U.S. patent application number 10/013205 was filed with the patent office on 2002-06-13 for human feeder cells that support proliferation of undifferentiated pluripotent stem cells.
Invention is credited to Gold, Joseph D., Xu, Chunhui.
Application Number | 20020072117 10/013205 |
Document ID | / |
Family ID | 42331357 |
Filed Date | 2002-06-13 |
United States Patent
Application |
20020072117 |
Kind Code |
A1 |
Xu, Chunhui ; et
al. |
June 13, 2002 |
Human feeder cells that support proliferation of undifferentiated
pluripotent stem cells
Abstract
This invention provides media that support the growth of primate
pluripotent stem cells in feeder-free culture, and cell lines
useful for producing such media and for other purposes, such as use
as a primary feeder layer. Conventionally, it has been necessary to
grow pluripotent embryonic cells on feeder layers of primary
embryonic fibroblasts, in order to prevent them from
differentiating. It has now been discovered that standard culture
media conditioned by special cell lines can be used to support
proliferation of pluripotent stem cells while inhibiting
differentiation in an environment free of feeder cells. This
invention includes mesenchymal and fibroblast-like cell lines
obtained from embryonic tissue or differentiated from embryonic
stem cells. Methods for deriving such cell lines, processing media,
and growing stem cells using the feeder cells or conditioned media
are described and illustrated in this disclosure.
Inventors: |
Xu, Chunhui; (Cupertino,
CA) ; Gold, Joseph D.; (San Francisco, CA) |
Correspondence
Address: |
GERON CORPORATION
230 CONSTITUTION DRIVE
MENLO PARK
CA
94025
|
Family ID: |
42331357 |
Appl. No.: |
10/013205 |
Filed: |
December 7, 2001 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
10013205 |
Dec 7, 2001 |
|
|
|
09900752 |
Jul 6, 2001 |
|
|
|
60216387 |
Jul 7, 2000 |
|
|
|
60220064 |
Jul 21, 2000 |
|
|
|
60175581 |
Jan 11, 2000 |
|
|
|
60303732 |
Jul 6, 2001 |
|
|
|
60322695 |
Sep 10, 2001 |
|
|
|
Current U.S.
Class: |
435/366 |
Current CPC
Class: |
C12N 2502/13 20130101;
C12N 15/1034 20130101; C12N 2501/39 20130101; C12N 2500/62
20130101; C12N 2501/385 20130101; C12N 2500/38 20130101; C12N
2500/14 20130101; C12N 2502/99 20130101; C12N 2501/13 20130101;
C12N 5/0606 20130101; C12N 2500/44 20130101; C12N 2501/155
20130101; C12N 15/1072 20130101; C12N 2503/02 20130101; C12N
2533/90 20130101; C12N 2500/30 20130101; C12N 2501/105 20130101;
A61K 35/12 20130101; C12N 2510/00 20130101; C12N 2510/04 20130101;
C12N 15/1096 20130101; C12N 2501/999 20130101 |
Class at
Publication: |
435/366 |
International
Class: |
C12N 005/08 |
Foreign Application Data
Date |
Code |
Application Number |
Jan 10, 2001 |
US |
PCT/US01/01030 |
Jul 19, 2001 |
WO |
01/51616 |
Claims
What is claimed as the invention is:
1. A composition of cells comprising: substantially
undifferentiated human embryonic stem (hES) cells; and cells from a
human cell line that are capable of maintaining human embryonic
stem cells in a substantially undifferentiated state through at
least 4 passages when the cells are cultured together.
2. The composition of claim 1, wherein the human cell line is a
line of mesenchymal cells or fibroblasts from a non-malignant
source.
3. The composition of claim 1, wherein the human cell line has been
obtained by differentiating human pluripotent stem cells.
4. The composition of claim 3, wherein the pluripotent stem cells
from which the human cell line has been obtained are embryonic stem
cells.
5. The composition of claim 1, wherein the human cell line is an
immortalized cell line.
6. The composition of claim 1, wherein the human cell line has been
genetically altered to express telomerase reverse transcriptase
(TERT) at an elevated level.
7. The composition of claim 3, wherein the human cell line has the
same genome as that of the hES cells in the composition.
8. The composition of claim 3, wherein the human cell line has a
different genome from that of the hES cells in the composition.
9. The composition of claim 1, wherein the cells of the human cell
line that are present in the composition have been inactivated to
prevent them from proliferating.
10. The composition of claim 9, wherein the inactivating comprises
irradiation or treatment with mitomycin c.
11. The composition of claim 1, wherein the human cell line has the
property that culturing it in a medium conditions the medium such
that hES cells can be maintained in a substantially
undifferentiated state in a growth environment essentially free of
feeder cells but containing the conditioned medium.
12. The composition of claim 1, wherein at least .about.60% of the
hES cells in the composition are undifferentiated.
13. A method of maintaining hES cells in a substantially
undifferentiated state, comprising culturing the composition of
claim 1.
14. A method of maintaining hES cells in a substantially
undifferentiated state, comprising coculturing the hES cells
through at least 4 passages with an immortalized human cell line
differentiated from human embryonic stem cells.
15. A method of obtaining a differentiated cell population,
comprising culturing the composition of claim 1, and subsequently
causing progeny of the hES cells to differentiate.
16. The method of claim 15, wherein the differentiated cell
population is a population of hepatocytes, neuronal cells, glial
cells, cardiomyocytes, or osteoblasts.
Description
RELATED APPLICATIONS
[0001] This application claims priority to pending U.S. application
Ser. No. 09/900,752, filed Jul. 6, 2001; and to provisional patent
applications 60/216,387, filed Jul. 7, 2000; and 60/220,064, filed
Jul. 21, 2000.
[0002] The aforelisted priority applications are hereby
incorporated herein by reference in their entirety, as are the
following: U.S. Ser. No. 60/175,581, filed Jan. 11, 2000; U.S. Ser.
No. 09/688,031, filed Oct. 10, 2000; U.S. Ser. No. 09/718,308,
filed Nov. 20, 2000; U.S. Ser. No. 60/303,732, filed Jul. 6, 2001;
U.S. Ser. No. 60/322,695, filed Sep. 10, 2001; and International
Patent Application PCT/US01/01030, filed Jan. 10, 2001, published
as WO 01/51616 on Jul. 19, 2001.
TECHNICAL FIELD
[0003] This invention relates generally to the field of cell
biology of embryonic cells. More specifically, it relates to the
derivation of cell lines producing conditioned culture media, which
in turn can be used to propagate human pluripotent stem cells in
feeder-free culture.
BACKGROUND
[0004] Recent discoveries have raised expectations that stem cells
may be a source of replacement cells and tissue for cells and
tissues that are damaged in the course of disease, infection, or
because of congenital abnormalities. Various types of putative stem
cells differentiate when they divide, maturing into cells that can
carry out the unique functions of particular tissues, such as the
heart, the liver, or the brain.
[0005] A particularly important discovery has been the development
of human pluripotent stem (hPS) cells (reviewed by R. A. Pedersen,
Scientif. Am. 280(4):68, 1999). These cells have the capacity to
differentiate into essentially all types of cells in the body. For
example, hPS cells have been used to generate cells that are
committed to a number of different cell lineages, which retain
their capacity to proliferate. Since these embryonic cells are
truly pluripotent, they have the potential to provide a stock
supply of different types of cells for regeneration of essentially
any type of failed tissue.
[0006] Early work on embryonic stem cells was done in mice
(reviewed in Robertson, Meth. Cell Biol. 75:173, 1997; and
Pedersen, Reprod. Fertil. Dev. 6:543, 1994). Desirable
characteristics of stem cells are that they be capable of
indefinite proliferation in vitro in an undifferentiated state,
retain a normal karyotype, and retain the potential to
differentiate to derivatives of all three embryonic germ layers
(endoderm, mesoderm, and ectoderm).
[0007] The usual method for culturing mouse stem cells is to grow
them on a layer of embryonic fibroblast feeder cells, such as the
STO cell line. Cells from a previous culture are dispersed into a
single cell suspension and cultured on the feeder cell layer. In
some experimental procedures, it is possible to grow the mouse stem
cells on gelatin plates without feeder cells, using recombinant
soluble leukemia inhibitory factor (LIF) to replace the activity
normally provided by the STO feeder cells (Robertson, supra). The
presence of LIF is sufficient to keep the mouse stem cells from
differentiating. By continuous passage of mouse stem cells, Berger
et al. (Growth Factors 14:145, 1997) selected out several cell
lines independent of exogenously added LIF.
[0008] The morphology, cell surface markers, and growth
requirements of embryonic cells from other species differ
significantly from mouse embryonic stem cells. Stem cells from
primate embryos are fragile, and it has been difficult to determine
conditions that allow them to grow in culture. Thomson et al. (U.S.
Pat. No. 5,843,780; Proc. Natl. Acad. Sci. USA 92:7844, 1995) were
the first to successfully culture 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 International Patent Publication WO 98/43679). hES cells have
the long-sought characteristics of hPS cells: they are capable of
long-term proliferation in vitro without differentiating, they
retain a normal karyotype, and they retain the capacity to
differentiate to a number of different derivatives.
[0009] A major obstacle to the use of such cells in human therapy
is that the originally described methods to propagate them involved
culturing on a layer of feeder cells. For example, when cultured in
a standard culture environment in the absence of mouse embryonic
fibroblasts as feeder cells, the hPS cells rapidly differentiate or
fail to survive. Unlike mouse ES cells, the presence of exogenously
added LIF does not prevent differentiation of the human ES cells.
Unfortunately, the use of feeder cells substantially increases the
cost of production, and makes scale-up of hPS cell culture
impractical. The feeder cells are metabolically inactivated to keep
them from outgrowing the stem cells, so it is necessary to have
fresh feeder cells for each splitting of the hES culture.
Furthermore, procedures are not yet developed for completely
separating feeder cell components away from embryonic cells
prepared in bulk culture. When embryonic cells are grown using
currently available culture methods, the presence of xenogeneic
components from the feeder cells complicates their potential use in
human therapy.
[0010] International Patent Publication WO/9920741 (Geron Corp.) is
entitled "Methods and Materials for the Growth of Primate-Derived
Primordial Stem Cells". In one embodiment, a cell culture medium is
provided for growing primate-derived primordial stem cells in a
substantially undifferentiated state, having a low osmotic pressure
and low endotoxin levels. The basic medium is combined with a
nutrient serum effective to support the growth of primate-derived
primordial stem cells and a substrate of feeder cells or an
extracellular matrix component derived from feeder cells. The
medium further includes non-essential amino acids, an anti-oxidant,
and growth factors that are either nucleosides or a pyruvate
salt.
[0011] New technology to facilitate growing undifferentiated hPS
cells without feeders would be a major achievement towards
realizing the full potential of embryonic cell therapy. In
particular, there is a need for media that support feeder-free
culture that is compatible with regulatory requirements for human
administration, and can be scaled up for commercial production.
SUMMARY
[0012] This invention provides media that support the growth of
primate pluripotent stem (pPS) cells in feeder-free culture, and
cell lines useful for production of such media.
[0013] One embodiment of this invention is a method for preparing a
conditioned medium suitable for culturing pPS cells in a growth
environment essentially free of feeder cells, comprising
conditioning medium by culturing cells in the medium, and then
harvesting the conditioned medium.
[0014] Another embodiment of this invention is a method for
culturing pPS cells, comprising providing a conditioned medium of
this invention, and culturing pPS cells in a feeder-free growth
environment containing the conditioned medium. In either of these
embodiments, the pPS cells can be human embryonic stem (hES)
cells.
[0015] The cells used for conditioning the medium are taken from a
cell line that can proliferate in culture for an extended period,
typically at least about 60 days. This does not necessarily mean
that the particular cells used for conditioning the medium be
long-lived; they can be irradiated or otherwise adapted to limit
replication. However, the parent cell line from which they are
taken will have the proliferative capacity stated. In particular
embodiments, the cell line is an immortalized mouse cell line, or a
human cell line, and may have characteristics of a fibroblast or
muscle cell line. Exemplary lines are obtained by differentiating
human embryonic stem cells ex vivo, and are euploid. Any of these
cell lines can be genetically altered to express a growth factor
(such as bFGF) or telomerase reverse transcriptase (TERT) at an
elevated level.
[0016] Another embodiment of this invention is conditioned medium
to support culturing pPS cells in a growth environment essentially
free of feeder cells, prepared according to a method of this
invention.
[0017] A further embodiment is a composition of proliferating pPS
cells or other cells in a growth environment that includes the
conditioned medium of this invention, optionally free of feeder
cells. In one variant of this embodiment, the medium is conditioned
by the cells in situ by culturing the pPS cells together with a
cell line of this invention that produce the factors that inhibit
differentiation. In this manner, the cell line acts like a
traditional feeder layer. In another variant of this embodiment,
the cell line is cultured alone to condition the medium in which it
is grown, and then the medium is harvested to support pPS cell
growth in a subsequent culture.
[0018] Yet another embodiment of this invention is a human cell
line obtained by differentiating a culture of human embryonic stem
(hES) cells into a population of differentiated cells that
comprises fibroblast-like or mesenchymal cells, and then selecting
such cells from the culture. Medium conditioned by culturing with
the selected cells supports growth of pPS cells in feeder-free
culture. If desired, the cell line can be genetically altered to
express TERT at an elevated level. Exemplary cell lines are
described in Examples 7-10, where they are referred to as human
feeder cells.
[0019] A further embodiment of the invention is a method for
screening cells suitable for producing conditioned medium that
supports growth of pPS cells, based on a feeder-free growth
environment in which growth of pPS cells without differentiation
can be promoted by medium conditioned by primary mouse embryonic
fibroblasts (mEF). In the screening method, a test medium
conditioned by cells being screened according to the method is used
instead, the ability of the test medium to support growth of the
pPS is assessed, and the suitability of the medium is correlated
with growth of the pPS without substantial differentiation.
[0020] Still another embodiment of the invention is a device for
preparing conditioned medium, comprising a culture chamber
containing cells from a cell line of this invention that can
condition medium to render it able to support growth of pPS cells
in feeder-free culture; and a port for withdrawing conditioned
medium from the culture chamber.
[0021] These and other embodiments of the invention will be
apparent from the description that follows.
DRAWINGS
[0022] FIG. 1 is a copy of photomicrographs showing the morphology
of hES cells in feeder-free culture. Panel A (Left Side) shows
morphology of hES cells cultured on feeder cells in regular culture
medium (mEF/RM), or on Matrigel.RTM., laminin, fibronectin, or
collagen IV in mEF conditioned medium. Panel B (Right Side) shows
morphology of hES cells maintained on Matrigel.RTM. in medium
conditioned by mEF, NHG190, STO and BJ cells, compared with
unconditioned regular medium (RM). The cells in RPE conditioned
medium differentiated within the first week of culture. The cells
in the other conditioned media all had hES colonies with
appropriate ES-morphology. Panel C is a bar graph showing integrin
expression measured in hES cells maintained on feeders in regular
medium (mEF/RM) or on Matrigel.RTM.; or on laminin in mEF
conditioned medium. Integrin components .alpha.6 and .beta.1 may
play a role in adherence of hES cells to extracellular matrix.
[0023] FIG. 2 shows surface marker expression in feeder-free cells
by FACS analysis. Panel A is a FACS scan profile showing expression
of SSEA-4 (sometimes present on undifferentiated cells) for hES
grown on feeders in regular medium (mEF/RM), or on extracellular
matrix with conditioned medium. Panel B is a bar graph showing
fluorescence intensity of surface markers for hES cells cultured on
different matrices. Panel C is a bar graph showing surface markers
for hES cells cultured on Matrigel.RTM. in conditioned medium from
different cell lines.
[0024] FIG. 3 is a reproduction of micrographs showing marker
expression detected by immuno-histochemistry for cells grown with
primary feeder cells (mEF) or on the extracellular matrices
Matrigel.RTM. or laminin. hES cells grown in feeder-free culture
have phenotypic markers similar to those of hES grown on mouse
fibroblast feeder cells.
[0025] FIG. 4 provides an analysis of Oct-4 and hTERT expression in
hES cells cultured with feeder cells (mEF) or extracellular matrix
(Matrigel.RTM. or laminin) with regular medium (RM) or conditioned
medium (CM). The upper panel is a copy of a gel showing Oct-4 and
hTERT expression at the mRNA level by RT-PCR. The lower panel is a
bar graph comparing the level of expression for cells grown on
different substrates, expressed as the ratio of Oct4 or hTERT to
the 18s standard. hES cells grown on Laminin and Matrigel.RTM. in
conditioned medium have similar expression patterns to those of
cells grown on a feeder layer.
[0026] FIG. 5, Panel A, is a copy of a photomicrograph showing the
morphology of a human fibroblast-like (hEF) cell line, derived from
human embryonic stem cells. These cells have been immortalized by
infecting with retrovirus pBABE containing an hTERT expression
cassette. Panel B (below) is a line graph showing the growth of the
hEF-like cells transduced for expression of the telomerase
catalytic subunit, compared with cells transduced with vector
control.
[0027] FIG. 6 is a copy of six photomicrographs, comparing human
embryonic stem cells maintained in different culture environments.
Panels A & D: hES cells grown directly on primary mouse
embryonic fibroblasts (mEF). Panels B & E: hES cells in
feeder-free culture supported by conditioned medium from primary
mEF. Panels C & F: hES cells in feeder-free culture supported
by medium from human embryonic fibroblast-like cells (FIG. 5). In
each of these conditions, healthy colonies of hES cells increased
in size, and had characteristic features of undifferentiated
embryonic stem cells.
[0028] FIG. 7 shows features of a second human cell line capable of
producing conditioned medium that supports hES cells in feeder-free
culture, derived from the H1 line of hES cells. Panel A is a copy
of a phase contrast micrograph, showing that the HEF1 cell line has
morphological characteristics of fibroblasts or mesenchymal cells.
Panel B (below) is a copy of the results of a TRAP assay, showing
that HEF1 cells transduced with a retroviral vector for telomerase
reverse transcriptase (hTERT) acquired telomerase activity.
[0029] FIG. 8 is a graph showing growth of hTERT-transduced HEF1
cells, and cells transduced with vector control. Both lines
initially doubled about once every 2 days. However, the control
cells stopped proliferating at 38 days, while the hTERT-transfected
cells continued proliferating over 60 days at a consistent growth
rate.
[0030] FIG. 9 is a copy of a micrograph of HEF1 cells with or
without transduction with hTERT, after staining for
senescence-associated .beta.-galactosidase, a known biomarker for
cellular aging. Transfection with hTERT extends the life-span of
the cell line and forestalls senescence.
[0031] FIG. 10 contains micrograph copies showing undifferentiated
colonies of hES cells grown on the hTERT immortalized HEF1 human
feeder cell line. Upper panels show the H9 line of hES cells
maintained on HEF1 cells (left), or under feeder-free conditions
using mEF cell conditioned medium. Lower panels show the H9.2
clonal sub-line. After 3-5 passages, the hES cells continued to
produce colonies with the morphological characteristics of
undifferentiated stem cells. This illustrates that the HEF1 feeder
cells inhibit hES cell differentiation, and can be used to maintain
hES cells in prolonged culture.
[0032] FIG. 11 shows colonies of hES cells after passaging into
conditioned medium produced by the same HEF1 cell line. Panel A is
a copy of a light micrograph, showing undifferentiated colonies in
cultures of hES cells maintained in medium conditioned by primary
mouse embryonic fibroblasts (mEF), or by the human fibroblast-like
cell line HEF1. Panel B (right) is a copy of the results of a TRAP,
showing that hES cells maintained using HEF1 conditioned medium
show telomerase activity characteristic of undifferentiated
cells.
DETAILED DESCRIPTION
[0033] This invention provides a system for preparing media that
support the growth of primate pluripotent stem (pPS) cells in the
absence of feeder cells. Cell lines have been developed that can
provide a reliable, effective supply of conditioned media for bulk
stem cell culture.
[0034] Conventionally, human embryonic stem cells are grown on
feeder layers of primary embryonic fibroblasts isolated from mouse
(mEFs). As an alternative, they can be grown in feeder-free culture
on an appropriate extracellular matrix, using medium conditioned by
primary mEFs (Example 2) to supply soluble components provided by
the mEFs in feeder culture. Fibroblasts derived from adults are
generally not used as feeder cells, suggesting that more mature
cells lose the ability to provide the factors requisite to support
stem cell growth.
[0035] It has now been discovered that feeder cells can be
immortalized and maintained in long-term culture without causing
them to lose the ability to produce high quality conditioned
medium. For example, primary mouse embryonic fibroblasts can be
immortalized by genetically altering them to express telomerase
reverse transcriptase (Examples 4 and 5). The telomerized mEF line
designated NHG190 supports the growth of hES cells in feeder-free
culture without differentiation through a number of passages (FIG.
1B), and can itself be perpetuated in culture, providing a ready
source of high-quality medium.
[0036] It has also been discovered that human cells suitable for
conditioning medium can be obtained by differentiating embryonic
stem cells in vitro. hES cells were differentiated by culturing in
suspension for 2-4 days, and then plating on gelatin-coated plates.
Fibroblast-like cells (hEF) were separated from the mixed
population and expanded (Example 7). The cell line was found to
lack substantial telomerase activity, and some of the cells were
transduced with a retrovirus expressing telomerase reverse
transcriptase (hTERT). Both the untransduced and the telomerized
cell lines proliferated in continual cell culture for over 7
population doublings in 50 days of culture (FIG. 5B).
[0037] Cultures of hES cells grown on Matrigel(D in hEF conditioned
medium formed colonies with morphology characteristic of
undifferentiated hES cells (FIG. 7, Panels C & F). The cultures
appeared indistinguishable from hES cells grown directly on a layer
of primary mEF feeders (Panels A & D). hES cells have
proliferated under these conditions for over 30 days without
differentiation.
[0038] This invention provides a significant advance in technology
for producing pluripotent stem cells suitable for commercial
distribution:
[0039] Availability of established cell lines supporting pPS
culture obviates the need to repeatedly prepare .primary feeder
cultures to continue the culture.
[0040] The cell lines of this invention facilitate producing pPS
cells on a commercial scale. In comparison with primary feeder
cells, they are easier and more economical to maintain and
expand.
[0041] Media produced by established cell lines are of consistently
high quality, and avoid the variability inherent in media obtained
from primary cell cultures.
[0042] In the context of human therapy, the use of telomerized
human cell lines to produce conditioned media is relatively
attractive from the perspective of regulatory scrutiny, since these
cells contain no xenogeneic components and no components of tumor
cell origin.
[0043] Those skilled in the art will readily appreciate that
pluripotent stem cells cultured using the cell lines and media of
this invention provide an important reserve for developing and
implementing new therapeutic strategies. A further description
follows.
[0044] Definitions
[0045] Prototype "primate Pluripotent Stem Cells" (pPS cells) are
pluripotent cells derived from pre-embryonic, embryonic, or fetal
tissue of the stated primate species at any time after gestation,
which have the characteristic of being capable under the right
conditions of producing progeny of several different cell types.
Preferred pPS cells are those capable of producing progeny that are
derivatives of all of the three germinal layers: endoderm,
mesoderm, and ectoderm, and capable of undergoing proliferation in
the absence of feeder cells, as described in this disclosure.
Non-limiting exemplars of pPS cells are rhesus and marmoset
embryonic stem cells, as described by Thompson et al., Proc. Natl.
Acad. Sci. USA 92:7844,1995, human embryonic stem (hES) cells, as
described by Thomson et al., Science 282:1145, 1998; and human
embryonic germ (hEG) cells, described in Shamblott et al., Proc.
Natl. Acad. Sci. USA 95:13726, 1998. Other types of non-malignant
pluripotent cells are included in the term. Specifically, any cells
that are fully pluripotent (that is, they are those capable of
producing progeny that are derivatives of all of the three germinal
layers) are included, regardless of whether they were derived from
embryonic tissue, fetal tissue, or adult tissue.
[0046] pPS cell cultures are said to be "substantially
undifferentiated" when they display morphology that clearly
distinguishes them from differentiated cells of embryo or adult
origin. pPS cells typically have high nuclear/cytoplasmic ratios,
prominent nucleoli, and compact colony formation with poorly
discernable cell junctions, and are easily recognized by those
skilled in the art. It is recognized that colonies of
undifferentiated cells can be surrounded by neighboring cells that
are differentiated. Nevertheless, the substantially
undifferentiated colony will persist when cultured under
appropriate conditions, and undifferentiated cells constitute a
prominent proportion of cells growing upon splitting of the
cultured cells. Useful cell populations described in this
disclosure contain any proportion of substantially undifferentiated
pPS having these criteria. Substantially undifferentiated cell
cultures may contain at least about 20%, 40%, 60%, or even 80%
undifferentiated pPS in order of increasing preference (in
percentage of total cells in the population).
[0047] 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 state according to the preceding definition.
Populations that proliferate through at least 4 passages 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.
[0048] "Feeder cells" or "feeders" are terms used to describe cells
of one tissue type that are co-cultured with cells of a tissue
type, to provide an environment in which the cells of the second
tissue type can grow. The feeder cells are optionally from a
different species as the cells they are supporting. For example,
certain types of pPS cells can be supported by primary cultures of
mouse embryonic fibroblasts, immortalized mouse embryonic
fibroblasts, or human fibroblast-like cells differentiated from hES
cells, as described later in this disclosure. In coculture with pPS
cells, feeder cells are typically inactivated by irradiation or
treatment with an anti-mitotic agent such as mitomycin c, to
prevent them from outgrowing the cells they are supporting. For use
in producing conditioned medium, inactivation of the cells is
optional, and depends in part on mechanical aspects of medium
production.
[0049] pPS cell populations are said to be "essentially free" of
feeder cells if the cells have been grown through at least one
round after splitting in which substantial fresh feeder cells are
not added to support the growth of the pPS. It is recognized that
if a previous culture containing feeder cells is used as a source
of pPS for the culture to which fresh feeders are not added, there
will be some feeder cells that survive the passage. For example,
hES cells are often cultured in a 9.6 cm.sup.2 well on a surface of
.about.375,000 primary irradiated embryonic fibroblasts near
confluence. By the end of the culture, perhaps 150,000 feeder cells
are still viable, and will be split and passaged along with hES
that have proliferated to a number of .about.1 to 1.5 million.
After a 1:6 split, the hES cells generally resume proliferation,
but the fibroblasts will not grow and only a small proportion will
be viable by the end of .about.6 days of culture. This culture is
"essentially free" of feeder cells, with compositions containing
less than about 5%, 1%, 0.2%, 0.05%, or 0.01% feeder cells
(expressed as % of total cells in the culture) being increasingly
more preferred.
[0050] Whenever a culture or cell population is referred to in this
disclosure as "feeder-free", what is meant is that the composition
is essentially free of feeder cells according to the preceding
definition, subject to further constraints only if explicitly
indicated. A "growth environment" is an environment in which cells
of interest will proliferate in vitro. Features of the environment
include the medium in which the cells are cultured, the
temperature, the partial pressure of O.sub.2 and CO.sub.2, and a
supporting structure (such as a substrate on a solid surface) if
present.
[0051] A "nutrient medium" is a medium for culturing cells
containing nutrients that promote proliferation. The nutrient
medium may contain any of the following in an appropriate
combination: isotonic saline, buffer, amino acids, antibiotics,
serum or serum replacement, and exogenously added factors.
[0052] 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.
[0053] The term "antibody" as used in this disclosure refers to
both polyclonal and monoclonal antibody. The ambit of the term
deliberately encompasses not only intact immunoglobulin molecules,
but also such fragments and derivatives of immunoglobulin molecules
(such as single chain Fv constructs), and fragments and derivatives
of immunoglobulin equivalents such as T-cell receptors, as may be
prepared by techniques known in the art, and retaining the desired
antigen binding specificity.
[0054] For the purposes of this disclosure, a "mesenchymal cell"
can be either a terminally differentiated cell or a proliferative
precursor cell committed to form cells of a mesenchymal tissue,
such as bone, dental tissue, cartilage, tendon, bone marrow stroma,
or muscle. Mesenchymal stem cells are included in the term, as are
terminally differentiated (post-mitotic) cells and more committed
replication-competent cells, such as osteoblast precursor
cells.
[0055] A cell is said to be "genetically altered" or "transformed"
when a polynucleotide has been transferred into the cell by any
suitable means of artificial manipulation, or where the cell is a
progeny of the originally altered cell that has inherited the
polynucleotide. The polynucleotide may contain a sequence that is
exogenous to the cell, it may contain native sequences in an
artificial arrangement (e.g., an encoding region linked to a
different promoter), or it may provide additional copies of a
native encoding sequence. Unless explicitly stated otherwise, the
process of transferring the polynucleotide into the cell can be
achieved by any technique suitable for the application at hand,
which may include but is not limited to electroporation or
liposome-mediated transfer, homologous recombination, transduction
or transfection using a viral or bacterial vector. The
polynucleotide will often comprise a transcribable sequence
encoding a protein of interest, which enables the cell to express
the protein at an elevated level. Also included are genetic
alterations by any means that result in functionally altering or
abolishing the action of an endogenous gene. Suitable methods for
effecting such alterations include homologous recombination using a
suitable targeting vector (U.S. Pat. Nos. 5,464,764, 5,631,153,
5,789,215, 5,589,369 and 5,776,774).
[0056] The genetic alteration is said to be "inheritable" if
progeny of the altered cell has the same alteration. Determination
of whether the genetic alteration is inheritable can be made by
detecting presence of the polynucleotide template (e.g., by PCR
amplification), or by detecting a phenotypic feature (such as
expression of a gene product or effect thereof) that depends on the
genetic alteration to be manifest.
[0057] A "cell line" is a population of cells that can be
propagated in culture through at least 10 passages. The population
can be phenotypically homogeneous, or the population can be a
mixture of measurably different phenotypes. Characteristics of the
cell line are those characteristics of the population as a whole
that are essentially unaltered after 10 passages.
[0058] A cell line is from a "non-malignant source" if it was
established from primary tissue that is not cancerous, nor from a
cell that was genetically altered with a known oncogene.
Immortalization of such cells by telomerization maintains their
non-malignant status.
[0059] A cell is described as "telomerized" if it has been
genetically altered with a nucleic acid encoding a telomerase
reverse transcriptase (TERT) of any species in such a manner that
the TERT is transcribed and translated in the cell. The term also
applies to progeny of the originally altered cell that have
inherited the ability to express the TERT encoding region at an
elevated level. The TERT encoding sequence is typically taken or
adapted from a mammalian TERT gene, exemplified by human and mouse
TERT, as indicated below.
[0060] A cell line is described as "permanent" or "immortalized" if
it has at least one of the following properties: 1) it has been
genetically altered for elevated expression of telomerase reverse
transcriptase (TERT), detectable, for example, as increased
telomerase activity in TRAP assay; 2) for cell lines otherwise
capable of no more than 15 population doublings, it has been
genetically altered to extend its replicative capacity under
suitable culture conditions to at least 20 population doublings; or
3) for cell lines otherwise capable of more than 15 population
doublings, it has been genetically altered to substantially extend
the replicative capacity of the cell line under typical culture
conditions. It is understood that cells meeting this definition
include not only the original genetically altered cells, but also
all progeny of such cells that meet the criteria of this
definition.
[0061] General Techniques
[0062] For further elaboration of general techniques useful in the
practice of this invention, the practitioner can refer to standard
textbooks and reviews in cell biology, tissue culture, and
embryology. Included are Teratocarcinomas and embryonic stem cells:
A practical approach (E. J. Robertson, ed., IRL Press Ltd. 1987);
Guide to Techniques in Mouse Development (P. M. Wasserman et al.
eds., Academic Press 1993); Embryonic Stem Cell Differentiation in
Vitro (M. V. Wiles, Meth. Enzymol. 225:900, 1993); Properties and
uses of Embryonic Stem Cells: Prospects for Application to Human
Biology and Gene Therapy (P. D. Rathjen et al., al.,1993).
Differentiation of stem cells is reviewed in Robertson, Meth. Cell
Biol. 75:173, 1997; and Pedersen, Reprod. Fertil. Dev. 10:31,
1998.
[0063] Methods in molecular genetics and genetic engineering are
described in Molecular Cloning: A Laboratory Manual, 2nd Ed.
(Sambrook et al., 1989); Oligonucleotide Synthesis (M. J. Gait,
ed., 1984); Animal Cell Culture (R. I. Freshney, ed., 1987); the
series Methods in Enzymology (Academic Press, Inc.); Gene Transfer
Vectors for Mammalian Cells (J. M. Miller & M. P. Calos, eds.,
1987); Current Protocols in Molecular Biology and Short Protocols
in Molecular Biology, 3rd Edition (F. M. Ausubel et al., eds., 1987
& 1995); and Recombinant DNA Methodology II (R. Wu ed.,
Academic Press 1995). Reagents, cloning vectors, and kits for
genetic manipulation referred to in this disclosure are available
from commercial vendors such as BioRad, Stratagene, Invitrogen, and
ClonTech.
[0064] General techniques used in raising, purifying and modifying
antibodies, and the design and execution of immunoassays including
immunohistochemistry, the reader is referred to Handbook of
Experimental Immunology (D. M. Weir & C. C. Blackwell, eds.);
Current Protocols in Immunology (J. E. Coligan et al., eds., 1991);
and R. Masseyeff, W. H. Albert, and N. A. Staines, eds., Methods of
Immunological Analysis (Weinheim: VCH Verlags GmbH, 1993).
[0065] General techniques in cell culture and media collection are
outlined in Large Scale Mammalian Cell Culture (Hu et al., Curr.
Opin. Biotechnol. 8:148, 1997); Serum-free Media (K. Kitano,
Biotechnology 17:73, 1991); Large Scale Mammalian Cell Culture
(Curr. Opin. Biotechnol. 2:375, 1991); and Suspension Culture of
Mammalian Cells (Birch et al., Bioprocess Technol. 19:251, 1990).
Other reading of interest includes Understanding Media (M. McLuhan,
Mentor N.Y., 1964) and The Medium is the Massage (M. McLuhan &
Q. Fiore, Bantam N.Y., 1967).
[0066] Preparation of Pluripotent Stem Cells from Primate Embryonic
Tissue
[0067] Conditioned media described in this application are useful
for culturing pluripotent stem cells in the presence or absence of
feeder cells. It is recognized that other types of cells may
benefit from being cultured in these media, and the compositions of
this invention may be used for such purposes without restriction.
However, the media (and the cells used to prepare media) have the
characteristic of being able to support the growth of primate
pluripotent stem cells in culture environments essentially free of
feeder cells.
[0068] Types of pluripotent stem (pPS) cells that may be used
include established lines of pluripotent cells derived from tissue
formed after gestation, including pre-embryonic tissue (such as a
blastocyst), embryonic tissue, or fetal tissue taken any time
during gestation, typically but not necessarily before .about.10-12
weeks gestation. Non-limiting exemplars are established lines of
hES or hEG cells, described in more detail below. Also contemplated
is use of the compositions of this disclosure during the initial
establishment or stabilization of such cells, in which case the
source cells would be primary pluripotent cells taken directly from
the source tissues. Also suitable are cells taken from a pPS cell
population already cultured in the absence of feeder cells.
[0069] Human embryonic stem (hES) cells can be prepared as
described by Thomson et al. (U.S. Pat. No. 5,843,780; Science
282:1145, 1998; Curr. Top. Dev. Biol. 38:133 ff., 1998; Proc. Natl.
Acad. Sci. USA 92:7844, 1995).
[0070] Briefly, human blastocysts are obtained from human in vivo
preimplantation embryos. Alternatively, in vitro fertilized (IVF)
embryos can be used, or one cell human embryos can be expanded to
the blastocyst stage (Bongso et al., Hum Reprod 4: 706, 1989).
Briefly, human embryos are cultured to the blastocyst stage in G1.2
and G2.2 medium (Gardner et al., Fertil. Steril. 69:84, 1998).
Blastocysts that develop are selected for ES cell isolation. The
zona pellucida is removed from blastocysts by brief exposure to
pronase (Sigma). The inner cell masses are isolated by
immunosurgery, in which blastocysts are exposed to a 1:50 dilution
of rabbit anti-human spleen cell antiserum for 30 minutes, then
washed for 5 minutes three times in DMEM, and exposed to a 1:5
dilution of Guinea pig complement (Gibco) for 3 min (see Solter et
al., Proc. Natl. Acad. Sci. USA 72:5099,1975). After two further
washes in DMEM, lysed trophectoderm cells are removed from the
intact inner cell mass (ICM) by gentle pipetting, and the ICM
plated on mEF feeder layers.
[0071] After 9 to 15 days, inner cell mass-derived outgrowths are
dissociated into clumps either by exposure to calcium and
magnesium-free phosphate-buffered saline (PBS) with 1 mM EDTA, by
exposure to dispase or trypsin, or by mechanical dissociation with
a micropipette; and then replated on mEF in fresh medium.
Dissociated cells are replated on mEF feeder layers in fresh ES
medium, and observed for colony formation. Colonies demonstrating
undifferentiated morphology are individually selected by
micropipette, mechanically dissociated into clumps, and replated.
ES-like morphology is characterized as compact colonies with a high
nucleus to cytoplasm ratio and prominent nucleoli. Resulting ES
cells are then routinely split every 1-2 weeks by brief
trypsinization, exposure to Dulbecco's PBS (without calcium or
magnesium and with 2 mM EDTA), exposure to type IV collagenase
(.about.200 U/mL; Gibco) or by selection of individual colonies by
micropipette. Clump sizes of about 50 to 2000 cells are
optimal.
[0072] Alternatively, primate PS cells can be passaged between
feeder-free cultures as a finer cell suspension, providing that an
appropriate enzyme and medium are chosen, and the plating density
is sufficiently high. By way of illustration, confluent human
embryonic stem cells cultured in the absence of feeders are removed
from the plates by incubating with a solution of 0.05% (wt/vol)
trypsin (Gibco) and 0.053 mM EDTA for 5-15 min at 37.degree. C.
With the use of pipette, the remaining cells in the plate are
removed and the cells are triturated with the pipette until the
cells are dispersed into a suspension comprising single cells and
some small clusters. The cells are then plated at densities of
50,000-200,000 cells/cm.sup.2 to promote survival and limit
differentiation. The phenotype of ES cells passaged by this
technique is similar to what is observed when cells are harvested
as clusters by collagen digestion. As another option, the cells can
be harvested without enzymes before the plate reaches confluence.
The cells are incubated .about.5 min in a solution of 0.5 mM EDTA
alone in PBS, washed from the culture vessel, and then plated into
a new culture without further dispersal.
[0073] Human Embryonic Germ (hEG) cells can be prepared from
primordial germ cells present in human fetal material taken about
8-11 weeks after the last menstrual period. Suitable preparation
methods are described in Shamblott et al., Proc. Natl. Acad. Sci.
USA 95:13726,1998 and International Patent Publication WO
98/43679.
[0074] Briefly, genital ridges are rinsed with isotonic buffer,
then placed into 0.1 mL 0.05% trypsin/0.53 mM sodium EDTA solution
(BRL) and cut into <1 mm.sup.3 chunks. The tissue is then
pipetted through a 100 .mu.L tip to further disaggregate the cells.
It is incubated at 37.degree. C. for .about.5 min, then .about.3.5
mL EG growth medium is added. EG growth medium is DMEM, 4500 mg/L
D-glucose, 2200 mg/L mM sodium bicarbonate; 15% ES qualified fetal
calf serum (BRL); 2 mM glutamine (BRL); 1 mM sodium pyruvate (BRL);
1000-2000 U/mL human recombinant leukemia inhibitory factor (LIF,
Genzyme); 1-2 ng/ml human recombinant basic fibroblast growth
factor (bFGF, Genzyme); and 10 .mu.M forskolin (in 10% DMSO).
[0075] Ninety-six well tissue culture plates are prepared with a
sub-confluent layer of feeder cells cultured for 3 days in modified
EG growth medium free of LIF, bFGF or forskolin, inactivated with
5000 rad .gamma.-irradiation. Suitable feeders are STO cells (ATCC
Accession No. CRL 1503). .about.0.2 mL of primary germ cell (PGC)
suspension is added to each of the wells. The first passage is
conducted after 7-10 days in EG growth medium, transferring each
well to one well of a 24-well culture dish previously prepared with
irradiated STO mouse fibroblasts. The cells are cultured with daily
replacement of medium until cell morphology consistent with EG
cells were observed, typically after 7-30 days or 1-4 passages.
[0076] Using Conditioned Media to Support Feeder-free Stem Cell
Cultures
[0077] pPS cells are typically cultured on a layer of feeder cells
that support the pPS cells in various ways. Without intending any
limitation to the claimed invention, it is a hypothesis of this
invention that feeder cells secrete soluble factors that promote
pPS cell survival or proliferation, or inhibit differentiation.
[0078] According to conventional methods, primate PS cells are
first derived and supported on primary embryonic fibroblasts of
mouse origin (Thomson et al., supra). As adapted in this
disclosure, mEFs are irradiated at a dose to inhibit proliferation
but permit synthesis of important factors that support hES cells
(.about.4000 rads .gamma.-irradiation), and then plated near
confluence. For example, 6-well culture plates can be coated by
incubation at 37.degree. C. with 1 mL 0.5% gelatin, and plated with
.about.375,000 irradiated mEFs per well. Feeder cell layers are
used 5 h to 1 week after plating, providing fresh hES medium just
before seeding the pPS cells.
[0079] As an alternative, it has been found that an artificial
growth environment can be used that is essentially free of feeder
cells, but nonetheless supports proliferation of pPS cells without
undergoing substantial differentiation, as defined earlier. The
growth of pPS cells in feeder-free culture without differentiation
is supported using a medium conditioned by culturing previously
with another cell type. Without intending any limitation of the
invention, it is hypothesized that the conditioning process
provides an opportunity for the cells to release into the medium
soluble factor that replace the role of the feeder cells in
promoting pPS cell survival or proliferation, or inhibiting
differentiation.
[0080] By way of illustration, conditioned medium can be prepared
by culturing irradiated primary mouse embryonic fibroblasts at a
density of 5.times.10.sup.5 cells per 9.6 cm.sup.2 well in a serum
replacement medium containing 4 ng/mL basic fibroblast growth
factor (bFGF). The culture supernatant is typically harvested after
1 day at 37.degree. C., and supplemented with additional growth
factors that benefit pPS cell culture.
[0081] Other features of the growth environment facilitate pPS
proliferation without differentiation. In the absence of feeder
cells, the pPS are plated onto a suitable culture substrate.
Particularly suitable are extracellular matrix components, such as
those derived from basement membrane or that may form part of
adhesion molecule receptor-ligand couplings. A commercial
preparation is available from Becton Dickenson under the name
Matrigel.RTM., and can be obtained in a Growth Factor Reduced
formulation. Both formulations are effective. Matrigel.RTM. is a
soluble preparation from Engelbreth-Holm-Swarm tumor cells that
gels at room temperature to form a reconstituted basement
membrane.
[0082] Other extracellular matrix components and component mixtures
are suitable as an alternative. Depending on the cell type being
proliferated, this may include laminin, fibronectin, proteoglycan,
entactin, heparan sulfate, and the like, alone or in various
combinations. Laminins are major components of all basal laminae in
vertebrates, which interact with integrin heterodimers such as
.alpha.6.beta.1 and .alpha.6.beta.4(specific for laminins) and
other heterodimers (that cross-reach with other matrices).
[0083] The pluripotent cells are plated onto the substrate in a
suitable distribution and in the presence of a medium that promotes
cell survival, propagation, and retention of the desirable
characteristics. All these characteristics benefit from careful
attention to the seeding distribution. One feature of the
distribution is the plating density. It has been found that plating
densities of at least about 20,000 cells cm.sup.-2 promote survival
and limit differentiation. Typically, a plating density of between
about 90,000 cm.sup.-2 and about 170,000 cm.sup.-2 is used.
[0084] Another feature is the dispersion of cells. The propagation
of mouse stem cells involves dispersing the cells into a
single-cell suspension (Robinson, Meth. Mol. Biol. 75:173, 1997 at
page 177). In contrast, the passage of pPS cells in the absence of
feeders benefits from preparing the pPS cells in small clusters.
Enzymatic digestion is halted before cells become completely
dispersed, and the cells are triturated with the pipette until they
are suspended as clumps of adherent cells, about 10-200 cells in
size. The clumps are then plated directly onto the substrate
without further dispersal.
[0085] Selecting cells suitable for conditioning media
[0086] Conditioned medium can be tested for its ability to support
pPS cells by culturing pPS cells in a feeder-free growth
environment where they grow without differentiation when supported
by medium conditioned by primary mouse embryonic fibroblasts (mEF),
but using the test medium instead. If pPS cells grow in a
substantially undifferentiated state, then the conditioned medium
can be characterized as supporting pPS cells in feeder free
culture.
[0087] A convenient way to determine whether pPS cells are
differentiating is to follow the morphological features of the
colonies. For example, characteristic morphological features of
undifferentiated hES cells are known by those skilled in the art,
and include high nuclear/cytoplasmic ratios, prominent nucleoli,
and compact colony formation with poorly discernable cell
junctions. During passage, some cells may differentiate
(particularly when replated as single cells, or when large clusters
are allowed to form). However, cultures typically reestablish a
larger proportion of undifferentiated cells during the culture
period. Ideally, the propagated cells will have a doubling time of
no more than about 20-40 hours.
[0088] According to this assay method, conditioned medium will be
considered capable of supporting growth of pPS cells if the
proportion of undifferentiated pPS cells in a subconfluent culture
(typically .about.5 days after passaging) does not substantially
decline through at least 4 passages in the conditioned medium
(optionally supplemented with additional growth factors or
otherwise processed as appropriate). Determination that the pPS
cells are undifferentiated can be done by morphological criteria,
and confirmed by demonstrating expression of Oct-4 and/or TERT.
[0089] If desired, a quantitative readout of this assay can be
obtained to estimate the quality or concentration of pPS cell
supportive factors in the medium. In one example, basal medium is
conditioned for various periods of time (say, 6 h, 12 h, 24 h, and
48 h), and the conditioned media are each tested for their ability
to support feeder-free pPS cell culture for consecutive 24 h
periods. Media rendered effective by briefer conditioning periods
are increasingly more preferred. In another example, basal medium
conditioned for 24 h is tested by dilution analysis (diluting in
basal medium, optionally supplemented with other nutrients) for its
ability to support pPS culture. Media that are effective after
greater dilution (for example, 1:1, 1:2, and 1:4 conditioned
medium:diluent medium) are increasingly more preferred. Other
variables that can be refined include the plating density of the
cells, whether or not they are inactivated (e.g., by irradiation or
with mitomycin c), and conditions for filtering the media.
[0090] Cell lines can be tested for their ability to produce
conditioned medium by culturing the cells in a basal medium for an
appropriate time, and then testing the medium for its ability to
support feeder-free pPS cell cultures as described above. If the
conditioned medium does not support feeder-free pPS cultures, the
method of conditioning can be adjusted in various parameters, such
as culture time, basal medium used, cell density, and possible
post-culture processing of the medium or supplementation with
additional additives. Adjustment of these and other parameters can
be performed empirically, and as a matter of routine
experimentation. A cell line will be considered to have passed the
test if it produces conditioned medium that support feeder-free pPS
cultures after routine optimization of any of the culture
parameters during conditioning.
[0091] The skilled reader will appreciate that cells capable of
producing conditioned medium will also be capable of maintaining
pPS cells in an undifferentiated form by culturing the cells
together. For this reason, the exemplary cell lines described in
Examples 7-10 are referred to as "human feeder cells". Accordingly,
differentiated cells of this invention can be identified in
coculture experiments. For example, the differentiated cell line
can be processed according to the traditional method of preparing
feeder cells described earlier, inactivated, and then overlaid with
pPS cells to determine whether they can support proliferation of
the pPS cells without differentiation (Example 10). Differentiated
cells that have this property are good candidates for producing
conditioned medium. Of course, if they pass the coculturing test,
they can be used to derive and maintain pPS cells as a feeder
layer, regardless of whether or not they are ideal for conditioning
medium.
[0092] If desired, the conditioned media and differentiated cells
of this invention can be further evaluated based on other
characteristics of the pPS cells they support. For example, pPS
cells can be further characterized based on expressed cell markers.
Tissue-specific markers can be detected using a suitable
immunological technique--such as flow cytometry for membrane-bound
markers, immunohistochemistry for extracellular or intracellular
markers, and enzyme-linked immunoassay, for markers secreted into
the medium. The expression of protein markers can also be detected
at the mRNA level by reverse transcriptase-PCR using
marker-specific primers. See U.S. Pat. No. 5,843,780 for further
details.
[0093] Under the microscope, ES cells appear with high
nuclear/cytoplasmic ratios, prominent nucleoli, and compact colony
formation with poorly discernable cell junctions. Primate ES cells
may express one or more of the stage-specific embryonic antigens
(SSEA) 3 and 4, and markers detectable using antibodies designated
Tra-1-60 and Tra-1-81 (Thomson et al., Science 282:1145, 1998).
Differentiation of hES cells in vitro results in the loss of
SSEA-4, Tra-1-60, and Tra-1-81 expression (if present) and
increased expression of SSEA-1. Undifferentiated hES cells
typically have alkaline phosphatase activity, which can be detected
by fixing the cells with 4% paraformaldehyde, and then developing
with Vector Red as a substrate, as described by the manufacturer
(Vector Laboratories, Burlingame, Calif.) Undifferentiated hES
cells also typically express Oct-4 and TERT, as detected by RT-PCR
(Example 3).
[0094] Another desirable phenotype of propagated pPS cells is a
potential to differentiate into cells of all three germinal layers:
endoderm, mesoderm, and ectoderm tissues. Pluripotency of hES cells
can be confirmed by injecting cells into SCID mice, fixing the
teratomas that form using 4% paraformaldehyde, and then examining
them histologically for evidence of cell types from the three germ
layers. Propagated pPS lines can be karyotyped using a standard
G-banding technique and compared to published karyotypes of the
corresponding primate species. 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 not noticeably
altered.
[0095] Exemplary Non-human Cell Lines
[0096] Feeder cells typically contain fibroblast type cells.
Primary embryonic or fetal feeder cell cultures are a mixed
population of cells, containing cells that have morphology of
fibroblasts and of early muscle cells. Different cells in the
population may play different roles in supporting pPS culture, and
the distribution and character of the culture may change.
[0097] More permanent feeder cell lines can be obtained for
producing medium according to this invention by obtaining embryonic
fibroblasts from a non-human species such as a mouse. The cells are
then genetically altered with an immortalizing gene, such as a gene
that expresses telomerase. The altered cell line can then be
passaged or frozen for future use in preparing cultured medium. To
use the cells in supporting pPS in a feeder culture, the feeder
cells are plated to near confluence, irradiated to prevent
proliferation, and then layered with small clumps of pPS cells that
have either been freshly isolated or passaged from previous
culture.
[0098] Mouse embryonic fibroblasts (mEF) can be obtained from
outbred CF1 mice (SASCO) or other suitable strains. In an
illustrative method, the abdomen of a mouse at 13 days of pregnancy
is swabbed with 70% ethanol, and the decidua is removed into
phosphate buffered saline (PBS). Embryos are harvested; placenta,
membranes, and soft tissues are removed; and the carcasses are
washed twice in PBS. They are then transferred to fresh 10 cm
bacterial dishes containing 2 mL trypsin/EDTA, and finely minced.
After incubating 5 min at 37.degree. C., the trypsin is inactivated
with 5 mL DMEM containing 10% FBS, and the mixture is transferred
to a 15 mL conical tube. Debris is allowed to settle for 2 min, the
supernatant is made up to a final volume of 10 mL, and plated onto
a 10 cm tissue culture plate or T75 flask. The flask is incubated
undisturbed for 24 h, after which the medium is replaced. When
flasks are confluent (.about.2-3 d), they are split 1:2 into new
flasks. Mouse embryonic fibroblasts can be propagated in mEF
medium, containing 90% DMEM (Gibco # 11965-092), 10% FBS (Hyclone #
30071-03), and 2 mM L-glutamine. T150 flasks are used (Corning #
430825), splitting the cells 1:2 every other day with trypsin, to
keep the cells subconfluent.
[0099] If desired, the cells used for conditioning medium can be
genetically altered to provide one or more additional features. For
example, for screening purposes, cells can be provided with drug
resistance genes for one or more antibiotics, such as neomycin,
hygromycin, or puromycin (Example 5). Cells can be provided with
marker genes, such as green fluorescent protein (Example 5),
.beta.-galactosidase, or certain cell-surface antigens (such as a
truncated NGF receptor) that provide a tag for immunoisolation.
Cells can also be provided with genes for the biosynthesis and
secretion of factors that supplement the potency of the medium for
supporting pPS culture. Exemplary is human basic fibroblast growth
factor (bFGF), and other nutritional supplements listed in this
disclosure.
[0100] To increase the replicative capacity of a cell line used for
conditioning medium, it can be telomerized. Cells are telomerized
by genetically altering them with a suitable vector, as described
elsewhere in this disclosure, so that they express the telomerase
catalytic component (TERT) at an elevated level. Particularly
suitable is the catalytic component of human telomerase (hTERT),
provided in International Patent Publication WO 98/14592. For some
applications, other TERT sequences can be used (mouse TERT is
provided in WO 99/27113).
[0101] Typically, the vector will comprise a TERT encoding region
under control of a heterologous promoter that will promote
transcription in the cell line. For example, sequences that can
drive expression of the TERT coding region include viral LTRs,
enhancers, and promoters (such as MPSV, SV40, MoLV, CMV, MSCV, HSV
TK), eukaryotic promoters (such as .beta.-actin, ubiquitin, EF1a,
PGK) or combinations thereof (for example, the CMV enhancer
combined with the .beta.-actin promoter). Expression of a marker
gene can be driven by the same promoter as the TERT gene, either as
a separate expression cassette, as part of a polycistronic
transcript (in which the coding regions of TERT and the marker gene
are separated by an IRES sequence, allowing both individual
proteins to be made from a single transcript driven by a single
promoter), or as part of the same cassette (a fusion between the
coding regions of both TERT and the marker gene, producing a
protein that provides the functions of both TERT and the marker
gene). Transfection and expression of telomerase in human cells is
described in Bodnar et al., Science 279:349, 1998 and Jiang et al.,
Nat. Genet. 21:111, 1999.
[0102] Before and after telomerization, telomerase activity and
hTERT expression can be determined using standard reagents and
methods. For example, pPS cells are evaluated for telomerase using
TRAP activity assay (Kim et al., Science 266:2011, 1997; Weinrich
et al., Nature Genetics 17:498, 1997). hTERT expression can also be
evaluated by RT-PCR.
[0103] Other methods of immortalizing cells are also contemplated,
such as genetically altering the cells with DNA encoding the SV40
large T antigen (U.S. Pat. No. 5,869,243, International Patent
Publication WO 97/32972), infecting with Epstein Bar Virus,
introducing oncogenes such as myc and ras, introducing viral
replication genes such as adenovirus E1a, and fusing cells having
the desired phenotype with an immortalized cell line. Transfection
with oncogenes or oncovirus products is usually less suitable when
the cells are to be used for therapeutic purposes.
[0104] Exemplary Human Cell Lines
[0105] It has been discovered that cells with particular
characteristics differentiated from human embryo derived cells can
be used to support a culture of undifferentiated hPS cells, either
as a feeder layer, or to produce conditioned medium for growing hPS
cells in feeder-free culture. Many types of fibroblast-like cells
derived from human embryo cells have this property, and can be
identified according to the assays described earlier.
[0106] An exemplary method for obtaining suitable cells involves
differentiating a culture of pPS cells (such as hES cells).
Differentiated cells with a particular phenotype are selected from
amongst the mixed differentiated cell population, and medium
conditioned by culturing with the selected cells is tested for its
ability to support growth of pPS cells in a culture environment
essentially free of feeder cells.
[0107] Differentiation of the pPS can be initiated by first forming
aggregates, for example, by overgrowth of a donor pPS cell culture,
or by culturing pPS cells in culture vessels having a substrate
with low adhesion properties that allows embryoid bodies (EB) to
form. Embryoid bodies can be made in suspension culture:
undifferentiated pPS cells are harvested by brief collagenase
digestion, dissociated into clusters, and plated in non-adherent
cell culture plates. The aggregates are fed every few days, and
then harvested after a suitable period, typically 4-8 days. The
cells are then cultured in a medium and/or on a substrate that
promotes enrichment of medium-conditioning cells. Alternatively,
the cells can be obtained by direct differentiation of pPS cells by
preparing a suspension of undifferentiated pPS cells, and then
plating directly onto a substrate that promotes regulated
differentiation into medium-conditioning cells. Suitable substrates
include glass or plastic coverslips that coated with a polycationic
substance, such as poly-ornithine, or an extracellular matrix.
[0108] Once differentiated, the population can be enriched for
medium-conditioning cells either according to markers they express
(for example, by immunolabeling and fluorescence sorting, by
sorting on magnetic beads, or by immune-specific lysis of
contaminating cells).
[0109] It has been discovered that mesenchymal or fibroblast-like
cells differentiated from hES cells are especially appropriate for
conditioning medium according to this invention. Mesenchymal cells
and fibroblasts can be recognized by morphological criteria.
Relatively undifferentiated mesenchymal cells have mononuclear
ovoid, stellate shape or spindle shape, with a round to oval
nucleus and a poorly defined cell border. The oval elongate nuclei
typically have prominent nucleoli and a mix of hetero- and
euchromatin. Fibroblast-like cells have a stellate or spindle
shape, with cytoplasmic processes that resemble those of
fibroblasts found in connective tissue. Further confirmation of the
mesenchymal or fibroblast nature of a cell can be obtained by
markers and secreted products of the cell, such as collagen matrix,
collagenase, and various isotypes of fibroblast growth factor,
particularly bFGF.
[0110] Where derived from an established line of hES cells,
differentiated cells with the foregoing properties will typically
have the same genome as the line from which they are derived. This
means that the chromosomal DNA will be over 90% identical between
the pPS cells and the derived cells, which can be inferred if the
differentiated cells are obtained from the undifferentiated line
through the course of normal mitotic division. Cells that have been
treated by recombinant methods to introduce a transgene (such as
TERT) or knock out an endogenous gene are still considered to have
the same genome as the line from which they are derived, since all
non-manipulated genetic elements are preserved. As illustrated in
Example 10, differentiated cells can be used to maintain hES cells
from the same line having the same genome (Examples 7-9) or hES
cells of a different line having a different genome (Example
10).
[0111] Differentiated pPS cells can then be tested according to the
assays outlined above, to determine if they are suitable for
conditioning medium in such a manner that the medium supports pPS
cell growth in feeder-free culture.
[0112] Cell lines differentiated and selected from hES in this
manner typically are capable of replicating in cell culture for at
least about 30 days (Example 7; FIG. 5(B)). In some embodiments,
the cells may replicate for at least .about.60 days or 120 days
(.about.10 doublings, 25 doublings, or 50 doublings). If desired,
the cells can also be genetically altered to express telomerase
reverse transcriptase at an elevated level, or otherwise
immortalized as described earlier.
[0113] Optionally, differentiated human ES cells suitable for
conditioning medium can be further adapted--for example, by
genetically altering to express a growth factor like bFGF, or to
express TERT, or to immortalize the cells, as described in the
previous section.
[0114] The reader will appreciate that cell lines derived from pPS
cells have a variety of potential commercial applications, of which
conditioning medium is only the first example. Fibroblast-like
cells and mesenchymal cells are useful for in vitro testing of
pharmaceutical compositions and genetic constructs, they can be
used to support culture of other cells, and they can be
incorporated into cellular compositions designed for tissue
regeneration in vivo.
[0115] Producing Conditioned Media
[0116] A conditioned medium of this invention is produced by
culturing cells in the medium, and then harvesting the conditioned
medium from the cell culture.
[0117] The cells used for the conditioning have the ability to
condition medium in a manner that gives it the capacity to support
pPS cells in feeder-free form, as described earlier. The base
medium used for conditioning can have any of several different
formulae, depending in part on the types of cells used. The medium
must be able to support culture of at least the cell line used for
the conditioning of the medium. It is convenient that the medium
also support culture of pPS after conditioning. However, as an
alternative, the medium can be supplemented with other factors or
otherwise processed after conditioning to adapt it for culturing
the pPS cells.
[0118] For supporting pPS cells in feeder-free culture, suitable
base media can be made from the following components: Dulbecco's
modified Eagle's medium (DMEM), Gibco # 11965-092; Knockout
Dulbecco's modified Eagle's medium (KO DMEM), Gibco # 10829-018;
Ham's F12/50% DMEM basal medium; 200 mM L-glutamine, Gibco #
15039-027; non-essential amino acid solution, Gibco 11140-050;
.beta.-mercaptoethanol, Sigma # M7522; human recombinant basic
fibroblast growth factor (bFGF), Gibco # 13256-029. Exemplary
serum-containing ES medium is made with 80% DMEM (typically KO
DMEM), 20% defined fetal bovine serum (FBS) not heat inactivated,
1% non-essential amino acids, 1 mM L-glutamine, and 0.1 mM
.beta.-mercaptoethanol. The medium is filtered and stored at
4.degree. C. for no longer than 2 weeks. Serum-free ES medium is
made with 80% KO DMEM, 20% serum replacement, 1% non-essential
amino acids, 1 mM L-glutamine, and 0.1 mM .beta.-mercaptoethanol.
Not all serum replacements work; an effective serum replacement is
Gibco # 10828-028 (proprietary formula; product obtainable from the
manufacturer). The medium is filtered and stored at
4.degree..degree. C. for no longer than 2 weeks. Just before
combining with the cells used for conditioning, human bFGF can be
added to a final concentration of 4 ng/mL.
[0119] The selected medium is then combined with the cells used for
conditioning in an environment that allows the cells to release
into the medium the components that support pPS cells. Optionally,
the cells can be inactivated (i.e., rendered incapable of
substantial replication) by radiation (e.g., .about.4,000 rads),
treatment with a chemical inactivator like mitomycin c, or by any
other effective method. The inactivation of the cells is not
necessary in instances where the medium is separated from the
conditioning cells before use in supporting hPS cell cultures.
[0120] The cells are cultured in the medium for sufficient time to
allow adequate concentration of released factors (or consumption of
media components) to produce a medium that supports the culturing
of pPS cells without differentiation. Typically, medium conditioned
by culturing for 24 h at 37.degree. C. produces medium that
supports pPS cell culture for 24 hours. However, the culturing
period can be adjusted upwards or downwards, determining
empirically (or by assaying for the concentration of essential
factors) what constitutes an adequate period. After collecting a
batch of conditioned medium, the cells can be used to condition a
further batch of medium over a further culture period, for as many
cycles as desired as long as the cells retain their ability to
condition the medium in an adequate fashion. For example,
fibroblast-like cells derived from differentiation of embryonic
stem cells can be used to condition medium over 1-day periods for
1-2 weeks (Example 8).
[0121] Selection of culture apparatus for conditioning medium can
be made based on the scale and purpose of medium collection. In
initial studies and for screening purposes, it is often convenient
to produce cultured medium in standard culture flasks or multi-well
plates. Large scale, automated, or GMP compliant production can
involve the use of specialized devices.
[0122] Continuous cell culture systems are reviewed by J. Furey
(Genetic Eng. News 20:10, May 15, 2000). Perfusion culture involves
removal of medium from the culture chamber, and replenishment with
fresh medium. In the spin basket system, a basket-like device is
attached to a drive shaft and covered by a porous screen through
which medium can be exchanged. In the external filter perfusion
system, a culture is circulated from a vessel, through a
hollow-fiber filter module, and back to the vessel, with a pump
attached to the loop to provide the circulation. A particular
perfusion system, the ATF System (available commercially from
Refine Technology, Edison, N.J.) consists of a diaphragm pump on
one end of a hollow-fiber housing, the other end of which is
connected to a bioreactor. Alternating tangential flow through the
fibers generates low shear laminar flow, which provides high flow
rates, scalability, and adaptability to different bioreactors.
[0123] Large-scale culture systems are also available from Aastrom
Sciences Inc., Ann Arbor Mich. The AastromReplicell.TM. System
provides for expansion from small starting cell populations (Koller
et al., Bone Marrow Transpl. 21:653, 1009; Koller et al., Blood
86:1784, 1995). Cellstasis.RTM. culture technology is marketed by
Genespan Corp., Bothell Wash. Cells reside in extracapilliary
spaces, and hollow fibers bring fresh media and oxygen into the
culture environment (R. Lewis, Genetic Eng. News 18(9), May 1,
1998). Any other suitable device can be used with this invention.
U.S. Pat. No. 4,501,815 describes a device for culturing
differentiated cells. U.S. Pat. No. 4,296,205 describes cell
culture and continuous dialysis flasks and their use. U.S. Pat. No.
5,994,129 describes a portable cassette for use in maintaining
biological cells. U.S. Pat. No. 5,362,642 describes a containment
system for storing, reconstituting, dispensing, and harvesting cell
culture media. U.S. Pat. No. 6,022,742 describes a culture device
and method.
[0124] A particular embodiment of this invention is a device
adapted for preparing conditioned medium, having a culture chamber
containing cells of this invention capable of conditioning medium,
and an outlet port that is optionally sealable for withdrawing
medium from the culture chamber after conditioning by the cells.
The device may also have a mass-transfer microporous surface in the
form of a plate, a hollow fiber, or other structure that partitions
the cultured cells from medium that has been conditioned, which
allows free passage of the medium, and which provides passage to
the outlet port. The device may also have one or more ports for
introducing fresh medium, introducing additional cells, or removing
expired cells and cell debris. For continuous flow systems, a pump
may be attached to the medium inlet or outlet port to provide
circulation.
[0125] In certain embodiments, the conditioned medium is
supplemented before use with additional growth factors that benefit
pPS cell culture. For hES cells, a growth factor like bFGF or FGF-4
is often used. It has been found that the ability of the medium to
support hES cells in feeder-free culture may benefit by adding bFGF
both before and after the conditioning of the medium (Example 6).
For hEG cells, culture medium may be supplemented with a growth
factor like bFGF, an activator of gp130, such as LIF, IL-6, or
Oncostatin-M, and perhaps a factor that elevates cyclic AMP levels,
such as forskolin or cholera toxin. Other types of pPS cells may
benefit from other factors in the medium, such as stem cell factor
(also known as Steel factor, c-kit ligand).
[0126] In one illustration, medium containing 20% serum replacement
plus 4 ng/mL bFGF is conditioned by culturing .about.24 h with the
irradiated conditioning cells of the HEF1 line (Example 9). The
medium is then filtered through a 0.22 .mu.m membrane, and
supplemented with further 8 ng/mL bFGF (adjusted to compensate for
bFGF adsorbed during filtering).
[0127] If desired, the conditioned medium can be processed further.
For example, it can be concentrated by salt filtration or selective
filtration, or it can be extracted to separate or store the
effective components. Medium extracts can then be reconstituted or
supplemented with fresh culture medium before use.
[0128] After preparation, the medium can be used to support pPS
cells in feeder-free culture, as described earlier, or stored
frozen for future use. It is also suitable for other purposes, and
can be used for such purposes without restriction. For example, the
medium can be added to pPS cultured in the presence of feeder
cells, in order to further support the proliferation of the cells
or limit differentiation. The medium can also be used to maintain
or promote proliferation of other types of cultured precursor cells
or terminally differentiated cells, as may be determined
empirically.
[0129] Use of pPS Cells Propagated with Conditioned Media
[0130] This description provides a system by which a sizeable
quantity of pluripotent cells can be produced commercially without
the need of feeder cells. These cell populations can be used for a
number of important purposes.
[0131] For example, pPS cells maintained without feeder cells can
be used to prepare antibody that is specific for embryo markers,
stem cell markers, germ cell markers, and other antigens that may
be expressed on the cells. Polyclonal antibodies can be prepared by
injecting a vertebrate animal with cells of this invention in an
immunogenic form. Production of monoclonal antibodies is described
in such standard references as U.S. Pat. Nos. 4,491,632, 4,472,500
and 4,444,887, and Methods in Enzymology 73B:3 (1981). Specific
antibody molecules can also be produced by contacting a library of
immunocompetent cells or viral particles with the target antigen,
and growing out positively selected clones. See Marks et al., New
Eng. J. Med. 335:730, 1996, and McGuiness et al., Nature
Biotechnol. 14:1449, 1996. A further alternative is reassembly of
random DNA fragments into antibody encoding regions, as described
in EP patent application 1,094,108 A.
[0132] pPS cells maintained without feeders can also be used to
prepare a cDNA library relatively uncontaminated with cDNA from
feeder cells. mRNA is prepared by standard techniques (Sambrook et
al., supra) from the pPS cells. After reverse transcribing into
cDNA, the preparation can be subtracted with nucleotides from
embryonic fibroblasts and other cells of undesired specificity, to
produce cDNA more specifically expressed in pPS cells.
[0133] pPS 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 cells in feeder-free culture. This system
has the advantage of not being complicated by a secondary effect
caused by perturbation of the feeder cells by the test compound. In
one application, growth affecting substances are tested by
withdrawing the, conditioned medium, and then culturing with
different cocktails of soluble factors to determine if the treated
cells are maintained and proliferate in a satisfactory manner.
Potential toxins can be tested in conditioned or regular medium by
determining whether cells treated are adversely affected. Potential
differentiation factors or conditions can be tested by treating the
cells according to the test protocol, and then determining whether
the treated cell develops functional or phenotypic characteristics
of a differentiated cell of a particular lineage.
[0134] pPS cells grown in feeder-free culture can also be
differentiated into restricted developmental lineage cells, or
terminally differentiated cells. Differentiation of the pPS can be
initiated by allowing overgrowth of undifferentiated pPS cell
cultures, forming embryoid bodies in suspension culture, or plating
pPS cells under conditions that promote differentiation in a
particular manner. Such conditions may include withdrawing or
adding nutrients, growth factors or cytokines to the medium,
changing the oxygen pressure, or altering the substrate on the
culture surface (for example, from an extracellular matrix to a
polycation such as polyornithine).
[0135] Scientists at Geron Corporation have discovered that
culturing pPS cells or embryoid body cells in the presence of
ligands that bind growth factor receptors promotes enrichment for
neural precursor cells. The growth environment may contain a neural
cell supportive extracellular matrix, such as fibronectin. Suitable
growth factors include EGF, bFGF, PDGF, IGF-1, noggin, and
follistatin. Under the appropriate circumstances, populations of
cells enriched for expression of the A2B5 marker have the capacity
to generate both neuronal cells (including mature neurons), and
glial cells (including astrocytes and oligodendrocytes).
[0136] Scientists at Geron Corporation have discovered that
culturing pPS cells or embryoid body cells in the presence of a
hepatocyte differentiation agent promotes enrichment for
hepatocyte-like cells. The growth environment may contain a
hepatocyte supportive extracellular matrix, such as collagen or
Matrigel.RTM.. Suitable differentiation agents include various
isomers of butyrate and their analogs, exemplified by n-butyrate.
The cultured cells are optionally cultured simultaneously or
sequentially with a hepatocyte maturation factor, such as an
organic solvent like dimethyl sulfoxide, or a cytokine or hormone
that promotes cell growth.
[0137] Scientists at Geron Corporation have also discovered that it
is possible to differentiate hPS cells into a highly enriched
population comprising cardiomyocytes or cardiomyocyte precursors.
The cardiomyocyte lineage cells can be obtained, for example, by
differentiating hES cells with 5-azacytidine. Spontaneously
contracting cells are separated from other cells in the population
by density centrifugation, and optionally cultured with creatine,
camitine, or taurine. It is also possible to differentiate hPS
cells into a highly enriched population comprising osteoprogenitors
or osteoblasts expressing osteocalcin and collagen-1. The cells can
be obtained by taking pPS-derived mesenchymal cells and
differentiating them in a medium containing BMP-4.
[0138] pPS-derived differentiated cells are of interest for
research, diagnostic, and therapeutic purposes, and can be used to
study the genomics of differentiation.
The Examples that Follow are Provided by way of Further
Illustration, and are not Meant to Imply any Limitation to the
Practice of the Claimed Invention.
EXAMPLES
Example 1
[0139] Culture of hES Cells
[0140] Undifferentiated hES cells that had been maintained on
primary mouse embryonic feeder cells were harvested, and then
maintained in the absence of feeders. The culture wells were coated
with Matrigel.RTM., and the cells were cultured in the presence of
conditioned nutrient medium obtained from a culture of irradiated
primary fibroblasts.
[0141] Preparation of Conditioned Media (CM) From Primary Mouse
Embryonic Fibroblasts (mEF)
[0142] Fibroblasts were harvested from T150 flasks by washing one
time with Ca.sup.++/Mg.sup.++ free PBS and incubating in 1.5-2 mL
trypsin/EDTA (Gibco) for about 5 min. After the fibroblasts
detached from the flask, they were collected in mEF media (DMEM
+10% FBS). The cells were irradiated at 4000 rad (508 sec at 140
kV: shelf setting 6 in a Torrex generator), counted and seeded at
about 55,000 cells cm.sup.-2 in mEF media (525,000 cells/well of a
6 well plate). After at least 4 hours, the media were exchanged
with SR containing ES media, using 0.3-0.4 mL cm.sup.-2 of plate
surface area. Before addition to the hES cultures, the conditioned
medium was supplemented with human bFGF (Gibco). Fibroblast
cultures were used in this system for about 1 week, before
replacing with newly prepared cells.
[0143] Matrigel.RTM. Coating
[0144] Growth Factor Reduced Matrigel.RTM. or regular Matrigel.RTM.
(Becton-Dickinson, Bedford, Mass.) was thawed at 4.degree. C. The
Matrigel.RTM. was diluted 1:10 to 1:500 (typically 1:30) in cold KO
DMEM. 1 mL of solution was added to each 9.6 cm.sup.2 well, and
incubated at room temp for 1 h. Plates were used within 2 h after
coating, or stored at 4.degree. C.
[0145] Human ES Culture
[0146] Undifferentiated hES colonies were harvested from hES
cultures on feeders as follows. Cultures were incubated in
.about.200 U/mL collagenase IV for about 5 minutes at 37.degree. C.
Colonies were harvested by picking individual colonies up with a 20
.mu.L pipet tip under a microscope or scraping and dissociating
into small clusters in CM. These cells were then seeded onto
Matrigel.RTM. in conditioned media at roughly 15 colonies to each
9.6 cm.sup.2 well (if 1 colony is .about.10,000 cells, then the
plating density is .about.15,000 cells cm.sup.-2).
[0147] The day after seeding on Matrigel.RTM., hES cells were
visible as small colonies (250-2,000 cells) and there were single
cells in-between the colonies that appeared to be differentiating
or dying. As the hES cells proliferated, the colonies became quite
large and very compact, representing the majority of surface area
of the culture dish. The hES cells in the colonies had a high
nucleus to cytoplasm ratio and had prominent nucleoli, similar to
hES cells maintained on feeder cells. At confluence, the
differentiated cells in between the colonies represented less than
10% of the cells in the culture.
[0148] Six days after seeding, the cultures had become almost
confluent. The cultures were split by incubating with 1
mL.about.200 U/mL Collagenase IV solution (1 mg/mL in KO DMEM) for
.about.5 minutes at 37.degree. C. The collagenase solution was
aspirated, 2 mL hES medium was added per well, and the hES cells
were scraped from the dish with a pipette. The cell suspension was
gently triturated to dissociate the cells into small clusters of
10-200 cells. The cells were then re-seeded on Matrigel.RTM. coated
plates in CM, as above. Cells were seeded at a 1:3 or 1:6 ratio,
approximately 90,000 to 170,000 cells cm.sup.-2, making up the
volume in each well to .about.3-4 mL. Medium was changed daily, and
the cells were split and passaged again at 13 and again at 19 d
after initial seeding, or when the culture was near confluency.
[0149] On day 19 after initial seeding, cells were harvested and
evaluated for surface marker expression by immunofluorescence cell
cytometry, using labeled antibodies specific for cell surface
markers. The results from this experiment are as follows:
1TABLE 1 Phenotype of hPS Cells Grown in the Absence of Feeder
Cells Percentage of Marker Specificity Cells Staining SSEA-4
undifferentiated cells 92% Tra-1-60 undifferentiated cells 92%
Tra-1-81 undifferentiated cells 83% SSEA-1 differentiated cells
12%
[0150] For the hES cells maintained in the absence of feeders, a
high percentage express SSEA-4, Tra-1-60 or Tra-1-81. These 3
markers are expressed on undifferentiated human ES cells that are
maintained on feeders (Thomson et al., 1998). In addition, there is
very little expression of SSEA-1, a glycolipid that is not
expressed (or expressed at low levels) on undifferentiated ES
cells. Immunohistochemical evaluation of SSEA-4, Tra-1-60 and
Tra-1-81 indicates that the expression of these markers in
localized to the ES colonies, not the differentiated cells in
between the colonies.
[0151] Cultures of hES cells have been grown in the absence of
feeder cells for over 147 days after initial seeding, with no
apparent change in the proliferative capacity or phenotype. Human
ES cells maintained on Matrigel.RTM. in mEF conditioned medium have
a doubling time of about 31-33 hours, similar to the proliferation
rate for hES cells grown on mEF feeder cells. H1 cells after 64
days of feeder-free culture showed a normal karyotype.
Example 2
[0152] Matrigel.RTM. and Laminin Support Growth of hES Cells in
Feeder-free Culture
[0153] The growth of the hES cells was followed on different matrix
components in medium conditioned using primary mouse embryonic
fibroblasts (mEF).
[0154] hES cultures were initially harvested from feeder cell
cultures maintained in ES medium (80% knockout DMEM (Gibco BRL,
Rockville, Md.), 20% knockout serum replacement (Gibco BRL,
Rockville, Md.), 1% Non-essential amino acids (Gibco BRL,
Rockville, Md.), 1 mM L-glutamine (Gibco), 0.1 mM
.beta.-mercaptoethanol (Sigma, St. Louis, Mo.), supplemented with 4
ng/mL recombinant human basic fibroblast growth factor (hbFGF;
Gibco)). Cultures were passaged by incubation in .about.200 U/mL
collagenase IV for about 5-10 minutes at 37.degree. C. Colonies are
then harvested by removing individual colonies up with a
Pipetman.TM. under a microscope or scraping, followed by gentle
dissociation into small clusters in conditioned medium and then
seeded onto matrix coated plates. Cells were seeded at a 1:3 or 1:6
ratio, approximately 90,000 to 170,000 cells cm.sup.-2). Seeding
dilution experiments indicated that the optimal minimum seeding
density under these conditions is about 20,000 cells cm.sup.-2.
[0155] Harvested hES cells were seeded onto Matrigel.RTM. or
gelatin in mEF conditioned medium. The day after seeding, cells
plated onto Matrigel.RTM. attached to the plate and formed small
colonies that were less compact than hES colonies on feeder layers.
Over the next few days, the colonies increased in size and the
cells became more compact. The resulting culture contained very
dense undifferentiated colonies surrounded by differentiated
cells.
[0156] About one week after seeding the cultures became confluent
and could be passaged. In contrast, cells seeded onto gelatin
showed poor survival and the cells that survived appeared
differentiated. Three hES cell lines, H1, H7 and H9 were cultured
on Matrigel.RTM. in mEF conditioned medium. Cultures maintained
under these conditions for over 180 days continued to display
ES-like morphology.
[0157] The major components of Matrigel.RTM. are laminin, collagen
IV and heparin sulfate proteoglycan. The ability of these
components to support hES cell culture was tested separately.
Laminin, collagen IV or fibronectin (all from Sigma) were diluted
to a final concentration of 20 .mu.g/mL, 10 .mu.g/mL and 5 .mu.g/mL
in PBS, respectively.
[0158] The hES cells seeded onto laminin, fibronectin or collagen
IV had colonies of undifferentiated hES cells, although the
cultures on fibronectin or collagen IV did not contain as many
undifferentiated colonies as the cultures on Matrigel.RTM. or
laminin. When cells on Matrigel.RTM. or laminin reached confluence,
the cells within the colonies became very compact, were
morphologically very similar to the cells maintained on feeders and
were serially passaged. After 40 days (6 passages), cells on
Matrigel.RTM. and laminin contained a high proportion of colonies
which continued to display ES-like morphology in long term culture.
However, cells maintained on fibronectin or collagen IV had fewer
colonies that displayed appropriate ES-morphology. As controls,
cells cultured on Matrigel.RTM. or laminin in non-conditioned
medium appeared to be proliferating more slowly and showed a
differentiated morphology after a few passages.
[0159] FIG. 1 shows the morphology of hES cells in feeder-free
culture. Panel A (Left Side) shows morphology of hES cells of the
H1 line cultured on feeder cells in non-conditioned medium
(mEF/RM), or on Matrigel.RTM., laminin, fibronectin, or collagen IV
in mEF conditioned medium. Panel B shows morphology of hES cells of
the H9 line maintained on Matrigel.RTM. in various types of
conditioned medium, described in Example 6.
[0160] Laminins are major components of all basal laminae in
vertebrates, which interact with integrin heterodimers, such as
.alpha.1.beta.1, .alpha.2.beta.1, .alpha.3.beta.1, .alpha.6.beta.1
and .alpha.6.beta.4, on cell surface and mediate cell growth and
migration during development. Among these integrins,
.alpha.6.beta.1 and .alpha.6.beta.4 are specific for laminins;
other integrins also interact with other matrices. Another
experiment tested whether laminin receptors are expressed on hES
cells and whether culturing hES on laminin or Matrigel.RTM. changes
the expression of laminin receptors expression. Expression of
integrins including .alpha.1, .alpha.2, .alpha.3, .alpha.6, .beta.1
and .beta.4 were examined by FACS analysis on cells maintained on
feeders, or on Matrigel.RTM. or laminin in conditioned medium. For
analyzing integrin expression, cells were stained with a panel of
integrin specific antibodies by the laminin-specific integrins
investigator kit (Chemicon International, Inc., Temecula, Calif.)
and analyzed by FACS as described below.
[0161] FIG. 1 Panel C shows integrin expression measured in HI hES
cells maintained on feeders in non-conditioned medium (mEF/RM) or
on Matrigel.RTM., or on laminin in mEF conditioned medium (CM).
[0162] Cells maintained in Matrigel.RTM./conditioned medium and
laminin/conditioned medium were cryopreserved as follows: The cells
were frozen in standard hES medium (not conditioned medium)
supplemented with 10% DMSO and additional 10% SR (total 30%). The
cells were thawed onto Matrigel.RTM. or laminin in conditioned
medium. Cells maintained normal karyotype after being thawed.
[0163] Human ES cells maintained on Matrigel.RTM. in mEF
conditioned medium showed a doubling time of about 31-33 hours,
similar to the proliferation rate for hES cells grown on mEF feeder
cells. H1 cells after 64 days of feeder-free culture showed a
normal karyotype.
Example 3
[0164] Phenotypic Markers of Undifferentiated hES Cells
[0165] Undifferentiated hES cells may express SSEA-4, Tra-1-60,
Tra-1-81, Oct-4, and hTERT under certain conditions. The expression
of these markers (if present) typically decreases upon
differentiation. In order to assess whether the cells maintained in
feeder-free conditions retained these markers, cells were evaluated
by immunostaining, reverse transcriptase PCR amplification, and
assay for telomerase activity.
[0166] FIG. 2 shows surface marker expression in feeder-free cells
by FACS analysis. Panel A: Expression of SSEA-4 in H1 cells
maintained on feeders in non-conditioned medium (mEF/RM), on
Matrigel.RTM., laminin, fibronectin and collagen IV in mEF
conditioned medium. Isotype controls are indicated by the dotted
lines. Panel B: Mean fluorescence intensity of SSEA-1, SSEA-4,
Tra-1-60 and Tra-1-81 in H1 cells cultured on different matrices.
Panel C: Mean fluorescence intensity of SSEA-1 , SSEA-4 , Tra-1-60
and Tra-1-81 in H9 cells cultured on Matrigel.RTM. in conditioned
medium from different cell lines.
[0167] For analysis by fluorescence-activated cell sorting (FACS),
the hES cells were dissociated in 0.5 mM EDTA in PBS and
resuspended to about 5.times.10.sup.5 cells in 50 .mu.L diluent
containing 0.1% BSA in PBS. For analyzing surface marker
expression, cells were incubated in the primary antibodies,
including IgG isotype control (0.5 .mu.g/test), IgM isotype control
(1:10), SSEA-1 (1:10), SSEA-4 (1:20), Tra-1-60 (1:40) and Tra-1-81
(1:80), diluted in the diluent at 4.degree. C. for 30 min. After
washing with the diluent, cells were incubated with rat anti-mouse
kappa chain antibodies conjugated with PE (Becton Dickinson, San
Jose, Calif.) at 4.degree. C. for 30 min. Cells were washed and
analyzed on FACSCalibur.TM. Flow Cytometer (Becton Dickinson, San
Jose, Calif.) using CellQuest.TM. software.
[0168] Similar to the hES cells on feeders, cells on Matrigel.RTM.,
laminin, fibronectin or collagen IV expressed SSEA-4, Tra-1-60 and
Tra-1-81. There was very little expression of SSEA-1, a glycolipid
that is not expressed by undifferentiated hES cells.
[0169] FIG. 3 shows marker expression detected by histochemistry.
For analysis by immunocytochemistry, cells were incubated with
primary antibodies, including SSEA-1 (1:10), SSEA-4 (1:20),
Tra-1-60 (1:40) and Tra-1-81 (1:80), diluted in knockout DMEM at
37.degree. C. for 30 min. Cells were then washed with warm knockout
DMEM and fixed in 2% paraformaldehyde for 15 min. After washing
with PBS, cells were incubated with 5% goat serum in PBS at RT for
30 min, followed by incubation with the FITC-conjugated goat
anti-mouse antibodies (1:125) (Sigma) at RT for 30 min. Cells were
washed, stained with DAPI and mounted. The staining was typically
performed .about.2 days after passaging. Cells were also examined
for expression of alkaline phosphatase, a marker for
undifferentiated ES cells. This was performed by culturing the
cells on chamber slides, fixing with 4% paraformaldehyde for 15
min, and then washing with PBS. Cells were then incubated with
alkaline phosphatase substrate (Vector Laboratories, Inc.,
Burlingame, Calif.) at room temperature in the dark for 1 h. Slides
were rinsed for 2-5 min in 100% ethanol before mounting.
[0170] The results show that SSEA-4, Tra-1-60, Tra-1-81, and
alkaline phosphatase were expressed by the hES colonies on
Matrigel.RTM. or laminin, as seen for the cells on feeders--but not
by the differentiated cells in between the colonies.
[0171] FIG. 4 shows Oct-4 and hTERT expression of H1 cells on
feeders and off feeders, as detected by reverse-transcriptase PCR
amplification. For radioactive relative quantification of
individual gene products, QuantumRNA.TM. Alternate18S Internal
Standard primers (Ambion, Austin Tex., USA) were employed according
to the manufacturer's instructions. Primers and amplification
conditions for particular markers are provided in International
patent application PCT/US01/01030.
[0172] Briefly, the linear range of amplification of a particular
primer pair was determined, then coamplified with the appropriate
mixture of alternate18S primers:competimers to yield PCR products
with coinciding linear ranges. Before addition of AmpliTaq.TM.
(Roche) to PCR reactions, the enzyme was pre-incubated with the
TaqStar.TM. antibody (ProMega) according to manufacturer's
instructions. Radioactive PCR reactions were analyzed on 5%
non-denaturing polyacrylamide gels, dried, and exposed to
phosphoimage screens (Molecular Dynamics) for 1 hour. Screens were
scanned with a Molecular Dynamics Storm 860 and band intensities
were quantified using ImageQuant.TM. software. Results are
expressed as the ratio of radioactivity incorporated into the hTERT
or Oct-4 band, standardized to the radioactivity incorporated into
the 18s band.
[0173] The transcription factor Oct-4 is normally expressed in the
undifferentiated hES cells and is down-regulated upon
differentiation. It was found that the cells maintained on
Matrigel.RTM. or laminin in conditioned medium (CM) for 21 days
express Oct-4, whereas cells maintained in Matrigel.RTM. in
unconditioned regular medium (RM) did not. Cells maintained on
fibronectin or collagen IV, which showed a large degree of
differentiation, expressed lower levels of Oct-4 compared to cells
on feeders, Matrigel.RTM. or laminin.
[0174] hTERT and Oct-4 expression was seen in all the culture
conditions except Matrigel.RTM. and regular medium. Furthermore,
after exposure of cells to retinoic acid (RA) or dimethyl sulfoxide
(DMSO), factors that promote cell differentiation, the expression
of hTERT was markedly decreased.
[0175] Pluripotency of the undifferentiated cells cultured without
feeders was determined by forming embryoid bodies in suspension
culture for 4 days, and then culturing on poly-ornithine coated
slides for 7 days. Immunocytochemistry showed staining patterns
consistent with cells of the neuron and cardiomyocyte lineages, and
cells staining for a-fetoprotein, a marker of endoderm lineage. The
undifferentiated cells were also tested for their ability to form
teratomas by intramuscular injection into SCID mice. Resulting
tumors were excised after 78-84 days. Cell types from all three
germ layers were identified by histological analysis.
Example 4
[0176] Preparation of Immortalized Mouse Feeder Cell Lines
[0177] Primary mouse embryonic fibroblasts (Robertson, supra) can
be immortalized by genetically altering them to express human
telomerase reverse transcriptase (hTERT). The fibroblasts (mEF) are
infected with a retroviral construct pBABE puro hTERT, containing
the hTERT coding sequence driven by the MoLV LTR and the
puromycin-resistance gene driven by the SV40 early promoter.
Isolates of primary mEFs are cultured in growth medium containing
10% fetal calf serum (HyClone), 2 mM glutamine (Gibco/BRL), and 90%
high glucose DMEM (Gibco/BRL). mEFs are split every 3 days at a
ratio of 1:2.
[0178] After 4 such splits, 5.times.10.sup.5 mEFs are plated onto a
100 mM dish. On the next day, cells are infected by replacing the
growth medium with a mixture containing 5 mL of retroviral stock
(1.times.10.sup.6 pfu/mL) and 4 .mu.g/mL polybrene, and incubating
at 37.degree. C. After 8 h, an additional 5 mL of the
retrovirus/polybrene mixture is added and the cells are incubated
at 37.degree. C.
[0179] On the next day, the retrovirus/polybrene mixture is removed
and replaced with fresh growth medium. After 4 hr, the mEFs are
removed from the plate with 0.5% trypsin/500 mM EDTA (Gibco/BRL)
and replated into 2 T150 flasks in 25 mL growth medium/flask. On
the next day, the medium is replaced with growth medium
supplemented with 0.5 micrograms/mL puromycin.
[0180] Cells are split once a week at a ratio of 1:4 for 8 weeks
and maintained in puromycin-containing medium. After 8 weeks, cells
are trypsinized and replated at a density of 2,000 cells per 150 mm
plate. Individual colonies are isolated with cloning cylinders 26
days later, expanded, and screened for telomerase activity.
Example 5
[0181] Preparation of the Mouse Feeder Cell Line NHG190
[0182] A permanent mouse cell line was established that is suitable
for conditioning medium for the culture of primate pluripotent stem
(pPS) cells. The NHG190 line is a mouse embryonic fibroblast cell
line immortalized with telomerase that is triple drug resistant,
and expresses green fluorescent protein (GFP).
[0183] Two mouse strains were obtained from Jackson Laboratory (Bar
Harbor, Me.) that have a transgene for resistance to the
antibiotics neomycin or hygromycin. The C57BL/6J
TgN(pPGKneobpA)3Ems mice and C57BL6J-TgN(pPWL512hyg)1 Ems mice from
Jackson Labs were cross-bred. Embryos that were both neomycin- and
hygromycin-resistant were dissected at day 13.5 post conception
according to standard protocols for preparing mouse embryonic
fibroblasts (mEF) for feeder layers (E. J. Robertson, pp. 71-112 in
Teratocarcinoma and Embryonic Stem Cell Lines, ed. E. J. Robertson,
Oxford: IRL Press, 1987). The derived mEF cells were stored
frozen.
[0184] The mEFs were thawed in growth medium containing 20% fetal
calf serum (HyClone), 2 mM L-glutamine (Gibco/BRL), 80% DMEM
(Gibco/BRL). The cells were expanded using 1:2 split ratios for 4
passages. Two flasks that had reached .about.75% confluency were
fed with fresh medium 4 h before electroporation. Cells were
removed from the flasks with 0.5% trypsin/500 mM EDTA (Gibco/BRL),
pelleted at 400.times.g for 5 min at room temperature, and
resuspended in the growth medium at a concentration of
4.times.10.sup.6 cells/mL.
[0185] The cell suspension was divided into two 500 .mu.L aliquots
and transferred to two 0.4 cm gap electroporation cuvettes
(BioRad). One cuvette received 5 .mu.g of the control plasmid
(pBS212; puromycin-resistance gene driven by the SV40 early
enhancer/promoter); the other received 5 micrograms of pGRN190,
comprising the murine telomerase reverse transcriptase (mTERT)
coding region driven by MPSV promoter plus puromycin resistance
gene driven by the SV40 early enhancer/promoter. The cells and DNA
were mixed by hand, and electroporated using a BioRad gene Pulser
with a BioRad capacitance extender at a setting of 300 V, 960
.mu.F.
[0186] Each aliquot of cells was transferred to an individual 150
cm plate containing 25 mL of growth medium. The medium on the
plates was exchanged on the following day, and on the next day,
growth medium was replaced by growth medium plus 0.5 .mu.g/mL
puromycin. The medium on the plates was exchanged for fresh
puromycin-containing medium every 48 hrs until 29 days after
electroporation. At this time, large individual colonies of
puromycin-resistant cells were evident in both the pBS212- and
pGRN190-electroporated plates. Ten colonies from the control plate
and 12 from the pGRN190-electroporated plate were isolated with
cloning cylinders and each colony was transferred to 1 well of a
48-well plate (1 well per colony).
[0187] One week later, all surviving colonies that had expanded to
reach confluence in the 48 well plate (three control colonies, 1
pGRN190-electroporated colony) were transferred individually to
wells of a 24 well plate. Six days later, the only colony that had
continued to expand was derived from the pGRN190-electroporated
plate, and was subsequently designated NH190. The cells were
maintained in growth medium plus 0.5 .mu./mL puromycin.
[0188] To facilitate monitoring of the cells in mixed culture
populations and in vivo, NH190 cells were further infected with a
retroviral construct conferring expression of green fluorescent
protein (GFP). The enhanced GFP sequence from plasmid pEGFP-1 is
one of the Living Colors.TM. fluorescent protein vectors, available
from ClonTech. It contains an enhanced GFP encoding region, with
changes that alter restriction nuclease cleavage sites, and shift
the excitation and emission wavelengths of the encoded protein. The
EGFP-1 sequence was cloned into the vector pMSCV.neo, ClonTech cat
# K1062-1. NH190 cells were transduced with the engineered vector,
and GFP positive cells were separated by FACS sorting. The GFP
expressing cell line was designated NHG190. These cells have been
carried in culture for over 3 months.
Example 6
[0189] Use of Conditioned Medium from Immortalized Mouse Cells to
Support Feeder-free Culture of Human ES Cells
[0190] Conditioned medium from several cell lines were tested for
their ability to support the growth of human embryonic stem cells
in feeder-free cultures. The media tested were from mEF (primary
mouse embryonic fibroblasts), STO (immortal mouse embryonic
fibroblast cell line), NHG190 (Example 5), BJ (human foreskin
fibroblast cell line immortalized with telomerase), and RPE (human
retinal epithelial cell line immortalized with telomerase).
[0191] Medium used for growing cells was as follows. 1. mEF medium:
90% DMEM (Gibco BRL, Rockville, Md.), 10% fetal bovine serum (FBS)
(heat inactivated) (Hyclone), and 2 mM L-glutamine. 2. STO medium:
mEF medium supplemented with 0.1 mM non-essential amino acids. 3.BJ
5ta medium: 90% DMEM and 10% Cosmic calf serum (not heat
inactivated). 3. NHG190 medium: mEF medium supplemented with
additional 10% FBS. 4. RPE medium: 90% DMEM/F12, 10% FBS (not heat
inactivated), 10 ml L-glutamine and 3.48 g/Lsodium bicarbonate. 5.
Differentiation medium: 80% knockout Dulbecco's modified Eagle's
medium (KO DMEM), 1 mM L-glutamine, 0.1 mM .beta.-mercaptoethanol
and 1% nonessential amino acids, supplemented with 20% FBS.
[0192] To prepare conditioned medium, the respective cell lines
were harvested by washing once with Ca.sup.++/Mg.sup.++ free PBS,
incubating in trypsin/EDTA (Gibco) for about 5 min, and suspending
in mEF medium. The cells were irradiated at .about.4000 rad
(.about.508 sec at 140 kV: shelf setting 6 in a Torrex generator,
EG&G Astrophysics Research Corp., Long Beach, Calif.). They
were then counted, and seeded at .about.55,000 cells/cm.sup.2 for
mEFs, .about.38,000/cm.sup.2 for NHG190 cells,
.about.95,000/cm.sup.2 for STO cells, .about.80,000/cm.sup.2 for BJ
cells, .about.90,000/cm .sup.2 for RPE cells. After at least 4 h,
the medium was exchanged with ES medium. Conditioned medium was
collected daily thereafter, and used for feeding of hES cultures.
Before addition to the hES cultures, each conditioned medium was
supplemented with 4 ng/mL of human basic fibroblast growth factor
(hbFGF; Gibco).
[0193] FIG. 1, Panel B (Right Side) shows morphology of hES cells
of the H9 line maintained on Matrigel.RTM. in medium conditioned by
mEF, NHG190, STO and BJ, compared with unconditioned regular medium
(RM). The cells in RPE conditioned medium differentiated within the
first week of culture. The cells in the other conditioned mediums
all had hES colonies with appropriate ES-morphology. Based on the
morphology, confluence of the culture, and the ratio of
differentiated to undifferentiated cells the conditioned medium can
be ranked in order of decreasing preference as follows: primary
mEF, NHG190, STO, and BJ.
[0194] Similar to cells maintained in conditioned medium from
primary mEF, cells on Matrigel.RTM. or laminin in medium
conditioned by other cell lines, including NHG190, STO and BJ,
expressed high levels of SSEA-4, Tra-1-60 and Tra-1-81 but low
levels of SSEA-1 as analyzed by FACS analysis (FIG. 2C). Cells on
Matrigel.RTM. or laminin in mEF conditioned medium or NHG190
conditioned medium were able to differentiate into three germ layer
cell types. Immunocytochemical analysis of the differentiated
cultures showed positive staining for .beta.-tubulin III consistent
with neurons (ectoderm lineage), cardiac troponin I consistent with
cardiomyocytes (mesoderm lineage), and .alpha.-fetoprotein,
consistent with cells of the endoderm lineage.
[0195] In Examples 1 and 2, medium was prepared by adding 4 ng/mL
hbFGF to the medium before conditioning with the mEFs, and then
again when the conditioned medium was collected and used for
feeding of the hES cells. To determine if both additions of hbFGF
to the medium are necessary to maintain the ES cells in the
undifferentiated state, experiments were performed in which one or
both additions of hbFGF were eliminated.
[0196] Cultures maintained in conditioned medium without the second
addition of hbFGF did not appear healthy at early passages, and
appeared differentiated after 29 days in culture. Cells maintained
in conditioned medium without the first addition of hbFGF displayed
mostly differentiated morphology, but still formed smaller
undifferentiated colonies after 27 days in culture. Cells
maintained in conditioned medium without either addition of hbFGF
completely differentiated after 18 days. In contrast, cells
cultured in conditioned medium prepared with both additions of bFGF
appeared healthy and undifferentiated in long-term culture. Thus,
preparing conditioned medium by adding bFGF both before and after
culturing with the feeder cells helps prevent differentiation of
hPS cells in the subsequent feeder-free culture.
[0197] Storage of conditioned medium was tested as follows: Batch
medium was prepared by conditioning for 1-2 days in mEF cell
cultures as described, and stored at 4.degree. C. in sealed culture
flasks. Feeder-free hES cell cultures were maintained with stored
medium exchanged daily. Characteristic morphological features of
undifferentiated stem cells were still present after at least 7
days, comparable to hES cells maintained in freshly conditioned
medium. In subsequent experiments, it was determined that mEF
conditioned medium can be stored frozen. Medium from days 1-5 of
the mEF culture was collected daily, pooled, and stored at
-20.degree. C. for >1 month. hES cells cultured for frozen and
fresh conditioned medium for 3 passages showed no differences.
[0198] To determine if leukemia inhibitory factor (LIF) can
substitute for conditioned medium in maintaining hES cells without
feeders, cells of the H1 and H9 line were cultured on Matrigel.RTM.
in ES medium containing LIF at a final concentration of 1500,
1,000, or 500 U/mL (recombinant LIF from R&D systems; Catalog #
250-L). Cells were simultaneously cultured in mEF conditioned
medium as the positive control, and unconditioned ES medium as
negative control. After one week, cultures in medium either with or
without LIF showed a large degree of differentiation, while
cultures maintained in mEF conditioned medium contained
predominately undifferentiated colonies. These data indicate that
LIF alone will not maintain hES cells in an undifferentiated state
in the absence of feeder cells.
Example 7
[0199] Preparation of Human Feeder Cell Lines
[0200] Cells were derived from hES cells that have the
morphological criteria of fibroblasts or mesenchymal cells. They
are capable of supporting hES cells in feeder-free culture.
[0201] The H9 hES cell line was obtained as described elsewhere in
this disclosure. To form embryoid bodies, the hES cells were
harvested after incubation with .about.200 U/mL collagenase IV at
37.degree. C. for 10 min, and dissociated into small clusters in
differentiation media and cultured in non-adherent cell culture
plates (Costar) to form aggregates in suspension.
.about.2.times.10.sup.6 cells were seeded into each well (9.6
cm.sup.2). After 2 days in suspension, the aggregates were
transferred into gelatin-coated plates. They attached to the plates
and continued to differentiate into cells with different
morphologies. Fibroblast-like cells were observed in clusters of
100-1000 cells in the mixed population of the differentiated cells
after an additional 11 days.
[0202] To isolate fibroblast-like cells, the culture was incubated
in .about.200 U/mL collagenase IV for 3 min at 37.degree. C. These
clusters of fibroblast-like cells were removed with a Pipetman.TM.
under a microscope and either transferred directly to a tube
containing the differentiation media or released into the
collagenase solution, and subsequently collected. The cells were
spun, resuspended in differentiation media and plated onto one well
of 6-well plate. The cells proliferated, and were serially
passaged. The cultures were switched to mEF media in the third
passage. In all procedures, cells were fed every 2-3 days.
[0203] To introduce telomerase into the fibroblast-like cells, they
were infected with retrovirus expressing hTERT as follows. Cells
were seeded onto 6-well plates at 8.6.times.10.sup.4 cells/well
(9.6 cm.sup.2) one day before infection, incubated with
virus-containing media supplemented with 4 .mu.g/mL polybrene for 8
h before changed to mEF medium. Different wells were infected with
pBABE-hTERT or a pBABE vector control.
[0204] The cells were cultured for additional 6 days, and selected
in puromycin at a final concentration of 1.6 .mu.g/mL for an
additional 8 days. The cells were then harvested and re-seeded in
mEF medium.
[0205] The cells were expanded and continued to display
fibroblast-like morphology for 50 days. Cells were collected for
TRAP assay 20 days after the infection. The cells were maintained
in mEF medium from day 0 to 27 and were switched to differentiation
media from day 28 to day 43. Cells were counted at each passage
after the selection and the population doubling was calculated.
[0206] FIG. 5 (Panel A) shows the morphology of the derived
hEF-like cell line, after transduction with retrovirus pBABE
containing an hTERT expression cassette. Panel B (below) shows the
growth curves of the hEF-like cells transduced for hTERT expression
(pBABE-hTERT=telomerized cells; pBABE (alone)=vector control). Both
cell lines proliferate in culture for at least 8 doublings,
presumably because they are derived from embryonic cells and have
not yet reached the Hayflick limit.
[0207] Telomerase activity of hEF cells was analyzed by TRAP assay
(Kim et al., Nucleic Acids Res. 25:2595, 1997). Cells transduced
with the hTERT expression cassette showed positive telomerase
activity, whereas the control cells did not show any telomerase
activity. The hTERT-hEF cells serially passaged with a similar
proliferation rate as that of the control cells.
Example 8
[0208] Harvesting Conditioned Medium from Human Feeder Cells
[0209] The hTERT-expressing human fibroblast-like cells prepared
according to the previous example were harvested by washing once
with Ca.sup.++/Mg.sup.++ free PBS and incubating in 1.5-2 mL
trypsin/EDTA (Gibco) for about 2 min. After the cells detached from
the plate, they were collected in mEF medium. The cells were
irradiated at 4000 rad, counted and seeded at about
3.7-5.times.10.sup.5 cells/well. After at least 16 h, the medium
was exchanged with hES media+4 ng/mL hbFGF (serum replacement
medium described above, with 4 ng/mL exogenously added human basic
fibroblast growth factor). Three to four mL were used per well of a
6 well plate.
[0210] Conditioned medium was collected daily for feeding of hES
cultures. Before addition to the hES cultures, this conditioned
medium was supplemented with 4 ng/mL of hbFGF (Gibco). The
hTERT-hEF cultures were used in this system for 1-2 weeks.
Example 9
[0211] Use of Conditioned Medium from Differentiated Human Cells to
Support Feeder-free Culture of Human Stem Cells
[0212] The hTERT-transduced hEF cells were tested for their ability
to support hES growth as follows. The H1 hES cell line maintained
on Matrigel.RTM. in medium conditioned using primary mouse
embryonic fibroblasts was dissociated and resuspended in medium
conditioned by the human fibroblast-like cells.
[0213] FIG. 6 shows hES maintained in hEF conditioned medium.
Cultures of hES cells replated in feeder-free culture on
Matrigel.RTM. supported by hEF conditioned medium (Panels C &
F), formed colonies with morphology characteristic of
undifferentiated hES cells. The cultures appeared indistinguishable
from hES cells grown directly on a layer of primary mEF feeder
cells (Panels A & D) or on Matrigel.RTM. in medium conditioned
by primary mEF. In each of these conditions, healthy colonies of
hES cells increased in size, and had characteristic features of
undifferentiated embryonic stem cells (Panels A, B, & C). A few
colonies showed a degree of differentiation (Panels D, E, & F),
but the extent of differentiation was similar under each of the
culture conditions.
[0214] Seven days after the seeding, the cultures had become almost
confluent and were split by incubating in .about.200 U/mL
collagenase IV for about 5 minutes at 37.degree. C. and scraping
the dish with a pipette. The cell suspension was transferred to a
15-mL conical tube and gently triturated to dissociate the cells
into small clusters of about 10-500 cells. The cells were then
re-seeded onto Matrigel.RTM.-coated plates in CM. Cells were seeded
at a 1:3 or 1:4 ratio, .about.130,000 to 170,00 cells cm.sup.-2.
Cells have been maintained under this condition for over 30 days
while displaying morphology characteristic of hES cells.
Example 10
[0215] A Human Feeder Cell Line Differentiated from hES Cells
Having a Different Genome
[0216] A second line of medium-conditioning cells was developed
from a different hES cell line designated H1. Embryoid bodies were
formed as before, and after 4 days in suspension culture were
plated onto gelatin-coated plates for an additional 9 days.
[0217] In this example, the cell population was developed from bulk
culture rather than being selected out by pipette. The cultures
were incubated in 2 mg/mL Collagenase type II in PBS for 30 min at
37.degree. C. The cells were harvested, dissociated, centrifuged,
resuspended in differentiation medium, and plated in a 6-well
plate. The proliferating cells were passaged in hEF medium (90%
DMEM, 10% heat-inactivated FBS, 0.1 mM non-essential amino acids,
and 2 mM L-glutamine), and fed every 2-3 days. After two passages,
the cell population appeared homogeneous with morphological
characteristics of fibroblasts or mesenchymal cells. This cell line
was designated HEF1.
[0218] Subpopulations were transduced with the retrovirus
telomerase expression vector (pBABE-hTERT), or with vector control,
as in Example 7.
[0219] FIG. 7 (Panel A) shows the morphology of the HEF1 cell line.
Panel B (below) shows telomerase activity, as measured in the TRAP
assay. Cells transduced with the hTERT expression cassette showed
positive telomerase activity at 20 or 65 days after transduction.
The untransduced cell line, or cells transduced with the vector
control showed no telomerase activity.
[0220] FIG. 8 shows the growth curves of the hTERT-transduced HEF1
cells, and cells transduced with vector control. Both lines doubled
about once every 2 days, until the 38 day point, when the control
cells stopped dividing (presumably because they had reached the
Hayflick limit). The hTERT-transfected cells continued
proliferating beyond the 60 day point (30 doublings) at a
consistent growth rate.
[0221] FIG. 9 is a micrograph of the hTERT transduced cells and
control cells, after staining for senescence-associated
.beta.-galactosidase, a known biomarker for cellular aging (Dimitri
et al., Proc. Natl. Acad. Sci. USA 92:9363, 1995). Cells grown on
chamber slides were fixed 2 min in 0.2% glutaraldehyde in PBS,
washed with PBS, and incubated overnight in 1 mg/mL
5-bromo-4-chloro-3-indolyl-D-galactosidase (X-gal), 5 mM
K.sub.4Fe(CN).sub.6, 5 mM K.sub.3Fe(CN).sub.6, 150 mM NaCl, 2 mL
MgCl.sub.2, in 40 mM citric acid phosphate buffer pH 6.0. The HEF1
control cells stained heavily for .beta.-galactosidase, whereas the
hTERT transduced cells did not. The combined results indicate that
the expression of hTERT extends the life-span characteristics of
the HEF1 cells.
[0222] To evaluate the efficacy of this new line as feeder cells
for maintaining embryonic stem cells in undifferentiated form, the
cells were first tested in coculture experiments. The HEF1 cells
were grown to near confluence, and inactivated by irradiation
(4,000 rads). The prepared wells were then used to passage hES
cells of the H9 line (passage 30), or its H9.2 cloned derivative
line (cloned at passage 29, then passaged 28 times). The medium was
the standard SR medium, not preconditioned with any other cell
type: 80% KO DMEM, 20% serum replacement, 1% non-essential amino
acids, 1 mM glutamine, and 0.1 mM .beta.-mercaptoethanol. Cultures
were then examined after 3 passages for the H9 line, or 5 passages
for the H9.2 line on the HEF1 cells.
[0223] FIG. 10 shows the results. The H9 cells maintained on HEF1
feeders show colonies with morphology characteristic of
undifferentiated hES cells (upper panels). The H9.2 line maintained
on telomerized HEF1 feeders also showed undifferentiated morphology
(lower panels), indistinguishable from hES cells concurrently grown
on Matrigel.RTM. in the absence of feeders in medium conditioned by
mouse embryonic fibroblasts.
[0224] The hES cells (H9 or H9.2) passaged on HEF1 feeders were
then assessed for their pluripotency. They were dissociated into
small clumps by incubating in 1 mg/mL collagenase IV at 37.degree.
C. for .about.5-10 min, and then suspension cultured in
differentiation medium (80% KO DMEM, 20% FBS, 1% non-essential
amino acids, 1 mM L-glutamine, and 0.1 mM .beta.-mercaptoethanol)
to form aggregates. After 4 days in suspension culture, the
embryoid bodies that had formed were transferred to gelatin-coated
plates. They attached to the surface, proliferated, and
differentiated into a heterogeneous cell population. Spontaneously
contracting cells were observed in various areas of the culture 8
days later, consistent with the formation of cardiomyocytes.
[0225] The ability of the HEF1 cell line to maintain hES cells
without differentiation in a coculture experiment implies that the
cell line is also a candidate for preparing conditioned medium.
Accordingly, ES medium was conditioned as in Example 8, using HEF1
cells irradiated at 6000 rad, and seeded at .about.4.1 to
5.5.times.10.sup.4 cells cm.sup.-2. The conditioned medium was then
tested for its ability to support growth of the H9 hES cells
cultured on a Matrigel.RTM. substrate.
[0226] FIG. 11 shows colonies of hES cells of the H9 line after
passaging into medium conditioned either by mouse embryonic
fibroblasts, or by the HEF1 cell line. The hES cells have been
maintained using the HEF1 conditioned medium for 4 passages,
continuing to display the morphology of undifferentiated ES cells.
The hES cells were found to maintain expression of hTERT and Oct-4.
As shown in Panel B, they also continued to demonstrate telomerase
activity, as measured in the TRAP assay, which is characteristic of
undifferentiated hES cells.
It is Understood that Certain Adaptations of the Invention
Described in this Disclosure are a Matter of Routine Optimization
for Those Skilled in the Art, and can be Implemented Without
Departing from the Spirit of the Invention, or the Scope of the
Appended Claims.
* * * * *