The
cells were cultured using a shaker flask instead of a spinner flask
under several different culture conditions, and the culture was
extended for over two months. [0126] H1 hES cells were harvested
from 6-well plates (growing on Matrigel.RTM. in mEF conditioned
medium) and seeded into shaker flasks under the following
conditions: [0127] Vessel: 100 mL Shaker Flask [0128] Inoculation
(seeding) density: 5.0.times.10.sup.5 cells/mL [0129] Medium
volume: 15 mL per shaker flask [0130] Agitation rate: 80 rpm
(Labline rotator/shaker in a 37.degree. C. CO.sub.2 incubator)
[0131] Medium exchange: Every other day initially, once every 2-3
days later The media used and culture periods were as follows:
[0132] A: mEF-CM+bFGF (8 ng/mL). Maintained for 98 days. [0133] B:
mEF-CM+bFGF (8 ng/mL)+laminin (33 .mu.g/mL to begin with, .about.10
.mu.g/mL thereafter for the rest of the culture). Maintained for 49
days. [0134] C: X-VIVO.TM. 10+FGF (40 ng/mL)+Flt-3 (75 ng/mL).
Maintained for 11 days. [0135] D: X-VIVO.TM. 10+FGF (40
ng/mL)+Flt-3 (75 ng/mL)+laminin (33 .mu.g/mL to begin with,
.about.10 .mu.g/mL thereafter for the rest of the culture).
Maintained for 11 days. Cell counts over the period of the culture
are shown in the following Table.
TABLE-US-00003 [0135] TABLE 3 Growth of hES Cells in Suspension
Cultures Medium A Medium B Medium C Medium D Days in (10.sup.5
(10.sup.5 (10.sup.5 (10.sup.5 culture cells/mL) cells/mL) cells/mL)
cells/mL) 0 5.0 5.0 5.0 5.0 1 (not counted) 7.5 6.9 7.3 3 5.9 5.0
2.8 2.8 11 2.7 2.3 2.5 2.4 32 0.3 0.15 -- -- 98 14 -- -- --
To determine whether the cells were maintaining the
undifferentiated phenotype, cells cultured for 4 weeks in Medium A
were plated back onto Matrigel.RTM. in mEF conditioned medium
containing bFGF.
[0136] FIG. 7 shows the results. After passaging, the cells derived
from the suspension culture were shown to grow and exhibit
characteristic undifferentiated hES cell colonies.
[0137] These data indicate that hES cells can be maintained in
suspension culture for at least three months, potentially
undergoing expansion by 3- to 40-fold after the culture becomes
fully established.
Example 5
Suspension Cultures Using Fresh Medium
[0138] The next experiment evaluates alternative additives for use
with fresh (unconditioned) medium in suspension cultures.
[0139] hES cells are harvested from a surface culture in fresh
medium under standard conditions (substrate of human laminin from
Sigma, coated onto 6-well plates at 2 .mu.g/cm.sup.2; X-VIVO 10.TM.
medium containing 80 ng/mL bFGF and 0.5 ng/mL TGF.beta.1). The
harvested cells are passaged into suspension culture in 100 mL
spinner flasks, using 50 mL per flask at an initial density of
.about.5.times.10.sup.5 cells/mL. The following medium alternatives
are evaluated:
[0140] 1) X-VIVO.TM. 10+bFGF (80 ng/mL)
[0141] 2) X-VIVO.TM. 10+bFGF (80 ng/mL)+TGF.beta.1 (0.5 ng/mL)
[0142] 3) X-VIVO.TM. 10+bFGF (40 ng/mL)+TGF.beta.1 (0.5 ng/mL)
[0143] 4) X-VIVO.TM. 10+bFGF (80 ng/mL)+TGF.beta.1 (0.5 ng/mL)+10
.mu.g/mL human laminin
[0144] 5) X-VIVO.TM. 10+bFGF (80 ng/mL)+TGF.beta.1 (0.5 ng/mL)+50
.mu.g/mL human serum albumin
[0145] Each spinner flask is placed on a Bellco carrier magnetic
stirrer (Bellco Biotechnology, Vineland N.J.) in a 37.degree. C.
CO.sub.2 incubator, at an initial agitation rate to 20 rpm. The
agitation rate is adjusted to keep the cells in suspension and
provide sufficient aeration, while minimizing shear forces. Medium
is changed every 2-3 days as before, monitoring cell count, and the
flasks are split as needed.
[0146] At regular intervals, cells are sampled from each flask and
plated back onto a laminin coated surface to evaluate morphology.
Cells returned to surface cultures and cells taken directly from
the suspension cultures are tested for pluripotency by
immunocytochemical staining of EB derived cells, as in Example 3
[0147] The compositions and procedures described above can be
effectively modified without departing from the claimed invention
and its equivalents.
* * * * *