U.S. patent application number 12/710078 was filed with the patent office on 2014-10-09 for primate pluripotent stem cell expansion without feeder cells and in the presence of fgf and matrigel or engelbreth-holm-swarm tumor cell preaparation.
This patent application is currently assigned to Geron Corporation. The applicant listed for this patent is Melissa K. Carpenter, Joseph D. Gold, Ramkumar Mandalam, Chunhui Xu. Invention is credited to Melissa K. Carpenter, Joseph D. Gold, Ramkumar Mandalam, Chunhui Xu.
Application Number | 20140302600 12/710078 |
Document ID | / |
Family ID | 27558647 |
Filed Date | 2014-10-09 |
United States Patent
Application |
20140302600 |
Kind Code |
A9 |
Mandalam; Ramkumar ; et
al. |
October 9, 2014 |
PRIMATE PLURIPOTENT STEM CELL EXPANSION WITHOUT FEEDER CELLS AND IN
THE PRESENCE OF FGF AND MATRIGEL OR ENGELBRETH-HOLM-SWARM TUMOR
CELL PREAPARATION
Abstract
This disclosure provides an improved system for culturing human
pluripotent stem cells. Traditionally, pluripotent stem cells are
cultured on a layer of feeder cells (such as mouse embryonic
fibroblasts) to prevent them from differentiating. In the system
described here, the role of feeder cells is replaced by components
added to the culture environment that support rapid proliferation
without differentiation. Effective features are a suitable support
structure for the cells, and an effective medium that can be added
fresh to the culture without being preconditioned by another cell
type. Culturing human embryonic stem cells in fresh medium
according to this invention causes the cells to expand surprisingly
rapidly, while retaining the ability to differentiate into cells
representing all three embryonic germ layers. This new culture
system allows for bulk proliferation of pPS cells for commercial
production of important products for use in drug screening and
human therapy.
Inventors: |
Mandalam; Ramkumar; (Union
City, CA) ; Xu; Chunhui; (Cupertino, CA) ;
Gold; Joseph D.; (San Francisco, CA) ; Carpenter;
Melissa K.; (Castro Valley, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Mandalam; Ramkumar
Xu; Chunhui
Gold; Joseph D.
Carpenter; Melissa K. |
Union City
Cupertino
San Francisco
Castro Valley |
CA
CA
CA
CA |
US
US
US
US |
|
|
Assignee: |
Geron Corporation
|
Prior
Publication: |
|
Document Identifier |
Publication Date |
|
US 20100317101 A1 |
December 16, 2010 |
|
|
Family ID: |
27558647 |
Appl. No.: |
12/710078 |
Filed: |
February 22, 2010 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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12170219 |
Jul 9, 2008 |
8637311 |
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12710078 |
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|
10235094 |
Sep 4, 2002 |
7410798 |
|
|
12170219 |
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|
10948956 |
Sep 24, 2004 |
7413904 |
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12170219 |
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09849022 |
May 4, 2001 |
|
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10948956 |
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09530346 |
Aug 29, 2000 |
6800480 |
|
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PCT/US98/22619 |
Oct 23, 1998 |
|
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09849022 |
|
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|
10235094 |
Sep 4, 2002 |
7410798 |
|
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10948956 |
|
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60317478 |
Sep 5, 2001 |
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60213740 |
Jun 22, 2000 |
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60213739 |
Jun 22, 2000 |
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60216387 |
Jul 7, 2000 |
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60220064 |
Jul 21, 2000 |
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Current U.S.
Class: |
435/363 |
Current CPC
Class: |
C12N 5/0693 20130101;
C12N 2501/2306 20130101; C12N 2501/998 20130101; C12N 2500/25
20130101; C12N 2501/065 20130101; C12N 2501/155 20130101; A61P
43/00 20180101; C12N 2502/13 20130101; C12N 2501/115 20130101; C12N
2506/02 20130101; C12N 5/068 20130101; C12N 2501/237 20130101; C12N
2510/04 20130101; C12N 5/0696 20130101; C12N 2501/105 20130101;
C12N 2500/98 20130101; C12N 5/0662 20130101; C12N 2503/02 20130101;
C12N 2510/00 20130101; C12N 5/0062 20130101; C12N 5/0672 20130101;
C12N 5/0692 20130101; C12N 11/04 20130101; C12N 2501/125 20130101;
C12N 2501/235 20130101; C12N 2533/50 20130101; C12N 5/0606
20130101; C12N 2501/119 20130101; C12N 2501/13 20130101; C12N
5/0671 20130101; C12N 5/0075 20130101; C12N 5/0678 20130101; C12N
2500/99 20130101; C12N 2533/90 20130101; C12N 5/0619 20130101; C12N
2502/99 20130101; C12N 2501/145 20130101; C12N 5/0068 20130101;
C12N 5/0607 20130101; C12N 15/1096 20130101; C12N 2501/26 20130101;
C12N 2500/90 20130101; C12N 5/0622 20130101; C12N 15/1034 20130101;
C12N 5/0603 20130101 |
Class at
Publication: |
435/363 |
International
Class: |
C12N 5/074 20100101
C12N005/074 |
Claims
1-20. (canceled)
21. A system for expanding primate pluripotent stem cells in vitro
without feeder cells comprising a nutrient media and a soluble
preparation from Engelbreth-Holm-Swarm tumor cells.
22. The system of claim 21, wherein the nutrient media is
conditioned by a feeder cell.
23. The system of claim 21, wherein the nutrient media is a fresh
media that is not conditioned by a feeder cell.
24. The system of claim 21, wherein the nutrient media comprises a
fibroblast growth factor.
25. A system for expanding a cell expressing the markers stage
specific antigen 4 (SSEA4), Tra-1-60 and Tra-1-81 in vitro without
feeder cells comprising a nutrient media and a soluble preparation
from Engelbreth-Holm-Swarm tumor cells.
26. A method of culturing primate pluripotent stem cells in vitro
without feeder cells comprising culturing the primate pluripotent
stem cells on a soluble preparation from Engelbreth-Holm-swarm
tumor cells and contacting the cells with a nutrient media.
27. The method of claim 26, wherein the nutrient media is
conditioned by a feeder cell.
28. The method of claim 26, wherein the nutrient media is a fresh
media that is not conditioned by a feeder cell.
29. The method of claim 26, wherein the nutrient media comprises a
fibroblast growth factor.
30. A method of culturing a cell expressing the markers stage
specific antigen 3 (SSEA3), stage specific antigen 4 (SSEA4),
Tra-1-60 and Tra-1-81 in vitro without feeder cells comprising
culturing the cell expressing the markers, stage specific antigen 4
(SSEA4), Tra-1-60 and Tra-1-81 on a soluble preparation from
Engelbreth-Holm-Swarm tumor cells and contacting the cell with a
nutrient media.
Description
REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. application Ser.
No. 12/170,219 which claims priority to U.S. Ser. No. 10/330,873,
now U.S. Pat. No. 7,413,902, filed Dec. 24, 2002, which is a
continuation of U.S. Ser. No. 09/530,346, filed Aug. 29, 2000 which
issued as U.S. Pat. No. 6,800,480, which claims priority to
PCT/US98/22619. This application is also a continuation of U.S.
Ser. No. 10/235,094, now U.S. Pat. No. 7,410,798, filed Sep. 4,
2002, which claims priority to provisional application U.S.
60/317,478, filed Sep. 5, 2001 and PCT Application PCT/US01/01030,
filed Jan. 10, 2001, which claims priority to U.S. Provisional
Application Nos. 60/175,581 filed Jan. 11, 2000 and 60/213,739
filed Jun. 22, 2000 all of which are hereby incorporated by
reference.
[0002] The priority documents are hereby incorporated herein by
reference in their entirety, along with PCT applications
PCT/US01/13471; PCT/US01/15861; PCT/US02/19477; PCT/US02/20998; and
PCT/US02/22245.
TECHNICAL FIELD
[0003] This invention relates generally to the field of cell
biology of embryonic cells. More specifically, it relates to the
propagation of pluripotent stem cells, and culture conditions and
materials that facilitate propagation and use of human embryonic
stem cells.
BACKGROUND
[0004] Considerable interest has been generated in the field of
regenerative medicine by recent work relating to the isolation and
propagation of human stem cells from the early embryo. These cells
have two very special properties: First, unlike other normal
mammalian cell types, they can be propagated in culture almost
indefinitely, providing a virtually unlimited supply. Second, they
can be used to generate a variety of tissue types of interest as a
source of replacement cells and tissues that are damaged in the
course of disease, infection, or because of congenital
abnormalities.
[0005] Early work on pluripotent stem cells was done in mice
(Robertson, Meth. Cell Biol. 75:173, 1997; and Pedersen, Reprod.
Fertil. Dev. 6:543, 1994). Experiments with human stem cells have
required overcoming a number of additional technical difficulties
and compilations. As a result, technology for culturing and
differentiating human pluripotent stem cells is considerably less
advanced.
[0006] Thomson et al. (U.S. Pat. No. 5,843,780; Proc. Natl. Acad.
Sci. USA 92:7844, 1995) were the first to successfully isolate and
propagate pluripotent stem cells from primates. They subsequently
derived human embryonic stem (hES) cell lines from human
blastocysts (Science 282:114, 1998). Gearhart and coworkers derived
human embryonic germ (hEG) cell lines from fetal gonadal tissue
(Shamblott et al., Proc. Natl. Acad. Sci. USA 95:13726, 1998; and
U.S. Pat. No. 6,090,622). Both hES and hEG cells have the
long-sought characteristics of pluripotent stem cells: they can be
cultured extensively without differentiating, they have a normal
karyotype, and they remain capable of producing a number of
important cell types.
[0007] A significant challenge to the use of pluripotent stem cells
for therapy is that they are traditionally cultured on a layer of
feeder cells to prevent differentiation (U.S. Pat. No. 5,843,780;
U.S. Pat. No. 6,090,622). According to Thomson et al. (Science
282:114, 1998), hPS cells cultured without feeders soon die, or
differentiate into a heterogeneous population of committed cells.
Leukemia inhibitory factor (LIF) inhibits differentiation of mouse
ES cells, but it does not replace the role of feeder cells in
preventing differentiation of human ES cells.
[0008] International Patent Publication WO 99/20741 (Geron Corp.)
is entitled Methods and materials for the growth of primate-derived
primordial stem cells. A cell culture medium is described for
growing primate-derived primordial stem cells in a substantially
undifferentiated state, having a low osmotic pressure and low
endotoxin levels. The basic medium can be combined with a serum
effective to support the growth of primate-derived primordial stem
cells on a substrate of feeder cells or a feeder cell matrix. The
medium may also include non-essential amino acids, an anti-oxidant,
and growth factors that are either nucleosides or a pyruvate
salt.
[0009] International Patent Publication WO 01/51616 (Geron Corp.)
is entitled Techniques for growth and differentiation of human
pluripotent stem cells. An article by Xu et al. (Nature
Biotechnology 19:971, 2001) is entitled Feeder-free growth of
undifferentiated human embryonic stem cells. An article by
Lebkowski et al. (Cancer J. 7 Suppl. 2:S83, 2001) is entitled Human
embryonic stem cells: culture, differentiation, and genetic
modification for regenerative medicine applications. These
publications report exemplary culture methods for propagating human
embryonic stem cells in an undifferentiated state, and their use in
preparing cells for human therapy.
[0010] New technology to facilitate growing and manipulating
undifferentiated pluripotent stem cells would be a substantial
achievement towards realizing the full commercial potential of
embryonic cell therapy.
SUMMARY OF THE INVENTION
[0011] This disclosure provides an improved system for expanding
primate pluripotent stem (pPS) cells. The technology allows the
user to rapidly produce high-quality pPS cells for use in therapy
are drug discovery, free of undesired contamination by cells of
other species and other tissue types.
[0012] Application of the technology involves introducing stem
cells into a culture environment containing components described
and exemplified in more detail in the sections that follow.
Typically, the environment will contain a support structure, a
culture medium, and one or more factors added to the medium that
support proliferation of the pPS cells in an undifferentiated
state. Exemplary support structures are made from isolated
extracellular matrix components. Exemplary culture media comprise
an isotonic buffer, a protein or amino acid source, and may also
comprise nucleotides, lipids, and hormones. An exemplary factor for
adding to the medium is a fibroblast growth factor. It has been
discovered that sufficient FGF in a suitable medium is sufficient
to maintain pPS cells in a substantially undifferentiated state
through extended culture. Other factors listed in this disclosure
can be added to improve the quality and expansion rate of the
culture when desired.
[0013] The culture environment can be essentially free of feeder
cells, since feeder cells are not required to keep the pPS cells
proliferating in an undifferentiated state. In this embodiment, the
cells consist essentially of undifferentiated pPS cells, and
progeny thereof that may have begun differentiation or adopted an
altered phenotype. Since they are all derived from the same pPS
cells, all of the cells in the culture will have the same genotype,
which means that the cells have the same chromosomal DNA (plus or
minus karyotype abnormalities or deliberate genetic alterations).
This can be ascertained by demonstrating that essentially all the
cells in the culture are derived from the same pPS cells. Included
are mixed populations made by combining different lines of pPS
cells and their progeny, as long as essentially each of the cells
in the culture are progeny of one of the starting cell lines.
[0014] An important virtue of this system is that there is no need
to condition the medium before combining it with the stem cells.
The skilled reader may wish to precondition the medium with other
cell lines in advance, but the medium can be added "fresh" to the
pPS cells and still support proliferation without differentiation.
This means that the medium has not been cultured with other cell
types before being added to the pPS cell culture (either by direct
substitution for spent medium, or in a gradual or continuous
exchange system).
[0015] Another virtue of the system is the ability to adjust
conditions so that the cells expand more rapidly (as much as 11/2
times faster) than they do when cultured on feeder cells according
to traditional techniques, or in conditioned medium. While the user
need not expand the cells rapidly in order to use this invention,
she has the option of growing the cells with a doubling time of as
little as 24 hours.
[0016] Using this culture system (optionally passaging the cells
into new culture environments when required), populations of
pluripotent stem cells can be obtained that are expanded 10-fold or
more when compared with the starting population. Even after
expansion, a high proportion of the cells are still
undifferentiated, according to morphological characteristics,
phenotypic markers, or the ability to differentiate into
derivatives of the three embryonic germ layers (endoderm, mesoderm,
and ectoderm).
[0017] Embodiments of this invention include the culture
environment in which the pPS cells are expanded and its use, the
combined composition of the environment and the pPS cells, and
various methods for expanding pPS cells using the reagents and
techniques described in this disclosure. This system can be used
with pPS cells of various types, exemplified by cells isolated or
propagated from human blastocysts, such as established human
embryonic stem cell lines and their equivalents.
[0018] This system can be used to generate genetically altered pPS
cells. The cells are transfected with a suitable vector for
effecting the desired genetic alteration, such as a DNA-lipid
complex. This is facilitated in the feeder-free culture systems of
this invention. The genetically altered cell population can be
expanded as already described, before or after genetic alteration
and/or selection of the altered genotype.
[0019] This system can also be used to generate differentiated cell
types of various kinds. After the undifferentiated pPS cells are
expanded to the desired number, they are caused to differentiate
according to any of a variety of differentiation paradigms provided
later in this disclosure. Differentiated populations can be
obtained in which at least 95% of the cells represent the same
tissue type or germ layer: for example, neural cells, hepatocytes,
cardiomyocytes, mesenchymal cells, or osteoblasts.
[0020] These and other aspects of the invention will be apparent
from the description that follows.
DRAWINGS
[0021] FIG. 1 shows 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 5Ta cells, compared with unconditioned regular
medium (RM). hES cells cultured in suitable conditioned media
contained colonies with appropriate morphology for undifferentiated
cells.
[0022] FIG. 2 shows marker expression detected by
immunocytochemistry for cells grown with primary feeder cells (mEF)
or on the extracellular matrices Matrigel.RTM. or laminin in
conditioned medium.
[0023] FIG. 3 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 shows OCT-4 and hTERT expression at
the mRNA level by RT-PCR. The lower panel compares the level of
expression for cells grown on different substrates, expressed as
the ratio of OCT-4 or hTERT to the 18s standard.
[0024] FIG. 4 is from an experiment in which hES were genetically
altered in feeder-free culture by lipofection. Panel A shows
morphology of hES cells on laminin after they have been transfected
for GFP expression. Panel B shows GFP expression in the same
colony. Panel C shows percentage of cells expressing GFP under
various conditions. Bright green cells were observed in
undifferentiated hES colonies of feeder-free cultures. In contrast,
very few green cells were found in hES cell colonies grown on
feeders.
[0025] FIG. 5 shows FAGS analysis for phenotypic markers on hES
cells grown in various culture environments. The H9 cell line was
maintained in a fresh (unconditioned) medium containing basic
fibroblast growth factor, stem cell factor (c-kit ligand), and
other factors that bind to receptors associated with gp130. The
levels of SSEA-1, SSEA-4, Tra 1-60 and Tra 1-81 (characteristic of
undifferentiated hES cells) were similar to cells maintained in
medium conditioned by mouse embryonic fibroblasts (MEF-CM). The
cultured cells expressed c-kit (a receptor for stem cell factor),
but not gp130 (associated with the LIF receptor). These cells can
still produce derivatives of all three embryonic germ layers.
[0026] FIG. 6 shows colonies of undifferentiated hES cells growing
in fresh ES medium containing basic fibroblast growth factor alone
at high (40 ng/mL) or low (8 ng/mL) concentration, or bFGF (40
ng/mL) in combination with SCF (15 ng/mL) or Flt-3 ligand (75
ng/mL). Shown for comparison are hES cells growing in ES medium
condoned by irradiated mouse embryonic fibroblasts. It has been
discovered that bFGF at concentrations of 40 ng/mL is sufficient to
maintain the hES cells in an undifferentiated form. The presence of
SCF or Flt-3 ligand under certain circumstances can improve the
proportion of undifferentiated cells in the culture.
[0027] FIG. 7 shows expression of SSEA-4 as evaluated by FACS
analysis in various growth factor combinations, described in
Example 6.
[0028] FIG. 8 shows colonies of hES cells after 6 passages
(sufficient for full adaptation) in different base media. (A) mEF
conditioned ES medium+bFGF (8 ng/mL); (B) X-VIVO.TM. 10+bFGF (40
ng/mL); (C) X-VIVO.TM. 10+bFGF (40 ng/mL)+stem cell factor (SCF,
Steel factor) (15 ng/mL); (D) X-VIVO.TM. 10+bFGF (40 ng/mL)+Flt3
ligand (75 ng/mL); (E) QBSF.TM.-60+bFGF (40 ng/mL). All three base
media (ES medium, X-VIVO.TM. 10, and QBSF.TM.-60) can be used to
expand hES cells in feeder-free culture. In this illustration, the
cells growing in combination shown in (C) expanded 8.2-fold per
passage, whereas those in conditioned medium expanded 2.2-fold. The
use of suitable fresh medium causes rapid expansion of
undifferentiated hES cells.
[0029] FIG. 9 shows the gene expression profile of hTERT and
Oct3/4, measured by real time RT-PCR, as described in Example
8.
[0030] FIG. 10 demonstrates that cells cultured in unconditioned
medium retain their pluripotency. hES cells passaged 7 times in mEF
conditioned medium, or unconditioned X-VIVO.TM. 10 medium
containing bFGF and SCF. The cells were then differentiated into
embryoid bodies, plated, and analyzed by immunocytochemistry for
phenotypic markers representing each of the three germ layers. The
cells stain for .alpha.-fetoprotein (representing ectoderm); muscle
actin (representing mesoderm), and .beta.-tubulin III (representing
endoderm). The cells grown in the culture system described in this
patent application are suitable for making a wide scope of
differentiated cell types.
DETAILED DESCRIPTION
[0031] Previous technology for culturing primate pluripotent stem
(pPS) cells has required that the cell culture environment contain
feeder cells in order to prevent them from differentiating. In
particular, the standard feeder cells used for culturing human
embryonic stem cell are irradiated mouse embryonic fibroblasts.
Unfortunately, using feeder cells increases production costs,
impairs scale-up, and produces mixed cell populations that
complicate quality control and regulatory approval for use in human
therapy.
[0032] This disclosure provides a system for rapidly expanding
primate pluripotent stem (pPS) cells in vitro without requiring a
layer of feeder cells to support the culture and inhibit
differentiation.
[0033] As a result of thorough investigation of the features
required, it has now been determined that the beneficial effect of
the feeder cells can be replaced by providing a suitable surface
and a suitable mixture of soluble factors. It turns out that more
intimate interaction between the pPS cells and the feeder cells is
not required, as long as the signal transduction pathways required
for undifferentiated growth are adequately activated by factors in
the culture environment.
[0034] In one version of the feeder-free culture system, the pPS
cells are grown in medium that has been preconditioned in a
separate culture of feeder cells of mouse or human origin (FIG. 1).
The feeder cells are grown to confluence in their own culture
environment, inactivated, and then cultured in one or more batches
of fresh medium to allow them to release an effective combination
of factors. The medium is then harvested, and used to support
growth of undifferentiated pPS cells plated onto a suitable
substrate. Doubling rate is comparable to hES grown on feeder
cells. Typically, the medium is changed daily, and the cells are
split and passaged every 6 or 7 days.
[0035] In an alternative version of the feeder-free culture system,
the pPS cells are grown in medium that has not been preconditioned,
but has been supplemented with ingredients that perform essentially
the same function as factors secreted from feeder cells. Certain
factor combinations comprising moderate to high levels of
fibroblast growth factors and other cells generate cultures that
can proliferate 20-fold or more through 6 or more passages, while
maintaining a majority of the cells in the culture in an
undifferentiated state (FIGS. 6 and 8). Near confluence, most of
the cells have morphological features of undifferentiated cells,
and express characteristic phenotypic markers: SSEA-4, Tra-1-60,
Tra-1-81, Oct-4, and telomerase reverse transcriptase (TERT)
[0036] Quite surprisingly, it was found that pPS cells grown in
unconditioned medium expand substantially more rapidly than pPS
cells grown on feeder cells or in conditioned medium. The reasons
for this are unclear; nor was it predictable based on what was
previously known about pPS cell culture. Nevertheless, this finding
is important, because it provides a rapid expansion method for
producing commercial grade undifferentiated pPS cells on a
commercial scale. Now that this technology is available, the
production of pPS cells for treating human patients in need of
tissue regeneration holds considerable promise.
[0037] The techniques provided in this invention represent an
important advance in the potential use of pluripotent stem cells
for research and therapeutic use. Further advantages of the
invention will be understood from the sections that follow.
Definitions
[0038] Prototype "primate Pluripotent Stem cells" (pPS cells) are
pluripotent cells derived from pre-embryonic, embryonic, or fetal
tissue at any time after fertilization, and have the characteristic
of being capable under the right conditions of producing progeny of
several different cell types. pPS cells are capable of producing
progeny that are derivatives of each of the three germ layers:
endoderm, mesoderm, and ectoderm, according to a standard
art-accepted test, such as the ability to form a teratoma in a
suitable host, or the ability to differentiate into cells stainable
for markers representing tissue types of all three germ layers in
culture.
[0039] Included in the definition of pPS cells are embryonic cells
of various types, exemplified by human embryonic stem (hES) cells,
defined below; embryonic stem cells from other primates, such as
Rhesus or marmoset stem cells (Thomson et al., Proc. Natl. Acad.
Sci. USA 92:7844, 1995; Developmental Biology 38:133, 1998); and
human embryonic germ (hEG) cells (Shamblott et al., Proc. Natl.
Acad. Sci. USA 95:13726, 1998). Other types of pluripotent cells
are also included in the term. Any cells of primate origin that are
capable of producing progeny that are derivatives of all three
germinal layers are included, regardless of whether they were
derived from embryonic tissue, fetal tissue, or other sources. It
is beneficial to use pPS cells that are karyotypically normal and
not derived from a malignant source.
[0040] Prototype "human Embryonic Stem cells" (hES cells) are
described by Thomson et al. (Science 282:1145, 1998; U.S. Pat. No.
6,200,806). The scope of the term covers pluripotent stem cells
that are derived from a human embryo at the blastocyst stage, or
before substantial differentiation of the cells into the three germ
layers. Those skilled in the art will appreciate that except where
explicitly required otherwise, the term includes primary tissue and
established lines that bear phenotypic characteristics of hES
cells, and derivatives of such lines that still have the capacity
of producing progeny of each of the three germ layers.
[0041] pPS cell cultures are described as "undifferentiated" or
"substantially undifferentiated" when a substantial proportion of
stem cells and their derivatives in the population display
morphological characteristics of undifferentiated cells, clearly
distinguishing them from differentiated cells of embryo or adult
origin. Undifferentiated pPS cells are easily recognized by those
skilled in the art, and typically appear in the two dimensions of a
microscopic view with high nuclear/cytoplasmic ratios and prominent
nucleoli. It is understood that colonies of undifferentiated cells
within the population will often be surrounded by neighboring cells
that are differentiated. Nevertheless, the undifferentiated
colonies persist when the population is cultured or passaged under
appropriate conditions, and individual undifferentiated cells
constitute a substantial proportion of the cell population.
Cultures that are substantially undifferentiated contain at least
20% undifferentiated pPS cells, and may contain at least 40%, 60%,
or 80% in order of increasing preference (in terms percentage of
cells with the same genotype that are undifferentiated).
[0042] Whenever a culture or cell population is referred to in this
disclosure as proliferating "without differentiation", what is
meant is that after proliferation, the composition is substantially
undifferentiated according to the preceding definition. Populations
that proliferate through at least four passages (.about.20
doublings) without differentiation will contain substantially the
same proportion of undifferentiated cells (or possibly a higher
proportion of undifferentiated cells) when evaluated at the same
degree of confluence as the originating culture.
[0043] "Feeder cells" or "feeders" are terms used to describe cells
of one type that are co-cultured with cells of another type, to
provide an environment in which the cells of the second type can
grow. 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 fresh feeder cells are not added to
support the growth of pPS cells. A feeder free culture will contain
less than about .about.5% feeder cells. Compositions containing
less than 1%, 0.2%, 0.05%, or 0.01% feeder cells (expressed as % of
total cells in the culture) are increasingly more preferred.
[0044] 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, and a
supporting structure (such as a substrate on a solid surface) if
present.
[0045] 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, serum or serum
replacement, and other exogenously added factors.
[0046] A "conditioned medium" is prepared by culturing a first
population of cells in a medium, and then harvesting the medium.
The conditioned medium (along with anything secreted into the
medium by the cells) may then be used to support the growth of a
second population of cells. Where a particular ingredient or factor
is described as having been added to the medium, what is meant is
that the factor (or a cell or particle engineered to secrete the
factor) has been mixed into the medium by deliberate
manipulation.
[0047] A "fresh medium" is a medium that has not been purposely
conditioned by culturing with a different cell type before being
used with the cell type it is ultimately designed to support.
Otherwise, no limitations are intended as to its manner of
preparation, storage, or use. It is added fresh (by exchange or
infusion) into the ultimate culture, where it may be consumed or
otherwise processed by the cell types that are present.
[0048] A cell is said to be "genetically altered", "transfected",
or "genetically 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 will often comprise a transcribable sequence
encoding a protein of interest, which enables the cell to express
the protein at an elevated level. The genetic alteration is said to
be "inheritable" if progeny of the altered cell have the same
alteration.
[0049] The term "antibody" as used in this disclosure refers to
both polyclonal and monoclonal antibody of any species. The ambit
of the term encompasses not only intact immunoglobulin molecules,
but also fragments and genetically engineered derivatives of
immunoglobulin molecules and equivalent antigen binding molecules
that retain the desired binding specificity.
General Techniques
[0050] General methods in molecular genetics and genetic
engineering are described in the current editions of Molecular
Cloning: A Laboratory Manual, (Sambrook et al., Cold Spring
Harbor); Gene Transfer Vectors for Mammalian Cells (Miller &
Calos eds.); and Current Protocols in Molecular Biology (F. M.
Ausubel et al. eds., Wiley & Sons). Cell biology, protein
chemistry, and antibody techniques can be found in Current
Protocols in Protein Science (J. E. Colligan et al. eds., Wiley
& Sons); Current Protocols in Cell Biology (J. S. Bonifacino et
al., Wiley & Sons) and Current Protocols in Immunology (J. E.
Colligan et al. eds., Wiley & Sons.). Reagents, cloning
vectors, and kits for genetic manipulation referred to in this
disclosure are available from commercial vendors such as BioRad,
Stratagene, Invitrogen, ClonTech, and Sigma-Aldrich Co.
[0051] Cell culture methods are described generally in the current
edition of Culture of Animal Cells: A Manual of Basic Technique (R.
I. Freshney ed., Wiley & Sons); General Techniques of Cell
Culture (M. A. Harrison & I. F. Rae, Cambridge Univ. Press),
and Embryonic Stem Cells: Methods and Protocols (K. Turksen ed.,
Humana Press). Other texts are Creating a High Performance Culture
(Aroselli, Hu. Res. Dev. Pr. 1996) and Limits to Growth (D. H.
Meadows et al., Universe Publ. 1974). Tissue culture supplies and
reagents are available from commercial vendors such as Gibco/BRL,
Nalgene-Nunc International, Sigma Chemical Co., and ICN
Biomedicals.
Sources of Pluripotent Stem Cells
[0052] Suitable source cells for culturing and differentiation
according to this invention include established lines of
pluripotent cells derived from tissue formed after gestation.
Exemplary primary tissue sources are embryonic tissue (such as a
blastocyst), or fetal tissue taken any time during gestation,
typically but not necessarily before 10 weeks gestation.
Non-limiting exemplars are established lines of primate embryonic
stem (ES) cells, exemplified below; and embryonic germ (EG) cells,
described in Shamblott et al., Proc. Natl. Acad. Sci. USA 95:13726,
1998; and U.S. Pat. No. 6,090,622. Also contemplated is use of the
techniques 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 tissues
listed.
Establishing Lines of Human Embryonic Stem (hES) Cells
[0053] Embryonic stem cells can be isolated from blastocysts of
members of the primate species (U.S. Pat. No. 5,843,780; Thomson et
al., Proc. Natl. Acad. Sci. USA 92:7844, 1995). Human embryonic
stem (hES) cells can be prepared from human blastocyst cells using
the techniques described by Thomson et al. (U.S. Pat. No.
6,200,806; Science 282:1145, 1998; Curr. Top. Dev. Biol. 38:133
ff., 1998) and Reubinoff et al, Nature Biotech. 18:399,2000.
Equivalent cell types to hES cells include their pluripotent
derivatives, such as primitive ectoderm-like (EPL) cells, as
outlined in WO 01/51610 (Bresagen).
[0054] Materials for the preparation of hES cell lines according to
traditional methods are as follows. Serum-containing ES medium is
made with 80% DMEM (typically knockout DMEM), 20% defined fetal
bovine serum (FBS), 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 (Gibco
#10828-028), 1% non-essential amino acids, 1 mM L-glutamine, and
0.1 mM .beta.-mercaptoethanol. Just before use, human bFGF is added
to a final concentration of 4-8 ng/mL.
[0055] Mouse embryonic fibroblasts (mEF) for use as feeder cells
can be obtained from outbred CF1 mice (SASCO) or other suitable
strains. 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.
[0056] Feeder cells are propagated in mEF medium, containing 90%
DMEM (Gibco #11965-092), 10% FBS (Hyclone #30071-03), and 2 mM
glutamine. mEFs are propagated in T150 flasks (Corning #430825),
splitting the cells 1:2 every other day with trypsin, keeping the
cells subconfluent. To prepare the feeder cell layer, cells are
irradiated at a dose to inhibit proliferation but permit synthesis
of important factors that support hES cells (.about.4000 rads gamma
irradiation). Six-well culture plates (such as Falcon #304) are
coated by incubation at 37.degree. C. with 1 mL 0.5% gelatin per
well overnight, and plated with 375,000 irradiated mEFs per well.
Feeder cell layers are used 5 h to 4 days after plating. The medium
is replaced with fresh hES medium just before seeding.
[0057] hES cells can be isolated from human blastocyst obtained
from human in vivo preimplantation embryos, in vitro fertilized
embryos, or one cell human embryos expanded to the blastocyst stage
(Bongso et al., Hum Reprod 4: 706, 1989). Human embryos can be
cultured to the blastocyst stage in G1.2 and G2.2 medium (Gardner
et al., Fertil. Steril. 69:84, 1998). The zona pellucida is removed
from blastocysts by brief exposure to pronase (Sigma). The inner
cell masses are isolated by immunosurgery or by mechanical
separation, and plated on mouse embryonic feeder layers, or in
feeder free culture as described below.
[0058] 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, collagenase 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 apparently 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.
Propagation of pPS Cells in the Absence of Feeder Cells
[0059] This invention allows pPS to be propagated in an
undifferentiated state, even in the absence of feeder cells.
Feeder-free pPS cell cultures can be obtained either by passaging
cells grown on feeder into feeder-free conditions, or by first
deriving the cells from blastocysts into a feeder-free
environment.
[0060] In the absence of feeders, the pPS cells are cultured in an
environment that supports proliferation without differentiation.
Aspects of culture that can affect differentiation include the
substrate upon which the cells are cultured, the medium in which
they are cultured, and the manner in which they are split and
passaged to new culture environments.
[0061] pPS cells may be supported in feeder-free culture on an
extracellular matrix. The matrix can be deposited by preculturing
and lysing a matrix-forming cell line (WO 99/20741), such as the
STO mouse fibroblast line (ATCC Accession No. CRL-1503), or human
placental fibroblasts. The matrix can also be coated directly into
the culture vessel with isolated matrix components. Matrigel.RTM.
is a soluble preparation from Engelbreth-Holm-Swarm tumor cells
that gels at room temperature to form a reconstituted basement
membrane. Other suitable extracellular matrix components may
include laminin, fibronectin, proteoglycan, entactin, heparan
sulfate, and so on, alone or in various combinations. Substrates
that can be tested using the experimental procedures described
herein include not only other extracellular matrix components, but
also polyamines, and other commercially available coatings. This
invention contemplates adding extracellular matrix to the fluid
phase of a culture at the time of passaging the cells or as part of
a regular feeding. This invention also contemplates extracellular
matrix deposited into the culture by cells within the culture (such
as pPS cells that have formed around the periphery of an
undifferentiated colony).
[0062] 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. 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.15,000 cells cm.sup.-2 (typically
90,000 cm.sup.-2 to 170,000 cm.sup.-2) promote survival and limit
differentiation.
[0063] Another consideration is cell dispersion. In one method,
enzymatic digestion is halted before cells become completely
dispersed (say, .about.5 min with collagenase IV). The plate is
then scraped gently with a pipette, and the cells are triturated
into clumps of adherent cells, about 10-2000 cells in size, which
are then passaged into the new culture environment. 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 0.05% (wt/vol) trypsin and 0.053 mM EDTA for 5-15 min at
37.degree. C. The remaining cells in the plate are removed,
triturated with the pipette until dispersed into single cells and
small clusters, and then replated. In another illustration, the
cells are harvested without enzymes before the plate reaches
confluence. The cells are incubated .about.5 min in 0.5 mM EDTA
alone in PBS, washed from the culture vessel, and then replated
without further dispersal.
[0064] pPS cells plated in the absence of fresh feeder cells
benefit from being cultured in a nutrient medium. The medium will
generally contain the usual components to enhance cell survival,
including isotonic buffer, essential minerals, and either serum or
a serum replacement of some kind. To inhibit differentiation, the
medium is formulated to supply some of the elements provided by
feeder cells or their equivalents.
[0065] The base nutrient medium used for conditioning can have any
of several different formulae. Exemplary serum-containing ES medium
is made with 80% DMEM (typically KO DMEM), 20% defined fetal bovine
serum (FBS), 1% non-essential amino acids, 1 mM L-glutamine, and
0.1 mM .beta.-mercaptoethanol. 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.
[0066] Other suitable base media are X-VIVO.TM. 10 expansion medium
(Biowhittaker) and QBSF.TM.-60 (Quality Biological Inc.) (Example
8). See also WO 98/30679 (Life Technologies Inc.) and U.S. Pat. No.
5,405,772 (Amgen). The medium will typically contain a protein
nutrient, in the form of serum (such as FBS), serum replacement,
albumin, or essential and non-essential amino acids in an effective
combination. It will also typically contain lipids, fatty acids, or
cholesterol as artificial additives or the HDL or LDL extract of
serum. Other beneficial factors that can be included are hormones
like insulin or transferrin, nucleosides or nucleotides, pyruvate,
and a reducing agent such as .beta.-mercaptoethanol.
Medium Additives
[0067] The nutrient medium used for culturing the pPS cells
comprises one or more factors that promote proliferation of the pPS
cells without differentiation. As will be apparent from the
following description, the supplementation can occur by
preculturing the medium with cells that secrete such factors, by
adding such factors to the medium artificially, or by both
techniques in combination.
[0068] Conditioned medium can be prepared by culturing irradiated
primary mouse embryonic fibroblasts (Example 1) or other cells
(Example 4) at a density of .about.5-6.times.10.sup.4 cm.sup.-2 in
a serum free medium such as KO DMEM supplemented with 20% serum
replacement and .about.4-8 ng/mL basic fibroblast growth factor
(bFGF). The culture supernatant is harvested after .about.1 day at
37.degree. C. The cells are cultured in the medium for sufficient
time to allow adequate concentration of released factors that
support pPS cell culture. Typically, medium conditioned by
culturing for 24 hours at 37.degree. C. contains a concentration of
factors that support pPS cell culture for at least 24 hours.
However, the culturing period can be adjusted upwards or downwards,
determining empirically what constitutes an adequate period. Medium
that has been conditioned for 1-2 days is typically used to support
pPS cell culture for 1-2 days, and then exchanged.
[0069] Non-conditioned medium that supports pPS cell growth in an
undifferentiated state can be created by adding to a suitable base
medium certain factors that invoke the appropriate signal
transduction pathways in undifferentiated cells.
[0070] It has been discovered that the fibroblast growth factor
family is especially effective in this regard. Exemplary are basic
FGF (FGF-2), and FGF-4, but other members of the family can also be
used. Also suitable are species homologs, artificial analogs,
antibodies to the respective FGF receptor, and other receptor
activator molecules. It has been determined from gene expression
analysis that undifferentiated hES cells express receptors for
acidic FGF (FGF-1). At a sufficient concentration (40 ng/mL,
depending on other conditions), FGF alone is sufficient to promote
growth of hES cells in an undifferentiated state (Examples 6 and
8).
[0071] This invention includes a method for determining additional
factors that facilitate the action of FGF and equivalents in their
support of undifferentiated pPS cell growth. The method involves
combining a plurality of factors into functional groups, and
culturing the cells with the groups in various combinations. Once
the effective groups are determined, the rest can be eliminated,
and the group can be dissected to determine the minimal effective
combination. This strategy is illustrated in Example 7.
[0072] As a supplement to FGF, ligands that bind c-kit, such as
stem cell factor (SCF, Steel factor), antibodies to c-kit, and
other activators of the same signal transduction pathway may also
be beneficial. SCF is dimeric and occurs in soluble and
membrane-bound forms. It transduces signals by ligand-mediated
dimerization of c-kit, which is a receptor tyrosine kinase related
to the receptors for platelet-derived growth factor (PDGF),
macrophage colony-stimulating factor, Flt-3 ligand and vascular
endothelial growth factor (VEGF). Also of interest are factors that
elevate cyclic AMP levels, such as forskolin. These factors or
their equivalents may be used individually or in an effective
combination with other influential factors in the medium, as
already described.
[0073] The formulations provided in the Example section below were
primarily designed for culturing hES cells. Where appropriate, the
illustrations in this disclosure can be adapted to other types of
pPS cells and multipotent cells by accommodating the known
properties of the cells. For example, the hEG cells claimed in U.S.
Pat. No. 6,090,622 are dependent on the presence of both bFGF and
an inducer of gp130 (such as LIF or Oncostatin-M). The culture
media for growing hEG cells can be adapted accordingly.
[0074] Each of the conditions described here can be optimized
independently, and certain combinations of conditions will prove
effective upon further testing. Such optimization is a matter of
routine experimentation, and does not depart from the spirit of the
invention provided in this disclosure.
Desirable Outcomes
[0075] A medium formulation can be tested for its ability to
support pPS cells by swapping it into a feeder-free culture system
in place of medium conditioned by primary mouse embryonic
fibroblasts (mEF), or some other proven standard (Examples 5-8). If
pPS cells grow in a substantially undifferentiated state, then the
medium can be characterized as supporting pPS cells in feeder free
culture.
[0076] One of the virtues of using fresh medium in this culture
system is the ability to adjust conditions so that the cells expand
more rapidly than they do when cultured on feeder cells according
to traditional techniques, or in conditioned medium. Populations of
pluripotent stem cells can be obtained that are 10-, 20-, 50-,
100-, or 1000-fold expanded when compared to the starting
population. Under suitable conditions, cells in the expanded
population will be 50%, 70% or more in the undifferentiated
state.
[0077] The degree of expansion per passage is calculated by
dividing the number of cells harvested at the end of the culture by
the number of cells originally seeded into the culture. Where
geometry of the culture environment is limiting or for other
reasons, the cells may optionally be passaged into a similar
culture environment for further expansion. The total expansion is
the product of all the expansions in each of the passages. Of
course, it is not necessary to retain all the expanded cells on
each passage. For example, if the cells expand 2-fold in each
culture, but only .about.50% of the cells are retained on each
passage, then approximately the same number of cells will be
carried forward. But after 4 cultures, the cells are said to have
undergone an expansion of 16-fold.
[0078] Cultures of hES cells on mouse embryonic fibroblast (mEF)
feeder cells, or in mEF conditioned medium, have a doubling time of
about 31-33 hours (Example 1). Certain culture environments of this
invention comprising fresh medium support doubling of hES cells in
less than .about.24 hours (Example 8), potentially in less than
.about.16 hours. In terms of expansion upon regular passaging in
standard culture wells, the system can be used to expand hES cells
by 10- to potentially 50-fold per week. Improved efficiency is
believed to be the result both of the more rapid doubling time, and
the higher proportion of pPS cells that take in the new environment
after passaging.
[0079] Of course, culture conditions inappropriate for pPS cells
will cause them to differentiate promptly. However, the reader
should be aware that marginally beneficial conditions may allow pPS
cells to go through a few passages while still retaining a
proportion of undifferentiated cells. In order to test whether
conditions are adequate for indefinite culture of pPS cells, it is
recommended that the cells be expanded at least 10- or 20-fold
though at least 4 passages. A higher degree of expansion and/or a
higher number of passages (e.g., at least 7 passages and 50- or
100-fold expansion) provides a more rigorous test. It is
permissible for a few phenotypic markers to undertake a
quantitative adjustment befitting adaptation to particular
conditions (say, up or down 2- or 5-fold)--they will typically
revert to previous levels when the cells are placed back into their
previous environment (Example 8). An effective test for whether a
cell is still pluripotent is the demonstration that the cell can
still be caused to differentiate into progeny that represents (or
bears antibody or PCR-detectable phenotypes) of each of the three
embryonic germ layers.
[0080] Nutrient medium and other culture characteristics formulated
according to this invention can be adapted to any culture device
suitable for growing pPS cells. Devices having a suitable surface
include regular tissue culture wells, T-flasks, roller bottles,
gas-permeable containers, and flat or parallel plate bioreactors.
Also contemplated are culture environments in which the pPS cells
are attached to microcarriers or particles kept in suspension in
stirred tank vessels. Fresh medium can be introduced into any of
these environments by batch exchange (replacement of spent medium
with fresh medium), fed-batch process (fresh medium added with no
removal), or ongoing exchange in which a proportion of the medium
is replaced with fresh medium on a continuous or periodic
basis.
Characteristics of Undifferentiated pPS Cells
[0081] Human ES cells have the characteristic morphological
features of undifferentiated stem cells. In the two dimensions of a
standard microscopic image, hES cells have high nuclear/cytoplasmic
ratios in the plane of the image, prominent nucleoli, and compact
colony formation with poorly discernable cell junctions. Cell lines
can be karyotyped using a standard G-banding technique (available
at many clinical diagnostics labs that provides routine karyotyping
services, such as the Cytogenetics Lab at Oakland Calif.) and
compared to published human karyotypes. It is desirable to obtain
cells that have a "normal karyotype", which means that the cells
are euploid, wherein all human chromosomes are present and are not
noticeably altered.
[0082] hES and hEG cells can also be characterized by expressed
cell markers detectable by antibody (flow cytometry or
immunocytochemistry) or by reverse transcriptase PCR. Human ES
cells typically have antibody-detectable SSEA-4, Tra-1-60, and
Tra-1-81, but little SSEA-1. pPS cells can also be characterized by
the presence of 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.). Expression
of hTERT and OCT-4 (detectable by RT-PCR) and telomerase activity
(detectable by TRAP assay) are also characteristic of many types of
undifferentiated pPS cells (Example 3).
[0083] Another desirable feature of propagated pPS cells is a
potential to differentiate into cells of all three germ layers:
endoderm, mesoderm, and ectoderm. Pluripotency of hES cells can be
confirmed by forming teratomas in SCID mice, and examining them for
representative tissues of all three germ layers. Alternatively,
pluripotency can be determined by allowing pPS cells to
differentiate non-specifically (for example, by forming embryoid
bodies), and then determining the cell types represented in the
culture by immunocytochemistry (FIG. 10). Potential of pPS cells to
differentiate into particular cell lines can be determined
according to procedures described later in this disclosure.
[0084] Certain cell populations described in this disclosure are
substantially undifferentiated, and can be passaged between
multiple cultures in the conditions described. During passage, some
cells may differentiate (particularly when replated as single cells
at low density, or when large clusters are allowed to form).
However, cultures typically reestablish a larger proportion of
undifferentiated cells as they reapproach confluence.
Genetic Alteration of Pluripotent Stem Cells
[0085] This disclosure also provides a system for obtaining pPS
cells that have been genetically altered, either in a transient or
stable fashion. The cells may be modified to give them desired
properties in the undifferentiated state, to give them desired
properties after differentiation into other cell types, or to
provide a method to positively or negatively select for particular
undifferentiated or differentiated phenotypes.
[0086] For therapeutic applications, it may be beneficial to modify
cells with therapeutic genes, or to render cells histocompatible
with the intended recipient. Genetic alteration can also be used to
prepare cells for sorting after differentiation. For example, the
hES cells are transfected with a drug susceptibility gene, such as
herpes simplex virus thymidine kinase (which renders cells
susceptible to ganciclovir), under control of a promoter specific
for undifferentiated cells, such as the OCT-4 promoter or the hTERT
promoter (WO 02/42445). After the culture has been made to
differentiate, residual undifferentiated cells can be eliminated
from the population using ganciclovir.
[0087] Suitable vector plasmids for transfecting into hES cells
include lipid/DNA complexes, such as those described in U.S. Pat.
Nos. 5,578,475; 6,020,202; and 6,051,429. Suitable reagents for
making DNA-lipid complexes include lipofectamine (Gibco/Life
Technologies #11668019) and FuGENE.TM. 6 (Roche Diagnostics Corp.
#1814443); and LipoTAXI.TM. (Invitrogen Corp., #204110). Viral
vector systems for producing hES cells with stable genetic
alterations can be based on adenovirus, retrovirus, or lentivirus,
prepared using commercially available virus components.
[0088] Genetic alteration of hES cells requires achieving
sufficiently high efficiency of genetic alteration, while not
promoting differentiation of the hES cells along an undesired
pathway.
[0089] Efficiencies of genetic alteration are rarely 100%, and it
is usually desirable to enrich the population for cells that have
been successfully altered. The genetically altered cells can be
enriched by taking advantage of a functional feature of the new
genotype. For example, where the pPS cells are transfected with a
label such as GFP, or with an immunostainable surface marker such
as NCAM, then the pPS cells can be suspended, separated by
fluorescence-activated cell sorting, and replated. The reader is
cautioned that complete dissociation of pPS cells usually promotes
differentiation.
[0090] A particularly effective way of enriching genetically
altered cells is positive selection using resistance to a drug such
as neomycin. To accomplish this, the cells can be genetically
altered by contacting simultaneously with vector systems for the
marker gene or gene of interest, and a vector system that provides
the drug resistance gene. If the proportion of drug resistance gene
in the mixture is low (say, 3:1), then most drug resistant cells
should also contain the gene of interest. Alternatively, the drug
resistance gene can be built into the same vector as the gene of
interest. After transfection has taken place, the cultures are
treated with the corresponding drug, and untransfected cells are
eliminated.
[0091] pPS cells are especially amenable to genetic alteration when
they are grown in feeder-free culture, elaborated throughout this
disclosure. Transient transfection using DNA/lipid complexes can be
as high as 60%. The cells are easier to manipulate, and there are
no feeder cells around to act as a sink for the vector. Drug
selection does not require availability of a drug-resistant feeder
cell. The number of undifferentiated pPS colonies that grow out
after transfection may also be improved.
[0092] Following genetic alteration and drug selection (on
drug-resistant feeders or feeder-free culture), it is possible to
pick colonies that demonstrate the altered phenotype, and culture
them separately. The picked colonies are dispersed into small
clumps of 25-100 cells, and replated in a suitable environment. It
is possible to achieve cultures of pPS cells in which a high
proportion (up to 90%) of the undifferentiated cells are
genetically altered.
Differentiation of Propagated pPS Cells
[0093] pPS cells cultured according to this invention can be used
to make differentiated cells of various commercially and
therapeutically important tissue types.
[0094] For example, scientists at Geron Corporation have discovered
methods for obtaining highly enriched populations of cells of the
neural lineage. Cells are changed to a culture medium containing
one or more neurotrophins (such as neurotrophin 3 or brain-derived
neurotrophic factor) and one or more mitogens (such as epidermal
growth factor, basic fibroblast growth factor, platelet-derived
growth factor, insulin-like growth factor 1, and erythropoietin).
Cultured cells are optionally separated based on whether they
express a marker such as A2B5 or NCAM. Neural precursors can be
obtained having the capacity to generate both neuronal cells
(including mature neurons), and glial cells (including astrocytes
and oligodendrocytes). Alternatively, replicative neuronal
precursors can be obtained that have the capacity to form
differentiated cell populations in which at least .about.5% of all
the cells in the population express tyrosine hydroxylase, a marker
of dopaminergic neurons. See PCT publication WO 01/88104 and PCT
application PCT/US01/15861.
[0095] Scientists at Geron Corporation have discovered that
culturing pPS cells or embryoid body cells in the presence of a
histone deacetylase inhibitor such as n-butyrate creates a
population of cells highly enriched for markers of the hepatocyte
lineage. The cultured cells are optionally cultured simultaneously
or sequentially with a hepatocyte maturation factor, such as EGF,
insulin, and FGF. Further details can be found in PCT publication
WO 01/81549.
[0096] Scientists at Geron Corporation have developed methods for
generating and purifying hES cell derived cells that have
characteristic markers of cardiomyocytes and spontaneous periodic
contractile activity. Differentiation is facilitated by nucleotide
analogs that affect DNA methylation (such as 5-aza-deoxy-cytidine),
growth factors, and bone morphogenic proteins. The cells can be
further enriched by density-based cell separation, and maintained
in media containing creatine, carnitine, and taurine. See PCT
application PCT/US02/22245.
[0097] Scientists at Geron Corporation have also discovered methods
for differentiating hES cells into mesenchymal cells in a medium
containing a bone morphogenic protein (BMP), a ligand for the human
TGF-.beta. receptor, or a ligand for the human vitamin D receptor.
The medium may further comprise dexamethasone, ascorbic
acid-2-phosphate, and sources of calcium and phosphate. Under
certain circumstances, derivative cells can have phenotypic
features of cells of the osteoblast lineage. See PCT application
PCT/US02/20998.
[0098] For therapeutic use, it is usually desirable that
differentiated cell populations be substantially free of
undifferentiated pPS cells. One way of depleting undifferentiated
stem cells from the population is to transfect them with a vector
in which an effector gene under control of a promoter (such s the
TERT promoter) that causes preferential expression in
undifferentiated cells. For further elaboration, the reader is
referred to PCT publication WO 02/42445.
Uses of Propagated pPS Cells and their Derivatives
[0099] This description provides a method by which large numbers of
pluripotent cells can be produced commercially without the need of
feeder cells, and then differentiated into committed precursor
cells or terminally differentiated cells. These cell populations
can be used for a number of important purposes. The use of pPS
cells for genomic analysis or to produce transcript libraries and
specific antibodies is further detailed in PCT publication WO
01/51616.
Screening Proliferation Factors, Differentiation Factors, and
Pharmaceuticals
[0100] 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 pPS cells in 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. The
conditioned medium is withdrawn from the culture and a simpler
medium (such as KO DMEM) is substituted. Different wells are then
treated with different cocktails of soluble factors that are
candidates for replacing the components of the conditioned medium.
Efficacy of each mixture is determined if the treated cells are
maintained and proliferate in a satisfactory manner, optimally as
well as in conditioned medium. 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.
[0101] Feeder-free pPS cultures can also be used for the testing of
pharmaceutical compounds in drug research. Assessment of the
activity of candidate pharmaceutical compounds generally involves
combining the differentiated cells of this invention with the
candidate compound, determining any resulting change, and then
correlating the effect of the compound with the observed change.
The screening may be done, for example, either because the compound
is designed to have a pharmacological effect on certain cell types,
or because a compound designed to have effects elsewhere may have
unintended side effects. Two or more drugs can be tested in
combination (by combining with the cells either simultaneously or
sequentially), to detect possible drug-drug interaction effects. In
some applications, compounds are screened initially for potential
toxicity (Castell et al., pp 375-410 in In vitro Methods in
Pharmaceutical Research, Academic Press, 1997). Cytotoxicity can be
determined by the effect on cell viability, survival, morphology,
on the expression or release of certain markers, receptors or
enzymes, on DNA synthesis or repair, measured by
[.sup.3H]-thymidine or BrdU incorporation, or on sister chromatid
exchange, determined by metaphase spread. The reader is referred
generally to the standard textbook In vitro Methods in
Pharmaceutical Research, Academic Press, 1997, and U.S. Pat. No.
5,030,015.
Therapeutic Compositions
[0102] Differentiated cells of this invention can also be used for
tissue reconstitution or regeneration in a human patient in need
thereof. The cells are administered in a manner that permits them
to graft to the intended tissue site and reconstitute or regenerate
the functionally deficient area.
[0103] In one example, neural stem cells are transplanted directly
into parenchymal or intrathecal sites of the central nervous
system, according to the disease being treated. Grafts are done
using single cell suspension or small aggregates at a density of
25,000-500,000 cells per .mu.L (U.S. Pat. No. 5,968,829). The
efficacy of neural cell transplants can be assessed in a rat model
for acutely injured spinal cord as described by McDonald et al.
(Nat. Med. 5:1410, 1999), and Kim et al. (Nature 418:50, 2002). A
successful transplant will show transplant-derived cells present in
the lesion 2-5 weeks later, differentiated into astrocytes,
oligodendrocytes, and/or neurons, and migrating along the cord from
the lesioned end, and an improvement in gait, coordination, and
weight-bearing.
[0104] The efficacy of cardiomyocytes can be assessed in an animal
model for cardiac cryoinjury, which causes 55% of the left
ventricular wall tissue to become scar tissue without treatment (Li
et al., Ann. Thorac. Surg. 62:654, 1996; Sakai et al., Ann. Thorac.
Surg. 8:2074, 1999, Sakai et al., J. Thorac. Cardiovasc. Surg.
118:715, 1999). Successful treatment will reduce the area of the
scar, limit scar expansion, and improve heart function as
determined by systolic, diastolic, and developed pressure. Cardiac
injury can also be modeled using an embolization coil in the distal
portion of the left anterior descending artery (Watanabe et al.,
Cell Transplant. 7:239, 1998), or by ligation of the left anterior
descending coronary artery (Min et al., J. Appl. Physiol. 92:288,
2002). Efficacy of treatment can be evaluated by histology and
cardiac function. Cardiomyocyte preparations embodied in this
invention can be used in therapy to regenerate cardiac muscle and
treat insufficient cardiac function (U.S. Pat. No. 5,919,449 and WO
99/03973).
[0105] Hepatocytes and hepatocyte precursors can be assessed in
animal models for ability to repair liver damage. One such example
is damage caused by intraperitoneal injection of D-galactosamine
(Dabeva et al., Am. J. Pathol. 143:1606, 1993). Efficacy of
treatment can be determined by immunocytochemical staining for
liver cell markers, microscopic determination of whether
canalicular structures form in growing tissue, and the ability of
the treatment to restore synthesis of liver-specific proteins.
Liver cells can be used in therapy by direct administration, or as
part of a bioassist device that provides temporary liver function
while the subject's liver tissue regenerates itself following
fulminant hepatic failure.
[0106] For purposes of commercial distribution, cells prepared
according to this invention are typically supplied in the form of a
pharmaceutical composition comprising an isotonic excipient, and
prepared under conditions that are sufficiently sterile for human
administration. For general principles in medicinal formulation of
cell compositions, the reader is referred to Cell Therapy: Stem
Cell Transplantation, Gene Therapy, and Cellular Immunotherapy, by
G. Morstyn & W. Sheridan eds, Cambridge University Press, 1996;
and Hematopoietic Stem Cell Therapy, E. D. Ball, J. Lister & P.
Law, Churchill Livingstone, 2000. The cells may be packaged in a
device or container suitable for distribution or clinical use,
optionally accompanied by information relating to use of the cells
in tissue regeneration, or restoring a therapeutically important
metabolic function.
[0107] The examples that follow are provided by way of further
illustration, and are not meant to imply any limitation in the
practice of the claimed invention.
Examples
Example 1
Growing hES Cells Without Feeder Cells in Conditioned Medium
[0108] In this example, undifferentiated hES cells that had been
maintained on primary mouse embryonic feeder cells were 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.
[0109] Conditioned medium (CM) was prepared as follows. The
fibroblasts were harvested from T150 flasks by washing once with
Ca.sup.++/Mg.sup.++ free PBS and incubating in trypsin/EDTA
(Gibco). After the fibroblasts detached from the flask, they were
collected in mEF medium (DMEM+10% FBS). The cells were irradiated
at 4000 rad, counted and seeded at about 55,000 cells cm.sup.-2 in
mEF medium. After at least 4 hours, the medium was exchanged with
SR containing ES medium. Conditioned medium was collected daily for
feeding of hES cultures. Alternatively, medium was prepared using
mEF plated in culture flasks, exchanging medium daily at 0.3-0.4 mL
cm.sup.-2. Before addition to the hES cultures, the conditioned
medium was supplemented with 4 ng/mL of human bFGF (Gibco).
Fibroblast cultures were used in this system for about 1 week,
before replacing with newly prepared cells.
[0110] 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 by scraping and dissociating
into small clusters in conditioned medium (CM). These cells were
then seeded onto Matrigel.RTM. coated plates (0.75-1 mL diluted
.about.1:30) in conditioned medium at 15 colonies to each 9.6
cm.sup.2 well.
[0111] The day after seeding on Matrigel.RTM., hES cells were
visible as small colonies and there were 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.
[0112] Six days after seeding, the cultures had become almost
confluent. The cultures were split using Collagenase IV, gently
triturated into small clusters of 10-2,000 cells, and then
re-seeded on Matrigel.RTM. coated plates in conditioned medium at
.about.90,000 to 170,000 cells cm.sup.-2. Medium was changed daily,
and the cells were split and passaged again at 13 and 19 days after
initial seeding.
[0113] 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.
[0114] 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 displaying 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.
[0115] 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), 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 4.
[0116] 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 2
Phenotypic Markers of hES Cells in Feeder-Free Culture
[0117] Undifferentiated hES cells express SSEA-4, Tra-1-60,
Tra-1-81, OCT-4, and hTERT. 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.
[0118] 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. They were labeled with specific primary
antibody and then fluorescent second antibody, and analyzed on a
Flow Cytometer.
[0119] 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.
[0120] FIG. 2 shows marker expression detected by
immunocytochemistry. Cells were incubated with primary antibody,
fixed in 2% paraformaldehyde, and then visualized with
FITC-conjugated goat anti-mouse immunoglobulin. 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.
[0121] Quantitative data on day 19 after initial seeding is shown
in the following table.
TABLE-US-00001 TABLE 1 Phenotype of hES Cells Grown in the Absence
of Feeder Cells Percentage of Cells Marker Specificity Staining
SSEA-4 undifferentiated cells 92% Tra-1-60 undifferentiated cells
92% Tra-1-81 undifferentiated cells 83% SSEA-1 differentiated cells
12%
[0122] FIG. 3 shows OCT-4 and hTERT expression of H1 cells grown on
feeders or in a feeder free environment, as detected by
reverse-transcriptase PCR amplification (detailed in WO
01/51616).
[0123] The POU transcription factor OCT-4 is normally expressed in
the undifferentiated hES cells and is down-regulated upon
differentiation. In this experiment, 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. hTERT and OCT-4
expression was seen in all the culture conditions except
Matrigel.RTM. and regular medium. After exposure of cells to
retinoic acid (RA) or dimethyl sulfoxide (DMSO), factors that
promote cell differentiation, the expression of hTERT was markedly
decreased.
[0124] Telomerase activity was measured by telomeric repeat
amplification protocol (TRAP assay: Kim et al., Science 266:2011,
1997; Weinrich et al., Nature Genetics 17:498, 1997). All the
cultures conditions showed positive telomerase activity after 40
days on Matrigel.RTM., laminin, fibronectin or collagen IV in mEF
conditioned medium.
Example 3
Pluripotency of hES Cells in Feeder-Free Culture
[0125] In vitro differentiation was induced in H1 hES cells
maintained in conditioned medium on Matrigel.RTM., laminin,
fibronectin or collagen IV for 26 days. The hES cells were
dissociated into small clumps by incubating in .about.200 U/mL
collagenase IV at 37.degree. C. for 10 min, and cultured in
suspension to form embryoid bodies (EBs) in medium containing DMEM,
20% FBS (Hyclone), 1 mM glutamine, 0.1 mM .beta.-mercaptoethanol,
and 1% non-essential amino acids (Gibco). After 4 days in
suspension, the aggregates were transferred onto
poly-ornithine-coated plates, and cultured for additional 7 days.
The cultures were then examined for the presence of beating cells,
and processed for immunocytochemistry.
[0126] The staining patterns were consistent with cells of the
neuron and cardiomyocyte lineages (.beta.-tubulin III and cardiac
troponin I, respectively). About 8 days after differentiation,
beating regions were identified in all cultures. There were also
cells staining for .alpha.-fetoprotein, a marker of endoderm
lineage.
[0127] hES cells were also tested for their ability to form
teratomas by intramuscular injection into SCID mice. Cells
maintained on feeders or off feeders were harvested, resuspended in
PBS and injected intramuscularly into SCID/beige mice
(5.times.10.sup.6 cells per site). Tumors were excised and
processed for histological analysis. Cystic epithelial structures,
probable dental component, cartilage and glandular epithelial or
neural components were found in teratomas derived from feeder-free
hES cultures.
Example 4
Sources of Conditioned Medium for Feeder-Free Culture
[0128] Media conditioned by several cell lines were tested for
their ability to support the growth of hES cells in feeder-free
culture. Isolation of primary mouse embryonic fibroblasts (mEF) is
described above. The NHG190 cell line is a telomerized mouse
embryonic fibroblast line described in WO 01/51616. STO is a
transformed mouse fibroblast line available from the ATCC. BJ 5ta
is a telomerized human foreskin fibroblast cell line. hTERT-RPE is
a telomerized human retinal epithelial cell line.
[0129] 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,
counted, and plated into culture vessels. After at least 4 h, the
medium was exchanged with ES medium containing 4 ng/mL bFGF.
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).
[0130] 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 5ta 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 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:
primary mEF, NHG190, STO, and BJ 5ta.
[0131] 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 5ta,
expressed high levels of SSEA-4, Tra-1-60 and Tra-1-81 but low
levels of SSEA-1 as analyzed by FAGS analysis. 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.
[0132] 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
at the concentrations tested, in the absence of feeder cells.
Example 5
Genetic Alteration of hES Cells in Feeder-Free Culture
[0133] hES cells maintained in feeder-free culture on laminin in
conditioned medium were genetically modified by transfecting with a
plasmid carrying green fluorescent protein (GFP) driven by the CMV
promoter.
[0134] hES cells of the H9 line maintained on laminin in
mEF-conditioned medium were transfected with a plasmid carrying GFP
driven by the CMV promoter (ClonTech cat. #6084-1) at 24 or 48 h
after plating. Initial experiments used a mixture of 5 .mu.g of
plasmid and 12 .mu.L of Lipofectamine 2000.TM. (Gibco, cat
#11668-019). Cells received 1 mL of DNA/lipid complex and were
incubated for 4 h at 37.degree. before the addition of 3 mL of
mEF-conditioned medium, and then monitored for GFP expression 24 h
after transfection.
[0135] FIG. 4 shows the results of this experiment. Panel A:
morphology of H9 cells maintained on laminin. Panel B: GFP-positive
cells observed in the same colony shown in A. Panel C: FACS
analysis of % GFP-positive cells in SSEA-4 high population
(undifferentiated cells). Cells were transfected 24 (bar 1 and 2)
or 48 h (bar 3 and 4) after the seeding and analyzed 24 (bar 1 and
3) or 48 h (bar 2 and 4) after the transfection. Bright green cells
were observed in compact areas of undifferentiated ES colonies on
laminin 24 h after transfection (Panels A & B). Transfection at
48 h after initial seeding gave the highest efficiency: 38% of the
cells were GFP-positive as determined by FACS analysis 24 h after
the transfection (Panel C).
[0136] To investigate whether the feeder-free hES cells can undergo
stable genetic modification, H1 hES cells maintained on
Matrigel.RTM. were cotransfected with a mixture of 7.5 .mu.g
plasmid carrying .beta.-galactosidase driven by the EF1a promoter,
and 2.5 .mu.g of plasmid carrying the PGK promoter driving the
neophosphotransferase gene. The cells were transfected 48 h after
plating on Matrigel.RTM. in mEF-conditioned medium. Ten pg of
plasmid plus 15 .mu.L of FuGENE.TM. (Roche Diagnostics Corp.) was
incubated with the cells in 1 mL for 4 h before adding 2.5 mL of
mEF-conditioned medium. After 48 h, medium was exchanged for
mEF-conditioned medium supplemented with 200 .mu.g/mL geneticin.
Cultures were maintained in selection medium with daily medium
exchange for over 21 days. All mock-transfected cultures (those
receiving FuGENE.TM. mixed with water rather than plasmid) died
within 48-72 h. Drug resistant colonies arose in the wells
transfected with both FuGENE.TM. and plasmid at a frequency of
about 1 in 10.sup.5 originally transfected cells. The colonies were
maintained in geneticin-containing mEF-conditioned medium and
expanded.
Example 6
Additives that Promote Undifferentiated ES Cell Growth in Fresh
Medium
[0137] Further experiments were conducted to investigate how
different growth factors influence the proliferation and
maintenance of undifferentiated hES cells of the H9 cell line.
[0138] hES medium contained 20% Serum Replacement (Gibco
#10828-028), 80% Knockout DMEM (Gibco #10829-018), 1% non-essential
amino acids (Gibco #11140-050), 1 mM L-glutamine (Gibco
#15039-027), and 2.5 mM .beta.-mercaptoethanol (Sigma #M7522). This
medium was supplemented with 40 ng/mL bFGF; 15 ng/mL stem cell
factor (SCF, R&D System #255SC); 100 ng/mL leukemia inhibitory
factor (LIF, Sigma #L5283 or Chemicon #LIF 1010); 50 ng/mL ciliary
neurotrophic factor (CNTF, R&D system #257-NT); 50 ng/mL
recombinant human Oncostatin M (OSM, Sigma #O9635); and 15 ng/mL
interleukin 6 (IL-6, R&D System #206-IL).
[0139] The H9 cell line (passage 31) was harvested from a culture
in conditioned medium, plated onto Matrigel.RTM., and cultured with
hES medium with the factors at the concentrations indicated above,
or 5- or 25-fold lower. Cells grown in the fully supplemented
medium displayed undifferentiated hES morphology. A higher degree
of differentiation was observed after 4 passages for the cultures
grown at lower concentrations of the growth factors, and the cells
maintained without growth factors were almost completely
differentiated. These cultures were terminated.
[0140] After 6 passages, cells from the full-strength cocktail were
replated onto Matrigel.RTM. as before, or onto laminin, which is
free of the growth factors contained in the Matrigel.RTM. matrix.
After 8 passages, a large percentage of cells (.about.50-70%) in
cultures grown on Matrigel.RTM. or laminin in this medium continued
to display undifferentiated hES morphology. Some cells on
Matrigel.RTM. or laminin were then passaged into hES medium
containing added 40 ng/mL bFGF; but not SCF, LIF, CNTF, OSM, or
IL-6. The cells continued to show an undifferentiated phenotype for
the next 4 passages.
[0141] FACS analysis for marker expression was conducted using the
following specific antibodies. SSEA 4, clone MC 813 mouse IgG3;
Tra-1 60, mouse IgM; Tra-1 81, mouse IgM; SSEA-1, mouse IgM; c-kit,
BD PharMingen #555714; R-PE labeled mouse anti-human CD117; mouse
IgG1, clone YB5.B8; gp130, R&D System #FAB 228P; R-PE labeled
mouse IgG1, clone 28123.111; R-PE labeled isotype control mouse
IgG1, PharMingen #33815; isotype control mouse IgG3, Sigma #M3645.
Goat anti-mouse IgG3 FITC labeled was obtained from Southern
Biotechnology #1102-02.
[0142] The cells were washed with warm PBS for 3-5 minutes,
incubated with 3 mL 0.5 mM EDTA at 37.degree. C. for 10 min, and
collected into a 15-mL tube containing 10 mL medium. They were spun
down at 1200 rpm (400 g), washed in 1% BSA/PBS, and suspended in
100 .mu.L of diluted primary antibody at 4.degree. C. for 30 min.
After rewashing in 1% BSA/PBS, they were incubated with goat
anti-mouse IgG3 FITC (1:100) at 4.degree. C. for 15-30 min, then
washed and resuspended in 500 .mu.L 1:1000 propidium iodine.
[0143] FIG. 5 (Upper Panel) shows the results of FACS analysis of
markers for undifferentiated phenotype for H9 cells maintained for
8 passages in the growth factor mixture. Expression patterns and
levels of surface markers, including SSEA-1, SSEA-4, Tra 1-60 and
Tra 1-81 in cultures maintained in high concentrations of growth
factors were similar to cells maintained in MEF conditioned medium
(MEF-CM).
[0144] These results confirm that conditioned medium contains
factors that promote stem cell growth without differentiation, and
that these factors can be either be secreted into the medium by
cells being used for the conditioning, or added to the medium
artificially.
[0145] FIG. 5 (Lower Panel) shows receptor-associated molecules
expressed by the H9 cells after 9 passages in MEF-CM (control), or
the artificial mixture of growth factors (GF) on either
Matrigel.RTM. or laminin. The hepatocellular carcinoma cell line
HepG2 serves as a positive control for gp130.
[0146] All cell lines stained positively for histocompatibility
Class I antigen (HLA-ABC), and were negative in the isotype control
(msIgG1). 50-70% of cells in cultures maintained in growth factors
or MEF-CM expressed c-kit (a receptor for stem cell factor) while
less than 20% of cells expressed gp130 (associated with the LIF
receptor). In contrast, almost 100% of HepG2 cells expressed gp130.
This pattern supports the hypothesis that ligands for c-kit help
support undifferentiated hES cell growth.
[0147] Cells passaged on Matrigel.RTM. in hES medium containing the
growth factor cocktail were evaluated for pluripotency. In
differentiation medium containing 80% KO-DMEM, 20% FBS, 1 mM
glutamine, 0.1 mM .beta.-mercaptoethanol, and 1% non-essential
amino acids, the cells readily formed embryoid bodies. After 5
days, the EBs were plated onto gelatin coated plates and
differentiated an additional 16 days. They were then fixed in 4%
paraformaldehyde, permeabilized with ethanol, blocked with 10%
normal goat serum, and then analyzed for phenotypic markers by
indirect antibody staining.
[0148] Immunocytochemical analysis showed the presence of
.beta.-tubulin III in cells having neuronal morphology. Other cells
stained for .alpha.-fetoprotein or smooth muscle actin. This
demonstrates that hES cells cultured in medium comprising SCF and
other factors have the capacity to differentiate into derivatives
of all three germ layers.
[0149] After 14 passages in the full-strength growth factor
cocktail (.about.70 population doublings), about 50-70% of cells
cultured on Matrigel.RTM. or laminin displayed morphology of
undifferentiated hES cells, and had a normal karyotype. Cells
cultured without any growth factor showed almost complete
differentiation after 4 weeks in culture. A high degree of
differentiation was also observed for cultures in which the growth
factors had been diluted by 5- or 25-fold.
[0150] Additional experiments were done to dissect the components
in the factor cocktail essential for hES cell growth. The H9 hES
cell line was cultured in non-conditioned ES medium supplemented
with bFGF alone at high (40 ng/mL) or low (8 ng/mL) concentration,
or bFGF (40 ng/mL) in combination with SCF (15 ng/mL) or Flt-3
ligand (75 ng/mL).
[0151] FIG. 6 shows the results. Cultures with high concentrations
of bFGF contained .about.30-50% cells having undifferentiated
morphology, with a higher proportion in cultures also containing
SCF or Flt-3 ligand. FACS analysis showed that .about.60% of the
cells in these cultures expressed SSEA-4. In comparison, cultures
in conditioned medium contained .about.80% with undifferentiated
morphology and .about.90% expressing SSEA-4.
[0152] In a subsequent experiment, the hES cell line H1 at passage
27 or the H7 line at passage 35 hES cells previously maintained in
conditioned medium were cultured in fresh ES medium containing
these growth factors.
TABLE-US-00002 TABLE 2 Factors added to fresh ES medium for hES
cell culture Condition bFGF Other Growth Factors A 8 ng/mL B C 40
ng/mL D 40 ng/mL SCF (15 ng/mL) E 40 ng/mL Flt-3L (75 ng/mL) F 40
ng/mL TPO (100 ng/mL) G 40 ng/mL LIF (100 ng/mL) H 40 ng/mL SCF (15
ng/mL), IL-6 (15 ng/mL), LIF (100 ng/mL), CNTF (50 ng/mL), OSM (50
ng/mL) I SCF (15 ng/mL) J SCF (100 ng/mL) K Flt-3L (75 ng/mL) L TPO
(100 ng/mL) M SCF (15 ng/mL), Flt-3L (75 ng/mL) N SCF (15 ng/mL),
TPO (100 ng/mL) O SCF (100 ng/mL), Flt-3L (100 ng/mL), IL-6 (15
ng/mL) P 40 ng/mL SCF (15 ng/mL), Flt-3L (75 ng/mL) Q 40 ng/mL SCF
(15 ng/mL), TPO (100 ng/mL)
[0153] Cultures were passaged in these conditions and evaluated on
an ongoing basis by morphological criteria. Many of the conditions
continued to maintain considerable numbers of undifferentiated
colonies. FIG. 7 shows expression of SSEA-4 as evaluated by flow
cytometry (gated for low or high staining level) at passage 9 (H7
cells) or passage 10 (H1 cells) after being transferred from
conditioned medium. H7 cells grown in any of these conditions
showed telomerase activity at passage 15.
Example 7
Other Base Media for Growing hES Cells in Feeder-Free Culture
[0154] hES cells passaged 29 times in conditioned medium were
weaned onto an alternative medium designed for proliferation and
development of hematopoietic cells.
[0155] Ex vivo expansion medium was obtained by arrangement with a
commercial supplier, and is thought to be based on the medium
described in U.S. Pat. No. 5,405,772 (Ponting, Amgen Inc.). The
Ponting medium comprises the following components: Iscove's
modified Dulbecco's medium; amino acids; vitamins, bovine albumin;
bovine transferrin (100 .mu.g/mL); lipids and cholesterol;
.beta.-mercaptoethanol; pyruvate; nucleotides; epidermal growth
factor (15 ng/mL); fibroblast growth factor (2 ng/mL);
platelet-derived growth factor (10 ng/mL); and insulin (10
.mu.g/mL). For use in the current experiments, the medium was
further supplemented with 2 mM L-glutamine, 1% non-essential amino
acids (Gibco), 0.1 mM .beta.-mercaptoethanol, and 8 ng/mL bFGF.
[0156] The cells were first passaged onto Matrigel.RTM. coated
plates using collagenase IV, and cultured for 2 days with
conditioned medium. On day 2, the 100% conditioned medium was
replaced with medium containing 80% conditioned medium plus 20%
fresh expansion medium. Cells were fed fresh daily and passaged
weekly. The proportion of expansion medium was increased by 20%
approximately every 2 days until the cells were completely weaned,
and then grown until they had been passaged a further 8 times.
[0157] At passages 1-4 in the expansion medium, the proportion of
cells with the morphology of undifferentiated phenotype appeared to
diminish slightly, but was restored by passage 8. When these cells
were passaged back to medium conditioned by primary mouse embryonic
fibroblasts, the cells were indistinguishable from those grown
throughout the period in conditioned medium by the second
passage.
[0158] To confirm that these cells retained their pluripotency,
embryoid bodies were formed and analyzed by immunocytochemistry for
phenotypic markers representing each of the three germ layers.
After passage 4 in expansion medium, the cells were dissociated
into small clumps using 200 U/mL collagenase IV at 37.degree. C.
for 10 min placed in suspension culture in differentiation medium
(DMEM+10% FBS) for 4 days, then transferred onto poly-L-ornithine
hydrobromide coated plates and cultured a further 10 days. They
were fixed in 4% paraformaldehyde, permeabilized, and labeled
alternately with mouse anti human .beta.-tubulin isotype III clone
SDL.3D10, mouse anti human muscle actin clone HHF35, or mouse anti
.alpha.-fetoprotein. Primary antibody was visualized using FITC
labeled goat anti-mouse IgG. Results showed that hES cells passaged
repeatedly in expansion medium (not previously conditioned), and
then differentiated, were positive for .beta.-tubulin and muscle
actin.
Example 8
Rapid Expansion Method for Producing Pluripotent Stem Cells
[0159] hES cells passaged 20 times in conditioned medium were
weaned onto an alternative medium designed for proliferation of
human hematopoietic cells. X-VIVO.TM. 10 expansion medium was
obtained from Biowhittaker; QBSF.TM.-60 was obtained from Quality
Biological Inc. The X-VIVO.TM. 10 formulation contains
pharmaceutical grade human albumin, recombinant human insulin and
pasteurized human transferrin. Exogenous growth factors, artificial
stimulators of cellular proliferation or undefined supplements are
not included in the X-VIVO.TM. 10 medium. They are also devoid of
any protein-kinase C stimulators. QBSF.TM.-60 is a serum-free
formulation that contains recombinant or pasteurized human
proteins. For use in these experiments, the X-VIVO.TM. 10 medium
was supplemented with 2 mM L-glutamine, 1% non-essential amino
acids (Gibco), 0.1 mM .beta.-mercaptoethanol, and 8 ng/mL bFGF. The
medium was further supplemented with 8 ng/mL or 40 ng/mL of bFGF
(Gibco); 40 ng/mL of bFGF and 15 ng/mL of SCF (R & D System);
or 40 ng/mL of bFGF and 75 ng/mL of Flt3 ligand (R & D System).
QBSF.TM.-60 medium was supplemented with 0.1 mM
.beta.-mercaptoethanol, 1% non-essential amino acids (Gibco) and 40
ng/mL of bFGF. hES cells cultured in mEF conditioned medium was
used as control in these experiments.
[0160] The hES cells were first passaged onto Matrigel.RTM. coated
plates using collagenase IV, and cultured for 2 days with
conditioned medium. On day 2, the conditioned medium was replaced
with 80% unconditioned ES medium plus 20% expansion medium. Cells
were fed fresh daily and passaged weekly. The proportion of
expansion medium was increased by 20% approximately every 2 days
until the cells were completely weaned, and then grown until they
had been passaged 6 more times.
[0161] FIG. 8 shows colonies of hES cell at the end of 6 passages
(sufficient for full adaptation) in the following media: (A) mEF
conditioned medium+bFGF (8 ng/mL); (B) X-VIVO.TM. 10+bFGF (40
ng/mL); (C) X-VIVO.TM. 10+bFGF (40 ng/mL)+stem cell factor (SCF,
Steel factor) (15 ng/mL); (D) X-VIVO.TM. 10+bFGF (40 ng/mL)+Flt3
ligand (75 ng/mL); (E) QBSF.TM.-60+bFGF (40 ng/mL).
[0162] The following table shows the average total cell expansion
per passage, for undifferentiated hES cells cultured for 4 passages
in mEF conditioned medium, or for 7 passages in X-VIVO.TM. 10 or
QBSF.TM.-60.
TABLE-US-00003 TABLE 3 Growth Rates for ES Cell Cultures Average
Cell Expansion Medium per Passage mEF conditioned medium 2.2 fold
X-VIVO .TM. 10 + bFGF (40 ng/mL) 6.0 fold X-VlVO .TM. 10 + bFGF (40
ng/mL) + SCF (15 ng/mL) 8.2 fold X-VIVO .TM. 10 + bFGF (40 ng/mL) +
Flt3 5.0 fold ligand (75 ng/mL) QBSF .TM.-60 + bFGF (40 ng/mL) 6.4
fold
[0163] The average expansion of cells per passage in X-VIVO.TM. 10
and QBSF.TM.-60 was greater than the cells cultured in mEF
conditioned medium culture. The cells in mEF conditioned medium
were passaged on average every 7 days, while the cells in
X-VIVO.TM. 10 and QBSF.TM.-60 were passaged on average every 5
days. Thus, the rate of expansion in unconditioned X-VIVO.TM. 10 or
QBSF.TM.-60 was .about.3.2 to 5.2 times faster than in mEF
conditioned ES medium.
[0164] FIG. 9 shows the gene expression profile of hTERT and
Oct3/4. The RNA was isolated from the cells using High Pure RNA
Isolation Kit (Roche Diagnostics) and evaluated by Taqman.TM. assay
(real time RT-PCR). The gene expression in each of the test
condition is plotted relative to expression in the control culture.
Taking into consideration the instrument error and assay
variability, differences in expression between the test and control
samples are only significant if greater than 2-fold. The analysis
shows expression of hTERT and Oct-3/4 decreases somewhat upon
adaptation to unconditioned X-VIVO.TM. 10 or QBSF.TM.-60 medium
(first four bars in each set), but returns to standard levels when
the cells are passaged back into mEF conditioned medium (last three
bars in each set).
[0165] To confirm that cells cultured in unconditioned medium
retain their pluripotency, embryoid bodies were formed and analyzed
by immunocytochemistry for phenotypic markers representing each of
the three germ layers. After passage 7 in expansion medium, the
cells were dissociated into small clumps using 200 U/mL collagenase
IV at 37.degree. C. for 10 min, placed in suspension culture in
differentiation medium (DMEM+10% FBS) for 4 days, then transferred
onto poly-L-ornithine hydrobromide coated plates for a further 10
days. They were fixed in 4% paraformaldehyde, permeabilized, and
labeled by immunocytochemistry.
[0166] FIG. 10 shows the results. hES cells passaged 7 times in
unconditioned X-VIVO.TM. 10 medium stained for .alpha.-fetoprotein
(representing endoderm); muscle actin (representing mesoderm), and
.beta.-tubulin III (representing ectoderm).
[0167] These results show that hES cells can be expanded in fresh
(non-conditioned) media in a feeder-free environment at a rapid
rate suitable for commercial production. The cells retain the
morphology of undifferentiated hES cells, and can be differentiated
into derivative cells representing all three germ layers.
[0168] The compositions and procedures provided in the description
can be effectively modified by those skilled in the art without
departing from the spirit of the invention embodied in the claims
that follow.
* * * * *