U.S. patent application number 10/839212 was filed with the patent office on 2005-06-09 for culture system and method for maintenance and proliferation of undifferentiated human embryonic stem cells.
This patent application is currently assigned to Industrial Technology Research. Invention is credited to Chang, Kuang-Ning, Chen, Wann-Hsin, Hsu, Lih-Tao, Huang, Jun-Jae, Kuo, Chao-Ying, Yang, Mei-Ju.
Application Number | 20050124063 10/839212 |
Document ID | / |
Family ID | 32906959 |
Filed Date | 2005-06-09 |
United States Patent
Application |
20050124063 |
Kind Code |
A1 |
Yang, Mei-Ju ; et
al. |
June 9, 2005 |
Culture system and method for maintenance and proliferation of
undifferentiated human embryonic stem cells
Abstract
The present invention discloses an improved culture system for
proliferative and undifferentiated growth of human embryonic stem
cells, comprising an extracellular matrix prepared from feeder
cells and a conditioned medium preconditioned by feeder cells. This
invention also relates to a culture method utilizing the foregoing
culture system.
Inventors: |
Yang, Mei-Ju; (Hsinchu City,
TW) ; Chang, Kuang-Ning; (Taoyuan County, TW)
; Hsu, Lih-Tao; (Taoyuan County, TW) ; Huang,
Jun-Jae; (Miaoli County, TW) ; Chen, Wann-Hsin;
(Hsinchu City, TW) ; Kuo, Chao-Ying; (Hsinchu
City, TW) |
Correspondence
Address: |
BACON & THOMAS, PLLC
625 SLATERS LANE
FOURTH FLOOR
ALEXANDRIA
VA
22314
|
Assignee: |
Industrial Technology
Research
Hsin Chu
TW
|
Family ID: |
32906959 |
Appl. No.: |
10/839212 |
Filed: |
May 6, 2004 |
Current U.S.
Class: |
435/366 ;
435/404 |
Current CPC
Class: |
C12N 5/0606 20130101;
C12N 2500/84 20130101; C12N 2502/13 20130101; C12N 2533/90
20130101 |
Class at
Publication: |
435/366 ;
435/404 |
International
Class: |
C12N 005/08 |
Foreign Application Data
Date |
Code |
Application Number |
Dec 9, 2003 |
TW |
92134707 |
Claims
What is claimed is:
1. A culture system for proliferation and undifferentiated growth
of human embryonic stem cells comprising: a culture matrix
comprising extracellular matrix prepared from feeder cells; and a
conditioned medium being preconditioned by feeder cells; wherein
said feeder cells are selected from primary mouse embryonic
fibroblasts (PMEF), a mouse embryonic fibroblast cell line (MEF),
murine fetal fibroblasts (MFF), human embryonic fibroblasts (HEF),
human fetal muscle (HFM), human fetal skin cells (HFS), human adult
skin cells, human foreskin fibroblasts (HFF), human adult fallopian
tubal epithelial cells (HAFT) or human marrow stromal cells
(hMSCs).
2. The culture system according to claim 1, wherein said feeder
cells are mouse embryonic fibroblasts or human foreskin
fibroblasts.
3. The culture system according to claim 1, wherein said culture
matrix is prepared by removing intracellular substances by means of
treating said feeder cells with NaOH or trinitrotoluene to obtain
extracellular matrix as culture matrix.
4. The culture system according to claim 3, wherein the
intracellular substances are removed by washing with water or
buffered solution.
5. The culture system according to claim 4, wherein said buffered
solution is phosphate buffered saline.
6. The culture system according to claim 1, wherein said
conditioned medium is prepared by the steps of: (a) inactivating
the feeder cells; (b) placing the cells obtained in step (a) in a
culture solution; and (c) collecting the cell culture solution as
conditioned medium.
7. The culture system according to claim 6, wherein said feeder
cells are inactivated by irradiation with gamma ray or treatment
with mitomycin C.
8. The culture system according to claim 7, wherein said feeder
cells are inactivated by treating with mitomycin C.
9. A method for culturing human embryonic stem cells, comprising
the steps of: obtaining human embryonic stem cells; culturing the
human embryonic stem cells in the culture system described
according to claims 1; and continuing the culture to maintain human
embryonic stem cells in a substantially proliferative and
undifferentiated state.
10. The method according to claim 9, wherein said human embryonic
stem cells are maintained in a substantially undifferentiated and
proliferative state over the course of at least five passages in
said culture system.
Description
BACKGROUND OF THE INVENTION
[0001] 1. Field of the Invention
[0002] The present invention relates to the field of human
embryonic stem (HES) cell culture, in particular undifferentiated
HES cell culture, and to methods for maintenance and proliferation
of such cells. More particularly, the invention relates to
maintenance of HES cells in a substantially proliferative and
undifferentiated status in a culture system without feeder
layer.
[0003] 2. Description of Related Art
[0004] Human embryonic stem (HES) cells are derived from the inner
cell mass of blastocyst. Nowadays, most HES cells can only be
maintained in culture in an undifferentiated state when grown on
inactivated feeder cells.
[0005] Previous studies have found that the important factors in
feeder cells that support the growth and proliferation of ES cells
include growth factors secreted by feeder cells in the culture
medium and extracellular matrix (ECM) constructed by the feeder
cells. The feeder cells can be a primary mouse embryonic fibroblast
(PMEF), a mouse embryonic fibroblast cell line (MEF), a murine
fetal fibroblast (MFF), a human embryonic fibroblast (HEF), a human
fetal muscle cell (HFM), a human fetal skin cell (HFS), a human
adult skin cell, a human foreskin fibroblast (HFF), a human adult
fallopian tubal epithelial cell (HAFT) and a human marrow stromal
cells (hMSCs) (WO 03/02944, WO 03/014313, J. H. Park et al., Biol
Reprod., 69: 2007-2017, 2003, M. Amit et al., Biol Reprod., 68 (6):
2150-2156, 2003, Outi Hovattal et al., Hum. Reprod., 18 (7):
1404-1409, 2003, Richards, M. et. Al, Nat Biotechnol., 20 (9):
933-936, 2002, James A. et al., Science, 282 (6): 1145-1147, 1998
and Linzhao Cheng et al., Stem Cells, 21: 131-142, 2003).
[0006] The extracellular matrix (ECM) is not merely a passive
structure. In the past few years, it has emerged that the matrix is
a dynamic action zone that functions to instruct cellular
phenotype. ECM proteins interact directly with cell surface
receptors to initiate signal transduction pathways and to modulate
those triggered by growth factors. ECM also controls the activity
and presentation of a wide range of growth factors. Thus modulation
of the ECM, by remodelling its structure and activity, has profound
effects on its function and the consequent behaviour of cells
residing on or within it. At present, the interacting mechanisms of
the ECM in establishment and maintenance of a ES cell culture is
not known. Possible roles for the ECM include the provision of ECM
components that provide attachment sites for the ES cells, trigger
signaling for cell renewal.
[0007] The feeder layer dependent culture system in scaling up and
impedes the mass production and clinical application of HES cells.
There are some problems in a feeder layer dependent culture system:
(1) the potential risks of transmission of pathogens from the
animal feeder cells to the HES cells and the fact that the current
system of propagation (human/animal or human/human co-culture) has
been construed as a xenotransplant, (2) feeder cells come mainly
from primary cells, while primary cells from different batches
offer different effect as feeder cells, rendering the quality
control of the cultured HES cells more difficult; (3) the limited
sources and numbers of feeder cells hamper the mass production and
applications of HES cells. Therefore, the method for the
maintenance and proliferation of undifferentiated HES cells without
feeder cells is critical for mass product and clinical application
of HES cells. (U.S. 2003/143736).
[0008] Xu et al. (Nat. Biotechnol., 19 (10): 971-974, 2001. WO
03/020920 and U.S. 2003/0017589) were the first to successfully
maintain undifferentiated HES cells in a feeder-free culture
system. In this system, HES cells are cultured on Matrigel from the
Engelbreth Holm Swarm (EHS) sarcoma or laminin in medium
conditioned by MEF. However, such synthetic matrices and
defined-matrix marcromolecules are not sufficient to mimic the more
complex cell-martix interactions provided by feeder cells. A study
has also indicated that this culture system is only suitable for
certain HES cell lines (e.g. H1 and H9), but unsustainable for
other HES cell lines (Outi Hovattal et al. Hum. Reprod., 18 (7):
1404-1409, 2003).
[0009] Accordingly, it is an object of the present invention to
provide an alternative feeder-free culture system to overcome some
of the disadvantages of the prior art.
SUMMARY OF THE INVENTION
[0010] The present invention provides methods and culture system
for culturing undifferentiated HES cells. The methods and culture
system described herein provide improved culture conditions that
allow the maintenance and proliferation of HES cells in a
substantially undifferentiated state.
[0011] In one aspect, the present invention provides a cell culture
system for growing HES cells in a substantially undifferentiated
state. The cell culture system of the invention comprises ECM as
culture matrix and conditioned medium.
[0012] The feeder cells to be used in the present invention can be,
for example, but not limited to primary mouse embryonic fibroblasts
(PMEF), a mouse embryonic fibroblast cell line (MEF), murine fetal
fibroblasts (MFF), human embryonic fibroblasts (HEF), human fetal
muscle cells (HFM), human fetal skin cells (HFS), human adult skin
cells, human foreskin fibroblasts (HFF), human adult fallopian
tubal epithelial cells (HAFT) and human marrow stromal cells
(hMSCs).
[0013] In another aspect, the present invention provides a culture
method for growing HES cells in a substantially undifferentiated
state, comprising culturing undifferentiated HES cells in a
feeder-free culture system of the invention. The HES cells cultured
in such culture system may be maintained in substantially
proliferative and undifferentiated state for at least five
passages.
BRIEF DESCRIPTION OF THE DRAWINGS
[0014] FIG. 1 shows the flow chart of culturing HES cells in the
feeder-free culture system according to this invention.
[0015] FIG. 2A shows the morphology of primary mouse embryonic
fibroblasts (PMEF) (300.times.).
[0016] FIG. 2B shows the structure of ECM of PMEF (300.times.).
[0017] FIG. 2C shows the macrofibril bundles and network structure
of ECM of PMEF observed under scanning electron microscope
(2000.times.).
[0018] FIG. 3A shows the morphology of human foreskin fibroblast
(HFF) (300.times.).
[0019] FIG. 3B shows the structure of ECM of HFF (300.times.).
[0020] FIG. 4A shows the morphology of HES cells (HES3) cultured on
ECM prepared from PMEF according to this invention (10.times.).
[0021] FIG. 4B shows the morphology of HES3 cells cultured on ECM
prepared from HFF according to this invention (10.times.).
[0022] FIG. 5A shows high alkaline phosphatase activity in HES3
cells cultured on ECM prepared from PMEF according to this
invention (10.times.).
[0023] FIG. 5B shows high alkaline phosphatase activity in HES3
cells cultured on ECM prepared from HFF according to this invention
(10.times.).
[0024] FIG. 6 shows the OCT-4 expression of HES3 cells cultured in
the feeder-free culture system according to this invention.
DETAILED DESCRIPTION OF THE INVENTION
[0025] In one aspect, the present invention relates to a cell
culture system for growing HES cells in a substantially
undifferentiated state. This invention is characterized by the
removal of feeder cells, while retaining the ECM structure and
nutrients secreted by the feeder cells, hence developing a
feeder-free culture system for HES cells, which breaks the
bottleneck facing the large scale production and clinical
application of HES cells.
[0026] Specifically, this invention provides a culture system for
growing HES cells, comprising a culture matrix consisting of ECM
prepared from feeder cells and a conditioned medium being
preconditioned by feeder cells.
[0027] The aforesaid culture matrix provides substratum for cell
attachment during culture and helps to maintain the HES cells in a
substantially undifferentiated state.
[0028] The aforesaid feeder cells may be fibroblasts or other types
of cells, which may be inactivated by large-dose radiation, such as
y-ray, or by drug, such as mitomycin C, so that the surviving cells
lose the capability to proliferate, but retain their physiological
functions, such as metabolism and synthesis of growth factors.
Specifically, the feeder cell may be selected from the group
consisting of primary mouse embryonic fibroblasts (PMEF), a mouse
embryonic fibroblast cell line (MEF), murine fetal fibroblasts
(MFF), human embryonic fibroblasts (HEF), human fetal muscle cells
(HFM), human fetal skin cells (HFS), human adult skin cells, human
foreskin fibroblasts (HFF), human adult fallopian tubal epithelial
cells (HAFT) and human marrow stromal cells (hMSCs). In the
preferred embodiments of the invention, the feeder cells are
derived from mouse embryonic fibroblasts (MEF) or human foreskin
fibroblasts (HFF).
[0029] In the preferred embodiment of the invention, said HES cells
are HES-3 or HES-4 cell lines.
[0030] The preparation of ECM may be performed by those skilled in
the art or refer to R. Ian Freshney (2000) Culture of Animal Cells:
A Manual of Basic Technique, 4th Edition, Wiley-Liss, Inc.
Basically, feeder cells were treated with NaOH or trinitrotoluene
(Triton) and followed by removing intracellular substances, such as
nucleus or organelles, so as to obtain the ECM as culture matrix.
The removal of intracellular substances may be achieved by washing
with buffered solution or water.
[0031] The aforesaid conditioned medium refers to culture medium in
which feeder cells have been cultivated already for a period of
time and thus is preconditioned by feeder cells. The preparation of
conditioned medium may be performed by those skilled in the art or
refer to U.S. Pat. Nos. 5,690,926, 2003/0008392, 2003/0073234, and
2002/0160509 as well as Reubinoff B. E. et al, Nat. Biotechnol., 18
(4): 399-404, 2002. The major ingredients of a conditioned medium
are typically amino acids, vitamins, carbohydrates, inorganic ions
and some other auxiliary substances. Growth factors that promote
cell growth or inhibit differentiation, e.g. leukemia inhibitory
factor (ILF), fibroblast growth factor (FGF), stem cell factor
(SCF), insulin-transferrin-selenium G supplement (ITS G supplement)
may also be added into the culture medium according to WO
03/020920, U.S. 2003/0017589, U.S. Pat. No. 5690926, U.S. Pat. No.
5,453,357, Xu, C. et al., Nat. Biotechnol., 19 (10): 971-974, 2001,
and Richards, M. et. Al, Nat Biotechnol., 20 (9): 933-936,
2002.
[0032] It is more specific that the preparation of the aforesaid
conditioned medium comprises of the steps of: (a) inactivating the
feeder cells; (b) placing the cells obtained in step (a) in a
culture solution; and (c) collecting the cell culture solution as
conditioned medium. In another aspect, the present invention
provides a culture method for growing HES cells in a substantially
undifferentiated state, comprising culturing undifferentiated HES
cells in the cell culture system of this invention as shown in FIG.
1. The method comprises the following steps: obtaining HES cells;
culturing the HES cells in the feeder-free culture system described
above; and maintaining the HES cells in substantially proliferative
and undifferentiated state, wherein HES cells in said feeder-free
culture system may be maintained in substantially proliferative and
undifferentiated state for at least five passages.
[0033] Definitions
[0034] The following terms will be defined as provided unless
otherwise stated. All other terminology used herein will be defined
with respect to its usage in the particular art to which it
pertains unless otherwise noted.
[0035] "Conditioned Medium"
[0036] Conditioned Medium as used for the purpose of describing the
present invention refers to the medium in which feeder cells have
been cultivated already for a period of time. The conditioned
medium of the present invention can be used for cultivation of HES
cells because it contains many mediator substances, such as growth
factors and cytokines, that were secreted by the feeder cells
cultivated previously and can thus help to promote the growth of
HES cells.
[0037] "Extracellular Matrix"
[0038] Extracellular Matrix or ECM or Defined Matrix occupies the
space between cells and establishes a complex network of different
combinations of collagens, proteoglycans, hyaluronic acid, laminin,
fibronectin, and many other glycoproteins including proteolytic
enzymes involved in degradation and remodeling of the ECM. ECM
plays an important structural and functional role in multicellular
organisms and is more than a scaffold that fills extracellular
spaces. Many of its components are engaged in processes mediating
cell-to-cell interactions. In the present invention, ECM serves as
culture matrix prepared from feeder cells so as to provide a
support in a feeder-free culture environment for HES cells.
[0039] "Feeder Cells"
[0040] Feeder Cells as used for the purposes of describing the
present invention refers to those used as a substratum on which
other cells are grown in a culture system. Feeder cells are usually
adherent growth-arrested but viable and bioactive cells (primary
cells or continuous cell lines) that have been incapacitated, for
example by irradiation.
[0041] All other acronyms and abbreviations have the corresponding
meaning as published in journals related to the arts of chemistry
and biology.
[0042] The following examples are presented in order to more fully
illustrate the preferred embodiments of the invention. They should
in no way be construed, however, as limiting the broad scope of the
invention. While the invention is described and illustrated herein
by references to various specific material, procedures and
examples, it is understood that the invention is not restricted to
the particular material combinations of material, and procedures
selected for that purpose. Numerous variations of such details can
be implied as will be appreciated by those skilled in the art.
EXAMPLE 1
Preparation of the Compositions of the Culture System
[0043] 1. Preparation of Conditioned Medium
[0044] Conditioned medium for maintaining HES cells was prepared
using the following procedure. Primary mouse embryonic fibroblasts
(PMEF) or human foreskin fibroblast (from Animal Technology
Research Institute, Taiwan) in the presence of a growth medium
prepared from 10% fetal bovine serum (FBS, from HyClone) and 90%
Dulbecco's Modified Eagle Medium (DMEM, from Gibco). When the cells
reach confluence, mitomycin C was added to inactivate the
fibroblasts. These fibroblasts were grown in the presence of a
growth medium prepared from 80% Dulbecco's modified eagle medium
(DMEM, from Gibco), 20% fetal bovine serum (FBS, from Hyclone), and
supplemented with 1 mM .beta.-mercaptoethanl (from Gibco), 1%
non-essential amino acids (from Gibco), 1% glutamine (from Gibco),
and 1% insulin-transferrin-selenium G supplement (ITS G supplement,
from Gibco) (refer to Richards, M. et al., Nat. Biotechnol., 20
(9): 933-936, 2002), in which fetal bovine serum may be substituted
by serum replacement to obtain serum-free culture medium. The ES
medium was collected and filter-sterilized (0.2 micron filter).
This medium was termed "conditioned ES medium". The conditioned ES
medium was used immediately or frozen at about -20.degree. C. until
needed. Based on the requirements of the cultured cells, the growth
medium may contain other ingredients without limited to those
discussed herein.
[0045] 2. Preparation of Culture Matrix
[0046] Culture matrix for maintaining HES cells was prepared using
the following procedure. Primary Mouse embryonic fibroblasts (PMEF)
or human foreskin fibroblasts (from Animal Technology Research
Institute, Taiwan) were grown to confluence in the presence of a
growth medium prepared from 10% fetal bovine serum (FBS, from
HyClone) and 90% Dulbecco's Modified Eagle Medium (DMEM, from
Gibco). When the cells reached confluence, mitomycin C was added to
inactivate the fibroblasts. For harvesting the culture matrix,
0.05N NaOH or 0.1% trinitrotoluene (from Sigma) was used to break
the cell membranes and followed by washing with pH 7.4 Dulbecco's
Phosphate Buffered Saline (1X, from Gibco) to remove organelle and
nucleus so as to harvest the culture matrix.
EXAMPLE 2
Culturing HES Cells using the Culture System of this Invention
[0047] HES cells (HES-3 or HES-4 cell lines) were plated onto the
culture matrix and incubated with conditioned medium mentioned
above and cultured in a 5% CO.sub.2 incubator under 37.degree. C.
The medium was changed every 1-2 days. After 7 days of culture, HES
cells were carried on subculture of cells.
EXAMPLE 3
Observing the Culture Matrix of Culture System
[0048] FIG. 2A shows the morphology of primary mouse embryonic
fibroblasts. The culture matrix derived from primary mouse
embryonic fibroblasts following the steps in Example 1 is shown in
FIG. 2B. When observed under scanning electron microscope, the
macrofibril bundles and network structure of said culture matrix
are visible as shown in FIG. 2C.
[0049] FIG. 3A and FIG. 3B shows respectively the morphology of
human foreskin fibroblast and the structure of culture matrix
prepared from human foreskin fibroblast according to the steps
described above.
EXAMPLE 4
Analyzing the Undifferentiated State of HES Cells
[0050] The effect of the culture system herein may be further
observed using biomarkers specifically expressed in
undifferentiated HES cells, e.g. alkaline phosphatase activity,
OCT-4, SSEA-3, SEA-4, TRA-1-60, and TRA-1-81 (refer to Thaomson J.
A. et al, Science, 282 (6): 1145-1147, 1998 or Reubinoff B. E. et
al. Nat Biotechnol. 18 (4): 399-404, 2000) to determine the
undifferentiated level of ES cells.
[0051] This invention uses the expression of alkaline phosphatase
activity and OCT-4 to evaluate the effect of the feeder-free
culture system herein. The assay of alkaline phosphatase activity
was employed according to the protocols provided within the
Alkaline phosphatase substrate kit (Vector Laboratories, Inc.). The
assay of OCT-4 was carried out according to the method described by
Richards, M. et. Al, Nat Biotechnol., 20 (9): 933-936, 2002. The
results are presented as follows:
[0052] 1. Observation of Cell Morphology:
[0053] FIG. 4A and FIG. 4B show the morphology of HES cells
cultured in the culture system herein using primary mouse embryonic
fibroblasts and human foreskin fibroblasts as culture matrix,
respectively.
[0054] 2. Assay for Alkaline Phosphatase Activity:
[0055] HES cells show high alkaline phosphatase activity before
differentiation, i.e., once they start to differentiate, they lose
the alkaline phosphatase activity. Thus the differentiation status
of cultured HES cells can be learned from the this enzyme activity.
The assay results show that HES cells cultured in this culture
system using either primary mouse embryonic fibroblasst or human
foreskin fibroblasts as culture matrix express high alkaline
phosphatase activity (in bright red color as a result of stain),
indicating that they were in a substantially undifferentiated state
(FIG. 5A and FIG. 5B). The results also showed that HES cells may
be continuously subcultured for at least five passages and
maintained the characteristics of substantial proliferation and
undifferentiation in the feeder-free culture system of the present
invention.
[0056] 3. Assay for Transcription Factor OCT-4:
[0057] As shown in FIG. 6 which compares the expression of OCT-4 in
HES cells cultured in various culture systems, lane 1 is feeder
cells derived from primary mouse embryonic fibroblasts; lane 2 is
feeder cells derived from human foreskin fibroblasts; lane 3 is
feeder-free culture system prepared according to this invention;
and lane 4 is a cell-free negative control. Transcription factor
OCT-4 can be observed in the ES cells cultured in the feeder-free
culture system herein (lane 3), indicating that this culture system
is able to maintain HES cells in substantially undifferentiated
state. Besides, the expression level of OCT-4 in feeder-free
culture system herein (lane 3) was comparable to that in feeder
layer culture systems (lane 1 and lane 2).
[0058] The feeder-free culture system for ES cells presented herein
offers the following advantages: (1) It prevents the potential
risks of transmission of pathogens from the animal/human feeder
cells to the human HES cells and the fact that the current system
of propagation (human/animal co-culture) has been construed as a
xenotransplant; (2) A better quality control of HES cells can be
reached; (3) A mass/bulk production of HES cells is feasible; and
(4) It sheds light on clinical application of HES cells.
[0059] The examples as disclosed above are used to provide detailed
description and are in no way to be considered to limit the scope
of the invention in any manner. All modifications and alterations
made by those familiar with the skill without departing from the
spirits of the invention and appended claims shall remain within
the protected scope and claims of the invention.
* * * * *